key: cord-0874999-xft4953n
authors: Qu, Yuanyuan; Zhang, Xueyan; Wang, Meiyu; Sun, Lina; Jiang, Yongzhong; Li, Cheng; Wu, Wei; Chen, Zhen; Yin, Qiangling; Jiang, Xiaolin; Liu, Yang; Li, Chuan; Li, Jiandong; Ying, Tianlei; Li, Dexin; Zhan, Faxian; Wang, Youchun; Guan, Wuxiang; Wang, Shiwen; Liang, Mifang
title: Antibody Cocktail Exhibits Broad Neutralization against SARS-CoV-2 and SARS-CoV-2 variants
date: 2021-04-16
journal: bioRxiv
DOI: 10.1101/2021.04.16.440083
sha: 9ee22fad6d0b3cbacd9366e6f4ca28a799cb04ff
doc_id: 874999
cord_uid: xft4953n

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated multiple variants resistant to therapeutic antibodies. In this study, 12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries. Of them, two RBD-binding antibodies (F61 and H121) showed high affinity neutralization against SARS-CoV-2, whereas three S2-target antibodies failed to neutralize SARS-CoV-2. Following structure analysis, F61 identified a linear epitope located in residues G446 -S494, which overlapped with angiotensin-converting enzyme 2 (ACE2) binding sites, while H121 recognized a conformational epitope located on the side face of RBD, outside from ACE2 binding domain. Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-CoV-2. Importantly, F61 and H121 exhibited efficient neutralizing activity against variants B.1.1.7 and B.1.351, those showed immune escape. Efficient neutralization of F61 and H121 against multiple mutations within RBD revealed a broad neutralizing activity against SARS-CoV-2 variants, which mitigated the risk of viral escape. Our findings defined the basis of therapeutic cocktails of F61 and H121 with broad neutralization and delivered a guideline for the current and future vaccine design, therapeutic antibody development, and antigen diagnosis of SARS-CoV-2 and its novel variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is acknowledged as the 53 novel coronavirus that causes the global pandemic of COVID-19 (Andersen et al., 2020) . Up 54 to 28 February 2021, over 100 million confirmed cases have been reported worldwide (World 55 Health Organization). SARS-CoV-2 grouped to the betacoronavirus genus (Wu et al., 2020) 56 is proved to share about 80% sequence identity to SARS-CoV and target the same cellular 57 receptor, angiotensin-converting enzyme 2 (ACE2) (Daniel et al., 2020) . ACE2 directly binds 58 to SARS-CoV-2 spike (S) protein which is consisted of S1 subunit and S2 subunit (Daniel 59 For recombinant human mAb production, the cDNA encoding mAb variable regions of 139 the heavy and light chains were cloned into expression plasmids containing the human IgG1 Enzyme-Linked Immunosorbent (ELISA) Assays and non-competitive ELISA assay 146 ELISA plates were coated with SARS-CoV-2 RBD protein, S1 protein, S2 protein, S 147 protein trimer and mutant S1 protein (Jiang-su East-Mab Biomedical Technology, CHN) at 4 148°C overnight. Following washing with PBST, serial dilutions of testing antibodies start at 149 1μg/ml or serial dilutions of plasma start at 1:100 were added to each well and incubated at 37 Surface plasmon resonance (SPR) assay 156 Purified antibodies targeting S1 were quantified with SPR assay using the BIAcore Virus neutralization assay. 164 The virus neutralization assay with pseudoviruses was conducted as described previously 165 (Nie et al., 2020) . Briefly, serially diluted antibodies were added into 96-well plates. After 166 that, 50 µl pseudoviruses were added to the plates, followed by incubation at 37°C for one 167 hour. Afterward, HuH-7 cells were added into the plates (2×10 4 cells/100 µl per well), 168 followed by 24h incubation at 37°C in a humidified atmosphere with 5% CO2. 169 Chemiluminescence detection was performed straight after, and the Reed-Muench method 170 was used to calculate the virus neutralization titer. The half-maximal inhibitory 171 concentrations (IC50) were determined using 4-parameter logistic regression (GraphPad 172 Prism version 8). 173 Authentic SARS-CoV-2 was used in the plaque reduction neutralization test (PRNT To isolate mAbs, we collected plasma and peripheral blood mononuclear cells (PBMCs) 227 from 15 confirmed COVID-19 convalescent patients in Hubei and Shandong Provence. We 228 evaluated antibodies titer in plasma to SARS-CoV-2 N protein and different fragments of S 229 protein, including RBD, S1, and S2 with ELISA ( Fig. 1A) and colloidal gold test (data not 230 shown). The plasma from donors 2, 10 and 11 showed higher IgG titer against RBD, S1 and 231 S2. Thus they were chosen for library construction by the pComb 3H vector system. The 232 library was established with a complexity of 1×10 8 estimated independent clones and 100% 233 Fab genes diversity after sequencing confirmation. Single clone screening was performed 234 with ELISA, and a total of 274 positive monoclonal were identified (Fig. 1B) . Based on 235 sequencing and ELISA results, four unique clones from RBD, five from S1, and three from 236 S2 were chosen as the candidates for further interrogation. 237 In order to analysis the characteristics of selected Fab antibodies, we recloned the Fab 238 antibodies into the IgG1 format. We further determined the binding specificity of the 12 239 candidate IgGs with purified SARS-CoV-2 virion and different S protein fragments (S1, S2, 240 RBD and S protein trimer) utilizing ELISA. All 12 antibodies were able to recognize purified 241 SARS-CoV-2 virion and S protein trimer. However, the binding strength of 12 antibodies to 242 purified SARS-CoV-2 virion varied. A199, B15, H278, B120 and H285 had a weak affinity 243 to virion. Nine antibodies (F61, F163, B15, H121, C25, A8, H184, B110 and A199) screened 244 with purified RBD and S1 were all positive to RBD. The rest three of them (H278, B120 and 245 H285) were found attached to S2 but not S1 nor RBD (Fig. 1C) . However, NTD specific 246 mAbs were not screened and identified.

Characterizing the binding profile of 12 candidate antibodies 249 The specificity of candidate SARS-CoV-2 specific IgGs were evaluated utilizing FACS. 250 All candidate IgGs (labeled by FITC) showed positive on the surface of HEK293 T cells 251 expressing SARS-CoV-2 S protein. In contrast, HBV mAb, as a negative control, 252 demonstrated no interaction with S protein ( Fig. 2A and Fig.S1 A) . Thus, all tested mAbs 253 were suggested binding specifically to SARS-CoV-2 S protein. 254 SPR assay were performed to evaluate the affinity of nine RBD-specific IgGs to S1 255 protein. F61, F163, H121, C25, H184, B110 and A199 showed a high affinity to S1 protein. 256 The KD values ranged from 1. Neutralizing capacity of 12 candidate IgGs were evaluated by authentic SARS-CoV-2 262 neutralization assay and pseudoviruses neutralization assay. F61, H121 and F163 exhibited 263 high neutralizing capacity with IC50 of 0.46 ug/ml, 0.48 ug/ml, and 0.64 ug/ml to authentic 264 SARS-CoV-2, and 0.027 ug/ml, 0.078 ug/ml, and 0.095 ug/ml to pseudoviruses, respectively. 265 However, A199 exhibited low neutralizing capacity, which which suggested not all 266 RBD-specific antibodies were neutralizing antibodies. S2-specific mAbs failed to neutralize 267 SARS-CoV-2 (data not shown). Moreover, the cocktail of F61 and H121 exhibited a 268 synergistic neutralization to authentic SARS-CoV-2 with the neutralizing capacity (0.13 269 ug/ml) increased four times compared to each one alone (Fig. 2C ). 270 To further characterize nine RBD-specific IgGs, we then analyzed antigenic epitopes of 271 nine RBD-specific antibodies by competitive ELISA. Antibodies were roughly classified into 272 three groups by competition percentages. Group one contained F61, F163 and B15. Group 273 two included H121, C25, A8, H184 and B110. Group one and group two were not 274 competitive, which suggested they bound to different antigenic epitopes. A199 from group 275 three had no competition with mAbs in group one yet a partial competition with those from 276 group two. A199 bound to a non-neutralization epitope on RBD (Fig. 3A) . 277 The inhibitory effect of these antibodies on the RBD-ACE2 interaction was investigated 278 by FACS using ACE2 expressing HEK293T cells. F61 and F163 from group one prevented 279 RBD from binding to ACE2 with no fluorescence signal from the antibody (FITC) or RBD 280 (Taxes red), which suggested they bound to an ACE2-competitive epitopes. B15 only partly 281 blocked the binding of RBD to ACE2 due to its low affinity and neutralization capacity. In to RBD with the highest proportion of double-positive cells (Fig. 3B) . 291 So far, we have obtained three types of antibodies, one of which is mainly represented by 292 F61 and F163 to recognize ACE2 receptor epitopes with high neutralization activity. One is 293 represented by H121 to identify epitopes which not overlap with ACE2 binding sites, but it 294 has high neutralization activity. And the last one is A199 which bound to a non-overlapping 295 epitope with ACE2 binding sites had no neutralization activity.

Interaction between mAbs and RBD via Computer Modeling and Docking. 297 F61 and H121 exhibited the high neutralization capacity and bound to different epitopes. 298 They were excellent candidate of antibody-based drugs to SARS-CoV-2. To precisely 299 delineate the interaction between antibody and antigen. We further investigated interaction 300 between F61/ H121 and RBD via computer simulation. Crystal structures that shared over 301 90% sequence similarity with F61 and H121 were used as the antibody template to build the 302 3D-structure of the two antibodies. Meanwhile, crystal structure 7DK3 (PDB) (Daniel et al., 303 2020) of SARS-CoV-2 RBD was used in the ZDOCK program and RDOCK program. 304 The outcomes demonstrated that F61 and H121 bind to diversified regions on RBD. F61 305 identified a linear epitope ranging from G446 to S494 within the RBM region involving 23 306 residues on RBD (Fig. 4A ). The predicted binding sites indicated both the light and the heavy 307 chain of F61. In specific, hydrogen-bond (H-bond) could be formed upon approaching RBD's 308 P479, C480，N481 and F486 with D108, N37 and S109 on the F61's light chain, as well as 309 F490 and L492 with G109 and R36, E484 with Y38 and Y114 on the heavy chain (Fig.4B,   310 upper panel). In comparison, H121 recognized a conformational epitope located remote from 311 the RBM region and mainly contributed by the heavy chain (Fig. 4A) . Specifically, R355, 312 G381 and L517 on RBD formed H-bond with S11A, N59 and Y109, L518 and A520 to Y37 313 on the heavy chain. R357 bond to Y38 on the light chain (Fig.4B, lower panel) . 314 The antibody-RBD complex structure was subsequently aligned with the ACE2-RBD 315 complex based on the RBD sequence ( Fig.4C upper panel) . Six residues within the F61 (Fig.5A) . 329 The change of binding activity between IgGs and mutant S1 was evaluated by ELISA. 330 The change of binding activity was defined by the value of OD450mutant S1 / OD450 S1. In this study, nine high affinity RBD-specific IgGs exhibited different neutralizing 365 activity. Two of them (F61 and H121) showed high neutralizing activity against 366 SARS-CoV-2. However, A199 failed to neutralize SARS-CoV-2, which suggested not all 367 RBD-specific antibodies were neutralizing antibodies. Meanwhile, our results from 368 competitive ELISA assay reviled that F61, F163 and B15 identified ACE2 competitive 369 epitopes. However, H121, C25, A8, H184 and B110 recognized epitopes which did not 370 overlap with ACE2 binding sites. A199 bound to a non-neutralization epitope that did not 371 overlap withremote from ACE2 binding sites. Results above suggested that neutralizing 372 capacities of antibodies did not rely on their ability to block RBD-ACE2 interaction. RBD 373 had three kinds of epitopes, thus, ACE2 competitive neutralization epitopes, ACE2 374 non-competitive neutralization epitopes, and non-neutralization epitopes. 375 Multiple mutations on S protein showed no resistant to F61 and H121. Therefore, the cocktail of F61 and H121 with broad neutralization improved treatment 420 efficacy by mitigating viral escape (Wang N. et al., 2021b) . While designing antibody-based, efficient biological drugs, it is essential to precisely 426 delineate the interaction between antibody and antigen structures. To achieve this, we used 427 computer modeling and docking technique, which is more rapid and accurate compared to 428 X-ray crystallography and cryo-electron microscopy (cryo-EM), for the binding structure 429 prediction of antibodies and RBD. Since antibodies have a highly conserved framework, 430 homology models building for antibodies can be reasonably accurate (Yamashita et al., 2014; 431 Leem et al., 2016) . Thus the models for F61 and H121 were highly reliable given that they 432 shared a similarity of more than 90% with the templates we used for homology modeling. The 

Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to 600

Broad neutralization of SARS-related viruses by human monoclonal 606 antibodies

Bamlanivimab does not 609 neutralize two SARS-CoV-2 variants carrying E484K in vitro

A 612 noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its 613 receptor ACE2 Science

Multivalent Nanobody Cocktails Efficiently Neutralize SARS-CoV-2

Kotai Antibody Builder: automated high-resolution structural modeling of antibodies

Spike mutations decrease

SARS-CoV-2 sensitivity to neutralizing antibodies but not ACE2-Ig in vitro

Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants

The D614G 627 mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity

Neutralization mutations of SARS-CoV-2 S protein and their effect on antibody 685 neutralizing activity. (A) Amino acid mutations on S protein

RBD-specific IgGs between mutant S1 protein and wild-type (WT) S1 protein was detected 688 by ELISA. The change of binding activity was defined by the ratio of OD450mutant

The dashed line indicated that the ratio was less than 0.5 or more than 1

The significant changes were marked red for decreased. (C)Neutralization activities of nine 691

RBD-specific IgGs towards mutations on S protein were measured by pseudovirus. The 692 changes in neutralization activity was showed in the ratio of IC50 between the variant and 693

The changes were marked with colored symbols, red 694 for decreased, blue for increased. (D)Neutralization activities of F61 and H121 towards 695 mutations K417N, E484K, and N501Y on S protein were measured by pseudovirus. The 696 changes in neutralization activity was showed in the ratio of IC50 between the variant and the 697

The changes were marked with colored symbols, red 698 for decreased, blue for increased.(E)Neutralization activities of F61 and C121 towards B1.1.7 699 and B1.351 were measured by pseudovirus and authentic SARS-CoV-2. The y-axis represents 700 the value of IC50. The dashed line indicated that the fold change of

 451 We thank XL. Shi from Tsinghua University for her technical help in human Antibodies 452 expressing; TY. Chen and his research from INNOVITA for supporting antibody detection