Abstract
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.
Highlights
Plasmodium vivax (Pv) is the second most important malaria parasite, which infects humans
A part of this Pv spz harvest was used for setting up assays with HepG2-A16 hepatocytes and part was vialed and cryopreserved in liquid nitrogen vapor phase (LNVP)
Pv spz were found to react with this monoclonal antibody (mAb) at a very high dilution, 1:1,024,000 (0.98 ng/mL)
Summary
Plasmodium vivax (Pv) is the second most important malaria parasite, which infects humans. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain for a long time as dormant hypnozoites [9,10]. Activation of hypnozoites after the primary attack causes additional blood stage infections called relapses. More recently, studies using microsatellite markers identified from the Pv genome sequence showed that relapse infections often result from activation of heterologous hypnozoites [13,14]. It has been quite difficult to reproducibly use these cell culture based platforms for screening new drugs against liver stages, because it has been difficult to obtain adequate quantities of Pv spz, and each time Pv spz are obtained they are from a different strain of Pv or a different infection of non-human primates with the same strain, in other words, a different lot of Pv spz
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