Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load

Background & Aims: Loss of serum HBsAg is a hallmark of spontaneous and therapy induced resolution of HBV infection, since it generally reflects a profound decrease in viral replication. However, integrated HBV DNA can contribute to HBsAg expression independent of viral replication. The relative contributions of these sources of HBsAg are not well understood. Specifically, it is not known whether actively transcribed HBV integration could spread throughout the entire liver. Methods: The relative distribution of HBsAg and HBV RNA in liver biopsy tissue from HBeAg-negative (HBe) patients was analyzed by immunohistochemistry and in situ hybridization (ISH), respectively. Frozen biopsy tissue was used for molecular analysis of intrahepatic viral RNA, virus-host chimeric transcripts and viral DNA. Results: Immunohistochemistry and ISH analysis revealed HBsAg and HBV RNA positivity in virtually all hepatocytes in the liver of some HBe- patients despite very low viremia. Reverse transcription quantitative PCR and RNA-sequencing analysis confirmed high expression levels of HBV envelope-encoding RNAs. However, the amount of viral transcriptional template (covalently closed circular (ccc)DNA) was too low to support this ubiquitous HBV RNA expression. In contrast, levels of total cellular HBV DNA were consistent with ubiquitous HBV integration. Finally, RNA-sequencing revealed the presence of many HBV-host chimeric transcripts with the potential for HBsAg expression. Conclusions: Transcriptionally active HBV integration can extend to the entire liver in some HBe- patients. This can lead to ubiquitous HBsAg expression independent of HBV replication. In such patients, HBsAg is probably not a clinically useful surrogate marker for viral resolution or functional cure. Lay summary: Loss of serum hepatitis B surface antigen (HBsAg) indicates resolution of HBV infection. However, integrated HBV DNA can contribute to HBsAg production independently of viral replication. We investigated the extent of HBsAg-producing viral integration in the livers of patients with low serum viral loads. Our findings suggest that transcriptionally active HBV integration can extend to the entire liver in some patients, questioning the clinical utility of HBsAg as a surrogate marker for viral replication. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.