Patent Application: US-46245300-A

Abstract:
the present invention provides cytotoxic epstein - barr virus t - cell epitopes derived from ebv structural antigens . preferred epitopes include yllemlwrl , yfleilwgl , ylleilwrl , ylqqnwwtl , lllallfwl , llvdllwll , lllialwnl , wlllflail , tllvdllwl , llwlllfla , illiialyl , vlfifgcll , rlgatiwql , ilyfiafal , slvivttfv , lmiiplinv , tlfigshvv , lipetvpyi , vlqwaslav and qltphtkav . the present invention also provides methods of treating or preventing ebv infection in subjects which involve administration of ebv cytotoxic t - cell epitopes .

Description:
lcls were established from sero - positive donors by exogenous virus transformation of peripheral b cells using the b95 . 8 ( type 1 ) or ag876 ( type 2 ) virus isolates . in addition , lcls transformed with the b95 . 8 isolate and expressing different hla a2 supertypes were also used in this study ( 12th histocompatability workshop cell panel ; eacc ). the peptide transporter ( tap )- negative b × t hybrid cell line 174 × cem . t2 ( referred to as t2 ) ( 22 ) were used for peptide stablization assays . all cell lines were routinely maintained in rpmi 1640 containing 2 mm glutamine , 100 iu / ml penicillin and 100 μg / ml streptomycin plus 10 % foetal calf serum ( fcs ) ( growth medium ). long - term cultures of ebv - negative normal b cell blasts were established as previously described using the cd40 system ( referred to as cd40 b cells ) ( 12 ). the burkitt &# 39 ; s lymphoma ( bl ) cell lines , bjab . gpti , bjab . mtlm6 , mutu cl . 59 and n = mutu cl . 216 were used in this study . these were derived from patients with non - endemic or endemic bl . bjab . mtlm6 and n = mutu cl . 59 have previously been shown to express lmp1 , while bjab . gpt1 and mutu cl . 216 are negative for lmp1 ( 5 , 27 ). these bl cell lines were routinely maintained in growth medium . to generate phytohaemagglutinin ( pha ) blasts , peripheral blood mononuclear ( pbmc ) cells were stimulated with pha ( commonwealth serum laboratories , melbourne ) and after 3 days , growth medium containing mla 144 supernatant and ril - 2 was added ( 2 ). pha blasts were propagated with bi - weekly replacement of il - 2 and nila supernatant ( no further pha added ) for up to 6 weeks . to isolate resident ebv , spontaneous lcls were established from a panel of 12 unrelated healthy ebv - seropositive donors by spontaneous outgrowth from pbmc cultured in the presence of 0 . 1 μg / ml cyclosporin a ( 19 ). in addition , virus isolates from eight different npc samples ( from southeast asia ) were directly sequenced from biopsy material . these isolates were classified as type 1 ebv based on the dna sequence divergence within the bam h1 wyh and e regions of the genome ( 23 , 24 ). specific oligonucleotide primers flanking the dna region encoding the lmp1 epitope were selected for pcr amplification . the resulting pcr products were purified using qiaquick spin columns ( qiagen inc . chatsworth , calif .) and sequenced in both directions using a prism ready reaction dyedeoxy terminator cycle sequencing kit ( applied biosystems inc ., foster city , calif .) following the manufacturer &# 39 ; s protocol . peptides synthesized by the merrifield solid phase method ( 16 ) were purchased from chiron mimotopes ( melbourne , australia ), dissolved in dimethyl sulphoxide and diluted in serum - free rpmi 1640 medium for use in standard ctl assays . “ to identify the potential hla a2 binding peptides within lmp1 , the hla binding predictions program of the bioinformnatics & amp ; molecular analysis section of the computational biosciences and engineering lab , center for information technology at the national institutes for health , 12 south drive , mcs 5624 ( building 12a , room 2033 ), bethesda md 20892 , usa , was employed as described elsewhere ( 18 ). these predicted peptides were then used in a standard mhc stablization assay using t2 cells as described earlier ( 1 ). briefly , t2 cells ( 2 × 10 5 ) were incubated with 200 μl of each of the peptides ( 200 μg / ml ) at 26 ° c . for 14 - 16h , followed by incubation at 37 ° c . for 2 - 3h . after the incubations , hla a2 expression was measured by facs using a monoclonal hla a2 - specific antibody ( ma2 . 1 ; atcc ). to generate polyclonal ctls , pbmc from hla a2 - positive donors were co - cultivated for seven days with the irradiated ( 8 , 000 rads ) t2 cells sensitized with synthetic peptides . on day 7 , these lymphocytes were restimulated with peptide - sensitized t2 cells . after 10 days of culture in growth medium , the cells were used as polyclonal effectors in a standard 51 cr - release assay against peptide - sensitized autologous pha blasts . to generate lmp1 - specific ctl clones , pbmc ( 10 6 / ml ) were cultivated with peptide sensitized autologous lymphocytes ( responder to stimulator ratio of 4 : 1 ) in 2 ml culture wells ( linbro ) for 3 days in growth medium . ctl clones , generated by seeding in 0 . 35 % agarose , were established and maintained in growth medium containing highly purified recombinant human il - 2 from e . coli ( 16 ), restimulating twice weekly with autologous lcls . these ctl clones were screened on a panel of recombinant vaccinia - infected autologous cd40 b cells to confirm the antigen specificity ( see below ). recombinant vaccinia constructs encoding ebv latent antigens and a vaccinia virus construct made by insertion of the psc11 vector alone and negative for thymidine kinase ( vacc . tk -) have been previously described ( 6 . 11 ). cd40 b cells were infected with recombinant vaccinia virus at a multiplicity of infection ( moi ) of 10 : 1 for 1 h at 37 ° c . as described earlier ( 6 , 11 ). after overnight infection , cells were washed with growth medium and processed for ctl assays or for immunoblotting to assess the expression of recombinant ebv antigens ( 12 ). target cells were either infected with recombinant vaccinia viruses or pre - sensitized with synthetic peptide epitopes ( wild - type or variant ) and then incubated with 51 cr for 90 min . following incubation , these cells were washed in growth medium and used as targets in a standard 5 h 51 cr - release assay ( 16 ). in some experiments , monoclonal antibodies ( moab ) specific for the non - polymorphic determinants on mhc class i ( w6 / 32 ) or class ii ( l243 ) antigens were added to define the mhc restriction of the ctl clones . pbmc from hla a2 - positive donors were distributed in graded numbers ( two fold dilutions ) from 6 . 25 × 103 to 5 × 104 cells per well in round - bottomed microtiter plates . approximately 5 × 10 4 k - irradiated ( 2 , 000 rads ) peptide sensitized ( 1 μg / ml ) auologous pbmc were added to give a total volume of 100 μl . twenty - four replicates were used at each concentration in each experiment . cultures were fed on days 4 and 7 with 50 μl of medium supplemented with 20u of ril - 2 and 30 % ( vol / vol ) supernatant from mla - 144 cultures . on day 10 , each ctl microculture was split into two replicates and used as effectors in a standard 5 h 51 cr - release assay against autologous pha blasts precoated with an lmp1 peptide or left uncoated . wells were scored as positive when the percent specific chromium release for peptide - sensitized target cells exceeded the mean release from untreated control wells by 3 sds . lda was performed by the method of maximum likelihood estimation ( 4 ). data from all experiments were compatible with the hypothesis of single - hit kinetics ( p & gt ; 0 . 4 ) and precursor estimates are given with 95 % confidence limits . “ to identify potential hla a2 - restricted epitopes within lmpi , the amino acid sequence was analyzed using the hla binding predictions program of the bioinformatics & amp ; molecular analysis section of the computational biosciences and engineering lab , center for information technology at the national institutes for health , 12 south drive , mcs 5624 ( building 12a , room 2033 ), bethesda md . 20892 , usa ( 18 ). a total of 11 peptides with an estimated half - time disassociation score of & gt ; 400 were selected ( table 1 ). these peptides were then tested for hla a2 bindin - efficiency using hla a2 - positive t2 cells . representative data from a series of experiments is presented in fig1 . this analysis showed that six , peptides significantly increased the expression of hla a2 on t2 cells suggesting that these peptides bind to this allele . the data presented above strongly suggested that lup1 includes sequences which can bind hla a2 molecules and are therefore potential targets for virus - specific ctls . to verify this hypothesis , pbmc from two hia a2 - positive ebv immune donors ( sb and as ) were stimulated with t2 cells sensitized with each of the hla a2 - binding peptides from lmp1 . on day 10 , these effector cells were tested against peptide - sensitized autologous pha blasts . represtative data from polyclonal ctls from donor sb are presented in fig2 . two peptides , ylqqnwwtl ( seq id no : 6 ) and yllemlwrl ( seq id no : 1 ), showed significant activation of polyclonal ctls ; however , the yllemewrl - stimulated ctls consistently showed significantly stronger ctl activity when compared to the ylqqnwvvtl - stimulated cells . similar data were also obtained for another hla a2 - positive donor ( as ) ( data not shown ). * to identify the potential hla a2 binding peptides within lmp1 , the hla binding predictions program of the bioinformatics & amp ; molecular analysis section of the computational bioscience and engineering lab , center for information technology at the national institutes for health , 12 south drive , mcs 5624 ( building 12a , room 2033 ), bethesda md 20892 , usa , was employed as described elsewhere ( 18 ). this program can be directly accessed through the world - wide web . to further characterize the ctl epitopes identified by the polyclonal ctls , vinis - specific ctl clones were generated using initial stimulation with peptide sensitized autologous pbnic followed by continous restimulation with irradiated autologous lcls . proliferating clones were screened for peptide recognition using the rapid visual assay for ctl specificity ( 2 ) and one clone , sb7 , clearly recognized the yllemlwrl ( seq id no : 1 ) peptide . no ctl clones specific for ylqqnwwtl ( seq id no : 6 ) were isolated using this procedure . to further confirm the antigen specificity of the sb7 clone , autologous cd40 - stimulated b cells were infected with recombinant vaccinia viruses encoding individual ebv antigens and then exposed to these ctls . the data presented in fig3 a clearly demonstrate that only target cells infected with the lmp1 - expressing vaccinia construct were recognized . in addition , only lmp1 - and hla a2 - positive bl cell lines ( bjab . mtlm16 or mutu cl . 59 ) were efficiently lysed by this clone , while bl cells negative for this antigen ( bjab . gpt1 and mutu cl . 216 ) were not recognized ( fig3 b ). these results confirm that yllelwrl ( seq ; id no : 1 ) is an lmp1 ctl epitope that is endogenously processed by virus - infected cells . in the next set of experiments we analyzed the frequency of ctl precursors ( ctlp ) for this epitope in pbmc from hla a2 - positive healthy ebv immune donors by limiting dilution analysis . yllemlwrl - specific ctlp were reactivated in vitro by stimulation of pbmcs from donors sb and as with autologous peptide - sensitized pbmcs . autologous pha blasts precoated with peptide yllemlwrl ( seq id no : 1 ) or untreated were used as target cells in a chromium release assay . the representative data in fig4 show that a very low frequency of memory ctl specific for the yllemelwrl ( seq id no : 1 ) epitope were detected in the hla a2 : positive donors as ( 1 : 223 , 535 +/− 107 , 437 ) and sb ( 1 : 252 , 650 +/− 122 , 875 ). to determine whether ebv - transformed lcls expressing different hla a2 supertypes could be recognized by clone sb7 , a panel of lcls expressing ten different supertypes of the hla a2 allele ( hla a * 0201 - hla a * 0210 ) were screened in a standard ctl assay . the data presented in fig5 clearly demonstrate that lcls expressing all the major hla a2 supertypes except hla a * 0205 were efficiently recognized by the ctl clone sb7 . this lysis was significantly inhibited by the hla class i - specific antibody w6 / 32 . surprisingly , hla a2 - positive and tap - negative t2 cells were also recognized by this clone suggesting that the yllemlwrl ( seq id no : 1 ) epitope is endogenously processed through a tap - independent pathway ( fig5 ). sequence analysis of the hla a2 - restricted lmp1 epitope in virus isolates from npc patients and healthy donors efficient presentation of the lmp1 epitope by h a * 0201 , hla a * 0203 and hia a * 0207 , which are common supertypes in the southeast asian ethnic population , raised the possibility that this epitope might be important as a potential target epitope for lmp1 - expressing npc . sequence analysis across this ctl epitope region in virus isolates from eight npc samples was carried out using lmp1 - specific primers . spontaneous lcls from healthy ebv immune donors were used as controls in this analysis . interestingly all ebv isolates from the npc samples displayed identical substitutions within this epitope ( table 2 ). in contrast , of the 12 virus isolates from healthy ebv immune donors , four encoded a sequence identical to that of the b95 . 8 isolate , while six displayed a different pattern of alterations within this epitope that differed from those found in the npc samples . in some isolates leucine at position 2 , methionine at position 5 and arginine at position 8 were substituted with phenylalanine , isoleucine and glycine , respectively ( table 2 ), while in other isolates only the methionine at position 5 was substituted with isoleucine . importantly , incubation of t2 cells with these variant peptides ( yfleilwgl ( seq id no : 32 ) and ylleilwrl ( seq id no : 33 )) significantly increased iihc expression on these cells ( fig6 a ), indicating efficient binding to hla a2 , the ylleilwrl ( seq id no : 33 ) peptide was efficiently recognized by the sb7 clone , while no ctl activity was seen in the presence of the yfleilwgl ( seq id no : 32 ) peptide ( fig6 b ). the latter result does not rule out the possibility that yfleilwgl ( seq id no : 32 ) is an epitope individuals infected with an ebv strain encoding this sequence . the loss of recognition by the clone sb7 is likely to be due an inappropriate t cell receptor interaction with the mhc - peptide complex rather than loss of mhc binding . thus it is possible that t cells expressing a different t cell receptors are capable of efficiently recognising this hla binding peptide ( 8 ). interestingly , virus isolates from the other two healthy ebv immune donors to determine whether npc cells can present endogenously expressed antigens to virus - specific ctls , tumour cells were either infected with recombinant vaccinia encoding ebna4 ( vacc . ebna4 ) or presensitized with synthetic peptide epitopes . fig7 illustrates the results from an experiment in which recombinant vaccinia - infected npc cells ( cl5 ) were compared with hla - matched type 2 lcls infected with vacc . ebna4 . following exposure to ebna4 - specific ctls , vacc . ebna4 - infected or peptide sensitized npc cells were efficiently recognised by both cm9 and cm29 ctl clones . the level of ctl lysis was comparable to that seen for lcls . these results clearly demonstrate that npc cells are able to transport sufficient levels of peptides into the er by tap - dependent mechanism and can efficiently transport mhc - peptide complexes from the er to the surface of the cell . normal antigen processing function in npc cells has significant implications for vaccines designed to control these tumours in vivo . earlier studies have demonstrated that the latent gene expression in npc is often limited to ebna1 and the transmembrane proteins , lmp1 and lmp2 ( 29 ). since ebna1 is not recognized by ebv - specific ctls , there is an increasing emphasis on designing strategies to control npc around epitopes known to be included within lmp1 ( 6 , 17 ). in view of the data presented in this study , it is reasonable to assume that lmp epitopes will be processed efficiently by npc cells . an effective approach to control npc cells in vivo may be to amplify lmp - specific ctl responses in these patients . this might be achieved by two different procedures . firstly npc patients might be immunised with synthetic peptides which include ctl epitopes from lmp1 and / or lmp2 . alternatively . lmp1 and lmp2 - specific ctls from hla matched healthy virus carriers may be adoptively transferred into npc patients in a manner analogous to that used to successfully treat ebv - associated polyclonal lymphomas in bone marrow transplant recipients ( 30 ). earlier work from various laboratories have shown that the viral phenotype of ebv - associated malignancies is likely to be a very important factor in reducing tumor susceptibility to virus - specific ctl surveillance , since viral antigen expression in these malignant cells in vivo is restricted to either ebna1 , or ebna1 and lmp1 ( 9 , 20 , 21 ). since it is now firmly established that ebna1 does not include class i - restricted ctl epitopes ( 6 , 17 ), considerable interest has been directed towards identifying potential epitopes within lmp1 . the present study was precisely designed to , address this issue . one of the limiting factors in identifying epitopes within lmp1 has been the fact that the ctl response to this antigen often constitutes as a minor component of the total virus - specific response ( 6 , 17 ). to overcome this problem we employed a modified protocol to identify potential hla a2 - restricted epitopes within lmp1 . an important step in this process was the use of a computer based program developed by parker and colleagues ( 18 ) designed to predict the potential hla binding peptides within various proteins from human pathogens . analysis of the lmp1 sequence from the b95 . 8 ebv isolate revealed a number of potential hla a2 binding peptides and a large proportion of these were then functionally shown to stabilize hla a2 molecules on t2 cells . stimulation of pbmcs with these peptides resulted in the activation of a strong polyclonal ctl response specific for the peptide yllemlwrl ( seq id no : 1 ), while a weaker response was seen for another peptide ylqqnwwtl ( seq id no : 6 ). the yllemlwrl ( seq id no : 1 ) sequence was confirmed as an lmp1 epitope by isolating a ctl clone ( sb7 ) specific for this peptide . the lmp1 specificity of the sb7 ctl clone was further confirmed by the recombinant vaccinia experiments and efficient lysis of lmp1 expressing h a2 - positive bl cells . the ctl response characterized in the present report is of interest not only because it is directed against a viral antigen constitutively expressed in many ebv - associated malignancies ( hd and npc ) but also because the hia a2 allele is common in virtually all human populations ( 13 ). niore importantly , ebv transformed lcls expressing all the major hla a2 supertypes were efficiently recognized by the lmp1 - specific ctl clone , a result that has important implications for anti - viral vaccine design aimed at protect different ethnic populations . it is important to mention here that the ctl response to the lmpi epitope in healthy seropositive individuals constitutes a minor component of the virus - specific ctl response and very low levels of ctl precursors are seen for this epitope . it may nevertheless be possible to amplify this component by vaccination with the relevant peptide or with adoptive transfer of in vitro activated lmp1 - specific ctls . such approaches may be of use in the control of hd and npc . efficient presentation of the yllemilwrl ( seq id no : 1 ) peptide by hla a * 0201 , hla a * 0203 and hla a * 0207 - positive lcls , which are common supertypes in the southeast asian population , raises the possibility that this epitope might be exploited as a potential target epitope for lmp1 - expressing npc . im patients , identified on clinical grounds and by heterophile antibody positivity , were bled during the first 5 - 10 days of illness and , in two cases , on a second occasion 24 - 36 months after the resolution of symptoms . these patients were hia typed for the hla a2 allele by serotyping in microcytotoxicity and by genotyping . three patients ( sb , lp and mg ) were identified as hia a2 - positive patients and this was subsequently confirmed by facs analysis using an hla a2 - specific monoclonal antibody ( atcc ). establishment and maintenance of cell lines : ebv - transformed lymphoblastoid cell lines ( lcls ) were established from a panel of im and healthy ebv - seropositive donors by exogenous virus transformation of peripheral b cells using type 1 ( b95 . 8 ) or type 2 ( ag876 ) ebv isolates ( 16 ), and were routinely maintained in rpmi 1640 containing 2 mm glutamine . 100 μg / ml penicillin and 100 μg / ml streptomycin plus 10 % foetal calf serum ( fcs ) ( growth medium ). in addition , the peptide transporter ( tap )- negative b × t hybrid cell line 174 × cem . t2 ( referred to as t2 ) ( 22 ) were used for peptide stablization assays . to generate phytohaemagglutinin ( pha ) blasts peripheral blood mononuclear cells ( pbmc ) were stimulated with pha ( commonwealth serum laboratories , melbourne ) and after 3 days , growth medium containing mla 144 supernatant and ril - 2 was added ( 36 ). pha blasts were propagated with bi - weekly replacement of il - 2 and nila supernatant ( no further pha added ) for up to 6 weeks . acute im pbmc effectors for use in ex vivo cytotoxicity assays were resuspended in growth medium supplemented with recombinant il2 and used directly in a cytotoxicity assay ( see below ). to generate polyclonal ctls , pbmcs from hla a2 - positive donors were co - cultivated for seven days with irradiated ( 8 , 000 rads ) t2 cells presensitized with synthetic peptides ( 37 ). on days 7 and 14 , these cultures were restimulated with peptide - sensitized t2 cells . after 18 days of culture in growth medium , the cells were used as polyclonal effectors in a standard 51cr - release assay against peptide - sensitized autologous pha blasts . peptides , synthesized by the merrifield solid phase method ! were purchased from chiron mimotopes ( melbourne , australia ), dissolved in dimethyl sulphoxide , and diluted in serum - free rpmi 1640 medium for use in standard ctl assays . mhc stabilisation assays hla a2 binding peptides within the gp85 and gp350 antigens were identified using a protocol as described in example 1 . these predicted peptides were then used in a standard mhc stablization assay using t2 cells . briefly , t2 cells ( 2 × 10 5 ) were incubated with 200 μl of each of the peptides ( 200 μg / ml ) at 26 ° c . for 14 - 16h , followed by incubation at 370 ° c . for 2 - 3h . after the incubations . hla a2 expression was measured by facs using a monoclonal hla a2 - specific antibody ( ma2 . 1 : atcc ). recombinant vaccinia constructs encoding the ebv structural antigens gp350 ( vacc . gp350 ) and gp85 ( vacc . gp85 ), and a vaccinia virus construct made by insertion of the psc11 vector alone and negative for thymidine kinase ( vacc . tk -) have been previously described ( 38 ). target cells were infected with recombinant vaccinia virus at a mutiplicity of infection ( moi ) of 10 : 1 for 1 h at 37 ° c ., as described earlier ( 6 , 12 ). after overnight infection , cells were washed with growth medium and processed for ctl assays or for immunoblotting to assess the expression of recombinant ebv antigens ( 11 ). target cells were either infected with recombinant vaccinia viruses or pre - sensitized with synthetic peptide epitopes and then incubated with 51cr for 90 min . following incubation , these cells were washed in growth medium and used as targets in a standard 5 h 51cr - release assay ( 16 ). immunisation of hla a2 / kb transgenic mice with gp350 and gp85 ctl epitopes hia a2 / k b transgenic mice used in this study have been described elsewhere ( 39 ). these mice express a chimeric class i molecule composed of the alpha 1 & amp ; 2 domains of the human a * 0201 allele and the alpha 3 domains of the mouse h - 2kb class i molecules . peptide immunizations were carried out as described by vitello and colleagues ( 40 ). briefy , these animals were twice immunized ( at a 14 day interval ) subcutaneously with 50 μg / mouse of ctl epitopes emulsified in ifa together with 5 μg of tetanus toxoid as a source of help . four weeks following peptide immunization , animals were assessed for gp350 - and gp85 - specific ctl response . for assessing these ctl responses , splenocytes ( 3 × 10 6 cells / ml ) were cocultured with syngeneic irradiated ( 2000 rad ) peptide - coated lps blasts ( 3 × 10 cells / ml ) and 3ug / ml human b2 - microglobulin . ctl activity was tested on day 6 using a standard 51cr - release assay . for protection experiments , groups of 8 weeks old female a2 / kb transgenic mice were immunized with ctl epitopes as described above . on day 28 , mice were challenged with vacc . gp85 and vacc . gp350 intraperitonealy ( 1 × 10 7 pfu in 100 μl pbs ). after four days of challenge , these animals were sacrificed and vaccinia titres measured in both ovaries by plaque assay on confluent cv1 cells . “ to identify potential hla a2 restricted epitopes within gp85 and gp350 , the amino acid sequence was analyzed using the hla binding predictions program of the bioinformatics & amp ; molecular analysis section of the computational biosciences and engineering lab , center for information technology at the national institutes for health , 12 south drive , mcs 5624 ( building 12a , room 2033 ), bethesda md 20892 , usa ( 18 ). a total of 20 peptides ( 13 from gp85 and 7 from gp350 ) with an estimated half - time disassociation score of & gt ; 100 for gp85 and & gt ; 50 for gp350 were selected ( table 3 ). these peptides were then tested for hla a2 binding efficiency using hla a2 - positive t2 cells . representative data from a series of experiments is presented in fig8 . this analysis showed that seven of these peptides significantly increased the expression of hla a2 on t2 cells * to identify the potential hla a2 binding peptides within gp85 and gp350 , the hla binding predictions program of the bioinformatics & amp ; molecular analysis section of the computational bioscience and engineering lab , center for information technology at the national institutes for health , 12 south drive , mcs 5624 ( building 12a , room 2033 ), bethesda md 20892 , usa , was employed as described elsewhere ( ref 18 ). this program can be directly accessed through the world - wide web . recognition of the gp85 and gp350 peptide epitopes by im effectors ex vivo the seven hia a2 - binding peptides , which included four peptides from gp85 and three peptides from gp350 were next tested for ctl recogniton by effectors from im patients . in addition , we also included an hla a2 - restricted ctl epitope from ebv latent mambrane protein ( lmp1 ) as a positive control ( 38 ). pbmcs from three hia a2 - positive im patients , sb , lp and mg were resuspended in il2 - supplemented growth medium and used as effectors in a standard 5lcr - release assay against hla - matched pha blasts sensitized with the gp85 , gp350 or lmp1 peptides . representative data from two different experiments is shown in fig9 ( a - c ). effectors from all three im patients showed clear recognition of the reference lmp1 peptide ( ylqqnwwtl ( seq id no : 6 )) consistent with our earlier finding that this peptide is recognized by ebv - specific ctls . more importantly , these im patients also showed strong recognition of target cells sensitized with selected gp85 or gp350 peptides . interestingly , each of these individuals showed a distinct pattern of reactivity against these peptides . im patient sb showed strong reactivity against peptides slv ( seq id no : 17 ) ( gp85 ) and vlqwaslav ( seq id no : 27 ) ( gp350 ) ( fig9 a ), while the lp and mg effectors recognised target cells preloaded with peptides lmiiplinv ( seq id no : 20 ) ( gp85 ) and vlqwaslav ( seq id no : 27 ) ( gp350 ) ( fig9 ( b - c )). furthermore , ex vivo effectors from patient lp also recognised target cells infected with vacc . gp350 and vacc . gp85 ( fig9 d ). in vitro expansion of gp85 and gp350 peptide epitope reactive ctls the data presented above clearly demonstrate that gp85 and gp350 include ctl determinants which can bind hia a2 molecules and are efficiently recognised by ex vivo effectors from im patients . to determine whether gp85 - or gp350 - reactive ctls can be detected following recovery from im , pbmcs from the two donors sb and lp were collected at 24 - 36 months post im respectively and were stimulated with t2 cells presensitized with each of the gp85 and gp350 peptides which showed strong hia a2 binding . on day 18 , these ctl effector were tested against peptide - sensitized autologous pha blasts . representative data from polyclonal ctls from donior sb are presented in fig1 . ctl effectors from donor sb not only showed strong reactivity against peptides slvivttfv ( seq id no : 17 ) and vlqwaslav ( seq id no : 27 ) but also recognized two other peptides from gp85 ( lmiiplinv ( seq id no : 20 ) and tlfigshvv ( seq id no : 24 )). donor lp also showed a similar pattern of ctl lysis . thus peptide tlfigshvv ( seq id no : 24 ) was a target for ebv - specific ctl recogntion in the memory response of these a2 - positive individuals , but this response was not detectable with ex vivo effectors during acute infection . another important point which needs to be highlighted here is that our attempts to activate gp85 - or gp350 - specific ctls with autologus lcls as stimulators were unsuccessful . this result is not surprising since it is well established that in latently infected b cells , gp350 or gp85 antigens are poorly expressed . the lcl - stimulated polyclonal t cell lines from these donors strongly reactive against latent antigens ( data not shown ). this obseravtion is consistent with our earlier studies which showed that ctl responses in healthy virus carriers is often dominated by reactivity to latent antigens ( 6 ). another explanation for an inability to detect gp350 - or gp85 - specific ctl reactivity following stimulation with the autologous lcls is that these responses may constitute a minor component of the total virus specific ctl - response in healthy virus carriers . indeed , limiting dilution analysis for ctl precursors specific for the 8p350 or gp85 peptide epitopes in post im donors sb and lp showed precursor frequencies of & gt ; 1 / 50 , 000 , while precursor frequencies for ctls that recognise ctl epitopes within the latent antigens were between 1 / 4 , 000 - 1 / 15 , 000 ( data not shown ). immunization of hla a2 / kb mice with gp85 and gp350 peptide epitopes induces specific ctl response having established that gp85 and gp350 includes ctl epitopes , we extended our studies to explore the possibility of using these peptide epitopes to induce specific ctl response in vivo . hla a2 / kb transgenic mice were used as an experimental model to address this issue . these mice express a chimeric class i molecule composed of the alpha 1 & amp ; 2 domains of the human a * 0201 allele and the alpha 3 domain of the mouse h - 2kb class i molecules . these animals were immunized subcutaneously with gp350 or gp85 ctl epitopes emulsified in ifa together with tetanus toxoid as a source of help . the slvivttfv ( seq id no : 17 ) and tlfigshvv ( seq id no : 24 ) peptides from gp85 and the vlqwaslav ( seq id no : 27 ) peptide from gp350 were used for immunisation . two weeks following immunization , specific ctl response was assessed in each mouse using splenocytes or pooled inguinal lymph node cells as effectors . data presented in fig1 ( a - c ) demonstrate that peptide epitopes from gp85 ( slvivttfv ( seq id no : 17 ) and tlfigshvv ( seq id no : 24 )) and gp350 ( vlqwaslav ( seq id no : 27 )) induced strong ctl response in splenocytes . interestingly , ctls activated from splenocytes with peptide tlfigshvv ( seq id no : 24 ) consistently showed strong lysis of targets , while splenocytes from slvivttfv ( seq id no : 17 ) and vlqwaslav ( seq id no : 27 ) immunized mice showed variable in vitro ctl lysis . a strong specific ctl activity was also noticed in pooled lymphocytes from inguinal lymph nodes ( fig1 d ). prior immunisation of hla a2 / kb mice with gp85 or gp350 ctl epitopes affords protection against recombinant vaccinia virus challenge four weeks after peptide immunization with gp85 or gp350 ctl epitopes , hla a2 / k b mice were challenged with 10 7 pfu of recombinant vaccinia virus encoding either gp85 or gp350 . after four days of challenge , these animals were sacrificed and vaccinia titres measured in both ovaries by plaque assay on confluent cv1 cells . data from one such experiment is presented in fig1 . animals immunised with gp85 and gp350 epitopes showed very low to undetectable virus in their ovaries , while in naive mice very high titres of vaccinia virus were detected . this protection correlated with strong induction of epitope - specific ctl responses detected in the splenocytes and lymph node cells collected four weeks after primary peptide vaccination in hla a2 / kb transgenic mice . there is increasing interest in formulating an effective vaccine against ebv , designed to not only limit the outgrowth of latently infected b cells in healthy individuals but to also block the development of many ebv - associated malignancies such as burkitt &# 39 ; s lymphoma ( bl ). nasopharyngeal carcinoma ( npc ) and hodgkin &# 39 ; s disease ( hd ). in western societies , the principle aim of such a vaccine would be to protect from im . in this context . virus load ( a large dose of orally transmitted virus and / or overexpansion of the virus - transformed b cell pool beyond a critical threshold ) may be a critical determinant of disease risk ( 7 ). therefore , a vaccine capable of either blocking primary ebv infection or significantly reducing the ebv load during primary infection may be adequate to avert clinical symptoms . a similar vaccine will also be able to reduce the immediate risk of lymphoproliferative disease in transplant patients receiving immunosuppressive therapy . on the other hand , ebv - associated malignancies such as bl , npc , and hd arise in patients years after their primary infection , and protection from these longer - term consequences would require a vaccine that ideally confers sterile immunity and prevents the establishment of the carrier state . ebv structural antigens , primarily gp350 , have long been considered as the potential candidates for an ebv vaccine . the suggestion that gp350 is a likely vaccine candidate was based initially upon the observation that this glycoprotein is the principal target of the virus - neutralizing antibody response ( 41 ). a number of recombinant formulations of gp350 , either presented as a subunit antigen or expressed from recombinant viral vectors , designed to induce high titre neutralizing antibodies , have shown significant protection against ebv - induced b cell lymphomas in cotton - top tamarins ( 31 ). however , development of neutralizing antibodies in vaccinated animals does not always shows limited correlation with protection from ebv infection , although recent results have suggested a role for gp350 - specific ctls in this protection ( 34 ). if the latter suggestion is correct , it is important to identify the potential ctl determinants within ebv structural proteins since it is now well established that immunization with whole viral proteins is unable to elicit an efficient ctl response . moreover , a vaccine based on ctl epitopes provides an opportunity to include determinants not only from gp350 but also from other structural antigens , such as gp85 . to address this issue we have used a novel protocol to successfully identify ctl epitopes within gp350 and gp85 . in the first set of experiments we identified hia a2 binding peptides within gp350 and gp85 . subsequent experiments were focussed on im patients with the hla a2 allele . using ex vivo primary effectors , we observed strong reactivity to three different gp350 and gp85 peptides . interestingly , individual im patients showed distinct patterns of reactivity to each of these peptides . strong reactivity against peptides slvivttfv ( seq id no : 17 ) ( gp85 ) and vlqwaslav ( seq id no : 27 ) ( gp350 ) was observed with ex vivo effectors from patient sb , while the lp and mg effectors recognized target cells preloaded with lmiiplinv ( seq id no : 20 ) ( gp85 ) and vlqwaslav ( seq id no : 27 ) ( gp350 ) peptides . more importantly , ex vivo effectors from patient lp also recognised target cells infected with vacc . gp350 and vacc . gp85 . interestingly , the level of ex vivo ctl lysis directed to epitopes from structural antigens was consistently higher than those seen in the same assays against hla a2 - restricted ctl epitopes from a latent antigen . these results are consistent with recent observations by steven and colleagues ( 42 ) that ex vivo ctl reactivity to lytic antigens in im patients is significantly higher compared to latent antigens . in the next set of experiments , we explored the possibility of detecting structural antigen - specific ctl responses in individuals following resolution of im symptoms . this follow up analysis was carried out 24 - 36 months post acute im . our initial attempts to isolate gp350 - or gp85 - specific ctls from post im donors by stimulating with the autologous lcl were unsuccessful . subsequently we used peptide loaded t2 cells as stimulators to generate gp350 - and gp85 - specific ctls . we have recently shown that this method can be successfully used to raise low frequency ebv - specific ctl precursors ( 38 ). stimulation of pbmc from donors sb and lp raised strong ctl responses to the gp85 and gp350 ctl epitopes . both donors sb and lp not only showed reactivity against peptides slvivttfv ( seq id no : 17 ), lmiiplinv ( seq id no : 20 ) and vlqwaslav ( seq id no : 27 ) but also recognized another peptide from gp85 , tlfigshvv ( seq id no : 24 ). it is interesting to note here that both donors showed no ex vivo ctl reactivity to tlfigshvv ( seq id no : 24 ) during acute im . one of the important conclusions drawn from these analyses is that , following recovery from acute im , there is a significant reduction in ctl precursors to the structural antigens , and the response becomes dominated by ctl reactive to the latent antigens . indeed , limiting dilution analysis for ctl precursors specific for the gp350 or gp85 peptide epitopes in donors sb and lp post im showed frequencies of & gt ; 1 / 50 , 000 , while precursor frequencies for ctl epitopes within latent antigens were between 1 / 4 , 000 - 1 / 15 , 000 . the detection of a strong ex vivo ctl response in im patients to the structural antigens has important implications for any future vaccine design . as mentioned above , to date , the major emphasis of vaccine design based on ebv structural antigens has been directed towards generating a strong neutralizing antibody response . however , these neutralizing antibody response fail to correlate with protection against ebv - induced polyclonal lymphomas in cotton - top marmosets . nevertheless , it is possible that this protection is mediated by structural antigen - specific ctl responses . to address this issue , we employed an experimental animal model system to determine whether gp350 or gp85 cm epitope immunized transgenic mice , expressing the human hla a2 antigen , are capable of ( a ) generating structural antigen - specific ctl responses and ( b ) reducing infection with a recombinant vaccinia virus infection expressing the gp350 or gp85 antigen . these mice not only showed induction of a strong ctl response following immunization but also acquired strong resistance to virus infection . it is important to mention here that although this experiment does not allow any firm conclusions on the efficacy of a gp350 and / or gp85 ctl epitope based vaccine in humans , it does clearly show that ctl epitopes from the ebv structural antigens can be used as immunogens to induce an efficient ctl response in vivo . moreover , this approach also overcomes limitations of whole gp350 or gp85 proteins which might be inefficient at eliciting ctl responses in humans . obviously one of the possible obstacles of any epitope - based approach to vaccination in humans is hla polymorphism because epitope choice is allele - specific . however , this obstacle might be overcome using appropriate mixtures of synthetic peptide epitope or by constructing vectors to express polypeptides in which the relevant epitope sequences are linearly joined together . indeed , earlier studies from our laboratory have shown that if such an ebv polyepitope sequence is expressed within cells from a recombinant vaccinia vector , all of the constituent epitopes are efficiently presented for ctl recognition ( 43 ), indicating the potential of this approach as a vaccine strategy . more recently , work in a murine model has also shown that each of several ctl epitopes combined in a polyepitope construct was capable of eliciting a ctl response in vivo and could protect the animals from subsequent challenge ( 44 ). in the long term , it may be possible to combine ctl epitopes from the ebv structural antigens , with latent antigen epitopes generating a chimeric protein that fuses the important immunogenic determinants from the two different types of antigens to design an effective vaccine . it will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described . the present embodiments are , therefore , to be considered in all respects as illustrative and not restrictive . 1 . burrows , j . m ., s . r . burrows , l . m . poulsen , t . b . sculley , d . j . moss , and r . khanna . 1996 . unusually high freqency of epstein - 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