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

摘要 向全球人口快速开发、分发和管理新冠肺炎疫苗 是平息这场大流行和未来再次发生的最有效的方法。必须接种疫苗 总的来说，在世界上资源丰富和资源贫乏的地区都是最有效的。我们提出了一种疫苗 基于植物病毒/聚合物混合物的输送装置。建议的制造方案将允许 及时规模化生产；最重要的是，疫苗输送装置在寒冷条件下将保持稳定。 链状，只需要单次给药。我们建议包装一种编码信使核糖核酸疫苗 SARS-CoV-2植物病毒样颗粒中的S蛋白(及其结构域) 病毒烟草花叶病毒(TMV)。烟草花叶病毒可以在体外或在植物体内重组以携带异源RNA 并且它在信使核糖核酸疫苗递送中的有效性已经被证明。植物VLP对免疫者来说是高度可见的 这些疫苗递送剂通过先天的多个受体传递信号来增强免疫力。 免疫系统；因此，VLP作为载体和明确定义的佐剂。此外，TMV提供了更高的 热稳定性--因此克服了冷链要求；但更重要的是，携带mRNA的TMV 候选疫苗具有高度的稳定性，可以经受熔融加工的严酷考验。 器件制造，因此允许配制缓释植入物或微针贴片 单一行政管理。我们将生产携带mRNA的TMV，然后应用熔融加工工具 制造缓释植入物。我们开发了一种用于制造蛋白质/聚合物的微挤出机。 允许将以植物病毒为基础的疫苗配制成缓释装置的混合物。我们已经这么做了 证明了以植物病毒为基础的疫苗可以经受住熔融加工的严格要求。在……任期内 在这个R21中，我们将把SARS-CoV-2衍生的mRNA盒包装成TMV，并验证mRNA的传递和蛋白质 免疫细胞表达(目标1)。我们将配制缓释VLP/聚合物(PLGA)共混物并确定 VLP/聚合物共混物中VLP(携带mRNA的TMV)的释放率(目标2)。我们将测定抗体效价 以及携带mRNA的TMV候选疫苗在小鼠体内的细胞抗SARS-CoV-2免疫应答 可溶性(主要增强)与植入型候选疫苗(单次给药)。我们将确定是否 疫苗接种产生中和SARS-CoV-2血清和细胞反应；体外和体内SARD-CoV-2 将进行挑战研究(目标3)。我们的技术带来了独特的属性，因为它不 需要冷链进行分发，并可在一次接种后接种疫苗。单人- 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 一个卫生倡议的目标，以防止未来的疫情。

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.