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

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: Molecular therapy. Nucleic acids

Article Title: A model system for antiviral siRNA therapeutics using exosome-based delivery.

doi: 10.1016/j.omtn.2022.08.011

Figure Lengend Snippet: Figure 2. Expression of viral antigens in vitro Expression constructs were delivered to HEK293T cells via EPM nanoplatform. Antigens introduced included the S1 subunit of the spike glycoprotein (A), the nucleocapsid (N) (B), and the RNA-dependent RNA polymerase (RdRp) (C). Expression of antigens was detected by western blot, and b-actin was used as a loading control. (D) Dose response of S1 expression, varying exosomal load, and pDNA payload after 48 h incubation. Fold change is indicated as compared to 75 mg exosomes, 40 mg PEI, and 1.5 mg pDNA, the selected standard reaction. The samples shown are pooled biological triplicates undergoing single replicate analysis. (E) Time course of N expression using the standard reaction. The blot shown is representative of 3 biological replicates analyzed individually. Fold change shown with respect to expression level after 12 h. (F) Time course of S1 expression using the standard reaction. The blot shown is representative of 3 biological replicates. Fold change shown with respect to expression level after 12 h. Data expression as means ± SDs for at least 3 biological replicates and significance determined by 1-way ANOVA with post-hoc comparison of all means to EPM-pDNA for target antigen alone (*p < 0.05, ***p < 0.001).

Article Snippet: Two different primary antibodies for the spike glycoprotein were obtained from Sino Biological, one polyclonal and one monoclonal antibody.

Techniques: Expressing, In Vitro, Construct, Western Blot, Control, Incubation, Comparison

Journal: PLoS ONE

Article Title: Superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein of Middle East respiratory syndrome coronavirus

doi: 10.1371/journal.pone.0220196

Figure Lengend Snippet: (A) Schematic representation of rAd/Spik, rAd/RBD, and rAd/NTD. A shuttle vector carrying the codon-optimized full-length MERS-CoV Spike gene, RBD gene, and NTD gene was constructed, as shown in the diagram. The vectors were used to generate recombinant replication-deficient adenovirus rAd/Spike, rAd/RBD, and rAd/NTD by homologous recombination with adenoviral genomic DNA. Numbers indicate amino acids. SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; TM, transmembrane domain. (B-D) Spike, RBD, and NTD expressions in the lysate (L) and supernatant (S) of HEK293 cells infected with (B) rAd/Spike, (C) rAd/RBD, and (D) rAd/NTD, respectively. The expressions of Spike and RBD were confirmed via immunoblotting assay using MERS-CoV spike protein S1 (aa 1–725) rabbit polyclonal antibody. NTD expression were confirmed by immunoblotting assay with anti-rAd/Spike immune serum.

Article Snippet: The expression and secretion of Spike and RBD proteins by rAd-infected HEK293 cells were verified via Western blot analysis by employing anti-MERS-CoV S1 protein rabbit polyclonal antibody [amino acids (aa) 1–725; 40069-T48; Sino Biological Inc., Beijing, China] and HRP-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, UK).

Techniques: Plasmid Preparation, Construct, Recombinant, Homologous Recombination, Binding Assay, Infection, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein of Middle East respiratory syndrome coronavirus

doi: 10.1371/journal.pone.0220196

Figure Lengend Snippet: Female BALB/c mice (4 mice/group) were immunized twice at week 0 and 2 with 1×10 7 PFUs of rAd/NTD, rAd/RBD, or rAd/Spike via the intranasal (IN) route, whereas control animals received 1×10 7 PFUs of rAd/mock. Sera and BALFs were collected for measuring antibody responses 7 days after the second immunization. (A) Systemic MERS S1-specific serum IgG antibody titers were determined by ELISA. (B) Mucosal MERS S1-specific IgA antibody titers in BALFs were measured by ELISA. (C) On day 7 after the last immunization, lung cells were harvested from mice and stained with K d /S291 tetramer, anti-CD8, and anti-CD44 antibody. The frequency of K d /S291 tetramer-positive and CD44-positive cells among the total lung CD8-positive cell population is indicated in the upper right quadrant. (D) The average percentages of K d /S291 tetramer-specific CD8 T cells in the lungs. Data are representative of at least two independent experiments with similar results and average SEM value of four mice. ns, not significant; *** p <0.001; ** p <0.01.

Article Snippet: The expression and secretion of Spike and RBD proteins by rAd-infected HEK293 cells were verified via Western blot analysis by employing anti-MERS-CoV S1 protein rabbit polyclonal antibody [amino acids (aa) 1–725; 40069-T48; Sino Biological Inc., Beijing, China] and HRP-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, UK).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: PLoS ONE

Article Title: Superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein of Middle East respiratory syndrome coronavirus

doi: 10.1371/journal.pone.0220196

Figure Lengend Snippet: Groups of mice were immunized as indicated in . On day 7 after the last immunization, sera and BALFs were collected from each group. Serially diluted sera and BALFs were evaluated for their neutralizing activity against MERS Spike pseudotyped virus in Huh 7.5 cells. The transduction percentage was calculated by measuring GFP expression in comparison with the expression in the control pseudovirus-infected cells. The dotted horizontal line indicates 50% neutralization. (A) Neutralization activities in serially diluted sera. (B) The IC 50 (50% inhibitory concentration) neutralization titers in the sera. (C) The neutralization activities in serially diluted BALFs. Data are representative of at least two independent experiments with similar results and average SEM value of four mice. (D) NHNE cells (10 6 cells/well) were inoculated with 300 μl of virus solution (MERS-CoV 10μl/PBS 10 ml) and N2 gene level of MERS-CoV was assessed at 1 day post-infection using real-time PCR. To prove the effect of neutralizing antibody for MERS-CoV spike protein, targeted antibody of MERS-CoV spike protein was diluted to 1:10, 1:50, 1:250 and the diluted antibodies were mixed with the virus solution at a ratio of 1:1 and maintained for 1 hour before infection to NHNE cells. Then, N2 gene level of MERS-CoV was assessed at 1 day post-infection using real-time PCR. The same experiments were carried out using mock antibody and the result was compared with that of targeted antibody for MERS-CoV spike protein. ns, not significant; *** p <0.001; ** p <0.01; * p <0.05.

Article Snippet: The expression and secretion of Spike and RBD proteins by rAd-infected HEK293 cells were verified via Western blot analysis by employing anti-MERS-CoV S1 protein rabbit polyclonal antibody [amino acids (aa) 1–725; 40069-T48; Sino Biological Inc., Beijing, China] and HRP-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, UK).

Techniques: Activity Assay, Virus, Transduction, Expressing, Comparison, Control, Infection, Neutralization, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein of Middle East respiratory syndrome coronavirus

doi: 10.1371/journal.pone.0220196

Figure Lengend Snippet: Groups of mice were immunized as indicated in . On day 7 after the last immunization, sera were collected from immune mice. (A) The NTD-specific, (B) RBD-specific, and (C) S2-specific IgG antibody responses were measured by ELISA. NTD, RBD, and S2 proteins were used as coating antigens (200 ng/well), and goat anti-mouse IgG-HPR was used as the detection antibody. (D) Protein competition neutralization assay. Sera collected from rAd/Spike immune mice were diluted at a ratio of 1:640 and were assayed for neutralization of MERS Spike pseudotyped virus in the presence of soluble NTD, RBD, S1, or S2 proteins at a concentration of 10 μg. Data are represented as mean ± SEM results of four mice from at least two independent experiments. ns, not significant; *** p <0.001; ** p <0.01; * p <0.05.

Article Snippet: The expression and secretion of Spike and RBD proteins by rAd-infected HEK293 cells were verified via Western blot analysis by employing anti-MERS-CoV S1 protein rabbit polyclonal antibody [amino acids (aa) 1–725; 40069-T48; Sino Biological Inc., Beijing, China] and HRP-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Virus, Concentration Assay

Journal: PLoS ONE

Article Title: Superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein of Middle East respiratory syndrome coronavirus

doi: 10.1371/journal.pone.0220196

Figure Lengend Snippet: Groups of mice were immunized as indicated in . Two weeks after last immunization, sera and BALFs were collected from immune mice and heat-inactivated at 56°C for 30 min before detecting neutralizing antibodies. (A) The neutralizing antibody titers in serially diluted sera were measured via MERS-CoV pseudovirus neutralization assay in Huh 7.5 cells. (B) IC 50 neutralization titer in the serum. (C) Neutralizing antibody titers in serially diluted BALFs. (D) IC 50 neutralization titer in BALFs. The data are presented as mean ± SEM values of four mice per group. ns, not significant; *** p <0.001.

Article Snippet: The expression and secretion of Spike and RBD proteins by rAd-infected HEK293 cells were verified via Western blot analysis by employing anti-MERS-CoV S1 protein rabbit polyclonal antibody [amino acids (aa) 1–725; 40069-T48; Sino Biological Inc., Beijing, China] and HRP-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, UK).

Techniques: Neutralization

Journal: PLoS ONE

Article Title: Superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein of Middle East respiratory syndrome coronavirus

doi: 10.1371/journal.pone.0220196

Figure Lengend Snippet: Female Balb/c mice were immunized three times at 0, 3, and 11 weeks with 1×10 7 PFU of rAd/Spike via IN route. Sera were collected for measuring antibody responses at 2, 6, 15, and 30 weeks. Control mice were immunized intranasally with 1×10 8 PFU of rAd/mock. (A) Immunization scheme. (B) Systemic RBD-specific IgG antibody titers were measured by ELISA after each immunization. (C) Mucosal RBD-specific IgA antibody titers in BALFs were measured 19 weeks after the last immunization by ELISA. (D) Neutralization antibody responses in sera were measured via MERS-CoV pseudovirus neutralization assay at 15 and 30 weeks. (E) Neutralizing activities in BALFs at 19 weeks after the third immunization. Data are presented as mean ± SEM values of four mice per group. *** p <0.001.

Article Snippet: The expression and secretion of Spike and RBD proteins by rAd-infected HEK293 cells were verified via Western blot analysis by employing anti-MERS-CoV S1 protein rabbit polyclonal antibody [amino acids (aa) 1–725; 40069-T48; Sino Biological Inc., Beijing, China] and HRP-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, UK).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Neutralization