Lentiviral vectors were produced in HEK293T cells by co-transfection using Lipofectamine® 2000 (Thermo Fisher, Darmstadt, Germany) as described previously [16 (link)]. Plasmids encoding HIV-1 gag/pol, rev, the luciferase-encoding lentiviral vector genome, and the SARS-CoV-2 wild-type 614D (#MN908947), the SARS-CoV-2 Delta (NC_045512.2), or the SARS-CoV-2 Omicron BA.1 (NC_045512.2) spike gene were transfected. The coronavirus spike genes were truncated, lacking the last 19 carboxy terminal amino acids, and were synthesized (Eurofins, Ebersberg, Germany; IDT, Leuven, Belgium) and cloned into the vector pcDNA 3.1(+) as described before [17 (link)]. After harvest, vector particles were used directly for neutralization assays. Pseudotyped vectors and serially diluted human serum (1:60 to 1:4860) were incubated for 45 min at 37 °C and used to transduce HEK293T-hACE2 cells in triplicate [12 (link)]. After 48 h, britelite plus luciferase substrate (PerkinElmer, Waltham, MA, USA) was added to measure luciferase activity. The reciprocal area under the curve (1/AUC) value calculated for each sample corresponds to the respective neutralization activity. Cut-off levels were determined with samples taken prior to the J&J vaccination, constituted by their mean values plus two standard deviations, and had a 1/AUC of 0.45 for WT, 0.44 for Delta, and 0.20 for Omicron.
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