The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.AMA1-RON2 | malaria | moving junction A picomplexa are a group of parasitic protozoa, including the human malaria parasite Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), that use apical secretory organelles to invade host cells. Invasion of Plasmodium spp. merozoites into erythrocytes begins with an initial weak attachment of the merozoite to the red blood cell (RBC) surface through yet-unidentified parasite receptor-RBC ligand interactions, followed by a reorientation that ultimately brings the apical end of the merozoite into close apposition with the RBC surface (1, 2). The merozoite then triggers the formation of a junction with the erythrocyte that by electron microscopy appears as a dense area below the erythrocyte membrane at the site of the merozoite's apposed apical end. In addition, the merozoite secretes its rhoptry contents into the RBC that may facilitate the invasion of the merozoite (2-4). The merozoite subsequently moves through the junction as it pulls itself into the RBC through connections between parasite surface proteins and its actin-myosin motor (5). Hence, the formation of the junction and its connection with the molecular motor through the cytoplasmic tail of parasite receptors is critical for invasion (6, 7). Formation of the parasitophorous vacuole, created by the inward flow of the RBC membrane (8-10), occurs coordinately with the entry of the parasite into the RBC (4). At the end of invasion, the electron-dense junction becomes part of the parasitophorous vacuole that surrounds the newly invaded parasite (2). In cytochalasin-treated merozoites where actin polymerization is disrupted, the parasites apically ...

Summary The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.

SummaryAntigenic diversity has posed a critical barrier to vaccine development against the pathogenic blood-stage infection of the human malaria parasite Plasmodium falciparum. To date, only strain-specific protection has been reported by trials of such vaccines in nonhuman primates. We recently showed that P. falciparum reticulocyte binding protein homolog 5 (PfRH5), a merozoite adhesin required for erythrocyte invasion, is highly susceptible to vaccine-inducible strain-transcending parasite-neutralizing antibody. In vivo efficacy of PfRH5-based vaccines has not previously been evaluated. Here, we demonstrate that PfRH5-based vaccines can protect Aotus monkeys against a virulent vaccine-heterologous P. falciparum challenge and show that such protection can be achieved by a human-compatible vaccine formulation. Protection was associated with anti-PfRH5 antibody concentration and in vitro parasite-neutralizing activity, supporting the use of this in vitro assay to predict the in vivo efficacy of future vaccine candidates. These data suggest that PfRH5-based vaccines have potential to achieve strain-transcending efficacy in humans.

Minassian et al. report that the RH5.1/AS01 B vaccine against blood-stage Plasmodium falciparum malaria is safe and immunogenic in a phase I/IIa clinical trial. They demonstrate a significantly reduced blood-stage parasite growth rate in vaccinees following controlled human malaria infection and identify that in vitro antibody-mediated growth inhibition activity is associated with challenge outcome.

are named inventors on patent applications relating to RH5 and/or other malaria vaccines and immunization regimens. L. Siani and S. Di Marco are employees of ReiThera (formerly Okairos), which is currently developing vectored vaccines for a number of diseases. J. Vekemans was an employee of GSK, which has acquired the ChAd63 vector from Okairos. R. Ashfield is a director of Ducentis and holds shares in the company, which is developing a therapy for autoimmune disease. A.M. Minassian has an immediate family member who is an inventor on patents relating to RH5 and/or other malaria vaccines and immunization regimens and who is a cofounder of, shareholder in, and consultant for SpyBiotech. S. Biswas is a cofounder and CEO of, and shareholder in, SpyBiotech and is a contributor in a patent application relating to multimerisation technology. J. Jin is a cofounder of and shareholder in SpyBiotech.