两种流行性乙型脑炎疫苗的安全性和免疫效果观察

目的观察乙型脑炎(乙脑)减毒活疫苗和Vero细胞乙型脑炎纯化疫苗(Vero细胞乙脑疫苗)的安全性和免疫效果。方法在湖南省沅江市和衡东县筛选8～20月龄无乙脑疫苗免疫史(基础免疫组)和1.5～3岁完成了基础免疫(加强免疫组)的二组健康儿童作为观察对象。沅江市的观察对象接种乙型脑炎减毒活疫苗,衡东县的观察对象接种Vero细胞乙脑疫苗。然后从每个市县的二组观察对象中抽取部分对象检测免疫前和免疫后1个月血清乙脑中和抗体滴度,并观察其接种不良反应。结果乙型脑炎减毒活疫苗接种不良反应总发生率为5.2%;Vero细胞乙脑疫苗接种不良反应总发生率为8.8%。两种疫苗的接种不良反应以全身发热为主,分别占不良反应的95.4%和92.3%,发热72h后恢复正常。乙型脑炎减毒活疫苗基础免疫后抗体阳性率为82.5%,抗体几何平均滴度(GMT)为1:23.1;加强免疫后抗体阳性率为93.2%,GMT为1:28.6。Vero细胞乙脑疫苗基础免疫后抗体阳性率为83.2%,GMT为1:25.1;加强免疫后抗体阳性率为93.7%,GMT为1:32.4。结论乙型脑炎减毒活疫苗和Vero细胞乙脑疫苗安全、有效,可在预防控制乙脑工作中推广使用。     

流行性乙型脑炎灭活疫苗与减毒活疫苗不同免疫策略效果研究

目的:为合理利用流行性乙型脑炎灭活疫苗和减毒活疫苗各自的优点,降低预防接种副反应的发生率,提高免疫效果。方法:初免疫2针均接种乙脑灭活疫苗,初免1年后实验组使用乙脑减毒活疫苗,对照组使用乙脑灭活疫苗。观察比较两种疫苗单一使用与联合使用的免疫效果和安全性。结果:接种乙脑减毒活疫苗24 h内副反应发生率为6.06%,未发生中重度反应;接种灭活疫苗副反应发生率为25.53%,中反应以上发生率为4.12%。加强免疫后1年中和抗体的GMT从1∶20.42降至1∶7.37。第3针免疫后,联合免疫组中和抗体阳性率为99.24%,单纯使用灭活疫苗组阳性率为95.86%,差异无显著性。结论:对儿童使用乙脑灭活疫苗进行基础免疫,用减毒活疫苗进行加强免疫有很好的免疫效果和安全性,是理想的免疫策略。     

冻干精制Vero细胞流行性乙型脑炎灭活疫苗在低流行区的人体反应及免疫效果

为了进一步观察冻干精制猴肾传代细胞(Vero细胞)流行性乙型脑炎(乙脑)灭活疫苗(Vero细胞乙脑疫苗)的接种反应及免疫效果,在乙脑的低流行区内蒙古自治区选择6～9岁未接种过乙脑疫苗的健康儿童为观察对象,分别接种Vero细胞乙脑疫苗、原代地鼠肾细胞(PHK)乙脑灭活疫苗和乙脑减毒活疫苗.结果显示接种第1剂Vero细胞乙脑疫苗后体温中、强反应率为5.6%,未见局部及其它全身反应,接种第2剂后只有1人为中反应;用Vero细胞乙脑疫苗基础免疫2针后1个月,血清中和抗体阳转率和几何平均滴度(GMT)分别为100.0%和1∶25.30,显著高于PHK乙脑灭活疫苗的56.8%和1∶8.83,亦显著高于乙脑减毒活疫苗1针免疫后的60.7%和1∶8.35;用Vero细胞乙脑疫苗基础免疫2针1年后中和抗体阳性率及GMT分别为50.0%和1∶7.42,加强免疫后中和抗体阳性率为100.0%,GMT达1∶220.1,≥4倍增长率为95.1%,加强免疫前后差异显著.由此可见,用Vero细胞乙脑疫苗对学龄儿童进行基础免疫或加强免疫,是安全有效的.     

流行性乙型脑炎减毒活疫苗免疫效果观察

目的评价流行性乙型脑炎减毒活疫苗的免疫效果。方法在北京市东城和昌平2个区随机选择满1周岁未接种过乙型脑炎疫苗的儿童99名，采集乙脑疫苗免疫前、免疫后1-2个月和2周岁加强免疫后1～2个月的外周静脉血各1份，分离血清检测血清乙型脑炎中和抗体。结果乙型脑炎减毒活疫苗的初次免疫成功率86．87％，95％CI（80．22％～93．52％），GMT1：17．86；加强免疫成功率97．33％，95％CI（93．68％～99．98％），GMT1：97．82。结论乙型脑炎减毒活疫苗具有较好的免疫原性，且该疫苗为单人份包装，更利于安全接种，适合基层推广。     

冻干精制Vero细胞流行性乙型脑炎灭活疫苗在流行区的人体反应及免疫效果

为了进一步观察冻干精制Vero细胞流行性乙型脑炎 (乙脑 )灭活疫苗 (Vero细胞乙脑疫苗 )的接种反应及免疫效果 ,在乙脑流行区江苏省苏州市吴中区选择 6～ 18月龄未接种过乙脑疫苗的健康婴幼儿为观察对象 ,分别接种Vero细胞乙脑疫苗、原代地鼠肾细胞 (PHK)乙脑灭活疫苗和乙脑减毒活疫苗。结果显示 :接种Vero细胞乙脑疫苗第 1剂后体温中、强反应率为 12 4 % ,未见局部及其它不良反应 ;接种第 2剂后无发热反应。用Vero细胞乙脑疫苗免疫 2针后 1个月 ,血清中和抗体阳转率和几何平均滴度 (GMT)分别为 10 0 0 %和 1∶2 7 7,显著高于PHK乙脑灭活疫苗和乙脑减毒活疫苗的免疫后血清学效果。用Vero细胞乙脑疫苗初免的儿童 ,1年后抗体阳性率与GMT分别为 88 3%和 1∶2 6 4 ,用同一种疫苗加强免疫后抗体阳性率与GMT分别为 10 0 0 %和 1∶2 80 1。此外 ,用Vero细胞乙脑疫苗对该区的 1～ 6岁已接种过乙脑疫苗的儿童进行加强免疫 ,结果显示 :免疫前阳性率与GMT分别为 6 8 2 %和 1∶10 2 ,免疫后阳性率为 10 0 0 % ,GMT达 1∶2 32 4 ,≥ 4倍增长率为 98 5 % ,加强免疫前后差异显著。由此可见 ,用Vero细胞乙脑疫苗对儿童进行初免或加强免疫 ,除初免第 1针有一过性发热外 ,无其它不良反应 ,血清学效果明显优于PHK乙     

乙脑减毒活疫苗两针法免疫效果观察

目的观察流行性乙型脑炎(乙脑)减毒活疫苗现行的2针免疫法免疫效果。方法在广州市海珠区选取未接种过乙脑疫苗且未患过乙脑的8~10月龄儿童176人,进行乙脑减毒活疫苗的初免以及14~18月后加强免疫;于每剂次免疫前后1个月采集静脉血检测乙脑中和抗体。结果初免前抗体阳性率9.6%,抗体几何平均滴度(GMT)为1∶1.27;初免后抗体阳性率86.9%,GMT为1∶19.35,抗体阳转率85.5%。加强免疫前抗体阳性率75.0%,GMT为1∶9.38;加强免疫后抗体阳性率96.0%,GMT为1∶40.92,加强免疫抗体阳转率84.1%,2针次后抗体总体阳转率95.6%。结论现行的乙脑减毒活疫苗的免疫程序能取得很好的免疫效果。     

两种流行性乙型脑炎疫苗基础免疫的近期免疫学效果和安全性观察

目的观察流行性乙型脑炎(下称乙脑)减毒活疫苗和灭活疫苗免疫学效果和安全性。方法对208名和176名12月—24月龄的儿童,分别用两种疫苗进行基础免疫,免疫前和免疫后一个月检测血清抗体,并观察其接种反应。结果208名经乙脑减毒活疫苗免疫的儿童,抗体阳性率由8.17%上升至89.42%,GMT由1:10.80上升至1:47.41;176名经乙脑灭活疫苗免疫的儿童,抗体阳性率由8.52%上升至67.61%,GMT由1:10.12上升至1:22.87。经乙脑减毒活疫苗免疫后抗体阳转率为88.48%,阳转儿童GMT为1:45.42;经乙脑灭活疫苗免疫后抗体阳转率为64.60%,阳转儿童GMT为1:21.89。安全性观察显示:两种疫苗均未发现异常反应发生。一般反应率减毒活疫苗为5.29%,其中轻、中、重的构成比分别为81.82%、9.09%、9.09%;灭活疫苗一般反应率为11.93%,其中轻、中、重的构成比分别为47.62%、38.10%、14.28%。结论乙脑减毒活疫苗的近期免疫学效果和安全性优于灭活疫苗,适宜推广使用。     

两种乙型脑炎疫苗的免疫效果观察

将 319名受隐性感染机会较少的 5～ 18月龄的婴幼儿分为两组 ,分别接种乙型脑炎 (乙脑 )灭活疫苗和减毒活疫苗。经间接免疫荧光法检测其血清抗体发现 ,免疫前血清抗体仅有 3.76%阳性。基础免疫后 ,两种疫苗的抗体阳性率分别增加至 59.56%和 56.0 8% ,但尚不能达到保护性水平。 1年后加强注射 1针 ,1个月后阳性率分别增加至 94 .62 %和 98.2 1% ,GMT也明显增加 ,半年后 ,阳性率改变为 72 .34 %和 94 .74 %。减毒活疫苗在加强免疫后的免疫效果及持久性明显高于灭活疫苗。根据观察结果认为 ,乙脑灭活疫苗免疫 ,应基础免疫两针 ,间隔 7～ 10天 ,第二年加强免疫1针之后 ,间隔 1年再加强 1针。减毒活疫苗基础免疫 1针 ,第二年加强 1针 ,经 4～ 5年后再加强注射。由于减毒活疫苗高的免疫效果 ,较少的接种次数以及较高的安全性 ,适宜于广大地区推广应用。     

流行性乙型脑炎减毒活疫苗与灭活疫苗免疫效果及人体接种反应观察

为比较流行性乙型脑炎 (乙脑 )减毒活疫苗和灭活疫苗的免疫效果和安全性 ,选择北京市 1～ 2周岁应初免儿童 2 6 4名 ,分组接种乙脑减毒活疫苗和乙脑灭活疫苗 ,观察人体接种反应 ,采用细胞蚀斑减少中和试验检测免疫前及免疫后血清乙脑中和抗体。结果显示 :减毒活疫苗组抗体阳转率和阳性抗体几何平均滴度 (GMT)分别为91 30 %和 1∶2 0 2 2 ;灭活疫苗组为 6 4 38%和 1∶16 5 1。经统计学分析 ,减毒活疫苗组抗体阳转率和阳性抗体GMT均高于灭活疫苗组。经观察 ,减毒活疫苗组和灭活疫苗组人体反应均为发热和接种部位轻度红肿 ,发热率分别为 8 5 1%和 8 13% ,中、强反应率为 2 13%和 0 81% ,红肿率为 1 4 2 %和 0 81%。所有发热及轻度红肿者均于2 4h恢复正常。提示 :乙脑减毒活疫苗免疫效果好 ,免疫针次少 ,安全性好 ,是现阶段预防乙脑比较理想的疫苗 ,适宜推广使用     

流行性乙型脑炎减毒活疫苗人群接种后中和抗体持久性观察

流行性乙型脑炎 (乙脑 )减毒活疫苗是一种安全、有效、免疫原性广的疫苗 ,其临床的保护效果得到多次观察结果证实。但乙脑活疫苗免疫持久性的观察由于工作难度大而还未见报道。为此 ,针对接种乙脑活疫苗后中和抗体持久性进行了 6年的血清学观察。结果显示 :接种疫苗前乙脑中和抗体 <1∶5的血清样本共 2 8份 ,初次免疫 1个月后中和抗体阳转率为 96 4%( 2 7/ 2 8,≥ 1∶10 )或 10 0 .0 %( 2 8/ 2 8,≥ 1∶5 ) ,加强免疫后 1个月为 10 0 0 %( 2 8/ 2 8,≥ 1∶10 ) ;加强免疫后 1年为 81 0 %( 17/ 2 1,≥ 1∶10 ) ;加强免疫后 6年抗体阳性率为 5 7 1%( 16 / 2 8,≥ 1∶10 )或75 0 %( 2 1/ 2 8,≥ 1∶5 )。表明乙脑活疫苗接种 1针 1个月后再加强免疫 1针至加强免疫后的第 6年 ,其中和抗体阳性率仍可维持在 5 7.1%～ 75 .0 %。表明乙脑活疫苗具有较好的免疫持久性。     

流行性乙型脑炎减毒活疫苗大面积接种后安全性和流行病学效果的5年观察

目的 考核乙脑减毒活疫苗大面积接种后的安全性和降低乙脑发病率,在乙脑高发区安徽省蒙城和涡阳县进行为期５年（１９９２￣１９９６年）的人群观察。方法 观察对象１￣６岁儿童。１岁初免１针,２岁加强１针。结果 通过５年连续观察共接种乙脑活疫苗３３５９４１人。 表明：①疫苗对小龄儿童接种后近期和５年内均未出现不良反应,表明疫苗是安全的。②当地乙脑平均总发病率有显著性下降,从接种观察前１９８７￣１９９１年的１     

流行性乙型脑炎减毒活疫苗大面积接种后安全性和流行病学效果的5年观察

目的　考核乙脑减毒活疫苗大面积接种后的安全性和降低乙脑发病率,在乙脑高发区安徽省蒙城和涡阳县进行为期５ 年（１９９２ ～１９９６ 年） 的人群观察。方法　观察对象为１ ～６ 岁儿童。１ 岁初免１ 针,２ 岁加强１ 针。结果　通过５ 年连续观察共接种乙脑活疫苗３３５ ９４１ 人。表明：①疫苗对小龄儿童接种后近期和５ 年内均未出现不良反应,表明疫苗是安全的。②当地乙脑平均总发病率有显著性下降,从接种观察前１９８７ ～１９９１ 年的１１ ．３４／１０ 万下降到１９９２ ～１９９６ 年的２ ．７４／１０ 万（ Ｐ＜ ０ ．００５） 。③１ ～６ 岁儿童发病专率观察前５ 年涡阳县平均为５６ ．２４／１０ 万,蒙城县为４４ ．５７／１０ 万;普种后５ 年,两县分别下降至１３ ．８３／１０ 万和１６ ．９４／１０ 万（ Ｐ＜ ０ ．００５） 。④有接种史儿童发病率明显低于无接种史的儿童（２ ．２１／１０ 万和３５８ ．１／１０ 万） 。１８ 例死亡病例全部为无接种史者。⑤疫苗免疫一针后中和抗体阳转率为８３ ．８７ ％ ～９４ ．７４ ％ 。结论　乙脑活疫苗安全有效,接种后可明显提高机体的免疫力,对降低乙脑发病控制流行效果显著。     

乙脑病毒SA14-14-2减毒株病毒外源因子污染检测分析

目的 检测乙脑病毒SA14- 14 -2减毒株的病毒外源因子.方法 用乙脑病毒免疫血清(阳性)将乙脑病毒SA 14 - 14 -2减毒株中和后,采用中和后的样品分别感染指示细胞(3T3、NRK、C3/36细胞),直接培养观察及红细胞吸附试验;中和后样品感染小鼠细胞系(3T3)培养14d的上清液再接种指示细胞(MRC -5、Vero、BHK21、3T3)培养观察及红细胞吸附试验;同时将流感病毒接种MDCK细胞,作为试验阳性对照.结果 乙脑病毒SA 14 - 14-2减毒株中和后的样品接种指示细胞(3T3、NRK、C6/36细胞),直接培养观察7d、14 d,细胞形态正常,无细胞病变征兆,红细胞吸附试验为阴性;中和后样品感染小鼠细胞系(3T3)培养14 d的上清液再接种指示细胞(MRC -5、Veto、BHK - 21、3T3)培养观察7d、14 d,细胞形态正常,无细胞病变征兆,红细胞吸附试验为阴性.结论 乙脑病毒SA14-14-2减毒株中未检测到人源、猴源、鼠源及其他病毒的污染,符合《WHO Technical Report Series,No.910,2002》的质量标准,可安全地用于乙脑减毒活疫苗的生产.     

A novel amino acid site closely associated with the neurovirulence of live,attenuated Japanese encephalitis vaccine (SA14-14-2 strain)

Japanese encephalitis (JE) poses a serious threat to the world’s public health yet without a cure, the only way to prevent Japanese encephalitis virus (JEV) infection is vaccination. Live attenuated vaccine (SA14-14-2 strain) is the most widely used JE vaccine, and clinical data have confirmed its safety and effectiveness. Eight sitesassociated with virulence in the Envelope (E) protein are often the focus of quality control of JE vaccine. However, sequences retrieved from NCBI, as well as our previous results showed that the wild strain SA14 may harbor two different amino acids at amino acid residue 244 of the E glycoprotein (E244), and it may be related to virulence. In this study, we introduced a single mutation at nt1708 (G → A) in the full-length cDNA clone of SA14-14-2, replacing a Gly with Glu at amino acid residue 244 of the E glycoprotein, and successfully constructed the mutant virus (JEV E244). JEV E244 exhibited a similar plaque morphology and growth characteristics to JEV SA14-14-2 in cell culture. However, it had lethal neurovirulence in mice and could enter the brain following intraperitoneal inoculation. Moreover, the virulence of JEV E244 in the context of vaccine in mice is significantly different from that of the JEV E244 alone. These results suggested that E244 site should be included in the assessment of the genetic stability of the attenuated JE vaccine. The detection of minor mutations in vaccine population and influence on the safety of vaccine is discussed.     

Development of a small animal peripheral challenge model of Japanese encephalitis virus using interferon deficient AG129 mice and the SA14-14-2 vaccine virus strain

Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern due to its recent geographic expansion. While commercial vaccines are available and used in some endemic countries, JEV continues to be a public health problem, with 50,000 cases reported annually. Research with virulent JEV in mouse models to develop new methods of prevention and treatment is restricted to BSL-3 containment facilities, confining these studies to investigators with access to these facilities. We have developed an adult small animal peripheral challenge model using interferon-deficient AG129 mice and the JEV live-attenuated vaccine SA14-14-2, thus requiring only BSL-2 containment. A low dose of virus (10 PFU/0.1 ml) induced 100% morbidity in infected mice. Increased body temperatures measured by implantable temperature transponders correlated with an increase in infectious virus and viral RNA in serum, spleen and brain as well as an increase in pro-inflammatory markers measured by a 58-biomarker multi-analyte profile (MAP) constructed during the course of infection. In the future, the MAP measurements can be used as a baseline for comparison in order to better assess the inhibition of disease progression by other prophylactic and therapeutic agents. The use of the AG129/JEV SA14-14-2 animal model makes vaccine and therapeutic studies feasible for laboratories with limited biocontainment facilities.     

Infection of mouse bone marrow-derived dendritic cells by live attenuated Japanese encephalitis virus induces cells maturation and triggers T cells activation

An attenuated Japanese encephalitis virus (JEV) strain SA14-14-2, generated from the wild strain SA14, is an effective live vaccine against JEV infection. It has led to a significant decrease in JEV infection around the world. Although it is highly effective, the mechanism for its robust immunity was not well investigated. In this study, the interaction of SA14-14-2 with bone marrow-derived dendritic cells (bmDCs) was investigated. Our results showed that the infection of bmDCs with SA14-14-2 resulted in viral replication and upregulation of bmDC maturation marker molecules (CD40, CD80, CD83 and MHC I). SA14-14-2 infection also stimulated the production of interferon-α (IFN-α), monocyte chemoattractant protein-1 (MCP-1/CCL2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of bmDC. Both MLR and ELISPOT assay showed an enhanced allostimulatory capacity of SA14-14-2-infected bmDCs. Furthermore, the SA14-14-2-infected bmDCs impaired the expansion of Foxp3+ regulatory T (Treg) cells with immunosuppressive potential, suggesting that SA14-14-2 infection induced antiviral immunity rather than immunosuppression. Taken together, our results indicated that SA14-14-2 infection caused bmDC maturation, changed the expression profiles of several cytokines, and triggered T cell activation. This offered an insight in the immunologic mechanisms associated with the high efficiency of the SA14-14-2 vaccine.     

A cost–effectiveness analysis of Japanese encephalitis vaccine in Cambodia

This study aimed to evaluate the cost and effectiveness of introducing a live, attenuated vaccine (SA 14-14-2) against Japanese encephalitis (JE) into the immunization program. The study demonstrated that SA 14-14-2 immunization is cost–effective in controlling JE in Cambodia compared to no vaccination. Averting one disability-adjusted life year, from a societal perspective, through the introduction of SA 14-14-2 through routine immunization, or a combination of routine immunization plus a campaign targeting children 1–5 or 1–10 years of age, costs US$22, US$34 and US$53, respectively. Sensitivity analyses confirmed that there was a high probability of SA 14-14-2 immunization being cost–effective under conditions of uncertainty.     

乙脑减毒活疫苗溶解稀释后稳定性观察

目的观察乙脑减毒活疫苗（SA14-14-2）（5人份/瓶）溶解稀释后病毒的稳定性。方法将该乙脑减毒活疫苗加入稀释液PBS（2.5ml/瓶）,溶解稀释后分别置于2℃～8℃、20℃～25℃、37℃存放,在设置的不同时间点,分别测定病毒滴度,观察其病毒滴度变化趋势。结果该乙脑减毒活疫苗溶解稀释后在2℃～8℃、20℃～25℃存放至24h,在37℃存放至14h,病毒滴度下降,但符合《中国药典》2005年版的要求（≥5.7LgPFU/ml）;在37℃存放大于14h后,病毒滴度下降明显,不符合《中国药典》2005年版的要求（≥5.7LgPFU/ml）。结论乙脑减毒活疫苗溶解稀释后在2℃～8℃、20℃～25℃可存放24h,在37℃可存放14h,其病毒滴度符合《中国药典》2005年版的要求（≥5.7LgPFU/ml）。     

6～12月龄婴儿接种流行性乙型脑炎活疫苗的临床安全性观察

目的考察流行性乙型脑炎减毒活疫苗在6～12月龄婴儿中接种时的安全性。方法选择41名6～12月龄婴儿,与同时接种的1岁以上儿童组进行比较,观察全身反应和局部反应。结果6～12月龄组和1～2岁组间疫苗反应无统计学意义(P>0.05)。结论乙脑减毒活疫苗在6～12龄婴儿中接种是安全的。     

Characterization of live-attenuated Japanese encephalitis vaccine virus SA14-14-2

The live attenuated Japanese encephalitis (JE) vaccine SA14-14-2 was licensed decades ago and now approved for clinical use in most JE endemic countries. Large-scale clinical trials demonstrate ideal safety and efficacy profile of this Chinese vaccine. The SA14-14-2 vaccine was derived from a virulent strain SA14 after hundreds of serial passaging in cells and animals, concern about virulence reversion remains exist. In the present study, to study the in vitro and in vivo genetic and attenuation stability of the vaccine virus, SA14-14-2 was serially passaged in Vero cells and mouse brain followed by sequence comparison and attenuation phenotype analysis. The results showed that no significant mutation was acquired after serial passaging in Vero cells except a single Ser66Leu mutation within capsid protein, which had no effect on viral virulence in mice. Importantly, serial passaging of SA14-14-2 in suckling mouse brain resulted in emergence of adaptive mutations and increased virulence in mice. Population and plaque-purified clone consensus sequence analysis showed four adaptive mutations in envelope (E) protein, F107L, K138E, T226R and I270T, sequentially occurred and become predominant during serial passaging in suckling mouse brain. Especially, these adaptive mutations were close related with the enhanced neurovirulence and neuroinvasiveness in mice. Our results provide experimental evidence of highly genetic and attenuation stability of SA14-14-2 following passaging in Vero cells, and reveal the potential virulence reversion during passaging in mouse brain in association with critical adaptive mutations in E protein. These findings are important for quality control and evaluation of live JE vaccines and will help understand the attenuation mechanism of flavivirus vaccine.