Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2

Abstract

Cost-effective, efficacious therapeutics are urgently needed to combat the COVID-19 pandemic. In this study, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (RBD). We discovered Nbs with picomolar to femtomolar affinities that inhibit viral infection at concentrations below the nanograms-per-milliliter level, and we determined a structure of one of the most potent Nbs in complex with the RBD. Structural proteomics and integrative modeling revealed multiple distinct and nonoverlapping epitopes and indicated an array of potential neutralization mechanisms. We bioengineered multivalent Nb constructs that achieved ultrahigh neutralization potency (half-maximal inhibitory concentration as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization and aerosolization.

Fig. 1. Production and characterizations of high-affinity RBD Nbs for SARS-CoV-2 neutralization.

(A) Binding affinities of 71 Nbs toward the RBD, as determined by ELISA. The pie chart shows the number of Nbs according to affinity and solubility. O.D., optical density. (B) Screening of 49 high-affinity Nbs with high-expression level, as determined by SARS-CoV-2–GFP pseudovirus neutralization assay. n = 1 for Nbs with neutralization potency IC₅₀ ≤ 50 nM; n = 2 for Nbs with neutralization potency IC₅₀ > 50 nM (n indicates the number of replicates). (C) Neutralization potency of 18 highly potent Nbs was calculated on the basis of the pseudotyped SARS-CoV-2 neutralization assay (luciferase). Purple, red, and yellow lines denote Nbs 20, 21, and 89 with IC₅₀ < 0.2 nM. Two different purifications of the pseudovirus were used. The average neutralization percentage was shown for each data point (n = 5 for Nbs 20 and 21; n = 2 for all other Nbs). (D) Neutralization potency of 14 neutralizing Nbs by SARS-CoV-2 plaque reduction neutralization test (PRNT). The average neutralization percentage was shown for each data point (n = 4 for Nbs 20, 21, and 89; n = 2 for other Nbs). (E) Summary table of pseudovirus and SARS-CoV-2 neutralization potencies of 18 Nbs. N/A, not tested. (F) SPR binding kinetics measurement for Nb21. Ka, acid dissociation constant; Kd, dissociation constant; K_D, equilibrium dissociation constant; RU, relative units.

Fig. 2. Nb epitope mapping by integrative structural proteomics.

(A) Summary of Nb epitopes on the basis of SEC analysis. Purple, Nbs that bind the same RBD epitope; green, Nbs of different epitopes; gray, not tested. (B) Representation of SEC profiling of the RBD, RBD-Nb21 complex, and RBD-Nb21-Nb105 complex. The y axis represents ultraviolet 280 nm absorbance units (mAu). (C) Cartoon model showing the localization of five Nbs that bind different epitopes: Nb20 (purple), Nb34 (green), Nb93 (dark pink), Nb105 (yellow), and Nb95 (light pink) in complex with the RBD (gray). Blue and red lines represent DSS cross-links shorter or longer than 28 Å, respectively. (D) Top-10-scoring cross-linking–based models for each Nb (cartoons) on top of the RBD surface.

Fig. 3. Crystal structure analysis of an ultrahigh-affinity Nb in complex with the RBD.

(A) Cartoon presentation of Nb20 in complex with the RBD. CDR1, -2, and -3 are in red, green, and orange, respectively. (B) Zoomed-in view of an extensive polar interaction network that centers on R35 of Nb20. (C) Zoomed-in view of hydrophobic interactions. (D) Surface presentation of the Nb20-RBD and hACE2-RBD complex (PDB ID 6M0J). Single-letter abbreviations for amino acid residues are as follows: A, Ala; E, Glu; F, Phe; L, Leu; M, Met; Q, Gln; R, Arg; V, Val.

Fig. 4. Potential mechanisms of SARS-CoV-2 neutralization by Nbs.

(A) hACE2 (blue) binding to spike trimer conformation (wheat, beige, and gray colors) with one RBD in the “up” conformation (PDB IDs 6VSB and 6LZG). (B) Nb20 (epitope I, purple) partially overlaps with the hACE2 binding site and can bind the closed spike conformation with all RBDs “down” (PDB ID 6VXX). (C) Summary of spike conformations accessible (+) to the Nbs of different epitopes. (D) Nb93 (epitope II, dark pink) partially overlaps with the hACE2 binding site and can bind to spike conformations with at least one RBD up (PDB ID 6VSB). (E and F) Nb34 (epitope III, green) and Nb95 (epitope IV, light pink) do not overlap with the hACE2 binding site and bind to spike conformations with at least two open RBDs (PDB ID 6XCN).

Fig. 5. Development of multivalent Nb cocktails for highly efficient SARS-CoV-2 neutralization.

(A) Pseudotyped SARS-CoV-2 neutralization assay of multivalent Nbs. The average neutralization percentage of each data point is shown (n = 2). ANTE-CoV2-Nab20T_GS/EK: homotrimeric Nb20 with the GS or EK linker; ANTE-CoV2-Nab21T_GS/EK: homotrimeric Nb21 with the GS or EK linker. (B) SARS-CoV-2 PRNT of monomeric and trimeric forms of Nbs 20 and 21. The average neutralization percentage of each data point is shown (n = 2 for the trimers; n = 4 for the monomers). (C) Summary table of the neutralization potency measurements of the multivalent Nbs. N/A, not tested. (D) Mapping mutations to localization of Nb epitopes on the RBD. The x axis corresponds to the RBD residue numbers (333 to 533). Rows in different colors represent different epitope residues. Epitope I: 351, 449 to 450, 452 to 453, 455 to 456, 470, 472, 483 to 486, and 488 to 496; epitope II: 403, 405 to 406, 408, 409, 413 to 417, 419 to 421, 424, 427, 455 to 461, 473 to 478, 487, 489, and 505; epitope III: 53, 355, 379 to 383, 392 to 393, 396, 412 to 413, 424 to 431, 460 to 466, and 514 to 520; epitope IV: 333 to 349, 351 to 359, 361, 394, 396 to 399, 464 to 466, 468, 510 to 511, and 516; epitope V: 353, 355 to 383, 387, 392 to 394, 396, 420, 426 to 431, 457, 459 to 468, 514, and 520.