一种KBMA炭疽疫苗候选株的研制

炭疽病是由炭疽芽胞杆菌Bacillus anthracis引起的一种人畜共患传染病,严重影响着人类的健康。近年来在细菌疫苗的研究中发现一种特殊的现象：细菌被杀死后,体内的代谢活性却仍然维持 (Killed but metabolically active,KBMA)。此发现为炭疽新型疫苗候选株的研制提供了新思路。先通过同源重组的方法,利用pMAD质粒和Cre-loxP重组酶系统完成对缺失两个毒性大质粒的炭疽芽胞杆菌减毒株AP422的uvrAB基因的敲除,得到AP422△uvrAB菌株,然后通过光化学处理 (包括长波紫外光的照射和8-甲氧基补骨脂素处理),使炭疽芽胞杆菌AP422△uvrAB失去繁殖能力。利用四氮唑化合物MTS检测其代谢活性,表明光化学处理杀死后的炭疽芽胞杆菌AP422△uvrAB在至少4 h内维持一个很高的代谢活性水平,即具备典型的KBMA特性。炭疽杆菌AP422 △uvrAB的KBMA菌株的成功研制为我们提供了一种新型炭疽疫苗候选株。     

炭疽活疫苗家兔免疫力与血清抗芽胞IgG关系的研究

炭疽疫苗是预防炭疽流行和炭疽生物恐怖的重要手段。已有动物实验表明,炭疽活疫苗的保护力优于以保护性抗原为主要成份的无细胞疫苗,但两类现行疫苗都有待重新评价和改进。炭疽疫苗的效力必须用适当的实验室方法进行检测与分析才能了解其性质和细节。试验中力图探寻炭疽活疫苗家兔免疫力与血清抗芽胞抗体水平的关系。用“皮上划痕人用炭疽活疫苗”免疫家兔,以特定制备的炭疽芽胞抗原用ELISA法检测血清抗炭疽芽胞IgG抗体水平,并用强毒炭疽杆菌攻击进行效力试验。免疫家兔血清几何平均抗芽胞IgG滴度在免疫后一个月内持续升高,14d达到206,28d时达到776,这时其抵抗20MLD毒菌攻击的保护率为80％,符合中国生物制品规程要求的保护力。一个月后抗体水平开始下降,42d时滴度降至223。实验所揭示的炭疽减毒活疫苗诱导的家兔抗芽胞IgG抗体与抗炭疽保护力之间的关系,既为评价现行疫苗提供了资料,也为研制新型疫苗建立了参考性指标。     

炭疽灭活和裂解芽胞抗原免疫家兔后血清中的IgG抗体应答

炭疽杆菌芽胞在炭疽免疫中发挥基本作用。实验中以炭疽活芽胞疫苗为原形,建立了制备灭活和裂解炭疽芽胞的方法,研究了各种灭活和裂解炭疽芽胞疫苗不同浓度、不同剂次免疫家兔的抗芽胞和毒素IgG应答,总结分析了各种灭活和裂解炭疽芽胞疫苗用于新疫苗成分之一的可能性。甲醛灭活炭疽芽胞疫苗设芽胞浓度2.5×108剂量组、5×108剂量组、1×109剂量组,于0、4、8周时3次免疫。在3剂免疫后血清抗炭疽芽胞IgG水平持续升高,首次免疫后4、8、12周时家兔血清中抗芽胞IgG几何平均滴度可达到600～16000。裂解炭疽芽胞疫苗的制备和动物免疫中,只采取了2.5×108芽胞浓度,两剂免疫,免疫时间为0、4周。在首次免疫后4、8、12周时家兔血清中抗芽胞IgG几何平均滴度分别为362、776和388。各时间点采集的家兔血清未能测出或只测出极微量的抗炭疽毒素IgG。通过上述研究认为,以裂解炭疽芽胞抗原作为炭疽疫苗成分之一,其抗原性和免疫原性是适宜的;免疫剂量可以设定为2.5×108芽胞浓度上下;免疫次数可定为2剂间隔1个月。     

PlcR在炭疽芽胞杆菌A16R中的功能研究

炭疽芽胞杆菌(Bacillus anthracis)、蜡样芽胞杆菌(B. cereus)和苏云金芽胞杆菌(B. thuringiensis)均属于蜡样芽胞杆菌群,在遗传学上有很高的相似性。PlcR (Phospholipase C regulator)在蜡样芽胞杆菌中是十分重要的调控因子,但plcR基因在炭疽芽胞杆菌中发生一个无义突变导致在炭疽芽胞杆菌中产生一个截短PlcR蛋白。为了研究plcR基因对炭疽芽胞杆菌功能的影响,文章以蜡样芽胞杆菌CMCC6330基因组为模板,构建重组表达质粒pBE2A-plcR后导入炭疽芽胞杆菌疫苗株A16R中获得重组菌株,对其进行表型分析。结果显示,炭疽芽胞杆菌重组菌株的溶血活性基本没有恢复,但恢复了部分神经鞘磷脂酶活性,表明将蜡样芽胞杆菌的plcR基因导入炭疽芽胞杆菌后,可以直接激活神经鞘磷脂酶活性。     

炭疽芽胞杆菌中CRISPR位点

【目的】考察炭疽芽胞杆菌中规律成簇的间隔短回文序列(Clustered regularly interspaced short palindromic repeats,CRISPR)位点多态性情况及基于CRISPR位点多态性的分子分型方法是否在炭疽芽胞杆菌分型中适用。【方法】下载NCBI数据库中6株炭疽芽胞杆菌基因组并截取其中CRISPR位点片段序列。根据炭疽芽胞杆菌内CRISPR位点信息,设计相关引物,以193株炭疽芽胞杆菌基因组为模板PCR扩增CRISPR位点片段,测序。本地Blast比对截取序列及测序结果,查看CRISPR位点在炭疽芽胞杆菌中的多态性情况,并比较炭疽芽胞杆菌与蜡样芽胞杆菌和苏云金芽胞杆菌内CRISPR位点情况。【结果】炭疽芽胞杆菌内CRISPR位点不存在多态性。【结论】基于CRISPR位点多态性的分子分型方法不适用于炭疽芽胞杆菌分型,但可以用于区分炭疽芽胞杆菌与蜡样芽胞杆菌和苏云金芽胞杆菌。     

炭疽芽胞杆菌BslA(260－652)蛋白的表达纯化与黏附功能鉴定

【目的】克隆表达炭疽芽胞杆菌BlsA的功能区片段并对其生物学功能进行鉴定。【方法】以炭疽芽胞杆菌A16R基因组DNA为模板PCR扩增bslA(260-652)基因片段,克隆至pET-28a(+)载体。将成功构建的重组质粒转化入大肠杆菌Rosetta(DE3)中,诱导表达后收集菌体经超声破碎后,对可溶表达部分用镍柱进行亲和层析纯化。以纯化后的蛋白为抗原,免疫BALB/c小鼠制备该蛋白的多抗,用ELISA和Western blot检测抗血清;使用间接免疫荧光实验和细菌黏附实验研究目标蛋白及其抗体的生物学功能。【结果】BslA(260-652)获得了可溶性表达,纯化后纯度约为87.4%。以纯化蛋白为抗原,免疫BALB/c小鼠制备的抗血清ELISA效价可达1∶20000。将BslA(260-652)蛋白与Hela细胞共孵育后,能够直接和Hela的细胞膜结合。细菌黏附实验表明BslA(260-652)蛋白及其相应的多抗血清都能够显著地抑制炭疽芽胞杆菌A16R对Hela细胞的黏附。【结论】大肠杆菌表达得到的炭疽芽胞杆菌BslA(260-652)蛋白具有与天然蛋白相似的生物活性,为深入研究BslA蛋白在炭疽芽胞杆菌致病过程中的作用奠定实验基础。     

炭疽菌保护性抗原受体结合区的表达及其多克隆抗体的制备

原核表达炭疽杆菌保护性抗原受体结合区并制备该蛋白的多克隆抗体.从炭疽芽胞杆菌A16R中经PCR扩增得到了炭疽菌保护性抗原(PA)受体结合区基因,即PA的第四结构域(PA-D4),将其克隆至含有6×His编码序列的原核表达载体pET-2b(+)中,将重组质粒转化大肠杆菌BL21(DE3),在IPTG诱导下进行蛋白表达;用HiTrapTM Chelating HP柱纯化重组蛋白,Western blot进一步鉴定;以纯化后的蛋白为抗原,免疫新西兰大耳白兔制备该蛋白的多克隆抗体;用ELISA和Western blot检测抗血清.结果表明,目的蛋白在大肠杆菌BL21(DE3)中获得了可溶性表达,纯化后纯度可达90%以上;制备了针对PA-D4融合蛋白的高效价抗血清,ELISA抗体滴度为1∶ 102 400;其抗体能特异性识别内源性的PA.PA-D4重组蛋白及其多克隆抗体的获得,为后续研究其功能和炭疽疫苗免疫保护机制奠定了基础.     

炭疽致死毒素中和试验检验标准

提出了一种毒素中和试验的操作细节标准。此实验室试验,测量含炭疽致死毒素抗体抗血清的特异性保护J774A．1细胞抗炭疽芽胞杆菌致死毒素的细胞毒性作用的能力,其比色试验建立在活细胞降解MTT的基础上,用炭疽吸附疫苗(AVA)制备的人和家兔抗血清验证这个试验结果。试验结果显示高水平的重复性和复现性,尤其是     

炭疽芽胞杆菌(Bacillus anthracis)检测质粒的构建及其应用

根据炭疽芽胞杆菌(Bacillus anthracis)毒性质粒pX01和pX02上的2个毒力相关基因cya和capA的序列特点,以pIJ2925为出发载体,采用一步重叠延伸PCR技术(One-step Overlap Extension PCR,简称OOE-PCR)构建了包含cya基因和capA基因保守区DNA片段的炭疽检测质粒pBIB2006。采用复合PCR对模拟炭疽危险品进行分析,结果表明pBIB2006可以为炭疽芽胞杆菌的检测提供准确、安全和方便的阳性参照品,从而为检测炭疽芽胞杆菌和炭疽芽胞杆菌灭活疫苗提供了便利。     

国内外炭疽研究的新进展

<正> 1966年10～11月在美国马里兰州召开的炭疽杆菌专题会议上,Nungester,W J(1967)提出了炭疽今后研究的15个问题,题目的范围很广,包括人和动物的炭疽流行病学。水土等外环境污染的消除。高原( 6000呎以上, 2000米以上)很少发病的原因( Manuel,1967),病原学包括炭疽杆菌的生活史,芽胞侵入机体的方式,芽胞发芽及感染机制,可溶性抗原的生物学和理化特性,炭疽病理生理学,类毒素和活菌苗的免疫应答,中和毒素的治疗作用等等。     

A novel immunogenic spore coat-associated protein in Bacillus anthracis: characterization via proteomics approaches and a vector-based vaccine system

New generation anthrax vaccines have been actively explored with the aim of enhancing efficacies and decreasing undesirable side effects that could be caused by licensed vaccines. Targeting novel antigens and/or eliminating the requirements for multiple needle injections and adjuvants are major objectives in the development of new anthrax vaccines. Using proteomics approaches, we identified a spore coat-associated protein (SCAP) in Bacillus anthracis. An Escherichia coli vector-based vaccine system was used to determine the immunogenicity of SCAP. Mice generated detectable SCAP antibodies three weeks after intranasal immunization with an intact particle of ultraviolet (UV)-irradiated E. coli vector overproducing SCAP. The production of SCAP antibodies was detected via western blotting and SCAP-spotted antigen-arrays. The adjuvant effect of a UV-irradiated E. coli vector eliminates the necessity of boosting and the use of other immunomodulators which will foster the screening and manufacturing of new generation anthrax vaccines. More importantly, the immunogenic SCAP may potentially be a new candidate for the development of anthrax vaccines.     

Induction of opsonic antibodies to the gamma-D-glutamic acid capsule of Bacillus anthracis by immunization with a synthetic peptide-carrier protein conjugate

The capsule of Bacillus anthracis, a polymer of gamma-D-glutamic acid, functions as a virulence determinant and is a poor immunogen. In this study we show that antibodies reactive with the B. anthracis capsule can be elicited in mice by immunization with a conjugate consisting of a synthetic gamma-D-glutamic acid nonamer peptide (gamma-D-glu9) covalently coupled to keyhole limpet hemocyanin. The serum response to gamma-D-glu9 was comprised primarily of IgG antibodies that recognized an epitope requiring a minimum of four gamma-linked D-glutamic acid residues. Antibodies to (gamma-D-glu9) bound to the surface of encapsulated B. anthracis cells and mediated opsonophagoctosis. These findings suggest that anti-capsular antibodies could mediate the clearance of vegetative B. anthracis cells in vivo. Thus, inclusion of an immunogenic capsular component as well as protective antigen in new anthrax vaccines would generate immune responses targeting both the bacteremic and toxigenic aspects of anthrax infection and thus may increase protective efficacy.     

Nasal immunization with the mixture of PA63, LF, and a PGA conjugate induced strong antibody responses against all three antigens

A new generation anthrax vaccine is expected to target not only the anthrax protective antigen (PA) protein, but also other virulent factors of Bacillus anthracis. It is also expected to be amenable for rapid mass immunization of a large number of people. This study aimed to address these needs by designing a prototypic triantigen nasal anthrax vaccine candidate that contained a truncated PA (rPA63), the anthrax lethal factor (LF), and the capsular poly-gamma-D-glutamic acid (gammaDPGA) as the antigens and a synthetic double-stranded RNA (dsRNA), polyriboinosinic-polyribocytodylic acid (poly(I:C)) as the adjuvant. This study identified the optimal dose of nasal poly(I:C) in mice, demonstrated that nasal immunization of mice with the LF was capable of inducing functional anti-LF antibodies (Abs), and showed that nasal immunization of mice with the prototypic triantigen vaccine candidate induced strong immune responses against all three antigens. The immune responses protected macrophages against an anthrax lethal toxin challenge in vitro and enabled the immunized mice to survive a lethal dose of anthrax lethal toxin challenge in vivo. The anti-PGA Abs were shown to have complement-mediated bacteriolytic activity. After further optimization, this triantigen nasal vaccine candidate is expected to become one of the newer generation anthrax vaccines.     

Recent progress in the development of anthrax vaccines

Bacillus anthracis is the etiological agent of anthrax. Although anthrax is primarily an epizootic disease; humans are at risk for contracting anthrax. The potential use of B. anthracis spores as biowarfare agent has led to immense attention. Prolonged vaccination schedule of current anthrax vaccine and variable protection conferred; often leading to failure of therapy. This highlights the need for alternative anthrax countermeasures. A number of approaches are being investigated to substitute or supplement the existing anthrax vaccines. These relied on expression of Protective antigen (PA), the key protective immunogen; in bacterial or plant systems; or utilization of attenuated strains of B. anthracis for immunization. Few studies have established potential of domain IV of PA for immunization. Other targets including the spore, capsule, S-layer and anthrax toxin components have been investigated for imparting protective immunity. It has been shown that co-immunization of PA with domain I of lethal factor that binds PA resulted in higher antibody responses. Of the epitope based vaccines, the loop neutralizing determinant, in particular; elicited robust neutralizing antibody response and conferred 97% protection upon challenge. DNA vaccination resulted in varying degree of protection and seems a promising approach. Additionally, the applicability of monoclonal and therapeutic antibodies in the treatment of anthrax has also been demonstrated. The recent progress in the direction of anthrax prophylaxis has been evaluated in this review.     

Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei: towards the development of an oral vaccine against anthrax

Bacillus anthracis is the causative organism of the disease anthrax. The ability of the organism to form resistant spores and infect via the aerosol route has led to it being considered as a potential biological warfare agent. The current available human vaccines are far from ideal, they are expensive to produce, require repeated doses and may invoke transient side-effects in some individuals. There is also evidence to suggest that they may not give full protection against all strains of B. anthracis. A new generation of anthrax vaccine is therefore needed. The use of Lactobacillus as a vector for expression of heterologous proteins from pathogens supplies us with a safe system, which can be given orally. Lactobacilli are commensals of the gut, generally regarded as safe and have intrinsic adjuvanticity. Oral vaccines may stimulate the mucosol immune system to produce local IgA responses in addition to systemic responses. These vectors are delivered at the mucosal surface, the site where the infection actually occurs and where the first line of defence lies. The gene encoding the protective antigen (PA) of B. anthracis, an immunogenic non-toxic component of the two toxins produced, is being cloned into different homologous vectors and subsequently transformed to various Lactobacillus strains. High intracellular expression levels for the PA in Lact. casei were achieved. Mucosal antigen presentation and humoral and cellular immune responses following immunization with transformants expressing PA in various ways (intracellular, surface-anchored and extracellular) are being studied.     

Proteomics reveals that proteins expressed during the early stage of Bacillus anthracis infection are potential targets for the development of vaccines and drugs

In this review, we advance a new concept in developing vaccines and/or drugs to target specific proteins expressed during the early stage of Bacillus anthracis (anthrax) infection and address existing challenges to this concept. Three proteins (immune inhibitor A, GPR-like spore protease, and alanine racemase) initially identified by proteomics in our laboratory were found to have differential expressions during anthrax spore germination and early outgrowth. Other studies of different bacillus strains indicate that these three proteins are involved in either germination or cytotoxicity of spores, suggesting that they may serve as potential targets for the design of anti-anthrax vaccines and drugs.     

Rabbit and nonhuman primate models of toxin-targeting human anthrax vaccines.

The intentional use of Bacillus anthracis, the etiological agent of anthrax, as a bioterrorist weapon in late 2001 made our society acutely aware of the importance of developing, testing, and stockpiling adequate countermeasures against biological attacks. Biodefense vaccines are an important component of our arsenal to be used during a biological attack. However, most of the agents considered significant threats either have been eradicated or rarely infect humans alive today. As such, vaccine efficacy cannot be determined in human clinical trials but must be extrapolated from experimental animal models. This article reviews the efficacy and immunogenicity of human anthrax vaccines in well-defined animal models and the progress toward developing a rugged immunologic correlate of protection. The ongoing evaluation of human anthrax vaccines will be dependent on animal efficacy data in the absence of human efficacy data for licensure by the U.S. Food and Drug Administration.     

Anthrax vaccine design: strategies to achieve comprehensive protection against spore,bacillus, and toxin

The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review.     

炭疽杆菌致病性研究进展

炭疽杆菌是人类历史上第一个被发现的病原菌。炭疽杆菌的研究在近几年取得了较大进展 ,特别是本年度其基因组序列测定已完成并向全世界公布 ,进一步深化了对炭疽杆菌的研究。炭疽杆菌致病性的研究一直是炭疽杆菌研究的重点 ,近年来此方面的研究取得了很多新进展 ,从基因组、致病物质及致病机制 3个方面对此作一个简单的介绍。