Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand.

Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum.

A defined mechanistic correlate of protection against Plasmodium falciparum malaria in non-human primates.

Malaria vaccine design and prioritization has been hindered by the lack of a mechanistic correlate of protection. We previously demonstrated a strong association between protection and merozoite-neutralizing antibody responses following vaccination of non-human primates against Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5). Here, we test the mechanism of protection. Using mutant human IgG1 Fc regions engineered not to engage complement or FcR-dependent effector mechanisms, we produce merozoite-neutralizing and non-neutralizing anti-PfRH5 chimeric monoclonal antibodies (mAbs) and perform a passive transfer-P. falciparum challenge study in Aotus nancymaae monkeys. At the highest dose tested, 6/6 animals given the neutralizing PfRH5-binding mAb c2AC7 survive the challenge without treatment, compared to 0/6 animals given non-neutralizing PfRH5-binding mAb c4BA7 and 0/6 animals given an isotype control mAb. Our results address the controversy regarding whether merozoite-neutralizing antibody can cause protection against P. falciparum blood-stage infections, and highlight the quantitative challenge of achieving such protection.

Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites.

In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites.

Malaria Vaccine Development: Focusing Field Erythrocyte Invasion Studies on Phenotypic Diversity: The West African Merozoite Invasion Network (WAMIN).

Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and proliferation. Invasion is mediated by multiple ligands, which could be promising vaccine targets. The usage and sequence of these ligands differs between parasites, yet most studies of them have been carried out in only a few laboratory-adapted lines. To understand the true extent of natural variation in invasion phenotypes and prioritize vaccine candidates on a relevant evidence base, we need to develop and apply standardized assays to large numbers of field isolates. The West African Merozoite Invasion Network (WAMIN) has been formed to meet these goals, expand training in Plasmodium phenotyping, and perform large-scale field phenotyping studies in order to prioritize blood stage vaccine candidates.

Basigin is a druggable target for host-oriented antimalarial interventions.

Plasmodium falciparum is the parasite responsible for the most lethal form of malaria, an infectious disease that causes a large proportion of childhood deaths and poses a significant barrier to socioeconomic development in many countries. Although antimalarial drugs exist, the repeated emergence and spread of drug-resistant parasites limit their useful lifespan. An alternative strategy that could limit the evolution of drug-resistant parasites is to target host factors that are essential and universally required for parasite growth. Host-targeted therapeutics have been successfully applied in other infectious diseases but have never been attempted for malaria. Here, we report the development of a recombinant chimeric antibody (Ab-1) against basigin, an erythrocyte receptor necessary for parasite invasion as a putative antimalarial therapeutic. Ab-1 inhibited the PfRH5-basigin interaction and potently blocked erythrocyte invasion by all parasite strains tested. Importantly, Ab-1 rapidly cleared an established P. falciparum blood-stage infection with no overt toxicity in an in vivo infection model. Collectively, our data demonstrate that antibodies or other therapeutics targeting host basigin could be an effective treatment for patients infected with multi-drug resistant P. falciparum.

Towards a comprehensive Plasmodium falciparum merozoite cell surface and secreted recombinant protein library.

BACKGROUND: Plasmodium falciparum is the aetiological agent for malaria, a deadly infectious disease for which no vaccine has yet been licensed. The proteins displayed on the merozoite cell surface have long been considered attractive vaccine targets because of their direct exposure to host antibodies; however, progress in understanding the functional role of these targets has been hindered by technical challenges associated with expressing these proteins in a functionally active recombinant form. To address this, a method that enables the systematic expression of functional extracellular Plasmodium proteins was previously developed, and used to create a library of 42 merozoite proteins. METHODS: To compile a more comprehensive library of recombinant proteins representing the repertoire of P. falciparum merozoite extracellular proteins for systematic vaccine and functional studies, genome-wide expression profiling was used to identify additional candidates. Candidate proteins were recombinantly produced and their integrity and expression levels were tested by Western blotting and ELISA. RESULTS: Twenty-five additional genes that were upregulated during late schizogony, and predicted to encode secreted and cell surface proteins, were identified and expressed as soluble recombinant proteins. A band consistent with the entire ectodomain was observed by immunoblotting for the majority of the proteins and their expression levels were quantified. By using sera from malaria-exposed immune adults, the immunoreactivity of 20 recombinant proteins was assessed, and most of the merozoite ligands were found to carry heat-labile epitopes. To facilitate systematic comparative studies across the entire library, multiple Plasmodium proteins were simultaneously purified using a custom-made platform. CONCLUSIONS: A library of recombinant P. falciparum secreted and cell surface proteins was expanded by 20 additional proteins, which were shown to express at usable levels and contain conformational epitopes. This resource of extracellular P. falciparum merozoite proteins, which now contains 62 full-length ectodomains, will be a valuable tool in elucidating the function of these proteins during the blood stages of infection, and facilitate the comparative assessment of blood stage vaccine candidates.