Antibodies targeting a quaternary site on SARS-CoV-2 spike glycoprotein prevent viral receptor engagement by conformational locking

. 2023 Oct 10;56(10):2442–2455.e8. doi: 10.1016/j.immuni.2023.09.003

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

CD3 PE-CF594	BD Biosciences	Cat#562280 RRID: AB_11153674
CD19 PE-Cy7	Biolegend	Cat#302216 RRID: AB_314246
CD20 APC-Cy7	Biolegend	Cat#302314 RRID: AB_314262
IgM V450	BD Biosciences	Cat#561286 RRID: AB_10611713
CD27 PerCP-Cy5.5	BD Biosciences	Cat#567181 RRID: AB_2916493
anti-His PE	Biolegend	Cat#362603 RRID: AB_2563634
TotalSeq™-C0253 anti-human Hashtag 3 Antibody	Biolegend	Cat#394665 RRID: AB_2801033
TotalSeq™-C0251 anti-human Hashtag 1 Antibody	Biolegend	Cat# 394661 RRID: AB_2801031
Peroxidase AffiniPure goat anti-human IgG Fcγ fragment–specific antibody	Jackson ImmunoResearch	Cat#109-035-170 RRID: AB_2810887
APC anti-human IgG Fc	BioLegend	Cat#366906 RRID: AB_2888847
Rabbit anti-SARS-Spike S1	Sino Biological	Cat#40591-T62 RRID: AB_2893171
Rabbit anti-SARS-Spike S2	Sino Biological	Cat#40590-T62
Horseradish peroxidase-conjugated anti-rabbit antibody	Cytiva	Cat#NA934-1ML RRID: AB_772206
12-16	This paper	N/A
12-19	This paper	N/A
S309	Pinto et al.⁴⁴	N/A
S2P6	Pinto et al.³⁴	N/A
2-15	Liu et al.²¹	N/A
REGN10987	Hansen et al.³⁹	N/A
5-7	Liu et al.²¹	N/A
4-33	Liu et al.²¹	N/A
4-8	Liu et al.²¹	N/A
4-18	Liu et al.²¹	N/A
CB6	Shi et al.⁴¹	N/A

Bacterial and virus strains

VSV-G pseudotyped ΔG-luciferase	Kerafast	Cat#EH1020-PM
hCoV-19/Hong_Kong/HKU-211129-001/2021	Shuai et al.⁶⁴	N/A
SARS-Related Coronavirus 2 Isolate USA-WA1/2020	BEI Resources	NR-52281
Isolate hCoV-19/USA/MD-HP20874/2021 (Lineage B.1.1.529; Omicron Variant)	BEI Resources	NR-56461

Biological samples

Patient 12 whole blood sample	This paper	N/A

Chemicals, peptides, and recombinant proteins

Polyethylenimine (PEI)	Polysciences Inc.	Cat#23966-100
hACE2-Fc	This paper	N/A
B.1.351 S2P spike trimer	This paper	N/A
D614G S2P spike trimer	This paper	N/A
XBB.1.16 S2P spike trimer	This paper	N/A

Critical commercial assays

Ni-NTA Agarose	Invitrogen	Cat#R90115
LIVE/DEAD Fixable Yellow Dead Cell Stain Kit	Invitrogen	Cat#L34959
Luciferase Assay System	Promega	Cat#E4550
QuikChange Lightning Site-Directed Mutagenesis Kit	Agilent	Cat#210518
Series S sensor chip CM5	Cytiva	Cat#BR100530
His-capture kit	Cytiva	Cat#28995056
rProtein A Sepharose	Cytiva	Cat#17127901
Pierce™ F(ab')2 Preparation Kit	ThermoFisher	Cat#44988
Endoproteinase Lys-C	Sigma-Aldrich	Cat#1142042900
3,3′,5,5′-tetramethylbenzidine (TMB) substrate	Sigma-Aldrich	Cat#T0440
One-Step Antibody Biotinylation Kit	Miltenyi Biotec	Cat#130-093-385
APC-streptavidin	Biolegend	Cat#405207
1 X LDS sample buffer	ThermoFisher	Cat# NP0008

Deposited data

4-33 heavy chain	GenBank	OQ787074.1
4-33 light chain	GenBank	OQ787075.1
12-16 heavy chain	GenBank	OQ787072.1
12-16 light chain	GenBank	OQ787073.1
12-19 heavy chain	GenBank	OQ787070.1
12-19 light chain	GenBank	OQ787071.1
4-33	Electron Microscopy Data Bank	EMDB: 26964
4-33	Protein Data Bank	PDB: 8CSJ
12-16	Electron Microscopy Data Bank	EMDB: 26583
12-16	Protein Data Bank	PDB: 7UKL
12-19	Electron Microscopy Data Bank	EMDB:26584
12-19	Protein Data Bank	PDB: 7UKM
Deep mutational scanning of SARS-CoV-2 Omicron BA.1 spike and mapping of escape variants for 12-19 antibody	N/A	https://github.com/dms-vep/SARS-CoV-2_Omicron_BA.1_spike_DMS_12-19
Interactive display of escape variants for 12-19	N/A	https://dms-vep.github.io/SARS-CoV-2_Omicron_BA.1_spike_DMS_12-19/

Experimental models: Cell lines

HEK293T	ATCC	Cat#CRL-3216; RRID: CVCL_0063
Vero-E6	ATCC	Cat#CRL-1586; RRID: CVCL_0574
Expi293 cells	Thermo Fisher Scientific	Cat#A14527

Experimental models: Organisms/strains

Syrian hamster model	N/A	N/A

Recombinant DNA

pRRLSIN.cPPT.PGK-GFP.WPRE	Addgene	RRID: Addgene_12252
pah-B.1.351 S2P	This paper	N/A
pah-D614G S2P	Hsieh et al.⁶⁵	RRID: Addgene_154754
pah-XBB.1.16 S2P	This paper	N/A
pCMV3-D614G spike	Liu et al.¹²	N/A
pCMV3-B.1.1.7 spike	Liu et al.¹²	N/A
pCMV3-B.1.351 spike	Liu et al.¹²	N/A
pCMV3-B.1.617.2 spike	Liu et al.¹²	N/A
pCMV3-BA.1 spike	Liu et al.¹²	N/A
pCMV3-BA.1.1 spike	Liu et al.¹²	N/A
pCMV3-BA.2 spike	Iketani et al.¹³	N/A
pCMV3-BA.2.12.1 spike	Wang et al.¹¹	N/A
pCMV3-BA.4/5 spike	Wang et al.¹¹	N/A
pCMV3-BA.2.75 spike	Wang et al.¹⁰	N/A
pCMV3-BA.2.75.2 spike	Wang et al.⁴⁷	N/A
pCMV3-BF.7 spike	Wang et al.¹⁹	N/A
pCMV3-BQ.1 spike	Wang et al.⁹	N/A
pCMV3-BQ.1.1 spike	Wang et al.⁹	N/A
pCMV3-XBB spike	Wang et al.⁹	N/A
pCMV3-XBB.1 spike	Wang et al.⁹	N/A
pCMV3-XBB.1.5 spike	Wang et al.⁶⁶	N/A
pCMV3-XBB.1.16 spike	This paper	N/A
pCMV3-XBB.2.3 spike	This paper	N/A
pCMV3-CH.1.1 spike	Wang et al.⁴⁷	N/A
pCMV3-DS.1 spike	Wang et al.⁴⁷	N/A
pCMV3-SARS-CoV spike	Liu et al.²⁷	N/A
pcDNA3-sACE2-WT(732)-IgG1	Chan et al.⁶⁷	RRID: Addgene_154104
pCMV3-BA.1 spike with single mutations	This paper	N/A

Software and algorithms

GraphPad Prism 9	GraphPad Software Inc	https://www.graphpad.com/scientific-software/prism/
PyMOL v.2.3.2	Schrödinger, LLC	https://pymol.org/2/#page-top
Biacore T200 Evaluation Software (Version 1.0)	Cytiva	N/A
Cell Ranger V(D)J pipeline	10X Genomics	https://support.10xgenomics.com/single-cell-vdj/software/pipelines/latest/using/vdj
FlowJo v10.4	BD Biosciences	https://www.flowjo.com/solutions/flowjo
cryoSPARC v3.3.1	Structura Biotechnology Inc.	https://cryosparc.com/