Journal: Vaccines

Article Title: Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.

doi: 10.3390/vaccines13010002

Figure Lengend Snippet: Figure 2. Mutations at S1/S2, S2′ positions were needed for SMERS to be expressed on the surface NDV particles. (A) The genomic arrangement of NDV-SMERS-S1/S2. Chicken-codon optimized SMERS with S1/S2 cleavage site mutation, 2P mutation, tPA secretion peptide, transmembrane domain (TMF) and cytoplasmic tail (CTF) of NDV F protein was inserted between P and M genes of NDV genome. (B) Allantoic fluid (ALF) harvested from embryonated chicken eggs inoculated with NDV-SMERS-S1/S2, and the ultracentrifuged virus samples (UC-virus) were subjected to a Western blot using anti-MERS-CoV

Article Snippet: Uninfected and NDV-infected DF-1 cells were fixed with 80% ice-cold acetone or 4% formaldehyde for 20 min. After 3 washes with PBS, samples were incubated with a blocking buffer (1% BSA and 10% FBS in PBS) for 1 h, followed by an incubation with mouse anti-MERS-CoV S1 monoclonal antibody (MAB10707, R&D system’s, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Mutagenesis, Virus, Western Blot

Journal: Vaccines

Article Title: Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.

doi: 10.3390/vaccines13010002

Figure Lengend Snippet: Figure 3. PCM1 and PCM2 did not improve SMERS expression. (A) A diagram of different versions of SMERS containing combinations of S1/S2, S2’, 2P, PCM1, PCM2 mutations. (B) HEK-293T/17 cells were transfected with 1 µg pCAGGS containing each version of SMERS gene. Cell lysates harvested at 72 h post transfection were subjected to a Western blot analysis using anti-MERS-CoV S2 and anti-β-actin antibodies. (C) The volumes of the ultracentrifuged virus samples were adjusted to equal TCID50/mL. The samples were then subjected to a Western blot analysis using anti-MERS-CoV S2 and anti-NDV N antibodies.

Article Snippet: Uninfected and NDV-infected DF-1 cells were fixed with 80% ice-cold acetone or 4% formaldehyde for 20 min. After 3 washes with PBS, samples were incubated with a blocking buffer (1% BSA and 10% FBS in PBS) for 1 h, followed by an incubation with mouse anti-MERS-CoV S1 monoclonal antibody (MAB10707, R&D system’s, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Expressing, Transfection, Western Blot, Virus

Journal: Vaccines

Article Title: Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.

doi: 10.3390/vaccines13010002

Figure Lengend Snippet: Figure 4. 2P mutation improved surface SMERS expression. (A) A diagram comparing the gene insert of NDV-SMERS-S1/S2, NDV-SMERS-S2’, and NDV-SMERS-S2’ no2P. (B) The ultracentrifuged virus samples were subjected to Western blot analysis using anti-MERS-CoV S2 and anti-NDV N antibodies, and (C) an MERS-CoV S1-sandwich ELISA. **** indicates significant difference (p < 0.0001). (D) The ultracentrifuged samples were stained with rabbit anti MERS-CoV S1 antibody, and then the 10-nm gold-conjugated goat anti-rabbit IgGs. Images were recorded under a TEM at 50,000× magnification.

Article Snippet: Uninfected and NDV-infected DF-1 cells were fixed with 80% ice-cold acetone or 4% formaldehyde for 20 min. After 3 washes with PBS, samples were incubated with a blocking buffer (1% BSA and 10% FBS in PBS) for 1 h, followed by an incubation with mouse anti-MERS-CoV S1 monoclonal antibody (MAB10707, R&D system’s, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Mutagenesis, Expressing, Virus, Western Blot, Sandwich ELISA, Staining

Journal: Vaccines

Article Title: Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.

doi: 10.3390/vaccines13010002

Figure Lengend Snippet: Figure 6. SMERS gene insert was stable after 5 egg-passages. (A) Viral RNAs of NDV-mCh and NDV- SMERS-S2’ from egg-passages 2–5 were purified and subjected to RT-PCRs using SMERS- and NNDV- specific primers. pCI.NDV-SMERS plasmid was used as positive controls. (B) The ultracentrifuged NDV-SMERS-S2’ samples from egg-passage 3–5 (E3, E4, and E5) were stained with rabbit anti MERS- CoV S1 antibody, followed by 10-nm gold-conjugated goat anti-rabbit IgGs. Images were recorded under a TEM at 50,000× magnification.

Article Snippet: Uninfected and NDV-infected DF-1 cells were fixed with 80% ice-cold acetone or 4% formaldehyde for 20 min. After 3 washes with PBS, samples were incubated with a blocking buffer (1% BSA and 10% FBS in PBS) for 1 h, followed by an incubation with mouse anti-MERS-CoV S1 monoclonal antibody (MAB10707, R&D system’s, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Purification, Plasmid Preparation, Staining

Journal: Journal of Immunological Methods

Article Title: Development and characterization of an indirect ELISA to detect SARS-CoV-2 spike protein-specific antibodies

doi: 10.1016/j.jim.2021.112958

Figure Lengend Snippet: Standard curve of the SARS-CoV-2 S1 ELISA a Standards (S1–S6) were prepared by two-fold serial dilutions of two human convalescent plasma samples starting at a dilution of 1:1010. A standard curve was created by 4PL regression analysis. The lowest standard (S6) was set to 100 AEUs, and the highest (S1) was set to 3200 AEUs. The R 2 value of the standard curves are depicted in the graph. b Stability of the standard solutions under different environmental conditions. We analyzed the stability upon storage for 96 h at 2–8 °C, room temperature (RT), 37 °C or upon 10 freeze and thaw cycles. c Stability of the standard solutions over time. The stability was analyzed upon storage for 7 months at 2–8 °C. d A newly thawed batch of the reference standard was compared with the old batch that had already been used for over 7 months. The dashed line depicts the cut-off for a positive antibody response, calculated as the mean OD value of the results of 120 negative human sera ( a) plus four standard deviations.

Article Snippet: The following antigens were used: 1 μg/ml HKU1 spike (S1) protein (Sino Biological, 40,021-V08H) or 0.5 μg/ml in PBS of MERS-CoV spike (S1) protein (Sino Biological, 40,069-V08H).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunological Methods

Article Title: Development and characterization of an indirect ELISA to detect SARS-CoV-2 spike protein-specific antibodies

doi: 10.1016/j.jim.2021.112958

Figure Lengend Snippet: Specificity of the SARS-CoV-2 S1 ELISA a 120 randomly selected anonymous human serum samples without SARS-CoV or SARS-CoV-2 history were analyzed by SARS-CoV-2 S1 ELISA (dilution 1:101). The cut-off of the assay (OD = 0.3) was set to the mean value (OD mean = 0.104) of the sera depicted here plus 4 standard deviations (SD = 0.049). b Serum sample #56 was analyzed for the detection of SARS-CoV-2 spike (S) and nucleoprotein (N) by Western blot (dilution 1:100). A low-sensitivity HRP substrate was used to detect the enzymatic activities of HRP-coupled secondary antibodies. The exposure times of 1 s and 30 s are displayed. The positive control was obtained from a COVID-19 patient (dilution 1:100, positive control, +). c Native Immunofluorescence analysis was performed to confirm the results. Microscopic analyses were performed at a magnification of 63× with a confocal laser scanning microscope (Leica SP5). All images were acquired with a laser intensity of 10% (488 nm excitation). Scale bar: 20 μM.

Article Snippet: The following antigens were used: 1 μg/ml HKU1 spike (S1) protein (Sino Biological, 40,021-V08H) or 0.5 μg/ml in PBS of MERS-CoV spike (S1) protein (Sino Biological, 40,069-V08H).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Positive Control, Immunofluorescence, Laser-Scanning Microscopy

Journal: Journal of Immunological Methods

Article Title: Development and characterization of an indirect ELISA to detect SARS-CoV-2 spike protein-specific antibodies

doi: 10.1016/j.jim.2021.112958

Figure Lengend Snippet: Variability of the results of the SARS-CoV-2 S1 ELISA a Intra-assay variability was analyzed using three different positive serum samples in five replicates in six different assays. Three operators performed the assays. The OD values are shown. The calculated coefficients of variability (CV) are shown in . b Exemplary standard curve of each operator. Based on these curves the AEU/ml were calculated for nine different positive human serum or plasma samples. c Inter-assay variability was analyzed using nine different positive serum samples in duplicates in six different assays performed by three operators. The OD values (left graph) and the calculated AEU/ml (right graph) are shown. White circles represent the values obtained by operator three.

Article Snippet: The following antigens were used: 1 μg/ml HKU1 spike (S1) protein (Sino Biological, 40,021-V08H) or 0.5 μg/ml in PBS of MERS-CoV spike (S1) protein (Sino Biological, 40,069-V08H).

Techniques: Enzyme-linked Immunosorbent Assay, Intra Assay, Inter Assay

Journal: Journal of Immunological Methods

Article Title: Development and characterization of an indirect ELISA to detect SARS-CoV-2 spike protein-specific antibodies

doi: 10.1016/j.jim.2021.112958

Figure Lengend Snippet: Cross-reactivity of antibodies (IgG) detecting other hCoV a 20 sera with MERS-CoV S1-specific or b 27 sera with HKU1 S1-specific antibodies were analyzed in the SARS-CoV-2 S1 ELISA and in the respective other hCoV ELISA.

Article Snippet: The following antigens were used: 1 μg/ml HKU1 spike (S1) protein (Sino Biological, 40,021-V08H) or 0.5 μg/ml in PBS of MERS-CoV spike (S1) protein (Sino Biological, 40,069-V08H).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunological Methods

Article Title: Development and characterization of an indirect ELISA to detect SARS-CoV-2 spike protein-specific antibodies

doi: 10.1016/j.jim.2021.112958

Figure Lengend Snippet: Detection of SARS-CoV-2 S1 protein-specific antibodies in COVID-19 patients a Consecutive serum or plasma samples from 23 COVID-19 patients (dilution 1:101) were analyzed using the SARS-CoV-2 S1 ELISA. The sample OD values are displayed to visualize the results of all samples. b Neutralization of authentic SARS-CoV-2 by the respective samples. c The same samples were analyzed using the commercially available SARS-CoV-2 S-specific IgG (upper panel) and IgA (lower panel) ELISAs.

Article Snippet: The following antigens were used: 1 μg/ml HKU1 spike (S1) protein (Sino Biological, 40,021-V08H) or 0.5 μg/ml in PBS of MERS-CoV spike (S1) protein (Sino Biological, 40,069-V08H).

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Journal of Immunological Methods

Article Title: Development and characterization of an indirect ELISA to detect SARS-CoV-2 spike protein-specific antibodies

doi: 10.1016/j.jim.2021.112958

Figure Lengend Snippet: Detection of SARS-CoV-2 S1 protein-specific antibodies and correlation analyses a - b Serum or plasma samples of 52 COVID-19 patients ( n = 88, dilution 1:101) were analyzed using the SARS-CoV-2 S1 in-house or EUROIMMUN IgG ELISA. Samples that were tested negative in the ELISA and positive in the neutralization test are represented by red dots. c Correlation of the in-house SARS-CoV-2 S1 ELISA and the EUROIMMUN (EI) IgG ELISA. d Correlation of binding antibodies detected by the in-house SARS-CoV-2 ELISA with live virus neutralizing titers (VNT100). Red lines reflect the best linear fit relationship between these variables, dotted lines visualize 95% confidence intervals. P and R values reflect two-tailed Spearman rank-correlation tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following antigens were used: 1 μg/ml HKU1 spike (S1) protein (Sino Biological, 40,021-V08H) or 0.5 μg/ml in PBS of MERS-CoV spike (S1) protein (Sino Biological, 40,069-V08H).

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Binding Assay, Two Tailed Test

Journal: Vaccines

Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

doi: 10.3390/vaccines8040634

Figure Lengend Snippet: Kinetics of serum and egg yolk anti-MERS COV-S IgY antibodies response of chickens after immunization with MERS COV-S recombinant protein compared with the adjuvant-immunized chicken (adjuvant control). Each week is represented by a pool of egg yolks of individual chicken in each group (S1-immunized and adjuvant-immunized).

Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

Techniques: Recombinant

Journal: Vaccines

Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

doi: 10.3390/vaccines8040634

Figure Lengend Snippet: Western blot analysis of anti-MERS-COV rS1 IgY antibodies. (Left) The S1 protein of MERS-COV was subjected to SDS-PAGE under reducing conditions; (Right) Western blot analysis of the anti-S1 IgY antibody response. SDS gels were electrically transferred onto nitrocellulose membranes and probed with IgY from immunized and nonimmunized hens (marker: molecular maker; lane A: S1-immunized IgY; lane B: adjuvant-immunized IgY). The strips were processed separately and pasted beside each other for documentation.

Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

Techniques: Western Blot, SDS Page, Marker

Journal: Vaccines

Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

doi: 10.3390/vaccines8040634

Figure Lengend Snippet: Dot blotting analysis. Purified anti-S1 IgY antibodies showed reactivity with different concentrations of the spike protein (S), S1, and receptor binding domain (RBD), but had no reactivity with nucleocapsid (NP) protein of MERS CoV.

Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

Techniques: Purification, Binding Assay

Journal: Vaccines

Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

doi: 10.3390/vaccines8040634

Figure Lengend Snippet: Recognition by anti-S1 IgY antibodies of viral antigen expressed in MERS-CoV-infected Vero E6 cells, using indirect immunofluorescence assay. ( A ) Vero E6 cells inoculated with MERS-CoV and stained with anti-S1 IgY antibodies and FITC-conjugated anti-chicken antibodies; and ( B ) control adjuvant IgY (Bright-field).

Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

Techniques: Infection, Immunofluorescence, Staining

Journal: Vaccines

Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

doi: 10.3390/vaccines8040634

Figure Lengend Snippet: Examples of different concentrations of anti-S1 IgY antibodies tested against MERS-CoV on Vero-E6 cells examined by CPE. The IC100 neutralization of the antibody were determined as the reciprocal of the highest dilution at which no CPE was observed.

Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

Techniques: Neutralization

Journal: Vaccines

Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

doi: 10.3390/vaccines8040634

Figure Lengend Snippet: Evaluation of the neutralizing potential of anti-S1 IgY antibodies, using plaque reduction neutralization test. ( A ) MERS-CoV (MOI 0.01) was incubated with different concentrations of anti-S1 IgY antibodies and added to Vero E6 cells. After virus adsorption, agar medium was added to the Vero E6 cells, and the plaques that formed were stained with crystal violet, each IgY concentration was tested in triplicate. ( B ) Percent inhibition of anti-S1 IgY antibodies with different concentrations. The best fit equation is:

Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

Techniques: Plaque Reduction Neutralization Test, Incubation, Adsorption, Staining, Concentration Assay, Inhibition

Journal: Vaccines

Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

doi: 10.3390/vaccines8040634

Figure Lengend Snippet: ( A ) Viral titer in the lungs of MERS-CoV mice treated with anti-SI IgY antibodies and control IgY (adjuvant). ( B ) Body weight changes after MERS-CoV infection between anti-SI IgY antibodies and IgY of adjuvant control group. ( C – F ) Histopathology of the lungs from human dipeptidyl peptidase 4 (hDPP4)-transgenic mice on day 8 after inoculation with MERS-CoV. Representative histopathological findings of mice with the highest cellular infiltration in alveoli by H&E staining ( C ) Massive mononuclear cell infiltrations including macrophages and lymphocytes with regenerated type II pneumocytes were seen in adjuvant control group (right column), but less in the anti-S1 IgY treated group (left column). Scale bars: 200 μm (upper row) and 20 μm (lower row). Al, alveoli; Br, bronchi; V, vessel. Detection of viral antigen in lung tissues of mice by immunohistochemistry ( D ) A few antigen positive cells were seen in the lungs of anti-S1 IgY treated group compared to adjuvant control group. Quantification of inflammation areas ( E ) The area of pulmonary lesion was determined based on the mean percentage of affected areas in each section of the collected lobes form each animal ( n = 8 or 6). Circles indicate averages from three observation lobes in each mouse. p = 0.1709 by Mann-Whitney test. Numbers of viral antigen positive cells in the alveoli ( F ) Data were obtained from 8 or 6 mice. Circles indicate averages of 5 observation fields in each mouse. * p = 0.0196 by Mann-Whitney test.

Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

Techniques: Infection, Histopathology, Transgenic Assay, Staining, Immunohistochemistry, MANN-WHITNEY