Fig. 3: SARS-CoV-2 S and endemic HCoV S-protein-specific cross-reactive IgG+ memory B cells from COVID donors and isolation and characterization of mAbs.

a–b. Flow cytometry analysis showing the single B-cell sorting strategy for COVID representative donor CC9 and frequencies of SARS-CoV-2 S and endemic β-HCoV, HCoV-HKU1 and α-HCoV, HCoV-NL63 S-protein-specific memory B cells in eight select COVID donors. The B cells were gated as SSL, CD3−, CD4−, CD8−, CD14−, IgD−CD19+, IgM−, IgG+. The frequencies of HCoV S-protein-specific IgG memory B cells were as follows; SARS-CoV-2 S (up to ~8%—range = ~1.6–8%), HCoV-HKU1 S (up to ~4.3%—range = ~0.2–4.3%), HCoV-NL63 S (up to ~0.6%–range = ~0.04–0.6%) protein single positive and SARS-CoV-2/HCoV-HKU1 S (up to ~2.4%–range = ~0.02–2.4%) and SARS-CoV-2/HCoV-NL63 S-protein (up to ~0.09%–range = ~0–0.09%) double positives. SARS-CoV-2-infected donors showed the presence of SARS-CoV-2/HCoV-HKU1 S-protein cross-reactive IgG memory B cells. Scatter dot plots show frequencies of S protein-specific B cells with a line at mean with SD. All differences between means with p values for each comparison are indicated. **p < 0.01; ***p < 0.001. A Mann–Whitney two-tailed test was used to compare the data groups. c Pie plots showing immunoglobulin heavy chain distribution of mAbs isolated from four COVID donors, CC9, CC10, CC36, and CC40. The majority of the mAbs were encoded by the IgVH3 immunoglobulin gene family. d Plots showing % nucleotide mutations in heavy (VH) and light (VL) chains of isolated mAbs across different individuals. The VH and VL mutations ranged from 0 to 11.6% and 0–4.4%, respectively, and are shown as scatter dot plots with a line at median. e CELISA-binding curves of isolated mAbs from four COVID donors with SARS-CoV-2 and HCoV-HKU1 spikes expressed on 293 T cells. Binding to HCoV spikes is recorded as % positive cells using a flow cytometry method. Five mAbs, three from the CC9 donor, and two from the CC40 donor show cross-reactive binding to SARS-CoV-2 and HCoV-HKU1 spikes. f Neutralization of SARS-CoV-2 by mAbs isolated from COVID donors. Four mAbs, two each from donors, CC36 and CC40, show neutralization of SARS-CoV-2. The neutralization experiments were performed in duplicate and repeated independently 1–2 times for reproducibility.