Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes

. 2022 Dec 13;55(12):2419–2435.e10. doi: 10.1016/j.immuni.2022.10.019

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Goat Anti-Human IgG(H+L)-HRP	SouthernBiotech	Cat# 2015-05; RRID:AB_2795588
Goat Anti-Human IgG Fc-HRP	SouthernBiotech	Cat# 2014-05; RRID:AB_2795580

Bacterial and virus strains

E. coli DH5 Alpha	Zymo Research	Cat# T3009
E. coli BL21-CodonPlus (DE3)-RIPL	Agilent Technology	Cat# 230280
SARS-CoV-2 pseudotyped reporter virus	BEI	Cat# NR-53817
SARS-CoV-2 Beta pseudotyped reporter virus	Scheid et al.³⁹	https://linkinghub.elsevier.com/retrieve/pii/S0092867421005353
SARS-CoV-2 Delta pseudotyped reporter virus	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
SARS-CoV-2 Omicron BA.1 pseudotyped reporter virus	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
SARS-CoV-2 Omicron BA.2 pseudotyped reporter virus	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
SARS-CoV-2 Omicron BA.2.12.1 pseudotyped reporter virus	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
SARS-CoV-2 Omicron BA.4/BA.5 pseudotyped reporter virus	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
SARS-CoV pseudotyped reporter virus	Robbiani et al.¹⁷	https://www.nature.com/articles/s41586-020-2456-9
WIV1-CoV pseudotyped reporter virus	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
SHC014-CoV pseudotyped reporter virus	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
BtKY72-CoV K493Y-T498W pseudotyped reporter virus	Starr et al.¹⁶	https://www.nature.com/articles/s41586-022-04464-z
Khosta2/SARS-CoV chimera	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
LYRa3/SARS-CoV chimera	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839

Chemicals, peptides, and recombinant proteins

Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	Cat# 11960-044
Fetal bovine serum (FBS)	Sigma-Aldrich	Cat# F4135
Gentamicin solution	Sigma-Aldrich	Cat# G1397 CAS:1405-41-0
Blasticidin S HCl	Gibco	Cat# A1113902 CAS:3513-03-9
Expi293™ Expression Medium	Gibco	Cat# A1435102
Expi293 Expression System Kit	Gibco	Cat# A14635
LB Broth (Miller)	Sigma-Aldrich	Cat# L3522
HBS-EP+ Buffer 20x	Teknova	Cat# H8022
BLI Mouse Plasma Cell Media	Berkeley Lights	Cat# 750-70004
BirA biotin-protein ligase standard reaction kit	Avidity	Cat# BirA500
SuperSignal ELISA Femto Maximum Sensitivity Substrate	Thermo Fisher Scientific	Cat# 37074
HRP-conjugated streptavidin	SouthernBiotech	Cat# 7105-05
Fluorinated octylmaltoside	Anatrace	Cat# O310F

Critical commercial assays

Luciferase Cell Culture Lysis 5X Reagent	Promega	Cat# E1531
Nano-Glo Luciferase Assay System	Promega	Cat# N1110
EZ-link NHS-PEG4 Biotinylation Kit	Thermo Fisher Scientific	Cat# 21455

Deposited data

SARS-CoV-2 WA1 S 6P + M8a-3 Fab complex coordinate	This paper	PDB: 7UZ4
SARS-CoV-2 WA1 S 6P + M8a-3 Fab complex cryo-EM map	This paper	EMDB: EMD-26878
SARS-CoV-2 WA1 S 6P + M8a-6 Fab complex coordinate	This paper	PDB: 7UZ5
SARS-CoV-2 WA1 S 6P + M8a-6 Fab complex cryo-EM map	This paper	EMDB: EMD-26879
SARS-CoV-2 WA1 S 6P + M8a-28 Fab complex coordinate	This paper	PDB: 7UZ6
SARS-CoV-2 WA1 S 6P + M8a-28 Fab complex cryo-EM map	This paper	EMDB: EMD-26880
SARS-CoV-2 WA1 S 6P + M8a-31 Fab complex coordinate	This paper	PDB: 7UZ7
SARS-CoV-2 WA1 S 6P + M8a-31 Fab complex cryo-EM map	This paper	EMDB: EMD-26881
SARS-CoV-2 WA1 S 6P + M8a-34 Fab complex coordinate	This paper	PDB: 7UZ9
SARS-CoV-2 WA1 S 6P + M8a-34 Fab complex cryo-EM map	This paper	EMDB: EMD-26883
SARS-CoV-2 WA1 S 6P + HSW-1 Fab complex coordinate	This paper	PDB: 7UZA
SARS-CoV-2 WA1 S 6P + HSW-1 Fab complex cryo-EM map	This paper	EMDB: EMD-26884
SARS-CoV-2 WA1 S 6P + HSW-2 Fab complex coordinate	This paper	PDB: 7UZB
SARS-CoV-2 WA1 S 6P + HSW-2 Fab complex cryo-EM map	This paper	EMDB: EMD-26885
SARS-CoV-2 Omicron BA.1 S 6P + M8a-31 Fab complex coordinate	This paper	PDB: 7UZ8
SARS-CoV-2 Omicron BA.1 S 6P + M8a-31 Fab complex cryo-EM map	This paper	EMDB: EMD-26882
SARS-CoV-2 RBD + M8a-34 Fab crystal structure	This paper	PDB: 7UZC
SARS-CoV-2 RBD + HSW-2 Fab crystal structure	This paper	PDB: 7UZD

Experimental models: Cell lines

HEK293T cells	Pear et al.⁴⁰	Cat# CCLV-RIE 1018 RRID: CVCL_0063
HEK293T_Ace2 cells	BEI	Cat# NR-52511
Expi293F cells	Gibco	Cat# A14527 RRID: CVCL_D615

Experimental models: Organisms/strains

C57BL/6 mice (4–6-week-old female)	Charles River	N/A

Recombinant DNA

pPPI4-SARS-CoV-2 S 6P	Hsieh et al.²⁵	N/A
p3BNC-SARS-CoV-2 RBD (residues 323-528)	This paper	N/A
p3BNC-SARS-CoV-2 Omicron BA.1 S 6P	This paper	N/A
p3BNC-M8a-3 IgG HC p3BNC-M8a-3 Fab HC p3BNC-M8a-3 LC	This paper	N/A
p3BNC-M8a-6 IgG HC p3BNC-M8a-6 Fab HC p3BNC-M8a-6 LC	This paper	N/A
p3BNC-M8a-28 IgG HC p3BNC-M8a-28 Fab HC p3BNC-M8a-28 LC	This paper	N/A
p3BNC-M8a-31 IgG HC p3BNC-M8a-31 Fab HC p3BNC-M8a-31 LC	This paper	N/A
p3BNC-M8a-34 IgG HC p3BNC-M8a-34 Fab HC p3BNC-M8a-34 LC	This paper	N/A
p3BNC-HSW-1 IgG HC p3BNC-HSW-1 Fab HC p3BNC-HSW-1 LC	This paper	N/A
p3BNC-HSW-2 IgG HC p3BNC-HSW-2 Fab HC p3BNC-HSW-2 LC	This paper	N/A
p3BNC-M8a-7 IgG HC p3BNC-M8a-7 LC	This paper	N/A
p3BNC-M8a-11 IgG HC p3BNC-M8a-11 LC	This paper	N/A
p3BNC-M8a-15 IgG HC p3BNC-M8a-15 LC	This paper	N/A
p3BNC-M8a-25 IgG HC p3BNC-M8a-25 LC	This paper	N/A
p3BNC-M8a-29 IgG HC p3BNC-M8a-29 LC	This paper	N/A
p3BNC-M8a-30 IgG HC p3BNC-M8a-30 LC	This paper	N/A
C102 IgG HC C102 LC	Barnes et al.¹⁰	https://www.nature.com/articles/s41586-020-2852-1
C144 IgG HC C144 LC	Robbiani et al.¹⁷	https://www.nature.com/articles/s41586-020-2456-9
S309 IgG HC S309 LC	Pinto et al.¹⁸	https://www.nature.com/articles/s41586-020-2349-y
C118 IgG HC C118 LC	Robbiani et al.¹⁷	https://www.nature.com/articles/s41586-020-2456-9
p3BNC-SARS-CoV-2 WA1 RBD SpyTag p3BNC-SARS-CoV-2 WA1 RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-SARS-CoV-2 Beta RBD SpyTag p3BNC-SARS-CoV-2 Beta RBD HisAvi	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
p3BNC-SARS-CoV-2 Delta RBD SpyTag p3BNC-SARS-CoV-2 Delta RBD HisAvi	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
p3BNC-SARS-CoV-2 Omicron BA.1 RBD SpyTag p3BNC-SARS-CoV-2 Omicron BA.1 RBD HisAvi	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
p3BNC-SARS-CoV-2 Omicron BA.2 RBD SpyTag p3BNC-SARS-CoV-2 Omicron BA.2 RBD HisAvi	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
p3BNC-SARS-CoV-2 Omicron BA.2.12.1 RBD SpyTag p3BNC-SARS-CoV-2 Omicron BA.2.12.1 RBD HisAvi	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
p3BNC-SARS-CoV-2 Omicron BA.4/BA.5 RBD SpyTag p3BNC-SARS-CoV-2 Omicron BA.4/BA.5 RBD HisAvi	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
p3BNC-RsSTT200-CoV RBD SpyTag p3BNC-RsSTT200-CoV RBD HisAvi	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839
p3BNC-Pang17-CoV RBD HisAvi p3BNC-Pang17-CoV RBD SpyTag	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-RaTG13-CoV RBD SpyTag p3BNC-RaTG13-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-SARS-CoV RBD SpyTag p3BNC-SARS-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-WIV1-CoV RBD SpyTag p3BNC-WIV1-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-SHC014-CoV RBD SpyTag p3BNC-SHC014-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-LYRa3-CoV RBD SpyTag p3BNC-LYRa3-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abq0839
p3BNC-C028 RBD SpyTag p3BNC-C028 RBD HisAvi	This paper	N/A
p3BNC-Rs4081-CoV RBD SpyTag p3BNC-Rs4081-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-RmYN02-CoV RBD SpyTag p3BNC-RmYN02-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-Rf1-CoV RBD SpyTag p3BNC-Rf1-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-Yun11-CoV RBD SpyTag p3BNC-Yun11-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-BM4831-CoV RBD SpyTag p3BNC-BM4831-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-BtKY72-CoV RBD SpyTag p3BNC-BtKY72-CoV RBD HisAvi	Cohen et al.¹⁵	https://www.science.org/10.1126/science.abf6840
p3BNC-Khosta2-CoV RBD SpyTag p3BNC-Khosta2-CoV RBD HisAvi	Cohen et al.¹²	https://www.science.org/10.1126/science.abq0839

Software and algorithms

GISAID	Elbe and Buckland-Merrett⁴¹ and Shu and McCauley⁴²	https://www.gisaid.org RRID:SCR_018251
Geneious	Geneious	https://www.geneious.com/
Prism v9.3.1	GraphPad	https://www.graphpad.com/scientific-software/prism/ RRID:SCR_002798
SerialEM 3.7	Mastronarde⁴³	https://bio3d.colorado.edu/SerialEM/ RRID:SCR_017293
cryoSPARC 3.2	Punjani et al.⁴⁴	https://www.cryosparc.com RRID:SCR_016501
UCSF Chimera	Goddard et al.⁴⁵ and Pettersen et al.⁴⁶	http://plato.cgl.ucsf.edu/chimera/ RRID:SCR_004097
UCSF ChimeraX	Goddard et al.⁴⁷ and Pettersen et al.⁴⁸	https://www.cgl.ucsf.edu/chimerax/ RRID:SCR_015872
XDS	Kabsch⁴⁹	http://xds.mpimf-heidelberg.mpg.de RRID:SCR_015652
PHASER	McCoy⁵⁰	https://www.phenix-online.org/documentation/reference/phaser.html RRID:SCR_014219
Phenix	Liebschner et al.⁵¹	https://www.phenix-online.org/ RRID:SCR_014224
Coot	Emsley et al.⁵²	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ RRID:SCR_014222
AIMLESS	Winn et al.⁵³	http://www.ccp4.ac.uk/html/aimless.html RRID:SCR_015747
MolProbity	Chen et al.⁵⁴	http://molprobity.biochem.duke.edu RRID:SCR_014226
PyMOL 2.4.0	Lilkova et al.⁵⁵	https://pymol.org/2/ RRID:SCR_000305
ConSurf Database	Landau et al.¹⁴	https://consurf.tau.ac.il RRID:SCR_002320
SAbDab	Dunbar et al.⁵⁶	http://opig.stats.ox.ac.uk/webapps/newsabdab/sabdab/
PDBePISA	Krissinel and Henrick²⁸	https://www.ebi.ac.uk/pdbe/pisa/
Bruker Sierra SPR-32 Pro analysis software	Bruker	https://www.bruker.com/en/products-and-solutions/surface-plasmon-resonance/sierra-spr-32-pro.html
Illustrator	Adobe	https://www.adobe.com

Other

384-well Nunc MaxiSorp ELISA plate	Millipore Sigma	Cat# P6491
100kDa cutoff Amicon concentrator	EMD Millipore	Cat# UFC910096
30kDa cutoff Amicon concentrator	EMD Millipore	Cat# UFC903096
10kDa cutoff Amicon concentrator	EMD Millipore	Cat# UFC901096
HisTrap HP	Cytiva	Cat# 17-5248-02
HiLoad 16/600 Superdex 200 pg	Cytiva	Cat# 28-9893-35
Superose 6 Increase 10/300 GL	Cytiva	Cat# 29-0915-96
HiTrap MabSelect SuRe	Cytiva	Cat# 11-0034-95
Superdex 200 Increase 10/300 GL	Cytiva	Cat# 28-9909-44
400 Mesh carbon-coated copper grids	Ted Pella	Cat# 01844-F
300 Mesh Quantifoil holey carbon 1.2/1.3 cooper grids	Electron Microscopy Sciences	Cat# Q350AR13A
High Capacity Amine Sensor chip	Bruker	Cat# 1862614
OptoSelect chip 11k	Berkeley Lights	Cat# 750-08090
BLI assay beads	Berkeley Lights	Cat# 520-00053