key: cord-352934-ypls4zau authors: Wan, Jinkai; Xing, Shenghui; Ding, Longfei; Wang, Yongheng; Gu, Chenjian; Wu, Yanling; Rong, Bowen; Li, Cheng; Wang, Siqing; Chen, Kun; He, Chenxi; Zhu, Dandan; Yuan, Songhua; Qiu, Chengli; Zhao, Chen; Nie, Lei; Gao, Zhangzhao; Jiao, Jingyu; Zhang, Xiaoyan; Wang, Xiangxi; Ying, Tianlei; Wang, Haibin; Xie, Youhua; Lu, Yanan; Xu, Jianqing; Lan, Fei title: Human IgG neutralizing monoclonal antibodies block SARS-CoV-2 infection date: 2020-07-03 journal: Cell Rep DOI: 10.1016/j.celrep.2020.107918 sha: doc_id: 352934 cord_uid: ypls4zau Summary COVID-19 has become a worldwide threat to humans, and neutralizing antibodies have therapeutic potential. We have purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain), and obtain 729 paired heavy and light chain fragments. Among these, 178 antibodies test positive for antigen binding, and the majority of the top 17 binders with EC50 below 1 nM are RBD binders. Furthermore, we identify 11 neutralizing antibodies, 8 of which show an IC50 within 10 nM, and the best one, 414-1, with IC50 of 1.75 nM. Through epitope mapping, we find 3 main epitopes in RBD recognized by these antibodies, and epitope B antibody 553-15 could substantially enhance the neutralizing abilities of most of the other antibodies. We also find that 515-5 could cross-neutralize the SARS-CoV pseudovirus. Altogether, our study provides 11 potent human neutralizing antibodies for COVID-19 as therapeutic candidates. COVID-19 has become a worldwide threat to humans, and neutralizing antibodies 35 have therapeutic potential. We have purified more than one thousand memory B cells 36 specific to SARS-CoV-2 S1 or RBD (receptor binding domain), and obtain 729 paired 37 heavy and light chain fragments. Among these, 178 antibodies test positive for 38 antigen binding, and the majority of the top 17 binders with EC 50 below 1 nM are RBD 39 binders. Furthermore, we identify 11 neutralizing antibodies, 8 of which show an IC 50 40 within 10 nM, and the best one, 414-1, with IC 50 of 1.75 nM. Through epitope 41 mapping, we find 3 main epitopes in RBD recognized by these antibodies, and 42 epitope B antibody 553-15 could substantially enhance the neutralizing abilities of 43 most of the other antibodies. We also find that 515-5 could cross-neutralize the 44 We screened sera samples from 11 patients recently recovered from COVID-19, and 119 found all individuals showed certain levels of serological responses, with #507 and 120 #501 being the weakest, to SARS-CoV-2 Spike RBD and S1 proteins ( Figure 1A ). We 121 also found that 10 sera, except for 507, showed neutralization abilities against 122 SARS-CoV-2 pseudoviral infection of HEK293T cells stably expressing human ACE2 123 ( Figure 1B ). Such observations, i.e. the sera from different individuals displayed a 124 wide range of antibody responses, were consistent with a recent report (Wu et al., 125 2020a). Of note, No.509 blood sample was obtained at the second day after 126 hospitalization (Table S2) , the sera already showed weak S1 antigen response and 127 pseudoviral neutralizing activities. 128 The RBD domain in the S1 region of SARS-CoV-2 spike protein is the critical region 129 mediating viral entry through host receptor ACE2. Using recombinant RBD and S1 130 antigens, we then isolated RBD and S1 bound memory B cells for antibody 131 identification using the PBMCs (peripheral blood mononuclear cells) from the 11 132 individuals by fluorescence activated cell sorting ( Figure S1A ). 133 Sequences encoding immunoglobulin heavy (IGH) and light (IGL) chains were 134 amplified from single B cell complementary DNA samples after reverse transcription 135 and then cloned through homologous recombination into mammalian expressing 136 vectors (Robbiani et al., 2017) . Overall, 729 naturally paired IGH and IGL clones were 137 obtained, and the numbers of clones derived from each individual were listed in Table 138 S1. 139 140 In order to screen for SARS-CoV-2 spike antigen specific monoclonal antibodies, we 143 used two primary assays based on ELISA (enzyme linked immunosorbent assay) and 144 FCA (flow cytometry assay), respectively. Among the 729 candidate antibodies expressed in HEK293E cells, 178 were positive for RBD or S1 binding ( Figure 2A ). All 146 the positive clones were then sequenced. Notably, almost all (98.6%) of the 147 sequences obtained were unique ones ( Figure 2B) , similarly as what were previously 148 reported in Ebola Zhang et al., 2016) and Yellow Fever (Calvert et 149 al., 2016) studies. 150 Based on the ranking of ELISA values and FCA positivities, we focused on 29 151 antibodies for further characterization. We first measured the precise values of EC 50 152 by ELISA, and identified 17 strong binders for S-ECD (extracellular domain) and RBD 153 with EC 50 below 1 nM, and the most potent one showing EC 50 at 0.057 nM (8.55 154 ng/ml) ( Figure 2C and Figure S2A ). Of note, among these 29 antibodies, 4 antibodies 155 (413-3, 414-4, 105-28 and 105-41) were FCA negative but bound recombinant RBD 156 relatively well in ELISA ( Figure 2C ). On the other hand, we also identified another 4 157 antibodies (505-8, 414-3-1, 505-17 and 515-5) showed strong FCA positivity but with 158 barely detectable ELISA signals ( Figure 2C ). These findings indicated certain 159 conformational differences might exist between the recombinant and 160 We also noticed that the majority of the 17 strong binders were RBD binders, except 162 for 413-2, 553-13 and 553-18 ( Figure 2C ). And we further confirmed that 553-13 and To identify neutralizing antibodies, we first employed pseudoviral infection assays 170 using HEK293T-ACE2 cells. From all the antibodies tested, we found a total of 16 171 pseudoviral neutralizing antibodies ( Figure 2C , and Figure 3A , column 3). Among 172 these, 11 could neutralize authentic virus entry into Vero-E6 cells, and 8 of them 173 showed potent IC 50 within 10 nM ( Figure 3A , column 1). We next characterized the 174 best one, 414-1, which was able to effectively block authentic viral entry at IC 50 of 1.75 nM ( Figure 3B , left). We also tested 414-1 expressing in CHO cells, and found it could 176 achieve ~ 300 mg/L without any optimization suggesting for a great potential in 177 therapeutic development. Furthermore, the RBD binding affinity of 414-1 was also 178 tested by BLI (bio-layer Interferometry assay), and showed comparable K d of 0.413 179 nM ( Figure 3B, right) . 180 Since CDR3 are the most critical region for antibody diversity, we then aligned the 181 CDR3 sequences of the heavy (CDR3 H ) and light (CDR3 L ) chains of the 11 authentic 182 viral neutralizing antibodies and found 9 unique ones ( Figure 3A To understand the neutralizing mechanism of the 11 antibodies, we performed 191 epitope mapping experiments for the 8 RBD binders (note, 505-8, 413-2 and 515-5 192 were non-RBD binders, Figure 2C and Figure 3A ). In order to do so, we first utilized 193 RBD-ACE2 blocking ELISA, and found that 6 of them could effectively compete ACE2 194 binding to RBD with IC 50 below 3 nM ( Figure 3A , column 6), indicating their binding 195 epitopes overlapping with ACE2 binding surface ( Figure 3C , middle left). 196 We then carried out BLI competition and mutagenesis assays for further analyses. 197 Due to the identical CDR3 sequence and high similarities among 505-3, 515-1 and 198 505-5 mentioned above, we only chose 505-3 as the representative in these assays. 199 Based on BLI competition results, the 8 RBD binders could be classified into 3 groups, replaced in the non-ACE2 binding surface ( Figure 3C , left) to locate these epitopes. 203 All epitope A antibodies were largely unaffected by these mutations ( Figure S3C, left) , 204 indicating that epitope A is limited within ACE2 binding surface ( Figure 3C , middle left) 205 considering that they competed ACE2 binding in blocking ELISA mentioned above. 206 Epitope B antibody, 553-15, was sensitive to F374A, A372T and C379A mutations 207 ( Figure S3C , middle) and also competed ACE2 in blocking ELISA ( Figure 3A , column 208 6), therefore we speculated that epitope B should include these residues and partially 209 overlap with ACE2 binding surface ( Figure 3C , middle right). Finally, residues critical 210 for epitope C antibody binding were shown in Figure 3C , right panel. Since they did 211 not compete ACE2 in blocking ELISA ( Figure 3A , column 6), we proposed epitope C 212 at the indicated area of RBD ( Figure 3C, right) . 213 Notable, despite that the epitope C and non-RBD binders could not block ACE2 214 binding for RBD ( Figure 3A The spike proteins of SARS-CoV-2 share 76% and 35% of amino acid identities with 231 SARS-CoV and MERS-CoV, respectively. Therefore, we wondered whether our 232 antibodies could cross-react with the S proteins of these two other coronaviruses. In 233 order to do so, we overexpressed the S proteins of SARS-CoV-2, SARS-CoV and MERS-CoV in HEK293T, and tested the cross-reactivities by flow cytometry analyses. 235 From this exercise, we found 3 antibodies, 415-5, 415-6 and 515-5, cross-recognizing 236 SARS-CoV S, but not MERS-CoV S ( Figure 4A and 4B). 415-5 and 515-5 shared 237 similar S protein affinities between SARS-CoV-2 and SARS-CoV, but 415-6 had 238 much lower affinity towards SARS-CoV S compared to SARS-CoV-2 S ( Figure 4C ). ELISA signals towards both RBD and S, but could robustly bind freshly expressed S 281 protein in A549 membrane ( Figure 2C, column 4, 6) . These included two neutralizing 282 antibodies 505-8 (mentioned above) and 515-5, indicating that the recombinant RBD 283 or S protein may differ from the membrane bound S in terms of 3D conformation. We 284 would have missed these antibodies if we only had used ELISA for antibody triage. 285 Therefore, future antibody study should consider multiple approaches for the initial 286 identification and quality control. The fluorescently labeled S1 bait was previously prepared by incubating 5 µg of His 422 tag-S1 protein with Anti His tag antibody-PE (Phycoerythrin) for at least 1 hr at 4 °C in 423 the dark, RBD bait performed as before. PBMCs were stained using 7AAD, Cocktail neutralization assay was performed with 2 antibodies by 1:1 (n:n), and 514 calculated IC 50 by total antibodies concentrate ion. 515 All experiments related to authentic virus were done in BSL-3. 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