Engineering Hepatic Microenvironments for Efficient and Predictive Pre-Clinical Plasmodium vivax Liver Stage Assays

No preclinical hepatic drug screening model is currently efficient and predictive enough to screen the thousands of compounds awaiting characterization for activity against Plasmodium vivax liver stage schizonts and hypnozoites. After finding simple addition of microfeatures to culture plastic results in stable long-term hepatocyte cultures suitable for P. vivax infection, we developed a novel fabrication process to emboss microfeatures onto the bottom surface of a 384-well polystyrene culture model. Careful downselection of protocol parameters, including surface activation of culture plastic, identification of competent lots of cryopreserved primary human hepatocytes, and refined isolation of P. vivax sporozoites result in a simple and robust method for production of over 100 liver forms from a 5,000 sporozoite inoculum in a 384-well microtiter plate format. Chemotherapeutic studies including reference compounds such as PI4K inhibitor s and Atovaquone confirm the model as an accurate predictor of form-specific prophylactic and radical cure activity. Screening performed on over 1500 compounds revealed several hits, including newly-described activity against hypnozoites, elucidating possible new approaches and targets for development of next-generation antimalarials with radical cure activity.