Developing Plasmodium cynomolgi culture as a model to investigate antimalarial effects on Plasmodium vivax

The ability to culture pathogenic organisms enables researchers to understand the biology of the organism which aids in the development of vaccines and drugs. Hence the establishment of the protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research into this important cause of human malaria. However, it is not possible to cultivate Plasmodium vivax, the most widely distributed and difficult to treat malaria parasite, due to its strict preference for reticulocytes. Plasmodium cynomolgi, a macaque infecting species phylogenetically close to P. vivax has been routinely used in animal models to study the dormant liver-stage forms of P. vivax. Cultivation of P. cynomolgi erythrocytic stage was reported in the early 1980s, has not been pursued further. In this study, we have revitalized the continuous culture of P. cynomolgi and systematically defined the optimal conditions for the tractable in vitro culture of this important P. vivax model. Importantly we have discovered that the successful in vitro culture of P. cynomolgi is strain dependent, with only one (Berok strain) of the three strains tested, is amenable to long term culture. Morphological and phenotypic characterisation of our P. cynomolgi (Berok strain) cultured parasites clearly show similarities with P. vivax. In addition to validating the potential of this culture system for high throughput drug screening in erythrocytic stages, a homogenous line was isolated from a single infected cell; paving the way for clean whole genome sequences. Understanding of hypnozoite biology of P. vivax remains elusive due to the lack of sustainable in vitro models for long term cultures of hepatocytes to capture the full liver stage cycle including the transition to blood stage parasites. To define the antimalarial drug effects in the full life cycle of P. vivax, we also developed a liver stage model using P. cynomolgi infected 3-dimensional (3D) hepatic spheroids which proved to be a predictive radical cure model which corresponds with outcomes using in vivo monkey models. The phosphatidylinositol-4-OH kinase (PI4K) inhibitor- KDU691 was used in this study as the exploratory tool compound to demonstrate the robustness of the 3D hepatic spheroid model in predicting in vivo outcomes. KDU691 was chosen as it was previously proven to be active in the causal prophylaxis model and delayed treatment model carried out in the conventional 2D monolayer based assays but failed in the in vivo radical cure model using rhesus monkeys. It is hoped that the in vitro tools developed using P. cynomolgi, will accelerate P. vivax vaccine and drug discovery efforts through better understanding of the mechanisms of its pathogenesis