Patent Application: US-92019501-A

Abstract:
the present invention provides cytotoxic epstein - barr virus t - cell epitopes . these epitopes are qvkwrmttl , vfsdgrvac , vpapagpiv , tysagivqi , lldfvrfmgv , qngalaintf , vssdgrvac , vssegrvac , vssdgrvpc , vssdglvac , vssdgqvac , vssdgrvvc , vpappvgpiv , veitpyeptg , veitpyeptw , veltpykptw , rriydlikl , rkiydliel and pylfwlagi . the present invention further provides vaccines including one or more of these epitopes , optionally with additional epitopes .

Description:
the ctl epitopes may be synthesised using techniques well known to those skilled in this field . for example , the ctl epitopes may be synthesised using solution synthesis or solid phase synthesis as described , for example , in chapter 9 entitled “ peptide synthesis ” by atherton and sheppard which is included in a publication entitled “ synthetic vaccines ” edited by nicholson and published by blackwell scientific publications . preferably a solid phase support is utilised which may be polystyrene gel beads wherein the polystyrene may be cross - linked with a small proportion of divinlylbenzene ( e . g . 1 %) which is further swollen by lipophilic solvents such as dichloromethane or more polar solvents such as dimethylformamide ( dmf ). the polystyrene may be functionalised with chloromethyl or anionomethyl groups . alternatively , cross - linked and functionalised polydimethyl - acrylamide gel is used which may be highly solvated and swollen by dmf and other dipolar aprolic solvents . other supports can be utilised based on polyethylene glycol which is usually grafted or otherwise attached to the surface of inert polystyrene beads . in a preferred form , use may be made of commercial solid supports or resins which are selected from pal - peg , pak - peg , ka , kr or tgr . in solid state synthesis , use is made of reversible blocking groups which have the dual function of masking unwanted reactivity in the a - amino , carboxy or side chain functional groups and of destroying the dipolar character of amino acids and peptides which render them inactive . such functional groups can be selected from t - butyl esters of the structure rco — ocme 3 — co — nhr which are known as t - butoxy carboxyl or roc derivatives . use may also be made of the corresponding benzyl esters having the structure rco — och 2 — c 6 h 5 and urethanes having the structure c6h5ch 2 o co — nhr which are known as the benzyloxycarboinyl or z - derivatives . use may also be made of derivatives of fluorenyl methanol and especially the fluorenyl - methoxy carbonyl or fmoc group . each of these types of protecting group is capable of independent cleavage in the presence of one other so that frequent use is made , for example , of boc - benzyl and fmoc - tertiary butyl protection strategies . reference also should be made to a condensing agent to link the amino and carboxy groups of protected amino acids or peptides . this may be done by activating the carboxy group so that it reacts spontaneously with a free primary or secondary amine . activated esters such as those derived from p - nitrophenol and pentafluorophenyl may be used for this purpose . their reactivity may be increased by addition of catalysts such as 1 - hydroxybenzotriazole . esters of triazine dhbt ( as discussed on page 215 - 216 of the abovementioned nicholson reference ) also may be used . other acylating species are formed in situ by treatment of the carboxylic acid ( i . e . the na - protected amino acid or peptide ) with a condensing reagent and are reacted immediately with the amino component ( the carboxy or c - protected amino acid or peptide ). dicyclohexylcarbodiimide , the bop reagent ( referred to on page 216 of the nicholson reference ), o &# 39 ; benzotriazole - n , n , n ′ n ′- tetra methyl - uronium hexaflurophosphate ( hbtu ) and its analogous tetrafluroborate are frequently used condensing agents . the attachment of the first amino acid to the solid phase support may be carried out using boc - amino acids in any suitable manner . in one method boc amino acids are attached to chloromethyl resin by warming the triethyl ammonium salts with the resin . fmoc - amino acids may be coupled to the p - alkoxybenzyl alcohol resin in similar manner . alternatively , use may be made of various linkage agents or “ handles ” to join the first amino acid to the resin . in this regard , p - hydroxymethyl phenylactic acid linked to aminomethyl polystyrene may be used for this purpose . details of the ctl epitopes of the present invention are set in tables 3 and 4 . as will be readily appreciated by those skilled in the art the cytotoxic t - cell epitopes and vaccines of the present invention can be used to protect against ebv . further given the possible greater involvement of type b ebv infection in immunocompromised individuals the present invention may have particular application in protection of individuals having decreased immune function , eg transplant patients . in order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following examples and figures . fig1 . immunoblot of recombinant vaccinia - ebna viruses : lane 1 : vacc - tk -, lane 2 : vacc - ebna - 3b ; lane 3 : vacc - ebna - 6b . m . wt . markers are on the left . fig2 . confirmation of specificity of ctl cultures b5 and a3 . ew - wil ( type a autologous lcl ) was infected with vacc - ebna - 6a , vacc - ebna - 6b or vacc - tk − and used as targets in a ctl assay ; ew - wil and ew - ag - 876 ( type b autologous lcl ) were used as control targets without vaccinia infection . fig3 a , b : identification of minimal epitope for culture b5 . targets : ew - wil lcl + peptide fig3 a : targets + peptides 7 - 12 of ebna 6b 20 - mer overlap fig3 b : targets + 9 - mer and 10 - mer overlapping peptides of the 20 - mer peptide no . 12 fig4 a , b : identification of minimal epitope for culture a3 . targets : ew - pha blasts + peptide fig4 b : targets + dilutions of four of the 10 - mer overlapping peptides of the 20 - mer peptide no . 19 fig5 : depletion experiment . targets : ew - c3 ( negative t cell culture ); effectors : ew type b epitope specific cultures pooled from lda cultures . ew = ctl treated with dynabeads only ; ew - cd - 8 : ctl depleted of cd4 t cells : ew - cd4 : ctl depleted of cd8 t cells fig6 . inhibition of type b epitope - positive ctl ( culture b - 15 ) by a - class i but not by a - class ii monoclonal antisera . peptide 74 = type b reactive peptide ; target cells = ew - c3 , a noncytolytic t cell culture . a 17 year old individual ( ew ) who initially presented with clinical and pathological ( ebv igm positive ) symptoms of acute im was used in this study . she was referred to this laboratory for investigation of her ebv status 7 months after the initial diagnosis as her clinical symptoms persisted . four bleeds were obtained from this donor over a period of 12 months during which time the symptoms fluctuated but did not completely resolve . the third and fourth bleeds were used for ctl analysis . dna was extracted from pbl by a small scale adaptation of a published extraction procedure ( 27 ). ebna 2a and 2b primers as previously described ( 20 ) were used to distinguish between type a and type b ebv by pcr . briefly , standard pcr reactions were carried out in a 25 μl volume using 1 μg genomic dna . the template was denatured for 2 min at 94 ° c . before subjecting to 35 pcr cycles ( 15 s at 94 ° c . 30 s at 56 ° c . and 15 s at 72 ° c .). pcr amplified products were identified on southern blots using digoxigenin - dutp ( dig ) labelled dna probes ( boehringer - mannheim australia ) generated by pcr using the same primers as above and either the purified m - aba bam hi insert from pm - bam h2 ( type a ) ( 36 ) or the purified jijoye bam hi / pst i insert from pj - hka7 ( type b ) ( 1 ) as templates . for the preparation of the probes , dig - dutp was present in the pcr reaction with 25 ng of each insert as template . recombinant vaccinia constructs ( designated vacc - ebna - 1 , vacc - ebna - 2 etc ) encoding ebnas - 1 , - 2b , - 4a and - 6a , the latent membrane proteins lmp1 and lmp2 , the lytic antigens bhrf1 and bzlf1 , and the control vacc - tk − have already been described ( 19 , 23 , 33 ). vacc - ebna - 3b and vacc - ebna - 6b ( i . e . vaccinia constructs encoding the type b sequences of ebna 3 and ebna 6 respectively ) were constructed from an ebv genomic library derived from purified viral genomes of the prototype type b strain ag - 876 ( 50 ). the viral dna was digested with hindiid and ligated into the λ - zap cloning system ( integrated sciences ). an 11 . 07 kbp hindiiie fragment encoding the ebna 3 family open reading frames ( orfs ) was subcloned into the vector puc19 . a 2 . 6 kbp bamhi - ceiii ( klenow - treated ) fragment within the region containing the berf1 orf of ebna - 3 ( 46 ) was isolated and ligated into a bamhi - hindii digest of pbcb07 . this vaccinia vector drives recombinant gene expression by the 7 . 5k vaccinia virus promoter ( 2 ). the ebna - 3b sequence encoded in the transfer plasmid was inserted into vaccinia virus by transfection and homologous recombination into the tk gene of vaccinia as described ( 2 ). a 4 kbp eco47iii - hindiii fragment within the ebv hindiiie region containing the berf2b orf of ebna - 6b was isolated and ligated into a hindii - hindiii digest of pbcb07 . this was subsequently processed as for ebna - 3b . integrity of the vacc - ebna - 3b and - 6b constructs was confirmed by immunoblot after infection of mouse cv - 1 cells . 2 × 10 5 cv - 1 cells infected with control vaccinia ( vacc - tk − ), vacc - 3b or vacc - 6b were lysed in protein sample buffer and separated on a 7 % sds - polyacrylamide gel in a mini - protean ii cell system ( bio - rad ). after transferring to nitro - cellulose the blot was incubated with a multivalent human serum ( mcr ) which contained ebna - specific antibodies . the reactions were visualised with the ecl detection system ( amersham ). fig1 illustrates the production of a 110 kda protein ( ebna - 3b ) or a 130 kda protein ( ebna - 6b ) corresponding to the expected products of ebna - 3b and ebna - 6b without the short first orfs . sequence analysis also confirmed the integrity of the constructs . ( a ) b cell cultures : lcls were established from ew by exogenous transformation of pbl by qimr - wil virus ( type a ; ( 38 )) and ag - 876 ( type b ). these lcls were designated ew - wil and ew ag - 876 . spontaneous lcls ( i . e . autologous lcls infected with the donor &# 39 ; s endogenous virus strain and designated ew - spon ) were established in the presence of cyclosporin ( 41 ). lcls from other donors were transformed by qimr - wil . b95 - 8 or iarc - bl74 virus ( 30 ) ( type a isolates ) or by ag - 876 or qimr - gor virus ( 37 ) ( type b isolates ). other lines used in this study include burkitt lymphorna cell lines ( chep and mutu ) and the spontaneous line qimr - ww2 - lcl ( 13 , 43 , 44 ). b cell lines were maintained in rpmi 1640 with 10 - 20 % fcs . ( b ) t cell cultures : unfractionated pbl were separated on ficoll - paque ( pharmacia , uppsala , sweden ) and stimulated by incubating with γ - irradiated ( 8000 rads ) autologous lcls ( ew - wil or ew - ag - 876 ; 2 × 10 6 cells per well in 2 ml growth medium without ril - 2 , responder : stimulator ratio 50 : 1 ) ( 28 ). after 3 days , the cultures were cloned in agarose ( seaplaque , fmc corp , rockland me .) and the colonies harvested 3 - 5 days later . once established , the t cells were maintained in t cell growth medium ( rpmi - 1640 supplemented with 15 % fcs , 30 % mla - 144 supernatant ( tib - 201 : american type culture collection , rockville , md . and ril - 2 . 20 units / ml ) ( 45 , 57 ). since the clonal nature of the t cells was not established , they are referred to in the text as cultures rather than clones . the cultures were routinely maintained in either 24 - well plates or tissue culture flasks and restimulated twice weekly with γ - irradiated ( 8000 rads ) autologous lcls . bulk t cell cultures were initially stimulated from the fourth bleed as for cloning but subcultured and maintained after 3 - 4 days as for t cell colonies . phytohaemagglutinin ( pha ) blasts were generated by stimulation of pbl with pha ( csl , melbourne , australia ) and subculturing after 4 days in t cell growth medium . cultures were regularly screened for mycoplasma contamination . three - colour cytofluorographic analysis of t cell cultures was performed by means of direct immunofluorescence using labelled monoclonal antibodies specific for cd3 ( tricolour - conjugated ), cd4 ( fluorescein - conjugated ), and cd8 ( phycoerythrin - conjugated ) and ( becton dickinson , california ). the labelled cells were analysed on a - facscan ( becton dickinson , lysis ii software ). t cells were assayed in modified 5 - hour 51 cr - release assays ( 30 ). briefly , target cells were labelled for 1 hour with 0 . 5 - 1 . 0 μc 51 cr , washed and resuspended at 10 5 cells / ml . 50 μl of target cells were incubated with an equal volume of effectors ( effector : target or e : t ratio generally 10 : 1 ) in 96 - well v - bottomed microtiter plates , centrifuged , and incubated at 37 ° c . for 5 hours . after centrifuging again , 25 μl supernatant was harvested and dried onto 96 - well solid scintillation microtiter plates before counting the radioactivity in a topcount microplate scintillation counter ( packard instrument company , meridan , conn .). for determination of antigenic specificity , target cells were infected with vaccinia construct ( multiplicity of infection approx . 10 : 1 ) and labelled with 51 cr by a modification of the previously described procedure ( 17 ). both infection and labelling occurred simultaneously for 2 . 5 hours before washing and using the cells in ctl assays . in some instances target cells were pretreated in v - wells with monoclonal antibodies to hla - class i ( w6 / 32 ) or class ii ( l243 ) for 30 min at room temperature before adding effector cells . the ebv - specific memory ctl response was assessed as previously described ( 31 ). limiting dilution cultures were established by distributing pbl from the fourth bleed in 24 replicates at 2 × 10 4 cells per well in 96 - well round - bottomed microtitre plates . doubling dilutions were made to a final concentration of 1 . 6 × 10 2 per well . approximately 1 × 10 4 γ - irradiated ( 8000 rads ) autologous type b ( ew - ag - 876 ) lcls were added to each well as both feeder and stimulator cells to a total volume of 100 μl . cultures were fed on days 4 and 7 with 50 μl of t cell growth medium and assayed on day 10 or later . the plates were subcultured and maintained in t cell growth medium and stimulated with γ - irradiated ew - ag - 876 lcls . initial assays were against type a and type b lcls and subsequently against peptide - coated pha - blasts or lcls . precursor frequencies were determined by a modification of the method of maximum likelihood estimation ( 11 ). assessment of peptide specific activity . for determination of the antigenic specificity at the peptide level , a set of 20 - mer overlapping peptides of ebna - 6b ( residues 100 - 1069 ( 46 )) was prepared using a kit and software distributed by chiron mimotopes ( chiron corporation , sydney , australia ). nine - mer and 10 - mer overlapping peptides with unblocked c and n termini were manufactured by chiron mimotopes . labelled target cells were preincubated with overlapping peptides in the 96 - well trays for 30 - 60 minutes before adding effectors . alternatively , labelled targets were preincubated with a high dose of peptide ( 40 - 80 μg / ml ) then washed twice before using in ctl assays . the washing procedure removes the complication of bystander killing in the latter type of experiments ( 6 ). t - cell cultures were depleted of either cd4 or cd8 cells by pretreating with saturating levels of okt4 ( mouse anti - cd4 ) or okt8 ( mouse anti - cd8 ) for 30 min on ice before washing twice and incubating with washed dynabeads m450 ( sheep anti - mouse igg coated beads , dynal , a . s , oslo , norway ) at 4 ° c . for 60 min with rotation . beads and adherent cells were removed magnetically and the remaining cells washed once before use in a ctl assay or facscan analysis . pcr analysis revealed the presence of both type a and type b ebv in blood from donor ew . the presence of both virus types was supported by the observation that both type a and type b spontaneous cell lines grew out although only the former ( referred to as ew - spon ) was established in long term culture . regression analysis indicated that the donor had memory ctl activity to both virus types . the number of cultures that could be assessed was therefore minimal compared with the t cell responses of most type a donors investigated by this laboratory . accordingly , 25 colonies from the third sample were assayed and seven of these selected for further study . three of these cultures ( b4 , b5 and b7 ) were type b specific i . e . they lysed autologous type b cells (& gt ; 15 % lysis ) but not type a (& lt ; 10 % lysis ); the other four ( a3 , a9 , b6 , and b9 ) were a - b specific i . e . they lysed both type a and type b autologous lcls (& gt ; 15 %) ( table 1 ). the seven selected cultures were assayed for ctl activity towards vaccinia - infected autologous type a lcls ( ew - wil ). four cultures ( a3 , a9 , b4 and b5 ) gave significant enhancement of ctl activity when vacc - ebna 6b infected lcls were compared with control cells or vacc - tk − infected target cells . furthermore , these cultures reacted poorly with other ebv sequences ( ebna - 1 , ebna - 2b , ebna - 3b , ebna - 4a , lmp1 , lmp2 , bzlf1 and bhrf1 ) expressed by recombinant vaccinia viruses thus indicating specificity for the vacc - ebna - 6b infected target . cultures b5 ( type b specific ) and a3 ( crossreactive ) were selected for more detailed analysis . the specificity of cultures a3 and b5 was confirmed in a separate experiment in which ew - wil target cells were infected with vacc - ebna - 6a and vacc - ebna - 6b . while the ctl activity of colony a3 was enhanced by infection of the target cells with either construct (& lt ; 10 % to 20 - 37 %). the effector function of colony bs was enhanced only when target cells were infected with vacc - ebna - 6b (& lt ; 10 % to 73 - 79 . 5 %) ( fig2 ). these results suggest that culture b5 recognised a type b specific epitope within ebna - 6 while culture a3 recognised a crossreactive epitope within ebna - 6 . the type b specific culture b5 recognised the peptide corresponding to ebna - 6b residues 210 - 229 ( peptide 12 of the ebna - 6b set ) when assayed against individual 20 - mer overlapping peptides preincubated with autologous pha blasts ( fig3 a ). nine - mer and 10 - mer overlapping peptides were then assayed to identify the minimal epitope as the 10 - mer qngalaintf ( residues 213 - 222 ; fig3 b ). the corresponding peptide from the type a overlapping sequence was not recognised by this culture , thus confirming the type b specificity of these t cells . culture a3 was assayed against pools of overlapping 20 - mer peptides from ebna 6b on autologous pha - blasts in order to identify its peptide target . a pool containing 6 × 20 - mer peptides ( peptides 19 - 24 in the ebna - 6b set ) from residue 280 - 349 was recognised by this ctl culture and these peptides were subsequently assayed individually to identify the reactive 20 - mer as peptide 19 , residues 280 - 299 ( fig4 a ). in fig4 b , the minimal epitope was identified as lldfvrfmgv corresponding to residues 284 - 293 of the ebna 6a and 6b sequences . this sequence is common to both type a and b of ebv and overlaps with the sequence of the b44 - restricted epitope eenlldfvrf ( residues 281 - 290 of ebna6a and 6b ; ( 7 )). as facs assays revealed that cultures recognising the ebna 6b - encoded sequence qngalaintf contained mixtures of cd4 + , cd4 + cd8 + and cd8 + cells when assayed initially , the possibility that the ctl activity was actually due to a cd4 component needed to be eliminated . several of these cultures also contained components that killed type a autologous targets , presumably because they were derived from the limiting dilution format and contained a mixture of cells . depletion and inhibition experiments confirmed that the cd8 + cells were the active ctls . in the depletion experiments , dynabeads were used to deplete either cd4 + or cd8 + cells from reactive cultures . when samples of three different epitope - reactive cultures were tested , depletion of the cd4 + cells ( which would have included the cd4 + cd8 + component ) did not reduce the ctl activity , but depletion of the cd8 + cells completely abrogated the epitope specific activity ( fig5 ). facs analysis in one of these experiments confirmed that the active fraction contained the cd8 + cells and that both the cd4 + and the cd4 + - cd8 + cells had been removed . fig6 illustrates the inhibition of the specific ctl activity of the mixed cultures by anti - class i monoclonal antiserum but not by the anti - class ii serum that was positive for dr , dp and dq alleles . it was concluded therefore that this epitope was class i - restricted . in order to identify the restricting allele , lcls , burkitt &# 39 ; s lymphoma cell lines or pha blasts of known hla specificity were incubated with minimalised peptides and washed before using as target cells in ctl assays . as table 2 indicates , the only target cells killed by the qngalaintf specific ctls were those that were both a201 and b62 ( b15 ) positive . in contrast , targets that were a201 but not b62 , and target cells that only shared a24 or b51 alleles with the donor were not killed . it was therefore concluded that the epitope is b62 ( b15 )- restricted . b62 which comprises at least 5 subtypes has recently been reclassified as part of the b15 family . in similar experiments , culture a3 consistently recognised cells bearing hla - a201 but not cells bearing a24 , b51 , or b62 . furthermore , the ctl activity of this culture was inhibited by anti - class i antibodies but not by anti - class ii antibodies . finally , the a3 minimal epitope corresponds to the motif already identified for hla - a201 restriction ( 39 ). it was therefore concluded that the epitope lldfvrfmgv corresponding to residues 284 - 293 in both ebna - 6a and ebna - 6b was hla class i a201 restricted . predominance of type b ctl response in limiting dilution cultures and bulk cultures limiting dilution culture methodology was used in order to investigate the magnitude of the type b specific ctl response . pbl stimulated by a feeder layer of irradiated autologous type b cells were assayed after 10 days against type a and type b autologous lcls . the results indicated a higher frequency of t cell precursors killing type b lcls than of those killing type a lcls . there were therefore a number of wells at limiting dilutions that were type b specific . in general , the wells manifesting type b specific activity retained this specificity . after minimalising the epitopes as described above , the cultures were retested for epitope specific activity . a frequency of 1 / 16395 was calculated for the type b epitope qngalaintf compared with 1 / 304 , 890 for the a - b specific epitope lldfvrfmgv when these cultures were assayed 12 weeks later . additional ebv ctl epitopes where obtained from other donors using similar methods . the identification of an ebv type b specific epitope represents the first demonstration of a ctl epitope encoded within type b specific regions of the ebv genome . the epitope was identified as qngalaintf corresponding to residues 213 - 222 of ebna - 6b . the collective data presented here indicates that stimulation of t cells from donor ew by autologous type b infected lcls reactivated a predominantly type b response rather than a cross - reactive response . the specificity of the type b response could be maintained on extended culture in vitro . clearly the response to qngalaintf would seem to be an immunodominant one as indicated by the number of reactive wells in the limiting dilution cultures . it is also interesting to note that a number of type b - specific ctl cultures were established for which specific epitopes could not be identified . it is possible that the target epitopes for these t cells lie within ebna - 4b or outside the regions included in the ebna - 2b , - 3b and - 6b constructs . the epitope qngalaintf appears to be restricted through hla b62 ( b15 ), although the subtype specificity has yet to be determined . motifs for b * 1501 have previously been published ( 39 ) and f is commonly found in the last anchor position ( 9 or 10 ) as in the epitope identified here . q or l appear to be the preferred residues at position 2 of b * 1501 ; for the new epitope , q is at position 1 . the corresponding type a sequence for this peptide ( qnaartlntf ) was nonreactive in ctl assays , thus confirming the type b specificity of the epitope . a second epitope lldfvrfmgv also represents a previously unrecognised ebv - encoded epitope . this sequence corresponds to residues 284 - 293 of both ebna - 6a and 6b and was shown to be restricted by hla - a201 . an interesting facet of this work was the detection of both type a and type b ebv in the one donor by pcr of both pbl and spontaneous cell lines . although the available evidence indicates a high prevalence of type b in the community , it is known that in healthy donors type a predominates as the one that is easiest to isolate . until now , responses to type a virus have therefore been easier to identify . it was noted in the results section that the donor did not give a particularly active cloning response . it is possible that the type b viruses are less immunogenic than type a , or even that type b ebv has evolved in part to evade the immune system . type b viruses could conceivably normally be resident in the epithelial compartment rather than the lymphocyte compartment due to their lower transformation efficiency . in this situation , type b epitopes may not be readily presented to the immune system . as already implied , the lower transformation efficiency of type b viruses ( 42 ) may hinder reinforcement over time of the immune response to the latency antigens . finally , it is important when considering the possibility of vaccinating against ebv to recognise that it may be necessary to vaccinate against both type a and type b . if a patient , particularly a transplant patient at risk of ebv - related post - transplant lymphoma ( 8 ), is protected against type a but not type b , there may even be greater risk of clinical disease if subsequently infected with the other virus type . it is particularly important to consider ebv type b epitopes in view of the finding that type b is commonly detected in immunosuppressed individuals . subunit vaccines against ebv are currently being tyialed . in the long term , such vaccines would ideally contain cross - protective specificities . definition of type b and cross - reactive epitopes allows development of a more effective vaccine , more effective in terms of the range of ebv specificities and hla types that are covered by the 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 . adldinger . h . k ., h . delius , u . k . freese , j . clarke , and g . w . bornkamm . 1985 . a putative transforming gene of jijoye virus differs from that of epstein - barr virus prototypes . virology 141 : 221 - 234 . 2 . andrew , m . e ., b . e . coupar , d . b . boyle , and g . l . ada . 1987 . the roles of influenza virus haemagglutinin and nucleoprotein in protection : analysis using vaccinia virus recombinants . scand . j . immunol . 25 : 21 - 28 . 3 . apolloni . a . and t . b . sculley . 1994 . detection of a - type and b - type epstein - barr virus in throat washings and lymphocytes . virology 202 : 978 - 981 . 4 . bogedain . c ., h . wolf , s . modrow , g . stuber . and w . jilg . 1995 . specific cytotoxic t lymphocytes recognize the immediate - early transactivator zta of epstein - barr virus . j . virol . 69 : 4872 - 4879 . 5 . buisson , m ., p . morand , o . genoulaz , m . j . bourgeat , m . micoud , and j . m . seigneurin . 1994 . changes in the dominant epstein - barr virus type during human immunodeficiency virus infection . j . gen . virol . 75 : 431 - 437 . 6 . burrows , s . r ., a . fernan , v . argaet , and a . suhrbier . 1993 . bystander apoptosis induced by cd8 + cytotoxic t cell ( ctl ) clones : implications for ctl lytic mechanisms . int . immunol . 5 : 1049 - 1058 . 7 . burrows . s . r ., i . s . misko , t . b . sculley , c . schmidt , and d . j . moss . 1990 . an epstein - barr virus - specific cytotoxic t - cell epitope present on a - and b - type transformants . j . virol . 64 : 3974 - 3976 . 8 . crawford . d . h ., j . a . thomas , g . janossy , p . sweny , o . n . fernando , j . f . moorhead , and j . h . thompson . 1980 . epstein barr virus nuclear antigen positive lymphoma after cyclosporin a treatment in patient with renal allograft . lancet 1 : 1355 - 1356 . 9 . dambaugh , t ., k . hennessy , l . chamnankit , and e . kieff . 1984 . u2 region of epstein - barr virus dna may encode epstein - barr nuclear antigen 2 . proc . natl . acad . sci . u . s a . 81 : 7632 - 7636 . 10 . doherty , p . c ., r . a . tripp , and j . w . sixbey . 1994 . evasion of host immune responses by tumours and viruses . ciba . found . symp . 187 : 245 - 256 . 11 . fazekas de st groth , s . 1982 . the evaluation of limiting dilution assays . j . immunol . methods 49 : r11 - 23 . 12 . gratama , j . w ., m . a . oosterveer . w . weimar , k . sintnicolaas , w . sizoo , r . l . bolhuis , and i . ernberg . 1994 . detection of multiple ‘ ebnotypes ’ in individual epstein - barr virus carriers following lymphocyte transformation by virus derived from peripheral blood and oropharynx . j . gen . virol . 75 : 85 - 94 . 13 . gregory , c . d ., m . rowe , and a . b . rickinson . 1990 . different epstein - barr virus - b cell interactions in phenotypically distinct clones of a burkitt &# 39 ; s lymphoma cell line . j . gen . virol . 71 : 1481 - 1495 . 14 . henle . g ., e . t . lennette , m . a . alspaugh , and w . henle . 1979 . rheumatoid factor as a cause of positive reactions in tests for epstein - barr virus - specific igm antibodies . clin . exp . immunol . 36 : 415 - 422 . 15 . henle . w . and g . henle . 1979 . seroepidemiology of the virus , p . 61 - 78 . in m . a . epstein and b . g . achong ( eds . ), the epstein - barr virus . springer - verlag , berlin . 16 . hu , l . f ., j . minarovits , s . l . cao , b . contreras - 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