Project Summary. Monoclonal antibodies are approved as therapeutics for a number of diseases. Antibody development in the infectious disease setting typically starts with pathogen-specific antigen (PSAg) discovery, followed by raising of PSAg-specific antibodies in animal models. Fully human, PSAg-specific antibodies encoded by circulating memory B cells from clinically immune donors present many advantages over antibodies raised in animals. There is no need for antibody humanization and it enables isolation of affinity- matured antibodies co-evolved with their native target antigens. In the case of Plasmodium falciparum, the malaria parasite studied here, antibody discovery is associated with several important additional challenges. Members of the antigen family PfEMP1 have been identified as key targets of acquired protective antibody- mediated immunity to malaria, but are fraught with substantial intra-clonal and inter-clonal variation. This greatly complicates identification of broadly cross-reactive and neutralizing antibodies. Furthermore, there is no practical animal model for the study of PfEMP1-specific acquired immunity (P. falciparum only infects humans). Finally, the low-throughput technologies so far available for interrogating the functionality of PfEMP1-specific antibodies have severely hampered the identification of broadly reactive, neutralizing antibodies. Human monoclonal antibodies (huMabs) therefore remain a largely unexplored tool in malaria antigen discovery. To address these challenges, we propose to explore the utility of a new technology for the discovery of BCR sequences encoding PSAg-specific antibodies (Specific Aim 1) and to assess the cross-reactivity and functionality of such antibodies in vitro (Specific Aim 2). Specifically, we will apply the recently described LIBRA-seq technology, which enables high-throughput mapping of antibody sequence to antigen specificity, using a specific type of PfEMP1 (VAR2CSA) that is centrally involved in the pathogenesis of a specific and severe complication to P. falciparum infection, called placental malaria. Correspondingly, VAR2CSA-specific antibodies have been identified as the key mechanism of naturally acquired protection from placental malaria. Subsequently, we will generate VAR2CSA-specific recombinant huMabs with the sequences identified by LIBRA-seq to verify their cross-reactivity and assess the functionality of the recombinant antibodies in a range of in vitro assays. The reason that we focus the initial studies proposed here on placental malaria is that the target antigen of acquired protective immunity to placental malaria (VAR2CSA-type PfEMP1) is well established, and because placental malaria is a major cause of malaria-related mortality and severe morbidity among pregnant women and their offspring in areas where transmission of P. falciparum occurs. These initial efforts will show the utility of our proposed approaches for identification of broadly neutralizing PfEMP1-specific antibodies. They will therefore provide a framework for antibody discovery efforts in other severe forms of P. falciparum malaria, e.g., cerebral malaria, and therefore will have a broad impact.

Project Narrative We propose to apply a newly developed technology to identify and sequence the antigen receptors of memory B cells recognizing VAR2CSA-type Plasmodium falciparum malaria parasite antigen (PfEMP1). VAR2CSA is responsible for placental malaria (PM) pathogenesis and a key target of antibody-mediated PM immunity. We will also generate VAR2CSA-specific affinity-matured monoclonal antibodies and characterize them in terms of cross-reactivity with geographically diverse sequence variants of VAR2CSA. The project is likely to identify new candidates for new antibody-based diagnosis and therapy. In addition, it will provide a framework for antibody discovery in other types of severe malaria, e.g., cerebral malaria.