Establishment of a pseudovirus neutralization assay based on SARS-CoV-2 S protein incorporated into lentiviral particles

•Scientific question: To establish the reliable and safe measurements of SARS-CoV-2 S pseudotyped virus infection system for entry inhibition and neutralization assays under BSL-2 conditions.•Evidence before this study: The coronavirus disease 2019 (COVID-19) variants are still disrupting the quality of human life globally, even with some success on effective vaccines and drugs targeting the SARS-CoV-2. Previously, researchers had successfully established several type S WT pseudovirus systems, but little was known of the difference between S WT and variants, especially in the neutralization assay.•New findings: We established a new SARS-CoV-2 S pseudovirus system that efficiently high packaging titer in S WT or variants pseudovirus. Furthermore, we used proteases inhibitors E-64d, camostat mesylate and antibodies targeted RBD to compare the inhibition potential of different S variants pseudovirus.•Significance of the study: The established SARS-CoV-2 S pseudovirus system seems to offer a safer, more convenient, and higher throughput way for conducting the SARS-CoV-2 viral entry research. In addition, this pseudovirus neutralization assay could benefit the availability of inhibitors and antibodies against SARS-CoV-2 variants. The coronavirus disease 2019 (COVID-19) is still causing a wide range of infections and deaths due to the high variability of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, it is necessary to establish a reliable and convenient pseudovirus-based neutralization assay to develop drug targeted variants of SARS-CoV-2. Based on the HIV-1 backbone, we generated a high titer luciferase (Luc)-expressing pseudovirus packaging system. Three dominant S mutant substitution pseudovirus were also established and identified compared to wide type in hACE2-overexpressing HEK-293T cells (293T-ACE2 cells). Compared to serine protease inhibitor camostat mesylate, the cysteine protease inhibitor E-64d could significantly block all SARS-CoV-2 mutant S pseudovirus infection in 293T-ACE2 cells. Furthermore, the neutralization ability of two antibodies targeted receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S) was evaluated, which showed different inhibition dose–effect curves among four types of S pseudovirus. Overall, we developed a pseudovirus-based neutralization assay for SARS-CoV-2, which would be readily adapted to SARS-CoV-2 variants for evaluating antibodies.