Broadly neutralizing antibodies target the coronavirus fusion peptide
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Abstract
The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2′ cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain–specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2′ cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.
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SciScore for 10.1101/2022.04.11.487879: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: All participants in the convalescent cohort provided informed consent for their blood products to be used for research purposes by signing the standard New York Blood Center (NYBC
IRB: For participants who received the SARS-CoV-2 mRNA-1273 vaccine (Moderna), whole blood, plasma and serum samples were obtained at the NIH Clinical Research Center in Bethesda, MD under protocols approved by the NIH Institutional Review Board, ClinicalTrials
IACUC: Animal ethics statement: Animal research was conducted under an IACUC approved protocols at the Integrated Research Facility, Frederick, Maryland, in compliance with the Animal Welfare Act and other federal statutes and regulations relating to …SciScore for 10.1101/2022.04.11.487879: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: All participants in the convalescent cohort provided informed consent for their blood products to be used for research purposes by signing the standard New York Blood Center (NYBC
IRB: For participants who received the SARS-CoV-2 mRNA-1273 vaccine (Moderna), whole blood, plasma and serum samples were obtained at the NIH Clinical Research Center in Bethesda, MD under protocols approved by the NIH Institutional Review Board, ClinicalTrials
IACUC: Animal ethics statement: Animal research was conducted under an IACUC approved protocols at the Integrated Research Facility, Frederick, Maryland, in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals.
Field Sample Permit: Animal ethics statement: Animal research was conducted under an IACUC approved protocols at the Integrated Research Facility, Frederick, Maryland, in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals.Sex as a biological variable Hamsterization of human monoclonal antibodies: Genomes corresponding to the mouse IgG2a heavy and light chains were aligned to the genome assembly MesAur1.0 (GCA_000349665.1) for a female Syrian golden hamster downloaded from Genbank. Randomization No randomization or blinding was applied to the analysis of participants’ plasma, serum or PBMC samples, but all samples were anonymized before being used in this study. Blinding No randomization or blinding was applied to the analysis of participants’ plasma, serum or PBMC samples, but all samples were anonymized before being used in this study. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Binding of secreted antibody to the beads was detected in the CY5 or TRED channels by capturing images at 6 min intervals over a 30 min time course. CY5suggested: NoneShotgun mutagenesis epitope mapping of antibodies by alanine scanning: Epitope mapping was performed essentially as previously described (43), using a SARS-CoV-2 (Wuhan Hu-1 strain) S2 subunit shotgun mutagenesis mutation library, made using a full-length expression construct for the SARS-CoV-2 spike glycoprotein. SARS-CoV-2 spike glycoprotein .suggested: NoneVaccinee and convalescent plasma binding to peptides: Polyclonal IgG antibodies from plasma or sera of vaccinated, convalescent, or naïve donors were purified using the Pierce Protein G Spin Plate (Thermo Scientific). Polyclonal IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Baculoviruses were produced by transfection of bacmid DNA into Sf9 cells and used to infect High Five cells (Life Technologies) at high (5 to 10) multiplicity of infection (MOI). Sf9suggested: NonePlasma IgG reactivity to human coronaviruses and donor selection: Multiplexed beads for SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E and HCoV-NL63 spike proteins, as well as CD4 as a negative control, were incubated with donor plasma diluted at 1/50, 1/250 or 1/1250 for 30 min at room temperature, then washed and stained with 2.5 μg/mL goat anti-human IgG Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-170). HCoV-229Esuggested: JCRB Cat# JCRB1838, RRID:CVCL_B3M4)mAbs were also expressed in-house by transient transfection of Expi293 cells (Gibco, A14527) using the ExpiFectamine 293 Transfection Kit (Gibco, A14524) according to manufacturer’s instructions. Expi293suggested: RRID:CVCL_D615)To generate green fluorescent protein (GFP)-tagged receptor cell lines, HeLa-ACE2 cells were transduced with lentivirus encoding GFP and sorted to collect the GFPhigh/ACE2high population. HeLa-ACE2suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)mAbs were added to the wells at a final concentration of 200 μg/mL and cultures were further incubated at 37 °C for 1h. 8,000 GFP+/ACE2+ HeLa cells were then added to each well and the co-cultures were maintained overnight to allow for syncytia development. HeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)A plasmid encoding cDNA for each spike protein mutant was transfected into HEK-293T cells and allowed to express for 22 h. HEK-293Tsuggested: NoneFor neutralization assays, 5 × 104 RD cells were inoculated at TCID75% OC43-GFP virus and incubated for 1h at 35°C. 4-fold serial dilutions (73 ng/mL - 300 μg/mL) of each mAb were incubated with TCID75 OC43-GFP virus for 1h at 35°C. 60 μL of mAb- virus mixture was used to inoculate each well containing 5 × 104 RD cells and cultures were incubated for 24 h at 35°C. RDsuggested: Nonetransducing plasmid pHR’ CMV-Luc, a TMPRSS2 plasmid and full-length spike plasmids from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63 and HCoV-229E into 293T cells using Lipofectamine 3000 transfection reagent (ThermoFisher Scientific, Asheville, NC, L3000-001) (49). 293 flpin-TMPRSS2-ACE2 cells (provided by Dr. Adrian Creanga, VRC/NIH) were used for SARS-CoV-2, SARS-CoV and hCoV-NL63 while HuH7.5 cells were used for MERS-CoV and hCoV-229E neutralization assay. HCoV-NL63suggested: None293Tsuggested: NoneHuH7.5suggested: RRID:CVCL_7927)SARS: Addgene #170447; SARS2 #170442; MERS #170448; NL63 #172666; alpha strain #170451; beta #170449; gamma #170450; delta #172320; omicron 180375) were co-transfected in HEK293T with Lipofectamine 2000 (ThermoFisher Scientific, 11668019) to produce single-round infection-competent pseudoviruses. HEK293Tsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources SARS: Addgene #170447; SARS2 #170442; MERS #170448; NL63 #172666; alpha strain #170451; beta #170449; gamma #170450; delta #172320; omicron 180375) were co-transfected in HEK293T with Lipofectamine 2000 (ThermoFisher Scientific, 11668019) to produce single-round infection-competent pseudoviruses. #172666suggested: NoneRecombinant DNA Sentences Resources Pre-fusion stabilized constructs for CCoV HuPn-2018 (Accession # QVL91811.1, aa1-1384 with E1140P and E1141P mutations) and PdCoV0081-4 ( Accession # MW685622.1, aa1-1092 with E854P and V855P mutations) were synthesized and cloned into pCDNA3.1- vectors (Genscript) with the following C-terminal modifications: T4 fibritin trimerization motif, HRV3C protease cleavage site, poly-GS linker, Avi-tag, and 8× His tag. pCDNA3.1-suggested: RRID:Addgene_52535)Briefly, the SARS-CoV-2 NTD and RBD were cloned into an in-house pFastBac vector. pFastBacsuggested: RRID:Addgene_1925)The spike S2 domain (699 to 1207 with F817P, A892P, A899P, A942P, K986P, V987P) was constructed into phCMV3 vector which contained an N-terminal secreting signal peptide, and C-terminal thrombin cleavage site and His6 tag. phCMV3suggested: RRID:Addgene_173431)SARS-CoV-2 RBD, SARS- CoV-2 NTD, SARS-CoV-1 spike and SARS-CoV-1 RBD, MERS-CoV spike, OC43-CoV spike, CCoV-HuPn-2018 spike, pPDCoV-0081-4 spike, HCoV-NL63 spike, HCoV-229E spike, HCoV- HKU1 spike, H1 HA and recombinant CD4 (gifted by Gavin Wright, (35)). pPDCoV-0081-4suggested: NoneSpike-containing lentiviral pseudovirions were produced by co-transfection of packaging plasmid pCMVdR8.2, pCMVdR8.2suggested: RRID:Addgene_8455)transducing plasmid pHR’ CMV-Luc, a TMPRSS2 plasmid and full-length spike plasmids from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63 and HCoV-229E into 293T cells using Lipofectamine 3000 transfection reagent (ThermoFisher Scientific, Asheville, NC, L3000-001) (49). 293 flpin-TMPRSS2-ACE2 cells (provided by Dr. Adrian Creanga, VRC/NIH) were used for SARS-CoV-2, SARS-CoV and hCoV-NL63 while HuH7.5 cells were used for MERS-CoV and hCoV-229E neutralization assay. pHR’suggested: NoneTMPRSS2suggested: RRID:Addgene_53887)2.5μg 2nd generation lentivirus backbone plasmid pCMV-dR8.2 dvpr (Addgene, 8455), 2μg pBOBI-FLuc (Addgene, 170674) and 1μg truncated coronavirus spike expressing plasmids ( pCMV-dR8.2suggested: RRID:Addgene_8455)pBOBI-FLucsuggested: RRID:Addgene_170674)Hamster genes with the highest homology to the mouse IgG2a heavy chain, lambda and kappa light chains genes were cloned into a pCDNA3.4 vector (Genscript) and expressed in Expi293 cells as described above. pCDNA3.4suggested: NoneSoftware and Algorithms Sentences Resources ) high-throughput flow cytometer and FACS data were analysed with FlowJo (Version 10.8.1 FlowJosuggested: (FlowJo, RRID:SCR_008520)Analyses of the VH and Vλ/Vκ genes, CDR3 sequences, and percentage of somatic mutations were carried out using Geneious Prime (Version 2021.0.3, https://www.geneious.com) and the International Immunogenetics Information System database (IMGT, http://www.imgt.org/) (40). https://www.geneious.comsuggested: (Geneious, RRID:SCR_010519)http://www.imgt.org/suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)Recombinant IgG mAbs were purified using HiTrap Protein A columns (Cytiva/GE Healthcare Life Sciences, 17040303). Cytiva/GE Healthcaresuggested: NonePhylogenetic tree generation: Full-length amino acid sequences of SARS-CoV-2 (accession #NC_045512.2), SARS-CoV (accession # AY278741.1), MERS-CoV (accession # NC_019843) , HCoV-NL63 (accession #NC_005831.2), HCoV-229E (accession #NC_002645.1), CCoV HuPn-2018 (accession #MW591993.2) and PDCov-0081-4 (accession #MW685622) were aligned using the L-INS-i method of MAFFT version 7. MAFFTsuggested: (MAFFT, RRID:SCR_011811)The sequence alignment was used to generate a sequence logo plot using the Weblogo 3.0 server and to color conserved amino acid residues on a pre-fusion stabilized spike protein (PDB 6VSB). Weblogosuggested: (WEBLOGO, RRID:SCR_010236)Images were acquired in A488, A568 and DAPI channels using a BZ-X fluorescence microscope (KEYENCE) and processed using Fiji ImageJ (42) ImageJsuggested: (ImageJ, RRID:SCR_003070)Iterative model building and refinement were carried out in Coot (46) and PHENIX (47), respectively. Cootsuggested: (Coot, RRID:SCR_014222)PHENIXsuggested: (Phenix, RRID:SCR_014224)Authentic OC43-CoV-GFP virus propagation and neutralization assay: Rhabdomyosarcoma cells (RD, ATCC CCL-136) were maintained at 37°C and 5% CO2 in No-glucose DMEM (Gibco, 11966-025), supplemented with 10% HI-FBS, 4500 mg/mL glucose, 1 mM sodium pyruvate (Gibco, 11360-070), 1 mM HEPES (Gibco, 15630-080) and 50 μg/mL gentamycin (Quality Biological, 120-098-661) Quality Biologicalsuggested: None50% neutralization titers (NT50) were calculated using the dose- response-inhibition model with 5-parameter Hill slope equation in GraphPad Prism 9. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Descriptive statistics (mean ± SEM or mean ± SD) and statistical analyses were performed using Prism version 9.3.1 (GraphPad). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)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: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT00001281 Recruiting Studies of Blood and Reproductive Fluids in HIV-Infected and… NCT05078905 Recruiting Vaccine Responses to SARS-CoV-2 and Other Emerging Infectiou… 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.
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