Abstract
Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines.
Highlights
More than 60 years ago, it was accidentally discovered that Gram-negative bacteria release Outer Membrane Vesicles (OMVs) (Bishop & Work, 1965; Chatterjee & Das, 1967)
Thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes-specific antibody titers and high frequencies of epitope-specific IFN-γ-producing CD8+ T cells
A total of 128 sequences were identified corresponding to 81 unique E. coli proteins. 2-DE-mass spectrometry analysis (MS) was coupled to bioinformatics analysis of the E. coli BL21(DE3) genome aimed at predicting all periplasmic and outer membrane proteins
Summary
More than 60 years ago, it was accidentally discovered that Gram-negative bacteria release Outer Membrane Vesicles (OMVs) (Bishop & Work, 1965; Chatterjee & Das, 1967). OMVs are closed spheroid particles of heterogeneous size, 50–300 nm in diameter, generated through a ‘budding out’ of the bacterial outer membrane. They represent a distinct secretory pathway with a multitude of functions, including inter and intra species cell-to-cell cross-talk, biofilm formation, genetic transformation, defence against host immune responses, and toxin and virulence factor delivery to host cells (Ellis & Kuehn, 2010; Kulp & Kuehn, 2010). OMV-based vaccines have already reached the market (Ladhani et al, 2016; Serruto et al, 2012), while others are in advanced clinical phases (Gerke et al, 2015; Rossi et al, 2016)
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