Do exosomes from natural infections can help in the development of a continuous in vitro culture system for Plasmodium vivax malaria?

Background Research on P. vivax is often neglected due to insufficient biological understanding of the parasite caused by the lack of research tools such as a robust in vitro culture system. P. vivax has a unique tropism for reticulocytes, young red cells which in their process to differentiation to mature red blood cells are known to secrete exosomes, nanovesicles of endocytic origin carrying selective cargo. In addition, our own data suggest that P. vivax-infected reticulocytes cytoadhere to the human spleen. Therefore, we hypothesize that plasma-derived exosomes from vivax patients may act as spleen communication signals inducing growth factors, which in turn might be needed for the exponential growth of the parasite in vitro. Material and Methods. Immortalized hHSCs (HuDEP2 cells) were expanded and differentiated to reticulocytes (Kurita et al, 2013). Plasma-derived exosomes from vivax patients (PvEX) and healthy donors (hEX) were purified by size exclusion chromatography and characterized by NTA, FACS, and proteomic analysis. For uptaken experiments, PKH67-labelled PvEX and PKH26-labeled hEX were incubated with human splenic fibroblasts (1010T cells) and observed by confocal microscopy. Results Expansion and differentiation of the HuDEP2 cell line proved highly variable and yielding circa 17% of reticulocytes with low viability. NTA analysis of plasma-derived exosomes revealed twice as much exosomes in patients as opposed to healthy donors. This was in agreement with quantitative FACS analysis of the transferrin receptor (CD71), an exosomal marker of reticulocytes. Mass spectrometry analysis identified parasite proteins in PvEX. Incubation of PvEX/hEX with 1010T cells showed that there was a significant higher uptake of PvEX as compared to hEX.