Serum HBsAg cannot distinguish cccDNA activity from integrated HBV DNA expression in chronic hepatitis B virus infection.

BACKGROUND & AIMS: HBsAg is currently used as the key serum biomarker to define functional cure of chronic hepatitis B (CHB). Declining HBsAg is often interpreted as evidence of cccDNA elimination or durable transcriptional silencing. However, HBsAg can also be produced from transcriptionally active HBV DNA integrated into the host genome (iDNA), which can make serum HBsAg an unreliable indicator of the cccDNA activity or amount. METHODS: We combined intrahepatic HBsAg immunostaining, quantitative serum HBsAg levels (qHBsAg), and digital droplet PCR-based profiling of HBV DNA and RNA in liver biopsies to identify patients in whom HBsAg expression was predominantly driven by cccDNA or by iDNA. We then compared HBsAg abundance, isoform composition, secretion, and intrahepatic staining patterns between these groups. RESULTS: All three envelope proteins (L, M, S) were detectable in liver tissue and serum in both groups. The relative abundance of L and S, assessed by western blot and immunofluorescence, did not differ between iDNA- and cccDNA-driven cases. Across the combined cohort, intrahepatic HBsAg burden correlated with serum qHBsAg, indicating similar intracellular retention and secretion behavior. Importantly, all established intrahepatic HBsAg staining patterns (membranous and cytoplasmic subtypes) occurred in both groups, with no pattern uniquely associated with either source. CONCLUSIONS: Thus, iDNA can sustain HBsAg expression profiles that are indistinguishable from those generated by active cccDNA. Serum HBsAg alone may therefore fail to identify patients who have achieved cccDNA elimination or durable silencing. These findings suggest that the use of serum qHBsAg as a component of the functional cure endpoint in clinical studies warrants re-evaluation. IMPACT AND IMPLICATIONS: Serum hepatitis B surface antigen (HBsAg) is the central biomarker used to define and monitor "functional cure" in chronic hepatitis B. This study shows that in patients with substantial HBV DNA integration, HBsAg is produced predominantly from integrated sequences, and that integration-derived HBsAg is indistinguishable from cccDNA-derived HBsAg in isoform composition, secretion behaviour, and histological distribution. Long-read sequencing confirmed that the integrated sequences generate full-length envelope transcripts. These findings are most relevant to virologists, hepatologists, clinical trialists, and regulators developing and evaluating HBV cure strategies. They indicate that serum HBsAg measurement cannot report cccDNA burden or transcriptional activity, and that focusing on HBsAg loss could miss "partial cure" states with favourable clinical outcomes. This warrants re-evaluation of HBsAg loss as a main functional cure endpoint, and supports the use of complementary or source-specific markers to assess treatment response.