human ace2 ectodomain  (Addgene inc)

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 1. Gel permeation chromatograms and SDS-Page analysis of different rh-s-ACE2 products. A) rh-s-ACE2, B) rh-s-ACE2i and C) rh-s-ACE2i-biotin, as pre pared (full line) and after 24 h storage at 4 ◦C in PBS buffer (dashed lines). Rh-s-ACEi reacted with biotin-C6-NHS and rh-s-ACE2i-biotin were also analyzed after mixing with ANANAS (ACE2:NP = 30 mol:mole) (dash-dot lines). Only if biotinylated the protein binds to ANANAS and elutes together with the nanoparticle fraction with a retention volume of about 8 mL; D) SDS-PAGE (4–15 %) analysis of the different rh-s-ACE2 products after 24 h storage at 4 ◦C, conducted in reducing (R) and non-reducing (NR) conditions. The expected protein (MW ~ 69500 Da) displays a band at around 70 kDa. The high MW smeared signal in the NR rh-s-ACE2 sample corresponds to protein multimers, which are reverted to the expected monomeric protein by the reducing (R) treatment.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: SDS Page

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 2. Pre-formulation studies. A) Size of rh-s-ACE2i-biotin:ANANAS assemblies at ACE2:NP molar ratio in solution between 15 and 120; B) Assemblies Z-potential as a function of rh-s-ACE2i-biotin:NP molar ratio in solution mixture. Values were measured in PBS buffer, where core ANANAS exhibit a slightly negative value of −15.43 mV; C) Gel permeation chromatograms of rh-s-ACE2i-biotin (5 μg/run) as such or when mixed with ANANAS at molar ratios ACE2:NP equal to 30, 60 and 90. The peak at retention volume of about 17 mL corresponds to the free protein, the peak at 8 mL corresponds to the nanoassembly; D) Number of rh-s-ACE2i-biotin molecules linked onto the NPs as a function of the ratio of ACE2i-biotin:NP in solution mixtures. Values were calculated from the chromatograms of panel C by comparing the areas of the rh-s-ACE2i-biotin peak in the assembly mixtures with that of the protein in the absence of the nanoparticles.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Formulation

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 3. Affinity of rh-s-ACE2-biotin in solution or multimerized onto the NP surface. Representative curves of binding to RBD of rh-s-ACE2-biotin in free form, and multimerized onto the surface of nanoparticles at ACE2:NPs molar ratios between 15:1 and 60:1 measured by ELISA; A) Wuhan, B) Omicron variants, C) dissociation constants calculated from the ELISA curves; D) dissociation constants of the rh-s-ACE2i-biotin:RBD interaction for ACE2i differently formulated relative to the protein in solution. An R value > 1 indicates lower affinity. Kds were computed using the GraphPad-Prism® software and the one site specific binding equation. Data indicate mean ± SD of 2–4 independent experiments each performed in duplicate. Full data are also reported in the Supplementary Information (S.I.) file (Table S1).

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 4. Inhibition of SARS-CoV-2 infectivity by monomeric and nanoassembled rh-s-ACE2i-biotin. A) Inhibition of SARS-CoV-2 infection in Calu-3 by rh-s- ACE2i-biotin in monomeric form as a function of its concentration. Infection (Wuhan variant) was performed at MOI (multiplicity of infection) of 0.1. The virus was pre-incubated for 10 min with rh-s-ACE2i-biotin prior to Calu-3 cells infection; B) Efficacy of inhibition of SARS-CoV-2 infection (Wuhan variant) by rh-s-ACE2i- biotin at 2.5 μg/mL in monomeric or multimerized form on the ANANAS core at different ACE2:NP molar ratios. To keep the concentration of ACE2 in the assay constant at 2.5 μg/mL, the concentration in nanoparticles was different in the different samples (12.9, 25.7 and 51.5 μg/mL for ACE2:NP-R60, ACE2:NP-R30 and ACE2:NP-R15, respectively). Non-functionalized nanoparticles were therefore tested as control at these three concentrations, together with rh-s-ACE2i-biotin at 2.5 μg/mL in monomeric form; C/D) Efficiency in viral (Wuhan/Omicron Variants) cycle inhibition of increasing ACE2:NP-R30 concentrations (0–5 μg/ml). Bars indicate the mean of n = 2 biological replicates. Each condition was tested in triplicate per replicate. Individual data points are shown as dots.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Inhibition, Infection, Concentration Assay, Variant Assay, Virus, Incubation, Control

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 5. Transmission Electron Microscopy. Representative images of A) SARS-CoV-2, B) the ANANAS:rh-s-ACEi2-30 (ACE2:NP-R30) nanoassembly and C1/C2) the mixture of SARS-CoV-2:ACE2:NP-R30 generated at PFU:NP molar ratio = 1:100. Samples were negatively stained with 1 % uranyl acetate, followed by biotin- nanogold (5 nm). The gold nanoparticles appear ad black dots. More images can be found in the Supplementary information file (Fig. S3).

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Transmission Assay, Electron Microscopy, Generated, Staining

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 6. Ex vivo imaging (A) Ex vivo optical imaging of excised organs from animals sacrificed 15′, 2 h, 24 h and 72 h after vehicle, ANANAS, and ACE2:NP-R30 administration. Br. = brain, Lu. = lungs, Li. = liver, Sp. = spleen, Ki. = kidneys. (B) Quantification of ex vivo optical imaging signal. Data are reported as mean ± SE. The data were analyzed by One-way ANOVA using Dunnet’s test. *p < 0.1, **p ≤0.01.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Ex Vivo, Imaging, Optical Imaging

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 7. Nanoparticles localization in tissues. Representative images of the tissue distribution of ANANAS or ACE2:NP-R30 in lungs 15′, 2, 24 and 72 h after treatment. The blue signal refers to the nuclei (Hoechst 33258 staining), green corresponds to the lysosomal component of macrophages (CD68 Antibody), red is associated with the alexa633 dye linked to the NPs, yellow corresponds to co-localized red and green signals. Scale bar = 100 μm. In the lower panel, representative images of higher magnification (15X) of lungs for each timepoint (yellow arrows: nanoparticles co-localized with macrophages, red arrows: nanoparticles alone). Scale bar = 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Staining

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 8. Histological evaluation by hematoxylin and eosin staining of lung tissue of mice treated with vehicle (CTR), ANANAS and ACE2:NP-R30 and sacrificed 15′, 2, 24, and 72 h after the treatment. Scale bar = 100 μm.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

Techniques: Staining

Journal: Biomaterials

Article Title: Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

doi: 10.1016/j.biomaterials.2023.122394

Figure Lengend Snippet: Fig. 9. Cytokine analysis in serum of untreated and treated animals. (A) IL-10, (B) INF-γ, and (C) TNF-α concentrations were measured in serum samples from treated animals and controls 72h after ANANAS or ACE2:NP-R30 administration. LDL = low detection limit. Data are reported as mean±SD. Statistical analysis was performed by One-way ANOVA, followed by Tukey post-hoc test. No differences were found comparing ANANAS or ACE2:NP-R30 with CTR mice, or directly comparing the two nanoformulations. Overall, these data indicate that the intranasal administration of these formulations to healthy mice does not stimulate an inflammatory response at least over the period of our analysis (72 h), confirming the evidence of lack of toxicity.

Article Snippet: Molecular cloning – The sequence encoding for human ACE2 ectodomain (Uniprot Q9BYF1 residues 18–615) was amplified from a pCEP4-myc-ACE2 (Addgene plasmid # 141185) [30] using polymerase chain reaction with oligonucleotides BclI-ACE2-Fw (aaaatgatcaTCCACCATTGAGGAACAGGCC) and ACE2-NotI-Rv (aaaagcggccgcGTCTGCATATGGACTCCAGTC).

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 , 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

Journal: Cell Reports

Article Title: Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail

doi: 10.1016/j.celrep.2023.112621

Figure Lengend Snippet: The E406W mutation remodels the SARS-CoV-2 RBD allosterically (A), Structural superimposition of the Wuhan-Hu-1 RBD (E406, gold, PDB: 6M0J , ACE2 not displayed) and the W406 RBD (light blue). (B and C) Structural superimposition of the REGN10987/REGN10933-bound Wuhan-Hu-1 RBD (E406, gold, PDB: 6XDG ) and the W406 RBD (light blue). Steric clashes indicated with red stars. (D) Structural superimposition of the ACE2-bound Wuhan-Hu-1 RBD (E406, gold, PDB: 6M0J ) and the W406 RBD (light blue). Hydrogen bonds are shown as dotted lines.

Article Snippet: Human ACE2 ectodomain , Twist Bioscience , N/A.

Techniques: Mutagenesis

Journal: Cell Reports

Article Title: Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail

doi: 10.1016/j.celrep.2023.112621

Figure Lengend Snippet: The E406W mutation dampens ACE2 binding severely (A–C) Biolayer interferometry binding analysis of monomeric human ACE2 to immobilized Wuhan-Hu-1 (A), Alpha (N501Y, B), or E406W (C) RBDs. (D) Mutation effects on avidity for dimeric human ACE2 as measured by yeast surface display for the E406W mutation and RBD mutations found in human-derived SARS-CoV-2 isolates deposited in GISAID as of September 27, 2021, across increasing frequency thresholds.

Article Snippet: Human ACE2 ectodomain , Twist Bioscience , N/A.

Techniques: Mutagenesis, Binding Assay, Derivative Assay

Journal: Cell Reports

Article Title: Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail

doi: 10.1016/j.celrep.2023.112621

Figure Lengend Snippet:

Article Snippet: Human ACE2 ectodomain , Twist Bioscience , N/A.

Techniques: Virus, Clinical Proteomics, Recombinant, Subcloning, Software, Electron Microscopy