A tracer-based method enables tracking of Plasmodium falciparum malaria parasites during human skin infection.

Introduction: The skin stage of malaria is a vital and vulnerable migratory life stage of the parasite. It has been characterised in rodent models, but remains wholly uninvestigated for human malaria parasites. To enable in depth analysis of not genetically modified (non-GMO) Plasmodium falciparum (Pf) sporozoite behaviour in human skin, we devised a labelling technology (Cy5M2, targeting the sporozoite mitochondrion) that supports tracking of individual non-GMO sporozoites in human skin. Methods: Sporozoite labelling with Cy5M2 was performed in vitro as well as via the feed of infected Anopheles mosquitos. Labelling was validated using confocal microscopy and flow cytometry and the fitness of labelled sporozoites was determined by analysis of infectivity to human hepatocytes in vitro, and in vivo in a rodent infection model. Using confocal video microscopy and custom software, single-sporozoite tracking studies in human skin-explants were performed. Results: Both in vitro and in mosquito labelling strategies yielded brightly fluorescent sporozoites of three different Plasmodium species. Cy5M2 uptake colocalized with MitoTracker® green and could be blocked using the known Translocator protein (TSPO)-inhibitor PK11195. This method supported the visualization and subsequent quantitative analysis of the migration patterns of individual non-GMO Pf sporozoites in human skin and did not affect the fitness of sporozoites. Conclusions: The ability to label and image non-GMO Plasmodium sporozoites provides the basis for detailed studies on the human skin stage of malaria with potential for in vivo translation. As such, it is an important tool for development of vaccines based on attenuated sporozoites and their route of administration.

Quantification of wild-type and radiation attenuated Plasmodium falciparum sporozoite motility in human skin.

Given the number of global malaria cases and deaths, the need for a vaccine against Plasmodium falciparum (Pf) remains pressing. Administration of live, radiation-attenuated Pf sporozoites can fully protect malaria-naïve individuals. Despite the fact that motility of these attenuated parasites is key to their infectivity and ultimately protective efficacy, sporozoite motility in human tissue (e.g. skin) remains wholly uncharacterized to date. We show that the ability to quantitatively address the complexity of sporozoite motility in human tissue provides an additional tool in the development of attenuated sporozoite vaccines. We imaged Pf movement in the skin of its natural host and compared wild-type and radiation-attenuated GFP-expressing Pf sporozoites. Using custom image analysis software and human skin explants we were able to quantitatively study their key motility features. This head-to-head comparison revealed that radiation attenuation impaired the capacity of sporozoites to vary their movement angle, velocity and direction, promoting less refined movement patterns. Understanding and overcoming these changes in motility will contribute to the development of an efficacious attenuated parasite malaria vaccine.