Leveraging access to parasite natural diversity to identify Plasmodium vivax culture-adaptable strain.

Plasmodium vivax (Pv) poses a significant challenge due to its extensive geographical distribution, resulting in a considerable burden of morbidity and mortality in regions where it is endemic. Diverging from Plasmodium falciparum (Pf), Pv manifests distinct characteristics, notably the presence of dormant liver stages and the inherent impediment to in vitro cultivation. These peculiarities present formidable obstacles to Pv malaria elimination. The development of a robust and reproducible in vitro culture system for Pv would be a game- changer to design efficacious anti-malarial strategies. The Phase I of our project aims to establish robust and long-term stable in vitro cultures of Pv clinical isolates. Our main advantage lies in having access to multiple clinical isolates thanks to our mobile laboratories installed in endemic regions of Cambodia, at the bedside of infected patients. This infrastructure enables us to systematically conduct in vitro culture tests on several hundred fresh and healthy Pv strains. This initial approach will facilitate the selection of Pv strains possessing a genetic background adapted for in vitro culture. The second part of the project will investigate optimal culture conditions, including, but not limited to, culture medium composition or impact of different concentrations of O2 and CO2 on in vitro survival of Pv using a specifically designed hypoxic chamber. We will explore hematopoietic stem cells as sources of reticulocytes and assess the potential of human mesenchymal stromal cells as support cells for Pv survival in vitro. Finally, we aim to establish a 3D organoid model capable of reproducing the specific conditions of the bone marrow, recognized as a primary site of Pv development. The third part of the project will utilize strains with the highest potential for in vitro culture to investigate the in vitro modification of Pv genome using Crispr Cas9. As a proof of concept, we will insert a gene encoding GFP. Regarding the Phase II of the project (R33) we will employ the genetic modification method developed in Phase I to establish different strains of genetically modified Pv. This aims to study various mechanisms of drug resistance and identify potential resistance markers. Our objective includes the study of the overexpression of the PvCRT or PvMDR1 genes and the expression of the V552I and Y976F variants in the Pvkelch12 and PvMDR-1 genes, respectively in order to explore resistance to the main drugs currently used in Pv treatment. This study has the potential not only to provide tools for a better understanding of Pv biology but also to identify potential drug resistance markers crucial for current malaria control strategies.