Engineering Genome‐Free Bacterial Cells for Effective <scp>SARS</scp> ‐ <scp>COV</scp> ‐2 Neutralisation

The COVID-19 pandemic has caused unparalleled impacts on global social dynamics, healthcare systems and economies, highlighting the urgent need for effective interventions to address current challenges and future pandemic preparedness. This study introduces a novel virus neutralisation platform based on SimCells (~1 μm) and mini-SimCells (100-200 nm), which are chromosome-free and non-replicating bacteria from an LPS-free Escherichia coli strain (ClearColi). SimCells and mini-SimCells were engineered to display nanobodies on their surface, specifically targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein - a critical immunogenic fragment essential for viral entry into host cells. It was demonstrated that nanobody-expressing SimCells achieved over 90% blocking efficiency against synthesised RBD from both the original Wuhan and the B.1.351 (Beta) variant using competitive enzyme-linked immunosorbent assay (ELISA) assay. More importantly, live virus neutralisation assays demonstrated that NB6 nanobody-presenting mini-SimCells effectively neutralised the live SARS-CoV-2 Victoria variant with an IC50 of 2.95 × 10⁹ ± 1.40 × 10⁸ mini-SimCells/mL. Similarly, VE nanobody-presenting mini-SimCells effectively neutralised the B.1.351 (Beta) variant of the SARS-CoV-2 virus with an IC50 of 5.68 × 10⁹ ± 9.94 × 10⁸ mini-SimCells/mL. The mini-SimCells successfully protected Vero cells, a cell line derived from the kidney of an African green monkey, from infection by the live virus of SARS-CoV-2 and its variants. These results suggest that SimCell-based neutralisation offers a promising strategy for the prevention and treatment of SARS-CoV-2, and potentially other viral infections.