Integrated HBV DNA and cccDNA maintain transcriptional activity in intrahepatic HBsAg‐positive patients with functional cure following PEG‐IFN‐based therapy

Summary Background Hepatitis B surface antigen (HBsAg) seroclearance marks regression of hepatitis B virus (HBV) infection. However, more than one‐fifth of patients with functional cure following pegylated interferon‐based therapy may experience HBsAg seroreversion. The mechanisms causing the HBV relapse remain unclear. Aim To investigate the level and origin of HBV transcripts in patients with functional cure and their role in predicting relapse. Methods Liver tissue obtained from patients with functional cure, as well as uncured and treatment‐naïve HBeAg‐negative patients with chronic hepatitis B (CHB) were analysed for intrahepatic HBV markers. HBV capture and RNA sequencing were used to detect HBV integration and chimeric transcripts. Results Covalently closed circular DNA (cccDNA) levels and the proportion of HBsAg‐positive hepatocytes in functionally cured patients were significantly lower than those in uncured and treatment‐naïve HBeAg‐negative patients. Integrated HBV DNA and chimeric transcripts declined in functionally cured patients compared to uncured patients. HBsAg‐positive hepatocytes present in 25.5% of functionally cured patients, while intrahepatic HBV RNA remained in 72.2%. The levels of intrahepatic HBV RNA, integrated HBV DNA, and chimeric transcripts were higher in functionally cured patients with intrahepatic HBsAg than in those without. The residual intrahepatic HBsAg in functionally cured patients was mainly derived from transcriptionally active integrated HBV DNA; meanwhile, trace transcriptional activity of cccDNA could also remain. Two out of four functionally cured patients with intrahepatic HBsAg and trace active cccDNA experienced HBV relapse. Conclusion Integrated HBV DNA and cccDNA maintain transcriptional activity and maybe involved in HBsAg seroreversion in intrahepatic HBsAg‐positive patients with functional cure and linked to virological relapse.