Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The inhibition of the interaction between the spike protein S1 receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor by kuwanon C (KC). Spike protein coated on a 96-well plate interacted with a preincubated mixture of the ACE2 receptor and ( A ) anti-SARS-CoV-2 spike S1 antibody as the positive control and ( B ) 0, 3.125, 6.25, 12.5, 25, 50, or 100 μM KC. The inhibition of the spike S1 RBD:ACE2 receptor interaction by KC was determined based on chemiluminescence measurements.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Inhibition, Binding Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The global kinetic analysis of KC binding to biotinylated ( A ) spike S1 RBD- and ( B ) ACE2 receptor-immobilized BLI sensors. The kinetics for the binding of KC to the spike S1 RBD or the ACE2 receptor were measured by the association of 0, 50, 200, and 400 μM of KC in PBS containing 1% DMSO with immobilized spike S1 or ACE2 receptor and the subsequent dissociation in PBS containing 1% DMSO.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The binding kinetics of KC to spike S1 RBD and ACE2 receptor.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: In silico docking simulation between KC and the spike protein/ACE2 receptor. KC was docked onto the SARS-CoV-2 spike protein and ACE2 receptor (PDB code: 6M0J) using AutoDock Vina. The pharmacophore of KC with each target proteins was analyzed using BIOVIA Discovery Studio Visualizer.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: In Silico

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC inhibits SARS-CoV-2 lentiviral pseudovirus infection in HEK293T cells stably expressing human ACE2 and TMPRSS2. ( A ) The cytotoxic effect of KC in HEK293T cells stably expressing human ACE2 and TMPRSS2 was determined using the MTT assay. HEK293T cells were cultured in 96-well plates (5 × 10 4 cells/well) for 18 h. ( B ) The ACE2 expression level in HEK293T cells was monitored during KC treatment using real-time quantitative PCR analysis. Then, WT or mutant (D614G) SARS-CoV-2 spike pseudovirus (at a final concentration of 1 × 10 4 TU/mL to each well) were mixed with different concentrations of KC (2 and 20 μM) or anti-SARS-CoV-2 antibody, and the mixtures were incubated at 37 °C for 1 h. Then, these mixtures were added to HEK293T cells. ( C , D ) Green fluorescent protein (GFP) expression levels using flow cytometry were assessed at 72 h after viral infection, scale bar = 100 μm. ( E , F ) The inhibitory effect of SARS-CoV-2 spike pseudovirus infection was determined by measuring GFP expression using flow cytometry and measured under a fluorescence microscope. Bar graph (mean ± SEM) statistics were determined from three experimental data sets using one-way ANOVA with Tukey’s post hoc test, *** p < 0.001, compared with the CON (KC-untreated) samples. ### p < 0.001, compared with the cell-only sample.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Stable Transfection, Expressing, MTT Assay, Cell Culture, Real-time Polymerase Chain Reaction, Mutagenesis, Concentration Assay, Incubation, Flow Cytometry, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC suppresses the infection of a clinical isolate of SARS-CoV-2 alpha strain (βCoV/Korea/KCDC03/2020) in Vero cells. Vero cells were cultured on 384-well plates (1.2 × 10 4 cells/well) for 24 h. Then, Vero cells were infected with SARS-CoV-2 (MOI 0.0125) immediately after being treated with serially diluted KC and incubated at 37 °C for 24 h. The cells were then stained using anti-SARS-CoV-2 nucleocapsid (N) primary antibody, Alexa Fluor 488-conjugated goat antirabbit IgG secondary antibody, and Hoechst 33342.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Cell Culture, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: Schematic of the blockade of the SARS-CoV-2 spike S1 RBD:ACE2 receptor interaction by KC.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques:

Journal: Cell Host & Microbe

Article Title: Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

doi: 10.1016/j.chom.2020.11.007

Figure Lengend Snippet: A Yeast-Display System to Completely Map SARS-CoV-2 RBD Antibody-Escape Mutations (A) Yeast display RBD on their surface. The RBD contains a c-Myc tag, enabling dual-fluorescent labeling to quantify RBD expression and antibody binding by flow cytometry. (B) RBD expression and antibody binding as measured by flow cytometry for yeast expressing unmutated RBD and an RBD mutant library. (C) Yeast expressing RBD mutant libraries are sorted to purge mutations that abolish ACE2 binding or RBD folding. These libraries are labeled with antibody, and cells expressing RBD mutants with decreased antibody binding are enriched by using FACS (the “antibody-escape” bin; see Figure S1 for gating). Deep-sequencing counts are used to compute the “escape fraction” for each mutation. Escape fractions are represented in logo plots, with tall letters indicating mutations that strongly escape antibody binding.

Article Snippet: Biotinylated human ACE2 , ACROBiosystems , Cat# AC2-H82E6.

Techniques: Labeling, Expressing, Binding Assay, Flow Cytometry, Mutagenesis, Sequencing

Journal: Cell Host & Microbe

Article Title: Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

doi: 10.1016/j.chom.2020.11.007

Figure Lengend Snippet: Complete Maps of Escape Mutations from 10 Human Monoclonal Antibodies (A) Properties of the antibodies as reported by Zost et al. (2020a) . SARS-CoV-2 neutralization potency is represented as a gradient from black (most potent) to white (non-neutralizing). Antibodies that bind SARS-CoV-1 spike or compete with RBD binding to ACE2 or rCR3022 are indicated in black. (B) Structure of the SARS-CoV-2 RBD (PDB: 6M0J ; Lan et al., 2020 ), with residues colored by whether they are in the core RBD distal from ACE2 (orange), in the receptor-binding motif (RBM, light blue), or in direct contact with ACE2 (dark blue). ACE2 is in gray. RBD sites where mutations escape antibodies are indicated with spheres. (C) Maps of escape mutations from each antibody. The line plots show the total escape at each RBD site (sum of escape fractions of all mutations at that site). Sites with strong escape mutations (indicated by purple at bottom of the line plots) are shown in the logo plots. Logo plots are colored by RBD region as in (B). Different sites are shown for the rCR3022-competing antibodies (top four) and all other antibodies (bottom six). For interactive escape maps, see https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_Crowe_antibodies . (D) Multidimensional scaling projection of the escape mutant maps, with antibodies having similar escape mutations drawn close together. Each antibody is shown with a pie chart that uses the color scale in (B) to indicate the RBD regions where it selects escape mutations. See also and and .

Article Snippet: Biotinylated human ACE2 , ACROBiosystems , Cat# AC2-H82E6.

Techniques: Bioprocessing, Neutralization, Binding Assay, Mutagenesis

Journal: Cell Host & Microbe

Article Title: Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

doi: 10.1016/j.chom.2020.11.007

Figure Lengend Snippet: Structural Mapping of Antibody Binding and Escape (A–D) For each antibody, the structure shows the RBD surface colored by the largest-effect escape mutation at each site, with white indicating no escape and red indicating the strongest escape mutation for that antibody. ACE2 contact residues are outlined in black. Antibodies are arranged by structural epitope: (A) core RBD, (B) ACE2-binding ridge, (C) the opposite edge of the RBM, or (D) the saddle of the RBM surface. (E) Crystal structure of the rCR3022-bound RBD (PDB: 6W41 ; Yuan et al., 2020 ), with Fab in purple, ACE2 contact sites outlined, and RBD colored according to sites of escape as in (A). (F) For 5 antibodies, Fab bound to SARS-CoV-2 spike ectodomain trimer was visualized by negative-stain EM. The modeled RBD is colored according to sites of escape as in (A). Fab chains are modeled in gold. Antibody names are colored according to Figure 2 B: core-binding, orange; RBM-binding, cyan; ACE2 contact site-binding, dark blue. See https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_Crowe_antibodies/ for interactive versions of the escape-colored structures in (A)–(D). See also .

Article Snippet: Biotinylated human ACE2 , ACROBiosystems , Cat# AC2-H82E6.

Techniques: Binding Assay, Mutagenesis, Staining

Journal: Cell Host & Microbe

Article Title: Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

doi: 10.1016/j.chom.2020.11.007

Figure Lengend Snippet: Functional and Evolutionary Constraint on Antibody-Escape Mutations (A) Variation at sites of antibody escape among currently circulating SARS-CoV-2 viruses. For each site of escape from at least one antibody, we counted sequences in GISAID with an amino-acid change. Sites with at least 5 GISAID variants (of 93,858 sequences at the time of analysis) are shown ordered by count; black cells indicate antibodies with escape mutations at that site. Sites are colored by RBD region. Antibodies are colored according to where the majority of their sites of escape fall. See also Figure S4 . (B) Escape maps (as in Figure 2 C), with letters colored according to how deleterious mutations are for ACE2 binding or RBD expression ( Starr et al., 2020 ). Only sites of escape mutations for each antibody are depicted. See Figure S5 for similar logo plots for all antibodies. (C) Mutational constraint on sites of escape. For each antibody, the mean effects of all possible amino acid mutations at sites of escape on ACE2 binding and RBD expression are shown. (D) Top: effective number of amino acids ( N eff ) in the sarbecovirus RBD alignment at sites of escape for each antibody. N eff is a measure of the variability of a site (the exponentiated Shannon entropy), and ranges from 1 for a position that is completely conserved to 20 for a site where all amino acids are present at equal frequency. Bottom: escape fraction for each sarbecovirus RBD homolog from the yeast display selections; 1 means complete escape (no binding), and 0 means no escape (complete binding).

Article Snippet: Biotinylated human ACE2 , ACROBiosystems , Cat# AC2-H82E6.

Techniques: Functional Assay, Binding Assay, Expressing

Journal: Cell Host & Microbe

Article Title: Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

doi: 10.1016/j.chom.2020.11.007

Figure Lengend Snippet: Viral Escape Mutant Selections with Individual Antibodies and Antibody Cocktails (A) Results of viral selections with five individual monoclonal antibodies. The number of replicates where escape variants were selected are indicated, color-coded according to whether escape was selected frequently (red) or rarely (white). Mutations present in the RBD of the selected escape variants are indicated. (B) Each point represents a different amino-acid mutation to the RBD, with the x axis indicating how strongly the mutation ablates antibody binding in our escape maps and the y axis indicating how the mutation affects ACE2 binding (negative values indicate impaired ACE2 binding). All selected mutations were accessible by single-nucleotide changes. The only accessible escape mutation from COV2-2165 that is not deleterious to ACE2 binding is D420Y, but this mutation is highly deleterious for RBD expression ( Figure 5 B; Figure S5 ). (C) Results of viral selections with antibody cocktails, indicating the number of replicates with escape out of the total tested. The data for the single antibodies are repeated from (A). In all panels, antibody names are colored according to where in the RBD the majority of their sites of escape fall: orange for the core RBD, light blue for the RBM, and dark blue for ACE2 contact residues. See also Figure S6 .

Article Snippet: Biotinylated human ACE2 , ACROBiosystems , Cat# AC2-H82E6.

Techniques: Mutagenesis, Bioprocessing, Binding Assay, Expressing

Journal: Cell Host & Microbe

Article Title: Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

doi: 10.1016/j.chom.2020.11.007

Figure Lengend Snippet:

Article Snippet: Biotinylated human ACE2 , ACROBiosystems , Cat# AC2-H82E6.

Techniques: Virus, Mutagenesis, Recombinant, Expressing, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Sequencing, Staining, Software, Chromatography, Microscopy, Cell Culture

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Identification of neutralizing antibodies with a PtY display platform. We first used our preconstructed naïve phage displayed human scFv library to screen binders with biotinylated SARS-CoV-2 RBD protein in the solution phase. After enrichment of phage binders, the scFv DNA from enriched binders was cloned into the yeast display plasmid, resulting in display of scFv on the yeast cell surface. We then performed FACS to isolate potential blocking antibodies that could prevent binding of the SARS-CoV-2 RBD to hACE2. The 0.013% gate contained blocking antibodies with high affinity toward RBD. That is, higher Y axis signal represented higher affinity to labeled RBD, whereas lower X signal represented higher potency in blocking the binding of differently labeled hACE2 to RBD. The potential blocking antibodies were sent for sequencing and transient expression. The purified antibodies were evaluated for affinity, blocking activity, biophysical properties, and virus-neutralizing activity

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Clone Assay, Plasmid Preparation, Blocking Assay, Binding Assay, Labeling, Sequencing, Expressing, Purification, Activity Assay

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characteristics of potential blocking antibodies

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Expressing, Binding Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of potential blocking antibodies. (a) Blocking assay was performed by immobilizing 1 µg/ml hACE2 on a plate. Serially diluted antibodies and biotinylated SARS-CoV-2 RBD protein were added for competitive binding to hACE2. IC 50 values were calculated with Prism V8.0 software using a four-parameter logistic curve fitting approach. (b) Epitope binning was carried out by BLI. Biotinylated SARS-CoV-2 RBD was immobilized onto the SA sensor, and a high concentration of the primary antibody was used to saturate its own binding site. Subsequently, a second antibody was applied to compete for the binding site on the SARS-CoV-2 RBD protein. Data were analyzed with Octet Data Analysis HT 11.0 software. (c) Neutralization activities of Ab2001.08 and Ab2001.10 were assessed by live virus assay. Live SARS-CoV-2 and serially diluted (3-fold) antibodies were added to VERO E6 cells. The PRNT 50 values were determined by plotting the plaque number (neutralization percentage) against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Binding Assay, Software, Concentration Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of JMB2002. Binding affinity of JMB2002 for the SARS-CoV-2 RBD (a)/S1 (b) prototype and its variants was determined by BLI. JMB2002 was loaded onto the AHC sensor, and serially diluted antigens were bound to JMB2002 on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. Blocking activity was assessed using ELISA with hACE2-coated plates. A mixture of biotinylated SARS-CoV-2 RBD (c)/S1 (d) proteins and JMB2002 was added for competitive binding to hACE2. IC 50 values were calculated by Prism V8.0 software using a four-parameter logistic curve fitting approach. Values are displayed as the mean ± standard deviations from three independent experiments. (e) The pseudovirus neutralization activity of JMB2002 was evaluated using a pseudotyped SARS-CoV-2 system, which contained a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to hACE2-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. IC 50 values were calculated by plotting the inhibition rate against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Binding Assay, Software, Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Luciferase, Expressing, Incubation, Infection, Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Targeting SARS-CoV-2 infection through CAR T cells and bispecific T cell engagers

doi: 10.1101/2022.01.19.476940

Figure Lengend Snippet: (A) Illustration of Spike protein localization on the surface of SARS-CoV-2 infected cells and (B) of full-length SARS-CoV-2 Spike protein mRNA expressing plasmid including the Endoplasmic Reticulum Retention Signal (ERRS) of Spike protein on C terminal. (C) 293 cells transfected with full-length Spike protein (blue histogram) or with VSV-G as a negative control (red histogram) expressing vectors. The cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, flow cytometry data overlays are shown. (D) 293 cells transduced with a lentivirus encoding a truncated Spike protein gene without the ERRS domain and Green Fluorescent Protein (GFP) as a reporter. Transduced cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, representative flow cytometry data plots are shown. (E) Illustration of ACE2 CAR and anti-SARS-CoV-2 Spike protein CAR constructs and their expression in CD8 T cells. A constitutive LTR promoter drives ACE2 or anti-Spike CAR and RFP genes separated by an Internal Ribosomal Entry Site (IRES). CAR constructs consist of CD8 alpha signal peptide, ACE2 or single chain variable fragment of an anti-Spike antibody, CD8 Hinge, CD8 transmembrane domain, 4-1BB (CD137) co-stimulatory domain and CD3ζ domain. Lentiviruses containing CARs were used to transduce primary CD8 T cells. (F) Expression of CAR constructs on CD8 T cells. Activated and transduced CD8 T cells were expanded for 10-12 days and stained with SARS-CoV-2 S1 protein fused to mouse Fc, and anti-mouse Fc secondary antibody. Flow cytometry plots showing ACE2 or anti-Spike surface expression versus RFP are shown. Anti-CD19 CAR expressing CD8 T cells were used as control. The experiments were replicated several times with similar results.

Article Snippet: For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems), then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).

Techniques: Infection, Expressing, Plasmid Preparation, Transfection, Negative Control, Staining, Flow Cytometry, Transduction, Construct

Journal: bioRxiv

Article Title: Targeting SARS-CoV-2 infection through CAR T cells and bispecific T cell engagers

doi: 10.1101/2022.01.19.476940

Figure Lengend Snippet: (A) Illustration of cytotoxicity assay against Spike-expressing target cells using ACE2 CAR or anti-Spike CAR expressing CD8 T cells as effector cells. (B) CAR engineered T cells cytotoxicity assays with Spike expressing 293 target cells at different Effector:Target ratios. CD8 T cells transduced with anti-CD19 CAR lentiviruses were used as control effector cells. Effector CD8 T cells were identified with CD8 staining while target cells were gated based on GFP (Spike) expression. Activation of effector cells and CAR expression were determined with CD25 expression after gating on CD8 T cells. (C) Percent cytotoxicity of ACE2 CAR (blue) and anti-Spike CAR (purple) T cells normalized to anti-CD19 CAR-T cells at different Effector:Target ratios and using Spike-expressing 293 cells as the target. (D) CAR engineered T cells cytotoxicity assays with Spike-expressing target B cell line (T2 cells). Wild-type CD8 T cells were used as negative control and anti-CD19 CAR expressing CD8 T cells were used as positive control. Panels show representative experiments replicated with similar results.

Article Snippet: For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems), then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).

Techniques: Cytotoxicity Assay, Expressing, Transduction, Staining, Activation Assay, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Targeting SARS-CoV-2 infection through CAR T cells and bispecific T cell engagers

doi: 10.1101/2022.01.19.476940

Figure Lengend Snippet: (A) . Illustration describing potential mechanism of action of ACE2-Bite. The extracellular domain (ECD) of ACE2 (blue) in ACE2-Bite binds to Spike protein (red) expressed on the surface of SARS-CoV-2 infected cells and the anti-CD3 fragment (orange) binds to CD3 molecule (purple) on T cells linking both cell types and inducing the activation of T cells which subsequently results in apoptosis of infected target cells. ACE2-Bite recombinant protein also contains a hemagglutinin (HA) tag at the C terminal. (B) Representation of ACE2-Bite construct and the protein production in 293 cells. A constitutive LTR promoter drives the expression of ACE2-Bite and RFP genes separated by an Internal Ribosomal Entry Site (IRES). ACE2-Bite cassette consists of ACE2 signal peptide (SP), ACE2 extracellular domain, a linker peptide, an anti-CD3 antibody single-chain variable fragment, a His-Tag, and a Hemagglutinin (HA) Tag. Lentiviruses expressing ACE2-Bite were used to transduce suspension 293 cells that produce and secrete ACE2-Bite protein in their culture supernatant. (C) Representation of the bead-based ACE2-Bite capture assay. Fluorescent beads coated with Spike-Receptor binding domain (S-RBD) were used to capture ACE2-Bite molecules which were detected via a recombinant CD3-Fc fusion protein and an anti-Fc antibody then subsequently analyzed via flow cytometry. ACE2-Fc molecules were also detected with S-RBD coated beads and anti-Fc antibody . (D) Detection of different concentrations of ACE2-Bite (1:10 and 1:300 dilutions were shown in orange and turquoise, respectively) and ACE2-Fc (3 μg/mL) (red) by bead-based ACE2-Bite capture assay. Wild-type 293 cell supernatant (Control supe, Blue) and staining buffer (None, Pink) were used as negative controls. (E) Binding of ACE2-Bite to Spike-GFP expressing T2 cell line and primary human T cells. HA staining of Spike-GFP expressing T2 cells (top panel) and CD8 T cells (bottom panel) when combined with ACE2-Bite (right plot) or control (Wild-type 293) (left plot) supernatant. (F) CD25 and GFP expression show activation and cytotoxicity state of resting CD8 T cells against Spike/GFP-expressing or control (transduced with GFP-expressing empty vector) 293 cells in the presence of ACE2-Bite or control supernatant. The experiments were replicated with similar results.

Article Snippet: For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems), then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).

Techniques: Infection, Activation Assay, Recombinant, Construct, Expressing, Transduction, Binding Assay, Flow Cytometry, Staining, Plasmid Preparation

Journal: bioRxiv

Article Title: Targeting SARS-CoV-2 infection through CAR T cells and bispecific T cell engagers

doi: 10.1101/2022.01.19.476940

Figure Lengend Snippet: Area under the curve (AUC) values of ACE2-Bite molecules in supernatants from different conditions. Supernatants from ACE2-Bite secreting and wild-type suspension 293 cells were collected at several timepoints representing different cell densities ranging from 3 to 7 million/mL. ACE2-Bite molecules taken from 3 million/mL cell culture supernatant were concentrated 5-folds and 30-folds. Flow through supernatant from the concentration process (Filter flow through) and wild-type control supernatant were used as controls. Fluorescent beads coated with Spike-Receptor binding domain (S-RBD) were used to capture ACE2-Bite molecules in supernatants titrated from 1:1 to 1:1000 by 10-fold serial dilutions were detected via a recombinant CD3-Fc fusion protein and an anti-Fc antibody. Geometric mean intensity of anti-Fc antibody fluorescence were used to generate curves which were used to calculate the area under the curve values.

Article Snippet: For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems), then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).

Techniques: Cell Culture, Concentration Assay, Binding Assay, Recombinant, Fluorescence

Journal: bioRxiv

Article Title: Targeting SARS-CoV-2 infection through CAR T cells and bispecific T cell engagers

doi: 10.1101/2022.01.19.476940

Figure Lengend Snippet: (A) Representation of ACE2-Bite binding to spike protein (wild-type or mutated) expressed on the cell surface membrane and its detection by immunostaining with an anti-HA antibody. Wild-type or mutant Spike proteins were expressed on 293 cells for ACE2-Bite/Spike binding assay. (B) Geometric mean intensity of anti-HA antibody staining used to detect ACE2-Bite molecules on mutant Spike-expressing cells. The cells were co-stained with an anti-Spike antibody and ACE2-Bite/anti-HA Tag antibody and analyzed via flow cytometry. For each condition, anti-Spike antibody-staining was used as a Spike protein marker and fluorescently equivalent gates were set before assessing the geometric mean fluorescent intensity of ACE2-Bite-stained cells to determine the quantitative value of ACE2-Bite fluorescence intensity per Spike protein. Unpaired t test was used to determine the statistical significance. (C) CD25 expression of CD8 T cells when co-cultured with wild-type and mutated Spike protein plasmid transfected 293 cells (used in B) in the presence of ACE2-Bite supernatant.

Article Snippet: For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems), then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).

Techniques: Binding Assay, Immunostaining, Mutagenesis, Staining, Expressing, Flow Cytometry, Marker, Fluorescence, Cell Culture, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Targeting SARS-CoV-2 infection through CAR T cells and bispecific T cell engagers

doi: 10.1101/2022.01.19.476940

Figure Lengend Snippet: (A) Schematic illustration of virus neutralization assay. ACE2-Bite and Spike (wild-type and mutated) pseudotyped lentiviruses are pre-incubated then added to ACE2-overexpressing 293 cells. (B) Representative FACS plots show neutralization data of the delta variant pseudotyped virus infection when pre-incubated with different concentrations of ACE2-Fc (top panel) or different dilutions of ACE2-Bite supernatant (bottom panel). The infection levels were determined 3 days later via flow cytometry based on GFP expression. (C) Line graph represents virus neutralization data of the lentiviruses pseudotyped with different Spike protein variants when pre-incubated with ACE2-Bite at different ACE2-Bite:virus ratios then added to ACE2-overexpressing 293s. These experiments were replicated twice with similar results.

Article Snippet: For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems), then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).

Techniques: Neutralization, Incubation, Variant Assay, Infection, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

doi: 10.1101/2021.07.14.452313

Figure Lengend Snippet: SPR-experiment testing for the presence of a two state reaction. 500 nM SARS-CoV-2 S1-S2 spike protein was injected over a constant immobilization level of 25 RU hACE2 and normalized to 100 % RU for the time point of injection phase end. Injection times were gradually increased (150 – 600 s).

Article Snippet: The biotinylated hACE2-fc (residues 18-740) was purchased by Acrobiosystems and contains a C-terminal IgG1 Fc-Tag (residues 100-330), followed by a biotinylated Avitag.

Techniques: Injection

Journal: bioRxiv

Article Title: Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

doi: 10.1101/2021.07.14.452313

Figure Lengend Snippet: (A) Multicycle experiment with SARS-CoV-2 RBD. hACE2-fc was immobilized on a protein A/G sensor chip and SARS-CoV-2 RBD was injected in concentration range of 3.9 – 1000 nM. The K D was globally fitted with a 1:1 Langmuir based interaction model. The kinetic parameters were determined with K D of 21.3 nM, k a of 4.25 E+5 +/- 2.2 E+2 [1/Ms] and k d of 9.1 E-3 +/- 4.2E-6 [1/s]. ( B ) Test on secondary state reaction. hACE2-fc was immobilized on a protein A sensor surface and 500 nM of SARS-CoV-2 was injected at a constant concentration for increasing contact intervals. Time points of injection phase end were normalized to 100 % and the dissociation starting point was aligned on the time scale.

Article Snippet: The biotinylated hACE2-fc (residues 18-740) was purchased by Acrobiosystems and contains a C-terminal IgG1 Fc-Tag (residues 100-330), followed by a biotinylated Avitag.

Techniques: Injection, Concentration Assay

Journal: bioRxiv

Article Title: Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

doi: 10.1101/2021.07.14.452313

Figure Lengend Snippet: (A) hACE2-fc was immobilized on a protein A/G sensor surface and CoV trimer proteins were injected in concentration range of 0.62 – 50 nM. The sensograms were globally fitted with the secondary state reaction model. (SARS-CoV 2002) : K D1 6.7 nM, k a1 6.72 E+5 ± 4.4E+3 [1/Ms], k d1 4.68 E-3 [1/s] ± 6.9E-5, k a2 8.80 E-3 ± 6.9E-5 [1/Ms], k d2 2.07E-4 ± 1.60 E-6 [1/s]; K D-total 160 pM (SARS-CoV-2 wt) : K D1 1.8 nM, k a1 7.82 E+5 ± 1.2E+3 [1/Ms], k d1 1.41 E-3 ± 3.75E-5 [1/s], k a2 1.05E-2 ± 1.8E-4 [1/Ms], k d2 2.44E-4 ± 3.5E-6 [1/s]; K D-total 41 pM. ( SARS-CoV-2 B.1.1.7 ) K D1 192.7 nM, k a1 1.31 E+6 ± 1.5E+4 [1/Ms], k d1 2.5 E-2 ± 1.5E-3 [1/s], k a2 5.3E-2 ± 1.5E-3 [1/Ms], k d2 1.1 E-4 ± 2.6E-6 [1/s]; K D-total 40 pM. For graphic representation of the distribution of primary and secondary state reaction, a component analysis was performed for each of the trimeric spike proteins using the injection concentration of 5.56 nM spike. The sensogram (total) is composed of the primary binding event (red), followed by a secondary transition event (blue) which results in a highly stable secondary complex ( B ) On–off chart for the kinetic values of the SARS-CoV spike trimers in the primary and secondary state.

Article Snippet: The biotinylated hACE2-fc (residues 18-740) was purchased by Acrobiosystems and contains a C-terminal IgG1 Fc-Tag (residues 100-330), followed by a biotinylated Avitag.

Techniques: Injection, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: Variable Induction of Pro-inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

doi: 10.1101/2021.05.26.445843

Figure Lengend Snippet: Cytokine responses in human PBMC induced by various commercial coronavirus spike proteins. ( A ) Rested PBMC were cultured with or without 2.0 μg/mL of raxibacumab (human anti-anthrax PA IgG used as a negative control), MERS S1-Fc, SARS CoV-1 S1-Fc, SARS CoV-2 S1-Fc from Vendor #2 (V#2 S1-Fc) and Vendor #1 (lot 24529-2003, V#1 S1-Fc 2003), and RBD-Fc from Vendor #2 (V#2 RBD-Fc) and Vendor #1 (lot 24530-2003, V#1 RBD-Fc 2003) for 24 hours. ( B ) Rested PBMC were cultured with or without plate-bound streptavidin (STAV) together with or without S1-biotin and RBD-biotin purchased from Vendor #2. The levels of IL-6 and TNFα were measured using the CBA human inflammatory cytokine kit and flow cytometric analysis. IL-8 levels were measured using an ELISA kit. The concentrations of the cytokines were calculated based on the standard curves, and the induction of cytokines were presented as stimulation indices. Data shown are statistical results (mean ± SE) generated from 8 ( A ) or 3 ( B ) healthy donors. Statistical analyses were performed by Excel using a two-tailed, Student’s T-test. *, **, *** and **** depict p < 0.05, 0.01, 0.005 and 0.001, respectively.

Article Snippet: S1-Fc (Catalog# 40591-V02H, lot LC14AP1605), RBD-Fc (Catalog# 40592-V02H, lot LC14MC2602), S1-biotin (Catalog# 40591-V27HB, lot LC14AU101), RBD-biotin (Catalog# 40592-V27B-B, lot MF14AP2302), ACE2 (Catalog# 10108-H08H, lot MB14JN0906), anti-S1 neutralizing antibody (Catalog# 40591-MM43, lot HB14AP2001) and anti-S1 non-neutralizing antibody (Catalog # 40591-MM42, lot HB14AP0701) were also purchased from Sino Biological (Vendor #2).

Techniques: Cell Culture, Negative Control, Enzyme-linked Immunosorbent Assay, Generated, Two Tailed Test

Journal: bioRxiv

Article Title: Variable Induction of Pro-inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

doi: 10.1101/2021.05.26.445843

Figure Lengend Snippet: LPS co-purifying with spike protein reagents induces proinflammatory cytokine production. ( A ) MERS S1-Fc, SARS CoV-1 S1-Fc, S1-Fc from Vendor #2 (V#2 S1-Fc), S1-Fc from Vendor #1 (lot 24056-2002-2 (V#1 S1-Fc 2002) and lot 24529-2003 (V#1 S1-Fc 2003)), RBD-Fc from Vendor #2 (V#2 RBD-Fc) and RBD-Fc from Vendor #1 (lot 25130-2004, V#1 RBD-Fc 2004), and ( B ) V#2 S1-Fc, V#1 S1-Fc 2002 and streptavidin (STAV) before and after treatment of endotoxin removal were assessed for the levels of endotoxin using the LAL Chromogenic Endotoxin Quantitation Kit. The concentrations of endotoxin are presented as EU/mg protein. Data shown are mean ± SE of the results from three independent experiments. ( C ) Time course of S1-Fc-induced cytokine responses. Rested PBMC were cultured with or without 2.0 μg/mL of S1-Fc (V#2 S1-Fc and V#1 S1-Fc 2002) for 1, 3, 6 or 24 hours. The levels of IL-6, IL-8 and TNFα in the supernatants of cultured PBMC were assessed using the CBA human inflammatory cytokine kit and flow cytometric analysis. ( D, E ) Blockade of S1-Fc-induced cytokine response by an LPS inhibitor, polymyxin B. Rested PBMC were cultured with or without 2.0 μg/mL of S1-Fc (V#2 S1-Fc) in the presence or absence of polymyxin B for 3 hours. The levels of IL-6, IL-8 and TNFα in the supernatants of cultured PBMC were measured using the CBA human inflammatory cytokine kit and flow cytometric analysis. Data shown in ( D ) are a representative of the flow cytometric results from three healthy donors and data shown in ( E ) are mean ± SE of the stimulation index derived from three healthy donors, which is calculated using mean fluorescent intensities (MFI) of the cytokines and the formula: cytokine MFI of treated PBMC/cytokine MFI of untreated PBMC. Statistical analyses were performed using a two-tailed, Student’s T-test. * and ** depict p < 0.05 and 0.01, respectively.

Article Snippet: S1-Fc (Catalog# 40591-V02H, lot LC14AP1605), RBD-Fc (Catalog# 40592-V02H, lot LC14MC2602), S1-biotin (Catalog# 40591-V27HB, lot LC14AU101), RBD-biotin (Catalog# 40592-V27B-B, lot MF14AP2302), ACE2 (Catalog# 10108-H08H, lot MB14JN0906), anti-S1 neutralizing antibody (Catalog# 40591-MM43, lot HB14AP2001) and anti-S1 non-neutralizing antibody (Catalog # 40591-MM42, lot HB14AP0701) were also purchased from Sino Biological (Vendor #2).

Techniques: Quantitation Assay, Cell Culture, Derivative Assay, Two Tailed Test

Journal: Immunity

Article Title: Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia

doi: 10.1016/j.immuni.2021.05.002

Figure Lengend Snippet: ACE2 expression in a CD4+ T cell subset increases after ex vivo stimulation (A) Comparison of immune features derived from each leukocyte subpopulation between experimental groups. A dot plot displaying the ES calculated in HAP versus COVID-19s (x axis) compared with the ES calculated in COVID-19m versus COVID-19s (y axis). Each dot represents one immunological feature. The red box highlights the immune feature focused in this figure. (B) Median expression of indicated markers in FlowSOM-derived clusters of unstimulated samples. (C) Median frequency and 25th and 75th percentiles of ACE2-positive cells in a subset of unstimulated CXCR3 + CCR6 + (Th1 Th17-enriched) CD4 + T cells. All TPs have been pooled. (D) Median frequency and 25th and 75th percentiles of CXCR3 + CCR6 + (Th1 Th17-enriched) CD4 + T cells at each TP. (E) Representative plot showing ACE2 and isotype staining within the T cell compartment of PMA and ionomycin-restimulated (5 h) COVID-19 samples. (F) Median frequency and 25th and 75th percentiles of ACE2-positive cells in FlowSOM-generated immune cell clusters after PMA and ionomycin restimulation (5 h). All TPs have been pooled. (G) Median expression of various markers in FlowSOM-derived clusters of PMA and ionomycin-restimulated (5 h) samples. (H) Median expression and 25th and 75th percentiles of PD-1 (left panel) and CTLA-4 (right panel) in FlowSOM-generated immune cell clusters after PMA and ionomycin restimulation (5 h). All TPs have been pooled. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, Mann-Whitney test, BH correction. See also Figure S7 .

Article Snippet: anti-human ACE2 (Biotin) (AC18F) , Adipogen Life sciences , Cat# AG-20A-0032B-C050; RRID: N/A.

Techniques: Expressing, Ex Vivo, Comparison, Derivative Assay, Staining, Generated, MANN-WHITNEY

Journal: Immunity

Article Title: Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia

doi: 10.1016/j.immuni.2021.05.002

Figure Lengend Snippet:

Article Snippet: anti-human ACE2 (Biotin) (AC18F) , Adipogen Life sciences , Cat# AG-20A-0032B-C050; RRID: N/A.

Techniques: Recombinant, Saline, Flow Cytometry, Software, Luminex