HBV-carrier Mice Research Articles

Enhanced hepatitis B virus-specific immunity by combined neutralizing antibody therapy and DNA vaccination in a murine model of chronic hepatitis B infection.

Successful treatment of chronic HBV infection remains a great challenge due to the difficulty in inducing efficient immune responses. Here, we investigated the therapeutic potential of DNA vaccination combined with a potent HBV broadly neutralizing antibody (bNAb) targeting the small surface viral antigen. C57BL/6 mice were transduced with adeno-associated virus (AAV)-HBV and were treated twice a week with HBV bNAb for 5 weeks. A DNA-based vaccine encoding the HBV core, envelope, and polymerase proteins was administered once to mice 3 weeks after initiating of antibody therapy. The antiviral effects and antigen-specific immune responses were evaluated before and for 8 weeks after therapeutic vaccination. Vaccine administration with or without antibody treatment induced the development of functional HBV-specific CD8+ T cells and envelope-specific resident memory T cells in the liver. The combination of antibody treatment and DNA vaccination enhanced the recruitment of B and CD8+ T lymphocytes into the liver of HBV-carrier mice two weeks after vaccination. However, although still detectable 2 months after vaccination, HBV-specific CD8+ T cells showed an exhausted phenotype, suggesting that they are dysfunctional. In contrast, a more effective control of antigenemia was observed following combination therapy, which was associated with the presence of HBs-specific memory B cells. Although the combination therapy did not result in a functional cure, our findings indicate it produced additive effects on the development of HBV-specific T cells in the liver immediately following treatment, offering a better insight into the mechanisms underlying hepatic tolerance.

Lymph node-targeted STING agonist nanovaccine against chronic HBV infection

Chronic hepatitis B virus (HBV) infection is a global health problem that substantially increases the risk of developing liver disease. The development of a novel strategy to induce anti-HB seroconversion and achieve a long-lasting immune response against chronic HBV infection remains challenging. Here, we found that chronic HBV infection affected the signaling pathway involved in STING-mediated induction of host immune responses in dendritic cells (DCs) and then generated a lymph node-targeted nanovaccine that co-delivered hepatitis B surface antigen (HBsAg) and cyclic diguanylate monophosphate (c-di-GMP) (named the PP-SG nanovaccine). The feasibility and efficiency of the PP-SG nanovaccine for CHB treatment were evaluated in HBV-carrier mice. Serum samples were analyzed for HBsAg, anti-HBs, HBV DNA, and alanine aminotransferase levels, and liver samples were evaluated for HBV DNA and RNA and HBcAg, accompanied by an analysis of HBV-specific cellular and humoral immune responses during PP-SG nanovaccine treatment. The PP-SG nanovaccine increased antigen phagocytosis and DC maturation, efficiently and safely eliminated HBV, achieved a long-lasting immune response against HBV reinjection, and disrupted chronic HBV infection-induced immune tolerance, as characterized by the generation and multifunctionality of HBV-specific CD8+ T and CD4+ T cells and the downregulation of immune checkpoint molecules. HBV-carrier mice immunized with the PP-SG nanovaccine achieved partial anti-HBs seroconversion. The PP-SG nanovaccine can induce sufficient and persistent viral suppression and achieve anti-HBs seroconversion, rendering it a promising vaccine candidate for clinical chronic hepatitis B therapy.

The lack of HBsAg secretion does neither facilitate induction of antiviral T cell responses nor Hepatitis B Virus clearance in mice

Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts is regarded as a key contributor. HBsAg is expressed in HBV-infected hepatocytes and those carrying an HBV integration. Whether either HBsAg secretion or the high antigen amount expressed in the liver determines its immunomodulatory properties, however, remains unclear. We, therefore, developed a novel HBV animal model that allowed us to study the role of secreted HBsAg. We introduced a previously described HBs mutation, C65S, abolishing HBsAg secretion into a replication-competent 1.3-overlength HBV genome and used adeno-associated virus vectors to deliver it to the mouse liver. The AAV-HBV established a carrier state of wildtype and C65S mutant HBV, respectively. We investigated antiviral B- and T-cell immunity in the HBV-carrier mice after therapeutic vaccination. Moreover, we compared the effect of a lacking HBsAg secretion with that of an antiviral siRNA. While missing HBsAg secretion allowed for higher levels of detectable anti-HBs antibodies after therapeutic vaccination, it did neither affect antiviral T-cell responses nor intrahepatic HBV gene expression, irrespective of the starting level. A treatment with HBV siRNA restricting viral antigen expression within hepatocytes, however, improved the antiviral efficacy of therapeutic vaccination, irrespective of the ability of HBV to secrete HBsAg. Our data indicate that clearing HBsAg from blood cannot significantly impact HBV persistence or T-cell immunity. This indicates that a restriction of hepatic viral antigen expression will be required to break HBV immunotolerance.

Mesencephalic Astrocyte-derived Neurotrophic Factor Supports Hepatitis B Virus-induced Immunotolerance

Background & AimsThe immune tolerance induced by Hepatitis B virus (HBV) is a major challenge for achieving effective viral clearance, and the mechanisms involved are not well understood. One potential factor involved in modulating immune responses is mesencephalic astrocyte-derived neurotrophic factor (MANF), which has been reported to be increased in patients with chronic hepatitis B. In this study, our objective is to examine the role of MANF in regulating immune responses to HBV. MethodsWe utilized a commonly used HBV-harboring mouse model, where mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. We assessed the HBV load by measuring the levels of various markers including HBsAg, HBeAg, HBcAg, HBV DNA, and HBV RNA. ResultsOur study revealed that following HBV infection, both myeloid cells and hepatocytes exhibited increased expression of MANF. Moreover, we observed that mice with myeloid-specific MANF knockout (ManfMye-/-) displayed reduced HBV load and improved HBV-specific T cell responses. The decreased HBV-induced tolerance in ManfMye-/- mice was associated with reduced accumulation of myeloid-derived suppressor cells (MDSCs) in the liver. Restoring MDSC levels in ManfMye-/- mice through MDSC adoptive transfer reinstated HBV-induced tolerance. Mechanistically, we found that MANF promoted MDSC expansion by activating the IL-6/STAT3 pathway. Importantly, our study demonstrated the effectiveness of a combination therapy involving an HBsAg vaccine and nanoparticle-encapsulated MANF siRNA in effectively clearing HBV in HBV-carrier mice. ConclusionThe current study reveals that MANF plays a previously unrecognized regulatory role in liver tolerance by expanding MDSCs in the liver through IL-6/ STAT3 signaling, leading to MDSC-mediated CD8+ T cell exhaustion.

Introducing adjuvant-loaded particulate hepatitis B core antigen as an alternative therapeutic hepatitis B vaccine component

Particulate hepatitis B core antigen (HBcoreAg) is a potent immunogen used as a vaccine carrier platform. HBcoreAg produced in E.coli encapsidates random bacterial RNA (bRNA). Using the heterologous protein-prime, viral-vector-boost therapeutic hepatitis B vaccine TherVacB, we compared the properties of different HBcoreAg forms. We explored how the content of HBcoreAg modulates antigen stability, immunogenicity, and antiviral efficacy. bRNA was removed from HBcoreAg by capsid disassembly, followed by reassembly in the absence or presence of specific nucleic acid-based adjuvants poly I:C or CpG. The morphology and structure of empty, bRNA-containing and adjuvant-loaded HBcoreAg were monitored by electron microscopy and nuclear magnetic resonance spectroscopy. Empty, bRNA-containing or adjuvant-loaded HBcoreAg were applied together with HBsAg and with or without nucleic acid-based external adjuvants within the TherVacB regimen in both wild-type and HBV-carrier mice. While HBcoreAg retained its structure upon bRNA removal, its stability and immunogenicity decreased significantly. Loading HBcoreAg with nucleic acid-based adjuvants re-established stability of the capsid-like antigen. Immunization with poly I:C- or CpG-loaded HBcoreAg induced high antibody titers against co-administered HBsAg. When applied within the TherVacB regimen, they activated vigorous HBcoreAg- and HBsAg-specific T-cell responses in wild-type and HBV-carrier mice, requiring a significantly lower dose of adjuvant compared to externally added adjuvant. Finally, immunization with adjuvant-loaded HBcoreAg mixed with HBsAg led to long-term control of persistent HBV replication in the HBV-carrier mice. Adjuvant-loaded HBcoreAg retained capsid integrity and stability, was as immunogenic invivo as externally adjuvanted HBcoreAg, requiring lower adjuvant levels, and supported immunity against co-administered, non-adjuvanted HBsAg. Thus, adjuvant-loaded HBcoreAg represents a promising novel platform for vaccine development. Hepatitis B core antigen (HBcoreAg) recapitulates the capsid of the HBV that hosts the viral genome. Produced recombinantly, it is not infectious but emerges as a potent immunogen in vaccine development. In this preclinical study, we show that loading HBcoreAg with defined nucleic-acid-based adjuvants on the one hand stabilizes the HBcoreAg with standardized capsid content and, on the other hand, efficiently promotes the immunity of HBcoreAg and a co-administered antigen, allowing for reduced adjuvant doses. Therefore, adjuvant-loaded HBcoreAg not only serves as an encouraging option for therapeutic hepatitis B vaccines, but could also act as an efficient adjuvant delivery system for other types of vaccine.

The hepatic GABAergic system promotes liver macrophage M2 polarization and mediates HBV replication in mice

Macrophages display functional phenotypic plasticity. Hepatitis B virus (HBV) infection induces polarizations of liver macrophages either to M1-like pro-inflammatory phenotype or to M2-like anti-inflammatory phenotype. Gamma-aminobutyric acid (GABA) signaling exists in various non-neuronal cells including hepatocytes and some immune cells. Here we report that macrophages express functional GABAergic signaling components and activation of type A GABA receptors (GABAARs) promotes M2-polarization thus advancing HBV replication. Notably, intraperitoneal injection of GABA or the GABAAR agonist muscimol increased HBV replication in HBV-carrier mice that were generated by hydrodynamical injection of adeno-associated virus/HBV1.2 plasmids (pAAV/HBV1.2). The GABA-augmented HBV replication in HBV-carrier mice was significantly reduced by the GABAAR inhibitor picrotoxin although picrotoxin had no significant effect on serum HBsAg levels in control HBV-carrier mice. Depletion of liver macrophages by liposomal clodronate treatment also significantly reduced the GABA-augmented HBV replication. Yet adoptive transfer of liver macrophages isolated from GABA-treated donor HBV-carrier mice into the liposomal clodronate-pretreated recipient HBV-carrier mice restored HBV replication. Moreover, GABA or muscimol treatment increased the expression of “M2” cytokines in macrophages, but had no direct effect on HBV replication in the HepG2.2.15 cells, HBV1.3-transfected Huh7, HepG2, or HepaRG cells, or HBV-infected Huh7-NTCP cells. Taken together, these results suggest that increasing GABA signaling in the liver promotes HBV replication in HBV-carrier mice by suppressing the immunity of liver macrophages, but not by increasing the susceptibility of hepatocytes to HBV infection. Our study shows that a previously unknown GABAergic system in liver macrophage has an essential role in HBV replication.

Activation of CD4 T cells during prime immunization determines the success of a therapeutic hepatitis B vaccine in HBV-carrier mouse models

We recently developed a heterologous therapeutic vaccination scheme (TherVacB) comprising a particulate protein prime followed by a modified vaccinia-virus Ankara (MVA)-vector boost for the treatment of HBV. However, the key determinants required to overcome HBV-specific immune tolerance remain unclear. Herein, we aimed to study new combination adjuvants and unravel factors that are essential for the antiviral efficacy of TherVacB. Recombinant hepatitis B surface and core antigen (HBsAg and HBcAg) particles were formulated with different liposome- or oil-in-water emulsion-based combination adjuvants containing saponin QS21 and monophosphoryl lipid A; these formulations were compared to STING-agonist c-di-AMP and conventional aluminium hydroxide formulations. Immunogenicity and the antiviral effects of protein antigen formulations and the MVA-vector boost within TherVacB were evaluated in adeno-associated virus-HBV-infected and HBV-transgenic mice. Combination adjuvant formulations preserved HBsAg and HBcAg integrity for ≥12 weeks, promoted human and mouse dendritic cell activation and, within TherVacB, elicited robust HBV-specific antibody and T-cell responses in wild-type and HBV-carrier mice. Combination adjuvants that prime a balanced HBV-specific type 1 and 2T helper response induced high-titer anti-HBs antibodies, cytotoxic T-cell responses and long-term control of HBV. In the absence of an MVA-vector boost or following selective CD8 T-cell depletion, HBsAg still declined (mediated mainly by anti-HBs antibodies) but HBV replication was not controlled. Selective CD4 T-cell depletion during the priming phase of TherVacB resulted in a complete loss of vaccine-induced immune responses and its therapeutic antiviral effect in mice. Our results identify CD4 T-cell activation during the priming phase of TherVacB as a key determinant of HBV-specific antibody and CD8 T-cell responses. Therapeutic vaccination is a potentially curative treatment option for chronic hepatitis B. However, it remains unclear which factors are essential for breaking immune tolerance in HBV carriers and determining successful outcomes. Our study provides the first direct evidence that efficient priming of HBV-specific CD4 T cells determines the success of therapeutic hepatitis B vaccination in two preclinical HBV-carrier mouse models. Applying an optimal formulation of HBV antigens that activates CD4 and CD8 T cells during prime immunization provided the foundation for an antiviral effect of therapeutic vaccination, while depletion of CD4 T cells led to a complete loss of vaccine-induced antiviral efficacy. Boosting CD8 T cells was important to finally control HBV in these mouse models. Our findings provide important insights into the rational design of therapeutic vaccines for the cure of chronic hepatitis B.

Cholesterol accumulation on dendritic cells reverses chronic hepatitis B virus infection-induced dysfunction.

Chronic hepatitis B (CHB) infection remains a serious public health problem worldwide; however, the relationship between cholesterol levels and CHB remains unclear. We isolated peripheral blood mononuclear cells from healthy blood donors and CHB patients to analyze free cholesterol levels, lipid raft formation, and cholesterol metabolism-related pathways. Hepatitis B virus (HBV)-carrier mice were generated and used to confirm changes in cholesterol metabolism and cell-surface lipid raft formation in dendritic cells (DCs) in the context of CHB. Additionally, HBV-carrier mice were immunized with a recombinant HBV vaccine (rHBVvac) combined with lipophilic statins and evaluated for vaccine efficacy against HBV. Serum samples were analyzed for HBsAg, anti-HBs, and alanine aminotransferase levels, and liver samples were evaluated for HBV DNA and RNA and HBcAg. CHB reduced free cholesterol levels and suppressed lipid raft formation on DCs in patients with CHB and HBV-carrier mice, whereas administration of lipophilic statins promoted free cholesterol accumulation and restored lipid rafts on DCs accompanied by an enhanced antigen-presentation ability in vitro and in vivo. Cholesterol accumulation on DCs improved the rHBVvac-mediated elimination of serum HBV DNA and intrahepatic HBV DNA, HBV RNA, and HBcAg and promoted the rHBVvac-mediated generation and polyfunctionality of HBV-specific CD11ahi CD8αlo cells, induction of the development of memory responses against HBV reinfection, and seroconversion from HBsAg to anti-HBs. The results demonstrated the important role of cholesterol levels in DC dysfunction during CHB, suggesting that strategies to increase cholesterol accumulation on DCs might enhance therapeutic vaccine efficacy against HBV and support development toward clinical CHB treatment.

Optimized Cationic Lipid-assisted Nanoparticle for Delivering CpG Oligodeoxynucleotides to Treat Hepatitis B Virus Infection.

Hepatitis B virus (HBV) infection is such a global health problem that hundreds of millions of people are HBV carriers. Current anti-viral agents can inhibit HBV replication, but can hardly eradicate HBV. Cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODNs) are an adjuvant that can activate plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) to induce therapeutic immunity for HBV eradication. However, efficient delivery of CpG ODNs into pDCs and cDCs remains a challenge. In this study, we constructed a series of cationic lipid-assisted nanoparticles (CLANs) using different cationic lipids to screen an optimal nanoparticle for delivering CpG ODNs into pDCs and cDCs. We constructed different CLANCpG using six cationic lipids and analyzed the cellular uptake of different CLANCpG by pDCs and cDCs in vitro and in vivo, and further analyzed the efficiency of different CLANCpG for activating pDCs and cDCs in both wild type mice and HBV-carrier mice. We found that CLAN fabricated with 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP) showed the highest efficiency for delivering CpG ODNs into pDCs and cDCs, resulting in strong therapeutic immunity in HBV-carrier mice. By using CLANCpG as an immune adjuvant in combination with the injection of recombinant hepatitis B surface antigen (rHBsAg), HBV was successfully eradicated and the chronic liver inflammation in HBV-carrier mice was reduced. We screened an optimized CLAN fabricated with DOTAP for efficient delivery of CpG ODNs to pDCs and cDCs, which can act as a therapeutic vaccine adjuvant for treating HBV infection.

CpG-C ODN M362 as an immunoadjuvant for HBV therapeutic vaccine reverses the systemic tolerance against HBV

Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both in vivo and in vitro, overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8+ and CD4+ T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5+ CD8+ T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.

Immunogenicity and Antiviral Response of Therapeutic Hepatitis B Vaccination in a Mouse Model of HBeAg-Negative, Persistent HBV Infection.

During the natural course of chronic hepatitis B virus (HBV) infection, the hepatitis B e antigen (HBeAg) is typically lost, while the direct transmission of HBeAg-negative HBV may result in fulminant hepatitis B. While the induction of HBV-specific immune responses by therapeutic vaccination is a promising, novel treatment option for chronic hepatitis B, it remains unclear whether a loss of HBeAg may influence its efficacy or tolerability. We therefore generated an adeno-associated virus (AAV)-vector that carries a 1.3-fold overlength HBV genome with a typical stop-codon mutation in the pre-core region and initiates the replication of HBeAg(−) HBV in mouse livers. Infection of C57BL/6 mice established persistent HBeAg(−) HBV-replication without any detectable anti-HBV immunity or liver damage. HBV-carrier mice were immunized with TherVacB, a therapeutic hepatitis B vaccine that uses a particulate HBV S and a core protein for prime vaccination, and a modified vaccinia Ankara (MVA) for boost vaccination. The TherVacB immunization of HBeAg(+) and HBeAg(−) HBV carrier mice resulted in the effective induction of HBV-specific antibodies and the loss of HBsAg but only mild liver damage. Intrahepatic, HBV-specific CD8 T cells induced in HBeAg(−) mice expressed more IFNγ but showed similar cytolytic activity. This indicates that the loss of HBeAg improves the performance of therapeutic vaccination by enhancing non-cytolytic effector functions.

NK cells contribute to hepatic CD8+ T cell failure in hepatitis B virus-carrier mice after alcohol consumption

Despite the fact that both Hepatitis B virus (HBV) infection and excessive alcohol consumption represent health problems worldwide, the mechanism by which alcohol affected the progression of HBV-associated liver disease are not completely understood. Therefore, we studied how alcohol affects the development of HBV infection and the role of T cells and NK cells in the antiviral response. Mononuclear cells (MNCs) derived from HBV-carrier mice and wild type (WT) mice were characterized for phenotype by flow cytometry, HBV antigen and gene expression were detected by Radio Immunoassay (RIA), immunohistochemistry and quantitative real-time (qRT)-PCR. Metabolomics changes were detected in mice liver tissue based on ultra high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOFMS). The mice after ethanol consumption shows higher levels of HBV surface Ag (HBsAg), HBV core antigen (HBcAg) and HBV 3.5 kb RNA expression, and a lower level of CD8+ T cells during HBV persistence, with an increased lymphocyte activation gene-3 (LAG-3) expression on CD8+ T cell. In addition, the energy metabolism was downregulated and the oxidative stress was upregulated in the liver tissue. Furthermore, NK cells depletion results in a lower levels of HBV surface Ag (HBsAg) and HBV 3.5 kb RNA expression, and a higher level of CD8+ T cells with reduced expression of LAG-3. In conclusion, alcohol abuse induces CD8+ T cells failure after acute HBV infection, but depletion of NK cells could retore CD8+ T cell activity. Moreover, downregulation of energy metabolism and upregulation of oxidative stress may also contribute to CD8+ T cell failure.

HBV X protein-based therapeutic vaccine accelerates viral antigen clearance by mobilizing monocyte infiltration into the liver in HBV carrier mice

BackgroundHepatitis B virus (HBV) persistently infected about 250 million people worldwide, and a curative treatment remains an unmet medical need. Among many approaches to treat chronic hepatitis B (CHB), therapeutic vaccines have been developed for two decades, but none have yielded promising results in clinical trials. Therefore, dissection of HBV clearance mechanisms during therapeutic vaccination in appropriate models, which could give rise to new curative therapies, is urgently needed. Growing evidence indicates that prolonged and intensive exposure of antigen-specific T cells to viral antigens is a major cause of T cell exhaustion, and decreases anti-HBV immunity efficacy of therapeutic vaccination. HBV X protein (HBx) is expressed at low levels, and the understanding of its immunogenicity and potential in therapeutic CHB vaccines is limited.MethodsHBV genome sequences from CHB patients were cloned into a pAAV plasmid backbone and transfected into immunocompetent mouse hepatocytes through hydrodynamic injection. Mice carrying > 500 IU/mL serum HBV surface antigen (HBs) for more than 4 weeks were considered HBV carriers mimicking human CHB and received 3 doses of weekly HBx vaccine by subcutaneous immunization. Serum HBV clearance was evaluated by monitoring serum HBs and HBV-DNA titers. Residual HBV in the liver was evaluated by western blotting for HBV core antigen. The splenic antigen-specific T cell response was quantified by a 15-mer overlapping peptide-stimulated interferon-γ enzyme-linked immunospot assay. Blood and hepatic immune cells were quantified by flow cytometric analysis.ResultsOur HBx-based vaccine induced systemic HBx-specific CD4+ and CD8+ T cell responses in HBV carrier mice and demonstrated significant HBs and HBV-DNA elimination. The protective effect persisted for at least 30 days without additional booster immunization. Different infiltrating myeloid cell subsets, each with distinctive roles during immune-mediated HBV clearance, were found in the liver of vaccinated mice. During vaccine therapy, inflammatory monocyte depletion resulted in sustained HBV clearance inhibition, whereas phagocytic monocyte-derived macrophage and Kupffer cell elimination resulted in only transient inhibition of vaccine-induced HBV clearance.ConclusionsWe report the potential role of HBx as a major immunogen in an HBV therapeutic vaccine and the significance of a liver-infiltrating monocyte subset during hepatic viral clearance.

Hepatitis B virus‐induced hyperactivation of B cells in chronic hepatitis B patients via TLR4

B cell hyperactivation and functional impairment were identified from patients with chronic hepatitis B virus (CHB) infection; however, the underlying mechanism remains unknown. Here, we aim to elucidate the mechanisms responsible for B cell hyperactivation during HBV infection. Peripheral CD19+ B cells isolated from 4 CHB patients and 4 healthy volunteers were analysed by RNA sequencing. A total of 1401 differentially expressed genes were identified from B cell transcriptome of CHB patients vs healthy volunteers. We found that B cells from CHB patients were functional impaired, with increased TLR4 expression, activated NF‐κB pathway and altered mitochondrial function. The expression of B cell activation‐related genes, including TLR4, was further validated using additional clinical samples. To further verify the role of TLR4 in B cell activation during CHB, B cell phenotypes were determined in wild‐type (WT) and TLR4−/− HBV‐carrier mice. Hyperactivated B cell and TLR4 signalling pathway were observed in WT HBV‐carrier mice, while TLR4 ablation failed to induce B cell hyperactivation, and downstream MyD88 and NF‐κB were also not altered. Taken together, TLR4 pathway plays a pivotal role in B cell hyperactivation during CHB, which might serve as a promising target for B cell function restoration.

Poly I:C-based rHBVvac therapeutic vaccine eliminates HBV via generation of HBV-specific CD8+ effector memory T cells

ObjectiveChronic hepatitis B (CHB) virus infection is a global health problem. Finding a cure for CHB remains a challenging task.DesignIn this study, poly I:C was employed as an adjuvant for...

Changes in natural killer cell subsets marked with CD27 and CD11b for HBV carrier mice

Objective: To evaluate the changes in natural killer cell subsets marked with CD27 and CD11b for HBV carrier mice. Methods: The pAAV-HBVl.2 plasmid was injected into the tail vein of C57BL/6 mice by hydrodynamic injection method to construct HBV-carrier model group and empty vector as the control group. Liver function and virological examination at different time points were used to judge the construction of HBV- plasmid carrier animal model. Flow cytometry was used to detect the frequency of NK cells and CD11b combined with CD27 NK cell subsets in spleen and liver. GraphPad Prism software was used for statistical analysis. Results: HBV-carrier mouse model was successfully constructed. There were no statistically significant difference in NK cell frequencies between spleen and liver of HBV carrier mice (P> 0.05), compared to control group. NK cells were divided into four subsets with in combination to CD27 and CD11b: CD11b(+)CD27(-)(CD11b(+)SP), CD11b(+)CD27(+)(DP), CD11b(-)CD27(+)(CD27(+)SP) and CD11b-CD27-(DN). Furthermore, the spleen of HBV-carrier mice had no statistically significant difference (P> 0.05) with the frequency of the four NK-cell subsets. The frequency of DN NK cell subsets was significantly increased in the liver of HBV carrier mice than control group (P< 0.001); however, the frequency of CD11b(+)SP cell subsets was significantly decreased (P < 0.05).There were no statistical significance in the frequency comparison between NK subgroups of DP and CD27(+)SP NK cell subsets (P> 0.05). Conclusion: HBV-carrier mice with abnormal distribution of hepatic NK cell subsets significantly increased and decreased the frequency of DN NK cell subsets and CD11b(+)SP cell subsets.

A unique B cell epitope-based particulate vaccine shows effective suppression of hepatitis B surface antigen in mice

ObjectiveThis study aimed to develop a novel therapeutic vaccine based on a unique B cell epitope and investigate its therapeutic potential against chronic hepatitis B (CHB) in animal models.MethodsA series...

Nasal route favors the induction of CD4+ T cell responses in the liver of HBV-carrier mice immunized with a recombinant hepatitis B surface- and core-based therapeutic vaccine

Immunization routes and number of doses remain largely unexplored in therapeutic vaccination. The aim of the present work is to evaluate their impact on immune responses in naïve and hepatitis B virus (HBV)-carrier mouse models following immunization with a non-adjuvanted recombinant vaccine comprising the hepatitis B surface (HBsAg) and core (HBcAg) antigens. Mice were immunized either by intranasal (i.n.), subcutaneous (s.c.) or simultaneous (i.n. + s.c.) routes. Humoral immunity was detected in all the animal models with the induction of a potent antibody (Ab) response against HBcAg, which was stronger than the anti-HBs response. In the HBV-carrier mouse model, the anti-HBs response was predominantly subtype-specific and preferentially induced by the i.n. route. However, the Ab titers were not sufficient to clear the high concentration of HBsAg present in the sera of these mice. The i.n. route was the most efficacious at inducing cellular immune responses, in particular CD4+ T cells. In naïve mice, cellular responses in spleen were strong and mainly due to CD4+ T cells whereas the CD8+ T-cell response was low. In HBV-carrier mice, high frequencies of HBs-specific CD4+ T cells secreting interferon (IFN)-γ, interleukin (IL)-2 and tumor necrosis factor (TNF)-α were found in liver only after i.n. immunization. Increased frequencies of CD4+ T cells expressing the integrin CD49a in liver suggest a role of nasal route in the cellular homing process. Multiple dose schedules appear to be a prerequisite for protein-based immunization in order to overcome immunotolerance in HBV-carrier mice. These findings provide new avenues for further preclinical and clinical development.

Kupffer Cells Support Hepatitis B Virus-Mediated CD8+ T Cell Exhaustion via Hepatitis B Core Antigen-TLR2 Interactions in Mice.

Hepatitis B virus (HBV) persistence is a fundamental process in chronic HBV infection and a key factor in all related liver diseases; however, the mechanisms have yet to be elucidated. We studied the role of TLR2 in HBV persistence using a well-established HBV-carrier mouse model generated by hydrodynamically injecting a phospho-adeno-associated virus/HBV1.2 plasmid into mice. We found that a genetic deficiency in TLR2 improves HBV elimination, whereas activating TLR2 led to more stable HBV persistence, suggesting that TLR2 activation is critical in HBV persistence. Furthermore, we noted that TLR2 activation could inhibit CD8(+) T cell function, causing the exhaustion phenotype in HBV-carrier mice, because TLR2 deficiency might rescue CD8(+) T cell function in a cellular adoptive experiment. TLR2 expression on Kupffer cells (KCs) was upregulated in HBV-carrier mice, which accounts for HBV persistence, because the difference in anti-HBV immunity between HBV-carrier wild-type and Tlr2(-/-) mice did not exist after KC depletion. In addition, similar to TLR2 deficiency, after KC depletion, CD8(+) T cells were more efficiently activated in HBV-carrier mice, leading to rapid HBV elimination. KCs produced more IL-10 upon TLR2 activation in response to direct hepatitis B core Ag stimulation, and the elevated IL-10 inhibited CD8(+) T cell function in HBV-carrier mice, because IL-10 deficiency or anti-IL-10R treatment resulted in CD8(+) T cells with stronger antiviral function. In conclusion, KCs support liver tolerance by inducing anti-HBV CD8(+) T cell exhaustion via IL-10 production after TLR2 activation by hepatitis B core Ag stimulation.

Prolonged suppression of HBV in mice by a novel antibody that targets a unique epitope on hepatitis B surface antigen

ObjectiveThis study aimed to investigate the therapeutic potential of monoclonal antibody (mAb) against HBV as a novel treatment approach to chronic hepatitis B (CHB) in mouse models.MethodsTherapeutic effects of mAbs...