Fig. 6: Development of a highly sensitive surrogate neutralization test for SARS-CoV-2.

a Scheme depicting interaction of SARS-CoV-2 spike on stimulator cell with ACE2-ζ chimeric receptor on biosensor cells. b Scheme depicting SARS-CoV-2 spike with N-terminal Strep-II-tag modification and stabilizing mutations (top) and ACE2-ζ chimeric receptor with N-terminal myc-tag (bottom). c Expression of SARS-CoV-2 spike on K562 RFP stimulator cells. d Expression of ACE2-ζ on JE6.1 NFκB-eGFP biosensor cells. e Fluorescence microscopy images representative of three replicates. K562 SARS-CoV-2 spike CD86 cells expressed RFP (red). JE6.1 NFκB-eGFP ACE2-ζ cells expressed eGFP upon activation (green). Stimulator cells were incubated with vaccinated donor serum at the indicated dilutions. Corresponding gMFI-values assessed by flow cytometry are provided in the Source Data file. f, g JE6.1 TPR ACE2-ζ biosensor cells were co-cultured with K562 SARS-CoV-2 spike stimulator cells. f Representative histograms showing reporter gene expression of JE6.1 TPR ACE2-ζ biosensor cells cultured with indicated stimulator cells (data presented as NFκB-eCFP FI, n = 1 experiment). g Stimulator cells were preincubated with sera obtained from a donor prior to and after vaccination with ChAdOX1. Each condition was performed in triplicates. Data is presented as individual values with mean ± SD of gMFI NFκB-eCFP (top). Each mean was then normalized to full stimulation without serum and non-linear regression curve fitting was performed. For the blocking serum an IC₅₀ value was calculated (bottom). h A cohort of 77 serum donors with reactivity to SARS-CoV-2 determined by an electroluminescence immunoassay was evaluated with the biosensor surrogate neutralization test. IC₅₀ values were then correlated to cut-off index (COI) values of the electroluminescence immunoassay (higher COI indicate higher antibody levels). Spearman correlation for non-normally distributed samples was performed (**** p < 0.0001, two-tailed). i A cohort of 12 serum donors was evaluated by a live-virus NT. IC₅₀ values determined by the biosensor surrogate neutralization test were correlated to the NT titers. Spearman correlation for non-normally distributed samples was performed (**p = 0.0021, two-tailed). Source data for this figure are provided as a Source Data file.