Journal: bioRxiv

Article Title: The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN

doi: 10.1101/2020.11.05.369264

Figure Lengend Snippet: a, Cells transfected with ACE2, L-SIGN, DC-SIGN, and CD147 (red line) or mock (shaded gray) were stained with SCoV2-NTD-Fc and RBD-Fc fusion protein (10 μg/mL). Each transfectant was also stained with a specific monoclonal antibody. b, Effect of mannan and anti-CD209 (anti-DC-SIGN) antibody on SCoV2-NTD-Fc binding to DC- and L-SIGN transfectants or mock (shaded gray). The transfectants were preincubated with mannan (light blue line) or anti-CD209 antibody (dark blue), followed by the staining with NTD-Fc fusion protein. c, Staining of DC- and L-SIGN transfectants (red line) or mock (shaded gray) with SCoV2-NTD-Fc and SCoV-NTD-Fc fusion proteins (10 μg/mL). d, Cells transfected with flag-tagged spike proteins of SCoV2, SCoV, human coronavirus OC43, or HKU1 (red line) or mock (shaded gray) were stained with DC-, L-SIGN-Fc fusion proteins or anti-Flag-tag antibody. Proportions of the stained cells are shown. Data are representative of three independent experiments.

Article Snippet: Mouse anti-CD209 mAb (clone 9E9A8), mouse IgG2a isotype control antibody (BioLegend, San Diego, CA, USA), mouse anti-L-SIGN mAb (clone 19F7), mouse anti-MHCII (clone WR18) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-human ACE2 mAb (R&D Systems, Minneapolis, MN, USA), rat anti-Flag mAb (Sigma-Aldrich, St Louis, MI, USA), mouse anti-human CD14-APC mAb (eBioscience, San Diego, CA, USA), Donkey anti-mouse IgG-APC mAb, anti-human IgG-Fc fragment specific-APC mAb, anti-rat IgG-APC mAb (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Transfection, Staining, Binding Assay, FLAG-tag

Journal: bioRxiv

Article Title: The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN

doi: 10.1101/2020.11.05.369264

Figure Lengend Snippet: a, SCoV2-PV infection on DC-SIGN, L-SIGN, mock, or ACE2 transfectants and Vero E6 cells. SCoV2-PV carrying a luciferase gene was used for the infection and luciferase activity was measured 24 h later. Asterisks indicate statistical significance derived from unpaired T-test; * P = 0.0018; ** P = 0.0003 b, DC-SIGN or L-SIGN transfectants were infected with recombinant SCoV2 with the NanoBiT luciferase gene. The luciferase activity was measured 24 h later. Asterisks indicate statistical significance derived from unpaired T-test; * P = 0.001; ** P = 0.0006. c, Cell-cell fusion assay of SCoV2 spike transfectants and DC- or L-SIGN transfectants. The effector cells expressing spike and T7 polymerase were cocultured with target cells expressing DC-SIGN, L-SIGN, mock, and T7 promoter-driven luciferase. Luciferase activities were measured after 24 h. Asterisks indicate statistical significance derived from unpaired T-test *** P <0.0001. d, Cell-cell fusion assay of SCoV2 spike transfectants (red) and DC-SIGN transfectants (green). Representative images are shown. Scale bar represents 50 μm in length. Data are representative of three independent experiments.

Article Snippet: Mouse anti-CD209 mAb (clone 9E9A8), mouse IgG2a isotype control antibody (BioLegend, San Diego, CA, USA), mouse anti-L-SIGN mAb (clone 19F7), mouse anti-MHCII (clone WR18) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-human ACE2 mAb (R&D Systems, Minneapolis, MN, USA), rat anti-Flag mAb (Sigma-Aldrich, St Louis, MI, USA), mouse anti-human CD14-APC mAb (eBioscience, San Diego, CA, USA), Donkey anti-mouse IgG-APC mAb, anti-human IgG-Fc fragment specific-APC mAb, anti-rat IgG-APC mAb (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Infection, Luciferase, Activity Assay, Derivative Assay, Recombinant, Cell-Cell Fusion Assay, Expressing

Journal: Immunology and Cell Biology

Article Title: The vaccinia‐based Sementis Copenhagen Vector coronavirus disease 2019 vaccine induces broad and durable cellular and humoral immune responses

doi: 10.1111/imcb.12539

Figure Lengend Snippet: Spike‐specific antibody and SARS‐CoV‐2 neutralization responses following SCV‐S vaccination. (a) S1 and S2 subunit endpoint IgM and IgG ELISA titers determined from serum of female C57BL/6J mice ( n = 3) at the indicated times after a single vaccination with 10 7 PFU of SCV‐S, with (b) ratio of S1‐ and S2‐specific IgG2c to IgG1 endpoint ELISA titers determined 28 days after vaccination. (c) S1 IgG ELISA binding titers (left panel) and cPass neutralization titers (right panel) in outbred ARC(s) and inbred C57BL/6J female mice ( n = 5) 21 days after a single vaccination with 10 7 PFU of SCV‐S or vector control, with (d) durability of response in the outbred cohort shown by S1‐specific endpoint IgG ELISA titers (left panel) and neutralization titers (right panel) at the indicated times. (e) S1‐specific IgG ELISA (left panel) and neutralization titers (right panel) 50 days after a single‐dose (day 0) or prime‐boost (day 0 and 28) vaccination of female C57BL/6J mice ( n = 5) with 10 7 PFU SCV‐S or control vector, with (f) neutralizing activity in ACE2 and RBD blocking ELISA, and (g) neutralizing activity (IC 80 titers) against lenti‐SARS‐CoV‐2‐S pseudoviruses bearing spike protein from the Wuhan reference strain, the alpha or beta variant, shown for prime‐boost samples. (h) Neutralization titers in young (6–8 weeks old; n = 5) and middle‐aged (9–10 months old; n = 10) C57BL/6J mice at the indicated times after prime‐boost vaccination with 10 7 PFU SCV‐S. Results shown are representative of four independent experiments (indicated above) with binding and neutralizing antibody levels comparable at similar doses and time points across all experiments. Symbols represent individual mice and bars show the mean ± s.e.m. from independent experiments. Data were log transformed and statistical significance determined using Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ACE2, angiotensin‐converting enzyme‐2; IC 80 , 80% inhibitory concentration; Ig, immunoglobulin; ns, not significant; PFU, plaque‐forming units; RBD, receptor‐binding domain; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SCV‐S, Sementis Copenhagen Vector spike protein.

Article Snippet: ACE2‐HEK293 recombinant cell line (catalog number 79951; BPS Bioscience, San Diego, CA, USA) was maintained in Dulbecco’s Modified Eagle Medium supplemented (Sigma Aldrich, MO, USA) with 10% fetal bovine serum, 2 mM l ‐glutamine and penicillin–streptomycin.

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Binding Assay, Plasmid Preparation, Activity Assay, Blocking Assay, Variant Assay, Transformation Assay, Concentration Assay