Preparation And Immune Characteristics Of Hepatitis C Virus Specific Exosome From Dendritic Cells

The hepatitis C virus (HCV) is a major pathogen, establishing persistent infection in more than four-fifths of those who contact it, and it is dramatically related with cirrhosis and hepatocellular carcinoma, which is a serious, worldwide public health problem. Up to now, there are no effective treatment measures available, and developments of a vaccine against HCV also have many impediments. Fortunately, dendritic cells (DCs), bridging innate and adaptive immunity, play a crucial role in clearing HCV, and exosome (Ex) derived from them is abundant of MHC class I and II complexes, hot shock protein 70 chaperones, costimulatory molecules. These properties provide exosome the necessary machinery, like Trojan horse, to transfer immune activation information and elicit antigen-specific immune responses. In theory, if we load HCV antigen messages to Ex, the immunogenicity of antigen would be highly promoted resulting in effectively eliciting cellular and humoral immune responses. This warrant researchers a novel strategy to develop HCV vaccine.Enlightened by the above academic hypothesis, we manage to demonstrate the potentiality of Ex. In this research, two parts of experiments were performed as following:1,Preparation and identification of DC-Ex.①Preparation of DC-Ex To investigate the effect of different concentrations of uric acid on DC-Ex, dendritic cells were cultured for 24 h in the medium supplemented with uric acid at 0.05, 0.1, 0.2 and 0.4 mmol/L respectively. To monitor the kinetics of exosome production from dendritic cells, a small volume (1 ml) of culture supernatant was harvested and analyzed for exosome surface specific maker CD80, CD86, using anti-CD80, anti-CD86 capture ELISA assay.The classical differential centrifugation isolation technique of exosome is a more lengthy and variable process. So we establish a novel method named ultrafiltration centrifugalization. Briefly, the culture supernatant of murine dendritic cells line (DC2.4) was collected and clarified through a hollow fiber cartridge to remove cells and their debris. The clarified supernatant was concentrated by Amicon ultrafiltration tube,and then the concentrated supernatant was purified when loaded into density gradient centrifugation to obtain the DC-Ex.Control group were applied by traditional differential centrifugation method.②Identification of DC-Ex Transmision electron microscope (TEM) was used to identify the morphology of exosome. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western Blot were employed to analyze their proteins. 2,Properties of immune responses elicited by HCV specific DC-Ex.Firstly, HCV core protein C was employed to stimulate DC2.4 cell lines to generate hepatitis C virus specific exosome in vitro. Subsequently, spleen cells of C57BL/6 mouse were cocultured with HCV specific DC-Ex, and finally, capture ELISA was applied to detect the level of IL-6, IL-12 and IFN-γin order to identify its'capacity of presenting antigen message, eliciting antigen-specific immune responses.Results of the experiments:1,Uric acid is a crucial regulatory factor significantly impacting the production of exosome in a dose-dependent manner. Especially when the uric acid concentration is 0.1 mmol/L, the exosome amount reaches peak amplitude.2,The technique we modified, termed"ultrafiltration centrifugalization", is a convenient and useful method for DC-Ex purification. As analyzed by electron microscopy, exosomes isolated by this method present a characteristic"cup shaped"morphology, a flattened sphere limited by a bi-lipidic layer, 60-90 nm in diameter, abundant of characteristic molecular markers MHC class I,MHC class II, CD80 and CD86. Compared with traditional method,ultrafiltration centrifugalization technique is a shorter and more producible process resulting in production being highly purified.3,HCV-specific exosome could efficiently present antigen message and induce spleen cells to generate high level IL-6, IL-12 and IFN-γ.Rudiment conclusion:Taken together, we concluded, under the induction of uric acid, ultrafiltration centrifugalization method could successfully obtain large quantities of highly purified exosome. HCV-specific exosome could efficiently present antigen message, elicit antigen-specific immune response. All those indicate that DC-Ex-based vaccine may be a potent candidate for HCV prevention and treatment.