NEW PEPTIDES AND THEIR USE

Abstract: A peptide comprising the amino acid sequence: U-O-X-glu-Z or O-X-glu-Z-ala-glu, wherein: U is an amino acid residue selected from val and ile; O is an amino acid residue selected from ala and thr; X is an amino acid residue selected from asp and glu; and Z is an amino acid residue selected from ile and val; the use of the peptide in the preparation of a vaccine; a composition for vaccination against malaria induced by Plasmodium falciparum, comprising such peptide in admixture with a pharmaceutically acceptable carrier; and a method of inducing immunity against malaria induced by Plasmodium falciparum, which comprises administering to a person in need of such immunity an effective amount of said composition.

Description: New peptidβs and their use. The present invention relates to new peptides, their use as active ingredients in vaccines and compositions con- + .ainir>σ same. BACKGROUND OF THE INVENTION Malaria is a wide spread disease, particularly in the developing countries, and scientists are constantly looking for new means to control the dangerous parasitic disease cau¬ sed by plasmodial parasites. Among these parasites Plasmo¬ dium falciparum causes the most severe disease, responsible for the major part of the mortality due to malaria. One strategy in combatting malaria resides in the use of a conventional vaccine based on attenuated or dead malaria parasites, but such approach has not been found to be practi¬ cally feasible. The alternatives are constituted by development of. modern techniques, such as the manufacture of proteins by chemical synthesis or by DNA technology, such proteins as components in subunit vaccines being able to induce protective immunity against the parasite infection. One strategy in the selection of antigenic sequences to be included in a potential subunit vaccine against Plasmo- dium falciparum malaria is to define the epitopes of antibo¬ dies which have the capacity to interfere with the parasite life cycle. Properly presented in immunogens these epitopes are expected to induce protective antibody responses. With regard to the asexual blood-stages of P . falciparum. the main attention in this context has been made to antibodies with capacity to inhibit merozoite reinvasion in vitro (Anders, R.F. (1985) Candidate antigens for an asexual blood-stage vaccine. Parasitol. Today i, 152-155) . However, ar-tibodies which inhibit the cytoadherence of infected erythrocytes to endothelial cells (Howard, R.J. (1988) Malarial proteins at the membrane of Plasmodium falciparum-infected erythrocytes and their involvement in cytoadherence to endothelial cells. Prog.Allergy 41, 98-147; Udomsangpetch, R. , Aikawa, M. , Ber- zins, K. , Wahlgren, M. and Perlmann, P. (1989) Cytoadherence of knobless Plasmodium falciparum-infβcted erythrocytes and its inhibition by a human monoclonal antibody. Nature 338, 763-765) or• inhibit rosette formation between uninfected and infected erythrocytes (Carlsson, J., Holmquist, G. # Taylor, D.W., Perlmann, P. and Wahlgren, M. (1990) Antibodies to a histidine-rich protein (PfHRPl) disrupt spontaneously formed Plasmodium falciparum erythrocyte rosettes. Proc .Natl .Acad. Sci. USA 87, 2511-2515) may also be of interest. Such antibo- dies are expected to interfere in vivo with the sequestration- of late-stage infected erythrocytes (Howard, R.J. (1988) Ma¬ larial proteins at the membrane of Plasmodium falciparum-in- fected erythrocytes and their involvement in cytoadherence to endothelial cells. Prog.Allergy 41, 98-147; Carlson, J., Holmquist, G., Taylor, D.W., Perlmann, P. and Wahlgren, M. (1990) Antibodies to a histidine-rich protein (PfHRPl) dis¬ rupt spontaneously formed Plasmodium falciparum erythrocyte rosettes. Proc .Natl .Acad.Sci . USA 87, 2511-2515) . The human monoclonal antibody (mAb) 33G2, obtained from an Epstein-Barr virus transformed B-cell originating from a Liberian P . falciparum-immune donor (Udomsangpetch,* R., Lundgren, . , Berzins, K., Wahlin, B., Perlmann, H., Troye- Blomberg, M. , Carlsson, J., Wahlgren, M. , Perlmann, P. and Bjorkman, A. (1986) Human monoclonal antibodies to P l55, a major antigen of malaria parasite Plasmodium falciparum. Science 231, 57-59) has several interesting biological pro¬ perties. It inhibits both P . falciparum erozoite reinvasion in in vitro cultures (Udomsangpetch, R., Lundgren, K. , Ber¬ zins, K. , Wάhlin, B. , Perlmann, H., Troye-Blomberg, M, Carls- son, J., Wahlgren, M., Perlmann, P. and Bjorkman, A. (1986) Human monoclonal antibodies to Pfl55. a major antigen of ma¬ laria parasite Plasmodium falciparum. Science 231, 57-59) as well as cytoadherence of infected erythrocytes to melanoma cells in vitro (Udomsangpetch, R., Aikawa, M., Berzins, K. , Wahlgren, M. and Perlmann, P. (1989) Cytoadherence of knob- less Plasmodium f lciparum-infected erythrocytes and its in- SUBSTITUTE SHEET i-l ition by a h mr.r- ιτ>o-ι_*cional artibociy. N u ? 338, 703- -765) . The mAb, thus, has the capacity to interfere with the parasite erythrocytic life cycle at two potential target si¬ tes for protective antibodies in vivo (Anders, R.F. (1985) Candidate i ens for an asexual blood-stage vaccine. Para- sitol. Today 1, 152-155) which makes the epitope recognized by the mAb of great interest with regard to vaccine develop¬ ment . The mAb 33G2 was initially selected due to its reacti- vity with Pfl55/RESA as detected by erythrocyte membrane im- unofluorescence (EMIF) and immunoblotting (Udomsangpetch, R., Lundgren, . , Berzins, K. , Wihlin, B., Perlmann, H. , Troye-Blomberg, M. , Carlsson, J. , Wahlgren, M. , Perlmann, P. and Bjorkman, A. (1986) Human monoclonal antibodies to Pfi55, a major antigen of malaria parasite Plasmodium falciparum. Science 231, 57-59) but subsequent analyses with recombinant fusion proteins and synthetic peptides revealed that the antibody showed reactivity with a family of cross-reacting P . f lciparum blood-stage antigens, including Pfi55/RESA, Pfll.l and Ag332 (Mattei, D., Berzins, K. , Wahlgren, M. , Udomsangpetch, R., Perlmann, P., Griesser, H.W., Scherf , A., Muller-Hill, B., Bonnefoy, S. , Guilotte, M., Langsley, G. , Pereira da Silva, L. and Mercereau-Puijalon, 0. (1989) Cross- -reactive antigenic determinants present on different Plas- modium falciparum blood-stage antigens. Parasite Immunol. 11, 15-30; Mercereau-Puijalon, 0., Langsley, G. , Mattei, D. , Guilotte, M. , Blisnick, T., Berzins, K. , Griesser, H.W., Scherf, A., Muller-Hill, B. and Pereira da Silva, L. (1987) Presence of cross-reacting determinants on several blood- -stage antigens of Plasmodium falciparum. UCLA Symp.Molec. Cell.Biol. 42, 343354; Udomsangpetch, R., Carlsson, J., Wahlin, B., Holmquist, G., Ozaki, L.S., Scherf, A., Mattei, D., Mercereau-Puijalon, 0., Uni, S., AikaW-, M., Perzins, K. and Perlmann, P. (1989) Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plas¬ modium falciparum antigens. J. Immunol. 142, 3620-3626) . A SUBSTITUTE SHEET feature shared between these antigens is their contents of several tandemly repeated amino acid sequences containing regularly spaced pairs of glutamic acid (Mattei, D. , Berzins, K. , Wahlgren, M., Udomsangpetch, R., Perlmann, P., Griesser, H.W. , Scherf, A., Muller-Hill, B. , Bonnefoy, S., Guillotte, M., Langsley, G., Pereira da Silva, L. and Mercereau-Puija¬ lon, 0. (1989) Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens. Pa¬ rasite Immunol. 11, 15-30; Favaloro, J.M., Coppel, R.L., Cor- coran, L.M., Foote, S.J., Brown, G.V., Anders, R.F. and Kemp, D.J. (1986) Structure of the RESA gene of Plasmodium falcipa¬ rum. Nucleic Acids Res. 14, 8265-8277; Scherf, A., Hilbich, C., Sieg, K., Mattei, D., Mercereau-Puijalon, 0. and Miiller- -Hill, B. (1988) The 11-1 gene of Plasmodium falciparum codes for distinct fast evolving repeats. EMB0 J. 7, 1129-1137). These dimerε of glutamic acid were suggested to be the struc¬ tures responsible for the antigenic cross-reactions seen bet¬ ween the three antigens (Mattei, D., Berzins, K., Wahlgren, M., Udomsangpetch, R., Perlmann, P. , Griesser, H.W., Scherf, A. , Muller-Hill, B., Bonnefoy, S., Guilotte, M., Langsley, G., Pereira da Silva, L. and Mercereau-Puijalon, 0. (1989) Cross-reac ive antigenic determinants present on different Plasmodium falciparum blood-stage antigens. Parasite Immunol. 11, 15-30; Merecereau-Puijalo , 0., Langsley, G. , Mattei, D. , Guilotte, M., Blisnick, T. , Berzins, K., Griesser, H.W. , Scherf, A., Muller-Hill, B. and Pereira da Silva, L. (1987) Presence of cross-reacting determinants on several blood- -stage antigens of Plasmodium falciparum. UCLA Symp.Molec. Cell.Biol. 42, 343-354; Udomsangpetch, R., Carlsson, J., Wahlin, B., Holmquist, G., Ozaki, L.S., Scherf, A., Mattei, D. , Mercereau-Puijalon, 0., Uni , S. , Aikawa, M. , Berzins, K. and Perlmann, P. (1989) Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plas¬ modium falciparum antigens. J. Immunol. 142, 3620-3626). Inhi- bition with synthetic peptides of the mAb 33G2 binding in EMIF showed that peptides corresponding to Ag332 repeat se- SUBSTITUTE SHEET quer-.-_-b wα;re '■;- - 3 me ;i. I Ag332 was the original target for the antibody (Udomsang¬ petch, R., Carlsson, J. ( Wahlin, B. , Holmquist, G. , Ozaki, L.S., Scherf, A., Mattei, D., Mercereau-Puijalon, 0., Uni, S., Aikε.wa, M. ( Berrins, K. and Fsrl.ϊfinn, D . (19f9) Reactivi¬ ty of the human monoclonal antibody 33G2 with repeated se¬ quences of three distinct Plasmodium falciparum antigens. J. Immunol. 142, 3620-3626) . The major object of the present invention is to provi- de new peptides capable of inducing immunity against malaria. Another object of the invention is to provide new com¬ positions for vaccination against malaria comprising such peptide. Yet another object of the invention is to provide a method of inducing immunity against malaria. SUMMARY OF THE INVENTION It has been found that a peptide comprising the amino acid sequence: U-O-X-glu-Z, wherein: U is an amino acid residue selected from val and ile; O is an amino acid residue selected from ala and thr; X is an amino acid residue selected from asp and glu; and 2 is an amino acid residue selected from ile and val is capable of providing protective immunity against malaria induced by Plasmodium falciparum. Such protective immunity can be provided also by a peptide comprising the amino acid . sequence : O-X-glu-Z-ala-glu, wherein 0, X and Z have the above meaning. Preferred embodiments of the peptide of the present invention are the following: SUBSTITUTE SHEET glu-ser-v l-thr-glu-glu-ile ; ser-v l-thr-glu-glu-ile-ala; val-thr-glu-glu-ile-ala-glu; ser-val-thr-glu-glu-ile; val-thr-glu-glu-ile-ala; val-thr-glu-glu-ile; ile-thr-glu-glu-ile; val-ala-glu-glu-ile; and ile-ala-asp-glu-ile . Particularly preferred are the following peptides val-thr-glu-glu-ile, thr-glu-glu-ile-ala-glu-glu, and thr-glu-glu-ile-ala-glu. Accordingly, the peptides of the present invention find medicinal use, particularly as acive ingredients in vaccines, such as vaccines against malaria. The peptides of the present invention are also useful in the preparation of vaccines, particularly vaccines for combatting malaria induced by Plasmodium falciparum. The invention also covers compositions for vaccination against malaria induced by Plasmodium falciparum. said compo¬ sition comprising a peptide selected among those defined or mentioned above in admixture with a pharmaceutically accep¬ table carrier. It is preferred to use carriers suitable for parenteral adminis ration. As is generally known within immunology the immunoge- nic response resulting from administra ion of a relatively small peptide can be enhanced in several ways. First, it is conceivable to contain the active peptide or principal in a larger molecule, wherein said peptide is present' in repeating units . Such polymerized form can be pre¬ pared using recombinant DNA techniques. Second, the peptide can be coupled to a macromolecular carrier, such as bovine serum albumin or other immunogenic SUBSTITUTE SHEET cerrier or -id i v nt th .? ir- -. ■ - • in ~ b p 'tsr imιτu'--e rssponπe against the peptide in view of the increased size of the molecule. The antigenic presentation of a small peptide in accordance with the invention can be improved for example by conjugation to a preformed iscσm as a carrier (for details see Journal of Im unological Methods, 98 (1987) 137-143, K. Ldfgren et al . ) . The composition of the present invention can be con¬ stituted by a solution, a suspension or other form of prepa- ration. Such solutions or suspension may take the form of sterilized aqueous isotonic preparations, such as isotonic saline solution or glucose solution. As indicated above pa- renteral administration is preferred. It goes without saying that although the peptides of the invention can be used alone, combinations of two or more of same can be contained in one and the same composition. Finally, the present invention provides a method of inducing immunity against malaria induced by Plasmodium fal¬ ciparum. said method comprising administering to a person in need of such immunity an effective amount of the composition as defined above. The method is particularly exercised in the form of parenteral injection. In the present disclosure the abbreviations used have the following meanings: A = ala = alanine; D = asp = aspartic acid; C = cys = cysteine E = glu = glutamic acid; I = ile = isoleucine; L = leu = leucine K = lys = lysine F = phe = phenylalanine P = pro = proline S = ser = serine T = thr = threonine; and W = trp = tryptophan Y = tyr = tyrosine V = val = val ine SUBSTITUTE SHE EXAMPLES The present invention will be described more detailed in the following specific examples. Said examples are not to be construed to limit the scope of the invention otherwise as defined in the appended claims. The examples are given with reference to the appended drawings, wherein: Fig. 1 shows antibody reactivity of peptides according to the invention expressed as absorbance at 405 ran; Fig.* 2 shows antibody reactivity to peptides according to the invention also expressed as the absorbance at 405 nm; Fig. 3 shows a replacement set analysis of 33G2 reac¬ tivity to an octapeptide; and Fig. 4 shows antibody reactivity to epitope analogs expressed as the absorbance at 405 nm. EXAMPLE 1 Establishment of a human B-cell line producing the monoclonal antibody 33G2. Periferal blood lymnphocytes (PBL) were isolated by standard procedures from heparinized blood of a Liberian do¬ nor known to be immune to malaria (Udomsangpetch, R. , Lund¬ gren, K., Berzins, K., Wahlin, B., Perlmann, H., Troye-Blom- berg, M., Carlsson, J., Wahlgren, M., Perlmann, P. and Bjork¬ man, A. (1986) Human monoclonal antibodies to Pfl55, a major antigen of malaria parasite Plasmodium falciparum. Science 231, 57-59) . The PBL were incubated for 3 days with P.falci- p rum parasite extract in order to activate the malaria spe¬ cific B-lymphocytes (Lundgren, K. , Wahlgren, M. , Troye-Blom- berg, M. , Berzins, K., Perlmann, H. and Perlmann, P. (1983) Monoclonal anti-parasite and anti-RBC antibodies produced by stable EBV-transformed B cell lines from malaria patients. J . Immunol . 131, 2000-2003) . For transformation of B-lymphocy¬ tes Epstein Barr virus (EBV) , contained in culture medium from the EBV-producing marmoset cell line B95-8, was added to the PBL and incubated for 2hr at 37°C. After washing, the cells were suspended at 2xl0^/ml in tissue culture medium SUBSTITUTE SHEET ( PP.V.I 164«J with 10!. fetal r lf rerun., 1 % gluta- mine and 25μg/ml gentamicin) containing 0.2 u-g/ml of cyclo- sporin A and then incubated in 10 ml roundbottomed tubes for 5 days at 37°C in air + 5% CO2. The cells were then transfer- r? to 50 ml ; :issu3 culture flasks for continued propagation for 9 days. Cyclosporin was present in all media during the first 2 weeks of cell propagation. Two weeks after transfor¬ mation, the cells were seeded in 96-well tissue culture pla¬ tes with 5xl0 5 irradiated (4500 rad) allogeneic PBL per well as feeder cells. Immunoglobulin producing cultures were de¬ tected by measuring in enzyme linked immunosorbent assay (ELISA) (Engvall & Perlmann, Immunochemistry 8, 871-874, 1971) the presence in the culture medium of the wells of im¬ munoglobulin. Immunoglobulin containing culture εupernatants were further analyzed for the presence of antibodies to the P . f lciparum antigen Pfl55/RESA by means of indirect immuno- fluorescence using glutaraldehyde fixed and air dried mono- layers of infected erythrocytes from P . falciparum in vitro cultures (Perlmann et al . J.Exp.Med. 159, 1686-1704, 1984). Cultures scoring positive in the latter assay were submitted to repeated cloning by limited dilution. Monoclonality of the antibodies produced was assessed by isoelectric focusing. The antibody producing clone 33G2 thus obtained showed high growth rate and antibody production (10-15 μg IgM/ml in 72 hours) (Udomsangpetch, R., Lundgren, K., Berzins, K., Wahlin, B., Perlmann, H., TroyeBlomberg, M., Carlsson, J., Wahlgren, J., Perlmann, P. and Bjorkman, A. (1986) Human monoclonal antibodies to Pfl55, a major antigen of malaria parasite Plasmodium falciparum. Science 231, 57-59). EXAMPLE 2 Inhibition in vitro of malaria parasite invasion into red blood cells . The capacity of the monoclonal antibody (mAb) 33G2 to inhibit parasite development in P . falciparum in vitro cultu¬ res was assayed by a procedure described by Wahlin et al . SUES7.7UTE SHEET (Proc .Nat .Acad. Sci . USA 81, 7912-7916, 1984) . P . falciparum cultures were diluted with normal 0+ erythrocytes to a para- sitemia of 0.5% and a hematocrit of 2% . Aliquots of the parasite suspension (100 μl) were seeded in quadruplicate in 96-well flat-bottomed microculture plates. The mAb 33G2 was added to the wells at differrent concentra¬ tions, either in culture supernatant or after purification on concanavalin A-Sepharose of ammonium sulfate precipitated culture supernatants (Kleine et al . Molec . Immunol . 16, 421-- 425, 1979). After incubation for 20 hr at 37°C in a candle jar (Trager & Jensen, Science 193, 673-675, 1976), the ery¬ throcytes from each well were washed in tris-buf ered Hanks solution (TH) and monolayers were prepared on eight-well mul- titest slides as follows. Erythrocyte suspensions were app- lied to slides treated with bicarbonate buffer (pH 9.6). Im¬ mediately after being washed in TH, the monolayers were fixed briefly (2x10 sec) in 1% glutaraldehyde in phosphate-buffered saline (pH 7.4), washed in distilled water and then air-dried extensively under a fan. The parasites were stained with acridine orange arid the number of parasitized erythrocytes was obtained by counting 4x10^ erythorcytes per sample. Both preparations of mAb 33G2 inhibited P . falciparum reinvasion efficiently in a concentration dependent manner, the culture supernatant contained mAb giving 50% inhibition of reinvasion at 14 μg/ml and the purified mAb giving 50% inhibition at 5.5 μg/ml (Udomsangpetch, R., Lundgren, K., Berzins, K. , Wahlin, B., Perlmann, H., Troye-Blomberg, M. , Carlsson, J., Wahlgren, M., Perlmann, P. and Bjorkman, A. (1986) Human monoclonal antibodies to Pfl55, a major antigen of malaria parasite Plasmodium falciparum. Science 231, 57-59) . EXAMPLE 3 Inhibition of cytoadherence of malaria infected ervthrocvtes to endothelial and melanoma cells in vitro. The capacity of mAb 33G2 to inhibit the cytoadherence SUBSTITUTE SHEE cf P ,jaΛ ; .paruτι in* .-. ^ L^d e'.-y hrccyteε to endoti-eliai cells was demonstrated in an assay using the melanoma cell line C32 as described by Udeinya et al . (Exp. Parasitol. 56, 207-214, 1983) . Melanoma cells grown on cover slips were fixed with 1% formaldehyde in phosphats-buffared saline i ph. 7.4) and then stored at 4°C until used. A suspension (2% hematocrit) of erythrocytes from a P . lciparum culture, containing mainly trophozoites and schizonts at 5-10% parasitemia, were incuba¬ ted with the fixed melanoma cells at room temperature on a rotating platform for 1 hour. Unbound erythrocytes were flushed away with phosphate-buffered saline. The coverslips were then fixed with 1% glutaraldehyde in phosphate-buffered saline, stained with Giemsa and examined in the light micro¬ scope. For assaying antibody mediated inhibition of cytoadhe- rence, pellets of infected erythrocytes (40-50 μl) were sus¬ pended in 100 μl of antibody solution (15-250 μg/ml) and in¬ cubated for 30 min at 37°C with agitation every 10 min . The erythrocytes were then diluted to a 2% hematocrit suspension and applied to the cytoadherence assay as described above. The number of bound erythrocytes per 100 melanoma cells was counted and expressed as the percentage of bound cells: The mAb 33G2 inhibited cytoadherence in a dose depen¬ dent manner, giving about 45% inhibition at the highest anti¬ body concentration tested (250 μg of purified mAb per ml) (Udomsangpetch, R. , Aikawa, M. , Berzins, K. , Wahlgren, M. and Perlmann, P. (1989) Cytoadherence of knobless Plasmodium fal¬ ciparum-infeeted erythrocytes and its inhibition by a human monoclonal antibody. Nature 338, 763-765) . EXAMPLE 4 Reactivity of monoclonal antibody 33G2 with cross-reactive P. falciparum antigens. The mAb 33G2 was initially selected due to its reacti¬ vity with the P . f lciparum antigen Pfl55/RESA as detected by immunofluorescence and immunoblotting (see Ex. 1 and ref . 1) . Analysis of antibody reactivity with different recombinant SUBSTITUTE SHEET P . alciparum blood stage antigens was performed by immuno- blotting using recombinant bacterial (E. coli)plagues (Mattei, D. , Berzins, K., Wahlgren, M. , Udomsangpetch, R., Perlmann, P., Griesser, H.W., Scherf, A., Muller-Hill, B., Bonnefoy, S., Guillotte, M., Langsley, G., Pereira da Silva, L. and Mercereau-Pui alon, 0. (1989) Cross-reactive antigenic deter¬ minants present on different Plasmodium falciparum blood- -stage antigens. Parasite Immunol. 11, 15-30). The mAb showed binding to bacterial plaques expressing parts of the P. fa.lci- parum antigens Pfll.l, Ag332 and Pfl55/RESA, showing the strongest reactivity with Ag332 expressing plaques (Mattei, D., Berzins, K., Wahlgren, M., Udomsangpetch, R., Perlmann, P., Griesser, H.W., Scherf, A., Muller-Hill, B., Bonnefoy, S., Guillott.e, M. , Langsley, G. , Pereira da Silva, L. and Mercereau-Pui alon, 0. ( 1989). Cross-reactive antigenic deter¬ minants present on different Plasmodium falciparum blood- -stage antigens. Parasite Immunol. 11, 15-30) . No binding was seen to bacterial plaques expressing the P . falciparum anti¬ gens FIRA or Ag281. The capacity of various synthetic pepti- des, corresponding to repeated sequences in the antigens Pfll.l, Ag332 and Pfll/RESA, to block the binding of mAb 33G2 to Pfll/RESA as detected by immunofluorescence was analysed (Udomsangpetch, R., Carlsson, J. , Wahlin, B., Holmquist, G., Ozaki, L.S., Scherf, A., Mattei, D., Mercereau-Puijalon, 0., Uni, S., Aikawa, M., Berzins, K. and Perlmann, P. (1989) Re¬ activity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens. J . Immunol . 142, 3620-3626). Different concentrations of the peptides (up to 200 μg/ml) were mixed with a fixed concentra- tion of the mAb, which then was used in the immuno luoresce assay (see Ex. 1) . The peptide Y (SVTEEIAEEDK)2 , correspon¬ ding to a dimer of amino acids 2-12 in antigen Ag332 (Mattei, D., Berzins, K., Wahlgren, M., Udomsangpetch, R., Perlmann, P., Griesser, H.W., Scherf, A., Muller-Hill, B., Bonnefoy, S., Guillotte, M., Langsley, G. , Pereira da Silva, L. and Mercereau-Puijalon, 0. (1989) Cross-reactive antigenic deter- SUBSTITUTE SHEET mi.-an ' -s preεe-.t 01. (.'ύTecent p lasmodiuιτ. faleipd.ιum b.lood- -stage antigens. Parasite Immunol. 11, 15-30), was the most efficient inhibitor of mAb binding, giving complete inhibi ¬ tion of immunofluorescence at 0.2 μg/ml (Udomsangpetch, R., Carlrεon, " * .. V hlin, B., Holmqui F , P., Ozaki, L.S., Scherf, A. , Mattei, D. , Mercereau-Puijalon, 0., Uni , S. , Aikawa, M., Berzins, K. and Perlmann, P. (1989) Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens. J . Immunol . 142, 3620-3626). Also some peptides corresponding to sequences in Pfll.l and PF155/RESA inhibited mAb 33G2 immunofluorescence but with considerably less efficiency, the Pfll.l peptide (EEVVEEVVP) 2 and the Pfl55/RESA peptide both giving complete inhibition at 100 μg/ml. The results show that mAb 33G2 re- cognizes a family of cross-reactive P . falciparum antigens including Pfll.l, Pfl55/RESA and Ag332, the latter antigen being the optimal target for the mAb (Udomsangpetch, R., Carlsson, J. , Wahlin, B., Holmquist, G. , Ozaki, L.S. , Scherf, A., Mattei, D., Mercereau-Puijalon, 0., Uni, S., Aikawa, M., Berzins, K. and Perlmann, P. (1989) Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens. . Immunol . 142, 3620-3626) . EXAMPLE 5 Determination of the epitope specificity of the monoclonal antibody 33G2. The detailed epitope specificity on the single amino acid level for the mAb 33G2 was performed using the multiple peptide synthesis technique (PEPSCAN) developed by Geysen et al . (J . Immunol .Methods 102, 259-274, 1987) . Peptides were synthesized on polyethylene rods on which polymers of poly- acrylic acid had been formed by irradiation. Polyethylene rods and Fmoc L-amino acids performed as active esthers (Cambridge Research Biochemicals, UK) were used for synthesis according to instructions of the manufacturer. The N-termi- SUBSTITUTE SHEET nals of all peptides were acetylated. As a basis for the mAb 33G2 epitope analysis, the sequence of amino acid residues 1-19 (ESVTEEIAEEDKSVIEEAV) of Ag332 (Mattei, D., Berzins, K., Wahlgren, M. , Udomsangpetch, R., Perlmann, P., Griesser, H.W., -Scherf, A., Muller-Hill, B., Bonnefoy, S., Guillotte, M. , Langsley, G., Pereira da Silva, L. and Mercereau-Pui a¬ lon, 0. (1989) Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens. Parasite Immunol. 11, 15-30) was used, containing sequences of the peptides with the highest reactivity with the mAb (Udomsangpetch, R., Carlsson, J., Wahlin, B., Holmquist, G., Ozaki, L.S., Scherf, A., Mattei, D., Mercereau-Puijalon, O., Uni, S., Aikawa, M. , Berzins, K. and Perlmann, P. (1989) Re¬ activity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens. J . Immunol . 142, 3620-3626) . All possible overlapping hepta¬ peptides, hexpeptides, pentapeptides and tetrapeptides cove¬ ring the mentioned sequence were synthesized and their re¬ activity with mAb 33G2 was analysed by ELISA as described by Geysen et al . (J. Immunol. Methods 102, 259-274, 1987) . Cultu¬ re supernatant containing mAb 33G2 (approx. 10 μg/ml), was diluted 1:100. Peptide containing rods were washed in phos¬ phate-buffered saline with 0.05% Tween 20 between all steps in ELISA. Bound antibodies were detected with a rabbit anti- -human IgM-alkaline phosphatase conjugate (Sigma, St. -Louis, MO) using p-nitrophenyl phosphate, disodium salt (Sigma) as substrate . The antibody showed reactivity with four heptapeptides corresponding to amino acids 1-7 (ESVTEEI), 2-8 (SVTEEIA), 3- -9 (VTEEIAE) and 4-10 (TEEIAEE) (Fig. la). When tested a- gainst hexapeptides the antibody recognized sequences corre¬ sponding to amino acids 2-7 (SVTEEI), 3-8 (VTEEIA) and 4-9 (TEEIAE) (Fig. lb). Reactivity to pentapeptides was restric¬ ted to one peptide, corresponding to amino acids 3-7 (VTEEI ) (Fig. lc), while no reactivity was seen with any of the tet¬ rapeptides (Fig. Id) . When tested against eight heptapeptides SUBSTITUTE SHEET ccrrjsp'in* v a ■ • i.i -aqυence ΣvSVTECIA ■ *.<A_..ιr-c a -ids 1-6), where one amino acid residue had been omitted in each pepti¬ de, the antibody could not recognize peptides where either V, T, E, E or I had been excluded (Fig. 2) . Tϊ.a ϊciΛb ?3G2 s'ϊt -tr lyzed f'r against octa- peptides based on the sequence ESVTEEIA, where single amino acid substitutions replaced each residue. Every residue in the parent peptide ESVTEEIA, which corresponds to residue 1-8 of the known sequence of Ag332, was replaced by the most co - mon 20 amino acids (Fig. 3) . The first (E), second (S) and last amino acid residue (A) were shown to be replaceable by any other amino acid without loosing the ability of the mono¬ clonal 33G2 to recognize the peptides. A linear, five amino acid, sequence (VTEEI) was shown to consist of amino acids which were either essential or replaceable mainly by amino acids of resembling chemical character. Subsitution of valine (V) by C, F, I, L, P, T, W and Y, and threonine (T) by A, C, I, L, P, S, and V, gave ELISA absorbance values of 20% or more compared to the values obtained with the parent octapep- tides. The pair of glutamic acids (E) , contained within the epitope, were the most essential residues. The first glutamic acid (E) was totally nonreplaceable while the second glutamic acid (E) was possible to replace with aspartic acid (D), a very conserved replacement, and to some extent with cysteine (C). The last amino acid within the epitope, isoleucine (I), was possible to replace with leucine (L) and valine (V) , two relatively conserved replacements. It could also be replaced to some degree by the positively charged amino acid histidine (H) . Based on the results in the replacement set analysis, pentapeptides corresponding to residue 3-7 of Ag332 were con¬ structed in which one or severala original amino acids had been replaced simultaneously. The results from this assay- showed that it was possible to replace several amino acids within the epitope, simultaneously, without loosing antibody reactivity (Fig. 4) . The antibody recognized most of the pep- SUBSTITUTE SHEET tides where the modi ications were consistent with the re¬ sults from the replacement set analysis; ITEEI, VAEEI, IADEI, IADEV, VVEEV and LVEEV. A decrease in antibody reacti¬ vity could be seen for some peptides where several amino acids were replaced. The antibody did not react with the pen- tapeptide, YLDEV, indicating that not all of the reactive single amino acid substitutions can be performed simul¬ taneously and still result in reactive peptides. SUEST . TUTE SHEET

Claims: CLAI MS 1. A peptide comprising the ammo acid sequence: U-O-X-glu-Z, wherein: ζ V is an a'.iino =L"id r-ci'i.'G ss-.ect"d rτι^ v= 1 s.n 1 . a,]?: 0 is an amino acid residue selected from ala and thr; X is an amino acid residue selected from asp and glu; and Z is an amino acid residue selected from ile and val. 0 2. A peptide comprising the amino acid sequence: O-X-glu-Z-ala-glu, wherein: 0, X and Z are as defined in claim 1. 3. A peptide according to claim 1, wherein said amino acid sequence is selected from: glu-ser-val-thr-glu-glu-ile; ser-val-thr-glu-glu-ile-ala; val-thr-glu-glu-ile-ala-glu; ser-val-thr-glu-glu-ile; val-thr-glu-glu-ile-ala; val-thr-glu-glu-ile; ile-thr-glu-glu-ile; val-ala-glu-glu-ile; and ile-ala-asp-glu-ile . 4. A peptide according to claim 3, wherein said amino acid sequence is: val-th'r-glu-glu-ile. 5. A peptide according to claim 2, wherein said amino acid sequence is selected from: thr-glu-glu-ile-ala-glu-glu; and thr-glu-glu-ile-ala-glu. 6. A peptide according to any preceding claim for me¬ dicinal use . 7. A peptide according to claim 6 for use as an active ingredient in a vaccine. 8. A peptide according to claim 7 for use as an active ingredient in a vaccine against malaria. 9. The use of the peptide according to any of claims 1 to 5 in the preparation of a vaccine. 10. The use of the peptide according to claim 9 in the preparation of a vaccine against malaria. 11. A composition for vaccination against malaria in- duced by Plasmodium falciparum. comprising a peptide accor¬ ding to any of claims 1 to 5 in admixture with a pharmaceuti¬ cally acceptable carrier. 12. A composition according to claim 11, wherein said carrier is suitable for parenteral administration. 13. A composition according to claim 11 or 12, wherein said peptide is comprised in a larger molecule, wherein it is present in repeating units. 14. A composition according to claim 11 or 12, wherein said peptide is coupled to a carrier molecule. 15. A composition according to claim 11 or 12, wherein said peptide is coupled to an immunogeniσ carrier. 16. A method of inducing immunity against malaria in¬ duced by Plasmodium falciparum. which comprises administering to a person in need of such immunity an effective amount of the composition of any of claims 11 to 15. 17. A method according to claim 16, wherein the admi¬ nistration is constituted by parenteral injection. SUBSTITUTE SHEET

Inventors: AHLBORG NIKLAS, BERZINS KLAVS, PERLMANN PETER, AHLBORG, NIKLAS, BERZINS, KLAVS, PERLMANN, PETER
Applicants: MALVAC FOUNDATION
Application year: 1991
Main classification: C07K7
IPC codes: A61K39, C07K14, C07K16