Joint host-pathogen genomic analysis identifies hepatitis B virus mutations associated with human NTCP and HLA class I variation.

Evolutionary changes in the hepatitis B virus (HBV) genome could reflect its adaptation to host-induced selective pressure. Leveraging paired human exome and ultra-deep HBV genome-sequencing data from 567 affected individuals with chronic hepatitis B, we comprehensively searched for the signatures of this evolutionary process by conducting "genome-to-genome" association tests between all human genetic variants and viral mutations. We identified significant associations between an East Asian-specific missense variant in the gene encoding the HBV entry receptor NTCP (rs2296651, NTCP S267F) and mutations within the receptor-binding region of HBV preS1. Through in silico modeling and in vitro preS1-NTCP binding assays, we observed that the associated HBV mutations are in proximity to the NTCP variant when bound and together partially increase binding affinity to NTCP S267F. Furthermore, we identified significant associations between HLA-A variation and viral mutations in HLA-A-restricted T cell epitopes. We used in silico binding prediction tools to evaluate the impact of the associated HBV mutations on HLA presentation and observed that mutations that result in weaker binding affinities to their cognate HLA alleles were enriched. Overall, our results suggest the emergence of HBV escape mutations that might alter the interaction between HBV PreS1 and its cellular receptor NTCP during viral entry into hepatocytes and confirm the role of HLA class I restriction in inducing HBV epitope variations.

Blocking viral entry with bulevirtide reduces the number of HDV-infected hepatocytes in human liver biopsies.

BACKGROUND & AIMS: Bulevirtide (BLV) is a first-in-class entry inhibitor and the only approved treatment for patients chronically infected with HDV in Europe. We aimed to investigate the efficacy of BLV treatment in paired liver biopsies obtained at baseline and after 24 or 48 weeks of treatment. METHODS: We performed a combined analysis of 126 paired liver biopsies derived from three clinical trials. In the phase II clinical trial MYR202, patients with chronic hepatitis D were randomised to receive 24 weeks of BLV at 2 mg, 5 mg or 10 mg/day. Patients in MYR203 (phase II) and MYR301 (phase III) received 48 weeks of BLV at 2 mg or 10 mg/day. Tenofovir disoproxil fumarate monotherapy or delayed treatment served as comparators. Virological parameters and infection-related host genes were assessed by qPCR and immunohistochemistry. RESULTS: At week 24, median intrahepatic HDV RNA decline from baseline was 0.9Log10 with 2 mg (n = 7), 1.1Log10 with 5 mg (n = 5) and 1.4 Log10 with 10 mg (n = 7) of BLV. At week 48, median reductions were 2.2Log10 with 2 mg (n = 27) and 2.7Log10 with 10 mg (n = 37) of BLV, while HDV RNA levels did not change in the comparator arms. Notably, a drastic decline in the number of hepatitis delta antigen-positive hepatocytes and a concomitant decrease in transcriptional levels of inflammatory chemokines and interferon-stimulated genes was determined in all BLV-treatment arms. Despite the abundance of HBsAg-positive hepatocytes, replication and covalently closed circular DNA levels of the helper virus HBV were low and remained unaffected by BLV treatment. CONCLUSION: Blocking viral entry diminishes signs of liver inflammation and promotes a strong reduction of HDV infection within the liver, thus suggesting that some patients may achieve HDV cure with long-term treatment. IMPACT AND IMPLICATIONS: Chronic infection with HDV causes the most severe form of viral hepatitis, affecting approximately 12 million people worldwide. The entry inhibitor bulevirtide (BLV) is the only recently approved anti-HDV drug, which has proven efficacious and safe in clinical trials and real-word data. Here, we investigated paired liver biopsies at baseline and after 24 or 48 weeks of treatment from three clinical trials to understand the effect of the drug on viral and host parameters in the liver, the site of viral replication. We found that BLV treatment strongly reduces the number of HDV-infected cells and signs of liver inflammation. This data implies that blocking viral entry ameliorates liver inflammation and that prolonged treatment regimens might lead to HDV cure in some patients. This concept will guide the further development of therapeutic strategies and combination treatments for patients with CHD. CLINICAL TRIAL NUMBERS: NCT03546621, NCT02888106, NCT03852719.

Efficacy and safety of sofosbuvir-velpatasvir and sofosbuvir-velpatasvir-voxilaprevir for hepatitis C in Korea: a Phase 3b study.

BACKGROUND/AIMS: Despite the availability of direct-acting antivirals (DAAs) for chronic hepatitis C virus (HCV) infection in Korea, need remains for pangenotypic regimens that can be used in the presence of hepatic impairment, comorbidities, or prior treatment failure. We investigated the efficacy and safety of sofosbuvir-velpatasvir and sofosbuvir-velpatasvir-voxilaprevir for 12 weeks in HCV-infected Korean adults. METHODS: This Phase 3b, multicenter, open-label study included 2 cohorts. In Cohort 1, participants with HCV genotype 1 or 2 and who were treatment-naive or treatment-experienced with interferon-based treatments, received sofosbuvir-velpatasvir 400/100 mg/day. In Cohort 2, HCV genotype 1 infected individuals who previously received an NS5A inhibitor-containing regimen ≥ 4 weeks received sofosbuvir-velpatasvir-voxilaprevir 400/100/100 mg/day. Decompensated cirrhosis was an exclusion criterion. The primary endpoint was SVR12, defined as HCV RNA < 15 IU/mL 12 weeks following treatment. RESULTS: Of 53 participants receiving sofosbuvir-velpatasvir, 52 (98.1%) achieved SVR12. The single participant who did not achieve SVR12 experienced an asymptomatic Grade 3 ASL/ALT elevation on day 15 and discontinued treatment. The event resolved without intervention. All 33 participants (100%) treated with sofosbuvir-velpatasvir-voxilaprevir achieved SVR 12. Overall, sofosbuvir-velpatasvir and sofosbuvir-velpatasvir-voxilaprevir were safe and well tolerated. Three participants (5.6%) in Cohort 1 and 1 participant (3.0%) in Cohort 2 had serious adverse events, but none were considered treatment-related. No deaths or grade 4 laboratory abnormalities were reported. CONCLUSION: Treatment with sofosbuvir-velpatasvir or sofosbuvir-velpatasvir-voxilaprevir was safe and resulted in high SVR12 rates in Korean HCV patients.

A Phase 3, Randomized Trial of Bulevirtide in Chronic Hepatitis D.

BACKGROUND: Coinfection with hepatitis D virus (HDV) accelerates the progression of liver disease associated with chronic hepatitis B. Bulevirtide inhibits the entry of HDV into hepatocytes. METHODS: In this ongoing phase 3 trial, patients with chronic hepatitis D, with or without compensated cirrhosis, were randomly assigned, in a 1:1:1 ratio, to receive bulevirtide subcutaneously at 2 mg per day (2-mg group) or 10 mg per day (10-mg group) for 144 weeks or to receive no treatment for 48 weeks followed by bulevirtide subcutaneously at 10 mg per day for 96 weeks (control group). Patients will complete 96 weeks of additional follow-up after the end of treatment. The primary end point was a combined response at week 48 of an undetectable HDV RNA level, or a level that decreased by at least 2 log10 IU per milliliter from baseline, and normalization of the alanine aminotransferase (ALT) level. The key secondary end point was an undetectable HDV RNA level at week 48, in a comparison between the 2-mg group and the 10-mg group. RESULTS: A total of 49 patients were assigned to the 2-mg group, 50 to the 10-mg group, and 51 to the control group. A primary end-point response occurred in 45% of patients in the 2-mg group, 48% in the 10-mg group, and 2% in the control group (P<0.001 for the comparison of each dose group with the control group). The HDV RNA level at week 48 was undetectable in 12% of patients in the 2-mg group and in 20% in the 10-mg group (P = 0.41). The ALT level normalized in 12% of patients in the control group, 51% in the 2-mg group (difference from control, 39 percentage points [95% confidence interval {CI}, 20 to 56]), and 56% in the 10-mg group (difference from control, 44 percentage points [95% CI, 26 to 60]). Loss of hepatitis B virus surface antigen (HBsAg) or an HBsAg level that decreased by at least 1 log10 IU per milliliter did not occur in the bulevirtide groups by week 48. Headache, pruritus, fatigue, eosinophilia, injection-site reactions, upper abdominal pain, arthralgia, and asthenia were more common in the 2-mg and 10-mg groups combined than in the control group. No treatment-related serious adverse events occurred. Dose-dependent increases in bile acid levels were noted in the 2-mg and 10-mg groups. CONCLUSIONS: After 48 weeks of bulevirtide treatment, HDV RNA and ALT levels were reduced in patients with chronic hepatitis D. (Funded by Gilead Sciences; MYR 301 ClinicalTrials.gov number, NCT03852719.).

No virologic resistance to bulevirtide monotherapy detected in patients through 24 weeks treatment in phase II and III clinical trials for chronic hepatitis delta.

BACKGROUND & AIMS: Bulevirtide (BLV) is a HDV/HBV entry inhibitor that is associated with virologic response (responders, HDV-RNA undetectable or ≥2 log10 IU/ml decrease from baseline) in >50% of patients after a 24-week treatment. However, some patients only achieve a <1 log10 IU/ml decline in HDV-RNA after the 24-week treatment (non-responders). Here, we report a viral resistance analysis in participants receiving BLV monotherapy who were non-responders or experienced virologic breakthrough (VB, i.e., two consecutive increases in HDV-RNA of ≥1 log10 IU/ml from nadir or two consecutive HDV-RNA detectable results if previously undetectable) from the phase II MYR202 and phase III MYR301 study. METHODS: Deep-sequencing of the BLV-corresponding region in HBV PreS1 and of the HDV HDAg gene, as well as in vitro phenotypic testing, were performed for the participant with VB (n = 1) and non-responders (n = 20) at baseline (BL) and Week 24 (WK24). RESULTS: No amino acid exchanges associated with reduced susceptibility to BLV within the BLV-corresponding region or within HDAg were identified in isolates from any of the 21 participants at BL or at WK24. Although variants (HBV n = 1; HDV n = 13) were detected at BL in some non-responders or in the participant with VB, none were associated with reduced sensitivity to BLV in vitro. Furthermore, the same variant was detected in virologic responders. A comprehensive phenotypic analysis demonstrated that the BLV EC50 values from 116 BL samples were similar across non-responders, partial responders (HDV RNA decline ≥1 but <2 log10 IU/ml), and responders regardless of the presence of HBV and/or HDV polymorphisms. CONCLUSIONS: No amino acid substitutions associated with reduced sensitivity to BLV monotherapy were detected at BL or WK24 in non-responders or the participant with VB after 24-week BLV treatment. IMPACT AND IMPLICATIONS: This is the first study investigating the development of resistance in patients treated with BLV. Excluding resistance to BLV as an explanation for an insufficient decrease in HDV-RNA levels during BLV therapy is an important finding for patients, clinicians, and researchers. It demonstrates that BLV has a high barrier to resistance, indicating it is safe and suitable for long-term treatment, although long-term surveillance for resistance should be performed. Our results hint at other still unknown mechanisms as an explanation for the persistence of serum HDV-RNA during inhibition of viral entry. CLINICAL TRIAL NUMBERS: NCT03546621 and NCT03852719.

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.

Hepatitis B virus haplotype number at baseline is a predictive marker of functional cure during antiviral therapy for patients with genotypes A and D HBeAg-positive chronic hepatitis B.

BACKGROUNDS AND AIMS: 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. RESULTS: 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. CONCLUSION: 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.

Sofosbuvir-velpatasvir in adults with hepatitis C virus infection and compensated cirrhosis in Japan.

BACKGROUND & PURPOSE: Protease-free regimens for chronic hepatitis C virus (HCV) infection are safe and effective for persons with either compensated or decompensated cirrhosis. We examined the efficacy and safety of sofosbuvir-velpatasvir in participants with HCV and compensated cirrhosis in Japan. METHODS: This was a Phase 3, multi-center, open-label study. At 20 sites, 37 individuals with chronic HCV infection of any genotype and compensated cirrhosis received sofosbuvir-velpatasvir (400 mg/100 mg) daily for 12 weeks. Participants were treatment-naïve or treatment-experienced with interferon-based treatments with or without HCV NS3/4A protease inhibitors. Prior exposure with HCV NS5A or NS5B inhibitors was prohibited. The primary study endpoint was sustained virologic response 12 weeks after treatment (SVR12). RESULTS: Among participants, 62% had HCV genotype 1 infection, and 38% had HCV genotype 2. More than three quarters (29/37, 78%) were HCV treatment naïve. All participants (37/37, 100%) achieved SVR12. Seventeen participants (46%) and three participants (8%) had pretreatment resistance-associated substitutions to HCV NS5A and NS5B nucleoside inhibitors respectively, yet no on-treatment breakthrough or relapse occurred. Sofosbuvir-velpatasvir for 12 weeks treatment was safe and well tolerated. The most commonly reported adverse events were headache (8%, 3/37) and diarrhea (5%, 2/37). One serious adverse event, patella fracture, occurred and was considered not treatment related. No participants discontinued study treatment due to an adverse event. Three participants (8%) had a Grade 3 laboratory abnormality; all were hyperglycemia. CONCLUSION: Sofosbuvir-velpatasvir resulted in high SVR rates and was well tolerated among Japanese patients with HCV and compensated cirrhosis. This single-tablet regimen offers a highly effective, protease-inhibitor free regimen for treating HCV. CLINICALTRIALS: gov Identifier: NCT04112303.

International multicenter validation of GES score for HCC risk stratification in chronic hepatitis C patients.

We have recently demonstrated the ability of a simple predictive model (GES) score to determine the risk of hepatocellular carcinoma (HCC) after using direct-acting antivirals. However, our results were restricted to Egyptian patients with hepatitis C virus (HCV) genotype 4. Therefore, we studied a large, independent cohort of multiethnic populations through our international collaborative activity. Depending on their GES scores, patients are stratified into low risk (≤ 6/12.5), intermediate risk (> 6-7.5/12.5), and high risk (> 7.5/12.5) for HCC. A total of 12,038 patients with chronic HCV were analyzed in this study, of whom 11,202 were recruited from 54 centers in France, Japan, India, the U.S., and Spain, and the remaining 836 were selected from the Gilead-sponsored randomized controlled trial conducted across the U.S., Europe, Canada, and Australia. Descriptive statistics and log-rank tests. The performance of the GES score was evaluated using Harrell's C-index (HCI). The GES score proved successful at stratifying all patients into 3 risk groups, namely low-risk, intermediate-risk, and high-risk. It also displayed significant predictive value for HCC development in all participants (p < .0001), with HCI ranging from 0.55 to 0.76 among all cohorts after adjusting for HCV genotypes and patient ethnicities. The GES score can be used to stratify HCV patients into 3 categories of risk for HCC, namely low-risk, intermediate-risk, and high-risk, irrespective of their ethnicities or HCV genotypes. This international multicenter validation may allow the use of GES score in individualized HCC risk-based surveillance programs.

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.

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.

Ledipasvir/Sofosbuvir for Patients Coinfected With Chronic Hepatitis C and Hepatitis B in Taiwan: Follow-up at 108 Weeks Posttreatment.

BACKGROUND: For patients coinfected with hepatitis C virus (HCV) and hepatitis B virus (HBV), HCV treatment with direct-acting antivirals can lead to HBV reactivation. We evaluated HBV reactivation during ledipasvir/sofosbuvir treatment and 108-week follow-up. METHODS: In Taiwan, 111 patients with HCV genotype 1 or 2 and HBV received ledipasvir/sofosbuvir (90mg/400mg) once daily for 12 weeks. HBV virologic reactivation was defined as postbaseline increase in HBV DNA from either less than the lower limit of quantification (LLOQ, 20 IU/mL) to equal to or more than LLOQ or equal to or more than LLOQ to >1 log10 IU/mL. HBV clinical reactivation was HBV virologic reactivation with alanine aminotransferase (ALT) >2× upper limit of normal. Factors associated with development of HBV virologic or clinical reactivation were evaluated with logistic regression analysis. RESULTS: All patients (100%, 111/111) maintained HCV suppression through 108 weeks after treatment. HBV virologic reactivation occurred in 73% of patients (81/111). Clinical reactivation occurred in 9% (10/111). The majority of HBV virologic reactivations (86%, 70/81) occurred by follow-up week 12, whereas clinical reactivation was generally more delayed. Eight (7%, 8/111) initiated HBV therapy. In regression analyses, baseline HBV DNA and hepatitis B surface antigen (HBsAg) levels were associated with HBV virologic reactivation and baseline ALT and HBV DNA, and HBsAg levels were associated with HBV clinical reactivation. CONCLUSION: Among HCV/HBV coinfected patients treated with direct-acting antivirals for HCV, HBV virologic reactivation occurred in a majority of patients during treatment and follow-up. In most patients, HBV virologic reactivation was asymptomatic; only a small proportion initiated HBV treatment. Notably, clinical reactivation may still occur >3 months after end of therapy. CLINICAL TRIALS REGISTRATION: NCT02613871.

Characterization of the liver immune microenvironment in liver biopsies from patients with chronic HBV infection.

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.

Prevalence of resistance-associated substitutions and phylogenetic analysis of hepatitis C virus infection in Russia.

OBJECTIVES: Due to limited hepatitis C virus (HCV) sequence availability from patients in Russia, the relationship between subtypes and baseline resistance-associated substitutions (RAS) to direct antiretroviral treatment outcome is not fully understood. METHODS: Deep sequencing of HCV NS3, NS5A, and NS5B sequences was performed on plasma HCV samples from 412 direct-acting antiviral (DAA)-naïve patients from Russia. Phylogenetic analysis was performed to investigate sequence similarities between HCV strains from Russia, Asia, Europe, and North America. Pretreatment HCV RAS was assessed with a 15% cutoff. RESULTS: HCV genotype GT1b and GT3a sequences in Russia were related to strains in Europe and Asia. The prevalence of GT1a and GT2a was low in Russia. In GT1b, the prevalence of NS5A Y93H was lower in Russia (6%) compared with Asia (15%). The prevalence of NS5B L159F was similar between Russia and Europe (26-39%). GT3a RAS prevalence was similar between Russia and Asia, Europe, and North America. The 2k/1b recombinant strain in Russia was related to strains from Europe. A higher prevalence of the NS5A RAS L31M (10%) was observed in 2k/1b sequences compared to GT1b (1-6%). CONCLUSIONS: The prevalence of RASs and the phylogenetic analysis showed similarities in HCV strains between Russia, Europe, and North America. This information may be useful for HCV regimens in Russia.

Analysis of Hepatitis B Virus Haplotype Diversity Detects Striking Sequence Conservation Across Genotypes and Chronic Disease Phase.

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.

Ledipasvir plus sofosbuvir in pregnant women with hepatitis C virus infection: a phase 1 pharmacokinetic study.

BACKGROUND: Hepatitis C virus (HCV) infection is increasing among pregnant women because of the opioid epidemic, yet there are no interventions to reduce perinatal HCV transmission or to treat HCV during pregnancy. Physiological changes in pregnancy alter the pharmacokinetics of some medications; thus, our aim was to compare the pharmacokinetic parameters of ledipasvir 90 mg plus sofosbuvir 400 mg during pregnancy with non-pregnant women. METHODS: This was an open-label, phase 1 study of pregnant women with genotype 1 HCV infection and their infants. A reference group of women who had participated in pharmacokinetic studies of ledipasvir-sofosbuvir during phase 2 and 3 trials was used. Participants were enrolled at Magee-Womens Hospital (Pittsburgh, PA, USA) between 23 and 24 weeks' gestation and had a 12-week course of oral ledipasvir-sofosbuvir (daily 90 mg ledipasvir plus 400 mg sofosbuvir). Three 12-h intensive pharmacokinetic visits were done at 25-26, 29-30, and 33-34 weeks' gestation and individual pharmacokinetics were summarised by geometric mean across the three visits. The primary outcome, analysed in all participants without suspected dosing errors, was the ledipasvir-sofosbuvir area under the concentration-time curve of the dosing interval (AUCtau) during pregnancy compared with the reference group by geometric mean ratio. This study is registered with ClinicalTrials.gov, NCT02683005. FINDINGS: From Oct 1, 2016, to Sept 30, 2018, 29 pregnant women were screened and nine (31%) were enrolled. Eight (89%) women were included in the primary analysis. Ledipasvir and sofosbuvir exposures were similar in the pregnant women versus the non-pregnant reference group (geometric mean ratio of AUCtau ledipasvir 89·3% [90% CI 68·7-116·1]; sofosbuvir 91·1% [78·0-106·3]). INTERPRETATION: Ledipasvir-sofosbuvir was safe and effective without clinically meaningful differences in drug exposure among pregnant versus non-pregnant women. FUNDING: National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Institutes of Health/Office of Research on Women's Health, and Gilead Sciences.

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.

Diminished hepatic IFN response following HCV clearance triggers HBV reactivation in coinfection.

In patients with HBV and HCV coinfection, HBV reactivation leading to severe hepatitis has been reported with the use of direct-acting antivirals (DAAs) to treat HCV infection. Here we studied the molecular mechanisms behind this viral interaction. In coinfected cell culture and humanized mice, HBV replication was suppressed by HCV coinfection. In vitro, HBV suppression was attenuated when interferon (IFN) signaling was blocked. In vivo, HBV viremia, after initial suppression by HCV superinfection, rebounded following HCV clearance by DAA treatment that was accompanied by a reduced hepatic IFN response. Using blood samples of coinfected patients, IFN-stimulated gene products including C-X-C motif chemokine 10 (CXCL10), C-C motif chemokine ligand 5 (CCL5), and alanine aminotransferase (ALT) were identified to have predictive value for HBV reactivation after HCV clearance. Taken together, our data suggest that HBV reactivation is a result of diminished hepatic IFN response following HCV clearance and identify serologic markers that can predict HBV reactivation in DAA-treated HBV-HCV-coinfected persons.

Switching from tenofovir disoproxil fumarate to tenofovir alafenamide in virologically suppressed patients with chronic hepatitis B: a randomised, double-blind, phase 3, multicentre non-inferiority study.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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 (both <1%) 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; p<0·0001]) 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; p<0·0001]), 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]; p <0·0001), 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. INTERPRETATION: 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. FUNDING: Gilead Sciences.

Discovery of Half-life of Circulating Hepatitis B Surface Antigen in Patients With Chronic Hepatitis B Infection Using Heavy Water Labeling.

In a pilot study, heavy water labeling was used to determine hepatitis B surface antigen (HBsAg) turnover rates in chronic hepatitis B (CHB) patients. The mean (standard deviation) half-life of HBsAg in blood was 6.7 (5.5) days, which reflects recent production in the liver and supports strategies aimed at reducing HBsAg production in CHB patients.