Flow cytometry-based evaluation of hepatic infection by non-fluorescent Plasmodium parasites

The complex life cycle of Plasmodium parasites, involving both liver and blood stages of infection in the mammalian host, presents significant challenges for malaria research. Although advances have been made in malaria vaccination and treatment strategies, important gaps in our understanding of the asymptomatic liver stage of Plasmodium infection remain. While reporter gene-expressing parasites are commonly used for drug screening and parasite biology studies during this phase of the Plasmodium life cycle, tools for assessing and quantifying hepatic infection in the absence of parasite-encoded reporter genes are limited. Here, we present a novel flow cytometry-based method that enables the quantitative assessment of infection of hepatic cells by non-fluorescent Plasmodium parasites. This method uses two parasite proteins, heat shock protein 70 (HSP70), found in the parasite cytoplasm, and upregulated in infectious sporozoites 4 (UIS4), located on the parasitophorous vacuole membrane, as markers for parasite detection and quantification. We demonstrate that the use of these markers facilitates the rapid and cost-effective quantification of hepatic infection and intracellular development of Plasmodium parasites devoid of fluorescent reporter genes. This method addresses critical regulatory and technical challenges to the evaluation of reporter-free whole-sporozoite vaccine candidates and could serve as a versatile tool for broader malaria research. Author SummaryPlasmodium parasites, the causative agents of malaria, initially infect their mammalian hosts liver, where they replicate silently before entering the bloodstream and triggering disease. The hepatic stage of infection is a critical target for vaccine and drug development, but remains technically challenging to study, particularly when using parasite lines that do not express fluorescent or luminescent reporter genes. Reporter-free parasite lines are often required for regulatory reasons, particularly in the context of whole-sporozoite vaccine research. To address this limitation, we developed a flow cytometry-based method that enables the detection and quantification of hepatic infection by reporter-free Plasmodium parasites. The approach relies on the detection of two parasite proteins, HSP70 and UIS4, enabling the quantification of infected cells and the assessment of intracellular parasite development. This method is rapid, scalable, and cost-effective, and can be applied to Plasmodium lines relevant for vaccine studies. By facilitating the analysis of hepatic infection in the absence of reporter genes, our approach expands the experimental toolkit available for malaria research and supports ongoing efforts to develop interventions that target this clinically silent but biologically essential stage of the parasites life cycle.