Received 11 January 1996/Accepted 17 April 1996

Transcription

1 JOURNAL OF VIROLOGY, Aug. 1996, p Vol. 70, No X/96/$ Copyright 1996, American Society for Microbiology Among All Human T-Cell Leukemia Virus Type 1 Proteins, Tax, Polymerase, and Envelope Proteins Are Predicted as Preferential Targets for the HLA-A2-Restricted Cytotoxic T-Cell Response CLAUDINE PIQUE, 1 * FRANCINE CONNAN, 2 JEAN-PIERRE LEVILAIN, 3 JEANNINE CHOPPIN, 2 AND MARIE-CHRISTINE DOKHÉLAR 1 URA 1156 CNRS 1 and Service d Immuno-Hématologie, 3 Institut Gustave Roussy, Villejuif, and INSERM U152, Institut Cochin de Génétique Moléculaire, Paris, 2 France Received 11 January 1996/Accepted 17 April 1996 The human T-cell leukemia virus type 1 (HTLV-1) is a human retrovirus associated with two diseases for which no successful treatment is yet available; the development of a vaccine is therefore an important issue. Since HTLV-1 is a persistent virus, an efficient vaccine will probably require a cytotoxic T-lymphocyte (CTL) response in addition to the production of antibodies. To identify potential CTL epitopes, we have selected, within all of the HTLV-1 proteins, nonapeptides containing anchor residues required for association with HLA-A2 molecules (residues at positions 2 and 9), which is the most frequently occurring A allele in all human populations. A set of 111 peptides was synthetized and tested in vitro in two assembly assays using processingdefective T2 cells. Anchor motifs selected were those containing two major anchor residues (L 2 /M 2 /I 2 -V 9 /L 9 /I 9 ) (one letter amino-acid code) and those including tolerated anchor residues (V 2 /A 2 /T 2 and/or A 9 /M 9 /T 9 ). The analysis of the binding capacity of the peptides confirms the high efficiency of the L 2 -V 9 anchor motif and shows that a systematic research of potential binding peptides should exclude peptides containing known detrimental residues rather than select only peptides with known favored residues. We show that 39 peptides representative of all the HTLV-1 proteins are able to bind to HLA-A2 molecules. Strong binder peptides which are very likely good CTL epitopes were identified in three HTLV-1 proteins, Tax, envelope, and polymerase. Three of the strong binder peptides correspond to previously described HLA-A2-restricted CTL epitopes in the Tax protein, and two others are localized in a domain of the viral envelope recognized by natural neutralizing antibodies. This latter result has important implications for the development of an anti-htlv-1 vaccine. Downloaded from The human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia (40, 49) and for a neuropathic disease, tropical spastic paraparesis/htlv-1-associated myelopathy (TSP/HAM) (3, 18), and is also associated with other clinical disorders (15, 19). HTLV-1 is endemic in Japan, Africa, South America, the Caribbean region, and Melanesia, where the seroprevalence varies from 5 to 15% (4, 9, 16, 22, 32, 43, 46). The total number of HTLV-1 carriers is estimated to be 10 million to 20 million worldwide (33). For HTLV-1-associated diseases, no treatment has been shown to be efficient in all patients, and the development of a specific vaccine, which should be feasible since HTLV-1 is a very conserved virus, is therefore an important issue. In the case of viruses persisting within cells, the humoral response could be insufficient to induce a long-term protection since residual infected cells represent potential sites for virus reactivation and disease development. Moreover, HTLV-1 replicates at very low levels in infected cells, and most proviruscontaining cells do not express viral proteins at the cell surface (14, 28). Major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) can eliminate such cells and should represent an important component of a protective response. In order to be effective, an HTLV-1 vaccine should therefore have the capacity to induce both the production of antibodies, including neutralizing antibodies, and a cellular * Corresponding author. Mailing address: URA 1156 CNRS, Institut Gustave Roussy, 39 rue C. Desmoulins, Villejuif, France. Phone: Fax: CTL response. HTLV-1 encodes the regulatory proteins Tax, Rex, Rof, and Tof and the structural proteins Gag, protease, polymerase, and envelope (5, 29, 34, 44). The global immune response is generally considered to be higher in TSP/HAM patients than in asymptomatic carriers (10, 23, 24), although this has been the matter of recent discussion for the CTL response (7). In TSP/HAM patients, moreover, HTLV-1-specific CTL can be detected in the blood as well as in the cerebrospinal fluid (10). The antiviral CTL are activated in vivo and are directed mainly to the HTLV-1 Tax regulatory protein and, at a lower frequency, to the structural proteins Gag, envelope, and polymerase (10, 25, 36). The immunodominance of the Tax protein might be due to its chronic expression, since the corresponding mrna is regularly detected in infected cells in vivo (17, 28). This predominant anti-tax response is probably very important in limiting the extent of active replication, since this regulatory protein is expressed early in cell infection. The epitopes recognized by the CTL are short peptides presented at the cell surface by MHC class I molecules. Peptides are produced in the cytosol by protein degradation and are then transported by the TAP1 and TAP2 proteins into the endoplasmic reticulum, where they associate with the MHC class I heavy chain/ 2 -microglobulin complexes (for a review, see reference 11). Each allelic form of class I proteins interacts with a specific set of peptides characterized by the presence of anchor residues at defined positions. The majority of peptides bound to the class I molecules have a restricted size of 9 1 amino acids and require free N-terminal and C-terminal groups. For the HLA-A2 molecule, the most frequent in the on October 2, 2018 by guest 4919

2 4920 PIQUE ET AL. J. VIROL. TABLE 1. Assembly with HLA-A2 molecules of HTLV-1 peptides detected by ELISA and fluorescence assay FIG. 1. Validation of the ELISA and the fluorescence assembly assays. The associations with HLA-A2 molecules of a known HLA-A2-restricted CTL epitope of HTLV-1 (the tax peptide) and two peptides (taxm12 and taxm19) in which anchor residues have been modified (residues underlined in panel A) were tested in the ELISA (A) and in the fluorescence assay (B) as described in Materials and Methods. (A) Final concentrations ranging from 0.6 to 60 M; (B) final concentrations of 60 M. The assembly ratios obtained for each peptide are indicated in parentheses. different human populations, the anchor residues have been characterized and are localized at positions 2 and 9 (X 2 -X 9 ). The canonical motif for binding to HLA-A2 molecules, which involves L or M (one-letter amino-acid code) at position 2 and V at position 9 (13), has been extended to I at position 2 and L or I at position 9 (20, 38). Other residues (V 2 /A 2 /T 2 and A 9 /M 9 /T 9 ) which are tolerated at anchor positions have been identified (27, 41). Nonanchor residues associated with good binding (favored residues) or with lack of binding (detrimental residues) have also been described (41). Recent works have demonstrated a direct relationship between the ability of a peptide to act as a CTL epitope and its affinity for class I molecules (30, 45). Therefore, the identification of the strongest binders is important for predicting the more immunogenic CTL epitopes in a given protein. In this study, we have tested the abilities of HTLV-1 peptides, each containing an HLA-A2 binding motif, to associate Peptide Sequence a to HLA-A2 molecules in vitro. This work allowed us (i) to make a global evaluation of the different HLA-A2-binding motifs currently available, which leads us to propose an efficient approach to select potential good binder peptides, and (ii) to identify strong HLA-A2-binding peptides in the HTLV-1 proteins, which very likely represent real HLA-A2- restricted CTL epitopes. MATERIALS AND METHODS Association with HLA-A2 molecules b ELISA Fluorescence assay gag I L I Q T Q A Q I gag F M Q T I R L A V gag L L L D L P A D I pol R L Q A L Q H L V pol S L P T T L A H L pol Q L A H I L Q P I pol S L I S H G L P V pol A L P E L Q A L L pol A L L G E I Q W V pol R L V Q T L P L L pol T L T G T T T V V pol Q L L A S A V L L pol V L L L D K Y T L pol I L S Q R S F P L pol Y L Y H Y L R T L pol L L S R K V V Y L pol T L Y R L H V W V pol S L L Q A I A Y L prot A L F S S N T P L prot K L T S L P V L I prot I L P I Q A P A V env S L Y L F P H W I env F L N T E P S Q L env Q L P P T A P P L NT env L L T L V Q L T L env V L Y S P N V S V env A L Q E Q C C F L env F L N I T N S H V env A L Q T G I T L V tax L L F G Y P V Y V tax T L G Q H L P T L tax T L S F P D P G L tax Y L Y Q L S P P I tax Q L G A F L T N V tax G L L P F H S T L tax L L F E E Y T N I rex I V T P Y W P P V tof L I I S P L P R V rof H L A F Q L S S I rof R L L S P L S P L a Peptides are classified by order of appearance of the proteins in the viral genome, and anchor residues are indicated in boldface type. b Nine mer peptides derived from all of the HTLV-1 proteins were selected and tested in the ELISA and fluorescence assembly assays at an estimated concentration of 100 M as described in Materials and Methods. Only peptides detected in both assays were considered as positive. NT, not tested. Cells and antibodies. The processing-defective cell line 174CEM.T2 (T2; HLA-A2.1, B51) used in the peptide-hla-a2 assembly assays (6, 35) was obtained from P. Cresswell (Howard Hughes Medical Institute, New Haven, Conn.). This cell line has a homologous deletion of the MHC class II region located on chromosome 6 which includes the TAP1 and TAP2 genes, which are involved in peptide transport from the cytosol to the endoplasmic reticulum (42). As a consequence of the absence of TAP1 and TAP2 proteins, the T2 cells

3 VOL. 70, 1996 HLA-A2-BINDING PEPTIDES IN THE HTLV-1 PROTEINS 4921 FIG. 2. Distribution of favored and detrimental residues in the binder and nonbinder HTLV-1 peptides. (A) Residues associated with good binding (favored residues) or with no binding (detrimental residues) according to Ruppert et al. (41) and distribution of these residues in the binder peptides; (B) distribution of favored and detrimental residues in the nonbinder peptides. Binder and nonbinder peptides are classified by order to appearance of the proteins in the viral genome. produce empty HLA-A2 molecules which can be loaded with synthetic peptides. The T2 cells were cultivated in RPMI 1640 supplemented with 10% fetal calf serum. Peptide selection and synthesis. The HTLV-1 sequences used is that of Seiki et al. (44). For the Tax, Rex, Rof, Tof, Gag, and Env proteins, the residues were numbered from the Met corresponding to the respective initiation codons. For the protease and polymerase proteins, residues were numbered from the start of the corresponding prot and pol open reading frames. Nonapeptides were selected in all of the HTLV-1 proteins, the regulatory proteins Tax, Rex, Rof and Tof and the structural proteins Gag, polymerase, protease, and envelope. All of the peptides containing two major anchor residues at positions 2 and 9 (L 2 /M 2 /I 2 - V 9 /L 9 /I 9 ) (13, 20, 38) were systematically selected in each viral protein. A set of 110 peptides was selected, but only 90 of them were tested since some peptides were too hydrophobic to be synthesized. Peptides containing one or two tolerated residues (27, 41) at position 2 (V 2 /A 2 /T 2 ) and/or position 9 (A 9 /M 9 /T 9 ) were selected in the proteins when only a few or no peptides containing major anchor residues were found. This is the case for the Rex and Tof proteins and the C-terminal part of the polymerase protein (21 peptides). Peptides with free N- and C-terminal groups were synthesized with the Multispin Synthesis System (Chiron Mimotopes, Clayton, Victoria, Australia). The

4 4922 PIQUE ET AL. J. VIROL. TABLE 2. Binding to HLA-A2 molecules of HTLV-1 peptides as a function of the nature of anchor residues a Anchor motif No. of peptides tested No. of binder peptides % of binders Major residues L 2 -V L 2 -I L 2 -L I 2 -L NS I 2 -V NS M 2 -L NS M 2 -V NS Total Tolerated residues V 2 /A 2 /T 2 -V 9 /L 9 /I NS V 2 /A 2 /T 2 -A 9 /M 9 /T NS Total a Nine-mer HTLV-1 peptides were tested in the two assembly assays at an estimated concentration of 100 M as described in Materials and Methods. Only peptides detected in both assays were considered positive. NS, not significant. amounts of peptides were estimated to be 1 mg by comparison with two controls of known concentrations. For taxm12 and taxm19 peptides and all positive peptides, the concentrations were determined by high-pressure liquid chromatography purification and quantitative amino acid determination. The mean of peptide concentrations obtained for the positive peptides is mg/ml, which shows that less than a twofold variation factor exists between the estimated and exact peptide concentrations. Peptides were stored at 20 C at a concentration of 2 mg/ml. Enzyme-linked immunosorbent assay (ELISA) for detection of peptides binding to HLA-A2. The peptide-hla-a2 assembly assay was previously described (6) and was used with minor modifications. Wells of microtiter plates were coated with 1 g of the anti-hla-a2 heavy chain monoclonal antibody BB7.2 in 100 l of phosphate-buffered saline (PBS) for2hat37 C and then saturated with 10 mg of bovine-serum albumin (BSA) per ml in PBS containing 0.05% Tween 20for1hat22 C. In parallel, T2 cells lysed in 62.5 l of lysis buffer (10 mm Tris-buffered saline [ph 7.5] containing 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 10 g of trypsin inhibitor per ml, 10 M pepstatin A, 10 M leupeptin, and 10 mm iodoacetamide) and were incubated with peptides (3.75 l of peptide in water) for 1hat37 C. Because of volume variation due to cell lysis, the final concentration of the diluted peptide was 100 M. After this incubation, 150 l of PBS containing 0.05% Tween 20, 10 mg of BSA per ml, and 1 mm phenylmethylsulfonyl fluoride added to the reaction mixture, and 100 l of this mixture was transferred to the precoated wells for a 2-h incubation at 37 C. After a wash with PBS containing 0.05% Tween 20, the anti- 2 -microglobulin monoclonal antibody M28 coupled to alkaline phosphatase (Pierce, Paris, France) was added (1 g/ml in PBS containing 0.05% Tween 20 and 2 mg ofbsaperml)for1hat37 C. After a wash, the alkaline phosphatase activity was detected by using p-nitrophenyl phosphate disodium hexahydrate substrate (Sigma ), and the resultant A 402/492 was measured. The absorbance assembly ratio was calculated as absorbance with peptide/absorbance without peptide. A cutoff of 1.3 was used to determine peptide positivity. This cutoff corresponds to the mean of the assembly ratios obtained for the 111 peptides ( ). Fluorescence assay for detection of peptide binding to HLA-A2. The assay was adapted from those described by Nijman et al. (35). Aliquots of T2 cells were incubated with 10 l of diluted peptides (final concentration, 100 M) or water in 200 l of RPMI without fetal calf serum in flat-bottom 96-well plates for 20hat37 C, 5% CO 2. Cells were washed twice in PBS containing 2% fetal calf serum (PBS-2%) and incubated with a saturating concentration of monoclonal antibody BB7.2 in 50 l of PBS-2% for 45 min at 4 C. After three washes with PBS-2%, cells were incubated with 50 l of a 1/50 dilution of fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin (Tebu, Paris, France) for 45 min at 4 C. Cells were washed three times, and fluorescence was measured at 488 nm in an EPICS C cell sorter (Coultronics, Margency, France). The fluorescence assembly ratio was calculated as mean fluorescence with peptide/mean fluorescence without peptide. A cutoff of 1.6, which corresponds to the mean of the assembly ratios obtained for the 111 peptides ( ), was used to determine peptide positivity. RESULTS One-third of peptides containing a binding motif associate with HLA-A2 molecules. We described previously an ELISA allowing the detection of peptides able to associate with HLA-A2 class I molecules obtained from T2 cell lysates (6). In the present work, we also used a second test adapted from reference 35 in which peptides added to live T2 cells stabilize surface-expressed HLA-A2 molecules further detected by indirect immunofluorescence. To validate these two assays, we first studied the HLA-A2 peptide assembly obtained with a known HLA-A2-restricted CTL epitope of HTLV-1, the tax peptide (26), and two peptides in which the anchor residues of the tax peptide were changed for hydrophilic ones (Fig. 1). As expected, the tax peptide promotes HLA-A2 molecule assembly, as detected by an increased absorbance in the ELISA (Fig. 1A) and by an increased mean fluorescence channel in the fluorescence assay (Fig. 1B). As shown in Fig. 1A, the association of the tax peptide with HLA-A2 molecules in the ELISA is observed with concentrations ranging from 60 to less than 0.6 M. In the fluorescence assay, the assembly of the HLA-A2 molecule with the tax peptide is observed with concentrations of peptide ranging from 60 to 6 M (data not shown). The taxm12 and taxm19 peptides, by contrast, present an inability or a reduced capacity to promote the HLA-A2 molecule assembly in both assays. In consequence, our two assays detect a specific assembly of peptides with HLA-A2 molecules, since the residue at position 2 and the residue at position 9 are important for the formation of the class I complexes. We next tested a set of 111 peptides representing all of the HTLV-1 proteins, each containing an HLA-A2 binding motif, in the ELISA at an estimated concentration of 100 M (Table 1). Forty peptides were found to be positive and were further tested in the fluorescence assay. All peptides except one (tax ) were also positive in the fluorescence assay. We considered that only the peptides giving a positive result (assembly ratio higher than 1.3) in the two assays were unambiguously able to associate with HLA-A2 molecules. Since a very low amount of the env peptide was obtained in the synthesis, this peptide was tested only in the ELISA and was considered to be positive. According to these criteria, 39 positive peptides were identified in the HTLV-1 proteins. Peptides containing the L 2 -V 9 anchor motif and no previously described detrimental residues bind better than others to HLA-A2 molecules. The peptide binding to HLA-A2 involves two major anchor residues at positions 2 and 9 but also other residues, as reported by Ruppert et al. (41) for peptides containing the L 2 /M 2 -V 9 /L 9 /I 9 anchor motif. Some residues located at various positions are associated with good binding (favored residues), and others are associated with lack of binding (detrimental residues) (Fig. 2). Our set of 111 peptides allowed us to evaluate the efficiency of peptides binding to HLA-A2 molecules as a function of the nature of the residues in the sequence. Results in Table 2 show that 38 of 90 peptides (42%) with major anchor residues bind to HLA-A2 molecules, compared with only 1 of 21 (5%) containing tolerated anchor residues. Among the peptides with a major anchor motif, 78% of all peptides containing the L 2 -V 9 anchor motif bind to HLA-A2 molecules, compared with 50% or less of peptides containing the L 2 -I 9 or L 2 -L 9 anchor motif. We next examined the frequency of favored and detrimental residues (highlighted in Fig. 2) in our set of binder peptides (37 peptides [Fig. 2A]) and nonbinder peptides (38 peptides [Fig. 2B]) containing a correct combination of anchor residues (L 2 -V 9,L 2 -L 9,L 2 -I 9, M 2 -V 9, and M 2 -L 9 ). Only 2 of 37 binder peptides contained detrimental residues without any favored residues (Fig. 2A), compared with 16 of 38 nonbinder peptides (Fig. 2B). Conversely, 20 of 37 binder peptides have favored residues without detrimental ones, compared with 5 of 38 nonbinder peptides. Our results clearly confirm the classification of Ruppert et al.

5 VOL. 70, 1996 HLA-A2-BINDING PEPTIDES IN THE HTLV-1 PROTEINS 4923 FIG. 3. Association of binder peptides with HLA-A2 molecules as a function of peptide concentrations. Binder peptides derived from the Tax protein (A), the Tof, Rof, and Rex proteins (B), the polymerase protein (C and D), the Gag and protease proteins (E), and the envelope protein (F) were tested at various concentrations in the ELISA, and the corresponding assembly ratios were determined. Assembly ratios 1.3 were considered to represent a specific assembly. (41) and underline the predictive value of the binding motif, taking into account anchor as well as nonanchor residues. The Tax, polymerase, and envelope proteins contain strong binder peptides. We have selected peptides containing an HLA-A2 binding motif in each of the HTLV-1 proteins. Results in Table 3 show the distribution of the binder peptides. It can be seen that all of the HTLV-1 proteins contain HLA-A2- binding peptides; 29 are located in the structural proteins Gag, polymerase, protease, and envelope, and 10 are located in the regulatory proteins Tax, Rex, Tof, and Rof. Since our goal was to identify peptides able to strongly associate with HLA-A2 molecules (strong binders), we next determined for each positive peptide the minimal concentration still able to promote HLA-A2 molecule assembly. Binder peptides were tested in the ELISA at various concentrations (Fig. 3). As a function of the lowest concentration promoting

6 4924 PIQUE ET AL. J. VIROL. TABLE 3. Distribution of the HLA-A2-binding peptides in the HTLV-1 proteins a TABLE 4. Strong, moderate, and poor binder peptides detected in the HTLV-1 proteins Viral protein No. of peptides tested No. of binder peptides Peptide Sequence Concn required for binding ( M) a Gag 8 3 Polymerase Protease 7 3 Envelope 22 8 Tax 15 6 Rex 9 1 Rof 10 1 Tof 4 2 a Ninemer HTLV-1 peptides were tested in the two assembly assays at an estimated concentration of 100 M as described in Materials and Methods. Only peptides detected in both assays were considered positive. HLA-A2 molecule assembly, binder peptides were grouped into poor (peptides positive at concentrations higher than 20 M), moderate (peptides positive at concentrations ranging from2to20 M), and strong (peptides positive at 1.5 M or less) binders. As shown in Table 4, poor and moderate binder peptides were detected in all of the HTLV-1 proteins. The concentrations required for the binding of these peptides range from 3.5 to 190 M; they are therefore very unlikely to represent, if used in a peptide-based vaccine, good epitopes for the generation of a strong CTL response. In contrast, strong binder peptides bind to HLA-A2 molecules at low concentrations (0.5 to 1.5 M). Such peptides were found in only three HTLV-1 proteins, Tax, polymerase, and envelope. Three of them (tax 11-19, tax , and tax ) are part of sequences described by others to represent natural HLA-A2- restricted CTL epitopes (26, 37). DISCUSSION Using two in vitro assembly assays, we have tested a set of 111 HTLV-1 peptides containing a specific binding motif and found that one-third of them, which are representative of all of the HTLV-1 proteins, associate with HLA-A2 molecules. Since a direct relationship between the high affinity of peptide binding to class I molecules and the ability to elicit a CTL response has been demonstrated (45), the identification of the strongest binders is important for predicting the immunogenic CTL epitopes. The binder peptides were therefore grouped into poor, moderate, and strong binders as a function of the lowest concentration able to promote HLA-A2 molecule assembly. By this approach, we have found 15 poor binders, 10 moderate binders, and 14 strong binders in the HTLV-1 proteins. Numerous studies have permitted the characterization of the anchor and nonanchor residues involved in peptide binding to HLA-A2 molecules, and several specific motifs are now available (13, 20, 27, 38, 41). Our work allowed us to make a global evaluation of the efficiency of these different motifs. Our results confirmed that the most frequent anchor motif is the L 2 -V 9 motif (78% of binder peptides), which is the original motif described by Falk et al. by sequencing natural peptides eluted from HLA-A2 molecules (13). This observation suggests that the direct sequencing of natural peptides is probably the most valid approach to determine a class I binding motif. Our results clearly accord with the classification of Ruppert et al. concerning favored and detrimental residues (41). Indeed, we have shown that 55% of the binder peptides contain favored residues, whereas only 5% contain detrimental ones. Moreover, we observed that favored residues are found in 10 of 14 (72%) of our strong binders, whereas only two (pol Strong binders env F L N I T N S H V 0.5 tax L L F G Y P V Y V 0.6 tax L L F E E Y T N I 0.6 env A L Q T G I T L V 0.6 pol L L S R K V V Y L 0.65 env V L Y S P N V S V 0.75 env F L N T E P S Q L 0.8 pol T L Y R L H V W V 0.9 pol S L L Q A I A Y L 0.9 tax Y L Y Q L S P P I 1 tax Q L G A F L T N V 1.4 pol Y L Y H Y L R T L 1.4 env A L Q E Q C C F L 1.4 env Q L P P T A P P L 1.5 Moderate binders gag F M Q T I R L A V 3.5 pol S L I S H G L P V 5 env L L T L V Q L T L 6 rof H L A F Q L S S I 6 tof L I I S P L P R V 10 prot I L P I Q A P A V 10 pol R L V Q T L P L L 12 pol I L S Q R S F P L 12 tax T L G Q H L P T L 13 tax G L L P F H S T L 19 Poor binders pol R L Q A L Q H L V 35 pol A L L G E I Q W V 35 pol T L T G T T T V V 60 env S L Y L F P H W I 60 pol Q L A H I L Q P I 80 gag I L I Q T Q A Q I 85 gag L L L D L P A D I 90 rex I V T P Y W P P V 100 pol Q L L A S A V L L 100 prot K L T S L P V L I 110 rof R L L S P L S P L 120 pol S L P T T L A H L 130 pol A L P E L Q A L L 140 pol V L L L D K Y T L 180 prot A L F S S N T P L 190 a Each binder peptide was tested at various dilutions in the ELISA (Fig. 3), and the lowest concentration able to promote HLA-A2 molecule assembly was used for classification into strong binders ( 1.5 M), moderate binders (2 to 20 M), and poor binders ( 20 M) and pol ) have detrimental residues (compare Fig. 2 and Table 4). According to our results, the a priori exclusion of peptides containing only detrimental residues would have missed 2 binder peptides among 37 and eliminated 16 nonbinder peptides among 38, whereas the a priori selection of peptides containing only favored residues would have missed 17 binder peptides and selected 5 nonbinder peptides. For a systematic research of potential binding peptides, it therefore seems more helpful to exclude peptides containing only detrimental residues than to select peptides containing only favored residues. Altogether, our results indicate a good approach to select potential HLA-A2 strong binder peptides: they should exhibit both the presence of the L 2 -V 9 anchor motif and, except for sequences containing favored residues, the absence of detrimental residues.

7 VOL. 70, 1996 HLA-A2-BINDING PEPTIDES IN THE HTLV-1 PROTEINS 4925 We used our classification into poor, moderate, and strong binder peptides to identify potential HLA-A2-restricted CTL epitopes in the HTLV-1 proteins. Moderate and low binder peptides bind to HLA-A2 molecules only at high concentrations (3.5 to 190 M) and are unlikely to represent good CTL epitopes. Strong binder peptides associate at concentrations ranging from 0.5 to 1.5 M and could represent potential CTL epitopes. Such peptides exist in only three HTLV-1 proteins, Tax, polymerase, and envelope. Antiviral CTL detected in HTLV-1-infected individuals are mainly directed to the Tax protein (24, 25), in which eight sequences containing HLA-A2-restricted epitopes have been described (26, 37). Among them, four (tax 11-19, tax 80-95, tax , and tax ) contain major HLA-A2 binding motifs. For the Tax protein, we have synthesized only peptides with major anchor residues and detected 6 binder peptides among 15, including 4 strong binders (tax 11-19, tax , tax , and tax ). Interestingly, three of them (tax 11-19, tax , and tax ) are included in the previously described CTL epitopes, tax 11-19, tax , and tax , respectively. This result suggests that the minimal sequences involved in HLA-A2 binding in the CTL epitopes tax and tax are the peptides tax and tax respectively. Furthermore, the fact that three sequences recognized by CTL correspond to strong binder peptides in our study underlines the usefulness of our approach for the prediction of CTL epitopes in a given protein. Parker et al. have reported that no CTL response against the C-terminal third of the Tax protein and especially against a 15-mer (X19, tax ), which contains the tax sequence, can be obtained by using fresh or phytohemagglutinin-stimulated CD8 T cells from HLA-A2-infected individuals (37). Interestingly, as mentioned above, tax is a strong binder peptide. Since it is highly probable that it can associate with HLA-A2 in vivo as well as in vitro, the absence of a CTL response against the tax sequence could be due either to a lack of a specific T-cell receptor in the CD8 population or to a lack of production of this peptide in infected cells. To answer this question, it will be interesting to identify the natural viral peptides presented by HLA-A2 molecules in HTLV- 1-infected cells as was done for other viruses (12, 48). Apart from the Tax protein, strong binder peptides were found in the polymerase and envelope proteins. CTL directed to these two proteins have been detected in infected individuals (10, 24, 36), but the epitopes recognized have not been identified yet. The strong binder peptides that we have identified in these two proteins are good candidates as epitopes recognized by CTL generated after infection. Moreover, the existence of a CTL response directed to the viral envelope is especially interesting because this protein is the major target for neutralizing antibodies. The viral envelope is synthesized as a precursor of 488 amino acids further cleaved into a surface protein (SU), involved in receptor binding, and a transmembrane protein (TM), involved in membrane fusion (21). Natural neutralizing antibodies obtained from TSP/HAM patients recognize at least three domains in the SU protein (env , env , and env ) (2). The first domain (env ) plays a crucial role in the envelope functions, as we have previously shown (8, 39), and is also a major target of the humoral and cellular immune response in humans. Indeed, this domain is the principal neutralizing B-cell epitope of the viral envelope (2), is recognized by antibodies with antibody-dependent cellular cytotoxicity activities (31), and contains epitopes recognized by helper T lymphocytes (1). Furthermore, the env sequence can protect rats and rabbits against a viral challenge with infected cells (47). In the present work, we show that two HLA-A2 strong binder peptides (env and env ) are located in this domain. Taken together with the previous findings, our results lead us to propose that the domain located between residues 175 and 200 of the SU protein represents a very good candidate for the development of an anti-htlv-1 vaccine able to induce both a humoral response, including the production of neutralizing antibodies, and a CTL response, each response being probably required for a longterm protection. ACKNOWLEDGMENTS We thank Yann Lécluse for cell sorter analysis. We are grateful to Linda Pritchard and Arielle Rosenberg for helpful suggestions and critical reading of the manuscript. This work was supported by the Agence Nationale de Recherche contre le SIDA (Paris, France). REFERENCES 1. Baba, E., M. Nakamura, K. Ohkuma, J. Kira, Y. Tanaka, S. Nakano, and Y. 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