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Published on: February 19, 2019
Spatial-quantitative profiling of intrahepatic HBsAg heterogeneity and clustered distribution in chronic HBV
Mingzhu Xu1, Ye Zheng2, Min Wu2
1Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, HuaShan Hospital, Shanghai Medical College Fudan University, Shanghai, China; Shanghai Public Health Clinical Center, Fudan University, Shanghai, China; School of Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
Insights
Clustered hepatitis B surface antigen (HBsAg)-positive hepatocytes are more common in HBeAg-negative individuals. These cells predominantly contain nuclear HBV DNA, offering insights into chronic hepatitis B persistence.
Area of Science:
- Hepatology
- Virology
- Immunology
Background:
- Hepatitis B surface antigen (HBsAg) expression in chronic hepatitis B (CHB) originates from both cccDNA and integrated HBV DNA.
- Intrahepatic HBsAg patterns vary, but their association with CHB phases and HBV DNA is not well understood.
Purpose of the Study:
- To investigate the spatial patterns of HBsAg in relation to HBV DNA across different phases of CHB.
- To understand the role of HBsAg distribution in viral persistence and host-virus interactions.
Main Methods:
- Analysis of 385 treatment-naive CHB patients using immunofluorescence and in situ hybridization (IF-ISH).
- Quantitative whole-slide imaging to assess HBsAg patterns and HBV DNA localization.
- Laser capture microdissection, Nanopore sequencing, and SMART-seq2 for HBV DNA and RNA analysis.
Main Results:
- Clustered HBsAg-positive hepatocytes were more frequent in HBeAg-negative (55.1%) than HBeAg-positive (34.5%) individuals.
- HBsAg-rich and HBV DNA-positive hepatocytes generally showed mutually exclusive patterns, with some colocalization.
- Nuclear HBV DNA was dominant in HBeAg-negative patients within clustered hepatocytes, while cytoplasmic HBV DNA predominated in HBeAg-positive patients.
Conclusions:
- Clustered HBsAg-rich hepatocytes are more prevalent in HBeAg-negative individuals and contain nuclear HBV DNA from integration and/or cccDNA.
- These findings reveal phase-specific intrahepatic viral heterogeneity in CHB.
- Understanding these spatial patterns offers insights into HBV persistence and potential strategies for functional cure.
Background & Aims:
Hepatitis B surface antigen (HBsAg), a hallmark of chronic hepatitis B virus (CHB) infection, is expressed from both covalently closed circular DNA (cccDNA) and integrated HBV DNA. HBsAg exhibits diffuse, scattered, or clustered intrahepatic patterns, yet their variation across disease phases and the relationship to HBV DNA remain unclear.
Methods:
Formalin-fixed paraffin-embedded sections from 385 treatment-naive patients with CHB were analyzed. The immunofluorescence and enhanced in situ hybridization (IF-ISH) assay was coupled with a whole-slide imaging-based quantitative pipeline to delineate the spatial patterns of HBsAg in the context of viral DNA. Laser capture microdissection isolated HBsAg-positive hepatocytes, followed by HBV-enriched Nanopore sequencing and SMART-seq2 based RNA sequencing.
Results:
HBeAg-negative individuals showed more clustered HBsAg-positive hepatocytes than HBeAg-positive individuals (55.1% vs. 34.5%). IF-ISH analysis with enhanced sensitivity for HBV DNA confirmed the general mutually exclusive pattern between HBsAg-rich and HBV DNA-positive hepatocytes, while also revealing a subset of colocalization events, together with phase-specific variations in the subcellular (nuclear vs. cytoplasmic) localization of viral DNA. Clustered HBsAg-rich hepatocytes predominantly exhibited cytoplasmic HBV DNA in patients who were HBeAg-positive (54.4%), whereas nuclear HBV DNA was dominant in patients who were HBeAg-negative (78.8%), harboring transcripts derived from integrant and/or cccDNA.
Conclusions:
This study provides quantitative evidence that clustered HBsAg-rich hepatocytes are more prevalent in individuals who are HBeAg-negative and predominantly harbor nuclear-localized HBV DNA originating from integration and/or cccDNA, thereby providing new insights into phase-specific intrahepatic viral heterogeneity and advancing understanding of host-virus interactions in HBV persistence.
Impact And Implications:
This study reveals that clustered HBsAg-positive hepatocytes, enriched in individuals who are HBeAg-negative, predominantly contain nuclear HBV DNA originating from integration and/or cccDNA. The mutually exclusive spatial patterns of HBsAg-rich and HBV DNA-positive hepatocytes reflect distinct viral reservoirs and dynamic changes across different phases of chronic HBV infection. These findings provide new insights into the intrahepatic landscape of HBV persistence and may inform phase-specific strategies for functional cure of chronic hepatitis B.
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