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

Background & Aims: Patterns of liver HBV antigen expression have been described but not quantified at single-cell resolution. We applied quantitative techniques to liver biopsies from individuals with chronic hepatitis B and evaluated sampling heterogeneity, effects of disease stage, and nucleos(t)ide (NUC) treatment, and correlations between liver and peripheral viral biomarkers. Methods: Hepatocytes positive for HBV core and HBsAg were quantified using a novel four-plex immunofluorescence assay and image analysis. Biopsies were analysed from HBeAg-positive (n = 39) and HBeAg-negative (n = 75) participants before and after NUC treatment. To evaluate sampling effects, duplicate biopsies collected at the same time point were compared. Serum or plasma samples were evaluated for levels of HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), and HBV RNA. Results: Diffusely distributed individual HBV core+ cells and foci of HBsAg+ cells were the most common staining patterns. Hepatocytes positive for both HBV core and HBsAg were rare. Paired biopsies revealed large local variation in HBV staining within participants, which was confirmed in a large liver resection. NUC treatment was associated with a >100-fold lower median frequency of HBV core+ cells in HBeAg-positive and HBeAg-negative participants, whereas reductions in HBsAg+ cells were not statistically significant. The frequency of HBV core+ hepatocytes was lower in HBeAg-negative participants than in HBeAg-positive participants at all time points evaluated. Total HBV+ hepatocyte burden correlated with HBcrAg, HBV DNA, and HBV RNA only in baseline HBeAg-positive samples. Conclusions: Reductions in HBV core+ hepatocytes were associated with HBeAg-negative status and NUC treatment. Variation in HBV positivity within individual livers was extensive. Correlations between the liver and the periphery were found only between biomarkers likely indicative of cccDNA (HBV core+ and HBcrAg, HBV DNA, and RNA). Impact and Implications: HBV infects liver hepatocyte cells, and its genome can exist in two forms that express different sets of viral proteins: a circular genome called cccDNA that can express all viral proteins, including the HBV core and HBsAg proteins, or a linear fragment that inserts into the host genome typically to express HBsAg, but not HBV core. We used new techniques to determine the percentage of hepatocytes expressing the HBV core and HBsAg proteins in a large set of liver biopsies. We find that abundance and patterns of expression differ across patient groups and even within a single liver and that NUC treatment greatly reduces the number of core-expressing hepatocytes.

Phenotypic CD8 T cell profiling in chronic hepatitis B to predict HBV-specific CD8 T cell susceptibility to functional restoration in vitro.

OBJECTIVE: Exhausted hepatitis B virus (HBV)-specific CD8 T cells in chronic HBV infection are broadly heterogeneous. Characterisation of their functional impairment may allow to distinguish patients with different capacity to control infection and reconstitute antiviral function. DESIGN: HBV dextramer+CD8 T cells were analysed ex vivo for coexpression of checkpoint/differentiation markers, transcription factors and cytokines in 35 patients with HLA-A2+chronic hepatitis B (CHB) and in 29 control HBsAg negative CHB patients who seroconverted after NUC treatment or spontaneously. Cytokine production was also evaluated in HBV peptide-stimulated T cell cultures, in the presence or absence of antioxidant, polyphenolic, PD-1/PD-L1 inhibitor and TLR-8 agonist compounds and the effect on HBV-specific responses was further validated on additional 24 HLA-A2 negative CHB patients. RESULTS: Severely exhausted HBV-specific CD8 T cell subsets with high expression of inhibitory receptors, such as PD-1, TOX and CD39, were detected only in a subgroup of chronic viraemic patients. Conversely, a large predominance of functionally more efficient HBV-specific CD8 T cell subsets with lower expression of coinhibitory molecules and better response to in vitro immune modulation, typically detected after resolution of infection, was also observed in a proportion of chronic viraemic HBV patients. Importantly, the same subset of patients who responded more efficiently to in vitro immune modulation identified by HBV-specific CD8 T cell analysis were also identified by staining total CD8 T cells with PD-1, TOX, CD127 and Bcl-2. CONCLUSIONS: The possibility to distinguish patient cohorts with different capacity to respond to immune modulatory compounds in vitro by a simple analysis of the phenotypic CD8 T cell exhaustion profile deserves evaluation of its clinical applicability.

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.

Background & Aims: 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. Methods: 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. Results: 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. Conclusions: 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. Lay summary: 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 vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

Sofosbuvir and risk of estimated glomerular filtration rate decline or end-stage renal disease in patients with renal impairment.

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.

Risk of hepatocellular carcinoma in treatment-naïve chronic hepatitis B patients receiving tenofovir disoproxil fumarate versus entecavir in the United States.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Ledipasvir-sofosbuvir in Adolescents With Chronic Hepatitis C and Hematological Malignancies Undergoing Chemotherapy.

OBJECTIVES: In children with hematological malignancies, chronic hepatitis C virus (HCV) infection has been associated with more rapid liver disease progression and higher risk of malignancy relapse due to chemotherapy interruption. We evaluated the safety and efficacy of ledipasvir-sofosbuvir for 12weeks in these patients. METHODS: In a phase 2, open-label study, at one site in Egypt, patients ages 12-<18years with chronic HCV genotype 1 or 4 infection undergoing maintenance chemotherapy for hematological malignancies received ledipasvir-sofosbuvir (90 mg/400 mg) once daily for 12weeks. The efficacy endpoint was sustained virologic response 12 weeks after treatment (SVR12). Safety was assessed by the incidence of adverse events and clinical and laboratory data, including HCV flares defined as alanine aminotransferase >3-fold increase from Day 1 and HCV RNA elevation >1 × log10 from Day 1. RESULTS: Of the 19 adolescents enrolled and treated, median age was 14 years (range 12-17), 84% (16/19) were male, and all had HCV genotype 4 and were HCV treatment naive. All patients completed treatment and achieved SVR12 (19/19, 100%, 95% confidence interval, 82-100). Common adverse events were pyrexia (5/19, 26%), diarrhea (4/19, 21%), and headache (4/19, 21%). Three patients experienced serious adverse events of pneumonia (two patients), and osteoarthritis and diarrhea (one patient); none were considered related to study drug. No patient experienced HCV flares. CONCLUSIONS: Ledipasvir-sofosbuvir was well-tolerated and efficacious in adolescents with chronic HCV genotype 4 and leukemia undergoing maintenance chemotherapy. These data support the use of this interferon and ribavirin-free regimen in adolescents with hematological malignancies.

Inhibition of Viral Replication Reduces Transcriptionally Active Distinct Hepatitis B Virus Integrations With Implications on Host Gene Dysregulation.

BACKGROUND & AIMS: 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. METHODS: 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 expression >2 standard deviations away from samples without viral integration. RESULTS: 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. CONCLUSION: 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.

Long-term Patient-Centered Outcomes in Cirrhotic Patients With Chronic Hepatitis C After Achieving Sustained Virologic Response.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Very low probability of significant liver inflammation in chronic hepatitis B patients with low ALT levels in the absence of liver fibrosis.

BACKGROUND: Guidelines recommend liver biopsy to rule out significant inflammatory activity in chronic hepatitis B (CHB) patients with elevated hepatitis B virus (HBV) DNA but without other indications for treatment. AIM: To study rates and determinants of clinically significant liver inflammation. METHODS: We selected patients with HBV DNA > 2000 IU/mL from the SONIC-B database. The presence of significant inflammation (METAVIR ≥ A2 or HAI ≥ 9) was assessed by liver biopsy and correlated with alanine aminotransferase (ALT) levels (according to AASLD upper limits of normal [ULN]) and stratified by the presence of significant liver fibrosis (Ishak ≥ 3 or METAVIR ≥ F2). RESULTS: The cohort included 2991 patients; 1672 were HBeAg-positive. ALT was < ULN in 270 (9%), 1-2 times ULN in 852 (29%) and > 2 times ULN in 1869 (63%). Significant fibrosis was found in 1419 (47%) and significant inflammatory activity in 630 (21%). Significant inflammatory activity was found in 34% of patients with liver fibrosis, compared to 9.5% of those without (P < 0.001). Among patients without fibrosis, significant inflammatory activity was detected in 3.6% of those with normal ALT, 5.0% of those with ALT 1-2 times ULN and in 13% of those with ALT > 2 times ULN (P < 0.001). ALT < 2 times ULN had a negative predictive value of 95% for ruling out significant inflammatory activity among patients without liver fibrosis. CONCLUSIONS: Among patients without significant fibrosis, an ALT level < 2 times ULN is associated with < 5% probability of significant inflammatory activity. If fibrosis can be ruled out using non-invasive methods, liver biopsy solely to assess inflammatory activity should be discouraged.

Effect of Remdesivir vs Standard Care on Clinical Status at 11 Days in Patients With Moderate COVID-19: A Randomized Clinical Trial.

Importance: Remdesivir demonstrated clinical benefit in a placebo-controlled trial in patients with severe coronavirus disease 2019 (COVID-19), but its effect in patients with moderate disease is unknown. Objective: To determine the efficacy of 5 or 10 days of remdesivir treatment compared with standard care on clinical status on day 11 after initiation of treatment. Design, Setting, and Participants: Randomized, open-label trial of hospitalized patients with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and moderate COVID-19 pneumonia (pulmonary infiltrates and room-air oxygen saturation >94%) enrolled from March 15 through April 18, 2020, at 105 hospitals in the United States, Europe, and Asia. The date of final follow-up was May 20, 2020. Interventions: Patients were randomized in a 1:1:1 ratio to receive a 10-day course of remdesivir (n = 197), a 5-day course of remdesivir (n = 199), or standard care (n = 200). Remdesivir was dosed intravenously at 200 mg on day 1 followed by 100 mg/d. Main Outcomes and Measures: The primary end point was clinical status on day 11 on a 7-point ordinal scale ranging from death (category 1) to discharged (category 7). Differences between remdesivir treatment groups and standard care were calculated using proportional odds models and expressed as odds ratios. An odds ratio greater than 1 indicates difference in clinical status distribution toward category 7 for the remdesivir group vs the standard care group. Results: Among 596 patients who were randomized, 584 began the study and received remdesivir or continued standard care (median age, 57 [interquartile range, 46-66] years; 227 [39%] women; 56% had cardiovascular disease, 42% hypertension, and 40% diabetes), and 533 (91%) completed the trial. Median length of treatment was 5 days for patients in the 5-day remdesivir group and 6 days for patients in the 10-day remdesivir group. On day 11, patients in the 5-day remdesivir group had statistically significantly higher odds of a better clinical status distribution than those receiving standard care (odds ratio, 1.65; 95% CI, 1.09-2.48; P = .02). The clinical status distribution on day 11 between the 10-day remdesivir and standard care groups was not significantly different (P = .18 by Wilcoxon rank sum test). By day 28, 9 patients had died: 2 (1%) in the 5-day remdesivir group, 3 (2%) in the 10-day remdesivir group, and 4 (2%) in the standard care group. Nausea (10% vs 3%), hypokalemia (6% vs 2%), and headache (5% vs 3%) were more frequent among remdesivir-treated patients compared with standard care. Conclusions and Relevance: Among patients with moderate COVID-19, those randomized to a 10-day course of remdesivir did not have a statistically significant difference in clinical status compared with standard care at 11 days after initiation of treatment. Patients randomized to a 5-day course of remdesivir had a statistically significant difference in clinical status compared with standard care, but the difference was of uncertain clinical importance. Trial Registration: ClinicalTrials.gov Identifier: NCT04292730.

aMAP risk score predicts hepatocellular carcinoma development in patients with chronic hepatitis.

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.

HBV variants are common in the 'immune-tolerant' phase of chronic hepatitis B.

Nucleos(t)ide analogues (NUC) treatment prevents progression of liver fibrosis in subjects with chronic hepatitis B (CHB). However, risk of 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-naïve IT subjects were 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 log10  IU/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 (<5%) drug resistance-associated amino acid substitutions in the HBV reverse transcriptase were detected in 9 (9%) subjects at pre-treatment but were not associated with reduced antiviral activity. Future studies should evaluate whether the detection of HBV variant during IT CHB is predictive of progression to immune clearance and poor prognosis, and whether early initiation of antiviral therapy during IT CHB to prevent the selection of HBV variants is clinically beneficial.

Remdesivir for 5 or 10 Days in Patients with Severe Covid-19.

BACKGROUND: Remdesivir is an RNA polymerase inhibitor with potent antiviral activity in vitro and efficacy in animal models of coronavirus disease 2019 (Covid-19). METHODS: We conducted a randomized, open-label, phase 3 trial involving hospitalized patients with confirmed SARS-CoV-2 infection, oxygen saturation of 94% or less while they were breathing ambient air, and radiologic evidence of pneumonia. Patients were randomly assigned in a 1:1 ratio to receive intravenous remdesivir for either 5 days or 10 days. All patients received 200 mg of remdesivir on day 1 and 100 mg once daily on subsequent days. The primary end point was clinical status on day 14, assessed on a 7-point ordinal scale. RESULTS: In total, 397 patients underwent randomization and began treatment (200 patients for 5 days and 197 for 10 days). The median duration of treatment was 5 days (interquartile range, 5 to 5) in the 5-day group and 9 days (interquartile range, 5 to 10) in the 10-day group. At baseline, patients randomly assigned to the 10-day group had significantly worse clinical status than those assigned to the 5-day group (P = 0.02). By day 14, a clinical improvement of 2 points or more on the ordinal scale occurred in 64% of patients in the 5-day group and in 54% in the 10-day group. After adjustment for baseline clinical status, patients in the 10-day group had a distribution in clinical status at day 14 that was similar to that among patients in the 5-day group (P = 0.14). The most common adverse events were nausea (9% of patients), worsening respiratory failure (8%), elevated alanine aminotransferase level (7%), and constipation (7%). CONCLUSIONS: In patients with severe Covid-19 not requiring mechanical ventilation, our trial did not show a significant difference between a 5-day course and a 10-day course of remdesivir. With no placebo control, however, the magnitude of benefit cannot be determined. (Funded by Gilead Sciences; GS-US-540-5773 ClinicalTrials.gov number, NCT04292899.).

Longitudinal analysis of serum microRNAs as predictors of cirrhosis regression during treatment of hepatitis B virus infection.

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.

Genomic modeling of hepatitis B virus integration frequency in the human genome.

Hepatitis B infection is a world-wide public health burden causing serious liver complications. Previous studies suggest that hepatitis B integration into the human genome plays a crucial role in triggering oncogenic process and may also constitutively produce viral antigens. Despite the progress in HBV biology and sequencing technology, our fundamental understanding of how many hepatocytes in the liver actually carry viral integrations is still lacking. Herein we provide evidence that the HBV virus integrates with a lower-bound frequency of 0.84 per diploid genome in hepatitis B positive hepatocellular cancer patients. Moreover, we calculate that integrated viral DNA generates ~80% of the HBsAg transcripts in these patients. These results underscore the need to re-evaluate the clinical end-point and treatment strategies for chronic hepatitis B patients.

Combined GS-4774 and Tenofovir Therapy Can Improve HBV-Specific T-Cell Responses in Patients With Chronic Hepatitis.

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.

Optimisation of the use of APRI and FIB-4 to rule out cirrhosis in patients with chronic hepatitis B: results from the SONIC-B study.

BACKGROUND: 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). METHODS: We applied established cutoffs to rule in (APRI >2·00; FIB-4 >3·25) or rule out (APRI <1·00; FIB-4 <1·45) 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: Foundation for Liver and Gastrointestinal Research, Rotterdam, Netherlands.

An OX40/OX40L interaction directs successful immunity to hepatitis B virus.

Depending on age of acquisition, hepatitis B virus (HBV) can induce a cell-mediated immune response that results in either cure or progressive liver injury. In adult-acquired infection, HBV antigens are usually cleared, whereas in infancy-acquired infection, they persist. Individuals infected during infancy therefore represent the majority of patients chronically infected with HBV (CHB). A therapy that can promote viral antigen clearance in most CHB patients has not been developed and would represent a major health care advance and cost mitigator. Using an age-dependent mouse model of HBV clearance and persistence in conjunction with human blood and liver tissue, we studied mechanisms of viral clearance to identify new therapeutic targets. We demonstrate that age-dependent expression of the costimulatory molecule OX40 ligand (OX40L) by hepatic innate immune cells is pivotal in determining HBV immunity, and that treatment with OX40 agonists leads to improved HBV antigen clearance in young mice, as well as increased strength of T cell responses in young mice and adult mice that were exposed to HBV when they were young and developed a CHB serological profile. Similarly, in humans, we show that hepatic OX40L transcript expression is age-dependent and that increased OX40 expression on peripheral CD4+ T cells in adults is associated with HBV clearance. These findings provide new mechanistic understanding of the immune pathways and cells necessary for HBV immunity and identify potential therapeutic targets for resolving CHB.

Differential Serum Cytokine Profiles in Patients with Chronic Hepatitis B, C, and Hepatocellular Carcinoma.

Cytokines play an important role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC), most cases of which are related to either hepatitis B virus (HBV) or hepatitis C virus (HCV). Prior studies have examined differences in individual cytokine levels in patients with chronic liver disease, but comprehensive cytokine profiling data across different clinical characteristics are lacking. We examined serum cytokine profiles of 411 patients with HCC (n = 102: 32% HBV, 54% HCV, 14% non-viral) and without HCC (n = 309: 39% HBV, 39% HCV, 22% non-viral). Multiplex analysis (Luminex 200 IS) was used to measure serum levels of 51 common cytokines. Random forest machine learning was used to obtain receiver operator characteristic curves and to determine individual cytokine importance using Z scores of mean fluorescence intensity for individual cytokines. Among HCC and non-HCC patients, cytokine profiles differed between HBV and HCV patients (area under curve (AUC) 0.82 for HCC, 0.90 for non-HCC). Cytokine profiles did not distinguish cirrhotic HBV patients with and without HCC (AUC 0.503) or HCV patients with and without HCC (AUC 0.63). In conclusion, patients with HBV or HCV infection, with or without HCC, have distinctly different cytokine profiles, suggesting potential differences in disease pathogenesis and/or disease characteristics.

Soluble intercellular adhesion molecule-1 is associated with hepatocellular carcinoma risk: multiplex analysis of serum markers.

Individualized assessment of hepatocellular carcinoma (HCC) risk in chronic liver disease remains challenging. Serum biomarkers including cytokines may offer helpful adjuncts to standard parameters for risk prediction. Our aim was to identify markers associated with increased HCC incidence. This was a prospective cohort study of 282 patients with both viral or non-viral chronic liver disease. Baseline serum cytokines and other markers were measured in multiplex with a commercially-available Luminex-based system. Patients were followed until death or HCC diagnosis. We performed Lasso-based survival analysis to determine parameters associated with HCC development. Cytokine mean florescence intensity (MFI) was the primary predictor and HCC development the primary outcome. 25 patients developed HCC with total follow-up of 1,363 person-years. Parameters associated with increased HCC incidence were cirrhosis, hepatic decompensation, and soluble serum intercellular adhesion molecule 1 (sICAM-1) MFI. No other molecules increased predictive power for HCC incidence. On univariate analysis, the parameters associated with HCC incidence in patients with cirrhosis were age, antiviral treatment, and high sICAM-1 MFI; on multivariate analysis, sICAM-1 remained associated with HCC development (adjusted HR = 2.75). On unbiased screening of serum cytokines and other markers in a diverse cohort, baseline sICAM-1 MFI is associated with HCC incidence.