Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains
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Abstract
The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human V H domain, F6, which we generated by sequentially panning large phage displayed V H libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized V H domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC 50 ) of 4.8 nM in vitro . Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.
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SciScore for 10.1101/2022.02.18.481058: (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 Secondary antibodies (anti-Flag-HRP or anti-Human Fc-HRP) were prepared with the same 5% milk at a dilution of 1:1000. 50 μL of secondary antibody was added into each well and incubated at room temperature for 1 hour. anti-Flag-HRPsuggested: (Shanghai Genomics Technology Cat# GNI4310-FG, RRID:AB_2885081)anti-Human Fc-HRPsuggested: NoneExperimental Models: Cell Lines Sentences Resources VH-Fc ab8, F6-ab8-Fc, and RBD-Fc were expressed with Expi293 cells as previously described[48, 62] Expi293suggested: RRID:CVCL_D615)HEK293T cells (ATCC, cat#CRL-3216) were used to produce pseudotyped retroviral particles as … SciScore for 10.1101/2022.02.18.481058: (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 Secondary antibodies (anti-Flag-HRP or anti-Human Fc-HRP) were prepared with the same 5% milk at a dilution of 1:1000. 50 μL of secondary antibody was added into each well and incubated at room temperature for 1 hour. anti-Flag-HRPsuggested: (Shanghai Genomics Technology Cat# GNI4310-FG, RRID:AB_2885081)anti-Human Fc-HRPsuggested: NoneExperimental Models: Cell Lines Sentences Resources VH-Fc ab8, F6-ab8-Fc, and RBD-Fc were expressed with Expi293 cells as previously described[48, 62] Expi293suggested: RRID:CVCL_D615)HEK293T cells (ATCC, cat#CRL-3216) were used to produce pseudotyped retroviral particles as described previously[79]. HEK293Tsuggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)HEK293T-ACE2-TMPRSS2 cells (BEI Resources cat# NR-55293) were seeded in 384-well plates at 20 000 cells for neutralization assays. HEK293T-ACE2-TMPRSS2suggested: NoneOne day before the assay, the Vero E6 cells (3 × 105 cells) were seeded in 24-well tissue culture plates per well. Vero E6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Evaluation of F6-ab8-Fc Prophylactic and Therapeutic Efficacy with SARS-CoV-2 mouse Models: Eleven to twelve-month old female immunocompetent BALB/c mice (Envigo, stock# 047) were used for SARS-CoV-2 in vivo Prophylactic and Therapeutic experiments as described previously[51]. BALB/csuggested: NoneRecombinant DNA Sentences Resources Pseudovirus Neutralization Assay: SARS-CoV-2 spike Wuhan-Hu-1 (+D614G), Alpha, Beta, Gamma, Delta, and Omicron protein genes were synthesized and inserted into pcDNA3.1 ( pcDNA3.1suggested: RRID:Addgene_79663)Software and Algorithms Sentences Resources The ELISA results were analyzed using GraphPad Prism 9.0.2. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)On-the-fly data pre-processing including patch mode motion correction (EER upsampling factor 1, EER number of fractions 40), patch mode CTF estimation, reference free particle picking, and particle extraction were carried out in cryoSPARC live. cryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)0.9.3[69], followed by iterative rounds of refinement in COOT and Phenix v. COOTsuggested: (Coot, RRID:SCR_014222)1.1.1[72], and PyMOL (v.2.2 Schrodinger, LLC) PyMOLsuggested: (PyMOL, RRID:SCR_000305)All MD simulations were performed using NAMD (version 2.13)[76] with the protocol adopted from earlier work[77]. NAMDsuggested: (NAMD, RRID:SCR_014894)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: Please consider improving the rainbow (“jet”) colormap(s) used on page 7. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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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