A molecular standard for circulating HBV RNA detection and quantification assays in patients with chronic hepatitis B.

Background & Aims: Circulating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of covalently closed circular DNA (cccDNA) and may help to evaluate HBV treatment activity. Different research assays have been proposed and, although two PCR-based research use only investigational assays have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays. Methods: HBV RNA-producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 unit length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP domain (Y63F) of the polymerase, and the ε-loop of the pregenomic (pg)RNA (mutation A1G). Results: The clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virion-like particles and only a minority in small extracellular vescicles. Nanopore sequencing of 5'RACE products shows that the majority of the Huh7-3D29-secreted HBV RNAs start at the 5' end of pgRNA and pgRNA-derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from one flask. Conclusion: We generated a clonal cell line that produces high quantities of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assay calibration. Impact and implications: Several investigational assays and two research use only assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of covalently closed circular DNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNA quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients' sera (e.g. pgRNA, HBx transcripts). This new RNA standard can be used to calibrate all PCR-based assays for circulating HBV RNA quantification to evaluate, in a non-invasive manner, the size of the transcriptionally active cccDNA pool and the activity of novel strategies aimed at curing HBV infection.

Effect of Peg-IFN on the viral kinetics of patients with HDV infection treated with bulevirtide.

Background & Aims: Bulevirtide is a first-in-class entry inhibitor antiviral treatment for chronic hepatitis D. The viral kinetics during bulevirtide therapy and the effect of combining bulevirtide with pegylated-interferon (Peg-IFN) are unknown. Methods: We used mathematical modelling to analyze the viral kinetics in two French observational cohorts of 183 patients receiving bulevirtide with or without Peg-IFN for 48 weeks. Results: The efficacy of bulevirtide in blocking cell infection was estimated to 90.3%, whereas Peg-IFN blocked viral production with an efficacy of 92.4%, albeit with large inter-individual variabilities. The addition of Peg-IFN to bulevirtide was associated with a more rapid virological decline, with a rate of virological response (>2 log of decline or undetectability) at week 48 of 86.9% (95% prediction interval [PI] = [79.7-95.0]), compared with 56.1% (95% PI = [46.4-66.7]) with bulevirtide only. The model was also used to predict the probability to achieve a cure of viral infection, with a rate of 8.8% (95% PI = [3.5-13.2]) with bulevirtide compared with 18.8% (95% PI = [11.6-29.0]) with bulevirtide + Peg-IFN. Mathematical modelling suggests that after 144 weeks of treatment, the rates of viral cure could be 42.1% (95% PI = [33.3-52.6]) with bulevirtide and 66.7% (95% PI = [56.5-76.8]) with bulevirtide + Peg-IFN. Conclusions: In this analysis of real-world data, Peg-IFN strongly enhanced the kinetics of viral decline in patients treated with bulevirtide. Randomized clinical trials are warranted to assess the virological and clinical benefit of this combination, and to identify predictors of poor response to treatment. Impact and implications: Bulevirtide has been approved for chronic HDV infection by regulatory agencies in Europe based on its good safety profile and rapid virological response after treatment initiation, but the optimal duration of treatment and the chance to achieve a sustained virological response remain unknown. The results presented in this study have a high impact for clinicians and investigators as they provide important knowledge on the long-term virological benefits of a combination of Peg-IFN and bulevirtide in patients with CHD. Clinical trials are now warranted to confirm those predictions.

After the Storm: Persistent Molecular Alterations Following HCV Cure.

The development of direct-acting antivirals (DAAs) against hepatitis C virus (HCV) has revolutionized the management of this pathology, as their use allows viral elimination in a large majority of patients. Nonetheless, HCV remains a major public health problem due to the multiple challenges associated with its diagnosis, treatment availability and development of a prophylactic vaccine. Moreover, HCV-cured patients still present an increased risk of developing hepatic complications such as hepatocellular carcinoma. In the present review, we aim to summarize the impact that HCV infection has on a wide variety of peripheral and intrahepatic cell populations, the alterations that remain following DAA treatment and the potential molecular mechanisms implicated in their long-term persistence. Finally, we consider how recent developments in single-cell multiomics could refine our understanding of this disease in each specific intrahepatic cell population and drive the field to explore new directions for the development of chemo-preventive strategies.

Lack of association between early on-treatment HBeAg seroclearance and development of hepatocellular carcinoma or decompensated cirrhosis.

Background & Aims: The association between hepatitis B envelope antigen (HBeAg) seroclearance during long-term nucleos(t)ide analogue (NA) treatment and the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB) remains unclear. Here, we aimed to investigate the association of HBeAg seroclearance during potent NA treatment with the development of HCC and decompensated cirrhosis. Methods: Using a multicenter historical cohort including 2,392 non-cirrhotic adult patients with HBeAg-positive CHB who initiated NA treatment with tenofovir or entecavir, the risk of HCC and decompensated cirrhosis was compared between patients who achieved HBeAg seroclearance within 36 months of NA treatment (the HBeAg-loss group) and those who did not (the HBeAg-maintained group), using inverse probability of treatment weighting. Results: Over a median of 6.6 years of NA treatment, 1,077 patients achieved HBeAg seroclearance (HBeAg loss rate = 6.0 per 100 person-years), 64 patients developed HCC (HCC incidence rate = 0.39 per 100 person-years), and 46 patients developed decompensated cirrhosis (decompensation incidence rate = 0.28 per 100 person-years). The HBeAg-loss and HBeAg-maintained groups had a similar risk of developing HCC (hazard ratio 0.89; 95% CI 0.47-1.68; p = 0.72) and decompensated cirrhosis (hazard ratio 0.98; 95% CI 0.48-1.81; p = 0.91). Compared with delayed HBeAg seroclearance beyond 10 years of NA treatment, the risk of HCC was comparable in those who achieved earlier HBeAg seroclearance at any time point within 10 years, regardless of baseline age and fibrotic burden. Conclusions: Early HBeAg seroclearance during NA treatment was not associated with a reduced risk of development of HCC or decompensated cirrhosis in non-cirrhotic HBeAg-positive patients with CHB. Impact and implications: The association between hepatitis B envelope antigen (HBeAg) seroclearance during long-term nucleos(t)ide analogue treatment and the risk of hepatocellular carcinoma in patients with chronic hepatitis B remains unclear. Our findings indicate that early on-treatment HBeAg seroclearance within 3 years was not associated with the development of hepatocellular carcinoma or decompensated cirrhosis. Achieving HBeAg seroclearance may not be an appropriate surrogate endpoint for preventing the development of liver-related outcomes in non-cirrhotic patients with HBeAg-positive chronic hepatitis B treated with nucleos(t)ide analogues.

A quick access to information on influenza burden and prevention in Lyon university hospital: A prospective QR code-based information campaign in 2022-2023.

BACKGROUND: Influenza vaccines are effective in decreasing hospitalizations and mortality related to influenza and its complications. However, the Vaccine Coverage Rate of influenza remains low and multifaceted efforts are required to improve it. The aim of this study was to assess the impact on influenza vaccine perception using a digital tool among outpatients and health care workers (HCWs). METHODS: A study was performed among outpatients and the HCWs of 23 hospital departments from 4 hospitals affiliated to Lyon university Hospitals (France), between October 2022 and February 2023. By scanning QR (Quick Response) codes, displayed on posters for patients, their companions, as well as in the letters sent to HCWs, users accessed anonymously to a web-application (ELEFIGHT®), which provided information on influenza and invited them to initiate a discussion on influenza prevention with their physicians during the consultation. Patients were also invited to complete a questionnaire regarding their perception of influenza vaccination before and after reading the information on ELEFIGHT®. The retention rate (RR = proportion of people who remain on the page for >2 s), the conversion rate (CR = proportion of people who click on the "Call-To-Action" button) and the absolute variation (difference in the perception before/after) and relative variation (absolute change as a percentage of the initial perception) in perception regarding influenza vaccination before and after consulting the application were calculated. RESULTS: 3791 scans were performed by 3298 patients and/or their companions with a RR of 52% and a CR of 55.1% and 253 scans by 221 HCWs with a RR of 71.2% and a CR of 115.3%. Participants spent an average of 47 s on the application. The questionnaire on influenza vaccination perception was completed by 1533 participants (46.5%); 1390 (90.7%) maintained the same position (neutral, favorable or unfavorable) on this vaccination before and after consulting the application. The relative variations in favor of vaccination were + 7.2% (unfavorable then favorable) and + 19.8% (neutral then favorable). CONCLUSION: This study suggests that a facilitated direct access to medical information through QR codes disseminated in health settings can help nudge people to foster their awareness of influenza and its prevention. Future deployments in a similar context or to other populations could be envisaged. Other vaccine-preventable and/or chronic diseases could also be the target of similar projects as part of public health programs.

Detection and Quantification of HBV Transcripts by Full-Length 5'RACE-PCR.

Chronic hepatitis B virus infection is a major health burden worldwide. Although efficient in maintaining the infection under control, current treatments are unable to fully eradicate the virus due to the persistence of its minichromosome, the so-called cccDNA. In the context of emerging antiviral and combinatorial therapies aiming at decreasing the cccDNA pool or at silencing its transcriptional activity, the detection and quantification of viral RNAs have gained increasing interest as a way to monitor the HBV reservoir. Here, we describe the protocol of the HBV full-length 5'RACE, a technique that allows to define the repertoire of the different HBV RNA species in vitro, intracellularly and extracellularly, and in vivo, in patients' serum in a qualitative or quantitative manner, depending on the choice of the post-analysis methodology.

Treatment with bulevirtide in HIV-infected patients with chronic hepatitis D: ANRS HD EP01 BuleDelta and compassionate cohort.

Background & Aims: In France, bulevirtide (BLV) became available in September 2019 through an early access program to treat patients with HDV. The aim of this analysis was to evaluate the efficacy and safety of BLV in patients with HIV and HDV coinfection. Methods: Patients received BLV 2 mg ± pegylated interferon-α (pegIFNα) according to the physician's decision. The primary endpoint (per-protocol analysis) was the virological response rate at Week 48, defined as the proportion of patients with undetectable serum HDV RNA or a HDV RNA decline >2 log10 IU/ml from baseline. Results: The characteristics of the 38 patients were as follows: 28 male, mean age 47.7 years, and mean baseline HDV RNA viral load 5.7 ± 1.2 log10 IU/ml. Median HIV viral load and mean CD4 count were 32 (30-65) copies/ml and 566 ± 307/mm3, respectively. Eight patients stopped treatment before Week 48. At Week 48, 10 of 19 patients (52.6%) in the 2 mg BLV group and five of seven patients (71.4%) in the 2 mg BLV + pegIFNɑ group had reached virological response (no HDV RNA available in four patients). At Week 48, seven of 19 patients in the 2 mg BLV group and three of six patients in the 2 mg BLV + pegIFNɑ group had a combined response (virological response and normal alanine aminotransferase level). Conclusions: Adults living with HIV coinfected with HDV can be treated by BLV with a virological response in more than 50% of patients. The combination of BLV and pegIFNɑ showed a strong virological response. Impact and implications: Bulevirtide is the only EMA-approved drug for HDV treatment, and we showed that it can be used in adults living with HIV, with an overall good tolerability. Bulevirtide induces a virological response in more than 50% of patients, suggesting that bulevirtide should be considered as a first-line therapy in this specific population. Bulevirtide in combination with pegIFNα could be used in patients without pegIFNα contraindication. No specific drug-drug interaction is reported. Bulevirtide is the only EMA-approved drug for HDV treatment, and we showed that it can be used in adults living with HIV, with an overall good tolerability. Bulevirtide induces a virological response in more than 50% of patients, suggesting that bulevirtide should be considered as a first-line therapy in this specific population. Bulevirtide in combination with pegIFNα could be used in patients without pegIFNα contraindication. No specific drug-drug interaction is reported. Bulevirtide is the only EMA-approved drug for HDV treatment, and we showed that it can be used in adults living with HIV, with an overall good tolerability. Bulevirtide induces a virological response in more than 50% of patients, suggesting that bulevirtide should be considered as a first-line therapy in this specific population. Bulevirtide in combination with pegIFNα could be used in patients without pegIFNα contraindication. No specific drug-drug interaction is reported.

Hepatitis B virus DNA integration: Implications for diagnostics, therapy, and outcome.

Hepatitis B virus (HBV) DNA integration - originally recognised as a non-functional byproduct of the HBV life cycle - has now been accepted as a significant contributor to HBV pathogenesis and hepatitis D virus (HDV) persistence. Integrated HBV DNA is derived from linear genomic DNA present in viral particles or produced from aberrantly processed relaxed circular genomic DNA following an infection, and can drive expression of hepatitis B surface antigen (HBsAg) and HBx. DNA integration events accumulate over the course of viral infection, ranging from a few percent during early phases to nearly 100 percent of infected cells after prolonged chronic infections. HBV DNA integration events have primarily been investigated in the context of hepatocellular carcinoma development as they can activate known oncogenes and other growth promoting genes, cause chromosomal instability and, presumably, induce epigenetic alterations, promoting tumour growth. More recent evidence suggests that HBsAg expression from integrated DNA might contribute to HBV pathogenesis by attenuating the immune response. Integrated DNA provides a source for envelope proteins required for HDV replication and hence represents a means for HDV persistence. Because integrated DNA is responsible for persistence of HBsAg in the absence of viral replication it impacts established criteria for the resolution of HBV infection, which rely on HBsAg as a diagnostic marker. Integrated HBV DNA has been useful in assessing the turnover of infected hepatocytes which occurs during all phases of chronic hepatitis B including the initial phase of infection historically termed immune tolerant. HBV DNA integration has also been shown to impact the development of novel therapies targeting viral RNAs.

Bulevirtide Combined with Pegylated Interferon for Chronic Hepatitis D.

BACKGROUND: In a phase 3 trial, bulevirtide monotherapy led to a virologic response in patients with chronic hepatitis D. Pegylated interferon (peginterferon) alfa-2a is recommended by guidelines as an off-label treatment for this disease. The role of combination therapy with bulevirtide and peginterferon alfa-2a, particularly with regard to finite treatment, is unclear. METHODS: In this phase 2b, open-label trial, we randomly assigned patients to receive peginterferon alfa-2a alone (180 µg per week) for 48 weeks; bulevirtide at a daily dose of 2 mg or 10 mg plus peginterferon alfa-2a (180 µg per week) for 48 weeks, followed by the same daily dose of bulevirtide for 48 weeks; or bulevirtide at a daily dose of 10 mg alone for 96 weeks. All the patients were followed for 48 weeks after the end of treatment. The primary end point was an undetectable level of hepatitis D virus (HDV) RNA at 24 weeks after the end of treatment. The primary comparison was between the 10-mg bulevirtide plus peginterferon alfa-2a group and the 10-mg bulevirtide monotherapy group. RESULTS: A total of 24 patients received peginterferon alfa-2a alone, 50 received 2 mg and 50 received 10 mg of bulevirtide plus peginterferon alfa-2a, and 50 received 10 mg of bulevirtide monotherapy. At 24 weeks after the end of treatment, HDV RNA was undetectable in 17% of the patients in the peginterferon alfa-2a group, in 32% of those in the 2-mg bulevirtide plus peginterferon alfa-2a group, in 46% of those in the 10-mg bulevirtide plus peginterferon alfa-2a group, and in 12% of those in the 10-mg bulevirtide group. For the primary comparison, the between-group difference was 34 percentage points (95% confidence interval, 15 to 50; P<0.001). At 48 weeks after the end of treatment, HDV RNA was undetectable in 25% of the patients in the peginterferon alfa-2a group, in 26% of those in the 2-mg bulevirtide plus peginterferon alfa-2a group, in 46% of those in the 10-mg bulevirtide plus peginterferon alfa-2a group, and in 12% of those in the 10-mg bulevirtide group. The most frequent adverse events were leukopenia, neutropenia, and thrombocytopenia. The majority of adverse events were of grade 1 or 2 in severity. CONCLUSIONS: The combination of 10-mg bulevirtide plus peginterferon alfa-2a was superior to bulevirtide monotherapy with regard to an undetectable HDV RNA level at 24 weeks after the end of treatment. (Funded by Gilead Sciences; MYR 204 ClinicalTrials.gov number, NCT03852433.).

Liver health in hemophilia in the era of gene therapy.

Gene therapy for hemophilia is a groundbreaking treatment approach with promising results and potential to reduce the burden of the disease. However, uncertainties remain, particularly regarding the liver side effects of AAV gene therapy, which are more common in hemophilia A. Unlike some other diseases, such as spinal muscular atrophy, where the target cell for gene therapy is different from the one affected by side effects, hemophilia gene therapy operates within the same cellular domain-the hepatocyte. This overlap is challenging and requires a targeted strategy to mitigate the risks associated with liver injury, which often requires temporary immunosuppressive therapy. A comprehensive approach is essential to increase the efficacy of gene therapy and reduce the likelihood of hepatocyte damage. Key components of this strategy include a thorough pre-gene therapy assessment of liver health, careful post-gene therapy liver monitoring, and prompt therapeutic intervention for loss of transgene expression and liver injury. Collaboration between hematologists and hepatologists is essential to ensure a well-coordinated management plan for patients undergoing hemophilia gene therapy. This review addresses the critical aspect of hepatic comorbidities in patients with hemophilia, emphasizing the need to identify and address these issues prior to initiating gene therapy. It examines the known mechanisms of liver damage and emphasizes the importance of liver monitoring after gene therapy. In addition, the review draws insights from experiences with other AAV-based gene therapies, providing valuable lessons that can guide hemophilia centers in effectively managing liver damage associated with hemophilia gene therapy.

TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism.

OBJECTIVE: Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment. DESIGN: We identified TLR8-expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients. RESULTS: Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12) and downregulated genes associated with the KC identity (eg, SPIC). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition. CONCLUSION: Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection.

The grade of obesity affects the noninvasive diagnosis of advanced fibrosis in individuals with MASLD.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD) is closely associated with obesity. We aimed to assess the impact of obesity on the performance of different noninvasive tests, including liver stiffness measurement (LSM) and Agile3+ (A3+), to detect advanced fibrosis (AF) in a population of patients with MASLD encompassing a wide range of BMI values. METHODS: A total of 479 patients with MASLD were consecutively included (Lyon Hepatology Institute). Clinical data and noninvasive tests, including FibroTest, LSM, A3+, Fibrosis-4 (FIB-4), magnetic resonance elastography, and liver biopsies, were collected. AF was determined by a composite endpoint, i.e., histological stage ≥ F3, overt diagnosis of cirrhosis by magnetic resonance elastography, or concordant LSM ≥ 9.6 kPa and FibroTest ≥ F3. RESULTS: The median BMI was 35.0 kg/m2, and the prevalence of AF was 28.6%. Patients with BMI ≥ 35 versus <35 had a lower proportion of AF, i.e., 19.3% versus 38.1% (p < 0.001), but higher indeterminate status for AF (34.2% vs. 15.4%; p < 0.001). In the case of BMI ≥ 35, LSM had lower specificity to rule in AF (77.9%) versus A3+ (90.4%), but A3+ had decreased sensitivity to rule out AF. A sequential LSM/A3+ strategy achieved high specificity to rule in AF and lowered the proportion of indeterminate cases in patients with BMI ≥ 35. CONCLUSIONS: The grade of obesity affects the detection of MASLD-related AF. A sequential use of LSM/A3+ could improve AF detection in patients with BMI ≥ 35.

Helicases DDX5 and DDX17 promote heterogeneity in HBV transcription termination in infected human hepatocytes.

BACKGROUND & AIMS: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients. RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

Co-Transcriptional Regulation of HBV Replication: RNA Quality Also Matters.

Chronic hepatitis B (CHB) virus infection is a major public health burden and the leading cause of hepatocellular carcinoma. Despite the efficacy of current treatments, hepatitis B virus (HBV) cannot be fully eradicated due to the persistence of its minichromosome, or covalently closed circular DNA (cccDNA). The HBV community is investing large human and financial resources to develop new therapeutic strategies that either silence or ideally degrade cccDNA, to cure HBV completely or functionally. cccDNA transcription is considered to be the key step for HBV replication. Transcription not only influences the levels of viral RNA produced, but also directly impacts their quality, generating multiple variants. Growing evidence advocates for the role of the co-transcriptional regulation of HBV RNAs during CHB and viral replication, paving the way for the development of novel therapies targeting these processes. This review focuses on the mechanisms controlling the different co-transcriptional processes that HBV RNAs undergo, and their contribution to both viral replication and HBV-induced liver pathogenesis.

Circulating HBV RNA and hepatitis B core-related antigen trajectories in persons with HIV/HBV coinfection and HBsAg loss on tenofovir therapy.

BACKGROUND: We evaluated long-term trajectories of circulating hepatitis B virus (HBV)-RNA and hepatitis B core-related antigen (HBcrAg) in persons with and without hepatitis B surface antigen (HBsAg) loss during tenofovir therapy in the Swiss HIV Cohort Study. METHODS: We included 29 persons with HIV (PWH) with HBsAg loss and 29 matched PWH without loss. We compared HBV-RNA and HBcrAg decline and assessed the cumulative proportions with undetectable HBV-RNA and HBcrAg levels during tenofovir therapy using Kaplan-Meier estimates. RESULTS: HBsAg loss occurred after a median of 4 years (IQR 1 - 8). All participants with HBsAg loss achieved suppressed HBV-DNA and undetectable HBV-RNA preceding undetectable qHBsAg levels, whereas 79% achieved negative HBcrAg. In comparison, 79% of the participants without HBsAg loss achieved undetectable HBV-RNA and 48% negative HBcrAg. After two years on tenofovir, an HBV RNA decline ≥1 log10 copies/ml had 100% sensitivity and 36.4% specificity for HBsAg loss, whereas an HBcrAg decline ≥1 log10 U/ml had 91.0% sensitivity and 64.5% specificity. CONCLUSIONS: HBV-RNA suppression preceded undetectable qHBsAg levels, and had high sensitivity but low specificity for HBsAg loss during tenofovir therapy in PWH. HBcrAg remained detectable in approximately 20% of persons with, and 50% of persons without HBsAg loss.

Cytosine base editing inhibits hepatitis B virus replication and reduces HBsAg expression in vitro and in vivo.

Chronic hepatitis B virus (HBV) infection remains a global health problem due to the lack of treatments that prevent viral rebound from HBV covalently closed circular (ccc)DNA. In addition, HBV DNA integrates in the human genome, serving as a source of hepatitis B surface antigen (HBsAg) expression, which impairs anti-HBV immune responses. Cytosine base editors (CBEs) enable precise conversion of a cytosine into a thymine within DNA. In this study, CBEs were used to introduce stop codons in HBV genes, HBs and Precore. Transfection with mRNA encoding a CBE and a combination of two guide RNAs led to robust cccDNA editing and sustained reduction of the viral markers in HBV-infected HepG2-NTCP cells and primary human hepatocytes. Furthermore, base editing efficiently reduced HBsAg expression from HBV sequences integrated within the genome of the PLC/PRF/5 and HepG2.2.15 cell lines. Finally, in the HBV minicircle mouse model, using lipid nanoparticulate delivery, we demonstrated antiviral efficacy of the base editing approach with a >3log10 reduction in serum HBV DNA and >2log10 reduction in HBsAg, and 4/5 mice showing HBsAg loss. Combined, these data indicate that base editing can introduce mutations in both cccDNA and integrated HBV DNA, abrogating HBV replication and silencing viral protein expression.

HBV DNA and HBsAg Levels at 24 Weeks Off-Treatment Predict Clinical Relapse and HBsAg Loss in HBeAg-Negative Patients Who Discontinued Antiviral Therapy.

BACKGROUND & AIMS: Patients who discontinue nucleo(s)tide analogue therapy are at risk of viral rebound and severe hepatitis flares, necessitating intensive off-treatment follow-up. METHODS: We studied the association between hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels at off-treatment follow-up week 24 (FU W24), with subsequent clinical relapse, and HBsAg loss in a multicenter cohort of hepatitis B e antigen (HBeAg)-negative patients with chronic hepatitis B who discontinued nucleo(s)tide analogue therapy. RESULTS: We studied 475 patients, 82% Asian, and 55% treated with entecavir. Patients with higher HBV DNA levels at FU W24 had a higher risk of clinical relapse (hazard ratio [HR], 1.576; P < .001) and a lower chance of HBsAg loss (HR, 0.454; P < .001). Similarly, patients with higher HBsAg levels at FU W24 had a higher risk of clinical relapse (HR, 1.579; P < .001) and a lower chance of HBsAg loss (HR, 0.263; P < .001). A combination of both HBsAg <100 IU/mL and HBV DNA <100 IU/mL at FU W24 identified patients with excellent outcomes (9.9% clinical relapse and 58% HBsAg loss at 216 weeks of follow-up). Conversely, relapse rates were high and HBsAg loss rates negligible among patients with both HBsAg >100 IU/mL and HBV DNA >100 IU/mL (P < .001). CONCLUSIONS: Among HBeAg-negative patients with chronic hepatitis B who discontinued antiviral therapy and who did not experience clinical relapse before FU W24, serum levels of HBV DNA and HBsAg at FU W24 can be used to predict subsequent clinical relapse and HBsAg clearance. A combination of HBsAg <100 IU/mL with HBV DNA <100 IU/mL identifies patients with a low risk of relapse and excellent chances of HBsAg loss and could potentially be used as an early surrogate end point for studies aiming at finite therapy in HBV.

Long-term quantitative hepatitis B surface antigen (HBsAg) trajectories in persons with and without HBsAg loss on tenofovir-containing antiretroviral therapy.

OBJECTIVES: Improving the understanding of the patterns of quantitative hepatitis B surface antigen (qHBsAg) trajectories associated with HBsAg loss is important in light of novel anti-hepatitis B virus agents being developed. We evaluated long-term qHBsAg trajectories in persons with HIV and HBV during tenofovir-containing antiretroviral therapy in the Swiss HIV Cohort Study. METHODS: We included 29 participants with and 29 without HBsAg loss, defined as qHBsAg <0.05 IU/mL. We assessed qHBsAg decline during therapy in both groups and used agglomerative hierarchical clustering to identify different qHBsAg trajectory profiles in persons with HBsAg loss. RESULTS: The median follow-up time was 11.9 years (IQR 8.4-14.1), and the median time to HBsAg loss was 48 months (IQR 12-96). Among participants with HBsAg loss, 79% had a qHBsAg decline ≥1 log10 IU/mL 2 years after starting tenofovir. The trajectories in qHBsAg levels during tenofovir therapy were heterogeneous, characterized by five distinct profiles. Among participants without HBsAg loss, only 7% had a qHBsAg decline ≥1 log10 IU/ml after 2 years. CONCLUSIONS: Most persons with HIV who experienced HBsAg loss had an early decline in qHBsAg levels, with diverse trajectories during long-term tenofovir therapy. In persons without HBsAg loss, qHBsAg levels remained remarkably stable over time.

Spatial transcriptomics reveals a low extent of transcriptionally active hepatitis B virus integration in patients with HBsAg loss.

OBJECTIVE: Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, contributing to the production of hepatitis B surface antigen (HBsAg) and to hepatocarcinogenesis. In this study, we aimed to explore whether transcriptionally active HBV integration events spread throughout the liver tissue in different phases of chronic HBV infection, especially in patients with HBsAg loss. DESIGN: We constructed high-resolution spatial transcriptomes of liver biopsies containing 13 059 tissue spots from 18 patients with chronic HBV infection to analyse the occurrence and relative distribution of transcriptionally active viral integration events. Immunohistochemistry was performed to evaluate the expression of HBsAg and HBV core antigen. Intrahepatic covalently closed circular DNA (cccDNA) levels were quantified by real-time qPCR. RESULTS: Spatial transcriptome sequencing identified the presence of 13 154 virus-host chimeric reads in 7.86% (1026 of 13 059) of liver tissue spots in all patients, including three patients with HBsAg loss. These HBV integration sites were randomly distributed on chromosomes and can localise in host genes involved in hepatocarcinogenesis, such as ALB, CLU and APOB. Patients who were receiving or had received antiviral treatment had a significantly lower percentage of viral integration-containing spots and significantly fewer chimeric reads than treatment-naïve patients. Intrahepatic cccDNA levels correlated well with viral integration events. CONCLUSION: Transcriptionally active HBV integration occurred in chronically HBV-infected patients at different phases, including in patients with HBsAg loss. Antiviral treatment was associated with a decreased number and extent of transcriptionally active viral integrations, implying that early treatment intervention may further reduce the number of viral integration events.

Quantification of circulating HBV RNA expressed from intrahepatic cccDNA in untreated and NUC treated patients with chronic hepatitis B.

OBJECTIVE: A convenient, reproducible biomarker of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) transcriptional activity is lacking. We measured circulating HBV RNA (cirB-RNA) in untreated and nucleos(t)ide analogues (NUC) treated chronic hepatitis B (CHB) patients to define its correlation with intrahepatic viral markers and HBV core-related antigen (HBcrAg). DESIGN: Paired liver biopsy and serum samples were collected from 122 untreated and 30 NUC-treated CHB patients. We measured cirB-RNA, HBV DNA, hepatitis B surface antigen (HBsAg), HBcrAg and alanine aminotransferase levels. cirB-RNA was quantified using an investigational HBV RNA assay for use on the cobas 6800 system. The test detects a region spanning the HBV canonical polyadenylation site. cccDNA and 3.5 kb RNA in liver tissue were assessed by quantitative PCR and droplet digital PCR. RESULTS: cirB-RNA was detectable in 100% of HBeAg(+) chronic hepatitis (CH), 57% and 14% of HBeAg(-) CH and chronic infection untreated patients and 47% of NUC-treated patients. cirB-RNA undetectability was associated with lower intrahepatic cccDNA transcriptional activity, as well as serum HBcrAg, but no significant differences in HBsAg, in both untreated and treated patients. In untreated HBeAg(-) patients, cirB-RNA correlated with intrahepatic 3.5 kb RNA and cccDNA transcriptional activity, serum HBV DNA and HBcrAg, but not with HBsAg or total cccDNA levels. Combined undetectability of both cirB-RNA and HBcrAg detection in untreated HBeAg(-) patients identified a subgroup with the lowest levels of intrahepatic transcriptionally active cccDNA. CONCLUSION: Our results support the usefulness of quantification of circulating HBV RNA expressed from cccDNA as an indicator of intrahepatic active viral reservoir in both untreated and NUC-treated CHB patients. TRIAL REGISTRATION NUMBER: NCT02602847.