Dong-Hua Zhang

N-glycosylation mutations within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape

BACKGROUND & AIMS HBV immune escape represents a challenge to prevention, diagnosis, and treatment of hepatitis B. Here, we analyzed the molecular and clinical characteristics of HBV immune escape mutants in a Chinese cohort of chronically infected patients. METHODS Two hundred sixteen patients with HBsAg and anti-HBs were studied, with one hundred eighty-two HBV carriers without anti-HBs as a control group. Recombinant HBsAg bearing the most frequent N-glycosylation mutations were expressed in CHO and HuH7 cells. After confirming N-glycosylation at the most frequent sites (129 and 131), together with inserted mutations, functional analysis were performed to study antigenicity and secretion capacity. RESULTS One hundred twenty-three patients had the wild-type HBs gene sequence, 93 patients (43%) had mutants in the major hydrophilic region (MHR), and 47 of the 93 patients had additional N-glycosylation mutations, which were transmitted horizontally to at least 2 patients, one of whom was efficiently vaccinated. The frequency of N-glycosylation mutation in the case group was much higher than that of the control group (47/216 vs. 1/182). Compared with wild-type HBsAg, HBsAg mutants reacted weakly with anti-HBs using a chemiluminescent microparticle enzyme immunoassay. Native gel analysis of secreted virion in supernatants of transfected HuH7 cells indicated that mutants had better virion enveloping and secretion capacity than wild-type HBV. CONCLUSIONS Our results suggest that specific HBsAg MHR N-glycosylation mutations are implicated in HBV immune escape in a high endemic area.

Clinical and molecular characterization of Wilson's disease in China: identification of 14 novel mutations

BackgroundWilsons disease (WND) is a rare autosomal recessive disorder. Here we have evaluated 62 WND cases (58 probands) from the Chinese Han population to expand our knowledge of ATP7B mutations and to more completely characterize WND in China.MethodsThe coding and promoter regions of the ATP7B gene were analyzed by direct sequencing in 62 Chinese patients (58 probands) with WND (male, n = 37; female, n = 25; age range, 2 ~ 61 years old).ResultsNeurologic manifestations were associated with older age at diagnosis (p < 0.0001) and longer diagnostic delay (p < 0.0001). Age at diagnosis was also correlated with urinary copper concentration (r = 0.58, p < 0.001). Forty different mutations, including 14 novel mutations, were identified in these patients. Common mutations included p.Arg778Leu (31.9%) and p.Pro992Leu (11.2%). Homozygous p.Arg778Leu and nonsense mutation/frameshift mutations were more often associated with primary hepatic manifestations (p = 0.0286 and p = 0.0383, respectively) and higher alanine transaminase levels at diagnosis (p = 0.0361 and p = 0.0047, respectively). Nonsense mutation/frameshift mutations were also associated with lower serum ceruloplasmin (p = 0.0065).ConclusionsWe identified 14 novel mutations and found that the spectrum of mutations of ATP7B in China is quite distinct from that of Western countries. The mutation type plays a role in predicting clinical manifestations. Genetic testing is a valuable tool to detect WND in young children, especially in patients younger than 8 years old. Four exons (8, 12, 13, and 16) and two mutations (p.Arg778Leu, p.Pro992Leu) should be considered high priority for cost-effective testing in China.

Association study of IFNAR2 and IL10RB genes with the susceptibility and interferon response in HBV infection.

Summary.  A recent genome‐wide association study discovered that two polymorphisms, interferon (IFN) alpha receptor 2 (IFNAR‐2) F8S and interleukin 10 receptor (IL10RB) K47E, were associated with susceptibility to hepatitis B virus (HBV) infection in Africa. Here, we reevaluate the effects of the two polymorphisms on HBV susceptibility in the Chinese Han population, and extended the study to look at their association with IFN response in chronic hepatitis B (CHB). We included 341 patients with CHB and 341 unrelated controls presenting with asymptotic HBV self‐limited infection, who were well matched in age and sex. In the CHB group, 101 patients had been treated with peg‐IFN‐alpha‐2a for 48 weeks and followed up for 24 weeks to determine the clinical response, resulting 34 individuals with sustained virological response (SVR) and 67 individuals with nonsustained response (NR). Subgroups in the CHB group were divided according to the viral loads, HBeAg and maternal HBsAg status. The association with the susceptibility to HBV infection was only observed for IL10RB K47E when we compared the individuals with persistent HBV infection through nonmaternal transmission to the controls with asymptomatic self‐limited HBV infection. Further, we found that the IFNAR2‐8SS genotype was associated with HBeAg negative patients (OR = 0.316, 95% CI: 0.121–0.825, P = 0.019) and that the IFNAR2‐8F allele was associated with the risk to high viral loads (OR = 1.667, 95% CI: 1.148–2.420, P = 0.007). In addition, the IFNAR2‐8FF genotype predisposed to higher MxA gene induction and correlated with sustained IFN response (OR = 0.348, 95% CI: 0.129–0.935, P = 0.036). Haplotype analysis based on polymorphisms of three single‐nucleotide polymorphisms, MxA −88 G/T, IFNAR‐2 F8S and IL10RB K47E showed that the haplotype distribution was significantly different between the SVR and NR groups (P = 0.040). This study suggests that IFNAR2 may play an important role in determining IFN response and clinical phenotypes of HBV infection in the Chinese Han population.

Serum WFA+‐M2BP levels for evaluation of early stages of liver fibrosis in patients with chronic hepatitis B virus infection

Accurate evaluation of liver fibrosis is crucial for predicting progression of chronic hepatitis B virus (HBV) infection. We assessed the utility of a novel fibrosis glycobiomarker Wisteria floribunda agglutinin‐positive Mac‐2‐binding protein (WFA+‐M2BP) for evaluating liver fibrosis and disease progression in patients with chronic HBV infection.

Assessment of Specific Antibodies to F Protein in Serum Samples from Chinese Hepatitis C Patients Treated with Interferon plus Ribavarin

ABSTRACT The hepatitis C virus (HCV) alternate reading frame protein or F protein of the HCV 1b genotype is a double-frameshift product of the HCV core protein. In order to assess the presence of antibodies specific for F protein and their clinical relevance in sera from HCV patients, we produced recombinant F protein and core protein of the HCV 1b genotype in Escherichia coli. An enzyme-linked immunosorbent assay was developed using purified recombinant HCV core, F protein, and a 99-residue synthetic F peptide (F99). The seroprevalences of anticore, anti-F protein, and anti-F99 synthetic peptide were 95%, 68%, and 36%, respectively, in 168 HCV patients. The prevalence of anti-F antibodies did not correlate with viral load, genotype, or alanine aminotransferase level. Interferon combination therapy induced a decline in the level of anti-F antibodies in 55 responders (P < 0.01). Thirteen responders (24%) lost their anti-F recombinant protein antibodies, and 17 (31%) lost their anti-F synthetic peptide antibodies, whereas no decrease was observed for the 17 nonresponders. These changes were significant between responders and nonresponders (P < 0.05). Meanwhile, no change was found in the anticore antibody titer of the 72 treated patients. The percentage of anti-F-protein-negative patients (15/15 [100%]) who achieved a sustained virological response (SVR) was higher than that of the anti-F-positive patients (70%) (P < 0.05). Based on these findings, HCV F protein elicits a specific antibody response other than the anticore protein response. Our data also suggest that the presence and level of anti-F antibody responses might be influenced by the treatment (interferon plus ribavirin) and associated with an SVR in Chinese hepatitis C patients.

Early serum hepatitis B virus large surface protein level: a stronger predictor of virological response to peginterferon alfa-2a than that to entecavir in HBeAg-positive patients with chronic hepatitis B.

BACKGROUND The response rate to antiviral therapy varies greatly among individuals, and its prediction is still very challenging. OBJECTIVES To evaluate the usefulness of serum hepatitis B virus large surface protein (LHBs) levels compared with HBsAg in prediction of the antiviral treatment effect. STUDY DESIGN Quantification of LHBs, HBsAg and HBV DNA was carried out at baseline and during antiviral therapy (weeks 4, 12, 24, 36 and 48) in HBeAg-positive patients treated with peginterferon alfa-2a (n = 21) or entecavir (n = 41). RESULTS The serum LHBs concentration was correlated positively with HBV DNA and HBsAg (r = 0.635 and 0.588, respectively). LHBs and HBV DNA levels decreased significantly in a biphasic manner and HBsAg level tended to decrease slowly in both treatment groups. In peginterferon alfa-2a group, the cutoff of 88.46 ng/ml in serum LHBs at week 4 gave the best AUC ( = 0.96) with positive and negative predictive values of 88.9% and 100%, in association with virological response (VR). Serum LHBs level at week 4 also showed an association with VR in entecavir group (AUC 0.78). The predictive model incorporating LHBs, HBsAg and HBV DNA could discriminate VR at baseline (AUC 0.79) and showed an association with serological response (SR) at week 12 (AUC 0.80) in peginterferon alfa-2a group. CONCLUSIONS On-treatment quantification of serum LHBs may be a more useful parameter for predicting VR in patients on peginterferon alfa-2a than those on entecavir. Combining LHBs, HBsAg and HBV DNA can predict VR and SR more effectively and earlier.

Characterization of the Specific CD4+ T Cell Response against the F Protein during Chronic Hepatitis C Virus Infection

Background The hepatitis C virus (HCV) Alternate Reading Frame Protein (ARFP or F protein) presents a double-frame shift product of the HCV core gene. We and others have previously reported that the specific antibodies against the F protein could be raised in the sera of HCV chronically infected patients. However, the specific CD4+ T cell responses against the F protein during HCV infection and the pathological implications remained unclear. In the current study, we screened the MHC class II-presenting epitopes of the F protein through HLA-transgenic mouse models and eventually validated the specific CD4+ T cell responses in HCV chronically infected patients. Methodology DNA vaccination in HLA-DR1 and-DP4 transgenic mouse models, proliferation assay to test the F protein specific T cell response, genotyping of Chronic HCV patients and testing the F-peptide stimulated T cell response in the peripheral blood mononuclear cell (PBMC) by in vitro expansion and interferon (IFN)- γ intracellular staining. Principal Findings At least three peptides within HCV F protein were identified as HLA-DR or HLA-DP4 presenting epitopes by the proliferation assays in mouse models. Further study with human PBMCs evidenced the specific CD4+ T cell responses against HCV F protein as well in patients chronically infected with HCV. Conclusion The current study provided the evidence for the first time that HCV F protein could elicit specific CD4+ T cell response, which may provide an insight into the immunopathogenesis during HCV chronic infection.

Characterization of gene expression profiles in HBV-related liver fibrosis patients and identification of ITGBL1 as a key regulator of fibrogenesis

Although hepatitis B virus (HBV) infection is the leading cause of liver fibrosis (LF), the mechanisms underlying liver fibrotic progression remain unclear. Here, we investigated the gene expression profiles of HBV-related LF patients. Whole genome expression arrays were used to detect gene expression in liver biopsy samples from chronically HBV infected patients. Through integrative data analysis, we identified several pathways and key genes involved in the initiation and exacerbation of liver fibrosis. Weight gene co-expression analysis revealed that integrin subunit β-like 1 (ITGBL1) was a key regulator of fibrogenesis. Functional experiments demonstrated that ITGBL1 was an upstream regulator of LF via interactions with transforming growth factor β1. In summary, we investigated the gene expression profiles of HBV-related LF patients and identified a key regulator ITGBL1. Our findings provide a foundation for future studies of gene functions and promote the development of novel antifibrotic therapies.

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

ABSTRACT Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.

A novel noninvasive algorithm for the assessment of liver fibrosis in patients with chronic hepatitis B virus infection

Several noninvasive blood biomarkers have been established for the assessment of liver fibrosis in patients with chronic hepatitis B virus (HBV) infection, but their clinical performance remains inconclusive. Here, we compared the diagnostic performance of these biomarkers and developed a novel algorithm for assessing liver fibrosis. Six hundred and sixteen chronically HBV‐infected and treatment‐naïve patients who underwent liver biopsy were enrolled and randomly divided into training (N=410) and internal validation cohorts (N=206). One hundred and fifty‐nine patients from another centre were recruited as an external validation cohort. Receiver operating characteristic (ROC) curves were used to analyse the performance of the gamma‐glutamyltransferase‐to‐platelet ratio (GPR), red cell volume distribution width‐to‐platelet ratio (RPR), FIB‐4 index, aspartate aminotransferase‐to‐platelet ratio index (APRI) and HBV DNA level against liver histology, and a novel algorithm was developed using the recursive partitioning and regression tree (RPART) method. In the training cohort, the area under the ROC curve of FIB‐4 was significantly higher than that of APRI (P=.038) but was comparable to those of GPR, RPR and HBV DNA; however, the performance of the biomarkers was similar among the validation cohort. The established RPR‐HBV DNA algorithm performed better in the training cohort than any individual blood biomarker, and the corresponding sensitivity, specificity, positive predictive value and negative predictive value were 63%, 90%, 72% and 80%, respectively. In the internal and external validation cohorts, the performance of the algorithm in assessing liver fibrosis was also superior to that of other biomarkers. These results suggest that the established RPR‐HBV DNA algorithm might improve the diagnostic accuracy of liver fibrosis in treatment‐naïve patients with chronic HBV infection, although additional studies are warranted to confirm these findings.