Journal: Acta Pharmaceutica Sinica. B

Article Title: Immunogenicity of mucosal COVID-19 vaccine candidates based on the highly attenuated vesicular stomatitis virus vector (VSV MT ) in golden syrian hamster

doi: 10.1016/j.apsb.2023.08.023

Figure Lengend Snippet: Characterization of the optimal cytoplasmic tail of SARS-CoV-2 spike protein for construction of retargeting VSV MT chimera virus. Single-cycle VSV S were pseudotyped with S mutants with different cytoplasmic tail lengths and were compared for their ability to mediate infection of ACE2-293T. (A) The spike protein conformations of WA1, Delta, and Omicron Variants. (B) SARS-CoV-2 S protein mutants with different cytoplasmic tail (CT) lengths. S FL , S Δ26 , S Δ25 , S Δ24 , S Δ23 , S Δ22 , S Δ21 , S Δ20 , and S Δ19 represent the full-length S protein or the S mutants with CTs truncated from 26 to 19 amino acids, respectively. The remaining CT regions are shown in red. SS, signal peptide sequence; ET, ectodomain; TM, transmembrane domain; CT, cytoplasmic tail. (C) Surface expression of S protein, S deletion mutants. At 24 h post-transfection, cells were subjected to a flow cytometry assay. (D) Intact antigenicity of the S Δ21 encoded by VSV vector. Binding activities of replication-defective VSV ΔG -GFP pseudotyped with S Δ21 were detected with neutralizing mAbs recognizing S protein, with the 2G1 against WA1 strain, REGN10987 or 8G3 recognizing Delta or Omicron variants, respectively. IC 50 of the above mAbs against different VSV pseudotypes were calculated. Data are shown as mean ± SD with three replicates. ns means P > 0.05. Statistical significance was determined using two-way ANOVA with multiple comparisons.

Article Snippet: Cells were pulsed with a 2 μg/mL peptide pool of SARS-CoV-2 spike protein PP003 (SinoBiological) at 37 °C in the CO 2 incubator.

Techniques: Virus, Infection, Sequencing, Expressing, Transfection, Flow Cytometry, Plasmid Preparation, Binding Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Immunogenicity of mucosal COVID-19 vaccine candidates based on the highly attenuated vesicular stomatitis virus vector (VSV MT ) in golden syrian hamster

doi: 10.1016/j.apsb.2023.08.023

Figure Lengend Snippet: Generation of retargeting VSV MT encoding S protein of various strains. (A) Diagram of retargeting VSV MT construct. Wild-type VSV has no S gene insertion; rVSV MT -S Δ21 encodes for SARS-CoV-2 S mutants of various strains with their genome. The parental VSV MT genome encodes the nucleoprotein (N), phosphoprotein (P), glycoprotein (G), RNA polymerase (L), and mutant M protein (MT) that harbors three mutations (ΔM51, V221F, and S226R). The gene for the truncated S protein of WA1, variants of Delta or Omicron (S ΔCT21 ) were used to replace the G gene of the VSV through Mlu I and XhoI enzyme digestion, eGFP gene was cloned into the sites with XhoI and NheI enzyme digestion. (B, C) Western blotting of rescued rVSV MT -S Δ21 chimera viruses. (B) S protein was detected using a specific antibody; (C) with VSV convalescent sera. The viruses were passaged in VeroE6 cells. Lanes 1, 2, and 3 represented collected cells infected with VSV MT -WA1 of passages 1, 5, and 10, respectively; Lanes 4, 5, and 6 represented those infected with VSV MT -Delta of passages 1, 5, 10; lines 7, 8, 9 represented those infected with VSV MT -Omicron of passages 1, 5, 10; line 10 represented VSV-GFP control. (D) Selective infection of hACE2-positive cell lines. hACE2-BHK21 and BHK21 cells were infected with rVSV MT -S Δ21 chimera viruses or VSV MT -GFP at MOI of 1. Twenty-four hours post-infection, GFP expression of infected cells was photographed by a fluorescence microscope. (E) Infectivity of different cell lines with rVSV MT -S Δ21 chimera viruses. Mean Fluorescent Intensity (MFI) was calculated. (F) Syncytia formation of rVSV MT -S Δ21 chimera viruses in different host cells. Virus-induced cytopathic effects are shown in the photomicrographs above. Confluent hACE-293T and VeroE6 cell monolayers were infected with VSV MT -WA1, VSV MT -Delta, and VSV MT -Omicron at an MOI of 1. Photographs were taken 24 h after infection. Syncytia mediated by the viruses were shown. (G) One step growth curve of rVSV MT -S Δ21 chimera viruses. VeroE6 cells were infected with VSV MT -WA1, VSV MT -Delta, and VSV MT -Omicron at MOI of 3. Plaque titrations from the indicated time points were performed with VeroE6 cells. (H) Plaque size formed by recombinant VSVs in VeroE6 cells. Plaques in Vero cells infected with VSVs. Ten individual plaques were selected randomly to calculate the areas. Images were acquired 24 h post-infection (h.p.i). (I) The average syncytium area (μm 2 ) was evaluated by Image J. ∗∗∗∗ P < 0.0001. Statistical significance was determined using two-way ANOVA with multiple comparisons. Above data are shown as means ± SD ( n = 3).

Article Snippet: Cells were pulsed with a 2 μg/mL peptide pool of SARS-CoV-2 spike protein PP003 (SinoBiological) at 37 °C in the CO 2 incubator.

Techniques: Construct, Mutagenesis, Clone Assay, Western Blot, Infection, Expressing, Fluorescence, Microscopy, Virus, Recombinant

Journal: Acta Pharmaceutica Sinica. B

Article Title: Immunogenicity of mucosal COVID-19 vaccine candidates based on the highly attenuated vesicular stomatitis virus vector (VSV MT ) in golden syrian hamster

doi: 10.1016/j.apsb.2023.08.023

Figure Lengend Snippet: Cross-reactivity among sera antibodies collected from animals vaccinated with VSV MT encoding various S proteins of various SARS-CoV-2 strains. Sera were collected from VSV MT -WA1 (A), VSV MT -Delta (B) or VSV MT -Omicron (C) administrated animals as above. Their neutralization titers against VSV MT -WA1, VSV MT -Delta, or VSV MT -Omicron were determined. The titers were expressed as the reciprocal of the highest dilution of antibody giving a 100% inhibition of cytopathic effect. The plots show the geometric mean with geometric standard deviation ( n = 12).

Article Snippet: Cells were pulsed with a 2 μg/mL peptide pool of SARS-CoV-2 spike protein PP003 (SinoBiological) at 37 °C in the CO 2 incubator.

Techniques: Neutralization, Inhibition, Standard Deviation

Journal: Phytomedicine

Article Title: Corilagin prevents SARS-CoV-2 infection by targeting RBD-ACE2 binding

doi: 10.1016/j.phymed.2021.153591

Figure Lengend Snippet: Computational docking prediction of corilagin. A) Molecular docking result showed the best binding pose of interaction, and B) the residues involved and types of interaction of corilagin with the receptor binding domain of the SARS-CoV-2 spike protein. C) Root mean square deviation (RMSD) plot of the SARS-CoV-2 spike protein receptor binding domain (RBD) backbone alone and in complex with corilagin during the 10ns molecular dynamics simulation.

Article Snippet: Purified SARS-CoV-2 RBD peptide (Sino Biological) were conjugated with biotin using EZ-Link™ Sulfo-NHS-Biotin (Genemore, Shanghai, China) according to the manufacturer's protocol.

Techniques: Binding Assay

Journal: Phytomedicine

Article Title: Corilagin prevents SARS-CoV-2 infection by targeting RBD-ACE2 binding

doi: 10.1016/j.phymed.2021.153591

Figure Lengend Snippet: Effect of corilagin on the interaction of SARS-CoV-2 spike RBD peptide and hACE2 receptor. A) The binding kinetics and steady-state analysis of the interaction between SARS-CoV-2 RBD protein and corilagin was monitored by biolayer interferometry (BLI). B) BLI was used to detect the binding association of hACE2 and corilagin at indicated concentrations. Representative results were shown from 3 independent experiments.

Article Snippet: Purified SARS-CoV-2 RBD peptide (Sino Biological) were conjugated with biotin using EZ-Link™ Sulfo-NHS-Biotin (Genemore, Shanghai, China) according to the manufacturer's protocol.

Techniques: Binding Assay

Journal: Phytomedicine

Article Title: Corilagin prevents SARS-CoV-2 infection by targeting RBD-ACE2 binding

doi: 10.1016/j.phymed.2021.153591

Figure Lengend Snippet: Corilagin blocks the binding of Spike-RBD peptide on hACE2 receptor. ELISA assay was used to examine the interference of corilagin in its binding to the SARS-CoV-2 RBD protein and hACE2 receptor. Data were expressed as mean ± S.D., representative results were evaluated from 3 independent experiments; * p < 0.05, one-way ANOVA analysis.

Article Snippet: Purified SARS-CoV-2 RBD peptide (Sino Biological) were conjugated with biotin using EZ-Link™ Sulfo-NHS-Biotin (Genemore, Shanghai, China) according to the manufacturer's protocol.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Phytomedicine

Article Title: Corilagin prevents SARS-CoV-2 infection by targeting RBD-ACE2 binding

doi: 10.1016/j.phymed.2021.153591

Figure Lengend Snippet: Corilagin suppresses the binding of Spike-RBD on ACE2 receptor in HEK293 cells. A) HEK293 cells were transfected with hACE2-EGFP (green). The transfected HEK293 cells were incubated with mFc-tagged SARS-CoV-2-RBD protein with or without corilagin (25–100 μM). Mouse IgG Fc TRITC antibody (red) was used to visualize the binding of SARS-CoV-2-RBD proteins on cell surface. All images were captured by confocal microscopy using a Leica SP8 (× 40 oil immersion objective lens). B) Images of Spike-RBD-ACE2 binding intensity were quantified by Image J. Data were expressed as mean ± S.D., n = 3; * p < 0.05, one-way ANOVA analysis.

Article Snippet: Purified SARS-CoV-2 RBD peptide (Sino Biological) were conjugated with biotin using EZ-Link™ Sulfo-NHS-Biotin (Genemore, Shanghai, China) according to the manufacturer's protocol.

Techniques: Binding Assay, Transfection, Incubation, Confocal Microscopy

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A, VH, VK and VL gene usage of antibodies from plasmablasts and memory B cells (MBCs). Up to the top four genes in each chart are shown with different colors (genes that were tied for 4 th and lower are not highlighted). B , Tukey’s plots showing heavy and light chain gene mutations of antibodies from plasmablasts and MBCs. Percent mutations were compared with the Mann-Whitney U-test. The middle line shows the median, and the box extends from the 1 st to 3 rd quartile. C , Top 21 neutralizing antibodies (IC50 <1 μg/mL) by antigen specificity (left), and neutralization curves of selected antibodies (right). D , SARS-CoV-2 neutralization potency versus heavy chain mutation levels of antibody panel. The 5 most potent antibodies are shown as solid circles. E , Neutralization potency of antibodies by cell type. Top values indicate percentages of non-neutralizing antibodies. Horizontal bars indicate mean values; Mann-Whitney U-test (non-neutralizing antibodies excluded from calculation). The 5 most potent antibodies are shown in color. F , SARS-CoV-2 neutralization curves of benchmark antibodies from different groups , , . G, Neutralization IC50 values of antibodies in our panel and benchmark antibodies in three different neutralization assays. Authentic SARS-CoV-2 FRNA values are from a single experiment done in quadruplicate, authentic SARS-CoV-2 (Scripps) values are an average of two experiments done in duplicate, pseudovirus (MLV) values are an average of 3 experiments in duplicate. The colors indicate the different sources of the antibodies: red, this study; blue, ref. 21; yellow, ref. 11; green, ref. 18.

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: MANN-WHITNEY, Neutralization, Mutagenesis

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , Isoaffinity plot of antibodies targeting SARS-CoV-2 RBD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. B , Neutralization potency versus affinity of anti-RBD antibodies. C , Isoaffinity plot of antibodies targeting SARS-CoV-2 NTD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. D , Neutralization potency versus affinity of anti-NTD antibodies. E , Epitope binning of anti-RBD antibodies (representative of n = 2 experiments). ACE2 was only used as an analyte (competitor) and not as a ligand, while all other antibodies were tested as both ligands and analytes. Solid lines indicate two-way competition while dotted lines indicate one-way competition. The number and percentage of neutralizing antibodies (IC50 < 10 µg/mL) in each bin are shown. F , Epitope bins represented by C135 (yellow bin), S309 (purple bin), ACE2 (red bin), CR3022 (cyan bin), as well as the NTD-specific antibody 4-8 (orange) modeled onto a SARS-CoV- 2 spike protein (white cartoon). Antibody 4-8 was not binned successfully in our experiments but binds to a similar region to 2-17 and 5-24 (ref. 5), which were binned. The epitope sites are color-coded the same as in and . N-glycans at the N343 glycan site are represented by sticks. G , Epitope binning of anti-NTD antibodies (representative of n = 2 experiments). All antibodies were tested as both ligands and analytes. Solid lines indicate two-way competition while dotted lines indicate one- way competition. The number and percentage of neutralizing antibodies (IC50 < 10 µg/mL) in each bin are shown. H , Binding of mAb panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). I , Neutralization potency of CV503, CV664 and CV993 against B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets, and numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type.

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: Neutralization, Binding Assay

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , CV503 binds to the ridge region of SARS-CoV-2 RBD. The heavy and light chains of CV503 are shown in orange and yellow, respectively. SARS-CoV-2 RBD is in white, where its ridge region (residues 471–491) is shown in blue. B, The ACE2/RBD complex structure (PDB ID: 6M0J) is superimposed on the CV503/RBD complex. The heavy chain of CV503 (orange) would clash with ACE2 (green) if bound to RBD simultaneously (indicated by red circle). C-D , Epitope of CV503. Epitope residues contacting the heavy chain are in dark blue and light chain are in light blue, while residues contacting both heavy and light chains are in ocean blue. In C , CDR loops that are directly involved in RBD-binding are labeled. In D , epitope residues are labeled. Epitope residues that are also involved in ACE2 binding are labeled in red. E, ACE2-binding site on the RBD are in light pink. ACE2 is represented as semi- transparent cartoon in pale green. Epitope residues and ACE2-interacting residues are defined as those with a buried surface area (BSA) > 0 Å 2 . F , F486 at the ridge region of SARS-CoV-2 RBD (blue) is clamped in a hydrophobic pocket formed by the heavy (orange) and light chains (yellow) of CV503.

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: Binding Assay, Labeling

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , Scheme of DVD-Ig TM . In our bispecific antibody naming system, the first name refers to the antibody used to make the outer binding site and the second refers to the antibody at the inner binding site. GS or EL refers to the type of linker connecting the two antigen-binding sites. See Materials and Methods for details. B , Binding of individual and bispecific antibodies to various domains from SARS-CoV-1 and SARS-CoV-2 (representative of n = 2 experiments). Area under the curve (AUC) values are shown after subtraction with the negative control antigen. C , Neutralization potency of bispecific antibodies with SARS-CoV-2 authentic and pseudotyped virus (MLV). Values are averaged from two experiments done in duplicate. D , Neutralization curves of CV1206_521_GS with SARS-CoV-2 authentic and pseudotyped virus. Curves are from a representative experiment, IC50 values for authentic virus are the average from two experiments and those for the pseudovirus are from an average of two (bispecific) or three (regular antibody) experiments. E, Neutralization potency of CV1206_521_GS versus a cocktail of CV1206 and CV521, with concentrations shown in the molar scale to enable a fair comparison. For the antibody combination, the values on the x-axis refers to the concentration of each antibody in the cocktail, e.g. 10 nM refers to 10 nM of CV1206 + 10 nM of CV521. F , 3D reconstruction of CV1206_521_GS from nsEM images. Two “one RBD up” models (PDB 6VYB) in green are docked into the reconstruction. Similarly, multiple mock scFv’s in orange and purple were docked to approximate the DVD-Ig molecule. O, outer binding site; I, inner binding site. G , Binding of bispecific antibody panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). H , Neutralization potency of bispecific antibodies against D614G, B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets: numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type. ND, not determined. I, Potency of CV503_664_EL versus individual component mAbs against wild-type and B.1.351 SARS-CoV-2 pseudotyped virus (lentivirus).

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: Binding Assay, Negative Control, Neutralization, Concentration Assay