Exosomes From Lentiviral Vector-encoding Ubiquitinated HBcAg Transduced Murine Bone Marrow-derived Dendritic Cells To Stimulate Cytotoxic T Lymphocytes Response In Vitro

Objective:Chronic infection of hepatitis B virus(HBV)is a serious public health problem.At present,there are about 290 million chronic HBV infected people in the world,and about 780,000 people die each year from HBV infection-related chronic hepatitis and its complications.At present,there is no effective method to completely remove the hepatitis B virus in the patient’s body.Specific cellular immune response plays a key role in controlling HBV infection.Generating specific cytotoxic T lymphocytes(CTL)throμgh antigen stimulation is an effective way to clear the virus in patients with chronic HBV infection.DC derived exosomes(Dexs)derived from dendritic cell(DC)can induce immune responses capable of eradicating the virus,but antigen-loaded exosomes have not yet shown therapeutic potential in HBV infection.Therefore,this study aimed to investigate the antiviral effect of DC-derived exosomes(Dexs-Ub-HBc Ag)loaded with ubiquitinated HBV core antigen(Ub-HBc Ag).Methods:The murine bone marrow-derived DCs were extracted and cultured,and their maturation was stimulated in vitro,and the DCs were transfected with the recombinant lentivirus(LV)carrying the Ub-HBc Ag fusion gene.The fluorescent expression of green fluorescence protein(GFP)was observed under a fluorescent microscope,and analyzed by Image J software,and the proportion of cells expressing high-level green fluorescent protein was quantified.High-purity Dexs was obtained by differential centrifugation.The size distribution curve of Dexs in each group was analyzed and evaluated by Zeta VIEW nanoparticle tracking analysis system,verified by transmission electron microscope photography,the total protein concentration of exosomes in each group was measured by BCA protein assay kit,and the extracted protein was detected by Western blot method,to verify the extracted exosomes.Stimulate mouse spleen-derived T lymphocytes with Dexs-Ub-HBc Ag,and detect the immune response mediated by specific T lymphocytes,including analyzing the expression of cytokines by enzyme-linked immunosorbent assay,and detecting the proliferation of T lymphocytes by CCK8 method,Lactate dehydrogenase release method to detect HBc Ag-specific CTL activity.Results:The experiment successfully extracted and cultured murine myeloid-derived DC,stimulated its maturation in vitro,and successfully infected the recombinant chronic disease vector LV-Ub-HBc Ag into DC,and the infection efficiency reached 50-60%.The high-purity Dexs obtained by ultracentrifugation,its structural state under the transmission electron microscope,the results of particle analysis,and the expression of the outer fluid marker marker protein.After the purity and concentration met the requirements of the continuous experiment,the protein band of HBc Ag was observed in the Dexs-Ub-HBc Ag group.Dexs-Ub-HBc Ag can effectively stimulate T lymphocytes in vitro to separate cytokines such as IL-4 and IFN-γ,and stimulate T lymphocytes to increase muscle.The CTL activity of T lymphocytes induced by Dexs-Ub-HBc Ag is significantly higher for pairs.Conclusion:Dexs-Ub-HBc Ag can promote T lymphocytes to secrete Th1 cytokines in vitro,thereby stimulating the proliferation of T lymphocytes and inducing the production of HBc Ag-specific CTL responses.These results suggest that Dexs-Ub-HBc Ag has the potential to be developed as a future therapeutic option for HBV elimination.