Synthetic
Compositions and methods for preventing and treating pneumococcal infections are provided. Compositions include novel polypeptides comprising an amino acid sequence corresponding to the R2 i or R22 domain of CbpA or a consensus sequence of one of these domains, and variants and fragments thereof, wherein the polypeptide is stabilized in a desired conformation, particularly a loop conformation. The polypeptides of the invention may be engineered to comprise a first and a second cysteine residue, thereby resulting in the formation of a disulfide bond that stabilizes the polypeptide in the desired conformation. Alternatively, a polypeptide of the invention may be modified to create a synthetic linkage between a first and second amino acid residue present within the polypeptide, wherein the synthetic linkage stabilizes the polypeptide in the desired conformation. The polypeptides of the invention may further comprise an amino acid sequence for a T cell epitope. Compositions further include isolated nucleic acid molecules that encode the polypeptides of the invention, immunogenic compositions and vaccines comprising the disclosed polypeptides, and antibodies specific for these polypeptides.
1. An isolated polypeptide comprising an R2 2 loop polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13, wherein the polypeptide comprises a synthetic linkage and the residues of the synthetic linkage are external to the amino acid sequence of SEQ ID NO: 13, wherein said synthetic linkage stabilizes SEQ ID NO: 13 in a loop confirmation, and said R2 2 loop polypeptide having said synthetic linkage produces a protective immune response against Streptococcus pneumonia infection.
2. The isolated polypeptide of claim 1 , wherein the synthetic linkage comprises at least a first cysteine residue and a second cysteine residue, wherein the first and the second cysteine residues form a disulfide bond such that the polypeptide is stabilized in said conformation.
3. The isolated polypeptide of claim 1 , wherein the synthetic linkage comprises a synthetic peptide bond between at least a first and a second amino acid residue present in the polypeptide, wherein the synthetic peptide bond stabilizes the polypeptide in said conformation.
4. The isolated polypeptide of claim 1 , wherein said amino acid sequence is selected from the group consisting of
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:1, 3, 5, 9, 27, or 31;
b) a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, 3, 5, 9, 27, or 31, wherein said polypeptide is immunogenic; and,
c) a polypeptide comprising an amino acid sequence comprising at least 25contiguous amino acids of SEQ ID NO: 1, 3, 5, 9, 27, or 31, wherein said polypeptide produces a protective immune response against Streptococcus pneumonia infection.
5. The isolated polypeptide of claim 2 , wherein the polypeptide comprising the first and the second cysteine residues is set forth in SEQ ID NO:17, 29, 33, 43, 71, 72, 73, or 74.
6. The isolated polypeptide of claim 2 , wherein
a) the first and said second cysteine residues or the first and the second amino acid residues forming the synthetic peptide bond flank the amino acid sequence of SEQ ID NO: 1, 3, 5, 9, 27, or 31;
b) at least one of the first or the second cysteine residues or the first and the second amino acid residues forming the synthetic peptide bond are internal to SEQ ID NO: 1, 3, 5, 9, 27, or 31; or,
c) both the first and the second cysteine residues or the first and the second amino acid residues forming the synthetic peptide bond are internal to SEQ ID NO: 1, 3, 5, 9, 27, or 31.
7. The polypeptide of claim 1 , wherein the polypeptide further comprises an amino acid sequence for a T cell epitope, and wherein the amino acid sequence for the T cell epitope comprises a polypeptide set forth in SEQ ID NO:49, 56, 57, 65 or 66.
8. An immunogenic composition comprising at least one polypeptide according to claim 1 and a pharmaceutically acceptable carrier.
9. The immunogenic composition of claim 8 , wherein the polypeptide is present in an amount effective to elicit production of antibodies against Streptococcus pneumoniae when administered to an animal.
10. The immunogenic composition of claim 8 , wherein the immunogenic composition comprises a second polypeptide comprising the amino acid sequences set forth in SEQ ID NO: 19.
11. A vaccine for treating or preventing a pneumococcal infection comprising at least one polypeptide according to claim 1 in a pharmaceutically acceptable carrier, wherein the polypeptide is present in an amount effective for treating or preventing a pneumococcal infection.
12. The vaccine of claim 11 further comprising an adjuvant.
13. The vaccine of claim 11 , wherein the polypeptide further comprises an amino acid sequence comprising a T cell epitope.
14. The vaccine of claim 11 , wherein the polypeptide is covalently conjugated to a protein carrier, wherein the protein carrier is tetanus toxoid or diphtheria toxoid.
15. The vaccine of claim 11 , wherein the vaccine comprises a second polypeptide.
16. The vaccine of claim 15 , wherein the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19.
17. The vaccine of claim 16 , wherein each polypeptide further comprises an amino acid sequence comprising a T cell epitope.
18. An isolated polypeptide comprising an R2 1 loop polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 19, wherein the polypeptide comprises a synthetic linkage and the residues of the synthetic linkage are external to the amino acid sequence of SEQ ID NO: 11, wherein said synthetic linkage stabilizes SEQ ID NO: 11 in a loop confirmation, and said R2 1 loop polypeptide having said synthetic linkage produces a protective immune response against Streptococcus pneumonia infection.
19. The isolated polypeptide of claim 18 , wherein the synthetic linkage comprises at least a first cysteine residue and a second cysteine residue, wherein the first and the second cysteine residues form a disulfide bond such that the polypeptide is stabilized in said conformation.
20. The isolated polypeptide of claim 18 , wherein the synthetic linkage comprises a synthetic peptide bond between at least a first and a second amino acid residue present in the polypeptide, wherein the synthetic peptide bond stabilizes the polypeptide in said conformation.
21. The isolated polypeptide of claim 19 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21.
22. The isolated polypeptide of claim 19 , wherein
a) the first and said second cysteine residues or the first and the second amino acid residues forming the synthetic peptide bond flank the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 19, or 23;
b) at least one of the first or the second cysteine residues or the first and the second amino acid residues forming the synthetic peptide bond are internal to SEQ ID NO: 1, 3, 5, 7, 19, or 23; or,
c) both the first and the second cysteine residues or the first and the second amino acid residues forming the synthetic peptide bond are internal to SEQ ID NO: 1, 3, 5, 7, 19, or 23.
23. The polypeptide of claim 20 , wherein the polypeptide comprises a synthetic peptide bond between the glutamic acid residue at position 6 of SEQ ID NO:19 and the lysine residue at position 22 of SEQ ID NO:19.
24. The polypeptide of claim 18 , wherein the polypeptide further comprises an amino acid sequence for a T cell epitope, and wherein the amino acid sequence for the T cell epitope comprises SEQ ID NO:49, 56, 57, 65 or 66.
25. An immunogenic composition comprising at least one polypeptide according to claim 18 and a pharmaceutically acceptable carrier.
26. The immunogenic composition of claim 25 , wherein the polypeptide is present in an amount effective to elicit production of antibodies against Streptococcus pneumoniae when administered to an animal.
27. A vaccine for treating or preventing a pneumococcal infection comprising at least one polypeptide according to claim 18 in a pharmaceutically acceptable carrier, wherein the polypeptide is present in an amount effective for treating or preventing a pneumococcal infection.
28. The vaccine of claim 27 further comprising an adjuvant.
29. The vaccine of claim 27 , wherein the polypeptide further comprises an amino acid sequence for a T cell epitope.
30. The vaccine of claim 27 , wherein the polypeptide is covalently conjugated to a protein carrier, wherein the protein carrier is tetanus toxoid or diphtheria toxoid.