This application proposes to describe the variant and invariant properties of CSA-binding proteins expressed by the malaria parasite, and to understand the mechanisms by which women develop humoral immunity which prevents parasite adhesion to CSA. Specifically, we will identify var gene products, unique transcripts, and CSA-binding proteins expressed by the malaria parasite: each of these approaches provides the opportunity to identify molecules involved in parasite-CSA interactions. RT-PCR will be employed to amplify var gene products from placental field isolates defined as CSA-binding; these amplified transcripts will be compared with var genes expressed by field isolated obtained from non-pregnant donors. Subtraction will employ subtractive PCR and subtraction libraries to identify gene transcripts unique to CSA-binding parasites from the placenta (and therefore not common with CD36-binding parasites from non-pregnant donors); these and other expression libraries can be screened with labeled CSA or with sera from multigravid women. CSA-binding proteins will be purified on CSA affinity columns, then employed in assays to characterize function as well as obtain peptide sequence; this information will be used to design oligonucleotide probes to screen libraries for full length sequence. We will then determine conserved and variant properties of these molecules, explore their contribution to parasite adhesion in the placenta, and identify epitopes targeted by naturally occurring antibodies known to inhibit parasite adhesion to CSA. Sequence variation and conserved motifs will be explored by aligning the molecules identified by the above methods. This information will be placed in the context of binding function by examining recombinant protein fragments for evidence of adhesion. We hypothesize that CSA- binding molecules expressed by the malaria parasite can take variant or distinct forms, but that motifs involved in binding are conserved, or that epitopes targeted by anti-adhesion antibodies are conserved or limited in their degree of variation. Therefore, in conjunction with the sequence and functional characterization of these molecules, we will determine the epitopic specificity of antibodies which block parasite binding to CSA. This information will provide the first full characterization of tissue-parasite adhesion in an accessible tissue, and may provide a model to examine other malaria syndromes. Further, the delineation of binding domains and B cell epitopes will provide the basis to design an anti-adhesion vaccine to protect primigravid and other women susceptible to placental infection with the CSA-binding parasites.
