A robust in vitro liver cell model for long-term study of Plasmodium vivax hypnozoites

Background Plasmodium vivax is the most prevalent species of human malaria parasites, affecting nearly half of the world’s population. Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax to tolerate most antimalarials, and later to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites, including hypnozoites. Experimentally, this form has previously been studied in the primary hepatocyte cell model, but this has some limitations due to its high cost and failures when used for long-term biological studies and high throughput drug screening. Methods Here, an immortalized human hepatocyte-like cell line (imHC) was infected with P. vivax sporozoites. The infected cultures were maintained for up to 28 days to obtain small liver-stage forms. A novel system to quickly enrich pure small liver-stage forms in culture was also developed. The susceptibility of the enriched small liver-stage forms, presumably hypnozoites, to known antimalarial drugs, atovaquone, primaquine, and tafenoquine, was examined. Results Small liver-stage forms of P. vivax could persist in long-term imHCs culture. These small forms had a single nucleus and could be enriched by treatment with DSM265, a compound active against growing parasites but not hypnozoites. Resistance to inhibition by atovaquone was consistent with the interpretation that the enriched small parasites represent hypnozoites. Primaquine and tafenoquine displayed poor activity at clearing these putative hypnozoites in vitro. Conclusions A robust cell-based model, with well-defined dormant liver-stage parasites in long-term stable human liver cells, allows us to follow hypnozoite formation and eventual reactivation to dividing parasites. This model is also well-suited to test radical cure efficacy of compounds against P. vivax hypnozoites. Thus, it will be of value for understanding hypnozoite biology and for drug discovery to eliminate dormant malaria. Keywords Malaria, Plasmodium vivax, Sporozoite, Liver stage, Hypnozoite, imHCs