Plasmodium falciparum causes over 1-2 million deaths annually in tropical and subtropical regions of the world. The asexual cycle is responsible for the immune pathology and the clinical manifestations of malaria. Vaccine studies have demonstrated protection against challenge infections using rhoptry antigens as the immunogen. Moreover, immune sera from individuals in an area hyperendemic for Falciparum malaria reacts with rhoptry proteins by immunoblot and immunofluorescence suggesting the availability of rhoptry proteins for immune attack in the infected host. We have cloned and sequenced DNA encoding the 3' end of Rhop-3/110 kd. The rationale for isolating Rhop-H genes is that regions responsible for erythrocyte binding and invasion can be more precisely mapped. Therefore, the goal of this study is to investigate the biological properties of the rhoptry protein complex of 140/130/110 kd (Rhop-H), and to determine their individual roles in erythrocyte recognition as a means of identifying erythrocyte components mediating rhoptry protein interaction. The initial objective of the study will be to screen a cDNA expression library from asexual stages using monospecific antisera prepared against the affinity purified rhoptry protein complex, Rhop-H. Protein products of the expression clones will be used in biological characterizations to determine the presence of erythrocyte binding and invasion specific domains. We will extend our characterizations of the encoded Rhop-3 recombinant protein by subcloning the isolated DNA into expression vectors to obtain recombinant proteins for use in biological characterizations and for production of monospecific antisera. The antisera will be tested for inhibitory activity in P. falciparum in vitro invasion assays to determine how the components function individually and together to promote parasite invasion. Hybridization analysis will be performed to determine the presence of multiple gene copy number, and the size of the mRNA encoding the proteins will be verified. The proposed study is part of a long term research goal to understand the biochemical and molecular basis for rhoptry protein interaction with the erythrocyte, and how immunity to these proteins might be utilized for vaccine development. The following specific aims will serve as guidelines to the research: Isolation and characterization of genes encoding Rhop-H proteins. 2. Expression of recombinant proteins, preparation of monospecific antisera and characterization of immunological responses for verification of expression product. 3. Biological characterization and functional significance of recombinant and native rhoptry proteins.
