Project Summary The protozoan parasite Plasmodium is the causative agent of malaria, which remains one of the most prominent public health challenges in the world today. Antibodies play a primary role in protection from severe disease caused by blood-stage infection. However, repeated infections over an extended period are required to induce protective humoral immunity. It is unclear if protective antibodies are derived from short-lived plasmablasts through an extrafollicular response or long-lived plasma cells via the germinal center. Induction of the latter is the primary goal of effective vaccines. While Plasmodium-specific antibodies are detectable in the serum of individuals with clinical malaria, the cell biology underlying these antibody responses remains unknown. Here we propose developing a Plasmodium-specific B cell tetramer to track antigen-specific B cell responses to close gaps in our understanding of the humoral response to Plasmodium. Using the blood-stage antigen, merozoite surface protein 2 (MSP2), a candidate antigen for a blood-stage malaria vaccine, we will generate two B cell tetramers, one containing a full-length recombinant P. falciparum MSP2 protein and another encompassing a chimeric PfMSP2/8 protein. The former can form fibrils in vitro, while the latter does not. Hence, we predict the chimeric protein will offer such benefits as increased stability over time and the identification of a greater number of MSP2-specific B cells that bind a broader number of epitopes across PfMSP2 than a tetramer composed solely of rPfMSP2. In sub-aim 1, we will monitor MSP2 fibril formation and determine each B cell tetramer's ability to recognize antigen-specific B cells ex vivo after immunization of mice with either rPfMSP2 or rPfMSP2/8. Then in sub-aim 2, we will confirm the specificity of the B cells for binding MSP2 by ELISA through the generation of monoclonal antibodies based on the B-cell receptor sequences of tetramer positive B cells. Confirmation of the B cell tetramers' binding specificity is essential before using them to identify MSP2-specific B cells in the blood of individuals from malaria endemic areas or individuals participating in vaccine studies.