Attune nxt instrument

The CDC activity of anti-SARS-CoV-2 S IgG antibodies was measured using SARS-CoV-2 spike–expressing Raji cells as previously described (Dufloo et al., 2021 (link)). Briefly, 5 × 10⁴ Raji-Spike cells were cultivated in the presence of 50% normal or heat-inactivated human serum, and with or without IgG antibodies (at 10 or 50 µg/ml and 10 consecutive 1:2 dilutions in PBS). After 24 h, the cells were washed with PBS and incubated for 30 min at 4°C with the live/dead fixable aqua dead cell marker (1:1,000 in PBS; Life Technologies) before fixation. Data were acquired on an Attune NxT instrument (Life Technologies). CDC was calculated using the following formula: 100 × (% of dead cells with serum − % of dead cells without serum)/(100 − % of dead cells without serum). Experiments were performed in duplicate in two independent experiments.

Laboratory-adapted (NL4.3, AD8 and YU-2) and T/F (WITO, CH058, CH077, CH0106, REJO and RHPA) (Ochsenbauer et al., 2012 (link)) viruses were produced from infectious molecular clones (NIH AIDS Reagent Program) as previously described (Casartelli et al., 2010 (link)). CEM-NKR-CCR5 cells (NIH AIDS Reagent Program) were infected with inocula of selected viruses, and adjusted to achieve 10%–30% of Gag⁺ cells at 48 h post infection (Bruel et al., 2016 (link), Bruel et al., 2017 ). Infected cells were incubated with IgG antibodies (at 15 μg/ml) in staining buffer (PBS, 0.5% BSA, 2mM EDTA) for 30 min at 37°C, washed and incubated 30 min at 4°C with AF647-conjugated anti-human IgG antibodies (1:400 dilution; Life technologies). Cells were then fixed with 4% paraformaldehyde and stained for intra-cellular Gag as previously described (Bruel et al., 2016 (link), Bruel et al., 2017 ). Data were acquired using an Attune Nxt instrument (Life Technologies) and analyzed using FlowJo software (v10.3; FlowJo LLC).

Complement-dependent cytotoxicity (CDC) of Raji cells was measured as previously described.³⁰ (link) Briefly Raji-Spike cells (5x10⁴) were cultivated in the presence of 50% normal (NHS) or heat-inactivated (HIHS) human serum and with or without pre-pandemic or COVID-19 sera (diluted 1:33 unless otherwise stated). After 24h, cells were washed with PBS and the live/dead fixable aqua dead cell marker (1:1,000 in PBS; Life Technologies) was added for 30 min at 4°C before fixation. Data were acquired on an Attune NxT instrument (Life Technologies). CDC was calculated using the following formula: 100 × (% of dead cells with serum − % of dead cells without serum)/(100 − % of dead cells without serum).

The S-Flow assay was performed as described [[26] , [27] (link), [28] ]. Briefly, HEK-293T (referred as 293T) cells were acquired from ATCC (ATCC Cat# CRL-3216, RRID:CVCL_0063) and tested negative for mycoplasma. 293T Cells stably expressing Spike or control cells were transferred into U-bottom 96-well plates (10⁵ cells/well). Cells were incubated at 4°C for 30 min with nasopharyngeal swabs (1:5 dilution) in PBS containing 0.5% BSA and 2 mM EDTA, washed with PBS, and stained using anti-IgG AF647 (Jackson ImmunoResearch cat# 109-605-170) or Anti-IgA AF647 (Jackson ImmunoResearch cat# 109-605-011). Cells were washed with PBS and fixed 10 min using 4% PFA. Data were acquired on an Attune Nxt instrument (Life Technologies). Stainings were also performed on control (293T Empty) cells. Results were analysed with FlowJo 10.7.1 (Becton Dickinson). The positivity of a sample was defined as a specific binding above 30%. The specific binding was calculated as follow: 100 x (% binding 293T Spike - % binding 293T Empty)/ (100 - % binding 293T Empty).

Laboratory-adapted (AD8 and YU2) and T/F (CH058, CH077, and THRO) viruses were produced from infectious molecular clones (NIH AIDS Reagent Program) as previously described (Bruel et al., 2016 (link); Bruel et al., 2017 (link)). CEM.NKR-CCR5 cells (NIH AIDS Reagent Program) were infected with inocula of selected viruses and adjusted to achieve 10–40% of Gag⁺ cells 48 h after infection (Bruel et al., 2016 (link); Bruel et al., 2017 (link)). Infected cells were incubated with IgG and IgA antibodies (at 15 µg/ml) in staining buffer (PBS, 0.5% bovine serum albumin, and 2 mM EDTA) for 30 min at 37°C, washed, and incubated for 30 min at 4°C with AF647-conjugated anti-human IgG antibodies (1:400 dilution; Life Technologies) or with mouse anti-human IgA HB200 antibodies (30 µg/ml final; Lorin and Mouquet, 2015 (link)) for 30 min and 1 h, respectively, at 4°C. Binding reactions using HB200 were revealed after washing using 1:400-diluted Alexa Fluor 647–conjugated goat anti-mouse antibodies (Thermo Fisher Scientific) for 30 min at 4°C. Cells were then fixed with 4% paraformaldehyde and stained for intracellular Gag as previously described (Bruel et al., 2016 (link); Bruel et al., 2017 (link)). Data were acquired using an Attune Nxt instrument (Life Technologies, Thermo Fisher Scientific) and analyzed using FlowJo software (v10.7.1).

AD8-infected CEM.NKR-CCR5 cells were first stained using the CellTrace Far Red cell proliferation kit (Invitrogen, Thermo Fisher Scientific) for 30 min at 37°C. 4 × 10⁴ target cells were then incubated for 5 min at room temperature with antibodies (at 15 µg/ml final concentration) and mixed 1:1 with 2, 4, or 8 × 10⁵ human monocytes and neutrophils. Following a brief spindown to promote cell contacts, mixtures were incubated for 4 h at 37°C. Cells were fixed and stained for intracellular Gag using KC57-RD1 antibody (diluted 1:500; Beckman Coulter). Data were acquired using an Attune Nxt instrument (Life Technologies, Thermo Fisher Scientific) and analyzed using FlowJo software (v10.7.1).