The apical organelles of Plasmodium falciparum merozoites, including the rhoptries and micronemes, are thought to play a role in erythrocyte invasion. The broad, long-term objective of this proposal is to test the hypothesis that rhoptry proteins, or parts of these proteins, may elicit protective or inhibitory anti-malarial antibodies when used as a vaccine in humans.
The specific aims of the project are threefold: 1. Identify the substantially invariant regions of rhoptry proteins by DNA sequence analysis: Using available sequence information, sequence the genes for selected rhoptry proteins from culture-adapted isolates of P. falciparum. From the deduced amino acid sequences, identify conserved and variable regions for these proteins among the different isolates. Analyze the genes from field isolates of the parasite following polymerase chain reaction amplification of the DNAs. 2. Identify inhibitory epitopes in vitro: Raise polyclonal antibodies and mouse monoclonal antibodies to recombinant (or authentic) rhoptry proteins. Assay the ability of these antibodies to inhibit in vitro growth of P. falciparum. Map the binding sites for the inhibitory antibodies. 3. Assess the diversity of the human immune response to the rhoptry antigens: Assay human immune sera for antibodies which bind the rhoptry proteins. Quantitate the in vitro response of primed human T cells by measuring cytokine release and 3H-thymidine incorporation after presentation of purified rhoptry proteins. These studies will initially concentrate on the 82 kDa rhoptry protein to: determine the frequency at which individuals develop anti-p82 antibodies in response to falciparum malaria; map epitopes on p82 which are inhibitory; assess whether these epitopes are conserved in different parasite isolates; and determine whether malaria-primed human T cells recognize p82. The results should identify functional domains of this rhoptry molecule, and the methods used should be invaluable for assessing the suitability of rhoptry proteins for a subunit, anti-malarial vaccine.
