Proteome‐minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform

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.

Strategies to Tackle Antimicrobial Resistance: The Example of Escherichia coli and Pseudomonas aeruginosa

“…Nevertheless, the high protein identity shared between E. coli species has raised concerns that E. coli OMV may induce an immune response against the commensal population [ 134 , 135 ]. Recently synthetic biology has been used to remove up to fifty-nine dispensable endogenous E. coli proteins that could potentially enhance the antigen specific immune response and at the same time they may theoretically reduce the cross recognition with natural flora [ 136 ]. Although, the manufacturing downstream process required for OMV production is potentially cheap and relatively unsophisticated, current bottlenecks to develop OMV vaccine candidate for Pseudomonas at the preclinical level, are the yields and the detoxification strategies.…”

Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine

“…The copyright holder for this preprint this version posted July 13, 2021. ; https://doi.org/10.1101/2021.07.12.452027 doi: bioRxiv preprint OMVs from BL21(DE3)Δ60(pET-FhuD2-RBM438-509), BL21(DE3) Δ60(pET-FhuD2-RBM438-462), BL21(DE3)Δ60(pET-FhuD2-RBM467-509) and BL21(DE3)Δ60(pET-FhuD2-RBM-BV438-509), were purified in an EZ control bioreactor (Applikon Biotechnology, Schiedam, Netherlands) as previously described (Zanella et al, 2021). Cultures were started at an OD600 of 0.1 and grown in LB medium at 30°C, pH 6.8 (±0.2), dO2 > 30%, 280-500 rpm until OD600=0.5, then the temperature was lowered to 25°C.…”

“…Patient-derived monoclonal antibodies (mAbs) binding epitopes in these chains of the RBD potently neutralize virus cell entry in vitro and are currently in clinical use or in advanced clinical development 18 . In the attempt of producing a vaccine eliciting neutralizing immunity against SARS-CoV-2, we generated OMVs decorated with the polypeptides derived from the RBM, fused with FhuD2, a Staphylococcus aureus lipoprotein shown to efficiently deliver heterologous protein domains to the E. coli outer membrane and to the vesicular compartment 10 .…”

“…To this end, the nucleotide sequence coding for 5 (RBM438-462) and 6 (RBM467-509) strands or a combination of the two (RBM438-509) were fused to the 3' end of the sequence encoding FhuD2 in plasmid pET-FhuD2 12 , thus generating the fusion proteins FhuD2-RBMs (Figure 1B). The resulting plasmids were used to transform E. coli60, a hyper-vesiculating E. coli BL21(DE3) derivative recently created in our laboratories 10 .…”

“…Since clinical evidence demonstrates that an accelerated induction of a Th1 cell response is associated with less severe cases of COVID-19 13,14 and that convalescent individuals develop strong memory CD4+ and CD8+ T cells 15 , the ability of OMVs to trigger Th1 represents a desired feature. Crucially, in addition to the simple and cost-effective setup required to produce and purify OMVs, the antigen-decorated vesicles are extremely stable for long-term storage at room temperature, making it a convenient vaccine to distribute all over the world 10 .…”