construct human ace2 hace2  (Addgene inc)

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a Kinetic analysis of B07, B09, and B10 VHHs on hACE2-Fc by BioLayer Interferometry (BLI) using various concentrations of VHHs (as indicated on the figure). AHC biosensors were used to immobilize hACE2-Fc. K D of each VHHs is indicated. One representative experiment out of two. b Epitope mapping. sACE2-Fc (5 µg/mL) was immobilized onto the AHC biosensors. After a baseline step, a first VHH was applied (VHH1; 5 µg/mL). The sensor was dipped in a mixture of VHH1 and the competitor VHH2 at the same concentration (5 µg/mL). An anti-IgM specific VHH was used as control (ctl). (Top) Immobilized sACE2 pre-bound to B07 (VHH1) and VHH2 being B09, B10, or IgM (ctl). (Middle) Immobilized sACE2 pre-bound to B09 (VHH1) and VHH2 being B07, B10, or IgM (ctl). (Bottom) Immobilized sACE2 pre-bound to B10 (VHH1) and VHH2 being B07, B09, or IgM (ctl). Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Concentration Assay, Control

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: Cells were incubated with the different VHHs (10 µg/mL), stained with an anti-myc antibody and a AF488-conjugated anti-mouse antibody, before being analyzed by flow cytometry. a B07, B09, B10 binding efficacy on HEK293-hACE2-expressing cells. Data are means ± SD of three independent experiments. Parental HEK293 cells were used as control. b B07, B09, B10 binding efficacy on cells expressing endogenous hACE2 (IGROV-1). Anti-IgE VHH was used as a control (ctl). Data are means ± SD, n = 6 (ctl, B07) or 3 (B09, B10) independent experiments. c Fluorescence diagram overlays: B07, B09, B10 efficacy on cells expressing exogenous (HEK293-hACE2) or endogenous hACE2 (IGROV-1). Background (blue) corresponds to the fluorescence intensity obtained on parental cells (HEK293) or using a VHH control (anti-IgE VHH) (IGROV-1). Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Incubation, Staining, Flow Cytometry, Binding Assay, Expressing, Control, Fluorescence

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: hACE2 activity in the presence of VHHs B07, B09, B10 or absence (-) was assayed using the SensoLyte 390 ACE2 Activity Assay Kit, which measures fluorogenic peptide cleavage. a Soluble hACE2 (sACE2) activity in the presence of 10 µM VHHs. An ACE2 inhibitor, provided with the kit, was used as control (DX600, 1 µM). Data are means ± SD of three independent experiments. Paired T test two-sided: ns, nonsignificant; ** p = 0.0053. b VHHs B07, B09, B10 were used at different doses (one representative experiment out of three performed in duplicates. Mean values of the duplicate). The mock control (buffer) was arbitrarily positioned at 10 -14 . c The ACE2 inhibitor DX600 was used at different doses. Data are means ± SD of seven independent experiments. The mock control (buffer) was arbitrarily positioned at 10 -14 . d Cell surface hACE2 activity in the presence of saturating concentration of VHHs ( > 20 µM) or 1 µM DX600. Data are means ± SD of four independent experiments. Paired T test two-sided: ns, nonsignificant; ** p = 0.0070. Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Activity Assay, Control, Concentration Assay

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a S-fuse assay. Inhibition by B07, B09, B10 VHHs after infection of S-Fuse cells (U2OS-hACE2 GFP1-10 and GFP11) with different SARS-CoV-2 variants (Delta B.1.617.2, BA.1, BQ.1.1, XBB.1.5, XBB1.16.1, EG.5.1.3, BA.2.86.1). Data are mean of two independent experiments. The dashed line indicates the limit of detection. An anti-IgM (or IgE) specific VHH was used as a control (ctl). IC50 values are indicated in the table. b Inhibition of 5 µg/mL spike binding to hACE2-expressing HEK293 cells by increasing concentrations of VHHs B07, B09 or B10. HEK293-hACE2 cells pretreated or not with the VHHs and incubated with soluble spike (S) protein (ancestral spike) were stained with an anti-S antibody. Results were normalized for nonspecific (0%) and specific binding in the absence of inhibitor (100%). Experiments were fitted to a one-site competitive binding model. Data are mean ± SD of three (B07, B09) independent experiments or mean of two independent experiments (B10). c Displacement of S (ancestral spike) binding by B07 (left) or B09 (right) for various concentrations of S (5 µg/mL, 8 µg/mL, 10 µg/mL). Experiments were carried out as in ( b ) (one representative experiment out of two - 10 µg/mL, or three - 5 µg/mL, 8 µg/mL). d Relationship between the observed IC50 values for B07 or B09 displacement of S binding and initial concentration of S (ancestral spike). Results represent means ± SD of three independent experiments (5 µg/mL, 8 µg/mL) or means of two independent experiments (10 µg/mL). Linear regression analysis: R B07 = 0.9005; R B09 = 0.7896. Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Inhibition, Infection, Control, Binding Assay, Expressing, Incubation, Staining, Concentration Assay

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a Crystal structure of hACE2 (light blue) in complex with B07 (purple) and B10 (yellow) (PDB: 9R19). b Surface representation of the complex between hACE2 (light blue) and B07 (purple) rotated to 90 ° C from a . For clarity B10 was not displayed. c Open-book representation and footprints (in white) of B07 on hACE2 (left) and hACE2 on B07 (right). The buried surfaces (BSA) are indicated under each surface. Residues of hACE2 in red correspond to those for which alanine substitutions compromise B07 VHH binding to hACE2 mutants ( f ). d B07 complementarity determining regions (CDRs) colored in green (CDR1), blue (CDR2), and red (CDR3). hACE2 footprint on B07 is represented. e Hydrogen bonds and salt bridges (yellow dashed lines) between hACE2 and B07. f Impact of hACE2 alanine substitution on B07 binding. Cells were transfected with myc tagged or untagged hACE2 alanine mutants, incubated with an anti-myc antibody or B07 (10 µg/mL), respectively and stained with a mouse anti-myc antibody and a AF647-conjugated anti-mouse antibody before being analyzed by flow cytometry. Data are mean ± SD of four (anti-myc) or three (B07) independent experiments. Source Data are provided as a Source Data file. g Electrostatic potential mapped on the surface of the structure of hACE2 and B07. h Superimposition of the binding area of B07 (purple) and SARS-CoV-2 RBD (PDB: 6M0J, green) on hACE2 (light blue). i Footprints of the site of interaction of B07 and RBD on hACE2. The contours are colored in purple (B07) or in green (RBD). The common area is striped.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Binding Assay, Transfection, Incubation, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a Kinetic analysis of VHH B07-Fc on hACE2-His by BioLayer Interferometry (BLI) using various concentrations of VHH B07-Fc (indicated on the figure). HIS2 biosensors were used to immobilize 1 µg/mL of hACE2-His. b VHH B07-Fc binding on cells expressing exogenous (HEK293-hACE2) or endogenous hACE2 (IGROV-1). Experiments were performed as in Fig. but using 0.1 µg/mL of VHH B07-Fc. Data are mean ± SD of six (HEK293) or four (IGROV-1) independent experiments. c Fluorescence diagram overlays as performed in Fig. . d hACE2, phalloidin (F-actin) and DAPI staining on primary human nasal epithelial cells (hNECs). Representative immunofluorescence staining (out of 4 independent experiments) of hACE2 (Red: B07-Fc staining) in combination with phalloidin (Cyan) and DAPI (Blue). Scale bars: ctl-Fc, 10 µm; B07-Fc, 5 µm. e Enzymatic activity of soluble hACE2 (sACE2) in the presence of VHH B07-Fc or absence using the SensoLyte 390 ACE2 Activity Assay Kit, as performed in Fig. . DX600 was an inhibitor control of the kit (one representative experiment out of two; mean values of duplicates). f Cell surface hACE2 activity in the presence of saturating concentration of VHHs (10 µM) or 1 µM DX600. Data are means ± SD of three independent experiments. Paired T test two-sided: ns, nonsignificant; ** p = 0.0070. g Inhibition of fusion (S-Fuse assay) by B07-Fc after infection of S-Fuse cells with different SARS-CoV-2 variants: Delta B.1.617.2, BA.1, BQ.1.1, XBB.1.5, XBB.1.16.1, EG.5.1.3, BA.2.86.1, JN.1.1, KP.3.3. The dashed line indicates the limit of detection. Data are mean of two independent experiments, except for BQ1.1 (three experiments). h Inhibition of infection quantified by counting SARS-CoV-2 nucleoprotein (N) positive cells. IGROV-1 cells were pre-incubated 1 h with serial dilutions of B07-Fc and infected with the indicated variants at 3 × 10 -2 infectious units per cell. Cells were stained with a pan-coronavirus anti-N antibody at day 1 pi. The percentage of inhibition is represented. One representative experiment performed in duplicates. i − k Impact of ACE2 genetic variation on VHH B07-Fc binding. i , j Cells were transfected with different ACE2 constructs ( i ) hACE2 polymorphism; ( j ) human, hamster, or mouse ACE2, incubated with B07-Fc (0.1 µg/mL or different doses) and stained with an AF647-conjugated anti-human antibody before being analyzed by flow cytometry. i Data are mean ± SD of independent experiments: n = 5 (S19P; I21T; K26R; T27A; E35K) or n = 4 (E37K; K68E; M82I, P84T) or n = 3 (H34N). j Data are mean ± SD of 5 (human ACE2) or 3 (hamster and mouse ACE2) independent experiments. k Kinetic analysis of B07-Fc on hamster ACE2-His by BioLayer Interferometry (BLI) using various concentrations of VHH B07-Fc (indicated on the figure). HIS2 biosensors were used to immobilize 2 µg/mL of His-ACE2. K D App is indicated. Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Binding Assay, Expressing, Fluorescence, Staining, Immunofluorescence, Activity Assay, Control, Concentration Assay, Inhibition, Infection, Incubation, Transfection, Construct, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a Schematic diagram showing the experimental design of B07-Fc prophylaxis in XBB.1.5 infected K18-hACE2 mice. Animals received intranasally (i.n.) 7 mg/kg VHH-B07-Fc (B07-Fc) or 7 mg/kg VHH-Fc ctl (ctl-Fc). Twenty-four hours later, they were infected with 10 5 PFU XBB.1.5. SARS-CoV-2 intranasally (i.n.). Three days post-infection, different tissues were collected for analysis. b Genomic RNA load measured by RT-qPCR of SARS-CoV-2 in lung. c SARS-CoV-2 infectious particles measured by conventional plaque assays. d Tubulin, phalloidin (F-actin), SARS-CoV-2 nucleocapsid (N) (viral replication) and DAPI staining on nasal respiratory epithelium, olfactory epithelium, and lung bronchioli extracted from K18-hACE2 uninfected mice, and animals that received VHH-Fc (Ctl-Fc or B07-Fc). Representative immunofluorescence staining of Tubulin (Green) in combination with phalloidin (Cyan), SARS-CoV-2 N protein (Red), and DAPI (Blue). This was performed on nasal, olfactory, and pulmonary epithelium of two VHH ctl-treated mice and four B07-Fc treated mice. Scale bars: 10 µm. e Relationship between viral genomic RNA load measured in the lung by RT-qPCR and B07-Fc concentration quantified in the lung by ELISA. f Schematic diagram showing the experimental design of B07-Fc prophylaxis in D614G infected K18-hACE2 mice. Animals received intranasally (i.n.) 5 mg/kg VHH-B07-Fc (B07-Fc) or 5 mg/kg VHH-Fc ctl (ctl-Fc). Twenty-four hours later, they were infected with 10 4 PFU D614G SARS-CoV-2 i.n. Mice were clinically monitored between days 3 to 6. Mice were euthanized on day 6 and lungs were collected for analysis. g Body weight measured over time post-infection in ctl-Fc ( n = 5) and B07-Fc-treated groups. Mice of the B07-Fc group were split into VHH_high ( n = 4) and VHH_low ( n = 6) according to the B07-Fc concentration measured in the lung on day 6 p.i. (higher or lower than 200 ng/g of lung, respectively). Data are presented as mean values +/- SD. Linear mixed model two sided (no adjustment for multiple comparisons): ns, nonsignificant (p = 0.26); ** p = 0.00378; *** p = 0.000396. h Relationship between body weight loss and B07-Fc concentration quantified in the lung by ELISA on day 6 p.i. Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Infection, Quantitative RT-PCR, Staining, Immunofluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature

Article Title: Neutralization, effector function and immune imprinting of Omicron variants

doi: 10.1038/s41586-023-06487-6

Figure Lengend Snippet: a , Schematic view of S mutations in SARS-CoV-2 variants evaluated in this study. Ins, insertion; SD1/2, subdomains 1 and 2. b , c , Equilibrium dissociation constants ( K d ) measured by BLI ( b ; n = 2 or 3 independent experiments) and SPR ( c ) for binding of the monomeric human ACE2 (hACE2) ectodomain to the indicated immobilized variant RBDs. d , Left, cell–cell fusion (indicated as the percentage of GFP + area) between cells expressing the indicated variant S glycoproteins and Vero E6-TMPRSS2 cells measured over an 18-h time-course experiment using a split-GFP system. Right, cell–cell fusion at 18 h (mean ± s.e.m.). Data are from six fields of view from a single experiment and representative of results from two biological replicates. Comparisons of fusogenicity mediated by BA.1, BA.2, or BA.4/5 S to BA.2.75.2, BQ.1.1, XBB.1 and XBB.1.5 S were completed using the one-sided Dunnett’s test; colours of asterisks indicate the reference group for the comparison (BA.1, gold; BA.2, green; BA.4/5, red). e, f , Relative entry of VSV pseudotyped with the indicated S variant in Vero E6-TMPRSS2 ( e ) or HEK293T-ACE2 ( f ) cells treated with 50 µM camostat, nafamostat or E64d. Normalized entry was calculated on the basis of entry values obtained for Vero E6-TMPRSS2 or HEK293T-ACE2 cells treated with DMSO only for each pseudovirus. Data are mean ± s.d. Twelve technical replicates were performed for each pseudovirus and inhibitor and one experiment representative of two independent biological replicates is shown. Comparison of relative entry values were made between Wu-G614 S VSV pseudovirus and each of the examined SARS-CoV-2 variant S VSV pseudoviruses using the one-sided Dunnett’s test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The monomeric human ACE2 ectodomain (residues 19–615) construct used for BLI contains an N-terminal signal peptide and a 10x His tag and was synthesized and inserted into pTwist-CMV by Twist Bioscience.

Techniques: Binding Assay, Variant Assay, Expressing, Comparison

Journal: Nature

Article Title: Neutralization, effector function and immune imprinting of Omicron variants

doi: 10.1038/s41586-023-06487-6

Figure Lengend Snippet: a , Biolayer interferometry binding curves obtained for monomeric human ACE2 binding to biotinylated Wu, BA.4/5, BA.2.75.2, BQ.1.1, XBB.1 or XBB.1.5 RBDs immobilized at the surface of streptavidin biosensors. Kinetic rate constants and affinities are shown in Supplementary Table . Fits are shown as solid black lines. b , Sensorgrams of monomeric human ACE2 binding to the Wu, BA.2.75.2, BA.4/5, BQ.1.1, XBB.1, XBB.1.5 and Wu E340A RBDs immobilized at the surface of an SPR chip coated with anti-Avi polyclonal Ab. Experiments were performed with serial dilutions of Fabs and run as single-cycle kinetics. Gray blocks denote the dissociation phase. Fits are shown as dashed grey lines. Kinetic rate constants and affinities are shown in Supplementary Table .

Article Snippet: The monomeric human ACE2 ectodomain (residues 19–615) construct used for BLI contains an N-terminal signal peptide and a 10x His tag and was synthesized and inserted into pTwist-CMV by Twist Bioscience.

Techniques: Binding Assay

Journal: Nature

Article Title: Neutralization, effector function and immune imprinting of Omicron variants

doi: 10.1038/s41586-023-06487-6

Figure Lengend Snippet: a , b , Cryo-EM structures of the BQ.1.1 RBD ( a ; cyan) or the XBB.1 RBD ( b ; pink) bound to the human ACE2 ectodomain (green) and the S309 Fab fragment (V h in purple and V l in magenta). Amino acid residues mutated relative to Omicron BA.2 are shown as red spheres. c , Zoomed-in view of the BQ.1.1 RBD interactions formed with human ACE2 with select amino acid residue side chains shown as sticks. N-linked glycans are shown as dark blue spheres in a – c . d , e , RBD-based superimposition of the LY-CoV1404-bound Wu RBD structure ( d ; purple, Protein Data Bank (PDB) ID: 7MMO ) or of the COV2-2130-bound Wu RBD structure ( e ; purple, PDB ID: 7L7E ) onto the BQ.1.1 RBD cryo-EM structure, highlighting the expected disruptions of electrostatic interactions with the monoclonal antibodies resulting from the K444T and the R346T RBD mutations. f , RBD-based superimpositions of the S309-bound BA.1 S (gold, PDB ID: 7TLY ), apo BA.2 S (green, PDB ID: 7UB0 ), S309- and ACE2-bound BQ.1.1 (cyan) and XBB.1 (pink) RBD cryo-EM structures. The N343 glycan along with select side chains are rendered as sticks. The expected N343 glycan clashes with BA.2 residues N370 and F371 (sticks) are indicated with a red star. Residues 368–373 are disordered in the XBB.1 RBD cryo-EM map, as is the case for the adjacent residues 380–392 and were not modelled. Select electrostatic interactions are highlighted with dotted lines in c – e .

Article Snippet: The monomeric human ACE2 ectodomain (residues 19–615) construct used for BLI contains an N-terminal signal peptide and a 10x His tag and was synthesized and inserted into pTwist-CMV by Twist Bioscience.

Techniques: Cryo-EM Sample Prep, Residue

Journal: Nature

Article Title: Neutralization, effector function and immune imprinting of Omicron variants

doi: 10.1038/s41586-023-06487-6

Figure Lengend Snippet: a - b , Electron micrographs representative of 6,487, 6,355, or 3,822 micrographs, respectively, (a) and 2D class averages (b) of the BQ.1.1 RBD (left), the XBB.1 RBD (middle) or BN.1 RBD (right) bound to the human ACE2 ectodomain and the S309 Fab fragment embedded in vitreous ice. The scale bar represents 100 nm (a) or 100 Å (b). c - d , Gold-standard Fourier shell correlation curves (c) and local resolution maps along with angular distribution heat maps calculated using cryoSPARC (d) for the 3D reconstructions of the BQ.1.1 RBD (left), the XBB.1 RBD (middle) or BN.1 RBD (right) bound to the human ACE2 ectodomain and the S309 Fab fragment. The 0.143 cutoff is indicated by a horizontal dashed line. e , Data processing flowchart. CTF: contrast transfer function; NUR: non-uniform refinement.

Article Snippet: The monomeric human ACE2 ectodomain (residues 19–615) construct used for BLI contains an N-terminal signal peptide and a 10x His tag and was synthesized and inserted into pTwist-CMV by Twist Bioscience.

Techniques:

Journal: Nature

Article Title: Neutralization, effector function and immune imprinting of Omicron variants

doi: 10.1038/s41586-023-06487-6

Figure Lengend Snippet: a , Sotrovimab-mediated neutralization of SARS-CoV-2 variant S VSV pseudoviruses presented as absolute potency (half-maximal inhibitory concentration (IC 50 )) (left) or relative to neutralization of Wu-D614 S VSV (right). Each symbol represents individual biological replicates ( n = 5–20). b , SPR analysis of S309 Fab binding to SARS-CoV-2 RBD variants. Each symbol represents K d values from independent experiments ( n = 3–10). c , Binding of sotrovimab immunoglobulin G (IgG) to cell-surface expressed SARS-CoV-2 S variants. d , Left, natural killer cell-mediated ADCC in the presence of sotrovimab or S309-GRLR. Data are presented as mean area under the curve (AUC) ± s.d. of percentage killing ( n = 4–10 donors). Right, ADCP of target cells via CD14 + peripheral blood mononuclear cells in the presence of sotrovimab or S309-GRLR. Data are presented as mean AUC ± s.d. ( n = 4–8 donors). e , Correlation of sotrovimab Fab binding affinity (from b ) with neutralizing activity (from a ) or ADCC (from d ). Dotted lines indicate the limit of detection for neutralization and binding affinity or the mean S309-GRLR AUCs for different variants. R 2 and P values are derived from two-tailed Pearson correlation. f , Body weight loss (left) and lung viral RNA load (right) on day 6 after infection of K18-hACE2 mice receiving S309, S309-GRLR or 30 mg kg −1 of an isotype-matched control antibody (anti-WVN ) one day before challenge. Solid lines represent the median; dotted lines represent the lower limit of quantification; n = 9–20 mice per group. Kruskal–Wallis ANOVA with Dunn’s post-test. g , Body weight (left), viral genomic RNA (middle) and replicating viral titres (right) measured in lungs on day 4 after infection of Syrian hamsters receiving S309 hamster IgG2a or 15 mg kg −1 of an isotype control (IC) monoclonal antibody (MPE8 IgG2a) one day before challenge. n = 6 hamsters per group. Kruskal–Wallis ANOVA with Dunn’s post-test between isotype control and S309. NS, not significant.

Article Snippet: The monomeric human ACE2 ectodomain (residues 19–615) construct used for BLI contains an N-terminal signal peptide and a 10x His tag and was synthesized and inserted into pTwist-CMV by Twist Bioscience.

Techniques: Neutralization, Variant Assay, Concentration Assay, Binding Assay, Activity Assay, Derivative Assay, Two Tailed Test, Infection

Journal: Nature

Article Title: Neutralization, effector function and immune imprinting of Omicron variants

doi: 10.1038/s41586-023-06487-6

Figure Lengend Snippet: a , Binding of the S2V29 monoclonal Ab to SARS-CoV-2 S variants expressed at the surface of ExpiCHO-S cells as measured by flow cytometry. S2V29 retains potent and equal binding against Wu-D614, BQ.1.1, XBB.1, XBB.1.5, BA.2, BN.1 and BA.2-E340A VSV pseudoviruses and was therefore used for quantifying cell-surface S expression. b , Correlation of sotrovimab Fab binding affinity with ADCP. The ADCP AUC values from Fig. are plotted on the y-axis and the binding affinity to each RBD variant obtained in Fig. is plotted on the x-axis. Dotted lines indicate the limit of detection for binding affinity and the mean of S309-GRLR AUCs from the different variants. c , ExpiCHO cells transiently transfected with S variants were incubated with the indicated concentrations of sotrovimab or S309-GRLR (G236R/L328R loss-of-function mutations introduced in the Fc domain of the human IgG1 heavy chain) and mixed with NK cells isolated from healthy donors in a range from 6:1 to 9:1 (NK:target cells). Target cell lysis was determined by a lactate dehydrogenase release assay. Data are presented as mean values +/– standard deviations (SD) from duplicates obtained using NK cells from two representative donors, both being homozygous for genotype V/V158 FcγRIIIa. d , ExpiCHO cells transiently transfected with S variants were fluorescently labelled with PKH67, incubated with the indicated concentrations of sotrovimab or S309-GRLR mAb and mixed with PBMCs labelled with CellTrace Violet from two healthy donors heterozygous for genotype R/H131 FcγRIIa at a ratio of 20:1 (PBMC:target cells). Association of CD14 + monocytes with S-expressing target cells (ADCP) was determined by flow cytometry. e , Eight-week-old female K18-hACE2 mice received 3, 10 or 30 mg/kg of S309 (parent of sotrovimab) or S309-GRLR or 30 mg/kg of an isotype-matched control monoclonal Ab (anti-West Nile virus hE16 ) by intraperitoneal injection one day before intranasal inoculation with 10 4 FFU of SARS-CoV-2 BQ.1.1. n = 9–20 animals per group. Tissues were collected at six days after infection. Lung live virus titer (left panel) and nasal turbinate (center panel) or nasal wash (right panel) viral RNA determined by RT-qPCR on day 6 are plotted (short, solid lines indicate the median; dotted lines indicate the LLOQ; n = 9–20 mice per group; Kruskal-Wallis ANOVA with Dunn’s post-test; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). f , Serum concentration of S309 hamster IgG2a measured by ELISA at day 4 post-infection. n = 6 hamsters per group.The horizontal bar represents the median.

Article Snippet: The monomeric human ACE2 ectodomain (residues 19–615) construct used for BLI contains an N-terminal signal peptide and a 10x His tag and was synthesized and inserted into pTwist-CMV by Twist Bioscience.

Techniques: Binding Assay, Flow Cytometry, Expressing, Variant Assay, Transfection, Incubation, Isolation, Lysis, Release Assay, Virus, Injection, Infection, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature

Article Title: Neutralization, effector function and immune imprinting of Omicron variants

doi: 10.1038/s41586-023-06487-6

Figure Lengend Snippet: a , b , Frequency of Wu RBD-binding (grey), Omicron (BA.1, BA.2 and BA.5) RBD pool-binding (red) and cross-reactive (blue) MBCs from donors of cohorts i–iv, as measured by flow cytometry. Data are individual frequencies for each donor ( a ) and mean frequency ± s.d. for each cohort ( n = 4–16 donors) ( b ). c , Analysis of cross-reactivity with the BQ.1.1 RBD of Omicron (BA.1, BA.2 and BA.5) RBD pool-binding (red bars in b ) and Wu/Omicron (BA.1, BA.2 and BA.5) RBD pool-cross-reactive (CR) (blue bars in b ) MBCs. Data are mean frequency ± s.d. for each cohort ( n = 4–16 donors). d , Cumulative cross-reactivity with the Wu RBD and the Omicron BA.1, BQ.1.1 or XBB.1 RBDs of IgGs secreted from in vitro-stimulated MBCs, as measured by ELISA. Data are mean absorbance values with the blank subtracted from n = 2 replicates of MBC cultures analysed from donors in cohorts vii and viii approximately 3 months after receiving their last vaccine dose. RBD-directed IgGs inhibiting binding of ACE2 to the Wu RBD are depicted in red. The total number ( n MBC ) and the number of ACE2-inhibiting (ACE2 inh ) RBD-directed IgG-positive cultures are indicated on top of each graph. Percentages of total (black) and ACE2-inhibiting (red) Wu-binding, Omicron-binding and Wu/Omicron-cross-reactive IgG-positive cultures are indicated within each quadrant. e , f , Individual frequencies ( e ) and mean (± s.d.) frequencies for each cohort ( n = 5–6 donors) ( f ) of Wu RBD-specific, Omicron-specific and RBD cross-reactive (BA.1, BQ.1.1 and XBB.1) IgG-positive cultures from donors of cohorts vii and viii.

Article Snippet: The monomeric human ACE2 ectodomain (residues 19–615) construct used for BLI contains an N-terminal signal peptide and a 10x His tag and was synthesized and inserted into pTwist-CMV by Twist Bioscience.

Techniques: Binding Assay, Flow Cytometry, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Nature

Article Title: Neutralization, effector function and immune imprinting of Omicron variants

doi: 10.1038/s41586-023-06487-6

Figure Lengend Snippet: a , b , Analysis of cross-reactivity with the BQ.1.1 RBD of Omicron (BA.1/BA.2/BA.5) RBD pool-specific (a) and Wu/Omicron (BA.1/BA.2/BA.5) RBD pool-cross-reactive (b) MBCs. Om.pool: MBCs reactive with the Omicron (BA.1/BA.2/BA.5) RBD pool in cohorts i–iv. c , Cumulative cross-reactivity with the Wu RBD and the Omicron BA.1, BQ.1.1 or XBB.1 RBDs of IgGs secreted from in vitro stimulated MBCs as measured by ELISA. Data represent average OD values with blank subtracted from n = 2 replicates of MBC cultures analyzed from donors of cohorts vii and viii at about 14 days after receiving the last vaccine dose. RBD-directed IgGs inhibiting binding of ACE2 to the Wu RBD are depicted in red. Number of total and ACE2-inhibiting (ACE2 inh ) RBD-directed IgG positive cultures are indicated on top of each graph. Percentages of Wu-specific, Omicron-specific and Wu/Omicron-cross-reactive IgG positive cultures are indicated within each quadrant. d , e , Individual frequencies (d) and mean frequencies ± SD for each cohort (n = 5-6) (e) of Wu RBD-specific (grey), Omicron-specific (red) and RBD cross-reactive (blue for BA.1, yellow for BQ.1.1 and purple for XBB.1) IgG positive cultures from donors of cohorts vii and viii at about 14 days after receiving the last vaccine dose. f , g , Frequency of Wu RBD-specific (grey), Omicron (BA.1/BA.2/BA.5) RBD pool-specific (red) and cross-reactive (blue) MBCs from donors of cohorts vii-viii at about 14 days after receiving the last vaccine dose, as measured by flow cytometry. Individual frequencies are shown in panels f and and mean frequencies ± SD for each cohort (n = 4–16) are shown in g. h , Analysis of cross-reactivity with the BQ.1.1 RBD of Omicron (BA.1/BA.2/BA.5) RBD pool-specific (red bars of panel g) and Wu/Omicron (BA.1/BA.2/BA.5) RBD pool-cross-reactive (blue bars of panel g) MBCs. Om.pool, MBCs recognizing the Omicron (BA.1/BA.2/BA.5) RBD pool. M ean frequencies ± SD are presented for each cohort (n = 5-6). i , j , Frequency of IgGs specific for the Wu RBD (grey), cross-reactive with the Wu/BA.5 RBDs (blue), the Wu/BQ.1.1 RBDs (orange), the Wu/XBB.1 RBDs (purple) or specific for either the BA.5, BQ.1.1 or XBB.1 RBD (red) as measured by ELISA after in vitro stimulation of MBCs from cohorts i–iv. Individual frequencies and mean ± SD (n = 4–16) are shown in panels i and j, respectively.

Article Snippet: The monomeric human ACE2 ectodomain (residues 19–615) construct used for BLI contains an N-terminal signal peptide and a 10x His tag and was synthesized and inserted into pTwist-CMV by Twist Bioscience.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry