Approximately 370 million people worldwide are chronically infected with hepatitis B virus (HBV). Despite the success of the prophylactic HBV vaccine, no therapeutic vaccine or other immunotherapy modality is available for treatment of chronically infected individuals. Clearance of HBV depends on robust, sustained CD8+ T activity; however, the limited numbers of therapeutic vaccines tested have not induced such a response. Most of these vaccines have relied on peptide prediction algorithms to identify MHC-I epitopes or characterization of T cell responses during acute infection. Here, we took an immunoproteomic approach to characterize MHC-I restricted epitopes from cells chronically infected with HBV and therefore more likely to represent the true targets of CD8+ T cells during chronic infection. In this study, we identified eight novel MHC-I restricted epitopes derived from a broad range of HBV proteins that were capable of activating CD8+ T cells. Furthermore, five of the eight epitopes were able to bind HLA-A2 and A24 alleles and activated HBV specific T cell responses. These epitopes also have potential as new tools to characterize T cell immunity in chronic HBV infection and may serve as candidate antigens for a therapeutic vaccine against HBV infection. 1. Introduction Hepatitis B virus (HBV) is a member of the Hepadnaviridae family of viruses which also includes woodchuck hepatitis virus (WHV) and duck hepatitis B virus. These viruses are primarily hepatotropic with infections characterized by fever, fatigue, muscle aches, and yellowing of the eyes and/or skin. The severity of these symptoms can vary with a proportion of cases being asymptomatic [1]. More than 2.5 billion people worldwide have been infected by HBV, but, for the vast majority of adults encountering the virus (>90%), the infection is acute and readily cleared by the immune system [2, 3]. For the remaining 5–10% of adults, and for neonates and unvaccinated children, HBV establishes a chronic infection. Approximately 370 million people worldwide are chronically infected and over 500,000 people die each year due to complications from HBV [1, 4]. These complications include liver cirrhosis, liver failure, and/or hepatocellular carcinoma (HCC) and it is estimated that up to 40% of chronically infected patients will develop at least one of these complications [5]. The primary determinant of whether hepatitis B virus is cleared or establishes a chronic infection is the robustness of the immune response, in particular the CD8+ T cell response [6–9]. Data from both animal models and
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