Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting

. 2022 Mar 7;38(12):110561. doi: 10.1016/j.celrep.2022.110561

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

CR3022	Produced in house (Yuan et al., 2020)	N/A
S309	Produced in house (Pinto et al., 2020	N/A
P2B-2F6	Produced in house (Ju et al., 2020	N/A
B38	Produced in house (Wu et al., 2020a, 2020b)	N/A
ADI-56046	Produced in house (Wec et al., 2020	N/A
ADI-55689	Produced in house (Wec et al., 2020)	N/A
ADI-55688	Produced in house (Wec et al., 2020	N/A
Ab15	This paper	N/A
Ab16	This paper	N/A
Ab17	This paper	N/A
Ab19	This paper	N/A
Ab20	This paper	N/A
HRP-conjugated rabbit anti-mouse IgG antibody	Abcam	CAT#ab97046
HRP-conjugated goat anti-mouse IgG, human/bovine/horse SP ads antibody	Southern Biotech	CAT#1013-05
CD3-BV786	BioLegend	CAT#100232
CD19-BV421	BioLegend	CAT#115549
IgM-BV605	BioLegend	CAT#406523
IgG-PerCP/Cy5.5	BioLegend	CAT#405314

Bacterial and virus strains

Sarbecovirus pseudotyped viruses	Produced in house (Garcia-Beltran et al., 2021a, 2021b)	N/A
WA1/2020 SARS-CoV-2 strain with a D614G mutation	(Chen et al., 2021)	N/A

Chemicals, peptides, and recombinant proteins

Monomeric and trimeric SARS-2, SARS-1, and WIV1 receptor binding domains	This paper	N/A
Monomeric SHC014 and RaTG13 receptor binding domains	This paper	N/A
SARS-2 two-proline stabilized spike ectodomain	Produced in house (Wrapp et al., 2020)	N/A
SARS-2^hg monomer and trimer	This paper	N/A
rsSARS-1^hg monomer and trimer	This paper	N/A
rsWIV1^hg monomer and trimer	This paper	N/A
RBM^hg monomer and trimer	This paper	N/A
Trimeric hemagglutinin head	This paper	N/A
Pierce HRV 3C protease	ThermoScientific	CAT#88946
Sigma Adjuvant System	SigmaAldrich	CAT#S6322
Streptavidin-PE conjugate	Invitrogen	CAT#12-4317-87
Aqua Live/Dead amine-reactive dye	ThermoFisher	CAT#L34957
Calcein, AM, cell-permeant dye	ThermoFisher	CAT#C3100MP
Streptavidin-APC/Cy7 conjugate	BioLegend	CAT#405208
Streptavidin-BV650 conjugate	BioLegend	CAT#405232
StrepTactin-PE conjugate	IBA Lifesciences	Item#6-5000-001
StrepTactin-APC conjugate	IBA Lifesciences	Item#6-5010-001
RNaseOUT	ThermoFisher	CAT#10777019
TALON Metal Affinity Resin	Takara	CAT#635652
Pierce Protein G Agarose	ThermoFisher	CAT#20399
1-Step ABTS substrate	ThermoFisher	Prod#37615

Critical commercial assays

FAB2G Biosensors	ForteBio	Item#18-5125
Ni-NTA Biosensors	ForteBio	Item#18-5101
SuperScript IV VILO MasterMix	ThermoFisher	CAT#11756050
Index crystal screen	Hampton Research	Item#HR2-144
MagMax mirVana Total RNA isolation kit	ThermoFisher	CAT#A27828

Deposited data

Atomic coordinates, Ab17 and SARS-2 RBD complex	Protein Data Bank	PDB 7TE1
Electron density map, Ab16 and SARS-2 spike complex	Electron Microscopy Data Bank	EMD-24894
Electron density map, Ab20 and SARS-2 spike complex	Electron Microscopy Data Bank	EMD-24895
Ab15 variable heavy chain	Genbank	OM407408
Ab16 variable heavy chain	Genbank	OM407409
Ab17 variable heavy chain	Genbank	OM407410
Ab19 variable heavy chain	Genbank	OM407411
Ab20 variable heavy chain	Genbank	OM407412
Ab15 variable light chain	Genbank	OM407413
Ab16 variable light chain	Genbank	OM407414
Ab17 variable light chain	Genbank	OM407415
Ab19 variable light chain	Genbank	OM407416
Ab20 variable light chain	Genbank	OM407417
B cell receptor sequences	Genbank	See Table S4 for accession numbers

Experimental models: Cell lines

Human: FreeStyle 293F	Thermo Fisher	Cat#R79007; RRID: CVCL_D603
Human: Expi293F	Thermo Fisher	Cat#A14527; RRID: CVCL_D615
Human: HEK 293T-humanACE2	(Moore et al., 2004)
Human: HEK293S GnTI-/-	ATCC	ATCC CRL-3022

Experimental models: Organisms/strains

C57BL/6 mice (strain: C57BL/6NCrl)	Charles River Laboratories	Strain code: 027
K18-hACE2 C57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J)	Jackson Laboratory	Strain #034860

Oligonucleotides

Murine B cell receptor sequencing primers	(Rohatgi et al., 2008; Tiller et al., 2009)	See Table S3 for oligo sequences

Software and algorithms

FlowJo v10	TreeStar	https://www.flowjo.com; RRID: SCR_008520
Prism v9	GraphPad	https://www.graphpad.com; RRID: SCR_002798
IMGT	International ImMunoGeneTics Information System	http://www.imgt.org; RRID: SCR_012780
Cloanalyst	(Kepler et al., 2014)	https://www.bu.edu/computationalimmunology/research/software/
RELION	(Scheres, 2012)	https://relion.readthedocs.io/en/release-3.1/
Chimera	(Pettersen et al., 2004)	https://www.cgl.ucsf.edu/chimera/
PHASER	(McCoy et al., 2007)	http://www.phaser.cimr.cam.ac.uk/index.php/Phaser_Crystallographic_Software
PHENIX	(Adams et al., 2010)	https://www.phenix-online.org/
COOT	(Emsley and Cowtan, 2004)	http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
PyMol	Schrödinger	http://www.pymol.org
MolProbity	(Chen et al., 2010)	http://molprobity.biochem.duke.edu
XDS	(Kabsch, 2010a, 2010b)	https://xds.mr.mpg.de

Other

Superdex 200 Increase 10/300 GL	GE Healthcare	Cytiva 28-9909-44