An effective blood stage vaccine against Plasmodium falciparum remains a research priority but the number of antigens that have been translated to candidates for testing in clinical trials remains limited. Investigations of the large number of potential targets found in the parasite proteome have been constrained by an inability to produce natively folded recombinant antigens for immunological studies. We overcame these constraints by generating a large library of demonstrably biochemically active merozoite surface and secreted full-length ectodomain proteins. We then systematically examined the antibody reactivity against these proteins in a cohort of Kenyan children (n=286) who were sampled at the start of a malaria transmission season and prospectively monitored for clinical episodes of malaria over the ensuing six months. We found that antibodies to previously untested or little-studied proteins had superior or equivalent potential protective efficacy to the handful of current leading malaria vaccine candidates. Moreover, cumulative responses to combinations comprising five of the ten top ranked antigens, including PF3D7_1136200, MSP2, RhopH3, P41, MSP11, MSP3, PF3D7_0606800, AMA1, Pf113 and MSRP1 were associated with 100% protection against clinical episodes of malaria. These data suggest that not only are there many more potential vaccine candidates for the vaccine development pipeline, but also that highly effective vaccination may be achieved through combining a selection of these antigens as observed in nature.

Highlights► PfRH5 is essential for Plasmodium falciparum erythrocyte invasion. ► Antibodies raised against full-length functional PfRH5 potently blocked invasion. ► Five common PfRH5 polymorphisms were identified across 290 clinical isolates. ► Antibodies raised against one variant inhibited all other common PfRH5 variants. ► Correctly folded recombinant PfRH5 is a strong P. falciparum vaccine candidate.

Plasmodium falciparum, the cause of almost all human malaria mortality, is a member of the Laverania subgenus which infects African great apes. Interestingly, Laverania parasites exhibit strict host specificity in their natural environment: P. reichenowi, P. billcollinsi, and P. gaboni infect only chimpanzees; P. praefalciparum, P. blacklocki, and P. adleri are restricted to gorillas, and P. falciparum is pandemic in humans. The molecular mechanism(s) responsible for these host restrictions are not understood, although the interaction between the parasite blood-stage invasion ligand EBA175 and the host erythrocyte receptor Glycophorin-A (GYPA) has been implicated previously. We reexamined the role of the EBA175-GYPA interaction in host tropism using recombinant proteins and biophysical assays and found that EBA175 orthologs from the chimpanzee-restricted parasites P. reichenowi and P. billcollinsi both bound to human GYPA with affinities similar to that of P. falciparum, suggesting that the EBA175-GYPA interaction is unlikely to be the sole determinant of Laverania host specificity. We next investigated the contribution of the recently discovered Reticulocyte-binding protein Homolog 5 (RH5)-Basigin (BSG) interaction in host-species selectivity and found that P. falciparum RH5 bound chimpanzee BSG with a significantly lower affinity than human BSG and did not bind gorilla BSG, mirroring the known host tropism of P. falciparum. Using site-directed mutagenesis, we identified residues in BSG that are responsible for the species specificity of PfRH5 binding. Consistent with the essential role of the PfRH5-BSG interaction in erythrocyte invasion, we conclude that species-specific differences in the BSG receptor provide a molecular explanation for the restriction of P. falciparum to its human host.surface plasmon resonance | protein interactions T he most deadly of the malaria parasites, Plasmodium falciparum, is highly divergent from the other species of Plasmodium known to infect humans (1-3), with its closest relatives comprising a group of chimpanzee and gorilla parasites from the subgenus Laverania (3-7). Despite the origin of P. falciparum as a zoonosis, and the continuing coexistence of humans and apes in West and Central Africa, extensive field studies have failed to detect P. falciparum in wild-living chimpanzees and gorillas (5). Although there are reports of P. falciparum infecting chimpanzees either in certain captive settings (2) or following splenectomy and deliberate transfer of P. falciparum-infected human blood (8-10), the resulting infections have low parasitemia and are not known to result in malignant tertian malaria, suggesting a host-specific barrier for replete infection. The existence of host-specific barriers within the Laverania subgenus is supported further by the strict host specificity exhibited by ape Laverania parasites in the wild: Plasmodium reichenowi, Plasmodium billcollinsi, and Plasmodium gaboni infect only chimpanzees, and Plasmodium praefalciparum, Plasmodium blacklocki, and Plasm...

Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.

Background: The GYPA-PfEBA175 interaction is important for erythrocyte invasion by the malaria parasite.Results: The entire ectodomain of EBA175 interacted with GYPA with different biochemical parameters to the previously determined GYPA-binding fragment containing two DBL domains.Conclusion: Regions outside of the tandem DBL domains contribute to GYPA binding by EBA175.Significance: These findings may assist the design of an EBA175-based malaria vaccine.