RESA

Acta Tropica 68 (1997) 37 – 51

T- and B-cell responses of malaria immune individuals to synthetic peptides corresponding to non-repeat sequences in the N-terminal region of the Plasmodium falciparum antigen Pf155/RESA Asli Kulane a,*, Abu Bakar Siddique a, Hedvig Perlmann a, Niklas Ahlborg a, Christian Roussilhon b, Adama Tall b, Alioune Dieye b, Peter Perlmann a, Marita Troye-Blomberg a a

Department of Immunology, Stockholm Uni6ersity, S-106 91 Stockholm, Sweden b Immunology Unit, Institut Pasteur de Dakar, B.P. 220 Dakar, Senegal

Received 6 February 1997; received in revised form 21 March 1997; accepted 20 April 1997

Abstract While the C-terminal repeat region of Pf155/RESA, a Plasmodium falciparum vaccine candidate has been extensively studied for B- and T-cell reactivities, little is so far known about the non-repeat region in this respect. The present study aimed at investigating the non-repeat sequence 171-227 of Pf155/RESA for T- and B-cell epitopes. Eight overlapping peptides were synthesised and assayed for their ability to stimulate peripheral blood mononuclear cells obtained from P. falciparum-immune donors to proliferate and to induce secretion of interferon-gamma (IFN-g) and/or interleukin 4 (IL-4) using the ELISPOT assay. The plasmas of the corresponding donors were tested for antibody reactivity with the same peptides in ELISA. The individual cellular responses to the different peptides varied and in general they were not correlated, emphasising the importance of including several parameters for T-cell activation. The most frequent T-cell responses (proliferation, IFN-g and/or IL-4) were seen with two partially overlapping peptides corresponding to the sequences 171 – 185 and 181–195 that induced responses in 71 and 62% of the donors, respectively. Although, the frequency of responders was high, the magnitude of the responses was generally low. Two

* Corresponding author. Fax: + 46 8 157356; e-mail: [email protected] 0001-706X/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 1 - 7 0 6 X ( 9 7 ) 0 0 0 7 0 - 3

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A. Kulane et al. / Acta Tropica 68 (1997) 37–51

overlapping peptides corresponding to the sequence 186 – 206 bound antibodies from a large number of plasma samples. IL-4 producing cells were frequently found in donors whose sera contained antibodies to the corresponding peptide. However, there was no absolute correlation and many donors having anti-peptide antibodies could also be induced to produce IFN-g. In conclusion, the non-repeat region of Pf155/RESA contains several epitopes inducing functionally distinct T-cell responses. The sequence 171 – 206 was found to contain both B- and T-cell epitopes recognised by almost all individuals naturally primed to malaria. Thus, this sequence should be a useful tool in future immuno-epidemiological studies and/or for inclusion into a subunit vaccine against the asexual blood stages of the P. falciparum parasite. © 1997 Elsevier Science B.V. Keywords: P. falciparum; Pf155/RESA; T-cell epitope; IFN-g; IL-4; ELISPOT

1. Introduction The development of effective protective immunity against the asexual blood stages of malaria has been shown to be both antibody-dependent and -independent (Weidanz and Long, 1988; Troye-Blomberg and Perlmann, 1994). With respect to the development of peptide-based subunit vaccines it is important to characterise epitopes that are recognised by human B-cells (antibodies) as well as epitopes that can activate appropriate CD4 + T-cells. An antigen generally considered to be an important candidate for a merozoite vaccine is the conserved antigen Pf155/RESA (Perlmann et al., 1984; Berzins and Perlmann, 1996). Several lines of evidence suggest that immune responses to Pf155/RESA are protective. Seroepidemiological studies have shown correlations between antibody levels, particularly against the C-terminal repeat sequences and reduced parasitemia in individuals from different study areas (Deloron and Cot, 1990; Petersen et al., 1990; Riley et al., 1991; Astagneau et al., 1994). Partial protection has been obtained in Aotus monkeys both by immunisation with recombinant fusion proteins containing Pf155/RESA repeat sequences (Collins et al., 1986) and by passive immunisation with human affinity purified antibodies reactive with Pf155/RESA repeats (Berzins et al., 1991). Furthermore, antibodies reactive with Pf155/RESA repeats are very efficient inhibitors of Plasmodium falciparum merozoite invasion of erythrocytes in vitro (Wa¨hlin et al., 1984; Perlmann et al., 1989). The C-terminal repeat region has been extensively studied for T-cell inducing capacity. We and others have shown that the repeat regions of this antigen contain several related and crossreacting T-cell activating sequences capable of inducing T-cells from P. falciparum- immune donors to proliferate (Rzepczyk et al., 1988), to secrete IFN-g and/or IL-4 (Troye-Blomberg et al., 1989; Fievet et al., 1995; El Ghazali et al., 1995). T-cell epitopes have also been identified in the N-terminal non-repeat region within residue 171-195 (Rzepczyk et al., 1988; Troye-Blomberg et al., 1991; Chougnet et al., 1991; Fievet et al., 1993; Migot et al., 1993). This latter region is also known to contain B-cell epitopes (Perlmann et al., 1989; Chougnet et al., 1991).

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39

The present investigation was undertaken to more in detail delineate T-cell epitopes in the sequence 171 – 227. For this purpose eight (13–15 amino acids long) overlapping peptides corresponding to the amino acid residues 171–227 were synthesised. The peptides were investigated for their capacity to induce cytokine (IFN-g and IL-4) producing cells, using ELISPOT assay and proliferation in vitro in peripheral blood mononuclear cells (PBMC) obtained from P. falciparum-primed donors living in Senegal, West Africa. The responses were analysed in relation to those obtained with recombinant (r) Pf155/RESA lacking the N-terminal part of the antigen. Plasma samples from individual donors were tested for the presence of antibodies to the same peptides as used for T-cell stimulation.

2. Materials and methods

2.1. Study subjects This study was carried out in Dielmo, Senegal, a malaria endemic area with a population of 250 inhabitants consisting of two major different ethnic groups Serere (78%), Mandigue (13%) and mixed groups (9%). The population of Dielmo is well characterised with regard to P. falciparum epidemiological and immunological criteria. The area has a high level of malaria prevalence with moderate seasonal variations (Trape et al., 1994). Samples of venous blood were obtained with informed consent from 21 malaria immune adults (mean age, 32.6 years; range, 18– 54 years) living all their life in Dielmo. None of the donors were clinically ill at the time of bleeding. For serological assays, plasma samples from the above donors and from an additional 21 randomly selected Swedish donors with no previous exposure to malaria were used. PBMC from Swedish donors not previously exposed to P. falciparum served as controls in the T-cell assays.

2.2. Peptides and antigen preparations Eight overlapping synthetic peptides corresponding to a non-repeat region of Pf155/RESA (residues 171 – 227) were synthesised, Pl-P5 by Boc chemistry (Houghten et al., 1986) and P6-P8 by Fmoc chemistry (Sa¨llberg et al., 1991). The purity of the peptides ranged from 65–95% according to high pressure liquid chromatography (Berzins et al., 1986). The sequences of the synthetic peptides and their location are shown in Table 1. All peptides were desalted and tested for cytotoxicity against PHA-activated blast cells, none were toxic. A recombinant (r) Pf155/RESA (lacking the N-terminal region) with a molecular mass of 89 kDa and representing approximately 70% of the C-terminal part of Pf155/RESA molecule was kindly provided by Drs P. Schools and D. Pye (CSL, Melbourne, Australia) and R. Anders (WEHI, Victoria 3050, Australia) and was included for comparison. A crude P. falciparum antigen extract was produced as described earlier (Kabilan et al., 1987) and used in ELISA.

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2.3. Preparation of peripheral blood mononuclear cells and plasma samples Heparinised venous blood (30–40 ml) were collected and a small fraction of plasma was taken for antibody detection using ELISA. PBMC were isolated by centrifugation on a Ficoll hypaque (Pharmacia-Upjohn, Stockholm, Sweden) density gradient.

2.4. T-cell proliferation assay PBMC were suspended (1×106/ml) in complete tissue culture medium (RPMIHEPES 1640 Gibco, Paisley, Scotland) supplemented with 2 mM L-glutamine, 25 mg/ml gentamycine and 10% heat inactivated AB + human serum and then seeded (1 × 105 cells/well) into 96-well round bottomed microculture plates (Linbro, Flow Laboratories) in triplicates per test. Antigens (2 mg/ml) were added at the initiation of the cultures. Medium alone or PHA (10 mg/ml) were used as negative and positive control, respectively. The cultures were incubated for 5 days at 37°C in an atmosphere of 5% CO2 and then pulsed with 1 mCi [3H]thymidine (specific activity 2 Ci/mmole; Amersham International, Buckinghamshire, UK) in complete culture medium for 18 h. Cellular incorporation of thymidine was measured in a liquid scintillation counter. Results are expressed as stimulation index (SI) defined as mean cpm of test cultures divided by mean cpm of control cultures. The range of cpms from unstimulated cultures were 688–2074. Tests having SI ] 2.5 were considered positive (Kabilan et al., 1988).

2.5. Re6erse ELISPOT assay for the enumeration of cytokine (IFN-g and IL-4) -secreting cells The number of cytokine secreting cells was determined by two-site reverse

Table 1 Synthetic peptides corresponding to non-repeat sequence of Pf155/RESA Peptide

Residuea nos.a

Sequenceb

P1 P2 P3 P4 P5 P6 P7 P8

171–185 176–190 181–195 186–200 192–206 199–211 207–221 213–227

LFDYNEKVDNLGRSG EKVDNLGRSGGDIIK LGRSGGDIIKKMQTL GDIIKKMQTLWDEIM MQTLWDEIMDINKRK IMDINKRKYDSLK YDSLKEKLQKTYSQY KLQKTYSQYKVQYDM

a b

Position according to Favaloro et al., 1986. Amino acid residues given in one letter code.

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ELISPOT techniques, as described earlier (El Ghazali et al., 1993, 1995). Briefly, PBMC were diluted in complete tissue culture medium to 2× 106/ml and incubated in 5 ml culture-tubes (Falcon) together with the antigens at a concentration of 2 mg/ml for 4 h in an atmosphere containing 5% CO2 at 37°C. Thereafter, 100 ml of the cell suspension (2×105 cells), in duplicates, were transferred to washed ELISPOT plates (Millilitre MAHA plates, Millipore, Bedford, MA) precoated with 15 mg/ml of mAb 1-DlK or IL-4-I (Mabtech AB, Stockholm, Sweden), specific for IFN-g and IL-4, respectively. The cells were further incubated for 34– 38 h under the same conditions. Thereafter, the plates were washed six times with phosphate buffered saline (PBS), followed by the addition of 100 ml/well of biotinylated mAbs 7-B6-1 or IL-4-II for IFN-g and IL-4, respectively (Mabtech AB) diluted in PBS to 1 mg/ml and incubated for 60–90 min at room temperature. The wells were then washed and incubated with streptavidin-alkaline phosphatase (Mabtech AB) for one h. Between all steps the plates were washed six times with PBS. Thereafter, the BCIP/NBT substrate solution (Bio-Rad Kit, Bio Rad Laboratories, Richmond, CA) (100 ml/well) was added and incubated in room temperature for 1 – 2 h or until dark blue spots began to appear. Colour development was stopped by washing with tap water. Plates were dried and spots were counted under dissection microscope ( ×40). Cytokine (IFN-g and IL-4) responses were considered positive when the number of spots was greater than the mean+ 2 S.E.M. of unstimulated wells for each individual, based on the finding that the mean error of duplicated spots for the ELISPOT assay was less than 20% of the counted spot numbers (Surcel et al., 1994).

2.6. Serology Plasma obtained from each donor was used for determination of anti-P. falciparum antibodies and anti-peptide antibodies in ELISA (1:1000 dilution) (Perlmann et al., 1989). Briefly, Micro ELISA plates (Costar, Cambridge, MA) were coated with 10 mg/ml of crude P. falciparum extract or with bovine serum albumin-conjugated peptides (1:2 w/w) (Berzins et al., 1986). Specifically bound antibodies were detected by alkaline-phosphatase-conjugated goat antibodies specific for human IgG (g-chains) with p-nitrophenyl phosphate as substrate. The concentration of specific IgG antibodies in the plasmas was determined from curves obtained with serial dilutions of human gamma globulin in wells coated with an antibody specific for HuIgG (g-chain) in all ELISA assays (Perlmann et al., 1989).

2.7. Statistics Student’s t-test was used for the analysis of the responses induced by different peptides and regression analysis for that of the relationship between antibody levels and peptides.

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3. Results

3.1. Antibodies to linear peptides in the non-repeat region of Pf 155 /RESA All Senegalese individuals had been exposed to P. falciparum malaria as reflected by elevated levels of IgG reactive with a crude P. falciparum antigen extract, but none of the subjects tested were clinically ill. As measured in an ELISA, the geometric mean IgG antibody levels for all the plasmas was 65.4 mg/ml (range, 24.8 – 188.5 mg/ml) (not shown), while in the non-exposed controls the background levels were 9 mg/ml (range, 6.3 – 11.1 mg/ml). Eight overlapping synthetic peptides corresponding to sequences in the N-terminal region (residue 171 – 227) of Pf155/RESA (Table 1) were used to analyse for the presence of specific antibodies. As can be seen in Fig. 1, the background levels in the control plasmas from the nonmalaria exposed individuals were generally low, usually below 1 mg/ml. For all peptides, except P3, the mean antibody levels were significantly higher in the P. falciparum-exposed donors as compared to the non-exposed, the clearest difference were seen with peptides P4 (unpaired t-test; PB 0.0013), P5 and P6 (P B 0.0002), respectively. For the peptides tested, the highest and most frequent responses were to P4 (Fig. 1). The mean geometric level to this peptide was 4.76 mg/ml. The individual antibody levels to this peptide were significantly correlated with that seen to the overlapping peptides P5 (r= 0.846; PB 0.0001) and P6 (r = 0.863; PB 0.0001). A good correlation was also seen with the other peptides except for P3.

3.2. Human T-cell responses to a r-Pf 155 /RESA fragment and peptides corresponding to the N-terminal non-repeat region Earlier results have suggested the existence of T-cell epitopes in the N-terminal region of Pf155/RESA (Rzepczyk et al., 1988; Troye-Blomberg et al., 1989). To further elucidate this, PBMC from immune adults living in a malaria endemic area (Dielmo) were assessed for their capacity to respond to a series of eight overlapping peptides (Table 1) in vitro. The assays used to measure T-cell activation were proliferation and ELISPOT, the latter enumerating the number of cytokine producing cells (IFN-g and IL-4) at the single cell level. A r-Pf155/RESA lacking the N-terminal region was included as a reference antigen. This antigen has earlier been shown to induce proliferation in 25–50% (Rzepczyk et al., 1988; Beck et al., 1995; Fievet et al., 1995), IFN-g and IL-4 in 30% (Fievet et al., 1995; El Ghazali et al., 1995) of individuals living in other malaria-endemic areas. The percentages of donors responding in this study by proliferation and cytokine production to the r-Pf155/RESA are shown in Table 2. Thirty-one percent of the donors responded by proliferation, 75% by IFN-g production and 21% by IL-4 production, respectively. When taking proliferation, IFN-g and IL-4 production as a total measure of lymphocyte activation 76% of the donors responded to the r-Pf155/RESA.

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Fig. 1. Scatter diagram of antibody levels from 21 malaria exposed donors and 21 non-malaria controls to peptides from the N-terminal region of Pf155/RESA (sequence 171 – 227), in ELISA. (“) Antibody levels in immune donors; () controls; thick bars indicate the geometric mean of the antibody levels in exposed donors while thin bars are of the non-exposed donors. Abscissa: peptides (P1 – P8) see Table 1. Ordinate: antibody levels (mg/ml).

Results of stimulation with the N-terminal peptides are shown in Table 2. All synthetic peptides elicited a positive response in at least some donors. On average, 8% of the donors responded by proliferation, 40% by IFN-g production and 30% by IL-4 production. However, as can be seen, the frequencies of positive responses induced by the different peptides varied. Because of the lack of lymphocytes available for testing, not all donors could be tested with each of the peptides in all assays (Table 2). The most frequent lymphoproliferative responses were seen with peptides P2 and P4 which induced positive responses in 13% of the individuals. Among the eight peptides tested, P1 induced IFN-g production in 70% of the

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Table 2 Lymphocyte responses after antigen or peptide stimulation Assays

Proliferationb (n= 16) IFN-g ELISPOT (n= 20) IL-4 ELISPOT (n= 14) Total (n= 21) a b

Peptides r-Pf155

P1

P2

P3

P4

P5

P6

P7

P8

31a 75 21 76

6 70 50 71

13 45 21 43

6 40 29 62

13 35 43 52

6 30 50 38

6 40 50 57

6 30 29 43

6 35 14 43

Values are percentage of positive responders. Number of donors tested in each assay.

donors, while 40 – 45% responded to P2, P3 and P6, respectively. The rest of the peptides induced IFN-g in more than 30% of the tested participants. Fifty percent of the donors responded with IL-4 production when stimulated with peptide P1, P5 and P6 while P4 induced IL-4 production in 43%, P3 and P7 in 29% of the donors, respectively. In agreement with earlier data (Troye-Blomberg et al., 1989) neither of the controls tested responded to either the recombinant protein or to the peptides (data not shown). Taken together, the overall responses to the peptides ranged from 38 to 71%, with P1, P3, P4 and P6 apparently being the most efficient stimulators (Table 2). The magnitude of the responses also varied considerably and was generally low (Table 3). When the relationship between the different T-cell responses to r-Pf155/RESA and the different peptides was investigated as exemplified in Table 4 for P1, no statistically significant correlation in individual donors was seen. IL-4 producing cells were frequently found in donors whose sera contained antibodies to the corresponding peptide. However, there was no significant correlation in individual donors between the presence of serum antibodies to the different peptides and the different T-cell responses (Fig. 2). The donors in Fig. 2 represent 12 individuals from whom enough lymphocytes were obtained to perform all the tests.

4. Discussion Pf155/RESA has earlier been shown to contain immunodominant T- and B-cell epitopes in the repetitive regions of the molecule (Rzepczyk et al., 1988; TroyeBlomberg et al., 1989; Kabilan et al., 1994; Udomsangpetch et al., 1994). Some of these studies (Rzepczyk et al., 1988; Troye-Blomberg et al., 1989; Fievet et al., 1995) suggested the existence of T-cell epitopes also in the N-terminal non-repeat region. The aim of the present study was to delineate antigenic determinants in this region recognised by T-cells or antibodies from clinically immune individuals. For

0.9 90.1 95 9 26 3.5 9 2

1.8 90.3 409 19 0.9 9 2

1.2 90.3 11 9 3 0.9 9 0.3

P2 0.9 90.2 992 1.0 9 0.3

P3 1.09 0.1 993 1.0 9 0.2

P4

1.09 0.2 6 92 2.09 0.8

P5

1.5 90.7 7 92 1.59 0.5

P6

1.09 0.6 892 0.89 0.2

P7

1.0 90.2 6 92 0.89 0.6

P8

Values are mean 9S.E.M.; SI, stimulation index; IFN-g, number of IFN-g secreting cells/2×105 PBMC; IL-4, number of IL-4 secreting cells/2×105 PBMC.

SI (n=16) IFN-g (n= 20) IL-4 (n= 14)

P1

r-Pfl55

Stimulants

Table 3 T-cell responses to different peptides

A. Kulane et al. / Acta Tropica 68 (1997) 37–51 45

Fig. 2. Individual responses against the r-Pf155/RESA and peptides (P1 – P8) from N-terminal region of the Pf155/RESA molecule. Threshold for SI, IFN-g and IL-4 release, peptide-specific antibodies are calculated as described in Section 2. Antibodies to repeat sequence covered by the r-Pf155RESA were extensively studied in previous reports (Perlmann et al., 1989). ( ) SI B 2.5, IFN-g B background, IL-4 B background, Ab B background; (a), SI = 2.5 –5, IFN-g= background −30, IL-4 = background − 5, Ab=background − 5; ( ), SI\ 5, IFN-g\ 30, IL-4\ 5, Ab\ 5.

46 A. Kulane et al. / Acta Tropica 68 (1997) 37–51

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this purpose, a panel of overlapping peptides corresponding to the Pf155/RESA sequence 171 – 227 was synthesised. The peptides were used to investigate proliferative T-cell responses as well as the number of IFN-g and IL-4 secreting cells in P. falciparum-primed individuals. All donors were from one village in Senegal, with a high level of malaria prevalence and moderate seasonal variations in transmission. All individuals were adults, had lived all their life in endemic areas and had elevated plasma levels of anti-malarial IgG antibodies. Antibodies reactive with conformational epitopes within an antigen are not detected by peptide-based epitope mapping. Still several of the peptides from the N-terminal non-repeat regions also contained antibody binding sequences. Out of the eight overlapping peptides two (P4: GDIIKKMQTLWDEIM and P5: MQTLWDEIMDINKRK) reacted significantly with most of the sera, indicative of the presence of a B-cell epitope within the amino acid sequence 192–200 (MQTLWDEIM) which is common to both P4 and P5. However, since each peptide might contain unique antibody binding structures further epitope mapping using truncated peptides is required to define that epitope. The most frequent and strongest T-cell responses were to the r-Pf155/RESA. With regard to proliferation and IFN-g production, a crude Pf155/RESA preparation has been shown to induce a response in 72% of the individuals living in a malaria endemic area of the Gambia (Troye-Blomberg et al., 1989). The individual responses to the various peptides varied markedly. Each donor could be stimulated above background by more than one peptide, but none of the peptides elicited a response in all donors. The overall frequency of responses to the peptides ranged between 38 and 71%. In general there was no correlation between the r-Pf155/RESA and peptides from the N-terminal part of Pf155. This was expected as r-Pf155/RESA lacks 30% of the Pf155/RESA sequence including the N-terminal region 171 – 227. The most frequent IFN-g responses were obtained with P1 (70%) while the most frequent IL-4 responses were obtained with P1, P4–P6 (43–50%). The peptides P2 and P4 induced proliferative responses in 13% of the donors tested. A peptide containing the sequence 176 – 199 has earlier been shown to induce proliferative

Table 4 Association between proliferation, IFN-g and IL-4 production induced by r-Pf155 and P1

Proliferation (n= 16) IFN-gELISPOT (n=20) IL-4 ELISPOT (n=14)

+/+

+/−

−/+

−/−

0 13 1

5 2 2

1 1 6

10 4 5

+/+, number of positive responders to both r-Pfl55/RESA and P1; −/−, number of non-responders to both r-Pfl55/RESA and P1; +/−, number of positive responders only to r-Pfl55/RESA; −/+, number of positive responders only to P1; n, total number of donors tested.

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responses in 30 – 50% of Melanesians (Rzepczyk et al., 1988). The higher percentage of responders in the Melanesian donors might indicate differences in transmission, genetics or that the peptide used in their study was longer and thus contained several epitopes. Although the frequency of responders was quite high, the magnitude of responses to the peptides was low in most individuals. This was probably due to the fact that the PBMCs were tested with short synthetic peptides, each representing a single T-cell epitope as contrasted to the larger antigens containing several epitopes. Whether the lack of response in some donors is a reflection of immune status, reallocation of cells outside the peripheral compartment (Hviid et al., 1991), insufficient sensitivity of the assays or, genetic restriction is presently unknown. Attempts to relate peptide responses to certain MHC class II haplotypes were difficult in the present study due to the great MHC class II polymorphism of the study group and in agreement with earlier findings (Sjo¨berg et al., 1992). IFN-g and IL-4 production are generally believed to be a reflection of a TH1 or TH2 type of responses, respectively (Mosmann et al., 1986; Romagnani, 1992). In the present study, no clear associations or dissociations were found between proliferation and production of the two investigated cytokines. Although IL-4 producing cells were frequently found in donors whose sera contained antibodies to the corresponding peptide, there was no absolute correlation in agreement with what has been reported earlier (Troye-Blomberg et al., 1990). Cells from many donors having anti-peptide antibodies could also be induced to produce IFN-g. Thus, proliferation, IFN-g and IL-4 production are not always mutually exclusive. However, whether this is a reflection of double producers of the THO phenotype as described for the mouse (Street et al., 1990) or the presence of cross-reacting T-cells primed to different antigens remains to be elucidated. In any event, these data emphasise the importance of including several parameters for T-cell activation, when evaluating the responses induced by different epitopes in a given population. In conclusion, the present study confirms and extends earlier investigations that the N-terminal region of Pf155/RESA contains several B- and T-cell epitopes recognised by antibodies and cells from many malaria-immune donors. This region is non-variant and preliminary results suggest that affinity purified antibodies recognising some of these sequences are efficient inhibitors of merozoite invasion into new erythrocytes in vitro. Thus, the N-terminal non-repeat region of Pf155/ RESA should be considered in the design of subunit vaccines against the asexual blood stages of P. falciparum and as a tool for future immunoepidemiological studies.

Acknowledgements This work was supported by grants from UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, Swedish Agency for Research Cooperation with Developing Countries (SIDA, SAREC), the Swedish

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Medical Research Council, the Swedish National Board for Laboratory Animals, Bergvall’s foundation and Ministere de la Cooperation et du Development (Paris). We would like to thank El Hadji Malik Fall and Ababacar Diouff for their excellent technical assistance.

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