Novel human cytomegalovirus (HCMV) cytotoxic T cell epitopes, polyepitopes, compositions comprising same and diagnostic and prophylactic and therapeutic uses therefor

Abstract

The present invention provides CTL epitope peptides and polyepitope peptides from 14 distinct antigens of human cytomegalovirus (HCMV) that are restricted through HLA the must commonly prevalent class I alleles in different ethnic populations of the world. These epitopes provide an important platform for CTL epitope-based vaccines against HCMV. The present invention further provides vaccine compositions comprising the subject epitope and polyepitope peptides and methods for vaccination of humans and for the adoptive transfer of HCMV-specific T cells to human subjects. The present invention further provides reagents and methods for determining the HCMV status or level of HCMV-specific immunity of a subject.

1 . An isolated peptide comprising a cytotoxic T-lymphocyte (CTL) epitope of an antigen of a cytomegalovirus of humans (HCMV) selected from the group consisting of pp28, pp50, pp65, pp71, pp150, gB, gH, IE-1, IE-2, US2, US3, US6, US11, and UL18 wherein said peptide consists of an amino acid sequence having about 9 to about 20 contiguous amino acids of said antigen and wherein:
 (i) said CTL epitope of pp65 consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 22 (SQEPMSIYVY); SEQ ID NO: 23 (ATVQGQNLKY); SEQ ID NO: 24 (IRETVELRQY); SEQ ID NO: 25 (IGDQYVKVY); SEQ ID NO: 26 (TVQGQNLKY); SEQ ID NO: 27 (YRIQGKLEY); SEQ ID NO: 28 (QVIGDQYVK); SEQ ID NO: 29 (LLLQRGPQY); SEQ ID NO: 30 (RVTGGGAMA); SEQ ID NO: 31 (GVMTRGRLK); SEQ ID NO: 32 (VYALPLKML); SEQ ID NO: 33 (QYDPVAALF); SEQ ID NO: 34 (VYYTSAFVF); SEQ ID NO: 35 (DVPSGKLFM); SEQ ID NO: 36 (DIDLLLQRG); SEQ ID NO: 37 (YVKVYLESF); SEQ ID NO: 38 (TVQGQNLKY); SEQ ID NO: 39 (EPMSIYVYAL); SEQ ID NO: 40 (HVRVSQPSL); SEQ ID NO: 41 (QARLTVSGL); SEQ ID NO: 42 (RRRHRQDAL); SEQ ID NO: 43 (QPKRRRHRQ); SEQ ID NO: 44 (LCPKSIPGL); SEQ ID NO: 47 (SEHPTFTSQY); SEQ ID NO: 48 (CEDVPSGKLF); SEQ ID NO: 49 (NEIHNPAVF); SEQ ID NO: 50 (RETVELRQY); SEQ ID NO: 51 (QEPMSIYVY); SEQ ID NO: 52 (QMWQARLTV); SEQ ID NO: 53 (LMNGQQIFL); SEQ ID NO: 54 (ILARNLVPM); SEQ ID NO: 56 (QEFFWDANDIY); SEQ ID NO: 57 (QEFFWDANDI); SEQ ID NO: 58 (QYRIQGKLE); SEQ ID NO: 59 (RKHRHLPVADAV); SEQ ID NO: 60 (DPVAALFFF); SEQ ID NO: 61 (PGKISHIMLDVA); SEQ ID NO: 62 (TRATKMQVI); SEQ ID NO: 63 (QAIRETVEL); SEQ ID NO: 64 (YHRTWDRHEGA); SEQ ID NO: 65 (FMRPHERNGFTV); SEQ ID NO: 66 (CPSQEPMSIYVY); SEQ ID NO: 67 (LNIPSINVHHYPSAAERKHR); SEQ ID NO: 68 (ATVQGQNLKYQEFFWDANDI); SEQ ID NO: 69 (QEFFWDANDIYRIFAELEGV); SEQ ID NO:70 (PQYSEHPTFTSQYRIQGKLE); SEQ ID NO:71 (SQYRIQGKLEYRHTWDRHDE); SEQ ID NO: 72 (VFTWPPWQAGILARNLVPMV); SEQ ID NO: 73 (ILARNLVPMVATVQGQNLKY); SEQ ID NO: 74 (DQYVKVYLESFCEDVPSGKL); SEQ ID NO: 75 (YPSAAERKHRHLPVADAVIH); SEQ ID NO: 76 (QYDPVAALFFFDIDLLLQRG); SEQ ID NO: 77 (IIKPGKISHIMLDVAFTSHE); SEQ ID NO: 78 (AHELVCSMENTRATKMQVIG); SEQ ID NO: 79 (TRATKMQVIGDQYVKVYLES); SEQ ID NO: 80 (MNGQQIFLEVQAIRETVELR); SEQ ID NO: 81 (QAIRETVELRQYDPVAALFF); SEQ ID NO: 82 (LTVSGLAWTRQQNQWKEPDV); SEQ ID NO: 83 (WQPAAQPKRRRHRQDALPGP); SEQ ID NO: 84 (YRHTWDRHDEGAAQGDDDVW); SEQ ID NO: 85 (TSAGRKRKSASSATACTSGV); SEQ ID NO: 86 (HRQDALPGPCIASTPKKHRG); SEQ ID NO: 87 (YYTSAFVFPTKDVALRHWC); SEQ ID NO: 88 (VTTERKTPRVTGGGAMAGAS); SEQ ID NO: 89 (QPFMRPHERNGFTVLCPKNM); SEQ ID NO: 90 (SICPSQEPMSIYVYALPLKM); SEQ ID NO: 91 (IYVYALPLKMLNIPSINVHH); SEQ ID NO: 92 (QQNQWKEPDVYYTSAFVFPT); SEQ ID NO: 93 (GAAQGDDDVWTSGSDSDEEL); SEQ ID NO: 94 (TGGGAMAGASTSAGRKRKSA); and SEQ ID NO: 95 (KDVALRHVVCAHELVCSMEN;   (ii) said CTL epitope of IE-1 consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 96 (SLLSEFCRV);   SEQ ID NO: 97 (VLAELVKQI); SEQ ID NO: 98 (ILGADPLRV); SEQ ID NO: 99 (TMYGGISLL); SEQ ID NO: 100 (LLSEFCRVL); SEQ ID NO: 101 (VLEETSVML); SEQ ID NO: 102 (CLQNALDIL); SEQ ID NO: 103 (ILDEERDKV); SEQ ID NO: 104 (IKEHMLKKY);   SEQ ID NO: 105 (DEEEAIVAY); SEQ ID NO: 106 (KLGGALQAK); SEQ ID NO: 107 (QYILGADPL); SEQ ID NO: 108 (KYTQTEEKF); SEQ ID NO: 109 (KARAKKDEL);   SEQ ID NO: 110 (VMKRRIEEI); SEQ ID NO: 111 (RHRIKEHML); SEQ ID NO: 112 (ELRRKMMYM); SEQ ID NO: 113 (QIKVRVDMV); SEQ ID NO: 114 (ELKRKMMYM);   SEQ ID NO: 115 (RRKMMYMCY); SEQ ID NO: 116 (AYAQKIFKIL); SEQ ID NO: 117 (CSPDEIMAYAQKIFKILDEE); SEQ ID NO: 118 (SEPVSEIEEVAPEEEEDGAE);   SEQ ID NO: 119 (VLCCYVLEETSVMLAKRPLI); SEQ ID NO: 120 (RRKMMYMCYRNIEFFTKNSA); and SEQ ID NO: 121 (NIEFFTKNSAFPKTTNGCSQ); and   (iii) said CTL epitope of pp150 consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 122 (GQTEPIAFV); SEQ ID NO: 130 (RPSTPRAAV); SEQ ID NO: 131 (SPWAPTAPL); SEQ ID NO: 132 (KARDHLAVL); SEQ ID NO: 133 (WPRERAWAL);   SEQ ID NO: 134 (NVRRSWEEL); SEQ ID NO: 138 (RIEENLEGV); SEQ ID NO: 139 (PLIPTTAVI); SEQ ID NO: 140 (LIEDFDIYV); SEQ ID NO: 141 (KMSVRETLV); SEQ ID NO: 142 (FLGARSPSL); SEQ ID NO: 143 (ALVNAVNKL); SEQ ID NO: 144 (ALVNFLRHL); SEQ ID NO: 145 (NILQKIEKI); SEQ ID NO: 146 (ERAWALKNPHLA); SEQ ID NO: 147 (WPRERAWALKNPHLAYNPFR); SEQ ID NO: 148 (STSQKPVLGKRVATPHASAR); and SEQ ID NO: 149 (HANTALVNAVNKLVYTGRLI).   
     
     
         2 - 100 . (canceled) 
     
     
         101 . An isolated polyepitope peptide comprising two or more cytotoxic T-lymphocyte (CTL) epitopes of the same or different antigen of a cytomegalovirus of humans (HCMV), wherein said antigen is selected from the group consisting of pp28, pp50, pp65, pp71, pp150, gB, gH, IE-1, IE-2, US2, US3, US6, US11, and UL18 and wherein said peptide consists of an amino acid sequence having about 9 to about 20 contiguous amino acids of said antigen and wherein:
 (i) said CTL epitope of pp65 consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 22 (SQEPMSIYVY); SEQ ID NO: 23 (ATVQGQNLKY); SEQ ID NO: 24 (IRETVELRQY); SEQ ID NO: 25 (IGDQYVKVY); SEQ ID NO: 26 (TVQGQNLKY); SEQ ID NO: 27 (YRIQGKLEY); SEQ ID NO: 28 (QVIGDQYVK); SEQ ID NO: 29 (LLLQRGPQY); SEQ ID NO: 30 (RVTGGGAMA); SEQ ID NO: 31 (GVMTRGRLK); SEQ ID NO: 32 (VYALPLKML); SEQ ID NO: 33 (QYDPVAALF); SEQ ID NO: 34 (VYYTSAFVF); SEQ ID NO: 35 (DVPSGKLFM); SEQ ID NO: 36 (DIDLLLQRG); SEQ ID NO: 37 (YVKVYLESF); SEQ ID NO: 38 (TVQGQNLKY); SEQ ID NO: 39 (EPMSIYVYAL); SEQ ID NO: 40 (HVRVSQPSL); SEQ ID NO: 41 (QARLTVSGL); SEQ ID NO: 42 (RRRHRQDAL); SEQ ID NO: 43 (QPKRRRHRQ); SEQ ID NO: 44 (LCPKSIPGL); SEQ ID NO: 47 (SEHPTFTSQY); SEQ ID NO: 48 (CEDVPSGKLF); SEQ ID NO: 49 (NEIHNPAVF); SEQ ID NO: 50 (RETVELRQY); SEQ ID NO: 51 (QEPMSIYVY); SEQ ID NO: 52 (QMWQARLTV); SEQ ID NO: 53 (LMNGQQIFL); SEQ ID NO: 54 (ILARNLVPM); SEQ ID NO: 56 (QEFFWDANDIY); SEQ ID NO: 57 (QEFFWDANDI); SEQ ID NO: 58 (QYRIQGKLE); SEQ ID NO: 59 (RKHRHLPVADAV); SEQ ID NO: 60 (DPVAALFFF); SEQ ID NO: 61 (PGKISHIMLDVA); SEQ ID NO: 62 (TRATKMQVI); SEQ ID NO: 63 (QAIRETVEL); SEQ ID NO: 64 (YHRTWDRHEGA); SEQ ID NO: 65 (FMRPHERNGFTV); SEQ ID NO: 66 (CPSQEPMSIYVY); SEQ ID NO: 67 (LNIPSINVHHYPSAAERKHR); SEQ ID NO: 68 (ATVQGQNLKYQEFFWDANDI); SEQ ID NO: 69 (QEFFWDANDIYRIFAELEGV); SEQ ID NO:70 (PQYSEHPTFTSQYRIQGKLE); SEQ ID NO:71 (SQYRIQGKLEYRHTWDRHDE); SEQ ID NO: 72 (VFTWPPWQAGILARNLVPMV); SEQ ID NO: 73 (ILARNLVPMVATVQGQNLKY); SEQ ID NO: 74 (DQYVKVYLESFCEDVPSGKL); SEQ ID NO: 75 (YPSAAERKHRHLPVADAVIH); SEQ ID NO: 76 (QYDPVAALFFFDIDLLLQRG); SEQ ID NO: 77 (IIKPGKISHIMLDVAFTSHE); SEQ ID NO: 78 (AHELVCSMENTRATKMQVIG); SEQ ID NO: 79 (TRATKMQVIGDQYVKVYLES); SEQ ID NO: 80 (MNGQQIFLEVQAIRETVELR); SEQ ID NO: 81 (QAIRETVELRQYDPVAALFF); SEQ ID NO: 82 (LTVSGLAWTRQQNQWKEPDV); SEQ ID NO: 83 (WQPAAQPKRRRHRQDALPGP); SEQ ID NO: 84 (YRHTWDRHDEGAAQGDDDVW); SEQ ID NO: 85 (TSAGRKRKSASSATACTSGV); SEQ ID NO: 86 (HRQDALPGPCIASTPKKHRG); SEQ ID NO: 87 (YYTSAFVFPTKDVALRHWC); SEQ ID NO: 88 (VTTERKTPRVTGGGAMAGAS);   SEQ ID NO: 89 (QPFMRPHERNGFTVLCPKNM); SEQ ID NO: 90 (SICPSQEPMSIYVYALPLKM); SEQ ID NO: 91 (IYVYALPLKMLNIPSINVHH); SEQ ID NO: 92 (QQNQWKEPDVYYTSAFVFPT); SEQ ID NO: 93 (GAAQGDDDVWTSGSDSDEEL); SEQ ID NO: 94 (TGGGAMAGASTSAGRKRKSA); and SEQ ID NO: 95 (KDVALRHWCAHELVCSMEN;   (ii) said CTL epitope of IE-1 consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 96 (SLLSEFCRV); SEQ ID NO: 97 (VLAELVKQI); SEQ ID NO: 98 (ILGADPLRV); SEQ ID NO: 99 (TMYGGISLL); SEQ ID NO: 100 (LLSEFCRVL); SEQ ID NO: 101 (VLEETSVML); SEQ ID NO: 102 (CLQNALDIL); SEQ ID NO: 103 (ILDEERDKV); SEQ ID NO: 104 (IKEHMLKKY); SEQ ID NO: 105 (DEEEAIVAY); SEQ ID NO: 106 (KLGGALQAK); SEQ ID NO: 107 (QYILGADPL); SEQ ID NO: 108 (KYTQTEEKF); SEQ ID NO: 109 (KARAKKDEL); SEQ ID NO: 110 (VMKRRIEEI); SEQ ID NO: 111 (RHRIKEHML); SEQ ID NO: 112 (ELRRKMMYM); SEQ ID NO: 113 (QIKVRVDMV); SEQ ID NO: 114 (ELKRKMMYM); SEQ ID NO: 115 (RRKMMYMCY); SEQ ID NO: 116 (AYAQKIFKIL); SEQ ID NO: 117 (CSPDEIMAYAQKIFKILDEE); SEQ ID NO: 118 (SEPVSEIEEVAPEEEEDGAE); SEQ ID NO: 119 (VLCCYVLEETSVMLAKRPLI); SEQ ID NO: 120 (RRKMMYMCYRNIEFFTKNSA); and SEQ ID NO: 121 (NIEFFTKNSAFPKTTNGCSQ); and   (iii) said CTL epitope of pp150 consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 122 (GQTEPIAFV); SEQ ID NO: 130 (RPSTPRAAV); SEQ ID NO: 131 (SPWAPTAPL); SEQ ID NO: 132 (KARDHLAVL); SEQ ID NO: 133 (WPRERAWAL);   SEQ ID NO: 134 (NVRRSWEEL); SEQ ID NO: 138 (RIEENLEGV); SEQ ID NO: 139 (PLIPTTAVI); SEQ ID NO: 140 (LIEDFDIYV); SEQ ID NO: 141 (KMSVRETLV);   SEQ ID NO: 142 (FLGARSPSL); SEQ ID NO: 143 (ALVNAVNKL); SEQ ID NO: 144 (ALVNFLRHL); SEQ ID NO: 145 (NILQKIEKI); SEQ ID NO: 146 (ERAWALKNPHLA); SEQ ID NO: 147 (WPRERAWALKNPHLAYNPFR); SEQ ID NO: 148 (STSQKPVLGKRVATPHASAR); and SEQ ID NO: 149 (HANTALVNAVNKLVYTGRLI).   
     
     
         102 - 113 . (canceled) 
     
     
         114 . A prophylactic or therapeutic vaccine composition for eliciting a cellular immune response in a human subject against HCMV, said composition comprising an effective amount of the isolated peptide of  claim 1  in combination with a pharmaceutically acceptable carrier, excipient, diluent or adjuvant. 
     
     
         115 - 116 . (canceled) 
     
     
         117 . A prophylactic or therapeutic vaccine composition for eliciting a cellular immune response in a human subject against HCMV, said composition comprising: (i) an effective amount of an isolated peptide consisting of about 9 to about 20 contiguous amino acids of a pp50 antigen of a cytomegalovirus of humans (HCMV) wherein said peptide comprises a cytotoxic T-lymphocyte (CTL) epitope of said antigen; and (ii) a pharmaceutically acceptable carrier, excipient, diluent or adjuvant. 
     
     
         118 . A prophylactic or therapeutic vaccine composition for eliciting a cellular immune response in a human subject against HCMV, said composition comprising:
 (i) an effective amount of an isolated peptide consisting of an amino acid sequence selected from the group consisting of:   SEQ ID NO: 163 (LLNCAVTKL); SEQ ID NO: 164 (QLRSVIRAL); SEQ ID NO: 165 (VTEHDTLLY); SEQ ID NO: 166 (RGDPFDKNY); SEQ ID NO: 167 (GLDRNSGNY); SEQ ID NO: 168 (TLLNCAVTK); SEQ ID NO: 169 (TVRSHCVSK); SEQ ID NO: 170 (YEQHKITSY); SEQ ID NO: 171 (TRVKRNVKK); SEQ ID NO: 172 (SEDSVTFEF); and SEQ ID NO: 173 (TRLSEPPTL); and   (ii) a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.   
     
     
         119 - 142 . (canceled) 
     
     
         143 . A prophylactic or therapeutic vaccine composition for eliciting a cellular immune response in a human subject against HCMV, said composition comprising: (i) an effective amount of the isolated polyepitope peptide of  claim 114 ; and (ii) a pharmaceutically acceptable carrier, excipient, diluent or adjuvant. 
     
     
         144 . The prophylactic or therapeutic vaccine composition of  claim 114  wherein the adjuvant comprises a saponified adjuvant comprising a saponin or a saponin fraction. 
     
     
         145 - 147 . (canceled) 
     
     
         148 . The prophylactic or therapeutic vaccine composition of  claim 143  wherein the adjuvant comprises a saponified adjuvant comprising a saponin or a saponin fraction. 
     
     
         149 . A method of enhancing the HCMV-specific cell mediated immunity of a human subject comprising administering an effective amount of the isolated peptide of  claim 1  or a polyepitope peptide or vaccine composition comprising said peptide sufficient to activate a CTL or a CTL precursor of said subject. 
     
     
         150 . (canceled) 
     
     
         151 . The method of  claim 149  or  150  wherein the subject is selected from the group consisting of: (i) a subject harboring a latent HCMV infection; (ii) a subject harboring an active HCMV infection; (iii) a subject who is immune suppressed; (iv) a subject who is immune compromised; (v) a transplant recipient; and (vi) a subject at risk of suffering from a complication arising from HCMV infection. 
     
     
         152 . The method of  claim 151  wherein the transplant recipient is a bone marrow transplant recipient. 
     
     
         153 . The method of  claim 151  wherein the subject is a female having reproductive capacity or a pregnant female. 
     
     
         154 . The method of  claim 149  or  150  further comprising determining CTL activation by performing a process comprising an assay selected from the group consisting of: MHC class 1 Tetramer assay, cytotoxicity assay, assay for IFN-γ production, cytokine assay, chromium release assay and ELISPOT. 
     
     
         155 . The method of  claim 149  wherein the peptide or polyepitope or vaccine composition is administered for a time and under conditions sufficient to elicit or enhance the expansion of CD8+ T cells. 
     
     
         156 . The method of  claim 149  wherein the peptide or polyepitope or vaccine composition is administered for a time and under conditions sufficient for HCMV-specific cell mediated immunity (CMI) to be enhanced in the subject. 
     
     
         157 . The method of  claim 149  wherein the peptide or polyepitope or vaccine composition is administered by injection. 
     
     
         158 - 166 . (canceled) 
     
     
         167 . A method of enhancing the HCMV-specific cell mediated immunity of a human subject, said method comprising contacting ex vivo a T cell obtained from a human subject with an effective amount of the isolated peptide of  claim 1  or a polyepitope peptide or vaccine composition comprising said peptide sufficient to confer HCMV reactivity on said T cells. 
     
     
         168 . (canceled) 
     
     
         169 . The method of  claim 167  further comprising introducing the reactivate T cells autologously to the subject or allogeneically to another human subject. 
     
     
         170 . The method of  claim 167  wherein the T cell is contained in a biological specimen obtained from a human subject. 
     
     
         171 . The method of  claim 170  wherein the biological specimen comprises bone marrow or thymus. 
     
     
         172 . The method of  claim 170  wherein the specimen comprises blood, peripheral blood mononuclear cells (PBMC) or buffy coat. 
     
     
         173 . The method of  claim 167  further comprising obtaining a specimen comprising the T cell from the subject. 
     
     
         174 - 180 . (canceled) 
     
     
         181 . A method of providing or enhancing immunity against HCMV in an uninfected human subject comprising administering to said subject an effective amount of the isolated peptide of  claim 1  or a polyepitope peptide or vaccine composition comprising said peptide sufficient to provide immunological memory against a future infection by HCMV. 
     
     
         182 - 192 . (canceled) 
     
     
         193 . A method of providing or enhancing immunity against HCMV in an uninfected human subject, said method comprising contacting ex vivo a T cell obtained from a human subject with an effective amount of the isolated peptide of  claim 1  or a polyepitope peptide or vaccine composition comprising said peptide sufficient to confer HCMV reactivity on said T cells. 
     
     
         194 - 206 . (canceled) 
     
     
         207 . A method for determining whether or not a subject has been previously infected with HCMV, said method comprising contacting ex vivo a T cell obtained from the subject with an antigen presenting cell (APC) primed with the isolated peptide of  claim 1  or a polyepitope peptide or vaccine composition comprising said peptide and determining the activation of a CTL or precursor CTL, wherein said activation of a CTL or precursor CTL indicates that the subject has been previously infected with HCMV. 
     
     
         208 - 230 . (canceled) 
     
     
         231 . A method for determining the level of HCMV-specific cell mediated immunity in a human subject, said method comprising contacting ex vivo a T cell obtained from the subject with an antigen presenting cell (APC) primed with the isolated peptide of  claim 1  or a polyepitope peptide or vaccine composition comprising said peptide and determining the level of activation of a CTL or precursor CTL, wherein the level of activation of a CTL or precursor CTL is correlated to the level of HCMV-specific cell mediated immunity of the subject. 
     
     
         232 - 254 . (canceled) 
     
     
         255 . A method of producing an HCMV-specific CTL comprising: (i) contacting a T cell with the isolated peptide of  claim 1  or a polyepitope peptide comprising said peptide or an antigen presenting cell (APC) primed with said peptide or polyepitope peptide or an autologous lymphoblastoid cell line (LCL) primed with said peptide or polyepitope peptide; (ii) culturing the T cell; and (iii) selecting T cells that proliferate. 
     
     
         256 - 261 . (canceled) 
     
     
         262 . An HCMV-specific CTL clone produced by the method of  claim 255 . 
     
     
         263 - 270 . (canceled)