key: cord-0905345-o5545c1x
authors: Tai, Wanbo; Zhang, Xiujuan; He, Yuxian; Jiang, Shibo; Du, Lanying
title: Identification of SARS-CoV RBD-targeting monoclonal antibodies with cross-reactive or neutralizing activity against SARS-CoV-2
date: 2020-05-13
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2020.104820
sha: 01275ec773b95b100c057480125039257da64d45
doc_id: 905345
cord_uid: o5545c1x

SARS-CoV-2-caused COVID-19 cases are growing globally, calling for developing effective therapeutics to control the current pandemic. SARS-CoV-2 and SARS-CoV recognize angiotensin-converting enzyme 2 (ACE2) receptor via the receptor-binding domain (RBD). Here, we identified six SARS-CoV RBD-specific neutralizing monoclonal antibodies (nAbs) that cross-reacted with SARS-CoV-2 RBD, two of which, 18F3 and 7B11, neutralized SARS-CoV-2 infection. 18F3 recognized conserved epitopes on SARS-CoV and SARS-CoV-2 RBDs, whereas 7B11 recognized epitopes on SARS-CoV RBD not fully conserved in SARS-CoV-2 RBD. The 18F3-recognizing epitopes on RBD did not overlap with the ACE2-binding sites, whereas those recognized by 7B11 were close to the ACE2-binding sites, explaining why 7B11 could, but 18F3 could not, block SARS-CoV or SARS-CoV-2 RBD binding to ACE2 receptor. Our study provides an alternative approach to prevent SARS-CoV-2 infection using anti-SARS-CoV nAbs.

which, 18F3 and 7B11, neutralized SARS-CoV-2 infection. 18F3 recognized conserved 23 epitopes on SARS-CoV and SARS-CoV-2 RBDs, whereas 7B11 recognized epitopes 24 on SARS-CoV RBD not fully conserved in SARS-CoV-2 RBD. The 18F3-recognizing 25 epitopes on RBD did not overlap with the ACE2-binding sites, whereas those 26 recognized by 7B11 were close to the ACE2-binding sites, explaining why 7B11 could, 27 but 18F3 could not, block SARS-CoV or SARS-CoV-2 RBD binding to ACE2 receptor. 28

Our study provides an alternative approach to prevent SARS-CoV-2 infection using 29

anti-SARS-CoV nAbs. controls. Briefly, ELISA plates were precoated with respective RBD proteins (1 99 μg/ml) overnight at 4℃, which were blocked with 2% fat-free milk in PBST for 2 h at 100 37℃. SARS-CoV RBD-specific mouse mAbs (10 and 1 μg/ml) were added to the 101 plates and incubated for 2 h at 37℃. After washes, the plates were further incubated 102 with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Fab specific, 103 1:3,000, Thermo Fisher Scientific) for 1 h at 37℃. Substrate 104 3,3',5,5'-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO) was the added to the 105 plates, and the reactions were stopped by addition of H 2 SO 4 (1N). The absorbance at 106 450 nm (A450) was measured using an ELISA plate reader (Tecan, San Jose, CA). 107

Mapping of epitopes of the selected SARS-CoV RBD-specific mAbs on 108 SARS-CoV RBD was performed using a protocol similar to that described above, 109 except for coating of the ELISA plates with respective SARS-CoV RBD WT and 110 mutant proteins (1 μg/ml), followed by the addition of mAbs at serial dilutions for 111 detection. binding affinity at 10 μg/ml ( Fig. 2A) . In contrast, all these mAbs could bind 153 SARS-CoV RBD protein, and most had strong binding affinity at 10 and 1 μg/ml (Fig.  154   2B) . These data suggest that SARS-CoV RBD-specific mAbs could cross-react with 155 SARS-CoV-2 RBD. pseudovirus infection with about 80% neutralization at 10 μg/ml (Fig. 2C) . However, 162 all these mAbs neutralized SARS-CoV pseudovirus infection, most of which had >50% 163 neutralizing ability at 10 μg/ml, and a few reached >50% neutralization at 1 μg/ml (Fig.  164   2D) . Notably, several mAbs, such as S28, 33G4, and 24F4, had potent neutralizing 165 activity against SARS-CoV pseudovirus, but failed to neutralize SARS-CoV-2 166 infection, even at 10 μg/ml (Fig. 2C-D) , suggesting that these mAbs may recognize 167 epitopes on the RBD of SARS-CoV different from those of SARS-CoV-2 RBD. 168 Therefore, we identified two SARS-CoV RBD-targeting nAbs with proven 169 cross-neutralizing ability against SARS-CoV-2 S protein-mediated viral entry. 170

We further detected the epitopes on SARS-CoV RBD potentially recognized by the two 172 cross-neutralizing mAbs, 7B11 and 18F3, and included two non-cross-neutralizing 173 mAbs, 13B6 and 33G4, as controls. We have previously shown that 13B6 may 174 recognize epitopes at residues R441 and D454 of SARS-CoV RBD (He et al., 2006a) . 175

Here, we constructed a series of SARS-CoV RBD mutant proteins based on the 176 interaction between the RBD and viral receptor (Li et al., 2005) , and performed an 177 ELISA to test binding ability of the above mAbs to these mutant proteins. Compared 178 with the binding to SARS-CoV RBD wild-type (WT) protein, neither 13B6 nor 18F3 179 bound to the RBD containing D392 and V394 mutations. Moreover, 13B6 did not bind 180 to the RBD containing D414 and F416 mutations, and 7B11 did not bind to the RBD 181 containing I428 and A430 mutations or the RBD containing K439 mutation (Fig. 3) . In 182 addition, 13B6 showed reduced binding to the RBDs containing V369/A371, 183 A371/K373, or Y481 mutations (Fig. 3) . This line of evidence suggests that the above 184 residues were the epitopes recognized by respective mAbs, among which residues 185 D392 and V394 in SARS-CoV RBD were conserved neutralizing epitopes 186 corresponding to residues D405 and V407 in SARS-CoV-2 RBD, and residues I428, 187 A430, and K439 in SARS-CoV RBD were neutralizing epitopes not fully conserved in 188 SARS-CoV-2 RBD (Fig. 4A) . receptor, the results revealed that 13B6 and 18F3 could not block such binding (Fig. 4B,  197 C). This might be due to that most or all epitopes recognized by 13B6 or 18F3 did not 198 overlap with the ACE2 binding sites on SARS-CoV or SARS-CoV-2 RBD (Li et al., 199 2005; Yan et al., 2020), while most epitopes recognized by 7B11 were very close to the 200 ACE2 binding sites (Fig. 4A ). In addition, control mAb 33G4 only blocked SARS-CoV 201 RBD, but not SARS-CoV-2 RBD, binding to the ACE2 receptor (Fig. 4B, C) , partially 202 explaining why this mAb did not neutralize SARS-CoV-2 infection. 203 Moreover, the two cross-neutralizing mAbs, 18F3 and 7B11, respectively recognized 227 two conserved, as well as several non-conserved, neutralizing epitopes on the RBDs of 228 SARS-CoV and SARS-CoV-2. While 18F3 could not block the binding between RBD 229 and ACE2 receptor, 7B11 did block this binding, indicating that they recognized 230 epitopes different from, or close to, the receptor binding sites on the RBDs. 231 232 Notably, 7B11 had a relatively higher neutralizing activity against SARS-CoV-2 233 infection than that against SARS-CoV infection, whereas its ability to inhibit the 234 SARS-CoV-2 RBD-ACE2 binding was relatively lower than that to inhibit the 235 SARS-CoV RBD-ACE2 binding, partially because that the neutralizing activity of 236 nAbs could not always be positively correlated with the inhibition of their binding to 237 the receptor. Other reasons might be due to that the binding between SARS-CoV-2 238 RBD and ACE2 receptor was much stronger than that between SARS-CoV RBD and 239 ACE2 (Tai et al., 2020) , potentially resulting in the reduced inhibition. 

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2020). (B) Inhibition of SARS-CoV RBD-specific mAbs for 411 the binding of SARS-CoV or SARS-CoV-2 RBD to ACE2 receptor by flow 412 cytometry analysis. Percent (%) inhibition was calculated based on the relative 413 fluorescence intensity with or without respective mAbs

SARS-CoV or SARS-CoV-2 RBD (2 μg/ml) was used for the binding to hACE2/293T

The experiments 416 were repeated twice and obtained similar results. (C) Representative flow cytometry 417 images of SARS-CoV RBD-specific mAbs (10 μg/ml) in inhibition of the binding 418 between SARS-CoV or SARS-CoV-2 RBD and ACE2 receptor

μg/ml) to hACE2/293T cells is shown in red line, 420 and the blockage of this binding by mAbs (33G4, 13B6, 18F3, and 7B11) is shown in 421 blue line. hIgG-Fc protein

The authors declare no competing interests. 257

Du, L., He, Y., Zhou, Y., Liu