Furthermore, some predominant SARS-CoV-2 variants, including B

Furthermore, some predominant SARS-CoV-2 variants, including B.1.351 and B.1.1.28 with K417N, E484K and N501Y mutations causing changes in antigenic sites overlapping with unique antigenic sites S1, S5 and S6, promote evasion of antibody-mediated immunity obtained by natural contamination or vaccination; however, no cross-binding mAbs displaying decreased binding activity to these variants have been reported (20, 22C24). anti-human IgG and goat anti-human IgM, then are statistically analyzed. (D) Recombinant monoclonal antibodies with SARS-CoV-2 RBD specificity are identified by ELISA. Gray line indicates limitation of anti-RBD Dp44mT antibodies detection. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?3: Phylogenetic analysis of heavy chain gene of SARS-CoV-2 RBD-specific mAbs. Maximum-likelihood phylogenetic tree of fully heavy chain of RBD-specific antibodies (N=77). Each color represents heavy chain sequence of SARS-CoV-2 RBD-specific mAbs from different convalescent individuals. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?4: Gene repertoire analysis of SARS-CoV-2 RBD-specific mAbs. V gene frequencies for heavy chain (A) and light chain (B) of SARS-CoV-2 RBD-specific antibodies. Colors indicate different convalescent individuals. Germline of VH is determined using the Immunogenetics (IMGT). DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?5: Binding activity to S protein and neutralizing capacity against SARS-CoV-2 pseudovirus of SARS-CoV-2 RBD-specific mAbs. (A, B) Binding activity of mAbs to SARS-CoV S protein are compared among individual in B, and to SARS-CoV-2 S protein in C. Black line indicates mean value of EC50. (C, D) Neutralizing capacity of mAbs against SARS-CoV-2 are compared among individual in (C) and blocking capacity of mAbs in (D). Neutralizing capacity are tested by SARS-CoV-2 pseudovirus. Blocking assay is performed by incubating mixture of antibodies and SARS-CoV-2 S protein with ACE2-expressing cells. Black line indicates mean IC50. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?6: Neutralization of SARS-CoV-2 RBD-specific mAbs against SARS-CoV-2 pseudovirus. Red indicates mAbs obtained from P03 convalescent individual. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?7: The Dp44mT correlation between neutralization potency and blocking capability of SARS-CoV-2 RBD-specific mAbs. The scatter plot depicting neutralizing capacity and blocking capacity of specific mAbs from different individuals annotated by colors. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?8: Analysis of CDRH3 length of antibodies derived from VH 3-53/66. (A) Repertoire information of RBD-specific antibodies composed of VH 3-53/66. (B) Length distribution of CDRH3 for RBD-specific antibodies derived from VH 3-53/66 by comparison with the remaining VH germline encoding antibodies. (C) Correlation of CDRH3 length and binding Rabbit Polyclonal to Akt activity to SARS-CoV-2 S protein is performed for specific antibodies derived from of VH 3-53/66. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?9: Correlation analysis for days after symptom onset and mean binding activity of SARS-CoV-2 RBD-specific mAbs from corresponding convalescent individuals using Spearman correlation test. r and P values of the correlation are indicated. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?10: Competition ELISA for neutralizing mAbs. Competition ELISA is performed by using naked mAbs to block HRP-coupled mAbs, and ELISA signal for each HRP-coupled mAb is usually normalized to the signal in the absence of naked mAbs. The heat map of competition ELISA data is usually shown, with parameters colored constantly from white (0, corresponding to 0% inhibition) to red (4, corresponding to 93.7% inhibition) in the scale bar. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?11: Epitope mapping of mAbs by Dp44mT clustering analysis and functional characterization. By competition ELISA data, neutralizing mAbs are clustered into six group, Cluster1-6, and corresponding epitopes to each mAb cluster are defined as Site1-6. The color ranging from red to blue represented blocking potency against other antibodies (4.321 corresponding to 95% blocking rate and 0.074 corresponding to 5% blocking rate). The source of information and neutralization potency of each mAb are also indicated by different colors. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?12: Analysis of blocking capability against SARS-CoV-2 S protein binding to ACE2. (A) Blocking capacity of nAbs targeting sites S2-6 are compared with that of nAbs recognizing S1. (B) The neutralizing capacity and blocking capacity of nAbs recognizing site S4 are analyzed, and nAbs ID are indicated in physique. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?13: Neutralization capacity of a combination of representative nAbs targeting sites S1-6 against the SARS-CoV-2 pseudovirus and SARS-CoV-2 pseudovirus. DataSheet_1.docx (3.4M) GUID:?0CBAC60C-A680-431F-8B70-FD5837C79723 Supplementary Figure?14: Identification of SARS-CoV-2 RBD critical residues recognized by nAbs using selected amino acid substitution. (A) Mutate residues of SARS-CoV-2 RBD shown in pink. (B) Mutation of residues leading to damaging effect on SARS-CoV-2 RBD activity. Dash line indicates 25% binding activity of mutant RBD relative to wild type RBD. (C) The selected amino acid of RBD is usually mutated to alanine or arginine on purpose. Binding activity of sites S1-6 representative mAbs to wild-type (WT) and mutant RBD was measured by ELISA. The binding capacity to mutate RBD is usually normalized by binding to wild type RBD. Lines denote 10% binding activity relative to wild type RBD and 25% binding activity relative to wild type.