细胞内快速连续传代培养甲肝病毒的研究

目的:研究甲肝病毒在细胞内快速连续传代培养病毒增殖情况,探索疫苗生产工艺改进的可能性。方法:将甲肝病毒吕8株感染KMB17细胞后形成带毒细胞,每7 d传代1次,检测每次收获物的甲肝病毒抗原(HAAg)滴度及感染性滴度,比较滴度的变化,同时检测一步生长曲线,分析病毒增殖特性有无变化。结果:甲肝病毒吕8株感染KMB17细胞后每7 d传代1次,其第1~8代抗原滴度为1 024~2 048 Eu/0.1 ml,感染性滴度为7.33~7.67 lg CCID50/ml,第9代开始下降,抗原滴度仅为256 Eu/0.1 ml,感染性滴度为6.83~7.00 lg CCID50/ml;在连续传10代内,细胞均未出现CPE现象;第10代与第1代相比,其一步生长曲线病毒增殖高峰均为20~25 d,没有明显变化。结论:甲肝病毒吕8株在KMB17细胞上每7 d传代1次,在第8代以内,其抗原滴度及感染性滴度均能达到较高的程度。经细胞内连续快速传代后,HAV吕8株生长的增殖高峰约为第20~25 d,与传代前样品没有明显差别。     

微量免疫荧光法检测甲肝减毒活疫苗病毒滴度

[目的]建立一种简便、灵敏的微量免疫荧光法检测甲肝减毒活疫苗感染性滴度。[方法]取适宜稀释度病毒接种细胞已成致密单层的96孔培养板,至增殖高峰时直接在板上固定细胞/病毒系统,用间接免疫荧光法观察结果,以Reed-Muench公式计算CCID50。[结果]随着培养时间的延长,感染性滴度逐渐增高,18d可达增长高峰,比常规组织培养ELISA法提前1周左右;此法的重复性良好,灵敏度与常规组织培养法相似(P>0.05);可用Leica Qwin荧光定量分析系统标定化判断结果。[结论]此法具有简便、灵敏、省时的优点,可代替常规组织培养ELISA法应用于甲肝减毒活疫苗感染性滴度检测中。     

水痘病毒在感染HAV2BS细胞中形态变化

目的分析水痘病毒(VZV)在感染甲肝病毒(HAV)的二倍体成纤维细胞(2BS细胞)中的形态发生过程.方法将已感染甲肝病毒21 d后的2BS细胞感染水痘病毒(实验组),分别于不同时间收获细胞,用超薄切片和负染的方法制备电镜样品;同时以单独感染HAV、VZV的2BS细胞做对照,进行电镜观察.结果实验组2BS细胞中可见甲肝病毒和不同形态的水痘病毒.结论VZV可以再度感染已被HAV感染的2BS细胞并有完整的子代病毒产生;VZV在已感染HAV的2BS细胞中的增殖受到甲肝病毒干扰,增殖数量少于VZV单独感染2BS细胞中的子代病毒量,并且复制周期延长.     

毛蚶中甲肝病毒净化实验研究

[目的 ]　研究被甲肝病毒污染的毛蚶净化方法。　 [方法 ]　采用二氧化氯 (ClO2 )、臭氧 (O3)和紫外线等物理和化学联合方法 ,通过循环水系统 ,净化被人工染毒的毛蚶。　 [结果 ]　应用ClO2 、O3和紫外线不同组合方法净化 4d能有效灭活甲肝病毒浓度 (TCID50 )为 1 0 4 的人工染毒毛蚶中的甲肝病毒 (HAV)。　 [结论 ]　ClO2 、O3和紫外线联合净化方法具有灭活毛蚶体内HAV的作用 ,但其效果与净化时间和毛蚶体内HAV浓度密切相关。     

海捕毛蚶中分离到甲型肝炎病毒

应用电镜和超薄切片技术、免疫荧光(IF)试验、酶联免疫试验、核酸分子杂交技术、病毒感染滴度测定及中和试验等方法,直接从海捕毛蚶中检测和分离到三株甲肝病毒(HAV),并在人二倍体(2BS)细胞上稳定传代,感染滴度在4.0~5.0TCID₅₀/ml之间。初步结果证实,海捕毛蚶受污染并携带甲肝病毒,可成为甲肝流行的潜在危险因素。     

SYB GRreenⅠ实时PCR检测甲肝病毒方法的建立与初步应用

目的：建立一种快速、特异的SYBR GreenⅠ实时PCR方法检测甲肝病毒。方法：对SYBR GreenⅠ实时PCR检测甲肝病毒方法的反应条件、反应体系进行优化,同时与传统RT-PCR方法进行灵敏度比较,提高该方法的特异性、灵敏性。并用该方法对甲肝病毒毒株、脊髓灰质炎病毒、柯萨奇病毒（1～6型）毒株、贝类水产品标本进行检测。结果：SYBR GreenⅠ实时PCR与传统RT-PCR检测甲肝病毒方法的灵敏度无明显区别,但检测时间缩短了1/2。该方法能特异检测出甲肝病毒,不能扩增脊髓灰质炎病毒、柯萨奇病毒（1～6型）毒株,对农贸市场40份贝类水产品进行甲肝病毒核酸检测,检出3份。结论：建立的SYBR GreenⅠ实时PCR方法用于甲肝病毒检测具有特异、灵敏、快速等优点,可用于甲肝的病原学监测。 基金 上海市医学重点学科建设项目（05Ⅲ029）     

SYBR Green Ⅰ实时PCR检测甲肝病毒方法的建立与初步应用

目的建立一种快速、特异的SYBR Green Ⅰ实时PCR方法检测甲肝病毒.方法对SYBR Green Ⅰ实时PCR检测甲肝病毒方法的反应条件、反应体系进行优化,同时与传统RT-PCR方法进行灵敏度比较,提高该方法的特异性、灵敏性.并用该方法对甲肝病毒毒株、脊髓灰质炎病毒、柯萨奇病毒(1～6型)毒株、贝类水产品标本进行检测.结果SYBR Green Ⅰ实时PCR与传统RT-PCR检测甲肝病毒方法的灵敏度无明显区别,但检测时间缩短了1/2.该方法能特异检测出甲肝病毒,不能扩增脊髓灰质炎病毒、柯萨奇病毒(1～6型)毒株,对农贸市场40份贝类水产品进行甲肝病毒核酸检测,检出3份.结论建立的SYBR Green Ⅰ实时PCR方法用于甲肝病毒检测具有特异、灵敏、快速等优点,可用于甲肝的病原学监测.     

人用SARS病毒灭活疫苗的研究

[目的]研制出安全、有效、质量可控的人用SARS病毒灭活疫苗。[方法]建立了生产疫苗用的Vero细胞库并进行了检定，分离培养可稳定传代的SARS病毒，对其生物学特性进行了研究；建立了适用于SARS疫苗生产的细胞库和可用于疫苗生产的病毒种子批；优化确定SARS疫苗的灭活工艺和纯化工艺；通过动物实验，对其安全性、有效性进行了研究。[结果]建立了符合SARS病毒灭活疫苗生产使用的细胞库；分离获得了可稳定传代的用于疫苗生产的SARS病毒株；建立了适用于产业化的SARS病毒的灭活工艺和纯化工艺；制备的人用SARS灭活疫苗经小鼠、豚鼠、家兔、恒河猴不同种动物试验，免疫后动物可产生较高SARS病毒中和抗体，可以有效防护SARS病毒对动物的攻击。安全性实验表明，该疫苗未见毒副反应。[结论]试验研制的人用SARS病毒灭活疫苗具有较强的免疫原性，设计的灭活疫苗的生产工艺是可行的，并获得了国家食品药品监督管理局颁发的临床研究批文，待进一步研究后，可望进行规模化生产。     

反转录聚合酶链反应检测水产品中甲型肝炎病毒的研究

目的：建立一种特异、灵敏的RT-PCR方法检测水产品中甲肝病毒。方法：采用抗体捕获RT-PCR和直接RNA提取RT-PCR对细胞传代的甲肝病毒、模拟染毒水产品中甲肝病毒、上海农贸市场水产品中的甲肝病毒进行检测。结果：2种RT-PCR检测细胞传代的甲肝病毒，检测灵敏度无明显区别；而对模拟染毒水产品中甲肝病毒的检测，两者检测灵敏度有显著差异。对农贸市场94份水产品中的甲肝病毒检测，检出率分别为2．13％、13．83％。结论：对于水产品中的甲肝病毒的检测，直接RNA提取RT-PCR要比抗体捕获RT-PCR方法灵敏。     

孩尔来福甲型肝炎灭活疫苗用于加强免疫的研究

为了观察孩尔来福 (Healive○R)甲型肝炎 (甲肝 )灭活疫苗用于加强免疫的效果 ,对 1～ 10岁 1年内曾接种过甲肝减毒活疫苗而血清学检测 [酶联免疫吸附试验 (ELISA) ]抗甲肝病毒抗体 (抗 HAV)阴性的 70名农村儿童 ,加强免疫 1剂Healive○R甲肝灭活疫苗 ,并于加强接种后 1个月采血检测 (ELISA)甲肝抗 HAV。结果显示 :所有加强免疫的儿童抗 HAV阳转率为 10 0 0 % ,几何平均滴度 (GMT)为 30 13mIU/ml;并且与接种甲肝减毒活疫苗的时间间隔不同抗 HAVGMT也不同 ,间隔时间长者高于间隔时间短者 ,与不同免疫程序接种 2剂Healive○R甲肝灭活疫苗的免疫效果规律一致 ,但显著低于 2剂Healive○R的抗 HAVGMT。Healive○R甲肝灭活疫苗对有甲肝减毒活疫苗接种史的儿童进行加强免疫 ,可使其产生较高的抗 HAVGMT。     

利用Labworks凝胶成像分析系统判断甲肝减毒活疫苗RT-PCR滴度

目的利用Labworks凝胶成像分析系统判断甲肝减毒活疫苗RT-PCR滴度,并比较此方法与常规组织培养法的相关性.方法通过对RT-PCR反应液的离子浓度、pH值等条件进行优化,建立了检测甲肝减毒活疫苗滴度的RT-PCR一步法;利用Labworks凝胶成像分析系统的半定量功能判断结果,并与常规组织培养法比较.结果利用Labworks凝胶成像分析系统判断结果的RT-PCR一步法与常规组织培养法检测甲肝减毒活疫苗滴度灵敏度相仿,检测结果无显著差异.结论此法与常规RT-PCR法相比具有更简便,判断标准更客观的优点,可代替组织培养法检测甲肝减毒活疫苗滴度.     

Development,Production, and Postmarketing Surveillance of Hepatitis A Vaccines in China

China has long experience using live attenuated and inactivated vaccines against hepatitis A virus (HAV) infection. We summarize this experience and provide recent data on adverse events after immunization (AEFIs) with hepatitis A vaccines in China. We reviewed the published literature (in Chinese and English) and the published Chinese regulatory documents on hepatitis A vaccine development, production, and postmarketing surveillance of AEFI. We described the safety, immunogenicity, and efficacy of hepatitis A vaccines and horizontal transmission of live HAV vaccine in China. In clinical trials, live HAV vaccine was associated with fever (0.4%–5% of vaccinees), rash (0%–1.1%), and elevated alanine aminotransferase (0.015%). Inactivated HAV vaccine was associated with fever (1%–8%), but no serious AEFIs were reported. Live HAV vaccine had seroconversion rates of 83% to 91%, while inactivated HAV vaccine had seroconversion rates of 95% to 100%. Community trials showed efficacy rates of 90% to 95% for live HAV and 95% to 100% for inactivated HAV vaccine. Postmarketing surveillance showed that HAV vaccination resulted in an AEFI incidence rate of 34 per million vaccinees, which accounted for 0.7% of adverse events reported to the China AEFI monitoring system. There was no difference in AEFI rates between live and inactivated HAV vaccines. Live and inactivated HAV vaccines manufactured in China were immunogenic, effective, and safe. Live HAV vaccine had substantial horizontal transmission due to vaccine virus shedding; thus, further monitoring of the safety of virus shedding is warranted.Key words: hepatitis A, vaccine, safety, efficacy     

转移因子对甲肝疫苗的热稳定性及免疫佐剂作用

转移因子(TF)具有增强甲肝疫苗热稳定性的作用,其效果与1M的MgCl₂相当,加入TF的甲肝疫苗在4～8℃保存6个月、室温保存21天、37℃保存7天,滴度基本不变,仍在6.5LgCCID₅₀/ml的合格范围内.当提高保存温度时,这种作用效果更明显;另外,TF还具有一定的免疫佐剂作用,甲肝疫苗与TF混合后免疫小白鼠,阳转率(10/10)比对照组(8/10)高,几何平均滴度是对照组的1.63倍.     

Detection of antibodies to HAV 3C proteinase in experimentally infected chimpanzees and in naturally infected children

Commercial assays for the diagnosis of hepatitis A detect antibody to hepatitis A virus (anti-HAV), but they cannot discriminate between antibody resulting from infection and antibody induced by inactivated vaccine. With the licensing and increasing use of inactivated hepatitis A vaccines, there is a need for a test to distinguish between infection and vaccination. Since antibodies to viral non-structural proteins are elicited by infection but not by vaccination with inactivated vaccine, we developed and evaluated a test for such antibodies. The antibody response to the non-structural 3C proteinase (anti-3C) of virus HAV was studied by ELISA in chimpanzees experimentally infected with virulent (wild type) or with attenuated HAV strains and in children who received inactivated HAV vaccine or placebo during a vaccination trial in Nicaragua. Anti-3C was detected in 89% of 18 chimpanzees infected with wild-type HAV strains and 27% of 26 chimpanzees infected with attenuated HAV strains. There was a direct correlation between severity of hepatitis and magnitude of the anti-3C response. In the vaccine trial, anti-3C was detected only in children who were infected with HAV during the study; IgG anti-3C persisted for at least 15 months after infection in one child. Vaccinated and uninfected children remained negative for anti-3C. The anti-3C response can be regarded as an indicator of viral replication. Its detection should be useful for distinguishing between antibody acquired in response to HAV infection and antibody induced by immunization with inactivated vaccine.     

氯灭活甲型肝炎病毒机理研究

目的：探讨氯灭活水中HAV机理及RT－PCR在评价消毒效果中的应用价值。方法：采用细胞培养、ELISA及大片段逐步步移RT－PCR对消毒前后HAV的感染性、抗原及核酸全序列进行检测。结果：在10或20mg/L的加氯量接触时间30min以上可完全灭活HAV的感染性,而病毒抗原性灭活需要10mg/L加氯量接触60min。HAV核酸5'非编码区（5'NTR）对氯最敏感,其灭活时间与病毒的感染性灭活一致。结论：氯灭活HAV是由于病毒核酸5'NTR的损伤。PCR技术既可用于消毒机理研究,也可用于病毒消毒效果评价。     

Immune memory at 17-years of follow-up of a single dose of live attenuated hepatitis A vaccine

BackgroundIn recent years, hepatitis A virus (HAV) infection has declined considerably in China, associated with wide deployment of HAV vaccines and improvement in socio-economic indicators. Towards the elimination of HA in the country, we assessed the duration and characteristics of immunity conferred by the widely used, locally manufactured HAV vaccine.MethodsThis is a longitudinal cohort study that followed recipients of a live attenuated HAV vaccine 17 years after the initial administration. Blood samples were collected from participants pre- and two-week post-booster HAV vaccine dose. Serum anti-HAV antibody was measured by ELISA method. Memory B and T cells were determined by ELISPOT and Flow Cytometry assays, respectively.ResultsA robust anamnestic response was observed two-week post-challenge. Both HAV-specific memory B cell and T cells remained, and responded quickly when re-encountering HAV. The magnitude of recall responses was present, regardless of the status of the serum anti-HAV antibody pre-booster.ConclusionsWe demonstrated long-term immunity from the live attenuated HAV vaccine, including antibody persistence and immunological memory. Considering the conditions that make elimination of infectious diseases feasible, following polio, hepatitis A could be targeted for elimination in China.     

国产甲肝减毒活疫苗(IA-1株)与进口甲肝灭活疫苗的2年免疫效果比较

目的　比较国产甲肝减毒活疫苗和进口甲肝灭活疫苗免疫效果。方法　在广西柳城县筛选出 6～ 1 2岁甲肝易感儿童 2 1 7人 ,以个体随机分为 4个组。B、C组接种国产甲肝减毒活疫苗 (LA - 1株 ,滴度 1 0 6 75 TCID5 0 ) ,接种程序分别为 0 - 6和 0 - 1 2 ;D组接种低滴度国产甲肝减毒活疫苗 (LA - 1株 ,滴度 1 0 6 0 TCID5 0 ) ,E组接种史克公司市售甲肝灭活疫苗 (贺福立适 ,72 0E1 .U) ,接种程序均为 0 - 6。各组于第 1针免后 1 ,6 ,1 2 ,2 4个月 ,第 2针免后 1个月采血 ,用美国ABBOTT公司的ImxmEIA试剂 ,检测甲肝抗体IgG。 结果　滴度 1 0 6 75 TCID5 0 的国产减毒活疫苗两种程序 ,除 0 - 6程序组在第 2针免后 1个月的抗体水平峰值较低外 ,其余各次随访的抗体反应结果均与进口甲肝灭活疫苗相似。低滴度减毒活疫苗 ( 1 0 6 0 TCID5 0 )的抗体阳性率和抗体水平均低于其他各组。结论　滴度 1 0 6 75 TCID5 0 国产甲肝减毒疫苗与进口甲肝灭活疫苗免疫效果相近 ,具有良好的免疫原性和很好的回忆反应 ,加强免疫可以提高疫苗的保护效果和延长保护年限。     

铝吸附甲型肝炎灭活疫苗抗原解离方法研究

目的建立铝吸附甲型肝炎（甲肝）灭活疫苗中甲肝病毒（HAV）抗原的解离方法。方法选择20％二乙醇胺1．25mL、10％TritonX．1000．2mL和PBS8．55mL混合液作为解离液A;pH7．2的3％（w／v）柠檬酸缓冲液作为解离液B;20％（w／v）柠檬酸三钠为解离液C。分别检测3种解离液在不同的解离条件下对铝佐剂甲型肝炎灭活疫苗的解离情况,用ELISA法检测解离后的HAV抗原含量,计算抗原回收率,确定最佳解离液及解离条件,并验证其可重复性。结果3种解离液HAV抗原解离试验显示,解离液C在室温（20~28℃）条件下解离1h,HAV抗原的回收率最高,可达95％及以上,重复试验的变异系数均〈10％。结论20％（w／v）柠檬酸三钠可作为铝佐剂甲肝灭活疫苗的首选抗原解离剂,该解离剂配方简单,操作简便,结果重复性好,准确度高。     

Dose range evaluation of a new inactivated hepatitis A vaccine administered as a single dose followed by a booster

A total of 242 healthy adults were immunised with a first dose of an investigational inactivated hepatitis A vaccine. Three concentrations (3, 6 and 12 EU [ELISA units]) of the experimental vaccine were used and compared to a licensed reference vaccine. The aim was to determine the antigenic concentration of the study vaccine inducing the highest seroconversion rate and anti-Hepatitis A virus (HAV) antibody response at 2 weeks after the primary immunisation. A booster dose was given at month 6. At 15 days after the primary immunisation the seroconversion rates in subjects vaccinated with the 6 and 12 EU vaccines were 78 and 94%, respectively. At 30 and 180 days after the primary immunisation the percentages of seropositivity were 100% for both groups. The antibody response to the 12 EU study vaccine was similar to that to the reference vaccine. The percentages of seropositivity at 15 and 180 days after the primary immunisation were 94 vs 93%, and 100 vs 93% in the experimental and reference vaccine respectively. Thus, because it induces early and lasting seroconversion, the 12 EU study vaccine seems to be the most effective as a high potency HAV vaccine.