2008—2012年麻疹减毒活疫苗的质量分析

目的对中国2008—2012年连续5年麻疹减毒活疫苗(简称麻疹疫苗)的批签发情况进行总结,评价其麻疹疫苗的总体质量。方法通过对送检样品的资料审查和关键项目的实验室检定,采用趋势分析法对病毒滴度等进行分析和比较,回顾麻疹疫苗质量的整体情况。结果中国麻疹疫苗整体质量较好,批签发通过率为98.6%。疫苗关键指标数据稳定,病毒滴度100%符合国家标准,其中8批疫苗病毒滴度由于超过警戒线企业主动撤检。结论中国麻疹减毒活疫苗的质量稳中有升。国家疫苗批签发程序对确保上市疫苗的质量发挥重要作用,趋势分析在批签发中的应用更加严格保证了上市疫苗的安全性和有效性。     

麻疹减毒活疫苗检定用Vero细胞代次对病毒滴度稳定性试验的影响

目的了解麻疹减毒活疫苗检定用Vero细胞代次的稳定性范围。方法用DMEM培养液将Vero细胞137代连续传至173代,检测其细胞活力,并观察Vero细胞的形态,接种麻疹减毒活疫苗参考品进行病毒滴定及热稳定性试验。结果 170代以下Vero细胞形态良好,细胞活力在85%以上,麻疹减毒活疫苗病毒滴度热稳定性试验的结果:137~169代Vero细胞活力及原麻疹减毒活疫苗病毒滴度热稳定性试验无统计学差异(P>0.05),170代以上代次的Vero细胞活力及原麻疹减毒活疫苗热稳定性试验有明显统计学差异(P<0.01)。结论 169代以下Vero细胞为检定用麻疹减毒活疫苗最佳细胞代次。     

BHK_(21)细胞对蚀斑法检测乙型脑炎减毒活疫苗结果的影响分析

目的分析BHK_(21)细胞对蚀斑法检测乙型脑炎减毒活疫苗系统的影响,降低检测系统误差。方法比较BHK_(21)细胞以不同频次传代培养时的细胞生长状态,以及以不同接种浓度进行疫苗病毒滴度检测时,对检测系统稳定性的影响。结果 BHK_(21)细胞培养4 d传代,其形态良好、边缘光滑、胞质透光性好、细胞分散均匀、细胞活率达到95%以上;BHK_(21)以105细胞/m L浓度接种时,乙型脑炎减毒活疫苗及其病毒滴度参考品的变异系数最小,分别为0.66%和0.64%。结论 BHK_(21)细胞培养4 d传代、以105个/m L浓度接种时用蚀斑法检测病毒滴度系统最稳定,可供乙型脑炎减毒活疫苗滴度检测参考。     

首批百日咳杆菌鼠源抗血清国家参考品的制备和标定

目的制备和标定百日咳杆菌鼠源抗血清国家参考品。方法按《中华人民共和国药典》2015版(三部)(简称《中国药典》)相关要求,制备候选百日咳杆菌鼠源抗血清参考品(简称候选参考品),以WHO百日咳杆菌鼠源抗血清标准品(编号97/642)为标准,组织3家单位进行协作标定,并采用热加速试验对候选参考品进行稳定性观察。结果共制备合格候选参考品300支,其外观、水分、分装精度均符合《中国药典》的相关要求;协作标定结果显示,室内和室间几何变异系数(geometric coefficient of variation,GCV)均低于20%;经统计学分析,最终确定候选参考品中含PT-Ig G、FHA-Ig G、PRN-Ig G分别为95 IU/m L、917 IU/m L、102 IU/m L,95%可信区间分别为91.7~103.7 IU/m L、900.2~944.5 IU/m L、99.4~106.1 IU/m L;每支安瓿分别含PT-Ig G 19 IU、FHA-Ig G 183 IU、PRN-Ig G21 IU;候选参考品于-20℃放置15个月及37℃放置7 d、14 d、21 d和28 d后,各种百日咳组分抗体的酶标单位值均变化不大,差异均无统计学意义(P0.05)。结论首批百日咳杆菌鼠源抗血清国家参考品均符合《中国药典》的各项要求,且一致性、稳定性良好,可用于百日咳组分疫苗小鼠效力血清学方法的质量控制评价。     

肠道病毒EV71疫苗效力国家参考品的协作标定

目的建立肠道病毒71型(Enterovirus 71,EV71)疫苗效力参考品,用于EV71疫苗效力的小鼠ED50检测质量控制。方法选取经Ⅲ期临床试验验证具有良好保护效果的一批EV71疫苗作为候选参考品,采用EV71小鼠ED50检测方法,对候选参考品进行效力的协作标定及适用性研究。结果候选参考品在各实验室的ED50均值分别为9.5~51.0 U,CV为3.8%~18.7%;5个实验室总均值为23.5 U,CV为60.9%。各实验室检测结果均符合正态分布;3个EV71灭活疫苗与参考品均表现出了良好的剂量效应关系,中和抗体阳转率均随疫苗稀释度的增加而降低,下降曲线形态相近。当以候选参考品为标准,3个灭活疫苗的体内效价比均值分别为2.7倍、0.8倍和0.9倍;CV分别为5.5%、27.5%和11.2%。结论建立的EV71疫苗效力国家参考品(320 U/0.5 m L),可用于EV71疫苗效力的小鼠ED_(50)检测质量控制。     

提高麻疹减毒活疫苗病毒滴度的研究

病毒滴度是麻疹减毒活疫苗的主要质量指标之一。本文结合生产实际,就有关提高麻疹疫苗病毒滴度的影响因素进行了探讨。试验说明,生产用毒种的质量十分重要,要保证麻疹疫苗的高滴度和生产的稳定,需要建立适合麻疹毒种的培养条件并保持培养条件一致,从而必须加强生产过程的全面质量管理。     

水痘减毒活疫苗生产场地变更质量可比性研究

目的通过可比性研究证明水痘减毒活疫苗生产场地变更后质量的产品一致性。方法从生产用原材料、生产工艺、产品质量和稳定性等几个方面进行可比性分析,比较水痘减毒活疫苗生产场地变更前后产品质量特性。结果确认水痘疫苗生产车间场地变更前后原材料未发生改变;生产工艺未发生变化,部分工艺参数由于设备更新进行了调整但未对工艺控制造成影响;对工艺用水、疫苗质量、疫苗稳定性的关键质量指标进行统计分析,其中新车间生产的原液病毒滴度为5.0 lgPFU/mL、半成品病毒滴度为4.6 lgPFU/mL,成品中水分含量为1.0%~1.4%、病毒滴度为7.8~8.0 lgPFU/mL、热稳定性试验为6.8 lgPFU/mL与老车间同步生产的成品结果(原液病毒滴度为4.9~5.0 lgPFU/mL,半成品病毒滴度为4.5~4.6 lgPFU/mL,成品中水分含量为1.1%~1.3%、病毒滴度为7.8~8.0 lgPFU/mL、热稳定性试验为6.8 lgPFU/mL)相似,且均在2014年老车间相对应检测项目均值±3 SD范围内,新老车间成品中的牛血清白蛋白残留量和乳糖酸红霉素残留量差异均无统计学意义(t=1.50,P0.05;t=1.00,P0.05);加速稳定性试验和长期稳定性试验结果显示,新老车间同步生产的各3批水痘疫苗成品的数据稳定性一致,各项检测指标均符合相关规定要求;疫苗安全性均符合相关要求,新老车间同步生产的疫苗试验结果一致。结论水痘减毒活疫苗生产场地变更前后产品质量特性高度相似,场地变更未对产品的安全性和有效性产生不良影响。     

猪流行性腹泻mRNA疫苗的制备与免疫原性评价

猪流行性腹泻(porcine epidemic diarrhea,PED)是一种可引起仔猪高致死率的传染性疾病,尽管已有灭活疫苗和减毒活疫苗上市,但由于病毒变异频繁导致保护效力欠佳,发病率依然居高不下。本研究首次制备了PED mRNA候选疫苗,并在小鼠和妊娠母猪上评价了其免疫原性,证明了基于病毒受体结合区异源二聚体的mRNA候选疫苗具有良好的免疫原性,可在小鼠上高效诱导体液和细胞免疫应答,单次免疫血清中和抗体滴度达到1:300以上,并在妊娠母猪上诱导了与灭活疫苗相近的中和抗体水平,实现了100%抗体转阳。本研究为PED mRNA疫苗的进一步产业化奠定了基础。     

第二代百日咳疫苗毒性国家标准品的建立

目的建立第二代百日咳疫苗毒性国家标准品(简称二代毒性标准品)。方法选用百日咳疫苗生产菌株(编号CS株CMCC58003)进行发酵罐培养,将收获的百日咳菌液灭活后分装冻干后,作为第二代百日咳国家毒性标准品的候选品(简称候选毒性标准品)。以第一代百日咳疫苗毒性国家标准品为标准(简称一代毒性标准品),由4个单位协作标定候选毒性标准品,采用热加速法考察候选毒性标准品的稳定性。结果共获得检定合格的候选毒性标准品1 800支;经4个实验室协作标定,每支二代毒性标准品LPU为21,HSU为41;且稳定性良好。结论候选毒性标准品各项指标均符合要求,可作为第二代百日咳疫苗毒性国家标准品使用。     

Report of a collaborative study for assessing the potency of hepatitis B vaccines

A collaborative study was carried out to examine the suitability of a hepatitis B vaccine derived from plasma as an immunogenicity reference for vaccines produced by recombinant DNA technology in yeast. The use of a plasma derived vaccine as reference appeared satisfactory, although the use of homologous reference improved agreement in potency estimates. The use of a recombinant standard did not however improve agreement for a recombinant vaccine produced by a different manufacturer. The variation in the dilution of vaccine required to induce antibodies in 50% of test animals and in potency estimates varied widely between laboratories (25-fold and 10-fold respectively). However this was similar to the variation found in a previous collaborative study.     

A collaborative study to assess the proficiency of laboratory estimates of potency of live measles vaccines.

A collaborative study was undertaken to assess the variability in estimates of the potency of measles vaccines. Overall a median variation of 2.0 log10 between estimates was observed. This was reduced to a median of 1.0 log10 when the potencies were expressed relative to a reference vaccine. A difference in the sensitivity between plaque assays and TCID50 assays was also reduced when relative potencies were used. The benefit of including a common reference preparation in vaccine assays was therefore demonstrated. For the vaccines assayed in this study, it was not necessary to use a measles reference of the same strain as the vaccines tested. We therefore recommend that measles vaccines be assayed against a single international reference preparation.     

A collaborative study on the use of single radial immunodiffusion for the assay of rabies virus glycoprotein

The single radial immunodiffusion (SRD) technique has been applied to the assay of the glycoprotein content of rabies vaccines produced in cell cultures. Fourteen laboratories in seven countries participated in a collaborative study to evaluate the reproducibility of the SRD technique; some laboratories also examined vaccines in the mouse protection (NIH) test and by enzyme immunoassay. Good agreement was found between potency estimates using the SRD technique: the geometric coefficients of variation for combined potency estimates of all laboratories were about 10%. SRD assays appear to have a role for the in vitro assay of antigen content of vaccine and could complement results obtained in in vivo assays which are subject to wide variability.     

A collaborative study to establish the International Reference Reagent for hepatitis B vaccine containing plasma-derived hepatitis B surface antigen

A collaborative study was conducted to establish a suitable international reference reagent for hepatitis B vaccine for use in immunogenicity assays. The limiting dilution required to induce antibodies in 50% of the test animals was determined for the proposed international reference reagent and three other plasma-derived hepatitis B vaccines. The minimum antigenic dose of these preparations varied widely (100-fold range) between laboratories. However, the expression of potencies of vaccines relative to the proposed International Reference Reagent reduced the variation between laboratories to within a 10-fold range. The reference reagent is intended for use in assays of hepatitis B vaccines in mouse (or guinea-pig) immunogenicity studies. For products made by different procedures, clinical trials in humans are necessary to establish a correlation between the immunogenic potency in animals and man.     

Standardization of methods reduces variability: explanation for historical discrepancies in biochemical screening

Over the past decade, many studies have argued the relative merits of different chromosome abnormality biochemical screening protocols in different labs. Results and interpretations have varied markedly (e.g., double vs. triple screening). In this study we sought to compare coefficients of variation (CV) among 12 laboratories in one system, using identical and different methodologies for the three parameters. Ten identical specimens were processed as part of the 1999 College of American Pathologists (CAP) (FP-A, FP-B) proficiency tests. Results were compared among 12 laboratories using the Abbott ELISA for alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG), and two methods for estriol [Diagnostic Services Laboratory (DSL) and an "in house" assay]. The range on the 10 specimens of means for AFP varied from 12.56 to 117.87; hCG 14.05-68.08; and estriol 0.61-2.73. CV for AFP specimens averaged 10%, hCG 8%, and estriol 35% (F = 22.4). However, when only DSL was used for estriol, the CV was reduced to 8.7%. Standardization of AFP and hCG across 12 labs has reduced CV to <10%, which is similar to accepted between run results. Wide variation of uE3 among different methods may explain the widely divergent results in the literature. With national standardization of all parameters, the widely divergent results seen in the literature should narrow, and regional medians will no longer be necessary.     

An international collaborative study of an anti-infectious bursal disease virus reference serum

Fourteen laboratories participated in a collaborative study of a freeze-dried preparation of anti-infectious bursal disease virus serum to assess the suitability of the serum as a standard for use in the infectious bursal disease virus neutralization test. Ten laboratories carried out micro-virus neutralization tests and six carried out plaque reduction tests, two laboratories carrying out both tests. When titres were expressed as a proportion of that obtained for a reference preparation there was a marked reduction in variation between results from different laboratories. The use of a reference preparation was therefore of value when comparing results from different laboratories. It is proposed that the reference preparation used in this study be used as a standard to facilitate the comparison of results from different laboratories. The proposed standard contains by definition 10,000 UK units.     

沪191麻疹疫苗免疫持久性和影响因素的评价

１９９１～１９９８年,我们对荆州区川店镇５０３名６～１５月龄儿童进行了现行沪１９１麻疹疫苗血清流行病学效果观察,结果表明,初次免疫后１个月麻疹ＩｇＧ抗体阳转率为９１６５％,ＧＭＴ为１∶２６６７４,达保护滴度者比例为４６５％。随着时间的推移,第４年上述指标迅速下降到４６８６％、１∶１２７４和１８５％,第６年时低至２９４３％、１∶４８９和１３６％。０２ｍｌ、０３ｍｌ和０５ｍｌ麻疹疫苗组的近期和远期效果是类似的,初免后１个月时ＩｇＧ滴度越高,其免疫持久性越好;初免月龄是影响麻苗免疫效果的主要原因,６月龄初免组的免疫效果明显低于≥８月龄组。结果提示麻苗８月龄初免是可行的。     

Rabies reference vaccine for use as a regional standard for Latin America and the Caribbean countries

A candidate rabies reference vaccine of suckling mouse brain (SMB) origin was prepared and standardized at the Pan American Zoonoses Center (PAHO/WHO) and evaluated in a collaborative study involving seven laboratories. On the basis of three different tests, its potency, immunogenicity, and stability were demonstrated to be satisfactory. The vaccine was proposed for consideration of the Latin American and Caribbean countries as a regional standard to determine the potency of SMB vaccines, the most widely used in the Region.     

CA16滴度微量检测方法的建立及其验证

目的建立用Vero细胞检测柯萨奇病毒A组16型(CA16)滴度的方法,并对其适用性进行初步验证。方法通过对细胞种类的选择、细胞接种浓度和细胞病变判定时间的优化,建立测定CA16滴度的半数细胞感染剂量法(CCID50法),并对其进行初步验证。结果通过对病毒感染后细胞病变情况的观察,确定了以Vero细胞作为CA16病毒滴度检定用细胞。Vero细胞的最佳接种浓度和结果判定时间分别为5×104~1×105细胞/mL(即96孔板内每孔加入的最终细胞量为5×103~1×104细胞)和7 d。由4组人员对6批CA16病毒液进行重复检测,实验结果表明不同实验人员测定结果的变异系数(CV)在1.739%~4.974%之间,说明该方法测定结果重复性好,准确度高。结论该法简便、稳定,可以灵敏、准确地检测CA16病毒的滴度,可用于相关疫苗生产过程中的质量控制。