Important contribution of p15 Gag-specific responses to the total Gag-specific CTL responses
Yu, Xu G.a,b; Shang, Hongb; Addo, Marylyn M.a; Eldridge, Robert L.a; Phillips, Mary N.a; Feeney, Margaret E.a; Strick, Darylda; Brander, Christiana; Goulder, Philip J. R.a,c; Rosenberg, Eric S.a; Walker, Bruce D.a; Altfeld, Marcus*; and the HIV Study Collaboration
From the a Partners AIDS Research Center and Infectious Disease Division, Massachusetts General Hospital and Harvard Medical School, Boston, USA, the b AIDS Research Center, First Affiliated Hospital China Medical University, Shenyang, China, and the c Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, UK. *See Appendix.
Requests for reprints to: M. Altfeld, MGH-East, CNY 5212, 149 13th Street Charlestown, MA 02129, USA.
Received: 3 August 2001;
revised: 1 October 2001; accepted: 3 October 2001.
Sponsorship: Supported by The Doris Duke Charitable Foundation, the National Institute of Health (R37 AI128568, R01 AI30914, R01 AI44656, R01 AI40873, U01 AI41535 and U01 AI41531), the Deutscher Akademischer Austauschdienst, the Deutsche Forschungsgemeinschaft, the Lloyd Foundation, the Partners/Fenway/Shattuck Center for AIDS Research (CFAR) and several private donors. B. D.W. is the recipient of a Doris Duke Distinguished Clinical Scientist Award and P. J. R. Goulder is an Elisabeth Glaser Scientist of the Pediatric AIDS Foundation.
Objectives: HIV-1 p15 Gag and its protease cleavage products, NCp7 and p6, are believed to play a major role in viral infectivity and assembly during the early and late stages of the retroviral life cycle. However, the extent to which p15 Gag is targeted by the host immune system in natural infection as well as precise cytotoxic T lymphocyte (CTL) epitopes within this protein remains to be defined.
Methods: In this study, 57 HIV-1 infected individuals and 10 HIV-1 negative controls were screened for CD8 and CD4 T-cell responses using overlapping peptides spanning the entire p15 Gag protein as well as the p17 Gag and p24 Gag proteins. Peptide-specific interferon-γ production was measured by Elispot assay and flow-based intracellular cytokine quantification, and cytotoxic activity was confirmed after isolation of peptide-specific CD8 T-cell lines.
Results: CD8 T lymphocytes specific to p15 Gag were found in 46% (26/57) of HIV-1 infected individuals studied and contributed on average 17% (range, 0–100%) to the total Gag-specific T-cell responses. Responses were clustered within three immunodominant regions of p15 Gag, mapping to important functional sites. These studies also include the description of the first three optimally defined CTL epitopes within p15 Gag.
Conclusions: These results indicate that p15 Gag is frequently recognized by HIV-1-specific CD8 T cells in HIV-1 infection and will be important in the comprehensive assessments of CTL responses in infected persons, as well as the design and testing of future HIV-1 vaccines and immunotherapeutic interventions.
HIV-1-specific cytotoxic T lymphocytes (CTL) and T-helper cells are considered to play a central role in controlling viral replication in HIV-1 infection [1–4]. Thus far, studies of HIV-1-specific cellular immune responses have been dominated by the analysis of responses directed against the structural proteins, in particular Gag. HIV-1 Gag-specific CD8 and CD4 T-cell responses have been shown to be negatively correlated to viral load in infected individuals [5–7]. Many optimal CD8 T-cell epitopes within p17 Gag and p24 Gag and CD4 T-cell epitopes within p24 Gag, as well as their restricting HLA alleles, have been described [7–10]. These epitopes have allowed for a more detailed assessment of the influence of cellular immune responses on viral load and disease progression, as well as for functional analysis of these responses [5,11–13]. However, very little is known about the role of cellular immune responses directed against p15 Gag, a protein that constitutes 27% of the total length of the HIV-1 Gag protein. P15 Gag and its protease cleavage products, NCp7 and p6, are believed to play a major role in viral infectivity and assembly during the early and late stages of retroviral life cycle .
In this study, a panel of 94 overlapping peptides spanning the entire Gag p15, p17 and p24 sequence was used in 57 patients to characterize cellular immune responses directed against HIV-1 Gag. It was shown that responses to p15 Gag contribute importantly to the total Gag-specific CD8 T-cell responses. In addition, the first three CTL epitopes within p15 Gag are reported here.
Materials and methods
Fifty-seven HIV-1-infected and 10 HIV-1-negative individuals were studied at the Massachusetts General Hospital (MGH). HIV-1 infected persons included: 37 subjects who were diagnosed and treated with highly active antiretroviral therapy (HAART) within 180 days of HIV-1 seroconversion ; 11 individuals with chronic treated HIV-1 infection; and nine HIV-1-infected individuals from the HIV-1 Controller Study Cohort (these patients have HIV-1 plasma viremia < 1000 RNA copies/ml in the absence of antiretroviral therapy and are referred to as ‘controllers'). At the time of first CTL analysis subjects on HAART had been treated for at least 6 months. The study was approved by the MGH Institutional Review Board and all individuals gave informed consent for participation in the studies.
Cell lines and media
Epstein–Barr virus-transformed B-lymphoblastoid cell lines (B-LCL) were established from the peripheral blood mononuclear cells (PBMC) of each subject and maintained in R20 medium [RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 2 mM l-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 10 mM HEPES, 20% heat-inactivated fetal calf serum (Sigma)] as described previously . For culture of CTL clones, medium containing 10% fetal calf serum (R10) supplemented with 50 U/ml recombinant interleukin-2 (rIL-2; kindly provided by M Gately, Hoffmann-La Roche, Nutley, New Jersey, USA) was used.
HLA class I typing
HLA class I typing was performed at the MGH Tissue Typing Laboratory using sequence-specific primer PCR .
Synthetic HIV-1 peptides
Peptides were synthesized on an automated peptide synthesizer (MBS 396, Advanced ChemTech, Louisville, Kentucky, USA) by using fluorenylmethoxycarbonyl chemistry. Ninety-four overlapping peptides spanning the HIV-1 SF2B clade p15, p17 and p24 Gag sequence (15 mers with 10 amino acid overlap) were generated. In addition, peptides corresponding to described optimal HIV-1 CTL epitopes were used .
Generation of CTL clones
CTL clones were isolated by limiting dilution as described previously , using the anti-CD3-specific monoclonal antibody (MAb) 12F6 as stimulus for T-cell proliferation. Developing clones were screened for HIV-1-specific CTL activity by 51Cr-release assay against autologous B-cell lines pulsed with the peptides recognized in the Elispot assays . HIV-1-specific clones were maintained by stimulation every 14–21 days with an anti-CD3 monoclonal antibody and irradiated allogeneic PBMC . HLA-restriction of CTL epitopes was determined using a panel of target cells matched through only one of the HLA-A, HLA-B or HLA-C class I alleles expressed by the effector cells .
Generation of peptide-specific CD8 T-cell lines
CD8 T cells were expanded non-specifically from PBMC over 10 days by using anti-CD3/CD4b bi-specific MAb . Peptide-specific CD8 T cells were subsequently isolated using an interferon (IFN)-γ catching assay, as described previously . Briefly, 10 × 106 CD8 T cells were incubated with 20 μM peptide and 1 μg/ml each of the anti-CD28 and anti-CD49d MAbs (Becton Dickinson, San Jose, California, USA) on 24-well plates at 37°C, 5% CO2, for 6–8 h. Cells were subsequently labeled with a bi-specific CD45/IFN-γ Catch Reagent and incubated for 45 min at 37°C, 5% CO2. After several washes, the IFN-γ producing cells were stained with a second IFN-γ detection antibody conjugated to phycoerythrin and separated by anti-phycoerythrin MicroBeads on a MACS separator. The isolated cells were then expanded for 10 days using autologous irradiated feeders, as described previously .
HIV-1-specific CD8 T-cell responses were quantified by Elispot assay as described previously . In brief, fresh PBMC were separated by Ficoll-Hypaque (Sigma) density gradient centrifugation and plated out at 50 000 to 100 000 cells per well with peptides at a final concentration of 1 × 10−5 M in 96-well polyvinylidene difluoride-backed plates (MAIP S45; Millipore, Bedford, Massachusetts, USA) precoated with 0.5 μg/ml anti-IFN-γ MAb, 1-DIK (Mabtech, Stockholm, Sweden) overnight at 4°C. For negative controls 100 000 PBMC were incubated with R10 alone. The plates were incubated overnight (14–16 h) at 37°C, 5% CO2 after which biotinylated anti-IFN-γ MAb 7-B6-1 biotin (Mabtech) was added at 0.5 μg/ml and incubation was continued for 90 min at room temperature. Following washing, 100 μl of 1 : 20 000 streptavidin-conjugated alkaline phosphatase (Mabtech) was added per well at room temperature for 45 min. Individual IFN-γ producing cells were detected as dark spots after a 20–30 min color reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium using an alkaline phosphatase-conjugated substrate (Bio Rad Labs, Hercules, California, USA). The number of specific IFN-γ secreting T cells was counted by direct visualization and calculated by subtracting the negative control value and expressed as spot-forming cells (SFC) per 1 × 106 input cells. Negative controls were always < 30 SFC/1 × 106 input cells. CD8 T-cell dependence of all responses to synthetic peptides was confirmed by CD4/CD8 T-cell depletion/enrichment studies using magnetic beads (MACS, Miltenyi Biotech, Germany), according to the manufacturer's protocol.
Fine mapping of optimal CTL epitopes
Fine-mapping of the CD8 T cell responses was achieved by Elispot assay using serial dilutions of truncated peptides . Between 50 000 and 100 000 freshly isolated PBMC per well were incubated with peptides of concentrations from 1 × 10−4 to 1 × 10−11 M overnight on the Elispot plate. All assays were run in duplicate. The optimal peptide was defined as the peptide that induced 50% maximal specific IFN-γ production by T cells at the lowest peptide concentration .
Flow-cytometric detection of antigen-induced intracellular IFN-γ secretion
Intracellular cytokine staining assays were performed as described elsewhere with minor modifications [12,22,23]. Briefly, 0.5–1 × 106 PBMC or CTL lines were incubated with 4 μM of peptide and 1 μg/ml each of the anti-CD28 and anti-CD49d MAbs (Becton Dickinson) at 37°C, 5% CO2 for 1 h before the addition of 10 mg/ml of brefeldin A (Sigma). Following a further 6 h incubation at 37°C, 5% CO2, the cells were placed at 4°C overnight. Cells were then washed and stained with surface antibodies, anti-CD8–peridinin chlorophyll-a protein, anti-CD4-allophycocyanin (Becton Dickinson) at 4°C for 30 min. After washing the cells were fixed and permeabilized using Caltag Fixation/Permeabilization Kit (Caltag, Burlinghame, California, USA) respectively for 15 min at room temperature in the dark and anti-IFN-γ–fluoresceine isothiocyanate was added (Becton Dickinson) at 4°C for 30 min. Cells were then washed and analyzed on a FACS Calibur flowcytometer (Becton Dickinson). Control conditions were established by the use of autologous PBMC, which were treated identically but without peptide stimulation. Assays using HLA-matched or mismatched B-LCL were run for the determination of HLA-restriction of responses as described : B-LCL that were pulsed with 10 μM peptide for 1 h were washed four times prior to incubation with effectors (1 × 105 B-LCL and 4 × 105 effectors) in 1 ml R10 medium. The anti-CD28 and anti-CD49d MAb were then added and the assay was run exactly as described above.
CD8 T-cell responses against HIV-1 p15 Gag peptides
Fifty-seven HIV-1-infected subjects were screened for T-cell responses against the HIV-1 p15 Gag protein using 26 overlapping peptides by IFN-γ Elispot assay. Among them, PBMC from 26 out of 57 (46%) recognized at least one overlapping p15 Gag peptide (median one overlapping peptide per responder; range, 1–3). Magnitudes of responses against p15 Gag peptides ranged from 40 to 480 SFC/1 × 106 PBMC (median, 110 SFC/1 × 106 PBMC). All responses were CD8 T-cell mediated as determined by CD4/CD8 T-cell depletion/enrichment and/or flow-based analysis of peptide-specific intracellular IFN-γ-production by CD8 T cells, except one response against the p15-8 peptide in individual AC-46, which was CD4 T-cell mediated. No response against p15 Gag peptides was observed in the 10 HIV-1 negative individuals used as controls. Three immunodominant regions within p15 Gag were frequently targeted by HIV-1-specific CD8 T cells of the tested individuals (Fig. 1). These regions correspond to functionally important sites within p15 Gag, including the zinc finger structures, the protease Gag cleavage site p7/p1, and the Vpr binding sites in p6 (Fig. 1). These data demonstrate that p15 Gag represents a frequent target for HIV-1-specific CD8 T cells in infected individuals and that these responses map to functionally important regions of the protein.
Contribution of p15 to total Gag CD8 T-cell response
In order to address how much CD8 T-cell response directed against p15 Gag contributed to the total Gag-specific CD8 T-cell activity directed against HIV-1, we screened a subset of 47 subjects from whom a sufficient number of cells were available with a total of 94 overlapping peptides spanning the entire Gag sequence, including p17, p24 and p15, in an IFN-γ Elispot assay. Overall, 36 out of 47 subjects (77%) recognized at least one of the HIV-1 Gag proteins. Subset analysis revealed that Gag-specific CD8 T-cell responses in chronically infected individuals were higher in magnitude (P = 0.02) than those observed in individuals treated during acute infection (Table 1), as described previously for other structural proteins . The magnitude of CD8 T-cell responses directed against p17 Gag and p24 Gag were higher than those directed against p15 Gag (P = 0.006 and P = 0.003, respectively) (Table 1). Overall, CD8 T-cell responses directed against p15 Gag contributed a mean of 17% to the total Gag-specific CD8 T-cell responses in these individuals (Table 1) and in four study individuals (8.5%), Gag-specific CD8 T-cell responses were directed entirely (100%) against p15 Gag. P15 Gag and p24 Gag were more frequently targeted in individuals with chronic and long-term non-progressive HIV-1 infection, but these differences did not reach statistical significance (Fischer's exact test, P > 0.08). In the entire cohort, p15 Gag was targeted less frequently than p24 Gag or p17 Gag by CD8 T cells of HIV-1-infected individuals. However, when the frequency of recognition was adjusted for the length of the protein, p15 Gag was recognized more frequently than p24 (Table 2). These differences did not, however, reach statistical significance by Fischer's exact test (Table 2). Taken together, Gag-specific CD8 T-cell responses would have been underestimated or completely missed in this cohort of 47 individuals without the assessment of p15 Gag-specific responses.
Identification of optimal CTL epitopes within HIV-1 p15
To date, 14 optimal epitopes within p17 Gag and 23 optimal epitopes within p24 Gag have been defined; however, no optimal CTL epitopes have been defined within p15 Gag . We therefore focused on the three immunodominant regions within p15 Gag and determined the optimal epitopes and their HLA class I restriction within these regions.
The responses to the p15-24 peptide in individuals AC-05, AC-39, AC-55 and 013572j were found to be restricted by HLA-B60 (HLA-B*4001) (Fig. 2a), an allele that is expressed in about 20% of the Caucasian population and is even more prevalent in Asian populations [26,27]. The optimal epitope was identified as KELYPLTSL (amino acid position p15 118–126) and exactly fits the published peptide-binding motif for HLA-B60 , with a glutamic acid at position 2 and a leucine at the C terminus. Four additional individuals recognized the p15-24 peptide but did not express HLA-B60, suggesting that additional epitopes restricted by other HLA class I alleles exist within this region. The precise epitopes targeted by these responses have not yet been defined.
A second CTL epitope appeared to be located in the overlapping region of peptides p15-8 and p15-9. The 9-mer peptide CRAPRKKGC (amino acid position p15 42–50) was defined as the optimal epitope, using serial dilutions of peptide truncations (Fig. 2b). This epitope was shown to be restricted by HLA-B14, which was expressed by both persons who recognized this epitope. The published peptide binding motif for B14 contains an arginine at both positions 2 and 5, and a leucine at the C terminus, and thus the novel epitope within p15 Gag matched sufficiently well with this motif. Overall, 33.3% (2/6) of individuals expressing HLA-B14 tested in the study recognized this novel epitope. This epitope is located within the structurally important linker region between the two zinc fingers in NCp7.
The most frequently recognized response among all subjects was found to be to the p15-14 peptide, which includes the highly conserved protease Gag cleavage site between p7 and p1 . Overall 25% (14/57) of studied individuals had detectable CD8 T-cell responses directed against this peptide (Fig. 1). An HLA-A*0201-restricted epitope (FLGKIWPSYK, amino acid position 70–79) was defined within this peptide (Fig. 2c). This epitope was recognized in 13 out of 24 (54%) individuals expressing the corresponding HLA class I allele.
Taken together, three novel CTL epitopes within immunodominant regions of p15 Gag were characterized in this study. All epitopes map to important functional sites within p15 Gag and were recognized frequently (33–100%) by individuals expressing the restricting HLA class I alleles, suggesting a strong immunogenecity of these epitopes.
Many CTL responses within p17 Gag and p24 Gag have been described so far, but little is known about the contribution of cellular immune responses directed against p15 Gag to the total Gag-specific responses. We therefore screened CD8 T-cell responses against HIV-1 p15 Gag in 57 HIV-1-infected individuals by using overlapping peptides spanning the entire p15 Gag sequence of HIV-1. The data showed that CD8 T-cell responses directed against p15 Gag were clustered within three immunodominant regions and contributed importantly to the CD8 T-cell responses against HIV-1 Gag. In addition, to our knowledge the first three optimal CTL epitopes within HIV-1 p15 Gag are reported in this study.
HIV-1-specific CTL responses directed against Gag have been reported to play an important role in control of HIV-1 infection. CD8 T-cell responses against an HLA-A2-restricted epitope in p17 Gag have been inversely correlated to viral load , as were p24 Gag-specific lymph-proliferative T-helper cell responses . However, p15 Gag, that constitutes 27% of the total length of the HIV-1 Gag protein, has been neglected so far in the analysis of HIV-1-specific immune responses. In this study we show that p15 Gag is frequently targeted by CD8 T cells during HIV-1 infection, with 46% of tested individuals having p15 Gag-specific responses. Adapted to length, p15 Gag was targeted even more frequently than p24 Gag. P15 Gag-specific responses were lower in magnitude than p17 Gag and p24 Gag-specific responses, but contributed up to 17% of total Gag-specific CD8 T-cell responses in infected individuals. In addition, four individuals (9%) had responses only against p15 Gag, but not against p17 Gag and p24 Gag. Taken together, Gag-specific CD8 T-cell responses would have been underestimated without assessment of p15 Gag-specific responses in the study individuals. This is of significance, as HIV-1 Gag is a component of several HIV-1 vaccines and immunogens, and p15 Gag-specific immune responses should be included in the assessment of immune responses induced by these.
In order to determine the HLA class I allele restricting the individual CD8 T-cell responses and to assess the impact of CTL-mediated immune pressure on sequence variation within the virus, the minimal epitopes inducing the CTL responses must be defined. We therefore fine-mapped the optimal CTL epitopes located within the three preferentially targeted regions of p15 Gag and determined their restricting HLA class I alleles. All three novel epitopes mapped to important functional sites within p15 Gag. The HLA-B14-restricted CTL epitope (CRAPRKKGC) covered the entire spatial structure between the two zinc fingers in NCp7, which folds into a globular structure and brings the two zinc fingers into close proximity . This globular structure has been shown to be crucial for viral infectivity and virion morphogenesis, as a single mutation in the linker region, which abolishes the globular structure, yields viruses that are non-infectious and have an immature morphology [29,30]. Not surprisingly, this region is highly conserved within the 64 clade B strains reported in the Los Alamos Database . However, CKAPRKKGC (R42K), CRAPRKR GC (K48R) and CKAPRKRGC (R42K/K48R) variants of this epitope are observed in 8%, 19% and 5% of reported clade B strains, respectively . We therefore tested the corresponding variant peptides to determine if these variants lead to the loss of CD8 T-cell recognition, using a CTL clone specific to the CRAPRKKGC peptide. All three variant peptides induced comparable lysis of labeled target cells by CTL, and required similar peptide concentrations for half-maximal lysis (data not shown) suggesting that CTL directed against this epitope demonstrate a large degree of cross-recognition of epitope variants, as described previously for other HLA-B14-restricted epitopes .
The HLA-B60-restricted CTL epitope KELYPLTSL overlaps another important functional region, in which it covers five amino acids of the eight amino acid-long second Vpr binding site in p6 Gag. Interestingly, all four individuals expressing HLA-B60 alleles included in this study recognized this epitope, demonstrating a high immunogenecity of this epitope in the context of B60. This epitope matched exactly the peptide binding motif described for HLA-B60 . In contrast, the most frequently recognized HLA-A*0201-restricted CTL epitope FLGKIWPSYK does not fit the described binding motif for HLA-A*0201 , as it has a lysine at the C-terminal end of the epitope instead of a leucin, isoleucin or valine. It was therefore not predicted as an HLA-A2-restricted epitope within HIV-1 using the HLA-A2 supertype motif . HLA-A*0201-restricted CTL epitopes that do not fit the described binding motif have also been described for other viral infections such as hepatitis C virus . These findings emphasize the importance of a comprehensive approach using overlapping peptides to characterize HIV-1-specific immune responses and to identify novel CTL epitopes, as these epitopes may otherwise be missed by approaches using epitope prediction based on peptide-binding motifs.
In conclusion, p15 Gag is as frequently targeted by T cells during HIV-1 infection as p17 Gag and p24 Gag, with 46% of individuals having p15-specific CD8 T-cell responses. Overall, p15 Gag-specific responses are lower in magnitude than p17 Gag- and p24 Gag-specific responses, but contributed an average of 17% of total Gag-specific CD8 T-cell responses in infected individuals. This report also includes the description of the first three optimal CTL epitopes defined within p15 Gag, including an HLA-A2-restricted epitope. HIV-1 p15 Gag should be included in the assessment of HIV-1-specific cellular immune responses and may represent an important target for future multicomponent HIV-1 vaccines.
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The HIV Study Collaboration
N. Basgoz, G. K. Robbins, B. Davis and S. A. Kalams, Partners AIDS Research Center and Infectious Disease Division, Massachusetts General Hospital and Harvard Medical School, Boston, USA. P. E. Sax, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. S. Boswell and D. S. Cohen, Fenway Community Health Center, Boston, Massachusetts, USA. Cited Here...
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HIV-specific T cell immunity across the entire HIV genome in Chinese men who have sex with men
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Early and late cytotoxic T lymphocyte responses in HIV infection
HIV-1; cytotoxic T lymphocytes; CTL epitopes; HIV-1 Gag; p15 Gag; HLA-A*0201
© 2002 Lippincott Williams & Wilkins, Inc.
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