Establishing strains and conditions for long-term P. vivax cultures - PROJECT ABSTRACT Plasmodium vivax (Pv) is responsible for a significant portion of the malaria cases outside of Africa and causes significant socioeconomic burden worldwide. New therapies to treat Pv are needed, but a continuous, high yield culture system that enables the study of Pv's biology has not been established. Without this tool, development of new therapies is stymied. The long-term goal of this research program is to study Pv biology to inform the development of new treatments for Pv. The objectives of this proposal are to (1) establish continuous, high yield culture systems for Pv's asexual stages and (2) to use this culture system to test triggers of gametocytogenesis so gametocytes can be generated in vitro for study. This research is needed because it will yield a key tool to study Pv asexual and sexual stages, thereby, enabling mechanistic research to identify and develop new therapies. The major bottlenecks to establishing continuous cultures have been (1) Pv preferentially invades a specific subset of reticulocytes, or immature erythrocytes, in culture, and (2) low parasite numbers due to poor development even when reticulocytes are supplemented for invasion. In preliminary studies, our team has used P. cynomolgi (Pcy), a closely-related malaria parasite to Pv, to develop a strategy for addressing these obstacles. We have shown that Pcy strains vary in their ability to grow in culture with some strains growing in culture immediately whereas others adapt with time, suggesting that intrinsic variation within strains exists and can be selected for to establish continuous cultures. Using our Pcy strains that grow in culture, we have also shown that a bone marrow (BM) mimetic composed of secreted factors from BM mesenchymal stromal/stem cells (MSCs) significantly improves Pcy development in culture. Intriguingly, our data also indicate that BM reticulocytes may trigger the development of gametocytes. Based on these data and the prior literature, we propose to test the hypothesis that continuous cultures of Pv can be achieved by establishing Pv strains that invade multiple reticulocyte subsets in culture using conditions that mimic the BM where Pv thrives in vivo. This proposal will test our hypothesis by pursuing three specific aims. During the R61 phase, the experiments in Aim 1 will establish Pv strain(s) that robustly invade reticulocytes in culture while the experiments in Aim 2 will improve Pv development in vitro by mimicking the BM microenvironment. Together, these aims are expected to yield a continuous culture system for Pv asexual stages. After achieving this milestone, Aim 3 will use this culture system to test triggers of gametocytogenesis during the R33 phase. The expected outcomes of this research are a continuous culture system for Pv and new insight into Pv invasion, development and gametocytogenesis. This research will have an immediate impact on Pv research because it will provide essential tools that will enable mechanistic insight into Pv biology and provide new knowledge that may lead to new therapies.
