重组乙肝病毒核心基因DNA与表面抗原-抗体共免疫应答研究

目的:了解小鼠对乙肝病毒核心抗原(HBcAg)基因免疫及与乙肝表面抗原-抗体复合物(HBsAg-抗HBs)联合免疫的应答。方法:用表达乙肝病毒核心抗原N端144个氨基酸的重组质粒DNA(简称pHBc144)100μg及含1μgHBsAg的重组乙肝表面抗原-鼠抗体组建的复合物(简称sIC)免疫Balb/c小鼠。用ELISA法检测血清抗体IgG和IgG1、IgG2a亚类的效价。用半定量PCR法分别检测鼠脾细胞IFN-γ及IL-4的mRNA。结果:pHBc144及sIC联合免疫鼠的抗HBs IgG、IgG1、IgG2a效价均显著高于sIC组(P＜0．05)。同时小鼠还可产生抗HBc及由HBsAg、HBcAg特异诱生的IFN-γ及IL-4。但与sIC共免疫,小鼠对HBcAg诱生的IFN-γ mRNA有所降低。结论:可用HBcAg基因与sIC共免疫,以获得针对乙肝病毒2种结构蛋白的免疫应答。     

不同剂量HBIG对母婴乙型肝炎病毒抗原抗体宫内传播的影响

目的探讨不同剂量乙肝免疫球蛋白（HBIG）对乙型肝炎病毒（HBV）抗原抗体宫内传播的影响。方法将母亲乙肝表面抗原（HBsAg）阳性的婴儿作为500例观察对象,根据出生前母亲是否用HBIG分为：观察1组：产前母亲孕末期28w、32w、36w各用200IU（蓉生）HBIG 200例;观察2组：产前母亲孕末期28w、32w、36w各用400IU（蓉生）HBIG 100例;对照1组：产前母亲孕末期不用HBIG 200例。观察生后12h内新生儿静脉血乙肝五项：HBsAg、乙型肝炎表面抗体（HBsAb）、乙型肝炎e抗原（HBeAg）、乙型肝炎e抗体（HBeAb）、乙型肝炎核心抗体（HBcAb）。结果观察1组200例新生儿HBsAg阳性1例,阳性率为0.5%,HBeAg阳性3例,（其中1例HBsAg同时阳性）阳性率为1.5%。对照组HBsAg阳性2例,阳性率为1%,HBeAg阳性8例,（其中2例HBsAg同时阳性）阳性率为4%。经统计学处理（HBsAg）χ2=0.336,P=0.562;（HBeAg）,χ2=2.337,P=0.126。观察1组与对照组生后24h内HBV抗原检测比较无显著差异。观察1组、观察2组与对照组HBsAb检测比较：观察1组新生儿HBsAb阳性率1%,观察2组新生儿HBsAb阳性率2%,对照组HBsAb阳性率1%,各组HBeAb和HBcAb检测比较,结果HBeAb和HBcAb检测母婴符合率均在97%-97.5%之间。结论孕妇HBV携带者产前孕末期用HBIG 200IU隔4w连用3次的方法对阻断乙肝病毒的宫内感染效果不显著。加大HBIG的用量400IU可基本阻断HBV垂直传播胎儿。但鉴于对照组宫内感染率仅4%,加大用量不适用所有HBV携带者孕妇,尤其是HBsAg单阳性孕妇。     

rhHSP70-HER2/neu抗原肽对小鼠乳腺癌的治疗作用研究

目的：使用在毕赤酵母中重组表达的rhHSP70与合成的胍R2／neu抗原肽在特定条件下形成复合物，探讨其用于肿瘤生物治疗的可行性。方法：在ADP存在的条件下，将rhHSP70与合成的HER2／neu抗原肽体外非共价结合形成复合物，并分次免疫BALB／c小鼠，检测该复合物诱导特异性CTL的能力和对小鼠乳腺癌的治疗作用。结果：rhHSP70-HER2／neu抗原肽复合物免疫小鼠后，可以诱导出肿瘤特异性CTL，并对小鼠的乳腺癌有明显的治疗作用。结论：在毕赤酵母中重组表达的rhHSP70可以在体外与一定的抗原肽非共价结合形成复合物，该复合物具有较强的诱导特异性CTL的能力。     

乙肝疫苗联合乙肝免疫球蛋白对阻断母婴垂直传播乙型肝炎病毒的临床分析

目的探讨孕妇产前用乙肝免疫球蛋白（HBIG）与乙型肝炎疫苗联合免疫阻断母婴传播的效果。方法将504例HBsAg（＋）孕妇分为A（预防组）,B（对照组）两组。A组：246名HBsAg阳性孕妇孕晚期每月分别注射基因重组型乙肝疫苗10μg、HBIG200IU（200IU/ml）,新生儿出生后采股静脉血,同时在出生后24h内注射HBIG200IU,然后在0、1、6月龄接种基因重组型乙肝疫苗,每次10μg。B组：258例产前未注射HBIG和基因重组型乙肝疫苗的HBsAg阳性孕妇,其所生新生儿在0、1、6（30μg、30μg、30μg）月龄只用基因重组型乙肝疫苗免疫。A、B两组婴儿都分别在0、3、6、9、12、24月龄静脉采血,用酶联免疫吸附试验（ELISA）检测HBV标志物,同时随访。结果A组的宫内感染率为3.25%,B组为4.16%,差异无统计学意义（χ^2=1.43,P〉0.05）。A组没有发生慢性HBV感染的婴儿,而B组中有7例婴儿发生慢性HBV感染,B组婴儿发生慢性HBV的感染率显著高于A组（χ^2=4.41,P〈0.05）。结论产前用HBIG和新生儿HBIG联合免疫可降低慢性HBV感染率,阻断宫内感染的慢性化,提高产程感染的阻断效果。     

CTL对同种异体靶细胞杀伤作用的实验研究

目的：研究特异性CTL杀伤的效应与HLA型别之间的关系。方法：用已知型别的EB病毒转化的B淋巴母样细胞（Epstein-Barr-transformed B lymphoblastoid cell line,EBV-LCL）与同种异体外周血单个核细胞（PBMC）共培养，激活同种异体抗原特异性细胞毒性T细胞（cytotoxicity T cell,CTL），然后利用同位素释放法观察CTL对HLA－I类表型不同的EBV－LCL的杀伤活性。结果：用EBV－LCL－1刺激自身PBMC－1所诱翌的CTL－1a，只能杀伤EBV－LCL－1，而不能杀伤HLA－I类表型不同的EBV－LCL－2；但用EBV－LCL－2刺激PBMC－1所诱导的CTL－1b，却能有效杀伤HLA－I类表型不同的EBV－LCL－2。结论：①MHC表型不同的免疫细胞之间可以发生相互作用；②TCR既不单独识别靶细胞表面的抗原肽，也不直接识别靶细胞表面的MHC分子（MHC特异性抗 原决定簇），而是识别MHC－抗原肽复合物的表达综合信息，后者可能是由MHC－抗原肽复合物表面的空间构象、电荷性质及其分布等信息所构成；③所谓CTL的特异性杀伤作用，是MHC－抗原肽复合物表面信息激活该信息性T细胞克隆的结果。     

HBV-前S_2抗原单克隆抗体制备、特性和应用

用HBsAg制备了一株分泌乙型肝炎病毒前S_2抗原单克隆抗体(MAb)的杂交瘤细胞株(1E_2),小鼠腹水中MAb效价达10~7。1E_2MAb有很强的抑制多聚人血清白蛋白(pHSA)和HBsAg中前S_2抗原结合的能力;与重组痘菌病毒表达的中分子蛋白(含S和前S_2抗原)而不与小分子蛋白(含S抗原)反应;与人工合成的前S_2肽段(N端12O-146氨基酸序列)特异性结合。应用1E_2MAb的酶免疫法检测乙肝病毒感染者血清中前S_2抗原,表明前S_2抗原滴度和HBsAg滴度有较好的相关性,在EBeAg阳性血清中更为明显;HBeAg阳性血清前S_2抗原的阳性率和几何平均滴度明显高于HBeAg阴性/HBsAg阳性血清。     

腺病毒六邻体蛋白型间线性化抗原位点的研究

目的 对人类腺病毒（adenovirus,AdV)六邻体型间抗原位点的特性进行研究。方法 通过计算机对腺病毒六邻体氨基酸序列进行比较分析，结合抗原性的预测结果和六邻体蛋白三维空间结构的肽段暴露状态，选定了保守性抗原位点进行多肽合成或通过构建重组质粒表达蛋白，将合成的六邻体肽段和表达纯化的蛋白抗原免疫动物后，用免疫印迹和间接免疫荧光方法检测抗血清的免疫特异性，结果 免疫印迹分析显示，抗多肽抗体和抗重组蛋白抗体均与腺病毒的六邻体蛋白特异性识别，间接免疫荧光显示，腺病毒感染HeLa细胞核内荧光着色，并且抗血清有较好的腺病毒型间反应性，合成多肽抗体与含有相同肽段的重组蛋白抗原产生特异性结合。结论 在腺病毒六邻体蛋白中存在有线性化的型间抗原位点，全盛的六邻体多肽和表达重组蛋白可用于诊断价值抗体的研制。     

B7-H1蛋白疫苗对HBV转基因小鼠HBsAg免疫效果影响的初步研究

目的:应用HBV转基因小鼠动物模型,研究B7-H1对HBsAg免疫效果的影响.方法:用重组人B7-H1与血源性HBsAg联合免疫HBV转基因小鼠,采用ELISA方法观察对转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的影响,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γ T细胞数量的影响,同时检测对小鼠淋巴细胞增殖及血清HBsAb水平的影响.结果:HBsAg组及HBsAg+B7-H1组免疫后脾细胞产生的Th1类细胞因子(IFN-γ、IL-2)、HBsAg特异性分泌IFN-γ T细胞、T细胞增殖及血清HBsAb水平较对照组显著增加(P<0.05),但HBsAg组及HBsAg+B7-H1组之间无显著性差异.结论:HBsAg可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子,并诱导小鼠产生特异性的体液免疫,打破免疫耐受.B7-H1对HBsAg在HBV转基因小鼠中的免疫效果无影响.     

重组白细胞α_1干扰素对乙型肝炎病毒感染的治疗

慢性乙型肝炎病人产生α干扰素能力有缺陷,这是作者用干扰素治疗此病的理由。本文提供用重组白细胞α_1干扰素治疗HBs-Ag阳性的慢性乙型肝炎的结果,以及对此药在HBsAg阳性慢性肝病临床使用问题进行了讨论。病人包括Ⅰ期治疗8人,Ⅱ期治疗24     

重组酵母菌产生的人体乙型肝炎疫苗

目前人体乙型肝炎疫苗都是用乙型肝炎病毒的表面抗原(HBsAg)来制备的。有几种哺乳动物细胞株(例如人体肝细胞瘤、感染SV40病毒的猴肾细胞以及小鼠L细胞等)与细菌可以表达此种抗原。但是前者可能致癌,后者表达甚微。有的用DNA重组术获得的疫苗其抗原效价以及对人体的完全性与效用均不肯定。1982年Valenzuela等首次报道酵母菌细胞不仅可表达HBsAg基因,而且可以把多肽装配成与人体血浆中分离者完全相同的颗粒。为此,作者采用啤酒酵母的发酵液来产生adw亚型的HBsAg。此菌携带用酵母乙醇脱氢酶Ⅰ为启动子的可表达的基因载     

Association of concurrent hepatitis B surface antigen and antibody to hepatitis B surface antigen with hepatocellular carcinoma in chronic hepatitis B virus infection

Antibody to hepatitis B surface antigen (HBsAg) (anti‐HBs) can exist in patients with chronic hepatitis B virus (HBV) infection. To date, little is known about the association of concurrent HBsAg and anti‐HBs (concurrent HBsAg/ anti‐HBs) with hepatocellular carcinoma (HCC). The aim of this study was to investigate the clinical relevance of concurrent HBsAg/anti‐HBs with preS deletion mutations and HCC in chronic HBV infection. A total of 755 patients with chronic HBV infection were included consecutively at a tertiary center. Logistic regression analysis was used to identify risk factors for HCC, and serum HBV DNA was amplified, followed by direct sequencing to detect preS deletions. The prevalence of concurrent HBsAg/anti‐HBs was 6.4% (48/755) and all HBVs tested were genotype C. HCC occurred more frequently in the concurrent HBsAg/anti‐HBs group than in the HBsAg only group [22.9% (11/48) vs. 7.9% (56/707), P = 0.002]. In multivariate analyses, age >40 years [odds ratio (OR), 14.712; 95% confidence interval (CI), 4.365–49.579; P < 0.001], male gender (OR 2.431; 95% CI, 1.226–4.820; P = 0.011), decompensated cirrhosis (OR, 3.642; 95% CI, 1.788–7.421; P < 0.001) and concurrent HBsAg/anti‐HBs (OR, 4.336; 95% CI, 1.956–9.613; P < 0.001) were associated independently with HCC. In molecular analysis, preS deletion mutations were more frequent in the concurrent HBsAg/anti‐HBs and HCC groups than in the HBsAg without HCC group (42.3% and 32.5% vs. 11.3%; P = 0.002 and 0.012, respectively). In conclusion, concurrent HBsAg/anti‐HBs is associated with preS deletion mutations and may be one of the risk factors for HCC in chronic HBV infection with genotype C. J. Med. Virol. 81:1531–1538, 2009. © 2009 Wiley‐Liss, Inc.     

The prevalence and long term outcome of occult hepatitis B virus infections in community based populations

Features of occult hepatitis B infection in community‐based populations have yet to be described. In this study we documented: (1) the prevalence and demographics, (2) associated serology and viral loads, and (3) clinical outcomes of occult hepatitis B infection in community‐based populations. Hepatitis B surface antigen (HBsAg)‐negative sera collected from three Northern Canadian communities (HBsAg prevalences: 11–12%) in 1983–1985 were tested for HBV‐DNA by nested stage polymerase chain reaction. Of 706 HBsAg negative sera, 9 (1.3%) were HBV‐DNA positive. The median age of occult hepatitis B infected patients at the time of sampling was 9.8 years (range 3.1–50.4 years) and six (67%) were female. Two (22%) individuals were anti‐HBs positive (in the absence of prior vaccination). Viral loads were undetectable in all but two samples (2.40 and 2.86 log₁₀ IU/ml). Only one of the five (20%) patients who were assessed clinically, remained HBV‐DNA positive at 25–30 year follow‐up. There was no clinical, biochemical or radiologic evidence of chronic hepatitis, cirrhosis or hepatocellular carcinoma in these individuals or on review of the charts from the remaining four infected patients. The results of this study suggest that in community‐based populations: (1) occult hepatitis B infection is not as common as HBsAg positive infection, (2) the majority of infected subjects are young females, (3) a minority are anti‐HBs positive, (4) viral loads are either undetectable or low, and (5) in the absence of concurrent liver disease, occult hepatitis B infection does not appear to be associated with long term adverse clinical outcomes. J. Med. Virol. 84:1369–1375, 2012. © 2012 Wiley Periodicals, Inc.     

282例HBV感染无症状表面抗原阳性者血清ALT与乙肝前S1抗原的检测

目的了解乙型肝炎病毒（HBV）感染无症状表面抗原阳性者血清ALT和乙肝前S1抗原的状况。方法用ELISA法检测血清标志物（HBsAg、抗HBs、HBeAg、抗HBe、抗HBc）和乙肝前S1抗原,用日立7060全自动生化分析仪检测血清ALT。结果 282例无症状表面抗原阳性者,乙肝前S1抗原总检出率为37.6%;ALT升高87例,占总人数的30.8%;ALT升高的87例患者中前S1抗原检出率为67.8%;ALT正常的195例患者中前S1抗原检出率为24.1%,两组比较差异有统计学意义（P〈0.01）。在282例无症状表面抗原阳性者中,HBeAg阳性组,乙肝前S1抗原检出率为73.9%,ALT异常阳性率为52.2%;HBeAg阴性组,乙肝前S1抗原检出率为31.0%,ALT异常阳性率为25.6%,两组比较差异有统计学意义（P〈0.01）。结论乙肝病毒携带者不能完全排除ALT正常情况下的病毒复制;血清中出现HBeAb并不一定表示HBV复制停止。因此,对于无症状表面抗原阳性者应进行定期复查,监测其乙肝血清标志物、ALT和前S1抗原。     

Seroprevalence of hepatitis B virus infection among children and adolescents in Singapore, 2008–2010

A national pediatric survey was undertaken to determine the prevalence of hepatitis B virus markers in Singapore. The aim was to assess the impact of the national childhood immunization program against hepatitis B implemented for all newborns since 1987. The survey involved prospective collection of residual sera from Singapore residents aged 1–17 years attending inpatient services or day surgery in two public hospitals between August 2008 and July 2010. A total of 1,200 sera were collected comprising 400 in each of the three age groups of 1–6, 7–12, and 13–17 years. The sera were tested for hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti‐HBs). Four of the 1,200 samples tested positive for HBsAg, giving an overall prevalence of 0.3%. One and three in the 7–12 years and 13–17 years age groups, respectively, were positive for HBsAg. About 40% possessed anti‐HBs (≥10 mIU/ml); the antibody prevalence decreased significantly from 63.8% in children aged 1–6 years to 32.8% in 7–12 year olds, and 23.5% in 13–17 year olds (P < 0.0005). The successful implementation of the national childhood hepatitis B immunization program over the last two decades has resulted in a low prevalence of HBsAg among children and adolescents. Singapore has achieved the World Health Organization Western Pacific Region's goal in reducing the prevalence of chronic HBV infection to below 2% among children aged 5 years and older by 2012 and to below 1% by 2017. J. Med. Virol. 85:583–588, 2013. © 2013 Wiley Periodicals, Inc.     

In vitro immune responses to hepatitis B surface antigen (pre-S2 and S) following remote infection by hepatitis B virus in humans

In this report we evaluate the human immune response to hepatitis B surface antigen (HBsAg) following remote infection with hepatitis B virus (HBV). HBsAg-reactive lymphocytes can be readily demonstrated in the peripheral blood of individuals with established immunity following infection with HBV.In vitro stimulation with small doses of plasma-derived HBsAg, yeast-derived HBsAg (S region) or pre-S2 peptide will induce specific IgG to HBsAg (anti-HBs) in the absence of a polyclonal increase in total IgG. The pre-S2 peptide will stimulate, in a T cell-dependent fashion, thein vitro production of anti-HBs with specificity for the S domain. This anti-HBs production is mediated by pre-S2-stimulated soluble T-cell factors. Peripheral blood mononuclear cells from individuals with established immunity proliferate to the yeast-derived HBsAg but not to the plasma-derived HBsAg or pre-S2 peptide. The chronic HBsAg carriers do not produce anti-HBs following stimulation with HBsAg regardless of the source or component of antigen used. Different study protocols failed to demonstrate HBsAg-specific responses in the peripheral blood mononuclear cells of chronic carriers.     

Reduced doses of hepatitis B immune globulin in the prevention of perinatal transmission of hepatitis B

From October 1982 to May 1983, newborn infants of 79 hepatitis B surface antigen (HBsAg)-positive women were enrolled in a study of the efficacy of hepatitis B immune globulin (HBIG) in the prophylaxis of perinatal transmission of hepatitis B virus (HBV) infection. HBIG 0.5 ml or 0.25 ml was given to the newborn within 15 minutes of birth and at 3 and 6 months. The mother-infant pairs were followed-up every 3 months for at least 9 months. Similar observations of untreated infants were used for comparison. Among infants of hepatitis B e antigen (HBeAg)-positive carrier mothers, the HBsAg carrier rates at 3 months were similar in the 0.5-ml and 0.25-ml HBIG dose groups. At 12 months the difference--17.7% of 17, 40% of 15--did not reach statistical significance, but the differences between these rates and that of the untreated control-85.7% of 35--did. Among infants of HBeAg-negative carrier mothers, HBV infection rates in both dose groups were similar to those of untreated infants. In the treated groups at 12 months about 45% of infants of HBeAg-positive mothers and 90% of infants of HBeAg-negative mothers were still negative for HBsAg and anti-HBs. Vaccination to induce active antibody is necessary to prevent postnatal infection and chronic carriage of HBV. To reduce the cost of combined passive and active hepatitis B immunoprophylaxis in children born to HBeAg-positive carrier mothers, 0.25 ml of HBIG could be used instead of the usually recommended 0.5 ml.     

山东省枣庄市乙型病毒性肝炎流行病学调查

目的　了解枣庄市人群中乙型肝炎的流行特征。方法　于 2 0 0 0年采用随机分层抽样 ,调查 312户家庭的 96 3人 ,以RIA法检测HBsAg、抗 HBs和抗 HBc。结果　HBsAg、抗 HBs、抗 HBc和HBV标化流行率分别为 7.0 8%、37.5 6 %、4 1.35 %和 4 4 .37%。HBsAg流行率男性高于女性 (P <0 .0 5 ) ,城区高于农村 (P <0 .0 1) ,在不同年龄及职业人群中差异无显著性 (P >0 .0 5 )。抗 HBs、抗 HBc和HBV感染率有随年龄增长而递增的趋势 (P <0 .0 1)。HBV总感染率男性高于女性 (P <0 .0 5 ) ,农村高于城市 (P <0 .0 5 )。结论　枣庄市人群HBV感染率较高 ,应积极采取预防和控制措施 ,减少发病。     

新生儿乙型肝炎疫苗免疫后13年效果观察

目的 研究乙型肝炎的远期免疫效果。方法 采用单纯随机方法连续13年对1986年出生并接种乙型肝炎疫苗的儿童进行隔年随访,采血检测HBsAg、抗-HBs、抗-HBc。结果 13年间HBsAg阳性率在0.46%-0.97%之间,未随免疫时间的延长而上升,乙型肝炎疫苗的远期保护效果为81.67%,与近期免疫效果相当。结论 免疫后13年仍无需加强免疫。     

Mutated epitopes of hepatitis B surface antigen fused to the core antigen of the virus induce antibodies that react with the native surface antigen

Fusion of peptide epitopes to the core antigen (HBcAg) of hepatitis B virus (HBV) enhances their immunogenicity, both quantitatively and qualitatively. In a number of vaccine-induced mutants of HBV, glycine₁₄₅ of the surface antigen S polypeptide (HBsAg) has been replaced by arginine, resulting in loss of cross-reactivity with antibodies to normal (wild-type) HBsAg. HBcAg fusion proteins carrying the immunodominant epitope of HBsAg, in which glycine₁₄₅ was replaced by arginine, glutamic acid, or lysine, were produced in Escherichia coli and formed particles that displayed HBc antigenicity and immunogenicity similar to that of HBcAg itself. The fusion proteins also elicited T-cell-proliferative responsiveness to HBcAg and HBsAg. Fusions carrying either wild-type or mutated epitopes of HBsAg showed HBs antigenicity in immunoblot analysis and antigen-capture immunoradiometric assay, but both mutant and wild-type derivatives induced antibodies that cross-reacted with wild-type HBsAg. The results emphasise the potential for HBcAg fusion proteins in vaccines by broadening the antibody response in a way that could confer protection against both wild-type and variant forms of HBV. J. Med. Virol. 51:159–166, 1997. © 1997 Wiley-Liss, Inc.