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

Abstract

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBD_SARS-CoV. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.

Fig. 1. The impact of RBD circulating variants on Nb binding and neutralization.

a ELISA binding of the spike variants (a summary heatmap). Data are shown as binding affinity fold change relative to that of RBD WT. b The fold change in neutralizing potencies of the Nbs against two dominant circulating variants (Alpha and Beta strains) relative to that of the wild-type SARS-CoV-2 pseudovirus particles. Negative values represent a loss in affinity or neutralization potency, and positive values represent a gain in affinity or neutralization potency. Based on the highest Nb concentration tested, reduction in affinity or neutralization potency greater than 1000-fold is represented as “<−1000”.

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

a Cryo-EM structure of the Nb21:S complex reveals 1-up and 2-down RBD conformations. b The involvement of three CDRs of Nb21 for RBD binding. c Additional Nb21:RBD interactions: side chains of R97, N52, and N55 (Nb21) form hydrogen bonds with the main chain carbonyl groups of L492 and Y449 and the side chain of T470 (RBD), respectively. The main-chain carbonyl group of A29 (Nb21) also forms a hydrogen bond with Q493 (RBD). Besides these polar interactions, F45 and L59 of Nb21 and V483 of RBD form a cluster of hydrophobic interactions, together, providing ultrahigh-affinity and selectivity for RBD binding. d Structural overlap of hACE2 with Nb21:RBD complex.

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

a Cryo-EM structures of Nbs 95 and 34 in complex with S. b Cryo-EM structure of the Nb105:Nb21:RBD complex. c Nb95: RBD interactions. Residues in pink denote Nb95 for RBD binding. d Nb105: RBD interactions. Residues in yellow denote Nb105 for RBD binding. e Class II Nb:RBD interactions are predominantly mediated by CDR3. Nbs are represented as ribbons. The CDR3 loops are shown as surface representations. f Steric effects of class II Nbs on hACE2:RBD interactions. N322 glycosylation (ACE2) is presented in red density.

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

a Cryo-EM structures of Nb17 in complex with S. b Nb17:RBD interactions are mediated by all three CDRs. c Cryo-EM structure of the Nb17:Nb105:RBD complex. d Nb17 structurally does not overlap with ACE2. e Cryo-EM structure of the Nb36:Nb21:RBD complex. f Epitope of Nb36 on the RBD surface. g Nb17 stacks on NTD via its framework, while isolated Nb36:RBD complex indicates Nb36 would clash with neighboring NTD on S. h ACE2 competition assay with the S.

Fig. 5. Class III Nbs bind semi-conserved epitopes unique to Nbs.

a Epitope clustering analysis of RBD Nbs and correlation with RBD sequence conservation and ACE2 binding sites. The conservation scores of SARS-CoV-2 Spike RBD amino acids were computed by ConSurf server using the empirical Bayesian method from the multiple sequence alignment and normalized by the z-score method. b Overview of three Nb classes binding to the RBD, RBD surface was colored based on conservation (ConSurf score). c–e. Structural comparison of different classes of Nbs with the closest mAbs for RBD binding.

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

a Localization of six RBD residues where major circulating variants mutate. b Buried surface area of Nbs by different RBD residues. c Buried surface area of Fabs by different RBD residues. d, e Representative structures of different classes of Nbs with major variant residues shown as spheres. Two Fab structures that bind similarly to Class I Nbs were shown on the side. f The boxplot showing the probability of epitope residues coinciding with the variant mutations (n = 24, 15, and 56 for Nbs, in vivo matured Nbs, and Fabs, respectively). Box plots indicate median (middle line), 25th, 75th percentile (box), and 5th and 95th percentile (whiskers) as well as outliers (single points). Statistical analysis was performed using a two-tailed student t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 7. Comparisons of RBD neutralizing Nbs and mAbs.

a Buried surface areas of RBD: Nb and RBD: Fab complexes. VH heavy chain, VL light chain. b Buried surface areas per-interface residue for Nbs and Fabs. c 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. d Quantification of interface cavity. Y-axis is the curvature value. (n = 25 and 60 for Nbs and Fabs, respectively). Box plots indicate median (middle line), 25th, 75th percentile (box), and 5th and 95th percentile (whiskers) as well as outliers (single points). Statistical analysis was performed using a two-tailed student t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. e Comparison of contributions from CDRs and FRs for RBD binding between in vivo matured Nbs and in vitro selected Nbs (n = 9 and 15 fpr in vitro selected Nbs and in vivo matured Nbs, respectively). f Representative structures of 7d showing different binding modes (epitope curvature) of an Nb and a Fab. Nbs target concave RBD surfaces to achieve high-affinity binding. g Representative structures of 7e showing the direct involvement of FR2 from an in vitro selected Nb (PDB# 7A29) for RBD interaction.