FIGURE 5.

The design of titration and pseudovirus neutralization assay (PVNA). (A,B) Infection, titration and PVNA was performed in a 96-well plate with 10-fold serial dilution of ppVSVΔG-SARS-CoV-2 S until a total of 8 or 10 dilutions and 6 or 8 replicates were obtained. Neutralization assay was also performed in a 96-well plate with 6 dilutions and 2 replicates; however, the sera samples were previously diluted and then mixed with ppVSVΔG-SARS-CoV-2 S 325 to 1,300 TCID₅₀/mL and incubated for 1 h at 37°C. (C) In titration, the cells containing the luciferase were lysed to perform the luciferase assay and a 96-well-plate luminometer was used. In the fluorescence method, the DAPI-stained cells containing GFP were analyzed by fluorescence microscope, and the images were analyzed with specialized software. An alternative method is by using a flow cytometer equipped with a 96-well-autosampler. In PVNA, the luciferase activity was determined by the relative light units (RLU) and the fluorescence by the number of GFP-positive cells. Percent neutralization must be normalized considering uninfected cells as 100% neutralization and infected cells with ppVSVΔG-SARS-CoV-2 S alone as 0% neutralization. (D) The 50% tissue culture infectious dose (TCID₅₀) and/or the 50% ppVSVΔG-SARS-CoV-2 S neutralizing doses (PVND₅₀) were calculated according to the Reed-Muench method and reference table.