Mitigating the looming vaccine crisis: production and delivery of plasmid-based vaccines

The exponentially growing human population and the emergence of new diseases are clear indications that the world can no longer depend solely on conventional vaccine technologies and production schemes. The race to find a new vaccine technology is crucial to help speed up and complement the World Health Organization (WHO) disease elimination program. The ultimate goal is to uncover fast and efficient production schemes in the event of a pandemic, and also to effectively fight deadly diseases such as malaria, bird flu, hepatitis, and human immunodeficiency virus (HIV). Plasmid DNA vaccines, if properly formulated, offer specific priming of the immune system and similar or even better prophylactic effects than conventional vaccines. This article discusses many of the critical issues that need to be considered when developing fast, effective, and reliable plasmid DNA vaccine manufacturing processes. Different modes of plasmid production via bacterial fermentation are compared. Plasmid purification by chromatography is specifically discussed as it is the most commercially viable bioprocess engineering technique for continuous purification of supercoiled plasmid DNA. Current techniques and progress covering the area of plasmid DNA vaccine design, formulation, and delivery are also put forward.     

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。     

电脉冲和布吡卡因增强A型肉毒毒素DNA核酸疫苗的免疫效果

目的：考查DNA疫苗注射免疫后电脉冲和布吡卡因佐剂化DNA疫苗递送方式对A型肉毒毒素DNA核酸疫苗免疫效果的影响。方法：A型肉毒毒素DNA复制子疫苗和传统DNA疫苗肌肉注射免疫小鼠后电脉冲和布吡卡因佐剂化DNA后再肌肉注射免疫小鼠;检测免疫小鼠的抗体和细胞水平,并分析抗体亚类。结果：电脉冲和布吡卡因这二种递送方式均增强DNA复制子疫苗和传统DNA疫苗的体液免疫和细胞免疫效果;电脉冲提高DNA疫苗免疫效果更为明显,并且电脉冲和布吡卡因组合这种递送方式增强DNA疫苗体液免疫和细胞免疫水平最高;与传统DNA疫苗相比,A型肉毒毒素DNA复制子疫苗在这些递送方式下均诱导产生了更好的特异性体液免疫和细胞免疫应答,并且这些递送方式没有改变DNA疫苗的Th1/Th2免疫应答特性,即DNA复制子疫苗诱导产生Th1/Th2混合免疫应答但偏向于Th2途经,而传统DNA疫苗则完全偏向于Th2途经。结论：电脉冲和布吡卡因增强DNA复制子疫苗和传统DNA疫苗的免疫效果,是提高DNA疫苗免疫原性的良好策略。     

Plasmid-DNA lipid and polymeric nanovaccines: a new strategic in vaccines development

Vaccination is the most effective and least expensive technique used for human diseases prevention and eradication. The need for more vaccine doses and the rapid establishment of facilities for the development of new vaccines are stimulating significate changes in the vaccine industry, which is gradually moving towards cell culture production. One approach is the third generation of vaccines, which are based on the use of plasmid DNA (pDNA) containing transgenes that encode an antigen capable of mimicking intracellular pathogenic infection and triggering both humoral and cellular immune responses. Plasmid DNA vaccination has distinct advantages over other vaccine technologies in terms of safety, ease of fabrication and stability. The effectiveness of pDNA vaccines against viruses, bacteria, parasites and cancer cells has been demonstrated in preclinical and clinical assays. Furthermore, currently there are a few veterinary pDNA vaccines in the market. The application of a simple formulation of naked pDNA as a vaccine is attractive, but a low transfection efficiency is often obtained. The use of nanoparticles to increase transfection efficiency is an approach that has been tested clinically. This review provides a summary of vaccine production, advances and major challenges associated with pDNA lipid and polymeric nanovaccines applications.     

Enhanced potency of individual and bivalent DNA replicon vaccines or conventional DNA vaccines by formulation with aluminum phosphate

DNA vaccines against botulinum neurotoxin (BoNTs) induce protective humoral immune responses in mouse model, but when compared with conventional vaccines such as toxoid and protein vaccines, DNA vaccines often induce lower antibody level and protective efficacy and are still necessary to increase their potency. In this study we evaluated the potency of aluminum phosphate as an adjuvant of DNA vaccines to enhance antibody responses and protective efficacy against botulinum neurotoxin serotypes A and B in Balb/c mice. The administration of these individual and bivalent plasmid DNA replicon vaccines against botulinum neurotoxin serotypes A and B in the presence of aluminum phosphate improved both antibody responses and protective efficacy. Furthermore, formulation of conventional plasmid DNA vaccines encoding the same Hc domains of botulinum neurotoxin serotypes A and B with aluminum phosphate adjuvant increased both antibody responses and protective efficacy. These results indicate aluminum phosphate is an effective adjuvant for these two types of DNA vaccines (i.e., plasmid DNA replicon vaccines and conventional plasmid DNA vaccines), and the vaccine formulation described here may be an excellent candidate for further vaccine development against botulinum neurotoxins.     

Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification

The past several years have witnessed a rapidly increasing number of reports on utilizing plasmid DNA as a vector for the introduction of genes into mammalian cells for use in both gene therapy and vaccine applications. “Naked DNA vaccines” allow the foreign genes to be transiently expressed in transfected cells, mimicking intracellular pathogenic infection and triggering both the humoral and cellular immune responses. While considerable attention has been paid to the potential of such vaccines to mitigate a number of infections, substantially less consideration has been given to the practical challenges of producing large amounts of plasmid DNA for therapeutic use in humans, for both clinical studies and, ultimately, full-scale manufacturing. Doses of naked DNA vaccines are on the order of milligrams, while typical small-scale Escherichia coli fermentations may routinely yield only a few mg/l of plasmid DNA. There have been many investigations towards optimizing production of heterologous proteins over the past three decades, but in these cases, the plasmid DNA was not the final product of interest. This review addresses the current state-of-the-art means for the production of plasmid DNA at large scale in compliance with existing regulatory guidelines. The impact of the nature of the plasmid vector on the choice of fermentation protocols is presented, along with the effect of varying cultivation conditions on final plasmid content. Practical considerations for the large-scale purification of plasmid DNA are also discussed.     

DNA疫苗的分子佐剂应用研究进展

DNA疫苗因在动物尤其是大型动物与人类中诱发较低的免疫反应而严重影响其推广应用。介绍提高与调节DNA疫苗诱导反应的策略：（1）以细胞因子表达质控为佐剂;（2）以质粒编码的趋化因子与共刺激分子为佐剂;（3）以CPG ODN为佐剂。     

Plasmid DNA vaccines

DNA vaccination is a novel approach for inducing an immune response. Purified plasmid DNA containing an antigen’s coding sequences and the necessary regulatory elements to expres them is introduced into the tissue via intramuscular injection or particle bombardment. Once the DNA reaches the tissue, the antigen is expressed in enough quantity to induce a potent and specific immune response and to confer protection against further infections. The effectiveness of DNA vaccines against viruses, parasites, and cancer cells has been demonstrated in numerous animal models. This new approach comes as an aid for the prevention of infectious diseases for which the conventional vaccines have failed. DNA vaccine research is providing new insights into some of the basic immunological mechanisms of vaccination such as antigen presentation, the role of effector cells, and immunoregulatory factors. In addition, DNA vaccines may enable us to manipulate the immune system in situations where the response to agents is inappropriate or ineffective. The study of the potential deleterious effects of DNA vaccines is furthering our knowledge regarding the relationship between bacterial DNA and the immune system, as well as its potential application for the study of neonatal tolerance and autoimmunity.     

DNA疫苗免疫佐剂的研究进展

DNA疫苗是最近几年从基因治疗研究领域发展起来的一种新型疫苗,它能诱导机体产生持久的体液免疫和细胞免疫应答,能够抗病毒,细菌和寄生虫的感染,对自身免疫性疾病和过敏性疾病有一定的疗效作用。但与传统的灭活疫苗相比,其免疫效价还比较低,最近的研究表明：联合使用DNA疫苗和疫苗佐剂如细胞因子,协同刺激分子等有助于提高DNA疫苗的免疫效价,这一发现有利于研制更有效的DNA疫苗,本文就通过使用免疫佐剂提高DNA免疫效价的最新进展做一综述。     

Transgenic plants for animal health: plant-made vaccine antigens for animal infectious disease control

A variety of plant species have been genetically modified to accumulate vaccine antigens for human and animal health and the first vaccine candidates are approaching the market. The regulatory burden for animal vaccines is less than that for human use and this has attracted the attention of researchers and companies, and investment in plant-made vaccines for animal infectious disease control is increasing. The dosage cost of vaccines for animal infectious diseases must be kept to a minimum, especially for non-lethal diseases that diminish animal welfare and growth, so efficient and economic production, storage and delivery are critical for commercialization. It has become clear that transgenic plants are an economic and efficient alternative to fermentation for large-scale production of vaccine antigens. The oral delivery of plant-made vaccines is particularly attractive since the expensive purification step can be avoided further reducing the cost per dose. This review covers the current status of plant-produced vaccines for the prevention of disease in animals and focuses on barriers to the development of such products and methods to overcome them.     

SIV DNA vaccine co-administered with IL-12 expression plasmid enhances CD8 SIV cellular immune responses in cynomolgus macaques

Current evidence suggests that a strong induced CD8 human immunodeficiency virus type 1 (HIV-1)-specific cell mediated immune response may be an important aspect of an HIV vaccine. The response rates and the magnitude of the CTL responses induced by current DNA vaccines in humans need to be improved and cellular immune responses to DNA vaccines can be enhanced in mice by co-delivering DNA plasmids expressing immune modulators. Two reported to work well in the mouse systems are interleukin (IL)-12 and CD40L. We sought to compare these molecular adjuvants in a primate model system. The cDNA for macaque IL-12 and CD40L were cloned into DNA vectors. Groups of cynomolgus macaques were immunized with 2 mg of plasmid expressing SIVgag alone or in combination with either IL-12 or CD40L. CD40L did not appear to enhance the cellular immune response to SIVgag antigen. However, more robust results were observed in animals co-injected with the IL-12 molecular adjuvant. The IL-12 expanded antigen-specific IFN-gamma positive effector cells as well as granzyme B production. The vaccine immune responses contained both a CD8 component as well a CD4 component. The adjuvanted DNA vaccines illustrate that IL-12 enhances a CD8 vaccine immune response, however, different cellular profiles.     

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。     

Approaches To New Vaccines

ABSTRACT:?

The explosive technological advances in the fields of immunology and molecular biology in the last 5 years had an enormous impact on the identification of candidate vaccines against diseases, which until a few years ago seemed uncontrollable. Increased knowledge of the immune system has helped to define the mechanisms that underlie successful immunization and is now being exploited to develop improved versions of existing vaccines and new vaccines against emerging pathogens, tumors, or autoimmune diseases. An understanding of the mechanisms of action of novel adjuvants and the development of new vector and delivery systems will have a major impact on vaccine strategies. The use of DNA encoding antigens from pathogenic viruses, bacteria, and parasites as vaccines is a new approach that is receiving considerable attention. This and other innovative approaches, including vaccine production in plants, are appraised in this review. The successful eradication of smallpox and the imminent eradication of poliomyelitis by worldwide immunization campaigns provide positive examples of how the vaccine-mediated approach can lead to disease elimination; with the advent of new vaccines and improved delivery systems, there is no scientific reason why these successes cannot be repeated.     

猪霍乱沙门氏菌递送的双启动子表达载体的构建

猪霍乱沙门氏菌C500株不仅可以作为预防猪沙门氏菌病的活疫苗,还可作为运送其他DNA疫苗的优良载体,并通过粘膜免疫诱导产生针对特定抗原的各种免疫应答。为增强其携带的DNA疫苗的免疫效力,本研究以真核表达载体pEGFP-C1为基础,将其真核启动子CMVie与原核启动子Ptrc串联,并在其多克隆位点MCS下游引入rrnbT1T2转录终止序列,构建了真、原核双启动子表达载体pEGFPPtrcR。用1×TSS法将其转化C500,得到工程菌C500/pEGFPPtrcR,通过SDS-PAGE和Westernblotting鉴定了报告基因EGFP的原核表达,该菌在荧光显微镜下能发出强烈绿色荧光,被证明在体外至少能稳定遗传20代;采用脂质体介导法将pEGFPPtrcR转染Vero细胞,EGFP在胞核和胞浆内表达,24h后观察可到明显绿色荧光。结果表明,双启动子表达载体pEGFPPtrcR构建成功,预示其携带的外源基因既可在C500中表达,又可在体细胞中表达,为研制以C500为载体的新型DNA疫苗的发展开辟了一个新的途径。     

鸡传染性喉气管炎病毒gB基因重组DNA疫苗的构建与免疫试验

本研究构建了编码ILTV主要抗原gB基因的重组DNA疫苗pcDNA-gB,质粒转染293-T细胞的间接免疫表明其表达的蛋白具有免疫反应性。为测定该DNA疫苗的免疫效果,将其与本室保存的重组鸡痘病毒rFPV-gB-gD-IgY分别以单独和混合的方式给4周龄非免疫鸡进行免疫,然后测定ILTV特异性抗体和T淋巴细胞增殖反应。结果表明这2种基因工程疫苗均能诱导鸡产生特异性的体液免疫及细胞免疫应答。其中以pcDNA-gB/rFPV-gB-gD-IgY联合免疫组的效果最好,其诱导的抗体水平已接近于常规弱毒疫苗,而细胞免疫水平则比后者高得多。上述研究结果为ILTV新型疫苗的研究奠定了基础。     

肿瘤DNA疫苗佐剂研究进展

对近年来在增加肿瘤DNA疫苗免疫原性、提高肿瘤DNA疫苗效力方面所取得的进展予以综述。简要阐述了以下几种较有效的增强肿瘤DNA疫苗效力策略的机制和进展：（1）以细胞因子表达质粒为佐剂;（2）以质粒编码的趋化因子、协同刺激分子、共刺激分子、补体为佐剂;（3）以CPGODN为佐剂;（4）其他一些佐剂。     

大肠杆菌菌蜕装载质粒DNA实验方法的优化

目的:优化大肠杆菌菌蜕装载质粒的效率,并将装载质粒的菌蜕转染抗原提呈细胞,以提高核酸疫苗的递送水平。方法:将质粒pHH43转化大肠杆菌DH5α,制备大肠杆菌菌蜕;优化菌蜕装载质粒时菌蜕、质粒和膜囊的比例,获得更高的装载效率,通过扫描及透射电镜、流式细胞术观察其形态变化及装载效率;将装载质粒的菌蜕与抗原提呈细胞——巨噬细胞RAW264.7和树突状细胞DC2.4共孵育,观察吞噬效果。结果:优化了大肠杆菌菌蜕装载质粒的效率,当菌蜕、质粒、膜囊的比例为7∶10∶4时效率达到最佳,装载DNA效率达98%以上;抗原提呈细胞吞噬装载了质粒的菌蜕,效率达100%。结论:大肠杆菌菌蜕可高效装载核酸疫苗,且高效被抗原提呈细胞捕获,有助于提高核酸疫苗的递送和免疫效果的提高。     

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.     

Prion疾病疫苗研究策略

朊病毒病是一类侵袭人类及多种动物中枢神经系统的致死性退行性脑病,目前缺乏有效的预防和治疗方法。朊病毒病的重组蛋白亚单位疫苗、DNA疫苗、合成肽疫苗、病毒样颗粒疫苗、树突状细胞疫苗、黏膜免疫疫苗等已取得一定进展,但现有的免疫策略仅能部分克服免疫耐受,诱导较低或中等滴度的抗体,对PrPSc感染动物模型只能提供部分保护,Prion疫苗研究任重而道远。     

DNA vaccines

Within the last decade bacterial plasmids encoding foreign antigens have revolutionized vaccine design. Although no DNA vaccine has yet been approved for routine human or veterinary use, the potential of this vaccine modality has been demonstrated in experimental animal models. Plasmid DNA vaccination has shown efficacy against viral, bacterial and parasitic infections, modulated the effects of autoimmune and allergic diseases and induced control over cancer progression. With a better understanding of the basic immune mechanisms that govern induction of protective or curative immune responses, plasmid DNA vaccines and their mode of delivery are continuously being optimized. Because of the simplicity and versatility of these vaccines, various routes and modes of delivery are possible to engage the desired immune responses. These may be T or B effector cell responses able to eliminate infectious agents or transformed cells. DNA vaccines may also induce an immunoregulatory/modulatory or immunosuppressive (tolerizing) response that interferes with the differentiation, expansion or effector functions of B and T cells. In this sense a DNA vaccine may be thought of as a 'negative' vaccine. Pre-clinical and initial small-scale clinical trials have shown DNA vaccines in either of these modes to be safe and well tolerated. Although DNA vaccines induce significant immune responses in small animal trials their efficacy in humans has so far been less promising thus necessitating additional optimizations of this novel vaccine approach.