DNA疫苗安全性的策略探究

DNA疫苗安全性问题主要来自DNA疫苗元件和疫苗工程菌,包括DNA疫苗与宿主基因组整合、接种后诱导宿主产生免疫耐受和自身免疫疾病、抗性基因的转移问题、疫苗中内毒素和抗生素及其他有害物质残留问题、宿主菌体内DNA复制与纯化过程中基因稳定性问题。本文针对这些问题进行策略上的探讨,为进一步开发安全的DNA疫苗提供帮助。     

减毒活疫苗作为DNA疫苗载体的研究进展

由于胞内感染菌与宿主细胞之间的相互作用,使相应的减毒活疫苗有可能成为更为理想的DNA疫苗载体,本文就减毒活疫苗作为理想的DNA疫苗的生物学基因,研究进展,存在的问题等方面进行综述。     

打开癌症的黑匣子(下)

<正>病毒中的逆转录现象带来了一个重要的后果,这就是当病毒的RNA转录为DNA时,后者就整合到了宿主细胞的DNA中。那么,整合以后将对宿主细胞带来什么样的效应呢?这就是"逆转录病毒癌基因的细胞起源的发现"。毕晓普和他的同事因此而荣获1989年的诺贝尔生理学或医学奖。     

打开癌症的黑匣子（下）

病毒中的逆转录现象带来了一个重要的后果,这就是当病毒的RNA转录为DNA时,后者就整合到了宿主细胞的DNA中。那么,整合以后将对宿主细胞带来什么样的效应呢？这就是“逆转录病毒癌基因的细胞起源的发现”。毕晓普和他的同事因此而荣获1989年的诺贝尔生理学或医学奖。     

一类新型疫苗—核酸疫苗

核酸疫苗是将编码某种抗原蛋白的外源基因（DNA或RNA）直接导入动物细胞内,并通过宿主细胞的转录系统合成抗原蛋白,诱导宿主产生对该抗原蛋白的免疫应答,以达到预防和治疗疾病的目的。因此,核酸疫苗又称基因疫苗或基因免疫。但目前研究最多的是DNA疫苗,由于其不需要任何化学载体,因此也称为裸DNA疫苗。1核酸疫苗的发现裸DNA疫苗最早是由沃尔夫（WOllf）等人于1990年在一次基因治疗的实验研究中意外发现的。他们用化学试剂处理小鼠骨骼肌细胞,以提高其摄入质粒DNA的能力。结果发现,未作任何处理的对照动物,其骨骼肌细胞也吸…     

用硫酸鱼精蛋白去除脊髓灰质炎传代细胞疫苗中的基质DNA

<正>用传代细胞代替原代细胞制备脊髓灰质炎疫苗的技术可大大改进和标化这些疫苗制品。关于疫苗的安全性人们主要考虑具有潜在致瘤性的基质DNA的污染。对灭活疫苗,可接受的细胞DNA含量为每剂量100ng以下,因而需要反复提纯。对减毒口服脊髓灰质炎疫苗(OPV)目前尚缺乏相应的评价标准。已建议对减毒口服脊髓灰质炎疫苗进行提纯以去除细胞DNA。     

核酸疫苗--一种新型疫苗

核酸疫苗是指将含有编码某种抗原蛋白基因序列的质粒载体作为疫苗,直接导入动物细胞内,从而通过宿主细胞的转录系统合成抗原蛋白,诱导宿主产生对该抗原蛋白的免疫应答,达到免疫的目的.核酸疫苗又称为基因疫苗或裸DNA疫苗,这种免疫称为核酸免疫、基因免疫、DNA介导的免疫以及遗传免疫等.     

基因工程改造的活病毒显示AIDS疫苗的可喜前景

去年有关AIDS疫苗研究的争论达到顶峰,由于实验室和临床应用方面的问题很多,使人们对AIDS疫苗的研究战略产生怀疑,对这方面已有详细的报道,其中具有代表性的人物是86岁的脊髓灰质炎和麻疹病毒疫苗发明者——微生物学家Albert Sabin,他认为脊灰病毒和麻疹病毒疫苗的成功是由于这些病毒是游离的,不寄宿于细胞内;而HIV不同,它可以在细胞内增殖,将病毒的基因产物整合到宿主细胞的DNA中去,但在受感染的细胞表面却没     

探索癌基因的成熟年代

1866年,Paul Broca 从他妻子家系的略图中,领悟到癌症的遗传因素。这一洞察所见,在他那时代似乎没有引起人们的注意(实际上,他的想法直至当代仍被 carl Segen 所忽视)。不过一个世纪以来,生物学家们都开始对肿瘤发生寻找遗传方面的解释。目前这个问题所获得的成果是:关于长期设想的“癌基因”已出现在我们的眼前。癌基因的发现首先是由于逆转病毒有转导细胞基因的偏好——正是在正常细胞 DNA 中寻找逆转病毒的癌基因使它实体化了。由此两个进一步的设想又增添到活跃的想象中去:一是逆转病毒DNA 的整合能够触动细胞基因,后者的激活明显地有引起肿瘤发生的作用;二是从各种肿瘤取得的 DNA 能够使啮盘类细胞发生瘤性生长,这种 DNA 似乎含有活     

大质粒DNA疫苗pSVK-CAVA高稳定性宿主菌的筛选与鉴定

目的:从多种大肠杆菌感受态细胞中筛选出适合该研究大分子质粒DNA疫苗的宿主菌,鉴定其达到中试要求。方法:将疫苗质粒pSVK-CAVA(14.7kb)转化4种大肠杆菌化学感受态细胞并提取质粒,通过琼脂糖凝胶电泳实验检测质粒的形态结构。对基因工程菌进行生化检测,并通过连续传代法和酶切鉴定进行稳定性检测,同时将质粒DNA瞬时转染至293T细胞中检测质粒表达能力。选取质粒含量最高和稳定性最好的宿主菌作为原始种子分装冻存,将原始种子库扩大培养,逐级建立好主种子库和工作种子库即三级种子库。通过摇瓶培养实验在4种常用基础培养基中挑选出最适合质粒生产的培养基。结果:确定了XL-10 Gold作为质粒DNA疫苗pSVK-CAVA的宿主菌,基因工程菌传代稳定性和结构稳定性良好,质粒能在293T细胞中体外表达。筛选出TB培养基为基础培养基,质粒容积产量达到9.9mg/L,比LB培养基提高了接近1倍。结论:该研究筛选出大肠杆菌XL-10 Gold作为质粒DNA疫苗的宿主菌,解决了大质粒在常用宿主菌中不稳定的难题,并对基础培养基进行了初步优化。     

Ensuring safety of DNA vaccines

In 1990 a new approach for vaccination was invented involving injection of plasmid DNA in vivo, which elicits an immune response to the encoded protein. DNA vaccination can overcome most disadvantages of conventional vaccine strategies and has potential for vaccines of the future. However, today 15 years on, a commercial product still has not reached the market. One possible explanation could be the technique's failure to induce an efficient immune response in humans, but safety may also be a fundamental issue. This review focuses on the safety of the genetic elements of DNA vaccines and on the safety of the microbial host for the production of plasmid DNA. We also propose candidates for the vaccine's genetic elements and for its microbial production host that can heighten the vaccine's safety and facilitate its entry to the market.     

Molecular characterization for food safety assessment of a genetically modified late blight resistant potato: an unusual case

Standard food safety assessments of genetically modified crops require a thorough molecular characterization of the novel DNA as inserted into the plant that is intended for commercialization, as well as a comparison of agronomic and nutritional characteristics of the genetically modified to the non-modified counterpart. These characterization data are used to identify any unintended changes in the inserted DNA or in the modified plant that would require assessment for safety in addition to the assessment of the intended modification. An unusual case of an unintended effect discovered from the molecular characterization of a genetically modified late blight resistant potato developed for growing in Bangladesh and Indonesia is presented here. Not only was a significant portion of the plasmid vector backbone DNA inserted into the plant along with the intended insertion of an R-gene for late blight resistance, but the inserted DNA was split into two separate fragments and inserted into two separate chromosomes. One fragment carries the R-gene and the other fragment carries the NPTII selectable marker gene and the plasmid backbone DNA. The implications of this for the food safety assessment of this late blight resistant potato are considered.

     

Issues associated with residual cell-substrate DNA in viral vaccines

The presence of some residual cellular DNA derived from the production-cell substrate in viral vaccines is inevitable. Whether this DNA represents a safety concern, particularly if the cell substrate is derived from a tumor or is tumorigenic, is unknown. DNA has two biological activities that need to be considered. First, DNA can be oncogenic; second, DNA can be infectious. As part of our studies to assess the risk of residual cell-substrate DNA in viral vaccines, we have established assays that can quantify the biological activities of DNA. From data obtained using these assays, we have estimated the risk of an oncogenic or an infectious event from DNA. Because these estimates were derived from the most sensitive assays identified so far, they likely represent worst-case estimates. In addition, methods that inactivate the biological activities of DNA can be assessed and estimations of risk reduction by these treatments can be made. In this paper, we discuss our approaches to address potential safety issues associated with residual cellular DNA from neoplastic cell substrates in viral vaccines, summarize the development of assays to quantify the oncogenic and infectivity activities of DNA, and discuss methods to reduce the biological activities of DNA.     

The National Institutes of Health system for enhancing the science,safety, and ethics of recombinant DNA research

Oversight of recombinant DNA research by the National Institutes of Health (NIH) is predicated on ethical and scientific responsibilities that are akin, in many ways, to those that pertain to the oversight of animal research. The NIH system of oversight, which originated more than 25 years ago, is managed by the NIH Office of Biotechnology Activities (OBA), which uses various tools to fulfill its oversight responsibilities. These tools include the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) and the Recombinant DNA Advisory Committee. The OBA also undertakes special initiatives to promote the analysis and dissemination of information key to our understanding of recombinant DNA, and in particular, human gene transfer research. These initiatives include a new query-capable database, an analytical board of scientific and medical experts, and conferences and symposia on timely scientific, safety, and policy issues. Veterinary scientists can play an important role in the oversight of recombinant DNA research and in enhancing our understanding of the many safety and scientific dimensions of the field. These roles include developing appropriate animal models, reporting key safety data, enhancing institutional biosafety review, and promoting compliance with the NIH Guidelines.     

Down-regulation of Prdx6 contributes to DNA vaccine induced vitiligo in mice

DNA vaccines are widely used against infectious agents for their ability to induce both humoral and cellular immune responses. However, safety concerns regarding autoimmune responses to DNA vaccines, particularly to certain plasmids, should not be neglected. In this study, we serendipitously found that mice inoculated with pcDNA3-ANXB1 (pcDNA3-b1) developed autoimmunity, which did not happen in pVAX-ANXB1 (pVAX-b1) inoculated mice. We also employed proteomics approaches to investigate the distinction between the two groups of DNA vaccine immunized mice. Five different proteins with three-fold or greater changes were separated and identified by two-dimensional electrophoresis. Our study verified the safety of the DNA vaccine and unveiled the underlying potential molecular mechanism of DNA vaccine delivery.     

Rational Vector Design for Efficient Non-viral Gene Delivery: Challenges Facing the Use of Plasmid DNA

Although non-viral gene delivery is a very straightforward technology, there are currently no FDA-approved gene medicinal products available. Therefore, improving potency, safety, and efficiency of current plasmid DNA vectors will be a major task for the near future. This article will provide an overview on factors influencing production yield and quality as well as safety issues that emerge from the vector design itself. Special focus will be on generating bacterial pDNA vectors by circumventing the use of antibiotic resistance genes, to generate safer gene medicinal products as well as smaller, more efficient DNA vectors.     

Technical and regulatory hurdles for DNA vaccines

DNA vaccines have been widely used in laboratory animals and non-human primates over the last decade to induce antibody and cellular immune responses. This approach has shown some promise, in models of infectious diseases of both bacterial and viral origin as well as in tumour models. Clinical trials have shown that DNA vaccines appear safe and well tolerated, but need to be made much more potent to be candidates for preventive immunisation of humans. This review describes recent work to improve the delivery of plasmid DNA vaccines and also to increase the immunogenicity of antigens expressed from the DNA vaccine plasmids, including various formulations and molecular adjuvants. Because DNA vaccines are relatively new and represent a novel vaccine technology, certain safety issues, such as the potential for induction of autoimmune disease and integration into the host genome, must be examined carefully. If potency can be improved and safety established, plasmid DNA vaccines offer advantages in speed, simplicity, and breadth of immune response that may be useful for the immunisation of humans against infectious diseases and cancers.     

DNA疫苗研究进展

DNA疫苗是20世纪90年代初出现的一种新型疫苗,近年来发展迅速,在预防和治疗病毒性疾病及肿瘤等方面效果显著。同传统的疫苗相比,DNA疫苗具有免疫效果好、生产成本低、临床应用方便等优点,但同样存在安全性的担忧。对DNA疫苗的发展及其作用机制、优势进行了综述,并对DNA疫苗的安全性提出了自己的观点与看法,可供DNA疫苗的研究者参考。     

Minicircle DNA immobilized in bacterial ghosts: in vivo production of safe non-viral DNA delivery vehicles

DNA as an active agent is among the most promising technologies for vaccination and therapy. However, plasmid backbone sequences needed for the production of pDNA in bacteria are dispensable, reduce the efficiency of the DNA agent and, most importantly, represent a biological safety risk. In this report we describe a novel technique where a site-specific recombination system based on the ParA resolvase was applied to a self-immobilizing plasmid system (SIP). In addition, this system was combined with the protein E-specific lysis technology to produce non-living bacterial carrier vehicles loaded with minicircle DNA. The in vivo recombination process completely divided an origin plasmid into a minicircle and a miniplasmid. The replicative miniplasmid containing the origin of replication and the antibiotic resistance gene was lost during the subsequently induced PhiX174 gene E-mediated lysis process, which results in bacterial ghosts. The minicircle DNA was retained in these empty bacterial cell envelopes during the lysis process via the specific interaction of a membrane anchored protein with the minicircle DNA. Using this novel platform technology, a DNA delivery vehicle--consisting of a safe bacterial carrier with known adjuvant properties and minicircle DNA with an optimized safety profile--can be produced in vivo in a continuous process. Furthermore, this study provides the basis for the development of an efficient in vitro minicircle purification process.