ENGINEERING ESCHERICHIA COLI OUTER MEMBRANE VESICLES AND OUTER MEMBRANE PROTEINS FOR SUBUNIT ANTIGEN DELIVERY AND VACCINE DESIGN

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Vaccines have been an incredible tool that has prevented infectious disease and saved countless of lives. Recent advances have led to the development of subunit vaccines, a more specific class of vaccines that contain only the antigenic components of the pathogen. However, these induce weaker immune responses compared to whole pathogen vaccines, requiring the use of adjuvants to boost the immune response. Bacterial outer membrane vesicles (OMVs) are not only natural adjuvants, but they can be engineered to present desired subunit antigens to immune cells, a technology backed by extensive research exploring their capabilities as a vaccine platform. We used OMVs derived from engineered ClearColi (CC), a non-pyrogenic E. coli strain previously engineered to hypervesiculate, to deliver pattern recognition receptor (PRR) agonists – particularly Toll-like receptor (TLR) agonists – that induce innate immunity in a tailored manner to mimic immune induction caused by pathogens. This was achieved by displaying biotin-binding domains (BBDs) on the surface of bacteria and docking biotinylated PRR agonists onto the BBD OMVs. We were able to successfully dock biotinylated poly(I:C) (TLR3), CpG oligonucleotides (TLR9), and flagellin (TLR5) onto BBD OMVs and demonstrate some respective TLR activity. We also used OMVs to deliver immunogenic regions of the SARS-CoV-2 nucleocapsid (N) protein. While immunization did not protect mice from a lethal SARS-CoV-2 challenge, immunization did yield high levels of long-term anti-antigen IgG antibodies with a balanced Th response. Additionally, we examined the bacterial protein cytolysin A (ClyA), a membrane protein often used for decorating OMVs, for their potential ability to deliver subunit antigens and induce immune responses. We found that ClyA fused with green fluorescent protein (GFP) did not form pores or kill cells at the same rate of ClyA while still inducing comparable immune cell response in vitro. An influenza challenge was conducted using a multimeric construct of influenza A matrix protein 2 (M2e4xHet) as the model antigen. The immunizations did not protect the mice, but ClyA-M2e4xHet protein induced comparable anti-M2e IgG titers comparable to those induced by ClyA-M2e4xHet OMVs. This research expands the capabilities of OMV vaccines, and the potential of new adjuvants derived from bacterial components.
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DeLisa, Matthew