Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes

Abstract

There is an urgent need to develop effective interventions resistant to the evolving variants of SARS-CoV-2. Nanobodies (Nbs) are stable and cost-effective agents that can be delivered by novel aerosolization route to treat SARS-CoV-2 infections efficiently. However, it remains unknown if they possess broadly neutralizing activities against the prevalent circulating strains. We found that potent neutralizing Nbs are highly resistant to the convergent variants of concern that evade a large panel of neutralizing antibodies (Abs) and significantly reduce the activities of convalescent or vaccine-elicited sera. Subsequent determination of 9 high-resolution structures involving 6 potent neutralizing Nbs by cryoelectron microscopy reveals conserved and novel epitopes on virus spike inaccessible to Abs. Systematic structural comparison of neutralizing Abs and Nbs provides critical insights into how Nbs uniquely target the spike to achieve high-affinity and broadly neutralizing activity against the evolving virus. Our study will inform the rational design of novel pan-coronavirus vaccines and therapeutics.

Figure 1.. The impacts of RBD circulating variants on Nb binding

1A: ELISA binding of the RBD mutants (a summary heatmap). Data shown as fold change of binding affinity relative to that towards RBD WT. 1B: The fold change of neutralizing potencies of the Nbs against two dominant circulating variants (UK and SA strains) compared to wild-type SARS-CoV-2 pseudovirus particles.

Figure 2.. Structure of an ultrapotent class I Nb (21)

2A: Cryo-EM structure of the Nb21:S complex reveals two main RBD conformations of “1-up and 2-down” and “2-up and 1-down”. 2B: The involvement of all three CDRs of Nb21 in RBD binding. 2C: Detailed Nb21:RBD interactions. 2D: Structural overlap of hACE2 with Nb21:RBD complex.

Figure 3.. Structures of class II Nbs (95, 34, and 105)

3A: Cryo-EM structures of Nbs 95 and 34 in complex with S. 3B: Cryo-EM structure of the Nb105:Nb21:RBD complex. 3C: Nb95: RBD interactions. Residues in pink denote Nb95 for RBD binding. 3D: Nb105: RBD interactions. Residues in yellow denote Nb105 for RBD binding. 3E: Class II Nb: RBD interactions are predominantly mediated by CDR3. Nbs are represented as ribbons. The CDR3 loops are shown as surface presentations. 3F: Steric effects of class II Nbs on hACE2:RBD interactions. N322 glycosylation (ACE2 ) is presented in red density.

Figure 4.. Structures of class III Nbs (17 and 36)

4A: Cryo-EM structures of Nb17 in complex with S. 4B: Nb17:RBD interactions are mediated by all three CDRs. 4C: Cryo-EM structure of the Nb17:Nb105:RBD complex. 4D: Nb17 structurally does not overlap with ACE2. 4E: Cryo-EM structure of the Nb36:Nb21:RBD complex. 4F: Epitope of Nb36 on the RBD surface. 4G: Nb17 stacks on NTD via its framework, while isolated Nb36:RBD complex indicates Nb36 would clash with neighboring NTD on S. 4H: ACE2 competition assay with the S.

Figure 5.. Class III Nbs bind novel and conserved neutralizing epitopes unique to Nbs

5A: Epitope clustering analysis of RBD Nbs and correlation with RBD sequence conservation and ACE2 binding sites. 5B: Overview of three Nb classes binding to the RBD, RBD surface was colored based on conservation (ConSurf score). 5C-E: Structural comparison of different classes of Nbs with the closest mAbs for RBD binding.

Figure 6.. mAbs and Nbs binding to RBD are differently affected by mutations in the circulating variants

6A: Localization of six RBD residues where major circulating variants mutate. 6B: Buried surface area of Nbs by different RBD residues. 6C: Buried surface area of Fabs by different RBD residues. 6D-E: Representative structures of different classes of Nbs with major variants residues shown as spheres. Two fab structures binding similarly to Class I Nbs were also shown on the side. 6F: The boxplot showing probability of an epitope residue to hit one of the mutations in variants.

Figure 7.. Comparisons of RBD neutralizing Nbs and mAbs

7A: Buried surface areas of RBD: Nb and RBD: Fab complexes. VH: heavy chain. VL: light chain. 7B: Buried surface areas per-interface residue for Nbs and Fabs. 7C: The contact contribution of CDRs and FRs of Nbs and Fabs in RBD binding (using a 6 Å cutoff). Contact contribution % was calculated as # of contacting residues on CDR or FR region/total # of contacting residues. 7D: Quantification of interface cavity. Y-axis is the curvature value. 7E: Comparison of contributions from CDRs and FRs for RBD binding between in vivo matured Nbs and in vitro selected Nbs. 7F: Representative structures showing different binding modes (epitope curvature) of Nb17 and a Fab. Nbs target concave RBD surfaces to achieve high-affinity binding. 7G: Representative structures showing the direct involvement of FR2 from an in vitro selected Nb (PDB# 7A29) for RBD interaction.