Xingwang Xie

In vitro inhibition of HBV replication by a novel compound, GLS4, and its efficacy against adefovir-dipivoxil-resistant HBV mutations.

BACKGROUND HBV infection continues to be an important worldwide cause of morbidity and mortality. Patients with chronic hepatitis B can be successfully treated using nucleoside/nucleotide analogues. However, drug-resistant HBV mutants frequently arise, leading to treatment failure and progression to liver disease. Here, we report the effects of GLS4, a non-nucleosidic inhibitor that exhibits a novel and highly specific anti-HBV activity. METHODS The median inhibitory concentrations (IC(50)s) of GLS4 on HBV were measured by Southern blotting. HBV capsid and core protein levels were detected by immunoblotting. To determine the antiviral activity of GLS4 against adefovir dipivoxil (ADV)-resistant HBV mutants, HepG2 cells transiently transfected with PUC-HBV1.2 plasmids that contained one of three major ADV-resistant mutations (rtA181T, rtA181V and rtN236T) were treated with GLS4. Intracellular HBV replicative intermediates were detected by Southern blotting. The effect on the in vitro assembly of HBV capsid protein was examined using dynamic light scattering and electron microscopy. RESULTS The IC(50) of GLS4 was 0.012 μM, which is significantly lower than that of lamivudine (0.325 μM). Immunoblot analysis of HepG2.2.15 cells and transiently transfected HepG2 cells indicated that GLS4 treatment interfered with the formation of core particles (assembly). The ADV-resistant HBV mutant strains were also sensitive to GLS4. Upon examining the in vitro assembly of HBV core protein 149 by electron microscopy, increased aberrant particles were observed after GLS4 treatment. CONCLUSIONS GLS4 is a new and unique potential anti-HBV agent that possesses a different mechanism of action than existing therapeutic drugs.

Peritumoural neutrophils negatively regulate adaptive immunity via the PD-L1/PD-1 signalling pathway in hepatocellular carcinoma

BackgroundPD-L1 expression on neutrophils contributes to the impaired immune response in infectious disease, but the detailed role of PD-L1 expression on neutrophils in HCC remains unclear.MethodsWe investigated the phenotype and morphology of neutrophils infiltrated in tumour tissues from both patients with HCC and hepatoma-bearing mice.ResultsWe found that neutrophils dominantly infiltrated in the peritumoural region. The neutrophil-to-T cell ratio (NLR) was higher in peritumoural tissue than that in the intratumoural tissue and was negatively correlated with the overall survival of patients with HCC. Infiltrating neutrophils displayed a phenotype of higher frequency of programmed cell death ligand 1 (PD-L1) positive neutrophils. The ratio of PD-L1+ neutrophils-to-PD-1+ T cells was higher in peritumoural tissue and better predicted the disease-free survival of patients with HCC. We further confirmed a higher frequency of PD-L1+ neutrophils and PD-1+ T cells in hepatoma-bearing mice. Functionally, the PD-L1+ neutrophils from patients with HCC effectively suppressed the proliferation and activation of T cells, which could be partially reversed by the blockade of PD-L1.ConclusionsOur results indicate that the tumour microenvironment induces impaired antitumour immunity via the modulation of PD-L1 expression on tumour infiltrating neutrophils.

FOXP3 gene polymorphism is associated with hepatitis B-related hepatocellular carcinoma in China

BackgroundPrevious evidence has shown that the FOXP3 gene was involved in the pathogenesis of several tumors; however, the correlation between single nucleotide polymorphisms (SNPs) in the FOXP3 gene and the susceptibility to hepatitis B-related hepatocellular carcinoma (HCC) remains unclear.MethodsWe analyzed two SNPs in the FOXP3 gene, rs2280883 and rs3761549, in 392 patients with HCC, 344 patients with chronic hepatitis B (CHB) and 372 matched healthy controls. Genotyping was performed by MALDI-TOF Mass Spectrometry for all donors.ResultsCompared to healthy controls, HCC patients had higher frequencies of the TT genotype (79.6%) at rs2280883 and the CC genotype (77.6%) at rs3761549 of the FOXP3 gene; CHB patients also had higher frequencies of the TT genotype (74.1%) at rs2280883 and the CC genotype (74.6%) at rs3761549. There were no significant differences in the distribution of FOXP3 genotypes between CHB donors and HCC donors. The TT genotype at rs2280883 was more frequent in patients with HCC than healthy donors (P = 0.01), but no significant difference was observed in this genotype between CHB and healthy donors (P = 0.479). C allele frequency at rs3761549 was higher in HCC patients than healthy donors (P = 0.03), but distribution of this allele was not significantly different between CHB patients and healthy donors (P = 0.11). Stratified analysis showed that the CC genotype at rs3761549 was significantly associated with a high incidence of portal vein tumor thrombus (P = 0.02) and that the TT/CT genotype at rs3761549 was significantly associated with an increased rate of tumor recurrence in HCC patients (P = 0.001).ConclusionsOur results suggested that the FOXP3 gene polymorphisms at rs2280883 and rs3761549 may be associated with hepatitis B-related HCC. At rs3761549, the CC genotype and the TT/CT genotype were associated with a high incidence of portal vein tumor thrombus and tumor recurrence, respectively.

Deep sequencing of hepatitis B virus basal core promoter and precore mutants in HBeAg-positive chronic hepatitis B patients

Mutants in the basal core promoter (BCP) and precore (PC) regions of hepatitis B virus (HBV) genome are associated with the progression of chronic hepatitis B (CHB) infection. However, quasispecies characteristics of naturally occurring mutants in those regions in HBeAg-positive CHB patients has not been well described, partly limited by quantitative assay. This study aimed to develop an Ion Torrent deep sequencing assay to determine BCP and PC mutant percentages in HBeAg-positive CHB patients who were treatment naïve and correlate them with different viral and host factors. Our results showed that Ion Torrent deep sequencing could achieve high accuracy (R2>0.99) within a dynamic range between 1% and 100%. Twelve hotspots with prevalence of greater than 20% were observed in EnhII/BCP/PC regions. G1719T, T1753V, A1762T and G1764A were genotype C related. BCP A1762T/G1764A double mutants were generally accompanied with PC 1896 wild type or lower PC G1896A mutant percentage. Lower serum HBeAg and HBsAg levels were associated with higher BCP A1762T/G1764A mutant percentages (≥50%). ALT levels were higher in patients with PC G1896A mutant percentage greater than 10%. In conclusion, deep sequencing such as Ion Torrent sequencing could accurately quantify HBV mutants for providing clinical relevant information during HBV infection.

Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection

Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1–6 was determined. We defined a domain comprising amino acids (aa) 192–221, 232–251, 262–281 and 292–331 of E1, and 421–543, 564–583, 594–618 and 634–673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444–463) and PUHI45 (aa 604–618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection.

Direct-acting Antiviral Agents Resistance-associated Polymorphisms in Chinese Treatment-naïve Patients Infected with Genotype 1b Hepatitis C Virus.

Background:It has been reported that several baseline polymorphisms of direct-acting antivirals (DAAs) agents resistance-associated variants (RAVs) would affect the treatment outcomes of patients chronically infected with hepatitis C virus (CHC). The aim of this study is to investigate the prevalence of DAAs RAVs in treatment-naïve GT1b CHC patients. Methods:Direct sequencing and ultra-deep sequencing of the HCV NS3, NS5A, and NS5B gene were performed in baseline serum samples of treatment-naïve patients infected with genotype 1b hepatitis C virus (HCVs). Results:One hundred and sixty CHC patients were studied. Complete sequence information was obtained for 145 patients (NS3), 148 patients (NS5A), and 137 patients (NS5B). Treatment-failure associated variants of DAAs were detected: 56.6% (82/145) of the patients presented S122G for simeprevir (NS3 protease inhibitor); 10.1% (14/148) of the patients presented Y93H for daclatasvir and ledipasvir (NS5A protein inhibitors); 94.2% (129/137) of the patients presented C316N for sofosbuvir (NS5B polymerase inhibitor). Nearly, all of the DAAs RAVs detected by ultra-deep sequencing could be detected by direct sequencing. Conclusions:The majority of genotype 1b CHC patients in China present a virus population carrying HCV DAAs RAVs. Pretreatment sequencing of HCV genome might need to be performed when patients infected with GT1b HCV receiving DAAs-containing regimens in China. Population sequencing would be quite quantified for the work.

Determination of the human antibody response to the neutralization epitopes encompassing amino acids 313-327 and 432-443 of hepatitis C virus E1E2 glycoproteins.

It has been reported that monoclonal antibodies (MAbs) to the E1E2 glycoproteins may have the potential to prevent hepatitis C virus (HCV) infection. The protective epitopes targeted by these MAbs have been mapped to the regionsencompassing amino acids 313–327 and 432–443. In this study, we synthesized these two peptides and tested the reactivity of serum samples from 336 patients, 210 of whichwere from Chronic Hepatitis C (CHC) patients infected with diverse HCV genotypes.The remaining 126 samples were isolated from patients who had spontaneously clearedHCV infection.In the chronic HCV-infected group (CHC group), the prevalence of human serum antibodies reactive to epitopes 313–327 and 432–443was 24.29%(51 of 210) and4.76%(10 of 210),respectively. In thespontaneousclearance group (SC group),the prevalence was 0.79%(1 of 126) and 12.70%(16 of 126), respectively.The positive serum samples that contained antibodies reactive to epitope 313–327 neutralizedHCV pseudoparticles (HCVpp) bearing the envelope glycoproteins of genotypes 1a or 1b and/or 4, but genotypes 2a, 3a, 5 and 6 were not neutralized. The neutralizing activity of these serum samples could not be inhibited by peptide 313–327. Six samples (SC17, SC38, SC86, SC92, CHC75 and CHC198) containing antibodies reactive to epitope 432–443 had cross-genotype neutralizing activities. Theneutralizing activityof SC38, SC86, SC92 and CHC75waspartiallyinhibited by peptide 432–443. However,the neutralizing activity of sample SC17 for genotype 4HCVpp and sample CHC198 for genotype 1b HCVppwere notinhibited by the peptide.This study identifies the neutralizing ability of endogenous anti-HCV antibodies and warrants the exploration of antibodies reactive to epitope432–443as sources for future antibody therapies.

2',3'-cyclic nucleotide 3'-phosphodiesterases inhibit hepatitis B virus replication.

2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV.

The transcription factor RFX5 is a transcriptional activator of the TPP1 gene in hepatocellular carcinoma

Regulatory factor X-5 (RFX5) was previously characterized as an essential and highly specific regulator of major histocompatibility class II (MHCII) gene expression in the immune system. We found that RFX5 is significantly upregulated in hepatocellular carcinoma (HCC) tumors and cell lines compared with non-tumor tissues in mRNA expression levels, but it fails to induce the expression of MHCII. However, RFX5 can strongly bind to the tripeptidyl peptidase 1 (TPP1) promoter region and then increase its transcriptional activity. We also found that manipulation the expression of RFX5 can significantly affect the expression of TPP1 in HepG2, which suggested that RFX5 can transcriptionally activate TPP1 in HCC. Moreover, TPP1 is overexpressed in HCC tissues and significantly correlated with poor prognosis of HCC patients, suggesting that it may have potential biological implications in HCC.

PPPDE1 is a novel deubiquitinase belonging to a cysteine isopeptidase family

Ubiquitinlation of proteins is prevalent and important in both normal and pathological cellular processes. Deubiquitinating enzymes (DUBs) can remove the ubiquitin tags on substrate proteins and dynamically regulate the ubiquitination process. The PPPDE family proteins were predicted to be a novel class of deubiquitinating peptidase, but this has not yet been experimentally proved. Here we validated the deubiquitinating activity of PPPDE1 and revealed its isopeptidase activity against ubiquitin conjugated through Lys 48 and Lys 63. We also identified ribosomal protein S7, RPS7, as a substrate protein of PPPDE1. Moreover, PPPDE1 could mediate the ubiquitin chain editing of RPS7, deubiquitinating Lys 48-linked ubiquitination, and finally stabilize RPS7 proteins. Taken together, we report that PPPDE1 is a novel deubiquitinase that belongs to a cysteine isopeptidase family.