Human Hepatitis B Virus Infection Research Articles

Attenuated effector T cells are linked to control of chronic HBV infection

Hepatitis B virus (HBV)-specific CD8+ T cells play a dominant role during acute-resolving HBV infection but are functionally impaired during chronic HBV infection in humans. These functional deficits have been linked with metabolic and phenotypic heterogeneity, but it has remained unclear to what extent different subsets of HBV-specific CD8+ T cells still suppress viral replication. We addressed this issue by deep profiling, functional testing and perturbation of HBV-specific CD8+ T cells during different phases of chronic HBV infection. Our data revealed a mechanism of effector CD8+ T cell attenuation that emerges alongside classical CD8+ T cell exhaustion. Attenuated HBV-specific CD8+ T cells were characterized by cytotoxic properties and a dampened effector differentiation program, determined by antigen recognition and TGFβ signaling, and were associated with viral control during chronic HBV infection. These observations identify a distinct subset of CD8+ T cells linked with immune efficacy in the context of a chronic human viral infection with immunotherapeutic potential.

Quantification of domestic cat hepadnavirus DNA in various body fluid specimens of cats: the potential viral shedding routes.

Domestic cat hepadnavirus (DCH) belongs to the Hepadnaviridae family together with human hepatitis B virus (HBV) that remains to be a major health problem worldwide. The transmission of HBV infectious virion has been one of the essential factors that contribute to high number of HBV infection in humans. It has been long known that various body fluid specimens of human with chronic HBV infection contain HBV DNA and demonstrated to be infectious. In contrast to this knowledge, the detection of DCH in various body fluid specimens of cats, has not been reported. This study explored the detection of DCH DNA in various body fluid specimens of cats by quantitative polymerase chain reaction (qPCR) and investigated whether the detection of DCH DNA from broader routes was correlated with any genomic diversity by phylogenetic analysis. A total of 1,209 body fluid specimens were included, and DCH DNA was detected not only in 4.70% (25/532) of blood samples; but also in 12.5% (1/8), 1.14% (1/88), 2.54% (10/394), and 1.65% (3/182) of auricular swab (AS), nasal swab (NS), oral swab (OS), and rectal swab (RS) specimens, respectively. Furthermore, the level of DCH DNA detected in the blood was significantly correlated with DCH DNA detection in OS (P = 0.02) and RS (P = 0.04) specimens. Genomic analysis revealed that there was no notable genomic diversity within the complete genome sequences obtained in this study. In conclusion, this study highlighted the presence of DCH DNA in various body fluid specimens of cats, and the potential role of these specimens in DCH horizontal transmission within the cat population warrants further studies.

Exploring the optimal vaccination strategy against hepatitis B virus in childhood (Review).

Vaccination against hepatitis B virus (HBV) remains the most effective strategy against HBV infection in humans. The present review summarized the optimal vaccination strategies against HBV in childhood. The following points are discussed: i) When and how the first HBV vaccines were developed; ii) the dosages, schedules and injection routes that are used for HBV vaccination; iii) the contraindications for HBV vaccination in the general paediatric population; iv) the challenges with the use of multivalent vaccines; v) the long-term immunogenicity and duration of protection against HBV; vi) the use of selective HBV vaccination and the hepatitis B immune globulin strategy in HBV-exposed infants; and vii) the effectiveness of the current HBV vaccination schemes. The present review is based on a Paediatric Virology Study Group (PVSG) webinar performed in the context of the 8th Workshop on Paediatric Virology.

Sera of Individuals Chronically Infected with Hepatitis B Virus (HBV) Contain Diverse RNA Types Produced by HBV Replication or Derived from Integrated HBV DNA.

This study aimed to better characterize the repertoire of serum hepatitis B virus (HBV) RNAs during chronic HBV infection in humans, which remains understudied. Using reverse transcription-PCR (RT-PCR), real-time quantitative PCR (RT-qPCR), RNA-sequencing, and immunoprecipitation, we found that (i) >50% of serum samples bore different amounts of HBV replication-derived RNAs (rd-RNAs); (ii) a few samples contained RNAs transcribed from integrated HBV DNA, including 5'-HBV-human-3' RNAs (integrant-derived RNAs [id-RNAs]) and 5'-human-HBV-3' transcripts, as a minority of serum HBV RNAs; (iii) spliced HBV RNAs were abundant in <50% of analyzed samples; (iv) most serum rd-RNAs were polyadenylated via conventional HBV polyadenylation signal; (v) pregenomic RNA (pgRNA) was the major component of the pool of serum RNAs; (vi) the area of HBV positions 1531 to 1739 had very high RNA read coverage and thus should be used as a target for detecting serum HBV RNAs; (vii) the vast majority of rd-RNAs and pgRNA were associated with HBV virions but not with unenveloped capsids, exosomes, classic microvesicles, or apoptotic vesicles and bodies; (viii) considerable rd-RNAs presence in the circulating immune complexes was found in a few samples; and (ix) serum relaxed circular DNA (rcDNA) and rd-RNAs should be quantified simultaneously to evaluate HBV replication status and efficacy of anti-HBV therapy with nucleos(t)ide analogs. In summary, sera contain various HBV RNA types of different origin, which are likely secreted via different mechanisms. In addition, since we previously showed that id-RNAs were abundant or predominant HBV RNAs in many of liver and hepatocellular carcinoma tissues as compared to rd-RNAs, there is likely a mechanism favoring the egress of replication-derived RNAs. IMPORTANCE The presence of integrant-derived RNAs (id-RNAs) and 5'-human-HBV-3' transcripts derived from integrated hepatitis B virus (HBV) DNA in sera was demonstrated for the first time. Thus, sera of individuals chronically infected with HBV contained both replication-derived and integrant-transcribed HBV RNAs. The majority of serum HBV RNAs were the transcripts produced by HBV genome replication, which were associated with HBV virions and not with other types of extracellular vesicles. These and other above-mentioned findings advanced our understanding of the HBV life cycle. In addition, the study suggested a promising target area on the HBV genome to increase sensitivity of the detection of serum HBV RNAs and supported the idea that simultaneous detection of replication-derived RNAs (rd-RNAs) and relaxed circular DNA (rcDNA) in serum provides more adequate evaluation of (i) the HBV genome replication status and (ii) the durability and efficiency of the therapy with anti-HBV nucleos(t)ide analogs, which could be useful for improvement of the diagnostics and treatment of HBV-infected individuals.

Identification of antibodies cross-reactive with woodchuck immune cells and activation of virus-specific and global cytotoxic T cell responses by anti-PD-1 and anti-PD-L1 in experimental chronic hepatitis B and persistent occult hepadnaviral infection.

Woodchuck (Marmota monax) infected with woodchuck hepatitis virus (WHV) is the most pathogenically compatible naturally occurring model of human hepatitis B virus (HBV) infection, chronic hepatitis B, and HBV-induced hepatocellular carcinoma. This system plays a crucial role in discovery and preclinical evaluation of anti-HBV therapies. Its utilization remains tempered by the relatively narrow range of validated immunologic and molecular tools. We evaluated commercial antibodies against immune cell phenotypic markers and T cell molecules for cross-reactivity with woodchuck antigenic equivalents. The confirmed antibodies against programed cell death protein-1 (PD-1) and its ligand (PD-L1) were examined for ex vivo ability to activate WHV-specific, global and bystander cytotoxic T cells (CTLs) in chronic hepatitis and asymptomatic infection persisting after self-resolved acute hepatitis. Examination of 65 antibodies led to identification or confirmation of 23 recognizing woodchuck T, regulatory T, B and natural killer cells, T cell-associated PD-1, PD-L1, CTLA-4 and TIM-3 molecules, CD25 and CD69 markers of T cell activation, and interferon gamma (IFNγ). Antibodies against woodchuck PD-1 and PD-L1 triggered in vitro highly individualized WHV-specific and global activation of CTLs in both chronic hepatitis and persistent occult infection. WHV-specific CTLs were more robustly augmented by anti-PD-1 than by anti-PD-L1 in chronic hepatitis, while global IFNγ-positive CTL response was significantly suppressed in chronic hepatitis compared to persistent occult infection. Anti-PD-1 and anti-PD-L1 also occasionally activated CTLs to specificities other than those tested suggesting their potency to trigger side effects. This was particularly apparent when T cells from chronic hepatitis were treated with anti-PD-L1. The current findings indicate that inhibition of the PD-1/PD-L1 pathway could reactivate virus-specific and global T cell responses in both chronic hepatitis and asymptomatic persistent infection. They suggest a mechanism of potential reactivation of clinically silent infection during anti-PD-1/PD-L1 treatment and indicate that this therapy may also subdue occult HBV infection.

Interferon and interferon-stimulated genes in HBV treatment.

Human hepatitis B virus (HBV) is a small enveloped DNA virus with a complex life cycle. It is the causative agent of acute and chronic hepatitis. HBV can resist immune system responses and often causes persistent chronic infections. HBV is the leading cause of liver cancer and cirrhosis. Interferons (IFNs) are cytokines with antiviral, immunomodulatory, and antitumor properties. IFNs are glycoproteins with a strong antiviral activity that plays an important role in adaptive and innate immune responses. They are classified into three categories (type I, II, and III) based on the structure of their cell-surface receptors. As an effective drug for controlling chronic viral infections, Type I IFNs are approved to be clinically used for the treatment of HBV infection. The therapeutic effect of interferon will be enhanced when combined with other drugs. IFNs play a biological function by inducing the expression of hundreds of IFN-stimulated genes (ISGs) in the host cells, which are responsible for the inhibiting of HBV replication, transcription, and other important processes. Animal models of HBV, such as chimpanzees, are also important tools for studying IFN treatment and ISG regulation. In the present review, we summarized the recent progress in IFN-HBV treatment and focused on its mechanism through the interaction between HBV and ISGs.

SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters.

Chronic hepatitis B (CHB) virus infection is one of the leading causes of cirrhosis and liver cancer. Although the major drugs against CHB including nucleos(t)ide analogs and PEG-interferon can effectively control human hepatitis B virus (HBV) infection, complete cure of HBV infection is quite rare. Targeting host factors involved in the viral life cycle contributes to developing innovative therapeutic strategies to improve HBV clearance. In this study, we found that the mRNA and protein levels of SIRT2, a class III histone deacetylase, were significantly upregulated in CHB patients, and that SIRT2 protein level was positively correlated with HBV viral load, HBsAg/HBeAg levels, HBcrAg, and ALT/AST levels. Functional analysis confirmed that ectopic SIRT2 overexpression markedly increased total HBV RNAs, 3.5-kb RNA and HBV core DNA in HBV-infected HepG2-Na+/taurocholate cotransporting polypeptide cells and primary human hepatocytes. In contrast, SIRT2 silencing inhibited HBV transcription and replication. In addition, we found a positive correlation between SIRT2 expression and HBV RNAs synthesis as well as HBV covalently closed circular DNA transcriptional activity. A mechanistic study suggested that SIRT2 enhances the activities of HBV enhancer I/HBx promoter (EnI/Xp) and enhancer II/HBc promoter (EnII/Cp) by targeting the transcription factor p53. The levels of HBV EnI/Xp and EnII/Cp-bound p53 were modulated by SIRT2. Both the mutation of p53 binding sites in EnI/Xp and EnII/Cp as well as overexpression of p53 abolished the effect of SIRT2 on HBV transcription and replication. In conclusion, our study reveals that, in terms of host factors, a SIRT2-targeted program might be a more effective therapeutic strategy for HBV infection.

Primary Tupaia Javanica Hepatocyte Culture as an In Vitro Model for Human Hepatitis B Virus Infection

Background: Hepatitis B virus (HBV) infection is still one of the biggest health problems in the world, which could lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Treatment for HBV infection has not yet achieved a functional cure. More studies are needed to investigate human HBV (HuHBV), but the scarcity of animal models for HuHBV infection became a barrier. Recently, many studies have shown that Tupaia are suitable for the study of HuHBV. The purpose of this study was to develop a primary tupaia hepatocyte (PTH) culture from T. javanica, a species of Tupaia found in Indonesia, and to prove that HuHBV can replicate in the PTH.Method: In vitro experimental study using PTH isolated from five wild adult T. javanica in Primate Research Center, IPB University. HuHBV was taken from humans with HBsAg and HBV-DNA (+). PTH cells then were infected with HuHBV after reaching 80% confluence. Observation on PTH cells was done everyday for 20 days. Qualitative and quantitative HBsAg were measured using a CMIA while HBV-DNA and cccDNA were measured by RT-PCR.Results: A cytopathic effect was seen on day post infection (DPI)-16. HBsAg and HBV-DNA were detected from DPI-2 until DPI-18, with HBV-DNA level peaked on DPI-12. cccDNA concentration was fluctuating from DPI-2 until DPI-20 with highest level on DPI-16.Conclusion: HuHBV could infect and replicate in PTH from T. javanica can be infected with HuHBV and HuHBV can replicate in the PTH from T. javanica.

Entry Inhibitors of Hepatitis B and D Viruses.

Human hepatitis B virus (HBV) and hepatitis D virus (HDV) cause acute and chronic infections. The latter poses a serious public health threat as it is the major cause of chronic hepatitis, liver failure, cirrhosis, and hepatocellular carcinoma (HCC). In nature, HBV and HDV have a narrow host range and highly hepatotropic, only infecting the hepatocytes of humans and a few primates. The elucidation of cell entry mechanism by HBV has made great progress in recent years, which strongly promotes the establishment of new HBV infection models and the development of viral entry inhibitors. The study of HBV entry inhibitors has culminated in the first direct antiviral treatment for HDV. This review provides a concise introduction on the progress of HBV/HDV entry inhibitors in the recent years.

Hepatitis B Virus DNA Integration and Clonal Expansion of Hepatocytes in the Chronically Infected Liver.

Human hepatitis B virus (HBV) can cause chronic, lifelong infection of the liver that may lead to persistent or episodic immune-mediated inflammation against virus-infected hepatocytes. This immune response results in elevated rates of killing of virus-infected hepatocytes, which may extend over many years or decades, lead to fibrosis and cirrhosis, and play a role in the high incidence of hepatocellular carcinoma (HCC) in HBV carriers. Immune-mediated inflammation appears to cause oxidative DNA damage to hepatocytes, which may also play a major role in hepatocarcinogenesis. An additional DNA damaging feature of chronic infections is random integration of HBV DNA into the chromosomal DNA of hepatocytes. While HBV DNA integration does not have a role in virus replication it may alter gene expression of the host cell. Indeed, most HCCs that arise in HBV carriers contain integrated HBV DNA and, in many, the integrant appears to have played a role in hepatocarcinogenesis. Clonal expansion of hepatocytes, which is a natural feature of liver biology, occurs because the hepatocyte population is self-renewing and therefore loses complexity due to random hepatocyte death and replacement by proliferation of surviving hepatocytes. This process may also represent a risk factor for the development of HCC. Interestingly, during chronic HBV infection, hepatocyte clones detected using integrated HBV DNA as lineage-specific markers, emerge that are larger than those expected to occur by random death and proliferation of hepatocytes. The emergence of these larger hepatocyte clones may reflect a survival advantage that could be explained by an ability to avoid the host immune response. While most of these larger hepatocyte clones are probably not preneoplastic, some may have already acquired preneoplastic changes. Thus, chronic inflammation in the HBV-infected liver may be responsible, at least in part, for both initiation of HCC via oxidative DNA damage and promotion of HCC via stimulation of hepatocyte proliferation through immune-mediated killing and compensatory division.

A systematic study of Tupaia as a model for human acute hepatitis B infection.

The molecular features of hepatitis B virus (HBV) infection, eradication, and pathogenesis are poorly understood, partly due to the lack of an adequate animal model that faithfully reproduces the course of infection. Although Tupaia belangeri were previously recognized as HBV-susceptible animals, the course of infection in adult tupaias remains obscure. Herein, we performed a longitudinal study and demonstrated that adult tupaias were efficiently infected (90% infection rate) with 108 copies of the HBV genome. HBV replicated vigorously, produced high levels of covalently closed circular DNA (cccDNA) in hepatocytes, and released hepatitis B surface antigen (HBsAg), hepatitis Be antigen (HBeAg), and HBV DNA into the serum at day 9 post-inoculation (p.i.), which then decreased on day 15 p.i. The kinetics were consistent with the expression of liver HBsAg and HBeAg, as determined with immunohistochemistry. The viral products in serum at day 9 and 15 p.i. represented de novo synthesized viral products, as treatment with a viral entry inhibitor completely abolished these products from the serum. Viral clearance and serological conversion occurred at day 21 p.i. and were accompanied by elevated alanine transaminase (ALT) levels and liver pathology, such as inflammatory infiltration and hepatocyte ballooning degeneration. Although ALT levels eventually returned to normal levels by day 42 p.i., the liver pathology persisted until at least day 120 p.i. The HBV infection process in tupaia, therefore, exhibits features similar to that of human acute HBV infection, including viral replication, viral eradication, ALT elevation, and liver pathology. Thus, adopting the tupaia model to study host-HBV interactions presents an important advance which could facilitate further investigation and understanding of human HBV infection, especially for features like cccDNA that current small-animal models cannot effectively model.

Innate and adaptive immunity associated with resolution of acute woodchuck hepatitis virus infection in adult woodchucks.

Viral and/or host factors that are directly responsible for the acute versus chronic outcome of hepatitis B virus (HBV) infection have not been identified yet. Information on immune response during the early stages of HBV infection in humans is mainly derived from blood samples of patients with acute hepatitis B (AHB), which are usually obtained after the onset of clinical symptoms. Features of intrahepatic immune response in these patients are less studied due to the difficulty of obtaining multiple liver biopsies. Woodchuck hepatitis virus (WHV) infection in woodchucks is a model for HBV infection in humans. In the present study, five adult woodchucks were experimentally infected with WHV and then followed for 18 weeks. Blood and liver tissues were frequently collected for assaying markers of WHV replication and innate and adaptive immune responses. Liver tissues were further analyzed for pathological changes and stained for important immune cell subsets and cytokines. The increase and subsequent decline of viral replication markers in serum and liver, the elicitation of antibodies against viral proteins, and the induction of virus-specific T-cell responses indicated eventual resolution of acute WHV infection in all animals. Intrahepatic innate immune makers stayed unchanged immediately after the infection, but increased markedly during resolution, as determined by changes in transcript levels. The presence of interferon-gamma and expression of natural killer (NK) cell markers suggested that a non-cytolytic response mechanism is involved in the initial viral control in liver. This was followed by the expression of T-cell markers and cytolytic effector molecules, indicating the induction of a cytolytic response mechanism. Parallel increases in regulatory T-cell markers suggested that this cell subset participates in the overall immune cell infiltration in liver and/or has a role in regulating AHB induced by the cytolytic response mechanism. Since the transcript levels of immune cell markers in blood, when detectable, were lower than in liver, and the kinetics, except for NK-cells and interferon-gamma, did not correlate well with their intrahepatic expression, this further indicated enrichment of immune cells within liver. Conclusion: The coordinated interplay of innate and adaptive immunity mediates viral clearance in the woodchuck animal model of HBV infection. The initial presence of NK-cell associated interferon-gamma response points to an important role of this cytokine in HBV resolution.

Evans Blue Inhibits HBV Replication Through a Dual Antiviral Mechanism by Targeting Virus Binding and Capsid Assembly.

Chronic hepatitis B (CHB) is a global health problem caused by human hepatitis B virus (HBV). Current treatment with interferons and nucleos(t)ide analogs (NAs) can cause population tolerance and drug resistance. Therefore, new antiviral drugs, especially those targeting host factors, are urgently needed. Here, we identified Evans blue as a new HBV inhibitor by screening an FDA drug library using Huh7DhNTCP cells and confirmed the antiviral activity in primary human hepatocytes and human sodium taurocholate cotransporting polypeptide (hNTCP)-transfected porcine primary hepatocytes. Our efficacy study showed that Evans blue has an IC50 of 2 μM against HBV infection in Huh7DhNTCP cells, and no apparent toxicity at up to 1000 μM. The IC50 of Evans blue against HBV in primary human hepatocytes was approximately 5 μM. Mechanism studies revealed that Evans blue has a dual anti-HBV effect. It inhibits both the binding of viral preS1 to host cells through the host factor NTCP and the virus capsid assembly by targeting the host factor BK channel. The KD of the direct interaction between Evans blue and NTCP is 8.82E-8 M. Evans blue can suppress capsid assembly at micromolar concentrations by reducing the cytosolic calcium ion concentration. Since the antiviral effects on HBV binding and assembly are both achieved through targeting host factors, Evans blue inhibits the infection of nucleos(t)ide analog drug-resistant HBV strains in Huh7DhNTCP cells. Taken together, our results suggest that Evans blue may be a promising anti-HBV drug candidate in the classes of both entry and assembly inhibitors.

Hepatitis B Virus Infection Alters Gut Microbiota Composition in Mice.

Gut microbiota composition is known to be associated with the progression of hepatitis B virus (HBV)-related liver cirrhosis in humans, outcome of HBV infection in mice, and seroconversion of HBV e-antigen in nucleot(s)ide analog-treated patients. The dynamic alteration of the gut microbiota following HBV infection is still unknown. In this study, a hydrodynamic injection mouse model mimicking acute or chronic HBV infection in humans with comparable virological and immunological features was used. The composition of gut microbiota in the control mice and mice with acute or chronic HBV infection was analyzed at different time points using the Illumina MiSeq platform. The expression of immune molecules in the colon was detected by real-time polymerase chain reaction. We found that the changes in gut microbiota composition, including the total operational taxonomic unit (OTU) count and Shannon-Weaver index, were significantly delayed in mice with HBV infection. Furthermore, the ratio of Bacteroidetes and Firmicutes was stable in the control mice, whereas remarkable dynamic patterns were observed in mice with HBV infection. Interestingly, the dynamic changes in Lactobacillus and Bifidobacterium were found to differ in acute or chronic HBV infection. In addition, the expression of IFN-γ and PD-L1 in the colon was found to be up-regulated early in mice with acute HBV infection, whereas the expression of PD-L1 in the colon of mice with chronic HBV infection was up-regulated later. These data indicate that HBV infection could hamper the development of the gut microbiota community and dynamically change the gut Firmicutes/Bacteroidetes ratio. These data improve our understanding of the relationship between gut microbiota and HBV infection.

HBV-Induced Immune Imbalance in the Development of HCC.

Chronic hepatitis B virus (HBV) infection is one of the high-risk factors for human HCC. Despite the integration of virus DNA and the oncoprotein HBx, chronic necroinflammation and hepatocellular regeneration account for hepatocarcinogenesis. As a non-cytopathic virus, HBV is extensively recognized to mediate chronic liver damage through abnormal immune attack. However, the mechanisms driving HBV infection to HCC are poorly understood. During chronic HBV infection in humans, the adaptive immunity changes from immune tolerance to progressive immune activation, inactivation, reactivation and exhaustion, all of which may be the immune pathogenic factors for the development of HCC. Recently, the immunopathogenic mechanisms were described in mouse HBV-induced HCC models, which is absolutely dependent on the presence of HBV-specific T cell response and NK cell-derived IFN-γ, findings which are consistent with the observations from CHB and HCC patients. In this review, we summarize recent research progression on the HBV-specific CD8+ T cells, and also CD4+ T cells, B cells and non-specific immune cells and molecules underlying chronic HBV infection and eventual HCC development to demonstrate the pathogenesis of HBV-induced immune imbalance. Based on the progression, we discussed the potential of immune-based therapies and their challenges in the treatment of HBV-related HCC, including the checkpoint inhibition, genetically modified T cell transfer, therapeutic vaccines and metabolic modulation.

The retinoic acid receptor (RAR) α-specific agonist Am80 (tamibarotene) and other RAR agonists potently inhibit hepatitis B virus transcription from cccDNA

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 IC50s < 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.

HBV infection-induced liver cirrhosis development in dual-humanised mice with human bone mesenchymal stem cell transplantation

ObjectiveDeveloping a small animal model that accurately delineates the natural history of hepatitis B virus (HBV) infection and immunopathophysiology is necessary to clarify the mechanisms of host-virus interactions and to...

The p.Ser267Phe variant of sodium taurocholate cotransporting polypeptide (NTCP) supports HBV infection with a low efficiency

Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for human hepatitis B virus (HBV) and its satellite virus Hepatitis D virus (HDV). Physiologically, NTCP is responsible for the majority of sodium-dependent bile acids uptake by hepatocytes. The p.Ser267Phe (S267F) variant of NTCP is a single nucleotide polymorphism (SNP) previously found to cause substantial loss of ability to support HBV and HDV infection and its taurocholic acid uptake function in vitro. Intriguingly, ten individuals were identified as S267F homozygotes in population studies of chronic hepatitis B (CHB) patients. In this study, we identified new HBV isolates from one homozygous S267F mutation carrier and confirmed new isolates also use wildtype-NTCP as a cellular receptor. Furthermore, we demonstrated S267F variant of NTCP, though inefficient, is still a functional receptor for HBV entry. This study advances our understanding of NTCP-mediated HBV infection.

Inhibitory effect of fasiglifam on hepatitis B virus infections through suppression of the sodium taurocholate cotransporting polypeptide

Fasiglifam is a selective partial agonist of G-protein–coupled receptor 40 (GPR40), which was developed for the treatment of type 2 diabetes mellitus. However, the clinical development of fasiglifam was voluntarily terminated during phase III clinical trials due to adverse liver effects. Fasiglifam showed an inhibitory effect on sodium taurocholate cotransporting polypeptide (NTCP) in human and rat hepatocytes. Recently, NTCP was reported to be a functional receptor for human hepatitis B virus (HBV) infections. Therefore, in this study, we hypothesised that fasiglifam would be a good candidate for a novel HBV entry inhibitor, and its effects were evaluated by using NTCP-overexpressing HepG2 cells, human hepatocyte cell lines and human hepatocytes (PXB cells) obtained from PXB mice. Pre-treatment with fasiglifam at a concentration of 30 μM prior to HBV infection significantly suppressed supernatant HBV DNA levels after HBV infection in NTCP-overexpressing HepG2 cells, human hepatocyte cell lines and PXB cells. Fasiglifam did not suppress supernatant HBV DNA levels up to 50 μM in HepG2.2.15.7 cells, which are stably transfected with a complete HBV genome without HBV infection. These results indicated that fasiglifam only affect on HBV infection via NTCP inhibition. For HBV treatment of fasiglifam, further investigation including additional non clinical research in addition to the evaluation of safety and efficacy in humans would be needed in the future study.

A cell-penetrating whole molecule antibody targeting intracellular HBx suppresses hepatitis B virus via TRIM21-dependent pathway.

Rationale: Monoclonal antibodies (mAbs) mostly targeting extracellular or cell surface molecules have been widely used in the treatment of various diseases. However, mAbs cannot pass through the cell membrane as efficiently as small compounds, thus limiting their use against intracellular targets. Methods to shuttle antibodies into living cells may largely expand research and application in areas based on mAbs. Hepatitis B virus X protein (HBx) is an important intracellular multi-functional viral protein in the life cycle of hepatitis B virus (HBV). HBx plays essential roles in virus infection and replication and is strongly associated with HBV-related carcinogenesis.Methods: In this study, we developed a cell-penetrating whole molecule antibody targeting HBx (9D11-Tat) by the fusion of a cell penetrating peptide (CPP) on the C-terminus of the heavy chain of a potent mAb specific to HBx (9D11). The anti-HBV effect and mechanism of 9D11-Tat were investigated in cell and mouse models mimicking chronic HBV infection.Results: Our results demonstrated that the recombinant 9D11-Tat antibody could efficiently internalize into living cells and significantly suppress viral transcription, replication, and protein production both in vitro and in vivo. Further analyses suggested the internalized 9D11-Tat antibody could greatly reduce intracellular HBx via Fc binding receptor TRIM21-mediated protein degradation. This process simultaneously stimulated the activations of NF-κB, AP-1, and IFN-β, which promoted an antiviral state of the host cell.Conclusion: In summary, our study offers a new approach to target intracellular pathogenesis-related protein by engineered cell-penetrating mAb expanding their potential for therapeutic applications. Moreover, the 9D11-Tat antibody may provide a novel therapeutic agent against human chronic HBV infection.