Identifying antibody escape mutations using the evolutionary capacity of trVLP-ASV

The continuous evolution of SARS-CoV-2 poses global health challenges. A safe, rapid, and versatile method for assessing functions of Spike protein mutations in ACE2 receptor binding and immune evasion would be highly valuable. To address this, we engineered a transcription- and replication-competent virus-like particle (trVLP) derived from the Sindbis virus, pseudotyped with the SARS-CoV-2 receptor binding domain (RBD). This trVLP exclusively propagates in BHK-21 cell engineered to express both RNA replicase and human ACE2, providing a controllable, safe model of SARS-CoV-2 RBD-ACE2 interaction mediated virus entry. The system enables characterization of RBD interactions with ACE2 from various mammalian hosts, demonstrating its utility for studying host-virus interactions. By leveraging the evolutionary capability of trVLP mediated by error-prone RNA replication, we screened for RBD variants that evade the antibody-mediated inhibition of cell entry. Together, these findings underscore the utility of the trVLP as a safe, rapid, and flexible platform for dissecting SARS-CoV-2 RBD evolution and identifying key adaptive mutations with implications for surveillance and countermeasure development. Overall design: In this experiment, we applied selection pressure with a range of antibodies, including REGN10987, LY-CoV1404, SA55, and six other antibodies, on the mutant library derived from the trVLP-ASV, enabling the identification of SARS-CoV-2 RBD mutants that can escape antibody neutralization.