PROTECTIVE PEPTIDE ANTIGEN.

Description

TITLE PROTECTIVE PfPTIDE ANTIGENF. INV. DES. DAVID R. COLNAN JOAN ELLIS S. NI GEL GODSON R'JTH S. NUSSEN7WF. IG VICTOR N. NUSSEN7WFIG DAVID H. SCHLESINGER PRIORITY GREAT BRITAIN SUNDAY aPFV OF ? P JANUARY 1ST SS SET. )98? Rome, there.................................................................................... Register A 27.08002.I2IT.4 Protocol No. 9343 TO / MINISTRY OF INDUSTRY * COMMERCE AND ART Provincial Office of Industry, Commerce and Crafts of Milan COPY OF THE MINUTES OF FILING FOR INDUSTRIAL INVENTION PATENT The year of nationality' 984 on the TWENTY-SEVENth day of the month of JANUARY NEW YORK UNIVERSITY with registered office in the United States of America, NEW YORK, NEW YORK (USA) through agents Eng. Glusapp* RIGHETTI - Eng. Franco ZORZOLI · Ini. Carlo GHIONI and with domicile for legal purposes at Milan - Via Carlo Farini, at Bugnion s.pji. presented to me, the undersigned: - Stamped application for the granting of a PATENT FOR INDUSTRIAL INVENTION having as TITLE. PROTECTIVE PEPTIDE ANTIGEN Designated Inventors: DAVID R. DOLMAN, JOAN ELLIS, G. N1GEL GODSÙN, RUTH S. NU SENZWE1G, VICTOR N. NUSSENZWEIG, DAVID H. SCHLESINGER Priority 1 of the patent application in: GREAT BRITAIN No. 8302399 of 28 January 1983 accompanied by: • Description in duplicate of η. 1 09 pages of writing. -Drawings, plates no. 9 (provisional) - Letter of Appointment · EKXRiiàKiiiHèXTKMWSAtH (with reservation) - Priority document and Italian translation (with reservation) of deed of transfer, and Italian translation (with reservation) - Proof of payment on postal account no. 00663004 in the name of the Tax and Concession Registry Office Rome of L. 868,000 issued by the Post Office of Milan 39 on 17 / 1 / 84 n. 909 The invention referred to in this application does not constitute the object of other filings of the same content, made anywhere on the same date, by the same owner. The application, descriptions and drawings listed above have been signed by the applicant and countersigned by me and stamped with the office seal. - Revenue stamp of L. 3,000,- For a copy conforming to the original **51 specify that for these questions and etlegoll Ι'ΙΒΝ postlo of bo A ι λ·.·._·Ίχ compliant (II, circular i. iverituoH Intact, Idyll The UI'FICIAL Busso p. the Director requests In coriLt'vii-*-r« CB, eoe rljerve 41 -ησ from the tlejsC “Benito Boschetto THE HEAD OF THE rrtCtO Raoul Holel (H) 9 343 A / 84 X Hon. Ministry of Industry, Commerce and Crafts CENTRAL PATENT OFFICE - ROME -OOOQO-The company NEW YORK UNIVERSITY of American nationality with headquarters in NEW YORK, NEW YORK (USA) and electively domiciled at the registered offices and domiciliaries lng. THE Giuseppe Righetti and others (see letter of appointment / of BUGNION SPA, Via C. Farini ,81 - MILAN, applies for a patent certificate for INVENTION fN INDUSTRIAL titled: PROTECTIVE PEPTIDE ANTIGEN Designated Inventors: DAVID R. COLMAN, JOAN ELLIS, G. NIGEL GODSON, RUTH S. NUSSENZWEIG, VICTOR N. NUS-, SEN2WEIG, DAVID H. SCHLESINGER Priority of patent application in: GREAT BRITAIN No. 8302349 of 28 January 1983 for this purpose the following are attached: ) Description of the finding in duplicate no. 105 pages 2) n. 9 drawing tables (provisional) 3) Att. of vers. in current account no. 00668004 of L. 868,000 4) letter of appointment (with reservation): ) Document. ) Act of .Milan, there more than the priority and Italian translation (with reservation) assignment and Italian translation (with reservation), January 1984 In Faith | Company NEW YORK UNIVERSITY JL MAN DATA RfW*' (no. Cerio Seoul Olirvi Wi 27.D8002.12IT.4 FZ / es mugniqh SpA, DESCRIPTION attached to a patent application for an INDUSTRIAL INVENTION with the title: “PROTECTIVE PEPTIDE ANTIGEN” Applicant: NEW YORK UNIVERSITY, of US nationality, located in NEW YORK (U.S.A.) Agents: Eng. Giuseppe RIGHETTI, Eng. Franco ZORZOLI, Eng. Carlo Raoul GHIONI of BUGNION SpA-Via Depositata il 198¾ _ o RIAS The present invention is based on the discovery that the protective CS sporozoite antigens of the genus Plasmodium possess a single immunodominant region composed of repeats of the same epitope. For P. knowlesi, the qoitope has been shown to be a dodecapeptide whose sequence is repeated several times within the structure of the CS protein. The repeating peptide has been chemically synthesized in both monomeric and dimeric forms. The synthetic repeating peptide is immunochemically reactive with polyclonal antibody preparations against P. knowlesi. Furthermore, all monoclonal antibodies against CS proteins that C. Farini, 81 - MILAN atN0 19343A / 84 SUMMARY neutralize the infectivity of sporozoites in vitro, F.\ ί; 1' f,, ί STA Spéij b They also react with the synthetic peptide. Therefore, the synthetic repeat peptide accounts for substantially all of the immunogenic activity exhibited by the naturally occurring sporozoite protective antigen of Pknowlesi. Several lines of evidence indicate that proteins CS proteins from Plasmodium um species infectious to rodents, monkeys, and humans are structurally similar. All possess an immunodominant region composed of similarly repeated epitopes. For each species, the repeating peptide of the sporozoite CS protein can be synthesized. The repeating peptide of a CS protein is immunogenic when administered in a composition and by administration methods known in the art to produce antibody. Based on the findings and teachings described herein, the structural determination and synthesis of the repeating peptide corresponding to any sporozoite of the Plasmodium um species, and the preparation of a vaccine composition incorporating said peptide and capable of eliciting protective immunity against said species, are now available to those skilled in the art. DESCRIPTION BACKGROUND OF THE INVENTION β U Ο Ν ί Ο i ν g. ρ Μ, The present invention relates to the field of antigens capable of providing protective immunity against malaria when incorporated into a vaccine. Malaria represents a global public health risk of enormous economic and medical significance. The disease contributes substantially to infant mortality in endemic areas and remains a serious and debilitating illness for those who suffer from it as adults. Despite improvements in mosquito reduction techniques and improved public health measures, the number of regions in the area where the disease is considered endemic is increasing. Furthermore, the risk of infection has substantially increased in some parts of the world due to the emergence of new drug-resistant mutations of the malaria parasite. The agent that causes malaria is a protozoan of the genus Plasmodium. Individual species within the genus appear to have a narrow host range for the animals they infect. For example, P. berghei and P. yoe 1 i are infectious for rodents, P,knowlesi and P. cynomolgi are primarily infectious to monkeys, while P.f alciparum, P. ivax, P. ovale and P. malariae are the species primarily infectious to humans. Despite species differences in host range, the life cycles, mode of infection, biochemistry, and genetics of the various Plasma spp. species are markedly similar. The life cycle of Plasmodium is complex, with the organism undergoing several distinct morphological changes involving both a mammalian host and a mosquito vector. The parasite, in the form of a sporozoite, is introduced into the mammalian host through the bite of the mosquito vector. The sporozoites rapidly disappear from the bloodstream and are subsequently found as intracellular parasites of liver parenchymal cells. This results in a blood infection characterized by the well-known clinical symptoms of malaria after a complex series of morphological and biochemical transitions. The parasite then resides in the red blood cells, where it continues its development. Substantial quantities of the parasite can be obtained from the red blood cells of infected patients. Vaccine development to provide protective immunity against malarial infection has been hampered by the fact that the parasite's life cycle in the mammalian host is primarily intracellular. Except for brief periods, the parasite is protected from the 5 ί. · ·. < from contact with the immune system. Two stages in the parasite's life cycle during which it is briefly exposed to the immune system are: 1) the interval following initial infection before sporozoites have successfully invaded liver cells and 2) the interval during which merozoites leave infected red blood cells and enter uninfected red blood cells. The transient exposure of merozoite forms to the extracellular environment has provided the basis for prior art attempts to develop host immunity to blood-borne forms of the parasite. Published European patent application No. 62924 discloses antigen proteins useful in the production of a vaccine to provide immunity against merozoite forms of the parasite. The usefulness of such a vaccine presumably lies in limiting or arresting the course of established malarial infection. An alternative approach, based on sporozoite antigens, has led to the discovery of antigenic and immunogenic proteins of sporozoites that are able to provide protective immunity against initial infection when administered as a vaccine. Cochrane, AH, et al., in Malaria, vol. 3, (J. Kreier, ed.) Academic Press NY (1980) p. 163-202; Nussenzweig, Λ ' 'i RS in Immunity to Animal Blood Parasites and Uomiηi, (L. Miller, J. Pino and J. McKelvey, eds.) Plenum, NY (1977) p. 75-87. Gwadz, R.W. et al., Bull, WHO Suppl. 1, 57, 165 (1979); Clyde, D.F., et al., Am. J, Trop, Med. and Hyg. 24, 397 (1975); McCarthy, V,,et al., Exp. Parasitol. 41, 167 (1977). These proteins are antigenically distinguishable for each Plasmodium species, but have several structural properties in common including chromatographic behavior, isoelectric point and electrophoretic mobility. Sporozoite antigens range in molecular weight from approximately 40,000 daltons to 70,000 daltons and have low isoelectric points, Santoro F. et al., J. Biol. Chem., 258, 3341, 1983. Comparison of tryptic extracts of purified sporozoite antigen proteins from different species of Plasmodium shows that several tryptic peptides have identical retention times on reversed-phase high-performance liquid chromatography, indicating that there is a high degree of homology between antigenic proteins of different species. Sporozoite antigens are components of the sporozoite surface coating. The presence of sporozoite antigen is indicated by a characteristic immunological reaction known as the circumsporozoite reaction, and by immunofluorescence tests. See Vanderberg, JP, et al. Mil. Med. (Suppl.) 134, 1183 (1969); and Nardin, E., et al., Nature 274, 55 (1978). These reactions make it possible to specifically detect sporozoite antigen for a given species of Piasmodium species, without resorting to time-consuming in vivo tests. This, in turn, made it possible to develop specific radioimmunoassays for sporozoite antigens, and ultimately the production of malaria antibodies directed against the sporozoite antigens of Piasmodium species. Antibodies against sporozoite antigens have been shown to provide protective immunity against the Plasmodium species from which they were derived, in rodents, monkeys, and human volunteers. The sporozoite protective antigen protein is referred to herein as CS protein, circumsporozoite protein, or sporozoite CS protein, these terms being deemed equivalent. A related U.S. patent application, Serial No. 234,096, filed February 12, 1981, discloses a vaccine based on a purified CS protein. That application, to which reference is made, is hereby deemed incorporated in its entirety as defined. The results presented here are based in part on techniques and concepts from the field of immunology. For convenience, some terms commonly used in the art are defined here. The term immunochemical reaction is used to indicate the specific interaction that occurs between an antigen and its corresponding antibody, regardless of the measurement procedure. This reaction is characterized by the noncovalent binding of one or more antibody molecules to one or more antigen molecules. The immunochemical reaction can be detected by a wide variety of immunoassays known in the art. The terms immunogenic or antigenic will be used herein to describe the ability of a given substance to stimulate the production of antibodies specifically immunoreactive to that substance when that substance is administered to a suitable test animal under conditions known to elicit antibody production. The term protective antigen refers to the ability of a given immunogen to confer resistance in an appropriate host against a given pathogen. The term epitope refers to a specific antibody that binds laterally to an antigen. Macromolecular antigens such as proteins typically have several epitopes with distinctive antibody-binding specificities. Different epitopes of the same antigen are recognizable with the help of monoclonal antibodies which, due to their high degree of specificity, are directed against single epitopes. Two different monoclonal antibodies directed against different epitopes on the same antigen can each bind the antigen without interfering with the other, unless the epitopes are so close together that binding of one spherically prevents binding of the other. The term immunodominant region refers to an area of ​​the antigen molecule that is primarily responsible for its antigenicity. SUMMARY OF THE INVENTION The present invention is based on the discovery that the protective CS sporozoite antigens of the genus Plasmodium possess a single immunodominant region composed of repeats of the same epitope. For P. knowlesi. The epitope has been shown to be a dodecapeptide whose sequence is repeated several times within the structure of the CS protein. The repeating peptide has been chemically synthesized into both monomeric and dimeric forms. The synthetic repeating peptide is immunochemically reactive with polyclonal antibody preparations against P. knowlesi. Furthermore, all monoclonal antibodies against CS proteins that neutralize sporozoite infectivity in vitro also react with the synthetic peptide. Therefore, the synthetic repeating peptide accounts for substantially all of the immunogenic activity exhibited by the naturally occurring sporozoite protective antigen of P. knowlesi. Several lines of evidence indicate that proteins CS proteins of Plasmodium species infectious to rodents, monkeys, and humans are structurally similar. All possess an immunodominant region composed of similarly repeated epitopes. For each species, the repeating peptide of the sporozoite CS protein can be synthesized. The repeating peptide of a CS protein is immunogenic when administered in a composition and by administration methods known in the art to result in antibody production. Based on the findings and teachings described herein, the structural determination and synthesis of the repeating peptide corresponding to any sporozoite of Plasmodium species, and the preparation of a vaccine composition incorporating said peptide and capable of eliciting protective immunity against said species, are now available to those skilled in the art. As further confirmation of the close relationships of CS proteins from different Plasmodium species, monoclonal antibodies against P.knowlesi sporozoites have been shown to cross-react with the Pf antigen from P. iparum, a species infectious to humans. It is therefore clear that the development of other synthetic peptides more specifically reactive with human malaria species are well within the capabilities of those ordinarily skilled in the art, who follow the teachings and revelations as set forth herein. DETAILED DESCRIPTION OF THE INVENTION In the following description, the materials used were commercially available, unless otherwise specified. The enzymes used in the cloning procedures were obtained from commercial sources. Restriction endonuclease reactions were performed according to the manufacturer's instructions. Unless otherwise specified, reaction conditions for other enzyme reactions were standard conditions used in the art, as described, for example, in Proceedings in Enz ^010915?', volume 60 (R. Wu, Ed.) Academic Press, (1980). Unless otherwise specified, the abbreviations used herein are common abbreviations acceptable for publication in scientific journals normally used by those skilled in the art to publish their results, such as those cited herein. In general, the experiments and the conclusions drawn from their results are summarized here. The approach considered here to clone a DNA segment encoding the sporozoite antigen protein was to clone cDNA made from mRNA obtained from infected mosquitoes. The cDNA approach was preferred in the initial cloning work since it was not known whether Plasmodium genomic DNA contained introns that prevented the expression of antigenically identifiable sporozoite proteins. Now that the short, repetitive nature of the 1' epitope is known, it is possible to select the DNA encoding the epitopes from a library of genomic Plasmodium DNA. Initial experiments were performed with cDNA from mRNA from infected mosquitoes, since it was only at that stage that Piasmodium was known to express sporozoite antigen. A cDNA library was constructed from Poly(A)RNA derived from P.knowlesi-infected mosquitoes. Double-base cDNAs, with poly-C residues at the end, were inserted into the plasmid pBR322, previously cut with Pst I and with poly-G at the end. Tetracycline-resistant transformed host cells were selected and single colonies of transformed cells were stored in microtiter dishes at -70°C. The cDNA clones were screened for their ability to express a protein containing the immunochemically reactive region of the sporozoite surface antigen. Once a cDNA encoding the sporozoite antigen was identified, others could easily be detected by hybridizing to the originally cloned cDNA as a probe. Clones derived either from cDNA libraries or genomic DNA could be identified in this manner, on the basis of homology between DNA segments encoding the sporozoite antigen of different Plasmodium species. Identification of clones expressing an immunoreactive protein was done by screening lysates of colonies of cells transformed (as above) with the cloned cDNA. Groups of 48 colonies were screened using a sensitive, double-sided immunoradiometric assay using monoclonal antibodies. This allowed the detection of CS protein in transformed cells. In summary, a monoclonal antibody to CS protein P. knowlesi was adsorbed to the wells of a microtiter plate. Lysates from the 48-colony clusters were each added to the wells and incubated for a sufficient time to allow the immunoreactive protein present in the lysate to bind to the adsorbed monoclonal antibody. The wells were then washed to remove any contaminating protein and a second monoclonal antibody radiolabeled to the CS P protein was added. knowlesi. The second labeled monoclonal antibody attaches to the antigenic protein that is already bound to the surface of the microtiter well by the first monoclonal antibody. If a group of 48 colonies was found to be positive, the colonies were screened individually in the same manner. In this way, positive clones were identified. Whenever an immunoreactive clone was detected, plasmid DNA was isolated from it and used to transform another host cell mutation, such as E. coli HB 101 or E. coli RR1. Mutants detected by tetracycline resistance were re-tested for the ability to express the chemically reactive immunoreactive protein, in order to confirm that the expression was a property of the plasmid DNA clone containing CS nucleotide sequences. Once a suitable plasmid DNA was obtained from the positive clones, the nucleotide sequence of the cDNA insert encoding at least the immunoreactive region of the CS protein was obtained (for cloning onto M1 3 ). Nucleotide sequence analysis procedures are well known in the art, including the procedure of Maxam and Gilbert, W. Proc. Nat. Acad. Sci. USA 74, 560 (1977) and the proceedings of Sanger, F. et al. Proc. Nat. Acad. Sci. USA 74, 5463 (1977). The latter procedure was employed in the present work. The complete nucleotide sequence of a segment of the CS protein Pknowlesi gene containing the immunochemically reactive locus is shown in Figure 1. A striking feature of the nucleotide sequence was that it was repetitive. In P.knowlesi, the sequence consisted of a 36-base-pair repeat, 8 complete units of which were represented in one clone (24 mer) together with partial units at both ends. In order to deduce the amino acid sequence encoded in DNA, it was necessary to identify the encoding base16 BU <·. ί VX 1 ί<* ca and, within the base, the correct reading frame. In this context, the advantage of using the Sanger supna sequencing procedure becomes evident and Coulson. A sequence vector, bacteriophage M13mp9, contains a betagalactosidase gene with a Pst I locus in the same reading frame as the Pst I cleavage locus of pBR322. Therefore, the reading frame can be deduced once the number of deoxy C-residues added during the tailing reaction is known and the sequence vector can express a betagalactosidase fusion protein comprising the immunochemically reactive part of the CS protein. Thus, two different recombinants were obtained M13mp9 with the 368 bp P. knowlesi DNA fragment inserted in opposite orientations. Only one of the two recombinants produced an immunochemically reactive betagalactosidase fusion protein, as measured by the radioimmunoassays described above. The clone producing the immunoreactive protein was used to identify the coding base and the direction of transcription of the P.knowlesi gene fragment. The correct reading frame was also deduced using immunological procedures. These demonstrated that the epitope defined by the monoclonal antibodies was destroyed by elastase, but not by trypsin or reducing agents, indicating that the epitope did not contain any lysine, arginine, or disulfide bonds but may contain alanine residues. Based on these experiments, it was deduced that the amino acid sequence of the repetitive peptide containing 12 amino acids was: H^N-ClnAlaGlnGlyAspGlyAlaAsnAlaGlyGlnPro-COOH To confirm the deduced amino acid sequence and the immunochemical reactivity of the above-described sequence, a dodecapeptide of the same amino acid sequence and its dimer were synthesized using an automated solid-phase peptide synthesis system. The synthetic monomer and dimer peptides were separately tested for immunochemical activity in the same type of radioimmunoassay used initially to screen the cDNA library. In this assay, two antibody binding loci must be present in the antigen, one for binding to the first monoclonal attached to the microtiter well and the second for binding to the added labeled antibody. Although the monomer peptide did not bind the labeled antibody, the dimer peptide was reactive, indicating that the dimer contained two complete or nearly complete antibody-binding loci. Furthermore, the same assay showed that the monomer was able to compete with and specifically inhibit the binding of P.knowlesi CS proteins to microtiter wells carrying the first monoclonal antibody. Therefore, the sequence illustrated above contains a sporozoite antigen epitope. Another important observation was that all monoclonal antibodies to Pknowlesi obtained, as well as all polyclonal antibodies obtained from monkeys immunized with irradiated sporozoites, also reacted with the synthetic peptides. Indeed, more than 70% of the antibodies to sporozoites found in the serum of immunized monkeys recognized this single epitope (Zavala, et al., J. Exp. Med. 157:1947, 1983). Thus a chemically synthesized dodecapeptide having an amino acid sequence identical to that repeated in a sporozoite membrane protein contains substantially all the antigenicities of the protein. CS that occurs naturally. It follows that based on the principles of immunology and following techniques and procedures known to those skilled in the art, a synthetic peptide based on the amino acid sequence s.pA of a known sporozoite CS protein can be incorporated into a vaccine composition capable of providing protective immunity in a host organism susceptible to a Piasmodium species from which the peptide sequence was derived. The following experiments, described generally, demonstrate the essential structure and functional similarities between sporozoite CS proteins of the species Plasmodium infectious for rodents, monkeys and humans. These similarities can be used to identify and synthesize antigenic peptides specific for all Plasmodium species, including, in particular, those infectious to humans. The structural determination and synthesis of the repeat peptide for each Plasmodium species may be performed by the procedures described herein or by equivalent procedures known in the art, or by procedures known in the art that accomplish what is described and taught in the present invention to eliminate some of the more tedious and time-consuming aspects of the original experiments. Significantly, cross-reactivity has been observed among monoclonal antibodies to the CS proteins of different Plasmodium species. For example, antibodies to the CS protein of P.knowlesi cross-react with the CS protein of Pcynomolgi and P. falciparum; antibodies to the CS protein of P. cynomolg cross-react with the sporozoite antigen of P. vivax; antibodies to the CS protein of P. yoeli nigeriensis cross-react with the sporozoite antigen of P. berghei, and in that case completely neutralize the infectivity of sporozoites of the latter species in mice. Additional immunochemical evidence has been adduced to demonstrate that all sporozoite CS proteins have a single immunodominant region and repetitive epitopes Zavala, et al., supra, The binding of several different monoclonal antibodies directed against the same protein has been demonstrated CS of sporozoites, measuring the inhibitory effects that binding of one could have on the other. Monoclonal antibodies directed against different sequences of an antigen should not interfere with their respective binding capacities. Conversely, if the monoclonal antibodies target the same epitope, or epitopes that are topographically close, they inhibit each other. In the case of P. knowlesi, each of the six monoclonal antibodies used strongly inhibited the binding of the others to the antigen. Υ-ΥΥ The same experiments were performed using monoclonal antibodies to the sporozoite antigen proteins of P.vivax, P.falciparum, P.malariae, P.cynomolgi and P.berghei, with identical results. Thus, these sporozoite CS proteins are all characterized by having a single immunodominant region. Of direct relevance to the development of a human malaria vaccine is the observation that antibodies in the serum of humans vaccinated and protected against sporozoites of Pfalciparum or P.vi vax are also directed against the same immunodominant region of the sporozoite CS protein. Pretreatment of a crude extract of sporozoites of either species with a single monoclonal antibody directed against the sporozoite antigen of the same species almost completely inhibited subsequent binding of the antigen (in the sporozoite extract) to polyclonal antibodies isolated from the serum of vaccinated human volunteers (Zavala, et al., supra). The fact that immunodominant regions of CS proteins contain repetitive epitopes was demonstrated by a two-locus solid-phase radioimmunoassay. In the assay, a monoclonal antibody was bound to the plastic surface of a microtiter well, antigen was added, and the antigen was found on the immobilized antibody. The well was then washed to remove all unbound material and a second monoclonal antibody, presumably directed against a different epitope of the same antigen, was added. The second antibody is labeled with a radioisotope to quantify the binding of the second antibody. The binding of the second antibody is proportional to the amount of antigen bound in the well. This immunoassay can only be performed if the antigen contains at least two epitopes. The first epitope binds to the antibody immobilized on the plastic plate, the second binds to the radiolabeled antibody. Surprisingly, for each CS protein, the two-site radioimmunoassay could be performed using a single monoclonal antibody. That is, the assay could be performed using a non-labeled monoclonal antibody A as the first monoclonal and the same monoclonal A as the second monoclonal. The result demonstrates that the sporozoite CS protein has at least two identical epitopes. A control experiment demonstrated that the result was not an artifact caused by aggregation. of the sporozoite antigen protein. P. knowlesi sporozoite extracts were dissolved in 2.0% (w / v) sodium dodecyl sulfate and 6 M urea and fractionated by ultracentrifuge in sucrose gradients. The existence of two epitopes was demonstrated in gradient fractions containing proteins of molecular weight 40,000, corresponding to the size of a CS protein monomer. Furthermore, there was no indication of the presence of CS protein aggregates. Identical results were obtained in experiments performed with extracts of P.vivax and P.falciparum (Zavala, et al., supra). It is therefore clear that all sporozoite CS proteins have a single immunodominant region comprising a peptide repeated many times within the protein. The repeated peptide contains the epitope, and each sporozoite CS protein is composed of a plurality of such repeated peptide epitopes. These epitopes are highly immunogenic in all animal species, including humans. Synthetic peptides containing the epitope of a given sporozoite CS protein are functionally identical to naturally occurring sporozoite antigens, with the obvious exception of two-locus xA radioimmunoassays, which require two epitopes on the same molecule. The functional behavior in two-locus assays is reproduced by synthetic dimers of the repeat peptide. It will therefore be readily appreciated that synthetic peptides, comprising an amino acid sequence corresponding to an epitope of a sporozoite CS protein in monomeric or multimeric form, can be incorporated into vaccines capable of inducing protective immunity against sporozoites of malaria parasites such as P. falciparum, P. vivax and P. malariae. Techniques to enhance the antigenicity of such repeat peptides include incorporation into a multimeric structure, binding to a highly immunogenic protein carrier, e.g., limpet hemocyanin, or diptera toxoid, and administration in combination with adjuvants or any other immune response facilitators. Furthermore, it will be understood that peptides specific for a plurality of Plasmodium stages and species can be incorporated into the same vaccine composition to provide a multivalent vaccine. Furthermore, the vaccine composition may include antigens to provide immunity against other diseases in addition to malaria. An amino acid sequence corresponding to an epitope of a CS (repeated peptide) protein can be obtained by chemical synthetic means or by purification from biological sources including genetically modified microorganisms or their culture media. The repeating peptide can be combined in an amino acid sequence with other peptides comprising fragments of other proteins, such as when synthesized as a fusion protein or linked to other antigenic or non-antigenic peptides of synthetic or biological origin. The term corresponding to an epitope of a CS protein will be understood to encompass the practical possibility that, in some cases, variations in the amino acid sequence of a naturally occurring repeat peptide may be antigenic and confer protective immunity against malaria sporozoite infection. Possible sequence variations include, but are not limited to, amino acid substitutions, extensions, deletions, interpolations, and combinations thereof. Such variations fall within the scope of the invention provided that the peptide containing them is antigenic and antibodies raised from such peptide cross-react with naturally occurring CS protein or non-variant repeat peptides. CS protein at a level sufficient to provide protective immunity when administered as a vaccine. These vaccine compositions will be combined with a physiologically acceptable medium. The routes of administration, antigen dose, and number and frequency of injections are all matters for optimization by a person skilled in the art, particularly given that there is experience in providing protective immunity with the injection of inactivated sporozoites. It is anticipated that the primary value of providing immunity to sporozoite infection will be for those individuals who have had no previous exposure to malaria, i.e., infants and children living in endemic and sub-endemic areas, and unexposed adults traveling to endemic areas. It is also anticipated that temporary immunity for infants may be provided by immunization of mothers during pregnancy. The details of the operation and practice of the present invention are set forth in the specific examples that follow. EXAMPLE 1 cDNA clone encoding a sporozoite antigen protein > .Ài Recombinant DNA technology techniques make extensive use of enzyme-catalyzed reactions. Purified enzymes for use in the practice of this invention are currently available from commercial sources. Unless otherwise specified, commercially available enzymes and reagents have been used. Restriction endonucleases, their nomenclature and locus specificities have been described in detail by Roberts, R.J., Nucl. Acids Res., 8, p. 63 (1980). The restriction enzymes used in this work were used in quantities and under reaction conditions specified by the manufacturer for each enzyme. Approximately 1000 P. knowlesi-infected mosquitoes grown, maintained, and collected as described by Cochrane et al., Proc. Natl. Acad. Sci. USA 79:5651-5655 (1982), were collected and dissected to obtain thoracic segments, which were stored on ice until dissection was completed. RNA was prepared from the thoraces essentially as described by Seeburg, P. H. et al; Celi, 12, 157 (1977), and by Chirgwin, J.M. et al; Biochemistry, 24, 5294 (1979). The tissue was homogenized in 10 ml of 5M guanidine thiocyanate, pH 5.0, 10 mM EDTA, and 0.1 M 2-mercaptoethanol until the tissue was dispersed. The solution was centrifuged at 10,000 rpm for 10 minutes and the supernatant was adjusted to 2% (w / v) Sarkosyl (ICN Pharmaceuticals Trademark, Plainview, New York), and heated to 65°C for two minutes. Cesium chloride (0.1 g / ml solution) was then added and the resulting solution was layered on 2 ml cushions of half-saturated 10 mM CsCl. EDTA in SW41 (Brand, Beckman Instruments, Fullerton, California) cellulose nitrate tubes. Centrifugation at 28,000 rpm was performed for approximately 2 hours at 20°C. The RNA pellet was dissolved in 5 mM EDTA, 0.5% {w / v)Sarkosyl and 5% (w / v) 2-mercaptoethanol, extracted with phenol and chloroform and precipitated with ethanol. Typically, 0.5-1 mg of RNA per g of tissue. The RNA was then passed over an oligo(dT)-cellulose column (Aviv, H; et al, Proc. Nat. Acad. Sci. USA, 69, 1408 (1972), to enrich the polyadenylate fraction. Alternatively, RNA can be prepared from mosquito thoraxes by the procedure modified by Liu, C.P. et al., Proc. Nat. Acad. Sci. 76:4503, 1979. According to this procedure, the tissue was homogenized in 8-10 vol. of 4M guanidine isothiocyanate pH 5.0 (with glacial acetic acid) and 0.1M 2-mercaptoethanol until the tissue was dispersed. Centrifugation was at 9,000 rpm for 3 minutes, and the supernatant was layered on 0.2 vol. of 5.7M CsCl in 0.10M EDTA (pH 6.5), in SW 41 cellulose nitrate tubes. Centrifugation was at 35,000 rpm for 16–20 hours at 20°C. Approximately 0.5–1 mg of RNA was obtained per gram of tissue. As described above, poly(A)+RNA was then chosen as the oligomer. A sample of mRNA isolated as described was translated in vitro using a translation system prepared from wheat germ (modified from Roberts, BE et al, Proc. Nat. Acad. Sci. USA, 70 2330 (1973) ). Proteins produced by in vitro translation were immunoprecipitated as described in Example 2, (alternatively, as illustrated by Goldman, BM and Biobei, G., Proc. Nati. Acad. Sci. 75:5066 (1978) ) and fractionated on an SDS-polyacrylamide gel (SDSPage) as described by Yoshida et al. J. Exp. Med 154, 1225 (1981) and in Example 2 of the co-pending U.S. patent application Serial No. 234,096 incorporated herein by reference. mRNA fractions containing sequences coding for CS proteins can be identified by this means. For preparative cDNA synthesis, the mRNA polyadenylate Total Η-bucket (approximately 20 / jg) was treated in a volume of 100 / jl with 1 mM methyl mercury (hereafter MeHg) (Aldrich Chemical, Milwaukee, Wisconsin) at room temperature for 5 minutes. The treatment was stopped by adding 0.5% (0.5 / Jl per 100 / Jl) undiluted α-mercaptoethanol and incubating at room temperature for 5 minutes. MeHg-treated polyadenylated mRNA was incubated in a 200 µl reaction containing 50 mM Tris-HCl pH 8.3, 10mM MgCl^, 20mM KCl, 5mM Dithiothreitol, 2mM each of dATP, dCTP, dGTP, and dTTP, 50 µCi PdCTP (specific activity, 800 Ci / mmol), 4µg oligo(dT)12-18, (Collaborative Research, Waltham, Massachusetts), 5µl RNasin (BIOTECH, Madison, Wisconsin) and approximately 200 reverse transcriptase units (from Beard, Life Sci., St. Petersburg, Fla.). Incubation was at 42° for 60 minutes. The reaction was stopped by extraction with phenol and chloroform (1:1), then with an equal volume of chloroform and precipitation with ethanol. The ethanol precipitate was dissolved in 50 µl 10 mM Tris-HCl, 1mM EDTA, pH=8 and fractionated on a Sephadex (Brand, Pharmacia, Ine., Uppsala, Sweden) G-75 column in a 1 ml Falcon plastic pipette (Brand, Falcon Plastics, Oxnard, California), using 10mM Tris-Hcl, pH 7.4 and 1mM EDTA as running buffer. The entrance peak of the non-excluded P counts was collected (about 300 μA) and adjusted to 0.3M NaOH and 1mM EDTA and incubated overnight at room temperature. After neutralization with 5 M sodium acetate pH 3.8 to a final pH of approximately 6.0, and ethanol precipitation, the second DNA mutation was synthesized in a 50 μA reaction containing the same buffer as described above, 500 μM each of dATP, dCTP, dGTIP, dTTP, 125 ^jCi PdCTP (800Ci / mmol), and 50 units of reverse transcriptase. Incubation was at 37°C for 90 minutes. The reaction products were extracted with phenol / chloroform and passed through a Sephadex 32 column. G-75 as described above and the excluded P peak was precipitated with ethanol before proceeding to treatment with nuclease S,. After fractionation on Sephadex G-75, synthesis of the second cDNA mutation was completed using the Klenow fragment of DNA polymerase I (BoehringerMannheim) in the presence of 50 mM Tris-HCl, pH 8.0, 7 mM MgCl^, and 1 mM dithiothreitol. The reaction mixture was incubated for 4 hours at 15°C, extracted with phenol-chloroform (1:1) and precipitated with ethanol as described above. The double-mutant cDNA was incubated with 300 units of S1 nuclease (Boehringer-Mannheim, Indianapolis, Indiana) in 24 µL of 0.3 mM NaCl, 30 mM Na acetate, pH 4.5, and 3 mM ZnCl^ at 41 °C for 5 minutes. The reaction was stopped by the addition of EDTA to 10mM and neutralized with Tris base. The P-labeled cDNA was size fractionated using a Sepharose CL-4B column (Brand, Pharmacia, Inc., Uppsala, Sweden) fitted into a 1ml Falcon plastic pipette and run in a buffer of 0.3M NaCl, 10mM Tris-HCl pH 8.0, 1mM EDTA. Various size classes of double-mutant cDNA were precipitated with ethanol and then fixed using young mammalian thymus terminal transferase (Enzo Biochem, Inc., New York, New York) in a volume of 100 for 1 min at 37°C in a buffer containing 100 mM K cacodylate, pH 7.6, 1 mM CoCl^, 0.1 mM DTT, 0.1 mM dCTP, 4 µm Ci of 3H-dCTP, 24 Ci / mmol) and approximately 20 units of terminal transferase / µm ds-cDNA. The reaction was stopped by adjusting the solution to 0.5M NaCl, 10mM EDTA and incubating at 65°C for 5 minutes. 1-5 micrograms of yeast + RNA were added before phenol:chloroform extraction and precipitated with ethanol twice. The fixation reaction UU Vii VI gio is generally described by Roychoudhury, R. et al? Nucl. Acids. Res., 3, 101 (1976). An alternative procedure, also implemented, is described by Land, H., et al. Nucl. Acids. Res. 9:2251, 1981. About 17 bases were added to the end of the 3' under these conditions. pBR322 plasmid cleaved by Pst I endonuclease and fixed with dG residues was obtained from a commercial source, New England Nuclear, Boston, Massachusetts. Equimolar amounts of dC-fixed cDNA and dG-fixed pBR322 were annealed at a concentration of 1 ^tg / ml, using sequential 2-hour incubations at 42°C, 30°C, and 14°C. The hybrid plasmid DNA was precipitated with ethanol and then used to transform ampicillin-resistant E. coli RR1 cells. Single-colony libraries were generated and stored in microtiter plates at -70°C. According to the alternative procedure, after 15–30 deoxy C residues were added to the cDNA and annealing took place at an equimolar concentration of dG-fixed pBR 322 (at 250 ng vector / ml), the annealing mixture was incubated at 68°C for 5 min, then at 42°C for 2 h, followed by slow cooling to room temperature for 2 h. The hybrid plasmid DNA was used to transform E. coli RR1 cells to tet resistance, as described by Dagert, M. et al, Gene 6:23, 1979. A library was generated and stored as individual colonies in Luria broth with 15% glycerol in microtiter plates at -70°C. A library of cDNA fragments (300-2000bp) was screened for colonies that expressed protein containing the immunochemically reactive region of the sporozoite surface antigen protein. Colonies were grown individually on a Petri dish containing agar-agar S (32g / liter tryptone, 5g / liter NaCl, 20g / liter yeast extract, 15g / liter Difeto agar, 0.2g / liter NaOH and 20mg / liter tetracycline). The plates were invaded with 2mls of 0.05M Tris-HCl, pH 7.5, and 0.5mg egg white lysosome (Sigma, St. Louis, Mo.) and scraped with a sterile spatula into a 15ml polypropylene tube (Fisher Scientific Supply). After incubation at room temperature for 30 min, followed by 60 min on melting ice in 95% ethanol and dry ice (~80°C) three times, and further incubation at 37°C for 10 min, crude cell extracts were treated with DNAse I (1 mg / ml), 4 mM CaCl^ and 4 mM MgCl^ at room temperature for 30 min. - 35 minutes and stored at -70°C for future use. The lysates were screened for the presence of an immunochemically reactive protein using monoclonal antibodies to P. knowlesi in a radioimmunoassay. In this procedure, the monoclonal antibody to iP. knowlesi was absorbed into the well of a microtiter plate to affinity purify all immunoreactive proteins present in the pooled cell culture medium. Lysates containing an immunoreactive protein were detected by reacting washed microtiter wells with a second anti-iP monoclonal antibody. 125 knowlesi marked I. To do this, microtiter plates were coated with 50 µL of anti-P.knowlesi monoclonal antibody (50 µg / ml) incubated at 4°C for 12–17 h, washed thoroughly with 1% (w / v) BSA saline solution, and then incubated with 50 µL of the pooled cell extract for 4–17 h at 4°C. After washing, a second anti125 x monoclonal antibody P, Knowlesi labeled . I was added to each well and incubated for 2 hours at room temperature. The washed wells were then individually tested for radioactivity. When a group of 48 colonies was found to be ι positive, the original single colonies that constituted the group were individually screened and the immunoreactive clones identified, isolated and genetically purified. Plasmid DNA was purified from 1 liter of cells containing an immunoreactive clone (plasmid pEG81). Cells were grown at 37° in Luria broth with 175 µg / ml tetracycline at approximately 5X10 µg / ml and the plasmid DNA amplified by adding 175 µg / ml chloramphenicol and incubating overnight (Clewell and Helinski, J. Bacteriol. 110, 1135 (1972) ). Plasmid DNA was extracted from cells using sodium dodecyl sulfate (SDS) (Godson and Vapnex, Biochim. Biophys. Acta 299, 516 (1973)) and purified using 5-20% (w / v) sucrose density gradients. This gave 500-1000 / jg of RF I plasmid DNA. This yug was used to transform other E.coli cells (HB101) to tetracycline resistance and their ability to express the immunoreactive protein was re-examined. pEG81 DNA was extracted with Pst 1 restriction endonuclease ligated with T4 DNA ligase to 0.5 µg of Pst I-cut sequence / cloning vector. M13mp9 at a 1:1 molar ratio and sequenced using the dideoxy chain termination procedure Sanger (Sanger and Coulson, supra) with a universal synthetic primer 5'-d(GTAAAACGACGGCCAGT)-3' (purchased from PL Biochemicals, Milwaukee, Wisc.). The complete nucleotide sequence of this segment of the P,knowlesi CS protein gene containing the immunoreactive locus is shown in Figure 1. An unexpected feature of the pEG81 fragment of Pknowlesi DNA is that it consists entirely of a 36-base pair repeat (8 full units plus one partial unit at either end). The coding base and the correct reading frame of the nucleotide sequence have been established as follows: (a) the reading frame of the Pst splitting site The pBR322 ampicillinase gene and the M13mp9 β -galactosidase genes are known to be iden- tics (5' XCTGCAGXX 3' ) . Met Thr Met Ile Thr Pro Ser Leu Ala Ala Gly M13mp9 ATG ACC ATG ATT ACG CCA AGC TTG GCT GCA GGT Pst I rift site Two different M13mp9 recombinants were obtained with the P. knowlesi DNA fragment inserted in opposite orientations. One of the recombinants produced an immunoreactive α-galactosidase fusion protein (M13mp9 / Pk 11) as measured by the radioimmunoassay. immunologically described above, the other clone did not. The M13mp9 / Pk 11 sequence therefore identified the coding base of the DNA. (b) The reading frame was further deduced from the fact that 17 AD residues were inserted between the ^-gal gene and the P.knowlesi gene fragment. (c) A possible reading frame encoded an alanine-rich peptide. That epitope likely contained alanine was verified by treating authentic P.knowlesi CS surface protein with porcine elastase, known to cleave peptides at alanine residues (Powers, J.C., et al. Biochem. Biophys. Acta, 485 : 156-166, (1977). Incubation of an extract of 10 sporozoites with 0.002 units of porcine elastase (Worthington Enzymes, infra) in Tris buffer, 0.05M pH 8.6, for 60 minutes at 37°C completely abrogated protein reactivity. CS with monoclonal antibodies as determined by a two-site immunoradiometric assay described in Example 4. Inactivation of the CS protein by elastase was reversed by the synthetic inhibitor OOCAla Ala Pro Ala (Powers, et al supra). Other possible reading frames encoded peptides that were excluded for various reasons: they were ρ? E, ' * ? ' 7 A too hydrophobic, or contained several cysteine, lysine, and arginine residues. The absence of these amino acid residues was determined in experiments showing that the epitope was trypsin resistant after complete reduction of the CS protein. Indeed, after incubation of another aliquot of the same sporozoite extract with 1 mg of TPCKtrypsin (Worthington Enzymes, Freehold, New Jersey) at 37°C for 30 min, followed by complete reduction and alkylation, the CS protein reacted completely with the monoclonal antibodies. The deduced amino acid sequence of the twelfth amino acid repeat is as follows based on the translation of the nucleotide sequence into the correct reading frame: Gln-Ala-Gln-Gly-Asp-Gly-Ala-Asn-Ala-Gly-Gln-Pro (All sequences are expressed from the end closest to the NH^ - terminus on the left to the end closest to the -COOH terminus on the right.) The immunoreactive part of the P.knowlesi protein is therefore contained within the 12 amino acid repeat. To confirm that the above amino acid sequence contains the immunoreactive locus, a dodecapeptide (with the same amino acid order . 1 '\ 1··Lj> ​​tu, as illustrated above) and a dimer of the dodecapeptide were synthesized using solid-phase resin synthesis (Marglin, H. and Merrifield, R.B., Ann. Rev. Biochem. 39:841-866 (1970). Sequence analysis performed by degradation of Edman's automated system confirmed that the peptide had been synthesized correctly. This provided final proof that this is the correct epitope. Rabbits were immunized with the dodecapeptide coupled to a carrier (bovine gamma globulin in complete Freunds adjuvant). Two weeks after injection, the rabbits were blood sampled and their serum tested for the presence of antibodies against the dodecapeptide and against sporozoite extracts. The results showed that the animals produced high titers (greater than 1:1000) of antibodies to the native CS protein present in parasite extracts. Once a clone expressing an immunochemically reactive protein has been identified, the inserted cDNA sequence can be used as a hybridization probe to identify cDNA encoding sporozoite antigen proteins from other species. Plasmodium. The cDNA clone can also be used to screen Plasmodium genomic DNA obtained, for example, from merozoites, to detect DNA sequences encoding the sporozoite antigen protein. Therefore, once the first cDNA sequence encoding a sporozoite antigen or a fragment thereof is cloned, the subsequent isolation and purification of other cDNA species is substantially simplified. EXAMPLE 2 Competition between monoclonal antibodies for specific antigen Monoclonal antibodies that bind to distinct regions of an antigen molecule do not interfere with each other; on the other hand, monoclonal antibodies directed against the same or topographically related epitopes or antigenic determinants will inhibit each other's activity. Therefore, it is possible to map the epitopes of an antigen. The number of CS protein epitopes reacting with monoclonal antibodies was determined by an immunoradiometric assay performed as follows: A) Preparation of plates coated with scrozoite extracts Sporozoites were purified from the salivary glands of infected mosquitoes as described by Yoshida, N. et al. Science 207, 71 (1980). They were suspended in phosphate buffered salt (PBS) UuGKìON S-pA k at a concentration of 10 / ml and subjected to acoustic treatment (100 W for 3 minutes), and further diluted 20-fold in PBS. Then, 50 ^j1 of the suspension was dispensed to the bottom of the wells of Falcon ”3911 microtiter plates, manufactured by Falcon Plastics, Oxnard, California. These were incubated overnight at 4°C and washed with PBS. The wells were then carefully washed with Tween20 (Brand, Atlas Europol SpA, Ternate, Italy, (0.05% w / v)) and incubated for 3 h in PBS containing 0.5% (w / v) bovine serum albumin (BSA) to saturate the hydrophobic loci of the plastic. B) Preparation of monoclonal antibodies Monoclonal antibodies were raised against different sporozoite species as described by Yoshida, et al. above; and Potocnjak, P., et al. J. Exp. Med. 151, 1504 (1980). Antibodies were isolated from the ascitic fluid of mice injected with hybridomas using standard chromatographic procedures (ion exchange chromatography and filtration through Sephadex G-200). Antibody purity was revealed by SDS-PAGE. The antibodies were then radiolabeled with 12 5 I used Iodogen (Pierce Chemical Co., Rockford, 111.) according to the manufacturer's instructions. The monoclonal activity was labeled with NTYAX >N'À specification varied between 10? - 3 X 10^cpm per / jg of protein. C) Monoclonal antibody titer The minimum concentration of a monoclonal antibody that saturates antigen loci in the bottom of microtiter plate wells was determined as follows: To a series of tubes containing a constant amount {0.5 ng) of radiolabeled monoclonal antibody diluted in PBS-BSA, increasing amounts of cold antibody were added, maintaining a constant total volume. Aliquots of the various mixtures containing the same number of counts, but different concentrations of antibody, were dispensed to the bottom of individual wells of microtiter plates pre-coated with specific antigen. After incubation for one hour at room temperature, the wells were washed with PBS-BSA and counted in a gamma counter. The highest concentration of monoclonal antibody that gives a maximum of bound counts represents the saturation dose of the monoclonal antibody, D) Competition between monoclonal antibodies for binding to the antigen Several monoclonal antibodies have been prepared against the CS proteins of P.knowlesi. The antibodies 125 I and the saturation dose determined as described above. Cross titrations were then performed as follows to determine whether each cold monoclonal antibody interfered with the binding of any other labeled monoclonal antibody to the solid-phase antigen. To a series of wells containing antigen, 50 µl of different concentrations of various cold monoclonal antibodies diluted in PBS-BSA were added. The plates were incubated for 1 hour at room temperature. Then, 50 µl of one of the radiolabeled monoclonal antibodies (e.g., 2G3) at twice the saturating concentration was added to all wells. After an additional hour of incubation, the wells were washed and counted. The number of antigen-specific counts bound was calculated as the number of counts bound in wells incubated with 2G3 alone at the saturating dose minus the number of counts bound in wells incubated with 2G3 in the presence of cold 2G3 at a concentration 10 times the saturating dose. The counts that could not be inhibited by the homologous cold antibody represent nonspecific binding. From these numbers, the percentages of inhibition of 2G3 binding by the other monoclonal antibodies were calculated. The titration is It was repeated for each labeled monoclonal antibody. The results are summarized in Table 1. It can be seen that all monoclonal antibodies to P.knowlesi strongly inhibit each other, indicating that they must bind to identical or closely related epitopes. TABLE I EVIDENCE FOR THE PRESENCE OF AN IMMUNODOMINANT REGION IN THE CS PROTEIN OF P.knowlesi Cold Inhibitor (Monoclonal Antibody) Percent Inhibition of Antigen Binding of Radiolabeled Monoclonal Ab 5H8 2G3 8B8 8A8 8E11 6B8 5H8 100 99 101 115 105 121 2G3 96 100 87 78 106 103 8B8 95 94 100 106 107 125 8A8 86 83 94 100 105 128 8E11 65 59 86 70 100 105 6B8 88 85 91 95 104 100 Monoclonal Control 6 0.4 0.5 8 0 0 PBS-BSA 0 0 0 0 0 0 An identical procedure was followed to study the specificities of monoclonal antibodies to P.vivax and P.falciparum (Tables II and III), P.malariae and P. berghei (not illustrated). The overall results demonstrate that there is a single immunodominant region in each CS protein. TABLE II EVIDENCE FOR THE PRESENCE OF AN IMMUNODOMINATING REGION IN THE P. vivax CS PROTEIN Cold Inhibitor (Monoclonal Antibody) Percent Inhibition of Antigen Binding of Radiolabeled Monoclonal Ab 3D10 5D9 3C1 4E8 2F2 3D10 100 102 99 101 59 5D9 83 100 98 101 65 3C1 113 103 100 102 67 4E8 105 100 99 100 60 2F2 100 101 100 116 100 Monoclonal Control 8 -6 -11 3 6 PBS-BSA 0 0 0 0 0 TABLE III EVIDENCE FOR THE PRESENCE OF AN IMMUNODOMINANT REGION IN THE P. falciparum CS PROTEIN Cold Inhibitor (Antibody - Monoclonal) Percent Inhibition of Antigen Binding of Radiolabeled Monoclonal Ab 3D6 2E7 1E9 2C11 2A10 3D6 100 98 99 95 75 2E7 98 100 102 99 90 1E9 100 103 100 100 99 2C11 104 100 99 100 100 2A10 110 102 106 100 100 Monoclonal Control 2 0 -4 8 0 PBS-BSA 0 0 0 0 0 EXAMPLE 3 Competition between monoclonal antibodies and polyclonal antisera for sporozoite antigens. A) Test for binding of polyclonal antibodies to sporozoite extracts The first step of this assay was essentially the same as described in Example 1; that is, microtiter plate wells were coated with acoustically treated crude extracts of sporozoites, washed with Tween-20, and saturated with PBS-BSA. Then, serial dilutions of polyclonal antibodies to the homologous sporozoite species were prepared in PBS-BSA, and 30 µl aliquots were dispensed to the bottom of individual microtiter wells. Controls consisted of wells incubated with dilutions of polyclonal antibodies to an unrelated antigen. After a 4-hour incubation at room temperature, the wells were washed. The presence of antibodies in the wells was detected with a second antibody (125 I-labeled and affinity purified) to the appropriate immunoglobulin species. For example, in the case of human polyclonal antibodies, the second antibody consisted of 50 µl (µg / ml) of an affinity-purified rabbit anti-human Ig. The rabbit antibodies were polyspecific, reacting with human kappa, gamma, and mu chains, and had been preabsorbed with mouse Ig. This adsorption was necessary to prevent interaction of the development reagent with the mouse monoclonal antibodies used in the inhibition assay described below. When monkey polyclonal antibodies were used, the development reagent was prepared similarly from a rabbit antiserum to monkey Ig. B) Inhibition of binding of polyclonal antibodies by monoclonal antibodies Wells coated with sporozoite extract were first incubated with a purified monoclonal antibody to the repetitive epitope of a CS protein at saturation levels (see Example 2 for determination of the saturation dose). After incubation for 1 hour at room temperature, dilutions of the polyclonal antibodies were added, and the assay proceeded as described above. Inhibition tests were performed in the following systems: 1) P.knowlesi sporozoite extracts reacting with monkey antisera to X-ray irradiated P.knowlesi sporozoites. Inhibitory monoclonal antibody 2G3 (Cochrane, AH et al. Proc. Nat. Acad. Sci. USA 79, 565 (1982) ) . 2) P.vivax sporozoite extracts reacting with serum from humans vaccinated by the bite of X-ray irradiated P.vi vax-infected mosquitoes. Inhibitory monoclonal antibody 2F2 (Nardin, EH et al. J. Exp. Med. 156:20 (1982) ). 3) P. falciparum sporozoite extracts reacting with sera from humans vaccinated by the bite of X-ray irradiated Pf alciparum-infected mosquitoes. Inhibitory monoclonal antibody 2A10 (Nardin, EH et al. supra ) . Typical results from these tests are shown in Figure 2. In each case, the monoclonal antibodies inhibited 70% or more of the interaction between the extracts and the polyclonal antibodies. Considering that the solid-phase antigen is prepared by acoustic treatment of whole sporozoites and likely contains plasma membrane as well as intracellular proteins, these results indicate that a large proportion of the immune response in the polyclonal sera was directed against the repetitive epitope of the immunodominant region of the CS protein. EXAMPLE 4 Presence of repetitive epitopes in the immunodominant region of CS proteins from several sporozoite species Experiments in Examples 2 and 3 showed that the CS proteins of four Plasmodium species contain a single immunodominant region. This section illustrates the evidence that 1) all immunodominant regions contain a repetitive epitope, and 2) all monoclonal antibodies react with a repetitive epitope present in the immunodominant region. The presence of repetitive epitopes in CS proteins is based on the observation that two-site immunoradiometric assays to measure CS proteins can be performed with a single monoclonal antibody. This is illustrated in Figure 3, for the CS proteins of P.viva, P.falciparum, and Pknowlesi. Identical results were obtained with the CS protein of P.berghe and the monoclonal 3D11 (not shown) and the protein CS of P.malariae. The tests were performed as follows: Wells of microtiter plates (Falcon 3911) were incubated overnight at 4°C with 50 µl of a 10 µg / ml solution of a monoclonal antibody in PBS. The wells were washed with PBS and incubated for 2 hours at room temperature with PBS-Tween 20 (0.05% w / v) and for 3 hours at room temperature with PBS-Tween 20-BSA (1% w / v). 30 / ml serial dilutions of sporozoite extracts were dispensed to the bottom of the wells, and the plates incubated overnight in the refrigerator. Extracts were prepared by treating purified salivary gland sporozoites (10 / ml) with 2% (w / v) NP-40 (Trademark, Particle Data Laboratories, Elmhurst, 111.) in PBS for hours at room temperature, followed by centrifugation at 100,000 g for one hour. Extract dilutions were made in PBS-BSA containing 0.1% (w / v) NP-40. After incubation, wells were washed with PBS-Tween 20-BSA. Then 50 (approximately 5-10 ng) of the 125 the same I-labeled monoclonal antibody, diluted in PBS-BSA-Tween 20, and incubated at room temperature for an additional hour. The wells were washed with PBS-Tween 20-BSA and counted. Controls consisted of wells initially coated with BSA only. As illustrated in Figure 3, specific binding was observed in every case using the homologous antigen. These experiments demonstrate that the various sporozoite extracts contain CS proteins that are at least bivalent, since they can bind two molecules of a single monoclonal antibody, one of them in the solid phase, bound to plastic, and the other in the fluid phase and radiolabeled. It could be argued, however, that the extracts contained aggregated CS protein. This possibility has been ruled out by the experiments described below, which directly illustrate that the molecular weight of the bivalent or multivalent antigen in the extracts corresponded to that of the CS protein monomers. The extracts were prepared as described above. 1'· - i ν,ν ι g.'i < . •.if.Xli treated with SDS 2% (w / v) - 6M urea, and subjected to ultracentrifugation on sucrose gradients at 5% (w / v) - 20% (w / v). The tests were performed in an ultracentrifuge, using a Beckman SW-20 50.1 rotor (Brand, Beckman Instrument Co., Fullerton, California) at 48,000 rpm for 20 hours. After centrifugation, the bottoms of the tubes were perforated and the drops collected in separate tubes. The fractions were analyzed for the presence of CS protein using two methods. 1) Fraction analysis using the two-site immunoradiometric assay. This was performed with several monoclonal antibodies for each extract, as previously described in this section. Test results are expressed as the number of sporozoite equivalents present in each fraction, as calculated from a common curve obtained on the same day of the experiment. 2) Analysis of fractions for inhibition of binding of monoclonal antibodies to antigen-coated plates. The gradient fractions were also analyzed by an assay that detects single epitopes on the CS protein. This test was performed as follows: antigen-coated plates were prepared, and the minimum saturating dose of the radiolabeled monoclonal antibody was determined as described in Example 2. Aliquots of gradient fractions were mixed with the radiolabeled antibody for one hour at room temperature, and then 30 µl of the mixtures were dispensed to wells containing the solid-phase antigen. After an additional hour of incubation, the wells were washed and counted. Binding inhibition is also expressed as the number of sporozoite equivalents present in the fraction, as calculated from a standard curve. The results of these experiments are summarized in Figures 4 and 5, which also illustrate the location of the protein-producing fractions. The results show that the CS antigen of P. vivax and P. knewlesi was detected by both assays and sedimented in a single peak between the ovalbumin and bovine serum albumin traces. The amount of CS protein found in this peak represented 95% or more of the original input. Since CS proteins and their precursors have molecular weights between 40,000 and 60,000, these results strongly suggest that the extracts contain primarily or exclusively monomers of these molecules. Furthermore, all monoclonal antibodies tested recognized a repetitive epitope on the CS protein, The simplest explanation for this occurrence is that all of these reacted with the same epitope. In summary, the present results and those of the previous examples demonstrate that the CS proteins of the human malaria parasites Ρ,ν i vax, P,falciparum and P. malariae contain a single immunodominant region and repetitive epitopes, as is the case for P.knowlesi. EXAMPLE 5 Cross-species reactivity of sporozoites Cross-reactions between monoclonal antibodies to repetitive epitopes of CS proteins from various PIasmod i um species were detected by circumsporoite reactions (CSP) or by indirect immunofluorescence assay. These tests are described in Nardi, EH et al. Nature 274, 55 (1978) and Danforth, HD et al. J. Parasitol, 64, 1123 (1978), and were performed with fio-clonal antibodies. For example, the CSP reaction is performed by incubating serum dilutions in PBS-BSA with purified viable salivary gland sporozoites at room temperature. After incubation for 10 minutes or more, the sporozoites are examined under a phase-contrast microscope. A positive reaction consists of a thread-like precipitate formed at the posterior end of the parasite. The CSP reaction does not occur in the cold, or with formaldehyde-fixed parasites. As demonstrated by Potocnjak, P., et al. supra, the CSP reaction is caused by cross-linking of the CS protein by antibodies. The indirect immunofluorescence test is performed with glutaraldehyde-fixed sporozoites. The parasites are treated with a 1% (w / v) solution of glutaraldehyde in PBS for 30 minutes at 0°C. They are then washed in PBS and incubated overnight with 0.1% (w / v) glycine in water. After washing per cent reflux, the resuspended sporozoites are deposited in 10^1 drops on microscopic slides, at a concentration of 2 X 10 / ml The drops are air-dried and kept at -70°C. The test is performed by incubating the sporozoites with dilutions of the immune serum for two hours at room temperature, followed by washing with PBS and a new incubation for 2 hours with a second, fluorescently labeled antibody directed against the Ig of the first immune serum. The second antibody (e.g., rabbit anti-human Ig) can be obtained from a commercial source (Cappel Laboratories, Cochranville, Pa.). After washing, the slides are viewed under a fluorescence microscope. Using both procedures, the following cross-reactions were observed between monoclonal antibodies to the repetitive epitopes of the CS protein: A) anti Pknowlesi ba reacted with P.falciparum and P. cynomolgus; B) anti-P.cynomolgi reacted with Pv i vax; C) anti-P.yoeli nigeriensis reacted with P.berghei. In this case, the cross-reactive monoclonal antibodies also neutralized the infectivity of the heterologous species; and D) anti-P.malariae reacted with P.brasilianum. EXAMPLE 6 Reactivity of synthetic peptides with monoclonal antibodies to the repetitive epitope of P.knowlesi The two synthetic peptides described in Example 1 were used in these studies. One of them consisted of the amino acid sequence 12. H^N-GlnAlaGlnGlyAspGlyAlaAsnAlaGlyGlnPro-COOH (12-MER) and the other was a dimer of the same sequence (24-MER). The 24-MER (but not the 12-MER) was shown directly by immunoradiometric assay to contain two kkT 1 ufi epitopes of the P.knowlesi CS protein. The assay was performed as follows: Wells of microtiter plates (Falcon 3911) were incubated overnight at 4°C with 50 µl of a 10 µL / ml solution in PBS of the 2G3 monoclonal anti-CS protein of P.knowlesi. The wells were washed with PBS and then incubated for 2 hours at room temperature, with P3S-Tween 20 (0.05% (w / v)), and then for an additional 3 hours with PBS-BSA (1% (w / v). Synthetic peptides were serially diluted in PBS-BSA-Tween 20, and 30 µl aliquots of the dilutions were dispensed to the bottom of individual wells. Plates were incubated overnight in the refrigerator, washed with PBS-BSA-Tween 20, and incubated with 5.0 µL (10 ng) 125 I-labeled monoclonal antibody 2G3 diluted in PBS-BSA-Tween 20 (approximately 100,000 cpm). Controls consisted of plates coated with an irrelevant monoclonal antibody of the same isotype as 2G3. The results are shown in Table IV. Specific counts were found only in wells incubated with 24-MER and were proportional to the dose of peptide added to the well. BUGtUON L-.pA TABLE IV Results of a two-site immunoradiometric test performed with 2G3 monoclonal antibody and synthetic peptides. Concentration of 12-MER Amount of radiolabeled 2G3 or 24-MER incubated with 1'an- (cpm) bound in wells incubates phase 2G3 body so 1 ida ti with: ( / jg / ml ) 12-MER 24-MER 500, 157 5517 50, 103 2056 5, 40 402 .5 0 93 .05 35 No specific 24-MER count was linked to wells where the solid-phase antibody was the monoclonal 3D11, which is directed against the CS protein of P . berghe i. These results strongly suggest that 24-MER contains two identical epitopes recognized by the 2G3 monoclonal and that 12-MER contains either one epitope or none. To distinguish between these two possibilities, a test was conducted to determine the capacity of the 12-MER inhibits the interaction between P.knowlesi CS proteins and the monoclonal antibody 2G3. The inhibition test was performed as follows: Wells of microtiter plates were coated with the monoclonal antibody 2G3, washed with Tween-20, and saturated with BSA as described above. In a first set of tubes, 12-MER was heavily diluted in PBS-BSA-Tween 20. In a second set of tubes, an extract of P.knowlesi sporozoites (prepared as described by Cochrane, et al., supra) was heavily diluted in PBS-BSA-Tween 20. Aliquots of each sporozoite dilution were mixed with equal volumes of all 12-MER dilutions. 30 µL of these mixtures were then added to the bottom of 2G3-coated wells. As positive and negative controls, 30 µl sporozoite dilution aliquots mixed with PBS-BSA-Tween were added to the other wells that had been precoated with 2G3 or an irrelevant monoclonal antibody. After an overnight incubation in the refrigerator, the wells were washed and 50 µl (10 µg) of I-labeled 2G3 diluted in PBS-BSA-Tween was added. 20. After an additional incubation at room temperature for one hour, the wells were washed and counted in a gamma counter. The results are shown in Table V. II 12-MER inhibited, in a dose-dependent manner, the interaction of the P.knowlesi CS protein with 2G3. TABLE V A synthetic dodecapeptide (12-MER) inhibits the binding of a monoclonal antibody (2G3) to the CS protein of P.knowlesi Concentration of extracts Amount of 2G3 binding (cpm) of P.knowlesi (sporozoites / ml) in the presence of inhibitor 12-MER at concentrations of 6 0 0.08 0.6 (mg / ml) 5.0 10 5582 3893 3018 1099 10 5 4390 1221 781 361 io 4 1353 102 70 3 10 3 156 72 0 37 0 0 0 0 0 The conclusion drawn from this experiment is that the 12-MER peptide contains an epitope of the CS protein of Ρ,knowlesi. The reactivity of the synthetic peptides was confirmed by radiolabeling of 24-MER and shows that it specifically bound a monoclonal antibody to Ρ, knowlesi (5H8}. This experiment was done as follows: A) Preparation of Sepharose-4B (Brand, Pharmacia, Ine. Uppsala, Sweden) coupled to the mcrc>clonal antibodies 5H8 and 3D11, directed against the CS proteins of the P.knowlesi and P.berghei . * ' 1J À Antibodies were coupled to CNBr Sepharose edges (Pharmacia Fine Chemicals Uppsala, Sweden) according to the manufacturer's instructions. After coupling, the edges (containing approximately 10 mg antibody / ml) were treated for one hour at room temperature with 0.5% glutaraldehyde (to prevent protein loss), then with a 10 mg / ml solution of glycine in PBS, and finally resuspended in a volume at 10 °C. 20% in PBS-BSA (1% w / v) Tween 20 (0.05% w / v). The Sepharose-coupled monoclonal antibodies were designated Septiarose-5H8 and Sepharose 3D11, respectively. B) The radio marking of the 24-MER was made with 125 I using the Bolton-Hunter reagent (Amersham International Ltd., Amersham, UK) according to the manufacturer's instructions, using 10 yul of a 24-MER solution (10 mg / ml) and 0.1 millicuri of the reagent Bolton Hunter. The peptide was purified after labeling on a Sephadex-G10 column equilibrated in PBS (0.2% w / v) gelatin. Assuming 100% of the peptide was recovered, the specific activity was 10 -cpm / ^jg of protein. C) Specific binding of radiolabeled 24-MER to Sepharose-5H8. Four 200 μΐ aliquots of the 20% (w / v) Sepharose-5H8 suspension were added to tubes containing 150 μL of a dilution of the labeled 24-MER in PBS-BSA Tween 20 (45,000 cpm). Two of the tubes were added with 50 µl of diluent. The other two tubes were added with 50 µl of cold 24MER (500 µg) diluted in PBS-BSA-Tween. The tubes were incubated overnight in the refrigerator. The edges were washed by centrifugation and counted. As controls, identical mixtures were prepared in tubes containing Sepharose-3D11. The results are shown in Table VI. These results demonstrate that the radiolabeled peptide bound specifically to the monoclonal antibody 5H8 against the CS protein of Pknowlesi. Furthermore, it appears that 24-MER may have interacted weakly with the 3D11 antibody. This is not surprising, considering that all CS proteins are structurally related and that, in these experiments, the molar ratio of antibody to binding peptide was very high. TABLE VI PUGIWN ίλρΛ Monoclonal antibody on Sepharose edges % inhibitor of added radiolabeled 24-MER that bound to edges (average of duplicates) 5H8 none 42.6 (anti—P,knowlesi) 24-WED 3D11 (anti-P.berghei) none 24-WED 6.6 2.5 EXAMPLE 7 Immunization with the synthetic repeat epitope of P.knowlesi (24-MER) Synthetic 24-MER is synthesized as described in Example 1, except that a cysteine ​​residue is added to the N-terminus. To determine if the synthesis has been performed correctly, an aliquot is subjected to acid hydrolysis under reduced pressure (6 M HCl, 110°C, 72 hours) and its amino acid composition is determined. The peptide is coupled to a carrier protein, either limpet hemocyanin or tetanus toxoid, via its N-terminal cysteine ​​residue, using m-maleimidolbenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling reagent (Ling, F.T., et al. Biochemistry 18, 690 (1979)). This is a bifunctional reagent that under appropriate conditions reacts specifically with the amino group of the support on one side and with the thiol group of the peptides on the other side. mg of the carrier protein in 0.25 ml of 0.05 M PO .4 buffer, pH 7.2, are reacted dropwise with 0.7 mg of MBS dissolved in dimethylformamide and stirred for 30 minutes at room temperature. The product, i.e., the MB support, was separated from the unreacted chemicals by passage through a Sephadex G-25 column equilibrated in 0.05M PO^ buffer, pH 6.0. The MB support is then reacted with 5 mg of the 24-MER containing cysteine, dissolved in PBS (PH 7.4). The mixture is stirred for 3 hours at room temperature. The coupling efficiency is checked with radioactive peptide; that is, a trace amount of the I-labeled 24-MER is mixed with cold peptide during the synthesis. Dialysis of the conjugate allows evaluation of the proportion of the incorporated moiety. The 24-MER group number for 100,000 MW support was estimated to be 10-14. Five rhesus monkeys were immunized with 200 mg of the conjugated protein absorbed onto aluminum hydroxide gel. Their serum was checked for the presence of antibodies to P. knowlesi CS proteins by the immunoradiometric assay described in Example 1. 3. That is, serum dilutions are incubated with antigen-coated wells of microtiter plates. The presence of monkey antibody bound to the solid-phase antigen is checked by incubation with affinity-purified rabbit anti-human Ig 125 labeled 1 (which cross-reacts strongly with rhesus monkey Ig). After 30 days, the monkeys' serum titers rise to titers greater than 1 / 1000. At this time, these monkeys (as well as five other control monkeys injected with unconjugated carrier protein adsorbed to aluminum hydroxide) are treated with 2,000 injectable P♦knowlesi sporozoites. The infection is monitored daily for a total of 30 days by microscopic examination of blood smears, starting one week after inoculation of the parasites. The results show that the five monkeys immunized with the vaccine (protein conjugate) are totally protected, that is, no parasites are found in their blood. In contrast, the control monkeys have P.knowlesi trophozoites in their circulation. 7-12 days after the change. EXAMPLE 8 Immunization with synthetic repeat epitopes of 6.^ P. vivax and P. falciparum The synthetic sequences corresponding to the repeated epitopes of P.vivax and P.falciparum are synthesized essentially as described in Example 1, except that cysteine ​​residues are added to the N termini as described in Example 7. To determine if the synthesis was performed correctly, aliquots are subjected to acid hydrolysis under reduced pressure (6 M HCl, 110°C, 72 hours) and their amino acid composition is determined. The peptides are coupled to a carrier protein, either limpet hemocyanin or tetanus toxoid, via its N-terminal cysteine ​​residue, using MBS as a coupling reagent, as described in Example 7. mg of the carrier protein in 0.25 ml of 0.05M PO^ buffer, pH 7.2, are reacted dropwise with 0.7 mg MBS dissolved in dimethylformamide, and stirred for 30 minutes at room temperature. The product, i.e., the MB-carrier, is separated from the unreacted chemicals by passing through a Sephadex G-25 column equilibrated in 0.05 M PO4 buffer, pH 6.0. The MB-carrier is then reacted with 5 mg of each cysteine-containing peptide, dissolved in PBS (pH 7.4). The mixture is stirred for 3 hours at room temperature. The coupling yield is checked with radioactive peptide; i.e., a trace amount of 125 The labeled compound is mixed with cold peptide during synthesis. Dialysis of the conjugate allows for the assessment of the proportion of the incorporated moiety. The number of synthetic peptides per 100,000 MW carrier is estimated to be about 10-14. Five chimpanzees were immunized with 200 μg of each of the conjugated proteins absorbed onto aluminum hydroxide gel. Their serum was checked for the presence of antibodies to the proteins. CS of P. vivax and P.falciparum from the immunoradiometric assay described in Example 3. That is, serum dilutions are incubated with antigen-coated wells of microtiter plates. The presence of chimpanzee antibody bound to the solid-phase antigen is checked by incubation with affinity-purified 12 5 I-labeled rabbit anti-human Ig (which cross-reacts strongly with chimpanzee Ig). After 30 days, the chimpanzee serum titers increased to titers greater than 1 / 1000. At this time, these chimpanzees (as well as five other control chimpanzees injected with unconjugated carrier protein adsorbed to aluminum hydroxide) were challenged with 2,000 viable P. vivax sporozoites. The infection is monitored daily for a total of 30 days by microscopic examination of blood smears, starting one week after inoculation of the parasites. The results show that the five chimpanzees immunized with the vaccine (protein conjugate) are completely protected, meaning no parasites are found in their blood. In contrast, control chimpanzees have P. vivax trophozoites in their circulation. 10-12 days after the test. Subsequently, the same chimpanzees were given a second challenge with 10,000 Pfalciparum sporozoites. Again, the vaccinated animals are protected, while the controls are all infected. Based on close similarities in the immunological responses of humans and chimpanzees and on the fact that protective immunity was obtained in humans by injection of inactivated sporozoites of Pf alciparum et P.vi vax, the results obtained by immunizing chimpanzees with the described synthetic peptide will also be obtained by following a similar treatment of human patients. A more precise identification of the immunodominant region in light of the immunodominant epitope itself is desirable for several reasons: first, such identification would be useful to provide further insight into the immunogenicity of the CS protein; second, a peptide with a shorter amino acid sequence identical or similar to that of the epitope is easier to prepare and / or purify, using either conventional peptide synthesis or recombinant DNA techniques. Such a peptide would be useful if it exhibits anti-CS binding activity similar to that of the dodecapeptide. Of even greater interest was whether the epitope was represented within an uninterrupted sequence of amino acids in the dodecapeptide or whether it was configurational, that is, formed by opposing residues by virtue of the higher-order structure of the dodecapeptide (12-peptide). Since several monoclonal (and polyclonal) antibodies to the CS protein neutralize the infectivity of sporozoites, if the epitope was represented in an uninterrupted sequence and consisted of a relatively short sequence, this peptide provides the basis for the development of a synthetic vaccine. The size and shape of epitopes found on carbohydrate antigens have been extensively studied, but less is known about the structure of epitopes from protein molecules. Some protein antigen epitopes have been defined at the level of their three-dimensional structure. In any case, the epitopes were formed not by the primary sequences alone, but by the juxtaposition of residues brought together by folding the polypeptide chain(s) of the native molecule. For example, monoclonal antibodies to sperm whale myoglobin did not react with any of the three CNBr cleavage fragments that collectively encompass the entire hemoglobin sequence (Berzofsky, J.A. et al., Topographical antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin (1982) J. Biol. Chem. 257:3189-3198; East; I.J. et al. Antigenic specificity of monoclonal antibodies to human myoglobin (1982) J. Biol. Chem. 2 57 : 3199). A monoclonal antibody linked to the epitope in lysozyme was found to comprise a region containing the silver 68 - silver 45 complex surrounding the catalytic locus (Smith-Gill, S.J. et al. Antigenic epitope map for a monoclonal antibody against lysozyme, (1982) J. Immunol. 128:314). Monoclonal antibodies recognizing the A chain ring (A 8-10) of insulin failed to bind to isolated A chains or synthetic peptides (Shnoer JA et al., (1983) Eur. J. Immunol. 13:693). The approach used here involved starting with the amino acid sequence of the repeat peptide of a sporozoite CS protein such as that of Plasmodium knowlesi, synthesizing its tandem repeat peptide (twice the number of amino acids) and its analogous peptides having progressively smaller sequences (with progressive omission of terminal amino acids), and determining the reactivity of monoclonal antibodies to the CS protein (monoclonal anti-CS) to these peptides and each of their analogs. The aim was to find the shortest analogue with high antibody reactivity, thereby simultaneously identifying the location of the epitope within the dodecapeptide (if the locus of such an epitope was in the primary amino acid sequence) and identifying a peptide having immunochemical reactivity against a monoclonal anti-CS similar to that of the dodecapeptide. The particular tandem repetitive polypeptide chosen for the experimental work described below was that of the CS protein of P.knowlesi. This repetitive polypeptide is a dodecapeptide having the amino acid sequence (N to C terminus) (Gln i-Ala 2-Gln 3-Gly 4-A Sp 5-Gly 6-Ala 7-Asn 8-Ala 9-Gly 10-Gln 11-Pro 12). Several monoclonal anti-CS antibodies have been raised against the CS protein of P.knowlesi (as described in patent application No. 234,096) and were available to test the immunoreactivity of peptides synthesized herein. However, the fact that both the peptides synthesized or employed in the present work and the antibodies are against P.knowlesi does not limit the applicability of the present invention to the CS protein of P.knowlesi. There is strong evidence that CS proteins from other Plasmodium species also contain tandem repetitive peptides and are related proteins to the CS protein of P.knowlesi (although their amino acid sequences may not be the same). For example, previously reported results of immunological tests indicated that only one area of ​​the CS molecule of Pv ivax, P, falciparum, P.knowlesi and P.berghei was recognized by all monoclonal anti-CS (elevation of the CS protein of the same species). Furthermore, cross-reactivity between CS proteins from different Plasmodium species and anti- 74 κιVGhìΟΝ has been reported. CS of P.knowlesi (Cochrane et al (1982) Proc. Nat ' 1 Acad. Sci. USA 79:5651). Furthermore, Santoro et al (1982) J. Biol. Chem. 258:3341, reported evidence that CS proteins from different malaria species are parts of a family of structurally related molecules. Therefore, it is anticipated that the present invention will be applicable to several species of Plasmodium, A synthetic dodecapeptide (corresponding to the protein P.knowlesi CS repeating peptide sequence) and a tetraeicosapeptide consisting of a tandem repeat of said dodecapeptide were prepared and tested for anti-CS binding activity with six antiCSs that had been produced after immunization of mice with intact sporozoites, but which were shown to be reactive with a single molecule, the CS protein: Cochrane, AH et al. Monoclonal antibodies identify protective antigens of Plasmodium knowlesi sporozoites (1982) Proc. Nat'l. Acad. Sci., USA, 79, 5651. The CS protein is stage and species specific (Vanderberg, et al. Military Medicine, 304:1183 (1969); Cochrane, AH et al. Antibody-induced ultrastructural changes of malaria sporozoites (1969), J. Immunol. 116, 859) uniformly distributed over the entire surface of the sporozoite and is shielded when ι ili 'tl U cross-linked by antibodies (Potocnjak P. et al. Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (1980) J. Exp. Med. 151, 1505.) Only one area of ​​the CS molecule from several species was recognized by all homologous monoclonal anti-CS and this immunodominant region was multivalent with respect to the expression of a single epitope: Zavala, F. et al. (1983), J. Exp. Med. 157 : 1947. DNA analysis showed that the immunogenic region of the P.knowlesi CS protein consists of a tandem ih repeat of 12 units of 12 amino acids each (see peptide antigen application). Furthermore, the epitope recognized by the first monoclonal anti-CS (2G3) was included within a single subunit. The synthetic dodecapeptide very effectively inhibited the anti-CS / CS reaction, while 24-MER interacted simultaneously with two 2G3 molecules. The present work demonstrated that epitopes that react with five other monoclonal anti-CS antibodies are also represented within the 24-MER. This was useful in determining whether the epitope lies within a 24-amino acid sequence, before testing whether the epitope was contained within the 12-amino acid sequence. , 0H JpÀ Four monoclonal antibodies (2G3, 5H8, 8B8, and 8E11) were used in the immunoactivity test of synthesized shorter length analogs of 12-peptide. The aim was the further positioning and identification of the epitope contained therein. An 11-amino acid analogue was first synthesized, specifically analogue (2-12) due to the omission of the N-terminal Gin residue. The reactivity of (2-12) was compared with that of (1-12) and was found to be substantially equivalent (see Table IX which will be shown below). Subsequently, the reactivities of 3-12, 4-12, and 5-12 were determined and found to be essentially identical to that of 1-12. However, the omission of the 5 C amino acid, Asp residue, resulted in a considerable drop in reactivity. It has been tentatively concluded that amino acid residues 1 to 3 and possibly 4 do not contribute to the immunoreactivity of the 12 peptide. Starting from the C terminus, analogues (1-11), (2-11) and (3-11) were not synthesized based on the hypothesis that amino acids 1, 2 and 3 did not contribute to immunoreactivity. (4-11) had immunoreactivity substantially equivalent to (actually, slightly greater than) that of (5-12). However, (5-11) showed a drop in reactivity commensurate with that observed when going from (5-12) to (6-12). Peptides of shorter length showed no reactivity, thus confirming that peptides (4-11) and (5-12) were those having high reactivity and shortest length. The above procedure of omitting one extreme amino acid at a time and testing the reactivity of the resulting peptide leads to epitope isolation in the minimum number of steps. Once it is shown that an amino acid residue does not participate in the epitope, it can be eliminated from further testing. As summarized in Table IX, the dodecapeptide NH^Gln-Ala-Gln-Gly-Asp-Gly-Ala-Asn-Ala-Gly-Gln-ProCONH^ (1-12), as well as peptides (2-12), (3-12), (4-12) and (5-12) were very effective inhibitors. However, after removal of Asp or Asp and Gly (positions 5 and 6), the degree of reactivity was considerably lower. This is consistent with the hypothesis that shorter peptides of 5-12 are not active. The role of amino acids from the C-terminal end of (1-12) has also been examined. Figure 8 is a graph illustrating the inhibition of binding of radiolabeled monoclonal antibody 5H8 to the CS protein of P.knowlesi in the presence of increasing concentrations of various peptides, said peptides being indicated as in Figure 2. As illustrated in part B of Figure 8, the removal of Pro (position 12) or Gin (position 11) from the dodecapeptide has a considerable effect on its reactivity with the 5H8 monoclonal. However, for monoclonals 2G3 and 8B8, the reactivity with 4-12 or 4-11 was almost identical, while 4-10 was much less reactive (part B, Figures 7 and 9). Figure 7 is a graph illustrating the inhibition of binding of the 2G3 monoclonal antibody to the protein CS of P.knowlesi by peptides that are analogues of the repeating peptide of CS proteins, these peptides being indicated by the position of their amino acid termini with the dodecapeptide sequence. Figure 9 illustrates the inhibition of binding of radiolabeled monoclonal antibody 8B8 to the CS protein of P. knowlesi in the presence of increasing concentrations of various peptides, indicated by their position within the dodecapeptide as in the previous figures. The dodecapeptide contains only one proline (at the C terminus) and this residue is not essential for reactivity with two monoclonal antibodies. The above results lead to the conclusion that: - 7 9 (a) Contrary to research findings on many other antigens, there is a single immunodominant region in the P.knowlesi CS protein and the epitope is positioned within a primary sequence (rather than a conformation) of 12 amino acids around residues 4-11; and (b) it is possible to obtain substantially equivalent immunoreactivity from a peptide having only a 8-residue sequence rather than a 12-residue one; that is, it is possible to obtain an immunoreactive peptide of a minimum amino acid length shorter than that of the repeating unit itself (dodecapeptide). From the above, it is clear that monoclonal antibodies react with a sequence of amino acids around residues (4-11). Figure 6 is a graph illustrating the relationship between the activity of the dodecapeptide (which constitutes the repeating unit of the repetitive peptide of the P·knowlesi CS protein) and a 24-peptide (which consists of a tandem repeat of said dodecapeptide) that inhibits the binding of different monoclonal antibodies to the P·knowlesi CS protein. Figure 6 illustrates that the specificity of 8E11 differs markedly from that of other monoclonal antibodies in that the interaction of 8E11 with the ι CS protein is inhibited only by (1-24) and not by (1-12). To examine the possibility that 8E11 recognizes an overlapping sequence between two tandem dodecapeptides 1-12, several additional peptides were synthesized and tested for inhibitory activity, with negative results. This indicates that 8E11 reacts with a configurational or topographical epitope formed by joining two dodecamers. Three of the four monoclonal antibodies used in the immunoreactive assays for the 12-p-heptide, 24-peptide, and synthetic analogs (2G3, 5H8, and 8E8) were recognized by the shortest reactive analogs of minimal length. Since the epitope is repeated twice within a sequence of 24 consecutive amino acids, the binding sites are unlikely to recognize secondary or tertiary structures of the polypeptide chain. The knockout immunogenicity of this epitope is most likely a reflection of the unusual structure of the CS protein, half of which consists of a tandem repeat of 12 amino acids, each repeat containing a potential epitope. It is noteworthy that the streptococcal M-type protein also contains a repeating peptide subunit, which contains the immunodominant epitope (Beachey, EH et al., Primary structure of protective antigens of streptococcal M protein type 24, (1980), J. Biol. Chem. 255 : 6284-6289. However, the CS protein is not known to bear any structural or other similarities to the M protein. In other studies, monoclonal antibodies 263 and 8E11 (or the corresponding Fab fragments) not only bound to the CS protein of P.knowlesi, but also neutralized the infectivity of sporozoites (Cochrane, AH et al. supra) . In light of the present results, it seemed possible that polyclonal antibodies to synthetic peptides representing the repetitive epitope of the protein CS of P.knowlesi could have biological activities ε imi 1 ari. Indeed, rabbits and mice have been successfully immunized against P.knowlesi with tetraeicosapeptide (1-24) conjugated to a carrier protein. Several animals produced antibodies that reacted with the sporozoite membrane and immunoprecipitated the CS protein. Furthermore, the sporozoites lost their infectivity when incubated in serum from one of the immunized rabbits. These observations raise hopes that if equivalent peptides from the CS proteins of human malaria parasites are found to be immunogenic in vivo, they can be used in the formulation of vaccines for humans. The general steps of the peptide synthesis techniques used herein are well known. Specific modifications made by the present inventors to adapt these techniques to the synthesis of the present peptides are described in the Examples. After synthesis, cleavage and purification, the peptides were tested for reactivity with anti Monoclonal CS, preferably partially purified. The anti-CS used in these trials was produced by inducing ascites tumor using hybridoma cells resulting from the fusion of plasmacytoma cells with spleen cells from a mouse immunized with the parasite, as described by Cochrane, AH et al. supra and as illustrated in the malaria vaccine patent application. The immunoactivity of the synthetic peptides was evaluated by measuring their ability to inhibit the reaction between the monoclonal anti-CS and the antigen {CS protein). The antigen has been purified from sporozoites, as illustrated in the malaria vaccine patent application and also as described by Vanderberg, supra, and Zavala, F., et al, supra, 1982, and is preferably 125 employed using labeled I antibody. β 3This aspect of the present invention is further described below in the following Examples, which are intended to illustrate but not limit its scope. Materials and sources Amino acid derivatives (and protecting groups) and benzhydrylamine resin: Beckman Instruments, Palo Alto, California. Hydroxybenzotriazole: Sigma Chemical Co., Ine. St. Louis, Mo. Sephadex G-25 and G-2OO: Pharmacia Fine Chemicals Co, Piscataway, NJ Bovine Serum Albumin: Sigma Chemical Co. St. Louis, MO. Iodogen: Pierce Chemical Co., Rockford, 111. Boulton-Hunter Reagent: Amersham Corp., Alrington Hgts. III EXAMPLE 9 Peptide synthesis The syntheses were performed using a benzhydrylamine (BHA) resin (0.654 meg / gm) on a template Vega 250C (Vega Biochemicals, Ing. Tuscon, Arizona) automatic synthesizer controlled by a Motorola computer with a program based on that of the Merrifield, R.B. Fed. Proc. 21:412 (1962), J. Chem. Soc. 85:2149, (1963). First, the dodecapeptide Gln-Ala-Gln-Gly-AspGly-Ala-Asn-Ala-Gly-Gln-Pro was assembled on 3.0 gms of the benzhydrylamine resin which had been suspended in CH^Cl^ and washed three times with CH^Cl^, three times with ethanol, and three times with CH^Cl^ ηθ1 synthesizer. The resin was washed for a total of two minutes and then treated with 50% trifluoroacetic acid containing 10% anisole in CH^Cl^ for 30 minutes, washed 10 times with CHCl, neutralized by washing twice with 10% diisopropylethylamine in D^Cl^. The first BOC-amino acid (BOC stands for tertiary butyloxycarbonyl) was coupled for one hour to the benzhydrylamine resin using a threefold molar excess of BOC-amino acid di-cyclohexylcarbodiimide in the presence of three moles excess CH^Cl^ and hydroxybenzotriazole. Additional aliquots, one each of hydroxybenzotriazole and diisopropylethylamine, were added to a three-fold molar excess of BOC amino acid for an additional hour. The resin was then washed with CH^Cl^ (three washes), absolute ethanol (three washes), and C^Cl^ (three washes), and an aliquot of the mixture was tested using the nihydrin procedure. Kaiser (Kaiser, E. et al. (1970), Analyt. Biochem. 34: 595) . resulting peptide was Boc-Gln(NPE)-Ala-Gln(NPE)Gly-Asp(OBZ)-Gly-Ala-Asn(NPE)-Ala-Gly-Gln(NPE)-Pro85 Co-BHA (ΝΡΕ stands for nitrophenyl ester and OBZ stands for benzyl). At this point in the synthesis, 50% of the protected peptide resin was removed and saved by HF cleavage and purification. The tandem repeat, i.e., the tetraeicosapeptide (1-24), was assembled by the sequential addition of protected amino acids in the same order as the dodecapeptide, using the procedure described above. After synthesis, 2.0 gms each of the (1-12) and (1-24) protected peptide resins were subjected to HF treatment as described below and the unprotected cleaved peptides were washed separately with anhydrous ether and extracted with alternating washes of glacial acetic acid and water. Cleavage of peptide resins (2 gms each) was accomplished in a Penninsula HF device (Penninsula Laboratories, San Carlos, California) in the presence of anisole (1.2 ml / mg resin) and methylethyl sulfur (1 ml / mg) at 10°C for 1 hour after which the mixture was carefully dried under high vacuum. The mixture was then washed with cold anhydrous ether, extracted with alternating washes of water and glacial acetic acid and freeze-dried. Crude peptides were then desalted by filtration through Sephadex G-25 gels (120 x 2.0 cm) in 200 mg aliquots. The column was equilibrated with 0.1 NH .HCO , pH 8.0, also used as a simple buffer. The column effluent was monitored by UV absorption at 254 and 206 nm with an LKB UVcord III control. The peptides thus synthesized were analyzed and characterized as follows: Samples were hydrolyzed in 5.7 N HCl for hours at 110°C, dried, reconstituted in 0.2 N sodium citrate, pH 2.2 and applied to the amino acid analyzer (Liquimat III) according to the Spackman procedure, DH et al., Anal. Chem., 30:1190, 1958. At selected steps during the synthesis, aliquots of the peptide resin were removed from the synthesizer reaction vessel, mixed with glass rims, subjected to solid-phase Edman degradation (Laursen, R; (1971) Eur. J. Biochem. 20:89) on a Sequemat Mini 15 peptide sequencer (Sequemat, Ine, Waltham, Mass.). After peptide cleaving from the resin, the purified crude and synthetic peptides were subjected to automated liquid-phase sequence analysis (Edman P. and Begg G., Eur. J. Biochem. 1:80-90 (1957) ) on a Beckman sequencer (Model 890) using a DMAA (dimethylaminoamine) peptide program to detect the presence of sequence faults and side chain protecting groups on the peptide not removed by HF cleavage. Quantification of the amount of error peptides and side chain protecting groups was achieved by sequence analysis, followed by identification and quantification of PTH amino acids by high-performance liquid chromatography, a well-accepted procedure of preliminary analysis, as illustrated by Niall, H.D. et al., Chemistry and biology of peptides: proceedings of the third American peptide symposium (1972) (J. Meienhofer, ed.) Ann Arbor Science Pub., 695, 1972; modified by Tregear, GW, Peptides: proceedings of the third European peptide symposium (Y. Wolman,ed.) (1974); and Tregear GW et al. Jr. Biochem. (1977) 16: 2817; as further modified by (Simmons, J. and Schlesinger, D.H., High-performance liquid chromatography of side-chain-protected amino acid phenylthiohydrantoins, (1980) Anal . Biochem., 104, 254; and Schlesinger, D.H. (1983) Meth. Enzymol. 91, 494. In addition to the dodecapeptide and 24-peptide, four groups of dodecapeptide analogues have been synthesized, each possessing as terminal C is the amino acid or Pro, Gin, Gly or Ala, which correspond to positions 12, 11, 10 and 9 of the dodecamer respectively (Table VII). Peptide analogues possessing Pro at their C-terminus were synthesized by removing dry protected peptide resin (approximately 10% in each step) at positions 10, 8, 7, 6, 5, 4, 3, and 2 during the synthesis of the dodecapeptide, as described above. This procedure yielded 9 protected peptide resins. * ' ί ·. j » .· ν 1Ά ' -g TABLE VII Amino acid composition and preliminary analysis of synthetic peptide segments of CS protein P.knowlesi Pro Jun Gly Ala peptide segment Preliminary 1 - 12 1.84(2) 0.95(1) 3.00(3) 3.00(3) 2.97(3) 1.4% 2-12 2.21(2) 0.85(1) 2.15(2) 3.00(3) 3.02(3) N.R. 3-12 2.18(2) 1.07(1 ) 2.00(2) 2.77(3) 1.88(2) N.R. 4-12 2.00(2) 1.10(1) 0.88(1 ) 2.84(3) 1.97(2) N.R. 5-12 2.18(2) 1.17(1) 1.00(1 ) 2.00(2) 1.93(2) N.R. 6-12 0.93(1 ) 1.00(1) 0.97(1 ) 2.13(2) 2.16(2) N.R. 7-12 0.90(1 ) 1.14(1 ) 1.03(1 ) 1.00(1) 2.00(2) N.R. 8-12 0.92(1 ) 1.05(1, 1.01(1, 1.00(1 ) 1.00(1) N.R. 10-12 — 1.10(1) 1.01(1) 1.00(1 ) — N.R. 1 - 24 4.15(4) 1.97(2) 6.14(6) 6,05(6) 5,95(6) 2,1% 4 - 11 2.16(2) — 0.88(1 ) 3.40(3) 2.00(2) N.R. 5-11 2.06(2) — 0.95(1 , 2.21(2) 2.01(2) N.R. 4-10 1.88(2, — — 3.00(3) 1.75(2) N.R. 4-9 2.00(2) — — 2.18(2) 1.97(2) N.R. 5-9 2.00(2) — — 0.81(1) 2.14(2) N.R. 6-9 1.00(1 ) — — 0,75(1) 2.15(2) N.R, 7-9 1.00(1) — — — 2.21(2) N.R. 7-6* 1.94(2) 0.89(1 ) 3.06(3) 2.95(3) 2.91(3) N.R. * peptide che collega due epitopi, cioè Ala 7Asn 8Ala 9Gly 1^ln )^>ro 12Gln 1Ala 2Gln 3Gly 4Asp 5Gly 6 Similarly, two peptides containing the C-terminal amino acid Gin were synthesized; i.e., peptides comprising residues at positions 5-11 and 4-11 of the dodecamer. Only one peptide containing Gly Γ at its C-terminus was synthesized and corresponded to positions 4-10 of the dodecamer. Finally, four peptides possessing Ala at their C-terminus (positions 7-9, 6-9, 5-9 and 4-9 of the dodecapeptide). A peptide bridging parts of the two epitopes, i.e., AlayAsng-Alag-Gly^-Gln.^ -Pro^-GlnyAla^-Gl n^-Gly^-Asp^-Gly^, was synthesized to determine whether the monoclonal antibody might not recognize the dodecapeptide or one of the predicted analogs might be directed against a sequence containing parts of the two tandem repeats. Those protected peptide resins were then cleaved and deprotected with HF, desalted on Sephadex G-25 and characterized by amino acid composition and sequence prediction analysis (see Table VIII, below). The peptides were u- TABLE VIII 125 Binding of the I-tagged tetraeicosa-peptide (24 amino acids) to several monoclonal antibodies raised against the sporozoite surface protein of P.knowlesi Edges carrying the following monoclonal antibodies against the surface protein of P. knowlesi cpm (% entry) of radiolabeled tetraeicosine peptide that bound to the edges suspended in: PBS-BSA PBS-BSA + excess of cold peptide tetrahexyl peptide 2G3 5H8 8B8 8A8 Control: borders carrying a monoclonal antibody (3D11) against the surface protein of P.berghei 2922 (25.8) 25 (0.2) 2332 (19.7) 20 (0.2) 31 55 (27.8) 20 (0.2) 1093 (10.3) 25 (0.2) (0.2) EXAMPLE 10 Reaction of monoclonal antibodies with tetraeicosapeptide Monoclonal antibodies against surface antigens of sporozoites of the monkey malaria parasite Plasmodium knowlesi were produced by fusion of plasmacytoma cells with spleen cells from a mouse immunized with the parasite as previously described by Cochrane et al., supra , (1982) and described in detail in the malaria vaccine patent application. Ε?.ζΙΟΝΙΟΝ kp.iV Monoclonal antibodies (4 idiotypes: 2G3, 8B8, 5H8 and 8E11) were partially purified from ascites of mice bearing hybridomas by 50% ammonium sulfate precipitation followed by molecular sieve chromatography on Sephadex G-200. All types of monoclonal antibodies that were raised against P.knowlesi and selected for reactivity with the parasite surface membrane (Cochrane et al., 1982) reacted with the tetraeicosapeptide. Competitive inhibition of the antigen-antibody reaction by the synthesized peptides was measured by the following radioimmunoassay. The CS protein from the sporozoite of P. knowlesi was used as the antigen. a) Antigen preparation: 3,000 sporozoites of £. Knowlesi purified (Vanderberg et al. Military Medicine 134:1183, 1969) in 50 µl of phosphate buffered salt (PBS) containing protease inhibitors, were dispensed to the bottom of polystyrene microtiter plates (Falcon plate 3911, BD and Co., Oxnard, Calif.) that were sealed with paraffin and frozen at -70°C for at least ten minutes. The plates were then allowed to slowly thaw at room temperature. This procedure was repeated six times. The plates were then incubated at 4°C overnight and at 56°C for five minutes. The P.knowlesi extract was then removed from the wells and these were washed three times with PBS containing 1% bovine serum albumin (BSA) and 0.1% NaN 3. The wells were then filled with PBS-BSA-NaN 3 and incubated for a few hours at room temperature. b) Preparation of peptides and two-site radioimmunoassay to determine the inhibitory activity of the analogues; (1) All synthetic peptides were dissolved in PBS at a concentration of 10 mM and subsequently serially diluted in PBS-1% BSA / 0.1% NaN. Twenty-five µL serial dilutions of each peptide were added to the antigen-coated wells. The control wells received only the diluent. So, 25 / ul of the 1' labeled monoclonal antibody 12 5 5 (approximately 5-10 ng, 10 μg) were added to the wells. The microtiter plates were incubated at 4°C for 18 hours, washed five times with PBS-BSA-NaN 3, and then the wells were counted for well-bound antibody. Antibodies (top) and peptides (bottom) were 125 I-labeled using Boulton-Hunter reagent or lodogen, according to the manufacturers' instructions. To determine whether the 24-peptide is recognized by all monoclonal antibodies, 5 mg of various types of purified monoclonal antibodies were linked to 1 ml of activated CNBr with Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, New Jersey, USA) in accordance with the manufacturer's instructions. 24 amino acid peptide was radiolabeled with 5 I with the Boulton-Hunter reagent at a specific activity of about 10 cpm / ug. Twenty µl of edges carrying the different antibodies were incubated for 60 minutes at room temperature in the presence of 100 µl of PBS-BSA containing either 1 µg of the radiolabeled peptide alone or radiolabeled peptide in the presence of 200 µg of cold tetraeicosapeptide. The edges were then washed for 100% reflux with PBS-BSA and counted in a gamma counter. Table 5 illustrates that the radiolabeled (1-24) bound specifically to monoclonal antibodies 2G3, 5H8, 8B8 and 8A8 coupled to Sepharose edges. Binding was completely inhibited by adding excess cold (1-24) to the incubation mixture. Similar results were obtained in other experiments using monoclonal antibodies 8E11 and 6B8 (not delusional). EXAMPLE 11 Localization of the epitope within the tetraeicosapeptide The inhibitory activity of (1-12) and (1-24) on the binding of radiolabeled antibodies to the CS protein of P.knowlesi sporozoite extracted as described in Example 10 was inspected. The antigen was immobilized on microtiter wells. Figure 1 demonstrates that for two of the three monoclonal antibodies tested (2G3, 5H8) the epitope must be contained within (1-12), since the inhibitory activities of (1-12) indicated by the white symbols - and (1-24) were almost identical. Similar results were obtained with 8B8 but are not illustrated in this figure. In contrast, binding of the 11' antibody 8E11 was inhibited only by (1-24). EXAMPLE 12 Epitope recognition by monoclonal antibodies 2G3, 5H8 and 8B8 The series of shorter analogues and a peptide that overlaps the two epitopes (7-6) illustrated in Table VII was used to analyze the specificity of antibodies 2G3, 5H8 and 8B8 using the inhibition immunoassays described in Examples 10 and 11. As illustrated in Figures 7, 8 and 9 and summarized in Table IX, the reactivity patterns of these antibodies were very similar. 96The peptides (1-12), (2-12), (3-12), (4-12), (b-12) were strongly inhibitory and as effective as (1-24), On a molar basis, their activities were similar, although in some cases, the larger peptides appeared to be slightly better inhibitors. However, a considerable drop in activity was observed in (6-12) and (7-12) compared to (5-12). These results indicate the presence of an immunodominant epitope around the Asp^-Gly-Ala-Asn-Ala-GlyGln-Pro segment. To determine the participation of the C-terminal amino acids, the activity of (4-10) and (4-11) was compared to that of (4-12). As also illustrated in Figures 7, 8 and 9, removal of proline (peptide 4-11) had little effect on the reactivity with monoclonal antibodies 2G3 and 8B8, while removal of both glutamine and proline (4-10) significantly decreased the ability of all three antibodies to inhibit the reaction with the antigen. The shortest peptides (4-9), (5-9), (6-9), (7-9), (8-12), 7GUGNiQN *PÀ TABLE IX Inhibitory effect of synthetic peptides on the binding of radiolabeled monoclonal antibodies to the CS protein of P.knowlesi Peptide Molar concentration of peptide (x ) required for 50% inhibition of monoclonal antibody binding 8B8 2G3 5H8 -12 -1 3 -1 2 1- 12 10 10 10 -12 -12 -11 2-12 10 10 10 -12 -12 -11 3-12 10 10 10 -12 -11 -11 4-12 10 10 10 -10 -10 -9 5 - 12 ' 10 10 10 -7 -6 -6 6-12 10 10 10 -10 -5 _-7 7-10 10 10 10 -12 -12 -10 4-10 10 10 10 4-11 10 10 10 EXAMPLE 13 Epitope recognition by monoclonal antibody 8E11 As mentioned above, the reaction of antibody 8E11 with the CS protein of P.knowlesi was inhibited by (1-24). Approximately 50% inhibition of binding of radiolabeled 8E11 to the CS protein was observed with -5 a concentration of (1-24) of 10 M. In contrast, (1-12) had no appreciable inhibitory effect -2 at a concentration of 10 M (see Figure 6). ì uy yìlAji EXAMPLE 14 The peptide: Ala 7-Asn 8-Al gGly io-Gln ii-Pro 12-Gln 1-Ala 2~Gln 3-Gly 4Asp -Gly„ was synthesized as described above. 6 Tested as in the above examples, this peptide was found to be inactive.

Claims

1. CLAIMS 1) Peptide characterized by comprising an epitope of a sporozoite CS protein of a member of the genus Piasmod i um. 2) Peptide according to claim 1, corresponding in amino acid sequence to a repeating peptide, said repeating peptide comprising the immunodominant region of the sporozoite CS protein of a member of the genus Plasmodium. 3) Peptide according to claim 1, characterized in that the member of the genus Piasmodium is selected from the group P.falciparum, P.vivax, Pknowlesi, P. malariae, P.cynomolgi, P.berghei, and P.yoeli nigeriensis. 4) Peptide according to claims 1 and 2, characterized in that it comprises the amino acid sequence (GlnAlaGlnGlyAspGlyAlaAsnAlaGlyGlnPro). 5) Malaria vaccine characterized in that it comprises as an active ingredient the peptide of claim 1 and a carrier. 9 9CUGhiiON ν.ρΛ 6) Vaccine according to claim 5, characterized in that said synthetic peptide is absorbed or covalently attached to a carrier protein. 7) Peptide comprising an amino acid sequence corresponding to an epitope of a CS protein of a sporozoite of a member of the genus Plasmodium. 8) Peptide according to claim 7, characterized in that the amino acid sequence corresponding to an epitope of a CS protein is chemically synthesized. 9) Peptide according to claim 7, characterized in that the member of the genus Plasmodium is selected from the group P.falciparum, P.vivax, P.knwolesi, P.cynomolgii, P.malariae, P.berghei, or P.yoeli ni gerì ersi 10) Peptide according to claim 9, characterized in that said amino acid sequence is (GlnAlaGlnGlyAspGlyAlaAsnAlaGlyGlnPro). 11) A malaria vaccine characterised by comprising a peptide according to claims 8 or 9 in a physiologically acceptable medium, 12) A vaccine for immunizing a mammal against malaria characterized in that it comprises a peptide according to claim 7, absorbed or covalently attached to a carrier protein, 100 in a physiologically acceptable medium. 13) Vaccine according to claim 12, characterized in that said peptide is immunochemically reactive with a monoclonal or polyclonal antibody to a CS protein of a sporozoite of the genus Piasmodium. 14) Fusion protein produced by a microorganism consisting essentially of: (a) a repeating peptide comprising an epitope of a sporozoite CS protein of a member of the genus Plasmodium, and (b) a part of a prokaryotic protein of that microorganism. 15) Fusion protein according to claim 14, characterized in that said member of the genus Plasmodium is selected from the group P.falciparum, P.vivax, P.knwolesi, P.cynomolgi, Pmalariae, P.berghei, or P.yoeli nigeriensis. 16) Fusion protein according to claim 15, characterized in that said peptide comprises the amino acid sequence (GlnAlaGlnGlyAspGlyAlaAsnAla GlyGlnPro). 17) Peptide according to claims 1-4, 7, 9 and 10 in purified form. 18) DNA base characterized in that it consists essentially of a deoxynucleotide sequence encoding the peptide of claims 1, 2 and 3. 19) DNA base consisting essentially of a deoxynucleotide sequence coding for the amino acid sequence (GlnAlaGlnGlyAspGlyAlaAsnAlaGlyGlnpro), 20) DNA transfer vector characterised in that it comprises an inserted DNA base consisting essentially of a deoxynucleotide sequence encoding the peptide of claims 1, 2, 3 and 4 21) DNA transfer vector characterized by comprising an inserted DNA base consisting essentially of a deoxynucleotide sequence coding for the amino acid sequence (GlnAlaGlnGlyAspGlyAlaAsnAlaGlyGlnPro). 22) DNA transfer vector according to claim 21, characterized in that said DNA base is inserted into a site suitable for the expression of the coding sequence, either directly or as a fusion protein. 23) Microorganism transformed by an expression vector comprising an inserted DNA base according to claim 22. 24) Microorganism transformed by an expression vector comprising an inserted DNA base according to claim 21. 25) Microorganism according to claim 23 comprising Escherichia coli. 26) Synthetic peptide characterized by the fact that it includes the amino acid sequence (GlnAlaGlnGlyAspGlyAlaAsnAlaGlyGlnPro). 27) Tandem repetitive peptide comprising an epitope from a CS protein of a member of the genus Plasmodium. 28) Peptide having an amino acid sequence consisting essentially of an amino acid subsequence, said subsequence defining an immunodominant epitope of a repeating unit of a tandemly repeating polypeptide of a Plasmodium circumsporozoite protein, said peptide being shorter in length than said repeating unit. 29) Peptide according to claim 28, characterized in that said circumsporozoite protein is from Plasmodium knowlesi. 30) Peptide according to claim 29, characterized in that said polypeptide has the amino acid sequence {Gln^-Al^-Gln^-Gly^-Asp^- Gly^- Ala^-Asn^Ala -Gly -Gin -Pro ) from the N terminus to the C terminus of said polypeptide. 31) Peptide according to claim 30, having an amino acid sequence comprising amino acid residues 5-11, excluding 1-3 and comprising at least one of 4 and 12. 103 32) Peptide according to claim 30 consisting of amino acid residues 5 to 12 of said sequence. 33) Peptide according to claim 30 consisting of amino acid residues 4 to 11 of said sequence. 34) Peptide according to claim 30, chemically synthesized. 35) Peptide according to claim 29, chemically synthesized. 36) Peptide having the amino acid sequence Asp-GlyAla-Asn-Ala-Gly-Gln-Pro. 37) Peptide having the amino acid sequence Gly-AspGly-Ala-Asn-Ala-Gly-Gln. 38) Peptide according to claim 28, said peptide being immunochemically reactive with at least two monoclonal antibodies to said circumsporozoite protein. 39) Antigenic protein of a sporozoite of a Plasmodium species in an essentially purified form. 40) Antigenic protein according to claim 39, characterized in that it comprises circumsporozoite protein of an element of the genus Plasmodium. 41) Antigenic protein according to claim 39, characterized in that it comprises Plasmodium falciparum circumsporozoite protein, 42) Antigenic protein according to claim 39, - 10 4 BueuiOi'i apA characterised in that it comprises P.vivax circumsporozoite protein. 43) Antigenic protein of a sporozoite of a Plasmodium species produced by a microorganism, said microorganism being transformed by an expression vector having inserted therein a deoxynucleotide sequence encoding said protein, the transformed microorganism being capable of producing said protein. 44) Protein according to claim 43, characterized in that it comprises circumsporozoite protein of an element of the genus Plasmodium. 45) Protein according to claim 43, characterized in that it comprises P.falciparum circumsporozoite protein. 46) Protein according to claim 43, characterized in that it comprises Pv ivax circumsporozoite protein. 47) Fusion protein produced by a microorganism comprising an antigenic protein from a sporozoite of a Piasmodium species as the C-terminal part of its amino acid sequence and a part of a prokaryotic protein of the microorganism as the N-terminal part of its amino acid sequence. 48) Fusion protein according to claim 47, 105 characterized in that the antigenic protein comprises P.falciparum circumsporozoite protein. 49) Fusion protein according to claim 47, characterized in that the antigenic protein comprises P.viva x circumsporozoite protein. 50) Fusion protein according to claim 47, characterized in that the antigenic protein comprises circumsporozoite protein of an element of the genus Plasmodium. pi of NEW YORK UNIVERSITY THE AUTHORIZED REPRESENTATIVE Eng. 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