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Hepatitis B surface antigen (HBsAg) loss or functional cure (FC) is considered the optimal therapeutic outcome for patients with chronic hepatitis B (CHB). However, the immune-pathological biomarkers and underlying mechanisms of FC remain unclear. In this study we comprehensively interrogate disease-associated cell states identified within intrahepatic tissue and matched PBMCs (peripheral blood mononuclear cells) from patients with CHB or after FC, at the resolution of single cells, to provide novel insights into putative mechanisms underlying FC. We combined single-cell transcriptomics (single-cell RNA sequencing) with multiparametric flow cytometry-based immune phenotyping, and multiplexed immunofluorescence to elucidate the immunopathological cell states associated with CHB vs. FC. We found that the intrahepatic environment in CHB and FC displays specific cell identities and molecular signatures that are distinct from those found in matched PBMCs. FC is associated with the emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes, and an activated innate response represented by liver-resident natural killer cells, specific Kupffer cell subtypes and marginated neutrophils. Surprisingly, we found MHC class II-expressing hepatocytes in patients achieving FC, as well as low but persistent levels of covalently closed circular DNA and pregenomic RNA, which may play an important role in FC. Our study provides conceptually novel insights into the immuno-pathological control of HBV cure, and opens exciting new avenues for clinical management, biomarker discovery and therapeutic development. We believe that the discoveries from this study, as it relates to the activation of an innate and altered immune response that may facilitate sustained, low-grade inflammation, may have broader implications in the resolution of chronic viral hepatitis. This study dissects the immuno-pathological cell states associated with functionally cured chronic hepatitis B (defined by the loss of HBV surface antigen or HBsAg). We identified the sustained presence of very low viral load, accessory antigen-presenting hepatocytes, adaptive-memory-like natural killer cells, and the emergence of helper CD4 T cells with cytotoxic or effector-like signatures associated with functional cure, suggesting previously unsuspected alterations in the adaptive immune response, as well as a key role for the innate immune response in achieving or maintaining functional cure. Overall, the insights generated from this study may provide new avenues for the development of alternative therapies as well as patient surveillance for better clinical management of chronic hepatitis B. [Display omitted] • An altered adaptive immune response – associated with CD4 cytotoxic T lymphocytes – emerges in functionally cured patients. • Increased recruitment and margination of neutrophils in the liver suggests a low grade immune active state linked to functional cure. • MHC class II-expressing hepatocytes were identified as accessory antigen-presenting cells. • Alterations in the immuno-pathological cell states identified in the liver environment are not always reflected in peripheral circulation. [ABSTRACT FROM AUTHOR]

Hepatitis B core-related antigen (HBcrAg) is a new biomarker for chronic hepatitis B (CHB) whose performance has not been critically or systematically appraised. Herein, we performed a systematic review to determine its clinical utility. We evaluated the biological pathway of HBcrAg and performed a systematic review of PubMed for clinical trials, cohort studies, and case-control studies that evaluated the clinical utility of HBcrAg. The effectiveness of HBcrAg in predicting HBV-specific clinical events (e.g. HBeAg seroconversion, phases of CHB, HBsAg loss, treatment response, and relapse after stopping therapy) was examined using receiver-operating characteristic curves. The correlation coefficients of HBcrAg with HBV DNA, quantitative HBsAg (qHBsAg), HBV RNA, and cccDNA were summarised from published studies. Median values were used as estimates. HBcrAg consists of three precore/core protein products: HBcAg, HBeAg, and a 22 kDa precore protein. HBcrAg assays have been associated with false-positive rates of 9.3% and false-negative rates of between 12-35% for CHB. The new iTACT-HBcrAg is more sensitive but does not reduce the false-positive rate. A PubMed search found 248 papers on HBcrAg, of which 59 were suitable for analysis. The clinical performance of HBcrAg was evaluated using AUROC analyses, with median AUROCs of 0.860 for HBeAg seroconversion, 0.867 for predicting HBeAg(-) hepatitis, 0.645 for HBsAg loss, 0.757 for treatment response, and 0.688 for relapse after stopping therapy. The median correlation coefficient (r) was 0.630 with HBV DNA, 0.414 with qHBsAg, 0.619 with HBV RNA and 0.550 with cccDNA. Correlation decreased during antiviral therapy, but combined biomarkers improved performance. HBcrAg has a mixed performance and has a poor correlation with HBsAg loss and antiviral therapy, hence HBcrAg results should be interpreted with caution. Hepatitis B core-related antigen (HBcrAg) has been used to assess management of patients with chronic hepatitis B (CHB) without a systematic and critical Sreview of its performance. Our finding that HBcrAg had a false-positive rate of 9% and a false-negative rate of 12-35% raises concerns, although larger studies are needed for validation. A systematic review showed that the performance of HBcrAg was variable depending on the CHB endpoint; it was excellent at predicting HBeAg seroconversion and HBeAg-negative chronic hepatitis (vs. chronic infection), which should be its main use, but it was poor for relapse after stopping antiviral therapy and for HBsAg loss. HBcrAg results should be interpreted with considerable caution, particularly by physicians, researchers, guideline committees and agencies that approve diagnostic tests. [Display omitted] • The biological pathway of HBcrAg is distinct to that of HBsAg. • HBcrAg are translation products from precore and pregenomic RNA comprising HBV core antigen, p22cr and HBeAg. • HBeAg contributes >70% to HBcrAg in HBeAg-positive patients, but its components are non-quantifiable in HBeAg-negative patients. • HBcrAg exhibits variable clinical performance. [ABSTRACT FROM AUTHOR]