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.

1 - 54 . (canceled) 
     
     
         55 . A vaccine comprising an isolated viral antigenic peptide and a peptide comprising a T cell epitope of HSP60, wherein the HSP60 peptide enhances the immunogenicity of the viral antigenic peptide by at least two fold compared to the peptide without the HSP60 peptide. 
     
     
         56 . The vaccine of  claim 55 , wherein the peptide comprising the T cell epitope of HSP60 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.   
     
     
         57 . A conjugate comprising a viral antigen covalently attached to a synthetic peptide carrier comprising a T cell epitope of HSP60 in which said synthetic peptide 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,   wherein the viral antigen is derived from a virus belonging to a virus family selected from the group consisting of flaviviridae and herpesviridae.   
     
     
         58 . The conjugate of  claim 57 , wherein the synthetic peptide 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. 
     
     
         59 . The conjugate of  claim 57 , wherein the viral antigen is derived from a betaherpesvirus. 
     
     
         60 . The conjugate of  claim 57 , wherein the viral antigen is derived from human cytomegalovirus. 
     
     
         61 . The conjugate of  claim 60 , wherein the viral antigen is derived from IE-1 protein. 
     
     
         62 . The conjugate of  claim 61 , wherein the viral antigen comprises a CTL epitope. 
     
     
         63 . The conjugate of  claim 57 , wherein the viral antigen is derived from a flavivirus. 
     
     
         64 . The conjugate of  claim 63 , wherein the viral antigen is selected from the group consisting of: West Nile virus (WNV), Yellow fever virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Kunjin virus, Japanese encephalitis virus, Dengue virus type 1, Dengue virus type 2, Dengue virus type 3 and Dengue virus type 4. 
     
     
         65 . The conjugate of  claim 64 , wherein the viral antigen is derived from West Nile Virus. 
     
     
         66 . The conjugate of  claim 64 , wherein the viral antigen is derived from the envelope protein of the virus. 
     
     
         67 . The conjugate of  claim 66 , wherein the viral antigen is derived from the E3 domain of said envelope protein. 
     
     
         68 . The conjugate of  claim 67 , wherein said viral antigen comprises a B cell epitope and a MHC II-restricted epitope. 
     
     
         69 . The conjugate of  claim 65 , wherein the viral antigen has an amino acid sequence as set forth in any one of SEQ ID NOS:11 and 12 and 21 and analogs, homologs, derivatives and salts thereof. 
     
     
         70 . The conjugate of  claim 69  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. 
     
     
         71 . The conjugate of  claim 64 , wherein the viral antigen has an amino acid sequence as set forth in any one of SEQ ID NOS:25-44. 
     
     
         72 . The conjugate of  claim 71  having an amino acid sequence as set forth in any one of SEQ ID NOS:56-64. 
     
     
         73 . A conjugate comprising a viral antigen covalently attached to a synthetic peptide carrier comprising a T cell epitope of HSP60 in which said synthetic peptide carrier has an amino acid sequence selected from the group consisting of KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO:76) and KKDRVTDALNATRAAVEEGI (Ec27h; SEQ ID NO:86) and analogs, derivatives and salts thereof. 
     
     
         74 . The conjugate of  claim 73 , wherein the viral antigen is derived from a virus belonging to a virus family selected from the group consisting of flaviviridae and herpesviridae. 
     
     
         75 . The conjugate of  claim 74 , wherein the viral antigen is derived from WNV. 
     
     
         76 . The conjugate of  claim 74 , wherein the viral antigen has an amino acid sequence as set forth in any one of SEQ ID NOS: 11-12, 25-44 and 21. 
     
     
         77 . The conjugate of  claim 76  having an amino acid sequence as set forth in any one of SEQ ID NOS:67-75 and 77-79. 
     
     
         78 . 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 SEQ ID NOS:25-44 and analogs, fragments, derivatives and salts thereof, other than the full length envelope protein of West Nile Virus or known fragments thereof. 
     
     
         79 . An isolated peptide antigen according to  claim 78  having an amino acid sequence as set forth in any one of SEQ ID NOS: 11, 12, 21 and 25-44. 
     
     
         80 . A conjugate comprising the peptide antigen of  claim 78  covalently attached to a carrier capable of enhancing the immunogenicity of said peptide. 
     
     
         81 . A nucleic acid sequence encoding the peptide of  claim 78 . 
     
     
         82 . The nucleic acid sequence of  claim 81 , wherein the sequence is as set forth in any one of SEQ ID NOS:19-20, 22 and 45-55. 
     
     
         83 . A vector comprising the nucleic acid molecule of  claim 82  operably linked to one or more transcription control sequences. 
     
     
         84 . A host cell comprising the vector of  claim 83 . 
     
     
         85 . A vaccine composition comprising a peptide antigen according to  claim 78  further comprising at least one pharmaceutically acceptable carrier, adjuvant, excipient or diluent. 
     
     
         86 . A synthetic peptide having an amino acid sequence as set forth in any one of SEQ ID NOS:76 and 86, and analogs and derivatives thereof. 
     
     
         87 . A vaccine composition comprising an antigen and a synthetic peptide adjuvant comprising a T cell epitope of HSP60 in which said synthetic peptide adjuvant has an amino acid sequence selected from the group consisting of KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO:76) and KKDRVTDALNATRAAVEEGI (Ec27h; SEQ ID NO:86) and analogs, homologs, derivatives and salts thereof. 
     
     
         88 . The composition of  claim 87 , wherein the antigen is selected from the group consisting of: a peptide, a peptide derivative, a protein, a polysaccharide, and an antibody. 
     
     
         89 . The composition of  claim 87 , wherein the antigen is a viral antigen. 
     
     
         90 . The composition of  claim 87 , wherein the antigen is covalently attached to said synthetic peptide adjuvant. 
     
     
         91 . The composition of  claim 87  comprising an admixture of the antigen and said synthetic peptide adjuvant. 
     
     
         92 . The composition of  claim 87 , wherein said viral antigen has 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. 
     
     
         93 . A diagnostic kit comprising at least one peptide antigen according to  claim 78 , and means for determining whether the peptide antigen binds specifically to a biological sample. 
     
     
         94 . A method of enhancing the immunogenicity of a viral antigen comprising covalently conjugating the antigen with a synthetic peptide carrier 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,   wherein the viral antigen is derived from a virus belonging to a virus family selected from the group consisting of flaviviridae and herpesviridae.   
     
     
         95 . A method of enhancing the immunogenicity of an antigen comprising covalently conjugating the antigen with a synthetic peptide carrier having an amino acid sequence as set forth in any one of SEQ ID NOS:76 and 86 and analogs and derivatives thereof. 
     
     
         96 . A method of immunizing a subject in need thereof against a viral infection comprising administering to the subject an effective amount of a vaccine composition comprising a conjugate according to  claim 57 . 
     
     
         97 . The method of  claim 96 , wherein the subject is selected from a group consisting of: humans, non-human mammals and non-mammalian animals. 
     
     
         98 . The method of  claim 97 , wherein the subject is human. 
     
     
         99 . The method according to  claim 96  further comprising steps prior to immunizing the subject comprising:
 isolating an antigen derived from a virus comprising at least one epitope selected from: a CTL epitope, a B cell epitope and a MHC II-restricted epitope;   covalently conjugating the antigen to said synthetic peptide carrier; and   preparing a vaccine composition comprising the conjugate and a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.   
     
     
         100 . A method for diagnosing exposure of a subject to a flavivirus, or for diagnosing a flavivirus infection in a subject comprising the steps of:
 (a) contacting a suitable biological sample with a viral antigen according to  claim 78  under conditions such that an immune reaction can occur;   (b) determining whether the peptide antigen binds specifically to the biological sample.   
     
     
         101 . A method according to  claim 100 , 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. 
     
     
         102 . A method according to  claim 100  for the differential diagnosis of a flavivirus infection. 
     
     
         103 . The method of  claim 95 , wherein the antigen is a viral antigen. 
     
     
         104 . A method of immunizing a subject in need thereof against a viral infection comprising administering to the subject an effective amount of a vaccine composition comprising a conjugate according to  claim 73 .