Plasmodium falciparum is the causative agent of the most severe form of malaria and accounted for almost all the 619,000 reported malaria deaths in 2021 (World Health Organisation, 2022). Parasite invasion of human erythrocytes is a complex multi-step process that ends with parasite internalisation. An essential step in this process involves leading blood-stage vaccine candidates, the ligand PfRh5 which forms a complex with the Cysteine-Rich Protective Antigen (CyRPA) and the PfRh5-interacting protein (PfRipr; RCR) and binds to basigin on the host erythrocyte. Recently, we showed that a disulphide-linked heterodimer consisting of the Plasmodium Thrombospondin-Related Apical Merozoite Protein (PfPTRAMP) and the Cysteine rich Small-Secreted protein (PfCSS; PTRAMP-CSS) binds RCR to form a pentameric complex, PCRCR, which anchors the parasite to the erythrocyte membrane during invasion (Scally et al., 2022). Invasion inhibitory nanobodies to both PTRAMP and CSS were identified but only had moderate efficacy. To further the rational design of PTRAMP-CSS immunogens, potent inhibitory epitopes on PTRAMP and CSS need to be identified.
Previous efforts have isolated nanobodies that were raised against the monomeric proteins, and to extend this we immunised alpacas with the PTRAMP-CSS heterodimer to determine if these nanobodies have neutralising potential. We have screened > 20 nanobodies and have identified three potent inhibitory nanobodies against PTRAMP-CSS. Using X-ray crystallography, we have identified novel neutralising and non-neutralising epitopes on PTRAMP and CSS. Through understanding the molecular definition of potent neutralising epitopes on PTRAMP-CSS a roadmap is provided for structure-guided development of these proteins for a blood-stage malaria vaccine that aims to relieve the disease’s current global burden.