Novel DNA-Launched Attenuated Vaccine for VEE Virus

描述(申请人提供)：委内瑞拉马脑炎病毒(VEEV)是一种危险的NIH/NIAID B类人类病原体和潜在的生物恐怖主义威胁。VEEV疫情发生在中美洲，此前已蔓延至美国。这种病毒在美国重新出现的潜在破坏性影响需要一种有效的疫苗来保护人口。目前，根据IND方案，使用TC-83减毒活疫苗为高危人员接种疫苗。疫苗会引起不良反应，而一些人不会产生中和抗体。开发新的VEEV疫苗的努力正在进行中。然而，由于疫苗开发是一个漫长的过程，而且TC-83疫苗的供应有限，美国可能很快就会经历VEEV疫苗的短缺，这可能会使美国人口得不到保护。我们提出了一种革命性的新技术，用于接种VEEV和潜在的其他病毒性疾病的疫苗。梅迪根公司最近开发出了“感染性DNA”(I-DNA)疫苗接种技术，它代表了常规DNA免疫与高效减毒活疫苗的独特结合。这项技术的关键特征是从携带VEEV疫苗分子克隆的I-DNA质粒体内发射减毒活病毒，具有增强的安全性和免疫原性。在初步研究中，我们已经证明，体内注射来自TC-83疫苗的原型I-DNA成功地在小鼠身上启动了减毒活疫苗。在此，我们在合理设计I-DNA克隆和I-DNA免疫技术的基础上，提出了用于安全高效接种VEEV的新型I-DNA VEEV疫苗的开发和可行性评价。在具体目标I中，我们将应用创新的“沉默突变”方法，在原型TC-83 I-DNA中引入额外的基因变化，以确保减毒表型的安全，并产生具有高水平安全性和免疫原性的I-DNA克隆。I-DNA克隆将被导入CHO细胞，并从培养上清液中获得减毒活病毒。I-DNA衍生病毒的安全性将与马里兰大学人类病毒学研究所合作在小鼠身上进行评估。在特定目的II中，我们将用I-DNA构建物免疫BALB/c小鼠，以诱导免疫应答，并评估体内的免疫原性。此外，最有希望的i-DNA VEEV疫苗的效力将在德克萨斯州圣安东尼奥的西南生物医学研究基金会(SFBR)的毒力VEEV挑战实验中进行评估。最后，SFBR还将进行体内逆转研究，以证明I-DNA衍生VEEV疫苗的遗传稳定性。这项为期两年的研究的目标是鉴定具有最佳安全性和免疫原性的i-DNA VEEV疫苗克隆，以便将来在非人类灵长类动物中进行评估和挑战实验。我们的初步结果表明，合理的疫苗设计和i-DNA技术可以通过提高安全性、遗传稳定性和免疫原性，并通过消除传统生产过程中许多昂贵的步骤，为VEEV疫苗提供革命性的解决方案。从本质上讲，减毒活疫苗将在接种疫苗的个体体内“制造”。这项技术还利用了DNA疫苗的许多优势(遗传同质性和稳定性，低制造、储存和运输成本，没有冷链)，更重要的是，增强了免疫原性。在细菌中产生的重组I-DNA克隆包含CpG基序，激活依赖TLR9和MyD88的信号通路，导致细胞和趋化因子的大量产生，从而诱导强大的启动效应和刺激获得性病毒特异性免疫反应。最终的i-DNA VEEV疫苗将代表一种新型疫苗，结合了DNA和减毒活疫苗的优点，并保留了具有人类使用历史的“经典”疫苗的骨干。I-DNA技术可以很容易地用于其他疫苗的开发，包括Weev、EEEV、其他甲型病毒和黄病毒的减毒活疫苗。如果成功，这项技术可能会改变许多病毒性疾病的减毒活疫苗领域。

DESCRIPTION (provided by applicant): Venezuelan Equine Encephalitis virus (VEEV) is a dangerous, NIH/NIAID category B human pathogen and a potential bioterrorism threat. Outbreaks of VEEV occur in Central America and have previously spread into the United States. The potentially devastating effects of the virus reemergence in the U.S. demand an effective vaccine to protect population. Currently, live attenuated TC-83 vaccine is used under IND protocol for vaccination of personnel at risk. The vaccine causes adverse effects, whereas some individuals do not develop neutralizing antibodies. The efforts to develop new VEEV vaccines are underway. However, because vaccine development is a lengthy process and the supply of TC-83 vaccine is limited, the U.S. may soon experience a shortage of VEEV vaccine, which can leave the U.S. population unprotected. We propose a revolutionary new technology for vaccination against VEEV and potentially, other viral diseases. Medigen has recently developed the "infectious DNA" (i-DNA) vaccination technology, which represents a unique combination of conventional DNA immunization with high efficacy of live attenuated vaccines. The key feature of this technology is that live attenuated virus is launched in vivo from i-DNA plasmid carrying a molecular clone of VEEV vaccine with enhanced safety and immunogenic features. In preliminary studies we have shown that injection in vivo of the prototype i-DNA derived from the TC-83 vaccine has successfully launched live attenuated vaccine in mice. Here we propose the development and feasibility evaluation of novel i-DNA VEEV vaccine for safe and efficient vaccination against VEEV based on the rational design of i-DNA clones and i-DNA immunization technology. In Specific Aim I, we will apply innovative "silent mutagenesis" method to introduce additional genetic changes into the prototype TC-83 i-DNA in order to secure attenuated phenotype and to generate i-DNA clones with high levels of safety and immunogenicity. The i-DNA clones will be transfected in CHO cells and live attenuated viruses will be harvested from culture media. Safety of i-DNA-derived viruses will be evaluated in mice in collaboration with the Institute of Human Virology, University of Maryland. In Specific Aim II we will vaccinate BALB/c mice with i-DNA constructs in order to induce immune response and to evaluate immunogenicity in vivo. Further, the efficacy of the most promising i-DNA VEEV vaccine will be evaluated in a virulent VEEV challenge experiment at Southwest Foundation for Biomedical Research (SFBR), San Antonio, TX. Finally, reversion studies in vivo will be also conducted at SFBR to demonstrate genetic stability of i-DNA - derived VEEV vaccine. The goal of this 2-year research is the identification of i- DNA VEEV vaccine clone with the optimal safety and immunogenicity profiles for future evaluation and challenge experiments in non-human primates. Our preliminary results suggest that the rational vaccine design and i-DNA technology can provide a revolutionary solution for VEEV vaccine by improving safety, genetic stability, and immunogenicity, and by eliminating many costly steps of the conventional manufacturing process. Essentially, live attenuated vaccine will be "manufactured" within the immunized individuals. This technology also utilizes many advantages of DNA vaccines (genetic homogeneity and stability, low cost of manufacturing, storage, and transportation, no cold chain) and, more importantly, enhances immunogenicity. Recombinant i-DNA clones produced in bacteria contains CpG motifs that activate TLR9 and MyD88-dependent signaling pathways resulting in robust production of cyto- and chemokines, which induce strong priming effects and stimulate acquired virus- specific immune responses. The final i-DNA VEEV vaccine will represent a novel class of vaccines combining the advantages of DNA and live attenuated vaccines and preserving the backbone of a "classic" vaccine with a history of human use. The i-DNA technology can be easily adapted for the development of other vaccines including live attenuated vaccines for WEEV, EEEV, other alphaviruses, and flaviviruses. If successful, this technology can potentially transform the field of live attenuated vaccines for many viral diseases.