Structural mapping of antibody landscapes to human betacoronavirus spike proteins

Abstract

Preexisting immunity against seasonal coronaviruses (CoVs) represents an important variable in predicting antibody responses and disease severity to severe acute respiratory syndrome CoV-2 (SARS-CoV-2) infections. We used electron microscopy-based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against β-CoV spike proteins in prepandemic (PP) sera or SARS-CoV-2 convalescent (SC) sera. We observed that most PP sera had antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the SC sera showed reactivity across all human β-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by preexisting antibodies and SC serum antibodies. Our studies provide an antigenic landscape to β-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-CoV-2-infected individuals.

Fig. 1.. Human serum reactivity to β-CoV spikes.

(A) ELISA EC₅₀ binding titers to OC43, HKU1, MERS, SARS, and SARS-CoV-2 spikes and median inhibitory concentration (IC₅₀) neutralization titers against OC43 virus and vesicular stomatitis virus (VSV)–pseudotyped SARS or SARS-CoV-2 virus for PP sera from eight healthy donors and SC sera from three SARS-CoV-2 donors. Ebola virus glycoprotein (EBOV GP) was used as a negative control for detecting nonspecific serum binding. Serum EC₅₀ or IC₅₀ titers are color-coded in gradients of orange or aquamarine, respectively. (B) Representative two-dimensional (2D) classes and side and top views of composite figures from ns-EMPEM analysis of polyclonal Fabs from eight PP sera with the OC43 spike. (C) Bar graph summary of OC43 spike epitopes targeted by PP donor sera. Antibodies to NTD-site 1 were observed in 2D class averages for donor 269 but did not reconstruct in 3D, as indicated by dotted lines. (D) Composite figures from ns-EMPEM analysis of polyclonal Fabs from donor 1412 with the HKU1 spike. The Fabs in (B) and (D) are color-coded on the basis of their epitope specificities as indicated at the bottom. OC43 or HKU1 spikes in (B) and (D) are represented in light gray or dark gray, respectively.

Fig. 2.. Cryo-EMPEM analysis of OC43 spike-polyclonal Fab complexes.

(A) High-resolution cryo-EMPEM reconstructions of OC43 spike complexed with polyclonal Fabs derived from PP sera from donors 269 (top left), 1051 (top right), or 1412 (bottom); the representative composite figures from ns-EMPEM from these donors are shown in the middle. Each map depicts a structurally unique polyclonal antibody class reconstructed at the indicated resolution with the Fabs colored according to the scheme used in Fig. 1. OC43 spike is represented in light gray. Fabs marked with a black dot were observed by ns-EMPEM but were not detected by cryo-EMPEM. Fab class from donor 269 marked with a star was resolved by cryo-EMPEM but not by ns-EMPEM. (B) Sapienic acid (aquamarine) binding within a hydrophobic pocket in the CTD-CTD interprotomeric interface. Protomers are colored in light pink, blue, or wheat and the interacting residues are shown in gray.

Fig. 3.. Cryo-EM structures of polyclonal Fabs targeting the OC43 spike.

(A to G) Tube or ribbon representation of atomic models of OC43 spike-Fab complexes along with zoomed-in views of epitope-paratope interactions. (A and B) Fab1 and Fab2 (red) target the NTD-site1 or RBS; (C) Fab3 (orange) targets NTD-site2 adjacent to RBS; (D to F) Fab4, Fab5, and Fab6 (yellow) target the CTD; and (G) Fab7 (blue) targets the NTD-CTD interface. The spike protomers are shown in light blue, light pink, or wheat (ribbon representation) with glycans in teal (sphere atom representation) and primary epitope contacts in gray. Detailed contact residues along with corresponding EM densities are shown in figs. S6 and S7. (H) Surface representation of OC43 spike (gray) showing collective epitopes of Fab1 to Fab10 colored on the basis of their binding site using the color scheme from Fig. 1.

Fig. 4.. ns- and cryo-EMPEM analysis of polyclonal Fabs from SC donor sera.

(A) Representative 2D classes and side and top views of composite figures from ns-EMPEM analysis of polyclonal Fabs from three SC donors complexed with β-CoV spikes. The donor numbers along with the corresponding CoV spikes are indicated above each panel in (A). The Fabs are color-coded on the basis of their epitope specificities as indicated at the bottom left. SARS-CoV-2, OC43, HKU1, and MERS spikes are represented in slate gray, light gray, dark gray, and beige, respectively. Three-dimensional reconstructions displaying potential self-reactive antibodies are shown in gray on the top right corners for both donors 1988 and donor 1999 in complex with SARS-CoV-2 spike. (B) Composite figure showing five unique antibody classes, Fab11 to Fab15 colored in shades of red, to SARS-CoV-2 spike NTD reconstructed using cryo-EMPEM analysis of polyclonal Fabs from donors 1988 and 1989 complexed with SARS-CoV-2–stabilized spikes. (C) Surface representation of SARS-CoV-2 spike showing epitopes of Fabs 11 to 15 from (B) on a single NTD (slate gray) with a zoomed-in view displaying the loop residues comprising each epitope. Loop 144 to 156 with the N149 glycan forms an immunodominant element commonly targeted by Fabs 11 to 14. The sub-epitope colors correspond to each Fab shown in (B).