Structure of Severe Acute Respiratory Syndrome Coronavirus Receptor-binding Domain Complexed with Neutralizing Antibody

The severe acute respiratory syndrome coronavirus (SARS-CoV, or SCV), which caused a world-wide epidemic in 2002 and 2003, binds to a receptor, angiotensin-converting enzyme 2 (ACE2), through the receptor-binding domain (RBD) of its envelope (spike, S) glycoprotein. The RBD is very immunogenic; it is a major SCV neutralization determinant and can elicit potent neutralizing antibodies capable of out-competing ACE2. However, the structural basis of RBD immunogenicity, RBD-mediated neutralization, and the role of RBD in entry steps following its binding to ACE2 have not been elucidated. By mimicking immune responses with the use of RBD as an antigen to screen a large human antibody library derived from healthy volunteers, we identified a novel potent cross-reactive SCV-neutralizing monoclonal antibody, m396, which competes with ACE2 for binding to RBD, and determined the crystal structure of the RBD-antibody complex at 2.3-Ä resolution. The antibody-bound RBD structure is completely defined, revealing two previously unresolved segments (residues 376–381 and 503–512) and a new disulfide bond (between residues 378 and 511). Interestingly, the overall structure of the m396-bound RBD is not significantly different from that of the ACE2-bound RBD. The antibody epitope is dominated by a 10-residue-long protruding β6–β7 loop with two putative ACE2-binding hotspot residues (Ile-489 and Tyr-491). These results provide a structural rationale for the function of a major determinant of SCV immunogenicity and neutralization, the development of SCV therapeutics based on the antibody paratope and epitope, and a retrovaccinology approach for the design of anti-SCV vaccines. The available structural information indicates that the SCV entry may not be mediated by ACE2-induced conformational changes in the RBD but may involve other conformational changes or/and yet to be identified coreceptors. The severe acute respiratory syndrome coronavirus (SARS-CoV, or SCV), which caused a world-wide epidemic in 2002 and 2003, binds to a receptor, angiotensin-converting enzyme 2 (ACE2), through the receptor-binding domain (RBD) of its envelope (spike, S) glycoprotein. The RBD is very immunogenic; it is a major SCV neutralization determinant and can elicit potent neutralizing antibodies capable of out-competing ACE2. However, the structural basis of RBD immunogenicity, RBD-mediated neutralization, and the role of RBD in entry steps following its binding to ACE2 have not been elucidated. By mimicking immune responses with the use of RBD as an antigen to screen a large human antibody library derived from healthy volunteers, we identified a novel potent cross-reactive SCV-neutralizing monoclonal antibody, m396, which competes with ACE2 for binding to RBD, and determined the crystal structure of the RBD-antibody complex at 2.3-Ä resolution. The antibody-bound RBD structure is completely defined, revealing two previously unresolved segments (residues 376–381 and 503–512) and a new disulfide bond (between residues 378 and 511). Interestingly, the overall structure of the m396-bound RBD is not significantly different from that of the ACE2-bound RBD. The antibody epitope is dominated by a 10-residue-long protruding β6–β7 loop with two putative ACE2-binding hotspot residues (Ile-489 and Tyr-491). These results provide a structural rationale for the function of a major determinant of SCV immunogenicity and neutralization, the development of SCV therapeutics based on the antibody paratope and epitope, and a retrovaccinology approach for the design of anti-SCV vaccines. The available structural information indicates that the SCV entry may not be mediated by ACE2-induced conformational changes in the RBD but may involve other conformational changes or/and yet to be identified coreceptors. The severe acute respiratory syndrome coronavirus (SARS-CoV, or SCV) 4The abbreviations used are: SCV, severe acute respiratory syndrome coronavirus (or SARS-CoV); mAb, monoclonal antibody; ACE2, angiotensin-converting enzyme 2; RBD, receptor-binding domain; Env, virus envelope glycoprotein; HIV, human immunodeficiency virus; MES, 4-morpholineethanesulfonic acid; CDR, complementarity-determining region; S glycoprotein; spike glycoprotein. infected more than 8000 humans with a fatality rate of ∼10% (1Ksiazek T.G. Erdman D. Goldsmith C.S. Zaki S.R. Peret T. Emery S. Tong S. Urbani C. Comer J.A. Lim W. Rollin P.E. Dowell S.F. Ling A.E. Humphrey C.D. Shieh W.J. Guarner J. Paddock C.D. Rota P. Fields B. DeRisi J. Yang J.Y. Cox N. Hughes J.M. LeDuc J.W. Bellini W.J. Anderson L.J. N. Engl. J. Med. 2003; 348: 1953-1966Crossref PubMed Scopus (3375) Google Scholar, 2Peiris J.S. Lai S.T. Poon L.L. Guan Y. Yam L.Y. Lim W. Nicholls J. Yee W.K. Yan W.W. Cheung M.T. Cheng V.C. Chan K.H. Tsang D.N. Yung R.W. Ng T.K. Yuen K.Y. Lancet. 2003; 361: 1319-1325Abstract Full Text Full Text PDF PubMed Scopus (2361) Google Scholar, 3Drosten C. Gunther S. Preiser W. van der W.S. Brodt H.R. Becker S. Rabenau H. Panning M. Kolesnikova L. Fouchier R.A. Berger A. Burguiere A.M. Cinatl J. Eickmann M. Escriou N. Grywna K. Kramme S. Manuguerra J.C. Muller S. Rickerts V. Sturmer M. Vieth S. Klenk H.D. Osterhaus A.D. Schmitz H. Doerr H.W. N. Engl. J. Med. 2003; 348: 1967-1976Crossref PubMed Scopus (3455) Google Scholar, 4Holmes K.V. N. Engl. J. Med. 2003; 348: 1948-1951Crossref PubMed Scopus (272) Google Scholar). Although there have been no recent outbreaks, the need to develop potent therapeutics and vaccines against a re-emerging SCV or a related virus remains of high priority. The amazingly rapid pace of SARS research for the last few years has resulted in a wealth of information for the virus, especially about its interactions with the host leading to disease and immune responses, which could also be helpful for the development of strategies to cope with other viral pathogens including influenza and HIV. Entry of viruses into animal cells is initiated by binding to cell-surface-associated receptors and can be prevented by neutralizing antibodies (nAbs) targeting the virus receptor-binding site (5Dimitrov D.S. Nat. Rev. Microbiol. 2004; 2: 109-122Crossref PubMed Scopus (393) Google Scholar, 6Smith A.E. Helenius A. Science. 2004; 304: 237-242Crossref PubMed Scopus (622) Google Scholar). In the case of SCV entry, the spike (S) glycoprotein (7Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar, 8Xiao X. Dimitrov D.S. Cell Mol. Life Sci. 2004; 61: 2428-2430Crossref PubMed Scopus (26) Google Scholar) binds to a receptor, angiotensin-converting enzyme 2 (ACE2) (9Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Crossref PubMed Scopus (4100) Google Scholar), through the receptor-binding site of its receptor-binding domain (RBD) (7Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar, 10Wong S.K. Li W. Moore M.J. Choe H. Farzan M. J. Biol. Chem. 2004; 279: 3197-3201Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 11Babcock G.J. Esshaki D.J. Thomas Jr., W.D. Ambrosino D.M. J. Virol. 2004; 78: 4552-4560Crossref PubMed Scopus (202) Google Scholar). The RBD is an attractive target for neutralizing antibodies that could prevent SCV entry by blocking the attachment of ACE2 (12He Y. Zhou Y. Liu S. Kou Z. Li W. Farzan M. Jiang S. Biochem. Biophys. Res. Commun. 2004; 324: 773-781Crossref PubMed Scopus (307) Google Scholar, 13Zhang M.Y. Choudhry V. Xiao X. Dimitrov D.S. Curr. Opin. Mol. Ther. 2005; 7: 151-156PubMed Google Scholar, 14Jiang S. He Y. Liu S. Emerg. Infect. Dis. 2005; 11: 1016-1020Crossref PubMed Scopus (174) Google Scholar, 15He Y. Lu H. Siddiqui P. Zhou Y. Jiang S. J. Immunol. 2005; 174: 4908-4915Crossref PubMed Scopus (204) Google Scholar, 16He Y. Zhu Q. Liu S. Zhou Y. Yang B. Li J. Jiang S. Virology. 2005; 334: 74-82Crossref PubMed Scopus (90) Google Scholar, 17Chen Z. Zhang L. Qin C. Ba L. Yi C.E. Zhang F. Wei Q. He T. Yu W. Yu J. Gao H. Tu X. Gettie A. Farzan M. Yuen K.Y. Ho D.D. J. Virol. 2005; 79: 2678-2688Crossref PubMed Scopus (160) Google Scholar, 18Yi C.E. Ba L. Zhang L. Ho D.D. Chen Z. J. Virol. 2005; 79: 11638-11646Crossref PubMed Scopus (50) Google Scholar). To understand the structural mechanisms underlying SCV immunogenicity and neutralization and help in the design of vaccines capable of eliciting predetermined highly effective neutralizing antibodies, we used a retrovaccinology (19Burton D.R. Nat. Rev. Immunol. 2002; 2: 706-713Crossref PubMed Scopus (504) Google Scholar) approach based on the combination of phage display and x-ray crystallography. The SCV is a member of the genus Coronavirus, which belongs to the Coronaviridae family of the order Nidovirales, which also includes families Arteriviridae and Roniviridae. Not only SCV but also other nidoviruses can infect humans and animals, resulting in a variety of severe diseases. The infection is initiated by the attachment of virus envelope glycoproteins (Envs) to receptors, which can be blocked by nAbs. The structural mechanisms of receptor recognition and neutralization by antibodies against any nidovirus were not previously known. Recently, the crystal structure of the SCV S RBD in complex with ACE2 was reported at 2.9-Ä resolution (20Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar). However, the structures of a receptor-free RBD and its complexes with nAbs are not known. Thus, fundamental questions related to the mechanism of SCV (and any other nidovirus) entry and neutralization, such as conformational changes induced by the binding of either the receptor or the nAbs, remain unanswered. To date, only a few structures of viral Envs complexed with nAbs are available, including Envs from influenza, picornaviruses, HIV, and West Nile virus (5Dimitrov D.S. Nat. Rev. Microbiol. 2004; 2: 109-122Crossref PubMed Scopus (393) Google Scholar, 21Nybakken G.E. Oliphant T. Johnson S. Burke S. Diamond M.S. Fremont D.H. Nature. 2005; 437: 764-769Crossref PubMed Scopus (298) Google Scholar); these structures have played an essential role in elucidating the mechanisms of viral neutralization. Here, we describe the identification of a potent cross-reactive SCV-neutralizing human monoclonal antibody, m396, and report the crystal structure of the antibody-antigen complex (Fab m396-SCV RBD) at 2.3-Ä resolution (Supplemental Table S1). The structure reveals a major neutralization determinant and its relationship with receptor recognition, providing structural insights into the mechanism of SCV entry and neutralization. Expression and Purification of the RBD—A fragment containing residues 317–518 from the S glycoprotein was cloned into pSecTag2B (Invitrogen) using BamHI and EcoRI restriction sites as previously described (7Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar, 22Chakraborti S. Prabakaran P. Xiao X. Dimitrov D.S. Virol. J. 2005; 2: 73Crossref PubMed Scopus (73) Google Scholar). The insert was further cloned into pAcGP67-A using the forward primer 5′-ACT GTC TAG ATG GTA CCG AGC TCG GAT CC-3′ (XbaI) and the reverse primer 5′-CAG TAG ATC TCG AGG CTG ATC AGC G-3′ (BglII). The pAcGP67-S was co-transfected with BaculoGold linearized baculovirus DNA into SF9 cells. High titer recombinant baculovirus stock was prepared by multiple amplifications. The protein was expressed in SF9 in and from with a The protein was further with a with and to in and Purification of the High and to human phage display library of was from cells of healthy Y. Zhu and D. S. in and used for of against RBD, to following a previously described Z. Dimitrov A.S. Choudhry V. A. Zhang M.Y. Y. Xiao X. Dimitrov D.S. J. Virol. PubMed Scopus Google Scholar). of were with and of RBD in a for 2 at the an of the of were from the infected and phage was used to of phage with high binding that to the RBD with were for further The and and the of and of these were The were for and the was as The used for was with a by a using containing and to its to the and were and in the by D. with the fragment with of by and SCV RBD were by using a The SCV RBD was a using was prepared for binding and of the of of or were at a rate of using containing and The and were to a by using the the were at and SCV complex was by in a and at were by The was of at only in the with a for the protein and the The of were with the 2 The was of and in to 2.3-Ä resolution were at for the complex and the from a at the of the was with the Z. W. PubMed Scopus Google Scholar). The structure was by with Biol. 2004; PubMed Scopus Google Scholar), using the SCV RBD from the receptor complex and of and and to the of and from different antibody structures for and for and for as The (residues of SCV RBD and of the of which were not in the were on the basis of The complex was with Full Text Full Text PDF PubMed Scopus Google Scholar) at 2.3-Ä resolution. of a and at were at the of the The and were and The structure was by with J. A. Scopus Google Scholar), using the of from the complex structure as the The the of The structure was using Full Text Full Text PDF PubMed Scopus Google Scholar), and a of was at the of the The and were and The M. PubMed Scopus (504) Google Scholar) was used for for and are in Table we identified S glycoprotein containing the RBD, which is a major SCV neutralization determinant (12He Y. Zhou Y. Liu S. Kou Z. Li W. Farzan M. Jiang S. Biochem. Biophys. Res. Commun. 2004; 324: 773-781Crossref PubMed Scopus (307) Google Scholar, 13Zhang M.Y. Choudhry V. Xiao X. Dimitrov D.S. Curr. Opin. Mol. Ther. 2005; 7: 151-156PubMed Google Scholar, 14Jiang S. He Y. Liu S. Emerg. Infect. Dis. 2005; 11: 1016-1020Crossref PubMed Scopus (174) Google Scholar, 15He Y. Lu H. Siddiqui P. Zhou Y. Jiang S. J. Immunol. 2005; 174: 4908-4915Crossref PubMed Scopus (204) Google Scholar, 16He Y. Zhu Q. Liu S. Zhou Y. Yang B. Li J. Jiang S. Virology. 2005; 334: 74-82Crossref PubMed Scopus (90) Google Scholar, 17Chen Z. Zhang L. Qin C. Ba L. Yi C.E. Zhang F. Wei Q. He T. Yu W. Yu J. Gao H. Tu X. Gettie A. Farzan M. Yuen K.Y. Ho D.D. J. Virol. 2005; 79: 2678-2688Crossref PubMed Scopus (160) Google Scholar, 18Yi C.E. Ba L. Zhang L. Ho D.D. Chen Z. J. Virol. 2005; 79: 11638-11646Crossref PubMed Scopus (50) Google Scholar), and residues for the binding of SCV to its receptor ACE2 (7Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar, 22Chakraborti S. Prabakaran P. Xiao X. Dimitrov D.S. Virol. J. 2005; 2: 73Crossref PubMed Scopus (73) Google Scholar). of these containing residues was cloned into a baculovirus expressed in and fragment was used as a antigen for of a large different human antibody which we from the of healthy Recently, library was also used for the of potent nAbs against and viruses Z. Dimitrov A.S. Choudhry V. A. Zhang M.Y. Y. Xiao X. Dimitrov D.S. J. Virol. PubMed Scopus Google Scholar). The with the binding to the RBD, m396, was to antibody and the binding rate and of the and the to SCV RBD in a (Supplemental S1). two we determined and and and for the and the The high for is to the effective of the antigen binding to the we that the antibody and entry mediated by the SCV S glycoprotein with an of and SCV entry mediated by the S glycoprotein from the which is not by other human monoclonal antibodies, including J. Li W. A. A. L.J. Wong S.K. Moore M.J. A.S. M. Choe H. Anderson L.J. Bellini W.J. Farzan M. Sci. S. A. 2004; PubMed Scopus Google Scholar, J. Li W. A. L.J. A. L. Wong S.K. K. Farzan M. J. Virol. 2005; 79: PubMed Scopus Google Scholar) and Becker S. K. Kolesnikova L. Y. A. Nat. Med. 2004; PubMed Scopus Google Scholar, K. A. G.J. Sci. S. A. 2005; PubMed Scopus Google Scholar), and virus from Urbani and with an of and and D. to be The structure of the SCV complex is in The overall RBD structure in the complex with is to that in the complex with ACE2 (20Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar). The the in the two RBD structures is However, the new RBD structure reveals a of residues in two segments (residues 376–381 and are in that were in the The antibody-bound RBD structure also reveals a new disulfide bond residues 378 and which was not in the in the RBD of a which includes a and and a which a in the The RBD structure that disulfide in the and in the of the and the The identified two segments of RBD in the complex are by and and structure of RBD. structural RBD structures based on RBD structures from the and ACE2 complexes are in and and segments in the structure the disulfide bond the RBD (between residues and which is as a and the at the segments and the β6–β7 loop that from the RBD and of the of and The a on the of the providing a binding into which the β6–β7 loop The of loop is a residues and is in the which is a in complexes Curr. Opin. Biol. PubMed Scopus Google Scholar). The the recognition of a residues has been at the of the structure M. Zhang M.Y. S. Dimitrov D.S. B. J. Science. 2005; PubMed Scopus Google Scholar), which is a major neutralizing determinant of HIV. residues in the β6–β7 loop with at the binding and residues and from the into the on the of the residues from the RBD and the in the of the RBD-antibody as by a of the two The J. Mol. Biol. PubMed Scopus Google Scholar), a of two for the RBD-antibody is a high of of is at the of the complex with from the two from the RBD and from the as determined with a The β6–β7 loop for of the RBD-antibody which indicates the role of the loop residues in the binding of the two The of the of the The of the binding is to the of other complexes D.R. Sci. S. A. PubMed Scopus Google Scholar). The interactions van der and or (Supplemental Table The of the at the the antibody and the SCV RBD are in Table The interactions in the complex are the β6–β7 loop of the RBD and the and of These interactions are with residues and of is in with the of in RBD is in the complex the of is in a bond with the of in ACE2. with other a role in RBD the is the of of RBD in the by the of the of and the of an bond with the of is the only from that with RBD it the residues that with RBD. The of a bond with the of the RBD with a of and an role in the of the RBD and the antibody in the and to the of the The for the has a high of which a highly for the and including of RBD Table of with a major in the of the two residues a of at the The binding sites on the RBD two residues in and the by and and residues and from the of these residues to antibody of have in the of the β6–β7 the of and bond to the of and the of a bond with the of and the from of and are these help the β6–β7 loop in the RBD. The RBD-antibody has two major the high of the and the of the putative major hotspot RBD into the antibody putative hotspot and of the RBD β6–β7 loop a protruding in on the RBD in The antibody binding includes in the by the in on the antibody in the paratope and the epitope structures are highly which could be a major for the high of of the RBD-antibody is the of the RBD into the of the binding at the antibody site residues and the RBD in Thus, the recognition of these two which the in the antibody, by is a of the SCV To the antibody any conformational changes RBD such as in the site or the we determined the crystal structure of at 2.3-Ä resolution. are two in the a in the the and structures of the is The in has a the in is The for is the for a human antibody A. B. J. Mol. Biol. PubMed Scopus Google Scholar). conformational an that may in the antibodies antigen which could a role as a mechanism Science. PubMed Scopus Google Scholar). However, we no conformational in the site the and for in of residues in antigen has a The major of are the identification of a potent SCV-neutralizing human monoclonal antibody, the of the structural mechanism of its and the of a high resolution structure of the SCV RBD. The the and the structures for the mechanism of SCV entry and the basis of neutralization. The antibody and the receptor a on the of RBD, of the β6–β7 loop and and these residues are for the binding of RBD to the antibody and the a of the RBD complexes with antibody and with receptor and the binding of the antibody and the receptor, on the and is that the neutralizing are in major of the β6–β7 the receptor ACE2 have of the loop on the of RBD. These that the antibody SCV by for the of residues in the β6–β7 loop of RBD and blocking the receptor binding Recently, were reported as a of W. Z. Yu M. W. C. H. Z. Zhang H. Zhang J. J. H. P. Zhang S. Science. 2005; PubMed Scopus Google Scholar, S.K. Li Y. H.W. Wong Wong Chan K.H. Yuen K.Y. Sci. S. A. 2005; PubMed Scopus Google Scholar). The of human and from of are residues and of SCV RBD, which are in the antibody are the a neutralizing of m396, be virus with the S glycoprotein from the with the residues in the RBD were in the including and the only the site as in the However, it is only in van der interactions with and the not significantly the neutralizing of the of available RBD of human SCV that the Thus, to SCV to be a cross-reactive antibody and may have for we that the RBD structure in the complex with antibody is to that in the complex with ACE2 2 and The RBD structure is However, it is very that binding of ACE2 and of could the conformational changes that to the RBD especially in of the but different binding sites of ACE2 and as as the of binding as However, the overall structure of the RBD is are in the of the that are in it remains to be conformational changes in other segments of an S glycoprotein The residues in the two segments (residues 376–381 and which were not in the complex (20Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar), are on the of RBD, to the neutralization and receptor-binding and also could conformational can only that the SCV entry is not through an mechanism that conformational changes in the RBD, it is that ACE2 could conformational changes in other of the S by or changes in the of the S glycoprotein through binding to ACE2. is that the ACE2 function is to the S glycoprotein by binding to that can conformational changes the of the S glycoprotein. have for the development of vaccines and therapeutics against SCV and for the mechanisms of virus neutralization and virus The identified antibody, m396, may have and we to it in animal on its could design other The structure of the antibody epitope could be used for the design of that are to elicit or antibodies retrovaccinology approach (19Burton D.R. Nat. Rev. Immunol. 2002; 2: 706-713Crossref PubMed Scopus (504) Google attractive is the use of the neutralizing the β6–β7 and based on its as protruding of in and by antibodies a role in neutralization and it is that it also binds other antibodies in to the receptor ACE2 and antibody the that the overall RBD structure in the complex with the antibody is the as the in the complex with the receptor further research to the fundamental of is the mechanism that the SCV entry into and there are other SCV receptors or the for were at the of the may be at