(en)A synthetic peptide comprising an amino acid sequence including the sequence Asp-Arg-Ala-X-Gly-Gln-Pro-Ala-Gly wherein X is selected from the group consisting of Asp and Ala, said peptide being capable of eliciting formation of antibodies recognizing the circumsporozoite protein of Plasmodium vivax sporozoites.

1.ApplicationNumber: US-4355087-A
1.PublishNumber: US-5700906-A
2.Date Publish: 19971223
3.Inventor: ARNOT; DAVID E.
ENEA; VINCENZO
NUSSENZWEIG; RUTH S.
NUSSENZWEIG; VICTOR

4.Inventor Harmonized: ARNOT DAVID E(US)
ENEA VINCENZO(US)
NUSSENZWEIG RUTH S(US)
NUSSENZWEIG VICTOR(US)

5.Country: US
6.Claims:
(en)A synthetic peptide comprising an amino acid sequence including the sequence Asp-Arg-Ala-X-Gly-Gln-Pro-Ala-Gly wherein X is selected from the group consisting of Asp and Ala, said peptide being capable of eliciting formation of antibodies recognizing the circumsporozoite protein of Plasmodium vivax sporozoites.

7.Description:
(en)The United States Government has rights in this invention based upon research support in the form of grant No. AID-DPE-0453-A-00-5012-00 from the Department of State, Agency for International Development.


This application is a Continuation-In-Part of application Ser. No. 06/754,645, filed Jul. 12, 1985, which was abandoned in favor of Continuation application Ser. No. 07/180,606, filed Apr. 5, 1988, now U.S. Pat. No. 4,957,869.


This application incorporates by reference the entire disclosure of the following copending commonly assigned U.S. Patents:
U.S. Pat. No. 4,466,917 corresponding to application Ser. No. 574,124, and U.S. Pat. No. 4,915,942 corresponding to application Ser. No. 649,903.
BACKGROUND OF INVENTION
The present invention relates to the field of antigens suitable for providing protective immunity against malaria when incorporated into a vaccine. Malaria constitutes a worldwide public health hazard of enormous economic and medical significance. The disease contributes substantially to infant mortality in endemic areas and remains a severe and debilitating illness for those who remain afflicted with it as adults. Despite advances in the techniques of mosquito abatement and improved public health measures, regions where the disease is considered endemic are increasing in area. Furthermore, the risk of infection was substantially increased in some parts of the world because of the occurrence of new drug-resistant strains of the malaria parasite.
This invention relates to the circumsporozoite (CS) protein of the sporozoite stage of Plasmodium vivax malaria parasite; to a DNA fragment coding for such protein; to immunogenic peptides and proteins comprising an amino acid sequence corresponding to the immunodominant epitope of said CS protein or immunologically active fragments thereof; and to DNA fragments coding for such peptides.
The aforementioned proteins and peptides are useful in inducing an immune response in humans and other animals and in conferring protective immunity in such hosts against infection by P. vivax sporozoites. The DNA fragments are useful in a method for preparing the protein and peptides of the present invention, using known recombinant DNA techniques.
DESCRIPTION OF THE PRIOR ART
The causative agent of malaria is a protozoan of the genus Plasmodium. Individual species within the genus appear to have a restricted host range for the animals they infect. For example, P. berghei and P. yoeli are infective to rodents, P. knowlesi and P. cynomolgi are primarily infective to monkeys, while P. falciparum, P. vivax, P. ovale and P. malariae are the species primarily infective to humans. Despite species differences in host range, the life cycles, mode of infection, biochemistry and genetics of the various Plasmodium species are markedly similar.
The life cycle of Plasmodium is complex, the organism undergoing several distinct morphological changes involving the participation of a mammalian host and a mosquito vector. The parasite, in the sporozoite form, is introduced to the mammalian host through the bite of the mosquito vector. The sporozoites rapidly disappear from the blood stream and are next found as intracellular parasites of liver parenchymal cells. A blood infection ensues, characterized by the well-known clinical symptoms of malaria after a complex series of morphological and biochemical transitions. The parasite is then found in the red blood cells, where it continues its development. Substantial amounts of the parasite may be obtained from the red blood cells of infected patients.
Vaccine development, to provide protective immunity against malaria infection has been thwarted by the fact that the parasite's life cycle in the mammalian host is primarily intracellular. Except for brief periods of time, the parasite is protected from contact with the immune system. Two stages in the parasite's life cycle during which it becomes briefly exposed to the immune system are; 1) the interval following initial infection before sporozoites have successfully invaded the cells of the liver; and 2) the interval during which merozoites leave infected red blood cells and enter uninfected red blood cells. The transient exposure of the merozoite forms in the extracellular milieu has provided the basis for prior art attempts to develop host immunity to blood forms of the parasite. European published Patent Application Number 62924, discloses antigenic proteins useful in the making of a vaccine to provide immunity against merozoite forms of the parasite. The utility of such a vaccine would presumably lie in limiting or arresting the course of the established malaria infection.
An alternative approach, based upon sporozoite antigens has led to the discovery of antigenic and immunogenic proteins of sporozoites that are capable of providing protective immunity against initial infection, when administered as a vaccine, Cochrane, A. H. et al., in Malaria, vol. 3, (J. Kreier, ed.) Academic Press N.Y. (1980) pp 163-202; Nussenzweig, R. S. in Immunity to Blood Parasites of Animals and Man, (L. Miller, J. Pino and J. McKelvey, eds.) Plenum, N.Y. (1977) pp. 75-87. Gwadz, R. W. et al., Bull, W. H. O. Supp. 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 numerous structural properties in common including chromatographic behavior, isoelectric point, and electrophoretic mobility. The 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.
The comparison of triptych digests of purified sporozoite antigen proteins of different Plasmodium species shows that several triptych peptides have identical retention times on reverse-phase high performance liquid chromatography, indicating that there is a high degree of homology between antigenic proteins of different species.
The sporozoite antigens are components of the sporozoite surface coat. The presence of the sporozoite antigens is indicated by a characteristic immunologic reaction known as the circumsporozoite reaction, and by immunofluorescence tests. See Vanderberg, J. P. 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 the sporozoite antigen for a given Plasmodium species, without resorting to time-consuming in vivo tests. This, in turn, has made it possible to develop specific radioimmunoassays for sporozoite antigens, and ultimately for the production of malaria antibodies directed against sporozoite antigens of Plasmodium species.
Antibodies against the sporozoite antigens have been shown to provide protective immunity against the Plasmodium species from which they were derived, in rodents, monkeys and in human volunteers. The sporozoite protective antigen protein is herein termed CS protein, circumsporozoite protein, or sporozoite CS protein, these terms being deemed equivalent. A co-pending U.S. application Ser. No. 234,096, filed Feb. 12, 1981, has been filed, disclosing a vaccine based upon purified CS protein. Said application (a copy of which is annexed hereto as Appendix A) is incorporated herein by reference as though set forth in full.
The results disclosed herein are based in part on the techniques and concepts of the field of immunology. For convenience, certain terms commonly used in the art are defined herein. The term "immunochemical reaction" is used to denote the specific interaction which occurs between an antigen and its corresponding antibody, regardless of the method of measurement. Such a reaction is characterized by a non-covalent binding of one or more antibody molecules to one or more antigen molecules. The immunochemical reaction may be detected by a large variety of immunoassays known in the art. The terms "immunogenic" or "antigenic" will be used here to describe the capacity 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 a suitable host against a given pathogen. The term "epitope" refers to a specific antibody binding site on an antigen. Macromolecular antigens, such as proteins, typically have several epitopes with distinctive antibody binding specificities. Different epitopes of the same antigen are distinguishable with the aid 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 may each bind the antigen without interfering with the other, unless the epitopes are so close together that the binding of one sterically inhibits the binding of the other. The term "immunodominant region" denotes an area of the antigen molecule which is mainly responsible for its antigenicity.
It has been shown that immunization of a host with inactivated sporozoites confers protective immunity in such host against infection with sporozoites of the same malarial species: Bruce-Chwatt, L. J. Essential Malariology, William Heineman Ltd. (Publ.) London, 1980. However, immunization with whole inactivated sporozoites is impractical because sporozoites are only available from the glands of infected mosquitoes and are therefore in very short supply.
The immunogenicity of sporozoites of any species resides largely, though not exclusively, in a single antigen, the circumsporozoite (CS) protein. In turn, the immunogenicity of this protein resides in a single epitope which is identically, or quasi-identically repeated many times in tandem.
CS proteins have been identified, analyzed and characterized for some plasmodium species including P. falciparum. Synthetic peptides (produced by chemical synthesis or biological methods) consisting of multiples or analogs of the repeating amino acid sequences have been shown to be immunogenic and are useful in the development of a malaria vaccine.
However, peptides derived from the immunodominant region of the different CS proteins display only species-specific antigenicity.
Therefore, in order to develop a malaria vaccine that would protect humans against infection by any human malaria species, it is necessary to identify, sequence and characterize the CS proteins (or the epitope regions thereof) for all the plasmodium species that infect humans.
Four species of parasitic protozoans cause malaria in humans. Two of these, P. falciparum and P. vivax, predominate. Vivax malaria occurs throughout the world and is the dominant form of malaria in China, Sri Lanka, and Central America. In some areas, such as the Amazon region and Southeast Asia, it is as common as falciparum malaria. Although causing much less mortality than falciparum malaria, vivax malaria with its characteristic relapsing fevers is an incapacitating disease and constitutes a serious public health problem and a barrier to economic development.
Therefore, there is an urgent need to prepare a malaria vaccine for humans, which would be protective against P. vivax infection.
OBJECTS OF THE INVENTION
One object of this invention is to identify and characterize the CS protein of P. vivax malaria parasite and the gene coding this protein.
Another object of this invention is to identify the amino acid sequence and primary structure of the P. vivax CS protein.
Yet another object of this invention is to provide a peptide or protein sequence associated with the P. vivax CS protein (i.e., comprising a subsequence thereof or an analog or homolog thereof) which is capable of inducing a protective immune response in humans.
Another object of this invention is to provide a peptide or protein comprising an amino acid sequence associated with the immunodominant epitope of the P. vivax CS protein capable of inducing a protective immune response in humans.
Another object of this invention is to provide a DNA sequence coding for immunogenic material capable of inducing a protective immune response in humans against P. vivax malaria infection.
A further object of this invention is to provide the aforementioned peptides and proteins which can be synthesized chemically or by recombinant DNA techniques and other biological methods.
It is also an object of the invention to develop a vaccine that confers protective immunity against infection by P. vivax malaria sporozoites.
These and other objects of this invention will become readily apparent to one of ordinary skill in the art in light of the present description, accompanying claims, and appended drawings.
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 repetitions 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 repeated peptide has been chemically synthesized in both monomeric and dimeric forms. The synthetic repeated peptide is immunochemically reactive with polyclonal antibody preparations against P. knowlesi. In addition, all monoclonal antibodies against CS proteins which neutralize the infectivity of sporozoites in vitro, also react with the synthetic peptide. Therefore, the synthetic repeated peptide constitutes substantially all of the immunogenic activity displayed by the naturally occurring sporozoite protective antigen of P. knowlesi.
Several lines of evidence indicate that CS proteins of the Plasmodium species infective to rodents, monkeys and humans are structurally similar. All possess an immunodominant region composed of similarly repeated epitopes. For each species, the repeated peptide of the sporozoite CS protein can be synthesized. The repeated peptide of a CS protein is immunogenic when administered in a composition, and by administration methods, known in the art to yield antibody production. On the basis of the discoveries and teachings herein described, structural determination and synthesis of the repeated peptide corresponding to any Plasmodium species sporozoite, and the preparation of a vaccine composition incorporating said peptide and capable of eliciting protective immunity against said species is now available to those of ordinary skill in the art.
As further confirmation of the close relationship of CS proteins of different Plasmodium species, monoclonal antibodies against P. knowlesi sporozoites have been shown to cross-react with P. falciparum antigen, a species infective to humans. It is therefore apparent that the development of other synthetic peptides more specifically reactive with human malaria species are well within the grasp of those ordinarily skilled in the art, following the teachings and disclosures as set forth herein.
One aspect of this invention relates to a peptide comprising the amino acid sequence Asp-Arg-Ala-X-Gly-Gln-Pro-Ala-Gly wherein X represents Asp or Ala, said peptide being capable of eliciting immune response in a host against P. vivax malaria infection.
Another aspect of this invention relates to a DNA fragment coding for the above peptide.
Another aspect of the invention relates to a protein comprising an amino acid sequence consisting essentially of the sequence Asp-Arg-Ala-X-Gly-Gln-Pro-Ala-Gly tandemly repeated at least twice.
Another aspect of this invention relates to a DNA sequence coding for such a protein.
Yet another aspect of the invention relates to a peptide or protein comprising an amino acid sequence corresponding to segments of the amino acid sequence of the CS protein of P. vivax that lie outside the bounds of the tandemly repeated domain of said protein and are substantially homologous to corresponding segments of CS proteins of other species; and to DNA sequences coding for such peptides and proteins.
Still another aspect of this invention relates to the P. vivax CS protein and to DNA coding for this protein.


BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an radioautograph of a genomic Southern blot of P. vivax DNA probed with a CDNA clone of P. cynomolgi.
FIG. 2 depicts a coarse and a fine structure map of the lambda phage clone VX1B containing the P. vivax CS gene.
FIG. 3 depicts the complete gene sequence encoding the CS protein of P. vivax, and its translation.
FIG. 4 depicts an alignment of the amino acid sequences encoded by the CS genes of P. vivax and P. cynomolgi and reveals the existence of substantial homology between them.
FIG. 5 is a graph depicting the binding to a peptide according to the present invention of: (a) anti-P. vivax monoclonal antibody, used alone or in the presence of competing concentrations of a peptide according to the present invention, or an unrelated peptide; and (b) an unrelated monoclonal antibody.
FIG. 6 depicts the complete gene sequence encoding the CS protein of P. cynomogli.
FIG. 7 depicts the a fusion protein containing the sequence of the immundominant region of the CS protein of P. knowlesi.
FIG. 8 shows the inhibitory effect of a monoclonal antibody to a CS protein on the binding of polyclonal antibodies to sporozoite extracts.
FIG. 9 depicts two-site immunoradiometric assays for detection of sporozoites performed with single monoclonal antibodies to CS proteins.
FIG. 10A shows the ultracentrifugation of P. vivax sporozoite extracts on sucrose gradients as a plot of gradient fraction v. amount of CS protein.
FIG. 10B shows ultracentrifugation of P. vivax sporozoite extracts on sucrose gradients, as a plot of gradient fraction v. inhibitory activity.
FIG. 11A shows the ultracentrifugation of P. knowlesi sporozoite extracts on sucrose gradients as a plot of gradient fraction v. amount of CS protein.
FIG. 11B shows the ultracentrifugation of P. knowlesi sporozoite extracts on sucrose gradients as a plot of gradient fraction v. amount of CS protein.


DETAILED DESCRIPTION OF THE INVENTION
In the following description, the materials employed were commercially available, unless otherwise specified. Enzymes used in the cloning procedures were obtained from commercial sources. Restriction endonuclease reactions were carried out according to the manufacturer's instructions. Unless otherwise specified, the reaction conditions for other enzyme reactions were standard conditions used in the art, as described, for example, in Methods in Enzymology, volume 60 (R. Wu, Ed.) Academic Press, (1980). Unless otherwise specified, the abbreviations herein are standard 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 outline, the experiments and conclusions following from the results thereof are set forth. The approach taken herein to clone a DNA segment coding for 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 because it was not known whether Plasmodium genomic DNA contained introns that might prevent the expression of antigenically identifiable sporozoite proteins. Now that the short and repeated nature of the epitope is known, it is feasible to select for DNA encoding the epitopes from a library of genomic Plasmodium DNA. The initial experiments were performed with cDNA from mRNA of infected mosquitoes, since it was only at that stage that the Plasmodium was known to express the sporozoite antigen. A cDNA library was constructed from Poly (A) + RNA derived from P. knowlesi-infected mosquitoes. Double stranded cDNA, tailed with poly-C residues, were inserted into the plasmid pBR322, previously cut with Pst I and tailed with poly-G. Host cells transformed to tetracycline resistance were selected and single colonies of transformed cells were stored in microtiter dishes at -70° C.
The cDNA clones were screened for the ability to express a protein that contained the immunochemically reactive region of the sporozoite surface antigen. Once a cDNA coding for the sporozoite antigen was identified, others could readily be detected by hybridization using the originally cloned cDNA as a probe. Clones derived either from cDNA or genomic DNA libraries could be identified in this manner, based upon the homology between DNA segments coding for 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. Pools of 48 colonies were screened using a sensitive, two-site immunoradiometric assay performed with monoclonal antibodies. This permitted the detection of the CS protein in the transformed cells.
In short, a monoclonal antibody to P. knowlesi CS protein was adsorbed to the wells of a microtiter plate. Lysates from pools of 48 colonies were each added to the wells and incubated for 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 radiolabelled second monocional antibody to P. knowlesi CS protein was added. The labelled second monoclonal antibody attaches to the antigenic protein that is already bound to the surface of the microtitre well by the first monoclonal antibody. If a pool of 48 colonies was found to be positive, the colonies were screened individually in the same fashion. In this manner, positive clones were identified.
Whenever an immunoreactive clone was detected, plasmid DNA was isolated from it and used to transform another host cell strain, such as E. coli HB 101 or E. coli RR1. Transformants detected by tetracycline resistance were rechecked for the ability to express the immunochemically reactive protein, in order to confirm that the expression was a property of the plasmid DNA clone containing CS nucleotide sequences. Once suitable plasmid DNA was obtained from the positive clones, the nucleotide sequence of the cDNA insert coding for at least the immunoreactive region of the CS protein was obtained (by cloning onto M13). Methods of nucleotide sequence analysis are well known in the art, including the method of Maxam and Gilbert, W. Proc. Nat. Acad. Sci. USA 74, 560 (1977) and the method of Sanger, P. et al, Proc. Nat. Acad. Sci. USA 74, 5463 (1977). The latter method was employed in the present work. The complete nucleotide sequence of a segment of the P. knowlesi CS protein gene that contains the immunochemically reactive site is shown in FIG. 7.
A surprising 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 either end. In order to deduce the amino acid sequence coded by the DNA, it was necessary to identify the coding strand and, within the strand, the correct reading frame. In this context, the advantage of using the Sanger and Coulson, supra sequencing method becomes evident. A sequencing vector, bacteriophage M13mp9, contains a betagalactosidase gene with a Pst I site in the same reading frame as the Pst I cleavage site 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 sequencing vector can express a beta-galactosidase fusion protein comprising the immunochemically reactive part of the CS protein. Therefore, two different M13mp9 recombinants were obtained, with the 368 bp P. knowlesi DNA fragment inserted in opposite orientations. Only one of the two recombinants produced immunochemically reactive betagalactosidase fusion protein, as measured by the above-described radioimmunoassays. The clone producing the immunoreactive protein was used to identify the coding strand and direction of transcription of the P. knowlesi gene fragment.
The correct reading frame was also deduced using immunological procedures. These showed that the epitope defined by the monoclonal antibodies was destroyed by elastase, but not by trypsin nor by reducing agents, indicating that the epitope did not contain any of lysine, arginine or disulfide bonds, but might contain alanine residues.
On the basis of such experiments, the amino acid sequence of the 12 amino acid-containing repetitive peptide was deduced to be:

H.sub.2 N-GlnAlaGlnGlyAspGlyAlaAsnAlaGlyGlnPro-COOH.

To confirm the deduced amino acid sequence and immunochemical reactivity of the above-described sequence, a dodecapeptide of the same amino acid sequence and a dimer thereof were synthesized using an automated solid phase peptide synthesis system.
The monomer and dimer synthetic peptides were separately tested for immunochemical activity in the same type of radioimmunoassay as was used initially to screen the cDNA library. In this test, two antibody binding sites must be present in the antigen, one for binding to the first monoclonal attached to the microtiter well and the second for binding the added labelled 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 sites. Furthermore, the same assay showed that the monomer was able to compete with and specifically inhibit the binding of CS proteins of P. knowlesi to the microtiter wells bearing the first monoclonal antibody. Therefore, the sequence shown above contains an epitope of the sporozoite antigen. Another important observation was that all monoclonal antibodies to P. knowlesi obtained to date, as well as all polyclonal antibodies obtained from monkeys immunized with irradiated sporozoites, also reacted with the synthetic peptides. Actually, more than 70% of the antibodies to sporozoites, found in the serum of the immunized monkeys, recognized this single epitope (Zavala et al., J. Exp. Med. 157:1947, 1983).
Therefore a chemically synthesized dodecapeptide having an amino acid sequence identical to that repeated in a sporozoite membrane protein contains substantially all of the antigenicity of the naturally occurring CS protein. It follows that based upon the principles of immunology and following techniques and procedures known to those of ordinary skill in the art, a synthetic peptide based upon the known amino acid sequence of a sporozoite CS protein can be incorporated into a vaccine composition capable of providing protective immunity in a host organism susceptible to a Plasmodium species from which the sequence of the peptide was derived. The following experiments, generally described, demonstrate the essential structural and functional similarities between the sporozoite CS proteins of the Plasmodium species infective to rodents, monkeys and humans. These similarities are exploitable to identify and synthesize the antigenic peptides specific for any Plasmodium species including, in particular, those infective to humans. The structural determination and synthesis of the repeated peptide for any Plasmodium species can be carried out by methods described herein or by equivalent methods known in the art, or by methods known in the art which exploit the disclosures and teachings of the present invention to eliminate some of the more time-consuming and tedious aspects of the original experiments. Of significance is that cross reactivity has been observed between 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 P. cynomolgi and P. falciparum; antibodies to the CS protein of P. cynomolgi cross react with the sporozoite antigen of P. vivax; antibodies to the CS protein of P. yoeli nigeriensis cross react with sporozoite antigen of P. berghei; and in that instance, completely neutralize the infectivity of sporozoites of the latter species, for 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 sporozoite CS protein was tested, measuring the inhibitory effects that the binding of one might have on the other. Monoclonal antibodies directed against different sequences within an antigen should not interfere with their respective binding capacities. Conversely, if monoclonals are directed against the same epitope, or epitopes which are topographically close, they inhibit each other. In the case of P. knowlesi, every one of the six monoclonals used, strongly inhibited the binding of the others to the antigen.
The same experiments were performed using monoclonals to the sporozoite antigen proteins of P. vivax, P. falciparum, P. malariae, P. cynomolgi and P. berghei, with identical results. Therefore, these sporozoite CS proteins all are characterized by having a single immunodominant region.
Of direct relevance to the development of a vaccine against human malaria is the observation that antibodies in the serum of humans vaccinated and protected against sporozoites of P. falciparum or P. vivax 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 the subsequent binding of the antigen (in the sporozoite extract) to polyclonal antibodies isolated from the serum of the vaccinated human volunteers (Zavala et al., supra).
The fact that immunodominant regions of the CS proteins contain repetitive epitopes was demonstrated by a solid phase two site radioimmunoassay. In the assay, a monoclonal antibody was bound to the plastic surface a microtiter well, antigen was added and the antigen was found to the immobilized antibody. The well was then washed to remove any unbound material and a second monoclonal antibody, presumably directed against a different epitope of the same antigen, was added. The second antibody is labelled with a radioisotope to quantitate the binding of the second antibody. Binding of the second antibody is proportional to the amount of antigen bound in the well. This immunoassay can be performed only if the antigen contains at least two epitopes. The first epitope binds to the antibody immobilized to the plastic of the plate, the second binds the radiolabelled antibody. Surprisingly, in the case of every CS protein, the two site radioimmunoassay could be performed using a single monoclonal antibody. That is to say, the assay could be performed using unlabelled 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. Extracts of P. knowlesi sporozoites were dissolved in 2.0% (w/v) sodium dodecylsulfate and 6M urea and fractionated by ultracentrifugation in sucrose gradients. The existence of two epitopes was demonstrated in fractions of the gradient 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 aggregates of CS proteins. Identical results were obtained in experiments performed with P. vivax and P. falciparum extracts (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 very immunogenic in all animal species, including man. 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 site radioimmunoassays requiring two epitopes on the same molecule. The functional behavior in two site assays is reproduced by synthetic dimers of the repeated peptide.
It will be readily appreciated therefore that synthetic peptides, comprising an amino acid sequence corresponding to an epitope of a sporozoite C8 protein in monomeric or multimeric form, can be incorporated into vaccines capable of inducing protective immunity against sporozoites of malaria parasites, e.g., P. falciparum, P. vivax and P. malarias. Techniques for enhancing the antigenicity of such repeated peptides include incorporation into a multimeric structure, binding to a highly immunogenic protein carrier, for example, keyhole limpet hemocyanin, or diptheria toxoid, and administration in combination with adjuvants or any other enhancers of immune response. Furthermore, it will be understood that peptides specific for a plurality of Plasmodium stages and species may be incorporated in the same vaccine composition to provide a multivalent vaccine. In addition, the vaccine composition may comprise antigens to provide immunity against other diseases in addition to malaria.
An amino acid sequence corresponding to an epitope of a CS protein (repeated peptide) may be obtained by chemical synthetic means or by purification from biological sources including genetically modified microorganisms or their culture media. The repeated peptide may be combined in an amino acid sequence with other peptides including fragments of other proteins, as for example, 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 include the practical possibility that, in some instances, amino acid sequence variations of a naturally occurring repeated peptide may be antigenic and confer protective immunity against malaria sporozoite infection. Possible sequence variations include, without limitation, amino acid substitutions, extensions, deletions, interpolations and combinations thereof. Such variations fall within the contemplated scope of the invention provided the peptide containing them is antigenic and antibodies elicited by such peptide cross-react with naturally occurring CS protein or non-variant repeated peptides of CS protein, to an extent sufficient to provide protective immunity when administered as a vaccine. Such vaccine compositions will be combined with a physiologically acceptable medium. Routes of administration, antigen dose, number and frequency of injections are all matters of optimization within the scope of ordinary skill in the art, particularly in view of the fact that there is experience in the art in providing protective immunity by the injection of inactivated sporozoites. It is anticipated that the principal value of providing immunity to sporozoite infection will be for those individuals who have had no previous exposure to malaria, e.g., infants and children who live in endemic and subendemic areas, and unexposed adults travelling into endemic areas. It is also anticipated that temporary immunity for infants may be provided by immunization of mothers during pregnancy. Details of the operation and practice of the present invention are set forth in the specific examples which follow.
One way to approach the problem of identification of the P. vivax CS protein would have been to make a cDNA clone, from DNA of either the sporozoite or the blood stage of P. vivax parasite. However, use of sporozoite material presents many difficulties as sporozoites are only available in scanty quantities and are contaminated with mosquito thoracic material. In addition, use of cDNA clones is time-consuming and may not reveal the complete structure of the protein of interest.
Although the CS gene does not appear to be well conserved across species, the following observations have been made:
(a) the presence of immunologically distinct CS epitopes is not a sufficient criterion for assessing overall homology between CS proteins; Enea, E. et al., Annali Instituto Superiore to Sanite, Rome (1985); Cochrane, A. H. et al., Molec. and Biochem. Parasitol. 14:111 (1985);
(b) at least five immunologically distinct CS proteins of P. cynomolgi strains bear highly homologous sequences outside of the repeating peptide sequence;
(c) the CS genes of P. cynomolgi and P. knowlesi are related; Arnot, D. E. et al., manuscript submitted, 1985; and
(d) P. vivax appears to be more closely related to the simian malarias than to the other common human malaria, P. falciparum; Dame, J. B. et al., Science, 225:593 (1984)--these authors are not affiliated with the assignee of this application.
Therefore, in attempting to identify and sequence the CS protein of P. vivax, the present inventors relied on gene probes of P. cynomolgi. The complete nucleotide sequence of the gene encoding the CS protein of P. cynomogli has been found by the present inventors and their co-workers and is disclosed in full in FIG. 6, along with its translation.
In search for the sequence of the P. vivax CS protein, DNA isolated from the blood stage of this parasite was size-fractionated, transferred on nitrocellulose, and hybridized with labeled DNA probes from P. cynomolgi CS genes. The probe used was a 700 base-pair Pst I fragment encoding the C-terminal domain and approximately 350 nucleotides of the 3' untranslated region of a cDNA clone of the gene encoding the P. cynomolgi CS protein. Specifically, the probe, designated P236-7, encompasses nucleotides 851 through 1827 (of the sequence shown in FIG. 6) flanked by C and G tails.
The foregoing P. cynomolgi probe was found to cross-hybridize under medium stringency conditions with single AccI (3.3 kilobases) HpaII(4.2 kb) and BglII (15 kb) fragments of P. vivax genomic DNA, as shown in lanes 1-4 of FIG. 1. Lane 5 contains P. cynomogli (Gombak) genomic DNA cut with BglII.
The procedure used for hybridization was as described in T. Maniatis et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory (1982), pages 382-389, incorporated by reference herein.
The 15 kb BGlII fragment was of a suitable size to be cloned in the bacteriophage lambda EMBL4 vectors according to methods described by Frischauf, A. M. et al., J. Mol. Biol. 170: 827 (1983), the disclosure of which is incorporated by reference in this application. Such vectors (or their DNA) are commercially available from sources such as Promega Biotec, Madison, Wis., or Vector Cloning Systems, San Diego, Calif.
Isolation of the lambda vector, packaging, handling of the genomic clone, and plaque hybridization, were conducted as described in Maniatis, T. et al., supra, pages 75-85; 256-268; 269-294, and 312-328. The entire disclosure of these sections is incorporated by reference.
Approximately 5000 plaques of a size-fractionated genomic library of P. vivax were screened using the Pst I fragment of the P. cynomogli (Gombak) cDNA clone as the probe; three positive plaques were obtained. The restriction map of one of these clones, indicated as lambda VX1B, is shown in the upper map of FIG. 2. This clone has a map consistent with the sizes of the cross-hybridizing fragments obtained in the genomic Southern blots (FIG. 1, lane 4). A series of plasmid subclones were therefore derived for further mapping and DNA sequence analysis.
A more detailed restriction map of the region cross-hybridizing with the P. cynomogli cDNA clone is shown in the lower map of FIG. 2. The hatched bar indicates the fragments cross-hybridizing with the cDNA probe.
DNA sequence analysis was initially carried out from the XbaI and the AccI sites, which also occur in the P. cynomolgi CS gene in the same position with respect to the coding sequence of the gene (see FIG. 2).
Sequence homology with the P. cynomolgi gene was detected around these sites and a complete sequence analysis of P. vivax genomic DNA in the region shown in the fine structure map of FIG. 2 was carried out. A continuous open reading frame occurs within this region encoding a protein with a central domain of repeating amino acids flanked by non-repetitive N and C terminal domains substantially homologous to the corresponding domains in the CS genes of P. cynomologi and P. knowlesi. The entire sequence of the P. vivax CS gene and its translation are shown in FIG. 3.
The amino acid sequence encoded by the repeating nucleotide sequence in the thus identified gene is Asp-Arg-Ala-X-Gly-Gln-Pro-Ala-Gly. X stands for either Asp or Ala as illustrated in FIG. 3. This amino acid sequence is repeated 19 times and constitutes 49% of the 373 amino acid protein.
In all the previously identified CS antigens of other plasmodium species, the repetitive amino acid sequence contained the immunodominant epitope of the CS protein. Therefore, it was postulated that the repeated amino acid sequence identified above would in fact compose the immunodominant epitope of the CS protein of P. vivax. This was tested and confirmed by exposing a peptide consisting of a tandem repeat (2x) of the repeating sequence to different concentrations of monoclonal antibody against P. vivax CS protein.
The monoclonal antibody quantitatively bound to a peptide consisting of two tandem repeats of the repeating amino acid sequence of FIG. 3.
More important, this peptide quantitatively inhibited the binding of monoclonal anti-CS to immobilized whole sporozoite extracts.
Based on these observations and on the behavior of peptides corresponding to the repeats of P. knowlesi and P. falciparum CS proteins, peptides containing this repeating sequence (preferably at least twice in tandem) will induce formation of antibodies against P. vivax CS protein, and therefore against P. vivax sporozoites, and will serve to confer protective immunity in humans against infection by this malaria parasite. The complete amino acid sequence of such peptides may also comprise segments other than the repeat unit. The only necessary condition is that the sequence corresponding to the repeating unit(s) of the P. vivax CS protein be available to induce immune response. Alternatively, amino acid sequences corresponding to the repeating unit of this CS protein could form part of whole proteins, provided that the immunogenic activity against P. vivax is conserved in such proteins.
As stated above, the immunological test results confirm that the synthetic peptide H(Asp-Gly-Gln-Pro-Ala-Gly-Asp-Arg-Ala) 2 contains the immunodominant epitope of the P. vivax CS protein.
Screening of P. vivax sporozoites from strains originating in Thailand, India, El Salvador, Brazil, Sri Lanka, North Korea and Papua, New Guinea with the 2F2 antibody indicated that the CS epitope recognized by this antibody is present in all isolates J. Immunol--1985, Zavala, F. et al., (in press). These experiments demonstrate that the peptide based on a Brazilian vivax strain protein sequence can compete with the binding of a monoclonal antibody raised against mixed S. E. Asian P. vivax sporozoites to extracts of sporozoites from a New Guinea vivax malaria. Thus, although the P. vivax CS gene was isolated from a Brazilian strain of P. vivax, the epitope deduced from the sequence of the cloned gene appears to be present in all P. vivax CS proteins so far studied. The characterization of this gene and its encoded amino acid sequence allows synthesis of large amounts of the repetitive epitope and other antigenically active regions of the P. vivax CS protein and development of a protective vaccine using peptides corresponding to such regions.
Peptides according to the present invention can be prepared by known chemical synthesis methods, if chain length and properties of the constituent amino acids permit, or by biological methods including recombinant DNA techniques.
Proteins according to the present invention can be prepared by known biological methods including recombinant DNA techniques.
Finally, homologs and analogs of repeating amino acid sequence or multiples thereof may be made in a manner analogous to copending commonly assigned U.S. patent application Ser. No. 695,257 of V. Nussenzweig et al., filed on Jan. 28, 1985 and entitled IMMUNODOMINANT EPITOPE OF THE SPOROZOITE SURFACE PROTEIN, incorporated by reference herein.
In addition to the repeating amino acid sequence of the P. vivax CS protein, two other regions of the P. vivax CS protein are of interest because of their extensive homology to the corresponding regions in the CS protein of P. cynomogli and P. knowlesi. These homologous sequences are aligned in FIG. 4 and compared with Gombak strain of P. cynomolgi.
Both the N-terminal and the C-terminal domains of the P. vivax and P. cynomogli protein sequences present substantial homology not only at the nucleotide level, but also at the amino acid level. The two proteins are almost completely identical in thirty amino acids at their N- and C-terminal, which probably constitute conserved hydrophobic leader and transmembrane sequences, respectively.
The P. knowlesi sequence is slightly less homologous to the P. vivax sequence.
In U.S. patent application Ser. No. 649,903 filed on Sep. 12, 1984 in the name of U. Vergara, et al., (the disclosure of which is incorporated by reference herein) the present inventors and their co-workers described a region of P. falciparum CS gene neighboring the repeating sequence region that had substantial homology with the corresponding P. knowlesi region, were exposed to the parasite surface, and possessed antigenicity. Similarly, the N 2 and C 2 regions of homology in the P. vivax CS protein are expected to be immunogenic and potentially useful in a malaria vaccine preparation because they also are highly conserved between different species.
The N 2 region comprises 20 amino acids flanking the repeats in the direction of the N-terminal region. The C 2 region lies within two sets of cystein residues which are located close to the C-terminal region.
EXAMPLE 1
DNA Isolation from the Blood Stage of P. vivax
Parasitized human red blood cells were purified from white cells by the method of Baggsly and Atkinson, Trans. Roy. Soc. Trop. Med. Hyg. 66:4 (1972), incorporated by reference.
Parasite pellets were thawed by stepwise addition of warm (50° C.) 0.1MEDTA (ethylenediaminetetraacetate), pH 8.0, and 0.5% Sarcosyl.
The ratio of buffer per volume of frozen parasite pellet was 10:1. Proteinase K was added to a final concentration of 100 micrograms/ml in the parasite-buffer mixture, which was then incubated at 50° C. for two hours.
Phenol (0.8 volumes, pH 8.2-8.5) was added followed by an equal volume of chloroform. The mixture was stirred and centrifuged at 3000 rpm (about 1,000 g) for five minutes at room temperature.
The lower phase was removed and the phenol/chloroform extraction was repeated as above until the organic phase was clear.
Sodium chloride was added to a final concentration of 0.2M. Two volumes of ethanol were added and the mixture was incubated at -20° C. for at least two hours.
The mixture was centrifuged at about 3000 g for ten minutes. The pellet was resuspended in a buffer containing 10 mM Tris and 10mM EDTA, pH 8 (0/10/10 buffer). RNase A was added to a final concentration of 20 micrograms/ml and RNase T1 was added to a final concentration of one unit/ml. The mixture was incubated at 37° C. for 60 minutes.
DNA was precipitated with ethanol and NaCl as above. The precipitated mixture was incubated at -20° C. for 30 minutes, centrifuged at 3,000 g for 10 minutes and the pellet was resuspended in 0/10/10 buffer. Ethanol precipitation was repeated. The precipitate was finally resuspended in 0/10/10 Tris EDTA.
The DNA was passed through a 22 G needle to reduce viscosity (from 5 to 15 times), extracted with phenol/chloroform (as above) two to three times, and precipitated with ethanol as above°
The mixture was centrifuged and the pellet was resuspended in 10 mM Tris, 1 mM EDTA at pH 8.0. The DNA concentration was estimated by running sequential dilutions on a 1% agarose gel along with known amounts of lambda phage DNA.
The DNA was size-fractionated by incubating a BglII digest with Carbowax 8,000 (polyethylene glycol) and NaCl at 7% (weight/volume) and 0.4M respectively at 37° C. overnight.
EXAMPLE 2
Preparation of Probe and Hybridization
Hybridization of the Southern blot was carried out by in 38% formamide, 6XSSC, 50 mM sodium phosphate buffer, pH 6.4, 10% dextran sulphate, 100 micrograms/ml salmon sperm DNA using 3×10 6 cpm of a probe nick translated using the standard method described in Maniatis et al., supra.
After overnight hybridizaton at 42° C., the filters were washed 4 times for 5 minutes at room temperature in 3X SSC, 50 mM sodium phosphate buffer pH6.4, containing 0.1% SDS and then for one minute at 42° C. in the same buffer. Radioautography was carried out at -70° C. with intensifying screens.
EXAMPLE 3
Ligation into Phage and Phage DNA Isolation and Sequencing
The DNA fragment isolated in Example 2 was ligated into EMBL3 as described in Maniatis, T., Supra, pages 75-85 and packaged in vitro, using the packaging extract available from Vector Cloning Systems according to the supplier's instructions. A genomic library was constructed as described on pages 269-293 and screened using the same P. cynomolgi probe as described on pages 320-322. The hybridization conditions were the same as those of Example 1.
Sequencing took place in accordance with the techniques of Maxam and Gilbert Meth. Enzymol. 65 (Part I): 497-559 (1980); and Sanger Nickler and Coulson, Proc. Nat'l Acad. Sci. (U.S.A.) 74:5456 (1977). The gene had an uninterrupted reading frame encoding a protein of 373 amino acids with a molecular weight of 37,253 daltons. The nineteen tandemly repeated units of 9 amino acids each are boxed in FIG. 3. These repeats constitute forty-nine percent (49%) of the total protein. Other general features of CS proteins can be noted, i.e., the central repetitive (immunodominant) epitope, an amino terminal signal sequence, a carboxy terminal hydrophobic transmembrane sequence and several groups of charged residues present in both the N and C terminal regions.
EXAMPLE 4
Peptide Synthesis
An 18-residue peptide amide was synthesized with a structure H-(Asp-Gly-Gln-Pro-Ala-Gly-Asp-Arg-Ala) 2 . The carboxyl terminus was an alpha carboxamide and not a free carboxylic acid.
The peptide was synthesized using the stepwise solid-phase method of R. B. Merrifield, J. Am. Chem. Soc. 85:2149 (1963) on a multidetachable benzhydrylamine resin (p-acyloxy benzhydrylamine copolystyrene 1% divinyl benzene resin) as described by Tam, J. P. et al., Tetrahedron Lett., 2851 (1981). Boc-Ala-p-acyloxybenzhydrylamine resin (0.4 mmol per gram substitution of resin) was placed into the reaction vessel of a Beckman 990M synthesizer and a double coupling protocol via dicyclohexylcarbodiimide was used to give a coupling efficiency more than 99.85% completion per step. The benzyl-based side chain protecting groups and tert-butoxycarbonyl (Boc) for the N-alpha terminus were used. The sequence Asp-Gly is prone to a cyclization reaction to form aspartimide during the synthesis and acid deprotection of the protected peptide-resin. To prevent base catalyzed aspartimide formation during the synthesis and the strong-acid catalyzed deprotection step, a new protection group, Asp (OcHex, aspartyl-Beta cyclohexyl ester) was used as described by Tam, J. P. et al., Tetrahedron Lett. 4033, (1979), incorporated herein by reference. The unpurified peptide, when examined by high pressure liquid chromatography gave a single symmetrical peak accounting for more than 83% of all peptide content. The crude peptide was purified by preparative low-pressure liquid chromatography (100-120 psi) on a reversed-phase C-18 column (2.5×30 cm) using aqueous CF 3 CO 2 H (0.05%) and acetonitrile gradient. The purified material gave a single symmetrical peak on analytical high pressure liquid chromatography and an amino acid analysis gave the correct theoretical values of amino acids. The overall yield based on the first alanine attached to the resin was 72%.
EXAMPLE 5
Recognition of the Peptide of the Present Invention by Monoclonal-Anti CS of P. vivax
Monoclonal-antibody 2F2 from Example 5 was serially diluted in PBS-BSA (indicated in FIG. 5 by black triangles); or in PBS-BSA containing 25 micrograins/ml of synthetic peptide (NANP) 3 (indicated by open triangles); or in PBS-BSA containing 25 micrograms/ml of the synthetic peptide of Example 4, i.e. (DRADGQPAG) 2 (indicated by black circles).
An unrelated monoclonal-antibody 2AlO (anti-falciparum CS protein) was serially diluted in PBS-BSA (indicated by open squares). 20-5 micrograms/ml of each dilution were delivered to the bottom of microtiter plates which had previously been coated with a synthetic peptide (DRADGQPAG) 2 saturated with 1% BSA. After 1 hour of incubation, the wells were washed with PBS-BSA in 50 microliters of 125 I-labelled affinity-purified goat anti-mouse IgG (10 5 cpm; specific activity about 2×10 6 cpm/microgram). After an additional hour of incubation, the wells were washed with PBS-BSA and counted. As shown in FIG. 5, 2F2 bound to the immobilized (DRADGQPAG) 2 peptide. By contrast, 2AlO did not bind. The binding of 2F2 was totally inhibited by (DRADGQPAG) 2 , but not affected by (NANP) 3 .
In other similar experiments, the binding of the anti-P. falciparum antibody 2AlO to the corresponding epitope (NANP) 3 was not inhibited by the synthetic peptide (DRADGQPAG) 2 . 1

Similar results are expected to be obtained if (DRAAGQPAG) 2 is used instead of (DRADGQPAG) 2 . In addition, similar results are expected if oligomers of these peptides or of peptides wherein X stands for both alanine and aspartic acid, as the force amino acid of each repeating unit.
EXAMPLE 6
Inhibition of the Binding of Labelled Monoclonal Antibody 2F2 to Immobilized P. vivax extracts by the Synthetic Peptide (Asp-Gly-Gln-Pro-Ala-Gly-Asp-Ala) 2
100,000 cpm of 2F2 antibody labelled by 125 I having a specific activity of about 2×10 6 cpm/microgram was diluted in PBS containing 1% BSA. The buffer also contained varying amounts of the synthetic peptide. The mixture was added to microtiter wells coated with P. vivax sporozoite extract. After one hour incubation in room temperature, the wells were washed three times with PBS containing 1% BSA and counted in the gamma counter. The results are summarized in Table I below.

TABLE I______________________________________Inhibition by the synthetic peptideH Asp--Gly--Gln--Pro--Ala--Gly--Asp--Ala!.sub.2 of the binding of.sup.125 I-labeled monoclonal antibody 2F2 to the immobilizedP. vivax extractsConcentration of Synthetic BoundPeptide (micrograms/ml) (cpm)______________________________________0 16,387 ± 1,085*200 84220 1,0912 2,1300.2 3,146______________________________________ *mean ± SD of counts in four wells.

In other preliminary experiments, the binding of the labeled 2F2 monoclonal-antibody to the P. vivax extract was shown to be specific since it was inhibited by diluting it in cold 2F2 antibody but not in cold non-relevant monoclonal antibody of the same subclass.
EXAMPLE 7
cDNA Clone Coding for a Sporozoite Antigen Protein
The techniques of recombinant DNA technology make extensive use of enzyme-catalyzed reactions. Purified enzymes for use in the practice of the present invention are currently available from commercial sources. Commercially available enzymes and reagents were employed unless otherwise specified. Restriction endonucleases, their nomenclature and site 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 amounts 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 harvested and dissected to obtain thoracic segments which were stored on ice until the dissection was completed. RNA was prepared from the thoraxes essentially as described by Seeburg, P. H. et al.; Cell, 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 O.IM 2-mercaptoethanol until all the tissue was dispersed. The solution was centrifuged at 10,000 rpm for 10 minutes and the supernatant adjusted to 2% (w/v) Sarkosyl (Trademark, ICN Pharmaceuticals, Plainview, N.Y.), and heated at 65° C. for two minutes. Cesium chloride was then added (0-19/ml of solution) and the resulting solution was layered over 2 ml cushions of half-saturated CsCi in 10 mm EDTA in SW41 (Trademark, Beckman Instruments, Fullerton, Calif.) cellulose nitrate tubes. Centrifugation was at 28,000 rpm for approximately 20 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. Usually, 0.5-1 mg of RNA were obtained 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 for the polyadenylated fraction. Alternatively, the RNA can be prepared from the mosquito thoraces by the procedure modified from Liu, C. P. et al., P Proc. Nat. Acad. Sci. 76:4503, 1979. According to this procedure, 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 took place at 9,000 rpm for 3 minutes, and the supernatant was layered over 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 @20° C. Approximately 0.5-1 mg of RNA was obtained per gram of tissue. Poly(A)+RNA was then oligo-dt selected as described above.
A sample of mRNA isolated as described was translated in vitro using a translation system prepared from wheat germ (modified from Roberts, B. E. et al., Proc. Nat. Acad. Sci. USA, 70, 2330 (1973)). Proteins produced by in vitro translation were immunoprecipitated as described in Example 7, (alternatively, as disclosed by Goldman, B. M., and Biobel, G., Proc. Natl. Acad. Sci., 75:5066 (1978)) and fractionated on an SDS-polyacrylamide gel (SDS-Page) as described by Yoshida, et al, J. Exp. Med 154, 1225 (1981) and in Example 2 of copending U.S. application Ser. No. 234,096, incorporated herein by reference. mRNA fractions containing sequences coding for the CS-proteins can be identified by this means.
For preparative cDNA synthesis total polyadenylated mRNA (approximately 20 μg) was treated in 100 μl volume with 1 mM methyl mercury (hereinafter MeHg) (Aldrich Chemical, Milwaukee, Wis.) at room temperature for 5 minutes. The treatment was stopped by adding 0.5% (0.5 μl per 100 μl) of undiluted β-mercaptoethanol and incubating at room temperature for 5 minutes. The MeHg treated polyadenylated mRNA was incubated in 200 ul reaction containing 50 mM Tris-HCl pH 8.3, 10 mM MgCl 2 , 20 mM KCl, 5 mM Dithiothreitol, 2 mM each of dATP, dCTP, dGTP and dTTP, 50 μCi, 32 PdCTP (specific activity, 800 Ci/mmol), 4 μg oligo(dT) 12-18, (Collaborative Research, Waltham, Mass.) 5 μl RNasin (BIOTECH, Madison, Wis.) and approximately 200 units reverse transcriptase (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 by ethanol. The ethanol precipitate was dissolved in 50 μl 10 mM Tris-HCl, 1 mM EDTA, pH=8 and fractionated on a column of Sephadex (Trademark, Pharmacia, Inc., Uppsala, Sweden) G-75 in a 1 ml Falcon (Trademark, Falcon Plastics, Oxnard, Calif.) plastic pipette, using 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA as the running buffer. The leading peak of unexcluded 32 P counts was collected (approximately 300 μl) and adjusted to 0.3M NAOH and 1 mM EDTA and incubated overnight at room temperature.
Following neutralization with 5M sodim acetate pH 3.8 to a final pH of approximately 6.0, and ethanol precipitation, the second DNA strand was synthesized in a 50 μl reaction containing the same buffer as described supra, 500 μM each of dATP, dCTP, dGTP, dTTP, 125 μci 32 PdCTP (800 Ci/mmole) and 50 units reverse transcriptase. Incubation was at 37° C. for 90 minutes. The reaction products were extracted with phenol/chloroform and passed over a Sephadex G-75 column as described above and the excluded 32 P peak was precipitated with ethanol before proceeding to treatment with S 1 nuclease.
After fractionation on Sephadex G-75, the second cDNA strand synthesis was completed using the Klenow fragment of DNA polymerase I (Boehringer-Mannheim) in the presence of 50 mM Tris-HCl, pH 8.0, 7 mm MgCl 2 and 1 mM dithiothreitol. The reaction mixture was incubated for 4 hours at 15° C., extracted with phenolchloroform (1:1) and precipitated with ethanol as described above.
The double stranded cDNA was incubated with 300 units of S1 nuclease (Boehringer-Mannheim, Indianapolis, Ind.) in 24 μl of 0.3 mM NaCl, 30 mM Na acetate, pH 4.5, and 3 mM ZnCl 2 at 41° C. for 5 minutes. The reaction was stopped by the addition of EDTA to 10 mM and neutralized with Tris base. The 32 P-labelled cDNA was size fractionated using a column of Sepharose CL-4B (Trademark, Pharmacia, Inc., Uppsala, Sweden) made up in a 1 ml Falcon plastic pipette and run in a buffer of 0.3M NaCl, 10 mM Tris-HCI pH 8.0, 1 mM EDTA. Various size classes of double-Btranded cDNA were precipitated with ethanol and then tailed using calf thymus terminal transferase (Enzo Biochem., Inc., New York, N.Y.) in a 100 μl volume for 1 minute at 37° C. in a buffer containing 100 mM K cacodylate, pH 7.6, 1 mM CoCl 2 , 0.1 mM DTT, 0.1 MM dCTP, 4 μCi of 3 H-dCTP, 24 Ci/mmole), and approximately 20 units terminal transferase/μm ds-cDNA. The reaction was stopped by adjusting the solution to 0.5M NaCl, 10 mM EDTA and incubating at 65° C. for 5 min. 1-5 micrograins of yeast+RNA were added prior to extraction with phenol: chloroform and precipitated with ethanol twice. The tailing reaction is described, generally, by Roychoudhury, R. et al., Nucl. Acids Res., 3, 101 (1976). An alternative procedure, also conducted, is described by Land, H. et al., Nucl. Acids. Res. 9:2251, 1981. Approximately 17 bases were added to the 3' end under these conditions. Plasmid pBR322 cleaved by Pst I endonuclease and tailed with dG residues was obtained from a commercial source, New England Nuclear, Boston, Mass. Equimolar amounts of dC tailed cDNA and dG tailed pBR322 were annealed at a concentration of 1 μg/ml, using sequential 2 hour incubations at 42° C., 30° C., and 14° C. The hybrid plasmid DNA was ethanol-precipitated and then used to transform E. coli RR1 cells to ampicillin resistance. Libraries of single colonies were generated and stored in microtiter dishes at -70° C.
According to the alternative procedure, after 15-30 deoxy C residues have been added to the cDNA and annealing to an equimolar concentration of dg-tailed pBR 322 has taken place (at 250 ng of vector/ml), the annealing mixture was incubated at 68° C. for 5 min, then at 42° C. for two hours, followed by slow cooling to room temperature for 2 hours. The hybrid plasmed 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 dishes at -70° C.
A library of (300-2000 bp) cDNA fragments was screened for colonies that expressed protein containing the immuno-chemically reactive region of the sporozoite surface antigen protein. Forty-eight colonies were grown individually on a petri dish containing S agar (32 g/liter tryprone, 5 g/liter NaCl, 20 g/liter yeast extract, 15 g/liter Difco agar 0.2 g/liter NaOH and 20 mg/liter tetraclycline). The plates were flooded with 2 mls of 0.05M Tris-HCl pH 7.5 and 0.5 mg egg-white lysozyme (Sigma, St. Louis, Mo.) and scraped with a sterile spatula into a 15 ml polypropylene tube (Fisher Scientific Supply). After incubation at room temperature for 30 minutes, followed by 60 minutes on ice freeze thawing in 95% ethanol and dry ice (-80° C.) three times, and further incubation at 37° C. for 10 minutes the crude cell extracts were treated with DNAse I (1 mg/ml), 4 mM CaCl 2 and 4 mM MgCl 2 at room temperature for 30 minutes and stored at -70° C. for future use.
The lysates were screened for the presence of any immunochemically reactive protein using P. knowlesi monoclonal antibodies, in a radioimmunoassay. In this method, anti-P. knowlesi monoclonal antibody adsorbed to the well of a microtiter dish was used to affinity purify any immunoreactive protein present in pooled cell lysate. Lysates containing an immunoreactive protein were detected by reacting the washed microtiter wells with a second 125 I-labelled anti-P. knowlesi monoclonal antibody. To do this, microtiter plates were coated with 50 μl anti-P. knowlesi monoclonal antibody (50 μ/ml) incubated at 4° C. for 12-17 hours, washed thoroughly with 1% (w/v) BSA-saline solution, and then incubated with 50 μl of the pooled cell extract for 4-17 hours at 4° C. After washing, a second 125 I-labelled anti-P. knowlesi monoclonal antibody was added to each well and incubated 2 hours at room temperature. The washed wells were then tested individually for radioactivity. When a pool of 48 colonies was found to be positive, the original single colonies that made up the pool were screened individually and the immunoreactive clones identified, isolated and genetically purified.
Plasmid DNA was purified from 1 liter of cells containing an immunoreactive clone (plasmid pEG81). The cells were grown at 37° in Luria broth with 15 μg/ml tetracycline to approximately 5×10 8 /cells per ml and the plasmid DNA amplified by adding 175 ug/ml of chloramphenicol and incubating overnight (Clewell and Helinski, J. Bacteriol. 110, 1135 (1972)). The plasmid DNA was extracted from the cells using sodium dodecyl sulphate (SDS) (Godson and Vapnek, Biochim. Biophys. Acta 299, 516 (1973) and purified using 5-20% (w/v) sucrose density gradients. This yielded 500-1000 μg plasmid RF I DNA 1 μg of this was used to transform other E. coli cells (HB101) to tetracycline resistance and their ability to express the immunoreactive protein was re-checked.
pEG81 DNA was digested with Pst I restriction endonuclease ligated with T4 DNA ligase to 0.5 μg of Pst I-cut cloning/sequencing vector M13mp9 at a 1:1 molar ratio and sequenced using the Sanger dideoxy chain termination method (Sanger and Coulson, supra) with a "universal" synthetic primer 5'-d GTAAAACGACGGCCAGT!-3' (purchased from PL Biochemicals, Milwaukee, Wis.). The complete nucleotide sequence of this segment of the P. knowlesi CS protein gene that contains the immunoreactive site is shown in FIG. 7.
An unexpected feature of the pEG81 fragment of P. knowlesi DNA is that it consisted entirely of a 36 base pair repeat (8 complete units plus a partial unit on either end). The coding strand and correct reading frame of the nucleotide sequence was established as follows:
(a) The reading frame of the Pst I cleavage site of pBR322 ampicillinase gene and of the M13mp9 β-galactosidase genes are known to be identical (5' X C T G C A G X X 3').
M13mp9 Met Thr Met Ile Thr Pro Ser Leu Ala Ala Gly ATG ACC ATG ATT ACG CCA AGC TTC, GCT GCA GGT
Pst I Cleaving Site
Two different M13mp9 recombinants were obtained with the P. knowlesi DNA fragment inserted in opposite orientations. One of the recombinants produced an immunoreactive -galactosidase fusion protein (M13mp9/Pk 11) as measured by the radioimmunoassay described supra, the other clone did not. The sequence of M13mp9/Pk 11 therefore identified the coding strand of the DNA.
(b) The reading frame was also deduced from the fact that 17 dC residues were inserted between the β-gal gene and the P. knowlesi gene fragment.
(c) One possible reading frame coded for an alanine-rich peptide. That the epitope probably 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 5 of an extract of 10 6 sporozoites with 0.002 units porcine elastase (Worthington Enzymes, infra) in Tris-buffer, 0.05M pH 8.6, for 60 minutes at 37° C., completely abrogated the reactivity of the CS protein 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 OOC-Ala Ala Pro Ala (Powers, et al supra).
Other possible reading frames coded for peptides which were excluded for various reasons: they were too hydrophobic or they contained several cysteine, lysine and arginine residues. The absence of such amino acid residues had been determined in experiments showing that the epitope was resistant to trypsin after complete reduction of the CS protein. Indeed, after incubation of another aliquot of the same sporozoite extract with 1 mg of TPCK-trypsin (Worthington Enzymes, Freehold, N.J.) at 37° C. for 30 minutes, followed by complete reduction and alkylation, the CS protein reacted fully with the monoclonal antibodies.
The deduced amino acid sequence of the twelve amino acid repeat is as follows based upon translation of the nucleotide sequence in the correct reading frame:

Gln-Ala-Gln-Gly-Asp-Gly-Ala-Asn-Ala-Gly-Gln-Pro

(All sequences are expressed from the end nearest NH 2 -terminus on the left to the end nearest the --COOH terminus on the right.) The immunoreactive portion of the P. knowlesi protein is therefore contained within the 12 amino acid repeat.
To confirm that the foregoing amino acid sequence contains the immunoreactive site, a dodecapeptide (with the same order of amino acids as shown above) and a dimer of the dodecapeptide have been synthesized, using solid phase resin synthesis (Marglin, H. and Merrifield, R. B., Ann. Rev. Biochem. 39:841-866 (1970). Sequence analysis performed by automated Edman degradation confirmed that the peptide had been correctly synthesized. The final proof that this is the correct epitope has been obtained. Rabbits were immunized with the dodecapeptide coupled to a carrier (bovine gamma globulin in complete Freunds adjuvant). Two weeks after the injection, the rabbits were bled and their serum assayed for the presence of antibodies against the dodecapeptide and against extracts of sporozoites. The results showed that the animals produced high titers (greater than 1:1000) of antibodies to the native CS protein present in the parasite extracts.
Once a clone expressing an immunochemically reactive protein has been identified, the inserted cDNA sequence can be employed as a hybridization probe to identify cDNA coding for sporozoite antigen proteins from other Plasmodium species. The cDNA clone can also be used to screen Plasmodium genomic DNA obtained, for example, from merozoites, to detect DNA sequences coding for sporozoite antigen protein. Therefore, once the first cDNA sequence coding for a sporozoite antigen or fragment thereof is cloned, the subsequent isolation and purification of other species cDNAs is substantially simplified.
EXAMPLE 8
Competition Between Monoclonals for Specific Antigen
Monoclonal antibodies which bind to distinct areas of an antigen molecule do not interfere with each other; on the other hand, monoclonal antibodies directed against the same or typographically related epitopes or antigenic determinants, will inhibit each other's activity. Thus, it is possible to map the epitopes of an antigen.
The number of epitopes of CS proteins which react with monoclonal antibodies was determined by an immunoradiometric assay performed as follows:
A) Preparation of plates coated with sporozoite extracts
Sporozoites were purified from salivary glands of infected mosquitoes as described by Yoshida, N. et al., Science 207, 71 (1980). They were suspended in phosphate-buffered saline (PBS) at a concentration of 10 6 /ml and subjected to sonication (100 W for 3 minutes), then further diluted 20-fold in PBS. Then 50 μl of the suspension were delivered to the bottom of wells of Falcon "3911" microtiter plates, manufactured by Falcon Plastics, Oxnard, Calif. These were incubated overnight at 4° C. and washed with PBS. The wells were then carefully washed with Tween-20 Trademark, Atlas Europol SpA, Ternate, Italy, (0.05% v/v) and incubated for 3 hours in PBS-containing 0.5% (w/v) bovine serum albumin (BSA) to saturate the hydrophobic sites of the plastic.
B) Preparation of monoclonal antibodies
Monoclonal antibodies were raised against different species of sporozoites as described by Yoshida et al., supra; and Potocnjak, P. et al., J. Exp. Med. 151, 1504 (1980). The antibodies were isolated from ascitic fluid of mice injected with the hybridomas by standard chromatographic procedures (ion exchange chromatography and filtration in Sephadex G-200). The purity of the antibodies was ascertained by SDS-PAGE. The antibodies were then radiolabeled with 125 I using Iodogen (Pierce Chemical Co., Rockford, Ill.) according to the instructions of the manufacturer. The specific activity varied between 10 7 -3×10 7 cpm per μg protein.
C) Titration of monoclonal antibodies
The minimal concentration of a monoclonal antibody which saturates the antigen sites in the bottom of wells of microtiter plates 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. 30 μl aliquots of the various mixtures containing the same number of counts, but different concentrations of antibody, were delivered to the bottom of individual wells of the microtiter plates precoated with specific antigen. After incubation for 1 hour at room temperature, the wells were washed with PBS-BSA and counted in a gamma counter. The greatest concentration of monoclonal antibody yielding a maximum of counts bound, represents the saturating dose of monoclonal antibody.
D) Competition between monoclonal antibodies for binding to the antigen
Several monoclonal antibodies were prepared against the CS proteins of P. knowlesi. The monoclonal antibodies were labeled with 125 I and the saturating dose determined as described, supra. Then, cross-titrations were 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 antigen-containing wells, 50 μl of different concentrations of the various cold monoclonals diluted in PBS-BSA were added. The plates were incubated for 1 hour at room temperature. Then, 50 μl of one of the radiolabeled monoclonals (for example, 2G3) at twice the saturating concentration, were added to all the wells. After an additional hour of incubation, the wells were washed and counted. The number of specific counts bound to antigen 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 3 times the saturating dose. The counts which could not be inhibited by the homologous cold antibody represent non-specific binding. From these numbers, the percentages of inhibition of binding of 2G3 by the other monoclonal antibodies were calculated. The titration was repeated for each labeled monoclonal antibody. The results are summarized in Table II. It can be seen that all monoclonal antibodies to P. knowlesi strongly inhibit each other, indicating that they must bind to closely related or identical epitopes.

TABLE II______________________________________Evidence for the Presence of an Immunodominant Region onthe CS Protein of P. knowlesiColdInhibitor % Inhibitor of Binding to Antigen of the(Monoclonal Radiolabeled Monoclonal AbAntibody) 5HB 2G3 BBB BAB BE11 6BB______________________________________5HB 100 99 101 115 105 1212G3 96 100 87 78 106 103BBB 95 94 100 106 107 125BAB 86 83 94 100 105 128BE11 65 59 86 70 100 1056BB 88 85 91 95 104 100Control 6 0.4 0.5 8 0 0MonoclonalPBS--BSA 0 0 0 0 0 0______________________________________

An identical procedure was followed to study the specificities of the monoclonal antibodies to P. vivax and P. falciparum (Tables II and III), P. malariae and P. berghei (not shown). The overall results demonstrate that there is a single immunodominant region in every CS protein.

TABLE III______________________________________Evidence for the Presence of an Immunodominant Region onthe CS Protein of P. vivax % Inhibitor of Binding to Antigen of theCold Inhibitor Radiolabeled Monoclonal Ab(Monoclonal Antibody) 3D10 5D9 3C1 4E8 2F2______________________________________3D10 100 102 99 101 595D9 83 100 98 101 653C1 113 103 100 102 674EB 105 100 99 100 602F2 100 101 100 116 100Control Monoclonal 8 -6 -11 3 6PBS--BSA 0 0 0 0 0______________________________________


TABLE IV______________________________________Evidence for the Presence of an Immunodominant Region onthe CS Protein of P. falciparum % Inhibitor of Binding to Antigen of theCold Inhibitor Radiolabeled Monoclonal Ab(Monoclonal Antibody) 3D6 2E7 1E9 2C11 2A10______________________________________3D6 100 98 99 95 752E7 98 100 102 99 901E9 100 103 100 100 992C11 104 100 99 100 1002A10 110 102 106 100 100Control Monoclonal 2 0 -4 8 0PBS--BSA 0 0 0 0 0______________________________________

EXAMPLE 9
Competition Between Monoclonal Antibodies and Polyclonal Antisera for Sorozoite Antigens
A) Assay for the binding of polyclonal antibodies to sporozoite extracts
The first step of this assay was essentially the same as that described in Example 8; that is, wells of microtiter plates were coated with crude sonicated extracts of sporozoites, washed with Tween-20 and saturated with PBS-BSA. Then, serial dilutions in PBS-BSA of the polyclonal antibodies to the homologous sporozoite species were prepared and aliquots of 30 μl delivered to the bottom of individual microtiter wells. Controls consisted of wells incubated with dilutions of polyclonal antibodies to an unrelated antigen. After an incubation of 4 hours 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 immunoglobulin of the appropriate 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 absorption was necessary to prevent the interaction of the developing reagent with the mouse monoclonal antibodies used in the inhibition assay described below. When monkey polyclonal antibodies were used, the developing reagent was similarly prepared from a rabbit antiserum to monkey Ig.
B) Inhibition of binding of polyclonal antibodies by monoclonal antibodies
The wells coated with sporozoite extract were first incubated with a purified monoclonal antibody to the repetitive epitope of a CS protein, at saturating levels (see Example 8 for the determination of the saturating dose). After incubation for 1 hour at room temperature, the dilutions of the polyclonal antibodies were added, and the assay proceeded as described above.
Inhibition assays have been performed in the following systems:
1) P. knowlesi sporozoite extracts reacting with monkey antisera to X-irradiated P. knowlesi sporozoites. Inhibitory monoclonal antibody2G3 (Cochrane, A. H. et al., Proc. Nat. Acad. Sci. USA 79, 565 (1982)).
2) P. vivax sporozoite extracts reacting with serum of humans vaccinated by the bite of X-irradiated P. vivax-infected mosquitoes. Inhibitory monoclonal antibody2F2 (Nardin, E. H. et al., J. Exp. Med. 156:20 (1982)).
3) P. falciparum sporozoite extracts reacting with serum of humans vaccinated by the bite of X-irradiated P. falciparum-infected mosquitoes. Inhibitory monoclonal antibody 2A10 (Nardin, E. E. et al., supra).
Typical results of these assays are illustrated in FIG. 8. In every 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 sonication of whole sporozoites and probably contains intracellular as well as plasma membrane 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 (see infra).
EXAMPLE 10
Presence of Repetitive Epitopes in the Immunodominant Region of CS Proteins from Several Species of Sporozoites
The experiments in Examples 8 and 9 showed that the CS proteins of four species of Plasmodium contain a single immunodominant region. Shown in this section is the evidence that 1) all immunodominant regions contain a repetitive epitope, and 2) all monoclonals 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 FIG. 9, for the CS proteins of P. vivax, P. falciparum and P. knowlesi. Identical results were obtained with the CS protein of P. berghei and the monoclonal 3D11 (not shown) and the CS protein of P. malariae. The assays were performed as follows:
Wells of microtiter plates (Falcon 3911) were incubated overnight at 4° C. with 50 μl of a 10 10 μg/ml solution in PBS of a monoclonal antibody. The wells were washed with PBS and incubated for 2 hours at room temperature with PBS-Tween 20 (0.05% v/v) and for 3 hours at room temperature with PBS-Tween 20-BSA (1% w/v). 30 μl of serial dilutions of extracts of sporozoites were delivered to the bottom of the wells, and the plates incubated overnight in the refrigerator. The extracts had been prepared by treating purified salivary gland sporozoites (10 7 /ml) with 2% (v/v)NP-40 (Trademark, Particle Data Laboratories, Elmhurst, Ill.) in PBS for 2 hours at room temperature, followed by centrifugation at 100,000 g for 1 hour. The dilutions of the extract were made in PBS-BSA containing 0.1% (v/v) NP-40. After incubation, the wells were washed with PBS-Tween 20-BSA. Then 50 μl (about 5-10 ng) of the same 125 I-labeled monoclonal antibody, diluted in PBS-BSA-Tween 20, were added, and incubation at room temperature proceeded for an additional hour. The wells were washed with PBS-Tween 20-BSA and counted. Controls consisted of wells initially coated with BSA alone. As shown in FIG. 9, specific binding was observed in every instance using the homologous antigen. These experiments demonstrate that the various extracts of sporozoites contain CS proteins which are at least divalent, since they can bind two molecules of a single monoclonal antibody, one of them in solid phase, attached to the plastic, and the other in fluid phase and radiolabeled.
It could be argued however, that the extracts contained aggregated CS protein. This possibility was excluded by the experiments described below, which show directly that the molecular weight of the divalent or multivalent antigen in the extracts corresponded to that of monomers of the CS proteins.
Extracts were prepared as described above treated with SDS 2% (w/v)--6M urea, and subjected to ultracentrifugation onto 5% (w/v)--20% (w/v) sucrose gradients. The runs were performed in an ultracentrifuge, using a Beckman SW-20 50.1 (Trademark, Beckman Instrument Co., Fullerton, Calif.) rotor at 48,000 rpm for 20 hours. After centrifugation, the bottoms of the tubes were perforated and drops collected in separate tubes. The fractions were analyzed by two methods for the presence of CS protein.
1) Analysis of the fractions by the two-site immunoradiometric assay. This was performed with several monoclonal antibodies for each extract, as described previously in this section. The results of the assays are expressed as number of sporozoite equivalents present in each fraction, as calculated from a standard curve obtained on the same day of the experiment.
2) Analysis of the fractions by inhibition of binding of monoclonals to antigen-coated plates. The gradient fractions were also analyzed by an assay which detects single epitopes on the CS protein. This assay was performed as follows: Antigen-coated plates were prepared, and the minimal saturating dose of radiolabeled monoclonal antibody was determined as described in Example 8. Aliquots of gradient fractions were mixed with the radiolabeled antibody for one hour at room temperature, and then 30 μl of the mixtures were delivered to the wells containing solid-phase antigen. After an additional hour of incubation, the wells were washed and counted. The inhibition of binding is also expressed as number of sporozoite equivalents present in the fraction, as calculated from a standard curve.
The results of these experiments are summarized in FIGS. 10 and 11, which also show the position in the fractions of marker proteins. The results show that the CS antigen of P. vivax and P. knowlesi was detected by both assays and sedimented in a single peak between the markers ovalbumin and bovine serum albumin. 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 mainly or exclusively, monomers of these molecules. Moreover, all monoclonal antibodies tested recognized a repetitive epitope on the CS protein. The simplest explanation for this finding is that all of them react with the same epitope.
In short, the present results and those of the previous examples, demonstrate that the CS proteins of the human malaria parasites P. vivax, P. falciparum and P. malariae contain a single immunodominant region and repetitive epitopes, as in the case of P. knowlesi.
EXAMPLE 11
Cross-reactivity between Species of Sporozoites
The cross-reactions between the monoclonal antibodies to the repetitive epitopes of the CS proteins from various species of Plasmodium were detected by circumsporozoite (CSP) reactions or by the indirect immunofluorescence test. These tests are described in Nardin, E. H. et al., Nature 274, 55 (1978) and Danforth, H. D. et al., J. Parasitol, 64, 1123 (1978), and were performed with monoclonal antibodies.
For example, the CSP reaction is performed by incubation of dilutions of serum in PBS-BSA with purified viable salivary gland sporozoites at room temperature. After 10 minutes or more of incubation, the sporozoites are examined by phase-contrast microscopy. 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 the cross-linking of the CS protein by antibodies.
The indirect immunofluorescence test is performed with glutaraldehyde-fixed sporozoites. The parasites are treated with 1% (v/v) glutaraldehyde solution in PBS for 30 minutes at 0° C. Then they are washed in PBS, and incubated overnight with 0.1% (w/v) glycine in water. After washing by centrifugation, the resuspended sporozoites are deposited within 10 μl droplets on microscopic slides, at a concentration of 2×10 6 /ml. The droplets are air-dried and kept at -70° C. The assay is performed by incubation of the sporozoites with dilutions of the immune serum for 2 hours at room temperature, followed by washings with PBS, and a new incubation for 2 hours with a second antibody, fluorescein-labeled, directed against the Ig of the first immune serum. The second antibody (for example, rabbit anti-human Ig) can be obtained from a commercial source (Cappel Laboratories, Cochranville, Pa.). After washing, the slides are viewed with 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-P. knowlesi reacted with P. falciparum and P. cynomolgi;
B) anti-P. cynomolgi reacted with P. vivax;
C) anti-P. yoeli nigeriensis reacted with P. berghei.
In this case the cross-reactive monoclonal antibodies even neutralized the infectivity of the heterologous species; and
D) anti-P. malariae reacted with P. brasilianum.
EXAMPLE 12
Reactivity of Synthetic Peptides with Monoclonal Antibodies to the Repetitive Epitope of P. knowlesi
The two synthetic peptides described in Example 7 were used in these studies. One of them was composed of the 12 amino acid sequence. H 2 N-GlnAIaGlnGlyAspGlyAlaAsnAlaGlyGlnPro-COOH (12-MER) and the other was a dimer of the same sequence (24-MER). The 24-MER (but not the 12-MER) was directly shown by immunoradiometric assay to contain two epitopes of the CS protein of P. knowlesi. The assay was performed as follows:
Wells of microtiter plates (Falcon 3911) were incubated overnight at 4° C. with 50 μl of a 10 μg/ml solution in PBS of the monoclonal 2G3 anti-CS protein of P. knowlesi. The wells were washed with PBS and then incubated for 2 hours at room temperature, with PBS-Tween 20 (0.05% (w/v), and then for 3 additional hours with PBS-BSA (1% (w/v)). The synthetic peptides were diluted serially in PBS-BSA-Tween 20, and 30 μl aliquots of the dilutions were delivered to the bottom of individual wells. The plates were incubated overnight in the refrigerator, washed with PBS-BSA-Tween 20, and incubated with 5.0 μl (10 ng) of 125 I-labeled monoclonal antibody 2G3 diluted in PBS-BSA-Tween 20 (about 100,000 cpm). Controls consisted of plates coated with a non-relevant monoclonal antibody of the same isotype as 2G3. The results are shown in Table V. Specific counts were found only in wells incubated with the 24-MER and they were proportional to the dose of peptide added to the well.
No specific counts of the 24-MER were bound to the wells in which the solid-phase antibody was the monoclonal 3D11, which is directed against the CS protein of P. berghei.

TABLE V______________________________________Results of two-site immunoradiometric assay performedwith monoclonal antibody 2G3 and synthetic peptidesConcentration of 12-Mer or 24-Mer Amount of radiolabeledincubated with the solid-phase 2G3 (cpm) bound inantibody 2G3 wells incubated with(ug/ml) 12-MER 24-MER______________________________________500. 157 551750. 103 20565. 40 402.5 0 93.05 -- 35______________________________________

These results strongly suggest that the 24-MER contains two identical epitopes recognized by the monoclonal 2G3, and that the 12-MER contains either one epitope or none. To distinguish between these possibilities, an assay was conducted to determine the ability of the 12-MER to inhibit the interaction between the CS proteins of P. knowlesi and the monoclonal antibody 2G3. The inhibition assay was performed as follows:
Wells of microtiter plates were coated with the monoclonal antibody 2G3 washed with Tween-20, saturated with BSA as described supra. In one series of tubes, the 12-MER was serially diluted in PBS-BSA-Tween 20. In a second series of tubes, an extract of P. knowlesi sporozoites (prepared as described by Cochrane et al., supra) was serially diluted in PBS-BSA-Tween 20. Aliquots of each dilution of sporozoites were mixed with equal volumes of all dilutions of 12-MER. 30 μI of these mixtures were then added to the bottom of the 2G3-coated wells. As positive and negative controls, 30 μl aliquots of sporozoite dilutions mixed with PBS-BSA-Tween 20 were added to other wells which had been precoated with 2G3 or with a non-relevant monoclonal antibody. After overnight incubation in the refrigerator, the wells were washed and 50 μl (10 μg) of 125 I-labeled 2G3 diluted in PBS-BSA-Tween 20 were added. Following an additional incubation at room temperature for 1 hour, the wells were washed and counted in a gamma counter. The results are shown in Table VI. The 12-MER inhibited, in a dose-dependent fashion, the interaction of the P. knowlesi CS protein with 2G3.

TABLE VI______________________________________Synthetic dodecapeptide (12-MER) inhibits binding of amonoclonal antibody (2G3) to the CS protein of P. knowlesi Amount of 2G3 bound (cpm) in the presence of 12-MERConcentration of P. knowlesi inhibitor at concentrationsextracts (sporozoites/ml) of (mg/ml):______________________________________ 0 0.08 0.6 5.010.sup.6 5582 3893 3018 109910.sup.5 4390 1221 781 36110.sup.4 1353 102 70 310.sup.3 156 72 0 370 0 0 0 0______________________________________

The conclusion drawn from this experiment is that the 12-MER peptide contains an epitope of the CS protein of P. knowlesi.
The reactivity of the synthetic peptides was confirmed by radiolabeling the 24-MER and showing that it bound specifically a monoclonal antibody to P. knowlesi (5HB). This experiment was performed as follows:
A) Preparation of Sepharose-4B (Trademark, Pharmacia, Inc. Uppsala, Sweden) coupled to the monoclonal antibodies 5H8 and 3D11, directed against the CS proteins of P. knowlesi and P. berghei respectively.
The antibodies were coupled to CNBR Sepharose beads (Pharmacia Fine Chemicals Uppsala, Sweden) following the instructions of the manufacturer. After coupling, the beads (containing about 10 mg antibody/ml) were treated for 1 hour at room temperature with 0.5% glutaraldehyde (to prevent leakage of the proteins), then with a solution of 10 mg/ml glycine in PBS, and finally resuspended at 20% volume in PBS-BSA (1% (w/v)) Tween 20 (0.05% (v/v)). The Sepharose-coupled monoclonals were designated Sepharose-5HB and Sepharose 3D11, respectively.
B) Radiolabeling of the 24-MER was performed with 125 I using the Bolton-Hunter reagent (Amersham International Ltd., Amersham, U.K.) according to the instructions of the manufacturer, using 10 μl of a solution of the 24-MER (10 mg/ml) and 0.1 millicuries of the Bolton-Hunter reagent. The peptide was purified after labeling in a Sephadex-GlO column equilibrated in PBS-gelatin (0.2% (w/v)). Presuming that 100% of the peptide was recovered, the specific activity was 10 5 -cpm/μg of protein.
C) Specific binding of the radiolabeled 24-MER to Sepharose-5H8.
Four 200 μl aliquots of the 20% (w/v) suspension of Sepharose-5HS were added to tubes containing 150 μl of a dilution of the labeled 24-MER in PBS-BSA-Tween 20 (45,000 cpm). To two of the tubes 50 μl of cold 24-MER (500 μg) diluted in PBS-BSA-Tween 20 were added. The tubes were incubated overnight in the refrigerator. The beads 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 anti-CS protein of P. knowlesi. In addition, it appears that the 24-MER may have interacted weakly with the 3D11 antibody. This is not surprising, considering the evidence that all CS proteins are structurally related, and that, in these experiments, the molar ratio of antibody to the ligand peptide was quite high.
EXAMPLE 13
Immunization with the Synthetic Repeated Epitope of P. knowlesi (24 -MER)
The synthetic 24-MER is synthesized as described in Example 7, except that a cysteine residue is added at the N-terminus. To determine whether the synthesis has been performed correctly, an aliquot is subjected to acid hydrolysis at reduced pressure (6M HCl, 110° C., 72 hours) and its amino acid composition is determined. The peptide is coupled to a carrier protein either keyhole limpet hemocyanin, or tetanus tozoid, through its N terminal cysteine residue, using m-maleimidolbenzoyl-N-hydroxy-succinimide ester (FIBS) as the coupling reagent (Ling, F. T. et al., Biochemistry 18, 690 (1979)). This is a bi-functional reagent which under appropriate conditions reacts specifically with the amino group of the carrier on the one hand, and with the thiol group of the peptides, on the other hand.
4 mg of the carrier protein in 0.25 ml of 0.05M PO 4 buffer, pH 7.2, is reacted dropwise with 0.7 mg MBS dissolved in dimethyl-formamide, and stirred for 30 min. at room temperature. The product, that is, MB-carrier, is separated from the unreacted chemicals by passage in a Sephadex G-25 column equilibrated in 0.05M PO 4 buffer, pH 6.0. The MB-carrier 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. Coupling efficiency is monitored with radioactive peptide; that is, a trace amount of 125 I-labeled 24-MER is mixed with cold peptide during the synthesis. Dialysis of the conjugate permits evaluation of the proportion of incorporated label. The number of 24-MER groups per 100,000 M.W. carrier was estimated to be about 10-14.
Five rhesus monkeys are immunized with 200 mg of the conjugated protein adsorbed to aluminum hydroxide gel. Their serum is monitored for the presence of antibodies to CS proteins of P. knowlesi by the immunoradiometric assay described in Example 9. 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 monitored by incubation with 125 I-labeled affinity-purified rabbit-antihuman Ig (which strongly cross-reacts with rhesus monkey Ig).
After30 days, the serum titers of the monkeys rise to titers of greater than 1/1000. At this time, these monkeys (as well as five other control monkeys injected with non-conjugated carrier protein adsorbed to aluminum hydroxide) are challenged with 2,000 viable P. knowlesi sporozoites. The infection is monitored daily for a total of 30 days by microscopic examination of blood smears, starting one week after the inoculation of the parasites. The results show that the five monkeys immunized with the vaccine (conjugated protein) are totally protected; that is, no parasites are found in their blood. In contrast, the control monkeys have trophozoites of P. knowlesi in the circulation 7-12 days after challenge.
EXAMPLE 14
Immunization with the Synthetic Repeated Epitopes of 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 7, except that cysteine residues are added at the N-terminals as described in Example 7. To determine whether the synthesis has been performed correctly, aliquots are subjected to acid hydrolysis at reduced pressure (6M HCI, 110° C., 72 hours) and their amino acid composition is determined. The peptides are coupled to a carrier protein either keyhole limpet hemocyanin, or tetanus toxoid, through its N terminal cysteine residue, by using MBS as the coupling reagent, as described in Example 7.
4 mg of the carrier protein in 0.25 ml of 0.05M PO 4 buffer, pH 7.2., is reacted dropwise with 0.7 mg MBS dissolved in dimethyl-formamide, and stirred for 30 min. at room temperature. The product, that is, MB-carrier, is separated from the unreacted chemicals by passage in a Sephadex G-25 column equilibrated in 0.05M PO 4 buffer, pH 6.0. The MB-carrier is then reacted with 5 mg of each peptide containing cysteine, dissolved in PBS (pH 7.4). The mixture is stirred for 3 hours at room temperature. Coupling efficiency is monitored with radioactive peptide; that is, a trace amount of 125 I-labelled is mixed with cold peptide during the synthesis. Dialysis of the conjugate permits evaluation of the proportion of incorporated label. The number of synthetic peptides per 100,000 H. W. Carrier is estimated to be about 10-14.
Five chimpanzees are immunized with 200 μg of each of the conjugated proteins adsorbed to aluminum hydroxide gel. Their serum is monitored for the presence of antibodies to CS proteins of P. vivax and P. falciparum by the immunoradiometric assay described in Example 9. That is, serum dilutions are incubated with antigencoated wells of microtiter plates. The presence of chimpanzee antibody bound to the solid-phase antigen is monitored by incubation with 125 I-labeled affinity-purified rabbit-antihuman Ig (which strongly cross-reacts with chimpanzee Ig).
After 30 days, the serum titers of the chimpanzees rise to titers of greater than 1/1000. At this time, these chimpanzees (as well as five other control chimpanzees injected with nonconjugated carrier protein adsorbed to aluminum hydroxide) are 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 the inoculation of the parasites. The results show that the five chimpanzees immunized with the vaccine (conjugated protein) are totally protected; that is, no parasites are found in their blood. In contrast, the control chimpanzees have trophozoites of P. vivax in the circulation 10-12 days after challenge.
Next, a second challenge with 10,000 P. falciparum sporozoites is given to the same chimpanzees. Again the vaccinated apes are protected, while the controls are all infected.
Based upon the close similarities of human and chimpanzee immune responses and on the fact that protective immunity has been obtained in humans by injection of inactivated sporozoites of P. falciparum and for P. vivax, the results obtained upon immunization of chimpanzees with the described synthetic peptide will also be obtained following similar treatment of human patients.