Chronic hepatitis B is caused by persistent hepatitis B virus infection and is commonly managed with long-term oral nucleos(t)ide analog therapy. Although these drugs can suppress viral replication in most adherent patients, loss of hepatitis B surface antigen, or HBsAg, remains uncommon during indefinite suppressive treatment. Add-on peginterferon alfa-2b is used in selected patients to stimulate an immune response that may promote HBsAg decline and increase the prospect of a functional cure, defined in the study as a step toward clearance rather than a guaranteed outcome. Its finite course can nevertheless be physically demanding: reported effects include fatigue, fever or chills, headache, muscle or joint pain, injection-site reactions, rash, itching, hair loss, appetite change, weight loss, sleep disturbance, mood symptoms and concern about blood-cell abnormalities.

The study recruited respondents from 1 to 13 May 2026 through hospital-based WeChat follow-up groups at West China Hospital of Sichuan University. Of 515 submitted questionnaires, 412 met the eligibility criteria: all had physician-diagnosed chronic hepatitis B, current or previous long-term oral antiviral treatment, and exposure to add-on peginterferon. At the time of the survey, 289 were receiving peginterferon, 84 had completed the recommended course and 39 reported premature discontinuation. The median age was 40 years, and 256 participants, or 62.1%, were men. Because investigators could not determine how many people received or viewed the invitation link, they could not calculate a formal response rate, making selection bias an important limitation.

Questionnaires assessed several domains with study-specific items: adverse-effect burden, belief in the need and value of treatment, physician–patient communication and a four-item persistence/value intention score covering willingness, confidence and willingness to tolerate discomfort to complete therapy. Adverse-effect burden was summarized from 15 items scored from 1 to 4, treatment belief from five items scored from 1 to 5, communication from three items scored from 1 to 5, and persistence/value intention from four items scored from 1 to 5. The Patient Health Questionnaire-4, or PHQ-4, separately measured anxiety, uncontrolled worry, loss of interest and depressed mood over the preceding two weeks on a scale of 0 to 12. Except for the PHQ-4, these measures were created for the study and were not formally pilot-tested or externally validated.

The most frequently reported moderate-to-severe effects or concerns were fatigue, affecting 37.1% of respondents, followed by rash or pruritus at 28.4%, alopecia at 27.4%, concern about leukocyte or platelet decline at 24.8%, myalgia or arthralgia at 24.0%, injection-site reactions at 22.3%, reduced appetite at 21.8%, worsened sleep at 20.6%, irritability at 18.9% and weight loss at 17.7%. Overall, 175 participants had wanted to stop peginterferon because of adverse effects, 136 had asked a physician about reducing the dose or stopping, and 39 reported actual premature discontinuation. Thus, thoughts of stopping were considerably more common than an all-cause early exit from therapy, which could also reflect a physician recommendation, perceived limited HBsAg response, inconvenience, cost or other reasons.

Multivariable logistic regression showed that each standard-deviation increase in the adverse-effect index was associated with higher odds of adverse-effect-related discontinuation thoughts, with an adjusted odds ratio of 1.86 and a 95% confidence interval of 1.37 to 2.51. Each standard-deviation increase in the PHQ-4 score was similarly associated with higher odds, with an adjusted odds ratio of 1.76 and a 95% confidence interval of 1.28 to 2.41. HBsAg feedback, oral antiviral duration, peginterferon duration, age and sex were not independently associated with those thoughts. Exploratory factor analysis separated the adverse-effect items into a broader symptom and daily-life component, including sleep, mood, skin, injection-site, laboratory and interference items, and a component dominated by fever or chills, fatigue, muscle or joint pain and headache. When both were entered together, only the broader component remained associated with discontinuation thoughts, indicating that the 15-item total should be interpreted as a heterogeneous burden index rather than a validated single dimension.

Psychological distress was also related to persistence/value intention among the 289 patients still receiving therapy. A stronger treatment-belief score was the largest positive correlate, with a standardized regression coefficient of 0.280 and a 95% confidence interval of 0.198 to 0.362. Better physician–patient communication had a smaller positive association, with a coefficient of 0.098 and a 95% confidence interval of 0.017 to 0.179, while higher PHQ-4 scores were associated with lower persistence/value intention, with a coefficient of −0.081 and a 95% confidence interval of −0.148 to −0.013. Adverse-effect burden and recalled HBsAg decline were not independently associated with the persistence score after adjustment. The model used heteroskedasticity-consistent HC3 standard errors because the intention score was bounded and concentrated toward its upper end.

Most participants recalled receiving an explanation of the treatment goal: 97.1% said they had been told that add-on peginterferon was intended to promote HBsAg decline or pursue clinical cure. In all, 76.9% knew their most recent HBsAg value and 95.9% knew either the value or its approximate trend; 78.2% reported that a physician had communicated an HBsAg decline. Yet these recalled messages were not independently associated with either discontinuation thoughts or persistence intention. The study did not verify laboratory results, and the coded feedback distinguished no decline or unclear feedback, a decline of uncertain magnitude and an obvious decline. The results therefore do not show that communicating HBsAg decline is ineffective; rather, they show that recalled decline feedback, as measured here, was not an independent correlate after accounting for other factors.

The distinction between intention and behavior is central to interpreting the results. Persistence/value intention was concentrated near the top of the five-point scale: 84.1% of currently treated respondents averaged at least 4, and 21.1% scored 5. Treatment belief and persistence intention were strongly correlated at 0.628 by Spearman’s rank correlation, and exploratory ordinal models supported partial rather than complete separation between them, indicating possible measurement overlap. Restricting the belief measure to three treatment-understanding items retained a positive association with persistence intention, with a standardized coefficient of 0.203. An exploratory interaction between belief and communication was not reproduced in sensitivity analyses and was therefore not treated as stable effect modification.

Several limitations constrain what can be concluded. The cross-sectional design does not establish temporal order or causality: the PHQ-4 captured symptoms only during the two weeks before questionnaire completion, whereas discontinuation thoughts could have occurred at any earlier point. The exploratory model of actual premature discontinuation contained only 39 events, and neither adverse-effect burden nor PHQ-4 score was independently associated with that all-cause outcome. Recruitment through hospital WeChat groups may also have favored patients more engaged in follow-up or more willing to remain in care. Although sensitivity analyses broadly supported the main associations in the 373 respondents who had not discontinued prematurely and in all 412 eligible participants, the findings remain associations in a selected survey population. Prospective studies are therefore needed to test whether structured education, symptom and distress screening and tiered follow-up improve treatment completion, ideally alongside verified HBsAg trajectories, missed injections, dose reductions and discontinuation records.

Subject of Research: Patient-reported treatment burden, communication and persistence during add-on peginterferon therapy for chronic hepatitis B

Article Title: Patient-reported treatment burden, communication, and persistence intention during add-on peginterferon alfa-2b therapy for chronic hepatitis B: a cross-sectional study

Article References: Han, N., Liu, H., Yan, L., & Tang, H. (2026). Patient-reported treatment burden, communication, and persistence intention during add-on peginterferon alfa-2b therapy for chronic hepatitis B: a cross-sectional study. Frontiers in Public Health, 14, Article 1972004. https://doi.org/10.3389/fpubh.2026.1972004

Image Credits: AI Generated

DOI: 10.3389/fpubh.2026.1972004

Keywords: chronic hepatitis B, peginterferon alfa-2b, treatment burden, treatment persistence, psychological distress, patient-reported outcomes, physician-patient communication, HBsAg decline, cross-sectional survey, Patient-reported, treatment, burden

The study enrolled 86 participants, including 21 healthy controls and 65 patients with treatment-naive chronic hepatitis B. Patients were divided by HBV DNA level into a group with levels at or below 10⁵ IU/mL, comprising 33 patients, and a group with levels above 10⁵ IU/mL, comprising 32 patients. Investigators also classified patients into European Association for the Study of the Liver clinical phenotypes using HBeAg status, HBV DNA and alanine aminotransferase levels. Serum CXCL-10 and CTLA-4 were measured by enzyme-linked immunosorbent assay, and investigators calculated laboratory-based inflammatory and fibrosis-related indices, including FIB-4 for all participants.

Serum concentrations of CXCL-10 and CTLA-4 differed significantly across the study groups and were highest in patients with elevated viral loads, with p-values below 0.001 for both comparisons. HBV DNA correlated with CXCL-10 at r = 0.786 and with CTLA-4 at r = 0.559. The two serum markers were also strongly correlated with one another, at r = 0.690; all three correlation coefficients had p-values below 0.001. These are measured associations in the enrolled cohort and do not establish that either marker causes changes in viral load or liver inflammation.

Histological data were available for 43 of the 65 patients, or 66.2% of the chronic hepatitis B group. Both serum markers correlated more strongly with the modified Ishak histological activity score than with fibrosis stage: r = 0.519 for CXCL-10 and r = 0.509 for CTLA-4, both with p-values below 0.001. CXCL-10 levels were elevated in EASL phase 2, identified in the abstract as HBeAg-positive chronic hepatitis, compared with phases 3 and 4, with p-values of below 0.001 and 0.005, respectively.

Descriptive receiver operating characteristic analyses showed moderate discrimination for CXCL-10 and CTLA-4, with areas under the curve of 0.78 and 0.76, respectively, and sensitivity of 47%. In a further exploratory analysis, CXCL-10 differentiated high histological activity, defined as a modified Ishak score of at least 7, from low activity, with an area under the curve of 0.77. The abstract does not report a validated diagnostic threshold or establish clinical use for either marker. Histological observations were limited to the 43 patients with biopsy data, and the reported analysis did not include longitudinal testing. The authors stated that the findings warrant confirmation in larger, longitudinal cohorts.

Subject of Research: Associations of serum CXCL-10 and CTLA-4 with viral load, liver injury and histological inflammation in treatment-naive chronic hepatitis B

Article Title: Serum CXCL-10 and CTLA-4 levels in treatment-naive chronic hepatitis B: associations with EASL-based clinical phenotypes, histological inflammatory activity, and non-invasive fibrosis indices

Article References: Uğraklı, S., Ezer, B., Kasapoğlu, E., Esen, H. S., Kökbudak, N., & Özdemir, M. (2026). Serum CXCL-10 and CTLA-4 levels in treatment-naive chronic hepatitis B: associations with EASL-based clinical phenotypes, histological inflammatory activity, and non-invasive fibrosis indices. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14501-1

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14501-1

Keywords: chronic hepatitis B, CXCL-10, CTLA-4, HBV DNA, liver inflammation, histological activity, fibrosis, immune markers, observational study, Serum, levels, treatment-naive

Researchers used least absolute shrinkage and selection operator regression followed by multivariable logistic regression to identify independent predictors of fibrosis. They then developed HEP-FIB from routinely available laboratory parameters, including platelet count, liver-function measures and lipid biomarkers. The score’s diagnostic performance was assessed with ROC-curve analysis, bootstrap internal validation and calibration analysis, and was compared with established non-invasive indices: APRI, FIB-4 and GPR. Ten machine-learning algorithms were also constructed and evaluated using measures of discrimination, classification performance, calibration and overall predictive ability. Decision curve analysis was used separately for HEP-FIB and the machine-learning models to examine clinical utility.

At an optimal HEP-FIB cutoff of 4 or higher, the score had a sensitivity of 78%, specificity of 89% and overall accuracy of 88%. Its AUC was higher than those for APRI, FIB-4 and GPR, which were 0.724, 0.654 and 0.773, respectively; all three comparisons had p values below 0.001 by the DeLong test. Bootstrap validation produced an optimism-corrected AUC of 0.881. Calibration analysis indicated good agreement between predicted and observed fibrosis probabilities, while decision curve analysis favored HEP-FIB over the default strategies assessed.

XGBoost performed best among the machine-learning approaches in the validation dataset, with an AUC of 0.914, accuracy of 0.88, sensitivity of 0.72, specificity of 0.93, balanced accuracy of 0.83 and Matthews correlation coefficient of 0.74. Its Brier score was 0.095, and the reported false-positive and false-negative rates were 0.062 and 0.275, respectively. The machine-learning models showed a marginal performance advantage over HEP-FIB and greater net clinical benefit than test-all and test-none strategies in decision curve analysis. These findings come from a retrospective cohort at one center and were supported only by internal bootstrap validation. The study did not establish performance in independent or diverse populations, so the results do not yet establish routine clinical use or replacement of existing fibrosis-assessment methods.

Subject of Research: Internal validation of HEP-FIB and machine-learning models for non-invasive prediction of significant hepatic fibrosis in chronic hepatitis B

Article Title: Novel HEP-FIB score combined with machine learning models for non-invasive prediction of significant hepatic fibrosis in chronic hepatitis B patients: a single-center, internally validated study

Article References: Bashir, A., Arora, R., Mehrotra, D., Bala, M., Parry, A. H., & Iqball, A. (2026). Novel HEP-FIB score combined with machine learning models for non-invasive prediction of significant hepatic fibrosis in chronic hepatitis B patients: a single-center, internally validated study. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14508-8

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14508-8

Keywords: chronic hepatitis B, hepatic fibrosis, HEP-FIB, machine learning, XGBoost, non-invasive testing, diagnostic accuracy, internal validation, clinical utility, Novel, score, combined

A major complication, however, is viral genetic diversity. Ten HBV genotypes, designated A through J, have been identified, and genotypes B and C dominate in East Asia. In China, more than 95 percent of hepatitis B patients carry genotype B or C virus, with genotype B more prevalent in the south and genotype C in the north. The preS1 sequence varies considerably across genotypes, producing genotype-specific immune responses in patients. Unfortunately, previously available kits for preS1 antibody detection did not account for this genotypic variation, which can yield false-negative outcomes and compromised accuracy when the assay confronts mismatched genotypes. A research team led by investigators at Jilin University has now reported in the journal iScience a quantitative ELISA built on a dual recombinant strategy designed to close this diagnostic gap, covering patients infected with either of the two major Chinese genotypes.

The core of the innovation is a fusion protein the authors call B-C-preS1. Starting from viral DNA isolated from the sera of patients infected with genotype B or genotype C HBV, the researchers amplified the DNA fragments encoding each genotype’s preS1 region by PCR, each yielding a band of roughly 1,200 base pairs on gel electrophoresis. Overlapping PCR then joined the two fragments into a single construct of approximately 2,400 base pairs. This DNA was cloned into a pcDNA3.1(−) vector, amplified in E. coli DH5α cells, and verified by restriction digestion, which released the expected vector and insert bands. The recombinant plasmid was then transfected into HEK293F cells for protein expression. After two rounds of affinity chromatography purification using Ni-NTA and Strep-Tactin resin, purified B-C-preS1 appeared as a single band of roughly 100 kilodaltons on SDS-PAGE. Western blotting with two previously reported genotype-specific antibodies confirmed that the fusion protein cross-reacts with antibodies raised against the preS1 regions of both genotype B and genotype C virus.

With the antigen in hand, the team needed a quantification standard: an antibody that recognizes preS1 epitopes from both genotypes. They immunized Balb/c mice with the B-C-preS1 fusion protein and monitored preS1-specific antibody levels by ELISA against plates coated separately with genotype B preS1, genotype C preS1, or the fusion protein. One mouse produced serum with high and comparable reactivity on all three plate types even after dilution to one part in a hundred million. Peripheral blood mononuclear cells from this animal were subjected to flow cytometric sorting using FITC-labeled B-C-preS1 and a PE-conjugated anti-mouse CD19 antibody to isolate antigen-specific B cells. The variable region genes from these cells were reverse-transcribed, amplified, and cloned into vectors carrying human IgG1 Fc for expression in HEK293F cells. Screening of the expressed antibodies identified one, named HB2, that cross-reacts with both genotype B and C preS1. SDS-PAGE showed a single band for the purified chimeric antibody, which separated into roughly 50- and 25-kilodalton bands after reduction, and size-exclusion chromatography confirmed that the monomer accounted for more than 95 percent of the preparation.

The binding properties of HB2 were characterized in detail. In ELISA, the half-maximal effective concentrations for detecting B-C-preS1, B-preS1, and C-preS1 were 177.27, 177.71, and 179.86 nanograms per milliliter, respectively, indicating essentially equivalent recognition of all three antigens. Surface plasmon resonance across a concentration range of 12.5 to 6,400 nanomolar demonstrated concentration-dependent binding to B-C-preS1, with a dissociation constant of 1.90 × 10⁻⁷ M and a slow dissociation rate of 1.25 × 10⁻³ per second, signifying a strong and stable interaction. These properties made HB2 an ideal calibrator for the assay. The team then systematically optimized every stage of the ELISA protocol through checkerboard titrations: the optimal coating antigen concentration was 2 micrograms per milliliter, the HRP-conjugated anti-human IgG detection antibody worked best at a 1:200,000 dilution, phosphate-buffered saline at pH 8.0 gave the highest positive-to-negative ratio as coating buffer, and overnight coating at 4 °C was optimal. Blocking with a mixture containing 0.2 percent bovine serum albumin, 0.25 percent Tween 20, 4 percent sucrose, and 8 percent goat serum for two hours at 37 °C maximized signal over background, and a 20-fold dilution of patient serum placed most readings within the linear range.

Performance metrics were rigorous. A serial dilution of HB2 produced a linear standard curve with an R² of 0.9939, and the quantitative detection range was set at 10 to 100 nanograms per milliliter. The qualitative cut-off, calculated from healthy donor sera as the mean plus two standard deviations, was 0.0775 optical density units, corresponding to a preS1 antibody concentration of 7.62 nanograms per milliliter, below the lower limit of the quantitative range. Intra-assay coefficients of variation across three independently manufactured kit batches ranged from 5.89 to 8.88 percent, while inter-assay variation ranged from 7.77 to 8.53 percent, both comfortably within the 15 percent precision requirement of Chinese guidelines. Recovery experiments, in which known amounts of HB2 were spiked into patient sera representing high, medium, and low antibody levels, yielded recovery rates between 90.80 and 102.58 percent, confirming good accuracy, particularly for medium-to-high antibody concentrations.

The validation panel comprised 156 patient sera confirmed positive by a commercial qualitative kit, plus 66 sera from healthy individuals. Genotyping by PCR amplification and sequencing of HBV DNA classified 29 sera as genotype B and 127 as genotype C. The home-made ELISA scored all 156 patient sera as positive and all 66 healthy sera as negative, producing an area under the ROC curve of 1. Cross-reactivity testing against hepatitis A virus, hepatitis C virus, and HBV genotypes A and D returned readings below the cut-off, indicating high specificity. Most revealing was the head-to-head comparison with a commercial quantitative kit that does not account for genotype. That kit correctly quantified the genotype C sera, correlating well with the new assay at an R² of 0.9519, but it returned negative results for all 29 genotype B sera, roughly 19 percent of the panel. In other words, the existing commercial assay systematically misses nearly one in five Chinese patients.

Stability testing addressed the practical demands of commercial deployment. Sealed kits stored at 4 °C for a full year retained 91.07 to 98.33 percent of their day-zero performance at month 12, well above the 80 percent threshold required in China. Accelerated testing at 37 °C for ten days showed readouts of 87.2 percent or better, with week-long values between 91.5 and 98.1 percent. Opened kits held at 4 °C retained 90.6 to 100.1 percent of baseline through seven days of use, and simulated transportation involving seven days of shaking followed by repeated drop tests produced no significant change in measured antibody levels. Taken together, the results describe an assay robust enough for real-world clinical logistics, with the authors noting that Beijing Abace Biotechnology has already received approval from China’s Food and Drug Administration for the kit.

Beyond hepatitis B, the authors frame the dual recombinant strategy as a general answer to a persistent problem in virology: rapid mutation and genotypic diversity undermine both nucleic acid detection, which depends on conserved primer and probe binding sites, and protein-based detection, which depends on conserved epitopes. By tandemly expressing key antigenic regions from multiple genotypes in a single molecule, the approach physically assembles characteristic epitopes into one broad-spectrum reagent, simplifying production compared with mixing separate genotype-specific proteins and widening patient coverage in a single reaction. The authors acknowledge limitations, including the need to know genotype sequences in advance, the requirement that mutation rates not be overwhelming, the need to optimize linker design and epitope spacing, and the small number of genotype B sera in this study, which came from northern China where genotype C predominates. Still, they argue the concept could extend to detection reagents, vaccines, and therapeutic antibodies for other highly variable viruses such as influenza, HIV, hepatitis C virus, and SARS-CoV-2, offering a template for keeping viral diagnostics ahead of viral evolution.

Subject of Research: Development and validation of a dual-genotype recombinant ELISA for quantifying hepatitis B virus preS1 antibodies in patients infected with genotype B or C virus

Article Title: A dual recombinant strategy to quantify PreS1 antibody in hepatitis B patients with genotype B or C virus by ELISA

Article References: Li, Y., Wen, X., Zhan, J., Cao, T., Chi, X., Cheng, H., Huang, Q., Zhang, W., Niu, J., Du, H., & Liu, Z. (2026). A dual recombinant strategy to quantify PreS1 antibody in hepatitis B patients with genotype B or C virus by ELISA. iScience, 29(11), Article 117857. https://doi.org/10.1016/j.isci.2026.117857

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117857

Keywords: hepatitis B virus, preS1 antibody, ELISA, genotype B, genotype C, recombinant protein, chimeric antibody, diagnostics, viral genotypes, serology, China, antibody quantification

The biological logic behind the study rests on a well-established principle of gene regulation. DNA methylation, the addition of methyl groups to cytosine bases within gene promoter regions, generally acts as a brake on transcription. When a promoter is heavily methylated, the chromatin adopts a closed configuration that limits access of transcriptional machinery, and the downstream gene is expressed at low levels. Conversely, loss of methylation, known as hypomethylation, often permits increased gene expression. For chemokines, which are potent signalling molecules that recruit immune cells to sites of infection or damage, the methylation state of the promoter can therefore dictate how strongly an inflammatory signal is broadcast. CXCL16, or C-X-C motif chemokine ligand 16, is one such chemokine, recognized as a key mediator in liver injury, yet its epigenetic regulation in chronic hepatitis B had remained unclear prior to this investigation.

To address that gap, the team assembled a study population of 217 patients with chronic hepatitis B, subdivided into 113 who were positive for hepatitis B e antigen (HBeAg) and 104 who were HBeAg-negative, alongside 27 healthy controls. HBeAg status is a clinically meaningful marker in HBV infection: HBeAg-positive patients are typically considered to carry a higher viral replicative activity and a distinct immunological profile compared with those who have seroconverted. By stratifying patients this way, the researchers could ask whether epigenetic changes at the CXCL16 promoter differed not only between diseased and healthy livers, but also between the two major immunological phases of chronic infection.

Across three molecular layers, the same pattern emerged. First, the percentage of methylation reference, a quantitative measure of promoter methylation, was significantly lower in chronic hepatitis B patients than in healthy controls, and the reduction was especially pronounced in HBeAg-positive cases. Second, mRNA levels of CXCL16 were markedly higher in the patient groups, again most prominently among those who were HBeAg-positive. Third, serum concentrations of the CXCL16 protein were elevated in patients relative to controls. The concordance across methylation, transcript and protein measurements is what gives the finding its mechanistic weight, because it suggests a coherent pathway from epigenetic derepression to enhanced chemokine production rather than an isolated correlation at a single molecular level.

The inverse relationship between methylation and expression was borne out statistically. CXCL16 promoter methylation was negatively correlated with CXCL16 expression, which is precisely what promoter methylation biology would predict: the less the promoter is methylated, the more the gene is transcribed and translated. Notably, methylation also showed negative correlations with several clinical and immunological markers. These included HBeAg status itself, the liver injury enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β). In practical terms, patients with the lowest CXCL16 promoter methylation tended to have the highest levels of transaminases, the canonical blood markers of hepatocyte damage, and the highest circulating concentrations of inflammatory cytokines that drive and amplify hepatic inflammation.

ALT and AST are enzymes released when hepatocytes are injured, and their serum activities form the backbone of non-invasive monitoring in viral hepatitis. TNF-α and IL-1β, meanwhile, are central cytokines in the innate immune response, capable of activating hepatic stellate cells, promoting leukocyte recruitment and sustaining the inflammatory milieu that, over years, leads to fibrosis and malignant transformation. The observation that CXCL16 promoter methylation aligns with all of these parameters simultaneously positions the epigenetic mark as a potential integrator of inflammatory burden. Rather than reflecting a single laboratory value, hypomethylation appears to sit upstream of a network of destructive processes in the HBV-infected liver.

To test whether the methylation measurement could do more than describe associations, the researchers performed receiver operating characteristic (ROC) analysis, a standard method for evaluating the discriminative capacity of a biomarker. The analysis demonstrated that CXCL16 promoter methylation had a favourable ability to distinguish HBeAg-positive patients, with an area under the curve (AUC) of 0.749. An AUC of 0.5 would indicate performance no better than chance, while 1.0 would indicate perfect discrimination, so a value of 0.749 represents a moderate-to-good level of diagnostic accuracy. Taken together with the significant correlations with inflammatory markers, the authors suggest that CXCL16 promoter methylation could serve as a complementary indicator for identifying chronic hepatitis B patients who carry a higher inflammatory burden.

The clinical appeal of such a marker lies in its non-invasive character. Assessing liver inflammation today often relies on serum transaminases, which can fluctuate and normalise even in the presence of ongoing disease, or on liver biopsy, which remains the gold standard for grading inflammation and staging fibrosis but carries procedural risks, sampling variability and patient reluctance. A blood-based epigenetic measurement that reflects the regulatory state of a chemokine gene could, if validated further, offer a repeatable and safer window into hepatic inflammatory activity. The authors conclude that CXCL16 promoter hypomethylation is closely associated with elevated CXCL16 expression and exacerbated hepatic inflammation in chronic hepatitis B patients, and propose it as a potential non-invasive biomarker for assessing CHB-related liver inflammation.

Beyond its diagnostic promise, the study enriches the mechanistic picture of how HBV infection translates into chronic liver damage. Viral persistence triggers innate and adaptive immune responses in the liver, and chemokines such as CXCL16 help orchestrate the recruitment of immune cells that both contain the virus and injure hepatocytes. The demonstration that this process is governed, at least in part, by promoter methylation adds an epigenetic layer to the known immunopathology of hepatitis B, and it identifies CXCL16 as a novel target for clinical evaluation and intervention. If future work confirms that modulating CXCL16 methylation or expression can influence inflammatory outcomes, the pathway could become more than a passive marker and evolve into a genuine therapeutic axis.

Several considerations temper immediate application. The study reports associations in a cross-sectional design, so causality between hypomethylation, chemokine expression and inflammation cannot be fully disentangled from the present data, and longitudinal follow-up would be needed to determine whether CXCL16 promoter methylation predicts disease progression, response to antiviral therapy or the later development of hepatocellular carcinoma. Moreover, methylation patterns can be influenced by age, environment and comorbidities, so the findings will require replication in larger and more diverse cohorts before any routine clinical use. The article was published open access on 14 October 2026 under a Creative Commons licence, and the authors, led by corresponding author Kai Wang, declare no competing interests. Even with those caveats, the convergence of methylation data, gene expression, protein levels and inflammatory correlations in a well-characterised cohort of 217 patients makes a compelling case that an epigenetic signature at the CXCL16 promoter mirrors the inflammatory fires burning in chronically HBV-infected livers, and offers researchers a fresh molecular handle on a disease that affects hundreds of millions of people worldwide.

Subject of Research: Epigenetic regulation of the CXCL16 chemokine gene in chronic hepatitis B liver inflammation

Article Title: CXCL16 gene promoter methylation is associated with inflammation in patients with chronic hepatitis B

Article References: Tian, Z., Xu, M., Li, H., Yao, Y., Fan, Y., Gao, S., & Wang, K. (2026). CXCL16 gene promoter methylation is associated with inflammation in patients with chronic hepatitis B. Virology Journal. https://doi.org/10.1186/s12985-026-03322-y

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03322-y

Keywords: chronic hepatitis B, HBV, CXCL16, DNA methylation, epigenetics, liver inflammation, biomarker, HBeAg, chemokine, TNF-alpha, IL-1beta, transaminases

The hepatitis B virus establishes its persistence through a peculiar molecular intermediate known as covalently closed circular DNA, or cccDNA. After the virus enters a hepatocyte, its partially double-stranded circular genome travels to the nucleus, where host repair enzymes convert it into a fully double-stranded, covalently closed circle. This minichromosome behaves remarkably like a cellular chromosome: it is wrapped in histone proteins, carries epigenetic marks, and serves as the template for all of the viral RNA transcripts that drive the production of new virions and, critically, of hepatitis B surface antigen, the viral protein that accumulates to enormous levels in the blood of chronically infected patients and is thought to exhaust and dysregulate antiviral immune responses. Because cccDNA is extraordinarily stable and can persist in dividing and non-dividing hepatocytes alike, clearing it has been the elusive goal of hepatitis B research for decades.

Existing treatments manage the consequences of cccDNA activity rather than the reservoir itself. Nucleoside and nucleotide analogues, such as tenofovir and entecavir, potently inhibit the viral polymerase and prevent new rounds of replication, but they leave cccDNA untouched, which is why discontinuation almost universally leads to viral rebound. Pegylated interferon-alpha can produce functional control in a minority of patients, likely through a combination of direct antiviral and immunomodulatory effects, but its tolerability is poor and its cure rates remain low. The result is that most patients face lifelong daily therapy, and the dream of a finite treatment course that leaves the liver free of active viral transcription has remained out of reach. Epigenetic silencing offers a conceptual alternative: if the viral minichromosome can be rendered permanently or durably transcriptionally silent, the virus may be functionally inactivated even though its DNA physically remains.

The investigational therapy described in the new study is built on this premise. It is designed as an epigenetic silencer, a construct that targets the hepatitis B viral DNA and recruits repressive chromatin-modifying machinery to the viral genome, thereby converting the active cccDNA minichromosome into a heterochromatic, transcriptionally inert state. In practical terms, the therapy aims to deposit repressive histone marks and promote DNA methylation patterns at the viral regulatory elements that normally drive transcription of all the viral RNAs, including the pregenomic RNA that seeds new replication and the abundant subgeniral RNAs that encode surface and core proteins. By shutting down transcription at its source, the approach addresses both the production of infectious virus and the antigenemia that blunts immune recovery.

The engineering challenge at the heart of such a therapy is specificity. The human liver contains roughly two hundred billion hepatocytes, and the silencing machinery must find the small fraction of cells harboring transcriptionally active cccDNA while leaving the host genome and its epigenetic landscape untouched. The reported system couples a DNA-binding module that recognizes sequences within the hepatitis B genome to effector domains capable of recruiting repressive complexes. Because the viral regulatory region, including the core promoter and enhancer elements, contains sequence features that are conserved across hepatitis B genotypes but absent from the human genome in the same configuration, the design exploits a natural sequence boundary between pathogen and host. Delivery to hepatocytes is achieved with a liver-directed formulation intended to concentrate the payload in the organ where more than ninety percent of viral replication occurs.

Epigenetic silencing of viral genomes is not an entirely new idea, but translating it into a viable therapy has required overcoming several biological realities. The first is that cccDNA is not a static target: it exists in variable copy numbers per cell, ranges from fully active to partially silenced states even within a single liver, and can be replenished from integrated viral DNA in some patients. The second is reversibility. Epigenetic marks are, by definition, modifiable, and a silenced minichromosome could in principle be reactivated if repressive marks decay or if the cell encounters inflammatory or proliferative signals that remodel chromatin. The investigators therefore designed the therapy not merely to deposit transient repressive marks but to establish a self-reinforcing silenced state, in which repressive chromatin at the viral promoter recruits further repressive machinery, creating a memory of inactivation that persists across time and, in dividing cells, across cell generations.

The functional consequences of such silencing would extend beyond simply lowering viral load. Hepatitis B surface antigen is now understood to be a principal driver of immune dysfunction in chronic infection, with circulating subviral particles reaching concentrations of micrograms per milliliter and contributing to the exhaustion of natural killer cells, T cells, and B cells that would otherwise clear infected hepatocytes. A therapy that transcriptionally inactivates cccDNA would be expected to produce a steep and sustained decline in surface antigen, removing this immunosuppressive burden and creating the conditions under which the patient’s own immune system can recognize and eliminate residual infected cells. In this sense, epigenetic silencing is best understood not as a standalone cure but as the keystone of a combination strategy, in which silencing of the reservoir is followed or accompanied by therapeutic vaccination, immune checkpoint modulation, or other immunorestorative interventions that consolidate the gain.

Safety considerations loom large for any therapy that deliberately manipulates chromatin. Broadly acting epigenetic drugs, such as the histone deacetylase inhibitors developed in oncology, carry systemic toxicity profiles that make them unattractive for chronic liver disease. The investigational silencer is therefore designed to act locally and selectively, with its repressive activity confined to the viral sequences it is targeted to. Another concern is the fate of integrated hepatitis B DNA, which in many chronic infections is scrambled into host chromosomes and cannot be circularized or fully silenced by the same mechanism; integrated DNA is a known source of surface antigen production and, in some contexts, of insertional mutagenesis that contributes to hepatocellular carcinoma. The therapy’s developers and independent commentators alike emphasize that silencing cccDNA addresses the replicative reservoir but does not by itself excise integrated sequences, underscoring that long-term monitoring for surface antigen rebound and for liver cancer risk will remain necessary even in successfully treated patients.

The publication arrives at a moment of unusual momentum in hepatitis B cure research. Dozens of candidates are advancing through clinical pipelines, including entry inhibitors, capsid assembly modulators, RNA interference therapeutics that degrade viral transcripts, surface antigen secretion inhibitors, and therapeutic vaccines. RNA interference drugs in particular have demonstrated that reducing viral antigen is feasible and well tolerated, and several have advanced to late-stage trials, though rebound after discontinuation has highlighted the persistence of the cccDNA reservoir that RNA-based approaches cannot touch. An epigenetic silencer occupies a distinctive position in this landscape because it acts on the reservoir itself, at the level of the minichromosome, rather than on the RNA or protein products that flow from it. If its silenced state proves durable, it could provide the reservoir control that RNA interference and direct-acting antivirals lack, while complementing the immune-directed agents needed to finish the job.

Much work remains before such a therapy could reach patients. The path from proof of concept in laboratory and animal models of hepatitis B infection to demonstrated safety and efficacy in humans is long, and epigenetic therapies will face scrutiny from regulators accustomed to evaluating small molecules and antibodies. Questions about the completeness of silencing across the enormous heterogeneity of cccDNA populations, the behavior of the therapy in patients with high viral loads and advanced fibrosis, the potential for rare escape variants in the targeted viral sequences, and the reversibility of the silenced state over years rather than weeks will all need rigorous answers. Nevertheless, the development of an investigational therapy designed to transcriptionally inactivate viral DNA marks a conceptual milestone: it treats the hepatitis B minichromosome not as an untouchable fixture of chronic infection but as an epigenetic state that can be rewritten. For the hundreds of millions of people living with chronic hepatitis B, the prospect of a finite course of treatment that leaves the virus silent, the immune system restored, and the liver protected represents a goal that has never been closer to serious experimental pursuit.

Subject of Research: Development of an investigational epigenetic silencer therapy that transcriptionally inactivates hepatitis B viral DNA in chronic hepatitis B

Article Title: Development of an investigational epigenetic silencer therapy to transcriptionally inactivate viral DNA in chronic hepatitis B

Article References: Anglero-Rodriguez, Y., Xiong, Q., Cappelluti, M. A., Voytek, S. B., Acosta, G., Choo-Wing, R., Hoffman, L., Khadka, A., Medina, J. A., Mugambwa, C., Pantano, C., Harel, S., DiPiazza, A., Abraham, S., Hildebrand, E. M., Ramirez, R. N., Zhai, A., Guo, X., Abubucker, S., … Marlowe, J. L. (2026). Development of an investigational epigenetic silencer therapy to transcriptionally inactivate viral DNA in chronic hepatitis B. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-026-01802-8

Image Credits: AI Generated

DOI: 10.1038/s41551-026-01802-8

Keywords: chronic hepatitis B, hepatitis B virus, cccDNA, epigenetic silencing, viral DNA, hepatitis B surface antigen, antiviral therapy, chromatin modification, functional cure, liver disease, RNA interference, transcriptional repression

Yet the numbers remain sobering. According to the review, even with the best currently available regimens, the functional cure rate among patients with chronic hepatitis B stays below 30 percent, and in many treatment settings it is far lower. Nucleos(t)ide analogues such as entecavir and tenofovir suppress viral replication with remarkable potency and an excellent safety profile, but they act on the reverse transcriptase step of the hepatitis B virus life cycle and leave the intracellular reservoirs of viral genetic material untouched. Pegylated interferon-alpha, the other pillar of standard therapy, can reinvigorate antiviral immune responses in a subset of patients, but response rates are limited and tolerability issues frequently force discontinuation. The result is a therapeutic plateau: viral suppression is easy to achieve, but genuine immune-mediated control of the infection remains the exception rather than the rule.

At the heart of this plateau, the authors identify the persistence of covalently closed circular DNA, or cccDNA, as the central virological obstacle. cccDNA is the circular, episodic form of the hepatitis B virus genome that resides in the nucleus of infected hepatocytes and serves as the transcriptional template for all viral RNAs, including the pregenomic RNA from which new viral particles are assembled. Because cccDNA is stable, long-lived and present in minute quantities per cell, it evades both the cytoplasmic degradation pathways triggered by nucleos(t)ide analogues and the surveillance of the immune system, which struggles to recognize infected hepatocytes that produce little or no viral antigen. Clearing cccDNA from the liver would require either eliminating every infected hepatocyte or inducing selective degradation of the minichromosome itself, and neither approach is currently achievable with acceptable safety in the overwhelming majority of patients.

Compounding the cccDNA problem is a second form of viral persistence that the review highlights in detail: the integration of hepatitis B virus DNA into the genome of the host hepatocyte. Integration occurs frequently during the chronic phase of infection, driven by the_error-prone nature of viral replication and the continual turnover of infected cells. Integrated viral sequences are typically rearranged and replication-defective, but they can act as persistent factories for hepatitis B surface antigen, sustaining high circulating levels of HBsAg even when cccDNA transcription has been effectively silenced. This has two major consequences. First, integrated DNA means that surface antigen loss cannot automatically be equated with the elimination of the viral reservoir, complicating the interpretation of treatment endpoints. Second, chromosomal integration is a recognized mutagenic event, and insertional disruption of host genes such as TERT has been implicated in the development of hepatocellular carcinoma, linking viral persistence directly to the most feared clinical outcome of chronic infection.

The review then turns to the immunological face of the problem, describing how prolonged exposure to elevated levels of viral antigens — above all hepatitis B surface antigen and hepatitis B e antigen — induces a state of immune tolerance that is sculpted from the very earliest stages of infection. In patients infected at birth or in early childhood, the developing immune system encounters enormous quantities of circulating antigen during the critical windows in which tolerance mechanisms are established. High antigen loads drive the deletion or functional silencing of HBV-specific T cells, promote the differentiation of regulatory T cells and exhaust antigen-presenting dendritic cells, so that by the time chronicity is established the immune system has been trained, in effect, to ignore the virus. Maternal antigen passage and the unique tolerogenic environment of the liver, which is continuously exposed to gut-derived material and normally biased toward immune non-responsiveness, reinforce this state and help explain why infection acquired early in life so rarely resolves spontaneously.

Even in patients whose T cells do persist, the review documents a landscape of immune exhaustion and dysfunction operating at multiple regulatory levels. HBV-specific CD8 T cells in chronic hepatitis B display the classic molecular signature of exhaustion: sustained expression of inhibitory receptors such as PD-1, CTLA-4, TIM-3 and LAG-3, shortened telomeres, impaired cytotoxic function and a shift toward a terminally differentiated, senescent phenotype. CD4 helper function is similarly compromised, weakening the support that CD8 cells require for sustained antiviral activity. Beyond the T cell compartment, natural killer cell function is dysregulated, B cell responses are blunted with defective production of neutralizing anti-HBs antibodies, and the intrahepatic cytokine milieu — enriched in suppressive mediators such as interleukin-10 and indoleamine 2,3-dioxygenase — actively restrains inflammatory responses. Exhausted T cells, the authors emphasize, are not simply passive casualties; they represent a partially reversible differentiation state, which is precisely why they have become the focal point of new immunotherapeutic strategies.

It is against this mechanistic backdrop that the review surveys the emerging arsenal of immunotherapy. Therapeutic vaccines aim to present HBV antigens in an immunogenic context that can break tolerance and prime new antiviral T cell responses. Checkpoint inhibitors, most notably antibodies directed against PD-1 or PD-L1, seek to reverse the exhausted state of HBV-specific T cells, an approach supported by incidental observations of HBsAg decline in cancer patients treated with such agents. Agonists of pattern-recognition receptors, including Toll-like receptor 7 and 8 agonists, are designed to reawaken innate antiviral defenses and restore interferon production within the liver. Additional strategies under investigation include engineered T cell technologies, cytokine modulation, and combinations that pair direct-acting antivirals with immune interventions to first reduce antigen load and then rebuild antiviral immunity. The authors stress, however, that no single agent has yet demonstrated the capacity to induce functional cure reliably, and that the field’s most promising direction lies in rational combination: reducing the antigenic burden, releasing the brakes on T cell function, and providing the innate cytokine signals needed to coordinate a durable multi-layered response.

The review also underscores why the liver’s unique immunobiology makes this task so difficult. The hepatic environment is constitutively tolerogenic, populated by liver sinusoidal endothelial cells, Kupffer cells and stellate cells that favor the induction of regulatory rather than effector T cell responses. Any curative strategy must therefore overcome not only the viral reservoirs and the exhaustion of the adaptive immune system, but also an organ-specific architecture that has evolved to suppress inflammation. Biomarker development is identified as an equally pressing need: quantitative HBsAg levels, hepatitis B RNA signatures and emerging measures of immune restoration are all being explored as tools to identify which patients are most likely to benefit from which combination, and to shorten the lengthy trials that current endpoints demand.

By synthesizing virological persistence mechanisms, the layered failure of antiviral immunity and the emerging immunotherapeutic toolkit into a single framework, Lin, Yu and Sun aim to provide both a theoretical foundation and practical critical perspective for the clinical translation of functional cure strategies. Their central message is one of mechanistic realism: the obstacles to curing chronic hepatitis B functionally are not a single barrier but an interlocking system of viral reservoirs, integrated DNA, antigen-driven tolerance and multi-level immune exhaustion. Understanding how these pieces reinforce one another — and designing combinations that dismantle them in the correct sequence — is, the authors conclude, the surest path toward turning a goal achieved by fewer than a third of patients today into a routine clinical outcome tomorrow.

Subject of Research: Immunological mechanisms and barriers to achieving a functional cure for chronic hepatitis B

Article Title: The core obstacles and immunological mechanisms in the study of functional cure for Chronic Hepatitis B

Article References: Lin, L., Yu, D., & Sun, S. (2026). The core obstacles and immunological mechanisms in the study of functional cure for Chronic Hepatitis B. Virology Journal. https://doi.org/10.1186/s12985-026-03310-2

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03310-2

Keywords: chronic hepatitis B, functional cure, HBV, cccDNA, HBsAg loss, immune exhaustion, viral integration, immunotherapy, T cell dysfunction, immune tolerance, liver disease, antiviral therapy

Hepatitis B virus (HBV) remains one of the world’s most consequential pathogens. An estimated 254 million people were living with chronic HBV infection in 2022, and roughly 1.1 million deaths were attributed to the virus that year. China carries the heaviest national burden, with approximately 75 million infected individuals. Chronic infection drives cirrhosis and hepatocellular carcinoma (HCC), but accumulating evidence also links chronic HBV to elevated risks of digestive system malignancies, kidney disease, and mortality from cardiovascular and cerebrovascular conditions compared with the general population. Against this backdrop, the World Health Organization has endorsed a global strategy to eliminate viral hepatitis by 2030, emphasizing both the prevention of new infections and the expansion of diagnosis and treatment to reduce mortality among those already infected.

Loss of HBsAg is the central therapeutic goal in chronic hepatitis B and underpins the treatment endpoint known as functional cure. Spontaneous HBsAg seroclearance also occurs during the natural course of infection, though infrequently — roughly 1 percent of patients per year. Prior research has consistently shown that patients who clear HBsAg experience substantially lower rates of cirrhosis and HCC. What remained uncertain, however, was whether seroclearance is also associated with overall survival and, in particular, with extrahepatic mortality from cancers and cardiovascular disease — a question of growing importance as the population of people living with chronic hepatitis B ages and accumulates comorbidities.

To address this gap, investigators drew on the Chronic Hepatitis B Infection and Liver Disease (HEPCARE) study, a multicenter, prospective, community-based cohort in Jiangsu Province. Participants were recruited through serological surveys conducted between September 2009 and November 2010 and were required to be HBsAg-positive, hepatitis C antibody-negative, and free of cirrhosis and liver cancer at enrollment. Because HBV DNA measurements began at the 2012 follow-up, the analysis defined 2012 as baseline and followed participants through December 2023, with assessments in 2012, 2013, 2014, 2016, 2018, 2020, and 2023. After exclusions, 6551 participants entered the analytical pool, and after propensity score matching on age, sex, region, and baseline antiviral treatment status, the final matched cohort comprised 1029 individuals who achieved sustained HBsAg seroclearance with HBV DNA below 100 IU/mL and 2931 who remained persistently HBsAg-positive.

A key methodological strength of the study lies in its handling of time. HBsAg seroclearance is a time-dependent event that occurs during follow-up, and conventional analyses that treat it as a fixed baseline characteristic are vulnerable to immortal time bias, which can distort survival estimates. By conducting regular interval surveillance and using time-dependent Cox regression, the researchers could allocate person-time accurately according to each participant’s changing serovirological status. The exposure itself was stringently defined: seroclearance required HBsAg negativity together with HBV DNA below the quantification limit of 100 IU/mL, confirmed at two or more consecutive follow-up visits, with participants whose profiles suggested occult hepatitis B infection excluded to avoid misclassification.

The results were striking across multiple outcome domains. Over a median follow-up of roughly 11 years, participants who achieved sustained seroclearance had significantly lower incidence rates of cirrhosis (2.02 versus 3.97 per 1000 person-years) and HCC (2.97 versus 4.97 per 1000 person-years). In fully adjusted time-dependent Cox models, seroclearance was associated with a 55 percent lower hazard of cirrhosis (adjusted hazard ratio 0.45) and a 48 percent lower hazard of HCC (adjusted hazard ratio 0.52). Mortality told a similar story: 64 deaths occurred in the seroclearance group compared with 405 in the persistent infection group, corresponding to incidence rates of 7.55 and 12.17 per 1000 person-years, and seroclearance was associated with a 53 percent lower hazard of all-cause mortality (adjusted hazard ratio 0.47).

Perhaps the most novel findings concerned cause-specific mortality. Among the 469 deaths in the matched population, 146 were attributed to extrahepatic cancers, 117 to cardiovascular disease, and 112 to liver-related causes. Sustained seroclearance was associated with lower hazards of liver-related mortality (adjusted hazard ratio 0.46), extrahepatic cancer-related mortality (0.51), and cardiovascular mortality (0.56), with the strongest association observed for digestive system cancer-related mortality. When competing risks were formally accounted for using Fine–Gray subdistribution hazard models, these associations persisted for cirrhosis, HCC, liver-related mortality, and extrahepatic cancer-related mortality, and model-standardized cumulative incidences at 10 years were consistently lower in the seroclearance state for all major endpoints examined.

The investigators then asked whether HBsAg loss carries prognostic information beyond viral DNA suppression alone — a clinically important question, since suppressing HBV DNA below 100 IU/mL is a conventional treatment goal. In an analysis treating serovirological status as a three-state time-varying variable, the HBsAg-negative state with very low HBV DNA was associated with lower hazards of cirrhosis, HCC, all-cause mortality, extrahepatic cancer mortality, and cardiovascular mortality compared with the HBsAg-positive state at similarly low viral loads. Conversely, isolated HBV DNA suppression without seroclearance was not associated with lower extrahepatic mortality. Additional weighting analyses incorporating baseline HBV DNA attenuated the HCC estimate somewhat, suggesting that part of that association depends on differences in baseline viral burden, but the associations with all-cause and extrahepatic cancer mortality remained statistically significant.

To translate the associations into population-level terms, the team estimated population attributable fractions and restricted mean survival time. Persistent infection was associated with attributable fractions of approximately 48 percent for cirrhosis, 47 percent for liver-related mortality, 42 percent for all-cause mortality, 41 percent for HCC, 40 percent for extrahepatic cancer mortality, and 35 percent for cardiovascular mortality. Using age as the time scale, restricted mean survival time was estimated at 83.21 years in the seroclearance state versus 78.04 years in the persistent infection state — a 5.17-year difference within the observed age range, with the survival advantage widening notably after age 60. Sex-stratified analyses suggested that these inverse associations were generally stronger in men, with statistically significant interactions for all-cause and liver-related mortality, echoing prior evidence that men with chronic hepatitis B tend to experience less favorable long-term outcomes than women.

The authors caution that most seroclearance events in the cohort occurred spontaneously rather than through antiviral therapy, so the findings primarily reflect the prognosis associated with spontaneous HBsAg loss, which may partly reflect a favorable underlying viral-host profile and overall health status. Residual confounding from unmeasured factors such as socioeconomic status and healthcare utilization cannot be excluded, the assay’s quantification limit of 100 IU/mL may have classified some low-level viremia as undetectable, and the single-province, community-based design warrants validation in other populations, including cohorts enriched for treatment-induced functional cure. Biologically, the authors propose that persistent viral antigen exposure and chronic immune activation may impair tumor immune surveillance and promote systemic inflammation, whereas HBsAg clearance accompanies a marked reduction in antigen burden and partial restoration of HBV-specific immune responses — hypothesis-generating mechanisms that require further study. Nevertheless, the consistency of the associations across competing-risk models, three-state analyses, and extensive sensitivity analyses, combined with the substantial attributable burden and survival differences, positions sustained HBsAg seroclearance with very low HBV DNA as an informative long-term prognostic marker in chronic hepatitis B, and underscores the potential population health value of broader attainment of this serovirological state.

Subject of Research: HBsAg seroclearance and long-term hepatic and extrahepatic mortality risks in chronic hepatitis B

Article Title: HBsAg seroclearance with HBV DNA <100 IU/mL and long-term risks of adverse liver events, all-cause mortality, and cause-specific mortality in chronic hepatitis B: a prospective cohort study

Article References: Zhang, Y., Yao, W., Jiang, J., Qian, J., Chen, X., Jiang, Q., Yan, Y., Wang, S., Bai, H., He, C., Zhu, L., Jiang, T., Hu, Z., Shen, H., Zhai, X., & Song, C. (2026). HBsAg seroclearance with HBV DNA <100 IU/mL and long-term risks of adverse liver events, all-cause mortality, and cause-specific mortality in chronic hepatitis B: a prospective cohort study. The Lancet Regional Health – Western Pacific, 74, Article 101983. https://doi.org/10.1016/j.lanwpc.2026.101983

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DOI: 10.1016/j.lanwpc.2026.101983

Keywords: hepatitis B, HBsAg seroclearance, functional cure, hepatocellular carcinoma, cirrhosis, all-cause mortality, extrahepatic cancer, cardiovascular mortality, HBV DNA, prospective cohort, population attributable fraction, chronic hepatitis B

The central obstacle to a cure is the remarkable persistence architecture of the hepatitis B virus. After the virus enters hepatocytes through the NTCP receptor, its relaxed circular DNA genome is repaired by host factors into covalently closed circular DNA, or cccDNA, a stable episomal minichromosome that persists in the nucleus and serves as the transcriptional template for all viral RNAs and proteins. In parallel, fragments of viral DNA integrate at random sites into the host genome as integrated DNA, or intDNA, which does not produce new virus but continuously secretes surface antigen and, in some cases, the oncogenic HBx protein, fueling immune tolerance and carcinogenesis. Current interferon-alpha and nucleos(t)ide analog therapies leave both reservoirs untouched, so viral rebound is common once treatment stops, and long-term nucleos(t)ide therapy carries risks such as nephrotoxicity.

The Groningen team frames the therapeutic landscape around two epigenetic strategies. The first, broad-acting epidrugs, globally modulate histone- and DNA-modifying enzymes; nine such agents are already FDA-approved in oncology. The second, epigenetic editing, fuses effector enzymes to programmable DNA-binding domains — zinc-finger proteins, transcription activator-like effectors, or deactivated CRISPR-Cas9 — to write repressive marks at specific viral loci. Because HBV gene activity on cccDNA is governed by histone acetylation and methylation, with HBx recruiting p300 to boost H3 and H4 acetylation while SETDB1-mediated H3K9me3 and PRMT1-mediated H4R3 methylation repress transcription, the viral minichromosome is an unusually tractable epigenetic target.

Among epidrugs, sirtuin 2 inhibitors have produced the most compelling results. The SIRT2 inhibitor AGK2 reduced HBV DNA, RNA, HBsAg and HBeAg by 15 to 30 percent in vitro and in HBV-transgenic mice without hepatotoxicity, and recent work showed the drug acts by recruiting repressive histone lysine methyltransferases to cccDNA, enriching H4K20me1, H3K27me3 and H3K9me3 while reducing RNA polymerase II occupancy. A more specific allosteric inhibitor, FLS-359, blocked the conversion of rcDNA into cccDNA entirely in primary human hepatocytes when given before infection, cutting cccDNA formation by more than half — though it was ineffective after infection was established, positioning it as a preventive rather than curative agent. Meanwhile, the DNA methyltransferase inhibitor 5-azacytidine reactivated interferon-stimulated genes and sensitized otherwise unresponsive cells to interferon-alpha, but it also raised NTCP expression and HBsAg levels, highlighting the double-edged nature of globally acting agents.

That lack of locus specificity is precisely what epigenetic editing is designed to fix, and the review catalogs five preclinical editing studies organized around two approaches. Indirect editing directs a Krüppel-associated box, or KRAB, repressor domain to key HBV regulatory elements; KRAB acts as a scaffold recruiting histone deacetylases, lysine methyltransferases and heterochromatin proteins to shut down transcription. Direct editing instead guides DNA methyltransferases such as DNMT3a or the bacterial M.SssI enzyme to CpG islands overlapping viral promoters, writing de novo methylation that can be mitotically inherited. Both routes reduced HBV RNA, DNA and antigens from cccDNA and, in some contexts, from intDNA, with far better specificity than epidrugs.

The zinc-finger platform, the most rigorously studied to date, illustrates both the promise and the limits. Zhao and colleagues built a six-finger ZFP-KRAB artificial transcription factor targeting the X gene enhancer that cut HBx RNA by roughly 60 percent in Hep3B cells, a line carrying integrated viral DNA. Luo and colleagues achieved the most durable result, with HBV DNA falling to about one-third of control levels in transgenic mice by day seven and remaining significantly lower at day 28, although HBsAg was unchanged. The direct approach by Xirong and colleagues, fusing DNMT3a to a ZFP targeting the X promoter CpG island, lowered HBsAg by 58 percent in mice and 90 percent in cells, with confirmed methylation at seven CpG sites — but the effect reversed by day 20, likely through passive or TET-mediated demethylation. A single TALE-based study by Bloom and colleagues delivered the fastest repression, cutting HBsAg and HBV RNA by 80 percent in Huh7 cells within 48 hours and by 80 to 95 percent in mice within five days, though attribution to KRAB was complicated by the absence of a TALE-only control.

The dCas9 platform, which replaces costly protein engineering with inexpensive single-guide RNAs, is now moving fastest toward patients. The only peer-reviewed dCas9 data for HBV come from a doctoral thesis by Rendón, who fused M.SssI to dCas9 and targeted the conserved CpG islands adjacent to the C and S gene promoters, achieving 2 to 15 percent increases in methylation that downregulated C and S gene transcription but faded within 48 hours. Nevertheless, two clinical trials of dCas9 epigenetic editing for hepatitis B are already underway — NCT06745973 and NCT06671093 — and parallel successes silencing HIV provirus with dCas9-KRAB, plus the demonstration that combined KRAB and DNA methyltransferase editors such as CRISPRoff produce durable, heritable silencing, provide strong proof of concept. Notably, dCas9 editing avoids the genotoxicity risk that shadows nuclease-based CRISPR-Cas9 approaches, whose guide RNAs can partially match human genomic sequences and whose double-strand breaks at the many, variable intDNA loci raise off-target mutagenesis concerns.

Industry has now taken up the challenge with three programs. Tune Therapeutics’ Tune-401, a liver-targeting lipid nanoparticle carrying a guide RNA to CGI2 and mRNA encoding a dCas9 fused to a methyltransferase and an undisclosed repressor, reported 99.99 percent repression of cccDNA-derived HBV RNA in primary human hepatocytes, strong repression of RNA and HBsAg from intDNA in Hep3B cells, epigenetic marks persisting through 275 rounds of cell division over 550 days, minimal off-target effects by RNA-seq, and a favorable safety profile in non-human primates — though these results have not yet been peer reviewed. A Phase 1b trial is recruiting in Hong Kong, Moldova and New Zealand. EpiGENIC’s Epi-003, a similar LNP platform, has entered a Phase 1 trial in China, and nChroma Bio’s CRMA-1001 has shown greater than 99 percent HBsAg reduction sustained for six months in preclinical models. A related Omega Therapeutics trial using LNP-delivered ZFP editors in liver cancer further validates liver-directed epigenetic delivery.

Significant hurdles remain before any of this translates into a functional cure. HBV genomes differ by more than 8 percent at the nucleotide level across genotypes, and the error-prone reverse transcriptase plus the chaotic nature of integration create sequence variability that could blunt editing efficacy, demanding bioinformatic surveillance and possibly personalized designs. Heterochromatin spreading from KRAB editors could silence neighboring host genes at unpredictable intDNA sites, while complete silencing of all antigen sources raises the paradox that eliminating HBsAg might allow exhausted immune cells to miss residual infection, arguing for integration with immune-reactivation strategies. Model limitations compound the problem: primary human hepatocytes rarely form intDNA, Hep3B lacks infection dynamics, and the field needs 3D organoid and humanized mouse systems that capture both reservoirs. Cost and equity loom as well, since most patients live in Southeast Asia and Sub-Saharan Africa while comparable gene therapies have been priced near two million dollars. Still, the authors conclude that with optimized effector combinations, LNP-based hit-and-run delivery — already shown to sustain liver gene silencing for over a year in mice and over 90 days in non-human primates in PCSK9 studies — and clinically relevant models, precision epigenetic editing could deliver what nucleos(t)ide analogs never could: a stable, reversible, non-genotoxic functional cure for chronic hepatitis B.

Subject of Research: Epigenetic editing strategies to silence hepatitis B virus cccDNA and integrated DNA for a functional cure of chronic hepatitis B

Article Title: The promise of epigenetic editing strategies in functionally curing chronic hepatitis B virus infections

Article References: Fonseca, S. G., Cortés-Mancera, F. M., Rots, M. G., Sarno, F., & van der Laan, M. (2026). The promise of epigenetic editing strategies in functionally curing chronic hepatitis B virus infections. Epigenetics Communications, 6(1), Article 4. https://doi.org/10.1186/s43682-025-00041-3

Image Credits: AI Generated

DOI: 10.1186/s43682-025-00041-3

Keywords: epigenetic editing, chronic hepatitis B, hepatitis B virus, cccDNA, integrated HBV DNA, dCas9, zinc-finger proteins, TALEs, KRAB, epidrugs, lipid nanoparticles, functional cure

The study began with a practical problem: researchers have lacked good tools to study the behavior of hepatitis B virus-specific T cells in a physiologically relevant setting. The team used a humanized mouse model carrying the human HLA-A11 molecule to identify a T cell receptor that recognizes HBc141-151, an epitope from the hepatitis B core antigen that is clinically relevant in human patients. This receptor, which the authors call HB-I, allowed them to generate transgenic mice whose entire CD8+ T cell population is specific for a single, human-relevant HBV epitope, providing a tractable platform for dissecting why these cells fail during chronic infection.

With that platform in hand, the researchers turned to an unbiased discovery strategy. Using a whole-genome CRISPR-Cas9 knockout library delivered into HBV-specific T cells, followed by screening under chronic antigen stimulation, they asked which genes, when deleted, would help T cells resist the dysfunctional state that normally develops. The screen converged on Ankrd11, a gene previously known as a chromatin regulator implicated in neural development and in Kabuki-like syndromes, but never before linked to T cell exhaustion. Loss of Ankrd11 consistently produced T cells that proliferated more vigorously and retained stronger effector characteristics.

The mechanistic picture that emerges from the paper is epigenetic. ANKRD11 appears to restrain the accessibility and acetylation of key effector genes. Using ATAC-seq, bulk RNA-seq and CUT&Tag profiling of the histone mark H3K27ac, the team showed that Ankrd11-deficient CD8+ T cells display increased chromatin openness and enhancer acetylation at loci encoding AP-1 family transcription factors, notably Fos and Fosb. Elevated AP-1 activity, in turn, drives a program of effector differentiation: the cells produce more granzyme B and interferon-gamma, resist the immunosuppressive conditions that normally silence them, and maintain function even when interleukin-2, a survival factor, is limiting.

Functionally, the consequences of losing this brake were striking. In mice carrying a replicating HBV plasmid or infected with recombinant HBV vectors, Ankrd11-deficient HBV-specific T cells expanded more robustly, infiltrated the liver more effectively, and drastically reduced serum levels of hepatitis B surface antigen, viral DNA and markers of liver damage. Parallel experiments using a second chronic infection model, lymphocytic choriomeningitis virus clone 13, confirmed the generality of the effect: adoptively transferred Ankrd11-deficient virus-specific P14 T cells showed enhanced granzyme expression, proliferation and viral control in the spleen and blood.

Perhaps the most conceptually interesting finding concerns the differentiation paths that exhausted T cells normally follow. In chronic infection and cancer, antigen-specific CD8+ T cells split into progenitor exhausted T cells, which express the transcription factor TCF-1 and serve as a self-renewing reservoir, and terminally exhausted T cells, which lose TCF-1 and are irreversibly dysfunctional. The authors found that Ankrd11 deficiency accelerates the conversion of progenitor exhausted cells into effector-like exhausted cells, but importantly, the resulting cells are cytotoxic and functional rather than inert. This suggests ANKRD11 does not simply maintain progenitor pools but actively gates how far exhausted cells differentiate toward effector competence.

The study also revealed a distinct route of reprogramming specific to chronic hepatitis B. A large fraction of HBV-specific CD8+ T cells in tolerant mice carries a PD-1-negative, TOX-negative phenotype that has resisted conventional checkpoint-based rescue strategies. When ANKRD11-mediated repression was relieved, these tolerant cells differentiated into PD-1-positive, KLRG1-positive effector cells armed with high granzyme levels, providing an explanation for the improved viral clearance. In other words, ANKRD11 deficiency unlocks an otherwise dormant pool of virus-specific cells that immune checkpoint blockade alone cannot reach.

The antitumor implications were tested directly in cancer models. Ankrd11-deficient CD8+ T cells promoted rejection of implanted tumors, enhanced intratumoral T cell activity, and acted synergistically with immune checkpoint blockade, potentiating the effect of PD-1 pathway inhibition. Given that the exhaustion of tumor-infiltrating lymphocytes is a central cause of immunotherapy failure, a single gene whose deletion reprograms effector differentiation under immunosuppressive conditions is an attractive candidate for therapeutic engineering, for example in chimeric antigen receptor or T cell receptor-engineered cell products. A provisional patent application has been filed based on the findings, underscoring the translational interest.

Caveats remain before ANKRD11 targeting reaches the clinic. ANKRD11 is a broad chromatin regulator with established roles in neural and cardiac development, so systemic inhibition is unlikely to be safe; the therapeutic window will probably lie in ex vivo engineering of T cells or in carefully targeted delivery. It will also be important to determine whether accelerated terminal differentiation comes at the cost of long-term memory formation. Nevertheless, the study delivers a clear conceptual advance: T cell dysfunction in chronic infection and cancer is not merely the product of inhibitory receptor signaling, but is epigenetically authored, and specific chromatin factors such as ANKRD11 can be removed to rewrite that script. For the hundreds of millions of people living with chronic hepatitis B, and for cancer patients whose T cells have gone quiet, that insight opens a new direction for immunotherapy design.

The concept of T cell exhaustion has shaped immunology for two decades. Since the landmark demonstration that chronically stimulated CD8+ T cells can regain function when inhibitory pathways are interrupted, researchers have catalogued a molecular signature of the dysfunctional state, and later work established the transcription factor TOX as a master architect of the exhaustion program, acting at both transcriptional and epigenetic levels. What has remained less clear is whether exhaustion is a fixed fate or a tunable state whose chromatin underpinnings can be deliberately rewritten. The new study adds weight to the second view by identifying a specific chromatin-associated protein whose removal shifts the balance of differentiation toward cytotoxic competence.

The methodological route to this finding is worth noting. Genome-wide CRISPR-Cas9 knockout screens have become a powerful way to uncover genes that constrain or enable T cell behavior, and previous efforts using this approach identified regulators such as REGNASE-1 and Roquin as suppressors of effector expansion and antitumor immunity. The present screen extends this logic into the setting of chronic hepatitis B virus infection, a context in which suppressive cues differ from those in tumors and in which conventional checkpoint blockade has shown only modest clinical benefit. That an unbiased screen converged on a gene with no prior immunological pedigree illustrates how phenotype-first discovery can bypass assumptions rooted in known pathways.

ANKRD11 itself carries an interesting dual history. It was first characterized as a chromatin regulator essential for neural development, and subsequent work showed it controls cardiac neural crest-mediated remodeling of the outflow tract. Its association with a Kabuki-like syndrome in humans reflects the pleiotropic consequences of disturbing a factor that operates broadly across tissues. This background both explains why the protein had escaped attention in immunology and reinforces the caution that any therapeutic interference must be confined to engineered cells rather than delivered systemically.

The AP-1 connection provides a mechanistic bridge to earlier T cell biology. AP-1 family transcription factors, built from Fos and Jun proteins, have long been recognized as immediate-early responders to T cell receptor signaling, and their activity is known to cooperate with other factors to specify effector genes. By showing that ANKRD11 restrains chromatin accessibility and H3K27 acetylation at AP-1 loci, the study suggests a route by which a chromatin regulator can gate a transcriptional program that is otherwise primed and waiting in naive and exhausted cells alike.

The authors have deposited the datasets underlying these conclusions in public repositories: bulk RNA-seq data under accession GSE299520, ATAC-seq data under GSE299519, and CUT&Tag data under GSE299521, with source data provided alongside the paper. No original code was generated. This transparency should allow groups working on T cell engineering to interrogate the chromatin changes directly and to test whether similar ANKRD11-sensitive programs operate in human tumor-infiltrating lymphocytes, a necessary step before the findings can inform next-generation cell therapy design.

Subject of Research: Epigenetic regulation of CD8+ T cell dysfunction in chronic viral infection and cancer

Article Title: ANKRD11 deficiency reprograms CD8+ T cell differentiation to enhance immunity in chronic infection and cancer

Article References: Xu, W., Guo, J., Cao, X., Li, L., Xiao, P., Zhang, X., Jin, Q., Zhang, F., Hou, B., Li, M., & Zhou, X. (2026). ANKRD11 deficiency reprograms CD8+ T cell differentiation to enhance immunity in chronic infection and cancer. Nature Immunology. https://doi.org/10.1038/s41590-026-02652-x

Image Credits: AI Generated

DOI: 10.1038/s41590-026-02652-x

Keywords: ANKRD11, CD8+ T cells, T cell exhaustion, chronic hepatitis B, epigenetics, immunotherapy, CRISPR screen, AP-1 transcription factors, granzyme B, immune checkpoint blockade, tumor immunity, T cell receptor