Potent human broadly SARS-CoV-2–neutralizing IgA and IgG antibodies effective against Omicron BA.1 and BA.2

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1. Version published to 10.1084/jem.20220638

   Jun 15, 2022
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   Apr 5, 2022

   SciScore for 10.1101/2022.04.01.486719: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   B cells were incubated for 30 min at 4°C with biotinylated tri-S and DyLight 650-coupled RBD, washed once with 1% FBS-PBS (FACS buffer), and incubated for 30 min at 4°C with a cocktail of mouse anti-human antibodies: CD19 Alexa 700 (HIB19, BD Biosciences, San Jose,	anti-human antibodies: CD19 suggested: None
   The cTfh antibody panel included: CD3 BV605 (SK7), CD4 PE-CF594 (RPA-T4), CD185/CXCR5 AF-488 (RF8B2), CD183/CXCR3 PE-Cy™5 (1C6/CXCR3), CD196/CCR6 PE-Cy™7 (11A9), CD197/CCR7 AF647 (3D12) (BD Biosciences), CD279/PD1 BV421 (EH12.2H7, BioLegend), and CD278/ICOS PE (ISA-3, Thermo Fisher Scientific).	CD3 sugg…

   SciScore for 10.1101/2022.04.01.486719: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   B cells were incubated for 30 min at 4°C with biotinylated tri-S and DyLight 650-coupled RBD, washed once with 1% FBS-PBS (FACS buffer), and incubated for 30 min at 4°C with a cocktail of mouse anti-human antibodies: CD19 Alexa 700 (HIB19, BD Biosciences, San Jose,	anti-human antibodies: CD19 suggested: None
   The cTfh antibody panel included: CD3 BV605 (SK7), CD4 PE-CF594 (RPA-T4), CD185/CXCR5 AF-488 (RF8B2), CD183/CXCR3 PE-Cy™5 (1C6/CXCR3), CD196/CCR6 PE-Cy™7 (11A9), CD197/CCR7 AF647 (3D12) (BD Biosciences), CD279/PD1 BV421 (EH12.2H7, BioLegend), and CD278/ICOS PE (ISA-3, Thermo Fisher Scientific).	CD3 suggested: (SouthernBiotech Cat# 9515-31, RRID:AB_2796843)
   The purified parental IgG1 antibody versions of benchmarked mAbs [REGN10933, REGN10987 (Hansen et al., 2020), CB6 (Shi et al., 2020), LY-CoV555 (Jones et al., 2021), CT-P59 (Kim et al., 2021), COV2-2196, COV2-2130 (Zost et al., 2020b), ADG-2 (Garrett Rappazzo et al., 2021) and S309 (Pinto et al., 2020)] were prepared as described above after cloning of synthetic DNA fragments (GeneArt, Thermo Fisher Scientific) coding for the immunoglobulin variable domains.	IgG1 suggested: None CB6 suggested: None LY-CoV555 suggested: None S309 suggested: None
   Comparative ELISA binding of Cv2.1169 IgG1 and IgA1 antibodies was performed at a concentration of 70 nM, and 7 consecutive dilutions in PBS.	IgA1 suggested: None
   To quantify blood-circulating human Cv2.1169 IgA1 and IgG1 in treated K18-hACE2 mice and golden hamsters, high-binding 96-well ELISA plates (Costar, Corning) were coated overnight with 250 ng/well of purified goat anti-human IgA or IgG antibody (Jackson ImmunoResearch, 0.8 µg/ml final).	purified goat anti-human IgA suggested: None IgG antibody suggested: None
   After washings, the plates were revealed by incubation for 1 h with goat HRP-conjugated anti-mice IgG, anti-golden hamster IgG, anti-human IgG or anti-human IgA antibodies (Jackson ImmunoReseach, 0.8 µg/ml final) and by adding 100 µl of HRP chromogenic substrate (ABTS solution, Euromedex) after washing steps.	anti-mice IgG suggested: None anti-golden hamster IgG suggested: None anti-human IgG suggested: None anti-human IgA suggested: None
   HEp-2 IFA assay: Recombinant SARS-CoV-2 S-specific and control IgG antibodies (mGO53 and ED38) at 100 µg/ml were analyzed by indirect immuno-fluorescence assay (IFA) on HEp-2 cells sections (ANA HEp-2 AeskuSlides®, Aesku.Diagnostics, Wendelsheim, Germany) using the kit’s controls and FITC-conjugated anti-human IgG antibodies as the tracer according to the manufacturer’ instructions.	control IgG suggested: None ED38 suggested: None
   Membranes were inserted into a Miniblot apparatus (Immunetics) and then incubated with human mAbs (at a concentration of 1 µg/ml) and mouse anti-Hisx6 antibody (1 µg/ml, BD Biosciences) in PBS-T 5% dry milk in each channel for 2 h.	anti-Hisx6 suggested: None
   Microarrays were blocked for 1 h in blocking solution (Thermo Fisher), washed and incubated for 1h30 with IgG antibodies at 2.5 µg/ml as previously described (Grzelak et al., 2020).	IgG suggested: None
   Antibodies (Cv2.1169, Cv2.1353, Cv2.3194, Cv2.3235 and Cv2.5213) and ACE2 ectodomain were covalently coupled to CM5 sensor chips (Biacore) using amino-coupling kit (Biacore) according to the manufacturer’s procedure.	ACE2 suggested: None
   Antibody-dependent cellular phagocytosis (ADCP) assay: PBMC were isolated from healthy donors’ blood (Etablissement Français du Sang) using Ficoll Plaque Plus (GE Healthcare)	Antibody-dependent cellular phagocytosis ( ADCP suggested: None
   Phagocytic scores were calculated by dividing the fluorescence signals (% FITC-positive cells x geometric MFI FITC-positive cells) given by anti-SARS-CoV-2 spike antibodies by the one of the negative control antibody mGO53.	anti-SARS-CoV-2 suggested: None
   Antibody-dependent cellular cytotoxicity (ADCC) assay: The ADCC activity of anti-SARS-CoV2 S IgG antibodies was determined using the ADCC Reporter Bioassay (Promega) as previously described (Dufloo et al., 2021).	anti-SARS-CoV2 S IgG suggested: None
   Briefly, 5×104 Raji-Spike cells were co-cultured with 5×104 Jurkat-CD16-NFAT-rLuc cells in presence or absence of SARS-CoV2 S-specific or control mGO53 IgG antibody at 10 µg/ml or 50 µg/ml and 10 consecutive 1:2 dilutions in PBS.	control mGO53 IgG suggested: None
   Crystallization and structure determinations: The Fab of anti-SARS-CoV-2 S antibody CR3022 (Ter Meulen et al., 2006), served as a crystallization chaperone molecule, and was produced and purified as described above (section with heading Single B-cell FACS sorting and expression-cloning of antibodies) (Koide, 2009).	anti-SARS-CoV-2 S suggested: None
   Six or 22 h post-inoculation, mice received an intraperitoneal (i.p.) injection of 5, 10, 20 or 40 mg/kg of Cv2.1169 IgG or IgA antibody, and of mGO53 control IgG or IgA antibody.	IgA suggested: None
   Four or 24 h post-intranasal inoculation, hamsters received an intraperitoneal (i.p.) injection of 10 or 5 mg/kg of Cv2.1169 IgG or IgA antibody, as well as the mGO53 control antibody or PBS.	mGO53 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Viruses were amplified by one or two passages in Vero E6 cell cultures and titrated.	Vero E6 suggested: RRID:CVCL_XD71)
   Drosophila S2 cells were stably co-transfected with pT350 and pCoPuro (for puromycin selection) plasmids.	S2 suggested: None
   HEp-2 IFA assay: Recombinant SARS-CoV-2 S-specific and control IgG antibodies (mGO53 and ED38) at 100 µg/ml were analyzed by indirect immuno-fluorescence assay (IFA) on HEp-2 cells sections (ANA HEp-2 AeskuSlides®, Aesku.Diagnostics, Wendelsheim, Germany) using the kit’s controls and FITC-conjugated anti-human IgG antibodies as the tracer according to the manufacturer’ instructions.	HEp-2 suggested: CLS Cat# 300397/p694_Hep-2, RRID:CVCL_1906)
   Briefly, 5×104 Raji-Spike cells were co-cultured with 5×104 Jurkat-CD16-NFAT-rLuc cells in presence or absence of SARS-CoV2 S-specific or control mGO53 IgG antibody at 10 µg/ml or 50 µg/ml and 10 consecutive 1:2 dilutions in PBS.	Jurkat-CD16-NFAT-rLuc suggested: None
   Briefly, 5×104 Raji-Spike cells were cultivated in the presence of 50% normal or heat-inactivated human serum, and with or without IgG antibodies (at 10 µg/ml or 50 µg/ml and 10 consecutive 1:2 dilutions in PBS).	Raji-Spike suggested: None
   The supernatants were titrated on Vero-E6 cells by classical plaque assays using semisolid overlays (Avicel, RC581-NFDR080I, DuPont) and expressed and PFU/100 mg of tissue (Baer and Kehn-Hall, 2014).	Vero-E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   SARS-CoV-2 infection and treatment in K18-hACE2 mice: B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (stock #034860) were imported from The Jackson Laboratory (Bar Harbor, ME, USA) and bred at the Institut Pasteur under strict SPF conditions.	B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: RRID:IMSR_JAX:034860)
   Recombinant DNA
   Sentences	Resources
   Expression and purification of viral proteins: Codon-optimized nucleotide fragments encoding stabilized versions of SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43-CoV, HKU1-CoV, 229E-CoV, NL63-CoV (2P) and BA.1 spike (HexaPro) (S) ectodomains, and SARS-CoV-2 S2 domain, followed by a foldon trimerization motif and C-terminal tags (Hisx8-tag, Strep-tag, and AviTag) were synthesized and cloned into pcDNA3.1/Zeo(+) expression vector (Thermo Fisher Scientific).	pcDNA3.1/Zeo(+ suggested: None
   For crystallographic experiments, a codon-optimized nucleotide fragment encoding the SARS-CoV-2 RBD protein (residues 331-528), followed by an enterokinase cleavage site and a C-terminal double strep-tag was cloned into a modified pMT/BiP expression vector (pT350, Invitrogen)	pMT/BiP suggested: None
   Drosophila S2 cells were stably co-transfected with pT350 and pCoPuro (for puromycin selection) plasmids.	pT350 suggested: None pCoPuro suggested: RRID:Addgene_17533)
   For Cryo-EM experiments, a codon-optimized nucleotide fragment encoding the SARS-CoV-2 spike (S) protein (residues 1-1208) was cloned with its endogenous signal peptide in pcDNA3.1(+) vector, and expressed as a stabilized trimeric prefusion construct with six proline substitutions (F817P, A892P, A899P, A942P, K986P, V987P), along with a GSAS substitution at the furin cleavage site (residues 682–685), followed by a Foldon trimerization motif (Hsieh et al., 2020), and C-terminal tags (Hisx8-tag, Strep-tag and AviTag).	pcDNA3.1 ( + ) suggested: None
   The dimeric form of Cv2.1169 IgA1 was produced by co-transfection of Freestyle™ 293-F cells with a human J chain pcDNA™3.1/Zeo(+) vector as previously described (Lorin and Mouquet, 2015)	pcDNA™3.1/Zeo(+ suggested: None
   To evaluate spike cross-reactivity, Freestyle™ 293-F were transfected with pUNO1-Spike-dfur expression vectors (Spike and SpikeV1 to V11 plasmids, Invivogen) (1.2 µg plasmid DNA per 106 cells) using PEI-precipitation method.	pUNO1-Spike-dfur suggested: None
   SARS-CoV-2 S-Fuse neutralization assay: S-Fuse cells (U2OS-ACE2 GFP1-10 or GFP 11 cells) were mixed (ratio 1:1) and plated at a density of 8 × 103 per well in a μClear 96-well plate (Greiner Bio-One) as previously described (Buchrieser et al., 2020).	GFP1-10 suggested: RRID:Addgene_68715)
   Software and Algorithms
   Sentences	Resources
   Cv2.1169 were also cloned into human Igγ1NA, Igγ1LALA [N297A and L234A/L235A mutations introduced by Site-Directed Mutagenesis (QuickChange, Agilent Technologies)], Igα1 and Fab-Igα1-expressing vectors (Lorin and Mouquet,	Agilent Technologies suggested: (Agilent Technologies, RRID:SCR_013575)
   Flow cytometry binding assays: SARS-CoV-2 specificity validation of cloned human IgG antibodies was performed using the S-Flow assay as previously described (Grzelak et al., 2020).	SARS-CoV-2 suggested: (Active Motif Cat# 91351, RRID:AB_2847848)
   Data were acquired using a CytoFLEX flow cytometer (Beckman Coulter), and analyzed using FlowJo software (v10.7.1; FlowJo LLC).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   HEp-2 sections were examined using the fluorescence microscope Axio Imager 2 (Zeiss, Jena, Germany), and pictures were taken at magnification x 40 with 5000 ms-acquisition using ZEN imaging software (Zen 2.0 blue version, Zeiss) at the Imagopole platform (Institut Pasteur)	Zen suggested: None
   For each antibody, Z-scores were calculated using ProtoArray® Prospector software (v5.2.3, Thermo Fisher Scientific), and deviation (σ) to the diagonal, and polyreactivity index (PI) values were calculated as previously described (Planchais et al., 2019).	ProtoArray® Prospector suggested: None
   The evaluation kinetic parameters of the studied interactions were performed by using BIAevaluation version 4.1.1 Software (Biacore).	BIAevaluation suggested: (BIAevaluation Software, RRID:SCR_015936)
   IC50 values were calculated using Prism software (v.9.3.1,	Prism suggested: (PRISM, RRID:SCR_005375)
   The final models were built by combining real space model building in Coot (Emsley et al., 2010) with reciprocal space refinement with phenix.refine.	Coot suggested: (Coot, RRID:SCR_014222)
   The final models were validated with Molprobity (Williams et al., 2018).	Molprobity suggested: (MolProbity, RRID:SCR_014226)
   Superpositions and figures were rendered using Pymol and UCSF Chimera (Pettersen et al., 2004)	Pymol suggested: (PyMOL, RRID:SCR_000305)
   CryoSPARC blob picker was used for automated particle picking and the resulting particles used to obtain initial 2D references, which were then used to auto-pick the micrographs.	CryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   Volcano plot comparing gene features (n=206 parameters) of tri-S+ B cells and normal memory B-cells (mB) was also performed using GraphPad Prism software (v.8.4,	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Circos plot linking antibody sequences with at least 75% identity within their CDRH3 was performed using online software at http://mkweb.bcgsc.ca/circos.	Circos suggested: (Circos, RRID:SCR_011798)
   GraphPad Prism Inc.)	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Principal component analysis (PCA) was performed using the prcomp() function in R Studio Server (v1.4.1103)	R Studio Server suggested: None

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04325646	Recruiting	Sero-epidemiological Study of the SARS-CoV-2 Virus Responsib…

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Version published to 10.1101/2022.04.01.486719v1 on bioRxiv

   Apr 1, 2022