Patent Application: US-1742108-A

Abstract:
the invention provides novel vaccination strategies based on a prime - boost vaccination regiment . the inventors have determined improved ways of boosting an immune response in a patient previously primed or exposed to a plurality of epitopes . the improved method requires the epitopes in the boosting phase to be administered individually , i . e . held on separate peptide constructs .

Description:
the dna vector psg2 , used throughout the study , was derived from prc / cmv ( invitrogen , paisley , uk ) by removing the bamh1 fragment that contains the sv40 origin of replication and neomycin resistance marker and replacing the cmv promoter with a longer version of the same promoter containing intron a . the resulting plasmid contains the cmv promoter with intron a for expression in eukaryotic cells , followed by a multiple cloning site and the bovine growth hormone poly - a sequence . the plasmid is incapable of replication in mammalian cells . the gene encoding the mel3 sequence ( table 1 ) was introduced into the multiple cloning site using standard methods . plasmid dna for injection was purified using anion - exchange chromatography ( qiagen , hilden , germany ) and diluted in phosphate buffered saline ( pbs ) at 1 mg dna / ml . recombinant and non - recombinant mva were routinely propagated and titrated in chicken embryo fibroblasts ( cef ) grown in minimal essential medium supplemented with 10 % foetal calf serum ( fcs ). recombinant mva were made as described by cloning the mel3 poly - epitope string ( table 1 ) into the vaccinia shuttle vector psc11 . cef infected with mva at a multiplicity of 0 . 05 pfu per cell were transfected with lipofectin ® transfection reagent ( gibco ) and shuttle plasmid as described 23 . the vaccinia p7 . 5 promoter drives expression of the polyepitope . recombinant mva were plaque purified 8 times . vaccinia viruses ( wr strain ) expressing mel3 , full length ny - eso - 1 ( kindly provided by dennis l . panicali , therion biologics corporation , ma 02142 , usa ) or tyrosinase were made by cloning the mel3 poly - epitope construct , the ny - eso - 1 and tyrosinase full length cdna into the thymidine kinase gene using the vector psc11 as previously described 24 . the mel3 poly - epitope string was cloned into the transfer vector psfv4 . 2 - mel3 . rna produced from this vector was used to construct recombinant sfvmel3 particles . recombinant sfv stocks were made and purified as described previously 16 . human ctl clones were isolated as described 25 . briefly , tetramer / cd8 double positive stained ctl cultures were sorted as single cells into u - bottom 96 - well plates , previously coated with anti - cd3 and anti cd28 both at 100 ng / ml in pbs , containing 10 5 irradiated b cells in iscove &# 39 ; s medium supplemented with 5 % human serum , 100 u / ml il - 2 . proliferating clones were expanded to & gt ; 10 7 cells and used as effectors for standard cr 51 release assays . the inventors used jy b cells infected with mva mel3 as targets for hla a2 restricted ctl ; xy b cells for hla - a1 restricted epitopes and h - 2b positive mc57 cells as targets for the d b restricted influenza nucleoprotein . we titrated ctl clones in three fold dilutions against targets fluorescent tetrameric hla - a2 . 1 / peptide complexes were synthesised as previously described 1 . a2 - kb / peptide complexes were synthesised in an analogous fashion using a chimeric heavy chain of α1 and α2 domain of the a2 . 1 molecule and the α3 domain , of the h - 2 d b molecule with human β2 - micro globulin . fresh pbl were isolated from blood taken from tail vein using red cell lysis buffer ( invitrogen , paisley , uk ). for tetramer stains 3 × 10 5 cells were resuspended in 20 μl rpmi 1640 ( sigma , st louis , mo .) supplemented with 10 % fcs . cells were incubated with tetramer for 15 minutes at 37 ° c . pbl were then incubated with rat anti mouse cd8α ( pharmingen , san diego , calif .) for 15 minutes at 4 ° c . cells were washed twice in pbs and resuspended in pbs for facscan ™ assay ( becton dickenson , mountain view , calif .) analysis . hhd mice express a transgenic monochain histocompatibility class i molecule in which the c terminus of the human β2m is covalently linked to the n terminus of a chimeric heavy chain ( hla - a2 . 1 α1 - α2 , h - 2d b α3 transmembrane and intracytoplasmic domains ) 20 . the h - 2d b and mouse β2m genes of these mice have been disrupted by homologous recombination resulting in complete lack of serologically detectable cell surface expression of mouse histocompatibility class i molecules . a2 - k b mice express chimeric heavy chain ( hla - a2 . 1 alpha 1 alpha 2 , h - 2 k b alpha 3 transmembrane and cytoplasmic domains ) in non - covalent association with mouse β2m . they additionally express a full set of c57bl / 6 - derived ( h - 2 b ) class 1a and 1b mouse histocompatibility molecules 26 . all a2 transgenic mice used were bred in the inventors &# 39 ; animal facility . female c57 / bl6 mice 4 - 6 weeks old were obtained from harlan orlac ( shaws farm , blackthorn , uk ). plasmid dna ( 25 - 50 μg / muscle ) was dissolved in pbs and injected into each musculus tibialis under general anaesthesia . 10 days after dna injection , mice were boosted with recombinant vaccinia viruses , which were diluted in pbs and 10 6 - 10 7 pfu and injected intravenously ( i . v .) into the lateral tail vein . alternatively freshly isolated spleen cells were separately infected ( multiplicity of infection of 5 ) with mel3 , full length tyrosinase and ny - eso - 1 vaccinia for 90 minutes in rpmi supplemented with 0 . 1 % bsa at 37 ° c . cells were washed 3 times and re suspended in sterile pbs at a concentration of 6 × 10 7 cells / ml combined and injected into lateral tail vein . mice received 6 × 10 6 spleen cells . mice primed with influenza virus a ( pr8 ) were infected by intra - nasal influenza injection ( 20 hau / mouse ). 30 days later mice were injected with dna . mel3 followed by mva . mel3 , as described above . for priming or boosting with sfv - mel3 10 8 virus particles were diluted in sterile pbs and injected into the lateral tail vein . freshly isolated spleen cells from hhd mice were separately incubated in rpmi medium with different peptides at a concentration of 10 − 6 m for 2 h . each cell pool was then labelled with a different concentration of carboxyfluorescein diacetate succinimidyl ester ( cfse , molecular probes , eugene , oregon ) to allow simultaneous tracking of the different populations in vivo 27 , hermans , i . f ., yang , j . and ronchese , f . unpublished results . labelled cells were pooled and injected at 10 7 cells / mouse into the tail vein . a control population without peptide that had been labelled with 5 -( and - 6 -)-((( 4 - chloromethyl ) benzoyl ) amino ) tetramethylrhodamine ( celltracker ® orange fluorescent probe , molecular probes , eugene , oregon ) was co - injected to assess killing of peptide pulsed targets relative to unpulsed cells . mice were bled at the time of injection of flurochrome labelled targets to determine their ctl frequencies to different epitopes . disappearance of peptide / flurochrome labelled cells was tracked using facs analysis of freshly isolated pbmc 5 h after the injection . percentage killing was calculated relatively to the unpulsed population labelled with celltracker ® orange fluorescent probe . 100 -( 100 ×(% pulsed /% unpulsed )). winmdi 2 . 8 and cellquest ™ 3 . 3 software was used to analyse the facs data . a string of 5 hla - a2 and 2 hla - a1 melanoma , epitopes was cloned into four distinct vectors : a ) naked plasmid dna ( mel3 . dna ); b ) vaccinia virus ( mel3 - vaccinia ); c ) modified vaccinia ankara virus ( mel3 . mva ); d ) semliki forest virus ( mel3 - sfv ). to ensure monitoring of ctl responses restricted by human and mouse class i molecules , the inventors introduced an additional epitope from the influenza nucleoprotein ( np ) restricted by h - 2 db class i molecules . since they had previously shown that presentation of amino - terminal np366 - 374 epitope can be affected by neighbouring amino acid residues 24 , the inventors decided to express the influenza np 366 - 374 epitope at the carboxyl terminal end of the poly - epitope construct . sequence of the poly - epitope constructs used in the paper are shown in table 1 . initial experiments were carried out , to compare the a2 binding affinity of each epitope contained within the mel . 3 construct . the results of these experiments demonstrated that mel3 peptide epitopes had a broad range of binding affinities for a2 molecules . the melan - a 26 - 35 peptide analogue 28 had the highest binding affinity , while the ny - eso - 1 157 - 165 and tyrosinase 1 - 9 peptides had a significantly lower affinity , as defined by their ability to inhibit at different concentrations presentation of the influenza matrix epitope 58 - 66 ( data not shown ). the inventors and others have previously demonstrated that optimal flanking residues are important to ensure presentation of class i restricted epitopes 24 , 29 . to establish that mel . 3 peptide epitopes were properly processed , and to assess that competition for binding to a2 molecules did not impair ctl recognition of lower affinity epitopes , they infected target cells with mel . 3 vaccinia and demonstrated that each of the 7 epitopes contained within the poly - epitope mel . 3 cassette was simultaneously presented to specific ctl ( fig1 ). the inventors had previously shown that proteasome dependent degradation impairs presentation of the mage3 a2 epitope 271 - 279 30 . it was therefore surprising to observe that infection of target cells with mel3 vaccinia was capable of sensitising them for lysis by mage3 271 - 279 specific ctl . further experiments demonstrated that processing of the mage3 271 - 279 epitope contained within the mel3 construct , unlike its processing within the full length mage3 protein , was lactacystein resistant and tap independent ( data not shown ), consistent with processing of mel . 3 construct by endoplasmic reticulum resident proteases . influenza np 366 - 374 specific ctl responses in mice vaccinated with poly - epitope encoded vaccines efficient presentation of the np366 - 374 epitope by mel3 vaccinia infected cells prompted the inventors to assess in c57 / b6 mice the ability of different vaccination strategies to induce a strong np366 - 374 specific ctl response ( fig2 ). ex - vivo monitoring of the np specific ctl response was carried out in pbl using db / influenza np366 - 374 tetramers . the inventors compared homologous vs heterologous prime boost vaccination protocols ( fig2 ), and analysed the kinetics of ctl induction by dna or mva priming ( data not shown ). the results of these experiments confirmed that heterologous vaccination strategies are capable of inducing long lasting vaccine driven ctl responses , with frequencies up to 100 times greater than frequencies obtained by strategies based on repeated injections of the same antigen delivery system ( fig2 ). to test the ability of the mel . 3 poly - epitope constructs to prime a2 restricted ctl responses in vivo , a2 transgenic mice were primed by dna - mel . 3 and boosted by mva - mel . 3 , vaccinia mel . 3 , or sfv - mel3 . initial experiments were carried out using a2 . 1 transgenic mice , which express chimeric a2 . 1 molecules containing the kb α3 domain , and endogenous d b and k b molecules ( a2 / kb mice ) 26 . to enable monitoring of the a2 restricted responses at the same time as the d b restricted influenza np366 - 374 response , the inventors employed novel a2 / kb tetramers , which were also capable of detecting the relevant ctl directly in pbl . simultaneous staining with a2 / kb and d b tetramers demonstrated that priming of a2 / kb mice with dna . mel3 , followed by mva - mel . 3 , induced melan - a 26 - 35 and influenza np 366 - 374 specific ctl responses ( fig3 ). in contrast , responses specific to other mel . 3 epitopes were not detectable by ex vivo tetramer stainings ( data not shown ). the ability to simultaneously monitor ctl responses against the influenza np epitope 366 - 374 and melan - a epitope 26 - 35 prompted the inventors to study whether previous exposure to influenza virus may compromise the ability of prime - boost protocols to expand melan - a 26 - 35 specific ctl in a2 - kb mice . in order to generate a strong np366 - 374 specific ctl response , a2 transgenic mice were immunised with influenza virus and subsequently received dna . mel3 followed by mva - mel3 ( fig3 ). the results of these experiments demonstrated that expansion of np366 - 374 specific ctl , prior vaccination with mel3 poly - epitope constructs , significantly reduced the expansion of melan - a specific ctl ( fig3 ). the inhibitory effect of pre - existing flu specific ctl on the ability of mel3 prime - boost to induce melan - a specific ctl response ( fig3 ) raised the possibility that t cell interference during heterologous vaccination strategies may compromise the induction of a broad range immune response . the presence of endogenous mouse class i molecules significantly narrows the a2 restricted repertoire in a2 kb mice , hence hampering the ability to study the interplay between a2 restricted ctl specific to different vaccine encoded determinants . this reasoning led the inventors to monitor the hierarchy of vaccine driven ctl responses upon prime boost protocols in the a2 transgenic mice hhd 20 . hhd mice , unlike a2kb transgenic mice , express a2 . 1 class i molecules linked to human β - 2 microglobulin in a db −/− and β - 2m −/− background , and have a much larger a2 restricted t cell repertoire than a2 / kb transgenic mice 20 . prime - boost vaccination of hhd mice induces large numbers of melanoma specific ctl priming of hhd mice with dna . mel3 led to the expansion of melan - a specific ctl to frequencies detectable by ex - vivo tetramer staining in all vaccinated mice ( data not shown ). in contrast , expansion of ny - eso - 1 and tyrosinase specific ctl was only detectable in a small proportion of immunised mice , while responses to the tyrosinase 1 - 9 and mage3 271 - 279 were not detected in blood tetramer stainings ( data not shown ). additional experiments confirmed that ny - eso - 1 specific ctl responses were primed by dna . mel3 injection , as shown by the significant ny - eso - 1 ctl response in dna . mel3 primed mice boosted with vaccinia virus encoding the full length ny - eso - 1 ( data not shown ). in contrast , injection of ny - eso - 1 vaccinia virus , without priming with dna . mel3 , led to a much lower frequency of ny - eso - 1 ctl . similar results were obtained upon injection of tyrosinase vaccinia in mice primed with dna mel3 ( data not shown ). the observation that the melan - a 26 - 35 specific ctl was the dominant vaccine driven ctl response after a single dna vaccination , presented an opportunity to study the interplay between ctl specific to different vaccine encoded determinants in prime - boost vaccination protocols . the inventors observed that boosting of dna . mel3 primed hhd mice with either vaccinia mel3 ( fig4 a ), mva . mel3 ( fig4 b ), or sfv - mel3 ( fig4 c ), led to the expansion of melan - a specific ctl , up to 70 - 80 % of total cd8 + t cells . although responses specific to ny - eso - 1 and tyrosinase epitopes were significantly lower than the melan - a specific responses , their frequencies ranged between 2 and 30 % of cd8 + t cells , confirming that dna priming and boosting with either replication competent ( i . e . vac . mel3 ) or incompetent viruses ( mva . mel . 3 and sfv - mel3 ) significantly enhance the frequency of ctl specific to three distinct melanoma specific epitopes . the inventors confirmed that vaccine driven ctl were cytolytic , as shown by their ability to kill fluorochrome labelled splenocytes pulsed with relevant peptides in vivo ( fig4 d ). these results demonstrated that the cumulative response specific to melan - a , ny - eso - 1 and tyrosinase in hhd mice primed with dna . mel3 and boosted with three distinct viral vectors accounted for the specificity of the majority of cd8 + population . since the inventors demonstrated the inhibitory effect of a pre - existing flu memory ctl response on the ability to induce melan - a specific ctl ( fig3 ), they sought to study whether the high numbers of melan - a specific ctl , dominating the immune response after dna priming , were capable of interfering with the expansion of ny - eso - 1 and tyrosinase specific ctl during the virus boosting . it is known that competition for antigen recognition on the surface of antigen presenting cells leads to the immunodominance of higher frequency ctl populations 31 - 33 . the inventors reasoned that the higher numbers of melan - a ctl after dna . mel 3 priming may lead either to rapid killing or shielding of mel3 vaccinia infected apc in vivo , resulting in a hampered stimulation of ctl specific to ny - eso - 1 and tyrosinase epitopes expressed by the same apc population . this reasoning led them to assess whether a higher frequency ny - eso - 1 and tyrosinase specific ctl responses could be obtained by separating the apc expressing ny - eso - 1 and tyrosinase proteins from the apc expressing the mel3 construct . the results of these experiments confirmed this hypothesis , as shown by : 1 ) expansion of ny - eso - 1 and tyrosinase specific ctl in dna . mel3 primed mice and boosted with a mixture of vaccinia viruses , encoding the full length tyrosinase and full length ny - eso - 1 proteins ( fig5 a ); 2 ) simultaneous expansion of melan - a , ny - eso - 1 and tyrosinase specific ctl upon adoptive transfer into dna . mel3 primed mice of three aliquotes of splenocytes infected ex - vivo with vaccinia viruses encoding full length ny - eso - 1 , tyrosinase and mel3 construct ( fig5 b , panels a , b and c ), while adoptive transfer of splenocytes infected with mel3 vaccinia led to the expansion of melan - a specific ctl ( fig5 b panels d , e and f ) the inventors have demonstrated that boosting of dna . mel3 primed mice with a mixture of recombinant viruses , encoding the full length tyrosinase , full length ny - eso - 1 and the mel3 construct , led to the simultaneous expansion of melan - a 26 - 35 , ny - eso - 1 157 - 165 tyrosinase 369 - 377 specific ctl ( fig6 a and b ). the identification of successful vaccination strategies to simultaneously expand large numbers of ctl with a broad specificity has important clinical applications , as we showed that t cell immunity induced by this type of optimised boosting strategies provides a more efficient in vivo killing of target cells than vaccinations based on poly - epitope prime boost strategies ( fig6 c and d ). immunodominance of melan - a specific ctl could be broken by separating the antigens during the boost . when separately infected splenocytes were used to boost a polyvalent response relevant peptides were separately presented and resulted in simultaneous expansion of melan - a , tyrasinase and ny - eso specific ctl . to simplify this approach the inventors used peptide pulsed dentritic cells to boost an mva . mel3 primed response . the cells used for boosting were either pulsed with a mixture of peptides ( fig7 a ) or separately pulsed ( fig7 b ). the inventors show that separate pulsing of apc is superior to pulsing apc with a mixture of peptides . this approach also demonstrates , that poly - epitope constructs encoded in vaccinia virus can efficiently prime and apc pulsed with peptide can efficiently boost a polyvalent ctl response . there is a tremendous momentum in vaccine development , as recent advances in the monitoring of antigen specific ctl responses in ex - vivo assays are rapidly improving our capacity to compare different vaccination protocols . in order to minimise the generation of tumour and virus antigen escape variants , it is important to ensure the expansion of vaccine driven ctl specific to several epitopes , including both dominant and subdominant determinants . several papers have dissected the causes responsible for immunodominance of ctl specific to viral 34 - 36 , and histocompatibility antigens 33 , 37 , 38 . however , it remains to be established how optimal vaccine strategies can lead to the simultaneous expansion of ctl specific to dominant and subdominant epitopes . to address these questions the inventors engineered 4 distinct vectors encoding a string of melanoma ctl epitopes ( table 1 ), and compared in a2 transgenic and wild type b6 mice the ability of different vaccination strategies to elicit vaccine specific ctl responses . in order to identify strategies capable of expanding ctl specific to dominant and subdominant determinants , peptide epitopes with high and low binding affinity for a2 molecules were linked in the same construct . more specifically , the inventors included the modified melan - a analogue 26 - 35 , previously shown to have an enhanced immunogenicity in vivo 28 and the ny - eso - 1 peptide 157 - 165 , shown to have a much lower binding affinity to a2 molecules 39 ( table 1 ). the inventors have developed a novel tetramer based technique to directly monitor the frequency of a2 restricted ctl expanded in a2 transgenic mice vaccinated in a prime - boost regimen . to increase the binding affinity of mouse cd8 to a2 molecules , they engineered a2 molecules containing the mouse h - 2k b alpha 3 domain ( a2 / k b molecules ), and demonstrated that tetrameric a2k b molecules have an increased staining efficiency for mouse a2 restricted ctl and can identify a2 restricted responses in a large proportion of a2 transgenic mice , as compared with tetrameric a2 molecules . while studying the immune response in a2kb mice , the inventors have demonstrated that expression of db molecules results in a strong influenza np366 - 374 specific response , which significantly impairs the expansion of ctl specific to other mel3 encoded epitopes . in order to study the interplay between a2 restricted ctl specific to different vaccine encoded determinants , the inventors immunised the a2 transgenic mice hhd 20 , which , unlike a2kb transgenic mice , express a2 molecules in a db −/− background . although several papers have recently studied the immune response in a2 transgenic mice vaccinated with poly - epitope constructs 19 , 20 , 40 , this is the first publication in which the hierarchy of poly - epitope vaccine driven ctl in a2 transgenic mice has been monitored by ex - vivo tetramer staining . the inventors compared several vaccination strategies and confirmed that immunisations based on the injection of non - cross reactive vectors ( heterologous prime - boost protocols ) ensure higher levels of vaccine specific immune responses than immunisations based on the injections of homologous vectors ( fig2 ). the presence of neutralising antibodies against viral structural proteins and the presence of ctl specific to viral proteins may account for the lower ctl responses in mice vaccinated with repeated injections of the same virus , as compared with ctl frequencies upon vaccination with non - cross reactive vectors ( fig2 ). it has been suggested that the limited number of proteins encoded by plasmid dna ensures that dna priming focuses the immune response towards the recombinant protein , while virus boosting successfully expand this response , resulting in high levels of ctl specific to the recombinant protein . however , the inventors have shown that priming with either mva . mel3 or sfv . mel3 led to a significant expansion of np366 - 374 specific ctl upon boosting with influenza virus or mva . mel3 , respectively ( fig2 ), demonstrating that the ability to prime ctl is not unique to dna vectors . in hhd a2 transgenic mice , due to the lack of ctl restricted by endogenous mouse class i molecules , heterologous prime boost resulted in a tremendous expansion of melan - a specific ctl up to 90 % of total cd8 + t cells ( fig4 a ), hence redirecting a large proportion of hhd mice &# 39 ; s a2 restricted repertoire towards vaccine encoded ctl determinants . several factors may contribute to the immunodominance of the melan - a specific ctl response . it is possible that a combination of an increased binding affinity for a2 molecules and for tcr , together with a favourable intracellular processing , may skew ctl responses towards the melan - a epitope 26 - 35 in dna . mel3 primed mice . previous studies have shown that “ suppression ” of t cell responses specific to non dominant epitopes by t cell responses specific to dominant epitopes is observed only when both types of determinants are presented on the same apc 32 , 33 , 38 . injection of large numbers of apc resulted in the expansion of t cells specific to the sub - dominant epitopes 41 . the inventors have demonstrated that immunodominance of the melan - a epitope 26 - 35 was overcome by boosting strategies based on the injection of either a mixture of different recombinant viruses ( fig5 a ) or splenocytes infected in vitro by individual vaccinia viruses encoding full length proteins , rather than a poly - epitope construct ( fig5 b ). these results are of importance , since several clinical trials are currently using heterologous prime boost vaccination protocols with poly - epitope constructs . while the inventors confirm the ability of heterologous prime - boost protocols in eliciting large numbers of vaccine specific ctl , they demonstrate that during heterologous prime - boost protocols , frequency of immunodominant ctl responses is significantly expanded over frequency of ctl responses specific to less dominant determinants . although there are numerous mechanisms which may account for a narrowing of the ctl repertoire which responds to a vaccine , the inventors &# 39 ; results are consistent with a model of immunodominance based on competition of t cells for antigen presenting cells ( apcs ) 32 , 33 , 38 . it is worth noting that the injection of vac . mel3 into naïve mice induces lymphocytocis with a ; shift of cd8 + frequency from 0 . 5 % up to 30 %, indicating a strong ctl response to the vaccinia virus . of the cd8 + t cells induced in the blood as many as 50 % are specific for melan - a 26 - 35 ( data not shown ), demonstrating that melan - a 26 - 35 is one of the most immunodominant epitopes expressed by the virus amongst more than 200 vaccinia proteins responsible for the viruses &# 39 ; structure , transcription and replication . this observation has important clinical implications for the design of viral - based vaccines encoding the melan - a epitope 26 - 35 the use of recombinant sfv in prime - boost protocols is very attractive , as the inventors have demonstrated that sfv can be used both as priming vector in combination with mva ( fig2 ) and for boosting in combination with dna ( fig4 c ). these results compliment a recently published report demonstrating in macaques the enhancement of simian immunodeficiency virus - specific immune responses induced by priming with recombinant sfv and boosting with mva 11 . the inventors demonstrated that pre - existing memory ctl responses significantly reduce ctl responses specific to other epitopes contained within the same construct ( fig3 b ). as several groups have used immunodominant influenza peptide epitopes as positive controls during vaccination trials , the inventors &# 39 ; result suggest that dna or virus based vaccines should not encode epitopes expressed by recurrent viruses , as pre - existing memory ctl response may compromise the induction of ctl responses specific to other vaccine encoded ctl determinants . the inventors have further shown that t cell competition is likely to play a role in modifying t cell responses in prime - boost vaccination strategies . the inventors &# 39 ; work strongly suggests that simultaneous presentation of different epitopes to a skewed repertoire of primed ctl leads to dominant expansion of a single ctl specificity . however , boosting the primed response with apc separately presenting the epitopes results in comparable expansion of ctl of multiple specificities to effective levels in vivo . thus , the present inventors have , amongst other things provided a novel system for dissecting the ability of different vaccination protocols to optimally induce polyvalent a2 - restricted ctl responses . in accordance with the present invention , methods for inducing a broad - based ctl response should restrict the use of polyvalent constructs to the priming phase and use separate vectors encoding individual epitopes , or separate proteins / peptides for the boosting phase . 1 . altman , j . d . et al . phenotypic analysis of antigen - 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