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The Journal of Immunology, 2006, 176: 5401-5408.
Copyright © 2006 by The American Association of Immunologists

Scanning the HIV Genome for CD4+ T Cell Epitopes Restricted to HLA-DP4, the Most Prevalent HLA Class II Molecule1

William M. Cohen*, Sandra Pouvelle-Moratille*, Xiao-Fei Wang*, Sandrine Farci*, Gaetan Munier*, Dominique Charron{dagger}, André Ménez*, Marc Busson{dagger} and Bernard Maillère2,*

* Protein Engineering and Research Department, Commissariat à l’Energie Atomique Saclay, Gif sur Yvette, France; and {dagger} Institut National de la Sante et de la Recherche Medicale U396, Hôpital St. Louis, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
HLA-DP4 alleles are carried by 75% of individuals and are the most frequent HLA II alleles worldwide. Because we have recently characterized the peptide-binding specificity of HLA-DP4 molecules, we developed a peptide-binding prediction method to identify HLA-DP4-restricted peptides in multiple Ags. CD4+ T cell response plays a key role in the immune control of HIV infection, but few HIV-specific T cell epitopes with multi-individual specificity have been identified. They are mostly restricted to HLA-DR molecules, which are very polymorphic molecules. We therefore looked for HLA-DP4-restricted CD4+ T cell epitopes in the whole genome of HIV. Twenty-one peptides were selected from the HXB2 HIV genome based on the prediction of binding to HLA-DP4 molecules. They were submitted to HLA-DP4-binding assays. Seventeen peptides bound to the HLA-DP401 molecule, whereas 15 peptides bound to HLA-DP402. Six peptides bound very tightly to HLA-DP401 and were investigated for their capacity to induce specific CD4+ T cell lines in vitro using dendritic cells and CD4+ T cells collected from eight seronegative HLA-DP4+ donors. Four peptides from env and reverse transcriptase proteins induced in vitro-specific T cell lines restricted to HLA-DP4 molecules. Peptide-induced T cells recognized variants other than the HXB2 sequence and were stimulated by native Ags processed by immature dendritic cells. The reverse transcriptase peptide is present in 65% of the isolated HIV variants. To our knowledge, we describe the first HIV epitopes restricted to HLA-DP4 molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Among all HLA II molecules, HLA-DP4 molecules have the unique property of being present at a high frequency worldwide. They comprise mainly two different molecules, namely HLA-DPA1*0103/DPB1*0401 (DP401) and HLA-DPA1*0103/DPB1*0402 (DP402), which differ by only 3 aas and have a very similar binding motif (1). HLA-DP4 gene frequency is ~50% in Europe, 60% in South America, 80% in North America, 60% in India, 25% in Africa, and only 15% in Japan (2). In the Caucasian population, HLA-DP4 molecules are found in ~76% percent of individuals and hence are as frequent as the HLA-A2 molecule, which is the predominant HLA I molecule. Multiple immunological studies have taken advantage of the high frequency of HLA-A2 to delineate relevant CTL epitopes, to evaluate vaccine efficiency or to document cellular immunity of patients affected by different disease stages. The high frequency of the HLA-DP4 molecules is fruitfully exploited to investigate the CD4 T cell response to tumors. HLA-DP4-restricted T cell epitopes were delineated by chance in the MAGE-3 (3) and NY-ESO-1 tumor Ags (4, 5). Melanoma patients have been vaccinated with dendritic cells (DC)3 loaded with the HLA-DP4-restricted peptide MAGE-3 (6, 7). Recently, functional HLA-DP4 multimers were produced with the MAGE-3 peptide and have been used to evaluate the frequencies of peptide-specific T cells induced by vaccination (8). Moreover, HLA-DP4-restricted T cell response is not limited to tumor Ags because several T cell lines and clones have been isolated from various pathogens, including viruses (9, 10, 11), demonstrating clearly the immune functionality of HLA-DP4 molecules.

In contrast, CD4 T cell response against HIV is a growing subject of interest. HIV infection promotes a progressive decline of CD4 T cells, which is successfully compensated by the highly active antiretroviral therapy (12). Virus load appears to diminish the proliferative capacity of HIV-specific T cells (13, 14) and hence the cell-mediated immunity against HIV (15). Ag persistence has been associated with a significant frequency of effector memory CD4+ T cells, which produce exclusively IFN-{gamma} and are short lived (16, 17, 18). In contrast, proliferative CD4+ T cell response to HIV components has been found to correlate inversely with viral load (19) and may result from the presence of central memory T cells, which produce IL-2 and are long lived (17, 18). Restoration of HIV CD4+ T cells by highly active antiretroviral therapy seems, however, to be limited and did not appear to involve central memory cells (12, 20). To document all of these cellular investigations and to propose peptide sequences for the design of epitope-based vaccines, CD4+ T cell epitopes from HIV have been identified. They derived from GAG (21, 22, 23, 24), env (25, 26), POL (21, 27, 28), and Nef (24, 29). They are almost all restricted to HLA-DR, very few epitopes being HLA-DQ-restricted (25, 29) and HLA-DP5-restricted (25). Some of these peptides were able to bind to multiple HLA-DR molecules and were recognized by T cell lines deriving from multiple individuals (21, 24, 26). However, none of them are restricted to the prevalent HLA-DP4 molecules.

Based on the previous binding data we obtained to characterize the binding specificity of HLA-DP4 molecules, we recently set up a new approach to delineate HLA-DP4-restricted T cell epitopes and applied it to the whole genome of HIV. Combining peptide-binding prediction, peptide-binding assays, and CD4+ T cell priming experiments allowed us to successfully find the first HLA-DP4-restricted T cell epitopes of HIV.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Peptides and Ags

Peptides were synthesized using standard Fmoc chemistry on a multiple peptide synthesizer APEX 396 (Advanced ChemTech). They were cleaved from the resin by 95% trifluoroacetic acid. Peptides, which exhibited a purity lower than 80%, were purified by reversed phase-HPLC on a C18 Vydac column (Interchim). The peptide bOxy 271-287 was biotinylated with biotinyl-6-aminocaproic acid (Fluka Chimie) on the N terminus before cleavage from the resin and HPLC purification. The sequence of each peptide was assessed by mass spectroscopy. Reverse transcriptase (RT) (recombinant HIV-1 HXB2 RT) and Gp120 (recombinant HIV-IIIB Gp120) are from the National Institute for Biological Standards and Control (NIBSC) Centralized Facility for AIDS Reagents (Herts, U.K.). RT is a gift from Dr. D. Stammer (GlaxoSmithKline, Research Triangle Park, NC).

HLA-DP4-specific peptide-binding assays

EBV homozygous cell lines PITOUT (DPA1*0103, DPB1*0401), HOM2 (DPA1*0103, DPB1*0401), and SCHU (DPA1*0103, DPB1*0402) were a gift from Dr. C. de Toma and J. Dausset (Centre d’Etude du Polymorphisme Humain, Paris, France). They were used as sources of human HLA-DP4 molecules. B7/21 hybridoma was a gift from Dr. Y. van de Wal (Department of Immunohematology and Blood Bank, Leiden, The Netherlands). mAb B7/21 was immunopurified from cell culture supernatants on Sepharose protein A gel as recommended by the manufacturer (Amersham Pharmacia Biotech). HLA-DP4 molecules were purified by affinity chromatography using B7/21 mAb coupled to protein A-Sepharose CL 4B gel (Pharmacia Biotech) as described previously for L243 mAb (30). Binding assays were performed by competitive ELISA as described previously (1). Briefly, 10 nM bOxy 271–287 peptide, an appropriate dilution of HLA-DP4 molecules, and serial dilutions of the peptide to be tested were incubated at 37°C for 24 h. Samples were then neutralized and applied to B7/21-coated plates for 2 h. Bound biotinylated peptide was detected by means of streptavidin-alkaline phosphatase conjugate (Amersham Biosciences) and 4-methylumbelliferyl phosphate substrate (Sigma-Aldrich). Emitted fluorescence was measured at 450 nm upon excitation at 365 nm in a Victor II spectrofluorimeter (PerkinElmer). Data were expressed as the peptide concentration that prevented binding of 50% of the labeled peptide (IC50). IC50 values of the Oxy 271–287 peptide served as a reference in each experiment.

Blood samples and HLA-DP genotyping

Blood cells were collected at the Etablissement Français du Sang (EFS) as buffy-coat preparations from anonymous healthy donors after informed consent and following the guidelines of EFS. Seronegativity of the donors was assessed using standard EFS protocols. PBMC were isolated by density centrifugation on Ficoll-Hypaque gradients (Sigma-Aldrich). Genotyping was performed using the Olerup SSP DPB1 typing kit (Olerup SSP) according to the manufacturer. HLA-DPB genotyping results were the following: donor 48 (0401/1301), donor 51 (0301/0401), donor 119 (0401/0601), donor 120 (03/0401), donor 123 (03/0401), donor 147 (0401), donor 156 (0401), donor 157 (0401).

Induction of CD4+ T cells with peptides

The induction of CD4+ T cells in vitro with HIV peptides was based on a protocol described previously (31, 32). Immature and mature DCs were generated from plastic-adherent PBMC by a 5-day culture in AIM-V medium supplemented with 1000 U/ml recombinant human GM-CSF (R&D Systems) and 1000 U/ml recombinant human IL-4 (R&D Systems). LPS (Sigma-Aldrich) (1 µg/ml) was used as maturation agent. CD4+ T lymphocytes were isolated from nonadherent PBMC by positive selection using an anti-CD4 mAb coupled to magnetic microbeads (Miltenyi Biotec). Mature DCs were incubated at 37°C, 5% CO2, for 4 h in IMDM (Invitrogen Life Technologies) supplemented with 24 mM glutamine, 55 mM asparagine, 150 mM arginine (all amino acids were obtained from Sigma-Aldrich), 50 U/ml penicillin and 50 µg/ml streptomycin (Invitrogen Life Technologies), and 10% human serum (hereafter referred to as complete IMDM) with a solution of the HIV peptide mixture (10 µg/ml each peptide). Pulsed mature DCs were added at 104 per round-bottom microwell to 105 autologous CD4+ lymphocytes in 200 µl of complete IMDM supplemented with 1000 U/ml IL-6 (R&D Systems) and 10 ng/ml IL-12 (R&D Systems). The CD4+ T lymphocytes were restimulated on days 7, 14, and 21 with autologous DCs freshly loaded with the HIV peptide mixture, and were grown in complete IMDM supplemented with 10 U/ml IL-2 (R&D Systems) and 5 ng/ml IL-7 (R&D Systems). The stimulated CD4+ T cells were analyzed for specificity in ELISPOT assays at least 6 days after the last stimulation. The frequency of MAGE-specific precursors was calculated on the basis of the Poisson distribution. Accordingly, an estimate of the precursor frequency is given by the following formula: precursor frequency = –ln((number of negative wells)/(number of wells tested).

IFN-{gamma} ELISPOT

Multiscreen HA plates (Millipore) were coated with 1 µg/ml mAb anti-human IFN-{gamma} (1-D1K; Mabtech) in PBS (Invitrogen Life Technologies) for 1 h at 37°C and saturated with complete IMDM. APCs were autologous immature DCs or HLA-DP4-transfected L cells (L-DP4 cells) (provided by Dr. H. Zarour, Pittsburgh University Cancer Institute, Pittsburgh, PA). HIV proteins were incubated for 4 h at 37°C at a concentration of 1 µM with immature DCs, which were subsequently washed before use. Peptides were directly added to the Multiscreen plates. Immature DCs (2 104/well) or L-DP4 cells (3 104/well) were distributed in Multiscreen plates together with 2 103 to 104 CD4+ T cells. After overnight incubation at 37°C, captured IFN-{gamma} was detected by subsequent addition of biotinylated mAb anti-hIFN-{gamma} (7-B6-1; Mabtech) (0.2 5 µg/ml), extravidin- phosphatase (Sigma-Aldrich) and NBT/5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich). Spot number was automatically determined by the AID EliSpot Reader System (AID). The t test was used for statistical evaluation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Selection of HLA-DP4-binding peptides in the HIV genome

We have recently characterized the peptide-binding motifs of HLA-DP401 and HLA-DP402 molecules. The amino acid selectivity of pockets 1, 4, 6, and 9 of their peptide-binding cleft was investigated by substituting the corresponding positions with various amino acids and by quantifying their effects on the binding (1). We completed this study with new binding data (M. Busson, F. A. Castelli, X.-F. Wang, W. M. Cohen, D. Charron, A. Ménez, and B. Maillère, manuscript in preparation) and built quantitative matrices of binding to HLA-DP4 molecules (Table I), as previously done for HLA-DR molecules by others (33, 34). To predict the peptide binders to HLA-DP4, we assigned to all 9-mer of a sequence a predicted IC50 by addition of the values in the P1, P4, P6, and P9 pockets for each corresponding amino acid of the peptide (Table I). Efficiency of the prediction was assessed using a set of 98 unrelated peptides, among which 17 bound to HLA-DP401. For an expected IC50 of 300 nM, we observed that eight were correctly predicted (47%) and two were predicted to be active, although they did not bind to HLA-DP401 (M. Busson, F. A. Castelli, X.-F. Wang, W. M. Cohen, D. Charron, A. Ménez, and B. Maillère, manuscript in preparation). In the HXB2 HIV sequence, we retained all the peptides, which exhibited an expected IC50 below 300 nM and excluded most of the peptides with a moderate predicted activity and a low frequency among HIV isolates. The 21 chosen peptides derived from five different proteins of HIV and showed various levels of conservation (Table II). They were synthesized and submitted to HLA-DP4-binding assays (Table II). On the basis of an activity threshold of 1000 nM, 17 peptides bound to HLA-DP401 and 15 peptides to HLA-DP402. Fourteen peptides bound to both molecules, in agreement with our previous observations that HLA-DP4 molecules share a large number of peptide binders (1). Six peptides (env 31–45, env 388–402, env 620–634, RT 338–352, Vpr 15–29, and Vpr 61–75) were very good binders to HLA-DP401 (IC50 below 100 nM) and were selected for cellular assays.


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  		Table I. Matrices of binding prediction to HLA-DP401 and HLA-DP402a

 

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  		Table II. Selection of peptides from the HIV genome for their capacity to bind HLA-DP4 moleculesa

 
Four HIV peptides are able to prime peptide-specific CD4+ T cell lines

Eight HLA-DP4+-seronegative donors were used to investigate the T cell priming capacity of the selected peptides. These assays were conducted as described previously (31, 32). Autologous mature DCs were loaded with a mixture of the six peptides for 4 h and distributed with purified CD4+ T lymphocytes in 60 different microwells. Microcultures were restimulated weekly with peptide-pulsed mature DCs and an appropriate mixture of cytokines. Using a peptide mixture makes it possible to increase the number of seeded wells for each peptide and hence to increase the probability for each peptide of encountering a peptide-specific T cell precursor. After three stimulations, peptide specificity of growing T lymphocytes was tested by IFN-{gamma} ELISPOT using HLA-DP4-transfected L cells (HLA-DP4) as APC. Twenty-two bulk CD4 T cell lines were specific for the peptide mixture (Table III). Their production of spots in the presence of the peptide mixture was at least three times higher than in the absence of peptide. Stimulation with each of the peptides revealed that four peptides are T cell stimulating, namely RT 338–352, env 31–45, env 388–402, and env 620–634. More precisely, among 22 T cell lines specific for the peptide mixture, one was specific for the peptide env 620–634, two were specific for env 388–402, five were specific for env 31–45, and 15 were specific for RT 388–402. Interestingly, the latter was immunogenic for almost all of the different tested donors. Only donor 156 did not give rise to T cell lines specific for this peptide. However, only one T cell line has been derived from this donor. In sharp contrast, no T cell line was specific for the peptides Vpr 15–29 and Vpr 61–75 (data not shown). Because human-activated CD4+ T cells express HLA II molecules and hence are able to present peptides to surrounding T cells, we investigated for 14 peptide-specific T cell lines whether stimulation did not result from these cell contacts but required L-DP4 cells. As shown in the last column of Table III, removal of the HLA-DP4-transfected cells abolished T cell stimulation. Peptide-specific T cells were therefore restricted to HLA-DP4 molecules.


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  		Table III. Peptide specificity and restriction of T cell lines induced by the HIV-1 peptide mixturea

 
RT 338–352- and env 31–45-specific T cell lines are stimulated by native Ags presented by DCs

We then investigated the presentation of the native proteins by DCs to peptide-specific T cell lines. Due to technical constraints, it was unfortunately possible to perform these experiments only on env 31–45- and RT 338–352-specific T cell lines. Autologous immature DCs were loaded with either HIV-1 RT or Gp120 proteins, washed and incubated with each of the four T cell lines (Fig. 1, left panels). Three T cell lines (157.49, 157.13, and 157.53) were specific for the RT 338–352 peptide and were stimulated by RT-loaded DCs. They were not stimulated by DCs loaded with either Gp120 or the env 31–45 peptide. In contrast, the 156.66 T cell line was specific for the Gp120-derived peptide env 31–45 and was stimulated by Gp120-loaded DCs. No response was observed with DCs loaded with either RT or the RT 338–352 peptide. We also evaluated the efficiency of peptide presentation using L-DP4 cells as APC and a dose range of each peptide (Fig. 1, right panels). The half-maximal stimulation of the T cell lines required a peptide concentration ranging from ~10–8 to 10–7 M. These T cells are therefore efficiently stimulated by the peptides and recognized the native Ags processed by the DCs.


Figure 1
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  		FIGURE 1. Recognition of the native proteins and the peptides env 31–45 and RT 338–352 by the peptide-specific T cell lines. T cell lines specific for env 31–45 (156.66) and RT 338–352 (157.49, 157.53, and 157.13) were obtained after three rounds of stimulations with the selected peptide mixture. Left panels, T cell lines (2 103 to 104 cells/well) were incubated in an IFN-{gamma} ELISPOT assay in the presence of autologous immature DCs (2 104/well) previously loaded with either recombinant RT protein or recombinant Gp120 protein or in the presence of immature autologous DCs (2 104/well) with or without env 31–45 or RT 338–352 peptide (10 µg/ml). Right panels, T cell lines (2 103 to 104 cells/well) were incubated in an IFN-{gamma} ELISPOT assay in the presence of L-DP4 cells (3 104/well) and a dose range of peptides env 31–45 and RT 338–352 ({blacksquare}) or without any peptide ({circ}). Each value represents the mean spot number of duplicates. ** and * indicate p < 0.01 and p < 0.05, respectively.

 
RT 338–352- and env 31–45-specific T cell lines can recognize other variants besides the HXB2 sequence

To assess the influence of natural mutations on binding to HLA-DP4 and on T cell stimulation, we then investigated the sequence variations of the RT 338–352 and env 31–45 peptides. The RT 338–352 peptide is highly conserved. There are 47 variants of this peptide among the 457 sequences retrieved from the Los Alamos database. Two hundred ninety-eight sequences (65%) are identical with that of HXB2. We have submitted some of the point variants, which are among the most frequent variants, to HLA-DP4-binding assays (Table IV) and to T cell stimulation assay (Fig. 2). In agreement with the peptide-binding motif (1), only the H346 variant exhibited a lower binding capacity to HLA-DP4 molecules as compared with the RT 338–352 peptide. This loss of binding results from the unfavorable accommodation of the histidine 346 in the P6 pocket (Table IV). Two variants (R350 and R352) were stimulating for both T cell lines, whereas four variants (I348, Y346, H346, F342) were stimulating for only one of them (Fig. 2). The env 31–45 peptide is not a predominant sequence because it is found at a frequency of only 3.9%. However, its core sequence, which is delimited by residues between the P1 and P9 positions, is highly conserved and is shared by 80% of the known sequences. We therefore investigated the influence of the main sequence variation in the flanking regions. The four synthesized peptides cover 20% of the sequences (Table IV). They tightly bound to HLA-DP4 molecules (Table III) and stimulated an env 31–45 T cell line (Fig. 2). As a result, the RT 338–352 and env 31–45 peptides elicit HLA-DP4-restricted T cells, which are able to recognize other variants besides the HXB2 sequence.


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  		Table IV. HLA-DP4-binding capacity of HIV natural variantsa

 

Figure 2
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  		FIGURE 2. Recognition of HIV variants of the env 31–45 and RT 338–352 peptides by peptide-specific T cell lines. T cell lines specific for env 31–45 (156.66) and RT 338–352 (157.53 and 157.13) were submitted to IFN-{gamma} ELISPOT assay. Two 103 to 104 cells/well were incubated in a IFN-{gamma} ELISPOT assay in the presence of L-DP4 cells (3 104/well) and 10 g/ml HIV peptide variants. Each bar represents the mean spot number of duplicates. ** and * indicate p < 0.01 and p < 0.05, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although HLA-DP4 molecules are the most prevalent HLA II molecules, until now HLA-DP4-restricted T cell epitopes have only been identified by chance and not in a deliberate manner. We have therefore set up an approach combining binding prediction, binding assays, and cellular assays to identify HLA-DP4-restricted T cell epitopes. This approach has been applied to the HIV genome. We identified 17 peptides that were good binders to HLA-DP401, four peptides that were T cell stimulating, and two peptides that were naturally processed epitopes of the native proteins.

In contrast to HLA-DQ molecules (35), the mode of binding to HLA-DP4 molecules relies on hydrophobic or aromatic primary anchor residues (1) and hence resembles that of HLA-DR molecules (33, 34, 36). In both HLA-DR and -DP4 molecules, the large hydrophobic P1 pocket accommodates aromatic or hydrophobic residues depending on the dimorphic residue located at position beta86. A second hydrophobic or aromatic primary anchor residue is located in the P6 pocket of HLA-DP4 molecules (1), whereas in HLA-DR molecules it is more variable in its location and nature (34, 36). One of the most successful approaches of delineation of HLA-DR-restricted T cell epitopes is the use of quantitative matrices (33, 34). We have therefore adapted this method to HLA-DP4 molecules and built quantitative matrices (M. Busson, F. A. Castelli, X.-F. Wang, W. M. Cohen, D. Charron, A. Ménez, and B. Maillère, manuscript in preparation) with the binding data recently obtained for substituted analogues of the naturally processed peptide Oxy 271–287 (1, 37). This prediction allowed us to successfully identify 17 good binders to HLA-DP401 molecules among 21 synthesized peptides. This is in agreement with the efficiency of the prediction observed on a set of unrelated peptides (M. Busson, F. A. Castelli, X.-F. Wang, W. M. Cohen, D. Charron, A. Ménez, and B. Maillère, manuscript in preparation). At a predicted threshold of 300 nM, it is estimated that we could miss 50% of the active peptides. A better coverage of the active peptides could be obtained at a threshold of 1000 nM, but it increases also the number of peptides to synthesize and the number of false positive peptides (M. Busson, F. A. Castelli, X.-F. Wang, W. M. Cohen, D. Charron, A. Ménez, and B. Maillère, manuscript in preparation). The six best peptides were submitted to in vitro CD4+ T cell-priming experiments, but other peptides with good binding IC50 values warrant investigation for their capacity to elicit a CD4+ T cell response. This is, for instance, the case for env 483–497, env 677–691, env 684–698, RT 346–360, and RT 403–417. Primary in vitro stimulations were performed using seronegative donor CD4+ T lymphocytes cocultured with autologous native DCs loaded with the mixture of peptides to be tested. This approach has been widely used to successfully identify T cell epitopes from tumor Ags, which were introduced as immunogens in clinical trials (31, 32). It has also been applied to identification of HIV CD4+ T cell epitopes (23, 29) and used to assess immunogenicity of therapeutic proteins (38). T cell epitopes identified by these assays have been found to be immunogenic in vaccination trials of naive individuals (39) and hence are expected to contribute to the design of prophylactic vaccines. Using these assays, we found four T cell-stimulating peptides, namely RT 338–352, env 31–45, env 388–402, and env 620–634. Two of them primed CD4+ T cells that recognized the processed epitopes of the native protein. In a pioneering study, the env 30–51 peptide, which includes env 31–45, was recognized by 2 of 15 asymptomatic seropositive responders. However, these responses were not documented for their HLA class II restriction (40).

With the exception of this study, no other investigations have highlighted the four peptides we identified as CD4+ T cell epitopes, although the HLA-DP4 molecules are very frequent worldwide. Peptide screening has been performed in HIV-1 seropositive donors by evaluating the proliferative responses of peptides spanning selected HIV Ags (17, 21, 22, 24, 26). This approach has identified many CD4+ T cell epitopes. Following natural infection, CD4+ T cell response to HIV components is highly variable and generally found in long-term nonprogressor patients (19) or in individuals treated soon after infection (41). The HIV-specific CD4+ T cell response is hampered by HIV infection (13, 14) and by exhaustion of memory CD4+ T cells (16, 17, 18). The response is raised against a reduced number of components (24, 42) and varies during HIV infection (16, 26, 43). Most of the CD4+ T cell epitopes characterized using seropositive donors are HLA-DR-restricted (17, 21, 24). These peptides have been selected as good binders to HLA-DR molecules (21), suggesting that a high affinity for HLA II molecules is a requisite to stimulate T cells upon HIV infection. Because we also selected the HLA-DP4-restricted peptides based on their binding capacities, it might be possible that they are also recognized by T lymphocytes of seropositive donors. Furthermore, priming with HLA-DP4-restricted peptides as RT 338–352 and env 31–45 gave rise to peptide-specific CD4+ T lymphocytes, which recognized naturally processed peptides of the native proteins. HIV-specific CD4+ T cell epitopes have been also delineated by repeated stimulation of seronegative PBMC with defined Ags. As an example, HIV RT protein has been used to prime CD4+ T lymphocytes in two different studies (27, 28). Both involved a limited numbers of naive donors and did not identify the RT 388–402 peptide. Because HLA-DP molecules are expressed less than HLA-DR molecules, we cannot exclude that upon infection or upon priming by the native Ag, the HLA-DP4-restricted T cells are poorly recruited. Nevertheless, a low priming of HLA-DP4-restricted CD4+ T cells during infection might be also an advantage for therapeutic vaccination because a pool of HIV-specific CD4+ T cells would be not affected by viremia and would be ready for priming by peptide injection. These findings should be considered for future vaccine development and cellular diagnosis.

Among the four HLA-DP4-restricted T cell-stimulating peptides we identified in this study, peptide RT 338–352 emerges as the most active. It was able to elicit 15 different T cell lines among 22 T cell lines and was immunogenic for seven naive individuals of eight investigated. This peptide is not the most active in binding to HLA-DP4 molecules, demonstrating that its T cell-stimulating ability does not entirely rely on its ability to bind to HLA-DP4 molecules. The high number of T cell lines specific for this peptide reflects the elevated number of T cell precursors able to recognize it and present in almost all naive individuals. In contrast, fewer naive precursors seem to be specific for the env 31–45, env 388–402, env 620–634 peptides, which might be less immunogenic. The four peptides we identified exhibit various degrees of conservation. Peptide RT 338–352 is common to 65% of HIV sequences, and two peptide-specific T cell lines are stimulated by other natural variants. In terms of inclusion in an epitope-based vaccine, this peptide therefore has two major advantages for a CD4 T cell epitope, namely conservation and population coverage. Its level is comparable to that of HLA-DR-restricted CD4 T cell epitopes (21). These peptides bind to many HLA-DR molecules and are expected to exhibit binding in at least 77% of the human population (21), a frequency similar to that of HLA-DP4 molecules. In contrast to the RT peptide, there is less conservation of env peptides, especially env 388–402 and env 620–634, which have multiple variants. Peptide env 31–45 is found in only 3.9% of isolates, but its core sequence, which is delimited by residues between the P1 and P9 positions, is common to 80% of variants. We also showed that the most frequent variants stimulated one T cell line specific for the env 31–45 peptide. Moreover, as shown previously in the NY-ESO-1 tumor Ag (1, 5), it is not excluded that the HLA-DP4 peptides we identified could be also presented by HLA-DR molecules, but HLA-DR restriction has not been investigated in this study.

Altogether, we describe in this study relevant HIV sequences of interest for epitope-based vaccines and cellular diagnosis. Recent advances in the treatment of HIV infection have underscored that a broad and vigorous CD4+ T cell response contributes to the control of viremia (19). These observations suggest that multiple T cell epitopes should be introduced into future therapeutic HIV vaccines such as the lipopeptide mixture approach (44). HLA-DR and -DP molecules are not in genetic disequilibrium and exhibit different peptide-binding specificity. The peptides that are restricted to them are therefore expected to recruit a large repertoire of peptide-specific T cells and hence to induce a large multiepitopic response.


   Acknowledgments
 
We thank J. Dausset and Dr. de Toma for their gift of EBV cell lines and Dr. D. Stammers (NIBSC) for the gift of HIV-1 RT. We are also grateful to Drs. Hassan Zarour and Christine Almunia for helpful discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the CEA and by the Agence Nationale de Recherches sur le SIDA Vaccine Network. The National Institute for Biological Standards and Control Centralized Facility for AIDS Reagents is supported by European Union Programme European Vaccine against AIDS/Medical Research Council (contract QLKZ-CT-1999-00609). Back

2 Address correspondence and reprint requests to Dr. Bernard Maillere, Protein Engineering and Research Department, bat 152, CEA-Saclay, 91191 Gif sur Yvette, France. E-mail address: bernard.maillere{at}cea.fr Back

3 Abbreviations used in this paper: DC, dendritic cell; RT, reverse transcriptase. Back

Received for publication November 22, 2005. Accepted for publication February 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Castelli, F. A., C. Buhot, A. Sanson, H. Zarour, S. Pouvelle-Moratille, C. Nonn, H. Gahery-Segard, J. G. Guillet, A. Menez, B. Georges, B. Maillere. 2002. HLA-DP4, the most frequent HLA II molecule, defines a new supertype of peptide-binding specificity. J. Immunol. 169: 6928-6934. [Abstract/Free Full Text]
  2. Charron, D., R. Fauchet, E. Albert, J. Bodmer, A. Cambon-Thomsen, L. Degos, J. Hors, A. Piazza, I. Schreuder. 1997. Genetic diversity of HLA, functional and medical Implication. D. Charron, ed. XIIth International Histocompatibility Workshop and conference EDK, Paris, France.
  3. Schultz, E. S., B. Lethe, C. L. Cambiaso, J. Van Snick, P. Chaux, J. Corthals, C. Heirman, K. Thielemans, T. Boon, P. van der Bruggen. 2000. A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes. Cancer Res. 60: 6272-6275. [Abstract/Free Full Text]
  4. Zeng, G., X. Wang, P. F. Robbins, S. A. Rosenberg, R. F. Wang. 2001. CD4+ T cell recognition of MHC class II-restricted epitopes from NY- ESO-1 presented by a prevalent HLA DP4 allele: association with NY-ESO- 1 antibody production. Proc. Natl. Acad. Sci. USA 98: 3964-3969. [Abstract/Free Full Text]
  5. Mandic, M., F. Castelli, B. Janjic, C. Almunia, P. Andrade, D. Gillet, V. Brusic, J. M. Kirkwood, B. Maillere, H. M. Zarour. 2005. One NY-ESO-1-derived epitope that promiscuously binds to multiple HLA-DR and HLA-DP4 molecules and stimulates autologous CD4+ T cells from patients with NY-ESO-1-expressing melanoma. J. Immunol. 174: 1633-1640.
  6. Schuler-Thurner, B., E. S. Schultz, T. G. Berger, G. Weinlich, S. Ebner, P. Woerl, A. Bender, B. Feuerstein, P. O. Fritsch, N. Romani, G. Schuler. 2002. Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells. J. Exp. Med. 195: 1279-1288. [Abstract/Free Full Text]
  7. Schultz, E. S., B. Schuler-Thurner, V. Stroobant, L. Jenne, T. G. Berger, K. Thielemanns, P. van der Bruggen, G. Schuler. 2004. Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy. J. Immunol. 172: 1304-1310. [Abstract/Free Full Text]
  8. Zhang, Y., N. Renkvist, Z. Sun, B. Schuler-Thurner, N. Glaichenhaus, G. Schuler, T. Boon, P. van der Bruggen, D. Colau. 2005. A polyclonal anti-vaccine CD4 T cell response detected with HLA-DP4 multimers in a melanoma patient vaccinated with MAGE-3.DP4-peptide-pulsed dendritic cells. Eur. J. Immunol. 35: 1066-1075. [Medline]
  9. Celis, E., R. W. Karr. 1989. Presentation of an immunodominant T-cell epitope of hepatitis B surface antigen by the HLA-DPw4 molecule. J. Virol. 63: 747-752. [Abstract/Free Full Text]
  10. Koelle, D. M., S. N. Reymond, H. Chen, W. W. Kwok, C. McClurkan, T. Gyaltsong, E. W. Petersdorf, W. Rotkis, A. R. Talley, D. A. Harrison. 2000. Tegument-specific, virus-reactive CD4 T cells localize to the cornea in herpes simplex virus interstitial keratitis in humans. J. Virol. 74: 10930-10938. [Abstract/Free Full Text]
  11. de Graaff, P. M., J. Heidema, M. C. Poelen, M. E. van Dijk, M. V. Lukens, S. P. van Gestel, J. Reinders, E. Rozemuller, M. Tilanus, P. Hoogerhout, et al 2004. HLA-DP4 presents an immunodominant peptide from the RSV G protein to CD4 T cells. Virology 326: 220-230. [Medline]
  12. Carcelain, G., P. Debre, B. Autran. 2001. Reconstitution of CD4+ T lymphocytes in HIV-infected individuals following antiretroviral therapy. Curr. Opin. Immunol. 13: 483-488. [Medline]
  13. McNeil, A. C., W. L. Shupert, C. A. Iyasere, C. W. Hallahan, J. A. Mican, R. T. Davey, Jr, M. Connors. 2001. High-level HIV-1 viremia suppresses viral antigen-specific CD4+ T cell proliferation. Proc. Natl. Acad. Sci. USA 98: 13878-13883. [Abstract/Free Full Text]
  14. Boritz, E., B. E. Palmer, C. C. Wilson. 2004. Human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cells that proliferate in vitro detected in samples from most viremic subjects and inversely associated with plasma HIV-1 levels. J. Virol. 78: 12638-12646. [Abstract/Free Full Text]
  15. Lichterfeld, M., D. E. Kaufmann, X. G. Yu, S. K. Mui, M. M. Addo, M. N. Johnston, D. Cohen, G. K. Robbins, E. Pae, G. Alter, et al 2004. Loss of HIV-1-specific CD8+ T cell proliferation after acute HIV-1 infection and restoration by vaccine-induced HIV-1-specific CD4+ T cells. J. Exp. Med. 200: 701-712. [Abstract/Free Full Text]
  16. Iyasere, C., J. C. Tilton, A. J. Johnson, S. Younes, B. Yassine-Diab, R. P. Sekaly, W. W. Kwok, S. A. Migueles, A. C. Laborico, W. L. Shupert, et al 2003. Diminished proliferation of human immunodeficiency virus-specific CD4+ T cells is associated with diminished interleukin-2 (IL-2) production and is recovered by exogenous IL-2. J. Virol. 77: 10900-10909. [Abstract/Free Full Text]
  17. Younes, S. A., B. Yassine-Diab, A. R. Dumont, M. R. Boulassel, Z. Grossman, J. P. Routy, R. P. Sekaly. 2003. HIV-1 viremia prevents the establishment of interleukin 2-producing HIV-specific memory CD4+ T cells endowed with proliferative capacity. J. Exp. Med. 198: 1909-1922. [Abstract/Free Full Text]
  18. Palmer, B. E., E. Boritz, C. C. Wilson. 2004. Effects of sustained HIV-1 plasma viremia on HIV-1 Gag-specific CD4+ T cell maturation and function. J. Immunol. 172: 3337-3347. [Abstract/Free Full Text]
  19. Rosenberg, E. S., J. M. Billingsley, A. M. Caliendo, S. L. Boswell, P. E. Sax, S. A. Kalams, B. D. Walker. 1997. Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia. Science 278: 1447-1450. [Abstract/Free Full Text]
  20. Elrefaei, M., M. D. McElroy, C. P. Preas, R. Hoh, S. Deeks, J. Martin, H. Cao. 2004. Central memory CD4+ T cell responses in chronic HIV infection are not restored by antiretroviral therapy. J. Immunol. 173: 2184-2189. [Abstract/Free Full Text]
  21. Wilson, C. C., B. Palmer, S. Southwood, J. Sidney, Y. Higashimoto, E. Appella, R. Chesnut, A. Sette, B. D. Livingston. 2001. Identification and antigenicity of broadly cross-reactive and conserved human immunodeficiency virus type 1-derived helper T-lymphocyte epitopes. J. Virol. 75: 4195-4207. [Abstract/Free Full Text]
  22. Malhotra, U., S. Holte, S. Dutta, M. M. Berrey, E. Delpit, D. M. Koelle, A. Sette, L. Corey, M. J. McElrath. 2001. Role for HLA class II molecules in HIV-1 suppression and cellular immunity following antiretroviral treatment. J. Clin. Invest. 107: 505-517. [Medline]
  23. Venturini, S., D. E. Mosier, D. R. Burton, P. Poignard. 2002. Characterization of human immunodeficiency virus type 1 (HIV-1) Gag- and Gag peptide-specific CD4+ T-cell clones from an HIV-1-seronegative donor following in vitro immunization. J. Virol. 76: 6987-6999. [Abstract/Free Full Text]
  24. Kaufmann, D. E., P. M. Bailey, J. Sidney, B. Wagner, P. J. Norris, M. N. Johnston, L. A. Cosimi, M. M. Addo, M. Lichterfeld, M. Altfeld, et al 2004. Comprehensive analysis of human immunodeficiency virus type 1-specific CD4 responses reveals marked immunodominance of gag and nef and the presence of broadly recognized peptides. J. Virol. 78: 4463-4477. [Abstract/Free Full Text]
  25. Wentworth, P. A., I. Bathurst, P. H. Feucht, H. A. Erlich, K. S. Steimer. 1994. Epitopes recognized by HIV-SF2 nef-specific CD4+ T-cell clones generated from HIV-1-uninfected donors. Vaccine 12: 117-128. [Medline]
  26. Malhotra, U., S. Holte, T. Zhu, E. Delpit, C. Huntsberry, A. Sette, R. Shankarappa, J. Maenza, L. Corey, M. J. McElrath. 2003. Early induction and maintenance of Env-specific T-helper cells following human immunodeficiency virus type 1 infection. J. Virol. 77: 2663-2674. [Abstract/Free Full Text]
  27. Manca, F., D. Fenoglio, M. T. Valle, G. Li Pira, A. Kunkl, A. Ferraris, D. Saverino, F. Lancia, L. Mortara, L. Lozzi, et al 1995. Human CD4+ T cells can discriminate the molecular and structural context of T epitopes of HIV gp120 and HIV p66. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 9: 227-237. [Medline]
  28. van der Burg, S. H., K. M. Kwappenberg, A. Geluk, M. van der Kruk, O. Pontesilli, E. Hovenkamp, K. L. Franken, K. E. van Meijgaarden, J. W. Drijfhout, T. H. Ottenhoff, et al 1999. Identification of a conserved universal Th epitope in HIV-1 reverse transcriptase that is processed and presented to HIV-specific CD4+ T cells by at least four unrelated HLA-DR molecules. J. Immunol. 162: 152-160. [Abstract/Free Full Text]
  29. Pancre, V., B. Georges, G. Angyalosi, F. Castelli, A. Delanoye, M. Delacre, E. Hachulla, B. Maillere, A. Bouzidi, C. Auriault. 2002. Novel promiscuous HLA-DQ HIV Nef peptide that induces IFN-{gamma}-producing memory CD4+ T cells. Clin. Exp. Immunol. 129: 429-437. [Medline]
  30. Texier, C., S. Pouvelle, M. Busson, M. Herve, D. Charron, A. Menez, B. Maillere. 2000. HLA-DR restricted peptide candidates for bee venom immunotherapy. J. Immunol. 164: 3177-3184. [Abstract/Free Full Text]
  31. Chaux, P., V. Vantomme, V. Stroobant, K. Thielemans, J. Corthals, R. Luiten, A. M. Eggermont, T. Boon, P. van der Bruggen. 1999. Identification of MAGE-3 epitopes presented by HLA-DR molecules to CD4+ T lymphocytes. J. Exp. Med. 189: 767-778. [Abstract/Free Full Text]
  32. Zarour, H. M., J. M. Kirkwood, L. S. Kierstead, W. Herr, V. Brusic, C. L. Slingluff, Jr, J. Sidney, A. Sette, W. J. Storkus. 2000. Melan-A/MART-151–73 represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4+ T cells. Proc. Natl. Acad. Sci. USA 97: 400-405. [Abstract/Free Full Text]
  33. Southwood, S., J. Sidney, A. Kondo, M. F. del Guercio, E. Appella, S. Hoffman, R. T. Kubo, R. W. Chesnut, H. M. Grey, A. Sette. 1998. Several common HLA-DR types share largely overlapping peptide binding repertoires. J. Immunol. 160: 3363-3373. [Abstract/Free Full Text]
  34. Sturniolo, T., E. Bono, J. Ding, L. Raddrizzani, O. Tuereci, U. Sahin, M. Braxenthaler, F. Gallazzi, M. P. Protti, F. Sinigaglia, J. Hammer. 1999. Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices [see comments]. Nat Biotechnol. 17: 555-561. [Medline]
  35. Raddrizzani, L., T. Sturniolo, J. Guenot, E. Bono, F. Gallazzi, Z. A. Nagy, F. Sinigaglia, J. Hammer. 1997. Different modes of peptide interaction enable HLA-DQ and HLA-DR molecules to bind diverse peptide repertoires. J. Immunol. 159: 703-711. [Abstract]
  36. Rammensee, H. G., T. Friede, S. Stevanoviic. 1995. MHC ligands and peptide motifs: first listing. Immunogenetics 41: 178-228. [Medline]
  37. Falk, K., O. Rotzschke, S. Stevanovic, G. Jung, H. G. Rammensee. 1994. Pool sequencing of natural HLA-DR, DQ, and DP ligands reveals detailed peptide motifs, constraints of processing, and general rules. Immunogenetics 39: 230-242. [Medline]
  38. Tangri, S., B. R. Mothe, J. Eisenbraun, J. Sidney, S. Southwood, K. Briggs, J. Zinckgraf, P. Bilsel, M. Newman, R. Chesnut, et al 2005. Rationally engineered therapeutic proteins with reduced immunogenicity. J. Immunol. 174: 3187-3196. [Abstract/Free Full Text]
  39. Jenkins, D. E., L. L. Yasukawa, R. Bergen, C. Benike, E. G. Engleman, A. M. Arvin. 1999. Comparison of primary sensitization of naive human T cells to varicella-zoster virus peptides by dendritic cells in vitro with responses elicited in vivo by varicella vaccination. J. Immunol. 162: 560-567. [Abstract/Free Full Text]
  40. Geretti, A. M., C. A. Van Baalen, J. C. Borleffs, C. A. Van Els, A. D. Osterhaus. 1994. Kinetics and specificities of the T helper-cell response to gp120 in the asymptomatic stage of HIV-1 infection. Scand. J. Immunol. 39: 355-362. [Medline]
  41. Alter, G., G. Hatzakis, C. M. Tsoukas, K. Pelley, D. Rouleau, R. LeBlanc, J. G. Baril, H. Dion, E. Lefebvre, R. Thomas, et al 2003. Longitudinal assessment of changes in HIV-specific effector activity in HIV-infected patients starting highly active antiretroviral therapy in primary infection. J. Immunol. 171: 477-488. [Abstract/Free Full Text]
  42. Peretz, Y., G. Alter, M. P. Boisvert, G. Hatzakis, C. M. Tsoukas, N. F. Bernard. 2005. Human immunodeficiency virus (HIV)-specific {gamma} interferon secretion directed against all expressed HIV genes: relationship to rate of CD4 decline. J. Virol. 79: 4908-4917. [Abstract/Free Full Text]
  43. Pitcher, C. J., C. Quittner, D. M. Peterson, M. Connors, R. A. Koup, V. C. Maino, L. J. Picker. 1999. HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression. Nat. Med. 5: 518-525. [Medline]
  44. Gahery-Segard, H., G. Pialoux, B. Charmeteau, S. Sermet, H. Poncelet, M. Raux, A. Tartar, J. P. Levy, H. Gras-Masse, J. G. Guillet. 2000. Multiepitopic B- and T-cell responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine. J. Virol. 74: 1694-1703. [Abstract/Free Full Text]



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