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Identification of novel consensus CD4 T-cell epitopes from clade B HIV-1 whole genome that are frequently recognized by HIV-1 infected patients

Fonseca, Simone Ga,b,c; Coutinho-Silva, Adrianaa,b,c; Fonseca, Luiz Augusto Ma,c; Segurado, Aluisio Cc,d; Moraes, Sandra La,c,e; Rodrigues, Hélciob; Hammer, Juergenf; Kallás, Esper Gc,g; Sidney, Johnh; Sette, Alessandroh; Kalil, Jorgea,b,c; Cunha-Neto, Edecioa,b,c

doi: 10.1097/01.aids.0000253353.48331.5f
Basic Science

Objective: To identify promiscuous and potentially protective human CD4 T-cell epitopes in most conserved regions within the protein-coding genome of HIV-1 clade B consensus sequence.

Design: We used the TEPITOPE algorithm to screen the most conserved regions of the whole genome of the HIV-1 subtype B consensus sequence to identify promiscuous human CD4 T-cell epitopes in HIV-1. The actual promiscuity of HLA binding of the 18 selected peptides was assessed by binding assays to nine prevalent HLA-DR molecules. Synthetic peptides were tested with interferon-γ ELISPOT assays on peripheral blood mononuclear cells (PBMC) from 38 HIV-1 infected patients and eight uninfected controls.

Results: Most peptides bound to multiple HLA-DR molecules. PBMC from 91% of chronically HIV-1 infected patients recognized at least one of the promiscuous peptides, while none of the healthy controls recognized peptides. All 18 peptides were recognized, and each peptide was recognized by at least 18% of patients; 44% of the patients recognized five or more peptides. This response was not associated to particular HLA-DR alleles. Similar responses were obtained in CD8 T-cell-depleted PBMC.

Conclusion: In silico prediction of promiscuous epitopes led to the identification of naturally immunodominant CD4 T-cell epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous patient population exposed to HIV-1. This combination of CD4 T-cell epitopes – 11 of them not described before – may have the potential for inclusion in a vaccine against HIV-1, allowing the immunization of genetically distinct populations.

From the aDivision of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, Brazil

bHeart Institute (InCor),University of São Paulo School of Medicine, Brazil

cInstitute for Investigation in Immunology – Millenium Institutes, Brazil

dDepartment of Infectious Diseases, University of São Paulo School of Medicine, Brazil

eInstitute of Tropical Medicine, University of São Paulo, Brazil

fDepartment of Genomic and Information Sciences, Hoffmann-La Roche Inc., Nutley, New Jersey, USA

gDivision of Infectious and Parasitic Diseases, Federal University of São Paulo, Brazil

hLa Jolla Institute for Allergy and Immunology, San Diego, USA.

Received 19 June, 2006

Accepted 28 July, 2006

Correspondence to E. Cunha Neto, Laboratory of Immunology – Heart Institute (Incor), University of São Paulo School of Medicine, Av. Dr. Enéas de Carvalho Aguiar, 44, Bloco 2, 9 andar, São Paulo, SP, 05403-000, Brazil. Tel: +55 11 3069 5906; fax: +55 11 3069 5953; e-mail:

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An effective vaccine appears to be the only means of halting the HIV-1 epidemic in developing countries that are unable to offer antiretroviral therapy (ART) to their patients. A major effort must be deployed to improve the design of new vaccine candidates, both at the level of epitope constitution, adjuvants and immunogen formulation, as well as their clinical evaluation, as no candidate vaccine tested in humans has so far shown significant protection [1]. In spite of the abundant evidence that cytotoxic CD8 T lymphocytes (CTL) are the primary anti-HIV-1 effectors [2–4], several studies have shown that a concomitant strong specific CD4 T-cell response is invariably needed [5,6]. The importance of virus-specific CD4 T cells and their association with a protective antiviral immune response has been demonstrated in humans [7]. The control of HIV-1 replication in the absence of ART has been associated with vigorous proliferative CD4 T-cell responses in HIV-1 positive patients [8]. T-cell help is important for priming the CD8 T-cell responses and for maintaining CD8 T-cell memory [9], and HIV-1-specific CD4 T cells play a functional role in response to viral antigen stimulation [10–12]. HIV-1-specific CD4 T-cell responses are more often detected in long-term non-progressor (LTNP) patients than in individuals with progressive disease [8,12]. In addition, control of viral replication by ART is associated with increases in HIV-1 specific CD4 T-cell responses [13]. Strong CD4 T-cell responses have also been observed in patients under ART with low-level viraemia and moderate numbers of CD4 T cells, the so-called partial controllers [14,15].

Although considerable information has been gathered on HIV-1 CD8 T-cell epitopes, comparatively few HIV-1 CD4 T-cell epitopes have been identified so far [16]. Given their major role in determining the functional status and memory of effector responses, appropriate HIV-1 CD4 T-cell epitopes should be an essential part of any candidate vaccine. It is thus necessary to identify the yet missing, frequently recognized CD4 T-cell HIV-1 epitopes recognized by the majority of individuals.

Until recently, the only available means for searching immunodominant epitopes was the direct testing of substantial numbers of overlapping peptides or peptide libraries. The identification of MHC-binding motifs allowed the prediction of potential T-cell epitopes [17], and such motifs were found to cluster in certain protein regions [18]. The TEPITOPE algorithm, that predicts binding to 25 distinct HLA-DR molecules based on quantitative matrices established from HLA-DR binding assays [19–23] leads to the selection of sequences enriched for high affinity-binding peptides, those with the highest chance of eliciting effective T-cell responses against immunogens [24]. Additionally, TEPITOPE also allows detection of sequences predicted to bind to several HLA-DR molecules simultaneously, opening the possibility of selection of promiscuous T-cell epitopes. This approach has been successfully used by our group [21–23,25] and others [26–31] to identify allele-specific and promiscuous T-cell epitopes. However, no study has addressed the primary peripheral blood mononuclear cell (PBMC) recognition of TEPITOPE-predicted promiscuous peptides in a genetically heterogeneous group previously exposed to HIV-1.

In order to identify the frequently recognized, immunodominant epitopes of all proteins of HIV-1, we used the TEPITOPE algorithm, to screen the most conserved regions within the protein-coding genome of HIV-1 clade B consensus sequence for multiple HLA-DR binding epitopes. Peptides were synthesized and subject to binding assays with nine prevalent HLA-DR molecules. T-cell recognition of the synthetic peptides was assessed with interferon (IFN)-γ ELISPOT assays in PBMC from chronically HIV-1-infected patients across the disease spectrum. We also performed depletion studies to ascertain the phenotype of responding T cells. Our approach has allowed us to identify multiple CD4 T-cell epitopes that were frequently recognized by HIV-1-positive patients, including several novel epitopes.

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Heparin-treated venous blood for PBMC isolation and EDTA-treated venous blood for DNA extraction were obtained from HIV-1-infected individuals from the HIV-1 outpatient clinics from the School of Medicine, University of São Paulo (n = 32) and Federal University of São Paulo (n = 6), as well as from healthy volunteers (n = 8). Eligible patients received antiretroviral treatment according to the accredited Brazilian Government Guidelines for Antiretroviral Therapy (ART), issued by the Brazilian Ministry of Health. Enrolled individuals included eight LTNP (HIV-1-infected for more than 8 years and ART-naive; > 500 CD4 T cells/μl) and 24 HIV-1-infected patients under ART in different disease stages: seven reconstituted patients (> 500 CD4 T cells/μl), nine partial controllers (250–500 CD4 T cells/μl; viral load < 10 000 HIV RNA copies/ml), and eight typical progressors (< 200 CD4 T cells/μl). Samples from six recently HIV-1 infected (as defined by the ‘detuned’ ELISA assay) individuals, not undergoing ART, were collected and used for certain experiments. Table 1 shows descriptive characteristics of study subjects. All subjects gave their written informed consent to participate in this study, which was approved by the Institutional Review Boards of the School of Medicine, University of São Paulo and the Federal University of São Paulo.

Table 1

Table 1

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Peptide selection

Since HIV-1 clade B is highly prevalent among HIV-1 seropositive patients in Brazil [32], we chose to focus on its sequences for our peptide selection. We scanned the conserved regions of consensus sequences (contiguous regions of at least 12 amino acid residues in which each position was conserved in at least 50% of the isolates) from the clade B HIV-1 protein-coding genome available at (version of December 2002) with the TEPITOPE algorithm ( The TEPITOPE algorithm can predict sequences that have the potential ability to bind to one or more of 25 different HLA-DR molecules which are present in most of the Caucasian population [19,33]. The algorithm also allows the selection of sequences predicted to bind simultaneously – thus, promiscuously – to several HLA-DR molecules, and a thorough study of the actual predictive ability of TEPITOPE for promiscuous CD4 T-cell epitopes has been described [22]. We synthesized HIV-1 peptides (Table 2) whose sequences were predicted to bind to at least 18 out of the 25 HLA-DR molecules in the TEPITOPE matrix as previously described [22]; corresponding to an inner nonamer core selected as the HLA-binding motif with flanking amino acids added when possible (e.g., conserved flanking sequences) at either or both N- and C-terminal ends, to increase the efficiency of in vitro peptide presentation to CD4 T cells [20]. A comparison with known CD4 epitopes was made using the HIV-1 CD4 T-cell epitope map in the Molecular Immunology section of the Los Alamos HIV database using the August 2005 update. A Brazilian patent application on the identified peptide sequences and their uses was filed on September 5, 2005 under serial number PI 0504117-1, as well as a corresponding PCT patent application.

Table 2

Table 2

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Peptide synthesis

Peptides were synthesized by solid phase technology using 9-fluorenylmethoxycarbonyl strategy, with the C-terminal carboxyl group in amide form. Peptide purity and quality were assessed by reverse-phase phase high performance liquid chromatography and mass spectrometry, as described [34].

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HLA class II peptide-binding assays

Peptide binding assays were performed by incubating purified human HLA class II molecules (5–500 nM) with various concentrations of unlabeled peptide inhibitors and 1–10 nM 125I-radiolabeled probe peptides for 48 h as described [35,36].

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CD8 T-cell depletion and flow cytometry

CD8 T cells were depleted from PBMC of 11 selected patients with Dynabeads M-450 (Dynal, Oslo, Norway), according to manufacturer's instructions. The efficiency of CD8 T-cell depletion was evaluated by flow cytometry analysis with a FACScalibur (Becton Dickinson, San Jose, California, USA). We used the anti-CD8-fluoresceine isothiocyanate (FITC) (Dakopatts, Glostrup, Denmark) antibody conjugate, with anti-β2 microglobulin-FITC and anti-Hepatitis B surface antigen (HBS)-FITC used as positive and negative controls, respectively. After CD8 T-cell depletion, the percentage of residual CD8 T cells was almost always < 2% and in no case > 10%.

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HLA class II typing

DNA extraction and low resolution PCR amplification with sequence-specific primers HLA-DR typing were performed as described [37–39].

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Enzyme-linked immunospot (ELISPOT) assay

To determine the frequency of IFN-γ producing cells, the ELISPOT assay was used as described previously [40]. Cryopreserved PBMC (105 cells/well) were incubated with 5 μM of each peptide on a 96-well ELISPOT plate (Multiscreen MAIPS4510, Millipore, Bedford, Massachusetts, USA) previously coated with anti-human IFN-γ monoclonal antibody (Endogen, Woburn Massachusetts, USA; 4 μg/ml), for 16 h. The reaction was developed as previously described [40]. Spots were counted using an automated stereomicroscope (KS ELISPOT, Zeiss, Oberkochem, Germany). The number of antigen-specific T cells, expressed as IFN-γ-spot-forming cells (SFC)/106 PBMC was calculated after subtracting negative control values (wells with cells in the absence of peptide) from the same subject. Positivity cut-off was ≥ 30 IFN-γ SFC/106 PBMC, which was above the mean ± 3 SD of the highest response found among all uninfected control subjects to any peptide (data not shown). ELISPOT assays were also performed after depletion of CD8 T cells to confirm if observed responses were indeed caused by the CD4 T cell activation.

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Statistical analysis

Statistical analysis was performed using GraphPad Prism version 3.0 package. Comparisons of the number of IFN-γ SFC/106 PBMC between the clinical groups were performed with the non-parametric One-Way ANOVA, Kruskal–Wallis and Dunn's Test. Spearman's Correlation was also used. Values of P < 0.05 were considered significant.

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Peptide selection and binding analysis

The sequences from whole clade B HIV-1 consensus protein-coding genome were scanned with TEPITOPE algorithm at 3% threshold, which led to the identification of regions predicted to bind significantly to multiple HLA-DR molecules. Peptides predicted to bind to 16 or more out of the 25 HLA-DR molecules tested by TEPITOPE, at the 3% threshold, were selected for synthesis (Table 2); we also synthesized the nef180–194 peptide, which showed predicted binding to 13 out of 25 HLA-DR molecules, because of the known high antigenicity of the nef molecule. Binding assays of the 18 selected peptides with the nine most prevalent HLA-DR molecules in the general population showed that p24131–150 and rev11–27 bound significantly to 100% (9/9) of the HLA-DR molecules, followed by integrase70–84, gp160188–201 and gp160481–498 that bound to 89% (8/9), and p1773–89, p632–46, vpr65–82 and vif144–158 that bound 78% (7/9) of the tested molecules (Table 3). Eleven out of the 18 identified, multiple MHC-binding peptides, protease7–21, protease80–94, integrase70–84, gp41261–276, gp16019–31, gp160174–185, gp160188–201, rev11–27, vpr58–72, vif144–158, vpu6–20, were novel CD4 T-cell epitopes, with overlaps shorter than 11 residues in comparison to known epitopes/HLA class II ligands present in the CD4 T-cell epitope listing available at the Los Alamos HIV immunology database (August 2005 update).

Table 3

Table 3

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Identification of HIV-1-derived epitopes recognized by PBMC from HIV-1-infected patients by the IFN-γ ELISPOT assay

PBMC samples from 32 HIV-1-infected patients in different clinical stages of disease were tested with the 18 selected peptides using the IFN-γ ELISPOT assay. The clinical characteristics of each patient are shown in Table 1. To establish the IFN-γ ELISPOT positivity cut-off, PBMC from eight HIV-1-seronegative controls were tested with the same peptides. Individual responses to each peptide ranged from 0 (60% of responses) to 20 IFN-γ SFC/106 PBMC (two different peptides in two different individuals) (data not shown). The mean number of IFN-γ SFC/106 PBMC + 3 SD induced by each peptide among seronegative individuals ranged from 2.4 to 26 IFN-γ SFC/106 PBMC. Based on these results, the positive response cut-off was established at ≥ 30 IFN-γ SFC/106 PBMC in our study, in excess of the highest peptide response plus + 3 SD observed among HIV-1 seronegative controls.

Table 4 shows the number of IFN-γ producing cells in response to the tested peptides in PBMC from chronically HIV-1-infected patients. All of the 18 selected peptides were recognized; each peptide was recognized by at least seven, and up to 14 out of the 32 patients. Significantly, 91% (29/32) of patients recognized at least one of the 18 peptides (Table 4). Two of the three patients who failed to recognize any tested peptide were HIV-1-progressor patients with < 200 CD4 T cells/μl. On average, each patient recognized five HIV-1 peptides (range, 0–18); 44% of patients recognized five or more peptides, and 19% of patients recognized 10 or more peptides (Table 4). The most frequently recognized peptide was protease7–21 (44% of patients) followed by p24131–150 and rev11–27 that were recognized by 40% and 34% of patients, respectively. We also observed that 75% (24/32) of the patients recognized the combination of the three most frequently recognized peptides (protease7–21, p24131–150, rev11–27). In addition, 91% of patients recognized a combination of eight peptides (protease7–21, p24131–150, rev11–27, gp160174–185, gp16019–31, p1773–89, vif144–158, vpu6–20), the same number of patients recognizing the full 18 peptide panel. These data indicate that the selected conserved consensus HIV-1 clade B peptides were frequently recognized among PBMC from HIV-1-infected patients belonging to distinct subgroups of clinical progression. IFN-γ ELISPOT assays were also performed with pools of all 18 peptides, in whole and CD8 T cell-depleted PBMC from HIV-1 infected patients, and positive responses were detected in the CD8-negative subset (representing CD4 T-cell responses) in eight of 11 tested individuals, although CD8 responses were simultaneously detected in several patients (Table 5).

Table 4

Table 4

Table 5

Table 5

The average magnitude of the response towards the combined 18 peptides among all patients was 717 IFN-γ SFC/106 PBMC (range, 30–4455). Significant positive correlations were observed between CD4 T-cell counts and CD4 nadir values with the magnitude of responses (P < 0.01, r, 0.43 and P < 0.001, r, 0.55, respectively). Furthermore, a significant positive correlation was observed between the magnitude and the breadth of the response (P < 0.0001, r, 0.79). There was no correlation between the breadth of the response and CD4 T-cell counts, CD4 T-cell nadir, or viral load. Furthermore, no correlation was found between viral load and the magnitude of the response, CD4 T-cell counts or CD4 T-cell nadir.

Analysis of the HLA-DR profile from responder (patients that responded to at least one peptide) and nonresponder patients indicated that a wide diversity of HLA-DR molecules (13 out of the possible 13) was represented among the responders for each peptide (data not shown). The frequencies of tested HLA-DR molecules among peptide responders in each clinical group also showed a wide diversity of HLA-DR molecules.

The magnitude of IFN-γ responses to all tested peptides by each individual (Table 4) was highest among LTNP patients (1489 ± 1348). Reconstituted patients (496 ± 549), partial controllers (535 ± 654) and progressors (386 ± 650) showed lower magnitudes, but LTNP patients showed significantly higher IFN-γ responses only in comparison to progressor patients (Table 4). We observed that LTNP recognized the highest number of peptides (7.5 ± 5.9), followed by partial controller patients (6.2 ± 3.4), progressor (4.0 ± 5.2) and reconstituted HIV-1+ patients (3.3 ± 2.0); however, there were no statistically significant differences in the numbers of recognized peptides between any of the groups.

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In the present study, we have identified a set of multiple HLA-DR binding CD4 T-cell epitopes derived from the whole clade B HIV-1 consensus protein-coding genome scanned with the TEPITOPE algorithm and recognized by primary CD4 T cells from HIV-1-infected patients. Furthermore, most of the CD4 T-cell epitopes from HIV-1 identified by this approach were previously unknown. This approach was able to identify epitopes in less studied HIV-1 proteins such as HIV-1 protease, integrase, and the small regulatory/accessory proteins vif, vpr and vpu [41]. This set of HIV-1 epitopes is recognized by 91% HIV-1-infected patients, and 43% recognized five or more peptides, indicating their immunodominance. Significantly, there were no previously known CD4 T-cell epitopes in HIV-1 protease, and the fact that the identified epitopes encompass sites of common drug resistance mutations may have a bearing on escape and drug resistance [42]. The fact that TEPITOPE-selected HIV-1 clade B consensus peptides were recognized by almost all HIV-1-infected patients tested (Table 4) indicates that the algorithm was able to successfully identify promiscuous epitopes in the whole protein-coding genome of HIV-1 clade B, in line with previous observations from our group and others [22,25,29]. The fact that most peptides selected with TEPITOPE in HIV-1 are indeed able to bind to multiple HLA-DR molecules in direct binding assays (Table 3) indicates that selected peptides are promiscuous ligands and can thus be presented by multiple HLA-DR molecules that could cover a genetically heterogeneous population. Our observation that HIV-1-infected patients recognize an average of five distinct peptides, and a significant proportion recognizes even higher numbers of epitopes (Table 4), supports their antigenicity and immunodominance.

DeGroot et al. (2004) [43] used another algorithm to identify 10 HIV-1 consensus T-cell epitopes, that were able to elicit IFN-γ Elispot responses in 10 out of 34 (29%) HIV-1-infected LTNP patients. The increased frequency of peptide recognition observed in our study could be due the intrinsic immunogenicity of our selected peptides as compared to those of DeGroot et al. or to patient variability. Our results also differ from another study by the same group, who identifed 20 epitopes from HIV-1 consensus sequences. Fifteen out of 20 such peptides were recognized by less than 18% of their patients, while all peptides in our study were individually recognized by 18% or more of tested patients [44]. Furthermore, we have shown that HIV-1-infected patients recognize on average five peptides, 43% of them responded to five or more epitopes, and six patients (19%) responded to 10 or more epitopes, corroborating their antigenicity and immunodominance.

The observation that a wide diversity of HLA-DR molecules was associated with recognition of each peptide (data not shown) suggests that peptides predicted to be promiscuous by TEPITOPE were indeed capable of being presented by multiple HLA-DR molecules. In addition, the frequency of allelic variation in the HLA-DR distribution of the patients tested is similar to the spectrum of the Brazilian mixed population [45].

The fact that we showed primary PBMC recognition of TEPITOPE-derived promiscuous HIV-1 peptides among HIV-1-infected patients is evidence that such epitopes were actually presented to T cells in the course of natural HIV-1 infection. Immunity triggered by such epitopes generated memory responses that can be boosted by contact with the same epitope or infectious agent, making these peptides candidate vaccinal epitopes. Furthermore, the fact that CD8 T-cell depletion reduced the PBMC responses against the HIV-1 peptide pool in several of the patients (Table 5) suggests the regions are recognized by CD8 T-cells as well. This is in line with the fact that more than 50% of the peptides contain known HIV-1 CD8 T-cell epitopes (Los Alamos HIV-1 Immunology database, data not shown). Recent data have suggested that single epitope-based vaccines are not powerful enough to induce full protective immunity. The combination of multiple CD4 and CD8 T-cell and B-cell epitopes as a pool or as a multiepitope polypeptide was shown to increase the immunogenicity [46,47]. In the present study, we observed that 75% of the patients recognized the combination of the three TEPITOPE selected promiscuous HIV-1 peptides (protease7–21, p24131–150, rev11–27). In addition, the combination of eight peptides (protease7–21, p24131–150, rev11–27, gp160174–185, gp16019–31, p1773–89, vif144–158, vpu6–20) induced positive IFN-γ responses by all 91% of patients responding to the full 18 peptide panel. Interestingly, five of the eight mentioned epitopes are novel. It is conceivable that inclusion of additional TEPITOPE-derived peptides to the above mentioned peptide combination may cover close to 100% of a genetically distinct population. The fact that PBMC from three HIV-1 patients failed to respond to the promiscuous HIV-1 consensus peptides tested was probably related to the stage of disease, since two of them belonged to the progressor group.

Considering that the peptides were selected from clade B HIV-1 consensus sequences, we searched for identity and homology between our peptide sequences and HIV-1 sequences described at a data bank ( For 14 of the 18 peptide sequences from HIV-1 clade B consensus, we found isolates matching their identical sequence among described sequences of HIV-1 clade B (data not shown). At any event, we found frequent ELISPOT responses (>18%) even for peptides encoding consensus sequences which were not represented among isolates (data not shown), indicating that patients' PBMC probably show cross-reactive recognition between sequences present in their own isolate and consensus sequences. Furthermore, sequences identical to several of our selected peptides also appear in frequencies above 50% among isolates from HIV-1 clades A, C, D and F deposited in the Los Alamos HIV sequence database (, data not shown). In other cases, prevalent sequences in other clades showed only one or two conservative amino acid changes from the clade B consensus, which did not alter the HLA-DR binding prediction profile of the sequence (data not shown). These observations reinforce the findings that our selected peptides – or sequences highly homologous to them – are broadly represented across several clades of HIV-1, raising the possibility that patients infected with other non-clade B HIV-1 may also recognize epitopes originally identified in HIV-1 clade B consensus.

Comparing the magnitudes of responses among the HIV-1 infected patient groups, a significantly stronger IFN-γ response was detected in LTNP patients as compared to progressors (PROG). In our study, no correlations between the viral load at the time of the study and either the magnitudes and breadths of IFN-γ responses were observed, in line with previous reports [48]. On the other hand, recent studies suggest an inverse correlation between viral load and the number of interleukin-2-secreting HIV-specific CD4 T cells [49,50], so the exclusive evaluation of IFN-γ producing cells may have underestimated the breadth and amplitude of peptide recognition in our study.

In conclusion, we have identified a group of highly promiscuous, frequently recognized, and conserved CD4 T-cell epitopes, derived from the whole HIV-1 clade B consensus protein-coding genome, including several novel, previously unknown sequences. Their properties suggest their use for the follow-up of T-cell immune responses in HIV-1-infected patients (e.g., along vaccine trials). One can speculate they may also have a potential for use as an immunogen, either as stand-alone or combined with an existing candidate HIV-1 vaccine. By virtue of being a polyepitopic, polyallelic, frequently recognized CD4 T-cell epitope combination based on conserved consensus epitopes of HIV-1, one would expect they should elicit a response with significant breadth and ample coverage, perhaps allowing for cross-clade immunization.

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We thank Dr Claudio Puschel and Mr. Washington Robert da Silva, B.Sc. for peptide synthesis and analysis, as well as Mrs Helena Tomiyama, B.Sc. and Ms Eliane Mairena, M.Sc. for their help with sample preparation and analysis and Drs. Sigrid Souza, Ho Li and Renata D'Agnolo for assistance in patient recruitment at the HIV Outpatient Clinic, Department of Infectious Diseases, School of Medicine, University of São Paulo.

Sponsorship: This work was supported by grants from FAPESP (São Paulo State funding Agency/Brazil) 01/00729-3; CNPq (Brazilian National Research council), scholarships to S.G.F (350273/2002-2), CNPq Investigator awards to E.C.N., A.C.S. and J.K. and NIH contract NO1-AI-95362 (to A.S. and J.S.).

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Olavo H. M. Leite from Department of Infectious Diseases, School of Medicine, University of São Paulo, São Paulo, Brazil and Leo K. Iwai from Heart Institute (InCor),University of São Paulo School of Medicine, São Paulo, Brazil are also authors of this paper.

50. Younes SA, Yassine-Diab B, Dumont AR, Boulassel MR, Grossman Z, Routy JP, et al. HIV-1 viremia prevents the establishment of interleukin 2-producing HIV-specific memory CD4+ T cells endowed with proliferative capacity. J Exp Med 2003; 198:1909–1922.

HIV-1; CD4+ T cell epitopes; HLA binding; vaccine; consensus; conserved

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