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.

A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

Microcompartments for Protection and Isolation of Nanoscale DNA Computing Elements.

Physical isolation of molecular computing elements holds the potential for increasing system complexity by enabling the reuse of standardized components and by protecting the components from environmental degradation. However, once elements have been compartmentalized, methods for communicating into these compartments are needed. We report the compartmentalization of steroid-responsive DNA aptamers within giant unilamellar vesicles (GUVs) that are permeable to steroid inputs. Monodisperse GUVs are loaded with aptamers using a microfluidic platform. We demonstrate the target-specific activation of individual aptamers within the GUVs and then load two noninterfering aptamers into the same GUV and demonstrate specific responses to all possible combinations of the two input steroids. Crucially, GUVs prevent the degradation of DNA components by nucleases, providing a potential mechanism for deploying nucleic acid components in vivo. Importantly, our compartments also prevent nonspecific cross-talk between complementary strands, thereby providing a method for parallel execution of cross-reacting molecular logic components. Thus, we provide a mechanism for spatially organizing molecular computing elements, which will increase system modularity by allowing standardized components to be reused.