A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2

Abstract

Neutralizing antibodies could potentially be used as antivirals against the coronavirus disease 2019 (COVID-19) pandemic. Here, we report isolation of four human-origin monoclonal antibodies from a convalescent patient, all of which display neutralization abilities. The antibodies B38 and H4 block binding between the spike glycoprotein receptor binding domain (RBD) of the virus and the cellular receptor angiotensin-converting enzyme 2 (ACE2). A competition assay indicated different epitopes on the RBD for these two antibodies, making them a potentially promising virus-targeting monoclonal antibody pair for avoiding immune escape in future clinical applications. Moreover, a therapeutic study in a mouse model validated that these antibodies can reduce virus titers in infected lungs. The RBD-B38 complex structure revealed that most residues on the epitope overlap with the RBD-ACE2 binding interface, explaining the blocking effect and neutralizing capacity. Our results highlight the promise of antibody-based therapeutics and provide a structural basis for rational vaccine design.

Fig. 1. Characterization of COVID-19 virus–specific neutralizing antibodies.

(A to D) The binding kinetics between four antibodies (B38, H4, B5, and H2) and COVID-19 virus RBD were measured using a single-cycle Biacore 8K system. (E to H) Competition binding to the COVID-19 virus RBD between antibodies and ACE2 was measured by BLI. Immobilized biotinylated COVID-19 virus RBD (10 μg/ml) was saturated with antibodies and then flowed with corresponding antibody in the presence of 300 nM soluble ACE2 (blue) or without ACE2 (red). As a control, the immobilized biotinylated RBD was flowed with buffer and then flowed with the equal molar concentration of ACE2 (black). The graphs show binding patterns after antibody saturation. (I) hACE2–enhanced green fluorescent protein (EGFP) was expressed on the HEK293T cell surface, and the cells were stained with 200 ng/ml COVID-19 virus RBD his-tag proteins preincubated with isotype IgG, B38, H4, B5, or H2. The percentages of anti-his-tag APC⁺ (allophycocyanin) cells and EGFP⁺ cells were calculated. (J and K) Competition binding to COVID-19 virus RBD between B38 and H4 was measured by BLI. Immobilized COVID-19 virus RBD (10 μg/ml) was saturated with 300 nM of the first antibody and then flowed with equal molar concentration of the first antibody in the presence of (blue) or without (red) the second antibody. Equal molar concentration of the second antibody was flowed on the immobilized RBD as a control (black). The graphs show binding patterns after saturation of the first antibody.

Fig. 2. Four antibodies can effectively neutralize COVID-19 virus, and two of them exhibit additive inhibition effect.

The mixtures of COVID-19 virus and serially diluted antibodies were added to Vero E6 cells. After 5 days of incubation, IC₅₀ values were calculated by fitting the cytopathic effect from serially diluted antibody to a sigmoidal dose-response curve. Medium containing 100 and 200 times the median tissue culture infectious dose of COVID-19 virus was used for testing the neutralizing abilities of individual antibody (A to D) and cocktail antibodies (E), respectively.

Fig. 3. The protection efficiency of mAbs in hACE2 mice model after infection with COVID-19 virus.

(A) Body weight loss was recorded for PBS, B38 treatment, and H4 treatment groups (for all groups, n = 4 mice). All the mice were challenged intranasally with COVID-19 virus, and a 25 mg/kg dose of antibodies was injected (intraperitoneally) 12 hours after infection. Equal volume of PBS was used as a control. The weight loss was recorded over 3 days, and a significant difference could be observed between the B38 group and the PBS group (unpaired t test, ***P < 0.001). (B) The virus titer in lungs of three groups was determined at 3 dpi by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The mAb treatment group reduced the viral load in the lungs of mice (unpaired t test, ***P < 0.001). (C to H) Representative histopathology of the lungs in COVID-19 virus–infected hACE2 mice (3 dpi). Severe bronchopneumonia and interstitial pneumonia was observed in the PBS group [(C) and (F)], with edema and bronchial epithelial cell desquamation (black arrow) and infiltration of lymphocytes within alveolar spaces (red arrow). Mild bronchopneumonia was observed in the H4 group [(E) and (H)], whereas no lesions were observed in the B38 group [(D) and (G)]. The images and areas of interest (red boxes) are magnified 100× and 400×, respectively.

Fig. 4. Structural analysis of B38 and COVID-19 virus RBD complex and the epitope comparison between B38 and hACE2.

(A) The overall structure of B38 Fab and COVID-19 virus RBD. The B38 heavy chain (cyan), light chain (green), and COVID-19 virus RBD (magenta) are shown in cartoon representation. (B) The epitope of B38 is shown in surface representation. The contact residues by heavy chain, light chain, or both are colored in cyan, green, and magenta, respectively. The residues on RBD involved in both B38 and hACE2 binding are labeled in red. (C) Superimposition of RBD-B38 and RBD-hACE2 [Protein Data Bank (PDB) ID 6LZG]. All molecules are shown in cartoon representation, with the same colors as in (A). hACE2 is colored in light pink. (D) The residues involved in hACE2-RBD binding are highlighted in light pink. The residues on RBD involved in both B38 and hACE2 binding are labeled in red. (E) The complex structure of SARS-CoV RBD (light blue) and hACE2 (yellow) (PDB ID 2AJF). (F) The residues in contact with hACE2 are colored in yellow. The residues are numbered according to SARS-CoV RBD. The residues involved in hACE2 binding of two RBDs are labeled in red. (G to I) The detailed interactions between COVID-19 virus RBD and CDR loops of the heavy chain. (J and K) The detailed interactions between COVID-19 virus RBD and CDR loops of the light chain. The residues are shown in stick representation, with the same colors as in (C). The water molecules are shown as red spheres. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.