New antigens appear on the surface of Plasmodium falciparum-infected erythrocytes (IEs). The new antigens may mediate essential functions in the parasite's life cycle and are potential targets of host immune responses leading to the death of intracellular parasites. Some antigens differ between isolates, others are conserved. The research objectives of this proposal are to identify and characterize IE surface antigens at the molecular level and to determine the genetic mechanisms which regulate their expression. The principal focus wil be on IE surfaced molecules involved in cytoadherence. A conserved antigen on the surface of IEs would be an attractive immunogen for vaccine-induced immunity to erythrocytic parasites.
The specific aims are: (1) To establish cloned parasite lines from different isolates and to manipulate the knob, cytoadherence, and antigen phenotypes of these cloned lines in vitro. The selective techniques of suspension in gelatin (for knobs), binding to melanoma cells (for cytoadherence), and culture in the presence of specific antibody (for antigenic change) will be used. (2) To isolate cDNA clones encoding the cytoadherence ligand/antigen. Lambda gt 11 cDN expression libraries will be screened with a panel of immunological reagents including monoclonal and polyclonal antibody; with thrombospondin and platelet glycoprotein, which are the putative receptors for IEs on endothelial cells; and with total cDNA probes from phenotypially different clones. (3) To prepare monospecific antibody to fusion proteins by the antibody select techniques and touse antibody to identify the native protein, confirm localization of the antigen on the surface of IEs, and to determine the function of the antigen. (4) To isolate cytoadherence antigen genes from two cloned parasite lines and to compare their structure by restriction mapping and DNA sequencing. The hypothesis that variant (immunogenic) and conserved (functional) domains exist in the gene will be tested. (5) To determine the copy number, genomic organization, chromosomal location, and linkages of cytoadherence genes, and to define the changes in genomic organization which accompany changes in knob, cytoadherence, and antigenic phenotype. The techniques of genomic and pulsed field Southern analyses employing cDNA and cloned genes as hybridization probes will be used.
