BACKGROUND
the development of an efficacious prophylactic vaccine for human immunodeficiency virus type  <dig>  is the goal of a concerted worldwide research effort  <cit> .
although the precise correlates of protective immunity against hiv- <dig> infection are not clearly defined, a large body of accumulated data suggests that an ideal hiv- <dig> vaccine will need to stimulate both humoral and cellular immune responses against the virus  <cit> .
while chronic untreated hiv- <dig> infection causes a profound immunodeficiency, the initial hiv- <dig> infection stimulates strong cellular and humoral immune responses against the virus  <cit> .
cd <dig> cytotoxic t lymphocytes  constitute a major component of the cellular arm of the immune response, and have a central role in the control of initial viremia immediately following hiv- <dig> infection and in the establishment of long-term aids free survival  <cit> .
the ability to rapidly and accurately characterize ctl responses in hiv- <dig> infected individuals has grown exponentially in recent years.
new technologies to detect ctl responses by measuring interferon-gamma  release, such as the enzyme linked immunospot  assay and cytokine flow cytometry , have been coupled with overlapping pooled peptide technology  to give detailed and precise analyses of hiv-1-specific cellular immune responses  <cit> .
the fine mapping of t cell epitopes and the identification of immunodominant regions of hiv- <dig> gene products is integral to vaccine design, to the development of immunotherapeutic strategies, and to the optimization of assays for assessing vaccine efficacy.
genetic diversity is the foremost obstacle for any screening method for hiv-1-specific cellular immunity  <cit> .
geographically defined epidemics can be characterized by the dominance of distinct genetic subtypes of hiv- <dig>  with at least  <dig> subtypes and  <dig> crfs of hiv- <dig> currently recognized  <cit> .
since intra-subtype amino acid sequence variation can be as high as 10–15%  and inter-subtype variation can be much higher, any single sequence of hiv- <dig> used for screening for ctl responses will differ considerably from the sequence of the infecting virus in an individual.
this is an important complicating factor for studying the ctl response, because ctl are primed in vivo in response to the autologous infecting virus.
although the use of autologous viral sequences has been employed in several studies, their general use is impractical and labor intensive since, not only does the viral sequence between individuals differ significantly, but the viral quasi-species in a single individual can also take many different forms  <cit> .
a proposed solution to this seemingly intractable problem has been to use olp sets based upon computationally derived consensus or ancestral sequences  <cit> .
viral consensus and ancestral sequences have the theoretical advantage of being more related on average to any given in vivo viral sequence than any arbitrary viral isolate would be.
a further complicating factor for olp screening is that practical peptide libraries usually comprise 15–20-mers overlapping by 10– <dig> amino acids, but the optimal length of peptides that bind to mhc class i molecules is 8– <dig> amino acids.
however, this problem is overcome in most cases by exogenous addition of the peptides at biologically excess concentrations  <cit> .
despite these compromises the utility of screening for ctl responses with consensus sequence based olp has been demonstrated in several recent studies, which have comprehensively analyzed the full breadth and magnitude of cellular immune responses to the entire hiv- <dig> proteome  <cit> .
although consensus sequence based olp have now become the de facto standard for studies of hiv- <dig> cellular immunity, the assumption that a subtype based consensus is implicitly better than a randomly chosen isolate from the same subtype and isolates from other subtypes has only recently been questioned formally  <cit> .
this study was undertaken to assess the ctl response to the gag and nef proteins of hiv- <dig> infected individuals in kenya, a predominantly hiv- <dig> subtype a endemic region, and to compare and contrast the ctl response measured with that determined by prior studies in subtype b and c epidemics.
ctl responses directed against the gag and nef gene products were selected because previous studies have shown that these two gene products contain the highest epitope density and are the most frequently recognized hiv- <dig> proteins in subtype b and c infection  <cit> .
detection of gag-specific ctl responses was performed using peptide libraries representing a subtype a consensus, a subtype a isolate, a subtype c isolate and a subtype d isolate, to gain an insight into subtype-specificity of ctl responses in a region of multiple subtype endemicity.
detection of nef-specific ctl responses was performed using peptide libraries representing a subtype a isolate, a subtype c isolate and a subtype d isolate.
after sequence analysis of the gag and nef genes from proviral dna confirmed that the cohort was predominantly hiv- <dig> subtype a infected, we integrated knowledge of the sequence of the infecting viral isolate, the hla-type of the subject and the ctl response made by that subject to:  determine the frequency of recognition and pattern of ctl immunodominance within the gag and nef proteins in subtype a infections;  compare the frequently recognized epitopes in subtype a infection with those previously determined for subtype b and c infections;  compare subtype matched and mismatched peptide based peptides sets for their ability to detect ctl responses;  characterize an immunodominant hla-cw <dig> restricted ctl epitope from hiv- <dig>  within the framework of this study we were able to directly test the hypothesis that in a given cohort of hiv-1-infected subjects consensus and isolate-based peptide libraries derived from the homologous subtype would detect significantly broader and greater magnitude ctl responses than heterologous subtype-derived peptide libraries.
RESULTS
hiv- <dig> gag and nef region subtype analysis and hla-typing
full-length gag and nef sequence analysis of pbmc proviral dna was performed to characterize the hiv- <dig> subtype distribution within the cohort.
a single full-length gag and nef sequence could be obtained from  <dig> of the  <dig> subjects .
in the gag region,  <dig> of the  <dig> subjects harbored hiv- <dig> subtype a ,  <dig> had subtype d. the remaining  <dig> gag sequences represented recombinant structures,  <dig> a1/d,  <dig> a2/d.
within the nef region,  <dig> of  <dig> subjects harbored hiv- <dig> subtype a , while the remaining  <dig> sequences represented  <dig> subtype d,  <dig> subtype c and  <dig> a1/d recombinant.
figure  <dig> shows phylogenetic trees of the gag  and nef  sequences obtained from the cohort, with the dna sequence from which the isolate based sequences were derived shown for clarity.
for both regions, the subtype a isolate shows closer branching to the majority of the cohort sequences than the subtype c or subtype d isolates.
a pair-wise comparison of the translated autologous virus protein sequences  with the sequence of each of the peptide sets to be used for the elispot screening was performed.
the autologous gag protein sequences have mean differences of  <dig> %,  <dig> %,  <dig> % and  <dig> % from the subtype a consensus, the subtype a isolate, subtype c isolate and subtype d isolate based gag peptide sets respectively.
the autologous nef protein sequences have mean differences of  <dig> %,  <dig> % and  <dig> % from the subtype a isolate, subtype c isolate and subtype d isolate based nef peptide sets respectively.
therefore, the cohort is predominantly infected with hiv- <dig> subtype a viruses, and as would be expected, the gag subtype a consensus and nef subtype a isolate based peptide sets are closest in sequence to the viruses present in the cohort.
the nucleotide sequences derived in this study were submitted to genbank and are available under the following accession numbers: .
all  <dig> subjects were high resolution hla class i genotyped to 4-digit accuracy.
the frequencies of the  <dig> most common hla-a alleles were: a <dig> ; a <dig> ; a <dig> ; a <dig> ; a <dig> ; a <dig> ; a <dig> ; a <dig> ; a <dig>  a <dig>  and the  <dig> most common hla-b alleles were: b <dig> ; b <dig> ; b <dig> ; b <dig> ; b <dig> ; b <dig> ; b <dig> ; b <dig> ; b <dig> ; b8101; and the  <dig> most common hla-c alleles were: cw <dig> ; cw <dig> ; cw <dig> ; cw <dig> ; cw <dig> ; cw <dig> ; cw <dig> ; cw <dig> ; cw <dig> ; cw <dig> .
the detected allele frequencies are in close agreement with published hla allele frequencies for kenya  <cit>  and hence, the cohort can be considered representative of the kenyan population.
elispot screening with overlapping peptide pools
the cohort was screened for ifnγ t cell responses against four sets of gag overlapping peptide pools and three sets of nef overlapping peptide pools.
the gag and nef genes were selected because these two gene products have been documented to contain the highest cd <dig> epitope density and are the most frequently recognized hiv- <dig> gene products  <cit> .
as described in the methods section, the sequences of the peptide sets used for the study were based upon homologous and heterologous subtype isolates, and on a computationally derived subtype a consensus sequence for the gag protein.
ifnγ elispot responses from cryopreserved pbmc were assessed by peptide matrix screening, followed by individual peptide confirmation assays in order to maximize the information from individual specimens.
in both the screening and confirmation assays, positive responses were defined as those where the test wells exceeded the 99% confidence interval of replicates of six negative control wells, while for the confirmation assays individual peptides were tested in triplicate.
the  <dig> subjects included in the study all demonstrated strong responses to the seb positive control .
table  <dig> summarizes the ifnγ elispot screening data in terms of the number of subjects responding to each peptide set and the number of discrete responses detected.
an epitope, or discrete epitopic region, was defined by a response to a single peptide or adjacent pair of overlapping peptides from an individual peptide set.
while  <dig> subjects responded to at least  <dig> peptide from the  <dig> sets of screening peptides,  <dig> subjects showed no detectable gag or nef peptide specific response.
of the  <dig> subjects responding to any peptide set   <dig> recognized at least  <dig> gag peptide set.
of interest, similar numbers of subjects recognized at least  <dig> peptide from the  <dig> gag peptide sets: the subtype a consensus was recognized by  <dig> subjects; the crf01_ae peptide set by  <dig> subjects; the subtype c peptide set by  <dig> subjects; and the subtype d peptide set by  <dig> subjects.
the total number of responses detected in all subjects was also evaluated.
among the gag responding subjects a total of  <dig> responses were detected , with between  <dig> and  <dig> epitopes recognized.
a slightly greater, although non-statistically significant, number of responses was detected by the subtype a consensus based peptide set compared with the isolate based peptide sets .
the subtype a gag consensus and subtype a gag isolate-based peptide sets detected  <dig> and  <dig> of the  <dig> total gag responses respectively, while the subtype c and d peptide sets each detected  <dig> responses.
the mean number of responses detected per responding subject was not notably different between the  <dig> gag peptide sets .
the total frequencies of cells, expressed as sfc/ <dig> pbmc, responding to each of the gag peptide sets is shown in figure 2a.
within the cohort the measured frequency of responding cells was similar for each peptide set and no statistical difference among the peptide sets was observed .
therefore, the ability to detect gag derived ctl epitopes in an elispot assay for ifnγ production was not significantly improved by using a set of peptides based upon the consensus of subtype a compared with randomly chosen homologous and heterologous subtype based isolate sequences.
1an epitope  was defined as a response to a single peptide or pair of adjacent peptides.
of the  <dig> subjects responding to any peptide set,  <dig> recognized at least  <dig> nef peptide set.
the  <dig> nef peptide sets were also recognized by equivalent numbers of subjects: the subtype a peptide set was recognized by  <dig> subjects; the subtype c peptide set by  <dig> subjects; and the subtype d peptide set by  <dig> subjects.
a total of  <dig> nef directed responses were detected among the nef responders with between  <dig> and  <dig> nef epitopes recognized by each subject .
the subtype a nef peptide set was significantly  better at detecting the nef-specific responses  compared with the heterologous subtype based peptide sets .
there was however, no statistically significant difference between the magnitude of sfc detected in response to each of the  <dig> nef peptide sets  .
multi-subtype reactive and subtype-specific ctl are detectable in hiv- <dig> subtype a infection
since the overall analysis showed a high degree of similarity and multi-subtype recognition, the cohort was examined for the number of responses detected across all subtypes and those detected by a subset of subtypes or a single subtype.
overlapping venn diagrams  were used to display the number of responses detected by the individual peptide sets and by the various combinations of peptide sets.
for example, figure 5a shows that of  <dig> identified gag responses,  <dig> were detected with all  <dig> peptide sets as shown by the central overlap of all  <dig> circles, each of which encompasses all responses detected by that peptide set.
by this analysis  <dig> of the  <dig> gag responses were detectable with at least  <dig> of the peptide sets, while the remaining  <dig> were detected with only  <dig> or  <dig> of the peptide sets.
strikingly, the consensus subtype a peptide set detected only  <dig> responses not detectable by any of the  <dig> isolate based peptide sets, and the isolate based peptide sets collectively, detected  <dig> responses not detected with the subtype a consensus peptide set.
moreover, the subtype c and d heterologous isolate derived peptide sets detected  <dig> responses not detected by either of the two subtype a based peptide sets, with  <dig> of the responses representing different epitopic regions since only  <dig> were against the same peptide.
it was reasoned that because the cohort contained  <dig> subtype c,  <dig> subtype d and  <dig> inter-subtype recombinant gag genes, then these subjects may be the source of the responses not detected with the subtype a derived peptide sets.
however, this was not the case, as only  <dig> of the  <dig> epitopes not detected with subtype a peptide sets could be attributed to subjects from which non-pure subtype a gag containing virus sequence was obtained.
this response was detected with the subtype c peptide set and was from a subject  carrying an inter-subtype recombinant gag sequence .
thus, the overall magnitude and breadth of the ctl response against subtype a gag was underestimated when screening with any single peptide set.
the use of a single peptide set derived from an homologous subtype gag sequence  did not give a complete representation of the repertoire of gag-specific epitopes recognized by any given subject in this study.
similar analysis for the nef protein showed that the homologous subtype a derived peptide set detected all but  <dig> of the nef responses detected in this study.
most of the nef responses were directed to the highly conserved central region of the protein with 26/ <dig> total responses detected by all  <dig> isolate derived peptide sets.
notably, the number of responses detected with the subtype a nef isolate peptide set  was significantly greater than that detected with either the subtype c nef  or d nef  peptide sets .
therefore in this cohort, the use of homologous subtype derived nef sequences did maximize the detection of nef ctl epitopes.
characteristics of positive and discordant peptides in relation to each other and to autologous virus sequences
the identified ctl responses were next compared and examined in greater detail.
all positively recognized peptides from all peptide sets were compared to the gag or nef sequence of autologous virus.
first, the total number of possible responses detected in the study was calculated by multiplying the number of discrete gag responses by  <dig> and the number of discrete nef responses by  <dig>  this calculation was performed since any response directed against gag had  <dig> possible hits , and any response against nef had  <dig> possible hits.
of a total of  <dig> possible responses directed against all gag peptide sets,  <dig> were detected, while of a possible  <dig> responses against all nef peptide sets,  <dig> were detected.
when the peptides representing the  <dig> detected gag and nef responses were compared to the autologous virus sequence obtained from each host, approximately one-third  were identical to the autologous virus sequence while approximately two-thirds  had at least one amino acid mismatch with the autologous sequence.
hence a greater number of responses were detected in subjects whose proviral dna contained a different sequence compared to the peptide used to detect the response.
to obtain a better impression of the cross-reactive nature of the ctl responses measured, the impact of "frame-of-epitope" and sequence variation between concordant and discordantly recognized peptides was also examined.
the "frame-of-epitope" refers to the offset position of a 9-mer minimal epitope within a given 15-mer screening peptide and the effect has been the subject of several recent studies  <cit> .
because the screening peptide sets were derived from quite disparate subtypes, there was significant frame offsetting between peptides mapping to the same region of the different peptide sets.
when a given epitope was detected with more than one peptide set, there were  <dig> examples of frame offsetting between the concordant  peptides .
this indicates that despite significant minimal epitope offsetting, cross-recognition of the peptide sets was frequent.
in addition, within the concordant peptides there were  <dig> examples of peptides with different sequences able to be recognized.
these most likely represent truly cross-reactive responses, since more than one peptide sequence can be recognized by ctl from a single individual.
1peptides from different peptide sets with the same sequence were scored once only in a given category.
next, the discordant peptides were compared to the corresponding positive peptides to identify the reason for non-cross-recognition.
the most likely reason for non-cross-recognition was a sequence difference between the peptides.
in  <dig> discrete epitopic regions a discordant peptide had a different sequence compared with the corresponding positive peptide, and of these  <dig> were in the same frame while  <dig> were offset in frame.
importantly, it was noted that in  <dig> discrete epitopic regions, discordant results were obtained between peptide sets despite sequence identity across the corresponding regions.
in  <dig> instances there was frame offsetting between the positive and discordant peptides, which could account for the disparity in recognition.
however, in  <dig> instances peptides of identical primary sequence were discordantly scored as positive.
in each of these  <dig> cases the positively scored peptide only marginally exceeded the cut-off for positivity.
however, it was impossible to determine if these discordantly scored peptides were a result of a false positive  or a false negative .
in summary, the majority of examples of discordant detection of ctl responses were due to sequence variation, while the position of a minimal epitope within a screening peptide inconsistently affected detection of ctl responses.
artifactual false positive or false negative detection of ctl responses was also encountered on rare occasions.
epitopes commonly targeted in subtype b and c are also targeted in subtype a infection
the most frequently recognized peptides  were examined for the possession of common hla alleles among the responder subjects and analyzed for known ctl epitopes.
table  <dig> outlines  <dig> commonly recognized peptides  or peptide overlaps  observed in the study and their probable hla-restrictions.
of these  <dig> epitopes  <dig> have been described before  <cit>  and are commonly recognized in both subtype b  <cit>  and subtype c  <cit>  infections.
the previously described minimal epitopes are also listed in table  <dig>  one nef peptide contains a previously defined hla-b* <dig> restricted epitope, or overlapping epitope, which is restricted by b*4201/ <dig> and/or b* <dig>  seven of the epitopes were detected with all peptide sets while  <dig> displayed variable cross-reactivity and  <dig> was subtype a nef-specific.
the well characterized and studied sl <dig> a*0201-restricted gag p <dig> epitope  was recognized by  <dig> of  <dig> a* <dig> subjects, but the variant  was recognized by only  <dig> of these  <dig> subjects, indicating sequence dependent modulation of immunodominance and cross-reactivity for this epitope.
variable cross-reactivity was also observed for another previously characterized epitope – the gag p <dig> kf <dig> b*5701/ <dig> restricted epitope – was recognized by  <dig> of  <dig> b*5701/ <dig> subjects, but the variant  was recognized by only  <dig> of these subjects.
the b* <dig> restricted gag p <dig> epitope tl <dig>  showed subtype specificity, in that of  <dig> b* <dig> subjects responding to this epitope only  <dig> cross-reacted with the peptides containing the variant tpqdlntml.
therefore, the m to t change in this epitope appears important for ctl recognition.
of the  <dig> nef responses which could be assigned to a particular hla-allele, only the potential a* <dig> restricted epitope  showed subtype specificity.
three a* <dig> subjects responded to this epitope in the a-nef peptide set, but none recognized the complementary peptides in the c- or d-nef peptide sets.
a deletion of  <dig> amino acids in the a-nef isolate sequence relative to the c- and d-nef isolates used in the study most likely accounts for this difference in recognition.
the  <dig> other nef epitopes which could assigned to hla-alleles had been defined previously and were recognized and conserved in the  <dig> peptide sets.
seven subjects recognized the p <dig> peptide frdyvdrffktlrae  in all of the peptide sets.
this sequence maps to a highly conserved region of p <dig> and is identical among  <dig> of the peptide sets and has a single substitution of f301y in the ug <dig>  peptide set.
in the subtype c peptide set,  <dig> peptides  have this sequence spread across their overlap, and both were recognized by all  <dig> subjects.
further characterization of this epitope is performed below.
1con = consensus peptide set
2iso = isolate peptide set
3nd = not defined previously
characterization of an immunodominant hla-cw* <dig> restricted gag epitope
the immunodominant, cross-subtype response directed against the conserved sequence frdyvdrffltlrae  was further characterized.
several epitopes have been previously mapped to this region and hence it was of interest to map the minimal epitope and to determine if this response was restricted by one, or multiple, hla alleles.
all of the seven subjects responding to this epitope expressed the hla-cw* <dig> allele .
while no exact match to the defined cw <dig> peptide-binding motif  <cit>  was found in the 15-mer, an internal 9-mer  had the best possible match of any minimal peptide within the  <dig> mer peptide.
therefore, hla restriction assays were carried out using blcl with a partial hla match with the test subjects and using blcl expressing cw* <dig>  in addition, both the 15-mer and 9-mer peptides  were tested directly for up-regulation of ifn-γ gene expression.
representative data for one of the six responders  is shown in figure 6a.
the 15-mer and yl <dig> peptides elicit an ifn-γ response from an almost identical number of cd8+ t cells .
the only common allele expressed by the blcl able to present the  <dig> mer peptide is hla-cw* <dig> demonstrating that cw* <dig> is the restricting allele for this epitope.
to confirm that the yl <dig> was the minimal epitope, ctl effector cells were generated from pbmc of a cw* <dig> responder subject  using an in vitro stimulation protocol with the 15-mer peptide.
following two rounds of in vitro stimulation the effector cells were used in a standard chromium release assay to test for cytotoxic activity against the 15-mer peptide # <dig> , two flanking peptides  to which no response was detected in the screening elispot assays, and the yl <dig> peptide.
figure 6b shows the efficient specific killing  of autologous blcl pulsed with yl <dig> or peptide # <dig> , but not peptide # <dig> which has the c-terminal l of yl <dig> truncated, nor peptide # <dig> which has the n-terminal y of tl <dig> truncated.
therefore the minimal epitope is yl <dig>  in addition the effector cells also demonstrated efficient killing of blcl pulsed with variants of yl <dig>  which were observed in the cohort.
as shown in figure 6b, blcl pulsed with each of three variants yl9-f6y, yl9-t8v and yl9-t8c were efficiently lysed.
the ability of related alleles within the cw <dig> family to present the yl <dig> peptide was also tested.
the effector cell line from knh <dig> was subjected to a further round of in vitro stimulation and the resultant cells used as effectors in a chromium release assay to test for cytotoxic activity against a panel of blcl expressing different hla-cw <dig> family alleles.
figure 6c shows that cw* <dig> and cw* <dig> can present yl <dig> to a cw*0304-restricted ctl line.
the peptide-pulsed cells are clearly lysed at a range of e:t ratios from 20: <dig> to  <dig> : <dig> compared with the sham-pulsed control cells.
the autologous blcl , and blcl which do not express any hla-cw <dig> alleles, were used as positive and negative controls respectively.
since the relative cross-reactivity of peptide variants can be over-estimated when using highly elevated concentrations of peptide, a titration of the response to yl <dig> and its variants was performed with pbmc from two yl <dig> responders.
figure 6d shows the sequence of the variants used and the number of occurrences of each variant in the proviral dna of the cohort.
in both subjects the response to yl <dig>  was of higher avidity than that to any of the three variants .
of note is that the yl9-t8c variant, the weakest stimulator of all the variants, was detected in the only one of nine cw* <dig> positive subjects that did not respond to the yl <dig> epitope.
therefore the yl <dig> epitope can be presented by most alleles within the hla-cw <dig> family, but in spite of sequence conservation potential escape mutants may already exist.
discussion
while hiv- <dig> subtype c is the predominant circulating pure subtype in sub-saharan africa, hiv- <dig> subtype a constitutes a significant proportion of the epidemic in eastern and central africa  <cit> .
recent studies have estimated that hiv- <dig> subtype a constitutes >90% of the epidemic in terms of pure subtype and recombinant forms in eastern africa, particularly in kenya  <cit> .
comprehensive studies have characterized ctl responses in hiv- <dig> subtype b and c infections, however few studies have mapped in detail ctl cell responses in hiv- <dig> subtype a infections.
our results show that, as has been observed and described for subtype b and c infections, the gag and nef proteins are frequent targets of the ctl response of hiv- <dig> subtype a infections.
although the frequency of responses detected in this cohort against individual peptides was not as great as that detected in studies of subtype b infection   <cit> , the pattern of ctl responses across the gag and nef proteins was remarkably similar to studies from other subtypes  <cit> .
hence, it appears likely that the n- and c-termini of p <dig>  much of p <dig> and the central conserved region of nef are immunodominant for ctl responses irrespective of the infecting subtype.
importantly, both the frequency of responding subjects, and the magnitude of the ctl responses detected against gag and nef using ifnγ elispot assay, was also consistent with results described in other studies of drug treatment naïve subjects from africa  <cit> .
given the disparity of the different peptide sets used  and the fact that the subtype a derived peptide sets were much more similar on average to the viral sequences obtained from the studied cohort, it was surprising to observe that both the magnitude and pattern of recognition of the peptide sets was so similar for both gag and nef.
ctl recognition is critically dependent upon peptide binding to mhc molecules and t cell receptor interactions with the peptide-mhc complex  <cit> ; hence it was reasonable to expect that peptides from non-subtype a origin should be recognized to a lesser extent than the subtype a derived peptides.
however, the results presented here demonstrate that viral sequence variability, and by extension the infecting subtype, does not predict the degree of subtype-specific responsiveness of ctl.
the subtype a based peptide sets only marginally improved our ability to detect ctl responses, with no single peptide set capable of detecting all ctl responses.
these observations are not without precedent, as we have demonstrated equivalent gag, env and nef multiple-subtype recognition by cd <dig> t cells using a recombinant vaccinia virus based expression system producing full-length gene products in a similar cohort of kenyan subjects  <cit> .
in addition, in studies on individuals from uganda, where subtype d and subtype a predominate in the hiv- <dig> epidemic, the lack of subtype-specificity and predictive capacity of the infecting subtype has also been described  <cit> .
a simplistic interpretation of these results is that broadly cross-reactive ctl are generated by any subtype of infection and these responses may be predictive of the ability of a single isolate, or consensus sequence, based vaccine to generate broadly cross-protective responses.
an important caveat associated with the experimental approach used in these studies need to be considered before such a profound conclusion can be reached with confidence.
the concentration of 15-mer peptides used in these studies is very high – usually in the low micro-molar range, whereas minimal peptides have been shown to have half-maximal ctl stimulatory capacity in the low nano-molar range to high pico-molar range  <cit> .
the excessive amounts of 15-mer peptide used in the screening assays may stimulate partially cross-reactive ctl, which might not recognize endogeneously processed and presented antigen on a virus infected cell.
titration of the hla-cw* <dig> restricted yl <dig> epitope variants performed in this study showed that even though cross-reactivity of the f6y variant was detected for all responding subjects  in the screening and confirmation assays, ctl have a much lower avidity for the f6y variant than the wild-type.
hence, true ctl cross-reactivity needs a more stringent definition – one that takes into account avidity for variant peptides, and translation of avidity measurements into biological relevance.
this effect may be even more pronounced on hiv- <dig> infected cells where surface expression of hla class i a- and b-alleles can be down-regulated by the nef protein  <cit> .
a further caveat associated with the use of 15-mer peptides is that minimal ctl epitopes – generally 9– <dig> amino acids in length – are embedded in the 15-mer peptides.
several recent studies have demonstrated that the relative position  of a minimal epitope within a 15-mer peptide can adversely affect the magnitude of the ctl response measured in response to a given 15-mer peptide  <cit> .
while the precise mechanism behind the "epitope-offsetting" effect has not been elucidated it is assumed that amino acid residues flanking the minimal epitope may impair the ability of the 15-mer to be bound by mhc, and/or processed, prior to ctl recognition.
as shown in the present study the epitope-offsetting effect can lead to artifactual false-negatives when comparing different sets of 15-mer olps.
hence, the high concentrations of 15-mer peptides used in screening assays may contribute to an over-estimation of cross-reactivity, and the offsetting of variant epitopes in different 15-mer peptide sets can lead to an under-estimation of cross-reactivity.
despite the potential to confound interpretation of our results, the epitope-offsetting effect was the exception rather than the rule, and as such we believe our overall interpretation of the results to be accurate.
in support of this are prior studies, which have shown that multi-subtype reactivity can be readily detected using recombinant vaccinia viruses to deliver gag, nef and env proteins into the cytoplasm – thereby enabling de novo antigen-processing and presentation to occur – avoiding concentration-dependent issues with peptide studies  <cit> .
reagent selection is a critical determinant for maximizing detection of ctl specific for different epitope variants of hiv- <dig>  while the use of reagents that match the autologous virus sequence in a given host would be the ideal reagent of choice, practical limitations preclude this approach for large-scale cross-sectional screening studies.
consensus and ancestral sequence based peptide sets have been proposed as a practical solution for maximizing ctl epitope detection.
however, as the data presented here shows, a consensus sequence representing a single subtype cannot fully capture the breadth of the ctl response.
utilization of multiple peptide sets clearly increases the breadth of detection of hiv-1-specific ctl, most likely because of the presence of different variants of many epitopes among the peptide sets.
using peptides representing different variants of epitopes – either in olp sets or as minimal peptide epitopes – therefore gives a better account of the true ctl breadth, especially in locations where multiple subtypes co-circulate.
the importance of revealing the true breadth of the ctl response in hiv- <dig> infection has been highlighted in the recent study of frahm et al.
<cit> , which showed that targeting of subdominant ctl epitopes was associated with better control of viral load.
if this result proves to be generalizable, then ctl-based vaccine and immunotherapeutic strategies will need to maximize the breadth of the induced ctl response in order to be efficacious.
this further highlights the importance of reagent selection for measuring ctl magnitude and breadth and for interpreting cross-reactivity in the setting of hiv- <dig> infection and vaccine trials.
while several hla-c restricted epitopes from hiv- <dig> have been defined  <cit> , this study presents, to our knowledge, the first evidence of hla-c restricted immunodominance at the population level.
interestingly, the minimal 9-mer  defined here  with a hla-cw* <dig> allelic restriction, has been defined with hla-b* <dig>  <cit>  and hla-a* <dig>  <cit>  restrictions in other populations.
however, the b* <dig> restriction analysis of this peptide defined by novitsky et al  <cit>  did not completely rule out the possibility that hla-c may have been the restricting locus because of the tight linkage disequilibrium of the hla-b* <dig> allele with cw <dig> alleles.
given that yl <dig> is recognized in the context of most members of the hla-cw <dig> allele family, and at least two other hla alleles, and is derived from a highly conserved region of the p <dig> protein, this peptide warrants inclusion in minimal epitope based vaccine strategies.
it should be noted that the most frequently observed peptide response and hla combination that we noted for the nef protein was also a hla-c allele: hla-cw* <dig> with peptide sequence kkrqeildlwvyhtq .
hla-c alleles may offer an attractive alternative to the more commonly proposed hla-a and -b loci for minimal epitope targeted vaccine strategies because they are refractory to down-regulation by the nef protein  <cit> .
CONCLUSIONS
in conclusion, we have shown that in subtype a infections the pattern of immunodominance and epitope clustering observed within the gag and nef proteins of hiv- <dig> is similar to that seen in subtype b and c infections.
importantly, similar epitope clustering patterns were observed using either a subtype matched consensus-based peptide set, or isolate-based peptide sets from heterologous subtypes.
in agreement with previous studies, knowledge of the predominant infecting subtype in a given population does not necessarily predict the subtype-specificity of the ctl response.
an extension of this observation was the finding that no single screening sequence, even a subtype matched consensus sequence, can fully capture the true breadth of the ctl response.
future studies aimed at capturing the true breadth and cross-reactivity of hiv-1-specific ctl responses in multiple-subtype endemic regions will require:  monitoring the viral quasi-species longitudinally to give a sense of what sequences may be driving a measured ctl response;  high resolution hla-typing data from the subjects studied,  titration studies with synthetic peptides representing minimal epitopes of multiple variants or peptides based upon the autologous viral sequence and;  an understanding of how closely the avidity of a ctl for a variant peptide needs to match that for the index peptide to be considered truly cross-reactive.
finally, vaccine trials offer the best setting for testing the true extent ctl cross-reactivity, since this is the only circumstance in which the exact sequence that primes a ctl response is known with certainty.

 the development of an efficacious prophylactic vaccine for human immunodeficiency virus type is the goal of a concerted worldwide research effort. although the precise correlates of protective immunity against hiv- infection are not clearly defined, a large body of accumulated data suggests that an ideal hiv- vaccine will need to stimulate both humoral and cellular immune responses against the virus. while chronic untreated hiv- infection causes a profound immunodeficiency, the initial hiv- infection stimulates strong cellular and humoral immune responses against the virus. cd cytotoxic t lymphocytes constitute a major component of the cellular arm of the immune response, and have a central role in the control of initial viremia immediately following hiv- infection and in the establishment of long-term aids free survival. the ability to rapidly and accurately characterize ctl responses in hiv- infected individuals has grown exponentially in recent years. new technologies to detect ctl responses by measuring interferon-gamma release, such as the enzyme linked immunospot assay and cytokine flow cytometry, have been coupled with overlapping pooled peptide technology to give detailed and precise analyses of hiv-1-specific cellular immune responses. the fine mapping of t cell epitopes and the identification of immunodominant regions of hiv- gene products is integral to vaccine design, to the development of immunotherapeutic strategies, and to the optimization of assays for assessing vaccine efficacy. genetic diversity is the foremost obstacle for any screening method for hiv-1-specific cellular immunity. geographically defined epidemics can be characterized by the dominance of distinct genetic subtypes of hiv- with at least subtypes and crfs of hiv- currently recognized. since intra-subtype amino acid sequence variation can be as high as 10–15% and inter-subtype variation can be much higher, any single sequence of hiv- used for screening for ctl responses will differ considerably from the sequence of the infecting virus in an individual. this is an important complicating factor for studying the ctl response, because ctl are primed in vivo in response to the autologous infecting virus. although the use of autologous viral sequences has been employed in several studies, their general use is impractical and labor intensive since, not only does the viral sequence between individuals differ significantly, but the viral quasi-species in a single individual can also take many different forms. a proposed solution to this seemingly intractable problem has been to use olp sets based upon computationally derived consensus or ancestral sequences. viral consensus and ancestral sequences have the theoretical advantage of being more related on average to any given in vivo viral sequence than any arbitrary viral isolate would be. a further complicating factor for olp screening is that practical peptide libraries usually comprise 15–20-mers overlapping by 10– amino acids, but the optimal length of peptides that bind to mhc class i molecules is 8– amino acids. however, this problem is overcome in most cases by exogenous addition of the peptides at biologically excess concentrations. despite these compromises the utility of screening for ctl responses with consensus sequence based olp has been demonstrated in several recent studies, which have comprehensively analyzed the full breadth and magnitude of cellular immune responses to the entire hiv- proteome. although consensus sequence based olp have now become the de facto standard for studies of hiv- cellular immunity, the assumption that a subtype based consensus is implicitly better than a randomly chosen isolate from the same subtype and isolates from other subtypes has only recently been questioned formally. this study was undertaken to assess the ctl response to the gag and nef proteins of hiv- infected individuals in kenya, a predominantly hiv- subtype a endemic region, and to compare and contrast the ctl response measured with that determined by prior studies in subtype b and c epidemics. ctl responses directed against the gag and nef gene products were selected because previous studies have shown that these two gene products contain the highest epitope density and are the most frequently recognized hiv- proteins in subtype b and c infection. detection of gag-specific ctl responses was performed using peptide libraries representing a subtype a consensus, a subtype a isolate, a subtype c isolate and a subtype d isolate, to gain an insight into subtype-specificity of ctl responses in a region of multiple subtype endemicity. detection of nef-specific ctl responses was performed using peptide libraries representing a subtype a isolate, a subtype c isolate and a subtype d isolate. after sequence analysis of the gag and nef genes from proviral dna confirmed that the cohort was predominantly hiv- subtype a infected, we integrated knowledge of the sequence of the infecting viral isolate, the hla-type of the subject and the ctl response made by that subject to: determine the frequency of recognition and pattern of ctl immunodominance within the gag and nef proteins in subtype a infections; compare the frequently recognized epitopes in subtype a infection with those previously determined for subtype b and c infections; compare subtype matched and mismatched peptide based peptides sets for their ability to detect ctl responses; characterize an immunodominant hla-cw restricted ctl epitope from hiv- within the framework of this study we were able to directly test the hypothesis that in a given cohort of hiv-1-infected subjects consensus and isolate-based peptide libraries derived from the homologous subtype would detect significantly broader and greater magnitude ctl responses than heterologous subtype-derived peptide libraries.  hiv- gag and nef region subtype analysis and hla-typing full-length gag and nef sequence analysis of pbmc proviral dna was performed to characterize the hiv- subtype distribution within the cohort. a single full-length gag and nef sequence could be obtained from of the subjects. in the gag region, of the subjects harbored hiv- subtype a, had subtype d. the remaining gag sequences represented recombinant structures, a1/d, a2/d. within the nef region, of subjects harbored hiv- subtype a, while the remaining sequences represented subtype d, subtype c and a1/d recombinant. figure shows phylogenetic trees of the gag and nef sequences obtained from the cohort, with the dna sequence from which the isolate based sequences were derived shown for clarity. for both regions, the subtype a isolate shows closer branching to the majority of the cohort sequences than the subtype c or subtype d isolates. a pair-wise comparison of the translated autologous virus protein sequences with the sequence of each of the peptide sets to be used for the elispot screening was performed. the autologous gag protein sequences have mean differences of %, %, % and % from the subtype a consensus, the subtype a isolate, subtype c isolate and subtype d isolate based gag peptide sets respectively. the autologous nef protein sequences have mean differences of %, % and % from the subtype a isolate, subtype c isolate and subtype d isolate based nef peptide sets respectively. therefore, the cohort is predominantly infected with hiv- subtype a viruses, and as would be expected, the gag subtype a consensus and nef subtype a isolate based peptide sets are closest in sequence to the viruses present in the cohort. the nucleotide sequences derived in this study were submitted to genbank and are available under the following accession numbers:. all subjects were high resolution hla class i genotyped to 4-digit accuracy. the frequencies of the most common hla-a alleles were: a; a; a; a; a; a; a; a; a a and the most common hla-b alleles were: b; b; b; b; b; b; b; b; b; b8101; and the most common hla-c alleles were: cw; cw; cw; cw; cw; cw; cw; cw; cw; cw. the detected allele frequencies are in close agreement with published hla allele frequencies for kenya and hence, the cohort can be considered representative of the kenyan population. elispot screening with overlapping peptide pools the cohort was screened for ifnγ t cell responses against four sets of gag overlapping peptide pools and three sets of nef overlapping peptide pools. the gag and nef genes were selected because these two gene products have been documented to contain the highest cd epitope density and are the most frequently recognized hiv- gene products. as described in the methods section, the sequences of the peptide sets used for the study were based upon homologous and heterologous subtype isolates, and on a computationally derived subtype a consensus sequence for the gag protein. ifnγ elispot responses from cryopreserved pbmc were assessed by peptide matrix screening, followed by individual peptide confirmation assays in order to maximize the information from individual specimens. in both the screening and confirmation assays, positive responses were defined as those where the test wells exceeded the 99% confidence interval of replicates of six negative control wells, while for the confirmation assays individual peptides were tested in triplicate. the subjects included in the study all demonstrated strong responses to the seb positive control. table summarizes the ifnγ elispot screening data in terms of the number of subjects responding to each peptide set and the number of discrete responses detected. an epitope, or discrete epitopic region, was defined by a response to a single peptide or adjacent pair of overlapping peptides from an individual peptide set. while subjects responded to at least peptide from the sets of screening peptides, subjects showed no detectable gag or nef peptide specific response. of the subjects responding to any peptide set recognized at least gag peptide set. of interest, similar numbers of subjects recognized at least peptide from the gag peptide sets: the subtype a consensus was recognized by subjects; the crf01_ae peptide set by subjects; the subtype c peptide set by subjects; and the subtype d peptide set by subjects. the total number of responses detected in all subjects was also evaluated. among the gag responding subjects a total of responses were detected, with between and epitopes recognized. a slightly greater, although non-statistically significant, number of responses was detected by the subtype a consensus based peptide set compared with the isolate based peptide sets. the subtype a gag consensus and subtype a gag isolate-based peptide sets detected and of the total gag responses respectively, while the subtype c and d peptide sets each detected responses. the mean number of responses detected per responding subject was not notably different between the gag peptide sets. the total frequencies of cells, expressed as sfc/ pbmc, responding to each of the gag peptide sets is shown in figure 2a. within the cohort the measured frequency of responding cells was similar for each peptide set and no statistical difference among the peptide sets was observed. therefore, the ability to detect gag derived ctl epitopes in an elispot assay for ifnγ production was not significantly improved by using a set of peptides based upon the consensus of subtype a compared with randomly chosen homologous and heterologous subtype based isolate sequences. 1an epitope was defined as a response to a single peptide or pair of adjacent peptides. of the subjects responding to any peptide set, recognized at least nef peptide set. the nef peptide sets were also recognized by equivalent numbers of subjects: the subtype a peptide set was recognized by subjects; the subtype c peptide set by subjects; and the subtype d peptide set by subjects. a total of nef directed responses were detected among the nef responders with between and nef epitopes recognized by each subject. the subtype a nef peptide set was significantly better at detecting the nef-specific responses compared with the heterologous subtype based peptide sets. there was however, no statistically significant difference between the magnitude of sfc detected in response to each of the nef peptide sets. multi-subtype reactive and subtype-specific ctl are detectable in hiv- subtype a infection since the overall analysis showed a high degree of similarity and multi-subtype recognition, the cohort was examined for the number of responses detected across all subtypes and those detected by a subset of subtypes or a single subtype. overlapping venn diagrams were used to display the number of responses detected by the individual peptide sets and by the various combinations of peptide sets. for example, figure 5a shows that of identified gag responses, were detected with all peptide sets as shown by the central overlap of all circles, each of which encompasses all responses detected by that peptide set. by this analysis of the gag responses were detectable with at least of the peptide sets, while the remaining were detected with only or of the peptide sets. strikingly, the consensus subtype a peptide set detected only responses not detectable by any of the isolate based peptide sets, and the isolate based peptide sets collectively, detected responses not detected with the subtype a consensus peptide set. moreover, the subtype c and d heterologous isolate derived peptide sets detected responses not detected by either of the two subtype a based peptide sets, with of the responses representing different epitopic regions since only were against the same peptide. it was reasoned that because the cohort contained subtype c, subtype d and inter-subtype recombinant gag genes, then these subjects may be the source of the responses not detected with the subtype a derived peptide sets. however, this was not the case, as only of the epitopes not detected with subtype a peptide sets could be attributed to subjects from which non-pure subtype a gag containing virus sequence was obtained. this response was detected with the subtype c peptide set and was from a subject carrying an inter-subtype recombinant gag sequence. thus, the overall magnitude and breadth of the ctl response against subtype a gag was underestimated when screening with any single peptide set. the use of a single peptide set derived from an homologous subtype gag sequence did not give a complete representation of the repertoire of gag-specific epitopes recognized by any given subject in this study. similar analysis for the nef protein showed that the homologous subtype a derived peptide set detected all but of the nef responses detected in this study. most of the nef responses were directed to the highly conserved central region of the protein with 26/ total responses detected by all isolate derived peptide sets. notably, the number of responses detected with the subtype a nef isolate peptide set was significantly greater than that detected with either the subtype c nef or d nef peptide sets. therefore in this cohort, the use of homologous subtype derived nef sequences did maximize the detection of nef ctl epitopes. characteristics of positive and discordant peptides in relation to each other and to autologous virus sequences the identified ctl responses were next compared and examined in greater detail. all positively recognized peptides from all peptide sets were compared to the gag or nef sequence of autologous virus. first, the total number of possible responses detected in the study was calculated by multiplying the number of discrete gag responses by and the number of discrete nef responses by this calculation was performed since any response directed against gag had possible hits, and any response against nef had possible hits. of a total of possible responses directed against all gag peptide sets, were detected, while of a possible responses against all nef peptide sets, were detected. when the peptides representing the detected gag and nef responses were compared to the autologous virus sequence obtained from each host, approximately one-third were identical to the autologous virus sequence while approximately two-thirds had at least one amino acid mismatch with the autologous sequence. hence a greater number of responses were detected in subjects whose proviral dna contained a different sequence compared to the peptide used to detect the response. to obtain a better impression of the cross-reactive nature of the ctl responses measured, the impact of "frame-of-epitope" and sequence variation between concordant and discordantly recognized peptides was also examined. the "frame-of-epitope" refers to the offset position of a 9-mer minimal epitope within a given 15-mer screening peptide and the effect has been the subject of several recent studies. because the screening peptide sets were derived from quite disparate subtypes, there was significant frame offsetting between peptides mapping to the same region of the different peptide sets. when a given epitope was detected with more than one peptide set, there were examples of frame offsetting between the concordant peptides. this indicates that despite significant minimal epitope offsetting, cross-recognition of the peptide sets was frequent. in addition, within the concordant peptides there were examples of peptides with different sequences able to be recognized. these most likely represent truly cross-reactive responses, since more than one peptide sequence can be recognized by ctl from a single individual. 1peptides from different peptide sets with the same sequence were scored once only in a given category. next, the discordant peptides were compared to the corresponding positive peptides to identify the reason for non-cross-recognition. the most likely reason for non-cross-recognition was a sequence difference between the peptides. in discrete epitopic regions a discordant peptide had a different sequence compared with the corresponding positive peptide, and of these were in the same frame while were offset in frame. importantly, it was noted that in discrete epitopic regions, discordant results were obtained between peptide sets despite sequence identity across the corresponding regions. in instances there was frame offsetting between the positive and discordant peptides, which could account for the disparity in recognition. however, in instances peptides of identical primary sequence were discordantly scored as positive. in each of these cases the positively scored peptide only marginally exceeded the cut-off for positivity. however, it was impossible to determine if these discordantly scored peptides were a result of a false positive or a false negative. in summary, the majority of examples of discordant detection of ctl responses were due to sequence variation, while the position of a minimal epitope within a screening peptide inconsistently affected detection of ctl responses. artifactual false positive or false negative detection of ctl responses was also encountered on rare occasions. epitopes commonly targeted in subtype b and c are also targeted in subtype a infection the most frequently recognized peptides were examined for the possession of common hla alleles among the responder subjects and analyzed for known ctl epitopes. table outlines commonly recognized peptides or peptide overlaps observed in the study and their probable hla-restrictions. of these epitopes have been described before and are commonly recognized in both subtype b and subtype c infections. the previously described minimal epitopes are also listed in table one nef peptide contains a previously defined hla-b* restricted epitope, or overlapping epitope, which is restricted by b*4201/ and/or b* seven of the epitopes were detected with all peptide sets while displayed variable cross-reactivity and was subtype a nef-specific. the well characterized and studied sl a*0201-restricted gag p epitope was recognized by of a* subjects, but the variant was recognized by only of these subjects, indicating sequence dependent modulation of immunodominance and cross-reactivity for this epitope. variable cross-reactivity was also observed for another previously characterized epitope – the gag p kf b*5701/ restricted epitope – was recognized by of b*5701/ subjects, but the variant was recognized by only of these subjects. the b* restricted gag p epitope tl showed subtype specificity, in that of b* subjects responding to this epitope only cross-reacted with the peptides containing the variant tpqdlntml. therefore, the m to t change in this epitope appears important for ctl recognition. of the nef responses which could be assigned to a particular hla-allele, only the potential a* restricted epitope showed subtype specificity. three a* subjects responded to this epitope in the a-nef peptide set, but none recognized the complementary peptides in the c- or d-nef peptide sets. a deletion of amino acids in the a-nef isolate sequence relative to the c- and d-nef isolates used in the study most likely accounts for this difference in recognition. the other nef epitopes which could assigned to hla-alleles had been defined previously and were recognized and conserved in the peptide sets. seven subjects recognized the p peptide frdyvdrffktlrae in all of the peptide sets. this sequence maps to a highly conserved region of p and is identical among of the peptide sets and has a single substitution of f301y in the ug peptide set. in the subtype c peptide set, peptides have this sequence spread across their overlap, and both were recognized by all subjects. further characterization of this epitope is performed below. 1con = consensus peptide set 2iso = isolate peptide set 3nd = not defined previously characterization of an immunodominant hla-cw* restricted gag epitope the immunodominant, cross-subtype response directed against the conserved sequence frdyvdrffltlrae was further characterized. several epitopes have been previously mapped to this region and hence it was of interest to map the minimal epitope and to determine if this response was restricted by one, or multiple, hla alleles. all of the seven subjects responding to this epitope expressed the hla-cw* allele. while no exact match to the defined cw peptide-binding motif was found in the 15-mer, an internal 9-mer had the best possible match of any minimal peptide within the mer peptide. therefore, hla restriction assays were carried out using blcl with a partial hla match with the test subjects and using blcl expressing cw* in addition, both the 15-mer and 9-mer peptides were tested directly for up-regulation of ifn-γ gene expression. representative data for one of the six responders is shown in figure 6a. the 15-mer and yl peptides elicit an ifn-γ response from an almost identical number of cd8+ t cells. the only common allele expressed by the blcl able to present the mer peptide is hla-cw* demonstrating that cw* is the restricting allele for this epitope. to confirm that the yl was the minimal epitope, ctl effector cells were generated from pbmc of a cw* responder subject using an in vitro stimulation protocol with the 15-mer peptide. following two rounds of in vitro stimulation the effector cells were used in a standard chromium release assay to test for cytotoxic activity against the 15-mer peptide #, two flanking peptides to which no response was detected in the screening elispot assays, and the yl peptide. figure 6b shows the efficient specific killing of autologous blcl pulsed with yl or peptide #, but not peptide # which has the c-terminal l of yl truncated, nor peptide # which has the n-terminal y of tl truncated. therefore the minimal epitope is yl in addition the effector cells also demonstrated efficient killing of blcl pulsed with variants of yl which were observed in the cohort. as shown in figure 6b, blcl pulsed with each of three variants yl9-f6y, yl9-t8v and yl9-t8c were efficiently lysed. the ability of related alleles within the cw family to present the yl peptide was also tested. the effector cell line from knh was subjected to a further round of in vitro stimulation and the resultant cells used as effectors in a chromium release assay to test for cytotoxic activity against a panel of blcl expressing different hla-cw family alleles. figure 6c shows that cw* and cw* can present yl to a cw*0304-restricted ctl line. the peptide-pulsed cells are clearly lysed at a range of e:t ratios from 20: to: compared with the sham-pulsed control cells. the autologous blcl, and blcl which do not express any hla-cw alleles, were used as positive and negative controls respectively. since the relative cross-reactivity of peptide variants can be over-estimated when using highly elevated concentrations of peptide, a titration of the response to yl and its variants was performed with pbmc from two yl responders. figure 6d shows the sequence of the variants used and the number of occurrences of each variant in the proviral dna of the cohort. in both subjects the response to yl was of higher avidity than that to any of the three variants. of note is that the yl9-t8c variant, the weakest stimulator of all the variants, was detected in the only one of nine cw* positive subjects that did not respond to the yl epitope. therefore the yl epitope can be presented by most alleles within the hla-cw family, but in spite of sequence conservation potential escape mutants may already exist. discussion while hiv- subtype c is the predominant circulating pure subtype in sub-saharan africa, hiv- subtype a constitutes a significant proportion of the epidemic in eastern and central africa. recent studies have estimated that hiv- subtype a constitutes >90% of the epidemic in terms of pure subtype and recombinant forms in eastern africa, particularly in kenya. comprehensive studies have characterized ctl responses in hiv- subtype b and c infections, however few studies have mapped in detail ctl cell responses in hiv- subtype a infections. our results show that, as has been observed and described for subtype b and c infections, the gag and nef proteins are frequent targets of the ctl response of hiv- subtype a infections. although the frequency of responses detected in this cohort against individual peptides was not as great as that detected in studies of subtype b infection, the pattern of ctl responses across the gag and nef proteins was remarkably similar to studies from other subtypes. hence, it appears likely that the n- and c-termini of p much of p and the central conserved region of nef are immunodominant for ctl responses irrespective of the infecting subtype. importantly, both the frequency of responding subjects, and the magnitude of the ctl responses detected against gag and nef using ifnγ elispot assay, was also consistent with results described in other studies of drug treatment naïve subjects from africa. given the disparity of the different peptide sets used and the fact that the subtype a derived peptide sets were much more similar on average to the viral sequences obtained from the studied cohort, it was surprising to observe that both the magnitude and pattern of recognition of the peptide sets was so similar for both gag and nef. ctl recognition is critically dependent upon peptide binding to mhc molecules and t cell receptor interactions with the peptide-mhc complex; hence it was reasonable to expect that peptides from non-subtype a origin should be recognized to a lesser extent than the subtype a derived peptides. however, the results presented here demonstrate that viral sequence variability, and by extension the infecting subtype, does not predict the degree of subtype-specific responsiveness of ctl. the subtype a based peptide sets only marginally improved our ability to detect ctl responses, with no single peptide set capable of detecting all ctl responses. these observations are not without precedent, as we have demonstrated equivalent gag, env and nef multiple-subtype recognition by cd t cells using a recombinant vaccinia virus based expression system producing full-length gene products in a similar cohort of kenyan subjects. in addition, in studies on individuals from uganda, where subtype d and subtype a predominate in the hiv- epidemic, the lack of subtype-specificity and predictive capacity of the infecting subtype has also been described. a simplistic interpretation of these results is that broadly cross-reactive ctl are generated by any subtype of infection and these responses may be predictive of the ability of a single isolate, or consensus sequence, based vaccine to generate broadly cross-protective responses. an important caveat associated with the experimental approach used in these studies need to be considered before such a profound conclusion can be reached with confidence. the concentration of 15-mer peptides used in these studies is very high – usually in the low micro-molar range, whereas minimal peptides have been shown to have half-maximal ctl stimulatory capacity in the low nano-molar range to high pico-molar range. the excessive amounts of 15-mer peptide used in the screening assays may stimulate partially cross-reactive ctl, which might not recognize endogeneously processed and presented antigen on a virus infected cell. titration of the hla-cw* restricted yl epitope variants performed in this study showed that even though cross-reactivity of the f6y variant was detected for all responding subjects in the screening and confirmation assays, ctl have a much lower avidity for the f6y variant than the wild-type. hence, true ctl cross-reactivity needs a more stringent definition – one that takes into account avidity for variant peptides, and translation of avidity measurements into biological relevance. this effect may be even more pronounced on hiv- infected cells where surface expression of hla class i a- and b-alleles can be down-regulated by the nef protein. a further caveat associated with the use of 15-mer peptides is that minimal ctl epitopes – generally 9– amino acids in length – are embedded in the 15-mer peptides. several recent studies have demonstrated that the relative position of a minimal epitope within a 15-mer peptide can adversely affect the magnitude of the ctl response measured in response to a given 15-mer peptide. while the precise mechanism behind the "epitope-offsetting" effect has not been elucidated it is assumed that amino acid residues flanking the minimal epitope may impair the ability of the 15-mer to be bound by mhc, and/or processed, prior to ctl recognition. as shown in the present study the epitope-offsetting effect can lead to artifactual false-negatives when comparing different sets of 15-mer olps. hence, the high concentrations of 15-mer peptides used in screening assays may contribute to an over-estimation of cross-reactivity, and the offsetting of variant epitopes in different 15-mer peptide sets can lead to an under-estimation of cross-reactivity. despite the potential to confound interpretation of our results, the epitope-offsetting effect was the exception rather than the rule, and as such we believe our overall interpretation of the results to be accurate. in support of this are prior studies, which have shown that multi-subtype reactivity can be readily detected using recombinant vaccinia viruses to deliver gag, nef and env proteins into the cytoplasm – thereby enabling de novo antigen-processing and presentation to occur – avoiding concentration-dependent issues with peptide studies. reagent selection is a critical determinant for maximizing detection of ctl specific for different epitope variants of hiv- while the use of reagents that match the autologous virus sequence in a given host would be the ideal reagent of choice, practical limitations preclude this approach for large-scale cross-sectional screening studies. consensus and ancestral sequence based peptide sets have been proposed as a practical solution for maximizing ctl epitope detection. however, as the data presented here shows, a consensus sequence representing a single subtype cannot fully capture the breadth of the ctl response. utilization of multiple peptide sets clearly increases the breadth of detection of hiv-1-specific ctl, most likely because of the presence of different variants of many epitopes among the peptide sets. using peptides representing different variants of epitopes – either in olp sets or as minimal peptide epitopes – therefore gives a better account of the true ctl breadth, especially in locations where multiple subtypes co-circulate. the importance of revealing the true breadth of the ctl response in hiv- infection has been highlighted in the recent study of frahm et al., which showed that targeting of subdominant ctl epitopes was associated with better control of viral load. if this result proves to be generalizable, then ctl-based vaccine and immunotherapeutic strategies will need to maximize the breadth of the induced ctl response in order to be efficacious. this further highlights the importance of reagent selection for measuring ctl magnitude and breadth and for interpreting cross-reactivity in the setting of hiv- infection and vaccine trials. while several hla-c restricted epitopes from hiv- have been defined, this study presents, to our knowledge, the first evidence of hla-c restricted immunodominance at the population level. interestingly, the minimal 9-mer defined here with a hla-cw* allelic restriction, has been defined with hla-b* and hla-a* restrictions in other populations. however, the b* restriction analysis of this peptide defined by novitsky et al did not completely rule out the possibility that hla-c may have been the restricting locus because of the tight linkage disequilibrium of the hla-b* allele with cw alleles. given that yl is recognized in the context of most members of the hla-cw allele family, and at least two other hla alleles, and is derived from a highly conserved region of the p protein, this peptide warrants inclusion in minimal epitope based vaccine strategies. it should be noted that the most frequently observed peptide response and hla combination that we noted for the nef protein was also a hla-c allele: hla-cw* with peptide sequence kkrqeildlwvyhtq. hla-c alleles may offer an attractive alternative to the more commonly proposed hla-a and -b loci for minimal epitope targeted vaccine strategies because they are refractory to down-regulation by the nef protein.  in conclusion, we have shown that in subtype a infections the pattern of immunodominance and epitope clustering observed within the gag and nef proteins of hiv- is similar to that seen in subtype b and c infections. importantly, similar epitope clustering patterns were observed using either a subtype matched consensus-based peptide set, or isolate-based peptide sets from heterologous subtypes. in agreement with previous studies, knowledge of the predominant infecting subtype in a given population does not necessarily predict the subtype-specificity of the ctl response. an extension of this observation was the finding that no single screening sequence, even a subtype matched consensus sequence, can fully capture the true breadth of the ctl response. future studies aimed at capturing the true breadth and cross-reactivity of hiv-1-specific ctl responses in multiple-subtype endemic regions will require: monitoring the viral quasi-species longitudinally to give a sense of what sequences may be driving a measured ctl response; high resolution hla-typing data from the subjects studied, titration studies with synthetic peptides representing minimal epitopes of multiple variants or peptides based upon the autologous viral sequence and; an understanding of how closely the avidity of a ctl for a variant peptide needs to match that for the index peptide to be considered truly cross-reactive. finally, vaccine trials offer the best setting for testing the true extent ctl cross-reactivity, since this is the only circumstance in which the exact sequence that primes a ctl response is known with certainty. 
while the different peptide sets detected a similar number of responses, each set was capable of detecting unique responses not identified with the other peptide sets. hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the hiv-1-specific ctl response. we have used the ifn-γ elispot assay and overlapping peptide pools to show that the pattern of ctl recognition of the gag and nef proteins in subtype a infection is similar to that seen in subtypes b and c. the p and p proteins of gag and the central conserved region of nef were targeted by ctl from hiv-1-infected kenyans. interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given ctl response. screening the cohort with peptide sets representing subtypes a, c and d, revealed that peptide sets based upon an homologous subtype only marginally improved the capacity to detect ctl responses. therefore, reagent choice and knowledge of the exact sequences that prime ctl responses will be important factors in experimentally defining cross-reactive ctl responses and their role in hiv- disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive ctl responses. while considerable effort has been focused upon mapping and defining immunodominant ctl epitopes in hiv- subtype b and subtype c infections, few epitope mapping studies have focused upon subtype a. several epitope/hla associations commonly seen in subtype b and c infection were also observed in subtype a infections.
 subtype a is a major strain in the hiv- pandemic in eastern europe, central asia and in certain regions of east africa, notably in rural kenya. while considerable effort has been focused upon mapping and defining immunodominant ctl epitopes in hiv- subtype b and subtype c infections, few epitope mapping studies have focused upon subtype a.  we have used the ifn-γ elispot assay and overlapping peptide pools to show that the pattern of ctl recognition of the gag and nef proteins in subtype a infection is similar to that seen in subtypes b and c. the p and p proteins of gag and the central conserved region of nef were targeted by ctl from hiv-1-infected kenyans. several epitope/hla associations commonly seen in subtype b and c infection were also observed in subtype a infections. notably, an immunodominant hla-c restricted epitope was observed, with 8/ hla-cw subjects responding to this epitope. screening the cohort with peptide sets representing subtypes a, c and d, revealed that peptide sets based upon an homologous subtype only marginally improved the capacity to detect ctl responses. while the different peptide sets detected a similar number of responses, each set was capable of detecting unique responses not identified with the other peptide sets.  hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the hiv-1-specific ctl response. interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given ctl response. therefore, reagent choice and knowledge of the exact sequences that prime ctl responses will be important factors in experimentally defining cross-reactive ctl responses and their role in hiv- disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive ctl responses. 