HLA-A*0201 restricted CD8+ T-lymphocyte responses to malaria: identification of new Plasmodium falciparum epitopes by IFN-gamma ELISPOT

Parasite Immunology, 2000: 22: 501±514

HLA-A*0201 restricted CD8‡ T-lymphocyte responses to malaria: identi®cation of new Plasmodium falciparum epitopes by IFN-g ELISPOT  LEZ 1, 2 , KATRIN PETER 2 , FULVIO ESPOSITO 3 , ISSA NEBIE 4 , JEAN-MARIE TIERCY 5 , JOHN MARIO GONZA ANILZA BONELO 1 , 2 , MYRIAM ARE VALO-HERRERA 1, 2 , DANILA VALMORI 6 , PEDRO ROMERO 6 ,  CRATES HERRERA 1 , GIAMPIETRO CORRADIN 2 & JOSE ALEJANDRO LO  PEZ 2 SO 1

Instituto de InmunologõÂa del Valle, Universidad del Valle, Cali, Colombia, Institut de Biochimie, Universite de Lausanne, Epalinges, Switzerland, 3 Dipartimento di Biologia Moleculare, Cellulare e Animale, Universita di Camerino, Italy, 4 Centre National de Lutte contre le Paludisme, Ouagadougou, Burkina Faso, 5 Transplantation Immunology Unit, HoÃpital Cantonal Universitaire, Geneva, Switzerland 6 Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, Centre Hospitalier Universitaire Vaudoise (CHUV), Lausanne, Switzerland 2

SUMMARY The role of antigen speci®c CD8‡ T-lymphocytes in mediating protection against sporozoite-induced malaria has been well established in murine models. In humans, indirect evidence has accumulated suggesting a similar protective role for antigen-speci®c CD8‡ T-lymphocytes. Nevertheless, the low frequency of circulating speci®c cells together with the lack of sensitive methods to quantify them has hampered the direct assessment of their function. Using a combination of short-term cell culture and IFN-g ELISPOT, we studied CD8‡ T-lymphocyte responses to a panel of HLA-A*0201 binding peptides. In addition to con®rming the response to already described epitopes, we also identi®ed ®ve new CD8‡ T-lymphocyte epitopes. These epitopes are presented in pre-erythrocytic stages gene products of Plasmodium falciparum 7G8 strain and correspond to the following protein segments: circumsporozoite (CS) 64-72, 104-113, 299-308 and 403-411; liver stage antigen (LSA-1) repeat region; sporozoite surface protein 2 or thrombospondin related anonymous protein (SSP2/TRAP) 78±88 and 504±513. Four of these peptides are conserved amongst all published sequences of P. falciparum strains. We conclude that the modi®ed IFN-g ELISPOT assay is a sensitive technique to monitor antigen-speci®c CD8‡ T-lymphocyte responses in human malaria which may help in the improvement and assessment of the ef®cacy of malaria subunit vaccines. Correspondence: Jose Alejandro LoÂpez, Mater Medical Research Institute, Aubigny Place, South Brisbane 4101, Australia Received: 10 December 1999 Accepted for publication: 15 June 2000 q 2000 Blackwell Science Ltd

Keywords cytotoxic T cell, epitopes, HLA-A*0201, Plasmodium falciparum, ELISPOT INTRODUCTION Inhibition of liver infection by sporozoites or blocking the development of Plasmodium pre-erythrocytic forms (liver stage) are some of the natural strategies employed to control malaria infection. To date, sterile immunity in malaria has been induced by immunization with X-ray attenuated sporozoites in mice (Nussenzweig et al. 1967) and humans (Clyde 1990). In mice, protection against malaria challenge has also been achieved upon vaccination with proteins and peptides derived from pre-erythrocytic stages of the parasite (Khusmith et al. 1991, Migliorini et al. 1993, Wang et al. 1996, Franke et al. 1997) as well as with DNA (Doolan et al. 1996, Hoffman et al. 1994, Sedegah et al. 1994, Rodrigues et al. 1997). This protection was achieved through the activation of different immune mechanisms such as antibodies (Rodrigues et al. 1993), CD4‡ T-lymphocytes (ReÂnia et al. 1991, Migliorini et al. 1993, Wang et al. 1996) and CD8‡ T-lymphocytes (Franke et al. 1997, Weiss et al. 1988, Romero et al. 1990, Renggli et al. 1995). The CD8‡ T-lymphocytes plays an important role in the immune response against malaria infection. Immunization against a de®ned cytotoxic T-lymphocyte (CTL) epitope derived from the circumsporozoite (CS) protein of P. berghei or P. yoelii was shown to protect upon sporozoite challenge in mice (Franke et al. 1997, Renggli et al. 1995, Schneider et al. 1998). Parasite-derived antigens are 501

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recognized by CD8‡ T-lymphocytes on the surface of infected hepatocytes presented in the context of MHC class I molecules (Hoffman & Franke 1996), triggering different effector mechanisms such as: (1) direct lysis of malaria infected hepatocytes by activated CD8‡ T-lymphocytes, a mechanism which has not been fully demonstrated (Romero et al. 1989, Weiss et al. 1990); (2) inhibition of parasite growth mediated by cytokines such as IFN-g released by stimulated T-lymphocytes (Scho®eld et al. 1987, NuÈssler et al. 1993, Green et al. 1994, Mellouk et al. 1994). In fact, we demonstrated that the protective effect of CD8‡ T-lymphocytes was not dependent upon direct cytotoxic activity (Renggli et al. 1997). Instead, it appeared to be mediated by cytokines such as IFN-g (Scheller et al. 1997). In humans, the association of certain HLA class I alleles with clinical protection from malaria was initially suggested by studies in Africa (Hill et al. 1992) and different CTL epitopes have been described in P. falciparum proteins expressed in pre-erythrocytic stages (CS, Exp-1, LSA-1, Pf16, SSP2/TRAP and STARP) (Hill et al. 1994, Aidoo et al. 1995, Blum-Tirouvanziam et al. 1995, Wizel et al. 1995b, Doolan et al. 1997). Moreover, while clear speci®c CTL responses can be detected in volunteers following immunization with irradiated P. falciparum sporozoites or DNA vaccines (Doolan et al. 1997, Malik et al. 1991, Wizel et al. 1995a, Wang et al. 1998), only weak malaria speci®c CTL responses can be observed in naturally exposed individuals living in highly endemic regions (Aidoo et al. 1995, Lalvani et al. 1996, Doolan et al. 1997, Plebanski et al. 1997). The identi®cation of CD8‡ speci®c T-lymphocytes in humans naturally exposed to malaria has been achieved using cytotoxic assays (Aidoo et al. 1995, Sedegah et al. 1992, Hill et al. 1994, Blum-Tirouvanziam et al. 1995). However, activated CD8‡ T-lymphocytes can produce IFN-g (Boehm et al. 1997), a soluble factor that can stimulate the production of nitric oxide (NO) by hepatocytes thereby inhibiting parasite growth (Green et al. 1994, Klotz et al. 1995, Scheller et al. 1997). Thus, the identi®cation of peptide speci®c IFN-g secreting CD8‡ T-lymphocytes constitutes an alternative approach to de®ne antigens potentially involved in protection and this is possible with the ELISPOT assay (Czerkinsky et al. 1988). The ELISPOT has been successfully used to assess CD8‡ T-lymphocyte responses to tumours and viral infections in humans (Di Fabio et al. 1994, Scheibenbogen et al. 1997) or the response to malaria infection by CD4‡ cells (Elghazali et al. 1995). Furthermore, CD8‡ T-lymphocyte responses in the rodent malaria have been also demonstrated by this method (Miyahira et al. 1995). It allows the detection of the IFN-g production at the single cell level and permits the enumeration of antigen speci®c T-lymphocytes present at relatively low frequencies in PBL. 502

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We selected the HLA-A*0201 encoded antigen, the most widely distributed HLA-A2 allele (Krausa et al. 1995), to determine restricted CD8‡ responses in samples from nonmalaria exposed individuals and donors living in a high transmission area in Burkina Faso (West Africa). The use of IFN-g ELISPOT revealed the presence of CD8‡ T lymphocytes reactive against previously described malaria HLA-A*0201 restricted epitopes derived from CS, LSA-1, SSP-2/TRAP and STARP proteins from P. falciparum. Furthermore, it allowed the identi®cation of novel CTL epitopes. MATERIALS AND METHODS Study populations and samples Two groups of individuals expressing HLA-A*0201 were studied: (i) donors without a history of malaria exposure, living in Switzerland for more than 3 years (n ˆ 9; average age: 39´0 years 6 16´0 SD; two females and seven males). (ii) Donors living in Goundry, Burkina Faso with a high level of malaria transmission (n ˆ 17; average age: 35´1 years 6 14´6 SD; ®ve females and 12 males) (Del Nero et al. 1994, Modiano et al. 1996). Malaria transmission in Burkina Faso is seasonal with a rainfall pattern, maximal transmission occurs from July to November (Del Nero et al. 1994, Modiano et al. 1996). Samples for this study were taken in February 1998, using `vacutainer CPT' tubes (Becton Dickinson, San Jose, CA, USA), processed according to the manufacturer's instructions and the cells used either for ELISPOT assays or were placed in culture within 36 h after bleeding. No malaria parasite infection was evidenced by thick smear in donors from Burkina Faso. In order to standardize the assay, PBL (4´0 ´ 108) were obtained through leucopheresis from two individuals that had not been exposed to malaria. Cells were frozen in DMEM with 10 mM HEPES, 1% nonessential amino acids and penicillin 100 U/ml and streptomycin 100 mg/ml (Life Technologies, Switzerland), here called complete medium (CM), supplemented with 10% AB human serum and 10% DMSO (Sigma, St Louis, MO, USA). Expression of the HLA-A*0201 class I molecules was evaluated with an initial screening by ¯ow cytometry using the speci®c monoclonal antibody BB7´2 and con®rmed by A2 group-speci®c polymerase chain reaction followed by hybridization with a panel of 15 sequence-speci®c oligonucleotide probes (level of resolution: A*0201-A*0221) (Tiercy et al. 1994). Peptides Proteins expressed in the pre-erythrocytic stages of P. falciparum were screened for the presence of the q 2000 Blackwell Science Ltd, Parasite Immunology, 22, 501±514

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peptide-binding motif to the HLA-A*0201 class I molecule (Falk et al. 1991). Using the Swissprot protein data bank, we selected a total of 18 peptides from the following proteins: circumsporozoite protein `CS', from the malaria strain 7G8 (Dame et al. 1984) and NF54 (De la Cruz et al. 1987); liver stage antigen 1 `LSA-1' (Zhu & Hollingdale 1991), sporozoite threonine and asparagine rich protein `STARP' (Fidock et al. 1994); sporozoite surface antigen 2 or thrombospondin related anonymous protein `SSP2/TRAP' (Robson et al. 1988) (Table 1). The sequence 104-113 from CS in the 7G8 strain differs from that in the NF54 strain at position 1 (K/T). The sequence 334±342 from the 7G8 strain, corresponding to 327±335 in the NF54 strain, differs in positions 1(K/Y), 3 (K/N), 6 (K/Q) and 9 (I/L) (Dame et al. 1984, de la Cruz et al. 1987, Lockyer & Schwarz 1987). Additionally, we synthesized a peptide corresponding to the 7G8 CS 334±342 sequence with a substitution at position 9 (I/L) (Table 1). We also studied a previously reported HLA-A*0202 restricted epitope from STARP protein (Aidoo et al. 1995). A well characterized HLA-A*0201 restricted CTL epitope derived from the in¯uenza virus matrix protein 55±68 `Flu MP' (Bednarek et al. 1991) was selected as positive control and an H-2 Kd CTL restricted epitope from the P. yoelii CS protein 280± 288 was used as a negative control (Rodrigues et al. 1991). Peptides were synthesized by solid phase Fmoc chemistry and the quality was controlled by HPLC and mass spectrometry. Stock solutions of peptides in DMSO (10±20 mg/ml) were stored at 20 8C. Peptide binding assay and HLA-A*0201-peptide complex stability Binding of peptides to the HLA-A*0201 class I molecules was evaluated using the HLA-A*0201 expression assay in the TAP de®cient T2 cell line. T2 cells were incubated overnight at room temperature in serum free medium (XVIVO 10 medium, Biowhittaker, Walkersville, MD, USA) containing 1 mg/ml b2 microglobulin (Sigma) and various concentrations of peptide (starting from 10 mg/ml). After washing, cells were incubated with FITC labelled BB7´2 mAb (10 mg/ml) for 30 min at 48C and ¯uorescence was measured by ¯ow cytometry (FACScan II, Becton Dickinson). The concentration of peptide required to obtain 50% of maximal increase in HLA-A*0201 antigen expression was determined from titration curves and normalized to that of the reference Flu MP 58-66 peptide (Valmori et al. 1998). To determine the stability of peptide/HLA-A*0201 complexes, T2 cells were incubated with saturating concentrations of different peptides (10 mM) and b2 microglobulin (1 mg/ml) in X-VIVO 10 medium overnight at room temperature. After peptide removal and the addition of 0´1 mM q 2000 Blackwell Science Ltd, Parasite Immunology, 22, 501±514

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emetine (Sigma) to block protein synthesis, cells were incubated at 37 8C for the indicated time periods (maximal 6 h). For each time point, an aliquot of cells was stained with mAb BB7´2. Thus, the decay of peptide-MHC complexes was measured and peptide half-life (h) was calculated using ®rst order kinetics. Enrichment of PBL for CD8‡ T lymphocytes CD8‡ T-lymphocytes from every PBL sample were positively selected by magnetic cell sorting (MiniMacs System, Miltneyi Biotec, Bergisch, Germany). Brie¯y, cells were incubated with 20 ml anti-CD8 magnetic beads in buffer PBS pH 7´4, EDTA 5 mM, and 1% BSA. The cell suspension was passed through a column ®xed in a magnetic ®eld, and the ¯ow-through cells were collected. To elute the CD8‡ population, 1 ml buffer was added after the column was removed from the magnetic ®eld. Purity of the cell population was assessed by ¯ow cytometry using antihuman CD4 and CD8 monoclonal antibodies (Becton Dickinson). The purity of the CD8‡ cell fractions ranged between 89±95% (data not shown). In-vitro stimulation with peptide Autologous peptide-pulsed PBL were used as stimulators. Brie¯y, cells in serum free medium containing 1 mg/ml of b2 microglobulin were g-irradiated (3000 rads) and incubated with either malaria or viral control peptides (1 mg/ml) in 48 well plates at a density of 1 ´ 106 cells/well (Costar, Cambridge, MA, USA) during 2 h at room temperature. After washing the peptide pulsed PBL, CD8‡ enriched T-lymphocytes were added (1 ´ 105) to each well. Cultures were incubated during 10 days at 37 8C, and 5% CO2 in CM containing 10% AB human serum and 10 ng/ml human recombinant IL-7 (a kind gift from Dr N. Vita, Sano® Research, France). Human recombinant IL-2 (Glaxo, Geneva, Switzerland) was added at 10 U/ml on day 3. IFN-g ELISPOT assay The IFN-g ELISPOT assay was performed on nitro-cellulose bottomed 96 wells microplates (Millipore MAHA S45, Bedford, MA, USA) using the IFN-g ELISPOT kit (Mabtech, Stockholm, Sweden). Plates were coated with D1 k-1 antibody at 5 mg/ml overnight at 48C. After blocking for 2 h at 37 8C with DMEM containing 5% FCS, plates were washed three times with sterile PBS pH 7´4 and CD8‡ enriched T-lymphocytes were then plated in 100 ml CM supplemented with 10% AB human serum and 10 U/ml human recombinant IL-2. T2 cells were pulsed individually with various peptides (1 mg/ml) in serum free medium 503

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containing b2 microglobulin (1 mg/ml) during 2 h at room temperature in separate 48-well-plate wells. Cells were washed three times with DMEM and added to plates (1 ´ 105/well). For experiments involving one week stimulated cell cultures, responding cells were plated at 5, 10 or 20 ´ 103/well; for the ex-vivo ELISPOT, 1 ´ 105 responding cells per well were plated and no cytokines were added to the medium. Plates were incubated for 20Ð24 h at 37 8C in the presence of 5% CO2, then washed three times with PBS pH 7´4 and 0´1% Tween 20, then three times with PBS alone. Biotinylated 7-B6±1 antibody (0´3 mg/ml) was added and incubated for 1 h at room temperature; plates were washed as described earlier and alkaline phosphatase-streptavidine (Boehringer Mannheim, Mannheim, Germany) was added (1 : 1000). The substrate used was BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; Sigma) diluted in water (50 ml/well). Plates were washed with water after spots were observed (10 min). Spots were enumerated by two independent readers using a stereomicroscope with a magni®cation of 15 ´. Only spots with dark centres and blurred borders were considered. Statistical analysis was implemented with an unpaired t-test (two tailed). P < 0´05 was considered statistically signi®cant for the results obtained with the IFN-g ELISPOT after one round of stimulation. Chromium release assay Target cells (T2) were peptide pulsed as described above and labelled with 51Cr (Nef Life Science Products, Belgium) in Tris-Dulbecco buffer supplemented with 0´2% BSA for 45 min at 37 8C. Effector CD8‡ T-lymphocytes were plated at various dilutions in V shaped 96-well microplates in DMEM 5% FCS, unlabeled K562 cells (5 ´ 104) were added and the plates were incubated for 15 min at 37 8C before adding 1 ´ 103 labelled T2 cells per well. 51Cr release was measured after 4 h culture at 37 8C. Percentage lysis was calculated as 100 ´ (experimental release ± spontaneous release)/(total release ± spontaneous release). A lytic unit was de®ned as the number of effector cells that generate 30% speci®c lysis. RESULTS Binding of Plasmodium falciparum peptides to HLA-A*0201, and stability of the complexes formed The addition of exogenous peptides to the TAP de®cient T2 cells in the presence of b2 microglobulin allows the stabilization of the peptide/MHC complexes and increases the level of expression of the HLA-A2 antigen on the cell surface, in a peptide dose dependent manner. This enables the determination of the binding capacity of peptides to the 504

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MHC class I molecule (Valmori et al. 1998). Among the 18 malaria peptides tested, three were found to bind HLAA*0201 with an ef®ciency comparable to that of the Flu MP peptide (binding `af®nity' 0´3±2´0); 10 presented an intermediate level of binding (binding `af®nity' 0´01Ð 0´3); and, ®ve exhibited no detectable binding (binding `af®nity' < 0´01, see Table 1). Evaluation of the CD8‡ T lymphocyte response to the Flu MP 58-66 peptide using IFN-g ELISPOT and chromium release assays In order to establish a more sensitive approach for the detection of low frequency CD8 T-lymphocytes we compared the chromium release assay with the IFN-g ELISPOT using a well de®ned HLA-A*0201 restricted epitope from the in¯uenza virus (Flu MP 58-66). Cells from HLAA*0201 donors were tested either ex-vivo or after three rounds of weekly stimulation. IFN-g producing cells were readily detectable in freshly isolated CD8‡ T-lymphocytes; after one round of stimulation, the number of cytokine producing cells increased approximately 20-fold, whereas only low cytotoxicity levels were detected with the chromium release assay (Figure 1a). In contrast, following the second and third stimulation, the level of target cell lysis increased considerably as well as the number of IFN-g producing cells. Interestingly, spot sizes also increased upon each stimulation (Figure 1b). The frequency of Flu MP speci®c CD8‡ T-lymphocytes in freshly isolated cells was 1 : 3348 for donor 1 and 1 : 2500 for donor 2 (Figure 1a), which after one round of stimulation reached 1 : 70 and 1 : 117, respectively. Establishment of an evaluation system for peptide speci®c CD8‡ T lymphocyte responses The frequency of peptide-speci®c IFN-g spot forming CD8‡ T-lymphocytes had been previously measured on freshly isolated PBL incubated in the presence or absence of peptides (Lalvani et al. 1997, Scheibenbogen et al. 1997). In our hands, this ex-vivo system generated variable levels of background that hampered interpretation of results. This problem could not be overcome by varying the number of responder cells (data not shown). In contrast, reliable results were regularly obtained using enriched CD8‡ T-lymphocytes instead of total PBL as responder cells, and TAP de®cient T2 cells as APC, as previously seen for the TNF-a ELISPOT (Herr et al. 1996). Following this protocol, we detected peptide-speci®c responses in HLA-A*0201 individuals, as illustrated in Figure 2. Finally, certain peptidespeci®c IFN-g responses became clearer after one week culture, as in the case of the Flu MP peptide. Reproducible q 2000 Blackwell Science Ltd, Parasite Immunology, 22, 501±514

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HLA-A*0201 restricted malaria epitopes

Table 1 Sequences of peptides evaluated as potential human CD8 epitopes

Amino acids

Sequence

Binding HLA-A*0201 relative activitya

Dissociation half life (h)

7±16 64±72 104±113 299±308 334±342 334±342 403±411

ILSVSSFLFV SLKKNSRSL KLRKPKHKKL NMPNDPNRNV KLKKIKNSI KLKKIKNSLb IMVLSFLFL

0´02