《内科学》课程教学资源（新冠文献）Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody.pdf_大学文库

LETTER Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirusspecific human monoclonal antibody Xiaolong Tiana#, Cheng Lia#, Ailing Huanga , Shuai Xiaa , Sicong Lua , Zhengli Shi b , Lu Lua , Shibo Jiang a , Zhenlin Yangc , Yanling Wua and Tianlei Ying a a MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of China; b CAS Key Laboratory of Special Pathogens, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, People’s Republic of China; c Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China ABSTRACT The newly identified 2019 novel coronavirus (2019-nCoV) has caused more than 11,900 laboratory-confirmed human infections, including 259 deaths, posing a serious threat to human health. Currently, however, there is no specific antiviral treatment or vaccine. Considering the relatively high identity of receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019- nCoV. Here, we report for the first time that a SARS-CoV-specific human monoclonal antibody, CR3022, could bind potently with 2019-nCoV RBD (KD of 6.3 nM). The epitope of CR3022 does not overlap with the ACE2 binding site within 2019-nCoV RBD. These results suggest that CR3022 may have the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019- nCoV infections. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, implying that the difference in the RBD of SARS-CoV and 2019-nCoV has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD. ARTICLE HISTORY Received 27 January 2020; Revised 2 February 2020; Accepted 3 February 2020 KEYWORDS 2019-nCoV; SARS-CoV; ACE2; monoclonal antibody; RBD Very recently, a novel coronavirus which was temporarily named “2019 novel coronavirus (2019-nCoV)” emerged in Wuhan, China [1]. As of 1 February 2020, 2019-nCoV has resulted in a total of 11,821 laboratory-confirmed human infections in China, including 259 deaths, and 132 exported cases in 23 countries outside of China (https://www.who.int/emergencies/ diseases/novel-coronavirus-2019/situation-reports). Currently, there is no vaccine or effective antiviral treatment against 2019-nCoV infection. Based on the phylogenetic analysis (GISAID accession no. EPI_ISL_402124) [2], 2019-nCoV belongs to lineage B betacoronavirus and shares high sequence identity with that of bat or human severe acute respiratory syndrome coronavirus-related coronavirus (SARSr-CoV) and bat SARS-like coronavirus (SLCoV) (Figure 1(a)). In previous studies, a number of potent monoclonal antibodies against SARS coronavirus (SARS-CoV) have been identified [3–7]. These antibodies target the spike protein (S) of SARS-CoV and SL-CoVs, which is a type I transmembrane glycoprotein and mediates the entrance to human respiratory epithelial cells by interacting with cell surface receptor angiotensin-converting enzyme 2 (ACE2) [8]. More specifically, the 193 amino acid length (N318-V510) receptor binding domain (RBD) within the S protein is the critical target for neutralizing antibodies [9]. Some of the antibodies recognize different epitopes on RBD; e.g. the SARS-CoV neutralizing antibodies CR3014 and CR3022 bound noncompetitively to the SARS-CoV RBD and neutralized the virus in a synergistic fashion [5]. We predicted the conformation of 2019-nCoV RBD as well as its complex structures with several neutralizing antibodies, and found that the modelling results support the interactions between 2019-nCoV RBD and certain SARS-CoV antibodies (Figure 1(b)). This could be due to the relatively high identity (73%) of RBD in 2019-nCoV and SARS-CoV (Figure 1(c)). For instance, residues in RBD of SARSCoV that make polar interactions with a neutralizing © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Tianlei Ying tlying@fudan.edu.cn Room 504, Fosun Building, 131 Dong-an Road, Shanghai 200032, People’s Republic of China; Yanling Wu yanlingwu@fudan.edu.cn Room 504, Fosun Building, 131 Dong-an Road, Shanghai 200032, People’s Republic of China; Zhenlin Yang yang_zhenlin@fudan.edu.cn Room 504, Fosun Building, 131 Dong-an Road, Shanghai 200032, People’s Republic of China # These authors contributed equally to this article. Supplemental data for this article can be accessed at https://doi.org/10.1080/22221751.2020.1729069. Emerging Microbes & Infections 2020, VOL. 9 https://doi.org/10.1080/22221751.2020.1729069

384©X.Tian et al antibody m396 as indicated by the complex crystal D95 ofm396-VL Concordantly,the elect as well as a MERS-CoV-spe action was also observed in the model of 2019-nCoV- antibody m336 developed by our laboratory [15],and RBD-m396, forming by R408 (RBD)and D95 that mos actions between antibody F26G19 or 8R 12] measured the binding kinetics using BLI.An irrelevant and the RBD in 2019-nCoV decreased significantly anti-CD40 antibody was used as a negative control due to the lack Similarly,the antibo ly m396,which .RBD-80 80-R162 ight bindi RBD (Figure 1(d)) only show of the goR-binding uc n the RBD of SARS CoV are not conserved on RBD of 2019-nCoV(Figure high-resolution structure of 2019-nCoV RBD and 1(c)),it is unlikely that the antibody 80R could effec understand why it could not be recognized by these tively recognize 2019-nCo t is urgent to antibodi SARS-C01 ctivity of ant hiCoanheipoha01pSeYoPoP was fo d to R rapid development of vaccines and therapeutic antibodies against 2019-nCov Koff of1.16× 10 s)binding kinetics. resulting in expressed ody was did no om blo (Figure 1(f) This ang e with th on of the to rcombinant S protein 5].Tofurther elucidate the binding epitopes of CR,we measured the compe and human ACE2 for the bin n)In both 20 120 COV RBD n biosensors 67loop form extensive contact,including at least human ACE2 in solution followed by the addition of seven pairs ofhydrogen bonds,with the receptor.Nota the test antibodies in the presence of ACE2.As bly,R426 on the forth a helix in SARS-CoV RBD builds shown in Figure 1(g),the antibody CR3022 did not 201-Co RBD Th the binding RBD)to on in 2019-nCov distinct from the SARs-CoV antibodies which recognizes an epitope that does not overlap with the ACE2 bind nt restingly,a lysine 019 largely from cement ues with D30 on ACE?which ver the hindin es in the ca hility to er ability.These data indicate that the RBD in S protein Some of the most poten Indeed,we r 0f2019- ACE2 ng anti ing o (BI found that 2019-nCov RBD hound potently to AcE2 ing that it is necessary to develop novel monoclonal The calculated affinity (K)of 2019-nCoV RBD with tibodies that could bind specifically to019-nCov human ACE2 was 15. 2nM (Figure 1()),which is com D Interestingly,it was reported that the antibody sp. pro wild-typ could be the ntial for the ne ml and the ndthattheexpressed019-nCoV RBD protein is func- be generated with CR3022 5).Furthermore,the mix tional [2]. ture of CR3022 and CR3014 neutralized SARS-CoV

antibody m396 as indicated by the complex crystal structure [10] are invariably conserved in 2019-nCoV RBD (Figure 1(d)). In the structure of SARS-CoVRBD-m396, R395 in RBD formed a salt bridge with D95 of m396-VL. Concordantly, the electrostatic interaction was also observed in the model of 2019-nCoVRBD-m396, forming by R408 (RBD) and D95 (m396-VL). This analysis suggests that some SARSCoV-specific monoclonal antibodies may be effective in neutralizing 2019-nCoV. In contrast, the interactions between antibody F26G19 [11] or 80R [12] and the RBD in 2019-nCoV decreased significantly due to the lack of salt bridges formed by R426-D56 in SARS-CoV-RBD-F26G19 or D480-R162 in SARSCoV-RBD-80R, respectively. Furthermore, while most of the 80R-binding residues on the RBD of SARSCoV are not conserved on RBD of 2019-nCoV (Figure 1(c)), it is unlikely that the antibody 80R could effectively recognize 2019-nCoV. Therefore, it is urgent to experimentally determine the cross-reactivity of antiSARS-CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. In this study, we first expressed and purified 2019- nCoV RBD protein. We also predicted the conformations of 2019-nCoV RBD and its complex with the putative receptor, human ACE2. Comparison of the interaction between the complex of ACE2 [13] and SARS-CoV RBD and homology model of ACE2 and 2019-nCoV RBD revealed similar binding modes (data not shown). In both complexes, β5–β6 loop and β6–β7 loop form extensive contact, including at least seven pairs of hydrogen bonds, with the receptor. Notably, R426 on the forth α helix in SARS-CoV RBD builds a salt bridge with E329 and a hydrogen bond with Q325 on ACE2. However, the arginine (R426 in SARS-CoV RBD) to asparagine (N439) mutation in 2019-nCoV RBD abolished the strong polar interactions, which may induce a decrease in the binding affinity between RBD and the receptor. Interestingly, a lysine (K417 in 2019-nCoV RBD) replacement of valine (V404 in SARS-CoV RBD) on β6 formed an extra salt bridge with D30 on ACE2, which may recover the binding ability. These data indicate that the RBD in S protein of 2019-nCoV may bind to ACE2 with a similar affinity as SARS-CoV RBD does. Indeed, we measured the binding of 2019-nCoV RBD to human ACE2 by the biolayer interferometry binding (BLI) assay, and found that 2019-nCoV RBD bound potently to ACE2. The calculated affinity (KD) of 2019-nCoV RBD with human ACE2 was 15.2 nM (Figure 1(f)), which is comparable to that of SARS-CoV spike protein with human ACE2 (15 nM) [14]. These results indicate that ACE2 could be the potential receptor for the new coronavirus, and that the expressed 2019-nCoV RBD protein is functional [2]. Next, we expressed and purified several representative SARS-CoV-specific antibodies which have been reported to target RBD and possess potent neutralizing activities, including m396 [3], CR3014 [4], CR3022 [5], as well as a MERS-CoV-specific human monoclonal antibody m336 developed by our laboratory [15], and measured their binding ability to 2019-nCoV RBD by ELISA (Figure 1(e)). Surprisingly, we found that most of these antibodies did not show evident binding to 2019-nCoV RBD. To confirm this result, we further measured the binding kinetics using BLI. An irrelevant anti-CD40 antibody was used as a negative control. Similarly, the antibody m396, which was predicted to bind 2019-nCoV RBD (Figure 1(d)), only showed slight binding at the highest measured concentration (2.0 µM). Further studies are needed to solve the high-resolution structure of 2019-nCoV RBD and understand why it could not be recognized by these antibodies. Notably, one SARS-CoV-specific antibody, CR3022, was found to bind potently with 2019-nCoV RBD as determined by ELISA and BLI (Figure 1(e,f)). It followed a fast-on (kon of 1.84 × 105 Ms−1 ) and slow-off (koff of 1.16 × 10−3 s −1 ) binding kinetics, resulting in a KD of 6.3 nM (Figure 1(f)). This antibody was isolated from blood of a convalescent SARS patient and did not compete with the antibody CR3014 for binding to recombinant S protein [5]. To further elucidate the binding epitopes of CR3022, we measured the competition of CR3022 and human ACE2 for the binding to 2019-nCoV RBD. The streptavidin biosensors labelled with biotinylated 2019-nCoV RBD were saturated with human ACE2 in solution, followed by the addition of the test antibodies in the presence of ACE2. As shown in Figure 1(g), the antibody CR3022 did not show any competition with ACE2 for the binding to 2019-nCoV RBD. These results suggest that CR3022, distinct from the other two SARS-CoV antibodies, recognizes an epitope that does not overlap with the ACE2 binding site of 2019-nCoV RBD. The RBD of 2019-nCoV differs largely from the SARS-CoV at the C-terminus residues (Figure 1(c)). Our results implied that such a difference did not result in drastic changes in the capability to engage the ACE2 receptor, but had a critical impact on the cross-reactivity of neutralizing antibodies. Some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the receptor binding site of SARSCoV failed to bind 2019-nCoV spike protein, indicating that it is necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD. Interestingly, it was reported that the antibody CR3022 completely neutralized both the wild-type SARS-CoV and the CR3014 escape viruses at a concentration of 23.5 μg/ml, and that no escape variants could be generated with CR3022 [5]. Furthermore, the mixture of CR3022 and CR3014 neutralized SARS-CoV 384 X. Tian et al

in a synergistic fashion by recognizing different epitopes on RBD [5]. These results suggest that CR3022 has the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-nCoV infections. We expect more cross-reactive antibodies against 2019-nCoV and SARS-CoV or other coronaviruses to be identified soon, facilitating the development of effective antiviral therapeutics and vaccines. Disclosure statement No potential conflict of interest was reported by the author (s). Funding This work was supported by grants from the National Natural Science Foundation of China (81822027, 81630090), National Key Research and Development Program of China (2019YFA0904400), National Megaprojects of China for Major Infectious Diseases (2018ZX10301403), Chinese Academy of Medical Sciences (2019PT350002), and the staff from Core Facility of Microbiology and Parasitology, Shanghai Medical College, Fudan University. ORCID Zhengli Shi http://orcid.org/0000-0001-8089-163X Shibo Jiang http://orcid.org/0000-0001-8283-7135 Tianlei Ying http://orcid.org/0000-0002-9597-2843 References [1] Chan JF, Kok KH, Zhu Z, et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect. 2020;9 (1):221–236. [2] Zhou P, Yang XL, Wang XG, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020. doi:10.1038/s41586-020- 2012-7. [Epub ahead of print] [3] Zhu Z, Chakraborti S, He Y, et al. Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies. Proc Natl Acad Sci USA. 2007;104(29):12123–12128. [4] ter Meulen J, Bakker ABH, van den Brink EN, et al. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets. The Lancet. 2004;363(9427):2139–2141. [5] ter Meulen J, Van Den Brink EN, Poon LLM, et al. Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants. PLoS Med. 2006;3(7):e237. [6] Sui J, Li W, Murakami A, et al. Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci USA. 2004;101 (8):2536–2541. [7] Traggiai E, Becker S, Subbarao K, et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 2004;10(8):871–875. [8] Gallagher TM, Buchmeier MJ. Coronavirus spike proteins in viral entry and pathogenesis. Virology. 2001;279(2):371–374. [9] Wong SK, Li W, Moore MJ, et al. A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem. 2004;279(5):3197–3201. [10] Prabakaran P, Gan J, Feng Y, et al. Structure of severe acute respiratory syndrome coronavirus receptorbinding domain complexed with neutralizing antibody. J Biol Chem. 2006;281(23):15829–15836. [11] Pak JE, Sharon C, Satkunarajah M, et al. Structural insights into immune recognition of the severe acute respiratory syndrome coronavirus S protein receptor binding domain. J Mol Biol. 2009;388 (4):815–823. [12] Hwang WC, Lin Y, Santelli E, et al. Structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80R. J Biol Chem. 2006;281(45):34610–34616. [13] Li F, Li W, Farzan M, et al. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science. 2005;309(5742):1864–1868. [14] Walls AC, Xiong X, Park Y-J, et al. Unexpected receptor functional mimicry elucidates activation of coronavirus fusion. Cell. 2019;176(5):1026– 1039.e15. [15] Ying T, Prabakaran P, Du L, et al. Junctional and allelespecific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody. Nat Commun. 2015;6:8223. Emerging Microbes & Infections 385