Appendix 1—key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | ArticExpress(DE3) | Agilent | Cat# 230192 | Competent cells, enabling efficient high-level expression of heterologous proteins. |
| Strain, strain background (vesicular stomatitis virus) | rVSV/SARS-CoV-2/GFP; WT2E1 | Schmidt et al., 2020 | Recombinant chimeric VSV/SARS-CoV-2 reporter virus.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-Y369N | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-G404E | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-D574N, E484K, Q493K | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-S371P, H66R, N969T | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-F490S, E484K, Q493K | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-Q493R, G252R | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-W64R, L452F | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-H245R, H1083Y | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-W64R, F490L, I931G | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-W64R, F490S | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-Y449H, F490S, Q787R | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-S494P | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-S172G | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-E484K | Schmidt et al., 2020 | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-W64R, S982R | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-T259K, K378Q | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-W64R, K378Q | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-F486S | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-T478R | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-T478I | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-Y508H | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-N354S | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-F486L | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-Y489H | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-K378Q | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-L452R | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-H245R, S349P, H1083Y | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-P384Q | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-E484G | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-W64R, Y170H, V705M | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-W64R, Y170H, Q787H | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (vesicular stomatitis virus) | 2E1-T315I | This study | Mutant rVSV/SARS-CoV-2/GFP derivative.Inquiries should be addressed to P. Bieniasz. | |
| Strain, strain background (betacoronavirus) | SARS-CoV-2, Isolate USA-WA1/2020 | BEI Resources | NR-52281 | Wild-type SARS-CoV-2. |
| Strain, strain background (betacoronavirus) | SARS-CoV-2, Isolate USA-WA (B.1.617.2) | R.Coler | Delta | SARS-CoV-2 delta variant of concern. |
| Biological sample (Lama glama) | Bone marrow aspirates | Capralogics | From two male llamas immunized with SARS-CoV-2 spike S1, RBD, and S2. | |
| Biological sample (L. glama) | Sera | Capralogics | From two male llamas immunized with SARS-CoV-2 spike S1, RBD, and S2. | |
| Cell line (Homo sapiens) | 293T/17 | ATCC | CRL-11268 | Human kidney epithelial cells. |
| Cell line (H. sapiens) | 293/ACE2cl.22 | Schmidt et al., 2020 | 293T cells expressing human ACE2 (single-cell clone). | |
| Cell line (H. sapiens) | 293T-ACE2 | Cawford et al., 2020 | BEI NR-52511 | 293T cells expressing human ACE2 (single-cell clone). |
| Cell line (H. sapiens) | Primary human airway epithelial cells | This study | Air-liquid interface culture system.Inquiries should be addressed to J. Debley. | |
| Cell line (Cercopithecus aethiops) | VERO C1008 [Vero 76, clone E6, Vero E6] | ATCC | CRL-1586 | Monkey kidney epithelial cells. |
| Cell line (C. aethiops) | TMPRSS2+ Vero E6 | R.Coler | Vero E6 cells expressing human TMPRSS2. | |
| Antibody | Anti-COVID-19 and SARS-CoV S glycoprotein [CR3022] (human monoclonal) | Absolute Antibody | Cat# Ab01680-10.0 | Flow cytometry (1:1000). |
| Antibody | Anti-human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (goat polyclonal) | Invitrogen | Cat# A-11013 | Flow cytometry (1:2000). |
| Recombinant DNA reagent | pET21-pelB | Fridy et al., 2014a | Expression plasmid for expressing nanobodies. | |
| Recombinant DNA reagent | pET21-pelB-SARS-CoV-2 Nanobody | This study; Fridy et al., 2014a |
See Table 1 and Table 2 | SARS-CoV-2 nanobody expression plasmids.Inquiries should be addressed to M. Rout. |
| Recombinant DNA reagent | pcDNA3.1+ SARS-1-S-C9 | T.Gallagher | SARS-CoV-1 spike expression plasmid. | |
| Recombinant DNA reagent | pcDNA3.1+ SARS-2-S-C9 WUHAN-1 | T.Gallagher | SARS-CoV-2 spike expression plasmid. | |
| Recombinant DNA reagent | pcDNA3.1+ SARS-2-B.1.1.7 | NIAID | SARS-CoV-2 spike expression plasmid for alpha variant. | |
| Recombinant DNA reagent | pHDM-SARS-CoV-2-Spike-B.1.351 | L.Stamatatos | SARS-CoV-2 spike expression plasmid for beta variant. | |
| Recombinant DNA reagent | pcDNA3.1+ SARS-2 P.1 | NIAID | SARS-CoV-2 spike expression plasmid for gamma variant. | |
| Recombinant DNA reagent | psPAX2 | D.Trono/Addgene | Plasmid #12260 | Second-generation lentiviral packaging plasmid. |
| Recombinant DNA reagent | pHAGE-CMV-Luc2-IRES-ZsGreen-W | J.Bloom / Cawford et al., 2020 |
NR-52516 | Lentiviral backbone plasmid that uses a CMV promoter to express luciferase followed by an IRES and ZsGreen. |
| Sequence-based reagent (primer) | 6N_CALL001 | This study | PCR and sequencing primer | NNNNNNGTCCTGGCTGCTCTTCTACAAGG |
| Sequence-based reagent (primer) | 6N_CALL001B | This study | PCR and sequencing primer | NNNNNNGTCCTGGCTGCTCTTTTACAAGG |
| Sequence-based reagent (primer) | 6N_VHH_SH_rev | This study | PCR and sequencing primer | NNNNNNCTGGGGTCTTCGCTGTGGTGC |
| Sequence-based reagent (primer) | 6N_VHH_LH_rev | This study | PCR and sequencing primer | NNNNNNGTGGTTGTGGTTTTGGTGTCTTGGG |
| Peptide, recombinant protein | Spike S1 (Wuhan Str.) | Sino Biological | Cat# 40591-V08H | For determining KDs. |
| Peptide, recombinant protein | Spike RBD | Sino Biological | Cat# 40592-VNAH | For determining KDs. |
| Peptide, recombinant protein | SARS-CoV-2 (2019-nCoV) Spike RBD-mFc Recombinant Protein | Sino Biological | Cat# 40592-V05H | For epitope mapping. |
| Peptide, recombinant protein | Spike S2 | Sino Biological | Cat# 40590-V08B | For immunization and for determining KDs. |
| Peptide, recombinant protein | Spike S1 (501Y.V1) | Sino Biological | Cat# 40591-V08H12 | For determining KDs. |
| Peptide, recombinant protein | Spike S1 (501Y.V2) | Sino Biological | Cat# 40591-V08H10 | For determining KDs. |
| Peptide, recombinant protein | SARS-CoV-2 Spike S1, Sheep Fc-Tag | The Native Antigen Co. | Cat# REC31806 | For immunization. |
| Peptide, recombinant protein | SARS-CoV-2 Spike S2, Sheep Fc-Tag | The Native Antigen Co. | Cat# REC31807 | For immunization. |
| Peptide, recombinant protein | Protein M | Grover et al., 2014 | Used to deplete light-chain containing IgGs. | |
| Peptide, recombinant protein | Thyroglobulin | Sigma-Aldrich | Cat# A8531-1V | Used to calibrate mass photometer. |
| Peptide, recombinant protein | Bovine serum albumin | Sigma-Aldrich | Cat# T9145-1VL | Used to calibrate mass photometer. |
| Peptide, recombinant protein | Beta-amylase | Sigma-Aldrich | Cat# A8781-1VL | Used to calibrate mass photometer. |
| Peptide, recombinant protein | FabRICATOR (IdeS) | Genovis | Cat# A0-FR1-050 | Protease to cleave VHH domain from the HCAb. |
| Commercial assay, kit | ProteOn Amine Coupling Kit | Bio-Rad | Cat# 1762410 | Used to couple VHH domain to beads. |
| Commercial assay, kit | TruSeq Nano DNA Low Throughput Library Prep Kit | Illumina | Cat# 20015964 | Used to sequence VHH. |
| Commercial assay, kit | Steady-GLO | Promega | Cat# E2520 | Used in pseudovirus assay. |
| Software, algorithm | Llama-Magic | Fridy et al., 2014a, | https://github.com/FenyoLab/llama-magic | For identifying nanobody sequences. |
| Software, algorithm | IMP, the Integrative Modeling Platform | Russel et al., 2012 | https://integrativemodeling.org | For integrative structural modeling. |
| Software, algorithm | UCSF ChimeraX | Pettersen et al., 2021 | https://www.rbvi.ucsf.edu/chimerax/download.html | For visualizing structural models. |
| Software, algorithm | synergy v0.4 | Wooten et al., 2021 | https://pypi.org/project/synergy/ | For observing synergy. |
| Software, algorithm | matplotlib v3.4.1 | Hunter, 2007 | https://pypi.org/project/matplotlib/ | For preparing figures. |
| Software, algorithm | seaborn v0.11.0 | Waskom, 2021 | https://seaborn.pydata.org/index.html | For preparing figures. |
| Software, algorithm | plotly v4.12.0 | 2019 Plotly, Inc | https://plotly.com/python/ | For preparing figures. |
| Software, algorithm | numpy v1.19.2 | Huo et al., 2020a | https://numpy.org | For data analysis. |
| Software, algorithm | pandas v1.1.2 | The pandas development team, 2020 | https://pandas.pydata.org | For data analysis. |
| Software, algorithm | scipy v1.5.0 | Virtanen et al., 2020 | https://www.scipy.org | For data analysis. |
| Software, algorithm | scikit-learn v0.23.2 | Pedregosa, 2011 | https://scikit-learn.org/stable/ | For data analysis. |
| Software, algorithm | python v3.8 | Van Rossum and Drake, 2009 | https://www.python.org | For data analysis and preparing figures. |
| Software, algorithm | Prism 9 | GraphPad | https://www.graphpad.com | For data analysis and preparing figures. |
| Software, algorithm | OCTET | Sartorius | For data analysis. | |
| Other | ProteOn GLC Sensor Chip | Bio-Rad | Cat# 176-5011 | For protein interaction analysis. |
| Other | Series S Sensor Chip CM5 | Cytiva | Cat# BR100530 | For protein interaction analysis. |
| Other | Hard-shell PCR plates, 96-well, thin-wall | Bio-Rad | Cat# HSP9661 | For differential scanning fluorimetry. |
| Other | Microseal ‘B’ seal | Bio-Rad | Cat# MSB1001 | For differential scanning fluorimetry. |
| Other | Precision Coverslips | Thorlabs | Cat# CG15KH1 | For mass photometry. |
| Other | CultureWell Reusable Gasket | Grace Bio-Labs | Cat# 103250 | For mass photometry. |
| Other | Protein A Sepharose 4B | Thermo Fisher | Cat# 101042 | For protein purification. |
| Other | Recombinant Protein G Sepharose 4B | Thermo Fisher | Cat# 101243 | For protein purification. |
| Other | CNBr-activated Sepharose 4 Fast Flow | Cytiva | Cat# 17098101 | For nanobody screening. |
| Other | SuperScript VILO Master Mix | Thermo Fisher | Cat# 11755250 | For viral escape analysis. |
| Other | Anti-mouse IgG Fc Capture Biosensors | Sartorius | Cat# 18-5088 | For epitope mapping. |
| Other | KOD Xtreme Hot Start DNA Polymerase | Sigma-Aldrich | Cat# 71975 | For viral escape analysis. |