Project Summary Chronic hepatitis B (CHB) affects over 250 million people worldwide, with ~1 million annual deaths due to liver disease and hepatocellular carcinoma. Up to 28% of persons living with HIV (PLWH) also have CHB. Since HIV increases liver disease progression from CHB and because liver disease is a leading cause of mortality in PLWH taking antiretroviral therapy, developing a HBV cure is imperative. Current nucleos(t)ide (NUC) therapy can control HBV replication but cannot cure CHB because it does not eradicate the stable covalently closed circular DNA (cccDNA), the template for HBV replication, from the hepatocyte. In addition, the US FDA defines HBV cure has elimination of total hepatitis B surface antigen (tHBsAg) from blood. The simplicity of this definition is belied by the complexity of the source of tHBsAg, which derives from either the cccDNA or HBV DNA that is integrated into the host genome (iDNA). Distinguishing the contribution of these two sources to HBsAg is important to target developing a cure. Further, our data using the novel techniques of droplet digital PCR (ddPCR) demonstrate that NUCs unexpectedly decrease transcription of pgRNA from cccDNA, but whether transcription of S mRNAs, the transcripts that encode for tHBsAg, is also reduced is unknown. To address these knowledge gaps, we propose to determine the contribution of cccDNA and iDNA to tHBsAg from 69 PLWH with different stages of CHB of whom 60 have paired biopsies. In a subset of these individuals, we will examine single hepatocytes to determine the proportions of hepatocytes with iDNA and cccDNA. The 69 individuals (129 biopsies since 60 have paired biopsies) in this proposal having varying stages of HBV infection including immune active CHB (HBeAg+ and HBeAg neg), inactive CHB, and occult hepatitis B. Aim 1 will use RNA seq on bulk liver tissue from 6 individuals with CHB to construct surface (S) mRNA maps, which will allow us to determine the proportion of S mRNA that originate from cccDNA versus iDNA. The latter are distinguished because iDNA will terminate in the human genome, truncating the viral sequence at its 3’ end. The maps will then be used to find major breakpoints in S mRNAs that occur with integration, allowing development of a multiplex ddPCR to study 69 bulk liver tissues and single hepatocytes from a subset of individuals. Aim 2 will interrogate liver biopsies from the 60 individuals with longitudinal biopsies during which time they were on NUCs to understand how NUCs affect these proportions. Data from Aims 1 and 2 will be correlated with plasma quantitative HBsAg and with circulating amounts of Large, Medium, and Small HBsAg. We will also determine if CD4+ T cell depletion affects the proportion of HBsAg that derives from iDNA. Our research will broadly impact the field by using novel techniques to determine the proportions of tHBsAg from iDNA or cccDNA, which will inform the rational design of therapies for HBV cure.