T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein

Microbes and Infection 7 (2005) 1184–1195 www.elsevier.com/locate/micinf

Original article

T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein Lúcia C.J. Abel a, Leo K. Iwai a,d,e, Wladia Viviani h, Angelina M. Bilate a,d, Kellen C. Faé a,d, Renata C. Ferreira a,d, Anna C. Goldberg a,d,1, Luiz Juliano e, Maria A. Juliano e, Bárbara Ianni b, Charles Mady b, Arthur Gruber g, Juergen Hammer f,2, Francesco Sinigaglia f, Jorge Kalil a,c,d, Edecio Cunha-Neto a,c,d,* a

Laboratory of Immunology, Heart Institute (InCor), University of São Paulo School of Medicine, Av. Dr. Enéas de Carvalho Aguiar, 44, Bloco II, 9° andar, São Paulo, SP 05403-000, Brazil b General Cardiopathies Division, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, SP 05403-000, Brazil c Division of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, SP 05403-000, Brazil d Institute for Investigation in Immunology, Millennium Institutes, Brazil e Department of Biophysics, Federal University of São Paulo/UNIFESP, São Paulo, Brazil f Bioxell, Milan, Italy g Department of Pathology, Faculty of Veterinary Medicine and Zoothechny, University of São Paulo, São Paulo, Brazil h Department of Biochemistry, Chemistry Institute, University of São Paulo, São Paulo, Brazil Received 11 January 2005; accepted 29 March 2005 Available online 12 May 2005

Abstract Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan parasites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas’ disease patients. Little is known about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on T cell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein by T cell lines, clones and PBMC from Chagas’ disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected individuals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7), -DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope [FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Y strain B13 protein; the underlined small residues GQA were the major HLA contact residues. Among natural B13 15-mer variant peptides, molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolished recognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas’ disease patients, S15.4 was preferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterization of T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoan repetitive antigens. © 2005 Elsevier SAS. All rights reserved. Keywords: T cell epitope; B13 protein; Trypanosoma cruzi; Tandemly repetitive proteins; Immunodominant antigens

Abbreviations: ASY, asymptomatic T. cruzi infected individuals; CCC, chronic Chagas’ disease cardiomyopathy; CLIP class II-associated Ii peptide; CPM, counts per minute; EBV, Epstein–Barr virus; ECG, electrocardiogram; HA, hemmagglutinin; LPS, lipopolysaccharide; N or CTRL, non-infected control individuals; PBMC, peripheral blood mononuclear cells; PHA, phytohaemagglutinin; SI, stimulation index. * Corresponding author. Tel.: +55 11 3069 5906/3069 5914; fax: +55 11 3069 5953. E-mail address: [email protected] (E. Cunha-Neto). 1 Department of Biochemistry, Chemistry Institute, University of São Paulo, São Paulo, Brazil 2 Department of Genomic and Information Sciences, Hoffmann-La Roche Inc., Nutley, NJ, USA 1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.03.033

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1. Introduction Proteins containing regions of tandemly repetitive amino acid sequences are present in several immunodominant protein antigens from pathogenic protozoa, including the agents of trypanosomiasis (Trypanosoma cruzi), malaria (Plasmodium spp.), toxoplasmosis (Toxoplasma gondii) and leishmaniasis (Leishmania spp.) [1–3]. The function of these regions is still poorly understood. It has been suggested that they are involved in binding to repetitive structures within the parasite and to host cell receptors. Also, repetitiveness may have defined physico-chemical or structural functions, as seen in the case of collagen, myosin, heat shock proteins or glue proteins [4–6]. Despite the diversity in size, number and distribution of the repetitive elements, several distinctive features are held in common: the property of repetitiveness itself, the bias in the component amino acids (hydrophobic residues rarely present), an unusual genetic and evolutionary history, and their immunodominance [1]. Little is known about the molecular mechanisms that lead to the immunodominance of repeated sequences, except for their multivalency which can make them activate B cells directly and behave, in some cases, as T-independent antigens [1]. It is known that the repetitiveness of a variety of agents causes T cell independent activation of B cells by crosslinking hapten-specific surface immunoglobulin [7]. There is much evidence that epitope conformation can be relevant for antigen–antibody binding [8,9], and antibodies against native protein epitopes preferentially recognize peptide sequences with conformational preferences [10]. Together, these reports suggested that structural features—which may be present in repetitive epitopes—might play a critical role in protein immunodominance. Several T. cruzi antigens contain tandemly repeated amino acid motifs. The repetitive units already described in T. cruzi have a variety of different sizes and may vary from 5 to 68 amino acids [11]. B13 is an immunodominant T. cruzi antigen recognized by IgG serum antibodies from 98% of T. cruzi infected individuals (both chronic Chagas’ disease cardiomyopathy, CCC, and asymptomatic/indeterminate (ASY)) in Latin America and encodes a partially degenerate tandemly repetitive 12-amino acid motif present in a 140 kDa membrane protein [12]. In has been shown that the multivalent protein epitopes of T. cruzi B13 antigen [13] and Plasmodium falciparum CS protein [14], display a similar conformation in each repetitive unit, leading authors to hypothesize that, at least in those cases, immunodominance was linked to their ability to be recognized by a single type of conformation-dependent antibody. Authors have speculated that amino acid sequences with conformational preferences and increased antigenic potential may have been selected for in other immunodominant tandemly repeated antigenic domains of protozoan proteins. Even though evidence from some systems supports that antibodies towards repetitive protozoan antigens may be T cell-independent [1], it cannot be excluded that, even in these

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cases, T cell help could boost antibody titers and affinity. Repetitive sequences in protozoan parasite antigens have also been shown to elicit T cell dependent immune responses. It has been reported that peripheral T cells from P. falciparum malaria patients recognize T cell epitopes in EB200, a repetitive region of the P. falciparum antigen Pf332 [15,16], the Pf155 repetitive domain of ring-infected erythrocyte surface antigen (RESA) [17] and the repetitive domain of circumsporozoite CS protein [18]. However, so far, there is no information on fine characterization of T cell epitopes in protozoan tandemly repetitive antigens. In order to study whether T cells from Chagas’ disease patients recognized immunodominant B13 protein, we analyzed the PBMC response to B13 from Chagas’ disease patients. To finely characterize the T cell recognition of the tandemly repetitive units of immunodominant B13 protein, we analyzed the HLA class II binding properties of B13 peptides and HLA association with PBMC responsiveness to B13, the minimal T cell epipope and the HLA contact residues, and explored the putative TCR contact residues. In order to search for differential recognition of variant B13 epitopes, we analyzed the proliferative responses of PBMC from CCC, asymptomatic T. cruzi infected (ASY) and non-infected control individuals (N), as well as a B13 epitope-specific T cell clone, against synthetic peptides encoding naturally occurring Y strain T. cruzi B13 variant sequence peptides. Our results identified frequent MHC-restricted T cell recognition of T cell epitopes from the repetitive domains in B13 protein.

2. Methods 2.1. Patients, materials and methods Heparinized venous blood was obtained from Chagas’ disease patients followed at the Heart Institute (InCor), University of São Paulo School of Medicine. Chagas’ cardiomyopathy patients (CCC) fulfilled the following diagnostic criteria: positive serology for T. cruzi, typical ECG abnormalities (left anterior hemiblock and/or right bundle branch hemiblock), varying degrees of ventricular dysfunction, with all other causes of ventricular dysfunction/heart failure excluded. Blood samples were also collected from asymptomatic “indeterminate” individuals (ASY), seropositive to T. cruzi, with normal ECG and bidimensional echocardiography. Peripheral blood samples were also collected from T. cruzi seronegative, age and sex-matched normal volunteers (N) as a control group. Sample collection procedures have been cleared by the Internal Review Board of the University of São Paulo School of Medicine. 2.2. B13 protein Recombinant T. cruzi B13 protein was obtained after expression of a recombinant clone obtained from a T. cruzi Y strain expression library screened with sera from chronic Cha-

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Table 1 Binding of synthetic B13 peptides scanning two 12-mer B13 tandemly repetitive units to different HLA class II molecules

gas’ disease patients [12]. The full amino acid sequence of the B13 insert in recombinant pMSgt11 plasmid is shown in Table 1 (GenBank accession number AY325808). The expressed b−galactosidase–B13 fusion protein was purified on p-aminophenyl-b-galactopyranoside agarose columns. B13 recombinant protein contains 19 tandemly repeated 12-amino acid motifs. Non-recombinant b-galactosidase (the fusion protein support) expressed from intact pMSgt11 plasmid was processed similarly and used in some experiments as a negative control. Endotoxin-depleted B13 protein obtained by treatment with Polimyxin B-Sepharose 4B (Sigma Chemical Co., St. Louis, MO, USA) was added to selected wells in control experiments [19]. 2.3. Synthetic peptides 13-mer overlapping synthetic peptides encompassing the whole 12-residue B13 motif including some sequence variants [12,20,21], as well as 10, 15-mer peptides containing the variant B13 sequences and centered HLA-DQ7-binding frames, from B13 protein (Genbank accession number AY325808.1) were obtained with Fmoc solid-phase chemistry [22] using multiple peptide synthesizers from Advanced ChemTech (model 396, Louisville, KY, USA) and Shimadzu (model PSSM-8, Tokyo, Japan). Overlapping 15-mer B13 peptide mixtures containing degenerate variant positions, were obtained with combinatorial chemistry by the addition of two or three fmoc-derivatized amino acid residues at appropriate steps during automated peptide synthesis. The indicator peptides GFKA7, GYRA2YA4, IAYDA5, UD4, HA, CLIP 89-101 (class II-associated Ii peptide) and TT 830-843 [23] were N-terminally biotinylated before cleaving them from resin by sequentially coupling two 6-aminocaproic acid spacers on the

N-terminus and one biotin molecule, using the abovedescribed standard procedure. Peptides were analyzed by MALDI-TOF mass spectrometry (Tof-Spec E, Micromass, Manchester, UK) and by analytical reverse-phase HPLC (Shimadzu, Tokyo, Japan) and were routinely > 80% pure. 2.4. Binding of peptides to HLA class II molecules MHC class II molecules were used from the following human HLA-homozygous lymphoblastoid cells: DR1 (DRB*0101) from HOM-2; DR2 (DRB1* 1501 + DRB5*0101), DR3 (DRB1*1701) from WT49; DR4 (DRB1*0401) from BSM; DR5 (DRB1*1101) from SWEIG; DR7 (DRB1*0701) from EKR and DR8 (DRB1*0801) from BM9. DR2 from transfected cells (DRB1*1501) was isolated from the transfectant cell line L466.1, a kind gift form Dr. R.W. Karr (Monsanto, St Louis, MO, USA). HLA-DR and -DQ Molecules were affinity-purified as described [24,25]. HLA-DQ7 (DQA1*0501/DQB1*0301) was purified from SWEIG lymphoblastoid cells using monoclonal antibody SPV-L3 [25]. B13 peptide-binding assays were performed with HLA molecules in high throughput competitive labeled-peptide ELISA-based assays [23]. Briefly, serially diluted peptides were incubated in the presence of N-terminal biotinylated indicator peptides as described and affinitypurified MHC class II molecules (100 ng protein) in a protease inhibitor cocktail, and transferred to transferred to an ELISA plate coated with monoclonal antibodies antiHLA-DQ SPV-L3 or anti-HLA-DR L243 (ATCC HB55), incubated with streptavidin-alkaline phosphatase, and 4-nitrophenylphosphate. Data are scored as the concentration of test peptide capable of inhibiting 50% of the binding of labeled-peptide to that HLA class II molecule (IC50%).

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Identification of B13 peptide contact residues to DQ7 (DQA1*0501/DQB1*0301) was performed using single lysine substitutions or double valine and histidine substitutions because they lack non-specific inhibitory effects on binding to HLA-DQ7 [23]. 2.5. HLA class II typing DNA was extracted alternatively by DTAB/CTAB or salting out methods [26]. DR typing was performed by low resolution PCR-SSP [27] as previously described [28]. DQA1 and DQB1 typing were performed by PCR-SSO using generic primers for exon-2 amplification [26]. 2.6. Peripheral blood mononuclear cell (PBMC) proliferation assays Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood by Ficoll–Hypaque density gradient centrifugation (d = 1.077), washed and incubated in Dulbecco’s modified Eagle’s medium (DMEM-GIBCO, Grand Island, NY, USA) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamicin, 10 mM HEPES buffer and 10% normal human serum (complete medium). In the proliferation assay, cell cultures from CCC, ASY and N individuals were carried out in triplicate in 96-well-flatbottom culture plates (105 cells per well; final volume 0.2 ml) with B13 protein (5 µg/ml) or B13 15-mer peptides (25 µM); phytohemagglutinin (PHA, 5 µg/ml; Sigma) was used as positive control, and complete medium as a negative control. b-galactosidase or endotoxin-depleted B13 protein was added to selected wells. Ideal concentrations for PBMC proliferation assays were previously established from dose-response curves for recombinant B13 protein [29] and 15-mer B13 peptides [30]. Plates were incubated in 5% CO2 at 37 °C for 5 days and cultures were pulsed with 1 µCi per well [3H]-thymidine (Amersham, Buckinghamshire, UK) for the final 18 h. [3H]-thymidine incorporation was measured at the Betaplate beta counter (Wallac Inc, Turku, Finland). Data are represented as the stimulation index (SI) defined as mean CPM experimental triplicates with antigen/mean CPM of triplicates of culture medium control. SI values ≥ 2.0 were considered positive. 2.7. Antigen-specific T cell lines and clones B13- and B13 peptide-specific T cell lines and clones were established from the peripheral blood of HLA-DQ7+ individuals (with a previous positive PBMC proliferative response to B13), by the addition of B13 protein (5 µg/ml) or B13 peptides (25 µg/ml) and incubation in complete medium containing IL-2 essentially as described [25]. The lines were expanded by re-stimulation every 15 days with phytohemagglutinin (PHA, 5 µg/ml), 40 U/ml IL-2 and irradiated (5000 rads) PBMC (106 well) and subjected to proliferation assays 10 days after the last PHA stimulus. Peptide-driven T

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cell lines were also cloned by limiting dilution essentially as described [31], with the addition of IL-7 and IL-15 (5 ng/ml) in the expansion phase. Proliferation assays of peptidedriven T cell line and clone were carried out incubating 3 × 104 T cell line and 105 irradiated HLA-DQ7 allogenic APC/well in triplicate in a 96-well-U-bottom culture plates (final volume 0.2 ml) with B13 protein (5 µg/ml) or B13 15-mer peptides (25 µM) and PHA at 5 µg/ml as a positive control (Sigma Chemical Co., St Louis, MO, USA). Data are represented as stimulation index (SI) defined above. SI values ≥ 2.0 were considered positive. 2.8. Mapping of the minimal B13 T cell epitope with combinatorial peptide libraries As an approach to mapping the T cell epitope in the degenerate 12-mer B13 repeats, we synthesized twelve 15-mer peptides overlapping with a 1-residue step and tested their recognition by a B13-specific T cell line. To allow for representation of all sequence variants reported in B13 and B13-like sequences [12,20,21], each 15-mer peptide was made degenerate in the variant positions, which was obtained, with combinatorial chemistry, by the addition of two or three fmocderivatized amino acid residues at appropriate steps during automated peptide synthesis [32]. Each 15-mer B13 mixturebased peptide library had 7–10 degenerate positions, with 288–5184 different sequences. Such “complex” mixture peptide libraries were diluted in culture medium, filtered and used at 250 µM total concentration; the concentration for each individual sequence was 0.05–1.00 µM in different mixtures. Fifteen-mer overlapping restricted combinatorial peptide libraries including only the variant sequences which were commonly encountered in all B13 and B13-like sequences [12,20,21] were also synthesized, and each “common” 15-mer B13 restricted combinatorial peptide library had 8–32 different sequences. “Common” restricted combinatorial peptide libraries were used at 20 µM total concentration, and the concentration for each individual sequence was 0.6–2.4 µM in different mixtures. Peptides were preincubated for 2 h with the irradiated (10,000 rad) EBV-B cell line SWEIG, which is homozygous to HLA-DQA1*0501/DQB1*0301. The B13driven T cell line from HLA-DQ7+ CCC patient SMC was incubated for 96 h in 5% CO2 at 37 °C, in triplicates of a flat-bottom 96 well culture plate, irradiated SWEIG EBV cells pulsed with the two series of 15-mer overlapping peptide libraries, and cultures were pulsed with 1 µCi per well [3H]-thymidine (Amersham, Buckinghamshire, UK) for the final 18 h. [3H]-thymidine incorporation was measured with the Betaplate beta counter (Wallac Inc, Turku, Finland). 2.9. Molecular modeling of the HLA-B13 peptide complexes Model building, visualization and quantitative study by Molecular Mechanics and Dynamics were carried out in an IBM workstation RS6000- 43P, using the INSIGHT-II pack-

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age (MS Inc., San Diego, USA). All potential energy evaluations were performed using CVFF forcefield, considering a pH=7.2 and a dielectric constant e = 80 in order to simulate implicitly the aqueous surroundings. Potential energy minimizations were carried until the energy derivative value was up to dE = 0.05 kcal/mol*A. The primary sequence of HLADQA1*0501/DQB1*0301 (DQ7) was manually aligned to the HLA-DR1 sequence and used for homology building (1DLH [33]). The resulting model was energy minimized and directly used for complexation of the 13-mer B13 peptide used for HLA-DQ7 binding assay: SPFGQAAAGDKPS. The docking step consisted in transposing the HLA-DR1complexed HA peptide main chain coordinates to the B13 peptide, since both are expected to have a polyprolinelike helix conformation. The template mouse hemoglobin Hb(64–76):Lys9 peptidic residue [34] was used as an unique reference point for B13 alignment, since the latter Lys11 residue can be expected to form a salt bridge with the HLA conserved aspartic residue: HLA-DQ7b:Asp55, corresponding to the pair Hb:Lys9/DR1b:Asp57. Therefore, the B13 model was fitted into the HLA-DQ7 model groove with an orientation analogue to the one exhibited by the template complex. The resulting HLA-DQ7-B13 peptide complex was then refined by a T = 400 K, t = 350 ps. Molecular dynamics simulation (including 50 ps for thermal equilibration), so that the overall structure and namely peptidic side chains and protein receptacle were allowed to mutually adapt. The mean structure assumed by the complex during this step was the one submitted to a simulated annealing experiment: starting at 300 K, lowering to 100 K, and then to 50 K, staying for a total of 65 ps (including 15 ps for thermal equilibration) at each temperature. The final structure was then energy optimized, and constitutes the refined HLA-DQ7/B13 peptide model. Models for each of the 10, 15-mer B13 peptide variants could be built with basis on the above-described HLADQ7/13-mer antigen model, by adding two residues to the original peptide and substituting, where needed, the original residues according to each variant sequence. New structures were then allowed to adjust by geometry optimization, considering at first a semi-rigid supermolecule where only the peptide substituted side chains were unfrozen, and therefore able to relax; then relaxing all peptide atoms, still keeping all protein atoms frozen; finally, submitting the fully relaxed complex structure to a molecular dynamics plus Simulated Annealing refinement. All these steps have been carried following the same protocol as reported above for the 13-mer peptide complex.

2.10. Data analysis Mann–Whitney’s rank sum test was used to compare continuous variables. Fisher’s exact test was used to compare frequencies of responder and non-responder individuals in T cell proliferation assays.

3. Results 3.1. T cell response against B13 protein: HLA restriction We found that PBMC proliferation to recombinant protein B13 was similar in frequency and intensity between the CCC and ASY groups (56% and 43% responders, respectively) and, surprisingly, even among the normal control individuals (60%). Average stimulation indexes for CCC, ASY and N groups were 4.8 ± 6.2, 3.1 ± 5.1 and 3.8 ± 4.4, respectively. PBMC from N individuals that responded to B13 protein failed to respond to b-galactosidase, the fusion protein support, and LPS depletion of recombinant B13 with immobilized polimyxin B failed to decrease B13-induced proliferation among N PBMC, ruling out the recognition of the support protein or contaminating bacterial products (data not shown) [28]. Table 1 shows that overlapping 13-mer B13-derived peptides scanning one 12-mer repetitive unit can bind, albeit with low affinity, to HLA-DQ7 (DQA1*0501/DQB1*0301) and HLA-DR1 (DRB1*0101) molecules. Peptides that shared the KP(S/P)(L/P)FGQAAG sequence also showed low affinity but significant binding to EBV-derived DR2b (DRB1*1501/ DRB5*0101, but not to DR2a (DRB1*1501) derived from transfectant cell L466.1, indicating that DR51 (DRB5*0101) may be able to bind B13 peptides. The specificity of binding to HLA-DQA1*0501/DQB1*0301 could be observed by the lack of detectable binding to DR5 (DRB1*1101) present in the same lymphoblastoid cell line from which the DQ7 molecule was extracted (SWEIG). Several different 13-mer peptides starting at different positions of the B13 tandem repeat can bind to HLA molecules, with the single exception of the peptide QAAAGDKPSLFGQ. It should also be mentioned that peptides in the third to fifth row, representing the common sequence variants of B13 [12,20,21], along with other tested variants (data not shown) bind with similar affinity to the HLA molecules. The full sequence of the B13 protein insert with its 19 partially degenerate tandem repeat units is also shown. Analyzing the HLA profile of individuals showing PBMC proliferative responses to B13 protein (Fig. 1), we observed that 84% of B13 responders carry at least one of the DR1, DR2, or DQA1*0501 B13-binding HLA alleles shown in Table 1, a statistically significant difference when compared to 65% among B13 non-responders (P = 0.048). Taken individually, DQA1*0501 was also significantly more represented among B13 responders than non-responders (DQA1*0501: 53% vs. 28%, P = 0.03); among Chagas’ disease patients, the difference in frequencies of DQA1*0501 among responders and non-responders was even more prominent (55% vs. 15%, P = 0.001), confirming its major role in T cell presentation of B13 epitopes (Fig. 1). 3.2. Mapping of the minimal DQ7-restricted B13 T cell epitope HLA DQA1*0501 is the most frequent B13-binding HLA allele in the general population—half of the times paired to

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Fig. 1. Proliferative response (105 cells per well) to B13 protein (5 µg/ml) in CCC (n = 27), ASY (n = 41) and normal control individuals (n = 23) sorted according to the expression of B13-binding HLA class II alleles DQA1*0501(DQB1*0301 or DQB1*0201), DR1/DR2, or none of them. Each point represents the SI value of a single individual. A few individuals carried both DQA1*0501 and DR1 or DR2 and were therefore depicted as points in both sections of the graph.

DQB1*0301, as stated above. Given the availability of the HLA-DQA1*0501/DQB1*0301 (HLA-DQ7) molecule for peptide-binding assays, we studied the minimal epitope recognized in the context of HLA-DQ7. Table 2 shows the proliferative response of B13-specific T cell line from HLADQ7+ CCC patient SMC to the two series of 15-mer overlapping library peptides degenerate at variant positions, one representing all possible variant positions (peptide librarTable 2 Proliferative response of anti-B13 T cell line SMC to overlapping 15-mer mixture peptides presented by HLA-DQ7+ EBV line SWEIG

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ies 1–12) and the second one only representing variants common to all isolates (peptide libraries 13–24), presented by homozygous EBV-B line SWEIG (DRB1*1101, DQA1*0501/DQB1*0301). The anti-B13 T cell line SMC recognized all 15-mer “complex” peptide libraries sharing the 10-mer sequence FGQAA(A/E)(G/A)(D/E)(K/R)(P/L), with an average stimulation index of 6.6 ± 0.3; negative peptides showed an average stimulation index of 1.1 ± 0.3 (Table 2). All 15-mer “common” peptide libraries including the 10-mer FGQAAAG(D/E)KP sequence, contained in the recognized “complex” peptide libraries, were recognized with an average stimulation index of 8.0 ± 0.1; negative peptides showed an average stimulation index of 1.4 ± 0.3 (Table 2). All 15-mer peptide libraries bound detectably to HLA-DQ7 (2–40 µM IC50% range, data not shown). B13 peptide-pulsed EBV-B lines WT-49 (DRB1*0301, DQA1*0501/DQB1*0201) and PRIESS (DRB1*0401, DQA1*0301/DQB1*0302) failed to stimulate anti-B13 T cell line SMC (data not shown), thus confirming the HLA restriction of the response. Results indicated that the HLA-DQ7 restricted minimal T cell epitope in B13 (FGQAAAG(D/E)KP) was a common sequence, present in 11 out of the 19 sequenced repetitive elements present in Y strain B13 protein as in Table 1. In the following experiments, we numbered B13 peptide sequences according to the minimal epitope, with the first residue (F) appearing as residue F1, and the C-terminal (P) as P10. For longer peptides residues extending from the N-terminal end had negative numbers. 3.3. Identification of critical HLA-DQ7 contact residues in B13 peptides Lysine-substituted analogues of B13 peptide SPFGQAAAGDKPS, comprised between residues S(-2) and S11, and containing the minimal DQ7-restricted B13 T cell epitope, were subjected to binding assays to HLA-DQ7. Table 3A indicates that lysine substitution of residues F1, G2 and A4 abolish peptide-binding to HLA-DQ7(SPFGQAAAGDKPS), while substituting residue A5 partially inhibits peptidebinding. In order to confirm these findings, we tested the binding of a B13 peptide in a different frame, comprised between D(-5) and D8 (DKPSPFGQAAAGD) and its valine-histidine double-substituted (VxH) analogues to HLA-DQ7. Table 3B shows that the double substitution of residues G2 and A4 (DKPSPFGQAAAGD) completely abolished binding. Further in support of this finding, Table 1 shows that the only peptide which failed to bind to HLA-DQ7 was the peptide comprised between Q(-10) and Q3, QAAAGDKPSLFGQ, devoid of the G2-Q3-A4 sequence. Together, data support the notion that G2 and A4 are the small-side chain major contact residues to HLA-DQ7, positioned at relative positions i and i + 2, in agreement with previously published data showing that DQ7-binding residues possess small-side chains and are located in the central part of an epitope [23], with a minor contribution for HLA-binding from residues F1 and A5.

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Table 3 HLA-DQ7 binding assay of substituted analogues of B13 peptides for the identification of HLA-DQ7 binding residues in B13. A. Single (lysine)substituted analogues and B. Double-(valine_histidine) substituted analogues

found 26 distinct combinations of the 10 B13-derived peptides recognized by single individuals (Table 4). Analyzing peptide reactivity according to clinical groups, we observed that peptide S15.9 was the most frequently recognized by CCC and ASY patients (71% and 63%, respectively), and had the highest average stimulation indexes among Chagas’ disease patients (4.6 and 3.5 by CCC and ASY, respectively) but was not recognized by normal individuals. Peptide S15.7 was the most frequently recognized among B13responsive, HLA-DQ7-positive normal individuals (55%). Peptide S15.4 (KPPPFGQAAAGDKPP) was frequently recognized by CCC patients, and threefold less recognized by ASY (64% vs. 18%, P = 0.07). Peptides S15.1 and S15.3 were the only ones more frequently recognized by ASY (27% and 46%) than CCC patients (9% and 18%) (P = 0.6 and P = 0.3, respectively) (Table 4 and Fig. 3). 3.5. Recognition of B13 variant peptides by S15.4-specific T cell clone

3.4. PBMC recognition of 15-mer natural variant Y strain T. cruzi B13 peptides among clinical groups Native 13-mer B13 peptides used in binding assays (Table 3A) or the 12-mer PFGQAAAGDKPS containing the minimal epitope were poor stimulants in PBMC proliferation assays, inducing responses in less than 15% of HLADQ7+ individuals that responded to recombinant B13 protein (data not shown). The mentioned peptides had the G2-Q3A4 binding cassette off-center, and lacked N-terminal flanking residues that could be important for T cell recognition [35]. Furthermore, as the 12-mer tandemly repetitive unit of B13 protein occurs in 19 imperfect copies, we synthesized 10 singular 15-mer B13 peptides (Fig. 2C), including a G2-Q3-A4 DQ7-binding motif positioned in the central region of the peptide and encompassing all natural sequence variants found in recombinant B13 protein from T. cruzi Y strain (see Table 1). We tested the recognition of the 10 singular B13-derived peptides by PBMC from HLA-DQ7 positive B13-responsive, HLA-DQ7-positive CCC and ASY patients, and normal controls (Table 4). Of the 31 B13-responsive individuals tested, 90% responded to at least one B13 peptide. The proliferative response to B13 peptides in most individuals in the CCC and N groups was similar in magnitude to that induced by recombinant B13 protein, while among ASY patients the response to B13 peptides used to be much lower than that directed to B13 protein. Similarly, CCC patients and N individuals recognized a significantly higher number of peptides than ASY patients (average of 3.2, 3.1 and 1.5 peptides/individual, respectively; CCC vs. ASY or N vs. ASY, P < 0.05). Profiles of peptide recognition varied significantly: out of 28 individuals recognizing any peptide, we

Proliferation assays with S15.4-specific T cell clone 3E5 against the other 15-mer B13 protein variant peptides showed that, at least for this T cell clone, positions p-3 to p-1, p7 and p11 maybe important HLA/TCR contact residues as amino acid substitutions in these positions abolished the recognition (Table 5). 3.6. Molecular modeling of the B13 peptide: HLA-DQ7 complex Molecular modeling of complexes between HLA-DQ7 and B13 peptides were performed in order to compare structural and functional (i.e. peptide-binding assays and T cell recognition) data. Molecular modeling of the 13-mer B13 peptide SPFGQAAAGDKPS, subject to the lysine substitutionDQ7 binding assay analysis, complexed to HLA-DQ7 showed that the region F1 to K9 fits into the peptide-binding groove. Side chains of residues at relative positions F1, G2, A4, A5, D8 fit into pockets in the antigen-binding groove, either at the a-helical portion (F1) or to its floor; G2 fits a shallower pocket allowing direct contact of the peptide main chain to the groove. It is of note that molecular modeling confirmed all four major and minor positions of HLA contact residues (F1, G2, A4, A5) identified by the lysine-substituted B13 peptide-binding assay. Interestingly, both major and minor HLA contact residues are conserved among all 19 sequenced repetitive elements present in Y strain B13 protein (Table 1). Molecular modeling of a complex between the B13 variant peptide S15.4 comprised between residues K(-4) and P11 (KPPPFGQAAAGDKPP) and HLA-DQ7 showed again that the region F1 to K9 fits into the peptide-binding groove (top view of peptide:HLA-DQ7 model, Fig. 2A). Solvent-exposed residues rendered in green were F1, G2, A6, K9, P11. The flanking N-terminal region [P(-2) to K(-4)] lies far from the peptide-binding groove, and the resulting high mobility precludes the attribution of a single energetically favorable side

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Fig. 2. Molecular modeling of B13 peptides docked to the antigen-binding groove of the HLA-DQ7 molecule after homology building with the HLA-DR1:HA peptide complex (1DLH [33]) as described in Section 2. A. Top view of complex between HLA-DQ7 and 15-mer B13 peptide S15.4. B13 peptide (white) and DQ7 molecule (blue) rendered as Van der Waals contours, peptide main chain without hydrogens displayed as red sticks, F1, Q3, A6, K9, P11 and A8 TCRexposed side chains with hydrogens, in green; putative N-terminal proline residues rendered in red with hydrogens. B. Side view of all 10 superimposed B13 variant peptides rendered as sticks on the antigen-binding groove of HLA-DQ7 molecule. Each peptide is rendered in a different color. Polymorphic positions are identified in each peptide. C. Alignment of B13 variant peptides, their TCR contact positions, and TCR contact sequence patterns.

chain configuration, but it can potentially interact with the solvent and the T cell receptor-particularly position -2 (Fig. 2A). Among the 10 natural sequence variants 15-mer B13 peptides docked to DQ7 tested above, several solventexposed (and therefore TCR-exposed) positions also show sequence variation, like positions 6 (A/E) and 11 (P/S/A), along with the apparently TCR-exposed position -2 (P/S/A) and potentially TCR-exposed position -3 (P/L) at the N-terminus region (Fig. 2A, B). Among the 10 variant B13 peptides, seven distinct six-residue solvent-exposed sequences are displayed, corresponding to each combination of polymorphic sequences at the exposed positions (Fig. 2C), with corresponding changes in the TCR-exposed side chains (Fig. 2A–C). 4. Discussion In this paper, we have finely characterized the T cell epitopes in protozoan tandemly repetitive antigen B13. We have shown that the tandemly repetitive sequences of T. cruzi B13 protein can be presented for T cell recognition in the

context of at least three distinct HLA class II molecules (Table 1 and Fig. 1), and identified the minimal T cell epitope recognized in the context of the most frequent of them, HLADQ7 (Table 2). Furthermore, we identified its HLA-DQ7 contact and putative TCR-exposed residues (Table 2 and Fig. 2), and demonstrated that variant B13 T. cruzi epitopes are differentially recognized between CCC and ASY patients (Table 4 and Fig. 3). The finding that common sequence variants of B13 protein peptides bind with similar IC50% values to HLADRB1*0101, DRB5*0101 DQA1*0501/DQB1*0301 indicates that each B13 protein unit can generate several HLAbinding epitopes. The low IC50% values observed in HLAbinding assays with peptides from tandemly repetitive B13 protein (Table 1) are in line with similar findings for the T1 peptide from the tandem repeats of P. falciparum CS protein [36] and may be related to their biased amino acid usage, favoring small and charged side chain residues [1]. The finding that such HLA alleles are significantly more represented among patients who show PBMC proliferative responses to recombinant protein B13 (Fig. 1) demonstrates that the B13-

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Table 4 Proliferative response of HLA-DQ7+ CCC, ASY and N individuals to recombinant B13 protein and variant B13 peptides

binding HLA class II molecules can indeed present B13 epitopes to T cells. A parallel study showed that PBMC from 92% of the individuals carrying the B13-binding HLA alleles presented proliferation, IFN-c or IL-4 production responses to recombinant B13 protein (data not shown). Together with the fact that 80% of individuals in the general population carry at least one of the B13-binding HLA alleles [28], results suggest that essentially all individuals carrying one of the B13-binding HLA alleles, or the majority of the population, can be taken as B13 responders. The identification of the 10-mer sequence FGQAAAG(D/E)KP as the minimal DQ7-restricted T cell epitope (Table 2), which contains the major antibody epitope FGQAAAGDK [37,38], may have implications in cognate T: B cell interactions. The identification of glycine and alanine as being the major contact residues of the B13 repetitive sequence peptides with HLA-DQ7, as reported by the substitution assays (Table 3A, B) is in line with published data for both DQ7 (DQA1*0501/DQB1*0301) [23] or DQA1*0301/ DQB1*0301 [39]. The fact that the four major and minor HLA

contact residues are conserved among the sequenced repetitive elements present in Y strain B13 protein (Table 1), indicates that B13 sequence polymorphism does not impair the ability of sequence variants to bind to HLA-DQ7. The fact that PBMC samples from 90% of B13-responsive tested individuals showed proliferative responses to at least one of the 15-mer variant B13 peptides, with magnitudes similar to those against recombinant B13 protein (Table 4) suggests that we have identified the major DQ7-restricted T cell epitopes of B13 protein. Several consistent differences were observed at the level of recognition of individual B13 peptides. The fact that two variant B13 peptide epitopes S15.9 and S15.10 (Fig. 3 and Table 4) were exclusively recognized by Chagas’ disease patients, indicate that in vivo presentation of B13 protein along infection was necessary for the appearance of the reactivities. Peptide S15.9 is probably the major “public” T cell epitope of B13 protein, since it is the most frequently recognized and with the highest stimulation indexes between both CCC and ASY patients. The fact that peptide S15.4 was recognized more frequently by CCC than ASY patients suggests

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Fig. 3. Proportion of responders to 15-mer variant B13 peptides among HLADQ7+ individuals responsive to recombinant B13 protein. A. CCC vs. ASY patients. B. Chagas’ disease vs. normal controls. Percent responders for a given peptide in a clinical group = number of individuals displaying SI ≥ 2.0/number of individuals in group.

that differential recognition of the peptides may be clinically relevant, given the demonstrated T cell cross-reactivity between B13 protein and cardiac myosin [40–42]. Recent data from our group has shown that a S15.4 specific T cell clone can cross-reactively recognize cardiac myosin epitopes [42]. One cannot exclude, however, that the differential recognition of B13 peptides is related to sequence polymorphisms of the T. cruzi B13 gene itself. The fact that the S15.4 can only be found in B13 from the Y strain, but not among available B13-like sequences from other T. cruzi strains [20,21] may suggest that selective recognition of S15.4 by CCC patients could be associated with infection by distinct T. cruzi strains bearing the respective epitopes. Similarly, one could not exclude that the low recognition by ASY patients of the B13 peptides tested here could be secondary to infection by a strain with different sequences. On the other hand, the similar PBMC proliferative responses to both recombinant

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B13 protein and B13 variant peptides S15.1 to S15.8 among Chagas’ disease patients and normal individuals (Table 4 and Fig. 3) indicates that individuals not exposed to T. cruzi are sensitized to sequences similar to B13 protein. This is in line with previous reports that non-exposed individuals may respond to antigens from different pathogens such as P. falciparum [43], Leishmania [44], along with another T. cruzi antigen [45] in primary T cell proliferation assays. In the case of B13, the possibility of its being a superantigenic effect is dismissed by the recognition of B13-derived synthetic peptides by N PBMC (Table 4 and Fig. 3). Molecular modeling and molecular dynamics of the HLADQ7:S15.4 B13 epitope complexes (Fig. 2A, B) indicated that certain solvent-exposed positions (p-1, p1, p3, p6, p9 and p11) are variant. Given the fact that amino acid variation in positions p-3 to p-1, p6, p7 and p11 (Table 5) abrogated recognition of S15.4-specific T cell clone 3E5, it is likely that amino acids at some of these positions are important T cell contact residues. Results from the B13 S15.4 peptidespecific T cell clone with lysine-substituted S15.4 peptides [42] are consistent with the observed results, indicating that residues at N-terminal region (p-3 to p-1) and at positions p6 and p11 are important T cell contact residues. The fact that 12-mer or 13-mer peptides (Table 3A) containing the minimal B13 epitope (Table 2) but lacking N-terminal flanking residues were not as efficiently recognized as the 15-mer B13 peptides (Table 4) by PBMC from HLA-DQ7+ individuals (data not shown) suggests that N-terminal flanking residues may also include important solvent-exposed TCR contacts. Assuming position p-2—not hindered by the HLADQ7 molecule—is another TCR contact, we have three polymorphic positions (p-1, p6 and p11) out of the six TCRexposed sites (p-1, p1, p3, p6, p9 and p11). In summary, we demonstrated for the first time down to structural detail that each tandemly repetitive T. cruzi B13 protein possesses several variant T cell epitopes that can be recognized in an MHC-restricted manner. Strikingly, MHCcontact residues are conserved in all repetitive regions, while solvent-exposed ones include variant positions, an ideal situation for T cell recognition by genetically heterogeneous hosts. Additionally, the increased molar concentration of individual B13 epitopes may compensate the low-avidity binding to HLA molecule. Thus, in spite of the detected lowavidity binding of B13 peptides to HLA class II molecule, we observed frequent T cell responses to B13 epitopes. However, we do not know yet whether this is a general mechanism that may be associated with antibody immunodominance of protozoan proteins or whether it was peculiar to the B13 protein. B13-derived 15-mer epitopes were shown to be antigenic and elicited peptide-specific T cells with proliferative capacity, usually associated with central T cell memory. These peptide-specific cells can hypothetically provide T cell help to B cells for antibody production, even in the case of repetitive antigens. To test the hypothesis that this is a general phenomenon, defined T cell epitopes in the repetitive regions of other immunodominant tandemly repetitive proto-

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Table 5 T cell clone 3E5 proliferation assays with B13-derived peptides

zoan antigens should be searched for, perhaps with the use of combinatorial peptide libraries.

[9]

Acknowledgments

[11]

This work has been supported by grants from São Paulo State Research Foundation (FAPESP) 94-1206-0 and 96/1440-7, Brazilian National Research Council (CNPq) 520533/97 (E.C.N.) and the Howard Hughes Medical Institute (J.K.). We thank Sandra Drigo for help in the HLA typing of critical samples.

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