2.6 SARS-CoV-2 Omicron BA.5 Pseudovirus Production and Neutralization Assay
Generation of pseudotyped SARS-CoV-2 Omicron BA.5 was performed as previously described.19, 20 Briefly, the backbone plasmid of pNL4-3.Luc.R-E- was cotransfected with the plasmid of pcDNA3.1-SARS-CoV-2 Omicron BA.5-S into HEK-293T cells by using VigoFect transfection reagent (Vigorous Biotechnology, Beijing, China). Cell culture supernatant containing the pseudoviruses after centrifuging at 3000 rpm for 10 min was harvested 48 h post-transfection. The pseudotyped viruses were stored at -80 °C until use. Detection of neutralizing antibodies against pseudotyped SARS-CoV-2 Omicron BA.5 variant was conducted by neutralization assay. Briefly, mouse sera were heat-inactivated at 56 °C for 30 min. Huh-7 cells were seeded in wells of a 96-well cell culture plate at a density of 1x104/well. Serially diluted mouse sera were incubated with the pseudotyped SARS-CoV-2 Omicron BA.5 for 30 min. Then, the mixture of mouse sera and pseudoviruses was transferred to a 96-well cell culture plate. After 12 h, the cell culture supernatant was discarded, and cell culturing continued with fresh DMEM containing 5% FBS for 48 h. Then, 1X lysis buffer was added to the wells for 30 min, the lysate was transferred to a 96-well half-area white plate, and luciferase activity was detected by using a Firefly luciferase assay kit (Promega, Madison, WI, USA). Luciferase values were measured using a SpectraMax i3x multi-mode microplate reader (Molecular Devices, USA). Neutralizing antibody titers (NT50) were defined as the serum dilutions that could reduce 50% relative luminescence units compared to virus control wells.