Development of a novel method to characterize and functionally analyze extracellular vesicles isolates from the plasma of HIV-1 positive patients.

ANDREA MORALES; PEREYRA GERBER, PEHUEN; DUETTE, GABRIEL; RUBIONE, JULIA; MATIAS OSTROWSKI

Mar del Plata

Congreso; LXIV Reunión anual de la Sociedad Argentina de Inmunología; 2016

Sociedad Argentina de Inmunología

Extracellular Vesicles (EVs) encompass a heterogeneous groupof structures surrounded by a lipid bilayer, which are produced andsecreted to the extracellular space by almost every cell type. Consequently,EVs are present in several biological fluids, including plasma.Given the capacity of EVs to transfer molecules from a donor to atarget cell, they are considered to mediate a novel and particulartype of intercellular communication, with involvement in a numberof physiological and pathological processes. The main hypothesisof our team is that EVs present in the plasma of patients chronicallyinfected with HIV-1 promote immune activation and inflammation, thuscontributing to the pathogenesis of the immune disorders observed inthese patients. A major limitation to address this question is obtaininglarge amounts of highly pure plasma-derived preparations of EVs.Here, we developed a method that combines the ability of SizeExclusionChromatography (SEC) to separate vesicles from solubleproteins from plasma followed by a high-molecular cut-off ultrafiltrationdevice to concentrate purified EVs. Finally, we set up a bead-basedimmunocapture assay to phenotypically characterize purified EVsusing a panel of antibodies against vesicle-enriched proteins by flowcytometry. Overall, this method is fast, reproducible, quantitative andinexpensive, allowing to overcome many of the actual technical limitationsin the field. Moreover, even if our main interest is to analyze EVsfrom HIV-1 chronically-infected individuals, this method could also beapplied to the analysis of EVs in other pathologies, such as cancer.