LAUSR

Significance Thanks to their potent built-in adjuvanticity, bacterial outer membrane vesicles (OMVs) represent an attractive vaccine platform. However, their full-blown exploitation relies on the availability of efficient engineering strategies. This work provides strong experimental evidence that OMVs can be successfully decorated with heterologous antigens by channeling them to the lipoprotein transport machinery. Not only did lipidated antigens accumulate in the vesicular compartment at high levels but they also interfered with the acylation process of lipid A, thus substantially reducing the lipopolysaccharide-mediated reactogenicity, a major hurdle for vaccine applications. The approach was validated with 5 Staphylococcus aureus antigens, and mice immunized with engineered OMVs were completely protected against S. aureus challenge, paving the way for development of vaccines against this important pathogen. Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigen-specific antibody titers when administered to mice in quantities as low as 0.2 μg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo-OMVs a promising vaccine candidate to be tested in clinics.