Hepatitis B Virus Mouse Model Research Articles

TCR-like bispecific antibodies toward eliminating infected hepatocytes in HBV mouse models

Therapeutics for eradicating hepatitis B virus (HBV) infection are still limited and current nucleos(t)ide analogs (NAs) and interferon are effective in controlling viral replication and improving liver health, but they cannot completely eradicate the hepatitis B virus and only a very small number of patients are cured of it. The TCR-like antibodies recognising viral peptides presented on human leukocyte antigens (HLA) provide possible tools for targeting and eliminating HBV-infected hepatocytes. Here, we generated three TCR-like antibodies targeting three different HLA-A2.1-presented peptides derived from HBV core and surface proteins. Bispecific antibodies (BsAbs) were developed by fusing variable fragments of these TCR-like mAbs with an anti-CD3ϵ antibody. Our data demonstrate that the BsAbs could act as T cell engagers, effectively redirecting and activating T cells to target HBV-infected hepatocytes in vitro and in vivo. In HBV-persistent mice expressing human HLA-A2.1, two infusions of BsAbs induced marked and sustained suppression in serum HBsAg levels and also reduced the numbers of HBV-positive hepatocytes. These findings highlighted the therapeutic potential of TCR-like BsAbs as a new strategy to cure hepatitis B.

Anti-HBc mirrors the activation of HBV-specific CD8+ T cell immune response and exhibits a direct effect on HBV control

BackgroundHepatitis B core antibody (anti-HBc) is commonly present in patients with chronic hepatitis B virus (HBV) infection and serves as a marker of humoral immunity. Herein, we aim to investigate the correlation between anti-HBc and antiviral immune response and its putative role in HBV control. MethodsQuantitative anti-HBc and levels of anti-HBc subtypes were measured in chronic hepatitis B (CHB) patients. The effects of anti-HBc on immune cells and HBV replication were evaluated using the HBV mouse models and human hepatoma cell lines. ResultsBaseline levels of IgG1 and IgG3 anti-HBc were elevated in CHB patients with favorable treatment response, and correlated with the virological response observed at week 52. Additionally, increased levels of IgM and IgG1 anti-HBc were observed exclusively in CHB patients with liver inflammation. Notably, significant correlations were identified between quantitative levels of anti-HBc and the frequencies of HBcAg-specific CD8+ T cells. Intriguingly, HBcAg efficiently activates T cells aided by B cells in vitro experiments. Moreover, anti-HBc inhibits HBV replication either by a direct effect or through complement-mediated cytotoxicity in HBV-producing cell lines. ConclusionsAnti-HBc reflects the activation of an HBV-specific CD8+ T cell immune response and may have anti-HBV activity.

Preclinical characterization of a novel potent core protein assembly modulator for the treatment of chronic hepatitis B viral infection

The hepatitis B virus (HBV) capsid or core protein is a promising drug target currently being investigated for potential curative therapies for chronic HBV infection. In this study, we performed extensive in vitro and in vivo characterization of a novel and potent HBV core protein assembly modulator (CpAM), CU15, for both anti-HBV activity and druggability properties. CU15 potently inhibited HBV DNA replication in in vitro HBV-infected HepG2.2.15 cells (EC50 of 8.6 nM), with a low serum shift. It was also effective in inhibiting HBV DNA and cccDNA formation in de novo HBV-infected primary human hepatocytes. Furthermore, CU15 was active across several HBV genotypes and across clinically relevant core protein variants. After oral administration to an in vivo HBV mouse model, CU15 significantly reduced plasma HBV DNA and RNA levels, at plasma exposure consistent with the estimated in vitro potency. In vitro, CU15 exhibited excellent passive permeability and relatively high metabolic stability in liver preparations across species (human > dog> rat). In vitro human liver microsomal studies suggest that the compound's major metabolic pathway is CYP3A-mediated oxidation. Consistent with the in vitro findings, CU15 is a compound with a low-to-moderate clearance and high oral bioavailability in rats and dogs. Based on the apparent in vitro–in vivo correlation observed, CU15 has the potential to exhibit low clearance and high oral bioavailability in humans. In addition, CU15 also showed low drug–drug interaction liability with an acceptable in vitro safety profile (IC50 > 10 µM).

Selective depletion of HBV-infected hepatocytes by class A capsid assembly modulators requires high levels of intrahepatic HBV core protein.

Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.

Hepatitis B virus-specific human stem cell memory T cells differentiate into cytotoxic T cells and eradicate HBV-infected hepatocytes in mice.

Chronic infection with the hepatitis B virus (HBV) induces progressive hepatic impairment. Achieving complete eradication of the virus remains a formidable challenge. Cytotoxic T lymphocytes, specific to viral antigens, either exhibit a numerical deficiency or succumb to an exhausted state in individuals chronically afflicted with HBV. The comprehension of the genesis and dissemination of stem cell memory T cells (TSCMs) targeting HBV remains inadequately elucidated. We identified TSCMs in subjects with chronic HBV infection and scrutinized their efficacy in a murine model with human hepatocyte transplants, specifically the TK-NOG mice. TSCMs were discerned in all subjects under examination. Introduction of TSCMs into the HBV mouse model precipitated a severe necro-inflammatory response, resulting in the elimination of human hepatocytes. TSCMs may constitute a valuable tool in the pursuit of a remedial therapy for HBV infection.

Early application of IFNγ mediated the persistence of HBV in an HBV mouse model

The antiviral activity of interferon gamma (IFNγ) against hepatitis B virus (HBV) was demonstrated both in vivo and in vitro in a previous study. IFNγ can suppress HBV replication by accelerating the decay of replication-competent nucleocapsids of HBV. However, in this study, we found that the direct application of the mouse IFNγ (mIFNγ) expression plasmid to the liver of an HBV hydrodynamic injection (HI) mouse model led to the persistence of HBV, as indicated by sustained HBsAg and HBeAg levels in the serum as well as an increased percentage of the HBsAg positive mice, whereas the level of HBV DNA in the serum and the expression of HBcAg in the liver were inhibited at the early stage after HI. Meanwhile, we found that the productions of both HBcAb and HBsAb were suppressed after the application of mIFNγ. In addition, we found that HBV could be effectively inhibited in mice immunized with HBsAg expression plasmid before the application of mIFNγ. Furthermore, mIFNγ showed antiviral effect and promoted the production of HBsAb when the mice subjected to the core-null HBV plasmid. These results indicate that the application of mIFNγ in the HBV HI mouse model, the mice showed defective HBcAg-specific immunity that impeded the production of HBcAb and HBsAb, finally allowing the persistence of the virus. Moreover, IFNγ-induced negative immune regulatory factors also play an important role in virus persistence.

In-text ref citation test High-pressure Hydrodynamic Injection as a Method of Establishing Hepatitis B Virus Infection in Mice

Abstract Among several existing mouse models for hepatitis B virus (HBV) infection, the high-pressure hydrodynamic injection (HDI) method is frequently used in HBV research due to its economic advantages and ease of implementation. The use of the HDI method is influenced by factors such as mouse genetic background, age, sex, and the type of HBV plasmid used. This overview provides a multidimensional analysis and comparison of various factors that influence the effectiveness of the HBV mouse model established through HDI. The goal is to provide a summary of information for researchers who create HBV models in mice.

High-pressure Hydrodynamic Injection as a Method of Establishing Hepatitis B Virus Infection in Mice.

Among several existing mouse models for hepatitis B virus (HBV) infection, the high-pressure hydrodynamic injection (HDI) method is frequently used in HBV research due to its economic advantages and ease of implementation. The use of the HDI method is influenced by factors such as mouse genetic background, age, sex, and the type of HBV plasmid used. This overview provides a multidimensional analysis and comparison of various factors that influence the effectiveness of the HBV mouse model established through HDI. The goal is to provide a summary of information for researchers who create HBV models in mice.

Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Antibiotic-induced gut bacteria depletion has no effect on HBV replication in HBV immune tolerance mouse model

Commensal microbiota is closely related to Hepatitis B virus (HBV) infection. Gut bacteria maturation accelerates HBV immune clearance in hydrodynamic injection (HDI) HBV mouse model. However, the effect of gut bacteria on HBV replication in recombinant adeno-associated virus (AAV)-HBV mouse model with immune tolerance remains obscure. We aim to investigate its role on HBV replication in AAV-HBV mouse model. C57BL/6 mice were administrated with broad-spectrum antibiotic mixtures (ABX) to deplete gut bacteria and intravenously injected with AAV-HBV to establish persistent HBV replication. Gut microbiota community was analyzed by fecal qPCR assay and 16S ribosomal RNA (rRNA) gene sequencing. HBV replication markers in blood and liver were determined by ELISA, qPCR assay and Western blot at indicated time points. Immune response in AAV-HBV mouse model was activated through HDI of HBV plasmid or poly(I:C) and then detected by quantifying the percentage of IFN-γ+/CD8+ T cells in the spleen via flow cytometry as well as the splenic IFN-γ mRNA level via qPCR assay. We found that antibiotic exposure remarkably decreased gut bacteria abundance and diversity. Antibiotic treatment failed to alter the levels of serological HBV antigens, intrahepatic HBV RNA transcripts and HBc protein in AAV-HBV mouse model, but contributed to HBsAg increase after breaking of immune tolerance. Overall, our data uncovered that antibiotic-induced gut bacteria depletion has no effect on HBV replication in immune tolerant AAV-HBV mouse model, providing new thoughts for elucidating the correlation between gut bacteria dysbiosis by antibiotic abuse and clinical chronic HBV infection.

Farnesoid X Receptor Activation Decreases Toll-like Receptor 2 Expression by Upregulating HBeAg Production

Background: Previous investigations have demonstrated that hepatitis B virus (HBV) infection leads to elevated serum bile acid levels, which is considered to cause liver damage. Thus, we suppose that bile acids may be of considerable significance in inducing immune tolerance. Methods: In this investigation, we explored the functions of the farnesoid X receptor (FXR), a nuclear receptor activated by bile acids, in modulating hepatitis B e antigen (HBeAg) production and toll-like receptor (TLR) expression in vitro and in vivo. Results: The results showed that FXR activation promoted secreted and intracellular HBeAg expression in HepG2 and HEK293T cells. However, FXR antagonist Z-guggulsterone (Z-g) decreased the bile acid-mediated HBeAg production. Meanwhile, TLR2 expression significantly reduced in HepG2 cells transfected with pAAV/HBV1.2 plasmid comprising whole HBV genome and treated with bile acids, but not with mutant pAAV/HBV1.2 plasmid with defected HBeAg product. In the hydrodynamic injection HBV mouse model, the level of serum HBeAg was decreased, but intrahepatic TLR2 expression was elevated in FXR-/- mice. Conclusions: In conclusion, FXR activation inhibits TLR2-mediated innate immunity by upregulating HBeAg production. Our data indicate that a mild elevation of serum bile acids may cause immune tolerance and lead to virus persistence in HBV-infected patients.

Antiviral Activity of CRISPR/Cas9 Ribonucleoprotein Complexes on a Hepatitis B Virus Model In Vivo

Chronic hepatitis B (CHB) is caused by hepatitis B virus (HBV) infection. This disease is a key issue for global health. Modern methods of therapy do not completely eliminate HBV from infected cells and do not cure chronic infection. The CRISPR/Cas9 systems of site-specific nucleases can effectively cleave do not target DNA including viral genomes. The cleavage of the major form of the HBV genome, i.e., covalently closed circular DNA (cccDNA), leads to a robust reduction in viral replication and degradation or mutational inactivation of cccDNA. CRISPR/Cas9-based approaches are one of the most promising ways to achieve a 'sterilizing' cure of CHB, i.e., complete elimination of the virus from the body. Here, the HBV mouse model in vivo has been used to analyze the antiviral activity of the high-specific Cas9 protein and sgRNA targeting HBV genome. We have found that a single injection of short-lived ribonucleoprotein complexes of CRISPR/Cas9 results in a ~10-fold reduction in HBV DNA levels in the serum and liver of mice as early as 48 h after the start of the experiment. The remaining HBV DNAs have been found to harbor rare indel mutations. Developing new antivirals for treating CHB based on CRISPR/Cas9 ribonucleoprotein complexes could substantially reduce the duration of CHB therapy and, potentially, achieve complete elimination of viral infection.

USP18 Attenuates the Anti-Hepatitis B Virus Effect of Ifn by Down Regulating JAK-STAT Pathway

Aim: USP18 is a type of IFN-stimulated gene, which is associated with virological responses to IFN therapy in HBV (hepatitis B virus). However, its detailed molecular mechanism needs to be explored. Materials & methods: With HBV replication cells and mouse models, the USP18 was overexpressed or inhibited, followed by treatment with IFN or Poly (I:C). The expressions of HBV DNA, HBsAg, HBeAg and protein factors in the samples were detected. Results: Overexpression of USP18 attenuates anti-HBV effect of IFN in vitro and in vivo by inhibiting JAK-STAT pathway and reducing the expression of MX1 and OAS. While, the inhibition of USP18 can promote to activate JAK-STAT pathway to enhance the antiviral effect of IFN. Conclusion: USP18 negatively regulates the anti-HBV effect of IFN by regulating JAK-STAT pathway. It may provide new insights into innate immunity mechanisms in CHB patients receiving IFN treatment.

Hepatocyte-targeted delivery of imiquimod reduces hepatitis B virus surface antigen.

Hepatitis B virus (HBV) can rapidly replicate in the hepatocytes after transmission, leading to chronic hepatitis, liver cirrhosis and eventually hepatocellular carcinoma. Interferon-α (IFN-α) is included in the standard treatment for chronic hepatitis B (CHB). However, this therapy causes serious side effects. Delivering IFN-α selectively to the liver may enhance its efficacy and safety. Imiquimod (IMQ), a Toll-Like Receptor (TLR) 7 agonist, stimulates the release of IFN-α that exhibits potent antiviral activity. However, the poor solubility and tissue selectivity of IMQ limits its clinical use. Here, we demonstrated the use of lipid-based nanoparticles (LNPs) to deliver IMQ and increase the production of IFN-α in the liver. We encapsulated IMQ in two liver-targeted LNP formulations: phospholipid-free small unilamellar vesicles (PFSUVs) and DSPG-liposomes targeting the hepatocytes and the Kupffer cells, respectively. In vitro drug release/retention, in vivo pharmacokinetics, intrahepatic distribution, IFN-α production, and suppression of serum HBV surface antigen (HBsAg) were evaluated and compared for these two formulations. PFSUVs provided >95% encapsulation efficiency for IMQ at a drug-to-lipid ratio (D/L) of 1/20 (w/w) and displayed stable drug retention in the presence of serum. DSPG-IMQ showed 79% encapsulation of IMQ at 1/20 (D/L) and exhibited ∼30% burst release when incubated with serum. Within the liver, PFSUVs showed high selectivity for the hepatocytes while DSPG-liposomes targeted the Kupffer cells. Finally, in an experimental HBV mouse model, PFSUVs significantly reduced serum levels of HBsAg by 12-, 6.3- and 2.2-fold compared to the control, IFN-α, and DSPG-IMQ groups, respectively. The results suggest that the hepatocyte-targeted PFSUVs loaded with IMQ exhibit significant potential for enhancing therapy of CHB.

Alpha-kinase 1 (ALPK1) agonist DF-006 demonstrates potent efficacy in mouse and primary human hepatocyte (PHH) models of hepatitis B.

In the treatment of chronic hepatitis B (CHB) infection, stimulation of innate immunity may lead to hepatitis B virus (HBV) cure. Alpha-kinase 1 (ALPK1) is a pattern recognition receptor (PRR) that activates the NF-κB pathway and stimulates innate immunity. Here we characterized the preclinical anti-HBV efficacy of DF-006, an orally active agonist of ALPK1 currently in clinical development for CHB. In adeno-associated virus (AAV)-HBV mouse models and primary human hepatocytes (PHHs) infected with HBV, we evaluated the antiviral efficacy of DF-006. In the mouse models, DF-006 rapidly reduced serum HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen levels using doses as low as 0.08 μg/kg, 1 μg/kg, and 5 μg/kg, respectively. DF-006 in combination with the HBV nucleoside reverse transcriptase inhibitor, entecavir, further reduced HBV DNA. Antiviral efficacy in mice was associated with an increase in immune cell infiltration and decrease of hepatitis B core antigen, encapsidated pregenomic RNA, and covalently closed circular DNA in liver. At subnanomolar concentrations, DF-006 also showed anti-HBV efficacy in PHH with significant reductions of HBV DNA. Following dosing with DF-006, there was upregulation of NF-κB-targeted genes that are involved in innate immunity. DF-006 was efficacious in mouse and PHH models of HBV without any indications of overt toxicity. In mice, DF-006 localized primarily to the liver where it potently activated innate immunity. The transcriptional response in mouse liver provides insights into mechanisms that mediate anti-HBV efficacy by DF-006.

In Vivo Mouse Models for Hepatitis B Virus Infection and Their Application.

Despite the availability of effective vaccination, hepatitis B virus (HBV) infection continues to be a major challenge worldwide. Research efforts are ongoing to find an effective cure for the estimated 250 million people chronically infected by HBV in recent years. The exceptionally limited host spectrum of HBV has limited the research progress. Thus, different HBV mouse models have been developed and used for studies on infection, immune responses, pathogenesis, and antiviral therapies. However, these mouse models have great limitations as no spread of HBV infection occurs in the mouse liver and no or only very mild hepatitis is present. Thus, the suitability of these mouse models for a given issue and the interpretation of the results need to be critically assessed. This review summarizes the currently available mouse models for HBV research, including hydrodynamic injection, viral vector-mediated transfection, recombinant covalently closed circular DNA (rc-cccDNA), transgenic, and liver humanized mouse models. We systematically discuss the characteristics of each model, with the main focus on hydrodynamic injection mouse model. The usefulness and limitations of each mouse model are discussed based on the published studies. This review summarizes the facts for considerations of the use and suitability of mouse model in future HBV studies.

Hepatocyte-Derived L-Carnitine Restricts Hepatitis B Surface Antigen Loss Through an Immunosuppressive Effect on Germinal Center-Related Immune Cells.

The outcome of hepatitis B virus (HBV) infection is significantly affected by host immune response; herein, we aim to dissect the effect of L-carnitine (L-Cn) on germinal center (GC)-related immune cells and the influence on the prognosis of HBV infection. In vitro and in vivo experiments were performed in patients with chronic HBV infection and a hydrodynamic injection mouse model. In vitro assays revealed that L-Cn significantly reduced GC-related immune responses and enhanced immunosuppressive profiles. Intriguingly, L-Cn released from lysed hepatocytes was associated with the degree of liver damage. Besides, the administration of L-Cn in an HBV mouse model resulted in delayed clearance of hepatitis B surface antigen (HBsAg) in serum and decreased GC formation in the spleen. Notably, patients with HBsAg loss showed decreased plasma L-Cn levels, and longitudinal observations found that low baseline levels of L-Cn were associated with a favorable treatment response in patients with chronic hepatitis B. The suppressive effect of hepatocyte-derived L-Cn on GC-related immune cells may contribute to the inability of HBsAg clearance in chronic HBV infection, indicating that L-Cn might serve as a potential therapeutic target for the treatment of HBV infection.

E3 ubiquitin ligase TRIM21 restricts hepatitis B virus replication by targeting HBx for proteasomal degradation

As a cytosol ubiquitin ligase and antibody receptor, Tripartite motif-containing 21 (TRIM21) has been reported to mediate the restriction of hepatitis B virus (HBV) through an HBx-antibody-dependent intracellular neutralization (ADIN) mechanism. However, whether TRIM21 limits HBV replication by targeting viral proteins remains unclarified. In this study, we demonstrate that TRIM21 inhibits HBV gene transcription and replication in HBV plasmid transfected and HBV-infected hepatoma cells. RING and PRY-SPRY domains are involved in this activity. TRIM21 interacts with HBx protein and targets HBx for ubiquitination and proteasomal degradation, leading to impaired HBx-mediated degradation of structural maintenance of chromosomes 6 (Smc6) and suppression of HBV replication. TRIM21 fails to restrict the replication of an HBx-deficient HBV. And knock-down of Smc6 largely impairs the anti-HBV activity of TRIM21 in HepG2 cells. In a hydrodynamic injection (HDI)-based HBV mouse model, we confirm an in vivo anti-HBV and anti-HBx therapeutic effect of TRIM21 by over-expression or knocking-out strategy. Our findings reveal a novel mechanism that TRIM21 restricts HBV replication through targeting HBx-Smc5/6 pathway, which may have an implication in the future TRIM21-based therapeutic application.

AdrA as a Potential Immunomodulatory Candidate for STING-Mediated Antiviral Therapy That Required Both Type I IFN and TNF-α Production.

Several dinucleotide cyclases, including cyclic GMP-AMP synthase, and their involvement in STING-mediated immunity have been extensively studied. In this study, we tested five bacterial diguanylate cyclases from the Gram-negative bacterium Salmonella Enteritidis, identifying AdrA as the most potent inducer of a STING-mediated IFN response. AdrA wild-type (wt) or its inactive version AdrA mutant (mut) were delivered by an adenovirus (Ad) vector. Dendritic cells obtained from wt mice and infected in vitro with Ad vector containing AdrA wt, but not mut, had increased activation markers and produced large amounts of several immunostimulatory cytokines. For dendritic cells derived from STING-deficient mice, no activation was detected. The potential antiviral activity of AdrA was addressed in hepatitis B virus (HBV)-transgenic and adenovirus-associated virus (AAV)-HBV mouse models. Viremia in serum of Ad AdrA wt-treated mice was reduced significantly compared with that in Ad AdrA mut-injected mice. The viral load in the liver at sacrifice was in line with this finding. To further elucidate the molecular mechanism(s) by which AdrA confers its antiviral function, the response in mice deficient in STING or its downstream effector molecules was analyzed. wt and IFN-αR (IFNAR)-/- animals were additionally treated with anti-TNF-α (Enbrel). Interestingly, albeit less pronounced than in wt mice, in IFNAR-/- and Enbrel-treated wt mice, a reduction of serum viremia was achieved-an observation that was lost in anti-TNF-α-treated IFNAR-/- animals. No effect of AdrA wt was seen in STING-deficient animals. Thus, although STING is indispensable for the antiviral activity of AdrA, type I IFN and TNF-α are both required and act synergistically.

Neddylation inhibitor MLN4924 has anti-HBV activity via modulating the ERK-HNF1α-C/EBPα-HNF4α axis.

Hepatitis B virus (HBV) infection is a major public health problem. The high levels of HBV DNA and HBsAg are positively associated with the development of secondary liver diseases, including hepatocellular carcinoma (HCC). Current treatment with nucleos(t)ide analogues mainly reduces viral DNA, but has minimal, if any, inhibitory effect on the viral antigen. Although IFN reduces both HBV DNA and HBsAg, the serious associated side effects limit its use in clinic. Thus, there is an urgent demanding for novel anti‐HBV therapy. In our study, viral parameters were determined in the supernatant of HepG2.2.15 cells, HBV‐expressing Huh7 and HepG2 cells which transfected with HBV plasmids and in the serum of HBV mouse models with hydrodynamic injection of pAAV‐HBV1.2 plasmid. RT‐qPCR and Southern blot were performed to detect 35kb mRNA and cccDNA. RT‐qPCR, Luciferase assay and Western blot were used to determine anti‐HBV effects of MLN4924 and the underlying mechanisms. We found that treatment with MLN4924, the first‐in‐class neddylation inhibitor currently in several phase II clinical trials for anti‐cancer application, effectively suppressed production of HBV DNA, HBsAg, 3.5kb HBV RNA as well as cccDNA. Mechanistically, MLN4924 blocks cullin neddylation and activates ERK to suppress the expression of several transcription factors required for HBV replication, including HNF1α, C/EBPα and HNF4α, leading to an effective blockage in the production of cccDNA and HBV antigen. Our study revealed that neddylation inhibitor MLN4924 has impressive anti‐HBV activity by inhibiting HBV replication, thus providing sound rationale for future MLN4924 clinical trial as a novel anti‐HBV therapy.