ACE2-binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies
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Abstract
The coronavirus spike glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus spike proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors. These mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and betacoronaviruses, including animal coronaviruses WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses showed that the fusion peptide–specific mAbs bound with different modalities to a cryptic epitope hidden in prefusion stabilized spike, which became exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs.
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SciScore for 10.1101/2022.03.30.486377: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The study protocols were approved by the Cantonal Ethics Committee of Ticino, Switzerland (CE-TI-3428, 2018-02166; CE-TI-3687, 2020-01572).
Consent: All blood donors provided written informed consent for participation in the study.Sex as a biological variable Female hamsters of 6-8 weeks old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 µl containing 1×104 TCID50 SARS-CoV-2 gamma variant (day 0). Randomization not detected. Blinding Tissue sections (5 μm) were analyzed after staining with hematoxylin and eosin and scored blindly for lung damage by an expert pathologist. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
… SciScore for 10.1101/2022.03.30.486377: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The study protocols were approved by the Cantonal Ethics Committee of Ticino, Switzerland (CE-TI-3428, 2018-02166; CE-TI-3687, 2020-01572).
Consent: All blood donors provided written informed consent for participation in the study.Sex as a biological variable Female hamsters of 6-8 weeks old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 µl containing 1×104 TCID50 SARS-CoV-2 gamma variant (day 0). Randomization not detected. Blinding Tissue sections (5 μm) were analyzed after staining with hematoxylin and eosin and scored blindly for lung damage by an expert pathologist. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Plates were washed 4 × in TBST and 30 μl of anti-human (Invitrogen) horseradish peroxidase-conjugated antibodies diluted 1:5,000 was added to each well and incubated at 37°C. anti-humansuggested: NoneAfter washing, secondary antibody goat anti-human IgG (H+L) DyLight680 (0.2 µg/ml) was incubated for 45 min at room temperature before reading on Innopsys InnoScan 710-IR Microarray Scanner. Generation of recombinant ACE2-mFc: Residues 18-615 of human ACE2 (UniProtKB - Q9BYF1) were synthesized by Genscript and cloned into pINFUSE-mIgG2b-Fc2 expression plasmid (InvivoGen) anti-human IgGsuggested: NoneACE2suggested: NoneAfter 2 h, infected cells were washed four times with DMEM before adding medium supplemented with anti-VSV-G antibody (I1- mouse hybridoma supernatant diluted 1 to 25, from CRL-2700, ATCC). anti-VSV-Gsuggested: NoneExperimental Models: Cell Lines Sentences Resources Recombinant protein was produced by transient transfection of Expi293 cells and purified using HiTrap Protein A column. Expi293suggested: RRID:CVCL_D615)Cell lines and media culture: 293T and A549 cells were cultured in high glucose DMEM (Gibco, catalog no. 61965-026) supplemented with 10% FBS, 1% (vol/vol) 293Tsuggested: NoneHuH-7 cell line was obtained from JCRB Cell Bank and cultured in DMEM (Gibco, catalog no. 31885-023) supplemented with 10% FBS, 1% (vol/vol) HuH-7suggested: NoneVero-TMPRSS2 cells were cultured in DMEM (Gibco, catalog no. 11995-040) supplemented with 10% FBS (VWR, catalog no. 97068-085) and PenStrep (Gibco, catalog no. 15140-122) (35). Vero-TMPRSS2suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)The lentivirus-containing pellet was resuspended in 100 μl media and was used to transduce 293T or A549 cell lines. A549suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)293T-ACE2, 293T-S, A549-ACE2 and A549-S cell lines were selected using 10 μg/ml puromycin (InvivoGen, catalog no. ant-pr-1) 4 days post-transduction. A549-Ssuggested: None293T-ACE2-TMPRSS2-GFP cell line was generated from 293T-ACE2 cells by subsequent transduction of pLVX-EF1a-TMPRSS2-IRES-ZsGreen1-containing lentivral prep and sorted using BD FACSAria III. 293T-ACE2suggested: NoneA549-ACE2-TMPRSS2 and Huh7-TMPRSS2 stable cell lines were generated using commercial lentivirus (Addgene, catalog no. 154982-LV) and selected using 10 μg/ml blasticidin (InvivoGen Huh7-TMPRSS2suggested: NoneTo produce NL63 VSV virus, HEK-293 cells were transfected with a pcDNA3.1 expression vector encoding full-length S harboring a truncation of the 20 C-terminal residues to improve membrane transport. HEK-293suggested: NoneFor NL63 S pseudotyped virus neutralization assay, Vero E6-TMPRSS2 cells maintained in DMEM supplemented with 10% FBS and 1% PenStrep, were seeded into white 96-well plates at 45,000 cells/well and cultured overnight at 37°C. Vero E6-TMPRSS2suggested: NoneInhibition of cell-to-cell fusion: For testing inhibition of spike-mediated cell–cell fusion, A549-S and A549-ACE2-TMPRSS2 cells were stained with CFSE (Thermo Fisher, catalog no. C1157) and CellTrace™ Far Red (Thermo Fisher, catalog no. C34572), respectively, according to manufacturer’s instruction. A549-ACE2-TMPRSS2suggested: NoneThe titer of the virus stock was determined by end-point dilution on Vero-E6 cells by the Reed and Muench method (76) Vero-E6suggested: NoneTo quantify infectious SARS-CoV-2 particles, endpoint titrations were performed on confluent Vero E6 cells in 96- well plates. Vero E6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Transient expression and monoclonal antibody staining of HCoV S-expressing 293 cells: For transient expression of HCoV spike proteins, 293T cells were co-transfected, with plasmid encoding ZsGreen (Bei Resources, catalog no. NR-52516) and corresponding HCoV spike proteins: SARS-CoV-2 Wuhan-Hu-1 S (catalog no. NR-52514) from Bei Resources; MERS-CoV S (VG40069-G-N) SARS-CoV-2 Wuhan-Hu-1 Ssuggested: NoneRecombinant DNA Sentences Resources SARS-CoV-2 S PentaPro gene, codon optimized for mammalian expression, was synthesized by GeneScript and cloned into pcDNA3.1 (-) expression vector. pcDNA3.1 ( - )suggested: NoneGeneration of overexpression cell lines: pLVX-puro-ACE2 transfer plasmid was kindly provided by Manfred Kopf (ETH Zurich). pLVX-puro-ACE2suggested: NonepLVX-EF1a-TMPRSS2-IRES-ZsGreen1 transfer plasmid was generated from the reference pWPI-IRES-Bla-Ak-TMPRSS2 plasmid (Addgene, catalog no. 154982). pLVX-EF1a-TMPRSS2-IRES-ZsGreen1suggested: NonepWPI-IRES-Bla-Ak-TMPRSS2suggested: RRID:Addgene_154982)pLVX-puro-spike transfer plasmid was generated from reference pHDM-SARS-CoV-2 spike (BEI resources, catalog no. NR-52514) pLVX-puro-spikesuggested: NoneBriefly, 293T cells at 70-80% confluency in T75 flask were co-transfected with transfer plasmids encoding genes of interest (ACE2, TMPRSS2 or SARS-CoV-2 S) and packaging plasmid psPax2 and envelope plasmid pMD. psPax2suggested: RRID:Addgene_12260)pMDsuggested: NoneBriefly, 293T cells were co-transfected with a lentiviral backbone encoding luciferase reporter (pHAGE-CMV-Luc2-IRES-ZsGreen-W (Bei Resources, catalog no. NR-52516), HIV-based packaging plasmids (Tat, Gag-Pol and Rev) (Bei Resources, catalog no. NR-52518, NR-52517 and NR-52519) and various spike expression plasmids using PEI in Optimem. pHAGE-CMV-Luc2-IRES-ZsGreen-Wsuggested: RRID:Addgene_164432)To produce NL63 VSV virus, HEK-293 cells were transfected with a pcDNA3.1 expression vector encoding full-length S harboring a truncation of the 20 C-terminal residues to improve membrane transport. pcDNA3.1suggested: RRID:Addgene_79663), NL63 S (VG40604-UT) from SinoBiological; SARS-CoV S (VG40150-G-N) from SinoBiological that was cloned into pHDM expression plasmid with 19 amino-acid C-terminal truncation (85), using PEI in Optimem as above. pHDMsuggested: NoneSoftware and Algorithms Sentences Resources Plates were immediately read at 450 nm on a BioTek plate reader and data plotted and fit in Prism 9 (GraphPad) using nonlinear regression sigmoidal, 4PL, X is the concentration to determine EC50 values from curve fits. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)First, the sequences were annotated using IgBlast version 1.16 (65) and IMGT as reference sequences (66). IgBlastsuggested: (IgBLAST, RRID:SCR_002873)Data was processed using GraphPad Prism v9.0. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Nine fields per well were imaged and were subsequently processed with Metaxpress and Powecore softwares. Metaxpresssuggested: (MetaXpress, RRID:SCR_016654)The variant was subjected to sequencing on a MinION platform (Oxford Nanopore) directly from the nasopharyngeal swabs; passage 2 virus on Vero E6 cells was used for the study described here. MinIONsuggested: (MinION, RRID:SCR_017985)Initial phases were obtained by molecular replacement in Phaser (81) on the CCP4 suite, using crystal structures of Fabs as search models. CCP4suggested: (CCP4, RRID:SCR_007255)Several subsequent rounds of model building and refinement were performed using Coot (82), Refmac5 (83) and Buster (84) to arrive to the final model for each complex. Cootsuggested: (Coot, RRID:SCR_014222)Cells were then washed and analyzed by flow cytometry using BD Symphony and FlowJo. FlowJosuggested: (FlowJo, RRID:SCR_008520)Results from OddPub: Thank you for sharing your data.
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: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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