Adaptation of a Novel RNA virus for vaccine use

描述(由申请人提供)：提出的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来创造一种新的RNA病毒疫苗，旨在克服安全、成本和其他减缓疫苗开发的限制。基于病毒的疫苗具有刺激先天免疫和获得性免疫的能力，与其他疫苗交付方法相比，可以提高疫苗效力。与DNA病毒疫苗相比，基于RNA病毒的疫苗提高了安全性，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，限制了疫苗的增强。我们的初步数据表明，通过消除所有天然的衣壳组装限制，只需将RNA和外壳蛋白混合在一起，就可以制造出安全性更高、疫苗稳定性更好、成本非常低的自组装疫苗。本研究计划的具体目的是：1)利用非天然衣壳的组装来源对昆虫鸡舍病毒RNA基因组进行修饰，并在体外证实颗粒的形成；2)在病毒启动子的控制下插入外源报告基因并评价其在细胞中的蛋白质积累；3)将转基因表达和纳米颗粒自组装结合起来，并证实两者都具有功能。由此产生的产品是一种病毒RNA疫苗，其包装特征独立于其天然衣壳，同时保留了使其成为良好疫苗抗原的病毒特征。这包括免疫细胞最佳抗原摄取的纳米颗粒大小，在室温下多年的稳定性，以及安全地将病毒转基因表达携带到免疫细胞中而不会有病毒重建的风险。鸡舍病毒被选为自组装病毒，因为它也具有许多理想的特性。它不是哺乳动物的病原体，因此将表现出更好的安全性。与目前用于疫苗开发的RNA病毒相比，它在哺乳动物细胞类型中具有非常高的抗原表达水平，并极大地减少了细胞死亡。鸡舍病毒以前没有被用于疫苗开发，因为天然的衣壳包装特性极大地限制了转基因插入的大小，而且病毒颗粒仍然需要在细胞培养中制造。这一应用的预期结果将是克服天然衣壳包装的局限性，并创造出基于鸡舍病毒基因组的自组装RNA纳米颗粒，具有高水平的抗原表达。我们的研究意义重大，因为它将实现我们的长期目标 创造具有成本效益、安全和强大的RNA疫苗，这是创新的，因为我们将验证任何具有理想特性的RNA病毒都可以用于纳米颗粒自组装，并加快RNA疫苗开发供人类使用的步伐。 公共卫生相关性：通过合理的设计改善疫苗特性是研究科学家和医学界的高度优先事项，因为疫苗在改善公共卫生方面发挥着关键作用。这项R03应用寻求通过测试一种新型的自组装RNA疫苗纳米颗粒来改善疫苗特性，这种纳米颗粒与目前批准的疫苗不同，制造迅速且容易，在室温下稳定，并且可以低成本地制造。我们的目标是实施可用于临床的改进的基于RNA的疫苗，这将对提高安全性、卓越的免疫激活和对传染病病原体和癌症的保护产生积极影响，并可能促进未来新疫苗的广泛采用。

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. PUBLIC HEALTH RELEVANCE: Improving vaccine characteristics by rational design is a high priority of research scientists and the medical community, because vaccines play a critical role in improving public health. This R03 application seeks to improve vaccine characteristics by testing a new type of self-assembling RNA vaccine nanoparticle, which unlike currently approved vaccines, is rapidly and easily made, is stable at room temperature, and can be made cost effectively. Our goal is to implement improved RNA based vaccines that can be used clinically, and this will have a positive impact on improved safety, superior immune activation and protection against infectious disease pathogens and cancer, and may facilitate widespread adoption of new vaccines in the future.