Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study

Abstract

Antibody-based therapeutics and vaccines are essential to combat COVID-19 morbidity and mortality after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Multiple mutations in SARS-CoV-2 that could impair antibody defenses propagated in human-to-human transmission and spillover or spillback events between humans and animals. To develop prevention and therapeutic strategies, we formed an international consortium to map the epitope landscape on the SARS-CoV-2 spike protein, defining and structurally illustrating seven receptor binding domain (RBD)–directed antibody communities with distinct footprints and competition profiles. Pseudovirion-based neutralization assays reveal spike mutations, individually and clustered together in variants, that affect antibody function among the communities. Key classes of RBD-targeted antibodies maintain neutralization activity against these emerging SARS-CoV-2 variants. These results provide a framework for selecting antibody treatment cocktails and understanding how viral variants might affect antibody therapeutic efficacy.

Fig. 1.. The antigenic landscape of the SARS-CoV-2 RBD can be divided into seven binding communities.

(A) HT-SPR was used to determine the competitive relationship between 186 RBD-directed mAbs. The dataset was analyzed by Carterra Epitope software to sort competition profiles of clones into related clusters, which are represented as regions of the dendrogram with shared color. The RBD epitope landscape can be broadly divided into seven communities containing mAbs that bind the RBM (RBD-1 through RBD-3), the outer face of the RBD (RBD-4 and RBD-5), or the inner face of the RBD (RBD-6 and RBD-7). Communities can be further divided into smaller clusters (e.g., RBD-2a and RBD-2b) and bins (e.g., RBD-2b.1, RBD-2b.2, and RBD-2b.3) on the basis of their discrete competition with other clusters and/or their ability to compete with ACE2 for spike binding. Black bars indicate single clones that were used in further analyses. Table S1 lists additional metrics (i.e., ACE2 blocking, kinetic analyses, and germline information) for the indicated mAbs; detailed information for the entire CoVIC panel can be found at covic.lji.org. (B) Binary heatmap matrix demonstrating the competition profile for the finer clusters and bins for the subset of single clones indicated by black bars in (A). The matrix here contains representative examples of each epitope community. RBD-2 can be divided into clusters a and b, which have varying ability to compete with mAbs in RBD-4 (e.g., RBD-2a mAbs do not compete, whereas most RBD-2b mAbs do). Cluster RBD-2b can be divided into three smaller bins that vary in their competition with both RBD-3 and RBD-4 mAbs: Those in 2b.1, but not 2b.2 or 2b.3, compete with RBD-3 mAbs, whereas mAbs in 2b.1 and 2b.2, but not 2b.3, compete with RBD-4 mAbs. RBD-4 contains mAbs that do (RBD-4a) and do not (RBD-4b) compete with ACE2. RBD-5 and RBD-7 have clusters of mAbs with lower neutralizing potency (i.e., RBD-5c, RBD-7b, and RBD-7c) relative to the other cluster in the same community (i.e., RBD-5a, RBD-5b, and RBD-7a). Rows and columns indicate the immobilized mAb and injected analyte mAb, respectively. Table S2 shows the complete matrix for competition among all 186 mAbs.

Fig. 2.. NS-EM analysis of representatives from each RBD-directed community.

(A) Location of important emerging mutations in a RBD. The spike trimer [adapted from PDB ID 7A94 (39)] viewed from the top with one “up” RBD is shown; individual spike monomers are colored white, gray, and black. The RBM can be topologically divided into three subsections: the “peak” that includes residues F486, S477, T478, and E484; the “valley” including residues Y453, K417, and L452; and the “mesa” with residue N501. Stars indicate residues on the central axis of the RBD. The “outer face” [exposed in the RBD down (closed) conformation] and “inner face” [buried inside the trimer in the RBD down (closed) conformation] define the lateral faces of the RBD and the “escarpment” (contains residues V367 and N439 and glycan 343). (B to D) NS-EM footprint of a representative antibody from each community mapped onto an RBD monomer. The colored shading corresponds to the community colors in Fig. 1. The ACE2 binding site is outlined with a dotted line. Side and top views of spike trimers show the Fab approach angle and binding stoichiometry for each representative. Table S3 shows NS-EM data for all of the 29 RBD-directed mAbs that we analyzed.

Fig. 3.. RBD-5, -6, and -7 antibodies retain neutralization activity against pseudovirus bearing mutations singly or together in VOCs.

Fold-change differences in potency for 38 RBD-directed antibodies and an ACE2-Fc fusion (CoVIC-069) are shown in a heatmap. In addition to VOCs, we also examined two pseudoviruses bearing clusters of mink-associated mutations: 4xM (G261D, Y453F, F486L, and N501T) and 5xM (G261D, Y453F, F486L, N501T, and V367F). Fig. S1 lists mutations represented in each variant. Fig. S10 shows neutralization curves for each virus–variant pair, and table S4 lists fold-change values corresponding to the heatmap. Single-letter abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 4.. NS-EM and neutralization analysis of mAbs targeting the NTD.

(A) Surface and cartoon [adapted from PDB ID 7A94 (39)] representation of the spike NTD. The residue positions of mutations and deletions in circulating VOCs are indicated in three views of the NTD. Fig. S1 lists mutations represented in each variant. (B) Footprints for three NTD-targeted antibodies, with the NTD “supersite” (26) outlined with a dashed line. The NTD-directed antibodies shown here define the approximate boundaries of the neutralizing epitope landscape. Additional NS-EM data are in table S3. (C) Fold-change in potency of pseudovirus neutralization experiments for each antibody–variant pair.