One approach toward development of a malaria vaccine is to elicit antibodies to the sexual stages of the malaria parasite, so that when an infectious blood meal is taken by the mosquito, the development of the sexual stages, and thereby transmission, of the parasite is blocked. Surface radio-iodination of mosquito midgut zygote stage proteins of the malaria parasite, Plasmodium falciparum, identifies three predominant proteins: Pfs230, a doublet Pfs48/45, and Pfs40. An additional, abundant zygote surface protein, Pfs25, is not efficiently labeled by iodination. Immune sera and mAbs recognizing these proteins reduce or block infectivity of malaria parasite sexual stages to the mosquito in membrane-feeding assays; therefore, all are candidates for study as transmission-blocking subunit components of a malaria cocktail vaccine. Undoubtedly, additional target antigens have yet to be identified. For example, with the rapid progress in the Malaria Genome Sequencing Project, it is possible to identify potential malaria vaccine targets by screening the databases for genes encoding likely extracellular proteins. Based upon putative protein domain structure it is possible to prioritize surface proteins, followed by lifecycle stage of expression studies to identify sexual stage candidates. It is important to critically evaluate the malaria parasite genome database for vaccine candidates - in addition to drug targets - and then transfer the knowledge gained to recombinant DNA-based and protein subunit-based vaccine studies. Development of a fertilization assay, at present unavailable, will aid in the characterization of sexual stage proteins and accelerate studies on the biology of the mosquito midgut stages of the Plasmodium lifecycle.DNA microarrays offer an emerging technology for tracking global changes in gene expression in cells under different physiological conditions. DNA microarrays are a logical complement to the Malaria Genome Project, to aid identification of sexual stage-specific gene expression. In a collaborative effort, a shot-gun microarray was constructed from a P. falciparum mung-bean nuclease genomic library and screened with cDNAs from asexual and gametocyte blood stage parasites. Differential gene expression between the asexual, trophozoite form of the parasite and the sexual, gametocyte form (stages IV-V) was described for 3,648 genes which were arrayed and screened. Immediately, this prototype array offers an unprecedented number of leads for transmission-blocking agents and for vaccines directed at blood stage antigens. Apicomplexan protozoa possess a family of micronemal and cell surface receptors composed of combinations of vertebrate von Willebrand factor-like A domains and thrombospondin type 1-like domains. In the malaria parasite, Plasmodium falciparum, the gene CTRP has 6 tandemly arrayed A domains followed by 7 thrombospondin type 1 domains. We have shown that CTRP is expressed in the micronemes of ookinetes, the motile stage that traverses and exits the mosquito midgut. Disruption of the P. falciparum CTRP gene by homologous integration demonstrates that it is essential during the ookinete-to-oocyst transition. Feeding of sexual stage CTRP disruptant lines to Anopheles mosquitoes results in production of mature ookinetes. Midgut wall oocysts, however, were never observed. CTRP is similar in structure to Plasmodium sporozoite-expressed TRAP, and we propose that CTRP similarly functions as a molecular winch in ookinete gliding motility, linking extracellular adhesion with an intracellular myosin/actin cytoskeletal motor. The differences in domain structure of CTRP and TRAP may reflect a distinction in extracellular binding targets within the midgut bloodmeal, the chitinous peritrophic membrane or the midgut epithelium. We are currently investigating CTRP as a potential transmission-blocking antigen. In general, the CTRP disruptions study serves as a technical model for further disruption studies with surface proteins identified via microarray or malaria genome database screens. - Malaria, Plasmodium falciparum, sexual stages, mosquito, transmission-blocking vaccine, gametocytes, ookinete, CTRP, microarray
