Compositions of hsp60 peptides and viral antigens for vaccination and diagnosis

Abstract

The present invention provides improved vaccines comprising an isolated viral antigenic peptide and a synthetic peptide derived from a T cell epitope of HSP60. The invention includes mixtures where the peptide serves as an adjuvant as well as conjugates where the peptide is covalently linked to the viral antigen. The known synthetic peptide carrier, p458, provides significantly improved immunogenicity for synthetic viral epitopes and analogs. Ec27 is a novel peptide derived from HSP60 which increases the immunogenicity substantially of the viral antigen both as a mixture or a covalent conjugate. Some of the isolated viral epitopes are novel and are claimed for diagnostic as well as therapeutic or prophylactic uses.

What is claimed is: 
     
         1 . An isolated peptide antigen having an amino acid sequence selected from the group consisting of: LVTVNPFVSVATANS (SEQ ID NO: 11), LVTVNPFVSVATANA (SEQ ID NO: 12), YIVVGRGEQQINHHWHK (SEQ ID NO: 21) and analogs, fragments, derivatives and salts thereof. 
     
     
         2 . The peptide antigen of  claim 1 , wherein the analogs have an amino acid sequence as set forth in any one of SEQ ID NOS: 25-44. 
     
     
         3 . A conjugate comprising the peptide antigen of  claim 1  covalently attached to a carrier capable of enhancing the immunogenicity of said peptide. 
     
     
         4 . The conjugate of  claim 3 , wherein the carrier is selected from the group of peptides consisting of:
 (a) NEDQKIGIEIIKRTLKI (p458h; SEQ ID NO: 1),   (b) NEDQKIGIEIIKRALKI (p458; SEQ ID NO: 2),   (c) EGDEATGANIVKVALEA (p458mt; SEQ ID NO: 3),   (d) NEDQNVGIKVALRAMEA (p458e; SEQ ID NO: 4),   (e) an analog or derivative of p458h (SEQ ID NO: 1) that has at least 70% of the electric and hydrophilicity/hydrophobicity characteristic of human HSP60 from position 458 to position 474, said peptide, analog or derivative being capable of increasing substantially the immunogenicity of the viral antigen when the conjugate is administered in vivo, preferably wherein the carrier is an analog of p458h (SEQ ID NO: 1):  458 NEDQKIGIEIIKRTLKI 474  in which the residue E 459  is either E or D; the residue D 460  is either D or E; the residue K 462  is either K or R or ornithine (Orn); the residue I 463  is either I or L, V, M, F, norleucine (Nle) or norvaline (Nva); the residue I 465  residue is either I or L, V, M, F, Nle or Nva; the residue E 466  is either E or D; the residue I 467  is either I or L, V, M, F, Nle or Nva; the residue I 468  is either I or L, V, M, F, Nle or Nva; the residue K 469  is either K or R or Orn; the residue R 470  is either R, K or Orn; the residue L 472  in either L or I, V, M, F, Nle or Nva; the residue K 473  is either K or R or Orn; and the residue I 474  is either I or L, V, M, F, Nle or Nva,   (f) KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO: 76) and analogs and derivatives thereof,   (g) KKDRVTDALNATRAAVEEGI (Ec27h; SEQ ID NO: 86) and analogs and derivatives thereof.   
     
     
         5 . The conjugate of  claim 4  having an amino acid sequence as set forth in any one of SEQ ID NOS: 13-16 and 23-24 and analogs, derivatives and salts thereof, or
 having an amino acid sequence as set forth in any one of SEQ ID NOS: 56-64, or 
 having an amino acid sequence as set forth in any one of SEQ ID NOS: 67-75 and 77-79. 
 
     
     
         6 . A nucleic acid sequence encoding the peptide of  claim 1 . 
     
     
         7 . The nucleic acid sequence of  claim 6 , wherein said sequence is as set forth in any one of SEQ ID NOS: 19-20, 22 and 45-55. 
     
     
         8 . A vector comprising the nucleic acid sequence of  claim 6  operably linked to one or more transcription control sequences. 
     
     
         9 . A host cell comprising the vector of  claim 8 . 
     
     
         10 . A vaccine composition comprising a peptide antigen according to  claim 1 , further comprising at least one pharmaceutically acceptable carrier, adjuvant, excipient or diluent. 
     
     
         11 . The vaccine of  claim 10  wherein the carrier is selected from the group consisting of:
 (a) NEDQKIGIEIIKRTLKI (p458h; SEQ ID NO: 1), 
 (b) NEDQKIGIEIIKRALKI (p458; SEQ ID NO:2), 
 (c) EGDEATGANIVKVALEA (p458mt; SEQ ID NO:3), 
 (d) NEDQNVGIKVALRAMEA (p458e; SEQ ID NO:4), 
 (e) an analog or derivative of p458h (SEQ ID NO: 1) that has at least 70% of the electric and hydrophilicity/hydrophobicity characteristic of human HSP60 from position 458 to position 474, said peptide, analog or derivative being capable of increasing substantially the immunogenicity of the viral antigen when the conjugate is administered in vivo, 
 (f) KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO:76) and analogs and derivatives thereof, 
 (g) KKDRVTDALNATRAAVEEGI (Ec27h; SEQ ID NO:86) and analogs and derivatives thereof. 
 
     
     
         12 . A vaccine composition comprising a conjugate according to  claim 3 . 
     
     
         13 . A method for immunizing a subject in need thereof against a viral infection, comprising administering to the subject a vaccine composition comprising a conjugate according to  claim 3 . 
     
     
         14 . The method of  claim 13  wherein the subject is selected from a group consisting of: humans, non-human mammals and non-mammalian animals. 
     
     
         15 . The method of  claim 14 , wherein said subject is human. 
     
     
         16 . A method for immunizing a subject in need thereof against a viral infection, comprising administering to the subject a vaccine composition comprising according to  claim 10 . 
     
     
         17 . A diagnostic kit comprising at least one peptide antigen having an amino acid sequence as set forth in any one of SEQ ID NOS:11-12, 21 and 25-44 and analogs, homologs, derivatives and salts thereof, and means for determining whether the peptide antigen binds specifically to a biological sample. 
     
     
         18 . A method for diagnosing exposure of a subject to a flavivirus or for diagnosing a flavivirus infection, comprising the steps of:
 (a) contacting a suitable biological sample with a viral antigen having an amino acid sequence as set forth in any one of SEQ ID NOS:11-12, 21 and 25-44 and analogs, homologs, derivatives and salts thereof under conditions such that an immune reaction can occur;   (b) determining whether the peptide antigen binds specifically to the biological sample.   
     
     
         19 . The method according to  claim 18 , wherein step (b) includes determining the extent of antigen-antibody complex formation, wherein an antigen-antibody complex formation level significantly higher than the level obtained for a sample obtained from a non-infected subject is indicative of exposure of the subject to the flavivirus. 
     
     
         20 . The method according to  claim 18  for the differential diagnosis of a flavivirus infection.