Journal: Frontiers in Molecular Biosciences

Article Title: Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites

doi: 10.3389/fmolb.2022.799703

Figure Lengend Snippet: Inhibition of HMOs on the binding between HepG2 cells and Spike protein RBD. Fluorescent labelled proteins were preincubated with 1 mg per ml 2′-FL, 6′-SL, and LNnT, respectively. The preincubation was performed at room temperature for 30 min. (A) Immunofluorescence for S protein RBD binding. The columns (from left to right) show staining of nuclear acid (Hoechst 33342), plasma membrane (CellMask™ Deep Red), S protein RBD, and merged image. Scale bar, 496 pixels. (B) Quantification of fluorescent intensity of spike protein RBD binding. Fluorescence intensity was quantified for selected cell area. Quantification was performed with software ImageJ. Asterisks indicate the statistical significance between groups compared (** p < 0.01%; *** p < 0.001%; ns p < 0.05).

Article Snippet: Recombinant human angiotensin-converting enzyme 2 (ACE2) (RayBiotech, Georgia, Product Number 230-30165), SARS-CoV-2 spike protein S1 Subunit RBD (Arg319-Phe541) (RayBiotech, Georgia, Product Number 230-30162) and spike protein S1 subunit (Val16-Arg685) (Sino Biological, China, Product Number 40591-V08H) were all derived from transfected human HEK293 cells.

Techniques: Inhibition, Binding Assay, Immunofluorescence, Staining, Fluorescence, Software

Journal: Frontiers in Molecular Biosciences

Article Title: Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites

doi: 10.3389/fmolb.2022.799703

Figure Lengend Snippet: Remodeling host glycome alters binding between host cells and spike protein RBD. 2F-Fucose (Fucosyltransferase Inhibitor); 3-F-Sia (Sialyltransferase Inhibitor). (A) Immunofluorescence for S protein RBD binding with modified HepG2 cells. The columns (from left to right) show staining of nuclear acid (Hoechst 33342), plasma membrane (CellMask™ Deep Red), S protein RBD, and merged image. Scale bar, 600 pixels. (B) Quantification of fluorescent intensity of spike protein RBD binding. Fluorescence intensity was quantified for selected cell area. Quantification was performed with software ImageJ. Asterisks indicate the statistical significance between groups compared (* p < 0.05%; ** p < 0.01%; *** p < 0.001%; ns p < 0.05).

Article Snippet: Recombinant human angiotensin-converting enzyme 2 (ACE2) (RayBiotech, Georgia, Product Number 230-30165), SARS-CoV-2 spike protein S1 Subunit RBD (Arg319-Phe541) (RayBiotech, Georgia, Product Number 230-30162) and spike protein S1 subunit (Val16-Arg685) (Sino Biological, China, Product Number 40591-V08H) were all derived from transfected human HEK293 cells.

Techniques: Binding Assay, Immunofluorescence, Modification, Staining, Fluorescence, Software

Journal: Frontiers in Molecular Biosciences

Article Title: Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites

doi: 10.3389/fmolb.2022.799703

Figure Lengend Snippet: Sialylated HMOs diminished binding of spike protein RBD to HepG2 Cells. Fluorescent labelled proteins were preincubated with 1 mg per ml 3′-FL (3′-sialyllactose) and 6′-SL (6′-sialyllactose) respectively at room temperature. (A) Immunofluorescence for S protein RBD binding with cells. The columns (from left to right) show staining of nuclear acid (Hoechst 33342), plasma membrane (CellMask™ Deep Red), S protein RBD, and merged image. Scale bar, 600 pixels. (B) Quantification of fluorescent intensity of spike protein RBD binding. Fluorescence intensity was quantified for selected cell area. Quantification was performed with software ImageJ. Asterisks indicate the statistical significance between groups compared (* p < 0.05%; ns p < 0.05).

Article Snippet: Recombinant human angiotensin-converting enzyme 2 (ACE2) (RayBiotech, Georgia, Product Number 230-30165), SARS-CoV-2 spike protein S1 Subunit RBD (Arg319-Phe541) (RayBiotech, Georgia, Product Number 230-30162) and spike protein S1 subunit (Val16-Arg685) (Sino Biological, China, Product Number 40591-V08H) were all derived from transfected human HEK293 cells.

Techniques: Binding Assay, Immunofluorescence, Staining, Fluorescence, Software

Journal: Frontiers in Molecular Biosciences

Article Title: Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites

doi: 10.3389/fmolb.2022.799703

Figure Lengend Snippet: Introducing high mannose glycans into viral binding. (A) Immunofluorescence for S protein RBD binding with modified cells. The columns (from left to right) show staining of nuclear acid (Hoechst 33342), plasma membrane (CellMask™ Deep Red), S protein RBD, and merged image. Scale bar, 600 pixels. Quantification of fluorescent intensity of spike protein RBD (B , C) or S1 subunit (D) binding. (C) Fluorescent labelled proteins were preincubated with purified high mannose at room temperature for 30 min before binding. Fluorescence intensity was quantified for selected cell area. Quantification was performed with software ImageJ. Asterisks indicate the statistical significance between groups compared (* p < 0.05%; ** p < 0.01%; *** p < 0.001%; **** p < 0.0001%; ns p < 0.05).

Article Snippet: Recombinant human angiotensin-converting enzyme 2 (ACE2) (RayBiotech, Georgia, Product Number 230-30165), SARS-CoV-2 spike protein S1 Subunit RBD (Arg319-Phe541) (RayBiotech, Georgia, Product Number 230-30162) and spike protein S1 subunit (Val16-Arg685) (Sino Biological, China, Product Number 40591-V08H) were all derived from transfected human HEK293 cells.

Techniques: Binding Assay, Immunofluorescence, Modification, Staining, Purification, Fluorescence, Software

Journal: Frontiers in Molecular Biosciences

Article Title: Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites

doi: 10.3389/fmolb.2022.799703

Figure Lengend Snippet: Modelled sialylated and desialylated ACE2-Covid S protein complexes. 3D structural modeling of glycosylated ACE2 interacting with S-protein. Results from glycomics and glycoproteomics of HEPG2 cell lines were used to generate (A) fully-desialylated and (B) fully-sialylated homologs of ACE2, interacting with S-protein.

Article Snippet: Recombinant human angiotensin-converting enzyme 2 (ACE2) (RayBiotech, Georgia, Product Number 230-30165), SARS-CoV-2 spike protein S1 Subunit RBD (Arg319-Phe541) (RayBiotech, Georgia, Product Number 230-30162) and spike protein S1 subunit (Val16-Arg685) (Sino Biological, China, Product Number 40591-V08H) were all derived from transfected human HEK293 cells.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites

doi: 10.3389/fmolb.2022.799703

Figure Lengend Snippet: Interactions of glycosylated ACE2 and S-protein were revealed using molecular dynamics simulations. (A) The number of intramolecular hydrogen bonds was drastically higher for each fully-sialylated N-glycan compared to the desialylated glycoform. (B) For Asn 90 and Asn 322, the sialic acid residue in the glycoform accounted for ∼10% of hydrogen bonds.

Article Snippet: Recombinant human angiotensin-converting enzyme 2 (ACE2) (RayBiotech, Georgia, Product Number 230-30165), SARS-CoV-2 spike protein S1 Subunit RBD (Arg319-Phe541) (RayBiotech, Georgia, Product Number 230-30162) and spike protein S1 subunit (Val16-Arg685) (Sino Biological, China, Product Number 40591-V08H) were all derived from transfected human HEK293 cells.

Techniques:

Journal: Viruses

Article Title: Clinical Utility of SARS-CoV-2 Antibody Titer Multiplied by Binding Avidity of Receptor-Binding Domain (RBD) in Monitoring Protective Immunity and Clinical Severity

doi: 10.3390/v15081662

Figure Lengend Snippet: Changes in anti-spike S1 IgG antibody titer and its RBD binding avidity in vaccinated subjects following 2–4 boost immunizations and in SARS-CoV-2-infected patients. ( A ) Antibody titer (BUg/mL). ( B ) RBD binding avidity (1/IC50, nM). Blood samples of healthy vaccinees ( n = 163, closed circles) were collected 3–4 weeks after each vaccination at the indicated times in and those of unvaccinated COVID-19 patients ( n = 339, open circles) on admission and 2–3 weeks after admission or at discharge. The median (interquartile range) values are in red. M: month.

Article Snippet: HEK293 expressing recombinant His Tag SARS-CoV-2 S1 of the original Wuhan strain (Accession# QHD43416.1, Val16–Arg685) and S protein RBD (Arg319–Phe541) were purchased from ACROBiosystems (Newark, DE, USA).

Techniques: Binding Assay, Infection

Journal: Viruses

Article Title: Clinical Utility of SARS-CoV-2 Antibody Titer Multiplied by Binding Avidity of Receptor-Binding Domain (RBD) in Monitoring Protective Immunity and Clinical Severity

doi: 10.3390/v15081662

Figure Lengend Snippet: Correlation between anti-SARS-CoV-2 IgG titers and their ABAT values at 3–4 weeks after 1st to 4th vaccinations showing a maturation of the antigen binding avidity. ( A ) After 1st vaccine, ( B ) after 2nd vaccine, ( C ) after 3rd vaccine, and ( D ) after 4th vaccine ( n = 163). Note the differences in the correlation regression lines for different antigen binding avidities.

Article Snippet: HEK293 expressing recombinant His Tag SARS-CoV-2 S1 of the original Wuhan strain (Accession# QHD43416.1, Val16–Arg685) and S protein RBD (Arg319–Phe541) were purchased from ACROBiosystems (Newark, DE, USA).

Techniques: Binding Assay

Journal: Viruses

Article Title: Clinical Utility of SARS-CoV-2 Antibody Titer Multiplied by Binding Avidity of Receptor-Binding Domain (RBD) in Monitoring Protective Immunity and Clinical Severity

doi: 10.3390/v15081662

Figure Lengend Snippet: ROC analysis to determine the cutoff values of serum anti-SARS-CoV-2 antibody level, IC50, and ABAT in vaccinated normal subjects and COVID-19 patients. ( A ) Anti-SARS-CoV-2 antibody level (BUg/mL), ( B ) IC50 (nM), ( C ) ABAT ((BUg/mL)/(IC50 nM)). AUC: area under the curve; CI: confidence interval.

Article Snippet: HEK293 expressing recombinant His Tag SARS-CoV-2 S1 of the original Wuhan strain (Accession# QHD43416.1, Val16–Arg685) and S protein RBD (Arg319–Phe541) were purchased from ACROBiosystems (Newark, DE, USA).

Techniques:

Journal: Viruses

Article Title: Clinical Utility of SARS-CoV-2 Antibody Titer Multiplied by Binding Avidity of Receptor-Binding Domain (RBD) in Monitoring Protective Immunity and Clinical Severity

doi: 10.3390/v15081662

Figure Lengend Snippet: Maturation of binding avidity with repeated vaccinations and determination of cutoff values of COVID-19 antibodies to avoid hospitalization. Correlation regression lines with 95% CI areas between anti-SARS-CoV-2 IgG titers (In BUg/mL) and ABAT In ((BUg/mL)/IC50 (nM)) after 1st ( A ), 2nd ( B ), and 3rd ( C ) vaccinations, and schematic diagram ( D ) showing the three areas of ABATs with different binding avidity maturation: high binding avidity ABAT area from ( C ), low binding avidity ABAT area from ( B ), and initial binding avidity ABAT area from ( A ). Dotted lines in ( C , D ) show cutoff values of S1-specific antibody titer and ABAT for predicting avoidance of hospitalization. Regions exceeding the above cutoff values are indicated by red box. Hollow circles, antibodies with high binding-avidity ABAT; Black solid circles, antibodies with low binding-avidity ABAT; Squares, antibodies with initial binding-avidity ABAT; Ab, antibody.

Article Snippet: HEK293 expressing recombinant His Tag SARS-CoV-2 S1 of the original Wuhan strain (Accession# QHD43416.1, Val16–Arg685) and S protein RBD (Arg319–Phe541) were purchased from ACROBiosystems (Newark, DE, USA).

Techniques: Binding Assay

Journal: Viruses

Article Title: Clinical Utility of SARS-CoV-2 Antibody Titer Multiplied by Binding Avidity of Receptor-Binding Domain (RBD) in Monitoring Protective Immunity and Clinical Severity

doi: 10.3390/v15081662

Figure Lengend Snippet: Correlation between anti-SARS-CoV-2 IgG antibodies with three different binding avidity maturations on admission and classification of severity of illness at 2–3 weeks after admission or at discharge. RBD binding avidity maturation of anti-SARS-CoV-2 IgG antibodies was measured and classified into three groups on admission, high binding avidity group ( n = 9), low binding avidity group ( n = 23), and initial binding avidity group ( n = 307) and classification of severity of illness at 2–3 weeks after admission or at discharge.

Article Snippet: HEK293 expressing recombinant His Tag SARS-CoV-2 S1 of the original Wuhan strain (Accession# QHD43416.1, Val16–Arg685) and S protein RBD (Arg319–Phe541) were purchased from ACROBiosystems (Newark, DE, USA).

Techniques: Binding Assay