Fig. 1: Reactivity of COVID and pre-pandemic human sera with cell surface-expressed human coronaviruses spikes and their soluble S-protein versions.

a Heatmap showing cell-based flow cytometry binding (CELISA) of COVID and pre-pandemic donor sera with 293 T-cell surface-expressed full-length spike proteins from β-(SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-OC43) and α-(HCoV-NL63 and HCoV-229E) human coronaviruses (HCoVs). Sera were titrated (six dilutions–starting at 1:30 dilution) and the extent of binding to cell surface-expressed HCoVs was recorded by % positive cells, as detected by PE-conjugated anti-human-Fc secondary Ab using flow cytometry. Area-under-the-curve (AUC) was calculated for each binding titration curve and the antibody titer levels are color-coded as indicated in the key. The binding of sera to vector-only plasmid (non-spike) transfected 293 T cells served as a control for non-specific binding. b ELISA binding of COVID and pre-pandemic donor sera to soluble S-proteins from β-(SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-OC43) and α-(HCoV-NL63 and HCoV-229E) HCoVs. Serum dilutions (eight dilutions–starting at 1:30 dilution) were titrated against the S-proteins and the binding was detected as OD405 absorbance. AUC representing the extent of binding was calculated from binding curves of COVID (left) and pre-pandemic (right) sera with S-proteins and comparisons of antibody binding titers are shown. The binding of sera with each protein is shown as scatter dot plots with a line at median. Binding to BSA served as a control for non-specific binding by the sera. The serum-binding experiments were carried out in duplicate and repeated independently at least once for reproducibility. Statistical comparisons between two groups were performed using a Mann–Whitney two-tailed test, (****p < 0.0001; ns p > 0.05).