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

HBV DNA integration originally recognized as a non-functional byproduct of the HBV life cycle has now been accepted as a significant contributor to HBV pathogenesis and HDV persistence. Integrated HBV DNA is derived from linear genomic DNA present in virus particles or produced from aberrantly processed relaxed circular genomic DNA following an infection, and can drive expression of 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 infection. HBV DNA integration events have primarily been investigated in the context of HCC development where they can activate known oncogenes and other growth promoting genes, cause chromosomal instability and presumably epigenetic alterations promoting tumor 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 relies 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 was also shown to impact the development of novel therapies targeting viral RNAs.

Rapid and Reliable Protein-Free HBV DNA Extraction and Sensitive Branched DNA Southern Blot Assay.

HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.

Method for Quantitative HDV RNA Detection: I, Manual Workflow (Serum and Liver Tissue) and II, Fully Automated High Throughput Workflow for Diagnostic Use.

The hepatitis delta virus (HDV) is a small RNA virus (1700 base pairs), which uses the surface proteins of the hepatitis B virus (HBV) as an envelope. Accurate and reliable quantitative detection of HDV RNA is central for scientific and translational clinical research or diagnostic purposes. However, HDV poses challenges for nucleic acid amplification techniques: (1) the circular genome displays high intramolecular base pairing; (2) high content of cytosine and guanine; and (3) enormous genomic diversity among the eight known HDV genotypes (GTs). Here, we provide step-by-step instructions for (A) a manual workflow to perform a quantitative HDV reverse transcription (RT)-PCR from serum and liver tissue and (B) a quantitative HDV RT-PCR assay with whole process control to be used for serum or plasma samples run on a fully automated system. Both assays target the conserved ribozyme region and demonstrate inclusivity for all eight HDV GTs. The choice of assay depends on the experimental needs and equipment availability. While the former is ideal for scientific research laboratories, the latter provides a useful tool in the field of translational research or diagnostics.

Multiscale modeling of HBV infection integrating intra- and intercellular viral propagation to analyze extracellular viral markers.

Chronic infection with hepatitis B virus (HBV) is caused by the persistence of closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. Despite available therapeutic anti-HBV agents, eliminating the cccDNA remains challenging. Thus, quantifying and understanding the dynamics of cccDNA are essential for developing effective treatment strategies and new drugs. However, such study requires repeated liver biopsy to measure the intrahepatic cccDNA, which is basically not accepted because liver biopsy is potentially morbid and not common during hepatitis B treatment. We here aimed to develop a noninvasive method for quantifying cccDNA in the liver using surrogate markers in peripheral blood. We constructed a multiscale mathematical model that explicitly incorporates both intracellular and intercellular HBV infection processes. The model, based on age-structured partial differential equations, integrates experimental data from in vitro and in vivo investigations. By applying this model, we roughly predicted the amount and dynamics of intrahepatic cccDNA within a certain range using specific viral markers in serum samples, including HBV DNA, HBsAg, HBeAg, and HBcrAg. Our study represents a significant step towards advancing the understanding of chronic HBV infection. The noninvasive quantification of cccDNA using our proposed method holds promise for improving clinical analyses and treatment strategies. By comprehensively describing the interactions of all components involved in HBV infection, our multiscale mathematical model provides a valuable framework for further research and the development of targeted interventions.

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.

Potent broadly neutralizing antibody VIR-3434 controls hepatitis B and D virus infection and reduces HBsAg in humanized mice.

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

Non-organ-specific autoantibodies with unspecific patterns are a frequent para-infectious feature of chronic hepatitis D.

Infections with hepatotropic viruses are associated with various immune phenomena. Hepatitis D virus (HDV) causes the most severe form of viral hepatitis. However, few recent data are available on non-disease-specific and non-organ-specific antibody (NOSA) titers and immunoglobulin G (IgG) levels in chronic hepatitis D (CHD) patients. Here, we examined the NOSA titers and IgG levels of 40 patients with CHD and different disease courses and compared them to 70 patients with chronic hepatitis B (CHB) infection. 43% of CHD patients had previously undergone treatment with pegylated interferon-α (IFN-α). The antibody display of 46 untreated patients diagnosed with autoimmune hepatitis (AIH) was used as a reference. The frequency of elevated NOSA titers (CHD 69% vs. CHB 43%, p < 0.01), and the median IgG levels (CHD 16.9 g/L vs. CHB 12.7 g/L, p < 0.01) were significantly higher in CHD patients than in patients with CHB, and highest in patients with AIH (96%, 19.5 g/L). Also, the antinuclear antibody pattern was homogeneous in many patients with AIH and unspecific in patients with viral hepatitis. Additionally, f-actin autoantibodies were only detectable in patients with AIH (39% of SMA). In CHD patients, IgG levels correlated with higher HDV viral loads, transaminases, and liver stiffness values. IgG levels and NOSA were similar in CHD patients irrespective of a previous IFN-α treatment. In summary, autoantibodies with an unspecific pattern are frequently detected in CHD patients with unclear clinical relevance.

Multiscale modeling of HBV infection integrating intra- and intercellular viral propagation for analyzing extracellular viral markers.

Chronic infection of hepatitis B virus (HBV) is caused by the persistence of closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. Despite available therapeutic anti-HBV agents, eliminating the cccDNA remains challenging. The quantifying and understanding dynamics of cccDNA are essential for developing effective treatment strategies and new drugs. However, it requires a liver biopsy to measure the intrahepatic cccDNA, which is basically not accepted because of the ethical aspect. We here aimed to develop a non-invasive method for quantifying cccDNA in the liver using surrogate markers present in peripheral blood. We constructed a multiscale mathematical model that explicitly incorporates both intracellular and intercellular HBV infection processes. The model, based on age-structured partial differential equations (PDEs), integrates experimental data from in vitro and in vivo investigations. By applying this model, we successfully predicted the amount and dynamics of intrahepatic cccDNA using specific viral markers in serum samples, including HBV DNA, HBsAg, HBeAg, and HBcrAg. Our study represents a significant step towards advancing the understanding of chronic HBV infection. The non-invasive quantification of cccDNA using our proposed methodology holds promise for improving clinical analyses and treatment strategies. By comprehensively describing the interactions of all components involved in HBV infection, our multiscale mathematical model provides a valuable framework for further research and the development of targeted interventions.

Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Impact and implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

A 3-Year Course Of Bulevirtide Monotherapy May Cure Hdv Infection In Cirrhotics.

Bulevirtide has been recently conditionally approved by the European Medicines Agency for the treatment of Chronic Hepatitis Delta, but the ideal duration of therapy is unknown. Here we describe the first case of cure of Hepatitis Delta following 3 years of Bulevirtide monotherapy in a patient with compensated cirrhosis and esophageal varices. During the 72-week off-Bulevirtide follow-up, virological and biochemical responses were maintained. In the off-therapy liver biopsy, intrahepatic HDV RNA and Hepatitis D antigen were undetectable, <1% hepatocytes were Hepatitis B surface antigen positive while hepatitis B core antigen was negative. Grading and staging improved compared to pre-treatment biopsy.

Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure.

OBJECTIVES: A major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices. DESIGN: Reference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/-PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB. RESULTS: Hirt and -PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA. CONCLUSIONS: We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.

Safety and efficacy of bulevirtide in combination with tenofovir disoproxil fumarate in patients with hepatitis B virus and hepatitis D virus coinfection (MYR202): a multicentre, randomised, parallel-group, open-label, phase 2 trial.

BACKGROUND: Bulevirtide is a first-in-class peptidic entry inhibitor for hepatitis B virus (HBV) and hepatitis D virus infection. In July, 2020, bulevirtide 2 mg received conditional marketing authorisation by the European Medical Agency for treatment of chronic hepatitis D virus infection. We investigated the antiviral activity of bulevirtide in patients chronically infected with HBV and hepatitis D virus. METHODS: MYR202 (ClinicalTrials.gov, NCT03546621; EudraCT, 2016-000395-13) was a multicentre, parallel-group, randomised, open-label, phase 2 trial. Adults (aged 18-65 years) with chronic hepatitis D virus infection, including patients with cirrhosis and patients who had contraindications to PegIFNα treatment or for whom treatment did not work, were eligible and were enrolled from four hospitals in Germany and 12 hospitals in Russia. Patients were randomly assigned (1:1:1:1) to receive 2 mg (n=28), 5 mg (n=32), or 10 mg (n=30) subcutaneous bulevirtide once per day with tenofovir disoproxil fumarate (TDF; 245 mg once per day orally) or TDF alone (245 mg once per day orally; n=30) for 24 weeks. Randomisation was done using a digital block scheme with stratification, consisting of 480 randomisation numbers separated into 30 blocks. The primary endpoint was undetectable hepatitis D virus RNA or 2 log10 IU/mL or higher decline in hepatitis D virus RNA at week 24, which was analysed in the modified intention-to-treat population, including patients who received study medication at least once after randomisation. Hepatitis D virus RNA concentrations were monitored until week 48. Safety was assessed for all patients who received at least one dose of bulevirtide or TDF. FINDINGS: Between Feb 16, 2016, and Dec 8, 2016, 171 patients with chronic hepatitis D virus infection were screened; 51 were ineligible based on the exclusion criteria and 120 patients (59 with cirrhosis) were enrolled. At week 24, 15 (54%, 95% CI 34-73) of 28 patients achieved undetectable hepatitis D virus RNA or a 2 log10 IU/mL or more decline in hepatitis D virus RNA (p<0·0001 vs TDF alone) with 2 mg bulevirtide, 16 (50%, 32-68) of 32 with 5 mg bulevirtide (p<0·0001), and 23 (77%, 58-90) of 30 with 10 mg bulevirtide (p<0·0001), versus one (4%, 0·1-18) of 28 with TDF alone. By week 48 (24 weeks after bulevirtide cessation), hepatitis D virus RNA concentrations had rebounded, with median changes from week 24 to week 48 of 1·923 log10 IU/mL (IQR 0·566-2·485) with 2 mg bulevirtide, 1·732 log10 (0·469-2·568) with 5 mg bulevirtide, and 2·030 log10 (1·262-2·903) with 10 mg bulevirtide. There were no deaths associated with treatment. Three (9%) patients in the bulevirtide 5 mg group, two (7%) patients in the bulevirtide 10 mg group, and one (4%) patient in the TDF group had serious adverse events. Common treatment-emergent adverse events included asymptomatic bile salt increases and increases in alanine aminotransferase and aspartate aminotransferase. INTERPRETATION: Bulevirtide induced a significant decline in hepatitis D virus RNA over 24 weeks. After cessation of bulevirtide, hepatitis D virus RNA concentrations rebounded. Longer treatment durations and combination therapies should be investigated. FUNDING: Hepatera LLC, MYR GmbH, and the German Centre for Infection Research, TTU Hepatitis.

A roadmap for serum biomarkers for hepatitis B virus: current status and future outlook.

Globally, 296 million people are infected with hepatitis B virus (HBV), and approximately one million people die annually from HBV-related causes, including liver cancer. Although there is a preventative vaccine and antiviral therapies suppressing HBV replication, there is no cure. Intensive efforts are under way to develop curative HBV therapies. Currently, only a few biomarkers are available for monitoring or predicting HBV disease progression and treatment response. As new therapies become available, new biomarkers to monitor viral and host responses are urgently needed. In October 2020, the International Coalition to Eliminate Hepatitis B Virus (ICE-HBV) held a virtual and interactive workshop on HBV biomarkers endorsed by the International HBV Meeting. Various stakeholders from academia, clinical practice and the pharmaceutical industry, with complementary expertise, presented and participated in panel discussions. The clinical utility of both classic and emerging viral and immunological serum biomarkers with respect to the course of infection, disease progression, and response to current and emerging treatments was appraised. The latest advances were discussed, and knowledge gaps in understanding and interpretation of HBV biomarkers were identified. This Roadmap summarizes the strengths, weaknesses, opportunities and challenges of HBV biomarkers.

Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo.

OBJECTIVE: Therapeutic strategies silencing and reducing the hepatitis B virus (HBV) reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo. DESIGN: HBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events. RESULTS: Both siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6. CONCLUSION: These results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.

Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system.

BACKGROUND & AIMS: Currently available HDV PCR assays are characterized by considerable run-to-run and inter-laboratory variability. Hence, we established a quantitative reverse transcription real-time PCR (RT-qPCR) assay on the open channel of a fully automated PCR platform (cobas6800, Roche) offering improved consistency and reliability. METHODS: A primer/probe-set targeting a highly conserved region upstream of the HDV antigen was adapted for use on the cobas6800. The lower limit of detection (LLOD) was determined using a dilution panel of the HDV WHO standard (n = 21/dilution). Linearity and inclusivity were tested by preparing 10-fold dilution series of cell culture-derived virus (genotype [GT]1-8; n = 5/dilution). Patient samples containing a variety of bloodborne viral pathogens were tested to confirm exclusivity (n = 60). RESULTS: The LLOD of the HDV utility-channel (HDV_UTC) assay was determined as 3.86 IU/ml (95% CI 2.95-5.05 IU/ml) with a linear range from 10-10ˆ8 IU/ml (GT1). Linear relationships were observed for all HDV GTs with slopes ranging from -3.481 to -4.134 cycles/log and R2 from 0.918 to 0.994. Inter-run and intra-run variability were 0.3 and 0.6 Ct (3xLLOD), respectively. No false-positive results were observed. To evaluate clinical performance, 110 serum samples of anti-HDV-Ab+ patients were analyzed using the HDV_UTC and CE-IVD RoboGene assays. 58/110 and 49/110 samples were concordant positive or negative, respectively (overall agreement 97.3%). Quantitative comparison demonstrated a strong correlation (R2 0.8733; 95% CI 0.8914-0.9609; p value <0.0001). CONCLUSION: The use of highly automated, sample-to-result solutions for molecular diagnostics holds many inherent benefits over manual workflows, including improved reliability, reproducibility and dynamic scaling of testing capacity. The assay we established showed excellent analytical and clinical performance, with inclusivity for all HDV GTs and a limit of quantification of 10 IU/ml, making it a sensitive new tool for HDV screening and viral load monitoring. LAY SUMMARY: The hepatitis delta virus (HDV) causes a severe form of inflammation in the liver. We developed a tool for molecular diagnostics, a polymerase chain reaction HDV assay that showed great performance. It can be used to improve diagnosis of HDV, as well as for monitoring treatment responses. The assay allows for quantification of the virus in the tested samples and is performed on a fully automated platform (cobas6800), which provides various benefits including less hands-on time and excellent comparability of test results.

Strong Replication Interference Between Hepatitis Delta Viruses in Human Liver Chimeric Mice.

BACKGROUND: Hepatitis D Virus (HDV) is classified into eight genotypes with distinct clinical outcomes. Despite the maintenance of highly conserved functional motifs, it is unknown whether sequence divergence between genotypes, such as HDV-1 and HDV-3, or viral interference mechanisms may affect co-infection in the same host and cell, thus hindering the development of HDV inter-genotypic recombinants. We aimed to investigate virological differences of HDV-1 and HDV-3 and assessed their capacity to infect and replicate within the same liver and human hepatocyte in vivo. METHODS: Human liver chimeric mice were infected with hepatitis B virus (HBV) and with one of the two HDV genotypes or with HDV-1 and HDV-3 simultaneously. In a second set of experiments, HBV-infected mice were first infected with HDV-1 and after 9 weeks with HDV-3, or vice versa. Also two distinct HDV-1 strains were used to infect mice simultaneously and sequentially. Virological parameters were determined by strain-specific qRT-PCR, RNA in situ hybridization and immunofluorescence staining. RESULTS: HBV/HDV co-infection studies indicated faster spreading kinetics and higher intrahepatic levels of HDV-3 compared to HDV-1. In mice that simultaneously received both HDV strains, HDV-3 became the dominant genotype. Interestingly, antigenomic HDV-1 and HDV-3 RNA were detected within the same liver but hardly within the same cell. Surprisingly, sequential super-infection experiments revealed a clear dominance of the HDV strain that was inoculated first, indicating that HDV-infected cells may acquire resistance to super-infection. CONCLUSION: Infection with two largely divergent HDV genotypes could be established in the same liver, but rarely within the same hepatocyte. Sequential super-infection with distinct HDV genotypes and even with two HDV-1 isolates was strongly impaired, suggesting that virus interference mechanisms hamper productive replication in the same cell and hence recombination events even in a system lacking adaptive immune responses.

High Rates of Liver Cirrhosis and Hepatocellular Carcinoma in Chronic Hepatitis B Patients with Metabolic and Cardiovascular Comorbidities.

BACKGROUND: The prevalence of metabolic and cardiovascular diseases is rising worldwide. However, little is known about the impact of such disorders on hepatic disease progression in chronic hepatitis B (CHB) during the era of potent nucleo(s)tide analogues (NAs). METHODS: We retrospectively analyzed a single-center cohort of 602 CHB patients, comparing the frequency of liver cirrhosis at baseline and incidences of liver-related events during follow-up (hepatocellular carcinoma, liver transplantation and liver-related death) between CHB patients with a history of diabetes, obesity, hypertension or coronary heart disease (CHD). RESULTS: Rates of cirrhosis at baseline and liver-related events during follow-up (median follow-up time: 2.51 years; NA-treated: 37%) were substantially higher in CHB patients with diabetes (11/23; 3/23), obesity (6/13; 2/13), CHD (7/11; 2/11) or hypertension (15/43; 4/43) compared to CHB patients without the indicated comorbidities (26/509; 6/509). Multivariate analysis identified diabetes as the most significant predictor for cirrhosis (p = 0.0105), while comorbidities did not correlate with liver-related events in pre-existing cirrhosis. CONCLUSION: The combination of metabolic diseases and CHB is associated with substantially increased rates of liver cirrhosis and secondary liver-related events compared to CHB alone, indicating that hepatitis B patients with metabolic comorbidities warrant particular attention in disease surveillance and evaluation of treatment indication.

Innate immunity in hepatitis B and D virus infection: consequences for viral persistence, inflammation, and T cell recognition.

Chronic infections with human hepatitis viruses continue to be a major health burden worldwide. Despite the availability of an effective prophylactic vaccine against the hepatitis B virus (HBV) and of antiviral agents efficiently suppressing HBV replication, more than 250 million people are currently chronically infected with this hepatotropic DNA virus, and resolution of chronic hepatitis B (CHB) is rarely achieved. Moreover, coinfection with the hepatitis D virus (HDV), a human RNA satellite virus requiring the envelope proteins of HBV for productive viral spreading, substantially aggravates the disease course of CHB. The molecular mechanisms by which these viruses interact with each other and with the intrinsic innate responses of the hepatocytes are not fully understood. While HBV appears to avoid innate immune recognition, HDV elicits a strong enhancement of innate responses. Notwithstanding, such induction does not hamper HDV replication but contributes to liver inflammation and pathogenesis. Intriguingly, HDV appears to influence the ability of T cells to recognize infected hepatocytes by boosting antigen presentation. This review focuses on current knowledge regarding how these viruses can shape and counteract the intrinsic innate responses of the hepatocytes, thus affecting the immune system and pathogenesis. Understanding the distinct strategies of persistence that HBV and HDV have evolved is central for advancing the development of curative therapies.

In Vivo Models of HDV Infection: Is Humanizing NTCP Enough?

The discovery of sodium taurocholate co-transporting polypeptide (NTCP) as a hepatitis B (HBV) and delta virus (HDV) entry receptor has encouraged the development of new animal models of infection. This review provides an overview of the different in vivo models that are currently available to study HDV either in the absence or presence of HBV. By presenting new advances and remaining drawbacks, we will discuss human host factors which, in addition to NTCP, need to be investigated or identified to enable a persistent HDV infection in murine hepatocytes. Detailed knowledge on species-specific factors involved in HDV persistence also shall contribute to the development of therapeutic strategies.