Structural Analysis of Neutralizing Epitopes of the SARS-CoV-2 Spike to Guide Therapy and Vaccine Design Strategies

Abstract

Coronavirus research has gained tremendous attention because of the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (nCoV or SARS-CoV-2). In this review, we highlight recent studies that provide atomic-resolution structural details important for the development of monoclonal antibodies (mAbs) that can be used therapeutically and prophylactically and for vaccines against SARS-CoV-2. Structural studies with SARS-CoV-2 neutralizing mAbs have revealed a diverse set of binding modes on the spike's receptor-binding domain and N-terminal domain and highlight alternative targets on the spike. We consider this structural work together with mAb effects in vivo to suggest correlations between structure and clinical applications. We also place mAbs against severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses in the context of the SARS-CoV-2 spike to suggest features that may be desirable to design mAbs or vaccines capable of conferring broad protection.

Keywords: COVID-19; SARS-CoV-2; coronavirus; immunogen; neutralizing antibodies; spike.

Figure 1

Structure and conformations of the SARS-CoV-2 trimeric spike protein. (A) Domain architecture of SARS-CoV-2 S, comprising the N-terminal domain (NTD), receptor-binding domain (RBD), receptor-binding motif (RBM), furin cleavage site (FCS), S2′, fusion peptide (FP), and heptad repeats 1 and 2 (HR1 and HR2), as they relate to the S1 and S2 subunits, as well as the transmembrane domain (TM) and cytoplasmic tail (CT). Glycosylation sites are numbered and marked with a Y. Regions of S that bind to known neutralizing monoclonal antibodies (mAbs) and nanobodies (VHHs) are indicated with green squares. (B) Conformational changes in the spike ectodomain during membrane fusion. Left: top view of prefusion SARS-CoV-2 S. Conformational changes in adjacent NTDs as the RBD of one protomer shifts into the up position are indicated with arrows. Middle-Left: Side view of prefusion SARS-CoV-2 S with 2 RBDs in the down conformation (RBMs hidden) and one RBD in the up conformation (RBM exposed), bound to the ACE2 receptor. Middle-Right: Side view of postfusion SARS-CoV-2 S with S1 shed and S2 subunits elongated towards the host cell membrane with FP inserted. Right: Side view of postfusion SARS-CoV-2 S after a collapse allowing HR2 to form a six-helix bundle with HR1, resulting in fusion of the viral membrane with the host cell membrane.

Figure 2

Mechanisms of antiviral protection by anti-coronavirus antibodies. Mechanisms include: (A) Inhibition of receptor binding: S interacts with heparan sulfate and ACE2 in the process of viral fusion. Antibodies obscure the ACE2 receptor-binding motif. (B) Antibody binding interferes with the prefusion to postfusion transition, preventing membrane fusion. The RBDs are shown in purple. (C) Antibody binding destabilizes the spike and leads to premature triggering of the postfusion conformation. (D) Antibodies bind multiple virions to form aggregates. (E) Fc-mediated functions include opsonization and activation of the complement system. Natural killer (NK) cell and phagocyte are shown.

Figure 3

Coronavirus spike epitopes. (A) SARS-CoV-2 RBM epitopes. Side view of SARS-CoV-2 trimer (PDB ID 6X2A [20]) shown as gray cartoon with one RBD in the up state (shown as purple cartoon with gray surface). ACE2 (yellow surface, PDB ID: 6M0J [22]) or representatives of RBM-specific antibodies are shown bound to a zoomed in view of the RBD. Antibody variable heavy chain (blue) and light chain (cyan) are shown; constant domains are omitted for clarity. Representative members are shown for each class: CC12.3 from RBM Class I, Cluster I (PDB ID 6XC4 [59]), COVA2-39 from RBM Class I, Cluster II (PDB ID 7JMP [65]), P2B-2F6 from RBM Class II (PDB ID 7BWJ [72]), S2M11 from RBM Class III (PDB ID 7K43 [71]), and CR3022 (left) and S309 (right) from RBD Core (PDB IDs 6W41 [86] and PDB ID 6WPS [80]). (B) RBM Class III antibodies that lock the trimer in a “closed” state. Side and top views are shown for a representative antibody (S2M11) bound to the SARS-CoV-2 trimer (PDB ID: 7K43 [71]). Glycans are shown as green sticks and the location of an N343 from a single protomer is indicated. (C) SARS-CoV-2 NTD epitope. Side view of SARS-CoV-2 trimer (PDB ID 6X2A [20]) shown as gray cartoon with one RBD in the up state (shown as purple cartoon with gray surface). A zoomed in view of the NTD is shown with a representative NTD-specific antibody (4A8, PDB ID 7C2L [85]) bound. Antibody variable heavy chain (blue) and light chain (cyan) are shown; constant domains are omitted for clarity. (D) Epitopes of coronaviruses. Representative antibodies are shown as colored surface: cyan for CC12.3 from RBM Class I (PDB ID 6XC4 [59]), green for P2B-2F6 from RBM Class II (PDB ID 7BWJ [72]), blue for S2M11 from RBM Class III (PDB ID 7K43 [71]), yellow for CR3022 and magenta for S309 from RBD Core (PDB IDs 6W41 [86] and PDB ID 6WPS [80]), orange for 4A8 from NTD (PDB ID 7C2L [85]), and purple for G4 from anti-MERS-CoV S2, (PDB ID 5W9H [87]). Glycans are shown as green sticks. Potential epitopes are indicated with red circles.

Figure 4

Surface conservation of S subdomains. (A) SARS-CoV-2 S NTD. Two side views of the S trimer are shown with the NTD zoomed in. Blue and red regions in the NTD of a single S protomer are conserved and variable, respectively, between SARS-CoV and SARS-CoV-2. Yellow regions are semi-conserved. Glycans are represented by green spheres. The rest of the trimer is in gray. PDB ID 7C2L used to generate the figure [85]. (B) SARS-CoV-2 S2 subunit. Two side views of the S trimer are shown. Coloring scheme is the same as in (A), with the S2 domain of a single protomer colored according to sequence conservation. PDB ID 7C2L used to generate the figure [85]. (C) SARS-CoV-2 S RBD. Two side views of the RBD and SD1 domains are shown with RBM at the top. Coloring scheme for sequence conservation is the same as in (A). PDB ID 7BZ5 used to generate the figure [66]. (D) MERS-CoV S NTD. Two side views of the S trimer are shown with the NTD zoomed in. Blue and red regions are conserved and variable, respectively, between MERS-CoV and SARS-CoV. Yellow regions are semi-conserved. Glycans are represented by green spheres. The rest of the trimer is in gray. PDB ID 5W9H used to generate the figure [87]. (E) MERS-CoV S2 subunit. Two side views of the S trimer are shown. Coloring scheme is the same as in (D), with the S2 domain of a single protomer colored according to sequence conservation. PDB ID 5W9H used to generate the figure [87]. mAb G4 binding site is indicated with a yellow star. (F) MERS-CoV S RBD. Two side views of the RBD and SD1 domains are shown with RBM at the top. Coloring scheme for sequence conservation is the same as in (D). PDB ID 5W9H used to generate the figure [87].