Methods and compositions for inducing immune responses against
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
(i) a binary toxin (CDT) portion having an amino acid sequence that compises SEO ID NO: 22 or an amino acid sequence having at least 90% homology to SEQ ID NO: 22;
(ii) a Toxin A portion having an amino acid sequence that comprises SEQ ID NO:24 or an amino acid sequence having at least 90% homology to SEQ ID NO: 24;
(iii) a first Toxin B portion having an amino acid sequence that comprises SEQ ID NO: 23 or an amino acid sequence having at least 90% homology to SEQ ID NO: 23; and
(iv) a second Toxin B portion having an amino acid sequence that comprises SEO ID NO: 25 or an amino acid sequence having at least 90% homology to SEQ ID NO: 25;
wherein each portion is from a C. difficile toxin protein.
2. The multivalent immunogenic polypeptide of claim 1 , wherein the Toxin A portion is between the two Toxin B portions.
3. The multivalent immunogenic polypeptide of claim 1 , wherein the CDT portion is N-terminal to one or both of the Toxin B portions.
4. 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.
5. The multivalent immunogenic peptide of claim 4 , 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).
6. The multivalent immunogenic polypeptide of claim 1 , wherein the polypeptide comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO: 21.
7. The multivalent immunogenic polypeptide of claim 1 , wherein the polypeptide comprises an amino acid sequence that is at least 97% homologous to SEQ ID NO: 21.
8. The multivalent immunogenic polypeptide of claim 1 , wherein the polypeptide comprises an amino acid sequence that is at least 99% homologous to SEQ ID NO: 21.
9. The multivalent immunogenic polypeptide of claim 1 , wherein:
(i) the CDT portion has an amino acid sequence that is at least 95% homologous to SEQ ID NO: 22;
(ii) the Toxin A portion has an amino acid sequence that is at least 95% homologous to SEQ ID NO: 24;
(iii) the first Toxin B portion has an amino acid sequence that is at least 95% homologous to SEQ ID NO: 23; and
(iv) the second Toxin B portion has an amino acid sequence that is at least 95% homologous to SEQ ID NO: 25.
10. The multivalent immunogenic polypeptide of claim 1 , wherein:
(i) the CDT portion has an amino acid sequence that is at least 97% homologous to SEQ ID NO: 22;
(ii) the Toxin A portion has an amino acid sequence that is at least 97% homologous to SEQ ID NO: 24;
(iii) the first Toxin B portion has an amino acid sequence that is at least 97% homologous to SEQ ID NO: 23; and
(iv) the second Toxin B portion has an amino acid sequence that is at least 97% homologous to SEQ ID NO: 25.
11. The multivalent immunogenic polypeptide of claim 1 , wherein:
(i) the CDT portion has an amino acid sequence that is at least 98% homologous to SEQ ID NO: 22;
(ii) the Toxin A portion has an amino acid sequence that is at least 98% homologous to SEQ ID NO: 24;
(iii) the first Toxin B portion has an amino acid sequence that is at least 98% homologous to SEQ ID NO: 23; and
(iv) the second Toxin B portion has an amino acid sequence that is at least 98% homologous to SEQ ID NO: 25.
12. A nucleic acid molecule, comprising a polynucleotide encoding the polypeptide of claim 1 .
13. A method of preparing the polypeptide of claim 1 , comprising:
(a) expressing the polypeptide in an insect host cell,
(b) isolating, the polypeptide from. the insect host cell in the presence of a non-ionic detergent.
14. The method of claim 13 , wherein the insect host is an Sf9 cell.
15. The method of claim 13 , wherein the insect host cell is transfected with a recombinant baculovirus construct under suitable conditions for expression of the polypeptide.
16. An immunogenic composition comprising the immunogenic polypeptide of claim 1 , and a pharmaceutically acceptable carrier, excipient, or diluent.
17. The immunogenic composition of claim 16 , wherein the composition comprises an adjuvant.
18. The immunogenic composition of claim 16 , wherein the adjuvant is a saponin-based adjuvant.
19. The immunogenic composition of claim 16 , wherein the saponin-based adjuvant contains Fraction A Matrix and Fraction C Matrix.
20. The immunogenic composition of claim 19 , wherein the amount of Fraction A Matrix is about 85% to about 92% by weight and the remainder is Fraction C Matrix.