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.
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 . 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 . 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.
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 .
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 (http://HIV-1-web.lanl.gov). 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 (http://www.hiv.lanl.gov/content/immunology/maps/maps.html, 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 . 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.
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.
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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.