Interplay between protein stability, binding to ACE2 and escape from neutralizing antibodies determines the natural selection of SARS-CoV-2 receptor binding domain variants
Emergence of new SARS-CoV-2 variants has raised concerns at the effectiveness of vaccines and antibody therapeutics developed against the unmutated wild-type virus. It is thus important to understand the emergence of mutants from an evolutionary viewpoint to be able to devise effective countermeasures against new variants. We examined the effect of 12 most commonly occurring mutations in the receptor binding domain (RBD) on its expression, stability, activity, and antibody escape potential- some of the factors that may influence the natural selection of mutants. Recombinant proteins were expressed in human cells. Stability was measured using thermal denaturation melts. Activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding of RBD variants to ACE2 and to a neutralizing human antibody CC12.1, respectively. Our results show that variants differ in their expression levels, suggesting that mutations can impact the availability of proteins for virus assembly. All variants have similar or higher stability than the wild-type, implying that increased RBD stability might be another important factor in virus evolution. In terms of ACE2 binding, when compared to the wild-type, only 3 out of 7 expressed variants show stronger affinity, 2 have similar affinity, whereas the other 2 have weaker affinity, indicating that increased affinity towards ACE2 is an important but not the sole factor in the natural selection of variants. In terms of CC12.1 binding, when compared to the wild-type, 4 out of 7 variants have weaker affinity, 2 have a similar affinity, and 1 variant has a stronger affinity. Taken together, these results indicate that multiple factors contribute towards the natural selection of variants, and all of these factors have to be considered in order to understand the natural selection of SARS-CoV-2 variants. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that particular variant.