Journal: Molecular Genetics and Metabolism

Article Title: In vitro activity of cysteamine against SARS-CoV-2 variants

doi: 10.1016/j.ymgme.2022.08.009

Figure Lengend Snippet: Binding of S1 protein to Vero-TMPRSS2 cells in the presence of cysteamine. (A) Binding of varying concentrations of S1 protein to the Vero-TMPRSS2 cells. The cells were incubated for 60 min at 37 °C with concentrations of S1 protein (Sino Biologicals) from 100 to 1000 ng/ml, followed by anti-S1-Alexa Fluor-488 antibody staining as described in Methods. Percent binding is the % of anti-S1+ cells after gating on singlets and live cells. The concentrations of 250 and 100 ng/ml were selected to perform the subsequent experiments based on the non-saturating level of S1 binding (54.5–85.7%). All titration experiments were replicated twice with similar outcome. (B) Scatter plots and (C) corresponding column graphs showing the effect of cysteamine on the binding of S1 protein to the Vero-TMPRSS2 cells. S1 protein (100 or 250 ng/ml) was preincubated with varying concentrations (0.31 to 10 mM) of cysteamine for 60 min at 37 °C. For control S 1 protein was incubated with FACS binding buffer without cysteamine. Cysteamine-treated or medium treated S1 protein solution was then transferred to a tube containing Vero-TMPRSS2 cells and the binding assay was performed as described in the Methods using anti-S1-Alexa Fluor-488 antibody staining. Percent binding represents the % of anti-S1+ cells after gating on singlets and live cells. All experiments were repeated twice with comparable outcome. (D) Scatter plots evaluated the effect of cysteamine on binding of S1 (250 ng/ml) using above condition. The gating strategy selected for the analysis was done using specific unstained cells treated with medium (control) or with 5 and 10 mM cysteamine-HCL for 60 min for the respective group.

Article Snippet: The cells were incubated for 60 min at 37 °C with concentrations of S1 protein (Sino Biologicals) from 100 to 1000 ng/ml, followed by anti-S1-Alexa Fluor-488 antibody staining as described in Methods.

Techniques: Binding Assay, Incubation, Staining, Titration

Journal: Molecular Genetics and Metabolism

Article Title: In vitro activity of cysteamine against SARS-CoV-2 variants

doi: 10.1016/j.ymgme.2022.08.009

Figure Lengend Snippet: Binding of virus to the Vero-TMPRSS2 in the presence of cysteamine-HCl. (A) Scatter plots obtained for the binding of S1 protein following infection with varying MOI of infection both in the absence or in the presence of 5 mM cysteamine HCl. Varying infectious units of SARS-CoV2 (Wild type SARS-CoV-2 (P4) isolate USA-WA1/2020) were pre-incubated with medium alone or with 5 mM cysteamine-HCl for 90 min at 37 °C in 5% CO2 and the mixture was incubated with Vero-TMPRSS2 cells for an additional 60 min at 37 °C to initiate viral infection. The level of bound S1 protein was then assayed by flow cytometry after staining with Alexa Four-488 conjugated anti-S1 antibody. Percent binding represents the % of anti-S1+ cells after gating on singlets and live cells. (B) Dose dependent inhibition of the virus binding to Vero-TMPRSS2 cells in the presence of cysteamine. Values were normalized with the background fluorescence obtained from stained uninfected cells under appropriate conditions.

Article Snippet: The cells were incubated for 60 min at 37 °C with concentrations of S1 protein (Sino Biologicals) from 100 to 1000 ng/ml, followed by anti-S1-Alexa Fluor-488 antibody staining as described in Methods.

Techniques: Binding Assay, Infection, Incubation, Flow Cytometry, Staining, Inhibition, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

doi: 10.2147/IJN.S427990

Figure Lengend Snippet: Panning of specific nanobodies against the SARS-CoV-2 S1 protein by naïve nanobody library. ( a ) Schematic diagram of nanobody screening. ( b ) The anti-SARS-CoV-2 spike S1 nanobodies were enriched 149-fold after three rounds of screening. ( c ) Phage ELISA was used to identify an anti-SARS-CoV-2 S1 individual clone after three rounds of bio-panning. ( d ) SARS-CoV-2 S1 specific nanobodies were identified from the top 20 clones that were specifically bound to SARS-CoV-2 S1. ( e ) Alignment of the amino acid sequence of three screened SARS-CoV-2 S1 nanobodies.

Article Snippet: To measure the affinity of Nb-H6 binding to spike proteins of SARS-CoV-2 variants (Alpha B.1.1.7, Delta B.1.617.2, Lambda C.37 (East Mab, China, 40591-V08H31 Sino Biological), Omicron BA.2 (40891-V08H43 Sino Biological, EVV00324 Antibody System), and Omicron BA.5 (40592-V08H130 Sino Biological), Biolayer Interferometry (BLI) assay was performed using the BLItz TM Label-free Protein Analysis System (ForteBio, USA) with High Precision Streptavidin (SAX) Biosensors (18–5117, Sartorius).

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Sequencing

Journal: International Journal of Nanomedicine

Article Title: A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

doi: 10.2147/IJN.S427990

Figure Lengend Snippet: Purification and identification of the SARS-CoV-2 S1-specific nanobody. The top three nanobodies with the highest affinity for SARS-CoV-2 S1 were identified and named Nb-H6, Nb-G7, and Nb-B7. ( a ) SDS-PAGE analysis of purified nanobodies Nb-H6, Nb-G7, and Nb-B7. (M) Marker. The molecular weight (kDa) is indicated on the left-hand side. ( b ) The affinity of Nb-H6, Nb-G7, and Nb-B7 to SARS-CoV-2 S1 (left panel) and SARS-CoV-2 RBD (right panel) at various concentrations (200, 50, 12.5, 3.12, 0.78, 0.19, 0.049, and 0.012 nM). The results are presented as mean absorbance values at OD 450 nm ± SD (n = 3).

Article Snippet: To measure the affinity of Nb-H6 binding to spike proteins of SARS-CoV-2 variants (Alpha B.1.1.7, Delta B.1.617.2, Lambda C.37 (East Mab, China, 40591-V08H31 Sino Biological), Omicron BA.2 (40891-V08H43 Sino Biological, EVV00324 Antibody System), and Omicron BA.5 (40592-V08H130 Sino Biological), Biolayer Interferometry (BLI) assay was performed using the BLItz TM Label-free Protein Analysis System (ForteBio, USA) with High Precision Streptavidin (SAX) Biosensors (18–5117, Sartorius).

Techniques: Purification, SDS Page, Marker, Molecular Weight

Journal: International Journal of Nanomedicine

Article Title: A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

doi: 10.2147/IJN.S427990

Figure Lengend Snippet: Characterization of the SARS-CoV-2 nanobody Nb-H6. ( a ) The binding kinetics of Nb-H6 with the immobilized SARS-CoV-2 S1 and RBD were measured using biolayer interferometry (BLI). ( b ) Competitive binding assays by ELISA. SARS-CoV-2 S1 (left panel) and RBD (right panel) bind to ACE2 after competitive blocking with serially diluted Nb-H6. IC 50 values were calculated by nonlinear regression fitting to a variable slope, a four-parameter dose-response model. Data are presented as mean ± SD of three technical replicates. ( c ) Table summarizing the affinity K D , association (K on ), and dissociation constants (K off ) determined by BLI as well as the competition assay IC 50 values for Nb-H6.

Article Snippet: To measure the affinity of Nb-H6 binding to spike proteins of SARS-CoV-2 variants (Alpha B.1.1.7, Delta B.1.617.2, Lambda C.37 (East Mab, China, 40591-V08H31 Sino Biological), Omicron BA.2 (40891-V08H43 Sino Biological, EVV00324 Antibody System), and Omicron BA.5 (40592-V08H130 Sino Biological), Biolayer Interferometry (BLI) assay was performed using the BLItz TM Label-free Protein Analysis System (ForteBio, USA) with High Precision Streptavidin (SAX) Biosensors (18–5117, Sartorius).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Competitive Binding Assay

Journal: International Journal of Nanomedicine

Article Title: A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

doi: 10.2147/IJN.S427990

Figure Lengend Snippet: Evaluation of the thermal and pH stability of Nb-H6. Nb-H6 was incubated at different temperatures or pH levels, and the affinity of Nb-H6 binding to SARS-CoV-2 S1 was determined by ELISA. ( a ) Nb-H6 was incubated at 4, 30, 60, and 90 °C for 5 minutes; ( b ) Nb-H6 was incubated at 90 °C for 5, 15, 30, 45, 60, 75, 90, and 105 minutes; and ( c ) Nb-H6 was incubated at different pHs for 2 hours and the binding capacity was measured using ELISA. Data are shown as mean ± SD, n=3.

Article Snippet: To measure the affinity of Nb-H6 binding to spike proteins of SARS-CoV-2 variants (Alpha B.1.1.7, Delta B.1.617.2, Lambda C.37 (East Mab, China, 40591-V08H31 Sino Biological), Omicron BA.2 (40891-V08H43 Sino Biological, EVV00324 Antibody System), and Omicron BA.5 (40592-V08H130 Sino Biological), Biolayer Interferometry (BLI) assay was performed using the BLItz TM Label-free Protein Analysis System (ForteBio, USA) with High Precision Streptavidin (SAX) Biosensors (18–5117, Sartorius).

Techniques: Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

doi: 10.2147/IJN.S427990

Figure Lengend Snippet: Nb-H6 suppresses SARS-CoV-2 infection in vitro. The SARS-CoV-2 spike pseudotyped GFP-luciferase lentivirus and authentic SARS-CoV-2 were mixed with serially diluted Nb-H6 and then infected host cells (HEK293-hACE2 and Vero E6, respectively) for infection. ( a ) Green fluorescence images were captured to visualize Nb-H6 neutralization against the SARS-CoV-2 pseudovirus (left panel), and the luciferase reaction assay was used to determine the IC 50 value (right panel). Data are shown as mean ± SD (n = 3). ( b ) The authentic virus neutralization assay was performed to verify the anti-authentic SARS-CoV-2 ability of Nb-H6. Anti-SARS-CoV-2 N protein antibody and HRP-linked secondary antibody (left panel) were used to label the infected cells (left panel). For the calculation of the IC 50 , the foci were counted by the focus formation assay (FFA) (right panel). The scale bar in each image represents 200 μm. Data are shown as mean ± SD (n = 6). All IC 50 values were calculated using a four-parameter logistic curve.

Article Snippet: To measure the affinity of Nb-H6 binding to spike proteins of SARS-CoV-2 variants (Alpha B.1.1.7, Delta B.1.617.2, Lambda C.37 (East Mab, China, 40591-V08H31 Sino Biological), Omicron BA.2 (40891-V08H43 Sino Biological, EVV00324 Antibody System), and Omicron BA.5 (40592-V08H130 Sino Biological), Biolayer Interferometry (BLI) assay was performed using the BLItz TM Label-free Protein Analysis System (ForteBio, USA) with High Precision Streptavidin (SAX) Biosensors (18–5117, Sartorius).

Techniques: Infection, In Vitro, Luciferase, Fluorescence, Neutralization, Virus, Tube Formation Assay

Journal: International Journal of Nanomedicine

Article Title: A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

doi: 10.2147/IJN.S427990

Figure Lengend Snippet: The binding activity of Nb-H6 with SARS-CoV-2 variants S1 and RBD. ( a ) The binding kinetics of Nb-H6 with the immobilized S1 of variants Alpha (B.1.1.7), Delta (B.1.617.2), Lambda (C.37), and Omicron (BA.2) were detected by BLI. ( b ) The binding capacity of Nb-H6 with RBD of variants Lambda (C.37), Delta (B.1.617.2), and Omicron (BA.2 and BA.5) was measured by BLI. (c) Table summarizing the affinity K D , association (K on ), and dissociation constants (K off ) determined by BLI.

Article Snippet: To measure the affinity of Nb-H6 binding to spike proteins of SARS-CoV-2 variants (Alpha B.1.1.7, Delta B.1.617.2, Lambda C.37 (East Mab, China, 40591-V08H31 Sino Biological), Omicron BA.2 (40891-V08H43 Sino Biological, EVV00324 Antibody System), and Omicron BA.5 (40592-V08H130 Sino Biological), Biolayer Interferometry (BLI) assay was performed using the BLItz TM Label-free Protein Analysis System (ForteBio, USA) with High Precision Streptavidin (SAX) Biosensors (18–5117, Sartorius).

Techniques: Binding Assay, Activity Assay

Journal: International Journal of Nanomedicine

Article Title: A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

doi: 10.2147/IJN.S427990

Figure Lengend Snippet: Nb-H6 inhibits SARS-CoV-2 variant pseudovirus infection. The SARS-CoV-2 variant spike pseudotyped GFP-luciferase lentivirus was incubated with different concentrations of Nb-H6 for 1 hour and subsequently infected HEK293-hACE2 cells for 6 hours to allow virus entry. Representative fluorescence images and dose-response curve for Nb-H6 neutralization of SARS-CoV-2 variants Alpha B.1.1.7 ( a ), Delta B.1.617.2 ( b ), Omicron BA.2 ( c ), and Omicron (BA.5) ( d ) pseudovirus at 48 hours post-infection. The scale bar in each image represents 200 μm. The IC 50 was calculated using a four-parameter logistic curve. Data are shown as mean ± SD (n =3).

Article Snippet: To measure the affinity of Nb-H6 binding to spike proteins of SARS-CoV-2 variants (Alpha B.1.1.7, Delta B.1.617.2, Lambda C.37 (East Mab, China, 40591-V08H31 Sino Biological), Omicron BA.2 (40891-V08H43 Sino Biological, EVV00324 Antibody System), and Omicron BA.5 (40592-V08H130 Sino Biological), Biolayer Interferometry (BLI) assay was performed using the BLItz TM Label-free Protein Analysis System (ForteBio, USA) with High Precision Streptavidin (SAX) Biosensors (18–5117, Sartorius).

Techniques: Variant Assay, Infection, Luciferase, Incubation, Virus, Fluorescence, Neutralization

Journal: International Journal of Nanomedicine

Article Title: A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

doi: 10.2147/IJN.S427990

Figure Lengend Snippet: The amino acid residues Asn72 and Arg97 of Nb-H6 are critical for binding to SARS-CoV-2 RBD. ( a ) Predicted amino acids involved in the binding interactions between SARS-CoV-2 spike RBD (gray) and Nb-H6 (Orange). The Nb-H6 amino acid residues Asn72 and Arg97 are shown to form hydrogen bonds with the SARS-CoV-2 residues Tyr449 and Tyr489, respectively. (b) Amino acid sequence alignment of RBM (blue box) of wild-type and variant RBD. The conserved sites Y449 and Y489 were highlighted with a red box. ( c ) Affinity of Nb-H6 N72A or Nb-H6 R97A binding to SARS-CoV-2 S1 and RBD at various concentrations (800, 266.67, 88.89, 29.63, 9.88, 3.29, 1.10, and 0.37 nM), with Nb-H6 as a positive control. The results are presented as mean absorbance values at OD 450 nm ± SD (n = 3). ( d ) Biolayer interferometry (BLI) was used to measure the binding kinetics of Nb-H6, Nb-H6 N72A, and Nb-H6 R97A with the immobilized SARS-CoV-2 spike S1 and RBD. ( e ) Competitive binding assays by ELISA. SARS-CoV-2 S1 (left panel) and RBD (right panel) bind to ACE2 after competitive blocking with serially diluted Nb-H6, Nb-H6 N72A, and Nb-H6 R97A. ( f ) The affinity of Nb-H6 binding to RBD mutants Y449A and Y489A was assessed by ELISA (left penal) and Biolayer interferometry (right penal). Nb-H6 was used at concentrations (400, 100, 25, 6.25, 1.56, 0.39, 0.10, and 0.02 nM) in ELISA, and results are presented as mean absorbance values at OD 450 nm ± SD (n = 3). Data are presented as mean ± SD of three technical replicates.

Article Snippet: To measure the affinity of Nb-H6 binding to spike proteins of SARS-CoV-2 variants (Alpha B.1.1.7, Delta B.1.617.2, Lambda C.37 (East Mab, China, 40591-V08H31 Sino Biological), Omicron BA.2 (40891-V08H43 Sino Biological, EVV00324 Antibody System), and Omicron BA.5 (40592-V08H130 Sino Biological), Biolayer Interferometry (BLI) assay was performed using the BLItz TM Label-free Protein Analysis System (ForteBio, USA) with High Precision Streptavidin (SAX) Biosensors (18–5117, Sartorius).

Techniques: Binding Assay, Sequencing, Variant Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Blocking Assay