A novel immortalized hepatocyte-like cell line (imHC) supports in vitro liver stage development of the human malarial parasite Plasmodium vivax.

BACKGROUND: Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, 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. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay. METHODS: A novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay. RESULTS: imHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening. CONCLUSIONS: imHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.

VIABILITY AND INFECTIVITY OF CRYOPRESERVED PLASMODIUM VIVAX SPOROZOITES.

Plasmodium vivax presents a great challenge to malaria control because of the ability of its dormant form in the liver, the hypnozoite, to cause relapse in otherwise fully recovered patient. Research efforts to better understand P. vivax hypnozoite biology have been hampered by the limited availability of its sporozoite form responsible for liver infection. Thus, the ability to cryopreserve and recover P. vivax sporozoites is an essential procedure. In this study, protective effects of hydroxyethyl starch (HES) alone and in combination with other cryoprotectants on P. vivax sporozoite recovery, viability and in vitro infectivity of a human liver HC-04 cell line were investigated. Sporozoites were harvested from P. vivax-infected female Anopheles mosquitoes and cryopreserved at a freezing rate of -1°C/minute to a final temperature of -80°C before being stored in a vapor phase liquid nitrogen tank. Cryopreserved sporozoites were thawed at 37°C and recovery of intact sporozoites assessed using a hemocytometer. Sporozoite viability and in vitro infectivity was measured using a gliding and an indirect immunofluorescence assay, respectively. A combination of 10% HES + 50% fetal bovine serum was the best cryopreservant compared to HES solution alone or mixed with cryopreservants such as dimethyl sulfoxide (DMSO) and sucrose. A mixture of bovine serum albumin, DMSO and sucrose in RPMI 1640 medium constituted an alternative cryopreservant. Sporozoites recovered from all cryopreservation media exhibited motility and infectivity of < 0.1% and < 0.001%, respectively. Thus, there is an urgent need for a vast improvement in cryopreservation procedures of viable and infective P. vivax sporozoites necessary for advancing research on hypnozoite biology.