Journal: Viruses

Article Title: Selective Electrochemical Detection of SARS-CoV-2 Using Deep Learning

doi: 10.3390/v14091930

Figure Lengend Snippet: The dataset used for initial manual data analysis with 2% signature rule.

Article Snippet: MERS-CoV , ZeptoMetrix NATMERS-ST [ ] , 400 , Negative.

Techniques:

Journal: Cell Reports Medicine

Article Title: Persistence of MERS-CoV-spike-specific B cells and antibodies after late third immunization with the MVA-MERS-S vaccine

doi: 10.1016/j.xcrm.2022.100685

Figure Lengend Snippet: Study design Study participants (n = 10) received two vaccinations wit MVA-MERS-S (V1 and V2) 28 days apart and a late third vaccination (V3) at 12 ± 4 months after prime. Blood was frequently sampled for up to 2 years after V3 at indicated time points (see also Table S1 ). Figure was created with BioRender.com .

Article Snippet: Plates were coated overnight at 4°C with 100 μL/well of either PBS containing anti-IgG capture antibody (15 μg/mL, Mabtech), MERS-CoV-S protein S1 or S2 subunit (10 μg/mL, SinoBiological), or PBS only.

Techniques:

Journal: Cell Reports Medicine

Article Title: Persistence of MERS-CoV-spike-specific B cells and antibodies after late third immunization with the MVA-MERS-S vaccine

doi: 10.1016/j.xcrm.2022.100685

Figure Lengend Snippet: MERS-CoV-S-specific humoral and T cell responses induced by three vaccinations with MVA-MERS-S (A) Longitudinal dynamics of S-specific IgG antibodies. Shown are the optical density (OD) values measured at 450–620 nm by ELISA. Data are represented as individual data points (mean of technical duplicates) and median ± interquartile range (IQR). (B) Neutralization activity of serum antibodies as measured by 50% plaque-reduction neutralization test (PRNT50). Data are represented as individual data points and median ± IQR. (C) Spearman correlation between S-specific IgG antibodies and serum neutralization activity. (D) T cell responses as measured by IFNγ ELISpot after stimulation with five overlapping peptide (OLP) pools (M1–M5), spanning the entire MERS-CoV-S amino acid sequence. Shown are the median values of spot forming units (SFUs; mean of technical triplicates) across all vaccinees (n = 10) for each OLP pool. Number of samples, median, and p values for each time point and all three assays are shown in Table S2 .

Article Snippet: Plates were coated overnight at 4°C with 100 μL/well of either PBS containing anti-IgG capture antibody (15 μg/mL, Mabtech), MERS-CoV-S protein S1 or S2 subunit (10 μg/mL, SinoBiological), or PBS only.

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Activity Assay, Plaque Reduction Neutralization Test, Enzyme-linked Immunospot, Sequencing

Journal: Cell Reports Medicine

Article Title: Persistence of MERS-CoV-spike-specific B cells and antibodies after late third immunization with the MVA-MERS-S vaccine

doi: 10.1016/j.xcrm.2022.100685

Figure Lengend Snippet: Isotype and subclass distribution within vaccine-induced MERS-CoV-S-specific antibodies S1- (A) and S2- (B) specific responses of IgM, IgG1–4, and IgA1–2 at different time points after vaccination, displayed as fold changes of median fluorescence intensities (MFIs; measured by bead-based ELISA) compared with baseline values at V1:day 0. Data are represented as individual data points (mean of technical duplicates) and median ± IQR. Number of samples and median fold changes are shown in Table S3 .

Article Snippet: Plates were coated overnight at 4°C with 100 μL/well of either PBS containing anti-IgG capture antibody (15 μg/mL, Mabtech), MERS-CoV-S protein S1 or S2 subunit (10 μg/mL, SinoBiological), or PBS only.

Techniques: Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Persistence of MERS-CoV-spike-specific B cells and antibodies after late third immunization with the MVA-MERS-S vaccine

doi: 10.1016/j.xcrm.2022.100685

Figure Lengend Snippet: Longitudinal dynamics of MERS-CoV-S-specific IgG1 and IgG3 antibodies Vaccine-induced S1/S2-specific IgG1 (A) and IgG3 (B) are displayed as MFI, measured by bead-based multiplex ELISA. Data are represented as individual data points (mean of technical duplicates) and median ± IQR. LLD, lower limit of detection. Number of samples, median, and p values for each time point are shown in Table S4 .

Article Snippet: Plates were coated overnight at 4°C with 100 μL/well of either PBS containing anti-IgG capture antibody (15 μg/mL, Mabtech), MERS-CoV-S protein S1 or S2 subunit (10 μg/mL, SinoBiological), or PBS only.

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Persistence of MERS-CoV-spike-specific B cells and antibodies after late third immunization with the MVA-MERS-S vaccine

doi: 10.1016/j.xcrm.2022.100685

Figure Lengend Snippet: Antigen-specific B cell responses induced by MVA-MERS-S vaccination (A) Representative IgG ELISpot images of antigen-specific and control wells for PBMCs taken before the first (V1:day 0) and after the third vaccination (V3:day 14). (B) Frequencies of vaccine-induced S1/S2-specific B cells displayed as SFUs/10 6 PBMCs as determined by IgG ELISpot. Data are represented as individual data points (mean of technical duplicates) and median ± IQR. The dotted line indicates the cutoff value (6.6 SFUs/10 6 PBMCs). (C) p values as determined by Wilcoxon signed rank test (between time points) and Mann Whitney U test (between S1 and S2 responses). Number of samples, median, and p values for each time point are shown in Table S5 .

Article Snippet: Plates were coated overnight at 4°C with 100 μL/well of either PBS containing anti-IgG capture antibody (15 μg/mL, Mabtech), MERS-CoV-S protein S1 or S2 subunit (10 μg/mL, SinoBiological), or PBS only.

Techniques: Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Persistence of MERS-CoV-spike-specific B cells and antibodies after late third immunization with the MVA-MERS-S vaccine

doi: 10.1016/j.xcrm.2022.100685

Figure Lengend Snippet: Characterization of vaccine-induced memory B cells (A) Gating strategy for analysis of isotypes and MERS-CoV-S-specific cells within the memory B cell (MBC) population (representative contour plots belong to time point V3:day 14 from one study participant; gating strategy for identification of MBCs within whole PBMCs shown in Figure S2 ). (B) Longitudinal dynamics of antigen-specific MBCs induced by three vaccinations with MVA-MERS-S (V1, V2, and V3). Data are displayed as frequencies of S-specific cells within IgM + /IgG + /IgA + MBCs. Boxplots indicate median ± IQR. Number of samples, median, and p values compared with V1:day 0 are shown in Table S6 . (C) Resting, intermediate, atypical, and activated MBC phenotypes as identified by expression of CD21 and CD27 (top left panel). Representative plots are shown for one study participant at V3:day 0 and V3:day 14 and depict overlaid contour plots of total IgG + MBCs and S-specific IgG + MBCs (bottom left panel). Longitudinal distributions of phenotypes within the S-specific IgG + MBC compartment are shown as mean values of all study participants.

Article Snippet: Plates were coated overnight at 4°C with 100 μL/well of either PBS containing anti-IgG capture antibody (15 μg/mL, Mabtech), MERS-CoV-S protein S1 or S2 subunit (10 μg/mL, SinoBiological), or PBS only.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: Persistence of MERS-CoV-spike-specific B cells and antibodies after late third immunization with the MVA-MERS-S vaccine

doi: 10.1016/j.xcrm.2022.100685

Figure Lengend Snippet: Correlation between vaccine-induced antibody and B cell responses Correlation analysis of MERS-CoV-S1- (A) and -S2- (B) specific IgG1, IgG3, and ASBC responses at different time points after vaccinations 1, 2, and 3 (V1, V2, and V3). S-specific MBC responses were included into correlograms of both the S1 and S2 antigen. Positive correlations are shown in blue and negative correlations in red, as indicated by the color bar. Sample numbers included into correlation analysis are provided in Table S7 .

Article Snippet: Plates were coated overnight at 4°C with 100 μL/well of either PBS containing anti-IgG capture antibody (15 μg/mL, Mabtech), MERS-CoV-S protein S1 or S2 subunit (10 μg/mL, SinoBiological), or PBS only.

Techniques:

Journal: Cell Reports Medicine

Article Title: Persistence of MERS-CoV-spike-specific B cells and antibodies after late third immunization with the MVA-MERS-S vaccine

doi: 10.1016/j.xcrm.2022.100685

Figure Lengend Snippet:

Article Snippet: Plates were coated overnight at 4°C with 100 μL/well of either PBS containing anti-IgG capture antibody (15 μg/mL, Mabtech), MERS-CoV-S protein S1 or S2 subunit (10 μg/mL, SinoBiological), or PBS only.

Techniques: Recombinant, Staining, Enzyme-linked Immunospot, Software

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: In vitro characterization of purified equine immunoglobulin against SARS-CoV-2. (A) The neutralizing titers of hyperimmune serum, purified IgG, and F(ab’) 2 derived from equine No. 15 and No. 16 were tested with wild type SARS-CoV-2 Wuhan 01. The serum neutralizing antibody titer was defined as the reciprocal of the highest dilution showing a 100% CPE reduction compared to the virus control. (B) The titers of purified SARS-CoV-2-specific IgG in equine sera were examined via RBD-capture ELISA. Two repeated tests were performed on each sample.

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: In Vitro, Purification, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Broad-spectrum neutralizing activity test against SARS-CoV-2 VOC and VOI. The neutralizing antibody titers were calculated as the highest dilution of sera that completely inhibited virus-caused CPE. The serum neutralizing antibody titer was defined as the reciprocal of the highest dilution showing a 100% CPE reduction compared to the virus control. (A) Neutralizing antibody titers of purified IgG and F(ab’) 2 of equine No.15 against SARS-CoV-2 VOC; (B) Neutralizing antibody titers of purified IgG and F(ab’) 2 of equine No.16 against SARS-CoV-2 VOC; (C) Neutralizing antibody titers of purified equine immunoglobulin of equine No.15 against SARS-CoV-2 VOI; (D) Neutralizing antibody titers of purified equine immunoglobulin of equine No.16 against SARS-CoV-2 VOI. Comparison to the wild type SARS-CoV-2 Wuhan01, the number above the column represented the fold by which the neutralizing titer of the IgG or F(ab’) 2 was weakened by the SARS-CoV-2 VOC and VOI. Samples were processed in triplicate, and error bars indicate standard error. Data are presented as the mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Activity Assay, Purification

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Evaluation of the protective efficacy of purified equine immunoglobulin in a mouse model. Groups of 13 BALB/c mice were administered with IgG or F(ab’) 2 at 1 day before mouse-adapted SARS-CoV-2 (BMA8) infection or 1 dpi with BMA8. Each mouse was given 250 µg of antibody at a dose of 10 mg/kg. BALB/c mice were challenged intranasally with a lethal dose 50 LD 50 of BMA8 before treatment or after administration. The survival rate, weight change, body temperature and clinical scores of BALB/c mice were monitored daily after SARS-CoV-2 BMA8 infection. (A) Schematic diagram of the administration of equine immunoglobulin drugs and virus challenge procedure; (B) Survival rate. (C) Percent weight change. (D) Body temperature change. Body weight change of mice in a with comparison to isotype control was measured by repeated measurements two-way analysis of variance (ANOVA) with Tukey’s post hoc test. Data are mean ± s.e.m. of each experimental group. (****P < 0.0001).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Purification, Infection

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Blood counts in SARS-CoV-2-infected mice. The hematological values of BALB/c mice were analysed, including lymphocyte (LYM), neutrophil percentage (Neu%), monocytes (Mon), platelet count (PLT) and white blood cell count (WBC), at 3 dpi after SARS-CoV-2 BMA8 infection. Four infected mice were sacrificed at 3 dpi to collect the whole blood for blood counts test. (A) White blood cell (WBC) count; (B) neutrophil (Neu) percentage; (C) lymphocyte (LYM) percentage; (D) platelet (PLT) (E) Monocyte(Mno). Data are presented as the mean ± SEM (n=4). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Infection, Cell Counting

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Histopathological and immunohistochemistry findings in SARS-CoV-2-infected mice. The lungs and spleens were collected from the control mice infected with SARS-CoV-2 without equine immunoglobulin drug injection at 3dpi, and the lungs, spleens, livers and kidneys were harvested from recovered mice. After each tissue was embedded in paraffin, the sections were sectioned for HE staining. (A, B, E, F) Lung tissue changes of control mice were characterized by more necrotic epithelial cells (blue arrow), a small amount of neutrophil infiltration, and perivascular edema with a small amount of inflammatory cell infiltration in the local alveolar cavity (yellow arrow). (C, D, G, H) Spleen tissue changes of control mice were characterized with spotted apoptosis of lymphocytes, nuclear pyknosis and deep staining or fragmentation in the spleen nodules (black arrows), and the expansion of germinal centers (yellow arrow), scattered neutrophils mostly seen in the red pulp granulocyte infiltration (red arrow), and more brown‒yellow particles in the red pulp (blue arrow). (I-L) The basically normal structure of the lung, spleen liver, and kidney tissues were found in administration groups given equine IgG or F(ab’) 2 . The figure showed immunohistochemistry (IHC) labeling against SARS-CoV-2 N. (M) Viral antigen was not detectable in prevention group given purified IgG; (N) Viral antigen was not detectable in prevention group given purified F(ab’) 2 ; (O) Viral antigen was detected for positive in prevention control group; (P) Viral antigen was not detectable in treatment group given purified IgG; (Q) Viral antigen was not detectable in treatment group given purified IgG F(ab’) 2 ; (R) Viral antigen was detected for positive in treatment control group. (scale bar = 100 μm).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Immunohistochemistry, Infection, Injection, Staining, Labeling, Purification

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Evaluation of the protective efficacy of purified equine immunoglobulin in the golden hamster model. Each golden hamster was given 500 µg of antibody at a dose of 10 mg/kg. Groups of golden hamsters were infected intranasally with 1,000 TCID 50 of wild-type SARS-CoV-2 Wuhan 01 before treatment and or after administration. The survival rate and weight change of BALB/c mice were monitored daily after SARS-CoV-2 Wuhan01 infection. Four infected golden hamsters in each group were sacrificed at 3 dpi, and the turbinate and lung samples were collected to analyze the viral RNA loads by RT‒qPCR and TCID 50 , respectively. (A) Schematic diagram of the administration of equine immunoglobulin drugs and virus challenge procedure. (B) Survival rate. (C) Percent weight change; Body weight change of mice in a with comparison to isotype control was measured by repeated measurements two-way analysis of variance (ANOVA) with Tukey’s post hoc test. Data are mean ± s.e.m. of each experimental group. (D) The viral loads of turbinate were quantified by RT‒qPCR at 3 dpi in each group; (E) The viral loads of lung were quantified by RT‒qPCR at 3 dpi in each group; (F) The viral loads of turbinate were determined by TCID 50 at 3 dpi in each group; (G) The viral loads of lung were determined by TCID 50 at 3 dpi in each group. Data are presented as the mean ± SEM (n=5). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Purification, Infection

Journal: Mikrochimica Acta

Article Title: Detection of SARS-CoV-2 receptor binding domain using fluorescence probe and DNA flowers enabled by rolling circle amplification

doi: 10.1007/s00604-023-05747-6

Figure Lengend Snippet: Evaluation of specificity of the RCA-enabled fluorometric ( a ) and colorimetric assay ( b ). The signal intensity generated from S protein RBD is compared with that from N protein, MERS protein RBD, SARS-Cov-2 B.1.617.2 spike protein RBD, and a mixture of the three proteins. The total protein concentration in each sample was 50 ng/mL

Article Snippet: Recombinant SARS-CoV-2 receptor-binding domain (RBD, source: HEK293-derived SARS-CoV-2 Spike RBD protein (Arg319-Phe541), with a 6x-His tag at C-terminal), recombinant MERS-CoV Spike RBD (Chinese Hamster Ovary-derived MERS-CoV Spike RBD protein (Glu367-Tyr606), with a 6x-His tag at C-terminal), recombinant SARS-CoV-2 B.1.617.2 RBD (source: HEK293-derived SARS-CoV-2 Spike RBD protein (Arg319-Phe541 (Leu452Arg, Thr478Lys), with a 6x-His tag at C-terminal), and recombinant SARS-CoV-2 nucleocapsid (source: Spodoptera frugiperda , Sf 21 (baculovirus)-derived SARS-CoV-2 nucleocapsid (Met1-Ala419), with a 6x-His tag at C-terminal) were purchased from Bio-Techne (Minneapolis, USA).

Techniques: Colorimetric Assay, Generated, Protein Concentration

Journal: Signal Transduction and Targeted Therapy

Article Title: Aptamers targeting SARS-CoV-2 nucleocapsid protein exhibit potential anti pan-coronavirus activity

doi: 10.1038/s41392-024-01748-w

Figure Lengend Snippet: Prediction of binding and affinity identification between the aptamers and the N proteins of SARS, MERS and -OC43. a Prediction of the binding affinity between the aptamers and N proteins of SARS, MERS and HCoV-OC43 via Zdock score. The N protein of SARS-CoV-2 and ALB protein were used as positive and negative control, respectively. The characterizations of affinity and specificity between aptamers and N proteins of ( b ) SARS, ( c ) MERS and ( d ) -OC43 were performed via CE. e The results of molecular docking to predict and simulate the binding sites of aptamer Seq-1022 to N proteins of SARS, MERS and -OC43 using Discovery Studio software

Article Snippet: SARS NP (40143-V08B), MERS NP (40068-V08B), RBD of spike protein of SARS-CoV-2 Omicron strain (40592-V08H121) and SARS-CoV-2 Nucleocapsid monoclonal antibody (40588-R0004) were purchased from Sino Biological Inc. Normal human serum was obtained from Beijing BioDee Biotechnology Co., Ltd. Human albumin was obtained from Sigma-Aldrich (St. Louis, MO).

Techniques: Binding Assay, Negative Control, Software

Journal: Vaccine

Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

doi: 10.1016/j.vaccine.2016.11.064

Figure Lengend Snippet: Description of the N-terminal domain (NTD) immunogen and vaccination schedule. (A) The location of the NTD protein on the Middle East respiratory syndrome coronavirus MERS-CoV spike (S) protein. The recombinant (r)NTD protein consists of 336 amino acid (aa) residues (18–353) of S protein. A gp67 signal peptide (SP) was added to the N terminus for expression of the rNTD protein. (B) Purified rNTD protein detected by SDS-PAGE (left) and Western blot (right). The purified rNTD protein was separated by a 10% SDS-PAGE and stained with 0.25% Coomassie brilliant blue. Anti-NTD polyclonal antibody and infrared ray-labeled secondary antibody were used for the Western blot assay. Lane 1: protein molecular weight marker; lane 2: purified rNTD protein. (C). Vaccination schedule and detection. Mice received three vaccinations consisting of 5 or 10 μg of rNTD protein combined with adjuvants at 4-week intervals. Sera were collected at the indicated times to analyze the humoral immune response. Six mice from each group were sacrificed 2 weeks after the last immunization. The spleens were harvested for enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and cytometric bead array (CBA) assays. In parallel experiments, the remaining mice were challenged with MERS-CoV to detect the protective effect elicited by the rNTD protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

Techniques: Recombinant, Expressing, Purification, SDS Page, Western Blot, Staining, Labeling, Molecular Weight, Marker, Enzyme-linked Immunospot

Journal: Vaccine

Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

doi: 10.1016/j.vaccine.2016.11.064

Figure Lengend Snippet: IHC detection of virus antigen expression in mouse tissue after challenge with MERS-CoV. Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.

Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

Techniques: Expressing

Journal: Nature Communications

Article Title: Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

doi: 10.1038/s41467-020-15562-9

Figure Lengend Snippet: a Diagram of full-length SARS-CoV-2 S protein with a 3xFLAG tag. S1, receptor-binding subunit; S2, membrane fusion subunit; TM, transmembrane domain; NTD, N-terminal domain; pFP, potential fusion peptide; HR-N, heptad repeat-N; HR-C, heptad repeat-C; b – f Detection of CoVs S protein in cells lysate by western blot. Mock, 293T cells transfected with empty vector. b Mouse monoclonal anti-FLAG M2 antibody; c Polyclonal goat anti-MHV-A59 S protein antibody AO4. d Polyclonal rabbit anti-SARS S1 antibodies T62. e Mouse monoclonal anti-SARS S1 antibody. f Mouse monoclonal anti-MERS-CoV S2 antibody. g – j Detection of CoVs S protein in pseudovirions by western blot.Gag-p24 served as a loading control. g Anti-FLAG M2. h Polyclonal goat anti-MHV-A59 S protein antibody AO4. i Polyclonal rabbit anti-SARS S1 antibodies T62. j Polyclonal anti-Gag-p24 antibodies. uncleaved S protein, about 180 kDa; cleaved S protein, about 90 kDa. Experiments were done twice and one is shown. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against MERS-CoV S2 antibody (#40070-MM11), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RP01) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Binding Assay, Western Blot, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

doi: 10.1038/s41467-020-15562-9

Figure Lengend Snippet: a , b Entry of SARS-CoV-2 S pseudovirions on indicated cell lines. Cells from human and animal origin were inoculated with SARS-CoV-2 S (red), SARS-CoV S (blue), or VSV-G (gray) pseudovirions. At 48 h post inoculation, transduction efficiency was measured according to luciferase activities. RS, Rhinolophus sinicus bat embryonic fibroblast; BHK/hAPN, BHK cells stably expressing hAPN, the hCoV-229E receptor; 293/hACE2, 293 cells stably expressing hACE2, the SARS-CoV receptor; HeLa/hDPP4, HeLa cells stably expressing hDPP4, the MERS-CoV receptor. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Binding of SARS-CoV S and SARS-CoV-2 S proteins to soluble hACE2. HEK293T cells transiently expressing SARS-CoV and SARS-CoV-2 S proteins were incubated with the soluble hACE2 on ice, followed by polyclonal goat anti-hACE2 antibody. Cells were analyzed by flow cytometry. The experiments were repeated at least three times. d Inhibition of SARS-CoV-2 S pseudovirion entry by soluble hACE2. SARS-CoV S, SARS-CoV-2 S, or VSV-G pseudovirions were pre-incubated with soluble hACE2, then mixture were added to 293/hACE2 cells. Cells were lysed 40 h later and pseudoviral transduction was measured. Experiments were done twice and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against MERS-CoV S2 antibody (#40070-MM11), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RP01) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Transduction, Luciferase, Stable Transfection, Expressing, Binding Assay, Incubation, Flow Cytometry, Inhibition

Journal: Nature Communications

Article Title: Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

doi: 10.1038/s41467-020-15562-9

Figure Lengend Snippet: a Inhibition of entry of SARS-CoV-2 S pseudovirion on 293/hACE2 by lysosomotropic agents (20 mM NH 4 Cl and 100 nM bafilomycin A). b Inhibition of entry of SARS-CoV, MERS-CoV, and MHV S pseudovirions by a PIKfyve inhibitor apilimod. HeLa/mCEACAM, 293/hACE2, HeLa/hDPP4 cells were pretreated with different concentrations of apilimod and transduced with MHV S, SARS-CoV S, MERS-CoV S pseudovirions, respectively. The luciferase activity was measured 40 h post transduction. VSV-G pseudovirions were used as a control. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Inhibition of MHV A59 infection by apilimod. The 17Cl.1 cells were pretreated with 3, 10, 30, 100, 300 nM apilimod for 30 min and infected by MHV A59 at MOI = 0.01. Viral infection and cell viability were determined by using qPCR and MTT assay, respectively. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. d , e Inhibition of entry of SARS-CoV-2 S protein pseudovirions by apilimod, YM201636, and tetrandrine. HEK 293/hACE2 cells were pretreated with either apilimod ( d ), YM201636 ( e ), or tetrandrine ( f ), then inoculated with SARS-CoV-2 S pseudovirons in the presence of drug. The luciferase activity were measured 40 h post transduction. YM201636, PIKfyve inhibitor; tetrandrine, TPC2 inhibitor. The experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM of technical triplicates. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against MERS-CoV S2 antibody (#40070-MM11), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RP01) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Inhibition, Transduction, Luciferase, Activity Assay, Infection, MTT Assay

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A, VH, VK and VL gene usage of antibodies from plasmablasts and memory B cells (MBCs). Up to the top four genes in each chart are shown with different colors (genes that were tied for 4 th and lower are not highlighted). B , Tukey’s plots showing heavy and light chain gene mutations of antibodies from plasmablasts and MBCs. Percent mutations were compared with the Mann-Whitney U-test. The middle line shows the median, and the box extends from the 1 st to 3 rd quartile. C , Top 21 neutralizing antibodies (IC 50 <1 μg/mL) by antigen specificity (left), and neutralization curves of selected antibodies (right). D , SARS-CoV-2 neutralization potency versus heavy chain mutation levels of antibody panel. The 5 most potent antibodies are shown as solid circles. E , Neutralization potency of antibodies by cell type. Top values indicate percentages of non-neutralizing antibodies. Horizontal bars indicate mean values; Mann-Whitney U-test (non-neutralizing antibodies excluded from calculation). The 5 most potent antibodies are shown in color. F , SARS-CoV-2 neutralization curves of benchmark antibodies from different groups , , . G, Neutralization IC 50 values of antibodies in our panel and benchmark antibodies in three different neutralization assays. Authentic SARS-CoV-2 FRNA values are from a single experiment done in quadruplicate, authentic SARS-CoV-2 (Scripps) values are an average of two experiments done in duplicate, pseudovirus (MLV) values are an average of 3 experiments in duplicate. The colors indicate the different sources of the antibodies: red, this study; blue, ref. ; yellow, ref. ; green, ref. .

Article Snippet: Following fixation, the cells were permeabilized with Triton X-100 and probed with a SARS-CoV/SARS-CoV-2 nucleoprotein-specific rabbit primary antibody (Sino Biological, Wayne, PA, USA, 40143-R001) followed by an Alexa Fluor 647-conjugated secondary antibody (Life Technologies, San Diego, CA, USA, A21245).

Techniques: MANN-WHITNEY, Neutralization, Mutagenesis

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , Isoaffinity plot of antibodies targeting SARS-CoV-2 RBD (representative of n = 2 experiments). The affinity (K D ) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a K D value of 10 pM. B , Neutralization potency versus affinity of anti-RBD antibodies. C , Isoaffinity plot of antibodies targeting SARS-CoV-2 NTD (representative of n = 2 experiments). The affinity (K D ) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a K D value of 10 pM. D , Neutralization potency versus affinity of anti-NTD antibodies. E , Epitope binning of anti-RBD antibodies (representative of n = 2 experiments). ACE2 was only used as an analyte (competitor) and not as a ligand, while all other antibodies were tested as both ligands and analytes. Solid lines indicate two-way competition while dotted lines indicate one-way competition. The number and percentage of neutralizing antibodies (IC 50 < 10 μg/mL) in each bin are shown. F , Epitope bins represented by C135 (yellow bin), S309 (purple bin), ACE2 (red bin), CR3022 (cyan bin), as well as the NTD-specific antibody 4–8 (orange) modeled onto a SARS-CoV-2 spike protein (white cartoon). Antibody 4–8 was not binned successfully in our experiments but binds to a similar region to 2–17 and 5–24 (ref. ), which were binned. The epitope sites are color-coded the same as in Fig. 2E and 2G. N-glycans at the N343 glycan site are represented by sticks. G , Epitope binning of anti-NTD antibodies (representative of n = 2 experiments). All antibodies were tested as both ligands and analytes. Solid lines indicate two-way competition while dotted lines indicate one-way competition. The number and percentage of neutralizing antibodies (IC 50 < 10 μg/mL) in each bin are shown. H , Binding of mAb panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). I , Neutralization potency of CV503, CV664 and CV993 against B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets, and numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type.

Article Snippet: Following fixation, the cells were permeabilized with Triton X-100 and probed with a SARS-CoV/SARS-CoV-2 nucleoprotein-specific rabbit primary antibody (Sino Biological, Wayne, PA, USA, 40143-R001) followed by an Alexa Fluor 647-conjugated secondary antibody (Life Technologies, San Diego, CA, USA, A21245).

Techniques: Neutralization, Binding Assay

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , CV503 binds to the ridge region of SARS-CoV-2 RBD. The heavy and light chains of CV503 are shown in orange and yellow, respectively. SARS-CoV-2 RBD is in white, where its ridge region (residues 471–491) is shown in blue. B, The ACE2/RBD complex structure (PDB ID: 6M0J) is superimposed on the CV503/RBD complex. The heavy chain of CV503 (orange) would clash with ACE2 (green) if bound to RBD simultaneously (indicated by red circle). C-D , Epitope of CV503. Epitope residues contacting the heavy chain are in dark blue and light chain are in light blue, while residues contacting both heavy and light chains are in ocean blue. In C , CDR loops that are directly involved in RBD-binding are labeled. In D , epitope residues are labeled. Epitope residues that are also involved in ACE2 binding are labeled in red. E, ACE2-binding site on the RBD are in light pink. ACE2 is represented as semi-transparent cartoon in pale green. Epitope residues and ACE2-interacting residues are defined as those with a buried surface area (BSA) > 0 Å 2 . F , F486 at the ridge region of SARS-CoV-2 RBD (blue) is clamped in a hydrophobic pocket formed by the heavy (orange) and light chains (yellow) of CV503.

Article Snippet: Following fixation, the cells were permeabilized with Triton X-100 and probed with a SARS-CoV/SARS-CoV-2 nucleoprotein-specific rabbit primary antibody (Sino Biological, Wayne, PA, USA, 40143-R001) followed by an Alexa Fluor 647-conjugated secondary antibody (Life Technologies, San Diego, CA, USA, A21245).

Techniques: Binding Assay, Labeling

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , Scheme of DVD-Ig ™ . In our bispecific antibody naming system, the first name refers to the antibody used to make the outer binding site and the second refers to the antibody at the inner binding site. GS or EL refers to the type of linker connecting the two antigen-binding sites. See for details. B , Binding of individual and bispecific antibodies to various domains from SARS-CoV-1 and SARS-CoV-2 (representative of n = 2 experiments). Area under the curve (AUC) values are shown after subtraction with the negative control antigen. C , Neutralization potency of bispecific antibodies with SARS-CoV-2 authentic and pseudotyped virus (MLV). Values are averaged from two experiments done in duplicate. D , Neutralization curves of CV1206_521_GS with SARS-CoV-2 authentic and pseudotyped virus. Curves are from a representative experiment, IC 50 values for authentic virus are the average from two experiments and those for the pseudovirus are from an average of two (bispecific) or three (regular antibody) experiments. E, Neutralization potency of CV1206_521_GS versus a cocktail of CV1206 and CV521, with concentrations shown in the molar scale to enable a fair comparison. For the antibody combination, the values on the x-axis refers to the concentration of each antibody in the cocktail, e.g. 10 nM refers to 10 nM of CV1206 + 10 nM of CV521. F , 3D reconstruction of CV1206_521_GS from nsEM images. Two ”one RBD up” models (PDB 6VYB) in green are docked into the reconstruction. Similarly, multiple mock scFv’s in orange and purple were docked to approximate the DVD-Ig molecule. O, outer binding site; I, inner binding site. G , Binding of bispecific antibody panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). H , Neutralization potency of bispecific antibodies against D614G, B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets: numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type. ND, not determined. I, Potency of CV503_664_EL versus individual component mAbs against wild-type and B.1.351 SARS-CoV-2 pseudotyped virus (lentivirus).

Article Snippet: Following fixation, the cells were permeabilized with Triton X-100 and probed with a SARS-CoV/SARS-CoV-2 nucleoprotein-specific rabbit primary antibody (Sino Biological, Wayne, PA, USA, 40143-R001) followed by an Alexa Fluor 647-conjugated secondary antibody (Life Technologies, San Diego, CA, USA, A21245).

Techniques: Binding Assay, Negative Control, Neutralization, Concentration Assay

Journal: Journal of Virological Methods

Article Title: Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses

doi: 10.1016/j.jviromet.2021.114111

Figure Lengend Snippet: Precision, Accuracy of all 9 CoV SeroAssay Capture Antigens. a

Article Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens. (a) Schematic illustration of microarray layout, and representative fluorescence images of the VaxArray CoV SeroAssay microarray in (b) through (l).

Techniques: Concentration Assay

Journal: Journal of Virological Methods

Article Title: Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses

doi: 10.1016/j.jviromet.2021.114111

Figure Lengend Snippet: Identifying Information for Nine Human Coronavirus Spike Antigens Represented on the CoV SeroAssay.

Article Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens. (a) Schematic illustration of microarray layout, and representative fluorescence images of the VaxArray CoV SeroAssay microarray in (b) through (l).

Techniques: Expressing, Binding Assay

Journal: Journal of Virological Methods

Article Title: Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses

doi: 10.1016/j.jviromet.2021.114111

Figure Lengend Snippet: Sensitivity, Linear Dynamic Range of all 9 CoV SeroAssay Capture Antigens.

Article Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens. (a) Schematic illustration of microarray layout, and representative fluorescence images of the VaxArray CoV SeroAssay microarray in (b) through (l).

Techniques:

Journal: Journal of Virological Methods

Article Title: Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses

doi: 10.1016/j.jviromet.2021.114111

Figure Lengend Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens.

Article Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens. (a) Schematic illustration of microarray layout, and representative fluorescence images of the VaxArray CoV SeroAssay microarray in (b) through (l).

Techniques: Fluorescence, Microarray, Binding Assay