Vero细胞在无血清条件下制备乙型脑炎灭活疫苗

目的 探索Vero细胞在无血清条件下制备乙型脑炎灭活疫苗的工艺. 方法 用无血清培养基培养Vero细胞,并将乙型脑炎病毒P3V2株毒种接种于培养的Vero细胞,观察细胞和病毒的生长情况.收获病毒液,通过灭活、纯化制备乙型脑炎灭活疫苗,检测疫苗各项指标,并与有血清培养工艺制备的疫苗(2013年生产的疫苗)进行比较.结果 无血清培养基培养的Vero细胞生长良好,培养5d的细胞呈致密单层生长.3批无血清培养工艺制备的疫苗的第1、2和3次收获液的平均病毒滴度分别为8.847、8.319和7.194 lgLD50/ml.3批无血清培养工艺制备的疫苗半成品的平均抗原含量(1.51 EU/ml)与有血清培养工艺制备的疫苗(1.41 EU/ml)相当,但前者的宿主细胞蛋白含量(19.69 μg/L)则明显低于后者(63.90 μg/L).结论 乙型脑炎病毒可在无血清Vero细胞中良好生长,Vero细胞无血清培养工艺制备乙型脑炎灭活疫苗是可行的.     

Vero细胞HCP试剂盒在冻干乙型脑炎灭活疫苗生产过程中的应用

目的 通过检测冻干乙型脑炎灭活疫苗（Vero细胞）生产过程各道工序中的细胞残余蛋白,达到控制疫苗中Vero细胞残余蛋白的目的.方法 采用Vero细胞宿主细胞蛋白（host cell protein,HCP）试剂盒,对2009-2012年共65批冻干乙型脑炎灭活疫苗（Vero细胞）在病毒液收获、浓缩、硫酸鱼精蛋白处理、纯化、脱糖和半成品配制阶段中的HCP含量进行检测.结果 2009-2012年,病毒液收获阶段Vero细胞HCP含量无明显差异.2010年工艺优化后,2010-2012年的HCP平均值与2009年相比,在浓缩、硫酸鱼精蛋白处理、纯化、脱糖、半成品配制阶段分别降低了78.83％、83.42％、78.62％、86.88％、86.40％.结论 通过Vero细胞HCP试剂盒的动态监测,可以对生产过程中Vero细胞的残余蛋白含量实施控制.     

乙型脑炎疫苗IC51

澳大利亚Intercell AG公司开发的乙型脑炎病毒(Japanese encephalitis virus,JEV)疫苗IC51,为培养于Vero细胞的SA14-14-2毒株经纯化和甲醛灭活制得,该疫苗于2009年上半年,分别获得澳大利亚治疗产品管理局(Therapeutic Goods Administration,TGA)、美国FDA和欧洲药品管理局(European medicines agency,EMEA)批准,用于成人预防乙型脑炎.     

无明胶保护剂冻干乙型脑炎灭活疫苗（Vero细胞）稳定性研究

目的  观察不含明胶的不同保护剂配方冻干乙型脑炎灭活疫苗（Vero细胞）的稳定性。方法  在现行冻干乙型脑炎灭活疫苗制备工艺的基础上,以乳糖替代明胶,并适当增加人血白蛋白用量至5、10和20 g/L,制成A、B、C三个保护剂配方疫苗。对疫苗进行25 ℃加速试验、37 ℃热稳定性试验和2～8 ℃长期稳定性试验,检测疫苗的有效抗原含量和效力（T值）,并与现行疫苗（对照）进行比较。结果  三个保护剂配方疫苗于25 ℃放置24周后,A、B疫苗有效抗原含量仅分别降低2.2%和5.9%,效力稳定;而C疫苗有效抗原含量降低13.6%,效力降低5.0%。37 ℃放置8周后,A、B、C疫苗有效抗原含量分别降低11.2%、13.2%、15.2%,效力分别降低5.8%、9.0%、7.2%。2～8 ℃放置48个月后,A、B、C疫苗有效抗原含量分别降低17.2%、18.4%、21.9%,效力均符合《冻干乙型脑炎灭活疫苗（Vero细胞）注册标准》。在上述试验中,A、B疫苗均优于或等于对照疫苗;C疫苗与对照疫苗差异较大,但均符合相关标准。结论  含A配方保护剂的冻干乙型脑炎灭活疫苗稳定性高且白蛋白用量少,因此,建议首选A配方保护剂。     

乙型脑炎病毒及疫苗研究进展

乙型脑炎病毒是黄病毒科中致病性最强的病毒之一,目前对该病毒的分子生物学研究进展较快.现阶段国内外主要使用三种类型疫苗,即鼠脑纯化灭活疫苗、原代地鼠肾细胞灭活疫苗和原代地鼠肾细胞减毒活疫苗.灭活疫苗和减毒活疫苗各有利弊.基因工程疫苗(包括载体重组活疫苗、嵌合减毒活疫苗、病毒样颗粒、亚单位疫苗及DNA疫苗)成为乙型脑炎疫苗发展的方向.     

乙型脑炎瘦苗IC51

澳大利亚IntercellAG公司开发的乙型脑炎病毒（Japaneseencephalitisvirus，JEV）疫苗IC51，由培养于Vero细胞的SA圹14-2毒株经纯化和甲醛灭活制得，该疫苗于2009年上半年分别获得澳大利亚治疗产品管理局（TherapeuticGoodsAdministration，TGA）、美国FDA和欧洲药品管理局（EuropeanMedivinesAgency，EMEA）批准，用于成人预防乙型脑炎。     

在Vero细胞中制备的纯化乙型脑炎灭活疫苗

目前只有一种经美国食品和药物管理局批准的乙型脑炎疫苗 ,即日本 BIKEN公司生产的鼠脑纯化灭活疫苗。尽管日本报道其安全性很好 ,但自 1986年以来美国、欧洲和澳大利亚接种者中副反应达 10 %～ 2 0 % ,如 :发热、头痛 ,局部红肿 ,并有较多的变态反应报告 ,如荨麻疹、面部血管神经性水肿伴呼吸困难。本文作者应用乙型脑炎减毒活疫苗SA14 - 14 - 2狗肾细胞株 (SA14 - 14 - 2 PDK)在Vero细胞传 5代适应后作为疫苗病毒株(JEV SA14 - 14 - 2 ,PDK- 8,Vero- 5 )。病毒接种 Vero细胞 35℃培养。接种后 3、5、7、9天收获病毒液。经过粗离…     

接种甲型肝炎减毒活疫苗致类甲肝反应1例

我县1998-07某街道接种甲型肝炎减毒活疫苗110人次,发生1例以黄疸为主的类肝炎反应.该儿童平时身体健康,无药物过敏史,未患过病毒性肝炎,亦无肝炎病接触史,在40d内无外出史及输血史,基本排除接种感染甲肝的可能性.患儿曾接种过卡介苗、乙型肝炎疫苗、百白破三联、麻疹减毒活疫苗、脊髓灰质炎疫苗、乙型脑炎疫苗和流脑疫苗,均无不良反应,附病历如下.     

WHO关于乙型脑炎疫苗的意见书

乙型脑炎是亚洲最重要的病毒性脑炎,目前大规模使用的乙型脑炎疫苗有鼠脑纯化灭活疫苗、细胞培养灭活疫苗和细胞培养减毒活疫苗3种类型.本文介绍了乙型脑炎病毒、疫苗的使用情况以及WHO关于疫苗及其使用方面的意见.     

冻干Vero细胞狂犬病纯化疫苗在120例门诊病人中的临床效果

目的:观察国产冻干非洲绿猴肾传代细胞(Vero细胞)为基质制备的狂犬病纯化疫苗临床应用的免疫效果和安全性。方法:选择门诊部就诊的犬致伤者240例,分为试验组和对照组。试验组接种冻干Vero细胞狂犬病纯化疫苗,对照组接种地鼠肾狂犬病纯化疫苗。2组人群均按照狂犬病疫苗暴露后常规免疫程序进行接种,于全程接种后15d测定中和抗体,并观察不良反应。结果:试验组抗体阳性率为96.7%,明显高于对照组的90.0%,P<0.05;不良反应发生率为10.8%,明显低于对照组的21.7%,P<0.05。结论:Vero细胞纯化疫苗抗体阳转率高、不良反应发生率低,临床应用优于地鼠肾纯化疫苗,适宜推广使用。     

Sabin株脊髓灰质炎病毒灭活疫苗的制备及免疫原性评价

目的  建立Sabin株毒种制备脊髓灰质炎灭活疫苗（inactivated poliovirus vaccine, IPV）生产工艺并评价其免疫原性。方法  采用Vero细胞培养Sabin株脊髓灰质炎Ⅰ、Ⅱ、Ⅲ型病毒,制备3价Sabin株IPV（Sabin strain IPV, sIPV）。通过ELISA检测D抗原含量。检测病毒滴度,电子显微镜下观察纯化病毒形态并电泳鉴定,分析抗原纯度,同时检测纯化原液宿主细胞残余DNA和蛋白含量。通过大鼠体内接种法比较sIPV和野生株IPV免疫原性,并观察中和抗体滴度变化。结果 Ⅰ、Ⅱ、Ⅲ型收获液D抗原含量分别为2 437、365、1 253 DU/ml,病毒滴度分别为每毫升8.4 、7.8和7.9 lg半数细胞培养物感染量。纯化病毒抗原纯度均﹥99%。电子显微镜下观察可见直径20~30 nm病毒颗粒,电泳鉴定正确。纯化原液的残余宿主细胞DNA和蛋白含量分别12.3 pg/500 μl和9.8 ng/ml。sIPV 3针免疫后在大鼠体内诱导产生抗Ⅰ、Ⅱ、Ⅲ型中和抗体的几何平均滴度分别为9 397.8、554.7和4 668.4,与野生株IPV相比Ⅰ型明显增高（t=-3.429,P=0.004）,Ⅱ、Ⅲ型无明显差别,初免后19周内均维持在较高水平。结论 建立了稳定的sIPV生产工艺,制备的sIPV免疫原性较好。     

1例儿童接种流行性乙型脑炎疫苗与川崎病相关性的调查分析

目的探讨接种灭活乙脑疫苗与川崎病发病的相关性,为疫苗安全接种提供理论依据。方法采用流行病学调查的方法对1名女童在菏泽市开发区岳程办事处某卫生室接种灭活乙脑疫苗后发生接种反应并被医院确诊为川崎病的事件进行调查分析。结果该女童接种灭活乙脑疫苗后引起了预防接种异常反应,该儿童很可能在接种灭活乙脑疫苗之前就处于川崎病发生的潜伏期,接种疫苗可能是其促发因素之一,或者川崎病只是接种灭活乙脑疫苗的偶合症,其具体原因,需要进一步考证。结论接种乙脑灭活疫苗不是本病例川崎病发生的直接原因,二者之间无必然联系。     

Immunogenicity of a scalable inactivated rotavirus vaccine in mice

Inactivated rotavirus vaccine is a safe and effective potential vaccine for the prevention of rotavirus infection among children, but no approved licensed vaccine is available now. In this study, a scalable inactivated rotavirus vaccine, prepared in Vero cells cultured by microcarrier fermentation, inactivated by formalin and absorbed by Al(OH)(3) adjuvant, was vaccinated into the six weeks-old female Balb/c mice by intramuscular injection. After twice immunization at interval of three weeks, both humoral and cell-mediated immune responses were assessed by ELISA, microneutralization assay and EISPOT assay. The results indicated that the scalable inactivated rotavirus vaccines induced not only high serum IgG antibody and neutralizing antibody responses, but Th1 and Th2 cytokine-secreting cell responses in mice immunized by the inactivated rotavirus vaccines. These results suggest that the scalable inactivated rotavirus vaccine has good immunogenicity, which provided the base for the scaled development of inactivated rotavirus vaccine in the future.     

埃博拉病毒疫苗最新研究进展

埃博拉病毒(Ebola virus,EBOV)是引起人类和非人灵长类动物严重出血热的最危险病原体之一,1976年首次得到报道.2014年撒哈拉以南非洲暴发的EBOV病的病死率高达90％.研究表明EBOV灭活疫苗无效,但多个新型EBOV候选疫苗已在临床前试验中显示有效,其中部分已进入临床试验.这些新型疫苗包括病毒载体疫苗、病毒样颗粒疫苗和核酸疫苗.此文对EBOV疫苗的研究进展做一综述.     

Immunogenicity of a thermally inactivated rotavirus vaccine in mice

Current approaches to the prevention of severe rotavirus diarrhea and deaths in children have all been through the use of live oral vaccines. To develop a safe and effective inactivated rotavirus vaccine (IRV), a new simple, rapid and robust method for the inactivation is critical and essential because chemical inactivation commonly used for a number of killed vaccines has been a challenge and problematic for rotavirus. We have examined an array of thermal conditions and demonstrated that purified YK-1 rotavirus in diluent buffer can be completely inactivated by heat treatment, as evidenced by the lack of virus growth in two successive passages in cell culture. Unlike chemical treatment that often causes physical and biochemical damages to viruses, thermally inactivated rotavirus particles maintained their structural, biochemical and antigenic integrity. A two-dose intramuscular administration of thermally inactivated YK-1 rotavirus without adjuvant resulted in high titers of total and neutralizing antibody in serum of mice. Adjuvant Al(OH)(3) further led to enhanced antibody titers and also dramatically lowered the amount of antigens in the vaccine formulation. Our results demonstrate the potential of heat inactivation as a novel approach to the manufacture of a safe and efficacious parenteral rotavirus vaccine, which should serve as an important addition to and back up for live oral rotavirus vaccine in children.     

A/H5N1 prepandemic influenza vaccine (whole virion, vero cell-derived, inactivated) [Vepacel®

The influenza A subtype H5N1 virus is a likely causative agent for the next human influenza pandemic. Pandemic influenza vaccine production can begin only after a novel pandemic virus emerges. Cell-based vaccine production has advantages over conventional egg-based methods, allowing more rapid large-scale vaccine production. A reliable Vero cell culture system is available for pandemic and prepandemic influenza vaccine production. Prepandemic influenza vaccines are an important component of influenza pandemic preparedness plans, as their targeted use in the pandemic alert period or early in a pandemic is likely to mitigate the consequences of an influenza outbreak. Vepacel? is a prepandemic influenza vaccine (whole virion, Vero cell-derived, inactivated) containing antigen of H5N1 strain A/Vietnam/1203/2004 and is approved for use in the EU. Clinical immunogenicity studies with the vaccine have demonstrated good rates of functional neutralizing antibody responses against the vaccine strain (A/Vietnam/1203/2004), meeting established immunogenicity criteria for seasonal influenza vaccines, and cross-reactivity against H5N1 strains from other clades. In phase I/II and III studies, a heterologous (A/Indonesia/05/2005) booster vaccine administered to healthy adult and elderly volunteers 6-24 months after the two-dose priming vaccine (A/Vietnam/1203/2004) regimen induced good immunogenic responses against both H5N1 strains, demonstrating strong immunological memory. Broadly similar, albeit less robust, responses were observed in two special risk cohorts of immunocompromised and chronically ill patients. In general, adverse events observed in clinical immunogenicity studies with H5N1 vaccine (A/Vietnam/1203/2004) were similar to those reported with non-adjuvanted, inactivated, seasonal influenza vaccines.     

乙型脑炎减毒活疫苗病毒群体的异质性分析

 目的  通过观察疫苗病毒群体中病毒个体的遗传和生物学特征,分析SA14-14-2株乙型脑炎减毒活疫苗病毒的均一性,为疫苗病毒的遗传稳定性提供实验证据。 方法   对3批疫苗病毒连续进行3次蚀斑纯化后,各挑选8个蚀斑纯化株,扩大培养一代。比较24个病毒纯化株的包膜（envelope,E）蛋白基因序列、单斑培养病毒滴度以及蚀斑特征。 结果   24个纯化株中共有6个病毒株（25%）的E蛋白核苷酸发生变化,其中2株（8.3%）的核苷酸变异导致其编码氨基酸发生改变,这些变异占总核苷酸变异数的28.6%。动物实验表明,这2个纯化株没有引起小鼠神经毒力变化。在所有24个纯化株中,我国2010年版药典规定的影响疫苗病毒遗传稳定性的8个E蛋白关键位点的氨基酸均未发生改变。24个纯化株病毒的蚀斑大小和形态以及单斑培养滴度均无明显差异。 结论   疫苗病毒具有典型的准种群体多样性特征,但在群体病毒中未检测到E蛋白氨基酸位点为野生型病毒位点的个体病毒,因此,疫苗病毒具有很好的减毒群体特征和遗传稳定性。     

Japanese encephalitis for a reference to international travelers

Japanese encephalitis (JE) is an inflammatory disease in the central nervous system caused by infection with Japanese encephalitis virus (JEV). JE is a disease with a high fatality rate and endemic and epidemic in East, Southeast, and South Asia. High morbidity is noted in children living in the endemic area. JEV is maintained mainly between vector mosquitoes and pigs in nature. The risk of JE increases as the number of vector mosquitoes increases. The expansion of JEV-endemic area depends on irrigated rice field and pig farming. These environments that are suitable for infectious cycle of JEV exist widely in Asia today. The effective and safe vaccine is available in endemic countries and for international travelers. JE vaccination is strongly recommended to those who visit the JEV-endemic regions, especially in the rainy season.