Kumari, G.; Chattopadhyay Pal, A.; Singh, P.; Ben Mamoun, C.

Babesia microti, the predominant cause of human babesiosis, has long eluded continuous cultivation in human erythrocytes, limiting our understanding of intraerythrocytic biology. Here we describe the development of the first in vitro culture system that supports continuous propagation of B. microti in human erythrocytes. Using this system, we assessed the intrinsic activity of clinically used antibabesial therapies and searched for key metabolic vulnerabilities that could be exploited for improved treatment. This approach identified an essential requirement for host-derived vitamin B5 during intraerythrocytic development. Leveraging this dependency, we identified MMV689258 as a potent inhibitor of parasite growth. Together, this continuous culture platform establishes a foundation for mechanistic and therapeutic studies of B. microti and provides a rational framework for developing improved interventions for human babesiosis.