Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells

Cao, Yunlong; Su, Bin; Guo, Xuesong; Sun, Wenjie; Deng, Yong-Qiang; Bao, Linlin; Zhu, Qinyu; Zhang, Xu; Zheng, Yao; Geng, Chenyang; Chai, Xiaoran; He, Runsheng; Li, Xiaofeng; Lv, Qi; Zhu, Hua; Deng, Wei; Xu, Yanfeng; Wang, Yanjun; Qiao, Li; Tan, Yafang; Song, Liyang; Wang, Guopeng; Du, Xiaoxia; Gao, Ning; Liu, Jiangning; Xiao, Junyu; Su, Xiao Dong; Du, Zongmin; Feng, Yingmei; Qin, Chuan; Qin, Cheng‐Feng; Jin, Ronghua; Xie, X. Sunney

doi:10.1016/j.cell.2020.05.025

Cited by 1,239 publications

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“…While all cryo-EM structural studies to date on SARS-CoV-2 Spike have identified only the "closed" or "one-up" RBD conformation (Cao et al, 2020;Pinto et al, 2020;Walls et al, 2020;Wrapp, Wang, et al, 2020), our split reporter assay identified a population of Spike in "two-up" or "three-up" conformation. The relative population of these RBD conformers and whether they exist in an ACE2-unbound state or emerge only upon ACE2 binding remains unknown ( Figure 6F).…”

Section: Discussionmentioning

confidence: 61%

Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity

Lui

Zhou

Lim

et al. 2020

Preprint

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A serious public health crisis is currently unfolding due to the SARS-CoV-2 pandemic. SARS-CoV-2 viral entry depends on an interaction between the receptor binding domain of the trimeric viral Spike protein (Spike-RBD) and the dimeric human angiotensin converting enzyme 2 (ACE2) receptor. While it is clear that strategies to block the Spike/ACE2 interaction are promising as anti-SARS-CoV-2 therapeutics, our current understanding is insufficient for the rational design of maximally effective therapeutic molecules. Here, we investigated the mechanism of Spike/ACE2 interaction by characterizing the binding affinity and kinetics of different multimeric forms of recombinant ACE2 and Spike-RBD domain. We also engineered ACE2 into a split Nanoluciferase-based reporter system to probe the conformational landscape of Spike-RBDs in the context of the Spike trimer. Interestingly, a dimeric form of ACE2, but not monomeric ACE2, binds with high affinity to Spike and blocks viral entry in pseudotyped virus and live SARS-CoV-2 virus neutralization assays. We show that dimeric ACE2 interacts with an RBD on Spike with limited intra-Spike avidity, which nonetheless contributes to the affinity of this interaction.Additionally, we demonstrate that a proportion of Spike can simultaneously interact with multiple ACE2 dimers, indicating that more than one RBD domain in a Spike trimer can adopt an ACE2accessible "up" conformation. Our findings have significant implications on the design strategies of therapeutic molecules that block the Spike/ACE2 interaction. The constructs we describe are freely available to the research community as molecular tools to further our understanding of SARS-CoV-2 biology. Introduction:In late 2019, a novel, pathogenic coronavirus (SARS-CoV-2) entered the human population and has since spread throughout the world. The number of people suffering from the associated disease (COVID-19) continues to rise, increasing the need for effective therapeutic interventions. SARS-CoV-1 and SARS-CoV-2 Spike proteins are highly homologous (~76% sequence identity). Similar to SARS-CoV-1, the interaction between the SARS-CoV-2 Spike protein and the angiotensinconverting enzyme 2 (ACE2) on human cells is critical for viral entry into host cells (Gralinski & Menachery, 2020;Tai et al., 2020;Wu et al., 2020). SARS-CoV-2 Spike is an obligate trimer, while ACE2 presents as a dimer on the cell surface (Chen, Liu, & Guo, 2020). Several highresolution structures of SARS-CoV-2 Spike receptor binding domain (Spike-RBD) bound to ACE2 have been published (Lan et al., 2020;Yan et al., 2020). However, as of this writing, structures of SARS-CoV-2 Spike trimer in complex with either the dimeric or monomeric form of ACE2 have not been reported, resulting in an incomplete understanding of the nature of this interaction.Structural studies of trimeric SARS-CoV-2 and SARS-CoV-1 Spike protein demonstrate that each of the Spike-RBDs, as in other coronaviruses, can undergo hinge-like movements to transition between "up" or "down" conformations. The...

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Section: Discussionmentioning

confidence: 61%

Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity

Lui

Zhou

Lim

et al. 2020

Preprint

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“…Of 294 RBD-targeting antibodies, 10% are encoded by IGHV3-53, as compared to only 0.5% to 2.6% in the repertoire of naïve healthy individuals ( 29 ) with a mean of 1.8% ( 30 ). The prevalence of IGHV3-53 in the antibody response in SARS-CoV-2 patients has also been recognized in some recent antibody studies ( 20, 22, 27 ). These observations indicate that IGHV3-53 represents a frequent and public antibody response to the SARS-CoV-2 RBD.…”

Section: Mainmentioning

confidence: 67%

Structural basis of a public antibody response to SARS-CoV-2

Yuan

Liu

et al. 2020

Preprint

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26Molecular-level understanding of human neutralizing antibody responses to SARS-CoV-27 2 could accelerate vaccine design and facilitate drug discovery. We analyzed 294 28 SARS-CoV-2 antibodies and found that IGHV3-53 is the most frequently used IGHV 29 gene for targeting the receptor binding domain (RBD) of the spike (S) protein. We 30 determined crystal structures of two IGHV3-53 neutralizing antibodies +/-Fab CR3022 31 ranging from 2.33 to 3.11 Å resolution. The germline-encoded residues of IGHV3-53 32 dominate binding to the ACE2 binding site epitope with no overlap with the CR3022 33 epitope. Moreover, IGHV3-53 is used in combination with a very short CDR H3 and 34 different light chains. Overall, IGHV3-53 represents a versatile public VH in neutralizing 35 SARS-CoV-2 antibodies, where their specific germline features and minimal affinity 36 maturation provide important insights for vaccine design and assessing outcomes.37 3 MAIN 38The ongoing COVID-19 pandemic, which is caused by severe acute respiratory 39 syndrome coronavirus 2 (SARS-CoV-2), is far from an end (1). The increasing global 40 health and socioeconomic damage require urgent development of an effective COVID-41 19 vaccine. While multiple vaccine candidates have entered clinical trials (2), the 42 molecular features that contribute to an effective antibody response are not clear. Over 43 the past decade, the concept of a public antibody response (also known as multidonor 44 class antibodies) to specified microbial pathogens has emerged. A public antibody 45 response describes antibodies that have shared genetic elements and modes of 46 recognition, and can be observed in multiple individuals against a given antigen. Such 47 responses to microbial pathogens have been observed against influenza (3), dengue (4), 48 malaria (5), and HIV (6). Identification of public antibody responses and characterization 49 of the molecular interactions with cognate antigen can provide insight into the 50 fundamental understanding of the immune repertoire and its ability to quickly respond to 51 novel microbial pathogens, as well as facilitate rational vaccine design against these 52 pathogens (7, 8). 54The spike (S) protein is the major surface antigen of SARS-CoV-2. The S protein utilizes 55 its receptor-binding domain (RBD) to engage the host receptor ACE2 for viral entry (9-56 12). Therefore, RBD-targeting antibodies could neutralize SARS-CoV-2 by blocking 57 ACE2 binding. A number of antibodies that target the RBD of SARS-CoV-2 have now 58 been discovered in very recent studies (13-28). We compiled a list of 294 SARS-CoV-2 59RBD-targeting antibodies where information on IGHV gene usage is available (17-28) 60 (Table S2), and found that IGHV3-53 is the most frequently used IGHV gene among 61 such antibodies ( Fig. 1A). Of 294 RBD-targeting antibodies, 10% are encoded by 62 IGHV3-53, as compared to only 0.5% to 2.6% in the repertoire of naïve healthy 63 4 individuals (29) with a mean of 1.8% (30). The prevalence of IGHV3-53 in the antibody 64 response in SARS-Co...

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“…Experimentally validated computational docking shows that five of them occupy the ACE2 binding site on the RBD, thus likely achieving direct inhibition of virus-ACE2 interaction. The binding sites of antibody BD368-2 18 and B38 55 also overlap with the RBD ACE2 binding interface. Interestingly, our antibody STE73-2G8 which has subnanomolar EC50 to RBD and potent inhibition and neutralization, binds to the N-terminal part of RBD, away from the ACE2 interface.…”

Section: Discussionmentioning

confidence: 97%

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

Bertoglio

Meier

Langreder

et al. 2020

Preprint

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COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein.The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronavirus has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naïve universal antibody gene libraries HAL9/10 anti SARS2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. Furthermore, these antibodies neutralized active SARS-Cov-2 virus infection of VeroE6 cells. All 17 were all able to bind the isolated RBD, four of them with sub-nanomolar EC50. Epitope analysis of the antibodies revealed that six bind at the RBD-ACE2 interface and two on the opposite side of the domain. Universal libraries from healthy donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation. 4/34 Main textIn 2015 Menachery et al. wrote: "Our work suggests a potential risk of SARS-CoV reemergence from viruses currently circulating in bat populations." 1 . Four years later, a novel coronavirus causing a severe pneumonia was discovered and later named SARS-CoV-2. The outbreak started on a sea food market in Wuhan, Hubei province (China) at the end of 2019. The disease was named COVID-19 (coronavirus disease 2019) by the World Health Organization (WHO). Sequencing showed high identity to bat corona viruses (CoV, in particular RaTG13), beta-CoV virus causing human diseases like SARS and MERS and, to a lesser extent, the seasonal CoV hCoV-OC43 and HCov-HKU1 2,3 . The spike (S) protein of SARS-CoV-2, as well as SARS-CoV, binds to the human zinc peptidase angiotensin-converting enzyme 2 (ACE2) which is expressed on lung cells, heart, kidney and intestine cells and acts as receptor for virus entry. S protein consists of the N-terminal S1 subunit, which includes the receptor binding domain (RBD), and the Cterminal S2 subunit which is anchored to the viral membrane and is required for trimerization and fusion of the virus and host membrane 4-6 . The membrane bound host protease TMPRSS2 is responsible for S protein priming by cleavage of specific sites between S1 and S2. In addition to proteolytic activation of the S2' site, conformational changes and viral entry 7-10 .Antibodies against the...

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Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells

Cited by 1,239 publications

References 78 publications

Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity

Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity

Structural basis of a public antibody response to SARS-CoV-2

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

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