Journal: medRxiv

Article Title: BNT162b2 Vaccine Induces Divergent B cell responses to SARS-CoV-2 S1 and S2

doi: 10.1101/2021.07.20.21260822

Figure Lengend Snippet: a-c , rmAb-antigen interaction intensities were measured by bio-layer interferometry with immobilized onto Octet sensors, and S protein subunits. a, RBD, b , S1n, and c , S2 in solution. Binding curves are shown for the indicated concentrations of antigen. d , Kd values of antibodies shown in a-c.

Article Snippet: For protein ELISAs, MaxiSorp 384-well plates (Thermo Fisher Scientific) were coated with 1 µg/ml recombinant SARS-CoV-2 S2 protein (Acro, S2N-C52H5), SARS-CoV-2 RBD (Acro, SPD-C52H3), or SARS-CoV-2 S1 (Acro, S1N-C52H3), HCoV-OC43 Spike protein (Sino Biological, 40607-V08B), HCoV-HKU1 Spike protein (Sino Biological, 40606-V08B), HCoV-229E Spike protein (Sino Biological, 40605-V08B), HCoV-NL63 Spike protein (Sino Biological, 40604-V08B) in carbonate-bicarbonate buffer at 4°C overnight.

Techniques: Binding Assay

Journal: medRxiv

Article Title: BNT162b2 Vaccine Induces Divergent B cell responses to SARS-CoV-2 S1 and S2

doi: 10.1101/2021.07.20.21260822

Figure Lengend Snippet: a , Binding of anti-S2 and anti-RBD rmAbs to SARS-CoV-2 S2, SARS-CoV-2 RBD, HCoV-OC43 spike, and HCoV-HKU1 spike, assessed by ELISA and represented as AUC of optical density measurements of serial dilutions. No binding was observed for HCoV-229E and HCoV-NL63 spike proteins. Threshold, represented as 0, was set to the average binding to BSA plus three times the standard deviation of background binding to BSA.

Article Snippet: For protein ELISAs, MaxiSorp 384-well plates (Thermo Fisher Scientific) were coated with 1 µg/ml recombinant SARS-CoV-2 S2 protein (Acro, S2N-C52H5), SARS-CoV-2 RBD (Acro, SPD-C52H3), or SARS-CoV-2 S1 (Acro, S1N-C52H3), HCoV-OC43 Spike protein (Sino Biological, 40607-V08B), HCoV-HKU1 Spike protein (Sino Biological, 40606-V08B), HCoV-229E Spike protein (Sino Biological, 40605-V08B), HCoV-NL63 Spike protein (Sino Biological, 40604-V08B) in carbonate-bicarbonate buffer at 4°C overnight.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nature

Article Title: PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection

doi: 10.1038/s41586-023-06322-y

Figure Lengend Snippet: a , b , Quantification of viral entry efficiency in Huh7.5 cells inoculated with pseudoviruses bearing fusion proteins from HCoV-229E ( n = 5), HCoV-OC43 ( n = 3), hCoV-NL63 ( n = 3), hCoV-HKU1 ( n = 3), EBoV (n = 3) and HCV ( n = 5). EBoV: Ebola virus, HCV: Hepatitis C virus. c , Left, relative amount of intracellular viral RNA in Huh7.5 cells infected with DENV (Dengue virus type I) at an MOI = 0.5 for 24 h. The amount of viral RNA in NC cells was normalized to 1. ( n = 4) Right: Quantification of % infected cells in HeLa cells infected with HSV-1 VP26-GFP at an MOI of 0.1 for 48 h. d , Quantification of the relative entry efficiency of the indicated pseudovirus in Huh7.5 cells overexpressing (OE) PLSCR1 or IFITM3. The luminescence intensity in vector group was normalized to 1. n = 4. e , Schematic showing the dissection of the cell entry route of SARS-CoV-2. f , g , Effect of the indicated compounds on SARS-CoV-2 entry in Huh7.5 cells (MOI = 1, 48 hpi, n = 3) ( f ) and A549-ACE2 cells (MOI = 0.2, 24 hpi, n = 3) ( g ). E-64d: 20 μM, Camostat: 30 μM, Bfa (Brefeldin a): 10 μM, HCQ: 10 μM. Cells were treated with indicated compounds 2 h before infection. h , Quantification of SARS-CoV-2 infection in E-64d (20 μM) treated or untreated Huh7.5 cells overexpressing vector or PLSCR1 with or without ectopic expression of TMPRSS2 (MOI = 1, 48 hpi). ( n = 4) i , Left, dose response of indicated compounds on SARS-CoV-2 infection in Calu-3 (MOI = 1, 24 hpi, n = 4). The amount of viral RNA in DMSO group was normalized to 1. E64-d and Camostat groups share the same DMSO control group. Right, quantification of SARS-CoV-2 infection in Control or PLSCR1 -KO Calu-3 (MOI = 1, 24 hpi, n = 4) treated with indicated compounds (E-64d: 20 μM, Camostat: 20 μM). Cells were treated with the indicated compounds 2 h before infection. The amount of viral RNA in NC-DMSO group was normalized to 1. Data are mean ± s.d. P values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test in a – c , d (HCoV-NL63 group), f , g , Brown–Forsythe and Welch ANOVA with Dunnett’s post-hoc test in d (SARS-CoV-2 and EBoV group), two-sided Student’s t -test in h , two-way ANOVA followed by Tukey’s multiple comparison test in i (left) or two-way ANOVA followed by Šídák’s multiple comparisons test in i (right). Experiments in this figure were performed three times.

Article Snippet: Expression plasmids of the glycoproteins for HcoV-229E (VG40605-UT), HcoV-OC43 (VG40607-UT), HcoV-HKU1 (VG40021-UT) and EboV (VG40304-CF) were purchased from Sino Biological. pLV-EF1α-Flag-IRES-Hygro was modified from pLV-EF1α-IRES-Hygro by inserting a Flag tag between the promoter region and the multiple cloning site.

Techniques: Infection, Plasmid Preparation, Dissection, Expressing

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: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Content of coronavirus antigen microarray.

Article Snippet: Coronavirus , OC43 , OC43 , HE , ATN39879.2 , HEK293 , Sino Biological , N-(AA16-394)-His-C , 40603-V08H.

Techniques: Microarray

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Coronavirus antigens on microarray.

Article Snippet: Coronavirus , OC43 , OC43 , HE , ATN39879.2 , HEK293 , Sino Biological , N-(AA16-394)-His-C , 40603-V08H.

Techniques: Microarray, Expressing, Construct

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Antibodies to coronaviral spikes in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients and age-matched controls (A) Mirror plots of the specific MFI increase of HEK293T cells expressing HCoV-OC43 spike (top) or SARS-CoV-2 spike (bottom) caused by individual sera. Each bar is an individual healthy control or patient. Samples are plotted according to the signal of antibodies to HCoV-OC43 spike and in the same position in the mirror plots. Antibody levels to SARS-CoV-2 spike in MIS-C patients are plotted on a different scale from the rest. (B) Mirror plots of the specific MFI increase of HEK293T cells expressing ERV3-1 (top) or HERV-K113 (bottom) envelope glycoproteins caused by individual sera. Samples are plotted in the same order as in (A).

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Expressing

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Prevalence of IgG antibodies to OC43 and SARS-CoV-2 spikes in JIA, JDM, and JSLE patients.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques:

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Antibodies to coronaviral spikes in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients and controls of different age Specific MFI increases of HEK293T cells expressing HCoV-OC43 spike (A and C) or SARS-CoV-2 spike (B and D) caused by individual sera from the indicated age and disease group. Each symbol is an individual healthy control or patient. Antibody levels to SARS-CoV-2 spike in MIS-C patients are plotted on a different scale from the rest. Red and blue numbers within the plots denote the p values of statistically significant increases and decreases, respectively, when comparing each disease group with the respective healthy control of the same age group. The older control group was also compared with the younger control group.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Expressing

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: SARS-CoV-2 neutralizing antibodies in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients (A) SARS-CoV-2-neutralizing antibody titers in the indicated age and disease group. Only patients with SARS-CoV-2 spike-binding antibodies detectable by flow cytometry were included. JIA, JDM, and JSLE patients of both age groups combined were compared with MIS-C patients by ANOVA on ranks tests. (B) Correlation of SARS-CoV-2-neutralizing antibody titers with levels of flow-cytometry-detectable antibodies to SARS-CoV-2 spike (left) or HCoV-OC43 spike (right). In (A) and (B), each symbol is an individual patient. In (B), one JSLE patient was removed from the regression analysis as an outlier for the HCoV-OC43 spike antibodies, based on the Kurtosis coefficient of the group.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Binding Assay, Flow Cytometry

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Antibodies to coronaviral nucleoproteins in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients and age-matched controls (A–C) Mirror plots of the specific MFI increase of HEK293T cells expressing HCoV-OC43 nucleoprotein (top) or SARS-CoV-2 nucleoprotein (bottom) caused by individual sera. Each bar is an individual child or adolescent healthy control or JIA, JDM, or JSLE patient (A), MIS-C patient (B), or adult healthy control (C). Samples are plotted according to the signal of antibodies to the HCoV-OC43 nucleoprotein and in the same position in the mirror plots. (D) Correlation of the proportion of total HCoV-OC43 nucleoprotein-binding antibodies represented by IgM (top) or IgG (bottom) classes, and the age of the donor or patient. Each symbol is an individual sample.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Expressing, Binding Assay

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Antibodies to coronaviral nucleoproteins in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients and controls of different ages Specific MFI increase of HEK293T cells expressing HCoV-OC43 nucleoprotein (A and C) or SARS-CoV-2 nucleoprotein (B and D) caused by individual sera from the indicated age and disease group. Each symbol is an individual healthy control or patient. Red and blue numbers within the plots denote the p values of statistically significant increases and decreases, respectively, when comparing each disease group with the respective healthy control of the same age group. The older control group was also compared with the younger control group.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Expressing

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Ratios of the levels of antibodies to coronaviral spikes and nucleoproteins in pediatric and adolescent JIA, JDM, and JSLE patients and age-matched controls (A) The log2-transformed ratios of total antibodies to the HCoV-OC43 spike to total antibodies to the HCoV-OC43 nucleoprotein (S: N) are plotted for the indicated age and disease group. Each symbol is an individual sample. Numbers within the plots denote the p values of statistically significant increases, when comparing each disease group with the respective healthy control of the same age group. (B) Heatmap of ranked S: N ratios in the same samples, with each column representing a patient or control. The sample annotations for disease; age; disease activity; and treatment with steroids, biologics, or disease-modifying anti-rheumatic drugs (DMARDs) are also indicated.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Transformation Assay, Activity Assay

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet:

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Recombinant, Software

Journal: bioRxiv

Article Title: Cell Surface Nucleocapsid Protein Expression: A Betacoronavirus Immunomodulatory Strategy

doi: 10.1101/2023.02.24.529952

Figure Lengend Snippet: (A) Maximum intensity projections of laser confocal microscopy z-stack images of infected HEK-293FT, MRC-5 and Vero cells with HCoV-OC43, stained live at 24 hpi (MOI = 1). Scale bar = 20 μm. Images are representative of at least three independent experiments with similar results. (B) Flow cytometry analyses of viable infected cells (MOI = 1), stained live at 24 hpi for HCoV-OC43 S and N proteins. Representative dot plots of flow cytometry analyses showing double staining of surface S and N, indicating the percentage of the gated cell population for each quadrant of the double staining. Data are representative of at least three independent experiments, each performed with triplicate samples. (C, D) Time course of surface S and N proteins expression in live infected cells with HCoV-OC43 at 24 and 48 hpi (MOI = 1). For each infected cell type, the following is shown: histogram overlays of surface S and N proteins, as well as the mean fluorescent intensity (MFI) is plotted showing mean +/- SEM (n = 3). One-way ANOVA and Dunnett s Multiple comparison test were used to compare infected conditions against mock-infected cells: ns (nonsignificant) p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are representative of one experiment out of at least three independent experiments performed in triplicate.

Article Snippet: Indicated amounts of recombinant GFP-His (Thermo Fisher # A42613) or HCoV-OC43 N-His (Sino Biological # 40643-V07E) were resuspended in 100 μl of DPBS, and cells were incubated for 15 min at 37° C and orbital shaking of 150 rpm.

Techniques: Confocal Microscopy, Infection, Staining, Flow Cytometry, Double Staining, Expressing

Journal: bioRxiv

Article Title: Cell Surface Nucleocapsid Protein Expression: A Betacoronavirus Immunomodulatory Strategy

doi: 10.1101/2023.02.24.529952

Figure Lengend Snippet: Flow cytometry analyses of human nasal (A) and bronchial (B) airway epithelial cells infected with HCoV-OC43 (MOI = 1), stained live at 72 hpi to detect cell surface S and N protein. Representative dot plots of flow cytometry analyses showing double staining of surface S and N, indicating the percentage of the gated cell population for each quadrant. For each infection, the following is shown: histogram overlays of surface S and N proteins, as well as the MFI is plotted showing mean +/- SEM (n = 2). * p < 0.05, ** p < 0.01 by Student s two-tailed unpaired t -test. Data are representative of one experiment out of two independent experiments performed in duplicate.

Article Snippet: Indicated amounts of recombinant GFP-His (Thermo Fisher # A42613) or HCoV-OC43 N-His (Sino Biological # 40643-V07E) were resuspended in 100 μl of DPBS, and cells were incubated for 15 min at 37° C and orbital shaking of 150 rpm.

Techniques: Flow Cytometry, Infection, Staining, Double Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Cell Surface Nucleocapsid Protein Expression: A Betacoronavirus Immunomodulatory Strategy

doi: 10.1101/2023.02.24.529952

Figure Lengend Snippet: (A) Histogram overlays of surface N protein expression of live HEK293-FT, BHK-21 and CHO-K1 cells transiently transfected with a plasmid encoding eGFP (negative control) or N protein, detected with Abs by flow cytometry. (B) Histogram overlays of exogenous rN binding to HEK293-FT, BHK-21 and CHO-K1 cells, incubated with recombinant eGFP (negative control) or N protein for 15 min, washed twice, stained live with Abs, and analyzed by flow cytometry. (C) Electric charge neutralization assay with a cationic polymer (polybrene) on infected cells. MRC-5 cells were infected with HCoV-OC43 (MOI = 10), washed twice, incubated with 10 μg/ml of polybrene, washed twice, stained live with Abs, and analyzed by flow at 24 hpi. (D) Electric charge neutralization assays with exogenous rN. HEK293-FT, BHK-21 and CHO-K1 cells were incubated with 50 ng of rN protein for 15 min, washed twice, incubated with 10 μg/ml of polybrene, washed twice, stained live with Abs and analyzed by flow. (E) GAG-deficient CHO cells were infected with HCoV-OC43 (MOI = 1), washed twice, incubated with 10 μg/ml of polybrene, washed twice, stained live with Abs, and analyzed by flow at 48 hpi. (F) GAG-deficient CHO cells were incubated with recombinant eGFP or rN protein for 15 min, washed twice, stained live with Abs, and analyzed by flow cytometry. (G) Heparinase treatment significantly abrogates the cell ability to bind and retain the N protein. Flow cytometry histogram semi-overlays of Vero, HEK293-FT, BHK-21 and CHO-K1 cells treated with heparinases for 1 h, washed twice, incubated with 50 ng of rN protein for 15 min, washed twice, stained live with Abs, and analyzed. (H) BLI sensorgrams from binding assays of sulfated GAGs to immobilized N or eGFP proteins. Streptavidin-coated biosensors were loaded with equivalent amounts of N or eGFP, measuring their ability to bind each GAG. Sensorgrams show association and dissociation phases, where the vertical dotted line indicates the end of the association step. In (C, D, E, F) the MFI of detected surface N protein from live cells is plotted, showing mean +/- SEM (n = 2). For (C, D) One-way ANOVA and Dunnett s Multiple comparison test were used to compare all conditions against mock cells: ns (nonsignificant) p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. In (E, F) , ns (nonsignificant statistically) p > 0.01, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Student s two-tailed unpaired t -test. The analyses were repeated with different protein preparations, and one representative assay out of at least three independent assays performed in duplicate is shown.

Article Snippet: Indicated amounts of recombinant GFP-His (Thermo Fisher # A42613) or HCoV-OC43 N-His (Sino Biological # 40643-V07E) were resuspended in 100 μl of DPBS, and cells were incubated for 15 min at 37° C and orbital shaking of 150 rpm.

Techniques: Expressing, Transfection, Plasmid Preparation, Negative Control, Flow Cytometry, Binding Assay, Incubation, Recombinant, Staining, Neutralization, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: Cell Surface Nucleocapsid Protein Expression: A Betacoronavirus Immunomodulatory Strategy

doi: 10.1101/2023.02.24.529952

Figure Lengend Snippet: (A, B) N binds human CKs with high affinity and inhibits in vitro CHK-mediated leukocyte migration. (A) BLI sensorgrams of binding assays showing association and dissociation phases of the interaction between N protein and 17 positively bound CKs at a concentration of 100 nM out of 64 human cytokines tested. The dotted line indicates the end of the association step. The analyses were repeated with different purified rN protein preparations. One representative assay of three independent assays is shown. (B) HCOV-OC43 N blocks CXCL12β chemotaxis of MonoMac-1 cells, similarly to SARS-CoV-2 N. (C) N proteins from other endemic HCoVs also block CXCL12β chemotaxis of MonoMac-1 cells. In (B, C) CXCL12β was incubated alone (migration baseline, green bars) or in the presence of the indicated viral protein, in the lower chamber of transwell migration devices. Migrated cells from the top chamber were detected in the lower chamber at the end of the experiment. The induction of migration shows mean +/- SEM (n = 3) from one representative assay performed in triplicate out of at least three independent assays. One-way ANOVA and Dunnett s Multiple comparison test were used to compare all conditions (except no CHK and viral protein alone conditions) against migration induced by CHK alone (green bars): ns (nonsignificant) p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (D) Cell surface HCoV-OC43 N protein is a target for Ab-based immunity. ADCC reporter bioassays were performed on HCoV-OC43-infected HEK-293FT, BHK-21, MRC-5 and Vero cells (24 hpi, MOI =1) using pooled sera from five mice immunized with HCoV-OC43 rN or from five naïve mice, and Jurkat effector-reporter cells. After overnight incubation, luciferase expression to gauge cell activation was measured. Data show mean +/- SEM (n = 3) of one representative assay out of three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student s two-tailed unpaired t -test.

Article Snippet: Indicated amounts of recombinant GFP-His (Thermo Fisher # A42613) or HCoV-OC43 N-His (Sino Biological # 40643-V07E) were resuspended in 100 μl of DPBS, and cells were incubated for 15 min at 37° C and orbital shaking of 150 rpm.

Techniques: In Vitro, Migration, Binding Assay, Concentration Assay, Purification, Chemotaxis Assay, Blocking Assay, Incubation, Infection, Luciferase, Expressing, Activation Assay, Two Tailed Test

Journal: STAR Protocols

Article Title: An antigen microarray protocol for COVID-19 serological analysis

doi: 10.1016/j.xpro.2021.100815

Figure Lengend Snippet: An overview of the observed response for 48 patient sera to the S1 fragment of spike protein for each of the four HCoV viruses as well as SARS-CoV-2

Article Snippet: Human coronavirus (HCoV-OC43) Spike S1 Protein (His Tag) , Sino Biological , Cat#40607-V08H1.

Techniques:

Journal: STAR Protocols

Article Title: An antigen microarray protocol for COVID-19 serological analysis

doi: 10.1016/j.xpro.2021.100815

Figure Lengend Snippet:

Article Snippet: Human coronavirus (HCoV-OC43) Spike S1 Protein (His Tag) , Sino Biological , Cat#40607-V08H1.

Techniques: Concentration Assay, Recombinant, Software, Microarray

Journal: Signal Transduction and Targeted Therapy

Article Title: A viral RNA-dependent RNA polymerase inhibitor VV116 broadly inhibits human coronaviruses and has synergistic potency with 3CLpro inhibitor nirmatrelvir

doi: 10.1038/s41392-023-01587-1

Figure Lengend Snippet: VV116 and its parent nucleoside X1 broadly inhibited human coronavirus. a The chemical structures of VV116 and X1. VV116 is efficiently metabolized to X1 after oral uptake, and then X1 is metabolized to active triphosphate X1-NTP in the cells. b–k The activity of VV116 in inhibiting SARS-CoV-2 variants (Delta, Omicron BA.1, and Omicron BA.5), HCoV-OC43, and HCoV-229E. The curves were fitted with a nonlinear regression model. VV116, X1, GS-441524, remdesivir (RDV), and NHC were compared head-to-head in Vero E6, RD, and Huh-7 cells ( b , d , f , h , j ), and then the antiviral activity of VV116, X1, and GS-441524 against the SARS-CoV-2 variants (Delta, Omicron BA.1, and Omicron BA.5), HCoV-OC43, and HcoV-229E were validated and compared in HEK293T-hACE2-TMPRSS2, Huh-7, and MRC-5 cells, respectively. Error bars denote mean ± sd of 3–6 independent replicates ( c , e , g , I , and k ). l The half-maximum effective concentration (EC 50 ) values of compounds against HCoVs. The EC 50 values were calculated using nonlinear regression in Prism version 7.00 (GraphPad software). The bar indicates the standard deviation (SD) from 3–6 independent experiments

Article Snippet: The cells were probed using anti-HCoV-OC43 nucleocapsid protein rabbit serum (ABclonal) as the primary antibody and HRP-conjugated Anti-Rabbit IgG (SA00001-2, Proteintech) as the secondary antibody and then stained with a DAB staining kit (PA110, TIANGEN).

Techniques: Activity Assay, Concentration Assay, Software, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: A viral RNA-dependent RNA polymerase inhibitor VV116 broadly inhibits human coronaviruses and has synergistic potency with 3CLpro inhibitor nirmatrelvir

doi: 10.1038/s41392-023-01587-1

Figure Lengend Snippet: In vitro quantification of the antiviral activity of combinations of two drugs to screen high potency drug combinations against HCoV-OC43. a–d The instantaneous inhibitory potential (IIP) of combinations of nucleoside analogs and proteinase inhibitors. The concentrations of the drug combinations were normalized by IC 50 values. D drug concentration. e–h Experimental IIP values (IIP com ) and corresponding theoretical IIP values predicted by Bliss independence (IIP Bcom ) of double drug combinations at 4 × IC 50 . i–l IIP com and IIP Bcom values of double drug combinations at 2 × IC 50 . The columns represent one of three independent experiments. m and n The landscapes for the interaction of VV116 and nirmatrelvir against HCoV-OC43 ( m ) and the SARS-CoV-2 Delta variant ( n ). The synergy δ-score was calculated using SynergyFinder with the zero-interaction potency (ZIP) model. Each point and column bar represent one of three independent experiments

Article Snippet: The cells were probed using anti-HCoV-OC43 nucleocapsid protein rabbit serum (ABclonal) as the primary antibody and HRP-conjugated Anti-Rabbit IgG (SA00001-2, Proteintech) as the secondary antibody and then stained with a DAB staining kit (PA110, TIANGEN).

Techniques: In Vitro, Activity Assay, Concentration Assay, Variant Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: A viral RNA-dependent RNA polymerase inhibitor VV116 broadly inhibits human coronaviruses and has synergistic potency with 3CLpro inhibitor nirmatrelvir

doi: 10.1038/s41392-023-01587-1

Figure Lengend Snippet: In vivo efficacy of VV116, nirmatrelvir, and the VV116 plus nirmatrelvir combination in 5-day-old suckling mice infected with HCoV-OC43. a Schematic of the experimental design for therapeutic treatment in suckling mice. Mice were intranasally challenged with 10 4 TCID50 of HCoV-OC43. Mice were divided into 9 groups ( n = 5 for each group): the vehicle group, the group receiving VV116 10 mpk, the group receiving VV116 25 mpk, the group receiving VV116 50 mpk, the group receiving nirmatrelvir 10 mpk with ritonavir 50 mpk, the group receiving nirmatrelvir 25 mpk with ritonavir 50 mpk, the group receiving drug combination of VV116 10 mpk and nirmatrelvir 10 mpk with ritonavir 50 mpk (Combo 1), the group receiving drug combination of VV116 25 mpk and nirmatrelvir 25 mpk with ritonavir 50 mpk (Combo 2), or the group receiving EIDD-2801 200 mpk. Vehicle or drug was administered at 2 h post-infection, and then quaque die (q.d.) from day 1 to day 4. Lung tissues were collected at 5 days post-infection ( n = 5). b Determination of viral RNA copies targeting nucleoprotein genes in the brains, spinal cords, lungs, and kidneys collected on day 5 by real-time fluorescence quantitative PCR. c Determination of viral titers in the brains, spinal cords, lungs, and kidneys collected on day 5 by immunoplaque assay. d Cytokine gene expression was measured in the brain, spinal cord, lungs, and kidneys at day 5. The relative gene expression of IL-1β, IL-6, IFNAR, TNF-α, CCL2, CXCL10, and ISG15 was compared to that of unchallenged mice. e–v Immunofluorescence staining of brains and spinal cords to detect the SARS-CoV-2 antigen. e , n Vehicle, f , o EIDD2801-200 mpk, g , p VV116-50 mpk, h , q VV116-10 mpk, i , r nirmatrelvir-10 mpk + rito-50 mpk, j , s Combo 1, k , t VV116-25 mpk, l , u nirmatrelvir-25 mpk with ritonavir-50 mpk, and m , v Combo 2. The scale bars on the pictures of tissue slides indicate 1000 µm. The data on viral copies and viral titers were statistically analyzed with Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001; and ns, not significant

Article Snippet: The cells were probed using anti-HCoV-OC43 nucleocapsid protein rabbit serum (ABclonal) as the primary antibody and HRP-conjugated Anti-Rabbit IgG (SA00001-2, Proteintech) as the secondary antibody and then stained with a DAB staining kit (PA110, TIANGEN).

Techniques: In Vivo, Infection, Fluorescence, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Staining