Malaria parasites are haploid for most of their life cycle, with zygote formation and meiosis occurring during the mosquito phase of development. The parasites can be analyzed genetically by transmitting mixtures of cloned parasites through mosquitoes to permit cross-fertilization of gametes to occur. A cross was made between two clones of Plasmodium falciparum differing in enzymes, drug sensitivity, antigens, and chromosome patterns. Parasites showing recombination between the parent clone markers were detected at a high frequency. Novel forms of certain chromosomes, detected by pulsed-field gradient gel electrophoresis, were produced readily, showing that extensive rearrangements occur in the parasite genome after cross-fertilization. Since patients are frequently infected with mixtures of genetically distinct parasites, mosquito transmission is likely to provide the principal mechanisms for generating parasites with novel genotypes.

Malaria vaccines are being developed against different stages in the parasite's life cycle, each increasing the opportunity to control malaria in its diverse settings. Sporozoite vaccines are designed to prevent mosquito-induced infection; first generation recombinant or synthetic peptide vaccines have been tested in humans. Asexual erythrocytic stage vaccines, developed to prevent or reduce the severity of disease, have been tested in animals and in humans. A third strategy is to produce sexual stage vaccines that would induce antibodies which would prevent infection of mosquitoes when ingested in a bloodmeal containing sexual stage parasites. Although not directly protective, the sexual stage vaccine combined with a sporozoite or asexual stage vaccine (protective component) could prolong the useful life of the protective component by reducing transmission of resistant vaccine-induced mutants. In areas of low endemnicity, the sexual stage vaccine could reduce transmission below the critical threshold required to maintain the infected population, thereby assisting in the control or eradication of malaria. Transmission of Plasmodium falciparum, the major human malaria, can be blocked by monoclonal antibodies against three sexual stage-specific antigens. We have cloned the gene encoding the surface protein of relative molecular mass Mr 25,000 (25K; Pfs25), expressed on zygotes and ookinetes of P. falciparum. The deduced amino-acid sequence consists of a signal sequence, a hydrophobic C-terminus, and four tandem epidermal growth factor EGF-like domains.

SummaryPfs25 is a sexual stage antigen of Plasmodiumfakiparum that is expressed on the surface of zygote and ookinete forms of the parasite. Monoclonal antibodies directed against native Pfs25 can block completely the development ofP. fakiparum oocysts in the midgut of the mosquito vector. Thus, this 25-kD protein is a potential vaccine candidate for eliciting transmission-blocking immunity in inhabitants of malaria endemic regions. We have synthesized, by secretion from yeast, a polypeptide analogue of Pfs25 that reacts with conformation-dependent monoclonal antibodies, and elicits transmission-blocking antibodies when used to immunize mice and monkeys in conjunction with a muramyl tripeptide adjuvant. Our results suggest the further evaluation of recombinant DNAo derived Pfs25 in transmission-blocking vaccination studies in humans. The increasing resistance to chemotherapy of the parasites responsible for human malaria has reaffirmed the requirement for an effective malaria vaccine. Subunit vaccine strategies using either peptide synthesis or recombinant DNA technologies have been focused primarily on the immunization of the human host against asexual forms of the parasite. For example, well-studied vaccination approaches have targeted the induction of neutralizing responses against the sporozoite form of the parasite that is injected by the mosquito vector during a blood meal. Also, merozoite and erythrocytic-stage antigens have been considered as immunogens for the suppression of parasitemia in susceptible individuals. An alternative approach for the control of endemic malaria in devdoping countries is that of transmission-blocking vaccination. Here, it has been proposed that such a strategy could prevent the transmission of viable sexual forms of the parasite from humans to mosquitoes. Foremost among transmission-blocking vaccine candidates is the Plasmodium fakiparum surface protein Pfs25. This 25-kD polypeptide is expressed on the surface of zygote and ookinete forms of the parasite as they develop in the midgut of the mosquito vector (1). mAbs to Pfs25 have been shown to block fully the development of sexual stages of the parasite (1, 2), and vaccinia virus recombinants that express Pfs25 can induce transmission-blocking antibodies in MHC-disparate congenic mice (3). Such studies have suggested that the production of large quantities of synthetic Pfs25 peptides, or recombinant Pfs25 protein, together with recreation of the appropriate conformation orB cell epitopes for immunological activity in vivo are required for the development of Pfs25 as a human vaccine. Here, we show that an analogue of Pfs25, synthesized by secretion from yeast, reacts with conformation-dependent mAbs, and can elicit transmission-blocking antibodies when used to immunize mice and monkeys in conjunction with a muramyl tripeptide (MTP) adjuvant. Materials and MethodsDNA Manipulations. Oligonucleotides were synthesized by the phosphoramidite method using DNA synthesizers (380]3; Applied Biosystems, Inc., Foster City, CA). Gene assem...

Protective immunity against malaria can be obtained by vaccination with irradiated sporozoites. The protective antigens known as circumsporozoite (CS) proteins, are polypeptides that cover the surface membrane of the parasite. The CS proteins contain species-specific immunodominant epitopes formed by tandem repeated sequences of amino acids. Here it is shown that the dominant epitope of Plasmodium falciparum is contained in the synthetic dodecapeptide Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Pro or (NANP)3. Monoclonal antibodies and most or all polyclonal human antibodies to the sporozoites react with (NANP)3, and polyclonal antibodies raised against the synthetic peptide (NANP)3 react with the surface of the parasite and neutralize its infectivity. Since (NANP)3 repeats are present in CS proteins of P. falciparum from many parts of the world, this epitope is a logical target for vaccine development.