mRNA COVID-19 Vaccines Delivered with Plant Virus/Polymer Devices

总结 为全球人口快速开发、分发和接种COVID-19疫苗 是遏制这一流行病和今后再次发生的最有效办法。疫苗接种必须进行 在世界上资源丰富和资源贫乏的地区，广泛地说，这是最有效的。我们提出了一种疫苗 基于植物病毒/聚合物共混物的递送装置。拟议的制造计划将允许 及时的大规模生产;最重要的是，疫苗输送装置在寒冷的外部是稳定的， 链，只需要单剂量给药。我们建议包装一种mRNA疫苗， 来源于植物的病毒样颗粒（VLP）中的SARS-CoV-2S蛋白（及其结构域） 烟草花叶病毒（TMV）。TMV可以在体外或植物中重组以携带异源RNA 并且已经证明了其在mRNA疫苗递送中的效用。植物VLP对免疫系统高度可见 系统，这些疫苗递送剂通过经由先天免疫系统的多个受体的信号传导来增强免疫力。 因此，VLP充当载体和明确定义的佐剂。此外，TMV提供高 热稳定性-因此克服了冷链要求;但更重要的是， 候选疫苗提供了高度的稳定性，它们可以承受熔融加工的严格条件。 因此，允许配制缓释植入物或微针贴剂， 单次给药。我们将生产载有mRNA的TMV，然后应用熔融加工工具， 制造缓释植入物。我们开发了一种微型挤出机，用于生产蛋白质/聚合物 混合物，允许将植物病毒疫苗配制成缓释装置。我们已经 证明了植物病毒疫苗可以承受严格的熔融加工。在…的任期内， 在R21中，我们将SARS-CoV-2衍生的mRNA盒包装到TMV中，并验证mRNA递送和蛋白质表达。 在免疫细胞中的表达（Aim 1）。我们将制定缓释VLP/聚合物（PLGA）混合物，并确定 VLP（载有mRNA的TMV）从VLP/聚合物共混物的释放速率（目的2）。我们将测定抗体滴度 和细胞抗SARS-CoV-2反应的mRNA-loaded TMV疫苗候选人在小鼠中，并比较 可溶性（初免-加强）与植入候选疫苗（单次给药）。我们将决定 疫苗接种产生中和SARS-CoV-2血清和细胞应答;体外和体内SARD-CoV-2 将进行攻毒研究（目标3）。我们的技术带来了独特的属性， 需要冷链进行配送，并且能够在单次给药时进行疫苗接种。单- 接种疫苗也可以使牲畜接种疫苗，这可能是向前迈出的一步，以满足 一个健康倡议的目标，以防止未来的爆发。

Summary The rapid development, distribution and administration of a COVID-19 vaccine to the global population is the most effective approach to quell this pandemic and future reoccurrence. Vaccination must occur broadly both in well-resourced and resource-poor areas of the world to be most effective. We propose a vaccine delivery device based on a plant virus/polymer blend. The manufacturing scheme proposed would allow for timely production at scale; most importantly, the vaccine delivery device would be stable outside of the cold- chain and only require a single-dose administration. We propose to package an mRNA vaccine encoding the SARS-CoV-2 S protein (and domains thereof) in virus-like particles (VLPs) derived from the plant virus Tobacco mosaic virus (TMV). TMV can be reconstituted in vitro or in plants to carry heterologous RNA and its utility in mRNA vaccine delivery has been demonstrated. Plant VLPs are highly visible to the immune system, these vaccine delivery agents boost immunity through signaling via multiple receptors of the innate immune system; therefore, the VLP serves as carrier and well-defined adjuvant. Furthermore, TMV offers high thermal stability – therefore overcoming cold chain requirements; but more importantly the mRNA-laden TMV vaccine candidates offer such a high degree of stability that they can withstand the rigors of melt-processed device manufacture, therefore allowing the formulation of slow-release implants or microneedle patches for single-administration. We will produce mRNA-laden TMV and then apply melt-processing tools manufacture the slow-release implants. We have developed a microextruder to manufacture protein/polymer blends that allows for the formulation of plant virus-based vaccines into slow-release devices. We have already demonstrated that plant virus-based vaccines can withstand the rigor of melt-processing. Under the tenure of this R21, we will package SARS-CoV-2 derived mRNA cassettes into TMV and verify mRNA delivery and protein expression in immune cells (Aim 1). We will formulate slow-release VLP/polymer (PLGA) blends and determine VLP (mRNA-laden TMV) release rates from the VLP/polymer blends (Aim 2). We will determine antibody titers and cellular anti-SARS-CoV-2 responses of the mRNA-laden TMV vaccine candidates in mice and compare soluble (prime-boost) vs. implant vaccine candidates (single administration). We will determine whether vaccination yields neutralizing SARS-CoV-2 sera and cellular responses; in vitro and in vivo SARD-CoV-2 challenge studies will be performed (Aim 3). Our technology brings unique attributes in that it does not require the cold chain for distribution and would enable vaccination upon single administration. Single- administration vaccines could also enable vaccination of livestock and this could be a step forward to meet the goals of the One Health Initiative to prevent future outbreaks.