CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: Use of multiple peptide sets increases the detectable breadth of the CTL response
© Currier et al; licensee BioMed Central Ltd. 2006
Received: 06 January 2006
Accepted: 18 April 2006
Published: 18 April 2006
Subtype A is a major strain in the HIV-1 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-1 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 p17 and p24 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 (Gag 296–304; YL9) was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa), revealed that peptide sets based upon an homologous subtype (either isolate or consensus) only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein), 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-1 disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive CTL responses.
The development of an efficacious prophylactic vaccine for human immunodeficiency virus type 1 (HIV-1) is the goal of a concerted worldwide research effort [1, 2]. Although the precise correlates of protective immunity against HIV-1 infection are not clearly defined, a large body of accumulated data suggests that an ideal HIV-1 vaccine will need to stimulate both humoral and cellular immune responses against the virus . While chronic untreated HIV-1 infection causes a profound immunodeficiency, the initial HIV-1 infection stimulates strong cellular and humoral immune responses against the virus [4, 5]. CD8 cytotoxic T lymphocytes (CTL) 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-1 infection and in the establishment of long-term AIDS free survival [6, 7]. The ability to rapidly and accurately characterize CTL responses in HIV-1 infected individuals has grown exponentially in recent years. New technologies to detect CTL responses by measuring interferon-gamma (IFNγ) release, such as the enzyme linked immunospot (ELIspot) assay and cytokine flow cytometry (CFC), have been coupled with overlapping pooled peptide technology (OLP) to give detailed and precise analyses of HIV-1-specific cellular immune responses [8–11]. The fine mapping of T cell epitopes and the identification of immunodominant regions of HIV-1 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 [12–14]. Geographically defined epidemics can be characterized by the dominance of distinct genetic subtypes of HIV-1, with at least 9 subtypes and 21 CRFs of HIV-1 currently recognized . Since intra-subtype amino acid sequence variation can be as high as 10–15% (depending on the viral gene product) and inter-subtype variation can be much higher, any single sequence of HIV-1 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 [16–18]. A proposed solution to this seemingly intractable problem has been to use OLP sets based upon computationally derived consensus or ancestral sequences [19, 20]. 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 10–12 amino acids, but the optimal length of peptides that bind to MHC class I molecules is 8–11 amino acids. However, this problem is overcome in most cases by exogenous addition of the peptides at biologically excess concentrations [21–23]. 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-1 proteome [10, 11, 24–26]. Although consensus sequence based OLP have now become the de facto standard for studies of HIV-1 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 [27–30].
This study was undertaken to assess the CTL response to the Gag and Nef proteins of HIV-1 infected individuals in Kenya, a predominantly HIV-1 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-1 proteins in subtype B and C infection [10, 11, 24–26, 31]. 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-1 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: (1) determine the frequency of recognition and pattern of CTL immunodominance within the Gag and Nef proteins in subtype A infections; (2) compare the frequently recognized epitopes in subtype A infection with those previously determined for subtype B and C infections; (3) compare subtype matched and mismatched peptide based peptides sets for their ability to detect CTL responses; (4) characterize an immunodominant HLA-CW03 restricted CTL epitope from HIV-1. 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-1 gag and nef region subtype analysis and HLA-typing
All 42 subjects were high resolution HLA class I genotyped to 4-digit accuracy. The frequencies of the 10 most common HLA-A alleles were: A6802 (28.6%); A0201 (21.4%); A0101 (19.0%); A3601 (14.3%); A7401 (14.3%); A3001 (11.9%); A3002 (9.5%); A2902 (9.5%); A2301 (9.5%) A6601 (7.1%) and the 10 most common HLA-B alleles were: B5301 (28.6%); B1503 (16.7%); B4202 (14.3%); B4501 (11.9%); B1510 (9.5%); B0801 (9.5%); B4901 (9.5%); B5703 (9.5%); B5101 (7.1%); B8101; and the 10 most common HLA-C alleles were: CW0401 (31.0%); CW0701 (26.2%); CW0304 (21.4%); CW0602 (21.4%); CW17(01–03) (21.4%); CW16(01,02) (16.7%); CW0202 (14.3%); CW18(01,02) (9.5%); CW0802 (7.1%); CW1502 (7.1%). The detected allele frequencies are in close agreement with published HLA allele frequencies for Kenya [32, 33] and hence, the cohort can be considered representative of the Kenyan population.
Elispot Screening with Overlapping Peptide Pools
Summary of responses detected against each of the peptide sets.
Number of Responders
Number of Responses Detected
Epitopes per Responder1
Subtype A Gag Consensus
Subtype A Gag Isolate (90CF402)
Subtype C Gag Isolate (DU422)
Subtype D Gag Isolate (98UG57143)
Subtype A Nef Isolate (92UG037)
Subtype C Nef Isolate (DU151)
Subtype D Nef Isolate (94UG114)
Of the 33 subjects responding to any peptide set, 24 recognized at least 1 Nef peptide set. The 3 Nef peptide sets were also recognized by equivalent numbers of subjects: the subtype A peptide set was recognized by 24 subjects; the subtype C peptide set by 22 subjects; and the subtype D peptide set by 22 subjects. A total of 40 Nef directed responses were detected among the Nef responders with between 1 and 3 Nef epitopes recognized by each subject (mean of 1.7 epitopes per responding subject). The subtype A Nef peptide set was significantly (χ2 test; p < 0.03) better at detecting the Nef-specific responses (detecting 38 of the 40 total responses) compared with the heterologous subtype based peptide sets (subtype C, 29 responses; and subtype D, 32 responses). There was however, no statistically significant difference between the magnitude of SFC detected in response to each of the 3 Nef peptide sets (Figure 2B) (Friedman Test and Kruskal-Wallis Test).
Multi-subtype reactive and subtype-specific CTL are detectable in HIV-1 subtype A infection
Characteristics of positive and discordant peptides in relation to each other and to autologous virus sequences
Comparison of concordant and discordant peptide responses.
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 76 discrete epitopic regions a discordant peptide had a different sequence compared with the corresponding positive peptide, and of these 52 were in the same frame while 24 were offset in frame. Importantly, it was noted that in 18 discrete epitopic regions, discordant results were obtained between peptide sets despite sequence identity across the corresponding regions. In 13 instances there was frame offsetting between the positive and discordant peptides, which could account for the disparity in recognition. However, in 5 instances peptides of identical primary sequence were discordantly scored as positive. In each of these 5 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 (peptide incorrectly scored positive) or a false negative (discordant peptide incorrectly scored 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
Summary of observed HLA associations of commonly recognized peptides and previously defined eptiopes contained within them.
Peptide set (protein)
Number of responders
HLA allele in common
Previously defined epitope
C W *0304
C W *0701
Characterization of an Immunodominant HLA-C w *0304 Restricted Gag Epitope
While HIV-1 subtype C is the predominant circulating pure subtype in sub-saharan Africa, HIV-1 subtype A constitutes a significant proportion of the epidemic in eastern and central Africa [35–37]. Recent studies have estimated that HIV-1 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-1 subtype B and C infections, however few studies have mapped in detail CTL cell responses in HIV-1 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-1 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 (up to 50% of subjects responding to a single Nef peptide) [10, 11], the pattern of CTL responses across the Gag and Nef proteins was remarkably similar to studies from other subtypes [10, 11, 26, 31]. Hence, it appears likely that the N- and C-termini of p17, much of p24 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 [27, 31].
Given the disparity of the different peptide sets used (derived from subtype A consensus, and subtype A, C and D isolates) 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 CD8 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-1 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 [40, 41]. 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-C W *0304 restricted YL9 epitope variants performed in this study showed that even though cross-reactivity of the F6Y variant was detected for all responding subjects (8/9) 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-1 infected cells where surface expression of HLA class I A- and B-alleles can be down-regulated by the Nef protein [42, 43].
A further caveat associated with the use of 15-mer peptides is that minimal CTL epitopes – generally 9–10 amino acids in length – are embedded in the 15-mer peptides. Several recent studies have demonstrated that the relative position (or offset) 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 [21, 23]. 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 [28, 39, 44].
Reagent selection is a critical determinant for maximizing detection of CTL specific for different epitope variants of HIV-1. 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-1 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-1 infection and vaccine trials.
While several HLA-C restricted epitopes from HIV-1 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 (YL9) defined here (and in reference ) with a HLA-C W *0304 allelic restriction, has been defined with HLA-B*1510  and HLA-A*0207  restrictions in other populations. However, the B*1510 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*1510 allele with C W 03 alleles. Given that YL9 is recognized in the context of most members of the HLA-C W 03 allele family, and at least two other HLA alleles, and is derived from a highly conserved region of the p24 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-C W *0701 with peptide sequence KKRQEILDLWVYHTQ (defined as KY11 associated with C W *0701 allele possession in reference ). 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 [42, 43].
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-1 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: (1) monitoring the viral quasi-species longitudinally to give a sense of what sequences may be driving a measured CTL response; (2) high resolution HLA-typing data from the subjects studied, (3) titration studies with synthetic peptides representing minimal epitopes of multiple variants or peptides based upon the autologous viral sequence and; (4) 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.
Anonymously donated HIV-1 positive blood units were collected between 1999 and 2000 from Kericho District Hospital (Kericho), Rift Valley Provincial Hospital (Nakuru) and Kenyatta National Hospital (Nairobi), all in southern Kenya, under a study approved by both Kenyan and U.S. based Institutional Review Boards. Names, personal information and medical conditions of the subjects were not available, but all subjects were antiretroviral treatment naïve. Blood units were identified only by an alphanumeric code and because of the nature of collection, CD4 counts and viral load data were not available. HIV-1 positivity was assessed by Serostrip (Saliva Diagnostic Systems, Medford NY, USA) and confirmed by ELISA (Organon Teknika/BioMerieux, Inc., Marcy l'Etiole, France).
PCR, cloning and sequencing of HIV-1 gag and nef
Peripheral blood mononuclear cell DNA was extracted using QIAamp blood extraction kit (QIAGEN, Valencia, CA). A nested PCR was employed to retrieve gag and nef region. The PCR mixtures and cycling conditions were described previously . For gag region, outer primers used to amplify gag (HXB2 positions 796 to 2381) were MSF12B (5'-AAATCTCTAGCAGTGGCGCCCGAACAG -3') and BJPOL3 (5'- GTTGACAGGTGTAGGTCCTAC-3'). Then either using F2NST (5'-GCGGAGGCTAGAAGGAGAGAGATGG -3') and SP3AS (5'-CCTCCAATTCCCCCTATCATTTTTGG-3') as the inner primers and directly sequenced the amplicon obtained, or performing the second-round PCR using two inner primer pairs, F2NST/SP3AS and GAG763 (5'-TGACTAGCGGAGGCTAGAAGGAGAGA-3')/JL80 (5'-TAATACTGTATCATCTGCTCCTGT-3'). The PCR products were pooled, purified and cloned into the pCR2.1-TOPO vector using a Topo TA cloning kit (Invitrogen Corp., Carlsbad, CA). Nef region from HXB2 positions 8797 to 9417 was amplified. Outer primers were JL106 (5'-TTCAGCTACCACCGCTTGCGAGACT-3') and UNINEF 7 (5'-GCACTCAAGGCAAGCTTTATTGAGGCTT-3'). The second-round PCR was done using two different inner primer pairs. The first pair was UVNEF1 (5'-GGGGTCGGGAACTGAAAATTAGTGC-3') and TATANEF (5'- GCAGCTGCTTATATGCAGGATCTGAGGG-3'). The second pair was UVNEF2 (5'-AGACAGGGCTTTGAAAGGGCTTT-3') and UNINEF7. The two PCR products obtained were pooled, purified and subjected to sequencing. Plasmid DNA or PCR product was directly sequenced using Big Dye terminator reaction kits and an Applied Biosystems 3100 capillary sequencer (Applies Biosystems Inc., Foster City, CA). The obtained sequences were aligned with reference sequences of the relevant subtypes and circulating recombinant forms (CRF). Phylogenetic analysis was performed to subtype designate the sequences of interest using SEQBOOT, DNADIST (Kimura 2-parameter, transition/transversion ratio = 2.0), NEIGHBOR, and CONSENSE modules of the PHYLIP Package. Deduced gag and nef protein were generated from the nucleotide sequences and aligned. Pair-wised distance matrix of proteins was calculated using maximum likelihood estimates based on the Dayhoff PAM matrix.
PCR-based HLA class I Typing
HLA typing was performed on all subjects at the University of Alabama (Birmingham, AL), using DNA extracted from either PBMC or autologous B lymphoblastoid cell lines (BLCL). Briefly, high molecular weight genomic DNA was extracted from immortalized B cells or PBMC using the QIAamp Blood Kit and protocols recommended by the manufacturer (Qiagen Inc., Valencia, California, USA). The 4-digit alleles at the HLA-A and -B loci were resolved by automated reference-strand conformation analyses (RSCA) of PCR amplicons corresponding to exon 2, intron 2, and exon 3 sequences (Pel-Freez Clinical Systems, Brown Deer, Wisconsin, USA). The 2-digit HLA-C specificities were defined separately by PCR with sequence-specific primers (SSP) (Pel-Freez Clinical Systems, Brown Deer, Wisconsin, USA).
PBMC were screened for HIV-specific CTL responses by stimulation with OLPs representing Gag and Nef from isolates of subtypes A, C and D. OLPs consisted of 15-mer peptides overlapping by 11 amino acids covering the entire Gag and Nef protein sequence of isolates 90CF402 (subtype A-gag, ACC# AAB38823), DU422 (subtype C-gag, ACC# CAD62240) 98UG57143 (subtype D-gag, ACC# AF484514), 92UG037 (subtype A-nef, ACC# AAC97549), DU151 (subtype C-nef, ACC# AAL05314) and 94UG114 (subtype D-nef, ACC# AAC97574). Peptides were synthesized using FMOC chemistry and standard solid-phase techniques with free amino termini. All peptides were >80% pure as determined by HPLC, mass spectroscopy, amino acid analysis and N-terminal sequencing. The A-Gag peptide set was synthesized at the Henry M Jackson Foundation and by Anaspec Inc. (San Jose, CA), while the C-Nef peptide set was a kind gift from Dr. Clive Gray (National institute for Communicable Diseases, Johannesburg, South Africa). All other peptide sets were synthesized at the Natural and Medical Sciences Institute (University of Tuebingen, Germany). Synthetic peptide epitopes of 9 amino acids in length with free amino termini were synthesized using FMOC chemistry and standard solid-phase techniques (Excel automated synthesizer; Waters, Milford MA). Syntheses were carried out in-house (Henry M. Jackson Foundation). All peptides were >80% pure as determined by HPLC, and verified for correct sequence by mass spectroscopy, amino acid analysis and N-terminal sequencing. The peptide pool matrices were of the following formats: 11 linear pools of 11 or 12 peptides and 11 pools of every 11th peptide for the Gag peptide sets; 7 linear pools of 7 or 8 peptides and 7 pools of every 7th peptide for the Nef peptide sets. Therefore, less than two whole 96-well ELIspot plates were required to screen all 7 peptides sets for each individual. See Additional file 1: Peptide Sequences for the list and sequence of all synthetic peptides used in the study.
Enzyme-linked immunospot (ELIspot) assay
All assays were performed using RPMI-1640 medium containing 10% normal human serum, 100 U/ml penicillin and 100 μg/ml streptomycin (complete medium; CM). Cryopreserved PBMC were recovered from liquid nitrogen, thawed rapidly, washed twice with CM and incubated at 2–5 × 106 cells/ml in CM for 14–16 hours. After the overnight rest, the PBMC were enumerated prior to addition to the ELIspot assay plates at 1–2 × 105 viable cells per well. Ninety-six well ELIspot plates (Multiscreen®-IP plates MAIP type plates, Millipore, MA) were prepared by pre-wetting with 50 μl of 70% ethanol (in water) per well and incubating for 5 minutes at room temperature followed by washing six times with PBS. Plates were coated with mouse anti-human IFNγ monoclonal antibody (1-D1K, Mabtech AB, Sweden) at 5 μg/ml in 100 μl PBS overnight at 4°C. Plates were washed six times with PBS and blocked with 100 μl of complete medium for one hour at 37°C. As a positive control for functional integrity of the cells, staphylococcal entertoxin-B (SEB) was added to the wells at 5 μg/ml final concentration and cells were incubated with CM only as a negative control. Peptides were used at a final concentration of 2 μg/ml and all ELIspot tests and controls were performed in triplicate wells. Plates were incubated for 20–24 hours at 37°C (5% CO2), washed six times with PBS/0.05% Tween 20 buffer and incubated for two hours with biotinylated mouse anti-human IFNγ monoclonal antibody (7B6-1-biotin, Mabtech AB, Sweden) at 2 μg/ml in PBS/0.05% Tween 20 buffer. ELIspot development consisted of a one hour incubation with an avidin horseradish peroxidase complex (Vectastain® ABC kit, Vector Labs, CA) in PBS/0.05% Tween 20 buffer followed by washing six times with PBS, and incubation with peroxidase substrate AEC for five minutes (AEC substrate Kit, Vector Labs, CA). ELIspot plates were examined under a stereomicroscope and spots were evaluated with an Automated Elispot Reader System using KS 4.3 software (Carl Zeiss, Thornwood, NY) by an independent scientist in a blinded fashion (Henry M. Jackson Foundation, Rockville, MD). Positive IFNγ spot forming units (SFU) representing single cells were counted and expressed as SFU per one million input PBMC. Assays were considered valid if responses of greater than 500 SFU per 1 million PBMC were detected with SEB. In the screening assays the seven peptide sets were tested in a matrix format consisting of 11 pools of 11 or 12 peptides for the Gag OLP sets and 7 pools of 7 or 8 peptides for the Nef OLP sets. After matrix deconvolution most of the individual peptides were screened in triplicate to confirm the response. Positive responses were defined as those where the mean of the test wells exceeded the 99% confidence interval of replicates of six negative control wells.
CTL effector cell generation
Effector cells were prepared by in vitro stimulation of thawed cryopreserved PBMC. Peptide pulsed (10 μg/ml overnight), irradiated (10000 rad) autologous BLCL (2 × 106 cells) were washed three times with CM and then co-cultured with 10 – 20 × 106 PBMC in 10 ml CM supplemented with 5 ng/ml rhIL-7 (R&D Systems, Minneapolis, MN) for 4 days. 5 ng/ml rhIL-2 (R&D Systems, Minneapolis, MN) was then added to the co-cultures, and the cultures were maintained and split with fresh CM and rhIL-2 for up to 24 days. Effector cell lines were then maintained by re-stimulation every 7–14 days with irradiated, peptide pulsed, autologous BLCL and 5 ng/ml rhIL-2 in CM.
Chromium release assay for cytotoxic activity
CTL effector cells prepared as above were screened for cytotoxic activity against peptide pulsed autologous or partially HLA matched BLCL. BLCL (1 × 106) were incubated overnight with 10 μg/ml of relevant peptide in the presence of 100 μCi of 51Cr-labelled sodium chromate (NEN, Boston, MA) and then washed three times with RPMI-1640 to remove excess 51Cr-sodium chromate and peptide. Non-peptide pulsed BLCL served as the negative control for cytotoxic activity. CTL activity was titrated at several effector to target (E:T) ratios and expressed as percentage of maximal specific lysis of 2500 51Cr-labelled target cells per well. Maximum release was determined by lysis of 51Cr-labelled target cells with 5% SDS. Percent specific cytotoxicity was calculated using the formula: 100 × [(test release - spontaneous release)/(maximum release - spontaneous release)]. Assays were considered valid if spontaneous release was <30%. All assay tests were performed in triplicate and a specific activity of >10% was considered to be positive.
Cytokine flow cytometry (CFC) assay
Cryopreserved PBMC were thawed rapidly, washed twice with CM and either used immediately, or incubated at 2–5 × 106 cells/ml in CM for 14–16 hours. PBMC were added at 0.5 to 1.0 × 106 cells per well into 96 well polypropylene tissue culture trays and stimulated either directly with SEB, or with autologous or allogeneic peptide-pulsed BLCL. BLCL were pulsed overnight with relevent peptide (10 μg/ml), washed five times after the overnight incubation, and distributed at 1 × 105 cells per well (ratio 10:1, PBMC:BLCL). As a positive control for functional integrity of the cryopreserved cells, SEB was added to a single well at 5 μg/ml final concentration. The assays were incubated for 6 hours at 37°C (5% CO2) in the presence of the protein transport inhibitor Brefeldin A (10 μg/ml; Sigma, St.Louis, MO), and were then terminated by transfer to 4°C overnight. Cells were stained for surface markers and intracellular IFNγ expression (CFC assay) the following day. Co-cultured PBMC (prepared above) were washed once with flow buffer (DPBS/0.1% BSA/0.1% sodium azide) and incubated in the 96-well tissue culture tray wells for 10 minutes in 200 μl flow buffer (containing 1 mM EDTA) at room temperature (same volume, temperature and base buffer used for all subsequent washings and incubations). Cells were washed once, fixed in 2% formaldehyde for 30 min and washed again. Fixed cells were permeabilized with 0.5% saponin (Sigma, St. Louis MO.) for 30 min, washed and resuspended in 0.5% saponin containing the following monoclonal antibodies: FITC-conjugated anti-interferon-γ (clone 25723.11); PE-conjugated anti-CD69 (clone L78); PerCP-Cy5.5-conjugated anti-CD8 (clone SK1); and APC-conjugated anti-CD3 (clone SK7)(BD Biosciences, San Jose, CA). After 30 min incubation, cells were washed three times with flow buffer and finally re-suspended in 200 μl of flow buffer. Stained cells were stored at 4°C and analyzed by flow cytometry within 24 hours. Data acquisition was performed on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). Initial gating was performed using a total lymphocyte gate based on forward and side scatter characteristics and acquisition of 50, 000 – 200, 000 cells within this gate. Color compensation was performed using similarly prepared cells from an HIV-1 sero-negative donor and staining singly labeled cells with anti-CD3 labeled with FITC, PE, PerCP-Cy5.5 and APC fluorochromes (BD Biosciences). Data sets were analyzed using FlowJo software (version 4) (TreeStar, Cupertino, CA). Test wells were considered positive if at least a two-fold increase in gated positive events was detected compared with the appropriate negative control. The mean background IFNγ positive CD8 T cells was 0.026% and 0.81% for the media only controls and sham-pulsed BLCL respectively.
We would like to thank Monique Wasunna and Usma Alam for expert assistance in the collection and processing of the blood bank samples. We are also grateful to James Tang (University of Alabama) for HLA typing of the cohort. Financial Support was provided by Department of Defense Collaborative agreement DAMD17-98-2-8007. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Departments of Army or Defense.
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