The SARS-CoV-2 Y453F mink variant displays a pronounced increase in ACE-2 affinity but does not challenge antibody neutralization

Abstract

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 from humans to animals has been reported for many domesticated species, including farmed minks. The identification of novel spike gene mutations appearing in minks has raised major concerns about potential immune evasion and challenges for the global vaccine strategy. One genetic variant, known as "cluster five," arose among farmed minks in Denmark and resulted in a complete shutdown of the world's largest mink production. However, the functional properties of this new variant are not established. Here we present functional data on the cluster-five variant, which contains a mutation resulting in a Y453F residue change in the receptor-binding domain (RBD) of the spike protein. Using an ELISA-based angiotensin-converting enzyme-2/RBD inhibition assay, we show that the Y453F variant does not decrease established humoral immunity from previously infected individuals or affect the neutralizing antibody response in a vaccine mouse model based on the original Wuhan strain RBD or spike as antigens. However, biolayer interferometry analysis demonstrates that it binds the human angiotensin-converting enzyme-2 receptor with a 4-fold higher affinity than the original strain, suggesting an enhanced transmission capacity and a possible challenge for viral control. These results also indicate that the rise in the frequency of the cluster-five variant in mink farms might be a result of the fitness advantage conferred by the receptor adaptation rather than evading immune responses.

Keywords: ACE-2; RBD; RNA virus; SARS-CoV-2; Virology; antibodies; immunology; mutant; receptor interaction; receptor structure–function.

Figure 1

Biophysical characterization of WT and Y453F RBD.A, details of the binding interface of SARS-COV-2 RBD (orange) and the human ACE-2 receptor (green). The residue 453 maps to the receptor-binding motif within the RBD (residue 438–506) and directly engages the N-terminal helix of the peptidase domain of ACE-2 (30). Dashed lines represent noncovalent interactions. Created with PDBe Molstar (https://molstar.org/). B, thermal denaturation curves of the WT and Y453F RBD. Data are represented as the 350:330 nm ratio of each replicate. C and D, WT (C) and Y453F (D) RBD binding response curves to ACE-2 determined by biolayer interferometry. ACE-2-Fc was immobilized onto anti-human IgG Fc capture sensors and dipped into serial dilutions of RBD (5-point 2-fold dilutions starting at 100 nM [WT] or 60 nM [Y453F]). Ti, inflection temperature; ACE-2, angiotensin-converting enzyme-2; RBD, receptor-binding domain.

Figure 2

Inhibition potency of COVID-19 convalescent patient sera against the WT and Y453F RBD. A, distribution of the inhibition potency of COVID-19 patient sera against WT and Y453F RBD (n = 141), analyzed using the Mann-Whitney test. Orange lines represent the median. B, linear regression and Spearman rank correlation analyses of the data in panel A. The trend line represents a linear regression. Negative inhibition values were normalized to 0. Ns, not significant; RBD, receptor-binding domain.

Figure 3

Inhibition potency of polyclonal sera and mAbs from mice immunized against SARS-CoV-2 WT spike or RBD. A, comparison of the best-fit IC₅₀ of polyclonal sera from mice immunized with SARS-CoV-2 WT RBD (RBD imm.) or spike (spike imm.) against WT RBD (black symbols) and the Y453F variant (gray symbols). A SARS-CoV-2 nonrelated immunization (control imm.) was used as the control. Connecting lines represent the nonlinear fit. Data are presented as the mean ± SEM. B, linear regression and Spearman rank correlation analyses of the inhibition potency (log[IC₅₀]) of the WT and Y453F RBD using individual mAbs raised against WT RBD (n = 10) or WT spike (n = 8). RBD, receptor-binding domain.