Two pan-SARS-CoV-2 nanobodies and their multivalent derivatives effectively prevent Omicron infections in mice

. 2023 Jan 12;4(2):100918. doi: 10.1016/j.xcrm.2023.100918

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins

DMEM medium	Invitrogen	Cat# C11995500BT
Gibco fetal bovine sera	Gibco	Cat# 10099141C
SMM 293-TII Expression Medium	SinoBiological	Cat# M293TII-1L
Sinofection Transfection Reagent	SinoBiological	Cat# STF02-5ML
SMS 293-SUPI cell culture supplement	SinoBiological	Cat# M293-SUPI-100mL
Polyethylenimine (PEI)	Polysciences	Cat# 24885-2

Antibodies

Mouse anti-his mAb	ZSGB-BIO	Cat# TA-02; RRID:AB_2801388
Goat anti-Mouse IgG (H&L)-HRP conjugated	Easybio	Cat# BE0102; RRID:AB_2923205
Goat anti-Llama IgG H&L (HRP)	Abcam	Cat# ab112786; RRID:AB_10858603
410,712 APC anti-human IgG Fc	BioLegend	Cat# 410712; RRID:AB_2565790
Anti-HIS-APC, GG11-8F3.5.1, 100 tests	Miltenyi Biotec	Cat# 130-119-782; RRID:AB_2751840
M13 Bacteriophage Antibody (HRP)	SinoBiological	Cat# 11973-MM05T-H; RRID:AB_2857928
Anti-Human IgM antibody [IM260]	Abcam	Cat# ab200541

Critical commercial assays

HisTrap HP 5 mL column	GE Healthcare	Cat# 17524802
Protein A HP 5 mL column	GE Healthcare	Cat#17040303
HiLoad 16/600 Superdex 200 pg	GE Healthcare	Cat# 28989335
Hitrap IgM purification 5×1ML	GE Healthcare	Cat# 17511001
Superose 6 Increase 10/300 GL	GE Healthcare	Cat# 29091596
NuPAGE 3–8% native PAGE	Invitrogen	Cat# EA03752BOX
Series S Sensor Chip SA	GE Healthcare	Cat# 29104992
Series S Sensor Chip Protein A	GE Healthcare	Cat#29127556

Deposited data

R14/SARS-CoV-2 RBD complex	This paper	PDB: 7WD1
S43/SARS-CoV-2 RBD complex	This paper	PDB: 7WD2

Experimental models: Cell lines

HEK293T cells	ATCC	Cat# CRL-3216; RRID:CVCL_0063
HEK293F cells	Gibco	Cat# 11625-019
BHK-21 cells	ATCC	Cat# CCL-10; RRID:CVCL_1915
Sf9 Cells	Invitrogen	Cat# 11496015
Hi5 cells	Invitrogen	Cat# B85502
Vero cells	ATCC	Cat# CCL-81; RRID:CVCL_0059

Bacterial and virus strains

Escherichia coli (E. coli) strain DH5α	Vazyme	Cat# C502-02
Live SARS-CoV-2 prototype strain hCoV-19/China/CAS-B001/2020	This paper	GISAID: EPI_ISL_514256-7
Live SARS-CoV-2 prototype BetaCoV/Wuhan/IVDC-HB-envF13/2020	This paper	GISAID: EPI_ISL_408511
Live SARS-CoV-2 Beta strain	This paper	NPRC 2.192100004
Live SARS-CoV-2 BA.1 strain	This paper	NPRC 2.192100007
Live SARS-CoV-2 BA.2 strain	This paper	NPRC 2.192100010
Live SARS-CoV-2 BA.4 strain	This paper	NPRC 2.192100012
Live SARS-CoV-2 BA.5 strain	This paper	NPRC 2.192100014

Experimental models: Organisms/strains

SPF BALB/c mice, female, 6–8 weeks	Vital River (Beijing, China)	Cat# 01011
SPF K18-hACE2 mice, female, 6–8 weeks	GemPharmatech Co., Ltd (Jiangsu, China)	Strain NO. T037657

Recombinant DNA

pCAGGS	MiaoLingPlasmid	Cat# P0165
pMES4	Addgene	Cat# 154837
pEGFP-N1	MiaoLingPlasmid	Cat# P0133
pFastbac1	Invitrogen	Cat# 10360014
Prototype RBD, residues 319-541	This paper	GenBank: MN908947
Prototype RBD with N501Y mutation, residues 319–541 (Alpha)	This paper	N/A
Prototype RBD with K417N, E484K, N501Y mutation, residues 319–541 (Beta)	This paper	N/A
Prototype RBD with K417T, E484K, N501Y mutation, residues 319–541 (Gamma)	This paper	N/A
Prototype RBD with L452R, T478K mutation, residues 319–541 (Delta)	This paper	N/A
Prototype RBD with K417N, L452R, T478K mutation, residues 319–541 (Delta plus)	This paper	N/A
Prototype RBD with L452R, E484Q mutation, residues 319–541 (Kappa)	This paper	N/A
Prototype RBD with L452Q, F490S mutation, residues 319–541 (Lambda)	This paper	N/A
prototype RBD with R346K, E484K, N501Y mutation, residues 319–541 (Mu)	This paper	N/A
Omicron BA.1 RBD, residues 319-541	This paper	GISAID: EPI_ISL_6640916
Omicron BA.2 RBD, residues 319-541	This paper	GISAID: EPI_ISL_9652748
Omicron BA.2 RBD with L452Q, residues 319–541 (BA.2.12.1)	This paper	N/A
Omicron BA.2 RBD with D339H, G446S, N460K, R493Q, residues 319–541 (BA.2.75)	This paper	N/A
Omicron BA.3 RBD, residues 319-541	This paper	GISAID: EPI_ISL_7605589
Omicron BA.4/5 RBD, residues 319-541	This paper	GISAID: EPI_ISL_12029894
SARS-CoV RBD, residues 306-527	This paper	GenBank: NC_004718
RaTG13 RBD, residues 319-541	This paper	GenBank: QHR63300.2
RshSTT182 RBD, residues 319-541	This paper	GISAID: EPI_ISL_852604
RacCS203 RBD, residues 319-541	This paper	GenBank: QQM18864.1
Rc-o319 RBD, residues 319-541	This paper	GenBank: BCG66627.1
RsYN04 RBD, residues 309-527	This paper	GISAID: EPI_ISL_1699444
GX/P2V/2017 RBD, residues 319-541	This paper	GISAID: EPI_ISL_410542
GD/1/2019 RBD, residues 319-541	This paper	GISAID: EPI_ISL_410721

Software and algorithms

FlowJo V10	FLOWJO	https://www.flowjo.com/solutions/flowjo/downloads
Graphpad Prism 7	GraphPad Software	https://www.graphpad.com/
BIAcore® 8K Evaluation software	GE Healthcare	N/A
PKSolver	Zhang et al.⁴¹	N/A
HKL2000	Otwinowski et al.⁴²	https://www.hkl-xray.com/hkl-2000
Phaser	Read R.J. et al.⁴³	N/A
COOT	Emsley et al.⁴⁴	http://www.mrc-lmb.cam.ac.uk/personal/peemsley/coot/
Phenix	Adams et al.⁴⁵	http://www.phenix-online.org/
MolProbity	Williams et al.⁴⁶	http://molprobity.biochem.duke.edu/index.php
PyMOL software	Molecular Graphics System, Version 1.8 Schrö dinger	https://pymol.org/2/