Continuous in vitro culture and genetics of Plasmodium vivax in a bespoke blood cell bioreactor

Project Summary Malaria resulting from infection of humans with the parasite Plasmodium vivax parasite remains a significant public health problem. Significant gaps remain in our understanding of P. vivax blood- and transmission-stage biology, as well as the development of drug and vaccine interventions. When compared to that of Plasmodium falciparum, this knowledge lags substantially because of the lack of an in vitro culture system. Indeed, much of our undersatnding of P. vivax is based on limited studies in human and animal model systems. We hypothesize that mimicking physiologically-relevant conditions experienced by P. vivax will support continuous culture in reticulocytes, the youngest of red blood cells and the host cell of the parasite. Our long-term objective is to continuously culture P. vivax parasites. We will utilize a bioreactor to control the culture environment to facilitate P. vivax growth in in vivo-like conditions, including in co-cultures with stromal cells from the bone marrow and splenic niches. In parallel, we will draw upon our expertise in in vitro genetics for both RBCs in vitro and Plasmodium parasites: we will engineer stem cells capable of producing reticulocytes more susceptible to P. vivax infection and recombinant parasites with enhanced ability to invade red blood cells. We thus will focus on optimizing parasite and reticulocyte health, as well as host-parasite interactions. Our specific aims in the R61 period are to optimize P. vivax culture in a bespoke bioreactor wherein we will mimic the in vivo physiological conditions of parasite proliferates. This includes: (1) the efficientproduction of host reticulocytes to support P. vivax culture; (2) the optimization of culture conditions in a bespoke bioreactor using iterative machine learning approaches; and (3) the use of diverse P. vivax parasites. In the R33 period, we will develop genetic methods for P. vivax, and use these to conduct a genetic screen to understand and overcome the restriction of P. vivax to reticulocytes. 01/09/2024