Molecular basis for antiviral activity of two pediatric neutralizing antibodies targeting SARS-CoV-2 Spike RBD

. 2022 Dec 9;26(1):105783. doi: 10.1016/j.isci.2022.105783

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Anti-SARS-CoV-2 RBD EH1	This paper	N/A
Anti-SARS-CoV-2 RBD EH2	This paper	N/A
Anti-SARS-CoV-2 RBD EH3	This paper	N/A
Anti-SARS-CoV-2 RBD EH5	This paper	N/A
Anti-SARS-CoV-2 RBD EH8	This paper	N/A
Brilliant Ultraviolet™ 395 anti-human CD3 (clone SK7)	BD Biosciences	Cat # 564001; RRID: AB_2744382
PE-Cy7 anti-human CD14 (clone M5E2)	BD Biosciences	Cat # 557742; RRID: AB_396848
PE anti-human CD19 (clone HIB19)	Biolegend	Cat # 302208; RRID: AB_314238
Brilliant Violet™ 786 anti-human IgG (clone G18-145)	BD Biosciences	Cat # 564230; RRID: AB_2738684
Brilliant Violet™ 650 anti-human IgD (clone IA6-2)	BD Biosciences	Cat # 740594; RRID: AB_2740295
Cross-reactive anti-SARS-CoV-1 RBD CR3022	ter Meulen et al.¹	RRID: AB_2848080
Anti-SARS-CoV-2 RBD CV3-1	Jennewein et al.²	N/A
Anti-SARS-CoV-2 S2 CV3-25	Jennewein et al.²	N/A
Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647	Invitrogen	Cat # A-21445; RRID: AB_2535862
Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, HRP	Invitrogen	Cat # A18823; RRID: AB_2535600
InVivoMAb human IgG1 isotype control	Bio X Cell	RRID: AB_2687817 Cat # BE0297
Rabbit anti-SARS-CoV-2 RBD polyclonal antibody	Li et al.³	N/A
Anti-SARS-CoV-2 RBD Casirivimab	Regeneron	N/A
Anti-SARS-CoV-2 RBD Imdevimab	Regeneron	N/A

Bacterial and virus strains

SARS-CoV-2, hCoV-19/USA/PHC658/2021 (B.1.617.2)	BEI Resources	Cat # NR-55611
SARS-CoV-2 Delta-nLuc VOC	This paper	N/A

Biological samples

Blood sample from a pediatric patient (Patient 12)	This paper	N/A
Plasma from SARS-CoV-2-infected donors (S002, S006)	Lu et al.⁴	N/A
Plasma from SARS-CoV-2 uninfected donors	Prévost et al.⁵	N/A
Primary human peripheral blood mononuclear cells (PBMCs)	FRQS AIDS network	N/A

Chemicals, peptides, and recombinant proteins

Dulbecco’s modified Eagle’s medium (DMEM)	Wisent	Cat# 319-005-CL
Penicillin/streptomycin	Wisent	Cat# 450-201-EL
Fetal bovine serum (FBS)	VWR	Cat# 97068-085
Bovine Serum Albumin (BSA)	BioShop	Cat# ALB001.100
Phosphate buffered saline (PBS)	Wisent	Cat# 311-010-CL
Gibco™ RPMI 1640 medium	Thermo Fisher Scientific	Cat # 11875093
Gibco™ MEM Non-essential amino acid (NEAA) solution	Thermo Fisher Scientific	Cat # 11140050
Sodium pyruvate	Life technologies	Ref # 11360-070
Tris-buffered saline (TBS)	Thermo Fisher Scientific	Cat # BP24711
Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate	Perkin Elmer Life Sciences	Cat # NEL105001EA
Tween20	Thermo Fisher Scientific	Cat # BP337-500
Passive lysis buffer	Promega	Cat # E1941
Puromycin dihydrochloride	Millipore Sigma	Cat # P8833
D-Luciferin potassium salt	Prolume	Cat # 306
Formaldehyde 37%	Thermo Fisher Scientific	Cat # F79-500
LIVE/DEAD Fixable AquaVivid Cell Stain	Thermo Fisher Scientific	Cat # L34957
Cell proliferation dye eFluor670	Thermo Fisher Scientific	Cat # 65-0840-85
Cell proliferation dye eFluor450	Thermo Fisher Scientific	Cat # 65-0842-85
FreeStyle 293F expression medium	ThermoFisher Scientific	Cat # 12338002
ExpiFectamine 293 transfection reagent	ThermoFisher Scientific	Cat # A14525
Protein A Sepharose CL-4B	Cytiva	Cat # 17096303
Ni-NTA agarose	Invitrogen	Cat # R90110
Papain-agarose resin	ThermoFisher Scientific	Cat # 20341
Gibco™ DMEM, high glucose, without L-Gln, L-Met and L-Cys	Thermo Fisher Scientific	Cat # 21013024
Gibco™ GlutaMax™ (100X)	Thermo Fisher Scientific	Cat #35050061
Gibco™ Dialyzed FBS	Thermo Fisher Scientific	Cat # 26400036
EDTA 0.5 M pH 8.0	Invitrogen	Cat # 15575020
Sodium chloride (NaCl)	Bioshop	Cat # SOD001.10
Sodium Phosphate Monobasic Anhydrous (NaH₂PO₄)	Fisher Scientific	Cat # BP329-1
Sodium Phosphate Dibasic Anhydrous (Na₂HPO₄)	Fisher Scientific	Cat #S374-1
Sodium dodecyl sulfate (SDS)	Fisher Scientific	Cat # BP166-500
Sodium deoxycholate (DOC)	Sigma-Aldrich	Cat # 30970-100G
Pierce™ Protease inhibitor Tablets	ThermoFisher Scientific	Cat # A32963
Avicel® Pharma Grade	FMC	Cat # RC-581 NF
Magnesium sulfate heptahydrate (MgSO₄ ·7H₂0)	Bioshop	Cat # MAG511.500
Potassium dihydrogen phosphate (KH2PO4)	Fisher Scientific	Cat # BP362-1
Adenosine 5′-triphosphate disodium salt hydrate (ATP)	Sigma-Aldrich	Cat # A3377-10G
Dithiothreitol (DTT)	Fisher Scientific	Cat # BP172-5
NP-40 (IGEPAL CA-630)	Sigma-Aldrich	Cat #I3021
Ficoll-Paque™	Cytiva	Cat # 17144003
7-AAD	BD Biosciences	Cat # 559925
Optiprep™	Sigma	Cat # D1556
Soluble human ACE2	Zhou et al.⁶	N/A
LD555-CD	Lumidyne Technologies	N/A
LD655-CoA	Lumidyne Technologies	N/A
SgrAI	New England Biolabs	Cat # R0603S
BsiWI-HF	New England Biolabs	Cat # R3553S
AgeI-HF	New England Biolabs	Cat # R3552S
NheI-HF	New England Biolabs	Cat # R3131S
Octet® Amine Reactive Second Generation (AR2G) biosensors	Sartorius	Cat # 18-5093
Octet® Amine Reactive Second Generation (AR2G) Reagent Kit	Sartorius	Cat # 18-5095
The PEG-it Virus precipitation solution (5X)	System Bioscience	Cat # LV810A-1
ProPlex™ HT-96 Eco Crystallography Screen	Molecular Dimensions	Cat # MD1-42-ECO
Index Crystallography Screen	Hampton Research	Cat # HR2-144
The LMB Crystallization Screen™	Molecular Dimensions	Cat # MD1-99

Critical commercial assays

Nano-Glo Luciferase Assay System (nanoluciferase substrate)	Promega	Cat # N1120
Pierce™ Gaussia Luciferase Glow Assay Kit	ThermoFisher Scientific	Cat # 16160
QuikChange II XL Site-Directed Mutagenesis Kit	Agilent Technologies	Cat # #200522
Alexa Fluor 488 Protein Labeling Kit	Invitrogen	Cat # A10235
Alexa Fluor 647 Protein Labeling Kit	Invitrogen	Cat # A20173
KAPA SYBR FAST qPCR Master Mix (2X) Kit	KAPA Biosystems	Cat # KK4600 and KK4601
Ambion DNase I (RNase-free)	ThermoFisher Scientific	Cat # AM2222
RNeasy Mini Kit (50)	Qiagen	Cat #/ID 74104
iScript advanced cDNA kit	Bio Rad	Cat #1725038
iQ Multiplex Powermix	Bio Rad	Cat # 1725848
Chromium Single Cell V(D)J Enrichment Kit, Human B Cell	10X Genomics	Cat # PN-1000016
EXPRE³⁵S³⁵S Protein Labeling Mix (³⁵S-L-methionine, ³⁵S-L-cysteine)	PerkinElmer	Cat # NEG072007MC

Deposited data

Crystal structure of SARS-CoV-2 RBD in complex with the neutralizing IGHV3-53-encoded antibody EH3 isolated from a nonvaccinated pediatric patient	This study	PDB 7UL1
Crystal structure of SARS-CoV-2 RBD in complex with the ridge-binding nAb EH8 isolated from a nonvaccinated pediatric patient	This study	PDB 7UL0

Experimental models: Cell lines

Vero E6	ATCC	Cat # CRL-1586; RRID: CVCL_0574
HEK 293T	ATCC	Cat # CRL-3216; RRID: CVCL_0063
Expi293 GnTI- Cells	ThermoFisher Scientific	Cat # A39240
293T-ACE2	Prevost et al.⁵	N/A
TZM-bl-ACE2	Ullah, Prévost et al.⁷	N/A
CEM.NKr-CCR5+	NIH AIDS Reagent Program	Cat # 4376; RRID: CVCL_X623
CEM.NKr-Spike	Anand et al.⁸	N/A
FreeStyle 293F cells	ThermoFisher Scientific	Cat # R79007; RRID: CVCL_D603

Experimental models: Organisms/strains

C57BL/6J.Cg-Tg(K18-ACE2)2Prlmn/J	The Jackson Laboratory	The Jackson Laboratory Stock No: 034860

Oligonucleotides

V(D)J library - enrichment primer 1 5′CTACACGACGCTCTTCCG ATCTAGCAGTGGTATCAACGCA-3′	This paper	N/A
V(D)J library - enrichment primer 2 5′CTACACGACGCTCTTCCGATCTAGC-3′	This paper	N/A
SARS-CoV-2 N F: 5′-ATGCTG CAATCGTGCTACAA-3′	Yale School of Medicine, W. M. Keck Foundation, Oligo Synthesis Resource	N/A
SARS-CoV-2 N R: 5′-GACTG CCGCCTCTGCTC-3′	Yale School of Medicine, W. M. Keck Foundation, Oligo Synthesis Resource	N/A
FAM-Gapdh	Bio Rad	Cat # 12001950
HEX-IL6	Bio Rad	Cat # 10031228
TEX615-CCL2	Bio Rad	Cat # 10031234
Cy5-CXCL10	Bio Rad	Cat # 10031231
Transgene Forward: GAC CCC TGA GGG TTT CAT ATA G	Yale School of Medicine, W. M. Keck Foundation, Oligo Synthesis Resource	#53437, Genotyping primers for K18-hACE2 mice. The Jackson Laboratory
Common: CAC CAA CAC AGT TTC CCA AC	Yale School of Medicine, W. M. Keck Foundation, Oligo Synthesis Resource	#53438, Genotyping primers for K18-hACE2 mice. The Jackson Laboratory
Wildtype forward: AAG TTG GAG AAG ATG CTG AAA GA	Yale School of Medicine, W. M. Keck Foundation, Oligo Synthesis Resource	#53439, Genotyping primers for K18-hACE2 mice. The Jackson Laboratory

Recombinant DNA

pcDNA3.1-SARS-CoV-2 RBD	Beaudoin-Bussieres et al.⁹	N/A
pcDNA3.1-SARS-CoV-2 RBD E484K	Gong et al.¹⁰	N/A
pNL4.3 R-E- Luc	NIH AIDS Reagent Program	Cat # 3418
pIRES2-eGFP vector	Clontech	Cat # 6029-1
pCG1-SARS-CoV-2 Spike WT (Wuhan-Hu-1)	Hoffmann et al., 2020¹¹	N/A
pCG1-SARS-CoV-2 Spike D614G	Beaudoin-Bussieres et al.⁹	N/A
pCG1-SARS-CoV-2 Spike B.1.429 variant	Li et al.³	N/A
pcDNA3.1-SARS-CoV-2 Spike B.1.1.7 variant	Tauzin et al.¹²	N/A
pcDNA3.1-SARS-CoV-2 Spike B.1.351 variant	Li et al.³	N/A
pcDNA3.1-SARS-CoV-2 Spike P.1 variant	Li et al.³	N/A
pcDNA3.1-SARS-CoV-2 Spike B.1.526 variant	Li et al.³	N/A
pCAGGS-SARS-CoV-2 Spike B.1.617.1 variant	Li et al.³	N/A
pCAGGS-SARS-CoV-2 Spike B.1.617.2 variant	Li et al.³	N/A
pCAGGS-SARS-CoV-2 Spike B.1.1.529 (BA.1) variant	Chatterjee et al.¹³	N/A
pCAGGS-SARS-CoV-2 Spike B.1.1.529 (BA.2) variant	Ding et al.¹⁴	N/A
pCG1-SARS-CoV-2 Spike D614G RBM mutants panel	Li et al.³	N/A
pLTR-Tat	Finzi et al.¹⁵	N/A
pCMV-S: pCMV3-SARS-CoV-2 Spike (S1+S2)-long	Sino Biological	Cat # VG40589-UT
pACP-tag(m)-2 ACE2-Fc fusion protein	Anand et al.¹⁶	N/A
pTRIOZ-hIgG1	InvivoGen	N/A
pTRIOZ-hIgG1-EH3 heavy chain	This paper	N/A
pTRIOZ-hIgG1-EH3 light chain	This paper	N/A
pTRIOZ-hIgG1-EH8 heavy chain	This paper	N/A
pTRIOZ-hIgG1-EH8 light chain	This paper	N/A
pACP-SARS-CoV-2 RBD_mini(329-527)	Chen et al.¹⁷	N/A
pACP-SARS-CoV-2 RBD (319-537)	Chen et al.¹⁷	N/A
pACP-SARS-CoV-2 RBD (319-537) B.1.1.7 variant	Chen et al.¹⁷	N/A
pACP-SARS-CoV-2 RBD_mini (329-527) B.1.351 variant	Chen et al.¹⁷	N/A
pACP-SARS-CoV-2 RBD (319-537) B.1.429 variant	InvivoGen, this paper	N/A
pACP-SARS-CoV-2 RBD (319-537) B.1.526 variant	InvivoGen, this paper	N/A
pACP-SARS-CoV-2 RBD (319-537) B.1.617.2 variant	InvivoGen, this paper	N/A
pACP-SARS-CoV-2 RBD (319-537) B.1.1.529.1 variant	GenScript, this paper	N/A

Software and algorithms

FlowJo v10.5.3	Treestar	https://www.flowjo.com/
Graphpad Prism v9.1.0	GraphPad Software	https://www.graphpad.com/
CFX Maestro™ Software (qPCR analyses)	Bio-rad Inc	N/A
Living Image v4.7.3	PerkinElmer	N/A
Phenix	Liebschner et al.⁵³	https://phenix-online.org/
Coot	Emsley et al.⁵⁴	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
UCSF ChimeraX	Goddard et al.⁵⁵	https://www.rbvi.ucsf.edu/chimerax/
PyMOL	The PyMOL Molecular Graphics System	https://www.schrodinger.com/products/pymol
MolProbity	Williams et al.⁵⁶	http://molprobity.biochem.duke.edu/
PISA	Krissinel and Henrick,¹⁹	https://www.ebi.ac.uk/pdbe/pisa/
MATLAB	Mathworks	https://www.mathworks.com
SPARTAN	St. Jude Children’s Research Hospital	https://www.scottcblanchardlab.com/software
BI-Aevaluation software	GE Healthcare	Cat # BR-1005-97

Others

TriStar LB 942 Multimode Microplate Reader and Luminometer	BERTHOLD TECHNOLOGIES GmbH & Co. KG	N/A
BD LSR II Flow Cytometer	BD Biosciences	N/A
MinION sequencer	Oxford Nanopore Technologies	N/A
FLO-MIN106 flowcell	Oxford Nanopore Technologies	N/A
BioAnalyzer 2100	Agilent Technologies	N/A
Maxisorp Nunc™ White 96-Well Flat-Bottom Microplate	ThermoFisher Scientific	Cat # 437796
Prism-based TIRF Microscope	Mothes Lab	N/A
ORCA-Flash4.0 V3 sCMOS camera	Hamamatsu	N/A
C1000 Touch thermal cycler	Bio-Rad	N/A
CFX Connect™ Real-Time PCR Detection System	Bio-Rad	N/A
Nanodrop Spectrophotometer ND-1000	ThermoFisher Scientific	N/A
96-well white plates for luciferase assays	Costar	Cat # 3917
XIC-3 animal isolation chamber	PerkinElmer	Cat # 123997
IVIS Spectrum	PerkinElmer	Cat # 124262
RAS-4 Rodent Gas Anesthesia System	PerkinElmer	Cat # CLS146737
BeadBug 6 homogenizer	Benchmark Scientific	Cat # D1036
Superose 6 10/300 GL	GE Healthcare	Cat # 17517201
Hiload 26/600 Superdex 200 pg	GE Healthcare	Cat # 28989335
Biacore 3000	Cytiva	N/A
Protein A sensor chip	Cytiva	Cat # 29127558
Octet RED96e System	ForteBio	N/A