载体介导的刚地弓形虫MIC疫苗的研制现状

刚地弓形虫引起的弓形虫病是一种严重危害人类健康的人兽共患寄生虫病,采用疫苗防治该病是当前研究的热点领域之一。MIC抗原是一种有效的疫苗候选分子,本研究综述了鼠伤寒沙门菌、卡介苗、格登链球菌、腺病毒血清型5和假狂犬病毒等载体介导的刚地弓形虫MIC疫苗的研制现状。     

弓形虫新基因wx2表位疫苗免疫小鼠的保护研究

采用牛物信息学方法对弓形虫新基因wx2进行表位分析预测,PCR扩增基因中编码2个表位的片段w2b和w2a,成功构建新基因的单表位疫苗质粒pcDNA3-W2b、pcDNA3-W2a和双表位疫苗pcDNA3-w2b2a,接种小鼠,观察表位疫苗的免疫保护作用.将表位疫苗分别通过肌肉注射免疫小鼠,对照组注射pcDNA3空质粒.ELJSA法检测血清IgG抗体水平,取脾细胞用流式细胞仪检测T淋巴细胞亚群.各组小鼠末次免疫后第4周每只小鼠经腹腔注射弓形虫速殖子500个,观察小鼠的生存时间.结果显示,pcDNA3-W2a2b双表位疫苗组小鼠血清IgG抗体水平显著高于对照组(P＜0.05),且pcDNA3-W2a2b双表位疫苗组小鼠脾细胞CD4 T与CD8 T淋巴细胞比值明显低于两单表位疫苗组,pcDNA3-W2a2b双表位疫苗组小鼠存活时间明显长于两单表位疫苗组(P＜0.05).实验结果表明,弓形虫新基因wz2表位疫苗能够诱导小鼠产生抗弓形虫感染保护性免疫,并且弓形虫poDNA3-W2b2a双表位疫苗的免疫保护性优于pcDNA3-W2b、pcDNA3-W2a两单表位疫苗.     

弓形虫主要表面抗原基因克隆的初步研究

弓形虫是能够引起人、畜感染的专性细胞内寄生的原虫,世界各地人群感染率一般在25％～50％,正常人感染可不显示任何临床症状而成为带虫者,但是胎儿感染常可导致胎儿畸形、神经系统发育障碍、流产甚至死胎;免疫功能低下或免疫功能缺陷的病人(如恶性肿瘤、接受器官移植、AIDS病人等)并发弓形虫感染,尤其神经系统感染常是引起死亡的主要原因之一。因此,弓形虫感染的早期诊断研究及弓形虫疫苗的开发利用,对于提高新生儿素质、预     

重组蜥蜴利什曼原虫载体疫苗的研制现状

蜥蜴利什曼原虫是一种新型疫苗载体,它已用于研发婴儿利什曼原虫、硕大利什曼原虫、刚地弓形虫、人免疫缺陷病毒1型、人乳头瘤病毒16型、丙型肝炎病毒疫苗。本文就重组蜥蜴利什曼原虫载体疫苗研制现状进行综述。     

刚地弓形虫亲环蛋白基因TgCyP真核表达质粒的构建及其在Hela细胞内的表达

亲环蛋白(cyclophilin,CyP)是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫(Toxoplasma gondii)速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用.本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHⅠ和EcoRⅠ酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因.PCR产物连接到pMD18-T克隆载体.用限制性内切酶BamHⅠ和EcoRⅠ双酶切克隆载体,将TgCyP目的基因克隆到载体pVAX1,构建真核表达质粒pVAX1-TgCyP.将真核表达质粒转染Hela细胞,利用间接免疫荧光检测其在Hela细胞内的表达情况.结果表明扩增的TgCyP基因与GenBank上相应基因序列(U04633.1)的一致性达100%,构建的真核表达质粒pVAX1-TgCyP能在转染的Hela细胞中表达,其表达产物与刚地弓形虫阳性血清具有免疫反应性.本研究表明TgCyP有望作为弓形虫疫苗的候选抗原,将为进一步研究该质粒的动物免疫试验奠定基础.     

宠物弓形虫的感染及其防控

弓形虫病是一种世界性分布的人兽共患寄生虫病,对人类,尤其是妇女、儿童危害很大,估计全世界有1/3人受到该病的威胁。孕妇感染弓形虫后导致早产、流产、胎儿发育畸形;弓形虫是免疫功能低下患者的主要死亡原因之一。犬、猫是弓形虫的中间宿主和终末宿主,是人类感染弓形虫的主要来源。随着我国经济的迅速发展和人民生活水平的不断提高,城市中饲养犬、猫作为宠物的人越来越多,人、宠物间的亲密接触增加了弓形虫病传播给人的机会。加强对宠物犬、猫弓形虫病的研究及防控势在必行。本文就弓形虫的危害、宠物犬猫弓形虫感染及其防控措施作以综述。     

实验动物血液及精液感染弓形虫动态研究

采用荧光定量PCR技术对实验动物家兔血液及精液感染弓形虫DNA检测实验及动态变化研究,阐明了弓形虫感染的发展和转归机理,为了解弓形虫与宿主动态关系,弓形虫经血液、精液传播提供理论依据.对性传播疾病、特别是艾滋病的预防提供重要参考价值.     

弓形虫感染实验动物模型的特点及建模方案的选择

弓形虫感染对人类生活和畜牧业发展构成严重威胁。弓形虫感染实验动物模型是进行弓形虫学相关研究的基础条件之一。在实际研究工作中,根据不同的实验目的、选取不同的实验动物和以不同的实验方法所建立的实验动物模型,呈现出复杂和多变的特点。这一方面可以满足不同实验的需要,但同时也在实验结果的评价上导致一定程度的不足。根据弓形虫感染实验动物模型的不同特点,针对特定的实验目的,选择适合方法建立适合的实验动物模型,是进行相关弓形虫学研究的有效基础和前提。     

制备间接血凝试验抗原用弓形虫速殖子的净化

弓形虫(Toxoplasma gondii)又名弓形体或弓浆虫,能引起人畜的弓形虫病。在弓形虫的生物学特性和免疫学等研究中,为了得到纯净的弓形虫速殖子,必须从感染小白鼠腹腔渗出物中去除多量的腹腔细胞和炎性细胞。国外有应用多孔玻璃滤器过滤,用超声波裂解腹腔细胞,再用胰蛋白酶消化细胞碎片,用梯度离心     

弓形虫与宿主细胞相互作用研究进展

刚地弓形虫(Toxoplasma gondii)在细胞内严格寄生,因此它能引起哺乳类宿主(包括人类)细胞的感染。凋亡在宿主细胞与弓形虫的相互作用中发挥着重要的作用。在未受感染的宿主细胞中,凋亡被间接机制所限制,因而宿主细胞能够对弓形虫发生炎症反应。与之相反,在被感染的宿主细胞中,由于凋亡信号级联反应直接受到了干扰,从而抑制了宿主细胞凋亡,这就有利于弓形虫在宿主细胞内的生存和发育。值得注意的是,弓形虫调节和抑制凋亡的两种能力,需要一个精密的调节系统来调控弓形虫和宿主细胞的相互作用,以维持弓形虫稳定的持续感染。重点从弓形虫有关的宿主细胞的凋亡方面进行了介绍。     

Development of a vaccine for toxoplasmosis: current status

The need for a vaccine for human toxoplasmosis is briefly discussed. Recent progress in knowledge of the protective immune response generated by Toxoplasma gondii and the current status of development of a vaccine for toxoplasmosis are highlighted.     

IL-15 prolongs the duration of CD8+ T cell-mediated immunity in mice infected with a vaccine strain of Toxoplasma gondii.

Immunization of mice with a vaccine (ts-4) strain of Toxoplasma gondii is known to induce complete protection against subsequent lethal infection. Ts-4-mediated protection has been reported to be primarily dependent on IFN-gamma-producing CD8+ T cells. However, duration of CD8+ T cell-mediated immunity in the ts-4-vaccinated animals is not known. In the present study, the kinetics of the CD8+ T cell response in mice immunized with the ts-4 strain of T. gondii was evaluated. Optimal CD8+ T cell immunity persisted at least 6 mo after vaccination, and mice at this time point continued to overcome lethal challenge with a more virulent strain. However, at 9 mo postimmunization, CD8+ T cell immunity was severely diminished and the mice succumbed to Toxoplasma challenge. Pretreatment of animals, vaccinated 9 mo earlier, with rIL-15 prevented the mortality induced by Toxoplasma challenge. The protective effect of IL-15 treatment was due to a rise in the frequency of Ag-specific CD8+ T cells. CD8+ T cells from IL-15-administered animals showed increased proliferation and IFN-gamma production in response to antigenic restimulation. These findings suggest that rIL-15 can reverse the decline in the long-term CD8+ T cell immune response in mice immunized with vaccine strain of T. gondii.     

Identification of potential apicoplast associated therapeutic targets in human and animal pathogen Toxoplasma gondii ME49

Toxoplasma gondii ME49 is an obligatory intracellular apicomplexa parasite that causes toxoplasmosis in humans, domesticated and wild animals. Waterborne outbreaks of acute toxoplasmosis worldwide reinforce the transmission of Toxoplasma gondii ME49 to humans through contaminated water and may have a greater epidemiological impact than previously believed. In the quest for drug and vaccine target identification subtractive genomics involving subtraction between the host and pathogen genome has been implemented for enlisting essential pathogen specific proteins. Using this approach, our analysis on both human and Toxoplasma gondii ME49 reveals that out of 7987 protein coding sequences of the pathogen, 950 represent essential non human-homologous proteins. Subcellular localization prediction & comparative-biochemical pathway analysis of these essential proteins gives a list of apicoplast-associated proteins having unique pathogen-specific metabolic pathway. These apicoplast-associated enzymes involved in fatty acid biosynthesis pathway of Toxoplasma gondii ME49, may be used as potential drug targets, as the pathway is vital for the protozoan's survival. Structure prediction of drug target proteins was done using fold based recognition method. Screening of the functional inhibitors against these novel targets may result in discovery of novel therapeutic compounds that can be effective against Toxoplasma gondii ME49. ABBREVIATIONS: DEG - Database of Essential Gene, KEGG - Kyoto Encyclopaedia of Genes and Genomes, KAAS - KEGG Automated Annotation Server, PFP - Protein Function Prediction, COG - Cluster of Orthologous Genes.     

Immunization against Toxoplasma gondii

The emergence of Toxoplasma gondii as a major opportunistic organism in immunocompromised individuals and the steady increase in economic losses due to animal toxoplasmosis have fuelled the interest in vaccine development. In this review, Fausto Araujo addresses aspects of vaccination against T. gondii with regard to the problem posed by this parasite to pregnant women and to severely immunocompromised individuals.     

Recombinant attenuated Toxoplasma gondii expressing the Plasmodium yoelii circumsporozoite protein provides highly effective priming for CD8+ T cell-dependent protective immunity against malaria

The protozoan parasite Toxoplasma gondii elicits strong cell-mediated immunity against itself as well as nonspecific resistance against other pathogens and tumors. For this reason, we asked whether recombinant Toxoplasma could be utilized as an effective vaccine vehicle for inducing immunity against heterologous microbial infections. The circumsporozoite protein (PyCSP) of Plasmodium yoelii was engineered into a T. gondii temperature-sensitive strain (ts-4), a mutant that induces complete protection against virulent Toxoplasma challenge. When administered to mice in a single dose, a recombinant ts-4 (CSC3) that both secretes and expresses surface PyCSP induced strong anti-CSP Ab responses, with an isotype distribution pattern similar to that stimulated by the T. gondii carrier. When challenged with P. yoelii sporozoites during the first month after CSC3 vaccination, these animals displayed substantial levels of nonspecific resistance attributable entirely to the T. gondii carrier. Nevertheless, after the nonspecific protection had waned, high levels (up to 79%) of specific immunity against sporozoite challenge were achieved by boosting the animals with recombinant vaccinia virus expressing PyCSP. These CSC3-primed PyCSP-vaccinia-boosted mice displayed high frequencies of splenic PyCSP-specific IFN-gamma-producing cells, as well as CD8+ T cell-dependent cytolytic activity. In vivo depletion of CD8+ lymphocytes at the time of challenge completely ablated protective immunity in the T. gondii-primed/vaccinia-boosted animals, while neutralization of IFN-gamma or IL-12 caused a partial but significant reduction in resistance. Together these findings establish the efficacy of recombinant attenuated Toxoplasma as a vaccine vehicle for priming CD8+-dependent cell-mediated immunity.     

中药大蒜素体外抗弓形虫效应及其机制的研究

目的 观察大蒜提取物体外抗弓形虫的作用效果及其机制.方法 将弓形虫RH株速殖子与兔肾细胞共同培养,加入不同浓度的大蒜素(实验组)和磺胺嘧啶(阳性对照组),培养不同时间后取出细胞,固定染色后观察细胞感染率及每个纳虫泡中弓形虫速殖子的数量;采用MTT比色法观察大蒜素对弓形虫速殖子侵袭细胞及其正常细胞增殖的影响;采用台盼兰着色法观察大蒜素对弓形虫速殖子活性的影响;采用TUNEL末端标记法检测弓形虫速殖子凋亡率,对药物的效应和机制进行探讨.结果 (1)10～80 μg/ml的大蒜素能抗弓形虫的感染,呈现剂量依赖性,与时间无明显的相关性.(2)40 μg/ml、80 μg/ml的大蒜素不能抑制细胞的增殖,对细胞无明显的毒副作用;160 μg/ml的大蒜素对细胞有明显的毒副作用.(3)大蒜素80 μg/ml时,台盼兰着色率最高,弓形虫的活力最低.(4)大蒜素80 μg/ml的浓度时,弓形虫速殖子凋亡率最高.结论 大蒜素可以抑制弓形虫速殖子的活力、对宿主细胞的感染力、在细胞内的增殖,无明显的毒副作用,最适宜浓度为80 μg/ml.大蒜素是一种良好的抗弓形虫药物,诱导弓形虫速殖子凋亡是其抗弓形虫机制之一.     

Expression of P23 of Cryptosporidium parvum in Toxoplasma gondii and evaluation of its protective effects

In this study, P23 of Cryptosporidium parvum sporozoites, an immunodominant surface protein, was stably expressed in Toxoplasma gondii (Tg/P23) and its protective effects were evaluated in a mouse model. The molecular weight and antigenic property of P23 expressed by Tg/P23 were similar to those of the native P23. Mice immunized with lysed Tg/P23 tachyzoites produced specific neutralizing antibodies against C. parvum. These findings indicate that the T. gondii vector may provide a new tool for the production of a recombinant vaccine against cryptosporidiosis in animals.     

Toxoplasma gondii: chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis

Toxoplasma gondii is responsible for fetopathy in farm animals and humans and severe disease in immunocompromised individuals (i.e. AIDS patients). Effective vaccines, inducing protective and long-lasting immunity to this global parasite, are still desired. In the work, we evaluated the immunogenic and immunoprotective activity of Escherichia coli chimeric Dr fimbriae bearing selected antigenic epitopes of three T. gondii antigens (SAG1, GRA1 and MAG1), in comparison with conventional recombinant antigens obtained in E. coli expression system. Our data demonstrate a very high protective efficacy of recombinant antigens supplemented with Freund's adjuvants, whereas chimeric Dr fimbriae as a vaccine proved non-protective. The recombinant antigen vaccine induced a strong specific antibody response and prevented the brain cysts formation by 89%. The results are promising and should be confirmed in further study on farm animals by use of less aggressive than Freund's adjuvant preparations.     

Initial characterization of an autoclaved Toxoplasma vaccine in mice

Toxoplasmosis is a zoonotic protozoal disease that has a major significance from the perspectives of public health and veterinary medicine. Therefore, an obvious long-term goal of many scientists would be the development of an effective vaccine. In this study, autoclaved vaccine was evaluated for its ability to protect mice against Toxoplasma gondii RH challenge as an acute infection model. Results showed that autoclaved Toxoplasma vaccine (ATV) when combined with BCG as an adjuvant was effective in triggering cell mediated immunity as shown by a significant increase in the percentage of splenic CD8+ T-lymphocytes. Following challenge, death of mice vaccinated with ATV was delayed for nine days. There was a significant decrease in parasite density in different organs, and a marked reduction of pathological changes in the liver suggesting that significant immune responses were mounted following vaccination. Future studies are warranted to test the vaccine against challenge with brain cysts as a chronic infection model and to evaluate it with other recent immunization strategies that can further enhance its immunogenicity.     

Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice

To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)-infection, T gondii SAG1 gene-transfectants were established by using RMA.S (H-2b), a murine transporter associated with the antigen processing (TAP) molecule-deficient lymphoma line, as a host antigen-presenting cell (APC). Immunization of C57BL/6 mice with the SAG1-transfected RMA.S induced CD8+ cytotoxic T lymphocytes (CTL) specific for not only SAG1-transfected RMA.S but also T gondii-infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.