Virus neutralization assays were performed on HEK293T cells that were transiently transfected with ACE2 and TMPRSS2 using a SARS-CoV-2 spike pseudotyped virus that expressed luciferase 33 . Pseudotyped virus was incubated with a serial dilution of heat-inactivated human serum samples or sera from individuals who were vaccinated in duplicate for 1 h at 37 °C. Virus-only and cell-only controls were also included. Then, freshly trypsinized HEK293T ACE2- and TMPRSS2-expressing cells were added to each well. After incubation for 48 h in a 5% CO2 environment at 37 °C, luminescence was measured using the Steady-Glo or Bright-Glo Luciferase assay system (Promega). Neutralization was calculated relative to virus-only controls. Dilution curves are shown as the mean ± s.e.m. neutralization. IDfifty values were calculated in GraphPad Prism. The ID50 values within groups were summarized as the GMT and statistical comparisons between groups were made with Wilxocon ranked-sign tests. In addition, the effects of the mutations on the neutralizing effect of the sera were expressed as fold change in ID50 of the wild-type compared to mutant pseudotyped virus. Statistical difference in the mean fold change between groups was determined using a two-tailed Student’s t-test.
IFN? FluoroSpot assays
Frozen peripheral blood mononuclear cells (PBMCs) were rapidly thawed, and the freezing medium was diluted into 10 ml of TexMACS medium (Miltenyi Biotech), centrifuged and resuspended in 10 ml of fresh medium with 10 U ml ?1 DNase (Benzonase, Merck-Millipore via Sigma-Aldrich), PBMCs were incubated at 37 °C for 1 h, followed by centrifugation and resuspension in fresh medium supplemented with 5% human serum (Sigma-Aldrich) before being counted. PBMCs were stained with 2 ?l of each antibody: anti-CD3–fluorescein isothiocyanate (FITC), clone UCHT1; anti-CD4–phycoerythrin (PE), clone RPA-T4; anti-CD8a–peridinin-chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5), clone https://www.datingranking.net/de/latin-dating-sites RPA-8a (all BioLegend, London, UK), LIVE/DEAD Fixable Far Red Dead Cell Stain Kit (Thermo Fisher Scientific). PBMC phenotyping was performed on the BD Accuri C6 flow cytometer. Data were analysed with FlowJo v.10 (Becton Dickinson). In brief, 1.5–2.5 ? 10 5 PBMCs were incubated in precoated Fluorospot plates (Human IFN? FLUOROSPOT (Mabtech)) in triplicate with peptide mixes specific to spike, nucleocapsid and membrane proteins of SARS-CoV-2 (final peptide concentration 1 ?g ml ?1 per peptide, Miltenyi Biotech) and an unstimulated and positive control mix (containing anti-CD3 (Mabtech), Staphylococcus Enterotoxin B, phytohaemagglutinin (all Sigma-Aldrich)) at 37 °C in a humidified CO2 atmosphere for 48 h. The cells and medium were decanted from the plate and the assay was developed following the manufacturer’s instructions. Developed plates were read using an AID iSpot reader (Oxford Biosystems) and counted using AID EliSpot v.7 software (Autoimmun Diagnostika). All data were then corrected for background cytokine production and expressed as spot-forming units per million PBMCs or CD3 + T cells.
Antibody finding and recombinant expression
Human monoclonal antibodies were isolated from plasma cells or memory B cells of donors who are immune to SARS-CoV or SARS-CoV-2 as previously described 34,35,36,37 . Recombinant antibodies were expressed in ExpiCHO cells at 37 °C and 8% CO2. Cells were transfected using ExpiFectamine. Transfected cells were supplemented 1 day after transfection with ExpiCHO Feed and ExpiFectamine CHO Enhancer. The cell culture supernatant was collected 8 days after transfection and filtered through a 0.2-?m filter. Recombinant antibodies were affinity purified on an AKTA xpress FPLC device using 5-ml HiTrap MabSelect PrismA columns followed by buffer exchange to histidine buffer (20 mM histidine, 8% sucrose, pH 6) using HiPrep desalting columns.
Pseudovirus neutralization assay having fun with monoclonal antibodies
MLV-based SARS-CoV-2 S-glycoprotein-pseudotyped viruses were prepared as previously described 35 . HEK293T/17 cells were cotransfected with a plasmid encoding the wild-type, B.1.1.7 or triple-mutant (spike(N501Y, E484K, K417N)) SARS-CoV-2 spike glycoprotein, an MLV Gag-Pol packaging construct and the MLV transfer vector encoding a luciferase reporter using X-tremeGENE HP transfection reagent (Roche) according to the manufacturer’s instructions. Cells were cultured for 72 h at 37 °C with 5% CO2 before collection of the supernatant. VeroE6 cells stably expressing human TMPRSS2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 1% penicillin–streptomycin (100 IU ml ?1 penicillin, 100 ?g ml ?1 ), 8 ?g ml ?1 puromycin and plated into 96-well plates for 16–24 h. Pseudovirus with a serial dilution of monoclonal antibodies was incubated for 1 h at 37 °C and then added to the wells after washing twice with DMEM. After 2–3 h, DMEM containing 20% fetal bovine serum and 2% penicillin–streptomycin was added to the cells. After 48–72 h of infection, Bio-Glo (Promega) was added to the cells and incubated in the dark for 15 min before the the luminescence was read using a Synergy H1 microplate reader (BioTek). Measurements were done in duplicate and relative luciferase units were converted to the percentage of neutralization and plotted with a nonlinear regression model to determine the IC50 values using GraphPad Prism software (v.9.0.0).