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End-of-treatment HBcrAg and HBsAb levels identify durable functional cure after Peg-IFN-based therapy in patients with CHB

  • Author Footnotes
    † Da Huang and Di Wu are co-first authors; they contributed equally to the work
    Da Huang
    Footnotes
    † Da Huang and Di Wu are co-first authors; they contributed equally to the work
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Author Footnotes
    † Da Huang and Di Wu are co-first authors; they contributed equally to the work
    Di Wu
    Footnotes
    † Da Huang and Di Wu are co-first authors; they contributed equally to the work
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Peng Wang
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Yongli Wang
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Wei Yuan
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Danqing Hu
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Junjian Hu
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Yaqi Wang
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Ran Tao
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Fang Xiao
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Xiaoping Zhang
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Xiaojing Wang
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Meifang Han
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Xiaoping Luo
    Correspondence
    Corresponding authors. Address: Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; National Medical Center for Major Public Health Events, Wuhan, China; State Key Laboratory for Zoonotic Diseases, Wuhan, China.
    Affiliations
    National Medical Center for Major Public Health Events, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Weiming Yan
    Correspondence
    Corresponding authors. Address: Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; National Medical Center for Major Public Health Events, Wuhan, China; State Key Laboratory for Zoonotic Diseases, Wuhan, China.
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Qin Ning
    Correspondence
    Corresponding authors. Address: Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; National Medical Center for Major Public Health Events, Wuhan, China; State Key Laboratory for Zoonotic Diseases, Wuhan, China.
    Affiliations
    Department and Institute of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    State Key Laboratory for Zoonotic Diseases, Wuhan, China

    Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
    Search for articles by this author
  • Author Footnotes
    † Da Huang and Di Wu are co-first authors; they contributed equally to the work
Open AccessPublished:February 08, 2022DOI:https://doi.org/10.1016/j.jhep.2022.01.021

      Highlights

      • HBsAb appearance is a surrogate endpoint that may complement HBsAg loss in reflecting functional cure of hepatitis B.
      • On-treatment HBsAg or HBcrAg levels correlated with HBsAg loss or HBsAb appearance with Peg-IFN-based treatment.
      • On-treatment HBsAg or HBsAb levels correlated with durable functional cure post Peg-IFN-based therapy.
      • EOT HBcrAg <4 log10U/ml and HBsAb >2 log10IU/L can identify responders likely to achieve durable functional cure.
      • HBcrAg and HBsAb levels partially reflect post-therapy anti-HBV immune responses in the host.

      Background & Aims

      Functional cure can be sustained in a proportion of patients with chronic hepatitis B (CHB) who lose hepatitis B surface antigen (HBsAg) after pegylated interferon alpha (Peg-IFN-ɑ)-based treatment. In this study, we aimed to identify biomarkers associated with a durable functional cure and to dissect potential immunological mechanisms.

      Methods

      Of 257 nucleos(t)ide analogue-suppressed patients with CHB in the ANCHOR study, 80 patients randomly assigned to 96-week Peg-IFN-α-based therapy with 24-week off-treatment follow-up were included in this parallel study. Virologic and immunological biomarkers were examined dynamically. A response was defined as HBsAg loss or hepatitis B surface antibody (HBsAb) appearance at the end of treatment (EOT). Sustained response (SR) or durable functional cure was defined as sustained HBsAg loss with or without the appearance of HBsAb at the end of follow-up (EOF).

      Results

      Thirty-six (45.0%) out of 80 patients achieved a response at EOT; 58.3% (21/36) of responders maintained SR at EOF. Quantitative hepatitis B core-related antigen (qHBcrAg) and HBsAb at EOT were associated with SR, with AUROCs of 0.697 (0.512-0.882, p = 0.047) and 0.744 (0.573-0.915, p = 0.013), respectively. A combination of HBcrAg <4 log10U/ml and HBsAb >2 log10IU/L at EOT had a positive predictive value of 100% for SR with an AUROC of 0.822 (0.684-0.961, p = 0.001). These patients showed maintained proportions of HBV envelope-specific CD8+T and B cells, a markedly increased proportion of T follicular helper cells after Peg-IFN-ɑ discontinuation, and significantly higher proportions of HBV polymerase-specific CD8+T and CD86+CD19+B cells at EOF.

      Conclusions

      Lower HBcrAg and higher HBsAb levels at EOT were associated with sustained cellular and humoral immune responses. They can be used to identify patients likely to achieve durable functional cure post Peg-IFN-based therapy.

      ClinicalTrials.gov identifier

      NCT02327416

      Lay summary

      Functional cure can be sustained in a proportion of patients with chronic hepatitis B after pegylated interferon alpha-based treatment. However, predicting who will achieve durable functional cure remains challenging. Herein, we show that low levels of hepatitis B core-related antigen and higher levels of hepatitis B surface antibodies at the end of treatment are linked to immunological responses and are associated with durable functional cure.

      Graphical abstract

      Keywords

      Introduction

      Sustained suppression of HBV replication is attainable with long-term nucleos(t)ide analogue (NUC) treatment, but it is still suboptimal. Hepatitis B surface antigen (HBsAg) loss with or without seroconversion to hepatitis B surface antibody (HBsAb) is considered a functional cure and the ideal endpoint of antiviral therapy for chronic hepatitis B (CHB).
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      However, few studies to date have focused on the association between HBcrAg and durable functional cure after Peg-IFN-based therapy.
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      • Jiang J.
      • Tan D.
      • Hou J.
      • et al.
      Switching from entecavir to PegIFN alfa-2a in patients with HBeAg-positive chronic hepatitis B: a randomised open-label trial (OSST trial).
      ,
      • Wu D.
      • Yan W.M.
      • Tan D.M.
      • Peng S.F.
      • Chen Y.P.
      • Jiang J.J.
      • et al.
      Combination of NA, Peg-IFN alpha-2b and GMCSF enhanced HBsAb production in NA suppressed CHB patients (the Anchor A Study): an interim analysis.
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      In this prospective study evaluating the efficacy of a 96-week Peg-IFN-based therapy in NUC-suppressed patients with CHB, we characterized the dynamics of HBV biomarkers during and post treatment. We identified factors associated with treatment response, particularly durable functional cure.

      Patients and methods

      Patients and study design

      Patients with CHB, aged 18-65 years, who had received NUC treatment for 1-5 years and had serum HBV DNA ≤1,000 copies/ml and HBsAg ≤3,000 IU/ml were eligible for inclusion. Exclusion criteria included receiving IFN or systemic antiviral therapy within the previous 6 months; coinfection with HIV, HCV or HDV; presence of cirrhosis and decompensated liver disease; pregnancy or lactation; and any other contraindication to IFN therapy. A response was defined as HBsAg loss or HBsAb appearance at the end of treatment (EOT). Sustained response (SR) or durable functional cure was characterized as sustained HBsAg loss with or without HBsAb appearance at the end of follow-up (EOF).
      This is a pilot study conducted from March 2014 to June 2019 in parallel with a multicentre randomized controlled trial (ANCHOR study). A total of 257 NUC-suppressed patients with CHB were enrolled in the ANCHOR study, and randomly assigned to receive either entecavir therapy or Peg-IFN-based combination therapy, of whom 80 consecutive patients receiving Peg-IFN-based therapy were included in the present study. Patients were treated with 48 weeks of Peg-IFN-ɑ (180 μg/week) and entecavir combination therapy followed by another 48 weeks of Peg-IFN-ɑ, then were followed up for 24 weeks. Blood samples were drawn at baseline and every 24 weeks until EOF. Paired liver biopsies were performed at baseline and week 48. Adverse events and laboratory test results were recorded according to the International Conference on Harmonisation Guideline Clinical Safety Data Management: Definitions and Standards for Expedited Reporting. Written informed consent was obtained from all participants. The study protocol was approved by an independent central ethics committee of Huazhong University of Science and Technology at Wuhan.

      Serum HBV marker measurements

      Serum HBV markers were detected in the Central Clinical Laboratory of Tongji Hospital. HBV DNA levels were assessed using a Cobas TaqMan assay kit (Roche Diagnostics, Branchburg, NJ). Quantitative HBsAg and HBsAb levels were determined using Elecsys HBsAg II and Anti-HBs II quantitative assays, respectively (Roche Diagnostics GmbH, Mannheim, Germany). The HBeAg concentration was quantitatively determined by electrochemiluminescence immunoassay (Abbott Laboratories, Chicago, IL). The HBcrAg concentration was determined using a fully automated lumipulse chemiluminescence enzyme immunoassay system (Fujirebio Inc, Tokyo, Japan; dynamic range 3-7 log10U/ml, lower detection limit 2 log10U/ml) according to the standard protocol. Serum samples with HBcrAg levels above the upper detection limit were serially diluted and retested.
      • Kimura T.
      • Rokuhara A.
      • Sakamoto Y.
      • Yagi S.
      • Tanaka E.
      • Kiyosawa K.
      • et al.
      Sensitive enzyme immunoassay for hepatitis B virus core-related antigens and their correlation to virus load.
      • Rokuhara A.
      • Tanaka E.
      • Matsumoto A.
      • Kimura T.
      • Yamaura T.
      • Orii K.
      • et al.
      Clinical evaluation of a new enzyme immunoassay for hepatitis B virus core-related antigen; a marker distinct from viral DNA for monitoring lamivudine treatment.
      • Kimura T.
      • Ohno N.
      • Terada N.
      • Rokuhara A.
      • Matsumoto A.
      • Yagi S.
      • et al.
      Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain.

      Intrahepatic HBV cccDNA measurement

      Quantitative intrahepatic HBV cccDNA was determined using real-time PCR. Total HBV DNA extracted from liver biopsy samples using the Qiagen QIAamp DNA Mini Kit (Qiagen, #51304, Germany) was digested with Plasmid-Safe ATP-dependent DNase (Epicentre, #e3101k, USA), which selectively removes linear double-stranded DNA and linear and closed-circular single-stranded DNA for subsequent HBV cccDNA detection. The sequences and positions of primers and probe targets to cccDNA are listed in the supplementary CTAT table. Serially diluted pHBV1.3-B6 plasmids (range of 5 to 5×10ˆ5 copies/μl) were used to establish standard curves, and the β-actin housekeeping gene was used as a quantitation standard to quantify cellular DNA.
      • Chen E.Q.
      • Feng S.
      • Wang M.L.
      • Liang L.B.
      • Zhou L.Y.
      • Du L.Y.
      • et al.
      Serum hepatitis B core-related antigen is a satisfactory surrogate marker of intrahepatic covalently closed circular DNA in chronic hepatitis B.

      Analysis of peripheral blood immunological indicators

      The proportions of HBV-specific CD8+ T cells, T follicular helper (Tfh) and B cells as well as the expression of CD86 and CD95 on B cells at EOT and EOF were analysed by flow cytometry. Monoclonal antibodies are shown in the supplementary CTAT table. Synthetic peptides and the corresponding phycoerythrin (PE)-labelled tetrameric peptide-HLA class I complexes representing the HLA-A2-restricted epitopes on the core peptide spanning amino acids 18 to 27 (FLPSDFFPSV), envelope peptides spanning amino acids 335 to 343 (WLSLLVPFV), and polymerase peptides spanning amino acids 575 to 583 (FLLSLGIHL) were purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA). Peripheral blood mononuclear cells were incubated with PE-labelled tetramers for 30 minutes and washed with fluorescence-activated cell sorting buffer, followed by staining with anti-CD8. Cell surface antigen staining was performed as per the protocol. The cells were analysed immediately on a BD FACS Canto II Flow Cytometer and FACS Diva Software.

      Statistical analysis

      Student’s t test or Mann-Whitney U test was performed for 2-level independent variables, and one-way analysis of variance was used for more than 2-level variables. The comparison of 2 ratios was processed with Crosstabs and Fisher’s exact test. Binary logistic regression and receiver-operating characteristic curves determined factors associated with treatment response and predictive efficacy. The optimal cut-off value of each biomarker was obtained using Youden’s index (sensitivity+specificity-1). A p value <0.05 was considered statistically significant. Statistical analysis was performed using SPSS 22.0 software.

      Results

      Baseline characteristics and treatment efficacy

      Of the 80 patients included in this study, 41.3% were HBeAg positive at baseline. The mean levels of HBsAg, HBcrAg, and HBeAg were 2.797±0.047 log10IU/ml, 4.710±0.129 log10U/ml and 0.155±0.098 log10S/CO, respectively. The median duration of prior NUC treatment was 2.49 years. At EOT, 36 (45.0%) patients achieved a response. The HBsAg loss and HBsAb appearance rates were 30.0% (24/80) and 43.8% (35/80), respectively. The mean titer of HBsAb was 139.0±33.9 IU/L. A total of 58.3% (21/36) of the responders maintained SR at EOF.
      No significant difference was observed in sex ratio, body mass index, HBeAg positive rate or HBV DNA undetectable rate, levels of alanine aminotransferase, total bilirubin, or liver stiffness at baseline between responders and non-responders. However, responders were significantly younger and had lower baseline HBsAg levels than non-responders. Among 36 responders, sustained and non-sustained responders had similar demographics and baseline characteristics (Table 1).
      Table 1Baseline characteristics and longitudinal changes in HBV parameters during and post treatment.
      All patients (n = 80)ResponseSustained response
      Responder (n = 36)Non-responder (n = 44)p valueSustained responder (n = 21)Non-sustained responder (n = 15)p value
      Males, n (%)66 (82.5%)27 (75.0%)39 (88.6%)0.14316 (76.2%)11 (73.3%)1.000
      Age, years34 (26-46)28 (23-36)41 (31-46)<0.001
      p <0.05 is considered significant difference between 2 groups.
      29 (23-36)27 (24-38)0.934
      Body mass index, kg/m223.12 (0.38)23.21 (0.60)23.05 (0.46)0.83723.18 (0.69)23.24 (1.11)0.962
      Duration of NUCs, years2.49 (1.78-3.89)2.19 (1.61-4.01)2.70 (1.86-3.83)0.8892.16 (1.80-3.41)3.36 (1.15-5.39)0.722
      Previous NUCs taken, n (%)0.805
       ETV39 (48.8%)15 (41.7%)24 (54.5%)
       LAM/LdT7 (8.8%)4 (11.1%)3 (6.8%)
       ADV9 (11.3%)5 (13.9%)4 (9.1%)
       LAM/LdT+ADV12 (15.0%)6 (16.7%)6 (13.6%)
       ETV+ADV/TDF13 (16.3%)6 (16.7%)7 (15.9%)
      HBV DNA positive, n (%)10 (12.5%)4 (11.1%)6 (13.6%)1.0002 (9.5%)2 (13.3%)1.000
      HBeAg positive, n (%)33 (41.3%)14 (38.9%)19 (43.2%)0.8208 (38.1%)6 (40.0%)1.000
      ALT, U/L
       Baseline20.0 (14.0-29.0)18.0 (13.0-26.5)22.0 (15.8-31.0)0.23218.0 (13.0-25.0)20.0 (13.0-30.0)0.709
       EOT31.5 (25.0-45.0)29.0 (24.3-40.8)35.0 (26.0-46.0)0.14730.0 (26.5-43.0)27.0 (24.0-34.0)0.251
       EOF20.0 (13.0-28.0)17.0 (12.0-23.8)23.0 (17.0-31.0)0.36213.0 (11.5-22.5)20.0 (14.0-24.0)0.275
      TBiL, μmol/L
       Baseline12.4 (10.1-16.1)11.5 (8.8-14.6)12.6 (11.0-18.2)0.08312.2 (9.6-14.4)10.3 (8.0-15.6)0.858
       EOT9.8 (8.4-13.1)9.6 (7.3-12.0)10.2 (8.6-14.7)0.0718.9 (6.9-12.3)9.8 (8.8-12.1)0.635
       EOF14.2 (10.9-16.4)14.1 (9.7-16.4)14.2 (11.7-18.5)0.47313.2 (9.5-16.3)14.8 (11.3-17.0)0.750
      Fibroscan, kPa
       Baseline5.1 (4.4-6.1)4.9 (4.4-6.0)5.4 (4.8-6.2)0.1664.9 (4.2-6.1)4.9 (4.4-6.0)0.770
       EOT5.9 (5.1-7.8)5.6 (4.9-6.6)6.1 (5.1-8.1)0.1335.5 (5.0-6.6)5.6 (4.9-7.1)0.954
       EOF5.9 (4.8-7.1)5.3 (4.4-6.7)6.6 (5.6-8.2)0.016
      p <0.05 is considered significant difference between 2 groups.
      5.4 (4.4-6.6)5.1 (4.4-6.7)0.584
      Longitudinal changes in HBV parameters
      cccDNA, copies/cell
       Baseline0.310 (0.061)0.279 (0.072)0.334 (0.095)0.8340.294 (0.109)0.260 (0.089)0.823
       Week 480.207 (0.052)0.131 (0.042)0.268 (0.088)0.2410.088 (0.033)0.196 (0.093)0.282
       cccDNA decline0.103 (0.066)0.149 (0.075)0.066 (0.102)0.6730.206 (0.096)0.062 (0.120)0.356
      HBsAg, log10 IU/ml
       Baseline2.797 (0.047)2.652 (0.072)2.915 (0.058)0.005
      p <0.05 is considered significant difference between 2 groups.
      2.533 (0.098)2.819 (0.093)0.092
       Week 241.537 (0.181)0.318 (0.259)2.535 (0.115)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      -0.448 (0.262)1.390 (0.352)<0.001
      p <0.05 is considered significant difference between 2 groups.
       Week 481.109 (0.198)-0.210 (0.246)2.188 (0.175)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      -0.952 (0.189)0.831 (0.398)<0.001
      p <0.05 is considered significant difference between 2 groups.
       Week 720.917 (0.197)-0.507 (0.207)2.049 (0.178)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      -1.261 (0.034)0.498 (0.338)<0.0001
      p <0.05 is considered significant difference between 2 groups.
       EOT0.680 (0.199)-0.828 (0.137)1.913 (0.204)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      -1.287 (0.014)-0.184 (0.247)<0.0001
      p <0.05 is considered significant difference between 2 groups.
       EOF1.134 (0.208)-0.108 (0.269)2.175 (0.203)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      <-1.301 (0.000)1.561 (0.306)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      HBsAg decline from baseline, log10 IU/ml
       Week 241.260 (0.162)2.334 (0.235)0.380 (0.104)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      2.981 (0.261)1.428 (0.309)<0.001
      p <0.05 is considered significant difference between 2 groups.
       Week 481.688 (0.180)2.862 (0.224)0.727 (0.165)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      3.486 (0.193)1.988 (0.364)<0.001
      p <0.05 is considered significant difference between 2 groups.
       Week 721.883 (0.178)3.162 (0.184)0.865 (0.164)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      3.794 (0.106)2.320 (0.289)<0.001
      p <0.05 is considered significant difference between 2 groups.
       EOT2.117 (0.184)3.480 (0.123)1.002 (0.196)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      3.821 (0.093)3.003 (0.214)0.003
      p <0.05 is considered significant difference between 2 groups.
       EOF1.665 (0.194)2.761 (0.251)0.748 (0.202)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      3.835 (0.098)1.257 (0.289)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      HBcrAg, log10 U/ml
       Baseline4.710 (0.129)4.593 (0.203)4.805 (0.166)0.4164.450 (0.276)4.794 (0.301)0.412
       Week 244.434 (0.115)4.174 (0.181)4.646 (0.142)0.041
      p <0.05 is considered significant difference between 2 groups.
      3.969 (0.231)4.461 (0.283)0.183
       Week 484.282 (0.116)4.013 (0.179)4.508 (0.144)0.032
      p <0.05 is considered significant difference between 2 groups.
      3.780 (0.228)4.337 (0.273)0.126
       Week 724.266 (0.107)4.055 (0.164)4.437 (0.138)0.0763.840 (0.210)4.327 (0.248)0.142
       EOT4.235 (0.120)3.876 (0.170)4.528 (0.155)0.006
      p <0.05 is considered significant difference between 2 groups.
      3.601 (0.204)4.260 (0.270)0.047
      p <0.05 is considered significant difference between 2 groups.
       EOF4.364 (0.170)3.986 (0.215)4.686 (0.249)0.039
      p <0.05 is considered significant difference between 2 groups.
      3.730 (0.193)4.370 (0.442)0.146
      HBcrAg decline from baseline, log10 U/ml
       Week 240.276 (0.050)0.419 (0.089)0.159 (0.050)0.004
      p <0.05 is considered significant difference between 2 groups.
      0.481 (0.136)0.332 (0.097)0.416
       Week 480.417 (0.058)0.580 (0.100)0.281 (0.060)0.005
      p <0.05 is considered significant difference between 2 groups.
      0.670 (0.150)0.456 (0.116)0.300
       Week 720.452 (0.065)0.596 (0.115)0.335 (0.067)0.041
      p <0.05 is considered significant difference between 2 groups.
      0.698 (0.182)0.467 (0.122)0.328
       EOT0.475 (0.078)0.717 (0.119)0.277 (0.094)0.004
      p <0.05 is considered significant difference between 2 groups.
      0.849 (0.180)0.533 (0.128)0.196
       EOF0.313 (0.126)0.624 (0.168)0.047 (0.176)0.022
      p <0.05 is considered significant difference between 2 groups.
      0.720 (0.211)0.479 (0.280)0.491
      HBV DNA, log10 IU/ml
       Baseline1.390 (0.038)1.361 (0.037)1.414 (0.062)0.6811.355 (0.053)1.371 (0.049)0.833
       Week 241.353 (0.015)1.354 (0.023)1.352 (0.020)0.9421.359 (0.036)1.348 (0.027)0.820
       Week 481.312 (0.007)1.302 (0.001)1.319 (0.012)0.3971.301 (0.000)1.304 (0.003)0.242
       Week 721.444 (0.051)1.351 (0.050)1.519 (0.082)0.019
      p <0.05 is considered significant difference between 2 groups.
      1.301 (0.000)1.417 (0.116)0.748
       EOT1.671 (0.112)1.301 (0.000)1.973 (0.193)0.002
      p <0.05 is considered significant difference between 2 groups.
      1.301 (0.000)1.301 (0.000)ns
       EOF1.955 (0.131)1.876 (0.223)2.021 (0.153)0.5841.301 (0.000)2.680 (0.468)0.001
      p <0.05 is considered significant difference between 2 groups.
      HBV DNA decline from baseline, log10 IU/ml
       Week 240.037 (0.039)0.007 (0.042)0.061 (0.063)0.752-0.004 (0.066)0.023 (0.045)0.758
       Week 480.079 (0.037)0.059 (0.036)0.094 (0.061)0.7280.054 (0.053)0.067 (0.047)0.860
       Week 72-0.053 (0.054)0.012 (0.046)-0.105 (0.089)0.2530.056 (0.056)-0.046 (0.079)0.282
       EOT-0.280 (0.115)0.060 (0.037)-0.559 (0.198)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      0.054 (0.053)0.070 (0.049)0.833
       EOF-0.563 (0.135)-0.514 (0.213)-0.604 (0.176)0.7430.054 (0.053)-1.310 (0.434)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      HBeAg, log10 S/CO
       Baseline0.155 (0.098)0.137 (0.151)0.169 (0.129)0.8710.071 (0.181)0.229 (0.265)0.613
       Week 24-0.005 (0.079)-0.078 (0.120)0.055 (0.105)0.406-0.196 (0.141)0.088 (0.208)0.250
       Week 48-0.033 (0.066)-0.098 (0.098)0.018 (0.088)0.381-0.179 (0.129)0.010 (0.150)0.346
       Week 72-0.032 (0.063)-0.087 (0.094)0.011 (0.085)0.445-0.198 (0.123)0.055 (0.141)0.183
       EOT-0.076 (0.066)-0.177 (0.081)0.007 (0.099)0.168-0.257 (0.102)-0.065 (0.130)0.247
       EOF0.049 (0.109)-0.099 (0.137)0.173 (0.164)0.216-0.253 (0.116)0.115 (0.283)0.378
      HBeAg decline from baseline, log10 S/CO
       Week 240.159 (0.034)0.215 (0.059)0.114 (0.039)0.1450.267 (0.088)0.141 (0.069)0.300
       Week 480.182 (0.046)0.222 (0.073)0.151 (0.058)0.4470.224 (0.089)0.219 (0.126)0.975
       Week 720.202 (0.053)0.259 (0.088)0.158 (0.064)0.3470.326 (0.108)0.174 (0.146)0.397
       EOT0.230 (0.066)0.314 (0.088)0.162 (0.095)0.2540.328 (0.109)0.294 (0.151)0.852
       EOF0.114 (0.082)0.236 (0.101)0.011 (0.122)0.1690.324 (0.102)0.114 (0.197)0.314
      HBsAb, IU/L
       Baseline<2.0<2.0<2.0ns<2.0<2.0ns
       Week 2411.7 (3.5)22.8 (7.4)2.7 (0.5)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      30.8 (11.5)11.6 (7.1)0.241
       Week 4840.1 (17.6)84.8 (38.1)3.6 (1.4)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      125.7 (64.0)27.5 (11.4)0.281
       Week 7269.3 (22.4)153.7 (47.1)2.0 (0.0)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      217.4 (70.6)68.8 (51.7)0.010
      p <0.05 is considered significant difference between 2 groups.
       EOT139.0 (33.9)306.5 (65.7)<2.0<0.0001
      p <0.05 is considered significant difference between 2 groups.
      418.9 (87.6)149.1 (86.8)0.012
      p <0.05 is considered significant difference between 2 groups.
       EOF109.6 (30.9)235.1 (60.9)<2.0<0.0001
      p <0.05 is considered significant difference between 2 groups.
      393.4 (90.1)13.6 (4.9)<0.0001
      p <0.05 is considered significant difference between 2 groups.
      Data are expressed as n (%), median (IQR) or mean (SEM).
      ADV, adefovir; ALT, alanine aminotransferase; cccDNA, covalently closed circular DNA; EOF, end of follow-up; EOT, end of treatment; ETV, entecavir; HBcrAg, hepatitis B core-related antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatits B surface antigen; HBsAb, hepatitis B surface antibody; LAM, lamivudine; LdT, telbivudine; NSR, non-sustained responder; NUC, nucleos(t)ide analogue;TBiL, total bilirubin; SR, sustained responder; TDF, tenofovir.
      p <0.05 is considered significant difference between 2 groups.

      Longitudinal changes in HBV biomarkers and factors associated with treatment response and SR

      The longitudinal changes in HBV biomarkers during and after treatment are shown in Table 1. The intrahepatic levels of cccDNA at baseline did not differ significantly between responders and non-responders. While only responders and sustained responders but not non-responders showed marked decreases in cccDNA levels from baseline to week 48 (Fig. S1). HBsAg levels were significantly lower and declined more during treatment in responders than non-responders, and such a difference in HBsAg levels was also observed between sustained and non-sustained responders. HBcrAg levels were significantly lower at weeks 24, 48, and EOT and declined more during treatment in responders than in non-responders. HBcrAg levels at EOT were significantly lower in sustained responders than in non-sustained responders. After NUC cessation, responders maintained virologic suppression, whereas non-responders had significant increases in HBV DNA levels. During the follow-up period, sustained responders maintained virologic suppression, while non-sustained responders experienced virologic relapse (1.301±0.000 vs. 2.680±0.468 log10IU/ml, p <0.001). HBeAg levels were comparable during and after treatment between responders and non-responders. HBsAb levels were significantly higher during treatment in responders, particularly sustained responders, than in non-responders.
      The association between baseline or on-treatment factors and treatment response or SR is shown in Table 2. Levels of alanine aminotransferase, HBV DNA, and HBeAg were not associated with response or SR. Age, HBsAg, and HBcrAg levels were associated with response. A series of receiver-operating characteristic curves further confirmed that age, HBsAg level at baseline, HBcrAg level at week 24 and HBsAg decline from baseline to week 24 could predict treatment response. Notably, a combination of age, baseline HBsAg level and HBsAg decline had a satisfactory predictive performance for treatment response with an AUROC of 0.924 (0.864-0.984, p <0.001). A slight improvement of predictive performance was observed when further combined with HBcrAg level at week 24, with an optimal AUROC of 0.938 (0.879-0.997, p <0.001) (Fig. S2).
      Table 2Baseline and on-treatment predictors of treatment response and sustained response.
      ResponseSustained response
      OR (95% CI)p valueOR (95% CI)p value
      Age, years0.913 (0.866–0.962)0.001
      p <0.05 is considered significant difference.
      1.003 (0.931–1.081)0.932
      ALT, U/L0.975 (0.936–1.016)0.2331.012 (0.952–1.077)0.701
      HBsAg, log10 IU/ml
       Baseline0.198 (0.058–0.667)0.009
      p <0.05 is considered significant difference.
      0.165 (0.025–1.083)0.060
       Week 240.250 (0.136–0.460)<0.001
      p <0.05 is considered significant difference.
      0.384 (0.211–0.697)0.002
      p <0.05 is considered significant difference.
       Week 480.362 (0.246–0.533)<0.001
      p <0.05 is considered significant difference.
      0.327 (0.154–0.693)0.004
      p <0.05 is considered significant difference.
       Week 720.297 (0.188–0.467)<0.000
      p <0.05 is considered significant difference.
      0.010 (0.000–0.864)0.043
      p <0.05 is considered significant difference.
      HBsAg decline from baseline, log10 IU/ml
       Week 244.457 (2.336–8.506)<0.001
      p <0.05 is considered significant difference.
      2.646 (1.389–5.038)0.003
      p <0.05 is considered significant difference.
       Week 482.988 (1.954–4.568)<0.001
      p <0.05 is considered significant difference.
      2.875 (1.396–5.920)0.004
      p <0.05 is considered significant difference.
       Week 723.937 (2.340–6.624)<0.001
      p <0.05 is considered significant difference.
      9.922 (2.055–47.916)0.004
      p <0.05 is considered significant difference.
      HBcrAg, log10 U/ml
       Baseline0.849 (0.575–1.254)0.4110.785 (0.446–1.382)0.402
       Week 240.625 (0.395–0.989)0.045
      p <0.05 is considered significant difference.
      0.642 (0.334–1.234)0.184
       Week 480.610 (0.385–0.968)0.036
      p <0.05 is considered significant difference.
      0.594 (0.303–1.163)0.129
       Week 720.634 (0.381–1.053)0.0780.564 (0.262–1.214)0.143
      HBcrAg decline from baseline, log10 U/ml
       Week 244.506 (1.300–15.622)0.018
      p <0.05 is considered significant difference.
      1.784 (0.452–7.047)0.409
       Week 483.531 (1.264–9.863)0.016
      p <0.05 is considered significant difference.
      1.925 (0.563–6.577)0.296
       Week 722.387 (0.992–5.744)0.0521.739 (0.583–5.185)0.321
      HBV DNA, log10 IU/ml
       Baseline0.598 (0.132–2.703)0.5040.714 (0.035–14.742)0.828
       Week 241.129 (0.044–28.920)0.9421.808 (0.013–251.108)0.814
       Week 480.000 (0.000–2.4×10ˆ6)0.345
       Week 720.333 (0.076–1.457)0.144
      HBV DNA decline from baseline, log10 IU/ml
       Week 240.612 (0.146–2.563)0.5010.650 (0.046–9.232)0.751
       Week 480.707 (0.164–3.036)0.6410.751 (0.035–16.220)0.855
       Week 721.765 (0.613–5.084)0.2925.886 (0.162–213.992)0.334
      HBeAg, log10 S/CO
       Baseline0.958 (0.576–1.595)0.8690.822 (0.393–1.720)0.603
       Week 240.758 (0.397–1.447)0.4020.568 (0.215–1.501)0.254
       Week 480.700 (0.317–1.545)0.3770.558 (0.167–1.861)0.342
       Week 720.720 (0.313–1.658)0.4400.405 (0.105–1.564)0.190
      HBeAg decline from baseline, log10 S/CO
       Week 243.084 (0.653–14.560)0.1553.321 (0.333–33.090)0.306
       Week 481.537 (0.511–4.620)0.4441.026 (0.213–4.953)0.974
       Week 721.600 (0.604–4.242)0.3441.890 (0.443–8.059)0.390
      HBsAb, log10 IU/L
       Week 242.211 (0.678–7.209)0.188
       Week 481.841 (0.746–4.545)0.186
       Week 723.055 (1.203–7.758)0.019
      p <0.05 is considered significant difference.
      Data are expressed as OR (95% CI).
      ALT, alanine aminotransferase; HBcrAg, hepatitis B core-related antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatits B surface antigen; HBsAb, hepatitis B surface antibody; OR, odds ratio.
      p <0.05 is considered significant difference.
      The association between EOT factors and SR is shown in Table 3. HBcrAg and HBsAb levels at EOT were independently associated with SR. The odds ratios (95% CI) were 0.389 (0.157-0.963, p = 0.041) and 3.862 (1.389-10.741, p = 0.010), respectively. Furthermore, the AUROC was estimated to be 0.697 (0.512-0.882, p = 0.047) for EOT HBcrAg, 0.744 (0.573-0.915, p = 0.013) for EOT HBsAb and 0.822 (0.684-0.961, p = 0.001) when combining these biomarkers. The optimal cut-off values of EOT HBcrAg and HBsAb were <4 log10U/ml and >2 log10IU/L, respectively. These 2 variates were combined to create a model called HBVCure crAb, which can be used to identify patients likely to achieve SR, with a positive predictive value of up to 100% (Fig. 1).
      Table 3End-of-treatment predictors of sustained response post treatment.
      Univariate analysisMultivariate analysis
      OR (95% CI)p valueOR (95% CI)p value
      HBeAg, log10 S/CO0.430 (0.102–1.805)0.249
      HBsAg, log10 IU/ml0.005 (0.000–2.235)0.089
      HBcrAg, log10 U/ml0.499 (0.240–1.038)0.0630.389 (0.157–0.963)0.041
      p <0.05 is considered significant difference.
      HBsAb, log10 IU/L3.153 (1.260–7.893)0.014
      p <0.05 is considered significant difference.
      3.862 (1.389–10.741)0.010
      p <0.05 is considered significant difference.
      Data are expressed as OR (95% CI).
      HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBcrAg, hepatitis B core-related antigen; HBsAb, hepatitis B surface antibody; OR, odds ratio.
      p <0.05 is considered significant difference.
      Figure thumbnail gr1
      Fig. 1The AUROC of end-of-treatment predictors of sustained response.
      p <0.05, ∗∗∗p <0.001. EOT, end of treatment; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBcrAg, hepatitis B core-related antigen; HBsAb, hepatitis B surface antibody; SR, sustained response.
      We further evaluated the association between biomarkers and treatment response in HBeAg-negative patients and HBeAg-positive patients (Table S1). HBsAg and HBsAb levels showed similar dynamics in both subgroups of patients. However, HBcrAg levels were significantly lower during treatment in HBeAg-negative patients than in HBeAg-positive patients (p <0.0001). Binary logistic regression analysis showed that the statistically significant association between HBcrAg levels and treatment response was only evident in HBeAg-negative patients (Table S2).

      Longitudinal changes in virologic and immunological biomarkers during follow-up according to various treatment outcomes

      As shown in Fig. 2A, HBV DNA, HBcrAg, and HBeAg levels were comparable at EOT and EOF between patients achieving HBsAg loss and those achieving HBsAb appearance at EOT. HBsAg levels were higher at EOT and EOF in patients who achieved HBsAb appearance than in those achieving HBsAg loss. HBsAg, HBV DNA, HBcrAg, and HBeAg levels at EOT and EOF did not differ significantly between patients achieving HBsAg loss and those with high HBsAb levels (>100 IU/L) at EOT.
      Figure thumbnail gr2
      Fig. 2The changes in virologic and immunological biomarkers during follow-up in patients with different treatment response.
      (A) Comparisons HBsAg, HBV DNA, HBcrAg and HBeAg. (B) Comparisons of HBV-specific CD8+T cells. (C) Comparisons of Tfh cells. (D) Comparisons of total (CD19+), memory (CD19+CD27+) and plasma (CD19+CD38+) B cells. (E) Comparisons of CD86 and CD95 expression on total and memory B cells. ∗p <0.05, ∗∗p <0.01, ∗∗∗∗p <0.0001. EOF, end of follow-up; EOT, end of treatment; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBcrAg, hepatitis B core-related antigen; HBsAb, hepatitis B surface antibody; Tfh, T follicular helper.
      During the follow-up period, the proportions of HBV-specific CD8+ T, total B (CD19+) and memory B (CD19+CD27+) cells, as well as CD86 and CD95 expression on total B cells, were relatively stable and comparable between patients achieving HBsAg loss and those achieving HBsAb appearance at the EOT. However, the proportions of HBV core- and envelope-specific CD8+ T cells, as well as CD86 and CD95 expression on total B cells, significantly declined in non-responders (Fig. 2B-E). These data suggested that treatment response defined either as HBsAg loss or as HBsAb appearance was related to stable post-therapy anti-HBV immunity.

      Serum HBcrAg and HBsAb correlated with other HBV biomarkers

      As shown in Fig. 3A, serum HBcrAg showed a positive correlation with HBsAg and HBV DNA at baseline, EOT, and EOF. At baseline, HBcrAg showed a positive correlation with intrahepatic cccDNA. At EOT, HBcrAg showed a negative correlation with HBsAb. Patients who were HBcrAg negative at baseline maintained virologic suppression even after NUC cessation and exhibited lower on-treatment levels of HBsAg and HBeAg than HBcrAg-positive patients (Fig. 3B) and tended to have lower cccDNA levels than those with positive HBcrAg (Fig. 3C). Patients with EOT HBcrAg <4 log10U/ml achieved durable virologic suppression and had significantly lower levels of HBV DNA, HBsAg and HBeAg at EOF than those with HBcrAg≥4 log10U/ml (Fig. 3D). HBsAb was negatively correlated with HBsAg and HBV DNA but not with HBeAg (Fig. 4A). Patients with EOT HBsAb >2 log10IU/L exhibited significantly lower HBsAg levels at EOT and EOF than those with HBsAb ≤2 log10IU/L (Fig. 4B). These findings indicated that patients with HBcrAg <4 log10U/ml or HBsAb >2 log10IU/L at EOT were more likely to achieve a sustained off-treatment response.
      Figure thumbnail gr3
      Fig. 3Serum HBcrAg levels are correlated with other HBV biomarkers.
      (A) Correlations of HBcrAg with HBsAg, HBeAg, HBV DNA, cccDNA and HBsAb. (B&C) Comparisons of on-treatment dynamic changes in HBV DNA, HBsAg, HBeAg, and cccDNA according to baseline HBcrAg levels. (D) Dynamic changes in HBV load during follow-up according to EOT HBcrAg levels. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. cccDNA, covalently closed circular DNA; EOF, end of follow-up; EOT, end of treatment; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBcrAg, hepatitis B core-related antigen; HBsAb, hepatitis B surface antibody.
      Figure thumbnail gr4
      Fig. 4Serum HBsAb levels correlated with other HBV biomarkers.
      (A) Correlations of HBsAb with HBsAg, HBV DNA and HBeAg. (B) Dynamic changes in HBV load during follow-up according to EOT HBsAb levels. ∗p <0.05, ∗∗p <0.01. EOF, end of follow-up; EOT, end of treatment; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBcrAg, hepatitis B core-related antigen; HBsAb, hepatitis B surface antibody.

      Lower HBcrAg and higher HBsAb at EOT correlated with sustained anti-HBV immune responses after treatment discontinuation

      The immunological alterations in patients with EOT HBcrAg <4 log10U/ml or HBsAb >2 log10IU/L after treatment discontinuation are shown in Fig. 5. The proportions of HBV-specific CD8+ T cells were relatively stable or higher in these 2 subgroups of patients than in the remaining patients (Fig. 5B). Tfh cell proportions were significantly increased in patients with EOT HBcrAg <4 log10U/ml, and EOT HBcrAg levels correlated with a decline in Tfh cell proportions during the follow-up period (Fig. 5C). The total B cell proportions were stable in patients with EOT HBcrAg <4 log10U/ml or HBsAb >2 log10IU/L, but significantly decreased in those with EOT HBsAb ≤2 log10IU/L. The expression of CD86 on total and memory B cells was stable in patients with EOT HBcrAg <4 log10U/ml or HBsAb >2 log10IU/L, but significantly decreased in the remaining patients (Fig. 5D). These results demonstrated sustained cellular and humoral immunity during follow-up in patients with EOT HBcrAg <4 log10U/ml or HBsAb >2 log10IU/L.
      Figure thumbnail gr5
      Fig. 5Dynamics of peripheral immunological indicators during follow-up according to EOT HBcrAg or HBsAb levels.
      (A) Representative flow cytometry staining of HBV-specific CD8+T, Tfh (CD4+CXCR5+) and B cells. (B) HBV-specific CD8+T cells. (C) Tfh cells. (D) B cells and its subtypes. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. EOF, end of follow-up; EOT, end of treatment; FSC, forward scatter; HBcrAg, hepatitis B core-related antigen; HBsAb, hepatitis B surface antibody; SSC, side scatter; Tfh, T follicular helper. (This figure appears in color on the web.)

      Discussion

      It has been reported that roughly 70-80% of patients who acchieve HBsAg loss following Peg-IFN-based therapy can sustain the response during the off-treatment follow-up period.
      • Ning Q.
      • Han M.
      • Sun Y.
      • Jiang J.
      • Tan D.
      • Hou J.
      • et al.
      Switching from entecavir to PegIFN alfa-2a in patients with HBeAg-positive chronic hepatitis B: a randomised open-label trial (OSST trial).
      ,
      • Wu D.
      • Wang P.
      • Han M.
      • Chen Y.
      • Chen X.
      • Xia Q.
      • et al.
      Sequential combination therapy with interferon, interleukin-2 and therapeutic vaccine in entecavir-suppressed chronic hepatitis B patients: the Endeavor study.
      ,
      • Hu P.
      • Shang J.
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      • Gong G.
      • Li Y.
      • Chen X.
      • et al.
      HBsAg loss with Peg-interferon Alfa-2a in hepatitis B patients with partial response to nucleos(t)ide analog: new switch study.
      However, factors associated with a durable functional cure remain unknown. The present pilot proof-of-concept study investigated the relationship between biomarkers and durable functional cure after completing Peg-IFN-based therapy. Our study demonstrated that patients who achieved either HBsAg loss or HBsAb response at EOT could maintain stable immunity against HBV and sustained off-treatment response to a similar extent. Age and on-treatment HBsAg and HBcrAg levels were independent predictors of HBsAg loss or HBsAb appearance induced by Peg-IFN-based treatment. The predictive effect of HBcrAg was significant in HBeAg-negative patients. Moreover, EOT HBcrAg <4 log10U/ml and HBsAb >2 log10IU/L were associated with sustained cellular and humoral immunity post Peg-IFN-based therapy and were identified as the optimal cut-off values for sustained response (HBVCure crAb model). This model may be used to identify Peg-IFN-α responders who are likely to achieve a durable functional cure.
      Intrahepatic cccDNA is a template for all HBV mRNA transcription and viral protein synthesis, representing a major obstacle to HBV eradication.
      • Nassal M.
      HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.
      Recently, Meier et al. reported that integrated HBV DNA rather than cccDNA may account for ubiquitous HBsAg production in some HBeAg-negative patients, challenging the clinical utility of HBsAg as a key marker for functional cure.
      • 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.
      Previous studies have demonstrated that serum HBcrAg reflects the transcriptional activity of intrahepatic cccDNA more accurately than HBsAg.
      • Erken R.
      • Zaaijer H.L.
      • Willemse S.B.
      • Bakker E.
      • Takkenberg B.B.
      • Reesink H.W.
      • et al.
      Hepatitis B core related antigen in relation to intrahepatic and circulating viral markers, before and after combination therapy.
      Our present study also confirmed a significant correlation between serum HBcrAg and intrahepatic cccDNA in NUC-suppressed patients. These findings supported the potential value of HBcrAg as a non-invasive surrogate biomarker of the intrahepatic cccDNA pool in virally suppressed patients.
      The present study showed that baseline and on-treatment HBsAg levels were associated with treatment response. These observations are consistent with previous studies, highlighting the clinical significance of HBsAg levels in predicting HBsAg loss.
      • Lim S.G.
      • Phyo W.W.
      • Ling J.Z.J.
      • Cloherty G.
      • Butler E.K.
      • Kuhns M.C.
      • et al.
      Comparative biomarkers for HBsAg loss with antiviral therapy shows dominant influence of quantitative HBsAg (qHBsAg).
      In addition to HBsAg, we also found that on-treatment HBcrAg levels could independently predict treatment response. HBcrAg levels were significantly lower and profoundly decreased in responders compared to non-responders. Intriguingly, such a significant difference was primarily observed in HBeAg-negative patients. HBcrAg levels were significantly higher but were more profoundly decreased in HBeAg-positive patients than in HBeAg-negative patients, which may partly be ascribed to the fact that HBeAg is a component of HBcrAg.
      • Hong X.
      • Hu J.
      Understanding HBcrAg components improves the interpretation of clinical HBcrAg assay results.
      ,
      • Hong X.
      • Luckenbaugh L.
      • Mendenhall M.
      • Walsh R.
      • Cabuang L.
      • Soppe S.
      • et al.
      Characterization of hepatitis B precore/core-related antigens.
      In line with this observation, a previous study reported that serum HBcrAg levels were significantly higher in HBeAg-positive patients than in HBeAg-negative patients without antiviral treatment.
      • Chen E.Q.
      • Feng S.
      • Wang M.L.
      • Liang L.B.
      • Zhou L.Y.
      • Du L.Y.
      • et al.
      Serum hepatitis B core-related antigen is a satisfactory surrogate marker of intrahepatic covalently closed circular DNA in chronic hepatitis B.
      ,
      • Maasoumy B.
      • Wiegand S.B.
      • Jaroszewicz J.
      • Bremer B.
      • Lehmann P.
      • Deterding K.
      • et al.
      Hepatitis B core-related antigen (HBcrAg) levels in the natural history of hepatitis B virus infection in a large European cohort predominantly infected with genotypes A and D.
      HBcrAg has previously been shown to be predictive of virologic relapse in HBeAg-negative patients.
      • Jung K.S.
      • Park J.Y.
      • Chon Y.E.
      • Kim H.S.
      • Kang W.
      • Kim B.K.
      • et al.
      Clinical outcomes and predictors for relapse after cessation of oral antiviral treatment in chronic hepatitis B patients.
      This current study indicated that the predictive effect of HBcrAg levels on treatment response was significant primarily in HBeAg-negative patients. The underlying mechanism remains unclear and deserves further exploration.
      After the withdrawal of Peg-IFN-based treatment, a small proportion of patients who lost HBsAg may experience HBsAg reversion. Therefore, reliable biomarkers associated with sustained off-treatment response are urgently needed to guide treatment decisions. Previous studies have suggested that HBcrAg can be used to predict virologic and clinical relapse after NUC discontinuation in HBeAg-negative patients.
      • Carey I.
      • Gersch J.
      • Wang B.
      • Moigboi C.
      • Kuhns M.
      • Cloherty G.
      • et al.
      Pregenomic HBV RNA and Hepatitis B core-related antigen predict outcomes in hepatitis B e antigen-negative chronic hepatitis B patients suppressed on nucleos(t)ide analogue therapy.
      EOT HBcrAg levels have been demonstrated to predict sustained HBV DNA suppression and HBeAg seroconversion.
      • Hsu Y.C.
      • Nguyen M.H.
      • Mo L.R.
      • Wu M.S.
      • Yang T.H.
      • Chen C.C.
      • et al.
      Combining hepatitis B core-related and surface antigens at end of nucleos(t)ide analogue treatment to predict off-therapy relapse risk.
      ,
      • Sonneveld M.J.
      • van Oord G.W.
      • van Campenhout M.J.
      • De Man R.A.
      • Janssen H.L.A.
      • de Knegt R.J.
      • et al.
      Relationship between hepatitis B core-related antigen levels and sustained HBeAg seroconversion in patients treated with nucleo(s)tide analogues.
      However, there are scarce data on serum biomarkers associated with durable functional cure after treatment discontinuation. Brakenhoff et al. found that HBV RNA alone may not adequately predict HBsAg loss after Peg-IFN-based treatment, but combining HBcrAg or HBsAg decline may significantly improve the predictive performance.
      • Brakenhoff S.M.
      • de Man R.A.
      • Boonstra A.
      • van Campenhout M.J.H.
      • de Knegt R.J.
      • van Bommel F.
      • et al.
      Hepatitis B virus RNA decline without concomitant viral antigen decrease is associated with a low probability of sustained response and hepatitis B surface antigen loss.
      In the present study of NUC-suppressed patients with CHB, EOT HBsAg and HBcrAg levels were significantly lower in sustained responders than in non-sustained responders. However, the multivariable analysis showed that EOT HBcrAg levels instead of EOT HBsAg levels were associated with sustained response. The potential superiority of EOT HBcrAg over HBsAg in identifying sustained responders might be partly explained by the findings of previous studies that quantification of HBcrAg could reflect residual transcriptional activity of cccDNA in HBeAg-negative patients or in those who lost HBsAg.
      • Testoni B.
      • Lebosse F.
      • Scholtes C.
      • Berby F.
      • Miaglia C.
      • Subic M.
      • et al.
      Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients.
      ,
      • Mak L.Y.
      • Cloherty G.
      • Wong D.K.
      • Gersch J.
      • Seto W.K.
      • Fung J.
      • et al.
      HBV RNA profiles in patients with chronic hepatitis B under different disease phases and antiviral therapy.
      Large-scale prospective studies are warranted to validate these results and draw a solid conclusion.
      In addition to low HBcrAg levels, high HBsAb levels might also be associated with sustained functional cure in HBeAg-negative patients who had achieved HBsAg loss with IFN treatment.
      • Li M.H.
      • Yi W.
      • Zhang L.
      • Lu Y.
      • Lu H.H.
      • Shen G.
      • et al.
      Predictors of sustained functional cure in hepatitis B envelope antigen-negative patients achieving hepatitis B surface antigen seroclearance with interferon-alpha-based therapy.
      High HBsAb titers have been considered to ensure adequate protection against HBV reactivation in patients undergoing biologic therapy for rheumatic diseases or direct-acting antivirals for chronic hepatitis C.
      • Tien Y.C.
      • Yen H.H.
      • Li C.F.
      • Liu M.P.
      • Hsue Y.T.
      • Hung M.H.
      • et al.
      Changes in hepatitis B virus surface antibody titer and risk of hepatitis B reactivation in HBsAg-negative/HBcAb-positive patients undergoing biologic therapy for rheumatic diseases: a prospective cohort study.
      ,
      • Poola S.
      • Sanaka S.
      • Sewell K.
      • Tillmann H.L.
      Hepatitis B surface antibody titers and hepatitis B reactivation with direct-acting antiviral therapy for hepatitis C.
      In contrast, the optimum titer of HBsAb to maintain functional cure deserves further exploration. The current study suggested that HBsAb levels negatively correlated with HBcrAg, HBsAg and HBV DNA at EOT. Intriguingly, the patients who achieved a HBsAb response or HBsAg loss at EOT both exhibited similar levels of sustained host immunity and durable off-treatment responses. EOT HBsAb >2 log10IU/L was determined to be the optimal cut-off value for durable off-treatment HBsAg loss. Patients with EOT HBsAb >2 log10IU/L showed a more sustained immune response than the remaining patients. Collectively, HBsAb and HBcrAg levels at EOT may serve as surrogate biomarkers of a durable functional cure.
      HBV-specific CD8+ T cells play a critical role in controlling HBV replication through the cytotoxic T cell-mediated killing of infected hepatocytes as well as IFNγ- and TNFα-mediated non-cytolytic mechanisms.
      • Phillips S.
      • Chokshi S.
      • Riva A.
      • Evans A.
      • Williams R.
      • Naoumov N.V.
      CD8(+) T cell control of hepatitis B virus replication: direct comparison between cytolytic and noncytolytic functions.
      A recent study revealed that an increased cytotoxic T-cell response was significantly associated with a rapid decline in HBsAg levels during tenofovir disoproxil fumarate and Peg-IFN add-on therapy.
      • Matsumoto A.
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      • Enomoto H.
      • Tanaka Y.
      • Shinkai N.
      • Okuse C.
      • et al.
      Pilot study of tenofovir disoproxil fumarate and pegylated interferon-alpha 2a add-on therapy in Japanese patients with chronic hepatitis B.
      In the present study, the proportion of HBV-specific CD8+ T cells was relatively stable during the follow-up period, and was significantly higher at EOF in the patients with EOT HBcrAg <4 log10U/ml or HBsAb >2 log10IU/L than in the remaining patients. A sustained HBV-specific CD8+ T-cell response was also evidenced in patients who achieved durable HBsAg loss at EOF (Fig. S3A), suggesting that sustained functional cure may be attributed to restorating the viral-specific T-cell response.
      Tfh and B cell responses may enhance HBsAb production and promote HBsAg clearance.
      • Wang X.
      • Dong Q.
      • Li Q.
      • Li Y.
      • Zhao D.
      • Sun J.
      • et al.
      Dysregulated response of follicular helper T cells to hepatitis B surface antigen promotes HBV persistence in mice and associates with outcomes of patients.
      ,
      • Zhang L.
      • Li H.
      • Ren H.
      • Hu P.
      Circulating PD-1(hi)CXCR5(+)CD4(+) T cells are associated with a decrease in hepatitis B surface antigen levels in patients with chronic hepatitis B who are receiving peginterferon-alpha therapy.
      In the present study, a significant increase in the proportion of Tfh cells was observed in patients with EOT HBcrAg <4 log10U/ml, and a higher EOT qHBcrAg level was significantly correlated with a decrease in the proportion of Tfh cells during the follow-up period. Several subtypes of B cells have previously been found to be associated with HBsAb production. CD19+CD27+ memory B cells had a significantly higher HBsAg-specific response than CD19+CD27- B cells in recently HBV-vaccinated individuals.
      • Poonia B.
      • Ayithan N.
      • Nandi M.
      • Masur H.
      • Kottilil S.
      HBV induces inhibitory FcRL receptor on B cells and dysregulates B cell-T follicular helper cell axis.
      CD19+CD38+ B cells were significantly increased and positively correlated with HBsAb titers in patients who achieved spontaneous HBsAg seroconversion.
      • Vyas A.K.
      • Sharma B.C.
      • Sarin S.K.
      • Trehanpati N.
      Immune correlates of hepatitis B surface antigen spontaneous seroconversion in hepatitis B e antigen negative chronic hepatitis B patients.
      CD86 is upregulated following activation of B cells.
      • Cyster J.G.
      • Allen C.D.C.
      B cell responses: cell interaction dynamics and decisions.
      A significant negative correlation occurred between the proportion of CD95+ B cells and HBV DNA viral load.
      • Zhao P.W.
      • Ma L.
      • Ji H.F.
      • Yu L.
      • Feng J.Y.
      • Wang J.
      • et al.
      The expression of TLR-9, CD86, and CD95 phenotypes in circulating B cells of patients with chronic viral hepatitis B or C before and after antiviral therapy.
      The present study showed that a stable proportion and activation of total and memory B cells after Peg-IFN-α cessation was evidenced in patients with EOT HBcrAg <4 log10U/ml or HBsAb >2 log10IU/L, or those who achieved HBsAg loss at EOF (Fig. S3C). In summary, we speculated that low HBcrAg and high HBsAb levels at EOT were associated with sustained off-treatment cellular and humoral immunity, which may favour durable functional cure.
      The current study has several limitations. First, a large-scale prospective cohort is required to validate the findings from this study, particularly the potential of EOT HBcrAg and HBsAb to identify durable functional cure after Peg-IFN-α discontinuation. Second, HBV genotype data were only available in 36 patients, with 32 (88.9%) genotype B and 4 (11.1%) genotype C. The impact of the HBV genotype on the durability of HBsAg loss warrants investigation. Third, the prolonged duration of Peg-IFN-based therapy limits its clinical utility due to the potential increased risk of adverse events (Table S3) as well as costly and inconvenient subcutaneous administration. Last, we cannot exclude the possibility that other novel biomarkers, e.g. HBV RNA, may be associated with durable functional cure, which also warrants further investigation.
      In conclusion, a lower HBcrAg level (<4 log10U/ml) and higher HBsAb level (>2 log10IU/L) at EOT, assembled as an HBVCure crAb model, were associated with sustained off-treatment cellular and humoral immunity. This model can be used to identify patients likely to achieve a durable functional cure after discontinuation of Peg-IFN-based treatment.

      Abbreviations

      cccDNA, covalently closed circular DNA; CHB, chronic hepatitis B; EOF, end of follow-up; EOT, end of treatment; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; HBsAb, hepatitis B surface antibody; NUC, nucleos(t)ide analogue; Peg-IFN-ɑ, pegylated interferon alpha; Tfh, T follicular helper; SR, sustained response.

      Financial support

      This work was supported by grants from the Chinese National Thirteenth Five Years Project in Science and Technology (2017ZX10202201), National Natural Science Foundation of China (NO. 81974075), and the National Key Research and Development Program of China (2021YFC2600200).

      Authors’ contributions

      All authors were involved in critical revision of manuscript. QN and WMY designed the study, had full access to all data, and take responsibility for the integrity and accuracy of the data analysis. DH, DW, PW, YLW, WY, DQH, JJH, YQW, RT, FX, XPZ, XJW, MFH enrolled patients and collected clinical data. DH, DW and WMY analyzed clinical data, completed virologic and immunological evaluation and drafted the manuscript. XPL had critical contributions to the revision of manuscript in discussion, data re-evaluation and presentation, and manuscript edition. All authors approved the final version of the manuscript, including the authorship list.

      Data availability statement

      The authors declare that all data included in this article will be made available upon request.

      Conflict of interest

      All authors declared that they have no conflict of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

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