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We investigated associations between hepatitis B virus (HBV) genome-length haplotype number (HN) at baseline in subjects with HBeAg-positive chronic hepatitis B (CHB), and the likelihood of achieving functional cure during direct-acting antiviral therapy METHOD: We analysed 86 HBeAg-positive baseline samples from patients with HBV genotypes A and D who were enrolled in a Phase II trial of tenofovir disoproxil fumarate (TDF) to determine if HN was a biomarker of HBsAg loss during therapy. Findings were validated using baseline samples from 181 patients with HBV genotypes A and D from an independent clinical trial utilising TDF or tenofovir alafenamide therapy in HBeAg-positive CHB.In the HBeAg-positive test cohort, patients with genotypes A or D and ≤2 haplotypes had a minimum of 21-fold higher likelihood of achieving HBsAg loss on TDF. Baseline HN (p 0.0001) was a stronger predictor of HBsAg loss on therapy than HBsAg titre (p = 0.03), HBeAg titre (p = 0.0002), or the presence of HBV basal core promoter (A1762T, p = 0.0379 and G1764A, p = 0.0176) or G1896A precore mutations (p = 0.0218). This finding was validated in the independent validation cohort. HN was statistically higher in patients with HBV genotypes B or C infection compared to genotypes A and D.Baseline HN ≤2 predicts which patients with HBV genotypes A or D will more likely progress to functional cure on current direct-acting antiviral therapy, with greater accuracy than current biomarkers including baseline HBsAg and HBeAg titre.

Background Serum hepatitis B surface antigen (HBsAg) levels correlate with the duration of chronic hepatitis B virus (HBV) infection and may predict the extent of hepatic fibrosis. Methods We analyzed data from the SONIC-B database, which contains data from 8 global randomized trials and 2 large hepatology centers. Relationship between HBsAg levels and presence of significant fibrosis (Ishak 3–4) or cirrhosis (Ishak 5–6) were explored, and clinically relevant cutoffs were identified to rule out cirrhosis. Results The dataset included 2779 patients: 1866 hepatitis B e antigen (HBeAg)-positive; 322 with cirrhosis. Among HBeAg-positive patients, lower HBsAg levels were associated with higher rates of significant fibrosis (odds ratio [OR], 0.419; P < .001) and cirrhosis (OR, 0.435; P < .001). No relationship was observed among HBeAg-negative patients. Among HBeAg-positive patients, genotype-specific HBsAg cutoffs had excellent negative predictive values (>97%) and low misclassification rates (≤7.1%) and may therefore have utility in ruling out cirrhosis. Diagnostic performance of the HBsAg cutoffs was comparable among patients in whom cirrhosis could not be ruled out with fibrosis 4 (FIB-4). Conclusions Hepatitis B virus genotype-specific HBsAg cutoffs may have utility in ruling out presence of cirrhosis in HBeAg-positive patients with genotypes B, C, and D and can be an adjunct to FIB-4 to reduce the need for further testing.

Chronic hepatitis B (CHB) is characterized by progression through different phases of hepatitis B virus (HBV) infection and disease. Although not necessary for HBV replication, there is increasing evidence that HBV splice variants are associated with liver disease progression and pathogenesis. However, there have been no studies till date on the frequency or diversity of splice variants for different HBV genotypes across the phases of CHB. Next generation sequencing data from 404 patient samples of HBV genotype A, B, C or D in Phase I, Phase II or Phase IV of CHB was analysed for HBV splice variants using an in house bioinformatics pipeline. HBV splice variants differed in frequency and type by genotype and phase of natural history. Splice variant Sp1 was the most frequently detected (206/404, 51% of patients), followed by Sp13 (151/404 37% of patients). The frequency of variants was generally highest in Phase II (123/165, 75% of patients), a phase typically associated with enhanced immune activation, followed by Phase I (69/99, 70% of patients). Splice variants were associated with reduced hepatitis B e antigen (HBeAg) levels and statistically reduced likelihood of achieving HBsAg loss (functional cure) in Phase II patients for Sp1 and Sp13 (p = .0014 and .0156, respectively). The frequency of HBV splice variants in patient serum differed markedly by HBV genotype and phase of CHB natural history. The increased levels of HBV splice variants detected in CHB phase II patients compared with the higher replicative Phase I in particular warrants further investigation.

Entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are the first-line treatment agents for chronic hepatitis B virus (HBV). Recently, whether the degree to which the risk of hepatocellular carcinoma (HCC) may be reduced by ETV vs TDF has been debated. We compared the incidence of HCC among treatment-naïve patients receiving TDF vs ETV in the United States.From a large administrative medical claims database of commercially insured patients, we identified 166,933 adults with a diagnosis of chronic hepatitis B and a minimum of 12 months of prior enrolment, of whom 3934 and 6127 initiated ETV and TDF respectively. Fine-Gray hazard regression models incorporating treatment propensity scores (PS) were used to estimate the risk of HCC incidence associated with TDF vs ETV; variables considered for adjustment included demographic characteristics, concomitant medication use and baseline comorbidities, as well as competing events including liver transplantation and medication changes.After PS weighting, the TDF and ETV groups were well-matched. During the follow-up, 90 patients developed HCC, including 50 receiving ETV and 40 receiving TDF, giving rise to crude incidence rates of 0.62 per 100 person-years (PY) and 0.30 per 100 PY respectively. In PS-weighted, multivariable analysis, TDF was associated with a subdistribution hazard ratio for HCC of 0.58 (95% confidence interval [CI]: 0.38-0.89) compared to ETV. Results were similar when patients ≥40 years and men and women were analysed separately.Among commercially insured, treatment-naïve patients with chronic hepatitis B in the United States, treatment with TDF was associated with significantly lower risk of HCC than ETV.

Background and aims In patients with chronic hepatitis B (CHB) infection, activation of toll-like receptor 8 may induce antiviral immunity and drive functional cure. Selgantolimod, a toll-like receptor 8 agonist, was evaluated in patients with CHB who were virally suppressed on oral antiviral treatment or viremic and not on oral antiviral treatment. Approach and results In this phase 1b study, patients were randomized 4:1 to receive either selgantolimod or placebo once weekly. Virally suppressed patients received either 1.5 mg (for 2 weeks) or 3 mg (for 2 weeks or 4 weeks). Viremic patients received 3 mg for 2 weeks. The primary endpoint was safety, as assessed by adverse events (AEs), laboratory abnormalities, and vital sign examination. Pharmacokinetic and pharmacodynamic parameters were assessed by plasma analysis. A total of 38 patients (28 virally suppressed, 10 viremic) were enrolled from six sites in Australia, New Zealand, and South Korea. Twenty patients (53%) experienced an AE and 32 (84%) had laboratory abnormalities, all of which were mild or moderate in severity. The most common AEs were headache (32%), nausea (24%), and dizziness (13%). With a half-life of 5 hours, no accumulation of selgantolimod was observed with multiple dosing. Selgantolimod induced transient dose-dependent increases in serum cytokines, including IL-12p40 and IL-1RA, which are important for the expansion and activity of multiple T- cell subsets and innate immunity. Conclusion Selgantolimod was safe and well-tolerated in virally suppressed and viremic patients with CHB and elicited cytokine responses consistent with target engagement. Further studies with longer durations of selgantolimod treatment are required to evaluate efficacy.

Hepatitis B virus (HBV) core protein-targeting compounds are in or entering clinical development without a standardized nomenclature. We propose a naming convention for these core-targeting antiviral products to provide clarity and accelerate HBV drug development.

More systematic analysis of hepatitis B virus (HBV) genome diversity, linked with tenofovir disoproxil fumarate (TDF) treatment and HBeAg seroconversion, are needed. GS-US-174-0115 was a double-blind, placebo-controlled, Phase 3, 192-week clinical trial that evaluated TDF in adolescents with chronic hepatitis B (CHB). HBV full-genome deep sequencing was performed using Illumina MiSeq at baseline (BL; n = 85), Week 8 (W8; n = 80), Week 72 (W72; PBO only, n = 42), and treatment-free follow-up (TDF only, n = 25). The viral diversity was calculated using Shannon entropy and population nucleotide diversity with a 2% variant cutoff. Our data showed (i) a higher viral diversity in the X region at baseline than the core/polymerase/surface regions, (ii) higher core/surface viral diversity at baseline for patients with seroconversion, (iii) an expected reduction in viral diversity after 8 weeks of TDF treatment, and (iv) a drop in viral diversity at W72 for patients receiving placebo with a seroconversion (n = 7). The higher viral diversity in X was associated with higher baseline alanine aminotransferase (ALT) levels (p

Introduction We evaluated 8, 12, or 24 weeks of ledipasvir/sofosbuvir in patients with hepatitis C virus and end-stage renal disease undergoing dialysis. Methods Primary efficacy end point was sustained virologic response 12 weeks after treatment. Primary safety end point was treatment discontinuation because of adverse events (AEs). Results Ninety-four percent (89/95) achieved sustained virologic response 12 weeks after treatment. Six patients died during treatment (n = 4) or before study completion (n = 2); no deaths were related to treatment. No patients discontinued treatment because of AEs. Thirteen percent had serious AEs; none were related to treatment. Discussion Treatment with ledipasvir/sofosbuvir was safe and effective in patients with end-stage renal disease undergoing dialysis.

Background: Sofosbuvir, a prodrug nucleoside inhibitor of hepatitis C virus, has a predominant circulating metabolite that is renally eliminated. Whether sofosbuvir is associated with chronic kidney disease (CKD) progression is not well understood.Methods: We performed a retrospective analysis of patients with estimated glomerular filtration rate (eGFR) 30-89 mL/min/1.73 m2 treated with sofosbuvir in 76 Phase 2/3 registrational trials. We evaluated eGFR at each study visit. Separately, we performed a retrospective analysis of an administrative claims database (IQVIA PharMetrics Plus™) to compare the risk of incident end-stage renal disease (ESRD) associated with the use of sofosbuvir or non-sofosbuvir regimens among patients with CKD using propensity score methods. Exposure, CKD status and outcomes were determined using diagnosis and medication claim codes. Cox proportional hazards methods were used to estimate ESRD risk.Results: Among 4642 trial participants with baseline stage 2 CKD (eGFR 60-89 ml/min/1.73 m2 ) and 682 trial participants with stage 3 CKD (eGFR 30-59 ml/min/1.73 m2 ) mean (SD) eGFR improved from baseline to 4 weeks post-treatment (+0.7 [9.3] and +2.6 [8.8] ml/min/1.73 m2 , respectively; p < 0.001 each). In the second analysis, among 2042 patients with CKD receiving sofosbuvir-based regimens compared to 431 receiving non-sofosbuvir-based regimens, after adjusting for baseline covariates and weighting based on treatment propensity scores, there was no significant difference in risk of ESRD (adjusted HR = 0.85, 95% CI: 0.51-1.42).Conclusions: Clinical trial participants with CKD did not experience worsening eGFR during sofosbuvir-based treatment, and sofosbuvir was not associated with an increased risk of ESRD in patients with CKD in a nationally-representative administrative claims database.

Aim The combination of ledipasvir and sofosbuvir (LDV/SOF) has been approved for the treatment of various hepatitis C virus (HCV) genotypes across many countries. This article presents an integrated analysis of three prospective phase II/III trials in the Asia-Pacific region to evaluate the efficacy and safety of 12 weeks of LDV/SOF in HCV genotype 2 patients without cirrhosis or with compensated cirrhosis. Methods A total of 200 patients were included in the integrated analysis. The primary end-point was the rate of sustained virologic response for 12 weeks after the end of therapy (SVR12), analyzed by fibrosis stage, treatment history, HCV genotype subtype, and presence of baseline resistance-associated substitutions (RAS). Safety was evaluated by adverse events and laboratory abnormalities. Results Twelve weeks of treatment with LDV/SOF was associated with high SVR12 rates (overall 98%) in patients with genotype 2 HCV, irrespective of fibrosis stage, treatment history, genotype 2 subtype, and presence of baseline non-structural protein 5A resistance-associated substitution (NS5A RAS), and LDV/SOF was well tolerated. Conclusions Twelve weeks of treatment with LDV/SOF provides a highly effective and safe treatment for patients with genotype 2 HCV, including those with advanced fibrosis. As a ribavirin-free and protease inhibitor-free regimen with minimal on-treatment monitoring requirements, LDV/SOF can potentially play a crucial role in achieving the WHO's goal of HCV elimination.

Background & Aims We aim to describe the liver immune microenvironment by analyzing liver biopsies from patients with chronic HBV infection (CHB). Host immune cell signatures and their corresponding localization were characterized by analyzing the intrahepatic transcriptome in combination with a custom multiplex immunofluorescence panel. Method Matching FFPE and fresh frozen liver biopsies were collected from immune active patients within the open-label phase IV study GS-US-174-0149. RNA-Seq was conducted on 53 CHB liver biopsies from 46 patients. Twenty-eight of the 53 samples had matched FFPE biopsies and were stained with a 12-plex panel including cell segmentation, immune and viral biomarkers. Corresponding serum samples were screened using the MSD Human V-plex Screen Service to identify peripheral correlates for the immune microenvironment. Results Using unsupervised clustering of the transcriptome, we reveal two unique liver immune signatures classified as immune high and immune low based on the quantification of the liver infiltrate gene signatures. Multiplex immunofluorescence analysis demonstrated large periportal lymphoid aggregates in immune high samples consisting of CD4 and CD8 T cells, B cells and macrophages. Differentiation of the high and low immune microenvironments was independent of HBeAg status and peripheral viral antigen levels. In addition, longitudinal analysis indicates that treatment and normalization of ALT correlates with a decrease in liver immune infiltrate and inflammation. Finally, we screened a panel of peripheral biomarkers and identified ICAM-1 and CXCL10 as biomarkers that strongly correlate with these unique immune microenvironments. Conclusion These data provide a description of immune phenotypes in patients with CHB and show that immune responses are downregulated in the liver following nucleotide analogue treatment. This may have important implications for both the safety and efficacy of immune modulator programs aimed at HBV cure. Lay summary Liver biopsies from patients with chronic hepatitis B were submitted to RNA-Seq and multiplex immunofluorescence and identified two different liver immune microenvironments: immune high and immune low. Immune high patients showed elevated immune pathways, including interferon signaling pathways, and increase presence of immune cells. Longitudinal analysis of biopsies from treatment experienced patients showed that treatment correlates with a marked decrease in inflammation and these findings may have important implications for both safety and efficacy of immune modulator programs for HBV cure., Graphical abstract, Highlights • Two different liver immune microenvironments were identified in patients with chronic hepatitis B: immune high and immune low. • Immune high patients had elevated immune pathway activity and immune cell signatures corresponding to B cells, T cells and macrophages. • Antiviral treatment and normalization of ALT correlates with a marked decrease in liver immune infiltrate and inflammation. • CXCL10 and ICAM-1 were identified as peripheral biomarkers that correlated with these differentiated immune microenvironments.

Background & Aims To evaluate the hypothesis that increasing T cell frequency and activity may provide durable control of hepatitis B virus (HBV), we administered nivolumab, a programmed death receptor 1 (PD-1) inhibitor, with or without GS-4774, an HBV therapeutic vaccine, in virally suppressed patients with HBV e antigen (HBeAg)-negative chronic HBV. Methods In a phase Ib study, patients received either a single dose of nivolumab at 0.1 mg/kg (n = 2) or 0.3 mg/kg (n = 12), or 40 yeast units of GS-4774 at baseline and week 4 and 0.3 mg/kg of nivolumab at week 4 (n = 10). The primary efficacy endpoint was mean change in HBV surface antigen (HBsAg) 12 weeks after nivolumab. Safety and immunologic changes were assessed through week 24. Results There were no grade 3 or 4 adverse events or serious adverse events. All assessed patients retained T cell PD-1 receptor occupancy 6–12 weeks post-infusion, with a mean total across 0.1 and 0.3 mg/kg cohorts of 76% (95% CI 75–77), and no significant differences were observed between cohorts (p = 0.839). Patients receiving 0.3 mg/kg nivolumab without and with GS-4774 had mean declines of −0.30 (95% CI −0.46 to −0.14) and −0.16 (95% CI −0.33 to 0.01) log10 IU/ml, respectively. Patients showed significant HBsAg declines from baseline (p = 0.035) with 3 patients experiencing declines of >0.5 log10 by the end of study. One patient, whose HBsAg went from baseline 1,173 IU/ml to undetectable at week 20, experienced an alanine aminotransferase flare (grade 3) at week 4 that resolved by week 8 and was accompanied by a significant increase in peripheral HBsAg-specific T cells at week 24. Conclusions In virally suppressed HBeAg-negative patients, checkpoint blockade was well-tolerated and led to HBsAg decline in most patients and sustained HBsAg loss in 1 patient. Lay summary Chronic hepatitis B virus infection (CHB) is characterized by a dysfunctional immune response. In patients with CHB, inhibitory receptors, such as programmed death receptor 1 (PD-1) are overexpressed on T cells, leading to an ineffective immune response in the liver. Herein, we show that the PD-1 inhibitor, nivolumab, is safe and effective for the treatment of virally suppressed patients with CHB. Australian New Zealand Clinical Trials Registry (http://www.anzctr.org.au/) number: ACTRN12615001133527.

International audience; In patients with chronic hepatitis B (CHB), loss of hepatitis B surface antigen (HBsAg) is considered a functional cure. However, HBsAg loss is uncommon with existing therapies, and predictive factors associated with HBsAg seroreversion are unknown. Using pooled data from three phase 3 clinical trials of patients with CHB treated with nucleos(t)ide analogue (NUC) monotherapy or peginterferon (Peg-IFN) ± NUC combination therapy, we conducted a retrospective analysis to characterize patients who achieved sustained HBsAg loss, the predictors of HBsAg seroreversion, and the impact of hepatitis B surface antibody (anti-HBs) seroconversion on durability of HBsAg loss. In these three international trials, 1,381 adults with CHB received either NUC monotherapy for up to 10 years or Peg-IFN-containing regimens for up to 1 year. A total of 55 patients had confirmed HBsAg loss, defined as two or more consecutive negative-qualitative HBsAg results, with a minimum of one repeat result after the end of treatment. Throughout a median of 96 (quartile [Q]1, Q3, 46, 135) weeks follow-up after HBsAg loss, HBsAg loss was durable in 82% (n = 45) of patients, with 10 patients experiencing HBsAg seroreversion. Anti-HBs seroconversion was observed during follow-up in 78% of patients who lost HBsAg and in 60% of those who subsequently seroreverted. In analyzing predictors of HBsAg seroreversion, study treatment was significant, yet anti-HBs seroconversion and treatment duration after initial HBsAg loss were not. Risk of HBsAg seroreversion was observed to be lower if HBsAg loss was sustained through the off-treatment week 24 visit (8/10 seroreversions occurred by posttreatment week 24). Conclusion: HBsAg loss after NUC or Peg-IFN-containing regimens was durable in 82% of patients with CHB. Anti-HBs seroconversion and treatment duration after initial HBsAg loss were not significantly associated with durability of HBsAg loss.

Ruling out the presence of cirrhosis is important for the management of chronic hepatitis B. We aimed to study and optimise the performance of two non-invasive indices for ruling out cirrhosis: the aspartate aminotransferase-platelet ratio index (APRI) and fibrosis score based on four factors (FIB-4).We applied established cutoffs to rule in (APRI2·00; FIB-43·25) or rule out (APRI1·00; FIB-41·45) cirrhosis to data from eight global randomised trials that required baseline biopsy, and identified new cutoffs aiming for a sensitivity for detection of cirrhosis greater than 90% and a negative predictive value (NPV) of greater than 95% in the same dataset. We externally validated the new cutoffs using data from all consecutive biopsied patients from two tertiary referral hospitals in the Netherlands and Canada.In the derivation dataset (n=2926; of whom 1750 were Asian); 340 (12%) individuals had cirrhosis. The validation cohort consisted of 1034 individuals (of whom 575 were Asian), with 155 (15%) individuals with cirrhosis. Application of conventional cutoffs for FIB-4 in the derivation dataset yielded unclassifiable results in 686 (23%) individuals, and 139 (41%) of the 340 patients with cirrhosis were misclassified as having no cirrhosis. Similarly, conventional cutoffs for APRI in the derivation dataset yielded unclassifiable results in 706 (24%) individuals, and 153 (45%) were misclassified as having no cirrhosis. An APRI of 0·45 or less had sensitivity of 91·5%, an NPV of 95·4%, and misclassified 29 (9%) of 340 individuals with cirrhosis in the derivation dataset, but performance was reduced in the validation set (22 [14%] of 155 individuals with cirrhosis misclassified). A FIB-4 score of 0·70 had a sensitivity of 90·9%, an NPV of 96·6%, and misclassified 31 (9%) of individuals with cirrhosis in the derivation dataset. In the validation cohort, the same score gave a sensitivity of 94·2%, an NPV of 97·3%, and misclassified nine (6%) of the individuals with cirrhosis. Subgroup analysis indicated that the new FIB-4 cutoff performed acceptably in all subgroups except for individuals aged 30 years or younger.Conventional cutoffs for APRI and FIB-4 should not be used to guide management of patients with chronic hepatitis B due to high rates of misclassification. A newly identified and externally validated cutoff for FIB-4 (≤0·70) can be used to exclude cirrhosis in patients over 30 years of age.Foundation for Liver and Gastrointestinal Research, Rotterdam, Netherlands.

Background & Aims: Hepatocellular carcinoma (HCC) is the leading cause of death in patients with chronic hepatitis. In this international collaboration, we sought to develop a global universal HCC risk score to predict the HCC development for patients with chronic hepatitis.Methods: A total of 17,374 patients, comprising 10,578 treated Asian patients with chronic hepatitis B (CHB), 2,510 treated Caucasian patients with CHB, 3,566 treated patients with hepatitis C virus (including 2,489 patients with cirrhosis achieving a sustained virological response) and 720 patients with non-viral hepatitis (NVH) from 11 international prospective observational cohorts or randomised controlled trials, were divided into a training cohort (3,688 Asian patients with CHB) and 9 validation cohorts with different aetiologies and ethnicities (n = 13,686).Results: We developed an HCC risk score, called the aMAP score (ranging from 0 to 100), that involves only age, male, albumin–bilirubin and platelets. This metric performed excellently in assessing HCC risk not only in patients with hepatitis of different aetiologies, but also in those with different ethnicities (C-index: 0.82–0.87). Cut-off values of 50 and 60 were best for discriminating HCC risk. The 3- or 5-year cumulative incidences of HCC were 0–0.8%, 1.5–4.8%, and 8.1–19.9% in the low- (n = 7,413, 43.6%), medium- (n = 6,529, 38.4%), and high-risk (n = 3,044, 17.9%) groups, respectively. The cut-off value of 50 was associated with a sensitivity of 85.7–100% and a negative predictive value of 99.3–100%. The cut-off value of 60 resulted in a specificity of 56.6–95.8% and a positive predictive value of 6.6–15.7%.Conclusions: This objective, simple, reliable risk score based on 5 common parameters accurately predicted HCC development, regardless of aetiology and ethnicity, which could help to establish a risk score-guided HCC surveillance strategy worldwide.Lay summary: In this international collaboration, we developed and externally validated a simple, objective and accurate prognostic tool (called the aMAP score), that involves only age, male, albumin–bilirubin and platelets. The aMAP score (ranged from 0 to 100) satisfactorily predicted the risk of hepatocellular carcinoma (HCC) development among over 17,000 patients with viral and non-viral hepatitis from 11 global prospective studies. Our findings show that the aMAP score had excellent discrimination and calibration in assessing the 5-year HCC risk among all the cohorts irrespective of aetiology and ethnicity.© 2020 European Association for the Study of the Liver. Published by Elsevier B.V. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

HBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms.DNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline.HBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations.Targeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC.Fresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations

Hepatocellular carcinogenesis of hepatitis B virus (HBV) infection may arise from integration of viral DNA into the host genome. We aimed to gauge the effect of viral inhibition on transcriptionally active HBV-host integration events and explore the correlation of viral integrations with host gene dysregulation.We leveraged data and biospecimens from an interventional trial, in which patients with HBV viremia above 2000 IU/mL and minimally raised serum liver enzyme were randomized to receive tenofovir disoproxil fumarate (TDF) or placebo for 3 years. Total RNA-sequencing was performed on paired liver biopsies taken before and after the 3-year intervention in 119 patients. Virus-host chimeric reads were captured to quantify the number of distinct viral integrations. Dysregulation of a host gene disrupted by viral integration was defined by aberrant expression2 standard deviations away from samples without viral integration.The TDF (n = 64) and placebo groups (n = 55) were comparable at baseline. Expressed viral integrations were detected in all pre- and posttreatment samples. The number of distinct viral integrations significantly correlated with circulatory biomarkers indicative of viral activities including HBV DNA, RNA, and viral antigens (P.0003 for all correlations). Moreover, TDF vs placebo achieved a significantly greater reduction in distinct viral integrations, with 3.28-fold and 1.81-fold decreases in the expressed integrations per million reads, respectively (analysis of covariance, P = .037). Besides, viral integrations significantly correlated with host gene dysregulation.Inhibition of viral replication reduces the number of transcriptionally active distinct HBV-host DNA integrations in patients with substantial viremia. Given the mutagenic potentials of viral integrations, such treatment effects should be considered in patient management.

Background & Aims: One strategy to treat chronic hepatitis B virus (HBV) infection could be to increase the functions of virus-specific T cells. We performed a multicenter phase 2 study to evaluate the safety and efficacy of GS-4774, a yeast-based therapeutic vaccine engineered to express HBV antigens, given with tenofovir disoproxil fumarate (TDF) to untreated patients with chronic HBV infection. Methods: We performed an open-label study at 34 sites in Canada, Italy, New Zealand, Romania, South Korea, and United States from July 2014 to August 2016. Adults who were positive for HB surface antigen (HBsAg) > 6 months and levels of HBV DNA ≥2000 IU/mL who had not received antiviral treatment for HBV within 3 months of screening were randomly assigned (1:2:2:2) to groups given oral TDF 300 mg daily alone (n = 27; controls) or with 2, 10, or 40 yeast units GS-4774 (n = 168), administered subcutaneously every 4 weeks until week 20 for a total of 6 doses. Blood samples were collected and analyzed and patients received regular physical examinations. Efficacy was measured by decrease in HBsAg from baseline to week 24. Specific responses to HBV (production of interferon gamma [IFNG], tumor necrosis factor [TNF], interleukin 2 [IL2], and degranulation) were measured in T cells derived from 12 HBeAg-negative patients with genotype D infections, after overnight or 10 days of stimulation of peripheral blood mononuclear cells with peptides from the entire HBV proteome. T-regulatory cells were analyzed for frequency and phenotype. Data from studies of immune cells were compared with data on reductions in HBsAg, HBV DNA, and alanine aminotransferase in blood samples from patients. Results: GS-4774 was safe and well tolerated but did not produce significant decreases in levels of HBsAg. Production of IFNG, TNF, and IL2 increased significantly at weeks 24 and 48, compared with baseline, in HBV-specific CD8+ T cells from patients given GS-4774 but not from controls. GS-4774 had greater effects on CD8+ than CD4+ T cells, which were not affected at all or very weakly by TDF with or without GS-4774. GS-4774 did not affect responses of T cells to other viruses tested. HBV core peptides induced the greatest production of IFNG by T cells following overnight stimulation, whereas HBV envelope antigens did not induce a response. Following 10 days of stimulation, production of IFNG and TNF increased with time of exposure to GS-4774; the greatest levels of responses were to HBV envelope antigens followed by core and polymerase peptides. We observed a correlation in patients given GS-4774 between increased T-cell functions and reductions in numbers of T-regulatory cells. Conclusions: In a phase 2 study of patients with chronic HBV infection given TDF with or without GS-4774, we found that vaccination can increase production of IFNG, TNF, and IL2 by CD8+ T cells exposed to antigenic peptides, with little effect on CD4+ T cells. Although GS-4774 did not reduce levels of HBsAg in patients, its strong immune stimulatory effect on CD8+ T cells might be used in combination with other antiviral agents to boost the antivirus immune response. Clinicaltrials.gov no: NCT02174276.

Achieving sustained virologic response (SVR) among patients with hepatitis C virus (HCV) leads to patient reported outcome (PRO) improvement. We aimed to assess the long-term post-SVR PRO trends in HCV patients with cirrhosis.Patients with HCV and cirrhosis treated in clinical trials with direct acting antiviral agents (DAAs) who achieved SVR-12 were prospectively enrolled in a long-term registry (clinicaltrials.gov #NCT02292706). PROs were collected every 24 weeks using the Short Form-36v2 (SF-36), CLDQ-HCV, and WPAI-HCV.Pre-treatment baseline data were available for 854 cirrhotic patients who achieved SVR after DAAs. Of these, 730 had compensated (CC) and 124 had decompensated cirrhosis (DCC) before treatment- patients with DCC reported severe impairment in their PROs in comparison to CC patients (by mean -5% to -16% of a PRO range size; p.05 for 16 out of 20 studied PROs]. After achieving SVR and registry enrollment, significant PRO improvements were noted from pre-treatment levels in 11/20 domains for those with DCC (+4% to +21%) and 19/20 PRO domains in patients with CC (+3% to +17%). Patients with baseline DCC had higher rates of hepatocellular carcinoma and mortality (P.05). In patients with CC, the PRO gains persisted up to 168 weeks (3.5 years) of registry follow-up. In patients with DCC, the improvements lasted for at least 96 weeks but a declining trend after year 2.Patients with HCV cirrhosis experience severe PRO impairment at baseline with sustainable improvement after SVR. Though those with DCC experience improvement, there is a decline after 2 years.

BACKGROUND AND AIMS: We conducted haplotype analysis of complete hepatitis B virus (HBV) genomes following deep sequencing from 368 patients across multiple phases of chronic hepatitis B (CHB) infection from four major genotypes (A-D), analyzing 4,110 haplotypes to identify viral variants associated with treatment outcome and disease progression. APPROACH AND RESULTS: Between 18.2% and 41.8% of nucleotides and between 5.9% and 34.3% of amino acids were 100% conserved in all genotypes and phases examined, depending on the region analyzed. Hepatitis B e antigen (HBeAg) loss by week 192 was associated with different haplotype populations at baseline. Haplotype populations differed across the HBV genome and CHB history, this being most pronounced in the precore/core gene. Mean number of haplotypes (frequency) per patient was higher in immune-active, HBeAg-positive chronic hepatitis phase 2 (11.8) and HBeAg-negative chronic hepatitis phase 4 (16.2) compared to subjects in the "immune-tolerant," HBeAg-positive chronic infection phase 1 (4.3, P< 0.0001). Haplotype frequency was lowest in genotype B (6.2, P< 0.0001) compared to the other genotypes (A = 11.8, C = 11.8, D = 13.6). Haplotype genetic diversity increased over the course of CHB history, being lowest in phase 1, increasing in phase 2, and highest in phase 4 in all genotypes except genotype C. HBeAg loss by week 192 of tenofovir therapy was associated with different haplotype populations at baseline. CONCLUSIONS: Despite a degree of HBV haplotype diversity and heterogeneity across the phases of CHB natural history, highly conserved sequences in key genes and regulatory regions were identified in multiple HBV genotypes that should be further investigated as targets for antiviral therapies and predictors of treatment response.

Background and aims Most patients with cirrhosis induced by chronic HBV infection experience fibrosis regression after long-term antiviral treatment, while some remain cirrhotic. Fibrosis regression is associated with lower odds of developing hepatic decompensation and hepatocellular carcinoma, but mechanisms impacting differential fibrosis regression between individuals are unclear. We asked whether soluble molecules, including serum microRNAs, could serve as biomarkers of fibrosis regression. Methods We analysed cryopreserved sera from clinical trials in which cirrhotic HBV-infected patients (baseline Ishak fibrosis score of 5-6) received 240 weeks of nucleotide analogue treatment. Liver biopsies at week 240 in these trials showed 71/96 patients (74%) had fibrosis regression (Ishak ≤ 4) while 25/96 (26%) remained cirrhotic (Ishak 5-6). We quantified inflammatory markers (CXCL10, soluble CD163) and miRNAs (n = 179) from serum at baseline, week 48 and week 240 of treatment in a sub-cohort of patients with (n = 14) or without (n = 14) fibrosis regression. Results CXCL10, sCD163 and miRNAs previously associated with HBV replication and inflammation decreased during treatment but did not differ based on fibrosis regression. Two miRNAs (miR-421 and miR-454-3p) had lower baseline expression in patients with subsequent fibrosis regression. In all, 27 miRNAs differed at week 240 and had higher expression in patients with fibrosis regression (eg miR-199a-3p, miR-423-3p, miR-142-3p, miR-let-7d-5p). Several miRNAs (miR-141-3p, let-7d-5p) that correlated with regression have previously been implicated in the pathophysiology of non-alcoholic steatohepatitis. Conclusions In cirrhotic patients with chronic HBV infection treated with antiviral therapy, serum miRNAs have differential expression based on fibrosis regression, suggesting potential utility as biomarkers.

Nucleos(t)ide analouges (NUC) treatment prevents progression of liver fibrosis in subjects with chronic hepatitis B (CHB). However, risk for hepatocellular carcinoma (HCC) persists despite viral suppression. Specific HBV variants have been associated with adverse outcomes, including HCC, however the frequency of these variants during the seemingly benign immunotolerant (IT) phase is unknown. Next generation sequencing and detailed virological characterization on a cohort of treatment-naive IT subjects was performed to determine the frequency of clinically relevant viral variants. Samples from 97 subjects (genotype B/C 55%/45%, median HBV-DNA 8.5 log10 IU/mL, median HBsAg 4.8 log10IU/mL, median HBeAg 3.6 log10 PEIU/mL) were analysed. Despite subjects being in the IT phase, clinically relevant HBV variants were common at baseline, particularly in the basal core promoter (BCP, overlaps the hepatitis B X (HBx) gene), precore, and PreS regions. BCP/HBx variants were independently associated with lower baseline HBeAg, HBsAg and HBV-DNA titres. Precore variants were independently associated with higher baseline ALT. Increased viral diversity was associated with increased age and lower HBV DNA, HBsAg and HBeAg levels. Low level (

Tenofovir disoproxil fumarate (TDF) is a recommended first-line therapy for both naïve and experienced patients with chronic hepatitis B (CHB), although reduced estimated glomerular filtration rate (eGFR), hypophosphatemia, hyperphosphaturia and Fanconi syndrome have been reported in some patients. Entecavir (ETV) could be considered as a rescue therapy for TDF-treated patients developing renal dysfunction, though patients with prior history of treatment with lamivudine (LAM) can develop ETV resistance strains, which can lead to potentially severe hepatitis flares. Tenofovir alafenamide (TAF), a new prodrug of tenofovir, has recently been developed to improve the renal and bone safety profile compared to TDF, while maintaining the same virologic efficacy. The recently published 48-week phase III TAF registration studies confirmed the superior safety profile. Here we describe a case of a 75-year-old woman with HBV mono-infection and compensated cirrhosis who developed ETV resistant strains and grade 3 chronic kidney disease after many years of LAM and adefovir (ADV) treatment and a TDF-induced Fanconi syndrome. The administration of 25mg/day of TAF, granted as part of a compassionate use program, rapidly suppressed viral replication to undetectable levels without worsening renal function or side effects.

Vesatolimod is an oral agonist of toll-like receptor 7 designed to minimize systemic exposure and side effects. We assessed the safety and efficacy of vesatolimod in viremic chronic hepatitis B (CHB) patients not currently on oral antiviral treatment (OAV) in a phase 2, multicentre, double-blind, randomized, placebo-controlled study. A total of 192 patients stratified by HBeAg status and alanine aminotransferase level were randomized 2:2:2:1 to receive oral vesatolimod (1-, 2- or 4-mg) or placebo once weekly for 12 weeks; tenofovir disoproxil fumarate (300-mg daily) was administered daily for 48 weeks. Efficacy was assessed by quantitative serum HBsAg decline at Week 24 from baseline. In addition to safety assessments, changes in whole-blood interferon-stimulated gene (ISG) transcripts and serum cytokines were explored. Most patients were male (64.1%) and HBeAg-negative (60.9%) at baseline. Among vesatolimod-treated patients, most (60.4%-69.1%) experienced ≥1 treatment-emergent adverse event; the majority were mild or moderate in severity. No clinically meaningful differences in HBsAg changes from baseline were observed between treatment groups. No patients experienced HBsAg loss, while 3 patients experienced HBeAg loss and hepatitis B e-antibody seroconversion at week 48. HBV DNA suppression rates were similar across all treatment arms at Week 24. ISG15 induction was dose-dependent and did not correlate with HBsAg changes. A small proportion of patients exhibited dose-dependent interferon-α induction that correlated with grade of influenza-like adverse events. Overall, vesatolimod is safe and well tolerated in CHB patients. Although consistent dose-dependent pharmacodynamic induction of ISGs was demonstrated, it did not result in clinically significant HBsAg decline.

Although there is substantial evidence suggesting poor health-related quality of life (HRQL) in patients with chronic hepatitis C (CHC), similar data in nonalcoholic steatohepatitis (NASH) have not been fully assessed. The aim is to compare HRQL scores in patients with CHC to those with NASH.Matched patients with advanced fibrosis (bridging fibrosis and compensated cirrhosis) due to CHC and NASH completed Short Form-36 (SF-36) questionnaire, Chronic Liver Disease Questionnaire (CLDQ), and Work Productivity and Activity Instrument questionnaire.We included 1,338 patients with NASH with advanced fibrosis (mean age 57.2 years, 47% men, 55% cirrhosis) and 1,338 matched patients with CHC. Patients with CHC and NASH had similar rates of employment and psychiatric disorders (P0.05). As expected, patients with NASH had higher body mass index (mean 33.7 vs 27.6) and more type 2 diabetes (74% vs 16%) (all P0.01). Patients with NASH had significantly lower HRQL scores related to physical health: Physical Functioning, Bodily Pain, General Health, Vitality, Physical Summary of SF-36, and Fatigue of CLDQ (P0.02). By contrast, patients with CHC had a lower Mental Health score of SF-36 and Emotional score of CLDQ and reported greater impairment in daily activities as measured by the Work Productivity and Activity Instrument questionnaire (P0.002). In multivariate analysis, after adjustment for demographic parameters, cirrhosis, and history of psychiatric disorders, having NASH was associated with lower physical HRQL scores and higher mental health-related scores (P0.05).Patients with NASH and advanced fibrosis have more impairment of their physical health-related scores than patients with CHC with advanced fibrosis. These data should dispel the misconception that NASH is an asymptomatic disease with little negative impact on patients' well-being.

Treatment with tenofovir disoproxil fumarate has been associated with renal toxicity or reductions in bone mineral density, or both, in some patients with chronic hepatitis B virus (HBV) infection. Tenofovir alafenamide is a tenofovir prodrug with high intrahepatic concentrations of active drug and reduced systemic tenofovir exposures compared with tenofovir disoproxil fumarate. In patients with chronic HBV, tenofovir alafenamide has shown efficacy non-inferior to that of tenofovir disoproxil fumarate with improved renal and bone safety. With this non-inferiority study, we aimed to evaluate the efficacy and safety of tenofovir alafenamide in patients with HBV infection switching from tenofovir disoproxil fumarate who are virally suppressed.Patients with chronic HBV infection who had been receiving tenofovir disoproxil fumarate for 48 weeks or more and who had HBV DNA less than the lower limit of quantification (LLOQ) for at least 12 weeks were recruited to this randomised, multicentre, double-blind, phase 3 non-inferiority study. Patients were randomly assigned in a 1:1 ratio to receive tenofovir alafenamide 25 mg once a day or to continue tenofovir disoproxil fumarate 300 mg once a day. The primary efficacy endpoint was loss of virological control, defined as the proportion of patients who received at least one dose of study drug who had HBV DNA of at least 20 IU/mL at week 48 by the modified US Food and Drug Administration (FDA) snapshot algorithm. Key safety endpoints were changes in hip and spine bone mineral density, estimated creatinine clearance by Cockcroft-Gault, and markers of bone turnover and renal tubular function. The study was powered for non-inferiority in efficacy of tenofovir alafenamide versus tenofovir disoproxil fumarate with a 4% margin. Investigators and patients were unaware of treatment allocation and on-treatment results. This trial is ongoing and is registered with ClinicalTrials.gov, number NCT02979613.Participants in this study were enrolled between Dec 29, 2016, and Oct 20, 2017. 541 patients were screened and 490 patients were randomly assigned to switch to tenofovir alafenamide or to stay on tenofovir disoproxil fumarate. Two patients assigned to receive tenofovir alafenamide did not receive treatment; thus the full analysis set for efficacy and safety analyses consisted of 243 patients in the tenofovir alafenamide group and 245 in the tenofovir disoproxil fumarate group. At week 48, one patient from each treatment group (both1%) had HBV DNA of at least 20 IU/mL (difference in proportion 0·0%, 95% CI -1·9 to 2·0), thereby showing non-inferior efficacy of tenofovir alafenamide to tenofovir disoproxil fumarate. Patients who received tenofovir alafenamide had significantly increased bone mineral density at hip (mean change 0·66% [SD 2·08] vs -0·51% [SD 1·91]; difference in least square means 1·17% [95% CI 0·80 to 1·54; p0·0001]) and at spine (mean change 1·74% [3·46] vs -0·11% [3·13]; difference in least square means 1·85% [1·24 to 2·46; p0·0001]), creatinine clearance by Cockcroft-Gault relative to tenofovir disoproxil fumarate (median change 0·94 mL/min [IQR -4·47 to 6·24] vs -2·74 mL/min [-7·89 to 1·88]; p0·0001), and improved markers of bone turnover and tubular function at week 48. The most common treatment-emergent adverse events were upper respiratory tract infection (18 [7%] of 243 patients in the tenofovir alafenamide group and 16 [7%] of 245 patients in the tenofovir disoproxil fumarate group) and nasopharyngitis (13 [5%] of 243 patients in the tenofovir alafenamide group and 12 [5%] of 245 patients in the tenofovir disoproxil fumarate group). The incidence of grade 3 and above adverse events and serious adverse events was low and similar between groups. No viral resistance was observed in patients who qualified for viral sequencing.These findings suggest that tenofovir alafenamide can be substituted for tenofovir disoproxil fumarate in patients with HBV infection for improved safety without a loss of efficacy.Gilead Sciences.

Background & Aims Tenofovir disoproxil fumarate (TDF) is a first‐line treatment for chronic hepatitis B (CHB). We aimed to describe the efficacy and safety profiles of TDF treatment for up to 10 years in a well‐described cohort of CHB patients. Methods Hepatitis B e antigen (HBeAg)‐negative and HBeAg‐positive patients from two randomised, double‐blind trials (ClinicalTrials. gov: NCT00117676 and NCT00116805) completed 48 weeks of randomised treatment with TDF or adefovir dipivoxil. A subset of these patients was then eligible to receive open‐label TDF treatment for up to 10 years. At Year 10, patients were assessed for virological suppression, alanine aminotransferase (ALT) normalisation, serological response, safety, and tolerability. Results Of 641 randomised and treated patients, 585 (91%) entered the open‐label extension phase with 203 (32%) patients completing Year 10 of the study. At Year 10, 118/118 (100%) of HBeAg‐negative patients and 78/80 (98%) of HBeAg‐positive patients with available data achieved hepatitis B virus (HBV) DNA

Background and aim Functional cure is the major goal of chronic hepatitis B (CHB) therapy though few biomarkers predict this outcome. HBsAg epitope occupancy can be influenced by therapeutic and immune pressure. The aim of this study was to map the HBsAg epitope profiles during long-term nucleos(t)ide analogue therapy in patients with genotype A CHB, in the context of HBsAg loss (SL)/seroconversion. Methods We evaluated 25 genotype A CHB patients in the GS-US-174-0103 trial of HBeAg-positive CHB patients treated with tenofovir or adefovir for 4 years, 14 who achieved SL whilst 11 had no change. We epitope mapped the major domains of HBsAg to identify those patients with HBsAg clearance profile (CP) (loss of binding at both loops 1 and 2 epitopes of the 'a' determinant) vs non-clearance profile (no change in epitope recognition, or loss of epitope binding at one loop only), correlating this to on-treatment HBsAg responses. Complexed anti-HBs was also measured. Results Analysis of the HBsAg epitope profiles of the 25 patients at baseline identified no predictive correlation with SL. In contrast, analysis at week 48 and end of study (week 192) or prior to SL identified significant predictive associations between development of HBsAg CPs and outcome of functional cure. The detection of a CP also correlated with the development of an alanine aminotransferase flare and detection of anti-HBs complexed with HBsAg. Conclusion The detection of HBsAg CPs by epitope mapping represents a novel viral biomarker, reflecting an emerging anti-HBs selection pressure prior to functional cure.

Background and Aims Tenofovir alafenamide (TAF) has similar efficacy to tenofovir disoproxil fumarate (TDF) but with improved renal and bone safety in chronic hepatitis B patients studied outside of China. We report 3-year results from two phase 3 studies with TAF in China (Clinicaltrials.gov: NCT02836249 and NCT02836236). Methods Chinese hepatitis B e antigen (HBeAg)-positive and -negative chronic hepatitis B patients with viremia and elevated alanine aminotransferase were randomized 2:1 to TAF or TDF treatment groups and treated in a double-blind fashion for 144 weeks (3 years). Efficacy responses were assessed by individual study while safety was assessed by a pooled analysis. Results Of the 334 patients (180 HBeAg-positive and 154 HBeAg-negative) randomized and treated, baseline characteristics were similar between groups. The overall mean age was 38 years and 73% were male. The mean HBV DNA was 6.4 log10 IU/mL. The median alanine aminotransferase was 88 U/L, and 37% had a history of antiviral use. At week 144, the proportion with HBV DNA

Background Patient knowledge about chronic diseases increases health-promoting behaviors and improves clinical outcomes. We assessed this association for patients with chronic viral hepatitis. Methods Untreated patients chronically infected with HBV (n = 500) or HCV (n = 500) were enrolled at 19 centers across India. A survey, adapted from the US CDC National Health and Nutrition Examination Survey (NHANES) questionnaire, was administered at a single visit to assess HBV/HCV knowledge, community disease awareness, treatment quality, and healthcare barriers. We developed the India Hepatitis Knowledge Index (IHKI), where a higher IHKI score (range 0–10) indicates increased hepatitis knowledge. Multivariate regression models evaluated demographic and disease factors. Results The overall mean IHKI score was 5.6 out of 10, with higher scores among patients with HBV (5.9) than HCV (5.3); p 2000 IU/ml. Among HCV patients, IHKI results had no significant associations with disease severity. Conclusions The association of IHKI with disease underscores the need to understand connections between hepatitis knowledge and progression and may guide efforts to address patient education and awareness of chronic viral hepatitis in India. Electronic supplementary material The online version of this article (doi:10.1007/s12072-016-9728-3) contains supplementary material, which is available to authorized users.