Unraveling sources of hepatitis B surface antigen before and after nucleos(t)ide analogue treatment in People with HIV

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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 stop 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. The US FDA defines HBV cure has elimination of hepatitis B surface antigen (HBsAg) from blood. The simplicity of this definition is belied by the complexity of the source of HBsAg, 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 single cell laser capture microdissection (scLCM) and droplet digital PCR (ddPCR) demonstrate that NUCs unexpectedly decrease transcription of pgRNA from cccDNA but whether transcription of S is also reduced is unknown. To address these knowledge gaps, we propose to determine the contribution of cccDNA and iDNA to HBsAg at the single hepatocyte level before and during NUC in 5 HIV-HBV co-infected individuals with paired archived liver tissue. This proposal will use archived paired liver tissue from 5 HIV-HBV co-infected individuals of whom 2 were not and 3 were on NUCs at biopsy 1. At biopsy 2, obtained ~3 years later, all 5 individuals were on NUCs. Aim 1 will use RNA seq on bulk liver tissue from biopsy 1 to construct surface (S) transcript maps, which will allow us to determine the proportion of S transcripts that originate from cccDNA versus iDNA. The latter are distinguished because iDNA will terminate in the human genome. The maps will then be used to find major breakpoints in S that occur with integration allowing development of a multiplex ddPCR to study single hepatocytes. This multiplex ddPCR will be applied to 100-200 single hepatocytes dissected with scLCM to uniquely quantify the S transcripts from cccDNA versus iDNA in each hepatocyte from biopsy 1. Aim 2 will then utilize hepatocytes from biopsy 2 to understand how NUCs affect these proportions. Data from Aims 1 and 2 will be correlated with plasma quantitative HBsAg. We will also determine if NUCs affect global cccDNA transcription or only for pgRNA by combining these data with our prior data from the same hepatocytes. Our research will broadly impact the field by using novel techniques to determine the proportions of HBsAg from cccDNA or iDNA, which will inform the rational design of therapies for HBV cure. In addition, this proposal will determine whether NUCs can silence cccDNA globally or are only specific for pgRNA.