Effects of tenofovir disoproxil fumarate on intrahepatic viral burden and liver immune microenvironment in patients with chronic hepatitis B.

Abstract

The impact of nucleos(t)ide analogues on intrahepatic viral burden and immune microenvironment in patients with chronic hepatitis B (CHB) is not clear. We aimed to characterise the effects of tenofovir disoproxil fumarate (TDF) on intrahepatic viral burden and the liver immune microenvironment in patients with CHB. Core liver biopsies were collected at baseline and year 3 from patients with CHB with minimally raised serum alanine aminotransferase in a double-blind placebo-controlled trial (NCT01522625). Paired biopsies were analysed by RNA-sequencing (n=119 pairs), a custom multiplex immunofluorescence assay (n=30 pairs), and HBV-targeted long-read DNA sequencing (n=49 pairs). Both non-integrated and integrated HBV DNA were present in all patients at baseline, with >65% having interchromosomal translocations. Treatment significantly reduced the frequency of HBV core+ hepatocytes and intrahepatic (integrated and non-integrated) HBV DNA, but had no effect on HBsAg+ hepatocytes. Clonally expanded integrations were enriched for HBsAg coding regions and showed dysregulation of nearby genes. At baseline, there was significant enrichment of intrahepatic CD8+ T cell proximity to HBV core+ hepatocytes, but not to HBsAg+ cells. The densities of T cells and B cells were significantly reduced by TDF. Transcriptomic analyses found TDF induced widespread downregulation of immune-related genes including inhibitory and regulatory genes. TDF significantly reduced intrahepatic integrated and non-integrated HBV DNA, exerting disparate effects on HBV core+ and HBsAg+ cells and on different immune cell subsets. Our data suggest there may be differential cytotoxic T cell-mediated killing of HBV core+ versus HBsAg+ hepatocytes, providing insights for HBV cure strategies.