Relative Mutant N501Y SARS-CoV-2 Spike Protein RBD Inhibition of Anti-Spike Protein IgG and ACE-2 Binding to Spike Protein Species
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Abstract
In the SARS-CoV-2 coronavirus pandemic of 2019 (COVID-19), it has become evident that the ACE-2 receptor-binding domain (RBD) of the viral spike protein (SP) is the target of neutralizing antibodies that comprise a critical element of protective immunity to the virus. The most definitive confirmation of this contention is that the two mRNA COVID-19 vaccines in general use, which elicit antibodies specific for the RBD, exhibit approximately 95% protective efficacy against COVID-19. A potential challenge to vaccine efficacy is the emergence of SARS-CoV-2 variants possessing multiple mutations affecting amino acid residues in the RBD. Of concern are variants that arose in the United Kingdom, Brazil and South Africa. One of the variants, designated B.1.351, has shown a higher transmissibility due to greater affinity for the ACE-2 receptor and decreased neutralization by convalescent plasma, therapeutic monoclonal antibodies, and post-vaccination plasma. Common to several of the variants is the N501Y mutation in the RBD, which may be responsible for at least part of the observed variant properties. To test this hypothesis, we measured the ability of the Y501 RBD to inhibit binding of the wild type RBD and full SP (S1 + S2) to the ACE-2 protein and a human monoclonal IgG antibody elicited to the wild type RBD, relative to the wild type RBD in two enzyme-linked immunosorbent assays (ELISAs). We found no significant difference in the IC 50 of the two RBD species’ inhibition of ACE-2 binding, but unexpectedly found that the IC 50 of the wild type RBD inhibition of antibody binding was nearly twice that of the Y501 RBD, reflecting a lower affinity. These results suggest that the individual N501Y mutation does not contribute to altered viral properties by itself, but may contribute to a collective conformational shift produced by multiple mutations.
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SciScore for 10.1101/2021.04.26.441517: (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. Table 2: Resources
Antibodies Sentences Resources A correction nomogram for this ELISA is reproduced in Figure 1A. ELISA for Human IgG Antibodies Specific for the SARS-CoV-2 Spike Protein Receptor-Binding Domain (RBD): This is a sandwich ELISA in which a capture antibody, rabbit anti-mouse IgG, is adsorbed onto microtititer wells, followed by antigen capture. Human IgGsuggested: Noneanti-mouse IgGsuggested: NoneELISA Inhibition by RBD Variants: Human anti-SP IgG or ACE-2 Fc was mixed with recombinant SARS-CoV-2 spike RBD His tag (R&D Systems) or recombinant SARS-CoV-2 … SciScore for 10.1101/2021.04.26.441517: (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. Table 2: Resources
Antibodies Sentences Resources A correction nomogram for this ELISA is reproduced in Figure 1A. ELISA for Human IgG Antibodies Specific for the SARS-CoV-2 Spike Protein Receptor-Binding Domain (RBD): This is a sandwich ELISA in which a capture antibody, rabbit anti-mouse IgG, is adsorbed onto microtititer wells, followed by antigen capture. Human IgGsuggested: Noneanti-mouse IgGsuggested: NoneELISA Inhibition by RBD Variants: Human anti-SP IgG or ACE-2 Fc was mixed with recombinant SARS-CoV-2 spike RBD His tag (R&D Systems) or recombinant SARS-CoV-2 spike RBD (N501Y)-His tag (Sino Biological, Wayne, PA), both comprising R319-F541 (MW 26 kDa), to produce serial dilutions of 0-500 ng/ml of RBD in the presence of 25 ng/ml of antibody standard for primary incubation following the blocking step of the rSP ELISA. anti-SP IgGsuggested: NoneN501Y)-His tagsuggested: NoneSoftware and Algorithms Sentences Resources IC50 values of inhibition curves were determined by exponential decay regression analysis using SigmaPlot 10 software, as (−0.693)/(-k), where k is the decay constant, expressed as molar concentration. SigmaPlotsuggested: (SigmaPlot, RRID:SCR_003210)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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