Establishment of an In Vitro Assay for Assessing the Effects of Drugs on the Liver Stages of Plasmodium vivax Malaria

Plasmodium vivax (Pv) is the second most important human malaria parasite. Recent data indicate that the impact of Pv malaria on the health and economies of the developing world has been dramatically underestimated. Pv has a unique feature in its life cycle. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain as dormant forms called hypnozoites, which cause relapses of malaria months to several years after the primary infection. Elimination of malaria caused by Pv will be facilitated by developing a safe, highly effective drug that eliminates Pv liver stages, including hypnozoites. Identification and development of such a drug would be facilitated by the development of a medium to high throughput assay for screening drugs against Pv liver stages. We undertook the present pilot study to (1) assess the feasibility of producing large quantities of purified, vialed, cryopreserved Pv sporozoites and (2) establish a system for culturing the liver stages of Pv in order to assess the effects of drugs on the liver stages of Pv. We used primaquine (PQ) to establish this assay model, because PQ is the only licensed drug known to clear all Pv hepatocyte stages, including hypnozoites, and the effect of PQ on Pv hepatocyte stage development in vitro has not previously been reported. We report that we have established the capacity to reproducibly infect hepatoma cells with purified, cyropreserved Pv spz from the same lot, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. These data provide the foundation for finalizing a medium throughput, high content assay to identify new drugs for the elimination of all Pv liver stages

Are these hypnozoites?

<p>HepG2-A16 cells were infected, maintained for 9 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#pone-0014275-g004" target="_blank">Fig 4</a> and stained with PvCSP mAb. Approximately 10% of the Pv liver stage parasites that expressed PvCSP were similar in size to 3-day trophozoites, and much smaller than the 9-day schizonts. Are these hypnozoites?</p

<i>In-vitro</i> development of liver stage parasites.

<p>25,000 Pv spz was used to infect 20,000 HepG2-A16 cells. Uninfected spz were washed off after three hrs and cells were maintained for 3 days with daily media change. Cells were fixed and Pv liver stage trophozoites (400 X magnification) were stained with mAb NVS3 against the PvCSP (20 µg/ml). <b>N</b>: Nucleus of HepG2-A16 cells, <b>C</b>: Cytoplasm of HepG2-A16 cells, <b>P</b>: Developing 3 day old Pv hepatocyte stage parasite (trophozoite).</p

Dose dependent effect of primaquine on the development of 9 day hepatocyte stage parasites.

<p>50,000 fresh Pv spz were added to wells containing 20,000 hepatoma cells and were cultured for 9 days in medium alone or in the presence of different concentrations of primaquine (PQ). The numbers of late liver stage schizonts (9 day assay) were counted by immunoflourescence microscopy after staining with the anti-PvCSP mAb, NVS3 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#s2" target="_blank">Materials and Methods</a>.</p

Hepatotoxic changes in HepG2-A16 cells after treatment with high dose primaquine for 9 days.

<p>Culturing HepG2-A16 cells with high concentrations of primaquine (10 µg/mL) induced hepatotoxic changes. <b>P</b>: Parasite; <b>White arrows</b>: Dense bodies in cytoplasm; <b>E</b>: Empty spaces due to focal cell loss in the cell culture well.</p

Immunofluorescence Assay.

<p>Air dried Pv spz were immunostained with monoclonal antibody against PvCSP (NVS3) at 0.98 ng/mL as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#s2" target="_blank">Materials and Methods</a>. The end point titer is 1∶1,024,000.</p

Multiple Pv liver stage parasites were seen in single hepatocyte in a 3 Day culture.

<p>HepG2-A16 cells were infected with Pv spz and the liver stage trophozoites were stained with the anti-PvCSP mAb, NVS3. Some HepG2-A16 cells were seen with multiple liver stage parasites (400X magnification). <b>N</b>: Nucleus of hepatocyte, C: Cytoplasm of hepatocyte, White Arrows: Individual 3 Day hepatocyte stage Pv.</p

<i>In vitro</i> development of hepatocyte stage parasites.

<p>25,000 Pv spz were added to wells containing 20,000 hepatoma cells and were cultured for 3 days and 50,000 Pv spz were added to wells containing 20,000 hepatoma cells and were cultured for 9 days, and the numbers of early liver stage trophozoites (3 day assay) or late liver stage schizonts (9 day assay) were counted by immunofluorescence microscopy after staining with the anti-PvCSP mAb, NVS3 (20 µg/mL).</p

No change in infectivity after cryopreservation of Pv spz.

<p>25,000 Pv spz (Fresh or thawed after cryopreserved in LNVP for one year) were added to wells containing 20,000 hepatoma cells and cultured for 3 days. The numbers of early liver stage trophozoites (3 day assay) were counted by immunofluorescence microscopy after staining with the anti-PvCSP mAb, NVS3, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#s2" target="_blank">Materials and Methods</a>.</p