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

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  • Supplementary Text
  • Figs. S1 to S23
  • Tables S1 to S8
  • Captions for Movies S1 to S22
  • References
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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.