Cell-free synthesis of influenza virus-like particles (VLPs) as prototyping platform for vaccine development and variant characterization

摘要 缺乏预防性和低成本的流感疫苗，生产速度缓慢 让我们对下一次全球大流行毫无准备。重组病毒样颗粒 (VLP)疫苗为我们提供了解决这些困难的独特机会，但它们的 重组合成策略仅限于细胞培养生产，这是有挑战的。 例如有限的吞吐量和获得的产品的体系结构异构性。在这个项目中， 我们的目标是将小麦胚芽无细胞蛋白表达平台与具有活性的微生物体相结合 不同来源(包括人类)合成糖基化流感病毒样颗粒(VLP)。 这些颗粒将进一步接受详细的形态和成分检查，使用 电子显微镜和天然质谱学来了解所获得的结构 并了解其组装和组成变化的机制。高- 分辨率断层扫描将被用来确定具有最大抗原的最佳VLP候选 入住率，从而最大限度地提高疫苗效率。我们假设这种方法将是 适用于稳定和精简的流感VLP，并将提供更多 灵活性和对其架构异构性的控制，并最终作为快速 流感疫苗研发的原型平台。目前的项目也将通知我们 关于使用这种方法进行病毒学和糖蛋白合成的效用。

SUMMARY The absence of prophylactic and low-cost influenza vaccines along with slow production rate makes us unprepared for the next pandemic outbreak worldwide. Recombinant virus-like particles (VLPs) vaccines provide us with unique opportunities to address these difficulties, but their recombinant synthesis strategies are limited to cell culture production, which has its challenges such as limited throughput and architectural heterogeneity of the obtained products. In this project, we aim to combine wheat germ cell-free protein expression platform with active microsomes of different origins (including human) to synthesize glycosylated influenza virus-like particles (VLPs). These particles will further undergo detailed morphological and compositional examination using electron microscopy and native mass spectrometry to understand the architecture of obtained products and understand the mechanisms of their assembly and composition variability. High- resolution tomography will be utilized to determine the best VLP candidates with maximal antigen occupancy and thus maximal vaccine efficiency. We hypothesize that this approach will be suitable for a stable and streamlined generation of Influenza VLPs and will offer much more flexibility and control over their architectural heterogeneity and eventually serve as a rapid prototyping platform for influenza vaccine development. The current project will also inform us about the utility of using such approaches for virology and for glycoprotein synthesis in general.