Claims
- 1. A hybrid polypeptide, comprising at least six different linked immunogenic peptides, wherein each peptide comprises an amino-terminal portion of a streptococcal M protein of at least 30 amino acids, and wherein the polypeptide has an amino-terminal peptide that is reiterated as a carboxy-terminal peptide and the polypeptide is capable of eliciting an immune response against more than one antigen of group A streptococci comprising at least M5, M6, M14, M19, M24, and M29.
- 2. The hybrid polypeptide according to claim 1 wherein the amino-terminal immunogenic peptide of the polypeptide is M24.
- 3. The hybrid polypeptide according to claim 2 wherein the polypeptide is recombinant and the immunogenic peptides are linked in tandem, the polypeptide having a structure of M24-M5-M6-M19-M29-M14-M24.
- 4. A hybrid polypeptide according to claim 3 wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.
- 5. A hybrid polypeptide, comprising at least seven different linked immunogenic peptides, wherein each peptide comprises an amino-terminal portion of a streptococcal M protein of at least 35 amino acids, and wherein the polypeptide has an amino-terminal peptide that is reiterated as a carboxy-terminal peptide and the polypeptide is capable of eliciting an immune response against more than one antigen of group A streptococci comprising at least M2, M11, M22, M33, M43, M59, and M94.
- 6. The hybrid polypeptide according to claim 5 wherein the amino-terminal immunogenic peptide of the polypeptide is M2.
- 7. The hybrid polypeptide according to claim 6 wherein the polypeptide is recombinant and the immunogenic peptides are linked in tandem, the polypeptide having a structure of M2-M43-M94-M22-M11-M59-M33-M2.
- 8. A hybrid polypeptide according to claim 7 wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:4.
- 9. A hybrid polypeptide, comprising at least seven different linked immunogenic peptides, wherein each peptide comprises an amino-terminal portion of a streptococcal M protein of at least 40 amino acids, and wherein the polypeptide has an amino-terminal peptide that is reiterated as a carboxy-terminal peptide and the polypeptide is capable of eliciting an immune response against more than one antigen of group A streptococci comprising at least M75, M76, M77, M89, M92, M101, and M114.
- 10. The hybrid polypeptide according to claim 9 wherein the amino-terminal immunogenic peptide of the polypeptide is M89.
- 11. The hybrid polypeptide according to claim 10 wherein the polypeptide is recombinant and the immunogenic peptides are linked in tandem, the polypeptide having a structure of M89-M 101-M77-M 114-M75-M76-M92-M89.
- 12. A hybrid polypeptide according to claim 11 wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:6.
- 13. A hybrid polypeptide, comprising at least seven different immunogenic peptides linked in tandem, wherein each peptide comprises an amino-terminal portion of a streptococcal M protein of at least 50 amino acids and, and wherein the polypeptide has an amino-terminal peptide that is reiterated as a carboxy-terminal peptide and the polypeptide is capable of eliciting an immune response against more than one antigen of group A streptococci comprising at least Spa, M1.0, M1.2, M3, M12, M18, and M28.
- 14. The hybrid polypeptide according to claim 13 wherein the amino-terminal immunogenic peptide of the polypeptide is M1.0.
- 15. The hybrid polypeptide according to claim 14 wherein the polypeptide is recombinant and the immunogenic peptides are linked in tandem, the polypeptide having a structure of M10.0-M12-Spa-M28-M3-M11.2-M18-M1.0.
- 16. A hybrid polypeptide according to claim 15 wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:8.
- 17. The hybrid polypeptide according to any one of claims 3, 7, 11, and 15 wherein the immunogenic peptides are linked by at least two amino acids encoded by a nucleic acid sequence that is a restriction enzyme recognition site.
- 18. The hybrid polypeptide according to claim 17 wherein the nucleic acid sequence is a restriction enzyme recognition site; the recognition site being at least one of BamHI, ClaI, EcoRI, HindIII, KpnI, NcoI, NheI, PmlI, PstI, SalI, and XhoI.
- 19. The hybrid polypeptide according to any one of claims 1-16 wherein the polypeptide is capable of eliciting at least one opsonic antibody that is not a tissue cross-reactive antibody in a subject.
- 20. The hybrid polypeptide according to claim 19 wherein the subject is a human or an animal.
- 21. The hybrid polypeptide according to any one of claims 1-16 further comprising a carboxy-terminal tag.
- 22. The hybrid polypeptide according to claim 21 wherein the carboxy-terminal tag is selected from the group consisting of alkaline phosphatase, β-galactosidase, hexahistidine, epitope tag DYKDDDDK (SEQ ID NO:18), epitope tag DLYDDDDK (SEQ ID NO:19), and GST.
- 23. The hybrid polypeptide according to claim 22 wherein the carboxy-terminal tag is hexahistidine.
- 24. The hybrid polypeptide according to any one of claims 1-16 further comprising at least one additional carboxy-terminal amino acid.
- 25. The hybrid polypeptide according to claim 24 wherein the additional carboxy-terminal amino acid is a D-amino acid.
- 26. The hybrid polypeptide according to claim 24 wherein the additional carboxy-terminal amino acid is cysteine.
- 27. The hybrid polypeptide according to claim 23 further comprising at least one additional carboxy-terminal amino acid.
- 28. The hybrid polypeptide according to claim 27 wherein the additional carboxy-terminal amino acid is cysteine.
- 29. A hybrid polypeptide comprising the amino acid sequence of SEQ ID NOS:2, 4, 6, or 8.
- 30. A nucleic acid molecule comprising a sequence encoding a hybrid polypeptide of SEQ ID NOS:2, 4, 6, or 8.
- 31. A nucleic acid expression construct comprising an expression control sequence operably linked to a polynucleotide encoding a hybrid polypeptide of SEQ ID NOS:2, 4, 6, or 8.
- 32. The construct according to claim 31 wherein the expression control sequence is an inducible promoter.
- 33. The construct according to claim 32 wherein the inducible promoter is selected from the group consisting of a lac, tac, trc, ara, trp, X phage, T7 phage, and T5 phage promoter.
- 34. The construct according to claim 32 wherein the inducible promoter is a T5 phage promoter.
- 35. The construct according to claim 31 wherein the construct comprises a nucleic acid expression vector selected from the group comprising a plasmid, phagemid, shuttle vector, cosmid, and virus.
- 36. The construct according to claim 31 wherein the construct comprises a nucleic acid expression vector, wherein the vector is a plasmid.
- 37. The construct according to claim 36 wherein the plasmid is pT5 (SEQ ID NO:17).
- 38. A host cell containing a construct according to any one of claims 31-37.
- 39. The host cell of claim 38 wherein the host cell is selected from the group consisting of a bacterium, a yeast cell, a nematode cell, an insect cell, and a mammalian cell.
- 40. The host cell of claim 39 wherein the host cell is a bacterium, wherein the bacterium is Escherichia coli.
- 41. A method of producing a hybrid polypeptide, comprising culturing a host cell containing a nucleic acid expression vector comprising at least one expression control sequence operably linked to a nucleic acid molecule encoding a hybrid polypeptide of SEQ ID NOS:2, 4, 6, or 8, under conditions and for a time sufficient for expression of the polypeptide.
- 42. The method of claim 41 wherein the expression control sequence is an inducible promoter.
- 43. The method according to claim 42 wherein the inducible promoter is selected from the group consisting of a lac, tac, trc, ara, trp, k phage, T7 phage, T5 phage promoter.
- 44. The method according to claim 42 wherein the inducible promoter is a T5 phage promoter.
- 45. The method according to claim 42 wherein the nucleic acid expression vector is pT5 (SEQ ID NO: 17).
- 46. A hybrid polypeptide produced according to the method of claim 45.
- 47. A composition, comprising a pharmaceutically acceptable carrier and a hybrid polypeptide according to any one of claims 1-16, 29, and 46.
- 48. A composition, comprising a pharmaceutically acceptable carrier and a mixture of at least two of the hybrid polypeptides according to claims 1, 5, 9, and 13.
- 49. The composition according to claim 48 wherein the polypeptides comprise immunogenic peptides linked in tandem, wherein said at least two polypeptides comprise a structure of M24-M5-M6-M19-M29-M14-M24, M2-M43-M94-M22-M11-M59-M33-M2, M89-M101-M77-M114-M75-M76-M92-M89, or M1.0-M12-Spa-M28-M3-M1.2-M18-M1.0.
- 50. A composition, comprising a pharmaceutically acceptable carrier and a mixture of the hybrid polypeptide according to claim 13 with at least one of the hybrid polypeptides according to any one of claims 1, 5, or 9.
- 51. The composition according to claim 50 wherein the polypeptides comprise immunogenic peptides linked in tandem, wherein the polypeptide according to claim 13 comprises a structure of M1.0-M12-Spa-M28-M3-M1.2-M18-M1.0 and said at least one other polypeptide comprise a structure of M24-M5-M6-M19-M29-M14-M24, M2-M43-M94-M22-M11-M59-M33-M2, or M89-M101-M77-M114-M75-M76-M92-M89.
- 52. A composition, comprising a pharmaceutically acceptable carrier and a mixture of at least three of the hybrid polypeptides according to claims 1, 5, 9, and 13.
- 53. The composition according to claim 52 wherein the polypeptides comprise immunogenic peptides linked in tandem, wherein said at least three polypeptides comprise a structure of M24-M5-M6-M19-M29-M14-M24, M2-M43-M94-M22-M11-M59-M33-M2, M89-M101-M77-M 114-M75-M76-M92-M89, or M1.0-M12-Spa-M28-M3-M1.2-M18-M1.0.
- 54. A composition, comprising a pharmaceutically acceptable carrier and a mixture of hybrid polypeptides according to claims 4, 8, 12, and 16.
- 55. The composition according to claim 47 wherein the polypeptides are in equimolar amounts.
- 56. The composition according to claim 47 wherein at least one of the hybrid polypeptides includes a Spa immunogenic peptide.
- 57. The composition according to claim 47 further comprising an adjuvant.
- 58. The composition according to claim 57 wherein the adjuvant is alum or Freund's.
- 59. The composition according to claim 54 further comprising an adjuvant, wherein said adjuvant is alum.
- 60. A method for preventing a microbial infection, comprising administering to a subject a composition according to claim 47 at a dose sufficient to elicit antibodies specific for one or more hybrid polypeptide, wherein the antibodies are opsonic and are not tissue cross-reactive.
- 61. The method for preventing an infection according to claim 60 wherein the microbial infection is a streptococcal infection.
- 62. The method for preventing an infection according to claim 60 wherein the streptococcal infection is a group A streptococcal infection.
- 63. The method for preventing an infection according to claim 60 wherein the hybrid polypeptide is administered by a route selected from the group consisting of enteral, parenteral, transdermal, transmucosal, and inhalation.
- 64. The method for preventing an infection according to claim 60 wherein the composition further comprises an adjuvant.
- 65. The method for preventing an infection according to claim 64 wherein the adjuvant is alum or Freund's.
- 66. The method for preventing an infection according to claim 60 wherein the subject is human or an animal.
- 67. A plurality of antibodies produced by the method according to claim 60.
- 68. The plurality of antibodies according to claim 67 wherein the antibodies further comprise at least one antibody specific for a M protein antigen not represented in a hybrid polypeptide.
- 69. The plurality of antibodies according to claim 68 wherein the M protein antigen not represented in a hybrid polypeptide is M4.
- 70. A method for treating or preventing a microbial infection, comprising administering to a subject a composition comprising a pharmaceutically acceptable carrier and a plurality of antibodies according to claims 67.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/348,434 filed Oct. 26, 2001. This provisional application is incorporated herein by reference in its entirety.
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under Grant No. AI-10085 awarded by the National Institutes of Health, and support from the Department of Veteran Affairs, Merit Review funds. The government may have certain rights in this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60348434 |
Oct 2001 |
US |