Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit
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Abstract
Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection. We previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 spike, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and binds to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is maintained against the Alpha, Delta, Gamma and Omicron variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region. Negative stain electron microscopy and a 1.74 Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs.
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SciScore for 10.1101/2021.08.02.454829: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable HCT-8 [HRT-18] cells (human male, ATCC CCL-244) were maintained in RPMI containing 10% horse serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Plates were washed 4X in wash buffer and the secondary antibody Goat anti-Human Ig-HRP (Southern Biotech, Cat# 2010-05), was added and incubated at 37°C for 1 hr. anti-Human Ig-HRPsuggested: (SouthernBiotech Cat# 2010-05, RRID:AB_2795564)After washing one time with PBS-T (PBS, 0.05% Tween-20), plate was incubated with rabbit … SciScore for 10.1101/2021.08.02.454829: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable HCT-8 [HRT-18] cells (human male, ATCC CCL-244) were maintained in RPMI containing 10% horse serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Plates were washed 4X in wash buffer and the secondary antibody Goat anti-Human Ig-HRP (Southern Biotech, Cat# 2010-05), was added and incubated at 37°C for 1 hr. anti-Human Ig-HRPsuggested: (SouthernBiotech Cat# 2010-05, RRID:AB_2795564)After washing one time with PBS-T (PBS, 0.05% Tween-20), plate was incubated with rabbit anti-nucleocapsid antibody (Sino Biological, Cat# 40643-T62) for 1h at room temperature on a plate shaker at 800 rpm. anti-nucleocapsidsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell Lines: All cell lines were incubated at 37°C in the presence of 5% CO2. 293-6E (human female, RRID:CVCL_HF20) and 293T cells (human female, RRID:CVCL_0063) cells were maintained in Freestyle 293 media with gentle shaking. thedetected: ( RRID:CVCL_HF20)293Tdetected: (CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)HCT-8 [HRT-18] cells (human male, ATCC CCL-244) were maintained in RPMI containing 10% horse serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. HCT-8suggested: NoneLLC-MK2 cells (Macaca mulatta, ATCC CCL-7) were maintained in cDMEM. LLC-MK2suggested: ATCC Cat# CCL-7, RRID:CVCL_3009)Huh7 cells (human male, a gift from Dr. Ram Savan, Department of Immunology University of Washington) were maintained in cDMEM. Huh7suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)The proteins were expressed in 293E cells and purified using Ni-NTA affinity resin followed by size exclusion chromatography on a superose 6 column as described in(Esposito et al., 2020) 293Esuggested: None, HIV-1 Tat (pHDM-tat1b, BEI resources NR-52518), SARS-CoV-2 spike (pHDM-SARS-CoV-2 Spike Wuhan-Hu-1, pHDM-SARS-CoV-2 Spike-B.1.1.7, SARS-CoV-2 Spike-P.1, pHDM-SARS-CoV-2 Spike-B.1.351 (Stamatatos et al., 2021), pCMV3-SARS-CoV-2-Spike-B.1.617.2, pTWist-WIV1-CoV (a gift from Alejandro Balazs (Addgene plasmid # 164438; http://n2t.net/addgene:164438; RRID:Addgene_164438), or pHDM-MERS-CoV Spike and a luciferase/GFP reporter (pHAGE-CMV-Luc2-IRES542 ZsGreen-W, BEI Resources Cat# NR-52516) were co-transfected into 293T cells at a 1:1:1:1.6:4.6 ratio using 293 Free transfection reagent according to the manufacturer’s instructions. 293Tsuggested: NonePseudoviron production was carried out at 32 °C for 72 hours after which the culture supernatant was harvested, clarified by centrifugation and frozen at −80°C. 293 cells stably expressing human HEK-293T-hACE2, for SARS-CoV-2 pseudoviruses, or Huh-7 cells for MERS-CoV pseudoviruses were seeded at a density of 4×103 cells/well in a 100 µL volume in 96-well flat bottom black-walled, clear bottom tissue culture plates (Greiner CELLSTAR Cat# 655090). 293suggested: NIH-ARP Cat# 103-306, RRID:CVCL_0045)Meanwhile 50 μL of cDMEM containing 6 µg/mL polybrene was added to each well of 293T-ACE2 or Huh-7 target cells and incubated for 30 min. Huh-7suggested: NoneRecombinant DNA Sentences Resources To generate a plasmid encoding the SARS-CoV-2 spike B.1.1. variant (pHDM-SARS-CoV-2-Spike-B.1.1.7) primers were designed that anneal 5’ of the H69 codon and just 3’ of the D1118 codon on the pHDM-SARS-CoV-2 Spike Wuhan-Hu-1 plasmid (BEI Resources Cat# NR-52514) and used to amplify cDNA corresponding to the N and C termini of the spike protein and the plasmid backbone using Platinum SuperFi II DNA Polymerase (Thermofisher Cat# 12368010) according to the manufacturer’s instructions. pHDM-SARS-CoV-2-Spike-B.1.1.7suggested: NoneThe synthesized DNA was cloned into the pHDM-SARS-CoV-2 Spike Wuhan-Hu-1 plasmid that was cut with EcoRI and HindIII and gel purified to remove the SARS-CoV-2 Spike cDNA using InFusion HD cloning Plus. Wuhan-Hu-1suggested: NoneBriefly, plasmids expressing the HIV-1 Gag and pol (pHDM540 Hgpm2, BEI Resources Cat# NR-52517), HIV-1Rev (pRC-CMV-rev1b, BEI Resources Cat# pRC-CMV-rev1bsuggested: RRID:Addgene_164443), HIV-1 Tat (pHDM-tat1b, BEI resources NR-52518), SARS-CoV-2 spike (pHDM-SARS-CoV-2 Spike Wuhan-Hu-1, pHDM-SARS-CoV-2 Spike-B.1.1.7, SARS-CoV-2 Spike-P.1, pHDM-SARS-CoV-2 Spike-B.1.351 (Stamatatos et al., 2021), pCMV3-SARS-CoV-2-Spike-B.1.617.2, pTWist-WIV1-CoV (a gift from Alejandro Balazs (Addgene plasmid # 164438; http://n2t.net/addgene:164438; RRID:Addgene_164438), or pHDM-MERS-CoV Spike and a luciferase/GFP reporter (pHAGE-CMV-Luc2-IRES542 ZsGreen-W, BEI Resources Cat# NR-52516) were co-transfected into 293T cells at a 1:1:1:1.6:4.6 ratio using 293 Free transfection reagent according to the manufacturer’s instructions. pCMV3-SARS-CoV-2-Spike-B.1.617.2suggested: Nonedetected: RRID:Addgene_164438)pHAGE-CMV-Luc2-IRES542suggested: NoneCell surface SARS-CoV-2 S binding assay: cDNA corresponding to AA 15-1336 of HCoV-OC43 was PCR amplified from pCAGGS-Flag-HCoV-OC43 Spike (a kind gift from Dr. Marceline Côté, University of Ottawa) and cloned into the pTT3 vector using InFusion cloning (Clontech). pCAGGS-Flag-HCoV-OC43suggested: NonepTT3suggested: NonecDNA for the HKU1 spike was PCR amplified from pCMV-HCoV-HKU1 (SinoBiological Cat# VG40606-UT) and subcloned into pTT3. pTT3-SARS-CoV-2-S (Seydoux et al., 2020), pHDM-MERS-CoV-Spike, pTWist-WIV1-CoV, pHDM-MERS-CoV-1 Spike, pTT3-HKU1 or pTT3-OC43 Spike were transfected into suspension-adapted 293T cells using 293 Free transfection reagent (EMD Millipore Cat# 72181) or PEI transfection reagent (PolySciences Inc. Cat# 23966) according to the manufacturer’s instructions. pCMV-HCoV-HKU1suggested: NonepHDM-MERS-CoV-Spikesuggested: NonepTT3-HKU1suggested: NonepTT3-OC43suggested: NoneSoftware and Algorithms Sentences Resources The antibody concentration that neutralized 50% or 80% of infectivity (IC50 and IC80 for mAbs) was interpolated from the neutralization curves determined using the log(inhibitor) vs. response -- Variable slope (four parameters) fit using automatic outlier detection in GraphPad Prism Software. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Model building was completed using COOT (Emsley and Cowtan, 2004) and refinement was performed in Phenix with the final refinement run through the PDB_REDO server (Joosten et al., 2014). COOTsuggested: (Coot, RRID:SCR_014222)Phenixsuggested: (Phenix, RRID:SCR_014224)Structural figures were made in Pymol (Schrodinger, LLC) Pymolsuggested: (PyMOL, RRID:SCR_000305)The plates were then washed once with 200µl of FACS buffer and stained with of PE-conjugated AffiniPure Fab fragment goat anti-human IgG (Jackson Immunoresearch Cat# 109-117-008) at a 1:100 dilution and live/dead green fluorescent reactive dye (Thermo Fisher Cat# L34970) at a 1:1000 dilution in 50 µl/well of 1X PBS. Thermo Fisher Cat#suggested: NoneResults 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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