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National AIDS Research Institute, Bhosari, Pune
Institute of Immunohaematology, Mumbai, India
Analysis of the human immunodeficiency virus type 1 (HIV-1) cytolytic T lymphocyte (CTL) epitopes recognized by the targeted population is critical for HIV-1 vaccine design. Peripheral blood mononuclear cells from 47 Indian subjects at different stages of HIV-1 infection were tested for HIV-1 Gag-, Nef-, and Env-specific T cell responses by interferon (IFN) enzyme-linked immunospot (ELISPOT) assay, using pools of overlapping peptides. The Gag and Nef antigens were targeted by 83% and 36% of responders. Five immunodominant regions, 4 in Gag and 1 in Nef, were identified in the study; these regions are conserved across clades, including the African subtype C clade. Three antigenic regions were also found to be recognized by CTLs of the study participants. These regions were not identified as immunodominant regions in studies performed in Africa, which highlights the importance of differential clustering of responses within HIV-1 subtype C. Twenty-six putative epitopes15 Gag (10 in p24 and 5 in p17), 10 Nef, and 1 Env (gp 41)were predicted using a combination of peptide matrix ELISPOT assay and CTL epitopeprediction software. Ninety percent of the predicted epitopes were clustered in the conserved immunodominant regions of the Gag and Nef antigens. Of 26 predicted epitopes, 8 were promiscuous, 3 of which were highly conserved across clades. Three Gag and 4 Nef epitopes were novel. The identification of conserved epitopes will be important in the planning of an HIV-1 vaccine strategy for subtype Caffected regions.
Class I major histocompatibility complex (MHC)restricted anti-HIV cytolytic T lymphocytes (CTLs) hold the key to the successful control of HIV infection [15]. A strong CTL response is often associated with better virus control and slower disease progression [68]. A potent CTL response was found to be associated with successful control of viremia in monkeys vaccinated with HIV/simian immunodeficiency virus (SIV) vaccines [9, 10]. In the SIV animal model, in vivo elimination of CD8+ T cells resulted in a dramatic increase in virus load [11].
CTLs recognize short peptidesthat is, epitopesin association with class I MHC molecules on the surface of infected cells. Thus, the class I human leukocyte antigen (HLA) profile of the infected population plays an important role in the recognition of CTL epitopes. Epitopes or epitope-rich immunodominant regions that can stimulate a broad range of HIV-specific CTLs may offer the best mode of protection [12, 13] and may be a preferred approach for HIV vaccine design.
The immunodominant regions and CTL epitopes have been well defined for HIV-1 subtype B [1417]. However, little information exists on CTL recognition in HIV-1 subtype C infection [12, 1823], which accounts for 90% HIV infections worldwide, including in India [24, 25]. Studies in South Africa identified immunodominant regions within the HIV-1 subtype C genome [12, 23]. Genetic diversity within subtype C strains in different geographic locations and the HLA profile of the population may influence the repertoire of epitopes recognized. In the present study, we examine the specificity of the CTL response in the context of recognition of individual epitopes. The data will be useful in understanding the basis for cross-clade and broadly reactive CTL responses. A combination of an in vitro assay (ELISPOT) and bioinformatics tools, such as the epitope-prediction programs Epitope Location Finder (ELF) and BioInformatics and Molecular Analysis Section (BIMAS), was used to identify the epitopes.
SUBJECTS, MATERIALS, AND METHODS
Study population.
Forty-seven HIV-1infected individuals enrolled in an ongoing study of clinical progression of HIV infection at the National AIDS Research Institute, Pune, were included in the study. The diagnosis of HIV-1 infection was made, using the kits approved by National AIDS Control Organization, India, by solid-phase combined ELISA and were confirmed by a rapid ELISA test. Blood samples were obtained at 1 of the scheduled visits, after written informed consent was obtained. The estimation of CD4+ T cell counts and plasma virus load was performed as described elsewhere [21]. Class I HLA-A, -B, and -C alleles were genotyped using DNA extracted from frozen peripheral blood mononuclear cells (PBMCs) by polymerase chain reaction (PCR) reverse-line sequence-specific oligonucleotide hybridization strips (Roche Molecular), as described elsewhere [26]. The ethics committees of the authors' institutions have approved the study.
Lymphocyte separation.
PBMCs were separated from freshly collected blood samples by the density-gradient centrifugation method. PBMCs and plasma were collected and stored frozen at -180°C and -70°C, respectively, for ELISPOT assay and plasma virus load estimation.
Interferon (IFN) ELISPOT assay.
Overlapping 20-mer peptides with a 10-aa overlap for HIV-1 subtype C Gag, HIV-1 subtype B Nef, and HIV-1 subtype B Env proteins were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. The lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO; Sigma) and then in RPMI medium at a concentration of 1 mg/mL. The overlapping peptides were pooled together in such a way that no pool contained >20 peptides. For 49 overlapping peptides of HIV-1 subtype C Gag (catalog no. 3993), 3 pools (G1, G2, and G3) were prepared containing peptides 116, 1732, and 3349, respectively. For HIV-1 subtype B Nef (catalog no. 4641), all 20 overlapping peptides were pooled in a single pool (N1); for 80 overlapping peptides of HIV-1 subtype B Env (catalog no. 3990), 4 pools were prepared: E1 (peptides 120), E2 (peptides 2140), E3 (peptides 4160), and E4 (peptides 6180). The final concentration of each peptide in any of the pools was 4 g/mL.
The PBMCs stored at -180°C were thawed and cultured at 37°C for 18 h in RPMI medium (Hi Media) with 10% fetal calf serum (Morgate), 200 mmol/L L-glutamine (Sigma), and antibiotics (100 U of penicillin and 100 g/mL streptomycin; Hi Media). Cultured cells (105) were added to each well in 96-well microtiter high-affinity plates (MAHA54510; Millipore) precoated with antiIFN- antibody (10 g/mL; Mabtech). The peptide pools were added at a concentration of 4 g/mL/peptide. Unstimulated and phytohemagglutinin-P (PHA-P; Murex)stimulated cells served as negative (mock) and positive controls, respectively. The assay was performed in duplicate. The plate was incubated for 16 h at 37°C in 5% CO2. The IFN-producing cells were then visualized with a biotinylated antiIFN- secondary antibody (Mabtech) and an enzyme-substrate complex, avidin-bound biotinylated horseradish peroxidase (Vectastain; Vector Laboratories), and the chromogen substrate (DAB; Research Genetics). The deep-browncolored spots with decreasing density radiating from the center were counted in a stereomicroscope (Stemi 2000 stereomicroscope; Carl Zeiss). The assay was considered valid only if positive (i.e., PHA-Pstimulated) control wells showed >100 spots/100,000 cells and if negative control wells showed <10 spots/100,000 cells. To determine the threshold background response to HIV-1 peptides, PBMCs from 10 HIV-1seronegative individuals were assessed by ELISPOT assay. The mean numbers of spots obtained for HIV-1seronegative individuals were 3 for HIV-1 Gag, 0 for HIV-1 Nef, and 5 for HIV-1 Env. The following 2 criteria were used to determine a positive response: (1) the mean number of spots against the respective antigen in HIV-1seronegative subjects plus 3 SDs was considered to be a cutoff for positivity and (2) peptide wells with >10 spots more than the background (i.e., the number of spots obtained in the negative control wells of the respective study subject) were considered to be positive. The results were expressed as spot-forming units (SFUs)/106 cells.
Epitope mapping.
A 2-step approach was employed for mapping the epitopes recognized by CTLs of study subjects (hereafter referred to as epitopes "recognized by study subjects"). In the first step, the study subjects responding to any of the peptide pools were identified in the screening ELISPOT assay. The lymphocytes from strong responders were subjected to a peptide matrix ELISPOT assay for identifying the individual peptide eliciting the response. The matrix pools were designed in such a way that each peptide is present in 2 different pools. Fourteen matrix pools for HIV-1 subtype C Gag peptides and 9 matrix pools for HIV-1 subtype B Nef peptides were used in the assay. Because of the large number of peptides for HIV-1 Env, 4 different sets of matrix pools were prepared, corresponding to each primary pool (E1E4), containing 9 pools/set. Peptides common in 2 pools eliciting responses were considered for further analysis.
The amino acid sequences of such peptides were submitted to 2 CTL epitope-prediction programs, ELF (available at: http://www.hiv.lanl.gov/content/hiv-db/ELF/epitope_analyzer.html) and BIMAS (available at: http://thr.cit.nih.gov/molbio/hla_bind/), along with the class I HLA types of the study subject recognizing that peptide. ELF defines the probable epitopes within the peptide of interest on the basis of HLA anchor motifs or previously described epitopes stored in the Los Alamos HIV Molecular Immunology Database. The BIMAS program allows users to locate and rank 8-mer, 9-mer, or 10-mer peptides that contain peptide-binding motifs for class I HLA molecules on the basis of a predicted half-time of dissociation to class I HLA molecules [27]. We predicted 8-mer and 9-mer epitopes by use of the BIMAS software.
To confirm that the predicted epitopes are able to induce INF- response in an class I HLArestricted manner, 5 of the peptides representing predicted epitopes were synthesized using 9-fluorenylmethoxy carbonylbased solid-phase chemistry (Xcyton; purity 100% by high-performance liquid chromatography). The peptides were tested in an ELISPOT assay on the PBMCs of the same patient who responded to the 20-mer peptide possessing the predicted epitope in the peptide-matrix ELISPOT.
Statistical analysis.
The correlation between HIV-1specific response and plasma virus load was assessed by nonparametric Spearman test. Similar analyses were performed for CD4+ T cell count. All data were analyzed using SPSS (version 10) software.
RESULTS
Forty-seven (29 male and 18 female) study subjects were enrolled in the study. The mean age of subjects was 31 years. The subjects had a median CD4+ T cell count of 422 cells/mm3 (range, 28938 cells/mm3) and a median virus load of 186,022 copies/mL (range, 4001,421,611 copies/mL). A significant negative association was found between CD4+ T cell count and plasma virus load (P < .05). The major HLA alleles expressed by the study subjects were A*11011 (31.25%), A*0211 (29.16%), B*3520 (10.52%), B*4006 (35.4%), B*4406 (27.1%), B*1807 (6.6%), Cw*0602 (37.5%), and Cw*1507 (22.91%).
Thirty (64%) of 47 patients showed a response to 1 HIV-1 antigens. Of 30 responders, 25 (83%) responded to HIV-1 subtype C Gag, 11 (36%) responded to HIV-1 subtype B Nef, and 6 (20%) responded to HIV-1 subtype B Env (figure 1). HIV-1 Gag was found to be the most targeted protein.
To confirm the CD8-mediated nature of the response, the ELISPOT assay was repeated on CD8-depleted cells in 3 responders from whom sufficient PBMCs were available. Complete abrogation of anti-HIV Gag responses was seen in 2 subjects, and 1 subject showed a 95% reduction in SFUs (figure 2).
The association of plasma virus load with the breadth of response (no. of HIV antigens recognized by the study subjects, as estimated in primary screening using peptide pools) and magnitude of response (no. of SFUs per 106 PBMCs) was studied. The response against Nef and p24 Gag antigen showed a trend of inverse correlation between the magnitude of the response and virus load, but the correlation was not statistically significant. No significant association was found between virus load and breadth of response (P > .05). The CD4+ T cell count also did not show any significant association with HIV-specific T cell response.
The study subjects showing responses of >200 SFUs/106 cells to Gag, Nef, and/or Env antigens in primary screening were considered to be strong responders. Peptide-matrix ELISPOT was performed on the PBMCs of 9 such study subjects. The amino acid sequences of the peptides identified in matrix were submitted to the prediction software along with the HLA types of the respective study subjects. By use of this algorithm, 26 potential epitopes were identified. The amino acid sequences of these epitopes, their location in the antigen, and their HLA restriction are shown in table 1.
Fifteen (10 in p24 and 5 in p17) of 26 potential epitopes were from the Gag antigen. The p24 epitopes were clustered in 2 regions: aa 955 (Gag aa 141190), aa 6595 (Gag aa 201230), and aa 135185 (Gag aa 261310). The most commonly identified p24 epitopes were AEWDRLHPV (recognized by 3 of 9 study subjects) and GPIAPGQM (recognized by 2 of 9 study subjects). The epitope AEWDRLHPV was also found to be recognized in context with multiple HLA alleles, including Cw*0602 and B*4006, in 1 patient (CPI 213). These 2 p24 epitopes showed >50% conservation across clades (obtained from http://www.hiv.lanl.gov/content/hiv-db/EPILIGN/EPI.html). The epitopes ILGLNKIV, DTINEEAAEW, and RMYSPVSIL were found to be highly conserved across clades, showing 90% conservation with Indian clade C sequences and >70% conservation with non-Indian clade C sequences (figure 3).
The epitopes predicted in the p17 region were found to be clustered in the region of aa 2250. This amino acid stretch exhibited >1 epitope. One of the 5 p17 epitopes, RPGGKKRYM, was recognized by 3 study subjects (table 1).
We identified 10 Nef epitopes, 8 of which were from the central regions (aa 102150) of the Nef protein. Two of these epitopes were recognized in association with at least 3 different class I HLA molecules (table 1). The epitope WIYHTQGYF showed multiple HLA specificities with B*35, Cw*06, and Cw*04 in 2 study subjects. All Nef epitopes showed 20%30% conservation with only subtype B sequences.
Of 26 identified epitopes, 7 (1 p24, 2 p17, and 4 Nef) epitopes were found to be novel. The p24 epitope, GPIAPGQM, and 1 of the p17 epitopes, HLVWASREL, were found to be conserved (figure 3), and the other p17 epitope (RPGGKKRYM) appeared to be subtype C specific, showing consensus only with subtype C sequences available in the database (figure 3).
Only 6 subjects showed an antiHIV Env response, of whom only 1 (CPI 197) exhibited a strong response (870 SFUs/106 cells) to pool E3. The epitope mapping performed for this subject predicted 1 gp 41 epitope. (RAIEAQQHML) (table 1). The epitope showed >50% conservation across clades (figure 3) and had multiple HLA specificities (table 1).
To confirm whether epitopes predicted using the algorithm of peptide-matrix ELISPOT and computer-assisted prediction were genuine, 5 peptides from among the 15 predicted Gag epitopes were synthesized. These peptides were applied in an ELISPOT assay using PBMCs from the same study subject who recognized the 20-mer peptide possessing the epitope in peptide-matrix ELISPOT. Four of these 5 epitopes were recognized by the respective study subjects (table 2). The peptides did not induce an INF- response in PBMCs of study subjects who did not possess the allele with which the epitope was predicted, thus establishing HLA restriction of the responses.
DISCUSSION
Although HIV-1 subtype C vaccine candidates based on the empirical selection of the viruses are likely to enter into clinical trials in different parts of the world, there is still need for a greater understanding of HIV-specific cellular immune responses in context with HLA profiles of the affected populations.
Putative CTL epitopes recognized by Indian patients, predicted by use of the combination of in vitro assay (peptide-matrix ELISPOT) and bioinformatics tools (epitope-prediction software), are reported here for the first time, to our knowledge. Since this prediction was based on certain assumptions, such as the strength of binding between the HLA molecule binding groove and anchor residues of the peptide, it was critical to ascertain that the predicted epitope can really induce the desired response. When we assessed the ability of 5 of 15 predicted Gag epitopes to stimulate IFN- release, 4 of 5 synthesized epitopes were able to elicit response.
Previous studies of HIV-1specific CTL responses have either used single peptides [5] or panels of optimal epitopes predicted in association with HLA types in ELISPOT assays [16, 17, 28, 29]. Such an approach may miss some of the epitopes that might be important in governing T cell responses. The algorithm using overlapping peptides spanning the whole antigen and the use of epitope-prediction software may increase the chance of recognizing a near-complete repertoire of the epitopes.
In the present study, Gag p24 and Nef were the most recognized antigens among the study population, with 83% responding to Gag and 33% responding to Nef. This is consistent with the findings of other studies of individuals infected with HIV-1 subtypes B, A, and C, irrespective of the nature of antigens used in the study [15, 29]. The broader and strong p24 response observed in the present study supports a dominant role of Gag-specific CTL epitopes in the natural course of HIV-1 subtype C infection.
Nef-specific responses are considered important in HIV-1 infection and are one of the earliest responses seen in acute HIV-1 infection [30]. The vigorous Nef-specific response has also been observed in individuals from India who recently experienced seroconversion [31]. The response to Nef antigen observed in chronic HIV infection in the present study highlights the need to understand the significance of these responses in the context of HIV disease progression.
The response to Env peptides was seen in only 6 study subjects (20%). This might be because of the genetic diversity in the Env antigens across clades [32, 33]. The low frequency of the response to Env may be attributed to the use of heterologous peptides.
No significant correlation was found between either CD4+ T cell count or plasma virus load and IFN- secretory response. Some previously reported studies have shown an inverse correlation between the frequency of HIV-specific CD8+ T cells and plasma virus load [1, 6, 29, 34, 35], whereas no such correlation was observed in other studies [16, 36, 37]. A study on targeting of different HIV antigens has shown that preferential targeting of Gag epitopes, rather than the breadth and magnitude of the response, may be an important marker of immune efficacy [22]. We have reported elsewhere a marginally significant correlation between CD8+ T cell IFN- response to subtype Cspecific Gag peptides and plasma virus load in patients during the first year of infection [21]. The patients in the present study may be at varied stages of infection, which may influence the correlation. All of these findings collectively indicate that factors other than just magnitude and breadth of the CTL response might be important in controlling HIV replication.
The epitopes predicted in the p17 region were found to be clustered in aa 2250 (RGGKLDKWEKIRLRPGGKKRYMIKHLVWASRELERF) and aa 6185 (MKQLQPALQTGTEELRSLYNTV), which were also identified as immunodominant regions recognized by South African patients with acute HIV infection [22] (figure 4). Most of the previously reported HIV-1 subtype B epitopes are also located in these regions [38].
The central domain of the Nef protein (aa 101150) appeared to be the immunodominant region, with 8 of 10 epitopes located in this region. The central domain of the Nef antigen has been reported as an epitope-rich region by other investigators, as well [22, 23, 39, 40]. The regions of aa 122141 and aa 92131 have been identified as immunodominant regions in HIV-1 subtype C Nef [22, 23]. Identification of such immunodominant regions may be important in designing a vaccine candidate for HIV-1 subtype Caffected populations.
However, our study also identified additional regions in HIV-1 Gag and Nef antigens that differ from previously reported immunodominant regions (figure 4); these additional regions include Gag aa 201230 (LKDTINEEAAEWDRLHPVHAGPIAPGQMRE), Nef aa 2140 (RAEPAADGVGAASRDLEKHGAITSSNTAAT), and Nef aa 181200 (GMDDPEREVLEWRFDSRLAFHHVARELHPE). Such additional regions have been identified in the other studies, as well [22, 23] (figure 4). The genetic diversity between the populations screened in different studies might influence the recognition of epitopes [20, 41, 42]. Also, isolates within HIV-1 clade C have been shown to be regionally clustered and, thus, might be different from each other [43]. Hence, the genomic variability in HIV-1 subtype C viral strains might also be responsible for such differences in recognition of epitopes. This observation highlights the importance of the identification of epitopes from different antigenic regions identified within subtypes.
As per the prediction of our algorithm, 2 p24, 5 Nef, and 1 gp41 epitopes were recognized by >1 HLA allele. Presentation of identical epitopes by closely similar class I HLA molecules has been described elsewhere [4446]. The epitopes recognized in the present study substantiate this observation.
We have shown that HLA alleles such as Cw*1507, B*4406, A*0101, Cw*12, and B*1801 are significantly associated with HIV infection in the study cohort [41]. Since these HLA alleles were not listed in the BIMAS epitope-prediction program database, epitopes associated with these HLA alleles could not be identified. It has also been shown that HLA-C alleles can present a number of HIV-derived epitopes [47]. This is especially important, because HIV Nef does not appear to down-regulate C alleles, and responses restricted by HLA-C may, thus, not be subject to the immune modulating effect. Hence, finer analysis of Nef CTL epitopes recognized by HLA-C alleles like Cw*1507 and Cw*12 will be important in assessing importance of Nef-specific responses in disease progression.
The HIV-1 Gag peptides used in the present study were from African HIV-1 subtype C (strain 96Z M651.8); the Gag sequence of this strain has 90% homology with all available Indian subtype C sequences. The peptides for Nef (HIV-1 BRU) and Env (HIV-1 MN) were based on subtype B antigens with 83% and 60% homology, respectively, with the available Indian HIV-1 subtype C sequences. The lower responses seen against Env peptides may be attributable to this genetic diversity. Hence, use of consensus Indian HIV-1 subtype C peptides might reveal subtype-Cspecific epitopes in Env antigens.
We have shown that the immunodominant regions identified in the present study are conserved across HIV-1 clades, including HIV-1 subtype C strains from South Africa. Targeting epitopes from multiple immunodominant regions might improve overall vaccine immunogenicity and minimize viral escape from immune recognition. We also identified epitopes from other antigenic segments, highlighting the importance of clustering of epitopes within subtypes. These observations need to be further substantiated with studies on larger populations and other HIV antigens. This may have critical importance for the development of vaccine candidates that would have universal applicability.
Acknowledgments
We thank Sanjay Mehendale, S. P. Tripathy, Manisha Ghate, Deepanjan Roy, Reva Kohali, and the staff of the National AIDS Research Institute clinic for providing clinical and demographic data on the study subjects. We also thank A. R. Risbud, Varsha Kale, and the staff of the Department of Serology for providing HIV diagnoses for the study subjects.
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