Identification and immunological characterization of the ligand domain of Plasmodium vivax reticulocyte binding protein 1

Background: Erythrocyte invasion by malaria parasites is essential for blood stage development and is an important determinant of host range. Parasite proteins that play a role in erythrocyte invasion are considered potential vaccine targets. Plasmodium vivax has a preference for invading human reticulocytes. Preference for this blood cell type is typically attributed to the reticulocyte-binding protein family (PvRBP), which mediate reticulocyte invasion by interacting with specific yet unknown receptors on the reticulocyte surface. Thus targeting the RBPs could prevent blood stage infection and disease. The RBPs are very large in size (250-300kDa), and very little is known about the functional domains of these proteins. A major challenge therefore is identifying the precise targets of neutralizing antibodies for inclusion in a vaccine. This study aims at immunologically and functionally characterizing the first member of this gene family (RBP1). Materials and methods: Recombinant proteins were produced from overlapping fragments ofRBP1 and their binding specificity to human erythrocytes determined using standard in vitrobinding assays. Immunogenicity in laboratory animals as well as naturally acquired antibody responses to these antigens was also evaluated using serum samples from individuals in endemic regions. Results: We have defined the specific binding domain of RBP1. We determined an N-terminal extracellular region, RBP1157-650 (RBP1-F8), as the specific binding domain for this protein. Recombinant RBP1-F8binds to both reticulocytes and normocytes, with a preference for immature reticulocytes. Mice and rabbits immunized with rRBP1-F8 generate high titer antibodies and these antibodies blocked RBP1-F8 erythrocyte binding. Most importantly, naturally acquired anti-RBP1 antibodies are prevalent in serum of P. vivax exposed individuals from Papua New Guinea (PNG) and Brazil, and these antibodies have anti-RBP1-F8 binding activity. Conclusion: Our data indicates that RBP1-F8 is naturally immunogenic and strongly supports its inclusion as candidate for a blood-stage vivax malaria vaccine. Further investigation is necessary to identify the specific epitopes and essential residues that make up the RBP1 ligand domain and its interacting partner(s) on human erythrocytes.