Tackling the elusive malaria liver stage using an improved human liver model

Go to the profile of Alison E. Roth
May 13, 2018
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The paper in Nature Communications is here: https://t.co/kfYTp2t2Q0

Malaria remains a significant global health burden causing over 200 million clinical cases and nearly half a million deaths each year. Transmitted by anopheline mosquitoes, infections with Plasmodium falciparum are the most common and deadliest, whilst Plasmodium vivax is the most widely distributed and hardest to control because of the dormant ‘hypnozoite’ liver stage. The goal of worldwide eradication depends on new antimalarial therapies, especially new combination drugs to combat emerging artemisinin resistance and new drugs or vaccines to eliminate the pre-erythrocytic stages in the liver, especially the P. vivax hypnozoite. 

Like many malaria researchers, we were frustrated by the lack of efficient experimental platforms to discover and evaluate new antimalarial drug and vaccines targeting the pre-erythrocytic stages. Standard methods for liver stage studies using large-format culture systems required more infective parasites with limited scalability and the reliance on hepatoma lines did not adequately support complete pre-erythrocytic stage development. To overcome these limitations, we focused this project on advancing use of primary human hepatocytes (PHHs) as a model platform in a 384-well format that could be scaled up for preclinical drug and vaccine screening. The decision to use the 384-well culture format was fortuitous since the format improved long-term cultivation of PHHs when combined with small modifications in culture methods and with suitably cryopreserved PHHs.

Most antimalarial drugs do not kill hypnozoites and the main anti-hypnozoite drug, primaquine, has potentially severe side effects limiting its use in malaria-endemic communities. Therefore, the desired liver model needed to support extended culture time to study reactivation of the P. vivax hypnozoite stages (>21 days) and allow complete development to blood-stage. The second important goal for P. vivax liver stage platform was to enable using it as a reliable source of blood-stage parasites since continuous in vitro blood culture of this parasite has not been possible.  

A key part of our successful liver model was finding a reliable source of cryopreserved PHH and identifying reliable biomarkers predictive of successful infection outcomes. Specifically, we found that formation of active bile canaliculi, high mitochondrial activity, and sustained hepatocyte health was a strict requirement for sporozoite invasion (Figure 1). Intriguingly, these biomarkers not only aided in selection of PHH donor lots but also revealed an association with developing Plasmodium schizont size (um2). In hindsight this makes a lot of sense that ‘happy’ physiologically-healthy hepatocytes are better for the parasite, too.

Using the new 384-well plate culture system, primary human hepatocytes (PHHs) maintain normal physiology with high functioning bile acid transport (green) and mitochondrial activity (red) long term (day 21 post-seed pictured). PHH nucleus is represented by blue. Scale bar (white) represents 100 microns.

In conjunction, with improving the health of the hepatocytes, we also focused on improving the health of infectious sporozoites. Previous studies had demonstrated that the dissection buffer in which sporozoites were isolated from mosquitoes could have a tremendous impact on their viability. Schneider’s insect medium without additives and a neutral pH is now our preferred dissection medium and we attribute its use to the higher rates of infection and development that we have achieved, including with cryopreserved sporozoites (Figure 2). Additional studies are underway to determine the basis of these enhancements in insect media and what further improvements can be accomplished

Using the new 384-well plate culture system, primary human hepatocytes (PHHs) maintain normal physiology with high functioning bile acid transport (green) and mitochondrial activity (red) long term (day 21 post-seed pictured). PHH nucleus is represented by blue. Scale bar (white) represents 100 microns.

Finally, we are especially pleased with the simplicity of our liver model giving global access to P. vivax and P. falciparum liver stages and allowing the assay to be established in any research laboratory with basic cell culture facilities. The basic materials are commercially available without IP restriction and no specialized equipment is needed. This ease of use has already facilitated establishment of multiple malaria research labs, including in endemic countries.

By Alison Roth and John Adams

The paper in Nature Communication is here: https://go.nature.com/2L0hZmT

Go to the profile of Alison E. Roth

Alison E. Roth

Research Associate, Center for Global Health and Infectious Diseases Research, University of South Florida

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