Outer Membrane Vesicles (OMVs) are a type of bacteria-derived membrane nanoparticles that range in size between 20 to 250 nm. OMVs were first discovered in the 1960s and are known to form naturally through a membrane blebbing process whereby a portion of the Gram-negative bacterial envelope pinches off, releasing nanovesicles. Although some control of OMVs’ specific cargo is suspected, their content is quite heterogeneous, having a variety of periplasmic components and even inner membrane and cytoplasmic molecules (Liu et al. 2022).
These nanoparticles are known to have various functional roles relevant to bacterial survival and performance, including support of cellular interactions, adaptation to environmental conditions, and delivery of virulence factors, among others (Schwechheimer and Kuehn, 2015). Of significance, in the context of bacterial infections, OMVs stimulate immune responses by virtue of their content, having several pathogen-associated molecular patterns (PAMPs), such as lipoprotein, lipopolysaccharide, nucleic acids, and peptidoglycan. Additionally, their capacity to serve as a delivery vehicle for specific antigens of interest has propelled their development for therapeutic applications, such as bacteria- and cancer vaccines.
Outer-Membrane Vesicles. Bacteria (Gram-negative and some Gram-positive) are known to produce membrane vesicles via different pathways. OMVs are derived from Gram-negative bacteria and may be formed through different mechanisms, including membrane blebbing. Therefore, OMVs are decorated with a range of molecules and contain heterogeneous cargo corresponding to the bacterial cell of origin. Retrieved without modifications from Liu et al. 2022. http://creativecommons.org/licenses/by/4.0/
Because OMVs contain PAMPs, they enable the combined activation of innate immune responses (e.g., Toll-like receptors or TLRs) and induce strong humoral and cell-mediated immunity. In addition, their small size enables these nanoparticles to access lymphatics for uptake by antigen-presenting cells. Other benefits, such as ease of manufacturing workflow, relatively low cost, and flexible antigen delivery, have stimulated interest in engineering and using OMVs as vehicles for bacteria-, viruses-, parasites-, and tumor-derived antigens.
Activation of Toll-like Receptors by OMV components. Retrieved without modifications from Mancini et al. 2020. http://creativecommons.org/licenses/by/4.0/
To effectively leverage OMVs for vaccine development, a commonly adopted engineering approach targets the bacterial blebbing process, enhancing OMV shedding. This high-yield hyperblebbing approach reduces cost and has been used in producing several OMV vaccines from pathogens, such as Salmonella, Neisseria meningitides, and Shigella (Mancini et al. 2020).
Additionally, while innate immunogenicity is a desirable and valuable inherent property of OMVs, engineering strategies are implemented to prevent adverse reactions. For example, an approach to reduce OMVs’ reactogenicity relies on modifying the Lipid A molecule, which constitutes the endotoxic component of lipopolysaccharides (Rossi et al. 2016).
In another approach, Flagellin, a protein in flagellated pathogens such as E. coli and Salmonella, has also been targeted for elimination in order to prevent unwanted inflammatory responses (Liu et al. 2016). Lastly, through engineering approaches, it’s possible for OMVs to carry specific antigens and even multiple antigens of interest on their surface, which enables their presentation in a relevant context and native conformation (Kashyap et al. 2022).
Despite the great potential of OMVs as an effective and safe vaccine platform, achieving consistent surface antigen expression remains challenging. Attaining optimal expression and surface targeting of recombinant proteins in bacteria could be especially difficult for large and complex antigens. Recognizing this significant limitation, Dr. Matthew DeLisa and colleagues at Cornell University, NY, developed a new strategy to more efficiently and consistently display different exogenous antigens on the surface of OMVs (Weyant et al. 2023). Their new platform, AvidVax, is enabled by expressing a “Synthetic Antigen-Binding Protein,” which Weyant and colleagues call “SNAP.”
For expression of SNAP constructs in OMVs, eMA fusions to ClyA, Lpp-OmpA, and the membrane-associated transporter domains of the autotransporters Int, Hbp, Ag43, and IgAP were codon-optimized for E. coli expression, synthesized, and cloned into plasmid pBAD2481 between EcoRI and SphI restriction sites with an NdeI site at the start codon by GenScript.” Weyant et al. 2023
SNAP consists of two main components, an outer membrane targeting domain and a biotin-binding domain, which is exposed on the bacterial surface and consequently on the generated OMVs. Therefore, SNAP-OMVs bypass some of the hurdles of recombinant protein expression in bacteria and provide a flexible platform for displaying a wide variety of biotinylated antigen types.
Synthetic Antigen-Binding Protein “SNAP”-OMV platform for antigen display. Recently developed by Matthew DeLisa’s team at Cornell University, the SNAP-OMV or AvidVax platform leverages avidin-biotin interactions for a universal antigen display strategy ideal for vaccine development. Figure 1, panel a, retrieved from Weyant et al. 2023.http://creativecommons.org/licenses/by/4.0/
Significantly, the platform enables the display of exogenous proteins carrying relevant post-translational modifications (e.g., CHO-expression systems). This is a definite advantage over approaches combining bacterial protein expression and OMV production, as such proteins often lack eukaryotic-like post-translational modifications.
Weyant and colleagues found that another advantage of the AvidVax platform is that it provides a good level of control over OMV antigen loading density, which is not as feasible with conventional bacterial recombinant expression. Lastly, the team showed that various antigens, such as biotinylated-GFP and biotinylated-linear peptides, docked onto SNAP-OMVs successfully elicited robust immune responses in mice. Simply mixing non-biotinylated-GFP with SNAP-OMVs was not nearly as effective in inducing IgG responses. Similarly, docking a melanoma neoantigen peptide onto SNAP-OMVs enabled a strong IgG response.
Overall, the newly developed AvidVax platform promises to facilitate the generation of OMV-based vaccines through a more straightforward and universal antigen docking workflow. This approach would solve the shortcomings of bacterial expression systems and reduce excessive troubleshooting needed to optimize the expression of new antigens in conventionally generated OMVs.
