Abstract：
Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell\'s outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.
摘要：
源自革兰氏阴性细菌的工程化外膜囊泡(OMV)是开发针对多种病原体的免疫力的有前途的疫苗技术。然而,由于蛋白质表达和定位到细菌宿主细胞外膜的瓶颈，OMV上的抗原展示可能是具有挑战性的控制和高度可变的，特别是对于庞大和复杂的抗原。这里,我们描述了与通用疫苗技术相关的方法，该技术称为AvidVax（基于抗生物素蛋白的疫苗抗原交联），用于在疫苗开发期间在OMV外部快速和简化的抗原组装。AvidVax平台涉及使用多个拷贝的合成抗原结合蛋白（SNAP）重塑OMV表面，它是一种工程融合蛋白，由与生物素结合蛋白连接的外膜支架蛋白组成。所产生的SNAP能够用生物素化亚基抗原的分子多样性阵列有效地装饰OMV，包括球状和膜蛋白，聚糖和糖缀合物，haptens,脂质，核酸,和短肽。我们详细介绍了AvidVax疫苗生产管道中的关键步骤，包括SNAP-OMV的制备和分离，生物素化和疫苗抗原的富集，以及负载抗原的SNAP-OMV的配制和表征。
