(56g) Fusion of Outer Membrane Vesicles: Surface-Display of Different Epitopes on a Single Vesicle

A sustained activation of B-cells, one hallmark of a strong and efficacious vaccine, is highly dependent on the direct crosslinking of the B-cell surface receptors by structures that display epitopes in high density [1]. These mechanistic characteristics of the immune system help inform the design criteria to better engineer coordinated antigen-adjuvant constructs that guide the adaptive immune response. Outer membrane vesicles (OMVs), non-reproductive bacteria-derived spherical nanostructures, integrate antigen and adjuvant into a single construct to help synchronize their interaction with cells of the immune system [2]. The display of antigens and epitopes from specific pathogens on OMVs through recombinant means is now prevalent in academic labs [3]. One challenge to the design of OMV-based vaccines is the display of multiple and different epitopes on the OMV surface. To date, engineered OMV vaccines have showcased surfaces with multiple but identical epitope or group of epitopes. The limitation is rooted in the lack of expression control over bacteria to simultaneously express multiple heterologous proteins as well as lack of control over their surface-display during OMV biogenesis.

To approach this challenge a different way, we considered membrane fusion, a process through which phospholipid bilayers, from identical or different structures, mix, collapse, and ultimately form a single bilayer. Through the fusion of OMVs, it is possible to achieve the surface-display of multiple different epitopes types on a single OMV surface. In this study, we show that fluorescently-labelled OMVs made from engineered Escherichia coli Nissle 1917, described in one of our previous studies [4], are capable of fusing under controllable conditions. Acidic pH and the presence of divalent cations such as Ca²⁺ and Mg²⁺ in the suspension tune the colloidal properties of suspended OMVs as well as modulate OMV aggregation and fusion kinetics. Quantitative measurements of the kinetics of the OMV bulk fusion show that the maximum fusion efficiency is increased with decreasing pH and increasing salt concentration but is fusion-limited at high salt concentrations, where particle aggregation begins to dominate.

[1] W. Liu and Y. H. Chen, “High epitope density in a single protein molecule significantly enhances antigenicity as well as immunogenecity: A novel strategy for modern vaccine development and a preliminary investigation about B cell discrimination of monomeric proteins,” Eur. J. Immunol., vol. 35, no. 2, pp. 505–514, 2005.

[2] L. van der Pol, M. Stork, and P. van der Ley, “Outer membrane vesicles as platform vaccine technology,” Biotechnol. J., vol. 10, no. 11, pp. 1689–1706, 2015.

[3] M. Kaparakis-Liaskos and R. L. Ferrero, “Immune modulation by bacterial outer membrane vesicles,” Nat. Rev. Immunol., vol. 15, no. 6, pp. 375–387, 2015.

[4] C. G. Rappazzo et al., “Recombinant M2e outer membrane vesicle vaccines protect against lethal influenza A challenge in BALB/c mice,” Vaccine, vol. 34, no. 10, pp. 1252–1258, 2016.