On-Demand Vaccine Production via Dock-and-Display of Biotinylated Antigens on Bacterial Extracellular Vesicles

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.