结核病DNA疫苗及其作用机制研究进展

为实现到2050年在全球范围内根除结核病(Tuberculosis,TB)这一宏伟目标,迫切需要开发新的TB疫苗和疫苗接种策略.虽然卡介苗(Bacille Calmette-Guérin,BCG)在保护儿童免受粟粒性结核和结核性脑膜炎等方面获得了成功,但其对成人肺结核(Pulmonary tuberculosis,PT...     

兽用DNA疫苗的研究进展

DNA疫苗即DNA免疫接种,是指将编码抗原的外源基因插入一表达载体中,直接接种到动物体内,于体内表达后,诱导产生特异性的细胞免疫和/或体液免疫的过程.由于DNA疫苗既拥有亚单位疫苗和灭活疫苗的安全性,又兼备只有减毒疫苗或重组疫苗才有的诱导保护性免疫应答的特点,因而受到越来越多的研究者青睐.在兽医领域,DNA疫苗已在家禽、家畜和小动物上进行了研究,研究的首选目标是传染性病原,如:病毒、细菌和寄生虫等,针对的疾病包括呼吸道、胃肠道、繁殖及全身性疾病等,自身免疫性疾病和肿瘤等的DNA疫苗则主要在一些伴侣动物上进行了研究,现将这方面的进展介绍如下.     

结核病疫苗的研究新进展

在全球范围内结核病又卷土重来 ,接种卡介苗 (ＢＣＧ)是目前预防结核病的主要措施。自 192 1年首次用于人体以来 ,大约有 3 0亿人口接种了ＢＣＧ。在接种ＢＣＧ 10～ 15年后 ,它对人体的保护效率会降低。随着迁移人口的增加、获得性免疫缺陷综合征 (ＡＩＤＳ)的流行、耐药菌株的出现 ,结核病又成为危害人类健康的头号杀手。现在全世界已有近 2 0亿人感染结核分支杆菌 ,每年新发病人数约 10 0 0万 ,死亡人数约3 0 0万[1] 。ＢＣＧ虽然安全有效副作用少 ,但它的保护效果不稳定 ,接种ＢＣＧ后会使结核菌素试验 (ＰＰＤ)阳性 ,对结核病的诊断产…     

预防与治疗性结核病疫苗的研究进展

迄今为止,结核病(Tuberculosis,TB)唯一被批准用于预防TB的疫苗是100多年前由牛分枝杆菌减毒而成的卡介苗(BCG)。BCG可预防小儿TB,但还无法预防成人感染TB。此外耐多药TB(MDR-TB)患者不断增多,这使全球TB感染的发病率及死亡率下降仍达不到世界卫生组织(WHO)的目标。因此,需要一种新的有效的TB疫苗和基础-增强免疫策略来替代BCG预防TB的感染。在这篇综述中,我们分析了目前已研发的预防和治疗性疫苗以及疫苗佐剂的研究进展,目的是使我们对TB的感染和防控的现状有全面的了解。     

疟疾DNA疫苗的研究进展

近年来，由于疟原虫及其媒蚊出现抗药性，疟疾重新泛滥，已证明蚊媒控制策略不能根除热带疟疾，抗疟形势面临严峻挑战，其防制策略必须转向个体防护研究。识别和分离疟原虫抗原的分子工具的发展，促使疟疾研究者们设法构建疫苗以抗疟感染，而DNA疫苗尤其是多基因DNA疫苗是一种划时代新技术，被誉为第三次疫苗革命，已用于抵抗多种病原体研究。核酸疫苗(Nucleic acid vaccine)是从基因治疗研究领域发展起来的一种全新疫苗，包括所有DNA或RNA疫苗以及合成核酸疫苗，但目前主要以DNA疫苗为主，故又称DNA疫苗。它直接把带有目的抗原基因的重组质粒转染或注射入细胞内，使之在细胞中持续表达天然抗原，经加工处理，与主要组织相容性复合体(MHC)形成复合物，并提呈到细胞表面，诱导特异性细胞毒性T细胞(CTL)及体液免疫应答。自1992年首先描述疟疾DNA疫苗以来，发展很快，本文就此研究状况综述如下。     

应用结核病DNA疫苗治疗小鼠耐多药结核病的研究

目的 研究结核分枝杆菌Ag85A质粒DNA疫苗单独或联合药物治疗小鼠耐多药结核病的效果,为建立耐多药结核病的免疫治疗新策略和新方案奠定基础.方法 用结核分枝杆菌高耐利福平、低耐异烟肼临床分离株HB361尾静脉注射17～19 g的6～8周龄雌性BALB/C小鼠后,将小鼠随机分为6组,每组10只.感染后第2天开始,分别用pVAX1载体(A组)、利福平(B组)、吡嗪酰胺(C组)、Ag85A质粒DNA疫苗(D组)、Ag85A质粒DNA疫苗联合利福平(E组)、Ag85A质粒DNA疫苗联合吡嗪酰胺(F组)治疗60 d.治疗结束后4周,分别取肺、肝和脾观察病理改变,称取重量,做菌落计数.结果 小鼠感染4周后,肺内菌量达到1.5×107 CFU,脾内菌量达到1.1×106 CFU.A、B组小鼠死亡率均为10%,其余各组小鼠均存活.治疗结束后4周,肺组织病理显示,各治疗组肺组织病变均有不同程度减轻,病变局限,可见正常的肺泡结构,肺泡轮廓相对清晰.与A组比较,C、D、E、F组肺组织菌落数分别减少了1.18、1.35、1.38、1.08 logs,脾脏菌落数分别减少了0.91、1.00、1.26、1.03 logs(P＜0.01).结论 结核分枝杆菌Ag85A质粒DNA疫苗单独或联合药物治疗小鼠耐多药结核病均有显著疗效.Ag85A质粒DNA疫苗与抗结核药物联合治疗是治疗耐多药结核病的最有前途的免疫策略.     

应用结核病DNA疫苗治疗小鼠耐多药结核病的研究

Objective To establish foundation for new strategy and program on immune therapy of multi-drug resistant tuberculosis (MDR-TB) by studying the therapeutic effects of Mycobacterium tuberculosis Ag85A plasmid DNA vaccine alone or combined with drugs on MDR-TB mice. Methods Sixty 6-8 weeks old female BALB/C mice were injected via tail vein with clinical isolate Mycobacterium tuberculosis HB361 which was highly resistant to rifampin (RFP) and lowly resistant to isoniazid. The mice were randomly divided into six groups, ten mice in each group. From the second day after infection,the mice respectively received pVAX1 vector (group A), RFP (group B), pyrazinamide (PZA) (group C),Ag85A plasmid DNA vaccine (group D), Ag85A plasmid DNA vaccine combined with RFP (group E),Ag85A plasmid DNA vaccine combined with PZA (group F) for sixty days. Four weeks after the end of treatment,the lung, liver and spleen of the mice were taken and their pathological changes, weight and colony count were examined. Results Four weeks after infection, the numbers of bacteria in lung and spleen of the mice reached up to 1.5×107 CFU and 1.1 × 106 CFU,respectively. The death rates of mice in group A and group B were both 10% ,and the mice in other groups were alive. Four weeks after the end of treatment,lung pathology in the treated groups showed that the lung lesions were slight and limited,normal alveolar structure were seen, and the profile of the alveoli was relatively clear. Compared with group A, group C, D, E, F reduced by 1.18,1.35,1.38,1.08 logs on the colony count of lung, and reduced by 0.91,1.00,1.26 and 1.03 logs on the colony count of spleen (P＜0.01), respectively. Conclusions Mycobacterium tuberculosis Ag85A plasmid DNA vaccine alone or combined with drugs has significant therapeutic effects on MDR-TB mice. Ag85A plasmid DNA vaccine combined with anti-tuberculosis drugs is the most promising immunization strategy for treatment of MDR-TB.     

应用结核病DNA疫苗治疗小鼠耐多药结核病的研究

Objective To establish foundation for new strategy and program on immune therapy of multi-drug resistant tuberculosis (MDR-TB) by studying the therapeutic effects of Mycobacterium tuberculosis Ag85A plasmid DNA vaccine alone or combined with drugs on MDR-TB mice. Methods Sixty 6-8 weeks old female BALB/C mice were injected via tail vein with clinical isolate Mycobacterium tuberculosis HB361 which was highly resistant to rifampin (RFP) and lowly resistant to isoniazid. The mice were randomly divided into six groups, ten mice in each group. From the second day after infection,the mice respectively received pVAX1 vector (group A), RFP (group B), pyrazinamide (PZA) (group C),Ag85A plasmid DNA vaccine (group D), Ag85A plasmid DNA vaccine combined with RFP (group E),Ag85A plasmid DNA vaccine combined with PZA (group F) for sixty days. Four weeks after the end of treatment,the lung, liver and spleen of the mice were taken and their pathological changes, weight and colony count were examined. Results Four weeks after infection, the numbers of bacteria in lung and spleen of the mice reached up to 1.5×107 CFU and 1.1 × 106 CFU,respectively. The death rates of mice in group A and group B were both 10% ,and the mice in other groups were alive. Four weeks after the end of treatment,lung pathology in the treated groups showed that the lung lesions were slight and limited,normal alveolar structure were seen, and the profile of the alveoli was relatively clear. Compared with group A, group C, D, E, F reduced by 1.18,1.35,1.38,1.08 logs on the colony count of lung, and reduced by 0.91,1.00,1.26 and 1.03 logs on the colony count of spleen (P＜0.01), respectively. Conclusions Mycobacterium tuberculosis Ag85A plasmid DNA vaccine alone or combined with drugs has significant therapeutic effects on MDR-TB mice. Ag85A plasmid DNA vaccine combined with anti-tuberculosis drugs is the most promising immunization strategy for treatment of MDR-TB.     

结核分枝杆菌DNA疫苗研究进展

20世纪 90年代初 ,有关质粒DNA诱导对其编码的抗原的免疫反应报道之后 ,DNA疫苗便成为疫苗技术增长最迅速的领域[1,2 ] 。现将DNA疫苗的概况及结核分枝杆菌DNA疫苗的研究现状介绍如下。一、DNA疫苗概况(一 )DNA疫苗作用原理与重组细菌或病毒疫苗相比 ,DNA疫苗仅由DNA(如质粒 )或RNA(如mRNA)组成 ,疫苗抗原的编码基因插入适当的真核细胞质粒DNA的始动子及终止密码之间 ,它可在细菌体内复制 ,而不能在人类宿主细胞内复制。此质粒可由大肠杆菌培养中纯化而得到。DNA被肌肉细胞摄取进入细胞核内 ,在此抗原基因被转录 ,mRNA被转…     

Development of vaccines against tuberculosis

Development of an effective vaccine against tuberculosis (TB) hinges on an improved understanding of the human immune responses to Mycobacterium tuberculosis. A successful vaccination strategy should be able to stimulate the appropriate arm of the immune system with concomitant generation of the memory cells. In the absence of a perfect strategy, while long term efforts of TB researchers continue to resolve the nature of protective immunity against TB and other related issues, the current approach, dictated by the urgency of a TB vaccine, employs available knowledge and technology to develop new TB vaccines and channel the promising ones to clinical trials. While Indian scientists have contributed in several areas towards the development of a TB vaccine, this review is an attempt to summarize their contributions mainly pertaining to the discovery of new antigens, immune responses elicited by antigens against TB and development of new vaccines and their evaluation in animal models.     

结核病黏膜疫苗的研究进展北大核心CSCD

呼吸道黏膜免疫是机体抵御结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染的第一道防线,是重要的抗MTB感染的保护性免疫,如何通过黏膜免疫,诱导黏膜免疫系统产生抗MTB感染的特异性免疫,是目前结核病疫苗研究的新方向。本文基于近年来结核病黏膜疫苗的研究,总结了不同类型疫苗的特点、黏膜免疫后的免疫学特性和免疫保护效果,展望了未来结核病黏膜疫苗的研究方向,希望能够对研发更加高效的结核病黏膜疫苗有所启示。     

寄生虫的核酸疫苗研究

所谓核酸疫苗，就是把外源基因克隆到真核质粒表达载体上，然后将重组的质粒ＤＮＡ直接注射到动物体内，使外源基因在活体内表达，产生的抗原激活机体的免疫系统，引发免疫反应。核酸疫苗具有突出的优点：（１）能诱导全方位的免疫应答，优于亚单位疫苗和灭活疫苗，既可产生细胞免疫又可诱导体液免疫; (2) 安全性, 没有减毒疫苗和灭活疫苗可能引起的致病作用; (3) 能表达经修饰的天然抗原, 由于抗原在真核细胞内表达, 能形成正确的折叠和进行翻译后修饰; (4) 长效性, 质粒DNA 可在细胞内存在两个月, 且单次免疫即可获得免疫保护作用达两年; (5) 能构建成多价疫苗并进行多级免疫; (6) 生产简便、成本低廉、稳定性好且贮运方便。所以, 核酸疫苗已在细菌、病毒、寄生虫等感染性疾病和肿瘤的预防和治疗中显示出巨大的潜力[1 ] , 部分感染性疾病的核酸疫苗已进入临床实验阶段[2 ]。核酸疫苗作为继灭活疫苗、减毒疫苗和基因工程亚单位疫苗之后的第三代疫苗, 正在成为一个新的研究热点。本文将重点介绍核酸疫苗的作用机理、研究的基本策略及其在寄生虫病防治中的应用。     

New tuberculosis vaccines

The current tuberculosis (TB) vaccine, bacille Calmette-Guerin (BCG), is a live vaccine used worldwide, as it protects against severe forms of the disease, saving thousands of lives every year, but its efficacy against pulmonary forms of TB, responsible for transmission of the diseases, is variable.For more than 80 years now no new TB vaccines have been successfully developed. Over the last decade the effort of the scientific community has resulted in the design and construction of promising vaccine candidates. The goal is to develop a new generation of vaccines effective against respiratory forms of the disease. We will focus this review on new prophylactic vaccine candidates that aim to prevent TB diseases.Two are the main strategies used to improve the immunity conferred by the current BCG vaccine, by boosting it with new subunit vaccines, and a second strategy is focused on the construction of new more effective live vaccines, capable to replace the current BCG and to be used as prime vaccines.After rigorous preclinical studies in different animal models new TB vaccine candidates enter in clinical trials in humans. First, a small Phase I for safety followed by immunological evaluation in Phase II trials and finally evaluated in large population Phase III efficacy trials in endemic countries. At present BCG prime and boost with different subunit vaccine candidates are the more advanced assessed in Phase II. Two prime vaccines (based on recombinant BCG) have been successfully evaluated for safety in Phase I trials. A short number of live attenuated vaccines are in advance preclinical studies and the candidates ready to enter Phase I safety trials are produced under current good manufacturing practices.     

Effectiveness of tuberculosis vaccines

BCG revaccination in the Sverdlovsk Region is performed thrice: at the age of 5, 10 and 15 years. The percent of children covered by tuberculin diagnosis and BCG vaccination is growing. Complications of BCG vaccination remain at the same level. Tuberculosis morbidity in children rises due to minor forms of tuberculosis. However, it is 2 times less than mean tuberculosis incidence in Russia. Primary infection is registered 4.5 times less frequently.     

结核杆菌Ag85A DNA疫苗在小鼠体内的免疫效应比较

目的探索增强结核杆菌DNA疫苗有效性的新方法，为研制并开发新一代高效结核杆菌DNA疫苗奠定基础。方法分别构建结核杆菌Ag85A抗原基因真核表达质粒及其与小鼠粒细胞-巨噬细胞集落刺激因子的真核嵌合表达质粒，体外转染COS7细胞检测两种重组质粒的表达活性后，将其分别用作DNA疫苗给BALB／c小鼠肌内注射，检测小鼠体内特异性体液免疫和细胞免疫应答相关指标，同时进行动物免疫保护性实验，比较分析两种DNA疫苗在小鼠体内的免疫效应。结果结核杆菌Ag85A抗原蛋白基因与小鼠粒细胞一巨噬细胞集落刺激因子的嵌合DNA疫苗在小鼠体内的免疫原性明显强于Ag85A抗原基因非嵌合DNA疫苗，但两种DNA疫苗的免疫保护性无明显差异。结论粒细胞-巨噬细胞集落刺激因子与结核杆菌免疫保护性抗原基因嵌合DNA疫苗能显著增强结核病DNA疫苗的免疫原性。     

New vaccines against tuberculosis

In September 2000, recognizing the effect of communicable diseases as obstacles to development in poorer countries, the European Commission assembled a special round table on 'accelerated action targeted at major communicable diseases within the context of poverty reduction'. The three major communicable diseases discussed were tuberculosis (TB), malaria and HIV. One outcome of this discussion was a workshop examining issues related to the fight against TB in Africa, which took place in Gorée, Sénégal, in May 2001. The timing was propitious, as new vaccines for TB (recombinant MVA and BCG, and adjuvanated recombinant fusion proteins or peptide constructs), are just beginning to enter human clinical trials. All but the last of these have shown promise in animal models, up to and including non-human primates, and all are strongly immunogenic and apparently safe. Humans trials for safety and efficacy are thus the logical next step. This review summarizes recent advances in tuberculosis vaccine development, with a special emphasis on issues raised at the Gorée meeting about testing and deploying new generation vaccines in TB-endemic areas such as Africa.     

DNA vaccines

BACKGROUND AND OBJECTIVES: Humoral and cellular immune responses to protein antigens can be efficiently primed by nucleic acid or DNA vaccination. In DNA-based vaccination, immunogenic proteins are expressed with correct posttranslational modification, conformation or oligomerization; this ensures the integrity of epitopes that stimulate neutralizing antibody (B cell) responses. DNA (or RNA) immunization is exceptionally potent in stimulating T cell responses because antigenic peptides are efficiently generated in (endogenous or exogenous) processing pathways (without interference by viral proteins) from intracellular or extracellular protein antigens expressed after transient in vivo transfection. Both features are difficult to achieve with recombinant subunit vaccines produced in eukaryotic or prokaryotic expression systems. The current state of vector designs, strategies for delivery of DNA vaccines, priming humoral and cellular immune responses by DNA vaccines, experimental strategies facilitated by DNA vaccines, unique advantages of DNA vaccination, experience of DNA vaccination in preclinical animal models and clinical trials, and potential risks of DNA vaccination are discussed. Excellent reviews on DNA-based vaccination have been published recently [1-3].     

结核分枝杆菌DNA疫苗对小鼠结核病免疫治疗作用的实验研究

目的探讨结核分枝杆菌DNA疫苗pcD85B、pcDMPT64以及小鼠白细胞介素12(IL-12)真核表达质粒psIL12对结核分枝杆菌感染的疗效与机制。方法将结核分枝杆菌H37Rv感染的C57BL/J6小鼠100只随机分成生理盐水对照组、pcDNA3.1对照组,psIL12治疗组,pcD85B、pcDMPT64、pcD85B+pcDMPT64、pcD85B+psIL12DNA疫苗治疗组。感染4周后分别给予生理盐水、空质粒、psIL-12及DNA疫苗,第1次治疗后2个月处死小鼠,检测器官荷菌量、脾淋巴细胞特异性γ干扰素(IFN-γ)、白细胞介素4(IL4)、肿瘤坏死因子-α(TNF-α)的分泌水平,并于第1次治疗后2个月、5个月观察小鼠肺、脾组织病理改变情况。结果pcD85B治疗组肺组织荷菌量(lg-1CFU/g)为6.99±0.40,比生理盐水对照组(8.15±0.37)、pCDNA3.1对照组(8.19±0.29)显著降低(P<0.01);脾组织荷菌量(lg-1CFU/g,x±s)为5.17±0.33,比生理盐水对照组(5.76±0.16)及空质粒对照组(5.88±0.21)显著降低(P<0.05)。psIL12治疗组的肺(7.41±0.50)、脾(5.31±0.21)荷菌量比对照组显著降低(P<0.05);pcD85B+psIL12治疗组肺、脾荷菌量与pcD85B组比较差别无统计学意义。pcD85B组脾淋巴细胞IFN-γ、TNFα水平比对照组显著升高(P<0.05),各组间IL4水平差异无统计学意义。生理盐水对照组、pCDNA3.1对照组肺组织