Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Mutations on the spike glycoprotein of SARS-CoV-2 Omicron subvariants. Residues that are not identical among Omicron subvariants are colored red. b , Workflow to generate cryo-EM structure of BA.2, BA.3, BA.2.13, BA.2.12.1, BA.4/5 spike glycoprotein trimer with S6P and R683A, R685A substitutions. c , Binding affinities of Omicron variants spike trimers to hACE2 measured by SPR. SPR analyses were conducted in biological duplicates. d , MD simulated interactions between hACE2 and RBD of Omicron variants. Structures of the RBD from Omicron variants and hACE2 are shown as ribbons.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Surface representation of S-trimers of BA.1, BA.2, BA.3, BA.2.13, BA.2.12.1 and BA.4/BA.5 (BA.4/5) variants. b , Structural interpretation and functional verification of the stability of the spike protein of BA.1, BA.2, BA.3, BA.2.13, BA.2.12.1 and BA.4/BA.5 variants. Left, superimposed structures of spike protein and the S2 domains of BA.1 (purple), BA.2 (red) and BA.4/BA.5 (blue). The binding surface areas between S2 subunits of the variants are calculated in the table on the right. c , Thermoflour analysis for these Omicron variants. Analyses were performed as biological duplicates. d , Binding affinities of RBDs of Omicron variants for hACE2 measured by SPR. Analyses were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Functional Assay, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a – d , Neutralizing titres against SARS-CoV-2 D614G, Omicron subvariants and SARS-CoV-1 pseudoviruses in plasma from vaccinated and convalescent individuals. a , Individuals who had received 3 doses of CoronaVac ( n = 40). b , Individuals who had received 2 doses of CoronaVac and 1 dose of ZF2001 ( n = 38). c , Individuals who, after receiving 3 doses of CoronaVac, had been infected with BA.1 and recovered ( n = 50). d , People who had recovered from SARS and received 2 doses of CoronaVac and 1 dose of ZF2001 ( n = 28). P -values were calculated using two-tailed Wilcoxon signed-rank tests of paired samples. The geometric mean titre is shown above each group of points. e , Neutralizing activity against SARS-CoV-2 variants and sarbecoviruses by therapeutic NAbs. Green, half-maximal inhibitory concentration (IC 50 ) ≤ 30 ng ml −1 ; white, 30 ng ml −1 < IC 50 < 1,000 ng ml −1 ; red, IC 50 ≥ 1,000 ng ml −1 ; *, IC 50 ≥ 10,000 ng ml −1 . All neutralization assays were performed as biological duplicates. * P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant ( P > 0.05).

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Infection, Two Tailed Test, Activity Assay, Concentration Assay, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: NT50 against SARS-CoV-2, SARS-CoV-1 D614G and Omicron subvariants spike-pseudotyped VSV by plasma samples from a , individuals who received 3 doses CoronaVac with (n = 50) or without (n = 40) BA.1 breakthrough infection; b , individuals who received 2 doses CoronaVac and ZF2001 booster with (n = 28) or without (n = 38) previous SARS-CoV-1 infection; c , individuals who received 3 doses CoronaVac (n = 40) or 2 doses CoronaVac with ZF2001 booster (n = 38). P-values were calculated using two-tailed Wilcoxon rank-sum tests and labeled above the bars. n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. Geometric means are labeled. Error bars refer to geometric standard deviations.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Infection, Two Tailed Test, Labeling, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , FACS analysis of pooled memory B cells (IgM − CD27 + ) from plasma of individuals who have recovered from BA.1 breakthrough infection after vaccination, vaccinated individuals and unvaccinated individuals who have recovered from BA.1 breakthrough infection. The percentage of cells recognizing WT or BA.1 RBD are shown. b , The heavy chain V domain somatic hypermutation (SHM) rate of BA.1-specific ( n = 968) and BA.1–WT cross-reactive ( n = 4,782) BCRs obtained from 10X scVDJ-seq from individuals who have recovered from BA.1 breakthrough infection after vaccination. Two-tailed Wilcoxon rank-sum test. Boxes show 25th percentile, median and 75th percentile, and violin plots show kernel density estimation curves of the distribution. c , t -SNE and unsupervised clustering of antibodies that bind WT SARS-CoV-2 RBD. Twelve epitope groups were identified on the basis of DMS of 1,538 antibodies. d , e , Epitope distribution and projection of antibodies from plasma of individuals who had recovered from infection with the WT virus, individuals who have recovered from BA.1 breakthrough infection after vaccination, and vaccinated individuals who had recovered from SARS. f , ACE2 competition level determined by competition ELISA ( n = 1,286) were projected onto the t -SNE. g , Neutralizing activity against SARS-CoV-2 D614G ( n = 1,509) and SARS-CoV-1 (HKU-39849; n = 1,457). h , Average mutational escape score projection of each epitope group on SARS-CoV-2 RBD (Protein Data Bank (PDB): 6M0J). All neutralization assays were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Infection, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Activity Assay, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: Neutralizing activity against SARS-CoV-2 D614G and Omicron subvariants pseudovirus by antibodies of each epitope group from BA.1 convalescents (BA.1-stimulated. A, n = 30; B, n = 41; C, n = 20; D1, n = 49; D2, n = 17; E1, n = 11; E2.1, n = 64; E2.2, n = 122; E3, n = 57; F1, n = 80; F2, n = 13; F3, n = 2), and from wildtype convalescents or vaccinees (WT-stimulated. A, n = 98; B, n = 55; C, n = 88; D1, n = 46; D2, n = 36; E1, n = 59; E2.1, n = 26; E2.2, n = 39; E3, n = 68; F1, n = 97; F2, n = 158; F3, n = 67). Geometric mean titers (GMT) are annotated above each group of points, and error bars indicate geometric standard deviation. P-values were calculated using two-tailed Wilcoxon rank-sum tests and labeled above the bars. n.s., not significant, p > 0.05. NAbs in the boxed epitope groups showed substantial neutralization potency changes against BA.2.12.1 or BA.4/5 compared to BA.1. All neutralization assays were conducted in biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Standard Deviation, Two Tailed Test, Labeling, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: Heavy chain V-J genes combination of a , WT-stimulated antibodies. b , BA.1-stimulated antibodies or each epitope group. The number of NAbs is annotated above the chord plot. IGHV genes are annotated only if the corresponding number of antibodies is greater than one.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques:

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Neutralizing activity against SARS-CoV-2 D614G and Omicron subvariants by BA.1-stimulated (A, n = 30; B, n = 41; C, n = 20) and WT-stimulated (A, n = 98; B, n = 55; C, n = 88) antibodies in Group A, B and C. Geometric mean of IC50 fold changes compared to IC50 against BA.2 are annotated above the bars. P-values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples, in comparison to IC50 against BA.2. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. b , Averaged escape maps at escape hotspots of BA.1-stimulated and WT-stimulated antibodies in group A, B and C, and corresponding MSA of various sarbecovirus RBDs. Height of each amino acid in the escape maps represents its mutation escape score. Mutated sites in Omicron variants are marked in bold.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Two Tailed Test, Neutralization, Mutagenesis

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Epitope of representative antibodies in group D1 (C110; PDB: 7K8V), D2 (LY-CoV1404; PDB: 7MMO), E2.1 (BD-744; PDB: 7EY0) and E2.2 (FC08; PDB: 7DX4). Residues highlighted in red indicate sites that are mutated in Omicron variants. b , Neutralizing activity of NAbs in group D1 ( n = 95), D2 ( n = 53), E2.1 ( n = 90) and E2.2 ( n = 161) against spike-pseudotyped SARS-CoV-2 variants. The geometric mean of the fold change in IC 50 relative to BA.2 is shown above each plot. Two-tailed Wilcoxon signed-rank test of paired samples, in comparison to IC 50 values versus BA.2. c , Neutralizing activity of representative potent NAbs in group D1 ( n = 24), D2 ( n = 12), E2.1 ( n = 23) and E2.2 ( n = 23) against SARS-CoV-2 spike L452 mutants. Geometric mean of the fold change in IC 50 relative to D614G is shown above each plot. Two-tailed Wilcoxon signed-rank test of paired samples. d , Average escape maps at escape hotspots of antibodies in epitope groups D1, D2, E2.1 and E2.2, and the corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron subvariants are marked in bold. All neutralization assays were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Two Tailed Test, Sequencing, Mutagenesis, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Neutralizing activity against SARS-CoV-2 D614G and Omicron subvariants by antibodies in group E3 (n = 125) and F1 (n = 177). Geometric mean of IC50 fold changes compared to BA.2 are annotated above the bars. P-values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples, in comparison to IC50 against BA.2. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. b , Epitope of representative antibodies in group E3 (S2H97, PDB: 7M7W) and F1 (S304, PDB: 7JW0). Residues highlighted in red indicate mutated sites in Omicron variants. c , Averaged escape maps at escape hotspots of antibodies in group E3 and F1, and corresponding MSA of various sarbecovirus RBDs. Height of each amino acid in the escape maps represents its mutation escape score. Mutated sites in Omicron variants are marked in bold.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Two Tailed Test, Neutralization, Mutagenesis

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a – c , Neutralizing activity against SARS-CoV-1 and SARS-CoV-2 subvariants by NAbs in group E1 ( a ; n = 70), F2 ( b ; n = 171) and F3 ( c ; n = 69). The geometric mean of the fold change in IC 50 relative to BA.2 is shown above each plot. P -values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples, compared with the IC 50 for BA.2. d , The epitope of Group E1 antibody BD55-3152 on the BA.1 RBD. e , Overlay of BD55-5840 in the complex with BA.1 or BA.2 RBD. f , g , The epitope and interactions on the binding interface of BD55-1239 (group F2) ( f ) and BD55-3372 (group F3) ( g ). Antibody residues are shown in blue, and RBD residues are in black or red. Residues highlighted in red indicate sites that are mutated in Omicron variants. h , Average escape maps of antibodies in epitope groups E1, F2 and F3, and the corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron subvariants are marked in bold. All neutralization assays were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Activity Assay, Two Tailed Test, Binding Assay, Sequencing, Mutagenesis, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Cartoon models of Cryo-EM structures of BD55-3152 in complex of BA.1 RBD, BD55-1239 in complex of BA.1 RBD, and BD55-3372 in complex of Delta RBD. b , Workflow to generate refined structural model of BD55-3152 and BD55-1239 in complex of BA.1 RBD, BD55-3372 in complex of Delta RBD, and BD55-5840 in complex of BA.2 RBD. c , Neutralizing activity of representative NAbs in group E1 (n = 68), F2 (n = 139) and F3 (n = 61) against SARS-CoV-2 D614G, in addition to D614G+D405N and D614G+R408S. Geometric mean of IC50 fold changes compared to IC50 against D614G are annotated above the bars. P-values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. d , Conformational comparison between BA.1 and BA.2 RBD regarding the 366-377 hairpin. e , Biolayer interferometry analysis of Group E1 antibodies S309 and BD55-5840 binding to Omicron BA.1 and BA.2 Spike trimer. Biolayer interferometry analyses were conducted in biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Cryo-EM Sample Prep, Activity Assay, Two Tailed Test, Neutralization, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Four epitope groups were identified among 102 BA.1-specific NAbs via k -means clustering and t -SNE of BA.1 RBD-based DMS profiles. b , c , Distribution of ACE2 competition level ( b ) and neutralizing activities ( c ) against BA.1. d , Neutralizing activities of BA.1-specific antibodies against pseudovirus with SARS-CoV-1 and SARS-CoV-2 spike variants (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. e , Average mutational escape score projection of each BA.1-specific epitope group on SARS-CoV-2 RBD (PDB: 7WPB). f , Averaged escape maps at escape hotspots of the 102 NAbs in the four epitope groups, and corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron variants are marked in bold. WT-related escaping mutations are highlighted. g , Neutralizing activities of BA.1-specific NAbs against BA.1- or BA.2-based pseudoviruses carrying single substitutions (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. Wilcoxon signed-rank test of paired samples, compared with IC 50 for BA.1. All neutralization assays were performed as biological duplicates.

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques: Sequencing, Mutagenesis, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: Heavy chain V-J gene combination of BA.1-specific neutralizing antibodies in BA.1-specific epitope groups A Omi , B Omi , D Omi and F3 Omi .

Article Snippet: Every 10 6 B cells in 100 μl solution were then stained with 3 μl FITC anti-human CD20 antibody (BioLegend, 302304), 3.5 μl Brilliant Violet 421 anti-human CD27 antibody (BioLegend, 302824), 2 μl PE/Cyanine7 anti-human IgM antibody (BioLegend, 314532), 2 μl PE/Cyanine7 anti-human IgD antibody (BioLegend, 348210), 0.13 μg biotinylated SARS-CoV-2 BA.1 protein (His and AVI Tag) (Sino Biological, 40592-V49H7-B) conjugated with PE-streptavidin or APC-streptavidin (TotalSeq-C0971 Streptavidin, BioLegend, 405271 and TotalSeq-C0972 Streptavidin, BioLegend, 405273), 0.13 μg SARS-CoV-2 WT biotinylated RBD protein (His and AVI Tag) conjugated with Brilliant Violet 605 Streptavidin and TotalSeq-C0973 Streptavidin (BioLegend, 405275) and TotalSeq-C0974 Streptavidin(BioLegend, 405277), 0.21 μg biotinylated Ovalbumin conjugated with TotalSeq-C0975 Streptavidin (BioLegend, 405279) and 5 μl 7-AAD (Invitrogen, 00-6993-50).

Techniques:

Journal: bioRxiv

Article Title: An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

doi: 10.1101/2020.06.02.130161

Figure Lengend Snippet: a , VSV G or SARS-CoV-2 spike pseudotyped lentivirus (PSV) was incubated with a dilution series of Ty1 or control nanobody (influenza NP-VHH1 ). Infectivity relative to cells infected with pseudotyped virus in the absence of nanobody is shown. Neutralization by Ty1 was repeated in duplicate across 6 assays, and the error bars represent the standard deviation. b , Cells were transfected with a plasmid harboring the SARS-CoV-2 spike for 24 h. Cells were fixed, permeabilized and stained with Ty1-AS635P (black and red) or left unstained (grey). Cells were analyzed by flow cytometry. Cell counts are presented as % of max (representative histogram). c , Vero E6 cells were infected with SARS-CoV-2 at a MOI of 1 for 24 hours. Cells were fixed, permeabilized and stained for DNA (blue), dsRNA (green) and with Ty1-AS635P (red). Pictures were taken by fluorescence microscopy and representative examples are shown. d , ACE2 expressing HEK293T cells were trypsinized, fixed and stained with RBD-AS635P alone, or preincubated with IAV NP-VHH1 or Ty1. Cells were analyzed by flow cytometry.

Article Snippet: SARS-CoV-2 S1-sheep-Fc (Native Antigen Company, SKU: REC31806) was used for the first two immunization, and SARS-CoV-2 RBD produced in FreeStyle 293F cells was used for the last two immunizations.

Techniques: Incubation, Infection, Virus, Neutralization, Standard Deviation, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Fluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

doi: 10.1101/2020.06.02.130161

Figure Lengend Snippet: SARS-CoV-2 spike pseudotyped lentivirus (PSV) was incubated with a dilution series of Ty1 or Ty1-Fc. Infectivity relative to cells infected with pseudotyped virus in the absence of nanobody is shown. Neutralization was repeated in duplicate across 3 assays, and the error bars represent the standard deviation

Article Snippet: SARS-CoV-2 S1-sheep-Fc (Native Antigen Company, SKU: REC31806) was used for the first two immunization, and SARS-CoV-2 RBD produced in FreeStyle 293F cells was used for the last two immunizations.

Techniques: Incubation, Infection, Virus, Neutralization, Standard Deviation