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RESA antigen
Immunology Letters, 19 (1988) 229-234
Elsevier IML 01115
T cell reactivity of defined peptides from a major Plasmodium falciparum vaccine candidate: the Pf155/RESA antigen M. T r o y e - B l o m b e r g 1, L. Kabilan ~, E. M. Riley 2, J. O r t l u n d ~, G. A n d e r s s o n 4, H. P e r l m a n n ~, O. O l e r u p 5, B. H 6 g h 3, E. Petersen 3, R. W. S n o w 2, A. B j 6 r k m a n 3, B. M. G r e e n w o o d 2 a n d P. P e r l m a n n 1 IDepartment of Immunology, University of Stockholm, S-106 91 Stockholm, Sweden," 2Medical Research Council Laboratories, P.O. Box 273, Fajara, Near Banjul, The Gambia; 3Yekepa Clinical Research Unit, Liberian Institute of Medical Research, Yekepa, Liberia and Department of Infectious Diseases, Karolinska Institute, Roslagstulls Hospital, S-114 89 Stockholm, Sweden; 4Research and Development, Biochemistry, Kabi Vitrum, S-112 87 Stockholm, Sweden; 5Centerfor Biotechnology and Dept. of Clinical Immunology, Karolinska Institute at Huddinge Hospital, S-141 86 Huddinge, Sweden
(Received 21 June 1988; accepted 22 August 1988)
1. Summary Several immunodominant B-cell epitopes of the P. f a l c i p a r u m antigen blood stage Pf155/RESA, a
major vaccine candidate antigen, are located in the molecular regions containing amino acid repeats. We started to map Pf155/RESA for T cell reactive epitopes. For this purpose, short synthetic peptides corresponding to the 3 '- and 5' repeat regions of the molecule as well as to non-repeated sequences outside these regions were prepared. T ceils from P. falciparum primed donors from two highly endemic areas o f Africa were tested for their responsiveness to the peptides by thymidine incorporation and/or interferon gamma (IFN-7) release. There was a considerable variation in the response to the different peptides. However, the strongest and most frequent responses were seen with a few peptides from the 3 'and 5'-repeat regions. Thus, the immunodominant B cell epitope regions of Pf155/RESA, contain several T cell epitopes. Since the repeat regions are known to be conserved in different P. f a l c i p a r u m strains, the T cell epitopes reported here may be suitable constituents o f a P. f a l c i p a r u m subunit vaccine.
2. Introduction
Plasmodium falciparum malaria, transmitted by the Anopheles mosquito, is a major parasitic disease in the developing world. It is estimated that between 200 and 400 million people are acutely infected every year. Although clinical immunity to malaria is acquired under natural conditions, it requires repeated infections and takes years to develop. The fact that immunity does develop, together with the rapid spread of resistance of the mosquito to insecticides and of the parasite to almost all available antimalarial drugs, has made development of efficient antimalarial vaccines a major goal in the present efforts to control the disease. Laboratory animals have been successfully vaccinated against malaria using crude parasite antigens [1]. However, such an approach is not feasible for humans, because of production and purification problems. Thus, a malaria vaccine suitable for human use will have to be a subunit vaccine using either recombinant DNA or synthetic peptide technology. Protection against malaria is, at least in part, antibody-mediated, since antibodies are believed to be important for the clearance of acute infections.
Key words." T cellepitopes; DefinedPlasmodiumfalciparum an-
tigens: Pf155/RESA Correspondence to: M. Troye-Blomberg,Dept. of Immunology, University of Stockholm, S-106 91 Stockholm, Sweden.
0165-2478 / 88 / $ 3.50 © 1988 ElsevierSciencePublishers B.V.(Biomedical Division)
229
However, it is well known that production of these antibodies is strictly regulated by the T cell system [2, 3]. Thus, helper T cells are essential for the induction of high levels of antimalarial antibodies. However, in certain rodent malaria models T celldependent but antibody-independent mechanisms appear to be solely responsible for immune protection [2]. Furthermore, recent studies in mice indicate that T cells may play a critical role for inhibition of parasite development in the liver following sporozoite infection [4, 5]. Likewise, T cells have also been shown to be important for the clearance of gametocytes and therefore for the development of transmission-blocking immunity [6]. Recent studies have demonstrated that antibody-independent sporozoite-induced immunity in normal mice resides in the CD8 (cytotoxic) subset of effector T cells [7, 8]. CD8 + T cells might function by being cytotoxic to infected liver cells or by releasing interferongamma (IFN-3,), known to block the development of parasites in the liver [4]. In general IFN-7 appears to be a good indicator of an existing T-cell immunity in P. falciparum malaria [9-11]. Thus, the capacity of an immunogen to induce efficient protective immunity to malaria depends largely on its T cellactivating potential, required both for eliciting efficient and long-lasting antibody formation and antibody-independent cell-mediated immunity. For this reason immunogens included in a subunit vaccine should contain both B and T cell-activating sites, preferably from the same molecule, to ensure anamnestic responses following reinfection after vaccination. 3. Pf155/RESA: a major vaccine candidate against the asexual blood stages of P. falciparum Pf155/RESA is generally believed to be a major candidate for a merozoite vaccine [12-14]. This antigen has been shown to contain two extensive blocks of tandemly repeated short amino acid sequences [15, 16]. The major units in its C-terminal repeat block are the sequence GluGluAsnValGluHisAspAla (EENVEHDA in one-letter code), tandemly repeated 5×, and the first half of this sequence (EENV) repeated > 30×, including a few variants and deletions [16]. The other repeat block in the middle of the molecule consists of AspAspGluHisValGluGluProThrValAla (DDEHVEEPTVA) repeated 230
2 x, and with major variations and deletions, another 5x. These repeat regions of Pf155 have been shown to contain some of the molecule's immunodominant B cell epitopes [17-19] and to be highly conserved in different P. falciparum strains [18]. To investigate the T cell activating capacity of Pf155 we have set up autologous T/B cooperation systems and lymphokine assays permitting assessment of both T cell stimulation and T celldependent secretion of anti-malarial antibodies in vitro [20, 21]. Intact Pf155 was shown to induce in vitro proliferation, interleukin 2 release, and IFN-7 production in T cells from donors primed to P. falciparum by previous infections [21]. In addition, responding T cells from Pf155-seropositive donors induced autologous B cells to secrete anti-Pf155 antibodies into culture supernatants [20]. These in vitro studies confirm that the intact Pf155 molecule possesses epitopes required for the stimulation of T helper cells and perhaps also for the induction of antibody independent cellular immunity. 4. T cell epitope mapping of the C-terminal repeat region of Pf155/RESA To define potential T cell activating epitopes in the immunodominant B cell regions in the C-terminal of Pf155, synthetic peptides representing repeated sequences from this region were prepared and studied for their capacity to induce in vitro proliferation of T cells from healthy African donors who were all primed to P. falciparum by previous infections. In this study 96 donors (adult males) were investigated in detail [22]. All were from an endemic P. falciparum area in Liberia [23], and all were seropositive when tested for anti-P falciparum antibodies by conventional immunofluorescence. Moreover, 75% had anti-Pf155 titers [12] ranging from 1:50-1:25 000. In the T cell proliferation assay, there was no correlation between these serum titers and the magnitude of the lymphocyte response. Totally, approximately 50% of the donors responded positively to intact Pfl 55, but only 6507oof the Pf155 responders showed positive T cell stimulation after exposure to any of the peptides, suggesting the existence of additional T cell epitopes outside the C-terminal repeat region. In most instances donors who did not respond to intact Pf155 did not respond to any of the peptides either.
The best and most frequent responses were obtained with a 20-mer consisting essentially o f 4 EENV-repeats surrounding the tripeptide EEV. Equivalent responses were obtained with other peptides based on the repeat unit EENV and variants thereof. These peptides were predicted to have high alpha-amphipathic scores and therefore to be potential T cell epitopes [24]. The minimal epitope involved in these reactions appears to comprise 3 EENV-units. Some of the EENV-responders also responded weakly to dimers o f the octapeptide EENVEHDA, with a low alpha-amphipathic score. However, a few donors who did not respond to EENV-based peptides nevertheless gave elevated responses to the octapeptide dimer. From these results it cannot be concluded to what extent the lymphocyte reactions obtained with the different peptides represent responses of different cells recognizing different epitopes or are crossreactions. As the peptides are very similar in sequences it is likely that they represent a few overlapping and cross-reacting epitopes. Current experiments with T cell clones support this conclusion. The fact that some donors who responded poorly to the EENV repeat-peptides were significantly stimulated by the EENVEHDA-dimer suggests that the latter formed distinct non-cross-reacting epitopes seen by the T cells o f only some Pf155 responders, perhaps because of differences in MHC-restriction. O f the above mentioned donors 31 were also studied in detail for IFN-7 release after in vitro T cell stimulation with intact Pf155 or synthetic peptides. These donors were selected because their T cells released significant amounts of IFN-7 when stimulated with the T cell mitogen leukoagglutinin (La). Without antigen stimulation, no or very small amounts of IFN-7 were detected in the supernatants. Approximately 50°7o o f the donors released IFN-7 significantly above background when incubated with intact Pf155 or synthetic peptides. As in the thymidine incorporation assay, only donors responding to intact protein also responded to the peptides. IFN-7 release was not correlated to antiPf155 serum titers. In accordance with previous results [9, 21], IFN-7 release also did not correlate with induction o f DNA-synthesis, suggesting that the two assays may measure stimulation o f primed T cells belonging to different subsets [25, 26]. The results indicate that the IFN-7 is an important com-
plement to the proliferation assay and should be included in the test panel whenever one wants to determine the degree o f T cell sensitization in different donor populations. 5. T cell epitopes outside the C-terminal repeat region of Pf155/RESA To define other possible T cell sites on Pf155/RESA, peptides (16-20 amino acids long) corresponding to repeated sequences from the 5 ' repeat region and to sequences outside the repeat regions were synthesized. The peptides were used for investigating proliferative T-cell responses and IFN7 production in vitro of Pfalciparum primed donors from one village with high but seasonal malaria transmission in the Gambia. During a two-week period at the end o f the malaria transmission period 46 adult males were studied; 67% of these donors had anti Pf155 serum titers (ranging from 1:50-1:25000). Even with this material there was a considerable variation in the responses to the different peptides. However, as exemplified with the results obtained with eight of the donors (Fig. 1), the strongest and most frequent responses were seen with peptides from the 3' - and the 5' -repeat regions, while peptides representing sequences outside the repeat region were less active. No correlation between proliferation and IFN-7 production was seen and neither o f these responses were correlated with Pf155 serum titers. The overall number of donors responding to Pf155 and the different peptides with IFN-3, was similar to that responding in the proliferation assay, but the responses were not correlated in individual donors, again emphasizing the importance o f including measurements of both proliferation and IFN- 7 production, when investigating T cell activation. Figure 2 shows a summary of the results. In this graph, the percentage o f donors responding above background by proliferation and/or IFN-3, release to each peptide is shown. As can be seen, 41°70 (range 38-69°7o) of the donors responded to the peptides from the 3' - and 5' -repeat regions, as compared to 31% (21 - 41%) that responded to regions outside the repeats. It may be noted that the peptides inducing the strongest response contain the 8-amino acid repeat unit E E N V E H D A and a dimer of the 4amino acid repeat unit EENV. The possibility that 231
Sl 1~5'~
r'
3' - -
3 I r----- 5'--~
'
i
60-
0
20
Peptldes
Fig. l. Ordinate: Mean stimulation of T cells from 8 donors primed to P..falciparum by natural infection expressed as stimulation index (SI) (= stimulation with peptides/stimulation in absence of stimulants). SI was calculated after 6 days o f culture with optimal concentrations o f Pf155 a n d uninfected RBC-ghost (Eo) (5 #g/ml) or peptides (0.1 or 1/~M). Abscissa: Peptides corresponding to repeat sequences located in the 5 ' - or 3'-repeat regions of P f I 5 5 / R E S A or located outside the repeats as indicated. [] = response to Pf155; [] = response to E o.
this peptide contains two non-crossreacting T cell epitopes is under investigation.
nl
P e p t ides
Fig. 2. Ordinate: % o f responding donors = expressed as donors with an SI _>2.5 a n d / o r IFN- 7 release above b a c k g r o u n d / n u m b e r o f donors tested with each peptide. SI was calculated after 6 days o f culture and IFN- 7 in culture supernatants after 5 days o f culture. For other explanations see Fig. I.
ed to establish this. At this stage, we cannot exclude that unresponsiveness may reflect the donors' immune status or a partial loss of antigenic sites from the test antigen during isolation.
7. Conclusions 6. MHC-restriction and lymphocyte responses MHC-restriction has important implication for malaria vaccine development [27-30]. We have started to address the question, how the T cell responses described here are affected by the donors' M H C type and, in particular, to what extent the apparent unresponsiveness o f certain donors reflects M H C (class II) restriction. Thus far, leukocyte material from 27 o f the Liberian donors has been fingerprinted in Southern blot analysis with class II-specific DNA restriction fragments. As expected, since these donors were randomly selected, very few had the same set-up o f M H C class II haplotypes, making evaluation o f the relationship between M H C type and malaria responsiveness very difficult. Presently, we are also typing the Gambian donors described above. This material contains samples from familyrelated donors and is, therefore, expected to facilitate analysis o f class II haplotypes. Thus far, available data do not prove that the apparent nonresponsiveness to Pf155 o f certain donors was due to genetic restriction; further experiments are need232
The present results show that the two repeat regions o f Pf155/RESA, known to contain some of the molecule's immunodominant B cell epitopes, also include some of its important T cell epitopes, capable o f stimulating T cells to proliferate and/or to release IFN-7. In this respect, Pf155 appears to be different from the CSP o f P. falciparum, where the immunodominant T cell domains have recently been shown to map to polymorphic regions outside its B cell immunodominant repeat regions [31]. Since the C-terminal repeat regions of Pf155 appear to be conserved among different P.falciparum strains [16, 18], the T cell activating sequences described here appear to be suitable components to be included in a subunit vaccine. They also provide a basis for epidemiological studies relating epitope-specific T cell responses in vitro to clinical immunity and malaria endemicity.
Acknowledgements We gratefully acknowledge the cooperation o f the blood donors from the Yekepa area o f the Liberian
Institute for Biomedical Research and the people of W a l l a l a n in T h e G a m b i a f o r t h e i r c o o p e r a t i o n . T h i s w o r k was s u p p o r t e d b y g r a n t s f r o m t h e R o c k e f e l l e r F o u n d a t i o n ( N e w York), t h e S w e d i s h M e d i c a l Research Council, the Swedish Agency for Research Cooperation with Developing Countries (SAREC), and the UNDP/World Bank/World Health Organiz a t i o n P r o g r a m m e for R e s e a r c h a n d T r a i n i n g in T r o p i c a l Diseases.
References [1] Desowitz, R. S. and Miller, L. H. (1980) WHO Bull. 6, 897. [2] Weidanz, W. P. and Long, C. A. (1988) Prog. Allergy 41,215. [3] Troye-Blomberg, M. and Perlmann, P. (1988) Prog. Allergy 41, 253. [4] Ferreira, A., Schofield, L., Enea, V., Schellekens, H., Van der Meide, P., Collins, W. E., Nussenzweig, R. S. and Nussenzweig, V. (1986) Science 232, 881. [5] Egan, J. E., Weber, J. L., Ballou, W., Majarian, W., Gordon, D.M., Hoffman, S.L., Wirtz, R.A., Schneider, I., Woollett, G., Hollingdale, M. R., Young, J. E and Hockmeyer, W. T. (1987) Science 236, 453. [6] Harte, P. G., Rogers, N. C. and Targett, G. A. T. (1985) Immunology 56, 1. [7] Schellekens, H., Nussenzweig, R. and Nussenzweig, V. (1987) Nature 330, 664. [8] Weiss, W. R., Sedegah, M., Beaudoin, R., Miller, L. H. and Good, M. E (1988) Proc. Natl. Acad. Sci. USA 85, 573. [9] Troye-Blomberg, M., Andersson, G., Stoczkowska, M., Shabo, R., Romero, P., Patarroyo, M.E., Wigzell, H. and Perlmann, P. (1985) J. Immunol. 135, 3498. [10] Riley, E. M., Jepsen, S., Andersson, G., Otoo, L. N. and Greenwood, B. M. (1988) Clin. Exp. Immunol. 71, 377. Ill] Riley, E. M., Andersson, G., Otoo, L. N., Jepsen, S. and Greenwood, B. M. (1988) Clin. Exp. Immunol. 73, 17. [12] Perlmann, H., Berzins, K., Wahlgren, M., Carlsson, J., Bj6rkman, A., Patarroyo, M. E. and Perlmann, P. (1986) J. Exp. Med. 159, 1686. [13] Coppel, R. L., Cowman, A. E, Anders, R. E, Bianco, A.E., Saint, R. B., Lingelbach, K. R., Kemp, D. J. and Brown, G. V. (1984) Nature (London) 310, 789. [14] Collins, M., Campbell, G. H., Brown, G. V., Kemp, D. J., Coppel, R. L., Skinner, J. C., Andrysiak, P. M., Favaloro, J. M., Corcoran, P. M., Broderson, J. R., Mitchell, G. E and Campbell, C. C. (1986) Nature (London) 232, 259. [15] Cowman, A. E, Coppel, R. L., Saint, R. B., Favaloro, J.,
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Crewther, P. E., Stahl, H. D., Bianco, A. E., Brown, G. V., Anders, R. E and Kemp, D. J. 0985) Mol. Biol. Med. 2, 207. Favaloro, J. M., Coppel, R. L., Corcoran, L. M., Foote, S. J., Brown, G. V., Anders, R. E and Kemp, D. J. 0986) Nucleic Acids Res. 14, 8265. Berzins, K., Perlmann, H., W~hlin, B., Carlsson, J., Wahlgren, M., Udomsangpetch, R., Bjorkman, A., Patarroyo, M. E. and Perlmann, P. (1986) Proc. Natl. Acad. Sci. USA 83, 1065. Perlmann, H. K., Berzins, K., W~thlin, B., Udomsangpetch, R., Ruangjirachuporn, W., Wahlgren, M. and Perlmann, H. P. 0987) J. Clin. Microbiol. 12, 2347. Perlmann, E, Berzins, K., Perlmann, H., Troye-Blomberg, M., Wahlgren, M. and W~hlin, B. (1988) in: Vaccines 1988 (R. A. Lerner, E Brown and H. Gin' burg, Eds.) pp. 183 - 187, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Kabilan, L., Troye-Blomberg, M., Andersson, G., Patarroyo, M. E. and Perlmann, P. 0987) Clin. Exp. Immunol. 68, 288. Troye-Blomberg, M., Kabilan, L., Andersson, G., Patarroyo, M. E. and Perlmann, P. (1987) in: Immune Regulation by Characterized Polypeptides, (G. Goldstein, J.-E Bach, and H. Wigzell, Eds.) Vol. 7, pp. 699-709, Liss, New York. Kabilan, L., Troye-Blomberg, M., Perlmann, P., Andersson, G., H6gh, B., Petersen, E., Bjorkman, A. and Perlmann, P. (1988) Proc. Natl. Acad. Sci. USA 85, 5659. Bj6rkman, A., Hedman, P., Brohult, P. O., Willcox, M., Diamant, I., Pehrsson, P. O., Rombo, L. and Bengtsson, E. 0985) Ann. Trop. Med. Parasit. 79, 239. Margaiit, H., Spouge, J. L., Cornette, J. L., Cease, K., DeLisi, C. and Berzofsky, J. A. (1987) J. Immunol. 138, 2213. Grabstein, K., Dower, S., Gillis, S., Urdal, D. and Larsen, A. (1987) J. Immunol. 136, 4503. Budd, R. C., Cerottini, J-C. and Mac Donald, H. R. (1987) J. Immunol. 138, 1245. Good, M. E, Berzofsky, J. A., Maloy, W. L., Hayashi, T., Fujii, N., Hockmeyer, W. T. and Miller, L. (1986) J. Exp. Med. 164, 665. Del Giudice, C., Cooper, J. A., Merino, J., Verdini, A. S., Pessi, A., Engers, H. D. and Corradin, G. (1986) J. Immunol. bert, P-H. (1986) J. Immunol. 137, 2952. Togna, A. R., Del Giudice, G., Verdini, A. S., Bonelli, E, Pessi, A., Engers, H. D. and Corradin, G. (1986) J. immunol. 137, 956. Guttinger, M., Caspers, P., Takacs, B., Trzeciak, A., GiUesen, D., Pink, J. R. and Sinigaglia, E (1988) EMBO J., 7, 2555. Good, M.F., Pombo, D., Quakyi, I.A., Riley, E.M., Houghten, R.A., Menon, A., Ailing, D. W., Berzofsky, J. A. and Miller, L. H. (1988) Proc. Natl. Acad. Sci. USA 85, 1199.
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Elsevier IML 01115
T cell reactivity of defined peptides from a major Plasmodium falciparum vaccine candidate: the Pf155/RESA antigen M. T r o y e - B l o m b e r g 1, L. Kabilan ~, E. M. Riley 2, J. O r t l u n d ~, G. A n d e r s s o n 4, H. P e r l m a n n ~, O. O l e r u p 5, B. H 6 g h 3, E. Petersen 3, R. W. S n o w 2, A. B j 6 r k m a n 3, B. M. G r e e n w o o d 2 a n d P. P e r l m a n n 1 IDepartment of Immunology, University of Stockholm, S-106 91 Stockholm, Sweden," 2Medical Research Council Laboratories, P.O. Box 273, Fajara, Near Banjul, The Gambia; 3Yekepa Clinical Research Unit, Liberian Institute of Medical Research, Yekepa, Liberia and Department of Infectious Diseases, Karolinska Institute, Roslagstulls Hospital, S-114 89 Stockholm, Sweden; 4Research and Development, Biochemistry, Kabi Vitrum, S-112 87 Stockholm, Sweden; 5Centerfor Biotechnology and Dept. of Clinical Immunology, Karolinska Institute at Huddinge Hospital, S-141 86 Huddinge, Sweden
(Received 21 June 1988; accepted 22 August 1988)
1. Summary Several immunodominant B-cell epitopes of the P. f a l c i p a r u m antigen blood stage Pf155/RESA, a
major vaccine candidate antigen, are located in the molecular regions containing amino acid repeats. We started to map Pf155/RESA for T cell reactive epitopes. For this purpose, short synthetic peptides corresponding to the 3 '- and 5' repeat regions of the molecule as well as to non-repeated sequences outside these regions were prepared. T ceils from P. falciparum primed donors from two highly endemic areas o f Africa were tested for their responsiveness to the peptides by thymidine incorporation and/or interferon gamma (IFN-7) release. There was a considerable variation in the response to the different peptides. However, the strongest and most frequent responses were seen with a few peptides from the 3 'and 5'-repeat regions. Thus, the immunodominant B cell epitope regions of Pf155/RESA, contain several T cell epitopes. Since the repeat regions are known to be conserved in different P. f a l c i p a r u m strains, the T cell epitopes reported here may be suitable constituents o f a P. f a l c i p a r u m subunit vaccine.
2. Introduction
Plasmodium falciparum malaria, transmitted by the Anopheles mosquito, is a major parasitic disease in the developing world. It is estimated that between 200 and 400 million people are acutely infected every year. Although clinical immunity to malaria is acquired under natural conditions, it requires repeated infections and takes years to develop. The fact that immunity does develop, together with the rapid spread of resistance of the mosquito to insecticides and of the parasite to almost all available antimalarial drugs, has made development of efficient antimalarial vaccines a major goal in the present efforts to control the disease. Laboratory animals have been successfully vaccinated against malaria using crude parasite antigens [1]. However, such an approach is not feasible for humans, because of production and purification problems. Thus, a malaria vaccine suitable for human use will have to be a subunit vaccine using either recombinant DNA or synthetic peptide technology. Protection against malaria is, at least in part, antibody-mediated, since antibodies are believed to be important for the clearance of acute infections.
Key words." T cellepitopes; DefinedPlasmodiumfalciparum an-
tigens: Pf155/RESA Correspondence to: M. Troye-Blomberg,Dept. of Immunology, University of Stockholm, S-106 91 Stockholm, Sweden.
0165-2478 / 88 / $ 3.50 © 1988 ElsevierSciencePublishers B.V.(Biomedical Division)
229
However, it is well known that production of these antibodies is strictly regulated by the T cell system [2, 3]. Thus, helper T cells are essential for the induction of high levels of antimalarial antibodies. However, in certain rodent malaria models T celldependent but antibody-independent mechanisms appear to be solely responsible for immune protection [2]. Furthermore, recent studies in mice indicate that T cells may play a critical role for inhibition of parasite development in the liver following sporozoite infection [4, 5]. Likewise, T cells have also been shown to be important for the clearance of gametocytes and therefore for the development of transmission-blocking immunity [6]. Recent studies have demonstrated that antibody-independent sporozoite-induced immunity in normal mice resides in the CD8 (cytotoxic) subset of effector T cells [7, 8]. CD8 + T cells might function by being cytotoxic to infected liver cells or by releasing interferongamma (IFN-3,), known to block the development of parasites in the liver [4]. In general IFN-7 appears to be a good indicator of an existing T-cell immunity in P. falciparum malaria [9-11]. Thus, the capacity of an immunogen to induce efficient protective immunity to malaria depends largely on its T cellactivating potential, required both for eliciting efficient and long-lasting antibody formation and antibody-independent cell-mediated immunity. For this reason immunogens included in a subunit vaccine should contain both B and T cell-activating sites, preferably from the same molecule, to ensure anamnestic responses following reinfection after vaccination. 3. Pf155/RESA: a major vaccine candidate against the asexual blood stages of P. falciparum Pf155/RESA is generally believed to be a major candidate for a merozoite vaccine [12-14]. This antigen has been shown to contain two extensive blocks of tandemly repeated short amino acid sequences [15, 16]. The major units in its C-terminal repeat block are the sequence GluGluAsnValGluHisAspAla (EENVEHDA in one-letter code), tandemly repeated 5×, and the first half of this sequence (EENV) repeated > 30×, including a few variants and deletions [16]. The other repeat block in the middle of the molecule consists of AspAspGluHisValGluGluProThrValAla (DDEHVEEPTVA) repeated 230
2 x, and with major variations and deletions, another 5x. These repeat regions of Pf155 have been shown to contain some of the molecule's immunodominant B cell epitopes [17-19] and to be highly conserved in different P. falciparum strains [18]. To investigate the T cell activating capacity of Pf155 we have set up autologous T/B cooperation systems and lymphokine assays permitting assessment of both T cell stimulation and T celldependent secretion of anti-malarial antibodies in vitro [20, 21]. Intact Pf155 was shown to induce in vitro proliferation, interleukin 2 release, and IFN-7 production in T cells from donors primed to P. falciparum by previous infections [21]. In addition, responding T cells from Pf155-seropositive donors induced autologous B cells to secrete anti-Pf155 antibodies into culture supernatants [20]. These in vitro studies confirm that the intact Pf155 molecule possesses epitopes required for the stimulation of T helper cells and perhaps also for the induction of antibody independent cellular immunity. 4. T cell epitope mapping of the C-terminal repeat region of Pf155/RESA To define potential T cell activating epitopes in the immunodominant B cell regions in the C-terminal of Pf155, synthetic peptides representing repeated sequences from this region were prepared and studied for their capacity to induce in vitro proliferation of T cells from healthy African donors who were all primed to P. falciparum by previous infections. In this study 96 donors (adult males) were investigated in detail [22]. All were from an endemic P. falciparum area in Liberia [23], and all were seropositive when tested for anti-P falciparum antibodies by conventional immunofluorescence. Moreover, 75% had anti-Pf155 titers [12] ranging from 1:50-1:25 000. In the T cell proliferation assay, there was no correlation between these serum titers and the magnitude of the lymphocyte response. Totally, approximately 50% of the donors responded positively to intact Pfl 55, but only 6507oof the Pf155 responders showed positive T cell stimulation after exposure to any of the peptides, suggesting the existence of additional T cell epitopes outside the C-terminal repeat region. In most instances donors who did not respond to intact Pf155 did not respond to any of the peptides either.
The best and most frequent responses were obtained with a 20-mer consisting essentially o f 4 EENV-repeats surrounding the tripeptide EEV. Equivalent responses were obtained with other peptides based on the repeat unit EENV and variants thereof. These peptides were predicted to have high alpha-amphipathic scores and therefore to be potential T cell epitopes [24]. The minimal epitope involved in these reactions appears to comprise 3 EENV-units. Some of the EENV-responders also responded weakly to dimers o f the octapeptide EENVEHDA, with a low alpha-amphipathic score. However, a few donors who did not respond to EENV-based peptides nevertheless gave elevated responses to the octapeptide dimer. From these results it cannot be concluded to what extent the lymphocyte reactions obtained with the different peptides represent responses of different cells recognizing different epitopes or are crossreactions. As the peptides are very similar in sequences it is likely that they represent a few overlapping and cross-reacting epitopes. Current experiments with T cell clones support this conclusion. The fact that some donors who responded poorly to the EENV repeat-peptides were significantly stimulated by the EENVEHDA-dimer suggests that the latter formed distinct non-cross-reacting epitopes seen by the T cells o f only some Pf155 responders, perhaps because of differences in MHC-restriction. O f the above mentioned donors 31 were also studied in detail for IFN-7 release after in vitro T cell stimulation with intact Pf155 or synthetic peptides. These donors were selected because their T cells released significant amounts of IFN-7 when stimulated with the T cell mitogen leukoagglutinin (La). Without antigen stimulation, no or very small amounts of IFN-7 were detected in the supernatants. Approximately 50°7o o f the donors released IFN-7 significantly above background when incubated with intact Pf155 or synthetic peptides. As in the thymidine incorporation assay, only donors responding to intact protein also responded to the peptides. IFN-7 release was not correlated to antiPf155 serum titers. In accordance with previous results [9, 21], IFN-7 release also did not correlate with induction o f DNA-synthesis, suggesting that the two assays may measure stimulation o f primed T cells belonging to different subsets [25, 26]. The results indicate that the IFN-7 is an important com-
plement to the proliferation assay and should be included in the test panel whenever one wants to determine the degree o f T cell sensitization in different donor populations. 5. T cell epitopes outside the C-terminal repeat region of Pf155/RESA To define other possible T cell sites on Pf155/RESA, peptides (16-20 amino acids long) corresponding to repeated sequences from the 5 ' repeat region and to sequences outside the repeat regions were synthesized. The peptides were used for investigating proliferative T-cell responses and IFN7 production in vitro of Pfalciparum primed donors from one village with high but seasonal malaria transmission in the Gambia. During a two-week period at the end o f the malaria transmission period 46 adult males were studied; 67% of these donors had anti Pf155 serum titers (ranging from 1:50-1:25000). Even with this material there was a considerable variation in the responses to the different peptides. However, as exemplified with the results obtained with eight of the donors (Fig. 1), the strongest and most frequent responses were seen with peptides from the 3' - and the 5' -repeat regions, while peptides representing sequences outside the repeat region were less active. No correlation between proliferation and IFN-7 production was seen and neither o f these responses were correlated with Pf155 serum titers. The overall number of donors responding to Pf155 and the different peptides with IFN-3, was similar to that responding in the proliferation assay, but the responses were not correlated in individual donors, again emphasizing the importance o f including measurements of both proliferation and IFN- 7 production, when investigating T cell activation. Figure 2 shows a summary of the results. In this graph, the percentage o f donors responding above background by proliferation and/or IFN-3, release to each peptide is shown. As can be seen, 41°70 (range 38-69°7o) of the donors responded to the peptides from the 3' - and 5' -repeat regions, as compared to 31% (21 - 41%) that responded to regions outside the repeats. It may be noted that the peptides inducing the strongest response contain the 8-amino acid repeat unit E E N V E H D A and a dimer of the 4amino acid repeat unit EENV. The possibility that 231
Sl 1~5'~
r'
3' - -
3 I r----- 5'--~
'
i
60-
0
20
Peptldes
Fig. l. Ordinate: Mean stimulation of T cells from 8 donors primed to P..falciparum by natural infection expressed as stimulation index (SI) (= stimulation with peptides/stimulation in absence of stimulants). SI was calculated after 6 days o f culture with optimal concentrations o f Pf155 a n d uninfected RBC-ghost (Eo) (5 #g/ml) or peptides (0.1 or 1/~M). Abscissa: Peptides corresponding to repeat sequences located in the 5 ' - or 3'-repeat regions of P f I 5 5 / R E S A or located outside the repeats as indicated. [] = response to Pf155; [] = response to E o.
this peptide contains two non-crossreacting T cell epitopes is under investigation.
nl
P e p t ides
Fig. 2. Ordinate: % o f responding donors = expressed as donors with an SI _>2.5 a n d / o r IFN- 7 release above b a c k g r o u n d / n u m b e r o f donors tested with each peptide. SI was calculated after 6 days o f culture and IFN- 7 in culture supernatants after 5 days o f culture. For other explanations see Fig. I.
ed to establish this. At this stage, we cannot exclude that unresponsiveness may reflect the donors' immune status or a partial loss of antigenic sites from the test antigen during isolation.
7. Conclusions 6. MHC-restriction and lymphocyte responses MHC-restriction has important implication for malaria vaccine development [27-30]. We have started to address the question, how the T cell responses described here are affected by the donors' M H C type and, in particular, to what extent the apparent unresponsiveness o f certain donors reflects M H C (class II) restriction. Thus far, leukocyte material from 27 o f the Liberian donors has been fingerprinted in Southern blot analysis with class II-specific DNA restriction fragments. As expected, since these donors were randomly selected, very few had the same set-up o f M H C class II haplotypes, making evaluation o f the relationship between M H C type and malaria responsiveness very difficult. Presently, we are also typing the Gambian donors described above. This material contains samples from familyrelated donors and is, therefore, expected to facilitate analysis o f class II haplotypes. Thus far, available data do not prove that the apparent nonresponsiveness to Pf155 o f certain donors was due to genetic restriction; further experiments are need232
The present results show that the two repeat regions o f Pf155/RESA, known to contain some of the molecule's immunodominant B cell epitopes, also include some of its important T cell epitopes, capable o f stimulating T cells to proliferate and/or to release IFN-7. In this respect, Pf155 appears to be different from the CSP o f P. falciparum, where the immunodominant T cell domains have recently been shown to map to polymorphic regions outside its B cell immunodominant repeat regions [31]. Since the C-terminal repeat regions of Pf155 appear to be conserved among different P.falciparum strains [16, 18], the T cell activating sequences described here appear to be suitable components to be included in a subunit vaccine. They also provide a basis for epidemiological studies relating epitope-specific T cell responses in vitro to clinical immunity and malaria endemicity.
Acknowledgements We gratefully acknowledge the cooperation o f the blood donors from the Yekepa area o f the Liberian
Institute for Biomedical Research and the people of W a l l a l a n in T h e G a m b i a f o r t h e i r c o o p e r a t i o n . T h i s w o r k was s u p p o r t e d b y g r a n t s f r o m t h e R o c k e f e l l e r F o u n d a t i o n ( N e w York), t h e S w e d i s h M e d i c a l Research Council, the Swedish Agency for Research Cooperation with Developing Countries (SAREC), and the UNDP/World Bank/World Health Organiz a t i o n P r o g r a m m e for R e s e a r c h a n d T r a i n i n g in T r o p i c a l Diseases.
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