Novel approaches for the development of live and inactivated viral vaccines

目前，对于大量历史上重要的病毒病原体和新的病毒病原体， 新出现的病毒性人畜共患病威胁。在某种程度上，这种情况是由于现有的不相容性造成的。 这些方法面临着各种新出现的病毒带来的挑战。衰减程度 通过诸如病毒的连续传代、病毒重组和定向分子生物学等方法介导 进化是不可预测的，这一特点导致了许多人的生产时间很长， 使用这些策略生产的疫苗。同样，使用这些构建的许多疫苗 方法已经证明了不良的安全性概况，由于固有的不确定性的数量， 引入衰减决定因素。在这项研究中，我们的目标是进一步定义一种新的策略， 构建减毒活病毒（LAV）疫苗，适用于开发针对多种 急性病毒性疾病我们的方法是基于对宿主策略的操纵， 蛋白质翻译我们假设使用稀有密码子作为疫苗的减毒决定因素， 候选人将产生更稳定的减毒病毒，这些病毒可以以可预测的方式进行合理改造 宿主细胞内的复制表型。此外，我们已经证明，这些取代可以 引入病毒基因组的各个位置，不像常规的氨基酸取代， 用于减弱病毒。我们还设计了第二个策略，以进一步提高 限制疫苗病毒在组织中复制的疫苗， 疾病病理学通过将组织特异性miRNA的靶序列引入病毒基因组中，我们已经证明了特异性阻断表达同源miRNA的靶细胞感染的能力，同时允许诱导保护性免疫应答所需的细胞群体中的正常不变的复制水平。重要的是，这一策略与现有疫苗的修改相容，以改善其安全性。本研究中定义的方法对快速开发各种疾病威胁的预防剂具有广泛的意义。

Currently, no vaccines exist for a significant number of historically important viral pathogens and newly emerging viral zoonotic disease threats. In part, this situation results from the incompatibility of existing methodologies with the challenges posed by a wide array of emerging viruses. The extent of attenuation mediated by approaches such as serial passaging of viruses, viral recombination and directed molecular evolution is unpredictable and this characteristic contributes to the lengthy production times for many vaccines made using these strategies. Likewise, a number of vaccines constructed using these methodologies have demonstrated poor safety profiles due to the inherent uncertainty in the number of attenuation determinants introduced. In this study we aim to further define a novel strategy to intuitively construct live-attenuated viral (LAV) vaccines applicable to the development of vaccines to a broad array of acute viral diseases. Our approach is based on the manipulation of host strategies for the regulation of protein translation. We hypothesize that use of rare codons as attenuation determinants for vaccine candidates will produce more stably attenuated viruses that can be rationally engineered with predictable replication phenotypes within host cells. Moreover, we have demonstrated that these substitutions can be introduced into various places in viral genomes, unlike amino acid substitutions which are conventionally employed to attenuate viruses. We have also designed a second strategy to further improve the safety of vaccines that restricts the replication of vaccine viruses in tissues that are responsible for engendering disease pathology. By introducing the target sequences for tissue-specific miRNAs into the viral genome we have demonstrated the ability to specifically block infection of target cells expressing the cognate miRNA, while permitting normal unaltered levels of replication in the cell populations required for the induction of a protective immune response. Importantly, this strategy is compatible with the modification of existing vaccines to improve their safety profiles. The approaches defined in this study have broad implications for the rapid development of prophylactic agents to a variety of disease threats.