Escherichia coli Nissle 1917 efficiently expresses the RBD domain of SARS-CoV-2 spike protein without codon optimization.

Bacterial outer membrane vesicles (OMVs) represent a promising and versatile platform for vaccine delivery. Their inherent self-adjuvant properties and the ability to be adorned with a wide range of heterogeneous antigens position them as a powerful tool in the fight against infectious diseases. Escherichia coli Nissle 1917 (EcN) stands out as a highly valuable probiotic strain because of its long history of safe use and proven clinical benefits in humans. The EcN strain was genetically engineered to derive OMVs displaying receptor binding domain (RBD) of SARS-CoV-2 spike protein on their surface. Although some research groups have previously expressed the SARS-CoV-2 viral spike protein or the RBD in E. coli, particularly in EcN, this study shows a maiden effort to utilize the gene encoding native RBD. The probiotic EcN exhibited a significant level of native RBD expression, demonstrating a more efficient codon usage pattern compared to commonly used bacterial expression systems such as BL21, and DH5α. EcN was engineered to display the native form of viral RBD on the surface using the Lpp-OmpA system. Cell fractionation studies clearly indicated the presence of RBD in the membrane fraction. OMVs displaying RBD on their surface were isolated using ultracentrifugation and the presence of RBD in the OMVs was confirmed by western blot followed by immunofluorescence analyses. Due to their preferential uptake by antigen presenting cells, OMVs derived from EcN bearing native form of RBD hold promise as a potential COVID-19 vaccine candidate.