The ACE2-binding interface of SARS-CoV-2 Spike inherently deflects immune recognition

Abstract

The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.

Two-dimensional multiplexed assay for rapid and deep SARS-CoV-2 serology profiling and for machine learning prediction of neutralization capacity

“…Interestingly, the correlations between neutralization titers and the IgG and IgM datasets to Spike-T and RBD-T, the designed mutant antigens harboring the triple mutants that disrupt the RBD-ACE2 interaction, were among the highest of all isotype-antigen pairs. These results are consistent with our previous results that revealed that the ACE2-interacting surface is not highly immunogenic (Hattori et al, 2021) and with other reports showing that a small fraction of antibodies target this surface of the spike protein (Voss et al, 2021).…”

“…Natural RBD mutants included RBD-V483A, RBD-V367F and RBD-G476S that were in circulation in the U.S. in 2020, and a designed triple mutant that disrupts the RBD-ACE2 interaction, N487K, Q493K and N501K in Spike (termed Spike-T hereafter) and RBD (termed RBD-T hereafter; Fig. 1D) (Hattori et al, 2021). These designed mutations disrupt the interaction of RBD with neutralizing antibodies that target the ACE2-interaction surface.…”

“…For Elastic Net, IgG-RBD-T, IgM-Spike-T and IgM-RBD-V483A were the top three. The selection of both RBD-T and Spike-T, which have mutations to eliminate antibody binding to the ACE2-interaction surface of the RBD (Hattori et al, 2021), may be rationalized based on our previous observation that a small fraction of RBD-binding antibodies target the ACE2-interaction surface, as discussed above, and by the notion that IgM binding to this surface should be captured in the data with RBD-V483A.…”

“…The analysis was based on full genome DNA sequences downloaded on April 13, 2020 (8,086 entries) and May 13, 2020 (24,681 entries). Vector construction has been described previously (Hattori et al, 2021).…”

“…Its receptor-binding domain (RBD) interacts with the canonical receptor, angiotensin-converting enzyme 2 (ACE2) (Hoffmann et al, 2020;Lan et al, 2020;Walls et al, 2020). Consequently, antibodies from infected subjects, as well as engineered reagents that interfere with the RBD-ACE2 interaction, can inhibit viral infection (Hansen et al, 2020;Hattori et al, 2021;Ju et al, 2020;Robbiani et al, 2020;Shi et al, 2020;Wec et al, 2020;Wu et al, 2020). Accordingly, most current vaccines utilize the prefusion-stabilized form of spike as the antigen (Baden et al, 2021;Polack et al, 2020).…”