High Throughput Analysis of Malarial Surface Antigens

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DESCRIPTION (provided by the applicant): The broad objective of this project is to develop array technologies to study the antigenicity and function of surface proteins of Plasmodium falciparum, the most virulent malaria species infecting humans. The variant surface protein PfEMPI is a key virulence molecule of P. falciparum that mediates parasite-infected erythrocyte adhesion as well as antigenic variation, and distinct forms of PfEMPI have been implicated in the pathogenesis of severe malaria and pregnancy malaria. Despite its key role in disease and protection, studies of PfEMPI variants are limited owing to its complexity, large size, and difficulty in expression. Sequencing of P. falciparum strain 3D7 genome is complete, and provides the basis for applying high throughput technologies to all gene products of interest. We will clone all constituent domains of 59 genes encoding PfEMPI in strain 3D7, as well as other current vaccine candidate antigens. We will determine the most efficient method for expression and preservation of antibody reactivity of these antigens, using cell-free translation and cell-surface expression. Antibody reactivity of recombinant antigens will be validated under native and denaturing conditions, using naturally acquired antibody from immune sera as well as conformation-sensitive monoclonal antibodies. Antigen arrays will be constructed using expressed proteins arrayed by self-assembling or spotting technologies, and compared with in situ cell-transfection or cell- spotting technologies on the surface of glass slides. Pilot studies will test the antigen arrays with serum samples collected in a longitudinal cohort study in Tanzania, and these will demonstrate the feasibility of protein array platforms for larger malaria immuno-epidemiology studies in the future. In addition, the conformation of recombinant antigens will also be validated in adhesion experiments with endothelial receptor molecules using arrayed PfEMPI domains. These experiments may also identify additional PfEMPI domain binding properties that could be investigated in detail in future studies. Immunological signatures or antibody profiles obtained from these assays may provide a rapid, efficient and systematic approach to identify candidate antigens for vaccines. In future, protein arrays can be expanded to the entire proteome, and used for functional studies including adhesion, protein-protein interactions, and variant-specific acquisition of antibodies against polymorphic antigens.