Discovery of nanobodies against SARS-CoV-2 and an uncommon neutralizing mechanism
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Abstract
SARS-CoV-2 and its variants continue to threaten public health. The virus recognizes the host cell by attaching its Spike receptor-binding domain (RBD) to the host receptor ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here we report the isolation, and biological and structural characterization of two single-chain antibodies (nanobodies, DL4 and DL28) from RBD-immunized alpaca. Both nanobodies bind Spike with affinities that exceeded the detection limit (picomolar) of the biolayer interferometry assay and neutralize the original SARS-CoV- 2 strain with IC 50 of 0.086 μg mL -1 (DL4) and 0.385 μg mL -1 (DL28). DL4 and a more potent, rationally designed mutant, neutralizes the Alpha variant as potently as the original strain but only displays marginal activity against the Beta variant. By contrast, the neutralizing activity of DL28, when in the Fc-fused divalent form, was less affected by the mutations in the Beta variant (IC 50 of 0.414 μg mL -1 for Alpha, 1.060 μg mL -1 for Beta). Crystal structure studies reveal that DL4 blocks ACE2-binding by direct competition, while DL28 neutralizes SARS-CoV-2 by an uncommon mechanism through which DL28 distorts the receptor-binding motif in RBD and hence prevents ACE2-binding. Our work provides two neutralizing nanobodies for potential therapeutic development and reveals an uncommon mechanism to design and screen novel neutralizing antibodies against SARS-CoV-2.
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SciScore for 10.1101/2021.07.20.453054: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Animal experiment and ethics: The alpaca immunization procedures were conducted in conformity with the institutional guidance for the care and use of laboratory animals, and the protocols were approved by the Institutional Committee of Ethics and Research of the Central Laboratory at Xinyang Agricultural and Forestry University. Sex as a biological variable The resulted emulsion was injected by the subcutaneous route at ten sites near the bow lymph node in the neck base of an adult female alpaca (3-years old). Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The plate was then … SciScore for 10.1101/2021.07.20.453054: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Animal experiment and ethics: The alpaca immunization procedures were conducted in conformity with the institutional guidance for the care and use of laboratory animals, and the protocols were approved by the Institutional Committee of Ethics and Research of the Central Laboratory at Xinyang Agricultural and Forestry University. Sex as a biological variable The resulted emulsion was injected by the subcutaneous route at ten sites near the bow lymph node in the neck base of an adult female alpaca (3-years old). Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The plate was then blocked by 0.5 %(w/v) BSA in TBS buffer for 30 min at RT and washed three times using TBS before incubated with anti-Myc antibodies at 1:2,000 dilution in TBS-BSA-T buffer (TBS supplemented with 0.5 %(w/v) BSA and 0.05 %(v/v anti-Mycsuggested: NoneFor simultaneous binding of DL28 and monoclonal antibodies (REGN10933, CV30, CB6, all in the IgG form) with RBD, the biotinylated RBD was coated as mentioned above. CV30suggested: NoneCB6suggested: NoneExperimental Models: Cell Lines Sentences Resources Protein expression and purification - Spike (S): The polypeptide containing, from N- to C-terminus, residues Met1 – Gln1208 (without the C-terminal transmembrane helix, Uniprot P0DTC2) of the SARS-CoV-2 S with mutations K986P/V987P, a GSAS linker substituting the furin sites (Arg682- Arg685), a C-terminal T4 fibritin trimerization motif (GYIPEAPRDGQAYVRKDGEWVLLSTFL), a TEV protease cleavage site, a FLAG tag and a polyhistidine tag 5 was encoded in a pCDNA3.1 backbone vector and overexpressed in Expi293 cells by transient transfection using polyethylenimine (PEI). Expi293suggested: RRID:CVCL_D615)Retroviral pseudotyped particles were generated by co-transfection of HEK293T cells using polyethylenimine with the expression vectors encoding the various viral envelope glycoproteins, the Murine leukemia virus core/packaging components (MLV Gag-Pol), and a retroviral transfer vector harboring the gene encoding the green fluorescent protein (GFP). HEK293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)VeroE6-hACE2 cells (104 cells/well) were seeded into a 48-well plate and infected 24 h later with 100 μL of virus supernatant in a final volume of 150 μL. VeroE6-hACE2suggested: NoneRecombinant DNA Sentences Resources Protein expression and purification - Spike (S): The polypeptide containing, from N- to C-terminus, residues Met1 – Gln1208 (without the C-terminal transmembrane helix, Uniprot P0DTC2) of the SARS-CoV-2 S with mutations K986P/V987P, a GSAS linker substituting the furin sites (Arg682- Arg685), a C-terminal T4 fibritin trimerization motif (GYIPEAPRDGQAYVRKDGEWVLLSTFL), a TEV protease cleavage site, a FLAG tag and a polyhistidine tag 5 was encoded in a pCDNA3.1 backbone vector and overexpressed in Expi293 cells by transient transfection using polyethylenimine (PEI). pCDNA3.1suggested: RRID:Addgene_79663)Protein expression and purification - RBD: The polypeptide containing, from N- to C-terminus, the honey bee melittin signal peptide (KFLVNVALVFMVVYISYIYAA), a Gly-Ser linker, residues 330-531 of the SARS-CoV-2 S, a Gly-Thr linker, the 3C protease site (LEVLFQGP), a Gly-Ser linker, the Avi tag (GLNDIFEAQKIEWHE), a Ser-Gly linker, and a deca-His tag was encoded in a pFastBac-backbone vector for overexpression in Trichoplusia ni High Five suspension cells. pFastBac-backbonesuggested: NoneBriefly, cells carrying nanobody-encoding pSb-init plasmids 46 were grown in Terrific Broth (TB, 0.17 M KH2PO4 and 0.72 M K2HPO4, 1.2 %(w/v) tryptone, 2.4 %(w/v) yeast extract, 0.5% (v/v) glycerol) supplemented with 25 mg L-1 chloramphenicol at 37 °C with shaking at 200 rpm. pSb-initsuggested: NoneOne microgram of the PCR product and 10 μg of the pDX_init vector 46 were digested separately with 50 units of BspQI (Cat. R0712L, New England Biolabs) for 1.5 h at 50 °C before heat inactivation at 80 °C for 10 min. pDX_initsuggested: RRID:Addgene_132697)The particles were eluted, and the phagemid was sub-cloned into pSb_init vector by fragment-exchange (FX) cloning and transformed into E. coli MC1061 cells for periplasmic expression and screening. pSb_initsuggested: RRID:Addgene_159422)SARS-CoV-2 pseudotypes for the Alpha (B1.1.7) and Beta (B1.351) variants were generated by incorporating the corresponding Spike mutations into the phCMV-SARS- CoV-2 plasmid. CoV-2suggested: NoneSoftware and Algorithms Sentences Resources The model was built with 2Fo-Fc maps in Coot 51, and refined using Phenix 52. Cootsuggested: (Coot, RRID:SCR_014222)Structures were visualized using PyMol. PyMolsuggested: (PyMOL, RRID:SCR_000305)Neutralization assay using SARS-CoV-2 SARS-CoV-2suggested: (BioLegend Cat# 944703, RRID:AB_2890874)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.
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