Targeting RIPR: a protein central to host cell invasion in Plasmodium knowlesi and Plasmodium vivax two neglected human malaria parasites.

Malaria is a significant and continuing health problem facing half of the world’s population resulting in ~600,000 mortalities annually, predominantly children. The causative agents are Plasmodium parasites, which are transmitted by mosquitoes and replicate in red blood cells (RBCs) of their host. Like other vector-borne diseases, malaria is predicted to increase its spread with the effects of climate change, making a meaningful intervention critical. Of the six human-infecting species Plasmodium falciparum (Pf) is the best studied; it is widespread, especially in Africa and most virulent. Groundbreakingly, two malaria vaccines targeting Pf have recently been approved. However, no vaccines are available against the prevalent Plasmodium vivax (Pv) which dominates outside of Africa or the South-East Asian parasite Plasmodium knowlesi (Pk). Neglected human malaria parasites Pk and Pv have proven difficult to control which hinders ongoing malaria eradication efforts. We are molecular parasitologists/vaccinologists targeting blood stages of Pf, Pv, Pk and have developed controlled human malaria infection trials (CHMI) allowing us to quickly progress vaccine candidates from the bench to clinical testing. As Pv cannot be maintained in vitro we established a transgenic parasite model using the closely related Pk to express orthologous Pv genes. Using CRISPR-Cas9 we can modify Pk genes to generate knockouts, whole gene or domain swops. Our combined expertise allows us to test antigens both as targets for neutralising antibodies and to explore their functional conservation for RBC invasion across Plasmodium species boundaries with the aim to translate these discoveries into cross-protective vaccines. A pentameric protein complex, comprised of PTRAMP, CSS, RIPR, CyRPA and RH5 called PCRCR is critical for invasion of RBCs by Pf and we recently identified part of this complex’s structure. The complex binds to basigin on the RBC via RH5. This interaction is essential for invasion. RH5 is the leading Pf blood-stage vaccine candidate with neutralising antibodies blocking RH5 from recognising basigin and recently showing efficacy against clinical malaria in young African children for the first time. However, RH5 is exclusive to Pf, whereas the other protein complex components show conservation in Pv and Pk. Preclinical testing of PfRIPR identified growth inhibitory epitopes within the RIPR tail, since validated by human vaccination in a Phase 1 clinical trial. We further discovered that PkRIPR is part of a protein complex with PkCSS and PkPTRAMP and essential for RBC invasion. Antibodies targeting PkRIPR also blocked RBC entry. This emphasises RIPR’s functional conservation for RBC entry across all Plasmodium species studied to date. Our proposal addresses the following complementary aims: 1.Validate and functionally explore additional protein interactors of the PkPCR+ complex and their conservation between Pk and Pv. 2.Characterise species-specific and cross-protective neutralising epitopes in PkRIPR and PvRIPR. 3.Define mechanisms how neutralising RIPR-specific mAbs block RBC entry by merozoites. This project will use reverse genetics, cell biology, protein biochemistry, proteomics and vaccinology approaches using Pk as a model for Pv. The study outcome will result in discoveries detailing the composition and essential function of the PCR+ complex for RBC invasion across Plasmodium species, which will significantly advance the generation of a Pv/Pk cross-protective malaria vaccine.