Structural insights for neutralization of Omicron variants BA.1, BA.2, BA.4, and BA.5 by a broadly neutralizing SARS-CoV-2 antibody

In this study, by characterizing several human monoclonal antibodies (mAbs) isolated from single B cells of the COVID-19–recovered individuals in India who experienced ancestral Wuhan strain (WA.1) of SARS-CoV-2 during early stages of the pandemic, we found a receptor binding domain (RBD)–specific mAb 002-S21F2 that has rare gene usage and potently neutralized live viral isolates of SARS-CoV-2 variants including Alpha, Beta, Gamma, Delta, and Omicron sublineages (BA.1, BA.2, BA.2.12.1, BA.4, and BA.5) with IC50 ranging from 0.02 to 0.13 μg/ml. Structural studies of 002-S21F2 in complex with spike trimers of Omicron and WA.1 showed that it targets a conformationally conserved epitope on the outer face of RBD (class 3 surface) outside the ACE2-binding motif, thereby providing a mechanistic insights for its broad neutralization activity. The discovery of 002-S21F2 and the broadly neutralizing epitope it targets have timely implications for developing a broad range of therapeutic and vaccine interventions against SARS-CoV-2 variants including Omicron sublineages.


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Figs. S1 to S9 Tables S1 to S3 Supplementary Figures   CoV-2 RBD and Spike-6p binding affinities of the mAb 002-S21F2.In these assays, 002-S21F2 mAb (5 ug/ml concentration) was captured on protein A sensors and its binding kinetics were tested with serial 2-fold diluted RBD (600 nM to 37.5 nM) and Spike hexapro protein (100 nM to 6.25 nM).Association was measured for 300 seconds followed by dissociation measurement for 600 seconds.
Fig. S1: Isolation and characterization of 92 SARS-CoV-2 RBD specific mAbs.(A) ELISA curves showing plasma IgG, IgM and IgA reactivity against SARS-CoV-2 RBD protein.The five subjects are COVID-19 recovered individuals recruited into a clinical study during the first wave of the pandemic in India.ELISA results with the plasma of a pre-pandemic healthy individual are shown for comparison.RBD-specific Max50 IgG binding titers for are shown in boxes.(B) Neutralization assay curves showing activity against ancestral SARS-CoV-2 Wuhan-I (WA.1) strain.The plasma dilution resulting in a 50% reduction in neutralization (FRNT-mNG50) is indicated in the boxes.(C) Flow cytometry plots show SARS-CoV-2 RBDspecific memory B cells in the peripheral blood lymphocytes of select SARS-CoV-2 recovered individuals.The plots shown were gated on CD19 positive and CD3 negative populations.Staining was performed using FITC conjugated RBD protein.(D) ELISA curves showing SARS-CoV-2 RBD binding by each of the purified mAbs (n=92).(E) The CDR3 length of each sequence was calculated and their distribution was plotted as a histogram.(F) Donut plots showing the heavy chain, lambda light chain and kappa light chain gene distribution of the SARS-CoV-2 RBD-specific mAbs (n=92).(G) Correlation plot of heavy chain and light chain SHM (%).Pearson correlation coefficient (R) and p-value have been shown.

Fig. S3 :
Fig.S3: Antibody 002-S21F2 showed high affinity to SARS-CoV-2 RBD and spike proteins in biolayer interferometry assays.(A) Octet BLI sensorgrams showing the SARS-CoV-2 RBD and Spike-6p binding affinities of the mAb 002-S21F2.In these assays, 002-S21F2 mAb (5 ug/ml concentration) was captured on protein A sensors and its binding kinetics were tested with serial 2-fold diluted RBD (600 nM to 37.5 nM) and Spike hexapro protein (100 nM to 6.25 nM).Association was measured for 300 seconds followed by dissociation measurement for 600 seconds.(B) Describing the KD (M), Kon (1/Ms) and Koff (1/s) values of the four potent mAbs with RBD and Spike hexapro proteins.
Fig.S3: Antibody 002-S21F2 showed high affinity to SARS-CoV-2 RBD and spike proteins in biolayer interferometry assays.(A) Octet BLI sensorgrams showing the SARS-CoV-2 RBD and Spike-6p binding affinities of the mAb 002-S21F2.In these assays, 002-S21F2 mAb (5 ug/ml concentration) was captured on protein A sensors and its binding kinetics were tested with serial 2-fold diluted RBD (600 nM to 37.5 nM) and Spike hexapro protein (100 nM to 6.25 nM).Association was measured for 300 seconds followed by dissociation measurement for 600 seconds.(B) Describing the KD (M), Kon (1/Ms) and Koff (1/s) values of the four potent mAbs with RBD and Spike hexapro proteins.
Fig. S5.Cryo-EM data analysis and validation for Omicron Spike-6P and 002-S21F2 complex.(A) Representative electron micrograph.(B) Representative 2D-class averages.2D classes showing head-to-head spike dimer are circled in red and shown below.(C) Classification scheme and refinement that yielded final cryo-EM map reconstruction.Boxed classes were selected for further processing and refinement.Boxed region containing one RBD complexed with one Fab in refined map masked for local refinement.(D) Gold standard Fourier shell correlation (FSC) curve of final overall (left) and locally refined (right) maps and resolution estimation based on 0.143 Fourier shell correlation criteria as indicated by a blue line.

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Fig. S6.SARS-CoV-2 RBD specific bnAb 002-S21F2 exhibits a rare immunogenetic make-up.(A) Tableshowingthe antibody heavy chain (HC) and light chain (LC) genetic information of the 002-S21F2 bnAb.(B) IMGT V-quest alignment analysis result of 002-S21F2 HC and LC gene with their respective germline sequence.Here, somatic mutations are marked as bold amino acids in black color.(C) CDRH3 amino acid sequence identity to 002-S21F2 is plotted against CDRL3 amino acid identity to 002-S21F2 for paired HC and LC sequences of SARS-CoV-2 mAbs banked in the CoV-AbDab database.SARS-CoV-2 mAbs with the same HC and LC germline gene as 002-S21F2 (IGHV5-51 and IGKV1-33) are shown in red color.MAbs using the same HC gene but different LC gene as 002-S21F2 are shown in blue color.MAbs using different HC genes but the same LC gene as 002-S21F2 are shown in orange color.MAbs using a different HC and LC combination as 002-S21F2 are shown in grey color.

Fig. S7 .
Fig. S7.Structural comparison of WA.1 (grey) and Omicron (cyan) spike-002-S21F2 mAb complex.(A) Overall structural alignment of RBD and Fab region of 002-S21F2 in WA.1 and Omicron complex structures.(B) Zoomed in view showing the local conformation change in the specified region of RBD in WA.1 vs Omicron.