Search

Chronic hepatitis D (CHD), a global health problem, manifests as the most severe form of viral hepatitis. The causative agent, HDV, is the smallest known human virus; it replicates its circular single-stranded RNA genome in the nucleus of hepatocytes. HDV requires HBV-encoded envelope proteins for dissemination and de novo cell entry. However, HDV can also spread through cell division. Following entry into hepatocytes, replicative intermediates of HDV RNA are sensed by the pattern recognition receptor MDA5 (melanoma differentiation antigen 5) resulting in interferon (IFN)-β/λ induction. This IFN response strongly suppresses cell division-mediated spread of HDV genomes, however, it only marginally affects HDV RNA replication in already infected, resting hepatocytes. Monotherapy with IFN-α/λ shows efficacy but rarely results in HDV clearance. Recent molecular insights into key determinants of HDV persistence and the accelerated development of specifically acting antivirals that interfere with the replication cycle have revealed promising new therapeutic perspectives. In this review, we briefly summarise our knowledge on replication/persistence of HDV, the newly discovered HDV-like agents, and the interplay of HDV with the IFN response and its consequences for persistence. Finally, we discuss the possible role of IFNs in combination with upcoming therapies aimed at HDV cure. [ABSTRACT FROM AUTHOR]

The human Hepatitis Delta Virus (HDV) is unique among all viral pathogens. Encoding only one protein (Hepatitis Delta Antigen; HDAg) within its viroid-like self-complementary RNA, HDV constitutes the smallest known virus in the animal kingdom. To disseminate in its host, HDV depends on a helper virus, the human Hepatitis B virus (HBV), which provides the envelope proteins required for HDV assembly. HDV affects an estimated 15-20 million out of the 240 million chronic HBV-carriers and disperses unequally in disparate geographical regions of the world. The disease it causes (chronic Hepatitis D) presents as the most severe form of viral hepatitis, leading to accelerated progression of liver dysfunction including cirrhosis and hepatocellular carcinoma and a high mortality rate. The lack of approved drugs interfering with specific steps of HDV replication poses a high burden for gaining insights into the molecular biology of the virus and, consequently, the development of specific novel medications that resiliently control HDV replication or, in the best case, functionally cure HDV infection or HBV/HDV co-infection. This review summarizes our current knowledge of HBV molecular biology, presents an update on novel cell culture and animal models to study the virus and provides updates on the clinical development of the three developmental drugs Lonafarnib, REP2139-Ca and Myrcludex B. [ABSTRACT FROM AUTHOR]

The author discusses the use of stem cell-derived hepatocytes for studying hepatitis B virus (HBV) infection. Topics discused include the comparison of the HBV cell culture system, the differentiation of liver-resident hepatic progenitor cell, human embryonic stem cell, and bone-marrow derived mesenchymal stem cells, and the expression of sodium (Na) taurocholate cotransporting polypeptide (NTCP) in differentiation of human hepatic progenitor (HepaRG) cell line.

Inhibition of virus entry has become a major concept in the development of new antiviral drugs. Entry inhibitors can either neutralize activities of viral surface proteins or target essential host factors such as (co)receptors. Due to its distinct tissue tropism and the highly specific viral and cellular factors involved in its entry, hepatitis B virus (HBV) is an ideal candidate for entry inhibition. Hepatitis B immunoglobulins neutralize infection by binding to the S-domain of HBV surface proteins and are used to prevent reinfection of the graft after liver transplantation. Novel S or preS-specific monoclonal antibodies are currently in development. The identification of sodium-taurocholate cotransporting polypeptide (NTCP) as a bona fide receptor has revealed a suitable target for HBV entry inhibition. NTCP receptor function is blocked by a variety of different agents including Myrcludex B, a synthetic N-acylated preS1-derived lipopeptide that inhibits HBV entry in vitro and in vivo with high efficacy. Current antiviral treatment for chronic HBV-infected patients focuses on the inhibition of the viral polymerase via nucleos(t)ide analogues (NA). Entry inhibitors in combination with NAs could block reinfection and shield naive hepatocytes that emerge from natural liver turnover, opening up new therapeutic options. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) is a major human pathogen causing about 750 000 deaths per year. The virion consists of a nucleocapsid and an envelope formed by lipids, and three integral membrane proteins. Although we have detailed structural insights into the organization of the HBV core, the arrangement of the envelope in virions and its interaction with the nucleocapsid is elusive. Here we show the ultrastructure of hepatitis B virions purified from patient serum. We identified two morphological phenotypes, which appear as compact and gapped particles with nucleocapsids in distinguishable conformations. The overall structures of these nucleocapsids resemble recombinant cores with two α-helical spikes per asymmetric unit. At the charged tips the spikes are contacted by defined protrusions of the envelope proteins, probably via electrostatic interactions. The HBV envelope in the two morphotypes is to some extent variable, but the surface proteins follow a general packing scheme with up to three surface protein dimers per asymmetric unit. The variability in the structure of the envelope indicates that the nucleocapsid does not firmly constrain the arrangement of the surface proteins, but provides a general template for the packing. [ABSTRACT FROM AUTHOR]

An approximate number of 250 million people worldwide are chronically infected with hepatitis B virus, making them susceptible to a coinfection with hepatitis D virus. The superinfection causes the most severe form of a viral hepatitis and thus drastically worsens the course of the disease. Until recently, the only available therapy consisted of interferon-α, only eligible for a minority of patients. In July 2020, the EMA granted Hepcludex conditional marketing authorization throughout the European Union. This first-in-class entry inhibitor offers the promise to prevent the spread in order to gain control and eventually participate in curing hepatitis B and D. Hepcludex is an example of how understanding the viral lifecycle can give rise to new therapy options. Sodium taurocholate co-transporting polypeptide, the virus receptor and the target of Hepcludex, and other targets of hepatitis D therapy currently researched are reviewed in this work. Farnesyltransferase inhibitors such as Lonafarnib, targeting another essential molecule in the HDV life cycle, represent a promising target for hepatitis D therapy. Farnesyltransferase attaches a farnesyl (isoprenyl) group to proteins carrying a C-terminal Ca1a2X (C: cysteine, a: aliphatic amino acid, X: C-terminal amino acid) motif like the large hepatitis D virus antigen. This modification enables the interaction of the HBV/HDV particle and the virus envelope proteins. Lonafarnib, which prevents this envelopment, has been tested in clinical trials. Targeting the lifecycle of the hepatitis B virus needs to be considered in hepatitis D therapy in order to cure a patient from both coexisting infections. Nucleic acid polymers target the hepatitis B lifecycle in a manner that is not yet understood. Understanding the possible targets of the hepatitis D virus therapy is inevitable for the improvement and development of a sufficient therapy that HDV patients are desperately in need of. [ABSTRACT FROM AUTHOR]

Chronic hepatitis D (CHD) is the most severe form of viral hepatitis, with rapid progression of liver-related diseases and high rates of development of hepatocellular carcinoma. The causative agent, hepatitis D virus (HDV), contains a small (approximately 1.7 kb) highly self-pairing single-strand circular RNA genome that assembles with the HDV antigen to form a ribonucleoprotein (RNP) complex. HDV depends on hepatitis B virus (HBV) envelope proteins for envelopment and de novo hepatocyte entry; however, its intracellular RNA replication is autonomous. In addition, HDV can amplify HBV independently through cell division. Cellular innate immune responses, mainly interferon (IFN) response, are crucial for controlling invading viruses, while viruses counteract these responses to favor their propagation. In contrast to HBV, HDV activates profound IFN response through the melanoma differentiation antigen 5 (MDA5) pathway. This cellular response efficiently suppresses cell-division-mediated HDV spread and, to some extent, early stages of HDV de novo infection, but only marginally impairs RNA replication in resting hepatocytes. In this review, we summarize the current knowledge on HDV structure, replication, and persistence and subsequently focus on the interplay between HDV and IFN response, including IFN activation, sensing, antiviral effects, and viral countermeasures. Finally, we discuss crosstalk with HBV. [ABSTRACT FROM AUTHOR]

The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV's evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (Marmota monax), white-tailed deer (Odocoileus virginianus), and lesser dog-like bat (Peropteryx macrotis) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus–host interaction of this unique group of animal viroid-like agents in relation to HDV. [ABSTRACT FROM AUTHOR]

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. 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. At week 24, median intrahepatic HDV RNA decline from baseline was 0.9Log 10 with 2 mg (n = 7), 1.1Log 10 with 5 mg (n = 5) and 1.4 Log 10 with 10 mg (n = 7) of BLV. At week 48, median reductions were 2.2Log 10 with 2 mg (n = 27) and 2.7Log 10 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. 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. 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. NCT03546621, NCT02888106, NCT03852719 [Display omitted] • Combined analysis of paired liver biopsies from three clinical trials investigating the entry inhibitor bulevirtide. • Bulevirtide strongly reduces intrahepatic HDV RNA levels and HDV-infected hepatocytes. • The reduction of infected cells reflects changes in viral loads in the serum. • Inflammatory gene expression is also decreased by bulevirtide treatment. • Intrahepatic levels of the helper virus HBV are not affected by the treatment. [ABSTRACT FROM AUTHOR]

Summary For almost three decades following the discovery of the human Hepatitis B Virus (HBV) the early events of virus infection (attachment to hepatocytes, specific binding to a receptor on hepatocytes) remained enigmatic. The gradual improvement of tissue culture systems for HBV has enabled the identification of viral determinants for viral infectivity and facilitated the discovery of the human sodium taurocholate co-transporting polypeptide (hNTCP) as a liver specific receptor of HBV and its satellite, the human Hepatitis Delta Virus (HDV). These findings are currently leading basic and clinical research activities in new directions. (1) Stable hNTCP-expressing cell lines have become a valuable platform to study the full HBV replication cycle from its native template, the cccDNA. (2) The suitability of NTCP complemented cell culture systems for high throughput screening approaches will facilitate identification of novel host factors involved in HBV replication (including those that determine the peculiar host specificity of HBV infection) and will enable identification and development of novel drug candidates for improved therapeutics. (3) Since NTCP is a major host-specific restriction factor for HBV and HDV, hNTCP-expressing animals provide the basis for future susceptible in vivo models. (4) The concept obtained with the entry inhibitor Myrcludex B demonstrates that NTCP is a suitable target for clinical interference with viral entry. This will foster further clinical approaches aiming at curative combination therapies. [ABSTRACT FROM AUTHOR]

The article presents a study which described a method to initiate the replication of hepatitis B virus (HBV), a DNA virus, using synthetic RNA. Topics discussed include a test of whether HBV pregenomic RNA (pgRNA) could initiate the virus life cycle, the ability of HBV launch to identify drug resistance mutations de novo, and synthesis of HBV pgRNA.

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. 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. 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. 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. 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. [Display omitted] • LGP2 binds HDV RNA. • LGP2 RNA binding and ATPase functions are essential for full MDA5-mediated IFN activation by HDV and viral suppression. • LGP2 Q425R predominates in Africans, mediating higher basal and faster HDV-induced IFN responses. • LGP2 Q425R leads to stronger HDV suppression and could explain the attenuated course of hepatitis D in African populations. • LGP2 Q425R promotes formation of stable MDA5–RNA complexes. [ABSTRACT FROM AUTHOR]

Besides HBV-dependent de novo infection, cell division-mediated spread contributes to HDV persistence and dampens the effect of antivirals that abrogate de novo infection. Nonetheless, the combination of these antivirals with interferons (IFNs) showed strong synergism in recent clinical trials, implying a complementary mode-of-action of IFNs. Therefore, we investigated the effect of IFN response on cell division-mediated HDV spread. Cells infected with HDV were passaged to undergo cell division. The effect of the IFN response was evaluated by blocking HDV-induced IFN activation, by applying different IFN treatment regimens, and by adjusting HDV infection doses. Cell division-mediated HDV spread was highly efficient following infection of HuH7NTCP cells (defective in IFN production), but profoundly restricted in infected IFN-competent HepaRGNTCP cells. Treatment with IFN-α/-λ1 inhibited HDV spread in dividing HuH7NTCP cells, but exhibited a marginal effect on HDV replication in resting cells. Blocking the HDV-induced IFN response with the JAK1/2 inhibitor ruxolitinib or knocking down MDA5 augmented HDV spread in dividing HepaRGNTCP cells. The virus-induced IFN response also destabilized HDV RNA in dividing cells. Moreover, the effect of exogenous IFNs on cell division-mediated HDV spread was more pronounced at low multiplicities of infection with weak virus-induced IFN responses. Both HDV-induced IFN response and exogenous IFN treatment suppress cell division-mediated HDV spread, presumably through acceleration of HDV RNA decay. Our findings demonstrate a novel mode-of-action of IFN, explain the more pronounced effect of IFN therapy in patients with lower HDV serum RNA levels, and provide insights for the development of combination therapies. Chronic hepatitis D is a major health problem. The causative pathogen hepatitis D virus (HDV) can propagate through viral particle-mediated infection and the division of infected cells. Although viral particle-dependent infection can be blocked by recently developed drugs, therapies addressing the cell division route have not been reported. Taking advantage of relevant cell culture models, we demonstrate that the widely used immune modulator interferon can efficiently suppress HDV spread through cell division. This work unveils a new function of interferon and sheds light on potentially curative combination therapies. [Display omitted] • Besides HBV-dependent de novo infection, HDV also propagates through cell division. • IFN response efficiently suppresses cell division-mediated HDV spread. • The effect of IFN treatment is more efficient when HDV-induced IFN response is low. • IFN response destabilizes HDV RNA during cell division. [ABSTRACT FROM AUTHOR]

The lack of an appropriate in vitro infection system for the major human pathogen hepatitis B virus (HBV) has prevented a molecular understanding of the early infection events of HBV. We used the novel HBV-infectible cell line HepaRG and primary human hepatocytes to investigate the interference of infection by HBV envelope protein-derived peptides. We found that a peptide consisting of the authentically myristoylated N-terminal 47 amino acids of the pre-S1 domain of the large viral envelope protein (L protein) specifically prevented HBV infection, with a 50% inhibitory concentration (IC50) of 8 nM. The replacement of myristic acid with other hydrophobic moieties resulted in changes in the inhibitory activity, most notably by a decrease in the IC50 to picomolar concentrations for longer unbranched fatty acids. The obstruction of HepaRG cell susceptibility to HBV infection after short preincubation times with the peptides suggested that the peptides efficiently target and inactivate a receptor at the hepatocyte surface. Our data both shed light on the molecular mechanism of HBV entry into hepatocytes and provide a basis for the development of potent hepadnaviral entry inhibitors as a novel therapeutic concept for the treatment of hepatitis B. [ABSTRACT FROM AUTHOR]

Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocytelike morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies. [ABSTRACT FROM AUTHOR]

Infection of hepatocytes by hepatitis B virus (HBV) depends on surface expression of its receptor Na+-taurocholate-cotransporting polypeptide (NTCP), but sufficient NTCP expression is lacking in most cell lines. NTCP can be introduced by plasmid transfection or transduction by viral vectors to render cells permissive for HBV. However, transient transfection of hepatocyte-derived cell lines is inefficient, resulting in inhomogeneous protein expression and does not allow to adapt the level of NTCP expression. We therefore utilized in vitro transcribed mRNA to introduce NTCP into cells. Optimization using alternative cap structures and nucleotide modifications rendered mRNA transfection into different non-hepatic and hepatic cell lines very efficient. After transfection of mRNA, surface expression and functionality of NTCP was demonstrated by staining with an N-terminal HBV-preS peptide and bile acid uptake. Introduction of NTCP by mRNA transfection increased susceptibility of hepatoma cells to HBV in a dose-dependent manner. Transfection of NTCP mRNA into non-liver cells, in contrast, supported bile acid uptake but did still not render the cells permissive for HBV, demonstrating the requirement for additional host factors. Introduction of candidate host factors by mRNA transfection will allow for fast and convenient analysis of the viral life cycle using a transient, but reliable expression system. [ABSTRACT FROM AUTHOR]

Chronic HDV infections cause the most severe form of viral hepatitis. HDV requires HBV envelope proteins for hepatocyte entry, particle assembly and release. Eight HDV and 8 HBV genotypes have been identified. However, there are limited data on the replication competence of different genotypes and the effect that different HBV envelopes have on virion assembly and infectivity. We subcloned complementary DNAs (cDNAs) of all HDV and HBV genotypes and systematically studied HDV replication, assembly and infectivity using northern blot, western blot, reverse-transcription quantitative PCR, and in-cell ELISA. The 8 HDV cDNA clones initiated HDV replication with noticeable differences regarding replication efficacy. The 8 HBV-HBsAg-encoding constructs all supported secretion of subviral particles, however variations in envelope protein stoichiometry and secretion efficacy were observed. Co-transfection of all HDV/HBV combinations supported particle assembly, however, the respective pseudo-typed HDVs differed with respect to assembly kinetics. The most productive combinations did not correlate with the natural geographic distribution, arguing against an evolutionary adaptation of HDV ribonucleoprotein complexes to HBV envelopes. All HDVs elicited robust and comparable innate immune responses. HBV envelope-dependent differences in the activity of the EMA-approved entry inhibitor bulevirtide were observed, however efficient inhibition could be achieved at therapeutically applied doses. Lonafarnib also showed pan-genotypic activity. HDVs from different genotypes replicate with variable efficacies. Variations in HDV genomes and HBV envelope proteins are both major determinants of HDV egress and entry efficacy, and consequently assembly inhibition by lonafarnib or entry inhibition by bulevirtide. These differences possibly influence HDV pathogenicity, immune responses and the efficacy of novel drug regimens. HDV requires the envelope protein of HBV for assembly and to infect human cells. We investigated the ability of different HDV genotypes to infect cells and replicate. We also assessed the effect that envelope proteins from different HBV genotypes had on HDV infectivity and replication. Herein, we confirmed that genotypic differences in HDV and HBV envelope proteins are major determinants of HDV assembly, de novo cell entry and consequently the efficacy of novel antivirals. [Display omitted] • HDV 1-8 presented marked differences in replication efficacy. • All HDV/HBV combinations supported HDV assembly with varied kinetics. • All HDVs elicited robust and comparable innate immune responses. • EMA-approved entry inhibitor bulevirtide showed pan-genotypic efficacy against HBV-enveloped HDV. • Lonafarnib, the assembly inhibitor showed pan-genotypic activity against HDV. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) is a globally-distributed pathogen and is a major cause of liver disease. HBV (or closely-related animal hepadnaviruses) can integrate into the host genome, but (unlike retroviruses) this integrated form is replication-defective. The specific role(s) of the integrated HBV DNA has been a long-standing topic of debate. Novel in vitro models of HBV infection combined with sensitive molecular assays now enable researchers to investigate this under-characterised phenomenon with greater ease and precision. This review covers the contributions these systems have made to understanding how HBV DNA integration induces liver cancer and facilitates viral persistence. We summarise the current findings into a working model of chronic HBV infection and discuss the clinical implications of this hypothetical framework on the upcoming therapeutic strategies used to curb HBV-associated pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Chronic hepatitis B and D virus (HBV/HDV) infections can cause cancer. Current HBV therapy using nucleoside analogues (NAs) is life-long and reduces but does not eliminate the risk of cancer. A hallmark of chronic hepatitis B is a dysfunctional HBV-specific T-cell response. We therefore designed an immunotherapy driven by naive healthy T cells specific for the HDV antigen (HDAg) to bypass the need for HBV-specific T cells in order to prime PreS1-specific T cells and PreS1 antibodies blocking HBV entry.Methods: Ten combinations of PreS1 and/or HDAg sequences were evaluated for induction of PreS1 antibodies and HBV- and HDV-specific T cells in vitro and in vivo. Neutralization of HBV by PreS1-specific murine and rabbit antibodies was evaluated in cell culture, and rabbit anti-PreS1 were tested for neutralization of HBV in mice repopulated with human hepatocytes.Results: The best vaccine candidate induced T cells to PreS1 and HDAg, and PreS1 antibodies blocking HBV entry in vitro. Importantly, adoptive transfer of PreS1 antibodies prevented, or modulated, HBV infection after a subsequent challenge in humanized mice.Conclusions: We here describe a novel immunotherapy for chronic HBV/HDV that targets viral entry to complement NAs and coming therapies inhibiting viral maturation. [ABSTRACT FROM AUTHOR]

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity. Author summary: Pyrin and HIN domain (PYHIN) proteins are best known for their role in immune sensing and inflammasome activation. However, most studies focused on the cytosolic DNA sensor AIM2. The human genome encodes four different PYHIN proteins, and recent studies revealed that the nuclear PYHIN protein IFI16 suppresses transcription of HIV and other viral pathogens. Here, we show that two other members of this protein family, MNDA and PYHIN1, that are also predominantly found in the nucleus share the antiretroviral activity of IFI16. Importantly, our data demonstrate that nuclear PYHIN proteins restrict HIV-1 in primary human macrophages and CD4+ T lymphocytes. Functional analyses revealed that the pyrin domains of these factors compete with DNA for binding of the transcription factor Sp1. Altogether, our results support a key role of nuclear PYHIN proteins in restricting HIV-1 and suggest that they are also active against other viral pathogens and retrotransposons. [ABSTRACT FROM AUTHOR]

Chronic infection with the human Hepatitis B virus (HBV) is a major global health problem. Hepatitis D virus (HDV) is a satellite of HBV that uses HBV envelope proteins for cell egress and entry. Using infection systems encoding the HBV/HDV receptor human sodium taurocholate co-transporting polypeptide (NTCP), we screened 1181 FDA-approved drugs applying markers for interference for HBV and HDV infection. As one primary hit we identified Acitretin, a retinoid, as an inhibitor of HBV replication and HDV release. Based on this, other retinoic acid receptor (RAR) agonists with different specificities were found to interfere with HBV replication, verifying that the retinoic acid receptor pathway regulates replication. Of the eight agonists investigated, RARα-specific agonist Am80 (tamibarotene) was most active. Am80 reduced secretion of HBeAg and HBsAg with IC 50 s < 10 nM in differentiated HepaRG-NTCP cells. Similar effects were observed in primary human hepatocytes. In HepG2-NTCP cells, profound Am80-mediated inhibition required prolonged treatment of up to 35 days. Am80 treatment of cells with an established HBV cccDNA pool resulted in a reduction of secreted HBsAg and HBeAg, which correlated with reduced intracellular viral RNA levels, but not cccDNA copy numbers. The effect lasted for >12 days after removal of the drug. HBV genotypes B, D, and E were equally inhibited. By contrast, Am80 did not affect HBV replication in transfected cells or HepG2.2.15 cells, which carry an integrated HBV genome. In conclusion, our results indicate a persistent inhibition of HBV transcription by Am80, which might be used for drug repositioning. • Feasibility of a high-content drug screening for inhibitors of HBV/HDV infection. • Clinically approved RAR agonists inhibit HBV infection in authentic cell lines. • The RARα-specific retinoid Am80 potently inhibits HBV transcription from cccDNA. [ABSTRACT FROM AUTHOR]

T cell therapy is a promising means to treat chronic HBV infection and HBV-associated hepatocellular carcinoma. T cells engineered to express an HBV-specific T cell receptor (TCR) may achieve cure of HBV infection upon adoptive transfer. We investigated the therapeutic potential and safety of T cells stably expressing high affinity HBV envelope- or core-specific TCRs recognizing European and Asian HLA-A2 subtypes. Both CD8+ and CD4+ T cells from healthy donors and from chronic hepatitis B patients became polyfunctional effector cells when grafted with HBV-specific TCRs and eliminated HBV from infected HepG2-NTCP cell cultures. A single transfer of TCR-grafted T cells into HBV-infected, humanized mice controlled HBV infection and virological markers declined 4-5 log or below detection limit. When - as in a typical clinical setting - only a minority of hepatocytes were infected, engineered T cells specifically cleared infected hepatocytes without damaging non-infected cells. Cell death was compensated by hepatocyte proliferation and alanine amino transferase levels peaking at day 5 to 7 normalized again thereafter. Co-treatment with the entry inhibitor Myrcludex B ensured long-term control of HBV infection. Thus, T cells stably transduced with highly functional TCRs have the potential to mediate clearance of HBV-infected cells causing limited liver injury. [ABSTRACT FROM AUTHOR]

Summary Chronic hepatitis delta represents the most severe form of chronic viral hepatitis. The current treatment of hepatitis delta virus (HDV) infection consists of the use of interferons and is largely unsatisfactory. Several new compounds are currently in development for the treatment of HDV infection. However, surrogate markers that can be used to develop clinical endpoints in HDV infection are not well defined. In the current manuscript, we aimed to evaluate the existing data on treatment of HDV infection and to suggest treatment goals (possible "trial endpoints") that could be used across different clinical trials. [ABSTRACT FROM AUTHOR]

Distinct populations of hepatocytes infected with hepatitis B virus (HBV) or only harboring HBV DNA integrations coexist within an HBV chronically infected liver. These hepatocytes express HBV antigens at different levels and with different intracellular localizations, but it is not known whether this heterogeneity of viral antigen expression could result in an uneven hepatic presentation of distinct HBV epitopes/HLA class I complexes triggering different levels of activation of HBV-specific CD8+ T cells. Using antibodies specific to two distinct HLA-A*02:01/HBV epitope complexes of HBV nucleocapsid and envelope proteins, we mapped their topological distributions in liver biopsy specimens of two anti-hepatitis B e antigen-positive (HBe+) chronic HBV (CHB) patients. We demonstrated that the core and envelope CD8+ T cell epitopes were not uniformly distributed in the liver parenchyma but preferentially located in distinct and sometimes mutually exclusive hepatic zones. The efficiency of HBV epitope presentation was then tested in vitro utilizing HLA-A*02:01/HBV epitope-specific antibodies and the corresponding CD8+ T cells in primary human hepatocyte and hepatoma cell lines either infected with HBV or harboring HBV DNA integration. We confirmed the existence of a marked variability in the efficiency of HLA class I/HBV epitope presentation among the different targets that was influenced by the presence of gamma interferon (IFN-γ) and availability of newly translated viral antigens. In conclusion, HBV antigen presentation can be heterogeneous within an HBV-infected liver. As a consequence, CD8+ T cells of different HBV specificities might have different antiviral efficacies. [ABSTRACT FROM AUTHOR]

Infection with the Hepatitis B Virus (HBV) is one of the strongest risk-factors for liver cancer (hepatocellular carcinoma, HCC). One of the reported drivers of HCC is the integration of HBV DNA into the host cell genome, which may induce pro-carcinogenic pathways. These reported pathways include: induction of chromosomal instability; generation of insertional mutagenesis in key cancer-associated genes; transcription of downstream cancer-associated cellular genes; and/or formation of a persistent source of viral protein expression (particularly HBV surface and X proteins). The contribution of each of these specific mechanisms towards carcinogenesis is currently unclear. Here, we review the current knowledge of specific sites of HBV DNA integration into the host genome, which sheds light on these mechanisms. We give an overview of previously-used methods to detect HBV DNA integration and the enrichment of integration events in specific functional and structural cellular genomic sites. Finally, we posit a theoretical model of HBV DNA integration during disease progression and highlight open questions in the field. [ABSTRACT FROM AUTHOR]

Chronic infection with the human hepatitis B virus (HBV) is a major health problem. Virus persistence requires the establishment and maintenance of covalently closed circular DNA (cccDNA), the episomal virus template in the nucleus of infected hepatocytes. Compared to replicative DNA intermediates (relaxed circular DNA [rcDNA]), copy numbers of cccDNA in infected hepatocytes are low. Accordingly, accurate analyses of cccDNA require enrichment of nuclear fractions and Southern blotting or selective quantitative PCR (qPCR) methods allowing discrimination of cccDNA and rcDNA. In this report, we analyzed cccDNA-specific primer pairs for their ability to amplify cccDNA selectively. Using mixtures of defined forms of HBV and genomic DNA, we determined the potential of different nucleases for targeted digestion of the open/relaxed circular DNA forms in the absence and presence of genomic DNA without affecting cccDNA. We found that the combination of T5 exonuclease with a primer set amplifying an approximately 1-kb fragment permits reliable quantification of cccDNA without the requirement of prior nucleus enrichment or Hirt extraction. We tested this method in four different in vitro infection systems and quantified cccDNA copy numbers at increasing multiplicities of inoculated genome equivalents. We further analyzed the kinetics of cccDNA formation and the effect of drugs (interferon, entry inhibitors, and capsid inhibitors) on cccDNA. Our method allows reliable cccDNA quantification at early stages of infection in the presence of a high excess of input virus and replicative intermediates and is thereby suitable for drug screening and investigation of cccDNA formation and maintenance. [ABSTRACT FROM AUTHOR]

Highlights • A novel HBV antigen combining S and L-derived epitopes was produced in lettuce. • The chimeric antigen induces humoral immune responses in orally-vaccinated mice. • Antibodies elicited by the chimeric antigen are strong inhibitors of HBV infection. Abstract Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S) envelope protein are currently used in universal vaccination programs and achieve protective immune response in more than 90% of recipients. However, new vaccination strategies are necessary for successful immunization of the remaining non- or low-responders. We have previously characterized a novel HBV chimeric antigen, which combines neutralization epitopes of the S and the preS1 domain of the large (L) envelope protein (genotype D). The S/preS121–47 chimera produced in mammalian cells and Nicotiana benthamiana plants, induced a significantly stronger immune response in parenterally vaccinated mice than the S protein. Here we describe the transient expression of the S/preS121–47 antigen in an edible plant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral administration of adjuvant-free Lactuca sativa expressing the S/preS121–47 antigen, three times, at 1 μg/dose, was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies were able to neutralize HBV infection in an NTCP-expressing infection system (HepG2-NTCP cell line) more efficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results support the S/preS121–47 antigen as a promising candidate for future development as an edible HBV vaccine. [ABSTRACT FROM AUTHOR]

Background & Aims Hepatitis B virus (HBV) and D virus (HDV) co-infections cause the most severe form of viral hepatitis. HDV induces an innate immune response, but it is unknown how the host cell senses HDV and if this defense affects HDV replication. We aim to characterize interferon (IFN) activation by HDV, identify the responsible sensor and evaluate the effect of IFN on HDV replication. Methods HDV and HBV susceptible hepatoma cell lines and primary human hepatocytes (PHH) were used for infection studies. Viral markers and cellular gene expression were analyzed at different time points after infection. Pattern recognition receptors (PRRs) required for HDV-mediated IFN activation and the impact on HDV replication were studied using stable knock-down or overexpression of the PRRs. Results Microarray analysis revealed that HDV but not HBV infection activated a broad range of interferon stimulated genes (ISGs) in HepG2 NTCP cells. HDV strongly activated IFN-β and IFN-λ in cell lines and PHH. HDV induced IFN levels remained unaltered upon RIG-I ( DDX58 ) or TLR3 knock-down, but were almost completely abolished upon MDA5 ( IFIH1 ) depletion. Conversely, overexpression of MDA5 but not RIG-I and TLR3 in HuH7.5 NTCP cells partially restored ISG induction. During long-term infection, IFN levels gradually diminished in both HepG2 NTCP and HepaRG NTCP cell lines. MDA5 depletion had little effect on HDV replication despite dampening HDV-induced IFN response. Moreover, treatment with type I or type III IFNs did not abolish HDV replication. Conclusion Active replication of HDV induces an IFN-β/λ response, which is predominantly mediated by MDA5. This IFN response and exogenous IFN treatment have only a moderate effect on HDV replication in vitro indicating the adaption of HDV replication to an IFN-activated state. Lay summary In contrast to hepatitis B virus, infection with hepatitis D virus induces a strong IFN-β/λ response in innate immune competent cell lines. MDA5 is the key sensor for the recognition of hepatitis D virus replicative intermediates. An IFN-activated state did not prevent hepatitis D virus replication in vitro, indicating that hepatitis D virus is resistant to self-induced innate immune responses and therapeutic IFN treatment. [ABSTRACT FROM AUTHOR]

Chronic infection by hepatitis B virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (covalently closed circular DNA [cccDNA]), integration of HBV DNA into the host cell genome is regularly observed in the liver in infected patients. While reported as a prooncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well understood, chiefly due to the lack of in vitro infection models that have detectable integration events. In this study, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10,000 cells, with the most consistent detection in Huh7-NTCP cells. The integration rate remained stable between 3 and 9 days postinfection. HBV DNA integration was efficiently blocked by treatment with a 200 nM concentration of the HBV entry inhibitor Myrcludex B, but not with 10 μM tenofovir, 100 U of interferon alpha, or a 1 μM concentration of the capsid assembly inhibitor GLS4. This suggests that integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV genome replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration. IMPORTANCE Hepatitis B virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the Hepadnaviridae family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer formation and persistence of virus infection. However, when and the mechanism(s) by which HBV DNA integration occurs are not clear. In this study, we have developed and characterized an in vitro system to reliably detect HBV DNA integrations that result from a true HBV infection event and that closely resemble those found in patient tissues. Using this model, we showed that integration occurs when the infection is first established. Importantly, we provide here a system to analyze molecular factors involved in HBV integration, which can be used to develop strategies to halt its formation. [ABSTRACT FROM AUTHOR]

Background & Aims Studying hepatitis delta virus (HDV) and developing new treatments is hampered by the limited availability of small animal models. Herein, a description of a robust mouse model of HDV infection that mimics several important characteristics of the human disease is presented. Methods HDV and hepatitis B virus (HBV) replication competent genomes were delivered to the mouse liver using adeno-associated viruses (AAV; AAV-HDV and AAV-HBV). Viral load, antigen expression and genomes were quantified at different time points after AAV injection. Furthermore, liver pathology, genome editing, and the activation of the innate immune response were evaluated. Results AAV-HDV infection initiated HDV replication in mouse hepatocytes. Genome editing was confirmed by the presence of small and large HDV antigens and sequencing. Viral replication was detected for 45 days, even after the AAV-HDV vector had almost disappeared. In the presence of HBV, HDV infectious particles were detected in serum. Furthermore, as observed in patients, co-infection was associated with the reduction of HBV antigen expression and the onset of liver damage that included the alteration of genes involved in the development of liver pathologies. HDV replication induced a sustained type I interferon response, which was significantly reduced in immunodeficient mice and almost absent in mitochondrial antiviral signaling protein (MAVS)-deficient mice. Conclusion The animal model described here reproduces important characteristics of human HDV infection and provides a valuable tool for characterizing the viral infection and for developing new treatments. Furthermore, MAVS was identified as a main player in HDV detection and adaptive immunity was found to be involved in the amplification of the innate immune response. Lay summary: Co-infection with hepatitis B and D virus (HBV and HDV, respectively) often causes a more severe disease condition than HBV alone. Gaining more insight into HDV and developing new treatments is hampered by limited availability of adequate immune competent small animal models and new ones are needed. Here, a mouse model of HDV infection is described, which mimics several important characteristics of the human disease, such as the initiation and maintenance of replication in murine hepatocytes, genome editing and, in the presence of HBV, generation of infectious particles. Lastly, the involvement of an adaptive immunity and the intracellular signaling molecule MAVS in mounting a strong and lasting innate response was shown. Thus, our model serves as a useful tool for the investigation of HDV biology and new treatments. [ABSTRACT FROM AUTHOR]

Chronic infection with the Hepatitis B Virus (HBV) is amajor cause of liver-relatedmorbidity and mortality. One peculiar observation in cells infected with HBV (or with closely-related animal hepadnaviruses) is the presence of viral DNA integration in the host cell genome, despite this form being a replicative dead-end for the virus. The frequent finding of somatic integration of viral DNA suggests an evolutionary benefit for the virus; however, the mechanism of integration, its functions, and the clinical implications remain unknown. Here we review the current body of knowledge of HBV DNA integration, with particular focus on the molecular mechanisms and its clinical implications (including the possible consequences of replication-independent antigen expression and its possible role in hepatocellular carcinoma). HBV DNA integration is likely to influence HBV replication, persistence, and pathogenesis, and so deserves greater attention in future studies. [ABSTRACT FROM AUTHOR]

Background & Aims The therapeutic option for patients with chronic hepatitis delta virus infection (CHD) is limited to interferon alpha with rare curative outcome. Myrcludex B is a first-in-class entry inhibitor inactivating the hepatitis B virus (HBV) and hepatitis D virus (HDV) receptor sodium taurocholate co-transporting polypeptide. We report the interim results of a pilot trial on chronically infected HDV patients treated with myrcludex B, or pegylated interferon alpha (PegIFNα-2a) or their combination. Methods Twenty-four patients with CHD infection were equally randomized (1:1:1) to receive myrcludex B, or PegIFNα-2a or their combination. Patients were evaluated for virological and biochemical response and tolerability of the study drugs at weeks 12 and 24. Results Myrcludex B was well tolerated and no serious adverse event occurred. Although hepatitis B surface antigen levels remained unchanged, HDV RNA significantly declined at week 24 in all cohorts. HDV RNA became negative in two patients each in the Myrcludex B and PegIFNα-2a cohorts, and in five patients of the Myrcludex B + PegIFNα-2a cohort. ALT decreased significantly in the Myrcludex B cohort (six of eight patients), and HBV DNA was significantly reduced at week 24 in the Myrcludex B + PegIFNα-2a cohort. Virus kinetic modeling suggested a strong synergistic effect of myrcludex B and PegIFNα-2a on both HDV and HBV. Conclusions Myrcludex B showed a strong effect on HDV RNA serum levels and induced ALT normalization under monotherapy. Synergistic antiviral effects on HDV RNA and HBV DNA in the Myr-IFN cohort indicated a benefit of the combination of entry inhibition with PegIFNα-2a to treat CHD patients. Lay summary Myrcludex B is a new drug to treat hepatitis B and D infection. After 24 weeks of treatment with myrcludex B and/or pegylated interferon α-2a, HDV R NA, a relevant marker for hepatitis D infection, decreased in all patients with chronic hepatitis B and D. Two of eight patients which received either myrcludex B or pegylated interferon α-2a, became negative for HDV RNA, and five of seven patients who received both drugs at the same time became negative. The drug was well tolerated. [ABSTRACT FROM AUTHOR]

Background & Aims Myrcludex B is a first-in-class compound, which blocks entry of hepatitis B and D virus into hepatocytes in vitro and in animal models. Based on the required preclinical data we aimed to translate this compound into the first application in humans. Methods Single ascending doses of myrcludex B, a 47 amino acid peptide, were administered up to 20 mg intravenously and 10 mg subcutaneously in a prospective open first-in-human, phase I clinical trial to 36 healthy volunteers. Safety, tolerability and plasma concentrations of myrcludex B were assessed and a pharmacokinetic model was derived. Results Myrcludex B was well tolerated and no serious or relevant AEs representing off-target effects, and no immunogenic effects were observed up to the highest applied dose of 20 mg (intravenously). Myrcludex B showed dose-dependent pharmacokinetics, best described by a 2-compartment target-mediated drug disposition model. Bioavailability of the subcutaneous application was large (85%). Interindividual variability was moderate. The pharmacokinetic model suggested that subcutaneous doses of 10 mg and above reach a target saturation of over 80% for at least 15 h. Conclusions Myrcludex B showed excellent tolerability up to high doses. Pharmacologic properties followed a 2-compartment target-mediated drug disposition model. These findings are vital for planning of further multiple dose efficacy trials in patients. Lay summary After showing antiviral activity in cell culture and animal models, myrcludex B, a new drug intended for the treatment of hepatitis B and D, has been administered the first time in humans. Healthy volunteers received the drug intravenously and subcutaneously up to high doses (20 mg). The drug was well tolerated and the characteristics of the drug determining its way in the human body could be described. These results will allow testing myrcludex B in hepatitis B and D patients. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) is a widespread human pathogen, responsible for chronic infections of ca. 240 million people worldwide. Until recently, the entry pathway of HBV into hepatocytes was only partially understood. The identification of human sodium taurocholate cotransporting polypeptide (NTCP) as a bona fide receptor of HBV has provided us with new tools to investigate this pathway in more details. Combined with advances in virus visualization techniques, approaches to directly visualize HBV cell attachment, NTCP interaction, virion internalization and intracellular transport are now becoming feasible. This review summarizes our current understanding of how HBV specifically enters hepatocytes, and describes possible visualization strategies applicable for a deeper understanding of the underlying cell biological processes. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) is a widespread human pathogen, responsible for chronic infections of ca. 240 million people worldwide. Until recently, the entry pathway of HBV into hepatocytes was only partially understood. The identification of human sodium taurocholate cotransporting polypeptide (NTCP) as a bona fide receptor of HBV has provided us with new tools to investigate this pathway in more details. Combined with advances in virus visualization techniques, approaches to directly visualize HBV cell attachment, NTCP interaction, virion internalization and intracellular transport are now becoming feasible. This review summarizes our current understanding of how HBV specifically enters hepatocytes, and describes possible visualization strategies applicable for a deeper understanding of the underlying cell biological processes. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) enters hepatocytes via its receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). So far, HBV infection has been achieved only in human hepatic cells reconstituted with hNTCP and not in cells of mouse origin. Here, the first mouse liver cell line (AML12) which gains susceptibility to HBV upon hNTCP expression is described. Thus, HBV infection of receptor-expressing mouse hepatocytes does not principally require a human cofactor but can be triggered by endogenous murine determinants. [ABSTRACT FROM AUTHOR]

Background & Aims Hepatitis B virus (HBV) is a major human pathogen restricted to hepatocytes. Expression of the specific receptor human sodium taurocholate cotransporting polypeptide (hNTCP) in mouse hepatocytes renders them susceptible to hepatitis delta virus (HDV), a satellite of HBV; however, HBV remains restricted at an early stage of replication. This study aims at clarifying whether this restriction is caused by the lack of a dependency factor or the activity of a restriction factor. Methods Six hNTCP-expressing mouse and human cell lines were generated and functionally characterized. By fusion with replication-supporting but non-infectable HepG2 cells, we analysed the ability of these heterokaryonic cells to fully support HBV replication by HBcAg expression and HBsAg/HBeAg secretion. Results While hNTCP expression in three mouse cell lines and the non-hepatic human HeLa cells conferred susceptibility to HDV, HBV replication was still restricted. Upon fusion of refractive cells to HepG2 cells, all heterokaryonic cells supported receptor-mediated infection with HBV. hNTCP was provided by the mouse cells and replication competence came from the HepG2 cell line. Transfection of a covalently closed circular DNA (cccDNA)-like molecule into non-susceptible cells promoted gene expression, indicating that the limiting step is upstream of cccDNA formation. Conclusions In addition to the expression of hNTCP, establishment of HBV infection in mouse and non-hepatocytic human cell lines requires supplementation with a dependency factor and is not limited by a restriction factor. This result opens new avenues for the development of a fully permissive immunocompetent HBV mouse model. [ABSTRACT FROM AUTHOR]

A novel analytical approach for the targeted profiling of bile acids (BAs) in human serum/plasma based on liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is presented. Reversed-phase chromatography enabled the baseline separation of 15 human BA species which could be readily detected by accurate mass analysis in negative ion mode. Blood proteins were removed by methanol precipitation in the presence of deuterium-labeled internal standards which allowed BA quantification in 50 μl plasma/serum. The assay was validated according to FDA guidance achieving quantification limits from 7.8 to 156 nM. Calibration curves prepared in charcoal-stripped serum/plasma showed excellent regression coefficients ( R > 0.997) and covered quantities from 7.8 to 10,000 nM depending on the analyzed species. Intra- and inter-day accuracy and precision were below 15 % for all analytes. Apparent extraction recoveries were above 97 %, and ion suppression rates were between 4 and 53 %. Mean BA level in serum/plasma from healthy volunteers ranged from 11 ± 4 nM (tauroursodeoxycholic acid) to 1321 ± 1442 nM (glycochenodeoxycholic acid). As a proof of concept, the assay was applied to plasma samples derived from a clinical phase I study of myrcludex B, a novel first-in-class virus entry inhibitor for the treatment of chronic hepatitis B and D. The results demonstrate that myrcludex-induced inhibition of the hepatic BA transporter Na-taurocholate cotransporting polypeptide (NTCP) significantly affects plasma BA level. These observations provide novel insights into drug-induced metabolic responses and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

CD8+ T cells are the main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems, such as low infection rates, restrict mechanistic studies of HBV-specific CD8+ T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2hNTCP cells that endogenously processed viral antigens and presented them to HBV-specific CD8+ T cells. This induced cytolytic and noncytolytic CD8+ T-cell effector functions and reduction of viral loads. [ABSTRACT FROM AUTHOR]

Background & Aims: Chronic hepatitis B and hepatitis D are global health problems caused by the human hepatitis B and hepatitis D virus. The myristoylated preS1 domain of the large envelope protein mediates specific binding to hepatocytes by sodium taurocholate co-transporting polypeptide (NTCP). NTCP is a bile salt transporter known to be inhibited by cyclosporin A. This study aimed to characterize the effect of cyclosporin A on HBV/HDV infection. Methods: HepaRG cells, primary human hepatocytes, and susceptible NTCP-expressing hepatoma cell lines were applied for infection experiments. The mode of action of cyclosporin A was studied by comparing the effect of different inhibitors, cyclophilin A/B/C-silenced cell lines as well as NTCP variants and mutants. Bile salt transporter and HBV receptor functions were investigated by taurocholate uptake and quantification of HBVpreS binding. Results: Cyclosporin A inhibited hepatitis B and D virus infections during and – less pronounced – prior to virus inoculation. Binding of HBVpreS to NTCP was blocked by cyclosporin A concentrations at 8μM. An NTCP variant deficient in HBVpreS binding but competent for bile salt transport showed resistance to cyclosporin A. Silencing of cyclophilins A/B/C did not abrogate transporter and receptor inhibition. In contrast, tacrolimus, a cyclophilin-independent calcineurin inhibitor, was inactive. Conclusions: HBV and HDV entry via sodium taurocholate co-transporting polypeptide is inhibited by cyclosporin A. The interaction between the drug and the viral receptor is direct and overlaps with a functional binding site of the preS1 domain, which mediates viral entry. [Copyright &y& Elsevier]

Background & Aims: Currently approved antivirals rarely cure hepatitis B virus (HBV) infection. Therefore additional therapeutic strategies interfering with other viral replication steps are needed. Using synthetic lipopeptides derived from the HBV envelope protein, we previously demonstrated prevention of de novo HBV infection in vivo. We aimed at investigating the ability of the lipopeptide Myrcludex-B to block HBV spreading post-infection. Methods: uPA/SCID mice reconstituted with human hepatocytes were infected with HBV. Daily subcutaneous Myrcludex-B administration was initiated either 3days, 3weeks or 8weeks post HBV inoculation. Viral loads were quantitated in serum and liver, and visualized by immunohistochemistry. Results: Myrcludex-B efficiently prevented viral spreading from the initially infected human hepatocytes, as demonstrated by the lack of increase in viremia, antigen levels and amount of HBcAg-positive human hepatocytes determined 6weeks after treatment. Myrcludex-B efficiently blocked HBV dissemination also when treatment was started in the ramp-up phase of infection, in mice displaying moderate levels of circulating virions (median 3×106 HBV DNA copies/ml). Notably, after 6weeks of treatment, not only the amount of HBcAg-positive hepatocytes, but also intrahepatic cccDNA loads, remained comparable to values found in mice sacrificed 3weeks post-infection. In none of the experimental settings, drug administration affected human hepatocyte half-life or altered virion productivity. Conclusions: Myrcludex-B efficiently not only prevented HBV spreading from infected human hepatocytes in vivo, but also hindered amplification of the cccDNA pool in initially infected hepatocytes. Administration of an entry inhibitor, possibly used in combination with current HBV drugs, may improve patients’ treatment outcome. [Copyright &y& Elsevier]

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved. [ABSTRACT FROM AUTHOR]

The pan-protein kinase C (PKC) inhibitor sotrastaurin (AEB071) is a novel immunosuppressant currently in phase II trials for immunosuppression after solid organ transplantation. Besides T-cell activation, PKC affects numerous cellular processes that are potentially important for the replication of hepatitis B virus (HBV) and hepatitis C virus (HCV), major blood-borne pathogens prevalent in solid organ transplant recipients. This study uses state of the art virological assays to assess the direct, non-immune mediated effects of sotrastaurin on HBV and HCV. Most importantly, sotrastaurin had no pro-viral effect on either HBV or HCV. In the presence of high concentrations of sotrastaurin, well above those used clinically and close to levels where cytotoxic effects become detectable, there was a reduction of HCV and HBV replication. This reduction is very likely due to cytotoxic and/or anti-proliferative effects rather than direct anti-viral activity of the drug. Replication cycle stages other than genome replication such as viral cell entry and spread of HCV infection directly between adjacent cells was clearly unaffected by sotrastaurin. These data support the evaluation of sotrastaurin in HBV and/or HCV infected transplant recipients. [ABSTRACT FROM AUTHOR]

Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). Synthetic peptides derived from the N-terminus of the large HBV envelope protein (L-protein) have been shown to efficiently block HBV entry. Myr-HBVpreS/2-78, the parent compound of these drugs, inhibits human HBV infection in vitro and in vivo. An efficient synthesis is required, as these peptides constitute a novel class of anti HBV drugs. Consequently, the solid phase synthesis of the N-terminal 77 amino acids of the viral L-protein was studied in detail. The peptide was N-terminally myristoylated to resemble the natural, postranslationally modified protein. The synthesis was monitored using the Fmoc cleavage pattern of the solid phase synthesis on a standard peptide synthesizer and by LC-MS analyses of the arising side products. "Difficult sequences" in the positions 42-47 of the peptide sequence complicate the efficient synthesis of the 77-mer peptide HBVpreS/2-78. Attempts were undertaken to optimize the synthesis by heating, double coupling or the use of pseudoproline dipeptides. HPLC-MS analyses showed that the efficiency of the synthesis could be increased best by temperature elevation. This resulted in a higher purity of the crude product after solid phase synthesis. It was possible to minimize the occurrence of side products due to the positive effects related to higher reaction temperature. In conclusion, the peptide is accessible by stepwise SPPS without the necessity of segment coupling. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different receptors on PHH. [ABSTRACT FROM AUTHOR]

360 million people are chronically infected with the human hepatitis B virus (HBV) and are consequently prone to develop liver cirrhosis and hepatocellular carcinoma. As approved therapeutic regimens—which modulate patients' antiviral defenses or inhibit the viral reverse transcriptase—are generally noncurative, strategies interfering with other HBV replication steps are required. Expanding on our demonstration that acylated peptides derived from the large HBV envelope protein block virus entry in vitro, we show their applicability to prevent HBV or woolly monkey hepatitis B virus infection in vivo, using immunodeficient urokinase-type plasminogen activator (uPA) mice repopulated with primary human or Tupaia belangeri hepatocytes. Accumulation of the peptides in the liver, their extraordinary inhibitory potency and specific mode of action permit subcutaneous delivery at very low doses. Inhibition of hepadnavirus entry thus constitutes a therapeutic approach to prevent primary HBV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients. [ABSTRACT FROM AUTHOR]