BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection methods
Aim. Evidence-backed execution summary for BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection methods from BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection.
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Main
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- The recent emergence and global spread of the SARS-CoV-2 variant Omicron (B.1.1.529) have posed a critical challenge to the efficacy of COVID-19 vaccines and neutralizing antibody (NAb) therapy -. Owing to multiple mutations to the spike protein, including in the receptor-binding domain (RBD) and N-terminal d...
Structural analyses of Omicron spike
reagent used in the protocol.
- Use
- Next, we measured the binding affinity between human ACE2 (hACE2) and S-trimers of the Omicron variants by surface plasmon resonance (SPR) (Extended Data Fig. ). The BA.4/BA.5 S-trimer showed a decreased binding affinity with hACE2 compared with those of the other Omicron subvariants; however, this measurement could...
NAb evasion by BA.2.12.1, BA.4 and BA.5
reagent used in the protocol.
- Use
- To probe NAb evasion by the recently emerged Omicron sublineages, we performed pseudovirus-neutralization assays using D614G, BA.1, BA.1.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA.4/BA.5 against plasma obtained from individuals who had received three doses of SARS-CoV-2 vaccine, vaccinated individuals who had recovere...
NAb evasion by BA.2.12.1, BA.4 and BA.5
reagent used in the protocol.
- Use
- Next, we examined the neutralizing activities of therapeutic antibodies against new Omicron subvariants (Fig. ). All seven tested Omicron subvariants displayed substantial evasion against neutralization by class 1 and class 2 RBD antibodies: the variants evaded neutralization by REGN-10933 (casirivimab), LY-CoV016 (...
NAb evasion by BA.2.12.1, BA.4 and BA.5
reagent used in the protocol.
- Use
- To delineate the underlying antibody-evasion mechanism of BA.2.13, BA.2.12.1 and BA.4/BA.5, in particular the escape from the humoral immunity induced by recovery from BA.1 infection or recovery from SARS in vaccinated individuals, we began by isolating RBD-targeting NAbs from such individuals, (Extended Data Fig....
Pseudovirus-neutralization assay
reagent used in the protocol.
- Use
- SARS-CoV-2 spike (GenBank: MN908947 ), Pangolin-GD spike (GISAID: EPI_ISL_410721), RaTG13 spike (GISAID: EPI_ISL_402131), SARS-CoV-1 spike (GenBank: AY278491 ), Omicron BA.1 spike (A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S...
Omicron escapes broad sarbecovirus NAbs
reagent used in the protocol.
- Use
- In total, five clusters of antibodies were found to exhibit broad sarbecovirus-neutralizing ability with diverse specificity, namely groups E1, E3, F1, F2 and F3 (Extended Data Fig. ). Whereas Group E3 and F1 antibodies demonstrated weak neutralizing activity against all variants owing to their highly conserved bind...
Omicron escapes broad sarbecovirus NAbs
reagent used in the protocol.
- Use
- The epitopes of group F2 and F3 antibodies cover a continuous surface on the back of the RBD and can only bind to RBDs in the up configuration (Fig. ). To probe how BA.2 escapes group F2 and F3 antibodies, we solved the cryo-EM structure of two representative BA.1-neutralizing antibodies-BD55-1239 from group F...
NAb evasion by BA.2.12.1, BA.4 and BA.5
To delineate the underlying antibody-evasion mechanism of BA.2.13, BA.2.12.1 and BA.4/BA.5, in particular the escape from the humoral immunity induced by recovery from BA.1 infection or recovery from SARS in vaccinated individuals, we began by isolating RBD-targeting NAbs from such individuals, (Extended Data Fig....
- Use
- To delineate the underlying antibody-evasion mechanism of BA.2.13, BA.2.12.1 and BA.4/BA.5, in particular the escape from the humoral immunity induced by recovery from BA.1 infection or recovery from SARS in vaccinated individuals, we began by isolating RBD-targeting NAbs from such individuals, (Extended Data Fig....
BA.1-specific NAbs exhibit narrow breadths
By combining high-throughput single-cell sequencing and high-throughput yeast display-based DMS, we have demonstrated the ability to decipher the complicated humoral immune repertoire elicited by Omicron infection and the underlying immune-evasion mechanism of L452 and F486 mutations. The ability to dissect the enti...
- Use
- By combining high-throughput single-cell sequencing and high-throughput yeast display-based DMS, we have demonstrated the ability to decipher the complicated humoral immune repertoire elicited by Omicron infection and the underlying immune-evasion mechanism of L452 and F486 mutations. The ability to dissect the enti...
Antibody isolation and recombinant production
SARS-CoV-2 BA.1 RBD-binding memory B cells were isolated from BA.1-infected convalescents who received SARS-CoV-2. In brief, CD19 + B cells were isolated with EasySep Human CD19 Positive Selection Kit II. Every 10 6 B cells in 100 µl solution were then stained with 3 µl FITC anti-human CD20 ant...
- Use
- SARS-CoV-2 BA.1 RBD-binding memory B cells were isolated from BA.1-infected convalescents who received SARS-CoV-2. In brief, CD19 + B cells were isolated with EasySep Human CD19 Positive Selection Kit II. Every 10 6 B cells in 100 µl solution were then stained with 3 µl FITC anti-human CD20 ant...
Antibody isolation and recombinant production
Sorted B cells were then processed with Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 following the manufacturer's user guide (10x Genomics, CG000208 ). In brief, sorted cells were resuspended in PBS after centrifugation. Gel beads-in-emulsion (GEMs) were obtained with 10X Chromium controller and then...
- Use
- Sorted B cells were then processed with Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 following the manufacturer's user guide (10x Genomics, CG000208 ). In brief, sorted cells were resuspended in PBS after centrifugation. Gel beads-in-emulsion (GEMs) were obtained with 10X Chromium controller and then...
Antibody sequence analysis
The antibody sequences obtained from 10X Genomics V(D)J sequencing were aligned to GRCh38 reference and assembled as immunoglobulin contigs by the Cell Ranger (v6.1.1) pipeline. Non-productive contigs and B cells that had multiple heavy chain or light chain contigs were filtered out of the analysis. V(D)J gene annot...
- Use
- The antibody sequences obtained from 10X Genomics V(D)J sequencing were aligned to GRCh38 reference and assembled as immunoglobulin contigs by the Cell Ranger (v6.1.1) pipeline. Non-productive contigs and B cells that had multiple heavy chain or light chain contigs were filtered out of the analysis. V(D)J gene annot...
DMS library construction
DMS libraries were constructed as previously described. In brief, SARS-CoV-2 RBD mutant libraries were constructed from Wuhan-Hu-1 RBD sequence (GenBank: MN908947, residues N331-T531), and Omicron RBD mutant libraries were created in a similar way based on Wuhan-Hu-1 RBD sequence with the addition of G339D,...
- Use
- DMS libraries were constructed as previously described. In brief, SARS-CoV-2 RBD mutant libraries were constructed from Wuhan-Hu-1 RBD sequence (GenBank: MN908947, residues N331-T531), and Omicron RBD mutant libraries were created in a similar way based on Wuhan-Hu-1 RBD sequence with the addition of G339D,...
High-throughput antibody-escape mutation profiling
The magnetic-activated cell sorting-based antibody-escape mutation profiling system, was used to characterize mutation escape profile for NAbs. In brief, ACE2-binding mutants were induced overnight for RBD expression and washed followed with two rounds of Protein A antibody-based negative selection and MYC tag-base...
- Use
- The magnetic-activated cell sorting-based antibody-escape mutation profiling system, was used to characterize mutation escape profile for NAbs. In brief, ACE2-binding mutants were induced overnight for RBD expression and washed followed with two rounds of Protein A antibody-based negative selection and MYC tag-base...
High-throughput antibody-escape mutation profiling
After three rounds of sequential cell sorting, the obtained cells were recovered overnight. Plasmids were extracted from pre- and post-sort yeast populations by 96-Well Plate Yeast Plasmid Preps Kit (Coolaber, PE053). The extracted plasmids were then used to amplify N26 barcode sequences by PCR. The final PCR produc...
- Use
- After three rounds of sequential cell sorting, the obtained cells were recovered overnight. Plasmids were extracted from pre- and post-sort yeast populations by 96-Well Plate Yeast Plasmid Preps Kit (Coolaber, PE053). The extracted plasmids were then used to amplify N26 barcode sequences by PCR. The final PCR produc...
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NAb evasion by BA.2.12.1, BA.4 and BA.5
To probe NAb evasion by the recently emerged Omicron sublineages, we performed pseudovirus-neutralization assays using D614G, BA.1, BA.1.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA.4/BA.5 against plasma obtained from individuals who had received three doses of SARS-CoV-2 vaccine, vaccinated individuals who had recovered from BA.1 infection, and vaccinated individuals who had recovered from severe acute respiratory syndrome (SARS) (Supplementary Table ). Plasma samples were collected four weeks after the booster shot or four weeks after discharge from hospital following COVID-19 illness. In plasma from individuals who had received an inactivated virus (CoronaVac) or RBD protein (ZF2001) booster six months after two doses of CoronaVac, BA.1, BA.1.1 and BA.2 showed no significant difference in resistance to neutralization by plasma (Fig. ), concordant with previous reports,. However, we f...
NAb evasion by BA.2.12.1, BA.4 and BA.5
Omicron subvariants evaded most WT-stimulated group A, B and C NAbs, although a subset of these antibodies showed broad effectiveness against Omicron (Extended Data Fig. ). These broad NAbs were largely enriched by BA.1 stimulation, are generally encoded by similar heavy chain V genes compared with WT-stimulated antibodies and display higher convergence (Extended Data Fig. ). These broad ACE2-competing NAbs in groups A, B and C have been shown to be enriched in individuals who received a booster dose of mRNA vaccine, which probably accounts for the high neutralizing activity against Omicron variants in plasma of individuals who had received three doses of mRNA vaccine. Nevertheless, BA.1-stimulated group B and C NAbs were significantly evaded by BA.4 owing to F486V and L452R RBD mutations, concordant with results from DMS (Extended Data Fig. ), which explains the strong humoral immun...
Methods
Blood samples were obtained from 40 volunteers who had received 3 doses of CoronaVac, 39 individuals who had received 2 doses of CoronaVac and 1 booster dose of ZF2001, 54 individuals who had recovered from BA.1 infection who had previously received 3 doses of CoronaVac,, and 30 individuals who had recovered from SARS who had received 2 doses of CoronaVac and 1 dose of ZF2001. The volunteers' blood samples were obtained four weeks after the booster shot or four weeks after discharge from the hospital following BA.1 infection.COVID-19 disease severity was defined as asymptomatic, mild, moderate, severe or critical according to the WHO Living Guidance for Clinical Management of COVID-19. Relevant experiments with plasma from SARS convalescents and SARS-CoV-2 vaccinees were approved by the Beijing Ditan Hospital Capital Medical University (ethics committee archiving no. LL-2021-...
High-throughput antibody-escape mutation profiling
The magnetic-activated cell sorting-based antibody-escape mutation profiling system, was used to characterize mutation escape profile for NAbs. In brief, ACE2-binding mutants were induced overnight for RBD expression and washed followed with two rounds of Protein A antibody-based negative selection and MYC tag-based positive selection to enrich RBD-expressing cells. Protein A antibody-conjugated products were prepared following the protocol for Dynabeads Protein A (Thermo Fisher, 10008D) and incubated with induced yeast libraries at room temperature for 30 min with shaking. MYC tag-based positive selection was performed according to the manufacturer's instructions (Thermo Fisher, 88843).
ELISA
To detect the broad-spectrum binding of the antibodies among Sarbecovirus, we used a panel of 20 synthesized sarbecovirus RBDs (Sino Biological Technology) (Supplementary Table ). According to the sequence of 20 RBDs, a set of nested primers was designed. The coding sequences were obtained by the overlap PCR with a 6 × His tag sequence to facilitate protein purification. The purified PCR products were ligated to the secretory expression vector pCMV3 with CMV promoter, and then transformed into Escherichia coli XL1-blue competent cells. Monoclones with correct transformation were cultured and expanded, and plasmids were extracted. Healthy HEK293F cells were passaged into a new cell culture and grown in suspension at 37 °C, 120 RPM, 8% CO2 to logarithmic growth phase and transfected with the recombinant constructs by using liposomal vesicles as DNA carrier. After transfec...
ELISA
A panel of 21 sarbecovirus RBDs (Supplementary Table ) in PBS was pre-coated onto ELISA plates (NEST, 514201) at 4 °C overnight. The plates were washed and blocked. Then 1 µg ml -1 purified antibodies or serially diluted antibodies were added and incubated at room temperature for 20 min. Next, Peroxidase-conjugated AffiniPure Goat Anti-Human IgG (H+L) (JACKSON, 109-035-003) was applied and incubated at room temperature for 15 min. Tetramethylbenzidine (TMB) (Solarbio, 54827-17-7) was added onto the plates. The reaction was terminated with 2 M H 2 SO 4 after 10 min incubation. Absorbance was measured at 450 nm using Ensight Multimode Plate Reader (PerkinElmer, HH3400). ELISA A 450 measurements at different antibody concentrations for a particular antibody-antigen pair were fit to the model y = Ac n /( c...
Antibody and ACE2 competition for RBD
Omicron RBD (Sino Biological, 40592-V08H121) protein in PBS was immobilized on the ELISA plates at 4 °C overnight. The coating solution was removed and washed 3 times with PBST and the plates were then blocked for 2 h. After blocking, the plates were washed 5 times, and the mixture of ACE2-biotin (Sino Biological, 10108-H27B-B) and serially diluted competitor antibodies was added followed by 30 min incubation at room temperature. Peroxidase-conjugated Streptavidin (Jackson ImmunoResearch, 016-030-084) was added into each well for another 20 min incubation at room temperature. After washing the plates five times, TMB (Solarbio, 54827-17-7) was added into each well. After 10 min, the reaction was terminated with 2 M H 2 SO 4. Absorbancewas measured at 450 nm using Ensight Multimode Plate Reader (PerkinElmer, HH3400). The ACE2 competition c...
Biolayer interferometry
Biolayer interferometry assays were performed on Octet RED 384 Protein Analysis System (Fortebio) according to the manufacturer's instructions. To measure the binding affinities, monoclonal antibodies were immobilized onto Protein A biosensors (Fortebio) and the fourfold serial dilutions of Omicron S-trimer (BA.1 and BA.2) in PBS were used as analytes. Data were collected with Octet Acquisition 9.0 (Fortebio) and analysed by Octet Analysis 9.0 (Fortebio) and Octet Analysis Studio 12.2 (Fortebio).
Measurement outputs
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Summarized information and experiment results of 1640 SARS-CoV-2 RBD antibodies involved in this study, including their sources, epitope groups, pseudovirus neutralizing IC50 an...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
We expressed and purified the prefusion-stabilized trimeric ectodomains of BA.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA.4/BA.5 spike (S-trimer). All the S-trimers contain Gly-Ser...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
To delineate the underlying antibody-evasion mechanism of BA.2.13, BA.2.12.1 and BA.4/BA.5, in particular the escape from the humoral immunity induced by recovery from BA.1 infe...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
To further specify the epitope distribution of NAbs elicited by post-vaccination BA.1 infection, we applied high-throughput yeast display-based DMS assays, and determined the m...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
To probe NAb evasion by the recently emerged Omicron sublineages, we performed pseudovirus-neutralization assays using D614G, BA.1, BA.1.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA.4/BA.5 against plasma obtained from individuals who had received three doses of SARS-CoV-2 vaccine,...
from paperScoring or quantification
Quantify the primary readouts for this experiment: Summarized information and experiment results of 1640 SARS-CoV-2 RBD antibodies involved in this study, including their sources, epitope groups, pseudovirus neutralizing IC50 an...; We expressed and purified the prefusion-stabilized trimeric ectodomains of BA.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA.4/BA.5 spike (S-trimer). All the S-trimers contain Gly-Ser...; To delineate the underlying antibody-evasion mechanism of BA.2.13, BA.2.12.1 and BA.4/BA.5, in particular the escape from the humoral immunity induced by recovery from BA.1 infe...; To further specify the epitope distribution of NAbs elicited by post-vaccination BA.1 infection, we applied high-throughput yeast display-based DMS assays, and determined the m....
from paperStatistical comparison
To probe NAb evasion by the recently emerged Omicron sublineages, we performed pseudovirus-neutralization assays using D614G, BA.1, BA.1.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA...; To delineate the underlying antibody-evasion mechanism of BA.2.13, BA.2.12.1 and BA.4/BA.5, in particular the escape from the humoral immunity induced by recovery from BA.1 infe...; Group D antibodies were most affected by the G446S mutation in BA.1, BA.1.1 and BA.3 (Fig. ); these NAbs therefore show higher potency against BA.2 (Fig. ). However, group D1 an...; In total, five clusters of antibodies were found to exhibit broad sarbecovirus-neutralizing ability with diverse specificity, namely groups E1, E3, F1, F2 and F3 (Extended Data...
from paperReporting output
Report representative outputs alongside summary comparisons for Summarized information and experiment results of 1640 SARS-CoV-2 RBD antibodies involved in this study, including their sources, epitope groups, pseudovirus neutralizing IC50 an..., We expressed and purified the prefusion-stabilized trimeric ectodomains of BA.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA.4/BA.5 spike (S-trimer). All the S-trimers contain Gly-Ser..., To delineate the underlying antibody-evasion mechanism of BA.2.13, BA.2.12.1 and BA.4/BA.5, in particular the escape from the humoral immunity induced by recovery from BA.1 infe..., To further specify the epitope distribution of NAbs elicited by post-vaccination BA.1 infection, we applied high-throughput yeast display-based DMS assays, and determined the m....
inferred from protocolStructured statistical methods
To probe NAb evasion by the recently emerged Omicron sublineages, we performed pseudovirus-neutralization assays using D614G, BA.1, BA.1.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA...; To delineate the underlying antibody-evasion mechanism of BA.2.13, BA.2.12.1 and BA.4/BA.5, in particular the escape from the humoral immunity induced by recovery from BA.1 infe...; Group D antibodies were most affected by the G446S mutation in BA.1, BA.1.1 and BA.3 (Fig. ); these NAbs therefore show higher potency against BA.2 (Fig. ). However, group D1 an...; In total, five clusters of antibodies were found to exhibit broad sarbecovirus-neutralizing ability with diverse specificity, namely groups E1, E3, F1, F2 and F3 (Extended Data...
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Evidence quotes (8)
To probe NAb evasion by the recently emerged Omicron sublineages, we performed pseudovirus-neutralization assays using D614G, BA.1, BA.1.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA.4/BA.5 against plasma obtained from individuals who had received three doses of SARS-CoV-2 vaccine, vaccinated individuals who had recovered from BA.1 infection, and vaccinated individuals who had recovered from severe acute respiratory syndrome (SARS) (Supplementary Table ). Plasma samples were collected four weeks after the booster shot or four weeks after discharge from hospital following COVID-19 illness. In plasma from individuals who had received an inactivated virus (CoronaVac) or RBD protein (ZF2001) booster six months after two doses of CoronaVac, BA.1, BA.1.1 and BA.2 showed no significant difference in resistance to neutralization by plasma (Fig. ), concordant with previous reports,. However, we found that BA.2 subvariants BA.2.13 and BA.2.12.1 showed increased immune-evasion capability over BA.2-with BA.2.12.1 exhibiting greater evasion than BA.2.13-and BA.4/BA.5 exhibiting even greater evasion (Fig. ). The decrease in neutralization was clearer in plasma obtained from individua...
Omicron subvariants evaded most WT-stimulated group A, B and C NAbs, although a subset of these antibodies showed broad effectiveness against Omicron (Extended Data Fig. ). These broad NAbs were largely enriched by BA.1 stimulation, are generally encoded by similar heavy chain V genes compared with WT-stimulated antibodies and display higher convergence (Extended Data Fig. ). These broad ACE2-competing NAbs in groups A, B and C have been shown to be enriched in individuals who received a booster dose of mRNA vaccine, which probably accounts for the high neutralizing activity against Omicron variants in plasma of individuals who had received three doses of mRNA vaccine. Nevertheless, BA.1-stimulated group B and C NAbs were significantly evaded by BA.4 owing to F486V and L452R RBD mutations, concordant with results from DMS (Extended Data Fig. ), which explains the strong humoral immune-evasion ability of BA.4/BA.5.
Blood samples were obtained from 40 volunteers who had received 3 doses of CoronaVac, 39 individuals who had received 2 doses of CoronaVac and 1 booster dose of ZF2001, 54 individuals who had recovered from BA.1 infection who had previously received 3 doses of CoronaVac,, and 30 individuals who had recovered from SARS who had received 2 doses of CoronaVac and 1 dose of ZF2001. The volunteers' blood samples were obtained four weeks after the booster shot or four weeks after discharge from the hospital following BA.1 infection.COVID-19 disease severity was defined as asymptomatic, mild, moderate, severe or critical according to the WHO Living Guidance for Clinical Management of COVID-19. Relevant experiments with plasma from SARS convalescents and SARS-CoV-2 vaccinees were approved by the Beijing Ditan Hospital Capital Medical University (ethics committee archiving no. LL-2021-024-02), the Tianjin Municipal Health Commission, and the ethics committee of Tianjin First Central Hospital (ethics committee archiving no. 2022N045KY). Written informed consent was obtained from each participant in accordance with the Declaration of Helsinki. All participants provided written info...
The magnetic-activated cell sorting-based antibody-escape mutation profiling system, was used to characterize mutation escape profile for NAbs. In brief, ACE2-binding mutants were induced overnight for RBD expression and washed followed with two rounds of Protein A antibody-based negative selection and MYC tag-based positive selection to enrich RBD-expressing cells. Protein A antibody-conjugated products were prepared following the protocol for Dynabeads Protein A (Thermo Fisher, 10008D) and incubated with induced yeast libraries at room temperature for 30 min with shaking. MYC tag-based positive selection was performed according to the manufacturer's instructions (Thermo Fisher, 88843).
To detect the broad-spectrum binding of the antibodies among Sarbecovirus, we used a panel of 20 synthesized sarbecovirus RBDs (Sino Biological Technology) (Supplementary Table ). According to the sequence of 20 RBDs, a set of nested primers was designed. The coding sequences were obtained by the overlap PCR with a 6 × His tag sequence to facilitate protein purification. The purified PCR products were ligated to the secretory expression vector pCMV3 with CMV promoter, and then transformed into Escherichia coli XL1-blue competent cells. Monoclones with correct transformation were cultured and expanded, and plasmids were extracted. Healthy HEK293F cells were passaged into a new cell culture and grown in suspension at 37 °C, 120 RPM, 8% CO2 to logarithmic growth phase and transfected with the recombinant constructs by using liposomal vesicles as DNA carrier. After transfection, the cell cultures were followed to assess the kinetics of cell growth and viability for 7 days. The cell expression supernatant was collected, and after centrifugation, passed through a Ni column for affinity purification. The molecular size and purity of eluted protein was confirmed by SDS...
A panel of 21 sarbecovirus RBDs (Supplementary Table ) in PBS was pre-coated onto ELISA plates (NEST, 514201) at 4 °C overnight. The plates were washed and blocked. Then 1 µg ml -1 purified antibodies or serially diluted antibodies were added and incubated at room temperature for 20 min. Next, Peroxidase-conjugated AffiniPure Goat Anti-Human IgG (H+L) (JACKSON, 109-035-003) was applied and incubated at room temperature for 15 min. Tetramethylbenzidine (TMB) (Solarbio, 54827-17-7) was added onto the plates. The reaction was terminated with 2 M H 2 SO 4 after 10 min incubation. Absorbance was measured at 450 nm using Ensight Multimode Plate Reader (PerkinElmer, HH3400). ELISA A 450 measurements at different antibody concentrations for a particular antibody-antigen pair were fit to the model y = Ac n /( c n + E n ) using the R package mosaic (v1.8.3), where y is the A 450 value and c is the corresponding antibody concentration. A, E and n are parameters, where E is the desired EC 50 value for the specific antibody and antigen.
Omicron RBD (Sino Biological, 40592-V08H121) protein in PBS was immobilized on the ELISA plates at 4 °C overnight. The coating solution was removed and washed 3 times with PBST and the plates were then blocked for 2 h. After blocking, the plates were washed 5 times, and the mixture of ACE2-biotin (Sino Biological, 10108-H27B-B) and serially diluted competitor antibodies was added followed by 30 min incubation at room temperature. Peroxidase-conjugated Streptavidin (Jackson ImmunoResearch, 016-030-084) was added into each well for another 20 min incubation at room temperature. After washing the plates five times, TMB (Solarbio, 54827-17-7) was added into each well. After 10 min, the reaction was terminated with 2 M H 2 SO 4. Absorbancewas measured at 450 nm using Ensight Multimode Plate Reader (PerkinElmer, HH3400). The ACE2 competition coefficient was calculated as ( B - A )/ B, where B is the A 450 value with 0.3 µg ml -1 antibody and A is the A 450 value with 6 µg ml -1 antibody.
Biolayer interferometry assays were performed on Octet RED 384 Protein Analysis System (Fortebio) according to the manufacturer's instructions. To measure the binding affinities, monoclonal antibodies were immobilized onto Protein A biosensors (Fortebio) and the fourfold serial dilutions of Omicron S-trimer (BA.1 and BA.2) in PBS were used as analytes. Data were collected with Octet Acquisition 9.0 (Fortebio) and analysed by Octet Analysis 9.0 (Fortebio) and Octet Analysis Studio 12.2 (Fortebio).
Machine-readable layer
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"name": "NAb evasion by BA.2.12.1, BA.4 and BA.5",
"text": "To probe NAb evasion by the recently emerged Omicron sublineages, we performed pseudovirus-neutralization assays using D614G, BA.1, BA.1.1, BA.2, BA.3, BA.2.12.1, BA.2.13 and BA.4/BA.5 against plasma obtained from individuals who had received three doses of SARS-CoV-2 vaccine, vaccinated individuals who had recovered from BA.1 infection, and vaccinated individuals who had recovered from severe acute respiratory syndrome (SARS) (Supplementary Table ). Plasma samples were collected four weeks after the booster shot or four weeks after discharge from hospital following COVID-19 illness. In plasma from individuals who had received an inactivated virus (CoronaVac) or RBD protein (ZF2001) booster six months after two doses of CoronaVac, BA.1, BA.1.1 and BA.2 showed no significant difference in resistance to neutralization by plasma (Fig. ), concordant with previous reports,. However, we f..."
},
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"name": "NAb evasion by BA.2.12.1, BA.4 and BA.5",
"text": "Omicron subvariants evaded most WT-stimulated group A, B and C NAbs, although a subset of these antibodies showed broad effectiveness against Omicron (Extended Data Fig. ). These broad NAbs were largely enriched by BA.1 stimulation, are generally encoded by similar heavy chain V genes compared with WT-stimulated antibodies and display higher convergence (Extended Data Fig. ). These broad ACE2-competing NAbs in groups A, B and C have been shown to be enriched in individuals who received a booster dose of mRNA vaccine, which probably accounts for the high neutralizing activity against Omicron variants in plasma of individuals who had received three doses of mRNA vaccine. Nevertheless, BA.1-stimulated group B and C NAbs were significantly evaded by BA.4 owing to F486V and L452R RBD mutations, concordant with results from DMS (Extended Data Fig. ), which explains the strong humoral immun..."
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"position": 3,
"name": "Methods",
"text": "Blood samples were obtained from 40 volunteers who had received 3 doses of CoronaVac, 39 individuals who had received 2 doses of CoronaVac and 1 booster dose of ZF2001, 54 individuals who had recovered from BA.1 infection who had previously received 3 doses of CoronaVac,, and 30 individuals who had recovered from SARS who had received 2 doses of CoronaVac and 1 dose of ZF2001. The volunteers' blood samples were obtained four weeks after the booster shot or four weeks after discharge from the hospital following BA.1 infection.COVID-19 disease severity was defined as asymptomatic, mild, moderate, severe or critical according to the WHO Living Guidance for Clinical Management of COVID-19. Relevant experiments with plasma from SARS convalescents and SARS-CoV-2 vaccinees were approved by the Beijing Ditan Hospital Capital Medical University (ethics committee archiving no. LL-2021-..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "High-throughput antibody-escape mutation profiling",
"text": "The magnetic-activated cell sorting-based antibody-escape mutation profiling system, was used to characterize mutation escape profile for NAbs. In brief, ACE2-binding mutants were induced overnight for RBD expression and washed followed with two rounds of Protein A antibody-based negative selection and MYC tag-based positive selection to enrich RBD-expressing cells. Protein A antibody-conjugated products were prepared following the protocol for Dynabeads Protein A (Thermo Fisher, 10008D) and incubated with induced yeast libraries at room temperature for 30 min with shaking. MYC tag-based positive selection was performed according to the manufacturer's instructions (Thermo Fisher, 88843)."
},
{
"@type": "HowToStep",
"position": 5,
"name": "ELISA",
"text": "To detect the broad-spectrum binding of the antibodies among Sarbecovirus, we used a panel of 20 synthesized sarbecovirus RBDs (Sino Biological Technology) (Supplementary Table ). According to the sequence of 20 RBDs, a set of nested primers was designed. The coding sequences were obtained by the overlap PCR with a 6 × His tag sequence to facilitate protein purification. The purified PCR products were ligated to the secretory expression vector pCMV3 with CMV promoter, and then transformed into Escherichia coli XL1-blue competent cells. Monoclones with correct transformation were cultured and expanded, and plasmids were extracted. Healthy HEK293F cells were passaged into a new cell culture and grown in suspension at 37 °C, 120 RPM, 8% CO2 to logarithmic growth phase and transfected with the recombinant constructs by using liposomal vesicles as DNA carrier. After transfec..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "ELISA",
"text": "A panel of 21 sarbecovirus RBDs (Supplementary Table ) in PBS was pre-coated onto ELISA plates (NEST, 514201) at 4 °C overnight. The plates were washed and blocked. Then 1 µg ml -1 purified antibodies or serially diluted antibodies were added and incubated at room temperature for 20 min. Next, Peroxidase-conjugated AffiniPure Goat Anti-Human IgG (H+L) (JACKSON, 109-035-003) was applied and incubated at room temperature for 15 min. Tetramethylbenzidine (TMB) (Solarbio, 54827-17-7) was added onto the plates. The reaction was terminated with 2 M H 2 SO 4 after 10 min incubation. Absorbance was measured at 450 nm using Ensight Multimode Plate Reader (PerkinElmer, HH3400). ELISA A 450 measurements at different antibody concentrations for a particular antibody-antigen pair were fit to the model y = Ac n /( c..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Antibody and ACE2 competition for RBD",
"text": "Omicron RBD (Sino Biological, 40592-V08H121) protein in PBS was immobilized on the ELISA plates at 4 °C overnight. The coating solution was removed and washed 3 times with PBST and the plates were then blocked for 2 h. After blocking, the plates were washed 5 times, and the mixture of ACE2-biotin (Sino Biological, 10108-H27B-B) and serially diluted competitor antibodies was added followed by 30 min incubation at room temperature. Peroxidase-conjugated Streptavidin (Jackson ImmunoResearch, 016-030-084) was added into each well for another 20 min incubation at room temperature. After washing the plates five times, TMB (Solarbio, 54827-17-7) was added into each well. After 10 min, the reaction was terminated with 2 M H 2 SO 4. Absorbancewas measured at 450 nm using Ensight Multimode Plate Reader (PerkinElmer, HH3400). The ACE2 competition c..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Biolayer interferometry",
"text": "Biolayer interferometry assays were performed on Octet RED 384 Protein Analysis System (Fortebio) according to the manufacturer's instructions. To measure the binding affinities, monoclonal antibodies were immobilized onto Protein A biosensors (Fortebio) and the fourfold serial dilutions of Omicron S-trimer (BA.1 and BA.2) in PBS were used as analytes. Data were collected with Octet Acquisition 9.0 (Fortebio) and analysed by Octet Analysis 9.0 (Fortebio) and Octet Analysis Studio 12.2 (Fortebio)."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "NAb evasion by BA.2.12.1, BA.4 and BA.5"
},
{
"@type": "HowToTool",
"name": "BA.1-specific NAbs exhibit narrow breadths"
},
{
"@type": "HowToTool",
"name": "Antibody isolation and recombinant production"
},
{
"@type": "HowToTool",
"name": "Antibody isolation and recombinant production"
},
{
"@type": "HowToTool",
"name": "Antibody sequence analysis"
},
{
"@type": "HowToTool",
"name": "DMS library construction"
},
{
"@type": "HowToTool",
"name": "High-throughput antibody-escape mutation profiling"
},
{
"@type": "HowToTool",
"name": "High-throughput antibody-escape mutation profiling"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Main"
},
{
"@type": "HowToSupply",
"name": "Structural analyses of Omicron spike"
},
{
"@type": "HowToSupply",
"name": "NAb evasion by BA.2.12.1, BA.4 and BA.5"
},
{
"@type": "HowToSupply",
"name": "NAb evasion by BA.2.12.1, BA.4 and BA.5"
},
{
"@type": "HowToSupply",
"name": "NAb evasion by BA.2.12.1, BA.4 and BA.5"
},
{
"@type": "HowToSupply",
"name": "Pseudovirus-neutralization assay"
},
{
"@type": "HowToSupply",
"name": "Omicron escapes broad sarbecovirus NAbs"
},
{
"@type": "HowToSupply",
"name": "Omicron escapes broad sarbecovirus NAbs"
}
],
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"@type": "ScholarlyArticle",
"headline": "BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection",
"datePublished": "2022",
"author": [
{
"@type": "Person",
"name": "Yunlong Cao"
},
{
"@type": "Person",
"name": "Ayijiang Yisimayi"
},
{
"@type": "Person",
"name": "Fanchong Jian"
},
{
"@type": "Person",
"name": "Weiliang Song"
},
{
"@type": "Person",
"name": "Tianhe Xiao"
},
{
"@type": "Person",
"name": "Lei Wang"
},
{
"@type": "Person",
"name": "Shuo Du"
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{
"@type": "Person",
"name": "Jing Wang"
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{
"@type": "Person",
"name": "Qianqian Li"
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{
"@type": "Person",
"name": "Xiaosu Chen"
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{
"@type": "Person",
"name": "Yuanling Yu"
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{
"@type": "Person",
"name": "Peng Wang"
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{
"@type": "Person",
"name": "Zhiying Zhang"
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{
"@type": "Person",
"name": "Pulan Liu"
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{
"@type": "Person",
"name": "Ran An"
},
{
"@type": "Person",
"name": "Xiaohua Hao"
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{
"@type": "Person",
"name": "Yao Wang"
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{
"@type": "Person",
"name": "Jing Wang"
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{
"@type": "Person",
"name": "Rui Feng"
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{
"@type": "Person",
"name": "Haiyan Sun"
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{
"@type": "Person",
"name": "Lijuan Zhao"
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{
"@type": "Person",
"name": "Wen Zhang"
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{
"@type": "Person",
"name": "Dong Zhao"
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{
"@type": "Person",
"name": "Jiang Zheng"
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{
"@type": "Person",
"name": "Lingling Yu"
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{
"@type": "Person",
"name": "Can Li"
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{
"@type": "Person",
"name": "Na Zhang"
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{
"@type": "Person",
"name": "Rui Wang"
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{
"@type": "Person",
"name": "Xiao Niu"
},
{
"@type": "Person",
"name": "Sijie Yang"
},
{
"@type": "Person",
"name": "Xuetao Song"
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{
"@type": "Person",
"name": "Yangyang Chai"
},
{
"@type": "Person",
"name": "Ye Hu"
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{
"@type": "Person",
"name": "Yansong Shi"
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{
"@type": "Person",
"name": "Linlin Zheng"
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{
"@type": "Person",
"name": "Zhiqiang Li"
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{
"@type": "Person",
"name": "Qingqing Gu"
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"@type": "Person",
"name": "Fei Shao"
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{
"@type": "Person",
"name": "Weijin Huang"
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"@type": "Person",
"name": "Ronghua Jin"
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"@type": "Person",
"name": "Zhongyang Shen"
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"@type": "Person",
"name": "Youchun Wang"
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{
"@type": "Person",
"name": "Xiangxi Wang"
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"@type": "Person",
"name": "Junyu Xiao"
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"name": "Xiaoliang Sunney Xie"
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"identifier": "10.1038/s41586-022-04980-y"
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