To identify well tolerated, new molecules active against the dormant liver-form (the hypnozoite) of Plasmodium vivax and P. ovale, MMV has a pragmatic test cascade in place. Large numbers of compounds known to be active against blood-stage parasites are screened first in an in vitro liver-stage assay, using rodent (P. berghei) or simian (P. cynomolgi) parasites, to determine if they have activity against liver-stage schizonts and hypnozoites (see Fig 1). Active compounds are then progressed to an in vivo assay to confirm their activity against the hypnozoites in a more physiologically relevant model.
The limitation of the current test cascade, however, is that we are not testing against parasites that infect humans (P. vivax and P. ovale) and so might miss some molecules. Additionally, the throughput of current assays looking at the P. cynomolgi hypnozoite is limited. To overcome this, MMV and the Bill & Melinda Gates Foundation (BMGF) are working with different research groups to develop a cost-effective P. vivax liver-stage assay.
In a major step towards that goal, a team of researchers from Mahidol University, Thailand, has now developed a P. vivax ‘hypnozoite’ cell-based in vitro assay able to screen up to 150 compounds a year.
1. What was involved in setting up the hypnozoite assay?
One of the issues with studying vivax and setting up an assay is that the blood-stage parasites can’t be cultured continuously in vitro. So you need to have access to new parasites, and therefore patients, on a regular basis. In Thailand, we have both main species of parasite – P. falciparum and P. vivax, so we are well situated for this kind of research. We have been able to develop P. vivax liverstage cultures in vitro successfully for up to 4 weeks. This time frame is in line with the typical time to relapse in Thailand.
The Center for Infectious Disease Research, formerly Seattle Biomed, a research institute in the USA, has recently developed reagents especially for vivax, enabling us to specifically identify the developing parasites in human cell lines. This really opened the door for more vivax research.
2. How does the assay work?
We culture human hepatoma cell lines (HC-04) in an 8-well plate. One day later we add P. vivax sporozoites into each well. The sporozoites come from mosquitoes from our insectary that have been fed on patient blood. We add the compounds to be investigated into the vivax cultures in triplicate wells and at a range of different concentrations, keeping some wells without compound as the control. Two weeks later, we count the small and large parasite forms, take the average and compare them with the control wells. With this assay we can perform two types of experiment. In the first, we mix the drug with the liver cells before – or at the same time – as the sporozoites are added and this allows us to study the chemopreventive ability of the compounds. In a second experiment, we add the compounds several days after addition of the sporozoites to the liver cells, allowing us to look at the ability of the compounds to kill hypnozoites.
3. Which compounds have you screened so far and what did you find?
The assay has been validated with primaquine and atovaquone as controls, to ensure it was working. We have also begun testing six out of 10 compounds from MMV’s portfolio. All the MMV compounds tested showed efficacy against the small forms in the prophylactic assay confirming the data obtained previously in the surrogate assay using P. cynomolgi. Interestingly, one of the compounds that was found to be weakly active in this assay was found to be highly active in the P. vivax liver-stages assay. This tells us that using this assay we are able to detect false negatives that would be missed by the P. cynomolgi assay.
4. What has it been like working as part of the BMGF liverstage consortium?
It has increased our ability to study the liver-stage parasite. By collaborating with other scientists, including those at MMV, we can access new tools and reagents for parasite identification and contribute P. vivax sporozoites to the group – a critical component of all experiments proposed by the consortium. Thanks to the generous support of the BMGF, by working together, every group in the consortium is able to move forward faster.
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