Protocol to express anti-viral nanobodies and antigenic proteins anchored on the bacterial cell surface through the engineering of probiotic Escherichia coli Nissle 1917

Here, we present a protocol to express anti-viral nanobodies or antigenic proteins anchored on the bacterial cell surface through the engineering of probiotic E. coli Nissle 1917 (EcN). We describe steps for bacterial transformation, protein validation, outer membrane vesicle (OMV) isolation, and in vivo administration. We then detail procedures for immunological assessment through ELISA, neutralization assay, and flow cytometry analysis. This protocol can be adapted for different therapeutic targets beyond SARS-CoV-2. For complete details on the use and execution of this protocol, please refer to Kamble et al. 1 • Steps for genetic engineering of probiotic E. coli Nissle 1917 for surface display • Instructions for outer membrane vesicle (OMV) isolation for nanobody transport • Oral dosing guidance for engineered EcN to safely induce targeted immune response • Procedure for functional assessment of pseudovirus neutralization post treatment Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol to express anti-viral nanobodies or antigenic proteins anchored on the bacterial cell surface through the engineering of probiotic E. coli Nissle 1917 (EcN). We describe steps for bacterial transformation, protein validation, outer membrane vesicle (OMV) isolation, and in vivo administration. We then detail procedures for immunological assessment through ELISA, neutralization assay, and flow cytometry analysis. This protocol can be adapted for different therapeutic targets beyond SARS-CoV-2.