CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

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.

Development of a single-domain antibody to target a G-quadruplex located on the hepatitis B virus covalently closed circular DNA genome.

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.

Highlights from the 2023 International Meeting on the Molecular Biology of Hepatitis B virus.

Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.

Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP.

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na+-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.

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.

Molecular determinants within the C-termini of L-HDAg that regulate hepatitis D virus replication and assembly.

Background & Aims: Hepatitis D virus (HDV) is the causative agent of chronic hepatitis delta, the most severe form of viral hepatitis. HDV encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus, which confer regulatory roles that are distinct from those of S-HDAg. Notably, these residues are divergent between different genotypes. We aimed to elucidate the molecular determinants within the C-termini that are essential for the regulatory role of L-HDAg in HDV replication and assembly. Methods: Northern blot, reverse-transcription quantitative PCR, and a newly established HDV trans-complementary system were used in this study. Results: C-termini of L-HDAg, albeit with high sequence variation among different genotypes, are interchangeable with respect to the trans-inhibitory function of L-HDAg and HDV assembly. The C-terminus of L-HDAg features a conserved prenylation CXXQ motif and is enriched with proline and hydrophobic residues. Abolishment of the CXXQ motif attenuated the inhibitory effect of L-HDAg on HDV replication. In contrast, the enrichment of proline and hydrophobic residues per se does not modify the trans-inhibitory function of L-HDAg. Nevertheless, these residues are essential for HDV assembly. Mechanistically, prolines and hydrophobic residues contribute to HDV assembly via a mode of action independent of the prenylated CXXQ motif. Conclusions: Within the C-terminus of L-HDAg, the CXXQ motif and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly. This intrinsic viral regulatory mechanism we elucidated deepens our understanding of the unique life cycle of HDV. Impact and implications: Hepatitis D virus (HDV) encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus. This C-terminal extension in L-HDAg confers regulatory roles in the HDV life cycle that are distinct from those of S-HDAg. Herein, we found that C-termini of L-HDAg, although with high sequence variation, are interchangeable among different HDV genotypes. Within the C-terminus of L-HDAg, the prenylation motif, and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly.

Hepatitis D infection induces IFN-ß-mediated NK cell activation and TRAIL-dependent cytotoxicity.

Background and aims: The co-infection of hepatitis B (HBV) patients with the hepatitis D virus (HDV) causes the most severe form of viral hepatitis and thus drastically worsens the course of the disease. Therapy options for HBV/HDV patients are still limited. Here, we investigated the potential of natural killer (NK) cells that are crucial drivers of the innate immune response against viruses to target HDV-infected hepatocytes. Methods: We established in vitro co-culture models using HDV-infected hepatoma cell lines and human peripheral blood NK cells. We determined NK cell activation by flow cytometry, transcriptome analysis, bead-based cytokine immunoassays, and NK cell-mediated effects on T cells by flow cytometry. We validated the mechanisms using CRISPR/Cas9-mediated gene deletions. Moreover, we assessed the frequencies and phenotype of NK cells in peripheral blood of HBV and HDV superinfected patients. Results: Upon co-culture with HDV-infected hepatic cell lines, NK cells upregulated activation markers, interferon-stimulated genes (ISGs) including the death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), produced interferon (IFN)-γ and eliminated HDV-infected cells via the TRAIL-TRAIL-R2 axis. We identified IFN-ß released by HDV-infected cells as an important enhancer of NK cell activity. In line with our in vitro data, we observed activation of peripheral blood NK cells from HBV/HDV co-infected, but not HBV mono-infected patients. Conclusion: Our data demonstrate NK cell activation in HDV infection and their potential to eliminate HDV-infected hepatoma cells via the TRAIL/TRAIL-R2 axis which implies a high relevance of NK cells for the design of novel anti-viral therapies.

Broad-spectrum activity of bulevirtide against clinical isolates of HDV and recombinant pan-genotypic combinations of HBV/HDV.

Background & Aims: Bulevirtide (BLV) is a small lipopeptide agent that specifically binds to the sodium taurocholate cotransporting polypeptide (NTCP) bile salt transporter and HBV/HDV receptor on the surface of human hepatocytes and inhibits HDV and HBV entry. As a satellite virus of HBV, HDV virions are formed after assembly of HDV RNA with the HBV envelope proteins (HBsAg). Because both viruses exist as eight different genotypes, this creates a potential for high diversity in the HBV/HDV combinations. To investigate the sensitivity of various combinations of HBV/HDV genotypes to BLV, clinical and laboratory strains were assessed. Methods: For the laboratory strains, the different envelopes from HBV genotypes A through H were combined with HDV genotypes 1-8 in cotransfection assays. Clinical plasma isolates were obtained from clinical studies and academic collaborations to maximise the diversity of HBV/HDV genotypes tested. Results: The mean BLV EC50 against HDV laboratory strains ranged from 0.44 to 0.64 nM. Regardless of HBV and HDV genotypes, the clinical isolates showed similar sensitivities to BLV with mean values that ranged from 0.2 to 0.73 nM. Conclusions: These data support the use of BLV in patients infected with any HBV/HDV genotypes. Impact and implications: This study describes the potent activity of BLV against multiple laboratory strains spanning all HBV/HDV A-H/1-8 genotype combinations and the most diverse collection of HDV clinical samples tested to date, including HBV/HDV genotype combinations less frequently observed in the clinic. Overall, all isolates and laboratory strains displayed similar in vitro nanomolar sensitivity to BLV. This broad-spectrum antiviral activity of BLV has direct implications on potential simplified treatment for any patient infected with HDV, regardless of genotype, and supports the new 2023 EASL Clinical Practice Guidelines on HDV that recommend antiviral treatment for all patients with CHD.

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.

CDK8 and CDK19: positive regulators of signal-induced transcription and negative regulators of Mediator complex proteins.

We have conducted a detailed transcriptomic, proteomic and phosphoproteomic analysis of CDK8 and its paralog CDK19, alternative enzymatic components of the kinase module associated with transcriptional Mediator complex and implicated in development and diseases. This analysis was performed using genetic modifications of CDK8 and CDK19, selective CDK8/19 small molecule kinase inhibitors and a potent CDK8/19 PROTAC degrader. CDK8/19 inhibition in cells exposed to serum or to agonists of NFκB or protein kinase C (PKC) reduced the induction of signal-responsive genes, indicating a pleiotropic role of Mediator kinases in signal-induced transcriptional reprogramming. CDK8/19 inhibition under basal conditions initially downregulated a small group of genes, most of which were inducible by serum or PKC stimulation. Prolonged CDK8/19 inhibition or mutagenesis upregulated a larger gene set, along with a post-transcriptional increase in the proteins comprising the core Mediator complex and its kinase module. Regulation of both RNA and protein expression required CDK8/19 kinase activities but both enzymes protected their binding partner cyclin C from proteolytic degradation in a kinase-independent manner. Analysis of isogenic cell populations expressing CDK8, CDK19 or their kinase-inactive mutants revealed that CDK8 and CDK19 have the same qualitative effects on protein phosphorylation and gene expression at the RNA and protein levels, whereas differential effects of CDK8 versus CDK19 knockouts were attributable to quantitative differences in their expression and activity rather than different functions.

In vitro cell culture models to study hepatitis B and D virus infection.

Chronic infection with the hepatitis B virus (HBV) and hepatitis D virus (HDV) can cause a major global health burden. Current medication regimens can repress viral replication and help to control disease progression, but a complete cure is hardly achieved due to the difficulties to eradicate viral templates (cccDNA and integrates). To develop novel curative antiviral therapies for HBV/HDV infection, it is vital to precisely understand the details of the molecular biology of both viruses and the virus-host interactions. One important prerequisite for gaining this aim is the availability of suitable in vitro models that support HBV/HDV infection, replicate both viruses via their authentic template and allow to adequately study host cell responses. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) receptor as the most crucial host factor promoted HBV/HDV research to a new era. Recently, the structure of human NTCP was solved, gaining a deeper understanding of HBV recognition as the bona fide receptor. After decades of continuous efforts, new progress has been achieved in the development of cell culture models supporting HBV/HDV study. This review summarizes the cell culture models currently available, discusses the advantages and disadvantages of each model, and highlights their future applications in HBV and HDV research.

Evaluation of the drug-drug interaction potential of the novel hepatitis B and D virus entry inhibitor bulevirtide at OATP1B in healthy volunteers.

Introduction: Bulevirtide is a first-in-class antiviral drug to treat chronic hepatitis B/D. We investigated the drug-drug interaction potential and pharmacokinetics of high-dose subcutaneous bulevirtide (5 mg twice daily) with organic anion transporting polypeptide 1B1 (OATP1B1) and cytochrome P450 (CYP) 3A4. Methods: This was a single-center, open-label, fixed-sequence drug-drug interaction trial in 19 healthy volunteers. Before and at bulevirtide steady state, participants ingested a single 40 mg dose of pravastatin. A midazolam microdose was applied to quantify CYP3A4 activity. Results: At bulevirtide steady state, pravastatin area under the concentration-time curve (AUC0-∞) increased 1.32-fold (90% CI 1.08-1.61). The 5 mg bulevirtide twice-daily treatment resulted in a mean AUC0-12 of 1210 h*ng/ml (95% CI 1040-1408) and remained essentially unchanged under the influence of pravastatin. CYP3A4 activity did not change to a clinically relevant extent. As expected, total bile acids increased substantially (35-fold) compared to baseline during bulevirtide treatment. All study medication was well tolerated. Discussion: The study demonstrated that high-dose bulevirtide inhibited OATP1B-mediated hepatic uptake of the marker substrate pravastatin but the extent is considered clinically not relevant. Changes in CYP3A4 activity were also not clinically relevant. In conclusion, this study suggests that OATP1B substrate drugs as well as CYP3A4 substrates may safely be used without dose adjustment in patients treated with bulevirtide. However, in patients using high statin doses and where concomitant factors potentially further increase statin exposure, caution may be required when using bulevirtide.

An RNA-based system to study hepatitis B virus replication and evaluate antivirals.

Hepatitis B virus (HBV) chronically infects an estimated 300 million people, and standard treatments are rarely curative. Infection increases the risk of liver cirrhosis and hepatocellular carcinoma, and consequently, nearly 1 million people die each year from chronic hepatitis B. Tools and approaches that bring insights into HBV biology and facilitate the discovery and evaluation of antiviral drugs are in demand. Here, we describe a method to initiate the replication of HBV, a DNA virus, using synthetic RNA. This approach eliminates contaminating background signals from input virus or plasmid DNA that plagues existing systems and can be used to study multiple stages of HBV replication. We further demonstrate that this method can be uniquely applied to identify sequence variants that confer resistance to antiviral drugs.

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.

Hepatitis delta: Epidemiology to recent advances in therapeutic agents.

Hepatitis D virus (HDV) was first described in 1977 and is dependent on the presence of hepatitis B surface antigen (HBsAg) for its entry into cells and on the human host for replication. Due to the envelopment with the hepatitis B virus (HBV) envelope, early phases of HDV entry resemble HBV infection. Unlike HBV, HDV activates innate immune responses. The global prevalence of HDV is estimated to be about 5% of HBsAg positive individuals. However, recent studies have described a wide range of prevalence between 12 to 72 million individuals. Infection can occur as super-infection or co-infection. The diagnosis of active HDV infection involves screening with anti HDV antibodies followed by quantitative PCR testing for HDV RNA in those who are HBsAg positive. The diagnostic studies have evolved over the years improving the validity and reliability of the tests performed. HDV infection is considered the most severe form of viral hepatitis and the HDV genotype may influence the disease course. There are eight major HDV genotypes with prevalence varying by geographic region. HDV treatment has been challenging as HDV strongly depends on the host cell for replication and provides few, if any viral targets. Better understanding of HDV virology has led to the development of several therapeutic agents currently being studied in different phase II and III clinical trials. There is increasing promise of effective therapies that will ameliorate the course of this devastating disease.

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.

Novel prime-boost immune-based therapy inhibiting both hepatitis B and D virus infections.

OBJECTIVE: Chronic HBV/HDV infections are a major cause of liver cancer. Current treatments can only rarely eliminate HBV and HDV. Our previously developed preS1-HDAg immunotherapy could induce neutralising antibodies to HBV in vivo and raise HBV/HDV-specific T-cells. Here, we further investigate if a heterologous prime-boost strategy can circumvent T-cell tolerance and preclude HDV superinfection in vivo. DESIGN: A DNA prime-protein boost strategy was evaluated for immunogenicity in mice and rabbits. Its ability to circumvent T-cell tolerance was assessed in immunocompetent hepatitis B surface antigen (HBsAg)-transgenic mice. Neutralisation of HBV and HDV was evaluated both in vitro and in immunodeficient human-liver chimeric mice upon adoptive transfer. RESULTS: The prime-boost strategy elicits robust HBV/HDV-specific T-cells and preS1-antibodies that can effectively prevent HBV and HDV (co-)infection in vitro and in vivo. In a mouse model representing the chronic HBsAg carrier state, active immunisation primes high levels of preS1-antibodies and HDAg-specific T-cells. Moreover, transfer of vaccine-induced antibodies completely protects HBV-infected human-liver chimeric mice from HDV superinfection. CONCLUSION: The herein described preS1-HDAg immunotherapy is shown to be immunogenic and vaccine-induced antibodies are highly effective at preventing HBV and HDV (super)infection both in vitro and in vivo. Our vaccine can complement current and future therapies for the control of chronic HBV and HDV infection.

Effect of variants in LGP2 on MDA5-mediated activation of interferon response and suppression of hepatitis D virus replication.

BACKGROUND & AIMS: Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), sense viral RNA to induce the antiviral interferon (IFN) response. LGP2, unable to activate the IFN response itself, modulates RIG-I and MDA5 signalling. HDV, a small RNA virus causing the most severe form of viral hepatitis, is sensed by MDA5. The mechanism underlying IFN induction and its effect on HDV replication is unclear. Here, we aimed to unveil the role of LGP2 and clinically relevant variants thereof in these processes. METHODS: RLRs were depleted in HDV susceptible HepaRGNTCP cells and primary human hepatocytes. Cells were reconstituted to express different LGP2 versions. HDV and IFN markers were quantified in a time-resolved manner. Interaction studies among LGP2, MDA5, and RNA were performed by pull-down assays. RESULTS: LGP2 is essential for the MDA5-mediated IFN response induced upon HDV infection. This induction requires both RNA binding and ATPase activities of LGP2. The IFN response only moderately reduced HDV replication in resting cells but profoundly suppressed cell division-mediated HDV spread. An LGP2 variant (Q425R), predominating in Africans who develop less severe chronic hepatitis D, mediated detectably higher basal and faster HDV-induced IFN response as well as stronger HDV suppression. Mechanistically, LGP2 RNA binding was a prerequisite for the formation of stable MDA5-RNA complexes. MDA5 binding to RNA was enhanced by the Q425R LGP2 variant. CONCLUSIONS: LGP2 is essential to mount an antiviral IFN response induced by HDV and stabilises MDA5-RNA interaction required for downstream signalling. The natural Q425R LGP2 is a gain-of-function variant and might contribute to an attenuated course of hepatitis D. IMPACT AND IMPLICATIONS: HDV is the causative pathogen of chronic hepatitis D, a severe form of viral hepatitis that can lead to cirrhosis and hepatocellular carcinoma. Upon infection, the human immune system senses HDV and mounts an antiviral interferon (IFN) response. Here, we demonstrate that the immune sensor LGP2 cooperates with MDA5 to mount an IFN response that represses HDV replication. We mapped LGP2 determinants required for IFN system activation and characterised several natural genetic variants of LGP2. One of them reported to predominate in sub-Saharan Africans can accelerate HDV-induced IFN responses, arguing that genetic determinants, possibly including LGP2, might contribute to slower disease progression in this population. Our results will hopefully prompt further studies on genetic variations in LGP2 and other components of the innate immune sensing system, including assessments of their possible impact on the course of viral infection.

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.