Compositions and methods for treating or preventing
Clostridium difficile is a leading cause of antibiotic-associated infection in hospitals worldwide. There is a need for a vaccine to Clostridium difficile that can target toxic proteins, or that can elicit adequate immunity to prevent infection or reduce the severity of infection. Modified Clostridium difficile toxin A and B (TcdA and TcdB) proteins are described herein, which comprise mutations that reduce toxin A and B toxicity compared to the native toxin. The proteins described are highly similar to the native toxin of Clostridium difficile , but toxicity is reduced.
1. A recombinant Clostridium toxin protein comprising a mutant of SEQ ID NO: 11 (AGISAGIPSLVNNEL), wherein the mutant comprises 1 to 4 mutations in LVNN of SEQ ID NO: 11.
2. The recombinant protein of claim 1 , wherein the mutation is L to K.
3. The recombinant protein of claim 1 , wherein the mutation is VNN to SAS.
4. The recombinant protein of claim 1 , wherein the mutant of SEQ ID NO: 11 comprises a mutant of SEQ ID NO: 12 (VPLAGISAGIPSLVNNELVL) or SEQ ID NO: 13 (LPIAGISAGIPSLVNNELIL).
5. The recombinant protein of claim 4 , wherein the mutant of SEQ ID NO:12 or SEQ ID NO: 13 comprises:
SEQ ID NO: 14 (VPLAGISAGIPSKVNNELVL);
SEQ ID NO: 15 (LPIAGISAGIPSKVNNELIL);
SEQ ID NO: 16 (VPLAGISAGIPSKSASELVL); or
SEQ ID NO: 17 (LPIAGISAGIPSKSASELIL).
6. A recombinant protein which is a Clostridium TcdA toxin protein (SEQ ID NO: 1) comprising a L1108K mutation.
7. A recombinant protein which is a Clostridium TcdB toxin protein (SEQ ID NO: 2) comprising a L1106K mutation.
8. A recombinant protein which is a Clostridium TcdA toxin protein (SEQ ID NO: 1) comprising mutations V1109S, N1110A, and N1111S.
9. A recombinant protein which is a Clostridium TcdB toxin protein (SEQ ID NO: 2) comprising mutations V1107S, N1108A, and N1109S.
10. The recombinant protein of claim 1 , wherein epithelial cell toxicity is reduced by 100-fold or greater, or 1000-fold or greater compared to wild-type Clostridium toxin.
11. The recombinant protein of claim 1 comprising:
a sequence of 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater identity to residues 958 to 1130 of TcdA (SEQ ID NO: 1); or
a sequence of 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater identity to residues 956 to 1128 of TcdB (SEQ ID NO: 2).
12. The recombinant protein of claim 1 encoded by a sequence of 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater identity to SEQ ID NO:4.
13. An immunogenic composition comprising a recombinant protein according to claim 1 , and a pharmaceutically acceptable excipient.
14. A vaccine comprising the immunogenic composition of claim 13 and a pharmaceutically acceptable adjuvant.