Project Summary There are approximately a quarter of a billion people worldwide chronically infected with hepatitis B virus (HBV) who are at risk of developing liver cirrhosis and hepatocellular carcinoma. The loss of life due to HBV-related complications each year is about 820,000 people. At the same time human immunodeficiency virus (HIV) infects about 38 million people worldwide. Among those, 7.4% globally and 15-28% in highly endemic areas are also infected with HBV. HIV co-infections pose a serious health burden as they accelerate the progression to liver cirrhosis and liver cancer with mechanisms that are still not well understood. Chronicity of HBV infections is linked to the stability and maintenance of the covalently closed circular DNA (cccDNA), which is the nuclear form of the HBV genome. As HBV infects human hepatocytes in the liver, the cccDNA is established in these cells and strategies that aim to inactivate or eliminate it are considered the solution to a functional cure. Current treatments against chronic HBV include nucleoside analogs and interferon α. While nucleoside analogs do not directly affect the cccDNA, a limited number of patients that receive interferon α or nucleoside analogs can resolve the infection and achieve a cure. The need for a global HBV cure is urgent, which is also why several approaches and investigational compounds aim for a cure. HBV is an exclusive hepatotropic virus that only infects human hepatocytes. HBV studies have been hindered by limited cell culture systems that include hepatoma cells after overexpression of the HBV receptor NTCP and primary human hepatocytes. The latter are the most physiologically relevant cells, but their availability and the inherent limitations of primary cells makes it difficult to use them to recapitulate the complete HBV lifecycle. To this end, we have developed cell culture systems based on primary human hepatocytes, isolated from humanized mice, that efficiently support long-term HBV infection and spread. Moreover, we developed a system of isolating these cells from HBV-infected humanized mice which provides unique opportunities for research. Specifically, cells are chronically infected with HBV and therefore carry high levels of cccDNA, which allows for modeling chronic HBV in vitro. Here we propose to use these innovative hepatocyte systems to establish robust co-cultures with CD4+ T cells for studying HIV/HBV co-infections in physiologically relevant primary cell culture systems. Overall, with our proposed work we will gain insights on the effects of HIV co-infection on HBV lifecycle and hepatocyte responses and we will establish a platform to identify novel antiviral strategies with the goal for a functional HBV cure.