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 ...

The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R h 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21-25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.

Transmission-blocking vaccines (TBVs) that target sexual stage parasite development could be an integral part of measures for malaria elimination. Pfs25 is a leading TBV candidate, and previous studies conducted in animals demonstrated an improvement of its functional immunogenicity after conjugation to EPA, a recombinant, detoxified ExoProtein A from Pseudomonas aeruginosa. In this report, we describe results of an open-label, dose-escalating Phase 1 trial to assess the safety and immunogenicity of Pfs25-EPA conjugates formulated with Alhydrogel®. Thirty malaria-naïve healthy adults received up to four doses of the conjugate vaccine, with 8, 16, or 47 μg of conjugated Pfs25 mass, at 0, 2, 4, and 10 months. Vaccinations were generally well tolerated. The majority of solicited adverse events were mild in severity with pain at the injection site the most common complaint. Anemia was the most common laboratory abnormality, but was considered possibly related to the study in only a minority of cases. No vaccine-related serious adverse events occurred. The peak geometric mean anti-Pfs25 antibody level in the highest dose group was 88 (95% CI 53, 147) μg/mL two weeks after the 4th vaccination, and declined to near baseline one year later. Antibody avidity increased over successive vaccinations. Transmission blocking activity demonstrated in a standard membrane feeding assay (SMFA) also increased from the second to the third dose, and correlated with antibody titer and, after the final dose, with antibody avidity. These results support the further evaluation of Pfs25-EPA/Alhydrogel® in a malaria-endemic population.