Novel DNA-Launched Attenuated Vaccine for VEE Virus

描述（由申请方提供）：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，具有潜在的生物恐怖主义威胁。VEEV的爆发发生在中美洲，以前曾传播到美国。该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，TC-83减毒活疫苗用于高危人员的接种。疫苗会引起不良反应，而有些人不会产生中和抗体。正在努力开发新的VEEV疫苗。然而，由于疫苗开发是一个漫长的过程，TC-83疫苗的供应有限，美国可能很快就会出现VEEV疫苗短缺的情况，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疫苗，并可能用于其他病毒性疾病。Medigen最近开发了“感染性DNA”（i-DNA）疫苗接种技术，该技术代表了传统DNA免疫与高效减毒活疫苗的独特组合。该技术的关键特征是活的减毒病毒在体内从携带VEEV疫苗的分子克隆的i-DNA质粒发射，具有增强的安全性和免疫原性特征。在初步研究中，我们已经表明，体内注射源自TC-83疫苗的原型i-DNA已经成功地在小鼠中启动了减毒活疫苗。本文基于i-DNA克隆的合理设计和i-DNA免疫技术，提出了新型i-DNA VEEV疫苗的研制和可行性评价。 在特定目标I中，我们将应用创新的“沉默诱变”方法将额外的遗传变化引入原型TC-83 i-DNA中，以确保减毒表型并产生具有高水平安全性和免疫原性的i-DNA克隆。将i-DNA克隆转染到CHO细胞中，并从培养基中收获减毒活病毒。将与马里兰州大学人类病毒学研究所合作，在小鼠中评价i-DNA衍生病毒的安全性。在Specific Aim II中，我们将用i-DNA构建体接种BALB/c小鼠，以诱导免疫应答并评价体内免疫原性。此外，将在西南生物医学研究基金会（SFBR）（圣安东尼奥，TX）的强毒VEEV攻毒实验中评价最有前途的i-DNA VEEV疫苗的效力。最后，还将在SFBR进行体内回复突变研究，以证明i-DNA衍生VEEV疫苗的遗传稳定性。这项为期2年的研究的目标是鉴定具有最佳安全性和免疫原性特征的i-DNA VEEV疫苗克隆，用于未来在非人灵长类动物中的评价和攻毒实验。 我们的初步结果表明，合理的疫苗设计和i-DNA技术可以提供一个革命性的解决方案，通过提高安全性，遗传稳定性和免疫原性，并通过消除许多昂贵的步骤，传统的生产工艺的VEEV疫苗。基本上，减毒活疫苗将在免疫个体内“制造”。该技术还利用了DNA疫苗的许多优点（遗传同质性和稳定性，生产、储存和运输成本低，无冷链），更重要的是增强了免疫原性。在细菌中产生的重组i-DNA克隆含有CpG基序，其激活TLR 9和MyD 88依赖性信号传导途径，导致细胞因子和趋化因子的稳健产生，其诱导强烈的引发效应并刺激获得性病毒特异性免疫应答。 最终的i-DNA VEEV疫苗将代表一类新型疫苗，其结合了DNA疫苗和减毒活疫苗的优点，并保留了具有人类使用历史的“经典”疫苗的骨架。i-DNA技术可以容易地适用于开发其他疫苗，包括WEEV、EEEV、其他甲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.