Identification of Plasmodium vivax proteins in plasma-derived exosomes from natural infections and analysis of their biodistribution in rodent models.
Introduction: Malaria caused by Plasmodium vivax is responsible for nearly 20 million yearly clinical cases including severe disease and death. Our present research effort is focused on the molecular characterization of plasma-derived exosomes. Exosomes are nanovesicles of endocytic origin recently shown to be involved in inter-cellular communication and whose selective-cargo is being explored as novel therapeutic agents and diagnostic markers in parasitic diseases (Marcilla et al., J Extracell Vesicles 3: 25040, 2014). Methods: Plasma was obtained from infected patients in Manaus (Brazil) as well as from healthy volunteers. Exosomes were isolated by Size exclusion chromatography (SEC) and characterized by NTA and bead-based assays following our own procedures (de Menezes- Neto et al., 2014 JEV). LC-MS/MS analysis was performed using different pre-treatments, digestions, and mass spectrometers. Biodistribution of NIR815-labelled exosomes was performed by retro-orbital injection of exosomes into black C57 mice and nude mice. Results: NTA analysis showed that plasma-derived exosomes were significantly more abundant in patients than in healthy volunteers. This was in agreement with quantitative FACS analysis of the transferrin receptor (CD71) contained in these exosomes. The proteomics analysis revealed the presence of 21 parasite proteins with an FDR<1% and at least two peptides in an individual sample or a single peptide in more than two samples. In vivo biodistribution of NIR815-labelled plasma-derived exosomes from infections in two different strains of mice demonstrated spleen-specific tropism. Conclusions: Plasma-derived exosomes from infections contained parasite proteins that can be explored as novel biomarkers of infection and antigens for vaccination. Plasmaderived exosomes from infections have spleen-specific tropism suggesting and as yet to be discovered intercellular communication role in this organ.