Structural basis for neutralization of Plasmodium vivax by naturally acquired human antibodies that target

1Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO, USA. 2Center for Global Health and Diseases, Case Western Reserve University, Cleveland, OH, USA. 3Department of Chemistry, Washington University in St Louis, St Louis, MO, USA. 4Department of Parasitology, University of Sao Paulo, Sao Paulo, Brazil. 5Malaria Molecular Epidemiology Unit, Pasteur Institute in Cambodia, Phnom Penh, Cambodia. 6Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. 7Department of Global Health, College of Public Health, University of South Florida, Tampa, FL, USA. 8Department of Chemistry, Washington University in St Louis, St Louis, MO, USA. 9Present address: Bioproduct Research and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA. *e-mail: niraj.tolia@nih.gov Malaria remains a life-threatening disease causing high morbidity and mortality1. Despite the long history of human battle against malaria, a viable vaccine is still desperately needed but remains elusive. This is in spite of the recognition that adults in malaria-endemic areas develop relative immunity to malaria infection2–4. Further, passive transfer of ɣ-globulins isolated from serum of patients exposed to malaria to infected non-immune children less than five years of age, demonstrated a significant reduction in parasitaemia2,5–7. Defining the structural correlates of naturally acquired immunity (NAI) is a critical factor for the design of a universal vaccine. The Plasmodium vivax Duffy-binding protein (DBP) is the most promising vaccine candidate for P. vivax malaria8–21. During reticulocyte invasion, P. vivax uses a Duffy-binding-like (DBL) domain in DBP, also known as region II (DBP-II) to engage Duffy antigen receptor for chemokines (DARC) on host reticulocytes8–12,14–18. DBP-II binds DARC via receptor-induced ligand dimerization, sandwiching DARC residues 19–30 between two DBP-II molecules17,18. DBP-II comprises three subdomains (1 to 3); subdomain 2 (SD2) is responsible for dimerization and receptor binding, which are required to engage DARC17,18. Rabbit and human antibodies that block the DBP–DARC interaction neutralize P. vivax, suggesting that a DBP-based vaccine will reduce infection5. However, the successful design of a DBP-II-based vaccine may be limited by strain-specific immune responses due to the polymorphic nature of DBP22,23 and the presence of immunodominant but non-protective epitopes within DBP24,25. Despite the polymorphic nature of DBP, broadly conserved epitopes of three inhibitory murine monoclonal antibodies have been identified in subdomain 3 of DBP-II19. These epitopes are distant from the dimer interface and DARC-binding site19. Furthermore, human vaccination with DBP-II elicits antibodies that block in vitro binding of four alleles of DBP to DARC, suggesting that broadly neutralizing epitopes of human antibodies may exist within DBP-II20,21. The identification of broadly conserved human neutralizing-antibody epitopes that contribute to naturally acquired immunity is essential for the improved rational design of potent strain-transcending DBP-based vaccines. Here we present the study of DBP-II in complex with two human neutralizing monoclonal antibodies, 053054 and 092096. These human monoclonal antibodies were produced by sorting individual DBP-II-specific B cells from a Cambodian donor with naturally acquired DBP-II-blocking antibodies and isolating, sequencing and cloning the variable regions from human ɣ-immunoglobulin (IgG) heavy and light chains. Structures of DBP-II–antibody complexes were determined by X-ray crystallography, and epitopes were further mapped by hydrogen–deuterium exchange mass spectrometry (HDX-MS) and mutational studies. Both antibodies inhibit binding of DBP to red blood cells, and 092096 neutralizes P. vivax in ex vivo Structural basis for neutralization of Plasmodium vivax by naturally acquired human antibodies that target DBP