Investigations on the Malaria Hypnozoite – IMHyp

Efforts to eradicate malaria are thwarted by the persistence of Plasmodium vivax, a most globally prevalent parasite species, in areas where control measures have brought P. falciparum to near-elimination. The resilience of P. vivax is principally due to its ability to form dormant liver forms (hypnozoites), some of which activate over the months and years after the mosquito bite to cause relapse episodes that maintain transmission. The only licenced drug that kills hypnozoites (primaquine) has potentially life-threatening side effects that severely restrict its large-scale use. Finding a non-toxic replacement or an efficient vaccine has become a priority. High throughput screening for lead compounds is precluded as it can only be done using non-human primates infected by P. cynomolgi, a parasite of macaques similar in most respects to P. vivax. Moreover, little is know on hypnozoite biology or biochemistry to guide this search.

This proposal aims at gathering a consortium combining distinct and complementary expertise to conduct the first detailed biological and molecular observations on the enigmatic hypnozoite. The proposed work will focus on the P. cynomolgi/Macaca fascicularis model, and will capitalises on recent advances in in vitro cultivation of blood and liver parasites, genetic manipulation of parasites, genetic analyses of single-cells, and in candidate vaccine discovery. Its is proposed to establish the model at the IDMIT (CEA, Fontenay-aux-Roses), where the newly built animal facilities associated with state-of-the art technical platforms are ideally suited for investigation on non-human primates, and in collaboration with researchers at the CEA/IMVA who have extensive expertise in fundamental and pre-clinical research on viral and bacterial pathogens in M. fascicularis. Three main aims are proposed. First, to set up the protocols and techniques needed to access all stages of the parasite’s life cycle, and to establish standard infection protocols, a necessary step for all future investigations. We will capitalise on the exclusive availability of a P. cynomolgi strain that can be maintained in vitro to develop a toolbox to generate the genetically modified parasites needed for conclusive functional studies. Second, to conduct investigations on the fundamental nature of hypnozoite biogenesis in order to establish whether sporozoites are programmed to form hypnozoites or to develop immediately upon hepatocyte invasion. This will be undertaken by population and single-cell transcriptomic and proteomic analyses of purified sporozoites from P. cynomolgi and other species that do not form hypnozoites. Finally, to assess the protective efficacy of a parasite antigens cocktail, identified as potential vaccine candidates in a mouse model, and to assess for the first time the effect of immunisation on the relapse patterns.

We are aware of the rather ambitious nature of the proposed work, and of the risks inherent to the use of novel technologies to investigate the challenging hypnozoite. However, we are confident to gather sufficient advances to spark and sustain future projects that will expand our understanding of the genesis and control of dormancy in the hypnozoite. Such advances should not only accelerate the quest for novel methods against P. vivax and P. ovale (the second parasite of humans widely prevalent in Africa that forms hypnozoites), but also provide fundamental insights into cellular quiescence, a phenomenon now implicated in diverse biological and pathological processes. Ultimately, this project will serve as the first step in establishing a centre of excellence for research on the hypnozoite, that could then be broadened to encompass other fundamental, clinical and immunological aspects of the infection that are best investigated in non-human primates.