Sleeping Beauty : tackling the dormant Plasmodium vivax hypnozoite

Abstract

Malaria, named for 'bad air' in Italian, is one of the oldest diseases known, and has brought down explorers, popes, kings and emperors through centuries. Yet, this mosquito-borne disease still evades all attempts at eradication, and puts almost half the global population at risk of infection. Malaria is most commonly known as a blood disease, but all malaria species have an initial obligate, yet clinically-silent development stage in the liver, before the symptomatic and cyclic infection of erythrocytes begins. It is during the liver stage that Plasmodium vivax (P. vivax), the most widely distributed human-infecting malaria species, harbors dormant forms called hypnozoites which can linger for weeks to months, and then relapse to cause recurrent blood stage infection. This dormant parasite reservoir is one of the biggest barriers to malaria eradication, yet very little is known about its biology.

Furthermore, there is a dire need for the development of new hypnozoite-killing drugs but phenotypic screens are hindered by a lack of in vitro platforms. In this work, I set out to develop an in vitro liver stage P. vivax model which could help elucidate the mysterious biology of hypnozoites and could serve as an antimalarial screening platform. As an added challenge, P. vivax parasites that are suitable for liver stage infection cannot be obtained outside of endemic settings. Thus the majority of the work in this thesis was performed in Thailand, where the entire liver stage of P. vivax was recapitulated using a multi-well culture format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs) using clinical P. vivax isolates. MPCCs feature key aspects of P. vivax biology, including establishment of persistent hypnozoites and growing schizonts, merosome release, and subsequent infection of red blood cells.

The platform was piloted as a tool to test existing and candidate anti-hypnozoite drugs, and further miniaturized to be suitable for high-throughput screening. Finally, a hybrid capture strategy and RNA sequencing was employed to describe the first transcriptome of any human malaria species and gain insight into hypnozoite biology. Taken together, the work presented here has already identified unique aspects of hypnozoite biology, a form that has remained a relative biological mystery since its discovery 3 decades ago. Future work offers the unique potential to gain further biological insights into P. vivax development in human hepatocytes, and represents a screening platform for candidate drugs directed against distinct stages of P. vivax.