Modular assembly of bacterial outer membrane vesicle-based vaccines and therapeutics

Abstract

Engineered outer membrane vesicles (OMVs) derived from laboratory strains of bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. As mimics of the bacterial cell surface, OMVs offer a molecularly-defined architecture for programming repetitive, high-density display of heterologous antigens in conformations that elicit strong B and T cell immune responses. However, antigen display on the surface of 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. To address this shortcoming, we demonstrate attachment of antigens utilizing the strong interactions between cohesin and dockerin protein domains, spontaneous isopeptide bond formation between the engineered domains SpyCatcher and SpyTag (or SnoopCatcher and SnoopTag), and a universal approach called AddVax (avidin-based dock-and-display for vaccine antigen cross (x)-linking), all of which constitute the linkage of tagged antigens to the exterior of OMVs that are remodeled to display multiple copies of a synthetic antigen receptor (SNARE). With AddVax, we show that SNARE-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 were injected in wild-type BALB/c mice, strong antigen-specific antibody responses were observed that depended on the physical coupling between the antigen and SNARE-OMV delivery vehicle. Next, we use AddVax to rapidly generate synthesized peptide antigen vaccines against a cancer model and SARS-CoV-2, with the latter generating potential neutralizing antibodies against the virus. Extending beyond vaccines, we then demonstrate modular attachment of antibodies to OMVs using general antibody-binding domains for cancer cell targeting and immune modulation, and we complete preliminary work on the combined incorporation of chemotherapeutic drugs and attachment of functional cancer-targeting antibodies on OMVs. Overall, our work on modular OMV designs has the potential to accelerate vaccine generation and simplify vaccine stockpiling, and it lays a foundation for OMV-based cell targeting and drug delivery.