Historically, basic research on P. vivax has lagged behind that for Plasmodium falciparum. This is partly because P. falciparum was considered the more deadly of the two and because P. vivax parasites were difficult to access and to work with. Even today the technical challenge is great; the blood-stage parasites still can’t be maintained in continuous culture.
Nevertheless, the discovery and development of anti-relapse molecules has always been a key research and development (R&D) focus for MMV. For example, over the years, investments have been made in testing new clinical candidates, developing tafenoquine with GlaxoSmithKline (GSK) and developing new clinical models in collaboration with the Indonesian army. Up to now, the major advances in cell biology have been made through Wellcome Trust co-funded collaborations with the Dutch Primate Centre, and Novartis-led discovery projects. Over the last year we have seen a significant change in the assays coming through, thanks to a continued effort on the part of our collaborators, complemented by new funding from the Bill & Melinda Gates Foundation to establish a P. vivax Malaria Consortium.
In 2014, a team of researchers from Mahidol University, Thailand, was able to establish a P. vivax ‘hypnozoite’ cell-based in vitro assay to screen up to 1,000 compounds a year. Dr Jetsumon Sattabongkot Prachumsri talks about the assay, what it has told us so far and what it’s like to work with the Consortium.
1. How does the assay work and what are its advantages?
The assay allows us to see the impact of various test compounds on small and large liver P. vivax forms. We culture the human liver cell line in an 8-well plate and then add P. vivax sporozoites (the liver-infective stages of the parasite) into each well a day later. We then add the test compounds to the cultures in triplicate at a range of concentrations. Six days later, we count the small and large parasite forms (believed to be hypnozoites and schizonts, respectively) take the average and compare them with the control wells.
Our system is in vitro and uses the human liver cell line, which means we can handle many more compounds and concentrations at the same time. As we use the same cell line, the findings are more robust, compared to a humanized mouse model, for example.
We have been working with 96-well plates, but are now moving to 384well plates, which will then enable us to screen yet more compounds in one go. With this assay, depending on the stage at which we add the compound, we can look at either the effect of the compound on the development of small and large forms (potential for chemoprevention) or the effect on fully developed forms (potential for radical cure – killing of hypnozoites and relapse prevention)
2. How many compounds have you been able to screen and what have you found so far?
We finished screening the first 25 compounds from MMV in the chemoprevention model and are in the process of testing 100 more. We have found some molecules with very good potential. Next, we will screen the Pathogen Box compounds (page 27). Our goal for the year is to validate the 96-well plate, initiate the 384-well plate format and screen up to 1,000 compounds.
3. What has it been like to work with MMV and the Gates’ P. vivax Malaria Consortium?
The project is managed with an industry-style approach so the timelines are tight which can be challenging, but I do enjoy it. I enjoy understanding the different research perspectives and because the funding is stable we can progress smoothly. If just one molecule screened from our system ultimately treats people it will be a goal fulfilled.