Induction Of Immune Response Against Hepatitis B Virus By Immunization With HBsAg Gene-modified Dendritic Cells Transduced With Recombinant Adenovirus

Hepatitis B virus (HBV) is the most common etiologic agent for infectious liver disease. Following initial infection, some individuals fail to resolve their infection and thereby become chronic carriers and there is a significant association between persistent infection and liver cirrhosis or hepatocellular carcinoma.CD8~+ T lymphocytes are an important component of host defense mechanisms responsible for HBV clearance in patients with acute hepatitis. Acutely infected patients characteristically produce a vigorous, polyclonal, and multispecific cytotoxic T lymphocytes (CTL) response that is usually sufficient to clear the infection, while persistently infected patients produce a weak or undetectable HBV-specific CTL response. It has been proposed that elimination of virus is due to CTL-mediated lysis of infected hepatocytes and/or antiviral effects of CTL-derived cytokines, such as IFN-y and TNF-a. Based on these observations, therapeutic enhancement of the T cell response to HBV has the potential to terminate chronic HBV infection.Chronic HBV infection is associated with T cell hyporesponsiveness or tolerance, although the precise mechanism responsible for this phenomenon is not entirely clear. Dendritic cells (DC) are the most potent antigen-presenting cells andplay a central role in the induction of antiviral immune responses. It has been shown that monocyte-derived DC (MoDC) from patients with chronic HBV infection are functionally impaired.HBV Transgenic mice (HBV-Tg) contain the complete HBV genome, express all HBV gene products, and replicate HBV in their hepatocytes similar to chronically infected patients, but they do not develop chronic hepatitis because they are immunologically tolerant to the viral Ags. The availability of these mice provides a model system to evaluate immunotherapeutic strategies to break tolerance and terminate persistent HBV infection.Thus far, several approaches to overcome CTL tolerance in HBV transgenic mice have been investigated. It has been demonstrated that HBsAg- or peptide-pulsed DCs can induce quantitatively normal HBsAg-specific CTL response, stronger than that induced by recombinant HBsAg vaccine or plasmid DNA immunization in HBV-Tg. However, they had no effect on viral replication and they did not cause hepatitis, suggesting that more efficient strategies must be developed to generate a functionally effective CTL response. On the other hand, it has been demonstrated that replication-defective recombinant adenovirus-transduced DCs more efficiently prime hepatitis C virus- or tumor antigen-specific cytotoxic T lymphocytes than specific antigen-pulsed DCs.In the present study, we constructed replication-defective recombinant adenovirus expressing HBsAg 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 HBsAg-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 HBsAg 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 adenovirusvector expressing HBsAgObjectiveTo construct a recombinant adenovirus vector expressing HBsAg and examine its efficiency of transfection and the expression of target antigen to explore the use of adenovirus vector in HBsAg gene-modified DCs in priming HBV-specific immune response.MethodsFor preparation of recombinant adenoviruses expressing HBsAg (Ad-S),β-galactosidase (Ad-lacZ) or green fluoresent protein (Ad-GFP), the AdEasy? XL Adenovirus Vector System was used. These vectors were replication-deficient adenovirus type 5 (Ad 5) deleted for the genes El and E3. Transgenes were first cloned into the p-shuttle-CMV vector (PSC-S, PSC-lacZ or PSC-GFP). The resultant plasmids were then linearized using Pme I and transformed into BJ5183-AD-1 cells to...