森林脑炎灭活疫苗和纯化疫苗及其不同免疫剂量的免疫效果研究

为了评价森林脑炎灭活疫苗的远期预防效果及森林脑炎纯化疫苗的人体免疫效果 ,并对 2种疫苗的安全性和免疫效果进行比较。 2种疫苗分别按要求进行免疫接种 ,观察局部反应和全身反应 ,并定期采集血清样本 ,分离血清用酶联免疫吸附法 (ELISA)和蚀斑减少试验法检测血清中和抗体效价。试验结果 :森林脑炎灭活疫苗局部反应发生率为 2 3.0 8% ,全身反应发生率为 15 .38% (16 /10 4 ) ,人群抗体在 2次接种后约 1/ 3人检出阳性 ,3次接种后约 1/ 2人检出阳性 ,多次免疫后 6 0 %检出阳性 ;森林脑炎纯化疫苗无 1例发生局部反应、全身反应 ,注射部位一…     

流行性乙型脑炎病毒血清抗体半微量空斑抑制法的改进

本文报道一种检测流行性乙型脑炎(简称乙脑)中和抗体的简化空斑抑制试验方法,用BHK21传代细胞在24孔塑料板上进行试验,在细胞未形成单层时,将病毒直接接种在刚消化的细胞悬液内,孵育后加甲基纤维素复盖物,再培养,细胞层经染色后即可计算空斑数。 用该法检查47名儿童乙脑疫苗免疫前后的中和抗体,与常规的鸡胚细胞全量法或BHK21传代细胞微量中和试验法作了比较,结果表明本法与其它常规法的敏感性一致,且具有简便快速、节省人力和原材料、空斑形成率高的优点,便于大量血清学检测。     

两种方法检测接种流行性出血热沙鼠肾细胞灭活疫苗后人体的中和抗体

本文应用空斑减少中和试验(PRNT)和细胞病变中和试验(cPENT)两种方法对出血热沙鼠肾细胞灭活疫苗扩大人体免疫后的血清进行中和抗体水平检测。根据两种方法对总计74人份的免疫后血清检测比较结果,两种方法检测的抗体阳转率和抗体水平(GMT)。CPENT法均高于PRNT法,经统计学处理均有显著性差异。不同免疫组的中和抗体水平比较结果,注射三针的阳转率(n=10,100％)高于两针组(n=10,20—30％);接种加氢氧化铝佐剂疫苗(n=13)较接种不加佐剂的两种疫苗(n=26)的抗体水平高,阳转率为92％—100％GMT为22—69;皮下途径(n=15)和肌肉途径(n=13)注射无明显差别,阳转率分别为87—93％和92—100％,GMT分别为29—46和22—61。以上结果进一步肯定沙鼠肾细胞疫苗的人体免疫性     

一种新型T细胞免疫原的原核表达及对口蹄疫疫苗的免疫增强作用

主要探讨了T细胞免疫原TI对口蹄疫疫苗的免疫增强作用。设计并原核表达产生了一种包含口蹄疫病毒VP1,VP4,3A和3D蛋白上多个T细胞表位与两个通用T细胞表位的T细胞免疫原,命名为TI;同时表达了O和Asia 1两个型口蹄疫病毒 VP1 蛋白的串联编码基因,表达产物命名为OA-VP1。将上述T细胞免疫原分别与OA-VP1和口蹄疫灭活疫苗按不同剂量组合免疫小鼠,于免疫后不同时间测定各组小鼠的体液与细胞免疫应答情况。采用微量中和试验检测小鼠O型和Asia1型中和抗体,采用流式细胞检测技术和测定γ-干扰素的水平来分析不同免疫组小鼠细胞免疫的水平。结果显示,与灭活疫苗或OA-VP1单独免疫组相比,添加TI抗原后灭活疫苗 (P＜0.01) 和OA-VP1免疫组(P＜0.05)小鼠均能产生高水平的特异性中和抗体;且CD4+ T细胞数量显著增多,IFN-γ产生水平显著升高 (P＜0.01)。说明TI抗原具有很好的诱导特异性体液与细胞免疫应答的作用,是一种很好的免疫增效剂,可作为口蹄疫蛋白亚单位疫苗和灭活疫苗中的一种有效成分,以提高疫苗的免疫效果。     

EV71与CA16双价灭活疫苗诱导小鼠免疫原性研究

目的评价EV71和CA16双价灭活疫苗免疫小鼠诱导的体液免疫和细胞免疫应答。方法分别应用EV71和CA16双价疫苗、EV71和CA16单价疫苗按单针、两针(0,14 d)的程序免疫小鼠,采用细胞病变法检测血清第1针免疫后21 d时中和抗体效价,应用LUMINEX液相芯片技术检测小鼠脾单个核细胞体外经抗原刺激分泌细胞因子的水平。结果双价疫苗与EV71单价疫苗单针、两针免疫相比,EV71中和抗体几何平均值相近(118.8∶84.0;159.5∶156.8,P0.05)。双价疫苗与CA16单价疫苗单针免疫诱导的CA16中和抗体均为阴性;两针免疫CA16中和抗体几何平均值一致(30.0∶26.9,P0.05)。加强免疫7 d后,小鼠脾MNC经EV71抗原刺激,双价疫苗较EV71单价疫苗诱导Th2类细胞因子IL-4、5、6、10和炎症因子TNF-α的分泌水平显著增高(P=0.020,P=0.027,P=0.038,P=0.019,P=0.026);MNC经CA16抗原刺激,双价疫苗与CA16单价疫苗相比诱导细胞因子水平差异无统计学意义(P0.05)。结论双价疫苗可诱导与单价疫苗相似的中和抗体应答水平,并可提高Th2类细胞因子应答,研究为EV71和CA16双价灭活疫苗的研发提供了实验依据。     

H7N9灭活疫苗联合MF59佐剂在小鼠体内诱导的长效体液免疫应答

以BALB/c小鼠为模型,探讨H7N9流感病毒灭活疫苗免疫小鼠后所诱导的长效体液免疫应答的动态变化。不同剂量的流感H7N9全病毒灭活疫苗单独或辅以MF59佐剂肌肉注射免疫小鼠一次。连续采集免疫后小鼠15个月的血清,用ELISA方法检测特异性IgG抗体水平,血凝抑制(hemagglutination inhibition,HI)试验和微量中和(microneutralization,MN)试验检测第15个月时的HI抗体和中和抗体效价。实验结果发现,小鼠血清中的特异性IgG抗体水平随时间变化持续缓慢上升,第5个月时达到顶峰,随后略有下降但一直持续平稳状态;IgG抗体滴度与疫苗剂量成正相关,且添加佐剂能提高抗体滴度。HI及MN抗体检测表明,免疫后第15个月产生的抗体能有效中和病毒,且抗体跟疫苗剂量成正比。以上研究表明,H7N9流感病毒灭活疫苗免疫小鼠一次诱导产生的特异性抗体能在较长期内保持比较平稳的抗体滴度,为小鼠提供免疫保护;增加抗原剂量和添加MF59佐剂能增加疫苗特异性抗体水平。该研究为H7N9流感疫苗产生的长期保护效应提供了一定的数据积累和参考。     

轮状病毒灭活疫苗和减毒活疫苗序贯免疫的体液免疫应答效果

目的:评价采用轮状病毒灭活疫苗进行初始免疫,减毒活疫苗进行加强免疫的序贯免疫方案的体液免疫应答效果。方法:将实验小鼠随机分为4组(口服疫苗组、序贯疫苗组、口服对照组及序贯对照组),按相应方案免疫后,ELISA检测血清轮状病毒特异性IgG和IgA、肠道轮状病毒特异性IgA;微量中和实验检测血清病毒特异性中和抗体;同时采用ELISA分析口服活疫苗后病毒排出情况。结果:与对照组相比,序贯疫苗组小鼠产生的轮状病毒特异性血清IgG、IgA、中和抗体及肠道IgA水平显著升高。与口服疫苗组相比,序贯疫苗组的免疫方案诱发的轮状病毒特异性血清IgG、IgA、中和抗体水平显著升高,肠道IgA水平两组间没有显著差异。同时,与口服疫苗组相比,序贯疫苗组中轮状病毒灭活疫苗进行的初始免疫未影响第一次口服活疫苗后病毒的排出量和排出时间,但序贯疫苗组第二次口服活疫苗后病毒的排出量迅速减少,排毒时间快速缩短,与口服疫苗组第三次服苗后病毒的排出量和排出时间相似。结论: 轮状病毒灭活疫苗和减毒活疫苗序贯免疫可有效诱发小鼠全身和黏膜局部的体液免疫应答,该方案将有可能成为轮状病毒疫苗临床应用的候选方案。     

病毒中和抗体检测方法研究进展

疫苗接种在病毒性传染病防控中发挥了重要的作用,有效的疫苗在机体内能够快速诱导高效价的中和抗体,中和抗体水平是衡量疫苗免疫保护效果的关键指标。为应对疫苗的快速研发、大规模临床试验检测以及疫苗接种后人群免疫原性检测的需求,急需建立高灵敏度、高通量的病毒中和抗体评价方法。目前病毒中和抗体检测方法除传统的以活病毒为基础的噬斑抑制法和细胞病变法外,结合新技术发展,已经有很多新的方法,如含有报告基因的重组活病毒的检测方法和以假病毒为基础的中和抗体检测方法等,现对病毒中和抗体检测方法的研究作一综述。     

重组PA4复制子病毒颗粒疫苗免疫原性的分析

目的:构建携带炭疽毒素保护性抗原第四结构域(PA4)基因的重组Semliki森林病毒(SFV)复制子病毒颗粒,并对其免疫原性进行研究。方法:将编码炭疽PA4的SFV复制子DNA载体pSCAR-SPA4,与辅助SFV DNA载体pSHCAR共转染BHK21细胞,制备表达PA4的重组复制子病毒颗粒;用重组复制子病毒颗粒疫苗免疫小鼠,并采用ELISA法检测其血清抗体水平和细胞因子IFN-γ和IL-4。结果:免疫小鼠血清中检测到较高的抗体水平,免疫小鼠的脾淋巴细胞经特异性抗原刺激后产生了明显的T细胞增殖反应并分泌产生了IFN-γ和IL-4。结论:重组PA4复制子病毒颗粒疫苗免疫小鼠后能够产生特异性的抗体反应和细胞免疫反应。制备的重组PA4复制子病毒颗粒极有潜力作为人用炭疽候选疫苗,为进一步研究新型炭疽疫苗奠定了基础。     

乙型脑炎病毒DNA初免-蛋白加强策略在小鼠模型上的免疫效应评价

为了评价基因Ⅰ型乙型脑炎病毒prM-E DNA疫苗与prM和EⅢ融合抗原亚单位疫苗采用DNA初免-蛋白加强免疫策略对小鼠的免疫效果,本研究将prM-E融合基因插入到pVAX1真核表达载体中,构建重组表达载体prM-E-pVAX1作为DNA疫苗进行初免,利用原核表达系统获得的prM和EⅢ融合抗原作为亚单位疫苗进行加强免疫。将32只4−6周龄雌性BALB/c小鼠随机分成4组,设置prM-E-pVAX1 DNA疫苗组、DNA初免-蛋白加强免疫组、prM和EⅢ融合抗原亚单位疫苗组及pVAX1载体对照组,通过ELISA检测血清中特异性抗体水平;通过噬斑减少中和试验滴定中和抗体滴度;通过细胞因子表达丰度和淋巴细胞增殖试验分析不同疫苗免疫组诱导产生的细胞免疫反应。结果表明,用DNA初免-蛋白加强策略免疫的小鼠诱导产生的中和抗体滴度略高于prM和EⅢ融合抗原亚单位疫苗免疫组,显著高于prM-E-pVAX1 DNA疫苗免疫组。DNA初免-蛋白加强策略在小鼠模型中诱导产生了有效的Th1/Th2型免疫反应,特别是显著诱导了Th1型细胞免疫反应。本研究为预防流行性乙型脑炎提供了新的免疫策略和理论参考依据。     

森林脑炎自然疫源地样本的监测及病毒的分离研究

为了解森林脑炎疫源地的分布变化趋势及样本分离病毒的特性,采集了森林脑炎高发区周边的森林全沟硬蜱、血蜱样本及森林脑炎患者的脑组织样本,用小白鼠脑内接种法检测、分离病毒分离的病毒经鉴别试验证明为森林脑炎病毒：蜱、脑两种标本检测的阳性率分别为50％和100％、结果表明森林脑炎的疫区有从林区向农业区扩散的趋势,且全沟硬蜱的带毒率较高;森脑患者的脑组织样本与蜱标本病毒的性状育差异     

白术多糖对小鼠免疫功能调节的研究

目的将白术多糖作为抗原刺激小鼠免疫系统,探讨其对免疫功能调节作用,为多糖在免疫调节功能方面的应用开发及在药物鉴定方面的应用提供科学依据。方法分别给予小鼠自术多糖、可溶性淀粉多糖、伤寒多糖抗原刺激,检测小鼠血清中的相应抗体及交叉抗体,探讨补益类水溶性多糖对小鼠免疫功能的调节作用。结果多糖抗原均能刺激机体产生特异性IgG类抗体（P〈0.05）;也能在一定程度上激发非特异性IgG类抗体即交叉抗体的产生;但不激发病理性抗体产生（RF阴性）。结论补益类水溶性多糖能激发免疫反应与其他多糖可能有共同的途径和机制。     

Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice

The smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have produced protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax). H3L, an intracellular mature virion envelope protein, was consistently recognized by high-titer antibodies in the majority of human donors, particularly after secondary immunization. We then focused on examining H3L as a valuable human antibody target. Purified human anti-H3L antibodies exhibited substantial vaccinia virus-neutralizing activity in vitro (50% plaque reduction neutralization test [PRNT50] = 44 microg/ml). Mice also make an immunodominant antibody response to H3L after vaccination with vaccinia virus, as determined by vaccinia virus protein microarray. Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed high-titer vaccinia virus-neutralizing antibodies (mean PRNT50 = 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD50) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02). Together, these data show that H3L is a major target of the human anti-poxvirus antibody response and is likely to be a key contributor to protection against poxvirus infection and disease.     

人乳头瘤病毒中和表位及抗体中和作用机制的研究进展

人乳头瘤病毒(Human papillomavirus,HPV)是一类无包膜的小DNA病毒,其衣壳蛋白由主要衣壳蛋白L1和次要衣壳蛋白L2组成,持续感染HPV将引起宫颈癌和尖锐湿疣等多种疾病。HPV衣壳蛋白L1和L2中分布着大量中和表位,并具有较强的免疫原性,HPV疫苗可诱导机体产生高滴度的中和抗体并阻碍病毒感染,进而预防宫颈癌等疾病的发生。分析阐述HPV衣壳蛋白中和表位及抗体的中和作用机理,有助于阐明HPV疫苗预防病毒感染的作用机制,为今后设计新一代保护范围更广的HPV疫苗奠定良好的基础。本文就HPV衣壳蛋白中和表位及抗体的中和作用机制进行综述。     

广谱流感疫苗的研究进展

广谱流感疫苗是指能够诱导针对流感病毒的广谱中和抗体或广谱的细胞免疫反应,从而保护动物或人类免受多数流感病毒毒株感染的疫苗。流感病毒广谱中和抗体的发现及机制研究为广谱流感疫苗的研发提供了新的思路。同时,流感病毒细胞免疫方面的研究进展、佐剂以及免疫策略等的研究进展都极大促进了广谱流感疫苗的研发。本文从以上几个方面介绍广谱流感疫苗的研究进展,并对未来的应用进行评价及展望。     

Evaluation of the European tick‐borne encephalitis vaccine against Omsk hemorrhagic fever virus

This study focused on the antigenic cross‐reactivity between tick‐borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) to assess the efficacy of the commercial TBE vaccine against OHFV infection. Neutralization tests performed on sera from OHFV‐ and TBEV‐infected mice showed that neutralizing antibodies are cross‐protective. The geometric mean titers of antibodies against TBEV and OHFV from TBEV‐infected mice were similar. However, the titers of anti‐TBEV antibodies in OHFV‐infected mice were significantly lower than those of anti‐OHFV antibodies in the same animals. In mouse vaccination and challenge tests, the TBE vaccine provided 100% protection against OHFV infection. Eighty‐six percent of vaccinees seroconverted against OHFV following complete vaccination, and the geometric mean titers of neutralizing antibodies against OHFV were comparable to those against TBEV. These data suggest that the TBE vaccine can prevent OHFV infection.     

A recombinant vaccine effectively induces c5a-specific neutralizing antibodies and prevents arthritis

Objectives

To develop and validate a recombinant vaccine to attenuate inflammation in arthritis by sustained neutralization of the anaphylatoxin C5a.

Methods

We constructed and expressed fusion protein of C5a and maltose binding protein. Efficacy of specific C5a neutralization was tested using the fusion protein as vaccine in three different arthritis mouse models: collagen induced arthritis (CIA), chronic relapsing CIA and collagen antibody induced arthritis (CAIA). Levels of anti-C5a antibodies and anti-collagen type II were measured by ELISA. C5a neutralization assay was done using a rat basophilic leukemia cell-line transfected with the human C5aR. Complement activity was determined using a hemolytic assay and joint morphology was assessed by histology.

Results

Vaccination of mice with MBP-C5a led to significant reduction of arthritis incidence and severity but not anti-collagen antibody synthesis. Histology of the MBP-C5a and control (MBP or PBS) vaccinated mice paws confirmed the vaccination effect. Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered.

Conclusions

Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases. Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity.     

Preclinical investigations of the safety, immunogenicity and efficacy of a purified, inactivated tick-borne encephalitis vaccine

A new tick-borne encephalitis (TBE) vaccine for human use has been developed. TBE virus (TBEV) was propagated in primary chick embryo cells, inactivated by formalin and purified by continuous-flow density gradient centrifugation. The TBE vaccine was tested for innocuity, immunogenicity and protective capacity in a series of laboratory tests. The results indicated that the vaccine is outstandingly well tolerated, highly immunogenic in various laboratory animals, and induces protective immunity in mice. These data suggest that this new vaccine should be studied in clinical trials.     

Zika virus-like particle vaccine protects AG129 mice and rhesus macaques against Zika virus

BackgroundZika virus (ZIKV), a mosquito-borne flavivirus, is a re-emerging virus that constitutes a public health threat due to its recent global spread, recurrent outbreaks, and infections that are associated with neurological abnormalities in developing fetuses and Guillain-Barré syndrome in adults. To date, there are no approved vaccines against ZIKV infection. Various preclinical and clinical development programs are currently ongoing in an effort to bring forward a vaccine for ZIKV.Methodology/Principle findingsWe have developed a ZIKV vaccine candidate based on Virus-Like-Particles (VLPs) produced in HEK293 mammalian cells using the prM (a precursor to M protein) and envelope (E) structural protein genes from ZIKV. Transient transfection of cells via plasmid and electroporation produced VLPs which were subsequently purified by column chromatography yielding approximately 2mg/L. Initially, immunogenicity and efficacy were evaluated in AG129 mice using a dose titration of VLP with and without Alhydrogel 2% (alum) adjuvant. We found that VLP with and without alum elicited ZIKV-specific serum neutralizing antibodies (nAbs) and that titers correlated with protection. A follow-up immunogenicity and efficacy study in rhesus macaques was performed using VLP formulated with alum. Multiple neutralization assay methods were performed on immune sera including a plaque reduction neutralization test, a microneutralization assay, and a Zika virus Renilla luciferase neutralization assay. All of these assays indicate that following immunization, VLP induces high titer nAbs which correlate with protection against ZIKV challenge.Conclusions/SignificanceThese studies confirm that ZIKV VLPs could be efficiently generated and purified. Upon VLP immunization, in both mice and NHPs, nAb was induced that correlate with protection against ZIKV challenge. These studies support translational efforts in developing a ZIKV VLP vaccine for evaluation in human clinical trials.     

Redundancy and plasticity of neutralizing antibody responses are cornerstone attributes of the human immune response to the smallpox vaccine

The smallpox vaccine is widely considered the gold standard for human vaccines, yet the key antibody targets in humans remain unclear. We endeavored to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans which could be used as a diagnostic serological marker of protective humoral immunity induced by the smallpox vaccine (vaccinia virus [VACV]). We have instead found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show that H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of immunoglobulin G (IgG) specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV. Comparable results were obtained for human (and mouse) anti-L1 IgG and even for anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen but instead by driving development of neutralizing antibodies to multiple viral proteins, resulting in a "safety net" of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose that this is a fundamental attribute of the smallpox vaccine.