Broad receptor tropism and immunogenicity of a clade 3 sarbecovirus

Although Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S) glycoproteins remain uncharacterized. Here, we show that the African Rinolophus bat clade 3 sarbecovirus PRD-0038 S has a broad ACE2 usage and that RBD mutations further expand receptor promiscuity and enable human ACE2 utilization. We determined a cryoEM structure of the PRD-0038 RBD bound to R. alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038 S using cryoEM and monoclonal antibody reactivity revealed its distinct antigenicity relative to SARS-CoV-2 and identified PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038 S vaccination elicited greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared to SARS-CoV-2 S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.


Figure S7
Conservation analysis of epitopes targeted by monoclonal antibodies between PRD-0038 S and SARS-CoV-2 S. (A) Epitope mapped onto PRD-0038 RBD structure with sequence alignment of key residues at the interface.The PRD-0038 RBD is shown in gray ( three RBDs are shown with distinct shades of gray for S2M11 which recognizes a quaternary epitope) and N-linked glycans are rendered as blue spheres.SARS-CoV-2 residue numbering is shown in gray and PRD-0038 residue numbering is shown in pink.(B) PRD-0038 S sequence conservation with SARS-CoV-2 S at key S2 fusion machinery epitopes (indicated with dashed lines).Residues are colored according to sequence identity (orange : not conserved, green : conserved).(C) Sequence alignment of corresponding S2 epitopes.SARS-CoV-2 residue numbering is shown in gray and PRD-0038 residue numbering is shown in pink.For (A) and (C) the RBM is depicted with a black outline and the monoclonal antibody epitopes are colored according to their antigenic sites (I, purple; II, red; IV, violet; V, green).

Figure
Figure S1 CryoEM data collection and refinement of the dimeric R. alcyone ACE2-bound PRD-0038 RBD complex.(A and B) Representative electron micrograph (A) and 2D class averages (B) of -the dimeric R. alcyone ACE2-bound PRD-0038 RBD embedded in vitreous ice.The scale bar represents 100 nm (A) or 100 Å (B).(C) Gold-standard Fourier shell correlation curves for the final cryoEM reconstructions of the dimeric -ACE2/RBD complex (solid gray line) and locally refined ACE2/RBD (solid black line) shown in (D).The 0.143 cutoff is indicated with a gray dashed line.(D) Local resolution map calculated using CryoSPARC and plotted onto the sharpened cryoEM map.(E) Data processing flowchart.CTF: contrast transfer function; NUR: non-uniform refinement.

Figure
Figure S3 Heatmaps of change in ACE2-binding avidity resulting from RBD mutations determined by DMS.

Figure
Figure S4 Binding data and Western blot analysis.(A and B) BLI binding analysis of 1 µM dimeric R. affinis (A) and R. sinicus (B) ACE2-Fc alleles to the biotinylated T487W PRD-0038 RBD immobilized on streptavidin biosensors.(C and D) BLI binding analysis of 1 µM dimeric R. affinis (C) and R. sinicus (D) ACE2-Fc alleles to the biotinylated K482Y PRD-0038 RBD immobilized on streptavidin biosensors.(E) Representative Western Blot of wildtype (WT) and mutant PRD-0038 S VSV pseudoviruses normalized based on the amount of incorporated S and VSV-M.Anti-VSV-M Antibody (Kerafast) and Monoclonal ANTI-FLAG® M2 antibody produced in mouse (Sigma) were used as the primary antibody against VSV backbone and S, respectively.Alexa Fluor® 680 AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch) was used as the secondary antibody.

Figure
Figure S5 CryoEM data collection and refinement of PRD-0038 S. (A and B) Representative electron micrograph (A) and 2D class averages (B) of PRD-0038 PentaPro S embedded in vitreous ice.The scale bar represents 100 nm (A) or 100Å (B).
(C) Gold-standard Fourier shell correlation curve for the cryoEM reconstruction.The 0.143 cutoff is indicated with a gray dashed line.(D) 3D reconstruction of PRD-0038 PentaPro S colored by local resolution as determined using cryoSPARC.(E) Data processing flowchart.CTF: contrast transfer function; NUR: non-uniform refinement.(F) 3D reconstruction obtained by local refinement of the PRD-0038 S NTD colored by local resolution as determined using cryoSPARC.(G) Gold-standard Fourier shell correlation curve.The 0.143 cutoff is indicated with a gray dashed line.

Figure
Figure S8 Dose-response curves of mouse serum neutralization before (A) and after (B) three immunizations with SARS-CoV-2 HexaPro S or PRD-0038 HexaPro S.

Figure
Figure S10 DMS epitope mapping of serum antibodies.(A) Evaluation of epitopes targeted by serum antibodies elicited by SARS-CoV-2 HexaPro S (mouse 1-3, 1-4, 1-5, and 1-6) or PRD-0038 HexaPro S (mouse 2-3 and 2-6) vaccination using yeast-displayed DMS of vaccine-matched and mismatched RBDs (indicated above each column).The average effect of mutations at each site are mapped to the SARS-CoV-2 structure, where blue and red indicate positions where mutations increase or decrease serum binding, respectively.

Table S3 Representative KD values determined from BLI binding analysis.
* N.D. -not determined due to weak binding