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Intrahepatic quantification of HBV antigens in chronic hepatitis B reveals heterogeneity and treatment-mediated reductions in HBV core-positive cells

Open AccessPublished:December 26, 2022DOI:https://doi.org/10.1016/j.jhepr.2022.100664

      Highlights

      • We applied a novel imaging technique to quantify HBsAg and HBV core antigen burden in liver biopsies from CHB patients
      • Frequency of HBV core+ hepatocytes was lower in HBeAg- negative versus HBeAg-positive subjects
      • NUC treatment was associated with a significant decline in HBV core+ cells but not HBsAg
      • Duplicate biopsies collected at the same timepoint revealed large local variation in HBV staining within subjects
      • HBV+ hepatocyte burden correlated with HBcrAg, HBV DNA and HBV RNA only in HBeAg- positive patients at baseline

      Abstract

      Background and Aims

      Patterns of liver Hepatitis B virus (HBV) antigen expression have been described but not quantified at single cell resolution. We applied quantitative techniques to liver biopsies from chronic hepatitis B (CHB) patients and evaluated sampling heterogeneity, effects of disease stage and nucleos(t)ide (NUC) treatment, and correlations between liver and peripheral viral biomarkers.

      Methods

      Hepatocytes positive for HBV core and HBsAg were quantified using a novel 4-plex immunofluorescence assay and image analysis. Biopsies were analyzed from HBeAg-positive (n=39) and HBeAg-negative (n=75) subjects before and after NUC treatment. Duplicate biopsies collected at the same timepoint were compared to evaluate sampling effects. Serum or plasma samples were evaluated for levels of HBV DNA, HBsAg, core-related antigen (HBcrAg), and HBV RNA.

      Results

      Diffusely distributed individual HBV core+ cells and foci of HBsAg+ cells were the most common staining patterns. Hepatocytes positive for both HBV core and HBsAg were rare. Paired biopsies revealed large local variation in HBV staining within subjects, which was confirmed in a large liver resection. NUC treatment was associated with a >100-fold lower median frequency of HBV core+ cells in HBeAg-positive and -negative subjects, while reductions in HBsAg+ cells were not statistically significant. The frequency of HBV core+ hepatocytes was lower in HBeAg-negative versus HBeAg-positive subjects at all timepoints evaluated. Total HBV+ hepatocyte burden correlated with HBcrAg, HBV DNA and HBV RNA only in baseline HBeAg-positive samples.

      Conclusions

      Reductions in HBV core+ hepatocytes were associated with HBeAg-negative status and NUC treatment. Variation in HBV positivity within individual livers was extensive. Correlations between the liver and periphery were found only between biomarkers likely indicative of cccDNA (HBV core+ and HBcrAg, HBV DNA and RNA).

      Lay Summary

      Hepatitis B virus infects liver hepatocyte cells and its genome can exist in two forms that express different sets of viral proteins: a circular genome called cccDNA that can express all viral proteins, including the HBV core and HBsAg proteins, or linear fragment which inserts into the host genome typically which express HBsAg but not HBV core. We used new techniques to determine the percentage of hepatocytes expressing the HBV core and HBsAg proteins in a large set of liver biopsies. We find that abundance and patterns of expression differ across patient groups and even within a single liver, and that NUC treatment greatly reduces the number of core-expressing hepatocytes.

      Graphical abstract

      Keywords

      Abbreviations:

      HBV (Hepatitis B virus), NUC (nucleo(t)side), Na+K+ ATPase (sodium potassium adenosine triphosphate-ase), HCC (Hepatocellular carcinoma), HBsAg (Hepatitis B surface antigen), HBV core (Hepatitis B core antigen), HBeAg (Hepatitis B e antigen), CHB (chronic hepatitis B), cccDNA (covalently closed circular DNA), TDF (tenofovir disoproxil fumarate), HBcrAg (core related antigen), ALT (alanine aminotransferase), ADV (adefovir), EASL (European Association for the Study of the Liver), mIF (multiplex immunofluorescence), ER (Epitope retrieval), QC (quality control)

      Introduction

      An estimated 260 million people worldwide are chronically infected with Hepatitis B virus HBV, resulting in more than 800,000 annual deaths from cirrhosis, liver failure, and hepatocellular carcinoma HCC

      World Health Organization: Global hepatitis report. 2017.

      . Current treatments for chronic hepatitis B (CHB) infection can efficiently suppress viral replication and improve patient outcome, but typically require life-long therapy and rarely lead to functional cure (defined as persistent HBsAg loss)
      • Kim G.A.
      • Lim Y.S.
      • An J.
      • Lee D.
      • Shim J.H.
      • Kim K.M.
      • et al.
      HBsAg seroclearance after nucleoside analogue therapy in patients with chronic hepatitis B: clinical outcomes and durability.
      . Consequently, curative therapies for CHB are sorely needed.
      During the HBV replication cycle, viral RNA is reverse-transcribed and processed into covalently closed circular DNA (cccDNA) that is transcribed to express all viral proteins
      • Tong S.
      • Revill P.
      Overview of hepatitis B viral replication and genetic variability.
      . However, the viral RNA can also generate a double-stranded linear form (dslDNA) as a consequence of failed primer-template switching during reverse transcription
      • Tu T.
      • Budzinska M.A.
      • Shackel N.A.
      • Urban S.
      HBV DNA Integration: Molecular Mechanisms and Clinical Implications.
      . HBV dslDNA can integrate into the host genome
      • Pollicino T.
      • Caminiti G.
      HBV-Integration Studies in the Clinic: Role in the Natural History of Infection.
      , resulting in a chromosomal template that is transcribed to express HBV surface antigen (HBsAg), but not the viral core protein (HBV core) or polymerase protein
      • Tu T.
      • Budzinska M.A.
      • Shackel N.A.
      • Urban S.
      HBV DNA Integration: Molecular Mechanisms and Clinical Implications.
      ,
      • van Buuren N.
      • Ramirez R.
      • Soulette C.
      • Suri V.
      • Han D.
      • May L.
      • et al.
      Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.
      . These hepatocytes with integrated HBV sequences can clonally expand
      • Jiang Z.
      • Jhunjhunwala S.
      • Liu J.
      • Haverty P.M.
      • Kennemer M.I.
      • Guan Y.
      • et al.
      The effects of hepatitis B virus integration into the genomes of hepatocellular carcinoma patients.
      • Mason W.S.
      • Gill U.S.
      • Litwin S.
      • Zhou Y.
      • Peri S.
      • Pop O.
      • et al.
      HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant.
      • Budzinska M.A.
      • Shackel N.A.
      • Urban S.
      • Tu T.
      Sequence analysis of integrated hepatitis B virus DNA during HBeAg-seroconversion.
      and may contribute to the development of HCC
      • Sung W.K.
      • Zheng H.
      • Li S.
      • Chen R.
      • Liu X.
      • Li Y.
      • et al.
      Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma.
      • Zhao L.H.
      • Liu X.
      • Yan H.X.
      • Li W.Y.
      • Zeng X.
      • Yang Y.
      • et al.
      Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma.
      • Peneau C.
      • Imbeaud S.
      • La Bella T.
      • Hirsch T.Z.
      • Caruso S.
      • Calderaro J.
      • et al.
      Hepatitis B virus integrations promote local and distant oncogenic driver alterations in hepatocellular carcinoma.
      . The relative proportion of HBV in the form of cccDNA or integrated viral DNA varies at different stages of CHB natural history, with cccDNA more abundant in HBeAg-positive (HBeAg+) subjects, while HBeAg-negative (HBeAg-) subjects have predominantly integrated HBV DNA
      • van Buuren N.
      • Ramirez R.
      • Soulette C.
      • Suri V.
      • Han D.
      • May L.
      • et al.
      Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.
      ,
      • Wooddell C.I.
      • Yuen M.F.
      • Chan H.L.
      • Gish R.G.
      • Locarnini S.A.
      • Chavez D.
      • et al.
      RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.
      ,
      • Meier M.A.
      • Calabrese D.
      • Suslov A.
      • Terracciano L.M.
      • Heim M.H.
      • Wieland S.
      Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load.
      . Sustained HBsAg loss in the periphery is the clinical marker of functional cure, so strategies that result in elimination of both cccDNA and integrated HBV are likely necessary to achieve this goal.
      Current HBV cure strategies aim to induce immune-mediated clearance of HBV-positive hepatocytes to eliminate otherwise stable viral DNA
      • Loglio A.
      • Vigano M.
      • Lampertico P.
      Novel Therapies That May Cure Chronic Hepatitis B Virus.
      . However, excessive immune-mediated hepatocyte killing could lead to potential adverse events in patients. Consequently, subjects with fewer HBV-positive cells may have a lower safety risk, however, there is no consensus on the fraction of HBV-positive hepatocytes in CHB populations
      • Wooddell C.I.
      • Yuen M.F.
      • Chan H.L.
      • Gish R.G.
      • Locarnini S.A.
      • Chavez D.
      • et al.
      RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.
      ,
      • Loglio A.
      • Vigano M.
      • Lampertico P.
      Novel Therapies That May Cure Chronic Hepatitis B Virus.
      . Quantification of the percentage of HBV-positive hepatocytes across disease stages and pre-/post-nucleos(t)ide (NUC) treatment requires a method that can accurately identify and quantify HBV-positive cells in liver samples, a sufficiently large liver biopsy collection to draw meaningful conclusions in different patient groups, and a greater understanding of how well individual biopsies represent the entire liver. We developed a multiplex immunofluorescence (mIF) assay and image analysis methods to describe the abundance of HBV antigen containing hepatocytes across a large liver biopsy collection to describe the HBV viral burden across different disease stages, as well as pre- and post-NUC treatment. We further evaluated correlations between HBV burden in the liver and well-established peripheral viral biomarkers.

      Materials and Methods

      Liver Biopsy Collection

      Liver biopsies were obtained from HBeAg+ and HBeAg- subjects enrolled in two randomized Phase 3 clinical trials of tenofovir disoproxil fumarate (TDF): GS-US-174-0102 (Clinicaltrials.gov: NCT00117676) and GS-US-174-0103 (Clinicaltrials.gov: NCT00116805)
      • Marcellin P.
      • Heathcote E.J.
      • Buti M.
      • Gane E.
      • de Man R.A.
      • Krastev Z.
      • et al.
      Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B.
      ,
      • Gordon S.C.
      • Krastev Z.
      • Horban A.
      • Petersen J.
      • Sperl J.
      • Dinh P.
      • et al.
      Efficacy of tenofovir disoproxil fumarate at 240 weeks in patients with chronic hepatitis B with high baseline viral load.
      . HBeAg- (GS-US-174-0102, n=375) and HBeAg+ (GS-US-174-0103, n=266) subjects with elevated ALT were randomized 2:1 to receive TDF or adefovir dipivoxil (ADV) for 48 weeks (Fig. S1). Based on the current European Association for the Study of Liver Diseases (EASL) guidelines
      European Association for the Study of the Liver
      European Association for the Study of the Liver: EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection.
      , the patient populations were classified as HBeAg+ chronic hepatitis (formerly Immune Active) and HBeAg- chronic hepatitis. Patients were predominantly genotypes A, B, C, and D (Table 1), consistent with the geographic distribution of HBV genotypes at study sites. Within each genotype, a mixture of HBeAg+ and HBeAg- patients were present (Table 1).
      Table 1Baseline Patient Population.
      HBeAg+ n=39HBeAg− n=75Total Cohort n=114
      Baseline demographics
      Age, y30 (24, 42)47 (39, 54)42 (31, 51)
      Men34 (87)64 (85)98 (86)
      Women5 (13)11 (15)16 (14)
      HBV genotype
      A6 (15)11 (15)17 (15)
      B5 (13)8 (11)13 (11)
      C10 (26)6 (8)16 (14)
      D16 (41)48 (64)64 (56)
      E1 (3)01 (1)
      F1 (3)01 (1)
      G01 (1)1 (1)
      U01 (1)1 (1)
      Baseline Peripheral biomarker values
      HBV DNA, log10 IU/mL8.0 (7.6, 8.7); n=396.0 (5.4, 7.3); n=697.1 (5.8, 8.0); n=108
      HBsAg, log10 IU/mL4.6 (4.1, 5.1); n=393.8 (3.3, 4.1); n=704.0 (3.6, 4.4); n=109
      HBV RNA, log10 IU/mL7.0 (6.5, 7.7); n=284.8 (4.2, 5.7); n=455.7 (4.6, 6.7); n=73
      HBcrAg, log10 IU/mL7.9 (7.3, 8.3); n=275.1 (4.3, 5.8); n=436.2 (4.9, 7.8); n=70
      ALT, U/L123 (80, 179); n=38116 (81, 234); n=71119 (81, 202); n=109
      *Data are n (%) or median (interquartile range [IQR]); statistical tests performed: Fisher’s exact test for categorical and Wilcoxon rank-sum test for continuous data.
      Optional core needle liver biopsies were collected at baseline and Week 44-48 (referred to as “Wk48”). Subjects who completed 48 weeks of treatment and provided a liver biopsy at Wk48 were given the option to begin (or continue) treatment with TDF for up to 7 additional years. A third optional liver biopsy was collected at Week 240 in these subjects who continued treatment. 431 liver biopsies were stained by multiplex immunofluorescence (mIF) assay. Subject samples analyzed were collected from twelve countries (USA, Great Britain, Germany, Canada, Greece, Bulgaria, Czech Republic, The Netherlands, France, Spain, Australia, Italy). All subjects signed an informed consent form prior to screening and in accordance with local regulatory and ethics committee requirements. Experimental protocol in these trials was approved by Gilead Sciences and all local regulatory agencies.

      Multiplex Immunofluorescence

      An automated multiplex IF assay was performed on n=431 core needle liver biopsies (n=133 stained by duplex IF for HBsAg and HBV core only; n=298 stained by 4-plex) from the GS-US-174-0102 and GS-US-174-0103 studies. Unmasking of antigens in formalin fixed, paraffin embedded tissue sections was achieved by heat-induced epitope retrieval. The multiplex antibody panel for HBV core (Gilead Sciences 366-2
      • Hong X.
      • Luckenbaugh L.
      • Mendenhall M.
      • Walsh R.
      • Cabuang L.
      • Soppe S.
      • et al.
      Characterization of Hepatitis B Precore/Core-Related Antigens.
      , Rabbit IgG), HBsAg (Gilead Sciences XTL17, Mouse IgG2a), Histone H3 (Abcam EPR16987, Rabbit IgG), and Na+K+-ATPase (Abcam EP1845Y, Rabbit IgG) as well as the HBsAg/HBV core duplex was developed using the same primary antibodies listed above, optimized and performed using the Opal technology workflow
      • Taube J.M.
      • Roman K.
      • Engle E.L.
      • Wang C.
      • Ballesteros-Merino C.
      • Jensen S.M.
      • et al.
      Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study.
      (Akoya Biosciences, Fig. S2) on a Bond RX autostainer (Leica Biosystem) and scanned on the Vectra Polaris (Akoya Biosciences). HBsAg detection antibody XTL17 has similar affinity for HBsAg of genotypes A, B, C, D, E, G, and H (0.3nM by ELISA). The HBV core detection antibody 366-2 binds to a linear epitope that is conserved across genotypes and present only in HBV Core antigen (not HBeAg)
      • Hong X.
      • Luckenbaugh L.
      • Mendenhall M.
      • Walsh R.
      • Cabuang L.
      • Soppe S.
      • et al.
      Characterization of Hepatitis B Precore/Core-Related Antigens.
      . Spectral DAPI (Akoya Biosciences) was used to detect nuclei in both assays. Assay performance was tested for nuclear dropout (loss of nuclear counterstain signal by DAPI, Fig. S3A), optimal antibody order, and appropriate positive and negative controls.

      Whole Slide Image Analysis

      Immunofluorescent whole slide images of liver biopsies were analyzed using Visiopharm software (Version 2020.05, Visiopharm Corporation). In order to automate and standardize the analysis, a customized multi-step algorithm was developed: 1) A decision forest algorithm was trained to detect the tissue from the background glass slide at 4x magnification in order to limit the high-resolution analysis to only the relevant regions; 2) A convolutional neural network (CNN, U-Net*)

      Ronneberger O, Fischer P, Brox T: U-Net: Convolutional Networks for Biomedical Image Segmentation. Medical Image Computing and Computer-Assisted Intervention – MICCAI 2015. Edited by Navab N, Hornegger J, Wells WM, Frangi AF. Cham: Springer International Publishing, 2015. pp. 234-241.

      was trained at 20x to segment and classify a) nuclei (for HBsAg/HBV core duplex stains, CNN trained on the nuclear DAPI channel) or b) cells (for 4-plex stains, CNN trained on the membrane marker Na+K+-ATPase to detect cell outlines and nuclear Histone H3 in addition to the DAPI channel to identify nuclei). Nuclear size exclusion was used in the duplex assay to distinguish hepatocytes from lymphocyte populations, whereas in the 4-plex assay both cytoplasmic- and nuclear-size exclusion were used. Hepatocytes were classified as HBsAg+ or HBV core+ individually, as well as “Total HBV+” if they stained for either HBsAg, HBV core or both viral antigens.

      Peripheral Biomarker Analysis

      Subject serum or plasma was collected longitudinally throughout the GS-US-174-0102 and GS-US-174-0103 clinical trials. HBV DNA and qualitative HBeAg were measured on-study at Labcorp (previously Covance Central Laboratories)
      • Marcellin P.
      • Heathcote E.J.
      • Buti M.
      • Gane E.
      • de Man R.A.
      • Krastev Z.
      • et al.
      Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B.
      . In subjects with sufficient residual sample volume at baseline, Wk48, and/or Wk240, the following viral biomarkers were evaluated retrospectively: HBV RNA
      • van Bommel F.
      • Bartens A.
      • Mysickova A.
      • Hofmann J.
      • Kruger D.H.
      • Berg T.
      • et al.
      Serum hepatitis B virus RNA levels as an early predictor of hepatitis B e antigen seroconversion during treatment with polymerase inhibitors.
      (DDL Diagnostics), HBcrAg (Fujiribio, Lumipulse, DDL Diagnostics), and quantitative HBsAg (Abbott Architect, i2000SR, Labcorp).

      Statistical Analysis

      All statistical analyses were performed using Graphpad Prism v8.0 and the statistical software R. Comparison of baseline characteristics between HBeAg+ and HBeAg- subjects was performed using a Fisher’s exact test for categorical and Wilcoxon rank-sum test for continuous data. Comparisons of % HBV+ hepatocytes (%HBsAg+, %HBV core+, % total HBV+ [HBsAg+ and/or HBV core+]) between timepoints and between HBeAg status were performed using a Mann-Whitney test or Wilcoxon test for paired and unpaired samples, respectively. A Pearson correlation was performed between % HBV+ hepatocytes and peripheral viral biomarkers. The corresponding p-values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method.

      Results

      Subject characteristics

      In total, 431 core needle liver biopsies (133 stained by duplex; 298 stained by 4-plex) were subjected to the multiplex IF assay. Final analysis was performed on 220 liver biopsies analyzed by the 4-plex IF assay that passed stringent quality control (QC) criteria, which included absence of extensive tissue folds, mechanical disruption, or tissue loss, strong autofluorescence in one or more of the signal channels, or dropout of nuclear DAPI/Histone H3 signal and/or Na+K+-ATPase membrane marker in majority of the biopsy. These 220 biopsies were obtained from 114 total individuals (75 HBeAg- and 39 HBeAg+) at baseline, Wk48, and/or Wk240 (Table 1, Supplemental Table 1). Statistically significant differences (p< 0.001) in baseline characteristics were observed for age, HBV DNA, HBsAg, HBcrAg and HBV RNA, but not ALT between HBeAg- and HBeAg+ subjects (Table 1). These differences are consistent with known differences between stages of HBV disease and the enrollment criteria for the studies
      • Marcellin P.
      • Heathcote E.J.
      • Buti M.
      • Gane E.
      • de Man R.A.
      • Krastev Z.
      • et al.
      Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B.
      ,
      • Montanari N.R.
      • Ramirez R.
      • Aggarwal A.
      • van Buuren N.
      • Doukas M.
      • Moon C.
      • et al.
      Multi-parametric analysis of human livers reveals variation in intrahepatic inflammation across phases of chronic hepatitis B infection.
      .

      Development of a 4-plex Assay to Accurately Quantify Biopsy HBV+ Hepatocytes

      To assess HBV+ hepatocyte burden in core needle liver biopsies, we initially developed a duplex IF assay using antibodies against HBsAg and HBV core in the liver. We identified multiple pitfalls of the duplex assay that resulted in challenges for downstream analysis, including high numbers of biopsies that failed QC, difficulty assigning viral antigens to individual hepatocytes, and inability to consistently and accurately quantify the total number of hepatocytes. A major reason for QC failure was dropout of nuclear DAPI signal in almost 40% of samples, potentially due to the archival nature of the biopsies in this study (Fig. S3A). We therefore expanded the multiplex panel to include Histone H3, which detected hepatocyte nuclei in many samples where DAPI failed (Fig. 1A). In addition, we expanded our multiplex panel to include Na+K+-ATPase, a heterodimeric surface membrane protein complex, to accurately detect individual hepatocytes and enable assignment of HBsAg and HBV core staining to individual cells (Fig. 1A, S3B). As a result, we were able to reduce the QC failure rate from 38% with the duplex IF assay to 23% with the 4-plex IF assay. In total, we applied the 4-plex IF assay and customized image analysis algorithm to a total of 298 core needle liver biopsies. Of those initially evaluated, 220 passed quality control. Remaining causes of QC failure included extensive tissue folds, mechanical disruption or tissue loss, strong autofluorescence in one or more of the signal channels, or dropout of nuclear DAPI/Histone H3 signal and/or Na+K+-ATPase membrane marker in majority of the biopsy.
      Figure thumbnail gr1
      Figure 1Development of a 4-plex IF assay to accurately quantify liver HBV burden. (A) Representative image of 4-plex displayed as single channels (right) or overlayed image (left). (B) Representative images of signal channels for cell membrane (Na+/K+-ATPase) and nuclear staining (histone H3) used to train convolutional neural network (U-Net; upper left) and corresponding probability maps to robustly segment cells (lower left); signal channels for HBV antigens (upper right) to accurately identify HBV-infected hepatocytes (lower right). DL, deep learning. (C) Representative image of multiplex IF showing diffuse HBcAg staining and foci of HBsAg staining. (D) Representative images of differential cell localization of HBsAg (left) and HBcAg (right) expression.
      A deep learning algorithm was developed to robustly detect cell membranes and nuclei (Fig. 1B). Compared to a threshold-based approach, the deep learning algorithm allowed for cell segmentation that was less affected by fluorescent intensity fluctuations. Nucleated and segmented cells were qualified as hepatocytes by their large size and were easily distinguished from leukocytes, which have significantly smaller nuclei and cytoplasm. Hepatocytes were classified as HBV+ if they stained for HBsAg and/or HBV core. This approach allowed for robust quantification of HBV positive cells as a percentage of total hepatocytes (Fig. 1B).
      The most common pattern observed across these biopsies was mutually exclusive HBsAg and HBV core staining, with diffusely distributed individual HBV core+ cells and foci of HBsAg+ cells (Fig. 1C-D). HBsAg was either localized to the cell membrane or cytoplasm within the hepatocyte (Fig. 1D). HBV core was present either as strong nuclear staining or as cytoplasmic staining within the hepatocyte (Fig. 1D). Although HBV core and HBsAg staining were typically non-overlapping, patches of mixed positive staining were observed in some samples and a subset of hepatocytes dual-positive for HBsAg and HBV core were identifiable. These dual-positive hepatocytes were infrequent, as previously reported
      • Montanari N.R.
      • Ramirez R.
      • Aggarwal A.
      • van Buuren N.
      • Doukas M.
      • Moon C.
      • et al.
      Multi-parametric analysis of human livers reveals variation in intrahepatic inflammation across phases of chronic hepatitis B infection.
      • Hsu H.C.
      • Lai M.Y.
      • Su I.J.
      • Chen D.S.
      • Chang M.H.
      • Yang P.M.
      • et al.
      Correlation of hepatocyte HBsAg expression with virus replication and liver pathology.
      • Zhang X.
      • Lu W.
      • Zheng Y.
      • Wang W.
      • Bai L.
      • Chen L.
      • et al.
      In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection.
      • Naumov N.V.
      • Mondelli M.
      • Alexander G.J.
      • Tedder R.S.
      • Eddleston A.L.
      • Williams R.
      Relationship between expression of hepatitis B virus antigens in isolated hepatocytes and autologous lymphocyte cytotoxicity in patients with chronic hepatitis B virus infection.
      • Hsu H.C.
      • Su I.J.
      • Lai M.Y.
      • Chen D.S.
      • Chang M.H.
      • Chuang S.M.
      • et al.
      Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection.
      • van Buuren N.
      • Ramirez R.
      • Turner S.
      • Chen D.
      • Suri V.
      • Aggarwal A.
      • et al.
      Characterization of the liver immune microenvironment in liver biopsies from patients with chronic HBV infection.
      (Fig. S4).

      Analysis of Duplicate Liver Biopsies Identifies Sampling Heterogeneity with Core Needle Biopsies

      We identified 15 instances where two core needle biopsies were collected from different portions of the liver at the same timepoint, either at baseline or week 48. This unique subset of samples allowed us to evaluate whether a single core needle biopsy is representative of the HBV burden across different areas of the liver.
      Surprisingly, we found no correlation in total HBV+ hepatocyte burden between two liver biopsies collected from the same individual and timepoint (Fig. 2A-B). This result suggests that there is extensive heterogeneity in HBV positivity within individual livers and hence a large effect of random sampling on the frequency of HBV-positive hepatocytes detected in individual core needle liver biopsies. When plotted separately, we found no correlation in HBsAg+ hepatocytes (Fig. S5A) and a statistically significant but poor correlation in HBV core+ hepatocytes (Fig. S5B) between two biopsies from the same subject-timepoint. To evaluate how biopsy sampling may impact analysis of a collection of individual biopsies, we plotted the total HBV+ hepatocyte burden from each of the two individual biopsies and compared it to the average of the duplicate biopsies (Fig 2C). Individual biopsy data produced a non-normal distribution of values, whereas the average of two biopsies approached a more normal distribution with fewer on the extreme high or low ends of the scale. This pattern suggested a “hot spot” effect of core needle biopsy sampling, in which the measured value tends to be higher or lower than the true tissue average because local regions of the liver tend to have either very high or very low percentages of HBV+ hepatocytes. To better visualize the heterogeneity in HBV positivity, we analyzed one large 12 mm x 14 mm wedge biopsy that was obtained from a single subject at week 48 post-NUC treatment (Fig. 2D). Staining in this sample was almost exclusively HBsAg+, with little to no HBV core+ hepatocytes. Heterogeneity in staining within this single biopsy was extensive, with large regions of no apparent HBV staining, areas with a small percentage of HBsAg+ hepatocytes, and focal regions of near-complete HBsAg positivity, consistent with observations above.
      Figure thumbnail gr2
      Figure 2Analysis of duplicate liver biopsies suggests inherent sampling heterogeneity for viral antigens. (A) Representative images from duplicate biopsies collected from the same patient-timepoint. Embedded legends indicate markers, corresponding color, and percent hepatocytes positive for viral antigens. (B) Correlation between % total HBV+ hepatocytes (defined as HBsAg+ and/or HBV core+) in biopsy #1 vs. biopsy #2 from the same patient-timepoint using Pearson’s correlation analysis. (C) Quantification of total HBV burden when duplicate biopsies are plotted individually (left) or averaged for the same patient-timepoint (right). (D) Representative image of a large wedge biopsy with regions of low (embedded image #1) and high (embedded image #2) % of HBsAg+ hepatocytes.
      Collectively, these data indicate that sampling heterogeneity has a large impact on the results obtained with single core needle biopsies, such that individual biopsies often skew toward very high or very low values that are likely not representative of the overall average across a patient’s liver.

      NUC-Treated, HBeAg- Subjects Have the Lowest HBV+ hepatocyte burden

      Since single core needle biopsies are not representative of the entire liver burden for individual patients, we focused on population-level analyses of HBV+ hepatocyte burden between HBeAg+ (n=39) and HBeAg- (n=75) CHB subjects at baseline and post-NUC treatment. Subjects for which two biopsies were collected at the same timepoint had their values averaged for analysis.
      NUC treatment for 48 weeks significantly reduced total HBV+ hepatocyte burden in both HBeAg+ and HBeAg- subjects (Fig. 3A, 3D). Comparable results were obtained in subjects that received TDF or ADV (Fig. S6A). This decline in total HBV+ hepatocyte burden with treatment was driven by large decreases in HBV core+ hepatocytes (Fig. 3C), while minimal changes in HBsAg+ hepatocytes were observed over time (Fig. 3B). HBeAg status also impacted HBV core positivity, as the frequency of HBV core+ hepatocytes was lower in HBeAg- subjects compared to HBeAg+ subjects at all timepoints evaluated (Fig. 3C). As previously mentioned, HBsAg+ HBV core+ cells were rare and only found in HBeAg+ subjects at baseline (Fig. S4). HBeAg-, NUC-treated subjects had the lowest HBV+ hepatocyte burden amongst the populations tested. A subset of subjects within our cohort had a complete longitudinal time course available pre- and post-NUC treatment (Supplemental Table 1). Paired analysis of this biopsy subset showed similar trends to the results observed with the entire cohort (Figs. S6B–D).
      Figure thumbnail gr3
      Figure 3HBeAg status and NUC treatment impact HBV+ hepatocyte burden. Quantification of % total HBV+ hepatocytes (A, defined as HBsAg+ and/or HBV core+), % HBsAg+ hepatocytes (B), and % HBV core+ hepatocytes (C) pre- and post- NUC treatment in HBeAg+ (light blue) and HBeAg- (dark blue) subjects. (D) Representative images from HBeAg+ (lower) and HBeAg- (upper) subjects throughout NUC-treatment. Embedded legend indicates markers and corresponding color. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Mann-Whitney test, only statistically significant differences shown.

      Correlations Between HBV+ hepatocyte burden and Peripheral Biomarkers are Dependent on Stage of Infection and Treatment Status

      Finally, we sought to understand if any correlations existed between the frequency of HBV+ hepatocytes and well-established peripheral viral biomarkers. In a subset of samples with available paired serum or plasma, we evaluated HBsAg, HBcrAg, HBV DNA, and HBV RNA (Fig. S7A) and performed correlation analyses of liver biopsy vs. peripheral biomarker data, stratified by HBeAg status.
      In HBeAg+ subjects, we identified statistically significant correlations between total HBV+ hepatocyte burden and peripheral HBcrAg, HBV DNA and HBV RNA at baseline, prior to NUC treatment (Table 2 and Fig. S8). Upon NUC treatment, however, most correlations between peripheral viral biomarkers and total HBV+ hepatocyte burden were lost. Correlations between peripheral biomarkers and HBsAg+ hepatocytes or HBV core+ hepatocytes were inconsistent when analyzed independently (Supplemental Table 2).
      Table 2Correlation Between Peripheral Viral Biomarkers and Total HBV+ Liver Burden.
      HBeAg StatusPeripheral viral biomarkerBaselineWeek 48Week 240
      R-valuep-valueAdjusted p-valueR-valuep-valueAdjusted p-valueR-valuep-valueAdjusted p-value
      HBeAg+HBsAg0.1950.3610.6290.1160.5890.7480.2790.150.355
      HBcrAg0.6380.002*0.0270.3490.2020.3960.050.8150.896
      HBV DNA0.6540.001*0.0180.6190.0320.139NR
      HBV RNA0.6510.001*0.0250.5190.0840.258-0.0160.9490.971
      HBeAg−HBsAg0.2790.1680.3710.1030.4350.6940.4330.004*0.033
      HBcrAg-0.1010.6820.835-0.1110.5520.721-0.0080.9710.97
      HBV DNA-0.1180.5570.721NRNR
      HBV RNA-0.1630.480.719-0.3980.1420.3350.0460.9320.97
      *Adjusted p<0.05: Pearson correlation analysis, p-values adjusted for false discovery rate using Benjamini-Hochberg procedure. NR, not reported due to insufficient (<5) data points for analysis. Bold text indicates statistical significance with adjusted p-value.

      Discussion

      Here we apply a novel 4-plex IF assay to quantify HBV antigens in a large cohort of HBV-infected patients and provide insight into viral antigen burden and distribution over time. Utilization of Histone H3/DAPI and Na+K+-ATPase allowed us to clearly define nuclei and cell membranes, which enabled training of a convolutional neural network to robustly segment cell membranes and nuclei. We identified population-level reductions in HBV core+ hepatocytes associated with NUC treatment and lower frequencies of HBV core+ hepatocytes in HBeAg+ vs. HBeAg- patients. Importantly, we demonstrated extensive regional variation in HBV positivity in liver samples. Significant correlations between peripheral biomarkers and total HBV+ hepatocyte burden were identified in untreated HBeAg+ patients, with few significant correlations observed in HBeAg- and/or NUC-treated patients.
      Consistent with existing literature, we observed strong HBV core and HBsAg staining in baseline liver samples from HBeAg+ subjects, while the HBeAg- liver samples demonstrated high HBsAg staining but little to no HBV core staining
      • Montanari N.R.
      • Ramirez R.
      • Aggarwal A.
      • van Buuren N.
      • Doukas M.
      • Moon C.
      • et al.
      Multi-parametric analysis of human livers reveals variation in intrahepatic inflammation across phases of chronic hepatitis B infection.
      ,
      • Hsu H.C.
      • Lai M.Y.
      • Su I.J.
      • Chen D.S.
      • Chang M.H.
      • Yang P.M.
      • et al.
      Correlation of hepatocyte HBsAg expression with virus replication and liver pathology.
      ,
      • Hsu H.C.
      • Su I.J.
      • Lai M.Y.
      • Chen D.S.
      • Chang M.H.
      • Chuang S.M.
      • et al.
      Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection.
      • van Buuren N.
      • Ramirez R.
      • Turner S.
      • Chen D.
      • Suri V.
      • Aggarwal A.
      • et al.
      Characterization of the liver immune microenvironment in liver biopsies from patients with chronic HBV infection.
      • Ramakrishna B.
      • Mukhopadhya A.
      • Kurian G.
      Correlation of hepatocyte expression of hepatitis B viral antigens with histological activity and viral titer in chronic hepatitis B virus infection: an immunohistochemical study.
      • Safaie P.
      • Poongkunran M.
      • Kuang P.P.
      • Javaid A.
      • Jacobs C.
      • Pohlmann R.
      • et al.
      Intrahepatic distribution of hepatitis B virus antigens in patients with and without hepatocellular carcinoma.
      . We observed relatively few cells in which both HBV core and HBsAg were detected at the same time, as previously reported
      • Hsu H.C.
      • Lai M.Y.
      • Su I.J.
      • Chen D.S.
      • Chang M.H.
      • Yang P.M.
      • et al.
      Correlation of hepatocyte HBsAg expression with virus replication and liver pathology.
      • Zhang X.
      • Lu W.
      • Zheng Y.
      • Wang W.
      • Bai L.
      • Chen L.
      • et al.
      In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection.
      • Naumov N.V.
      • Mondelli M.
      • Alexander G.J.
      • Tedder R.S.
      • Eddleston A.L.
      • Williams R.
      Relationship between expression of hepatitis B virus antigens in isolated hepatocytes and autologous lymphocyte cytotoxicity in patients with chronic hepatitis B virus infection.
      • Hsu H.C.
      • Su I.J.
      • Lai M.Y.
      • Chen D.S.
      • Chang M.H.
      • Chuang S.M.
      • et al.
      Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection.
      • van Buuren N.
      • Ramirez R.
      • Turner S.
      • Chen D.
      • Suri V.
      • Aggarwal A.
      • et al.
      Characterization of the liver immune microenvironment in liver biopsies from patients with chronic HBV infection.
      . The presence of HBsAg+/HBV core-cells is consistent with literature characterizing hepatocytes containing integrated HBV DNA
      • Tu T.
      • Budzinska M.A.
      • Shackel N.A.
      • Urban S.
      HBV DNA Integration: Molecular Mechanisms and Clinical Implications.
      ,
      • Meier M.A.
      • Calabrese D.
      • Suslov A.
      • Terracciano L.M.
      • Heim M.H.
      • Wieland S.
      Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load.
      . Double-stranded linear HBV DNA has been shown to express HBsAg, but not HBV core
      • Yang W.
      • Summers J.
      Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor.
      • Freitas N.
      • Abe K.
      • Cunha C.
      • Menne S.
      • Gudima S.O.
      Support of the infectivity of hepatitis delta virus particles by the envelope proteins of different genotypes of hepatitis B virus.
      • Budzinska M.A.
      • Shackel N.A.
      • Urban S.
      • Tu T.
      Cellular Genomic Sites of Hepatitis B Virus DNA Integration.
      , and targeted long-read sequencing showed that many HBV integrations are clonally expanded, consistent with the foci of HBsAg+/HBV core-cells
      • van Buuren N.
      • Ramirez R.
      • Soulette C.
      • Suri V.
      • Han D.
      • May L.
      • et al.
      Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.
      ,
      • Mason W.S.
      • Gill U.S.
      • Litwin S.
      • Zhou Y.
      • Peri S.
      • Pop O.
      • et al.
      HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant.
      . Additionally, HBsAg+/HBV core-cells were dominant in HBeAg- patients, where most HBV transcripts derive from integrated DNA
      • van Buuren N.
      • Ramirez R.
      • Soulette C.
      • Suri V.
      • Han D.
      • May L.
      • et al.
      Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.
      .
      In contrast, HBsAg-/HBV core+ staining is less well defined, as infected hepatocytes containing cccDNA should be able to co-express HBV core and HBsAg. However, the fact that HBsAg-/HBV core+ cells were most common in HBeAg+, untreated subjects is informative, as most HBV transcripts in this population have been shown to derive from cccDNA
      • van Buuren N.
      • Ramirez R.
      • Soulette C.
      • Suri V.
      • Han D.
      • May L.
      • et al.
      Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.
      . We similarly observed that HBeAg+ subjects contained significantly more HBV core+ hepatocytes compared to HBeAg- patients at all timepoints. Consequently, it is likely that HBsAg-/HBV core+ cells represent cccDNA-containing cells and the level of intracellular HBsAg simply falls below the limit of assay detection in many cases, as previously reported
      • Gowans E.J.
      • Burrell C.J.
      Widespread presence of cytoplasmic HBcAg in hepatitis B infected liver detected by improved immunochemical methods.
      . This would imply that the amount of intracellular HBsAg is lower in most cccDNA+ cells than in those with integrated HBV. This could occur if cccDNA+ cells produce lower amounts of HBsAg than cells with integrated HBV and/or if HBsAg is more efficiently secreted from cccDNA+ cells. Indeed, HBV integration has been reported to change the ratios of large, medium, and small HBsAg production, and thereby disrupt efficient particle secretion and cause HBsAg accumulation in the cytoplasm
      • Chang H.-Y.
      • Tsai H.-W.
      • Teng C.-F.
      • Hui-Ching Wang L.
      • Huang W.
      • Su I.-J.
      Ground glass hepatocytes provide targets for therapy or prevention of hepatitis B virus-related hepatocellular carcinoma.
      . A subset of cells with cccDNA may have sufficient HBsAg for detection, explaining the occasional presence of dual HBsAg+/HBV core+ hepatocytes, as well as their disappearance upon NUC treatment. This scenario is consistent with all the staining patterns observed here and with the literature cited above.
      We determined that individual core needle biopsies are typically not representative of the entire liver, hence caution is needed in extrapolating biopsy measurements to the entire organ
      • Meier M.A.
      • Calabrese D.
      • Suslov A.
      • Terracciano L.M.
      • Heim M.H.
      • Wieland S.
      Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load.
      . In 15 individuals who had two biopsies collected from different parts of the liver at the same timepoint, there were poor correlations between HBV antigen staining in the two biopsies. Many samples contained “hot spots” of HBsAg+ hepatocytes, presumed to be clonal expansions harboring integrated HBV DNA. The associated focal pattern of HBsAg staining could explain the tendency observed here for needle biopsies to yield liver HBV antigen measurements that were frequently much higher or lower than the average burden. This conclusion is supported by the heterogeneous distribution in HBV antigen staining in a single large wedge biopsy. These data suggest the need for caution when interpreting biopsy results for individual patients. However, in our sub-group analysis, the median frequency of HBV+ hepatocytes was similar whether single biopsy values or averages of two biopsies were used to measure liver HBV positivity, suggesting that comparisons of population medians is reliable in spite of the observed liver sampling effects on an individual patient level. Consequently, we focused our analyses on population-level, rather than individual-patient comparisons.
      A major finding of this study is that NUC treatment resulted in large reductions in HBV core+, but not HBsAg+, hepatocytes in both HBeAg+ and HBeAg- subjects. To our knowledge, only a single previous report has documented this pattern of NUC effects in the liver and was semi-quantitative
      • Tsai H.W.
      • Lin Y.J.
      • Wu H.C.
      • Chang T.T.
      • Wu I.C.
      • Cheng P.N.
      • et al.
      Resistance of ground glass hepatocytes to oral antivirals in chronic hepatitis B patients and implication for the development of hepatocellular carcinoma.
      . We confirm and extend that observation with quantitative analysis of a much larger biopsy set. Our results suggest that NUC treatment reduces HBV core burden (likely cccDNA+) by reducing HBV replication (and hence viral spread) but does not significantly impact antigen expression from pre-existing integrated HBV DNA. The recently reported effect of NUCs on reducing integration burden
      • Hsu Y.C.
      • Suri V.
      • Nguyen M.H.
      • Huang Y.T.
      • Chen C.Y.
      • Chang I.W.
      • et al.
      Inhibition of Viral Replication Reduces Transcriptionally Active Distinct Hepatitis B Virus Integrations With Implications on Host Gene Dysregulation.
      likely results from preventing formation of new integrations, rather than direct effects on existing integrants. Accordingly, in the periphery we observed a multi-log10 decrease in HBV DNA with NUC treatment versus a half-log10 median HBsAg decline
      • Marcellin P.
      • Heathcote E.J.
      • Buti M.
      • Gane E.
      • de Man R.A.
      • Krastev Z.
      • et al.
      Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B.
      , consistent with integrated HBV DNA maintaining HBsAg production during NUC treatment
      • Grudda T.
      • Hwang H.S.
      • Taddese M.
      • Quinn J.
      • Sulkowski M.S.
      • Sterling R.K.
      • et al.
      Integrated hepatitis B virus DNA maintains surface antigen production during antivirals.
      . Collectively, these results suggest a moderate half-life of cccDNA+ hepatocytes, as the HBV core+ hepatocyte population is reduced by >95% over 48 weeks of treatment. This occurs even though NUC treatment does not completely block viral replication
      • Burdette D.L.
      • Lazerwith S.
      • Yang J.
      • Chan H.L.Y.
      • Delaney Iv W.E.
      • Fletcher S.P.
      • et al.
      Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.
      , indicating that the level of residual infectious virus during NUC treatment is too low to compensate for natural hepatocyte turnover and/or immune-mediated pressure on HBV core+ cells. A moderate cccDNA half-life is also supported by previous reports that a year of NUC treatment eliminates over 80% of cccDNA
      • Werle-Lapostolle B.
      • Bowden S.
      • Locarnini S.
      • Wursthorn K.
      • Petersen J.
      • Lau G.
      • et al.
      Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.
      and that in cases of lamivudine resistance, a majority of cccDNA can be replaced with resistant genomes in a span of approximately 6 months
      • Huang Q.
      • Zhou B.
      • Cai D.
      • Zong Y.
      • Wu Y.
      • Liu S.
      • et al.
      Rapid Turnover of Hepatitis B Virus Covalently Closed Circular DNA Indicated by Monitoring Emergence and Reversion of Signature-Mutation in Treated Chronic Hepatitis B Patients.
      .
      Statistically significant correlations between total detected HBV+ hepatocyte frequency and the peripheral biomarkers HBcrAg, HBV DNA, and HBV RNA were present only in HBeAg+ patients, where a large portion of HBV+ hepatocytes represent cccDNA+ infected cells that may distribute more evenly in the liver than clonal integrations. In contrast, correlations between detected HBV+ hepatocyte frequency and peripheral viral biomarkers were largely absent in HBeAg- and NUC-treated patients, where the large majority of HBV antigen staining in biopsies represents integrations
      • Mason W.S.
      • Gill U.S.
      • Litwin S.
      • Zhou Y.
      • Peri S.
      • Pop O.
      • et al.
      HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant.
      ,
      • Wooddell C.I.
      • Yuen M.F.
      • Chan H.L.
      • Gish R.G.
      • Locarnini S.A.
      • Chavez D.
      • et al.
      RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.
      ,
      • Montanari N.R.
      • Ramirez R.
      • Aggarwal A.
      • van Buuren N.
      • Doukas M.
      • Moon C.
      • et al.
      Multi-parametric analysis of human livers reveals variation in intrahepatic inflammation across phases of chronic hepatitis B infection.
      which do not produce HBV DNA or HBcrAg. As previously reported, correlations between biopsy HBsAg and peripheral HBsAg were largely absent across patient groups
      • Lau J.Y.
      • Bain V.G.
      • Davies S.E.
      • Alexander G.J.
      • Williams R.
      Export of intracellular HBsAg in chronic hepatitis B virus infection is related to viral replication.
      , likely due to the biopsy sampling heterogeneity and foci of HBsAg+ hepatocytes demonstrated in our study. The potential presence of HBV+ hepatocytes below the limits of detection by immunohistochemistry could also have impacted correlation analyses.
      We also found inconsistent correlations between peripheral biomarkers and either HBsAg+ hepatocytes or HBV core+ hepatocytes analyzed individually. Earlier efforts to identify such correlations have also yielded conflicting results. We identified a significant correlation between HBsAg+ hepatocytes and peripheral HBV DNA in untreated HBeAg+ but not HBeAg- patients. One previous report found the same
      • Thompson A.J.
      • Nguyen T.
      • Iser D.
      • Ayres A.
      • Jackson K.
      • Littlejohn M.
      • et al.
      Serum hepatitis B surface antigen and hepatitis B e antigen titers: disease phase influences correlation with viral load and intrahepatic hepatitis B virus markers.
      , another found no correlations in either group
      • Ramakrishna B.
      • Mukhopadhya A.
      • Kurian G.
      Correlation of hepatocyte expression of hepatitis B viral antigens with histological activity and viral titer in chronic hepatitis B virus infection: an immunohistochemical study.
      , and a third found an inverse correlation between intrahepatic and serum HBsAg
      • Lau J.Y.
      • Bain V.G.
      • Davies S.E.
      • Alexander G.J.
      • Williams R.
      Export of intracellular HBsAg in chronic hepatitis B virus infection is related to viral replication.
      . Similarly, we identified no clear patterns of correlation between peripheral markers and HBV core+ hepatocytes, despite a previous report correlating liver HBV core staining with serum HBV DNA
      • Ramakrishna B.
      • Mukhopadhya A.
      • Kurian G.
      Correlation of hepatocyte expression of hepatitis B viral antigens with histological activity and viral titer in chronic hepatitis B virus infection: an immunohistochemical study.
      . Notably, our analysis algorithm did not differentiate between nuclear vs. cytoplasmic HBV core, which vary based on HBeAg status and inflammation, or membranous vs. cytoplasmic HBsAg, which may differ between cccDNA and integrated HBV
      • Hsu H.C.
      • Su I.J.
      • Lai M.Y.
      • Chen D.S.
      • Chang M.H.
      • Chuang S.M.
      • et al.
      Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection.
      ,
      • Chu C.M.
      • Liaw Y.F.
      Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis.
      ,
      • Chu C.-M.
      • Liaw Y.-F.
      Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection.
      . These variables, differences in patient groups, and differences in methods for quantifying viral antigens may explain the disparate results of correlation analyses here and in the literature.
      An accurate understanding of HBV+ hepatocyte burden across disease stage and treatment status may help inform clinical development of novel HBV cure approaches. Many HBV cure strategies now aim to achieve immune-mediated elimination of the HBV-positive cells and safety may be linked to the number of hepatocytes that need to be killed and the speed with which they are eliminated. Identification of subjects with a low HBV+ hepatocyte burden would be the safest way to initiate such studies. Our current study suggests that at a population level, HBeAg- subjects on NUC treatment have the lowest HBV+ hepatocyte burden and may represent the best starting population for clinical development of some HBV cure agents. In our study cohort, serum viral biomarkers were not successful in identifying a population with lower HBV+ hepatocyte burden than HBeAg- NUC-treated patients overall. Our results also suggest that cytolytic mechanisms capable of killing cells expressing HBsAg are likely to be required to achieve cure, since HBV core+ (likely cccDNA+) cells represent only a small fraction of the HBV+ hepatocytes in NUC-treated patients.

      Financial support

      This work was funded by Gilead Sciences Inc.

      Conflict of interest

      At the time this study was conducted, AA, PO, JB, NvB, CM, SC, MVA, VS, TT, ST, AG, SF, LD, BF and SB were employees and stockholders of Gilead Sciences, Inc.

      Author contributions

      AA, PO, LD, BF and SB designed the study, MA, SC, ST and TT coordinated sample inventories and databases, CM and NvB designed and performed the experiments; AA, PO, JB, NvB, and SS analyzed the data. AA, PO, NvB, SPF, and SB wrote the manuscript. VS, PM, MB, and AG coordinated the clinical trial and biopsy collection. All authors approved the final version of the manuscript.

      Data statement

      The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

      Acknowledgements

      We would like to thank Sarah Tse from Bioscience Communications for the high-quality figures.

      Appendix A. Supplementary data

      The following is the supplementary data to this article:

      References

      1. World Health Organization: Global hepatitis report. 2017.

        • Kim G.A.
        • Lim Y.S.
        • An J.
        • Lee D.
        • Shim J.H.
        • Kim K.M.
        • et al.
        HBsAg seroclearance after nucleoside analogue therapy in patients with chronic hepatitis B: clinical outcomes and durability.
        Gut. 2014; 63: 1325-1332
        • Tong S.
        • Revill P.
        Overview of hepatitis B viral replication and genetic variability.
        J Hepatol. 2016; 64: S4-S16
        • Tu T.
        • Budzinska M.A.
        • Shackel N.A.
        • Urban S.
        HBV DNA Integration: Molecular Mechanisms and Clinical Implications.
        Viruses. 2017; 9: 75
        • Pollicino T.
        • Caminiti G.
        HBV-Integration Studies in the Clinic: Role in the Natural History of Infection.
        Viruses. 2021; 13: 368
        • van Buuren N.
        • Ramirez R.
        • Soulette C.
        • Suri V.
        • Han D.
        • May L.
        • et al.
        Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.
        JHEP Rep. 2022; 4100449
        • Jiang Z.
        • Jhunjhunwala S.
        • Liu J.
        • Haverty P.M.
        • Kennemer M.I.
        • Guan Y.
        • et al.
        The effects of hepatitis B virus integration into the genomes of hepatocellular carcinoma patients.
        Genome Res. 2012; 22: 593-601
        • Mason W.S.
        • Gill U.S.
        • Litwin S.
        • Zhou Y.
        • Peri S.
        • Pop O.
        • et al.
        HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant.
        Gastroenterology. 2016; 151: 986-998 e4
        • Budzinska M.A.
        • Shackel N.A.
        • Urban S.
        • Tu T.
        Sequence analysis of integrated hepatitis B virus DNA during HBeAg-seroconversion.
        Emerg Microbes Infect. 2018; 7: 142
        • Sung W.K.
        • Zheng H.
        • Li S.
        • Chen R.
        • Liu X.
        • Li Y.
        • et al.
        Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma.
        Nat Genet. 2012; 44: 765-769
        • Zhao L.H.
        • Liu X.
        • Yan H.X.
        • Li W.Y.
        • Zeng X.
        • Yang Y.
        • et al.
        Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma.
        Nat Commun. 2016; 712992
        • Peneau C.
        • Imbeaud S.
        • La Bella T.
        • Hirsch T.Z.
        • Caruso S.
        • Calderaro J.
        • et al.
        Hepatitis B virus integrations promote local and distant oncogenic driver alterations in hepatocellular carcinoma.
        Gut. 2022; 71: 616-626
        • Wooddell C.I.
        • Yuen M.F.
        • Chan H.L.
        • Gish R.G.
        • Locarnini S.A.
        • Chavez D.
        • et al.
        RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.
        Sci Transl Med. 2017; 9
        • Meier M.A.
        • Calabrese D.
        • Suslov A.
        • Terracciano L.M.
        • Heim M.H.
        • Wieland S.
        Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load.
        J Hepatol. 2021; 75: 840-847
        • Loglio A.
        • Vigano M.
        • Lampertico P.
        Novel Therapies That May Cure Chronic Hepatitis B Virus.
        Clin Liver Dis. 2021; 25: 875-899
        • Marcellin P.
        • Heathcote E.J.
        • Buti M.
        • Gane E.
        • de Man R.A.
        • Krastev Z.
        • et al.
        Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B.
        N Engl J Med. 2008; 359: 2442-2455
        • Gordon S.C.
        • Krastev Z.
        • Horban A.
        • Petersen J.
        • Sperl J.
        • Dinh P.
        • et al.
        Efficacy of tenofovir disoproxil fumarate at 240 weeks in patients with chronic hepatitis B with high baseline viral load.
        Hepatology. 2013; 58: 505-513
        • European Association for the Study of the Liver
        European Association for the Study of the Liver: EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection.
        J Hepatol. 2017; 67: 370-398
        • Hong X.
        • Luckenbaugh L.
        • Mendenhall M.
        • Walsh R.
        • Cabuang L.
        • Soppe S.
        • et al.
        Characterization of Hepatitis B Precore/Core-Related Antigens.
        J Virol. 2021; : 95
        • Taube J.M.
        • Roman K.
        • Engle E.L.
        • Wang C.
        • Ballesteros-Merino C.
        • Jensen S.M.
        • et al.
        Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study.
        J Immunother Cancer. 2021; : 9
      2. Ronneberger O, Fischer P, Brox T: U-Net: Convolutional Networks for Biomedical Image Segmentation. Medical Image Computing and Computer-Assisted Intervention – MICCAI 2015. Edited by Navab N, Hornegger J, Wells WM, Frangi AF. Cham: Springer International Publishing, 2015. pp. 234-241.

        • van Bommel F.
        • Bartens A.
        • Mysickova A.
        • Hofmann J.
        • Kruger D.H.
        • Berg T.
        • et al.
        Serum hepatitis B virus RNA levels as an early predictor of hepatitis B e antigen seroconversion during treatment with polymerase inhibitors.
        Hepatology. 2015; 61: 66-76
        • Montanari N.R.
        • Ramirez R.
        • Aggarwal A.
        • van Buuren N.
        • Doukas M.
        • Moon C.
        • et al.
        Multi-parametric analysis of human livers reveals variation in intrahepatic inflammation across phases of chronic hepatitis B infection.
        J Hepatol. 2022; 77: 332-343
        • Hsu H.C.
        • Lai M.Y.
        • Su I.J.
        • Chen D.S.
        • Chang M.H.
        • Yang P.M.
        • et al.
        Correlation of hepatocyte HBsAg expression with virus replication and liver pathology.
        Hepatology. 1988; 8: 749-754
        • Zhang X.
        • Lu W.
        • Zheng Y.
        • Wang W.
        • Bai L.
        • Chen L.
        • et al.
        In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection.
        J Clin Invest. 2016; 126: 1079-1092
        • Naumov N.V.
        • Mondelli M.
        • Alexander G.J.
        • Tedder R.S.
        • Eddleston A.L.
        • Williams R.
        Relationship between expression of hepatitis B virus antigens in isolated hepatocytes and autologous lymphocyte cytotoxicity in patients with chronic hepatitis B virus infection.
        Hepatology. 1984; 4: 63-68
        • Hsu H.C.
        • Su I.J.
        • Lai M.Y.
        • Chen D.S.
        • Chang M.H.
        • Chuang S.M.
        • et al.
        Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection.
        J Hepatol. 1987; 5: 45-50
        • van Buuren N.
        • Ramirez R.
        • Turner S.
        • Chen D.
        • Suri V.
        • Aggarwal A.
        • et al.
        Characterization of the liver immune microenvironment in liver biopsies from patients with chronic HBV infection.
        JHEP Rep. 2022; 4100388
        • Ramakrishna B.
        • Mukhopadhya A.
        • Kurian G.
        Correlation of hepatocyte expression of hepatitis B viral antigens with histological activity and viral titer in chronic hepatitis B virus infection: an immunohistochemical study.
        J Gastroenterol Hepatol. 2008; 23: 1734-1738
        • Safaie P.
        • Poongkunran M.
        • Kuang P.P.
        • Javaid A.
        • Jacobs C.
        • Pohlmann R.
        • et al.
        Intrahepatic distribution of hepatitis B virus antigens in patients with and without hepatocellular carcinoma.
        World J Gastroenterol. 2016; 22: 3404-3411
        • Yang W.
        • Summers J.
        Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor.
        J Virol. 1999; 73: 9710-9717
        • Freitas N.
        • Abe K.
        • Cunha C.
        • Menne S.
        • Gudima S.O.
        Support of the infectivity of hepatitis delta virus particles by the envelope proteins of different genotypes of hepatitis B virus.
        J Virol. 2014; 88: 6255-6267
        • Budzinska M.A.
        • Shackel N.A.
        • Urban S.
        • Tu T.
        Cellular Genomic Sites of Hepatitis B Virus DNA Integration.
        Genes (Basel). 2018; 9
        • Gowans E.J.
        • Burrell C.J.
        Widespread presence of cytoplasmic HBcAg in hepatitis B infected liver detected by improved immunochemical methods.
        J Clin Pathol. 1985; 38: 393-398
        • Chang H.-Y.
        • Tsai H.-W.
        • Teng C.-F.
        • Hui-Ching Wang L.
        • Huang W.
        • Su I.-J.
        Ground glass hepatocytes provide targets for therapy or prevention of hepatitis B virus-related hepatocellular carcinoma.
        AIMS Medical Science. 2018; 5: 90-101
        • Tsai H.W.
        • Lin Y.J.
        • Wu H.C.
        • Chang T.T.
        • Wu I.C.
        • Cheng P.N.
        • et al.
        Resistance of ground glass hepatocytes to oral antivirals in chronic hepatitis B patients and implication for the development of hepatocellular carcinoma.
        Oncotarget. 2016; 7: 27724-27734
        • Hsu Y.C.
        • Suri V.
        • Nguyen M.H.
        • Huang Y.T.
        • Chen C.Y.
        • Chang I.W.
        • et al.
        Inhibition of Viral Replication Reduces Transcriptionally Active Distinct Hepatitis B Virus Integrations With Implications on Host Gene Dysregulation.
        Gastroenterology. 2022; 162: 1160-11670 e1
        • Grudda T.
        • Hwang H.S.
        • Taddese M.
        • Quinn J.
        • Sulkowski M.S.
        • Sterling R.K.
        • et al.
        Integrated hepatitis B virus DNA maintains surface antigen production during antivirals.
        J Clin Invest. 2022; 132e161818
        • Burdette D.L.
        • Lazerwith S.
        • Yang J.
        • Chan H.L.Y.
        • Delaney Iv W.E.
        • Fletcher S.P.
        • et al.
        Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.
        PLoS One. 2022; 17e0262516
        • Werle-Lapostolle B.
        • Bowden S.
        • Locarnini S.
        • Wursthorn K.
        • Petersen J.
        • Lau G.
        • et al.
        Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.
        Gastroenterology. 2004; 126: 1750-1758
        • Huang Q.
        • Zhou B.
        • Cai D.
        • Zong Y.
        • Wu Y.
        • Liu S.
        • et al.
        Rapid Turnover of Hepatitis B Virus Covalently Closed Circular DNA Indicated by Monitoring Emergence and Reversion of Signature-Mutation in Treated Chronic Hepatitis B Patients.
        Hepatology. 2021; 73: 41-52
        • Lau J.Y.
        • Bain V.G.
        • Davies S.E.
        • Alexander G.J.
        • Williams R.
        Export of intracellular HBsAg in chronic hepatitis B virus infection is related to viral replication.
        Hepatology. 1991; 14: 416-421
        • Thompson A.J.
        • Nguyen T.
        • Iser D.
        • Ayres A.
        • Jackson K.
        • Littlejohn M.
        • et al.
        Serum hepatitis B surface antigen and hepatitis B e antigen titers: disease phase influences correlation with viral load and intrahepatic hepatitis B virus markers.
        Hepatology. 2010; 51: 1933-1944
        • Chu C.M.
        • Liaw Y.F.
        Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis.
        Gastroenterology. 1987; 92: 220-225
        • Chu C.-M.
        • Liaw Y.-F.
        Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection.
        Gastroenterology. 1987; 92: 220-225