Journal: bioRxiv

Article Title: Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

doi: 10.1101/2020.07.31.231274

Figure Lengend Snippet: a. Diagram showing the domain architecture of SARS-CoV-2 nsp13. b. Size exclusion chromatography (Superdex 200 Increase) of purified nsp13. c. SDS-PAGE gel (Coomassie stain) showing nsp13 purity. d. Differential scanning fluorimetry (DSF) of SARS-CoV-2 nsp13 in the absence and presence of ATP analogs (1 mM). Melting temperatures are: APO- 40.5 °C, ATPγS- 45.5 °C, ADP:AlF 3 - 52.5 °C, and ADP- 41.0 °C for n=2 independent measurements from two separate protein preparations. e. Fluorescence anisotropy measurements for fluorescence DNA and RNA partial duplexes (10 nM) with and without nsp13 (3.4 μM) added. Data shown as mean standard ± deviation (SD) for n=2 independent measurements from two separate protein preparations.

Article Snippet: These experiments were carried out on a C1000 Touch Thermal cycler CFX-96 instrument (GE Healthcare). recombinant constructs were diluted to a final concentration of 4 μM in DSF buffer (25 mM HEPES-KOH pH 7.5, 25 mM KCl, 5 mM MgCl 2 , 1 mM TCEP, 0.005% triton-X) and assayed in a 96-well plate (Hard-shell HSP9665 Bio-Rad).

Techniques: Size-exclusion Chromatography, Purification, SDS Page, Staining, Fluorescence, Standard Deviation

Journal: Journal of Virology

Article Title: Characterization of a Novel ACE2-Based Therapeutic with Enhanced Rather than Reduced Activity against SARS-CoV-2 Variants

doi: 10.1128/JVI.00685-21

Figure Lengend Snippet: Biophysical characterization of SARS-CoV-2 S1 variants. (A) Particle size distribution of recombinant purified SARS-CoV-2 S1 variants analyzed via MADLS. (B) Thermal stability analysis of purified SARS-CoV-2 variants showing increased T m for B.1.1.7, B.1.351, and P.1 variants compared to that of WT S1. (C) Kinetic affinity of catalytically active ACE2-Fc with SARS-CoV-2 S1 WT, D614G, B1.1.7, B.1.351, and P.1. All sensograms fitted with 1:1 Langmuir binding model. Analyte starting concentration 250 nM with 2-fold serial dilutions. 3-fold and 2.3-fold higher affinity detected for S1 of B.1.1.7 and P.1 variants, respectively. Physical particle number determined via p24 ELISA (D) and infectious viral titer (E) comparison for SARS-CoV-1, SARS-CoV-2 Wuhan, SARS-CoV-2 D614G, SARS-CoV-2 B1.1.7, and SARS-CoV-2 B1.351 pseudotyped vectors, showing comparable particle concentration but diverse infectivity capacity (mean ± standard deviation [SD]). One-way analysis of variance (ANOVA) Dunnett’s multiple comparisons test (F = 105.2, df = 4, 10).

Article Snippet: Protein labeled cells were stained with biotinylated SARS-CoV-2 S1 (ACRO biosystems, S1N-C82E8) and detected with streptavidin AF647 (Invitrogen, S21374).

Techniques: Recombinant, Purification, Binding Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, Standard Deviation

Journal: Journal of Virology

Article Title: Characterization of a Novel ACE2-Based Therapeutic with Enhanced Rather than Reduced Activity against SARS-CoV-2 Variants

doi: 10.1128/JVI.00685-21

Figure Lengend Snippet: Characterization of ACE2-Fc receptor decoy. (A) Schematic representation of ACE2-Fc molecule with a streamlined antibody-like expression/purification process and biophysical characterization. (B) Three-dimensional (3D) structure of SARS-CoV-2 spike S1 domain (green) in complex with ACE2 (blue) (PDB 6M0J ). Inset, zoomed section of the ACE2 catalytic site showing the H374 and H378 residues (blue) in complex with Zn (red), and the H374N and H378N mutations (orange). (C) Enzymatic activity of active (blue) and H374N, H378N mutated (orange) ACE2-Fc using Mca-APK(Dnp) fluorogenic peptide (mean ± SD). (D) Binding kinetics of active (top) and inactive (bottom) ACE2-Fc with Ang II. (E) ELISA of SARS-CoV-2 active spike trimer (left) or S1 domain (center) against WT active and ACE2(HH:NN)-Fc, showing comparable binding capacity. No binding detected with control antigen (right) or negative-control antibody (mean ± SD). EC 50 , half maximal effective concentration; RFU, relative fluorescent units; OD, optical density. Unpaired t test of AUC (left t = 1.086, df = 24; center t = 1.79, df = 24).

Article Snippet: Protein labeled cells were stained with biotinylated SARS-CoV-2 S1 (ACRO biosystems, S1N-C82E8) and detected with streptavidin AF647 (Invitrogen, S21374).

Techniques: Expressing, Purification, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Concentration Assay

Journal: Journal of Virology

Article Title: Characterization of a Novel ACE2-Based Therapeutic with Enhanced Rather than Reduced Activity against SARS-CoV-2 Variants

doi: 10.1128/JVI.00685-21

Figure Lengend Snippet: Characterization of Fc effector functions. (A) ELISA of SARS-CoV-2 active spike trimer (left) or S1 domain (right) with ACE2(HH:NN) WT Fc (blue), LALA Fc (green), or LALA-PG Fc (orange), showing comparable binding capacity (mean ± SD). One-way ANOVA of AUC with Tukey’s multiple comparisons (left F = 0.3121, df = 2, 72; right F = 34.17, df = 2, 72, compared to blue). (B) Binding capacity on SupT1 cell line expressing SARS-CoV-2 full-length spike, by flow cytometry with ACE2(HH:NN) WT Fc (blue), LALA Fc (green), or LALA-PG Fc (orange) (mean ± SD). One-way ANOVA of AUC with Tukey’s multiple comparisons (blue versus green, F = 3.986, df = 2, 54). MFI, mean fluorescence intensity. (C) Fc-mediated binding capacity to U937, K562, and SupT1 of ACE2(HH:NN) WT Fc (blue), LALA Fc (green), or LALA-PG Fc (orange), detected with biotinylated SARS-CoV-2 S1 and streptavidin conjugated secondary agent. No binding was detected with ACE2-Fc constructs carrying the LALA or LALA-PG mutations (mean ± SD). (D) Representative flow cytometry of Fc-mediated binding of ACE2(HH:NN) WT Fc (blue) and LALA-PG Fc (orange) on human monocyte-derived M1 macrophages. No binding detected with Fc carrying the LALA-PG mutation ( n = 4). (E) SPR binding kinetic of ACE2(HH:NN) WT Fc, LALA Fc, or LALA-PG Fc on human FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb. LALA-PG mutations mediated a complete abrogation of FcγR interaction. Sensograms fitted with 1:1 Langmuir binding model.

Article Snippet: Protein labeled cells were stained with biotinylated SARS-CoV-2 S1 (ACRO biosystems, S1N-C82E8) and detected with streptavidin AF647 (Invitrogen, S21374).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Expressing, Flow Cytometry, Fluorescence, Construct, Derivative Assay, Mutagenesis

Journal: Journal of Virology

Article Title: Characterization of a Novel ACE2-Based Therapeutic with Enhanced Rather than Reduced Activity against SARS-CoV-2 Variants

doi: 10.1128/JVI.00685-21

Figure Lengend Snippet: SARS-CoV-2 spike binding and neutralization. (A) SPR binding kinetics of ACE2(HH:NN) WT Fc, LALA-PG Fc, LY-CoV555, REGN10933, and REGN10987 against SARS-CoV-1, SARS-CoV-2 variants (Wuhan, D614G, B1.1.7, B.1.351, and P.1), and HCoV-NL63 S1 domains. ACE2(HH:NN) Fc and ACE2(HH:NN) Fc LALA-PG were able to efficiently bind all spike protein tested. All sensograms were fitted with Langmuir 1:1 binding model, except for SARS-CoV-1 S1 kinetics, which were fitted with two-state kinetics. Two-fold serial dilutions starting from 250 nM (500 nM for HCoV-NL63 S1). (B) Cell microarray screening of human cell-membrane proteome with ACE2-Fc (LALA-PG), control Fc (LALA-PG), CTLA4-hFc, or PBS. Depicted is a selection of antigens (key legend on the right panel). ACE2-Fc (LALA-PG) shows strong specific interaction with SARS-CoV-2 spike protein only. See also Table S1. (C) Neutralization assay of authentic SARS-CoV-2 virus with ACE2(HH:NN) WT Fc (blue) and LALA-PG Fc (orange). Both variants show comparable neutralization efficiencies (mean ± SD). Unpaired t test of AUC ( t = 1.695, df = 28). (D) Neutralization assay of SARS-CoV-1, SARS-CoV-2 Wuhan, SARS-CoV-2 D614G, SARS-CoV-2 B1.1.7, and SARS-CoV-2 B1.351 pseudotyped vectors with ACE2(HH:NN)-Fc (LALA-PG), LY-CoV-55, REGN10933/REGN10987 cocktail, REGN10933, and REGN10987. Marked decrease of neutralization capacity for SARS-CoV-2 B1.351 detected for LY-CoV555, REGN10933/REGN10987 cocktail, REGN10933, and REGN10987. No loss of neutralization capacity detected for ACE2(HH:NN)-Fc (LALA-PG) receptor decoy (mean ± SD). One-way ANOVA of AUC with Dunnett’s multiple comparisons to blue (F = 369.2, df = 4,88). Bottom right panel, fold change of neutralization capacity based on NT 50 values. *, unmeasurable NT 50 value.

Article Snippet: Protein labeled cells were stained with biotinylated SARS-CoV-2 S1 (ACRO biosystems, S1N-C82E8) and detected with streptavidin AF647 (Invitrogen, S21374).

Techniques: Binding Assay, Neutralization, Microarray, Selection

Journal: Journal of Virology

Article Title: Characterization of a Novel ACE2-Based Therapeutic with Enhanced Rather than Reduced Activity against SARS-CoV-2 Variants

doi: 10.1128/JVI.00685-21

Figure Lengend Snippet: In vivo SARS-CoV-2 neutralization. (A) Fc-mediated binding capacity of ACE2(HH:NN) WT Fc (blue), LALA-PG Fc (orange), or ACE2(HH:NN) hamster Fc (green) to HEK293T cells expressing hamster FcγRI, FcγRIIa, FcγRIIb, and FcγRIII receptors, detected with biotinylated SARS-CoV-2 S1 and streptavidin conjugated secondary agent. No binding was detected with ACE2(HH:NN)-Fc constructs carrying the LALA-PG mutations on the hamster FcγRs, while limited binding was detected with the ACE2(HH:NN) WT Fc. (B) Syrian hamster serum concentration of i.p. injected ACE2(HH:NN)-Fc LALA-PG at 5 and 50 mg/kg doses, over the course of 28 days ( n = 6/group, 3 animals per time point). C max , maximum detected concentration; T max , peak concentration time; t 1/2 , half-life; V z , volume of distribution; Cl, clearance rate; MRT, mean residence time. (C to I) Syrian hamster intranasally challenged with authentic SARS-CoV-2. ACE2(HH:NN)-Fc (LALA-PG) administered i.p. at day 1 postchallenge at 5 mg/kg, 50 mg/kg, or placebo (PBS) ( n = 6 per group). (C) Body weight change (%) relative to that on the day of viral inoculation. Day of therapeutic administration marked with arrow. Significant reduction of body weight change relative to that of the placebo, detected for both treatment regimens (mean ± SD). Individual day comparison placebo versus 50 mg/kg dose two-way ANOVA with Sidak’s multiple comparisons compared to placebo group. *, P = 0.01; **, P = 0.004; ***, P = 0.0001; ****, P < 0.0001. One-way ANOVA of AUC with Tukey’s multiple comparisons (F = 9.379, df = 2, 225). (D) Hamster activity monitoring (wheel rotation) showing faster recovery trend at 5 to 6 DPI for the treated groups. (E) Clinical symptoms scoring per group per day, based on fur appearance, nasal/ocular discharge, posture, breathing, activity, and body weight. (F) Total E RNA and subgenomic RNA PCR assay from lung extracts at 7 DPI. Limit of detection 3.28 RNA copies. Samples with undetectable RNA were assigned a value of 1 (mean ± SD). (G) Subgenomic RNA PCR swab test. Limit of detection 2.88 RNA copies; samples with undetectable RNA were assigned a value of 1 (mean ± SD). Two-way ANOVA with Dunnett’s multiple comparisons. (H) Necropsy pathology lung score (categories 1 to 4) showing reduction in lung damage for ACE2(HH:NN)-Fc LALA-PG treated groups. Bottom, representative lung damage for grade scores 1, 2, 3, and 4. (D) Human IgG1 Fc concentration in hamster sera at day −5 and day 7 relative to viral inoculation. Limit of detection 4 ng/ml. Samples with undetectable levels were assigned a value of 1 (mean ± SD). Two-way ANOVA with Sidak’s multiple comparisons (F = 39.2, df = 2, 22).

Article Snippet: Protein labeled cells were stained with biotinylated SARS-CoV-2 S1 (ACRO biosystems, S1N-C82E8) and detected with streptavidin AF647 (Invitrogen, S21374).

Techniques: In Vivo, Neutralization, Binding Assay, Expressing, Construct, Concentration Assay, Injection, Activity Assay

Journal: Journal of Virology

Article Title: Characterization of a Novel ACE2-Based Therapeutic with Enhanced Rather than Reduced Activity against SARS-CoV-2 Variants

doi: 10.1128/JVI.00685-21

Figure Lengend Snippet: ACE2-Fc formulation optimization. (A) Thermal stability analysis via nanoDSF of ACE2(HH:NN)-Fc in PBS at pH 7.4 or in 20 mM His buffer with pH range of 3.5 to 7. Highest stability obtained with 20 mM His (pH 6.5). (B) Thermal stability of ACE2(HH:NN)-Fc in 20 mM His pH 6.5 (orange) and 3.5 (green) following 2 h of incubation at RT. Full stability could be recovered following buffer exchange of ACE2(HH:NN)-Fc from pH 3.5 to pH 6.5 (blue). (C) Particle size distribution analysis via MADLS of ACE2(HH:NN)-Fc at 1 mg/ml in PBS (pH 7.4), 20 mM His *(pH 3.5, 6.5), or buffer exchanged from pH 3.5 to 6.5. Increase of particle size of sample at pH 3.5 was partially recovered upon buffer exchange in 20 mM His (pH 6.5) (mean ± SD). (D) ACE2(HH:NN)-Fc binding capacity for SARS-CoV-2 S1 in ELISA in PBS (pH 7.4; blue), 20 mM His (pH 6.5; orange), or 20 mM His (pH 3.5) followed by buffer exchange in 20 mM His (pH 6.5; green) (mean ± SD). (E) Thermal stability analysis via nanoDSF of ACE2(HH:NN)-Fc (LALA-PG) in PBS at pH 7.4 (blue) or in 20 mM His (pH 6.5; orange) showing a 3.9°C T m shift. (F) Particle size distribution analysis via MADLS of ACE2(HH:NN)-Fc (LALA-PG) at 1 mg/ml in PBS (pH 7.4; blue) and 20 mM His (pH 6.5; orange) showing comparable profile (mean ± SD).

Article Snippet: Protein labeled cells were stained with biotinylated SARS-CoV-2 S1 (ACRO biosystems, S1N-C82E8) and detected with streptavidin AF647 (Invitrogen, S21374).

Techniques: Nano Differential Scanning Fluorimetry, Incubation, Buffer Exchange, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Infection and Immunity

Article Title: GigC, a LysR Family Transcription Regulator, Is Required for Cysteine Metabolism and Virulence in Acinetobacter baumannii

doi: 10.1128/IAI.00180-20

Figure Lengend Snippet: Differential scanning fluorimetry analysis of GigC. Differential scanning fluorimetry (DSF) assays were performed on recombinant GigC alone (A and B) and in the presence of the indicated concentrations of l-cysteine (l-Cys) (C and D). DSF data are plotted as the first derivative of the raw fluorescence value over the indicated temperature range. The DSF experiment was repeated three times, and results from a representative experiment are shown.

Article Snippet: Briefly, 20-μl reaction mixtures were prepared in clear 96-well microtiter plates (Applied Biosystems) in DSF buffer (9.25 mM HEPES, 140 mM NaCl, 7.5× SYPRO orange [Sigma-Aldrich] [pH 7.0]).

Techniques: Recombinant, Fluorescence