Britelite plus luciferase substrate

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.

An Ebola ADCC assay previously developed in our laboratory (60 (link)) was used to assess the cytotoxic potential of Ebola VLP-exposed cells. Briefly, 293 Trex cells (target cells), stably transfected with luciferase and EBOV GP genes, were induced with 2.0 μg/mL of doxycycline for 24 hours. Cells were harvested, washed, and mixed with PBMCs (effector cells), either unexposed (control) or those exposed to Ebola VLPs for 48 hours, at different effector/target cell ratios (10:1, 5:1, and 2:1) in complete media in 96-well plates. All the wells received 1:200 diluted rhesus macaque anti-EBOV GP plasma (71 (link)). Plates were incubated at 37°C, 6% CO₂ for 6 hours. Following incubation, cells were washed thrice with PBS and cell pellets resuspended in 100 μL/well of complete media. Cells were lysed with the addition of 100 μL/well of Britelite Plus luciferase substrate (PerkinElmer), and luciferase activity (relative luciferase units, RLU) was measured on a BioTek Synergy HTX reader. Percentage target cell killing was calculated by the following formula: ([RLU_control – RLU_{anti-EBOV GP plasma}] × 100)/RLU_control.