Potent human broadly SARS-CoV-2–neutralizing IgA and IgG antibodies effective against Omicron BA.1 and BA.2
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Abstract
Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in Wuhan COVID-19 convalescents combining serological, cellular, and monoclonal antibody explorations revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor-binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.
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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: CD19suggested: NoneThe 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). CD3sugg…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: CD19suggested: NoneThe 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). CD3suggested: (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. IgG1suggested: NoneCB6suggested: NoneLY-CoV555suggested: NoneS309suggested: NoneComparative ELISA binding of Cv2.1169 IgG1 and IgA1 antibodies was performed at a concentration of 70 nM, and 7 consecutive dilutions in PBS. IgA1suggested: NoneTo 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 IgAsuggested: NoneIgG antibodysuggested: NoneAfter 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 IgGsuggested: Noneanti-golden hamster IgGsuggested: Noneanti-human IgGsuggested: Noneanti-human IgAsuggested: NoneHEp-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 IgGsuggested: NoneED38suggested: NoneMembranes 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-Hisx6suggested: NoneMicroarrays 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). IgGsuggested: NoneAntibodies (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. ACE2suggested: NoneAntibody-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 ( ADCPsuggested: NonePhagocytic 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-2suggested: NoneAntibody-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 IgGsuggested: NoneBriefly, 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 IgGsuggested: NoneCrystallization 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 Ssuggested: NoneSix 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. IgAsuggested: NoneFour 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. mGO53suggested: NoneExperimental Models: Cell Lines Sentences Resources Viruses were amplified by one or two passages in Vero E6 cell cultures and titrated. Vero E6suggested: RRID:CVCL_XD71)Drosophila S2 cells were stably co-transfected with pT350 and pCoPuro (for puromycin selection) plasmids. S2suggested: NoneHEp-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-2suggested: 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-rLucsuggested: NoneBriefly, 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-Spikesuggested: NoneThe 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-E6suggested: NoneExperimental 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/Jsuggested: 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: NoneFor 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/BiPsuggested: NoneDrosophila S2 cells were stably co-transfected with pT350 and pCoPuro (for puromycin selection) plasmids. pT350suggested: NonepCoPurosuggested: 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: NoneThe 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: NoneTo 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-dfursuggested: NoneSARS-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). GFP1-10suggested: 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 Technologiessuggested: (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-2suggested: (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). FlowJosuggested: (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) Zensuggested: NoneFor 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® Prospectorsuggested: NoneThe evaluation kinetic parameters of the studied interactions were performed by using BIAevaluation version 4.1.1 Software (Biacore). BIAevaluationsuggested: (BIAevaluation Software, RRID:SCR_015936)IC50 values were calculated using Prism software (v.9.3.1, Prismsuggested: (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. Cootsuggested: (Coot, RRID:SCR_014222)The final models were validated with Molprobity (Williams et al., 2018). Molprobitysuggested: (MolProbity, RRID:SCR_014226)Superpositions and figures were rendered using Pymol and UCSF Chimera (Pettersen et al., 2004) Pymolsuggested: (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. CryoSPARCsuggested: (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 Prismsuggested: (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. Circossuggested: (Circos, RRID:SCR_011798)GraphPad Prism Inc.) GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Principal component analysis (PCA) was performed using the prcomp() function in R Studio Server (v1.4.1103) R Studio Serversuggested: NoneResults 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).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.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… Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
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