6p-stabilized sars-cov-2 s ectodomain  (GenScript corporation)

Journal: Science Immunology

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern

doi: 10.1126/sciimmunol.abp9312

Figure Lengend Snippet: 87G7 potently neutralizes Omicron and other SARS-CoV-2 variants (A) S protein schematic with mutations indicated that are found in Alpha, Beta, Gamma, Delta and Omicron (BA.1 and BA.2) variants of concern (VOCs), and Lambda and Mu variants of interest (VOIs), relative to ancestral SARS-CoV-2. The S N-terminal domain (NTD, in blue) and receptor-binding domain (RBD, in orange), and S1/S2 junction are indicated. (B) Neutralizing activity of 87G7 against virus particles pseudotyped with ancestral SARS-CoV-2 S (Wuhan-Hu-1 strain) or S proteins of Alpha, Beta, Delta and Omicron. Error bars indicate standard deviation between at least two independent replicates. (C and D) 87G7 mediated neutralization of live SARS-CoV-2 and variants. Neutralizing potency of 87G7 and REGN10933 against the D614G SARS-CoV-2, and Alpha, Beta, Delta, Gamma and BA.1 and BA.2 Omicron VOCs (C) and against Lambda and Mu SARS-CoV-2 VOIs (D) . Error bars indicate standard deviation between at least two independent replicates. (E) Inhibitory Concentrations 50% (IC50) of 87G7 against SARS-CoV-2 variants calculated from the neutralization curves displayed in panel b, c and d. NT: not tested.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( )(S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Binding Assay, Activity Assay, Standard Deviation, Neutralization

Journal: Science Immunology

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern

doi: 10.1126/sciimmunol.abp9312

Figure Lengend Snippet: Structural basis for binding and neutralization by 87G7 (A) Composite cryo-EM density map for the SARS-CoV-2 spike ectodomain in complex with the 87G7 antibody Fab fragment. The spike protomers are colored blue, gray, and pink, and the 87G7 light- and heavy-chain variable domains colored purple and yellow, respectively. (B) Surface representation of the 87G7-bound RBD overlaid with the RBD-bound ACE2 (PDB ID: 6M0J). (C) Surface representation of the RBD colored according to the Kyte-Doolittle scale, where the most hydrophobic residues are colored tan and the most hydrophilic residues are colored blue. The residues which make up the 87G7 core epitope and the ACE2 footprint are outlined. (D) Close-up view showing selected interactions formed between 87G7 and the SARS-CoV-2 RBD (E) ELISA binding of 87G7 to plate-immobilized WT, F456A, F486A and Y489A S1 domains. (F) 87G7 neutralizing activity against pseudoviruses with Wuhan-Hu-1 S and S F486A . REGN10933 and REGN10987 were taken along as a reference in panel E and F. (G) Side-by-side comparison of the SARS-CoV-2 RBD bound to 87G7, WRAIR-2125 (PDB ID: 7N4L), 58G6 (PDB ID: 7E3L), P5C3 (PDB ID: 7PHG), COV2-2196 (PDB ID: 7L7D), S2E12 (PDB ID: 7K45) and A23-58.1 (PDB ID: 7LRS). (H) Germline origins and CDR3 sequences of heavy and light chains of 87G7 and other F486-directed SARS-CoV-2 mAbs with broad neutralization capacity. PDB entries of spike-antibody structures and references are indicated. NA: not applicable.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( )(S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Binding Assay, Neutralization, Cryo-EM Sample Prep, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Science Immunology

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern

doi: 10.1126/sciimmunol.abp9312

Figure Lengend Snippet: 87G7 recognizes a conserved epitope in SARS-CoV-2 RBD (A) Surface representation of the SARS-CoV-2 S RBD with mutations colored red that are found in Omicron BA.1 (left panel) and Omicron BA.2 (middle panel). The right panel displays the set of mutations surrounding the 87G7 core epitope that are present in Alpha, Beta, Gamma, Delta, Lambda or Mu (see also ). The 87G7 core epitope residues are colored green. (B) 87G7 neutralizing activity against pseudoviruses with S variants carrying single residue substitutions found in the SARS-CoV-2 variants of concern. The REGN10933 and REGN10987 therapeutic mAbs were used for benchmarking. Data are shown as mean (± SEM) of two independent experiments with technical triplicates, and corresponding IC50 titers are presented in the lower panel.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( )(S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Activity Assay

Journal: Science Immunology

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern

doi: 10.1126/sciimmunol.abp9312

Figure Lengend Snippet: Prophylactic and therapeutic treatment was assessed in the K18-hACE2 SARS-CoV-2 mouse model. 87G7 or isotype control mAb was administered intraperitoneally (10 mg/kg body weight) into groups of mice (n = 5) at 24 hours before (A, B, C) or after virus challenge (D, E, F) . Mice were challenged intranasally with 10 5 PFU of SARS-CoV-2 (D614G, Alpha, Beta, Gamma or Delta) and monitored daily for weight loss (A and D). Five days after challenge lungs were collected from all mice, and lung viral antigen levels were determined by immunohistochemistry (B and E; Table S2, Figure S6) , and infectious SARS-CoV-2 loads in lung tissue were measured by plaque assay (C and F) . The mean values ± SEM of all data points were shown. Dashed line indicates assay limits of detection. Mann-Whitney U test was used to evaluate the statistical difference between the 87G7 and isotype-treated groups (**p<0.01, *p<0.05, ns p>0.05).

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( )(S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Immunohistochemistry, Plaque Assay, MANN-WHITNEY

Journal: Science Immunology

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern

doi: 10.1126/sciimmunol.abp9312

Figure Lengend Snippet: 87G7 or isotype control mAb was administered intraperitoneally (10 mg/kg body weight or 20mg/kg for Omicron-challenged hamsters) into groups of Syrian hamsters (n = 5, and n = 6 for Omicron groups) at 24 hours before (A, B, C) or 12 hours after virus challenge (D, E, F) . Hamsters were challenged intranasally with 10 4 TCID50 of D614G SARS-CoV-2, Beta, Gamma or Omicron. Four days after challenge hamsters were euthanized, and infectious SARS-CoV-2 titer in lung homogenates and nasal cavity were evaluated by TCID50 measurement (A and D) . Lung and nasal cavity were examined for lesions by histopathological scoring and presence of viral antigen by immunohistochemistry (B, C and E, F; Table S3-S5, Figure S7-S10) . The mean values ± SEM of all data points were shown. Mann-Whitney U test was used to evaluate the statistical difference between the 87G7 and isotype-treated groups (**p<0.01, *p<0.05, ns p>0.05).

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( )(S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Immunohistochemistry, MANN-WHITNEY

Journal: bioRxiv

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

doi: 10.1101/2022.02.17.480751

Figure Lengend Snippet: (A) S protein schematic with mutations indicated that are found in Alpha, Beta, Gamma, Delta and Omicron (BA.1 and BA.2) variants of concern (VOCs), and Lambda and Mu variants of interest (VOIs), relative to ancestral SARS-CoV-2. The S N-terminal domain (NTD, in blue) and receptor-binding domain (RBD, in orange), and S1/S2 junction are indicated. SARS-CoV-2 lineage naming according to WHO (World Health Organization) and PANGO (Phylogenetic Assignment of Named Global Outbreak). (B) Neutralizing activity of 87G7 against virus particles pseudotyped with ancestral SARS-CoV-2 S (Wuhan-Hu-1 strain) or S proteins of Alpha, Beta, Delta and Omicron. Error bars indicate standard deviation between at least two independent replicates. (C and D) 87G7 mediated neutralization of live SARS-CoV-2 and variants. Neutralizing potency of 87G7 and REGN10933 against the D614G SARS-CoV-2, and Alpha, Beta, Delta, Gamma and BA.1 and BA.2 Omicron VOCs (C) and against Lambda and Mu SARS-CoV-2 VOIs (D) . Error bars indicate standard deviation between at least two independent replicates. (E) Inhibitory Concentrations 50% (IC50) of 87G7 against SARS-CoV-2 variants calculated from the neutralization curves displayed in panel b, c and d. NT: not tested.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( ) (S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Binding Assay, Activity Assay, Standard Deviation, Neutralization

Journal: bioRxiv

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

doi: 10.1101/2022.02.17.480751

Figure Lengend Snippet: (A) ELISA binding curves of 87G7 to the plate-immobilized N-terminal domain (NTD), the receptor-binding domain (RBD) or ectodomain of SARS-CoV-2 S. (B) Binding kinetics of 87G7 to SARS-CoV-2 S measured by biolayer interferometry (BLI). 87G7 mAb was loaded at optimal concentration (21 nM) onto the anti-human Fc biosensor for 10 mins, after which association of antigen was achieved by incubating the sensor with a 2-fold dilution series of recombinant SARS-CoV-2 S1 monomer or S ectodomain trimer for 10 min, followed by a dissociation step in PBS for 30 min. K D : equilibrium dissociation constant. k on : association rate constant, k off : dissociation rate constant. K D App reflects the ‘apparent affinity’ between IgG antibodies and spike trimer. (C) Binding competition between 87G7 and benchmarking antibodies to SARS-CoV-2 S ectodomain trimer evaluated using BLI. The strep-tagged SARS-CoV-2 S antigen was loaded to the anti-human Fc biosensor bound with antibodies against Strep tag. The competitor antibody was bound to spike (step 1) before incubation with the analyte antibody (step 2) as indicated, and percent competition bins are indicated in the table (dark red: >90% competition, light red; 40-80% competition, white: <10% competition). Data from a representative experiment (out of two) are shown. Benchmarking antibodies tested for binding competition with 87G7 are shown in complex with SARS-CoV-2 RBD and include REGN10933 (PDB: 6XDG), REGN10987 (PDB: 6XDG), S309 (parent of VIR-7831, PDB: 6WPS), CR3022 (PDB: 6W41) and 47D11 (PDB: 7AKD) (D) BLI-based receptor-binding inhibition assay. SARS-CoV-2 S ectodomain bound to the sensor was pre-incubated with 87G7 or two control mAbs REGN10933 (ACE2 binding competitor) or S309 (non-ACE2 competing), followed by a washing step and subsequent exposure to soluble human ACE2 receptor. The experiment was performed twice, data from a representative experiment is shown. (E) ELISA-based receptor-binding inhibition assay. SARS-CoV-2 S ectodomain pre-incubated with serially diluted 87G7 or two control mAbs REGN10933 or REGN10987 (both ACE2 binding competitors) was added to ELISA plates coated with soluble human ACE2. Spike binding to ACE2 was detected using an HRP-conjugated antibody recognizing the C-terminal Strep-tag on SARS-CoV-2 S ectodomain. Data points represent the average ± SDM, for n = 3 replicates from two independent experiments.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( ) (S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Recombinant, Strep-tag, Incubation, Inhibition

Journal: bioRxiv

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

doi: 10.1101/2022.02.17.480751

Figure Lengend Snippet: (A) Representative motion-corrected micrograph of the 87G7-bound SARS-CoV-2 spike ectodomains embedded in vitreous ice. Scale bar = 50 nm. (B) As shown in A, for the sample incubated with 0.2% fluorinated octyl maltoside. (C) Representative reference-free 2D class averages generated in cryoSPARC. (D) As shown in C, for the sample incubated with 0.2% fluorinated octyl maltoside. (E) DeepEMhancer filtered EM density maps for the globally refined spike-87G7 complex and locally refined RBD-87G7 complex, colored according to local resolution which was calculated in Relion3.1. The outline of the local refinement mask is overlaid with the globally refined map. Representative density and fitted atomic coordinates for the S2 region of the globally refined map is shown on the left. (F) Gold-standard Fourier shell correlation (FSC) curves generated from the independent half maps contributing to the 2.9 Å resolution global refinement and 4.9 Å resolution local refinement. (G) Angular distribution calculated in cryoSPARC for particle projections in the local refinement. (H) Cryo-EM density for the locally refined 87G7 epitope-paratope region with the fitted atomic coordinates. RBD residues are colored blue and the light- and heavy-chain variable domains are colored purple and yellow, respectively.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( ) (S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Incubation, Generated, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

doi: 10.1101/2022.02.17.480751

Figure Lengend Snippet: (A) Composite cryo-EM density map for the SARS-CoV-2 spike ectodomain in complex with the 87G7 antibody Fab fragment. The spike protomers are colored blue, gray, and pink, and the 87G7 light- and heavy-chain variable domains colored purple and yellow, respectively. (B) Surface representation of the 87G7-bound RBD overlaid with the RBD-bound ACE2 (PDB ID: 6M0J). (C) Surface representation of the RBD colored according to the Kyte-Doolittle scale, where the most hydrophobic residues are colored tan and the most hydrophilic residues are colored blue. The residues which make up the 87G7 core epitope and the ACE2 footprint are outlined. (D) Close-up view showing selected interactions formed between 87G7 and the SARS-CoV-2 RBD (E) ELISA binding of 87G7 to plate-immobilized WT, F456A, F486A and Y489A S1 domains. (F) 87G7 neutralizing activity against pseudoviruses with Wuhan-Hu-1 S and S F486A . REGN10933 and REGN10987 were taken along as a reference in panel E and F. (G) Side-by-side comparison of the SARS-CoV-2 RBD bound to 87G7, WRAIR-2125 (PDB ID: 7N4L), 58G6 (PDB ID: 7E3L), P5C3 (PDB ID: 7PHG), COV2-2196 (PDB ID: 7L7D), S2E12 (PDB ID: 7K45) and A23-58.1 (PDB ID: 7LRS). (H) Germline origins of 87G7 and other F486-directed SARS-CoV-2 mAbs with broad neutralization capacity. NA: not applicable.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( ) (S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Cryo-EM Sample Prep, Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Neutralization

Journal: bioRxiv

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

doi: 10.1101/2022.02.17.480751

Figure Lengend Snippet: (A) The top panel shows a surface representation of the WRAIR-2125 Fab fragment variable domains, rendered at 6 Å resolution, in complex with SARS-CoV-2 RBD residues 470-494 (generated using PDB ID: 7N4L). The CDR H2, H3 and L3 loop are labelled in italics. The Fab heavy and light chain are colored yellow and purple and RBD residues are colored blue. The bottom panel shows a close-up view showing selected interactions formed between WRAIR-2125 residues and the SARS-CoV-2 RBD residues Y489 and F489. The unmodelled region of the CDR H3 loop is indicated with a dashed line. (B) As shown in A for 87G7.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( ) (S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Generated

Journal: bioRxiv

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

doi: 10.1101/2022.02.17.480751

Figure Lengend Snippet: (A) Surface representation of the SARS-CoV-2 S RBD with mutations colored red that are found in Omicron BA.1 (left panel) and Omicron BA.2 (middle panel). The right panel displays the set of mutations surrounding the 87G7 core epitope that are present in Alpha, Beta, Gamma, Delta, Lambda or Mu (see also ). The 87G7 core epitope residues are colored green. (B) 87G7 neutralizing activity against pseudoviruses with S variants carrying single residue substitutions found in the SARS-CoV-2 variants of concern. The REGN10933 and REGN10987 therapeutic mAbs were used for benchmarking. Data are shown as mean (± SEM) of two independent experiments with technical triplicates, and corresponding IC50 titers are presented in the lower panel.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( ) (S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

doi: 10.1101/2022.02.17.480751

Figure Lengend Snippet: Prophylactic and therapeutic treatment was assessed in the K18-hACE2 SARS-CoV-2 mouse model. 87G7 or isotype control mAb was administered intraperitoneally (10 mg/kg body weight) into groups of mice (n = 5) at 24 h before (A, B, C) or after virus challenge (D, E, F) . Mice were challenged intranasally with 10 5 PFU of SARS-CoV-2 (D614G, Alpha, Beta, Gamma or Delta) and monitored daily for weight loss (A and D). Five days after challenge lungs were collected from all mice, and lung viral antigen levels were determined by immunohistochemistry (B and E; ) , and infectious SARS-CoV-2 loads in lung tissue were measured by plaque assay (C and G) . The mean values ± SEM of all data points were shown. Dashed line indicates assay limits of detection. Non-parametric Mann-Whitney U tests were used to evaluate the statistical difference between the 87G7 and isotype-treated groups (**p<0.01, *p<0.05, ns p>0.05).

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( ) (S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Immunohistochemistry, Plaque Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

doi: 10.1101/2022.02.17.480751

Figure Lengend Snippet: 87G7 or isotype control mAb was administered intraperitoneally (10 mg/kg body weight or 20mg/kg for Omicron-challenged hamsters) into groups of Syrian hamsters (n = 5, and n = 6 for Omicron groups) at 24 h before (A, B, C) or 12 h after virus challenge (D, E, F) . Hamsters were challenged intranasally with 10 4 TCID50 of D614G SARS-CoV-2, Beta, Gamma or Omicron. Four days after challenge hamsters were euthanized, and infectious SARS-CoV-2 titer in lung homogenates and nasal cavity were evaluated by TCID50 measurement (A and D) . Lung and nasal cavity were examined for lesions by histopathological scoring and presence of viral antigen by immunohistochemistry (B, C and E, F; - ) . The mean values ± SEM of all data points were shown.

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( ) (S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

doi: 10.1101/2022.02.17.480751

Figure Lengend Snippet:

Article Snippet: Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct ( ) (S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® ( ).

Techniques: Immunohistochemistry