GENE FOR ENCODING A HUMAN MALARIA VACCINE ANTIGEN

Abstract: The invention is a gene for expressing antigens for producing a human malaria vaccine. The gene includes a cloned nucleotide sequence or segment for encoding the 25kDa surface protein of zygotes and ookinetes of Plasmodium falciparum.

Description: GENE FOR ENCODING A HUMAN MALARIA VACCINE ANTIGEN BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to genes for encoding malarial vaccine antigens. More specifically, this invention relates to a gene for encoding the Plasmodium falciparum 25 kDA ookinete surface antigen. 2. Description of the Background Art Malaria continues to exact a heavy toll from mankind. Approximately 25 percent of all deaths of children in rural Africa between the ages of one to four years are caused by Malaria. This death rate continues despite the sensitivity of local parasites to chloroquine. Mosquito control is difficult in this setting. The greatest hope at present for reducing this mortality rate is a protective vaccine that reduces the incidence of the disease and death by suppressing the replication of the parasite. The major cause of malaria in humans is the parasite . . Plasmodium falciparum. The value of various vaccines to combat malaria is appreciated through an understanding of the life -cycle of the parasite. Infection in man begins when young malarial parasites or "sporozoites" are injected into the blood stream of a human by the mosquito. After injection the parasite localizes in liver cells. After approximately one week the parasites or "merozoites" are released into the blood stream. The entry of the parasites into the blood stream begins the "erythrccytic" phase. Each parasite - enters a red blood cell in order to grow and develop. When " the erozoite matures in the red blood cell, it is known as _ at-, trophσ^oite and schizont. A schizont is the stage when 2 micϊear: dϋv sion. occurs to form individual merozoites which are released to invade other red cells. After several schizogonic cycles, some parasites, instead of becoming schizonts through asexual reproduction, develop into large uninucleate parasites. These parasites undergo sexual development. ? Sexual development of the malaria parasites involves " the female or "macrogametocyte" and the male parasite or " icrogametocyte". These gametocytes do net undergo any further development in man. Upon ingestion of the gametocytes into the mosquito, the complicated sexual cycle begins in the idgut of the mosquito. The red blood cells disintegrate in the midgut of the mosquito after 10 to 20 minutes. The microgametocyte continues to develop through exflagellation and releases 8 highly flagellated microgametes. Fertilization occurs with the fusion of the ιαicr-agamete into a macrogaι_.ete. The fertilized parasite is known?, as a zygote that develops into an "ookinete". The ook- nete penetrates the midgut wall of the mosquito and transforms into the oocyst within which many small sporozoites form. When the oocyst ruptures the sporozoites migrate to the salivary gland of the mosquito via the haemolymph. Once in the saliva of the mosquito, the parasite can be injected into a host. Malaria vaccines are being developed against different stages in the parasite's life-cycle which includes the sporozoite, asexual erythrocyte, and sexual stage. Each development increases the opportunity to control malaria in the many diverse settings within which the disease occurs. Sporozoite vaccines would prevent mosquito-induced infections . First generation vaccines of this type have been tested in humans. Asexual erythrocytic stage vaccines would be useful in reducing the severity of the disease. Multiple candidate antigens have been cloned and tested in animals and in humans. Sexual stage vaccines would induce antibodies which, when ingested in a bloodmeal containing sexual stage parasites, would prevent infection of mosquitoes. Although not directly protective against infection or disease, the sexual stage vaccine combined with a protective vaccine such as a sporozoite or asexual stage vaccine would reduce the chance of transmission of vaccine-induced mutants resistant to the protective component. In this manner, the useful life of the protective component would be lengthened. In some geographical areas the sexual stage vaccine could reduce transmission below the critical threshold required to maintain the infected population. This reduced transmission would be useful in assisting in the control or eradication of malaria. U.S. Patent Number 4,632,909 to Carter and Miller, herein incorporated by reference, discloses monoclonal antibodies that bind with one or more proteins located on the surface of gametes or zygotes of malaria parasites and are targets for sexual stage vaccines . These antibodies ~ are specific for antigens on mosquito midgut stages of 3 malaria parasite and sterilize the parasites in mosquitoes - ' - otherwise- capable of transmitting the disease . The 7 monoclonal antibodies are specific for the 255 , 59 and 53 . - surface proteins on Plasmodium falciparum and for the 25 K. surface protein on zygotes and ookinetes of Plasmodium gallinaceum. This invention includes a process for blocking transmissions of malaria paras ites . The process 0 involves the feeding of mosquitoes carrying the malaria 1 paras it e= monoclonal antibodies specific for a glycoprotein _ oir. the; su fe-ce of the malaria parasite zygote . The giycop otei'_r has a molecular weight of 24-30 K. The -- proces s is effective in a zygote up to about 3 hours after " fertilization . This invention does not involve a cloned 6 gene to induce transmis sion blocking immunity to alaria nor a deduced pep tide from the gene for use in a zalarial vaccine . A study to identify antigens useful to develop malaria transmission blocking immunity is disclosed in the article, Carter et al., "Target Antigens in Malaria Transmission Blocking Immunity," Phil. Trans. R. Soc. Land. 3 307:201-213 (1984), herein incorporated by reference. This article describes the phases of development of malaria parasites wherein transmission blocking immunity occurs. Target antigens on gametes and newly fertilized zygotes and target antigens of post-fertilization transmission blocking immunity- are identified in the article. This article does not disclose a cloned gene to induce transmission blocking immunity to malaria nor a deduced peptide from the gene for use in a malarial vaccine. An article, Grotendorst et al. , "A Surface Procein Expressed During the Transformation of Zygotes of Plasmodium gallinaceum is a Target of Transmission-Blocking Antibodies," Infection and Immunity, Vol. 45, No. 3, p. 775-777 (1984), herein incorporated by reference, discloses a specific protein suitable for use as an antigen. This article identifies materials and procedures that are useful in isolating and identifying an antigen which is a surface protein of M r 26,000 synthesized by zygotes of I\_ gallinaceum. Monoclonal antibodies, having properties of anti-ookinete serum, were found in certain examples to suppress infectivity of fertilized parasites to mosquitoes. An analogous 25kDa surface protein synthesized by zygotes and ookinetes of Plasmodium falciparum is described by Vermeulen et al. , "Sequential Expression of Antigens on Sexual Stages of Plasmodium Falciparum accessible to Transmission-blocking Antibodies in the Mosquito," J. EXP. Med. 162:1460-1476 (1985), herein incorporated by reference. These articles do not disclose a cloned gene of P. falciparum to induce transmission blocking immunity to malaria nor a deduced peptide from the gene for use in a malarial vaccine. The industry is lacking a gene which can produce a vaccine designed to induce transmission blocking immunity to Plasmodium falciparum 25kDa surface protein (herein after Pfs25) and other sexual stage antigens. The industry is also lacking a synthetic peptide that can be expressed from the above gene and used in a pharmaceutical composition to produce a malarial vaccine. Vaccines derived from such genes would prolong the usefulness of other protective malarial vaccines as well as reduce the spread of malaria in areas of low transmission. 1 SUMMARY OF THE INVENTION 22 The invention is a gene for expressing antigens for 33 producing a human malaria vaccine. The gene includes a 4 cloned nucleotide sequence or segment for encoding the 5 25kDa surface protein of zygotes and ookinetes of 6 Plasmodium falciparum. The segment of the gene encoding 7 the protein is 8. ATG AAT AAA CTT TAC AGT TTG TTT CTT TTC CTT TTC ATT CAA CTT 99 GC-ATAΛAAA TAT AAT AAT GCG AAA GTT ACC GTG GAT ACT GTA TGC 100 AAA AGA- GGA TTT TTA ATT CAG ATG AGT GGT CAT TTG GAA TGT AAA li: TGT GAA AAT GAT TTG GTG TTA GTA AAT GAA GAA ACA TGT GAA GAA 12 " AAA GTT CTG AAA TGT GAC GAA AAG ACT GTA AAT AAA CCA TGT GGA 13 GAT TTT TCC AAA TGT ATT AAA ATA GAT GGA AAT CCC GTT TCA TAC 14 GCT TGT AAA TGT AAT CTT GGA TAT GAT ATG GTA AAT AAT GTT TGT 15 ATA CCA AAT GAA TGT AAG AAT GTA ACT TGT GGT AAC GGT AAA TGT 15- ATA TTA GAT ACA AGC AAT CCT GTT AAA ACT GGA GTT TGC TCA TGT 17 - AAT ATA GGC AAA GTT CCC AAT GTA CAA GAT CAA AAT AAA TGT TCA 18 AAA GAT GGA GAA ACC AAA TGC TCA TTA AAA TGC TTA AAA GAA AAT 19 GAA ACC TGT AAA GCT GTT GAT GGA ATT TAT AAA TGT GAT TGT AAA 20 GAT GGA TTT ATA ATA GAT AAT GAA AGC TCT ATA TGT ACT GCT TTT 21 TCA GCA TAT AAT ATT TTA AAT CTA AGC ATT ATG TTT ATA CTA TTT 22 TCA GTA TGC TTT TTT ATA ATG TAA. 23 The invention includes a synthetic protein which is 24 useful finr preparing a malaria vaccine. The synthetic 25 proteirr off the cloned gene is 26 M Asn Lys Leu Tyr Ser Leu Phe Leu Phe Leu Phe lie Gin Leu 27 Ser lie Lys Tyr Asn Asn Ala Lys Val Thr Val Asp Thr Val Cys 28 Lys Arg Gly Phe Leu lie Gin Met Ser Gly His Leu Glu Cys Lys 29 Cys Glu Asn Asp Leu Val Leu Val Asn Glu Glu Thr Cys Glu Glu Lys Val Leu Lys Cys Asp Glu Lys Thr Val Asn Lys Pro Cys Gly Asp Phe Ser Lys Cys He Lys He Asp Gly Asn Pro Val Ser Tyr Ala Cys Lys Cys Asn Leu Gly Tyr Asp Met Val Asn Asn Val Cys He Pro Asn Glu Cys Lys Asn Val Thr Cys Gly Asn Gly Lys Cys He Leu Asp Thr Ser Asn Pro Val Lys Thr Gly Val Cys Ser Cys Asn He Gly Lys Val Pro Asn Val Gin Asp Gin Asn Lys Cys Ser Lys Asp Gly Glu Thr Lys Cys Ser Leu Lys Cys Leu Lys Glu Asn Glu Thr Cys Lys Ala Val Asp Gly He Tyr Lys Cys Asp Cys Lys Asp Gly Phe He He Asp Asn Glu Ser Ser He Cys Thr Ala Phe Ser Ala Tyr Asn He Leu Asn Leu Ser He Met Phe He Leu Phe Ser Val Cys Phe Phe He Met. The invention includes a pharmaceutical composition having the synthetic protein and the method to make an anti-malarial vaccine including the synthetic protein. 3RIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the neucleotide and predicted a ino acid sequence of Pfs25. Figure 2 illustrates a Northern blot analysis of asexual and sexual stage RNA. Figure 3 illustrates a protein structure of Pfs25 arranged to emphasize the relatedness of the EGF-like domains. DETAILED DESCRIPTION OF THE INVENTION The invention is the isolated and cloned gene for encoding the 25 kDa surface protein (Pfs25) of zygotes and ookinetes of Plasmodium falciparum. The deduced amino acid sequence of this gene consists of a signal sequence, a hydrophobic C- erminus, and four tandem epidermal growth factor (EGF)-like domains. The cloned gene of this invention, therefore, provides a useful composition that 3 can express an antigen that is useful in preparing a 4 malarial vaccine. The antigen is also a useful product of 5 this invention. The antigen is a polypeptide that can be 0 used in therapeutic quantities to prepare pharmaceutical ~ compositions which are suitable as malaria vaccines. The genes for encoding the three sexual stage-specific antigens of P. falciparum have not been cloned to date. 00 This is in part do to the fact that monoclonal antibody 11 defined: e=p±tαpes are dependent on disulfide bonds and large 22 quantities σf parasites and purified protein needed for 33 peptide sequencing are difficult to obtain. The gene of &4 this invention is obtained by purifying Pfs25 from zygotes 5 of P. falciparum. In the preferred embodiment of the 6 invention the 3D7 clone of NF54 P. falciparum is used. The 7 Pfs25 is purified by using immunoaffinity chromatography 8 " and SDS-PAGE. The microsequence of the protein is then 9 performed in order to make oligonucleotide probes to screen 0 ~ genomic DNA libraries.. The purified Pfs25 is digested 1 with trypsin, because the amino-terminus was blocked. The 2 resulting peptides are fractionated by HPLC. Five peptide 3 sequences are obtained from this process. These peptide 4 sequences are identified in Figure 1. 5 A highly degenerate oligonucleotide probe is 6 constructed from tryptic peptide sequence. This 7 oligonucleotide is used to clone a 600 bp Dra I fragment 8 (pS R 2) of genomic DNA. The Dra I fragment contains one 9 long open reading frame but no termination codon. 0 Therefore, a 3.5 kb Hind III fragment (ρNF4.13) is cloned. 1 Both cloned fragments can be used to determine the 2 nucleotide sequence of Pfs25. Figure 1 illustrates the amino acid sequence for Pfs25 as deduced by the above process. This amino acid sequence is shown above the nucleotide sequence. The gene has a single exon that codes for a polypeptide of 217 amino acids. The predicted coding region of the nucleotide sequence is capitalized and consists of 654 base pairs. The N-terminus is blocked; therefore, the start of the mature protein has not been determined. Solid lines represent tryptic peptide sequences determined by microsequencing. Asterisks represent the sequence determined by microsequencing radiolabellec peptides. The dotted line represents the indeterminate amino acid residue of the microsequenced peptide. The double solid line represents the tryptic peptide sequence used to construct oligonucleotide probes. The open circles represent the sites of possible asparagine-linked glycosylation. The broken lines represent the hydrophobic regions. First and most important in providing evidence that the gene for the the 25kDa ookinete surface antigen is cloned is that five of the microsequenced tryptic peptides of Pfs25 are found within the deduced amino acid sequence of P£s25 as shown in Figure 1. A preparation of (35s)-labelled ?fs25 can be immunopurified• from zygotes that had been metabolically labelled with 35s-methionine and 35g-cysteir.e. The resulting tryptic peptides of Pfs25 are separated by HPLC and the fraction containing the most radioactivity is microsequenced. The radioactive peaks for cysteine and methionine in the tryptic peptide of Pfs25 perfectly match the position of these residues in the deduced amino acid sequence of the Pfs25 gene of Figure 1 marked by asterisks. A malarial vaccine can be produced from the gene of Figure 1 or a portion thereof. The gene car. be modified using known techniques to delete the signal sequence, the 1 hydrophobic anchor, and/or other portions of the gene. 2 These modifications to the gene continue to produce a 33 suitable antigen for use in the vaccine. 4- ' Those skilled in the art can understand that certain 5" codons presented in the sequence of Figure 1 can be 6 substituted by other codons and produce an equivalent 7 segment. The polypeptide produced by the equivalent 8 segment has a corresponding change in its amino acid 9 - sequence, but has an equivalent function to the preferred 100 polypeptide. For example, a substitution of glutamic acid 111 £ό_r- gjLyc.uπe_ azt amino acid 131 does not alter the function 122 o ' the-- poϊypept de. 132 Second, the gene of this invention is expressed 14 preferentially in the sexual stages of P. falciparum. This 15 is the stage in which Pfs25 is synthesized. The mRNA from 16 gametocytes (not shown) and five hour old zygotes as 12. illustrated in Figure 2, lane 5, of P. falciparum using the 18S 3D7 clone of NF 54, have an abundance of an approximately 19S 1.4 kb species that hybridizes to pSKR 2. In contrast, the 20 mR.NA from asexual stage parasites of 3D7 as illustrated in 21 Figure 2, lane A, gives only a weak signal. The weak 22 signal from the asexual stage parasites is most likely due 23 to the presence of some gametocytes in the preparation of 24 parasites. For example pSKR 2 does not hybridize with the 25- mRNA.as illustrated in Figure 2, lane L, from a P^ 26 falciparum parasite that produces no gametocytes such as 27 the LF4 clone of a Liberian isolate of P. falciparum. 28 Third, when gametes, zygotes, or ookinetes of P^ 29 falciparum are metabolically labeled, multiple sexual stage 30 proteins incorporate 35g-. me thioni e and Pfs25 is not a 31 major radiolabelled product. In contrast, Pfs25 is the 32- predominate product incorporating 35g-cysteine as known by tests which are standard in the art. The deduced amino acid sequence contains 11 percent cysteine which correlates with these results. Fourth, the structure of the protein from the deduced amino acid sequence is consistent with previous reports as cited above of the biochemistry of the 25 kDa surface glycoprotein. The deduced sequence contains a putative signal peptide at the N-terminus and a short hydrophobic anchor at the C-terminus. It has four potential glycosylation sites for N-linked sugars and encodes for a polypeptide of approximately 24 kDa. The deduced sequence also has a short hydrophobic anchor of 15 amino acids and the lack of a potential cytoplasmic hydrophilic region at the C-terminus. The organization of the cysteines between the signal sequence and the hydrophobic C-terminus is similar to the domains in EGF 1 as illustrated in Figure 3B. Based on the position of the cysteines in the exons of human EGF precursor and human LDL receptor, three cysteine residues precede the consensus sequence Y/F-x-C x-C x-x--G-Y/F and one follows it as illustrated in Figure 3B. The EGF-like domain is also found in invertebrates such as notch in Drosophilia melanogaster and lin-12 in Caenorhabditis elegans. This invention provides the first report of the presence of EGF-like domains in proteins of unicellular organisms. The presence of EGF-like domains in the protein of this invention can be expected to have a growth factor effect on higher organisms including the mosquito. EXAMPLE The following example provides the procedure for obtaining the gene of the invention. This example represents the preferred embodiment of the invention. 2 A 3D7 clone of an NF 54 isolate of P. falciparum was ; cultured in vitro and zygotes were prepared as described - above. Pfs25 from Triton X-100 extracts of five hour old 2 zygotes (10 ) was immunoaffinity purified using monoclonal " antibody 1C7 covalently linked to Sepharose 4B beads (CnBr-activated) . Pfs25 had been etabolically labeled with trans S35 which has 707= 35s-methionine, 20% - 35s-cysteine, and 10% other 35s-compounds and is 0 commercially available from ICN Radiochemicals, Inc. The 11 be-a-ds- contained Pfs25 and were resuspended in SDS-PAGE 1 sample; bluffer having 5% SDS, 62.5mM Tris at pH 6.8, 0.002% 1 Bromophenol blue, and 8 M urea. These were heated at 68°C for five minutes. The eluted protein in the sample buffer 5 was loaded onto a 12% SDS-polyacrylamide gel under 6 nonreducing conditions. The Pfs25 was the only radiolabelled protein identified on the gel and was -1 recovered from the gel by passive diffusion. The 1 lyophilized sample was resolubilized in 0.5 M Tris-HCl pH 8.5, 6 M guanidine hydrochlorid , 0.3 mM EDTA buffer containing 64 mM dithiothreitol, and incubated for 2 hours at 37°C in a £ atmosphere. Iodoacetamide was added to a final concentration of 174 mM and reacted for 1 hour at 25°C in the dark. An excess of 2-mercaptoethanol was added followed by 10 volumes of absolute ethanol. The reduced and alkylated protein was allowed to precipitate at -20°C for 4 hours. The remaining pellet was resuspended in 50 mM NH4HCO3, pH 7.9 and digested with two 0.5 ug doses of TPCK treated trypsin (Sigma), each dose being followed by a 6 hour incubation at 37°C. The digestion was terminated by heating the sample for 10 minutes at 65oc The tryptic peptides were fractionated on a reverse phase HPLC (RP-300, Applied Biosystems, Inc.) using an 50% acetonitrile gradient with 0.1% trifluoracetic acid. Peptide microsequencing was performed on a model 470A gas phase sequencer from Applied Biosyste s, Inc. 40% of each cycle's product was analyzed on an attached model 120A PTH analyzer using the manufacturer's program, C3RPTH. For one peptide, the radioactivity of the remaining 60% of each cycle's product was determined. Pvadioactive peaks (***) were found in cycle numbers 1, 7, 12, and 17. The peptide sequence identified by double solid lines was used to construct highly degenerate oligonucleotide probes. A group of oligonucleotides of 512 degeneracy hybridized to a 1.4 kb band of zygote RNA. By varying the codon for proline, the probe was divided into four groups of 128 degeneracy each, one group of which hybridized to a 1.4 kb band of RNA as well as a 600 bp Dra I fragment of genomic DNA. 20,000 plaques, of a size-selected Dra I fragment library in lambda gt 10, were screened with this oligonucleotide probe. One clone was identified and subcloned into Blue-script SK (pSKR 2) . pSKR 2 was used to identify a 3.5 kb Hind III fragment in a size selected library in the vector pSP64. Both strands of each clone were sequenced by the dideoxynucleo ide terminator method. In Figure 2 the Northern blot analysis of asexual and sexual stage RNA is shown. Total cellular RNA (20 ug/lane) was prepared from asexual parasites (L) of LF 4, asexual parasites (A) of 3D7, or 5 hour old zygotes (S) of 3D7 were electrophores-ed through a 1% agarose/formaldehyde gel and transferred to a nylon membrane. The filter was hybridized at 55°C overnight with randomly primed pSKR 2 insert (specific activity 109 c.p.m. ug~l) and washed as described Size markers are 0.24-9.5 kb RNA ladder (BRL) . Figure 3 illustrates in part A protein structure of Pfs25 arranged to emphasize the relatedness of the EGF-like oma ns. e oxes represent cyste ne resi ues and other identical or related amino acids. Part B illustrates _ EGΕ^-like repeat consensus sequence from Pfs25, .lin-12, -- notch-,. EGF, and human LDL receptor. The boxes represent 5 cysteine. residues. The double box represents a core of 2 consensus sequence. The letters designate the following 7 amino acids: C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; I, 2 He; K; Lys; L, Leu; N, Asn; T, Thr; Y s Tyr; x, any amino acid; and unspecified number of a ino acids.

Claims: 1. A gene for expressing antigens for producing a human malaria vaccine consisting essentially of a cloned gene for encoding the 25 kDa surface protein or a portion thereof of zygotes and ookinetes of Plasmodium falciparum. 2. The coding region of said gene of claim 1 wherein said nucleotide segment is ATG AAT AAA CTT TAC AGT TTG TTT CTT TTC CTT TTC ATT CAA CTT AGC ATA AAA TAT AAT AAT GCG AAA GTT ACC GTG GAT ACT GTA TGC AAA AGA GGA TTT TTA ATT CAG ATG AGT GGT CAT TTG GAA TGT AAA TGT GAA AAT GAT TTG GTG TTA GTA AAT GAA GAA ACA TGT GAA GAA AAA GTT CTG AAA TGT GAC GAA AAG ACT GTA AAT AAA CCA TGT GGA GAT TTT TCC AAA TGT ATT AAA ATA GAT GGA AAT CCC GTT TCA TAC GCT TGT AAA TGT AAT CTT GGA TAT GAT ATG GTA AAT AAT GTT TGT ATA CCA AAT GAA TGT AAG AAT GTA ACT TGT GGT AAC GGT AAA TGT ATA TTA GAT ACA AGC AAT CCT GTT AAA ACT GGA GTT TGC TCA TGT AAT ATA GGC AAA GTT CCC AAT GTA CAA GAT CAA AAT AAA TGT TCA AAA GAT GGA GAA ACC AAA TGC TCA TTA AAA TGC TTA AAA GAA AAT GAA ACC TGT AAA GCT GTT GAT GGA ATT TAT AAA TGT GAT TGT AAA GAT GGA TTT ATA ATA GAT AAT GAA AGC TCT ATA TGT ACT GCT TTT TCA GCA TAT AAT ATT TTA AAT CTA AGC ATT ATG TTT ATA CTA TTT TCA GTA TGC TTT TTT ATA ATG TAA. 3. The gene of claim 2 wherein said nucleotide segment encodes said 25kDa surface protein of zygotes and ookinetes, said surface protein having a signal sequence, a hydrophobic C-terminus, and four tandem epidermal growth factor-like domains. 4. A malarial vaccine comprising said surface protein of claim 3. . synt et c pept e or pro uc ng a human malaria 2 vaccine consisting essentially of the 25 kDa surface 3J protein or a portion thereof of zygotes and ookinetes of 44 Plasmodium falciparum produced by a cloned gene. 5 " 6. The gene of claim 5 wherein said surface protein 6 has a deduced amino acid segment is 7 Met Asn Lys Leu Tyr Ser Leu Phe Leu Phe Leu Phe He Gin Leu 8 Ser He Lys Tyr Asn Asn Ala Lys Val Thr Val Asp Thr Val Cys 9) Lys Arg Gly Phe Leu He Gin Met Ser Gly His Leu Glu Cys Lys 10" Cys, Glu-Asn Asp Leu Val Leu Val Asn Glu Glu Thr Cys Glu Glu 11. Lys ■ Vail L nt Lys Cys Asp Glu Lys Thr Val Asn Lys Pro Cys Gly 12" Asp Phe Ser- Lys Cys He Lys He Asp Gly Asn Pro Val Ser Tyr 13 Ala Cys Lys Cys Asn Leu Gly Tyr Asp Met Val Asn Asn Val Cys 14 He Pro Asn Glu Cys Lys Asn Val Thr Cys Gly Asn Gly Lys Cys 15 He Leu Asp Thr Ser Asn Pro Val Lys Thr Gly Val Cys Ser Cys 16 Asn He Gly Lys Val Pro Asn Val Gin Asp Gin Asn Lys Cys Ser 17 ' Lys Asp Gly Glu Thr Lys Cys Ser Leu Lys Cys Leu Lys Glu Asn IS. Glu Thr Cys Lys Ala Val Asp Gly He Tyr Lys Cys Asp Cys Lys 19 ' - Asp Gly Phe He He Asp Asn Glu Ser Ser He Cys Thr Ala Phe 20 Ser Ala Tyr Asn He Leu Asn Leu Ser He Met Phe He Leu Phe 21 Ser Val Cys Phe Phe He Met. 2 7. A malarial vaccine comprising said surface protein 23 of claim 6. 4 ' 8. A pharmaceutical composition comprising the 25kDa 5 surface protein or portion thereof of zygotes and ookinetes 6 o± Plasmodium falciparum produced by a cloned gene, said 7 protein being in a therapeutically effective concentration 8 to provide a growth factor effect on higher organisms.

Inventors: KASLOW DAVID C, MILLER LOUIS H, KASLOW, DAVID, C., MILLER, LOUIS, H.
Applicants: US HEALTH, THE UNITED STATES OF AMERICA AS REPRESENTED BY THE
Application year: 1989
Main classification: C07K7
IPC codes: A61K38, A61K39, C07K14, C12N15