Development and validation of a model for hepatitis B e antigen seroconversion in entecavir-treated patients with chronic hepatitis B

Achieving hepatitis B e antigen (HBeAg) seroconversion is a satisfactory endpoint during antiviral treatment for chronic hepatitis B (CHB). This study aimed to develop and validate a novel scoring system to predict HBeAg seroconversion during entecavir (ETV) treatment. A total of 526 patients with HBeAg-positive CHB treated with ETV for at least 1 year were randomly assigned to the training and validation cohorts. Baseline parameters including hepatitis B virus DNA, hepatitis B surface antigen (HBsAg), hepatitis B core antibody (HBcAb), and alanine aminotransferase level were quantified. Patients who achieved HBeAg seroconversion were compared with those without HBeAg seroconversion. A prediction model was established to predict HBeAg seroconversion during ETV treatment. After a median follow up of 2.67 years, 93 (36.0%) and 87 (32.5%) patients in the training and validation cohorts developed HBeAg seroconversion. A prediction score composed of age, HBsAg and HBcAb quantification was derived. Areas under receiver operating characteristic curve at 5 years of this prediction score were 0.70 and 0.72 in the training and validation cohorts. By using the dual cutoff values of 0.28 and 0.58, the model was endowed with high sensitivity and specificity to exclude or identify patients developing HBeAg seroconversion (90.3% sensitivity and 90.2% specificity in the training cohort as well as 92.8% sensitivity and 84.4% specificity in the validation cohort, respectively). A novel prediction score that uses baseline clinical variables was developed and validated. The score accurately estimates the probabilities of developing HBeAg seroconversion at 5-years ETV therapy in patients with CHB.     

Clearance of serum HBsAg and anti‐HBs seroconversion following antiviral therapy for chronic hepatitis B

In this study, we have analyzed the evolution of serum HBsAg levels in 16 patients with chronic hepatitis B who showed an HBsAg seroconversion following antiviral therapy. The data showed that the clearance of serum HBsAg is slower than that of serum HBV DNA, which may reflect a slow kinetics of clearance of infected hepatocytes. Interestingly, HBsAg was detectable for a longer time using the Architect assay than with the Bio‐Rad assay. As viremia suppression is achieved in most patients under therapy with the new generation of nucleoside analogs, these data suggest that the quantitative monitoring of serum HBsAg may represent a novel tool for the assessment of antiviral therapy efficacy. J. Med. Virol. 81:1336–1342, 2009. © 2009 Wiley‐Liss, Inc.     

HBcAg-induced upregulated 4-1BB ligand on B cells contributes to B-cell hyperactivation during chronic hepatitis B infection

During the natural history of chronic hepatitis B infection (CHB), the function of B cells is still obscure. Several limited studies have suggested that B cells are highly active in patients with CHB. In the present study, we reported that the 4-1BB ligand (4-1BBL) expression on B cells was significantly higher in patients with CHB than that in healthy subjects, meanwhile, the patients with CHB had higher serological IgG levels. Further, after being stimulated with sCD40L or hepatitis B core antigen (HBcAg), B cells had higher levels of 4-1BBL. After being cocultured with 4-1BBL+ B cells, the expressions of CD69 and 4-1BB on CD4+ T cells were significantly higher than that cocultured with 4-1BB− B cells. Cytokines including interleukin (IL)-2, IL-4, and IL-6 were significantly higher in the supernatant from 4-1BBL+ B cells coculture group than those from coculture group of 4-1BBL− B cell group, respectively; while IFN-γ and TNF-α in cocultured supernatant of 4-1BBL+ B cell group were significantly lower. Consistently, the total IgG levels in culture supernatant were significantly higher in 4-1BBL+ B cell group. Thus, hyperactive status of B cells in patients with CHB could be partially derived from the higher 4-1BBL expression on B cells triggered by HBcAg. 4-1BBL signaling pathway is involved in B cells activation, and further regulates B cell-T cell interaction by modulating the cytokines secretion, which might be critical in B cells dysfunction during CHB infection.     

Genetic heterogeneity in the precore region of hepatitis B virus in hepatitis B e antigen-negative chronic hepatitis B Patients: Spontaneous seroconversion and interferon-induced seroconversion

To elucidate the relationship between the clinical severity of chronic liver disease and the precore mutations in hepatitis B e antigen (HBeAg)-nega-tive hepatitis B virus (HBV) carriers, mutations were investigated in the precore region of HBV DNA in 20 chronic hepatitis B patients who sero-converted either spontaneously or after the administration of α-interferon (IFN), and 5 asymptomatic carriers. The precore mutation with a stop codon at nucleotide 1896 was found in all patients, irrespective of the histology and in all asymptomatic carriers. The second mutation at nucleotide 1899 was found in 40% of cases studied but always followed by the first mutation at nucleotide 1896. The mixed viral infection of precore mutant and wild-type HBV virus was found in 40% of seroconverted cases after IFN treatment and in sera of HBV carriers obtained within a year after the spontaneous Seroconversion. These data suggest that the precore mutants prevail over wild-type HBV in all HBeAg-negative HBV carriers within several years after the sero-conversion, but their prevalence could not confine the clinical severity of chronic liver disease. © 1995 Wiley-Liss, Inc.     

PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients

Programmed death-1 (PD-1) is demonstrated to have an increased expression on antigen-specific T cells during chronic virus infections, and the blockage of PD-1/PD-ligand (PD-L1) pathway could restore the function of exhausted T cells. We measured the PD-1 expression levels on HBV-specific CD8 T cells and investigated the role of PD-1/PD-L1 pathway in T-cell responses of patients with different HBV infection statuses. Compared to the patients with convalescent acute hepatitis B, PD-1 expression on total CD8 T cells from chronic hepatitis B (CHB) patients was significantly upregulated, especially on the HBV pentamer-positive CD8 T cells. And PD-L1, but not PD-L2, was also significantly upregulated on PBMC from CHB patients. In CHB patients, HBV-specific T cells and cellular proliferation could be observed under the recombinant HBV-Ag stimulation in vitro, and blockade of PD-1 pathway significantly enhanced the IFN-gamma production and cellular proliferation of PBMC. Furthermore, PD-1 expression level on HBV-pentamers positive CD8 T cells was positively associated with plasma viral load in CHB patients. Thus, PD-1 upregulation on HBV-specific CD8 T cells is engaged in the dysfunction of T cells and high viraemia in CHB patients, and the antiviral T-cell responses could be improved by the blockade of this inhibitory PD-1/PD-L1 pathway.     

Longitudinal study of hepatitis activity and viral replication before and after HBeAg seroconversion in chronic hepatitis B patients infected with genotypes B and C

The aims of this study were to compare chronic hepatitis B (CHB) patients with genotypes B and C for the probability of HBeAg seroconversion, hepatitis activity, and viral replication before and after HBeAg seroconversion and to compare the prevalence of core promoter and precore mutations. A total of 180 asymptomatic Chinese patients with CHB were monitored for a median of 53.8 months, and 38 patients with cirrhosis-related complications were studied. Hepatitis B virus (HBV) DNA levels were measured in 16 patients with HBeAg seroconversion at 3 months before, during, and 3 months after HBeAg seroconversion and in all patients at the last follow-up. Hepatitis B genotypes and core promoter and precore mutations were determined. Compared to patients with genotype C (n = 109), patients with subtype Ba (n = 69) had a higher rate of anti-HBe positivity on presentation (P = 0.05). HBeAg-positive patients with subtype Ba had a higher cumulative rate of HBeAg seroconversion than patients with genotype C (P = 0.02). However, there were no differences between the two groups with regard to the median HBV DNA levels before, during, and after HBeAg seroconversion; the probability of having persistently normal or elevated aminotransferase levels; and the median HBV DNA levels and liver biochemistry at the last follow-up. There was no difference in the prevalence of genotypes and core promoter and precore mutations between patients with and without cirrhosis-related complications. Though patients with subtype Ba had earlier HBeAg seroconversion, the liver biochemistry, HBV DNA levels in different phases of the disease, and the probability of development of cirrhosis-related complications were the same with genotypes Ba and C.     

抗原活化慢性乙型肝炎患者外周血树突状细胞的对比研究

目的 探讨不同抗原在体外活化慢性乙型肝炎患者外周血树突状细胞(DC)及诱导特异性T细胞应答的能力.方法 用无血清培养基从慢性乙型肝炎患者外周血中分离培养DC,在DC成熟前,分别加入HBsAg多肽、HBcAg多肽刺激,用流式细胞仪检测DC表型,用液闪计数仪观察DC对T细胞的增殖作用,用ELISA法检测混合淋巴细胞反应(MLR)中IL-12的分泌水平.结果 经HBcAg多肽刺激DC的CD86表达率为(92.20±5.18)%,明显高于HBsAg多肽刺激组(76.19±3.90)%和未加抗原组(62.37±4.24)%,P＜0.01;经HBcAg多肽刺激组DC诱导同种异体静止T细胞增殖的能力每分钟液闪计数值cpm为34 326±3088,明显高于HBsAg多肽刺激组20 306±2897和单个核细胞组3454±409,P＜0.01;经HBcAg多肽刺激组DC MLR中IL-12(348±42.8)ng/L,分别高于HBsAg多肽刺激组(226±30.6)ng/L和未加抗原组(116±15.6)ng/L,P＜0.01.结论 使用HBcAg多肽刺激DC可比HBsAg多肽更有效地提呈病毒抗原,提高诱导特异性T细胞应答的能力.     

慢性乙型肝炎患者血清HBsAg水平与肝脏炎症和纤维化程度相关性研究

目的 探讨慢性乙型肝炎患者HBsAg水平与肝脏炎症和纤维化的关系.方法 301例确诊为慢性乙型肝炎的患者纳入研究,同时进行肝脏活检术检查和生物化学、铁蛋白、血清HBsAg、HBV DNA定量检测.Spearman等级相关分析血清HBsAg水平与肝脏炎症分级和纤维化分期间的关系;logistic回归分析法分析相关指标的诊断意义,受试者工作曲线法评价血清HBsAg水平预测肝脏炎症分级和纤维化分期的准确性.结果 体质量指数、年龄、性别、基因型和家族史对CHB患者肝脏炎症和纤维化无明显影响(P＜0.05).AST、ALT随着炎症、纤维化进展而升高,差异具有统计学意义(x2=71.193、96.344、47.847、63.981;P =0.000、0.000、0.000、0.000).纤维化为S4时,HBV DNA明显下降(x2=33.322,P=0.000).HBsAg水平随着炎症和纤维化程度加重而逐步下降(x2=68.173,15.719;P =0.000,0.000).HBsAg水平预测≤G3和≤S3的曲线下面积分别为0.732和0.793,特异度分别为0.778、0.891,灵敏度依次为0.685、0.633.结论 中国CHB患者HBsAg水平随着肝脏炎症分级和纤维化分期程度加重而逐渐下降.血清HBsAg水平对CHB患者肝脏炎症≤G3和纤维化≤S3的预测具有较高的特异性,且对纤维化的预测优于炎症.     

Detection of hepatitis B surface antigen (HBsAg) in peripheral blood mononuclear cells of patients with acute and chronic active hepatitis B

Seventy five patients with acute and chronic active hepatitis (CAH) were studied by indirect immunofluorescence with monoclonal antibodies for the presence of hepatitis B surface antigen (HBsAg) on peripheral blood mononuclear cells (PBMC). The viral surface antigen was detected in the PBMC of all the patients with hepatitis B virus (HBV)-induced CAH and in acute patients with more than 2 months of evolution. No HBsAg was detected in the samples obtained from 12 normal controls or from 14 non-A, non-B CAH patients. Analysis of PBMC subsets revealed that HBsAg was present in non-T cells; dual fluorescence studies showed HBsAg on surface Ig-positive lymphocytes. The binding of anti-HBs monoclonal antibodies was higher than that of a goat anti-HBs serum, and the highest reactivity was observed with an antibody against the pre-S(2)-region sequence. Both HBsAg and hepatitis B core antigen (HBcAg) were also detected in lysates of PBMC by dot blot analysis.     

IL-28B基因突变与慢性丙型肝炎治疗应答的相关性研究

目的 探讨白介素28B(IL-28B)基因型与慢性丙型肝炎(CHC)患者抗病毒治疗反应的相关性.方法 220例CHC患者均接受聚乙二醇干扰素(peg-IFN)联合利巴韦林(RBV)治疗48周,随访至停药后24周.检测IL-28B(rs8099917)位点,比较IL-28B基因型与抗病毒疗效的关系,评估IL-28B单核苷酸多态性(SNP)在CHC患者治疗应答中的作用.结果 TT、TG和GG基因型在持续应答组(SVR)中的比例分别是71.4％、25.0％和3.6％;在无应答组(NR)分别是15.8％、60.5％和23.7％;在复发组(RP)分别是38.1％、52.3％和9.6％;三组之间比较差异具有统计学意义(P＜0.001).NR与SVR组内基因型比较,TG vsTT的OR为7.67(P＜0.001,95％ CI:2.91～20.56),差异有统计学意义.RP与SVR组内基因型比较,TG vsTT的OR为3.10(P＜0.01,95％ CI:1.41～6.36),差异有统计学意义.结论 IL-28B(rs8099917)基因型与慢丙肝患者抗病毒应答密切相关,可作为治疗前的一个重要的预测因子.     

阿德福韦酯治疗慢性乙型肝炎HBeAg血清学转换与基因型、HBV特异性CTL的关系

目的 探讨阿德福韦酯治疗慢性乙型肝炎患者发生HBeAg血清学转换与HBV基因型、HBV特异性CTL的关系.方法 70例慢性乙型肝炎患者HBV DNA阳性（HBV DNA≥1×104拷贝/ml）,HBeAg阳性45例,其中B基因型23例（51.11%）,C基因型22例（48.89%）.ALT＞2×正常参考值上限（ULN）、人白细胞抗原（HLA）-A2阳性,用阿德福韦酯（正大天睛药业公司生产）10 mg,口服,每日1次.观察治疗12个月后HBeAg血清学转换与HBV基因型、HBV特异性CTL的关系.结果 阿德福韦酯治疗12个月后,HBV特异性CTL（0.68%±0.11%）高于治疗前（0.33%±0.11%）,t=8.36 P＜0.001,HBV DNA（3.01±0.2）log10拷贝/ml低于治疗前[（6.27 ±0.70）log10拷贝/ml]t=12.63 P＜0.001,HBV DNA阴转（＜500拷贝/ml）43例（61.43%）.HBeAg阳性45例中发生HBeAg转阴13例（28.89%）,HBeAg血清学转换8例（17.78%）,HBeAg血清学转换者HBV特异性CTL（0.86 ±0.05%）高于无HBeAg血清学转换者（37例,82.22%）的HBV特异性CTL（0.61%±0.07%）,t=7.88,P＜0.001.HBeAg血清学转换8例中,B基因型7例（占B基因型30.43%）,C基因型1例（占C基因型4.55%）,x2=5.15,P＜0.05.结论 阿德福韦酯能提高CHB患者的HBV特异性细胞免疫功能,治疗后HBeAg血清学转换的发生与HBV特异性CTL水平升高有关,并可能与基因型有关.     

The precore mutation is associated with severity of liver damage in Brazilian patients with chronic hepatitis B.

BACKGROUND: The clinical relevance of the G1896A precore mutation in chronic hepatitis B is still poorly understood. OBJECTIVES: To assess the frequency of G1896A precore mutation in Brazilian patients with chronic hepatitis B, as well as its relation to the viral genotype, serum HBV-DNA levels and liver damage. STUDY DESIGN: Fifty chronic hepatitis B patients (29 HBeAg-negative and 21 HBeAg-positive) were studied. HBV-DNA was quantified by the Amplicor HBV Monitor test and precore region and S gene were amplified and submitted to automatic sequencing. The histological activity index (HAI), degrees of hepatic fibrosis and distribution of core antigen (HBcAg) in hepatocytes were determined. RESULTS: Precore mutation occurred in 1/21 (4.8%) HBeAg-positive patients and in 17/29 (58.6%) HBeAg-negative (p < 0.0001). Genotype D was identified in 56.5%, genotype A in 41.3%, and genotype F in 2.2%. The frequency of genotypes D and A, as well as serum levels of ALT and HBV-DNA were similar in patients infected with wild type and with precore mutant. Patients infected with precore mutant presented a higher frequency of moderate/severe HAI (p: 0.03) and moderate/severe fibrosis and cirrhosis (p: 0.03) than those infected with wild type. There was no association between G1896A mutation and cytoplasmic expression of HBcAg. CONCLUSIONS: Precore mutation was frequent among Brazilian subjects with chronic hepatitis B and its presence was associated with greater severity of liver disease.     

HBsAg seroclearance in Chinese patients receiving lamivudine therapy for chronic hepatitis B virus infection

We report two Chinese patients in whom lamivudine treatment resulted in HBsAg seroclearance. One patient received lamivudine, and another patient received 12-week famciclovir treatment followed by lamivudine. Lamivudine was maintained after HBeAg seroconversion. These two patients lost HBsAg at 24 and 27 months (ages, 23 and 19.3 years, respectively) and developed measurable titer of anti-HBs after 65 and 71 months of therapy, respectively. The liver biochemistry was normal after HBeAg seroconversion. The serum hepatitis B virus (HBV) DNA levels were undetectable (<200 copies/ml) both at the time of HBeAg seroconversion and at the last follow-up. Liver biopsy of one patient showed nearly normal histology, with undetectable intrahepatic total HBV DNA and covalently closed circular DNA. In conclusion, lamivudine therapy can result in HBsAg seroclearance at an early age even though the phenomenon is rare.