Converting non-neutralizing SARS-CoV-2 antibodies targeting conserved epitopes into broad-spectrum inhibitors through receptor blockade
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Abstract
All but one of the authorized monoclonal antibody-based treatments for SARS-CoV-2 are largely ineffective against Omicron, highlighting the critical need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective therapeutic antibodies target epitopes that are not highly conserved. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to antibodies that are known to be non-neutralizing, but which target highly conserved epitopes in the viral spike protein. These inhibitors, called Re ceptor-blocking co nserved n on- n eutralizing A nti b o d ies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOC), including Omicron. Neutralization potency is dependent on both the binding and inhibitory ReconnAb components as activity is lost when the linker joining the two is severed. In addition, a bifunctional ReconnAb, made by linking ACE2 to a bispecific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad- spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.
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SciScore for 10.1101/2022.01.24.477625: (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 not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Library Design: A library of antibodies directed against SARS-CoV-2 Spike (S) protein was developed using paired antibody sequences, meaning antibody sequences for which the heavy and light chain are both known, from the Coronavirus Antibody Database, CoV-AbDab57. CoV-AbDab57suggested: NoneCrossMAb, antibody, hFc-ACE2, and Lentiviral plasmids were maxi prepped in the same fashion. hFc-ACE2suggested: NoneTwo microliters of mouse anti-hexa His antibody (BioLegend) were added to the 10 mL … SciScore for 10.1101/2022.01.24.477625: (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 not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Library Design: A library of antibodies directed against SARS-CoV-2 Spike (S) protein was developed using paired antibody sequences, meaning antibody sequences for which the heavy and light chain are both known, from the Coronavirus Antibody Database, CoV-AbDab57. CoV-AbDab57suggested: NoneCrossMAb, antibody, hFc-ACE2, and Lentiviral plasmids were maxi prepped in the same fashion. hFc-ACE2suggested: NoneTwo microliters of mouse anti-hexa His antibody (BioLegend) were added to the 10 mL sample and incubated for 1 h at room temperature. anti-hexasuggested: NoneExperimental Models: Cell Lines Sentences Resources Viral transfections were done in HEK293T cells using calcium phosphate transfection reagent. HEK293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)The resultant 10mL was added to plated HEK cells from which the medium had been removed. HEKsuggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)Neutralization: The target cells used for infection in viral neutralization assays were from a HeLa cell line stably overexpressing the SARS-CoV-2 receptor, ACE2, as well as the protease known to process SARS-CoV-2, TMPRSS2. HeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)ACE2/TMPRSS2/HeLa cells were plated one day prior to infection at 5,000 cells per well. ACE2/TMPRSS2/HeLasuggested: NoneRecombinant DNA Sentences Resources Equimolar aliquots of each scFv plasmid were pooled and the resultant pool was amplified using primers which annealed to the hexa-his Tag (reverse primer) or signal peptide (forward primer) and had a 50bp overlap with the pPNL6 vector digested with NheI and BamHI. scFvsuggested: NoneYeast were resuspended in electroporation buffer (10 mM Tris Base, 250 mM sucrose, 2mM MgCl2) containing the gel extracted library amplification and digested pPNL6 vector. pPNL6suggested: NoneLight chain (LC) and LC-ACE2 fusion proteins: Antibody sequences were cloned into the CMV/R plasmid backbone for expression under a CMV promoter. CMV/Rsuggested: NonehCoV spike proteins constructs: Spike proteins from six hCoVs were cloned into a pADD2 vector between the rBeta-globin intron and β-globin poly(A). pADD2suggested: NoneLentivirus plasmids: Plasmids encoding the full-length spike proteins with native signal peptides were cloned into the background of the HDM-SARS2-Spike-delta21 plasmid (Addgene Plasmid #155130). HDM-SARS2-Spike-delta21suggested: RRID:Addgene_155130)They are: pHAGE-Luc2- IRS-ZsGreen (NR-52516), HDM-Hgpm2 (NR-52517), pRC-CMV-Rev1b (NR-52519), and HDM- tat1b (NR-52518). pHAGE-Luc2- IRS-ZsGreensuggested: NoneThe EtOH solution was then added to a geneJET plasmid miniprep column and the regular wash steps and elution steps were followed. geneJETsuggested: NoneDigested FAbs were purified by SP/AKTA using 50 mM NaOAc, pH5 with gradient NaCl elution (using 50 mM NaOAc + 1M NaCl, pH5). pH5suggested: NoneThe plasmids were added to D10 medium in the following ratios: 10 µg pHAGE-Luc2-IRS-ZsGreen, 3.4 µg FL Spike, 2.2 µg HDM-Hgpm2, 2.2 µg HDM-Tat1b, 2.2 µg pRC-CMV-Rev1b in a final volume of 1000 µL. pRC-CMV-Rev1bsuggested: RRID:Addgene_164443)Software and Algorithms Sentences Resources The sequence alignment was again produced using MUSCLE31 and sequence identity was calculated using Geneious Geneioussuggested: (Geneious, RRID:SCR_010519)The sequences used were: 6VXX, UniRef90_U5NJG5, _L7UP8, _A0A7U3W1C7, _K9N5Q8, _A0A2I6PIW5, _A0A3Q8AKM0, _U5WHZ7, _A0A5H2WTJ3, _A0A0U1WJY8, _A0A166ZND9, _A0A678TRJ7, _A0A2R4KP93, _A0A2Z4EVK1, _A0A7R6WCE7, _E0ZN36, _A0A6M3G9R1, _F1DAZ9, _A0A0U1UYX4, _A0A2R3SUW7, _A0A2Z4EVN5, _A0A2Z4EVN2, _U5LMM7, _A0A5Q0TVR4, _E0XIZ3, _A0A023Y9K3, _A0A2R4KP86, _A0A088DJY6, _A0A7G6UAJ9, _S4X276, _A0A4Y6GL90, _A3EXG6, _F1BYL9, _E0ZN60, _A0A0K1Z054, _A0A0U1WHI2, and NCBI accession numbers: YP_009047204.1, QLR06867.1, AAK32191.1, AGZ48828.1, AAT84362.1, QHR63300.2, ABD75513.1, YP_003767.1. NCBIsuggested: (NCBI, RRID:SCR_006472)The sequences were first aligned using the MUSCLE algorithm, and then two phylogenetic trees were made, both using PhyML 3.3.20180621. MUSCLEsuggested: (MUSCLE, RRID:SCR_011812)PhyMLsuggested: (PhyML, RRID:SCR_014629)The sequences were then analyzed by sequence alignment using SnapGene software. SnapGenesuggested: (SnapGene, RRID:SCR_015052)FAb Production from IgGs: 1/10 volume of 1M Tris, pH 8 was added to IgGs at ∼2 mg/mL in PBS. 2 µL of a 1 mg/mL stock of Lys-C (stock stored at -70C) was added for each mg of human IgG1 and digested for 1 hour at 37 °C with moderate rotation. IgGssuggested: NoneFinal data analysis was done in Prism. Prismsuggested: (PRISM, RRID:SCR_005375)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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