Compositions relating to a mutant Clostridium difficile toxin and methods thereof

Abstract

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.

The invention claimed is: 
     
       1. A composition comprising (a) a first polypeptide, which comprises the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 of SEQ ID NO: 4 is not present, wherein a side chain of a lysine residue of the first polypeptide is crosslinked to a beta-alanine moiety, and wherein the first polypeptide further comprises a crosslink between a side chain of an aspartic acid residue of the first polypeptide and a glycine moiety, and a crosslink between a side chain of a glutamic acid residue of the first polypeptide and a glycine moiety; and (b) a second polypeptide, which comprises the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 of SEQ ID NO: 6 is not present, wherein a side chain of a lysine residue of the second polypeptide is crosslinked to a beta-alanine moiety, and wherein the second polypeptide further comprises a crosslink between a side chain of an aspartic acid residue of the second polypeptide and a glycine moiety, and a crosslink between a side chain of a glutamic acid residue of the second polypeptide and a glycine moiety. 
     
     
       2. The composition according to  claim 1 , wherein the second polypeptide further comprises a dehydroalanine moiety. 
     
     
       3. The composition according to  claim 1 , wherein the composition is immunogenic. 
     
     
       4. The composition according to  claim 1 , wherein the composition comprises an immunologically effective amount of the first polypeptide and an immunologically effective amount of the second polypeptide. 
     
     
       5. The composition according to  claim 1 , wherein the composition is lyophilized. 
     
     
       6. The composition according to  claim 1 , wherein the composition further comprises an adjuvant. 
     
     
       7. The composition according to  claim 1 , wherein the composition further comprises aluminum hydroxide. 
     
     
       8. The composition according to  claim 1 , wherein the composition does not further comprise an adjuvant. 
     
     
       9. The composition according to  claim 1 , wherein the composition further comprises trehalose. 
     
     
       10. The composition according to  claim 1 , wherein the composition further comprises polysorbate-80. 
     
     
       11. A composition comprising (a) a first polypeptide, which comprises the amino acid sequence set forth in SEQ ID NO: 4, wherein the methionine residue at position 1 of SEQ ID NO: 4 is not present, wherein a side chain of a first lysine residue of the first polypeptide is crosslinked to a beta-alanine moiety, and wherein a second lysine residue of the first polypeptide is crosslinked to an aspartic acid residue or to a glutamic acid residue of the first polypeptide; and (b) a second polypeptide, which comprises the amino acid sequence set forth in SEQ ID NO: 6, wherein the methionine residue at position 1 of SEQ ID NO: 6 is not present, wherein a side chain of a lysine residue of the second polypeptide is crosslinked to a beta-alanine moiety, and wherein a second lysine residue of the second polypeptide is crosslinked to an aspartic acid residue or to a glutamic acid residue of the second polypeptide. 
     
     
       12. The composition according to  claim 11 , wherein the second polypeptide further comprises a dehydroalanine moiety. 
     
     
       13. The composition according to  claim 11 , wherein the composition is immunogenic. 
     
     
       14. The composition according to  claim 11 , wherein the composition comprises an immunologically effective amount of the first polypeptide and an immunologically effective amount of the second polypeptide. 
     
     
       15. The composition according to  claim 11 , wherein the composition is lyophilized. 
     
     
       16. The composition according to  claim 11 , wherein the composition further comprises an adjuvant. 
     
     
       17. The composition according to  claim 11 , wherein the composition further comprises aluminum hydroxide. 
     
     
       18. The composition according to  claim 11 , wherein the composition does not further comprise an adjuvant. 
     
     
       19. The composition according to  claim 11 , wherein the composition further comprises trehalose. 
     
     
       20. The composition according to  claim 11 , wherein the composition further comprises polysorbate-80.