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HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection. HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed. We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid. Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection. HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles. [Display omitted] • The infectivity of splicing-deficient HBV was significantly decreased compared to WT HBV in hu-FRG mice. • SP1 RNA encodes HBcSP1, which is found in 10 to 50% of nucleocapsids in patients with CHB and cell line-derived WT HBV. • The splicing-deficient mutant A487C, lacking HBcSP1, reduces the efficiency of viral entry kinetics. • Disulfide bonds between Cys48 and Cys183 in the A487C capsid led to a locked conformation that disfavored viral entry. [ABSTRACT FROM AUTHOR]

Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-β1 signaling. The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice. We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-β1 (TGF-β1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-β1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-β1-induced fibrogenesis. Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-β1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection. HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-β1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection. [Display omitted] • HIV and HIV gp120 increase HBV-induced profibrogenic gene expression in hepatocytes and hepatic stellate cells. • HIV coinfection increases HBV-induced liver fibrogenesis through upregulation of the HIF-1α and TGF-β1 pathway. • The HIF-1α inhibitor (acriflavine) can block HIV-, HBV- and TGF-β1-induced liver fibrogenesis. • HIF-1α represents a novel target for antifibrotic therapy in patients with HIV/HBV coinfection. [ABSTRACT FROM AUTHOR]

Mechanisms behind the impaired response of antigen-specific B cells to therapeutic vaccination in chronic hepatitis B virus (HBV) infection remain unclear. The development of vaccines or strategies to overcome this obstacle is vital for advancing the management of chronic hepatitis B. A mouse model, denominated as E6F6-B, was engineered to feature a knock-in of a B-cell receptor (BCR) that specifically recognizes HBsAg. This model served as a valuable tool for investigating the temporal and spatial dynamics of humoral responses following therapeutic vaccination under continuous antigen exposure. Using a suite of immunological techniques, we elucidated the differentiation trajectory of HBsAg-specific B cells post-therapeutic vaccination in HBV carrier mice. Utilizing the E6F6-B transfer model, we observed a marked decline in antibody-secreting cells 2 weeks after vaccination. A dysfunctional and atypical pre-plasma cell population (BLIMP-1+ IRF4+ CD40- CD138- BCMA-) emerged, manifested by sustained BCR signaling. By deploying an antibody to purge persistent HBsAg, we effectively prompted the therapeutic vaccine to provoke conventional plasma cell differentiation. This resulted in an enhanced anti-HBs antibody response and facilitated HBsAg clearance. Sustained high levels of HBsAg limit the ability of therapeutic hepatitis B vaccines to induce the canonical plasma cell differentiation necessary for anti-HBs antibody production. Employing a strategy combining antibodies with vaccines can surmount this altered humoral response associated with atypical pre-plasma cells, leading to improved therapeutic efficacy in HBV carrier mice. Therapeutic vaccines aimed at combatting HBV encounter suboptimal humoral responses in clinical settings, and the mechanisms impeding their effectiveness have remained obscure. Our research, utilizing the innovative E6F6-B mouse transfer model, reveals that the persistence of HBsAg can lead to the emergence of an atypical pre-plasma cell population, which proves to be relevant to the potency of therapeutic HBV vaccines. Targeting the aberrant differentiation process of these atypical pre-plasma cells stands out as a critical strategy to amplify the humoral response elicited by HBV therapeutic vaccines in carrier mouse models. This discovery suggests a compelling avenue for further study in the context of human chronic hepatitis B. Encouragingly, our findings indicate that synergistic therapy combining HBV-specific antibodies with vaccines offers a promising approach that could significantly advance the pursuit of a functional cure for HBV. [Display omitted] • The HBsAg-specific BCR knock-in mouse was constructed to study the characteristics of B cells in chronic HBV infection. • An atypical pre-plasma cell population was identified post therapeutic vaccination in chronic HBV carrier mice. • Atypical pre-plasma cells display enhanced BCR signaling, ER stress and apoptosis-related gene expression. • Combination therapy (antibody + vaccine) partially overcame abnormal B-cell differentiation and led to superior HBsAg suppression. [ABSTRACT FROM AUTHOR]

The use of tenofovir disoproxil fumarate (TDF) is associated with a reduction in bone mineral density and an increase in bone metabolism biomarkers. However, data on clinical bone fractures remain limited. We evaluated the impact of TDF compared to entecavir on the risk of fracture in elderly patients with chronic hepatitis B (CHB). Patients with CHB aged ≥60 years receiving entecavir or TDF between January 2008 and December 2022 were identified using a territory-wide database in Hong Kong. The risk of incident fracture in entecavir- and TDF-treated patients before and after month 24 were compared after propensity score matching. A total of 41,531 patients with CHB (mean age 69.8±7.8 years, 61.6% male) receiving entecavir (n = 39,897 [96.1%]) and TDF (n = 1,634 [3.9%]) were analysed. At a median follow-up of 25.3 (9.1–58.5) months, 1,733 (4.2%) patients developed incident fracture. Patients with incident fracture were more likely to have diabetes, hypertension, congestive heart failure, rheumatoid arthritis, osteoporosis, and a history of fracture. Compared with propensity score-matched entecavir-treated patients, the risk of incident fracture in TDF-treated patients was comparable in the first 24 months (weighted subdistribution hazard ratio [sHR] 0.99, 95% CI 0.56–1.73, p = 0.960) but increased after month 24 (weighted sHR 1.80, 95% CI 1.11–2.93, p = 0.019). The 24-, 60-, and 96-month cumulative incidences (95% CI) of fracture in TDF-treated and entecavir-treated patients were 2.3% (1.6%–3.4%) vs. 2.6% (1.9%–3.5%), 6.4% (5.0%–8.2%) vs. 4.7% (3.8%–6.0%), and 10.2% (8.3%–12.6%) vs. 6.8% (5.4%–8.5%), respectively. The risk of fracture increased with TDF treatment for ≥24 months in elderly patients with CHB. Selection of nucleos(t)ide analogues should be individualised based on age and comorbidities. Previous literature suggested that the use of tenofovir disoproxil fumarate (TDF) is associated with a decrease in bone mineral density. However, data on the impact of TDF on long-term incident clinical fracture remains scarce. In this real-world territory-wide study of 41,531 treated patients with chronic hepatitis B in Hong Kong, patients who received TDF were at a higher risk of fracture after 2 years of treatment than those who received entecavir. Given the ageing population of patients with chronic hepatitis B and the rising prevalence of comorbidities, our findings support the current treatment guidelines that recommend selecting antiviral treatment based on age and comorbidities. [Display omitted] • Long-term fracture risk associated with tenofovir disoproxil fumarate treatment is unclear. • Tenofovir disoproxil fumarate was associated with a higher fracture risk after 2 years in patients aged ≥60. • Results remained robust on propensity score analysis in patients aged ≥65 and ≥70 years. • Selection of antiviral therapy should be customised based on age and comorbidities. [ABSTRACT FROM AUTHOR]

Sub-Saharan African (SSA) ethnicity has been associated with a higher risk of hepatocellular carcinoma (HCC) among individuals with chronic hepatitis B in cross-sectional studies. However, the incidence of HCC and performance of HCC risk scores in this population are unknown. We conducted an international multicenter retrospective cohort study of all consecutive HBV-monoinfected individuals of SSA or Afro-Surinamese (AS) ethnicity managed at sites in the Netherlands, the United Kingdom and Spain. We assessed the 5- and 10-year cumulative incidences of HCC in the overall study population, among different clinically relevant subgroups and across (m)PAGE-B subgroups. Next, we explored the different risk factors for HCC. During a median follow-up of 8 years, we analyzed 1,473 individuals of whom 34 developed HCC. The 5- and 10-year cumulative incidences of HCC were 1% and 2.4%. The 10-year cumulative incidence of HCC was 0.7% among individuals without advanced fibrosis at baseline, compared to 12.1% among individuals with advanced fibrosis (p <0.001). Higher age (adjusted hazard ratio [aHR] 1.05), lower platelet count (aHR 0.98), lower albumin level (aHR 0.90) and higher HBV DNA log10 (aHR 1.21) were significantly associated with HCC development. The 10-year cumulative incidence of HCC was 0.5% among individuals with a low PAGE-B score, compared to 2.9% in the intermediate- and 15.9% in the high-risk groups (p <0.001). In this unique international multicenter cohort of SSA and AS individuals with chronic hepatitis B, we observed 5- and 10-year cumulative HCC risks of 1% and 2.4%, respectively. The risk of HCC was negligible for individuals without advanced fibrosis at baseline, and among individuals with low baseline (m)PAGE-B scores. These findings can be used to guide HCC surveillance strategies. Sub-Saharan African ethnicity has been associated with a higher risk of hepatocellular carcinoma among individuals with chronic hepatitis B. In this international multicenter cohort study of sub-Saharan African and Afro-Surinamese individuals living with chronic hepatitis B in Europe, we observed 5- and 10-year cumulative incidences of hepatocellular carcinoma of 1% and 2.4%, respectively. The risk was negligible among individuals without advanced fibrosis and a low baseline (m)PAGE-B score. These findings can be used to guide HCC surveillance strategies in this population. [Display omitted] • We found a 5- and 10-year cumulative HCC incidence of 1% and 2.4%. • Older age, higher HBV DNA levels, lower platelet count, and lower albumin levels increased the risk of HCC. • The 10-year HCC risk was negligible for individuals without advanced fibrosis at baseline. • The 10-year HCC risk was negligible for individuals with low baseline (m)PAGE-B scores. • These findings suggest opportunities for individualized HCC surveillance strategies. [ABSTRACT FROM AUTHOR]

HBsAg loss is only observed in a small proportion of patients with chronic hepatitis B (CHB) who undergo interferon treatment. Investigating the host factors crucial for functional cure of CHB can aid in identifying individuals who would benefit from peginterferon-α (Peg-IFNα) therapy. We conducted a genome-wide association study (GWAS) by enrolling 48 patients with CHB who achieved HBsAg loss after Peg-IFNα treatment and 47 patients who didn't. In the validation stage, we included 224 patients, of whom 90 had achieved HBsAg loss, to validate the identified significant single nucleotide polymorphisms. To verify the functional involvement of the candidate genes identified, we performed a series of in vitro and in vivo experiments. GWAS results indicated a significant association between the rs7519753 C allele and serum HBsAg loss in patients with CHB after Peg-IFNα treatment (p = 4.85 × 10-8, odds ratio = 14.47). This association was also observed in two independent validation cohorts. Expression quantitative trait locus analysis revealed higher hepatic TP53BP2 expression in individuals carrying the rs7519753 C allele (p = 2.90 × 10-6). RNA-sequencing of liver biopsies from patients with CHB after Peg-IFNα treatment revealed that hepatic TP53BP2 levels were significantly higher in the HBsAg loss group compared to the HBsAg persistence group (p = 0.035). In vitro and in vivo experiments demonstrated that loss of TP53BP2 decreased interferon-stimulated gene levels and the anti-HBV effect of IFN-α. Mechanistically, TP53BP2 was found to downregulate SOCS2, thereby facilitating JAK/STAT signaling. The rs7519753 C allele is associated with elevated hepatic TP53BP2 expression and an increased probability of serum HBsAg loss post-Peg-IFNα treatment in patients with CHB. TP53BP2 enhances the response of the hepatocyte to IFN-α by suppressing SOCS2 expression. Chronic hepatitis B (CHB) remains a global public health issue. Although current antiviral therapies are more effective in halting disease progression, only a few patients achieve functional cure for hepatitis B with HBsAg loss, highlighting the urgent need for a cure for CHB. This study revealed that the rs7519753 C allele, which is associated with high expression of hepatic TP53BP2, significantly increases the likelihood of serum HBsAg loss in patients with CHB undergoing Peg-IFNα treatment. This finding not only provides a promising predictor for HBsAg loss but identifies a potential therapeutic target for Peg-IFNα treatment. We believe our results are of great interest to a wide range of stakeholders based on their potential clinical implications. [Display omitted] • GWAS indicated rs7519753 is associated with HBsAg loss after Peg-IFNα treatment. • Rs7519753 C allele was strongly associated with higher hepatic TP53BP2 expression. • TP53BP2 promoted the response of hepatocytes to IFN-α in vitro and in vivo. • Loss of TP53BP2 decreased the anti-HBV effect of IFN-α in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Immunotherapy for chronic hepatitis B virus (HBV) infection has not yet demonstrated sufficient efficacy. We developed a non-integrative lentiviral-vectored therapeutic vaccine for chronic hepatitis B and tested its antiviral effects in HBV-persistent mice and two inactive HBsAg carriers. Lentiviral vectors (LVs) encoding the core, preS1, or large HBsAg (LHBs) proteins of HBV were evaluated for immunogenicity in HBV-naïve mice and therapeutic efficacy in a murine model of chronic HBV infection. In addition, two inactive HBsAg carriers each received two doses of 5×107 transduction units (TU) or 1×108 TU of lentiviral-vectored LHBs (LV-LHBs), respectively. The endpoints were safety, LHBs-specific T-cell responses, and serum HBsAg levels during a 24-week follow-up. In the mouse models, LV-LHBs was the most promising in eliciting robust antigen-specific T cells and in reducing the levels of serum HBsAg and viral load. By the end of the 34-week observation period, six out of ten (60%) HBV-persistent mice vaccinated with LV-LHBs achieved serum HBsAg loss and significant depletion of HBV-positive hepatocytes in the liver. In the two inactive HBsAg carriers, vaccination with LV-LHBs induced a considerable increase in the number of peripheral LHBs-specific T cells in one patient, and a weak but detectable response in the other, accompanied by a sustained reduction of HBsAg (-0.31 log 10 IU/ml and -0.46 log 10 IU/ml, respectively) from baseline to nadir. A lentiviral-vectored therapeutic vaccine for chronic HBV infection demonstrated the potential to improve HBV-specific T-cell responses and deplete HBV-positive hepatocytes, leading to a sustained loss or reduction of serum HBsAg. Chronic HBV infection is characterized by an extremely low number and profound hypo-responsiveness of HBV-specific T cells. Therapeutic vaccines are designed to improve HBV-specific T-cell responses. We show that immunization with a lentiviral-vectored therapeutic HBV vaccine was able to expand HBV-specific T cells in vivo , leading to reductions of HBV-positive hepatocytes and serum HBsAg. [Display omitted] • A lentiviral-vectored therapeutic vaccine was able to improve HBV-specific T cell responses in mice with chronic HBV infection. • Lentiviral vector expressing the large HBs protein (LV-LHBs) was the most effective for the resolution of chronic infection. • Vaccination with LV-LHBs induced intrahepatic lymphoid aggregates but not bystander T-cell activation. • LV-LHBs was well tolerated and led to a sustained reduction of serum HBsAg in two inactive HBsAg carriers. [ABSTRACT FROM AUTHOR]

Recent studies reported that moderate HBV DNA levels are significantly associated with hepatocellular carcinoma (HCC) risk in hepatitis B e antigen (HBeAg)-positive, non-cirrhotic patients with chronic hepatitis B (CHB). We aimed to develop and validate a new risk score to predict HCC development using baseline moderate HBV DNA levels in patients entering into HBeAg-positive CHB from chronic infection. This multicenter cohort study recruited 3,585 HBeAg-positive, non-cirrhotic patients who started antiviral treatment with entecavir or tenofovir disoproxil fumarate at phase change into CHB from chronic infection in 23 tertiary university-affiliated hospitals of South Korea (2012–2020). A new HCC risk score (PAGED-B) was developed (training cohort, n = 2,367) based on multivariable Cox models. Internal validation using bootstrap sampling and external validation (validation cohort, n = 1,218) were performed. Sixty (1.7%) patients developed HCC (median follow-up, 5.4 years). In the training cohort, age, gender, platelets, diabetes and moderate HBV DNA levels (5.00–7.99 log 10 IU/ml) were independently associated with HCC development; the PAGED-B score (based on these five predictors) showed a time-dependent AUROC of 0.81 for the prediction of HCC development at 5 years. In the validation cohort, the AUROC of PAGED-B was 0.85, significantly higher than for other risk scores (PAGE-B, mPAGE-B, CAMD, and REAL-B). When stratified by the PAGED-B score, the HCC risk was significantly higher in high-risk patients than in low-risk patients (sub-distribution hazard ratio = 8.43 in the training and 11.59 in the validation cohorts, all p <0.001). The newly established PAGED-B score may enable risk stratification for HCC at the time of transition into HBeAg-positive CHB. In this study, we developed and validated a new risk score to predict hepatocellular carcinoma (HCC) development in patients entering into hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) from chronic infection. The newly established PAGED-B score, which included baseline moderate HBV DNA levels (5–8 log 10 IU/ml), improved on the predictive performance of prior risk scores. Based on a patient's age, gender, diabetic status, platelet count, and moderate DNA levels (5–8 log 10 IU/ml) at the phase change into CHB from chronic infection, the PAGED-B score represents a reliable and easily available risk score to predict HCC development during the first 5 years of antiviral treatment in HBeAg-positive patients entering into CHB. With a scoring range from 0 to 12 points, the PAGED-B score significantly differentiated the 5-year HCC risk: low <7 points and high ≥7 points. [Display omitted] • A new HCC risk prediction score was developed in patients entering into HBeAg-positive CHB from chronic infection. • PAGED-B score incorporated moderate HBV DNA levels and diabetes status into the original PAGE-B score. • PAGED-B score predicted the 5-year risk of HCC with high accuracy. [ABSTRACT FROM AUTHOR]

RG6346 is an N -acetyl-D-galactosamine (GalNAc)-conjugated, double-stranded RNA interference agent targeting the HBV genome S-region. We investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of RG6346 in healthy volunteers and patients with chronic HBV infection (CHB). This first-in-human, adaptive, randomized, double-blinded, phase I study recruited three groups of participants: Group A, 30 healthy volunteers received single-dose RG6346 at 0.1, 1.5, 3.0, 6.0, or 12.0 mg/kg, or placebo; Group B, nucleos(t)ide analogue-naïve participants with CHB received single-dose RG6346 at 3.0 mg/kg (n = 6) or placebo (n = 3); Group C, participants with nucleos(t)ide-suppressed CHB received four doses (every 28 days) of RG6346 at 1.5, 3.0, or 6.0 mg/kg (n = 4 in each cohort) or placebo (n = 6). RG6346 treatment for up to 4 months was safe and well tolerated. The most common adverse event was a mild injection site reaction. Several nucleos(t)ide-naïve participants exhibited self-resolving transaminase elevations with preserved liver function. By the end of RG6346 treatment in Group C (Day 112), the mean reduction from baseline in hepatitis B surface antigen (HBsAg) was 1.39, 1.80, and 1.64 log 10 IU/ml in the 1.5, 3.0, and 6.0 mg/kg cohorts, respectively. Of the 12 participants in Group C, 11 (91.7%) achieved a ≥1 log 10 IU/ml reduction in HBsAg (3 of 11 [27.3%] had the response sustained at conditional follow-up Day 448). No dose-response relationship was apparent between RG6346 and serum HBsAg levels. The RG6346-induced HBsAg response was independent of hepatitis B e antigen status. Moderate-to-marked sustained reductions of hepatitis B core-related antigen, HBV RNA, HBV DNA (in nucleos[t]ide analogue-naïve participants), and hepatitis B e antigen levels were observed. These favorable safety and pharmacodynamic data support the clinical development of RG6346 as the backbone of a finite antiviral treatment regimen, with the goal of sustained HBsAg loss (functional cure) in patients with CHB. ClinicalTrials.gov NCT03772249. Currently available therapies for chronic HBV infection are associated with low rates of functional cure and new, more efficacious treatments are needed. This first-in-human study of RG6346, an RNA interference therapy, showed a favorable safety profile as well as marked and durable reductions in hepatitis B surface antigen levels. These results support the continued development of RG6346 as the backbone of a finite treatment regimen targeting high functional cure rates and are important for HBV researchers and physicians. [Display omitted] • Chronic hepatitis B virus infection remains a major public health concern. • Long-term detection of HBsAg defines the persistence of CHB. • CHB treatment with RG6346 every 4 weeks for ≤4 months was safe and well tolerated. • RG6346 induced significant and durable reductions in HBsAg in patients with CHB. • These data support the clinical development of RG6346 for CHB treatment. [ABSTRACT FROM AUTHOR]

Acute-on-chronic liver failure (ACLF) is a clinical syndrome associated with high short-term mortality in patients with chronic liver disease. Chronic hepatitis B is the main cause of ACLF (HBV-ACLF) in China and other Asian countries. To improve disease management and survival for patients with ACLF, we aimed to discover novel biomarkers to enhance HBV-ACLF diagnosis and prognostication. We performed a metabolomics profiling of 1,024 plasma samples collected from patients with HBV-related chronic liver disease with acute exacerbation at hospital admission in a multi-year and multi-center prospective study (367 ACLF and 657 non-ACLF). The samples were randomly separated into equal halves as a discovery set and a validation set. We identified metabolites associated with 90-day mortality in the ACLF group and the progression to ACLF within 28 days in the non-ACLF group (pre-ACLF) using statistical analysis and machine learning. We developed diagnostic algorithms in the discovery set and used these to assess the findings in the validation set. ACLF significantly altered the plasma metabolome, particularly in membrane lipid metabolism, steroid hormones, oxidative stress pathways, and energy metabolism. Numerous metabolites were significantly associated with 90-day mortality in the ACLF group and/or pre-ACLF in the non-ACLF group. We developed algorithms for the prediction of 90-day mortality in patients with ACLF (area under the curve 0.87 and 0.83 for the discovery set and validation set, respectively) and the diagnosis of pre-ACLF (area under the curve 0.94 and 0.88 for the discovery set and validation set, respectively). To translate our discoveries into practical clinical tests, we developed targeted assays using liquid chromatography-mass spectrometry. Based on novel metabolite biomarkers, we established tests for HBV-related ACLF with higher accuracy than existing methods. NCT02457637 and NCT03641872. Acute-on-chronic liver failure (ACLF) is a clinical syndrome associated with high short-term mortality affecting 25% of patients hospitalized with cirrhosis. Chronic hepatitis B is the main etiology of ACLF in China and other Asian counties. There is currently no effective therapy. Early diagnosis and accurate prognostication are critical for improving clinical outcomes in patients with ACLF. Based on novel metabolite biomarkers, we developed liquid chromatography-mass spectrometry tests with improved accuracy for the early diagnosis and prognostication of HBV-related ACLF. The liquid chromatography-mass spectrometry tests can be implemented in clinical labs and used by physicians to triage patients with HBV-related ACLF to ensure optimized clinical management. [Display omitted] • Metabolomics uncovered profound metabolic perturbations associated with HBV-related ACLF in patient plasma. • We identified novel metabolite biomarkers that can be used to enhance ACLF prognostication and early diagnosis. • We developed and validated LCMS-based clinical tests with improved accuracy for ACLF prognostication and early diagnosis. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B is a global public health problem, and coinfection with hepatitis delta virus (HDV) worsens disease outcome. Here, we describe a hepatitis B virus (HBV) surface antigen (HBsAg)-targeting monoclonal antibody (mAb) with the potential to treat chronic hepatitis B and chronic hepatitis D. HBsAg-specific mAbs were isolated from memory B cells of HBV vaccinated individuals. In vitro neutralization was determined against HBV and HDV enveloped with HBsAg representing eight HBV genotypes. Human liver-chimeric mice were treated twice weekly with a candidate mAb starting 3 weeks post HBV inoculation (spreading phase) or during stable HBV or HBV/HDV coinfection (chronic phase). From a panel of human anti-HBs mAbs, VIR-3434 was selected and engineered for pre-clinical development. VIR-3434 targets a conserved, conformational epitope within the antigenic loop of HBsAg and neutralized HBV and HDV infection with higher potency than hepatitis B immunoglobulins in vitro. Neutralization was pan-genotypic against strains representative of HBV genotypes A-H. In the spreading phase of HBV infection in human liver-chimeric mice, a parental mAb of VIR-3434 (HBC34) prevented HBV dissemination and the increase in intrahepatic HBV RNA and covalently closed circular DNA. In the chronic phase of HBV infection or co-infection with HDV, HBC34 treatment decreased circulating HBsAg by >1 log and HDV RNA by >2 logs. The potently neutralizing anti-HBs mAb VIR-3434 reduces circulating HBsAg and HBV/HDV viremia in human liver-chimeric mice. VIR-3434 is currently in clinical development for treatment of patients with chronic hepatitis B or D. Chronic infection with hepatitis B virus and co-infection with hepatitis D virus place approximately 290 million individuals worldwide at risk of severe liver disease and cancer. Available treatments result in low rates of functional cure or require lifelong therapy that does not eliminate the risk of liver disease. We isolated and characterized a potent human antibody that neutralizes hepatitis B and D viruses and reduces infection in a mouse model. This antibody could provide a new treatment for patients with chronic hepatitis B and D. [Display omitted] • Identification of a human mAb VIR-3434 that potently neutralizes HBV and HDV. • VIR-3434 targets a conserved, conformational epitope of the HBsAg antigenic loop. • VIR-3434 treatment blocks intrahepatic HBV spread in human liver-chimeric mice. • VIR-3434 treatment reduces circulating HBsAg and HDV RNA in co-infected mice. • Data have enabled clinical development of VIR-3434 against chronic hepatitis B/D. [ABSTRACT FROM AUTHOR]

Representatives from academia, industry, regulatory agencies, and patient advocacy groups convened under the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) in June 2022 with the primary goal of achieving consensus on chronic HBV and HDV treatment endpoints to guide clinical trials aiming to "cure" HBV and HDV. Conference participants reached an agreement on some key points. The preferred primary endpoint for phase II/III trials evaluating finite treatments for chronic hepatitis B (CHB) is a "functional" cure, defined as sustained HBsAg loss and HBV DNA less than the lower limit of quantitation (LLOQ) 24 weeks off-treatment. An alternate endpoint would be "partial cure" defined as sustained HBsAg level <100 IU/mL and HBV DNA

Current hepatocellular carcinoma (HCC) risk scores do not reflect changes in HCC risk resulting from liver disease progression/regression over time. We aimed to develop and validate two novel prediction models using multivariate longitudinal data, with or without cell-free DNA (cfDNA) signatures. A total of 13,728 patients from two nationwide multicenter prospective observational cohorts, the majority of whom had chronic hepatitis B, were enrolled. aMAP score, as one of the most promising HCC prediction models, was evaluated for each patient. Low-pass whole-genome sequencing was used to derive multi-modal cfDNA fragmentomics features. A longitudinal discriminant analysis algorithm was used to model longitudinal profiles of patient biomarkers and estimate the risk of HCC development. We developed and externally validated two novel HCC prediction models with a greater accuracy, termed aMAP-2 and aMAP-2 Plus scores. The aMAP-2 score, calculated with longitudinal data on the aMAP score and alpha-fetoprotein values during an up to 8-year follow-up, performed superbly in the training and external validation cohorts (AUC 0.83–0.84). The aMAP-2 score showed further improvement and accurately divided aMAP-defined high-risk patients into two groups with 5-year cumulative HCC incidences of 23.4% and 4.1%, respectively (p = 0.0065). The aMAP-2 Plus score, which incorporates cfDNA signatures (nucleosome, fragment and motif scores), optimized the prediction of HCC development, especially for patients with cirrhosis (AUC 0.85–0.89). Importantly, the stepwise approach (aMAP -> aMAP-2 -> aMAP-2 Plus) stratified patients with cirrhosis into two groups, comprising 90% and 10% of the cohort, with an annual HCC incidence of 0.8% and 12.5%, respectively (p < 0.0001). aMAP-2 and aMAP-2 Plus scores are highly accurate in predicting HCC. The stepwise application of aMAP scores provides an improved enrichment strategy, identifying patients at a high risk of HCC, which could effectively guide individualized HCC surveillance. In this multicenter nationwide cohort study, we developed and externally validated two novel hepatocellular carcinoma (HCC) risk prediction models (called aMAP-2 and aMAP-2 Plus scores), using longitudinal discriminant analysis algorithm and longitudinal data (i.e. , aMAP and alpha-fetoprotein) with or without the addition of cell-free DNA signatures, based on 13,728 patients from 61 centers across mainland China. Our findings demonstrated that the performance of aMAP-2 and aMAP-2 Plus scores was markedly better than the original aMAP score, and any other existing HCC risk scores across all subsets, especially for patients with cirrhosis. More importantly, the stepwise application of aMAP scores (aMAP -> aMAP-2 -> aMAP-2 Plus) provides an improved enrichment strategy, identifying patients at high risk of HCC, which could effectively guide individualized HCC surveillance. [Display omitted] • We established two novel, high accuracy prediction models of HCC, called aMAP-2 and aMAP-2 Plus scores. • aMAP-2 and aMAP-2 Plus scores could overcome the limitations of the original aMAP score and the other existing HCC risk scores. • The stepwise application of aMAP scores provides an improved enrichment strategy, identifying patients at high risk of HCC. [ABSTRACT FROM AUTHOR]

Current therapy for chronic hepatitis B virus (cHBV) infection involves lifelong treatment. New treatments that enable HBV functional cure would represent a clinically meaningful advance. ALN-HBV and VIR-2218 are investigational RNA interference therapeutics that target all major HBV transcripts. We report on: i) the safety of single doses of VIR-2218 (modified from ALN-HBV by enhanced stabilization chemistry plus technology to reduce off-target, seed-mediated binding while maintaining on-target antiviral activity) and ALN-HBV in humanized mice; ii) a cross-study comparison of the safety of single doses of VIR-2218 and ALN-HBV in healthy human volunteers (n = 24 and n = 49, respectively); and iii) the antiviral activity of two doses of 20, 50, 100, 200 mg of VIR-2218 (total n = 24) vs. placebo (n = 8), given 4 weeks apart, in participants with cHBV infection. In humanized mice, alanine aminotransferase (ALT) levels were markedly lower following administration of VIR-2218 compared with ALN-HBV. In healthy volunteers, post-treatment ALT elevations occurred in 28% of participants receiving ALN-HBV compared with none in those receiving VIR-2218. In participants with cHBV infection, VIR-2218 was associated with dose-dependent reductions in hepatitis B surface antigen (HBsAg). The greatest mean reduction of HBsAg at Week 20 in participants receiving 200 mg was 1.65 log IU/ml. The HBsAg reduction was maintained at 0.87 log IU/ml at Week 48. No participants had serum HBsAg loss or hepatitis B surface antibody seroconversion. VIR-2218 demonstrated an encouraging hepatic safety profile in preclinical and clinical studies as well as dose-dependent HBsAg reductions in patients with cHBV infection. These data support future studies with VIR-2218 as part of combination regimens with a goal of HBV functional cure. ClinicalTrials.gov identifiers: NCT02826018 and NCT03672188. A significant unmet need exists for therapies for chronic HBV (cHBV) infection that achieve functional cure. We report clinical and non-clinical data on two investigational small-interfering RNAs that target HBx, ALN-HBV and VIR-2218, demonstrating that incorporation of enhanced stabilization chemistry plus technology in VIR-2218 reduces its propensity to cause ALT elevations relative to its parent compound, ALN-HBV. We also show that VIR-2218 reduces hepatitis B surface antigen levels in a dose-dependent manner in participants with cHBV infection. These studies support the continued development of VIR-2218 as part of therapeutic regimens for cHBV infection, with the goal of a functional cure, and are important for HBV researchers and physicians. [Display omitted] • VIR-2218 and ALN-HBV are siRNAs targeting all HBV mRNA transcripts. • VIR-2218 is a glycol nucleic acid–modified version of ALN-HBV. • ALT elevations were more pronounced with ALN-HBV than VIR-2218 in mice and humans. • VIR-2218 reduced HBsAg levels in participants with chronic HBV infection. • VIR-2218 had a positive hepatic safety profile in preclinical and clinical studies. [ABSTRACT FROM AUTHOR]

Nucleos(t)ide analogues (NUCs) are the standard and mostly lifelong treatment for chronic HBeAg-negative hepatitis B, as functional cure (loss of HBsAg) is rarely achieved. Discontinuation of NUC treatment may lead to functional cure; however, to date, the evidence for this has been based on small or non-randomized clinical trials. The STOP-NUC trial was designed with the aim of increasing the HBsAg loss rate using a NUC treatment interruption approach. In this multicenter, randomized-controlled trial, 166 HBeAg-negative patients with chronic hepatitis B on continuous long-term NUC treatment, with HBV DNA <172 IU/ml (1,000 copies/ml) for ≥4 years, were randomized to either stop (Arm A) or continue NUC treatment (Arm B) for a 96-week observation period. In total, 158 patients were available for final analysis, 79 per arm. The primary endpoint was sustained HBsAg loss up to week 96. Our study met its primary objective by demonstrating HBsAg loss in eight patients (10.1%, 95% CI 4.8%–19.5%) in Arm A and in no patient in Arm B (p = 0.006). Among patients with baseline HBsAg levels <1,000 IU/ml, seven (28%) achieved HBsAg loss. In Arm A, re-therapy was initiated in 11 (13.9%) patients, whereas 32 (40.5%) patients achieved sustained remission. A decrease of HBsAg >1 log IU/ml was observed in 16 patients (20.3%) in Arm A and in one patient (1.3%) in Arm B. No serious adverse events related to treatment cessation occurred. Cessation of NUC treatment was associated with a significantly higher rate of HBsAg loss than continued NUC treatment, which was largely restricted to patients with end of treatment HBsAg levels <1,000 IU/ml. As HBeAg-negative patients with chronic hepatitis B on nucleos(t)ide analogues (NUCs) rarely achieve functional cure, treatment is almost always lifelong. The STOP-NUC trial was conducted to investigate whether discontinuing long-term NUC treatment can increase the cure rate. We found that some patients achieved functional cure after stopping NUCs, which was especially pronounced in patients with HBsAg levels <1,000 at the end of NUC treatment, and that many did not need to resume therapy. The results of the Stop-NUC trial provide evidence for the concept of stopping NUC treatment as a therapeutic option that can induce functional cure. [Display omitted] • Stopping long-term NUC treatment can induce functional cure in HBeAg-negative patients. • Functional cure is associated with HBsAg levels <1,000 IU/ml at the time point of NUC treatment cessation. • All patients showed relapses in HBV DNA levels after NUC treatment discontinuation. But re-treatment with NUCs was only required in 14% of patients over a 96-week follow-up and no patient suffered hepatic decompensation. [ABSTRACT FROM AUTHOR]

Bulevirtide recently received conditional approval from the EMA for the treatment of chronic hepatitis delta, but the ideal duration of therapy is unknown. Herein, we describe the first case of hepatitis delta cure following 3 years of bulevirtide monotherapy in a patient with compensated cirrhosis and esophageal varices. During the 72-week off-bulevirtide follow-up, virological and biochemical responses were maintained. In the off-therapy liver biopsy, intrahepatic HDV RNA and hepatitis D antigen were undetectable, <1% of hepatocytes were hepatitis B surface antigen positive and all were negative for hepatitis B core antigen. Ishak grading and staging were improved following treatment. [ABSTRACT FROM AUTHOR]

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

We recently developed a heterologous therapeutic vaccination scheme (TherVacB) comprising a particulate protein prime followed by a modified vaccinia-virus Ankara (MVA)-vector boost for the treatment of HBV. However, the key determinants required to overcome HBV-specific immune tolerance remain unclear. Herein, we aimed to study new combination adjuvants and unravel factors that are essential for the antiviral efficacy of TherVacB. Recombinant hepatitis B surface and core antigen (HBsAg and HBcAg) particles were formulated with different liposome- or oil-in-water emulsion-based combination adjuvants containing saponin QS21 and monophosphoryl lipid A; these formulations were compared to STING-agonist c-di-AMP and conventional aluminium hydroxide formulations. Immunogenicity and the antiviral effects of protein antigen formulations and the MVA-vector boost within TherVacB were evaluated in adeno-associated virus-HBV-infected and HBV-transgenic mice. Combination adjuvant formulations preserved HBsAg and HBcAg integrity for ≥12 weeks, promoted human and mouse dendritic cell activation and, within TherVacB, elicited robust HBV-specific antibody and T-cell responses in wild-type and HBV-carrier mice. Combination adjuvants that prime a balanced HBV-specific type 1 and 2 T helper response induced high-titer anti-HBs antibodies, cytotoxic T-cell responses and long-term control of HBV. In the absence of an MVA-vector boost or following selective CD8 T-cell depletion, HBsAg still declined (mediated mainly by anti-HBs antibodies) but HBV replication was not controlled. Selective CD4 T-cell depletion during the priming phase of TherVacB resulted in a complete loss of vaccine-induced immune responses and its therapeutic antiviral effect in mice. Our results identify CD4 T-cell activation during the priming phase of TherVacB as a key determinant of HBV-specific antibody and CD8 T-cell responses. Therapeutic vaccination is a potentially curative treatment option for chronic hepatitis B. However, it remains unclear which factors are essential for breaking immune tolerance in HBV carriers and determining successful outcomes. Our study provides the first direct evidence that efficient priming of HBV-specific CD4 T cells determines the success of therapeutic hepatitis B vaccination in two preclinical HBV-carrier mouse models. Applying an optimal formulation of HBV antigens that activates CD4 and CD8 T cells during prime immunization provided the foundation for an antiviral effect of therapeutic vaccination, while depletion of CD4 T cells led to a complete loss of vaccine-induced antiviral efficacy. Boosting CD8 T cells was important to finally control HBV in these mouse models. Our findings provide important insights into the rational design of therapeutic vaccines for the cure of chronic hepatitis B. [Display omitted] • Rational design of therapeutic hepatitis B vaccines is key to improving their antiviral effect. • A heterologous prime-boost vaccine with particulate antigen, an optimized adjuvant and a vector-boost is most promising. • Activation of CD4 T cells during prime vaccination is the key to break immune tolerance and control HBV. • HBV antigens formulated with potent adjuvants activating Th1 type CD4 T cells promote antibody and CD8 T-cell responses. • Antiviral efficacy is lost when CD4 or CD8 T cells are depleted during prime vaccination. [ABSTRACT FROM AUTHOR]

The comparative risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB) receiving tenofovir disoproxil fumarate (TDF) vs. entecavir (ETV) remains controversial. In this individual patient data (IPD) meta-analysis, we aimed to compare HCC risk between the two drugs and identify subgroups who may benefit more from one treatment than the other. Published meta-analyses, electronic databases and congress proceedings were searched to identify eligible studies through January 2021. We compared HCC risk between the two drugs using a multivariable Cox proportional hazards model with anonymised IPD from treatment-naïve patients with CHB receiving TDF or ETV for ≥1 year. Treatment effect consistency was explored in propensity score matching (PSM), weighting (PSW) and subgroup analyses for age, sex, hepatitis B e-antigen (HBeAg) positivity, cirrhosis and diabetes status. We included 11 studies from Korea, Taiwan and Hong Kong involving 42,939 patients receiving TDF (n = 6,979) or ETV (n = 35,960) monotherapy. Patients receiving TDF had significantly lower HCC risk (adjusted hazard ratio [HR] 0.77; 95% CI 0.61–0.98; p = 0.03). Lower HCC risk with TDF was consistently observed in PSM (HR 0.73; 95% CI 0.59–0.88; p <0.01) and PSW (HR 0.83; 95% CI 0.67–1.03; p = 0.10) analyses and in all subgroups, with statistical significance in the ≥50 years of age (HR 0.76; 95% CI 0.58–1.00; p <0.05), male (HR 0.74; 95% CI 0.58–0.96; p = 0.02), HBeAg-positive (HR 0.69; 95% CI 0.49–0.97; p = 0.03) and non-diabetic (HR 0.79; 95% CI 0.63–1.00; p <0.05) subgroups. TDF was associated with significantly lower HCC risk than ETV in patients with CHB, particularly those with HBeAg positivity. Longer follow-up may be needed to better define incidence differences between the treatments in various subgroups. Previous aggregate data meta-analyses have reported inconsistent conclusions on the relative effectiveness of tenofovir disoproxil fumarate and entecavir in reducing hepatocellular carcinoma risk in patients with chronic hepatitis B (CHB). This individual patient data meta-analysis on 11 studies involving 42,939 patients from Korea, Taiwan and Hong Kong suggested that tenofovir disoproxil fumarate-treated patients have a significantly lower hepatocellular carcinoma risk than entecavir-treated patients, which was observed in all subgroups of clinical interest and by different analytical methodologies. These findings should be taken into account by healthcare providers when determining the optimal course of treatment for patients with CHB and may be considered in ensuring that treatment guidelines for CHB remain pertinent. [Display omitted] • Relative HCC risk for TDF vs. ETV treatment is undetermined in patients with CHB. • Prior meta-analyses are limited by heterogeneity of observational studies. • Using individual patient data enables a consistent analytic approach across studies. • TDF was consistently associated with lower HCC risk than ETV. [ABSTRACT FROM AUTHOR]

We examined the long-term incidence of hepatocellular carcinoma (HCC) and hepatic decompensation among chronic hepatitis B (CHB) patients who have achieved hepatitis B surface antigen (HBsAg) seroclearance. All adult CHB-monoinfected patients who cleared HBsAg between January 2000 and December 2020 were identified using a territory-wide database in Hong Kong. Patients who underwent liver transplantation and/or developed HCC before HBsAg seroclearance or less than 6 months follow-up were excluded. The primary and secondary endpoints were HCC and hepatic decompensation respectively. We identified 9,769 patients with CHB who achieved HBsAg seroclearance (mean age 57 years, 60.0% male, 13.2% cirrhosis); most had compensated liver function at HBsAg loss. At a median (25th–75th percentile) follow-up of 4.6 (2.2–8.4) years, 106 (1.1%) patients developed HCC. Patients who developed HCC were older, more likely to be male and have cirrhosis, and had higher alanine aminotransferase and lower platelets at the time of HBsAg loss than patients without HCC. The cumulative incidence of HCC remained steady 0–7 and 8–12 years after HBsAg loss (p = 0.898) (crude annual incidence drop: −0.04%, 95% CI −0.13% to 0.04%, p = 0.265). Moreover, 124/9,640 (1.3%) patients developed hepatic decompensation. The growth in cumulative incidence of hepatic decompensation decelerated 8–12 years after HBsAg loss (p = 0.009) (crude annual incidence drop: −0.23%, 95% CI −0.40% to −0.06%, p = 0.012). In multivariable analysis, HBsAg loss for over 7 years was associated with a reduced risk of hepatic decompensation (adjusted subdistribution hazard ratio [aSHR] 0.55, 95% CI 0.31–0.97, p = 0.039) but not HCC (aSHR 1.35, 95% CI 0.83–2.19, p = 0.230). HCC risk persists in patients after HBsAg loss, whereas the risk of hepatic decompensation decreases over time. Patients with chronic hepatitis B (CHB) still have a non-negligible risk of hepatocellular carcinoma (HCC) after 12 years of HBsAg seroclearance, especially among those with cirrhosis. The risk of developing hepatic decompensation decreases over time after HBsAg seroclearance. In clinical practice, although patients with CHB who cleared HBsAg have a more favourable clinical outcome than those who remain chronically infected, long-term HCC surveillance would still be necessary for patients with cirrhosis and other high-risk subgroups after HBsAg seroclearance. [Display omitted] • HCC risk persists over time in patients with HBsAg loss. • Risk of hepatic decompensation decreases after 7 years of HBsAg seroclearance. • Age, male sex, cirrhosis, and low platelet count were risk factors for HCC after HBsAg loss. • HBsAg seroreversion was not a risk factor for HCC on multivariable analysis. [ABSTRACT FROM AUTHOR]

Selgantolimod (GS-9688) is a Toll-like receptor 8 (TLR8) agonist that suppresses HBV in vitro. In a phase II study, we evaluated the safety and efficacy of weekly selgantolimod treatment in virally suppressed individuals with chronic HBV taking oral antiviral treatment. Forty-eight patients were randomized into two cohorts (hepatitis B e antigen [HBeAg]-positive and -negative [n = 24 each]) to receive oral selgantolimod 3 mg, 1.5 mg, or placebo (2:2:1) once weekly for 24 weeks while maintaining oral antivirals. The primary efficacy endpoint was the percentage of patients with a ≥1 log10 IU/ml decline in hepatitis B surface antigen (HBsAg) from baseline to week 24. Post-treatment, patients continued on oral antivirals for 24 weeks. The primary endpoint was reached by one participant, who was HBeAg-negative and received selgantolimod 1.5 mg. In contrast with placebo-treated patients (n = 9), only selgantolimod-treated patients (n = 39 total) had HBsAg declines greater than 0.1 log 10 IU/ml at weeks 24 (18%, 7/39) and 48 (26%, 10/39), HBsAg loss (5%, 2/39 through 48 weeks), or HBeAg loss (16%, 3/19 through 48 weeks). The most common adverse events in selgantolimod-treated groups were nausea (46%), upper respiratory tract infection (23%), and vomiting (23%). Gastrointestinal disorders were mostly mild and transient. Selgantolimod induced transient dose-dependent increases in serum cytokines, including IL-12p40, IFN-γ, and IL-1RA, as well as rapid redistribution of some circulating immune cell subsets. Oral selgantolimod up to 3 mg once weekly for 24 weeks was generally safe and well tolerated and led to serologic changes associated with progression to durable cure in two individuals by week 48. NCT03491553. The only robust criterion for stopping treatment in chronic hepatitis B is loss of hepatitis B surface antigen (known as functional cure), which is rare during nucleos(t)ide analogue therapy. It is likely that novel antiviral and immunomodulatory therapies will be needed to achieve finite functional cure. Selgantolimod is an oral Toll-like receptor 8 agonist that has shown antiviral activity in vitro as well as safety in a phase I clinical trial with weekly dosing. In this phase II study, selgantolimod therapy was associated with transient increases in serum cytokines, rapid redistribution of circulating immune cell subsets, modest reductions in HBsAg and HBeAg levels, and occasional loss of HBsAg (5%) and HBeAg (16%) among participants with chronic hepatitis B on nucleos(t)ide analogue therapy with viral suppression. Our results support continued development of selgantolimod as a component of a future hepatitis B cure regimen. [Display omitted] • Currently there is no finite cure for chronic HBV infection. • Combination therapy with immune modulators and other HBV-specific agents are being explored. • The immune modulator selgantolimod is an oral agonist of Toll-like receptor 8. • Selgantolimod once-weekly was given to individuals with chronic HBV for 6 months. • Six months after treatment, a minority experienced serologic changes associated with durable cure. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, in part because of inadequate early detection strategies. Current recommendations for screening consist of semi-annual abdominal ultrasound with or without serum alpha-fetoprotein in patients with cirrhosis and in demographic subgroups with chronic hepatitis B infection. However, this screening strategy has several deficiencies, including suboptimal early-stage sensitivity, false positives with subsequent harms, inter-operator variability in ultrasound performance, and poor adherence. A blood-based biomarker with sufficient performance characteristics for early-stage disease could overcome several of these barriers to improving early-stage detection. However, prior to use of a biomarker for screening in clinical practice, a multistep validation is required in order to understand test performance characteristics. These steps include case-control validation, followed by validation in prospective cohorts of at-risk patients. Until recently, we lacked adequate longitudinal validation cohorts for early HCC detection; however, several validation cohorts are maturing, including the Hepatocellular Carcinoma Early Detection Study and the Texas Hepatocellular Carcinoma Consortium, which will allow for rigorous validation of candidate biomarkers. While there are several promising biomarkers awaiting validation, in order to supplant abdominal ultrasound, a candidate biomarker must show adequate test performance and overcome practical hurdles to ensure adoption in clinical practice. The promise of blood-based biomarkers is significant, especially given the limitations of ultrasound-based screening; however, they require adequate validation and several logistical obstacles must be overcome prior to clinical implementation. [ABSTRACT FROM AUTHOR]

Antiviral therapy improves the clinical outcomes of patients with chronic hepatitis B (CHB), including those with cirrhosis. In the present study, we validated the Baveno VII definition of recompensation and explored the criteria for stable improvement of liver function tests in entecavir-treated patients with CHB-related decompensated cirrhosis. In this multicentre prospective study, patients with decompensated (ascites) CHB-related cirrhosis were enrolled and treated with entecavir for 120 weeks. Patients were followed up for clinical events, viral and biochemical tests, and ultrasonography every 6 months. The recompensation rate per Baveno VII criteria was calculated. Multivariate regression models were used to identify the predictors of recompensation. Finally, the criteria for stable improvement of liver function tests were explored. Of the 320 recruited patients, 283 completed the 120-week study, with 261/283 (92.2%) achieving HBV DNA levels <20 IU/ml and 171/283 (60.4%) achieving resolution of ascites, encephalopathy, and absence of recurrent variceal bleeding for at least 12 months. We identified model for end-stage liver disease <10 and/or liver function tests within Child-Pugh Class A (albumin >35 g/L, international normalised ratio <1.50 and total bilirubin <34 μmol/L) as the criteria for stable improvement of liver function tests. Accordingly, 56.2% (159/283) of patients fulfilled the Baveno VII definition of recompensation with a stable improvement of liver function tests defined by the current study. Our study defined the criteria for a stable improvement of liver function tests required by the Baveno VII definition of recompensation in patients with CHB-related decompensated cirrhosis on antiviral therapy. The criteria derived from this multicentre prospective study warrant further validation in patients with cirrhosis of other aetiologies. Decompensation of cirrhosis marks the point at which the liver is no longer able to function normally (and symptoms become apparent). Recently the idea of recompensation was proposed for individuals who may experience an improvement in liver function if the underlying cause of their liver disease is addressed (e.g. antivirals for viral cirrhosis). Herein, we show that over 50% of patients with hepatitis B-related decompensated cirrhosis treated with antivirals could recompensate and we propose laboratory criteria which could be used to define recompensation. [Display omitted] • This is the first prospective validation of the Baveno VII recompensation definition in patients with HBV-related cirrhosis. • A stable improvement of liver function tests was defined as MELD <10 and/or ALB & INR & TBIL within Child-Pugh A. • On-treatment MELD scores may be more predictive of the probability of recompensation than baseline MELD scores. • Prompt antiviral therapy is effective for patients with HBV-related cirrhosis, even under severe conditions. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B (CHB) and liver fibrosis are associated with a high risk of hepatocellular carcinoma (HCC) development. We assessed whether entecavir (ETV) plus Biejia-Ruangan compound (BRC), an anti-fibrotic traditional Chinese medicine, can further reduce the risk of HCC in treatment-naïve Chinese patients with CHB and an Ishak fibrosis score of ≥3 points derived from our parent double-blind randomized placebo-controlled trial. After a 72-week comparison between ETV+BRC and ETV+placebo treatment, participants were eligible to enter an open-label treatment phase and were followed up every 6 months. The primary [secondary] endpoints were the incidence of HCC [liver-related deaths, non-HCC events, and non-liver-related deaths]. Modified intention-to-treat (mITT), intention-to-treat (ITT), and per-protocol (PP) populations were defined for the time-to-event analysis. A total of 1,000 patients were recruited; the median age was 42.0 years; 69.9% were male and 58.3% were HBeAg positive. In the mITT population, the 7-year cumulative incidence of HCC [liver-related deaths] was 4.7% [0.2%] for ETV+BRC, which was significantly lower than 9.3% [2.2%] for ETV monotherapy (p = 0.008 [ p = 0.030]). Notably, ETV+BRC treatment yielded a lower incidence of HCC in those who did not achieve regression of fibrosis at week 72 than ETV monotherapy (p = 0.018). There were no differences in the other 2 secondary endpoints or safety profiles between the groups. Multivariable Cox proportional regression analysis, including the treatment allocation as a parameter, also demonstrated that ETV+BRC treatment was associated with a reduced incidence of HCC. The ITT and PP analyses showed consistent results. ETV plus BRC combination treatment could further reduce the risk of HCC and liver-related deaths in patients with CHB and advanced fibrosis or cirrhosis, which may have important clinical implications for HCC prevention. Patients with chronic hepatitis B virus infection are at an increased risk of developing liver cancer (specifically hepatocellular carcinoma [HCC]). While there are effective antiviral treatments that can suppress the virus in chronically infected patients, the risk of HCC remains. Herein, we show that adding a traditional Chinese medicine called Biejia-Ruangan compound to an antiviral reduced the risk of HCC in patients with chronic hepatitis B. [Display omitted] • This RCT-based prospective cohort study included 1,000 patients and lasted for 7 years. • Compared with ETV treatment, the combination of ETV + Biejia-Ruangan compound could further reduce the incidence of HCC. • Biejia-Ruangan compound in addition to ETV should be considered for patients with CHB and advanced fibrosis or cirrhosis. [ABSTRACT FROM AUTHOR]

RNA interference therapy has been shown to reduce hepatitis B surface antigen (HBsAg) levels in preclinical models, which could confer functional cure in patients with chronic hepatitis B. This phase IIa trial (ClinicalTrials.gov Identifier: NCT03365947) assessed the safety and efficacy of the small-interfering RNA JNJ-73763989 (JNJ-3989) plus a nucleos(t)ide analogue (NA), with/without the capsid assembly modulator JNJ-56136379 (JNJ-6379) in patients with chronic hepatitis B. Treatment-naïve and NA-suppressed patients received 3 subcutaneous JNJ-3989 doses every week (QW; 100, 200, or 300 mg), 2 weeks (Q2W; 100 mg) or 4 weeks (Q4W; 25, 50, 100, 200, 300, or 400 mg), or JNJ-3989 Q4W (200 mg) plus oral JNJ-6379 250 mg daily for 12 weeks. Patients received NAs throughout. Eighty-four patients were recruited. All treatments were well tolerated, with all 5 serious adverse events considered unrelated to study drugs. JNJ-3989 100 to 400 mg Q4W resulted in HBsAg reductions ≥1 log 10 IU/ml from baseline in 39/40 (97.5%) patients at the nadir. All patients receiving the triple combination (n = 12) had HBsAg reductions ≥1 log 10 IU/ml from baseline at the nadir. HBsAg reductions were similar for HBeAg-positive (n = 21) and HBeAg-negative (n = 47) patients in all JNJ-3989 Q4W treatment arms, including the triple combination (n = 68). Smaller HBsAg reductions were seen with 25 mg (n = 8) and 50 mg (n = 8) than with 100 to 400 mg (n = 40). Shorter dosing intervals (QW [n = 12] and Q2W [n = 4]) did not improve response vs. Q4W dosing. HBsAg reductions ≥1 log 10 IU/ml from baseline persisted in 38% of patients 336 days after the last JNJ-3989 dose. JNJ-3989 plus an NA, with/without JNJ-6379, was well tolerated and resulted in HBsAg reductions up to 336 days after the last JNJ-3989 Q4W dose. NCT03365947. Hepatitis B virus affects people's livers and produces particles called hepatitis B surface antigen (HBsAg) that damage a person's liver and can help the virus infect a person for a long time, known as chronic hepatitis B (CHB). In this study, a new treatment called JNJ-3989 was assessed (in combination with normal treatment known as nucleos(t)ide analogues), for its safety and effectiveness in reducing the number of HBsAg particles in people with CHB. The results of this study showed that treatment with JNJ-3989 could be safe for people with CHB, lowered their HBsAg levels, and kept HBsAg levels lowered for 336 days in 38% of patients after receiving their last dose of JNJ-3989. [Display omitted] • Small-interfering RNA (siRNA) lowers HBsAg levels in patients with CHB. • Safety and efficacy of 3 siRNA JNJ-3989 doses + a nucleos(t)ide analogue was studied. • All JNJ-3989 doses were well tolerated in patients with CHB in this study. • Many patients experienced HBsAg declines ≥1 log 10 IU/ml from baseline. • HBsAg reduction persisted 336 days after the last JNJ-3989 dose in many patients. [ABSTRACT FROM AUTHOR]

Nucleos(t)ide reverse transcriptase inhibitors do not completely suppress HBV DNA in chronic HBV infection (cHBV). Vebicorvir (VBR) is an investigational core inhibitor that interferes with multiple aspects of HBV replication. This phase II trial evaluated the safety and efficacy of VBR in combination with entecavir (ETV) in treatment-naïve patients with cHBV. HBeAg-positive, treatment-naïve patients without cirrhosis were randomised 1:1 in a double-blind manner to once-daily VBR 300 mg+ETV 0.5 mg or placebo (PBO)+ETV 0.5 mg for 24 weeks. The primary endpoint was change in mean log 10 HBV DNA from Baseline to Week 12 and 24. All patients in both treatment groups (PBO+ETV: 12/12; VBR+ETV: 13/13) completed the study. At Week 12, VBR+ETV led to a greater mean (SD) reduction from Baseline in log 10 IU/ml HBV DNA (–4.45 [1.03]) vs. PBO+ETV (–3.30 [1.18]; p = 0.0077). At Week 24, VBR+ETV led to a greater reduction from Baseline in log 10 IU/ml HBV DNA (–5.33 [1.59]) vs. PBO+ETV (–4.20 [0.98]; p = 0.0084). Greater mean reductions in pregenomic RNA were observed at Week 12 and 24 in patients receiving VBR+ETV vs. PBO+ETV (p < 0.0001 and p < 0.0001). Changes in viral antigens were similar in both groups. No drug interaction between VBR and ETV was observed. Two patients experienced HBV DNA rebound during treatment, with no resistance breakthrough detected. The safety of VBR+ETV was similar to PBO+ETV. All treatment-emergent adverse events and laboratory abnormalities were Grade 1/2. There were no deaths, serious adverse events, or evidence of drug-induced liver injury. In this 24-week study, VBR+ETV provided additive antiviral activity over PBO+ETV in treatment-naïve patients with cHBV, with a favourable safety and tolerability profile. NCT03577171 Hepatitis B is a long-lasting viral infection of the liver. Current treatments can suppress hepatitis B virus but do not offer the opportunity of cure, hence, new treatment approaches are required. Herein, we show that the combination of the novel core inhibitor vebicorvir with an existing antiviral (entecavir) in treatment-naïve patients chronically infected with hepatitis B virus demonstrated greater antiviral activity than entecavir alone. Additionally, vebicorvir was safe and well tolerated. Thus, further studies evaluating its potential role in the treatment of chronic hepatitis B are warranted. [Display omitted] • Complete suppression of HBV replication is essential for finite treatment regimens • Vebicorvir (VBR) is an HBV core protein inhibitor developed to treat chronic HBV • VBR interferes with 2 additional steps in HBV replication and thus complements NrtIs • VBR+ETV led to a significantly deeper reduction in HBV DNA and pregenomic RNA than ETV alone • VBR+ETV was associated with favorable safety and tolerability during 24 weeks of treatment [ABSTRACT FROM AUTHOR]

With or without antiviral treatment, few individuals achieve sustained functional cure of chronic hepatitis B virus (HBV) infection. A better definition of what mediates functional cure is essential for improving immunotherapeutic strategies. We aimed to compare HBV-specific T cell responses in patients with different degrees of viral control. We obtained blood from 124 HBV-infected individuals, including those with acute self-limiting HBV infection, chronic infection, and chronic infection with functional cure. We screened for HBV-specific T cell specificities by ELISpot, assessed the function of HBV-specific T cells using intracellular cytokine staining , and characterized HBV-specific CD4 T cells using human leukocyte antigen (HLA) class II tetramer staining, all directly ex vivo. ELISpot screening readily identified HBV-specific CD4 and CD8 T cell responses in acute resolving infection compared with more limited reactivity in chronic infection. Applying more sensitive assays revealed higher frequencies of functional HBV-specific CD4 T cells, but not CD8 T cells, in functional cure compared to chronic infection. Function independent analysis using HLA multimers also identified more HBV-specific CD4 T cell responses in functional cure compared to chronic infection, with the emergence of CD4 T cell memory both after acute and chronic infection. Functional cure is associated with higher frequencies of functional HBV-specific CD4 memory T cell responses. Thus, immunotherapeutic approaches designed to induce HBV functional cure should also aim to improve CD4 T cell responses. Immunotherapy is a form of treatment that relies on harnessing the power of an individual's immune system to target a specific disease or pathogen. Such approaches are being developed for patients with chronic HBV infection, in an attempt to mimic the immune response in patients who control HBV infection spontaneously, achieving a so-called functional cure. However, what exactly defines protective immune responses remains unclear. Herein, we show that functional cure is associated with robust responses by HBV-specific CD4 T cells (a type of immune cell). [Display omitted] • HBV-specific CD4 T cells can be analysed directly ex vivo , without the need for in vitro expansion. • Functional HBV-specific CD4 T cell responses are stronger in functional cure vs. chronic infection. • MHC-class II multimers identify HBV-specific CD4 T cells reliably in HBsAg- patients, but rarely when HBsAg is present. • CD4 T cell immunity should be considered as a target for HBV immunotherapies. [ABSTRACT FROM AUTHOR]

Bepirovirsen, an antisense oligonucleotide targeting pregenomic and mRNA transcripts of HBV, has been conjugated to N -acetyl galactosamine (GSK3389404) to enhance hepatocyte delivery. This dose-finding study was the first to assess GSK3389404 for chronic HBV infection. This phase IIa, randomised, double-blind, placebo-controlled, 2-part study was conducted in 22 centres in Asia (NCT03020745). Pharmacokinetic findings from Part 1 informed Part 2 dosing. In Part 2, patients with chronic hepatitis B on nucleos(t)ide analogue therapy were randomised 11:2 to GSK3389404 (30, 60, 120 mg weekly or 120 mg bi-weekly) or placebo until Day 85. Coprimary endpoints included HBsAg response (≥1.5 log 10 IU/ml reduction from baseline) rate, safety and pharmacokinetics. Parts 1 and 2 included 12 (9 GSK3389404, 3 placebo) and 66 patients (56 GSK3389404, 10 placebo), respectively. In Part 2, one patient each in the 60 mg weekly, 120 mg weekly and 120 mg bi-weekly arms achieved a HBsAg response. HBsAg reductions were dose-dependent (Day 85: mean 0.34 [60 mg weekly] to 0.75 log 10 IU/ml [120 mg weekly]) and occurred in hepatitis B e antigen-positive and -negative patients. No patient achieved HBsAg seroclearance. 43/56 (77%) GSK3389404- and 9/10 (90%) placebo-treated patients reported adverse events. No deaths were reported. Alanine aminotransferase flares (>2x upper limit of normal) occurred in 2 GSK3389404-treated patients (120 mg weekly, 120 mg bi-weekly); both were associated with decreased HBsAg, but neither was considered a responder. GSK3389404 plasma concentrations peaked 2–4 hours post dose; mean plasma half-life was 3–5 hours. GSK3389404 showed an acceptable safety profile and target engagement, with dose-dependent reductions in HBsAg. However, no efficacious dosing regimen was identified. NCT03020745. Hepatitis B virus (HBV) can result in chronic HBV infection, which may ultimately lead to chronic liver disease, primary liver cancer and death; HBV proteins may prevent the immune system from successfully controlling the virus. GSK3389404 is an investigational agent that targets HBV RNA, resulting in reduced viral protein production. This study assessed the safety of GSK3389404 and its ability to reduce the viral proteins in patients with chronic HBV infection. GSK3389404 showed dose-dependent reduction in hepatitis B surface antigen, with an acceptable safety profile. While no clear optimal dose was identified, the findings from this study may help in the development of improved treatment options for patients with chronic HBV infections. [Display omitted] • GSK3389404 is a GalNAc-conjugated antisense oligonucleotide targeting HBV pregenomic and mRNA transcripts. • GSK3389404 treatment for 12 weeks led to a dose-dependent reduction of HBsAg in patients with chronic HBV infection. • HBsAg reduction occurred in both HBeAg+/- patients, indicating the target site is away from the integration hotspot. • Only 3 of 56 patients had a >1.5 log IU/ml reduction in HBsAg and no HBsAg seroclearance was achieved. • GSK3389404 had an acceptable safety profile with no unexpected safety signals. [ABSTRACT FROM AUTHOR]

It is unknown whether HBsAg seroclearance affects the risk of hepatocellular carcinoma (HCC) recurrence after liver resection. We aimed to investigate the impact of HBsAg seroclearance on the recurrence of HCC after curative liver resection, with a focus on late recurrence. This study comprised 2,520 consecutive patients who received curative liver resection for HBV-related HCC of Barcelona Clinic Liver Cancer stage 0 or A in Korea between 2000 and 2017. To focus on late recurrence, patients with recurrence or a follow-up duration less than 2 years were excluded. The impact of HBsAg seroclearance on HCC recurrence was assessed by landmark analysis (2-, 5-and 8-year after liver resection), time-dependent Cox and multistate modeling. The mean patient age was 54.4 years and 75.7% were men. A total of 891 (35.4%) patients developed HCC recurrence at rates of 11.2%, 25.5%, and 46.8% at 3, 5, and 10 years after resection. HBsAg seroclearance was achieved in 172 (6.8%) patients during a median follow-up duration of 6.9 years after resection. HBsAg seroclearance, compared with persistent HBsAg positivity, was associated with a lower risk of late HCC recurrence in the 2-, 5-, and 8-year landmark analysis (p = 0.04, p = 0.02 and p = 0.03, respectively) and on time-dependent multivariable Cox modeling (adjusted hazard ratio 0.62; p = 0.005). Based on a 3-state unidirectional illness–death model, patients without HBsAg seroclearance transitioned to HCC recurrence more rapidly than patients who experienced HBsAg seroclearance. HBsAg seroclearance is associated with a lower risk of late recurrence of HBV-related HCC among Korean patients who undergo curative liver resection. Hepatitis B virus (HBV) infection is a leading cause of chronic liver disease and hepatocellular carcinoma (HCC). Suppression of HBV replication is known to lower the risk of HCC recurrence after liver resection (a procedure used to treat and in some cases cure HCC). However, whether the loss of a specific HBV protein (hepatitis B surface antigen or HBsAg) has an impact on recurrence after liver resection remains unknown. Herein, we show that loss of HBsAg is associated with a reduce risk of late recurrence of HCC after liver resection in patients with HBV-related HCC. [Display omitted] • Whether HBsAg seroclearance affects the risk of late (≥2 years) HCC recurrence after liver resection remains unanswered. • 2,520 consecutive Korean patients who underwent curative-intent liver resection for HBV-related HCC of BCLC 0 or A were analyzed. • HBsAg seroclearance after liver resection was independently associated with a reduced risk of late (≥2 years) HCC recurrence. [ABSTRACT FROM AUTHOR]

Addressing HBV is vital to meeting the World Health Organization (WHO)'s viral hepatitis elimination goals, as 47% of viral hepatitis complications can be attributed to HBV. The objective of this study is to develop an agent-based model determining which integrated strategies involving vaccination, screening, and treatment would achieve the WHO's goals. We developed an agent-based model to characterize the HBV epidemic in a high-income country with ongoing immigration. The spread of HBV was simulated through sexual networks and perinatal transmission. Model parameters were estimated from the literature and calibrated against historical HBV data. Sensitivity analyses were performed to assess the uncertainty. We predict that under the current strategies, the incidence of acute hepatitis B, and HBV-attributable decompensated cirrhosis and hepatocellular carcinoma would decrease by 64.5%, 9.4%, and 10.5% between 2015–2030, respectively. However, the incidence of chronic hepatitis B and liver-related deaths would increase by 26.6% and 1.0% between 2015–2030, respectively. Results were sensitive to the number of immigrants and HBV prevalence in immigrants. The results suggest that the current vaccination, screening, and treatment strategies will be inadequate to achieve WHO elimination goals. Even with extensive integrated scale-up in vaccination, screening, and treatment, the morbidity and mortality targets may not be reachable, highlighting the need for a re-evaluation of the global strategy for HBV, the importance of developing curative therapy for HBV, and of establishing tailored strategies to prevent long-term sequelae and improve health in immigrants. We have developed a model that reflects the dynamics of hepatitis B virus (HBV) transmission in a high-income country with ongoing immigration, which enabled us to forecast the epidemiology of HBV for policy-level decision making. Our analysis suggests that current vaccination, screening, and treatment strategies are inadequate to achieve the WHO goals of eliminating chronic hepatitis B. Even with extensive integrated scale-up in vaccination, screening, and treatment, the morbidity and mortality targets may not be reachable. [Display omitted] • We developed an agent-based model reflecting the dynamics of HBV transmission in a high-income country with ongoing immigration. • Current and scaled-up vaccination, screening, and treatment strategies are inadequate to achieve the WHO goals. • Further medical research on curable treatments for CHB is urgently needed. • For an immigrant-receiving country, the WHO goals may not be realistic. • Formulation of immigration screening policies would allow for a reduction in HBV-related complications. [ABSTRACT FROM AUTHOR]

HBV nucleos(t)ide reverse transcriptase inhibitors (NrtIs) do not completely suppress HBV replication. Previous reports indicate persistent viremia during NrtI treatment despite HBV DNA being undetectable. HBV core inhibitors may enhance viral suppression when combined with NrtIs. This phase II trial (NCT03576066) evaluated the efficacy and safety of the investigational core inhibitor, vebicorvir (VBR), in virologically- suppressed patients on NrtIs. Non-cirrhotic, NrtI-suppressed patients with chronic HBV were randomised to VBR 300 mg once daily or matching placebo (PBO) for 24 weeks. Treatment was stratified by hepatitis B e antigen (HBeAg) status. The primary endpoint was change from Baseline in serum HBeAg or hepatitis B surface antigen (HBsAg) after 24 weeks. Of 73 patients enrolled, 47 were HBeAg positive and 26 were HBeAg negative. In HBeAg-positive and -negative patients, there were no differences in the change from Baseline at Week 24 for HBsAg or HBeAg. Using a novel, high-sensitivity assay to detect HBV DNA, a greater proportion of patients with detectable HBV DNA at Baseline achieved undetectable HBV DNA at Week 24 in the VBR+NrtI vs. PBO+NrtI group. In HBeAg-positive patients, a greater change from Baseline in HBV pregenomic (pg)RNA was observed at Week 24 with VBR+NrtI vs. PBO+NrtI. Treatment-emergent adverse events (TEAEs) in VBR+NrtI patients included upper respiratory tract infection, nausea, and pruritus. No serious adverse events, Grade 4 TEAEs, or deaths were reported. In this 24-week study, VBR+NrtI demonstrated a favourable safety and tolerability profile. While there were no significant changes in viral antigen levels, enhanced viral suppression was demonstrated by greater changes in DNA and pgRNA with the addition of VBR compared to NrtI alone. NCT03576066. Core inhibitors represent a novel approach for the treatment of chronic hepatitis B virus (HBV) infection, with mechanisms of action distinct from existing treatments. In this study, vebicorvir added to existing therapy reduced HBV replication to a greater extent than existing treatment and was generally safe and well tolerated. [Display omitted] • Complete suppression of HBV replication is essential for finite treatment regimens. • Vebicorvir (VBR) is a novel inhibitor of the HBV core protein. • VBR interferes with two additional steps in HBV replication than NrtIs. • Added to NrtI, VBR did not significantly change mean HBV antigens over 24 weeks. • Added to NrtI, VBR further reduced HBV DNA and pgRNA by high-sensitivity PCR assays. [ABSTRACT FROM AUTHOR]

After hepatitis B surface antigen (HBsAg) seroclearance, the risk of hepatocellular carcinoma (HCC) remains, and the optimal surveillance strategy has yet to be determined. Herein, we aimed to evaluate incidence and risk factors for HCC and establish a novel prediction model for HCC development after HBsAg seroclearance. A total of 1,443 patients with chronic hepatitis B who achieved HBsAg seroclearance between 1991 and 2020 were retrospectively screened for study eligibility. The data from 831 of these patients were included in the final analysis. A prediction model was developed based on multivariable Cox models. Harrell's C-index and a time-dependent AUROC were used for discrimination. Bootstrap analysis was performed for internal validation. Overall, 40 patients (4.8%) developed HCC after HBsAg seroclearance during a follow-up of 4,644 person-years (0.86%/year). Age at HBsAg seroclearance, presence of cirrhosis, family history of HCC, and more-than-moderate alcohol consumption were independently predictive of HCC, and these 4 independent variables were used to develop the prediction model. The C-index of the model was 0.804. The time-dependent AUROCs of the score for HCC prediction at 5, 10, and 15 years were 0.799, 0.835, and 0.817, respectively. The score also showed good discrimination in the internal validation and sensitivity analysis. The novel prediction model based on age, cirrhosis, family history of HCC, and alcohol consumption enables reliable risk estimation of HCC after HBsAg seroclearance and may serve as a useful reference for decision-making in HCC surveillance for HBsAg-cleared patients. After spontaneous hepatitis B surface antigen (HBsAg) seroclearance, the risk of hepatocellular carcinoma (HCC) remains. Age at HBsAg seroclearance, presence of cirrhosis, family history of HCC, and more-than-moderate alcohol consumption were independently associated with HCC development after HBsAg seroclearance. The novel prediction model using these 4 variables enables reliable risk estimation of HCC and serves as a useful reference for decision-making in HCC surveillance and management for HBsAg-cleared patients. [Display omitted] • Age, cirrhosis, family history of HCC, and alcohol consumption independently predicted HCC development after HBsAg seroclearance. • A simple risk score using these 4 variables showed good discriminative performance and calibration for HCC prediction. • The first prediction model derived from HBsAg-cleared patients may be a useful reference for decision-making in surveillance. [ABSTRACT FROM AUTHOR]

Chronic HBV is clinically categorized into 4 phases by a combination of serum HBV DNA levels, HBeAg status and alanine aminotransferase (ALT): immunotolerant (IT), immune-active (IA), inactive carrier (IC) and HBeAg-negative hepatitis (ENEG). Immune and virological measurements in the blood have proven useful but are insufficient to explain the interrelation between the immune system and the virus since immune dynamics differ in the blood and liver. Furthermore, the inflammatory response in the liver and parenchymal cells cannot be fully captured in blood. Immunological composition and transcriptional profiles of core needle liver-biopsies in chronic HBV phases were compared to those of healthy controls by multiplex immunofluorescence and RNA-sequencing (n = 37 and 78, respectively) analyses. Irrespective of the phase-specific serological profiles, increased immune-gene expression and frequency was observed in chronic HBV compared to healthy livers. Greater transcriptomic deregulation was seen in IA and ENEG (172 vs. 243 DEGs) than in IT and IC (13 vs. 35 DEGs) livers. Interferon-stimulated genes, immune-activation and exhaustion genes (ICOS , CTLA4 , PDCD1) together with chemokine genes (CXCL10 , CXCL9) were significantly induced in IA and ENEG livers. Moreover, distinct immune profiles associated with ALT elevation and a more accentuated immune-exhaustion profile (CTLA4 , TOX , SLAMF6 , FOXP3) were observed in ENEG, which set it apart from the IA phase (LGALS9 , PDCD1). Interestingly, all HBV phases showed downregulation of metabolic pathways vs. healthy livers (fatty and bile acid metabolism). Finally, increased leukocyte infiltrate correlated with serum ALT, but not with HBV DNA or viral proteins. Our comprehensive multi-parametric analysis of human livers revealed distinct inflammatory profiles and pronounced differences in intrahepatic gene profiles across all chronic HBV phases in comparison to healthy liver. Immunological studies on chronic HBV remain largely restricted to assessment of peripheral responses due to the limited access to the site of infection, the liver. In this study, we comprehensively analyzed livers from a well-defined cohort of patients with chronic HBV and uninfected controls with state-of-the-art techniques, and evaluated the differences in gene expression profiles and inflammation characteristics across distinct disease phases in patients with chronic HBV. [Display omitted] • RNA-Seq and multiplex immunofluorescence (mIF) was performed on liver biopsies to characterize the HBV clinical phases. • Chronic HBV livers were characterized by higher immune-gene expression and leukocyte infiltrate than healthy livers. • ALT determined immune gene expression profiles in livers of IA and ENEG patients. • Immune-exhaustion profiles were observed in livers of IA and ENEG patients. • Leukocyte infiltrate correlated with serum ALT, but not with HBV DNA or intrahepatic HBcAg and HBsAg. [ABSTRACT FROM AUTHOR]

There have been unprecedented advances in the identification of new treatment targets for chronic hepatitis B that are being developed with the goal of achieving functional cure in patients who would otherwise require lifelong nucleoside analogue treatment. Many of the new investigational therapies either directly target the immune system or are anticipated to impact immunity indirectly through modulation of the viral lifecycle and antigen production. While new viral biomarkers (HBV RNA, HBcAg, small, middle, large HBs isoforms) are proceeding through validation steps in clinical studies, immunological biomarkers are non-existent outside of clinical assays for antibodies to HBs, HBc and HBe. To develop clinically applicable immunological biomarkers to measure mechanisms of action, inform logical combination strategies, and guide clinical management for use and discontinuation of immune-targeting drugs, immune assays must be incorporated into phase I/II clinical trials. This paper will discuss the importance of sample collection, the assays available for immunological analyses, their advantages/disadvantages and suggestions for their implementation in clinical trials. Careful consideration must be given to ensure appropriate immunological studies are included as a primary component of the trial with deeper immunological analysis provided by ancillary studies. Standardising immunological assays and data obtained from clinical trials will identify biomarkers that can be deployed in the clinic, independently of specialised immunology laboratories. [ABSTRACT FROM AUTHOR]

Nucleoside analogues are the mainstay of treatment for patients with chronic HBV infection but have no direct effect on covalently closed circular DNA. Long-term HBV viral suppression is now routine, but the desirable endpoint of functional cure is rarely achieved. Newer therapies, targeting other aspects of the replicative life cycle of HBV, present opportunities to deliver finite therapy and HBV 'cure'. This is an area of keen focus for the HBV community. We describe a severe case of hepatitis B reactivation, occurring shortly after the withdrawal of a nucleoside analogue within the protocol of a clinical trial (REEF-2). Despite best supportive care and prompt re-introduction of tenofovir, the patient developed subacute liver failure, requiring emergency orthotopic liver transplantation. As we strive to achieve HBV cure, this case highlights the potential risks of finite therapy and highlights the need for improved biomarker-driven strategies and re-evaluation of study protocols. [ABSTRACT FROM AUTHOR]

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. 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). 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 log 10 U/ml and HBsAb >2 log 10 IU/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. 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. NCT02327416 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. [Display omitted] • 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 log 10 U/ml and HBsAb >2 log 10 IU/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. [ABSTRACT FROM AUTHOR]

Nucleo(s)tide analogue (NUC) withdrawal may result in HBsAg clearance in a subset of patients. However, predictors of HBsAg loss after NUC withdrawal remain ill-defined. We studied predictors of HBsAg loss in a global cohort of HBeAg-negative patients with undetectable HBV DNA who discontinued long-term NUC therapy. Patients requiring retreatment after treatment cessation were considered non-responders. We enrolled 1,216 patients (991 with genotype data); 98 (8.1%) achieved HBsAg loss. The probability of HBsAg loss was higher in non-Asian patients (adjusted hazard ratio [aHR] 8.26, p < 0.001), and in patients with lower HBsAg (aHR 0.243, p < 0.001) and HBV core-related antigen (HBcrAg) (aHR 0.718, p = 0.001) levels. Combining HBsAg (<10, 10-100 or >100 IU/ml) and HBcrAg (<2log vs. ≥2 log) levels improved prediction of HBsAg loss, with extremely low rates observed in patients with HBsAg >100 IU/ml with detectable HBcrAg. HBsAg loss rates also varied with HBV genotype; the highest rates were observed for genotypes A and D, and none of the patients with HBV genotype E experienced HBsAg loss (p < 0.001 for the overall comparison across genotypes; p < 0.001 for genotypes A/D vs. genotypes B/C). HBV genotype C was independently associated with a higher probability of HBsAg loss when compared to genotype B among Asian patients (aHR 2.494; 95% CI 1.490–4.174, p = 0.001). The probability of HBsAg loss after NUC cessation varies according to patient ethnicity, HBV genotype and end-of-treatment viral antigen levels. Patients with low HBsAg (<100 IU/ml) and/or undetectable HBcrAg levels, particularly if non-Asian or infected with HBV genotype C, appear to be the best candidates for treatment withdrawal. A subset of patients may achieve clearance of hepatitis B surface antigen (HBsAg) – so-called functional cure – after withdrawal of nucleo(s)tide analogue therapy. In this multicentre study of 1,216 patients who discontinued antiviral therapy, we identified non-Asian ethnicity, HBV genotype C, and low hepatitis B surface antigen and hepatitis B core-related antigen levels as factors associated with an increased chance of HBsAg loss. [Display omitted] • A minority of patients with chronic hepatitis B may achieve HBsAg clearance after withdrawal of antiviral therapy. • In this multicenter study comprising 1,216 patients, non-Asian ethnicity was associated with the highest chance of HBsAg loss. • Among Asian patients, genotype C was associated with a higher chance of HBsAg loss. • Low HBsAg levels (<100 IU/ml) and undetectable HBcrAg levels were associated with a higher chance of HBsAg loss. [ABSTRACT FROM AUTHOR]

The entry inhibitor bulevirtide (BLV) received conditional approval from the EMA in July 2020 for the treatment of adult patients with compensated chronic hepatitis delta. However, the effectiveness and safety of BLV administered as monotherapy beyond 48 weeks in difficult-to-treat patients with HDV-related cirrhosis is presently unknown. Herein, we describe the first patients with HDV-related compensated cirrhosis who were treated with BLV (10 mg/day as a starting dose) for up to 3 years on a compassionate use program. Patients were also monitored for HBcrAg and HBV RNA levels, and HDV- and HBV-specific T-cell markers. In the patient who stopped BLV at week 48, after achieving a virological and biochemical response, the initial virological and biochemical rebound was followed by alanine aminotransferase normalization coupled with low HDV RNA and HBsAg levels. In the 2 patients treated continuously for 3 years, virological and biochemical responses were maintained throughout the treatment period even after dose reduction. In a patient with advanced compensated cirrhosis, liver function tests significantly improved, esophageal varices disappeared, and histological/laboratory features of autoimmune hepatitis resolved. Overall, no safety issues were recorded, as bile salt increase was asymptomatic. While serum HBV RNA levels remained undetectable in all patients, HBV core-related antigen levels showed a progressive, yet modest decline during long-term BLV treatment. No HDV-specific interferon-γ-producing T cells were detected, neither after HDV reactivation (after BLV withdrawn in Patient 1) nor during 3 years of BLV treatment. In conclusion, this report shows that continuous administration of BLV monotherapy for 3 years leads to excellent virological and clinical responses in patients with HDV-related cirrhosis who had contraindications to interferon-based therapies. [ABSTRACT FROM AUTHOR]

Several models have recently been developed to predict risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). Our aims were to develop and validate an artificial intelligence-assisted prediction model of HCC risk. Using a gradient-boosting machine (GBM) algorithm, a model was developed using 6,051 patients with CHB who received entecavir or tenofovir therapy from 4 hospitals in Korea. Two external validation cohorts were independently established: Korean (5,817 patients from 14 Korean centers) and Caucasian (1,640 from 11 Western centers) PAGE-B cohorts. The primary outcome was HCC development. In the derivation cohort and the 2 validation cohorts, cirrhosis was present in 26.9%–50.2% of patients at baseline. A model using 10 parameters at baseline was derived and showed good predictive performance (c-index 0.79). This model showed significantly better discrimination than previous models (PAGE-B, modified PAGE-B, REACH-B, and CU-HCC) in both the Korean (c-index 0.79 vs. 0.64–0.74; all p <0.001) and Caucasian validation cohorts (c-index 0.81 vs. 0.57–0.79; all p <0.05 except modified PAGE-B, p = 0.42). A calibration plot showed a satisfactory calibration function. When the patients were grouped into 4 risk groups, the minimal-risk group (11.2% of the Korean cohort and 8.8% of the Caucasian cohort) had a less than 0.5% risk of HCC during 8 years of follow-up. This GBM-based model provides the best predictive power for HCC risk in Korean and Caucasian patients with CHB treated with entecavir or tenofovir. Risk scores have been developed to predict the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B. We developed and validated a new risk prediction model using machine learning algorithms in 13,508 antiviral-treated patients with chronic hepatitis B. Our new model, based on 10 common baseline characteristics, demonstrated superior performance in risk stratification compared with previous risk scores. This model also identified a group of patients at minimal risk of developing HCC, who could be indicated for less intensive HCC surveillance. [Display omitted] • A new HCC prediction model (PLAN-B) was developed using machine learning algorithms in antiviral-treated patients with chronic hepatitis B. • The utility of the model was validated in independent Korean and Caucasian cohorts. • PLAN-B comprises 10 baseline parameters: cirrhosis, age, platelet count, ETV/TDF, sex, serum ALT and HBV DNA, albumin and bilirubin levels, and HBeAg status. • The PLAN-B model demonstrated satisfactory predictive performance for HCC development and outperformed other risk scores. [ABSTRACT FROM AUTHOR]

Guidelines recommend hepatocellular carcinoma (HCC) surveillance in patients with chronic HBV infection. Several HCC risk prediction models are available to guide surveillance decisions, but their comparative performance remains unclear. Using a retrospective cohort of patients with HBV treated with nucleos(t)ide analogues at 130 Veterans Administration facilities between 9/1/2008 and 12/31/2018, we calculated risk scores from 10 HCC risk prediction models (REACH-B, PAGE-B, m-PAGE-B, CU-HCC, HCC-RESCUE, CAMD, APA-B, REAL-B, AASL-HCC, RWS-HCC). We estimated the models' discrimination and calibration. We calculated HCC incidence in risk categories defined by the reported cut-offs for all models. Of 3,101 patients with HBV (32.2% with cirrhosis), 47.0% were treated with entecavir, 40.6% tenofovir, and 12.4% received both. During a median follow-up of 4.5 years, 113 patients developed HCC at an incidence of 0.75/100 person-years. AUC values for 3-year HCC risk were the highest for RWS-HCC, APA-B, REAL-B, and AASL-HCC (all >0.80). Of these, 3 (APA-B, RWS-HCC, REAL-B) incorporated alpha-fetoprotein. AUC values for the other models ranged from 0.73 for PAGE-B to 0.79 for CAMD and HCC-RESCUE. Of the 7 models with AUC >0.75, only APA-B was poorly calibrated. In total, 10–20% of the cohort was deemed low-risk based on the published cut-offs. None of the patients in the low-risk groups defined by PAGE-B, m-PAGE-B, AASL-HCC, and REAL-B developed HCC during the study timeframe. In this national cohort of US-based patients with HBV on antiviral treatment, most models performed well in predicting HCC risk. A low-risk group, in which no cases of HCC occurred within a 3-year timeframe, was identified by several models (PAGE-B, m-PAGE-B, CAMD, AASL-HCC, REAL-B). Further studies are warranted to examine whether these patients could be excluded from HCC surveillance. Risk prediction models for hepatocellular carcinoma (HCC) in patients infected with hepatitis B virus (HBV) could guide HCC surveillance decisions. In this large cohort of US-based patients receiving treatment for HBV, most published models discriminated between those who did or did not develop HCC, although the RWS-HCC, REAL-B, and AASL-HCC performed the best. If confirmed in future studies, these models could help identify a low-risk subset of patients on antiviral treatment who could be excluded from HCC surveillance. [Display omitted] • CAMD, APA-B, HCC-RESCUE, CU-HCC, AASL-HCC, RWS-HCC, REAL-B models identified patients at high risk of developing HCC. • RWS-HCC, REAL-B, and AASL-HCC had the highest AUCs and also predicted the actual risk of HCC closely. • No low-risk patients (∼10-20% of cohort) defined by PAGE-B, m-PAGE-B, AASL-HCC, CMAD and REAL-B models developed HCC within 3 years. • Further studies are warranted to examine whether this low-risk group may be excluded from HCC surveillance. [ABSTRACT FROM AUTHOR]

Despite several recent meta-analyses on the topic, the comparative risk of hepatocellular carcinoma in patients with chronic hepatitis B (CHB) receiving entecavir (ETV) or tenofovir disoproxil fumarate (TDF) remains controversial. The controversy partly results from the arbitrary nature of significance levels leading to contradictory conclusions from very similar datasets. However, the use of observational data, which is prone to both within- and between-study heterogeneity of patient characteristics, also lends additional uncertainty. The asynchronous introduction of ETV and TDF in East Asia, where the majority of these studies have been conducted, further complicates analyses, as does the ensuing difference in follow-up time between ETV and TDF cohorts. Researchers conducting meta-analyses in this area must make many methodological decisions to mitigate bias but are ultimately limited to the methodologies of the included studies. It is therefore important for researchers, as well as the audience of published meta-analyses, to be aware of the quality of observational studies and meta-analyses in terms of patient characteristics, study design and statistical methodologies. In this review, we aim to help clinicians navigate the published meta-analyses on this topic and to provide researchers with recommendations for future work. [ABSTRACT FROM AUTHOR]

Regulatory T cell (Treg) depletion increases antitumor immunity. However, severe autoimmunity can occur following systemic loss of Tregs, which could be avoided by selectively depleting intratumoral Tregs. Herein, we aimed to investigate the role of tumor-infiltrating CCR4+ Tregs in hepatocellular carcinoma (HCC) and to provide a potential target strategy for immunotherapy. CCR4+ Tregs were analyzed by flow cytometry in murine models and clinical samples. The function of tumor-infiltrating and induced CCR4+ Tregs was interrogated by genetic and epigenetic approaches. To block CCR4+ Treg chemotaxis, we developed an N-terminus recombinant protein of CCR4 (N-CCR4-Fc) as a neutralizing pseudo-receptor that effectively bound to its ligand CCL22. The efficacy of CCR4 antagonism as an immunotherapeutic agent was evaluated by tumor weights, growth kinetics and survival curves. CCR4+ Tregs were the predominant type of Tregs recruited to hepatitis B-associated HCC (HBV+ HCC), correlating with sorafenib resistance and HBV load titers. Compared with CCR4- Tregs, CCR4+ Tregs exhibited increased IL-10 and IL-35 expression, and enhanced functionality in suppressing CD8+ T cells. CCR4+ Tregs also displayed PD-1+TCF1+ stem-like properties. ATAC-seq data revealed substantial chromatin remodeling between tumor-infiltrating Tregs (TIL-Tregs) and induced Tregs, suggesting that long-term chromatin reprogramming accounted for the acquisition of enhanced immunosuppressive stem-like specificity by CCR4+ TIL-Tregs. Treatment with a CCR4 antagonist or N-CCR4-Fc blocked intratumoral Treg accumulation, overcame sorafenib resistance, and sensitized tumors to PD-1 checkpoint blockade. Intratumoral stem-like CCR4+ Tregs orchestrated immunosuppressive resource cells in the tumor microenvironment. CCR4 could be targeted to enhance antitumor immunity by specifically blocking infiltration of Tregs into the tumor microenvironment and inhibiting maintenance of the TIL-Treg pool. Targeting regulatory T cells is a promising approach in cancer immunotherapy; however, severe autoimmunity can occur following systemic regulatory T cell loss. This could be avoided by selectively depleting intratumoral regulatory T cells. Herein, targeting intratumoral stem-like CCR4+ regulatory T cells helped to overcome sorafenib resistance and sensitize tumors to immune checkpoint blockade in mouse models of liver cancer. This approach could have wide clinical applicability. [Display omitted] • CCR4+ Tregs, as the predominant type of Treg in HBV+HCC, orchestrate immunosuppressive resource cells. • CCR4+ Tregs exhibit specific cytokine-driven PD-1+TCF1+ stem-like properties. • N-CCR4-Fc, as a neutralizing pseudo-receptor, effectively blocks CCR4-mediated chemotaxis to enhance antitumor immunity. • Antagonism of CCR4 could be used to overcome sorafenib resistance and sensitize tumors to PD-1 checkpoint blockade. [ABSTRACT FROM AUTHOR]

HBsAg-specific antibody responses are difficult to detect during chronic hepatitis B infection (CHB) and are often overlooked. The aim of this study was to examine whether anti-HBs may be involved in functional cure (FC) by profiling anti-HBs responses in patients with CHB using a panel of specific assays. Longitudinal serum samples were obtained from 25 patients with CHB who were infected with HBV genotype A and were undergoing nucleos(t)ide analogue (NA) treatment: 14 achieved FC while 11 remained infected (non-FC). Anti-HBs immune complexes (HBsAg-IC), FcγRIIIa dimer binding, epitope specificity and neutralisation efficacy were measured. HBsAg-IC peaks were detected prior to HBsAg loss in 10/14 FC patients. These HBsAg-IC peaks overlapped with either an alanine aminotransferase (ALT) flare (8/10 patients), or a rise in ALT (2/10 patients). HBsAg-IC peaks were detected in 7/11 non-FC patients, but were not associated with an ALT flare. FCγRIIIa binding was detected in 9/14 FC patients, independent from detection of overlapping HBsAg-IC/ALT peaks. FC patients had stable HBsAg epitope occupancy across the study, whereas non-FC patients had a reduction in HBsAg epitope occupancy within the first 12–24 weeks of NA treatment. Convalescent sera from FC patients recognised more HBsAg epitopes and neutralised HBV infection more potently than anti-HBs derived from vaccinees. Neutralisation potency appeared to increase post-HBsAg loss in 4/5 FC patients examined. Using these assays, we confirm that anti-HBs responses are present and fluctuate over time in this cohort of patients with HBeAg+ CHB, who were infected with HBV genotype A and treated with NAs. Key anti-HBs profiles associated with either FC or failure to achieve FC were also identified, suggesting a role for anti-HBs responses in FC. Using a panel of assays to characterise hepatitis B surface antibody (anti-HBs) responses in a group of patients with chronic hepatitis B, we identified anti-HBs profiles associated with either functional cure, or failure to achieve functional cure. Functional cure was associated with immune complex peaks which overlapped with alanine aminotransferase flares. Conversely, in those who did not achieve functional cure, immune complex peaks were present, but were not associated with alanine aminotransferase flares, and a decline in anti-HBs diversity was observed early during treatment. [Display omitted] • HBsAg-IC peaks that coincided with either an ALT flare or a rise in ALT were detected before HBs-loss in 10/14 FC patients. • FcγRIIIa dimer binding was detected in 9/14 FC patients, independent of detection of overlapping HBsAg-IC/ALT peaks. • A reduction in HBsAg epitope occupancy was observed between 12 and 24 weeks of treatment in non-FC patients. • Convalescent sera recognised more HBsAg epitopes and neutralized HBV infection more potently than vaccinees' sera. • Neutralisation potency of sera increased up to 100 weeks after HBsAg loss in 4/5 FC patients examined. [ABSTRACT FROM AUTHOR]

Current antiviral therapies control but rarely eliminate HBV, leaving chronic HBV carriers at risk of developing hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring antiviral T-cell immunity could lead to HBV elimination and cure of chronically infected patients. We constructed bispecific T-cell engager antibodies that are designed to induce antiviral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and CD3 or CD28 on T cells. T-cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of the T-cell response was determined by detection of pro-inflammatory cytokines, effector function (by cytotoxicity) and antiviral effects. To study in vivo efficacy, immune-deficient mice were transplanted with HBVenv-positive and -negative hepatoma cells. The 2 T-cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent antiviral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. In vivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T-cell activation, tumor infiltration and reduction of tumor burden. This study demonstrates that the administration of HBVenv-targeting T-cell engager antibodies facilitates a robust T-cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC. T-cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies, they bind envelope proteins on the surface of the hepatitis B virus (HBV) and CD3 or CD28 on T cells. This way, they induce a potent antiviral and cytotoxic T-cell response that leads to the elimination of HBV-positive cells. These bispecific T-cell engager antibodies are exciting therapeutic candidates for chronic hepatitis B and HBV-associated hepatocellular carcinoma. [Display omitted] • HBV cure most likely requires reconstitution of anti-HBV immunity. • We designed T-cell engagers that bind HBV envelope proteins and CD3/CD28 on T cells. • HBV-specific T-cell engagers activate T cells and stimulate their antiviral activity. • Engaged T cells control HBV replication and eliminate HBV-positive hepatoma in vivo. [ABSTRACT FROM AUTHOR]

Loss of serum HBsAg is a hallmark of spontaneous and therapy induced resolution of HBV infection, since it generally reflects a profound decrease in viral replication. However, integrated HBV DNA can contribute to HBsAg expression independent of viral replication. The relative contributions of these sources of HBsAg are not well understood. Specifically, it is not known whether actively transcribed HBV integration could spread throughout the entire liver. The relative distribution of HBsAg and HBV RNA in liver biopsy tissue from HBeAg-negative (HBe−) patients was analyzed by immunohistochemistry and in situ hybridization (ISH), respectively. Frozen biopsy tissue was used for molecular analysis of intrahepatic viral RNA, virus-host chimeric transcripts and viral DNA. Immunohistochemistry and ISH analysis revealed HBsAg and HBV RNA positivity in virtually all hepatocytes in the liver of some HBe− patients despite very low viremia. Reverse transcription quantitative PCR and RNA-sequencing analysis confirmed high expression levels of HBV envelope-encoding RNAs. However, the amount of viral transcriptional template (covalently closed circular (ccc)DNA) was too low to support this ubiquitous HBV RNA expression. In contrast, levels of total cellular HBV DNA were consistent with ubiquitous HBV integration. Finally, RNA-sequencing revealed the presence of many HBV-host chimeric transcripts with the potential for HBsAg expression. Transcriptionally active HBV integration can extend to the entire liver in some HBe− patients. This can lead to ubiquitous HBsAg expression independent of HBV replication. In such patients, HBsAg is probably not a clinically useful surrogate marker for viral resolution or functional cure. Loss of serum hepatitis B surface antigen (HBsAg) indicates resolution of HBV infection. However, integrated HBV DNA can contribute to HBsAg production independently of viral replication. We investigated the extent of HBsAg-producing viral integration in the livers of patients with low serum viral loads. Our findings suggest that transcriptionally active HBV integration can extend to the entire liver in some patients, questioning the clinical utility of HBsAg as a surrogate marker for viral replication. [Display omitted] • A unique set of liver biopsies from patients with HBeAg-negative chronic hepatitis B infection was studied. • HBV RNA and antigen expression are ubiquitous in the liver despite low viremia. • Levels of the viral transcriptional template cccDNA are not sufficient for ubiquitous HBV RNA and antigen expression. • Ubiquitous viral antigen expression is associated with virus-host chimeric transcription. • Intrahepatic viral DNA and RNA levels are consistent with widespread HBV integration. [ABSTRACT FROM AUTHOR]

Current antiviral therapies, such as pegylated interferon-α and nucleos(t)ide analogues, effectively improve the quality of life of patients with chronic hepatitis B. However, they can only control the infection rather than curing infected hepatocytes. Complete HBV cure is hampered by the lack of therapies that can directly affect the viral minichromosome (in the form of covalently closed circular DNA [cccDNA]). Approaches currently under investigation in early clinical trials are aimed at achieving a functional cure, defined as the loss of HBsAg and undetectable HBV DNA levels in serum. However, achieving a complete HBV cure requires therapies that can directly target the cccDNA pool, either via degradation, lethal mutations or functional silencing. In this review, we discuss cutting-edge technologies that could lead to non-cytolytic direct cccDNA targeting and cure of infected hepatocytes. [ABSTRACT FROM AUTHOR]