HCV C-FC Engineered Mice Dendritic Cells In Enhancing Immune Response Against HCV

Hepatitis C virus (HCV) is the major causative agent of non-A, non-B hepatitis throughout the world with more than 170 million people infected . The majority of infected patients are unable to clear the virus and many develop chronic liver disease, cirrhosis and hepatocellular carcinoma . It is not clearly known how an individual develops a chronic hepatitis virus carrier state; however, a defective immune response of the host is thought to play a critical role in the underlying pathogenetic mechanism.Dendritic cells and Langerhans cells are specialized for the recognition of pathogens and have a pivotal role in the control of immunity.Inadequate Ag presentation by APCs contributes to the failure of the human immune system to mount effective immune responses against chronic infection and tumors. Accumulating evidence indicates that a vaccine or immunotherapy, which can induce combined CD4+ and CD8+ T cell and B cell immune responses, is likely the most effective one to prevent or control chronic infections such as hepatitis virus infection, or tumors.Efficient Ag presentation is essential to induce effective cellular and humoral immune responses. Thus, one central goal of current immunotherapy and vaccine development is to enhance Ag presentation to induce potent and broad immune responses. Here, a novel Ag presentation strategy is developed by transducing dendritic cells (DCs) to produce an Ag for presentation as an exogenous Ag to efficiently induce both humoral and cellular immunity. The principle of this strategy is illustrated by genetically modifying DCs to secrete a model hepatitis C virus Ag fused with a cell-binding domain and to process the fusion Ag as an exogenous Ag after receptor-mediated internalization for MHC class I and II presentation. Vigorous Ag-specific CD4+ helper and CD8+ cytotoxic T cell, as well as B cell, responses were induced by the transduced DCs in mouse models. Thus, this novel strategy uses a receptor-mediated internalization process to efficiently induce all arms of the adaptive immunity and may provide a powerful means to develop potent vaccines and immunotherapies.In this study, we design a novel Ag presentation strategy by transducing DCs to produce and secrete a fusion protein consisting of a hepatitis C virus (HCV) core protein HCV C Ag fused with a cell-binding domain such as the Fc fragment of IgG. The secreted fusion proteins, in addition to inducing Ab responses, are transported back to DCs via receptor-mediated internalization. It has been demonstrated that Ag presentation by receptor-mediated internalization of DCs can be enhanced up to 1000-fold, compared with fluid phase Ag pinocytosis. As a result, the fusion Ags are processed in the endosomal pathway and presented by DCs as exogenous for MHC-II presentation to induce CD4+ Th cells. The internalized Ags can also be presented to MHC-I (cross-priming) byDCs to directly activate CTLs. Thus, this strategy uses a unifying mechanism to activate all arms of the adaptive immunity. In this study, we demonstrate that this receptor-mediated Ag presentation strategy is able to induce vigorous Th cell, CTL, and B cell responses against the model HCV nucleocapsid protein in mouse models. The results are as following:1 Construction and expression of fusion proteins HCV C-FcTo construct a recombinant cherimal plasmid of HCV-Fc which can express HCV core gene and IgG Fc gene ,which should be useful to transfect dendritic cells and changed into dendritic cell vaccince. The IgG Fc gene derived from the plasmid pCMVsFc by using polymerase chain reaction(PCR) was inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of HCV C plasmid(pcDNA3HCV C) then the recombinant plasmid pcDNA3.HCV-Fc was obtained. Resules showed that the insert DNA of pcDNA3 HCV-Fc was conformed HCV core and Fc gene by endonuclease, PCR and sequencing. HCV core gene and Fc gene was expressed transiently with Lipofectamine 2000 coated in human hepatoblastoma 7721 cells which conformed by immunofluorescence.Recombina...