key: cord-0320519-mwo384hh
authors: Zhang, Xinghai; Luo, Feiyang; Zhang, huajun; Guo, Hangtian; Zhou, Junhui; Li, Tingting; Chen, Shaohong; Song, Shuyi; Shen, Meiying; Wu, Yan; Gao, Yan; Han, Xiaojian; Wang, Yingming; Hu, Chao; Lu, Yuchi; Wang, Wei; Wang, Kai; Tang, Ni; Jin, Tengchuan; Yang, Chengyong; Cheng, Guofeng; Yang, Haitao; Jin, Aishun; Ji, Xiaoyun; Gong, Rui; Chiu, Sandra; Huang, Ailong
title: A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA.1 and BA.2
date: 2022-04-26
journal: bioRxiv
DOI: 10.1101/2022.04.26.489529
sha: 67c97e789f26f51bde6ce6f3deea737f5d7e6b95
doc_id: 320519
cord_uid: mwo384hh

Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC50) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 μg of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.

The coronavirus disease 2019 pandemic caused by severe acute 71 respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 has lasted for over two years.

Although several antiviral agents (e.g., vaccines and neutralizing antibodies (NAbs)) 73 have been authorized for the prevention and treatment of SARS-CoV-2 infection, the 74 rapid emergence of variants of concern (VOCs), such as the Omicron variant, 75 significantly reduces the efficacies of these antiviral agents and worsens the clinical 76 outcomes achieved with them 2,3 . Furthermore, it was recently reported that no The SARS-CoV-2 spike (S) protein is a typical class I viral fusion protein that 84 contains a surface subunit (S1) and a transmembrane subunit (S2). The entry of 85 SARS-CoV-2 into host cells relies on the interaction between the receptor-binding 86 domain (RBD) in S1 and its obligate receptor, angiotensin-converting enzyme 2 87 (ACE2) 6 . Structural determination of the RBD/ACE2 complex revealed that the 88 SARS-CoV-2 RBD contains a receptor-binding motif (RBM) that is the core region 89 for recognition 7 . The RBD in the S protein has two distinct conformations, with "up" 90 or "open" representing the receptor-accessible state and "down" or "closed" 91 representing the receptor-inaccessible state [8] [9] [10] . Because the RBD is critical for viral 92 entry and is highly antigenic 11 , it is an attractive target for drug and vaccine In a previous study, we isolated a panel of NAbs with strong neutralizing activities 115 against SARS-CoV-2 15 . Among these NAbs, 58G6 was recently found to inhibit all 116 authentic VOCs, including the Delta and Omicron variants, with half-maximal 117 inhibitory concentration (IC 50 ) values of 1.69 ng/ml and 54.31 ng/mL, respectively. 118 Importantly, this NAb has been shown to provide protection against the Delta and 119 Omicron variants in vivo (under review). Here, we report a newly identified NAb 120 termed 55A8 that can broadly neutralize wild-type (WT) SARS-CoV-2 (defined as 121 SARS-CoV-2 unless otherwise indicated), as well as Omicron BA.1 and BA.2 with 122 extreme potency. In addition, 55A8 and 58G6 showed synergetic effects both in vitro 123 and in vivo that enhanced the SARS-CoV-2 neutralizing potency and breadth. 124 Structural analysis revealed that 55A8 is a Class III NAb that recognizes a conserved 125 epitope. Therefore, a cocktail of 55A8 and 58G6 (2-cocktail) could be developed to 126 combat currently circulating VOCs (e.g., Omicron) and possible emerging VOCs in 127 the future. 

55A8 strongly binds to S proteins from SARS-CoV-2 and different variants 131 55A8 was isolated by sorting single B cells producing antibodies with potent 132 neutralizing activity against SARS-CoV-2 and the Beta (B. enzyme-linked immunosorbent assay (ELISA) (Fig. 1a) . The binding of 58G6, the 137 previously identified NAb mentioned above, was also tested (under review).

Furthermore, we found that 55A8 and 58G6 could efficiently recognize other S 139 proteins from a panel of SARS-CoV-2 variants (Extended Data Fig. 1 ). The 2-antibody cocktail of 58G6 and 55A8 provides synergetic protection in vivo 174 To preliminarily assess whether the combination of 58G6 and 55A8 (2-antibody 175 cocktail, 2-cocktail) could induce synergistic effector function responses in vivo, 176 hamsters were intranasally infected with 10 4 plaque-forming units (PFU) of Omicron 177 BA.1 and treated with 58G6 (1500 µg), 55A8 (500 μg), or 58G6 and 55A8 mixture 178 (2-cocktail, 1000 μg of 58G6 and 300 μg of 55A8) at 1 h pre-infection and 24 and 48 h 179 post-infection (Extended Data Fig. 3a ). On day 3 post-infection, the animals were 180 sacrificed, and the turbinates, trachea and lungs were harvested. We observed that all 181 the treatments led to robust viral clearance (Extended Data Fig. 3b To refine the synergetic effect, more animal protection experiments were performed.

To assess the protective efficacy of 55A8 against the Omicron variant, we intranasally in combination with 58G6 (Group 4, 500 μg for each antibody) (Fig. 3a) . Another 198 group (Group 5) was treated following the regimen for Group 1 but administered 199 buffer instead of the antibody (Fig. 3a) . We measured the effects of 55A8 alone or in 200 combination with 58G6 on Omicron viral replication in clinically relevant tissues (i.e., 201 the nasal turbinates, trachea and lungs), which were collected on day 3 post-infection.

Omicron viral replication was detected by RT-qPCR and plaque assays. As expected, hamsters treated with buffer had significant viral RNA copy numbers 205 and viral titers in the turbinates and lungs. Pretreatment with 55A8 or the 2-cocktail at 206 1 h pre-infection followed by two-dose administration lowered the Omicron viral 207 RNA copy number in the lungs by 4-5 logs (Fig. 3a ). More notably, in the turbinates, 208 pretreatment with the 55A8 and 58G6 mixture 1 h pre-infection followed by two-dose 209 administration (Group 3) reduced the Omicron viral RNA copy number by six orders 210 of magnitude, producing a copy number that was significantly lower than that in 211 Group 1 (Fig. 3b) . Consistently, the viral titers in Group 3 at 3 days post-infection 212 (dpi) were significantly lower than those in the other groups or under the limit the To further investigate the mechanism by which 55A8 neutralizes the Omicron variant, When 55A8 Fabs were bound to the Omicron BA.1 S protein, the S trimer adopted a 241 "1-up/2-down" conformation (Class 1) or a "2-up/1-down" conformation (Class 2) 242 ( Fig. 5a and b) . We refined both to an overall resolution of 3.4 Å (Extended Data Fig.   243 6e and f), with the majority of selected particle images representing a overlap between the 55A8 Fab and ACE2 on the same bound RBD, which explained 348 why the mAb 55A8 was not able to compete with ACE2 for Omicron RBD binding.

When 55A8 Fabs were bound to BA.1 S trimers, the S trimer adopted a "1-up/2-down" 350 conformation or a "2-up/1-down" conformation. In the "1-up/2-down" conformation, 351 two 55A8 Fabs were bound to two down RBDs and occluded ACE2 binding to the 352 remaining up RBD. In the "2-up/1-down" conformation, one 55A8 Fab bound to one 353 down RBD and could occlude ACE2 binding to an adjacent up RBD while freeing the 354 other up RBD for binding to ACE2, which explains the partial competition between 355 55A8 and ACE2 for Omicron BA.1 S trimer binding.

To further evaluate the potential of the combination of 58G6 and 55A8, we 358 determined the cryo-EM structures of Omicron BA.1 S trimers in complex with 55A8 359 and 58G6 Fabs. Consistent with using 55A8 alone, the BA.1 S trimer also adopted a For the mAb competition experiments, biotinylated S proteins were loaded at 1 nm 576 onto SA biosensors, and mAb association was performed at 20 μg/mL for 300 s. For 577 ACE2 competition experiments, the biotinylated RBD and S were loaded at 1 nm and 578 3 nm, respectively, at 1 μg/mL onto SA biosensors. The first antibody was allowed to 579 associate for 600 s at 20 μg/mL, and the second protein (ACE2 (50 μg/mL) or a 580 mixture of equal amounts of antibodies and ACE2) was allowed to associate for 300 581 s. To build the structures of the Omicron S-55A8/58G6 Fab complex, the previously 749 described structure of the Omicron S-55A8 complex model was placed and rigid-body 750 fitted into cryo-EM electron density maps using UCSF Chimera. The 58G6 Fab 751 model 15 was manually built in Coot 0.9 with the guidance of the cryo-EM electron 752 density maps, and overall real-space refinements were performed using Phenix 1.19.

The data validation statistics are shown in Extended Data Table 2 . Statistical analyses were performed using GraphPad Prism software v.9.2.0.

Comparisons between two groups were performed using unpaired Student's t tests.

Comparisons among multiple groups were performed using one-way ANOVA 759 followed by Tukey's multiple comparison post hoc test. P < 0.05 was considered 760 significant (significance is denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, 761 and ****P < 0.0001). Outliers (%) 0.00 0.00 0.00

The coordinates and cryo-EM map files for the 55A8-BA.1 S complexes and 764

1 S complexes have been deposited in the Protein Data Bank (PDB) 765 under accession number 7WWI, 7WWJ, 7WWK, 7XJ6, 7XJ8 and 7XJ9

We thank the Center for Animal Experiment and BSL-3 laboratory

Chinese 771 Academy of Sciences; and the National Virus Resource Center for resource support

This work was jointly supported by the Natural Science Foundation of Hubei 773

the National Natural Science Foundation of China 774 (32170949, 81871639, 92169109, 81871656 and 8181101099); the National Science 775 and Technology Major Project

Aishun Jin and Haitao Yang conceived and designed the 784 project. For biological function analysis of the NAbs

Yingming Wang and Chao Hu screened and cloned the 786 antibodies, and expressed and purified the antibodies; Feiyang Luo

Wei Wang were responsible for BLI assays for the binding ability, the affinity and the 788 competition experiment of NAbs

Kai Wang 789 and Ni Tang prepared various pseudovirus and conducted the pseudovirus 790 neutralization assays. For the efficacy test of the NAbs in vitro and in vivo

Yan Wu and Rui Gong 792 performed authentic SARS-CoV-2 neutralization assays and animal experiments. For 793 structure analysis

the cryo-EM data, and built and refined the structure model; Xiaoyun Ji, Haitao Yang 796 and Tengchuan Jin analyzed and discussed the cryo-EM data

Rui Gong and Xinghai Zhang wrote the manuscript. All 798 authors revised and reviewed the final manuscript