MTN for SARS-CoV-2
The MTN for SARS-CoV-2 was performed in BSL-3 laboratories. All samples were first heat inactivated by incubation at 56°C for 30 min, and then 2-fold dilutions were prepared in Dulbecco’s modified Eagle’s medium (DMEM). The MTN was performed using a modification of a previously described protocol (Gaudreault et al., 2020). Briefly, serum samples were initially diluted 1:10, and then 50 μL per well of each serum sample, in duplicate, was subjected to 2-fold serial dilutions (1:20–1:320) in 50 μL of culture medium. Then, 50 μL of a 100 TCID50 of hCoV-19/Italy/CAM-INMI-32803-66/2020 (EPI_ISL_493333) was added to each well containing the serially diluted serum and incubated for 1 h at 37°C. Finally, 100 μL of Vero E6 cell suspension, adjusted with maintenance medium to 105 cells/mL, was added to each well and incubated at 37°C and 5% CO2. After 4–5 days of incubation, the cells were fixed and stained with a solution of 0.1% crystal violet in 5% paraformaldehyde (PFA) for 30 min to detect cytopathic effects (CPE).
The neutralisation endpoint titre was determined as the highest serum dilution at which at least 50% of the wells showed CPE. An MNT titre ≥20 was considered positive (Gaudreault et al., 2020).