A modular platform for on-demand vaccine self-assembly enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

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 created a universal approach called AddVax (avidin-based dock-and-display for vaccine antigen cross (x)-linking) whereby virtually any antigen that is amenable to biotinylation can be linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen receptor (SNARE) comprised of an outer membrane scaffold protein fused to a member of the avidin family. 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. Overall, these results demonstrate AddVax as a modular platform for rapid self-assembly of antigen-studded OMVs with the potential to accelerate vaccine generation, respond rapidly to pathogen threats in humans and animals, and simplify vaccine stockpiling.

Biological Nanoparticles in Vaccine Development

“…In comparison to whole-pathogen vaccines, such as live-attenuated and inactivated vaccines, biologically-derived nanoparticles have the potential for more favorable safety profiles, as they are non-infectious and unable to replicate; therefore, incomplete inactivation or pathogenic reversion is not a concern. Genetic modification or chemical linkage allows surface display of many heterologous antigens, and novel coupling strategies such as sortase-mediated conjugation ( Thérien et al, 2017 ) and Addvax ( Weyant et al, 2021 ) provide additional flexibility to further modify these highly versatile platforms for use against various diseases.…”

“…The presentation of antigen on the surface of OMVs presents its own level of difficulty, because most proteins do not naturally localize to the outer membrane. Therefore, antigen fusion with outer membrane proteins or autotransporters, such as cytolysin A (ClyA) ( Chen et al, 2010 ; Rappazzo et al, 2016 ), Sec-dependent signal peptide (spPelB) ( Weyant et al, 2021 ), and hemoglobin protease ( Daleke-Schermerhorn et al, 2014 ), is commonly used to display antigen on the OMV outer surface. Chemical conjugation of the antigen to the exterior of the OMV can also be used to present antigen (or additional adjuvant) on the surface of the OMV, as was done for the commercially available vaccine PedVaxHIB ® .…”

“…Due to the inherent flexibility and capability of the OMV platform, there is interest in expressing heterologous antigens from different pathogens on the same OMV to create novel combined vaccines ( König et al, 2021 ). One recently reported strategy is a clever universal approach termed “Addvax,” avidin-based dock-and-display for vaccine antigen cross-linking ( Weyant et al, 2021 ). The Addvax approach utilizes an avidin binding moiety fused with an outer membrane protein so that the biotin binding domain may be expressed on the OMV exterior ( Figure 8 ).…”

“… The Addvax approach for external modification of outer membrane vesicles (OMVs). The Addvax approach, developed by Weyant et al (2021) , consists of a scaffold protein inserted into the OMV membrane that is fused to a biotin-binding domain, such as avidin. These avidin-decorated OMVs can then be reacted with any biotinylated compound, including proteins, peptides, carbohydrates, haptens, lipids, and nucleic acids, for conjugation to the OMV.…”