Induction Of Immune Response Against Hepatitis B Virus By HBcAg Gene-modified Dendritic Cells Vaccine

Chronic Hepatitis B virus (HBV) is one if the most common infectious diseases worldwide and leads to high morbidity and mortality due to the development of cirrhosis and hepatocellular carcinoma.The formation of immune tolerance is the center of chronicity of Hepatitis B, and is related to HBV and host. The difference of immune response to HBV determines the chronicity of Hepatitis B. Antigen, antigen presenting cells (APC) and lymphcytes are important factors of determination of immune response.HBV persistently infected patients produce a weak or undetectable HBV-specific CTL response. It has been shown that monocyte-derived DC (MoDC) from patients with chronic HBV infection are functionally impaired. So, therapeutic enhancement of the T cell response to HBV has the potential to terminate chronic HBV infection.HBV Transgenic mice (HBV-Tg) are the model of persistent HBV infection and provide a model system to evaluate immunotherapeutic strategies to break tolerance and terminate persistent HBV infection.The particulate HBcAg is extremely immunogenic and can function both as aT-cell-dependent and T-cell-independent antigen. Immunization with HBcAg preferentially primes the Thl-type cellular immune response. During chronic HBV infection, HBcAg is the only antigen that was known to elicit a prominent immune response.In the present study, we constructed replication-defective recombinant adenovirus expressing HBcAg and studied the potential of DCs transduced with the recombinant adenovirus in priming HBV-specific immune response in transgenic and nontransgenic mice, compared its antiviral effect with that of HBcAg-pulsed DCs and plasmid DNA immunization, and evaluated its feasibility of adenovirus-transduced DCs as an immunotherapeutic vaccine. The results we obtained may provide more information about HBcAg gene-modified DCs and lay ground for explore more effective HBV therapeutic vaccine. This study includes three parts.Part IConstruction and examination of a recombinant adenovirus vectorexpressing HbcAgObjectiveTo construct a recombinant adenovirus vector expressing HBcAg and examine its efficiency of transfection and the expression of target antigen to explore the use of adenovirus vector in HBcAg gene-modified DCs in priming HBV-specific immune response.MethodsFor preparation of recombinant adenoviruses expressing HBcAg (Ad-C), theAdEasy^? XL Adenovirus Vector System was used. The vector wasreplication-deficient adenovirus type 5 (Ad 5) deleted for the genes El and E3. Transgenes were first cloned into the p-shuttle-CMV vector (PSC-C). The resultant plasmid was then linearized using Pme I and transformed into BJ5183-AD-1 cells to produce recombinant adenovirus (rAd) plasmids, which were identified as positive by Pac I digestion. These purified Ad plasmids were linearized with Pac I and subsequently transfected into Ad-293 cells to produce rAd. Further amplification of virus stocks was achieved by infection in Ad-293 cells. Finally, the virus particle titre was determined by Reed-Muench assay. The expression of GFP by Ad-GFP-transduced DCs was detected by flow cytometry, and the expression of HBcAg by Ad-C-transduced DCs, CHOand P815 cells was determined by RIA and Western Blotting.Results1. PCR and sequence analysis all provided the evidence that the PSC-C vector and the Ad-C plasmid we obtained contained the right and intact HBV C gene sequence.2. The virus particle titre of Ad-C was determined by Reed-Muench assay with resulting 50% tissue culture infective doses (TCID 50/mL) of 1 × 10⁸.3. The expression of GFP by Ad-GFP-transduced DC was 93.72 + 3.01 (%), which was detected by flow cytometry as the efficiency of transfection of rAd into DCs at an MOI of 100.4. The expressions of HBcAg were all positive in the cell supernatants ruptured membrane of Ad-C-transduced DCs, CHO and P815 cells, but the culture supernatants were almost negative, which were determined by RIA and Western Blotting.Conclusions1. The replication-defective recombinant adenovirus which expressed HBcAg(Ad-C) was constructed succ...