Compositions relating to a mutant
In one aspect, the invention relates to an immunogenic composition that includes a mutant Clostridium difficile toxin A and/or a mutant Clostridium difficile toxin B. Each mutant toxin includes a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation, relative to the corresponding wild-type C. difficile toxin. The mutant toxins may further include at least one amino acid that is chemically crosslinked. In another aspect, the invention relates to antibodies or binding fragments thereof that binds to said immunogenic compositions. In further aspects, the invention relates to isolated nucleotide sequences that encode any of the foregoing, and methods of use of any of the foregoing compositions.
1. A method for eliciting an immune response against Clostridium difficile in a mammal, said method comprising administering to the mammal an effective dose of a composition, which comprises a modified C. difficile toxin that is produced by contacting a wild-type C. difficile toxin with 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) and N-hydroxysuccinimide (NHS); wherein a side chain of a lysine residue of the modified C. difficile toxin is crosslinked to a beta-alanine moiety.
2. The method according to claim 1 , wherein the modified C. difficile toxin has been further contacted with glycine.
3. The method according to claim 1 , wherein the modified C. difficile toxin has been further contacted with alanine or glycine methyl ester.
4. The method according to claim 1 , wherein the modified C. difficile toxin comprises at least 500 contiguous amino acids of SEQ ID NO: 1.
5. The method according to claim 1 , wherein the modified C. difficile toxin comprises at least 500 contiguous amino acids of SEQ ID NO: 2.
6. The method according to claim 1 , wherein the method comprises administering two doses of the composition.
7. The method according to claim 6 , wherein the second dose is administered about 1 week after the first dose.
8. The method according to claim 6 , wherein the second dose is administered about 2 weeks after the first dose.
9. The method according to claim 6 , wherein the second dose is administered about 4 weeks after the first dose.
10. The method according to claim 6 , wherein the method comprises administering three doses of the composition.
11. The method according to claim 7 , wherein the method comprises administering four doses of the composition.
12. The method according to claim 1 , wherein the modified C. difficile toxin is purified.
13. The method according to claim 1 , wherein the composition further comprises an adjuvant.
14. The method according to claim 13 , wherein the adjuvant comprises an aluminum adjuvant.
15. The method according to claim 1 , wherein the modified C. difficile toxin comprises a crosslink between a second lysine residue of the modified C. difficile toxin and a side chain of an aspartic acid residue of the modified C. difficile toxin.
16. The method according to claim 1 , wherein the immune response against C. difficile is sustained for at least 4 weeks.
17. The method according to claim 1 , wherein the immune response elicited is sufficient to prevent a C. difficile infection in the mammal.
18. The method according to claim 1 , wherein the mammal is a human.
19. The method according to claim 13 , wherein the adjuvant comprises aluminum phosphate.
20. The method according to claim 13 , wherein the adjuvant comprises aluminum hydroxide.
21. The method according to claim 13 , wherein the adjuvant comprises a CpG oligonucleotide.
22. The method according to claim 1 , wherein the composition further comprises a carbohydrate selected from the group consisting of sorbitol, mannitol, starch, dextran, sucrose, trehalose, lactose, and glucose.
23. The method according to claim 1 , wherein the composition further comprises a surfactant.
24. The method according to claim 1 , wherein the composition further comprises polysorbate 80.
25. An immunogenic composition comprising an adjuvant and a modified C. difficile toxin that is produced by contacting a wild-type C. difficile toxin with 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) and N-hydroxysuccinimide (NHS); wherein a side chain of a lysine residue of the modified C. difficile toxin is crosslinked to a beta-alanine moiety.
26. The composition according to claim 25 , wherein a second side chain of a lysine residue of the modified C. difficile toxin is crosslinked to an aspartic acid residue of the modified C. difficile toxin.
27. The composition according to claim 25 , wherein the adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, and a CpG oligonucleotide.
28. The composition according to claim 25 , wherein the composition further comprises a carbohydrate selected from the group consisting of sorbitol, mannitol, starch, dextran, sucrose, trehalose, lactose, and glucose.
29. The composition according to claim 25 , wherein the composition further comprises a surfactant.
30. The composition according to claim 25 , wherein the composition further comprises polysorbate 80.
31. The composition according to claim 26 , wherein the composition further comprises polysorbate 80.