New models to discover new molecules active against the dormant liver stage of P. vivax malaria

2016
Prof. Dennis Kyle, Distinguished University Health Professor, University of South Florida, USA

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.

Following on from the 2014 work by a team of researchers from Mahidol University, in 2015, a second team of researchers was also able to establish a P. vivax ‘hypnozoite’ cell-based in vitro assay. Prof. Dennis Kyle explains the progress, the advantages of their assay and what it’s been like working with the Consortium.

1. How is your work to establish a P. vivax ‘hypnozoite’ cell-based  in vitro assay progressing?

It’s really exciting, we’ve had several breakthroughs in the last couple of years. We are now able to culture primary human hepatocytes for more than 4 weeks, infect them with sporozoites and get parasite development all the way to invading reticulocytes.

All of this has now been achieved in a 384-well plate format with which we can attempt high-throughput screening for the first time. We have begun by screening 27 of MMV’s compounds several times to help refine the assay. We expect to screen at least 1,000 compounds this year for MMV.

2. What are the advantages of this assay?

Because we can maintain the primary human hepatocytes in culture for more than 4 weeks, we can routinely do radical cure screens and assess hypnozoite activity from days 5–9 post-sporozoite invasion. We can also look for relapse in vitro from as early as day 21 and as late as days 33–35.

We have found that human hepatocytes prefer to be confined in a 3D space. So by optimizing the ‘microfeature’ design at the bottom of each well we can promote optimal development of hepatocytes and the parasite. As this is all conducted in a 384-well format our screening is both high-content and high-throughput.

3. What has it been like to work with MMV and the Gates’ P. vivax Malaria Consortium?

It’s a good example of how sharing information early can be very helpful. It’s a high-risk project so working individually through independent grant systems would not have been successful. Although, in a sense, the groups are competitors, we routinely share information through interactions in the Consortium. When we have something that does or doesn’t work, we inform the other groups and vice versa. It works very well – for example, we got confirmation of what the hypnozoite looks like in two different  in vitro models and a mouse model. It’s an example of how the Gates’ Consortium model can work to solve big problems.

Working with other members of the Consortium has been critical. The biggest challenge is getting access to the sporozoites so that we can actually do the assays. We are grateful for the contributions of Jetsumon Prachumsri, Chiara Andolini and Francois Nosten in the field in Thailand who provide us with infected mosquitoes.

MMV was involved right from the beginning as advisors and contributors to all the discussions. They are the leader in the field of malaria drug discovery. Being able to access all the different compounds and test systems in their network was invaluable in refining and validating our assay. We hope they can now reap the rewards by using the models to find the next drug to kill the hypnozoite.