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Recombinant, Escherichia coli-derived outer membrane vesicles (rOMVs), which display heterologous protein subunits, have potential as a vaccine adjuvant platform. One drawback to rOMVs is their lipopolysaccharide (LPS) content, limiting their translatability to the clinic due to potential adverse effects. Here, we explore a unique rOMV construct with structurally remodeled lipids containing only the lipid IVa portion of LPS, which does not stimulate human TLR4. The rOMVs are derived from a genetically engineered B strain of E. coli, ClearColi, which produces lipid IVa, and which was further engineered in our laboratory to hypervesiculate and make rOMVs. We report that rOMVs derived from this lipid IVa strain have substantially attenuated pyrogenicity yet retain high levels of immunogenicity, promote dendritic cell maturation, and generate a balanced Th1/Th2 humoral response. Additionally, an influenza A virus matrix 2 protein-based antigen displayed on these rOMVs resulted in 100% survival against a lethal challenge with two influenza A virus strains (H1N1 and H3N2) in mice with different genetic backgrounds (BALB/c, C57BL/6, and DBA/2J). Additionally, a two-log reduction of lung viral titer was achieved in a ferret model of influenza infection with human pandemic H1N1. The rOMVs reported herein represent a potentially safe and simple subunit vaccine delivery platform., (Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.)

Currently approved influenza vaccines predominantly protect through antibodies directed against the highly variable glycoprotein hemagglutinin (HA), necessitating annual redesign and formulation based on epidemiological prediction of predominant circulating strains. More conserved influenza protein sequences, such as the ectodomain of the influenza M2 protein, or M2e, show promise as a component of a universal influenza A vaccine, but require a Th1-biased immune response for activity. Recently, recombinant, bacterially derived outer membrane vesicles (OMVs) demonstrated potential as a platform to promote a Th1-biased immune response to subunit antigens. Here, we engineer three M2e-OMV vaccines and show that all elicit strong IgG titers, with high IgG2a:IgG1 ratios, in BALB/c mice. Additionally, the administration of one M2e-OMV construct containing tandem heterologous M2e peptides (M2e4xHet-OMV) resulted in 100% survival against lethal doses of the mouse-adapted H1N1 influenza strain PR8. Passive transfer of antibodies from M2e4xHet-OMV vaccinated mice to unvaccinated mice also resulted in 100% survival to challenge, indicating that protection is driven largely via antibody-mediated immunity. The potential mechanism through which M2e-OMVs initiated the immune response was explored and it was found that the constructs triggered TLR1/2, TLR4, and TLR5. Our data indicate that OMVs have potential as a platform for influenza A vaccine development due to their unique adjuvant profile and intrinsic pathogen-mimetic nature., (Copyright © 2016 Elsevier Ltd. All rights reserved.)