Ejuan Zhang

Modulation of Hepatitis B Virus Replication and Hepatocyte Differentiation by MicroRNA-1

MicroRNAs (miRNAs) are highly conserved small noncoding RNAs participating in regulation of various cellular processes. Viruses have been shown to utilize cellular miRNAs to increase their replication in host cells. Until now, the role of miRNAs in hepatitis B virus (HBV) replication has remained largely unknown. In this study, a number of miRNA mimics were transfected into hepatoma cell lines with HBV replication. It was noted that microRNA‐1 (miR‐1) transfection resulted in a marked increase of HBV replication, accompanied with up‐regulated HBV transcription, antigen expression, and progeny secretion. However, bioinformatics and luciferase reporter analysis suggested that miR‐1 may not target the HBV genome directly but regulate the expression of host genes to enhance HBV replication. Further studies showed that miR‐1 was able to enhance the HBV core promoter transcription activity by augmenting farnesoid X receptor α expression. In addition, miR‐1 arrested the cell cycle at the G1 phase and inhibited cell proliferation by targeting histone deacetylase 4 and E2F transcription factor 5. Analysis of the cellular gene expression profile indicated that miR‐1 transfected hepatoma cells developed a differentiated phenotype of hepatocytes. Conclusion: MiR‐1 regulates the expression of several host genes to enhance HBV replication and reverse cancer cell phenotype, which is apparently beneficial for HBV replication. Our findings provide a novel perspective on the role of miRNAs in host‐virus interactions in HBV infection. (HEPATOLOGY 2011;)

Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.

Toll-like receptor-induced innate immune responses in non-parenchymal liver cells are cell type-specific

Little is known of how the Toll‐like receptor (TLR) system can modulate the function of non‐parenchymal liver cells (NPC) as a major component of the innate and adaptive immune system of the liver. To investigate the diversification of TLR signalling pathways in NPC, we isolated Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC) from wild‐type C57BL/6 mice and examined their responses to TLR1 to TLR9 agonists. The data show that KC respond to all TLR ligands by producing tumour necrosis factor‐α (TNF‐α) or interleukin‐6 (IL‐6), to TLR3 and TLR4 ligands only by producing interferon‐β (IFN‐β), to TLR1 and TLR8 ligands by significantly up‐regulating major histocompatibility complex (MHC) class II and costimulatory molecules, and to TLR1, ‐2, ‐4 and ‐6 ligands by inducing high levels of T‐cell proliferation and IFN‐γ production in the mixed lymphocyte reaction (MLR). Similarly, LSEC respond to TLR1 to ‐4, ‐6, ‐8 and ‐9 ligands by producing TNF‐α, to TLR3 and ‐4 ligands by producing IL‐6, and to TLR3 ligands by producing IFN‐β. Interestingly, despite significant up‐regulation of MHC class II and co‐stimulatory molecules in response to TLR8 ligands, LSEC stimulated by TLR1, ‐2 or ‐6 could stimulate allogeneic T cells as assessed by MLR. By contrast, myeloid dendritic cells, used as positive control for classical antigen‐presenting cells, respond to TLR1, ‐2, ‐4 and ‐9 ligands by both up‐regulation of CD40 and activation of allogeneic T cells. In conclusion, NPC display a restricted TLR‐mediated activation profile when compared with ‘classical’ antigen‐presenting cells which may, at least in part, explain their tolerogenic function in the liver.

Combination of DNA Prime – Adenovirus Boost Immunization with Entecavir Elicits Sustained Control of Chronic Hepatitis B in the Woodchuck Model

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients.

Contribution of Toll-like receptors to the control of hepatitis B virus infection by initiating antiviral innate responses and promoting specific adaptive immune responses

It is well accepted that adaptive immunity plays a key role in the control of hepatitis B virus (HBV) infection. In contrast, the contribution of innate immunity has only received attention in recent years. Toll-like receptors (TLRs) sense pathogen-associated molecule patterns and activate antiviral mechanisms, including intracellular antiviral pathways and the production of antiviral effector interferons (IFNs) and pro-inflammatory cytokines. Experimental results from in vitro and in vivo models have demonstrated that TLRs mediate the activation of cellular signaling pathways and the production of antiviral cytokines, resulting in a suppression of HBV replication. However, HBV infection is associated with downregulation of TLR expression on host cells and blockade of the activation of downstream signaling pathways. In primary HBV infection, TLRs may slow down HBV infection, but contribute only indirectly to viral clearance. Importantly, TLRs may modulate HBV-specific T- and B-cell responses in vivo, which are essential for the termination of HBV infection. Thus, TLR agonists are promising candidates to act as immunomodulators for the treatment of chronic HBV infection. Antiviral treatment may recover TLR expression and function in chronic HBV infection and may increase the efficacy of therapeutic approaches based on TLR activation. A combined therapeutic strategy with antiviral treatment and TLR activation could facilitate the restoration of HBV-specific immune responses and thereby, achieve viral clearance in chronically infected HBV patients.Cellular & Molecular Immunology advance online publication, 24 November 2014; doi:10.1038/cmi.2014.112

Toll-like receptor (TLR)-mediated innate immune responses in the control of hepatitis B virus (HBV) infection

AbstractnThe role of adaptive immune responses in the control of hepatitis B virus (HBV) infection is well accepted. The contribution of innate immune responses to the viral control is recognized but yet not fully understood. Toll-like receptors (TLRs) sense pathogen-associated molecule patterns and activate antiviral mechanisms including the intracellular antiviral pathways and the production of antiviral effectors like interferons (IFNs) and pro-inflammatory cytokines. Activation of the TLR3 pathway and the production of IFN-β represent one of the major mechanisms leading to the suppression of HBV replication in the liver, as shown in different in vitro and in vivo models. TLR4 signaling and TLR2 signaling result in the activation of intracellular pathways including MAPK and PI-3xa0K/Akt in hepatocytes and reduce HBV replication in an IFN-independent manner. HBV is able to counteract the actions of TLR3 and TLR2/4 through downregulation of TLR expression and attenuation of the cellular signaling pathways. Thus, TLR ligands are promising candidates as immunomodulators and therapeutics for the treatment of chronic HBV infection. Specific antiviral treatment against HBV could recover the TLR functions in chronic HBV infection and increase the effectiveness of therapeutic approaches based on TLR activation.

Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of Hepatitis B Virus in a Hydrodynamic Injection Mouse Model

ABSTRACT We have previously shown that poly(I:C) activates murine hepatic cells to produce interferon (IFN) and suppresses hepatitis B virus (HBV) replication in vitro. Therefore, we addressed whether poly(I:C) is able to induce the clearance of HBV in vivo. The chronic HBV replication mouse model was established by the hydrodynamic injection (HI) of pAAV-HBV1.2 into the tail veins of wild-type and IFN-α/βR-, IFN-γ-, and CXCR3-deficient C57BL/6 mice. Fourteen days post-HI of pAAV-HBV1.2, mice were administered poly(I:C) by intraperitoneal injection, intramuscular injection, or HI. Only treatment of poly(I:C) by HI led to HBV clearance in wild-type C57BL/6 mice. Serum HBsAg disappeared within 40 days postinfection (dpi) in mice that received poly(I:C) by HI, and this was accompanied by the appearance of anti-HBs antibodies. HBV-specific T-cell and antibody responses were significantly enhanced by HI of poly(I:C). HBV replication intermediates and HBcAg-positive hepatocytes were eliminated in the liver. HI of poly(I:C) induced the production of IFNs in mice and enhanced the levels of cytokines, IFN-stimulated genes, and T-cell markers in the liver. Importantly, poly(I:C)-induced HBV clearance was impaired in IFN-α/βR-, IFN-γ-, and CXCR3-deficient mice, indicating that the induction of type I IFN and the stimulation and recruitment of T cells into the liver are essential for HBV clearance in this model. Taken together, the application of poly(I:C) by HI into the liver enhances innate and adaptive immune responses and leads to HBV clearance in an HBV mouse model, implicating the potential of intrahepatic Toll-like receptor 3 (TLR3) activation for the treatment of chronic hepatitis B patients. IMPORTANCE It has become well accepted that immunomodulation is a potentially useful approach to treat chronic viral infection. Recently, combinations of antiviral treatment and therapeutic vaccinations were evaluated for therapies of chronic hepatitis B virus (HBV) infection. Activation of the innate immune branch may also be important for viral control and contributes to HBV clearance. Our present study demonstrated that hepatic TLR3 activation led to clearance of hepatitis B virus in an HBV mouse model. For the first time, we showed that HBV clearance in this model is dependent not only on type I interferon (IFN) but also on type II IFN, indicating a coordinated action of innate and adaptive immune responses. T-cell recruitment appeared to be critical for the success of TLR3-mediated antiviral action. These findings implicate the potential of intrahepatic TLR3 activation for the treatment of chronic HBV infection.

DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV) core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic.

Background Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4) primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV) clearance. Principal Findings Plasmids expressing HBV core protein (HBcAg) or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc), CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI) of pAAV/HBV1.2. HBV surface antigen (HBsAg) and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance. Conclusion Viral clearance could be efficiently achieved by Th1/Th2-balanced immune response, with a small but significant shift in T-cell and B-cell immune responses.

Coexistence of Hepatitis B Virus Quasispecies Enhances Viral Replication and the Ability To Induce Host Antibody and Cellular Immune Responses

ABSTRACT Hepatitis B virus (HBV) quasispecies contain a large number of variants that serve as a reservoir for viral selection under antiviral treatment and the immune response, leading to the acute exacerbation and subsequent development of liver failure. However, there is no clear experimental evidence for a significant role of HBV quasispecies in viral pathogenesis. In the present study, HBV sequences were amplified from a patient with severe liver disease and used for construction of HBV replication-competent plasmids. Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining were performed to analyze the expression, secretion, and subcellular localization of viral proteins in vitro. Viral replication intermediates were detected by Southern blotting. HBV gene expression and replication and the induction of specific immune responses in an HBV hydrodynamic injection (HI) mouse model were investigated. The results demonstrated that two naturally occurring HBV variants, SH and SH-DPS, were identified. The variant SH-DPS expressed only a nonexportable hepatitis B virus surface antigen (HBsAg) with abnormal intracellular accumulation. The coexistence of the HBV variants at a ratio of 1 to 4 (SH to SH-DPS) increased HBV replication. Significantly stronger intrahepatic cytotoxic T lymphocyte (CTL) responses and antibody responses specific to HBsAg were induced in mice by the HBV variants when coapplied by HI. These findings uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses, representing a novel mechanism for the immunopathogenesis of HBV infection. IMPORTANCE Hepatitis B virus (HBV) is an important human pathogen. HBV quasispecies with genetically heterogenous variants are thought to play a role in the progression of HBV-associated liver diseases. So far, direct evidence is available in only a few cases to confirm the proposed role of HBV variants in the pathogenesis. We report here that the coexistence of two naturally occurring HBV variants at a ratio of 1 to 4 increased HBV replication and induced significantly stronger intrahepatic cytotoxic T lymphocyte responses and antibody responses specific to HBV surface antigen (HBsAg) in mice. Our discovery uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses and may enhance immune-mediated liver damage under some circumstances, representing a novel mechanism for the immunopathogenesis of HBV infection.

DNA Prime — Adenovirus Boost Immunization Induces a Vigorous and Multifunctional T-cell response against Hepadnaviral Proteins in the Mouse and Woodchuck Model

ABSTRACT Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8+ T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.