Nanobodies against Plasmodium vivax AMA1 block parasite invasion

Plasmodium vivax is the most widespread malaria species to infect human populations. P. vivax invades young red cells called reticulocytes to replicate and proliferate. An essential protein in parasite invasion is the Apical Membrane Antigen 1, PvAMA1, which forms the tight junction between parasite and red cell membrane through its interaction with Rhoptry Neck Protein 2, PvRON2. Here we report the identification of PvAMA1 nanobodies using phage display in combination with Sanger sequencing, next-generation sequencing and structure prediction tools. We show that these nanobodies bind with high affinities and across several different epitopes on PvAMA1. Several nanobodies recognize PvAMA1 in merozoites of late-stage schizonts and inhibit parasite invasion of red blood cells. These growth inhibitory nanobodies bind to conformational epitopes of PvAMA1 and most bind to a site distinct from a known potent human monoclonal antibody 826827. Crystal structures of two inhibitory nanobodies in complex with PvAMA1 show that they bind novel neutralizing epitopes which are outside the hydrophobic groove that contacts PvRON2. Furthermore, we show that two other PvAMA1 nanobodies also inhibit Plasmodium falciparum invasion. These results demonstrate the potential of PvAMA1 nanobodies to inhibit malaria parasite invasion across multiple Plasmodium species.