Adaptation of a Novel RNA virus for vaccine use

描述（由申请人提供）：拟议研究的总体目标是使用一种新的自组装纳米颗粒包装方法创建一种新的RNA病毒疫苗，旨在克服安全性、成本和其他阻碍疫苗开发的限制。基于病毒的疫苗具有刺激先天免疫和适应性免疫的能力，与其他疫苗递送方法相比，这可以提高疫苗效力。与DNA病毒疫苗相比，基于RNA病毒的疫苗具有更高的安全性，因为它们不能整合到宿主基因组中，但仍然受到基于细胞的衣壳包装方法的限制，这些方法成本高昂，并且限制了疫苗的增强。我们的初步数据表明，通过去除所有天然衣壳组装限制，只需将RNA和外壳蛋白混合在一起，就可以以非常低的成本制造出安全性更高、疫苗稳定性更好的自组装疫苗。本研究计划的具体目的是：1)利用非天然衣壳起源组装修饰昆虫鸡舍病毒RNA基因组，并确认其体外颗粒形成；2)在病毒启动子控制下插入外源报告基因，并评估其在细胞内的蛋白质积累；3)将转基因表达与纳米颗粒自组装结合起来，并确认两者的功能。最终产物是一种病毒RNA疫苗，具有独立于其天然衣壳的包装特性，同时保留了使其成为良好疫苗抗原的病毒特性。这包括纳米颗粒大小，免疫细胞对抗原的最佳摄取，在室温下多年的稳定性，以及安全携带病毒转基因表达进入免疫细胞而不存在病毒重组的风险。选择禽舍病毒进行自组装是因为它也具有许多理想的特性。它不是哺乳动物病原体，因此将表现出更高的安全性。它在哺乳动物细胞类型中具有非常高的抗原表达水平，与目前用于疫苗开发的RNA病毒相比，它大大减少了细胞死亡。鸡舍病毒以前没有被用于疫苗开发，因为天然衣壳包装特性极大地限制了转基因插入物的大小，而且病毒颗粒仍然需要在细胞培养中制造。该应用的预期结果将是克服天然衣壳包装的限制，并基于禽舍病毒基因组创建具有高水平抗原表达的自组装RNA纳米颗粒。我们的研究意义重大，因为它将实现我们的长期目标

DESCRIPTION (provided by applicant): The overall objective of the research proposed is to create a new RNA virus vaccine using a novel self-assembling nanoparticle packaging method, designed to overcome safety, cost and other limitations that slow vaccine development. Virus-based vaccines have the ability to stimulate both innate and adaptive immunity, which can improve vaccine potency compared to other methods of vaccine delivery. RNA virus based vaccines have improved safety compared to DNA virus vaccines because they can't integrate into the host genome, but are still limited by cell-based capsid packaging methods that are costly and limit vaccine boosting. Our preliminary data show that by removing all native capsid assembly constraints self-assembling vaccines can be made with improved safety, vaccine stability, and at very low cost, simply by mixing RNA and coat protein together. The Specific Aims of this research plan are 1) to modify the insect Flock House Virus RNA genome with a non-native capsid origin of assembly and confirm in vitro particle formation, 2) to insert a foreig reporter transgene under the control of a virus promoter and evaluate the protein accumulation in cells, and 3) to combine the transgene expression and nanoparticle self-assembly, and to confirm that both are functional. The resulting product is a viral RNA vaccine with packaging characteristics independent of its native capsid, while retaining the virus characteristics that make it a good vaccine antigen. That includes nM particulate size for optimal antigen uptake by immune cells, stability at room temperature for years, and the ability to safely carry viral transgene expression into immune cells without the risk of virus reconstitution. Flock House Virus was selected for self-assembly because it also has many desirable characteristics. It is not a mammalian pathogen, and thus will exhibit improved safety. It has very high levels of antigen expression in mammalian cell types, and greatly reduced cell death compared to RNA viruses currently used in vaccine development. Flock House Virus has not been previously exploited for vaccine development because the native capsid packaging characteristics significantly limit transgene insert size and the virus particles still need to be made in cell culture. The expected outcome of this application will be to overcome native capsid packaging limitations, and create a self-assembling RNA nanoparticle based on the Flock House Virus genome, with high level antigen expression. Our research is significant, because it will fulfill our long-term objective to create cost effective, safe and robust RNA vaccines, and it is innovative because we will validate that any RNA virus with desirable characteristics can be adapted for nanoparticle self- assembly, and increase the pace of RNA vaccine development for human use.