Journal: bioRxiv

Article Title: Antibody-mediated cell entry of SARS-CoV-2

doi: 10.1101/2023.02.20.529249

Figure Lengend Snippet: ( A ) Binding site locations of selected monoclonal antibodies and ACE2 binding site. Surface regions of the SARS-CoV-2 spike trimer in a top view targeted by eight selected antibodies on the RBD and NTD are highlighted by ellipses. Various domains of one protomer are colored (RBM in magenta, the rest of RBD in red and NTD in blue); the other two protomers in white and gray, respectively. The ACE2 binding site is marked with a yellow dashed line. ( B ) Schematic representation of antibody IgG (heavy and light chains) and FcγRI (α and γ chains) constructs, as well as a diagram for how FcγRI captures an IgG antibody on the surface of membrane based on the crystal structure PDB ID: 4W4O . ( C ) HEK293T cells with or without expressing FcγRI were decorated with eight selected monoclonal antibodies and tested for membrane fusion with the full-length G614 S protein expressing cells in our standard cell-cell fusion assay. Cell-cell fusion led to reconstitution of α and μ fragments of β-galactosidase yielding an active enzyme and thus the fusion activity was quantified by a chemiluminescent assay. Cells only and cells expressing FcγRI with no antibody added were negative controls. ( D ) The cell-cell fusion assay was used to analyze seven polyclonal IgG antibodies purified from serum samples of vaccinated convalescent individuals reported previously .

Article Snippet: The SARS-CoV-2 G614 seed virus (the clinical isolate New York-PV09158/2020; ATCC #NR-53516) was obtained through BEI Resources (Manassas, VA) and amplified in TMPRSS2-E6 cells.

Techniques: Binding Assay, Bioprocessing, Construct, Membrane, Expressing, Cell-Cell Fusion Assay, Activity Assay, Purification

Journal: bioRxiv

Article Title: Antibody-mediated cell entry of SARS-CoV-2

doi: 10.1101/2023.02.20.529249

Figure Lengend Snippet: ( A ) Schematic representation of the full-length human ACE2 and design of antibody-based expression constructs. Various segments for ACE2 include: catalytic peptidase domain, neck domain; TM, transmembrane anchor; and CT, cytoplasmic tail. Expression constructs of antibody-ACE2 chimera, the Fab heavy chain of an antibody is fused with the neck domain, TM and CT of ACE2, coexpressed with the Fab light chain. A diagram showing how Fab is presented on the cell surfaces by the ACE2 TM anchor. ( B ) HEK293T cells transfected with eight different antibody-ACE2 chimeric constructs were tested for membrane fusion with the full-length S protein (G614 or Omicron subvariant BA.2) expressing cells in the β-galactosidase-based cell-cell fusion assay. The wildtype ACE2 was a positive control; no receptor/no S a negative control. ( C ) Infection of HEK293T cells transfected with either ACE2 or various antibody-ACE2 chimeric constructs by HIV-based pseudotyped viruses using the full-length G614 and BA.1 S constructs in a single cycle. Empty vector was used as a negative control. ( D ) Virus foci in MDCK cells transfected with either ACE2 or various antibody-ACE2 chimeric constructs followed by infection of the authentic SARS-CoV-2 G614 isolate. Empty vector was used as a negative control.

Article Snippet: The SARS-CoV-2 G614 seed virus (the clinical isolate New York-PV09158/2020; ATCC #NR-53516) was obtained through BEI Resources (Manassas, VA) and amplified in TMPRSS2-E6 cells.

Techniques: Expressing, Construct, Transfection, Membrane, Cell-Cell Fusion Assay, Positive Control, Negative Control, Infection, Plasmid Preparation, Virus

Journal: bioRxiv

Article Title: Antibody-mediated cell entry of SARS-CoV-2

doi: 10.1101/2023.02.20.529249

Figure Lengend Snippet: ( A ) Schematic representation of the membrane-bound B cell receptor (BCR) expression constructs and a diagram showing how Fab is presented on the cell surfaces by the BCR TM anchor. ( B ) HEK293T cells transfected with eight different membrane-bound BCR constructs were tested for membrane fusion with the full-length S protein (G614 or Omicron subvariant BA.2) expressing cells in the β-galactosidase-based cell-cell fusion assay. The wildtype ACE2 was a positive control and no receptor/no S a negative control. ( C ) Infection of HEK293T cells transfected with either ACE2 or various BCR constructs by HIV-based pseudotyped viruses using the full-length G614 and Omicron BA.1 S constructs in a single cycle. Empty vector was used as a negative control. ( D ) Virus foci in MDCK cells transfected with either ACE2 or various antibody-ACE2 chimeric constructs followed by infection of the authentic SARS-CoV-2 G614 isolate. Empty vector was used as a negative control.

Article Snippet: The SARS-CoV-2 G614 seed virus (the clinical isolate New York-PV09158/2020; ATCC #NR-53516) was obtained through BEI Resources (Manassas, VA) and amplified in TMPRSS2-E6 cells.

Techniques: Membrane, Expressing, Construct, Transfection, Cell-Cell Fusion Assay, Positive Control, Negative Control, Infection, Plasmid Preparation, Virus

Journal: bioRxiv

Article Title: Antibody-mediated cell entry of SARS-CoV-2

doi: 10.1101/2023.02.20.529249

Figure Lengend Snippet: ( A ) and ( B ) Inhibition of viral infectivity by protease inhibitors. Pseudovirus (G614 S) infection of HEK293T cells transfected with ACE2 or antibody constructs with/without TMPRSS2 were treated with either E-64d (a cathepsin L inhibitor) or Camostat (TMPRSS2 inhibitor). DMSO, organic solvent used to dissolve E-64d. ( C - E ) Antibody neutralization of pseudoviruses containing the G614 S protein was determined using IgG antibodies, C63C8, SP1-77, S2H97 and C63C7 in red; G32B6 and C12A2 in magenta; and C12C9 in blue.

Article Snippet: The SARS-CoV-2 G614 seed virus (the clinical isolate New York-PV09158/2020; ATCC #NR-53516) was obtained through BEI Resources (Manassas, VA) and amplified in TMPRSS2-E6 cells.

Techniques: Inhibition, Infection, Transfection, Construct, Solvent, Neutralization