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SARS-CoV-2 pseudotyped particles were generated as previously described (Robbiani et al., 2020 (link); Schmidt et al., 2020 ). Briefly, 293T cells were transfected with pNL4–3ΔEnv-nanoluc and pSARS-CoV-2-S_Δ19. For generation of RBD-mutant pseudoviruses, pSARS-CoV-2-S_Δ19 carrying indicated spike mutations was used instead (Muecksch et al., 2021 ; Wang et al., 2021 ; Weisblum et al., 2020 ). Particles were harvested 48 h post-transfection, filtered and stored at −80°C.
Four-fold serially diluted mAbs were incubated with SARS-CoV-2 pseudotyped virus for 1 h at 37 °C. The mixture was subsequently incubated with HT1080_Ace2 cl14 cells (Schmidt et al., 2020 ) for 48 h after which cells were washed with PBS and lysed with Luciferase Cell Culture Lysis 5× reagent (Promega). Nanoluc Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (Promega) with the Glomax Navigator (Promega). The obtained relative luminescence units were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped virus in the absence of mAbs. The half-maximal and 90% inhibitory concentrations (IC₅₀ and IC₉₀) were determined using four-parameter nonlinear regression (least squares regression method without weighting; constraints: top = 1, bottom = 0) (GraphPad Prism).

Fourfold serially diluted pre-pandemic negative control plasma from healthy donors, plasma from individuals who received mRNA vaccines or monoclonal antibodies were incubated with SARS-CoV-2 pseudotyped virus for 1 hour at 37 °C. The mixture was subsequently incubated with 293T_Ace2 cells^{16 (link)} (for all WT neutralization assays) or HT1080Ace2 cl14 (for all mutant panels and variant neutralization assays) cells^{5 (link)} for 48 hours after which cells were washed with PBS and lysed with Luciferase Cell Culture Lysis 5× reagent (Promega). Nanoluc Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (Promega) with the Glomax Navigator (Promega). The relative luminescence units were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped virus in the absence of plasma or monoclonal antibodies. The half-maximal neutralization titers for plasma (NT₅₀) or half-maximal and 90% inhibitory concentrations for monoclonal antibodies (IC₅₀ and IC₉₀) were determined using four-parameter nonlinear regression (least squares regression method without weighting; constraints: top=1, bottom=0) (GraphPad Prism).