Abstract

Bacterial Outer Membrane Vesicles (OMVs) are naturally produced by all Gram-negative bacteria and play a key role in their biology and pathogenesis. Over the last few years, OMVs have become an increasingly attractive vaccine platform for three main reasons. First, they contain several Microbe-Associated-Molecular Patterns (MAMPs), crucial for stimulating innate immunity and promoting adaptive immune responses. Second, they can be easily purified from the culture supernatant, thus making their production process inexpensive and scalable. Third, OMVs can be engineered with foreign antigens. However, the OMV platform requires some optimization for a full-blown exploitation. First, OMVs carry a number of endogenous proteins that would be useful to eliminate to avoid possible interference of immune responses toward the vaccine antigens. Second, OMVs carry abundant quantities of lipopolysaccharide (LPS). LPS is a potent stimulator of the immune system, therefore is essential for OMV adjuvaticity, but such adjuvanticity has to be modulated to avoid reactogenicity. In this study, we have addressed the two issues by creating a strain releasing OMVs with a minimal amount of endogenous proteins and containing a detoxified LPS. In particular, we first developed a CRISPR/Cas9-based genome editing tool which allows the inactivation of any “dispensable” gene in two working days. The efficacy and robustness of this tool was validated on 78 “dispensable genes”. Using our CRISPR/Cas9 protocol, an OMV proteome-minimized E. coli strain, named E. coli BL21(DE3)Δ58, deprived of 58 OMV associated proteins was created. We demonstrated that E. coli BL21(DE3)Δ58 had growth kinetics similar to the progenitor strain and featured a remarkable increase in OMV production. Two additional genes involved in the LPS biosynthetic pathway (msbB and pagP) were subsequently inactivated creating E. coli BL21(DE3)Δ60 which released OMVs with a substantially reduced reactogenicity. The exploitation of the two strains in vaccine applications was finally validated. We successfully engineered E. coli BL21(DE3)Δ58 and E. coli BL21(DE3)Δ60 with several different antigens, demonstrating that such antigens compartmentalized with high efficiency in the OMVs. We also demonstrated that the engineered OMVs from E. coli BL21(DE3)Δ58 and E. coli BL21(DE3)Δ60-derived OMVs elicited high antigen-specific antibody and T cell responses.