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tyo:4502
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Takeda Pharmaceutical
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Nov 17th, 2020 12:00AM
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Aug 3rd, 2017 12:00AM
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https://www.uspto.gov?id=US10835597-20201117
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Compositions and methods for stabilizing flaviviruses with improved formulations
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Embodiments herein relate to compositions and methods for stabilizing Flaviviruses. In certain embodiments, compositions and methods disclosed herein concern stabilizing live, attenuated or unattenuated (e.g. live whole) flaviviruses. Other embodiments relate to compositions and methods for reducing degradation of live, attenuated or unattenuated flaviviruses. Other embodiments relate to improved formulations for prolonging stabilization of live attenuated or unattenuated Flaviviruses during manufacturing, storage, accelerated storage and transport. Yet other embodiments relate to uses of compositions disclosed herein in kits for transportable applications and methods.
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10835597
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1. A flavivirus composition comprising:
one or more live flaviviruses;
at least one of trehalose in a concentration ranging from 5.0% to 15.0% (w/v) and sucrose in a concentration ranging from 5.0% to 15.0% (w/v);
urea;
mannitol; and
human serum albumin.
2. The flavivirus composition according to claim 1, wherein the composition further comprises a base buffer, the base buffer comprises phosphate buffered saline (PBS), HEPES buffer, histidine buffer or Tris buffer.
3. The flavivirus composition according to claim 1, comprising at least one salt comprising sodium chloride (NaCl), sodium phosphate (Na2HPO4), potassium chloride (KCl) or potassium phosphate (KH2PO4).
4. The flavivirus composition according to claim 3, wherein the at least one salt comprises NaCl at a concentration of 10 mM to 200 mM.
5. The flavivirus composition according to claim 1 further comprising, one or more amino acids or derivatives thereof or salts, esters or amide derivatives thereof.
6. The flavivirus composition according to claim 5, wherein the one or more amino acids or derivatives thereof or salts, esters or amide derivatives thereof comprises methionine, arginine, alanine or a combination thereof.
7. The flavivirus composition according to claim 5, wherein the one or more amino acids or derivatives thereof or salts, esters or amide derivatives thereof comprises monosodium glutamate (MSG).
8. The flavivirus composition according to claim 5, wherein the one or more amino acids or salts, esters or amide derivatives thereof comprise alanine and methionine.
9. The flavivirus composition according to claim 1, further comprising a protein agent comprising dextran, polyol polymer or gelatin.
10. The flavivirus composition according to claim 1, wherein the one or more live flaviviruses is selected from the group consisting of dengue virus, West Nile virus, tick-borne encephalitis virus, yellow fever virus, Japanese encephalitis virus, Kunjin virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Zika virus, or any related flavivirus thereof.
11. The flavivirus composition according to claim 1, wherein the one or more live flaviviruses comprises live, attenuated flaviviruses.
12. The flavivirus composition according to claim 1, wherein the one or more live flaviviruses comprises dengue virus.
13. The flavivirus composition according to claim 1, wherein the composition does not contain magnesium chloride (MgCl2).
14. The flavivirus composition according to claim 9, wherein the one or more protein agents have a concentration from 0.05% to 1.0% (w/v).
15. The flavivirus composition according to claim 1, wherein the trehalose, sucrose, or combination of trehalose and sucrose is present in the composition at a total concentration ranging from 5.0% to 15.0% (w/v).
16. The flavivirus composition according to claim 1, wherein the trehalose, sucrose, or combination of trehalose and sucrose is present in the composition at a total concentration of less than 10.0% (w/v).
17. The flavivirus composition according to claim 5, wherein the one or more amino acids or derivatives thereof or salts, esters or amide derivatives is present in the composition at a concentration ranging from 1.0 mM to 25.0 mM.
18. The flavivirus composition according to claim 1, wherein the urea concentration in the composition is from 0.01% to 1.0% (w/v).
19. The flavivirus composition according to claim 1, wherein the composition further comprises alanine, methionine and MSG.
20. The flavivirus composition according to claim 19, wherein the HSA concentration is from 0.05% to 0.5% (w/v); wherein the sucrose, trehalose or combination of sucrose and trehalose concentration is from 5.0% to 15.0% (w/v); wherein the mannitol concentration is from 1.0% to 15.0% (w/v); wherein the alanine concentration is from 5.0 mM to 25.0 mM; wherein the methionine concentration is from 1.0 mM to 5.0 mM; wherein the MSG concentration is from 1.0 mM to 20 mM; and wherein the urea concentration is from 0.01% to 0.5% (w/v).
21. The flavivirus composition according to claim 1, wherein the composition comprises sucrose and alanine.
22. The flavivirus composition according to claim 21, wherein the HSA concentration is from 0.05% to 0.5% (w/v); wherein the sucrose concentration is from 0.5% to 15.0% (w/v); wherein the alanine concentration is from 5.0 mM to 25.0 mM; and wherein the urea concentration is from 0.01% to 0.5% (w/v).
23. The flavivirus composition according to claim 1, wherein the composition comprises sucrose and methionine.
24. The flavivirus composition according to claim 23, wherein the HSA concentration is from 0.05% to 0.5% (w/v); wherein the methionine concentration is from 1.0 mM to 5.0 mM; and wherein the urea concentration is from 0.01% to 0.5% (w/v).
25. The flavivirus composition according to claim 1, wherein the composition comprises arginine.
26. The flavivirus composition according to claim 25, wherein the HSA concentration is from 0.05% to 0.5% (w/v); wherein the sucrose, trehalose or combination of sucrose and trehalose concentration is from 5.0% to 15.0% (w/v); wherein the arginine concentration is from 1.0 mM to about 20.0 mM; and wherein the urea concentration is from 0.1% to 0.3% (w/v).
27. The flavivirus composition according to claim 1, wherein the composition further comprises MSG.
28. The flavivirus composition according to claim 27, wherein the HSA concentration is from 0.05% to 0.5% (w/v); wherein the trehalose concentration is from 0.5% to 15.0% (w/v); wherein the MSG concentration is from 1.0 mM to 20 mM; and wherein the urea concentration is from 0.01% to 0.5% (w/v).
29. A flavivirus composition comprising, one or more live flaviviruses;
at least one of trehalose in a concentration ranging from 5.0% to 15.0% (w/v) and sucrose in a concentration ranging from 5.0% to 15.0% (w/v);
at least one of mannitol or MSG; and
human serum albumin.
30. The flavivirus composition according to claim 29, wherein the flavivirus composition further comprises a base buffer, the base buffer comprises phosphate buffered saline (PBS), HEPES buffer, histidine buffer or Tris buffer.
31. The flavivirus composition according to claim 29 further comprising, one or more amino acids or derivatives thereof or salts, esters or amide derivatives thereof.
32. The flavivirus composition according to claim 31, wherein the one or more amino acids or derivatives thereof or salts, esters or amide derivatives thereof comprises methionine, arginine, alanine or a combination thereof.
33. The flavivirus composition according to claim 31, wherein the flavivirus composition comprises sucrose, methionine, alanine and MSG.
34. A method for stabilizing live flaviviruses, the method comprising:
combining one or more live flaviviruses with a composition comprising:
at least one of trehalose and sucrose; and urea;
wherein the composition stabilizes the one or more live flaviviruses.
35. The method according to claim 34, wherein the one or more live flaviviruses are selected from the group consisting of dengue virus, West Nile virus, tick-borne encephalitis virus, yellow fever virus, Japanese encephalitis virus, Kunjin virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Zika virus, or any related virus thereof.
36. The method according to claim 35, wherein the one or more live flaviviruses comprises dengue virus.
37. The method according to claim 34, wherein flavivirus composition is lyophilized.
38. The method according to claim 34, wherein the lyophilized flavivirus composition is stored at 25° C. for extended periods.
39. The method according to claim 37, further comprising partially or wholly rehydrating the composition prior to administration of the composition to a subject as part of an immunogenic composition.
40. The method according to claim 34, further comprising a buffer.
41. The method according to claim 34, further comprising one or more protein agents selected from the group consisting of a serum albumin, a human serum albumin (HSA), a bovine serum albumin (BSA), a recombinant serum albumin, a bovine serum, gelatin, dextran, a polyol polymer, or combinations thereof.
42. The method according to claim 34 further comprising, one or more amino acids or derivatives thereof or salts, esters or amide derivatives thereof.
43. The method according to claim 42, wherein the one or more amino acids or derivatives thereof or salts, esters or amide derivatives thereof are selected from the group consisting of alanine, arginine, methionine, MSG or combinations thereof.
44. The method according to claim 34, wherein the urea is present in the composition at a concentration ranging from 0.01% to 0.5% (w/v).
45. A kit for stabilizing live flaviviruses comprising:
a composition according to claim 1; and
at least one container.
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PRIORITY
This is a National Phase Application filed under 35 U.S.C. 371 as a national stage of PCT/US2017/045375, filed Aug. 3, 2017, an application claiming the benefit of U.S. Provisional Application No. 62/370,611, filed Aug. 3, 2016, the content of each of which is hereby incorporated by reference in its entirety.
FIELD
Embodiments herein relate to compositions and methods for stabilizing flaviviruses. In certain embodiments, compositions and methods disclosed herein concern stabilizing live, attenuated or unattenuated (e.g. live whole) flaviviruses. Other embodiments relate to compositions and methods for reducing degradation of live, attenuated or unattenuated flaviviruses. Other embodiments relate to improved formulations for prolonging stabilization of live attenuated or unattenuated flaviviruses during manufacturing, storage, accelerated storage, transport and delivery. Yet other embodiments relate to uses of compositions disclosed herein in kits for transportable applications and administration methods.
BACKGROUND
Vaccines and vaccine formulations have been shown to be important for protecting humans and animals from the detrimental effects of a wide variety of diseases, such as those caused by viruses. One of the most successful prophylactic technologies for viral vaccines is to immunize animals or humans with a weakened or attenuated strain of the virus (a “live, attenuated virus”). Appropriately attenuated viral strains that are part of immunogenic compositions exhibit limited replication after immunization, and therefore, do not give rise to disease. However, the limited viral replication of the attenuated virus is sufficient to express the full repertoire of viral antigens and generates potent and long-lasting immune responses to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the immunized subject has a reduced chance of developing disease. These live, attenuated viral vaccines are among the most successful vaccines used in public health.
In order for live, attenuated viral vaccines to be effective, they must be capable of replicating after immunization, and the viruses themselves must be protected from degradation during preparation as well as transport prior to administration to a subject, in order to ensure for example, that the correct dosage concentration is delivered to the subject. Some vaccines are sensitive to temperature extremes; either excessive heat or accidental freezing can inactivate the vaccine. Maintaining this “cold chain” throughout distribution is particularly difficult in the developing world. Therefore, there remains a need for improving the stability of both existing and newly developed live, attenuated viruses and live viruses in order to improve manufacturing of vaccines, as well as, transport and delivery of the formulations.
SUMMARY
Embodiments herein relate to compositions and methods for stabilizing live flaviviruses. In certain embodiments, compositions and methods disclosed herein concern stabilizing live, attenuated or unattenuated whole flaviviruses. Other embodiments relate to compositions and methods for reducing degradation of live, attenuated or unattenuated flaviviruses. Yet other embodiments relate to improved formulations for prolonging stabilization of live attenuated or unattenuated flaviviruses during manufacturing (e.g. vaccines or anti-viral treatments), storage, transport and administration to a subject. Yet other embodiments relate to uses of compositions or formulations disclosed herein in kits for transportable applications and methods.
Embodiments herein relate to formulations for stabilizing live viruses that can include one or more live flaviviruses, one or more carbohydrate agents, and one or more amino acids or salts, esters or amide derivatives thereof. In accordance with these embodiments, formulations of use herein stabilize live flaviviruses. In other embodiments, formulations of use herein stabilize live flaviviruses for commercial use.
In certain embodiments, formulations disclosed herein can include, but are not limited to, formulations for stabilizing flaviviruses. Flaviviruses can include, but are not limited to, dengue virus, West Nile virus, tick-borne encephalitis virus, yellow fever virus, Japanese encephalitis virus, Kunjin virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Zika virus, or any related virus thereof.
In some embodiments, formulations disclosed herein can further include a buffer. In accordance with these embodiments, the buffer can include, but is not limited to, phosphate buffered saline (PBS), TRIS buffer, HEPES buffer or the like. In accordance with these embodiments, the buffer can include at least one salt of sodium chloride (NaCl), monosodium and/or disodium phosphate (Na2HPO4), potassium chloride (KCl), and potassium phosphate (KH2PO4). In some embodiments, the buffer of formulations disclosed herein can include sodium chloride (NaCl) having a concentration of about 10.0 mM to about 200.0 mM. In other embodiments, formulations for stabilizing live or live, attenuated flaviviruses do not contain magnesium chloride (MgCl2).
In certain embodiments, formulations disclosed herein can further include one or more protein agents. In accordance with these embodiments, the one or more protein agents can include, but is not limited to, albumin, gelatin and/or dextran. For example, the albumin can include, but is not limited to, a recombinant albumin, a native albumin, human serum albumin (HSA), recombinant human serum albumin (rHSA), native human serum albumin (nHSA), or an albumin-like agent. In other embodiments, the one or more protein agents in the composition can include, but are not limited to, any serum albumin, a human serum albumin (HSA), a bovine serum albumin (BSA), any comparable mammalian serum, gelatin, dextran, a polyol polymer, or combinations thereof. In accordance with these embodiments, one or more protein agents in a formulation disclosed herein can include albumin, such as human serum albumin or recombinant albumin having a concentration of about 0.01% to about 2.0% (w/v). In one embodiment, a formulation disclosed herein can include human serum albumin (HSA) having a concentration of about 0.01% to about 2.0% (w/v).
In other embodiments, the one or more carbohydrate agents of formulations disclosed herein can include, but are not limited to, trehalose (e.g. D-trehalose dehydrate), galactose, fructose, lactose, sucrose, chitosan, mannitol or combinations thereof. In accordance with these embodiments, the one or more carbohydrate agent concentration can be from about 0.5% to about 15.0% (w/v). In certain embodiments, the one or more carbohydrate agents in a formulation disclosed herein can include trehalose and/or sucrose. In other embodiments, the one or more carbohydrate agents in a formulation disclosed herein can include mannitol in combination with at least one of trehalose and sucrose. In other embodiments, the one or more carbohydrate agents in the composition can include mannitol in combination with both trehalose and sucrose. In certain embodiments, formulations disclosed herein do not include sorbitol. In other embodiments, some formulations disclosed herein do not contain poloxamer 407 or other poloxamer.
In some embodiments, for example, the one or more amino acids in a formulation can include, but are not limited to, alanine, arginine, methionine, or combinations thereof. In certain embodiments, one or more amino acids can include an amino acid derivative or salt thereof. In yet other embodiments, an amino acid derivative can include monosodium glutamate (MSG) or potassium glutamate. In certain embodiments, the one or more amino acids or derivatives thereof are present in a formulation at a concentration of about 0.5 mM to about 150.0 mM.
In some embodiments, formulations disclosed herein can include one or more osmolytes. In accordance with these embodiments, one or more osmolytes can include urea (for example a carbamide) or other substitutable agent, for example, caprolactam, PEG 400 or PEG 600. In accordance with these embodiments, the one or more osmolyte concentration (e.g. urea) can be about 0.01% to about 0.5% (w/v).
In certain embodiments, formulations disclosed herein can include sucrose and/or trehalose in a buffer and urea. In other embodiments, the formulation can further include one or more amino acids. In certain embodiments, the one or more amino acids can include at least one of alanine and methionine. In accordance with these embodiments, these formulations can further include at least one of mannitol and MSG. In certain embodiments, a formulation can include trehalose and mannitol in a buffer without urea or amino acids. In accordance with these embodiments, the buffer can include PBS (or TRIS, HEPES or other suitable buffer), NaCl and albumin (e.g., HSA).
In some embodiments, such as those according to any of paragraphs, a formulation can include a base buffer, sucrose, albumin, mannitol, alanine, methionine, MSG and/or urea in any combination. In accordance with these embodiments, certain formulations can include recombinant HSA having a concentration of about 0.01% to about 2.0% (w/v), sucrose concentration having a concentration of about 1.0% to about 15.0% (w/v), mannitol concentration having a concentration of about 0.1% to about 15.0% (w/v), alanine having a concentration of about 0.5 mM to about 100 mM, methionine concentration having a concentration of about 1.0 mM to 5.0 mM, MSG concentration having a concentration of about 1.0 mM to about 50.0 mM, and urea concentration having a concentration of about 0.05% to about 1.0% (w/v).
In some embodiments, formulations contemplated herein can include recombinant albumin or HSA, trehalose and/or sucrose in a base buffer, mannitol, alanine, methionine, MSG and urea.
In some embodiments, recombinant HSA concentration can be about 0.01% to about 2.0% (w/v), trehalose concentration can be about 1.0% to about 10.0% (w/v), sucrose concentration can be about 1.0% to about 15.0% (w/v), mannitol concentration can be about 0.1% to about 15.0% (w/v), alanine can be about 0.5 mM to about 100 mM, methionine concentration can be about 1.0 mM to 5.0 mM, MSG concentration can be about 5.0 mM to about 100.0 mM, and urea concentration can be about 0.05% to about 1.0% (w/v).
In some embodiments, formulations can include recombinant HSA, sucrose, alanine and urea. In other embodiments, recombinant HSA concentration can be about 0.01% to about 0.2%, sucrose can be a concentration can be about 1.0% to about 15.0% (w/v), alanine can be about 0.5 mM to about 100 mM, and urea concentration can be about 0.05% to about 1.0% (w/v). Alternatively, sucrose can be replaced with trehalose at a concentration of about 1.0% to about 10% (w/v).
In some embodiments, stabilizing one or more live flaviviruses can include reducing loss or reducing degradation of the one or more live flaviviruses upon freezing, freeze drying, at refrigeration temperatures, at room temperature and at about 25° C., as compared to a composition not containing one or more of these agents. In some embodiments, stabilizing one or more live flaviviruses as contemplated herein can include obtaining a 10%, a 20%, a 30%, a 40% or a 50% or more reduction in loss or degradation of the live or live, attenuated flaviviruses.
In some embodiments, methods disclosed herein can include partially or wholly dehydrating the formulation that includes the live flavivirus. In other embodiments, methods can include partially or wholly rehydrating the formulation that includes the live or live, attenuated flaviviruses prior to administration to a subject as part of an immunogenic composition (e.g. vaccine composition). In yet other embodiments, methods can include freezing live or live, attenuated flavivirus compositions disclosed herein.
Other embodiments relate to kits for stabilizing live or live, attenuated flaviviruses. In accordance with these embodiments, kits can include any composition or formulation disclosed herein and at least one container.
Certain embodiments concern live, attenuated flavivirus compositions disclosed herein and methods directed to manufacturing an immunogenic composition (e.g. a vaccine) capable of reducing or preventing onset of a medical condition such as an infection or disease caused by one or more of the flaviviruses. In certain embodiments, one or more live, attenuated flaviviruses can be part of a pharmaceutical composition that can include a pharmaceutically acceptable excipient or carrier. In accordance with these embodiments, the pharmaceutical composition can be administered to a subject in order to reduce onset of a condition caused by a flavivirus infection when administered to a subject, and/or prepared for administration to a subject. A subject can be a mammal for example, a domesticated animal a pet, livestock, other animal or a human subject (e.g. an adult, adolescent or child).
In some embodiments, compositions can include recombinant HSA or HSA, sucrose, methionine and urea. In accordance with these embodiments, recombinant HSA or HSA can have a concentration ranging from about 0.01% to about 2.0% (w/v), sucrose can have a concentration ranging from about 0.5% to about 10.0% (w/v), methionine can have a concentration ranging from about 1.0 mM to about 5.0 mM, and urea can have a concentration ranging from about 0.05% to about 0.5% (w/v). In other embodiments, a formulation having recombinant HSA or HSA, sucrose, methionine and urea can further include one or more of alanine at about 5.0 mM to about 25.0 mM, mannitol at about 1.0% to about 15% (w/v) and monosodium glutamate (MSG) at about 1.0 mM to about 20.0 mM. In certain embodiments, the compositions can be in a sucrose base buffer disclosed herein. In yet other embodiments, sucrose can be replaced with trehalose in these compositions.
In other embodiments, a composition can include recombinant HSA, sucrose and/or trehalose, arginine and urea. In accordance with these embodiments, recombinant HSA or HSA can have a concentration ranging from about 0.05% to about 0.5% (w/v), sucrose and/or trehalose can have a concentration ranging from about 0.5% to about 10.0% (w/v), arginine can have a concentration ranging from about 1.0 mM to about 50.0 mM, and urea can have a concentration ranging from about 0.01% to about 0.5% (w/v).
In some embodiments, a composition can include recombinant HSA, sucrose and/or trehalose, MSG (monosodium glutamate) and urea. In accordance with these embodiments, recombinant HSA or HSA can have a concentration ranging from about 0.05% to about 2.0% (w/v), sucrose and/or trehalose can have a concentration ranging from about 0.5% to about 10.0% (w/v), MSG can have a concentration ranging from about 1.0 mM to about 20.0 mM and urea can have a concentration ranging from about 0.01% to about 0.5% (w/v).
In some embodiments, compositions and methods disclosed herein can include combining one or more live or live, attenuated flaviviruses with a formulation that includes one or more carbohydrate agents and one or more amino acids or salts, esters or amide derivatives thereof. In accordance with these methods, formulations disclosed herein can stabilize one or more live or live, attenuated flaviviruses from degradation. Formulations disclosed herein are capable of stabilizing for example any serotype or strain of flavivirus. In certain embodiments, flaviviruses include enveloped viruses, for example, dengue virus (e.g. serotypes 1-4), yellow fever virus, West Nile virus and Zika virus.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the instant specification and are included to further demonstrate certain aspects of some embodiments disclosed herein. Certain embodiments may be better understood by reference to one or more of these drawings in combination with the detailed description presented herein.
FIG. 1 represents an exemplary histogram plot illustrating data obtained from experiments performed while analyzing stability of live flaviviruses in various formulations, according to some embodiments disclosed herein.
FIG. 2 represents an exemplary bar graph illustrating data obtained from experiments performed while analyzing stability of live flaviviruses in various formulations, before lyophilization, after lyophilization, and after lyophilization and storage at room temperature (e.g. 25° C.) for about 5 weeks, according to some embodiments disclosed herein.
FIGS. 3A and 3B are exemplary graphs representing data from experiments using various formulations for testing stability of live flaviviruses after exposure to room temperature (e.g. 25° C.) for 5 weeks after lyophilization, log loss (3A) and total log loss (3B) at both a 50% bulk drug substance (BDS dose) (solid triangles) and a current dose (CTM Dose) (solid squares), in one embodiment disclosed herein.
FIGS. 4A and 4B are exemplary graphs representing data from experiments using various formulations for testing stability of live flaviviruses after exposure to room temperature (e.g. 25° C.) for 5 weeks after lyophilization, log loss (4A) and total log loss (4B) at both a 50% BDS (solid triangles) and a CTM (solid squares), in one embodiment disclosed herein.
FIGS. 5A and 5B are exemplary graphs representing data from experiments using various formulations for testing stability of live flaviviruses after exposure to room temperature (e.g. 25° C.) for 5 weeks after lyophilization, log loss (5A) and total log loss (5B) at both a 50% BDS (solid triangles) and a CTM (solid squares), in one embodiment disclosed herein.
FIGS. 6A and 6B are exemplary graphs representing data from experiments using various formulations for testing stability of live flaviviruses after exposure to room temperature (e.g. 25° C.) for 5 weeks after lyophilization, log loss (6A) and total log loss (6B) at both a 50% BDS (solid triangles) and a CTM (solid squares), in one embodiment disclosed herein.
FIGS. 7A-7D represent exemplary bar graphs illustrating effects of single excipients (in a base buffer containing trehalose) regarding a flavivirus viral potency in both liquid and lyophilized forms, in one embodiment disclosed herein.
FIGS. 8A-8B represent exemplary graphs illustrating effects of single excipients (in a base buffer containing sucrose) regarding a flavivirus viral potency in both liquid and lyophilized forms, stability loss (8A) and lyophilized log loss (8B) in one embodiment disclosed herein.
FIGS. 9A-9B represent exemplary graphs illustrating effects of single excipients and excipient combinations (in a base buffer containing trehalose) regarding a flavivirus viral potency in both liquid and lyophilized forms, lyophilized log loss (9A) and stability loss (9B) in one embodiment disclosed herein.
FIGS. 10A-10B are exemplary graphs representing data from screening for the effect of different agents, carbohydrates (e.g. trehalose and sucrose) and mannitol (in a base buffer) on flaviviral stability and log loss, in one embodiment disclosed herein.
FIGS. 11A-11B are exemplary summaries of excipient screening data sets on flaviviruses potency losses, in one embodiment disclosed herein.
FIGS. 12A-12C are exemplary bar graphs illustrating Definitive Screening Design (DSD) design of experiments formulations for a flavivirus formulated for titer in liquid, lyophilized (stored at −80° C.) and overall stability (12A) and stability trend (12B) of the samples for transport (lyophilized formulation stored at 25° C.) and log drop under the various conditions tested (12C), in some embodiments disclosed herein.
FIGS. 13A-13C are exemplary bar graphs representing flavivirus serotypes (in a tetravalent immunogenic composition) regarding formulation design and viral titer losses, in some embodiments disclosed herein.
FIGS. 14A-14D are exemplary line graphs representing different flaviviruses (e.g. dengue-1, -2, -3 and -4) as lyophilized immunogenic formulations stored at refrigeration temperatures (e.g. 4° C.) for about 39 weeks, in some embodiments disclosed herein.
FIGS. 15A-15B are exemplary bar graphs representing data from screening for the effect of various excipient combinations on flavivirus potency, lyophilized log loss (15A) and stability log loss (15B), in some embodiments disclosed herein.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Definitions
As used herein, “a” or “an” can mean one or more than one of an item, component or element.
As used herein, “about” can mean up to and including plus or minus five percent, for example, about 100 mM can mean 95 mM and up to 105 mM.
As used herein “TDV” refers to exemplary dengue viruses (e.g. a live, attenuated dengue virus serotype, TDV-1, dengue-dengue chimeras, dengue chimera with a dengue-2 backbone (e.g. dengue-2 PDK-53 or modified version thereof with at least 95% homology to PDK-53 etc.).
As used herein “DMEM” can mean Dulbecco's modified minimal essential medium.
As used herein “PBS” can mean Phosphate Buffered Saline.
As used herein “FBS” can mean Fetal Bovine Serum.
As used herein “HSA” can mean Human Serum Albumin.
As used herein “Lyo” can mean lyophilized or dehydrated depending on the frame of reference.
As used herein the specification, “subject” or “subjects” can include, but are not limited to, mammals such as humans (e.g. adult, adolescents, young children or infant) or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g., hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, goats, horses, cattle, other livestock, prairie dogs, wild rodents, or zoo animals.
As used herein, the terms “virus chimera,” “chimeric virus,” “flavivirus chimera” and “chimeric flavivirus” can mean a chimera having at least 2 different viruses represented in a construct for example a construct of a flavivirus chimera has 2 different flaviviruses represented by including non-structural and structural elements from each of flavivirus. Examples of flavivirus chimeras can include, but are not limited to, dengue virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus, yellow fever virus, Zika virus and any combination thereof. For example, a dengue-dengue, dengue-Zika, or a yellow-fever/dengue chimera is contemplated.
As used herein, the term “dengue-dengue chimera” can mean at least two different dengue virus serotypes make up the dengue-dengue chimera.
As used herein, “nucleic acid chimera” can mean a construct of the present disclosure including a nucleic acid sequence from at least two different viruses for example, a chimeric flavivirus or a dengue-dengue chimera disclosed herein can be a nucleic acid chimera.
As used herein, “a live flavivirus” can mean a wild-type live flavivirus (e.g. for use in manufacturing live, attenuated flaviviruses). “Live, attenuated flavivirus” can mean a live flavivirus having a mutation, a flavivirus chimera or other selected for traits where the virus has reduced to no infectivity and reduced expansion or is unable to be transmitted from one host to another (e.g. via mosquito) and is of use in manufacturing immunogenic compositions and where other traits can include reduced virulence, increased safety, increased efficacy or improved growth, etc.
DETAILED DESCRIPTION
In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one of skill in the relevant art that practicing the various embodiments does not require employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details may be modified through routine experimentation. In some embodiments, well known methods or components have not been included in the description.
In certain embodiments, stability of live flaviviruses of use in immunogenic or vaccine formulations have been studied in various formulations disclosed herein. In certain embodiments, formulations which increases flavivirus stability to reduce loss of titer of liquid, frozen, lyophilized, partially lyophilized and re-hydrated live flavivirus formulations has been demonstrated.
In some embodiments, formulations disclosed herein can include, but are not limited to, formulations for stabilizing flaviviruses. Flaviviruses can include, but are not limited to, dengue virus, West Nile virus, tick-borne encephalitis virus, yellow fever virus, Japanese encephalitis virus, Kunjin virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Zika virus, or any related virus thereof.
In some embodiments, formulations disclosed herein can include a buffer. In accordance with these embodiments, the buffer can include, but is not limited to, phosphate buffered saline (PBS), Histidine, TRIS, 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid (HEPES) or similar buffer. In accordance with these embodiments, the buffer can include a salt of sodium chloride (NaCl), monosodium and/or disodium phosphate (Na2HPO4), potassium chloride (KCl), and potassium phosphate (KH2PO4) or combination thereof. In some embodiments, the buffer of formulations disclosed herein can include NaCl, monosodium and/or disodium phosphate (Na2HPO4), potassium chloride (KCl), and potassium phosphate (KH2PO4) or combination thereof having a concentration of about 10.0 mM to about 200.0 mM; or about 10 mM to about 150 mM; or about 15 mM to about 75 mM or about 15 mM to about 50 mM. In other embodiments, formulations for stabilizing live flaviviruses do not contain magnesium chloride (MgCl2).
In certain embodiments, formulations disclosed herein can further include one or more protein agents. In accordance with these embodiments, the one or more protein agents can include, but is not limited to, albumin, gelatin and/or dextran. For example, the albumin can include, but is not limited to, a recombinant albumin, a native albumin, human serum albumin (HSA) or an albumin-like agent. In other embodiments, the one or more protein agents in the composition can include, but is not limited to, any serum albumin, a human serum albumin (HSA), a bovine serum albumin (BSA), any comparable mammalian serum, gelatin, dextran, a polyol polymer, or combinations thereof.
In some embodiments, one or more protein agents in a formulation disclosed herein can include albumin, such as human serum albumin or recombinant albumin, having a concentration of about 0.01% to about 2.0% (w/v); or about 0.05% to about 1.0% (w/v); or 0.05% to about 0.5% (w/v); or 0.075% to 0.2% (w/v); or 0.1% to 0.3% (w/v). In accordance with these embodiments, any formulation disclosed herein can include human serum albumin (HSA) having a concentration of about 0.01% to about 2.0%.
In other embodiments, the one or more carbohydrate agents of formulations disclosed herein can include, but are not limited to, trehalose, galactose, fructose, lactose, sucrose, chitosan, mannitol or combinations thereof. In accordance with these embodiments, the one or more carbohydrate agent concentration can be from about 0.5% to about 15.0% (w/v); or about 1.0% to about 10% or about 5.0% to about 8.0%. In certain embodiments, the one or more carbohydrate agents in the composition can include trehalose and/or sucrose. In other embodiments, the one or more carbohydrate agents in the composition can include mannitol in combination with at least one of trehalose and sucrose. In yet other embodiments, the one or more carbohydrate agents in the composition can include mannitol in combination with both trehalose and sucrose. In certain embodiments, the one or more carbohydrate agents of the formulations disclosed herein have 10% (w/v) or less carbohydrate concentration. In other embodiments, the one or more carbohydrate agents of the formulations disclosed herein have less than 10% (w/v) carbohydrate concentration. In certain embodiments, the formulations disclosed herein do not include sorbitol. In other embodiments, formulations disclosed herein do not contain poloxamer 407 or other poloxamer.
In some embodiments, the one or more amino acids in a formulation can include, but are not limited to, alanine, arginine, methionine, or combinations thereof. In certain embodiments, one or more amino acids can include an amino acid derivative or salt thereof. In yet other embodiments, an amino acid derivative can include monosodium glutamate (MSG) or potassium glutamate. In certain embodiments, the one or more amino acids or derivatives thereof are present in a formulation at a concentration of about 0.5 mM to about 150.0 mM; or about 0.5 mM to 100 mM; or about 1 mM to about 50 mM or about 1.0 mM to about 15.0 mM or about 2.5 mM to 10 mM.
In some embodiments, formulations disclosed herein can include one or more osmolytes. In accordance with these embodiments, one or more osmolytes can include urea (for example a carbamide) or other substitutable agent, for example, caprolactam, PEG 400 or PEG 600. In accordance with these embodiments, the one or more osmolyte concentration can be about 0.01% to about 1.0% (w/v); or about 0.01% to about 0.5% (w/v); or about 0.05% to about 0.5% (w/v) or about 0.075% to about 0.4% (w/v) or about 0.125% (w/v).
In certain embodiments, formulations disclosed herein can further include, sucrose or trehalose, one or more amino acids and urea. In accordance with these embodiments, these formulations can further include at least one of mannitol, MSG and urea. In certain embodiments, a formulation can include trehalose in a suitable buffer and mannitol in a buffer. In accordance with these embodiments, the buffer can include PBS or TRIS or HEPES or other suitable buffer, NaCl and albumin (e.g., HSA). In certain embodiments, the buffer is PBS or HEPES. In certain embodiments, the one or more amino acids can include alanine and/or methionine. In other embodiments a formulation can include sucrose in a suitable buffer and urea.
In certain embodiments, flavivirus formulations can include, but are not limited to, one or more live flaviviruses, buffer, one or more amino acids, and one or more carbohydrate agents. For example, immunogenic compositions and vaccine formulations disclosed herein can include buffers having concentrations of about 1.0 to 30.0 mM Phosphate Buffer, about 1.0 to 30.0 HEPES Buffer, about 1.0 to 30.0 Histidine Buffer, about 1.0 to 30.0 Tris Buffer, with or without salt and trehalose, sucrose or a combination of trehalose and sucrose. Other exemplary immunogenic compositions or vaccine formulations can include about 1.0 to 30.0 mM Phosphate Buffered Saline (PBS) with about 10.0 to about 100 mM sodium chloride at a pH of about 7.2, See Table 1 below.
TABLE 1
Exemplary formulations of immunogenic compositions
No
Sugar
Buffer
NaCl
1
Trehalose (10%) +
10 mM Phosphate Buffer (pH: 7.2)
—
2
0.1% HSA
10 mM Phosphate Buffer (pH: 7.2)
50 mM
3
10 mM HEPES Buffer (pH: 7.2)
—
4
10 mM HEPES Buffer (pH: 7.2)
50 mM
5
10 mM Histidine Buffer (pH: 7.2)
—
6
10 mM Histidine Buffer (pH: 7.2)
50 mM
7
10 mM Tris Buffer (pH: 7.2)
—
8
10 mM Tris Buffer (pH: 7.2)
50 mM
9
Sucrose (10%) +
10 mM Phosphate Buffer (pH: 7.2)
—
10
0.1% HSA
10 mM Phosphate Buffer (pH: 7.2)
50 mM
11
10 mM HEPES Buffer (pH: 7.2)
—
12
10 mM HEPES Buffer (pH: 7.2)
50 mM
13
10 mM Histidine Buffer (pH: 7.2)
—
14
10 mM Histidine Buffer (pH: 7.2)
50 mM
15
10 mM Tris Buffer (pH: 7.2)
—
16
10 mM Tris Buffer (pH: 7.2)
50 mM
17
Control, Reference
10 mM Phosphate Buffer (pH: 7.4)
137 mM
Formulation (15%
Trehalose + 0.1%
HSA + % Pluronic
F127 ®)
In other embodiments, a formulation can include a base buffer, sucrose, albumin, mannitol, alanine, methionine, MSG and/or urea in any combination. In accordance with these embodiments, certain formulations can include recombinant HSA having a concentration of about 0.01% to about 2.0% (w/v) or about 0.05% to about 1.0% (w/v), sucrose concentration having a concentration of about 1.0% to about 15% (w/v) or less than 10% (w/v), mannitol concentration having a concentration of about 0.5% to about 15.0% (w/v), alanine having a concentration of about 0.5 mM to about 100 mM or about 5.0 mM to about 50 mM, methionine concentration having a concentration of about 1.0 mM to 5.0 mM or about 1.5 mM to about 3.0 mM, MSG concentration having a concentration of about 5.0 mM to about 100.0 mM or about 5.0 mM to about 25 mM, and urea concentration having a concentration of about 0.05% to about 1.0% (w/v) or about 0.1% to about 0.5% (w/v).
In some embodiments, formulations contemplated herein can include recombinant albumin or HSA, trehalose, mannitol, alanine, methionine, MSG and urea. In accordance with these embodiments, recombinant HSA concentration can be about 0.01% to about 2.0% (w/v) or about 0.05 to about 1.0%, trehalose concentration can be about 1.0% to about 15.0% (w/v) or about 2.5% to less than 10%, mannitol concentration can be about 0.1% to about 10.0% (w/v), alanine can be about 1.0 mM to about 50 mM or about 5 mM to about 25 mM, methionine concentration can be about 0.05 mM to about 5.0 mM or about 0.1 mM to about 4.0 mM, MSG concentration can be about 1.0 mM to about 50.0 mM or about 5.0 mM to about 25.0 mM, and urea concentration can be about 0.05% to about 0.5% (w/v) or about 0.05 to about 0.3% (w/v).
In certain embodiments, formulations disclosed herein can include recombinant HSA, sucrose, alanine and urea. In other embodiments, recombinant HSA concentration can be about 0.01% to about 2.0% (w/v) or about 0.05% to about 0.5% (w/v), sucrose can be a concentration of about 1.0% to about 10% or about 5%, alanine concentration can be about 1.0 mM to about 50 mM, and urea is concentration can be about 0.05% to about 0.5% (w/v) or about 0.05 to about 0.3% (w/v).
In some embodiments, stabilizing one or more live flaviviruses can include reducing loss or reducing degradation of the one or more live flaviviruses upon freezing, freeze drying, at refrigeration temperatures, at room temperature and at about 25° C., as compared to a composition not containing one or more of these agents. In some embodiments, stabilizing one or more live flaviviruses as contemplated herein can include obtaining a 10%, a 20%, a 30%, a 40% or a 50% or more reduction in loss of the live or live, attenuated flaviviruses when formulated by compositions and methods disclosed herein.
In some embodiments, methods disclosed herein can include partially or wholly dehydrating the formulation that includes the live flavivirus. In other embodiments, methods can include partially or wholly rehydrating the formulation that includes the live or live, attenuated flaviviruses prior to administration to a subject as part of an immunogenic composition (e.g. vaccine composition). In yet other embodiments, methods can include freezing live or live, attenuated flavivirus compositions disclosed herein.
Certain embodiments disclosed herein concern live, attenuated flavivirus compositions and methods directed to manufacturing an immunogenic composition (e.g. a vaccine) capable of reducing or preventing onset of a medical condition such as an infection or disease caused by one or more of the flaviviruses contemplated herein. In certain embodiments, one or more live, attenuated flaviviruses can be part of a pharmaceutical composition that can include a pharmaceutically acceptable excipient or carrier. In accordance with these embodiments, the pharmaceutical composition can be administered to a subject in order to reduce onset of a condition caused by a flavivirus infection when administered to a subject, and/or prepared for administration to a subject. A subject can be a mammal for example, a domesticated animal a pet, livestock, other animal or a human subject (e.g. an adult, adolescent or child).
In some embodiments, a composition disclosed herein can include recombinant HSA or HSA, sucrose, methionine and urea. In accordance with these embodiments, recombinant HSA can have a concentration ranging from about 0.05% to about 0.5% (w/v), sucrose can have a concentration ranging from about 0.5% to about 10.0% (w/v), methionine can have a concentration ranging from about 1.0 mM to about 5.0 mM, and urea can have a concentration ranging from about 0.05% to about 0.5% (w/v). In other embodiments, a formulation having recombinant HSA or HSA, sucrose, methionine and urea can further include one or more of alanine at about 5.0 mM to about 25.0 mM, mannitol at about 1.0% to about 15% (w/v) and monosodium glutamate (MSG) at about 1.0 mM to about 20.0 mM. In certain embodiments, the compositions can be in a sucrose base buffer disclosed herein.
In other embodiments, a composition can include recombinant HSA, sucrose and/or trehalose, arginine and urea. In accordance with these embodiments, recombinant HSA or HSA can have a concentration ranging from about 0.05% to about 1.0% (w/v), sucrose and/or trehalose can have a concentration ranging from about 0.5% to about 10.0% (w/v), arginine can have a concentration ranging from about 1.0 mM to about 50.0 mM, and urea can have a concentration ranging from about 0.01% to about 0.5% (w/v) or about 0.05 to about 0.3% (w/v).
In some embodiments, a composition can include recombinant HSA, sucrose and/or trehalose, MSG (monosodium glutamate) and urea. In accordance with these embodiments, recombinant HSA or HSA can have a concentration ranging from about 0.05% to about 0.5% (w/v), sucrose and/or trehalose can have a concentration ranging from about 0.5% to about 15.0% (w/v) or less than 10% (w/v), MSG can have a concentration ranging from about 1.0 mM to about 20.0 mM and urea can have a concentration ranging from about 0.01% to about 0.5% (w/v) or about 0.05 to about 0.3% (w/v).
In some embodiments, compositions and methods disclosed herein can include combining one or more live or live, attenuated flaviviruses with a formulation that includes one or more carbohydrate agents and one or more amino acids or salts, esters or amide derivatives thereof. In accordance with these methods, formulations disclosed herein can stabilize one or more live or live, attenuated flaviviruses from degradation. Formulations disclosed herein are capable of stabilizing for example any serotype or strain of flavivirus. In certain embodiments, flaviviruses include enveloped viruses, for example, dengue virus (e.g. serotypes 1-4), yellow fever virus, West Nile virus and Zika virus.
In other embodiments, a composition disclosed herein can include a flavivirus in a buffer, urea and one or more amino acids that can include, but are not limited to, alanine, arginine, methionine or combinations thereof, and one or more carbohydrate agents that can include, but is not limited to, at least one of sucrose, trehalose and/or mannitol. Certain compositions disclosed herein also can include salt or a salt solution. In accordance with these embodiments, these flavivirus formulations can be used for liquid, frozen or lyophilized storage of a live flaviviruses or live, attenuated flaviviruses at about −80° C. to about 40° C. or above without significant loss of the viral titer in the composition. For example, long-term storage at 4° C. and 25° C. can be achieved using formulations disclosed herein. In certain embodiments, compositions contemplated herein can be partially or wholly dehydrated and/or hydrated. In accordance with these embodiments, long term storage of flavivirus formulations disclosed herein can include accelerated storage of lyophilized formulations and transport or storage at room temperature for long periods of time, greater than 5 weeks.
In some embodiments, live flaviviruses contemplated herein to benefit from compositions and formulations of the present disclosure can include, but are not limited to, dengue virus, West Nile virus, tick-borne encephalitis virus, yellow fever virus, Japanese encephalitis virus, Kunjin virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Zika virus, or any related flavivirus thereof. In accordance with these embodiments, live flaviviruses or live, attenuated flaviviruses of use in vaccines can be stored at various temperatures where formulations disclosed herein increase stability of the formulation by, for example, reducing degradation of the flavivirus such as reduction in titer loss of the flaviviruses in the formulation. Therefore, use of these formulations to preserve flaviviruses provides solutions regarding efficient manufacturing, transport and delivery of known doses of flaviviruses in the form of immunogenic compositions at reduced costs and time for production. The disclosed formulations allow for more accurate delivery of a live flavivirus or live, attenuated flavivirus immunogenic composition to a patient in need of such a formulation.
In certain embodiments, compositions disclosed herein can be used in methods for preparing vaccine compositions of use for administration to a subject in need thereof. In certain embodiments, immunogenic compositions against flavivirus infection or complications thereof are provided. For example, compositions disclosed herein can be used in order to manufacture vaccines or immunogenic compositions where improved storage conditions and reduced flavivirus degradation are sought. In other embodiments, one or more or all four dengue virus serotypes can be manufactured into an immunogenic composition disclosed herein to reduce degradation of the flavivirus or preserve viral titer in order to more accurately deliver a known quantity of flavivirus to a subject. In certain embodiments, when combined, immunogenic compositions can include multivalent vaccine compositions (e.g., bi-, tri- and tetravalent) to more accurately confer simultaneous protection against infection by more than one species or strain of flavivirus. In some embodiments, flaviviruses can include live flaviviruses and/or live, attenuated flaviviruses. In other embodiments, more than one serotype or strain of a flavivirus or different flaviviruses or chimeras can be combined in an immunogenic composition. In accordance with these embodiments, compositions disclosed herein can provide a stabilizing formulation to more accurate delivery of immunogenic compositions against flaviviruses where transport and storage survival of the live flaviviruses is improved.
In certain compositions, live, attenuated flaviviruses can include nucleic acids encoding one or more proteins from 2 or more different flaviviruses such as a chimera. In other embodiments, immunogenic compositions and formulations disclosed herein can include one or more dengue serotypes referenced as TDV-1 (e.g. dengue serotype 1, a dengue 1/2 chimera), TDV-2 (e.g. dengue serotype 2, a modified live, attenuated dengue-2 virus), TDV-3 (e.g. dengue serotype 3, a dengue 3/2 chimera), and TDV-4 (e.g. dengue serotype 4, a dengue 4/2 chimera), and combinations thereof. It is contemplated that any dengue virus construct (e.g. any flavivirus chimera) or live attenuated dengue virus can be stabilized by formulations disclosed herein. In some embodiments, live, attenuated dengue virus chimeras provided herein can contain the nonstructural protein genes of one dengue virus (e.g. a dengue-2 virus), or the equivalent thereof, and one or more of the structural protein genes or immunogenic portions thereof of at least a second flavivirus (e.g. yellow-fever, other dengue serotype, Zika virus). For example, certain embodiments concern dengue-dengue, dengue-yellow fever, dengue-Zika, dengue-West Nile, dengue-JEV or other flavivirus chimera as mono-, di-, tri- or tetravalent formulations against one or more flaviviruses.
Some embodiments concern methods for decreasing inactivation of a live flavivirus or live, attenuated flaviviruses including, but not limited to, combining one or more flaviviruses with a composition capable of reducing inactivation of the flaviviruses disclosed herein wherein the composition decreases inactivation of the flavivirus. In accordance with these embodiments, the live flaviviruses can include particular flaviviruses, such as those having similar features as the enveloped flaviviruses such as dengue virus serotypes, other flaviviruses having a similar genetic make-up, structural and non-structural protein layout and/or secondary structure or the like to flaviviruses such as dengue virus, Zika virus and yellow fever virus. For example, these formulations are capable of reducing degradation of flaviviruses during particular processes such as encapsidation, manufacturing, transport and administration of a formulated immunogenic composition to a subject, etc. Flaviviruses share several common features such as a common size (40-65 nm), symmetry (enveloped, icosahedral nucleocapsid), nucleic acid (positive-sense, single-stranded RNA of around 10,000-11,000 bases). It is contemplated that any flavivirus or RNA-enveloped virus can be stabilized by the compositions disclosed herein.
In certain embodiments, a live, attenuated flavivirus or flavivirus chimera contemplated herein can be formulated into a pharmaceutical composition wherein the pharmaceutical composition can be administered alone or in combination with other immunogenic agents, and/or can be used to prepare a vaccine composition to be administered to a subject. In certain embodiments, mono-, bi-, tri or tetravalent dengue virus compositions can be formulated into a pharmaceutical composition using compositions disclosed herein to reduce degradation of the formulation, for example, during transport. In accordance with these embodiments, flaviviruses can be lyophilized in any formulation disclosed herein and transported at room temperature without significant loss of titer (See the Examples for some exemplary formulations and methods).
Embodiments herein concern methods and compositions to reduce or prevent degradation and/or inactivation of live, attenuated flaviviruses. In accordance with these embodiments, certain compositions can include combinations of components that reduce degradation and/or inactivation of live flaviviruses and/or live, attenuated flaviviruses. Other embodiments herein concern combinations of agents capable of enhancing stability these flaviviruses. Yet other compositions and methods herein are directed to reducing the need for lower temperatures (e.g., refrigerated or frozen storage) while increasing the shelf life of aqueous and/or reconstituted live flaviviruses or live, attenuated flaviviruses.
In accordance with these embodiments, one or more live or live, attenuated flaviviruses can be combined with one or more amino acids and one or more carbohydrates. In certain embodiments, flavivirus formulations disclosed herein include at least three components: a PBS-based buffer or other buffer and urea. In other embodiments, a salt agent can be added to these compositions in order to enhance buffering capacity or other property of the formulation.
In certain embodiments, carbohydrate agents contemplated of use in compositions herein can include, but are not limited to, sucrose, fructose, galactose, trehalose, mannitol or other similar carbohydrate. In other embodiments, amino acids or derivatives thereof contemplated of use in the compositions and formulations disclosed herein include, but are not limited to, alanine, arginine, methionine, glutamate, monosodium glutamate (MSG) or salts, esters or amide derivatives thereof. In still other embodiments, protein agents of use in the compositions and formulations disclosed herein include, but are not limited to, serum albumin, a human serum albumin (HSA), recombinant form of albumin, a bovine serum albumin (BSA), a fetal bovine serum (FBS), gelatin, dextran, a polyol polymer, or combinations thereof. In some embodiments, the protein agent comprises an albumin at a concentration from about 0.05% to about 2.0% of the particular immunogenic composition.
In certain embodiments, formulations and compositions disclosed herein can include a phosphate buffered saline (PBS) base buffer, with at least one of sodium chloride (NaCl), monosodium and/or disodium phosphate (Na2HPO4). In some embodiments, the base buffer can also include potassium chloride (KCl) and/or potassium phosphate (KH2PO4). In other embodiments, a buffer for example, a base buffer, can include sodium chloride at a concentration of about 10.0 mM to about 200.0 mM. It is contemplated that MgCl2 is not included in any of the formulations disclosed herein as it was demonstrated to adversely affect stability of the live flaviviruses.
In certain embodiments, compositions disclosed herein can be used to lyophilize and/or rehydrate live or live, attenuated flaviviruses; transport the flaviviruses at various temperatures from frozen to room temperature and other storage features with improved stability. Compositions contemplated herein can increase stabilization and/or reduce inactivation and/or degradation of a live or live, attenuated flaviviruses at various storage temperatures, during processing and purification, during transport and delivery and during freeze-thaws cycles.
In certain embodiments, compositions disclosed herein can be partially or wholly dehydrated or hydrated. Further, compositions disclosed herein can be used during and after lyophilization of a live flavivirus or live, attenuated flavivirus compositions (see for example, Table 2 below). In accordance with these embodiments, a composition can be 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more dehydrated. Compositions described herein are capable of increasing shelf-life of an aqueous or rehydrated flavivirus composition. In accordance with these embodiments, compositions disclosed herein can increase stability of live flaviviruses or live, attenuated flaviviruses at a wide-range of temperatures such as room temperature, sub-zero temperatures, elevated temperatures (e.g., from −80° C. to 40° C. and above) under lyophilized or liquid/frozen conditions. In other embodiments, compositions disclosed herein can increase stability of a live flavivirus or live, attenuated flaviviruses 2-fold, 4-fold, 10-fold or more than a live, attenuated flavivirus composition not formulated in at least a composition of a base buffer (e.g., PBS-based buffer, TRIS, HEPES or Histidine etc.), one or more carbohydrate agents, and one or more amino acids.
TABLE 2
Exemplary Lyophilization Parameters
of Formulations Disclosed Herein
Primary/Secondary
Freeze
Drying
Final
Step
1
2
3
1
2
1
Shelf Temp (° C.)
−45
−45
−45
−37
+25
4
Ramp Rate (° C./
—
1.7
—
0.1
0.1
1.0
min)
Time (min)
—
120
120
2400
360
HOLD
Vacuum (mT)
—
—
50
50
50
50
In certain embodiments, compositions contemplated herein can decrease inactivation and/or degradation of a hydrated live flavivirus for greater than 24 hours at various temperatures (e.g., about 20° C. to about 25° C. or even as high as 40° C.) or refrigeration temperatures (e.g., about 0° to about 10° C. or up to 20° C.). In some embodiments, compositions disclosed herein can maintain stability for 90% or greater flaviviruses for 24 hours or more. In addition, formulations and compositions contemplated herein can reduce inactivation of a hydrated flavivirus during at least 2, at least 3, at least 4, at least 5, at least 6 and more freeze-thaws cycles. Other compositions and methods concern formulations and compositions capable of reducing inactivation of a hydrated live flavivirus or live, attenuated flaviviruses for about 24 hours to about 26 weeks or greater at refrigeration temperatures (e.g., about 0° to about 10° C.).
Still other methods concern using formulations and compositions for 50% or more reduction in the loss of flavivirus titer, for example, after lyophilization and/or after a certain period of time and/or after exposure to a certain temperature. In certain embodiments, the methods disclosed herein provide formulations and compositions capable of reducing the loss of flavivirus titer for a period of 5, or 6, or 7 or more weeks, post-lyophilization and exposure to 25° C. temperatures, as compared to a composition or formulation without formulations disclosed herein.
In certain aspects, formulations can include hydrolyzed gelatin or dextran instead of serum albumin as a protein agent. However, in certain formulations gelatin can be excluded for a variety of reasons including the fact that it is an animal product. Other considerations are that gelatin can cause allergic reactions in immunized children and adults and could be a cause of vaccine-related adverse events. Additionally, gelatin can be sourced from bovine or porcine bones and spinal material. These sources can include extraneous agents and raise safety concerns. Additionally, albumin can be collected from humans, which also poses a potential risk of introducing unsafe extraneous agents. Therefore, certain compositions concern using agents other than animal by-products that have equally stabilizing effects on live attenuated or unattenuated flaviviruses is contemplated. In certain embodiments, recombinant albumin can used in formulations disclosed herein to reduce adverse effects of other albumins.
Compositions disclosed herein can provide for increased protection of live flaviviruses from for example, freezing and/or thawing, and/or elevated temperatures. In certain embodiments, compositions disclosed herein can stabilize, reduce deterioration and/or prevent inactivation of dehydrated live, attenuated viral products in room temperature conditions (e.g., about 25° C.). In other embodiments, formulations and compositions contemplated herein can stabilize, reduce deterioration and/or prevent inactivation of aqueous live, attenuated viral products at about 25° C. or up to or about 40° C. Compositions and methods disclosed herein can facilitate the storage, distribution, delivery and administration of viral vaccines in developed and underdeveloped regions.
Those skilled in the art will recognize that compositions or formulas herein relate to viruses that are unattenuated or attenuated by any method, including but not limited to, cell culture passage, reassortment, incorporation of mutations in infectious clones, reverse genetics, insertions, deletions, other recombinant DNA or RNA manipulation. In addition, those skilled in the art will recognize that other embodiments relate to viruses that are engineered to express any other proteins or flavivirus RNA including, but not limited to, recombinant flaviviruses. Such viruses may be used as vaccines for infectious diseases, vaccines to treat oncological conditions, or viruses to introduce express proteins or RNA (e.g., gene therapy, antisense therapy, ribozyme therapy or small inhibitory RNA therapy) to treat disorders.
Pharmaceutical Compositions
Some embodiments herein relate to pharmaceutical compositions for live or live, attenuated flaviviruses in aqueous or lyophilized form. Those skilled in the art will recognize based on the present disclosure that formulations that improve viral stability (e.g. thermal) and prevent freeze-thaw inactivation of pharmaceutical compositions disclosed herein can improve products that are liquid, powdered, freeze-dried or lyophilized and can be prepared by methods known in the art. After reconstitution, such stabilized vaccines formulations and immunogenic compositions can be administered by a variety routes, including, but not limited to intradermal administration, subcutaneous administration, intramuscular administration, intranasal administration, by inhalation, pulmonary administration or oral administration. In other embodiments, delivery devices to administer any pharmaceutical compositions disclosed herein are contemplated. In accordance with these embodiments, a variety of devices for vaccine delivery are known in the art, including, but not limited to, syringe and needle injection, bifurcated needle administration, administration by intradermal patches or pumps, intradermal needle-free jet delivery (intradermal, etc.), intradermal particle delivery, or aerosol powder delivery.
In certain embodiments, compositions can be described that typically include a physiologically acceptable buffer. Those skilled in the art recognize that PBS-based buffers, TRIS-based buffers, HEPES-based buffers, Histidine-based buffers and buffer systems were found to have unexpected stabilizing effect on the flavivirus formulations and compositions disclosed herein. In addition, those skilled in the art recognize that adjusting salt concentrations to near physiological levels (e.g., saline or 0.15 M total salt) may be optimal for parenteral administration of compositions to prevent cellular damage and/or pain at the site of injection. Those skilled in the art also will recognize that as carbohydrate concentrations increase, salt concentrations can be decreased to maintain equivalent osmolarity and osmolality to the formulation. In certain embodiments, a buffering media with pH greater than 6.0 to about pH 10 is contemplated. In other embodiments, a buffering media with a pH greater than 6.8 to about 8.0 is contemplated. In yet other embodiments, a buffering media with a pH greater than 7.0 to about 7.5 is contemplated.
Embodiments herein provide for administration of immunogenic compositions and vaccine formulations to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. “Biologically compatible form suitable for administration in vivo” refers to a form of the active agent (e.g., live, attenuated flavivirus compositions) to be administered in which any toxic effects are outweighed by the therapeutic effects of the active agent. Administration of a therapeutically active amount of the therapeutic compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve a desired result. For example, a therapeutically active amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability formulations to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response.
An active agent, such as a vaccine, may be administered to a subject in an appropriate carrier or diluent, which is co-administered with the agent. The term “pharmaceutically acceptable carrier” as used herein is intended to include diluents such as saline and aqueous buffer solutions. The active agent may also be administered parenterally or intraperitoneally. Pharmaceutical compositions suitable for injection may be administered by means known in the art. For example, sterile aqueous solutions (water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion may be used. In all cases, compositions and formulations can be sterile and can be fluid to the extent that that can be easy ejected from a syringe.
Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Compositions and formulations may further be treated to prevent against the contaminating action of microorganisms such as bacteria and fungi. Sterile injectable solutions can be prepared by incorporating active compound in an amount with an appropriate solvent or with one or a combination of ingredients enumerated above, as required, followed by sterilization.
In certain embodiments, compositions and formulations in liquid forms (solutions) can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. In accordance with these embodiments, the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. It is contemplated that slow release capsules, timed-release microparticles, and the like can also be employed for administering compositions herein. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In some embodiments, formulations disclosed herein can be administered before, during and/or after exposure to a flavivirus of the present disclosure.
Analytical Methods
In certain embodiments, method for identifying an excipient that improves stability of one or more live viruses can include generating an experimental virus stability dataset; constructing an exploratory statistical model by performing univariate regression and multivariate data analyses on the stability dataset; identifying at least one excipient, wherein the at least one excipient increases stability of one or more live viruses. In accordance with these methods, hits and levels of the at least one excipient can be integrated into a definitive screening design (DSD). In other methods, optimal excipient concentrations can be identified for the at least one excipient, wherein the optimal excipient concentration reduces viral potency loss compared to viruses without the at least one excipient. In yet other embodiments, experimental virus stability dataset can be modeled using stepwise regression tools available in JMP 11.2.0 intended for response surface methods (RSM). In accordance with these methods, one or more live viruses can include flaviviruses.
Further methods for identifying optimum conditions for stability of flaviviruses can include evaluating excipients that include, but are not limited to, trehalose, galactose, fructose, sucrose, chitosan, sorbitol, mannitol, serum albumin, human serum albumin (HSA), bovine serum albumin (BSA), fetal bovine serum (FBS), gelatin, dextran, a polyol polymer, alanine, arginine, methionine, MSG and urea. These methods are contemplated to be of use for identifying stabilizing formulations for any virus or live, attenuated virus formulation.
Kits
Other embodiments disclosed herein concern kits for use with compositions and methods as disclosed herein. Compositions and live, attenuated virus formulations may be provided in a kit for use in scientific studies or for delivery and/or use to administer to a subject. In accordance with these embodiments, kits can include, but are not limited to, a suitable container, live or live, attenuated flavivirus compositions, including live, attenuated flaviviruses, and one or more additional agents such as other anti-viral agents, anti-fungal or anti-bacterial agents.
In other embodiments, kits can further include a suitably aliquoted composition of use in a subject to be vaccinated or treated, as appropriate. In addition, compositions and formulations disclosed herein may be partially or wholly dehydrated or may be aqueous. Kits contemplated herein may be stored at room temperatures or at refrigerated temperatures as disclosed herein, depending on the particular formulation.
In other embodiments, kits can generally include at least one vial, test tube, flask, bottle, syringe or other container, into which a composition can be placed, and suitably aliquoted. Where an additional component is provided, kits can also generally contain one or more additional containers into which this agent or component may be placed. Kits herein can also include a composition and any other reagent(s) in containers for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained (e.g., immunogenic or vaccine compositions).
EXAMPLES
The following examples are included to demonstrate certain embodiments presented herein. It is appreciated by those of skill in the art that the techniques disclosed in the Examples which follow represent techniques discovered to function well in the practices disclosed herein, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope herein.
Example 1
Buffer Screen
In certain exemplary methods, liquid compositions and lyophilizable compositions suitable for preclinical and clinical testing and use of flavivirus immunogenic compositions or vaccines were identified. Certain experiments were performed to initially identify candidate buffers and excipients to be used in subsequent stabilization studies. Buffers tested included HEPES, PBS and Tris-based buffers. Excipients tested included various carbohydrates, such as trehalose, galactose, fructose, sorbitol, lactose, sucrose, chitosan or combinations thereof. Other excipients tested included various protein agents, serum albumin, a human serum albumin (HSA), recombinant HSA, a bovine serum albumin (BSA), a fetal bovine serum (FBS), gelatin, dextran, a polyol polymer, or combinations thereof. Yet other excipients screened included various amino acids or salts, esters or amide derivatives thereof, such as alanine, arginine, glutamate (e.g. monosodium glutamate (MSG)) methionine, or combinations thereof. Other components used in these initial testes included various polyols/polymers (e.g., mannitol, gelatin, dextran), osmolytes (e.g., urea), salts (e.g., magnesium chloride, sodium chloride), and chelating agents (e.g., EDTA).
In some exemplary methods, live, attenuated dengue viruses were used as an exemplary flaviviruses in various compositions for pre-clinical and clinical testing. Compositions for these methods are provided herein. In one exemplary experiment, predetermined amounts of a live, attenuated dengue virus immunogenic compositions were used. In another exemplary experiment, a live, attenuated dengue serotype 3 immunogenic composition was used. It is noted that this serotype can be considered the least stable and least immunogenic and was therefore used to develop stabilizing compositions that can be used with the other dengue virus serotypes in addition to one that is less stable. It is contemplated that any attenuated or unattenuated flavivirus can be used in these exemplary compositions to increase stability of the flaviviruses and reduce degradation.
Example 2
Screening for Stabilizing Formulations
In some flavivirus formulations disclosed herein, hydrolyzed gelatin can be used instead of serum albumin as a protein source. However, gelatin, while providing stability to the live, attenuated viruses, can cause allergic reactions when administered to a subject and can sometimes be a cause of vaccine-related adverse events. Certain compositions disclosed herein can include combinations of components that provide comparable outcomes to reduce deterioration of live, attenuated flaviviruses while providing formulations having reduced allergic reactions when administered to a subject. For example, polyols can be used as an additional or alternative source of a protein agent in flavivirus formulations disclosed herein. In some examples, formulations having no albumin or gelatin that are comparable or can provide increased stability to live, attenuated flaviviruses were created. These formulations include, but are not limited to, various concentrations of agents such as a PBS-based buffer, one or more carbohydrate agents, one or more amino acids, one or more protein agents, and/or one or more salt agents.
Stability of live, attenuated flavivirus immunogenic compositions were tested as a function of potency loss using various stabilizing formulations (e.g., titer loss or Log10 PFU/dose). Samples of dengue virus serotype 3 (TDV-3) were lyophilized and then exposed to 25° C. for a five week period. Potency loss, or a reduction in the loss of titer over this period, was evaluated for the various formulations, as illustrated in FIG. 1. One base buffer used was PBS with 50 mM NaCl, 0.1% HSA, and 10% carbohydrate. Alanine was present at 10 mM, methionine was present at 2.5 mM, MSG was present at 10 mM, mannitol was present at 5%, and urea was present at 0.25%. A previously-developed formulation that included F-127, trehalose, and albumin (FTA, positive control formulation) was used as a reference. As demonstrated, the various formulations illustrated in FIG. 2 reduce potency loss even more than reference (that was previously demonstrated to improve flavivirus stability), and therefore, provide improved means for stabilizing flavivirus vaccines.
Virus potency observations before/after lyophilization and during accelerated storage stability of the lyophilized flavivirus (e.g. dengue serotype-3, TDV-3) samples are illustrated in Table 3 below. For each formulation, the log10 loss in virus potency due to lyophilization was calculated by subtracting the titer of lyophilized virus samples stored at −80° C. from the initial titer (frozen liquid control stored at −80° C.). In addition, for each formulation, log10 loss in potency during accelerated stability was calculated by subtracting the titer after storage of the lyophilized formulation at 25° C. from same formulation that was stored at −80° C. at 5 weeks.
Most of the formulations demonstrated improved lyophilization yields during the lyophilization process compared to the positive reference control, FTA. After 5 weeks stability study at 25° C., the following trends in virus stability were observed. In one example, flavivirus (e.g. dengue serotype-3, TDV-3) formulations containing high trehalose/sucrose alone (10%), virus potency loss was in the following order: 10% sugar+0.1% HSA>10% sugar+0.1% HSA+Met+MSG+Ala>10% sugar+0.1% HSA+Urea. In another example, flavivirus (e.g. dengue serotype-3, TDV-3) formulations containing combinations of low trehalose/sucrose (1%) and mannitol (4%), virus potency loss was in the following order: 1% sugar+4% Mannitol+0.1% HSA>1% sugar+4% Mannitol+0.1% HSA+Urea>1% sugar+4% Mannitol+0.1% HSA+Met+MSG+Ala. In flavivirus (e.g. dengue serotype-3, TDV-3) formulations containing mannitol alone, virus potency loss was in the following order: 5% Mannitol+0.1% HSA>5% Mannitol+0.1% HSA+Urea>5% Mannitol+0.1% HSA+Met+MSG+Ala.
TABLE 3
Pre-lyophilization, post-lyophilization and post-stability flavivirus (e.g. dengue serotype-3, TDV-3) potency
No.
Lyo
Stability
Total
(corresponds
log
log loss
log
to Table 14)
Samples
liquid
SD
−80° C.
SD
25° C.
SD
loss
(5 W)
loss
1
1% Trehalose + 0.1%
4.54
0.07
4.31
0.10
4.25
0.05
0.23
0.06
0.29
HSA + 4% Mannitol +
Met + MSG + Ala
5
10% Sucrose + 0.1%
5.06
0.12
4.82
0.03
4.38
0.09
0.24
0.44
0.68
HSA + Urea
3
1% Sucrose + 0.1%
4.63
0.05
4.48
0.07
3.99
0.10
0.15
0.49
0.64
HSA + 4% Mannitol +
Met + MSG + Ala
2
10% Trehalose + 0.1%
4.71
0.08
4.68
0.08
4.17
0.07
0.03
0.51
0.54
HSA + Urea
10
5% Mannitol + 0.1%
4.57
0.11
4.12
0.01
3.42
0.07
0.45
0.70
1.15
HSA + Met + MSG + Ala
6
1% Sucrose + 0.1% HSA +
4.66
0.13
4.58
0.15
3.87
0.12
0.08
0.71
0.79
4% Mannitol
4
1% Trehalose + 0.1%
4.22
0.05
4.32
0.10
3.57
0.07
−0.1
0.75
0.65
HSA + 4% Mannitol + Urea
13
10% Trehalose + 0.1%
5.04
0.08
4.53
0.06
3.78
0.10
0.51
0.75
1.26
HSA
9
1% Sucrose + 0.1%
4.77
0.04
4.56
0.07
3.78
0.10
0.21
0.78
0.99
HSA + 4% Mannitol + Urea
8
10% Sucrose + 0.1% HSA
5.02
0.06
4.84
0.08
4.05
0.11
0.18
0.79
0.97
14
Control, reference
5.03
0.04
4.57
0.04
3.76
0.04
0.56
0.81
1.37
formulation
12
10% Sucrose + 0.1%
5.20
0.23
4.85
0.11
4.00
0.08
0.35
0.85
1.2
HSA + Met + MSG + Ala
7
1% Trehalose + 0.1%
4.55
0.12
4.52
0.14
3.65
0.15
0.03
0.87
0.9
HSA + 4% Mannitol
11
10% Trehalose + 0.1%
4.90
0.04
4.60
0.04
3.72
0.03
0.30
0.88
1.18
HSA + Met + MSG + Ala
15
5% Mannitol + 0.1%
3.62
0.13
3.84
0.02
0.00
n/a
−0.22
3.84
3.62
HSA + Urea
16
5% Mannitol + 0.1% HSA
4.01
0.14
4.08
0.08
0.00
n/a
−0.07
4.08
4.01
Example 3
Prediction Profiling
As an additional method for evaluating the efficacy of various formulations on flavivirus vaccine stabilization, prediction profiling methods can be used as a way to down-select various excipients included in the formulation and/or identify excipients that are more effective than others. The effects of various excipients were tested using expression profiling, (data not shown) after lyophilization, five weeks after lyophilization, and five weeks after lyophilization and exposure to 25° C. The data obtained from the prediction profiling were compared to a desirability value, which was used as a guideline for determining the best retention of virus potency after lyophilization and after exposure to 25° C. for 5 weeks. Sucrose and trehalose were found to be interchangeable using these prediction profiling methods. Positive interactions were observed between excipients that can result in increased flavivirus stability profiles after lyophilization and incubation at 25° C. for 5 weeks (data not shown).
Example 4
Lyophilization Stabilization Study
To evaluate effects of various excipients on the stability of a dengue virus serotype 3 vaccine, sample formulations of flavivirus (e.g. dengue serotype-3, TDV-3) vaccines were lyophilized and exposed to 25° C. for 5 weeks, as illustrated in FIG. 2 (see also, Table 4 below). Titers were obtained prior to lyophilization or pre-lyophilization (solid black bar in each triplicate, bar to the left of each test), after lyophilization or post-lyophilization (solid light gray bar in each triplicate, bar in the middle of each test), and after lyophilization and exposure to 25° C. for 5 weeks (solid medium-gray bar to the right of each test; two separate vials were titrated in triplicate). A control, reference formulation that included F-127, trehalose, and albumin (FTA) was used to assess potential improved formulation characteristics.
Statistically significant improvement was observed in virus potency retention after incubation at 25° C. for 5 weeks. Combinations of these excipients demonstrate higher tolerance to thermal stress in the solid state over time compared to the reference/control formulation.
TABLE 4
Exemplary compositions of various lyophilized flavivirus
(e.g. dengue serotype-3, TDV-3) formulations All of
the formulations in this table were prepared in 10 mM
sodium phosphate buffer with 30 mM NaCl, pH 7.2.
Formulation
No.
Composition
(corresponds
Trehalose
Mannitol
Ala
HSA
Urea
to FIG. 2)
%
(%)
(mM)
(%)
(%)
1
10
1
20
0.3
0
2
10
1
10
0
0.250
3
1
1
0
0.3
0.125
4
1
1
20
0.1
0.250
5
10
5
0
0.1
0.000
6
10
3
0
0.3
0.250
7
5.5
3
10
0.1
0.125
8
1
5
0
0
0.250
9
5.5
5
20
0.3
0.250
10
1
3
20
0
0.000
11
10
5
20
0
0.125
12
5.5
1
0
0
0.000
13
1
5
10
0.3
0.000
14
Control, Reference sample (15% Trehalose + 0.1% HSA +
1% Pluronic F127 ®)
Example 5
Modeling Stabilization Study with Trehalose and HSA
Potential impact of two excipients, trehalose and HSA, were assessed on stabilizing formulations for subsequent use in a definitive screen. Effects of 0.3% (w/v) HSA and 5% (w/v) trehalose concentrations on stability of exemplary live, attenuated flaviviruses after solid-state lyophilization were assessed (data not shown). These experiments demonstrated that effective stabilization formulations can include a lower concentration of Trehalose, 5% (w/v) and HSA at 0.3% (w/v), as indicated in Table 5 below. In addition, it is noted that mannitol and urea were also analyzed for their ability to stabilize live, attenuated viruses such as flaviviruses and demonstrated to have positive effects on flavivirus stabilization.
TABLE 5
Exemplary compositions of various lyophilized flavivirus
(e.g. dengue serotype-3, TDV-3) formulations
Max desirability
Component
Concentration
Mannitol
3.0% (w/v)
Trehalose
5.0% (w/v)
Urea
0.125% (w/v)
HSA
0.3% (w/v)
Example 6
Screening for Exemplary Tetravalent Stabilizing Formulations
Data obtained for the stabilization formulations for flavivirus (e.g. dengue serotype-3, TDV-3) (above) can be applied to other dengue virus serotypes, including as part of tetravalent dengue virus formulations. Two different target doses were evaluated for each dengue virus serotype (TDV-1, TDV-2, TDV-3 and TDV-4) illustrated in FIG. 3 to FIG. 6, a 50% BDS (triangles) and a CTM (squares). The bulk dose target formulation included about 50% of the bulk drug substance (BDS) for each dengue virus serotype, and the current dose target formulation included about 2.5% of bulk drug substance for each dengue virus serotype (also referred to as the Current Dose or CD). Both the current and bulk dose target formulations also included mannitol (3.0% (w/v)), trehalose (5% (w/v)), and urea (0.125% (w/v)). Table 6 below lists the formulations used to evaluate tetravalent virus stability.
TABLE 6
Exemplary tetravalent dengue virus vaccine formulations
Final excipient concentrations after formulation with BDS
CD
Native
Form.
PF 127
HSA
rHSA
Trehalose
Mannitol
Urea
Dextran
No.
Composition
(%)
(%)
(%)
(%)
(%)
(%)
(%)
Form 1
Mannitol(3%) + Trehalose
0.02
0.002
5
3
0.125
1
CD
(5%) + Urea(0.125%) + Dextran
(1%) (low dextran)
Form 2
Mannitol(3%) + Trehalose
0.02
0.002
5
3
0.125
5
CD
(5%) + Urea(0.125%) + Dextran
(5%) (high dextran)
Form 3
Mannitol(3%) + Trehalose
0.02
0.002
0.3
5
3
0.125
CD
(5%) + Urea(0.125%) +
recombinant HSA(0.3%)
Form 4
Mannitol(3%) + Trehalose
0.02
0.3
5
3
0.125
CD
(5%) + Urea(0.125%) + native
HSA(0.3%)
FTA_CD
FTA
1
0.1
15
Form 5
Mannitol(3%) + Trehalose
0.5
0.05
7.5
3
0.125
1
50% BDS
(7.5%) + Urea(0.125%) + Dextran
(1%) (low dextran)
Form 6
Mannitol(3%) + Trehalose
0.5
0.05
7.5
3
0.125
5
50% BDS
(7.5%) + Urea(0.125%) + Dextran
(5%) (high dextran)
Form 7
Mannitol(3%) + Trehalose
0.5
0.05
0.3
7.5
3
0.125
50% BDS
(7.5%) + Urea(0.125%) +
recombinant HSA(0.3%)
Form 8
Mannitol(3%) + Trehalose
0.5
0.3
7.5
3
0.125
50% BDS
(7.5%) + Urea(0.125%) + native
HSA(0.3%)
CON_50%
Reference/control
1
0.1
15
BDS
Target viral titers for the tetravalent dengue virus formulations are illustrated below in Table 7.
TABLE 7
Target viral titers for tetravalent dengue virus vaccine formulations
Current dose target titer(CD)
50% BDS target titer(50% BDS)
PFU/dose
PFU/dose
TDV-1
2.00E+04
5.09E+05
TDV-2
5.00E+03
1.27E+05
TDV-3
1.00E+05
2.54E+06
TDV-4
3.00E+05
8.03E+06
As illustrated in FIGS. 3A to 3B to FIGS. 6A to 6B, excipients tested were dextran at 1% (w/v) and 5% (w/v) (as an alternative to HSA), recombinant HSA at 0.3% (w/v), and natural HSA at 0.3% (w/v), along with a FTA positive reference formulation. The effects of each were evaluated after exposure to 25° C. for 5 weeks after lyophilization (FIGS. 3A, 4A, 5A and 6A) and after total loss (FIGS. 3B, 4B, 5B and 6B), which is the summation of potency loss after lyophilization (liquid to solid) and loss after exposure to 25° C. for 5 weeks. Generally, the lower the titer loss and the more overlap between the doses, the higher the stabilizing effect of the excipient.
Example 7
Modeling Stabilization Study with Trehalose and HSA
Based on the above data pertaining to the concentrations of trehalose and HSA, modeling studies were used to reassess the design space for Flavivirus stabilizing formulations (data not shown). Modeling data tested the impact of 0.2% HSA and 5% trehalose concentrations on stability of exemplary live, attenuated Flaviviruses after lyophilization, according to one embodiment disclosed herein, indicated that stabilization formulations can include Trehalose at 5% and HSA at 0.2% (data not shown).
Example 8
Effects of Various Agents on Formulation Cake Integrity and Virus Stability
Various formulations were evaluated at different concentrations and in combinations in lyophilized formulations for their ability to improve dengue virus stability (e.g. dengue virus serotype 3) as well as to form freeze-dried cakes with suitable appearance and suitable physical integrity for a variety of intended purposes. As described herein, lyophilized cakes were rated on a subjective scale from 1 to 3, where 1=bad, 2=fair, and 3=good. Cakes with a rating of 1 were distinguished as having extensive structural collapse, meltback on the bottom and sides, significant shrinkage and retraction from all sides, and/or appearance of granules. Cakes with a rating of 2 had partial shrinkage and retraction from the sides, moderate meltback on the bottom, and/or large cracks or fissures running through the cake horizontally or vertically. Cakes with a rating of 3 were pharmaceutically elegant, appearing as compact, (mostly white, excipient combination depending) cake structures with generally flat surfaces and no or minimal shrinkage or retraction of the cake from the top of the cake or sides of the vial. Effects of varying ratios of carbohydrate such as sugars and mannitol were tested for effects on cake integrity using placebo formulations. An optimized sugar/mannitol ratio was selected for studying the effect of sugar/mannitol (e.g. trehalose or sucrose to mannitol) on virus stability with respect to lyophilization and stability of the lyophilized virus. In this example, this experiment was performed with formulated bulk drug substance a dengue virus serotype 3 (e.g. TDV3).
An exemplary list of placebo formulations evaluated is provided in Table 8 below. Based on results of screening the placebo formulations, three different sugar to mannitol ratios were selected for screening virus stability: sugar (10% (w/v)) alone; sugar to mannitol ratio of 1:4; and mannitol alone (5% (w/v)). Stabilizing excipients (urea and amino acids) identified from previous studies were also evaluated in combinations with different sugar to mannitol ratios.
TABLE 8
Sugar/Mannitol formulations examined during
lyophilization stability and cake studies
Sugar/Mannitol
Formulation
Sugar (w/v)
Mannitol (w/v)
No.
Sugar
(%)
(%)
1
Trehalose
1.0
4.0
2
Trehalose
2.0
4.0
3
Trehalose
2.0
3.0
4
Trehalose
10.0
0
5
Sucrose
1.0
4.0
6
Sucrose
2.0
4.0
7
Sucrose
2.0
3.0
8
Sucrose
10.0
0
9
No sugar
0
5.0
All of the formulations were prepared in 10 mM sodium phosphate buffer with 30 mM NaCl, pH 7.2 and 0.1% (w/v) HSA. The list of virus-containing formulations that were evaluated as a part of this study is provided in Table 9.
TABLE 9
Formulations evaluated for dengue virus (dengue virus serotype
3) lyophilization and accelerated stability studies
Form.
No.
(corresponds
to Table 3)
Composition
1
10% Trehalose + 0.1% HSA
2
10% Trehalose + 0.1% HSA + 0.25% Urea
3
10% Trehalose + 0.1% HSA + 10 mM MSG +
10 mM Alanine + 2.5 mM Methionine
4
10% Sucrose + 0.1% HSA
5
10% Sucrose + 0.1% HSA + 0.25% Urea
6
10% Sucrose + 0.1% HSA + 10 mM MSG +
10 mM Alanine + 2.5 mM Methionine
7
1% Trehalose + 4% Mannitol + 0.1% HSA
8
1% Trehalose + 4% Mannitol + 0.1% HSA +
0.25% Urea
9
1% Trehalose + 4% Mannitol + 0.1% HSA +
10 mMMSG + 10 mM Alanine + 2.5 mM Methionine
10
1% Sucrose + 4% Mannitol + 0.1% HSA
11
1% Sucrose + 4% Mannitol + 0.1% HSA +
0.25% Urea
12
1% Sucrose + 4% Mannitol + 0.1% HSA +
10 mM MSG + 10 mM Alanine + 2.5 mM Methionine
13
5% Mannitol + 0.1% HSA
14
5% Mannitol + 0.1% HSA + 0.25% Urea
15
5% Mannitol + 0.1% HSA + 10 mM MSG + 10 mM
Alanine + 2.5 mM Methionine
16
FTA(15% Trehalose + 0.1% HSA + 1% PF127)
In certain methods, effects of varying the ratio of sugar (e.g., trehalose and/or sucrose) to mannitol on cake appearance of lyophilized samples and subsequently on flavivirus (e.g. dengue virus) stability was evaluated. In one method, to evaluate effect of sugar to mannitol ratio on cake integrity and appearance, placebo formulations were prepared and lyophilized. Following lyophilization, the physical integrity and appearance of cake formation was evaluated (data not shown). Cake integrity appeared to be good with mannitol alone, sugar to mannitol ratio of 1:4 and 2:4, however, partial collapse was observed with formulations prepared with a sugar to mannitol ratio of about 2:3. There was no notable difference in cake integrity observed between trehalose and sucrose formulations. When sugar alone was used, the cake integrity did not appear and test as well was observed using mannitol alone. Based on the cake integrity observations of this exemplary study, the following combinations were selected for virus stability studies: sugar alone, sugar to mannitol ratio of 1:4, and mannitol alone.
In one example, a flavivirus (dengue serotype 3) composition with the selected sugar to mannitol ratios was prepared in combination with the amino acid mixture or urea as additional excipients (see Table 8). Following lyophilization, the physical integrity of cakes was evaluated (data not shown). In examples of formulations containing sugar alone, the cake integrity was similar to control FTA formulations. Formulations containing mannitol alone and sugar to mannitol ratio of 1:4 formed cakes with what appeared to be acceptable appearances and good physical integrity. Addition of urea or the amino acid mixture did not significantly affect cake integrity. These proven formulations appear to be useful for pharmaceutical compositions of use in immunogenic formulations.
Example 9
Combined Semi-Empirical Screening and Design of Experiments (DoE)
To experimentally approach formulation development of a live virus vaccine candidate with improved stability, one would ideally determine the causes and mechanisms of virus degradation and then design formulations to minimize their occurrence. Although this approach has been demonstrated with several viral vaccines it has focused primarily on liquid based formulations and not lyophilized formulations. More empirical approaches are often required including traditional excipient screening studies, quality by design (QbD) approaches (including systematic integration of risk management and prior scientific knowledge with Design of Experiments, DoE), or a combination of both approaches. In this study, a combination approach was demonstrated in a step-wise fashion utilizing a live attenuated dengue vaccine candidate as a model live virus vaccine. To identify formulations that achieve a target product profile of a parenterally administered, lyophilized, multivalent live virus vaccine was tested with sufficient long-term storage at 2-8° C. First, semi-empirical screening was performed to identify virus stabilizers followed by a more detailed evaluation of the top stabilizing excipient “hits” using and exploratory data analysis that acted as guidance for the design of a predictive statistical model by DoE. The step-wise use of a combination approach provides a basis for a systematic, stage appropriate approach to live virus vaccine formulation development during early stage product development.
Preliminary Screening of Pharmaceutical Excipients on Flavivirus (e.g. Dengue Serotype-3, TDV-3) Stability
Preliminary screening identified a number of potential stabilizing excipients, in terms of maintaining viral potency during lyophilization and an accelerated stability, while producing acceptable physical integrity of the dried cake after freezing. (See FIGS. 7 and 8). For this initial excipient screening, flavivirus (e.g. dengue serotype-3, TDV-3) formulations were prepared in a base buffer that contained either trehalose or sucrose in 10 mM phosphate buffer with 30 mM NaCl, pH 7.2. As outlined above, other buffers were also evaluated, including HEPES, Tris, and histidine (with and without NaCl). No differences were observed in the storage stability of the aforementioned buffers; however, flavivirus (e.g. dengue serotype-3, TDV-3) appeared to have additional stability when NaCl was included (data not shown). Lyophilized flavivirus (e.g. dengue serotype-3, TDV-3) formulations were prepared using various excipients from different known categories of pharmaceutical additives. Lyophilized flavivirus (e.g. dengue serotype-3, TDV-3) formulations were prepared using various excipients (see Table 12). These additives were selected based on a combination of experience, literature searches of formulation composition of live virus vaccines (marketed and investigational) and scientific rationale. The cake appearance of the lyophilized samples was assessed (Tables 10 and 11) based on a rating system (1-3, where 1 is the worst cake profile and 3 is the best cake profile by observation and physical characteristics).
FIGS. 7A-7D and Table 10 depict lyophilized Flavivirus (e.g. dengue serotype-3, TDV-3) formulations and virus titers at pre and post-lyophilization with respect to different stabilizers in a trehalose base buffer. Loss in virus titer was calculated following lyophilization (FIGS. 7A-7D) and the cake appearance of the lyophilized samples was also assessed (Table 10) based on a visual rating system (as described above). These results demonstrated that many excipients were able to stabilize flaviviruses (e.g. dengue serotype-3, TDV-3) during lyophilization (in the base buffer) including urea, EDTA, polyols, proteins, polymers and amino acids, which resulted in <0.2 log loss in titer. Certain excipients did not individually protect against lyophilization (e.g. MgCl2 and poloxamer 407) while others (sorbitol) could stabilize virus during lyophilization, but failed to produce an elegant cake structure and were excluded from further analysis. Therefore, these agents were not used in the disclosed formulations herein. Similar excipient trends were observed when sucrose replaced trehalose in the base buffer (FIGS. 8A-8B).
In this example, FIG. 7A illustrates flavivirus (e.g. dengue serotype-3, TDV-3) viral titers in pre and post lyophilized samples and FIG. 7B illustrates flavivirus (e.g. dengue serotype-3, TDV-3) titer loss following lyophilization. Data are represented as mean values±s.d. Virus titers are provided in log10 pfu/0.5 ml. * represents titers below LOD. Base buffer of these examples contained 10% trehalose in 10 mM phosphate buffer with 30 mM NaCl, pH 7.2. With respect to these exemplary experiments wherein trehalose was replaced by sucrose, FIG. 8A illustrates flavivirus (e.g. dengue serotype-3, TDV-3) viral titer loss following lyophilization. Data are represented as mean values±s.d. Virus titers are given in log10 pfu/ml.
Particular excipient candidates from exemplary experiments illustrated in FIGS. 7A & 8A were further evaluated for their ability to stabilize flavivirus (e.g. dengue serotype-3, TDV-3) via short-term accelerated storage stability at 25° C. for 2 weeks. Virus potency, as measured by viral titers in a plaque assay, and the loss in virus titer over time (expressed as stability log10 loss), are illustrated in FIGS. 7C and 7D and FIG. 8B for trehalose and sucrose containing base buffer formulations, respectively. FIG. 7C illustrates flavivirus (e.g. dengue serotype-3, TDV-3) viral titers in lyophilized samples after an accelerated stability study, and FIG. 7D illustrates flavivirus (e.g. dengue serotype-3, TDV-3) titer loss after 2 weeks at room temperature (˜25° C.). FIG. 8B illustrates flavivirus (e.g. dengue serotype-3, TDV-3) viral titer loss after lyophilized samples were stored for 5 weeks at room temperature (˜25° C.). These results depict that formulations containing HSA, urea, alanine, methionine, MSG, mannitol, or gelatin displayed similar trends in both the base formulations and displayed improved virus stabilization during storage at 25° C. These results indicate that HSA, urea, alanine, methionine, MSG, and mannitol are flavivirus (e.g. dengue serotype-3, TDV-3) stabilizers and were selected for further screening using the same trehalose/-sucrose base buffer formulations.
TABLE 10
The visual cake integrity rating of lyophilized formulations
based on visual appearance for flavivirus (e.g. dengue
serotype-3, TDV-3) lyophilized formulations containing
trehalose (as illustrated in FIG. 7A-7D) (as detailed
above, 1-3, where 1 is the worst and 3 is the best).
Average Cake
Lyophilized formulation
Appearance
MgCl2
1
Sorbitol
2
Mannitol
2
Polysorbate 80
2
Lactose
3
Sucrose
3
HSA
3
Urea
3
Alanine
3
EDTA
3
Methionine
3
Gelatin
3
MSG
3
Dextran 40
3
Reference agent, poloxamer 407
3
TABLE 11
The visual cake integrity rating of lyophilized formulations based
on visual appearance for flavivirus (e.g. dengue serotype-3, TDV-3)
lyophilized formulations containing sucrose (as illustrated in
FIG. 8A-8B) (as detailed above, 1-3, where 1 has the worst and
3 has the best cake appearance and physical attributes).
Average Cake
Lyophilized formulation
Appearance
Mannitol
1
Sorbitol
1
MgCl2
1
Alanine
2
Urea
2
HSA
3
Lactose
3
Trehalose
3
MSG
3
Methionine
3
EDTA
3
PS80
3
Gelatin
3
Dextran 40
3
PF127
3
These results demonstrated that many excipients listed in Table 12 were able to stabilize flavivirus (e.g. dengue serotype-3, TDV-3) during lyophilization (in the base buffer) including urea, EDTA, polyols, proteins, polymers and amino acids, which resulted in <0.2 log loss in titer. The additives MgCl2 and poloxamer 407 imparted the lowest individual agent virus stabilizing potential during lyophilization with >2 log loss individually (however, poloxamer 407 had a synergistic stabilizing effect in the presence of other excipients; see below for more details). Certain excipients (e.g., MgCl2 and poloxamer 407) did not individually protect against lyophilization while others (e.g., sorbitol) could stabilize virus during lyophilization. Similar excipient trends were observed when sucrose replaced trehalose in the base buffer (FIG. 8).
TABLE 12
Summary list of excipients screened for ability to stabilize
Flavivirus (e.g. dengue serotype-3, TDV-3) virus during
lyophilization and accelerated storage as well as to form
pharmaceutically elegant lyophilized cakes. Excipients were
evaluated in presence of various base buffers containing
additional additives as described in the text.
Category
Excipients
Proteins/Polymers
Human serum albumin and rHSA (0.1,
0.3, and 2%)
Gelatin (3.0%)
Dextran 40 (1-10%)
Sugars
Sucrose (1-10%)
D-Trehalose dihydrate (1-15%)
Lactose (4%)
Polyols
Sorbitol (3.0%)
Mannitol (1, 3, 5%)
Surfactants
Polysorbate-80 (0.05%), PF127 (1.0%)
Amino acids
Monosodium 1-glutamate (10 mM)
Methionine (2.5 mM)
Alanine (10 mM)
Arginine (10 mM)
Osmolytes
Urea (0.125 and 0.25%)
Salts
Magnesium chloride (100 mM)
Chelating agents
EDTA (1 mM)
Some of the exemplary excipient candidates were further evaluated for their ability to stabilize flaviviruses (e.g. dengue serotype-3, TDV-3) via short-term accelerated storage stability at 25° C. for 2 weeks. Virus potency as measured by viral titers in a plaque assay, and the loss in virus titer over time (expressed as stability log10 loss), are shown in FIGS. 7C and 7D for trehalose containing base buffer formulations, and in FIGS. 8A and 8B for sucrose containing base buffer formulations. Formulations containing, for example, HSA, urea, alanine, methionine, MSG, mannitol, and/or gelatin displayed similar trends in both the base formulations and displayed improved virus stabilization during storage at 25° C. Interestingly, dextran displayed superior stability with trehalose but not with sucrose base buffer. Despite polysorbate 80 and lactose displaying good lyoprotection, these excipients were not able to stabilize flaviviruses (e.g. dengue serotype-3, TDV-3) during storage in the dried state at 25° C. In summary, HSA, urea, alanine, methionine, MSG, and mannitol were identified as potential flavivirus (e.g. dengue serotype-3, TDV-3) stabilizers and were selected for further screening using the same trehalose/sucrose base buffer formulations.
Screening of Excipient Combinations for Flavivirus (e.g. Dengue Serotype-3, TDV-3) Stability
The effect of combining the lead excipients at the different concentrations on flavivirus (e.g. dengue serotype-3, TDV-3) titers before and after lyophilization and accelerated stability was evaluated. Referring now to FIG. 9 and FIG. 15, base buffer contained 10% trehalose in 10 mM phosphate buffer with 30 mM NaCl, pH 7.2, and 0.1% HSA. FIGS. 9A and 15A illustrate flavivirus (e.g. dengue serotype-3, TDV-3) titer loss following lyophilization; and FIG. 9B and FIG. 15B illustrate flavivirus (e.g. dengue serotype-3, TDV-3) viral titer loss after lyophilized samples were stored for 5 weeks at room temperature (˜25° C.). In these figures, data are represented as mean values±s.d. Virus titers are given in log10 pfu/ml.
For these studies, HSA (at 0.1 and 0.2% w/v) was added to the base formulations because in certain methods related to the initial screen indicated HSA functioned as an effective lyoprotectant (FIGS. 9 and 15). Loss (<0.2 log loss) of flaviviruses (e.g. dengue serotype-3, TDV-3) titer post-lyophilization at both HSA concentrations was observed in base formulations containing either 10% sucrose or trehalose, respectively (FIGS. 9A and 15A). Therefore, subsequent base buffer formulations were prepared containing phosphate buffer, NaCl, 10% sugar (trehalose or sucrose) with 0.1% HSA and indicated additional excipients. Most of the excipient combinations in either of these base formulations displayed improved virus stability in both post lyophilization and accelerated stability samples (FIGS. 9 and 15). Maximum lyoprotection was demonstrated by using a combination of amino acids and urea added in the base formulations. After 5 weeks at 25° C., either of the base formulations with mannitol and urea was added, either with or without amino acids such as alanine, arginine, MSG and methionine displayed a highly improved viral stability (FIGS. 9 and 15). Increasing the sugar concentrations in the base formulations to 15% did not appear to provide any additional advantage in terms of protecting viral titers.
TABLE 13
The visual cake integrity rating of lyophilized formulations
based on visual appearance for flavivirus (e.g. dengue serotype-3,
TDV-3) lyophilized formulations (as seen in FIG. 9A-9B) (as detailed
above, 1-3, where 1 represents the worst and 3 represents the
best cake appearance and physical features).
Average Cake
Lyophilized formulation
Appearance
Met + MSG + Ala + Urea + Mann
2
15% Tre + 0.1% HSA
2
Met
2
Mann
3
10% Tre + 2% HSA
3
10% Tre + 0.1% HSA
3
Met + MSG + Ala + Urea
3
MSG
3
Ala
3
Urea
3
Met + MSG + Ala
3
Met + MSG
3
Met + Ala
3
MSG + Ala
3
Met + Urea
3
MSG + Urea
3
Ala + Urea
3
Arg
3
Arg + Urea
3
The final screening experiments investigated the effect of varying sugar to mannitol ratios on flavivirus (e.g. dengue serotype-3, TDV-3) potency and lyophilized cake integrity. Referring now to the composition formulations detailed in Table 9 and FIG. 10 A to 10B. FIG. 10A illustrates flavivirus (e.g. dengue serotype-3, TDV-3) titer loss following lyophilization, and FIG. 10B illustrates flavivirus (e.g. dengue serotype-3, TDV-3) titer loss after lyophilized samples were stored for 5 weeks at room temperature (˜25° C.). Base buffer contained 10 mM phosphate buffer with 30 mM NaCl, pH 7.2, and 0.1% HSA. Data are represented as mean values±s.d. Virus titers are given in log10 pfu/ml. * N/A below LOD.
Flavivirus (e.g. dengue serotype-3, TDV-3) formulations with the selected sugar to mannitol ratios were prepared in combination with the amino acid mixture or urea as additional excipients in a base buffer (Table 9). Most of the formulations (as found in Tables 3 & 9) demonstrated improved viral titer yields after lyophilization (Table 3). The addition of urea demonstrated improved virus stabilizing potential, compared with the amino acid mixture, in base buffer containing either sucrose or trehalose after storage at 25° C. for 5 weeks (Tables 3 & 9, Formulation 5 and 6; FIG. 10A). Interestingly, when mannitol was added to the base formulations, the amino acid mixture demonstrated increased virus stabilizing potential compared to urea (Tables 3 & 9, Formulation 9 and 10; FIG. 10A). Nevertheless, certain formulations (e.g., 13, 14, and 15) containing mannitol alone demonstrated poor virus stabilizing effect irrespective of addition of urea or amino acid mixture. These observations demonstrate an important effect of amorphous sugar on virus stability, and these experiments further demonstrate sugar and mannitol interactions as important excipients in terms of stabilizing flaviviruses. To further study the effects of these promising stabilizing excipients on flaviviruses (e.g. dengue serotype-3, TDV-3) during freeze-drying and accelerated storage, a rational design of experiment approach was employed as described below.
Design of Experiment
Referring now to FIG. 11, illustrates a comprehensive viral potency loss dataset for every formulation analyzed during screening studies. Data were sorted into three stages of screening represented by Screening Groups 1, 2, and 3 coinciding with data presented in FIGS. 8-10, and FIG. 15. These data illustrate log10 pfu/0.5 mL titers for liquid (pre-lyo), post-lyo, and 5 weeks at 25° C. stability samples. FIGS. 11A and 11B illustrate identification of statistically significant terms that provide protection from freeze-drying and thermal challenge (Log10 lyo loss (FIG. 11A) and log10 drop 5 weeks at 25° C. (FIG. 11B)). Data were modeled using stepwise regression to determine which terms were significantly impacting potency as statistically observed. In addition to using the Bayesian Information stopping Criterion to help converge on a best fit model, main effect and interaction terms were considered significant if their probability value was <0.05. The adequacy of the model fit was also assessed using the R2 values.
The semi-empirical screening of excipients and formulations generated a large dataset of frozen liquid (pre-lyo), post-lyo, and accelerated stability (5 weeks at 25° C.) viral potency measurements for 85 individually prepared formulations of flaviviruses (e.g. dengue serotype-3, TDV-3) (data not shown). Analyzing the comprehensive dataset visually reveals that, with each sequential screening experiment, there is an increase in retention of flavivirus (e.g. dengue serotype-3, TDV-3) potency for each condition in addition to improved cake appearance. Varying excipient concentrations and their combinations throughout the screening exercises assembled an empirically derived data set that could be utilized using regression modeling to construct an exploratory statistical model.
To determine which excipients (or terms) were individually contributing or interacting to affect flavivirus (e.g. dengue serotype-3, TDV-3) potency losses, the dataset was modeled using stepwise regression tools available in JMP 11.2.0 intended for response surface methods (RSM). The regression analysis utilized the minimum Bayesian Information Stopping Criterion to arrive at the best model and terms and interactions that did not show significance (probability statistic <0.05) were excluded. Model fits with R2 values of 0.44 and 0.73 were determined for log10 lyo loss and log10 drop 5 weeks at 25° C., respectively (FIGS. 11A and 11B). Excipients including HSA, urea, mannitol, and the sugars sucrose and trehalose were identified as being statistically significant for limiting flavivirus (e.g. dengue serotype-3, TDV-3) potency loss after lyophilization (data not shown). Similar sets of excipients were demonstrated to protect flavivirus (e.g. dengue serotype-3, TDV-3) potency loss during accelerated storage stability. Certain additives: urea, mannitol, and HSA demonstrated single term significance while combinations of trehalose or sucrose with urea, and trehalose with mannitol were identified as having significant two term interactions. Amino acids, alanine, methionine, and MSG were included in the model because they seemed to provide stabilizing effects during the screening stages, but were shown in this analysis to not be statistically significant. Utilizing these exploratory models as guidance, the additives HSA, urea, mannitol and trehalose or sucrose were chosen as promising candidates for further study.
TABLE 14
DOE formulation design
DOE
Composition
Form.
Trehalose
Mannitol
Ala
HSA
Urea
No.
%
(%)
(mM)
(%)
(%)
1
10
1
20
0.3
0
2
10
1
10
0
0.250
3
1
1
0
0.3
0.125
4
1
1
20
0.1
0.250
5
10
5
0
0.1
0.000
6
10
3
0
0.3
0.250
7
5.5
3
10
0.1
0.125
8
1
5
0
0
0.250
9
5.5
5
20
0.3
0.250
10
1
3
20
0
0.000
11
10
5
20
0
0.125
12
5.5
1
0
0
0.000
13
1
5
10
0.3
0.000
Referring now to Table 14 and FIG. 12, the Table and Figures provide as follows: Table 14: DOE formulation design FIG. 12A: Flavivirus (e.g. dengue serotype-3, TDV-3) viral titers in liquid, lyophilized (stored at −80° C.) and stability samples (lyophilized formulations stored at 25° C., 5 weeks), FIG. 12B: Flavivirus (e.g. dengue serotype-3, TDV-3) titer loss during lyophilization and 5 weeks stability at 25° C. All formulations contained 10 mM sodium phosphate, 30 mM NaCl, pH 7.2 as an exemplary buffer. FIG. 12C represents three Sorted Estimates Charts generated by analysis of DSD data using forward stepwise regression and the stopping rule set to minimize the corrected Akaike's Information Criterion (AICc) using JMP 11.2.0. Main effect and interaction terms were considered significant if their probability value was <0.05. The adequacy of the model fit was also assessed using the R2 values.
The lead excipient hits and levels identified by the exploratory univariate regression and multivariate data analyses were integrated into a definitive screening design (DSD) (Table 14) in order to confirm by design the significance of their impact to stability and to explore the optimal concentration range for each excipient. Although displaying similar statistical impacts, trehalose was chosen over sucrose because of its additional interactions identified by the exploratory model analysis. Although alanine was not found to be a significant, it was included in the DSD as a representative amino acid, which were present in the most promising formulations during screening. The choice of excipient concentration ranges was informed by the response surface model prediction profiler (data not shown) and the DSD was constructed using the Design of Experiments platform in JMP 11.2.0 and generated 13 individual formulations for assessment. Flaviviruses (e.g. dengue serotype-3, TDV-3) were added to each formulation outlined in Table 14, lyophilized, and challenged at 25° C. for 5 weeks. Definitive Screening Design (DSD) design of experiments formulations for each flavivirus (e.g. dengue serotype-3, TDV-3) and titer in liquid, lyophilized (stored at 80° C.) and stability samples (lyophilized formulations stored at 25° C.) were illustrated. (FIGS. 11A and 11B) Main effect and interaction terms were considered significant if their probability value was <0.05. Solid vertical lines represent p value threshold and any bar crossing these lines demonstrates significant effect. The adequacy of the model fit was also assessed using the R2 values. * N/A below LOD. Formulation details are as follows: F1: 10% Tre+1% Mann+Ala(20 mM)+0.3% HSA; F2:10% Tre+1% Mann+Ala(10 mM)+0.25% U; F3:1% Tre+1% Mann+0.3% HSA+0.125% U; F4:1% Tre+1% Mann+Ala(20 mM)+0.1% HSA+0.25% U; F5:10% Tre+5% Mann+0.1% HSA; F6:10% Tre+3% Mann+0.3% HSA+0.25% U; F7:5.5% Tre+3% Mann+Ala(10 mM)+0.1% HSA+0.125% U; F8:1% Tre+5% Mann+0.250% U; F9:5.5% Tre+5% Mann+Ala (20 mM)+0.3% HSA+0.25% U; F10:1% Tre+3% Mann+Ala(20 mM); F11:10% Tre+5% Mann+Ala(20 mM)+0.125% U; F12: 5.5% Tre+1% Mann; F13: 1% Tre+5% Mann+Ala (10 mM)+0.3% HSA. Mann, mannitol; Tre, trehalose dihydrate; HSA, human serum albumin; Ala, alanine; and U, urea.
Frozen liquid (pre-lyo), post lyo (time 0), and 5 week stability samples were measured for viral potency by plaque assay (FIGS. 12A and 12B). The data set generated by the DSD was analyzed using forward stepwise regression and the stopping rule set this time to minimize the corrected Akaike's Information Criterion (AICc) in JMP 11.2.0. This stepwise regression approach identified the most significant terms and further illuminated the design space for each of the excipients tested (FIG. 12C). Model fits with R2 values of 0.79, 0.89, and 0.99 for each of the responses, including log10 total potency loss. The analysis of losses in viral potency highlighted that mid to high concentrations of trehalose and HSA significantly stabilized potency loss when in concert with low to mid concentrations of mannitol and mid to high concentrations of urea. It is noted that alanine demonstrated no statistically significant effect (not identified by the model).
A contour profiler showing optimized design space for the Definitive Screen Design response surface model for two factor terms, trehalose and HSA, with Mannitol and urea having locked mixture values of 3% and 0.125%, respectively, was generated using JMP 11.2.0. Utilization of JMP's prediction profiler (data not shown). The contour profiler was generated from the predictive DSD models for each of the responses identified the optimal excipient concentrations that protect against viral potency losses as being: 5% trehalose, 3% mannitol, 0.3% HSA, and 0.125% urea.
Evaluate Excipient Combinations for Tetravalent Dengue Vaccine Dosage Form
TABLE 15
Formulation design using DOE
DOE
Formulation
No.
Composition
Form_1
Mannitol (3%) + Trehalose (5%) + Urea (0.125%) +
Dextran (1% (low dextran)
Form_2
Mannitol (3%) + Trehalose (5%) + Urea (0.125%) +
Dextran (5%) (high dextran)
Form_3
Mannitol (3%) + Trehalose (5%) + Urea (0.125%) +
recombinant HSA (0.3%)
Form_4
Mannitol (3%) + Trehalose (5%) + Urea (0.125%) +
native HSA (0.3%)
Referring now to Table 15 and FIG. 13, panels provides as follows: Table 15: Formulation design using DOE, (FIG. 13A: Titer loss of tetravalent formulation: TDV serotypes 1-4 (an immunogenic formulation) after lyophilization, FIG. 13B: Titer loss of TDV serotypes 1-4 (in a tetravalent vaccine formulation) after lyophilized samples were stored at 25° C. for 5 weeks, FIG. 13C: Total viral titer loss of TDV serotypes 1-4 after lyophilization and 25° C. storage for 5 weeks. All DOE formulations also contained 10 mM sodium phosphate, 30 mM NaCl, pH 7.2 * Not calculated (below LLOD).
Tetravalent DOE formulations (TDV-1, -2, -3, -4) were prepared by varying the concentration of dextran (DOE Formulations 1 & 2), rHSA (DOE Formulation 3), and nHSA (DOE Formulation 4) in the base formulation containing trehalose, mannitol, phosphate buffer and NaCl (Table 15). DOE formulations were evaluated and rated as a function of lyophilized cake integrity (data not shown) and combined log10 loss in virus titer (FIG. 13B) following lyophilization and accelerated stability. DOE formulations containing rHSA or nHSA displayed improved virus potency yields when compared to dextran-containing formulations. DOE formulations containing rHSA displayed the highest viral potency recovery for all serotypes with <0.3 log loss for TDV-2 to TDV-4 and 0.5 log loss for TDV-1 (FIG. 13B). Partial cake collapse, however, was observed with the HSA containing formulations (data not shown). Interestingly, DOE formulations containing dextran were more elegant in terms of cake physical appearance, but were not able to preserve virus potency after lyophilization (FIG. 13B).
Real Time Stability Study of Tetravalent Dengue Vaccine Formulations
In addition to the accelerated stability study with the tetravalent TDV at 25° C., a 4° C. stability study was initiated to generate real-time stability at the target long-term storage temperature. Each DOE formulation was stored at 4° C. for up to 39 weeks with viral potency measurements taken at time zero, 5, 13, and 39 weeks. Referring now to FIG. 14, DOE formulations 1-4, contained 5% trehalose, 3% mannitol, 0.125% urea, in 10 mM sodium phosphate with 30 mM NaCl pH 7.2 and the following additives: 1% dextran (FIG. 14A), 5% dextran (FIG. 14B), 0.3% recombinant HSA (FIG. 14C), or 0.3% native HSA (FIG. 14D). Lyophilized samples were stored at 4° C. and reconstituted for viral potency measurements at 0, 5, 13, and 39 weeks. Highlighted region represents the upper and lower 95% Confidence Interval determined in JMP 11.2.0. The integrated table in each graph contains Log10 pfu/0.5 mL titer data for each serotype for liquid (Pre-Lyo), time 0 (Post-Lyo (T0)), and the predicted titer at 78 weeks (Pred. 78 wks). (Of note: the predicted titer was determined by use of the Adapt2 Version 3.4.1 Tool developed by PRISM Training and Consultancy Limited, UK).
All candidate tetravalent TDV formulations demonstrated minimal to no loss in virus potency for all serotypes after 39 weeks at 4° C. (FIGS. 14A-14D). Apparent increases in viral potency over time can be interpreted as zero loss given assay variability (based on in-house observations). Longitudinal time point virus titers were predicted by extrapolation from the best fit line at 78 weeks (2× the last real-time data point) (FIGS. 14A-14D) and demonstrated minimal loss in viral potency predicted over time. However, formulations containing dextran appeared to lose significant amounts of virus titer after lyophilization, compared to those with either HSA, making the total net loss in potency higher for the formulations containing dextran than HSA. These real-time data suggest that the identified and optimized candidate formulations exhibit the desired solid state stability profiles at the target storage temperature.
TABLE 16
The cake appearance of the lyophilized samples was
assessed (FIG. 15A-15B) based on a visual rating system
(1-3, where 1 is the worst and 3 is the best).
Average Cake
Lyophilized formulation
Appearance
Met + MSG + Ala + Urea + Mann
3
Met + MSG + Ala + Urea
3
MSG + Ala
3
Met + Urea
3
MSG + Urea
3
Ala + Urea
3
15% Suc + 0.1% HSA
3
10% Suc + 2.0% HSA
3
10% Suc + 0.1% HSA
3
Met
3
MSG
3
Ala
3
Urea
3
Mann
3
Met + MSG + Ala
3
Met + MSG
3
Met + Ala
3
Arg
3
Arg + Urea
3
In certain experiments, the effect of combining the lead excipients at the different concentrations on flavivirus (e.g. dengue serotype-3, TDV-3) titers before and after lyophilization and accelerated stability were evaluated. For these studies, HSA (at 0.1 and 0.2% w/v) was added to the base formulations described above because the results of the initial screen indicated HSA functioned as an effective lyoprotectant (FIG. 15). FIG. 15 demonstrates the results from screening of the effect of various excipient combinations (in a base buffer containing trehalose) on flavivirus (e.g. dengue serotype-3, TDV-3) viral potency. Base buffer contained 10% trehalose in 10 mM phosphate buffer with 30 mM NaCl, pH 7.2. Results indicate (FIG. 15A) flavivirus (e.g. dengue serotype-3, TDV-3) titer loss following lyophilization, (FIG. 15B) flavivirus (e.g. dengue serotype-3, TDV-3) titer loss after lyophilized samples were stored for 5 weeks at room temperature (˜25° C.). Data are represented as mean values±s.d. Virus titers are given in log 10 pfu/ml.
As illustrated in FIG. 15, a <0.2 log loss of flavivirus (e.g. dengue serotype-3, TDV-3) titer post-lyophilization was observed at both HSA concentrations in base formulations containing either 10% sucrose or trehalose, respectively (FIG. 15A). Therefore, subsequent base buffer formulations were prepared containing phosphate buffer, NaCl, 10% sugar (trehalose or sucrose) with 0.1% HSA and indicated additional excipients. Most of the excipient combinations in either of these base formulations displayed improved virus stability in both post lyophilization and accelerated stability samples (FIGS. 15A-15B). Maximum lyoprotection was demonstrated by using a combination of amino acids and urea added in the base formulations. After 5 weeks at 25° C., either of the base formulations with mannitol and urea added, either with or without amino acids such as alanine, arginine, MSG and methionine demonstrated vastly improved viral stability (FIGS. 15A-15B). Increasing the sugar concentrations in the base formulations to 15% did not appear to provide any additional advantage in terms of protecting viral titers.
Preservation of virus potency in a live attenuated viral vaccine is vital for successful immunization across the shelf life of the product, which in turn, is largely dependent on the composition of the formulation and the type of final pharmaceutical dosage form (i.e., liquid vs lyophilized). Since lyophilization involves freezing and desiccation stresses, single excipients are often not sufficient for virus stabilization and hence a combination of additives is often required to protect against different degradation mechanisms. Sugars such as sucrose or trehalose, appeared to be very important in these lyophilized formulation for lyoprotection of the flavivirus titers and for other live virus vaccines. For example, trehalose in combination with gelatin was demonstrated to have better stabilization of a live-attenuated mumps vaccine compared to a combination of sucrose and sorbitol. Conversely, sucrose and lactalbumin afforded better thermal protection to a live, attenuated peste des petits ruminants (PPR) vaccine as opposed to trehalose alone.
In certain exemplary methods, the effect of both the sucrose and trehalose was systematically studied in combination with other excipients on the stability of flaviviruses (e.g. dengue serotype-3, TDV-3). Formulations containing urea, EDTA, polyols, proteins, polymers and amino acids displayed high virus recoveries post lyophilization with <0.2 log loss in flavivirus (e.g. dengue serotype-3, TDV-3) titer independent of sugar type (trehalose vs. sucrose). Among the studied excipients, viral potency losses were observed to be significant after lyophilization with formulations containing MgCl2 and poloxamer 407 compared to other formulations. MgCl2 is well known for its stabilization of poliovirus; however, the instant methods provide support of detrimental effects on flavivirus stability. Stabilizing effects of MgCl2 may be virus dependent.
Once the lead excipients of these methods were identified, selected excipient combinations were tested on their ability to preserve flaviviruses (e.g. dengue serotype-3, TDV-3) potency. Formulations containing certain combinations of amino acids as well as urea displayed significant observable flavivirus lyoprotection, and when further combined with mannitol, imparted the highest virus potency stabilization after 25° C. storage for 5 weeks. While not wishing to be bound by any strict formulation, urea appears to have an important role in protecting flavivirus integrity/stability during dehydration. Addition of amino acids improved stability of various proteins in sucrose containing formulations during storage and could potentially play a role in stabilization via preferential hydration and/or increased solvent surface tension. Mannitol also demonstrated improved stabilizing effects on flaviviruses.
One approach adopted for formulation design of lyophilized live flavivirus vaccine candidates was a combination of semi-empirical experimentation and enhanced exploratory data analyses. The starting list of pharmaceutical excipients to examine for this work was reduced to 18 additives using scientific rationale based on literature review, pharmaceutical experience and considerations of the final target product profile (see Table 9). The empirical screening of excipient combinations generated a historical dataset that was interrogated by multivariate analyses and visualizations to generate exploratory graphs from initial models that guided selection of key excipients, their potential ranges, as well as excipient interactions in terms of their virus stabilization effects. Building an experimental dataset from the start instead of designing multivariate screening experiments, avoided adding excipients that had no effect and/or the use of inappropriate ranges for the excipients in a designed study.
Building an experimental virus stability dataset, performing an exploratory data analysis and retroactively fitting the data into ‘initial’ regression models is not without its limitations. This is why the models generated at this stage were described as exploratory (FIGS. 11A and 11B) in that they helped guide the statistically designed experiment likely to result in more reliable predictive models (DSD; Table 15). Historical datasets not statistically designed for precise, predictive modeling may contain non-essential data points, inconsistent response measurements, and/or incomplete measurement data for response parameters. These types of dataset artifacts are unlikely to contribute to accurate and reliable statistical models. This is illustrated by the low R2 value for the model fit of log10 loss in titer after lyophilization (FIG. 11A). This may have been in part due to the difficulty in modeling the space between the two concentrations of HSA tested (0.1% and 2%), which contributed to artificially inflating the significance of the HSA*HSA interaction (FIG. 12A), thus identifying possible misleading curvilinear terms. Having a clear scientific understanding of the dataset imparts the ability to rationally utilize the exploratory data analysis and initial statistical models as guidance for selection of excipients and their concentrations.
Selection of the lead excipients and their potential working concentrations was essential to feed into and maximize the effectiveness of the predictive model derived from the designed experiment. Examination of the response data in the DSD screening models (FIG. 12C), and identifying the most desirable settings and ranges via the prediction profiler in JMP culminated in the creation of a contour plot (data not shown) capable of illustrating and mapping the design space of the optimal formulation for stabilization of lyophilized Flavivirus (e.g. dengue serotype-3, TDV-3). The DSD identified significant interactions between trehalose and HSA, found the stabilizing characteristics to be independent of alanine, and was able to confirm the optimal concentrations of mannitol and urea. The DSD model has good fits with high R2 values that provided confidence in the reliability of the model allowing final selection of the lead formulation.
The models were authenticated as predictive and verified when the lead lyophilized formulation was prepared containing flaviviruses (e.g. a tetravalent mixture of TDV-1, -2, -3, and -4) and challenged at accelerated (25° C. for 5 weeks) and real time stability studies (4° C. for 39 weeks). As predicted by the DSD models, limited loss in virus potency after lyophilization and storage at 25° C. was found for Formulations 3 and 4. Despite elevated losses in potency after lyophilization for dextran containing formulations, all four formulations were predicted to maintain minimal to no loss of potency during storage in solid state for all serotypes at 4° C. for up to 78 weeks (2× the last stability time point), therefore, demonstrating that the candidate lyophilized formulations identified for stabilization of monovalent flaviviruses (e.g. dengue serotype-3, TDV-3) at 25° C. are stabilizing for all four dengue virus serotypes. Currently, many vaccine manufactures wish to replace human derived HSA with rHSA to avoid potential risks associated with human derived products. The substitution of dextran and rHSA for native HSA was an attempt to address this concern.
The instant study provides a case study of the efficiency with which a combined empirical screening and statistical modeling analysis can be used to identify and confirm the primary contributors (and their optimal concentrations) for stabilization and formulation development of candidate lyophilized live virus vaccines. This combined approach to formulation development of lyophilized live virus vaccines not only can save both money and time, but also enables building an enhanced viral stability data package that can support parallel process and formulation development efforts as well as submissions to regulatory agencies who recommend a Quality by Design approach to vaccine product development.
Materials and Methods
In one exemplary method, frozen dengue virus stock (e.g. TDV-3) stocks: ˜7.30×107 pfu/mL) (for example, in certain methods Lot no: DMV013-3 was used) in FTA buffer (1% Pluronic F127® (F) poloxamer 407, 15% Trehalose (T), and 0.1% HSA (A) in PBS buffer, pH 7.4) were stored at −80° C. Stocks were thawed in a 37° C. water bath and aliquoted (100 μL in 1.5 mL tubes). The aliquots were stored at −80° C. until use. In certain methods, aliquots can be frozen and thawed more than one time. For example, the thermal history of these aliquots equates to two freeze thaw cycles. Frozen dengue virus stocks were thawed and mixed by gentle vortexing. Samples were prepared in the following buffers: 10 mM Tris (pH 7.2), 10 mM histidine (pH 7.2), 10 mM HEPES (pH 7.2), 10 mM sodium phosphate (pH 7.2), all with and without 50 mM NaCl, as well as 10 mM phosphate with 137 mM NaCl (PBS). All excipients stock solutions were prepared in their respective buffers and the pH was adjusted to 7.2 or 7.4. To prepare certain virus formulations, virus stock was mixed with the formulation buffer to achieve a final virus concentration of approximately 2×105 pfu/mL.
In one example, six hundred and fifty micro-liters (650 μL) of each of the dengue virus formulations (e.g. tetravalent dengue formulations) were then aliquoted into 2 mL vials, and the vials were either lyophilized or stored at −80° C. as a pre-lyophylization control. A conservative lyophilization cycle was used to ensure that candidate formulations being evaluated were properly freeze-dried. All the samples were loaded in a pre-cooled device to −45° C. Thermocouples were placed in reference (e.g. FTA) control/placebo vials. Thermocouples were placed in following fashion within the shelf: one in the middle, one in the front, two in the back. Once the cycle was completed, the chamber was backfilled with nitrogen until chamber pressure reached 580 Torr and vials were fully stoppered. The chamber was allowed to reach atmospheric pressure (760 Torr). At this point, the release valve was opened and the tray containing the stoppered, lyophilized vials was removed. All vials were then sealed. The freeze-dried vials were then visually assessed and photographed with a digital camera (8-megapixel) to record the cake appearance and structure. The lyophilized virus formulations were then stored at −80° C.
All placebo and virus samples were prepared in a base buffer containing 10 mM sodium phosphate (pH 7.2) with 30 mM NaCl and 0.1% HSA. FTA was prepared in 10 mM PBS (phosphate buffered saline with 137 mM NaCl (pH 7.4). All excipients stock solutions were prepared in their respective buffers and the pH was adjusted to about 7.0 to about 8.0 or about 7.2 to about 7.4. Both pre and post lyophilized virus samples were evaluated for potency using a microtiter IFA assay. Stability studies for selected virus and lyophilized formulations were performed at about room temperature (e.g. 25° C.) (four vials) for five weeks. Samples stored at −80° C. (four vials) for five weeks were used as controls.
In another example, frozen dengue virus stocks (e.g. TDV-1, TDV-2, and TDV-4 virus) in phosphate buffer, 15% Trehalose, 1% poloxamer 407 (F127®) and 0.1% HSA—were supplied and stored frozen at −80° C. until further use. Sucrose and trehalose were purchased from Pfanstiehl Laboratories (Waukegan, Ill.). L-Arginine, methionine, sodium L-glutamate, EDTA, dextran 40, magnesium chloride, mannitol, sorbitol, gelatin were purchased from Sigma Aldrich (St. Louis, Mo.). poloxamer 407 (F127®) sample was received from BASF. Salts for preparing buffers, polysorbate 80, urea were purchased from Fisher Scientific and Acros. Human serum albumin was purchased from Octapharma and Recombinant HSA (rHSA) was obtained from Novazymes.
In other examples, virus titers were determined by plaque assay using a 96-well microtiter method. Briefly, an adherent Vero cell monolayer was infected with dilutions of either monovalent or tetravalent virus, followed by the addition of a viscous overlay. Plates were then incubated at 37° C. for 2 or 3 days depending on serotype, then washed and fixed for staining. Staining was performed using serotype-specific monoclonal antibodies. The assay has a working variable range of ±0.34, 0.17, 0.19, and 0.19 log10 pfu/0.5 mL (dose) for serotypes 1 through 4, respectively as determined by trending control analysis. Each sample was measured in triplicate and the results were reported as an average of the values.
Virus Formulation and Lyophilization.
Bulk solutions of each excipient were prepared in phosphate buffer containing 30 mM NaCl and the pH was adjusted to 7.2. Desired amounts of excipients were mixed with phosphate buffer with 30 mM NaCl to obtain the final concentrations and then appropriate amount of the virus stock was added to achieve 2×105 pfu/ml. All viral preparation work was carried out in a class II biosafety cabinet (Labconco, Kans. City, Mo.).
In certain methods, about 650 μl of viral vaccine formulations were dispensed in sterile 2 mL vials and partially sealed with sterile rubber stoppers. A conservative lyophilization cycle with the process ramping the temperature from −45 to 25° C. at a pressure of 50 mTorr over the course of over 2 days was used to produce lyophilized formulations. Samples were loaded in a FTS Lyo Star II (SP Scientific) that had pre-cooled shelves set to −45° C. Following a freeze step at −45° C. for 2 h and a ramp down to reduced pressure of 50 mTorr (about 2 hr.), the temperature was increased to −37° C. with ramp rate of 0.1° C./min. At the end of primary drying, temperature ramped up to 25° C. with ramp rate of 0.1° C./min and continued drying for 6 hr. Following completion of secondary drying, shelf was cooled to 4° C. and held at 4° C. until the cycle was completed (see Table 2 for more details). Upon cycle completion, vials were backfilled with nitrogen and stoppered under vacuum (580,000 Torr) prior to being removed from the chamber, and then tightly sealed with aluminum seals. The lyophilization cycle was monitored by placing thermocouples in the bottom center of at least three placebo vials per batch and using comparative pressure measurement (pirani vs. capacitance manometer).
Thermo Stability Testing of Lyophilized Virus Formulations.
Selected lyophilized vaccine formulations were stored at 4° C. in refrigerator and at 25° C. in a stability chamber. After indicated lengths of time, vials were sampled and rehydrated with 0.5 ml of sterile water for injection. Viral infectivity of the reconstituted vaccine was assessed using microtiter assay. To evaluate virus stability following lyophilization, infectious potency of rehydrated vaccine was determined by microtiter assay. Loss in virus titer following lyophilization was calculated by subtracting the titer of lyophilized virus samples stored at −80° C. from the initial titer (the frozen liquid control sample stored at −80° C.). Similarly, the loss of virus potency during real time and accelerated storage was determined by comparing the viral infectivity of the incubated samples with the infectivity of post lyophilization samples that were stored at −80° C. Errors for losses were calculated by propagation of error method using following equation SE(C)=√{square root over (SE(A){circumflex over ( )}2+SE(B){circumflex over ( )}2)}. For each condition, samples were titrated in triplicate and average log10 titer was calculated.
Statistical Analysis.
Response Surface methods were fit using stepwise regression tools and the Definitive Screening Design was generated using the Design of Experiments platform available in JMP profiler v11.2.0 (JMP Corporation, Cary, N.C.).
Pharmaceutical Cake Quality Determination.
Lyophilized cakes were rated on a subjective scale from 1 to 3, where 1=bad, 2=fair, and 3=good. Cakes with a rating of 1 were distinguished as having extensive structural collapse, meltback on the bottom and sides, significant shrinkage and retraction from all sides, and/or appearance of granules. Cakes with a rating of 2 had partial shrinkage and retraction from the sides, moderate meltback on the bottom, and/or large cracks or fissures running through the cake horizontally or vertically. Cakes with a rating of 3 were pharmaceutically elegant, appearing as compact, (mostly white, excipient combination depending) cake structures with generally flat surfaces and no or minimal shrinkage or retraction of the cake from the top of the cake or sides of the vial.
Abbreviations
CM: Capacitance manometer
DOE: Design of experiment
HSA: Human serum albumin
HEPES: 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid
IFA: Immunofocus Assay
MSG: Monosodium glutamate
NaCl: Sodium chloride
PBS: Phosphate buffered saline
rHSA: Recombinant human serum albumin
THAM/Tris: Tris (hydroxymethyl) aminomethane
Pfu: Plaque forming unit
PF127: Pluronic F-127®, poloxamer 407
All of the COMPOSITIONS and METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of preferred embodiments, it is apparent to those of skill in the art that variations may be applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.
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16322791
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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tyo:4502
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Takeda Pharmaceutical
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Jun 9th, 2020 12:00AM
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Dec 8th, 2017 12:00AM
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https://www.uspto.gov?id=US10675341-20200609
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Parenteral norovirus vaccine formulations
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The present invention relates to single dose parental vaccine compositions comprising mixtures of monovalent Norovirus virus-like particles. Methods of conferring protective immunity against Norovirus infections in a human subject by administering such compositions are also disclosed.
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10675341
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1. A method of eliciting protective immunity against Norovirus in a human comprising administering parenterally to the human no more than a single dose of a vaccine composition, said composition comprising
(i) genogroup I Norovirus virus-like particles (VLPs), wherein said genogroup I Norovirus VLPs comprise a capsid protein derived from a genogroup I viral strain, and
(ii) genogroup II Norovirus VLPs, wherein said genogroup II Norovirus VLPs comprise a capsid protein derived from a genogroup II viral strain,
wherein said genogroup I Norovirus VLPs and genogroup II Norovirus VLPs are present in the composition in different amounts, wherein said composition comprises about 15 μg to 50 μg of genogroup I Norovirus VLPs and about 50 μg to 150 μg of genogroup II Norovirus VLPs,
wherein said composition induces at least a three-fold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the composition.
2. The method of claim 1, wherein the composition comprises about 15 μg of said genogroup I Norovirus VLPs.
3. The method of claim 1, wherein the composition comprises about 50 μg of said genogroup II Norovirus VLPs.
4. The method of claim 1, wherein the composition comprises about 15 μg of said genogroup I Norovirus VLPs and about 50 μg of said genogroup II Norovirus VLPs.
5. The method of claim 1, wherein the composition induces at least a six-fold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the composition.
6. The method of claim 1, wherein said Norovirus VLPs are monovalent VLPs or multivalent VLPs.
7. The method of claim 1, wherein said genogroup I Norovirus VLPs are Norwalk virus VLPs and said genogroup II Norovirus VLPs are VLPs generated from expression of a consensus sequence of genogroup II Norovirus.
8. The method of claim 1, wherein the composition further comprises at least one adjuvant.
9. The method of claim 8, wherein said at least one adjuvant is a toll-like receptor agonist.
10. The method of claim 8, wherein the adjuvant is selected from monophosphoryl lipid A and aluminum hydroxide.
11. The method of claim 1, wherein said composition further comprises a buffer.
12. The method of claim 11, wherein said buffer is selected from the group consisting of L-histidine, imidazole, succinic acid, tris, and citric acid.
13. The method of claim 1, wherein the vaccine composition is administered to the human by an intravenous, subcutaneous, intradermal, or intramuscular route of administration.
14. The method of claim 13, wherein the vaccine composition is administered to the human by an intramuscular route of administration.
15. The method of claim 1, wherein the vaccine composition is formulated as a liquid.
16. The method of claim 1, wherein the increase in Norovirus-specific antibody titer is induced within seven days of administration of the single dose of the composition.
17. The method of claim 1, wherein said vaccine composition confers protection from one or more symptoms of Norovirus infection.
18. The method of claim 8, wherein said at least one adjuvant is an aluminum hydroxide.
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is the Divisional of application Ser. No. 14/796,614, filed Jul. 10, 2015, which is a Continuation of application Ser. No. 13/840,403, filed Mar. 15, 2013, now U.S. Pat. No. 9,801,934, which is a Continuation of PCT/US2012/046222, filed Jul. 11, 2012, which claims the benefit of Provisional Application 61/506,447, filed on Jul. 11, 2011, the contents of each are herein incorporated by reference in their entireties.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: LIGO_024_03US_SeqList_ST25, date recorded on Jan. 30, 2018, file size 5 kilobytes).
FIELD OF THE INVENTION
The invention is in the field of vaccines, particularly vaccines for Noroviruses. In addition, the invention relates to methods of preparing vaccine compositions and methods of inducing and evaluating protective immune responses against Norovirus in humans.
BACKGROUND OF THE INVENTION
Noroviruses are non-cultivatable human Caliciviruses that have emerged as the single most important cause of epidemic outbreaks of nonbacterial gastroenteritis (Glass et al., 2000; Hardy et al., 1999). The clinical significance of Noroviruses was under-appreciated prior to the development of sensitive molecular diagnostic assays. The cloning of the prototype genogroup I Norwalk virus (NV) genome and the production of virus-like particles (VLPs) from a recombinant Baculovirus expression system led to the development of assays that revealed widespread Norovirus infections (Jiang et al. 1990; 1992).
Noroviruses are single-stranded, positive sense RNA viruses that contain a non-segmented RNA genome. The viral genome encodes three open reading frames, of which the latter two specify the production of the major capsid protein and a minor structural protein, respectively (Glass et al. 2000). When expressed at high levels in eukaryotic expression systems, the capsid protein of NV, and certain other Noroviruses, self-assembles into VLPs that structurally mimic native Norovirus virions. When viewed by transmission electron microscopy, the VLPs are morphologically indistinguishable from infectious virions isolated from human stool samples.
Immune responses to Noroviruses are complex, and the correlates of protection are just now being elucidated. Human volunteer studies performed with native virus demonstrated that mucosally-derived memory immune responses provided short-term protection from infection and suggested that vaccine-mediated protection is feasible (Lindesmith et al. 2003; Parrino et al. 1977; Wyatt et al., 1974).
Although Norovirus cannot be cultivated in vitro, due to the availability of VLPs and their ability to be produced in large quantities, considerable progress has been made in defining the antigenic and structural topography of the Norovirus capsid. VLPs preserve the authentic confirmation of the viral capsid protein while lacking the infectious genetic material. Consequently, VLPs mimic the functional interactions of the virus with cellular receptors, thereby eliciting an appropriate host immune response while lacking the ability to reproduce or cause infection. In conjunction with the NIH, Baylor College of Medicine studied the humoral, mucosal and cellular immune responses to NV VLPs in human volunteers in an academic, investigator-sponsored Phase I clinical trial. Orally administered VLPs were safe and immunogenic in healthy adults (Ball et al. 1999; Tacket et al. 2003). But, multiple doses of a relatively high amount of VLPs were required to observe an immune response. At other academic centers, preclinical experiments in animal models have demonstrated enhancement of immune responses to VLPs when administered intranasally with bacterial exotoxin adjuvants (Guerrero et al. 2001; Nicollier-Jamot et al. 2004; Periwal et al. 2003; Souza et al. (2007) Vaccine, Vol. 25(50):8448-59). However, protective immunity against Norovirus in humans remains elusive because the indicators of a protective immune response in humans have still not been clearly identified (Herbst-Kralovetz et al. (2010) Expert Rev. Vaccines 9(3), 299-307).
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery that a single dose of a Norovirus vaccine elicits a rapid, robust protective immune response against Norovirus in humans when administered parenterally. Accordingly, the present invention provides a method of eliciting protective immunity against Norovirus in a human comprising administering parenterally to the human no more than a single dose of a vaccine composition, said composition comprising genogroup I and/or genogroup II Norovirus VLPs, wherein said composition induces at least a three-fold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the composition. In certain embodiments, the increase in Norovirus-specific antibody titer is induced within seven days of administration of the single dose of the composition. In some embodiments, the vaccine composition is administered to the human via an intravenous, subcutaenous, intradermal, or intramuscular route of administration. In one embodiment, the vaccine composition is administered to the human by an intramuscular route of administration.
The single dose vaccine compositions can comprise doses of about 5 μg to about 150 of genogroup I Norovirus VLPs, genogroup II Norovirus VLPs, or both. In embodiments in which the single dose vaccine compositions comprise both genogroup I and genogroup II Norovirus VLPs, the dose of each VLP can be the same or different. In one embodiment, the composition comprises no more than 50 μg of genogroup I Norovirus VLPs. In another embodiment, the composition comprises no more than 25 μg of genogroup I Norovirus VLPs. In yet another embodiment, the composition comprises no more than 150 μg of genogroup II Norovirus VLPs. In still another embodiment, the composition comprises no more than 50 μg of genogroup II Norovirus VLPs. The Norovirus VLPs can be monovalent VLPs or multivalent VLPs.
In some aspects of the invention, genogroup I Norovirus VLPs in the vaccine compositions comprise a capsid protein derived from a genogroup I viral strain. In one embodiment, the genogroup I Norovirus VLPs comprise a capsid protein from a genogroup I, genotype 1 Norovirus. In another embodiment, the genogroup I Norovirus VLPs comprise a capsid protein from Norwalk virus. In other aspects of the invention, genogroup II Norovirus VLPs in the vaccine compositions comprise a capsid protein derived from a genogroup II viral strain. In some embodiments, the genogroup II Norovirus VLPs comprise a capsid protein from a genogroup II, genotype 4 Norovirus. In certain embodiments, the genogroup II Norovirus VLPs are VLPs generated from expression of a consensus sequence of genogroup II Norovirus. In one particular embodiment, the genogroup II Norovirus VLPs comprise a capsid protein having a sequence of SEQ ID NO: 1.
In certain embodiments, the vaccine composition further comprises at least one adjuvant. The adjuvant is preferably not a bacterial exotoxin adjuvant. In one embodiment, the adjuvant is a toll-like receptor agonist, such as monophosphoryl lipid A (MPL), flagellin, or CpG. In another embodiment, the adjuvant is aluminum hydroxide (e.g. alum). In certain embodiments, the vaccine composition comprises two adjuvants, such as MPL and aluminum hydroxide. In some embodiments, the vaccine composition may further comprise a buffer, such as L-histidine, imidazole, succinic acid, tris, and citric acid. The vaccine composition can be formulated as a dry powder or a liquid. In one embodiment, the vaccine composition is formulated as a liquid (e.g. aqueous formulation).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-FIG. 1B. Results of pan-ELISA assays measuring combined serum IgG, IgA, and IgM levels from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, 50, or 150 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The geometric mean titer for anti-GI.1 (FIG. 1A) and anti-GII.4 (FIG. 1B) antibodies is shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28.
FIG. 2A-FIG. 2B. Results of pan-ELISA assays measuring combined serum IgG, IgA, and IgM levels from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, 50, or 150 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The geometric mean fold rise for anti-GI.1 (FIG. 2A) and anti-GII.4 (FIG. 2B) antibodies is shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28.
FIG. 3A-FIG. 3B. Results of pan-ELISA assays measuring combined serum IgG, IgA, and IgM levels from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, 50, or 150 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The percent seroresponse rates (i.e. four-fold increase in antibody titer compared to pre-immunization titers) for anti-GI.1 (FIG. 3A) and anti-GII.4 (FIG. 3B) antibodies are shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28.
FIG. 4A-FIG. 4B. Results of ELISA assays measuring serum IgA from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, or 50 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The geometric mean titer for anti-GI.1 (FIG. 4A) and anti-GII.4 (FIG. 4B) antibodies is shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28.
FIG. 5A-FIG. 5B. Results of ELISA assays measuring serum IgA from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, or 50 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The geometric mean fold rise for anti-GI.1 (FIG. 5A) and anti-GII.4 (FIG. 5B) antibodies is shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28.
FIG. 6A-FIG. 6B. Results of ELISA assays measuring serum IgA from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, or 50 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The percent seroresponse rates (i.e. four-fold increase in antibody titer compared to pre-immunization titers) for anti-GI.1 (FIG. 6A) and anti-GII.4 (FIG. 6B) antibodies are shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28.
FIG. 7A-FIG. 7B. Results of ELISA assays measuring serum IgG from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, or 50 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The geometric mean titer for anti-GI.1 (FIG. 7A) and anti-GII.4 (FIG. 7B) antibodies is shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28.
FIG. 8A-FIG. 8B. Results of ELISA assays measuring serum IgG from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, or 50 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The geometric mean fold rise for anti-GI.1 (FIG. 8A) and anti-GII.4 (FIG. 8B) antibodies is shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28.
FIG. 9A-FIG. 9B. Results of ELISA assays measuring serum IgG from human volunteers immunized intramuscularly with placebo (saline) or a vaccine formulation containing 5, 15, or 50 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The percent seroresponse rates (i.e. four-fold increase in antibody titer compared to pre-immunization titers) for anti-GI.1 (FIG. 9A) and anti-GII.4 (FIG. 9B) antibodies are shown for each of the dosage levels at 7 and 21 days after the first immunization and 7 and 28 days after the second immunization. Volunteers received immunizations on study days 0 and 28.
FIG. 10. Results of pan-ELISA assays measuring combined serum IgG, IgA, and IgM levels from human volunteers immunized with either a Norovirus intranasal, monovalent vaccine as described in El Kamary et al. (2010) J Infect Dis, Vol. 202(11): 1649-1658 (LV01-103 groups) or a Norovirus intramuscular, bivalent vaccine as described in Example 1 (LV03-104 groups) at the indicated time points. Human volunteers received either placebo or two doses of either the intramuscular or intranasal vaccine formulation. The intramuscular, bivalent Norovirus vaccine contained 5 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 Norovirus VLP. The intranasal, monovalent vaccine contained 100 μg of a genogroup I.1 Norovirus. Volunteers receiving the intranasal vaccine or placebo were challenged with live Norovirus following the second immunization.
FIG. 11A-FIG. 11B. FACS analysis of peripheral blood mononuclear cells obtained from human volunteers on Day 0 prior to immunization with either a 5 μg dose of Norovirus intramuscular, bivalent vaccine (FIG. 11A) or placebo (FIG. 11B) and Day 7 post-immunization. CD19+ PBMC are mucosally targeted as evidenced of expression of alpha 4/beta7 homing receptor and chemokine CCR10 receptor.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of eliciting a protective immunity to Norovirus infections in a subject. In particular, the present invention provides methods of eliciting a protective immunity against Norovirus in a human by parenterally administering to the human no more than a single dose of a vaccine comprising Norovirus VLPs and optionally at least one adjuvant, wherein the vaccine confers protection from or amelioration of at least one symptom of Norovirus infection. The inventors have surprisingly discovered that intramuscular administration of no more than a single dose of a vaccine composition comprising Norovirus VLPs to humans induces a rapid (i.e. within 7 days of immunization) serum seroconversion (i.e. at least a three-fold increase in antigen-specific serum antibody titers above pre-vaccination levels) that is indicative of a protective immune response against Norovirus infection and illness. The immune responses induced by this single dose vaccine composition plateau at high antibody titers similar to that observed with natural infection by administration of live virus in human challenge studies. Interestingly, a boost dose of the vaccine is not required as the immune response is not increased upon further administration of an additional vaccine dose.
The invention provides a vaccine composition comprising one or more Norovirus antigens. By “Norovirus,” “Norovirus (NOR),” “norovirus,” and grammatical equivalents herein, are meant members of the genus Norovirus of the family Caliciviridae. In some embodiments, a Norovirus can include a group of related, positive-sense single-stranded RNA, nonenveloped viruses that can be infectious to human or non-human mammalian species. In some embodiments, a Norovirus can cause acute gastroenteritis in humans. Noroviruses also can be referred to as small round structured viruses (SRSVs) having a defined surface structure or ragged edge when viewed by electron microscopy.
Included within the Noroviruses are at least five genogroups (GI, GII, GIII, GIV, and GV). GI, GII, and GIV Noroviruses are infectious in humans, while GIII Noroviruses primarily infect bovine species. GV has recently been isolated from mice (Zheng et al. (2006) Virology, Vol 346: 312-323). Representative of GIII are the Jena and Newbury strains, while the Alphatron, Fort Lauderdale, and Saint Cloud strains are representative of GIV. The GI and GII groups may be further segregated into genetic clusters or genotypes based on genetic classification (Ando et al. (2000) J. Infectious Diseases, Vol. 181(Supp2):5336-5348; Lindell et al. (2005) J. Clin. Microbiol., Vol. 43(3): 1086-1092). As used herein, the term genetic clusters is used interchangeably with the term genotypes. Within genogroup I, there are 8 GI clusters known to date (with prototype virus strain name): GI.1 (Norwalk (NV-USA93)); GI.2 (Southhampton (SOV-GBR93)); GI.3 (Desert Shield (DSV-USA93)); GI.4 (Cruise Ship virus/Chiba (Chiba-JPN00)); GI.5 (318/Musgrove (Musgrov-GBR00)); GI.6 (Hesse (Hesse-DEU98)); GI.7 (Wnchest-GBR00); and GI.8 (Boxer-USA02). Within genogroup II, there are 19 GII clusters known to date (with prototype virus strain name): GII.1 (Hawaii (Hawaii-USA94)); GII.2 (Snow Mountain/Melksham (Msham-GBR95)); GII.3 (Toronto (Toronto-CAN93)); GII.4 (Bristol/Lordsdale (Bristol-GBR93)); GII.5 (290/Hillingdon (Hilingd-GBR00)); GII.6 (269/Seacroft (Seacrof-GBR00)); GII.7 (273/Leeds (Leeds-GBR00)); GII.8 (539/Amsterdam (Amstdam-NLD99)); GII.9 (378 (VABeach-USA01)), GII.10 (Erfurt-DEU01); GII.11 (SW9180JPN01); GII.12 (Wortley-GBR00); GII.13 (Faytvil-USA02); GII.14 (M7-USA03); GII.15 (J23-USA02); GII.16 (Tiffin-USA03); GII.17 (CSE1-USA03); GII.18 (QW101/2003/US) and GII.19 (QW170/2003/US).
By “Norovirus” also herein is meant recombinant Norovirus virus-like particles (rNOR VLPs). In some embodiments, recombinant expression of at least the Norovirus capsid protein encoded by ORF2 in cells, e.g., from a baculovirus vector in Sf9 cells, can result in spontaneous self-assembly of the capsid protein into VLPs. In some embodiments, recombinant expression of at least the Norovirus proteins encoded by ORF1 and ORF2 in cells, e.g., from a baculovirus vector in 519 cells, can result in spontaneous self-assembly of the capsid protein into VLPs. VLPs are structurally similar to Noroviruses but lack the viral RNA genome and therefore are not infectious. Accordingly, “Norovirus” includes virions that can be infectious or non-infectious particles, which include defective particles.
Non-limiting examples of Noroviruses include Norovirus genogroup 1 strain Hu/NoV/West Chester/2001/USA, GenBank Accession No. AY502016; Chiba virus (CHV, GenBank AB042808); Norovirus genogroup 2 strain Hu/NoV/Braddock Heights/1999/USA, GenBank Accession No. AY502015; Norovirus genogroup 2 strain Hu/NoV/Fayette/1999/USA, GenBank Accession No. AY502014; Norovirus genogroup 2 strain Hu/NoV/Fairfield/1999/USA, GenBank Accession No. AY502013; Norovirus genogroup 2 strain Hu/NoV/Sandusky/1999/USA, GenBank Accession No. AY502012; Norovirus genogroup 2 strain Hu/NoV/Canton/1999/USA, GenBank Accession No. AY502011; Norovirus genogroup 2 strain Hu/NoV/Tiffin/1999/USA, GenBank Accession No. AY502010; Norovirus genogroup 2 strain Hu/NoV/CS-E1/2002/USA, GenBank Accession No. AY50200; Norovirus genogroup 1 strain Hu/NoV/Wisconsin/2001/USA, GenBank Accession No. AY502008; Norovirus genogroup 1 strain Hu/NoV/CS-841/2001/USA, GenBank Accession No. AY502007; Norovirus genogroup 2 strain Hu/NoV/Hiram/2000/USA, GenBank Accession No. AY502006; Norovirus genogroup 2 strain Hu/NoV/Tontogany/1999/USA, GenBank Accession No. AY502005; Norwalk virus, complete genome, GenBank Accession No. NC.sub.-001959; Norovirus Hu/GI/Otofuke/1979/JP genomic RNA, complete genome, GenBank Accession No. AB187514; Norovirus Hu/Hokkaido/133/2003/JP, GenBank Accession No. AB212306; Norovirus Sydney 2212, GenBank Accession No. AY588132; Norwalk virus strain SN2000JA, GenBank Accession No. AB190457; Lordsdale virus complete genome, GenBank Accession No. X86557; Norwalk-like virus genomic RNA, Gifu'96, GenBank Accession No. AB045603; Norwalk virus strain Vietnam 026, complete genome, GenBank Accession No. AF504671; Norovirus Hu/GII.4/2004/N/L, GenBank Accession No. AY883096; Norovirus Hu/GII/Hokushin/03/JP, GenBank Accession No. AB195227; Norovirus Hu/GII/Kamo/03/JP, GenBank Accession No. AB195228; Norovirus Hu/GII/Sinsiro/97/JP, GenBank Accession No. AB195226; Norovirus Hu/GII/Ina/02/JP, GenBank Accession No. AB 195225; Norovirus Hu/NLV/GII/Neustrelitz260/2000/DE, GenBank Accession No. AY772730; Norovirus Hu/NLV/Dresden174/pUS-NorII/1997/GE, GenBank Accession No. AY741811; Norovirus Hu/NLV/Oxford/B2S16/2002/UK, GenBank Accession No. AY587989; Norovirus Hu/NLV/Oxford/B4S7/2002/UK, GenBank Accession No. AY587987; Norovirus Hu/NLV/Witney/B7S2/2003/UK, GenBank Accession No. AY588030; Norovirus Hu/NLV/Banbury/B9S23/2003/UK, GenBank Accession No. AY588029; Norovirus Hu/NLV/ChippingNorton/2003/UK, GenBank Accession No. AY588028; Norovirus Hu/NLV/Didcot/B9S2/2003/UK, GenBank Accession No. AY588027; Norovirus Hu/NLV/Oxford/B8S5/2002/UK, GenBank Accession No. AY588026; Norovirus Hu/NLV/Oxford/B6S4/2003/UK, GenBank Accession No. AY588025; Norovirus Hu/NLV/Oxford/B6S5/2003/UK, GenBank Accession No. AY588024; Norovirus Hu/NLV/Oxford/B5S23/2003/UK, GenBank Accession No. AY588023; Norovirus Hu/NLV/Oxford/B6S2/2003/UK, GenBank Accession No. AY588022; Norovirus Hu/NLV/Oxford/B6S6/2003/UK, GenBank Accession No. AY588021; Norwalk-like virus isolate Bo/Thirsk10/00/UK, GenBank Accession No. AY126468; Norwalk-like virus isolate Bo/Penrith55/00/UK, GenBank Accession No. AY126476; Norwalk-like virus isolate Bo/Aberystwyth24/00/UK, GenBank Accession No. AY126475; Norwalk-like virus isolate Bo/Dumfries/94/UK, GenBank Accession No. AY126474; Norovirus NLV/IF2036/2003/Iraq, GenBank Accession No. AY675555; Norovirus NLV/IF1998/2003/Iraq, GenBank Accession No. AY675554; Norovirus NLV/BUDS/2002/USA, GenBank Accession No. AY660568; Norovirus NLV/Paris Island/2003/USA, GenBank Accession No. AY652979; Snow Mountain virus, complete genome, GenBank Accession No. AY134748; Norwalk-like virus NLV/Fort Lauderdale/560/1998/US, GenBank Accession No. AF414426; Hu/Norovirus/hiroshima/1999/JP(9912-02F), GenBank Accession No. AB044366; Norwalk-like virus strain 11MSU-MW, GenBank Accession No. AY274820; Norwalk-like virus strain B-1SVD, GenBank Accession No. AY274819; Norovirus genogroup 2 strain Hu/NoV/Farmington Hills/2002/USA, GenBank Accession No. AY502023; Norovirus genogroup 2 strain Hu/NoV/CS-G4/2002/USA, GenBank Accession No. AY502022; Norovirus genogroup 2 strain Hu/NoV/CS-G2/2002/USA, GenBank Accession No. AY502021; Norovirus genogroup 2 strain Hu/NoV/CS-G12002/USA, GenBank Accession No. AY502020; Norovirus genogroup 2 strain Hu/NoV/Anchorage/2002/USA, GenBank Accession No. AY502019; Norovirus genogroup 2 strain Hu/NoV/CS-D1/2002/CAN, GenBank Accession No. AY502018; Norovirus genogroup 2 strain Hu/NoV/Germanton/2002/USA, GenBank Accession No. AY502017; Human calicivirus NLV/GII/Langen1061/2002/DE, complete genome, GenBank Accession No. AY485642; Murine norovirus 1 polyprotein, GenBank Accession No. AY228235; Norwalk virus, GenBank Accession No. AB067536; Human calicivirus NLV/Mex7076/1999, GenBank Accession No. AF542090; Human calicivirus NLV/Oberhausen 455/01/DE, GenBank Accession No. AF539440; Human calicivirus NLV/Herzberg 385/01/DE, GenBank Accession No. AF539439; Human calicivirus NLV/Boxer/2001/US, GenBank Accession No. AF538679; Norwalk-like virus genomic RNA, complete genome, GenBank Accession No. AB081723; Norwalk-like virus genomic RNA, complete genome, isolate:Saitama U201, GenBank Accession No. AB039782; Norwalk-like virus genomic RNA, complete genome, isolate:Saitama U18, GenBank Accession No. AB039781; Norwalk-like virus genomic RNA, complete genome, isolate:Saitama U25, GenBank Accession No. AB039780; Norwalk virus strain:U25G11, GenBank Accession No. AB067543; Norwalk virus strain:U201 GII, GenBank Accession No. AB067542; Norwalk-like viruses strain 416/97003156/1996/LA, GenBank Accession No. AF080559; Norwalk-like viruses strain 408/97003012/1996/FL, GenBank Accession No. AF080558; Norwalk-like virus NLV/Burwash Landing/331/1995/US, GenBank Accession No. AF414425; Norwalk-like virus NLV/Miami Beach/326/1995/US, GenBank Accession No. AF414424; Norwalk-like virus NLV/White River/290/1994/US, GenBank Accession No. AF414423; Norwalk-like virus NLV/New Orleans/306/1994/US, GenBank Accession No. AF414422; Norwalk-like virus NLV/Port Canaveral/301/1994/US, GenBank Accession No. AF414421; Norwalk-like virus NLV/Honolulu/314/1994/US, GenBank Accession No. AF414420; Norwalk-like virus NLV/Richmond/283/1994/US, GenBank Accession No. AF414419; Norwalk-like virus NLV/Westover/302/1994/US, GenBank Accession No. AF414418; Norwalk-like virus NLV/UK3-17/12700/1992/GB, GenBank Accession No. AF414417; Norwalk-like virus NLV/Miami/81/1986/US, GenBank Accession No. AF414416; Snow Mountain strain, GenBank Accession No. U70059; Desert Shield virus DSV395, GenBank Accession No. U04469; Norwalk virus, complete genome, GenBank Accession No. AF093797; Hawaii calicivirus, GenBank Accession No. U07611; Southampton virus, GenBank Accession No. L07418; Norwalk virus (SRSV-KY-89/89/J), GenBank Accession No. L23828; Norwalk virus (SRSV-SMA/76/US), GenBank Accession No. L23831; Camberwell virus, GenBank Accession No. U46500; Human calicivirus strain Melksham, GenBank Accession No. X81879; Human calicivirus strain MX, GenBank Accession No. U22498; Minireovirus TV24, GenBank Accession No. U02030; and Norwalk-like virus NLV/G nedd/273/1994/US, GenBank Accession No. AF414409; sequences of all of which (as entered by the date of filing of this application) are herein incorporated by reference. Additional Norovirus sequences are disclosed in the following patent publications: WO 2005/030806, WO 2000/79280, JP2002020399, US2003129588, U.S. Pat. No. 6,572,862, WO 1994/05700, and WO 05/032457, all of which are herein incorporated by reference in their entireties. See also Green et al. (2000) J. Infect. Dis., Vol. 181(Suppl. 2):5322-330; Wang et al. (1994) J. Virol., Vol. 68:5982-5990; Chen et al. (2004) J. Virol., Vol. 78: 6469-6479; Chakravarty et al. (2005) J. Virol., Vol. 79: 554-568; Hansman et al. (2006) J. Gen. Virol., Vol. 87:909-919; Bull et al. (2006) J. Clin. Micro., Vol. 44(2):327-333; Siebenga, et al. (2007) J. Virol., Vol. 81(18):9932-9941, and Fankhauser et al. (1998) J. Infect. Dis., Vol. 178:1571-1578; for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Noroviruses. The nucleic acid and corresponding amino acid sequences of each are all incorporated by reference in their entirety. In some embodiments, a cryptogram can be used for identification purposes and is organized: host species from which the virus was isolated/genus abbreviation/species abbreviation/strain name/year of occurrence/country of origin. (Green et al., Human Caliciviruses, in Fields Virology Vol. 1 841-874 (Knipe and Howley, editors-in-chief, 4th ed., Lippincott Williams & Wilkins 2001)). Genogroup II, genotype 4 (GII.4) viral strains (e.g., Houston, Minerva (also known as Den Haag), and Laurens (also known as Yerseke) strains) are preferred in some embodiments. As new strains are identified and their genetic sequences are made available, one skilled in the art would be able to employ VLPs using these contemporary strains in the compositions and methods of the present invention using ordinary skill. Thus, the present invention contemplates VLPs made from such strains as suitable antigens for use in the compositions and methods described herein.
The Norovirus antigen may be in the form of peptides, proteins, or virus-like particles (VLPs). In a preferred embodiment, the Norovirus antigen comprises VLPs. As used herein, “virus-like particle(s) or VLPs” refer to a virus-like particle(s), fragment(s), aggregates, or portion(s) thereof produced from the capsid protein coding sequence of Norovirus and comprising antigenic characteristic(s) similar to those of infectious Norovirus particles. Norovirus antigens may also be in the form of capsid monomers, capsid multimers, protein or peptide fragments of VLPs, or aggregates or mixtures thereof. The Norovirus antigenic proteins or peptides may also be in a denatured form, produced using methods known in the art.
The VLPs of the present invention can be formed from either the full length Norovirus capsid protein such as VP1 and/or VP2 proteins or certain VP1 or VP2 derivatives using standard methods in the art. Alternatively, the capsid protein used to form the VLP is a truncated capsid protein. In some embodiments, for example, at least one of the VLPs comprises a truncated VP1 protein. In other embodiments, all the VLPs comprise truncated VP1 proteins. The truncation may be an N- or C-terminal truncation. Truncated capsid proteins are suitably functional capsid protein derivatives. Functional capsid protein derivatives are capable of raising an immune response (if necessary, when suitably adjuvanted) in the same way as the immune response is raised by a VLP consisting of the full length capsid protein.
VLPs may contain major VP1 proteins and/or minor VP2 proteins. In some embodiments, each VLP contains VP1 and/or VP2 protein from only one Norovirus genogroup giving rise to a monovalent VLP. As used herein, the term “monovalent” means the antigenic proteins are derived from a single Norovirus genogroup. For example, the VLPs contain VP1 and/or VP2 from a virus strain of genogroup I (e.g., VP1 and VP2 from Norwalk virus). Preferably the VLP is comprised of predominantly VP1 proteins. In one embodiment of the invention, the antigen is a mixture of monovalent VLPs wherein the composition includes VLPs comprised of VP1 and VP2 from a single Norovirus genogroup mixed with VLPs comprised of VP1 and VP2 from a different Norovirus genogroup (e.g. Norwalk virus and Houston virus) taken from multiple viral strains. Purely by way of example the composition can contain monovalent VLPs from one or more strains of Norovirus genogroup I together with monovalent VLPs from one or more strains of Norovirus genogroup II. Strains may be selected based on their predominance of circulation at a given time. In certain embodiments, the Norovirus VLP mixture is composed of GI.1 and GII.4 viral strains. More preferably, the Norovirus VLP mixture is composed of the strains of Norwalk and a consensus capsid sequence derived from genogroup II Noroviruses. Consensus capsid sequences derived from circulating Norovirus sequences and VLPs made with such sequences are described in WO 2010/017542, which is herein incorporated by reference in its entirety. For instance, in one embodiment, a consensus capsid sequence derived from genogroup II, genotype 4 (GII.4) viral strains comprises a sequence of SEQ ID NO: 1. Thus, in some embodiments, the vaccine composition comprises a mixture of monovalent VLPs, wherein one monovalent VLP comprises a capsid protein from a genogroup I Norovirus (e.g. Norwalk) and the other monovalent VLP comprises a consensus capsid protein comprising a sequence of SEQ ID NO: 1.
(SEQ ID NO: 1)
M K M A S S D A N P S D G S T A N L V P E V N N E V M A L E P V
V G A A I A A P V A G Q Q N V I D P W I R N N F V Q A P G G E F
T V S P R N A P G E I L W S A P L G P D L N P Y L S H L A R M Y
N G Y A G G F E V Q V I L A G N A F T A G K I I F A A V P P N F
P T E G L S P S Q V T M F P H I I V D V R Q L E P V L I P L P D
V R N N F Y H Y N Q S N D P T I K L I A M L Y T P L R A N N A G
D D V F T V S C R V L T R P S P D F D F I F L V P P T V E S R T
K P F T V P I L T V E E M T N S R F P I P L E K L F T G P S G A
F V V Q P Q N G R C T T D G V L L G T T Q L S P V N I C T F R G
D V T H I A G T Q E Y T M N L A S Q N W N N Y D P T E E I P A P
L G T P D F V G K I Q G V L T Q T T R G D G S T R G H K A T V S
T G S V H F T P K L G S V Q F S T D T S N D F E T G Q N T K F T
P V G V V Q D G S T T H Q N E P Q Q W V L P D Y S G R D S H N V
H L A P A V A P T F P G E Q L L F F R S T M P G C S G Y P N M N
L D C L L P Q E W V Q H F Y Q E A A P A Q S D V A L L R F V N P
D T G R V L F E C K L H K S G Y V T V A H T G Q H D L V I P P N
G Y F R F D S W V N Q F Y T L A P M G N G T G R R R A L
However, in an alternative embodiment of the invention, the VLPs may be multivalent VLPs that comprise, for example, VP1 and/or VP2 proteins from one Norovirus genogroup intermixed with VP1 and/or VP2 proteins from a second Norovirus genogroup, wherein the different VP1 and VP2 proteins are not chimeric VP1 and VP2 proteins, but associate together within the same capsid structure to form immunogenic VLPs. As used herein, the term “multivalent” means that the antigenic proteins are derived from two or more Norovirus genogroups or strains. Multivalent VLPs may contain VLP antigens taken from two or more viral strains. Purely by way of example the composition can contain multivalent VLPs comprised of capsid monomers or multimers from one or more strains of Norovirus genogroup I (e.g. Norwalk virus) together with capsid monomers or multimers from one or more strains of Norovirus genogroup II (e.g. Houston virus). Preferably, the multivalent VLPs contain capsid proteins from the strains of Norwalk and Houston Noroviruses, or other predominantly circulating strains at a given time.
The combination of monovalent or multivalent VLPs within the composition preferably would not reduce the immunogenicity of each VLP type. In particular it is preferred that there is no interference between Norovirus VLPs in the combination of the invention, such that the combined VLP composition of the invention is able to elicit immunity against infection by each Norovirus genotype represented in the vaccine. Suitably the immune response against a given VLP type in the combination is at least 50% of the immune response of that same VLP type when measured individually, preferably 100% or substantially 100%. The immune response may suitably be measured, for example, by antibody responses, as illustrated in the examples herein.
As used herein, “genogroup I Norovirus VLPs” refer to either monovalent or multivalent VLPs that comprise a capsid protein derived from one or more genogroup I Norovirus strains. In some embodiments, genogroup I Norovirus VLPs comprise a full length capsid protein from a genogroup I Norovirus (e.g. Norwalk virus). In other embodiments, genogroup I Norovirus VLPs comprise a consensus capsid protein derived from various genogroup I strains. The genogroup I strains from which the consensus capsid sequence is derived can be within the same genotype or genetic cluster or from different genotypes or genetic clusters. Similarly, as used herein, “genogroup II Norovirus VLPs” refer to either monovalent or multivalent VLPs that comprise a capsid protein derived from one or more genogroup II Norovirus strains. In some embodiments, genogroup II Norovirus VLPs comprise a full length capsid protein from a genogroup II Norovirus (e.g. Laurens or Minerva virus). In other embodiments, genogroup II Norovirus VLPs comprise a consensus capsid protein derived from various genogroup II strains. The genogroup II strains from which the consensus capsid sequence is derived can be within the same genotype or genetic cluster or from different genotypes or genetic clusters. In one embodiment, the genogroup II Norovirus VLPs comprise a capsid consensus sequence of genogroup II, genotype 4 (GII.4) Norovirus. Thus, in some embodiments, the genogroup II Norovirus VLPs comprise a capsid sequence of SEQ ID NO: 1.
Multivalent VLPs may be produced by separate expression of the individual capsid proteins followed by combination to form VLPs. Alternatively multiple capsid proteins may be expressed within the same cell, from one or more DNA constructs. For example, multiple DNA constructs may be transformed or transfected into host cells, each vector encoding a different capsid protein. Alternatively a single vector having multiple capsid genes, controlled by a shared promoter or multiple individual promoters, may be used. IRES elements may also be incorporated into the vector, where appropriate. Using such expression strategies, the co-expressed capsid proteins may be co-purified for subsequent VLP formation, or may spontaneously form multivalent VLPs which can then be purified.
A preferred process for multivalent VLP production comprises preparation of VLP capsid proteins or derivatives, such as VP1 proteins, from different Norovirus genotypes, mixing the proteins, and assembly of the proteins to produce multivalent VLPs. The VP1 proteins may be in the form of a crude extract, be partially purified or purified prior to mixing. Assembled monovalent VLPs of different genogroups may be disassembled, mixed together and reassembled into multivalent VLPs. Preferably the proteins or VLPs are at least partially purified before being combined. Optionally, further purification of the multivalent VLPs may be carried out after assembly.
Suitably the VLPs of the invention are made by disassembly and reassembly of VLPs, to provide homogenous and pure VLPs. In one embodiment multivalent VLPs may be made by disassembly of two or more VLPs, followed by combination of the disassembled VLP components at any suitable point prior to reassembly. This approach is suitable when VLPs spontaneously form from expressed VP1 protein, as occurs for example, in some yeast strains. Where the expression of the VP1 protein does not lead to spontaneous VLP formation, preparations of VP1 proteins or capsomers may be combined before assembly into VLPs.
Where multivalent VLPs are used, preferably the components of the VLPs are mixed in the proportions in which they are desired in the final mixed VLP. For example, a mixture of the same amount of a partially purified VP1 protein from Norwalk and Houston viruses (or other Norovirus strains) provides a multivalent VLP with approximately equal amounts of each protein.
Compositions comprising multivalent VLPs may be stabilized by solutions known in the art, such as those of WO 98/44944, WO 00/45841, incorporated herein by reference.
Compositions of the invention may comprise other proteins or protein fragments in addition to Norovirus VP1 and VP2 proteins or derivatives. Other proteins or peptides may also be co-administered with the composition of the invention. Optionally the composition may also be formulated or co-administered with non-Norovirus antigens. Suitably these antigens can provide protection against other diseases.
The VP1 protein or functional protein derivative is suitably able to form a VLP, and VLP formation can be assessed by standard techniques such as, for example, size exclusion chromatography, electron microscopy and dynamic laser light scattering.
The antigenic molecules of the present invention can be prepared by isolation and purification from the organisms in which they occur naturally, or they may be prepared by recombinant techniques. Preferably the Norovirus VLP antigens are prepared from insect cells such as 519 or H5 cells, although any suitable cells such as E. coli or yeast cells, for example, S. cerevisiae, S. pombe, Pichia pastori or other Pichia expression systems, mammalian cell expression such as CHO or HEK systems may also be used. When prepared by a recombinant method or by synthesis, one or more insertions, deletions, inversions or substitutions of the amino acids constituting the peptide may be made. Each of the aforementioned antigens is preferably used in the substantially pure state.
The procedures of production of norovirus VLPs in insect cell culture have been previously disclosed in U.S. Pat. No. 6,942,865, which is incorporated herein by reference in its entirety. Briefly, a cDNA from the 3′ end of the genome containing the viral capsid gene (ORF2) and a minor structural gene (ORF3) is cloned. The recombinant baculoviruses carrying the viral capsid genes is constructed from the cloned cDNAs. Norovirus VLPs are produced in 519 or H5 insect cell cultures.
In some embodiments, the vaccine composition comprises one or more adjuvants in combination with the Norovirus antigen. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as Bordatella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Pifco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; and Quil A.
Suitable adjuvants also include, but are not limited to, toll-like receptor (TLR) agonists, particularly toll-like receptor type 4 (TLR-4) agonists (e.g., monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs), aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, liposomes, oil-in-water emulsions, MF59, and squalene. In some embodiments, the adjuvants are not bacterially-derived exotoxins. Preferred adjuvants include adjuvants which stimulate a Th1 type response such as 3DMPL or QS21.
Monophosphoryl Lipid A (MPL), a non-toxic derivative of lipid A from Salmonella, is a potent TLR-4 agonist that has been developed as a vaccine adjuvant (Evans et al. 2003). In pre-clinical murine studies intranasal MPL has been shown to enhance secretory, as well as systemic, humoral responses (Baldridge et al. 2000; Yang et al. 2002). It has also been proven to be safe and effective as a vaccine adjuvant in clinical studies of greater than 120,000 patients (Baldrick et al., 2002; Baldridge et al. 2004). MPL stimulates the induction of innate immunity through the TLR-4 receptor and is thus capable of eliciting nonspecific immune responses against a wide range of infectious pathogens, including both gram negative and gram positive bacteria, viruses, and parasites (Baldridge et al. 2004; Persing et al. 2002). Inclusion of MPL in vaccine formulations should provide rapid induction of innate responses, eliciting nonspecific immune responses from viral challenge while enhancing the specific responses generated by the antigenic components of the vaccine.
In one embodiment, the present invention provides a composition comprising monophosphoryl lipid A (MPL) or 3 De-O-acylated monophosphoryl lipid A (3D-MPL) as an enhancer of adaptive and innate immunity. Chemically 3D-MPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA), which is incorporated herein by reference. In another embodiment, the present invention provides a composition comprising synthetic lipid A, lipid A mimetics or analogs, such as BioMira's PET Lipid A, or synthetic derivatives designed to function like TLR-4 agonists.
In certain embodiments, the vaccine composition comprises two adjuvants. A combination of adjuvants may be selected from those described above. In one particular embodiment, the two adjuvants are MPL and aluminum hydroxide (e.g., alum). In another particular embodiment, the two adjuvants are MPL and oil.
The term “effective adjuvant amount” or “effective amount of adjuvant” will be well understood by those skilled in the art, and includes an amount of one or more adjuvants which is capable of stimulating the immune response to an administered antigen, i.e., an amount that increases the immune response of an administered antigen composition, as measured in terms of the IgA levels in the nasal washings, serum IgG or IgM levels, or B and T-Cell proliferation. Suitably effective increases in immunoglobulin levels include by more than 5%, preferably by more than 25%, and in particular by more than 50%, as compared to the same antigen composition without any adjuvant.
In one embodiment, the present invention provides a vaccine composition formulated for parenteral administration, wherein the composition includes at least two types of Norovirus VLPs in combination with aluminum hydroxide and a buffer. The buffer can be selected from the group consisting of L-histidine, imidazole, succinic acid, tris, citric acid, bis-tris, pipes, mes, hepes, glycine amide, and tricine. In one embodiment, the buffer is L-histidine or imidazole. Preferably, the buffer is present in a concentration from about 15 mM to about 50 mM, more preferably from about 18 mM to about 40 mM, or most preferably about 20 mM to about 25 mM. In some embodiments, the pH of the antigenic or vaccine composition is from about 6.0 to about 7.0, or from about 6.2 to about 6.8, or about 6.5. The vaccine composition can be an aqueous formulation. In some embodiments, the vaccine composition is a lyophilized powder and reconstituted to an aqueous formulation.
In certain embodiments, the vaccine composition further comprises at least one adjuvant in addition to the two or more types of Norovirus VLPs, aluminum hydroxide, and a buffer. For instance, the adjuvant can be a toll-like receptor agonist, such as MPL, flagellin, CpG oligos, synthetic lipid A or lipid A mimetics or analogs. In one particular embodiment, the adjuvant is MPL.
The Norovirus VLPs included in the vaccine compositions of the invention can be any of the VLPs described herein. In one embodiment, the two types of Norovirus VLPs each comprise a capsid protein from different genogroups (e.g., genogroup I and genogroup II). For instance, one type of Norovirus VLP comprises a capsid protein derived from a genogroup I Norovirus and the other type of Norovirus VLP comprises a capsid protein derived from a genogroup II Norovirus. In one embodiment, one type of Norovirus VLP comprises a capsid protein from Norwalk virus and the other type of Norovirus VLP comprises a consensus capsid protein derived from genogroup II, genotype 4 Noroviruses (e.g., a capsid protein comprising a sequence of SEQ ID NO: 1). The vaccine composition can comprise about 5 μg to about 200 μg of each Norovirus VLP, more preferably about 15 μg to about 50 μg of each Norovirus VLP. In some embodiments, the dose of one type of Norovirus VLP is different than the dose of the other type of Norovirus VLP. For instance, in certain embodiments, the vaccine composition comprises about 5 μg to about 15 μg of a genogroup I VLP and about 15 μg to about 50 μg of a genogroup II VLP. In other embodiments, the vaccine composition comprises about 15 μg to about 50 μg of a genogroup I VLP and about 50 μg to about 150 μg of a genogroup II VLP.
In some embodiments, the vaccine compositions further comprise a pharmaceutically acceptable salt, including, but not limited to, sodium chloride, potassium chloride, sodium sulfate, ammonium sulfate, and sodium citrate. In one embodiment, the pharmaceutically acceptable salt is sodium chloride. The concentration of the pharmaceutically acceptable salt can be from about 10 mM to about 200 mM, with preferred concentrations in the range of from about 100 mM to about 150 mM. Preferably, the vaccine compositions of the invention contain less than 2 mM of free phosphate. In some embodiments, the vaccine compositions comprise less than 1 mM of free phosphate. The vaccine compositions may also further comprise other pharmaceutically acceptable excipients, such as sugars (e.g., sucrose, trehalose, mannitol) and surfactants.
As discussed herein, the compositions of the invention can be formulated for administration as vaccines formulations. As used herein, the term “vaccine” refers to a formulation which contains Norovirus VLPs or other Norovirus antigens of the present invention as described above, which is in a form that is capable of being administered to a vertebrate, particularly a human, and which induces a protective immune response sufficient to induce immunity to prevent and/or ameliorate a Norovirus infection or Norovirus-induced illness and/or to reduce at least one symptom of a Norovirus infection or illness.
As used herein, the term “immune response” refers to both the humoral immune response and the cell-mediated immune response. The humoral immune response involves the stimulation of the production of antibodies by B lymphocytes that, for example, neutralize infectious agents, block infectious agents from entering cells, block replication of said infectious agents, and/or protect host cells from infection and destruction. The cell-mediated immune response refers to an immune response that is mediated by T-lymphocytes and/or other cells, such as macrophages, against an infectious agent, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates infection or reduces at least one symptom thereof. In particular, “protective immunity” or “protective immune response” refers to immunity or eliciting an immune response against an infectious agent, which is exhibited by a vertebrate (e.g., a human), that prevents or ameliorates an infection or reduces at least one symptom thereof. Specifically, induction of a protective immune response from administration of the vaccine is evident by elimination or reduction of the presence of one or more symptoms of acute gastroenteritis or a reduction in the duration or severity of such symptoms. Clinical symptoms of gastroenteritis from Norovirus include nausea, diarrhea, loose stool, vomiting, fever, and general malaise. A protective immune response that reduces or eliminates disease symptoms will reduce or stop the spread of a Norovirus outbreak in a population. Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995) Plenum Press New York). The compositions of the present invention can be formulated, for example, for delivery to one or more of the oral, gastro-intestinal, and respiratory (e.g. nasal) mucosa. The compositions of the present invention can be formulated, for example, for delivery by injection, such as parenteral injection (e.g., intravenous, subcutaneous, intradermal, or intramuscular injection).
Where the composition is intended for delivery to the respiratory (e.g. nasal) mucosa, typically it is formulated as an aqueous solution for administration as an aerosol or nasal drops, or alternatively, as a dry powder, e.g. for rapid deposition within the nasal passage. Compositions for administration as nasal drops may contain one or more excipients of the type usually included in such compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents, and the like. Viscosity agents can be microcrystalline cellulose, chitosan, starches, polysaccharides, and the like. Compositions for administration as dry powder may also contain one or more excipients usually included in such compositions, for example, mucoadhesive agents, bulking agents, and agents to deliver appropriate powder flow and size characteristics. Bulking and powder flow and size agents may include mannitol, sucrose, trehalose, and xylitol.
Where the composition is intended for parenteral injection, such as intravenous (i.v.), subcutaneous (s.c.), intradermal, or intramuscular (i.m.) injection, it is typically formulated as a liquid suspension (i.e. aqueous formulation) comprised of at least one type of Norovirus VLP and optionally at least one adjuvant. In one embodiment, the adjuvant may be MPL. In another embodiment, liquid vaccine formulated for parenteral administration may have more than one adjuvant. In a preferred embodiment, a parenterally-formulated (e.g., i.m., i.v., or s.c.-formulated) liquid vaccine comprises Norovirus genogroup I and/or genogroup II VLPs with aluminum hydroxide (e.g. alum) and monophosphoryl lipid A (MPL) as adjuvants. In one embodiment, a liquid formulation for parenteral administration comprises Norovirus genogroup antigen(s), such as one or more types of Norovirus VLPs as described herein, MPL, aluminum hydroxide, and a buffer. In another embodiment, a liquid formulation for parenteral administration comprises Norovirus genogroup antigen(s), MPL, oil, and a buffer. In certain embodiments, the buffer in the parenteral vaccine formulations is L-histidine or imidazole. Parenteral administration of liquid vaccines can be by needle and syringe, as is well known in the art.
In certain embodiments, a vaccine composition of the invention for eliciting a protective immune response against Norovirus in humans comprises genogroup I and/or genogroup II Norovirus VLPs at a dose of no more than 150 μg. For instance, in some embodiments, the vaccine composition comprises no more than 150 μg, no more than 100 μg, no more than 50 μg, no more than 25 μg, no more than 15 μg, or no more than 10 μg of genogroup I Norovirus VLPs. In other embodiments, the vaccine composition comprises no more than 150 μg, no more than 100 μg, no more than 50 μg, no more than 25 μg, no more than 15 μg, or no more than 10 μg of genogroup II Norovirus VLPs. In certain embodiments, the vaccine composition comprises no more than 150 μg of each genogroup I and genogroup II Norovirus VLPs. In such embodiments, the dose of genogroup I Norovirus VLPs and genogroup II VLPs can be the same or different. For instance, in one embodiment, the vaccine composition may comprise no more than 50 μg of genogroup I Norovirus VLPs and no more than 150 μg of genogroup II Norovirus VLPs. In another embodiment, the vaccine composition may comprise no more than 25 μg of genogroup I Norovirus VLPs and no more than 50 μg of genogroup II Norovirus VLPs. In other embodiments, the vaccine composition may comprise no more than 15 μg of genogroup I Norovirus VLPs and no more than 50 μg of genogroup II Norovirus VLPs. In still other embodiments, the vaccine composition may comprise no more than 25 μg of genogroup I Norovirus VLPs and no more than 150 μg of genogroup II Norovirus VLPs.
The genogroup I and genogroup II Norovirus VLPs can be derived from any of the Norovirus strains described herein. In one embodiment, the genogroup I Norovirus VLPs are genogroup I, genotype 1 (GI.1) VLPs (i.e. comprise a capsid protein from a GI.1 Norovirus). In another embodiment, the genogroup I Norovirus VLPs are Norwalk VLPs. In another embodiment, the genogroup II Norovirus VLPs are genogroup II, genotype 4 (GII.4) VLPs. In still another embodiment, the genogroup II Norovirus VLPs are VLPs generated from expression of a consensus sequence of genogroup II Norovirus. In a particular embodiment, the genogroup II Norovirus VLPs comprise a capsid protein having a sequence of SEQ ID NO: 1.
The vaccine compositions hereinbefore described may be lyophilized and stored anhydrous until they are ready to be used, at which point they are reconstituted with diluent. Alternatively, different components of the composition may be stored separately in a kit (any or all components being lyophilized). The components may remain in lyophilized form for dry formulation or be reconstituted for liquid formulations, and either mixed prior to use or administered separately to the patient. In some embodiments, the vaccine compositions are stored in kits in liquid formulations and may be accompanied by delivery devices, such as syringes equipped with needles. In other embodiments, the liquid vaccine compositions may be stored within the delivery devices in a kit. For example, a kit may comprise pre-filled syringes, autoinjectors, or injection pen devices containing a liquid formulation of a vaccine composition described herein.
The lyophilization of vaccines is well known in the art. Typically the liquid antigen is freeze dried in the presence of agents to protect the antigen during the lyophilization process and to yield a cake with desirable powder characteristics. Sugars such as sucrose, mannitol, trehalose, or lactose (present at an initial concentration of 10-200 mg/mL) are commonly used for cryoprotection of protein antigens and to yield lyophilized cake with desirable powder characteristics. Lyophilizing the compositions theoretically results in a more stable composition.
The amount of antigen in each vaccine composition is selected as an amount which induces a robust immune response without significant, adverse side effects. Such amount will vary depending upon which specific antigen(s) is employed, route of administration, and adjuvants used. In general, the dose administered to a patient, in the context of the present invention should be sufficient to effect a protective immune response in the patient over time, or to induce the production of antigen-specific antibodies. Thus, the composition is administered to a patient in an amount sufficient to elicit an immune response to the specific antigens and/or to prevent, alleviate, reduce, or cure symptoms and/or complications from the disease or infection, and thus reduce or stop the spread of a Norovirus outbreak in a population. An amount adequate to accomplish this is defined as a “therapeutically effective dose.”
The vaccine compositions of the present invention may be administered via a non-mucosal or mucosal route. These administrations may include in vivo administration via parenteral injection (e.g. intravenous, subcutaneous, intradermal, and intramuscular) or other traditional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or intranasal routes or directly into a specific tissue. Other suitable routes of administration include transcutaneous, subdermal, and via suppository. In one embodiment, the vaccine is administered by a parenteral route of administration, such as intravenous, subcutaneous, intradermal, or intramuscular. In certain embodiments, the vaccine is administered by an intramuscular route of administration. Administration may be accomplished simply by direct administration using a needle, catheter or related device (e.g. pre-filled syringes or autoinjectors), at a single time point or at multiple time points. Other parenteral formulations may be delivered subcutaneously or intradermally by microinjection or skin patch delivery methods.
The present invention provides methods for eliciting protective immunity against Norovirus in a subject comprising parenterally administering to the subject no more than a single dose of a vaccine composition of the invention, wherein said vaccine comprises genogroup I and/or genogroup II Norovirus VLPs as described herein and optionally at least one adjuvant. In such embodiments, the single dose vaccine composition induces at least a three-fold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the composition. In some embodiments, the single dose vaccine composition induces at least a six-fold increase in Norovirus-specific serum antibody titer as compared to the titer in the human prior to administration of the composition. In other embodiments, the single dose vaccine composition induces a Norovirus-specific serum antibody titer comparable to the antibody titer induced by exposure to live Norovirus in a natural infection—i.e., a greater than ten-fold increase in Norovirus-specific serum antibody as compared to the titer in the human prior to administration of the composition. In certain embodiments, the single dose vaccine composition induces the increase in Norovirus-specific serum antibody titer within seven days of administration of the composition. Preferably, the single dose vaccine composition is administered by an intravenous, subcutaneous, or intramuscular route of administration. In a certain embodiment, the single dose vaccine composition is administered intramuscularly to the human.
As described herein, in some embodiments, the single dose vaccine compositions suitable for use in the method comprise no more than 150 μg each of genogroup I and/or genogroup II Noroviruses. For instance, in some embodiments, the vaccine composition comprises no more than 150 μs, no more than 100 μs, no more than 50 μs, no more than 25 μs, no more than 15 μs, or no more than 10 μg of genogroup I Norovirus VLPs. In other embodiments, the vaccine composition comprises no more than 150 μs, no more than 100 μs, no more than 50 μs, no more than 25 μs, no more than 15 μg, or no more than 10 μg of genogroup II Norovirus VLPs. In certain embodiments, the vaccine composition comprises no more than 50 μg of each genogroup I and genogroup II Norovirus VLPs. In embodiments in which the single dose vaccine composition comprises both genogroup I and genogroup II Norovirus VLPs, the dose of genogroup I Norovirus VLPs and genogroup II VLPs can be the same or different. For instance, in one embodiment, the vaccine composition may comprise no more than 50 μg of genogroup I Norovirus VLPs and no more than 150 μg of genogroup II Norovirus VLPs. In another embodiment, the vaccine composition may comprise no more than 25 μg of genogroup I Norovirus VLPs and no more than 50 μg of genogroup II Norovirus VLPs. In other embodiments, the vaccine composition may comprise no more than 15 μg of genogroup I Norovirus VLPs and no more than 50 μg of genogroup II Norovirus VLPs. In still other embodiments, the vaccine composition may comprise no more than 25 μg of genogroup I Norovirus VLPs and no more than 150 μg of genogroup II Norovirus VLPs.
In one embodiment of the method, the subject is a human and the vaccine confers protection from one or more symptoms of Norovirus infection. Although others have reported methods of inducing an immune response with Norovirus antigens (see U.S. Patent Application Publication No. US 2007/0207526), the indicators of a protective immune response against Norovirus in humans have still not been clearly identified (Herbst-Kralovetz et al. (2010) Expert Rev. Vaccines 9(3), 299-307). Unlike several vaccines currently licensed in the U.S. where effectiveness of the vaccine correlates with serum antibodies, studies have shown that markers of an immune response, such as increased titers of serum IgG antibodies against Norwalk virus, are not associated with protective immunity in humans (Johnson et al. (1990) J. Infectious Diseases 161: 18-21). Moreover, another study examining Norwalk viral challenge in humans indicated that susceptibility to Norwalk infection was multifactorial and included factors such as secretor status and memory mucosal immune response (Lindesmith et al. (2003) Nature Medicine 9: 548-553).
Because Norovirus is not able to be cultured in vitro, no viral neutralization assays are currently available. A functional assay which serves as a substitute for the neutralization assay is the hemagglutination inhibition (HAI) assay (see Example 1). HAI measures the ability of Norovirus vaccine-induced antibodies to inhibit the agglutination of antigen-coated red blood cells by Norovirus VLPs because Norovirus VLPs bind to red blood cell antigens (e.g. histo-blood group antigens). This assay is also known as a carbohydrate blocking assay, as it is indicative of the functional ability of antibodies to block binding of the virus or VLPs to blood group antigen carbohydrates on a red blood cell. In this assay, a fixed amount of Norovirus VLPs is mixed with a fixed amount of red blood cells and serum from immunized subjects. If the serum sample contains functional antibodies, the antibodies will bind to the VLPs, thereby inhibiting the agglutination of the red blood cells. As used herein, “functional antibodies” refer to antibodies that are capable of inhibiting the interaction between Norovirus particles and red blood cell antigens. In other words, functional antibody titer is equivalent to histo-blood group antigen (HBGA) or carbohydrate blocking antibody titer. The serum titer of Norovirus-specific functional antibodies can be measured by the HAI assay described above. The serum titer of Norovirus-specific functional antibodies can also be measured using an ELISA-based assay in which a carbohydrate H antigen is bound to microtiter wells and Norovirus VLP binding to H antigen is detected in the presence of serum (see Example 1 and Reeck et al. (2010) J Infect Dis, Vol. 202(8):1212-1218). An increase in the level of Norovirus-specific functional antibodies can be an indicator of a protective immune response. Thus, in one embodiment, the administration of the vaccine elicits a protective immunity comprising an increase in the serum titer of Norovirus-specific functional antibodies as compared to the serum titer in a human not receiving the vaccine. The serum titer of Norovirus-specific functional antibodies indicative of a protective immune response is preferably a geometric mean titer greater than 40, 50, 75, 100, 125, 150, 175, 200 as measured by the HAI assay or blocking titer (BT)50 (50% inhibition of H antigen binding by Norovirus VLPs) geometric mean titer of greater than 100, 150, 200, 250, 300, 350, 400, 450, or 500 as measured by the H antigen binding assay. In one embodiment, the serum titer of Norovirus-specific functional antibodies is a geometric mean titer greater than 40 as measured by the HAI assay. In another embodiment, the serum titer of Norovirus-specific functional antibodies is a geometric mean titer greater than 100 as measured by the HAI assay. In another embodiment, the serum titer of Norovirus-specific functional antibodies is a BT50 geometric mean titer greater than 100 as measured by the H antigen binding assay. In still another embodiment, the serum titer of Norovirus-specific functional antibodies is a BT50 geometric mean titer greater than 200 as measured by the H antigen binding assay.
In a further aspect, the administration of the vaccine elicits a protective immunity comprising an IgA mucosal immune response and an IgG systemic immune response by administering parenterally (preferably intramuscularly) to the subject no more than a single dose of an antigenic or vaccine composition comprising one or more types of Norovirus antigens and optionally at least one effective adjuvant. The inventors have surprisingly found that parenteral administration of the Norovirus vaccine compositions described herein induces a robust IgA response in addition to a strong IgG response. Typically, strong IgA responses are only observed when vaccines are administered through a mucosal route of administration.
In certain embodiments, the administration of the vaccine elicits a protective immunity comprising an increase in the level of IgA Norovirus-specific antibody secreting cells in the blood as compared to the level in a human not receiving the vaccine. In some embodiments, the administration of the vaccine elicits a protective immunity comprising an increase in the level of IgA Norovirus-specific antibody secreting cells in the blood as compared to the level in the human before receiving the vaccine. In one embodiment, the IgA Norovirus-specific antibody secreting cells are CCR10+, CD19+, CD27+, CD62L+, and α4β7+. Antibody secreting cells with this marker profile are capable of homing to both peripheral lymphoid tissue, such as Peyer's patch in the gut, and mucosal lymphoid tissue, such as the gut mucosa. In one embodiment, the number of CCR10+, CD19+, CD27+, CD62L+, and α4β7+ IgA antibody secreting cells is greater than about 500, about 700, about 1,000, about 1,500, or greater than about 2,000 cells per 1×106 peripheral blood monocytes. In another embodiment, the IgA Norovirus-specific antibody secreting cells are CCR10+, CD19+, CD27+, CD62L−, and α4β7+. Antibody secreting cells with this marker profile generally exhibit homing only to mucosal sites and can be indicative of a memory B-cell response. In some embodiments in which the vaccine is administered intramuscularly, the number of CCR10+, CD19+, CD27+, CD62L−, and α4β7+ IgA antibody secreting cells is greater than about 5,000, about 6,500, about 7,000, about 10,000, about 13,000, about 15,000, or greater than about 20,000 cells per 1×106 peripheral blood monocytes.
Similar findings have been observed with vaccines for other viruses, such as rotavirus. For rotavirus vaccines, there is controversy over whether serum antibodies are directly involved in protection or merely reflect recent infection (Jiang, 2002; Franco, 2006). Defining such correlates of protection is particularly difficult in the context of diarrheal diseases such as rotavirus or norovirus, where preclinical studies inferring protection may be multifaceted with contributions from mucosal immunity (such as intestinal IgA), cytokine elaboration, and cell mediated immunity. The difficulty in measuring such immune responses during clinical development, and the lack of correlation to serum antibody measurements, requires that the effectiveness of a vaccine for these types of viruses can only be demonstrated through human clinical challenge experiments.
As mentioned above, administration of a vaccine composition of the present invention prevents and/or reduces at least one symptom of Norovirus infection. Symptoms of Norovirus infection are well known in the art and include nausea, vomiting, diarrhea, and stomach cramping. Additionally, a patient with a Norovirus infection may have a low-grade fever, headache, chills, muscle aches, and fatigue. The invention also encompasses a method of inducing a protective immune response in a subject experiencing a Norovirus infection by administering to the subject a vaccine formulation of the invention such that at least one symptom associated with the Norovirus infection is alleviated and/or reduced. A reduction in a symptom may be determined subjectively or objectively, e.g., self assessment by a subject, by a clinician's assessment or by conducting an appropriate assay or measurement (e.g. body temperature), including, e.g., a quality of life assessment, a slowed progression of a Norovirus infection or additional symptoms, a reduced severity of Norovirus symptoms or suitable assays (e.g. antibody titer, RT-PCR antigen detection, and/or B-cell or T-cell activation assay). An effective response may also be determined by directly measuring (e.g., RT-PCR) virus load in stool samples, which reflects the amount of virus shed from the intestines). The objective assessment comprises both animal and human assessments.
The invention also provides a method of generating antibodies to one or more Norovirus antigens, said method comprising administration of a vaccine composition of the invention as described above to a subject. These antibodies can be isolated and purified by routine methods in the art. The isolated antibodies specific for Norovirus antigens can be used in the development of diagnostic immunological assays. These assays could be employed to detect a Norovirus in clinical samples and identify the particular virus causing the infection (e.g. Norwalk, Houston, Snow Mountain, etc.). Alternatively, the isolated antibodies can be administered to subjects susceptible to Norovirus infection to confer passive or short-term immunity.
The invention will now be illustrated in greater detail by reference to the specific embodiments described in the following examples. The examples are intended to be purely illustrative of the invention and are not intended to limit its scope in any way.
EXAMPLES
Example 1. Dose Escalation, Safety and Immunogenicity Study of Intramuscular Norovirus Bivalent Virus-Like-Particle (VLP) Vaccine in Humans (LV03-104 Study), Cohort A
This example describes Cohort A of a randomized, multi-site, dose-escalation study of the safety and immunogenicity of four dosage levels of an intramuscular (IM) Norovirus Bivalent VLP Vaccine adjuvanted with monophosphoryl lipid A (MPL) and aluminum hydroxide (AlOH) compared to placebo in adult subjects. Approximately 48 subjects 18 to 49 years of age were enrolled in the cohort. Subjects received two doses of the vaccine or placebo, by intramuscular (IM) injection, 28 days apart using a 1.5 inch (38 mm) needle.
The Norovirus Bivalent VLP Vaccine contained genogroup I, genotype 1 (GI.1) and genogroup II, genotype IV (GII.4) VLPs as the antigens, and Monophosphoryl Lipid A (MPL) and aluminum hydroxide (AlOH) as adjuvants, sodium chloride (NaCl) and L-histidine (L-His) as buffer (pH 6.3-6.7), ethanol and water for injection. The composition of the intramuscular Norovirus Bivalent VLP Vaccine is summarized in Table 1. The GII.4 VLPs comprised a capsid sequence of SEQ ID NO: 1, which was derived from three GII.4 strains.
TABLE 1
Final Drug Product Composition for Four IM Norovirus
Bivalent VLP Vaccine Formulations per 0.5 mL
Formulation
GI.1-
GII.4
VLP
VLP
MPL
Al*
NaCl
L-His
Ethanol
(μg)
(μg)
(μg)
(mg)
(mg)
(mg)
(mg)
10 μg
5
5
50
0.5
4.38
1.55
19.7
Dosage
30 μg
15
15
50
0.5
4.38
1.55
19.7
Dosage
100 μg
50
50
50
0.5
4.38
1.55
19.7
Dosage
300 μg
150
150
50
0.5
4.38
1.55
19.7
Dosage
*as Aluminum Hydroxide
Placebo was sterile normal saline for injection (0.9% NaCl and preservative-free). The dose escalation of the vaccine was conducted as follows: after appropriate screening for good health, subjects in Cohort A were enrolled sequentially into each of four dosage groups of ˜12 subjects each (Dosage Groups A1, A2, A3, and A4). Dosage Groups A1, A2, A3, and A4 represent bivalent antigenic dosages of 5/5 μg, 15/15 μg, 50/50 μg, and 150/150 μg, respectively, of the G I.1 and GII.4 norovirus. Subjects in each dosage group were randomized 5:1 to receive vaccine or placebo. Subjects in Dosage Group A1 received their respective randomized treatment (10 subjects received 5/5 μg vaccine and 2 subjects received placebo). Subjects were followed for safety assessment by review of the symptoms recorded on the memory aid (Days 0-7) and interim medical histories from the Day 7, 21, 28, 35, and 56 visits. Safety data was reviewed by the Central Safety Monitor (CSM). After the 7-day post Dose 2 safety data (Study Day 35) were available for review from subjects in Dosage Group A1 and considered acceptable, subjects in Dosage Group A2 were eligible to receive their initial dose. The same rule applied for dosing in the subsequent dosage groups; that is, after the 7-day post Dose 2 safety data (Study Day 35) were available for review from a dosage group, the next dosage group was eligible to receive their initial dose.
At the end of enrollment in Cohort A, approximately 10 subjects in each Dosage Group received vaccine (total of 40 vaccinees) and 2 subjects in each group received saline (total of approximately 8 saline control recipients).
The subjects kept a daily memory aid of solicited symptoms including four local injection site reactions, such as pain, tenderness, redness, and swelling, and 10 systemic signs or symptoms including daily oral temperature, headache, fatigue, muscle aches, chills, joint aches and gastrointestinal symptoms of nausea, vomiting, diarrhea, abdominal cramps/pain for Days 0 through 7 after each dose of IM Norovirus Bivalent VLP Vaccine or control. The redness and swelling at the injection site was measured and recorded daily for 7 days after each injection.
Interim medical histories were obtained at each follow-up visit on Days 7+3, 21+3, 28+3, 35+3, 56+7, 180+14, and 393+14 and at the follow-up telephone call on Day 265+14; subjects were queried about interim illness, doctor's visits, any serious adverse events (SAEs), and onset of any significant new medical conditions. Subjects had a CBC with WBC differential and platelet count, and serum BUN, creatinine, glucose, AST, and ALT assessed at screening and on Days 21 and 35 (˜7 days after each dose) to assess continuing eligibility and safety, respectively.
Blood from subjects was collected before vaccination on Day 0 and on Days 7+3, 21+3, 28+3, 35+3, 56+7, 180+14, and 393+14 to measure serum antibodies (IgG, IgA, and IgM separately and combined) to IM Norovirus Bivalent VLP Vaccine by enzyme-linked immunosorbent assays (ELISA). Serum carbohydrate blocking activity and serum HAI antibodies were also measured. For subjects in Cohort A, antibody secreting cells (ASCs), homing markers, memory B cells and cellular immune responses were assayed.
The following methods were used to analyze the blood samples collected from immunized individuals or individuals receiving the placebo.
Serum Antibody Measurements by ELISA
Measurement of antibodies to norovirus by ELISA was performed for all subjects, using purified recombinant Norovirus VLPs (GI.1 and GII.4 separately) as target antigens to screen the coded specimens. Briefly, norovirus VLPs in carbonate coating buffer pH 9.6 were used to coat microtiter plates. Coated plates were washed, blocked, and incubated with serial two-fold dilutions of test serum followed by washing and incubation with enzyme-conjugated secondary antibody reagents specific for human total IgG, IgG1, IgG2, IgG3, IgG4, IgA and IgM. Appropriate substrate solutions were added, color developed, plates read and the IgG, IgA and IgM endpoint titers determined in comparison to a reference standard curve for each antibody class. Geometric mean titers (GMTs), geometric mean fold rises (GMFRs) and seroresponse rates for each group was determined. Seroresponse was defined as a 4-fold increase in antibody titer compared to pre-immunization titers.
Norovirus Carbohydrate Histo-Blood-Group Antigens (HBGA) Blocking Activity
Blocking assays to measure the ability of serum antibodies to inhibit NV VLP binding to H type 1 or H type 3 synthetic carbohydrates were performed as previously described (Reeck et al. (2010) J Infect Dis, Vol. 202(8):1212-1218). Briefly, NV VLPs for the blocking assays were incubated with an equal volume of serum, and serially two-fold diluted from a starting dilution of 1:25. Neutravidin-coated, 96-well microtiter plates were washed and coated with 2.5 μg/mL of either synthetic polyvalent H type 1-PAA-biotin or polyvalent H type 3-PAA-biotin. The sera-VLP solutions were added. Plates were washed and rabbit polyclonal sera specific to NV VLPs was added, washed, and followed by incubation with horseradish peroxidase conjugated goat anti-rabbit IgG. The color was developed with tetramethylbenzidine peroxidase liquid substrate and stopped with 1M phosphoric acid. Optical density was measured at 450. Positive and negative controls were performed. Fifty-percent blocking titers (BT50) were determined, defined as the titer at which OD readings (after subtraction of the blank) are 50% of the positive control. A value of 12.5 was assigned to samples with a BT50 less than 25. Geometric mean titers (GMTs), geometric mean fold rises (GMFRs) and seroresponse rates for each group were determined. Seroresponse was defined as a 4-fold increase in antibody titer compared to pre-immunization titers. A blocking control serum sample was used as an internal control. An assay to confirm the specificity of the blocking was performed using the same protocol for the blocking assay with the following exceptions: after coating with carbohydrate, sera was incubated directly on the plate without first pre-incubating with VLP. After washing, VLPs were incubated on the plate and detected as for the blocking assay.
Norovirus Hemagglutination Antibody Inhibition (HAI) Assay
Vaccine-induced antibodies were examined for the capacity to inhibit hemagglutination of 0-type human RBCs by the norovirus VLPs as previously described (El Kamary et al. (2010) J Infect Dis, Vol. 202(11): 1649-58). HAI titers were calculated as the inverse of the highest dilution that inhibited hemagglutination with a compact negative RBC pattern and are presented as GMTs, GMFRs and ≥4-fold rises.
Norovirus GI.1 and GII.4 VLPs were separately serially diluted and incubated with an equal volume of a 0.5% human RBC suspension in a 96-well V bottom plate. The amount of norovirus VLP antigens corresponding to 4 HA units were determined and confirmed by back titration. Test sera were heat inactivated at 56 C for 30 minutes and treated with freshly prepared 25% Kaolin suspension. To eliminate serum inhibitors, test samples were pre-adsorbed with RBCs. The HAI assay were performed as follows: pre-treated sera (diluted 2-fold in PBS pH 5.5) were added to 96 well V-plates and incubated with an equal volume of Norovirus GI.1 and GII.4 VLP antigen, respectively, containing 4 HA units. A suspension of 0.5% RBCs was added to each well and plates incubated for an additional 90 minutes at 4 C. Wells containing only PBS or antigen without serum served as negative and positive controls, respectively. Geometric mean titers (GMTs), geometric mean fold rises (GMFRs) and seroresponse rates for each group were determined. Seroresponse was defined as a 4-fold increase in antibody titer compared to pre-immunization titers.
Antibody Secreting Cell Assays
PBMCs were isolated from approximately 60 mL of anti-coagulated blood on Days 0, 7+3, 28+3, and 35+3 after administration of IM Norovirus Bivalent VLP Vaccine or placebo. Approximately 25 mL of blood for fresh PBMC assays and 35 mL of blood for cryopreservation of PBMCs was obtained. ASC assays detect cells secreting antibodies to norovirus VLPs (Tacket et al. (2000) J. Infect. Dis., Vol. 182:302-305; Tacket et al. (2003) Clin. Immunol., Vol. 108:241-247; El Kamary et al. (2010) J Infect Dis, Vol. 202(11): 1649-58). Fresh PBMCs were evaluated for ASC frequency and determination of homing markers from a subset of subjects. Cryopreserved PBMCs from subjects participating in Cohort A were evaluated for ASC frequency. The response rate and mean number of ASC per 106 PBMCs at each time point for each group are described. A positive response is defined as a post-vaccination ASC count per 106 PBMCs that is at least 3 standard deviations (SD) above the mean pre-vaccination count for all subjects (in the log metric) and at least 8 ASC spots, which corresponds to the mean of medium-stimulated negative control wells (2 spots) plus 3 SD as determined in similar assays.
Measurement of Norovirus Virus-Specific Memory B-Cells
Anti-coagulated blood was collected only in Cohort A subjects (approximately 25 mL on Days 0, 28, 56 and 180) to measure memory B cells on days 0, 28, 56 and 180 after vaccination using an ELISpot assay preceded by in vitro antigen stimulation (Crotty et al. (2004) J. Immunol. Methods, Vol. 286:111-122.; Li et al. (2006) J. Immunol. Methods, Vol. 313:110-118). Peripheral blood mononuclear cells (5×106 cells/mL, 1 mL/well in 24-well plates) were incubated for 4 days with norovirus GI.1 and GII.4 VLP antigens separately to allow for clonal expansion of antigen-specific memory B cells and differentiation into antibody secreting cells. Controls included cells incubated in the same conditions in the absence of antigen and/or cells incubated with an unrelated antigen. Following stimulation, cells were washed, counted, and transferred to ELISpot plates coated with Norwalk VLP. To determine frequency of virus-specific memory B cells per total Ig-secreting B lymphocytes, expanded B cells were also added to wells coated with anti-human IgG and anti-human IgA antibodies. Bound antibodies were revealed with HRP-labeled anti-human IgG or anti-human IgA followed by appropriate substrate. Conjugates to IgA and IgG subclasses (IgA1, IgA2 and IgG1-4) are also used to determine antigen-specific subclass responses that may be related with distinct effector mechanisms and locations of immune priming. Spots were counted with an ELISpot reader. The expanded cell populations for each subject were examined by flow cytometry to confirm their memory B cell phenotype, i.e. CD19+, CD27+, IgG+, IgM+, CD38+, IgD, among others (Crotty et al. (2004) J. Immunol. Methods, Vol. 286:111-122.; Li et al. (2006) J. Immunol. Methods, Vol. 313:110-118).
Cellular Immune Responses
Anti-coagulated blood (approximately 25 mL on Days 0, 28, 56, and 180) from subjects in Cohort A were collected as coded specimens and the PBMCs isolated and cryopreserved in liquid nitrogen for possible future evaluation of CMI responses to norovirus GI.1 and GII.4 VLP antigens. Assays that are performed include PBMC proliferative and cytokine responses to norovirus GI.1 and GII.4 VLP antigens by measuring interferon (IFN)-γ and interleukin (IL)-4 levels among others according to established techniques (Samandari et al. (2000) J. Immunol., Vol. 164:2221-2232; Tacket et al. (2003) Clin. Immunol., Vol. 108:241-247). T cell responses are also evaluated.
Results
Safety assessment included local and systemic solicited symptoms for 7 days and unsolicited symptoms for 28 days after each dose. Serious Adverse Events are monitored for 12 months. Immunogenicity was assessed with serum obtained prior to and after each vaccination for Pan-ELISA antibodies (IgG, IgA and IgM combined) and peripheral blood mononuclear cells (PBMCs) for IgG and IgA antibody secreting cells (ASC) via Elispot.
All four dosage groups have been enrolled for Cohort A with post dose two safety data available from all four dosage groups (40 vaccinees total). Among the 40 vaccinees, pain or tenderness were the most common local symptoms reported after either dose, whereas swelling or redness was infrequent. No severe local symptoms were reported. Systemic symptoms of headache, myalgia, or malaise after either dose were reported by less than half of the vaccinees. No vaccinees reported fever. No related SAEs were reported.
As shown in FIGS. 1A-3B, robust anamnestic Pan-ELISA antibody responses (combined IgG, IgA, and IgM) were observed to both VLP antigens 7 days after the first dose of the lowest dosage (5 μg GI.1+5 μg of GII.4 VLPs). The second dose did not boost the post dose one responses. Similar results were observed for antigen-specific serum IgG and serum IgA responses measured separately (FIGS. 4A-9B). Dose-dependent responses were observed for the antibody responses to both antigens (FIGS. 1A-9B). However, the maximal response to the GI.1 VLP appeared to be achieved with a lower dose than the maximal response to the GII.4 VLP (15 μg vs. 50 μg). Interestingly, the single dose of the intramuscularly administered Norovirus bivalent vaccine induced a surprisingly, significantly greater antigen-specific antibody titer than the titer induced by two doses of an intranasally administered monovalent VLP vaccine comprising a 20 fold higher VLP dose (FIG. 10; compare LV03-104 5 μg group to LV01-103 100 μg group). Moreover, the low dose (5 μg), IM bivalent Norovirus vaccine produced a Norovirus-specific antibody titer similar to that induced in humans exposed to the native Norovirus (FIG. 10).
Robust IgG and IgA Elispot responses were also observed at 7 days after the first dose of the lowest dosage (5 μg) for both VLP antigens (Table 2). Notably, the antibody secreting cell (ASC) responses were biased to IgA vs. IgG and ASCs exhibited a mucosal homing (alpha 4/beta7) and chemokine (CCR10) receptor phenotype as assessed by flow cytometry (FIGS. 11A-11B; Table 3). As shown in Table 3, a greater number of ASCs exhibit mucosal homing markers (beta 7+, CD62L−) as compared to dual mucosal/peripheral homing markers (beta 7+, CD62L+). Table 4 shows the percentage of memory B cells per 106 peripheral blood monocytes that respond to the VLP antigens. A larger percentage of antigen-specific memory B cells also express mucosal homing markers as compared to the dual mucosal/peripheral or peripheral homing markers. Similar responses were also observed in recipients who received the 15 μg and 50 μg doses (Tables 2-4).
TABLE 2
Day 7, Characterization of PBMC response. Approximation
of Antibody Secreting Cells (ASCs)/million CD19+ cells.
ASC/million CD19+ cells - Day 7
Vaccine Response Percent
Vaccine Specific
Norovirus-specific
IgA
IgG
IgA
IgG
Specific to
Specific to
Percent of Total
B cells
GI.1
GI.1
GII.4
GII.4
GI.1
GII.4
Circulating PBMC
Geometric mean A1
30947
13807
10947
3945
4.48%
1.49%
5.96%
5 μg dose (n = 5)
Standard
6674
9780
3651
2261
Deviation A1
5 μg dose
Geometric mean A2
25296
17004
7108
4336
4.23%
1.14%
5.37%
15 μg dose (n = 4)
Standard
10846
18770
6055
5697
Deviation A2
15 μg dose
Geometric mean A3
36158
20572
14103
2549
5.67%
1.67%
7.34%
50 μg dose (n = 4)
Standard
11470
418
7627
2230
Deviation A3
50 μg dose
Geometric mean A4
34183
9566
26213
11310
4.37%
3.75%
8.13%
150 μg dose (n = 4)
Standard
32938
4466
89769
15226
Deviation A4
150 μg dose
Placebo (n = 2)
0
152
0
108
0.02%
0.01%
0.03%
TABLE 3
ASC markers in vaccine and placebo recipients by flow cytometry-Day 7
Percent Total
Vaccine Specific
Vaccine Specific
% of Total
% of % Total
% of % Total
ASC
Total per million cells*
Percent of Total*
CD19+ B
CD27+, CD38+,
CD27+, CD38+,
CD27+, CD38+,
CD27+, CD38+,
Circulating
cells that are
CCR10+_Beta 7+,
CCR10+ Beta 7+,
CCR10+, Beta 7+
CCR10+, Beta 7+
PBMC Mucosal
CD27+ & CD38+
CD62L−
CD62L+
CD62L(+)&(−)
CD62L(+)&(−)
Homing
Geometric mean A1
25.10%
6.86%
1.06%
2.78%
1656
0.17%
5 μg dose (n = 5)
Standard
10.45
3.13
1.01
Deviation A1
5 μg dose
Geometric mean A2
12.99%
16.98%
2.43%
4.63%
1355
0.14%
15 μg dose (n = 4)
Standard
9.13
1.56
0.23
Deviation A2
15 μg dose
Geometric mean A3
31.71%
26.43%
3.63%
12.01%
23915
2.39%
50 μg dose (n = 4)
Standard
6.32
1.82
1.38
Deviation A3
50 μg dose
Geometric mean A4
33.46%
30.06%
5.68%
15.74%
31350
3.14%
150 μg dose (n = 4)
Standard
9.86
2.97
1.70
Deviation A4
150 μg dose
Placebo (n = 2)
1.26%
22.00%
0.87%
1.20%
5
0.001%
*Assumes the majority of ASCs are norovirus-specific.
TABLE 4
Memory B cell responses in vaccine and placebo recipients-Day 7
Percent Total
Vaccine Specific
% of Total
% of % Total
Memory
Total per million cells*
Vaccine Specific
CD19+ B
% of % Total
CD27+, CD38+,
CD27+, CD38+,
CD27+, CD38+,
Percent of Total*
cells that are
CD27+, CD38+,
CD138+,
CD138+, CCR10+,
CD138+, CCR10+,
Circulating
CD27+, CD38+,
CD138+, CCR10+
CCR10+_Beta 7+,
Beta 7+
Beta 7+
PBMC Mucosal
CD138+
Beta 7+, CD62L−
CD62L+
CD62L(+)&(−)
CD62L(+)&(−)
Homing
Geometric mean A1
N/D
N/D
N/D
N/D
N/D
N/D
5 μg dose
Geometric mean A2
1.54%
11.58%
1.92%
0.21%
61
0.01%
15 μg dose (n = 4)
Standard
1.54
3.94
0.94
Deviation A2
15 μg dose
Geometric mean A3
3.31%
16.10%
4.60%
0.68%
1364
0.14%
50 μg dose (n = 4)
Standard
1.11
2.16
0.97
Deviation A3
50 μg dose
Geometric mean A4
1.56%
16.90%
8.10%
0.39%
778
0.08%
150 μg dose (n = 4)
Standard
0.22
3.26
4.57
Deviation A4
150 μg dose
Placebo (n = 1)
0.10%
12.50%
0.00%
0.01%
0
0%
*Assumes the majority of ASCs are norovirus-specific.
In the absence of an available direct viral neutralization assay due to the inability to culture Norovirus in vitro, functional assays which serve as substitutes for viral neutralization assays were conducted to measure functional antibodies in vaccinees.
Using the carbohydrate H antigen blocking activity assay described above, the inhibition of GI.1 VLP binding to H antigen mediated by vaccine-induced serum antibodies was measured. Data are presented as geometric mean fold rise (GMFR) and seroresponse (4-fold rise) in Table 5, and as geometric mean titer (GMT) in Table 6. Surprisingly, after just one intramuscular injection of the vaccine formulation, significant carbohydrate blocking activity was observed in all dose groups; in fact, the administration of a second dose of vaccine did not significantly increase blocking activity compared to post-dose 1 levels. The inhibition of binding activity was maintained throughout the testing period, up to 56 days post dose 1.
TABLE 5
Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovirus GI.1 Geometric Mean Fold Rise (GMFR) and Seroresponse (4-Fold Rise)
Study Day
28 Days Post Dose 1
7 Days Post Dose 2
28 Days Post Dose 2
7 Days Post Dose 1
21 Days Post Dose 1
(Pre-Dose 2)
(35 Days Post Dose 1)
(56 Days Post Dose 1)
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
Treatment
(95%
Rise
(95%
Rise
(95%
Rise
(95%
Rise
(95%
Rise
Group
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
5/5 mcg
9
26.6
88.9
9
25.1
88.9
9
19.7
100.0
9
20
88.9
9
16.6
77.8
VLP Vaccine
(8.3,
(51.8,
(8.9,
(51.8,
(8.2,
(66.4,
(7.7,
(51.8,
(5.7,
(40.0,
85.1)
99.7)
70.3)
99.7)
47.1)
100.0)
51.7)
99.7)
48.1)
97.2)
15/15 mcg
8
33.2
100.0
8
25.5
100.0
8
18.5
100.0
7
22.2
100.0
7
8.4
57.1
VLP Vaccine
(13.6,
(63.1,
(10.5,
(63.1,
(8.4,
(63.1,
(8.8,
(59.0,
(2.4,
(18.4,
80.8)
100.0)
61.8)
100.0)
40.6)
100.0)
56)
100.0)
29.6)
90.1)
50/50 mcg
10
38.6
100.0
10
27.9
100.0
10
20.9
100.0
10
19
100.0
9
10.2
77.8
VLP Vaccine
(18.3,
(69.2,
(13.4,
(69.2,
(10,
(69.2,
(9.9,
(69.2,
(4.6,
(40.0,
81.6)
100.0)
58)
100.0)
43.5)
100.0)
36.4)
100.0)
22.8)
97.2)
150/150 mcg
7
30.6
100.0
8
19.4
100.0
8
16.3
100.0
8
18.8
100.0
8
23.8
100.0
VLP Vaccine
(16.3,
(59.0,
(13.1,
(63.1,
(11.7,
(63.1,
(12.8,
(63.1,
(17,
(63.1,
57.6)
100.0)
28.5)
100.0)
22.6)
100.0)
27.5)
100.0)
33.3)
100.0)
Placebo
8
0.9
0.0
8
0.8
0.0
8
0.8
0.0
8
0.8
0.0
8
0.6
0.0
(0.8,
(0.0,
(0.7,
(0.0,
(0.6,
(0.0,
(0.7,
(0.0,
(0.3,
(0.0,
1)
36.9)
1.1)
36.9)
1.1)
36.9)
1.1)
36.9)
1.2)
36.9)
Results based on all subjects receiving both doses of study product.
Two subjects' data points are excluded due to a possible mix-up of specimens; one of these data points is a baseline specimen resulting in the subject not having fold rise data available for any time point.
TABLE 6
Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovirus GI.1 Geometric Mean Titer (GMT)
Study Day
28 Days Post Dose 1
7 Days Post Dose 2
28 Days Post Dose 2
Pre-Dose 1
7 Days Post Dose 1
21 Days Post Dose 1
(Pre-Dose 2)
(35 Days Post Dose 1)
(56 Days Post Dose 1)
Treatment
GMT
GMT
GMT
GMT
GMT
GMT
Group
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
5/5 mcg
9
28.9
9
768.5
9
723.7
9
568
9
577.1
9
478.3
VLP Vaccine
(12.7,
(344.1,
(398.1,
(321.8,
(351.2,
(293.3,
65.9)
1716)
1316)
1003)
948.3)
780.1)
15/15 mcg
8
24.9
8
826.1
8
634.3
8
459.8
7
610.3
7
230.9
VLP Vaccine
(12.7,
(524.9,
(285.9,
(225.3,
(354.6,
(105.2,
48.7)
1300)
1407)
938.6)
1050)
506.7)
50/50 mcg
10
17.3
10
669.2
10
483.7
10
362.4
10
328.9
9
184
VLP Vaccine
(9.9,
(329.1,
(258.7,
(192.9,
(191.9,
(97.2,
30.3)
1361)
904.2)
680.7)
563.8)
348.3)
150/150 mcg
8
15.5
7
435
8
300.7
8
252.7
8
291.5
8
369.7
VLP Vaccine
(11.1,
(262.5,
(173.9,
(146.7,
(171.5,
(233.8,
21.8)
720.8)
520)
435.2)
495.4)
584.6)
Placebo
8
29
9
24.6
9
22.5
9
22.2
8
24.6
8
18.3
(9.1,
(9.8,
(8.8,
(8.9,
(8.4,
(10.1,
92.8)
62.1)
57.3)
55.5)
72.6)
33.3)
Results based on all subjects receiving both doses of study product.
Two subjects' data points are excluded due to a possible mix-up of specimens.
Similarly, carbohydrate blocking activity of serum antibodies against GII.4 VLPs was measured. A significant response was observed in all dosing groups as measured by GMFR and seroresponse (Table 7) as well as GMT (Table 8). Similar to the antibody-mediated blocking of GI.1 binding described above, robust blocking of GII.4 VLP carbohydrate binding activity was detected after just one dose, and a second dose did not appear to enhance the blocking activity.
TABLE 7
Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovirus GII.4 Geometric Mean Fold Rise (GMFR) and Seroresponse (4-Fold Rise)
Study Day
28 Days Post Dose 1
7 Days Post Dose 2
28 Days Post Dose 2
7 Days Post Dose 1
21 Days Post Dose 1
(Pre-Dose 2)
(35 Days Post Dose 1)
(56 Days Post Dose 1)
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
Treatment
(95%
Rise
(95%
Rise
(95%
Rise
(95%
Rise
(95%
Rise
Group
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
5/5 mcg
9
5
33.3
9
5.9
55.6
9
4.7
44.4
9
4.7
44.4
9
5
55.6
VLP Vaccine
(1.6,
(7.5,
(1.7,
(21.2,
(1.4,
(13.7,
(1.6,
(13.7,
(1.6,
(21.2,
16.1)
70.1)
20.3)
86.3)
15.7)
78.8)
13.8)
78.8)
15.9)
86.3)
15/15 mcg
8
11
62.5
8
9.2
62.5
8
7.4
62.5
7
7
57.1
7
5.6
57.1
VLP Vaccine
(2.7,
(24.5,
(3,
(24.5,
(2.5,
(24.5,
(2.1,
(18.4,
(2.3,
(18.4,
45.3)
91.5)
27.9)
91.5)
21.8)
91.5)
23)
90.1)
14)
90.1)
50/50 mcg
10
18.6
70.0
10
12.2
70.0
10
8.4
70.0
10
8.7
70.0
9
5.2
66.7
VLP Vaccine
(4.9,
(34.8,
(3.8,
(34.8,
(2.9,
(34.8,
(2.9,
(34.8,
(2.2,
(29.9,
70.8)
93.3)
39.4)
93.3)
24.1)
93.3)
26.1)
93.3)
12)
92.5)
150/150 mcg
7
10.1
57.1
8
5.5
50.0
8
4.4
50.0
8
4.3
37.5
8
3.1
25.0
VLP Vaccine
(2,
(18.4,
(1.9,
(15.7,
(1.6,
(15.7,
(1.7,
(8.5,
(1.3,
(3.2,
51.8)
90.1)
16.5)
84.3)
12.2)
84.3)
10.7)
75.5)
7.2)
65.1)
Placebo
8
1
0.0
8
1.1
0.0
8
1.3
12.5
8
1.8
12.5
8
2
12.5
(0.9,
(0.0,
(1,
(0.0,
(0.9,
(0.3,
(0.5,
(0.3,
(0.7,
(0.3,
1.1)
36.9)
1.3)
36.9)
2.1)
52.7)
6.7)
52.7)
6.1)
52.7)
Results based on all subjects receiving both doses of study product.
Two subjects' data points are excluded due to a possible mix-up of specimens; one of these data points is a baseline specimen resulting in the subject not having fold rise data available for any time point.
TABLE 8
Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovirus GII.4 Geometric Mean Titer (GMT)
Study Day
28 Days Post Dose 1
7 Days Post Dose 2
28 Days Post Dose 2
Pre-Dose 1
7 Days Post Dose 1
21 Days Post Dose 1
(Pre-Dose 2)
(35 Days Post Dose 1)
(56 Days Post Dose 1)
Treatment
GMT
GMT
GMT
GMT
GMT
GMT
Group
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
5/5 mcg
9
40.3
9
202.1
9
236.9
9
189.7
9
188.7
9
201.6
VLP Vaccine
(18,
(106.3,
(133.4,
(108.6,
(118.7,
(116.4,
90)
384.3)
420.6)
331.3)
300.1)
349.5)
15/15 mcg
8
23.7
8
260.1
8
218.1
8
175.4
7
182.3
7
146.3
VLP Vaccine
(12.8,
(95.1,
(104.2,
(82.7,
(89.1,
(92.4,
43.8)
711.1)
456.3)
372)
372.8)
231.5)
50/50 mcg
10
28.4
10
527.2
10
345.2
10
238.3
10
246.5
9
160.2
VLP Vaccine
(13.1,
(271.1,
(195.5,
(139.4,
(138.7,
(107.4,
61.5)
1025)
609.6)
407.3)
438.2)
238.8)
150/150 mcg
8
63
7
721.8
8
347.7
8
277
8
267.9
8
193.5
VLP Vaccine
(24.8,
(344.6,
(186.1,
(145.6,
(158.7,
(121.6,
160.4)
1512)
649.5)
527)
452.2)
308.2)
Placebo
8
24.1
9
22.8
9
24.9
9
29.1
8
44
8
48.2
(12.9,
(12.6,
(12.7,
(15.1,
(12.6,
(16.7,
45)
41.6)
48.7)
56.1)
154.2)
139.5)
Results based on all subjects receiving both doses of study product.
Two subjects' data points are excluded due to a possible mix-up of specimens.
Hemagglutination Inhibition assays (HAI) were also utilized to test the response of serum antibodies from vaccinated subjects against target Norovirus VLP antigens. Similar to carbohydrate H antigen binding studies, just one dose of VLP vaccine induced antibodies that inhibited hemagglutination in all dosing groups, as measured by GMFR (Table 9), 4-fold rise (Table 9), and GMT (Table 10). Though the level of inhibition of hemagglutination was maintained through the last day tested (28 days post dose 2, 56 days post dose 1), the second dose of VLP vaccine did not appear to enhance vaccine-induced antibody-mediated inhibition of hemagglutination.
TABLE 9
Hemagglutination Inhibition Assay Anti-Norovirus GI.1 Geometric Mean Fold Rise (GMFR) and Seroresponse (4-Fold Rise)
Study Day
28 Days Post Dose 1
7 Days Post Dose 2
28 Days Post Dose 2
7 Days Post Dose 1
21 Days Post Dose 1
(Pre-Dose 2)
(35 Days Post Dose 1)
(56 Days Post Dose 1)
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
Treatment
(95%
Rise
(95%
Rise
(95%
Rise
(95%
Rise
(95%
Rise
Group
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
5/5 mcg
9
5.4
77.8
9
7
88.9
9
6.1
88.9
9
6
77.8
9
6.3
88.9
VLP Vaccine
(3,
(40.0,
(4.6,
(51.8,
(4.1,
(51.8,
(3.7,
(40.0,
(3.9,
(51.8,
9.8)
97.2)
10.7)
99.7)
9.3)
99.7)
9.5)
97.2)
10.3)
99.7)
15/15 mcg
8
8.9
100.0
8
9.5
87.5
8
7.1
75.0
7
8.5
85.7
7
8.1
100.0
VLP Vaccine
(4.4,
(63.1,
(4,
(47.3,
(3.1,
(34.9,
(4.1,
(42.1,
(4.4,
(59.0,
18)
100.0)
22.5)
99.7)
16.1)
96.8)
17.7)
99.6)
15.2)
100.0)
50/50 mcg
10
22.4
100.0
10
16.7
100.0
10
13.9
100.0
10
14.5
100.0
9
11.8
100.0
VLP Vaccine
(11.6,
(69.2,
(9.3,
(69.2,
(8.1,
(69.2,
(9.3,
(69.2,
(6.3,
(66.4,
43)
100.0)
29.8)
100.0)
24)
100.0)
22.7)
100.0)
21.9)
100.0)
150/150 mcg
7
12.6
85.7
8
11.1
100.0
8
8.4
100.0
8
8.4
100.0
8
7.3
100.0
VLP Vaccine
(5.7,
(42.1,
(6.4,
(63.1,
(5,
(63.1,
(5,
(63.1,
(4.5,
(63.1,
28)
99.6)
19.3)
100.0)
14)
100.0)
14)
100.0)
11.9)
100.0)
Placebo
8
1
0.0
8
1
0.0
8
1
0.0
8
0.9
0.0
8
1
0.0
(0.8,
(0.0,
(0.9,
(0.0,
(0.9,
(0.0,
(0.7,
(0.0,
(0.8,
(0.0,
1.2)
36.9)
1.2)
36.9)
1.1)
36.9)
1.1)
36.9)
1.2)
36.9)
Results based on all subjects receiving both doses of study product.
Two subjects' data points are excluded due to a possible mix-up of specimens; one of these data points is a baseline specimen resulting in the subject not having fold rise data available for any time point.
TABLE 10
Hemagglutination Inhibition Assay Anti-Norovirus GI.1 Geometric Mean Titer (GMT)
Study Day
28 Days Post Dose 1
7 Days Post Dose 2
28 Days Post Dose 2
Pre-Dose 1
7 Days Post Dose 1
21 Days Post Dose 1
(Pre-Dose 2)
(35 Days Post Dose 1)
(56 Days Post Dose 1)
Treatment
GMT
GMT
GMT
GMT
GMT
GMT
Group
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
5/5 mcg
9
26.4
9
143.5
9
185.3
9
162.1
9
157
9
167.4
VLP Vaccine
(14.1,
(53.6,
(88.6,
(83.3,
(80.3,
(88.4,
49.4)
383.8)
387.6)
315.6)
307.1)
316.8)
15/15 mcg
8
11.9
8
105.3
8
113.1
8
84.2
7
103.3
7
99.2
VLP Vaccine
(6.1,
(56.1,
(42.5,
(31.1,
(43.3,
(46.9,
23.4)
197.6)
301.3)
227.6)
246.6)
209.7)
50/50 mcg
10
7.2
10
160
10
119.2
10
99.7
10
103.8
9
81.1
VLP Vaccine
(5.3,
(79.4,
(60.4,
(51.8,
(58.4,
(38.8,
9.6)
322.6)
235.1)
191.8)
184.5)
169.5)
150/150 mcg
8
9.2
7
114.1
8
101.6
8
77.2
8
77.2
8
67.3
VLP Vaccine
(7.5,
(50.9,
(62,
(51.8,
(51.8,
(44.7,
11.3)
255.7)
166.4)
115)
115)
101.3)
Placebo
8
16.8
9
16.6
9
16.1
9
16.6
8
15.4
8
16.2
(10.1,
(11.7,
(10.7,
(11,
(10,
(10.8,
28.1)
23.7)
24.1)
25)
23.7)
24.4)
Results based on all subjects receiving both doses of study product.
Two subjects' data points are excluded due to a possible mix-up of specimens.
Inhibition of hemagglutination was also achieved when the target VLP was a mismatched virus. Vaccine-induced serum antibodies inhibited hemagglutination by a Houston virus strain VLP, as measured by GMFR and seroresponse (Table 11) as well as GMT (Table 12). In this case, the higher VLP vaccine doses afforded stronger responses, particularly as measured by 4-fold rise or GMT. GMFR and 4-fold rise were also significantly increased when the target VLP was the 2003 Cincinnati virus strain, as measured just 7 days post dose 1 (Table 13).
TABLE 11
Hemagglutination Inhibition Assay (Houston Virus Strain VLP), Anti-Norovirus
GII.4 Geometric Mean Fold Rise (GMFR) and Seroresponse (4-Fold Rise)
Study Day
28 Days Post Dose 1
7 Days Post Dose 2
28 Days Post Dose 2
7 Days Post Dose 1
21 Days Post Dose 1
(Pre-Dose 2)
(35 Days Post Dose 1)
(56 Days Post Dose1)
GMFR
4-Fold
GMFR
4-Fold
GMFR
4-Fold
4-Fold
GMFR
4-Fold
Treatment
(95%
Rise
(95%
Rise
(95%
Rise
GMFR
Rise
(95%
Rise
Group
N
CI)
(95% CI)
N
CI)
(95% CI)
N
CI)
(95% CI)
N
(95% CI)
(95% CI)
N
CI)
(95% CI)
5/5 mcg
9
1.2
0.0
9
1.3
0.0
9
1.3
0.0
9
1.3
0.0
9
1.3
0.0
VLP Vaccine
(1,
(0.0,
(0.9,
(0.0,
(0.9,
(0.0,
(1,
(0.0,
(1,
(0.0,
1.5)
33.6)
1.7)
33.6)
1.7)
33.6)
1.7)
33.6)
1.6)
33.6)
15/15 mcg
8
1.7
12.5
8
1.6
12.5
8
1.5
0.0
7
1.5
0.0
7
1.6
0.0
VLP Vaccine
(0.9,
(0.3,
(1.1,
(0.3,
(1.1,
(0.0,
(1,
(0.0,
(1.1,
(0.0,
3)
52.7)
2.4)
52.7)
2.2)
36.9)
2.2)
41.0)
2.4)
41.0)
50/50 mcg
10
2
10.0
10
1.6
10.0
10
1.7
10.0
10
1.5
10.0
9
1.3
11.1
VLP Vaccine
(0.9,
(0.3,
(0.8,
(0.3,
(0.9,
(0.3,
(0.9,
(0.3,
(0.8,
(0.3,
4.3)
44.5)
3.1)
44.5)
3.1)
44.5)
2.4)
44.5)
2)
48.2)
150/150 mcg
7
4.3
57.1
8
2.6
50.0
8
1.9
12.5
8
1.9
12.5
8
1.7
12.5
VLP Vaccine
(1.5,
(18.4,
(1.2,
(15.7,
(1.1,
(0.3,
(1.1,
(0.3,
(1.1,
(0.3,
12.4)
90.1)
5.8)
84.3)
3.3)
52.7)
3.5)
52.7)
2.6)
52.7)
Placebo
8
1
0.0
8
1.1
0.0
8
1.1
0.0
8
1.2
12.5
8
1.1
12.5
(0.9,
(0.0,
(0.9,
(0.0,
(1,
(0.0,
(0.8,
(0.3,
(0.7,
(0.3,
1.1)
36.9)
1.3)
36.9)
1.2)
36.9)
1.9)
52.7)
1.8)
52.7)
Results based on all subjects receiving both doses of study product.
Two subjects' data points are excluded due to a possible mix-up of specimens; one of these data points is a baseline specimen resulting in the subject not having fold rise data available for any time point.
TABLE 12
Hemagglutination Inhibition Assay (Houston Virus Strain VLP), Anti-Norovirus GII.4 Geometric Mean Titer (GMT)
Study Day
28 Days Post Dose 1
7 Days Post Dose 2
28 Days Post Dose 2
Pre-Dose 1
7 Days Post Dose 1
21 Days Post Dose 1
(Pre-Dose 2)
(35 Days Post Dose 1)
(56 Days Post Dose 1)
Treatment
GMT
GMT
GMT
GMT
GMT
GMT
Group
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
N
(95% CI)
5/5 mcg
9
143.5
9
177.4
9
180.8
9
180.8
9
189.1
9
180.8
VLP Vaccine
(97.9,
(122.7,
(114.4,
(114.4,
(119.2,
(122.7,
210.3)
256.5)
285.6)
285.6)
299.9)
266.3)
15/15 mcg
8
129.8
8
218.3
8
207.5
8
200.2
7
169.5
7
187.2
VLP Vaccine
(86.4,
(129.2,
(134.8,
(132.8,
(114.1,
(119.1,
194.9)
368.8)
319.3)
301.7)
252)
294.2)
50/50 mcg
10
161.9
10
323.8
10
252.5
10
273.9
10
242.5
9
195.2
VLP Vaccine
(119.7,
(156.5,
(130.4,
(150.9,
(150.2,
(125,
219)
669.8)
489.2)
497.2)
391.5)
304.9)
150/150 mcg
8
210.6
7
853.5
8
546.2
8
406.3
8
406.3
8
354.1
VLP Vaccine
(108.8,
(297.4,
(237.6,
(201.5,
(180.3,
(184.6,
407.5)
2449)
1255)
819.1)
915.4)
679.5)
Placebo
8
148.9
9
150.1
9
157
9
167.4
8
183.6
8
162.4
(73.8,
(80.1,
(77.9,
(91.4,
(95,
(88.5,
300.3)
281.1)
316.5)
306.4)
354.5)
297.9)
Results based on all subjects receiving both doses of study product.
Two subjects' data points are excluded due to a possible mix-up of specimens.
TABLE 13
Inhibition of Hemagglutination in placebo versus 50/50 μg VLP vaccine
Hemagglutination Inhibition Assay (2003 Cincinnati Virus Strain
VLP), Anti-Norovirus GII.4 Geometric Mean Fold Rise (GMFR)
and Geometric Seroresponse (4-Fold Rise)
Results by Treatment Group 7 Days Post Dose 1
Treatment Group
N
GMFR (95% CI)
4-Fold Rise (95% CI)
Placebo
2
1.0 (0.6, 1.8)
0.0 (0.0, 84.2)
50/50 μg VLP Vaccine
10
4.4 (1.6, 11.9)
50.0 (18.7, 81.3)
The results from this study demonstrated that the Bivalent IM Norovirus VLP vaccine was generally well tolerated. The immunogenicity data suggested that a single vaccine dose may be sufficient to protect seropositive human adults. The results from the carbohydrate blocking activity and hemagglutination inhibition assays provided further evidence that a single vaccine dose induced serum antibodies with potent anti-Norovirus activity. The magnitude and rapidity of the observed immune responses following a single parenteral dose in humans were dramatic when compared to earlier immune responses reported by multiple nasal VLP vaccine administrations at much higher VLP dosages (El Kamary et al. (2010) J Infect Dis, Vol. 202(11): 1649-1658). These responses were also superior to those induced by orally administered Norovirus VLPs (Tacket et al. (2003) Clin Immunol 108:241-247; Ball et al. (1999, Gastroenterology 117:40-48) as well as those induced by Norovirus VLPs produced by transgenic plants (Tacket et al. (2000) J Infect Dis 182:302-305). In particular, this intramuscular vaccine formulation produced anamnestic responses within seven days of immunization and maximal serum antibody responses were observed after a single dose, including a significant IgA response and functional carbohydrate blocking activity and hemagglutination inhibition activity. Thus, this Norovirus bivalent vaccine induced a strong, protective immune response in humans that was superior to immune responses induced by any currently available Norovirus vaccine.
Example 2. Dose Escalation, Safety and Immunogenicity Study of Intramuscular Norovirus Bivalent Virus-Like-Particle (VLP) Vaccine in Humans (LV03-104 Study)
The following example provides the remaining planned portion of the clinical study described in Example 1, wherein a randomized, multi-site, dose-escalation study is conducted in adults ≥18 years of age of the safety and immunogenicity of four dosage levels of an intramuscular (IM) Norovirus Bivalent VLP Vaccine adjuvanted with monophosphoryl lipid A (MPL) and aluminum hydroxide (AlOH), compared to placebo. Subjects will receive two doses of the vaccine or placebo, by intramuscular (IM) injection, 28 days apart using a 1.5 inch (38 mm) needle. This example is intended to further illustrate the principles of the present invention.
Cohort A has completed enrollment in the study and was described above in Example 1. Cohort B contains ˜20 subjects 50-64 years of age. Cohort C contains ˜30 subjects 65-85 years of age. Approximately 98 subjects are enrolled in the study as a whole.
In Cohort B, ˜20 subjects 50-64 years of age are enrolled and randomized 1:1 to receive vaccine (N=10) or placebo (N=10). After the 7-day post Dose 2 safety data (Study Day 35) are available for review from subjects in Cohort B, subjects in Cohort C are eligible to receive their initial dose. In Cohort C, ˜30 subjects 65 to 85 years of age are enrolled and randomized 1:1:1 to receive vaccine adjuvanted with MPL and AlOH (N=10), or vaccine adjuvanted with AlOH alone, i.e. no MPL (N=10), or placebo (N=10). The antigen concentrations of the norovirus VLPs and of the AlOH in the two vaccine formulations to be evaluated in Cohort C are identical; only the presence or absence of MPL is different.
The Norovirus Bivalent VLP Vaccine contains genogroup I, genotype 1 (GI.1) and genogroup II, genotype IV (GII.4) VLPs as the antigens, and Monophosphoryl Lipid A (MPL) and aluminum hydroxide (AlOH) as adjuvants, sodium chloride (NaCl) and L-histidine (L-His) as buffer (pH 6.3-6.7), ethanol and water for injection. The GII.4 VLPs comprised a capsid sequence of SEQ ID NO: 1, which was derived from three GII.4 strains.
The single dosage of vaccine selected for further evaluation in Cohorts B and C is the lowest dosage in Cohort A that results in the most robust and reproducible immune response that is also generally well tolerated. The Day 56 safety and immunogenicity data from subjects in Cohort A is reviewed by the CSM/SMC and the bivalent dosage is selected for evaluation in Cohorts B and C.
The subjects keep a daily memory aid of solicited symptoms including four local injection site reactions, such as pain, tenderness, redness, and swelling, and 10 systemic signs or symptoms including daily oral temperature, headache, fatigue, muscle aches, chills, joint aches and gastrointestinal symptoms of nausea, vomiting, diarrhea, abdominal cramps/pain for Days 0 through 7 after each dose of IM Norovirus Bivalent VLP Vaccine or control. The redness and swelling at the injection site are measured and recorded daily for 7 days after each injection.
Interim medical histories are obtained at each follow-up visit on Days 7+3, 21+3, 28+3, 35+3, 56+7, 180+14, and 393+14 and at the follow-up telephone call on Day 265+14; subjects are queried about interim illness, doctor's visits, any serious adverse events (SAEs), and onset of any significant new medical conditions. Subjects have a CBC with WBC differential and platelet count, and serum BUN, creatinine, glucose, AST, and ALT assessed at screening and on Days 21 and 35 (˜7 days after each dose) to assess continuing eligibility and safety, respectively.
Blood from subjects is collected before vaccination on Day 0 and on Days 7+3, 21+3, 28+3, 35+3, 56+7, 180+14, and 393+14 to measure serum antibodies (IgG, IgA, and IgM separately and combined) to IM Norovirus Bivalent VLP Vaccine by enzyme-linked immunosorbent assays (ELISA). Serum carbohydrate blocking activity and serum HAI antibodies are also measured.
The methods described above for Cohort A are used to analyze the blood samples collected from immunized individuals or individuals receiving the placebo. The results of the study will be employed in the development of a clinical protocol for administration of the vaccine formulations of the invention.
The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings using no more than routine experimentation. Such modifications and equivalents are intended to fall within the scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.
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15836030
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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tyo:4502
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Takeda Pharmaceutical
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Nov 20th, 2018 12:00AM
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Sep 18th, 2008 12:00AM
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https://www.uspto.gov?id=US10130696-20181120
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Method of conferring a protective immune response to norovirus
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The present invention relates to vaccine compositions comprising Norovirus antigens and adjuvants, in particular, mixtures of monovalent VLPs and mixtures of multivalent VLPs, and to methods of conferring protective immunity to Norovirus infections in a human subject.
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10130696
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1. A method of eliciting protective immunity to a Norovirus infection in a human comprising administering to the human a vaccine comprising at least one Norovirus virus-like particle (VLP) wherein the content of each Norovirus VLP in the vaccine is from about 1 μg to about 50 μg.
2. The method of claim 1, wherein said Norovirus VLPs are selected from the group consisting of Norovirus genogroup I and genogroup II viral strains.
3. The method of claim 1, wherein said Norovirus VLPs are monovalent VLPs.
4. The method of claim 1, wherein said Norovirus VLPs are multivalent VLPs.
5. The method of claim 3, wherein said Norovirus VLPs are monovalent VLPs from different genogroups.
6. The method of claim 5, wherein said Norovirus VLPs comprise Norwalk virus VLPs and Houston virus VLPs.
7. The method of claim 1, wherein said vaccine further comprises a delivery agent.
8. The method of claim 7, wherein the delivery agent is a bioadhesive.
9. The method of claim 8, wherein said bioadhesive is a mucoadhesive.
10. The method of claim 9, wherein said mucoadhesive is selected from the group consisting of dermatan sulfate, chondroitin, pectin, mucin, alginate, cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides, hydroxypropyl methylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose.
11. The method of claim 10, wherein said mucoadhesive is a polysaccharide.
12. The method of claim 11, wherein said polysaccharide is chitosan, chitosan salt, or chitosan base.
13. The method of claim 1, wherein the vaccine further comprises an adjuvant that is not a bacterially-derived exotoxin.
14. The method of claim 13, wherein the adjvuant is selected from the group consisting of toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, and liposomes.
15. The method of claim 14, wherein the adjuvant is a toll-like receptor (TLR) agonist.
16. The method of claim 14, wherein the adjuvant is MPL.
17. The method of claim 14, wherein the adjuvant is alum.
18. The method of claim 14, wherein the adjuvant comprises MPL and alum.
19. The method of claim 1, wherein the vaccine is in a powder formulation.
20. The method of claim 1, wherein the vaccine is in a liquid formulation.
21. The method of claim 1, wherein said vaccine is administered to the human by a route selected from the group consisting of mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, and transdermal routes of administration.
22. The method of claim 21, wherein said vaccine is administered intranasally.
23. The method of claim 22, wherein said vaccine is administered to the nasal mucosa by rapid deposition within the nasal passage from one or more devices comprising the vaccine held close to the nasal passageway.
24. The method of claim 23, wherein said vaccine is administered to one or both nostrils.
25. The method of claim 21, wherein said vaccine is administered intramuscularly.
26. The method of claim 1, wherein said vaccine confers protection from one or more symptoms of Norovirus infection.
27. A method of eliciting protective immunity to a Norovirus infection in a human comprising administering to the human a vaccine comprising at least one Norovirus virus-like particle (VLP) wherein the content of each Norovirus VLP in the vaccine is from about 1 μg to about 50 μg, and wherein said vaccine is administered intramuscularly.
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27
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage application of International Application No. PCT/US2008/076763, filed Sep. 18, 2008, which claims the benefit of priority of U.S. Provisional Application No. 60/973,389, filed Sep. 18, 2007 and U.S. Provisional Application No. 60/986,826, filed Nov. 9, 2007, which are herein incorporated by reference in their entireties.
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with Government support under W81XWH-05-C-0135 awarded by the U.S. Army. The Government has certain rights in this invention.
FIELD OF THE INVENTION
The invention is in the field of vaccines, particularly vaccines for Noroviruses. In addition, the invention relates to methods of preparing vaccine compositions and methods of inducing a protective immune response.
BACKGROUND OF THE INVENTION
Noroviruses are non-cultivatable human Caliciviruses that have emerged as the single most important cause of epidemic outbreaks of nonbacterial gastroenteritis (Glass et al., 2000; Hardy et al., 1999). The clinical significance of Noroviruses was under-appreciated prior to the development of sensitive molecular diagnostic assays. The cloning of the prototype genogroup I Norwalk virus (NV) genome and the production of virus-like particles (VLPs) from a recombinant Baculovirus expression system led to the development of assays that revealed widespread Norovirus infections (Jiang et al. 1990; 1992).
Noroviruses are single-stranded, positive sense RNA viruses that contain a non-segmented RNA genome. The viral genome encodes three open reading frames, of which the latter two specify the production of the major capsid protein and a minor structural protein, respectively (Glass et al. 2000). When expressed at high levels in eukaryotic expression systems, the capsid protein of NV, and certain other Noroviruses, self-assembles into VLPs that structurally mimic native Norovirus virions. When viewed by transmission electron microscopy, the VLPs are morphologically indistinguishable from infectious virions isolated from human stool samples.
Immune responses to Noroviruses are complex, and the correlates of protection are just now being elucidated. Human volunteer studies performed with native virus demonstrated that mucosally-derived memory immune responses provided short-term protection from infection and suggested that vaccine-mediated protection is feasible (Lindesmith et al. 2003; Parrino et al. 1997; Wyatt et al., 1974).
Although Norovirus cannot be cultivated in vitro, due to the availability of VLPs and their ability to be produced in large quantities, considerable progress has been made in defining the antigenic and structural topography of the Norovirus capsid. VLPs preserve the authentic confirmation of the viral capsid protein while lacking the infectious genetic material. Consequently, VLPs mimic the functional interactions of the virus with cellular receptors, thereby eliciting an appropriate host immune response while lacking the ability to reproduce or cause infection. In conjunction with the NIH, Baylor College of Medicine studied the humoral, mucosal and cellular immune responses to NV VLPs in human volunteers in an academic, investigator-sponsored Phase I clinical trial. Orally administered VLPs were safe and immunogenic in healthy adults (Ball et al. 1999; Tacket et al. 2003). At other academic centers, preclinical experiments in animal models have demonstrated enhancement of immune responses to VLPs when administered intranasally with bacterial exotoxin adjuvants (Guerrero et al. 2001; Nicollier-Jamot et al. 2004; Periwal et al. 2003; Souza et al. (2007) Vaccine, doi: 10.1016/j.vaccine.2007.09.040). However, no studies have reported being able to achieve protective immunity against Norovirus using any Norovirus vaccine.
SUMMARY OF THE INVENTION
The present invention provides methods of inducing protective immunity to a Norovirus infection in a subject, in particular a human subject, comprising administering a vaccine comprising at least one Norovirus antigen. In one embodiment, the antigen is a Norovirus virus-like particle (VLP). Vaccines used in the methods of the invention may further comprise one or more adjuvants. The Norovirus VLPs can be selected from genogroup I or genogroup II virus or a mixture thereof. In one embodiment, the vaccine comprises Norovirus VLPs in a concentration from about 0.01% to about 80% by weight. In another embodiment, the vaccine comprises dosages of Norovirus VLPs from about 1 μg to about 100 mg per dose.
In some embodiments, the vaccine further comprises a delivery agent, which functions to enhance antigen uptake, provide a depot effect, increase antigen retention time at the site of delivery, or enhance the immune response through relaxation of cellular tight junctions at the delivery site. The delivery agent can be a bioadhesive, preferably a mucoadhesive selected from the group consisting of dermatan sulfate, chondroitin, pectin, mucin, alginate, cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides, hydroxypropyl methylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose. Preferably, the mucoadhesive is a polysaccharide. More preferably, the mucoadhesive is chitosan, or a mixture containing chitosan, such as a chitosan salt or chitosan base.
In other embodiments, the vaccine comprises an adjuvant. The adjuvant may be selected from the group consisting of toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL®), synthetic lipid A, lipid A mimetics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, endotoxins, for instance bacterial endotoxins and liposomes. Preferably, the adjuvant is a toll-like receptor (TLR) agonist. More preferably, the adjuvant is MPL®.
The methods of the present invention include administering Norovirus vaccines formulated as a liquid or a dry powder. Dry power formulations may contain an average particle size from about 10 to about 500 micrometers in diameter. Suitable routes for administering the vaccine include mucosal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal. In particular, the route of administration may be intramuscular or mucosal, with preferred routes of mucosal administration including intranasal, oral, or vaginal routes of administration. In another embodiment, the vaccine is formulated as a nasal spray, nasal drops, or dry powder, wherein the vaccine is administered by rapid deposition within the nasal passage from a device containing the vaccine held close to the nasal passageway. In another embodiment, the vaccine is administrated to one or both nostrils.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows that Norwalk Virus (NV)-specific IgG is elicited in rabbits immunized with dry powder VLPs. Rabbits were dosed 3 times, via the intranasal route of administration, on days 1, 22 and 43 (arrows) with 50 μg NV-VLP+50 μg MPL. Serum from each rabbit was tested for NV-VLP-specific IgG by ELISA on the days indicated. Only the VLP vaccinated rabbits had NV-VLP-specific IgG, whereas the untreated and placebo treatment groups had no detectable antigen-specific antibodies (data not shown). Arithmetic means of the responses are shown and expressed in U/mL (1 U˜1 μg). Bars indicate the standard error of the mean.
FIG. 2 depicts the results of ELISA assays measuring serum IgA (panel A) and IgG (panel B) levels from human volunteers immunized with control (adjuvant/excipient) or a vaccine formulation containing one of three doses of Norwalk Virus VLPs (5, 15, or 50 μg). The geometric mean fold-increase in anti-VLP titer is shown for each of the dosage levels at 35 days after the second immunization (day 56). Volunteers received immunizations on days 0 and 21.
FIG. 3 shows the levels of IgA (panel A) and IgG (panel B) antibody secreting cells (ASCs) in human volunteers receiving vaccine formulations with the 50 μg dose of Norwalk Virus VLPs or control (adjuvant/excipient). The geometric mean (GMN) of ASCs per 106 peripheral blood mononuclear cells (PBMCs) is plotted versus study day (day 7 or day 28), specifically seven days post immunization. Volunteers received immunizations on days 0 and 21.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of eliciting a protective immunity to Norovirus infections in a subject. In particular, the present invention provides methods of administering a vaccine comprising Norovirus VLPs and at least one adjuvant to a human, wherein the vaccine confers protection from at least one symptom of Norovirus infection. Additionally or alternatively, the vaccine may further comprise at least one delivery agent.
Norovirus Antigens
The invention provides a composition comprising one or more Norovirus antigens. By “Norovirus,” “Norovirus (NOR),” “norovirus,” and grammatical equivalents herein, are meant members of the genus Norovirus of the family Caliciviridae. In some embodiments, a Norovirus can include a group of related, positive-sense single-stranded RNA, nonenveloped viruses that can be infectious to human or non-human mammalian species. In some embodiments, a Norovirus can cause acute gastroenteritis in humans. Noroviruses also can be referred to as small round structured viruses (SRSVs) having a defined surface structure or ragged edge when viewed by electron microscopy. Included within the Noroviruses are at least four genogroups (GI-IV) defined by nucleic acid and amino acid sequences, which comprise 15 genetic clusters. The major genogroups are GI and GII. GIII and GIV are proposed but generally accepted. Representative of GIII is the bovine, Jena strain. GIV contains one virus, Alphatron, at this time. For a further description of Noroviruses see Vinje et al. J. Clin. Micro. 41:1423-1433 (2003). By “Norovirus” also herein is meant recombinant Norovirus virus-like particles (rNOR VLPs). In some embodiments, recombinant expression of at least the Norovirus capsid protein encoded by ORF2 in cells, e.g., from a baculovirus vector in Sf9 cells, can result in spontaneous self-assembly of the capsid protein into VLPs. In some embodiments, recombinant expression of at least the Norovirus proteins encoded by ORF1 and ORF2 in cells, e.g., from a baculovirus vector in Sf9 cells, can result in spontaneous self-assembly of the capsid protein into VLPs. VLPs are structurally similar to Noroviruses but lack the viral RNA genome and therefore are not infectious. Accordingly, “Norovirus” includes virions that can be infectious or non-infectious particles, which include defective particles.
Non-limiting examples of Noroviruses include Norwalk virus (NV, GenBank M87661, NP056821), Southampton virus (SHV, GenBank L07418), Desert Shield virus (DSV, U04469), Hesse virus (HSV), Chiba virus (CHV, GenBank AB042808), Hawaii virus (HV, GenBank U0761 1), Snow Mountain virus (SMV, GenBank U70059), Toronto virus (TV, Leite et al., Arch. Virol. 141:865-875), Bristol virus (BV), Jena virus (JV, AJ01099), Maryland virus (MV, AY032605), Seto virus (SV, GenBank AB031013), Camberwell (CV, AF145896), Lordsdale virus (LV, GenBank X86557), Grimsby virus (GrV, AJ004864), Mexico virus (MXV, GenBank U22498), Boxer (AF538679), C59 (AF435807), VA115 (AY038598), BUDS (AY660568), Houston virus (HoV, AY502023), MOH (AF397156), Parris Island (PiV; AY652979), VA387 (AY038600), VA207 (AY038599), and Operation Iraqi Freedom (OIF, AY675554). The nucleic acid and corresponding amino acid sequences of each are all incorporated by reference in their entirety. In some embodiments, a cryptogram can be used for identification purposes and is organized: host species from which the virus was isolated/genus abbreviation/species abbreviation/strain name/year of occurrence/country of origin. (Green et al., Human Caliciviruses, in Fields Virology Vol. 1 841-874 (Knipe and Howley, editors-in-chief, 4th ed., Lippincott Williams & Wilkins 2001)). Norwalk virus, Snow Mountain virus, and Houston virus are preferred in some embodiments.
The Norovirus antigen may be in the form of peptides, proteins, or virus-like particles (VLPs). In a preferred embodiment, the Norovirus antigen comprises VLPs. As used herein, “virus-like particle(s) or VLPs” refer to a virus-like particle(s), fragment(s), aggregates, or portion(s) thereof produced from the capsid protein coding sequence of Norovirus and comprising antigenic characteristic(s) similar to those of infectious Norovirus particles. Norovirus antigens may also be in the form of capsid monomers, capsid multimers, protein or peptide fragments of VLPs, or aggregates or mixtures thereof. The Norovirus antigenic proteins or peptides may also be in a denatured form, produced using methods known in the art.
The VLPs of the present invention can be formed from either the full length Norovirus capsid protein such as VP1 and/or VP2 proteins or certain VP1 or VP2 derivatives using standard methods in the art. Alternatively, the capsid protein used to form the VLP is a truncated capsid protein. In some embodiments, for example, at least one of the VLPs comprises a truncated VP1 protein. In other embodiments, all the VLPs comprise truncated VP1 proteins. The truncation may be an N- or C-terminal truncation. Truncated capsid proteins are suitably functional capsid protein derivatives. Functional capsid protein derivatives are capable of raising an immune response (if necessary, when suitably adjuvanted) in the same way as the immune response is raised by a VLP consisting of the full length capsid protein.
VLPs may contain major VP1 proteins and/or minor VP2 proteins. Preferably each VLP contains VP1 and/or VP2 protein from only one Norovirus genogroup giving rise to a monovalent VLP. As used herein, the term “monovalent” means the antigenic proteins are derived from a single Norovirus genogroup. For example, the VLPs contain VP1 and/or VP2 from a virus strain of genogroup I (e.g., VP1 and VP2 from Norwalk virus). Preferably the VLP is comprised of predominantly VP1 proteins. In one embodiment of the invention, the antigen is a mixture of monovalent VLPs wherein the composition includes VLPs comprised of VP1 and VP2 from a single Norovirus genogroup mixed with VLPs comprised of VP1 and VP2 from a different Norovirus genogroup (e.g. Norwalk virus and Houston virus) taken from multiple viral strains. Purely by way of example the composition can contain monovalent VLPs from one or more strains of Norovirus genogroup I together with monovalent VLPs from one or more strains of Norovirus genogroup II. Preferably, the Norovirus VLP mixture is composed of the strains of Norwalk and Houston Noroviruses.
However, in an alternative embodiment of the invention, the VLPs may be multivalent VLPs that comprise, for example, VP1 and/or VP2 proteins from one Norovirus genogroup intermixed with VP1 and/or VP2 proteins from a second Norovirus genogroup, wherein the different VP1 and VP2 proteins are not chimeric VP1 and VP2 proteins, but associate together within the same capsid structure to form immunogenic VLPs. As used herein, the term “multivalent” means that the antigenic proteins are derived from two or more Norovirus genogroups or strains. Multivalent VLPs may contain VLP antigens taken from two or more viral strains. Purely by way of example the composition can contain multivalent VLPs comprised of capsid monomers or multimers from one or more strains of Norovirus genogroup I (e.g. Norwalk virus) together with capsid monomers or multimers from one or more strains of Norovirus genogroup II (e.g. Houston virus). Preferably, the multivalent VLPs contain capsid proteins from the strains of Norwalk and Houston Noroviruses.
The combination of monovalent or multivalent VLPs within the composition preferably would not reduce the immunogenicity of each VLP type. In particular it is preferred that there is no interference between Norovirus VLPs in the combination of the invention, such that the combined VLP composition of the invention is able to elicit immunity against infection by each Norovirus genotype represented in the vaccine. Suitably the immune response against a given VLP type in the combination is at least 50% of the immune response of that same VLP type when measured individually, preferably 100% or substantially 100%. The immune response may suitably be measured, for example, by antibody responses, as illustrated in the examples herein.
Multivalent VLPs may be produced by separate expression of the individual capsid proteins followed by combination to form VLPs. Alternatively multiple capsid proteins may be expressed within the same cell, from one or more DNA constructs. For example, multiple DNA constructs may be transformed or transfected into host cells, each vector encoding a different capsid protein. Alternatively a single vector having multiple capsid genes, controlled by a shared promoter or multiple individual promoters, may be used. IRES elements may also be incorporated into the vector, where appropriate. Using such expression strategies, the co-expressed capsid proteins may be co-purified for subsequent VLP formation, or may spontaneously form multivalent VLPs which can then be purified.
A preferred process for multivalent VLP production comprises preparation of VLP capsid proteins or derivatives, such as VP1 proteins, from different Norovirus genotypes, mixing the proteins, and assembly of the proteins to produce multivalent VLPs. The VP1 proteins may be in the form of a crude extract, be partially purified or purified prior to mixing. Assembled monovalent VLPs of different genogroups may be disassembled, mixed together and reassembled into multivalent VLPs. Preferably the proteins or VLPs are at least partially purified before being combined. Optionally, further purification of the multivalent VLPs may be carried out after assembly.
Suitably the VLPs of the invention are made by disassembly and reassembly of VLPs, to provide homogenous and pure VLPs. In one embodiment multivalent VLPs may be made by disassembly of two or more VLPs, followed by combination of the disassembled VLP components at any suitable point prior to reassembly. This approach is suitable when VLPs spontaneously form from expressed VP1 protein, as occurs for example, in some yeast strains. Where the expression of the VP1 protein does not lead to spontaneous VLP formation, preparations of VP1 proteins or capsomers may be combined before assembly into VLPs.
Where multivalent VLPs are used, preferably the components of the VLPs are mixed in the proportions in which they are desired in the final mixed VLP. For example, a mixture of the same amount of a partially purified VP1 protein from Norwalk and Houston viruses (or other Norovirus strains) provides a multivalent VLP with approximately equal amounts of each protein.
Compositions comprising multivalent VLPs may be stabilized by solutions known in the art, such as those of WO 98/44944, WO 00/45841, incorporated herein by reference.
Compositions of the invention may comprise other proteins or protein fragments in addition to VP1 and VP2 proteins or derivatives. Other proteins or peptides may also be co-administered with the composition of the invention. Optionally the composition may also be formulated or co-administered with non-Norovirus antigens. Suitably these antigens can provide protection against other diseases.
The VP1 protein or functional protein derivative is suitably able to form a VLP, and VLP formation can be assessed by standard techniques such as, for example, electron microscopy and dynamic laser light scattering.
Antigen Preparation
The antigenic molecules of the present invention can be prepared by isolation and purification from the organisms in which they occur naturally, or they may be prepared by recombinant techniques. Preferably the Norovirus VLP antigens are prepared from insect cells such as Sf9 or H5 cells, although any suitable cells such as E. coli or yeast cells, for example, S. cerevisiae, S. pombe, Pichia pastori or other Pichia expression systems, mammalian cell expression such as CHO or HEK systems may also be used. When prepared by a recombinant method or by synthesis, one or more insertions, deletions, inversions or substitutions of the amino acids constituting the peptide may be made. Each of the aforementioned antigens is preferably used in the substantially pure state.
The procedures of production of norovirus VLPs in insect cell culture have been previously disclosed in U.S. Pat. No. 6,942,865, which is incorporated herein by reference in its entirety. Briefly, a cDNA from the 3′ end of the genome containing the viral capsid gene (ORF2) and a minor structural gene (ORF3) were cloned. The recombinant baculoviruses carrying the viral capsid genes were constructed from the cloned cDNAs. Norovirus VLPs were produced in Sf9 or H5 insect cell cultures.
Adjuvants
The invention further provides a composition comprising adjuvants for use with the Norovirus antigen. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as Bordatella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Pifco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; and Quil A.
Suitable adjuvants also include, but are not limited to, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, and liposomes. Preferably, the adjuvants are bacterially-derived exotoxins. Also preferred are adjuvants which stimulate a Th1 type response such as 3DMPL or QS21.
Monophosphoryl Lipid A (MPL), a non-toxic derivative of lipid A from Salmonella, is a potent TLR-4 agonist that has been developed as a vaccine adjuvant (Evans et al. 2003). In preclinical murine studies intranasal MPL has been shown to enhance secretory, as well as systemic, humoral responses (Baldridge et al. 2000; Yang et al. 2002). It has also been proven to be safe and effective as a vaccine adjuvant in clinical studies of greater than 120,000 patients (Baldrick et al., 2002; 2004). MPL stimulates the induction of innate immunity through the TLR-4 receptor and is thus capable of eliciting nonspecific immune responses against a wide range of infectious pathogens, including both gram negative and gram positive bacteria, viruses, and parasites (Baldrick et al. 2004; Persing et al. 2002). Inclusion of MPL in intranasal formulations should provide rapid induction of innate responses, eliciting nonspecific immune responses from viral challenge while enhancing the specific responses generated by the antigenic components of the vaccine.
Accordingly, in one embodiment, the present invention provides a composition comprising monophosphoryl lipid A (MPL®) or 3 De-O-acylated monophosphoryl lipid A (3D-MPL®) as an enhancer of adaptive and innate immunity. Chemically 3D-MPL® is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA), which is incorporated herein by reference. In another embodiment, the present invention provides a composition comprising synthetic lipid A, lipid A mimetics or analogs, such as BioMira's PET Lipid A, or synthetic derivatives designed to function like TLR-4 agonists.
The term “effective adjuvant amount” or “effective amount of adjuvant” will be well understood by those skilled in the art, and includes an amount of one or more adjuvants which is capable of stimulating the immune response to an administered antigen, i.e., an amount that increases the immune response of an administered antigen composition, as measured in terms of the IgA levels in the nasal washings, serum IgG or IgM levels, or B and T-Cell proliferation). Suitably effective increases in immunoglobulin levels include by more than 5%, preferably by more than 25%, and in particular by more than 50%, as compared to the same antigen composition without any adjuvant.
Delivery Agent
The invention also provides a composition comprising a delivery agent which functions to enhance antigen uptake, provide a depot effect, or increase antigen retention time at the site of delivery (e.g., delay expulsion of the antigen). Such a delivery agent may be a bioadhesive agent. In particular, the bioadhesive may be a mucoadhesive agent such as chitosan, a chitosan salt, or chitosan base (e.g. chitosan glutamate).
Chitosan, a positively charged linear polysaccharide derived from chitin in the shells of crustaceans, is a bioadhesive for epithelial cells and their overlaying mucus layer. Formulation of antigens with chitosan increases their contact time with the nasal membrane, thus increasing uptake by virtue of a depot effect (Illum et al. 2001; 2003; Davis et al. 1999; Bacon et al. 2000; van der Lubben et al. 2001; 2001; Lim et al. 2001). Chitosan has been tested as a nasal delivery system for several vaccines, including influenza, pertussis and diphtheria, in both animal models and humans (Illum et al. 2001; 2003; Bacon et al. 2000; Jabbal-Gill et al. 1998; Mills et al. 2003; McNeela et al. 2004). In these trials, chitosan was shown to enhance systemic immune responses to levels equivalent to parenteral vaccination. In addition, significant antigen-specific IgA levels were also measured in mucosal secretions. Thus, chitosan can greatly enhance a nasal vaccine's effectiveness. Moreover, due to its physical characteristics, chitosan is particularly well suited to intranasal vaccines formulated as powders (van der Lubben et al. 2001; Mikszta et al. 2005; Huang et al. 2004).
Accordingly, in one embodiment, the present invention provides an antigenic or vaccine composition adapted for intranasal administration, wherein the composition includes antigen and an effective amount of adjuvant. In preferred embodiments, the invention provides an antigenic or vaccine composition comprising Norovirus antigen such as Norovirus VLP, in combination with at least one delivery agent, such as chitosan, and at least one adjuvant, such as MPL®, CPGs, imiquimod, gardiquimod, or synthetic lipid A or lipid A mimetics or analogs.
The molecular weight of the chitosan may be between 10 kDa and 800 kDa, preferably between 100 kDa and 700 kDa and more preferably between 200 kDa and 600 kDa. The concentration of chitosan in the composition will typically be up to about 80% (w/w), for example, 5%, 10%, 30%, 50%, 70% or 80%. The chitosan is one which is preferably at least 75% deacetylated, for example 80-90%, more preferably 82-88% deacetylated, particular examples being 83%, 84%, 85%, 86% and 87% deacetylation.
Vaccine and Antigenic Formulations
The compositions of the invention can be formulated for administration as vaccines or antigenic formulations. As used herein, the term “vaccine” refers to a formulation which contains Norovirus VLPs or other Norovirus antigens of the present invention as described above, which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity to prevent and/or ameliorate an infection and/or to reduce at least one symptom of an infection and/or to enhance the efficacy of another dose of VLPs or antigen. As used herein, the term “antigenic formulation” or “antigenic composition” refers to a preparation which, when administered to a vertebrate, e.g. a mammal, will induce an immune response. As used herein, the term “immune response” refers to both the humoral immune response and the cell-mediated immune response. The humoral immune response involves the stimulation of the production of antibodies by B lymphocytes that, for example, neutralize infectious agents, block infectious agents from entering cells, block replication of said infectious agents, and/or protect host cells from infection and destruction. The cell-mediated immune response refers to an immune response that is mediated by T-lymphocytes and/or other cells, such as macrophages, against an infectious agent, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates infection or reduces at least one symptom thereof. In particular, “protective immunity” or “protective immune response” refers to immunity or eliciting an immune response against an infectious agent, which is exhibited by a vertebrate (e.g., a human), that prevents or ameliorates an infection or reduces at least one symptom thereof. Specifically, induction of a protective immune response from administration of the vaccine is evident by elimination or reduction of the presence of one or more symptoms of gastroenteritis or a reduction in the duration or severity of such symptoms. Clinical symptoms of gastroenteritis from Norovirus include nausea, diarrhea, loose stool, vomiting, fever, and general malaise. A protective immune response that reduces or eliminates disease symptoms will reduce or stop the spread of a Norovirus outbreak in a population. Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995) Plenum Press New York). The compositions of the present invention can be formulated, for example, for delivery to one or more of the oral, gastro-intestinal, and respiratory (e.g. nasal) mucosa.
Where the composition is intended for delivery to the respiratory (e.g. nasal) mucosa, typically it is formulated as an aqueous solution for administration as an aerosol or nasal drops, or alternatively, as a dry powder, e.g. for rapid deposition within the nasal passage. Compositions for administration as nasal drops may contain one or more excipients of the type usually included in such compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents, and the like. Viscosity agents can be microcrystalline cellulose, chitosan, starches, polysaccharides, and the like. Compositions for administration as dry powder may also contain one or more excipients usually included in such compositions, for example, mucoadhesive agents, bulking agents, and agents to deliver appropriate powder flow and size characteristics. Bulking and powder flow and size agents may include mannitol, sucrose, trehalose, and xylitol.
In one embodiment, the Norovirus vaccine or antigenic formulation of the present invention contains one or more Norovirus genogroup antigen(s) as the immunogen, an adjuvant such as MPL®, a biopolymer such as chitosan to promote adhesion to mucosal surfaces, and bulking agents such as mannitol and sucrose. For example, the Norovirus vaccine may be formulated as 10 mg of a dry powder containing one or more Norovirus genogroup antigen(s) (e.g., Norwalk virus, Houston virus, Snow Mountain virus), MPL® adjuvant, chitosan mucoadhesive, and mannitol and sucrose as bulking agents and to provide proper flow characteristics. The formulation may comprise about 7.0 mg (25 to 90% w/w range) chitosan, about 1.5 mg mannitol (0 to 50% w/w range), about 1.5 mg sucrose (0 to 50% w/w range), about 25 μg MPL® (0.1 to 5% w/w range), and about 100 μg Norovirus antigen (0.05 to 5% w/w range).
Norovirus antigen may be present in a concentration of from about 0.01% (w/w) to about 80% (w/w). In one embodiment, Norovirus antigens can be formulated at dosages of about 5 μg, about 15 μg, about 25 μg, about 50 μg, about 100 μg, about 200 μg, about 500 μg, and about 1 mg per 10 mg dry powder formulation (0.05, 0.15, 0.25, 0.5, 1.0, 2.0, 5.0, and 10.0% w/w) for administration into both nostrils (10 mg per nostril) or about 10 μg, about 30 μg, about 50 μg, about 100 μg, about 200 μg, about 400 μg, about 1 mg, and about 2 mgs (0.1, 0.3, 0.5, 1.0, 2.0, 4.0, 10.0 and 20.0% w/w) per 20 mg dry powder formulation for administration into one nostril. The formulation may be given in one or both nostrils during each administration. There may be a booster administration 1 to 12 weeks after the first administration to improve the immune response. The content of each Norovirus antigen in the vaccine and antigenic formulations may be in the range of 1 μg to 100 mg, preferably in the range 1-1000 μg, more preferably 5-500 μg, most typically in the range 10-200 μg. Total Norovirus antigen administered at each dose can be either about 10 μg, about 30 μg, about 200 μg, about 250 μg, about 400 μg, about 500 μg, or about 1000 μg. The total vaccine dose can be administered into one nostril or can be split in half for administration to both nostrils. Dry powder characteristics are such that less than 10% of the particles are less than 10 μm in diameter. Mean particle sizes range from 10 to 500 μm in diameter.
In another embodiment, the antigenic and vaccine compositions can be formulated as a liquid for subsequent administration to a subject. A liquid formulation intended for intranasal administration would comprise Norovirus genogroup antigen(s), adjuvant, and a delivery agent such as chitosan. Liquid formulations for intramuscular (i.m.) administration would comprise Norovirus genogroup antigen(s), adjuvant, and a buffer, without a delivery agent (e.g., chitosan).
Preferably the antigenic and vaccine compositions hereinbefore described are lyophilized and stored anhydrous until they are ready to be used, at which point they are reconstituted with diluent. Alternatively, different components of the composition may be stored separately in a kit (any or all components being lyophilized). The components may remain in lyophilized form for dry formulation or be reconstituted for liquid formulations, and either mixed prior to use or administered separately to the patient. For dry powder administration, the vaccine or antigenic formulation may be preloaded into an intranasal delivery device and stored until use. Preferably, such intranasal delivery device would protect and ensure the stability of its contents.
The lyophilization of antigenic formulations and vaccines is well known in the art. Typically the liquid antigen is freeze dried in the presence of agents to protect the antigen during the lyophilization process and to yield a cake with desirable powder characteristics. Sugars such as sucrose, mannitol, trehalose, or lactose (present at an initial concentration of 10-200 mg/mL) are commonly used for cryoprotection of protein antigens and to yield lyophilized cake with desirable powder characteristics. Lyophilizing the compositions theoretically results in a more stable composition. While the goal of most formulation processes is to minimize protein aggregation and degradation, the inventors have discovered that the presence of aggregated antigen enhances the immune response to Norovirus VLPs (see Examples 3 and 4). Therefore, the inventors have developed methods by which the percentage of aggregation of the antigen can be controlled during the lyophilization process to produce an optimal ratio of aggregated antigen to intact antigen to induce a maximal immune response.
Thus, the invention also encompasses a method of making Norovirus antigen formulations comprising (a) preparing a pre-lyophilization solution comprising Norovirus antigen, sucrose, and chitosan, wherein the ratios of sucrose to chitosan are from about 0:1 to about 10:1; (b) freezing the solution with liquid nitrogen; and (c) lyophilizing the frozen solution at ambient temperature for 48-72 hours, wherein the final lyophilized product contains a percentage of said Norovirus antigen in aggregated form. In one embodiment, the pre-lyophilization solution further comprises a bulking agent. In another embodiment, said bulking agent is mannitol.
Appropriate ratios of sucrose and chitosan to yield desired percentages of aggregation can be determined by the following guidelines. A pre-lyophilization mixture containing a weight ratio of sucrose to chitosan in a range from about 2.5:1 to about 10:1 will yield greater than 95% intact Norovirus antigen post-lyophilization (i.e. less than 5% aggregated antigen; see Example 13). A range of sucrose to chitosan weight ratios of about 1:1 to about 2.1:1 will yield about 50% to about 90% intact Norovirus antigen (i.e. about 10% to about 50% aggregated antigen). Weight ratios of 0:1 sucrose to chitosan will produce less than 30% of intact Norovirus antigen. Omission of both sucrose and chitosan will produce less than 5% intact antigen (i.e. greater than 95% aggregated antigen). Using these guidelines, the skilled artisan could adjust the sucrose to chitosan weight ratios in the pre-lyophilization mixture to obtain the desired amount of aggregation necessary to produce an optimal immune response.
In addition, the inclusion of sucrose and chitosan to the pre-lyophilization solution promotes the stability of the intact Norovirus antigen over time. The ratio of aggregated antigen/intact antigen in the formulation does not increase when stored as a dry powder for a period of about 12 months or greater (see Example 10). Thus, this lyophilization procedure ensures stable formulations with predictable and controllable ratios of aggregated to intact Norovirus antigen.
Methods of Stimulating an Immune Response
The amount of antigen in each antigenic or vaccine formulation dose is selected as an amount which induces a robust immune response without significant, adverse side effects. Such amount will vary depending upon which specific antigen(s) is employed, route of administration, and adjuvants used. In general, the dose administered to a patient, in the context of the present invention should be sufficient to effect a protective immune response in the patient over time, or to induce the production of antigen-specific antibodies. Thus, the composition is administered to a patient in an amount sufficient to elicit an immune response to the specific antigens and/or to prevent, alleviate, reduce, or cure symptoms and/or complications from the disease or infection, and thus reduce or stop the spread of a Norovirus outbreak in a population. An amount adequate to accomplish this is defined as a “therapeutically effective dose.”
For a substantially pure form of the Norovirus antigen, it is expected that each dose will comprise about 1 μg to 10 mg, preferably about 15-500 μg for each Norovirus antigen in the formulation. In a typical immunization regime employing the antigenic preparations of the present invention, the formulations may be administered in several doses (e.g. 1-4), each dose containing 1-1000 μg of each antigen. The dose will be determined by the immunological activity the composition produced and the condition of the patient, as well as the body weight or surface areas of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular composition in a particular patient.
The antigenic and vaccine formulations of the present invention may be administered via a non-mucosal or mucosal route. These administrations may include in vivo administration via parenteral injection (e.g. intravenous, subcutaneous, and intramuscular) or other traditional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or intranasal routes or directly into a specific tissue. Alternatively, the vaccines of the invention may be administered by any of a variety of routes such as oral, topical, subcutaneous, mucosal, intravenous, intramuscular, intranasal, sublingual, transcutaneous, subdermal, intradermal and via suppository. Administration may be accomplished simply by direct administration using a needle, catheter or related device, at a single time point or at multiple time points.
In a preferred embodiment, the antigenic and vaccine formulations of the present invention are administered by the intranasal route. Immunization via the mucosal surfaces offers numerous potential advantages over other routes of immunization. The most obvious benefits are 1) mucosal immunization does not require needles or highly-trained personnel for administration, and 2) immune responses are raised at the site(s) of pathogen entry, as well as systemically (Isaka et al. 1999; Kozlowski et al. 1997; Mestecky et al. 1997; Wu et al. 1997).
In a further aspect, the invention provides a method of eliciting an IgA mucosal immune response and an IgG systemic immune response by administering (preferably intranasally) to a mucosal surface of the patient an antigenic or vaccine composition comprising one or more Norovirus antigens, at least one effective adjuvant and/or at least one delivery agent.
The present invention also contemplates the provision of means for dispensing intranasal formulations of Norovirus antigens hereinbefore defined, and at least one adjuvant or at least one delivery agent as hereinbefore defined. A dispensing device may, for example, take the form of an aerosol delivery system, and may be arranged to dispense only a single dose, or a multiplicity of doses. Such a device would deliver a metered dose of the vaccine or antigenic formulation to the nasal passage. Other examples of appropriate devices include, but are not limited to, droppers, swabs, aerosolizers, insufflators (e.g. Valois Monopowder Nasal Administration Device, single dose Bespak UniDose DP dry powder intranasal delivery device), nebulizers, and inhalers. The devices may deliver the antigenic or vaccine formulation by passive means requiring the subject to inhale the formulation into the nasal cavity. Alternatively, the device may actively deliver the formulation by pumping or spraying a dose into the nasal cavity. The antigenic formulation or vaccine may be delivered into one or both nostrils by one or more such devices. Administration could include two devices per subject (one device per nostril). Actual dose of active ingredient (Norovirus antigen) may be about 5-1000 μg. In a preferred embodiment, the antigenic or vaccine formulation is administered to the nasal mucosa by rapid deposition within the nasal passage from a device containing the formulation held close to the nasal passageway.
The invention also provides a method of generating antibodies to one or more Norovirus antigens, said method comprising administration of a vaccine or antigenic formulation of the invention as described above to a subject. These antibodies can be isolated and purified by routine methods in the art. The isolated antibodies specific for Norovirus antigens can be used in the development of diagnostic immunological assays. These assays could be employed to detect a Norovirus in clinical samples and identify the particular virus causing the infection (e.g. Norwalk, Houston, Snow Mountain, etc.). Alternatively, the isolated antibodies can be administered to subjects susceptible to Norovirus infection to confer passive or short-term immunity.
The invention provides methods for eliciting protective immunity to a Norovirus infection in a subject comprising administering a vaccine to the subject, wherein said vaccine comprises Norovirus VLPs and at least one adjuvant. In one embodiment, the subject is a human and the vaccine confers protection from one or more symptoms of Norovirus infection. Although others have reported methods of inducing an immune response with Norovirus antigens (see U.S. Patent Application Publication No. US 2007/0207526), no one has demonstrated the induction of a protective immune response in humans. Unlike several vaccines currently licensed in the U.S. where effectiveness of the vaccine correlates with serum antibodies, studies have shown that markers of an immune response, such as increased titers of serum antibodies against Norwalk virus, are not associated with protective immunity in humans (Johnson et al. (1990) J. Infectious Diseases 161: 18-21). Moreover, another study examining Norwalk viral challenge in humans indicated that susceptibility to Norwalk infection was multifactorial and included factors such as secretor status and memory mucosal immune response (Lindesmith et al. (2003) Nature Medicine 9: 548-553). Because Norovirus is not able to be cultured in vitro, no viral neutralization assays are currently available. A functional assay which serves as a substitute for the neutralization assay is the hemagglutination inhibition (HAI) assay. HAI measures the ability of Norovirus vaccine-induced antibodies to inhibit the agglutination of antigen-coated red blood cells by Norovirus VLPs because Norovirus VLPs bind to red blood cell antigens. In this assay, a fixed amount of Norovirus VLPs is mixed with a fixed amount of red blood cells and serum from immunized subjects. If the serum sample contains functional antibodies, the antibodies will compete with the VLPs for binding to the red blood cells, thereby inhibiting the agglutination of the red blood cells.
Similar findings have been observed with vaccines for other viruses, such as rotavirus. For rotavirus vaccines, there is controversy over whether serum antibodies are directly involved in protection or merely reflect recent infection (Jiang, 2002; Franco, 2006). Defining such correlates of protection is particularly difficult in the context of diarrheal diseases such as rotavirus or norovirus, where preclinical studies inferring protection may be multifaceted with contributions from mucosal immunity (such as intestinal IgA), cytokine elaboration, and cell mediated immunity. The difficulty in measuring such immune responses during clinical development, and the lack of correlation to serum antibody measurements, requires that the effectiveness of a vaccine for these types of viruses can only be demonstrated through human clinical challenge experiments.
As mentioned above, administration of the vaccine of the present invention prevents and/or reduces at least one symptom of Norovirus infection. Symptoms of Norovirus infection are well known in the art and include nausea, vomiting, diarrhea, and stomach cramping. Additionally, a patient with a Norovirus infection may have a low-grade fever, headache, chills, muscle aches, and fatigue. The invention also encompasses a method of inducing a protective immune response in a subject experiencing a Norovirus infection by administering to the subject a vaccine formulation of the invention such that at least one symptom associated with the Norovirus infection is alleviated and/or reduced. A reduction in a symptom may be determined subjectively or objectively, e.g., self assessment by a subject, by a clinician's assessment or by conducting an appropriate assay or measurement (e.g. body temperature), including, e.g., a quality of life assessment, a slowed progression of a Norovirus infection or additional symptoms, a reduced severity of Norovirus symptoms or suitable assays (e.g. antibody titer, RT-PCR antigen detection, and/or B-cell or T-cell activation assay). An effective response may also be determined by directly measuring (e.g., RT-PCR) virus load in stool samples, which reflects the amount of virus shed from the intestines). The objective assessment comprises both animal and human assessments.
Stability and efficacy in animal models of the vaccine and antigenic formulations disclosed herein are reported in International Application No. PCT/US07/79929, which is herein incorporated by reference in its entirety.
EXAMPLES
The invention will now be illustrated in greater detail by reference to the specific embodiments described in the following examples. The examples are intended to be purely illustrative of the invention and are not intended to limit its scope in any way.
Example 1. GLP Toxicity Study of Norovirus Vaccine Formulations in Rabbits
The purpose of this study was to evaluate the potential toxicity of a Norwalk virus-virus-like particle (NV-VLP) vaccine following three intranasal doses in rabbits. The NV-VLP vaccine contained (per 10 mg dry powder) 25 μg of a Genogroup I VLP, 25 μg MPL, 7 mg chitosan glutamate, 1.475 mg mannitol, and 1.475 mg sucrose. The study was conducted over an eight week period. The persistence, reversibility, or delayed onset of any effects were assessed after a four-week, no-treatment recovery interval. Sixty New Zealand White rabbits (30/sex) were randomly assigned to three groups (10 rabbits/sex/group). Group 1 animals were not dosed (i.e. naïve). Group 2 animals were administered 10 mg/nostril (20 mg total) of placebo (i.e. adjuvant/excipient: MPL, chitosan, sucrose, and mannitol). Group 3 animals were administered 10 mg/nostril (20 mg total) of NV-VLP vaccine, which represented 25 μg of antigen per nostril (50 μg total). Animals in groups 2 and 3 were dosed on study day (SD) 1, 22, and 43 by intranasal administration using the Bespak Unidose intranasal dry powder device. Animals (5/group/sex) were subjected to a full gross necropsy on SD 46 and 74. Parameters evaluated during the study included mortality, clinical and cageside observations, body weights, body weight changes, food consumption, body temperature, ophthalmology examinations, clinical pathology (clinical chemistry, hematology, and urinalysis), gross pathology, organ weight data, and histopathology. The study outline is summarized in Table 1. The conclusions of the study are summarized in Table 2.
TABLE 1
Study Parameters for GLP Toxicity Study of
Norwalk Vaccine Formulation
SPF New Zealand White Rabbits
Species
with ear tag IDs
No. Animals/Sex/
10 males and 10 females/group
Dose Group
Total Number of
60
Animals in Study
Group 1
Non-treated controls
Group 2
Adjuvant/Excipient
Group 3
1x maximum human dose
VLPs in Adjuvant/Excipient
TABLE 2
Safety and Toxicology Findings for Norwalk Vaccine Formulation
Observations
No treatment related effects on mortality,
clinical or cageside observations.
Body weight and
No adverse effect on body weights or body
body weight changes
weight changes.
Food consumption
No treatment related adverse effect on
food consumption.
Body temperature
No treatment related adverse effect on
body temperature.
Opthamology
No ocular lesions were noted in any animal
over the course of the study.
Clinical Pathology
Polyclonal activation of B lymphocyte
populations in rabbits receiving NV-VLP
Vaccine or Adjuvant/Excipient was noted
days 3-76.
Absolute monocyte values were elevated
in rabbits receiving NV-VLP Vaccine or
Adjuvant/Excipient on days 3-46.
There were no treatment effects on selected
urinalysis parameters.
Gross Pathology
No treatment related observations.
Organ weights
No adverse effects on absolute or relative
organ weights.
Histopathology
Varying degrees of inflammatory infiltrates,
either within the lamina propria of nasal
turbinates or free within the nasal passages,
and/or hemorrhage within the nasal passages of
rabbits receiving NV-VLP Vaccine or
Adjuvant/Excipient. The observed lesions are
those that would be expected in an immunologic
reaction. Lesions in both groups were limited in
nature and resolved completely by SD 74.
Cage side observations revealed no significant findings. Hematological measures (increases in globulin and total protein) were typical of B lymphocyte polyclonal activation and may be attributable to adjuvant effects. Histopathology findings consisted of varying degrees of inflammatory infiltrates, either within the lamina propria of nasal turbinates or free within the nasal passages, and/or mild hemorrhage in the nasal passages of rabbits in both groups. The observed lesions would be expected in an immunologic reaction. Lesions in both groups were limited in nature and resolved completely by study day 74.
Serological samples analyzed by ELISA for NV-VLP specific IgG showed measurable anti-NV-VLP titers in 30% of the immunized animals on day 10 following a single dose (see FIG. 1). Boost treatments on days 22 and 43 increased both the number of seroconverted animals and levels of product-specific antibodies, and by day 73, 90% of the immunized animals seroconverted. None of the naïve or matrix treated controls had quantifiable levels of NV-VLP specific antibodies (data not shown).
The immune response was further characterized by evaluating memory B-cell responses in an additional set of rabbits immunized intranasally with the same formulation on days 1, 15 and 29. Memory B-cell responses were measured as described in International Application No. PCT/US07/79929, which is herein incorporated by reference in its entirety. Tissues collected 156 days after the last boost showed the presence of NV-VLP-specific memory B-cells in the peripheral blood, the spleen, and most notably, in the mesenteric lymph nodes. The antigen-specific memory B-cells in the mesenteric lymph nodes were IgA positive. Additionally, NV-VLP-specific antibody-secreting long-lived plasma cells were present in the bone marrow.
Example 2. Dose Escalation Safety Study of Norwalk Vaccine Formulation in Humans
A double-blind, controlled, dose-escalation phase 1 study of the safety and immunogenicity of a Norovirus genogroup 1 vaccine was conducted. The vaccine consisted of lyophilized Norwalk virus-like particles (VLPs) in a dry powder matrix designed for intranasal administration. Vaccinees included healthy adult volunteers who were H type 1 antigen secretors. The rationale for enrollment of H type 1 antigen secretors is that H type 1 antigen secretors are susceptible to Norwalk viral infections while non-secretors are resistant. As a control, 2 additional volunteers at each dosage level received matrix alone. The dry powder matrix included 25 μg MPL® adjuvant, 7 mg chitosan, 1.5 mg mannitol, and 1.5 mg sucrose. Volunteers were dosed on days 0 and 21 and were required to keep a 7-day diary of symptoms after each dose. Blood for serology, antibody secreting cells (ASC), and stool and saliva samples for mucosal antibody evaluation were collected.
The components of the Norwalk VLP vaccine are listed in Table 3. The vaccine is packaged in an intranasal delivery device. Single administrations of Norwalk VLP Vaccine were packaged in a single dose Bespak (Milton Keynes, UK) UniDose DP dry powder intranasal delivery device. Each device delivered 10 mg of the dry powder vaccine formulation. Each dose of vaccine consisted of two delivery devices, one in each nostril. The total vaccine dose was 20 mg of dry power. The formulation of Adjuvant/Excipient is the same as the Norwalk VLP Vaccine except that no Norwalk VLP antigen is included in the formulation. The formulation of the Adjuvant/Excipient (also referred to as dry powder matrix) is summarized in Table 4.
TABLE 3
Norwalk VLP Vaccine Composition
Quantity per
Molecular
10 mg dry
% of Final
Component
class
powder
Formulation
Norwalk VLP
Recombinant
2.5, 7.5, 25,
0.025, 0.075, 0.25, or
protein
or 50
μg
0.50%
Monophosphoryl
Phospholipid
25
μg
0.25%
Lipid A
Chitosan
Polysaccharide
7.0
mg
70%
Mannitol
Sugar
1.5
mg
15%*
Sucrose
Sugar
1.5
mg
15%
TABLE 4
Adjuvant/Excipient (dry powder matrix)
Quantity per
10 mg dry
% of Final
Component
Molecular class
powder
Formulation
Monophosphoryl
Phospholipid
25
μg
0.25%
Lipid A
Chitosan
Polysaccharide
7.0
mg
70%
Mannitol
Sugar
1.5
mg
15%
Sucrose
Sugar
1.5
mg
15%
Specifically, the dose escalation of the vaccine was conducted as follows: After appropriate screening for good health, a group of 3 volunteers was randomized to receive either 5 μg Norwalk VLP Vaccine plus dry powder matrix (n=2) or dry powder matrix alone (n=1) by the intranasal route. These 3 volunteers were followed for safety for 21 days and their safety data reviewed by the Independent Safety Monitor (ISM). Upon approval of the ISM, these individuals received their second dose of Vaccine or matrix on day 21, and 4 additional volunteers were randomized to receive either 5 μg VLP protein plus dry powder matrix (n=3) or matrix alone (n=1) by the intranasal route. The ISM reviewed the safety data from this second group and upon approval of the ISM, the second intranasal dose was given 21 days after the first dose. Volunteers kept a 7-day diary of symptoms after each dose. After the ISM determined that escalation to the next higher dose was acceptable, another group of 7 volunteers was randomized to receive either Norwalk VLP Vaccine containing 15 μg VLP protein (n=5) or dry powder matrix alone (n=2) by the intranasal route at day 0 and day 21. Again, 7-day symptom diaries were recorded and reviewed by the ISM before the second dose at day 21. Finally, after review of the safety data from the first two dosage cohorts, the ISM determined that dose escalation was acceptable and a final group of 7 volunteers were randomized to receive either Norwalk VLP Vaccine containing 50 μg VLP protein (n=5) or dry powder matrix alone (n=2) by the intranasal route on day 0 and day 21. Seven-day symptom diaries and other safety data were again reviewed by the ISM before the second dose at day 21.
The volunteers kept a daily diary of symptoms (including local symptoms such as: nasal discharge, nasal pain/discomfort, nasal congestion, runny nose, nasal itching, nose bleed, headache and systemic symptoms such as: daily oral temperature, myalgia, nausea, vomiting, abdominal cramps, diarrhea, and loss of appetite) for 7 days after receiving Norwalk VLP Vaccine or dry powder matrix alone. Interim medical histories were obtained at each follow-up visit (days 7±1, 21±2, 28±2, 56±2 and 180±14); volunteers were queried about interim illness, medications, and doctor's visits. Volunteers were asked to report all serious or severe adverse events including events that were not solicited during follow up visits. Volunteers had CBC and serum creatinine, glucose, AST, and ALT assessed on days 7 and 28 (7 days after each immunization) and, if abnormal, the abnormal laboratory test was followed until the test became normal or stabilized.
The blinded data indicated that of the volunteers that received the low dose (n=5) or matrix (n=2), 4 of 7 reported some or all of the following: nasal discharge, nasal pain, stuffiness, itching, sneezing, headache, and/or sore throat in the first 24 hours after vaccination. One volunteer reported a minor nosebleed on each of days 1 and 6. Of the volunteers that received the middle dose (n=5) or matrix (n=2), 5 of 7 reported mild nasal discharge, stuffiness, itching, sneezing, and/or headache in the first 24 hours. Symptoms generally resolved in the first 72 hours, but stuffiness persisted to day 7 in one volunteer. A summary of the findings on the unblinded data is presented in Table 5 below, which also includes adverse events reported in the high dose. These findings indicate that intranasal Norovirus VLP vaccine is associated with local, usually mild, short-lived symptoms that appeared to be independent of VLP concentration. No differences were seen between the adjuvant/excipient (or matrix) control group and the Norwalk VLP vaccine groups for adverse events, hematology, blood chemistry and/or physical examination results.
TABLE 5
Number of Volunteers with Adverse Events to
Norwalk VLP Vaccine or Adjuvant/Excipient
Adjuvant/
Low
Mid
High
Excipient
Dose
Dose
Dose
Reported Adverse Events
(N = 6)
(N = 5)
(N = 5)
(N = 5)*
Nose and Throat
Nasal Stuffiness
4
2
3
1
Nasal Itching
3
3
2
2
Nasal Discharge
3
3
4
3
Nasal Pain
—
2
1
2
Sneezing
3
2
1
3
Nose Bleed
—
1
1
—
Sore Throat/URI
—
1
—
1
Itchy Sore Throat
—
1
—
—
Burning in Nose/Throat
—
1
—
1
Chest
Cough
2
—
—
—
Chest discomfort
—
—
—
1
Systemic
Headache
2
2
1
1
Malaise
3
2
—
1
Nausea
—
1
—
1
Abdominal Cramp
1
—
—
1
Laboratory
ALT/AST
—
1
—
—
AST
1
—
—
—
ALT
—
—
—
1
Alk Phos
—
—
—
1
Gastrointestinal
Diarrhea
—
1
1
Loss of appetite
1
—
1
—
No Adverse Events Reported
—
—
1
2
*One subject in cohort 3 did not receive the second dose
Blood was collected before immunization and on days 7±1, 21±2, 28±2, 56±2, and 180±14 to measure serum antibodies to Norwalk VLP Vaccine by enzyme-linked immunosorbent assays (ELISA). Before and on day 7 after administration of each dose of Vaccine or dry powder matrix alone peripheral blood lymphocytes were collected to detect antibody secreting cells by ELISPOT assay. Before and on days 21±2, 56±2 and 180±14 after vaccination, whole blood was obtained to separate cells and freeze for future studies of cell mediated immunity, including cytokine production in response to Norwalk VLP antigen, and lymphoproliferation. Whole stool samples were collected before immunization and on days 7±1, 21±2, 28±2, 56±2, and day 180±14 for anti-Norwalk VLP sIgA screening. Saliva was collected with a commercially available device (Salivette, Sarstedt, Newton, N.C.) before immunization and on days 7±1, 21±2, 28±2, 56±2, and if positive for mucosal antibodies at day 56, a day 180±14 sample was collected and screened for anti-Norwalk VLP sIgA. Finally blood from volunteers receiving the highest dose of Norwalk VLPs (50 μg, third cohort described above) was screened for memory B-cells on days 0, 21, 56 and 180.
The following methods were used to analyze the blood, stool, and saliva samples collected from immunized individuals or individuals receiving the dry powder matrix alone:
A. Serum Antibody Measurements by ELISA
Twenty mL of blood were collected before and at multiple time points after vaccination for measurement of antibodies to Norwalk virus by ELISA, using purified recombinant Norwalk VLPs as target antigen to screen the coded specimens. Briefly, Norwalk VLPs in carbonate coating buffer pH 9.6 were used to coat microtiter plates. Coated plates were washed, blocked, and incubated with serial two-fold dilutions of test serum followed by washing and incubation with enzyme-conjugated secondary antibody reagents specific for human IgG, IgM, and IgA. Appropriate substrate solutions were added, color developed, plates read, and the IgG, IgM, and IgA endpoint titers were determined in comparison to a reference standard curve for each antibody class. A positive response was defined as a 4-fold rise in titer after vaccination. The serum titers at day 56 (35 days after the second immunization) for each of the vaccine doses are shown in FIG. 2. The results show a dose-dependent increase in serum titers for IgG and IgA. A significant serum titer for both IgG and IgA was observed in volunteers receiving the vaccine containing 50 μg of Norovirus antigen.
B. Antibody Secreting Cell Assays
PBMCs were collected from heparinized blood (30 mL for cohorts 1 and 2, 25 mL for cohort 3) for ASC assays to detect cells secreting antibodies to Norwalk VLPs. These assays were performed on days 0, 7±1, 21±2, and 28±2 after administration of Norwalk VLP Vaccine or dry powder matrix alone. The response rate and mean number of ASC per 106 PBMC at each time point for each dosage were described. A positive response was defined as a post-vaccination ASC count per 106 PBMCs that is at least 3 standard deviations (SD) above the mean pre-vaccination count for all subjects (in the log metric) and at least 8 ASC spots, which corresponds to the mean of medium-stimulated negative control wells (2 spots) plus 3 SD as determined in similar assays.
The results of the ASC assays for the 50 μg dose of Norwalk VLPs are depicted in FIG. 3. Circulating IgG and IgA antibody secreting cells were observed seven days after initial and boost vaccinations, suggesting that the vaccine is immunogenic.
C. Measurement of Functional Antibody Response
Serum collected as described in paragraph B, above, was further analyzed to determine the functional properties of the anti-Norwalk virus antibodies. Serial two-fold dilutions of test serum were analyzed with respect to their ability to inhibit hemagglutination of red blood cells by Norwalk VLPs (a functional assay to indicate protective immune responses). A positive response was defined as a 4-fold rise in titer after vaccination. The serum titers and hemagglutination inhibition titers at day 56 (35 days post boost) for five subjects who received the 50 μg dose of the Norwalk VLPs vaccine are shown in Table 6. The results show that seventy five percent (75%) of the individuals who exhibited a seroconversion response as measured by serum IgG titers also developed a functional antibody response capable of blocking the binding receptor on human red blood cells as measured by hemagglutination inhibition.
TABLE 6
Serum IgG and Hemagglutination Inhibition (HAI)
(functional) Titers on Day 0 and Day 35 Post Boost
(35PB) for Five Human Volunteers.
Subject Reference
Day 0
Day 35PB
Serum IgG Titers
A
2,444.6
37,185.9
B
4,462.1
23,508.4
C
7,735.7
13,357.8
D
884.5
4,577.5
E
12,719.0
91,710.8
Hemagglutination Inhibition (HAI) Titers
A
8
256
B
8
256
C
512
512
D
<8
8
E
128
1024
D. Measurement of Norwalk Virus-Specific Memory B-Cells
Heparinized blood was collected from cohort 3 (30 mL days 0 and 21, 50 mL days 56 and 180) to measure memory B cells on days 0, 21, 56 and 180 after vaccination using an ELISpot assay preceded by an in vitro antigen stimulation. A similar assay was successfully used to measure frequency of memory B cells elicited by Norwalk VLP formulations in rabbits (See International Application No. PCT/US07/79929, herein incorporated by reference). Peripheral blood mononuclear cells (5×106 cells/mL, 1 mL/well in 24-well plates) are incubated for 4 days with Norwalk VLP antigen (2-10 μg/mL) to allow for clonal expansion of antigen-specific memory B cells and differentiation into antibody secreting cells. Controls include cells incubated in the same conditions in the absence of antigen and/or cells incubated with an unrelated antigen. Following stimulation, cells are washed, counted and transferred to ELISpot plates coated with Norwalk virus VLP. To determine frequency of virus-specific memory B cells per total Ig-secreting B lymphocytes, expanded B cells are also added to wells coated with anti-human IgG and anti-human IgA antibodies. Bound antibodies are revealed with HRP-labeled anti-human IgG or anti-human IgA followed by True Blue substrate. Conjugates to IgA and IgG subclasses (IgA1, IgA2 and IgG1-4) may also be used to determine antigen-specific subclass responses which may be related with distinct effector mechanisms and locations of immune priming. Spots are counted with an ELISpot reader. The expanded cell populations for each volunteer are examined by flow cytometry to confirm their memory B cell phenotype, i.e. CD19+, CD27+, IgG+, IgM+, CD38+, IgD−.
E. Cellular Immune Responses
Heparinized blood (50 mL cohorts 1 and 2, 25 mL cohort 3) was collected as coded specimens and the peripheral blood mononuclear cells (PBMC) isolated and cryopreserved in liquid nitrogen for possible future evaluation of CMI responses to Norwalk VLP antigen. Assays that may be performed include PBMC proliferative and cytokine responses to Norwalk VLP antigen and can be determined by measuring interferon (IFN)-γ and interleukin (IL)-4 levels according to established techniques.
F. Collections of Stool and Saliva for Anti-Norwalk VLP sIgA
Anti-recombinant Norwalk Virus IgA is measured in stool and saliva samples. Saliva specimens are treated with protease inhibitors (i.e. AEBSF, leupeptin, bestatin, and aprotinin) (Sigma, St. Louis, Mo.), stored at −70° C., and assayed using a modification of a previously described assay (Mills et al. (2003) Infect. Immun. 71: 726-732). Stool is collected on multiple days after vaccination and specimens stored at −70° C. until analysis. The specimens are thawed, and protease inhibitor buffer added to prepare a 10% w/v stool suspension. Stool supernatants are assayed for recombinant Norwalk Virus (rNV)-specific mucosal IgA by ELISA, as described below.
Approximately 2-3 mL of whole saliva was collected before and at multiple time points after vaccination. Saliva was collected by a commercially available device (Salivette, Sarstedt, Newton, N.C.), in which a Salivette swab is chewed or placed under the tongue for 30-45 seconds until saturated with saliva. Saliva was collected from the swab by centrifugation.
G. Measurement of Anti-Norwalk VLP in Stool and Saliva
ELISAs, utilizing plates coated with either anti-human IgA antibody reagents or target rNV VLP antigen coatings, are performed to determine total IgA and to titer the specific anti-VLP IgA responses for each specimen. Total or specific IgA are revealed with HRP-labeled anti-human IgA as described above. An internal total IgA standard curve is included to quantify the IgA content. Response is defined as a 4-fold rise in specific antibody.
Example 3. Safety and Immunogenicity Study of Two Dosages of Intranasal Norwalk VLP Vaccine in Humans
A randomized, double blind study in healthy adults is conducted to compare the safety and immunogenicity of two dosage levels of a Norwalk virus-like particle (VLP) vaccine with adjuvant/excipients and placebo controls (empty device). The vaccine consists of Norwalk virus-like particles (VLPs) in a dry powder matrix designed for intranasal administration as described in Example 2. Vaccinees include healthy adult volunteers who are H type 1 antigen secretors. The human volunteers are randomly assigned to one of four groups and each group receives one of the following treatments: a 50 μg dose of the Norwalk VLP vaccine, a 100 μg dose of the Norwalk VLP vaccine, the adjuvant/excipient, or placebo. Volunteers are dosed on days 0 and 21 and are required to keep a 7-day diary of symptoms after each dose. Blood for serology, antibody secreting cells (ASC), and stool and saliva samples for mucosal antibody evaluation are collected.
The components of the vaccine are listed in Table 3 in Example 2. The vaccine is packaged in an intranasal delivery device. Single administrations of the Norwalk VLP vaccine are packaged in a single dose Bespak (Milton Keynes, UK) UniDose DP dry powder intranasal delivery device. Each device delivers 10 mg of the dry powder vaccine formulation. Each dose of vaccine consists of two delivery devices, one in each nostril. The total vaccine dose is 20 mg of dry power. Therefore, the 50 μg vaccine dose consists of two devices that each deliver 10 mg of dry powder formulation, wherein each 10 mg of dry powder formulation consists of 25 μg of Norwalk VLP, 25 μg MPL® adjuvant, 7 mg chitosan, 1.5 mg mannitol, and 1.5 mg sucrose. Similarly, the 100 μg vaccine dose consists of two devices that each deliver 10 mg of dry powder formulation, wherein each 10 mg of dry powder formulation consists of 50 μg of Norwalk VLP, 25 μg MPL® adjuvant, 7 mg chitosan, 1.5 mg mannitol, and 1.5 mg sucrose. The formulation of Adjuvant/Excipient is the same as the Norwalk VLP vaccine except that no Norwalk VLP antigen is included in the formulation. The formulation of the Adjuvant/Excipient (also referred to as dry powder matrix) is summarized in Table 4 in Example 2. The placebo group receives two empty devices.
The volunteers keep a daily diary of symptoms (including local symptoms such as: nasal discharge, nasal pain/discomfort, nasal congestion, runny nose, nasal itching, nose bleed, headache and systemic symptoms such as: daily oral temperature, myalgia, nausea, vomiting, abdominal cramps, diarrhea, and loss of appetite) for 7 days after receiving either one of two doses of the Norwalk VLP vaccine, dry powder matrix alone, or the placebo. Interim medical histories are obtained at each follow-up visit (days 7+1, 21+2, 28+2, 56+2 and 180+14); volunteers are queried about interim illness, medications, and doctor's visits. Volunteers are asked to report all serious or severe adverse events including events that are not solicited during follow up visits. Volunteers have CBC and serum creatinine, glucose, AST, and ALT assessed on days 7 and 28 (7 days after each immunization) and, if abnormal, the abnormal laboratory test is followed until the test becomes normal or stabilizes.
Blood is collected before immunization and on days 7+1, 21+2, 28+2, 56+2, and 180+14 to measure serum antibodies to the Norwalk VLP vaccine by enzyme-linked immunosorbent assays (ELISA). Before and on day 7 after administration of each dose of vaccine, dry powder matrix alone, or placebo, peripheral blood lymphocytes are collected to detect antibody secreting cells by ELISPOT assay. Before and on days 21+2, 56+2 and 180+14 after vaccination, whole blood is obtained to separate cells and freeze for future studies of cell mediated immunity, including cytokine production in response to Norwalk VLP antigen, and lymphoproliferation. Whole stool samples are collected before immunization and on days 7+1, 21+2, 28+2, 56+2, and day 180+14 for anti-Norwalk VLP sIgA screening. Saliva is collected with a commercially available device (Salivette, Sarstedt, Newton, N.C.) before immunization and on days 7+1, 21+2, 28+2, 56+2, and if positive for mucosal antibodies at day 56, a day 180+14 sample is collected and screened for anti-Norwalk VLP sIgA. Blood is also screened for memory B-cells on days 0, 21, 56 and 180.
Methods used to analyze the blood, stool, and saliva samples collected from immunized individuals, or individuals receiving the dry powder matrix alone or placebo are described in detail in Example 2.
Example 4. Norwalk Virus Challenge Study in Humans Immunized with Norwalk Virus VLP Vaccine Formulation
A multi-site, randomized, double-blind, placebo-controlled Phase 1-2 challenge study is conducted in 80 human volunteers immunized with the Norwalk VLP vaccine described in Example 2 above. Eligible subjects include those 18-50 years of age, in good health, who express the H type-1 oligosaccharide (as measured by positive salivary secretor status) and who are other than Type B or AB blood type. Subjects who are non H type-1 secretors or who have Type B or AB blood are reported to be more resistant to infection with Norwalk virus and are excluded from the study. At least 80% of volunteers are expected to be eligible based on these two criteria.
Following screening, eligible volunteers who meet all acceptance criteria are randomized (1:1) into one of two equal sized cohorts with approximately 40 volunteers in each cohort. Cohort 1 is immunized with Norwalk VLP and cohort 2 receives placebo. Volunteers are immunized with 10 mg Norwalk VLP vaccine in each nostril (20 mg total dry powder) or placebo. Each 10 mg of Norwalk VLP vaccine contains 50 μg of Norwalk VLP, 7 mg chitosan, 25 μg MPL®, 1.5 mg of sucrose and approximately 1.5 mg of mannitol. Thus, each volunteer in cohort 1 receives a total dosage of 100 μg of Norwalk VLP antigen at each immunization. Volunteers receive vaccine or placebo on study days 0 and 21.
The safety of the Norwalk virus VLP vaccine compared to placebo is assessed. Volunteers keep a diary for 7 days following each immunization with the vaccine or placebo to document the severity and duration of adverse events. Serious adverse events (SAEs) and the occurrence of any significant new medical conditions is followed for 6 months after the last dose of vaccine or placebo and for 4 months after the challenge with infectious virus.
All volunteers are challenged with infectious Norwalk virus between 21 to 42 days after the second dose of vaccine or placebo (between study days 42 and 56). Each volunteer receives at or >than the 50% Human Infectious Dose (HID 50), i.e. the amount of infectious virus that is expected to cause disease in at least 50% of volunteers in the placebo group. The HID 50 is between about 48 and about 480 viral equivalents of the Norwalk virus. The Norwalk virus is mixed with sterile water and given orally. The inoculation is preceded by ingestion of 500 mg sodium bicarbonate in water, to prevent breakdown of the virus by stomach acid and pepsin. A second ingestion of sodium bicarbonate solution (500 mg sodium bicarbonate in water) is taken 5 minutes after oral inoculation of the infectious virus. The volunteers remain at the challenge facility for at least 4 days and at least 18 hours after symptoms/signs of acute gastroenteritis (vomiting, diarrhea, loose stool, abdominal pain, nausea, and fever) are absent.
Several metrics are monitored to determine the efficacy of the Norwalk VLP vaccine in preventing or reducing symptoms/signs of acute gastroenteritis induced by the viral challenge. All volunteers record their clinical symptoms of acute gastroenteritis and these symptoms are documented by the research staff at the study sites. Disease symptoms/signs from cohort 1 receiving the vaccine are compared to cohort 2 placebo recipients.
Sera and stool samples are routinely collected from all volunteers prior to immunization with the vaccine or placebo, and after challenge. Serum samples are analyzed by ELISA for IgA and IgG, titers against the Norwalk VLPs. The Norwalk antigen and Norwalk RNA are tested in stool samples by ELISA and PCR, respectively, which indicate the presence of virus, the amount of virus shed from the intestines, and the duration of viral shedding. Subjects who become ill after challenge, are subject to additional laboratory studies including serum chemistries, BUN, creatinine, and liver function tests until symptoms/signs resolve.
Results from the vaccine group (cohort 1) and the placebo group (cohort 2) are compared to assess the protective efficacy of the vaccine against Norovirus disease overall (primary endpoint), and/or its efficacy in ameliorating the symptoms/signs (severity and # of days of illness) and/or the reduction of the presence, the amount and/or the duration of virus shedding (secondary endpoints).
The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings using no more than routine experimentation. Such modifications and equivalents are intended to fall within the scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.
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36. GSK Press Room.
37. Corixa Press Room.
38. BioMira Web Site.
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12678813
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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tyo:4502
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Takeda Pharmaceutical
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May 12th, 2015 12:00AM
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Nov 24th, 2010 12:00AM
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https://www.uspto.gov?id=US09028809-20150512
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Compositions, methods and uses for expression of enterobacterium-associated peptides
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Embodiments of the present invention generally disclose methods, compositions and uses for generating and expressing enterobacterial-associated peptides. In some embodiments, enterobacterial-associated peptides include, but are not limited to plague-associated peptides. In certain embodiments, methods generally relate to making and using compositions of constructs including, but not limited to, attenuated or modified vaccinia virus vectors expressing enterobacterial-associated peptides. In other embodiments, vaccine compositions are reported of use in a subject.
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9028809
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1. An immunogenic composition comprising:
one or more constructs of live attenuated, modified vaccinia Ankara (MVA) viruses encoding:
at least one V307 peptide which is the C-terminally truncated low-calcium response V (LcrV) protein antigen of Yersinia pestis lacking its C-terminal amino acids 308 to 326 and
a mammalian secretory signal sequence and a viral translational control sequence, wherein the mammalian secretory signal sequence is tissue plasminogen activator (tPA) secretory signal and the viral translational control sequence is a viral internal ribosomal entry site (IRES), wherein the one or more constructs in the composition are capable of inducing a protective immune response to one more strains of Yersinia pestis in a mammalian subject.
2. The composition of claim 1, wherein the protective immune response induced is against encapsulated and non-encapsulated strains of Yersinia pestis.
3. The composition of claim 1, wherein the viral IRES is from encephalomyocarditis virus (EMCV).
4. The composition of claim 1, wherein the at least one V307 peptide lacks immunosuppressive sequences of the LcrV protein antigen.
5. The composition of claim 1, wherein the composition comprises a pharmaceutically acceptable carrier.
6. A kit comprising the composition of claim 1 and at least one container.
7. The kit of claim 6, further comprising a delivery device for delivery to the subject.
8. A method of inducing a protective immune response in a mammalian subject comprising administering the composition of claim 1 to the subject.
9. An immunogenic composition for administration to a mammalian subject comprising a pharmaceutically acceptable carrier and
one or more constructs of live attenuated, modified vaccinia Ankara (MVA) viruses encoding:
at least one V307 peptide which is the C-terminally truncated low-calcium response V (LcrV) protein antigen of Yersinia pestis lacking its C-terminal amino acids 308 to 326 and
a mammalian secretory signal sequence and a viral translational control sequence, wherein the mammalian secretory signal sequence is tissue plasminogen activator (tPA) secretory signal and the viral translational control sequence is encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES), wherein the one or more constructs in the composition are capable of inducing a protective immune response to one more strains of Yersinia pestis in the subject.
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9
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CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a national phase application, filed pursuant to 35 U.S.C. 371, that claims the benefit of PCT application No. PCT/US10/58094, filed on Nov. 24, 2010, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/264,144, filed on Nov. 24, 2009. Pursuant to 35 U.S.C. 119, the prior applications are incorporated herein by reference in their entirety for all purposes.
FEDERALLY FUNDED RESEARCH
Some embodiments disclosed herein were supported in part by grant number 1R43A1061940-01 from the National Institutes of Health. The government may have certain rights in this invention.
FIELD
Embodiments of the present invention report methods, compositions and uses for generating and expressing constructs having enterobacterial-associated peptides. In some embodiments, enterobacterial-associated peptides include, but are not limited to, plague-associated peptides. In certain embodiments, the present invention discloses making and using constructs including, but not limited to, attenuated or modified vaccinia virus vectors expressing enterobacterial-associated peptides.
BACKGROUND
Vaccines to protect against viral infections have been effectively used to reduce the incidence of human disease. One of the most successful technologies for viral vaccines is to immunize animals or humans with a weakened or attenuated strain of the virus (a “live, attenuated virus”). Due to limited replication after immunization, the attenuated strain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and generates potent and long-lasting immune responses to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the immunized individual is protected from disease. These live, attenuated viral vaccines are among the most successful vaccines used in public health.
Yersinia is a genus of bacteria in the family of Enterobacteriaceae. Yersinia are facultative anaerobes. Some members of Yersinia are pathogenic in humans. Often, rodents are the natural reservoirs of Yersinia; less frequently other mammals may serve as a host to these bacteria. Infection can occur either through arthopod bite, exposure to blood, aerosol transmission (e.g. Y. pestis), or by, for example, consumption of food products (e.g. vegetables, milk-derived products and meat) contaminated with the bacteria. Other modes likely exist (e.g. via protozoonitic mechanisms) for transmission. The Yersinia family is rather large, but only two have been linked to water-borne outbreaks of disease, Y. pseudotuberculosis and Y. enterocolitica. Yersinia species are found all over the world in animal reservoirs (e.g., rodent reservoirs for Y. pestis), isolated in well-water, water treatment plants, rivers and lakes. Yersinia pestis (also referred to as Pasteurella pestis) is the most famous member of the Yersinia species and is the causative organism of plague.
SUMMARY
Embodiments of the present invention generally relate to methods, compositions and uses for expressing enterobacterial-associated peptides. In some embodiments, enterobacterial-associated peptides include, but are not limited to, plague-associated peptides. Certain embodiments report making and using constructs of the present invention for treating or protecting a subject having been exposed or likely to be exposed to an Enterobacteria. In accordance with these embodiments, constructs may include, but are not limited to, attenuated or modified vaccinia virus vectors expressing enterobacterial-associated peptides. In other embodiments, methods and compositions report making and using compositions having constructs including, but not limited to, attenuated or modified vaccinia virus vectors expressing Yersinia spp-associated peptides, for example, in order to induce an immune response in a subject against the Yersinia spp. Some of these embodiments address a solution for a potential threat of using Yersinia spp. as a bioweapon or potential for a Yersinia spp. outbreak with its global health implications.
Certain embodiments report composition having constructs with antigens or peptides associated with Yersinia spp. including, but not limited to, F1, V, truncated V or YopD polypeptides, or combinations thereof. Other embodiments may include one or more low-calcium response (V) antigens with a C-terminal truncation. In accordance with these embodiments, C-terminal truncation of low-calcium response (V) antigens may include, but are not limited to, a truncation that suppresses expression of a pro-inflammatory cytokine, truncations that remove immunosuppressive sequences, truncations that are less immunosuppressive than corresponding full-length or unmodified LcrV protein, deletion is of up to 163 contiguous residues of LcrV, internal deletions, internal deletion up to 90 contiguous residues, internal deletion extending into the region spanning amino acids 240 to 325 of LcrV protein, C-terminal deletions of up to 50 contiguous residues, an LcrV protein of at least 275 residues in length or combinations thereof. Some embodiments report vaccine compositions capable of reducing or preventing infection in a subject caused by exposure to enterobacteria (e.g. Yersinia spp), including, for example, protection from encapsulated and unencapsulated forms of the organism.
In some aspects, constructs of use as vaccine compositions, can include one or more secretory signal sequences alone or in combination with one or more translation control region sequences. In accordance with these embodiments, a secretory signal sequence can be one or more signal sequences functional in mammalian cells. In other embodiments, a secretory signal sequence includes, but is not limited to, tissue plasminogen activator (tPA) leader sequence, the co-factor leader sequence, the pre-proinsulin leader sequence, the invertase leader sequence, the immunoglobulin A leader sequence, the ovalbumin leader sequence, and the P-globin leader sequence or other proleader sequences known in the art.
Vaccine compositions disclosed herein can be administered by any method known in the art. In certain embodiments, a vaccine can be administered intradermally, intramuscularly, by inhalation, intranasally, intravenously or by any other route known in the art. Some compositions can be administered by time-release or other formulations as assessed by a health provider.
Other embodiments concern kits for making or using compositions disclosed. It is reported that a kit may include constructs having a modified vaccinia viral vector and one or more enterobacterial-derived antigen. Other kits can include methods for making a construct contemplated herein.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain embodiments. Some embodiments may be better understood by reference to one or more of these drawings alone or in combination with the detailed description of specific embodiments presented.
FIG. 1 represents an exemplary construct of modified vaccinia virus and a Yersinia-associated peptide.
FIGS. 2A-2E represent exemplary electrophoretic separations and analyses of expression patterns from clonal recombinant viruses.
FIG. 3A represents a histogram of an immune response in mice to vaccine of construct compositions of some embodiments disclosed herein, pre-boost and pre-challenge.
FIG. 3B represents an exemplary histogram of an immune response in mice to vaccine of construct compositions of some embodiments disclosed herein, pre-boost and pre-challenge.
FIG. 4A represents an exemplary plot illustrating survival rates of mice immunized with an exemplary enterobacterial-directed vaccines or control formulations following intranasal challenge with enterobacterium.
FIG. 4B represents an exemplary plot illustrating survival rates of mice immunized with an exemplary enterobacterial-directed vaccines or control formulations following intraperitoneal challenge with enterobacterium.
FIG. 5 represents an exemplary assessment of safety of enterobacterial-directed vaccines inoculated in immunocompromised mice.
FIG. 6 represents an exemplary plot of antibody titers measured and plotted after IM (intramuscular) or ID (intradermal) pre and post boosts of vaccines of some embodiments disclosed herein.
FIG. 7 represents a compilation of construct data as Table 3.
DEFINITIONS
As used herein, “a” or “an” may mean one or more than one of an item.
As used herein, vessel can include, but is not limited to, test tube, mini- or microfuge tube, channel, vial, microtiter plate or container.
As used herein the specification, “subject” or “subjects” may include, but are not limited to, mammals such as humans or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g. hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairie dogs, wild rodents, or zoo animals.
As used herein, “about” can mean plus or minus ten percent.
As used herein, “attenuated virus” can mean a virus that demonstrates reduced or no clinical signs of disease when administered to a subject such as a mammal (e.g. human or an animal).
As used herein, “MSC” can mean multiple cloning site.
As used herein, “dSP” can mean divergent vaccinia promoter.
As used herein, “MVA” can mean modified vaccinia Ankara.
As used herein, “EMCV” can mean encephalomyocarditis virus.
As used herein, “IRES” can mean internal ribosome entry site from encephalomyocarditis virus or other viruses.
As used herein, “IRES(A7)” can mean IRES from encephalomyocarditis virus with 7 adenosine residues in bifurcation loop; source-pCITE-1.
As used herein, “IRES(A6)” can mean IRES from encephalomyocarditis virus mutated to have 6 adenosine residues in bifuraction loop.
As used herein, “pDIIIgfp” can mean MVA del III gfp marker transfer plasmid.
As used herein, “pI*” can mean transfer vector plasmids.
As used herein, “tPA” can mean secretory signal from tissue plaminogen activator.
As used herein, “se/l” can mean synthetic optimized early late poxvirus promoter.
As used herein, “H6” can mean the vaccinia gene H6 early/late native poxvirus promoter.
As used herein, “F1” can mean Y. pestis capsular protein.
As used herein, “V” can mean Y. pestis virulence factor LcrV.
As used herein, “V307” or “V307” can mean C-terminal LcrV truncation of amino acids 308-326 of Y. pestis. V protein.
As used herein, “YopD” can mean Y. pestis outer protein D.
As used herein, “del III” can mean modified vaccinia Ankara deletion region III.
As used herein, “GFP” can mean enhanced green fluorescent protein.
As used herein, “CEF” can mean chicken embryo fibroblasts.
DESCRIPTION
In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the details outlined herein, but rather that concentrations, times and other details may be modified through routine experimentation. In some cases, well-known methods or components have not been included in the description.
Some embodiments of the present invention report vaccine compositions including, but not limited to, vaccine compositions having one or more construct comprising modified or attenuated vaccinia virus and one or more enterobacterial-associated peptides. In certain embodiments, a vaccine composition may include a recombinant modified vaccinia Ankara (MVA) vector associated with one or more enterobacterial-associated peptides. In other embodiments, a vaccine composition may include a recombinant modified vaccinia Ankara (MVA) vector associated with one or more enterobacterial-associated peptides where at least one of the enterobacterial-associated peptides includes one or more Yersinia-associated peptides. For example, one vaccine composition can include recombinant modified vaccinia Ankara (MVA) vector expressing Yersinia pestis antigens. In accordance with this vaccine composition, an MVA construct expressing one or more Yersinia pestis-associated antigens may be generated (e.g. V, F1, LcrV protein or mutants or fragments thereof).
Yersinia
Plague is primarily a disease of wild rodents transmitted by fleas, but it can also afflict humans, domestic pets, and wild animals. The disease has devastated human and animal populations throughout history. In recent years, it has caused severe epidemics in many parts of the world, resulting in human deaths and severe economic losses. Yersinia pestis is widespread throughout wild rodent populations in the southwestern United States, Southeast Asia, Eastern Europe, central and southern Africa, as well as South America, and human populations in these areas are highly susceptible. In the United States, plague has spread throughout the Western states, causing significant mortality in squirrels, wild mice, and prairie dogs. Domestic cats are also susceptible to Y. pestis infections, and they have been identified as the source of infection in many recent cases of plague in humans.
Because of its pathogenicity in humans, particularly the pneumonic form of the disease, and its potential for human-to-human transmission, Y. pestis is considered a potential candidate for biowarfare. It is believed that Mongols first used plague as a crude bioweapon in the 1300s during the siege of Kaffa as plague victims were launched over the wall of the city. During World War II, a secret branch of the Japanese army is reported to have dropped plague-infected fleas over China causing several outbreaks in humans. In following years, research programs in biological weapons in the US and Soviet Union successfully developed methods for aerosolization of plague. More recently, evidence that scientists from the former Soviet Union worked with plague serves notice that Y. pestis is still considered a feasible bioweapon. In 1970, the World Health Organization estimated that intentional release of 50 kg of the plague bacterium, Y. pestis, over a city of 5 million people could result in as many as 150,000 clinical cases and 36,000 deaths. A 2001 U.S. Congressional Office of Technology report estimated that a deliberate aerosol release of Y. pestis could cause more than 9,000 clinical cases and at least 2,000 deaths. Therefore, there is an immediate need for novel vaccines that can protect troops, health professionals and first-responder personnel from the threat of Y. pestis bioweapons that can limit the spread of disease by vaccination of individuals at risk after a bioterrorist attack and that can limit disease outbreaks in endemic countries.
Certain embodiments of the present invention report compositions having constructs directed against Yersini spp. For example, vaccine compositions may be directed to the prevention or reduced incidence of plague.
In recent years the development of novel plague vaccines has been the focus of extensive research, because commercially licensed vaccines based on heat or formaldehyde killed suspension of Y. pestis, were found to be unsafe. Capsular F1 (17.5 kDa) and V (35 kDa) antigens are natural virulence factors produced by Y. pestis. Both antigens impact innate immune responses required to control bacterial spread at the early stages of infection. F1-based vaccines are immunogenic but these vaccines have failed to provide protection against naturally occurring non-encapsulated strains of Y. pestis. Some embodiments of the present invention, may include secreted V antigen which plays for example, a role in delivery of other Yersinia outer proteins (Yops) and stimulates secretion of IL-10 (an anti-inflammatory cytokine) associated with the suppression of TNF-α and IFN-γ, a hallmark of plague.
Antigens of use for vaccines against Yersinia spp infection may include LcrV (low-calcium-response V or V antigen) or other plasmid-encoded, virulence proteins (e.g. Yops, or Yersinia outer proteins) which are essential for survival in mammalian hosts. Yops and LcrV are secreted by a type III mechanism (Ysc), and Yops are unidirectionally targeted into the cytosol of associated eukaryotic cells in a tissue culture infection model. LcrV is required for Yops targeting, and recent findings have revealed that it can localize to the bacterial surface. Therefore, some of the coding sequences of use in some embodiments of the present invention can include coding sequences for Y. pestis antigens capsular protein F1, full length virulence factor LcrV (V), a truncated form of LcrV (V307), other carboxyterminal truncations of LcrV and effector protein YopD.
Other embodiments may include one or more low-calcium response (V) antigens with a C-terminal truncation. In accordance with these embodiments, C-terminal truncation of low-calcium response (V) antigens may include, but are not limited to, a truncation that suppresses expression of a pro-inflammatory cytokine, truncations that remove immunosuppressive sequences, truncations that are less immunosuppressive that corresponding full-length or unmodified LcrV protein, deletion is of up to 163 contiguous residues of LcrV, internal deletions, internal deletion up to 90 contiguous residues, internal deletion extending into the region spanning amino acids 240 to 325 of LcrV protein, C-terminal deletions of up to 50 contiguous residues, an LcrV protein of at least 275 residues in length (e.g. Schneedind et. al U.S. patent application Ser. No. 11/293,024 filed Dec. 2, 2005, incorporated herein by reference in its entirety for all purposes). In certain embodiments, a modified LcrV protein may suppress expression of a pro-inflammatory cytokine to a lesser extent than a corresponding unmodified LcrV protein. In some embodiments, the pro-inflammatory cytokine may be TNF-α or other known pro-inflammatory cytokines known in the art. In other embodiments, a deletion can include residues 271 to 300 of LcrV protein (rV10). It is contemplated that the same or similar amino acids corresponding to this region of LcrV from Yersinia pestis may be deleted in other LcrV proteins. In one example, amino acids 271-300 in Yersinia pestis correspond to 280-309 in Y. enterocolitica.
In other embodiments, other enterobacteria-derived proteins or peptides can be of use in vaccine constructs contemplated herein for administration to a subject to reduce incidence of or prevent a condition. Certain embodiments report compositions having constructs directed against any pathogenic enterobacterium. For example, vaccine compositions may be directed to the prevention or reduced incidence of an infection in a subject caused by exposure or suspected exposure to a pathogenic enterobacteria. Other enterobacteria can include, but are not limited to Salmonella spp., Shigella spp, Escherichia coli strains or other pathogenic enterobacteria.
Poxviridae
Poxviruses (members of the family Poxviridae) are viruses that can, as a family, infect both vertebrate and invertebrate animals. There are four known genera of poxviruses that may infect humans: orthopox, parapox, yatapox, molluscipox. Orthopox include, but are not limited to, variola virus, vaccinia virus, cowpox virus, monkeypox virus, and smallpox. Parapox include, but are not limited to, orf virus, pseudocowpox, bovine papular stomatitis virus; Yatapox: tanapox virus, yaba monkey tumor virus. Molluscipox include, but are not limited to, molluscum contagiosum virus (MCV). Some of the more common oixviruses are vaccinia and molluscum contagiousum, but monkeypox infections seem to be on the rise.
Poxvirus family, vaccinia virus, has been used to successfully vaccinate against smallpox virus. Vaccinia virus is also used as an effective tool for foreign protein expression to elicit strong host immune response. Vaccinia virus enters cells mainly by cell fusion, although currently the receptor is not known. Virus contains three classes of genes, early, intermediate and late, transcribed by viral RNA polymerase and associated transcription factors. Diseases caused by poxviruses have been known about for centuries.
Orthopoxviruses
Certain embodiments of the present invention may include using modified or attenuated orthopoxviruses in vaccine compositions. Orthopoxvirus is a genus of the Poxviridae family, that includes many agents isolated from mammals, including, but not limited to, vaccinia, monkeypox, cowpox, camelpox, seal poxvirus, buffalo poxvirus, raccoon poxvirus, skunk poxvirus, vole poxvirus and ectromelia viruses. Members of Poxviridae have large linear double-stranded DNA, with genome sizes ranging from 130 to 300 kbp. One of the members of the genus is variola virus, which causes smallpox. Smallpoxwas previously eradicated using another orthopoxvirus, the vaccinia virus, as a vaccine.
Modified Vaccinia Virus Ankara (MVA)
Some embodiments in the present invention report compositions and methods of use of recombinant vaccinia viruses derived from attenuated poxviruses (e.g., modified vaccinia virus Ankara (MVA), NYVAC, LC16m8 or CVI-78) that are capable of expressing predetermined genes or gene segments. Those skilled in the art recognize that other attenuated poxviruses can be generated by for example, serial passage in cell culture or by deliberate deletion of poxviral genes or other methods known in the art. In certain embodiments, predetermined genes may be inserted at the site of a naturally occurring deletion in the MVA genome. In other embodiments, recombinant MVA viruses can be used, for example, for the production of polypeptides (e.g. antigens) or for encoding antigens of use for vaccine compositions capable of inducing an immune response in a subject administered the vaccine compositions.
In certain embodiments, modified or attenuated poxviruses (e.g. modified vaccinia Ankara (MVA), NYVAC, LC16m8, or CVI-78), can be used in a subject (e.g. mammals such as humans) as a delivery system. Previously, MVA was administered to over 120,000 individuals and proven to be a safe and effective vaccine against small pox. In other embodiments, recombinant MVA vaccine candidates have been shown to induce protective humoral and cellular immunity against diseases caused by viruses, bacteria, parasites, or tumors from which antigens or peptides were derived. Additional features that make MVA a suitable vector include its ability to induce protective immune responses when administered by different routes and its genetic and physical stability properties.
Tranlational Control Sequences
Some embodiments may include an optional enhancer, for example, a translation control sequence. In certain embodiments, a translation control sequence may include an internal ribosomal entry site (IRES) (e.g. EMCV-IRES). Viral IRESs can be classified into four groups: Group 1 (Cricket paralysis virus (CrPV), Plautia stali intestine virus (PSIV) and Taura syndrome virus (TSV)); Group 2 (Hepatitis C virus, (HCV), classical swine fever virus (CSFV) and porcine teschovirus 1 (PTV-1)); Group 3 (encephalomyocarditis virus (EMCV), foot-and-mouth-disease virus (FMDV) and Theiler's Murine Encephalomyelitis virus (TMEV)); and Group 4 (poliovirus (PV) and rhinovirus (RV)). In other embodiments, viral untranslated regions (UTRs) found 5′ to viral coding sequences can be used to direct translation. Any translation control sequence of use in viral constructs known in the art is contemplated. In certain embodiments, a viral internal ribosome entry site (IRES) may be used to increase expression of plague antigens contemplated herein. An IRES sequence can be positioned after a stop codon in a messenger RNA molecule and a ribosome can re-attach to the mRNA and a second protein can be translated from the same RNA. Thus, the IRES sequence can be used to express multiple antigens, for instance multiple enterobacterial antigens or an enterobacterial antigen and an antigen from another pathogenic virus or bacterium. For example, if the second protein is a selectable marker, then use of this marker will increase the probability that the gene of interest (placed between the promoter and the IRES) will be expressed by detection of the selectable marker. A number of bicistronic vectors have been produced based on these concepts. Use of any of bicistronic vectors known in the art are contemplated herein.
Secretory Signals
Alternatively, embodiments of the present invention may include constructs having one or more secretory signal sequences. Secretory signals of use can include, but are not limited to, a mammalian secretory signal sequence. Translation control sequences and/or secretory signals were demonstrated to increase efficacy of some vaccines. In some embodiments, one or more secretory signal sequences may include a proleader sequence. In certain examples, a tPA pre-proleader sequence may be used, where a leader sequence includes, but is not limited to, tissue plasminogen activator (tPA) leader sequence, oc-factor leader sequence, pre-proinsulin leader sequence, invertase leader sequence, immunoglobulin A leader sequence, ovalbumin leader sequence, and P-globin leader sequence or other proleader sequences known in the art or a combination thereof.
In some embodiments, designing a construct, such that a protein is expressed, it may be necessary to incorporate into a first nucleic acid region a DNA sequence encoding a signal sequence, for example, in cleavable form, where the expressed protein is desired to be secreted. Without limiting embodiments of the present invention to any one theory or mode of action, a signal sequence can be a peptide that is present on proteins destined either to be secreted or to be membrane bound. These signal sequences are normally located at the N-terminus of the protein and are generally cleaved from the mature protein. The signal sequence generally interacts with the signal recognition particle and directs the ribosome to the endoplasmic reticulum where co-translational insertion takes place. Where the signal sequence is cleavable, it is generally removed by for example, a signal peptidase. The choice of signal sequence which is to be utilized may depend on the requirements of the particular situation and can be determined by the person of skill in the art. In the context of the exemplification provided herein, but without being limited in that regard, tPA may be used to facilitate secretion of a peptide, protein or construct of interest. If a membrane protein is desired, both a 5′ cleavable signal sequence at the amino end of the protein and a non-cleavable membrane anchor at the 3′ (carboxy) end of the protein may be needed. These could be provided within the vector or one or both could be encoded by the DNA of the protein of interest.
Some embodiments of the present invention include, but are not limited to, compositions including one or more constructs. For example, a construct may be designed to produce proteins or peptides that are cytoplasmically retained, secreted or membrane bound. Deciding what form a protein or peptide of interest may need to take can depend on functional requirements. For example, anchored cell surface expression of a protein of interest provides a convenient means for screening for molecules that interact with the protein of interest such as antibodies, antagonists, agonists or the like particularly to the extent that the protein is expressed on the membrane of an adherent cell type. Still further embodiments concern membrane anchored forms of proteins or peptide that may be suitable for administration to a subject for example, for generating monoclonal antibodies to the protein or peptide. This may be due to host cells providing a source of the protein or peptide that can be correctly folded and have appropriate post-translational modifications, for example, glycosylation and disulphide bond formation. In addition, a host cell may provide adjuvant properties, for example, antigenic differences from a recipient subject, notably in major histocompatibility complexes (MHC).
Alternatively, secreted proteins can be suitable where a protein or peptide is to be harvested and purified. A nucleic acid molecule encoding a signal sequence to the extent that one is utilized, may be positioned in the construct at any suitable location which can be determined as a matter of routine procedure by the person of skill in the art. In some embodiments, a signal sequence may be positioned immediately 5′ to the nucleic acid sequence encoding a peptide, protein or construct of interest (such that it can be expressed as an immediately adjacent fusion with the protein of interest) but 3′ to a promoter such that expression of a signal sequence is placed under control of the promoter. A nucleic acid sequence encoding a signal sequence can form part of a first nucleic acid region of a construct.
Selection Markers
In certain embodiments, additional selection markers may be used, for example, one may insert any number of selection markers which may be designed, for example, to facilitate the use of the vectors in a variety of ways, such as purification of a molecule of interest. For example, glutathione S-transferase (OST) gene fusion system provides a convenient means of harvesting a construct, protein or peptide of interest. Without limiting to any one theory or mode of action, a GST-fusion protein can be purified, by virtue of the OST tag, using giutathione agarose beads. Embodiments of the present invention should be understood to extend to constructs encoding a secretable GST-molecule fusion. This could be achieved, for example, by designing the sequence of a first nucleic acid region such that it encodes a cleavable signal sequence fused to a cleavable GST which is, in turn, fused to the molecule of interest. In another example, a fusion tag could be used which is itself a fusion between 360 bp of protein A (allowing purification of the secreted product) and beta lactamase (a bacterial enzyme which allows testing of supernatants by a simple colour reaction). Beta lactamase facilitates selection of an assay for a molecule of interest in the absence of an assay for molecule of interest. The protein A/beta lactamase fusion can be separated from the molecule of interest by a cleavage site to facilitate cleavage, so that after the molecule is purified, the tag can be easily removed. Any other selection marker known in the art may be used.
Other fusion tags that could be included to facilitate purification of a molecule or construct of interest include, but are not limited to, staphylococcal protein A, streptococcal protein G, hexahistidine, calmodulin-binding peptides and maltose-binding protein (e.g. the latter is also useful to help ensure correct folding of a molecule of interest). Yet another selectable marker may include an antibiotic resistance gene. Other embodiments may include an antibiotic resistance gene. These genes have previously been utilized in the context of bicistronic vectors as the selection marker or HAT-based selectable bicistronic vector may be used.
Electrophoresis
Electrophoresis ma be used to separate molecules (e.g. large molecules such as proteins or nucleic acids) based on their size and electrical charge. There are many variations of electrophoresis known in the art. A solution through which the molecules move may be free, usually in capillary tubes, or it may be embedded in a matrix. Common matrices include polyacrylamide gels, agarose gels, and filter paper.
Proteins, peptides and/or antibodies or fragments thereof may be purified, partially purified, detected or analyzed by any means known in the art. In certain embodiments, methods for separating and analyzing molecules may be used such as gel electrophoresis or column chromatography methods.
Any method known in the art for detecting, analyzing and/or measuring levels of antibodies may be used in embodiments reported herein. For example, assays for antibodies or antibody fragments may include, but are not limited to, ELISA assays, chemiluminescence assays, flow cytometry and other techniques known in the art.
Imaging Agents and Radioisotopes
In certain embodiments, constructs having proteins or peptides may be linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a fluorescent, a luminescent, or a colored product upon contact with a substrate. Examples of suitable enzymes include luciferase, green fluorescent protein, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. The use and identification of such labels is well known to those of skill in the art.
In other embodiments, labels or molecules capable of detecting peptides, antigens, constructs, antibodies or antibody fragments may include using aptamers. Methods for making and using aptamers are well known in the art and these methods and uses are contemplated herein.
Some embodiments can include methods for detecting and/or making polyclonal or monoclonal antibodies produced by a subject exposed to vaccine compositions disclosed in some embodiments of the present invention. For example, antibodies or antibody fragments produced capable of inducing passive immunity to a subject may be isolated, analyzed and/or produced as a whole antibody or fragment thereof, or a polyclonal or a monoclonal antibody. Any means for producing or analyzing these antibodies known in the art are contemplated.
Nucleic Acid Amplification
Nucleic acid sequences used as a template for amplification can be isolated from viruses, bacteria, cells or cellular components contained in the biological sample, according to standard methodologies. A nucleic acid sequence may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary cDNA. In one embodiment, the RNA is whole cell RNA and is used directly as the template for amplification. Any method known in the art for amplifying nucleic acid molecules are contemplated (e.g. PCR, LCR, Qbeta Replicase).
Expressed Proteins or Peptides
Genes or gene segments can be expressed in any number of different recombinant DNA expression systems to generate large amounts of the polypeptide product, which can then be purified and used in methods and compositions reported herein. Any method known in the art for generating and using constructs is contemplated. In certain embodiments, genes or gene fragments encoding one or more polypeptide mays be inserted into an expression vector by standard cloning or subcloning techniques known in the art.
Some embodiments, using a gene or gene fragment encoding a polypeptide may be inserted into an expression vector by standard subcloning techniques. An expression vector may be used which produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification of a peptide or protein. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, N.J.), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6× His system (Qiagen, Chatsworth, Calif.).
Pharmaceutical Compositions and Routes of Administration
Aqueous compositions of some embodiments herein can include an effective amount of a therapeutic protein, peptide, construct, epitopic core region, stimulator, inhibitor, and the like, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Aqueous compositions of vectors expressing any of the foregoing are also contemplated. The phrases “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
Aqueous compositions of some embodiments herein can include an effective amount of a therapeutic protein, peptide, construct, an effective amount of the compound, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions can also be referred to as inocula. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
The biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. The active compounds or constructs will then generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, intranasal or even intraperitoneal routes. Any route used for vaccination or boost of a subject can be used. The preparation of an aqueous composition that contains an active component or ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use in preparing solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
Pharmaceutical forms suitable for injectable use can include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
If formulations or constructs disclosed herein are used as a therapeutic to boost an immune response in a subject, a therapeutic agent can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
A carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
Upon formulation, solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but slow release capsules or microparticles and microspheres and the like can also be employed.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion.
The term “unit dose” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the construct composition or boost compositions calculated to produce desired responses, discussed above, in association with its administration, e.g., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments or vaccinations and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. For example, a subject may be administered a construct composition disclosed herein on a daily or weekly basis for a time period or on a monthly, bi-yearly or yearly basis depending on need or exposure to a pathogenic organism or to a condition in the subject (e.g. cancer).
The active therapeutic agents may be formulated within a mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Alternatively active agents (e.g. constructs) may be formulated to comprise a certain number of constructs per dose known to produce a desired effect in a subject. Multiple doses can also be administered.
In addition to the compounds formulated for parenteral administration, such as intravenous, intradermal or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; biodegradable and any other form currently used.
One may also use intranasal or inhalable solutions or sprays, aerosols or inhalants. Nasal solutions can be aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions can be prepared so that they are similar in many respects to nasal secretions. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation. Various commercial nasal preparations are known and can include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.
Additional formulations which are suitable for other modes of administration can include suppositories and pessaries. A rectal pessary or suppository may also be used. In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%.
Oral formulations can include excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. In certain defined embodiments, oral pharmaceutical compositions will comprise an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such compositions is such that a suitable dosage can be obtained.
The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
Kits
Further embodiments concern kits of use with methods and compositions described herein. Some embodiments concern kits having vaccine compositions of use to prevent or treat subjects having or exposed to an enterobacteria. Kits can be portable, for example, able to be transported and used in remote areas. Other kits may be of use in a health facility to treat a subject having been exposed to an enterobacteria or suspected of being at risk of exposure to an enterobacteria (e.g. Yersinia spp).
Other embodiments can concern kits for making and using molecular constructs described herein. In certain embodiments, compositions can include constructs having attenuated or modified MVA and Yersinia spp.-associated antigens (e.g. V307). Other constructs can also include at least one secretory signal sequence. Yet other embodiments can have a construct that includes translation control sequences (e.g. IRES, UTRs). Other reagents for making and using constructs are contemplated.
Kits can also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the constructs, vaccine compositions and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Optionally, one or more additional agents such as other anti-viral agents, anti-fungal or anti-bacterial agents may be needed for compositions described, for example, for compositions of use as a vaccine.
Dose ranges used during vaccination can vary depending on the nature of the live attenuated vaccine and viral vector used. For recombinant poxviruses these doses can range between 105-107 PFUs. In certain embodiments of the present invention, immunogenic doses can be as low as 102 pfu. Frequency of vaccination can vary depending on the nature of the vaccine and also the route of administration used. One regimen can include a primary immunization (prime) followed up by a boost administration four to six weeks post-prime immunization. In certain embodiments of the present invention, improvements in antigen translation and expression can permit fewer and/or lower doses to be administered to a subject.
Compositions disclosed herein may be administered by any means known in the art. For example, compositions may include administration to a subject intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, via a catheter, via a lavage, in a creme, or in a lipid composition. Certain compositions can be administered by one route for one boost and another route for a second or additional boost of a composition, as can be pre-determined for a condition or prevention of an infection. In accordance with these embodiments, one boost can be administered intramuscularly and another boost can be administered intradermally or a combinations thereof, depending on a subject's circumstances, as well as dose and frequency determinations for a particular composition.
Preparations
Any method known to one skilled in the art may be used for large scale production of recombinant MVA. For example, master and working seed stocks may be prepared under GMP conditions in qualified primary CEFs or by other methods. Cells may be plated on large surface area flasks, grown to near confluence and infected at selected MOI and vaccine virus purified. Cells may be harvested and intracellular virus released by mechanical disruption, cell debris removed by large-pore depth filtration and host cell DNA digested with endonuclease. Virus particles may be subsequently purified and concentrated by tangential-flow filtration, followed by diafiltration. The resulting concentrated bulk vaccine may be formulated by dilution with a buffer containing stabilizers, filled into vials, and lyophilized. Compositions and formulations may be stored for later use. For use, lyophilized vaccine may be reconstituted by addition of diluent.
Poxviruses are known for their stability. The ability to lyophilize vaccinia for long term, room temperature storage and distribution was one of the key attributes that permitted widespread use of the vaccine and eradication of smallpox. Recently, it was demonstrated that Dryvax vaccinia virus stockpiled in the 60's was still potent after several decades. Procedures for lyophilization and storage of poxviruses are well known in the art and could be applied to the recombinant poxvirus vaccines for some embodiments disclosed herein.
The following examples are included to demonstrate certain embodiments presented herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered to function well in the practices disclosed herein. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the certain embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope herein.
EXAMPLES
Example 1
Protein Expression
In some exemplary methods, expression of the F1, V and V307 antigens of Y. pestis was assessed by immuno-blot analyses of proteins from cells infected with the MVA/Y. pestis recombinant viruses. Expression levels were evaluated in whole cell extracts and in cell culture supernatants after infection of MVA permissive CEF cells and non-permissive mammalian Vero cells (FIGS. 2A-2E). Expression of the F1 protein by the MVA/F1 recombinant was detected in both the cellular pellet and cell culture supernatants. In MVA/F1 infected CEF cells, the F1 capsular antigen was observed as two lower molecular weight forms consistent with the predicted protein (approximately 18 kilodaltons, kd; predicted protein) and a higher molecular weight form at approximately 23 kd (FIG. 2A). The 23 kd form was more prevalent in the cell supernatant, suggesting that it is preferentially secreted. The capsular F1 protein has one predicted N-glycosylation site and six predicted o-glycosylation sites for vertebrate cells. Thus, it was postulated that the 23 kd form could represent a glycosylated protein. Deglycosylation of CEF-expressed F1 eliminated the 23 kd form and only the 18 kd form remained (FIG. 2E). Higher molecular weight forms of approximately 34 kd and above also were observed in the cell pellets. These forms are consistent with dimers and other multimers; the F1 capsular protein avidly forms multimeric structures upon secretion. These forms were not observed upon extensive denaturation (see for example FIG. 2E). In CEF cells infected with the MVA/IRES/tPA/F1 virus, only low levels of expression of the lower molecular weight forms could be observed in the cell pellets.
In mammalian Vero cells infected with the MVA/F1 recombinant virus, the lower molecular weight form of 18 kd was more predominant than the 23 kd glycosylated form in both the cell pellet and the cell supernatant (FIG. 2B). Again, the 23 kd form disappeared upon deglycosylation (data not shown). Higher molecular weight forms of apparent molecular weights 33, 36 and 39 kd also were detected and were relatively more prominent in the culture supernatants. In Vero cells infected with the MVA/IRES/tPA/F1 recombinant virus, only low levels of expression of the F1 antigen were detected in the cell pellets. The reduced level of expression directed by the IRES/tPA constructs is in contrast to similar constructs made in raccoon poxvirus, previously described. In that case, IRES/tPA directed higher levels of expression and secretion in infected cells.
Expression patterns of V and V307 antigens were simpler and were similar in both infected CEF and Vero cells (see for example, FIGS. 2C and 2D). Cells infected with either MVA/V or MVA/V307 recombinant viruses expressed a single form of approximately 36 and 35 kd, respectively, consistent with predicted sizes (37 and 35 kd). Cells infected with MVA/IRES/tPA/V expressed two molecular forms of approximately 36 and 40 kd. The higher molecular weight form was consistent with the size of tPA/V fusion and was preferentially secreted. In contrast, cells infected with the MVA/IRES/tPA/V307 recombinant virus expressed only a single form of 35 kd. The predicted V and V307 open reading frames do not encode consensus glycosylation sequences for vertebrate cells. In the case of V antigen expression, the addition of IRES/tPA slightly reduced protein expression levels. In infected CEF cells, the ratio of secreted to cellular V antigen seemed slightly enhanced with the addition of the IRES/tPA sequence (FIG. 2C). However, the ratios seemed consistent between all the constructs in infected Vero cells (FIG. 2D).
FIGS. 2A-2E illustrates experiments concerning monolayers of CEF or Vero cells transfected with recombinant MVA-plague viruses at MOI of 0.5 pfu/cell. After 48 h post transfection, cells were harvested, cellular and supernatant extracts were prepared and subjected to SDS-PAGE followed by western blot analysis as described in the methods. (A) F1 expression in CEF cellular extracts (c) and supernatant (s) fractions. (B) F1 expression in Vero cell extracts (c) and supernatant (s) fractions. (C) V and V307 expression in CEF cell (c) and supernatant (s) fractions. (D) V and V307 expression in Vero cell (c) and supernatant (s) fractions. (E) Effect of glycosidase treatment on molecular forms of F1. F1 expression from CEF cell and supernatant (sup) fractions treated with glycosidase (+) or without treatment (−). The F1 control lane contains 10 ng expressed in E. coli from a caf1 operon expression vector (approximate mass 18 kd).
The expression of the F1, V and V307 antigens directed by the recombinant MVA viruses was assessed by immuno-blot analyses. Antigen expression from the different MVA recombinants was evaluated in MVA permissive CEF cells and non-permissive mammalian Vero cells (FIGS. 2A and 2B). MVA/F1 recombinants caused expression of F1 proteins of in both cellular extracts and cell supernatants. Due to aggregation of the cF1 capsular antigen, we typically observe both monomer and dimer forms of the F1 protein in SDS-PAGE. Supernatant forms are of higher molecular weight, presumably due to glycosylation of the F1 protein. (FIGS. 2A and B) MVA/V constructs showed significantly higher expression in CEF (FIG. 2C) as compared to Vero (FIG. 2D). MVA/V and MVA/V307 had similar expression levels in CEF (FIG. 2C). Although MVA/IRES/tPA/V307 expression was lower compared to MVA/V or MVA/V307, the V307 protein was more efficiently secreted. (FIG. 2C).
Overview
In one example, a construct composition including a truncated version of the low-calcium response V (V307) antigen from Yersinia pestis under translational control of encephalomyocarditis virus (EMVC) internal ribosomal entry site (IRES) and with the tissue plasminogen activator (tPA) secretory signals was administered to mice. The construct composition conferred enhanced immunogenicity and consistently conferred significant protection in mice (87.5%-100%) against intranasal or intraperitoneal challenge with CO92 (encapsulated) or Java 9 (non-encapsulated) strains of Y. pestis, respectively. Although the MVA construct expressing the full version of V antigen was highly immunogenic it provided significantly less protection (37.5%) against CO92 or Java 9 strains, respectively in this experiment. An MVA construct expressing the capsular protein (F1) failed to elicit detectable antibodies but conferred 50% and 25% protection against CO92 or Java 9 challenge, respectively. All the MVA vectored plague vaccines tested in this study were shown to be completely safe in severe combined immuno-deficient (SCID) mice. MVA has been stockpiled for use as a second-generation smallpox vaccine, with superior safety to the original live, attenuated vaccinia strains. Thus, a recombinant MVA/IRES/tPA/V307 vaccine has the potential to simultaneously provide protection against smallpox and plague.
In these examples, the following were tested: i) the suitability of MVA to express Y. pestis F1 and V antigens; ii) the immunogenicity and protective capacity of MVA-based recombinants in mice; iii) the influence of an internal ribosomal entry site (IRES) of encephalomyocarditis virus in combination with the secretory signal of tissue plasminogen activator (tPA) on the immunogenicity and protective capacity of MVA-based vaccine candidates; and iv) the safety of MVA recombinants in immunocompromised mice. The findings demonstrated that a recombinant MVA virus expressing a truncated form of Y. pestis V antigen in the presence of the IRES and tPA (MVA/IRES/tPA/V307) provided increased immunogenicity, safety, and protection against challenge with different strains of Y. pestis in mice.
In addition, a study of the safety and efficacy of MVA-vectored candidate vaccines that can express and export F1 and V antigens of Y. pestis are described. In immunogenicity studies in BALB/c mice, the MVA-vectored vaccines expressing the V antigen elicited robust antibody responses. Given the strong immunogenic potential of these MVA/V constructs subsequent studies focused on this antigen. The V protein is required for human or animal infectious disease by the three pathogenic Yersinia species, (e.g., Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis). In one example, MVA expressing a variant of the V antigen in which the V antigen is truncated to remove the segment associated with the suppression of endogenous IL-12, TNF-α and IFN-γ in vivo, was tested for immunogenicity and protection. Moreover, effect of combining the IRES translational control sequence with the tPA secretory signal on the immunogenicity and protective capacity of the truncated V antigen was examined. All the MVA constructs expressing the V antigen were found to be immunogenic. When the protective capacities of all generated MVA constructs were tested in a mouse model of pneumonic plague, the MVA/IRES/tPA/V307 consistently induced protection against lethal challenge with the highly virulent Y. pestis CO92 strain at 35LD50 as well as at 350LD50. The protection observed with the MVA/V307 construct was significantly lower (P<0.05) than that induced by the MVA/IRES/tPA/V307 vaccine. Thus, the IRES/tPA processing signals appear to potentiate the protective immune response
In these studies, MVA/F1 vaccine failed to elicit a significant antibody response against the F1 antigen. However, 50% of immunized mice were protected against challenge with Y. pestis CO92 strain with a median survival time of 11 days. Either very low antibody response to F1 or a cellular immune response to the protein may contribute to protect from Y. pestis infection.
Production of high levels of antigen-specific antibodies elicited by the MVA/IRES/tPA/V307 construct may account for the high level of protection observed in these studies. However, the MVA/V construct generated high anti-V antibody titers yet less protection from challenge.
Naturally-occurring non-encapsulated variants of Y. pestis have been shown to be virulent. An effective plague vaccine should be able to protect against infection with both encapsulated and non-encapsulated variants of Y. pestis. In these studies, a vaccine candidate construct having MVA/IRES/tPA/V307 conferred complete protection against a non-encapsulated strain of Y. pestis (Java 9). Preliminary data suggest that this protection was partially mediated by antibodies; passive transfer of anti-MVA/IRES/tPA/V307 antibodies protected from Java 9 challenge.
These MVA vectored plague vaccines were tested for safety in immunocompromised (SCID) mice. Infection of SCID mice with replication competent poxviruses causes significant weight loss and poxvirus lesions analogous to the disseminated viremia that can occur in vaccinated individuals with underlying immune deficiencies. In these studies, it was demonstrated that the MVA constructs expressing plague antigens are safe in SCID mice and fail to induce systemic disease even in the absence of effective B or T cell immunity.
These studies demonstrate the potential of MVA to effectively express Y. pestis antigens and generate protective immune responses. The MVA constructs expressing the V307 antigen in conjunction with signal sequences was shown to be very immunogenic, safe, conferring protection against intranasal or intraperitoneal challenge with Y. pestis.
All the MVA-V constructs were immunogenic, but when tested for protection against plague in mice, the MVA/IRES/tPA/V307 induced 87.5 to 100% protection against lethal challenge with the highly virulent Y. pestis CO92 strain. This finding is in agreement with earlier reports indicating improved immunogenic properties of a subunit candidate vaccine based on V307 in offering enhanced protection against plague
These findings will lead to the development of new vaccination strategies, for example, for biodefense since MVA has been stockpiled for use as a second-generation smallpox vaccine. A vaccine that simultaneously generates protective immune responses to two biological threats, smallpox and plague, may be a valuable biodefense tool.
FIG. 1 illustrates construction of rMVA/Y. pestis antigen viruses. In this example, expression cassettes for each of the Y. pestis antigens, F1, V and V307 were inserted into pdIIIGFP. Another cassette contained the EMCV-EMCV IRES sequence followed by the tPA secretory signal was fused to the V307 antigen coding sequence. The cassettes were generated by PCR to contain the SmaI and the BamHI restriction sites. Each expression cassette was inserted into a plasmid that contained DNA segments (flank 1 and flank 2) adjacent to deletion III within the HindIII A fragment of MVA. The plasmid also contained a strong synthetic early/late vaccinia virus promoter upstream of a multiple cloning site (MCS) and coding sequences for GFP under the control of a divergent synthetic vaccinia virus early/late promoter.
Example 2
Immunogenicity of MVA Constructs
In another exemplary method, groups of BALB/c mice were immunized intramuscularly with MVA/Y. pestis constructs encoding the F1, V or V307 antigens. Antibody titers after a single immunization (pre-boost) and after two immunizations (pre-challenge) were assessed by ELISA analysis. Pre-challenge antibody titers elicited by the MVA/V construct were significantly higher (P<0.05) than the MVA/V307 construct, however, there was no significant difference between pre-boost titers induced by these constructs (FIG. 3B). The effect of the IRES and tPA sequences on immunogenicity was examined. As shown in FIG. 3B, expression of V307 under the control of IRES and secretory signals (MVA/IRES/tPA/V307) significantly enhanced its immunogenicity. Both pre-boost and pre-challenge antibody titers were significantly higher (P<0.05) in mice immunized with MVA/IRES/tPA/V307 than with MVA/V307. Analysis of IgG subclasses elicited by the MVA/IRES/tPA/V307 and MVA/V307 constructs showed a balanced response between IgG1 and IgG2a subclasses (data not shown). MVA constructs expressing the V antigen induced a significant booster effect (P<0.05) on antibody responses in all immunized mice as compared to primary immunization (FIG. 3B). No booster effect was detected in mice immunized with the MVA/F1 or MVA/IRES/tPA/F1 constructs. Moreover, pre-challenge antibody titers elicited by the MVA/F1 or MVA/IRES/tPA/F1 constructs were significantly lower (P>0.05) than titers induced by constructs expressing the V antigen (FIGS. 3A and 3B).
FIG. 3 illustrates immune responses to MVA/plague vaccines in mice. Groups of eight 4-6 week-old BALB/c mice were vaccinated intramuscularly with MVA-plague vaccines. Following two immunizations separated by 28 days, serum samples collected on days 28 and 42 post-initial vaccinations and analyzed by ELISA to determine humoral immune responses to F1 or V antigens. (A) Antibody responses to F1 antigen. (B) Antibody responses to V antigen from mice immunized with MVA/plague vaccines.
Example 3
Protection Against Plague Challenge
In other examples, an animal model was used to assess protection against plague after introduction of some constructs disclosed herein. All mice vaccinated with the MVA/IRES/tPA/V307 survived lethal plague challenge with either the CO92 (35 LD50) or Java 9 (100 LD50) strain of Y. pestis (FIGS. 4A and 4B). Moreover, passive transfer of pooled immune serum from mice immunized with MVA/IRES/tPA/V307 to naïve BALB/c mice conferred significant protection (P<0.05) against the Java 9 strain of Y. pestis as compared to the control mice (data not shown). In contrast, only 25%, 37.5% to 50% of mice immunized with MVA/V307, MVA/V, or MVA/IRES/tPA/V survived challenge with the CO92 strains of Y. pestis, respectively (FIG. 4A). Mice immunized with MVA/F1 or MVA/IRES/tPA/F1 had 50% or 25% survival rate against challenge with CO92 or Java 9 strain of Y. pestis, respectively (FIGS. 4A and 4B).
FIG. 4 illustrates a Kaplan-Meier survival analysis of mice immunized with MVA/plague vaccines. Two weeks following booster immunizations, mice were challenged (A) intranasally with 1×105 pfu (35LD50) of Y. pestis (CO92) or (B) intraperitoneally with 100 cfu (100 LD50) of Y. pestis (Java 9) and survival rates were recorded over a period of 2 weeks.
Example 4
Minimal Protective Dose of MVA/IRES/tPA/V307 Vaccine
To establish the minimal protective dose of the lead candidate vaccine, groups of 8 BALB/c mice were immunized (prime and boost) with increasing doses (5×105 pfu, 5×106 pfu or 5×107 pfu) of MVA/IRES/tPA/V307 and then challenged with either 35 or 350 LD50s of the CO92 Y. pestis strain. Mice immunized with increasing doses of MVA/IRES/tPA/V307 elicited corresponding increased immune responses with pre-challenge antibody titers of 3.38, 3.75 or 4.25 (log 10), respectively. The highest immunization dose (5×107) elicited significantly higher antibody titer (P<0.05) compared to the lower doses and it conferred significant protection (87.5%) against challenge with either 35 or 350 LD50s of the CO92 Y. pestis strain, respectively (Table 2, below). There was no significant difference (P>0.05) between survival rates of mice immunized with the two lower doses of the MVA/IRES/tPA/V307 vaccine following challenge with 35LD50 of Y. pestis (Table 2). A 10-fold increase in the challenge dose (350 LD50) reduced the survival conferred by the lowest dose (5×105 pfu) of the MVA/IRES/tPA/V307 vaccine and was not significantly different (P>0.05) from that of the MVA/GFP control group (Table 2). However, significant protection (P=0.01) was conferred on mice immunized with 5×106 pfu of the vaccine against challenge with 350LD50 of Y. pestis compared to the control group.
TABLE 2
Survival rate of mice immunized with increasing doses of
MVA/IRES/tPA/V307 and subsequently challenged via the
intranasal route with CO92 strain of Y. pestis
% Survival
Vaccination
Challenge
(14 days
Median Survival
Dose (pfu)
(LD50)
post-challenge)
Time (Days)a
5 × 105
35
62.5
N/A
5 × 106
35
37.5
10.5
5 × 107
35
87.5
N/A
5 × 107 (MVA)
35
12.5
3.0
5 × 105
350
12.5
3.0
5 × 106
350
37.5
6.0
5 × 107
350
87.5
N/A
5 × 107 (MVA/gfp)
350
0.0
3.0
aMedian survival time is the time at which 50% of animals have died. This value is not applicable (N/A) for groups with >50% survival rates.
Safety of MVA/Plague Vaccine Candidates in Immunocompromised Mice
All mice in the vaccinia-Wyeth inoculated-group developed clinical disease symptoms characterized by pox lesions on their tails and feet and persistent weight loss; they died within 5-7 weeks post-infection. None of the animals from the MVAwt or MVA/Y. pestis vaccine constructs developed any pox lesions. Weight loss in the vaccinia-Wyeth group was significantly greater (P<0.0001) than in groups that were infected with MVAwt or MVA/plague vaccine constructs (FIG. 5).
Example 5
Antibody Titers
FIG. 6 represents groups of mice (n=10) were immunized via the intramuscular or intradermal route with 5×107 PFU of MVA/plague vaccines expressing different secretory signals. Antibody titers (Mean±SD) following prime and boost immunization with MVA/Plague vaccines were analyzed by Elisa. Post-boost titers from mice immunized with MVA/tPA/V307 (IM) or MVA/IRES/tPA/V307 (ID) were significantly higher than MVA/C13L/V307 or MVA/IRES/tPA/V307 (IM) groups (P values **<0.01, *<0.05).
There were no significant differences between pre-boost antibody titers from the various immunized groups of mice. The co-expression of tPA (MVA/tPA/V307) significantly improved the immunogenicity of intramuscularly administered MVA/plague vaccine compared to vaccine co-expressing the C13L secretory signal (MVA/C13L/V307, P<0.01) or additionally expressing the IRES translational enhancer (MVA/IRES/tPA/V307, P<0.05). Intradermal administration of MVA/IRES/tPA/V307 significantly increased (P<0.05) the immunogenicity of the vaccine.
Materials and Methods
Construction of MVA Recombinant Vaccines
The transfer plasmid pdIIIGFP (provided) was used to generate recombinant MVA expressing Y. pestis antigens. This plasmid contained: 1) DNA segments (flank 1 and flank 2) adjacent to deletion III within the HindIII A fragment of MVA, 2) a strong synthetic early/late (SEL) vaccinia virus promoter upstream to a multiple cloning site (MCS), and 3) the green fluorescent protein (GFP) gene under the control of a divergent SEL promoter (FIG. 1). A second transfer plasmid, pdIIIGFP/IRES/tPA, containing the ECMV IRES sequence followed by the tPA secretory signal was generated by insertion of an IRES/tPA cassette into pdIIIGFP. Expression cassettes for each of the Y. pestis antigens, F1, full lengthV and V truncated at aa 307 (V307), were inserted into pdIIIGFP or pdIIIGFP/IRES/tPA. Expression cassettes were generated by PCR (Table 1) to contain appropriate restriction sites for insertion into pdIIIGFP or pdIIIGFP/IRES/tPA. The PCR products were cloned into the MCS of pdIIIGFP or pdIIIGFP/IRES/tPA and the resulting plasmids were designated as pdIIIGFP/F1, pdIIIGFP/IRES/tPA/F1, pdIIIGFP/V, pdIIIGFP/IRES/tPA/V, pdIIIGFP/V307 and pdIIIGFP/IRES/tPA/V307.
TABLE 1
Yersinia pestis antigen PCR primer sequences. Restriction Enzyme sites
capitalized.
PCR Primer Sequence
RE Site
5′F1
5′-gtgaGTCGACatgaaaaaaatcagttccgttatc-3′ (SEQ. ID. NO: 1)
SalI
3′F1
5′-gcGAATTCttattggttagatacggttacggt-3′ (SEQ. ID. NO: 2)
EcoRI
5′V native
5′-gtgaGTCGACatgattagagcctacgaacaaaacc-3′ (SEQ. ID. NO: 3)
SalI
5′V I/t
5′-tgacGCCGGCattagagcctac-3′ (SEQ. ID. NO: 4)
NgoMIV
3′V full
5′-cgcGAATTCtcatttaccagacgtgtcatc-3′ (SEQ. ID. NO: 5)
EcoRI
3′V307
5′-gcGAATTCtcaacggttcagtgcttcaatag-3′ (SEQ. ID. NO: 6)
EcoRI
Recombinant MVA-plague viruses were generated as described previously. Briefly, chicken embryo fibroblasts (CEF), were infected with wild type MVA at a multiplicity of 0.05 and one hour (h) later the cells were transfected with each of the transfer vectors using Lipofectamine™ (Invitrogen, Carlsbad, Calif.). At 48-72 h post-transfection, monolayers were harvested, centrifuged at 500 RCF for 5 minutes at 4° C. and cells disrupted by freeze-thaw and sonication (2 times for 15 seconds using a Virtis600 at setting 3). The disrupted cell extracts containing possible recombinant viruses expressing GFP were plated onto fresh CEF cells and overlaid with 0.8% agarose. After 48-72 h, recombinant virus-generated plaques were detected by fluorescence and picked into media with a glass pipette. The cell/virus samples were sonicated and plated as described above. After three consecutive rounds of plaque isolation, high titer virus stocks were prepared in CEF cells for subsequent in vitro and in vivo characterization.
In Vitro Expression of Y. pestis Antigens
The in vitro expression of recombinant MVA viruses containing the F1, V, V307 or IRES/tPA/F1, IRES/tPA/V, IRES/tPA/V307 antigens was determined by immuno-blot analyses. CEF or Vero cells were plated into 6-well plates and infected with the recombinant MVA/F1, MVA/IRES/tPA/F1, MVA/V, MVA/IRES/tPA/V, MVA/V307 or MVA/IRES/tPA/V307 viruses at an MOI of 0.5 or 5, respectively in serum free conditions. At 48 h post-infection, the infected cells were harvested in the presence of a protease inhibitor cocktail (Mini Protease tabs, Roche Diagnostics, Indianapolis, Ind.), washed, resuspended in 1× loading buffer and heated to 95° C. for 5 min. The supernatants from the infected cells were centrifuged and concentrated by ultrafiltration with a 3 kDa cutoff membrane (Nanosep 3K Omega, Pall, Inc., East Hills, N.Y.). The supernatants were then combined with an equal volume of 2× loading buffer and heated to 95° C. for 5 min. Supernatant and cell samples were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane for immuno-blot analysis using polyclonal rabbit anti-F1 or anti-V serum produced in-house. The polyclonal antibodies were generated by inoculating specific pathogen free (SPF) rabbits with purified F1 (from caf1 operon expression system) or V (ATCC, BEI Resources, Manassas, Va., cat# NR-3832) proteins. These antibodies showed minor background to MVA wild type expressed from CEF or Vero when used in immunoblot analyses.
The F1 glycosylation state was analyzed using a protein deglycosylation enzyme mix (New England Biolabs (NEB), Ipswich, Mass., cat# P6039S). Briefly, Vero and CEF cells were infected with MVA/F1 or MVA/IRES/tPA/F1 and harvested as previously described. The cell pellets, suspended in 30 ul H2O, and concentrated supernatants were processed using the NEB kit protocol. 18 μl of the resuspended cell pellet and concentrated supernatant samples, were denatured for 10 minutes at 100° C. G7 reaction buffer containing 10% NP40 was added to bring the reaction volume to 50 μl. Each reaction was split and half was treated with 2.5 ul NEB deglycosylation enzyme cocktail and incubated at 37° C. for 4 hr. 10 ul, of each reaction, was subjected to electrophoresis and analyzed by Western using the polyclonal rabbit anti-F1 serum.
Yersinia pestis Cultures
Preparation of Y. pestis cultures and subsequent animal challenge experiments were conducted as previously described. Briefly, to prepare a working stock, 75 μL of the frozen Y. pestis isolate was thawed, vortexed, spread onto blood agar plates (Remel, Lenexa, Kans.), and incubated at 28° C. for 48 h. The bacterial lawn was scraped from the agar plates into 200 ml Heart Infusion Broth (Difco Laboratories, Detroit, Mich.) with 0.2% xylose and incubated at 28° C. for 48 h. Final stocks were prepared by adding 20% glycerol to the broth culture (v/v) and were stored in aliquots at −80° C. The bacterial strains CO92 and Java 9 of Y. pestis were provided. The F− strain of choice for these studies would have been the C12 strain (this one was not available at the time); so F1− Java 9 which was readily available was used.
Immunization and Challenge
Groups of eight 4-6 week-old female BALB/c mice (Harlan Sprague Dawley, Indianapolis, Ind.) received primary and booster immunizations (28 days apart) with each vaccine candidate via intramuscular injections into the hind legs. A dose of 5×107 plaque forming units (pfu) in 50 μl was used for both injections. Control groups were immunized with either recombinant F1 protein (rF1—40 μg), empty MVA vector (MVA/GFP—5×107 pfu) or with phosphate buffered saline (PBS—50 μL). At two weeks post-boost, all animals were challenged with the wild-type Y. pestis CO92 strain by intranasal instillation of 10 μL (5 μL into each nostril) of inoculum containing 1×105 colony forming units (cfu) (35LD50) of the bacteria. Animals challenged with the Java 9 strain of Y. pestis received 100 μl inoculum containing 100 cfu (100LD50) intraperitoneally. The isolate of Java 9 used in this study was less virulent via the intranasal route but highly virulent when used intraperitoneally. Challenged animals were monitored for two weeks.
A group of eight 4-6 week-old naïve mice was passively immunized intraperitoneally with 100 μl of post-boost pooled serum with a titer of 100,000 from mice immunized with two doses of MVA/IRES/tPA/V307. The passively immunized mice were challenged intraperitoneally with a 100 μl inoculum containing 100 cfu (100LD50) of the Java 9 strain of Y. pestis. Challenged animals were monitored for two weeks. The stability and virulent phenotype of the Y. pestis CO92 or Java 9 frozen stock cultures were validated by testing aliquots for bacterial counts and lethal doses. The number of colony-forming units was determined by plating 100 μL of each dilution onto blood agar and incubating at 28° C. for 48 h. Lethal dose (LD50) values were determined by inoculating groups of 11-12 week-old BALB/c mice intranasally (10 μL) or intraperitoneally (100 μL) with 10-fold dilutions of the Y. pestis CO92 or Java 9 bacterial cultures, respectively. Following inoculation, animals were monitored for 14 days, mortalities were recorded and LD50 was calculated by a method known in the art.
Serology
Serum samples were collected on day 28 post-primary vaccination and day 14 post-boost (pre-challenge) to assess antibody titers against Y. pestis F1 or V antigens. Serum total IgG as well as IgG1 and IgG2a subclass titers were measured by enzyme-linked immunosorbent assay (ELISA) as described previously. Briefly, 96-well ELISA plates were coated with purified recombinant F1 or V antigen (0.1 μg in 100 μL carbonate buffer, pH 9.6 per well) at 4° C. overnight. Coated plates were washed twice with 0.05% TWEEN 20 in PBS (washing buffer) and rinsed with blocking buffer (1% BSA in PBS) for 1 h at room temperature (RT). Serum samples then were serially diluted from 1:100-1:100,000 in ELISA diluent (0.1% BSA in washing buffer) and added in triplicate to the prepared ELISA plates. Known negative and positive serum samples from mice inoculated with recombinant F1 or V antigen from previous studies were used as controls and plates were incubated for 1 h at RT. After washing, 100 μL per well of a 1:10,000 dilution of horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Abcam Inc, Cambridge, Mass.) was added to each well and incubated for 1 h at RT. Plates were washed, and 100 μL per well of tetra-methyl-benzidine (TMB) chromogen (Invitrogen, Calsbad, Calif.) was added to each well and incubated in the dark for 5 minutes. The reaction was then stopped by adding 100 μL per well of 2 mM H2SO4 (Sigma, St Louis, Mo.). Colorimetry was assessed using a microplate reader (ELx800-BioTek, Winooski, Vt.) at test wavelength of 450 nm and a reference wavelength of 630 nm. The highest dilution that was positive (exceeded the mean of known negative serum samples plus three standard deviations) was considered the endpoint, and its reciprocal value was recorded as the titer.
Groups of six, five-week old BALB/c SCID mice (Harlan Sprague Dawley, Indianapolis, Ind.) were inoculated intraperitoneally with 1×108 pfu of MVA/F1, MVA/V, MVA/V307, MVA/IRES/tPA/V307 or wild type MVA (MVAwt). An additional group received 1×106 pfu of the vaccinia Wyeth strain via the same route. Mice were monitored daily for 3 months and their weight was recorded weekly. Mice died naturally or were euthanized when showing body-conditioning score less than two (BCS<2) as previously described.
Statistical Analysis
One way ANOVA was used to evaluate the vaccine group effects on pre-boost and pre-challenge antibody titers. If the vaccine group effect was statistically significant (P<0.05 by Kruskal-Wallis test), an all pair-wise comparison among groups was performed using an unadjusted P-value of 0.05. Survival analysis was performed to assess vaccine effectiveness against challenge with either CO92 or Java 9; reported P-values are from Fisher's exact test. Probability values<0.05 were considered significant using the GraphPad Prism 5 software (La Jolla, Calif.) for all statistical analyses.
All of the COMPOSITIONS and METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of preferred embodiments, it is apparent to those of skill in the art that variations maybe applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.
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13511652
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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424/ 93.6
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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tyo:4502
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Takeda Pharmaceutical
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Mar 3rd, 2015 12:00AM
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Jun 10th, 2012 12:00AM
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https://www.uspto.gov?id=US08968996-20150303
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Compositions and methods for rapid immunization against dengue virus
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Embodiments of the present invention report compositions and methods for vaccinating a subject against all dengue virus serotypes. In some embodiments, multiple vaccine compositions may be administered to a subject in different anatomical locations in order to induce a rapid response to all dengue virus serotypes. In certain embodiments, administration of two or more vaccine compositions to a subject against all dengue virus serotypes may include two or more routes of administration.
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8968996
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1. A method for inducing neutralizing antibodies in a subject against three or more dengue virus serotypes, comprising, administering two or more doses of a single immunogenic composition of a mixture of three or more different dengue-dengue chimeras of live, attenuated dengue viruses to the subject at two or more anatomical locations on the same day, inducing neutralizing antibodies in the subject against three or more dengue virus serotypes.
2. The method of claim 1, further comprising administering at least one additional booster administration of an immunogenic composition of live, attenuated dengue viruses 1 to 180 days after the same day administrations of the immunogenic composition claim 1.
3. The method of claim 1, wherein the single immunogenic composition comprises a predetermined ratio of live, attenuated dengue viruses representing the three or more dengue virus serotypes in the single composition.
4. The method of claim 1, wherein the single immunogenic composition comprises equivalent ratios of live, attenuated dengue viruses representing the three or more dengue virus serotypes in the single composition.
5. The method of claim 2, wherein the immunogenic composition used for at least one additional booster administration is identical to the single immunogenic composition used for the same day administrations of the immunogenic composition of claim 1.
6. The method of claim 2, wherein the immunogenic composition used for at least one additional booster administration is different than the single immunogenic composition used for the same day administrations of claim 1 and comprises pre-determined concentrations of one or more monovalent live, attenuated dengue virus serotypes.
7. The method of claim 6, wherein the pre-determined concentration of dengue virus serotypes includes a higher concentration of one or more live, attenuated dengue virus serotypes than the single immunogenic composition used for the same day administrations of claim 1.
8. The method of claim 7, wherein the higher concentration is 2 to 100,000 fold greater concentration than used in the single immunogenic composition according to claim 1.
9. The method of claim 1, wherein the two or more anatomical sites comprise different anatomical locations using the same mode of administration.
10. The method of claim 1, wherein the two or more anatomical sites comprise different anatomical locations using different modes of administration.
11. The method of claim 1, wherein modes of administration of the single immunogenic composition comprise subcutaneous (SC), intradermal (ID), or intramuscular (IM).
12. The method of claim 2, wherein at least one additional booster is administered to the subject within 30 days after the same day single immunogenic composition administrations.
13. The method of claim 1, further comprising administering at least one immunogenic agent to the subject.
14. The method of claim 1, wherein the single immunogenic composition comprises all four dengue virus serotypes.
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14
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PRIORITY
This application is a continuation-in-part application and claims the benefit under 35 USC §120 of U.S. Non-Provisional application Ser. No. 12/790,511 filed May 28, 2010 which claims priority under 35 USC §119(e) to U.S. Provisional Application Ser. No. 61/183,020 filed on Jun. 1, 2009. All prior applications are incorporated herein by reference in their entirety for all purposes.
FIELD
Embodiments of the present invention report compositions and methods for administering a vaccine to a subject against all dengue virus strains. In some embodiments, vaccine compositions may be administered by subcutaneous, intradermal, intramuscular or other injection or introduction methods. In certain embodiments, injection in a subject of a vaccine against all dengue virus types includes multiple anatomical sites at day 0. Other embodiments include follow-on injections from within days of the first treatment to up to 12 months after initial injection(s). In other embodiments, no additional injections are needed other than the day 0 treatment. In certain embodiments, subcutaneous, intradermal, intramuscular or other modes of introducing to a subject, a vaccine composition against dengue virus to provide protection against three or more of the dengue serotypes DEN-1, DEN-2, DEN-3 and DEN-4 upon administration at day 0.
BACKGROUND
Vaccines for protection against viral infections have been effectively used to reduce the incidence of human disease. One of the most successful technologies for viral vaccines is to immunize animals or humans with a weakened or attenuated strain of the virus (a “live, attenuated virus”). Due to limited replication after immunization, the attenuated strain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and can generate potent and long-lasting immune responses to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the immunized individual is protected from disease. These live, attenuated viral vaccines are among the most successful vaccines used in public health.
SUMMARY
Embodiments of the present invention generally relate to methods and compositions for inducing protection in a subject against multiple dengue viruses by, for example, administering a multivalent dengue vaccine to a subject. Some embodiments can include introducing a vaccine composition to a subject via intradermal (ID) injection. In accordance with these embodiments, the vaccine composition can be introduced to a subject intradermally to, for example, to induce neutralizing antibodies against three or more dengue virus serotypes. In certain embodiments, a vaccine composition can include, but is not limited to, a single dose of one formulation of a multivalent dengue serotype vaccine having a predetermined ratio administered to a subject. In other embodiments, a vaccine composition may include, but is not limited to; an initial dose of one formulation of dengue vaccine (e.g. tetravalent formulations such as DENVax™) and then one or more boosts of the same, or a different formulation can be administered to a subject.
Other aspects herein can concern inducing a humoral or cellular immune response in a subject by, for example, introducing a vaccine composition to a subject via an intradermal route wherein the vaccine composition includes, but is not limited to, a dengue virus vaccine. In accordance with these embodiments, compositions disclosed can be administered intradermally to a subject for modulating neutralizing antibody production in the subject against three or more dengue virus serotypes. Some aspects concern predetermined composition ratios (e.g. 1:1:1, 10:1 1:2:2, 1:10; 10:1, 3:4:3:3, 1:4:1; 5:5:4:5; or any ratio of three or more serotypes is contemplated) of the various serotypes of dengue virus or fragments thereof or attenuated compositions thereof in a single vaccine composition in order to increase cross protection and levels of neutralizing antibodies in a subject against at least three dengue virus serotypes when the subject is administered the single vaccine composition.
In certain embodiments, some advantages of using intradermal introduction of a vaccine against dengue virus can include, but are not limited to, multiple protection (cross protection) against some or all dengue virus serotypes in a subject, reduced cost by using reduced volumes of vaccine doses compared to subcutaneous injection, modulation of antibodies produced against some or all dengue virus serotypes in a subject and reduced pain at a site of administration in a subject administered a composition of vaccine against dengue virus.
In some embodiments, a single dose vaccine against dengue virus can include one or more dengue virus serotype(s). In addition, certain embodiments concern treating a subject with at least one additional injection(s) of a vaccine containing multiple dengue viruses administered at a separate site from the first injection, for example, in close proximity to the initial injection or in a distant anatomical site on the subject. In addition, at least one additional intradermal injection(s) may be performed less than 30 days after the first administration to the subject while others are performed 30 days and up to 12 months after the first administration of the vaccine.
Other embodiments disclosed herein relate to methods and compositions for inducing protection in a subject against all dengue virus serotypes by, for example, administering a vaccine to a subject against all dengue virus serotypes in two or more doses on one or more than one anatomical location consecutively within a short interval of time. Some embodiments can include introducing a vaccine composition to a subject via intradermal (ID), subcutaneous (SC), or intramuscular (IM) injection in one location and consecutively in another anatomical location by ID, SC, IM or by other introduction method at a second different anatomical location. Other embodiments include using any combination of modes of administration for introducing a dengue virus vaccine of all dengue virus serotypes to a subject where administration of the vaccine occurs at two or more anatomical sites or by two or more different routes consecutively on the same day to the subject.
Some embodiments include treating a subject in need of dengue virus tetravalent vaccinations consecutively at two or more anatomical locations. In certain embodiments, a subject may need two consecutive administrations in a single day to induce adequate levels of neutralizing antibodies which will protect against dengue infection. In other embodiments, a subject may be administered dengue virus multivalent vaccinations consecutively at two or more anatomical locations, then the subject can be administered at least a third vaccine within 30 days such as about 7, about 14, about 21 or about 28 days later with a composition comprising dengue virus serotypes which may or may not have all serotypes. In other embodiments, a subject may be administered dengue virus tetravalent vaccinations consecutively at two or more anatomical locations on day 0, then the subject can be administered at least a third vaccine within 30 days such as about 7, about 14, about 21 or about 28 days later with a composition comprising dengue virus serotypes which may or may not have all serotypes. Vaccine compositions of these and other embodiments disclosed herein may include two or more dengue virus serotypes at a predetermined ratio for the subsequent administrations beyond the initial dual vaccination. These subsequent vaccinations may depend on personalized titers of antibodies post dual injection or other criteria such as results of test populations. In certain embodiments, a subsequent vaccination may only include a single dengue serotype (e.g. DEN-4).
In certain embodiments, the composition introduced to the subject comprises vaccines against all dengue virus serotypes, for example tetravalent DENVax™ or another similar formulation. DENVax™ comprises a tetravalent dengue vaccine of predetermined ratio where the vaccine is made up of constructs on an attenuated DEN-2 backbone (see for example, PCT Application Number PCT/US01/05142 filed on Feb. 16, 2001 incorporated herein by reference in its entirety for all purposes). In other compositions, all dengue vaccine virus serotypes are in equal proportions in the composition. In yet other compositions, each dengue vaccine virus serotype may be in a particular ratio to one another such that introduction of the composition induces sufficient levels of neutralizing antibodies which would provide the subject with sufficient protection against infection with three or more dengue viruses (e.g. DEN-1, DEN-2, DEN-3 and/or DEN-4). For example, if a subject, after receiving two or more compositions consecutively at two or more anatomical locations and the subject has lower protection to one or more particular dengue virus serotypes, then a booster for that subject can contain a multiple (more than two) vaccine components or a single vaccine component to improve immune responses to all four dengue viruses in the subject. In accordance with these embodiments, samples from a subject may be analyzed for resistance to dengue infection using standard means known in the art.
In certain embodiments, the vaccine composition can be introduced to a subject by any route in multiple anatomical locations to, for example, protect against three or more dengue serotypes after consecutive administrations. In certain embodiments, a vaccine composition can include, but is not limited to, a single dose of a formulation containing all serotypes of dengue virus (e.g. DENVax™) administered to a subject capable of providing protection against at least three dengue virus serotypes. In other embodiments, a vaccine composition can include attenuated dengue virus serotypes in combination with other anti-pathogenic compositions (e.g. Japanese encephalitis, yellow fever, West Nile, influenza, Chikungunya or other). Compositions contemplated herein can be administered by any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, ingested, and any other method. Introduction in two or more anatomical sites can include any combination administration including by the same mode in two or more anatomical sites or by two or more different modes that include two or more separate anatomical sites. In accordance with these embodiments, two or more anatomical sites can include different limbs. In other embodiments, vaccinations can be delivered to a subject using any device known in the art including, but not limited to, a needle and syringe, jet injection, microneedle injection, patch delivery (e.g. skin), intradermal delivery devices, inhalation device, intranasal device, slow release microparticles, and any other acceptable vaccine-delivery device.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain embodiments. Some embodiments may be better understood by reference to one or more of these drawings alone or in combination with the detailed description of specific embodiments presented.
FIG. 1 represents an example of an intradermal injection device currently available.
FIG. 2 represents examples of injection sites in a non-human primate subject having intradermal introduction of a vaccine against dengue virus.
FIG. 3 represents a bar graph comparison of neutralizing antibody titer produced against different ratios of dengue virus serotypes after a one (primary) administration via the subcutaneous (SC) versus intradermal (ID) route of injection of a vaccine against dengue virus.
FIG. 4 represents a bar graph comparison of neutralizing antibody titer produced against different dengue virus serotypes after a second, boosting administration via the subcutaneous (SC) versus intradermal (ID) injection of a vaccine against dengue virus.
FIG. 5 represents a histogram plot of neutralizing antibody titers after subcutaneous and intradermal immunizations with a vaccine against a dengue virus serotype-4 in mice.
FIGS. 6A and 6B represent graphic depictions of mouse survival after vaccination with a dengue vaccine followed by a challenge with wild-type dengue virus. Mice were vaccinated by SC or ID route of infection with a dengue vaccine (e.g. DENVax-4) or a buffer/placebo (e.g. TFA).
FIG. 7 represents neutralizing antibody titers for DEN-1, DEN-2, DEN-3 and DEN-4 at day 28 and day 56 after two day-0; or 1 day-0 and 1 day-42 injections (e.g. DENVax™; 4:3:4:5 ratio).
FIG. 8 represents neutralizing antibody titers for DEN-1, DEN-2, DEN-3 and DEN-4 at day 28 and day 56 after two day-0; or 1 day-0 and 1 day-42 injections (e.g. DENVax™; 3:3:3:3, approximately equivalent amounts used).
FIGS. 9A-9D represent graphs comparing neutralizing antibody titers achieved in non-human primates after SC immunization with tetravalent dengue virus vaccines. Two groups were vaccinated with the needle-free device via the subcutaneous route either twice on the same day (0,0) or once on day 0 and again on day 60 (0,60).
FIGS. 10A-10B represent data obtained from a human clinical trial. Seronegative humans (humans demonstrating little to no antibodies to dengue virus serotypes at the onset of the trial) were given two doses of a tetravalent serotype formulation of dengue vaccine either subcutaneously or intradermally (day 0 and day 90). Antibody levels against each of the dengue serotypes were analyzed on days 0, 30, 60, 90 and 120.
FIGS. 11A-11D represent a graph comparing neutralizing antibody titers achieved in non-human primates after subcutaneous immunization with a tetravalent serotype dengue vaccine. Two groups were vaccinated either twice on the same day (0,0) or once on day 0 and again on day 60 (0,60). Serum was analyzed for presence of antibodies on days 0, 28, 58, 73 and 90, and the detection of antibodies against all four dengue serotypes were analyzed (DEN-1, DEN-2, DEN-3, DEN-4).
DEFINITIONS
As used herein, “a” or “an” may mean one or more than one of an item.
As used herein, vessel can include, but is not limited to, test tube, mini- or micro-fuge tube, channel, vial, microtiter plate or container.
As used herein the specification, “subject” or “subjects” may include but are not limited mammals such as humans or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g., hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairie dogs, or zoo animals.
As used herein, “about” or “approximately” can mean plus or minus ten percent.
As used herein, “attenuated virus” can mean a virus that demonstrates reduced or no clinical signs of disease when administered to a subject such as a mammal (e.g., human or an animal).
As used herein, “consecutively” can mean in close temporal proximity, usually within a single patient visit and within 24 hours.
As used herein, “administration” can mean delivery of a vaccine or therapy to an individual animal or human by any one of many methods such as intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, oral, buccal, by inhalation, intranasally, or any others known in the art.
DESCRIPTION
In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the details outlined herein, but rather that concentrations, times and other details may be modified through routine experimentation. In some cases, well-known methods or components have not been included in the description.
Certain aspects of the present invention include, but are not limited to, administration of vaccine compositions against dengue virus.
Embodiments of the present invention generally relate to methods and compositions for inducing protective neutralizing antibodies in a subject against three or more dengue virus serotypes. Other embodiments can include introducing a vaccine composition to a subject via any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, orally, intranasally, vaginal, intravenous, ingested, and any other method wherein the vaccine composition so introduced induces neutralizing antibodies against three or more dengue virus serotypes. In certain embodiments, the vaccine composition comprises a dose of a vaccine against three or more dengue virus serotypes administered to a subject. In other embodiments, the vaccine composition comprises an initial dose against all four dengue serotypes then, one or more other vaccine compositions administered to a subject.
Other aspects of the present invention include modulating an immune response to a vaccine against dengue virus administered intradermally compared to subcutaneously to a subject. Vaccines against dengue virus may include a composition comprising predetermined ratios of all four live, attenuated dengue vaccine viruses, recombinant dengue vaccine viruses, chimeric viruses or mutants thereof. The ratios of various dengue serotypes may be equivalent or nearly equal in representation or certain serotypes may be represented at higher concentrations than others depending on need or ability to induce a balanced neutralizing antibody response in the subject. In accordance with these embodiments, ratios of different dengue vaccines may differ by 2 to 100,000 fold (e.g. plaque forming units) between any two serotypes. This can depend on, for example, number of serotypes represented in the formulation, predetermined response and desired effect. It is contemplated that any dengue vaccine virus serotype formulation may be used to generate a vaccine (e.g. attenuated virus etc.) of use in consecutive administration to a subject in need thereof where the composition includes, but is not limited to, three or more dengue virus serotypes.
In other embodiments, compositions of dengue virus vaccine formulations may be introduced to a subject prior to, during or after exposure to dengue virus by the subject. In accordance with these embodiments, a subject may receive more than one administration consecutively or more than one administration comprising a dengue virus formulation, optionally, followed by one or more additional administrations at a later time. Intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, oral, and any other method of applications of formulations described herein may be combined with any other anti-viral treatment. In some embodiments, it is contemplated that intradermal, subcutaneous, intramuscular introduction of a formulation contemplated herein may be administered to any appropriate region of a subject's body (e.g. arm, shoulder, hip, intranasally etc). In addition, parenteral administration of vaccine formulations may be combined with other modes of administration such as intranasal, pulmonary, oral, buccal, or vaginal in consecutive administrations. In some embodiments, it is contemplated that, after consecutive administrations as described herein primary or booster administrations may occur consecutively on the same day, consecutive days, weekly, monthly, bi-monthly or other appropriate treatment regimen.
Dengue is endemic in Asia, Central and South America including Colombia, the Caribbean, the Pacific Islands, and parts of Africa and Australia. It is estimated that 3.6 billion people (55% of the world's population) live in areas at risk of dengue virus transmission (DVI). Infection with a dengue virus can result in a range of symptoms, from subclinical disease to debilitating but transient dengue fever to life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Currently, there is no therapeutic treatment or prophylactic vaccine for dengue fever. Given the impact of dengue on populations in endemic countries and on travelers to those regions, a vaccine to prevent dengue is needed.
Dengue is a mosquito borne viral disease, transmitted from human to human primarily by the mosquito, Aedes aegypti. Dengue viruses (DEN) contain a single-stranded, positive-sense RNA genome of approximately 11 kb. The genome consists of three structural proteins, capsid (C), premembrane (prM), and envelope (E), and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. There are four different serotypes of dengue viruses, DEN-1, DEN-2, DEN-3 and DEN-4. Primary infection with a given serotype induces lifelong serotype specific immunity. However, there is no long-term cross-protective immunity against the other three dengue virus serotypes, and subsequent infection with an alternate serotype leads to increased probability of more severe disease, such as DHF or DSS.
Due to the disease enhancement associated with secondary DENV infections, a multivalent (e.g. tetravalent) vaccine that stimulates immunity against more than one and up to all four serotypes of DENV is needed. Several DENV vaccine candidates attenuated by classical serial passage in cell culture have proven unsafe or poorly immunogenic. Chimeric live-attenuated, recombinant DENV vaccines candidates, including viruses based on the attenuated genetic background of yellow fever 17D (YF-17D) vaccine virus, DENV-2 PDK-53 vaccine virus, or DENV-4 containing a 30-nucleotide 3′ non-coding region (NCR) deletion are known in the art.
A challenging issue in the development of an effective live-attenuated dengue virus (DENV) vaccine is the interference between the four dengue vaccine viruses when administered as a tetravalent formulation. Interference is manifest when one or more components of a multivalent mixture will induce lower immune responses than those elicited by each individual monovalent vaccine. Interference has been observed with vaccines for diseases with multiple pathogenic serotypes, such as polio, dengue or others. Due in part to this interference, it was previously discovered that three dose regimen of oral polio vaccine is required to induce adequate immune responses to the three key serotypes. Historically studies with live attenuated tetravalent dengue vaccines have shown that the DENY serotype that elicits the strongest neutralizing antibody response when administered alone tends to dominate immune responses when administered in the context of a multivalent formulation containing other serotypes. As an example, tetravalent mixtures of four different live, attenuated dengue vaccines showed dominant responses to the DEN-3 component and reduced immune responses to DEN-1, -2 and -4 (see for example, Sabchareon, et al., 2002, Kitchener, et al. 2006). As a result of this dominance, clinical development of the tetravalent mixtures was suspended. Interference has been seen with recombinant, live attenuated viruses as well. Interference was documented in tetravalent mixtures of dengue/yellow fever chimeras (Guy, et al. 2009. Evaluation of Interferences between Dengue Vaccine Serotypes in a Monkey Model. Am. J. Trop Med. Hyg. 80: 3012-311). In these studies, two serotypes were found to dominate the responses in tetravalent formulations of ChimeriVax vaccine strains. Interference could be overcome by administering two bivalent vaccine formulations, either in separate anatomical locations or sequentially in time, or by a third administration of the tetravalent formulation after one year. Similarly, it was demonstrated that improved multivalent responses with tetravalent recombinant vaccine strains (in this case, formulations containing DENV or chimeric DENV with deletions in the 3′ non-coding region) could be obtained only with a prolonged four month internal between the first and second administration. (Blaney, et al., 2005. Recombinant, Live-Attenuated Tetravalent Dengue Virus Vaccine Formulations Induce a Balanced, Broad, and Protective Neutralizing Antibody Response against Each of the Four Serotypes in Rhesus Monkeys. J. Virology 79: 5516-5528).
Successful vaccination often requires vaccine delivery to closely mimic natural infection. To date, all clinical trials of dengue candidate vaccines have utilized the SC route using needle and syringe. The natural route of dengue infection is through mosquito transmission in the dermis. The skin is thought to be an immuno-competent organ functioning as an immune barrier to infections A highly dense network of specialized antigen-presenting cells (APCs, such as Langerhan's cells and dendritic cells) are present in the epidermis and serve to protect the host against infectious pathogens through efficient uptake and presentation of antigens to the regional lymph nodes. Both these subsets of APCs together with resident macrophages have been shown to be natural targets of dengue virus infection. Given the fact that the epidermis is rich in immunocompetent cells, it was contemplated herein that the use of intradermal route for dengue virus vaccine delivery will favor the induction of more potent and balanced immune responses to all four dengue virus serotypes. Specifically, the presence of an increased number of natural host cells in the skin for virus replication may reduce interference and permit replication of the less dominant viruses in tetravalent formulations. In certain embodiments, intradermal immunization of multivalent, live, attenuated dengue vaccines can be used to induce more balanced immune responses to dengue virus exposure in a subject.
Certain embodiments disclosed herein concern DENVax™. DENVax™ is a dengue vaccine that consists of a mixture of four recombinant dengue virus strains designed to generate immune responses to the four dengue serotypes (DEN-1, DEN-2, DEN-3 and DEN-4). Not to be bound by any limitations to a particular tetravalent formulation, DENVax™, the dengue serotype 2 vaccine component (DENVax-2) corresponds to an attenuated DEN-2 PDK-53 strain. This construct has already been investigated in many clinical studies. The other dengue vaccine strains (DENVax-1, DENVax-3 and DENVax-4) are chimeras consisting of the DEN-1, DEN-3 or DEN-4 structural pre-membrane (prM) and envelope (E) protein genes cloned into a DEN-2 PDK-53 non-structural gene backbone. These recombinant viruses express the surface antigens of DEN-1, DEN-3 or DEN-4 and retain the genetic alterations responsible for the attenuation of the DEN-2 PDK-53 strain. In certain embodiments, DENVax™ can be used as an example of a multivalent live, attenuated dengue vaccine having all four dengue virus serotypes represented in one vaccine composition at various ratios. Other embodiments relate to optimizing tetravalent vaccine administrations. Yet other embodiments relate to DENVax™ immunization methods.
During the course of exploring intradermal delivery of multivalent dengue vaccines, it was discovered that administration of more than one dose of a multivalent vaccine in at least two separate anatomical sites induced neutralizing antibody responses that were approximately equivalent or superior to administering multiple doses separated by time. Further, it was discovered that the benefit of multiple site administration was independent of the route of immunization.
This finding was unexpected. Information was previously disclosed regarding multiple subcutaneous administrations of a tetravalent vaccine based on deleted, attenuated and/or recombinant viruses. It was reported that a second administration of a tetravalent administration 30 days after the first administration failed to increase neutralizing antibody titers. In contrast, a second administration 120 days after the first, improved neutralizing antibody titers to all four dengue serotypes. Similar information was reported in clinical trials of yellow fever/dengue recombinant vaccines (Poo, et al. 2011 Ped. Inf. Dis J. 30: 1-9) It was also suggested that a three month interval between administrations was suboptimal for generating neutralizing antibody response against multiple dengue viruses. (Capeding et al. 2011 Vaccine 29: 3863-3872) These reports regarding two clinical studies suggested that longer intervals such as 6-9 months are required to generate better multivalent immune responses. Lastly, in early human challenge studies, it was reported that wild type dengue viruses elicit broadly cross-reactive antibodies that persist for up to 6 months after initial infection. These data support the concept that a short immunization regimen are suboptimal for live attenuated vaccines: the transient cross-reactive antibodies previously observed would effectively neutralize any of the live, attenuated vaccine components in a multivalent formulation. Until the instant disclosure, immunization regimens with multivalent, live attenuated vaccines at shorter intervals in more than one anatomical site were not considered a viable option for treating a subject in need of such a treatment. It is contemplated herein that multiple site administration, by accessing larger numbers of antigen presenting cells and/or more than one draining lymph node, permits immune responses to less dominant components of a multivalent, live attenuated vaccine and effectively reduces vaccine interference.
In certain embodiments, the composition introduced to the subject comprises vaccines against all dengue virus serotypes (DEN-1, DEN-2, DEN-3, DEN-4). In other embodiments, a composition contemplated herein can include DENVax™ or other similar formulation. In some compositions, vaccine viruses against all dengue serotypes are in equal proportions in the composition. In yet other compositions, each dengue vaccine virus serotype may be in a particular ratio to one another such that introduction of the composition provides the subject with sufficient levels of neutralizing antibodies against all dengue viruses (e.g. DEN-1, DEN-2, DEN-3, DEN-4).
Certain embodiments disclosed herein relate to methods and compositions for a rapid induction of protection in a subject against all dengue virus serotypes by, for example, administering a vaccine to a subject against all dengue virus serotypes in more than one anatomical location consecutively on the same day. Some embodiments can include introducing a vaccine composition to a subject via intradermal (ID) or subcutaneous (SC) injection or other administration mode in one anatomical location then introducing at least a second vaccine composition at another anatomical location by ID, SC or other administration mode. Some embodiments include using any combination of modes of administration for introducing a dengue virus vaccine of all dengue virus serotypes to a subject where administration of the vaccine occurs at two or more anatomical sites or by two or more different routes on day 0 to the subject. Some embodiments include using the same mode of administration but at different anatomical locations.
Some dengue virus vaccine compositions described herein range in dosage from from 102 to 5×106 PFU for each serotype in a composition. Other compositions (e.g. follow-on vaccinations) contemplated herein include compositions that have dosages less than or more than this range based on immune response in the subject after primary immunization. In certain embodiments, ratios can vary for the various Dengue vaccine virus serotypes depending on need and immune response in a subject.
In certain embodiments, compositions introduced on the first vaccination or in any follow-on vaccination contemplated herein may include one tetravalent dengue virus composition. In accordance with these embodiments, the composition can include DENVax™ or other similar tetravalent formulation of equal or equivalent ratios or at predetermined serotype ratios. Other embodiments, can include using different formulations (e.g. serotype ratios) for each of the vaccine compositions administered at the primary vaccination or any follow-on vaccinations (e.g. less than 30 days later).
Some embodiments herein include treating a subject in need of such a vaccine, on day 0 at two or more anatomical locations then administering at least a second vaccine within 30 days such as about 7, about 14, about 21 or about 28 days later with a composition comprising dengue virus serotypes which may or may not have all serotypes. In certain embodiments, each vaccination has all dengue virus serotypes represented in the vaccine formulation. Vaccine compositions of follow-on administration disclosed herein may include two or more dengue virus serotypes at a predetermined ratio for the subsequent administration(s).
In certain embodiments, the composition introduced to the subject comprises all dengue virus serotypes. In some embodiments, vaccine compositions comprise various formulations of DENVax™ or other similar formulation. In certain vaccine compositions, the ratio of DEN-1:DEN-2:DEN-3:DEN-4 can be 3:3:3:3, 4:3:4:5, 5:4:5:5, 5:4:5:5, 5:5:5:5, 10:1:10:100 or other ratio where the ratio between 2 serotypes can be about 2 to about 100,000 fold difference (e.g. DENVax4:3:4:5™ etc.) in a single composition. In certain embodiments a dengue serotype ratio can be DEN-1 at 2×104: DEN-2 at 5×104: DEN-3 at 1×105: DEN-4 at 3×105 PFUs or DEN-1 at 8×103: DEN-2 at 5×103: DEN-3 at 1×104: DEN-4 at 2×105 PFUs. In some compositions, all dengue vaccine virus serotypes are in equal proportions in the composition. In yet other compositions, each dengue vaccine virus serotype may be in a particular ratio to another serotype such that introduction of the composition provides the subject with adequate or more than adequate levels of neutralizing antibodies which confer protection against all dengue viruses (e.g. Dengue 1, 2, 3 and 4). For example, if after receiving two or more consecutive vaccinations on day 0 at two or more anatomical locations, the subject has lower protection to one or more particular dengue virus serotypes, then a booster for that subject can contain an increased concentration of the one or more dengue vaccine virus serotype (that demonstrated lower neutralizing antibodies) to provide better protection against all dengue virus types. In accordance with these embodiments, samples from a subject may be analyzed for an immune response to dengue serotype infection (e.g. Dengue-1, -2, -3, -4) using standard means known in the art.
In certain embodiments, the vaccine composition can be simultaneously or consecutively introduced to a subject intradermally in multiple anatomical locations to, for example, protect against all dengue serotypes (e.g. cross protection). In certain embodiments, a vaccine composition can include, but is not limited to, a single formulation of all dengue vaccine virus serotypes (e.g. DENVax™) administered to a subject capable of providing full protection against infection by all dengue virus serotypes. In other embodiments, a vaccine composition can include attenuated dengue virus serotypes in combination with other anti-pathogenic compositions (e.g. Japanese encephalitis, West Nile, influenza etc.). Compositions contemplated herein can be administered by any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, ingested, and any other method. Introduction in two or more anatomical sites can include any combination administration including by the same mode in two or more anatomical sites or by two different modes that include two separate anatomical sites. In accordance with these embodiments, two or more anatomical sites can include different limbs.
For example, if a subject, after receiving two or more consecutive vaccinations on day 0 at two or more anatomical locations and the subject does not induce poor levels of neutralizing antibodies to one or more particular dengue virus serotypes, then a booster vaccination for that subject can contain an increased concentration of the one or more dengue vaccine virus serotype (that demonstrated lower levels of neutralizing antibodies) to provide complete protection against infection by all dengue virus types. In accordance with these embodiments, samples from a subject may be analyzed for resistance to dengue infection using standard means known in the art.
In certain embodiments, two doses of the vaccine composition can be consecutively introduced to a subject in multiple anatomical locations to, for example, to protect against all dengue serotypes (e.g. cross protection) at day 0. In certain embodiments, a vaccine composition can include, but is not limited to, a single dose of dengue vaccine viruses for all serotypes (e.g. DENVax™) administered to a subject capable of inducing neutralizing antibodies to levels which would provide full protection against infection by all dengue virus serotypes. Thus, a particular subject may need to visit a clinic only one time to receive enough protection to visit or remain in a region having dengue virus for a predetermined period of time (e.g. 30 days). In other embodiments, a vaccine composition can include attenuated dengue virus serotypes in combination with vaccine compositions against other pathogens (e.g. flaviviruses such as Japanese encephalitis, West Nile, or other viruses such as influenza etc.). Compositions contemplated herein can be administered by any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, ingested, and any other method. Introduction in two or more anatomical sites can include any combination administration including by the same mode in two or more anatomical sites or by two or more different modes that include two or more separate anatomical sites. In accordance with these embodiments, two or more anatomical sites can include different limbs, different tissues, intranasally, as drops (e.g. for the eye), intramuscular in two or more locations.
In certain embodiments, vaccine compositions disclosed herein can be chimeric constructs that can include a mixture of constructs that make up at least 3 dengue serotypes in a vaccine composition for administration to a subject. In other embodiments, dengue virus vaccines can include constructs having an attenuated flavivirus backbone with various dengue serotype substitutions representing each of the four serotypes where the constructs can be mixed in a composition for administration as a vaccine.
Tetravalent formulations, e.g. DENVax™, can be prepared by mixing predetermined amounts of each monovalent vaccine component. Based on input titer of each vaccine component, a defined volume of monovalent vaccines can be added to a final volume of either 0.1 mL (e.g. for intradermal) or 0.5 mL (e.g. for subcutaneous) vaccine formulation. The remaining volume of the tetravalent DENVax™ vaccine can be composed of diluent containing Trehalose (15%) F127 (1%) and human serum albumin (0.1%) in a saline buffer to stabilize the live, attenuated vaccine formulation.
Methods
Nucleic Acid Amplification
Nucleic acids may be used in any formulation or used to generate any formulation contemplated herein. Nucleic acid sequences used as a template for amplification can be isolated viruses (e.g. dengue viruses), according to standard methodologies. A nucleic acid sequence may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary cDNA. In some embodiments, the RNA is whole cell RNA and is used directly as the template for amplification. Any method known in the art for amplifying nucleic acid molecules is contemplated (e.g., PCR, LCR, Qbeta Replicase, etc).
Expressed Proteins or Peptides
Genes can be expressed in any number of different recombinant DNA expression systems to generate large amounts of the polypeptide product, which can then be purified and used in methods and compositions reported herein. Any method known in the art for generating and using constructs is contemplated. In certain embodiments, genes or gene fragments encoding one or more polypeptide may be inserted into an expression vector by standard cloning or subcloning techniques known in the art.
Proteins, peptides and/or antibodies or fragments thereof may be detected or analyzed by any means known in the art. In certain embodiments, methods for separating and analyzing molecules may be used such as gel electrophoresis or column chromatography methods.
Electrophoresis
Electrophoresis may be used to separate molecules (e.g., large molecules such as proteins or nucleic acids) based on their size and electrical charge. There are many variations of electrophoresis known in the art. A solution through which the molecules move may be free, usually in capillary tubes or it may be embedded in a matrix or other material known in the art. Common matrices can include, but are not limited to, polyacrylamide gels, agarose gels, mass spec, blotting and filter paper.
Some embodiments, using a gene or gene fragment encoding a polypeptide may be inserted into an expression vector by standard subcloning techniques. An expression vector may be used which produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification of a peptide or protein. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, N.J.), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×His system (Qiagen, Chatsworth, Calif.).
Pharmaceutical Formulations
Any pharmaceutical formulation known in the art for a vaccine is contemplated herein. In certain embodiments, a formulation can contain one or more dengue virus serotype in various ratios in a single vaccine. It is contemplated that formulations can contain other agents of use in vaccination of a subject including, but not limited to other active or inactive ingredients or compositions known to one skilled in the art.
All contemplated vaccinal viruses herein can be administered in the form of vaccinal compositions which can be prepared by any method known to one skilled in the art. In certain embodiments, the virus compositions are lyophilized and are mixed with a pharmaceutically acceptable excipient (e.g. water, phosphate buffered saline (PBS), wetting agents etc.) In other embodiments, vaccine compositions can include stabilizers that are known to reduce degradation of the formulation and prolong shelf-life of the compositions.
In other embodiments, an adjuvant may be added to the composition to induce, increase, stimulate or strengthen a cellular or humoral immune response to administration of a vaccination described herein. Any adjuvant known in the art that is compatible with compositions disclosed herein is contemplated.
Some embodiments herein concern amounts or doses or volumes of administration of a tetravalent dengue virus composition and the amount or dose can depend on route of administration and other specifications such as the subject getting the vaccine (e.g. age, health condition, weight etc.).
It is contemplated herein that compositions described can be administered to a subject living in an area having dengue virus, a subject traveling to an area having dengue virus or other subject such as any human or animal capable of getting dengue fever or other dengue virus condition. In certain embodiments, it may be recommended that a subject traveling to an area having dengue virus is administered one or more vaccine compositions (e.g. two or more on Day 0) about 1 to about 3 months prior to dengue virus exposure. Vaccines herein can be administered as a prophylactic treatment to prevent infection in adults and children. A subject can be naïve or non-naïve subject with respect to exposure to dengue virus and vaccine regimens disclosed herein.
Kits
Other embodiments concern kits of use with the methods (e.g. methods of application or administration of a vaccine) and compositions described herein. Some embodiments concern kits having vaccine compositions of use to prevent or treat subjects having been exposed or suspected of being exposed to one or more dengue viruses. In certain embodiments, a kit may contain one or more than one formulation of dengue virus serotype(s) (e.g. attenuated vaccines, trivalent or tetravalent formulations, DENVax™) at predetermined ratios. Kits can be portable, for example, able to be transported and used in remote areas such as military installations or remote villages in dengue endemic areas. Other kits may be of use in a health facility to treat a subject having been exposed to one or more dengue viruses or suspected of being at risk of exposure to dengue virus.
Kits can also include a suitable container, for example, a vessel, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the agent (e.g. a vessel), composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Optionally, one or more additional agents such as immunogenic agents or other anti-viral agents, anti-fungal or anti-bacterial agents may be needed for compositions described, for example, for compositions of use as a vaccine against one or more additional microorganisms.
In other embodiments, kits can include devices for administering one or more vaccination to a subject such as an ID, SQ, IM, an inhaler, intranasal applicator or other device for administering a vaccine composition disclosed herein.
The following examples are included to demonstrate certain embodiments presented herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered to function well in the practices disclosed herein. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the certain embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope herein.
EXAMPLES
Example 1
Previous studies revealed that natural infection with each DENV (dengue virus) serotype leads to long-lived protection against dengue fever caused by the homologous serotype. In certain embodiments, administration of an effective dengue vaccine closely mimics natural infection and can serve as a mode for administering vaccines against Dengue virus. Embodiments reported herein can concern a natural infection route of dengue virus (DENV) infection, similar to intradermal delivery by the transporting host, a mosquito bite. In certain embodiments, intradermal injection to deposit the vaccine viruses into the same tissue can be used. Skin is a highly accessible organ and represents an effective immune barrier, mainly attributed to the presence of Langerhans cells (LCs) residing in the epidermis. Skin immunization elicits a broad range of immune responses, including humoral, cellular, and mucosal and has the potential to bypass the effect of pre-existing immunity on the immunogenicity of administered vaccines.
Some embodiments for intradermal (ID) administration of the tetravalent dengue vaccines in a subject in need of such a treatment are reported. One exemplary method of intradermal administration was performed on four Cynomologous macaques administered a DENVax™ ((DENVax-1: 1×105 PFU, DENVax-2; 1×105 PFU, DENVax3: 1×105 PFU, DENVax4: 1×105 PFU) Dengue virus vaccine) by intradermal administration. To achieve an equivalent dose of virus, 0.15 ml of vaccine was deposited ID in three closely spaced sites using a needle-free jet injector (see FIGS. 1 and 2, below). FIG. 1 represents an intradermal inject (e.g., PharmaJet® or other intradermal device) device used for intradermal inoculations.
FIG. 2 illustrates inoculation sites on Cynomolgus macaques post vaccination with PharmaJet device. Animals were boosted 60 days later with the same formulation by the same route. Serum samples were collected at predetermined intervals, days 15, 30, 58, 74, and 91 and were tested for the presence of neutralizing antibodies directed against the four Dengue serotypes. PRNT (plaque reduction neutralization test, known in the art for quantifying levels of anti-DEN neutralizing antibodies) were performed on the sera samples.
It was demonstrated that the neutralizing antibody titers are significantly higher after ID administration as compared to SC administration (p<0.05 for DENV-1 and DENV-2) after a primary administration (see FIG. 3) or after a secondary administration (p<0.05 for all DENV serotypes) (See FIG. 4). Since the sites were closely spaced in the same area, and each innoculum consists of all four viruses, this mode of vaccine delivery closely resembles a single administration of DENVax™. FIG. 3 illustrates 50% PRNT (plaque reduction neutralization titers) Geometric Mean Titers at Day 58 (58 days after the primary administration). FIG. 4 illustrates 50% PRNT Geometric Mean Titers at Day 74 (14 days after the secondary administration on Day 60). As can be seen in the figures, the neutralizing antibody titers to all four dengue viruses were higher after intradermal versus subcutaneous administration. In addition, the number of animals that demonstrated neutralizing antibody responses (“seroconversion” defined as PRNT >10) was greater after the first dose of vaccine (see Table 1, the percentage of animals that seroconverted to each of the four Dengue serotypes is shown after primary and secondary immunization).
TABLE 1
Seroconversion of non-human primates after dengue immunization
% Seroconversion
DENVax
DEN-1
DEN-2
DEN-3
DEN-4
Formulation
Prime
Boost
Prime
Boost
Prime
Boost
Prime
Boost
5:5:5:5 SC
87.5%
100.0%
100.0%
100.0%
75.0%
100.0%
50.0%
100.0%
5:5:5:5 ID
100.0%
100.0%
100.0%
100.0%
100.0%
100.0%
100.0%
100.0%
The immunized animals were tested for protection against challenge with wild type dengue viruses. In cynomolgus macaques, wild type dengue virus infection leads to virus replication and viremia, but no clinical signs. At day 91, two monkeys were challenged with DENV-1 (Dengue virus serotype 1) and two monkeys challenged with DEN-2 (Dengue virus serotype 2). Serum samples were collected daily for 11 days after challenge. Levels of dengue virus RNA were measured in the samples by quantitative real-time polymerase chain reaction technology (q-rtPCR) and titers of viable virus were measured by virus isolation and plaque formation on Vero cells. The results are shown in Tables 2 and 3. Neutralizing antibodies against DEN-1 at Day 91, just prior to challenge (“Pre-Challenge”) and Day 105, 14 days after challenge (“Post”). Viremia is given as the number of days that live DEN-1 virus could be isolated from blood samples (“Duration”) and the log 10 of the peak titer isolated from each animal. Viral RNA is given as the number of days viral RNA could be detected in the serum samples (“Duration”) and peak viral RNA levels in each monkey, expressed as the log 10 of the number of viral RNA genomes detected.
TABLE 2
Responses after challenge with DEN-1
DEN-1 PRNT
Viremia
Viral RNA
Monkey
Formulation
Pre-Challenge
Post
Duration
Peak
Duration
Peak
CY0174
5:5:5:5 SC
240
240
0
0
0
0
CY0181
5:5:5:5 SC
640
61440
0
0
5
5.6
CY0192
5:5:5:5 ID
1920
1280
0
0
0
0
CY0194
5:5:5:5 ID
7680
1920
0
0
0
0
CY0061
Controls
1
2560
6
2.0
9
5.7
CY0193
Controls
25
2560
3
2.7
7
6.4
CY0058
Controls
1
640
5
2.9
7
5.5
CY0073
Controls
1
1280
5
3.6
10
6.2
TABLE 3
Responses after challenge with DEN-2
DEN-2 PRNT
Viremia
Viral RNA
Monkey
Formulation
Pre-Challenge
Post
Duration
Peak
Duration
Peak
CY0172
5:5:5:5 SC
3413
3413
0
0
1
3.9
CY0177
5:5:5:5 SC
853
533
0
0
0
0
CY0198
5:5:5:5 ID
240
320
0
0
0
0
CY0201
5:5:5:5 ID
1920
1600
0
0
0
0
CY0088
Controls
6
10240
6
2.3
8
5.1
CY0199
Controls
1
3640
5
1.8
9
4.7
CY0065
Controls
1
10240
5
2.9
8
5.8
CY0104
Controls
1
10240
4
2.4
8
5.7
After challenge, the SC and ID immunized animals were completely protected from DEN-1 or DEN-2 induced viremia (compared to the control animals that demonstrated significant viremia of long duration). In all of the ID immunized animals, but not all of the SC immunized animals, there was also an absence of viral RNA replication and a lack of an increase in antibody titer after challenge (compare the ID animals to SC injected CY0181, CY0172 or the control animals). These data suggest that protection is “sterilizing” and prevents any virus replication after challenge.
Example 2
In another example, an optimized DENVax™ formulation delivered in different locations and with different timings will be tested in non-human primates. Groups of eight Cynomolgus macaques will be immunized with a DENVax™ formulation containing 1×105 plaque forming units (pfu), 1×104 pfu, 1×105 pfu and 1×105 pfu of DENVax™-1, DENVax™-2, DENVax™-3 and DENVax™-4, respectively (abbreviated 5:4:5:5). Two doses will be administered in 0.1 ml ID. Groups will be immunized with either one dose in each arm at Day 0, one dose in one arm at Day 0 and one dose in the other arm at Day 7, or one dose in one arm at Day 0 and one dose in the other arm at Day 60. These groups will be compared to a group that receives the same dose (5:4:5:5) in three sites in the same are on Day 0 and three sites in the other arm on Day 60 as well as a group that receives the same dose in a single 0.5 ml SC immunization in one arm at Day 0 and in the other arm at Day 60. A control group will be immunized with vaccine excipients only (no vaccine viruses). Following immunization, blood samples will be collected on days 0, 7 (for peak viremia), 15, 30, 60, and 90 to test the neutralizing antibodies against the four Dengue virus serotypes by PRNT50. PBMCs collected on days 30, 60, 90 will be also monitored for IFN-γ secretion by an ELISPOT assay. On day 90, two animals from each group will be challenged with wild type of DEN-1, DEN-2, DEN-3, or DEN-4 viruses. Challenged animals will be monitored for clinical signs and temperature (twice daily), changes in food consumption (once daily) and body weight (weekly). In addition, all animals will be bled daily for 11 days post-challenge to monitor viremia and hematological parameters. Again, the speed and duration of PRNT responses to all four DEN viruses and protection after day 90 challenge will be assessed. It is believed that intradermal administration in multiple sites and in distinct anatomical locations may be more effective than subcutaneous administration as a single bolus. Multiple sites can provide exposure of the vaccine to more antigen presenting cells. Distinct anatomical locations can permit vaccine access to multiple lymph nodes. In addition, booster immunizations of Dengue vaccines have only been administered after the development of antibody responses in mice, primates and human clinical trials, thirty days or longer. At this time, neutralizing antibodies inhibit the response to the live viral vaccines. It was previously shown that boosting primates one month after primary immunization was less effective than dosing four months after primary immunization. It was speculated that high levels of homologous and heterologous antibodies that circulate after the initial immunization can inhibit viral replication in a second dose. While prolonged (two months or longer) immunization may circumvent this inhibition, it has not been tested whether accelerated immunization regimen with shorter immunization intervals, before the development of potent neutralizing antibody responses may be advantageous. Such a shortened regimen may be an advantage in endemic countries or for travelers, where exposure to Dengue viruses in between the immunizations may put them at risk of disease.
Example 3
In another example, a human clinical trial has been initiated, studying the safety and immunogenicity of two DENVax™ formulations, administered in 0.1 ml either by ID or SC injection. Groups of 12 individuals will be immunized with for example, a low dose DENVax™ formulation (8×103 pfu, 5×103 pfu, 1×104 pfu and 2×105 pfu of DENVax™-1, -2, -3 and -4, respectively) or a high dose (2×104 pfu, 5×104 pfu, 1×105 pfu and 3×105 pfu of DENVax™-1, -2, -3 and -4, respectively) of DENVax™ ID or SC on Days 0 and 90. Two control groups will be injected SC or ID with phosphate-buffered saline. Patients will be monitored for any adverse events, and for any significant changes in hematological or blood chemistry parameters. Serum samples will be collected to measure vaccine virus replication and neutralizing antibody responses at periodic intervals.
Example 4
Immunogenicity and efficacy of DENVax™ administered intradermally in AG129 mice. In another example, two studies were performed to compare the effect of route of administration on immunogenicity and efficacy of DENVax™ in AG129 mice. In one example, the immunogenicity of monovalent DENVax™-4 (e.g. vaccine against one Dengue virus serotype) was compared in AG129 mice by measuring the neutralizing antibody responses following SC injection under the skin on the back or ID injection into the foot pad using a needle and syringe. Groups of 8 AG129 mice were injected ID or SC with 105 PFU/dose of chimeric DENVax™-4 vaccine in 50 μl and 100 μl final volume, respectively. Six weeks after priming, animals from each treatment group were boosted via the corresponding ID or SC route with 105 PFU of DENVax™-4 or TFA. Mice were bled on Day 31 and 58 and collected sera were pooled to measure neutralizing antibody responses.
Immunization of DENVax™-4 via the ID route elicited a 5-fold higher neutralizing antibody response to DEN-4 after the boost compared to the response induced via the SC route (see for example, FIG. 4). The anti-DEN-4 response elicited by either route of immunization had a marked cross-neutralizing activity against DEN-3 but not against DEN-1 or DEN-2 serotypes. FIG. 4 represents neutralizing antibody responses following primary and secondary immunization of AG129 mice with chimeric DENVax™-4. Mice were bled on Day 31 and 58 and collected sera were pooled to measure neutralizing antibody responses using the plaque reduction assay (PRNT50).
Two weeks after the boost animals from each group were split in to two groups and challenged with 106 PFU of DEN-1 (Mochizuki virus strain) or DEN-2 (New Guinea C strain) viruses. Challenged animals were monitored for clinical signs of disease and survival rates were recorded over a period of 5 weeks. Mice immunized via the ID route showed no signs of disease after DEN-1 challenge (FIG. 5A). In the SC immunized group only one mouse succumbed to infection while the rest of animals had no any apparent signs of infection (FIG. 5B). In contrast, all control animals succumbed to infection by day 13 after DEN-1 challenge (FIG. 5A). Following DEN-2 challenge, all animals immunized with only DENVax™-4 via the route succumbed to infection by day 25 with mean survival time (MST) of 19.5 days as compared to the control (FTA) mice that all succumbed by day 17 (MST=12.5 days) post-challenge (FIG. 5B). In contrast, fifty percent of ID DENVax™-4 immunized mice survived the infection until the end of the 5 week monitoring period (FIG. 5B). FIGS. 5A and 5B represent survivals of DENVax™-4 immune AG129 mice following challenge with DEN-1 (a) or DEN-2 (b) viruses. Challenged animals were monitored for clinical signs of disease and survival rates were recorded over a period of 5 weeks.
In a second study, immunogenicity of tetravalent DENVax™ vaccine administered SC or ID in mice (e.g. AG129) was tested. Groups of AG129 mice, six per group were injected SC or ID with the DENVax™ in 100 μl or 50 μl (final volume), respectively. Mice were immunized with DENVax™ at a 5:4:5:5 (105 PFU of DENVax™-1, -3 and -4 and 104 PFU of DENVax™-2) dose level of composite chimeric vaccines. All immunized animals received a booster injection of 5:4:5:5 DENVax™ (105 PFU of DENVax™-1, -3 and -4 and 104 PFU of DENVax™-2) 42 days' post-primary inoculation. Blood samples were collected on days 42 and 56 to measure neutralizing antibody responses to each DEN virus serotype.
As represented in Table 4, both primary and secondary neutralizing antibody responses to all four DEN serotypes were induced. Following the boost, the neutralizing anti-DEN-1, DEN-3 and DEN-4 antibody titers were increased by 2, 5 and 2 fold, respectively in the group of mice injected ID as compared to the SC immunized animals. Neutralizing responses to DEN-2 virus were comparable in both groups. Immunization via the SC route resulted in a profile of dominant neutralizing antibody responses against DEN-1>DEN-2>DEN-3>DEN-4, with neutralizing titers 5120, 1280, 640 and 80, respectively. The hierarchy of neutralizing antibody responses after ID administration had shifted as follows; DEN-1>DEN-3>DEN-2>DEN-4 with neutralizing antibody titers 10240, 3840, 1280 and 160, respectively.
TABLE 4
Comparison of the immunogenicity of tetravalent DENVax ™ bearing
the ratio 5:4:5:5 PFU of each composite chimeric virus (105 PFU of
DENVax ™-1, -3 and -4 and 104 PFU of DENVax ™-2) after SC
or ID immunization of mice. Blood samples were collected on days 42 and
56 to measure neutralizing antibody responses to each DEN virus serotype.
Neutralizing Antibody Titers (GMT)
DENVax ™
DEN-1
DEN-2
DEN-3
DEN-4
Formulation
Prime
Boost
Prime
Boost
Prime
Boost
Prime
Boost
5:4:5:5/SC
1920
5120
3200
1280
1280
640
80
80
5:4:5:5/ID
2560
10240
1280
1280
1600
3840
120
160
Materials and Methods
Mice: AG129 mice have an “intact” immune system; deficient for the interferon (IFN)-α/β and -γ receptors. Dengue infection has been described for this model. Other studies: pathogenesis, cell tropism, and ADE have also been examined. This model permits challenge with DEN-1 and DEN-2.
Non-human primates: Cynomolgus, rhesus macaques carry virus (viremia), but no disease manifests.
Rapid Dosing Study
Example 5
In one exemplary study, immune responses to tetravalent Dengue vaccines were evaluated for different routes of administration and dosing regimens in the non-human primate model comparing vaccine delivery by conventional needle injection to needle-free administration. The quantifiable endpoints for the nonhuman primate study are i) the route for greatest geometric mean neutralizing antibody titer against each of the four dengue serotypes in non-human primates and ii) the protection from challenge with two of the dengue serotypes.
Two dosing schedules were evaluated in this study—two consecutive doses on Day 0 (at different anatomical sites) were compared to administration of two doses given 60 days apart (0.60). The high dose formulation of the tetravalent formulation (e.g. DENVax™) was used for immunization in this study. This vaccine lot is the same material used for two Phase 1 studies being conducted. The high dose tetravalent formulation vaccine consists of 2×104 pfu of DEN-1, 5×104 pfu of DEN-2, 1×105 pfu DEN-3 and 3×105 pfu DEN-4. The study design for the nonhuman primate study is shown in Table 5.
TABLE 5
Non Human Primate Study
No. of
Route/
Site(s) for
Challenge on Day 90,
Method of
Group1
Treatment
Immunizations
Dosing
SC route
Administration
1
High dose
Day 0
Two
3 animals with wt DENV-2,
ID
DENVax
(both arms)
3 animals with wt DENV-4
PharmaJet
Injector
2
High dose
Day 0, Day 60
One
3 animals with wt DENV-2,
ID
DENVax
(alternate
3 animals with wt DENV-4
PharmaJet
arms)
Injector
3
High dose
Day 0, Day 60
One
3 animals with wt DENV-2,
ID
DENVax
(alternate
3 animals with wt DENV-4
Needle/Syringe
arms)
4
High dose
Day 0
Two
3 animals with wt DENV-2,
SC
DENVax
(both arms)
3 animals with wt DENV-4
PharmaJet
Injector
5
High dose
Day 0, Day 60
One
3 animals with wt DENV-2,
SC
DENVax
(alternate
3 animals with wt DENV-4
PharmaJet
arms)
Injector
6
High dose
Day 0, Day 60
One
3 animals with wt DENV-2,
SC
DENVax
(alternate
3 animals with wt DENV-4
Needle/Syringe
arms)
7
PBS
Day 0, Day 60
One
3 animals with wt DENV-2,
ID
(alternate
3 animals with wt DENV-4
PharmaJet
arms)
Injector
Serum samples were collected after each vaccination and wild type dengue virus challenge on Days 0, 3, 5, 7, 10, 12, 14, 53, 64, 67, 88, 91, 93, 95, 97, 99, 101, 102 and 104 to analyze the samples for dengue viremia. Serum samples were also collected on Days 0, 30, 53, 75, 88 and 104 to determine the levels of neutralizing antibodies induced by the tetravalent formulation administered by needle/syringe or the ID injector.
Serum samples were collected at specified intervals during the course of the study. Sera collected on Days 0, Day 30 and Day 88 (pre-boost) have been assayed for neutralizing antibodies to Dengue-1, Dengue-2, Dengue-3 and Dengue-4. The GMT antibody titers are shown below in Table 6.
TABLE 6
Neutralizing antibody titers to all four dengue serotypes for Days
30, 53, 75 and 88 after one or two immunizations with DENVax.
Dose Schedule/
Route/Method of
Day 30 Post-Dose 1,
Day 53 Post-Dose 1,
Administration/
Reciprocal GMTs
Reciprocal GMTs
Group
Treatment
DEN-1
DEN-2
DEN-3
DEN-4
DEN-1
DEN-2
DEN-3
DEN-4
1
2 doses (Day 0),
80
1280
127
36
63
1280
40
13
PJ ID, DENVax
2
2 doses (Day 0, 60),
18
14
101
11
32
14
22
10
PJ ID, DENVax
3
2 doses (Day 0, 60),
160
36
64
9
45
40
10
5
N/S ID, DENVax
4
2 doses (Day 0),
1016
1016
403
80
640
1280
127
45
PJ SC, DENVax
5
2 doses (Day 0, 60),
154
1816
226
64
285
1280
57
22
PJ SC, DENVax
6
2 doses (Day 0, 60),
80
1140
113
11
80
806
20
28
N/S SC, DENVax
7
2 doses (Day 0, 60),
10
5
6
5
7
5
5
5
PJ ID, PBS
Neutralizing antibody titers of <10 are reported as “5”. Serum dilutions started at 1:10
Seroconversion (values in parenthesis) is defined as titer >10 over Day 0 < 10 baseline titer or
a >4-fold rise in titer if baseline titer on Day 0 was >10.
Dose Schedule/
Route/Method of
Day 75 Post-Dose 1,
Day 88 Post-Dose 1,
Administration/
Reciprocal GMTs
Reciprocal GMTs
Group
Treatment
DEN-1
DEN-2
DEN-3
DEN-4
DEN-1
DEN-2
DEN-3
DEN-4
1
2 doses (Day 0),
40
806
25
25
57
1016
25
25
PJ ID, DENVax
2
2 doses (Day 0, 60),
113
28
63
71
160
90
80
45
PJ ID, DENVax
3
2 doses (Day 0, 60),
160
40
57
36
90
101
57
36
N/S ID, DENVax
4
2 doses (Day 0),
403
718
90
57
254
640
90
64
PJ SC, DENVax
5
2 doses (Day 0, 60),
508
1810
226
113
403
1280
127
80
PJ SC, DENVax
6
2 doses (Day 0, 60),
143
806
71
57
113
4064
32
40
N/S SC, DENVax
7
2 doses (Day 0, 60),
5
5
5
5
5
5
5
5
PJ ID, PBS
Neutralizing antibody titers of <10 are reported as a value of “5”.
Serum dilutions for analysis started at 1:10.
Results were generated from duplicates or triplicates.
PJ = exemplary PharmaJet needle-free injector,
N/S = needle/syringe;
GMT = Geometric mean titer;
ID = intradermal;
SC = subcutaneous
Data are presented as geometric mean titer (GMT) ± standard error (SE)
All 42 animals in the study were seronegative at the start of the study and displayed no neutralizing antibody titers to any of the four dengue serotypes on Day 0. The results on Day 30 after priming the animals with DENVax™ showed that animals receiving two doses of DENVax™ on Day 0 (one dose in each arm) by either the ID or SC route of administration, displayed a high neutralizing antibody titer to Dengue-1, Dengue-2 and Dengue-4 (Groups 1 and 4). Seroconversion rates by day 30 were 100% for both groups as compared to groups 2 and 3. Both groups maintained high levels of neutralizing antibody responses up to day 88 just prior to virus challenge.
For live attenuated vaccines, vaccine virus replication after immunization is an important measure of vaccine uptake and vaccine safety. Vaccine virus replication in the nonhuman primates was evaluated after the first and second immunization with a live attenuated tetravalent formulation vaccine (DENVax™). Serum samples collected on Days 0, 3, 5, 7, 10, 12, 14 after the first immunization were tested for the presence of viral RNA from the vaccine strains using a qRT-PCR assay (see Table 7).
TABLE 7
DENVax-2 RNA detected in the serum after primary immunization with DENVax.
No. Animals Positive for Viral RNA, DENVax-2 Viral
RNA, Log10 GE/mL
Group
Dosing Schedule
Day 5
Day 7
Day 10
Day 12
Day 14
1
2 doses (Day 0),
—
3/6
4/6
5/6
3/6
PJ ID
(3.9-4.8)
(4.5-5.3)
(3.9-5.2)
(3.8-5.1)
2
2 doses (Day 0, 60),
—
—
—
—
—
PJ ID
3
2 doses (Day 0, 60),
—
—
1/6
1/6
1/6
N/S ID
(3.8)
(3.8)
(3.8)
4
2 doses (Day 0),
1/6 (3.8)
5/6
5/6
1/6
—
PJ SC
(3.8-5.0)
(3.7-5.3)
(4.0)
5
2 doses (Day 0, 60),
1/6 (3.8)
5/6
5/6
5/6
3/6
PJ SC
(4.5-5.4)
(3.8-5.4)
(3.2-4.8)
(3.7-5.0)
6
2 doses (Day 0, 60),
—
3/6
4/6
3/6
2/6
N/S SC
(3.9-5.6)
(3.8-5.4)
(4.3-5.0)
(3.7-4.2)
7
2 doses of PBS (Day
—
—
—
—
—
0, 60), PJ ID
Results are averages from duplicate or triplicate data.
Samples with titers <log10 3.6 were considered negative.
GE/mL = genome equivalents/mL
N/S = needle/syringe;
PJ = PharmaJet needle-free injector
Viral RNA was not detected on Day 0 (pre-vaccination) and Day 3 (post-immunization). For all groups, viral RNA was detected only for the Dengue-2 serotype from day 5 to day 14 post-vaccination after the first immunization. For Groups 1, 3, 5 and 6 endpoint titers were not observed by 14 days post-immunization. Peak titers were observed on Day 10 for Groups 1 and 4, and on Days 7 and 10 for Groups 5 and 6 (Table 7). Viral RNA was not detected for any of the groups after the second immunization evaluated on Days 64 and 67 (4 and 7 days post-dose 2).
On Day 90, three animals from each group were challenged with either wild-type Dengue-2 or Dengue-4 to demonstrate efficacy upon immunization with the tetravalent formulation. Protected animals should exhibit a lack of wild-type Dengue virus infection and replication. Wild-type challenge virus (Dengue-2 and Dengue-4) replication was analyzed for all of the groups after challenge with 106 PFU wild-type Dengue 2 (New Guinea C strain) and Dengue 4 (814669 strain) viruses on Days 91, 93, 95, 97, 99, 101, 102 and 104 (Table 8) Dengue vaccine (e.g. DENVax™): post-challenge viremia
TABLE 8
Protection of DENVax-immunized NHPs from wt DENV challenge.
Pre-Challenge
Antibody
Post-Challenge
Vaccination &
Titers (GMT), Day 88
Viremia (log10 GE/mL)
Challenge Regimen
DEN-1
DEN-2
DEN-3
DEN-4
Day 3
Day 5
Day 7
2 doses on Day 0,
DENV-2
57
1016
25
25
—
—
—
PJ ID, DENVax
DENV-4
—
—
—
2 doses on Day 0,
DENV-2
160
90
80
45
—
—
—
60, PJ ID, DENVax
DENV-4
—
—
—
2 doses on Day 0, 60,
DENV-2
90
101
57
36
—
—
—
N/S ID, DENVax
DENV-4
—
—
—
2 doses on Day 0,
DENV-2
254
640
90
64
—
—
—
PJ SC, DENVax
DENV-4
—
—
—
2 doses on Day 0, 60,
DENV-2
403
1280
127
80
—
—
—
PJ SC, DENVax
DENV-4
—
—
—
2 doses on Day 0, 60,
DENV-2
113
4064
32
40
—
—
—
N/S SC, DENVax
DENV-4
—
—
—
Viral RNA of the wild-type challenge viruses was detected only in Group 7 that had received PBS. For Dengue-2, viral RNA was detected in 3 of 3 animals on Days 93 to 97. For Dengue-4, viral RNA was detected in only 1 of 3 animals on Day 95. One important observation of the groups that were immunized with the tetravalent formulation is that no viral RNA for either the Dengue-2 or the Dengue-4 challenge viruses was observed. These results suggested that the tetravalent formulations immunization by any of the dosing schedules tested conferred immune protection against challenge of both Dengue-2 and Dengue-4 wild-type viruses.
Overall, this nonhuman primate study clearly showed that the novel dosing schedule of administering two doses of a tetravalent formulation on Day 0 at two distinct sites (e.g. different arms) induced levels of neutralizing antibodies that were equivalent or higher than those observed for more traditional dosing schedules of delivering the prime and boost immunization 2 to 3 months apart. The onset of the immune responses was more rapid for the groups that received two doses on Day 0 and long lasting. The application of the needle-free ID or SC injector enhanced the immune responses such that higher titers were observed.
Example 6
AG129 Mouse Studies on Rapid Immunization
In another exemplary study, novel dosing schedules were designed that explore either administration of two vaccine doses at two distinct sites on a single occasion or shorter dosing intervals between two doses of vaccine which will enhance compliance of vaccinated subjects to return for the second immunization. Standard Dengue vaccines developed previously typically require three doses over the course of a year to achieve robust multivalent Dengue immune responses. With respect to vaccination schedules presented herein, response was evaluated for immunization occurring in at least two anatomical two sites, and administering, in certain embodiments, a full dose (see Table 9) at each site intradermally. This protocol was performed in part to activate immune cells and antigen presenting cells in two different lymph nodes on Day 0 to induce higher levels and more robust dengue-specific immune responses compared to administering two doses intradermally 7, 14 or 42 days apart. In one study two routes of administration were compared, SC and ID routes using a conventional 42-day interval between vaccinations. The mice were immunized with a low dose formulation of a tetravalent formulation (DENVax™; 3:3:3:3 ratio of each of the serotypes) which consisted of 103 PFU of each Dengue-1, -2, -3, and -4 (e.g. DENVax™-1, -2, -3 and -4) in a 0.05 mL volume given via the intradermal route (in the foot pad). The in live portion of this study was conducted prior to initiation of this contract. The study design is shown in Table 9 below.
TABLE 9
Study design for AG129 mouse study DEN-012.
Number of
Groups
Dose
Number of Immunizations/Route
Animals
A
DENVax ™
1 (day 0)/ID
6
(3:3:3:3)
B
DENVax ™
2 (day 0)/ID, giving a full dose
6
(3:3:3:3)
into each of two footpads
C
DENVax ™
2 (7 days apart)/ID
6
(3:3:3:3)
D
DENVax ™
2 (14 days apart)/ID
6
(3:3:3:3)
E
DENVax ™
2 (42 days apart)/ID
6
(3:3:3:3)
F
FTA (negative
2 (14 days apart)/ID
6
control group)
G
DENVax ™
2 (42 days apart)/SC
6
(3:3:3:3)
The neutralizing antibody titers to Dengue 1-4 present in the collected mouse sera were determined by a microneutralization assay. Sera were collected at specified time points throughout the study and the longevity of the immune responses was studied by maintaining the study groups until Day 160 (longer than 5 months after study start). The results obtained from sera collected on Days 28 and 56 post-immunization are illustrated in Table 10.
TABLE 10
Neutralizing antibody titers (GMTs) to DEN-1, -2, -3 and -4
GMT, Day 28
Group
Treatment Groups
DEN-1
DEN-2
DEN-3
DEN-4
A
1 (day 0)/ID
400
100
200
40
B
2 (day 0)/ID, giving
800
200
800
160
a full dose into each
of two footpads
C
2 (42 days apart)/ID
400
100
200
40
D
2 (14 days apart)/ID
<20
<20
<20
<20
(negative control)
GMT, Day 56
Group
DEN-1
DEN-2
DEN-3
DEN-4
A
800
200
400
40
B
3200
400
1600
160
C
1600
200
800
40
D
<20
<20
<20
<20
In previous studies, a conventional dosing schedule of priming animals was used on Day 0 and then administering a booster vaccination on Day 42 to evaluate the immune responses for the tetravalent dengue vaccine. Both prime and boost vaccinations were administered by the subcutaneous (SC) route. This dosing schedule was included in the study for comparison to the novel dosing schedules. Initially, one study (represented in Table 10) compared the SC and ID routes of administration using the conventional dosing interval of giving two doses 42 days apart. The results indicate that there is no significant difference between the SC and ID routes with respect to neutralizing antibodies induced in this mouse model. This study further explored whether two doses administered on Day 0 at two anatomical sites (one dose into each of two foot pads) could induce neutralizing antibody levels similar to the standard dosing schedule (2 doses 42 days apart) described above. The results show that immunization on Day 0 at two sites each, with a full dose of a tetravalent formulation of DENVax™, via the ID route induced neutralizing antibody levels to all four dengue serotypes that are equivalent in magnitude to the conventional dosing schedule. The effect of a single vaccine dose administered by the ID route was also studied (Group A). Administration of a single dose of DENVax™ on Day 0 resulted in antibody responses that trended slightly lower compared to two doses on Day 0 (compare Groups A and B). Increasing the interval between the two doses from 7 to 42 days did increase antibody responses beyond the levels observed. Evaluation of the longevity of the Dengue immune response revealed that neutralizing antibody titers to all four dengue serotypes remained at high levels at Day 160 post-immunization independent of route of administration and dosing schedule (data not shown).
Overall, the results suggested that the intradermal route of administration induces neutralizing antibody levels equivalent to those observed for the subcutaneous route. Further, the administration of two doses on Day 0 at two different sites by the ID route induced a robust neutralizing antibody response equivalent to conventional dosing schedules. The antibody responses induced were long lasting and decreased only slightly. The animals did not display increased morbidity and mortality. This study demonstrated that administration of two vaccine doses at two distinct sites is a viable option for immunization as the resulting antibody titers and duration of immune responses are equivalent in magnitude to those resulting from two doses given 42 days apart. These dosing regimens will be beneficial for travelers to dengue endemic regions and others in need of fast protection from dengue virus exposure.
Example 7
Another Rapid Immunization Study in AG129 Mice
The objective of this study was to determine whether administering two doses at two sites ID on Day 0 will induce higher levels and more robust Dengue-specific immune responses compared to administering two doses ID 42 days apart. The hypothesis to be tested was whether administration of a full vaccine dose to each of two sites intradermally will activate immune cells and antigen presenting cells that traffic to two different lymph nodes, thereby reducing interference between the four DENVax™ vaccine components. The design for this AG129 mouse study is shown below in Table 11.
TABLE 11
Design of Example 7 AG129 mouse study
Number of
Group
Dose
Number of Immunizations/Route
Animals
1
DENVax ™
2 doses (day 0)/ID using both
8
3:3:3:3
footpads
2
DENVax ™
2 doses (day 0, day 42)/ID using
8
3:3:3:3
both footpads
3
DENVax ™
2 doses (day 0)/ID using both
8
4:3:4:5
footpads
4
DENVax ™
2 doses (day 0, day 42)/ID using
8
4:3:4:5
both footpads
5
FTA (negative
2 doses (day 0)/ID using both
8
control group)
footpads
In this exemplary method, two different vaccine dose levels (low and medium dose), were used for immunization using the novel dosing schedule of administering two doses on Day 0 compared to two doses 42 days apart. The mice were dosed with either a low dose formulation of DENVax™ (3:3:3:3) which consisted of 103 PFU of each of DENVax™-1, -2, -3, and 4 in a 0.05 mL volume given via the intradermal route (in the foot pad) or a medium dose formulation of DENVax™ (4:3:4:5) which contained 104 PFU of DENVax™-1, 103 PFU of DENVax™-2, 104 PFU of DENVax™-3, and 105 PFU of DENVax™-4 in a 0.05 mL volume. On Day 0 all mice were immunized and Groups 2 and 4 were boosted on Day 42. Sera for antibody analysis were collected on Days 14, 41 and 56 post-primary vaccination and analyzed using a plaque reduction microneutralization assay to determine the neutralizing antibody levels to all four dengue serotypes. Immunogenicity results obtained from pooled mouse serum samples are shown in Table 12.
TABLE 12
Neutralizing antibody titers for AG129 mouse study DEN-013
Reciprocal Neutralizing Antibody Titers (PRNT50)
14 Days p.i.1
41 Days p.i1
56 Days p.i.1
Group
DEN-1
DEN-2
DEN-3
DEN-4
DEN-1
DEN-2
DEN-3
DEN-4
DEN-1
DEN-2
DEN-3
DEN-4
1
320
160
80
20
640
640
640
80
800
1600
800
40
2
320
320
40
20
640
640
320
80
800
800
400
40
3
640
80
80
40
2560
640
1280
160
3200
800
800
40
4
320
80
40
20
320
160
640
80
1600
800
800
80
5
20
20
20
20
20
20
20
20
20
20
20
20
1p.i.—post-infection
In this example, immunization with either the low or medium dose tetravalent vaccine (e.g. DENVax™) formulation induced neutralizing antibodies to all four dengue serotypes at the early check on Day 14 post-vaccination, independent of administration of one vs. two doses on Day 0. The medium dose DENVax™ formulation induced slightly higher neutralizing antibody titers by Day 28 for Groups 1 and 3 particularly for DEN-1 and DEN-3, that received two doses on Day 0 compared to groups that received only a single dose on Day 0 (Groups 2 and 4). The antibody titers obtained from sera collected on Day 56 indicate that the neutralizing antibody responses persisted and did not wane regardless of whether the animals were boosted on Day 42 or received vaccine only on Day 0. The results obtained in this study further support the application of the novel dosing schedule of administering two doses on Day 0 at two distinct sites (e.g. immunologically).
TABLE 13
Neutralizing antibody titers for AG129 mouse study DEN-013
DEN-1
DEN-2
DEN-3
DEN-4
Day 28
Day 56
Day 28
Day 56
Day 28
Day 56
Day 28
Day 56
Study 1
3:3:3:3
2 (d0)
640
800
320
1600
160
800
20
40
2 (d0, 42)
320
800
320
800
160
400
40
40
4:3:4:5
2 (d0)
1280
3200
320
800
640
800
80
40
2 (d0, 42)
320
1600
160
800
80
800
40
80
FTA
2 (d0)
40
20
<20
20
20
20
<20
20
NMS
20
20
20
20
20
20
<20
20
Study 2
3:3:3:3
2 (d0)
800
3200
200
400
800
1600
160
160
2 (d0, 42)
400
1600
100
200
200
800
40
40
FTA
2 (d0, 14)
20
<20
<20
<20
20
20
20
<20
NMS
20
20
40
20
20
20
20
20
ELISPOT dengue virus neutralizing titers calculated using 50% NMS cutoff at a starting dilution of 1:20. Serum from individual animals within a group were pooled and tested in triplicate.
Example 8
FIGS. 9A-9D represent graphs comparing neutralizing antibody titers achieved in non-human primates after immunization with tetravalent DENVax containing DENVax-1 (1×105 pfu); DENVax-2 (1×104 pfu); DENVax-3 (1×105 pfu); DENVax-4 (1×106 pfu). Two groups were vaccinated with the needle-free PharmaJet device via the subcutaneous route either twice on the same day (0,0) or once on day 0 and again on day 60 (0,60). Serum was analyzed for presence of antibodies on days 0, 30, 53, 75 and 88, and the detection of antibodies against four dengue serotypes were analyzed (DEN-1, DEN-2, DEN-3, DEN-4).
In another example, seronegative human subjects were immunized with two doses of a tetravalent formulation of DENVax containing DENVax-1 (1×104 pfu); DENVax-22 (1×103 pfu); DENVax3 (1×104 pfu); DENVax-4 (1×105 pfu). The route of immunization was subcutaneous or intradermal, and the vaccinations were given 90 days apart. Antibody levels against each of the dengue serotypes were analyzed on days 0, 30, 60, 90 and 120. The vaccine induced neutralizing antibodies to all four serotypes. However, the levels of seroconversion were different when comparing the routes of immunization. Overall, the intradermal route of immunization produced appeared to be more “balanced” immune responses in this study, with the levels of antibodies being more equivalent as compared to the subcutaneous route.
FIG. 10 represents the data obtained from a human clinical trial in Colombia. Seronegative humans were given two doses of a tetravalent formulation of DENVax containing DENVax-1 (1×104 pfu); DENVax-2 (1×103 pfu); DENVax-3 (1×104 pfu); DENVax-4 (1×105 pfu) subcutaneously or intradermally. Antibody levels against each of the dengue serotypes were analyzed on days 0, 30, 60, 90 and 120.
In this exemplary method, non-human primates were immunized with two doses of a tetravalent vaccine (e.g. DENVax™ DENVax-1: 2×104 pfu, DENVax-2: 5×104 pfu, DENVax-3: 1×105 pfu, DENVax-4: 3×106 pfu) either simultaneously on Day 0, or two separate doses on days 0 and 60. The vaccine induced neutralizing antibodies to all four Dengue serotypes. By day 90 post vaccination, the neutralizing antibody titers of the two groups were relatively equal (FIG. 11). However, the kinetics of the immune response was more rapid in the group which received two immunizations on day 0. The results obtained in this study further support the application of the novel dosing schedule of administering two doses on Day 0 at two immunologically distinct sites.
FIG. 11 represents a graph comparing neutralizing antibody titers achieved in non-human primates after subcutaneous immunization with tetravalent DENVax containing DENVax-1 (1×105 pfu); DENVax-2 (1×104 pfu); DENVax-3 (1×105 pfu); DENVax-4 (1×106 pfu). Two groups were vaccinated either twice on the same day (0,0) or once on day 0 and again on day 60 (0,60). Serum was analyzed for presence of antibodies on days 0, 28, 58, 73 and 90, and the detection of antibodies against four dengue serotypes were analyzed (DEN-1, DEN-2, DEN-3, DEN-4).
All of the COMPOSITIONS and METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of preferred embodiments, it is apparent to those of skill in the art that variations maybe applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.
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13492884
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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435/5
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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tyo:4502
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Takeda Pharmaceutical
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Dec 15th, 2015 12:00AM
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May 28th, 2010 12:00AM
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https://www.uspto.gov?id=US09211323-20151215
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Compositions and methods for administration of vaccines against dengue virus
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Embodiments of the present invention report compositions and methods for vaccinating a subject against dengue viruses. In some embodiments, vaccine compositions may be administered by intradermal introduction. In certain embodiments, intradermal introduction in a subject of a vaccine against dengue virus may include one or more intradermal boosts after initial vaccination. Other embodiments include intradermal injection of a vaccine composition against dengue virus wherein the composition provides protection against two or more of DEN-1, DEN-2, DEN-3 and DEN-4.
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9211323
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1. A method for inducing an immune response in a subject against three or more dengue virus serotypes, comprising,
administering to the subject by at least a first intradermal introduction, a first immunogenic composition of a mixture of three or more live, attenuated dengue viruses, the composition comprising at least one dengue-dengue chimera and three or more different dengue virus serotype in the mixture, and administering at least a second intradermal introduction(s) of a second immunogenic composition against one or more dengue virus serotypes within 30 days after the first administration of the first immunogenic composition to the subject and inducing an immune response in the subject against three or more dengue virus serotypes.
2. The method of claim 1, wherein administering the at least a second intradermal introduction(s) of the second immunogenic composition occurs in the same anatomical area as the first intradermal introduction of the first immunogenic composition.
3. The method of claim 1, wherein administering the at least a second intradermal introduction(s) of the second immunogenic composition occurs at a separate anatomical site on the subject as the first intradermal introduction, and wherein the second intradermally-administered composition engages different lymph nodes than those engaged by the first intradermally-administered composition.
4. The method of claim 1, wherein the first immunogenic composition comprises at least three dengue virus serotypes at a predetermined ratio.
5. The method of claim 1, wherein the first immunogenic composition comprises all four dengue virus serotypes, a tetravalent formulation.
6. The method of claim 1, further comprising administering at least one additional immunogenic agent to the subject as part of, or separately from the first immunogenic composition.
7. The method of claim 6, wherein the additional immunogenic agent comprises at least one Toll receptor (TLR) ligand(s) and the additional immunogenic agent is added to the first immunogenic composition prior to administration to the subject.
8. The method of claim 1, wherein the mixture of three or more live, attenuated dengue viruses comprises a live, attenuated dengue-2 virus, and at least one dengue-dengue chimeric virus wherein capsid and non-structural proteins are from the live, attenuated dengue-2 virus, and wherein the backbone of the dengue-dengue chimera is dengue-2; and pre-membrane and envelope proteins are from at least a second, different dengue virus serotype.
9. The method of claim 8, wherein the pre-membrane and envelope proteins of the at least a second dengue virus are from dengue-1, dengue-3 or dengue-4.
10. The method of claim 8, wherein the at least one dengue-dengue chimeric viruses are two or more of dengue-2/dengue-1, dengue-2/dengue-3 and dengue-2/dengue-4.
11. A method for inducing an immune response in a subject against dengue virus serotypes, comprising,
administering to the subject by at least a first intradermal introduction, a single immunogenic composition of four live, attenuated dengue viruses wherein the composition comprises live, attenuated dengue-2 virus wherein the live, attenuated dengue-2 virus is not chimeric; and three dengue-dengue chimeras of live, attenuated dengue viruses of dengue 1, dengue 3 and dengue 4 and inducing an immune response in the subject against all four dengue virus serotypes.
12. The method of claim 11 administering at least a second immunogenic composition in the same anatomical area as the first intradermal introduction.
13. The method of claim 11, administering at least a second immunogenic composition intradermally at a separate anatomical area on the subject from the first intradermal introduction, and wherein the second intradermally-administered composition engages different lymph nodes than engaged by the first intradermally-administered composition.
14. The method of claim 11, wherein the dengue serotypes of the immunogenic composition comprise a predetermined ratio of one dengue virus serotype to another dengue virus serotype in the single composition.
15. The method of claim 1, wherein the first immunogenic composition and the second immunogenic composition comprise the same formulation.
16. The method of claim 1, wherein the first immunogenic composition and the second immunogenic composition comprise the same formulation, and wherein dengue-4 is present in the formulation at a concentration that is higher than the concentration of dengue-2 in the formulation.
17. The method of claim 1, wherein the first immunogenic composition and the second immunogenic composition comprise different formulations.
18. The method of claim 1, further comprising administering at least one additional immunogenic agent to the subject as part of, or separately from the second immunogenic composition.
19. The method of claim 11, further comprising administering at least a second immunogenic composition against dengue virus within 30 days after the first administration of the single immunogenic composition.
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19
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PRIORITY
This application claims the benefit under 35 USC §119(e) of provisional U.S. patent application Ser. No. 61/183,020 filed on Jun. 1, 2009, which is incorporated herein by reference in its entirety.
FEDERALLY FUNDED RESEARCH
This invention was made with Government support under U01 AI070443 awarded by the National Institutes of Health. The Government has certain rights in this invention.
FIELD
Embodiments of the present invention report compositions and methods for administering a vaccine to a subject against dengue viruses. In some embodiments, vaccine compositions may be administered by intradermal injection. In certain embodiments, intradermal injection in a subject of a vaccine against dengue virus may include one or more intradermal boosts after initial vaccination. Other embodiments include intradermal injection of a vaccine composition against dengue virus wherein the composition provides protection against more than one serotype of dengue virus, such as DEN-1, DEN-2, DEN-3 and DEN-4.
BACKGROUND
Vaccines for protection against viral infections have been effectively used to reduce the incidence of human disease. One of the most successful technologies for viral vaccines is to immunize animals or humans with a weakened or attenuated strain of the virus (a “live, attenuated virus”). Due to limited replication after immunization, the attenuated strain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and can generate potent and long-lasting immune responses to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the immunized individual is protected from disease. These live, attenuated viral vaccines are among the most successful vaccines used in public health.
SUMMARY
Embodiments of the present invention generally relate to methods and compositions for inducing protection in a subject against dengue virus by, for example, administering a vaccine to a subject against dengue viruses. Some embodiments can include introducing a vaccine composition to a subject via intradermal (ID) injection. In accordance with these embodiments, the vaccine composition can be introduced to a subject intradermally to, for example, protect against one or more than one dengue serotype (e.g. cross protection). In certain embodiments, a vaccine composition can include, but is not limited to, a single dose of one serotype of dengue virus (e.g. DENVax 4) administered to a subject. In other embodiments, a vaccine composition may include, but is not limited to; an initial dose of one serotype of dengue virus (e.g. DENVax 4 or other serotype) and then one or more boosts of the same, a combination or a different serotype can be administered to a subject.
Other aspects herein can concern inducing a cellular immune response in a subject by, for example, introducing a vaccine composition to a subject via intradermal introduction wherein the vaccine composition includes, but is not limited to, a dengue virus vaccine. In accordance with these embodiments, compositions disclosed can be administered intradermally to a subject for modulating neutralizing antibody production in the subject against dengue virus serotypes. Some aspects concern predetermined composition ratios (e.g. 1:1, 1:2, 1:4, any ratio of two or more serotypes is contemplated) of the various serotypes of dengue virus or fragments thereof or attenuated compositions thereof in a single vaccine composition in order to increase cross protection in a subject against some or all dengue virus serotypes when the subject is administered the single vaccine composition intradermally.
In certain embodiments, some advantages of using intradermal introduction of a vaccine against dengue virus can include, but are not limited to, multiple protection against some or all dengue virus serotypes in a subject, reduced cost by using small doses compared to subcutaneous injection, modulation of antibodies produced against some or all dengue virus serotypes in a subject and reduced pain at a site of administration in a subject administered a composition of vaccine against dengue virus.
In some embodiments, a single dose vaccine against dengue virus can include one or more dengue virus serotype(s). In accordance with these embodiments, a subject may be treated with at least one additional intradermal injection(s) administered at a separate site from the first injection, for example, next to or in a separate anatomical site on the subject. In addition, at least one additional intradermal injection(s) may be performed less than 30 days after the first administration to the subject. Vaccine compositions of these and other embodiments disclosed herein may include two or more dengue virus serotypes at a predetermined ratio.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain embodiments. Some embodiments may be better understood by reference to one or more of these drawings alone or in combination with the detailed description of specific embodiments presented.
FIG. 1 represents an example of an intradermal injection device currently available.
FIG. 2 represents examples of injection sites in a subject having intradermal introduction of a vaccine against dengue virus.
FIG. 3 represents a bar graph comparison of subcutaneous versus intradermal injection of a vaccine against dengue virus and neutralizing antibody titer produced against different dengue virus serotypes after a primary administration.
FIG. 4 represents a bar graph comparison of subcutaneous versus intradermal injection of a vaccine against dengue virus and neutralizing antibody titer produced against different dengue virus serotypes after a second, boosting administration.
FIG. 5 represents a histogram plot of subcutaneous and intradermal immunizations with a vaccine against a dengue virus serotype in mice.
FIGS. 6A and 6B represent graphic depictions of a challenge experiment using two different dengue virus serotypes on a dengue serotype-immune mouse population following vaccination of the mice with another dengue virus serotype.
DEFINITIONS
As used herein, “a” or “an” may mean one or more than one of an item.
As used herein, vessel can include, but is not limited to, test tube, mini- or micro-fuge tube, channel, vial, microtiter plate or container.
As used herein the specification, “subject” or “subjects” may include but are not limited mammals such as humans or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g., hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairie dogs, or zoo animals.
As used herein, “about” can mean plus or minus ten percent.
As used herein, “attenuated virus” can mean a virus that demonstrates reduced or no clinical signs of disease when administered to a subject such as a mammal (e.g., human or an animal).
DESCRIPTION
In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details may be modified through routine experimentation. In some cases, well-known methods or components have not been included in the description.
Certain aspects of the present invention include, but are not limited to, administration of vaccine compositions against dengue virus.
Embodiments of the present invention generally relate to methods and compositions for inducing protection in a subject against dengue virus serotypes. Other embodiments can include introducing a vaccine composition to a subject via intradermal (ID) injection wherein the vaccine composition introduced intradermally induces cross protection against some or all dengue serotypes. In certain embodiments, the vaccine composition comprises a single dose of a vaccine against dengue virus serotype 4 (DENVax 4) administered to a subject. In other embodiments, the vaccine composition comprises an initial dose of DENVax 4 then, one or more boosts of the vaccine administered to a subject.
Other aspects of the present invention include modulating an immune response to a vaccine against dengue virus administered intradermally compared to subcutaneously to a subject. Vaccines against dengue virus may include a composition comprising ratios of serotypes of dengue virus, live attenuated dengue virus, or fragments thereof such as proteins or nucleic acids derived or obtained from dengue virus serotypes. Ratios of various serotypes may be equal or certain serotypes may be represented more than others depending on need or exposure or potential exposure to the virus. In accordance with these embodiments, a ratio may be a 1:2, 1:3, 1:4, 1:10, 1:20; 1:1:1, 1:2:2, 1:2:1, 1:1:1:1, 1:2:1:2; 1:3:1:3, 2:3:3:3, 5:4:5:5, 1:2:2 or any ratio for any of serotypes 1, 2, 3 and/or 4, depending on for example, number of serotypes represented in the formulation, predetermined response and effect desired. It is contemplated that any dengue virus serotype formulation may be used to generate a vaccine (e.g. attenuated virus etc.) of use in intradermal administration to a subject in need thereof. It is contemplated that some formulations may be more effective than others when introduced intradermally than other formulations.
In other embodiments, compositions of dengue virus vaccine formulations may be introduced intradermally to a subject prior to, during or after exposure to dengue virus by the subject. In accordance with these embodiments, a subject may receive a single intradermal injection or more than one injection comprising a dengue virus formulation, optionally, followed by one or more additional injections. Intradermal applications of formulations described herein may be combined with any other anti-viral treatment or administration mode of vaccine (e.g. subcutaneous injection) to a subject. In some embodiments, it is contemplated that intradermal introduction of a formulation contemplated herein may be administered to any appropriate region of a subject's body (e.g. arm, hip, etc). In addition, intradermal administration of vaccine formulations herein as primary or boost administrations may occur in the same day, consecutive days, weekly, monthly, bi-monthly or other appropriate treatment regimen.
Methods
Nucleic Acid Amplification
Nucleic acids may be used in any formulation or used to generate any formulation contemplated herein. Nucleic acid sequences used as a template for amplification can be isolated viruses (e.g. dengue viruses), according to standard methodologies. A nucleic acid sequence may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary cDNA. In some embodiments, the RNA is whole cell RNA and is used directly as the template for amplification. Any method known in the art for amplifying nucleic acid molecules is contemplated (e.g., PCR, LCR, Replicase, etc).
Expressed Proteins or Peptides
Genes can be expressed in any number of different recombinant DNA expression systems to generate large amounts of the polypeptide product, which can then be purified and used in methods and compositions reported herein. Any method known in the art for generating and using constructs is contemplated. In certain embodiments, genes or gene fragments encoding one or more polypeptide may be inserted into an expression vector by standard cloning or subcloning techniques known in the art.
Proteins, peptides and/or antibodies or fragments thereof may be detected or analyzed by any means known in the art. In certain embodiments, methods for separating and analyzing molecules may be used such as gel electrophoresis or column chromatography methods.
Electrophoresis
Electrophoresis may be used to separate molecules (e.g., large molecules such as proteins or nucleic acids) based on their size and electrical charge. There are many variations of electrophoresis known in the art. A solution through which the molecules move may be free, usually in capillary tubes, or it may be embedded in a matrix or other material known in the art. Common matrices can include, but are not limited to, polyacrylamide gels, agarose gels, mass spec, blotting and filter paper.
Some embodiments, using a gene or gene fragment encoding a polypeptide may be inserted into an expression vector by standard subcloning techniques. An expression vector may be used which produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification of a peptide or protein. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, N.J.), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×His system (Qiagen, Chatsworth, Calif.).
Pharmaceutical Formulations
Any pharmaceutical formulation known in the art for a vaccine is contemplated herein. In certain embodiments, a formulation can contain one or more DEN serotype in various ratios, depending on predetermined exposure, to or existence of dengue virus subtypes. It is contemplated that formulations can contain other agents of use in vaccination of a subject including, but not limited to other active, or inactive ingredients or compositions known to one skilled in the art.
Kits
Other embodiments concern kits of use with the methods (e.g. methods of application or administration of a vaccine) and compositions described herein. Some embodiments concern kits having vaccine compositions of use to prevent or treat subjects having, exposed or suspected of being exposed to one or more dengue viruses. In certain embodiments, a kit may contain one or more than one formulation of dengue virus serotype(s) (e.g. attenuated vaccines) at predetermined ratios. Kits can be portable, for example, able to be transported and used in remote areas such as military installations or remote villages. Other kits may be of use in a health facility to treat a subject having been exposed to one or more dengue viruses or suspected of being at risk of exposure to dengue virus.
Kits can also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the agent, composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Optionally, one or more additional agents such as immunogenic agents or other anti-viral agents, anti-fungal or anti-bacterial agents may be needed for compositions described, for example, for compositions of use as a vaccine against one or more additional microorganisms.
The following examples are included to demonstrate certain embodiments presented herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered to function well in the practices disclosed herein. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the certain embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope herein.
EXAMPLES
Example 1
Previous studies revealed that natural infection with each DEN (dengue virus) serotype leads to long-lived protection against dengue lever caused by the homologous serotype. In certain embodiments, administration of an effective dengue vaccine closely mimics natural infection and can serve as a mode for administering vaccines against dengue virus. Embodiments reported herein can concern a natural infection route of dengue virus (DEN) infection, similar to intradermal delivery by the transporting host, a mosquito bite. In certain embodiments, intradermal injection to deposit the vaccine viruses into the same tissue can be used. Skin is a highly accessible organ and represents an effective immune barrier, mainly attributed to the presence of Langerhans cells (LCs) residing in the epidermis. Skin immunization elicits a broad range of immune responses, including humoral, cellular, and mucosal and has the potential to bypass the effect of pre-existing immunity on the immunogenicity of administered vaccines.
Some embodiments for intradermal (ID) administration of the tetravalent dengue vaccines in a subject in need of such a treatment are reported. One exemplary method of intradermal administration was performed on four Cynomolgous macaques administered a 5:5:5:5 DENVax (dengue virus vaccine) by intradermal administration. To achieve an equivalent dose of virus, 0.15 ml of vaccine was deposited. ID in three closely spaced sites using a needle-free jet injector (see FIGS. 1 and 2, below), FIG. 1 represents an intradermal inject (e.g., PharmaJet®) device used for intradermal inoculations.
FIG. 2 illustrates inoculation sites on Cynomolgus macaques post vaccination with PharmaJet device. Animals were boosted 60 days later with the same formulation by the same route. Serum samples were collected at predetermined intervals, days 15, 30, 58, 74, and 91 and were tested for the presence of neutralizing antibodies directed against the four dengue serotypes. PRNT (plaque reduction neutralization test, known in the art for quantifying levels of anti-DEN neutralizing antibodies) were performed on the sera samples.
It was demonstrated that the neutralizing antibody titers are significantly higher after ID administration as compared to SC administration after a primary administration (see FIG. 3) or after a secondary administration (See FIG. 4). Since the sites were closely spaced in the same area, and each inoculum consists of all four viruses, this mode of vaccine delivery closely resembles a single administration of DENVax. FIG. 3 illustrates 50% PRNT Geometric Mean Titers at Day 58 (58 days after the primary administration). FIG. 4 illustrates 50% PRNT Geometric Mean Titers at Day 74 (14 days after the secondary administration on Day 60). As can be seen in the figures, the neutralizing antibody titers to all four dengue viruses were higher after intradermal versus subcutaneous administration. In addition, the number of animals that demonstrated neutralizing antibody responses (“seroconversion” defined as PRNT>10) was greater after the first dose of vaccine (see Table 1, the percentage of animals that seroconverted to each of the four dengue serotypes is shown after primary and secondary immunization).
TABLE 1
Seroconversion of non-human primates after dengue immunization
% Seroconversion
DENVax
DEN-1
DEN-2
DEN-3
DEN-4
Formyiation
Prime
Boost
Prime
Boost
Prime
Boost
Prime
Boost
5:5:5:5 SC
87.5%
100.0%
100.0%
100.0%
75.0%
100.0%
50.0%
100.0%
5:5:5:5 ID
100.0%
100.0%
100.0%
100.0%
100.0%
100.0%
100.0%
100.0%
The immunized animals were tested for protection against challenge with wild type dengue viruses. In cynomolgus macaques, wild type dengue virus infection leads to virus replication and viremia, but no clinical signs. At day 91, two monkeys were challenged with DENV-1 (dengue virus serotype 1) and two monkeys challenged with DEN-2 (dengue virus serotype 2). Serum samples were collected daily for 11 days after challenge. Levels of dengue virus RNA were measured in the samples by quantitative real-time polymerase chain reaction technology (q-rtPCR) and titers of viable virus were measured by virus isolation and plaque formation on Vero cells. The results are shown in Tables 2 and 3. Neutralizing antibodies against DEN-1 at Day 91, just prior to challenge (“Pre-Challenge”) and Day 105, 14 days after challenge (“Post”). Viremia is given as the number of days that live DEN-1 virus could be isolated from blood samples (“Duration”) and the log 10 of the peak titer isolated from each animal. Viral RNA is given as the number of days viral RNA could be detected in the serum samples (“Duration”) and peak viral RNA levels in each monkey, expressed as the log 10 of the number of viral RNA genomes detected.
TABLE 2
Responses after challenge with DEN-1
DEN-1 PRNT
Viremia
Viral RNA
Monkey
Formulation
Pre-Challenge
Post
Duration
Peak
Duration
Peak
CY0174
5:5:5:5 SC
240
240
0
0
0
0
CY0181
5:5:5:5 SC
640
61440
0
0
5
5.6
CY0192
5:5:5:5 ID
1920
1280
0
0
0
0
CY0194
5:5:5:5 ID
7680
1920
0
0
0
0
CY0061
Controls
1
2560
6
2.0
9
5.7
CY0193
Controls
25
2580
3
2.7
7
6.4
CY0058
Controls
1
640
5
2.9
7
5.5
CY0073
Controls
1
1280
5
3.6
10
6.2
TABLE 3
Responses after challenge with DEN-2
DEN-2 PRNT
Viremia
Viral RNA
Monkey
Formulation
Pre-Challenge
Post
Duration
Peak
Duration
Peak
CY0172
5:5:5:5 SC
3413
3413
0
0
1
3.9
CY0177
5:5:5:5 SC
853
533
0
0
0
0
CY0198
5:5:5:5 ID
240
320
0
0
0
0
CY0201
5:5:5:5 ID
1920
1600
0
0
0
0
CY0088
Controls
6
10240
6
2.3
8
5.1
CY0199
Controls
1
3840
5
1.8
9
4.7
CY0065
Controls
1
10240
5
2.9
8
5.8
CY0104
Controls
1
10240
4
2.4
8
5.7
After challenge, the SC and ID immunized animals were completely protected from DEN-1 or DEN-2 induced viremia (compared to the control animals that demonstrated significant viremia of long duration). In all of the ID immunized animals, but not all of the SC immunized animals, there was also an absence of viral RNA replication and a lack of an increase in antibody titer after challenge (compare the ID animals to SC injected CY0181, CY0.172 or the control animals). These data suggest that protection is “sterilizing” and prevents any virus replication after challenge.
Example 2
in another example, an optimized DENVax formulation delivered in different locations and with different timings will be tested in non-human primates, Groups of eight Cynomolgus macaques will be immunized with a DENVax formulation containing 1×105 plaque forming units (pfu), 1×104 pfu, 1×105 pfu and 1×105 pfu of DENVAx-1, DENVax-2, DENVax-3 and DENVax-4, respectively (abbreviated 5:4:5:5). Two doses will be administered in 0.1 ml ID. Groups will be immunized with either one dose in each arm at Day 0, one dose in one arm at Day 0 and one dose in the other arm at Day 7, or one dose in one arm at Day 0 and one dose in the other arm at Day 60, These groups will be compared to a group that receives the same dose (5:4:5:5) in three sites in the same are on Day 0 and three sites in the other arm on Day 60 as well as a group that receives the same dose in a single 0.5 ml SC immunization in one arm at Day 0 and in the other arm at Day 60. A control group will be immunized with vaccine excipients only (no vaccine viruses). Following immunization, blood samples will be collected on days 0, 7 (for peak viremia), 15, 30, 60, and 90 to test the neutralizing antibodies against the four dengue virus serotypes by PRNT50. PBMCs collected on days 30, 60, 90 will be also monitored for IFN-γ secretion by an ELISPOT assay. On day 90, two animals from each group will be challenged with wild type of DEN-1, DEN-2, DEN-3, or DEN-4 viruses. Challenged animals will be monitored for clinical signs and, temperature (twice daily), changes in food consumption (once daily) and body weight (weekly). In addition, all animals will be bled daily for 11 days post-challenge to monitor viremia and hematological parameters. Again, the speed and duration of PRNT responses to all four DEN viruses and protection after day 90 challenge will be assessed. It is believed that intradermal administration in multiple sites and in distinct anatomical locations may be more effective than subcutaneous administration as a single bolus. Multiple sites can provide exposure of the vaccine to more antigen presenting cells. Distinct anatomical locations can permit vaccine access to multiple lymph nodes. In addition, booster immunizations of dengue vaccines have only been administered after the development of antibody responses in mice, primates and human clinical trials, thirty days or longer. At this time, neutralizing antibodies inhibit the response to the live viral vaccines. It was previously shown that boosting primates one month after primary immunization was less effective than dosing four months after primary immunization. It was speculated that high levels of homologous and heterologous antibodies that circulate after the initial immunization can inhibit viral replication in a second dose. While prolonged (two months or longer) immunization may circumvent this inhibition, it has not been tested whether accelerated immunization regimen with shorter immunization intervals, before the development of potent neutralizing antibody responses may be advantageous. Such a shortened regimen may be an advantage in endemic countries or for travelers, where exposure to dengue viruses in between the immunizations may put them at risk of disease.
Example 3
In another example, a human clinical trial has been initiated, studying the safety and immunogenicity of two DENVax formulations, administered in 0.1 ml either by ID or SC injection. Groups of 12 individuals will be immunized with for example, a low dose DENVax formulation (8×103 pfu, 5×103 pfu, 1×104 pfu and 2×1 pfu of DENVax-1, -2, -3 and -4, respectively) or a high dose (2×104 pfu, 5×104 pfu, 1×105 pfu and 3×105 pfu of DENVax-1, -2, -3 and -4, respectively) of DENVax ID or SC on Days 0 and 90. Two control groups will be injected SC or ID with phosphate-buffered saline. Patients will be monitored for any adverse events, and for any significant changes in hematological or blood chemistry parameters. Serum samples will be collected to measure vaccine virus replication and neutralizing antibody responses at periodic intervals.
Example 4
Immunogenicity and efficacy of DENVax administered intradermally in AG129 mice. In another example, two studies were performed to compare the effect of route of administration on immunogenicity and efficacy of DENVax in AG129 mice. In one example, the immunogenicity of monovalent DENVax-4 (e.g. vaccine against one Dengue virus serotype) was compared in AG129 mice by measuring the neutralizing antibody responses following SC injection under the skin on the back or ID injection into the foot pad using a needle and syringe. Groups of 8 AG129 mice were injected ID or SC with 105 PFU/dose of chimeric DENVax-4 vaccine in 50 μl and 100 μl final volume, respectively. Six weeks after priming, animals from each treatment group were boosted via the corresponding ID or SC route with 105 PFU of DENVax-4 or TEA. Mice were bled on Day 31 and 58 and collected sera were pooled to measure neutralizing antibody responses.
Immunization of DENVax-4 via the ID route elicited a 5-fold higher neutralizing antibody response to DEN-4 after the boost compared to the response induced via the SC route (see for example, FIG. 4). The anti-DEN-4 response elicited by either route of immunization had a marked cross-neutralizing activity against DEN-3 but not against DEN-1 or DEN-2 serotypes. FIG. 4 represents neutralizing antibody responses following primary and secondary immunization of AG129 mice with chimeric DENVax-4. Mice were bled on Day 31 and 58 and collected sera were pooled to measure neutralizing antibody responses using the plaque reduction assay (PRNT50).
Two weeks after the boost animals from each group were split in to two groups and challenged with 106 PFU of DEN-1 (Mochizuki virus strain) or DEN-2 (New Guinea C strain) viruses. Challenged animals were monitored for clinical signs of disease and survival rates were recorded over a period of 5 weeks. Mice immunized via the ID route showed no signs of disease after DEN-1 challenge (FIG. 5A). In the SC immunized group only one mouse succumbed to infection while the rest of animals had no any apparent signs of infection (FIG. 5B). In contrast, all control animals succumbed to infection by day 13 after DEN-1 challenge (FIG. 5A). Following DEN-2 challenge, all animals immunized with only DENVax-4 via the SC route succumbed to infection by day 2.5 with mean survival time (MST) of 19.5 days as compared to the control (TFA) mice that all succumbed by day 17 (MST=12.5 days) post-challenge (FIG. 5B). In contrast, fifty percent of ID DENVax-4 immunized mice survived the infection until the cud of the 5 week monitoring period (FIG. 5B). FIGS. 5A and 5B represent survivals of DENVax-4 immune AG129 mice following challenge with DEN-1 (a) or DEN-2 (b) viruses. Challenged animals were monitored for clinical signs of disease and survival rates were recorded over a period of 5 weeks.
In a second study, immunogenicity of tetravalent DENVax vaccine administered SC or ID in mice (e.g. AG129) was tested. Groups of AG129 mice, six per group were injected SC or ID with the DENVax in 100 μl or 50 μl (final volume), respectively. Mice were immunized with DENVax at a 5:4:5:5 (105 PFU of DENVax-1, -3 and -4 and 104 PFU of DENVax-2) dose level of composite chimeric vaccines. All immunized animals received a booster injection of 5:4:5:5 DENVax 42 days' post-primary inoculation. Blood samples were collected on days 42 and 56 to measure neutralizing antibody responses to each DEN virus serotype.
As represented in Table 4, both primary and secondary neutralizing antibody responses to all four DEN serotypes were induced. Following the boost, the neutralizing anti-DEN-1, DEN-3 and DEN-4 antibody titers were increased by 2, 5 and 2 fold, respectively in the group of mice injected ID as compared to the SC immunized animals. Neutralizing responses to DEN-2 virus were comparable in both groups. Immunization via the SC route resulted in a profile of dominant neutralizing antibody responses against DEN-1>DEN-2>DEN-3>DEN-4, with neutralizing titers 5120, 1280, 640 and 80, respectively. The hierarchy of neutralizing antibody responses after ID administration had shifted as follows; DEN-1>DEN-3>DEN-2>DEN-4 with neutralizing antibody titers 10240, 3840, 1280 and 160, respectively.
TABLE 4
Comparison of the immunogenicity of tetravalent DENVax
bearing the ratio 5:4:5:5 PFU of each composite chimeric virus
(105 PFU of DENVax-1, -3 and -4 and 104 PFU of DENVax-2)
after SC or ID immunization of mice.
Neutralizing Antibody Titers (GMT)
DENVax
DEN-1
DEN-2
DEN-3
DEN-4
Formulation
Prime
Boost
Prime
Boost
Prime
Boost
Prime
Boost
5:4:5:5/SC
1920
5120
3200
1280
1280
640
80
80
5:4:5:5/ID
2560
10240
1280
1280
1600
3840
120
160
Blood samples were collected on days 42 and 56 to measure neutralizing antibody responses to each DEN virus serotype.
Materials and Methods
Mice: AG129 mice have an “intact” immune system; deficient for the interferon (IFN)-α/β and -γ receptors. Dengue infection has been described. Other studies: pathogenesis, cell tropism, and ADE have been examined. This model permits challenge with DEN-1 and DEN-2.
Non-human primates: Cynomolgus, rhesus macaques carry virus (viremia), but no disease manifests.
All of the COMPOSITIONS and METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of preferred embodiments, it is apparent to those of skill in the art that variations maybe applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.
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12790511
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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tyo:4502
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Takeda Pharmaceutical
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Oct 20th, 2020 12:00AM
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Oct 12th, 2018 12:00AM
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https://www.uspto.gov?id=US10806781-20201020
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Compositions and methods for live, attenuated alphavirus formulations
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Embodiments herein relate to compositions of and methods for live attenuated alphaviruses. In certain embodiments, a live, attenuated virus composition includes, but is not limited to, one or more live, attenuated alphaviruses and compositions to reduce inactivation and/or degradation of the live, attenuated alphavirus. In other embodiments, the live, attenuated virus composition may be a vaccine composition. In yet other compositions, a live, attenuated alphavirus composition may include HEPES buffer. In other embodiments, the HEPES buffer may further include a carbohydrate and gelatin and/or a salt.
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10806781
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1. A live attenuated alphavirus virus composition comprising:
one or more live, attenuated alphaviruses selected from the group consisting of chikungunya (CHIK) virus, o'nyong'nyong virus, Eastern equine encephalitis, Western equine encephalitis, and Venezuelan equine encephalitis;
10.0 to 20.0 mM HEPES buffer;
one or more carbohydrate agents having a concentration from 1.0% to 15% (w/v) selected from the group consisting of: trehalose, sucrose, mannitol, sorbitol, and galactose; and
0.1% to 1.0% (w/v) gelatin,
wherein the composition stabilizes live attenuated alphavirus compositions.
2. The virus composition of claim 1, wherein the live, attenuated alphaviruses are Chikungunya (CHIK) viruses.
3. The virus composition of claim 1, wherein the composition is in aqueous form.
4. The virus composition of claim 1, wherein the composition is partially or wholly dehydrated.
5. The virus composition of claim 1, wherein the composition comprises 10.0 to 20.0 mM HEPES, sucrose and gelatin.
6. The virus composition of claim 1, wherein the gelatin concentration comprises 0.5% to 1.0% (w/v).
7. The virus composition of claim 1, wherein the HEPES buffer concentration is 15 mM and the gelatin concentration is 0.5% to 1.0% (w/v).
8. The virus composition of claim 1, further comprising 10 to 200 mM salt.
9. A method for decreasing inactivation of a live, attenuated alphavirus composition comprising, combining one or more live attenuated alphaviruses selected from the group consisting of chikungunya (CHIK) virus, o'nyong'nyong virus, Eastern equine encephalitis, Western equine encephalitis, and Venezuelan equine encephalitis with a composition comprising: 10 mM to 20 mM HEPES buffer; one or more carbohydrate agents at a concentration of 1.0% to 15% (w/v) selected from the group consisting of: trehalose, sucrose, mannitol, sorbitol, and galactose; and gelatin having a concentration of 0.1% to 1.0% (w/v), wherein the composition decreases inactivation of the live, attenuated alphavirus compositions.
10. The method of claim 9, further comprising partially or wholly dehydrating the combination.
11. The method of claim 9, further comprising partially or wholly re-hydrating the composition prior to administration.
12. The method of claim 9, wherein the composition increases the shelf-life of an aqueous virus composition.
13. The method of claim 9, wherein the concentration of HEPES buffer is 15 mM; and the gelatin concentration is 0.5% to 1.0% (w/v).
14. The method of claim 9, wherein the live, attenuated alphavirus composition is formulated for use as a medicament for administration to a subject to reduce the onset of a health condition.
15. A kit for decreasing the inactivation of a live, attenuated alphavirus composition
comprising:
at least one container;
a composition comprising 10 mM to 20 mM HEPES buffer; one or more carbohydrate agents at a concentration of 1.0% to 15% (w/v) selected from the group consisting of: trehalose, sucrose, mannitol, sorbitol, and galactose;
0.1% to 1.0% (w/v) gelatin; and
an alphavirus, wherein the alphaviruses are selected from the group consisting of chikungunya (CHIK) virus, o'nyong'nyong virus, Eastern equine encephalitis, Western equine encephalitis, and Venezuelan equine encephalitis.
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15
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PRIORITY
This application is a divisional of U.S. patent application Ser. No. 14/209,921 filed Mar. 13, 2014, which claims benefit of U.S. Provisional Application Ser. No. 61/784,122 filed Mar. 14, 2013. The entire contents of the aforementioned applications are herein incorporated by reference.
FIELD
Embodiments herein relate to compositions and methods for stabilizing live, attenuated viruses. Other embodiments relate to compositions and methods for reducing degradation of live, attenuated viruses. Still other embodiments relate to uses of these compositions in kits for portable applications and methods.
BACKGROUND
Vaccines to protect against viral infections have been effectively used to reduce the incidence of human or animal disease. One of the most successful technologies for viral vaccines is to immunize animals or humans with a weakened or attenuated strain of the virus (a “live, attenuated virus”). Due to limited replication after immunization, the attenuated strain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and generates potent and long-lasting immune responses to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the immunized individual is protected from disease. These live, attenuated viral vaccines are among the most successful vaccines used in public health.
The majority of viral vaccines approved for sale in the U.S. are live, attenuated viruses. Highly successful live viral vaccines include the yellow fever 17D virus, Sabin poliovirus types 1, 2 and 3, measles, mumps, rubella, varicella and vaccinia viruses. Use of the vaccinia virus vaccine to control smallpox outbreaks led to the first and only eradication of a human disease. The Sabin poliovirus vaccine has helped prevent crippling disease throughout the world and is being used in the efforts to eradicate polio. Childhood vaccination with measles, mumps, rubella and varicella vaccines prevent millions of deaths and illnesses internationally.
Chikungunya fever, a mosquito-borne viral disease that recently re-emerged to cause millions of cases of severe and often chronic arthralgia in Africa and Asia. Chikungunya has recently emerged in the Caribbean, demonstrating spread to the Western Hemisphere. Vaccines against this condition will not only prevent disease in endemic parts of the world, but will reduce the risk of importation into the U.S. and other parts of the Americas.
Recent technical advances, such as reassortment, reverse genetics and cold adaptation, have led to the licensure of live, attenuated viruses for influenza and rotavirus. A number of live, viral vaccines developed with recombinant DNA technologies are in animal and human clinical testing. These recombinant viral vaccines rely on manipulation of well-characterized attenuated viral vaccines. The safe, attenuated viruses are genetically engineered to express protective antigens for other viral or bacterial pathogens.
In order for live, attenuated viral vaccines to be effective, they must be capable of replicating after immunization. Thus, any factors that inactivate the virus can cripple the vaccine. In addition to freeze-drying, various additives have been identified that can help stabilize the viruses in live, attenuated viral vaccines (See for example Burke, Hsu et al 1999).
Other commonly used vaccines are sensitive to temperature extremes; either excessive heat or accidental freezing can inactivate the vaccine. Maintaining this “cold chain” throughout distribution is particularly difficult in the developing world. Thus, there remains a need for improving the stability of both existing and newly developed live, attenuated viral vaccine formulations.
SUMMARY
Embodiments herein concern methods and compositions to reduce or prevent deterioration or inactivation of live attenuated Alphavirus compositions. Certain compositions disclosed can include combinations of components that reduce deterioration of a live, attenuated alphaviruses. Other embodiments herein concern combinations of excipients that greatly enhance the stability of live, attenuated alphaviruses. Yet other compositions and methods herein are directed to reducing the need for lower temperatures (e.g. refrigerated or frozen storage) while increasing the shelf life of aqueous and/or reconstituted live attenuated, alphaviruses. In accordance with these embodiments, a live, attenuated alphavirus composition can be used to induce an immune response to the alphavirus in a subject wherein the subject can have a reduced incidence of infection caused by the alphavirus.
Some embodiments, directed to compositions, can include, but are not limited to, one or more live, attenuated alphaviruses, such as one or more live, attenuated alphavirus in combination with HEPES buffer, one or more carbohydrates and gelatin. In accordance with these embodiments, any HEPES buffer, and any gelatin product of use in a subject can be used in the composition. The sources of gelatin can vary from those derived from a mammalian origin to synthetically generated gelatin forms. Carbohydrates of use in the composition include but are not limited to sucrose, lactose galactose, trehalose, fructose, sorbitol, dextrose, mannitol and other carbohydrate sources. In certain embodiments, all three components are required to stabilize a live, attenuated alphavirus composition. In other embodiments, a salt can be added to the composition to provide salinity or osmolality to the composition (e.g. sodium chloride or other salt). In certain embodiments, a composition contemplated herein can include, but is not limited to, buffered HEPES about pH 6.0 to pH 10.0 at about 1 to 40 mM HEPES, one or more carbohydrate agents at about 1 to 25% w/v, and one or more protein agents that includes gelatin at about 0.01 to 5.0% w/v, wherein the composition decreases inactivation and/or degradation of a live, attenuated alphavirus.
Compositions contemplated herein can increase the stabilization and/or reduce the inactivation and/or degradation of a live, attenuated alphavirus including, but not limited to, chikungunya virus, o'nyong'nyong virus, Ross River virus, eastern equine encephalitis, Venezuelan Equine Encephalitis Virus and western equine encephalitis or other alphaviruses in the Coronaviridae and Togaviridae families. Other Semliki Forest virus complexes include, but are not limited to, Bebaru virus, Mayaro virus, Subtype: Una virus, O'Nyong Nyong virus: Subtype: Igbo-Ora virus, Ross River virus: Subtype: Bebaru virus; Subtype: Getah virus; Subtype: Sagiyama virus, Semliki Forest virus: Subtype: Me Tri virus.
Chikungunya virus is an alphavirus with a positive sense single-stranded RNA genome of approximately 11.6 kb. It is a member of the Semliki Forest Virus complex and is closely related to Ross River Virus, O'Nyong Nyong virus and Semliki Forest Virus. Compositions disclosed herein can be used for any member of the Semliki Forest Virus complex to increase stability or reduce degradation of a live, attenuated virus of use in vaccine compositions.
Human epithelial, endothelial, primary fibroblasts and monocyte-derived macrophages are permissive for chikungunya virus in vitro and viral replication is highly cytopathic but susceptible to type I and II interferon. In vivo, chikungunya virus appears to replicate in fibroblasts, skeletal muscle progenitor cells and myofibers
Other embodiments concern live, attenuated virus compositions and methods directed to vaccine or immunogenic compositions capable of reducing or preventing onset of a medical condition caused by one or more of the alphaviruses contemplated herein. Pharmaceutical compositions disclosed herein concern compositions that are prepared for or formulated for introduction to a subject such as a human, an animal such as a domesticated animal or live-stock.
In certain embodiments, compositions contemplated herein can be partially or wholly dehydrated or hydrated. Further, compositions disclosed herein can be used during and after lyophilization of a live, attenuated alphavirus composition. In accordance with these embodiments, a composition may be 20% or more; 30% or more; 40% or more; 50% or more; 60% or more; 70% or more; 80% or more; or 90% or 95% or more dehydrated. Compositions described herein are capable of increasing the shelf life of an aqueous or rehydrated live attenuated alphavirus. Compositions disclosed herein increase stability of live, attenuated alphavirus at a wide-range of temperatures such as room temperature, sub-zero temperatures, elevated temperatures (e.g. −80° C.-37° C. and above) under lyophilized or liquid/frozen conditions. In certain embodiments, compositions disclosed herein can increase stability of a live, attenuated alphavirus 2 fold, 4 fold, 10 fold or more than a live, attenuated alphavirus composition not exposed to at least a composition of HEPES buffer, carbohydrate and gelatin.
Other embodiments concern methods for decreasing inactivation of a live, attenuated alphaviruses including, but not limited to, combining one or more live attenuated alphaviruses with a composition capable of reducing inactivation of a live, attenuated virus including, but not limited to, one or more protein agents; one or more saccharides or polyols agents; and one or more buffers, wherein the composition decreases inactivation of the live attenuated virus. In accordance with these embodiments, the live attenuated virus may include, but is not limited to, a Togavirus or Coronavirus, or in certain embodiments, any Alphavirus.
In certain embodiments, compositions contemplated herein are capable of decreasing inactivation and/or degradation of a hydrated live attenuated Alphavirus for greater than 12 to 24 hours at room temperatures (e.g. about 20° C. to about 25° C. or even as high as 37° C.) or refrigeration temperatures (e.g. about 0° to about 10° C.). In some embodiments, a combination composition is capable of maintaining about 100 percent of the live attenuated Alphavirus for greater than 24 hours. In addition, combination compositions contemplated herein are capable of reducing inactivation of a hydrated live attenuated virus during at least 2 freeze, at least 3, at least 4, at least 5, at least 6 and more thaw cycles. Other methods concern combination compositions capable of reducing inactivation of a hydrated live attenuated virus for about 24 hours to about 50 days at refrigeration temperatures (e.g. about 0° to about 10° C.). Compositions contemplated in these methods, can include, but are not limited to, a buffer, HEPES buffer, one or more carbohydrates such as sucrose or trehalose and one or more protein agents including gelatin. In certain embodiments, the live, attenuated virus composition remains at about 100% viral titer after greater than 20 hours at approximately 37° C. and about 100% viral titer after 50 days at refrigeration temperatures around 4° C. Other embodiments herein may include live, attenuated alphavirus composition remaining at about 90%, or about 80% viral titer after 7 days at approximately 21° C. and about 90%, or about 80% viral titer after 50 days at refrigeration temperatures around 4° C. Other embodiments contemplated include live, attenuated virus compositions remaining at about 3× to about 10× the concentration of viral titer after several hours (e.g. 20 hours) at approximately 37° C. compared to other compositions known in the art. (See for example, FIGS. 3 and 4). Compositions disclosed herein reduce degradation of the live, attenuated alphavirus when the composition is stored at approximately 37° C.
Other embodiments concern kits for decreasing the inactivation of a live, attenuated virus composition including, but not limited to, a container; and a composition including, but not limited to, buffered HEPES about pH 6.0 to pH 10.0 at about 1 to 30 mM HEPES, one or more carbohydrate agents (e.g. sucrose and/or trehalose) at about 1 to 25% w/v, and one or more protein agents that includes gelatin at about 0.01 to 5.0% w/v, wherein the composition decreases inactivation and/or degradation of a live, attenuated Alphavirus. In accordance with these embodiments, a kit may further include one or more live, attenuated alphaviruses. In other embodiments, a kit may further include a salt or salt solution (e.g. sodium chloride).
In other embodiments, compositions contemplated herein may contain trace amounts or no divalent cations. For example, compositions contemplated herein may have trace amounts or no calcium/magnesium (Ca+2/Mg+2).
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the instant specification and are included to further demonstrate certain aspects of particular embodiments herein. The embodiments may be better understood by reference to one or more of these drawings in combination with the detailed description presented herein.
FIG. 1 represents an exemplary histogram of experiments using various compositions for testing the stability of exemplary attenuated Alphavirus compositions at 37° C.
FIG. 2 represents an exemplary histogram of experiments using compositions having different carbohydrate agents for testing the stability of exemplary attenuated Alphavirus compositions at 4° C.
FIG. 3 represents an exemplary histogram of experiments using various compositions for testing the stability of exemplary attenuated Alphavirus compositions at 37° C.
FIG. 4 represents an exemplary histogram of experiments using various compositions for testing the stability of exemplary attenuated Alphavirus compositions at 37° C.
FIG. 5 represents an exemplary graph plotting data from experiments using various liquid compositions for testing the stability of exemplary attenuated Alphavirus compositions at 4° C.
FIG. 6 represents an exemplary graph plotting data from experiments using various liquid compositions for testing the stability of exemplary attenuated Alphavirus compositions at −80° C.
FIG. 7 represents an exemplary graph plotting data from experiments using various lyophilized compositions for testing the stability of exemplary attenuated Alphavirus compositions at 4° C.
FIG. 8 represents an exemplary graph plotting data from experiments using various lyophilized compositions represents an exemplary histogram of exemplary attenuated Alphavirus compositions at −80° C.
FIG. 9 represents an exemplary histogram of experiments using various compositions having different gelatin formulations for testing the stability of exemplary attenuated Alphavirus compositions.
FIG. 10 represents an exemplary histogram of experiments using various compositions having different gelatin formulations for testing the stability of exemplary attenuated Alphavirus compositions after freeze-thaw treatment.
FIG. 11 represents an exemplary histogram of experiments using various compositions having different gelatin formulations for testing the stability of exemplary attenuated Alphavirus compositions after lyophilization.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Definitions
As used herein, “a” or “an” may mean one or more than one of an item.
As used herein, “about” may mean up to and including plus or minus five percent, for example, about 100 may mean 95 and up to 105.
As used herein, “carbohydrate” agents can mean one or more monosaccharides, (e.g. glucose, galactose, ribose, mannose, rhamnose, talose, xylose, or allose arabinose.), one or more disaccharides (e.g. trehalose, sucrose, maltose, isomaltose, cellibiose, galactose gentiobiose, laminaribose, xylobiose, mannobiose, lactose, or fructose.), trisaccharides (e.g. acarbose, raffinose, melizitose, panose, or cellotriose) or sugar polymers (e.g. dextran, xanthan, pullulan, cyclodextrins, amylose, amylopectin, starch, celloologosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan, or chitin).
As used herein CHIKV can mean Chikungunya Virus.
As used herein TCID50 can mean 50% Tissue Culture Infective Dose.
As used herein HB can mean HEPES Buffer Saline.
As used herein HBS can mean HEPES Buffer Saline+Sucrose.
As used herein HSG can mean HEPES Buffer Saline+Sucrose+Gelatin.
As used herein IRES can mean Internal Ribosomal Entry Site.
As used herein DMEM can mean Dulbecco's modified minimal essential medium.
As used herein MCT can mean Microcentrifuge Tubes.
As used herein PBS can mean Phosphate Buffered Saline.
As used herein FBS can mean Fetal Bovine Serum.
As used herein Pre-MVS can mean Pre-Master Virus Seed.
As used herein Lyo can mean lyophilized or dehydrated depending on the formulation of reference.
As uses herein gelatin can be a translucent, colorless, brittle (when dry), flavorless solid substance, derived from collagen obtained from various animal by-products or other. It is commonly used as a gelling agent and is commercially available. Any commercially available, isolated or synthetic gelatin agent is contemplated herein.
As used herein, “attenuated virus” can mean a virus that demonstrates reduced or no clinical signs of disease when administered to an animal.
DETAILED DESCRIPTIONS
In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details may be modified through routine experimentation. In some cases, well known methods or components have not been included in the description.
Stability of alphavirus vaccines has been assessed in certain embodiments disclosed herein. In certain embodiments, a formulation which confers significant protective effect from loss of titer of liquid, frozen, lyophilized and re-hydrated live, attenuated alphavirus formulations has been demonstrated. In certain embodiments, compositions disclosed herein concern a combination of two or more or all three components of HEPES buffer, one or more protein agents that include gelatin and one or more carbohydrate agents. In certain embodiments, a composition disclosed herein can include an alphavirus in a HEPES buffer, a carbohydrate that includes at least one of sucrose or trehalose and a gelatin derived from any source (e.g. pharmaceutical grade or a grade capable of being introduced to a subject). Certain compositions disclosed herein include salt or a salt solution. These formulations can be used for liquid, frozen or lyophilized storage of a live, attenuated alphavirus at about −80° C. to about 37° C. or above storage without significant loss of the CHIK vaccine. For example, long-term storage at 4° C. is also a possibility for this formulation.
Embodiments herein concern methods and compositions to reduce or prevent deterioration or inactivation of live attenuated Alphavirus compositions. Certain compositions disclosed can include combinations of components that reduce deterioration of a live attenuated virus. Other embodiments herein concern combinations of excipients that greatly enhance the stability of live attenuated viruses. Yet other compositions and methods herein are directed to reducing the need for lower temperatures (e.g. refrigerated or frozen storage) while increasing the shelf life of aqueous and/or reconstituted live attenuated alphavirus.
In accordance with these embodiments, certain live attenuated viruses are directed to alphaviruses. Some embodiments, directed to compositions, can include, but are not limited to, one or more live, attenuated alphaviruses, such as one or more live, attenuated alphavirus in combination with HEPES buffer, one or more carbohydrates and/or one or more protein agent that includes gelatin. In certain embodiments, alphavirus formulations disclosed herein include at least all three components. In other embodiments, a salt can be added in order to increase buffering capacity of the formulation.
Compositions contemplated herein can increase the stabilization and/or reduce the inactivation and/or degradation of a live attenuated alphavirus including, but not limited to, a live attenuated alphaviruses that include but are not limited to, chikungunya virus, o'nyong'nyong virus, Ross River virus, eastern equine encephalitis, Venezuelan Equine Encephalitis Virus and western equine encephalitis or other alphaviruses in the Coronaviridae and Togaviridae families.
Other embodiments concern live, attenuated virus compositions and methods directed to a vaccine compositions capable of reducing or preventing onset of a medical condition caused by one or more of the alphaviruses contemplated herein. In certain embodiments, a live, attenuated alphavirus is one that is incapable of replicating in mosquitoes. In other embodiments, a live, attenuated alphavirus contemplated herein is manipulated to be under eukaryotic control (e.g. insertion of an IRES sequence)
In certain embodiments, compositions contemplated herein can be partially or wholly dehydrated or hydrated. In other embodiments, carbohydrate agents contemplated of use in compositions herein can include, but are not limited to, sucrose, fructose, galactose and trehalose.
In certain embodiments, HEPES buffer is from about 1 mM to about 40 mM; a carbohydrate concentration is about 1 to about 25% w/v; and gelatin is about 0.01% to about 5%. In other embodiments, HEPES buffer is from about 1 mM to about 20 mM; a carbohydrate concentration is about 5 to about 20% w/v; and gelatin is about 0.1% to about 2%. In yet other embodiments, HEPES buffer is from about 5 mM to about 15 mM; a carbohydrate concentration is about 5 to about 25% w/v; and gelatin is about 0.5% to about 1.5%. In certain embodiments, formulations can further include 10-150 mM salt (e.g. sodium chloride or other appropriate salt known in the art). Other buffering agents can be used in certain compositions herein in combination with the required three components above.
Some embodiments herein concern partially or wholly dehydrated live, attenuated alphavirus compositions. In accordance with these embodiments, a composition may be 20% or more; 30% or more; 40% or more; 50% or more; 60% or more; 70% or more; 80% or more; or 90% or more dehydrated. In yet other embodiments, a composition disclosed herein can be a fully lyophilized composition.
Other embodiments concern methods for decreasing inactivation of a live attenuated alphaviruses including, but not limited to, combining one or more live attenuated alphaviruses with a composition capable of reducing inactivation of a live, attenuated alphavirus including, but not limited to, one or more protein agents; one or more carbohydrate, saccharides or polyols agents; and a HEPES buffer, wherein the composition decreases inactivation of the live, attenuated alphavirus. In accordance with these embodiments, the live attenuated virus may include, particular alphaviruses, such as those related to CHIK (e.g. Semliki Forest complex viruses).
Additionally, methods and compositions disclosed herein can include freeze drying or other dehydrating methods for the combination. In accordance with these methods and compositions, the methods and compositions decrease inactivation of the freeze dried or partially or wholly dehydrated live attenuated virus. In other methods, compositions for decreasing inactivation of a live attenuated virus may include an aqueous composition or may comprise a rehydrated composition after dehydration. Compositions described herein are capable of increasing the shelf life of an aqueous or rehydrated live attenuated alphavirus.
In certain embodiments, compositions contemplated herein are capable of decreasing inactivation and/or degradation of a hydrated live attenuated alphavirus for greater than 12 to 24 hours at room temperatures (e.g. about 20° C. to about 25° C. or even as high as 37° C.) or refrigeration temperatures (e.g. about 0° to about 10° C.). In some embodiments, a combination composition is capable of maintaining about 100 percent of the live attenuated Alphavirus for greater than 24 hours. In addition, combination compositions contemplated herein are capable of reducing inactivation of a hydrated live attenuated virus during at least 2 freeze and thaw cycles (or 3 or 4 or 5 etc.). Other methods concern combination compositions capable of reducing inactivation of a hydrated live attenuated virus for about 24 hours to more than 50 days at refrigeration temperatures (e.g. about 0° to about 10° C.). Compositions contemplated in these methods, can include, but are not limited to, a buffer, HEPES buffer, one or more carbohydrates such as sucrose or trehalose and one or more protein agents including gelatin. In certain embodiments, the live, attenuated virus composition remains at about 100% viral titer after greater than 20 hours at approximately 37° C. and about 100% viral titer after more than 50 days at refrigeration temperatures around 4° C. Other embodiments herein may include live, attenuated alphavirus composition remaining at about 90%, or about 80% viral titer after 7 days at approximately 21° C. and about 90%, or about 80% viral titer after 50 days at refrigeration temperatures around 4° C. Other embodiments contemplated include live, attenuated virus compositions remaining at about 3× to about 10× the concentration of viral titer after several hours (e.g. 20 hours) at approximately 37° C. compared to other compositions known in the art. (See the Example Section). Compositions disclosed herein reduce degradation of the live, attenuated alphavirus when the composition is stored at approximately 37° C. as well as other temperatures.
Other embodiments concern kits for decreasing the inactivation of a live, attenuated virus composition including, but not limited to, a container; and a composition including, but not limited to, buffered HEPES about pH 6.0 to pH 10.0, one or more carbohydrate agents (e.g. sucrose and/or trehalose), and one or more protein agents that includes gelatin, wherein the composition decreases inactivation and/or degradation of a live, attenuated Alphavirus. In accordance with these embodiments, a kit may further include one or more live, attenuated alphaviruses. buffered HEPES about pH 6.0 to pH 10.0 at about 1 to 40 mM HEPES, one or more carbohydrate agents at about 1 to 25% w/v, and one or more protein agents that includes gelatin at about 0.01 to 5.0% w/v, wherein the composition decreases inactivation and/or degradation of a live, attenuated alphavirus.
In other embodiments, compositions contemplated herein may contain trace amounts or no divalent cations. For example, compositions contemplated herein may have trace amounts or no calcium/magnesium (Ca+2/Mg+2).
No formulation for a live, attenuated Alphavirus vaccine has been identified that provides long term stability of lyophilized formulations at temperatures greater than 2-8° C. In addition, no formulation has been described that prevents loss of titer, stabilizes or reduces degradation of aqueous vaccines for greater than a few hours.
Formulations for other live, attenuated viruses have also been described (see for example Burke, Hsu et al. 1999). One common stabilizer, referred to as SPGA is a mixture of 2 to 10% sucrose, phosphate, potassium glutamate and 0.5 to 2% serum albumin (see for example Bovarnick, Miller et al. 1950). Various modifications of this basic formulation have been identified with different cations, with substitutions of starch hydrolysate or dextran for sucrose, and with substitutions of casein hydrolysate or poly-vinyl pyrrolidone for serum albumin. Other formulations use hydrolyzed gelatin instead of serum albumin as a protein source (Burke, Hsu et al 1999). However, gelatin can cause allergic reactions in immunized children and could be a cause of vaccine-related adverse events. U.S. Pat. No. 6,210,683 describes the substitution of recombinant human serum albumin for albumin purified from human serum in vaccine formulations.
Embodiments herein disclose compositions that enhance the stability of and/or reduce deterioration of live, attenuated virus vaccines compared to those in the prior art. Certain compositions disclosed herein provide stability of aqueous viruses for up to 2 hours; up to 3 hours; up to 4 hours and greater than 21 hours at or about 37° C. Certain compositions disclosed herein provide stability of aqueous viruses for up to 1 day to about 1 week or more, at or about room temperature (e.g. 25° C.). Embodiments contemplated herein provide increased protection of a live, attenuated virus from for example, freezing and/or thawing, and/or elevated temperatures. In certain embodiments, compositions herein can stabilize, reduce deterioration and/or prevent inactivation of dehydrated live, attenuated viral products in room temperature conditions (e.g. about 25° C.). In other embodiments, compositions contemplated herein can stabilize, reduce deterioration and/or prevent inactivation of aqueous live, attenuated viral products at about 25° C. or up to or about 37° C. Compositions and methods disclosed herein can facilitate the storage, distribution, delivery and administration of viral vaccines in developed and under developed regions.
Those skilled in the art will recognize that compositions or formulas herein relate to viruses that are attenuated by any means, including but not limited to, cell culture passage, reassortment, incorporation of mutations in infectious clones, reverse genetics, other recombinant DNA or RNA manipulation. In addition, those skilled in the art will recognize that other embodiments relate to viruses that are engineered to express any other proteins or RNA including, but not limited to, recombinant alphaviruses. Such viruses may be used as vaccines for infectious diseases, vaccines to treat oncological conditions, or viruses to introduce express proteins or RNA (e.g., gene therapy, antisense therapy, ribozyme therapy or small inhibitory RNA therapy) to treat disorders.
In some embodiments, compositions herein can contain one or more viruses with membrane envelopes (e.g., enveloped viruses) of the Togavirus, or Coronavirus, or any Alphavirus of the Togavirus family. In other embodiments, compositions herein can contain one or more enveloped, positive strand RNA virus of the Togavirus, or Coronavirus families. In certain embodiments, compositions can contain one or more live, attenuated alphavirus (e.g. Chikungunya) having one or more insertion, deletion or mutation to induce attenuation of the virus for use in a vaccine composition.
In certain embodiments, live attenuated alphavirus compositions can include one or more live attenuated Alphavirus constructs described in U. S. App No. PCT/US2009/000458, Filed Jan. 23, 2009 entitled: ATTENUATED RECOMBINANT ALPHAVIRUSES INCAPABLE OF REPLICATING IN MOSQUITOES AND USES THEREOF and U.S. patent application Ser. No. 12/804,535 filed Jul. 23, 2010, both applications and continuations and divisionals thereof are incorporated by reference for all purposes in their entirety.
Some embodiments herein relate to compositions for live, attenuated viruses in aqueous or lyophilized form. Those skilled in the art will recognize that formulations that improve thermal viral stability and prevent freeze-thaw inactivation will improve products that are liquid, powdered, freeze-dried or lyophilized and prepared by methods known in the art. After reconstitution, such stabilized vaccines can be administered by a variety routes, including, but not limited to intradermal administration, subcutaneous administration, intramuscular administration, intranasal administration, pulmonary administration or oral administration. A variety of devices are known in the art for delivery of the vaccine including, but not limited to, syringe and needle injection, bifurcated needle administration, administration by intradermal patches or pumps, intradermal needle-free jet delivery (intradermal etc), intradermal particle delivery, or aerosol powder delivery.
Embodiments can include compositions consisting of one or more live attenuated viruses (as described above) and a mixture of HEPES buffer or similar buffer; one or more carbohydrates and one or more proteins that include(s) gelatin. In certain embodiments, compositions include, but are not limited to one or more live attenuated alphaviruses, HEPES buffer or similar buffer; one or more of sucrose or trehalose and one or more proteins that include gelatin.
In some embodiments, the carbohydrate is a sugar or a polyol. Sugars can include, but are not limited to, monosaccharides, (e.g. glucose, galactose, ribose, mannose, rhamnose, talose, xylose or allose arabinose), disaccharides (e.g. trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, or fructose.), trisaccharides (e.g. acarbose, raffinose, melizitose, panose, or cellotriose) or sugar polymers (e.g. dextran, xanthan, pullulan, cyclodextrins, amylose, amylopectin, starch, celloologosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan, or chitin). Polyols can include, but are not limited to, mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, and glycerol.
Anhydrobiotic organisms that can tolerate low water conditions contain large amounts of trehalose. Trehalose has been shown to prevent both membrane fusion events and phase transitions that can cause membrane destabilization during drying. Structural analysis suggests that trehalose fits well between the polar head groups in lipid by layers. Trehalose also prevents denaturation of labile proteins during drying. It is thought that trehalose stabilizes proteins by hydrogen bonding with polar protein residues. Trehalose is a disaccharide consisting of two glucose molecules in a 1:1 linkage. Due to the 1:1 linkage, trehalose has little or no reducing power and is thus essentially non-reactive with amino acids and proteins. This lack of reducing activity may improve the stabilizing affect of trehalose on proteins. In certain embodiments, trehalose provides stability to live, attenuated viruses. This activity of trehalose may be due to its ability to stabilize both the membranes and coat proteins of the viruses.
In certain embodiments, compositions can be described that typically include a physiologically acceptable buffer. Those skilled in the art recognize that HEPES was found to have unexpected stabilizing effect on the alphavirus compositions disclosed herein. In addition, those skilled in the art recognize that adjusting salt concentrations to near physiological levels (e.g., saline or 0.15 M total salt) may be optimal for parenteral administration of compositions to prevent cellular damage and/or pain at the site of injection. Those skilled in the art also will recognize that as carbohydrate concentrations increase, salt concentrations can be decreased to maintain equivalent osmolarity to the formulation. In certain embodiments, a buffering media with pH greater than 6.8 to about pH 10.0 is contemplated; some live, attenuated viruses (e.g. alphaviruses) are unstable at low pH.
Some live, attenuated viral vaccine compositions herein concern compositions that increase stability and/or reduce deterioration of live, attenuated virus in addition to having reduced immunogenicity or are non-immunogenic.
Pharmaceutical Compositions
Embodiments herein provide for administration of compositions to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By “biologically compatible form suitable for administration in vivo” is meant a form of the active agent (e.g. live, attenuated virus composition of the embodiments) to be administered in which any toxic effects are outweighed by the therapeutic effects of the active agent. Administration of a therapeutically active amount of the therapeutic compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve a desired result. For example, a therapeutically active amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability formulations to elicit a desired response in the individual. Dosage regima may be adjusted to provide the optimum therapeutic response.
In some embodiments, composition (e.g. pharmaceutical chemical, protein, peptide of an embodiment) may be administered in a convenient manner such as subcutaneous, intravenous, by oral administration, inhalation, transdermal application, intravaginal application, topical application, intranasal or rectal administration. In a more particular embodiment, the compound may be orally or subcutaneously administered. In another embodiment, the compound may be administered intravenously. In one embodiment, the compound may be administered intranasally, such as inhalation.
A compound may be administered to a subject in an appropriate carrier or diluent, co-administered with the composition. The term “pharmaceutically acceptable carrier” as used herein is intended to include diluents such as saline and aqueous buffer solutions. The active agent may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use may be administered by means known in the art. For example, sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion may be used. In all cases, the composition can be sterile and can be fluid to the extent that easy syringability exists. It may further be preserved against the contaminating action of microorganisms such as bacteria and fungi. The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
Sterile injectable solutions can be prepared by incorporating active compound in an amount with an appropriate solvent or with one or a combination of ingredients enumerated above, as required, followed by sterilization.
Upon formulation, solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. It is contemplated that slow release capsules, timed-release microparticles, and the like can also be employed for administering compositions herein. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In some embodiments, formulations disclosed herein can be administered before, during and/or after exposure to an alphavirus of the instant invention.
In another embodiment, nasal solutions or sprays, aerosols or inhalants may be used to deliver the compound of interest. Additional formulations that are suitable for other modes of administration include suppositories and pessaries. A rectal pessary or suppository may also be used.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. In certain embodiments, oral pharmaceutical compositions can include an inert diluent or assimilable edible carrier, or may be enclosed in hard or soft shell gelatin capsule, or may be compressed into tablets, or may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage can be obtained.
Kits
Further embodiments concerns kits for use with methods and compositions described herein. Compositions and live virus formulations may be provided in the kit. The kits can also include a suitable container, live, attenuated virus compositions detailed herein and optionally one or more additional agents such as other anti-viral agents, anti-fungal or anti-bacterial agents.
The kits may further include a suitably aliquoted composition of use in a subject in need thereof. In addition, compositions herein may be partially or wholly dehydrated or aqueous. Kits contemplated herein may be stored at room temperatures or at refrigerated temperatures as disclosed herein depending on the particular formulation.
The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a composition may be placed, and preferably, suitably aliquoted. Where an additional component is provided, the kit will also generally contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the agent, composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
EXAMPLES
The following examples are included to demonstrate certain embodiments presented herein. It should be appreciated by those of skill in the art that the techniques disclosed in the Examples which follow represent techniques discovered to function well in the practices disclosed herein, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope herein.
Example 1
Buffer Screen
In certain exemplary method, liquid composition and lyophilizable compositions suitable for preclinical and clinical testing and use of alphavirus vaccines are identified. One consideration regarding a liquid composition in accordance with these exemplary compositions is that some alphaviruses are pH sensitive (e.g. to low pH). Therefore, components of a compositions disclosed herein include careful considerations regarding pH. In certain exemplary compositions, the pH of the formulations was about pH 6 to about 10 with many formulations around pH 6.5 to 7.5 and up to around 9.5.
In some methods, attenuated Chikungunya Viruses (hereinafter CHIK) are used as an example of an alphavirus composition for pre-clinical and clinical testing. Compositions for these methods are provided. In one exemplary experiment, a predetermined amount of CHIK-IRES vaccine (pMVS) where this attenuated virus is under control of an IRES insertion. Any attenuated alphavirus can be used in these exemplary compositions to increase stability of the composition and reduce degradation. Initially, many different base buffers were tested such as DMEM, PBS, HEPES and others.
Certain tests were performed, such as incubation for up to 21 hours at 37° C. to test stability of the attenuated virus formulation. Samples were taken to titrate for the presence of infectious virus by TCID50 in 96 well plates on Vero cells. A percentage of the remaining virus as compared to an input (un-incubated) vaccine control was calculated. Incubation of 105 TCID50 of the CHIK virus vaccine in compositions containing PBS alone, 20% DMEM or DMEM buffered Dextrose demonstrated a rapid loss in potency. Certain exemplary compositions were found to be effective at stabilizing attenuated alphaviruses such as CHIK virus vaccine, for example, a composition containing various concentrations of HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (data not shown) such as about 1 to about 30 mM HEPES. In one example, a composition containing 150 mM NaCl and 15 mM HEPES (HEPES Buffer Saline—HS) was found to provide increased stability to the attenuated alphavirus vaccine compared to a control (FIG. 1).
FIGS. 1 and 2 represent exemplary histograms illustrating potency (indicated as percentage of total virus remaining after the time period indicated) of the attenuated alphavirus, CHIKV, vaccine remaining after incubation in various compositions for ˜21 hours at 37° C. Compositions containing different concentrations of HEPES increased stability of the CHIK vaccine significantly compared to other buffer compositions (20%-55% vs. less than 10%, data not shown). In FIG. 1, compositions containing 15 mM HEPES having 150 mM NaCl or 15 mM HEPES demonstrated significant affects on vaccine stability and potency compared to others.
Example 2
In some other exemplary methods, a long term stability experiment at 4° C. was performed to analyze effects of various carbohydrates (e.g. sugars) on alphavirus vaccine stability, for example the CHIK virus vaccine, based on observations that including one or more carbohydrates had a positive effect on CHIK vaccine stability. Compositions containing HEPES and a carbohydrate, such as sucrose, lactose, trehalose, galactose, fructose, D-sorbitol, Dextrose and D-Mannitol, were generated. Individual aliquots of a predetermined concentration of CHIK-IRES vaccine (pMVS) were formulated in these compositions, and incubated for over 12 weeks at 4° C. Samples were collected at time points indicated in FIG. 2 and titrated on Vero cells. As illustrated in FIG. 2, compositions of about 15% Trehalose; 15% Sucrose or 10% D-Manitol in combination with HEPES Buffered Saline (HB) demonstrated about an equal improvement in virus stability, better than other compositions.
In certain exemplary methods, formulations that included either sucrose or trehalose were examined for properties regarding increased stability of alphavirus vaccine and other formulations. In certain methods, 105 TCID50 of a CHIK virus vaccine was incubated in various compositions of HEPES buffer (HB) with increasing concentrations of sucrose or trehalose in the presence or absence of a protein at room temperature, 37° C., and analyzed for stability for up to 21 hours. As illustrated in the histogram plot in FIG. 3, compositions containing HEPES buffer with 5% sucrose (referred to as HBS) or HEPES buffer with 15% trehalose more stability than compositions of HEPES buffer and human serum albumin or at other carbohydrate concentrations.
FIG. 2 is an exemplary graph demonstrating stability of a liquid alphavirus composition, CHIKV vaccine composition containing HEPES buffered saline and various carbohydrates over 12 weeks at 4° C. FIG. 2 represents an exemplary histogram plot illustrating percentage of total virus remaining after an incubation in compositions containing HEPES buffer150 mM NaCl and various concentrations of carbohydrates, e.g. sucrose or trehalose, in the presence or absence of 0.1% HSA for weeks at 4° C.
Example 3
Screening for Protein Induced Stability Formulations
In other exemplary methods, different protein agents were analyzed for increased stability of alphavirus formulations compared to controls, without protein or with against other proteins. Compositions containing HB or HBS with target protein agents were analyzed. After incubation (37° C. for ˜21 hrs) of about 105 per experimental condition of the attenuated CHIK vaccine composition, aliquots were removed and titrated for growth in Vero cells by TCID50. Then, the percentage of remaining virus titer was assessed. As illustrated in FIG. 4, the addition of gelatin to the formulations with or without carbohydrate increased alphavirus vaccine stability at 37° C. (see FIG. 4).
FIG. 4 represents an exemplary histogram illustrating the percent total of CHIK virus titer remaining after incubation in compositions containing HEPES Buffered Saline and a protein, such as Lactoferrin, Tripton, Lactalbumin, and Gelatin, for ˜21 hours at 37° C. Among all tested composions, the composition containing Gelatin and HB buffer demonstrated increased stability by reducing degradation of the alphavirus at room temperature on vaccine stability. The effect was observed to be more than additive when a carbohydrate such as sucrose was included in the composition.
Significant increase in stability of the alphavirus vaccine as compared to the vaccine stored in culture medium containing FBS or PBS alone was observed. One exemplary formulation which produced a very stabile virus vaccine was determined to be a HEPES buffer, sucrose and gelatin formation. Including recombinant gelatin in the formulation, greatly decreased the lability of this alphavirus vaccine.
Example 4
Long Term Stability Study
In some exemplary methods, a concentration range of gelatin was analyzed to determine which concentration of gelatin had the best stabilizing property for an alphavirus composition. In one method, two concentrations of gelatin were selected for combinatory use with HBS (HBS+0.5% and HBS+1% Gelatin) in certain compositions. Then, a long term stability study evaluating the liquid CHIK vaccine at 4° C. or −80° C. was conducted (Table 1) with compositions containing Gelatin and HBS. Examples of these compositions are provided below.
Exemplary Compositions:
1. HB—HEPES Buffer Saline 15 mM HEPES and 150 mM NaCl
2. HBS—HEPES Buffer Saline with 5% Sucrose
3. HSG (0.5% Gelatin)—HEPES Buffer Saline with 5% Sucrose and 0.5% Gelatin
4. HSG (1% Gelatin)—HEPES Buffer Saline with 5% Sucrose and 1% Gelatin
TABLE 1
Long Term Stability Study Designs
weeks
0
1
3
4
8
12
24
36
4° C.
x
x
x
x
x
x
x
x
−80° C.
x
x
x
x
x
x
x
x
Vaccine samples formulated in these compositions were stored in 5004 volume into 1.5 mL MCT. 15 samples were stored in 4° C. (Micro Climate Chamber; Model# MCB-12-33-33-H/AC) and 15 samples were stored in −80° C. (Thermo; Model# ULT2186-6-D43) per formulation.
Samples were taken for potency evaluation at the time points indicated in Table 1 and FIGS. 5-6. Samples incubated at 4° C. (FIG. 5) were analyzed in parallel with samples incubated at −80° C. (FIG. 6) to demonstrate the trend of the titer over 36 weeks. As illustrated in the graphs in FIG. 5, vaccines formulated in these compostions had significantly reduced titer loss up to week 12 at 4° C. After incubation for 24 weeks, loss of 1 log10 TCID50 or more of the virus titer was observed. The addition of gelatin demonstrated significant positive effects on stabilization of alphavirus vaccine formulation (attenuated CHIK). The alphavirus composition was stable at −80° C. in all compositions tested for the duration of the study (FIG. 6).
Example 4
Lyophilized Formulations
In another exemplary method, long term stability of a lyophilized attenuated alphavirus formulation (e.g. CHIK vaccine formulation) were evaluated. The lyophilized vaccine formulations were stored at 4° C. (FIG. 7) or −80° C. (FIG. 8). Samples taken at the indicated time points were reconstituted and titrated in Vero cells by TCID50. The exemplary attenuated CHIK vaccine formulated in HSG (both 0.5% and 1% Gelatin) demonstrated minimal loss of virus titer for greater than 80 weeks at 4° C. while HB or HBS composition lost about 1 log10 TCID50 of the virus titer after 24 weeks (FIG. 7). The CHIK vaccine was very stable at −80° C. in all compositions tested for the duration of 80 weeks and more (FIG. 8).
In one other exemplary method, Gelatin from different sources was compared for the ability to stabilize the CHIK vaccine. No differences were observed (FIG. 9) between any manufacturers including Sigma, Merck, Tekni and Gelita.
FIGS. 5-6 represent exemplary graphs demonstrating increased stability of the liquid an attenuated alphavirus vaccine formulation (e.g. CHIKV) stored in compositions containing HB, HBS or HSG for up to 80-90 weeks at 4° C. (FIG. 5) or at −80° C. (FIG. 6). FIGS. 7-8 represent exemplary graphs demonstrating potency of the CHIKV vaccine lyophilized in compositions containing HB, HBS or HSG for 80-90 weeks at 4° C. (FIG. 7) or at −80° C. (FIG. 8).
FIG. 9 provides an exemplary histogram comparing effect of Gelatin from different sources on stabilizing the CHIK vaccine.
FIG. 10 represents an exemplary histogram plot comparing effects of gelatin from different sources on CHIK vaccine stability after freeze-thaw (F-T) treatment. CHIK vaccine compositions include HEPES (HS) buffer with 0.5% gelatin. Gelatins from five different sources were tested including Sigma, Merck, Tekni, Gelita, and Nitta. CHIK vaccine compositions were exposed to one (1×), three (3×), or five (5×) rounds of F-T treatment. No significant differences were observed among the different sources of gelatin. Therefore, this data supports that any source of gelatin (e.g. capable of being introduced to a subject, such as a pharmaceutical grade) can be used in the instant formulations to increase stability of the live, attenuated virus in the compositions disclosed herein.
FIG. 11 illustrates an exemplary histogram plot comparing effect of gelatin from different sources on CHIK vaccine stability after lyophilization, as compared to liquid cultures. CHIK vaccine compositions include HEPES (HS) buffer with either 0.5% or 1.0% gelatin. Gelatin from Merck and Nitta (beMatrix) were tested. No significant differences were observed between gelatin from Merck and Nitta. Both CHIK vaccine compositions produced a stable lyophilized cake, which retained a significant titer after reconstitution from lyophilization compared to liquid formulations.
TABLE 2
List of Abbreviation
CHIKV
Chikungunya Virus
TCID50
50% Tissue Culture Infective Dose
HB
Hepes Buffer Saline
HBS
Hepes Buffer Saline + Sucrose
HSG
Hepes Buffer Saline + Sucrose + Gelatin
IRES
Internal Ribosomal Entry Site
DMEM
Dulbecco’s modified minimal essential medium
MCT
Microcentrifuge Tubes
PBS
Phosphate Buffered Saline
FBS
Fetal Bovine Serum
Pre-MVS
Pre-Master Virus Seed
Materials and Methods
Individual aliquots of a predetermined dose of CHIK-IRES vaccine (pre-MVS) were formulated in compositions containing buffers including Hepes buffered saline (HB), Hepes Buffered Saline containing sucrose (HBS), Hepes Buffered saline containing sucrose and gelatin (HSG) at varying concentrations of gelatin (e.g. 0.5% and 1% Gelatin). Formulated hydrated or liquid vaccine was incubated at certain temperatures such as room temperature 37° C., frozen 4° C. or flash frozen, −80° C. Samples were taken from these formulations at predetermined intervals, and titrated for the presence of infectious virus by TCID50 in for example, 96 well plates with Vero cells.
Cell Lines and Tissue Culture
A research-grade Vero cell bank derived from the applicant's cGMP Working Cell Bank was prepared to perform these experiments. Vero cells were obtained: Vero (WHO) Working Cell Bank passage: 142 (lot#INV-VERO-WCB-001; 5×106), and was stored in liquid nitrogen. A vial was rapidly thawed in a water bath and directly inoculated into pre-warmed cDMEM (Dulbecco's modified minimal essential medium), about 19 mls containing penicillin-streptomycin, 40 mM L-glutamine and 10% FBS) in a T-75 cm2 flask and incubated at 37° C., 5% CO2. Cells were allowed to grow to confluency, and subcultured using PBS, Trypsin (HyClone, for example, cat#SH30042.01) and cDMEM-10. This flask was expanded to two T-185 cm2 flasks and grown until the cells reached 100% confluency. Cells were harvested by trypsinization, centrifuged at 800×g for 10 minutes, and resuspended in DMEM containing 20% FBS and 10% DMSO at a concentration of 1×107 cells/mL. These cells (20 mL total) were aliquoted into cryovials (20×1 mL) and labeled: Vero WHO WCB p#142-2 (Waisman) (WWCB) 1 ml 1×107 cells/mL, 13Jan12 LV and stored in liquid nitrogen.
Vero WWCBI WHO cells were grown and maintained in Dulbecco's modified minimal essential medium (DMEM) containing penicillin-streptomycin and 10% FBS (HyClone) (DMEM-10%-FBS). Trypsin was used to maintain cells. Two days before viral adsorption, 96-well plates were plated with 1.4×105 cells/mL in 100 uL per well of DMEM-FBS-10%. Incubators were monitored daily to maintain indicated temperatures. Virus dilutions, adsorption and TCID50 assays were performed in cDMEM-FBS 2%.
CHIK Attenuated virus
Molecular generation of CHIK vaccine used in various methods described is designated CHIK-002 (previously described). CHIK vaccine was generated and propagated in Vero cells. A pre-Master Virus seed stock was used for these experiments at a concentration of 105 TCID50/mL. Briefly, the CHIK pre-MVS was generated after infection of monolayers of Vero cells. Vaccine-virus is secreted into the supernatant, and the virus is harvested from the medium after clarification/removal of the dead Vero cells. The CHIK-pre-MVS was stabilized in DMEM containing 10% FBS, and stored at −80° C.
Assay Method
TCID50 assay methods were used to quantify the amount of infectious virus present (potency or stability) in the vaccine preparations. TCID50 is defined as the level of dilution of a virus at which half of a series of replicates of infected wells in the 96-well plate shows signs of virus infectivity, as evidenced by for example, CPE (Cytopathic Effect). Vero cells (WWCBI WHO) were grown and maintained in Dulbecco's modified minimal essential medium (DMEM) containing penicillin-streptomycin, L-glutamine and 10% FBS (HyClone) (DMEM 10%-FBS). Aliquots of the formulated samples were rapidly thawed in a water bath and mixed. An initial dilution of pre-MVS into a working concentration was performed, and ten-fold dilution series of these samples were made in for example, cDMEM-2% FBS in 96-well plates. Diluted viruses were maintained at 4° C. prior to inoculation of the Vero cell monolayers. At the time of assay, the growth medium was aspirated from the 96-well plate, and 100 μL of each virus dilution was added to the wells. The plates were incubated for 3-5 days at 37° C. and 5% CO2. Titer was calculated using the Spearman-Karber method.
Vaccine Formulations
Stability experiments were prepared with vaccines including research-grade vaccine preparations, and the CHIK-IRES pMVS derived at Inviragen. For screening of excipients and stability studies using various compositions provided herein, vaccine formulations were prepared in a final volume of 500 μL containing 105 TCID50/mL virus per sample. Samples were prepared in bulk in indicated buffers/formulations and input samples were taken before the study was initiated as a measure of initial titer. Samples were aliquoted into MCT and stored for the indicated time and temperature. Each of the four formulations were prepared for 5004 final with 105 TCID50/mL virus per sample. 60 samples per formulation were prepared in bulk and input samples were taken before they were aliquotted into 1.5 mL MCT containing 500 uL.
Formulated Vaccine Storage
Vaccine formulations were stored at 4° C. (Micro Climate Chamber; Model# MCB-12-33-33-H/AC) and at −80° C. (REVCO Elite Plus; Model# ULT2186-6-D43). Both systems were monitored with Dickson Wizard2—900 MHZ Logger (Model#WT-220 for 4° C. and WT-240 for −80° C.).
All of the COMPOSITIONS and METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of preferred embodiments, it is apparent to those of skill in the art that variations maybe applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.
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16159221
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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tyo:4502
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Takeda Pharmaceutical
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Jan 9th, 2018 12:00AM
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Jul 10th, 2015 12:00AM
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https://www.uspto.gov?id=US09861691-20180109
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Norovirus vaccine formulations
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The present invention relates to antigenic and vaccine compositions comprising Norovirus antigens and adjuvants, in particular, mixtures of monovalent VLPs and mixtures of multivalent VLPs, and to a process for the production of both monovalent and multivalent VLPs, the VLPs comprising capsid proteins from one or more Norovirus genogroups.
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9861691
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1. A composition comprising two or more Norovirus antigens and a mucoadhesive, wherein at least one of the Norovirus antigens is from genogroup I and at least one of the Norovirus antigens is from genogroup II, and wherein the amount of each of the two or more Norovirus antigens is from about 1 μg to about 100 μg per dose.
2. The composition of claim 1, wherein the antigen comprises Norovirus virus-like particles (VLPs).
3. The composition of claim 2, wherein the Norovirus VLPs comprise a capsid protein.
4. The composition of claim 3, wherein the capsid protein is VP1 and/or VP2.
5. The composition of claim 2, wherein the VLPs are monovalent VLPs.
6. The composition of claim 2, wherein the VLPs are multivalent VLPs.
7. The composition of claim 1, wherein the genogroup I is GI.1 and the genogroup II is GII.4.
8. The composition of claim 1, wherein the mucoadhesive is selected from the group consisting of chitosan, chitosan salt, chitosan base, chitosan glutamate, polysaccharides, glycosaminoglycans, chondroitin sulfate, dermatan sulfate chondroitin, keratan sulfate, heparin, heparan sulfate, hyaluronan, pectin, alginate, glycogen, amylase, amylopectin, cellulose, chitin, stachyose, unulin, dextrin, dextran, cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, mucin, mucopolysaccharides, cellulose derivatives, hydroxypropyl methylcellulose, carboxymethylcellulose, lectins, fimbrial proteins, and deoxyribonucleic acid.
9. The composition of claim 8, wherein the mucoadhesive is a polysaccharide.
10. The composition of claim 1, further comprising an adjuvant.
11. The composition of claim 10, wherein the adjuvant is selected from the group consisting of toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, and liposomes.
12. The composition of claim 11, wherein the adjuvant is a toll-like receptor (TLR) agonist.
13. The composition of claim 12, wherein the adjuvant is MPL.
14. A method of generating antibodies to Norovirus antigens, comprising administering to a subject the composition of claim 1.
15. The method of claim 14, wherein the composition is administered to the subject by a route selected from the group consisting of mucosal, intranasal, sublingual, oral, rectal, vaginal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, and transdermal routes of administration.
16. The method of claim 15, wherein the route of administration is intramuscular.
17. The method of claim 15, wherein the route of administration is intranasal.
18. The method of claim 17, wherein the composition is administered using an inhaler.
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/837,885, filed Mar. 15, 2013, which is a continuation of U.S. patent application Ser. No. 13/330,854, filed Dec. 20, 2011, which is a continuation of U.S. patent application Ser. No. 12/816,495, filed Jul. 16, 2010, which issued as U.S. Pat. No. 8,431,116 on Apr. 30, 2013 and which is a continuation of U.S. patent application Ser. No. 12/093,921, filed May 15, 2008, which issued as U.S. Pat. No. 7,955,603 on Jun. 7, 2011 and which is a national stage application of International Application No. PCT/US2007/079929, filed Sep. 28, 2007, which claims priority to U.S. Patent Application Ser. No. 60/847,912, filed Sep. 29, 2006, and U.S. Patent Application Ser. No. 60/973,392, filed Sep. 18, 2007, all of which are herein incorporated by reference in their entireties.
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with Government support under DAMD17-01-C-0400 and W81XWH-05-C-0135 awarded by the U.S. Army. The Government has certain rights in this invention.
FIELD OF THE INVENTION
The invention is in the field of vaccines, particularly vaccines for Noroviruses. In addition, the invention relates to methods of preparing vaccine compositions and methods of inducing an immunogenic response.
BACKGROUND OF THE INVENTION
Noroviruses are non-cultivatable human Caliciviruses that have emerged as the single most important cause of epidemic outbreaks of nonbacterial gastroenteritis (Glass et al., 2000; Hardy et al., 1999). The clinical significance of Noroviruses was under-appreciated prior to the development of sensitive molecular diagnostic assays. The cloning of the prototype genogroup I Norwalk virus (NV) genome and the production of virus-like particles (VLPs) from a recombinant Baculovirus expression system led to the development of assays that revealed widespread Norovirus infections (Jiang et al. 1990; 1992).
Noroviruses are single-stranded, positive sense RNA viruses that contain a non-segmented RNA genome. The viral genome encodes three open reading frames, of which the latter two specify the production of the major capsid protein and a minor structural protein, respectively (Glass et al. 2000). When expressed at high levels in eukaryotic expression systems, the capsid protein of NV, and certain other Noroviruses, self-assembles into VLPs that structurally mimic native Norovirus virions. When viewed by transmission electron microscopy, the VLPs are morphologically indistinguishable from infectious virions isolated from human stool samples.
Immune responses to Noroviruses are complex, and the correlates of protection are just now being elucidated. Human volunteer studies performed with native virus demonstrated that mucosally-derived memory immune responses provided short-term protection from infection and suggested that vaccine-mediated protection is feasible (Lindesmith et al. 2003; Parrino et al. 1997; Wyatt et al., 1974).
Although Norovirus cannot be cultivated in vitro, due to the availability of VLPs and their ability to be produced in large quantities, considerable progress has been made in defining the antigenic and structural topography of the Norovirus capsid. VLPs preserve the authentic confirmation of the viral capsid protein while lacking the infectious genetic material. Consequently, VLPs mimic the functional interactions of the virus with cellular receptors, thereby eliciting an appropriate host immune response while lacking the ability to reproduce or cause infection. In conjunction with the NIH, Baylor College of Medicine studied the humoral, mucosal and cellular immune responses to NV VLPs in human volunteers in an academic, investigator-sponsored Phase I clinical trial. Orally administered VLPs were safe and immunogenic in healthy adults (Ball et al. 1999; Tacket et al. 2003). At other academic centers, preclinical experiments in animal models have demonstrated enhancement of immune responses to VLPs when administered intranasally with bacterial exotoxin adjuvants (Guerrero et al. 2001; Nicollier-Jamot et al. 2004; Periwal et al. 2003). Collectively, these data suggest that a vaccine consisting of properly formulated VLPs represents a viable strategy to immunize against Norovirus infection.
SUMMARY OF THE INVENTION
The present invention provides antigenic and vaccine formulations comprising a Norovirus antigen. In one embodiment, the formulations further comprise at least one adjuvant. The Norovirus antigen can be derived from genogroup I or genogroup II viral sequences or a consensus viral sequence. The Norovirus formulations comprise antigenic peptides, proteins or virus-like particles (VLPs). In one embodiment, the VLPs may be denatured. In another embodiment, the antigenic peptides and proteins are selected from the group consisting of capsid monomers, capsid multimers, protein aggregates, and mixtures thereof. In another embodiment, the Norovirus antigen is present in a concentration from about 0.01% to about 80% by weight. The dosage of Norovirus antigen is present in an amount from about 1 μg to about 100 mg per dose.
In another embodiment, the Norovirus VLPs are recombinant VLPs produced in an expression system using a Norovirus nucleic acid sequence, which encodes at least one capsid protein or fragment thereof. The capsid protein is selected from the group consisting of VP1 and VP2 or a combination thereof. The expression system can be a recombinant cellular expression system such as a yeast, bacterial, insect, mammalian expression system, or a baculovirus-infected cellular expression system.
In still another embodiment, the composition further comprises a delivery agent, which functions to enhance antigen uptake by providing a depot effect, increase antigen retention time at the site of delivery, or enhance the immune response through relaxation of cellular tight junctions at the delivery site. The delivery agent can be a bioadhesive, preferably a mucoadhesive selected from the group consisting of glycosaminoglycans (e.g., chondroitin sulfate, dermatan sulfate chondroitin, keratan sulfate, heparin, heparan sulfate, hyaluronan), carbohydrate polymers (e.g., pectin, alginate, glycogen, amylase, amylopectin, cellulose, chitin, stachyose, unulin, dextrin, dextran), cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (including mucin and other mucopolysaccharides) cellulose derivatives (e.g., hydroxypropyl methylcellulose, carboxymethylcellulose), proteins (e.g. lectins, fimbrial proteins), and deoxyribonucleic acid. Preferably, the mucoadhesive is a polysaccharide. More preferably, the mucoadhesive is chitosan, or a mixture containing chitosan, such as a chitosan salt or chitosan base.
In yet another embodiment, the present invention provides a composition further comprising an adjuvant. The adjuvant may be selected from the group consisting of toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL®), synthetic lipid A, lipid A mimetics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, endotoxins, for instance bacterial endotoxins and liposomes. Preferably, the adjuvant is a toll-like receptor (TLR) agonist. More preferably, the adjuvant is MPL®.
The compositions of the present invention may be provided as a liquid formulation or a dry powder formulation. Dry power formulations of the present invention may contain an average particle size from about 10 to about 500 micrometers in diameter. In one embodiment, the composition is an antigenic formulation. In another embodiment, the composition is formulated for administration as a vaccine. Suitable routes of administration include mucosal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal. In particular, the route of administration may be intramuscular or mucosal, with preferred routes of mucosal administration including intranasal, oral, or vaginal routes of administration. In another embodiment, the composition is formulated as a nasal spray, nasal drops, or dry powder, wherein the formulation is administered by rapid deposition within the nasal passage from a device containing the formulation held close to or inserted into the nasal passageway. In another embodiment, the formulation is administrated to one or both nostrils.
The present invention also provides methods for generating an immune response to Norovirus in a subject, comprising administering to the subject an antigenic formulation or a vaccine comprising the Norovirus composition. In one embodiment, the antigenic formulations and vaccines comprising the Norovirus composition find use in generating antibodies to one or more Norovirus antigens. In another embodiment, the Norovirus vaccine formulations may be used to treat Norovirus infections.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates an in vitro antigen-specific proliferation assay of murine cervical lymph node cells following in vivo intranasal immunization with 10 μg VLP.
FIG. 2 illustrates in vitro antigen-specific proliferation assay of splenocytes following in vivo intranasal immunization with 10 μg VLP.
FIG. 3 illustrates in vitro antigen-specific proliferation assay of splenocytes following in vivo intraperitoneal immunization with 25 μg VLP.
FIG. 4 illustrates VLP-specific IgG or IgA from antibody secreting cells (ASCs) measured by ELISPOT assay.
FIG. 5 illustrates VLP-specific IgG measured by ELISA.
FIG. 6 illustrates the result of a potency assay for serum IgG response against Norwalk VLPs.
FIG. 7 depicts the results of a potency assay comparing serum IgG responses against Norwalk VLPs in mice immunized with either a liquid formulation of the antigen or a formulation reconstituted from dry powder. The graph shows potency versus concentration of Norwalk VLPs in the different formulations.
FIG. 8 shows the serum IgG response in rabbits on day 21 (left panel) and day 42 (right panel) following administration of different formulations of Norovirus VLP vaccine.
FIG. 9 illustrates the serum IgG response in rabbits immunized intranasally with either a liquid formulation or a dry powder formulation of Norwalk VLPs.
FIG. 10 depicts the stability of dry powder formulation as measured by quantitative SDS-PAGE analysis and size exclusion chromatography (SEC). Regression analysis indicates no statistical trends in either the total or intact μg VLP per 10 mg dry powder over 1 year. The percent aggregate is a calculation assuming that VLP protein not detected by SEC, compared to the total VLP protein by quantitative SDS-PAGE, is aggregated.
FIG. 11 illustrates the results of an ELISA assay of anti-Norovirus antibody response in mice immunized i.p. with multiple Norovirus antigens. The thin arrows indicate booster injections with formulations containing only Norwalk VLPs. The thick arrows denote booster injections with formulations containing both Norwalk and Houston VLPs.
FIG. 12 illustrates an ELISA assay of anti-Norovirus antibody response in mice immunized i.p. with either Norwalk VLPs, Houston VLPs, or a combination of Norwalk and Houston VLPs.
FIG. 13 shows the presence of Norwalk VLP-specific long-lived plasma cells in splenocytes (A), cervical lymph nodes (B), and bone marrow (C) in mice 114 days after intranasal immunization with Norwalk VLPs in mice.
FIG. 14 depicts the Norwalk-specific memory B cell response in splenocytes of mice immunized intranasally with Norwalk VLPs. Panel A shows IgA antibody secreting cells on day 0 (left graph) and day 4 in culture with Norwalk VLPs (right graph). Panel B shows the IgG antibody secreting cells on day 0 (left graph) and day 4 in culture with Norwalk VLPs (right graph). The difference in the number of cells between day 0 and day 4 indicates the level of memory B cell expansion and differentiation.
FIG. 15 shows the ELISPOT assay results of peripheral blood mononuclear cells isolated from rabbits immunized intranasally with a Norwalk VLP vaccine formulation. The left panel shows the number of Norwalk VLP-specific antigen secreting cells (ASCs) at day 0 (day of tissue harvest), while the right panel illustrates the number of Norwalk VLP-specific ASCs after 4 days in culture with Norwalk VLPs. The difference in the number of cells between day 0 and day 4 indicates the memory B cell response.
FIG. 16 shows the ELISPOT assay results of splenocytes harvested from rabbits immunized intranasally with a Norwalk VLP vaccine formulation. The left panel shows the number of Norwalk VLP-specific antigen secreting cells (ASCs) at day 0 (day of tissue harvest), while the right panel illustrates the number of Norwalk VLP-specific ASCs after 4 days in culture with Norwalk VLPs. The difference in the number of cells between day 0 and day 4 indicates the memory B cell response.
FIG. 17 shows the ELISPOT assay results of bone marrow cells harvested from the tibias of rabbits immunized intranasally with a Norwalk VLP vaccine formulation. The left panel shows the number of Norwalk VLP-specific antigen secreting cells (ASCs) at day 0 (day of tissue harvest), while the right panel illustrates the number of Norwalk VLP-specific ASCs after 4 days in culture with Norwalk VLPs. The presence of ASCs at day 0 indicates the presence of long-lived plasma cells. The difference in the number of cells between day 0 and day 4 indicates the memory B cell response.
FIG. 18 shows the ELISPOT assay results of mesenteric lymph node cells harvested from rabbits immunized intranasally with a Norwalk VLP vaccine formulation. Panel A shows IgG positive antibody secreting cells (ASCs) specific for Norwalk VLPs. Panel B shows IgA positive ASCs specific for Norwalk VLPs. The left panels show the number of Norwalk VLP-specific ASCs at day 0 (day of tissue harvest), while the right panels illustrate the number of Norwalk VLP-specific ASCs after 4 days in culture with Norwalk VLPs. The presence of ASCs at day 0 indicates the presence of long-lived plasma cells. The difference in the number of cells between day 0 and day 4 indicates the memory B cell response.
FIG. 19 illustrates in vitro antigen-specific proliferation assay of splenocytes following in vivo intranasal immunization in rabbits. The left panel shows T cell proliferation upon restimulation with Norwalk VLPs in unfractionated splenocytes, while the right panel shows CD4+ T cell proliferation upon restimulation with Norwalk VLPs.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to Norovirus antigenic and vaccine compositions and methods of preparing the compositions. In particular, the present invention provides a composition that comprises a Norovirus antigen and at least one adjuvant. Additionally or alternatively, the composition may further comprise at least one delivery agent. The invention also provides methods of administering the composition to an animal to produce an immune response or generate antibodies to Norovirus antigens.
Norovirus Antigens
The invention provides a composition comprising one or more Norovirus antigens. By “Norovirus,” “Norovirus (NOR),” “norovirus,” and grammatical equivalents herein, are meant members of the genus Norovirus of the family Caliciviridae. In some embodiments, a Norovirus can include a group of related, positive-sense single-stranded RNA, nonenveloped viruses that can be infectious to human or non-human mammalian species. In some embodiments, a Norovirus can cause acute gastroenteritis in humans. Noroviruses also can be referred to as small round structured viruses (SRSVs) having a defined surface structure or ragged edge when viewed by electron microscopy. Included within the Noroviruses are at least four genogroups (GI-IV) defined by nucleic acid and amino acid sequences, which comprise 15 genetic clusters. The major genogroups are GI and GII. GIII and GIV are proposed but generally accepted. Representative of GIII is the bovine, Jena strain. GIV contains one virus, Alphatron, at this time. For a further description of Noroviruses see Vinje et al. J. Clin. Micro. 41:1423-1433 (2003). By “Norovirus” also herein is meant recombinant Norovirus virus-like particles (rNOR VLPs). In some embodiments, the recombinant Norovirus VLPs are produced in an expression system using a Norovirus nucleic acid sequence, which encodes at least one capsid protein or fragment thereof. In other embodiments, recombinant expression of at least the Norovirus capsid protein encoded by ORF2 in cells, e.g., from a baculovirus vector in Sf9 cells, can result in spontaneous self-assembly of the capsid protein into VLPs. In yet other embodiments, recombinant expression of at least the Norovirus proteins encoded by ORF1 and ORF2 in cells, e.g., from a baculovirus vector in Sf9 cells, can result in spontaneous self-assembly of the capsid protein into VLPs. The Norovirus nucleic acid sequence may also be a consensus sequence comprising various Norovirus strains or a synthetic construct modified to enhance yields or stability, or improve antigenic or immunogenic properties of the encoded antigen. VLPs are structurally similar to Noroviruses but lack the viral RNA genome and therefore are not infectious. Accordingly, “Norovirus” includes virions that can be infectious or non-infectious particles, which include defective particles.
Non-limiting examples of Noroviruses include Norwalk virus (NV, GenBank M87661, NP056821), Southampton virus (SHV, GenBank L07418), Desert Shield virus (DSV, U04469), Hesse virus (HSV), Chiba virus (CHV, GenBank AB042808), Hawaii virus (HV, GenBank U07611), Snow Mountain virus (SMV, GenBank U70059), Toronto virus (TV, Leite et al., Arch. Virol. 141:865-875), Bristol virus (BV), Jena virus (JV, AJ01099), Maryland virus (MV, AY032605), Seto virus (SV, GenBank AB031013), Camberwell (CV, AF145896), Lordsdale virus (LV, GenBank X86557), Grimsby virus (GrV, AJ004864), Mexico virus (MXV, GenBank U22498), Boxer (AF538679), C59 (AF435807), VA115 (AY038598), BUDS (AY660568), Houston virus (HoV), Minerva strain (EF126963.1), Laurens strain (EF126966.1), MOH (AF397156), Parris Island (PiV; AY652979), VA387 (AY038600), VA207 (AY038599), and Operation Iraqi Freedom (OIF, AY675554). The nucleic acid and corresponding amino acid sequences of each are all incorporated by reference in their entirety. In some embodiments, a cryptogram can be used for identification purposes and is organized: host species from which the virus was isolated/genus abbreviation/species abbreviation/strain name/year of occurrence/country of origin. (Green et al., Human Caliciviruses, in Fields Virology Vol. 1 841-874 (Knipe and Howley, editors-in-chief, 4th ed., Lippincott Williams & Wilkins 2001)). Use of a combination of Norovirus genogroups such as a genogroup I.1 (Norwalk virus) and 11.4 (Houston virus) or other commonly circulating strains, or synthetic constructs representing combinations or portions thereof are preferred in some embodiments. New strains of Noroviruses are routinely identified (Centers for Disease Control, Morbidity and Mortality Weekly Report, 56(33):842-846 (2007)) and consensus sequences of two or more viral strains may also be used to express Norovirus antigens.
The Norovirus antigen may be in the form of peptides, proteins, or virus-like particles (VLPs). In a preferred embodiment, the Norovirus antigen comprises VLPs. As used herein, “virus-like particle(s) or VLPs” refer to a virus-like particle(s), fragment(s), aggregates, or portion(s) thereof produced from the capsid protein coding sequence of Norovirus and comprising antigenic characteristic(s) similar to those of infectious Norovirus particles. Norovirus antigens may also be in the form of capsid monomers, capsid multimers, protein or peptide fragments of VLPs, or aggregates or mixtures thereof. The Norovirus antigenic proteins or peptides may also be in a denatured form, produced using methods known in the art.
Norovirus antigens may also include variants of the said capsid proteins or fragments thereof expressed on or in the VLPs of the invention. The variants may contain alterations in the amino acid sequences of the constituent proteins. The term “variant” with respect to a polypeptide refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence. The variant can have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. Alternatively, a variant can have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software.
General texts which describe molecular biological techniques, which are applicable to the present invention, such as cloning, mutation, and the like, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (“Ausubel”). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, e.g., the cloning and mutating of capsid proteins of Norovirus. Thus, the invention also encompasses using known methods of protein engineering and recombinant DNA technology to improve or alter the characteristics of the proteins expressed on or in the VLPs of the invention. Various types of mutagenesis can be used to produce and/or isolate variant nucleic acids including concensus sequences that encode for protein molecules and/or to further modify/mutate the proteins in or on the VLPs of the invention. They include but are not limited to site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like.
The VLPs of the present invention can be formed from either the full length Norovirus capsid protein such as VP1 and/or VP2 proteins or certain VP1 or VP2 derivatives using standard methods in the art. Alternatively, the capsid protein used to form the VLP is a truncated capsid protein. In some embodiments, for example, at least one of the VLPs comprises a truncated VP1 protein. In other embodiments, all the VLPs comprise truncated VP1 proteins. The truncation may be an N- or C-terminal truncation. Truncated capsid proteins are suitably functional capsid protein derivatives. Functional capsid protein derivatives are capable of raising an immune response (if necessary, when suitably adjuvanted) in the same way as the immune response is raised by a VLP consisting of the full length capsid protein.
VLPs may contain major VP1 proteins and/or minor VP2 proteins. Preferably each VLP contains VP1 and/or VP2 protein from only one Norovirus genogroup giving rise to a monovalent VLP. As used herein, the term “monovalent” means the antigenic proteins are derived from a single Norovirus genogroup. For example, the VLPs contain VP1 and/or VP2 from a virus strain of genogroup I (e.g., VP1 and VP2 from Norwalk virus). Preferably the VLP is comprised of predominantly VP1 proteins. In one embodiment of the invention, the antigen is a mixture of monovalent VLPs wherein the composition includes VLPs comprised of VP1 and/or VP2 from a single Norovirus genogroup mixed with VLPs comprised of VP1 and/or VP2 from a different Norovirus genogroup taken from multiple viral strains (e.g. Norwalk virus and Houston virus). Purely by way of example the composition can contain monovalent VLPs from one or more strains of Norovirus genogroup I together with monovalent VLPs from one or more strains of Norovirus genogroup II. Preferably, the Norovirus VLP mixture is composed of the strains of Norwalk and Houston Noroviruses.
However, in an alternative embodiment of the invention, the VLPs may be multivalent VLPs that comprise, for example, VP1 and/or VP2 proteins from one Norovirus genogroup intermixed with VP1 and/or VP2 proteins from a second Norovirus genogroup, wherein the different VP1 and VP2 proteins are not chimeric VP1 and VP2 proteins, but associate together within the same capsid structure to form immunogenic VLPs. As used herein, the term “multivalent” means that the antigenic proteins are derived from two or more Norovirus genogroups. Multivalent VLPs may contain VLP antigens taken from two or more viral strains. Purely by way of example the composition can contain multivalent VLPs comprised of capsid monomers or multimers from one or more strains of Norovirus genogroup I together with capsid monomers or multimers from one or more strains of Norovirus genogroup II. Preferably, the multivalent VLPs contain capsid proteins from the strains of Norwalk and Houston Noroviruses.
The combination of monovalent or multivalent VLPs within the composition preferably would not block the immunogenicity of each VLP type. In particular it is preferred that there is no interference between Norovirus VLPs in the combination of the invention, such that the combined VLP composition of the invention is able to elicit immunity against infection by each Norovirus genotype represented in the vaccine. Suitably the immune response against a given VLP type in the combination is at least 50% of the immune response of that same VLP type when measured individually, preferably 100% or substantially 100%. The immune response may suitably be measured, for example, by antibody responses, as illustrated in the examples herein.
Multivalent VLPs may be produced by separate expression of the individual capsid proteins followed by combination to form VLPs. Alternatively multiple capsid proteins may be expressed within the same cell, from one or more DNA constructs. For example, multiple DNA constructs may be transformed or transfected into host cells, each vector encoding a different capsid protein. Alternatively a single vector having multiple capsid genes, controlled by a shared promoter or multiple individual promoters, may be used. IRES elements may also be incorporated into the vector, where appropriate. Using such expression strategies, the co-expressed capsid proteins may be co-purified for subsequent VLP formation, or may spontaneously form multivalent VLPs which can then be purified.
A preferred process for multivalent VLP production comprises preparation of VLP capsid proteins or derivatives, such as VP1 and/or VP2 proteins, from different Norovirus genotypes, mixing the proteins, and assembly of the proteins to produce multivalent VLPs. The capsid proteins may be in the form of a crude extract, be partially purified or purified prior to mixing. Assembled monovalent VLPs of different genogroups may be disassembled, mixed together and reassembled into multivalent VLPs. Preferably the proteins or VLPs are at least partially purified before being combined. Optionally, further purification of the multivalent VLPs may be carried out after assembly.
Suitably the VLPs of the invention are made by disassembly and reassembly of VLPs, to provide homogenous and pure VLPs. In one embodiment multivalent VLPs may be made by disassembly of two or more VLPs, followed by combination of the disassembled VLP components at any suitable point prior to reassembly. This approach is suitable when VLPs spontaneously form from expressed VP1 protein, as occurs for example, in some yeast strains. Where the expression of the VP1 protein does not lead to spontaneous VLP formation, preparations of VP1 proteins or capsomers may be combined before assembly into VLPs.
Where multivalent VLPs are used, preferably the components of the VLPs are mixed in the proportions in which they are desired in the final mixed VLP. For example, a mixture of the same amount of a partially purified VP1 protein from Norwalk and Houston viruses (or other Norovirus strains) provides a multivalent VLP with approximately equal amounts of each protein.
Compositions comprising multivalent VLPs may be stabilized by solutions known in the art, such as those of WO 98/44944, WO0045841, incorporated herein by reference.
Compositions of the invention may comprise other proteins or protein fragments in addition to VP1 and VP2 proteins or derivatives. Other proteins or peptides may also be co-administered with the composition of the invention. Optionally the composition may also be formulated or co-administered with non-Norovirus antigens. Suitably these antigens can provide protection against other diseases.
The VP1 protein or functional protein derivative is suitably able to form a VLP, and VLP formation can be assessed by standard techniques such as, for example, electron microscopy and dynamic laser light scattering.
Antigen Preparation
The antigenic molecules of the present invention can be prepared by isolation and purification from the organisms in which they occur naturally, or they may be prepared by recombinant techniques. Preferably the Norovirus VLP antigens are prepared from insect cells such as Sf9 or H5 cells, although any suitable cells such as E. coli or yeast cells, for example, S. cerevisiae, S. pombe, Pichia pastori or other Pichia expression systems, mammalian cell expression such as CHO or HEK systems may also be used. When prepared by a recombinant method or by synthesis, one or more insertions, deletions, inversions or substitutions of the amino acids constituting the peptide may be made. Each of the aforementioned antigens is preferably used in the substantially pure state.
The procedures of production of norovirus VLPs in insect cell culture have been previously disclosed in U.S. Pat. No. 6,942,865, which is incorporated herein by reference in its entirety. Briefly, a cDNA from the 3′ end of the genome containing the viral capsid gene (ORF2) and a minor structural gene (ORF3) were cloned. The recombinant baculoviruses carrying the viral capsid genes were constructed from the cloned cDNAs. Norovirus VLPs were produced in Sf9 or H5 insect cell cultures.
Adjuvants
The invention further provides a composition comprising adjuvants for use with the Norovirus antigen. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as Bordatella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Pifco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; and Quil A.
Suitable adjuvants also include, but are not limited to, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, and liposomes. Preferably, the adjuvants are not bacterially-derived exotoxins. Preferred adjuvants are those which stimulate a Th1 type response such as 3DMPL or QS21.
Monophosphoryl Lipid A (MPL), a non-toxic derivative of lipid A from Salmonella, is a potent TLR-4 agonist that has been developed as a vaccine adjuvant (Evans et al. 2003). In preclinical murine studies intranasal MPL has been shown to enhance secretory, as well as systemic, humoral responses (Baldridge et al. 2000; Yang et al. 2002). It has also been proven to be safe and effective as a vaccine adjuvant in clinical studies of greater than 120,000 patients (Baldrick et al., 2002; 2004). MPL stimulates the induction of innate immunity through the TLR-4 receptor and is thus capable of eliciting nonspecific immune responses against a wide range of infectious pathogens, including both gram negative and gram positive bacteria, viruses, and parasites (Baldrick et al. 2004; Persing et al. 2002). Inclusion of MPL in intranasal formulations should provide rapid induction of innate responses, eliciting nonspecific immune responses from viral challenge while enhancing the specific responses generated by the antigenic components of the vaccine.
Accordingly, in one embodiment, the present invention provides a composition comprising monophosphoryl lipid A (MPL®) or 3 De-O-acylated monophosphoryl lipid A (3D-MPL®) as an enhancer of adaptive and innate immunity. Chemically 3D-MPL® is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA), which is incorporated herein by reference. In another embodiment, the present invention provides a composition comprising synthetic lipid A, lipid A mimetics or analogs, such as BioMira's PET Lipid A, or synthetic derivatives designed to function like TLR-4 agonists.
The term “effective adjuvant amount” or “effective amount of adjuvant” will be well understood by those skilled in the art, and includes an amount of one or more adjuvants which is capable of stimulating the immune response to an administered antigen, i.e., an amount that increases the immune response of an administered antigen composition, as measured in terms of the IgA levels in the nasal washings, serum IgG or IgM levels, or B and T-Cell proliferation. Suitably effective increases in immunoglobulin levels include by more than 5%, preferably by more than 25%, and in particular by more than 50%, as compared to the same antigen composition without any adjuvant.
Deliver Agent
The invention also provides a composition comprising a delivery agent which functions to enhance antigen uptake based upon, but not restricted to, increased fluid viscosity due to the single or combined effect of partial dehydration of host mucopolysaccharides, the physical properties of the delivery agent, or through ionic interactions between the delivery agent and host tissues at the site of exposure, which provides a depot effect. Alternatively, the delivery agent can increase antigen retention time at the site of delivery (e.g., delay expulsion of the antigen). Such a delivery agent may be a bioadhesive agent. In particular, the bioadhesive may be a mucoadhesive agent selected from the group consisting of glycosaminoglycans (e.g., chondroitin sulfate, dermatan sulfate chondroitin, keratan sulfate, heparin, heparan sulfate, hyaluronan), carbohydrate polymers (e.g., pectin, alginate, glycogen, amylase, amylopectin, cellulose, chitin, stachyose, unulin, dextrin, dextran), cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (including mucin and other mucopolysaccharides) cellulose derivatives (e.g., hydroxypropyl methylcellulose, carboxymethylcellulose), proteins (e.g. lectins, fimbrial proteins), and deoxyribonucleic acid. Preferably, the mucoadhesive agent is a polysaccharide, such as chitosan, a chitosan salt, or chitosan base (e.g. chitosan glutamate).
Chitosan, a positively charged linear polysaccharide derived from chitin in the shells of crustaceans, is a bioadhesive for epithelial cells and their overlaying mucus layer. Formulation of antigens with chitosan increases their contact time with the nasal membrane, thus increasing uptake by virtue of a depot effect (Illum et al. 2001; 2003; Davis et al. 1999; Bacon et al. 2000; van der Lubben et al. 2001; 2001; Lim et al. 2001). Chitosan has been tested as a nasal delivery system for several vaccines, including influenza, pertussis and diphtheria, in both animal models and humans (Illum et al. 2001; 2003; Bacon et al. 2000; Jabbal-Gill et al. 1998; Mills et al. 2003; McNeela et al. 2004). In these trials, chitosan was shown to enhance systemic immune responses to levels equivalent to parenteral vaccination. In addition, significant antigen-specific IgA levels were also measured in mucosal secretions. Thus, chitosan can greatly enhance a nasal vaccine's effectiveness. Moreover, due to its physical characteristics, chitosan is particularly well suited to intranasal vaccines formulated as powders (van der Lubben et al. 2001; Mikszta et al. 2005; Huang et al. 2004).
Accordingly, in one embodiment, the present invention provides an antigenic or vaccine composition adapted for intranasal administration, wherein the composition includes antigen and optionally an effective amount of adjuvant. In preferred embodiments, the invention provides an antigenic or vaccine composition comprising Norovirus antigen such as Norovirus VLP, in combination with at least one delivery agent, such as chitosan, and at least one adjuvant, such as MPL®, CPGs, imiquimod, gardiquimod, or synthetic lipid A or lipid A mimetics or analogs.
The molecular weight of the chitosan may be between 10 kDa and 800 kDa, preferably between 100 kDa and 700 kDa and more preferably between 200 kDa and 600 kDa. The concentration of chitosan in the composition will typically be up to about 80% (w/w), for example, 5%, 10%, 30%, 50%, 70% or 80%. The chitosan is one which is preferably at least 75% deacetylated, for example 80-90%, more preferably 82-88% deacetylated, particular examples being 83%, 84%, 85%, 86% and 87% deacetylation.
Vaccine and Antigenic Formulations
The compositions of the invention can be formulated for administration as vaccines or antigenic formulations. As used herein, the term “vaccine” refers to a formulation which contains Norovirus VLPs or other Norovirus antigens of the present invention as described above, which is in a form that is capable of being administered to a vertebrate and which induces an immune response sufficient to induce a therapeutic immunity to ameliorate an infection and/or to reduce at least one symptom of an infection and/or to enhance the efficacy of another dose of VLPs or antigen. As used herein, the term “antigenic formulation” or “antigenic composition” refers to a preparation which, when administered to a vertebrate, e.g. a mammal, will induce an immune response. As used herein, the term “immune response” refers to both the humoral immune response and the cell-mediated immune response. The humoral immune response involves the stimulation of the production of antibodies by B lymphocytes that, for example, neutralize infectious agents, block infectious agents from entering cells, block replication of said infectious agents, and/or protect host cells from infection and destruction. The cell-mediated immune response refers to an immune response that is mediated by T-lymphocytes and/or other cells, such as macrophages, against an infectious agent, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates infection or reduces at least one symptom thereof. Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995) Plenum Press New York). The compositions of the present invention can be formulated, for example, for delivery to one or more of the oral, gastro-intestinal, and respiratory (e.g. nasal) mucosa.
Where the composition is intended for delivery to the respiratory (e.g. nasal) mucosa, typically it is formulated as an aqueous solution for administration as an aerosol or nasal drops, or alternatively, as a dry powder, e.g. for rapid deposition within the nasal passage. Compositions for administration as nasal drops may contain one or more excipients of the type usually included in such compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents, and the like. Viscosity agents can be microcrystalline cellulose, chitosan, starches, polysaccharides, and the like. Compositions for administration as dry powder may also contain one or more excipients usually included in such compositions, for example, mucoadhesive agents, bulking agents, and agents to deliver appropriate powder flow and size characteristics. Bulking and powder flow and size agents may include mannitol, sucrose, trehalose, and xylitol.
In one embodiment, the Norovirus vaccine or antigenic formulation of the present invention may be formulated as a dry powder containing one or more Norovirus genogroup antigen(s) as the immunogen, an adjuvant such as MPL®, a biopolymer such as chitosan to promote adhesion to mucosal surfaces, and bulking agents such as mannitol and sucrose. For example, the Norovirus vaccine may be formulated as 10 mg of a dry powder containing one or more Norovirus genogroup antigen(s) (e.g., Norwalk virus, Houston virus, Snow Mountain virus), MPL® adjuvant, chitosan mucoadhesive, and mannitol and sucrose as bulking agents and to provide proper flow characteristics. The formulation may comprise about 7.0 mg (25 to 90% w/w range) chitosan, about 1.5 mg mannitol (0 to 50% w/w range), about 1.5 mg sucrose (0 to 50% w/w range), about 25 μg MPL® (0.1 to 5% w/w range), and about 100 μg Norovirus antigen (0.05 to 5% w/w range).
Norovirus antigen may be present in a concentration of from about 0.01% (w/w) to about 80% (w/w). In one embodiment, Norovirus antigens can be formulated at dosages of about 5 μg, about 15 μg, and about 50 μg per 10 mg dry powder formulation (0.025, 0.075 and 0.25% w/w) for administration into both nostrils or about 10 μg, about 30 μg, and about 100 μg (0.1, 0.3 and 1.0% w/w) for administration into one nostril. The formulation may be given in one or both nostrils during each administration. There may be a booster administration 1 to 12 weeks after the first administration to improve the immune response. The content of the Norovirus antigens in the vaccine and antigenic formulations may be in the range of 1 μg to 100 mg, preferably in the range 1-500 μg, more preferably 5-200 μg, most typically in the range 10-100 μg. Total Norovirus antigen administered at each dose will be either about 10 μg, about 30 μg, or about 100 μg in a total of 20 mg dry powder when administered to both nostrils or 10 mg dry powder when administered to one nostril. Dry powder characteristics are such that less than 10% of the particles are less than 10 μm in diameter. Mean particle sizes range from 10 to 500 μm in diameter.
In another embodiment, the antigenic and vaccine compositions can be formulated as a liquid for subsequent administration to a subject. A liquid formulation intended for intranasal administration would comprise Norovirus genogroup antigen(s), adjuvant, and a delivery agent such as chitosan. Liquid formulations for intramuscular (i.m.) or oral administration would comprise Norovirus genogroup antigen(s), adjuvant, and a buffer, without a delivery agent (e.g., chitosan).
Preferably the antigenic and vaccine compositions hereinbefore described are lyophilized and stored anhydrous until they are ready to be used, at which point they are reconstituted with diluent, if used in a liquid formulation. Alternatively, different components of the composition may be stored separately in a kit or device (any or all components being lyophilized). The components may remain in lyophilized form for dry formulation or be reconstituted for liquid formulations, and either mixed prior to use or administered separately to the patient. For dry powder administration the vaccine or antigenic formulation may be preloaded into an intranasal delivery device or topical (e.g., dermal) delivery patch and stored until used. Preferably, such delivery device and associated packaging would protect and ensure the stability of its contents.
The lyophilization of antigenic formulations and vaccines is well known in the art. Typically the liquid antigen is freeze dried in the presence of agents to protect the antigen during the lyophilization process and to yield powders with desirable characteristics. Sugars such as sucrose, mannitol, trehalose, or lactose (present at an initial concentration of 10-200 mg/mL) are commonly used for cryoprotection and lyoprotection of protein antigens and to yield lyophilized cake or powders with desirable characteristics. Lyophilized compositions are theoretically more stable. Other drying technologies, such as spray drying or spray freeze drying may also be used. While the goal of most formulation processes is to minimize protein aggregation and degradation, the inventors have demonstrated that the presence of aggregated antigen enhances the immune response to Norovirus VLPs (see Examples 3 and 4 in animal models). Therefore, the inventors have developed methods by which the percentage of aggregation of the antigen can be controlled during the lyophilization process to produce an optimal ratio of aggregated antigen to intact antigen to induce a maximal immune response in animal models.
Thus, the invention also encompasses a method of making Norovirus antigen formulations comprising (a) preparing a pre-lyophilization solution comprising Norovirus antigen, sucrose, and chitosan, wherein the ratios of sucrose to chitosan are from about 0:1 to about 10:1; (b) freezing the solution; and (c) lyophilizing the frozen solution for 30-72 hours, wherein the final lyophilized product contains a percentage of said Norovirus antigen in aggregated form. The lyophilization may occur at ambient temperature, reduced temperature, or proceed in cycles at various temperatures. For illustration purposes only, lyophilization may occur over a series of steps, for instance a cycle starting at −69° C., gradually adjusting to −24° C. over 3 hours, then retaining this temperature for 18 hours, then gradually adjusting to −16° C. over 1 hour, then retaining this temperature for 6 hours, then gradually adjusting to +34° C. over 3 hours, and finally retaining this temperature over 9 hours In one embodiment, the pre-lyophilization solution further comprises a bulking agent. In another embodiment, said bulking agent is mannitol.
Appropriate ratios of sucrose and chitosan to yield desired percentages of aggregation can be determined by the following guidelines. A pre-lyophilization mixture containing mass ratios of sucrose to chitosan in a range from about 2:1 to about 10:1 will yield a range of about 50% to 100% intact Norovirus antigen (i.e. 0% to 50% aggregated antigen) post-lyophilization depending on pre-lyophilization solution concentrations (see Example 13). Mass ratios of 0:1 sucrose to chitosan will produce less than 30% of intact Norovirus antigen (i.e. greater than 70% aggregated antigen). Omission of both sucrose and chitosan and use of only a bulking agent, such as mannitol, will produce less than 10% intact antigen (i.e. greater than 90% aggregated antigen depending on pre-lyophilization solution concentrations). Using these guidelines, the skilled artisan could adjust the sucrose to chitosan mass ratios and concentrations in the pre-lyophilization mixture to obtain the desired amount of aggregation necessary to produce an optimal immune response.
In addition, the inclusion of sucrose, chitosan, and mannitol in the pre-lyophilization solution has no negative effect on the stability of the intact Norovirus antigen over time, i.e. the ratio of aggregated antigen/intact antigen in the formulation does not increase when stored as a dry powder for a period of about 12 months or greater (see Example 10). Thus, this lyophilization procedure ensures stable formulations with predictable and controllable ratios of aggregated to intact Norovirus antigen.
Methods of Stimulating an Immune Response
The amount of antigen in each antigenic or vaccine formulation dose is selected as an amount which induces a robust immune response without significant, adverse side effects. Such amount will vary depending upon which specific antigen(s) is employed, route of administration, and adjuvants used. In general, the dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time, or to induce the production of antigen-specific antibodies. Thus, the composition is administered to a patient in an amount sufficient to elicit an immune response to the specific antigens and/or to alleviate, reduce, or cure symptoms and/or complications from the disease or infection. An amount adequate to accomplish this is defined as a “therapeutically effective dose.”
For a substantially pure form of the Norovirus antigen, it is expected that each dose will comprise about 1 μg to 10 mg, preferably about 2-50 μg for each Norovirus antigen in the formulation. In a typical immunization regime employing the antigenic preparations of the present invention, the formulations may be administered in several doses (e.g. 1-4), each dose containing 1-100 μg of each antigen. The dose will be determined by the immunological activity the composition produced and the condition of the patient, as well as the body weight or surface areas of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular composition in a particular patient.
The antigenic and vaccine formulations of the present invention may be administered via a non-mucosal or mucosal route. These administrations may include in vivo administration via parenteral injection (e.g. intravenous, subcutaneous, and intramuscular) or other traditional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or intranasal routes or directly into a specific tissue. Alternatively, the vaccines of the invention may be administered by any of a variety of routes such as oral, topical, subcutaneous, mucosal, intravenous, intramuscular, intranasal, sublingual, transcutaneous, subdermal, intradermal and via suppository. Administration may be accomplished simply by direct administration using a patch, needle, catheter or related device, at a single time point or at multiple time points.
In a preferred embodiment, the antigenic and vaccine formulations of the present invention are administered to a mucosal surface. Immunization via the mucosal surfaces offers numerous potential advantages over other routes of immunization. The most obvious benefits are 1) mucosal immunization does not require needles or highly-trained personnel for administration, and 2) immune responses are raised at the site(s) of pathogen entry, as well as systemically (Isaka et al. 1999; Kozlowski et al. 1997; Mestecky et al. 1997; Wu et al. 1997).
In a further aspect, the invention provides a method of eliciting an IgA mucosal immune response and an IgG systemic immune response by administering (preferably intranasally or orally) to a mucosal surface of the patient an antigenic or vaccine composition comprising one or more Norovirus antigens, at least one effective adjuvant and/or at least one delivery agent.
The present invention also contemplates the provision of means for dispensing intranasal formulations of Norovirus antigens hereinbefore defined, and at least one adjuvant or at least one delivery agent as hereinbefore defined. A dispensing device may, for example, take the form of an aerosol delivery system, and may be arranged to dispense only a single dose, or a multiplicity of doses. Such a device would deliver a metered dose of the vaccine or antigenic formulation to the nasal passage. Other examples of appropriate devices include, but are not limited to, droppers, swabs, aerosolizers, insufflators (e.g. Valois Monopowder Nasal Administration Device, Bespak UniDose DP), nebulizers, and inhalers. The devices may deliver the antigenic or vaccine formulation by passive means requiring the subject to inhale the formulation into the nasal cavity. Alternatively, the device may actively deliver the formulation by pumping or spraying a dose into the nasal cavity. The antigenic formulation or vaccine may be delivered into one or both nostrils by one or more such devices. Administration could include two devices per subject (one device per nostril). Actual dose of active ingredient (Norovirus antigen) may be about 5-1000 μg. In a preferred embodiment, the antigenic or vaccine formulation is administered to the nasal mucosa by rapid deposition within the nasal passage from a device containing the formulation held close to or inserted into the nasal passageway.
The invention also provides a method of generating antibodies to one or more Norovirus antigens, said method comprising administration of a vaccine or antigenic formulation of the invention as described above to a subject. These antibodies can be isolated and purified by routine methods in the art. The isolated antibodies specific for Norovirus antigens can be used in the development of diagnostic immunological assays. These assays could be employed to detect a Norovirus in clinical samples and identify the particular virus causing the infection (e.g. Norwalk, Houston, Snow Mountain, etc.). Alternatively, the isolated antibodies can be administered to subjects susceptible to Norovirus infection to confer passive or short-term immunity.
As mentioned above, the vaccine formulations of the invention may be administered to a subject to treat symptoms of a Norovirus infection. Symptoms of Norovirus infection are well known in the art and include nausea, vomiting, diarrhea, and stomach cramping. Additionally, a patient with a Norovirus infection may have a low-grade fever, headache, chills, muscle aches, and fatigue. The invention encompasses a method of inducing an immune response in a subject experiencing a Norovirus infection by administering to the subject a vaccine formulation of the invention such that at least one symptom associated with the Norovirus infection is alleviated and/or reduced. A reduction in a symptom may be determined subjectively or objectively, e.g., self assessment by a subject, by a clinician's assessment or by conducting an appropriate assay or measurement (e.g. body temperature), including, e.g., a quality of life assessment, a slowed progression of a Norovirus infection or additional symptoms, a reduced severity of Norovirus symptoms or suitable assays (e.g. antibody titer and/or T-cell activation assay). The objective assessment comprises both animal and human assessments.
EXAMPLES
The invention will now be illustrated in greater detail by reference to the specific embodiments described in the following examples. The examples are intended to be purely illustrative of the invention and are not intended to limit its scope in any way.
Example 1. Investigations into Immune Responses to Different Norovirus Antigen Forms
To investigate the efficacy of the vaccine formulations, mice were immunized intranasally (i.n.) with liquid suspension vaccine formulation by micropipette. Mice received only a single vaccine dose (prime).
For the experiment, three vaccine formulations were prepared. The first, referred to as 100% aggregate, was prepared by lyophilization of VLPs under conditions that disrupt the native structure of the VLP and induce aggregation. The second, 100% intact, was prepared with rehydrated lyophilized placebo, spiked with 100% native monodisperse VLPs from non-lyophilized VLP stock. The third formulation, 50/50 Mix, is made either by mixing the previous two formulations at a ratio of 1:1, or by lyophilizing under conditions that yield ˜50% intact and 50% aggregated VLPs. The structural state and concentration of the intact native VLP was assayed by size exclusion high performance liquid chromatography (SE-HPLC) and ultraviolet (UV) absorbance. The total protein concentration (which includes the aggregate) of the formulations was determined by quantitative staining of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-resolved proteins. Percent aggregated/intact was calculated as the ratio of intact native VLP to total protein.
TABLE 1
Mixtures shown below were prepared for Experiment
605.125, mouse i.n. liquid vaccination.
Group
Chitosan
Mannitol
Sucrose
MPL
Norwalk VLP
number
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
1
3.5
0.750
0.750
1.0
1.0
2
3.5
0.750
0.750
1.0
1.0
3
3.5
0.750
0.750
1.0
1.0
4
3.5
0.750
0.750
1.0
0
Table 1. Prime for exp 605.125 (mouse i.n.) Values indicate final concentrations of the formulations.
Dose: 20 μL per mouse, 10 μL per nare.
Group 1, 100% Agg: rehydrated 100% aggregated VLP
Group 2, 100% Intact: rehydrated lyophilized placebo, spiked with 100% intact VLPs from non-lyophilized VLP stock.
Group 3, 50/50 mix: 1:1 mixture of solutions from Groups 1 and 2.
Group 4, Naïve: rehydrated lyophilized placebo
This experiment measures the immune response in mice to different Norovirus VLP formulations. Groups of mice (5 per group) were vaccinated intranasally (i.n.) once with rehydrated dry powder formulations shown in Table 1. Animals vaccinated with VLP-containing formulations received the same amount of total protein. 100% Agg (100% aggregated VLP protein); 100% Intact (100% native, monodisperse VLPs); 50/50 Mix (1:1 mixture of monodisperse and aggregated VLP); Naïve (no VLP protein). On day 14 following i.n. immunization, mice were euthanized, the cervical lymph nodes and spleens were harvested, and a single cell suspension was prepared for in vitro antigen-specific cell proliferation assays. In these assays the response of cervical lymph node cells or splenocytes were assessed to determine immunogenic responses against the antigen following in vivo immunization. Cervical lymph node cells or splenocytes were restimulated with either native monodisperse VLPs (native VLP, black bars) or heat-denatured VLP protein (ΔVLP, white bars) and the extent of cellular proliferation from each antigen form (100% Agg, 100% Intact, 50/50 Mix, or naïve) was measured by tritiated thymidine incorporation as indicated on the ordinate axis (CPM) (FIG. 1, cervical lymph node cells; FIG. 2, splenocytes).
Example 2. In Vitro Antigen-Specific Proliferation Assay
To further investigate the potency of the vaccine formulations, mice were immunized intraperitoneally (i.p.) with liquid suspension vaccine formulation. Mice received only a single vaccine dose (prime).
Similar to Example 1, groups of mice (5 per group) were vaccinated, but this time intraperitoneally (i.p.), once with rehydrated dry powder formulations shown in Table 2. Again, animals vaccinated with VLP-containing formulations received the same amount of total protein. 100% Agg (100% aggregated VLP protein); 100% Intact (100% native, intact VLPs); 50/50 Mix (1:1 mixture of intact and aggregated VLP); Naïve (no VLP protein).
TABLE 2
Mixtures shown below were prepared for 605.127,
mouse i.p. liquid immunization.
Group
Chitosan
Mannitol
Sucrose
MPL
Norwalk VLP
number
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
(mg/mL)
1
7
1.475
1.475
0.025
0.025
2
7
1.475
1.475
0.025
0.025
3
7
1.475
1.475
0.025
0.025
4
7
1.475
1.475
0.025
0
Prime for exp 605.127 (mouse i.p.) Values indicate final concentrations of the formulations and are equivalent to a single 10 mg delivery device.
Dose: 1 mL per mouse i.p.
Group 1, 100% Agg: rehydrated 100% aggregated VLP
Group 2, 100% Intact: rehydrated lyophilized placebo, spiked with 100% intact VLPs from non-lyophilized VLP stock.
Group 3, 50/50 mix: rehydrated from lyophilized 50/50 intact VLP/Aggregate
Group 4, Naïve: rehydrated lyophilized placebo
In this assay, response of different murine cells to VLPs following in vivo immunization was measured. On day 14 following immunization, mice were euthanized, the spleens were harvested, and a single cell suspension was prepared. Splenocytes were restimulated with either intact, native VLPs (native VLP, dotted bars) or heat-denatured VLP protein (ΔVLP, white bars) and the extent of cellular proliferation from each antigen form (100% Agg, 100% Intact, 50/50 Mix, or naïve) was measured by tritiated thymidine incorporation as indicated on the ordinate axis (CPM) (FIG. 3). These data indicate that different biophysical forms of the VLPs prepared in the vaccine formulations elicit comparable T cell responses.
Example 3. VLP-Specific ELISPOT Assay
VLP-specific antibody-secreting cell (ASC) responses were measured from mice immunized intraperitoneally with different NV-VLP formulations described in Example 2. Groups of mice (5 per group) were vaccinated i.p. once with rehydrated dry powder formulations shown in Table 2 (Example 2). Animals vaccinated with VLP-containing formulations received the same amount of total protein. 100% Agg (100% aggregated VLP protein); 100% Intact (100% native, intact VLPs); 50/50 Mix (1:1 mixture of intact and aggregated VLP); Naïve (no VLP protein). On day 14, the mice were euthanized and the cervical lymph nodes were harvested. The cervical lymph node cells were cultured overnight on native, intact VLP-coated ELISPOT plates and were developed for either IgG or IgA-specific ELISPOTS using the appropriate HRP-conjugated secondary antibodies (FIG. 4). These data show that the three VLP antigen formulations all elicit an antigen-specific B cell response. The group immunized with 100% Agg VLPs exhibited the greatest immune response.
Example 4. VLP-Specific ELISA
Serum IgG levels were measured from mice immunized i.p. with different NV-VLP formulations. Groups of mice (5 per group) were vaccinated i.p. once with rehydrated dry powder formulations shown in Table 2 (Example 2). Animals vaccinated with VLP-containing formulations received the same amount of total protein. 100% Agg (100% aggregated VLP protein); 100% Intact (100% native, intact VLPs); 50/50 Mix (1:1 mixture of intact and aggregated VLP); Naïve (no VLP protein). On day 14, serum was collected and assayed by ELISA for anti-VLP-specific serum IgG (FIG. 5). These data correlate with the results shown in Example 3, indicating that the three VLP antigen formulations all elicit an antigen-specific B cell response. Again, the group immunized with 100% Agg VLPs showed the greatest immune response.
Example 5. Vaccine Formulations in Rabbits
Formulations were administered intranasally (i.n.) in rabbits using the Valois Monopowder Nasal Administration Device. The dry powder formulations are shown in Tables 3 and 4.
TABLE 3
Formulations described below were prepared for 605.129,
rabbit i.n. dry powder (DP) vaccination.
Prime formulations for exp 605.129 (Rabbit i.n.)
(final amounts for DP vaccines).
Norwalk
Chitosan
Mannitol
Sucrose
MPL
VLP
Group
(mg/
(mg/
(mg/
(mg/
(mg/
number
10 mg DP)
10 mg)
10 mg)
10 mg)
10 mg)
1
7
1.475
1.475
0.025
0.025
2
7
1.475
1.475
0.025
0.025
3
7
1.475
1.475
0.025
0.025
4
7
1.475
1.475
0.025
0
Values indicate final concentrations of the formulations based on a single device (10 mg DP) which is ½ total dose.
Dose: 20 mg DP per animal, 10 mg per nare.
Group 1, 100% Agg: 100% aggregated lyophilized VLP
Group 2, 100% Intact: 100% intact lyophilized VLP
Group 3, 50/50 mix: 50/50 intact/aggregate lyophilized VLP (not a mixture of 1 & 2)
Group 4, Naïve: placebo
TABLE 4
Formulations shown below were prepared for 605.129,
rabbit i.n. dry powder (DP) vaccination.
Boost formulations for exp 605.129 (Rabbit i.n.)
(final amounts for DP vaccines).
Norwalk
Chitosan
Mannitol
Sucrose
MPL
VLP
Group
(mg/
(mg/
(mg/
(mg/
(mg/
number
10 mg DP)
10 mg)
10 mg)
10 mg)
10 mg)
1
7
1.475
1.475
0.025
0.025
2
7
0
2.95
0.025
0.025
3
7
1.475
1.475
0.025
0.025
4
7
1.475
1.475
0.025
0
Values indicate final concentrations of the formulations based on a single device (10 mg DP) which is ½ total dose.
Dose: 20 mg DP per animal, 10 mg per nare.
Group 1, 100% Agg: 100% lyophilized aggregated VLP
Group 2, 100% Intact: 100% intact** VLP*
Group 3, 50/50 mix: 50/50 intact/aggregate lyophilized VLP (not a mixture of 1 & 2)
Group 4, Naïve: lyophilized placebo
*Formulated without mannitol to increase amount of intact VLP post lyophilization.
**Preparation yielded only ~80% intact VLP.
Example 6. Potency Assay of Norovirus Vaccine Formulation in Mice
Female C57B16 mice were immunized intraperitoneally (i.p.) on day 0 with different dilutions of a reconstituted Norwalk VLP dry powder vaccine (containing Norwalk VLP, MPL and chitosan). Each animal was injected with 100 μL of the formulations indicated Serum was collected weekly and serum anti-VLP IgG measured by ELISA. Values for serum collected 3 weeks following immunization are shown in FIG. 6.
The value for each individual mouse is represented, with bars indicating the group mean. Serum anti-VLP IgG values correlated with the dose of vaccine indicated. This experimental design has been refined and developed as a potency assay required for the release of GMP manufactured vaccines for human clinical trials (FIG. 6).
Example 7. Potency of Liquid Vs. Reconstituted Norovirus Formulations in Mice
Female C57B16 mice were immunized i.p. on day 0 with formulations that contained chitosan, mannitol, MPL, and various concentrations of Norwalk VLP (Table 5) in a volume of 100 μL. An internal standard curve was generated (groups 1-5) by solubilizing 10 mg/mL of dry powder matrix (mannitol, MPL, and chitosan) in purified water and adding the specified amounts of liquid Norwalk VLP. In contrast, the GMP VLP lots were previously lyophilized and then solubilized in 1.0 ml of purified water (groups 6-8). Serum was collected from mice on days 14, 21 and 30, and serum anti-Norwalk VLP IgG was measured by ELISA.
TABLE 5
Liquid and Reconstituted Norwalk Formulations
used to immunize mice (i.p.).
Calculated Potency
Group
Treatment
95% CI
Potency
Min
Max
1
5 μg VLP in Placebo
0.173
58.0
39.0
86.3
2
2.5 μg VLP in Placebo
0.192
23.3
15.0
36.3
3
1.25 μg VLP in Placebo
0.182
11.2
7.4
17.0
4
0.63 μg VLP in Placebo
0.287
5.4
2.8
10.4
5
0.31 μg VLP in Placebo
0.114
3.8
2.9
4.9
6
2.5 μg GMP lot
0.276
11.3
6.0
21.3
7
7.5 μg GMP lot
0.221
96.8
58.2
161.0
8
25 μg GMP lot
0.147
113.6
80.9
159.5
The relative potency for each formulation was calculated using the following formula: Inv Log (Ave.—Y intercept/slope). Potency is plotted against VLP concentration in the formulations and reported in relation to the standard curve generated using known amounts of VLP spiked into the matrix background (FIG. 7). The results shown are representative of 3 separate serum collection time points. These data indicate that the Norwalk VLP formulation reconstituted from dry powder has an overall higher potency than the liquid formulations.
Example 8. Potency of Dry Powder Formulation in Rabbits
Forty-three female New Zealand White rabbits were intranasally (i.n.) immunized using the Valois Monopowder Nasal Administration Device with either 5 μg (Low) or 25 μg (Hi) of Norwalk VLPs±MPL and ±chitosan formulated into dry powders. One group received the Hi dose of VLPs and MPL formulated as a liquid and administered intramuscularly (i.m.). Rabbits were vaccinated on days 0 and 21. MPL, when used, was used at the same dose as the VLPs (i.e., 5 μg Norwalk VLPs and 5 μg MPL). Chitosan, when used, was 7 mg/dose.
Serum IgG specific for the Norwalk VLPs (as determined by ELISA) is shown in FIG. 8. Mean values for each treatment groups are shown for day 21 (left panel, collected just prior to administration of the booster immunization) and day 42 (right panel). Values are reported in U/mL of VLP-specific IgG, with 1 U approximating 1 μg. Standard deviations are indicated by bars. All treatment groups had 6 animals, except the negative control group (3 rabbits) and the intramuscularly immunized group (4 animals). These data show that generally the higher VLP dose results in greater serum anti-VLP IgG levels. Chitosan, in particular, enhances responses to intranasal vaccines. The i.m. immunized group showed the greatest responses. However, VLP-specific IgG levels in the intranasally immunized groups were also quite robust.
Example 9. Potency of Liquid Vs. Dry Norovirus Formulations Given Intranasally in Rabbits
Female New Zealand White rabbits were intranasally immunized using the Valois Monopowder Nasal Administration Device with 50 μg of Norwalk VLPs+50 μg MPL+14 mg chitosan formulated into either a dry powder or a liquid. The vaccine content was identical, except for the physical state. Immunizations were on days 0 and 21 (weeks 0 and 3), with serum collected prior to the boost at 3 weeks, and again at 6 weeks following the initial vaccination. Serum IgG specific for Norwalk VLPs was measured by ELISA, and the results are shown in FIG. 9.
Group means are indicated, with the bars representing standard deviations. The dry powder immunization group had 6 rabbits, and the liquid immunization group had 10 rabbits. Eight negative control rabbits are represented. Little difference was seen between the liquid and dry powder immunization groups at 3 weeks; however, at 6 weeks following the initial immunization, rabbits immunized with the dry powder formulation had superior serum anti-VLP IgG responses compared to the liquid immunization group.
Example 10. Stability of Norovirus Dry Powder Formulations
To investigate the stability of the dry powder VLP formulation, bulk drug product was prepared by mixing (per 10 mg drug product) 25 μg of a Genogroup I VLP in solution with 25 μg MPL, 700 μg chitosan glutamate, 1.475 mg mannitol, and 1.475 mg sucrose. The solution was lyophilized, blended with an additional 6.3 mg chitosan glutamate (per 10 mg drug product), filled into Bespak unidose devices at a nominal 10 mg of dry powder, and stored in sealed foil pouches with desiccant capsules. Total VLP content was measured using Imperial stained SDS-PAGE and scanning densitometry, while size exclusion chromatography (SEC) was used to quantify intact VLP content. These measurements indicated that, within experimental error, no change in either total or intact VLP was detectable over the 12 month period (FIG. 10). Assuming that the lower VLP protein recovery by SEC, when compared to SDS-PAGE results, was due to aggregation, the calculated % aggregate did not increase with time but rather remained constant or decreased throughout the 12 months of storage. One of the more common stability issues with proteins is increased aggregation with storage. Based on the results in FIG. 10, it can be concluded that the formulation results in a stable percentage of intact VLPs allowing the product to be manufactured and used over at least a one year period.
Example 11. Multiple Norovirus Antigens
Eight C57Bl/6 mice (female, 9 weeks of age) were immunized intraperitoneally (IP) on days 0 and 14 with 2.5 μg Norwalk VLP formulated with 0.7 mg chitosan, 2.5 μg MPL and 0.3 mg mannitol brought to 0.1 mL with water. Two control mice were immunized with saline. On days 28 and 49, they were immunized again IP with 2.5 μg Norwalk VLP+2.5 μg Houston VLP formulated with 0.7 mg chitosan, 2.5 μg MPL and 0.3 mg mannitol brought to 0.1 mL with water. The control mice again received saline. Serum samples were collected weekly beginning on week 5 (day 35) and analyzed by ELISA for reactivity with Norwalk VLPs or Houston VLPs. The time of boost with the Norwalk only mixtures are indicated by the thin arrows, and the Norwalk+Houston VLP mixtures are indicated by thick arrows. Individual serum IgG responses specific for Norwalk VLPs (top panel) or Houston VLPs (bottom panel) in U/mL (with 1 U approximating 1 μg of IgG) are shown. Means are indicated by bars. Note that the Y-axis scales are different, as the anti-Norwalk responses were much more robust due to two previous immunizations on days 0 and 14 (weeks 0 and 2). However, the responses against Houston VLPs are quite robust, with a large increase appearing in the second week after the boost. These data demonstrate that specific immune responses can be generated against different antigenic strains of Norovirus VLPs in the same immunizing mixture. (FIG. 11).
Example 12. Immune Response to Different Norovirus Antigens
Female C57B16 mice were immunized intraperitoneally (IP) on days 0 and 14 with 25 μg Norwalk VLP, 25 μg Houston VLP, or a combination of 25 μg of each Norwalk and Houston VLP. Serum was collected weekly and serum anti-VLP IgG measured by ELISA. Values for serum collected 4 weeks following immunization are shown in FIG. 12.
VLP content of the immunizations is indicated on the X axis. The value for each individual mouse is represented, with bars indicating the group mean. Antibody levels are represented in U/mL, with 1 U approximating 1 μg of serum IgG. Values in the left panel were determined using Norwalk VLPs as the capture agent, while Houston VLPs were used to coat ELISA plates in order to measure the values on the right panel. These data show that immunization with Norwalk VLP does not lead to serum antibodies that are able to recognize Houston VLPs, or vice versa.
Example 13. Mixtures of Sucrose and Chitosan Preserve Norovirus VLP Structure in Dry Powder Formulations
The following experiments examined the effects of sucrose, chitosan, and mannitol, alone or in combination, in pre-lyophilization solutions on the native Norwalk VLP quaternary structure during lyophilization. Table 6 is a composite of several experiments showing pre-lyophilization solution concentrations of the constituents of interest, the total volume of the mixture, and the corresponding mass ratios. All solutions were manually swirled and gently vortexed to homogeneity, then shell frozen in liquid nitrogen and lyophilized external to the unit using side-arm vessels for times ranging from about 30 to 60 hours.
TABLE 6
Pre-lyophilization solution mixtures used for testing the effects of different
concentrations and combinations of sucrose, chitosan glutamate (chitosan) and
mannitol on the structure of the quaternary structure the Norwalk VLP.
Mass equivalents
S = sucrose
Solution concentrations of constituents pre-
Total
C = chitosan
Experiment
lyophilization (mg/mL)
Volume
M = mannitol
and Sample
Sucrose
Chitosan
Mannitol
VLP (protein)
(mL)
S
C
M
LE1
0
0
100
0.83
0.30
0
0
1
LE2
0
0
75.0
0.62
0.40
LE3
0
0
50.0
0.42
0.60
LE4
0
0
25.0
0.21
1.20
LE5
0
0
10.0
0.08
3.00
LG1-LG3
0
7.83
0
0.20
1.28
0
1
0
LG4-LG6
0
5.06
0
0.13
1.98
LG7-LG9
0
2.09
0
0.05
4.78
LG10
19.32
1.93
0
0.05
5.18
10
1
0
LG11
10.05
2.01
0
0.05
4.98
5
1
LG12
5.13
2.05
0
0.05
4.88
2.5
1
LG13
9.52
0.00
0
0.09
2.63
1
0
LJ1-LJ2
5.29
2.51
0
0.09
2.79
2
1
0
LJ3-LJ4
4.17
1.98
0
0.07
3.54
LJ5-LJ6
3.65
1.73
0
0.06
4.04
LJ7-LJ8
2.93
1.39
0
0.05
5.04
LJ9-LJ10
5.25
2.49
0
0.09
2.81
LJ11-LJ12
4.14
1.97
0
0.07
3.56
LJ13-LJ14
3.63
1.72
0
0.06
4.06
LIG1d-Sa
2.98
1.42
0.00
1.12
4.94
2
1
0
LIG1d-S1
12.89
6.12
0.00
1.12
2.29
2
1
0
LIG1d-S2
12.26
5.82
12.26
0.67
2.41
1
0.5
1
LIG1d-Sb
2.95
1.40
2.95
0.67
5.00
1
0.5
1
LIG1d-S3
29.32
0.00
29.32
0.83
1.01
0
0
1
Table 7 shows the results from size exclusion-high performance liquid chromatography (SE-HPLC) analysis of the lyophilized samples shown in Table 6. Lyophilized samples were reconstituted with water and analyzed by SE-HPLC. Unprocessed NV-VLPs, analyzed concurrently, were used as a reference standard to quantify the NV-VLP content of the reconstituted test samples. Both UV and fluorescence detectors were used for quantification (data shown are from the fluorescence detector). The SE-HPLC was conducted using a Superose™ 6 10-300 column, with mobile phase consisting of 10 mM sodium phosphate, 10 mM citric acid, pH 5, and 500 mM NaCl, at a flow rate of 0.5 mL/min. Protein concentrations were quantified using integrated areas of elution peaks. “VLP” is the peak that eluted at about 15 min from the column, and any preceding shoulder and/or peak tail within the approximate elution time window of the reference standard NV-VLP analyzed concurrently. The VLP fragment that elutes from the column at around 32 min is a highly stable single species that results from destabilization and consequent disassembly of the VLP. Intermediate and smaller fragments were not observed.
The results show that combinations of sucrose and chitosan produced a wide range of native monodisperse NV-VLP recoveries including the highest (approximately 100% recovery) post-lyophilization (samples LG10-LG12). Moreover, the NV-VLP elution peak shapes from these samples were identical to the unprocessed NV-VLP reference standard indicating high preservation of native structure. Samples containing sucrose only exhibited peak shapes similar to the reference standard, though NV-VLP recoveries were lower (approx. 60% recovery (sample LG13) Samples that contained only mannitol resulted in nearly completely aggregated VLPs (samples LE1-LE6 and LIG1d-S3). The deleterious effects of mannitol on NV-VLP structure were counteracted by the presence of chitosan and sucrose (samples LIG1d-S2 and LIG1d-SB).
TABLE 7
Experiment and sample identification, and results for testing the effect
of sucrose, chitosan, and mannitol or combinations thereof on stability
of NV-VLP structure during freezing and lyophilization.
Measured SE-HPLC
mean protein
concentration and peak
Mean percent values of
elution time
recovered protein as
Mass equivalents
VLP
percent of theoretical
S = sucrose
Theoretical
“VLP”
Fragment
Total
C = chitosan
Experiment
VLP conc
~15 min
~32 min
protein
“VLP”
M = mannitol
and Sample
(mg/mL)
N
(mg/mL)
(mg/mL)
(%)
(%)
S
C
M
LE1-LE5
0.25
5
0.02
0.12
56.0
6.3
0
0
1
LG1-LG9
0.25
9
0.06
0.00
24.0
24.0
0
1
0
LG10
0.25
1
0.25
0.00
101
101
10
1
0
LG11
0.25
1
0.25
0.00
101
101
5
1
0
LG12
0.25
1
0.25
0.00
100
100
2.5
1
0
LG13
0.25
1
0.16
0.00
65
65
1
0
0
LJ1-LJ14
0.25
14
0.22
0
85.4
85.4
2
1
0
LIG1d-S1
0.25
1
0.21
0
88
88
2
1
0
LIG1d-Sa
0.25
1
0.12
0
50
50
2
1
0
LIG1d-S2
0.25
1
92
0
92
92
1
0.5
1
LIG1d-Sb
0.25
1
60
0
60
60
1
0.5
1
LIG1d-S3
0.25
1
<1
<1
<1
<1
0
0
1
Example 14. Induction of Norovirus-Specific Long-Lived Plasma Cells and Memory B Cells in Mice Immunized Intranasally
A. Norwalk VLP-Specific Long-Lived Plasma Cells
BALB/c mice were immunized intranasally with Norovirus VLPs and an adjuvant. Naïve controls were administered the adjuvant alone. At 114 days after immunization, spleen, cervical lymph nodes, and bone marrow were harvested from both groups of mice. On the day of harvesting the tissues (day 0), cells were assayed using an ELISPOT assay for the presence of antigen-specific antibody-secreting cells (ASCs). The results are presented in FIG. 13A-C for the different tissues. The detection of immunoglobulins (IgG, IgA, and IgM) in these tissues indicates the presence of Norovirus-specific long-lived plasma cells.
B. Norwalk VLP-Specific Memory B Cells
An in vitro assay was developed to detect the presence of Norwalk VLP-specific memory B-cells from mice immunized intranasally with Norwalk VLPs. Various lymphoid tissues or whole blood (peripheral blood mononuclear cells, splenocytes, lymph node cells, etc.) can serve as the source of cells that can be assayed for the presence of memory B-cells using this assay.
In this experiment, the spleen was harvested and processed from immunized and naïve animals (controls), and splenocytes were cultured for four days in the presence or absence (controls) of Norwalk VLPs (20 μg/ml). An initial VLP-specific ELISPOT assay was performed on the day of tissue harvest (day 0) to establish background levels of ASCs (see Section A above). After four days in culture the cells were harvested and assayed again in an ELISPOT assay to quantify the number of VLP-specific ASCs. The difference in VLP-specific ASC numbers between the day 0 and the day 4 assays represent the antigen-specific memory B-cell population. The results of this experiment are shown in FIGS. 14A and B.
Example 15. Norovirus Memory B Cell Responses in Rabbits
Two female New Zealand White rabbits were immunized intranasally with a dry powder formulation consisting of 25 μg Norwalk VLP, 25 μg MPL, 1.5 mg mannitol, 1.5 mg sucrose, and 7.0 mg chitosan per 10 mg of dry powder loaded into Valois Mark 4 intranasal delivery devices. The two rabbits received a total of three immunizations at 14 day intervals. For these experiments, a non-immunized female rabbit was used as a naïve control.
A. Collection and Processing of Rabbit Tissues
Peripheral blood mononuclear cells (PBMCs): Whole blood (˜50 mL) was obtained from rabbits in collection tubes containing EDTA to prevent coagulation. The whole blood was diluted 1:3 with sterile D-PBS and ˜35 mL of diluted whole blood was layered onto 15 mL of Lympholyte Separation Medium in a sterile 50-mL centrifuge tube. The tubes were centrifuged at 800×g for 20 minutes at room temperature. The buffy coat layer containing the PBMCs was carefully removed using a sterile 5 mL pipette and the cells were washed twice with D-PBS. If necessary, contaminating red blood cells were removed by ACK lysis. The cells were resuspended in RPMI-1640-10% FBS (1640-C) and counted in a hemocytometer using a Trypan exclusion method.
Mesenteric lymph node cells: The lymph nodes were aseptically collected from each rabbit separately following euthanasia. The tissues were maintained in a sterile plastic Petri dish containing ˜10 mL of RPMI-1640-No Serum (1640-NS). The lymph nodes were pressed through a sterile mesh screen using a sterile pestle to disperse the tissue and obtain a single cell suspension of lymph node cells. The cells were collected, washed twice with 1640-NS, and finally filtered through a sterile 70 μm filter to remove clumps and debris. The cells were resuspended in 1640-C and counted in a hemocytometer using a Trypan blue exclusion method.
Splenocytes: Spleens were aseptically obtained from each rabbit following euthanasia. The spleens were placed in sterile Petri dishes containing approximately 10 mL of 1640-NS. Using a sterile 22-guage needle and syringe the media was repeatedly injected into the tissue to disrupt the splenic capsule and elaborate the cells. Sterile forceps were then used to tease apart the remaining tissue fragments. The contents of the Petri dish were transferred to a sterile centrifuge tube and the cell suspension and disrupted splenic tissue was allowed to sit for 6-8 minutes to allow for the settling of large tissue fragments. The single cell suspension was transferred to a second sterile centrifuge tube and the cells were washed once with 1640-NS. The red blood cells in the splenocyte prep were removed by an ACK lysis (8 mL ACK buffer, 8 minutes, room temperature) and the cells were washed one more time with 1640-NS and finally filtered through a sterile 70 μm filter to remove clumps and debris. The final cell pellet was resuspended in 1640-Complete and counted in a hemocytometer using a Trypan blue exclusion method.
Bone marrow cells: The tibia bones in the lower legs were removed from individual rabbits following euthanasia. To remove the bone marrow cells the ends of the bones were aseptically cut off using a bone saw and the contents of the bone were flushed out by repeated injections of 1640-NS medium. The bone marrow cells were pipetted up and down repeatedly to break up and disperse clumps of cells. The cells were washed once with 1640-NS; the red blood cells were lysed with ACK, and the cells were washed one more time with 1640-NS. Finally, the cells were filtered through a sterile 70 μm filter to remove clumps and debris. The final cell pellet was resuspended in 1640-Complete and counted in a hemocytometer using a Trypan blue exclusion method.
B. ELISPOT Assays
Following pre-wetting and washing, 96-well Millipore PVDF filter plates were coated with a sterile solution of native Norwalk VLPs at a concentration of 40 μg/ml in a final volume of 50 μl/well. The plates were incubated overnight at 4° C., washed with D-PBS, and blocked with the addition of 1640-C. Mesenteric lymph node cells, splenocytes, and bone marrow cells from the immunized rabbits and from the naïve control rabbit were added to the wells at varying concentrations (1×106, 5×105, 2×105, and 1×105 cells/well) and the plates were incubated overnight at 37° C. The plates were washed thoroughly with PBS-Tween and secondary reagents specific for rabbit IgG and IgA were added to the wells and incubated for an additional 2 hours at room temperature. Following extensive washing the plates were developed with DAB chromagen/substrate and read in an ELISPOT plate reader. Spots appearing on wells from naïve control animals were subtracted from the experimental groups. The data is expressed as Norwalk VLP-specific antibody-secreting cells (ASCs) and is normalized per 1×106 cells.
C. Norwalk VLP-Specific Memory B-Cell Assay
Isolated lymphoid cells from the various tissues described above were resuspended in 1640-C medium in the presence of Norwalk VLPs (10 μg/mL) at a density of 5×106 cells per mL. The cells were incubated in 24-well plates in 1-mL volumes for four days at 37° C. VLP-specific ELISPOT assays were performed on these cells at the time of culturing. After four days in culture the cells were harvested, washed twice with 1640-NS medium, resuspended in 1640-Complete, and counted in a hemocytometer using a Trypan blue exclusion method. The cells were tested once again in a Norwalk VLP-specific ELISPOT assay. The data obtained from the ELISPOT assays performed on the day of tissue harvest is referred to as day 0 (background) ASC activity. Any spots detected at the day 0 time point are assumed to be actively-secreting plasma cells or long-lived plasma cells (LLPCs). The data obtained from the ELISPOT assay performed on the 4-day cultured cells is referred to as day 4 ASC activity, and the memory B-cell activity is represented by the difference between day 4 ASC activity and day 0 ASC activity.
D. Norwalk VLP-Specific Memory B-Cells are Present in the Peripheral Blood of Intranasally Immunized Rabbits
Whole blood was obtained from two immunized rabbits (RB735, RB1411) 141 days following the last of three intranasal immunizations with a dry powder formulation vaccine containing Norwalk VLPs as described above. Blood was also obtained from an non-immunized, naïve rabbit. The blood was processed to obtain peripheral blood mononuclear cells (PBMCs) and the PBMCs were placed in a Norwalk VLP-Specific memory B-Cell assay (section C above). The results are shown in FIG. 15. The left panel shows results of the initial ELISPOT assay at the time of tissue harvest (day 0 ASCs). The right panel shows the results of the ELISPOT assay after 4 days in culture with Norwalk VLPs (day 4 ASCs).
The day 0 ELISPOT results (FIG. 15, left panel) illustrate that there are no VLP-specific plasma cells remaining in the peripheral blood approximately 140 days after the last boost with Norwalk VLP dry powder vaccine. The right panel of FIG. 15 shows the ELISPOT assay results from PBMCs cultured for four days in vitro with Norwalk VLPs. In the two immunized rabbits, a significant number of PBMCs, presumably a subpopulation of memory B-cells, have matured into active IgG-secreting Norwalk VLP-specific plasma cells. Although assays for IgA-secreting memory B-cells were conducted, only IgG-secreting memory B-cells were detected in the PBMC population. As expected, the naïve animal showed no antigen-specific memory B-cells. Thus, VLP-specific memory B-cells were found in the peripheral circulation of rabbits 140+ days following the last of three intranasal immunizations.
E. Norwalk VLP-Specific Memory B-Cells are Present in the Spleen of Intranasally Immunized Rabbits
Splenocytes were obtained from the spleens of the two vaccine immunized rabbits and the non-immunized control rabbit. Norwalk VLP-specific memory B-cell assays (described above) were performed on these cells and the results are shown in FIG. 16. As observed for the PBMC population the day 0 ELISPOT assay shows that there are no antigen-specific plasma cells present in the spleen (FIG. 16, left panel). However, following a four day in vitro incubation with Norwalk VLPs, IgG-secreting Norwalk VLP-specific memory B-cells are apparent in the splenocyte population. Thus, the spleen represents one site for the migration of memory B-cells following intranasal immunization.
F. A Population of Norwalk VLP-Specific Long-Lived Plasma Cells is Found in the Bone Marrow but No Memory B-Cells are Present
Bone marrow cells were obtained from the tibias of the experimental rabbits and assayed for the presence of long-lived plasma cells and memory B-cells. The results are presented in FIG. 17. The left panel of FIG. 17 shows that rabbit 1411 still had a significant population of antigen-specific plasma cells in the bone marrow. Plasma cells that migrate to the bone marrow and reside there for a significant period of time following immunization are referred to as long-lived plasma cells (LLPCs). Rabbit 735 did not show a high number of LLPCs. No LLPCs were found in the bone marrow of the naïve rabbit. The bone marrow cells were cultured in a memory B-cell assay and re-tested for the presence of memory B-cells. The right panel of FIG. 17 shows that there are essentially no antigen-specific memory B-cells present in the bone marrow. Thus, long-lived plasma cells migrate to the bone marrow but no memory B-cells are found there.
G. Both IgG-Secreting and IgA-Secreting Norwalk VLP-Specific Memory B-Cells are Present in the Mesenteric Lymph Nodes of Intranasally Immunized Rabbits
The mesenteric lymph nodes were obtained from all of the experimental rabbits and the isolated cells were assayed for LLPCs and memory B-cells. The results from this assay are shown in FIG. 18A. As with most of the lymphoid tissue analyzed, except bone marrow, no LLPCs (FIG. 18A left panels) were found in the mesenteric nodes. Following in vitro incubation with Norwalk VLPs, a very high number of IgG-secreting VLP-specific memory B-cells were evident in the mesenteric lymph node population (FIG. 18A, right panel). The numbers of memory B-cells observed in the mesenteric lymph nodes were significantly higher than those observed for the other lymphoid tissues assayed.
Numerous researchers have shown that immunization at a mucosal inductive site, such as the nasal passages or the gut, is capable of eliciting a so-called mucosal immune response. This response has generally been characterized by the presence of IgA+ B-cells and IgA-secreting plasma cells localized in the mucosal lymphoid tissue. For this reason the mesenteric lymph node cells were also assayed for the presence of IgA-secreting LLPCs or memory B-cells. The results from these assays are shown in FIG. 18B. Once again, no IgA+ LLPCs were found in the mesenteric lymph node population (FIG. 18B, left panel). However, IgA-secreting memory B-cells were detected in this tissue (FIG. 18B, right panel). Thus, intranasal immunization with a dry powder Norwalk VLP vaccine formulation elicited a mucosal immune response that resulted in the migration of both IgG+ and IgA+ antigen-specific memory B-cells to the gut-associated lymphoid tissue. The production of antigen-specific memory B cells induced by immunization with the Norwalk vaccine formulation is a possible indicator of vaccine effectiveness. The presence of memory B cells is one marker of long-lasting immunity.
H. VLP-Specific CD4+ Memory T Cells
Splenocytes harvested from immunized rabbits were restimulated with intact Norwalk VLPs and the extent of cellular proliferation was measured by tritiated thymidine incorporation as indicated on the ordinate axis (CPM) (FIG. 19). The left panel shows cellular proliferation of an unfractionated population of splenocytes, while the right panel shows cellular proliferation of CD4+ T cells.
The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings using no more than routine experimentation. Such modifications and equivalents are intended to fall within the scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.
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14796714
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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tyo:4502
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Takeda Pharmaceutical
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Dec 13th, 2016 12:00AM
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Jul 25th, 2014 12:00AM
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https://www.uspto.gov?id=US09518096-20161213
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Virus-like particles comprising composite capsid amino acid sequences for enhanced cross reactivity
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The present invention provides polypeptides having a composite amino acid sequence derived from a consensus sequence representing the capsid proteins of two or more circulating strains of a non-enveloped virus. In particular, the invention provides virus-like particles comprising at least one composite polypeptide. Such virus-like particles have antigenic epitopes of two or more circulating strains of a non-enveloped virus and produce an increase in antisera cross-reactivity to one or more circulating strains of the non-enveloped virus. Methods of making composite virus-like particles and vaccine formulations comprising composite virus-like particles are also disclosed.
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9518096
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1. An isolated polypeptide or fragment thereof having a composite amino acid sequence, wherein said composite amino acid sequence comprises SEQ ID NO: 1 or SEQ ID NO: 22.
2. A vaccine formulation comprising the polypeptide of claim 1.
3. The vaccine formulation of claim 2, further comprising a second polypeptide, wherein said second polypeptide comprises a capsid protein from a Norovirus.
4. The vaccine formulation of claim 3, wherein said Norovirus is a genogroup I or genogroup II Norovirus.
5. The vaccine formulation of claim 2 further comprising an adjuvant.
6. The vaccine formulation of claim 5, wherein the adjuvant is selected from the group consisting of toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, liposomes, oil-in-water emulsion, MF59, and squalene.
7. The vaccine formulation of claim 5, further comprising a delivery agent.
8. The vaccine formulation of claim 7, wherein the delivery agent is a mucoadhesive.
9. The vaccine formulation of claim 8, wherein the mucoadhesive is selected from the group consisting of glycosaminoglycans, carbohydrate polymers, polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides, polyions, cellulose derivatives, proteins, and deoxyribonucleic acid.
10. The vaccine formulation of claim 9, wherein the mucoadhesive is a polysaccharide.
11. The vaccine formulation of claim 10, wherein said polysaccharide is chitosan, chitosan salt, or chitosan base.
12. The vaccine formulation of claim 5, wherein the vaccine formulation is a liquid formulation.
13. The vaccine formulation of claim 5, wherein the vaccine formulation is a dry powder formulation.
14. The dry powder formulation of claim 13 in combination with one or more devices for administering one or more doses of said formulation.
15. The dry powder formulation of claim 14, wherein said one or more doses are unit doses.
16. The dry powder formulation of claim 14, wherein the device is a single-use nasal administration device.
17. The vaccine formulation of claim 2, wherein said formulation is administered to a subject by a route selected from the group consisting of mucosal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, and transdermal routes of administration.
18. The vaccine formulation of claim 17, wherein said mucosal administration is intranasal, oral, or vaginal.
19. The vaccine formulation of claim 18, wherein the formulation is in the form of a nasal spray, nasal drops or dry powder.
20. A method of inducing a protective immunity to a norovirus viral infection in a subject comprising administering to the subject the vaccine formulation of claim 2.
21. The method of claim 20, wherein said vaccine formulation confers protection from one or more symptoms of Norovirus infection.
22. A method of making a norovirus virus-like particle comprising expressing the polypeptide of claim 1 in a host cell; growing the cell in conditions in which the virus like particle is formed; and isolating the virus-like particle.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 13/023,363, filed Feb. 8, 2011, now U.S. Pat. No. 8,841,120, which is a continuation-in-part of International Application No. PCT/US2009/053249, filed Aug. 10, 2009, which claims the benefit of U.S. Provisional Application No. 61/087,504, filed Aug. 8, 2008, and U.S. Provisional Application No. 61/218,603, filed Jun. 19, 2009, all of which are herein incorporated by reference in their entireties.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: LIGO_022_02US_SeqList_ST25.txt, date recorded: Feb. 8, 2011, file size 120 kilobytes).
FIELD OF THE INVENTION
The invention is in the field of vaccines, particularly vaccines comprising virus-like particles with a composite amino acid sequence derived from a consensus sequence representing two or more capsid proteins from non-enveloped viruses. In addition, the invention relates to methods of preparing vaccine compositions and methods of inducing a protective immune response using the vaccine compositions of the invention.
BACKGROUND OF THE INVENTION
The prevalent approach to preparing vaccines for viruses with seasonal or year-to-year patterns is modeled by commercial Influenza vaccines which require the anticipation, publication, and subsequent synthesis of a new vaccine when the virus evolves to present a different antigenic profile. This approach causes significant timeline delays and cost as new antigens are synthesized in anticipation of the next years viral strain. Further, as evidenced by the failings of the 2008 influenza vaccine, errors in the predicted strain can result in significant disease related costs as patients are under-protected. Thus, improved methods for designing and preparing vaccines to protect against multiple circulating strains of disease-causing virus is desirable.
Noroviruses are non-cultivatable human Caliciviruses that have emerged as the single most important cause of epidemic outbreaks of nonbacterial gastroenteritis (Glass et al. (2000) J Infect Dis, Vol. 181 (Sup 2): S254-S261; Hardy et al. (1999) Clin Lab Med, Vol. 19(3): 675-90). These viruses have been grouped into five different genogroups of which genogroups I and II are further subdivided into greater than 25 genotypes and are the agents for the vast majority of illness in humans attributed to this virus. There are significant challenges to the development of vaccines against Norovirus, including the inability to propagate the virus in culture and suitable animal models of acute gastroenteritis. Standard virologic techniques including viral attenuation or in vitro neutralization assays are therefore not possible today.
Noroviruses contain a 7.5 Kb single strand positive sense RNA genome that contains three open reading frames. The major viral capsid protein (VP1) is encoded by ORF2 and expression of this protein results in the spontaneous assembly of virus-like particles (VLPs), which mimic the structure of the virus but are incapable of replication. This structure is composed of 180 monomeric subunits of VP1 and are candidate vaccines to prevent acute gastroenteritis. The VP1 monomer has two domains: a shell (S) domain that forms the inner viral core and a prominent protruding (P) domain linked by a flexible hinge. The P domain is further subdivided into two subdomains P1 and P2, which is the most surface exposed region and is thought to contain important cell recognition and antigenic sites. Homology analysis indicates that the majority of the hypervariable amino acid regions of VP1 are located in the P2 domain (Allen et al. (2008) PLoS One, Vol. 1: 1-9).
Recent epidemiology studies have lead to the hypothesis that Norovirus evolution is epochal with periods of stasis followed by emergence of novel epidemic strains, similar to that observed for Influenza virus. Most recent outbreaks appear to be related to emergence of variant virus in the GII.4 genotype at a persistence interval of around two years. There is a need in the art for a vaccine candidate that provides antigenic epitopes that would be cross protective for multiple Norovirus, or other non-enveloped virus strains, which would obviate the need for construction of vaccines for each contemporary outbreak strain.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery that a polypeptide comprising a composite capsid sequence, which combines epitopes from a number of circulating viral strains, can be used to produce a more robust immunological response to multiple viral strains. Such a polypeptide can be used to prepare vaccine formulations that are protective against several circulating strains of the virus, and therefore improve strain-to-strain and year-to-year protection.
The present invention provides at least one polypeptide having a composite amino acid sequence, wherein said composite amino acid sequence is derived from a consensus sequence representing the capsid proteins of two or more circulating strains of a non-enveloped virus, and wherein the at least one polypeptide forms a virus-like particle when expressed in a host cell and contains at least 1 different amino acid as compared to each of the capsid sequences of said two or more circulating strains. In one embodiment, the virus-like particle comprising the at least one composite polypeptide has antigenic properties of the two or more circulating strains of the non-enveloped virus. In another embodiment, the composite polypeptide or composite virus-like particle provides an increase in antisera cross-reactivity to one or more circulating strains of the non-enveloped virus as compared to the antisera cross-reactivity obtained by immunizing with a virus-like particle containing only protein from said one or more circulating strains.
The virus-like particle may comprise at least one polypeptide having a composite amino acid sequence derived from a consensus sequence representing capsid proteins of two or more circulating strains of a non-enveloped virus, wherein the non-enveloped virus is selected from the group consisting of Calicivirus, Picornavirus, Astrovirus, Adenovirus, Reovirus, Polyomavirus, Papillomavirus, Parvovirus, and Hepatitis E virus. In one embodiment, the non-enveloped virus is a Calicivirus. In another embodiment, the Calicivirus is a Norovirus or Sapovirus. The Norovirus may be a genogroup I or genogroup II Norovirus.
The consensus sequence may be derived from two or more Norovirus strains classified in the same genogroup and genotype. In one embodiment, the consensus sequence is derived from genogroup II, genotype 4 Norovirus strains, such as Houston, Minerva, and Laurens strains. In another embodiment, the consensus sequence is derived from Norovirus strains from at least two different genotypes within a genogroup. In still another embodiment, the consensus sequence is derived from Norovirus strains from at least two different genogroups.
The present invention also encompasses a virus-like particle comprising at least one composite polypeptide derived from two or more circulating Calicivirus strains and a capsid protein from a second non-enveloped virus, such as Norovirus. The capsid protein may be a VP1 and/or VP2 protein from a genogroup I or genogroup II Norovirus. In another embodiment, the virus-like particle comprises at least one composite polypeptide derived from two or more circulating strains of a Calicivirus and a second composite polypeptide derived from two or more circulating strains of a second Calicivirus. Preferably, the virus-like particle has antigenic properties of the two or more circulating strains of the first Calicivirus and the two or more circulating strains of the second Calicivirus.
The present invention also provides an isolated polypeptide or fragment thereof having a composite amino acid sequence, wherein said composite amino acid sequence is derived from a consensus sequence representing the capsid proteins of two or more circulating strains of a non-enveloped virus, and wherein the polypeptide contains at least 1 different amino acid as compared to each of the capsid sequences of said two or more circulating strains. The non-enveloped virus may be a Calicivirus, such as a Sapovirus or Norovirus. Alternatively, the non-enveloped virus may be a Papillomavirus.
The present invention contemplates vaccine formulations comprising one or more composite polypeptides or composite virus-like particles of the invention. Each of the composite virus-like particles comprises at least one polypeptide having a composite amino acid sequence derived from a consensus sequence representing the capsid proteins from two or more circulating strains of a non-enveloped virus. The non-enveloped virus may be a genogroup I or genogroup II Norovirus. In some embodiments, the vaccine formulation further comprises an adjuvant. In other embodiments, the vaccine formulation further comprises a delivery agent. In still other embodiments, the vaccine formulation further comprises a pharmaceutically acceptable carrier. The vaccine formulation may be a liquid formulation or a dry powder formulation.
The invention also provides a method of inducing a protective immunity to a viral infection in a subject comprising administering to the subject a vaccine formulation disclosed herein. In one embodiment, the viral infection is a Norovirus infection. In another embodiment, the vaccine formulation confers protection from one or more symptoms of Norovirus infection.
The present invention also contemplates a method of making a composite virus-like particle. In one embodiment, the method comprises aligning amino acid sequences of capsid proteins from two or more circulating strains of a non-enveloped virus; determining a consensus sequence from said aligned amino acid sequences; preparing a composite sequence based on said consensus sequence that contains at least 1 different amino acid as compared to each of the capsid sequences of said two or more circulating strains; and expressing said composite sequence in a host cell, thereby producing a virus-like particle. The non-enveloped virus may be a Calicivirus, Picornavirus, Astrovirus, Adenovirus, Reovirus, Polyomavirus, Papillomavirus, Parvovirus, and Hepatitis E virus.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Amino acid consensus sequence of VP1 proteins from genogroup II, genotype 4 Norovirus (SEQ ID NO: 2). The consensus sequence was determined from an alignment of Houston, Minerva, and Laurens strains.
FIG. 2. Nucleotide sequence encoding the composite VP1 protein from genogroup II, genotype 4 Norovirus (SEQ ID NO: 3). The amino acid sequence encoded by this nucleotide sequence is provided as SEQ ID NO: 22.
FIG. 3. SDS-PAGE/Coomassie analysis of sucrose gradient purified composite VLPs.
FIG. 4. HPLC SEC chromatogram of readings at 220 nm (top) and 280 nm (bottom) of composite expression cell culture supernatant purified by sucrose gradient.
FIG. 5. SDS-PAGE/Silver-stain analysis of composite sequence VLPs purified by column chromatography.
FIG. 6. HPLC SEC chromatogram of readings at 280 nm of composite VLPs.
FIG. 7. Immunization with composite VLP (CVLP) elicits antigen-specific IgG. Groups of 7 mice were immunized (i.p.) with various concentrations of CVLP (indicated on the X axis) on days 0 and 7. Serum was collected on day 14 and CVLP-specific IgG was measured by ELISA. Horizontal lines indicate geometric means of each treatment group.
FIG. 8. Immunization with composite VLP/Norwalk VLP (CVLP/NVLP) combination elicits NVLP-specific IgG. Groups of 7 mice were immunized (i.p.) with various concentrations of NVLP alone (purple bars) or in combination with equal amounts of CVLP (black bars) on days 0 and 14. Serum was collected on day 21 and NVLP-specific IgG was measured by ELISA. Data is reported as the mean+standard error of the mean (SEM).
FIG. 9. Immunization with composite VLP/Norwalk VLP (CVLP/NVLP) combination elicits CVLP-specific IgG. Groups of 7 mice were immunized (i.p.) with various concentrations of either composite VLP alone (green bars) or in combination with equal amounts of NVLP (black bars) on days 0 and 14. Serum was collected on day 21 and CVLP-specific IgG was measured by ELISA. Data is reported as the mean+standard error of the mean (SEM).
FIG. 10. CVLP-specific IgG cross-reacts with other Norovirus isolates. Antibody titers measured 21 days after a single immunization with the either Composite VLPs or GII.4 2002 VLPs show that Composite VLPs elicit ˜10 fold higher titers as compared to the GII.4 2002 VLPs. Antibody titers for animals immunized with all GII.4 VLPs show poor cross reactivity to GI.1 VLPs. Data are expressed as geometric mean+standard error of the mean (SEM).
FIG. 11. Rabbits were immunized IM on day 0 and 21 with equal amounts of Norwalk VLP (NVLP) and composite VLP (CVLP). Serum was collected on day 28 and VLP-specific IgG was evaluated. The resulting data was log transformed and evaluated by linear regression analysis. IgG titers are expressed as reciprocal dilutions and shown as geometric mean titers.
FIG. 12. Rabbits were immunized IM on day 0 and 21 with equal amounts of Norwalk VLP (NVLP) and composite VLP (CVLP). Spleens were collected on day 75 and unfractionated cells were stimulated in culture for 5 days with either NVLP or CVLP and the amount of thymidine incorporation was measured. The mean and SD are shown for each rabbit in the treatment groups indicated on the X axis. Data are expressed as mean+SD.
FIG. 13. Rabbits were immunized IM on day 0 and 21 with equal amounts of Norwalk VLP (NVLP) and composite VLP (CVLP). Spleens and mesenteric lymph nodes (LN) were collected on day 75 and analyzed for the presence of VLP-specific memory B-cells by ELISPOT. Individual responses are shown for NVLP and CVLP. Data are represented as the number of VLP-specific IgG secreting cells per million cells present.
FIG. 14. Rabbits were immunized IM on days 0, 14, and 21 with equal amounts of Norwalk VLP (NVLP) and composite VLP (CVLP) as indicated in the legend. Serum was collected on day 21 and 35 and NVLP-specific IgG and IgA was measured by ELISA. Results are displayed as geometric group means+SEM.
FIG. 15. Rabbits were immunized IM on days 0, 14, and 21 with equal amounts of Norwalk VLP (NVLP) and composite VLP (CVLP) as indicated in the legend. Serum was collected on day 21 and 35 and CVLP-specific IgG and IgA was measured by ELISA. Results are displayed as geometric group means+SEM.
FIG. 16. Rabbits were immunized IM on days 0, 14, and 21 with equal amounts of Norwalk VLP (NVLP) and composite VLP (CVLP). Spleens were collected on day 35 and unfractionated cells were stimulated in vitro for 5 days. Splenocytes were stimulated with various VLPs from the two genogroups as indicated in the graph legend. Results are displayed as geometric group means+SD.
FIG. 17. Mice were immunized IP on days 0 and 7 with equal amounts of Norwalk VLP (NVLP) and composite VLP (CVLP) as indicated on the X axis. Serum was collected on day 14 and analyzed for the presence of VLP-specific IgG by ELISA. Individual responses are shown and titers are expressed as reciprocal dilutions. Horizontal bars represent geometric group means.
FIG. 18. Mice were immunized IP on days 0 and 7 with equal amounts of Norwalk VLP (NVLP) and composite VLP (CVLP) as indicated on the X axis. Serum was collected on day 14 and analyzed for the presence of antibodies capable of inhibiting hemagglutination of human red blood cells (type 0 positive). Individual responses are shown and titers are expressed as reciprocal dilutions. Horizontal bars represent geometric group means.
FIG. 19. Serum anti-VLP IgG in rabbits intranasally immunized on days 0 and 21 with 50 μg of VLP vaccine formulation (Norwalk VLPs+composite GII.4 VLPs). Individual responses are shown and expressed in μg/mL from serum collected on day 35. Bars indicate the geometric group means.
FIG. 20. Amino acid consensus sequence of VP1 proteins from genogroup II Norovirus (SEQ ID NO: 7). The consensus sequence was determined from an alignment of GII.1 (Accession Number: AAL13001), GII.2 Snow Mountain (Accession Number: AAB61685), and GII.3 (Accession Number: AAL12998) strains. The “x” indicates positions in which the amino acid differed among all three strains.
FIG. 21. Amino acid consensus sequence of VP1 proteins from genogroup I Norovirus (SEQ ID NO: 12). The consensus sequence was determined from an alignment of Norwalk virus (Accession Number: M87661), Southampton (Accession Number: Q04542), and Chiba virus (Accession Number: BAB18267) strains. The “x” indicates positions in which the amino acid differed among all three strains.
FIG. 22. Amino acid consensus sequence of L1 proteins from Human Papillomavirus (SEQ ID NO: 17). The consensus sequence was determined from an alignment of HPV-11, HPV-16, and HPV-18 viral strains. The “x” indicates positions in which the amino acid differed among all three strains.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides vaccine formulations comprising a polypeptide having a composite amino acid sequence, wherein the composite amino acid sequence is derived from capsid sequences of circulating strains of non-enveloped virus. Virus-like particles produced from such polypeptide sequences provide antigenic epitopes for several viral strains and can be used to induce an immune response that is protective against viral infection from multiple strains. Accordingly, the present invention provides a virus-like particle comprising at least one polypeptide having a composite amino acid sequence. A “composite amino acid sequence” or “composite sequence”, as used herein, is a sequence derived from a consensus sequence of at least two viral protein sequences. In one embodiment, the viral protein sequences are capsid sequences. A composite amino acid sequence may be derived from a consensus sequence by selecting one of two or more amino acids at the variable positions in the consensus sequence.
As used herein, a “consensus sequence” is a sequence containing one or more variable amino acids, and is determined by aligning and comparing the viral protein sequences of two or more viruses. A consensus sequence may also be determined by aligning and comparing the nucleotide sequences of two or more viruses. The consensus sequence may be determined from protein or nucleotide sequences of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more circulating strains of a non-enveloped virus.
The polypeptide having a composite amino acid sequence may contain at least one different, at least two different, at least three different, at least four different, at least five different, at least six different, at least seven different, at least eight different, at least nine different, at least ten different, at least fifteen different, at least twenty different, at least twenty-five different, at least thirty different, at least thirty-five different, at least forty different, at least forty-five different, or at least fifty different amino acids as compared to each of the protein sequences of the two or more circulating strains used to determine the consensus sequence. In some embodiments, the polypeptide having a composite amino acid sequence may form a virus-like particle when expressed in a host cell.
In one embodiment of the invention, the virus-like particle (VLP) comprises at least one polypeptide having a composite amino acid sequence, wherein said composite amino acid sequence is derived from a consensus sequence representing the capsid proteins of two or more circulating strains of a non-enveloped virus, and wherein the at least one polypeptide forms a virus-like particle when expressed in a host cell and contains at least 1 different amino acid as compared to each of the capsid sequences of said two or more circulating strains. Preferably, the virus-like particle has antigenic properties of the two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more circulating strains of a non-enveloped virus. In some embodiments, the virus-like particle provides an increase in antisera cross-reactivity to one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more circulating strains of the non-enveloped virus as compared to the antisera cross-reactivity obtained by immunizing with a virus-like particle containing only protein from one or more circulating strains. In one embodiment, the virus-like particle provides at least a two-fold increase in antisera cross-reactivity.
In another embodiment, the virus-like particle comprises at least one polypeptide having a composite amino acid sequence derived from a consensus sequence representing the capsid proteins of two or more circulating strains of a non-enveloped virus, wherein the non-enveloped virus is selected from the group consisting of Calicivirus, Picornavirus, Astrovirus, Adenovirus, Reovirus, Polyomavirus, Papillomavirus, Parvovirus, and Hepatitis E virus. The invention also includes strains of non-enveloped viruses that have not yet been characterized or discovered at the time of filing. In some embodiments, among others, the non-enveloped virus is a Calicivirus. Caliciviruses are divided into four genera: Norovirus and Sapovirus, which cause infection in humans, and Lagovirus and Vesivirus, which are associated with veterinary infections. In preferred embodiments, the Calicivirus is a Sapovirus or Norovirus.
The Norovirus genus is split primarily into two major genogroups (GI and GII). Two other genogroups (GIII and GIV) are proposed, but generally accepted. Representative of GIII is the bovine, Jena strain. GIV contains one virus, Alphatron, at this time. The GI and GII groups may be further segregated into clusters or genotypes based on genetic classification (Ando et al. (2000) J. Infectious Diseases, Vol. 181(Supp2):S336-S348; Lindell et al. (2005) J. Clin. Microbiol., Vol. 43(3): 1086-1092). As used herein, the term genetic clusters is used interchangeably with the term genotypes. Within genogroup I, there are 6 GI clusters (with prototype virus strain name): GI.1 (Norwalk); GI.2 (Southhampton); GI.3 (Desert Shield); GI.4 (Cruise Ship virus/Chiba); GI.5 (318/Musgrove); and GI.6 (Hesse). Within genogroup II, there are 9 GII clusters (with prototype virus strain name): GII.1 (Hawaii); GII.2 (Snow Mountain/Melksham); GII.3 (Toronto); GII.4 (Bristol/Lordsdale); GII.5 (290/Hillingdon); GII.6 (269/Seacroft); GII.7 (273/Leeds); GII.8 (539/Amsterdam); and GII.9 (378). The circulating Norovirus strains are classified through comparison to prototype strains belonging to these genetic clusters. The most prevalent circulating strains belong to genogroup II.
Nucleic acid and protein sequences for a number of Norovirus isolates are known. Additional representative, non-limiting sequences, including sequences of ORF1, ORF2, ORF3, and their encoded polypeptides from Norovirus isolates are listed in the National Center for Biotechnology Information (NCBI) database. In one embodiment of the invention, the Norovirus may be a genogroup I or genogroup II Norovirus. Composite and consensus amino acid sequences may be determined from any of the known Norovirus strains. See, for example, GenBank entries: Norovirus genogroup 1 strain Hu/NoV/West Chester/2001/USA, GenBank Accession No. AY502016; Norovirus genogroup 2 strain Hu/NoV/Braddock Heights/1999/USA, GenBank Accession No. AY502015; Norovirus genogroup 2 strain Hu/NoV/Fayette/1999/USA, GenBank Accession No. AY502014; Norovirus genogroup 2 strain Hu/NoV/Fairfield/1999/USA, GenBank Accession No. AY502013; Norovirus genogroup 2 strain Hu/NoV/Sandusky/1999/USA, GenBank Accession No. AY502012; Norovirus genogroup 2 strain Hu/NoV/Canton/1999/USA, GenBank Accession No. AY502011; Norovirus genogroup 2 strain Hu/NoV/Tiffin/1999/USA, GenBank Accession No. AY502010; Norovirus genogroup 2 strain Hu/NoV/CS-E1/2002/USA, GenBank Accession No. AY50200; Norovirus genogroup 1 strain Hu/NoV/Wisconsin/2001/USA, GenBank Accession No. AY502008; Norovirus genogroup 1 strain Hu/NoV/CS-841/2001/USA, GenBank Accession No. AY502007; Norovirus genogroup 2 strain Hu/NoV/Hiram/2000/USA, GenBank Accession No. AY502006; Norovirus genogroup 2 strain Hu/NoV/Tontogany/1999/USA, GenBank Accession No. AY502005; Norwalk virus, complete genome, GenBank Accession No. NC.sub.--001959; Norovirus Hu/GI/Otofuke/1979/JP genomic RNA, complete genome, GenBank Accession No. AB187514; Norovirus Hu/Hokkaido/133/2003/JP, GenBank Accession No. AB212306; Norovirus Sydney 2212, GenBank Accession No. AY588132; Norwalk virus strain SN2000JA, GenBank Accession No. AB190457; Lordsdale virus complete genome, GenBank Accession No. X86557; Norwalk-like virus genomic RNA, Gifu′96, GenBank Accession No. AB045603; Norwalk virus strain Vietnam 026, complete genome, GenBank Accession No. AF504671; Norovirus Hu/GII.4/2004/N/L, GenBank Accession No. AY883096; Norovirus Hu/GII/Hokushin/03/JP, GenBank Accession No. AB195227; Norovirus Hu/GII/Kamo/03/JP, GenBank Accession No. AB195228; Norovirus Hu/GII/Sinsiro/97/JP, GenBank Accession No. AB195226; Norovirus Hu/GII/Ina/02/JP, GenBank Accession No. AB195225; Norovirus Hu/NLV/GII/Neustrelitz260/2000/DE, GenBank Accession No. AY772730; Norovirus Hu/NLV/Dresdenl74/pUS-NorII/1997/GE, GenBank Accession No. AY741811; Norovirus Hu/NLV/Oxford/B2S16/2002/UK, GenBank Accession No. AY587989; Norovirus Hu/NLV/Oxford/B4S7/2002/UK, GenBank Accession No. AY587987; Norovirus Hu/NLV/Witney/B7S2/2003/UK, GenBank Accession No. AY588030; Norovirus Hu/NLV/Banbury/B9S23/2003/UK, GenBank Accession No. AY588029; Norovirus Hu/NLV/ChippingNorton/2003/UK, GenBank Accession No. AY588028; Norovirus Hu/NLV/Didcot/B9S2/2003/UK, GenBank Accession No. AY588027; Norovirus Hu/NLV/Oxford/B8S5/2002/UK, GenBank Accession No. AY588026; Norovirus Hu/NLV/Oxford/B6S4/2003/UK, GenBank Accession No. AY588025; Norovirus Hu/NLV/Oxford/B6S5/2003/UK, GenBank Accession No. AY588024; Norovirus Hu/NLV/Oxford/B5S23/2003/UK, GenBank Accession No. AY588023; Norovirus Hu/NLV/Oxford/B6S2/2003/UK, GenBank Accession No. AY588022; Norovirus Hu/NLV/Oxford/B6S6/2003/UK, GenBank Accession No. AY588021; Norwalk-like virus isolate Bo/Thirskl0/00/UK, GenBank Accession No. AY126468; Norwalk-like virus isolate Bo/Penrith55/00/UK, GenBank Accession No. AY126476; Norwalk-like virus isolate Bo/Aberystwyth24/00/UK, GenBank Accession No. AY126475; Norwalk-like virus isolate Bo/Dumfries/94/UK, GenBank Accession No. AY126474; Norovirus NLV/IF2036/2003/Iraq, GenBank Accession No. AY675555; Norovirus NLV/IF1998/2003/Iraq, GenBank Accession No. AY675554; Norovirus NLV/BUDS/2002/USA, GenBank Accession No. AY660568; Norovirus NLV/Paris Island/2003/USA, GenBank Accession No. AY652979; Snow Mountain virus, complete genome, GenBank Accession No. AY134748; Norwalk-like virus NLV/Fort Lauderdale/560/1998/US, GenBank Accession No. AF414426; Hu/Norovirus/hiroshima/1999/JP(9912-02F), GenBank Accession No. AB044366; Norwalk-like virus strain 11MSU-MW, GenBank Accession No. AY274820; Norwalk-like virus strain B-1SVD, GenBank Accession No. AY274819; Norovirus genogroup 2 strain Hu/NoV/Farmington Hills/2002/USA, GenBank Accession No. AY502023; Norovirus genogroup 2 strain Hu/NoV/CS-G4/2002/USA, GenBank Accession No. AY502022; Norovirus genogroup 2 strain Hu/NoV/CS-G2/2002/USA, GenBank Accession No. AY502021; Norovirus genogroup 2 strain Hu/NoV/CS-G12002/USA, GenBank Accession No. AY502020; Norovirus genogroup 2 strain Hu/NoV/Anchorage/2002/USA, GenBank Accession No. AY502019; Norovirus genogroup 2 strain Hu/NoV/CS-D1/2002/CAN, GenBank Accession No. AY502018; Norovirus genogroup 2 strain Hu/NoV/Germanton/2002/USA, GenBank Accession No. AY502017; Human calicivirus NLV/GII/Langen1061/2002/DE, complete genome, GenBank Accession No. AY485642; Murine norovirus 1 polyprotein, GenBank Accession No. AY228235; Norwalk virus, GenBank Accession No. AB067536; Human calicivirus NLV/Mex7076/1999, GenBank Accession No. AF542090; Human calicivirus NLV/Oberhausen 455/01/DE, GenBank Accession No. AF539440; Human calicivirus NLV/Herzberg 385/01/DE, GenBank Accession No. AF539439; Human calicivirus NLV/Boxer/2001/US, GenBank Accession No. AF538679; Norwalk-like virus genomic RNA, complete genome, GenBank Accession No. AB081723; Norwalk-like virus genomic RNA, complete genome, isolate:Saitama U201, GenBank Accession No. AB039782; Norwalk-like virus genomic RNA, complete genome, isolate:Saitama U18, GenBank Accession No. AB039781; Norwalk-like virus genomic RNA, complete genome, isolate:Saitama U25, GenBank Accession No. AB039780; Norwalk virus strain:U25GII, GenBank Accession No. AB067543; Norwalk virus strain:U201 GII, GenBank Accession No. AB067542; Norwalk-like viruses strain 416/97003156/1996/LA, GenBank Accession No. AF080559; Norwalk-like viruses strain 408/97003012/1996/FL, GenBank Accession No. AF080558; Norwalk-like virus NLV/Burwash Landing/331/1995/US, GenBank Accession No. AF414425; Norwalk-like virus NLV/Miami Beach/326/1995/US, GenBank Accession No. AF414424; Norwalk-like virus NLV/White River/290/1994/US, GenBank Accession No. AF414423; Norwalk-like virus NLV/New Orleans/306/1994/US, GenBank Accession No. AF414422; Norwalk-like virus NLV/Port Canaveral/301/1994/US, GenBank Accession No. AF414421; Norwalk-like virus NLV/Honolulu/314/1994/US, GenBank Accession No. AF414420; Norwalk-like virus NLV/Richmond/283/1994/US, GenBank Accession No. AF414419; Norwalk-like virus NLV/Westover/302/1994/US, GenBank Accession No. AF414418; Norwalk-like virus NLV/UK3-17/12700/1992/GB, GenBank Accession No. AF414417; Norwalk-like virus NLV/Miami/81/1986/US, GenBank Accession No. AF414416; Snow Mountain strain, GenBank Accession No. U70059; Desert Shield virus DSV395, GenBank Accession No. U04469; Norwalk virus, complete genome, GenBank Accession No. AF093797; Hawaii calicivirus, GenBank Accession No. U07611; Southampton virus, GenBank Accession No. L07418; Norwalk virus (SRSV-KY-89/89/J), GenBank Accession No. L23828; Norwalk virus (SRSV-SMA/76/US), GenBank Accession No. L23831; Camberwell virus, GenBank Accession No. U46500; Human calicivirus strain Melksham, GenBank Accession No. X81879; Human calicivirus strain MX, GenBank Accession No. U22498; Minireovirus TV24, GenBank Accession No. UO2030; and Norwalk-like virus NLV/G nedd/273/1994/US, GenBank Accession No. AF414409; sequences of all of which (as entered by the date of filing of this application) are herein incorporated by reference. Additional Norovirus sequences are disclosed in the following patent publications: WO 2005/030806, WO 2000/79280, JP2002020399, US2003129588, U.S. Pat. No. 6,572,862, WO 1994/05700, and WO 05/032457, all of which are herein incorporated by reference in their entireties. See also Green et al. (2000) J. Infect. Dis., Vol. 181(Suppl. 2):5322-330; Wang et al. (1994) J. Virol., Vol. 68:5982-5990; Chen et al. (2004) J. Virol., Vol. 78: 6469-6479; Chakravarty et al. (2005) J. Virol., Vol. 79: 554-568; Hansman et al. (2006) J. Gen. Virol., Vol. 87:909-919; Bull et al. (2006) J. Clin. Micro., Vol. 44(2):327-333; Siebenga, et al. (2007) J. Virol., Vol. 81(18):9932-9941, and Fankhauser et al. (1998) J. Infect. Dis., Vol. 178:1571-1578; for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Noroviruses.
Nucleic acid and protein sequences for a number of Sapovirus isolates are also known. Representative Sapovirus sequences, including sequences of ORF1 and ORF2, and their encoded polypeptides from Sapovirus isolates are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, GenBank entries: Sapovirus Mc10, GenBank Accession No. NC.sub.--010624; Sapporo virus, GenBank Accession No. U65427; Sapovirus Mc10, GenBank Accession No. AY237420; Sapovirus SaKaeo-15/Thailand, GenBank Accession No. AY646855; Sapporo virus, GenBank Accession No. NC.sub.--006269; Sapovirus C12, GenBank Accession No. NC.sub.--006554; Sapovirus C12, GenBank Accession No. AY603425; Sapovirus Hu/Dresden/pJG-Sap01/DE, GenBank Accession No. AY694184; Human calicivirus SLV/cruise ship/2000/USA, GenBank Accession No. AY289804; Human calicivirus SLV/Arg39, GenBank Accession No. AY289803; Porcine enteric calicivirus strain LL14, GenBank Accession No. AY425671; Porcine enteric calicivirus, GenBank Accession No. NC.sub.--000940; Human calicivirus strain Mc37, GenBank Accession No. AY237415; Mink enteric calicivirus strain Canada 151A, GenBank Accession No. AY144337; Human calicivirus SLV/Hou7-1181, GenBank Accession No. AF435814; Human calicivirus SLV/Mex14917/2000, GenBank Accession No. AF435813; Human calicivirus SLV/Mex340/1990, GenBank Accession No. AF435812; Porcine enteric calicivirus, GenBank Accession No. AF182760; Sapporo virus-London/29845, GenBank Accession No. U95645; Sapporo virus-Manchester, GenBank Accession No. X86560; Sapporo virus-Houston/86, GenBank Accession No. U95643; Sapporo virus-Houston/90, GenBank Accession No. U95644; and Human calicivirus strain HuCV/Potsdam/2000/DEU, GenBank Accession No. AF294739; sequences of all of which (as entered by the date of filing of this application) are herein incorporated by reference. See also Schuffenecker et al. (2001) Arch Virol., Vol. 146(11):2115-2132; Zintz et al. (2005) Infect. Genet. Evol., Vol. 5:281-290; Farkas et al. (2004) Arch. Virol., Vol. 149:1309-1323; for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Sapoviruses.
The composite and consensus amino acid sequences may be derived from capsid sequences of at least two Norovirus genogroup I or genogroup II strains. In one embodiment, the VLP comprises a polypeptide having a composite sequence derived from a consensus sequence of the capsid proteins from two or more genogroup II, genotype 4 Norovirus strains. Non-limiting examples of genogroup II, genotype 4 Norovirus strains include Houston strain, Minerva strain, Laurens strain, Bristol strain, Lordsdale strain, Farmington Hills strain, Hunter strain, Carlow strain, and the US95/96-US, 2006a, and 2006b strains.
In another embodiment of the invention, the virus-like particle is comprised of at least one composite polypeptide wherein the sequence of the composite polypeptide is derived from the VP1 sequences of Houston, Minerva, and Laurens. In another embodiment, the composite sequence comprises or consists of SEQ ID NO: 1 or SEQ ID NO: 22. In still another embodiment, composite sequences based on Houston, Minerva, and Laurens may be derived from the consensus sequence defined by SEQ ID NO: 2.
In some embodiments, the consensus sequence may be determined from Norovirus strains from at least two different genotypes or at least two different genogroups. In one embodiment of the present invention the virus-like particle is comprised of at least one polypeptide having a composite amino acid sequence, wherein the composite amino acid sequence is derived from a consensus sequence of capsid proteins of Norovirus strains from at least two different genotypes within a genogroup. By way of example, the consensus sequence may be derived from the capsid sequences of genogroup II, genotype 2 and genogroup II, genotype 4 Norovirus strains. In another embodiment, the consensus sequence may be derived from the capsid sequences of three or more genotypes within a genogroup.
In other embodiments, the consensus sequence may be determined from Norovirus strains from at least two different genogroups. One such embodiment, among others, would be a VLP comprising a polypeptide having a composite amino acid sequence, wherein said composite amino acid sequence is derived from a consensus sequence of capsid proteins of genogroup I, genotype 1 and genogroup II, genotype 4 Norovirus strains.
The present invention also provides a virus-like particle (VLP) comprising a composite polypeptide derived from a consensus sequence of capsid proteins from two or more circulating strains of Norovirus and a capsid protein from a second Norovirus. The second Norovirus may be a genogroup I or genogroup II Norovirus. The capsid protein from the second Norovirus can be the major capsid protein, VP1, which is encoded by ORF 2, or the minor capsid protein, VP2, which is encoded by ORF 3, or combinations of VP1 and VP2. In one embodiment, the capsid protein from the second Norovirus is a VP1 protein from a genogroup I Norovirus.
In another embodiment, the invention provides a VLP comprising a composite polypeptide derived from a consensus sequence representing the capsid proteins of two or more circulating strains of Calicivirus and a second polypeptide having a second composite amino acid sequence, wherein said second composite amino acid sequence is derived from a consensus sequence representing the capsid proteins of two or more circulating strains of a second Calicivirus. Preferably, the virus-like particle has antigenic properties of the two or more circulating strains of the first Calicivirus and the two or more circulating strains of the second Calicivirus.
The second polypeptide contains at least one different, at least three different, at least five different, at least ten different, at least fifteen different, at least twenty different, at least twenty-five different, at least thirty different, at least thirty-five different, at least forty different, at least forty-five different, or at least fifty different amino acids as compared to each of the capsid sequences of said two or more circulating strains of the second Calicivirus. In some embodiments, the second polypeptide forms a virus-like particle when expressed in a host cell. In another embodiment, the second Calicivirus is a Norovirus. In another embodiment, the Norovirus is a genogroup I Norovirus. The genogroup I Norovirus may be any of the genogroup I strains disclosed herein. In one embodiment, the genogroup I Norovirus is selected from the group consisting of Norwalk virus, Southampton virus, Hesse virus, and Chiba virus.
The present invention also encompasses isolated polypeptides or fragments thereof having the composite amino acid sequences defined here in, as well as nucleic acids or vectors encoding the same. In one embodiment, the isolated polypeptide or fragment thereof has a composite amino acid sequence, wherein said composite amino acid sequence is derived from a consensus sequence representing the capsid proteins of two or more circulating strains of a non-enveloped virus, and wherein the polypeptide contains at least 1 different amino acid as compared to each of the capsid sequences of said two or more circulating strains. In another embodiment, the composite sequence contains at least 3 different amino acids compared to the capsid sequence of one or more circulating strains of the non-enveloped virus. In another embodiment, the composite sequence contains 5-50 different amino acids compared to the capsid sequence of one or more circulating strains of the non-enveloped virus. In still another embodiment, the consensus sequence is SEQ ID NO: 2.
The composite polypeptide may have a sequence derived from two or more circulating strains of any non-enveloped virus disclosed herein. In one embodiment, the non-enveloped virus is a Calicivirus. In another embodiment, the Calicivirus is a Norovirus or Sapovirus. In another embodiment, the Norovirus is a genogroup I or genogroup II Norovirus, or combinations thereof. In yet another embodiment, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 22.
In one embodiment, the present invention provides an isolated nucleic acid encoding the polypeptide having a composite amino acid sequence, wherein said composite amino acid sequence is derived from a consensus sequence representing the capsid proteins of two or more circulating strains of a non-enveloped virus, and wherein the polypeptide contains at least 1 different amino acid as compared to each of the capsid sequences of said two or more circulating strains. In another embodiment, the nucleic acid has the sequence of SEQ ID NO: 3. In another embodiment, the invention provides a vector comprising an isolated nucleic acid encoding a composite polypeptide. In yet another embodiment, the invention provides a host cell comprising a vector encoding a composite polypeptide.
The antigenic molecules of the present invention (e.g. VLPs, polypeptides, and fragments thereof) can be prepared by isolation and purification from the organisms in which they occur naturally, or they may be prepared by recombinant techniques. Once coding sequences for the desired particle-forming polypeptides have been isolated or synthesized, they can be cloned into any suitable vector or replicon for expression. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is within the skill of an ordinary artisan. The vector is then used to transform an appropriate host cell. Suitable recombinant expression systems include, but are not limited to, bacterial (e.g. E. coli, Bacillus subtilis, and Streptococcus), baculovirus/insect, vaccinia, Semliki Forest virus (SFV), Alphaviruses (such as, Sindbis, Venezuelan Equine Encephalitis (VEE)), mammalian (e.g. Chinese hamster ovary (CHO) cells, HEK-293 cells, HeLa cells, baby hamster kidney (BHK) cells, mouse myeloma (SB20), and monkey kidney cells (COS)), yeast (e.g. S. cerevisiae, S. pombe, Pichia pastori and other Pichia expression systems), plant, and Xenopus expression systems, as well as others known in the art. Particularly preferred expression systems are mammalian cell lines, bacteria, insect cells, and yeast expression systems.
Each of the aforementioned antigens (e.g. VLPs, polypeptides, or fragments thereof) is preferably used in the substantially pure state. Depending on the expression system and host selected, VLPs are produced by growing host cells transformed by an expression vector under conditions whereby the particle-forming polypeptide is expressed and VLPs can be formed. The selection of the appropriate growth conditions is within the skill of the art.
Preferably the VLP antigens are prepared from insect cells such as Sf9, High Five, TniPro, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni. The procedures for producing VLPs in insect cell culture is well known in the art (see, for example, U.S. Pat. No. 6,942,865, which is incorporated herein by reference in its entirety). Briefly, the recombinant baculoviruses carrying the composite capsid sequence are constructed from the sythetic cDNAs. The recombinant baculovirus are then used to infect insect cell cultures (e.g. Sf9, High Five and TniPro cells) and composite VLPs can be isolated from the cell culture. A “composite VLP” is a VLP comprising at least one polypeptide having a composite amino acid sequence derived from a consensus sequence representing the capsid proteins of two or more circulating strains of a non-enveloped virus.
If the VLPs are formed intracellularly, the cells are then disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the VLPs substantially intact. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (E. L. V. Harris and S. Angal, Eds., 1990).
The particles are then isolated (or substantially purified) using methods that preserve the integrity thereof, such as, by density gradient centrifugation, e.g., sucrose gradients, PEG-precipitation, pelleting, and the like (see, e.g., Kirnbauer et al. J. Virol. (1993) 67:6929-6936), as well as standard purification techniques including, e.g., ion exchange and gel filtration chromatography.
General texts which describe molecular biological techniques, which are applicable to the present invention, such as cloning, mutation, and the like, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (“Ausubel”). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, e.g., the cloning and expression of capsid proteins of non-enveloped viruses, such as Calicivirus.
In some embodiments, the antigenic molecules of the present invention (e.g. VLPs, polypeptides, and fragments thereof) are produced in vivo by administration of a vector comprising an isolated nucleic acid encoding a composite polypeptide. Suitable vectors include, but are not limited to, viral vectors, such as Vesicular Stomatitis Virus (VSV) vector, Equine Encephalitis Virus (EEV) vector, Poxvirus vector, Adenovirus vector, Adeno-Associated Virus (AAV), retrovirus vector, and expression plasmids, such as pFastBacl, pWINEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors will be readily apparent to the skilled artisan.
The present invention also encompasses a vaccine formulation comprising the VLPs, polypeptides, or nucleic acids described herein. In one embodiment, the vaccine formulation comprises a composite VLP and a second virus-like particle, wherein said second virus-like particle comprises a capsid protein from a Norovirus. The second VLP may comprise a native capsid protein from a genogroup I or genogroup II Norovirus. The second VLP may comprise a full length Norovirus capsid protein such as VP1 and/or VP2 protein or certain VP1 or VP2 derivatives. Alternatively, the second VLP comprises a truncated capsid protein, such as a truncated VP1 protein. The truncation may be an N- or C-terminal truncation. Truncated capsid proteins are suitably functional capsid protein derivatives. Functional capsid protein derivatives are capable of raising an immune response in the same way as the immune response is raised by a VLP consisting of the full length capsid protein. Vaccine formulations comprising mixtures of VLPs are described in WO 2008/042789, which is herein incorporated by reference in its entirety. Purely by way of example the vaccine formulation can contain VLPs from one or more strains of Norovirus genogroup I together with VLPs comprising a composite protein from one or more strains of Norovirus genogroup II. Preferably, the Norovirus VLP mixture is composed of the strains of Norwalk and genogroup II, genotype 4 Noroviruses. In another embodiment, the vaccine formulation comprises a composite VLP and a Norwalk VLP, wherein the composite VLP comprises a polypeptide having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 22. In still another embodiment, the vaccine formulation comprises a first composite VLP and a second composite VLP, wherein said first and second composite VLPs comprise at least one polypeptide derived from different consensus sequences. For instance, a first composite VLP comprises a composite protein from one or more strains of Norovirus genogroup I and a second composite VLP comprises a composite protein from one or more strains of Norovirus genogroup II. In one embodiment, the first composite VLP comprises a composite protein from one or more strains of Norovirus genogroup I, genotype 1 (GI.1) and a second composite VLP comprises a composite protein from one or more strains of Norovirus genogroup II, genotype 4 (GII.4).
In some embodiments, the vaccine formulation further comprises an adjuvant. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as Bordatella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Pifco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); mineral salts, including aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate and salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; and Quil A.
Suitable adjuvants also include, but are not limited to, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, liposomes, oil-in-water emulsion, MF59, and squalene. In some embodiments, the adjuvants are bacterially-derived exotoxins. In other embodiments, adjuvants which stimulate a Thl type response, such as 3DMPL or QS21, may be used. In certain embodiments, the adjuvant is a combination of MPL and aluminum hydroxide.
In some embodiments, the adjuvant is monophosphoryl lipid A (MPL). MPL is a non-toxic derivative of lipid A from Salmonella, is a potent TLR-4 agonist that has been developed as a vaccine adjuvant (Evans et al. (2003) Expert Rev Vaccines, Vol. 2: 219-229). In pre-clinical murine studies intranasal MPL has been shown to enhance secretory, as well as systemic, humoral responses (Baldridge et al. (2000) Vaccine, Vol. 18: 2416-2425; Yang et al. (2002) Infect Immun., Vol. 70: 3557-3565). It has also been proven to be safe and effective as a vaccine adjuvant in clinical studies of greater than 120,000 patients (Baldrick et al. (2002) Regul Toxicol Pharmacol, Vol. 35: 398-413). MPL stimulates the induction of innate immunity through the TLR-4 receptor and is thus capable of eliciting nonspecific immune responses against a wide range of infectious pathogens, including both gram negative and gram positive bacteria, viruses, and parasites (Persing et al. (2002) Trends Microbiol, Vol. 10: S32-37). Inclusion of MPL in intranasal formulations should provide rapid induction of innate responses, eliciting nonspecific immune responses from viral challenge while enhancing the specific responses generated by the antigenic components of the vaccine. In some embodiments, MPL can be combined with one or more additional adjuvants. For instance, MPL can be combined with aluminum hydroxide to create a suitable adjuvant for intramuscular administration of a vaccine formulation.
In other embodiments, the adjuvant is a naturally occurring oil, such as squalene. Squalene is a triterpenoid hydrocarbon oil (C30H50) produced by plants and is present in many foods. Squalene is also produced abundantly by human beings, for whom it serves as a precursor of cholesterol and steroid hormones. It is synthesized in the liver and the skin, transported in the blood by very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL), and secreted in large amounts by sebaceous glands.
Since it is a natural component of the human body and is biodegradable, squalene has been used as a component of vaccine adjuvants. One of these squalene adjuvants is MF59, an oil-in-water emulsion developed by Chiron. MF59 has been shown in various preclinical and clinical studies to significantly enhance the immune response to a wide variety of vaccine antigens. MF59 is a part of an influenza subunit vaccine, which has been licensed in various European countries since 1997. More than 20 million doses of this vaccine have been given, and it has been shown to have an excellent safety profile. The safety of vaccines with the MF59 adjuvant has also been shown by various investigational clinical studies using recombinant antigens from hepatitis B virus, hepatitis C virus, cytomegalovirus, herpes simplex virus, human immunodeficiency virus, uropathogenic Escherichia coli, etc., in various age groups, including 1- to 3-day-old newborns.
The term “effective adjuvant amount” or “effective amount of adjuvant” will be well understood by those skilled in the art, and includes an amount of one or more adjuvants which is capable of stimulating the immune response to an administered antigen, i.e., an amount that increases the immune response of an administered antigen composition, as measured in terms of the IgA levels in the nasal washings, serum IgG or IgM levels, or B and T-Cell proliferation. Suitably effective increases in immunoglobulin levels include by more than 5%, preferably by more than 25%, and in particular by more than 50%, as compared to the same antigen composition without any adjuvant.
In another embodiment of the invention, the vaccine formulation may further comprise a delivery agent, which functions to enhance antigen uptake based upon, but not restricted to, increased fluid viscosity due to the single or combined effect of partial dehydration of host mucopolysaccharides, the physical properties of the delivery agent, or through ionic interactions between the delivery agent and host tissues at the site of exposure, which provides a depot effect. Alternatively, the delivery agent can increase antigen retention time at the site of delivery (e.g., delay expulsion of the antigen). Such a delivery agent may be a bioadhesive agent. In some embodiments, the bioadhesive may be a mucoadhesive agent selected from the group consisting of glycosaminoglycans (e.g., chondroitin sulfate, dermatan sulfate chondroitin, keratan sulfate, heparin, heparan sulfate, hyaluronan), carbohydrate polymers (e.g., pectin, alginate, glycogen, amylase, amylopectin, cellulose, chitin, stachyose, unulin, dextrin, dextran), cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (including mucin, other mucopolysaccharides, and GelSite®, a natural acidic polysaccharide extracted from the aloe plant), polyions, cellulose derivatives (e.g., hydroxypropyl methylcellulose, carboxymethylcellulose), proteins (e.g. lectins, fimbrial proteins), and deoxyribonucleic acid. In one embodiment, the vaccine formulations comprise a polysaccharide such as chitosan, chitosan salt, chitosan base, or a natural polysaccharide (e.g. GelSite®).
Chitosan, a positively charged linear polysaccharide derived from chitin in the shells of crustaceans, is a bioadhesive for epithelial cells and their overlaying mucus layer. Formulation of antigens with chitosan increases their contact time with the nasal membrane, thus increasing uptake by virtue of a depot effect (Illum et al. (2001) Adv Drug Deliv Rev, Vol. 51: 81-96; Illum et al. (2003) J Control Release, Vol. 87: 187-198; Davis et al. (1999) Pharm Sci Technol Today, Vol. 2: 450-456; Bacon et al. (2000) Infect Immun., Vol. 68: 5764-5770; van der Lubben et al. (2001) Adv Drug Deliv Rev, Vol. 52: 139-144; van der Lubben et al. (2001) Eur J Pharm Sci, Vol. 14: 201-207; Lim et al. (2001) AAPS Pharm Sci Tech, Vol. 2: 20). Chitosan has been tested as a nasal delivery system for several vaccines, including influenza, pertussis and diphtheria, in both animal models and humans (Illum et al. (2001) Adv Drug Deliv Rev, Vol. 51: 81-96; Illum et al. (2003) J Control Release, Vol. 87: 187-198; Bacon et al. (2000) Infect Immun., Vol. 68: 5764-5770; Jabbal-Gill et al. (1998) Vaccine, Vol. 16: 2039-2046; Mills et al. (2003) A Infect Immun, Vol. 71: 726-732; McNeela et al. (2004) Vaccine, Vol. 22: 909-914). In these trials, chitosan was shown to enhance systemic immune responses to levels equivalent to parenteral vaccination. In addition, significant antigen-specific IgA levels were also measured in mucosal secretions. Thus, chitosan can greatly enhance a nasal vaccine's effectiveness. Moreover, due to its physical characteristics, chitosan is particularly well suited to intranasal vaccines formulated as powders (van der Lubben et al. (2001) Eur J Pharm Sci, Vol. 14: 201-207; Mikszta et al. (2005) J Infect Dis, Vol. 191: 278-288; Huang et al. (2004) Vaccine, Vol. 23: 794-801).
In another embodiment of the invention, the vaccine formulation may further comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier, including any suitable diluent or excipient, includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to the subject receiving the vaccine formulation, and which may be administered without undue toxicity. As used herein, the term “pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopia, European Pharmacopia or other generally recognized pharmacopia for use in mammals, and more particularly in humans. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (Mack Pub. Co. N.J. current edition). The formulation should suit the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans, preferably the formulation is sterile, non-particulate and/or non-pyrogenic. The vaccine formulation, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
In some embodiments of the present invention, among others, vaccine formulations comprise chitosan, a chitosan salt, or a chitosan base. The molecular weight of the chitosan may be between 10 kDa and 800 kDa, preferably between 100 kDa and 700 kDa and more preferably between 200 kDa and 600 kDa. The concentration of chitosan in the composition will typically be up to about 80% (w/w), for example, 5%, 10%, 30%, 50%, 70% or 80%. The chitosan is one which is preferably at least 75% deacetylated, for example 80-90%, more preferably 82-88% deacetylated, particular examples being 83%, 84%, 85%, 86% and 87% deacetylation.
The compositions of the invention can be formulated for administration as vaccines or antigenic formulations. As used herein, the term “vaccine” refers to a formulation which contains VLPs or other antigens of the present invention as described above, which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity to prevent and/or ameliorate an infection and/or to reduce at least one symptom of an infection and/or to enhance the efficacy of another dose of VLPs or antigen. As used herein, the term “antigenic formulation” or “antigenic composition” refers to a preparation which, when administered to a vertebrate, e.g. a mammal, will induce an immune response. As used herein, the term “immune response” refers to both the humoral immune response and the cell-mediated immune response. The humoral immune response involves the stimulation of the production of antibodies by B lymphocytes that, for example, neutralize infectious agents, block infectious agents from entering cells, block replication of said infectious agents, and/or protect host cells from infection and destruction. The cell-mediated immune response refers to an immune response that is mediated by T-lymphocytes and/or other cells, such as macrophages, against an infectious agent, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates infection or reduces at least one symptom thereof. In particular, “protective immunity” or “protective immune response” refers to immunity or eliciting an immune response against an infectious agent, which is exhibited by a vertebrate (e.g., a human), that prevents or ameliorates an infection or reduces at least one symptom thereof. Specifically, induction of a protective immune response from administration of the vaccine is evident by elimination or reduction of the presence of one or more symptoms of gastroenteritis or a reduction in the duration or severity of such symptoms. Clinical symptoms of gastroenteritis from Norovirus include nausea, diarrhea, loose stool, vomiting, fever, and general malaise. A protective immune response that reduces or eliminates disease symptoms will reduce or stop the spread of a Norovirus outbreak in a population. Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995) Plenum Press New York). The compositions of the present invention can be formulated, for example, for administration to a subject by mucosal or parenteral (e.g. intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal) routes of administration. Such mucosal administration could be, but is not limited to, through gastro-intestinal, intranasal, oral, or vaginal delivery. In one embodiment, the vaccine formulation is in the form of a nasal spray, nasal drops or dry powder. In another embodiment, the vaccine formulation is in a form suitable for intramuscular administration.
Vaccine formulations of the invention may be liquid formulations or dry powder formulations. Where the composition is intended for delivery to the respiratory (e.g. nasal) mucosa, typically it is formulated as an aqueous solution for administration as an aerosol or nasal drops, or alternatively, as a dry powder, e.g. for rapid deposition within the nasal passage. Compositions for administration as nasal drops may contain one or more excipients of the type usually included in such compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents, and the like. Viscosity agents can be microcrystalline cellulose, chitosan, starches, polysaccharides, and the like. Compositions for administration as dry powder may also contain one or more excipients usually included in such compositions, for example, mucoadhesive agents, bulking agents, and agents to deliver appropriate powder flow and size characteristics. Bulking and powder flow and size agents may include mannitol, sucrose, trehalose, and xylitol.
In one embodiment, the vaccine formulation contains one or more composite VLPs as the immunogen, an adjuvant such as MPL®, squalene, or MF59®, a biopolymer such as chitosan or GelSite® to promote adhesion to mucosal surfaces, and bulking agents such as mannitol and sucrose.
For example, a vaccine may be formulated as 10 mg of a dry powder containing one or more composite VPLs as discussed herein, such as the GII.4 composite VPL, MPL® adjuvant, chitosan mucoadhesive, and mannitol and sucrose as bulking agents and to provide proper flow characteristics. The formulation may comprise about 7.0 mg (25 to 90% w/w range) chitosan, about 1.5 mg mannitol (0 to 50% w/w range), about 1.5 mg sucrose (0 to 50% w/w range), about 25 μg MPL® (0.1 to 5% w/w range), and about 100 μg composite VLP antigen (0.05 to 5% w/w range).
Composite VLPs/antigens may be present in a concentration of from about 0.01% (w/w) to about 80% (w/w). In one embodiment, VLPs can be formulated at dosages of about 5 μg, about 15 μg, about 25 μg, about 50 μg, about 100 μg, about 200 μg, about 500 μg, and about 1 mg per 10 mg dry powder formulation (0.05, 0.15, 0.25, 0.5, 1.0, 2.0, 5.0, and 10.0% w/w) for administration into both nostrils (10 mg per nostril) or about 10 μg, about 30 μg, about 50 μg, about 100 μg, about 200 μg, about 400 μg, about 1 mg, and about 2 mgs (0.1, 0.3, 0.5, 1.0, 2.0, 4.0, 10.0 and 20.0% w/w) per 20 mg dry powder formulation for administration into one nostril. The formulation may be given in one or both nostrils during each administration. There may be a booster administration 1 to 12 weeks after the first administration to improve the immune response. The content of each VLP/antigen in the vaccine and antigenic formulations may be in the range of 1 μg to 100 mg, preferably in the range 1-1000 μg, more preferably 5-500 μg, most typically in the range 10-200 μg. Total VLP/antigen administered at each dose can be either about 10 μg, about 30 μg, about 200 μg, about 250 μg, about 400 μg, about 500 μg, or about 1000 μg. The total vaccine dose can be administered into one nostril or can be split in half for administration to both nostrils. Dry powder characteristics are such that less than 10% of the particles are less than 10 μm in diameter. Mean particle sizes range from 10 to 500 μm in diameter.
In another embodiment of the invention, the dry powder formulation may be in combination with one or more devices for administering one or more doses of the formulation. Such a device may be a single-use nasal administrative device. In another embodiment, one or more doses are unit doses.
In some embodiments, the antigenic and vaccine formulations are liquid formulations for subsequent administration to a subject. A liquid formulation intended for intranasal administration would comprise composite VLP/antigen(s), adjuvant, and a delivery agent such as chitosan. Liquid formulations for parenteral (e.g., subcutaneous, intradermal, or intramuscular (i.m.)) administration would comprise composite VLP/antigen(s), adjuvant, and a buffer, without a delivery agent (e.g., chitosan).
Preferably the antigenic and vaccine formulations hereinbefore described are lyophilized and stored anhydrous until they are ready to be used, at which point they are reconstituted with diluent. Alternatively, different components of the composition may be stored separately in a kit (any or all components being lyophilized). The components may remain in lyophilized form for dry formulation or be reconstituted for liquid formulations, and either mixed prior to use or administered separately to the patient. For dry powder administration, the vaccine or antigenic formulation may be preloaded into an intranasal delivery device and stored until use. Preferably, such intranasal delivery device would protect and ensure the stability of its contents.
The invention also encompasses compositions comprising one or more of the immunogenic nucleic acids, polypeptides, and/or VLPs, described herein. Different polypeptides, including composite polypeptides and capsid polypeptides or fragments thereof may be mixed together in a single formulation. Within such combinations, an antigen of the immunogenic composition may be present in more than one polypeptide, or multiple epitope polypeptide.
The immunogenic compositions may comprise a mixture of composite polypeptides and nucleic acids encoding composite polypeptides, which in turn may be delivered using the same or different vehicles. Antigens may be administered individually or in combination, in e.g., prophylactic (i.e., to prevent infection) or therapeutic (to treat infection) immunogenic compositions. The immunogenic composition may be given more than once (e.g., a “prime” administration followed by one or more “boosts”) to achieve the desired effects. The same composition can be administered in one or more priming and one or more boosting steps. Alternatively, different compositions can be used for priming and boosting.
The present invention also contemplates a method of inducing protective immunity to a viral infection in a subject comprising administering any of the vaccine formulations described herein. In one embodiment, the viral infection is a Norovirus infection. In another embodiment, the vaccine formulation confers protection from one or more symptoms of Norovirus infection.
The present invention also provides a method for making a VLP comprising a composite polypeptide. In one embodiment, the method comprises aligning amino acid sequences of capsid proteins from two or more circulating strains of a non-enveloped virus; determining a consensus sequence from said aligned amino acid sequences; preparing a composite sequence based on said consensus sequence that contains at least one different amino acid as compared to each of the capsid sequences of said two or more circulating strains; and expressing said composite sequence in a host cell, thereby producing a virus-like particle. In another embodiment, the composite sequence contains at least three different amino acids as compared to each of the capsid sequences of said two or more circulating strains. In another embodiment, the composite sequence contains at least five different amino acids as compared to each of the capsid sequences of said two or more circulating strains. In yet another embodiment, the composite sequence contains at least nine different amino acids as compared to each of the capsid sequences of said two or more circulating strains. In some embodiments, the consensus sequence may be determined from aligning nucleotide sequences of capsid proteins from two or more circulating strains of a non-enveloped virus; and preparing a composite nucleotide sequence based on said consensus sequence. Non-limiting examples of a non-enveloped virus suitable for use in the method are Calicivirus, Picornavirus, Astrovirus, Adenovirus, Reovirus, Polyomavirus, Papillomavirus, Parvovirus, and Hepatitis E virus. In some embodiments, the non-enveloped virus is a Calicivirus. The Calicivirus may be a Norovirus or Sapovirus. In another embodiment, the Norovirus is a genogroup I or genogroup II Norovirus.
The invention will now be illustrated in greater detail by reference to the specific embodiments described in the following examples. The examples are intended to be purely illustrative of the invention and are not intended to limit its scope in any way.
EXAMPLES
Example 1
Design of a Norovirus GII.4 Consensus Gene
A consensus amino acid sequence for the major capsid protein (VP1) of genogroup II, genotype 4 (GII.4) Norovirus was determined by homology comparison of two recently circulating GII.4 Strains, Minerva, AKA 2006-a; and Laurens, AKA 2006-b, with a GII.4 Houston strain obtained in 2002. The alignment of the three different Norovirus GII.4 isolates is shown below. The consensus sequence (SEQ ID NO: 2) determined from the homology comparison of the three GII.4 strains is shown in FIG. 1.
A composite sequence was derived from the consensus sequence by selecting amino acids from the Minerva sequence in variable positions of the consensus sequence where all three strains differed. The chosen amino acids were present in antigenic regions near to but not including the proposed carbohydrate binding domain. The composite GII.4 sequence was used for the production of a synthetic gene encoding a composite GII.4 Norovirus VP1 protein (SEQ ID NO: 1). The GII.4 composite VP1 amino acid sequence (GII.4 Comp) is shown in the alignment below as SEQ ID NO: 1 with the amino acid sequences of the VP1 proteins from Houston, Minerva, and Laurens virus (SEQ ID NOs: 4, 5, and 6, respectively). The DNA sequence encoding the GII.4 composite VP1 (SEQ ID NO: 3) is shown in FIG. 2.
Houston
53
Minerva
MKMASSDANPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIR
53
Laurens
53
GII.4 Comp
MKMASSDANPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIR
53
Houston
NNFVQAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQ
106
Minerva
NNFVQAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQ
106
Laurens
NNFVQAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQ
106
GII.4 Comp
NNFVQAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQ
106
Houston
VILAGNAFTAGKIIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLP
159
Minerva
VILAGNAFTAGKIIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLP
159
Laurens
VILAGNAFTAGKIIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLP
159
GII.4 Comp
VILAGNAFTAGKIIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLP
159
Houston
DVRNNFYHYNQSNDPTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDF
212
Minerva
DVRNNFYHYNQSNDPTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDF
212
Laurens
212
GII.4 Comp
DVRNNFYHYNQSNDPTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDF
212
Houston
IFLVPPTVESRTKPFTVPILTVEEMTNSRFPIPLEKLFTGPSGAFVVQPQNGR
265
Minerva
265
Laurens
IFLVPPTVESRTKPFTVPILTVEEMTNSRFPIPLEKLFTGPSGAFVVQPQNGR
265
GII.4 Comp
IFLVPPTVESRTKPFTVPILTVEEMTNSRFPIPLEKLFTGPSGAFVVQPQNGR
265
Houston
318
Minerva
318
Laurens
318
GII.4 Comp
318
Houston
371
Minerva
371
Laurens
371
GII.4 Comp
APLGTPDFVGKIQGVLTQTTRGDGSTRGHKATVSTGSVHFTPKLGSVQFSTDT
371
Houston
424
Minerva
424
Laurens
424
GII.4 Comp
424
Houston
477
Minerva
477
Laurens
477
GII.4 Comp
TFPGEQLLFFRSTMPGCSGYPNMNLDCLLPQEWVQHFYQEAAPAQSDVALLRF
477
Houston
530
Minerva
VNPDTGRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYTLAPM
530
Laurens
VNPDTGRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYTLAPM
530
GII.4 Comp
VNPDTGRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYTLAPM
530
Houston
539
Minerva
GNGTGRRRA (SEQ ID NO: 5)
539
Laurens
GNGTGRRRA (SEQ ID NO: 6)
539
GII.4 Comp
GNGTGRRRA (SEQ ID NO: 1)
539
Example 2
Purification of Composite VLPs
Synthetic gene construct of Norovirus GII.4 composite sequence for capsid domains described in Example 1 was cloned into recombinant Baculovirus. Infection of insect cells demonstrated high yield of production of VLP. A 40 mL aliquot of a P2 pFastBac recombinant baculovirus stock for the composite VLP VP1 gene was processed with a sucrose gradient to verify the expression and assembly of composite VLPs. The conditioned media was first layered onto a 30% sucrose cushion and then centrifuged at 140 K×g to pellet the VLP. The pellet was resuspended, layered onto a sucrose gradient and then centrifuged at 140 K×g. A visible white layer was observed within the gradient after centrifugation. 500 μL fractions from the gradient were collected and then analyzed by SDS-PAGE/Coomassie gel (FIG. 3). The expected banding pattern for composite VLP at ˜56 kDa was observed within the sucrose gradient fractions.
Using a high pressure liquid chromatography system with a running buffer of 20 mM Tris 150 mM NaCl pH 7 at a flow rate of 0.5 mL/minute, a 50 μL aliquot of the composite expression cell culture supernatant was loaded on to a Superose-6 size exclusion column. An intact VLP peak was observed at ˜15.3 minutes at 280 nm and 220 nm confirming integrity of the composite VLPs (FIG. 4).
Composite VLPs were also purified from conditioned media using column chromatography. Conditioned media was processed by cation exchange chromatography. The cation exchange elution fraction was further purified by hydrophobic interaction chromatography (HIC). The HIC elution fraction was concentrated and buffer exchanged by tangential flow filtration. The final product was sterile filtered and stored at 4° C. 500 ng of the purified composite VLPs (CM3 lot) was analyzed by silver-stained SDS-PAGE (FIG. 5).
Using a high pressure liquid chromatography system with a running buffer of 20 mM Tris 150 mM NaCl pH 7 at a flow rate of 1.0 mL/minute, a 50 μL aliquot of the purified CM3 composite VLPs was loaded on to a Superose-6 size exclusion column. An intact VLP peak was observed at ˜7.5 minutes at 280 nm confirming integrity of the composite VLPs (FIG. 6).
Example 3
Composite Immunogenicity
Female C57BL/6 mice approximately 8-10 weeks of age were immunized intraperitoneally with decreasing concentrations of composite VLP (CVLP) starting with 50 μg and decreasing 2 fold to 0.19 μg. The CVLP contained a polypeptide having the sequence of SEQ ID NO: 1 as described in Example 1. A group of animals immunized with PBS alone was included as a negative control. Serum samples were collected and analyzed for the presence of CVLP-specific IgG by ELISA (FIG. 7). The results from this experiment indicate that the linear range of the dose curve is between approximately 6 μg and 0.2 μg. Doses above 6.25 μg of CVLP do not appear to enhance immune responses in a dose-dependent manner. The EC50 value (defined as the effective dose yielding a 50% response) was calculated to be approximately 1.0 μg/mL using Softmax Pro software (Molecular Devices Corporation, Sunnyvale, Calif.).
Example 4
Multiple Antigen Effect of Composite VLPs
Female C57BL/6 mice (8-10 weeks of age) were immunized intraperitoneally with varying doses of either Norwalk VLP alone (NVLP), composite VLP (CVLP) alone or in combination. A group of animals immunized with PBS alone was included as a negative control. Serum samples were collected and analyzed for the presence of antigen-specific IgG by ELISA (FIGS. 8 and 9). The results indicate that immunizing with the combination of the CVLP and NVLP enhances the immune response such that a higher IgG level is achieved with a lower dose of antigen. For example, immunizing with 1 μg of each NVLP and CVLP elicits a more robust immune response then administering with either VLP alone. The antibodies from animals immunized with CVLP did not cross-react with NVLP and vise versa (data not shown).
Example 5
Composite VLPs Elicit Cross-Reactivite Antibodies
Female C57/BL6 mice, approximately 10-12 weeks of age, were immunized intraperitoneally with either 30 μg Houston VLPs or composite VLPs formulated with MPL (20 μg) as an adjuvant. The composite VLPs contained a polypeptide having the sequence of SEQ ID NO: 1 as described in Example 1. The mice were bled on day 21 following immunization and the sera were assayed in an antigen-specific ELISA to determine antibody titers for composite, Houston, Laurens, and Norwalk VLPs. The data are shown in FIG. 10. Immunization with composite VLP induces a broader response across more serotypes as evidenced by the greater response to the Laurens strain while maintaining response to the Houston strain. Immunization with Houston VLPs also induces cross-reactive antibodies against composite and Laurens but the magnitude of the response is not as great as that observed with immunization with the composite VLPs. There was no detectable response to Norwalk VLP, which is a GI.1 Norovirus.
Example 6
Efficacy of Bivalent Vaccine in Rabbits
A study was performed to evaluate the efficacy of a bivalent Norovirus vaccine comprising Norovirus GII.4 composite VLPs (CVLPS) as described in Example 2 and Norwalk VLPs (NVLPs, GI.1). Rabbits were intramuscularly immunized with this bivalent vaccine on days 0 and 21. VLP doses ranged from 20 μg to 0.002 μg of each type of VLP and each vaccine formulation contained 25 μg MPL and 250 μg AlOH. Serum was collected from each rabbit on day 28 and VLP-specific IgG was evaluated. Spleens and mesenteric lymph nodes were collected on day 75 and evaluated for the presence antigen-specific cellular immunity.
Serum IgG titers were measured by ELISA using microtiter plates coated with either NVLP or CVLP as a capture. Titers are expressed as reciprocal dilutions (FIG. 11). Antigen-specific T-cell responsiveness was evaluated by tritiated thymidine incorporation after a 5-day in vitro stimulation with 5 μg of either NVLP or CVLP (FIG. 12). Memory B-cells were evaluated by VLP-specific ELISPOT and results are expressed as antibody-secreting cells per million cells (FIG. 13).
The results of this study demonstrate that the IM bivalent norovirus vaccine formulated with the adjuvants MPL and AlOH elicits high VLP-specific IgG responses, responsive T-cells and memory B-cells capable of responding to stimulation with both NVLP and CVLP.
Example 7
High-Dose Bivalent Vaccination in Rabbits
This example outlines experiments designed to determine if high doses of the composite and Norwalk VLPs in the bivalent vaccine would lead to any adverse events. Rabbits were intramuscularly immunized with the bivlaent vaccine (see Example 6) on days 0, 14, and 21. VLP doses ranged from 150 μg to 5 μg of each VLP (Norwalk and composite) and each formulation contained 50 μg MPL and 500 μg AlOH. The general health, coat condition, and injection site of the immunized rabbits were monitored every 12 hours for the first 72 hours and then daily thereafter. Serum was collected from each rabbit on day 21 and day 35 and Norwalk VLP (NVLP)-specific (FIG. 14) and composite VLP (CVLP)-specific (FIG. 15) IgG and IgA were evaluated. Spleens were also harvested on day 35 and evaluated for the presence of antigen-specific cellular immunity (FIG. 16).
Serum IgG titers were measured by ELISA using microtiter plates coated with either NVLP or CVLP as a capture. Titers are expressed as reciprocal dilutions. Antigen-specific T-cell responsiveness was evaluated by tritiated thymidine incorporation after a 5-day in vitro stimulation with the indicated antigens (e.g. composite VLPs, GII.4 (2002) VLPs, GII.4 (2006 VLPs, and Norwalk VLPs).
The results from this study shows that the Norovirus bivalent vaccine is safe at the tested doses as evidenced by the fact that all rabbits appeared healthy throughout the study duration and no injection site reactions were observed. The immune responses measured from vaccinated rabbits confirm that the bivalent Norovirus vaccine is effective for eliciting both VLP-specific antibodies as well as VLP-responsive T-cells.
Example 8
Mouse Potency Assay for Norovirus Vaccine Efficacy
This example outlines the development of a mouse potency assay to evaluate the potency of the bivalent Norovirus vaccine. Mice were immunized IP on day 0 and 7 with equal concentrations ranging from 0.002 μg to 30 μg of Norwalk VLP (NVLP) and composite VLP (CVLP). Serum was collected from each mouse on day 14 and VLP-specific IgG was evaluated (FIG. 17). The neutralizing activity of the antibodies was also measured by hemagglutination inhibition assay (HAI) using Type O positive human red blood cells (FIG. 18). Only Norwalk-specific HAI titers could be assessed because the GII.4 genotypes do not hemagglutinate red blood cells.
Serum IgG titers were measured by ELISA using microtiter plates coated with either NVLP or CVLP as a capture. Titers are expressed as reciprocal dilutions. HAI titers were measured by using a standard hemagglutination assay.
The results from this study indicate that vaccination with the bivalent Norovirus vaccine elicits potent and functional IgG titers such that they are capable of inhibiting hemagglutination of human red blood cells. These results are of particular importance because they demonstrate that the antibodies elicited in response to the vaccination have functionality, which may lead to neutralization of the actual virus during an infection.
Example 9
Chitosan Formulations of a Norovirus Bivalent Vaccine
A study was performed in rabbits with the bivalent Norovirus VLP vaccine to evaluate the role of chitosan in this vaccine formulation. The formulation contained equal amounts of a Norwalk VP1 VLP and a composite GII.4 VLP (see Example 2). Rabbits were intranasally immunized with dry powder formulations on days 0 and 21. VLP doses ranged from 150 μg to 5 μg of each type of VLP and each formulation contained 50 μg MPL. Chitosan concentration was varied for each dose range (7 mg, 0.7 mg and 0 mg) to determine its role in immunogenicity. Serum was collected from each rabbit and VLP-specific IgG was evaluated (FIG. 19).
Serum IgG titers were measured by ELISA using microtiter plates coated with VLP as a capture. Serial dilutions of a proprietary in-house rabbit anti-VLP serum were used to generate standard curves. Titers are expressed in Units anti-VLP/mL (one Unit is approximately equal to 1 μg).
Results from these experiments indicate that chitosan at the highest dose (7 mg) is required to achieve maximum immunogenicity. The IgG data for the 50 μg dose is shown in FIG. 19 and results are represented as U/ml. The IgA antibody response is shown below in Table 1.
TABLE 1
Antigen-Specific IgA Responses.
VLP
50
50
50
(μg)
Chitosan
7
0.7
0
(mg)
Geometric Mean
770 (474, 1253)
67 (32, 142)
83 (38,179)
IgA Titers
(95% CI)
Example 10
Design of a Norovirus GII Consensus Gene
The methods of the present invention may also be used to generate capsid consensus sequences amongst Norovirus GII isolates from different GII genotypes, GII.1, GII.2, GII.3. The following alignment was generated from VP1 sequences from three different Norovirus GII isolates. The consensus sequence (SEQ ID NO: 7) determined from the homology comparison of the three GII strains is shown in FIG. 20.
A composite sequence is derived from the consensus sequence by selecting amino acids from a sequence of one of the strains for variable positions of the consensus sequence where two or more strains differ. Preferably the sequence from which amino acids are selected is a recently circulating strain, or a strain that is more commonly associated with disease or more commonly occurring amongst the strains being evaluated. In this Example, amino acids were selected from the Snow Mountain sequence at variable positions of the consensus sequence at which all three strains differed to generate a composite VP1 GII sequence. The composite GII sequence is used for production of a synthetic gene encoding a composite GII VP1 protein for induction of cross-immunity amongst GII Norovirus isolates.
The composite GII VP1 amino acid sequence (Composite) is shown in the alignment below as SEQ ID NO: 11 with the amino acid sequences of the VP1 proteins from GII.1 (Accession Number: AAL13001), GII.2 Snow Mountain (Accession Number: AAB61685), and GII.3 virus (Accession Number: AAL12998) (SEQ ID NOs: 8, 9, and 10, respectively).
Composite
53
GII.1
53
GII.2 Snow
53
GII.3
53
Composite
106
GII.1
106
GII.2 Snow
106
GII.3
106
Composite
159
GII.1
159
GII.2 Snow
159
GII.3
159
Composite
212
GII.1
212
GII.2 Snow
212
GII.3
212
Composite
265
GII.1
265
GII.2 Snow
265
GII.3
265
Composite
318
GII.1
306
GII.2 Snow
306
GII.3
318
Composite
371
GII.1
352
GII.2 Snow
359
GII.3
364
Composite
424
GII.1
403
GII.2 Snow
410
GII.3
416
Composite
477
GII.1
456
GII.2 Snow
463
GII.3
469
Composite
530
GII.1
509
GII.2 Snow
516
GII.3
522
Composite
FRFDSWVNQFYSLAPMGTGNGRRRI (SEQ ID NO: 11)
555
GII.1
534
GII.2 Snow
FRFDSWVNQFYSLAPMGTGNGRRRI (SEQ ID NO: 9)
541
GII.3
547
Example 11
Design of a Norovirus GI Consensus Gene
The methods of the present invention may also be used to generate capsid consensus sequences amongst Norovirus GI isolates. The following alignment was generated from VP1 sequences from three different Norovirus GI isolates. The consensus GI sequence (SEQ ID NO: 12) determined from the homology comparison of the three GI strains is shown in FIG. 21.
A composite sequence is derived from the consensus sequence by selecting amino acids from a sequence of one of the strains for variable positions of the consensus sequence where two or more strains differ. Preferably the sequence from which amino acids are selected is a recently circulating strain, or a strain that is more commonly associated with disease or more commonly occurring amongst the strains being evaluated. In this Example, amino acids were selected from the Southampton sequence at variable positions of the consensus sequence at which all three strains differed to generate a composite VP1 GI sequence. The composite GI sequence is used for production of a synthetic gene encoding a composite GI VP1 protein for induction of cross-immunity amongst GI Norovirus isolates.
The composite GI VP1 amino acid sequence (Composite) is shown in the alignment below as SEQ ID NO: 16 with the amino acid sequences of the VP1 proteins from Norwalk virus (Accession Number: M87661), Southampton (Accession Number: Q04542), and Chiba virus (Accession Number: BAB18267) (SEQ ID NOs: 13, 14, and 15, respectively).
Composite
53
Norwalk VP
53
Southampto
53
Chiba VP1
53
Composite
PWIINNFVQAPQGEFTISPNNTPGDVLFDLQLGPHLNPFLSHLSQMYNGWVGN
106
Norwalk VP
106
Southampto
106
Chiba VP1
PWIINNFVQAPQGEFTISPNNTPGDVLFDLQLGPHLNPFLSHLSQMYNGWVGN
106
Composite
159
Norwalk VP
159
Southampto
159
Chiba VP1
159
Composite
212
Norwalk VP
210
Southampto
211
Chiba VP1
211
Composite
265
Norwalk VP
263
Southampto
264
Chiba VP1
264
Composite
318
Norwalk VP
314
Southampto
317
Chiba VP1
317
Composite
371
Norwalk VP
362
Southampto
370
Chiba VP1
370
Composite
424
Norwalk VP
412
Southampto
423
Chiba VP1
422
Composite
477
Norwalk VP
461
Southampto
476
Chiba VP1
475
Composite
530
Norwalk VP
514
Southampto
529
Chiba VP1
528
Composite
546
Norwalk VP
530
Southampto
545
Chiba VP1
544
Example 12
Design of a Human Papillomavirus Consensus Gene for L1
The methods of the present invention may also be used to generate consensus sequences amongst other non-enveloped viruses. The following alignment was generated from three Human Papillomavirus (HPV): HPV-11, HPV-16, and HPV-18. The consensus L1 capsid protein sequence (SEQ ID NO: 17) determined from the homology comparison of the three HPV strains is shown in FIG. 22.
A composite sequence is derived from the consensus sequence by selecting amino acids from a sequence of one of the strains for variable positions of the consensus sequence where two or more strains differ. Preferably the sequence from which amino acids are selected is a recently circulating strain, or a strain that is more commonly associated with disease or more commonly occurring amongst the strains being evaluated. In this Example, amino acids were selected from the HPV-18 sequence at variable positions of the consensus sequence at which all three strains differed to generate a composite L1 HPV sequence. The composite HPV sequence is used for production of a synthetic gene encoding a composite L1 polypeptide for induction of cross-immunity amongst a variety of HPV strains.
The composite HPV L1 amino acid sequence (Composite) is shown in the alignment below as SEQ ID NO: 21 with the amino acid sequences of the L1 proteins from HPV-11, HPV-16, and HPV-18 virus (SEQ ID NOs: 18, 19, and 20, respectively).
Composite
53
HPV16 L1
18
HPV18 L1
53
Composite
106
HPV11 L1
43
HPV16 L1
70
HPV18 L1
105
Composite
159
HPV11 L1
94
HPV16 L1
123
HPV18 L1
158
Composite
212
HPV11 L1
147
HPV16 L1
176
HPV18 L1
211
Composite
265
HPV11 L1
200
HPV16 L1
229
HPV18 L1
264
Composite
318
HPV11 L1
253
HPV16 L1
282
HPV18 L1
317
Composite
371
HPV11 L1
306
HPV16 L1
335
HPV18 L1
370
Composite
424
HPV11 L1
357
HPV16 L1
387
HPV18 L1
423
Composite
477
HPV11 L1
410
HPV16 L1
440
HPV18 L1
476
Composite
530
HPV11 L1
463
HPV16 L1
493
HPV18 L1
529
Composite
569
HPV11 L1
500
HPV16 L1
531
HPV18 L1
567
Example 13
Dose Escalation Safety Study of Composite VLP Vaccine Formulation in Humans
A double-blind, controlled, dose-escalation phase 1 study of the safety and immunogenicity of a Norovirus vaccine is conducted. The vaccine consists of composite Norovirus virus-like particles (VLPs) in a dry powder matrix designed for intranasal administration. The composite VLPs contain a polypeptide having the amino acid sequence of SEQ ID NO: 1. Vaccinees include healthy adult volunteers who are H type 1 antigen secretors. The rationale for enrollment of H type 1 antigen secretors is that H type 1 antigen secretors are susceptible to Norovirus infections while non-secretors are resistant. As a control, 2 additional volunteers at each dosage level receive matrix alone. The dry powder matrix includes 25 μg MPL® adjuvant, 7 mg chitosan, 1.5 mg mannitol, and 1.5 mg sucrose. Volunteers are dosed on days 0 and 21 and are required to keep a 7-day diary of symptoms after each dose. Blood for serology, antibody secreting cells (ASC), and stool and saliva samples for mucosal antibody evaluation are collected.
The components of the vaccine are listed in Table 2. The vaccine is packaged in an intranasal delivery device. Single administrations of the composite VLP vaccine are packaged in a single dose Bespak (Milton Keynes, UK) UniDose DP dry powder intranasal delivery device. Each device delivers 10 mg of the dry powder vaccine formulation. Each dose of vaccine consists of two delivery devices, one in each nostril. The total vaccine dose is 20 mg of dry power. The formulation of Adjuvant/Excipient is the same as the composite VLP vaccine except that no composite VLP antigen is included in the formulation. The formulation of the Adjuvant/Excipient (also referred to as dry powder matrix) is summarized in Table 3.
TABLE 2
Composite VLP Vaccine Composition
Quantity per
10 mg dry
% of Final
Component
Molecular class
powder
Formulation
Composite VLP
Recombinant
2.5, 7.5 or
0.025, 0.075, 0.25, or
protein
25, 50 μg
0.50%
Monophosphoryl
Phospholipid
25 μg
0.25%
Lipid A
Chitosan
Polysaccharide
7.0 mg
70%
Mannitol
Sugar
1.5 mg
15%*
Sucrose
Sugar
1.5 mg
15%
TABLE 3
Adjuvant/Excipient (dry powder matrix)
Quantity per
10 mg dry
% of Final
Component
Molecular class
powder
Formulation
Monophosphoryl
Phospholipid
25 μg
0.25%
Lipid A
Chitosan
Polysaccharide
7.0 mg
70%
Mannitol
Sugar
1.5 mg
15%
Sucrose
Sugar
1.5 mg
15%
Specifically, the dose escalation of the vaccine is conducted as follows: After appropriate screening for good health, a group of 3 volunteers is randomized to receive either 5 μg of the composite VLP vaccine plus dry powder matrix (n=2) or dry powder matrix alone (n=1) by the intranasal route. These 3 volunteers are followed for safety for 21 days, and the Independent Safety Monitor (ISM) reviews their safety data. After approval of the ISM, these individuals receive their second dose of vaccine or matrix on day 21, and 4 additional volunteers are randomized to receive either 5 μg VLP protein plus dry powder matrix (n=3) or matrix alone (n=1) by the intranasal route. The ISM reviews the safety data from this second group and after approval of the ISM, the second intranasal dose is given 21 days after the first dose. Volunteers keep a 7-day diary of symptoms after each dose. After the ISM determines that escalation to the next higher dose is acceptable, another group of 7 volunteers is randomized to receive either the composite VLP vaccine containing 15 μg VLP protein (n=5) or dry powder matrix alone (n=2) by the intranasal route at day 0 and day 21. Again, 7-day symptom diaries are recorded and reviewed by the ISM before the second dose at day 21. Finally, after review of the safety data from the first two dosage cohorts, the ISM determines if dose escalation is acceptable and a final group of 7 volunteers is randomized to receive either the composite VLP vaccine containing 50 μg VLP protein (n=5) or dry powder matrix alone (n=2) by the intranasal route on day 0 and day 21. Again, the ISM reviews seven-day symptom diaries and other safety data before the second dose at day 21.
The volunteers keep a daily diary of symptoms (including local symptoms such as: nasal discharge, nasal pain/discomfort, nasal congestion, runny nose, nasal itching, nose bleed, headache and systemic symptoms such as: daily oral temperature, myalgia, nausea, vomiting, abdominal cramps, diarrhea, and loss of appetite) for 7 days after receiving the composite VLP vaccine or dry powder matrix alone. Interim medical histories are obtained at each follow-up visit (days 7±1, 21±2, 28±2, 56±2 and 180±14); volunteers are queried about interim illness, medications, and doctor's visits. Volunteers are asked to report all serious or severe adverse events including events that are not solicited during follow up visits. Volunteers have CBC and serum creatinine, glucose, AST, and ALT assessed on days 7 and 28 (7 days after each immunization) and, if abnormal, the abnormal laboratory test is followed until the test becomes normal or stabilizes.
Blood is collected before immunization and on days 7±1, 21±2, 28±2, 56±2, and 180±14 to measure serum antibodies to the composite VLP vaccine by enzyme-linked immunosorbent assays (ELISA). Before and on day 7 after administration of each dose of vaccine or dry powder matrix alone peripheral blood lymphocytes are collected to detect antibody secreting cells by ELISPOT assay. Before and on days 21±2, 56±2 and 180±14 after vaccination, whole blood is obtained to separate cells and freeze for future studies of cell mediated immunity, including cytokine production in response to composite VLP antigen, and lymphoproliferation. Whole stool samples are collected before immunization and on days 7±1, 21±2, 28±2, 56±2, and day 180±14 for anti-composite VLP sIgA screening. Saliva is collected with a commercially available device (Salivette, Sarstedt, Newton, N.C.) before immunization and on days 7±1, 21±2, 28±2, 56±2, and if positive for mucosal antibodies at day 56, a day 180±14 sample is collected and screened for anti-composite VLP sIgA. Finally blood from volunteers receiving the highest dose of composite VLPs (50 μg, third cohort described above) is screened for memory B-cells on days 0, 21, 56 and 180.
The following methods are used to analyze the blood, stool, and saliva samples collected from immunized individuals or individuals receiving the dry powder matrix alone:
A. Serum Antibody Measurements By ELISA
Twenty mL of blood are collected before and at multiple time points after vaccination for measurement of antibodies to the composite VLP by ELISA, using purified recombinant composite VLPs as target antigen to screen the coded specimens. Briefly, composite VLPs in carbonate coating buffer pH 9.6 are used to coat microtiter plates. Coated plates are washed, blocked, and incubated with serial two-fold dilutions of test serum followed by washing and incubation with enzyme-conjugated secondary antibody reagents specific for human IgG, IgM, and IgA. Appropriate substrate solutions are added, color developed, plates read, and the IgG, IgM, and IgA endpoint titers are determined in comparison to a reference standard curve for each antibody class. A positive response is defined as a 4-fold rise in titer after vaccination.
B. Antibody Secreting Cell Assays
Peripheral blood mononuclear cells (PMBCs) are collected from thirty mL of heparinized blood for ASC assays to detect cells secreting antibodies to composite VLPs. These assays are performed on days 0, 7±1, 21±2, and 28±2 after administration of the composite VLP vaccine or dry powder matrix alone. A positive response is defined as a post-vaccination ASC count per 106 PBMCs that is at least 3 standard deviations (SD) above the mean pre-vaccination count for all subjects (in the log metric) and at least 8 ASC spots, which corresponds to the mean of medium-stimulated negative control wells (2 spots) plus 3 SD as determined in similar assays.
C. Measurement of Composite VLP-Specific Memory B-Cells
Heparinized blood is collected from cohort 3 (30 mL days 0 and 21, 50 mL days 56 and 180) to measure memory B cells on days 0, 21, 56 and 180 after vaccination using an ELISpot assay preceded by an in vitro antigen stimulation. A similar assay was successfully used to measure frequency of memory B cells elicited by Norwalk VLP formulations in rabbits (See WO 2008/042789, herein incorporated by reference in its entirety). Peripheral blood mononuclear cells (5×106 cells/mL, 1 mL/well in 24-well plates) are incubated for 4 days with composite VLP antigen (2-10 μg/mL) to allow for clonal expansion of antigen-specific memory B cells and differentiation into antibody secreting cells. Controls include cells incubated in the same conditions in the absence of antigen and/or cells incubated with an unrelated antigen. Following stimulation, cells are washed, counted and transferred to ELISpot plates coated with composite VLP. To determine frequency of VLP-specific memory B cells per total Ig-secreting B lymphocytes, expanded B cells are also added to wells coated with anti-human IgG and anti-human IgA antibodies. Bound antibodies are revealed with HRP-labeled anti-human IgG or anti-human IgA followed by True Blue substrate. Conjugates to IgA and IgG subclasses (IgA1, IgA2 and IgG1-4) may also be used to determine antigen-specific subclass responses which may be related with distinct effector mechanisms and locations of immune priming. Spots are counted with an ELISpot reader. The expanded cell populations for each volunteer are examined by flow cytometry to confirm their memory B cell phenotype, i.e. CD19+, CD27+, IgG+, IgM+, CD38+, IgD−.
D. Cellular Immune Responses
Heparinized blood (50 mL cohorts 1 and 2, 25 mL cohort 3) is collected as coded specimens and the PBMCs isolated and cryopreserved in liquid nitrogen for possible future evaluation of cell-mediated immune (CMI) responses to composite VLP antigen. Assays that may be performed include PBMC proliferative and cytokine responses to composite VLP antigen and can be determined by measuring interferon (IFN)-γ and interleukin (IL)-4 levels according to established techniques.
E. Collections of Stool and Saliva for Anti-Composite VLP sIgA
Anti-composite VLP IgA is measured in stool and saliva samples. Saliva specimens are treated with protease inhibitors (i.e. AEBSF, leupeptin, bestatin, and aprotinin) (Sigma, St. Louis, Mo.), stored at −70° C., and assayed using a modification of a previously described assay (Mills et al. (2003) Infect. Immun. 71: 726-732). Stool is collected on multiple days after vaccination and specimens stored at −70° C. until analysis. The specimens are thawed, and protease inhibitor buffer added to prepare a 10% w/v stool suspension. Stool supernatants are assayed for composite VLP-specific mucosal IgA by ELISA, as described below.
Approximately 2-3 mL of whole saliva is collected before and at multiple time points after vaccination. Saliva is collected by a commercially available device (Salivette, Sarstedt, Newton, N.C.), in which a Salivette swab is chewed or placed under the tongue for 30-45 seconds until saturated with saliva. Saliva is collected from the swab by centrifugation.
F. Measurement of Anti-Composite VLP in Stool and Saliva
ELISAs, utilizing plates coated with either anti-human IgA antibody reagents or target composite VLP antigen coatings, are performed to determine total IgA and to titer the specific anti-VLP IgA responses for each specimen. Total or specific IgA are revealed with HRP-labeled anti-human IgA as described above. An internal total IgA standard curve is included to quantify the IgA content. Response is defined as a 4-fold rise in specific antibody.
Example 14
Safety and Immunogenicity Study of Two Dosages of Intranasal Composite VLP Vaccine in Humans
A randomized, double blind study in healthy adults is conducted to compare the safety and immunogenicity of two dosage levels of a composite Norovirus virus-like particle (VLP) vaccine with adjuvant/excipients and placebo controls (empty device). The vaccine consists of composite Norovirus virus-like particles (VLPs) in a dry powder matrix designed for intranasal administration as described in Example 13. Vaccinees include healthy adult volunteers who are H type 1 antigen secretors. The human volunteers are randomly assigned to one of four groups and each group receives one of the following treatments: a 50 μg dose of the composite VLP vaccine, a 100 μg dose of the composite VLP vaccine, the adjuvant/excipient, or placebo. Volunteers are dosed on days 0 and 21 and are required to keep a 7-day diary of symptoms after each dose. Blood for serology, antibody secreting cells (ASC), and stool and saliva samples for mucosal antibody evaluation are collected.
The components of the vaccine are listed in Table 2 in Example 13. The vaccine is packaged in an intranasal delivery device. Single administrations of the composite VLP vaccine are packaged in a single dose Bespak (Milton Keynes, UK) UniDose DP dry powder intranasal delivery device. Each device delivers 10 mg of the dry powder vaccine formulation. Each dose of vaccine consists of two delivery devices, one in each nostril. The total vaccine dose is 20 mg of dry power. Therefore, the 50 μg vaccine dose consists of two devices that each deliver 10 mg of dry powder formulation, wherein each 10 mg of dry powder formulation consists of 25 μg of composite VLP, 25 μg MPL® adjuvant, 7 mg chitosan, 1.5 mg mannitol, and 1.5 mg sucrose. Similarly, the 100 μg vaccine dose consists of two devices that each deliver 10 mg of dry powder formulation, wherein each 10 mg of dry powder formulation consists of 50 μg of composite VLP, 25 μg MPL® adjuvant, 7 mg chitosan, 1.5 mg mannitol, and 1.5 mg sucrose. The formulation of Adjuvant/Excipient is the same as the composite VLP vaccine except that no composite VLP antigen is included in the formulation. The formulation of the Adjuvant/Excipient (also referred to as dry powder matrix) is summarized in Table 3 in Example 13. The placebo group receives two empty devices.
The volunteers keep a daily diary of symptoms (including local symptoms such as: nasal discharge, nasal pain/discomfort, nasal congestion, runny nose, nasal itching, nose bleed, headache and systemic symptoms such as: daily oral temperature, myalgia, nausea, vomiting, abdominal cramps, diarrhea, and loss of appetite) for 7 days after receiving either one of two doses of the composite VLP vaccine, dry powder matrix alone, or the placebo. Interim medical histories are obtained at each follow-up visit (days 7+1, 21+2, 28+2, 56+2 and 180+14); volunteers are queried about interim illness, medications, and doctor's visits. Volunteers are asked to report all serious or severe adverse events including events that are not solicited during follow up visits. Volunteers have CBC and serum creatinine, glucose, AST, and ALT assessed on days 7 and 28 (7 days after each immunization) and, if abnormal, the abnormal laboratory test is followed until the test becomes normal or stabilizes.
Blood is collected before immunization and on days 7+1, 21+2, 28+2, 56+2, and 180+14 to measure serum antibodies to the composite VLP vaccine by enzyme-linked immunosorbent assays (ELISA). Before and on day 7 after administration of each dose of vaccine, dry powder matrix alone, or placebo, peripheral blood lymphocytes are collected to detect antibody secreting cells by ELISPOT assay. Before and on days 21+2, 56+2 and 180+14 after vaccination, whole blood is obtained to separate cells and freeze for future studies of cell mediated immunity, including cytokine production in response to composite VLP antigen, and lymphoproliferation. Whole stool samples are collected before immunization and on days 7+1, 21+2, 28+2, 56+2, and day 180+14 for anti-composite VLP sIgA screening. Saliva is collected with a commercially available device (Salivette, Sarstedt, Newton, N.C.) before immunization and on days 7+1, 21+2, 28+2, 56+2, and if positive for mucosal antibodies at day 56, a day 180+14 sample is collected and screened for anti-composite VLP sIgA. Blood is also screened for memory B-cells on days 0, 21, 56 and 180.
Methods used to analyze the blood, stool, and saliva samples collected from immunized individuals, or individuals receiving the dry powder matrix alone or placebo are described in detail in Example 13.
Example 15
Experimental Human Challenge Study with Infectious Norovirus Following Vaccination with Composite Norovirus VLP Vaccine
A multi-site, randomized, double-blind, placebo-controlled Phase 1-2 challenge study is conducted in 80 human volunteers immunized with the composite Norovirus VLP vaccine. Eligible subjects include those 18-50 years of age, in good health, who express the H type-1 oligosaccharide (as measured by positive salivary secretor status) and who are other than Type B or AB blood type. Subjects who are non H type-1 secretors or who have Type B or AB blood are reported to be more resistant to infection with Norwalk virus and are excluded from the study. At least 80% of volunteers are expected to be eligible based on these two criteria.
Following screening, eligible volunteers who meet all acceptance criteria are randomized (1:1) into one of two equal sized cohorts with approximately 40 volunteers in each cohort. Cohort 1 is immunized with composite VLP and cohort 2 receives placebo. Volunteers are immunized with 10 mg composite VLP vaccine in each nostril (20 mg total dry powder) or placebo. Each 10 mg of composite VLP vaccine contains 50 μg of Composite VLP, 7 mg chitosan, 25 μg MPL®, 1.5 mg of sucrose and approximately 1.5 mg of mannitol. Thus, each volunteer in cohort 1 receives a total dosage of 100 μg of composite VLP antigen at each immunization. Volunteers receive vaccine or placebo on study days 0 and 21.
The safety of the composite virus VLP vaccine compared to placebo is assessed. Volunteers keep a diary for 7 days following each immunization with the vaccine or placebo to document the severity and duration of adverse events. Serious adverse events (SAEs) and the occurrence of any significant new medical conditions is followed for 6 months after the last dose of vaccine or placebo and for 4 months after the challenge with infectious virus.
All volunteers are challenged with infectious Norovirus between 21 to 42 days after the second dose of vaccine or placebo (between study days 42 and 56). Each volunteer receives at or >than the 50% Human Infectious Dose (HID 50), i.e. the amount of infectious virus that is expected to cause disease in at least 50% of volunteers in the placebo group. The HID 50 is between about 48 and about 480 viral equivalents of the challenge virus strain. The challenge Norovirus is mixed with sterile water and given orally. The inoculation is preceded by ingestion of 500 mg sodium bicarbonate in water, to prevent breakdown of the virus by stomach acid and pepsin. A second ingestion of sodium bicarbonate solution (500 mg sodium bicarbonate in water) is taken 5 minutes after oral inoculation of the infectious virus. The volunteers remain at the challenge facility for at least 4 days and at least 18 hours after symptoms/signs of acute gastroenteritis (vomiting, diarrhea, loose stool, abdominal pain, nausea, and fever) are absent.
Several metrics are monitored to determine the efficacy of the composite VLP vaccine in preventing or reducing symptoms/signs of acute gastroenteritis induced by the viral challenge. All volunteers record their clinical symptoms of acute gastroenteritis and these symptoms are documented by the research staff at the study sites. Disease symptoms/signs from cohort 1 receiving the vaccine are compared to cohort 2 placebo recipients.
Sera and stool samples are routinely collected from all volunteers prior to immunization with the vaccine or placebo, and after challenge. Serum samples are analyzed by ELISA for IgA and IgG, titers against the challenge VLPs. The challenge virus antigen and challenge virus RNA are tested in stool samples by ELISA and PCR, respectively, which indicate the presence of virus, the amount of virus shed from the intestines, and the duration of viral shedding. Subjects who become ill after challenge, are subject to additional laboratory studies including serum chemistries, BUN, creatinine, and liver function tests until symptoms/signs resolve.
Results from the vaccine group (cohort 1) and the placebo group (cohort 2) are compared to assess the protective efficacy of the vaccine against Norovirus disease overall (primary endpoint), and/or its efficacy in ameliorating the symptoms/signs (severity and # of days of illness) and/or the reduction of the presence, the amount and/or the duration of virus shedding (secondary endpoints).
The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings using no more than routine experimentation. Such modifications and equivalents are intended to fall within the scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.
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14341375
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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tyo:4502
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Takeda Pharmaceutical
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Feb 16th, 2021 12:00AM
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Jan 15th, 2016 12:00AM
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https://www.uspto.gov?id=US10920288-20210216
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Detection of particle-contained reverse transcriptase activity
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The present invention relates to methods and kits for detecting in a sample the presence of a virus particle or a virus-like particle that has reverse transcriptase activity and methods for preparing a retroviral contaminant-free substance. An aspect of the present invention is a method for detecting the presence of a virus particle in a sample of a Virus-like Particle (VLP) drug substance comprising a step of performing PCR-based reverse transcriptase (PBRT) on a sample of the VLP drug substance that has been treated with a protease.
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10920288
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1. A method for detecting the presence of virus particle-contained reverse transcriptase activity in a test sample comprising steps of:
(1) adding to the test sample a protease and a detergent in an amount that is insufficient to disrupt an intact virus particle and incubating the resultant solution under conditions that allow the protease to digest any soluble reverse transcriptase present in the resultant solution, thereby producing a digested solution;
(2) inactivating the protease in the digested solution, thereby producing an inactivated protease solution;
(3) adding a detergent in an amount that is sufficient to disrupt an intact virus particle, thereby producing a detergent-containing solution;
(4) adding into the detergent-containing solution or into a fraction of the detergent-containing solution (i) an isolated RNA molecule, (ii) a first primer that hybridizes to a nucleic acid sequence corresponding to a first part of the isolated RNA molecule, (iii) a second primer that hybridizes to the complement of a second part of the isolated RNA molecule, and (iv) a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution;
(5) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if a virus particle-contained reverse transcriptase is present in the test sample; and
(6) identifying the PCR-amplified product, thereby detecting the presence of the virus particle-contained reverse transcriptase in the test sample.
2. The method of claim 1, wherein an isolated nucleic acid probe comprising a detectable label which hybridizes to a nucleic acid sequence corresponding to the isolated RNA molecule is added after step (3) or during step (4).
3. The method of claim 1, wherein the test sample is diluted with a buffer.
4. The method of claim 1, wherein the detergent is added during step (1) such that the detergent concentration in the digested solution is less than about 0.002%.
5. The method of claim 1, wherein the detergent is added during step (3) such that the detergent concentration in the detergent-containing solution or in the PBRT assay solution is between about 0.1% and 0.3%.
6. The method of claim 1, wherein the step of inactivating the protease comprises adding at least one protease inhibitor.
7. The method of claim 1, further comprising a step of concentrating the inactivated protease solution prior to step (3).
8. The method of claim 1, wherein a positive control sample comprising a soluble reverse transcriptase or a particle that contains a reverse transcriptase is obtained and processed according to steps (1) to (6), (2) to (6), (3) to (6), or (4) to (6).
9. The method of claim 8, wherein the positive control sample comprises the test sample.
10. The method of claim 1, wherein a sample is obtained and processed according to steps (1) to (6) but without adding an isolated RNA molecule in step (4), thereby producing a negative control sample.
11. The method of claim 2, wherein the detectable label is selected from the group consisting of biotin, a colloidal particle, digoxigenin, an electron-dense reagent, an enzyme, a fluorescent dye, hapten, a magnetic bead, a metallic bead, and a radioactive isotope.
12. The method of claim 1, wherein the test sample is a Virus-like Particle (VLP) drug substance.
13. The method of claim 12, wherein the VLP drug substance is a norovirus VLP drug substance.
14. The method of claim 13, wherein the norovirus is the Norwalk virus.
15. The method of claim 1, wherein the isolated RNA molecule is at least about 95% identical to a fragment of Dengue virus type 4 genome which has the sequence of SEQ ID NO: 1.
16. The method of claim 15, wherein the isolated RNA molecule is at least about 95% identical to the sequence of SEQ ID NO: 5.
17. The method of claim 1, wherein the first primer comprises the sequence of SEQ ID NO: 3.
18. The method of claim 1, wherein the second primer comprises the sequence of SEQ ID NO: 2.
19. A method for detecting the presence of an enveloped virus particle in a Virus-like Particle (VLP) drug substance comprising steps of:
(1) obtaining a sample of the VLP drug substance;
(2) diluting the sample in Proteinase K buffer which is supplemented with Triton X-100 detergent thereby obtaining a diluted sample,
wherein the Triton X-100 is present in an amount that is insufficient to disrupt an intact virus particle;
(3) adding Proteinase K to the diluted sample and incubating the resultant solution under conditions that allow the Proteinase K to digest any soluble reverse transcriptase present in the resultant solution but not to digest all reverse transcriptase contained in virus particles, thereby producing a digested solution;
(4) inactivating the Proteinase K in the digested solution by addition of Phenylmethylsulfonyl fluoride (PMSF), thereby producing an inactivated protease solution;
(5) centrifuging the inactivated protease solution by high speed centrifugation through a sucrose cushion, thereby producing a concentrated solution;
(6) adding a detergent in an amount that is sufficient to disrupt an intact virus particle, thereby producing a detergent-containing concentrated solution;
(7) adding to the detergent-containing concentrated solution or to a fraction of the detergent-containing concentrated solution an isolated RNA molecule, a first primer that hybridizes to a 5′ end of a nucleic acid sequence corresponding to the isolated RNA molecule, a second primer that hybridizes to the 3′ end of the isolated RNA molecule, an isolated nucleic acid probe which hybridizes to the isolated RNA molecule, and a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution;
(8) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if a virus particle containing a reverse transcriptase is present in the PBRT assay solution; and
(9) identifying the PCR-amplified product, thereby detecting the presence of the enveloped virus particle in the VLP drug substance.
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19
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RELATED APPLICATION
This application claims priority to and benefit of U.S. Provisional Application No. 62/104,252, filed Jan. 16, 2015. The contents of the aforementioned patent application are herein incorporated by reference in their entireties.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 13, 2016, is named LIGO-02701WO_ST25.txt and is 15,131 bytes in size.
BACKGROUND OF THE INVENTION
Retroviruses are important infectious agents in humans and animals and are responsible for a large number of diseases. A retrovirus may be passed to a subject via contact with a biological sample, e.g., an organ transplant, blood transfusion, and vaccine. Reverse transcriptase (RT) activity is a hallmark of retroviruses and therefore its presence in a biological sample can indicate the presence of a retrovirus particle in the sample.
Cell lines are commonly used for the propagation of recombinant viruses for production of biological products, e.g., vaccines. Such cell lines, which often contain integrated retrovirus-like elements, release RT following cell lysis. Therefore, when a biological product obtained from such a cell line has RT activity, it is unclear whether the RT activity is due to RT released by the cell or due to contamination of the biological product by a viral particle that contains RT; it is import to be able to distinguish between virus particle-contained and free RT activity since the presence of free RT in a biological product is of less significance than the presence of an intact retrovirus particle.
Accordingly, there is an unmet need for methods and kits capable of identifying RT activity in a sample that is due to a virus particle contaminant.
SUMMARY OF THE INVENTION
Provided herein are methods and kits for detecting in a sample the presence of a virus particle or a virus-like particle that has reverse transcriptase activity and methods for preparing a retroviral contaminant-free substance.
An aspect of the present invention is a method for detecting the presence of a virus particle in a sample of a Virus-like Particle (VLP) drug substance comprising a step of performing PCR-based reverse transcriptase (PBRT) on a sample of the VLP drug substance that has been treated with a protease.
Another aspect of the present invention relates to a method for detecting the presence of virus particle-contained reverse transcriptase activity in a test sample. The method includes the steps of: (1) adding a protease to the test sample and incubating the resultant solution under conditions that allow the protease to digest any soluble reverse transcriptase present in the resultant solution, thereby producing a digested solution; (2) inactivating the protease in the digested solution, thereby producing an inactivated protease solution; (3) adding a detergent in an amount that is sufficient to disrupt an intact virus particle, thereby producing a detergent-containing solution; (4) adding to the detergent-containing solution or to a fraction of the detergent-containing solution an isolated RNA molecule, a first primer that hybridizes to a nucleic acid sequence corresponding to a first part of the isolated RNA molecule, a second primer that hybridizes to the complement of a second part of the isolated RNA molecule, and a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution; (5) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if a virus particle-contained reverse transcriptase is present in the test sample; and (6) identifying the PCR-amplified product, thereby detecting the presence of the virus particle-contained reverse transcriptase in the test sample. An isolated nucleic acid probe which hybridizes to the nucleic acid sequence corresponding to the isolated RNA molecule may be added during or prior to step (4).
In the above aspect, when the isolated RNA molecule is added in step (4), substantially no DNA template for the isolated RNA molecule is concurrently added. To ensure this, a DNase may be added to the isolated RNA molecule prior to step (4); if added, the DNase is inactivated prior to step (4). Step (4) of the above aspect may further include adding one or more of dNTPs, a reaction buffer, water, Mg2+, and Mn2+. The DNA polymerase can be Taq Polymerase, a high-fidelity DNA polymerase, or another polymerase known in the art. In some embodiments, a detergent is added during step (1) of the above aspect in an amount that is insufficient to disrupt an intact virus particle.
Some embodiments of the above aspect include a step of concentrating the inactivated protease solution prior to step (3), e.g., by one or more of centrifugation, dialysis, precipitation, or passing through a column. For example, this step may involve subjecting the inactivated protease solution to high speed centrifugation through a sucrose cushion.
In embodiments of the above aspect, at least one sample is obtained and processed according to steps (1) to (6), (2) to (6), (3) to (6), or (4) to (6). In embodiments, positive control samples comprising a soluble reverse transcriptase, e.g., a recombinant reverse transcriptase (e.g., Avian Myeloblastosis virus αβ holoenzyme (AMV RT)), is obtained and processed according to steps (1) to (6), (2) to (6), (3) to (6), or (4) to (6); these positive control samples may include the test sample. In embodiments, positive control samples to which has been added a particle that contains a reverse transcriptase, e.g., a murine leukemia virus (MLV) particle, is obtained and processed according to steps (1) to (6), (2) to (6), (3) to (6), or (4) to (6); these positive control samples may include the test sample. In embodiments, at least one sample is obtained and processed according to steps (1) to (6), (2) to (6), (3) to (6), or (4) to (6) but without adding an isolated RNA molecule in step (4), thereby producing a negative control sample(s); these negative control samples may include the test sample.
In yet another aspect of the present invention is a method for detecting the presence of an RT-containing virus particle in a sample comprising a step of performing PCR-based reverse transcriptase (PBRT) on the sample that has been treated with a protease.
In another aspect of the present invention relates to a method for detecting the presence of an RT-containing virus particle in a Virus-like Particle (VLP) drug substance. The method includes steps of: (1) obtaining a sample of the VLP drug substance; (2) diluting the sample in Proteinase K buffer which is supplemented with Triton X-100 detergent (in an amount that is insufficient to disrupt an intact virus particle) thereby obtaining a diluted sample; (3) adding Proteinase K to the diluted sample and incubating the resultant solution under conditions that allow the Proteinase K to digest any soluble reverse transcriptase present in the resultant solution but not to digest all reverse transcriptase contained in virus particles, thereby producing a digested solution; (4) inactivating the Proteinase K in the digested solution by addition of phenylmethylsulfonyl fluoride (PMSF), thereby producing an inactivated protease solution; (5) centrifuging the inactivated protease solution by high speed centrifugation through a sucrose cushion, thereby producing a concentrated solution; (6) adding a detergent in an amount that is sufficient to disrupt an intact virus particle, thereby producing a detergent-containing concentrated solution; (7) adding to the detergent-containing concentrated solution or to a fraction of the detergent-containing concentrated solution an isolated RNA molecule, a first primer that hybridizes to a 5′ end of a nucleic acid sequence corresponding to the isolated RNA molecule, a second primer that hybridizes to the 3′ end of the nucleic acid sequence corresponding to the isolated RNA molecule, an isolated nucleic acid probe which hybridizes to the nucleic acid sequence corresponding to the isolated RNA molecule, and a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution; (8) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if a virus particle-protected and released reverse transcriptase is present in the PBRT assay solution; and (9) identifying the PCR-amplified product, thereby detecting the presence of the RT-containing virus particle in the VLP drug substance.
Another aspect of the present invention is a retroviral contaminant-free Virus-like Particle (VLP) drug substance. This substance is identified by a method of an above aspect including its embodiments.
An aspect of the present invention relates to a method for detecting the presence of virus particle-contained reverse transcriptase activity in a test sample. The method includes steps of: (1) adding a protease to the test sample and incubating the resultant solution under conditions that allow the protease to digest any soluble reverse transcriptase present in the resultant solution, thereby producing a digested solution; (2) inactivating the protease in the digested solution, thereby producing an inactivated protease solution; (3) adding a detergent in an amount that is sufficient to disrupt an intact enveloped virus particle, thereby producing a detergent-containing solution; (4) adding to the detergent-containing solution or to a fraction of the detergent-containing solution an isolated RNA molecule, a first primer that hybridizes to a nucleic acid sequence corresponding to a first part of the isolated RNA molecule, a second primer that hybridizes to the complement of a second part of the isolated RNA molecule, and a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution; (5) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if a virus particle-protected and released reverse transcriptase is present in the PBRT assay solution; and (6) identifying the PCR-amplified product, thereby detecting the presence of the virus particle-contained reverse transcriptase in the test sample. An isolated nucleic acid probe which hybridizes to the nucleic acid sequence corresponding to the isolated RNA molecule may be added during or prior to step (4).
In some embodiments, a detergent is added during step (1) in the above aspect of the present invention in an amount that is insufficient to disrupt an intact enveloped virus particle such that the detergent concentration is less than about 0.002%. In some embodiments, a detergent is added during step (3) such that the detergent concentration in the detergent-containing solution or in the PBRT assay solution is between about 0.1% and 0.3%.
Another aspect of the present invention relates to a method for detecting the presence of an enveloped virus particle in a Virus-like Particle (VLP) drug substance. The method includes steps of: (1) obtaining a sample of the VLP drug substance; (2) diluting the sample in Proteinase K buffer which is supplemented with Triton X-100 detergent (in an amount that is insufficient to disrupt an intact enveloped virus particle) thereby obtaining a diluted sample; (3) adding Proteinase K to the diluted sample and incubating the resultant solution under conditions that allow the Proteinase K to digest any soluble reverse transcriptase present in the resultant solution but not to digest all reverse transcriptase contained in enveloped virus particles, thereby producing a digested solution; (4) inactivating the Proteinase K in the digested solution by addition of Phenylmethylsulfonyl fluoride (PMSF), thereby producing an inactivated protease solution; (5) centrifuging the inactivated protease solution by high speed centrifugation through a sucrose cushion, thereby producing a concentrated solution; (6) adding a detergent in an amount that is sufficient to disrupt an intact virus particle, thereby producing a detergent-containing concentrated solution; (7) adding to the detergent-containing concentrated solution or to a fraction of the detergent-containing concentrated solution an isolated RNA molecule, a first primer that hybridizes to a 5′ end of a nucleic acid sequence corresponding to the isolated RNA molecule, a second primer that hybridizes to the 3′ end of a nucleic acid corresponding to the isolated RNA molecule, an isolated nucleic acid probe which hybridizes to a nucleic acid corresponding to the isolated RNA molecule, and a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution; (8) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if an enveloped virus particle-released reverse transcriptase is present in the PBRT assay solution; and (9) identifying the PCR-amplified product, thereby detecting the presence of the virus particle in the VLP drug substance.
In some embodiments, Triton X-100 is added during step (2) in the above aspect of the present invention such that the detergent concentration is less than about 0.002%. In some embodiments, a detergent is added during step (6) such that the detergent concentration in the detergent-containing concentrated solution or in the PBRT assay solution is between about 0.1% and 0.3%.
Another aspect of the present invention is a method for detecting the presence of a virus particle in a sample comprising a step of performing PCR-based reverse transcriptase (PBRT) on the sample that has been treated with a protease.
Another aspect of the present invention is a method for detecting the presence of an enveloped virus particle containing reverse transcriptase in a sample of a Virus-like Particle (VLP) drug substance comprising a step of performing PCR-based reverse transcriptase (PBRT) on a sample of the VLP drug substance that has been treated with a protease.
The invention includes kits for performing a method of an above aspect including its embodiments. The kits include instructions for use.
Another aspect of the present invention is a retroviral contaminant-free Virus-like Particle (VLP) drug substance. This substance is identified by a method of an above aspect including its embodiments.
In some embodiments, an isolated nucleic acid probe, e.g., having a detectable label, is used which hybridizes to a nucleic acid sequence corresponding to the isolated RNA molecule is added to the inactivated protease solution.
In some embodiments, a test sample is diluted with a buffer, e.g., about one- to about 20-fold (e.g., about 10-fold).
In some embodiments, the detergent is Triton X-100.
In some embodiments, the protease is Proteinase K.
In some embodiments, the protease inhibitor is PMSF.
In some embodiments, the test sample is a Virus-like Particle (VLP) drug substance, e.g., a norovirus (e.g., Norwalk virus) VLP drug substance.
In some embodiments, the isolated RNA molecule is an in vitro-transcribed RNA molecule.
In some embodiments along with the DNA polymerase is added one or more of dNTPs, a reaction buffer, water, Mg2+, and Mn2+. The DNA polymerase can be Taq Polymerase, a high-fidelity DNA polymerase, or another polymerase known in the art.
In some embodiments, the isolated RNA molecule is at least about 95% identical to a fragment of Dengue virus type 4 genome which has the sequence of SEQ ID NO: 1. The isolated RNA molecule can be at least about 95% identical to the sequence of SEQ ID NO: 5. The first primer may include the sequence of SEQ ID NO: 3 and the second primer may include the sequence of SEQ ID NO: 2. The isolated nucleic acid probe may include the sequence of SEQ ID NO: 4.
Exemplary detergents include Brij-35, Brij-58, CHAPS, CHAPSO, n-Dodecyl-beta-D-Maltoside, NP-40, Octyl glucoside, Octyl thioglucoside, SDS, Sodium Deoxycholate, Triton X-100, Triton X-114, Tween 20, or Tween 80. Preferably, the detergent is Triton X-100.
Examples of a protease includes but is not limited to bromelain, caspase, cathepsins, chymotrypsin, elastase, endoproteinase AspN, endoproteinase GluC, enterokinase (enteropeptidase), Factor Xa, furin, papain, pepsin, Proteinase K, subtilisin, thrombin, or trypsin. Preferably, the protease is Proteinase K. The step of inactivating the protease includes adding one or more protease inhibitors, for example AEBSF-HCl, Aprotinin, Bestatin, E-64, EDTA, Leupeptin, Pepstatin A, and/or Phenylmethylsulfonyl fluoride (PMSF). Preferably, the protease inhibitor is PMSF.
Examples of the detectable label includes but is not limited to biotin, a colloidal particle, digoxigenin, an electron-dense reagent, an enzyme, a fluorescent dye, hapten, a magnetic bead, a metallic bead, and a radioactive isotope. Preferably, the detectable label is a florescent dye. Examples of the florescent dye include 6-FAM, ABY®, Acridine, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, BioSearch Blue, Coumarin, Cy® 3, Cy® 3.5, Cy® 5, Cy® 5.5, FAM™, FITC®, GPF (and variants thereof), HEX™, JOE™, JUN®, Marina Blue, NED™, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PET®, Pulsar®, Quasar® 570, Quasar® 670, Quasar® 705, Rhodamine Green, Rhodamine Red, ROX™, SYBR® Green, TAMRA™, TET™, Texas Red®, TRITC, and VIC®.
The invention includes kits for performing a method of an above aspect including its embodiments. The kits include instructions for use.
Any of the above aspects, embodiments, features, or examples can be combined with any other aspect, embodiment, feature, or example.
Other features and advantages of the invention will be apparent from the Detailed Description, the drawings, and the claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods and kits including a highly-sensitive PCR-based reverse transcriptase (PBRT) assay for detecting retrovirus particle contaminants in a sample. The invention relies on the sensitivity of soluble reverse transcriptase (RT) activity to proteinase K digestion and substantially insensitivity of virus particle-contained RT activity; thus, a sample showing RT activity that is sensitive to proteinase K digestion likely does not have a retrovirus contamination whereas a sample showing RT activity that is substantially insensitive to proteinase K digestion likely has retrovirus contamination.
Without being bound by theory, experimental evidence disclosed herein shows that one or more of the following features of the present invention leads to unexpectedly superior results:
1. Dilution of a sample to be tested into a larger volume of buffer, e.g., Proteinase K buffer, in order to dilute out the high concentration of protein-based components in the sample, allows for a more complete proteolytic degradation of RT not contained in virus particles.
2. Addition of a very small amount of detergent, e.g., Triton X-100, during proteolytic digestion, e.g., by Proteinase K, of virus particles helps facilitate complete digestion. Moreover, the amount of detergent used does not diminish virus particle-contained RT activity.
3. Inactivation of the protease, e.g., Proteinase K, by the addition of a protease inhibitor, e.g., PMSF, is performed following the proteolytic digestion step.
4. Collection and concentration, e.g., high speed centrifugation through a sucrose cushion, of viral particles following the inactivation of the protease, is performed in the presence of the protease inhibitor to ensure that protease activity does not persists during the PBRT reaction.
Definitions
As used herein, the terms “virus”, “enveloped virus”, “virus-particle” are used interchangeably and refer to a structure that in one attribute resembles a virus but which may or may not been demonstrated to be infectious and/or capable of replication. On the other hand, a “virus-like particle (VLP)” refers to a structure that in one attribute resembles a virus but lacks genetic material. Thus, a VLP is not infectious and/or capable of replication; thus, a VLP is not harmful to a subject. However, an immune response is generated when the subject is vaccinated with a VLP as if the immune system has been presented with a virus.
Examples of Human retroviruses include but are not limited to HIV-1, HIV-2, HTLV-I, and HTLV-II; simian retroviruses include but are not limited to SIV-1 and SIV-2; other mammalian retroviruses include but are not limited to MLV, FeLV, BLV, and MMTV; and bird retroviruses include but are not limited to ALV and ASV.
Any virus, e.g., retrovirus, which comprises RT can be detected by the present methods and kits.
As used herein, a sample is any sample (e.g., a test sample) that is suspected of containing a virus particle, e.g., as a contaminant. The sample may be a protein solution, a peptide solution, a salt solution, an intravenous (IV) solution, a drug substance, a culture medium, a cell suspension, a suspension comprising an explanted tissue from a subject (e.g., a tissue biopsy or an organ), a blood product, a biological product, an antibody-containing solution (e.g., a monoclonal antibody), a vaccine (e.g., an inactivated viral vaccine and a live virus vaccine), or VLP preparation.
A “biological product,” as used herein, refers to a product or material that is produced by a cell or organism. The biological product may be a natural product of the organism, or may be produced by an organism that has been altered in some way such that it produces the biological product. Examples of biological products include, but are not limited to, vaccines, antibodies (e.g., monoclonal antibodies), therapeutic proteins, viruses (e.g., recombinant viruses for gene therapy such as, for example, adenovirus, vaccinia virus, pox virus, adeno-associated virus, and herpes virus), enzymes, growth factors, polysactharides, nucleic acids including DNA and RNA, and particles (e.g., virus-like particles). Other non-limiting examples of a biological product include blood (and a blood component), semen, urine, saliva, sputum, and cerebrospinal fluid obtained from a subject. Examples of a blood component include whole blood, serum, plasma, and platelets.
The subject may be from a mammal. The mammal can be a human or a non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, ox, buffalo, sheep, or pig. The subject can also be a bird or fowl. In embodiments, the mammal is a human.
The term “drug substance”, as used herein, refers to the material that contains a therapeutic drug and that is used to formulate, along with excipients, a pharmaceutical composition or drug product.
The terms “isolated”, “purified”, or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. An isolated product can be obtained from a natural source or can be synthetically obtained; for example, a nucleic acid molecule can be obtained from a cell expressing a gene or coding sequence (i.e., in vitro) and/or can be obtained from a machine that synthesizes the molecule. In other words, a nucleic acid used herein can be naturally-produced or synthetically-produced.
As used herein, the term “primer” refers to a string of linked nucleotide residues comprising a sufficient number of residues to be used in a PCR reaction or in a reverse transcription reaction. A primer may be used to amplify, copy, reverse-transcribe, confirm, or reveal the presence of an identical, similar, or complementary DNA or RNA in a sample. As used herein, the term “probe” refers to a nucleic acid sequence used in the detection of identical, similar, or complementary nucleic acid sequences. A primer can be used to bind to RNA, DNA, or cDNA.
As used herein, a sample is “substantially free” of a virus particle means that the sample does not comprise a detectable level of the virus particle as measured by a PCR or RT-PCR assay or the like.
In some embodiments, the methods and kits described herein utilize enzymes to degrade RNA, such as ribonucleases (RNases). RNases are nucleases that degrade RNA. In some embodiments, RNases provide a thorough means to degrade a non-encapsulated RNA that might otherwise be resistant to degradation due to aggregation. RNases can be endoribonucleases such as RNase A, RNase H, RNase III, RNase I, and others; or exoribonucleases such as RNase II, RNase R, exoribunuclease I, or others. Ribonuclease A (RNase A) is a pancreatic ribonuclease often used in research, and specifically cleaves single-stranded RNA.
The term “DNase” refers to an enzyme that degrades DNA. Any DNase can be used in aspects of the invention. A wide variety of DNases are known and can be categorized including but not limited to DNase I, DNase II, DNase III, DNase IV, DNase V, DNase VI, DNase VII, and DNase VIII.
The term “% identical” as in “95% identical” or “at least about 95% identical” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotides occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The terms “identical” or percent “identity,” in the context of two or more nucleic acid sequences, refer to two or more sequences or subsequences that are the same sequences. Sequences are “substantially identical” if two sequences have a specified percentage of nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, or across the entire sequence where not indicated
Unless specifically stated or obvious from context, as used herein, the term “about”, is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
Throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the methods or processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the terms “a,” “an,” and “the” are understood to be singular or plural. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.
Methods for isolating, amplifying, or detecting nucleic acids, including methods for detecting and amplifying target RNA or DNA sequences (for example, by polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), or PCR-based reverse transcriptase (PBRT)) are well known in the art.
One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18th edition (1990). These texts can, of course, also be referred to in making or using an aspect of the invention.
All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.
In the below examples, the methods of the present invention are used to detect reverse transcriptase (RT) activity in a norovirus virus-like particle (VLP) drug substance that was purified from recombinant baculovirus-infected Sf9 cells, i.e., a cell line used for the propagation of recombinant baculoviruses for production of biologicals and which is known to contain integrated insect retrovirus-like elements and to release RT activity following baculovirus-induced lysis. Trace levels of RT activity present in VLP drug substance was demonstrated to be sensitive to proteinase K digestion and therefore not particle-associated. In contrast, Sf9 cell end-of-production-harvest material was shown to contain significant amounts of RT activity; only a very small fraction of this activity was proteinase K-resistant and therefore, this very small fraction is particle-associated. These data demonstrate that the vast majority of RT activity in harvest material is not particle-associated and that there is no evidence for retroviral particle contamination in purified drug substance.
EXAMPLES
Example 1
PCR-Based Reverse Transcriptase (PBRT) Assay can Quantify RT Levels in a Sample Having as Few as Six RT Molecules
In order to measure small quantities of RT activity a highly sensitive PCR-based RT (PBRT) assay was required. In separate studies involving the development of arbovirus RT-PCR assays, a particular sequence in the Dengue 4 genome was identified that demonstrates reproducible performance in RT-PCR experiments. In vitro-transcribed RNA containing this sequence as an RNA template was used for the detection of RT activity in a PBRT assay. Table 1 shows primer and probe sequences used in the employed in the Den4 RT-PCR assay.
TABLE 1
Sequence
Nucleotide
SEQ ID
(5′ to 3′)
Fluorophore
Quencher
Position
NO:
Dengue virus
AF326573
1-10649
1
type 4 strain
814669, complete
Den4 Fwd
aaaccgtgctgcctgtagct
10325-10344
2
Den4 Rev
ggtctcctctaaccgctagtcca
10415-10437
3
(rc)
Den4 Probe
cggaagctgtacgcgtg
FAM
MGB-NFQ
10392-10408
4
113 nt Den 4
aaaccgtgctgcctgtagctcc
10325-10437
5
sequence
gccaataatgggaggcgtaata
atccccagggaggccatgcgc
cacggaagctgtacgcgtggc
atattggactagcggttagagg
agacc
A 113 nt synthetic DNA sequence representing the above-mentioned portion of the Den4 genome was cloned into plasmid pcDNA 3.1 between the NotI and KpnI sites. The resulting clone was linearized with PstI. Linearized DNA was used in an in vitro transcription reaction to generate an approximate a 1.5 kb RNA molecule containing the 113 nt Den4 synthetic sequence which served as the template RNA for the PBRT assay.
For the template RNA to be useful in a PBRT assay it is helpful to eliminate all plasmid DNA template used in the in vitro transcription reaction since residual DNA would effectively yield a PCR signal in the absence of added RT. This was achieved by employing multiple rounds of Turbo™ DNase Treatment (Life Technologies™; Carlsbad, Calif., USA) followed by a final purification step employing the RNeasy Mini Kit (Qiagen; Venlo, Limburg, NL). The final purified RNA product showed no evidence of degradation when analyzed by glyoxal agarose gel electrophoresis.
The positive control RT enzyme employed in the PBRT assay was the Avian Myeloblastosis virus αβ holoenzyme of molecular weight 157,000 daltons supplied by Life Sciences, Advanced Technologies, Inc. (St Petersburg, Fla., USA). This is a highly purified enzyme preparation free of nucleases with a specific activity of 62,686 units/mg. A standard curve of this enzyme was established ranging from 0.1 nanounits (nu) to 10,000 nu per μL and 1 μL of each standard curve sample was added to a PBRT standard curve reaction which resulted in a range of 6 molecules to 600,000 molecules of RT in the PBRT standard curve. All PBRT reactions employed 5 ng of Den4 template RNA as well as the Den4 primers and probe and buffer components. Two reactions containing no added RT served as controls for background signals arising from residual DNA in the RNA template preparation. A “no template” control lacking the Den4 RNA was also run. Table 2 shows that when samples are run in triplicate for 40 cycles, a linear RT standard curve is observed with an R2 value of 0.986. The R2 value was slightly less than 0.99 due to a small amount of scatter in the 0.1 nu standard curve triplicate samples (6 molecules of RT per reaction). Importantly, the “No RT” samples exhibited Ct values only slightly less than 40, which demonstrates success in eliminating background signals due to residual DNA.
TABLE 2
Sample
10,000
1000
100
10
1
0.1
No
No
No
nu RT
nu RT
nu RT
nu RT
nu RT
nu RT
RT
RT
Template
Ct Value
17.54
20.77
24.10
27.53
30.65
34.73
39.02
39.01
≥40
These data verify the ability of the present method to quantify RT levels down to 0.1 nu or six molecules per sample.
The above-mentioned PBRT assays were run using the TaqMan RNA-to-CT 1-Step Kit (Life Technologies™, catalog numbers 4392653, 4392938, and 4392656) with the exception that the RT enzyme from the kit was not added to the reactions since the assays were being used to detect the presence of contaminating RT or RT associated with the AMV RT and MLV spikes. Cycle times and temperatures were those described by the kit manufacturer.
Example 2
The PBRT Assay can Distinguish Between Particle-Associated and Free RT Activity in Test Samples
Proteinase K (PK) was selected as an exemplary protease for distinguishing between particle-contained and free RT enzymatic activity. It was posited that under appropriate conditions PK would effectively eliminate all non-particle-contained RT activity in a given sample whereas the RT activity in intact, enveloped retroviral particles would be protected from proteolytic digestion. Conditions for this methodology were developed using norovirus VLP drug substance samples as a model. As a control for free (non-virus particle-contained) enzymatic activity samples of the VLP drug substance were supplemented with avian myeloblastosis virus (AMV) RT. As a control for RT contained in intact retrovirus particles, samples of the VLP drug substance were supplemented with murine leukemia virus (MLV) particles. An assay was deemed successful when RT activity due to AMV RT supplementation was eliminated by PK activity whereas RT activity from MLV particle supplementation was protected (i.e., RT activity persisted). It appeared that a crucial part of the assay was the ability to completely inactivate PK activity following PK treatment so that proteolytic activity would not be carried over into the PBRT assay steps and thereby reduce the RT-PCR performances. Also, it was determined that a PK inhibitor must not interfere with the PBRT assay.
It was discovered that a successful method involved one or more of the following steps:
1. Dilution of a sample to be tested into a larger volume of Proteinase K buffer, in order to dilute out the high concentration of virus-like particles in the sample, allowing for a more complete proteolytic degradation of RT not contained in virus particles.
2. Addition of a very small amount of Triton X-100 during Proteinase K digestion of virus particles helps facilitate complete digestion. Moreover, the amount of Triton X-100 used does not diminish virus particle-contained RT activity.
3. Inactivation of the Proteinase K, by the addition of PMSF, is necessary following the proteolytic digestion step.
4. Collection and concentration by high speed centrifugation through a sucrose cushion, of viral particles following the inactivation of Proteinase K should occur in the presence of the PMSF to ensure that Proteinase K activity does not persists during the PBRT reaction.
Example 3
The PBRT Assay can Distinguish in a Test Sample Activity Due to Endogenous RT, Activity Due to Supplemented RT, and Activity Due to Supplemented Virus Particle-Contained RT
Three 0.5 mL samples of Norwalk VLP drug substance were analyzed according to the PBRT assay of Example 1. Sample 1 was not supplemented. Samples 2 and 3, which served as positive controls, were supplemented with AMV RT or MLV, respectively. Following supplementation, 25 μL of each sample was set aside to determine baseline RT activities, i.e., prior to proteolytic digestion. The remainder of each sample was subjected to PK digestion, followed by recovery of intact virus particles by high speed centrifugation. Prior to assaying for RT activity, all samples were diluted 1:2 with 2× AMV dilution buffer (a source of non-ionic detergent) to lyse (disrupt) any enveloped particles, thereby ensuring that all RT activity was being measured. The PK digested and set aside samples were assayed for RT activity using the PBRT assay described in Example 1. Data from this experiment are shown in Table 3
TABLE 3
Ct Values from PBRT (40 cycles)
Sample 1 -
Sample 2 -
Sample 3 -
No supple-
supplemented with
supplemented with
mentation
AMV RT
MLV particles
Baseline RT
33.3
17.6
18.4
Post-PK and
39.7
39.4
16.5
Recovery
No RT Control
38.5
As shown in Table 3, test sample 1, which was not supplemented, exhibited a small level of baseline RT activity (Ct value=33.3) prior to PK treatment. However, following PK treatment and recovery, the Ct value increased to 39.7, which is similar to “No RT control” levels. These data indicate that the baseline RT activity is not particle associated, is susceptible to PK digestion, and is not recoverable by high speed centrifugation. Sample 2, which was supplemented with AMV RT, behaved as expected in that it exhibited very strong RT baseline activity (Ct=17.6) prior to PK digestion but after PK digestion and high speed centrifugation RT activity returned to “No RT Control” levels. This indicates that both the endogenous and supplemented RT activities were degraded by PK. Similar to sample 2, sample 3, which was supplemented with MLV particles, exhibited strong baseline RT activity (Ct value=18.4). However, unlike sample 2, in sample 3, PK digestion and high speed recovery of intact particles actually resulted in an increase in RT activity (Ct value=16.5) due to concentration of the PK-resistant, intact MLV particles by the centrifugation recovery step.
These data support a premise that small amounts of measurable RT activity is present in Norwalk VLP drug substance which is not particle associated and is susceptible to proteolytic degradation. In other words, this endogenous RT activity is not due to intact retrovirus particle contamination.
The experiments described above were replicated with similar results observed. See, Table 4.
TABLE 4
Ct Values from PBRT (40 cycles)
Sample 1 -
Sample 2 -
Sample 3 -
No supple-
supplemented with
supplemented with
mentation
AMV RT
MLV particles
Baseline RT
35.5
35.5
35.5
Post-PK and
39.0
39.0
39.0
Recovery
No RT Control
40.0
In control experiments for the data presented in Tables 3 and 4, every sample tested in the PBRT reactions was also tested following supplementation with AMV RT in order to test for the possibility of protease carry over into the PBRT reactions. In this case every “spiked” sample showed a similarly strong RT signal demonstrating good performance of the PBRT reaction which was indicative of the absence of protease carryover.
Example 4
End of Production Harvest Material Fractions have Greater RT Activity Relative to Earlier Harvest Fractions
It has been shown that particle-associated errantivirus RNA sequences are more prevalent in end of production (EOP) harvest material fractions (i.e., baculovirus-lysed Sf9 cells) relative to medium harvested from healthy, non-baculovirus-infected Sf9 cells; it is believed that EOP harvest material fractions contain greater amounts both free and particle-associated RT than medium harvested from healthy Sf9 cells.
EOP harvest test samples were tested as described in Example 3 samples except that the EOP samples were diluted 10-fold in PK buffer; this was because a preliminary experiment demonstrated exceptionally high initial RT levels in EOP test samples, which would hinder distinguishing RT activity among samples. Table 5 shows the results of this experiment.
TABLE 5
Ct Values from PBRT (40 cycles)
EOP
EOP
EOP
Sample 1 -
Sample 2 -
Sample 3 -
No supple-
supplemented with
supplemented with
mentation
AMV RT
MLV particles
Baseline RT
19.7
18.7
18.4
Post-PK and
30.7
30.4
19.4
Recovery
No RT Control
39.4
As can be seen in Table 5, EOP Sample 1, the un-supplemented sample, had a strong baseline RT activity. Baseline RT activity for EOP Sample 2, the AMV RT-supplemented sample, was only about two-fold greater. EOP Samples 1 and 2 exhibited essentially identical Ct values as each other following PK digestion and their recovery that fell to within the RT standard curve range, which indicates the presence of a PK-resistant, particle-contained fraction of RT activity in EOP harvest material. On the other hand, EOP Sample 3, the MLV-supplemented sample, showed strong baseline RT activity and activity after PK digestion; this is consistent with the PK-resistant-MLV spike which served as a control for the successful recovery of RT from intact retrovirus particles.
Data from the three EOP samples are consistent with the presence of PK-resistant, particle-contained RT in EOP harvest material. However, the particle-contained RT is clearly a small fraction with respect to the total amount of RT activity in the initial harvest sample since the pre- and post-PK signals differ by over three orders of magnitude when compared to the RT standard curve. This observation is consistent with the absence of detectable particle-associated RT activity in purified drug substance, as described in Example 3. The low level of particle-associated RT activity in harvest material to begin with, coupled with the use of a detergent step in the VLP purification process, are likely important factors leading to the absence of detectable particle-associated RT activity in the final drug substance.
Example 5
Alternative Divalent Cation Test
Data presented in the prior Examples were generated in experiments employing Mg2+ ions in the RT-PCR buffer. In order to allow for the possibility of detecting novel RT activity from enzymes requiring Mn2+ ions, the drug substance evaluation experiment was repeated with the addition of 3 mM MnCl2 in the RT-PCR master mix buffer. Results of these experiments are shown in Table 6.
TABLE 6
Ct Values from PBRT (40 cycles)
(MnCl2 supplementation)
EOP
EOP
EOP
Sample 1 -
Sample 2 -
Sample 3 -
No supple-
supplemented with
supplemented with
mentation
AMV RT
MLV particles
Baseline RT
40.0
29.3
25.4
Post-PK and
40.0
40.0
24.5
Recovery
No RT Control
40.0
Table 6 shows that the outcomes are qualitatively similar to the results shown in Tables 3 and 4 with the exception that all of the Ct values were skewed upward Therefore, Mn2+ ions did not stimulate the detection of a novel Mn2+-dependent RT that might have been present in an as of yet unidentified contaminating retrovirus. RT activity in drug substance and the RT and MLV spikes resulted in the same patterns previously observed.
EQUIVALENTS
The invention can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
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15542265
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takeda vaccines, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:05PM
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Apr 1st, 2022 06:05PM
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Takeda Pharmaceutical
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Health Care
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Pharmaceuticals & Biotechnology
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