Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

Tuberculosis is an ancient scourge of mankind. According to statistics, there are more than 10 million new cases of tuberculosis each year and the annual death toll for tuberculosis exceeds three millions. The current available BCG is of questionable efficacy, and its protection ranges from 0 to 85%. Therefore, developing a safe and effective vaccine against this scourge is very important. Previous studies have shown that the secreted proteins of Mycobacterium tuberculosis (M. TB) can induc…     

结核分枝杆菌组合DNA疫苗的免疫效果

以编码结核分枝杆菌分泌蛋白Ag85B、ESAT 6和MPT6 3的基因为插入片段 ,构建核酸疫苗联合免疫小鼠。以原核表达纯化的抗原为检测物 ,检测了抗原特异性的抗体和γ 干扰素的形成。研究表明 ,组合核酸疫苗第 3次免疫后 2 1天 ,实验小鼠血清中Ag85B抗体滴度达到 10 5以上 ,Ag85B抗原刺激产生的特异性γ 干扰素达到 (17.0± 7.0 )u/ml。组合疫苗虽然没有提高小鼠血清中ESAT 6及MPT6 3蛋白的特异性抗体滴度 ,但仍显著刺激产生了两种特异性的 γ 干扰素。攻毒实验表明 ,经组合疫苗免疫后小鼠肺部结核杆菌数量显著下降。肺部切片显示 ,免疫小鼠病理状况较对照组有明显改善。因此 ,研究提示Ag85B等组合核酸疫苗具有较好的结核病预防效果。     

结核杆菌融合蛋白DNA疫苗构建及免疫保护研究

目的:评价结核DNA疫苗免疫鼠产生细胞因子和抵抗结核分枝杆菌攻击的能力。方法:将结核菌Mtb8.4基因和谷胱甘肽S转移酶基因插入pVAX1载体,构建表达Mtb8.4和GST融合蛋白的DNA疫苗pVS8.4G。小鼠分成5组,用pVS8.4G、pVAX1、pIL2S 100μg和PBS 0.1mL各免疫3次,间隔2w。另一组用BCG免疫1次。每组10只鼠在加强后,无菌取脾培养。另外10只小鼠用H37Rv攻击,2w后取脾、肝和肺培养结核菌并计数。结果:pVS8.4G免疫鼠脾细胞培养上清mIL-2和mIFN-γ平均为380.9和422.1pg/mL,显著高于阴性对照组,与BCG组无显著差异。5个组的平均mIL-6和mIL-10无显著差异。pVS8.4G免疫小鼠脾、肝和肺的平均结核菌载量分别为42 093.2、43 264.1和37 264.8CFU/g,低于pVAX1、pIL2S和PBS组相应器官的载量。结论:DNA疫苗pVS8.4G能刺激产生Th1型免疫应答,免疫鼠抵抗H37Rv攻击的能力增强。     

MPT63核酸疫苗的制备及其免疫原性

扩增结核分枝杆菌分泌蛋白MPT63编码序列 ,克隆于真核表达载体pJW 4 30 3中 .转染COS 7细胞 ,用Western印迹检测表明该基因在细胞内得到正确表达 .用该质粒连续免疫C5 7BL 6小鼠 3次后 ,用纯化的重组蛋白MPT63检测小鼠血清中的抗体 ,发现抗体滴度达到 10 5,免疫动物中γ干扰素的含量达到 2 5 8± 0 2U ml ,为空载体DNA免疫对照组的 2 0倍以上 ,说明MPT63核酸疫苗在实验动物体内引起了较强的免疫应答     

Differential immunogenicity and protective efficacy of DNA vaccines expressing proteins of Mycobacterium tuberculosis in a mouse model

BALB/c mice were vaccinated three times (2-week intervals) with plasmid DNA separately encoding antigen Ag85B, ESAT-6 or Ag85A from Mycobacterium tuberculosis. The protective efficacy of these DNA vaccines against intravenous M. tuberculosis H37Rv challenge infection was measured by counting bacterial loads in spleen and lung and recording changes in lung pathology. The splenocyte proliferative response to the corresponding antigens and antigen-specific interferon (IFN)-γ secreted by splenocytes of the vaccinated mice were also detected. We found a clear hierarchy of protective efficacies among the three DNA vaccines tested in this study. Plasmid DNA encoding Ag85A provided the strongest protection and showed the least change in lung pathology, followed by plasmid DNAs encoding Ag85B and ESAT-6. However, DNA-85B reduced comparative bacterial load in lung tissue, as did DNA-85A. Compared to the control group, protective efficacies conferred by different DNA vaccines were consistent with the lymphoproliferative responses to the corresponding antigens as well as the secretions of antigen-specific IFN-γ. Our study demonstrates that both Ag85A and Ag85B are the most promising of the candidate antigens tested for future TB vaccine development.     

结核杆菌抗原85A与小鼠白细胞介素21共表达DNA疫苗的构建及其免疫效应研究

目的:利用真核表达质粒pRSC,构建结核杆菌抗原85A(Ag85A)与小鼠白细胞介素21(mIL21)共表达重组体pRSC-mIL21-Ag85A,为研究新型结核杆菌DNA疫苗提供新的策略。方法:从质粒pcDNA3.1-mIL21中经PCR扩增出mIL21基因,并插入质粒pRSC中构成pRSC-mIL21;再从pIRES-Ag85A质粒中经PCR扩增出Ag85A基因,构建于pRSC-mIL21重组质粒上,成为共表达DNA疫苗pRSC-mIL21-Ag85A。结果:经酶切、基因测序证实,该疫苗构建正确并能成功表达目的基因。共表达DNA疫苗免疫小鼠后,CTL活性、特异性淋巴细胞增殖水平及小鼠血清特异性抗体均呈有意义的提高。结论:结核杆菌Ag85A与mIL21共表达DNA疫苗能诱导小鼠免疫反应,为进一步研究DNA疫苗抗结核杆菌攻击的免疫防护效应奠定了基础。     

佐剂 DDA 和 MPL 对提高结核杆菌组合DNA 疫苗免疫效果的比较研究

编码结核杆菌 3 种抗原 Ag85B , MPT64 , MPT83 的基因片段插入真核表达载体作为组合疫苗免疫小鼠, DDA 和 MPL 作为佐剂分别提高了此三价苗的免疫原性和免疫保护效果,且相比之下 DDA 优于 MPL. 添加 DDA 后, Ag85B , MPT64 , MPT83 抗原特异的 IFN- γ含量分别为 (265.37±79.2) U/ml , (185.31 ±58.3) U/ml, (108.13±54.4) U/ml ,分别比非佐剂组的高 16 U/ml , 45 U/ml 和 2 U/ml ,与 MPL 组 3 种抗原特异性 IFN- γ的含量无显著差异 . IL-4 的含量在各组中无显著差异 . 攻毒后细菌计数结果显示,添加佐剂的三价苗组小鼠的肺脏和脾脏的载菌量分别比空载体组降低了 2~3 个数量级,且佐剂 DDA 组显著优于佐剂 MPL 组和未加佐剂组 . 病理切片结果与载菌量数据相一致,添加佐剂组,特别是 DDA 组小鼠肺部淋巴细胞相对减少,巨噬细胞增多 . 因此, DDA 作为佐剂能显著提高核酸疫苗的免疫效率,佐剂 MPL 不能提高结核杆菌多价核酸疫苗的免疫效率 .     

Immunogenicity and protective efficacy of three DNA vaccines encoding pre-erythrocytic- and erythrocytic-stage antigens of Plasmodium cynomolgi in rhesus monkeys

Although several malaria vaccine candidate antigens have been identified, the most suitable methods for their delivery are still being investigated. In this regard, direct immunization with DNA encoding these vaccine target antigens is an attractive alternative. Here, we have investigated the immune responses to DNA immunization with three major vaccine target antigens: the apical membrane antigen-1 and the 19-kDa C-terminal fragment of merozoite surface protein-1 from the erythrocytic stage, and the thrombospondin-related adhesive protein from the pre-erythrocytic stage of Plasmodium cynomolgi in rhesus monkeys. Antigen-specific antibodies were developed in all the immunized monkeys and peripheral blood mononuclear cells from all immunized monkeys proliferated to different extents upon in vitro stimulation with the corresponding recombinant proteins. The immunized monkeys were challenged with P. cynomolgi sporozoites. All of the immunized animals developed infection but although there was no significant difference between the control and vaccinated animals in terms of pre-patent period, total duration of patency and primary peak parasitemia, the vaccinated animals had significantly lower secondary peak parasitemia than the control animals.     

Assuring the quality, safety, and efficacy of DNA vaccines

Scientists in academia whose research is aimed at the development of a novel vaccine or approach to vaccination may not always be fully aware of the regulatory process by which a candidate vaccine becomes a licensed product. It is useful for such scientists to be aware of these processes as the development of a novel vaccine could be problematic owing to the starting material often being developed in a research laboratory under ill-defined conditions. This paper examines the regulatory process with respect to the development of a DNA vaccine. DNA vaccines present unusual safety considerations that must be addressed during preclinical safety studies, including adverse immunopathology, genotoxicity through integration into a vaccinees chromosomes, and the potential for the formation of anti-DNA antibodies.     

针对结核分枝杆菌潜伏感染的DNA疫苗V569免疫原性及保护性的初步研究

为研究针对结核分枝杆菌潜伏感染的DNA疫苗,基于质粒A39构建了p-VAX1-Ag85B-Rv3425-Rv2029c-PPE26 (V569)质粒DNA,并对其免疫原性及保护性进行初步研究。免疫性评价试验共分6组：PBS、p-VAX1-Ag85B(A)、p-VAX1-Ag85B-Rv3425(A3)、A39、V569和BCG,采用左后腿肌内注射C57BL/6小鼠,用流式细胞术和酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)分别检测细胞免疫和体液免疫水平;构建斑马鱼-海分枝杆菌潜伏感染模型,将PBS、A、A3、A39、BCG、V569分别通过腹腔注射免疫斑马鱼后,每日注射地塞米松10ug诱导海分枝杆菌复发感染,对斑马鱼肝脏进行菌落计数并绘制生存曲线。结果显示,与BCG组相比,V569能引发实验小鼠强烈的细胞免疫反应(IFN-γ高水平分泌),外周血CD4^＋/CD8^＋ T细胞比例明显增加。在斑马鱼-海分枝杆菌潜伏感染复发模型中,与BCG 免疫组相比,V569免疫斑马鱼后可显著减少其肝脏中海分枝杆菌数量,斑马鱼存活情况得到显著改善,表明V569 DNA疫苗可能是一种抗结核潜伏感染的候选DNA疫苗。     

Protection of mice with a divalent tuberculosis DNA vaccine encoding antigens Ag85B and MPT64

Tuberculosis (TB) remains to be a major challenge tothe public health in the world. It is estimated that, through-out the world, 15 individuals are affected by TB and 6 ofthem die from it every minute [1]. Drug resistance andcoinfection with HIV, which ut…     

Paleopathological and biomolecular study of tuberculosis in a medieval skeletal collection from England

Nine human skeletons of medieval date from a rural English burial site show signs of skeletal tuberculosis. They were subject to polymerase chain reaction (PCR) assays aimed at detecting traces of DNA from infecting mycobacteria, with the purpose both of confirming the paleopathological diagnosis of tuberculosis and determining in individual cases whether disease was due to M. tuberculosis or M. bovis. In all nine cases, evidence for M. tuberculosis complex DNA was found, and in all instances it appeared that disease was due to M. tuberculosis rather than M. bovis. The significance of the findings for understanding tuberculous infection in rural agrarian communities in medieval England is discussed.     

Toward novel vaccines against tuberculosis: current hopes and obstacles

Approximately 2 million people die of tuberculosis (TB) each year. The current vaccine, Bacille Calmette-Guérin (BCG), albeit widely employed, does not protect against adult pulmonary disease, and new vaccines are urgently needed to reduce the incidence of TB worldwide. New insights into the cellular and molecular mechanisms that underlie the interactions between Mycobacterium tuberculosis and its host have been exploited to develop novel vaccine candidates that recently have entered clinical trials. This review provides a brief overview of different approaches toward a new vaccination strategy and summarizes major challenges for the next decade.     

Biodegradable microcapsules with entrapped DNA for development of new DNA vaccines

In this study an universal method for preparation of biodegradable microcapsules for antigen entrapment was proposed and optimized. The multilayer microcapsules were prepared by layer-by-layer adsorption of various polyelectrolytes (such as alginate, poly-L-lysine, κ-carrageenan, chitosan and dextran derivatives). High entrapment efficiency of protein and plasmid DNA (non less than 90%) was shown. To carry out in vivo tests, a set of microcapsules with entrapped pTKShi plasmid encoding the E2 polypeptide of classical swine fever was prepared. It was shown that an injection of these microcapsules into mice induced an immune response. The highest antibody titers of mouse blood sera were got after immunization by microcapsules based on modified dextran/carrageenan and modified chitosan/carrageenan. The proposed method for antigen entrapment in biodegradable microcapsules could be used for development of encapsulated vaccines of a new generation (DNA-vaccines).     

Immunogenicity of DNA and recombinant Sendai virus vaccines expressing the HIV-1 gag gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens. Foundation item: National 863 project (2003AA219070)     

保护性病毒抗原的筛选策略及其在新型疫苗研发中的应用

随着疫苗研发技术的发展,新型疫苗在传染病的预防中得到了广泛应用。由于新型疫苗安全性良好,因此其在烈性病疫苗的应用中有着得天独厚的优势,然而研制新型疫苗的前提是筛选出保护性抗原。随着各种组学研究的发展,针对真核生物的多种生物信息学方法代表着最前沿的技术手段。相对于真核细胞,病毒具有更为简单的结构,对应着相对简单的研究方法,未来的保护性抗原筛选策略,需要结合生物信息学和传统分子生物学方法的优势。本文分别从宿主和病毒入手,论述了病毒保护性抗原的筛选策略,列举了一系列基于真核细胞开发的可能用于保护性抗原筛选的生物信息学方法,并总结了应用保护性抗原进行新型疫苗设计的案例,以便加深对病毒保护性抗原筛选策略的认知,为新型疫苗的研发提供借鉴。     

结核疫苗保护力评价用感染菌液的制备和保藏研究

实验研究中对结合分枝杆菌感染菌液进行活菌数量与毒力的稳定性观察。以活菌计数与感染豚鼠后的肝、脾、肺病变指数为评判指标进行观察,结果显示低温保藏菌液在32个月内,其活菌数量处于6.7～18×105CFU/ml之间,感染豚鼠的肝、脾、肺病变指数处于42～51之间。说明低温保藏菌液具有很好的稳定性,可作为结核分枝杆菌感染豚鼠模型标准化用菌液。     

Novel use of guanidinium isothiocyanate in the isolation of Mycobacterium tuberculosis DNA from clinical material

Nucleic acid amplification technologies offer great promise for the rapid, sensitive and specific diagnosis of tuberculosis. However, the isolation of inhibitor-free DNA from biological specimens is a bottleneck of the PCR assay. Here we describe a simple method for the isolation of PCR-amplifiable DNA of Mycobacterium tuberculosis from all types of samples of pulmonary and extrapulmonary origin tested. Briefly, it involves concentration of the bacilli by high-speed centrifugation, removal of PCR inhibitors by a wash solution containing guanidinium isothiocyanate and the release of bacterial DNA by heating in the presence of detergents and Chelex-100 resin. The entire process is accomplished within approximately 3 h. The method has been validated on 780 samples of human, bovine and guinea pig origin including sputum, cerebrospinal fluid, pulmonary fluids, pus, fine needle aspirate, tissue, blood and milk.     

百日咳杆菌粘附素对猪源支气管败血波氏杆菌的保护性抗原的筛选

摘要：【目的】本研究通过百日咳杆菌黏附素(PRN)基因的分段克隆表达及其在BALB/c小鼠的主动和被动免疫保护试验筛选PRN中的保护性抗原肽。【方法和结果】利用大肠杆菌进行PRN的完整蛋白、N端和C端多肽及其RI和RII区域多肽（双拷贝）的表达,命名为GST-PRN、GST-PN、GST-PC、GST-2PRI和GST-2PRII。Western blot检测证实5种表达产物均具有良好的反应原性。在主动免疫保护试验中,5种表达产物均能诱导小鼠产生较高的PRN抗体水平;当使用3 LD50的支气管败血波氏杆菌