Translational Project

Johns Hopkins University, Baltimore MD

Investigators

Abstract

Hepatitis B virus (HBV) infects 300 million people chronically and is the leading cause of end-stage liver disease and hepatocellular carcinoma, outcomes which are worse in the ~10% of people with HIV who also have chronic hepatitis B. Thus, finding an HBV cure, which requires loss of hepatitis B surface antigen (HBsAg), is urgently needed for people with HBV mono-infection (PWHB) and people with HBV/HIV (PWHHB). We address the two critical limbs of an HBV functional cure: the persistence of HBsAg and the transcriptional activity of the covalently closed circular DNA (cccDNA) viral genome. HBsAg can derive from cccDNA or HBV DNA integrated into host chromosomes (iDNA), complicating its response to nucleos(t)ide analogue (NUC) antivirals, the mainstay of treatment. Using single-cell and novel molecular approaches, we previously showed that a) cccDNA activity declines with NUCs and b) iDNA-derived transcription explains HBsAg maintenance during NUCs. In the Translational Project of BICC, we propose to characterize the mechanisms underlying HBsAg persistence and cccDNA activity in liver tissues taken from PWHB and PWHHB from diverse geographic settings and of distinct viral genotypes: our overall goal is to identify the major roadblocks impeding an HBV functional cure. In aim one, we will test if the burden of iDNA-derived HBsAg determines the response of HBsAg to NUCs: we use a combination of long-read DNA/RNA sequencing and novel molecular tools to quantify iDNA-derived HBsAg in liver tissues, applying single-cell laser capture microdissection (scLCM) and droplet digital PCR (ddPCR) to characterize the composition of cells producing HBsAg and their dynamics during NUCs. Novel virus-host mRNA hybrids will be fed into the Clinical Project as potential biomarkers. In aim two we apply scLCM/ddPCR to characterize cccDNA inactivity during NUCs in thousands of individual hepatocytes to test the hypothesis that HBV exhibits viral quorum sensing, enhancing or dampening its transcriptional activity in response to its overall intrahepatic burden. We will assemble viral landscapes (viroscapes) that uncover clustered islands of HBV persistence in situ. We will perform cas9-Nanopore and single-molecule epigenetic sequencing on cccDNAs isolated from liver tissues to test if methylation of cccDNA CpG islands is associated with its transcriptional regulation. In aim three we work with the Multiomics Core to identify host factors that regulate cccDNA activity using the 10x Visium Spatial Gene Expression Analysis (VSGEA) platform and our newly developed viral pipeline to extract HBV RNAs and map them in situ. We will sequence cccDNAs to characterize mutations associated with transcriptional activity. We will enrich the 10x VSGEA for T and B cell receptors, interrogating this dataset for intrahepatic HBV-specific clonotypes that are also sequenced in the periphery through the Immunology Project. We will determine if the quantity, proximity, or phenotype of T and B cell clonotypes are associated with constraints on HBV spread and transcription. These aims, in collaboration with the larger BICC, are focused on the major impediments to an HBV cure.