Quantitative hepatitis B core antibody and quantitative hepatitis B surface antigen:Novel viral biomarkers for chronic hepatitis B management

The management of hepatitis B virus(HBV)infection now involves regular and appropriate monitoring of viral activity,disease progression,and treatment response.Traditional HBV infection biomarkers are limited in their ability to predict clinical outcomes or therapeutic effectiveness.Quantitation of HBV core antibodies(qAnti-HBc)is a novel non-invasive biomarker that may help with a variety of diagnostic issues.It was shown to correlate strongly with infection stages,hepatic inflammation and fibrosis,chronic infection exacerbations,and the presence of occult infection.Furthermore,qAnti-HBc levels were shown to be predictive of spontaneous or treatment-induced HBeAg and HBsAg seroclearance,relapse after medication termination,re-infection following liver transplantation,and viral reactivation in the presence of immunosuppression.qAnti-HBc,on the other hand,cannot be relied on as a single diagnostic test to address all problems,and its diagnostic and prognostic potential may be greatly increased when paired with qHBsAg.Commercial qAnti-HBc diagnostic kits are currently not widely available.Because many methodologies are only semi-quantitative,comparing data from various studies and defining universal cut-off values remains difficult.This review focuses on the clinical utility of qAnti-HBc and qHBsAg in chronic hepatitis B management.

Enhancement of CTLs induced by DCs loaded with ubiquitinated hepatitis B virus core antigen

AIM： To investigate whether hepatitis B virus （HBV） could induce a hepatitis B virus core antigen （HBcAg）specific cytotoxic T lymphocyte （CTL） response in vitro by dendritic cells （DCs） transduced with lentiviral vector-encoding ubiquitinated hepatitis B virus core antigen （LV-Ub-HBcAg）.METHODS： Recombinant LV-Ub-HBcAg were transfected into highly susceptible 293 T cells to obtain high virus titres, Bone marrow-derived DCs isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin （IL）-4. LV-Ub-HBcAg, lentiviral vector-encoding hepatitis B virus core antigen （LV-HBcAg）, lentiviral vector （LV） or lipopolysaccharide were added to induce DC maturation, and the DC phenotypes were analyzed by flow cytometry. The level of IL-12 in the supernatant was detected by enzyme-linked immunosorbent assay （ELISA）. T lymphocytes were proliferated using Cell Counting Kit-8. DCs were cultured and induced to mature using different LVs, and co-cultured with allogeneic T cells to detect the secretion levels of IL-2, IL-4, IL-10and interferon-γ in the supernatants of T cells by ELISA. Intracellular cytokines of proliferative T cells were analyzed by flow cytometry, and specific CTL activity was measured by a lactate dehydrogenase release assay.RESULTS： LV-Ub-HBcAg-induced DCs secreted more IL-12 and upregulated the expression of CD80, CD86 and major histocompatibility class ]I, DCs sensitised by different LVs effectively promoted cytokine secretion; the levels of IL-2 and interferon-y induced by LV-Ub- HBcAg were higher than those induced by LV-HBcAg, Compared with LV-HBcAg-transduced DCs, LV-Ub- HBcAg-transduced DCs more efficiently stimulated the proliferation of T lymphocytes and generated HBcAgspecific cytotoxic T lymphocytes.CONCLUSION： LV-Ub-HBcAg effectively induced DC maturation. The mature DCs efficiently induced T cell polarisation to Thl and generated HBcAg-specific CTLs.

Serum hepatitis B core-related antigen as a surrogate marker of hepatitis B e antigen seroconversion in chronic hepatitis B

BACKGROUND Quantitative hepatitis B core-related antigen(qHBcrAg)has a better correlation with intrahepatic hepatitis B virus(HBV)covalently closed circular DNA(cccDNA)than HBV DNA or hepatitis B e antigen(HBeAg),but data are still lacking for its clinical application.AIM The aim was to investigate serum qHBcrAg levels in patients with chronic hepatitis B and assess the correlation of serum qHBcrAg with pregenomic RNA(pgRNA),cccDNA,and HBeAg seroconversion.METHODS This study was a secondary analysis of patients who underwent percutaneous liver biopsy between July 2014 and June 2019 in two multicenter randomized controlled clinical trials of peginterferon vs nucleos(t)ide analog(NUC)-based therapy(NCT03509688 and NCT03546530).Serum qHBcrAg,pgRNA,HBV DNA,hepatitis B core antigen,HBeAg,liver cccDNA,and HBV DNA were measured.The correlations of serum qHBcrAg with other biomarkers were analyzed.RESULTS A total of 139 patients were included.The mean qHBcrAg levels were 5.32±1.18 log10 U/mL at baseline and decreased during treatment(all P<0.0001).Serum qHBcrAg levels were positively correlated with pgRNA(r=0.597,P<0.0001)and cccDNA(r=0.527,P<0.0001)levels.The correlation of serum qHBcrAg level and intrahepatic HBV DNA levels at baseline was weak but significant(r=0.399,P<0.0001).HBcrAg predicted HBeAg seroconversion,with areas under the receiver operating characteristics curve of 0.788 at 24 wk and 0.825 at 48 wk.Log HBcrAg at wk 24 and 48 was independently associated with HBeAg seroconversion[odds ratio(OR)=2.402,95%confidence interval(CI):1.314-4.391,P=0.004;OR=3.587,95%CI:1.315-9.784,P=0.013].CONCLUSION Serum HBcrAg levels were correlated with HBV virological markers and could be used to predict HBeAg seroconversion.

On-treatment monitoring of liver fibrosis with serum hepatitis B core-related antigen in chronic hepatitis B

BACKGROUND Non-invasive evaluation for liver fibrosis is clinically important,especially in patients with undetectable hepatitis B virus(HBV)DNA treated with nucleoside analogs.AIM To clarify the monitoring power of hepatitis B core-related antigen(HBcrAg)for hepatic histologic changes in patients with chronic hepatitis B(CHB)treated with entecavir.METHODS This prospective multicenter study used multiple ordinal and multivariate logistics regression analysis to assess variables associated with Ishak fibrosis score and regression for fibrosis regression,respectively,in 403 CHB patients,including 374 with entecavir for 72 weeks(291 underwent paired liver biopsy)and 29 as controls.RESULTS Level of HBcrAg correlated negatively with liver fibrosis staging(γ=-0.357,P<0.001)in hepatitis B e antigen(HBeAg)-positive patients,and positively with liver fibrosis staging in HBeAg-negative patients.Higher HBcrAg concentration was associated with younger age,HBeAg positive status,high HBV DNA loads,high level of hepatitis B surface antigen(HBsAg)and higher necroinflammation,but not with HBV genotype.Serum concentration of HBcrAg,basal core promoter/precore(BCP/PC)mutant,quantitation of HBsAg(qHBsAg)and platelet counts were independently associated with Ishak fibrosis score on multiple ordinal regression.HBV DNA was undetectable in 88.37%of patients treated with entecavir at week 72,while their level of HBcrAg was still detectable.A greater reduction in post-treatment HBcrAg concentration was associated with the regression of hepatic fibrosis and histological improvement.HBcrAg concentration>6.33 log IU/mL at baseline and logarithmic reduction>1.03 log IU/mL at week 72 were associated with a higher chance of regression of liver fibrosis and histological improvement,respectively.CONCLUSION HBcrAg level is associated with liver fibrosis progression.HBcrAg is an excellent monitor of hepatic histological changes,especially in CHB patients treated with nucleoside analogs.

Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus

AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier. METHODS: One to 6 tandem copies of HBV preS1 (21-47) fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E.coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric particles was detected with immuno-capture PCR. RESULTS: Recombinant antigens CI, CII, CIII carrying 1-3 copies of HBV preSl (21-47) individually could form virus-like particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preSl (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CI, CII, CIII could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CI, CII and CIII) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CI, CII and CIII were able to capture HBV virions in immuno-capture PCR assay in vitro. CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

Mutations of pre-core and basal core promoter before and after hepatitis B e antigen seroconversion

AIM: To investigate the role of pre-core and basal core promoter(BCP) mutations before and after hepatitis Be antigen(HBe Ag) seroconversion.METHODS: The proportion of pre-core(G1896A) and basal core promoter(A1762T and G1764A) mutant viruses and serum levels of hepatitis B virus(HBV) DNA, hepatitis B surface antigen(HBs Ag), and HB core-related antigen were analyzed in chronic hepatitis B patients before and after HBe Ag seroconversion(n = 25), in those who were persistently HBe Ag positive(n = 18), and in those who were persistently anti-HBe positive(n = 43). All patients were infected with HBV genotype C and were followed for a median of 9 years.RESULTS: Although the pre-core mutant became predominant(24% to 65%, P = 0.022) in the HBe Ag seroconversion group during follow-up, the proportion of the basal core promoter mutation did not change. Median HBV viral markers were significantly higher in patients without the mutations in an HBe Ag positive status(HBV DNA: P = 0.003; HBs Ag: P < 0.001; HB core-related antigen: P = 0.001). In contrast, HBV DNA(P = 0.012) and HBs Ag(P = 0.041) levels were significantly higher in patients with the pre-core mutation in an anti-HBe positive status.CONCLUSION: There is an opposite association of the pre-core mutation with viral load before and after HBe Ag seroconversion in patients with HBV infection.

Humoral and cellular immunogenecity of DNA vaccine based on hepatitis B core gene in rhesus monkeys

INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant association among persistentinfection, liver cirrhosis and hepatocellularcarcinoma[1-3].

Precore/basal core promoter mutants quantification throughout phases of hepatitis B virus infection by Simpleprobe

AIM:To investigate precore/basal core promoter(PC/BCP) mutants throughout hepatitis B virus(HBV) infection and to determine their relationship to hepatitis B early antigen(HBeA g) titers.METHODS:We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012.None of the patients received antiviral therapy.HBV DNA from serum,was quantified by real-time PCR.The HBV genotype was determined by direct sequencing of the S gene.We used the Simpleprobe ultrasensitivequantitative method to detect PC/BCP mutants in each patient.We compared the strain number,percentage,and the changes in PC/BCP mutants in different phases,and analyzed the relationship between PC/BCP mutants and HBe Ag by multiple linear regression and logistic regression.RESULTS:Patients with HBV infection(n = 191) were assigned to groups by phase:Immune tolerance(IT) = 55,Immune clearance(IC) = 67,Low-replicative(LR) = 49,and HBeA g-negative hepatitis(ENH) = 20.Of the patients(male,112; female,79) enrolled,122 were HBe Ag-positive and 69 were HBe Ag-negative.The median age was 33 years(range:18-78 years).PC and BCP mutation detection rates were 84.82%(162/191) and 96.86%(185/191),respectively.In five HBe Ag-negative cases,we detected double mutation G1896A/G1899 A.The logarithm value of PC mutant quantities(log10 PC) significantly differed in IT,IC,and LR phases,as well as in the ENH phase(F = 49.350,P < 0.001).The logarithm value of BCP mutant quantities(log10 BCP) also differed during the four phases(F = 25.530,P < 0.001).Log10 PC and log10 BCP values were high in the IT and IC phases,decreased in the LR phase,and increased in the ENH phase,although the absolute value at this point remained lower than that in the IT and IC phases.PC mutant quantity per total viral load(PC%) and BCP mutant quantity per total viral load(BCP%) differed between phases(F = 20.040,P < 0.001; F = 10.830,P < 0.001),with PC% and BCP% gradually increasing in successive phases.HBeA g titers negatively correlated with PC%(Spearman's rho =-0.354,P < 0.001) and BCP%(Spearman's rho =-0.395,P < 0.001).The negative correlation between PC% and HBeA g status was significant(B =-5.281,P = 0.001),but there was no such correlation between BCP% and HBeA g status(B =-0.523,P = 0.552).CONCLUSION:PC/BCP mutants become predominant in a dynamic and continuous process.Log10 PC,log10 BCP,PC% and BCP% might be combined to evaluate disease progression.PC% determines HBeA g status.

Precore/basal core promoter mutants and hepatitis B viral DNA levels as predictors for liver deaths and hepatocellular carcinoma

AIM： To conduct a retrospective study in 400 chronic hepatitis B patients in order to identify hepatitis B viral factors associated with complications of liver disease or development of hepatocellular carcinoma. METHODS： The mean follow-up time was 83.6 ± 39.6 mo. Alpha-fetoprotein test and abdominal ultrasound were used for cancer surveillance. Hepatitis B basal core promoter mutants, precore mutants, genotypes, hepatitis B viral DNA （HBV DNA） level and hepatitis B e antigen （HBeAg） were measured. Univariate analysis and logistic regression were used to assess odds ratios for viral factors related to liver deaths and hepatocellular carcinoma development. RESULTS： During follow-up, 38 patients had liver deaths not related to hepatocellular carcinoma. On multivariate analysis, older age [odds ratio： 95.74 （12.13-891.31）, P 〈 0.0001], male sex [odds ratio： 7.61 （2.20-47.95）; P = 0.006], and higher Iogzo HBV DNA [odds ratio： 4.69 （1.16-20.43）; P 〈 0.0001] were independently predictive for these liver related deaths. Also, 31 patients developed hepatocellular carcinoma. Multivariate analysis showed that older age [odds ratio： 26.51 （2.36-381.47）; P = 0.007], presence of precore mutants [odds ratio： 4.23 （1.53-19.58）, P = 0.02] and presence of basal core promoter mutants [odds ratio： 2.93 （1.24-7.57）; P = 0.02] were independent predictors for progression to hepatocellular carcinoma. CONCLUSION： Our results show that high levels of baseline serum HBV DNA are associated with non- hepatocellular carcinoma-related deaths of liver failure, while genetic mutations in the basal core promoter and precore regions are predictive for development of hepatocellular carcinoma.

Intrahepatic distribution of hepatitis B virus antigens in patients with and without hepatocellular carcinoma

AIM: To study the intrahepatic expression of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in chronic hepatitis B patients with and without hepatocellular carcinoma.METHODS: A total of 33 chronic hepatitis B patients (mean age of 40.3 &#x000b1; 2.5 years), comprising of 14 HBeAg positive and 19 HBeAg negative patients; and 13 patients with hepatitis B virus related hepatocellular carcinoma (mean age of 49.6 &#x000b1; 4.7 years), were included in our study. Immunohistochemical staining for HBcAg and HBsAg was done using standard streptavidin-biotin-immunoperoxidase technique on paraffin-embedded liver biopsies. The HBcAg and HBsAg staining distributions and patterns were described according to a modified classification system.RESULTS: Compared to the HBeAg negative patients, the HBeAg positive patients were younger, had higher mean HBV DNA and alanine transaminases levels. All the HBeAg positive patients had intrahepatic HBcAg staining; predominantly with &#x0201c;diffuse&#x0201d; distribution (79%) and &#x0201c;mixed cytoplasmic/nuclear&#x0201d; pattern (79%). In comparison, only 5% of the HBeAg-negative patients had intrahepatic HBcAg staining. However, the intrahepatic HBsAg staining has wider distribution among the HBeAg negative patients, namely; majority of the HBeAg negative cases had &#x0201c;patchy&#x0201d; HBsAg distribution compared to &#x0201c;rare&#x0201d; distribution among the HBeAg positive cases. All but one patient with HCC were HBeAg negative with either undetectable HBV DNA or very low level of viremia. Intrahepatic HBcAg and HBsAg were seen in 13 (100%) and 10 (77%) of the HCC patients respectively. Interestingly, among the 9 HCC patients on anti-viral therapy with suppressed HBV DNA, HBcAg and HBsAg were detected in tumor tissues but not the adjacent liver in 4 (44%) and 1 (11%) patient respectively.CONCLUSION: Isolated intrahepatic HBcAg and HBsAg can be present in tumors of patients with suppressed HBV DNA on antiviral therapy; that may predispose them to cancer development.

Novel DNA vaccine based on hepatitis B virus core gene induces specific immune responses in Balb/c mice

AIM： To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS： A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes （CTLs） of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS： HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1：97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector：target ratio of 20：1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION： pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.

Mutations in hepatitis B virus core regions correlate with hepatocellular injury in Chinese patients with chronic hepatitis B

AIM: To elucidate the relationship between the frequency of core mutations and the clinical activity of hepatitis B virus (HBV)-related liver disease and to characterize the amino acid changes in the core region of HBV.METHODS: We studied 17 Chinese patients with chronic hepatitis B according to their clinical courses and patterns of the entire core region of HBV.RESULTS: Amino acid changes often appeared in the HBV core region of the HBV gene in patients with high values of alanine aminotransferase (ALT) or with the seroconversion from HbeAg to anti-HBe. The HBV core region with amino acid changes had high frequency sites that corresponded to HLA Ⅰ/Ⅱ restricted recognition epitopes reported by some investigators.CONCLUSION: The core amino acid changes of this study occur due to influence of host immune system. The presence of mutations in the HBV core region seems to be important for predicting the clinical activity of hepatitis B in Chinese patients.

Review on hepatitis B virus precore/core promoter mutations and their correlation with genotypes and liver disease severity

Of 350 million people worldwide are chronically infected with hepatitis B virus(HBV)and are at risk of developing cirrhosis and hepatocellular carcinoma(HCC)later in life.HBV is the most diverse DNA virus,and its genome is composed of four open reading frames:Presurface antigen/surface antigen gene(preS/S),precore/core gene(preC/C),polymerase gene(P),and theχgene(χ).HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate.The virus has 10 genotypes(A to J)with different geographical distributions.There are various HBV mutations in the HBV genome,including preC/C mutations,preS/S mutations,P gene mutations,andχgene mutations.The core promoter region plays a vital part in the replication,morphogenesis and pathogenesis of the virus.The precore region also plays a crucial role in viral replication.Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity.preC/C mutations are associated with liver disease progression.Precore mutations stop hepatitis B e antigen(HBeAg)production and basal core promoter mutations downregulate HBeAg production.Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC.The emergence of antiviral-resistant mutations is the main reason for treatment failure.This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity.Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.

Association of core promoter mutations of hepatitis B virus and viral load is different in HBeAg(+) and HBeAg(-) patients

AIM:To identify the prevalence of hepatitis B e antigen (HBeAg) and to assess the association of hepatitis B virus (HBV) core promoter mutations and viral load in Indonesian patients.METHODS:Sixty-four patients with chronic hepatitis,65 with liver cirrhosis and 50 with hepatocellular carcinoma were included in this study.HBeAg and hepatitis B e antibody (HBeAb) tests were performed using enzyme-linked immunosorbent assay and the mutations were analyzed by sequencing.Viral load was measured by real-time polymerase chain reaction.RESULTS:Of 179 patients,108 (60.3%) were HBeAg(-) and 86 (79.6%) of these HBeAg(-) patients had been seroconverted.The A1896 mutation was not found in HBeAg(+) patients,however,this mutation was detected in 70.7% of HBeAg(-) patients.This mutation was frequently found when HBeAg was not expressed (87.7%),compared to that found in HBeAg seroconverted patients (65.1%).The A1899 mutation was also more prevalent in HBeAg(-) than in HBeAg(+) patients (P=0.004).The T1762/A1764 mutation was frequently found in both HBeAg(+) and HBeAg(-) patients,however,the prevalence of this mutation did not significantly differ among the two groups (P=0.054).In HBeAg(+) patients,the T1762/A1764 mutation was correlated with lower HBV DNA (P < 0.001).The A1899 mutation did not correlate with HBV DNA (P=0.609).In HBeAg(-) patients,the T1762/A1764 mutation alone was not correlated with HBV DNA (P=0.095),however,the presence of either the T1762/A1764 or A1896 mutations was associated with increased HBV DNA (P < 0.001).CONCLUSION:The percentage of HBeAg(-) patients is high in Indonesia,and most of the HBeAg(-) patients had been seroconverted.The A1896 mutation was most likely the major cause of HBeAg loss.The T1762/A1764 mutation alone was associated with lower viral loads in HBeAg(+) patients,but not in HBeAg(-) patients.

Cloning and characterization of a novel hepatitis B virus core binding protein C12

AIM： To elucidate the biological function of HBV core antigen （HBcAg） on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS： HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA （cDNA） library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） and synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） containing X-α-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. M-FI reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. RESULTS： C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION： The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression. 2005 The W.IG Press and Elsevier Inc. All rights reserved

HBV pgRNA联合HBcrAg对慢性乙型肝炎患者停药后复发的预测价值

目的分析乙型肝炎病毒(hepatitis B virus,HBV)前基因组RNA(pregenomic RNA,pgRNA)水平联合HBV核心相关抗原(hepatitis B virus core-related antigen,HBcrAg)定量对慢性乙型肝炎(chronic viral hepatitis B,CHB)患者停药后复发风险的预测价值。方法选取中国人民解放军联勤保障部队第926医院2020年6月至2021年6月收治的113例CHB患者为研究对象,所有患者均已给予足疗程的正规抗病毒治疗,停药前均检测批pgRNA与HBcrAg。根据患者停药1年内复发情况分为复发组(38例)和未复发组(70例),比较两组患者的一般资料、肝功能、肾功能、甲胎蛋白(alphafetoprotein,AFP)、pgRNA及HBcAg水平等指标。应用多因素Logistic回归分析CHB患者停药后复发的影响因素。应用受试者工作特征(receiver operator characteristic,ROC)曲线分析pgRNA联合HBcrAg对CHB患者停药后复发风险的预测价值。结果复发组患者饮酒史比例[47.37%(18/38)比22.86%(16/70)]、AFP[(29.64±7.18)μg/L比(20.38±6.46)μg/L]、pgRNA[(7.97±1.99)lg拷贝/ml比(4.97±1.24)lg拷贝/ml]和HBcrAg[(7.04±1.76)lg IU/ml比(5.11±1.28)lg IU/ml]水平均显著高于未复发组(P均<0.05)。多因素Logistic回归分析表明,饮酒史(OR=5.354,95%CI:1.055~68.858,P=0.046)、AFP(OR=1.189,95%CI:1.036~1.468,P=0.015)、pgRNA(OR=1.047,95%CI:1.117~8.109,P=0.007)和HBcrAg(OR=2.152,95%CI:1.154~4.308,P=0.021)是CHB患者停药后复发的独立危险因素。pgRNA与HBcrAg联合预测CHB患者停药后复发的ROC曲线下面积为0.954,最佳截点为>0.128,此时敏感度为98.9%,特异度为97.1%。结论pgRNA和HBcrAg与CHB患者停药后复发风险密切相关,早期监测两者水平有助于发现停药后复发高风险的患者,早期调整治疗方案。

Inhibition of hepatitis B virus by oxymatrine in vivo

AIM To investigate the anti-HBV effect ofoxymatrine (oxy) in vivo.METHODS HBV transgenic mice were producedby micro-injection of a 4.2kb fragmentcontaining the complete HBV genomes.Expression level of HBsAg and HBcAg in thetransgenic mice liver was determined byimmunohistochemical assay.RESULTS Four groups (6 mice in each group)were injected intraperitoneally with oxy at thedosage of 100,200, and 300 mg/kg or with salineonce a day for 30 days. Both HBsAg and HBcAgwere positive in livers of all the six mice in thecontrol group (injected with saline), and werepositive in livers of two mice in 100 mg/kg groupand 300mg/kg group. In 200mg/kg group,HBsAg and HBcAg were negative in livers of allthe six mice. Based on the results, 200 mg/kg isthe ideal dosage to explore the effect of oxy atdifferent time points. According to the oxytreatment time, mice were divided into fourgroups: 10 d, 20 d, 30 d and 60 d (4 mice in eachgroup). Each mouse underwent liver biopsy twoweeks before the treatment of oxy. Down-regulation of HBsAg and HBcAg appeared aftertreatment of oxymatrine for 10 d and 20 d, Dane-like particles disappeared after the treatment ofoxy for 20d under electron microscopy,however, the expression level of HBsAg andHBcAg returned to normal 60 d later after oxytreatment.CONCLUSION oxymatrine can reduce thecontents of HBsAg and HBcAg in transgenic miceliver, longer treatment time and larger dosagedo not yield better effects.

苏州地区HBV感染患者血清中乙型肝炎核心相关抗原表达特点分析

目的探究乙型肝炎核心相关抗原(hepatitis B core-related antigen,HBcrAg)在乙型肝炎病毒感染患者血清中的表达特点,为HBcrAg在临床上的深入应用提供理论依据。方法本研究纳入200例因怀疑乙型肝炎病毒感染在苏州市第五人民医院就诊的患者为研究对象,均进行血清中HBcrAg、HBeAg和HBV DNA检测,对结果进行统计以及分组分析。结果以HBeAg、HBV DNA检测结果为参考标准进行比较,HBcrAg检测的灵敏度为82.8%,特异性为89.7%,阴性/阳性总体符合率为86.5%,具有较好的一致性(Kappa=0.728,95%CI 63.2%~82.3%),且阳性检出率为3者中最高。对不同表型患者的分组研究发现,HBeAg/HBV DNA双阳性患者血清中HBcrAg的阳性检出率和表达量均最高。在不同乙肝两对半模式患者中,135阳性患者血清中HBcrAg的阳性检出率和表达量均显著高于145阳性及其他模式患者,且HBcrAg的表达量与HBV DNA的表达量具有较好的正相关性(r=0.81,P<0.001)。结论HBcrAg是一种可靠的反映HBV病毒复制状态的新型标志物,且在HBeAg/HBV DNA双阳性患者和乙肝两对半135阳性患者血清中阳性检出率和表达量较高,这些特征能为HBcrAg在临床上进一步的深入应用丰富理论基础,为临床上评估HBV感染患者治疗效果及判断治疗终点等提供参考价值。

HBeAg negative variants and their role in the natural history of chronic hepatitis B virus infection

Molecular virology methods including polymerase chain reaction, cloning and sequencing have revolutionised our understanding of viral genome variation. In the case of hepatitis B virus (HBV), sequencing studies have identified a number of virus variants normally found during the natural course of chronic infection. The appearance of the precore stop codon (with G-for-A substitution at position 1896) and basal core promoter (BCP) (with A-for-T and G-for-A, at positions 1762 and 1764, respectively) variants which reduce or abrogate hepatitis B e antigen (HBeAg) production, heralds the initiation of the seroconversion phase from HBeAg to anti-HBe positivity. The gradual removal of the tolerogenic effect of HBeAg leads to the awakening of the immune response (immune clearance phase). Most patients after HBeAg seroconversion become &#x0201c;inactive HBsAg carriers&#x0201d;. However during the course of infection precore and/or BCP variants may emerge and be selected leading to HBeAg negative chronic hepatitis B (CHB) with high viremia levels (reactivation phase). The prevalence of HBeAg negative CHB has been increasing over the last few decades and has become the commonest type of HBV infection in many countries of the world. This probably reflects the aging of existing HBV carriers and the effective prevention measures restricting new HBV infections. Frequent acute exacerbations accompanied by high viral replication, elevated alanine aminotransferase levels and histological activity are a common feature of HBeAg negative CHB leading to cirrhosis much faster than in HBeAg positive CHB patients.

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes

INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).