A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern

Abstract

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.

Stabilizing proteins through saturation suppressor mutagenesis

“…Combing individual mutants from deep mutational scans has limitations and that there are often trade offs between enhancements in stability and binding (47). However, by combining putative stabilizing mutations which are not close to each other or active-site residues we believe that significant stabilization is possible without negatively impacting binding affinity as we have demonstrated both for CcdB in the present work and for the RBD for SARS-CoV-2 in another study (38). Overall the present methodology offers a robust pathway to identify stabilizing mutations in any protein of interest for which a surface display based binding screen is available.…”

“…In related work, we have stabilized the receptor binding domain of the Spike S protein of SARS-COV-2 using saturation suppressor mutagenesis (38). We identified several stabilizing mutations and could stabilize the protein by 8 °C.…”

Mechanistic insights into global suppressors of protein folding defects

“…These suppressors, D389E, L390M and P527I are located on the protein surface (Figure 5G). When individually introduced into WT mRBD, they show a ΔT m of ∼3 °C (Figure 5H) (11). The suppressors were also subjected to unfolding (in 3 M GdnCl) and refolding (in 0.5 M GdnCl) kinetic studies.…”

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies Against Delta and Omicron in Mice

“…In addition to efforts to induce higher antibody levels in individuals (for example through booster vaccination with homologous antigens), another approach is regular vaccine matching to current circulating variants, and/or customisation of vaccine formulations to include wild-type sequences and any relevant mutations in antigenic sites. The mRBD1-3.2 + mRBD1-3.2-beta antigen used in the formulation of Vaccine 3 in this study [10,11] was prepared using wild-type and Beta variant sequences at the time when the latter was a major concern. Although not statistically significant, there was a slightly stronger neutralisation of Omicron BA.1.1 by serum samples collected from mice immunised with this antigen, compared to only WT antigen.…”

“…This vaccine is due to enter Phase I human clinical trials soon, and elicits high antibody titers in mice that received prime-boost immunisations on Days 0, 21 and 42 against the wildtype SARS-CoV-2 virus. We have previously shown that ELISA and neutralisation titers are virtually identical post the first and second boosts [11].…”