Baoju Wang

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.

Immunostaining of PD-1/PD-Ls in liver tissues of patients with hepatitis and hepatocellular carcinoma

AIM To investigate the expression of programmed death (PD)-1, PD ligand 1 (PD-L1) and PD-L2 in liver tissues in the context of chronic hepatitis and hepatocellular carcinoma (HCC). METHODS Liver biopsies and HCC specimens from patients were collected and histologically examined. The expression of PD-1, PD-L1, and PD-L2 in biopsy specimens of chronic hepatitis and HCC specimens was evaluated by immunohistochemical staining. The association between the expression level of PD-1, PD-L1, and PD-L2 and clinical and pathological variables was analyzed statistically. RESULTS Expression of PD-1 was found in liver-infiltrating lymphocytes. In contrast, PD-L1 and PD-L2 were expressed in non-parenchyma liver cells and tumor cells. The expression of PD-L1 was significantly correlated with hepatitis B virus infection (1.42 ± 1.165 vs 0.50 ± 0.756, P = 0.047) and with the stage of HCC (7.50 ± 2.121 vs 1.75 ± 1.500 vs 3.00 ± 0.001, P = 0.018). PD-1 and PD-Ls were significantly up-regulated in HCC specimens (1.40 ± 1.536 vs 5.71 ± 4.051, P = 0.000; 1.05 ± 1.099 vs 4.29 ± 3.885, P = 0.004; 1.80 ± 1.473 vs 3.81 ± 3.400, P = 0.020). CONCLUSION PD-L1 may contribute to negative regulation of the immune response in chronic hepatitis B. PD-1 and PD-Ls may play a role in immune evasion of tumors.

Improvement of inflammatory responses associated with NF-κB pathway in kidneys from diabetic rats

Abstract.Objective:The effects of fenofibrate on the kidneys of diabetic rats were investigated by measuring the inflammatory responses associated with transcription factor nuclear factor kappa-B (NF-κB) pathway.Methods:Male Wistar rats were randomly divided into 3 groups: normal control, diabetes control and diabetes + fenofibrate (10 in each group). The expression of NF-κB p65, plasminogen activator inhibitor-1 (PAI-1), and intercellular adhesion molecule-1 (ICAM-1) in renal cortex was detected by Western blot, RT-PCR, and immunohistochemistry, respectively. Blood lipid profiles, glucose, and urine albumin were measured as well.Results:The expression of NF-kB p65, PAI-1, and ICAM-1 was significantly higher in the diabetes control than those in the normal control, and treatment with fenofibrate inhibited the increased expression of these factors in kidneys by 48.18 %, 35.04 %, and 26.41 %, respectively when compared with the diabetes control, although they were still higher in diabetes + fenofibrate than those in the normal control. Correspondingly, the profiles of lipid were significantly elevated in the diabetes control compared with the normal control, and decreased significantly in diabetes + fenofibrate.Conclusions:Fenofibrate exhibited a downregulating effect on the NF-κB pathway in diabetic kidneys, implying that fenofibrate could be a potential treatment for diabetic nephropathy.

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.

Isothiafludine, a novel non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation.

Aim:To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo.Methods:HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg−1·d−1) for 15 d.Results:NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 μmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 μmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, 20 μmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks.Conclusion:NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.

The expression of PD-1 ligands and their involvement in regulation of T cell functions in acute and chronic woodchuck hepatitis virus infection.

Background The programmed cell death 1 (PD-1)/programmed death-1 ligand 1 (PD-L1) system may play a role in the negative regulation of T cell functions in hepatitis B virus (HBV) infection. Thus, it is important to study its role in the widely used animal model for HBV infection of woodchucks with woodchuck hepatitis virus (WHV). Methods Woodchuck PD-L1 (wPD-L1) and -L2 (wPD-L2) were cloned and characterized. The levels of wPD-L1 expression in primary woodchuck hepatocytes (PWH), peripheral blood mononuclear cells (PBMCs), and liver tissue of naive and WHV-infected woodchucks were examined by real time reverse transcription (RT)-PCR and flow cytometry. Using antibodies against wPD-L1 and -L2, the effect of blocking PD-1/PD-L1/PD-L2 interaction on the proliferation and degranulation of woodchuck PBMCs was examined. Principal Findings Both wPD-L1 and -L2 showed a high homology to their counterparts of other mammalian species and humans. WPD-L1 expression in PWH and PBMCs of naive animals was low but could be stimulated by Toll-like receptor (TLR) ligands and interferons (IFN). WPD-L1 expression in liver tissue was significantly higher than that measured in PWHs and was slightly elevated during acute and chronic WHV infection. However, wPD-1 and wPD-L1 expression on PBMCs was strongly up-regulated during acute and chronic infection. In vitro blockade with antibodies against wPD-L1 and -L2 partially enhanced proliferation and degranulation of PBMCs from WHV-infected woodchucks. Conclusions Our results demonstrated that wPD-1/wPD-L1 expression in hepatocytes and PBMCs can be induced by different inflammatory stimuli and is up-regulated mainly on PBMCs during WHV infection. A blockade of the woodchuck PD-1/PD-L pathway could partially enhance T cell functions in WHV infection.

An Unenumerative DNA Computing Model for Vertex Coloring Problem

The solution space exponential explosion caused by the enumeration of the candidate solutions maybe is the biggest obstacle in DNA computing. In the paper, a new unenumerative DNA computing model for graph vertex coloring problem is presented based on two techniques: 1) ordering the vertex sequence for a given graph in such a way that any two consecutive labeled vertices i and i+1 should be adjacent in the graph as much as possible; 2) reducing the number of encodings representing colors according to the construture of the given graph. A graph with 12 vertices without triangles is solved and its initial solution space includes only 283 DNA strands, which is 0.0532 of 312 (the worst complexity).

Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance

Summary.  Seroconversion from hepatitis B surface antigen (HBsAg) to antibodies against HBsAg (anti‐HBs) usually indicates resolution of hepatitis B virus (HBV) infection. Here, two HBV‐infected patients with seroconversion to anti‐HBs were found to be persistently positive for HBeAg and HBV DNA. Immunohistology of liver biopsies confirmed the expression of HBV proteins in the liver of one patient. The neutralizing ability of anti‐HBs in patient sera was demonstrated by blocking HBV infection of primary tupaia hepatocytes. Analysis of the HBsAg‐encoding region of HBV isolates from patients indicated the coexistence of heterogeneous HBV genomes in patients. The majority of recombinant variant HBsAg was reactive in HBsAg assays and was able to bind to anti‐HBs. Circulating immune complexes (CIC) of HBsAg in patient sera could be detected by polyethylene glycol precipitation and trypsin digestion. Thus, neutralizing anti‐HBs may appear in chronic HBV carriers for long periods but does not necessarily lead to complete viral clearance.

Establishing a new animal model for hepadnaviral infection: susceptibility of Chinese Marmota-species to woodchuck hepatitis virus infection

Hepatitis B virus infection (HBV) is a major medical problem in China. The lack of a suitable infection model in China is recognized as an obstacle for research on HBV in China. Chinese Marmota-species is phylogenetically closely related to Marmota monax, thus, it might be suitable to serve as an animal model for HBV infection. Therefore, we attempted to prove the claim about the existence of woodchuck hepatitis virus (WHV)-like viruses in Chinese Marmota-species and to determine the susceptibility of these species to experimental WHV infection. In the present study, 653 sera from three Chinese Marmota-species, Marmota himalayana, Marmota baibacina and Marmota bobak, were screened for WHV-like viruses by serological and molecular assays. The susceptibility to WHV of three species was investigated by experimental infection and monitored by testing of anti-WHc and WHsAg by ELISA, detection of WHV DNA by PCR, and detection of WHV replication intermediates and antigens in liver samples. No evidence for the existence of a genetically closely related virus to WHV in three Chinese Marmota-species was found by serological assays and PCR. M. himalayana was susceptible to WHV infection as inoculated animals became positive for anti-WHc, WHsAg and WHV DNA. Further, WHV replication intermediates and proteins were detected in liver samples. In contrast, M. baibacina remained negative for tested virological parameters. M. bobak species showed a limited susceptibility to WHV. Our data do not support early reports about WHV-like viruses in China. M. himalayana is suitable for the establishment of a model for hepadnaviral infection.

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.