Fig. 1
Experimental workflow for multispecies drug and vaccine preclinical assays. a A single vial of a pre-established cryopreserved primary human hepatocyte (PHH) donor is sufficient to seed a full 384-well plate and run 360 single-point therapeutic candidates. By day 3 post-seed, PHHs show in vivo-like phenotypes and are suitable for Plasmodium sporozoite infection. Defined preclinical assay modes are standardized using semi-automated hand pipettes and a 384-well pin tool. Upon experiment fixation, plates are fluorescently stain with a specific LS biomarker and imaged on a high content imaging (HCI) system. b Plasmodium sporozoites exhibit gliding motility while traveling from the injection site in the dermis to liver. Contact with liver components, including Kupffer cells, highly sulfated heparan sulfate proteoglycans, and liver endothelium triggers proteolytic processing of surface proteins (i.e., circumsporozoite protein) and activation of hepatocyte invasion machinery (i.e., TRAP, TREP, P36). Antibody inhibition of invasion pathways can be assessed by pre-incubation of infectious sporozoites with test serum or purified antibody in vitro prior to infection of plated hepatocytes (ILSDA). Following successful invasion, including formation of a parasitophorous vacuole membrane, P. vivax differentiates into hypnozoites and liver schizonts, which complete development after day 9. Day 5 or 6 is an ideal endpoint for both P. falciparum and P. vivax, for ILSDA and prophylactic drug response assays, as P. falciparum completes development shortly thereafter, and P. vivax schizonts are then distinguishable from hypnozoites based on size and morphology. Radical cure drug response assays are ended at day 8 to allow for treatment and clearance of susceptible liver forms
