Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: (A) Average force profiles of WT (red), Alpha (blue), Beta (orange), Gamma (sky blue), Epsilon (green), Kappa (pink), and Delta (gray) variants as a function of the distance between the centers of mass of RBD and ACE2. (B) Initial snapshot of WT. Residues subjected to each mutation are shown as solid sticks (N501, K417, E484, L452, and T478). RBD and ACE2 are, respectively, colored in light gray and yellow. All N-glycans, water, and ions are hidden for clarity. (C) Initial snapshot of WT with clockwise 90° rotation along the normal from (B). All N-glycans are depicted in different colors. Any other residues, water, and ions are not shown for clarity.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Mutagenesis

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: Two-dimensional contact maps at D = 53 Å. (A) Interacting residue pairs between RBD WT and ACE2. RBD residues subjected to mutation are shown in colored boxes at the bottom: (B) blue for Alpha, (C) orange for Beta, and (D) green for Epsilon. The contact frequency is numbered with colors from light blue to dark blue. Dark red and yellow colors on the map, respectively, represent increased and decreased interactions between RBD and ACE2 upon mutations.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Mutagenesis

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: (A) The average number of contacts between RBD residue 501 and ACE2. (B, C) Representative snapshots at D = 53 Å of (B) Alpha variant and (C) WT. (D) Average number of contacts between RBD residue 417 and ACE2 and (E, F) their interacting residue pairs at D = 53 Å of (E) Beta and (F) Alpha variants. (G) Average number of contacts between RBD residue 478 and ACE2 and (H, I) key interaction pairs at D = 78 Å of (H) Delta and (I) Epsilon variants. The overall color scheme is the same as in Figure , and each mutated residue in each variant is shown using the same colors (i.e., red for WT, blue for Alpha, orange for Beta, green for Epsilon, and gray for Delta). Interacting residues are depicted as solid sticks, and residues losing their interactions are shown as transparent sticks. RBD and ACE2 are presented in light gray and yellow, respectively.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Variant Assay

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: Binding affinities between RBD variants and ACE2 and its comparison with the simulation results. K d is obtained from microscale thermophoresis experiments. F WT / F is a ratio, where F WT and F are the respective maximum pulling force of WT and of each variant obtained from the SMD simulations.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Binding Assay, Microscale Thermophoresis, Variant Assay

Journal: Immunology and Cell Biology

Article Title: The vaccinia‐based Sementis Copenhagen Vector coronavirus disease 2019 vaccine induces broad and durable cellular and humoral immune responses

doi: 10.1111/imcb.12539

Figure Lengend Snippet: Spike‐specific antibody and SARS‐CoV‐2 neutralization responses following SCV‐S vaccination. (a) S1 and S2 subunit endpoint IgM and IgG ELISA titers determined from serum of female C57BL/6J mice ( n = 3) at the indicated times after a single vaccination with 10 7 PFU of SCV‐S, with (b) ratio of S1‐ and S2‐specific IgG2c to IgG1 endpoint ELISA titers determined 28 days after vaccination. (c) S1 IgG ELISA binding titers (left panel) and cPass neutralization titers (right panel) in outbred ARC(s) and inbred C57BL/6J female mice ( n = 5) 21 days after a single vaccination with 10 7 PFU of SCV‐S or vector control, with (d) durability of response in the outbred cohort shown by S1‐specific endpoint IgG ELISA titers (left panel) and neutralization titers (right panel) at the indicated times. (e) S1‐specific IgG ELISA (left panel) and neutralization titers (right panel) 50 days after a single‐dose (day 0) or prime‐boost (day 0 and 28) vaccination of female C57BL/6J mice ( n = 5) with 10 7 PFU SCV‐S or control vector, with (f) neutralizing activity in ACE2 and RBD blocking ELISA, and (g) neutralizing activity (IC 80 titers) against lenti‐SARS‐CoV‐2‐S pseudoviruses bearing spike protein from the Wuhan reference strain, the alpha or beta variant, shown for prime‐boost samples. (h) Neutralization titers in young (6–8 weeks old; n = 5) and middle‐aged (9–10 months old; n = 10) C57BL/6J mice at the indicated times after prime‐boost vaccination with 10 7 PFU SCV‐S. Results shown are representative of four independent experiments (indicated above) with binding and neutralizing antibody levels comparable at similar doses and time points across all experiments. Symbols represent individual mice and bars show the mean ± s.e.m. from independent experiments. Data were log transformed and statistical significance determined using Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ACE2, angiotensin‐converting enzyme‐2; IC 80 , 80% inhibitory concentration; Ig, immunoglobulin; ns, not significant; PFU, plaque‐forming units; RBD, receptor‐binding domain; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SCV‐S, Sementis Copenhagen Vector spike protein.

Article Snippet: ACE2‐HEK293 recombinant cell line (catalog number 79951; BPS Bioscience, San Diego, CA, USA) was maintained in Dulbecco’s Modified Eagle Medium supplemented (Sigma Aldrich, MO, USA) with 10% fetal bovine serum, 2 mM l ‐glutamine and penicillin–streptomycin.

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Binding Assay, Plasmid Preparation, Activity Assay, Blocking Assay, Variant Assay, Transformation Assay, Concentration Assay

Journal: Nature Communications

Article Title: Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies

doi: 10.1038/s41467-022-31300-9

Figure Lengend Snippet: a Tri-part NanoLuc® peptide fragments are individually fused to recombinant S protein (purple) and ACE2 (tan). Interaction of S protein and ACE2 induces complementation of the split-luciferase and ‘turns on’ luminescence (left). In the presence of neutralizing antibodies, the interaction between S protein and ACE2 is blocked, preventing luminescence (right). Figure generated using BioRender. b Molecular model of the predicted refolding of NanoLuc® (PDB ID: 5IBO ) is shown (green) after complementation of fragments β10 and β9 is driven by the interaction between full spike protein (trimer) and ACE2 (PDB ID: 7A97 ).

Article Snippet: As such, luciferase fragments can be fused at either terminus of RBD and N-terminus of ACE2. b (S)RBD-β9 and β10-ACE2 binder pair was screened with increasing concentrations of neutralizing Ab (NAb, Sino Biological 40592-MM57) in human serum. c Using (S)RBD-β9 and β10-ACE2 binder pair, the signal between 0 μg/mL of NAb (darker color) vs. 100 μg/mL of NAb (lighter color adjacent bar) shows a 10-fold decrease upon neutralization. d Testing 18 patient plasma samples: (S)RBD-β9 and β10-ACE2 were mixed directly with plasma samples, followed by the addition of the detection solution (Δ11S and substrate).

Techniques: Recombinant, Luciferase, Generated

Journal: Nature Communications

Article Title: Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies

doi: 10.1038/s41467-022-31300-9

Figure Lengend Snippet: a Molecular modeling of the distances between N-terminus of ACE2 to the N-terminus of RBD is ~60 Å and to the C-terminus is ~53 Å (PDB ID: 6M0J ). As such, luciferase fragments can be fused at either terminus of RBD and N-terminus of ACE2. b (S)RBD-β9 and β10-ACE2 binder pair was screened with increasing concentrations of neutralizing Ab (NAb, Sino Biological 40592-MM57) in human serum. c Using (S)RBD-β9 and β10-ACE2 binder pair, the signal between 0 μg/mL of NAb (darker color) vs. 100 μg/mL of NAb (lighter color adjacent bar) shows a 10-fold decrease upon neutralization. d Testing 18 patient plasma samples: (S)RBD-β9 and β10-ACE2 were mixed directly with plasma samples, followed by the addition of the detection solution (Δ11S and substrate). Samples 16, 17, and 18 (indicated by ‡ above bar) are known to be convalescent whereas other samples are from ICU patients with unknown antibody presence/levels. Source data are provided as a Source data file.

Article Snippet: As such, luciferase fragments can be fused at either terminus of RBD and N-terminus of ACE2. b (S)RBD-β9 and β10-ACE2 binder pair was screened with increasing concentrations of neutralizing Ab (NAb, Sino Biological 40592-MM57) in human serum. c Using (S)RBD-β9 and β10-ACE2 binder pair, the signal between 0 μg/mL of NAb (darker color) vs. 100 μg/mL of NAb (lighter color adjacent bar) shows a 10-fold decrease upon neutralization. d Testing 18 patient plasma samples: (S)RBD-β9 and β10-ACE2 were mixed directly with plasma samples, followed by the addition of the detection solution (Δ11S and substrate).

Techniques: Luciferase, Neutralization

Journal: Nature Communications

Article Title: Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies

doi: 10.1038/s41467-022-31300-9

Figure Lengend Snippet: a Molecular modeling of the distances between N-terminus of ACE2 to the N-termini of nearest S protein is ~48 Å and ~88 Å, respectively (PDB ID: 7A97 ). b Comparison of signal and background for the full spike (wild type and variants) and ACE2 pairs. c Serial dilution of a commercially available NAb (Sino Biological, 40592-R001) in the presence of β10-(S)WT and β9-ACE2-Fc pair. d Serum samples that have been tested previously on two different COVID-19 detection assays were also tested using Neu-SATiN. Wild type full spike protein with β10 tag (β10-(S)WT) and ACE2 with β9 tag (β9-ACE2-Fc) was used as the binders. Signals from patient samples were normalized to the signal from normal human serum with the binders alone (no neutralizing antibodies). The red dotted line at 10% activity (i.e., 90% neutralization) indicates the cutoff line to distinguish neutralizing samples from the non-neutralizing samples; ‘+’ and ‘−‘ signs below the sample number indicate the result from two prior tests detecting anti-SARS-CoV-2 antibodies (COV2G Siemens 1st Gen and EUROIMMUN EIA). e Activity measured in protein-based surrogate neutralization assay (pbSNA) versus neu-SATiN for the wild-type (WT) variant shows high correlation with a Pearson’s correlation test r value of 0.88. N = 66. f Neutralization efficacy of patient samples against WT and variant S proteins. Samples used in ( d ) ( n = 43) were tested with an additional n = 35 patient samples that were positive for anti-SARS-CoV-2 antibodies. Luminescence signals from each patient serum were measured and normalized to human serum (no NAb). Known post-vaccination, post-infection, and negative (no known infection or vaccination) samples were plotted separately. Red dotted line at 10% indicates the cutoff between the negative samples and the positive samples for distinction of neutralization. g Comparison of serum data from patients that were vaccinated to patients that were vaccinated after a documented infection ( n = 40). All patient samples were collected before November 2021, prior to any known Omicron infections. Source data are provided as a Source data file.

Article Snippet: As such, luciferase fragments can be fused at either terminus of RBD and N-terminus of ACE2. b (S)RBD-β9 and β10-ACE2 binder pair was screened with increasing concentrations of neutralizing Ab (NAb, Sino Biological 40592-MM57) in human serum. c Using (S)RBD-β9 and β10-ACE2 binder pair, the signal between 0 μg/mL of NAb (darker color) vs. 100 μg/mL of NAb (lighter color adjacent bar) shows a 10-fold decrease upon neutralization. d Testing 18 patient plasma samples: (S)RBD-β9 and β10-ACE2 were mixed directly with plasma samples, followed by the addition of the detection solution (Δ11S and substrate).

Techniques: Comparison, Serial Dilution, Activity Assay, Neutralization, Variant Assay, Infection

Journal: Scientific Reports

Article Title: HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells

doi: 10.1038/srep15085

Figure Lengend Snippet: ( A ) DU145 prostate tumor cells, incubated with recombinant human HMGB1 (rhHMGB1) for 24–48 h, were lysed and analyzed by western blot for presence of clusterin, and for β-actin for equal loading. ( B ) DU145 tumor cells were treated with docetaxel (DTX) for 1–4 days and their supernatants were evaluated for HMGB1 by ELISA. ( C ) DU145 tumor cells were treated with the indicated chemotherapeutic agents for 24 h and the supernatants analyzed for HMGB1.

Article Snippet: DU145 cells or PC3 cells were treated with docetaxel (DTX) for 1–4 days or were treated with the indicated chemotherapeutic agents for 24 h. Supernatants were collected and analyzed for the presence of human HMGB1 by an ELISA kit (LS-F4038) from LifeSpan Biosciences.

Techniques: Incubation, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay