Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease

Abstract

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.

Figure. 1.. Binding and neutralization properties of S2 stem-helix mAbs.

a. Dot plots showing ELISA binding (OD₄₀₅) reactivity of immune sera from COVID-19 convalescent donors (n = 15), spike mRNA-vaccinated donors (n = 10) and SARS-CoV-2 recovered-vaccinated donors (n = 15) to 25-mer peptides corresponding to spike S2 stem-helix regions of human β-(sarbecoviruses: SARS-CoV-1 or 2; merbecovirus: MERS-CoV; embecoviruses: HCoV-HKU1, HCoV-OC43) and α-(HCoV-NL63 and HCoV-229E) coronaviruses. 12 out of 15 (80%) SARS-CoV-2 recovered-vaccinated donor sera show cross-reactive binding to β-CoV spike stem-helix peptides. b. Correlation between binding of infected-vaccinated sera to SARS-CoV-2 stem-helix peptide and the other β-CoV (MERS-CoV, HCoV-HKU1 and HCoV-OC43) stem-helix peptides. Responses for binding to two stem-helix peptides were compared by nonparametric Spearman correlation two-tailed test with 95% confidence interval and the Spearman correlation coefficient (r) and the p-value are indicated. c. A total of 40 S2 stem-helix mAbs were isolated from 9 SARS-CoV-2 recovered-vaccinated donors (CC9, CC24, CC25, CC67, CC68, CC84, CC92, CC95 and CC99). MAbs were isolated by single B cell sorting using SARS-CoV-2 and MERS-CoV S-proteins as baits. Heatmap showing IGVH germline gene usage (colored: VH1–46 (green), VH3–23 (plum) and other V-genes (grey)), lineage information (unique (sky) and expanded (tangerine) lineages) and V-gene nucleotide somatic hypermutations (SHMs). EC₅₀ ELISA binding titers of mAbs with β- and α-HCoV spike S2 stem-helix region peptides. MAbs showed binding to β- but not α-HCoV derived stem-helix peptides. IC₅₀ neutralization of mAbs against pseudoviruses of clade1a (SARS-CoV-2 and Pang17), clade 1b (SARS-CoV-1, WIV1, SHC014) sarbecoviruses and MERS-CoV. Spike S2 stem-helix bnAbs, CC40.8, S2P6 and CV3–25 were used as controls for binding and neutralization assays. d. 32 of 40 stem-helix bnAbs were unique clones that neutralized all ACE2-utilizing sarbecoviruses and 23 out of 32 unique mAb neutralized MERS-CoV, in addition to sarbecoviruses.

Figure. 2.. Neutralization of SARS-CoV-2 VOCs, and immunogenetic properties of S2 β-CoV spike stem-helix bnAbs.

a. Neutralization of 10 select S2 stem-helix bnAbs against SARS-CoV-2 (WT) and five major SARS-CoV-2 variants of concern [B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron)]. b-c. Pie plots showing IGHV and IGKV/IGLV gene usage distribution of isolated stem-helix mAbs. Enriched heavy (IGHV1–46 (green) and IGHV3–23 (plum)) (b) and light (IGKV3–20 (sky) and IGLV1–51 (cantaloupe)) (c) gene families are colored. Dot plots showing % nucleotide mutations (SHMs) in the heavy (VH) or light (VL) chains of isolated stem-helix mAbs. The mAbs are grouped by neutralization against sarbecoviruses or sarbecoviruses + MERS-CoV. d-e. CDRH3 (d) or CDRL3 (e) length distributions of isolated mAbs across sarbecovirus broadly neutralizing and sarbecovirus + MERS-CoV broadly neutralizing mAb groups compared to human baseline germline reference. MAbs with 10- and 11- amino acid-CDRH3s or mAbs with 9- and 11- amino CDRL3s, enriched in S2 stem-helix bnAbs compared to baseline germline reference, are indicated by red arrows. f. Sequence conservation logos of 9 (n = 8) and 11 (n = 18) amino acid long CDRL3-bearing stem-helix bnAbs show enrichment of certain J-gene encoded residues.

Figure. 3.. Binding kinetics and fine epitope specificities of S2 β-CoV spike stem-helix bnAbs.

a. BLI binding kinetics of S2 stem-helix mature bnAbs and their inferred germline (iGL) versions to monomeric SARS-CoV-2 and MERS-CoV stem-helix peptides. Binding kinetics were obtained using a 1:1 binding kinetics fitting model on ForteBio Data Analysis software and maximum binding responses, dissociations constants (K_D) and on-rate (k_on) and off-rate constants (k_off) for each antibody-protein interaction are compared. K_D, k_on and k_off values were calculated only for antibody-antigen interactions where a maximum binding response of 0.2nm was obtained. The S2 stem-helix bnAb iGL Abs showed substantially reduced binding to stem-helix peptides compared to their corresponding mature versions. Statistical comparisons between two groups were performed using a Mann-Whitney two-tailed test, (***p < 0.001, ****p < 0.0001; ns- p >0.05). b. Association of S2 stem-helix peptide binding by S2 bnAbs and their iGLs with CDRH3 and CDRL3 motifs. The top line shows characteristics of mAbs with (red) and without (blue) RG motifs in CDRH3s. The bottom line shows characteristics of mAbs with (red) and without (blue) WD motifs in CDRL3s. The left column shows the numbers of mAbs with and without CDR3 motifs with respect to two most common V genes: IGHV1–46 and IGHV3–23 for RG motifs in CDRH3, IGKV3–20 and IGLV1–51 for WD motifs in CDRL3s. The middle and right columns show the responses of iGL and mature mAbs to stem peptides of SARS-CoV-2 and MERS-CoV, respectively. P-values of associations between RG / WD motifs and the responses are shown on tops of the plots and denoted as follows: ns≥0.05, *<0.05, **<0.005. P-values are computed using linear regression. c. ELISA-based epitope mapping of S2 stem-helix bnAbs with SARS-CoV-2 stem alanine scan peptides (25mer). Heatmap shows fold-changes in EC₅₀ binding titers of mAb binding to SARS-CoV-2 stem-helix peptide alanine mutants compared with the WT peptide. SARS-CoV-2 stem-helix residue positions targeted (2-fold or higher decrease in EC₅₀ binding titer compared to WT stem peptide) is indicated in colors. Three hydrophobic residues, F¹¹⁴⁸, L¹¹⁵² and F¹¹⁵⁶, were commonly targeted by stem-helix bnAbs and that form the core of the bnAb epitope. Association of dependence on the stem bnAb core epitope residues with heavy (IGHV1–46 and IGHV3–23) and light (IGKV3–20 and IGLV1–51) chain genes usage and CDRL3 lengths is shown. d-g. A SARS-CoV-2 spike protein cartoon depicts the S2-stem epitope region in green at the base of the prefusion spike ectodomain (d). Sequence conservation of stem-helix hydrophobic core epitope residues (F¹¹⁴⁸, L¹¹⁵² and F¹¹⁵⁶) across β-coronavirus spikes (PDB: 6XR8) (e). D¹¹⁴⁶ stem-helix residue is also indicated. Side (f) and top (g) views of spike stem-helix region highlight the core epitope residues. h.i. Interactions between SARS-CoV-2 S2 stem helix with (h) S2P6 and (i) CC40.8 highlighting the contribution of antibody germline-encoded residues in recognition of hydrophobic stem-helix core epitope. The SARS-CoV-2 S2 stem helices are shown in green, while heavy and light chains of antibodies in orange and yellow, respectively. Germline gene encoded residues are highlighted in red. Structures with PDB codes 7RNJ and 7SJS are used for S2P6 and CC40.8, respectively.

Figure. 4.. Prophylactic treatment of aged mice with S2 stem-helix bnAbs protects against challenge with diverse betacoronaviruses.

a. Two S2 stem-helix bnAbs (CC68.109 and CC99.103) individually, or a DEN3 control antibody were administered intra-peritoneally (i.p.) at 300μg per animal into 9 groups of aged mice (10 animals per group). Each group of animals was challenged intra-nasally (i.n.) 12h after antibody infusion with one of 3 mouse-adapted (MA) betacoronaviruses, (MA10-SARS-2 = SARS-CoV-2; 1 × 10³ plaque forming units (PFU), MA15-SARS1 = SARS-CoV-1; 1 × 10⁵ PFU or M35c4-MERS = MERS-CoV; 1 × 10³ PFU). As a control, groups of mice were exposed only to PBS in the absence of virus. b., e., h. Percent weight change in S2 stem-helix bnAbs or DEN3 control antibody-treated animals after challenge with mouse-adapted betacoronaviruses. Percent weight change was calculated from day 0 starting weight for all animals. c., f., i. Day 2 post-infection Hemorrhage (Gross Pathology score) scored at tissue harvest in mice prophylactically treated with S2 stem-helix bnAbs or DEN3 control mAb. d., g., j. Day 2 post-infection pulmonary function (shown as Penh score) was measured by whole body plethysmography in mice prophylactically treated with S2 stem-helix bnAbs or DEN3 control mAb. Statistical comparisons between groups were performed using a Kruskal-Wallis non-parametric test and significance was calculated with Dunnett’s multiple comparisons test between each experimental group and the DEN3 control Ab group. (p <0.05, **p <0.01, ***p <0.001; ****p < 0.0001; ns- p >0.05).