犬用口服狂犬病减毒活疫苗猴体安全性试验研究

目的:进一步研究犬用口服狂犬病减毒活疫苗CTN-22毒株的安全性。方法:用高于现场用量4倍浓度疫苗肌肉注射猴体后,采血进行中和抗体检测。结果:猴体肌肉注射疫苗后,均健康存活,中和抗体1个月为1∶13.8～19.5,2个月上升为1∶33.5～1∶37.6。结论:迸一步证实了CTN-22毒株制备的犬用口服狂犬病减毒活疫苗是安全的、可靠的。     

犬用口服狂犬病减毒活疫苗安全实验研究

目的:探讨犬用口服狂犬病减毒活疫苗(CTN-22毒株)的安全性。方法:依照WHO 专家会议关于犬和野生食肉动物口服狂犬病疫苗接种现场试验要求和标准的报告,以及美国动物与动物制品法规—兽医生物制品标准,进行本疫苗的安全性试验。结果:口服免疫的5只猫和口服、注射免疫的各5只黄毛鼠,观察21d,无任何症状发生,均健康存活;用本疫苗10个现场使用量的1ml,各对5只家犬肌肉和大隐神经及其周围组织浸润注射后,观察3～35d,无狂犬病症状,并按方法中所规定时间淘汰犬,剖取颈淋巴结、脑、唾液腺等组织分离病毒,均为阴性;3月龄10只犬以10个现场剂量口服免疫后,1、2、3d连续3次取唾液进行病毒分离亦均阴性,免疫后观察6个月未显示狂犬病任何征象,均健康存活。结论:证实CTN-22毒株制备的犬用口服狂犬病减毒活疫苗是安全的。     

戊型肝炎实验动物模型研究

选用1～3岁龄的国产恒河猴(Macaca mulatta)22只(其中有2只作为对照)进行了戊型肝炎病毒(Hepatitis E Virus简称HEV)的实验感染.第一代感染两批动物:第一批感染4只,有3只发病,第二批感染4只全部发病.而后用第二批感染猴的粪便和肝脏悬液作为第一代,又连续进行传代实验。第二代传代选用的4只猴也全部发病.用肝悬液传代的1只猴也发病.第三代传代选用7只猴,有5只猴发病其中有1只猴(M-34号)是用第二代传代猴的胆汁感染的.所有发病猴血清丙氨酸转氨酶(ALT)水平均有不同程度的升高,一般高于正常值的3～6倍,最高可达10倍左右。肝活检可见肝细胞炎症和坏死。猴的粪便标本用免疫电镜(IEM)检测可见到直径为27～32nm的病毒颗粒。     

人二倍体细胞2BS株小儿麻痹活疫苗Ⅰ型安全性和免疫原性观察

用人二倍体细胞2BS株作基质制备的Ⅰ型aca～aae小儿麻痹减毒活疫苗,经现场服用观察获得可喜的效果。实验分小、中、大量三组进行,小、中量组设有原猴肾疫苗对照,小量组采双份血清和半数儿童的大便标本,按常规法检测血清抗体及分离病毒。服苗后其体温反应轻微,一般临床症状很少,未发生与疫苗有关的病例,证明了该疫苗的安全性。免疫后1个月血清中和抗体阳转率为78.83％,GMT=290,免疫原性令人满意。排毒率     

甲型肝炎减毒活疫苗（H2株）的猴体保护试验

采用恒河猴进行甲型肝炎(简称甲肝)活病毒减毒株(H2株)即活疫苗株的保护试验．当疫苗组(5只猴)于接种5个月后已产生效价为1：10～1：20抗甲肝抗体时，与对照组(6只猴)同时接受甲肝病毒强毒株“合32”的攻击，结果发现对照组动物在1～7周均产生效价为1：20～1：1280的抗体。并出现丙氨酸转氨酶(全部动物)和乳酸脱氨酶同功酶(5／6动物)的异常升高；而疫苗组除抗体水平稿有上升外，并没有上述两种酶的异常升高。这说明活疫苗株(H2株)具有良好的免疫原性和弱毒性质，可保护猴体耐受甲肝病毒强毒株“合-32”的攻击，并且还髓明恒河猴可作为甲肝病毒动物模型的可靠性。     

海口市犬狂犬病病毒携带及疫苗接种后免疫效果分析

目的了解海口市犬狂犬病毒携带和接种疫苗后的免疫状况。方法采集本市动物门诊、养犬场等犬只的唾液和血清样品各226份，用ELISA方法检测唾液中狂犬病带毒率和血清中的抗体阳性率。结果犬唾液的带毒率为0，血清抗体阳性率为71．68％，且进口疫苗免疫后的抗体阳性率为91．59％，明显高于国产疫苗的53．78％。结论海口市未发现带狂犬病毒犬只，接种进口狂犬疫苗的免疫效果比国产狂犬疫苗好。     

狂犬疫苗免疫后血清抗体阳转情况观察

<正> 注射狂犬疫苗是被动物咬伤者预防狂犬病的有效措施,注射后能否产生抗狂犬病毒中和抗体(以下简称狂抗)是防止发病的关键。我站从1991年起对部分接种狂犬疫苗者进行狂抗检测,对阴性者加强接种井观察阳转情况,现将结果报告如下。 1 材料与方法 1.1 检测对象:被动物咬伤后到我站注射狂犬疫苗者。 1.2 免疫方法:狂犬疫苗统一由我站到上级站购进,多厂家多批号。免疫程序采用肌肉注射5针法,于0、3、7、14、30天各肌肉注射一针,最后一针注射完15天后抽静脉血检测血清中和抗体。阴性者加强注射2针,2针之间间隔10天,最后1针注射后10天再抽静脉血检测血清中和抗体。     

黏膜感染SIV病毒的恒河猴在生殖道、肠和淋巴组织特征性的细胞因子、化学因子和Foxp3表达

[目的]阐明SARS病毒感染后能否再次感染,疫苗产生的抗体中长期保护效果,被动免疫是否真正安全有效等,为防治SARS提供实验依据。[方法]实验分4组,分别为一组(SARS恒河猴恢复组):用感染SARS-CoV发病12月后的4只恒河猴,均有中和抗体产生。二组(SARS食蟹猴恢复组):用感染SARS-CoV发病12月后的3只食蟹猴,均有中和抗体产生。三组(SARS血清输入恒河猴组):3只恒河猴,病毒接种时中和抗体阴性。病毒接种两天后输入抗体阳性血清(恒河猴血清,感染获得,效价为:1:128),用量10ml/只,分别经肌肉和静脉输入,各5ml。四组(恒河猴SARS-CoV感染组):2只恒河猴,病毒接种时中和抗体阴性。SARSCo-V经鼻腔接种,在感染的第1天开始到7天安乐死时,不同时间取咽拭子、血液和脏器,进行病毒分离,RT-PCR检测和中和抗体测定。[结果]一组(SARS恒河猴恢复组):接种SARS-CoV后未见发热等异常临床表现。血清生化无ALT、LDH、CK、总蛋白和血清白蛋白异常。3只猴在接种病毒后的咽拭子中,RT-PCR分别未检出、第1天检出、第1-3天中检出病毒。第2、5、7天咽拭子中、7天安乐死时血、肺、肝、脾和淋巴结等组织中病毒分离均为阴性。2只猴肺组织病理学检查见轻度肺炎。二组(SARS食蟹猴恢复组):接种3只未见任何不良临床表现,血清生化5项正常。3只猴在接种病毒后的咽拭子标本中,RT-PCR分别未检出SARS病毒、在第1-2天检出、在第1-3天中检出病毒。第2、5、7天咽拭子中、7天安乐死时血、肺、肝、脾和淋巴结等组织中病毒分离均为阴性。3只猴肺组织病理学检查见轻度肺炎等。三组(SARS血清输入恒河猴组):3只恒河猴在病毒接种的第2-5天时有一过性的体温升高,3940℃。血清生化5项正常。3只猴在接种病毒后的咽拭子标本中,RT-PCR分别在第1-3、第1-4天和第1-2天检出SARS病毒。2只猴第7天咽拭子中病毒分离阳性。另外1只在第2、5、7天咽拭子中、7天安乐死时血、肺、肝、脾和淋巴结等组织中病毒分离均为阴性。3只猴肺组织病理学检查见轻度肺炎等。四组(SARS恒河猴SARS-CoV感染组):2只猴病毒接种后,第2-4天时有一过性的体温升高,3940℃。2只进行接种病毒后1-7天安乐死时,RT-PCR在恒河猴的咽拭子标本中连续检出SARS病毒。在第2、5天咽拭子中、7天安乐死时肺组织中病毒分离阳性。2只猴肺等组织病理学检查发现肺组织表面局部有轻度发灰实变现象,可见到间质性肺炎病变,内皮细胞受损,出血和水肿。大多数肺泡没有完整的内衬细胞残留,肺泡间隔变宽并被以吞噬细胞为主的单核炎症细胞浸润,同SARS肺炎改变。实验表明,前期感染产生中和抗体的恒河猴、食蟹猴再次感染病毒,和模型对照猴比较,动物肺组织等只出现轻微或无病理变化,RT-PCR检出时间大大缩短,病毒培养未能分离出病毒,所有这些指标,均证实中和抗体有明显的保护作用,是有效的。被动免疫从结果来看,有一定的作用,但保护作用弱。     

几种狂犬病疫苗对犬只免疫效果的评价

目的为了加强犬只免疫，选择更优质的疫苗对犬进行预防接种。方法引进荷兰Interver，法国Virbac兽用疫苗与国产人用、兽用狂犬病疫苗进行对照研究，分别在免疫前及免疫后15、30、90、180、360d抽血并分离血清，进行狂犬病毒抗体检测。结果引进的两种兽用疫苗与国产人用疫苗均能在免疫后15～30d产生保护性抗狂犬病抗体。结论引进的Interver和Virbac兽用疫苗、国产人纯化狂犬病疫苗对犬均有较好的保护作用。     

乙肝病毒疫苗鸡蛋抗体HBsAg-IgY的制备及体外中和作用

目的:制备特异性的乙肝病毒疫苗鸡蛋抗体(HBsAg-IgY),探索抗体检测条件并进行体外中和作用试验。方法:用重组酵母乙型肝炎疫苗(HBsAg)免疫产蛋母鸡,收集鸡蛋,用鸡卵黄抗体IgY提取系统提取鸡卵黄抗体IgY;用改进的ELISA法检测抗体IgY含量,探索最佳检测条件;采用ELISA法研究IgY对Hepg2.2.15细胞系分泌HBsAg的中和试验。结果:在免疫后第1～3周,鸡卵黄抗体IgY含量开始上升,第6周达到高峰,并可持续2个月。抗HBsAg IgY可中和HepG2.2.15细胞系分泌的HBsAg,抗体在稀释度为1∶200时中和作用最强。结论:采用重组酵母乙型肝炎疫苗免疫母鸡,可持续性获得特异性抗HBsAg IgY;体外实验证实,抗HBsAg-IgY对HepG2.2.15细胞HBsAg具有中和作用。     

2010-2011年绵阳市犬密度及犬只狂犬病病毒带毒调查报告

目的了解绵阳市犬只密度和狂犬病病毒带毒感染状况,为防制狂犬病提供科学依据。方法北川、平武未报告过狂犬病,随机抽取2个村进行调查;在其余7个报告狂犬病病例的县市区分别抽取1个报病村和1个未报病村进行调查。调查内容包括犬密度、免疫和拴养等情况。在9个县市区采集50份犬脑组织检测狂犬病毒抗原。结果共调查9个县市区18个村,平均犬密度为0.41只/户,免疫率为81.9%,拴养率67.55%。报病村,其免疫率和拴养率均较未报病村低,但差异无统计学意义。50份犬脑组织未发现狂犬病毒抗原阳性者。结论绵阳市犬只密度较高,免疫率和拴养率低,需加强犬只管理和免疫,有效预防和控制狂犬病的发生。     

Humoral immune responses in rabbits induced by an experimental inactivated severe acute respiratory syndrome coronavirus vaccine prepared from F69 strain

Background The etiologic agent of severe acute respiratory syndrome (SARS) has been confirmed to be a novel coronavirus (CoV), namely SARS-CoV. Developing safe and effective SARS-CoV vaccines is essential for us to prevent the possible reemergence of its epidemic. Previous experiences indicate that inactivated vaccine is conventional and more hopeful to be successfully developed.Immunogenicity evaluation of an experimental inactivated SARS-CoV vaccine in rabbits was conducted and reported in this paper.Methods The large-scale cultured SARS-CoV F69 strain was inactivated with 0.4% formaldehyde and purified, then used as the immunogen combined with Freund‘s adjuvant. Eight adult New Zealand rabbits were immunized four times with this experimental inactivated vaccine. Twelve sets of rabbit serum were sampled from the third day to the seventy-fourth day after the first vaccination. The titers of specific anti-SARS-CoV IgG antibody were determined by indirect enzyme-linked immunosorbent assay, and the neutralizing antibody titers were detected with micro-cytopathic effect neutralization test.Results Rapid and potent humoral immune responses were induced by the inactivated SARS-CoV vaccine in all the eight test rabbits. Titers of both specific IgG antibody and neutralizing antibody peaked at about six weeks after first vaccination, with the maximum value of 1 : 81 920 and 1 : 20 480,respectively. After that, serum antibody levels remained at a plateau or had a slight decrease,though two boosters were given in the succedent 4 to 5 weeks. Cross neutralization response existed between SARS-CoV F69 strain and Z2-Y3 strain.Conclusions The inactivated SARS-CoVvaccine made from F69 strain owns strong immunogenicity, and the cross neutralization response between the two different SARS-CoV strains gives a hint of the similar neutralizing epitopes, which provide stable bases for the development of inactivated SARS-CoV vaccines.     

Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC

Objective In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. Methods A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. Results Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1:105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. Conclusion An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.     

全人源抗狂犬病病毒G蛋白单链抗体制备及中和活性鉴定

目的:构建全人源抗狂犬病病毒scFv噬菌体抗体库,筛选针对狂犬病病毒G蛋白不同表位的单链抗体(scFv)并鉴定其中和活性?方法:分离130例经狂犬病疫苗免疫接种志愿者的外周血淋巴细胞,提取总RNA,逆转录制备cDNA,构建全人源抗狂犬病病毒scFv噬菌体抗体库?以纯化的狂犬病病毒G蛋白包板筛选,对阳性克隆进行可溶性表达,并鉴定中和活性?结果:构建全人源抗狂犬病病毒scFv噬菌体抗体库,库容为5.0 × 107,经核酸序列分析,证实插入片段为scFv?经过5轮筛选,从富集的次级抗体库中随机挑出140个克隆,通过phage-ELISA鉴定得到4株核酸序列不同的scFv抗体?经His-Trap纯化后进行中和活性鉴定,获得1株有中和活性的scFv,其中和效价为0.13 IU/mg?结论:构建全人源抗狂犬病病毒scFv噬菌体抗体库,获得1株具有中和活性的抗狂犬病病毒G蛋白scFv抗体,为进一步研发狂犬病治疗性抗体药物奠定了基础?     

Immunogenicities of Env glycoproteins from circulating HIV-1 isolates in China focusing on the strategy of ＂DNA prime plus protein boost＂

Background The adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes. Methods Most circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48（AE）, GX79（AE）, NX22（BC）, GS22（BC）, HN24（Thai-B） were constructed. Expression of gp120 from these DNA vaccines was detected by Western blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of ＂DNA prime plus protein boost＂ and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains. Results Response of gp120-specific antibody was relatively low after DNA primes （mean titer=10^4.72）; however, the titer of gp120-specific antibody went up with 2 protein boosts （mean titer=10^6.81）. Above all, neutralizing antibody （Nab） titers induced by this combined approach were much better than those elicited by DNA or protein used alone （P 〈0.01）. Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses. Conclusion Polyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.     

Efficacy of inactivated vaccine containing cyto-hemagglutinin against epidemic hemorrhagic fever in rabbits

Using the Z-10 strain of epidemic hemorrhagic fever virus (EHFV) as seed, and the primary cell of Meriones unguiculatus kidney tissue as incubation cell, a propiolactone inactivated epidemic hemorrhagic fever (EHF) vaccine was prepared, according to a similar procedure required for the production of biological products such as the Japanese B encephalitis vaccine. Besides the EHFV antigen detected by ELISA or reversed passive hemagglutination test (RPHA) as were used for the formalin inactivated vaccine, higher titres (1:128-1:1024) of EHFV hemagglutinin antigen was also detected in this EHFV vaccine. Immunization with twice intramuscular injection of this vaccine produced high titred (1:20-1:160) neutralizing antibody and low titred (1:10-1:20) hemagglutination antibody, in addition to the immunofluorescence (IF) and reversed passive hemagglutination inhibition (RPHI) antibodies. These results indicated an apparent difference in the immunogenicity between the beta-propiolactone and formalin inactivated EHF vaccines. With the approval of the Ministry of Health, human test is now underway in this laboratory.     

新型冠状病毒中和抗体酶联免疫检测试剂盒的制备及应用

目的 开发一种新型冠状病毒中和抗体酶联免疫检测试剂盒,分析新型冠状病毒中和抗体在新冠疫苗效果监测中的临床应用价值。方法 表达纯化SARS-CoV-2病毒S蛋白RBD结构域和人ACE2蛋白,将RBD蛋白偶联HRP、ACE2蛋白偶联生物素(Bio),制备新型冠状病毒中和抗体检测试剂盒;收集15例注射新冠疫苗志愿者血清,检测新冠疫苗注射前后机体中和抗体水平。结果 注射新冠疫苗前SARS-CoV-2中和抗体阳性率为0.00%,第1、2、3针新冠疫苗注射后11～15 d,阳性率分别为13.33%、93.33%、100.00%,第2针注射后第180～190 d,阳性率降低到33.33%;性能检测分析显示,最低检出限为0.06μg/mL,批内CV<5%,批间CV<10%,线性检测范围为0.030～3.125μg/mL,分析灵敏度验证值为0.040μg/mL,与微量细胞中和试验进行对比,确认与其具有高度一致性。结论 新型冠状病毒中和抗体酶联免疫检测试剂盒性能评价良好,检测方法具有快速、方便、易操作等特点,可以用于评估新冠疫苗的保护效果。     

Vaccination of School Children With Live Mumps Virus Vaccine

Live, attenuated mumps virus vaccine (Mumpsvax) was administered to 146 school children 6 to 9 years of age. One child developed clinical mumps nine days after vaccination; epidemiological and serological data strongly suggest that this child had become infected before vaccination. Apart from this single instance there were no apparent clinical reactions that could be ascribed to the administration of the vaccine. Sixty-three of the 146 children with no clinical history of mumps had an initial serum neutralizing antibody titre of less than 1:2. Specific antibodies to mumps virus were detected in 93.5% of the sera of the susceptible children 28 days after vaccination, and the geometric mean antibody titre of these sera was low (1:6). Of the 80 initially seropositive children 21 (26.2%) showed a significant antibody response to the vaccine and this was influenced by the pre-existing antibody level. These data have further demonstrated the safety and efficacy of the live mumps vaccine in children.