key: cord-0998939-5a5a8ifz
authors: Wu, Yan; Wang, Feiran; Shen, Chenguang; Peng, Weiyu; Li, Delin; Zhao, Cheng; Li, Zhaohui; Li, Shihua; Bi, Yuhai; Yang, Yang; Gong, Yuhuan; Xiao, Haixia; Fan, Zheng; Tan, Shuguang; Wu, Guizhen; Tan, Wenjie; Lu, Xuancheng; Fan, Changfa; Wang, Qihui; Liu, Yingxia; Zhang, Chen; Qi, Jianxun; Gao, George Fu; Gao, Feng; Liu, Lei
title: A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2
date: 2020-05-13
journal: Science
DOI: 10.1126/science.abc2241
sha: ce199fb5c60f3982c0cc38a6bbc74ce303e21e5d
doc_id: 998939
cord_uid: 5a5a8ifz

Neutralizing antibodies could be antivirals against COVID-19 pandemics. Here, we report isolation of four human-origin monoclonal antibodies from a convalescent patient, all of which display neutralization abilities. B38 and H4 block the binding between virus S-protein RBD and cellular receptor ACE2. A competition assay indicates their different epitopes on the RBD, making them a potential virus-targeting MAb-pair to avoid 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.

The COVID-19 caused by the novel coronavirus COVID-19 virus has become a pandemic. The virus has spread worldwide, causing fever, severe respiratory illness and pneumonia (1, 2). Phylogenetic analysis indicates that the virus is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV) (3- 5) , but it appears to be more easily transmitted from person to person (6) . To date, no specific drugs or vaccines are available.

COVID-19 virus belongs to the betacoronavirus genus, which includes five pathogens that infect humans (7, 8) . Among them, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) are two highly pathogenic viruses. As with other coronaviruses, the spike glycoprotein (S) homotrimer on the COVID-19 virus surface plays an essential role in receptor binding and virus entry. The S protein is a class I fusion protein-each S protomer consists of S1 and S2 domains (9) with the receptor binding domain (RBD) located on the S1 domain (8) . Previous studies revealed that the COVID-19 virus, similarly to SARS-CoV, uses the ACE2 receptor for cell entry (3, [10] [11] [12] [13] . Numerous neutralizing antibodies have been found targeting the RBDs of SARS-CoV or MERS (14) (15) (16) . Therefore, screening for neutralizing antibodies that target the COVID-19 virus RBD is a priority.

We expressed COVID-19 virus RBD protein as bait to isolate specific single memory B-cells from COVID-19 patient peripheral blood mononuclear cells (PBMCs). The variable regions encoding the heavy and light chain were each amplified from separate single B cells, and then were cloned into a pCAGGS vector with the constant region to produce IgG1 antibodies as described previously (17) . Seventeen paired B cell clones were amplified, three of which were identical (B5, B59 and H1). To identify the antibody binding abilities, the plasmids containing the paired heavy and light chains were cotransfected into HEK 293T cells for monoclonal antibody (MAbs) production. The supernatants were then screened for binding to the RBD by bio-layer interferometry (BLI). An irrelevant anti-SFTSV Gn antibody and a SARS specific antibody were used as controls (18) . The supernatants from four different antibodies (B5, B38, H2 and H4) bound to COVID-19 virus RBD, but not to SARS-CoV RBD (fig. S1), suggesting the epitopes of the two RBDs are immunologically distinct. The usage of heavy chain (V H ) and light chain (V L ) variable genes in these four antibodies are listed in table S1.

The dissociation constants (K d ) for the four antibodies binding to COVID-19 virus RBD, measured using surface plasmon resonance (SPR) ranged from 10 −7 to 10 −9 M (Fig. 1, A to D). We next studied the neutralizing activities of these four antibodies against COVID-19 virus (BetaCoV/Shenzhen/SZTH-003/2020). All exhibited neutralizing activities, with IC 50 values ranging from 0.177 μg/ml to 1.375 μg/ml (Fig.  2 , A to D). Cocktail B38 and H4 exhibit synergetic neutralizing ability, even in the presence of a higher titer of virus (Fig.  2E) .

To evaluate the ability of each antibody to inhibit binding between RBD and ACE2, we performed a competition assay using BLI and a blocking assay using fluorescence-activated cell sorting (FACS). For the BLI assay, streptavidin biosensors labeled with biotinylated RBD were saturated with antibodies, and then the test antibodies were flowed through in the presence of soluble ACE2. B38 and H4 showed complete competition with ACE2 for binding to RBD. In contrast, B5 displayed partial competition, while H2 did not compete with ACE2 for RBD binding (Fig. 1, E to H). The blocking assay by FACS presented the same result (Fig. 1I ). To determine whether B38 and H4 target the same epitope, we performed an epitope competition assay by BLI. The Ni-NTA sensor labeled with the RBD was saturated with B38 IgG and H4 IgG was flowed through, or the sensor was first saturated with H4 IgG and B38 IgG was flowed through. Although RBD was saturated with the first antibody, the second antibody could still bind to RBD, but with some inhibition. This suggests that B38 and H4 recognize different epitopes on RBD with partial overlap (Fig. 1 , J and K).

To explore the protection efficacy of B38 and H4 against challenge with COVID-19 virus in vivo, hACE2 transgenic mice were administered a single dose of 25 mg/kg of B38 or H4 12 hours after viral challenge. The body weight of B38 group decreased slowly and recovered at 3 days post infection (dpi) compared with the PBS control group and the H4 group (Fig. 3A ). The number of viral RNA copies in the lung were also measured at 3 dpi. The RNA copies of both B38 group and H4 group were significantly lower than the PBS group, with a reduction of 32.8% and 26%, respectively (Fig. 3B ). These results show the same trends as the neutralization abilities. Histopathological examination indicated that severe bronchopneumonia and interstitial pneumonia can be observed in the mice of PBS control group, with edema and bronchial epithelia cell desquamation and infiltration of lymphocytes within alveolar spaces (Fig. 3, C and F) . Mild bronchopneumonia was observed in the H4 group (Fig. 3 , E and H), while no lesions were observed in the B38 group (Fig. 3 , D and G).

Consistent with the binding affinity between RBD and B38 or H4, stable complexes were obtained in both RBD-B38 and RBD-H4 mixtures ( fig. S2 ). Complex crystal structure of RBD-B38 Fab was solved at 1.9 Å resolution (table S2) . Three complementarity-determining regions (CDRs) on the heavy chain and two CDRs on the light chain are involved in interaction with RBD (Fig. 4, A, B , and G to K). The buried surface area of heavy and light chain on the epitope is 713.9 Å and 497.7 Å, respectively. There are 36 residues in the RBD involved in the interaction with B38, in which 21 residues and 15 residues interact with heavy and light chain, respectively (table S3 and Fig. 4B ). Sequence alignment indicates that only 15 of the 36 residues in the epitope (defined as residues buried by B38) are conserved between COVID-19 virus and SARS-CoV (Fig. 4, D to F, and fig. S3 ). Notably, most contacts in the interface between B38 and RBD are hydrophilic interactions (table S4) . Water molecules play important role in the binding between COVID-19 RBD and B38 (Fig. 4 , G and I to K). These difference explain the B38 specific binding to COVID-19 rather than SARS-CoV.

To explore the structural basis for B38 blocking the interaction between COVID-19 virus RBD and ACE2, the complex structures of RBD/B38-Fab and RBD/hACE2 were superimposed. Both the VH and V L of B38 would in sterically hinder ACE2 binding (Fig. 4C) . Notably, the RBD in B38-bound form and hACE2-bound form have no significant conformational changes with a Cα root mean squared deviation (RMSD) of 0.489 Å (for 194 atoms). Further analysis indicated that 18 of the 21 amino acids on the RBD are involved in binding both B38 and ACE2 (Fig. 4D) , which explains why B38 abolishes the binding between COVID-19 virus RBD and the receptor.

As the COVID-19 outbreak continues to spread, characterization of the epitopes on the COVID-19 virus RBD will provide valuable information for vaccine development. Furthermore, the molecular features of the neutralizing antibody targeting epitopes are helpful for the development of small molecule or peptide drugs/inhibitors. The neutralizing antibodies themselves are also promising candidates for prophylactic and therapeutic treatment against COVID-19 virus. and coordinated the project. Y.W., F.G. and G. were measured by BLI. Immobilized biotinylated COVID-19 virus RBD (10 μg/mL) were 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 of ACE2 (black). The grams show binding patterns after antibody saturation. (I) hACE2-EGFP was expressed on the HEK293T cell surface, and the cells were stained with 200 ng/mL COVID-19 virus RBD his-tag proteins pre-incubated with isotype IgG, B38, H4, B5 or H2. The percentage of both antihistag APC + 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) were saturated with 300 nM of the first antibody, and then flowed with equal molar of the first antibody in the presence of (blue) or without (red) the second antibody. Equal molar of the second antibody flowed on the immobilized RBD as a control (black). The grams show binding patterns after saturation of the first antibody. 

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We thank the staff of the BL17U1 beamline at Shanghai Synchrotron Radiation Facility (SSRF) for data collection. We thank Dr. Honglei Sun for discussion the histopathology experiment.