Inorganic polyatomic oxyanions for protecting against antigenic damage during pathogen inactivation for vaccine production
Provided are methods for rapidly inactivating a pathogen, or for producing a vaccine composition containing an inactivated noninfectious pathogen having retained antigenicity and/or immunogenicity, comprising exposing the pathogen to a chemical inactivating agent (e.g., one or more chemical oxidizing, alkylating or crosslinking agents) in the presence of inorganic polyatomic oxyanions in an amount and for a time sufficient to render the pathogen noninfectious while enhancing retention of pathogen antigenicity and/or immunogenicity relative to that retained by contacting the pathogen with the chemical inactivating agent alone. The methods are broadly applicable to pathogens having RNA or DNA genomes (e.g., including viruses, bacteria, fungi, and parasites). Also provided are vaccine compositions (medicaments) containing a pathogen inactivated by exposure to an inactivating agent in the presence of elevated concentrations of inorganic polyatomic oxyanions, and methods for eliciting an immune response in a subject by administering the vaccine compositions.
1. A method for producing an immunogenic vaccine composition comprising an inactivated pathogen, the method comprising:
contacting a pathogen, having an RNA or DNA genome, with a chemical inactivating agent in the presence of one or more inorganic polyatomic oxyanions in an amount and for a time-period sufficient for the chemical inactivating agent to render the pathogen noninfectious while enhancing retention of pathogen immunogenicity relative to that retained by contacting the pathogen with the chemical inactivating agent without the one or more inorganic polyatomic oxyanions, wherein the inorganic polyatomic oxyanions comprise one or more of:
phosphate (HPO 4 2− ) at a level of at least 50 mM;
sulfate (SO 4 2− ) at a level of at least 25 mM;
trimetaphosphate (P 3 O 9 3− ) at a level of at least 0.10 mM; or
triphosphate (P 3 O 10 5− ) at a level of at least 0.05 mM, and wherein the chemical inactivating agent comprises one or more of a chemical oxidizing, alkylating, or crosslinking agent.
2. The method of claim 1 , wherein the chemical inactivating agent comprises one or more of hydrogen peroxide, formaldehyde, β-propiolactone (BPL), binary ethylenimine (BEI) inactivation, or Fenton reagent(s) comprising hydrogen peroxide in combination with a transition metal.
3. The method of claim 1 , wherein the inorganic polyatomic oxyanion is a polyatomic oxyanion selected from one or more of sodium phosphate (Na 2 HPO 4 ) at a level of at least 50 mM; sodium sulfate (Na 2 SO 4 ) at a level of at least 25 mM; sodium trimetaphosphate (Na 3 P 3 O 9 ) at a level of at least 0.10 mM; sodium triphosphate (Na 5 P 3 O 10 ) at a level of at least 0.05 mM; or magnesium sulfate (MgSO 4 ) at a level of at least 150 mM.
4. The method of claim 3 , wherein the inorganic polyatomic oxyanion is one or more of sodium phosphate (Na 2 HPO 4 ) at a level of at least 100 mM, at least 500 mM, at least 750 mM, at least 1000 mM, and at least 1500 mM; sodium sulfate (Na 2 SO 4 ) at a level of at least 50 mM, at least 100 mM, at least 500 mM, at least 750 mM, at least 1000 mM, and at least 1500 mM; sodium trimetaphosphate (Na 3 P 3 O 9 ) at a level of at least 0.5 mM, at least 1.5 mM, at least 3 mM, at least 10 mM, at least 15 mM, at least 30 mM, or at least 60 mM; sodium triphosphate (Na 5 P 3 O 10 ) at a level of at least 0.1 mM, at least 0.5 mM, at least 1.5 mM, at least 3 mM, at least 10 mM, at least 15 mM, or at least 30 mM; or magnesium sulfate (MgSO 4 ) at a level of at least 250 mM, at least 500 mM, at least 750 mM, at least 1000 mM, and at least 1500 mM.
5. The method of claim 1 , further comprising verifying immunogenicity of the noninfectious pathogen using pathogen-specific antibody, B cell or T cell immunoassays, agglutination assays, or antigen binding assays, wherein producing an immunogenic vaccine composition comprising an inactivated pathogen is afforded.
6. The method of claim 2 , wherein the Fenton reagent comprises hydrogen peroxide in combination with at least one transition metal ion selected from ions of Cu, and/or Fe.
7. The method of claim 6 , wherein a mixture of different transition metal ions are used in combination with hydrogen peroxide.
8. The method of claim 1 , wherein the pathogen is a virus, or a bacterium.
9. The method of claim 8 , wherein the pathogen is a virus.
10. The method of claim 9 , wherein the virus is from Family Togaviridae, Flaviviridae, Poxviridae, or Orthomyxoviridae.
11. The method of claim 9 , wherein the virus is from Family: Togaviridae, Genus: Alphavirus, Family: Flaviviridae, Genus: Flavivirus, Family: Poxviridae, Genus: Orthopoxvirus, or Family: Orthomyxoviridae, Genus: Influenzavirus.
12. The method of claim 9 , wherein the virus is chikungunya virus (CHIKV, Family: Togaviridae, Genus: Alphavirus), dengue virus serotypes 1-4 (DENV 1-4), and yellow fever virus (YFV, Family: Flaviviridae, Genus: Flavivirus), vaccinia virus (VV, Family: Poxviridae, Genus: Orthopoxvirus), or influenza virus (Family: Orthomyxoviridae, Genus: Influenzavirus).
13. The method of claim 8 , wherein the pathogen is a bacterium.
14. The method of claim 13 , wherein the bacterium is Campylobacter.
15. The method of claim 14 , wherein the Campylobacter is C. coli or C. jejuni.
16. The method of claim 13 , wherein the bacterium is Shigella spp.
17. The method of claim 13 , wherein the bacterium is Listeria spp.
18. The method of claim 1 , wherein the pathogen is isolated or purified prior to contacting with the inactivating reagent.
19. The method of claim 2 , wherein contacting the pathogen comprises contacting the pathogen with hydrogen peroxide or with the Fenton reagent, in the presence of the one or more inorganic polyatomic oxyanions, and with a compound having formula I:
wherein R 1 is independently H or C1-C4 alkyl optionally substituted with —OH; wherein R 2 is independently H, C1-C2 alkyl optionally substituted with —OH or with aryl; and wherein X is independently H or halogen; and pharmaceutically acceptable salts thereof.
20. The method of claim 19 , wherein X and R 2 are H; and wherein R 1 is H (isatin β-thiosemicarbazone), —CH 3 (N-methyl-isatin β-thiosemicarbazone (methisazone)), or propyl (N-propyl-isatin β-thiosemicarbazone).
21. The method of claim 20 , wherein X is H, and R 1 is —CH 3 (N-methyl-isatin β-thiosemicarbazone (methisazone))
22. The method of claim 2 , wherein contacting the pathogen comprises contacting the pathogen with the Fenton reagent and one or more compounds each having one of formulas II-V:
wherein R 1 is H or C1-C4 alkyl optionally substituted with —OH; and wherein X is independently H or halogen; and salts, including pharmaceutically acceptable salts thereof;
wherein R 1 is H or C1-C4 alkyl optionally substituted with —OH; wherein X is independently H or halogen; and wherein R 2 is independently H, C1-C2 alkyl optionally substituted with —OH, or with aryl; and salts, including pharmaceutically acceptable salts thereof; and
wherein R 2 and R 3 are independently H, C1-C2 alkyl optionally substituted with —OH, or with aryl; and salts, including pharmaceutically acceptable salts thereof; and combinations thereof.
23. The method of claim 22 , wherein X of formula (II) is H, and R 1 of formula (II) is H (isatin), —CH 3 (N-methyl-isatin), or propyl (N-propyl-isatin); wherein X, R 1 , and R 2 of formula (III) are H (indole, 2,3-dione, 3-hydrazone); wherein R 2 and R 3 of formula (IV) are H (thiosemicarbazide); and wherein R 2 and R 3 of formula (V) are H (semicarbazide).
24. The method of claim 22 , wherein contacting the pathogen comprises contacting the pathogen with the Fenton reagent, thiosemicarbazide, and a compound having formula VI:
wherein R 1 is H or C1-C4 alkyl.
25. The method of claim 24 , wherein R 1 is H (isatin), —CH 3 (N-methyl-isatin), or propyl (N-propyl-isatin).
26. The method of claim 24 , wherein R 1 is H (isatin).
27. An immunogenic vaccine composition having an inactivated pathogen, produced by the method of claim 1 .
28. A method of eliciting an immune response against a pathogen, the method comprising:
obtaining an immunogenic vaccine composition having an inactivated pathogen prepared by the method of claim 27 ; and
administering the immunogenic vaccine composition to a subject, thereby eliciting in the subject an immune response against the pathogen.
29. A method for inactivating a pathogen, the method comprising:
contacting a pathogen having an RNA or DNA genome with hydrogen peroxide, or a Fenton reagent containing hydrogen peroxide in combination with a transition metal, in the presence of one or more inorganic polyatomic oxyanions, in an amount and for a time-period sufficient for the hydrogen peroxide or the Fenton reagent to render the pathogen noninfectious while retaining pathogen immunogenicity, wherein the inorganic polyatomic oxyanions comprise one or more of:
phosphate (HPO 4 2− ) at a level of at least 50 mM;
sulfate (SO 4 2− ) at a level of at least 25 mM;
trimetaphosphate (P 3 O 9 3− ) at a level of at least 0.10 mM; or
triphosphate (P 3 O 10 5− ) at a level of at least 0.05 mM.
30. The method of claim 29 , wherein the contacting is with the Fenton reagent comprising hydrogen peroxide in combination with a transition metal, in the presence of the one or more inorganic polyatomic oxyanions.
31. The method of claim 29 , wherein the inorganic polyatomic oxyanion is a polyatomic oxyanion selected from one or more of sodium phosphate (Na 2 HPO 4 ) at a level of at least 50 mM; sodium sulfate (Na 2 SO 4 ) at a level of at least 25 mM; sodium trimetaphosphate (Na 3 P 3 O 9 ) at a level of at least 0.10 mM; sodium triphosphate (Na 5 P 3 O 10 ) at a level of at least 0.05 mM; or magnesium sulfate (MgSO 4 ) at a level of at least 150 mM.
32. The method of claim 31 , wherein the inorganic polyatomic oxyanion is one or more of sodium phosphate (Na 2 HPO 4 ) at a level of at least 100 mM, at least 500 mM, at least 750 mM, at least 1000 mM, and at least 1500 mM; sodium sulfate (Na 2 SO 4 ) at a level of at least 50 mM, at least 100 mM, at least 500 mM, at least 750 mM, at least 1000 mM, and at least 1500 mM; sodium trimetaphosphate (Na 3 P 3 O 9 ) at a level of at least 0.5 mM, at least 1.5 mM, at least 3 mM, at least 10 mM, at least 15 mM, at least 30 mM, or at least 60 mM; sodium triphosphate (Na 5 P 3 O 10 ) at a level of at least 0.1 mM, at least 0.5 mM, at least 1.5 mM, at least 3 mM, at least 10 mM, at least 15 mM, or at least 30 mM; or magnesium sulfate (MgSO 4 ) at a level of at least 250 mM, at least 500 mM, at least 750 mM, at least 1000 mM, and at least 1500 mM.
33. The method of claim 29 , wherein the Fenton reagent comprises hydrogen peroxide in combination with at least one transition metal ion selected from the group consisting of Cu, and Fe.
34. The method of claim 29 , wherein a mixture of different transition metal ions are used in combination with hydrogen peroxide.
35. The method of claim 29 , wherein the pathogen is a virus, or a bacterium.
36. The method of claim 35 , wherein the pathogen is a virus.
37. The method of claim 36 , wherein the virus is from Family Togaviridae, Flaviviridae, Poxviridae, or Orthomyxoviridae.
38. The method of claim 36 , wherein the virus is from Family: Togaviridae, Genus: Alphavirus, Family: Flaviviridae, Genus: Flavivirus, Family: Poxviridae, Genus: Orthopoxvirus, or Family: Orthomyxoviridae, Genus: Influenzavirus.
39. The method of claim 38 , wherein the virus is chikungunya virus (CHIKV, Family: Togaviridae, Genus: Alphavirus), dengue virus serotypes 1-4 and yellow fever virus (DENV 1-4, YFV, Family: Flaviviridae, Genus: Flavivirus), vaccinia virus (VV, Family: Poxviridae, Genus: Orthopoxvirus), or influenza virus (Family: Orthomyxoviridae, Genus: Influenzavirus).
40. The method of claim 35 , wherein the pathogen is a bacterium.
41. The method of claim 40 , wherein the bacterium is Campylobacter.
42. The method of claim 41 , wherein the Campylobacter is C. coli or C. jejuni.
43. The method of claim 40 , wherein the bacterium is Shigella spp.
44. The method of claim 40 , wherein the bacterium is Listeria spp.
45. The method of claim 29 , wherein the pathogen is isolated or purified prior to the contacting.
46. The method of claim 29 , wherein contacting the pathogen further comprises contacting the pathogen with a compound having formula I:
wherein R 1 is independently H or C1-C4 alkyl optionally substituted with —OH; wherein R 2 is independently H, C1-C2 alkyl optionally substituted with —OH or with aryl; and wherein X is independently H or halogen; and pharmaceutically acceptable salts thereof.
47. The method of claim 46 , wherein X and R 2 are H; and wherein R 1 is H (isatin β-thiosemicarbazone), —CH 3 (N-methyl-isatin β-thiosemicarbazone (methisazone)), or propyl (N-propyl-isatin β-thiosemicarbazone).
48. The method of claim 47 , wherein R 1 is —CH 3 (N-methyl-isatin β-thiosemicarbazone (methisazone)).
49. The method of claim 29 , wherein contacting the pathogen further comprises contacting the pathogen with one or more compounds each having one of formulas II-V:
wherein R 1 is H or C1-C4 alkyl optionally substituted with —OH; and wherein X is independently H or halogen; and salts, including pharmaceutically acceptable salts thereof;
wherein R 1 is H or C1-C4 alkyl optionally substituted with —OH; wherein X is independently H or halogen; and wherein R 2 is independently H, C1-C2 alkyl optionally substituted with —OH, or with aryl; and salts, including pharmaceutically acceptable salts thereof, and
wherein R 2 and R 3 are independently H, C1-C2 alkyl optionally substituted with —OH, or with aryl; and salts, including pharmaceutically acceptable salts thereof; and combinations thereof.
50. The method of claim 49 , wherein X of formula II is H, and R 1 of formula (II) is H (isatin), —CH 3 (N-methyl-isatin), or propyl (N-propyl-isatin); wherein X, R 1 , and R 2 of formula (III) are H (indole, 2,3-dione, 3-hydrazone); wherein R 2 and R 3 of formula (IV) are H (thiosemicarbazide); and wherein R 2 and R 3 of formula (V) are H (semicarbazide).
51. The method of claim 49 , wherein the methisazone reagent comprises thiosemicarbazide and a compound having formula VI:
wherein R 1 is H or C1-C4 alkyl.
52. The method of claim 51 , wherein R 1 is H (isatin), —CH 3 (N-methyl-isatin), or propyl (N-propyl-isatin).
53. The method of claim 51 , wherein R 1 is H (isatin).