Kanjo, K and Chattopadhyay, G and Malladi, SK and Singh, RK and Jayatheertha, S and Varadarajan, R (2022) Biophysical Correlates of Enhanced Immunogenicity of a Stabilized Variant of the Receptor Binding Domain of SARS-CoV-2. In: Journal of Physical Chemistry B .
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Abstract
The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We have previously reported the design and characterization of a mammalian cell expressed RBD derivative, mRBD1-3.2, that has higher thermal stability and greatly enhanced immunogenicity relative to the wild type mRBD. The protein is highly thermotolerant and immunogenic and is being explored for use in room temperature stable Covid-19 vaccine formulations. In the current study, we have investigated the folding pathway of both WT and stabilized RBD. It was found that chemical denaturation of RBD proceeds through a stable equilibrium intermediate. Thermal and chemical denaturation is reversible, as assayed by binding to the receptor ACE2. Unusually, in its native state, RBD binds to the hydrophobic probe ANS, and enhanced ANS binding is observed for the equilibrium intermediate state. Further characterization of the folding of mRBD1-3.2, both in solution and after reconstitution of lyophilized protein stored for a month at 37 °C, revealed a higher stability represented by higher Cm, faster refolding, slower unfolding, and enhanced resistance to proteolytic cleavage relative to WT. In contrast to WT RBD, the mutant showed decreased interaction with the hydrophobic moiety linoleic acid. Collectively, these data suggest that the enhanced immunogenicity results from reduced conformational fluctuations that likely enhance in vivo half-life as well as reduce the exposure of irrelevant non-neutralizing epitopes to the immune system. © 2023 The Authors. Published by American Chemical Society.
| Item Type: | Journal Article |
|---|---|
| Publication: | Journal of Physical Chemistry B |
| Publisher: | American Chemical Society |
| Additional Information: | The copyright of this article belongs to the Authors. |
| Keywords: | Antibodies; Hydrophobicity; Linoleic acid; Mammals; Chemical denaturation; Domain derivative; Equilibrium intermediate; High thermal stability; Immunogenicity; Mammalian cells; Neutralizing antibodies; Receptor-binding domains; Thermotolerant; Wild types; Coronavirus |
| Department/Centre: | Division of Biological Sciences > Molecular Biophysics Unit |
| Date Deposited: | 10 Mar 2023 05:37 |
| Last Modified: | 10 Mar 2023 05:37 |
| URI: | https://eprints.iisc.ac.in/id/eprint/80931 |
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