Dissection-independent production of Plasmodium sporozoites from whole mosquitoes.

Progress towards a protective vaccine against malaria remains slow. To date, only limited protection has been routinely achieved following immunisation with either whole-parasite (sporozoite) or subunit-based vaccines. One major roadblock to vaccine progress, and to pre-erythrocytic parasite biology in general, is the continued reliance on manual salivary gland dissection for sporozoite isolation from infected mosquitoes. Here, we report development of a multi-step method, based on batch processing of homogenised whole mosquitoes, slurry, and density-gradient filtration, which combined with free-flow electrophoresis rapidly produces a pure, infective sporozoite inoculum. Human-infective Plasmodium falciparum and rodent-infective Plasmodium berghei sporozoites produced in this way are two- to threefold more infective than salivary gland dissection sporozoites in in vitro hepatocyte infection assays. In an in vivo rodent malaria model, the same P. berghei sporozoites confer sterile protection from mosquito-bite challenge when immunisation is delivered intravenously or 60-70% protection when delivered intramuscularly. By improving purity, infectivity, and immunogenicity, this method represents a key advancement in capacity to produce research-grade sporozoites, which should impact delivery of a whole-parasite based malaria vaccine at scale in the future.

Plasmodium UIS3 sequesters host LC3 to avoid elimination by autophagy in hepatocytes.

The causative agent of malaria, Plasmodium, replicates inside a membrane-bound parasitophorous vacuole (PV), which shields this intracellular parasite from the cytosol of the host cell 1 . One common threat for intracellular pathogens is the homeostatic process of autophagy, through which cells capture unwanted intracellular material for lysosomal degradation 2 . During the liver stage of a malaria infection, Plasmodium parasites are targeted by the autophagy machinery of the host cell, and the PV membrane (PVM) becomes decorated with several autophagy markers, including LC3 (microtubule-associated protein 1 light chain 3) 3,4 . Here we show that Plasmodium berghei parasites infecting hepatic cells rely on the PVM transmembrane protein UIS3 to avoid elimination by host-cell-mediated autophagy. We found that UIS3 binds host LC3 through a non-canonical interaction with a specialized surface on LC3 where host proteins with essential functions during autophagy also bind. UIS3 acts as a bona fide autophagy inhibitor by competing with host LC3-interacting proteins for LC3 binding. Our work identifies UIS3, one of the most promising candidates for a genetically attenuated vaccine against malaria 5 , as a unique and potent mediator of autophagy evasion in Plasmodium. We propose that the protein-protein interaction between UIS3 and host LC3 represents a target for antimalarial drug development.

Kinome-wide RNAi screen implicates at least 5 host hepatocyte kinases in Plasmodium sporozoite infection.

Plasmodium sporozoites, the causative agent of malaria, are injected into their vertebrate host through the bite of an infected Anopheles mosquito, homing to the liver where they invade hepatocytes to proliferate and develop into merozoites that, upon reaching the bloodstream, give rise to the clinical phase of infection. To investigate how host cell signal transduction pathways affect hepatocyte infection, we used RNAi to systematically test the entire kinome and associated genes in human Huh7 hepatoma cells for their potential roles during infection by P. berghei sporozoites. The three-phase screen covered 727 genes, which were tested with a total of 2,307 individual siRNAs using an automated microscopy assay to quantify infection rates and qRT-PCR to assess silencing levels. Five protein kinases thereby emerged as top hits, all of which caused significant reductions in infection when silenced by RNAi. Follow-up validation experiments on one of these hits, PKCsigma (PKCzeta), confirmed the physiological relevance of our findings by reproducing the inhibitory effect on P. berghei infection in adult mice treated systemically with liposome-formulated PKCsigma-targeting siRNAs. Additional cell-based analyses using a pseudo-substrate inhibitor of PKCsigma added further RNAi-independent support, indicating a role for host PKCsigma on the invasion of hepatocytes by sporozoites. This study represents the first comprehensive, functional genomics-driven identification of novel host factors involved in Plasmodium sporozoite infection.