Adhesin as immunogen against enterotoxigenic Escherichia coli

• US 20060153878A1
• Filed: 01/10/2006
• Published: 07/13/2006
• Est. Priority Date: 01/11/2005
• Status: Active Grant

• Current Assignee: Naval Medical Research Center, the united states of america as represented by the secretary of the navy
• Original Assignee: the united states of america as represented by the secretary of the navy
• Inventors: Savarino, Stephen J.
• Granted Patent: US 9,079,945 B2
• Time in Patent Office: Days
• US Class Current: 424/241.100
• CPC Class Codes: A61K 2039/523 expressing foreign proteins
• CPC Class Codes: A61K 2039/545 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/0258 Escherichia
• CPC Class Codes: A61P 1/12 Antidiarrhoeals
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 16/1232 from Escherichia (G)
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/35 containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The inventive subject matter relates to the methods for the induction of immunity and prevention of diarrhea resulting from Escherichia coli. The inventive subject matter also relates to the use Escherichia coli adhesins as immunogens and to the construction of conformationally stability and protease resistant Escherichia coli adhesin constructs useful for inducing immunity to Escherichia coli pathogenic bacteria. The methods provide for the induction of B-cell mediated immunity and for the induction of antibody capable of inhibiting the adherence and colonization of Escherichia coli including enterotoxigenic Escherichia coli, to human cells.

Claims

1. A method of inducing an immune response comprising the steps:

a. administrating a priming dose of an immunogen comprising whole or an antigenic polypeptide fragment of Escherichia coli fimbriae or whole or antigenic polypeptide fragement of Escherichia coli fibrillae with unit dose range of 50 μ

g to 1 mg of immunogen in a buffered aqueous solution;

b. administrating of boosting doses with first dose at least 1 week after priming dose with a unit dose range of 50 μ

g to 1 mg of immunogen in a buffered aqueous solution, wherein an immune response is elicited.

2. The method of claim 1, wherein said whole or antigenic polypeptide fragment of Escherichia coli fimbriae is a class 5 fimbriae.

3. The method of claim 1, wherein said Escherichia coli fimbriae is selected from the group consisting of colonization factor antigen I, CS4, CS14, CS1, PCF071, CS17, CS19 and CS2.

4. The method of claim 1, wherein said antigen polypeptide fragment of enterotoxigenic Escherichia coli fimbriae or fibrillae is a monomer or polymer of Escherichia coli adhesin polypeptides.

5. The method of claim 4, wherein said Escherichia coli fimbriae adhesin is selected from the group consisting of CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD or CotD.

6. The method of claim 1, wherein said immune response is induction of immunoglobulin molecules capable of binding to said enterotoxigenic Escherichia coli fimbriae or fibrillae.

7. The method of claim 1, wherein said immune response inhibits enterotoxigenic Escherichia coli fimbriae or fibrillae adherence to human cells.

8. The method of claim 7, wherein said human cells are mucosal epithelial cells.

9. The method of claim 1, wherein said immune response reduces or prevents diarrhea in humans.

10. The method of claim 1, wherein said immunogen is administered subcutaneously, transdermally, intramuscularly, orally, transcutaneously or nasally.

11. The method of claim 1, wherein said whole or antigenic polypeptide fragment of Escherichia coli fibrillae is coli surface antigen 3.

12. The method of claim 11, wherein said antigenic polypeptide of Escherichia coli fibrillae is an CstH polypeptide.

13. An immunogenic composition wherein said composition is a complex comprising an amino acid sequence encoding a whole or an antigenic polypeptide fragment of Escherichia coli fimbrial adhesin linked at the C-terminal end of said fimbrial adhesin to a linker which is operatively linked at the C-terminus of said linker to a whole or an antigenic polypeptide fragment of Escherichia coli major structural fimbrial subunit.

14. The immunogenic composition of claim 13, wherein said whole or an antigenic polypeptide fragment of Escherichia coli fimbrial adhesin and whole or an antigenic polypeptide fragment of Escherichia coli major structural fimbrial subunit are derived from class 5 fimbriae.

15. The immunogenic composition of claim 13, wherein said whole or an antigenic polypeptide fragment of Escherichia coli fimbrial adhesin is from Escherichia coli fimbriae selected from the group consisting of colonization factor antigen I, CS4, CS14, CS1, PCF071, CS17, CS19 and CS2.

16. The immunogenic composition of claim 13, wherein said whole or an antigenic polypeptide fragment of Escherichia coli fimbrial adhesin is a monomer or polymer of adhesin polypeptides.

17. The immunogenic composition of claim 13, wherein said whole or an antigenic polypeptide fragment of Escherichia coli fimbrial adhesin is selected from the group consisting of CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD and CotD.

18. The immunogenic composition of claim 13, wherein said linker is composed of the amino acid sequence of SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or operable fragments thereof.

19. The immunogenic composition of claim 13, wherein said whole or an antigenic polypeptide fragment of Escherichia coli fimbriae adhesin is a CfaE polypeptide composed of amino acid sequence of SEQ ID No. 4 or antigenic fragments thereof.

20. The immunogenic composition of claim 13, wherein said whole or an antigenic polypeptide fragment of Escherichia coli major structural fimbrial subunit is selected from the group consisting of CfaB, CsfA, CsuA1, CsuA2, CooA, CosA, CsbA, CsdA, and CotB.

21. The immunogenic composition of claim 20, wherein said polypeptide fragment is selected from the group consisting of SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15.

22. The immunogenic composition of claim 13, wherein said whole or an antigenic polypeptide fragment of Escherichia coli fimbriae major structural subunit confers conformational stability and protease resistance to said whole or an antigenic polypeptide fragment of Escherichia coli fimbrial adhesin.

23. The immunogenic composition of claim 13, wherein said whole or an antigenic polypeptide fragment of Escherichia coli fimbriae major structural fimbrial subunit is CfaB with SEQ ID No. 5 or SEQ ID No. 6 or antigenic fragment thereof.

24. A method of inducing an immune response comprising the steps:

a. administering said immunogenic composition as in claim 13 at a unit dose range of 50 μ

g to 1 mg;

b. administering a boosting dose of said immunogenic composition at least 1 week after priming dose with unit dose range of 50 μ

g to 1 mg in a buffered aqueous solution, wherein an immune response is elicited.

25. The method of claim 24, wherein said immune response is induced production of immunoglobulin molecules capable of binding to whole or an antigenic polypeptide fragment of Escherichia coli fimbriae.

26. The method of claim 24, wherein said immune response inhibits Escherichia coli adherence to human cells.

27. The method of claim 24, wherein said human cells are mucosal epithelial cells.

28. The method of claim 24, wherein said immunogenic composition is administered subcutaneously, transdermally, intramuscularly, orally, transcutaneously or nasally.

29. The method of claim 24, wherein said immune response reduces or prevents diarrhea in humans.

30. The method of claim 24, wherein said immune response inhibits adherence of diarrheagenic bacteria to human cells.

31. The method of claim 24, wherein said immunity reduces colonization of diarrheagenic bacteria.

32. A method of inducing an immune response comprising the steps:

a. administering a priming dose of an immunogen containing a host bacterial cell containing said immunogenic composition as in claim 13 such that adequate numbers of host bacterial cells are administered such that expressed recombinant adhesin polypeptide of said immunogenic composition is 50 μ

g to 1 mg per dose; and

b. administering 1 to 4 boosting doses of said host bacterial cell containing said immunogenic composition as in claim 13 with the first boosting dose at least 1 week after priming dose of said immunogen such that the immunogen is administered in adequate numbers of host bacterial cells per dose such that the expressed recombinant adhesin polypeptide of said immunogenic composition is 50 μ

g to 1 mg per dose, wherein an immune response is elicited.

33. The method of claim 32, wherein said host bacterial cell is a killed or live attenuated bacteria.

34. The method of claim 33, wherein said host bacterial cell is selected from the group consisting of Escherichia coli, member of the genus Shigella, member of the genus Campylobacter, member of the genus Salmonella, member of the genus Vibrio.

35. The method of claim 32, wherein said immune response is induction of immunoglobulin molecules capable of binding to said Escherichia coli fimbriae.

36. The method of claim 32, wherein said immune response inhibits Escherichia coli fimbriae adherence to human cells.

37. The method of claim 32, wherein said immune response inhibits Escherichia coli colonization to human cells

38. The method of claim 36 wherein said human cells are mucosal epithelial cells.

39. The method of claim 32, wherein said immunogen is administered orally.

40. The method of claim 32, wherein said immunity prevents diarrhea in humans.

41. The method of claim 32, wherein said immunity prevents diarrhea in humans caused by bacteria selected from the group consisting of Escherichia coli, member of the genus Shigella, member of the genus Campylobacter, member of the genus Salmonella, member of the genus Vibrio.

42. An immunogenic composition wherein said composition is a complex comprising an amino acid sequence encoding a whole or an antigenic polypeptide fragment of Escherichia coli CstH linked at the C-terminal end of said CstH to a linker which is operatively linked at the C-terminus of said linker to a donor strand polypeptide of CstH.

43. The immunogenic composition of claim 42, wherein said CstH is composed of SEQ ID No. 20 or antigenic polypeptide fragments thereof.

44. The immunogenic composition of claim 42, wherein said linker is composed of the amino acid sequence of SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or operable fragments thereof.

45. The immunogenic composition of claim 42, wherein said donor strand polypeptide duplicated polypeptide of CstH is N-terminal region of CstH composed of sequence SEQ ID No. 21.

46. The immunogenic composition of claim 42, wherein said complex comprising an amino acid sequence is SEQ ID No. 22.

47. The immunogenic composition of claim 42, wherein said complex comprising an amino acid sequence is SEQ ID No. 23.

48. A method of inducing an immune response comprising the steps:

a. administering a priming dose of an immunogen containing a host bacterial cell containing said immunogenic composition as in claim 42 such that adequate numbers of host bacterial cells are administered such that expressed recombinant adhesin polypeptide of said immunogenic composition is 50 μ

g to 1 mg per dose; and

b. administering 1 to 4 boosting doses of said host bacterial cell containing said mmunogenic composition as in claim 42 with the first boosting dose at least 1 week after priming dose of said immunogen such that the immunogen is administered in adequate numbers of host bacterial cells per dose such that the expressed recombinant adhesin polypeptide of said immunogenic composition is 50 μ

g to 1 mg per dose, wherein an immune response is elicited.

49. The method of claim 48, wherein said host bacterial cell is a killed or live attenuated bacteria.

50. The method of claim 48, wherein said host bacterial cell is selected from the group consisting of Escherichia coli, member of the genus Shigella, member of the genus Campylobacter, member of the genus Salmonella, member of the genus Vibrio.

51. The method of claim 48, wherein said immune response is induction of immunoglobulin molecules capable of binding to said Escherichia coli fimbriae.

52. The method of claim 48, wherein said immune response inhibits Escherichia coli fimbriae adherence to human cells.

53. The method of claim 48, wherein said immune response inhibits Escherichia coli colonization to human cells

54. The method of claim 52, wherein said human cells are mucosal epithelial cells.

55. The method of claim 48, wherein said immunogen is administered orally.

56. The method of claim 48, wherein said immunity prevents diarrhea in humans.

57. The method of claim 48, wherein said immunity prevents diarrhea in humans caused by bacteria selected from the group consisting of Escherichia coli, member of the genus Shigella, member of the genus Campylobacter, member of the genus Salmonella, member of the genus Vibrio.