Discovery and characterization of a pan-betacoronavirus S2-binding antibody

Abstract

The continued emergence of deadly human coronaviruses from animal reservoirs highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq), we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryoelectron microscopy (cryo-EM) structure of 54043-5 bound to the prefusion S2 subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses in vitro, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

Keywords: LALA-PG; LIBRA-seq; S2 subunit; antibodies; betacoronaviruses; coronaviruses; cryo-EM; spike protein.

Figure 1.. Identifying broadly reactive antibodies using a diverse CoV spike LIBRA-seq panel.

(A) Panel of antibodies identified by LIBRA-seq (rows), with corresponding V- and J-genes and percent nucleotide identities, CDR3 lengths and amino acid sequences, and isotypes (columns). LIBRA-seq scores for each antigen are shown alongside each antibody as a heatmap from −2 (tan) to 2 (purple). (B) 50 IgG or IgA cells identified by LIBRA-seq are shown as circles, with their respective LIBRA-seq scores (LSSs) for SARS-CoV-2 spike (x-axis), SARS-CoV spike (y-axis), and MERS-CoV spike (color heatmap). (C) Binding of antibodies to a panel of human coronavirus spike proteins. ELISA area under the curve (AUC) values are shown as heatmaps from minimum (white) to maximum (dark teal) binding. Antibodies that bound to the SARS-CoV-2 index strain spike are shown. Full ELISA binding curves and controls are shown in Figure S1.

Figure 2.. 54043–5 is a broadly reactive, S2-binding antibody.

(A) Phylogenetic tree of spikes from select members of the Orthocoronavirinae subfamily. Members of the alpha- (α) beta- (β) and deltacoronavirus (δ) genera are indicated by color. The betacoronavirus genus contains five lineages: A-D and the Hibecovirus lineage (H). Spikes included in LIBRA-seq experiments and binding assays are underlined, and those included exclusively in binding assays are boldly labeled. Scale bar denotes amino acid phylogenetic distance. (B) ELISA curves for 54043–5 binding to spikes from selected CoVs (left) and associated AUC values for each (right). An “*” denotes a non-human coronavirus. Influenza (NC99) HA = negative control. Results are shown as mean ± SEM (C) ELISA curves for 54043–5 binding to spikes from SARS-CoV-2 variants (left) and associated AUC values for each (right). (D) ELISA AUCs for 54043–5 binding to the S1 and S2 domains of spikes from SARS-CoV-2 and MERS-CoV, or positive control antibodies CR3022 and 1F8. Full ELISA binding curves and controls are shown in Figure S1.(E) SPR sensorgrams for 54043–5 Fab binding to the S2 subunit of the SARS-CoV-2 spike. Binding curves are colored black, and data fit to a 1:1 binding model is colored red.

Figure 3.. Cryo-EM structure of Fab 54043–5 bound to the SARS-CoV-2 S2 subunit.

(A) Side and top-down views of the 3.0 Å 3D reconstruction of Fab 54043–5 bound to S2. S2 protomers are colored green, blue, or salmon. 54043–5 heavy and light chains are purple and white, respectively. (B) 54043–5 binds an epitope at the apex of S2, spanning the junction between heptad repeat 1 (HR1) and the central helix (CH). The S2 subunit bound to one Fab is shown as cartoons (left), with two protomers fit in the corresponding EM map and colored gray. One protomer is colored according to the gene schematic (bottom) and 54043–5 is colored as in (A). Zoomed-in views of the 54043–5 Fab-S2 interface (right) are shown as cartoons, with important residues shown as sticks. Phe100F is shown with a partially transparent surface, illustrating space filling in a hydrophobic pocket within the epitope. Oxygen atoms: red, nitrogen atoms: blue, sulfur atoms: yellow, hydrogen bonds: light blue dashed lines. (C) Top and side views of the SARS-CoV-2 spike protein, with the S2 subunit shown as cartoons and colored as in (A). 54043–5 Fab is shown bound to one S2 protomer. The S1 subunit, shown as a partially transparent gray surface, clashes with bound 54043–5.

Figure 4.. Antibody sequence feature uniqueness and conservation of epitope across CoVs.

(A) Sequence feature analysis of 54043–5 compared to human coronavirus antibodies in the CoVAbDab. Plot displays the percent amino acid identity of the CDRH3 (x-axis) and CDRL3 (y-axis) of a subset of antibodies to those of 54043–5. Antibodies shown have at least one identical variable (V) gene or ≥50% CDR3 sequence identity. Colors denote shared V gene usage with 54043–5. (B) The count of similar public clones for each S2-binding antibody with characterized epitopes, based on epitope group. Each antibody is represented as a point, with its x-axis coordinate reflecting the number of antibodies in the CoVAbDab sharing both heavy and light V genes and having a CDRH3 with ≥50% amino acid sequence identity. The y-axis coordinate corresponds to the number of antibodies with the same V genes and a CDRL3 sequence with ≥50% identity. (C) Sequence conservation among betacoronaviruses within the S2 subunit, mapped onto a surface representation of SARS-CoV-2 S2 colored from least conserved (white) to most conserved (dark purple). Sequence alignment can be found in Table S4. The 54043–5 epitope is outlined in yellow. A zoomed-in view of the S2 apex is shown as a cartoon, with 54043–5 epitope residues shown as sticks. (D) Phylogenetic tree of betacoronavirus strains and coronavirus strains from other genera used in binding assays. Phylogenetic distance is based on 54043–5 epitope residues within the SARS-CoV-2 spike protein compared to the corresponding residues in each strain. Red boxes are beside each strain tested for binding, filled with a color gradient (white to dark teal) corresponding to the AUC values from Figure 2B. (E) Relative epitope buried surface area (BSA) and conservation across the Orthocoronavirinae genera. Two conserved and structurally characterized S2-epitopes are shown compared to the 54043–5 epitope, with relative BSA shown as a color gradient from white (0 BSA) to golden yellow (highest BSA within the row). The percent conservation of each epitope residue among the four genera is shown as a line graph.

Figure 5.. Fc-mediated effector functional characteristics of lead candidates in bead-based and cell-based assays.

(A) Lead cross-reactive antibody candidates were tested for antibody-dependent cellular phagocytosis (ADCP) of SARS-CoV-2 spike-coated beads, compared to positive control antibody CR3022 and negative control palivizumab (specific for RSV F). (B) Antibody-dependent monocyte phagocytosis (ADMP) of SARS-CoV-2-infected cells, compared to positive control antibody CR3022 and a no-antibody negative control. (C) Antibody-dependent cellular trogocytosis (ADCT) for SARS-CoV-2, compared to positive control antibody CR3022 and palivizumab (negative control). (D) Antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2, compared to positive control antibody CR3022 and palivizumab (negative control). (E). Antibody-dependent cellular phagocytosis of OC43 spike-coated beads compared to OC43-specific positive control antibody 54044–5 and palivizumab (negative control). AUC values were calculated based on scores reported in Figure S4E–H. All data are shown as mean ±SDs.

Figure 6.. Treatment of mice with 54043–5 as pre- or post-exposure treatment of SARS-CoV-2 infection.

Mice were treated, according to group, either 24 hours prior to (prophylactic) or following (therapeutic) intranasal SARS-CoV-2 challenge. Control antibodies were chosen based on availability at the time of the assays performed. Survival results are expressed as percent survival with all points plotted. All other results are expressed as absolute mean values +SEM. (A, B) Body weight (A) and survival (B) were monitored daily for K18-hACE2 mice treated prophylactically with assigned antibody for 14 days following challenge with SARS-CoV-2 (WA1/2020). Statistical significance of body weight differences at days 5–7 post-infection (p.i.) are reported in Figure S6B. (C) Three mice per group in the prophylactic study were sacrificed 3 days p.i. and viral titers in the lung tissue were measured. Lung viral titer is expressed as the logarithm of the viral genomic equivalents (GEq) per mL of homogenized lung tissue. (D, E) Body weight (D) and clinical score (E) were monitored daily for BALB/c mice treated therapeutically with assigned antibody for five days following challenge with SARS-CoV-2 (MA10). (F) Four mice per group in the therapeutic study were sacrificed on days 2 and 5 p.i. and viral titers in the lung tissue were measured. Lung viral titer is expressed as the logarithm of the 50% tissue culture infectious dose of virus (TCID50) per gram of lung tissue. ****, p<0.0001; ***, p<0.001.