Deep Mutational Engineering of broadly-neutralizing and picomolar affinity nanobodies to accommodate SARS-CoV-1 & 2 antigenic polymorphism
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Abstract
We report in this study the molecular engineering of nanobodies that bind with picomolar affinity to both SARS-CoV-1 and SARS-CoV-2 Receptor Binding Domains (RBD) and are highly neutralizing. We applied Deep Mutational Engineering to VHH72, a nanobody initially specific for SARS-CoV-1 RBD with little cross-reactivity to SARS-CoV-2 antigen. We first identified all the individual VHH substitutions that increase binding to SARS-CoV-2 RBD and then screened highly focused combinatorial libraries to isolate engineered nanobodies with improved properties. The corresponding VHH-Fc molecules show high affinities for SARS-CoV-2 antigens from various emerging variants and SARS-CoV-1, block the interaction between ACE2 and RBD and neutralize the virus with high efficiency. Its rare specificity across sarbecovirus relies on its peculiar epitope outside the immunodominant regions. The engineered nanobodies share a common motif of three amino acids, which contribute to the broad specificity of recognition. These nanobodies appears as promising therapeutic candidates to fight SARS-CoV-2 infection.
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SciScore for 10.1101/2021.12.07.471597: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Cells were incubated on ice for 15 minutes with anti- HA antibody (Invitrogen HA Tag Mouse anti-Tag, DyLight® 650 conjugate, Clone: 2-2.2.14; 1:100 dilution) and Streptavidin-PE (Thermo Fisher scientific; catalog number S866; 1:100 dilution). anti- HAsuggested: (Claes Ãrvel lab; Karolinska Institute Cat# anti-MuV rabbit 144, RRID:AB_2747378)anti-Tagsuggested: NoneCompetitive indirect enzyme linked immunoassay (competitive ELISA): Nunc™ MaxiSorp 96-well Immuno-Plates (Thermo Fisher Scientific, Illkirch, France) were coated with 200 μL/well of AffinityPure goat anti-mouse IgG+IgM (H+L) antibody (Jackson … SciScore for 10.1101/2021.12.07.471597: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Cells were incubated on ice for 15 minutes with anti- HA antibody (Invitrogen HA Tag Mouse anti-Tag, DyLight® 650 conjugate, Clone: 2-2.2.14; 1:100 dilution) and Streptavidin-PE (Thermo Fisher scientific; catalog number S866; 1:100 dilution). anti- HAsuggested: (Claes Ãrvel lab; Karolinska Institute Cat# anti-MuV rabbit 144, RRID:AB_2747378)anti-Tagsuggested: NoneCompetitive indirect enzyme linked immunoassay (competitive ELISA): Nunc™ MaxiSorp 96-well Immuno-Plates (Thermo Fisher Scientific, Illkirch, France) were coated with 200 μL/well of AffinityPure goat anti-mouse IgG+IgM (H+L) antibody (Jackson Immuno Research Laboratories Inc., Pennsylvania, USA) at 10 μg/mL in 50 mM potassium phosphate buffer, and incubated overnight (16h) at 22 ± 2 °C. anti-mouse IgG+IgMsuggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293 Freestyle™ were transiently transfected at a density of 2.5 106 cells/mL in 100mL Freestyle medium (Thermo-Fisher) by addition of 150 μg plasmid and 1.8 mL of linear polyethylenimine (PEI, 0.5 mg/ml) (Polysciences). HEK293suggested: NoneThen, the culture medium of each plate containing the VERO E6 cells is removed and 500 μL of each VHH/virus mixture is added to each well in duplicate. VERO E6suggested: NoneRecombinant DNA Sentences Resources Gap repair transformations were made in plasmid pNT VHH72 between restriction sites NheI and NotI with 1 μg of digested vector and a molar ratio of 12:1 (library/digested vector). pNTsuggested: NoneGenes coding for the various RBD domains were cloned in the pCAGGS RBD-SARS-CoV-2 plasmid, which was a kind gift from Florian Krammer lab. pCAGGS RBD-SARS-CoV-2suggested: NoneSoftware and Algorithms Sentences Resources Reads were demultiplexed and each sample was processed separately using the Galaxy platform (https://usegalaxy.org/) using the functions described in Blankenberg et al 45. Galaxysuggested: (Galaxy, RRID:SCR_006281)After seven days at 37°C, 120 rpm, 8% CO2, supernatant was purified using HiTrap Protein A for VHH-Fc constructs or HisTrap Excel for RBD constructs, following the manufacturer’s instructions (GE Healthcare). HisTrap Excelsuggested: NoneAffinity measurement by BioLayer Interferometry: Binding kinetics were determined using an Octet RED96 instrument (ForteBio). BioLayersuggested: (Harvard Medical School Center for Macromolecular Interactions Core Facility, RRID:SCR_018270)The initial structure of VHH72 was built by homology modelling using the MODELLER software 46 and the coordinates of VHH72 in PDB structure 6WAQ as template 18. MODELLERsuggested: (MODELLER, RRID:SCR_008395)Analysis of the trajectories was achieved using in-house scripts written in the macrolanguage of CHARMM v42b153. CHARMMsuggested: (CHARMM, RRID:SCR_014892)Figures were produced with PyMol [PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.] and Gnuplot 5.1. PyMOLsuggested: (PyMOL, RRID:SCR_000305)Gnuplotsuggested: (Gnuplot, RRID:SCR_008619)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: We detected the following sentences addressing limitations in the study:As a result, a second generation of antibodies might be required to overcome these limitations and could be obtained by reshaping the initial sequences by conferring them the necessary properties, such as affinity and selectivity, to have optimal therapeutic efficacy for treatment in humans. From this perspective, many teams have proposed a wide range of methods to generate candidates with the expected properties, mostly increased affinity.29-32 Affinity maturation aims at improving biological activity by adjusting the kinetic parameters of the binding to the target, which in turn may confer greater therapeutic efficacy.25,33 However, the magnitude of this effect depends largely on the epitope recognized by the antibody and the initial affinity along with the format of the antibody and its valence.25 In the context of the current COVID-19 pandemic, several studies have described affinity maturation of VHH or conventional antibodies to enhance their binding to SARS-CoV-2 antigens, by CDR-swapping approaches 34, saturation mutagenesis in CDRs 35,36 or light-chain shuffling36. In recent years, Deep Mutational Scanning (DMS) approaches have emerged as a powerful tool for understanding protein/protein interactions. Deep Mutational Scanning (DMS) explores in a selected protein all possible unique substitutions, i.e. all unique mutations for each position. DMS defines mutational landscape of the protein and helps to understand the interaction modalities as recently shown for the RBD...
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.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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