Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease
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SciScore for 10.1101/2022.03.04.479488: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: COVID-19 infected-vaccinated donors: Sera and PBMC samples from convalescent COVID-19 donors, vaccinated donors, and COVID-19-recovered vaccinated donors, were provided through the “Collection of Biospecimens from Persons Under Investigation for 2019-Novel Coronavirus Infection to Understand Viral Shedding and Immune Response Study” UCSD IRB# 200236 as reported earlier (35).
Consent: All human donors were assessed for medical decision-making capacity using a standardized, approved assessment, and voluntarily gave informed consent prior to being enrolled in the
IACUC: S Animal Welfare Assurance Number: D16-00256 (A3410-01), under approved IACUC protocols.
Euthanasia Agents: …SciScore for 10.1101/2022.03.04.479488: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: COVID-19 infected-vaccinated donors: Sera and PBMC samples from convalescent COVID-19 donors, vaccinated donors, and COVID-19-recovered vaccinated donors, were provided through the “Collection of Biospecimens from Persons Under Investigation for 2019-Novel Coronavirus Infection to Understand Viral Shedding and Immune Response Study” UCSD IRB# 200236 as reported earlier (35).
Consent: All human donors were assessed for medical decision-making capacity using a standardized, approved assessment, and voluntarily gave informed consent prior to being enrolled in the
IACUC: S Animal Welfare Assurance Number: D16-00256 (A3410-01), under approved IACUC protocols.
Euthanasia Agents: Immediately prior to infection, mice were anesthetized by injection of ketamine and xylazine intraperitoneally and weighed.Sex as a biological variable 12-month-old female Balb/c mice (strain 047) were purchased from Envigo for Sarbecovirus challenge experiments (65, 85). Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Fluorescently labeled antibodies specific for cell surface markers were prepared as 1:100 dilution as a master mix in FACS buffer, to stain the PBMC samples for CD3 (APC-Cy7, BD Pharmingen cat.# CD3suggested: NoneFor recombinant protein ELISA, mouse anti-His antibody (Invitrogen cat. anti-Hissuggested: NoneAfter another washing, alkaline phosphatase-conjugated goat anti-human IgG Fc secondary antibody (Jackson ImmunoResearch cat.# anti-human IgGsuggested: None902360) were used to determine the reactivity of monoclonal antibodies to human epithelial type 2 (HEp2) by indirect immunofluorescence. human epithelial type 2suggested: NoneExperimental Models: Cell Lines Sentences Resources Cell lines: FreeStyle293-F cells (Thermo Fisher Scientific cat.# FreeStyle293-Fsuggested: RRID:CVCL_D603)R79007) were grown in FreeStyl 293 Expression Medium (Gibco cat.# 12338018), and Expi293F cells (Gibco cat.# Expi293Fsuggested: RRID:CVCL_D615)Adherent HEK293T cells and HeLa-ACE2 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% heat-inactivated FBS, 4mM L-Glutamine and 1% P/S, maintaining in the incubator at 37°C, 5% CO2. HeLa-ACE2suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)The stable hACE2 or hDPP4-expressing HeLa cell line was generated using an ACE2 lentivirus protocol previously described (7). HeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)ELISA using peptides or recombinant proteins: N-terminal biotinylated peptides corresponding to stem helix of SARS-CoV-1/2, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E and HCoV-NL63 were synthesized at A&A Labs ( HCoV-229Esuggested: JCRB Cat# JCRB1838, RRID:CVCL_B3M4)Pseudovirus production: HIV-based lentivirus backbone plasmid pCMV-dR8.2 dvpr (Addgene #8455), pBOB-Luciferase (Addgene #170674) were co-transfected into HEK293T cells along with full-length or variously truncated SARS-CoV1, WIV1, SHC014, Pang17, SARS-COV2, SARS-CoV-2 variants of concern [(B.1.1.7(alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta) and B.1.1.529 (Omicron)] and MERS-CoV spike using Lipofectamine 2000 (ThermoFisher Scientific cat.# 11668019) to produce single-round infection-competent pseudoviruses ( HEK293Tsuggested: NoneHEp2 epithelial cell polyreactive assay: According to manufacturer’s instructions, HEp2 slides (Hemagen cat.# HEp2suggested: CLS Cat# 300397/p694_Hep-2, RRID:CVCL_1906)Mock-transfected 293T cells were used as a negative control. 293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)Virus titration: SARS-CoV-2-MA10, SARS-CoV-1-MA15 and MERS-CoV-M35c4 were grown and titered using VeroE6 cells as previously described (87). VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Experimental Models: Organisms/Strains Sentences Resources 12-month-old female Balb/c mice (strain 047) were purchased from Envigo for Sarbecovirus challenge experiments (65, 85). Balb/csuggested: NoneC57Bl/6 288/330+/+ mice, which encode two human codons in the mouse dipeptidyl peptidase gene, were used for MERS-CoV mouse adapted challenge experiments (66). C57Bl/6 288/330+/+suggested: NoneRecombinant DNA Sentences Resources Plasmid construction: To generate soluble S ectodomain proteins from SARS-CoV-1 (residues 1-1190; GenBank: AAP13567) ,SARS-CoV-2 (residues 1-1208; GenBank: MN908947) , HCoV-HKU1 (residue 1-1295; GenBank: YP_173238.1) , HCoV-OC43 (residues 1-1300; GenBank: AAX84792.1), MERS-CoV (residues 1-1291; GenBank: APB87319.1), HCoV-229E (residues 1-1110; GenBank: NP_073551.1) and HCoV-NL63 (residues 1-1291; GenBank: YP_003767.1) , we synthesized the DNA fragments from GeneArt (Life Technologies) and cloned them into the phCMV3 vector (Genlantis cat.# phCMV3suggested: RRID:Addgene_173431)Briefly, the pBOB-hACE2 or hDPP4 plasmid and lentiviral packaging plasmids ( pBOB-hACE2suggested: NonehDPP4suggested: NonepMDL, pREV, and pVSV-G (Addgene #12251, #12253, #8454)) were co-transfected into HeLa cells using Lipofectamine 2000 reagent (ThermoFisher Scientific cat.# 11668019). pMDLsuggested: NonepREVsuggested: NonepVSV-Gsuggested: RRID:Addgene_138479)Pseudovirus production: HIV-based lentivirus backbone plasmid pCMV-dR8.2 dvpr (Addgene #8455), pBOB-Luciferase (Addgene #170674) were co-transfected into HEK293T cells along with full-length or variously truncated SARS-CoV1, WIV1, SHC014, Pang17, SARS-COV2, SARS-CoV-2 variants of concern [(B.1.1.7(alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta) and B.1.1.529 (Omicron)] and MERS-CoV spike using Lipofectamine 2000 (ThermoFisher Scientific cat.# 11668019) to produce single-round infection-competent pseudoviruses ( pCMV-dR8.2suggested: RRID:Addgene_8455)pBOB-Luciferasesuggested: NoneSoftware and Algorithms Sentences Resources The protocol was approved by the UCSD Human Research Protection Program. UCSD Human Research Protection Programsuggested: None08-771-23) and sorted for S protein-specific memory B cells using BD FACSMelody sorter. BD FACSMelodysuggested: NoneSingle colonies were picked for sequencing and analysis on IMGT V-Quest online tool (http://www.imgt.org) and downstream plasmid production. http://www.imgt.orgsuggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)Fifty percent maximal response concentrations (EC50) were calculated using the Asymmetrical dose-response model of Richard’s version in GraphPad Prism 7 (GraphPad Software). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Following three washes with FACS buffer, the cells were resuspended and analyzed by flow cytometry (BD Lyrics cytometer), and the binding data were generated by calculating the Mean Fluorescence Intensity using FlowJo 10 software. FlowJosuggested: (FlowJo, RRID:SCR_008520)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).
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.
- 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.
Results from scite Reference Check: We found no unreliable references.
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