Data from both animal models  and HIV-infected subjects support an important role for CD8 T lymphocytes in the control of virus in early infection [2–5]. Despite this, strong CD8 T cell responses in HIV-infected individuals do not correlate with the duration of chronic disease or progression to AIDS.
The HIV-1 virus can survive in the setting of strong selective forces, by virtue of its unique ability to accommodate a large number of mutations in its genome. As these changes frequently map to regions containing both antibody  and cytotoxic T lymphocyte (CTL) epitopes [7–10], it is not surprising that disease progression is highly variable in HIV-positive subjects.
The genetic background of the host also influences disease progression, and a number of factors have been identified. Of note are the chemokine receptor polymorphisms, of which a number have been characterized [11–15]. The major histocompatibility complex (MHC) also represents a crucial factor influencing disease progression [16,17]. Since each MHC molecule allows the selection of a different antigenic peptide for presentation to T lymphocytes, class I genotypes dictate the quality of the immune response directed against the virus. Maximum heterozygosity at the MHC class I loci favours a delayed onset of disease, whereas homozygosity is associated with a less-favourable outcome. Individual alleles are also associated with prognosis. Most notably, the B*35–Cw4 haplotype in Caucasians shows a strong association with rapid progression whereas the alleles B*27 and B*57 are commonly associated with a slower onset of disease [18–23].
The Caucasian allele B*5701, and its closely related African counterpart B*5703, are associated with slow progression to disease in the Amsterdam Cohort and Rwandan Women's Cohort, respectively. The p24 epitope KAFSPEVIPMF (KAFS) evokes strong CD8 T cell responses in B*5701 individuals . This epitope is highly conserved in B clade isolates and also represents the A clade consensus sequence. KAFS spans the boundary between the α-helix 1 and the α-helix 2 (H1–H2) and H2 of the p24 capsid. Both helices play a crucial role in the formation of p24 dimer interfaces and stabilize HIV capsid assembly and maturation [25,26]. Despite tight structural constraints, which limit diversity in this region, sequence variants of KAFS have been reported in the Los Alamos database (HIV web site-http://www.hiv-lanl.gov). Although the total number is small, there is considerable variation in these isolates. As some changes could give rise to escape variants that evade immune recognition, this study investigates the degree to which CD8 T cells from B*5701+ and B*5703+ donors tolerate variations in KAFS. First, a combination of functional assays was used to examine the degree to which naturally occurring clade variants of the KAFS epitope are recognized by clones generated from HIV-1-infected B*5701+ and B*5703+ donors. Second, peripheral blood mononuclear cells (PBMC) from B*5701+ and B*5703+ subjects were used to investigated whether they have similar patterns of cross-clade recognition.
Five B*5703+ donors were enrolled through a dedicated clinic for sex workers in Nairobi, Kenya, and one B*5701+ donor from the John Warin Ward, Churchill Hospital, Oxford, UK [27,28]. HLA class I typing was performed by the amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) using sequence-specific primers . All patients have been HIV positive for over 8 years in the absence of antiretroviral therapy but although the cohort fulfilled the standard criteria for categorization as slow progressors, recent CD4 cell counts for some patients were low (< 400 × 106 cells/l). The patients were selected as they generated strong KAFS-restricted CD8 T cell responses in cellular assays. At the time of sampling, CD4 cell counts ranged from 259 to 737 × 106 cells/l.
Isolation of peripheral blood mononuclear cells
PBMC were isolated from heparinized venous blood by Ficoll-Hypaque (NYCOMED PHARMA AS, Oslo, Norway) density gradient centrifugation. Isolated cells were washed and maintained in RMPI 1640 (Sigma-Aldrich, Dorset, UK) supplemented with 10% fetal calf serum, 100 IU/l penicillin (Gibco, BRL, Paisley, Scotland), 100 mg/l streptomycin (Gibco, BRL) (referred to as R10) prior to cellular analysis.
Peptides were synthesized by F-moc chemistry using a Zinner analytical synthesizer (Advanced Chemtech, Louisville, Kentucky, USA). Peptide purity was determined by high-performance liquid chromatography. The majority of clade variants reported in the Los Alamos database were included in this investigation.
Generation of MHC class I tetrameric complexes
The B*5701–biotinylation substrate motif-tagged plasmid was a kind gift from Dr Michel Klein and Dr George Gao. This plasmid was used as a template for the generation of the B*5703 construct, which was carried out by performing two rounds of PCR-dependent Quikchange Site-Directed PCR-based Mutagenesis Kit (Stratagene Cloning Systems, La Jolla, California, USA). The mutagenized constructs were screened by sequence analysis. The expression of MHC class I heavy chain, β2-microglobulin, the refolding of B*5701/B*5703 and the biotinylation and tetramerization of complexes were carried out according to previously described protocols [30,31]. The fluorochrome conjugates used to label biotinylated complexes include avidin–phycoerythrin (PE) and streptavidin–Quantum Red (QR) (Sigma-Aldrich).
Generation of cytotoxic T lymphocyte lines
Cytotoxic CD8 T cell lines were generated according to methods described previously. In brief, 5 × 106 cells/l PBMC were incubated with peptide at a final concentration of 100 μmol/l at 37°C for 1 h. Following incubation, cells were resuspended at a concentration of 2.5 × 106 cells/ml in RPMI supplemented with 25 ng/ml interleukin 7. On day 3, cells were fed by addition of recombinant interleukin 2, in the form of Lymphocult T (Biotest AG, Dreiech, Germany), at a concentration of 10 IU/ml. Cells were screened for their ability to lyse peptide-pulsed B lymphoblastoid cells (BCL) on day 14.
T cells clones
Tetramer staining and magnetic (MACS) bead selection were employed to generate CD8 T cell clones. Up to 1 × 106 cells, from a day 14 CTL line, were stained with 0.5 μg sterile B*5701KAFS–PE conjugated tetramer at 37°C for 15 min and subsequently washed in cold phosphate-buffered saline. The cells were then incubated in 20 μl anti-PE microbeads (MACS Miltenyl Biotec, Surrey, UK) for 15 min at 4°C, washed and resuspended in 500 μl cold RPMI. Meanwhile, MS+/MR+ columns were attached to column adaptors and placed in the magnetic field of a MACS separator. Columns were washed three times with 500 μl cold RPMI, prior to the addition of magnetically labelled cells. The flow through, containing negative cell fractions, was discarded. Columns were washed three times with pre-cooled RPMI and removed from the magnetic field. Tetramer-reactive cells were retrieved by the addition of 1 ml RPMI. Cell viability was determined using Trypan blue (Sigma). Purified tetramer-reactive cells, at concentrations of 1 and 3 cells/well, were cloned into round-bottomed 96-well plates containing 1.5 × 104peptide-pulsed irradiated autologous BCL, 1 × 105 irradiated mixed allogeneic PMBC and a 1 in 200 dilution of phytohaemagglutinin (PHA; stock 9 mg/ml). Clones were supplemented with 10 IU/ml interleukin 2 on day 3 and their specificity was assessed by tetramer staining and cytolytic activity on day 14.
T cell cytotoxicity assays
Standard chromium-51 release assays were performed according to previously described protocols. In brief, either autologous or HLA-matched BCL were labelled with chromium-51 (51CR) for 1 h at 37°C. Following extensive washing, BCL were incubated with peptide antigens for 1 h at 37°C. Following further washes, 5 × 103 cells were plated in round-bottomed 96-well plates. For the peptide titration assays, peptide were added to target cells and remained in the assays throughout. For the peptide binding assays, target cells were labelled with peptide for 1 h at 37°C and washed extensively thereafter. The cells were then divided equally, and one half was labelled with 51CR as described. The remainder were maintained at 37°C, labelled with 51CR 24 h later and tested for their ability to induce cytolytic activity. Effector cells, at ratios of 3:1 (indirect assay) or 1:1 (peptide titration assay), were added, and the final incubation volumes were adjusted to 200 μl. All assays were performed in duplicate, and specific 51CR was calculated as ([experimental release − spontaneous]/[maximum release − spontaneous release]) × 100%.
Staining of CD8 T cells
CD8 cells were stained by incubating 5 × 105 cells with 0.5 μg tetramer for 15 min at 37°C. Following a wash step, cells were incubated with 0.5 μg anti-CD8 Tricolor antibody (CALTAG Laboratories, Burlingame, California, USA) for 15 min at room temperature. Subsequently, cells were washed and fixed in 5% formaldehyde. For cross-reactivity experiments, 0.5 μg index PE and variant QR tetramers were mixed prior to staining experiments. The staining procedure was performed as described.
Peptide-based assay for interleukin-γ
A standard ELISpot assay was used to detect interleukin-γ (IFNγ) release by PBMC, as described previously . PBMC were plated at a density of 5 × 104 cells in the presence of 20 μmol/l peptide. Control wells included PBMC alone and PBMC incubated with PHA. Responses were reported as spot-forming units (SFU) per million PBMC. To fulfil criteria for a positive result, SFU in the peptide wells needed to be at least double that observed with media alone, and exceed 20 per million PBMC.
Sequencing of the p24 KAFS epitope
Genomic DNA was isolated from PBMC using the PureGene DNA Isolation Kit (Gentra Systems, Minneapolis, Minnasota, USA) and used as a source of proviral DNA from which the p24 KAFS epitope was amplified by nested PCR. The p24 5′ outer primer-GAGATA(A/C)(A/G)AGACACCAA(A/G)GAAGC and the 3′ outer primer TCACTTCCCCTTGGTTC TCTC were used to amplify 100 ng purified DNA in the first round of PCR. From the first reaction, 1 μl was amplified in a subsequent PCR amplification using the nested primers p24 3′ B clade inner primer-TGCATGGCTGCTTGATGTCCC, p24 3′ A clade inner primer-TGCATAGCTGCCTGGTGTCCC and 5′ primer-CAGCCAAAATTACCCTATAGTGC. Following amplification, nested p24 PCR products were cloned using the commercially available TOPO TA Cloning Kit (Invitrogen, Groningen, the Netherlands). Colonies containing the p24 sequence inserts were expanded in Luria Bertani broth and plasmid DNA was isolated using a QIAprep Miniprep Spin Kit (QIAGEN, West Surrey, UK).
KAFS-reactive T cell clone cross-reactivity
B*5701 KAFS-restricted clones were generated from a Caucasian donor (AG). Two clones were tested for their ability to lyse the index peptide and the most common clade variant [A2G, S4N] in a concentration-dependent manner (Fig. 1a,b). Representative data clearly demonstrate that B*5701-restricted clones selected on the basis of recognition of KAFS also recognized the [A2G, S4N] variant containing two amino acid substitutions. These clones displayed similar levels of lytic ability at saturating concentrations of peptide; however, at lower concentrations, cytotoxicity was lower against the variant epitopes.
Presentation of KAFS and [A2G, S4N] by B*5703
The common African subtype B*5703 is also associated with slow HIV-1 disease progression. To investigate whether the KAFS and [A2G, S4N] epitopes are presented by this, B*5703 BCL were pulsed with both epitopes and tested in a CTL assay using the B*5701-restricted clones described (Fig. 1c,d). As shown, B*5703 can present both clade variants to B*5701-restricted CD8 T lymphocytes. Indeed, the [A2G, S4N] variant appeared to be presented more efficiently by B*5703 than by B*5701.
Cross-reactivity of the KAFS-restricted response
Although the frequency of KAFS variant viral isolates reported in the Los Alamos database is low (Table 1), these viruses come from Sub-Saharan countries where B*5703 is a common allele. To investigate the ability of clones grown on the index peptide to recognize epitope variants, a combination of CTL assays and tetramer staining was performed. The cytolytic data indicate that the vast majority of peptides induce the lysis of peptide-pulsed targets in a peptide concentration-dependent manner (Fig. 2), often with very similar titration curves. The representative data in Fig. 2 depict two clones generated from the same donor. There is discrepancy in the recognition of the [S4N, V7I] peptide variant between clones 1.4 and 2.2, probably because of differences in the T cell receptor expressed by the clones. Neither clone recognized the P5Q variant.
Differences in cross-reactivity
To date, the majority of variant epitopes that evade T cell recognition acquire mutations that affect their binding to MHC. Consequently, the ability of variant epitopes to bind stably to the B*57 molecules were tested over time. Since there is evidence to suggest the immunogenicity of an epitope may correlate more closely with its dissociation rate from class I molecules rather than binding affinity, a binding assay protocol was chose that had previously discriminated between agonist and antagonist epitopes . The ability of peptide-pulsed BCL to remain CTL targets up to 24 h after initial exposure to peptide was tested. Target cells were first labelled with peptide and then washed thoroughly. Figure 3 shows results for clone 2.2, which indicates that all variants remained bound to B*5701 for at least 24 h. There are, however, differences between the lytic activities at 4 and 24 h for individual epitopes. This is particularly evident for [A2G, S4N] and P5Q variants, which induced approximately 50% of cytolytic activity at 24 h compared with that at time 0. When this assay was performed to assess binding of [A2G, S4N] to B*5703, similar levels of cytolytic activity were detected 4 or 24 h after peptide pulsing (unpublished observation). The poor binding of P5Q may explain its weak recognition in cytolytic assays.
Binding of refolded B*5703 tetramers
The binding of variant B*57 tetramers to T cell clones was also investigated. B*5701 and B*5703 are highly similar class I molecules, and their amino acids differences do not affect their ability to bind identical populations of T lymphocytes (unpublished observations). The B*5703 molecule was used as it proved less problematic to refold ex vivo. The binding of B*5703 variant tetramers to both B*5701- and B*5703-restricted T cell clones was assessed. Data depicted in Figure 4 highlight results obtained using a panel of variant tetramers on one B*5701 and three B*5703 KAFS-restricted T cell clones and clearly demonstrates the binding of variant tetramers to all clones. The staining intensities for M10V, [S4N, V71] and F3L were almost identical between the different clones, with one exception. The V7I tetramer stained clones with a lower fluorescence intensity; however, as this peptide induced strong cytolytic activity, we believe that this merely reflects the quality of the tetramer.
Broad cross-clade reactivity extends to peripheral blood mononuclear cells
As CTL clones are propagated in vitro, it is possible that they are not representative of KAF-reactive T lymphocytes present in fresh PBMC. Consequently, cross-reactivity of KAFS-reactive T lymphocytes was assessed in freshly isolated or cryopreserved PBMC, examining both IFNγ production (Fig. 5) and, where possible, tetramer binding to the T cell receptor (Fig. 6). The ability of KAFS and clade variants to induce IFNγ production were assessed in one B*5701 and five B*5703 HIV-infected subjects (Fig. 5). Proviral DNA was isolated from PBMC and the region of p24 encompassing KAFS was sequenced (Table 2). In most instances, KAFS represented the dominant viral epitope. The exceptions were donor ML525, who primarily harboured the [A2G, S4N] clade variant, and ML1295 from whom a RGFSPEVIPMF variant was isolated. The majority of donors produced IFNγ in response to clade-variant epitopes. There was, however, heterogeneity amongst individuals with respect to the degree of cross-reactivity observed. In the B*5701 donor, all variant epitopes induced IFNγ production, and the numbers of cells that produced cytokine were almost identical to or, in one case, even greater than observed for the index epitope. There was some evidence of differential effector functions induced by different peptides. For example, in subject AG, peptide P5Q induced cytokine production but this peptide was poorly recognized in CTL assays by clones propagated from this subject (Fig. 2). Broad cross-clade reactivity was also observed in ML1295 and most epitopes induced cytokine production at over 50% the value obtained using the KAFS peptide. Interestingly, the sequence isolated from this individual was a [K1R, A2G] variant, and KAFS was not detected in proviral DNA. Despite this, broad responses to variant epitopes were evident. In this instance, the P5Q variant failed to induce cytokine production. Donors ML684, ML713 and ML768 differed in terms of the breath of cross-clade activity observed for IFNγ. ML684 responded to all clade variants, but responses to some variants were low. The remaining variants induced IFNγ production which approximated that recorded for KAFS; indeed, M10V induced cytokine production in a larger number of cells than that observed for KAFS. Donor ML713 displayed a more restricted response to KAFS variants and three peptides, namely [A2G, S4N], F3L and P5Q, failed to activate T lymphocytes. ML684 resembled ML713 in that V7I, M10V and A2N were recognized. Donor ML768 recognized five of the seven variant epitopes. Like donor ML713, responses to peptides [A2G, S4N] and [S4N, V7I] were absent. Unlike ML713, however, small responses to peptides F3L and P5Q were detected and variants V7I, A2N and F3L proved more potent inducers of cytokine production. Finally, donor ML525 displayed a very limited response to the KAFS variants and primarily responded to peptides [A2G, A4N] and [S4N, V71]. Responses to the other variants were either low or absent. ML525 differed from the other subjects in that the main viral epitope isolated from her proviral DNA at this timepoint was [A2G, S4N].
To determine whether the responses observed by ELISpot were genuinely cross-reactive, tetrameric complexes were generated, although technical difficulties prevented the synthesis of all possible variant tetramers. PBMC from a number of donors were stained simultaneously with both the index and variant tetramers. In the case of donor ML684, [A2G, S4N], M10V and F3L tetramers were used. The results (Fig. 6) clearly demonstrate co-staining of the same T cell population by B*5703 tetramers refolded with either the KAFS or variant epitopes (R3 denotes double-positive cells and R8 represents single-positive cells). This shows that the same population, rather than two distinct populations, of T cells is responsible for the cross-reactivity seen in functional assays. The M10V tetramer bound 100% of KAFS-reactive cells and this result is reflected by the ability of this peptide to induce IFNγ production in all KAFS-restricted T cells. The F3L tetramer only bound a small percentage of KAFS-reactive cells and this result agrees with the low IFNγ response to this epitope. Function, however, did not always correlate with tetramer binding. For instance, the [A2G, S4N] tetramer bound 100% of KAFS-restricted cells but only induced IFNγ production in a small number of T cells. This result has important implications and reiterates the importance of combining a number of immunological methods to assess T cell function. Tetramer binding, taken in isolation, does not reflect the ability of a T cell to react functionally to the altered ligand. A similar staining profile was observed in donor ML768, where the [A2G, S4N] epitope failed to induce cytokine production yet the tetrameric complex bound all KAFS-reactive T lymphocytes (R3 denotes single-positive populations whereas R4 represents double-positive populations) . The M10V and F3L tetrameric complexes also bound KAFS-restricted T lymphocytes but in this instance the levels of cross-reactivity correlated with IFNγ production. For donor ML1295, the staining profiles reflected the functional data obtained by ELISpot, and all [A2G, S4N]-, M10V- and V7I-reactive T cells comprised KAFS-reactive T cells. In all instances a small population of KAFS-restricted cells failed to bind variant tetramers, and this agrees with the data obtained by ELISpot. Interestingly, KAFS does not appear to represent the index viral epitope in donor ML1295, yet broad cross-reactivity to KAFS and other variants was evident.
The MHC class I alleles B*5701 and B*5703 are associated with delayed HIV-1 disease progression [19,33]. Both alleles are closely related and their products present identical epitopes, in particular the p24 KAFS epitope, to CD8 T lymphocytes . KAFS spans H1 and H2 of the p24 capsid protein in a structurally conserved region fundamental for capsid assembly. Yet despite structural constraints, variants of KAFS have been reported. As some could potentially represent viral escape mutants, we have investigated the ability of B*57 KAFS-restricted CD8 T cells to recognize database-derived variant epitopes. First, cross-reactivity was assessed using KAFS-restricted CD8 T cell clones directed against the majority of KAFS variants reported in the Los Alamos database. Most peptide variants adequately primed B*5701 target cells for killing. The A2N and [A2G, S4N] epitopes contain amino acid changes in the P2 anchor position of the KAFS epitope. Asparagine is not a defined P2 anchor for B*57 but may be tolerated given that it is polar. Similarly, a small hydrophobic amino acid, such as glycine, may also be accommodated. Only variant P5Q failed to induce strong lysis of target cells. Collectively, the cytolytic data demonstrate broad recognition of peptide variants. Binding to KAFS-restricted T cell clones was observed using a panel of variant tetrameric complexes. It has been reported that tetramers refolded around escape variant epitopes bind T cell clones grown on the index peptide at 4°C but not at 37°C . Our variant complexes demonstrated strong binding at 37°C and are, therefore, likely to represent physiological interactions.
By extending the study to include PBMC, populations of cross-reactive T lymphocytes were detected in all donors. Interestingly, the P5Q variant induced IFNγ production from PBMC yet demonstrated a diminished capacity to sensitize target cells for lysis by T cell clones derived from the same donor. In general, however, P5Q represented a weak ligand. Finally, the panel of variant tetrameric complexes was used to demonstrate that single populations of CD8 T lymphocytes were responsible for the functional cross-reactivity. The percentage of variant tetrameric complexes that bound KAFS-reactive T lymphocytes correlated with cytokine production. The exception proved to be [A2G, S4N] as the tetramer reacted with 100% of KAFS-reactive T lymphocytes in two donors yet this variant failed to induce significant IFNγ secretion. A plausible explanation is that although the [A2G, S4N] tetramer has an affinity for KAFS-restricted T cells, the epitope is unable to activate all downstream effector functions. For donor ML684, KAFS-restricted T cell clones also recognized [A2G, S4N] (data not shown), yet PBMC failed to secrete significant levels of IFNγ. Altered peptide ligands vary in terms of the functions they induce , and [A2G, S4N] may represent an example.
By scanning KAFS sequences from the Los Alamos database, it is evident that certain amino acids are conserved. Most notable are glutamic acid, isoleucine, proline and phenylalanine at positions 35, 37, 38 and 40 in the mature p24 capsid protein, which represent amino acids 6, 8, 9 and 11, respectively, of the epitope. In a crystallographic structure where p24 adopts a typical N–N dimer form, the H1–H2 helices play a crucial role in the intermolecular packing of the capsid . In a study where p24 formed an atypical N–C dimer complex, the importance of the H1–H2 helices in the formation of a H9 interface is demonstrated. The interactions important for this include methionine at position 39 (amino acid 10 of KAFS) and a variety of residues surrounding KAFS. Certain amino acids, which include lysine position 30 and glutamic acid position 35 (amino acids 1 and 5 of KAFS, respectively), are also thought to form part of an intramolecular charged network, stabilizing the monomeric form of p24 .
In summary, broad cross-clade reactivity can be demonstrated in B*5701 and B*5703 individuals using a variety of immunological assays. As KAFS-restricted T lymphocytes tend to be broadly cross-reactive and recognize the majority of structurally compatible mutations, there is, perhaps, little selection pressure on this epitope. This, combined with the inability of viral progeny to tolerate diverse escape mutations in this structurally important region, could help to explain the association of these alleles with good prognosis.
It has recently been demonstrated that the highly conserved B*2705-restricted p24 epitope KRWII LGLNK (GAG amino acids 263–272) requires a series of compensatory mutations to escape immune recognition. An arginine to lysine mutation at position 264, which diminishes binding to MHC resulting in immune evasion by CTL , is strongly associated with a leucine to methionine change at position 268 and also requires an additional compensatory mutation outside the epitope . KRWIILGLNK is an immunodominant epitope in B*2705 individuals, and infants vertically infected with virus containing escape variant epitopes target B*2705 subdominant peptides but fail to control HIV replication . We predict that KAFS is dominant epitope associated with viral control in B*5701+ and B*5703+ subjects, and that many variants of this epitope would be tolerated by the original T cell population. Some, however, such as P5Q, may evade recognition by KAFS-reactive T cells. Viral isolates containing this sequence are rare; so perhaps a complex series of compensatory mutations, as described for the B*27-restricted epitope, are required to produce viable progeny. [A2G, S4N] represents a more common variant, as indicated by the frequency of isolates in the database and in our patient group. [A2G, S4N] might retain the ability to induce cytolytic activity in KAFS-reactive T cells but fail to activate other effector functions. Most epitopes in this study showed good binding to the B*57 molecules. This is important considering that most escape variants evade immune recognition by acquiring mutations that affect binding to MHC [9,39,40].
B*5701 donors who progress at normal rates have been described , and it is important to establish whether their progression relates to the outgrowth of KAFS or other B*5701-restricted variants. Finally, our findings have implications for HIV-1 vaccine design, and our data pertaining to the [A2G, S4N] epitope highlights the importance of incorporating clade variant sequences into epitope-based vaccine constructs.
We are grateful to the donors in this study for their participation and to the clinical staff.
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Keywords:© 2002 Lippincott Williams & Wilkins, Inc.
cellular immunity; CD8; infection diseases; retrovirus; HIV sequence variability; viral infection