Immunizing compositions and methods of use
The present invention provides compositions including siderophore receptor polypeptides and porins from gram negative microbes, and preferably, lipopolysaccarhide at a concentration of no greater than about 10.0 endotoxin units per milliliter. The present invention also provides methods of making and methods of using such compositions.
1. A method for inducing the production of an antibody that specifically binds at least one SRP in a human, the method comprising administering to a human an effective amount of a composition comprising:
at least two SRPs isolated from a gram negative microbe;
LPS at a concentration of no greater than about 10.0 EU/ml; and
a pharmaceutically acceptable carrier, wherein the composition induces in the human antibody that specifically binds at least one SRP.
2. The method of claim 1 wherein the gram negative microbe is an enteropathogen.
3. The method of claim 1 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.
4. The method of claim 1 wherein the gram negative microbe is a member of the tribe Escherichieae or Salmonelleae.
5. The method of claim 1 wherein the gram negative microbe is Salmonella spp., Escherichia coli, Actinobacillus spp., Haemophilus spp., or Campylobacter spp.
6. The method of claim 1 wherein the at least two SRPs have molecular weights of between about 60 kDa and about 100 kDa as determined by sodium dodecyl-polyacrylamide gel electrophoresis.
7. The method of claim 1 further comprising at least one porin having a molecular weight of between about 30 kDa and about 43 kDa as determined by sodium dodecyl-polyacrylamide gel electrophoresis.
8. The method of claim 3 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Klebsiella spp., Serratia spp., or Yersinia spp.
9. The method of claim 3 wherein the member of the family Vibrionaceae is Vibrio cholerae.
10. The method of claim 3 wherein the member of the family Pasteurellaceae is Pasturella spp.
11. The method of claim 10 wherein the Pasturella spp, is Pasturella multocida or Pasteurella haemolytica.
12. The method of claim 3 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.
13. The method of claim 12 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.
14. The method of claim 1 wherein the gram negative microbe is a first gram negative microbe, wherein the antibody specifically binds an SRP present on the surface of a second gram negative microbe.
15. The method of claim 14 wherein the second gram negative microbe is an enteropathogen.
16. The method of claim 14 wherein the second gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.
17. The method of claim 16 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Klebsiella spp., Serratia spp., or Yersinia spp.
18. The method of claim 16 wherein the member of the family Vibrionaceae is Vibrio cholerae.
19. The method of claim 16 wherein the member of the family Pasteurellaceae is Pasturella spp.
20. The method of claim 19 wherein the Pasturella spp, is Pasturella multocida or Pasteurella haemolytica.
21. The method of claim 16 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.
22. The method of claim 21 wherein the Pseudomonas spp, is Pseudomonas aeruginosa.
23. The method of claim 14 wherein the first and second gram negative microbes are different species.
24. The method of claim 14 wherein the first and second gram negative microbes are different genera.
25. The method of claim 14 wherein the second gram negative microbe is a member of the tribe Escherichieae or Salmonelleae.
26. The method of claim 14 wherein the second gram negative microbe is Salmonella spp., Escherichia coli, Actinobacillus spp., Haemophilus spp., or Campylobacter spp.
27. The method of claim 1 wherein the human has or is at risk of having a gram negative infection.
28. The method of claim 1 wherein the method further comprises collecting the antibody.
29. The method of claim 28 wherein the method further comprises administering the collected antibody to a second human.
30. The method of claim 29 wherein the administering the collected antibody comprises administration of colostrum to the second human.
31. The method of claim 28 wherein the collecting comprises collecting serum, plasma, blood, or colostrum.
32. The method of claim 1 wherein the method further comprises isolating the antibody.
33. A method for inducing the production of an antibody that specifically binds at least one SRP in a human, the method comprising administering to a human an effective amount of a siderophore receptor polypeptide (SRP) composition comprising:
at least two SRPs isolated from a gram negative microbe;
lipopolysaccharide (LPS) at a concentration of no greater than the concentration of LPS in a reference composition comprising: the at least two siderophore receptor polypeptides isolated from the gram negative microbe and lipopolysaccharide; wherein the reference composition is produced by a process comprising:
providing the gram negative microbe;
disrupting the gram negative microbe in a buffer;
solubilizing the disrupted gram negative microbe for greater than about 24 hours in a solution comprising sarcosine to result in solubilized and insoluble cellular material, wherein a ratio of the sarcosine to gram weight of disrupted gram negative microbe is between about 0.8 gram sarcosine per about 4.5 grams of disrupted gram negative microbe and about 1.2 grams sarcosine per about 4.5 grams of disrupted gram negative microbe; and
isolating molecules of the gram negative microbe, wherein the isolated molecules comprise the at least two SRPs and LPS; and
a pharmaceutically acceptable carrier, wherein the SRP composition induces in the human antibody that specifically binds at least one SRP.
34. The method of claim 33 wherein the gram negative microbe is an enteropathogen.
35. The method of claim 33 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.
36. The method of claim 33 wherein the gram negative microbe is a member of the tribe Escherichieae or Salmonelleae.
37. The method of claim 33 wherein the gram negative microbe is Salmonella spp., Escherichia coli, Actinobacillus spp., Haemophilus spp., or Campylobacter spp.
38. The method of claim 33 wherein the at least two SRPs have molecular weights of between about 60 kDa and about 100 kDa as determined by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
39. The method of claim 33 wherein the SRP composition further comprises at least one porin having a molecular weight of between about 30 kDa and about 43 kDa as determined by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
40. The method of claim 35 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Klebsiella spp., Serratia spp., or Yersinia spp.
41. The method of claim 35 wherein the member of the family Vibrionaceae is Vibrio cholerae.
42. The method of claim 35 wherein the member of the family Pasteurellaceae is Pasturella spp.
43. The method of claim 42 wherein the Pasturella spp, is Pasturella multocida or Pasteurella haemolytica.
44. The method of claim 35 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.
45. The method of claim 44 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.
46. The method of claim 33 wherein the gram negative microbe is a first gram negative microbe, wherein the antibody specifically binds an SRP present on the surface of a second gram negative microbe.
47. The method of claim 46 wherein the second gram negative microbe is an enteropathogen.
48. The method of claim 46 wherein the second gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.
49. The method of claim 48 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Klebsiella spp., Serratia spp., or Yersinia spp.
50. The method of claim 48 wherein the member of the family Vibrionaceae is Vibrio cholerae.
51. The method of claim 48 wherein the member of the family Pasteurellaceae is Pasturella spp.
52. The method of claim 51 wherein the Pasturella spp, is Pasturella multocida or Pasteurella haemolytica.
53. The method of claim 48 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.
54. The method of claim 53 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.
55. The method of claim 48 wherein the first and second gram negative microbes are different species.
56. The method of claim 48 wherein the first and second gram negative microbes are different genera.
57. The method of claim 48 wherein the second gram negative microbe is a member of the tribe Escherichieae or Salmonelleae.
58. The method of claim 48 wherein the second gram negative microbe is Salmonella spp., Escherichia coli, Actinobacillus spp., Haemophilus spp., or Campylobacter spp.
59. The method of claim 48 wherein the human has or is at risk of having a gram negative infection.
60. The method of claim 48 wherein the method further comprises collecting the antibody.
61. The method of claim 60 wherein the method further comprises administering the collected antibody to a second human.
62. The method of claim 60 wherein the administering the collected antibody comprises administration of colostrum to the second human.
63. The method of claim 60 wherein the collecting comprises collecting serum, plasma, blood, or colostrum.
64. The method of claim 33 wherein the method further comprises isolating the antibody.
65. The method of claim 1 wherein the concentration of LPS of the composition is determined by a gel-clot test comprising a Limulus amebocyte lysate.
66. The method of claim 65 wherein the gel-clot test comprises contacting the composition with a lysate of horseshoe crab circulating amebocytes to form a mixture, wherein the pH of the composition is between 6 and 8, incubating the mixture for about 1 hour at about 37° C., and determining if there is gelation, wherein gelation indicates the presence of LPS.
67. The method of claim 33 wherein the concentration of LPS of the composition is determined by a gel-clot test comprising a Limulus amebocyte lysate.
68. The method of claim 67 wherein the gel-clot test comprises contacting the composition with a lysate of horseshoe crab circulating amebocytes to form a mixture, wherein the pH of the composition is between 6 and 8, incubating the mixture for about 1 hour at about 37° C., and determining if there is gelation, wherein gelation indicates the presence of LPS.