Methods and compositions for inducing immune responses against Clostridium difficile
Disclosed herein are methods and compositions for treating or preventing bacterial infection. In particular, the methods and compositions are directed towards C. difficile infection. In particular aspects, the compositions are vaccines containing multimeric polypeptides containing portions of multiple toxins from bacteria. The polypeptides induce effective immune responses thus treating or preventing infection.
1. A multivalent immunogenic polypeptide comprising portions of at least four Clostridium difficile ( C. difficile ) toxin proteins, wherein the portions of at least four C. difficile toxin proteins are:
(i) a binary toxin (CDT) portion comprising the receptor binding component of binary toxin (CDTb);
(ii) a Toxin A portion comprising the truncated receptor binding domain of Toxin A (TcdA) protein containing 19 repeats;
(iii) a first Toxin B portion comprising the receptor binding domain of a first toxin B protein; and
(iv) a second Toxin B portion comprising the receptor binding domain of a second toxin B protein; wherein the first toxin B protein and the second toxin B protein are from distinct C. difficile strains.
2. The multivalent immunogenic polypeptide of claim 1 , wherein at least one Toxin B protein is from an epidemic strain.
3. The multivalent immunogenic polypeptide from claim 2 , wherein the epidemic strain is the NAP1 strain.
4. The multivalent immunogenic polypeptide of claim 1 , wherein one of the Toxin B proteins is from the 630 strain.
5. The multivalent immunogenic polypeptide of claim 1 , wherein the portions are oriented with the Toxin A portion between the two Toxin B portions.
6. The multivalent immunogenic polypeptide of claim 1 , wherein the CDT portion is N-terminal to one or both of the Toxin B portions.
7. The multivalent immunogenic polypeptide of claim 1 , wherein the CDT portion has an amino acid sequence that comprises or consists of SEQ ID NO:22 or an amino acid sequence having at least 90% homology to the sequence.
8. The multivalent immunogenic polypeptide of claim 1 , wherein one of the Toxin B portions has an amino acid sequence that comprises or consists of SEQ ID NO:23 or an amino acid sequence having at least 90% homology to the sequence.
9. The multivalent immunogenic polypeptide of claim 1 , wherein the second Toxin B portion has an amino acid sequence that comprises or consists of SEQ ID NO:25 or an amino acid sequence having at least 90% homology to the sequence.
10. The multivalent immunogenic polypeptide of claim 1 , wherein the Toxin A portion has an amino acid sequence that comprises or consists of SEQ ID NO:24 or an amino acid sequence having at least 90% homology to the sequence.
11. The multivalent immunogenic peptide of claim 1 , wherein each portion is separated by a two amino acid linker, a three amino acid linker, or a four amino acid linker.
12. The multivalent immunogenic peptide of claim 11 , wherein the portions are separated by a two amino acid linker and the linker is selected from the group consisting of Alanine-Serine (AS), Leucine-Glutamic acid (LE), and Serine-Arginine (SR).
13. The multivalent immunogenic polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:21.
14. A nucleic acid molecule, comprising a polynucleotide encoding the polypeptide of claim 1 .
15. A method of preparing the polypeptide of claim 1 , comprising:
(a) expressing the polypeptide in an insect host cell,
(b) purifying the polypeptide in the presence of a non-ionic detergent in the form of a nanoparticle, and
(c) suspending the nanoparticle in a pharmaceutically acceptable carrier, excipient, or diluent.
16. The method of claim 15 , wherein the insect host is an Sf9 cell.
17. The method of claim 15 , wherein the insect host cell is transfected with a recombinant baculovirus construct under suitable conditions for expression of the polypeptide.
18. An immunogenic composition comprising the immunogenic polypeptide of claim 1 , and a pharmaceutically acceptable carrier, excipient, or diluent.
19. The immunogenic composition of claim 18 , wherein the composition comprises an adjuvant.
20. The immunogenic composition of claim 18 , wherein the adjuvant is a saponin-based adjuvant.
21. The immunogenic composition of claim 18 , wherein the saponin-based adjuvant contains Fraction A Matrix and Fraction C Matrix.
22. The immunogenic composition of claim 18 , wherein the amount of Fraction A Matrix is about 85% to about 92% by weight and the remainder is Fraction C Matrix.