Supplementary Materials
Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational
escape
Paul-Albert Koenig, Hrishikesh Das, Hejun Liu, Beate M. Kümmerer, Florian N. Gohr, Lea-Marie Jenster, Lisa D. J. Schiffelers, Yonas M. Tesfamariam, Miki Uchima, Jennifer D. Wuerth, Karl Gatterdam, Natalia Ruetalo, Maria H. Christensen, Caroline I. Fandrey, Sabine Normann, Jan M. P. Tödtmann, Steffen Pritzl, Leo Hanke, Jannik Boos, Meng Yuan, Xueyong Zhu, Jonathan L. Schmid-Burgk, Hiroki Kato, Michael Schindler, Ian A. Wilson, Matthias Geyer, Kerstin U. Ludwig, B. Martin Hällberg, Nicholas C. Wu, Florian I. Schmidt
Materials/Methods, Supplementary Text, Tables, Figures, and/or References
- Download Supplement
- Supplementary Text
- Figs. S1 to S23
- Tables S1 to S8
- Captions for Movies S1 to S22
- References
- MDAR Reproducibility Checklist
Images, Video, and Other Media
- Movie S1
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD neutralize infection. Caco-2 cells were mock infected and microscopically followed over time.
- Movie S2
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD neutralize infection. Caco-2 cells were infected with mNeonGreen-expressing icSARS-CoV-2-mNG and microscopically followed over time.
- Movie S3
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD neutralize infection. Caco-2 cells were infected with mNeonGreen-expressing icSARS-CoV-2-mNG in the presence of 250 nM VHH E and microscopically followed over time.
- Movie S4
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD neutralize infection. Caco-2 cells were infected with mNeonGreen-expressing icSARS-CoV-2-mNG in the presence of 250 nM VHH U and microscopically followed over time.
- Movie S5
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD neutralize infection. Caco-2 cells were infected with mNeonGreen-expressing icSARS-CoV-2-mNG in the presence of 250 nM VHH V and microscopically followed over time.
- Movie S6
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD neutralize infection. Caco-2 cells were infected with mNeonGreen-expressing icSARS-CoV-2-mNG in the presence of 250 nM VHH W and microscopically followed over time.
- Movie S7
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD neutralize infection. Caco-2 cells were infected with mNeonGreen-expressing icSARS-CoV-2-mNG in the presence of 250 nM VHH LaM-4 and microscopically followed over time.
- Movie S8
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH LaM-4 and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S9
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH E and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S10
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH U and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S11
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH V and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S12
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH W and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S13
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH E and Sigma Protease Inhibitor Cocktail for tissue culture media (1:200); microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S14
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH EV and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S15
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH VE and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S16
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH EW and microscopy images were recorded every 20 min for 14h at 37° C. Time stamp represents hh:mm.
- Movie S17
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH WE and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S18
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH EE and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S19
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH EEE and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S20
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH VV and microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S21
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH E and 3 uM VHH VV; microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
- Movie S22
- Camelid nanobodies against two epitopes on SARS-CoV-2 Spike RBD activate spike fusion. HEK 293 cells inducibly expressing SARS-CoV-2 S Δ18 and either eGFP or tagRFP-t were seeded in a 1:1 ratio and induced with 1 μg/mL doxycycline for 20 h. Cells were treated with 1 μM VHH U and 3 uM VHH EEE; microscopy images were recorded every 20 min for 14 h at 37°C. Time stamp represents hh:mm.
