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 293T 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 (6 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. Binding of sera to vector-only plasmid (non-spike) transfected 293T 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 (8 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. Binding to BSA served as a control for non-specific binding by the sera. Statistical comparisons between two groups were performed using a Mann-Whitney test, (**p <0.01; ***p < 0.001, ****p < 0.0001; ns- p >0.05).