Plasma-derived exosomes from Plasmodium vivax infected liver-chimeric (huHep) FRG mice as novel biomarkers of liver infection

Background: Increasing evidence is emerging regarding the importance of extracellular vesicles in malaria infections. Exosomes are extracellular vesicles that carry biomarkers of disease, including liver diseases, and regulate physiological processes. A unique aspect of Plasmodium vivax is the presence of hypnozoites, latent hepatic stages causing clinical relapses.The human liver-chimeric (huHep) FRG mice are a robust P. vivax infection model that allows complete exo-erythrocytic development of liver stages including merozoites maturation and the capacity to invade human reticulocytes. Most relevant, the huHep mice support latency and growth of dormant liver stages sensitive to primaquine. Here, we employed a proteomics approach to characterize the content of plasma-derived exosomes in this P. vivax humanized mouse liver model. Methods: Exosomes were purified from plasma of FRG huHep mice infected with P. vivax at different infection time-points by size exclusion chromatography (SEC). Exosomes were characterized by nanoparticle tracking analysis for estimating size and concentration and by bead-based flow cytometry for the presence of CD5L, a novel exosomal marker. SEC fractions containing CD5L-positive exosomes were analyzed by Liquid Chromatography (nanoLCULTRA-EKSIGENT) followed by mass spectrometry (LC-MS/MS). Peptides were searched against P. vivax, human and mouse protein databases. Protein identification was based on the presence of unique peptides and a False Discovery Rate <1%. Results: Molecular analysis of the exosomal marker CD5L showed that purified exosomes from plasma of FRG huHep mice were enriched in SEC fractions (7-8-9-10) showing a mean size of 121.21 nm and 2.29x1011 particles/ml. Proteomic analysis of these fractions showed the presence of 348 and 281 proteins from mouse and human origin, respectively, including canonical exosomal markers. Remarkably, we identified P. vivax proteins including blood and liver stage as well as uncharacterized proteins. Candidate proteins are being expressed as GST-fusion-proteins in a cell-free wheat germ system to generate tools that will facilitate multiplex studies with sera from P. vivax infected patients, including sera from true relapsing patients. Conclusion: This study represents proof-of-principle that plasma-derived exosomes from P. vivax FRG huHep mice contain parasite and human hepatocyte proteins with the potential to unveil biological features of liver infection and to identify hypnozoite biomarkers.