Development of a SARS-CoV-2 neutralization assay based on a pseudotyped virus using a HIV system

Regarding the extensive global attention to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that constitutes an international public health emergency, pseudovirus neutralization assays have been widely applied due to their advantages of being able to be conducted in biosafety level 2 laboratories and having a high safety factor. In this study, by adding a blue fluorescent protein (AmCyan) gene to the HIV system pSG3-oenv backbone plasmid HpaI and truncating the C-terminal 21 amino acids of the SARS-CoV-2 spike protein (S), high-titer SARS-CoV-2-Sdel21-AmCyan fluorescent pseudovirus was successfully packaged. The fluorescent pseudovirus was used to establish a neutralization assay in a 96-well plate using 293T cells stably transfected with the AF cells. Then, parameters such as the ratio of backbone and membrane plasmid, sensitive cells, inoculation of cells and virus, as well as incubation and detection time were optimized. The pseudovirus neutralization assay demonstrated high accuracy, sensitivity, repeatability, and a strong correlation with the luminescent pseudovirus neutralization assay. Additionally, we scaled up the neutralizing antibody determination method by increasing the plate size from 96 wells to 384 wells. We have established a robust fluorescent pseudotyped virus neutralization assay for SARS-CoV-2 using the HIV system, providing a foundation for serum neutralization antibody detection, monoclonal antibody screening, and vaccine development. Regarding the extensive global attention to SARS-CoV-2 that constitutes an international public health emergency, pseudovirus neutralization assays have been widely applied due to their advantages of being able to be conducted in biosafety level 2 laboratories and having a high safety factor. In this study, by adding a blue fluorescent protein (AmCyan) gene to the HIV system pSG3-oenv backbone plasmid HpaI and truncating the C-terminal 21 amino acids of the SARS-CoV-2 spike protein (S), high-titer SARS-CoV-2-Sdel21-AmCyan fluorescent pseudovirus was successfully packaged. The fluorescent pseudovirus was used to establish a neutralization assay in a 96-well plate using 293T cells stably transfected with the ACE2 receptor and Furin protease (AF cells). Then, parameters such as the ratio of backbone and membrane plasmid, sensitive cells, cell addition, virus inoculation, incubation time, and detection time were optimized. The pseudovirus neutralization assay showed good accuracy, sensitivity, repeatability, and a good correlation with the luminescent pseudovirus neutralization assay. Additionally, we further developed a high-throughput and automated 384-well plate neutralization assay based on the 96-well plate. In conclusion, we have established a robust fluorescent pseudovirus neutralization assay for SARS-CoV-2 using the HIV system, providing a foundation for serum neutralization antibody detection, monoclonal antibody screening, and vaccine development. image