The goal of this project is to produce the full length outer-membrane proteins from different stages of the malaria parasite in a secreting yeast expression system. The purpose for the production of these recombinant proteins is to provide sufficient quantities of the outer-membrane proteins from both the sporozoite stage and the sexual stage of Plasmodium falciparum to test as vaccine candidates. Previous results have indicated that irradiated sporozoites injected into human subjects provide protection malaria challenge. Attempts at using the tetrapeptide repeats of the circumsporozoite protein (CSP), the major antigen on the infective sporozoite, resulted in limited success in generating antibody to the sequences, and failure in providing predictable protection to humans. It has 1, been postulated that other primary sequences in the CSP may be necessary as T-helper sites, hepatocyte receptor-binding sites or other B-cell epitopes. To study the function, immunogenicity, and potential suitability of the full length peptide for vaccine use, we have ligated the CSP gene from P. falciparum into a specially constructed vector which promotes maturation of the CSP through the plasma membrane of Saccharomyces cerevisiae. The advantage of this approach is not only to produce a full length peptide but also to produce a peptide which will not require detergent extraction, will have a more native conformation and secondary structure, and will contain post-translational modifications such as N-- glycosylation. We have found three forms of the recombinant DNA-derived CSP which have been secreted from the yeast cell (approx. 80% yield). ELISA and RIPA results indicate that these forms are antigenically similar to the authentic CSP. The major species of this protein, 50-55 kDa, does not appear to be glycosylated. A second antigen, the 25 kDa protein, represents a major antigen of the zygote land ookinete form of the sexual stage. A gene representing the truncated form of The 25 kDa protein is being cloned into a secreting yeast system and into an adenoviral vector. This recombinant protein may be important in stimulating transmission blocking antibodies in humans.
