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Genetic crosses are most powerful for linkage analysis when progeny numbers are high, parental alleles segregate evenly and numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. We report on two genetic crosses—(1) an allopatric cross between a laboratory-adapted parasite (NF54) of African origin and a recently patient-derived Asian parasite, and (2) a sympatric cross between two recently patient-derived Asian parasites. We generated 144 unique recombinant clones from the two crosses, doubling the number of unique recombinant progeny generated in the previous 30 years. The allopatric African/Asian cross has minimal levels of inbreeding and extreme segregation distortion, while in the sympatric Asian cross, inbred progeny predominate and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny provide the power to map small-effect mutations and epistatic interactions. The segregation distortion in the allopatric cross slightly erodes power to detect linkage in several genome regions. We greatly increase the power and the precision to map biomedically important traits with these new large progeny panels., Button-Simons et al. report on two different genetic crosses of Plasmodium falciparum derived from Asian and African origin parasites, and generate large numbers of recombinant progeny. These crosses are characterized and have the potential to map biologically important traits across parasite genomes.

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.

Malaria, a disease caused by Plasmodium parasites, kills nearly 600,000 people annually. Inoculated by an infectious mosquito bite, Plasmodium sporozoites travel to the liver and infect hepatocytes. Here, they develop into merozoites that are released into the blood to cause malaria associated mortality and morbidity. No fully protective malaria vaccine exists, but immunizations with genetically attenuated parasites (GAPs) that arrest in the liver confer sterile protection from challenge in mice. Moreover, using a novel super-infection assay, we observed that GAP immunization induces a potent type I IFN response to control liver stage infection. However, the factors required for the induction and propagation of the IFN response, and the influence of this response on adaptive immunity had not been elucidated. To identify the upstream inducers of IFN signaling, we immunized mice with either mosquito debris or GAPs and measured gene expression in hepatocytes by RNA-Seq. 800 genes were significantly upregulated upon GAP immunization, including transcripts for cytosolic RNA and DNA sensors. To examine the influence of the IFN response on adaptive immunity, WT, IRF3−/− and IFNAR−/− mice were immunized with a suboptimal dose of GAP, and challenged 4 weeks later with WT parasite. The immunization regimen elicited sterile protection in 20% of the WT mice, while 100% protection was achieved in IRF3−/− or IFNAR−/− mice. Taken together, the data suggest that hepatocytes utilize RNA and DNA sensors to induce a type I IFN response that is necessary for early control of liver stage infection, yet detrimental to adaptive immunity. Current studies are examining the mechanisms behind the IFN-mediated suppression of adaptive immunity.

Protracted sterile protection conferred by a P. yoelii genetically attenuated parasite (PyGAP) vaccine was found to be completely dependent on CD8+ T lymphocytes. Immunization studies with perforin and IFN-γ knock out mice indicated that the protection was largely dependent on perforin as compared to IFN-γ. Both liver and spleen CD8+ T cells from PyGAP immunized mice induced massive apoptosis of liver stage (LS)-infected hepatocytes in vitro without the release of detectable IFN-γ and TNF-a, which directly correlated with GAP vaccine efficacy in vivo. Most importantly, we demonstrated that CD8+ T cells isolated from naïve mice that had survived wild-type P. yoelii sporozoite infection targeted mainly sporozoite-traversed and uninfected hepatocytes, revealing an immune evasion strategy that might have been used by wild-type parasites to subvert host immune responses. Differential processing of the parasite antigens in liver of immunized or naturally infected mice may be responsible for contrasting outcomes of their CTL responses. A controversial role of circumsporozoite protein (CSP) in host immune protection versus immune evasion against malaria is also highlighted by our findings. This research was supported by SBRI innovation grant.