Novel approaches for the development of live and inactivated viral vaccines

目前，没有针对大量历史上重要的病毒病原体和新的 新出现的病毒性人畜共患病威胁。这种情况在一定程度上是由于现有的 应对各种新出现的病毒构成的挑战的方法。衰减的程度 通过病毒连续传代、病毒重组和定向分子等途径介导 演变是不可预测的，这一特点导致许多人的生产时间很长 使用这些策略制造的疫苗。同样，一些使用这些材料构建的疫苗 方法学已经证明了较差的安全概况，因为在数量上的固有不确定性 引入了衰减决定因素。在这项研究中，我们的目标是进一步定义一种新的策略，以直观地 构建适用于开发多种疫苗的减毒活病毒(LAV)疫苗 急性病毒性疾病。我们的方法是基于对宿主策略的操纵来调节 蛋白质翻译。我们假设使用稀有密码子作为疫苗的衰减决定因素 候选者将产生更稳定的减毒病毒，这些病毒可以合理地设计成可预测的 宿主细胞内的复制表型。此外，我们已经证明了这些替换可以是 被引入到病毒基因组的不同位置，而不像传统的氨基酸替换 用来减弱病毒的。我们还设计了第二个战略，以进一步提高 限制疫苗病毒在导致病毒产生的组织中复制的疫苗 疾病病理学。通过将组织特异性miRNAs的目标序列引入病毒基因组，我们已经证明了特定地阻止表达同源miRNA的目标细胞的感染的能力，同时允许诱导保护性免疫反应所需的细胞群体中正常的未改变的复制水平。重要的是，这一战略与对现有疫苗的修改相兼容，以改善其安全性。这项研究中定义的方法对于快速开发预防药物以应对各种疾病威胁具有广泛的意义。

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.