The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

Outer membrane vesicles (OMVs) are nanoscale proteoliposomes that are ubiquitously secreted by Gram-negative bacteria. Interest in these bioparticles has escalated over the years, leading to discoveries regarding their composition, production, and vaccine potential. Given that many steps in vesicle biogenesis are ‘engineerable,’ it is now possible to tailor OMVs for specific applications. Such tailoring involves modifying the OMV-producing bacterium through protein, pathway, or genome engineering in a manner that specifically alters the final OMV product. For example, targeted deletion or upregulation of genes associated with the cell envelope can modulate vesicle production or remodel the composition of vesicle components such as lipopolysaccharide. Likewise, bacteria can be reprogrammed to incorporate heterologously expressed proteins into either the membrane or lumenal compartment of OMVs. We anticipate that further research in the field of OMV engineering will enable continued design and biosynthesis of specialized vesicles for numerous biotechnological purposes ranging from the delivery of vaccines to the deconstruction of cellulosic substrates.

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