Gene transfer of immunomodulatory cytokinesBiological and functional characterization of adenoassociated virus expressing Interferon-alpha and Interleukin-15 in a HBV transgenic mouse model

Resumen

Worldwide 350 million people suffer from HBV infection and approximately 1 million people die annually because of HBV-induced liver pathologies. The host immune response to HBV antigens is a critical factor determining the outcome of the infection. In this thesis we have developed an immune-gene therapy strategy for the treatment of CHB. As gene vehicle, we employed recombinant AAV viruses and the expression of two immunostimulatory citokines, IFN-α and IL-15. In the first part, we tested the functionality of both virus in vivo and determine the optimal dosage. Next, we tested the antiviral activity of both vectors in a highly tolerant animal model of CHB, HBV transgenic mice. We found that AAVIL-15 therapy increased serum levels of IL-15 bound to IL-15Rα leading to robust intrahepatic expansion of IFN-gamma-producing lymphocytes and increased HBV-specific CD8+ T-lymphocytes which, however, lacked cytolytic activity. Contrariwise, AAVIFN-α stimulated cytotoxic functions of HBV-specific CD8+T-cells without increasing their number. Notably, administration of AAVIL-15+AAVIFN-α to transgenic mice increased both the number and the cytolytic activity of anti-HBV CD8+T cells within the liver causing extensive hepatocellular necrosis and viral clearance. AAVIL-15-treated transgenic mice showed IFN-gamma-dependent hepatic PD-L1 upregulation and a high proportion of PD-1+ intrahepatic T-cells. Combining AAVIFN-α with AAVIL-15 reduced PD-1 and PD-L1 expression allowing HBV-specific T-lymphocytes to kill target cells. Accordingly, treatment of HBV transgenic mice with AAVIL-15 plus anti-PD-L1 antibody caused intense cytolysis and viral clearance. Our study defines a potent new approach to break immune tolerance with broad applications in chronic viral infections and neoplastic conditions. Abbreviation: AAV: Adeno-associated virus; CHB: Chronic hepatitis B; IFN-α: Interferon-alpha; IL-15: Interleukin-15; HBV: Human hepatitis B virus; PD-1: Programmed-death 1; PD-L1: Programmed-death ligand 1.