Immunizing compositions and methods of use
The present invention provides compositions including siderophore receptor polypeptides 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 composition comprising:
at least two siderophore receptor polypeptides (SRPs) isolated from a gram negative microbe; and
lipopolysaccharide (LPS) at a concentration of no greater than about 10.0 endotoxin units per milliliter (EU/ml).
2. The composition of claim 1 further comprising a pharmaceutically acceptable carrier.
3. The composition of claim 1 wherein the gram negative microbe is an enteropathogen.
4. The composition of claim 1 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.
5. The composition of claim 1 wherein the gram negative microbe is a member of the tribe Escherichieae or Salmonelleae.
6. The composition of claim 1 wherein the gram negative microbe is Salmonella spp., Escherichia coli , Actinobacillus spp., Haemophilus spp., or Campylobacter spp.
7. The composition of claim 1 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.
8. The composition 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 separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
9. A siderophore receptor polypeptide (SRP) composition comprising:
at least two SRPs isolated from a gram negative microbe; and
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.
10. The SRP composition of claim 9 further comprising a pharmaceutically acceptable carrier.
11. The SRP composition of claim 9 wherein the gram negative microbe is an enteropathogen.
12. The SRP composition of claim 9 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.
13. The SRP composition of claim 9 wherein the gram negative microbe is a member of the tribe Escherichieae or Salmonelleae.
14. The SRP composition of claim 9 wherein the gram negative microbe is Salmonella spp., Escherichia coli , Actinobacillus spp., Haemophilus spp., or Campylobacter spp.
15. The SRP composition of claim 9 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.
16. The SRP composition of claim 9 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.
17. The composition of claim 1 wherein the concentration of LPS is determined by a gel-clot test comprising a Limulus amebocyte lysate.
18. The composition of claim 17 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.
19. The composition of claim 9 wherein the concentration of LPS is determined by a gel-clot test comprising a Limulus amebocyte lysate.
20. The composition of claim 19 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.
21. The composition of claim 4 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Klebsiella spp., Serratia spp., Yersinia spp., Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacter spp., Flavobacterium spp., or Aeromonas spp.
22. The composition of claim 4 wherein the member of the family Vibrionaceae is Vibrio cholerae.
23. The composition of claim 4 wherein the member of the family Pasteurellaceae is Pasturella spp.
24. The composition of claim 23 wherein the Pasturella spp. is Pasturella multocida or Pasteurella haemolytica.
25. The composition of claim 4 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.
26. The composition of claim 25 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.
27. The composition of claim 12 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Klebsiella spp., Serratia spp., Yersinia spp., Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacter spp., Flavobacterium spp., or Aeromonas spp.
28. The composition of claim 12 wherein the member of the family Vibrionaceae is Vibrio cholerae.
29. The composition of claim 12 wherein the member of the family Pasteurellaceae is Pasturella spp.
30. The composition of claim 29 wherein the Pasturella spp. is Pasturella multocida or Pasteurella haemolytica.
31. The composition of claim 12 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.
32. The composition of claim 31 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.