Bright glo luciferase kit

HIV-1 particles pseudotyped with SARS-CoV-2 spike were made as previously described.^{31 (link)} Briefly, a T75 flask was seeded the day before with 3 million HEK293T/17 cells in 10 ml complete Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin. Cells were transfected using 60 μg of PEI-Max (Polysciences) with a mix of three plasmids: 9.1 μg HIV-1 luciferase reporter vector,^{32 (link)} 9.1 μg HIV p8.91 packaging construct and 1.4 μg wild-type SARS-CoV-2 Spike expression vector.^{32 (link)} Supernatants containing pseudotyped virions were harvested 48 h post-transfection, filtered through a 0.45-μm filter. Infectivity was titrated by serial dilution of supernatant in DMEM (10% FBS and 1% penicillin–streptomycin) followed by addition to HeLa cells (10 000 cells per 100 µl per well) that stably express ACE2 (provided by J. E. Voss, Scripps Institute). After 48–72 h luminescence was assessed as a proxy of infection by lysing cells with the Bright-Glo luciferase kit (Promega), using a Glomax plate reader (Promega).

Serial dilutions of heat-inactivated plasma samples were prepared with DMEM media (10% FBS and 1% Pen/Strep) and incubated with pseudotype virus for 1-hour at 37°C in 96-well plates. Next, Hela cells stably expressing the ACE2 receptor (provided by Dr James Voss, The Scripps Research Institute) were added (10,000 cells/25 μL per well) and the plates were left for 72 hr. Infection level was assessed in lysed cells with the Bright-Glo luciferase kit (Promega), using a Victor™ X3 multilabel reader (Perkin Elmer). Measurements were performed in duplicate and duplicates used to calculate the ID₅₀.

293T, 293T-hACE2 stable cells, Huh-7 (human liver cells), and BHKs were maintained under standard cell culture conditions in DMEM with L-glutamine, antibiotics, and 10% Fetal Bovine Serum. Single-cycle, Vesicular Stomatitis Virus (VSV) pseudotype assays were performed as previously described [1 (link)]. Briefly, 293T “producer cells” were transfected with spike plasmids or empty vector as a “no spike” control and infected 24-hours later with VSV-ΔG-luc/GFP particles, generating chimeric-spike pseudotyped particles that were harvested 72 hours post-transfection and stored at -80°C. Target cells were plated in 96-well format, and spin-infected in quadruplicate with equivalent volumes of viral pseudotypes at 1200xG for 1 hour at 4°C. Infected cells were incubated for approximately 18–20 hours and luciferase was measured using the Promega BrightGlo luciferase kit following manufacturers’ instructions (Promega). Entry signal was normalized to the average signal for the “no spike” control. Plates were measured and analyzed in triplicate. Data are representative of four complete biological replicates. All graphed data points in this study are available in S1 Data.

Neutralization assays were conducted as previously described (Carter et al., 2020 (link); Monin et al., 2021 (link); Seow et al., 2020 (link)). Serial dilutions of serum samples (heat inactivated at 56°C for 30mins) or mAbs were prepared with DMEM-C media and incubated with pseudotyped virus for 1 h at 37°C in 96-well plates. Next, HeLa cells stably expressing the ACE2 receptor (provided by Dr James Voss, Scripps Research, La Jolla, CA) were added (12,500 cells/50μL per well) and the plates were left for 72 h. The amount of infection was assessed in lysed cells with the Bright-Glo luciferase kit (Promega), using a Victor™ X3 multilabel reader (PerkinElmer). Measurements were performed in duplicate and duplicates used to calculate the ID₅₀.

Neutralization assays were performed as described recently^{49 (link)}. Briefly, a 1:20 dilution of heat-inactivated serum was serially diluted in 3-fold steps and mixed with Env-pseudotyped virus. After a 1 h incubation at RT the mix was added to TZM-bl cells which were seeded to a density of 17.000 cells/well a day prior. After 72 h the cell medium was removed, the cells were lysed and luciferase was measured using the Bright Glo Luciferase kit (Promega). Luciferase activity was measured using a Glomax plate reader. Neutralization titers (ID₅₀-values) were determined as the serum dilution at which infectivity was inhibited by 50%.

Serial dilutions of serum samples (heat-inactivated at 56°C for 30 min) were prepared with DMEM (25 μL) (10% FBS and 1% Pen/Strep) and incubated with pseudotyped virus (25 μL) for 1 h at 37°C in half-area 96-well plates. Next, HeLa cells stably expressing the ACE2 receptor were added (10,000 cells/25 μL per well), and the plates were left for 72 h. Infection levels were assessed in lysed cells with the Bright-Glo luciferase kit (Promega), using a Victor X3 multilabel reader (Perkin Elmer). Each serum sample was run in duplicate and was measured against the five SARS-CoV-2 variants within the same experiment using the same dilution series.