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.

1 - 36 . (canceled) 
     
     
         37 . An immunogenic composition comprising a mutant  Clostridium difficile  toxin A, which comprises a glucosyltransferase domain having at least two mutations and a cysteine protease domain having at least one mutation, according to the numbering of SEQ ID NO:1, and wherein the methionine at position 1 of SEQ ID NO:1 is absent. 
     
     
         38 . The immunogenic composition of  claim 37 , wherein the glucosyltransferase domain comprises at least two mutations present at amino acid positions 101, 269, 272, 285, 287, 460, 462, 541, or 542. 
     
     
         39 . The immunogenic composition of  claim 37 , wherein the glucosyltransferase domain comprises at least two mutations selected from W101A, D269A, R272A, D285A, D287A, E460A, R462A, S541A, and L542G. 
     
     
         40 . The immunogenic composition of  claim 37 , wherein the glucosyltransferase domain comprises mutations D285A and D287A. 
     
     
         41 . The immunogenic composition of  claim 37 , wherein the cysteine protease domain comprises at least one mutation present at amino acid positions 543, 589, 655, or 700. 
     
     
         42 . The immunogenic composition of  claim 37 , wherein the cysteine protease domain comprises at least one mutation selected from S543A, D589A, D589N, H655A, and C700A. 
     
     
         43 . The immunogenic composition of  claim 37 , wherein the cysteine protease domain comprises mutation C700A. 
     
     
         44 . An immunogenic composition comprising a mutant  Clostridium difficile  toxin B, which comprises a glucosyltransferase domain having at least two mutations and a cysteine protease domain having at least one mutation, according to the numbering of SEQ ID NO:2, and wherein the methionine at position 1 of SEQ ID NO:2 is absent. 
     
     
         45 . The immunogenic composition of  claim 44 , wherein the glucosyltransferase domain comprises at least two mutations present at amino acid positions 102, 270, 273, 286, 288, 384, 461, 463, 520, or 543. 
     
     
         46 . The immunogenic composition of  claim 44 , wherein the glucosyltransferase domain comprises at least two mutations selected from W102A, D270A, D270N, R273A, D286A, D288A, N384A, D461A, D461R, K463A, K463E, W520A, and L543A. 
     
     
         47 . The immunogenic composition of  claim 44 , wherein the glucosyltransferase domain comprises mutations D286A and D288A. 
     
     
         48 . The immunogenic composition of  claim 44 , wherein the cysteine protease domain comprises at least one mutation present at amino acid positions 544, 587, 653, or 698. 
     
     
         49 . The immunogenic composition of  claim 44 , wherein the cysteine protease domain comprises at least one mutation selected from G544A, D587A, D587N, H653A and C698A. 
     
     
         50 . The immunogenic composition of  claim 44 , wherein the cysteine protease domain comprises mutation C698A. 
     
     
         51 . An immunogenic composition comprising a mutant  Clostridium difficile  toxin A, which comprises a mutation at amino acid positions 285, 287, and 700, according to the numbering of SEQ ID NO:1, and wherein the methionine at position 1 of SEQ ID NO:1 is absent, and a mutant  Clostridium difficile  toxin B, which comprises a mutation at amino acid positions 286, 288, and 698, as compared to SEQ ID NO:2, and wherein the methionine at position 1 of SEQ ID NO:2 is absent. 
     
     
         52 . The immunogenic composition of  claim 51 , wherein the mutant  C. difficile  toxin A comprises the mutations D285A, D287A, and a C700A, and the mutant  C. difficile  toxin B comprises mutations D286A, D288A, and C698A. 
     
     
         53 . The immunogenic composition of  claim 51 , wherein each mutant  C. difficile  toxin comprises at least one of:
 a) a crosslink between a side chain of an aspartic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide;   b) a crosslink between a side chain of a glutamic acid residue of the polypeptide and a side chain of a lysine residue of the polypeptide;   c) a beta-alanine moiety linked to a side chain of at least one lysine residue of the polypeptide; and   d) a glycine moiety linked to a side chain of at least one aspartic acid residue of the polypeptide or to a side chain of at least one glutamic acid residue of the polypeptide.   
     
     
         54 . The immunogenic composition of  claim 51 , wherein at least one amino acid is chemically crosslinked. 
     
     
         55 . The immunogenic composition of  claim 54 , wherein the at least one amino acid is crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). 
     
     
         56 . The immunogenic composition of  claim 54 , wherein the at least one amino acid is crosslinked by N-hydroxysuccinimide (NHS).