Xin Zheng

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.

Targeting Murine Immune Responses to Selected T Cell- or Antibody-Defined Determinants of the Hepatitis B Surface Antigen by Plasmid DNA Vaccines Encoding Chimeric Antigen

By exchanging sequences from the middle-surface (MS) and small-surface (S) Ag of hepatitis B virus (HBV) with corresponding sequences of the MS Ag of woodchuck hepatitis virus, we constructed chimeric MS variants. Using these constructs as DNA vaccines in mice, we selectively primed highly specific (non-cross-reactive) Ab responses to pre-S2 of the HBV MS Ag and the “a” determinant of the HBV S Ag, as well as Ld- or Kb-restricted CTL responses to HBV S epitopes. In transgenic mice that constitutively express large amounts of HBV surface Ag in the liver we could successfully suppress serum antigenemia (but not Ag production in the liver) by adoptive transfer of anti-pre-S2 or anti-“a” immunity but not CTL immunity. DNA vaccines greatly facilitate construction of chimeric fusion Ags that efficiently prime specific, high-affinity Ab and CTL responses. Such vaccines, in which sequences of an Ag of interest are exchanged between different but related viruses, are interesting tools for focusing humoral or cellular immunity on selected antigenic determinants and elucidating their biological role.

Antiviral therapy for chronic hepatitis B in China

Abstract The vaccination program against hepatitis B virus (HBV) has greatly reduced the incidence of HBV infection. However, almost one-fourth of the HBV infected patients worldwide are still located in China. The healthcare burden from chronic HBV infection is a big challenge for the Chinese government and clinicians. Antiviral therapy plays a central role in controlling chronic HBV infection and preventing the disease progression. However, due to the specific economic and medical system issues, the first-line antiviral agents recommended by the AASLD and EASL have not been widely used for Chinese patients. In this review, we will discuss some key issues in the area of antiviral treatment for chronic hepatitis B in China.

Inhibition of hepatitis B virus (HBV) gene expression and replication by HBx gene silencing in a hydrodynamic injection mouse model with a new clone of HBV genotype B

BackgroundIt has been suggested that different hepatitis B virus (HBV) genotypes may have distinct virological characteristics that correlate with clinical outcomes during antiviral therapy and the natural course of infection. Hydrodynamic injection (HI) of HBV in the mouse model is a useful tool for study of HBV replication in vivo. However, only HBV genotype A has been used for studies with HI.MethodsWe constructed 3 replication-competent clones containing 1.1, 1.2 and 1.3 fold overlength of a HBV genotype B genome and tested them both in vitro and in vivo. Moreover, A HBV genotype B clone based on the pAAV-MCS vector was constructed with the 1.3 fold HBV genome, resulting in the plasmid pAAV-HBV1.3B and tested by HI in C57BL/6 mice. Application of siRNA against HBx gene was tested in HBV genotype B HI mouse model.ResultsThe 1.3 fold HBV clone showed higher replication and gene expression than the 1.1 and 1.2 fold HBV clones. Compared with pAAV-HBV1.2 (genotype A), the mice HI with pAAV-HBV1.3B showed higher HBsAg and HBeAg expression as well as HBV DNA replication level but a higher clearance rate. Application of two plasmids pSB-HBxi285 and pSR-HBxi285 expressing a small/short interfering RNA (siRNA) to the HBx gene in HBV genotype B HI mouse model, leading to an inhibition of HBV gene expression and replication. However, HBV gene expression may resume in some mice despite an initial delay, suggesting that transient suppression of HBV replication by siRNA may be insufficient to prevent viral spread, particularly if the gene silencing is not highly effective.ConclusionsTaken together, the HI mouse model with a HBV genotype B genome was successfully established and showed different characteristics in vivo compared with the genotype A genome. The effectiveness of gene silencing against HBx gene determines whether HBV replication may be sustainably inhibited by siRNA in vivo.

Susceptibility of Different Hepatitis B Virus Isolates to Interferon-Alpha in a Mouse Model Based on Hydrodynamic Injection

Interferon alpha (IFN-α) is commonly used for the treatment of chronic hepatitis B (CHB) patients. Many factors including viral genetics may determine the outcome of IFN-α therapy. In this study, we tested whether the expression of IFN-α directly in the liver inhibits HBV gene expression and replication using a HBV hydrodynamic injection (HI) mouse model. Two replication-competent clones from different HBV isolates that belonging to HBV genotype A and B based on a pAAV vector (pAAV-HBV-A and pAAV-HBV-B) were compared for their susceptibility to IFN-α. HBV clones were injected into mice either alone or in combination with a murine (m) IFN-α expression plasmid (pmIFN-α). HBsAg and HBeAg concentrations and HBV DNA levels in mice differed after injection of these two HBV clones. Co-application of pmIFN-α together with the two distinct isolates resulted in markedly different kinetics of decline of HBsAg, HBeAg, and HBV DNA levels in the mice. Immunohistochemical staining of liver sections with anti-HBc showed that mIFN-α application completely inhibited the expression of HBcAg in mice inoculated with pAAV-HBV-B, whereas the expression of HBcAg was only reduced in mice with pAAV-HBV-A. Consistently, mice injected with pAAV-HBV-B and pmIFN-α showed higher expression levels of the IFN-stimulated genes (ISGs) ISG15, OAS, PKR as well as proinflammatory cytokine IL-6 in the liver. In addition, expression levels of anti-inflammatory cytokine IL-10 was down-regulated significantly in liver of the mice injected with pAAV-HBV-B and pmIFN-α. Our data demonstrate that IFN-α exerts antiviral activity in HBV mouse model, but different HBV isolates may have diverse susceptibility to IFN-α.

Immunosuppressive drugs modulate the replication of hepatitis B virus (HBV) in a hydrodynamic injection mouse model.

Hepatitis B virus (HBV) reactivation and recurrence are common in patients under immunosuppression and can be controlled by hepatitis B immunoglobulin, antivirals, and hepatitis B vaccine. However, the detailed analysis of HBV infection under immunosuppression is essential for the prophylaxis and therapy for HBV reactivation and recurrence. In this study, HBV replication and T cell responses were analyzed in a HBV-transfected mouse model under immunosuppressive therapy. During the treatment, HBV replication was at a high level in mice treated with dexamethasone, cyclosporine, and cyclophosphamide, whereas was terminated in mice treated with mycophenolate mofetil. After the withdrawal, HBV replication was at low or high levels in the dexamethasone-treated mice or in both cyclosporine- and cyclophosphamide-treated mice. The early withdrawal of cyclosporine allowed the recovery of suppressed T cell responses and led to subsequent HBV clearance, while the adoptive immune transfer to the mice with HBV persistence led to HBV suppression. Taken together, long-term HBV persistence under immunosuppression depends on the immunosuppressive drugs used and on the treatment duration and is mediated by the suppressed intrahepatic CD8 T cell response. These data may be helpful for individualized immunosuppressive therapy in patients with high risk of HBV reactivation and recurrence, and the mouse system is suitable for studying HBV reactivation and recurrence under immunosuppression.

Decreased monocyte subsets and TLR4-mediated functions in patients with acute severe fever with thrombocytopenia syndrome (SFTS)

OBJECTIVES The role of a newly discovered bunyavirus, the severe fever with thrombocytopenia syndrome virus (SFTSV), in the pathogenesis of severe fever with thrombocytopenia syndrome (SFTS) is poorly understood. In this study, it was hypothesized that peripheral monocytes, which are constantly exposed to viral infection in the blood, are likely targeted by the causative virus in SFTS patients. METHODS Fifty-three patients and 25 healthy volunteers were enrolled in the study. Monocyte counts in the peripheral blood of all human subjects were monitored throughout the progress of the disease. SFTSV viral load and the expression of monocyte genes were investigated by real-time RT-PCR. Cytokine production of monocytes in SFTS patients upon lipopolysaccharide (LPS) stimulation was examined by ELISA. RESULTS In comparison to SFTS patients in the convalescent stage and healthy controls, monocyte cell counts and percentages in patients at the acute stage were significantly lower. Decreased monocyte cell counts and subsets were positively correlated with SFTSV viral loads in the serum samples from SFTS patients. Despite their higher basal toll-like receptor 4 (TLR4) expression, monocytes from patients in the acute phase were shown to be compromised regarding the production of tumor necrosis factor alpha, but not interleukin 10, upon LPS stimulation. CONCLUSIONS These data strongly suggest that monocytes could be a major target during SFTSV infection. The decreased population and dysfunction of monocytes in acute SFTS patients may contribute to the disease severity.

Decreased myeloid dendritic cells indicate a poor prognosis in patients with severe fever with thrombocytopenia syndrome

OBJECTIVES Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by a novel bunyavirus in which host immune system suppression is thought to be crucial in the development of disease. This study was designed to study the frequencies and activation status of dendritic cells (DCs) at different stages of SFTS and their association with disease severity. METHODS All confirmed SFTS patients (N=115) were recruited from the Wuhan Union Hospital in 2015; routine laboratory parameters were collected. The frequencies, phenotypes, and subsets of circulating DCs, including myeloid and plasmacytoid dendritic cells (mDCs and pDCs), were analyzed by flow cytometry. Serum levels of interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNF-α) were detected by ELISA. The laboratory parameters and other clinical events related to mortality were assessed by multivariate logistic regression analysis and receiver operating characteristic (ROC) curves. RESULTS The frequency of circulating mDCs, especially from day 9 after disease onset, could serve as a valuable prognostic biomarker for the outcome in SFTS patients (area under the curve=0.929, p<0.0001). In addition, persistent down-regulation of the co-stimulatory molecules CD80/CD86 on the mDCs was observed during the disease process. Moreover, levels of mDCs were inversely correlated with the production of IL-6, IL-10, and TNF-α and with viral load at admission. CONCLUSIONS The present results indicate that DCs might be functionally impaired in SFTS. A decreased level of circulating mDCs was closely correlated with the severity of SFTS.

A simple and practical score model for predicting the mortality of severe fever with thrombocytopenia syndrome patients.

AbstractSevere fever with thrombocytopenia syndrome (SFTS) is an emerging disease with a high fatality rate. The risk factors for death are not clearly identified, and there is no clinical score model to predict the prognosis. We retrospectively collected the clinical information of clinical symptoms and laboratory parameters of SFTS patients on admission. After analyzing the clinical characteristics of 179 SFTS patients, we found that an elevated level of neurologic symptoms, respiratory symptoms, viral load, and a lower level of monocyte percentage were the critical risk factors for mortality. We used the 4 variables to assemble a score formula named the SFTS index [SFTSI = 5 × Neurologic symptoms-level + 4 × Respiratory symptoms-level + 3 × LG10 Viral load – 2 × LN Monocyte% – 7]. The AURC of this model was 0.964, which was higher than the AURC 0.913 of the viral load especially among the patients with higher viral loads (0.936 vs 0.821). We identified that the neurologic symptoms, respiratory symptoms, viral load, and monocyte percentage were the critical risk factors for SFTS mortality. The clinical score model of SFTSI provides a practical method for clinicians to stratify patients with SFTS and to adopt prompt effective treatment strategies.

Multidrug resistance protein 4 is a critical protein associated with the antiviral efficacy of nucleos(t)ide analogues.

Multidrug resistance protein 4 (MRP4) has been associated with nucleos(t)ide analogue (NA) antiretroviral therapy failure, though is unclear if MRP4 is also correlated with the failure of anti‐hepatitis B virus (HBV) therapy.