Divergent roles for the RH5 complex components, CyRPA and RIPR in human-infective malaria parasites.

Malaria is caused by Plasmodium parasites, which invade and replicate in erythrocytes. For Plasmodium falciparum, the major cause of severe malaria in humans, a heterotrimeric complex comprised of the secreted parasite proteins, PfCyRPA, PfRIPR and PfRH5 is essential for erythrocyte invasion, mediated by the interaction between PfRH5 and erythrocyte receptor basigin (BSG). However, whilst CyRPA and RIPR are present in most Plasmodium species, RH5 is found only in the small Laverania subgenus. Existence of a complex analogous to PfRH5-PfCyRPA-PfRIPR targeting BSG, and involvement of CyRPA and RIPR in invasion, however, has not been addressed in non-Laverania parasites. Here, we establish that unlike P. falciparum, P. knowlesi and P. vivax do not universally require BSG as a host cell invasion receptor. Although we show that both PkCyRPA and PkRIPR are essential for successful invasion of erythrocytes by P. knowlesi parasites in vitro, neither protein forms a complex with each other or with an RH5-like molecule. Instead, PkRIPR is part of a different trimeric protein complex whereas PkCyRPA appears to function without other parasite binding partners. It therefore appears that in the absence of RH5, outside of the Laverania subgenus, RIPR and CyRPA have different, independent functions crucial for parasite survival.

Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion.

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. Super-resolution microscopy and real-time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites.

Design of a basigin-mimicking inhibitor targeting the malaria invasion protein RH5.

Many human pathogens use host cell-surface receptors to attach and invade cells. Often, the host-pathogen interaction affinity is low, presenting opportunities to block invasion using a soluble, high-affinity mimic of the host protein. The Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) provides an exciting candidate for mimicry: it is highly conserved and its moderate affinity binding to the human receptor basigin (KD ≥1 µM) is an essential step in erythrocyte invasion by this malaria parasite. We used deep mutational scanning of a soluble fragment of human basigin to systematically characterize point mutations that enhance basigin affinity for RH5 and then used Rosetta to design a variant within the sequence space of affinity-enhancing mutations. The resulting seven-mutation design exhibited 1900-fold higher affinity (KD approximately 1 nM) for RH5 with a very slow binding off rate (0.23 h-1 ) and reduced the effective Plasmodium growth-inhibitory concentration by at least 10-fold compared to human basigin. The design provides a favorable starting point for engineering on-rate improvements that are likely to be essential to reach therapeutically effective growth inhibition.

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex.

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

Cellular dissection of malaria parasite invasion of human erythrocytes using viable Plasmodium knowlesi merozoites.

Plasmodium knowlesi, a zoonotic parasite causing severe-to-lethal malaria disease in humans, has only recently been adapted to continuous culture with human red blood cells (RBCs). In comparison with the most virulent human malaria, Plasmodium falciparum, there are, however, few cellular tools available to study its biology, in particular direct investigation of RBC invasion by blood-stage P. knowlesi merozoites. This leaves our current understanding of biological differences across pathogenic Plasmodium spp. incomplete. Here, we report a robust method for isolating viable and invasive P. knowlesi merozoites to high purity and yield. Using this approach, we present detailed comparative dissection of merozoite invasion (using a variety of microscopy platforms) and direct assessment of kinetic differences between knowlesi and falciparum merozoites. We go on to assess the inhibitory potential of molecules targeting discrete steps of invasion in either species via a quantitative invasion inhibition assay, identifying a class of polysulfonate polymer able to efficiently inhibit invasion in both, providing a foundation for pan-Plasmodium merozoite inhibitor development. Given the close evolutionary relationship between P. knowlesi and P. vivax, the second leading cause of malaria-related morbidity, this study paves the way for inter-specific dissection of invasion by all three major pathogenic malaria species.