Parasite Immunology, 2002, 24, 161–169
Identification of HLA-A2 restricted CD8+ T-lymphocyte responses to Plasmodium vivax circumsporozoite protein in individuals naturally exposed to malaria
Plasmodium ORIGINAL vivax ARTICLE CS Blackwell Science, LtdCD8+ T-lymphocyte epitopes
MYRIAM ARÉVALO-HERRERA1, ANAIS ZULLY VALENCIA1, JUANA VERGARA1, ANILZA BONELO1, KATHARINA FLEISCHHAUER2, JOHN MARIO GONZÁLEZ1, JUAN CARLOS RESTREPO3, JOSÉ ALEJANDRO LÓPEZ4, DANILA VALMORI5, GIAMPIETRO CORRADIN6 & SÓCRATES HERRERA1 1
Instituto de Inmunología del Valle, Universidad del Valle, Cali, Colombia, 2Tissue Typing Laboratory, Department of Biology and Biotechnology, Instituto Scientifico HS Rafaele, DIBIT, Milan, Italy, 3Radiotherapy Service, Hospital Universitario del Valle, Cali, Colombia, 4 Mater Medical Research Institute, Brisbane, Australia, 5Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, Centre Hospitalier Universitaire Vaudoise (CHUV), Lausanne, Switzerland and 6Institut de Biochimie, Université de Lausanne, Epalinges, Switzerland
SUMMARY Specific CD8− T-lymphocyte (CTL) activity against Plasmodium pre-erythrocytic stages (P-ES) derived antigens is considered one of the most important mechanisms for malaria protection. Plasmodium vivax is the second most prevalent human malaria parasite species distributed worldwide. Although several CTL epitopes have been identified in Plasmodium falciparum P-ES derived antigens, none has been described for P. vivax to date. In this study, we analysed HLA-A*0201 specific CD8− T-lymphocyte responses to the P. vivax circumsporozoite (CS) protein in both malaria exposed and nonexposed populations from the Colombian Pacific Coast. First, we analysed the prevalence of HLA-A2 allele in the study populations and found that approximately 38% of the individuals expressed this molecule and that 50% of them were HLAA*0201. We then selected, on the P. vivax CS, five peptide sequences containing the HLA-A*0201 binding motifs and used the corresponding synthetic peptides to evaluate the CD8− T-lymphocyte interferon (IFN)-γ response. Peripheral blood mononuclear cells from the HLA-A*0201 donors were in vitro stimulated with these peptides and IFN-γ production was determined by an ELISPOT assay. Specific CD8− Tlymphocyte responses were detected for three peptides located in the C-terminal region of the protein. Specific responses to these peptides were also detected in several individuals expressing different HLA-A*02 subtypes. The potential of these peptides to induce specific cytolysis and that of long synthetic Correspondence: Dr Myriam Arévalo-Herrera, Instituto de Inmunología del Valle, Universidad del Valle, AA 2388 Cali, Colombia (e-mail:
[email protected]). Received: 29 May 2001 Accepted for publication: 21 February 2002 ).
© 2002 Blackwell Science Ltd
peptides comprising these epitopes as P. vivax malaria vaccine subunits are being studied. Keywords Plasmodium vivax, CD8− T lymphocytes, HLAA*0201, IFN-γ, malaria
INTRODUCTION Malaria is caused by a protozoan parasite of the genus Plasmodium, which can infect multiple vertebrate species ranging from reptiles to primates. Four species can cause disease in humans, with Plasmodium falciparum and Plasmodium vivax comprising the two prevalent species. P. falciparum is the most widespread and dangerous parasite, particularly in the African continent, where it frequently causes death (1) both in children less than 5 years of age and pregnant women (2). Although P. falciparum also exists in Asia, Oceania and the American continents, they are mainly affected by P. vivax transmission with approximately 35 million cases reported per year (3). The disease caused by P. vivax is clinically less severe than that associated with P. falciparum and is rarely lethal; however, it has an important socio-economic impact due to the incapacitating symptoms and the clinical relapses that characterize infection with this parasite species. Eradication of parasite transmission has been intensively attempted for several decades through the use of insecticides and chemotherapy (4). However, the efficacy of such strategies is limited by the emergence of mosquito resistance to insecticides (5) and parasite resistance to anti-malarial drugs (1). Vaccination is currently considered feasible and the most promising cost-effective control strategy (6). This idea is supported by the observation that populations
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exposed to malaria transmission for a long time in endemic areas can reach solid clinical immunity that prevents disease complications and death, even without necessarily becoming resistant to the infection (7). In addition, pioneering immunization with radiation attenuated sporozoites provided the first evidence that protective immunity can be achieved through vaccination (8–10). Both humoral and cellular immune responses have been shown to be involved in malaria protection (11,12); however, at pre-erythrocytic level, CD8+ T-lymphocytes activity mediated by interferon (IFN)-γ release is considered the most important effector mechanism for malaria parasite killing (13). In rodents, it has been shown that in vivo depletion of CD8+ T-lymphocytes completely abolishes malaria immunity (14) and that passive transfer of cytotoxic T-cell clones recognizing the Plasmodium berghei circumsporozoite (CS) protein completely protects mice against sporozoite challenge (15). Based on these observations, great efforts are being invested at identifying parasite specific targets of protective immune responses that may be useful for malaria vaccine development. Several CD8+ T-lymphocyte epitopes derived from P. falciparum antigens and restricted by different HLA class I molecules, including HLA-A*0201, have been identified in humans (16 –19). However, due to the lack of efficient parasite in vitro culture systems and to the scarcity of suitable animal models, only few studies have focused on P. vivax. Although P. vivax CS protein is the best characterized preerythrocytic P. vivax antigen and multiple B and T helper epitopes have been identified (18,20–22), no CD8 + Tlymphocyte epitopes have been described to date. In the present study, we report for the first time the identification of three HLA-A2 restricted CD8+ epitopes that could be incorporated as components of an anti-P. vivax vaccine.
matrix protein (Flu-MA), was used as reference CD8+ CTL epitope (26). Peptides were synthesized by the solid phase F-moc techniques as previously described (27). After highperformance liquid chromatography purification and mass spectrometry analysis, peptides were diluted in dimethyl sulphoxide and stocked at −20°C at a concentration of 20 mg/ml.
HLA-A*0201 T2 cell binding assay The binding capacity of the peptides to the HLA-A*0201 molecule was assessed using the TAP deficient T2 cell line as described previously (28). Serial three-fold dilutions of the P. vivax and Flu-MA peptides were prepared from initial concentrations of 100 µg/ml and 10 µg/ml, respectively, in X-VIVO serum free medium (Biowhittaker, Walkersville, MD, USA) containing 3 µg/ml of β-2 microglobulin (Sigma, St Louis, MO, USA). T2 cells (1 × 105) were added and plates incubated overnight at room temperature. After centrifugation, peptide/major histocompatibility complex (MHC) molecule complexes were stained with 50 µl (1 mg/ ml) of an anti-HLA-A2-fluorescein isothiocyanate (FITC) monoclonal antibody (clone BB7·2) for 30 min at 4°C. The HLA-A*0201 expression was determined by flow cytometry analysis (FAC-Sort Becton Dickinson, San José, CA, USA). The results were expressed as relative fluorescence index (= mean fluorescence of samples/mean fluorescence of background). The Flu-MA peptide was used as reference epitope because of its high binding affinity to the HLAA*0201 molecule (26). The concentration of this peptide, required to obtain 50% of maximal increase of HLA-A*0201 surface expression on the T2 cells, was given the value of one unit. The binding capacity of malaria peptide was considered high when values were between 0·3 and 2 units, intermediate from 0·01 to < 0·3 units and low < 0·01 units.
MATERIALS AND METHODS Peptide/HLA-A*0201 complex stability Peptide synthesis The CS protein sequence corresponding to the P. vivax Belem strain (23) was screened for the presence of HLA-A2 binding motifs. The search was performed by a direct alignment of the complete sequence obtained from the Swissprot protein data bank with the putative HLA-A2 binding motifs L2V9 and L2L9−10 previously described (24,25) using the findpattern of the GCG program (Genetic Computer Group Software, Madison, WI, USA). The program retrieved five sequences containing the putative HLA-A*0201 binding motif. Two of them also contained the HLA-A*0202 binding motif. Four of the selected peptides (PV1, PV3, PV5 and PV7) were located within the C-terminal part of the protein and one peptide (PV6) was located in the N-terminal region (23). Peptide 58– 66 (GILGFVFTL) of the influenza virus
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The stability of the peptides/HLA-A*0201 complexes was determined using the T2 cell line as previously described (29). Briefly, saturating concentrations of each peptide (100 µg/ml for P. vivax peptides and 10 µg/ml for Flu-MA peptide) were added to T2 cells (1 × 105) diluted in X-VIVO serum free medium (Biowhittaker) containing 3 µg/ml of β-2 microglobulin. After 18–24 h of incubation at room temperature (18°C), cells were centrifuged for peptide removal and the protein synthesis was blocked by the addition of emetine (10−4 ) (Sigma). The cells were then incubated at 30 min, 1, 2, 4 and 6 h time points at 37°C and stained with the antiHLA-A2-FITC-monoclonal antibody (BB7·2) (American Tissue Culture Collection, Manassas, VA, USA) and analysed by flow cytometry (28). The peptide Flu-MA was used as internal standard. The decay of peptide/MHC complex © 2002 Blackwell Science Ltd, Parasite Immunology, 24, 161–169
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was measured and peptide half-life (h) was calculated using first-order kinetics (19).
Plasmodium vivax CS CD8 + T-lymphocyte epitopes
blood were obtained and used for peripheral blood lymphocyte separation as described below. Fractions of plasma were also maintained at −70°C for malaria serology.
Study population Two groups of individuals from Colombia were studied: one group was composed of individuals naturally exposed to P. vivax in malaria endemic areas of the Pacific Coast and the other of non-exposed individuals from the urban area of Cali, a non-malaria endemic region. Individuals from different communities of endemic regions, as well as individuals from Cali, were initially screened to determine the allelic distribution of the HLA-A2 molecule. HLA-A*02 subtyping was performed in volunteers from two endemic villages near Buenaventura, the main port on the Colombian Pacific Coast. Zacarias is a community with approximately 600 inhabitants located 5 km from Buenaventura by road, and Punta Soldado has nearly 300 people and is located aproximately 50 km away by boat. The Pacific Coast has a population comprising mostly (90%) individuals of African origin, living in rural settlings. It has a high rain precipitation rate (375 mm3) with a rather stable malaria transmission through the whole year; however, slight peaks of malaria occur during the dry season, at the beginning of the year (February to May). The individuals selected for this study were adults who had been permanently exposed for more than 20 years to P. vivax and P. falciparum transmission (30). Individuals from Cali were all adult mestizos without previous malaria infections. The malaria exposure was determined both by clinical history as well as by the presence of anti-malarial antibodies. Antibodies to the P. vivax parasite blood forms were tested by an immunofluorescent antibody test and antisporozoite antibodies were assessed by an enzyme-linked immunosorbent assay technique using CS protein derived peptides as antigen (22). Additionally, the presence of P. vivax and P. falciparum parasites at the time of the bleeding was determined by thick smear, and positive individuals were not included in the study.
Blood collection and HLA typing After receiving informed consent, blood was collected from 508 adult individuals and processed for HLA-A2 typing by flow cytometry (28). A total of 149 HLA-A2 positive donors, 75 naturally exposed to malaria (45 from Zacarias and 30 from Punta Soldado) and 51 non-exposed (from Cali) were selected for HLA-A*02 subtyping by the PCRSSOP technique (31). Fourteen individuals from the endemic areas, expressing the HLA-A*0201 allele (six from Zacarias and eight from Punta Soldado) as well as nine from Cali were selected to determine their malaria specific CD8+ CTL responses. From these individuals, 30 ml of © 2002 Blackwell Science Ltd, Parasite Immunology, 24, 161–169
CD8+ T-lymphocyte stimulation with P. vivax derived peptides Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hystopaque (Sigma) density-gradient and further CD8+ T-lymphocyte purification using the magnetic cell sorting system was performed according to manufacturer instruction (Miltenyi Biotech GmbH, Bergisch, Gladbach, Germany). PBMC from each donor were incubated with an anti-CD8+ magnetic beads for 15 min at 4°C. After washing, PBMC were loaded into the magnetic column and the flow-through was collected. The CD8+ purified population was eluted from the column and the purity was assessed by flow cytometry using specific anti-CD4+ and anti-CD8+ monoclonal antibodies (Becton Dickinson, San José, CA, USA). After washing, purified CD8+ T-lymphocytes (1– 2 × 105) were resuspended in complete RPMI medium supplemented with 10% of AB+ human serum and 10 ng/ml of interleukin (IL)-7 (kindly gift of Dr Vitta, Sanoffi Research, France). Irradiated (3 krad) autologous PBMC prepulsed with the corresponding P. vivax derived peptide (1 µ) was added and cultures incubated for 10 days at 37°C and 5% CO2. After 3 days, 20 U/ml of IL-2 (Glaxo-Wellcome, Geneva, Switzerland) was added to each culture.
Evaluation of the peptide specific activity by IFN-γγ ELISPOT The frequency of CD8+ T-lymphocytes producing IFN-γ was evaluated using a commercial kit for IFN-γ ELISPOT determination (Mabtech, Stockholm, Sweden). Microtitre plate wells (MAHA S45, Millipore, Bedford, MA, USA) were coated with anti-IFN-γ monoclonal antibody at 5 µg / ml overnight at room temperature (18°C). Wells were washed and blocked with PBS-1% BSA for 2 h at 37°C. After standardization of the number of cells per well, CD8+ T-lymphocytes stimulated with P. vivax peptides (20 × 103 per well) or Flu-MA peptide (2·5 × 103 per well) were plated. Peptide-pulsed (1 µ) and non-pulsed irradiated TAPdeficient T2 cells (106 per well) were diluted in X-VIVO serum free medium in the presence of 3 µg/ml of β-2 microglobulin and used as APC. Microplates were incubated for 24 h at 35°C and 5% CO2 and, after washing with PBS containing 0·05% Tween 20, a biotinilated anti-IFN-γ antibody (7-B6-1) was added at 3 µg/ml and incubated for 1 h at room temperature. After washing, alkaline phosphatasestreptavidine (1 mg/ml) diluted 1 : 1000 was added and the reaction revealed with 50 µl/well of the substrate BCIP/NBT
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HLA-A2 Donors
Communities
na
Exposed
Zacarias Pta Soldado Zavaletas El Coco Cali
134 123 20 67 164
Total
508
Non-exposed
Table 1 Prevalence of HLA-A2 positive donors
A*0201 %
nb
Positive
%
45 56 10 23 72
33·6 45·5 10·0 34·3 43·9
45 30 ND 23 51
17 15 ND 7 33
37·7 50·0 ND 30·4 64·7
198
38·9
149
72
48·3
Positive
a
Number of donors analysed. HLA-A2 expression was determined by flow cytometry using FITC labelled monclonal antibody BB7·2. bNumber of HLA-A2 positive donors analysed for HLA-A0201 expression by PCR-SSOP technique. ND, Not done.
(5-bromo-2-chloro-3-indolyl phosphatase/nitroblue tetrazolium, Sigma). After the appearance of dark blue spots, the plates were washed three times and read in a stereo-microscope. The number of spots present in the duplicate wells, with and without peptide, were counted by two different observers and the results analysed in an unpaired t-test (two tail) where P < 0·05 was considered statitically significant, as described previously (19).
RESULTS Prevalence of HLA-A2 alleles in different Colombian communities Expression of HLA-A2 molecules was analysed in individuals from different communities from a malaria endemic region of the Colombian Pacific Coast, as well as in the non-endemic city of Cali. As shown in Table 1, 38·9% (198 of 508) of the white blood cell samples analysed by a fluorescence-activated cell sorter (FACS) expressed the HLAA2 molecules. However, the frequency of HLA-A2 positive donors significantly varied among the different communities, ranging from 10% in Zavaletas community to 45% in Punta Soldado. Blood samples from HLA-A2 positive individuals were used to determine the prevalence of the different HLA-A*02 subtypes. Although the anti-HLA-A2 monoclonal antibody (BB7·2) used in the FACS analysis also crossreacted with the A28 molecule, we found that all individuals recognized by this monoclonal were in fact HLA-A2. The most frequent allele expressed both in exposed and nonexposed populations was the HLA-A*0201 (37·7% and 64·7%, respectively) as determined using the PCR-SSOP technique (32). Although almost one-half of the positive HLA-A2 individuals studied in the different endemic regions expressed the HLA*0201 subtype, we selected the Zacarias and Punta Soldado communities to assess the recognition of the P. vivax derived peptides based on their geographical accessibility to
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Cali, where the samples were processed, their degree of exposure to malaria and their acceptance of multiple bleedings for results confirmation.
Selection and analyses of CS derived peptides containing HLA-A2 binding motifs The HLA-A*0201 binding capacity of the selected peptides was evaluated by analysing their ability to induce HLAA*0201 over-expression upon overnight incubation with the human TAP deficient T2 cell line using the Flu-MA peptide as internal control. As shown in Table 2, decapeptides PV1 and PV5 bound to HLA-A*0201 with intermediate affinity; however, low to undetectable binding was observed for peptides PV3, PV6 and PV7. Since peptides PV3 and PV6 also contain the binding motif when synthesized as nonapeptides (PV3n and PV6n), we decided to test these nonapeptides and found a remarkable increase in their binding capacity. In agreement with the binding data, the stability of peptide/MHC complexes indicated that decapeptides PV1, PV5 as well as nonapeptides PV3n and PV6n, were able to form relatively stable peptide/MHC complexes (2–5 h) compared to peptide/MHC complexes formed by peptide Flu-MA (> 6·0 h).
Assessment of human CD8+ T-lymphocyte responses to CS derived peptides To assess CD8+ T-lymphocyte responses of human donors to the CS selected peptides, the CD8+ T-cell enriched fraction was cultured for 10 days in the presence of autologous irradiated (10 krad) antigen presenting cells prepulsed during 1 h with each of the peptides (1 µ) and exogenously added cytokines. At the end of the culture period, specific CD8+ positive responses were evaluated by IFN-γ ELISPOT assay, currently considered one of the most sensitive method for the detection and enumeration of antigen specific © 2002 Blackwell Science Ltd, Parasite Immunology, 24, 161–169
Plasmodium vivax CS CD8 + T-lymphocyte epitopes
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Table 2 Localization and binding of P. vivax peptides to HLA-A2 molecules
Code
Position
1
2
3
4
5
6
7
8
9
PV1 PV3 PV3n PV5 PV6 PV6n PV7 Flu-MA
301–309 365 –374 365 –373 341–349 6 –15 6 –14 367–376 58 – 66
Y S S T L L G G
L L L L L L L I
D G G N A A V L
K L L D V V I G
V V V L S S L F
R I I E S S L V
A L L T I I V F
T L L D L L L T
V V V V L L A L
10
L
L L
Binding motifs
Relative binding capacity (Units)a
Dissociation half-lifeb
A*0201 A*0201 A*0201 A*0201 A*0201/02 A*0201/02 A*0201/02 A*0201
0·12 NDB 0·50 0·06 < 0·01 0·40 NDB 1·00
3·0 NA 4·6 2·7 NA 3·8 NA > 6·0
The anchor residues for the HLA-A2 binding motifs in position 2 and 9–10 are underlined. NDB, not detectable binding. NA, not applicable. aRelative binding capacity of malaria peptides to the T2 cell line expressing the HLA-A*0201 allele. The concentration of the Flu-MA peptide required to obtain 50% of maximal increase of HLA-A*02 antigen surface expression was given the value of 1 unit. The binding capacity of malaria peptide was considered high when values were between 0·3 –2 units, intermediate between 0·01 to < 0 units and low < 0·1 units. bThe peptide/HLA-A*0201 complex stability was calculated by dissociation peptide half-life (h) using first-order kinetics.
IFN-γ secreting CD8+ T-lymphocytes (33). A total of 14 samples (six from Zacarias and eight from Punta Soldado) from HLA-A2 adult individuals, previously exposed to frequent P. vivax infections and who agreed to be repeatedly bled, were analysed. All individuals were negative for circulating malaria parasites by thick smear but all of them presented high antibody titres against P. vivax sporozoites and blood forms at the moment of the study. As shown in Table 3, a significant proportion of IFN-γ producing cells was detected upon stimulation with peptides PV1, PV3 and PV5 in donors from Punta Soldado. One of the eight individuals from this community (PS-036) responded to peptide PV1, two of them (PS-009 and 085) responded to peptide PV3, whereas five individuals (PS-009, 036, 081, 085 and 086) responded to peptide PV5. In addition, 50% of the individuals responded to the Flu-MA peptide (PS-008, 015, 039 and 081). Although, CD8+ T-lymphocytes from some of the responding individuals were tested by limiting dilution analysis in the chromium release assay, no detectable specific lysis could be observed (data not shown). The frequency of P. vivax CS specific CD8+ IFN-γ producing precursors ranging between 1 : 600 and 1 : 2950. None of the individuals from Zacarias significantly responded to stimulation with any P. vivax derived peptides; however, four of six individuals responded to peptide Flu-MA. Except for a borderline non-specific response to PV3 peptide in one of the nonimmune donors (SV), none of the non-exposed individuals recognized the P. vivax derived peptides (Table 4). None of the exposed individuals recognized peptides PV6 and PV7 (data not shown). Remarkably, a specific response to peptides PV1, PV3 and PV5 was also detected for some malaria exposed individuals expressing either HLA-A*0213, A*0211 or A*0205 alleles (Figure 1). © 2002 Blackwell Science Ltd, Parasite Immunology, 24, 161–169
Table 3 Recognition of P. virax derived peptides by HLA-A*0201 malaria exposed individuals PV1
PV5
PV3
Flu-MA
Code
–
+
–
+
–
+
–
+
PS-008 PS-009 PS-015 PS-036 PS-039 PS-081 PS-085 PS-086 Zac-236 Zac-094 Zac-218 Zac-238 Zac-236 Zac-185
31 3 41 26 29 40 3 31 22 41 22 22 30 34
36 1 35 85 28 38 1 33 18 41 19 18 33 40
48 6 37 30 41 39 7 28 30 10 9 19 30 43
44 30 46 35 43 40 30 34 30 14 7 21 27 42
36 4 40 17 37 25 9 19 61 16 23 31 30 23
36 18 43 31 39 37 26 32 59 15 23 28 32 22
32 20 30 27 27 37 1 23 16 43 20 53 80 28
61 12 64 33 46 74 0 36 73 53 57 250 173 125
IFN-γ-ELISPOT of stimulated CD8+ T-lymphocytes from HLAA*0201 naturally exposed individuals (PS: Punta Soldado and Zac: Zacarias). Results are expressed as number of spots in 2 × 104 cells cultured without (–) or with P. vivax derived or 2·5 × 103 Flu-MA peptides (+). The mean values ± SD were calculated from duplicates from at least two independent observers and analysed in a t-test (two-tailed). P < 0·05 was considered statistically significant (underlined values).
DISCUSSION It is currently accepted that a malaria vaccine aimed at blocking parasite development in the liver should induce vigorous CD8+ T-lymphocyte responses specific for liver stage antigens. Evidence of the existence of these T-cells in
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Table 4 Recognition of P. virax derived peptides by HLA-A*0201 malaria non-exposed individuals PV1
PV5
PV3
Flu-MA
Code
–
+
–
+
–
+
–
+
SV MA ZV GQ DJ PA GI LO EA
33 23 56 62 42 30 28 44 10
30 27 53 61 39 36 29 34 5
22 11 57 53 52 20 26 62 17
31 13 61 57 59 29 42 54 19
8 11 75 43 44 24 55 41 21
11 11 68 53 44 27 50 31 12
36 8 95 85 52 30 0 22 3
198 410 122 163 77 167 0 234 140
IFN-γ-ELISPOT of CD8+ T-lymphocytes from HLA-A*0201 nonexposed individuals. Results are expressed as number of spots in 2 × 104 cells cultured without (–) or with P. vivax derived or 2·5 × 103 Flu-MA peptides (+). The mean values ± SD were calculated from duplicates from at least two independent observers and analysed in a t-test (two-tailed). P < 0·05 was considered statistically significant (underlined values).
P. vivax infection has not been reported to date. We describe here, for the first time, three peptides derived from the P. vivax pre-erythrocytic CS protein which are recognized by HLA-A2 restricted CD8+ T-lymphocytes from individuals living in malaria endemic areas and induce the production of IFN-γ. Our findings are very relevant in view of HLAA2 being the most frequent allele worldwide (34,35). We found a high prevalence of this allele in the HispanoAmerican Mestizo population (38·9%) and the HLA-A*0201 subtype in both malaria exposed (39·3%) and non-exposed (64·7%) donors (32,36). Although the five sequences represented by PV1, PV3, PV5, PV6 and PV7 contained the putative HLA-A*0201 binding motifs L2V9 and L2L9−10, PV6 and PV7 also contained anchor residues for HLA-A*0202 (24,25). A good correlation was found between the binding affinity and the complex dissociation half-life of the five peptides. PV1 and PV5, which showed intermediate binding affinity (0·12 and 0·06, respectively), formed the most stable peptide/MHC complexes (3·0 h and 2·7 h, respectively). PV1, PV3 and PV5 were frequently recognized by individuals from Punta Soldado, as indicated by the IFN-γ production of CD8+ T-lymphocytes upon peptide in vitro stimulation, and PV5 was the most frequently recognized (five of eight individuals tested). While detection of CD8+ T-lymphocyte responses to PV1 and PV5 was in agreement with binding data, detection of PV3 specific responses was more puzzling due to the lack of significant binding of PV3 in the T2 overexpression assay. However, when PV3 was synthesized as a nonapeptide (365 –373), also containing the HLA-A*0201
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binding motif (SLGLVILLV), the affinity to the HLAA*0201 molecule was increased (0·5 units). The differential recognition of PV3 could be explained by the cleavage of the carboxyl-terminal residue (leucine) by serum proteases present in the lymphocyte cultures, leading to the generation of the nonapeptide 365–373. This phenomenon would not be observed in the T2 assay since it was performed in the absence of serum (37). Similarly, we observed a high affinity of the nonamer PV6 (0·4 units). Further studies to determine the immunogenicity of these peptides (PV3 and PV6) in HLA-A*0201/Kb transgenic mice and the human CD8+ T-lymphocyte recognition will need to be performed. Although a low number of P. vivax CS IFN-γ producing specific CD8+ T-lymphocytes (1 : 600 to 1 : 2950) after 10 days stimulation was found in individuals from Punta Soldado, they were comparable to those specific to P. falciparum CS in highly endemic areas of Africa (19,38). These low numbers could be due to the short exposure of Plasmodium antigens to the immune system. Although in P. falciparum endemic areas individuals could be frequently exposed to highly infected mosquito bites (200–300 bites per month) (39), the presence of the target CS protein in the hepatic cells during each infection is very short (4 h) (40). In other diseases, such as HIV, hepatitis B and cancer, antigen persistence is likely to be much longer and result in an increased recruitment of specific CD8+ CTL precursor (26). Indeed, individuals frequently exposed to malaria irradiated sporozoites have also higher frequencies of CD8+ CTL precursors than individuals naturally exposed in endemic areas (41). It has been demonstrated that mice inoculated with P. berghei irradiated sporozoites are able to develop a great number and long lasting liver schizonts (blocked forms) (28%) than those inoculated with non-irradiated sporozoites (1–3%) (40). This effect could be a prerequisite to increase the number of malaria specific CD8+ CTL precursors to induce an efficient parasite clearance in the liver. Thus, the inability to find specific lysis by the chromium release assay might be due to the limited sensitivity of this method (42). Although the number of IFN-γ producing cells was low, statistical analysis indicated significant differences (P < 0·05) between the number of spots present in wells with and without peptide. Similar to previous studies in P. falciparum, our naive donors presented a borderline response to P. vivax peptides. This might be explained by the presence of CD8+ IFN-γ producing precursors induced by exposure to other common microorganisms (43), which may induce the presence of memory T-cell that recognize malaria peptides as promiscuous epitopes (44,45). We also found detectable CD8+ T-lymphocyte responses to some of the peptides in three individuals expressing nonHLA-A*0201 alleles (HLA-A*0211, A*0205 and A*0213). This observation is consistent with previous studies where © 2002 Blackwell Science Ltd, Parasite Immunology, 24, 161–169
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Plasmodium vivax CS CD8 + T-lymphocyte epitopes
Figure 1 IFN-γ-ELISPOT detection of CD8+ T-lymphocytes in malaria exposed individuals with various HLA-A*02 subtypes. The results are expressed as number of spots in 20 × 103 cultured cells without (open bars) or with P. vivax derived (full bars). For the Flu-MA peptides, the number of spots was read in 2·5 × 103 stimulated CD8+ T-cell lymphocytes. Donor PS-065 did not respond to Flu-MA peptide. P < 0·05 was considered statistically significant.
CD8+ stimulating peptides derived from P. falciparum were recognized by CD8+ CTL in the context of more than one HLA class I molecule (degenerated recognition) (46). It has also been demonstrated in hepatitis B that HLA-A*0201 binding peptides are able to cross-bind a broad group of HLA-A2 subtypes defining the A2-like superfamily (A*0201, 0202, 0203, 0205, 6801 and 6902) (47). Here, we found that individuals expressing the A*0211 and A*0213 alleles, not yet included into the A2 super-family group, were able to recognize the P. vivax peptides. This cross-reactivity may be due to the similarity in the residues that modulate the © 2002 Blackwell Science Ltd, Parasite Immunology, 24, 161–169
binding affinity of the different HLA-A2 alleles, which are evolutionarily derived from a common ancestor (48,49). HLA-A*02 alleles can be divided in two subgroups, A*0201 and A*0205, based on the presence of different residues in the α1 and α2 domains of the binding groove (A*0201: F9, Q43, V95, L156 and A*0205: Y9, R43, L95 W156). Peptides binding to A*0201 are likely to cross-bind to other members of the HLA-A*0201 subgroups. The HLA-A*0211 and A*0213 belong to A*0201 subgroup (50). All the peptides recognized by malaria exposed individuals, are located in the CS C-terminal region. PV1 and
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PV3 map in a homologueous position to two HLA-A*02 restricted CTL epitopes previously described in P. falciparum (CSPf 334 –342 and CSPf 394 – 402) (16,46). It has been demonstrated that the P. falciparum CS C-terminal region is highly polymorphic (51) and that this polymorphism can abrogate the recognition of CTL epitopes (52). However, studies performed in more than 16 P. vivax isolates collected worldwide, including the Belem strain, have shown a limited sequence variation in this region of the protein (23,53,54). Overall, the results obtained in this study are important for the development of a P. vivax malaria vaccine that is able to elicit pre-erythrocytic stage specific CD8+ IFN-γ responses. Several human trials have demonstrated good immunogenicity (55,56) as well as short-lived protective efficacy of a P. falciparum vaccine based in the carboxyl region of the CS protein (57,58). These results confirm the feasibility of a vaccine targeting the pre-erythrocytic parasite stages. In P. vivax, a protective vaccine targeting this phase of the parasite development is likely to prevent relapses due to liver hypnozoites, one of the most undesirable effects of P. vivax infection.
ACKNOWLEDGEMENTS We acknowledge the participation of the malaria endemic communities in the Colombian Pacific Coast. We also thank Asoclinic for providing us with donors from Cali and Dr Natalio Vitta from Sanoffi Research, France, for providing the recombinant IL-7. This work was supported by the WHO-TDR Special Programme and the Colombian Research Council, COLCIENCIAS.
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