口蹄疫细胞免疫研究进展

口蹄疫是世界性重大动物疫病之一,接种疫苗是预防该病的重要策略之一.随着对口蹄疫疫苗及其免疫特性的深入研究和探讨,许多学者对口蹄疫细胞免疫机制的研究更进了一步,就参与口蹄疫细胞免疫的各类细胞及细胞免疫与新疫苗设计的研究进展作一综述.     

口蹄疫新型疫苗及其免疫特性研究进展

口蹄疫是世界性重大动物疫病,接种疫苗是预防该病的重要策略之一。随着现代分子生物学技术的发展,对一些新型口蹄疫疫苗如亚单位疫苗、活载体疫苗、核酸疫苗、可饲疫苗、多表位疫苗等的研究和探索已全面展开。我们简要介绍了近年涌现的口蹄疫新型疫苗,以为口蹄疫分子疫苗的设计提供参考。     

口蹄疫病毒单克隆抗体的制备及应用研究进展

口蹄疫是严重影响全球政治经济的烈性动物传染病,快速诊断及有效防治对口蹄疫的防控具有重要意义。单克隆抗体具有高特异性、均质、活性单一等优点,在生物医学领域中有广泛用途。目前,国内外学者制备了多种抗口蹄疫病毒的单克隆抗体,并应用于口蹄疫病毒抗原定型、疫苗量化、抗体水平监测、自然感染与疫苗免疫动物的鉴别诊断,以及口蹄疫病毒抗原表位分析等方面。我们简要综述口蹄疫病毒单克隆抗体的制备及应用进展。     

口蹄疫病毒重组蛋白表达及其亚单位疫苗研究进展

口蹄疫(FMD)是偶蹄动物的一种急性、热性传染病,曾多次在世界上发生过大流行,研制和生产新型口蹄疫疫苗是防制该病爆发的有效措施之一。目前,基因工程疫苗成为该领域的研究热点。就口蹄疫病毒(FMDV)结构蛋白和非结构蛋白在大肠杆菌、昆虫细胞、酵母菌、哺乳动物细胞和转基因植物等系统中的表达现状及其基因工程亚单位疫苗研究进展进行综述。     

口蹄疫新型疫苗研究进展

口蹄疫(FMD)是一种严重威胁畜牧业发展的重要传染病,目前世界上许多国家和地区都有该病的流行与发生.其控制措施主要是疫苗免疫,虽然传统疫苗在该病的防控中起了重要的作用,但也存在着诸多的缺点.因此研制新型的FMD疫苗是今后的发展方向.本文结合实验室在FMD新型疫苗研究方面所开展的探索性研究工作,综述了国内外在FMD基因工程弱毒苗或灭活苗、蛋白质和合成肽疫苗、空衣壳疫苗、细胞因子增强型疫苗等研究领域所取得的进展.     

铁蛋白与口蹄疫病毒VP1在大肠杆菌中融合表达及纳米颗粒自组装

口蹄疫是由口蹄疫病毒引起的世界上最重要的畜牧疾病之一,严重影响世界畜牧业的发展,而疫苗免疫仍然是对疫情预防和控制的最有效手段。铁蛋白具有自组装和生物修饰的特性,在纳米疫苗等领域具有广阔的应用前景。选用O型口蹄疫病毒的vp1基因和幽门螺杆菌铁蛋白基因,通过融合PCR将vp1基因构建到铁蛋白亚基基因前端,在大肠杆菌中表达后通过His标签进行镍柱亲和层析纯化。将纯化好的重组蛋白进行Western blotting检测、质谱分析和透射电镜观察,发现重组蛋白VP1-Ferritin在大肠杆菌中获得表达,并可自组装成纳米颗粒。     

近年来出现的几类新概念动物疫苗

动物疫病流行广泛、传播迅速,严重危害养殖业的发展。疫苗接种是预防和控制动物传染病最有效的策略之一。目前,随着生物技术的发展和疫病防控的需要,安全、高效、广谱、用量少、具有标记特征的新型疫苗成为研发重点。文中就近年来出现的黏膜疫苗、长效与速效疫苗、嵌合疫苗、纳米颗粒疫苗等新概念动物疫苗的发展、应用及优缺点进行了评述,并提出了其发展方向,以期为动物疫苗的研发提供借鉴。     

禽流感病毒新型疫苗的研究进展

疫苗免疫是禽流感防控的主要措施之一,随着生物技术的不断发展,基因工程亚单位疫苗、活载体疫苗、DNA疫苗等新型疫苗得以研究和开发,这为禽流感的防控提供了新的手段。新型疫苗除具有传统疫苗的保护效果外,在生物安全和普遍防控等方面也具有广泛的优势,是禽流感疫苗发展的新方向。     

型内嵌合口蹄疫病毒全长感染性cDNA克隆的构建

【目的】近年来,O型口蹄疫的不断暴发严重危害了我国畜牧业的发展,其病原——O型口蹄疫病毒已演化出3种谱系:中国型猪毒系、泛亚系和缅甸98系。其中中国型猪毒系病毒高度嗜猪,对养猪业危害最大。目前应用的疫苗已不能有效保护中国型猪毒系变异株的流行,这给我国猪口蹄疫的防控带来了极大的困难。为了进一步发展免疫原性好、抗原谱广的猪O型口蹄疫疫苗候选株,本研究以O/HN/93现用疫苗毒株的感染性克隆为骨架,用流行的新猪毒系病毒的部分VP3和VP1基因(主要是替换VP1蛋白上的B-C环和G-H环)替换疫苗毒株的相应部分,构建了嵌合的FMDV全长cDNA克隆。【方法】线化的嵌合全长质粒和表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,体内转录拯救嵌合病毒。【结果】嵌合全长质粒转染BHK-21细胞36h后,出现明显的FMDV致细胞病变效应。对收获的病毒分别用RT-PCR、间接免疫荧光、电子显微镜观察结果证实成功拯救到嵌合的FMDV。拯救的病毒乳鼠致病性试验结果表明该拯救病毒对乳鼠的致病力减弱。该嵌合病毒的成功拯救为研制口蹄疫新型疫苗等奠定了基础。     

保护剂及其在口蹄疫疫苗中的研究进展

保护剂在动物疫苗中具有重要的作用,直接影响疫苗的质量.以保护剂为核心介绍了在疫苗保存前病毒抗原和保护剂的准备,保护剂的选用原则,分类、作用机理,着重讨论了保护剂在口蹄疫疫苗中的研究进展以及当前存在的问题,并对其应用前景提出展望.     

Saponin Adjuvants. IV. Evaluation of The Adjuvant Quil a in the Vaccination of Cattle Against Foot-And-Mouth Disease

The saponin adjuvant Quil A has been investigated in the vaccination of cattle against foot-and-mouth disease. Using a Frenkel type vaccine a dose-response relationship has been established between Quil A and neutralizing antibody titres. Ten ml of vaccine was combined with 0, 50, 200, 800, and 3200 µg of Quil A. The combinations were each injected into 4 animals. The local reaction on the site of injection produced by injection of the vaccine alone and in combination with different doses of Quil A has been estimated. On this basis a therapeutical dose at 1 mg of Quil A has been estimated to combine maximum adjuvant effect with a minimum of adverse reactions. This dose has been tested in the vaccination of cattle with FMD vaccines derived from BHK suspension cell virus of type O and A respectively. The vaccines were tested in 10 ml and 5 ml doses with or without Quil A, and each in 4 animals. It is concluded that Quil A is a valuable adjuvant for use in the induction of neutralizing antibodies against foot-and-mouth disease in cattle.     

Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves

The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R² = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.     

The stability and potency of vaccines prepared from inactivated foot-and-mouth disease virus concentrates

The stability of 146S particles in concentrates of foot-and-mouth disease virus stored at 4 degrees C was similar to that of 146S particles in a conventional virus preparation. Proteolytic degradation of VPl was not observed in the stored conventional virus preparation or inhibitor-supplemented concentrate but was observed in a supplement-free concentrate. The potencies of vaccines made from the conventional and concentrated preparations and stored in parallel at 4 degrees C appeared to decrease after 16 weeks. The vaccines made from the supplement-free concentrate and the Trasylol supplemented concentrate appeared to be at least as potent as the conventional vaccine and were clearly superior to vaccine made from ox serum supplemented concentrate.     

口蹄疫病毒氨基酸改造对其病毒样颗粒稳定性的影响

病毒样颗粒 (Virus like particles,VLPs) 的稳定性是目前影响口蹄疫VLPs疫苗质量的主要因素。为进一步提升口蹄疫VLPs疫苗的质量,基于口蹄疫病毒三维空间结构,通过动力学分析软件设计并筛选出3个氨基酸改造位点。经点突变试剂盒成功制备出上述3种突变型重组质粒,转化大肠杆菌Escherichia coli BL21菌株后经体外诱导表达,Ni离子层析柱纯化后,SDS-PAGE结果证明3种氨基酸突变不影响目标蛋白的表达。体外组装获得的3种突变型VLPs稳定性研究结果发现,内部疏水性侧链氨基酸的引入使VLPs的形态变得更加均一 (N4017W),且其稳定性比其他两种VLPs明显提高。结果表明,衣壳内部疏水性作用力有助于VLPs的形成且有助于维持衣壳的稳定性,为提高VLPs疫苗质量提供了新的研究思路,有助于推进VLPs疫苗的发展。     

A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants.

A large-scale vaccination experiment involving a total of 138 cattle was carried out to evaluate the potential of synthetic peptides as vaccines against foot-and-mouth disease. Four types of peptides representing sequences of foot-and-mouth disease virus (FMDV) C3 Argentina 85 were tested: A, which includes the G-H loop of capsid protein VP1 (site A); AT, in which a T-cell epitope has been added to site A; AC, composed of site A and the carboxy-terminal region of VP1 (site C); and ACT, in which the three previous capsid motifs are colinearly represented. Induction of neutralizing antibodies, lymphoproliferation in response to viral antigens, and protection against challenge with homologous infectious virus were examined. None of the tested peptides, at several doses and vaccination schedules, afforded protection above 40%. Protection showed limited correlation with serum neutralization activity and lymphoproliferation in response to whole virus. In 12 of 29 lesions from vaccinated cattle that were challenged with homologous virus, mutant FMDVs with amino acid substitutions at antigenic site A were identified. This finding suggests the rapid generation and selection of FMDV antigenic variants in vivo. In contrast with previous studies, this large-scale vaccination experiment with an important FMDV host reveals considerable difficulties for vaccines based on synthetic peptides to achieve the required levels of efficacy. Possible modifications of the vaccine formulations to increase protective activity are discussed.     

Potency assessment of foot-and-mouth disease vaccines in cattle by means of antibody assays.

A tendency has emerged for some years to replace the challenge infection of cattle for the assessment of foot-and-mouth disease (FMD) vaccine potency. This can be actually evaluated by means of antibody assays on cattle sera, at about 3/4 weeks after the vaccination. Serological results can be worked out as single titres (to be compared with a pre-determined threshold level) or as mean antibody titres induced by different vaccine dilutions. However, the assessment of FMDV-specific antibody titres would not fully depict the extent and the efficacy of the immune response of cattle; moreover, the antibody response would not be proportional if potent vaccines are used (greater than or equal to 10-12 PD50). Thus, a particular approach is suggested for the serological procedures, which enable credible estimates of potent FMD vaccines to be formulated.     

Engineering viable foot-and-mouth disease viruses with increased thermostability as a step in the development of improved vaccines

We have rationally engineered foot-and-mouth disease virus to increase its stability against thermal dissociation into subunits without disrupting the many biological functions needed for its infectivity. Amino acid side chains located near the capsid intersubunit interfaces and either predicted or found to be dispensable for infectivity were replaced by others that could establish new disulfide bonds or electrostatic interactions between subunits. Two engineered viruses were normally infectious, genetically stable, and antigenically indistinguishable from the natural virus but showed substantially increased stability against irreversible dissociation. Electrostatic interactions mediated this stabilizing effect. For foot-and-mouth disease virus and other viruses, some evidence had suggested that an increase in virion stability could be linked to an impairment of infectivity. The results of the present study show, in fact, that virion thermostability against dissociation into subunits may not be selectively constrained by functional requirements for infectivity. The thermostable viruses obtained, and others similarly engineered, could be used for the production, using current procedures, of foot-and-mouth disease vaccines that are less dependent on a faultless cold chain. In addition, introduction of those stabilizing mutations in empty (nucleic acid-free) capsids could facilitate the production of infection-risk-free vaccines against the disease, one of the economically most important animal diseases worldwide.     

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.     

伪狂犬病新型疫苗研究进展

伪狂犬病是多种家畜和野生动物的一种重要传染病,给世界畜牧业特别是养猪业造成了巨大的经济损失,疫苗免疫是预防控制该病的主要手段。综述了伪狂犬病亚单位疫苗,核酸疫苗,重组疫苗,基因缺失疫苗等新型疫苗的研究进展。