An invariant, "universal" T-cell epitope in the P. falciparum circumsporozoite protein.

An invariant, "universal" T-cell epitope in the P. falciparum circumsporozoite protein* F. Sinigaglia, M. Guttinger, H. Matile, & J.R.L. Pink Although there are important obstacles to malaria vaccine development, we believe they might be overcome by a strategy of searching for conserved regions of a vaccine candidate that are recognized in association with many different HLA molecules and, if necessary, deliberately modifying the conserved sequences to improve their immunogenicity for T cells. This approach is illustrated by work on the circumsporozoite (CS) protein of Plasmodium falciparum, which covers the surface of the malaria sporo- zoite and is the best characterized of current vaccine candidates. Introduction Researchers who are attempting to produce vaccines against malaria are confronted with major difficulties that fall into two main categories (1). (a) The vari- ability of the pathogen, a well-known problem with influenza and definitely a potential problem with malaria, where a vaccine would be useless if directed against highly variable malaria epitopes. (b) The vari- ability of the human population's ability to respond. Many if not most peptides have limited abilities to bind to or be presented in association with different HLA molecules. What is needed in a synthetic vaccine is a peptide which binds to a large number of different HLA variants and to which most or all individuals can respond. Taking a slightly unusual approach, we have found a peptide derived from the circumsporozoite (CS) protein, the coat protein of the malaria sporo- zoite, whose characteristics offer possible solutions to both of these problems, and which is therefore a good candidate for inclusion in a vaccine. Methods and results 1. A peptide to which most Individuals can respond We started by testing the primary in vitro responses of unimmunized donors against three CS-protein- derived peptides which were predicted to contain T-cell epitopes (2). The majority of individuals re- sponded to a peptide, CS.T3, whose sequence, DIEK- KIAKMEKASSVFVVNS, corresponds to residues 378-398 of the CS protein except that two cysteines at positions 384 and 389 were substituted with alanine residues. This suggested that the CS.T3 peptide could * From the Central Research Units, Bld. 69/211, F. Hoffmann-La Roche Ltd, CH-4002 Basle, Switzerland. Correspondence to Dr F. Sinigaglia at this address. associate with many different MHC molecules and might be a good candidate for a "universal" T-cell epitope. We therefore challenged mononuclear cells from the peripheral blood of 20 malaria non-immune donors with the peptide in vitro. From the stimulated cells of 8 donors, close to 300 CS.T3-specific T-cell clones were derived. Analysis of the restriction specifi- city of the clones revealed that the peptide could be recognized in combination with at least 8 different DR molecules including DR1, 2, 4, 5, W6, 7, 9 (3) and 8 (F.S., unpublished results). Because of the wide range of DR antigens able to present the CS.T3 peptide, it was possible that the peptide might contain more than one distinct T-cell epitope. In fact, the analysis of the responses of T-cell clones to a series of peptides truncated at either the N or C-terminus of the CS.T3 sequence indicated that closely overlapping epitopes between residues 380 and 396 were recognized in slightly different ways in association with different DR antigens (Fig. 1). We confirmed that peptide CS.T3 could bind to different DR antigens by testing it and several other peptides (comprising together about 50% of the CS protein) in a peptide competition assay (4). The com- petition assay asks whether the "test peptide" can inhibit the response of a T-cell clone to its specific antigen. We used two T-cell clones, one that recog- nized peptide 325-341 of the CS protein in association with DR5, and one specific for peptide 260-273 of the P. falciparum blood-stage p190 protein associated with DRw6 molecules. We found that CS.T3 was the only peptide able to compete with the binding of the stimulator peptides to DR5 and DRw6. CS.T3 was also the only peptide out of the five tested to bind to DRI using a direct binding assay on soluble DR1 molecules (12). It is possible that the modified 378-398 sequence binds more strongly to certain DR antigens than does the natural sequence (although this has not been tested directly), since as shown in Fig. 2 the responses of T-cell clones, restricted to particular DR types and 94 Bulletin of the World Health Organization, 68 (Suppl.): 94-98 (1990) Wide responsiveness to an Invariant CS sequence Fig. 1. Definition of determinants In peptide CS.T3 recognized by T-cell clones restricted by different DR alleles (3). D I EKK I AKMEKAS SVFNVVNS < to the CS.T3 peptide, are stronger than to the un- modified sequence. Such a result would be interesting, as it suggests that naturally occurring sequences can be improved as vaccine candidates by judicious selection of cross-reactive, more immunogenic variant peptides. H. The CS 378-398 sequence Is conserved As pointed out by De La Cruz et al. (5), one possible difficulty for the development of a P. falciparum mal- aria vaccine based on the CS protein is the variability of candidate T-cell epitopes in different strains of the parasite. Inspection of the sequences of the CS pro- teins of three isolates of P. falciparum revealed the presence of variation in only 9 aminoacids (5, 6), all of them in the regions flanking the repeats. Most vari- ation was seen in regions containing human T-cell epitopes (7), suggesting that the pressure for variation was due to the responses of immune T cells. Support for this idea came from the observation that T-cell clones recognizing a polymorphic region (residues 330-341) of the CS protein are sensitive to some of the naturally occurring amino acid substitutions (8). The five T-cell clones tested failed to recognize a peptide in which the Lys at position 339 was replaced by Gln, a naturally occurring variant. Similarly, De La Cruz et al. have shown that animals immunized with peptides based on one sequence (7G8 isolate) could not respond when challenged with variant peptides (LE5 and Wel isolates) (9). On the basis of these results and other evidence it has been argued that polymorphism may be advantageous to the parasite by enabling it to escape the T-cell-mediated immunity stimulated by genetically different isolates (1, 5). An analysis of 5 different isolates of P.falciparum (Wel, LE5, T4R, NF54 and 7G8), however, showed that the sequence of residues 378-398 is completely conserved (10). Thus, if the CS.T3 peptide does func- tion to induce responses against the parasite, the protection it offers will not be limited to only a small subset of malaria isolates. Ill. The peptide can act as a helper determinant To examine whether the CS.T3 peptide could induce helper T-cell function in vivo, we coupled it to the B cell determinant (11) (NANP)3. Mice from several strains, which were normally unable to respond to the NANP repeats, produced antibodies to the repeats, as well as to sporozoites when injected with the CS.T3- (NANP)3 conjugate (3). This shows that the CS.T3 sequence is also recognized in association with many different mouse Ia molecules, and that it can indeed function, at least in mice, as a carrier determinant for responses to other parts of the CS protein. A putative vaccine is of course useless if the T cells elicited by it recognize the physiologically processed products of the natural pathogen poorly or not at all. However, because we know very little about the physiological pathways for "processing" a native protein antigen, or about the specificity of the pro- teolytic enzymes which generate the fragments de- stined to bind to the MHC class II molecules, it is WHO Bulletin OMS: Supplement Vol. 68 1990 DR1 DR2 DR4 DR5 DRw6 DR7 DR8 DR9 DRI DR2 DR4 DR5 DRw6 DR7 DR8 DR9 70 '9S F. SInigagia et al. Fig. 2. Effect of Ala-Cys substitutions on the response of T-cell clones to peptlde CS.T3. The response of clones BH.16 and DP.13, restricted to DR1 and DR4 respectively, was measured as 3H-thymidine incorporation of 2 x 104 cells after 2 days culture in the presence of autologous irradiated antigen-presenting cells and peptide CS.T3 (A) or its Cys- containing-homologue (A), whose sequence corresponds exactly to that of residues 378-398 of the CS protein. The Cys- containing homologue is still recognized by both clones, although at concentrations of about 100-fold higher than CS.T3. 30 20 0 40 E C) 0 10I 04- .01 20 0 E 0. 0 10 0-+ .01 BH.16 (DR1) .1 1 10 Peptide conc. (gg/mI) .1 1 Peptide conc. (gg/mi) 1 00 1 001 0 WHO Bulletin OMS: Supplement Vol. 68 199096 Wide responsiveness to an invariant CS sequence Fig. 3. Pre-lmnwnlzatlon with CS.T3 boot the antibody response to a subsequent Injection of sporozoltes. BALB/c mice (5 per group) were immunized with (U, A) or without (O, A) 50pg of peptide CS.T3 in complete Freund's adjuvant Four weeks later, all mice were injected i.v. with 105 P. falciparum sporozoites. Following sporozoite injection (day 0), the IgM (0, U) and IgG (A, A) antibody responses of each group to (NANP)50 were measured at various times. Antibody responses are given as mean optical density (OD) measured in an ELISA assay with individual mouse sera diluted 1:3000. 2.0- cn -j1.0 0.0 0 20 40 60 80 100 120 Day difficult to predict whether synthetic peptides, which by trial and error are found to stimulate T cells from immune individuals in vitro, are generated in vivo. We found that T-cell clones recognizing CS.T3 in associ- ation with several DR antigens also responded in vitro to the parasite-derived CS protein (2, 4, 8), as well as to a recombinant polypeptide containing the 378-398 sequence with the native protein's cysteine residues (4). In addition, BALB/c mice primed with the CS.T3 peptide generate a secondary response when boosted with sporozoites (Fig. 3; H. Matile et al., unpublished results), showing that in spite of the alanine substitu- tions the 378-398 region is seen in the native protein presented by sporozoites. We conclude that a P. falciparum vaccine containing the CS.T3 epitope linked covalently to a NANP polymer should be able to induce parasite-specific humoral and cellular immunity in the genetically diverse human popula- tion, and this immunity should be boosted by natural exposure to sporozoites. References 1. Good, M.F. et al. The real difficulties for malaria sporozoite vaccine development: nonresponsiveness and antigenic variation. Immunol. today, 9: 351-355 (1988). 2. Slnigaglia, F. et al. Epitopes recognized by human T lymphocytes on malaria circumsporozoite protein. Eur. j. immunol., 18: 633-636 (1988). 3. SinIgaila, F. at al. A malaria T-cell epitope recog- nized in association with most mouse and human MHC class 11 molecules. Nature, 336: 778-780 (1988). 4. Kligus, J. at al. Vaccine T-cell epitope selection by a peptide competition assay. Proc. Natl Acad. Sci., USA, 86: 1629-1633 (1989). 5. De La Cruz, V.F. et al. Sequence variation in putative functional domains of the circumsporozoite protein of Plasmodium falciparum: implication for vaccine de- velopment. J. biol. chem., 262: 11935-11939 (1987). 6. Del Porillo, H.A. at al. Circumsporozoite gene of a Plasmodium falciparum strain from Thailand. Mol. biochem. parasitol., 24: 289-294 (1987). WHO Bulletin OMS: Supplement Vol. 68 1990 97 F. Sinigaglia et al. 7. Good, M.F. et al. Human T-cell recognition of the circumsporozoite protein of Plasmodium falciparum: immunodominant T-cell domains map to the poly- morphic regions of the molecule. Proc. NatlAcad. Sci., USA, 85: 1199-1203 (1988). 8. Guttnger, M. et al. Human T cells recognize poly- morphic and non-polymorphic regions of the Plasmo- dium falciparum circumsporozoite protein. Eur. Mol. Biol. Org. (EMBO) j., 7: 2555-2558 (1988). 9. De La Cruz, V.F. at al. Lack of cross-reactivity between variant T-cell determinants from malaria circum- sporozoite protein. J. immunol., 141: 2456-2460 (1988). 10. Caspers, P. et al. The circumsporozoite protein gene from NF54, a Plasmodium falciparum isolate used in malaria vaccine trials. Mol. biochem. parasitol., 35: 185-190 (1989). 11. Zavala, F. et al. Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. J. exp. med., 157: 1947-1957 (1983). 12. Jardetzky, T.S. et al. Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding. EMBO j., 9: 1797-1803 (1990). 9w WHO Bulletin OMS: Supplement Vol. 68 1990