Desarrollo de nuevas estrategias de vacunacion frente a la infeccion por el virus de la hepatitis de la marmota (whv). Estudio de posibles mecanismos de evasion del whv

Resumen

TITULO: DESARROLLO DE NUEVAS ESTRATEGIAS DE VACUNACIÓN FRENTE A LA INFECCIÓN POR EL ¿ VIRUS DE LA HEPATITIS DE LA MARMOTA (WHV). ESTUDIO DE POSIBLES MECANISMOS DE EVASIÓN DEL WHV RESUMEN: The primary objective of this work is the development of new therapeutic strategies against viral hepatitis chronic infection. The reason for the chronicity of hepatitis B virus (HBV) infection is widely assume to be related with a suboptimal immune response against the virus. Thus, the induction of a strong and multispecific Ti type immune response could represent an effective treatment modality against chronic hepatitis infection. One of our main aims is the development of therapeutic vaccine strategies to improve chronic hepatitis B treatment outcome, using the best animal model for chronic hbv infection: woodchucks chronically infected with the woodchuck hepatitis virus (whv). First as preliminary step we tested our different vaccination strategies in mice: a) Dendritic Cells (dcs) pul sed with viral antigens, b) Recombinant viral vectors expressing viral antigens, c) Non viral vectors like Polyethilenimina (PEÍ) carrying plasmids expressing viral antigens (core or surface protein of the whv). To evaluate the efficacy of different protocols of immunization in BALB/c mice, we have determined several CD8 epitopes from whv core and surface antigens. Next we analysed CD4 and CD8 immune response obtained after immunization with the protocols previously described. We have also tested the capacity of the different strategies of immunization to induce protection against a viral or tumor challenge. Our results showed that the best protocol of immunization was the administraron of DCs transduced with first generation ¿ adenovirus expressing whv core antigen. %&/on the other hand our data indicate that the expression of WHV core protein in WHV-infected APCs do not affect their immunostimulatory function, causing normal priming of CD4 lymphocytes. However, the immune response and the protection against viral challenge were very low in comparison with the immune response obtained after transduction of DCs with other viral antigens. Also we show that Adcore markedly attenuates hepatocellular apoptosis and the increase in serum transaminase levéis after concanavalin a challenge. The antiapoptotic status of infected liver cells may represent a mechanism ravoring viral persistence. Study of mecanisms implicates on this antiapoptotic state of HBV-infected hepatocites could offer a new opportunity for effectively manaqing persistent HBV infection. Finally, we generated and characterized DCs from woodchuck peripheral blood mononuclear cells (wdcs) by culturing them in the presence of human IL-4 and GM-CSF. Our results demonstrated that wdcs are functional and induced antigen specific immune response against adenovirus expressed protein. Following transfection with a recombinant adenovirus wDCs can be used as a feasible and effective tool for eliciting WHV-specific T-cell responses indication their potential to serve as prophylactic and therapeutic vaccines. The significance of our results resides in the importance of woodchucks as the best predi ni cal animal model for the development of new prophylactic and therapeutic strategies against HBV infection and also the development of hepatocellular carcinoma (HCC).