Background and Aims
Plasmodium vivax invades reticulocytes exclusively to cause malaria in humans. The interactions between the parasite adhesin P. vivax reticulocyte binding protein 2b (PvRBP2b) with Transferrin receptor 1 (TfR1) enables host cell recognition, which is critical for successful invasion. We aim to isolate naturally acquired human monoclonal antibodies to PvRBP2b and map the epitopes bound by inhibitory antibodies to facilitate vaccine design.
Single B cells that recognized PvRBP2b were isolated using fluorescence activated cell sorting of peripheral blood mononuclear cells from adult Cambodian individuals. Antibody sequences were cloned and human monoclonal antibodies were expressed and purified by Protein A column affinity chromatography followed with size exclusion chromatography. Inhibition of the PvRBP2b-TfR1 interaction was examined using a flow cytometry-based red blood cell binding assay. Epitopes bound by the human monoclonal antibodies were mapped using a collection of PvRBP2b recombinant fragments. Competition ELISAs were performed using a panel of well characterized inhibitory mouse monoclonal antibodies to determine if the human antibodies have different structural modes of action.
Sequencing of 94 antibody heavy chains from PvRBP2b specific memory B cells from three Cambodian individuals has yielded 36 distinct clonal groups. Currently, we have expressed human monoclonal antibodies from 16 different clonal groups, four of which abolish PvRBP2b binding to reticulocytes and four that moderately inhibit PvRBP2b binding. Seven of these inhibitory human monoclonal antibodies compete with our collection of inhibitory mouse monoclonal antibodies whereas one human monoclonal antibody binds a completely new inhibitory epitope.
Interrogating the human antibody repertoire generated from natural infection of P. vivax has revealed the presence of several inhibitory monoclonal antibodies to PvRBP2b with different structural modes of action. The characterization of inhibitory epitopes will guide the rational design of vaccine antigens.