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Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 variants via multiplex assay
Ester Lopez, … , Wai-Hong Tham, Amy W. Chung
Ester Lopez, … , Wai-Hong Tham, Amy W. Chung
Published July 12, 2021
Citation Information: JCI Insight. 2021;6(16):e150012. https://doi.org/10.1172/jci.insight.150012.
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Resource and Technical Advance COVID-19

Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 variants via multiplex assay

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Abstract

The SARS-CoV-2 receptor binding domain (RBD) is both the principal target of neutralizing antibodies and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations, limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralizing capacity of antibodies at the angiotensin-converting enzyme 2–RBD (ACE2-RBD) interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P, and N501Y to the ACE2 receptor and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research, informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.

Authors

Ester Lopez, Ebene R. Haycroft, Amy Adair, Francesca L. Mordant, Matthew T. O’Neill, Phillip Pymm, Samuel J. Redmond, Wen Shi Lee, Nicholas A. Gherardin, Adam K. Wheatley, Jennifer A. Juno, Kevin J. Selva, Samantha K. Davis, Samantha L. Grimley, Leigh Harty, Damian F.J. Purcell, Kanta Subbarao, Dale I. Godfrey, Stephen J. Kent, Wai-Hong Tham, Amy W. Chung

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Figure 3

Characterization of RBD variants in the multiplex array.

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Characterization of RBD variants in the multiplex array.
(A) Structure o...
(A) Structure of novel coronavirus spike RBD complexed with its receptor, ACE2 (PDB ID code 6LZG), highlighting the amino acid positions of 24 RBD natural variants selected from the GISAID repository in June 2020, with the subsequent inclusion of S477N, and their proximity to the RBD-ACE2 interface. The structural illustration was generated using PyMol. Variants with a significantly higher affinity to ACE2 as determined in our multiplex assay are highlighted in blue. (B) Cumulative frequency of variants ranked from most frequently isolated to less frequent included in the multiplex array. Frequency of genomes is based on high-quality 2019-nCov genome sequences reported by the GISAID database as of May 25, 2021. Variants with a significantly higher affinity to ACE2 as determined in our multiplex assay are highlighted in blue. (C) Relative EC50 values (μg/mL) obtained for each of the variants. Values are color-coded blue for EC50 less than 10 μg/mL and red for EC50 values more than 10 μg/mL. (D) Mean fold difference for each RBD variant EC50 value relative to WT RBD. Data are presented as dot plots of (n = 4) independent experiments, with the mean ± SEM. Bars for variants with more than a 2-fold difference in affinity to the WT RBD are colored blue. Bars for variants with a slightly increased affinity (< 2-fold) are colored white. Variants with a weaker EC50 value relative to WT are colored red. (E) Bio-layer interferometry sensograms of immobilized ACE2 with 2-fold 6–100 nM serial dilutions of SARS-CoV-2 RBD variants in solution. Binding curves representative of 2 independent experiments for each variant are plotted (solid-colored lines), globally fitted to a 1:1 binding model (black line).

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