key: cord-0716467-9a2pyqrf authors: Ye, Lilin; Chen, Xiangyu; Li, Ren; Pan, Zhiwei; Qian, Chunfeng; Yang, Yang; You, Renrong; Zhao, Jing; Gao, Leiqong; Li, Zhirong; Huang, Qizhao; Xu, Lifan; Tang, Jianfang; Tian, Qin; Yao, Wei; Hu, Li; Yan, Xiaofeng; Zhou, Xinyuan; Liu, Pinghuang; Wu, Yuzhang; Deng, Kai; Zhang, Zheng; Chen, Yaokai; Qian, Zhaohui title: Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2 receptor date: 2020-04-11 journal: nan DOI: 10.1101/2020.04.06.20055475 sha: ffda4b565052650f13871b577db7aef4ecef7821 doc_id: 716467 cord_uid: 9a2pyqrf The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). To date, no prophylactic vaccines or approved therapeutic agents are available for preventing and treating this highly transmittable disease. Here we report two monoclonal antibodies (mAbs) cloned from memory B cells of patients recently recovered from COVID-19, and both mAbs specifically bind to the spike (S) protein of SARS-CoV-2, block the binding of receptor binding domain (RBD) of SARS-CoV-2 to human angiotensin converting enzyme 2 (hACE2), and effectively neutralize S protein-pseudotyped virus infection. These human mAbs hold the promise for the prevention and treatment of the ongoing pandemic of COVID-19. The! severe! acute! respiratory! syndrome! coronavirus! 2! (SARS^CoV^2)! has! caused! a! global!pandemic!of!novel!corona!virus!disease!(COVID^19).!To!date,!no!prophylactic! vaccines!or!approved!therapeutic!agents!are!available!for!preventing!and!treating!this! highly! transmittable! disease.! Here! we! report! two! monoclonal! antibodies! (mAbs)! cloned!from!memory!B!cells!of!patients!recently!recovered!from!COVID^19,!and!both! mAbs!specifically!bind!to!the!spike!(S)!protein!of!SARS^CoV^2,!block!the!binding!of! receptor! binding! domain! (RBD)! of! SARS^CoV^2! to! human! angiotensin! converting! enzyme!2!(hACE2),!and!effectively!neutralize!S!protein^pseudotyped!virus!infection.! These!human!mAbs!hold!the!promise!for!the!prevention!and!treatment!of!the!ongoing! pandemic!of!COVID^19.! ! ! ! All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi. org/10.1101 org/10. /2020 Main&text& According! to! World! Health! Organization! (WHO)! newly! updated! situation! report! on! March! 18 th ,! 2020,! the! corona! virus! disease! 2019! (COVID^19)! pandemic! has! confirmed!191,127!cases!and!claimed!7,807!deaths!worldwide 1 .!The!etiological!agent! of!COVID^19!has!been!identified!as!a!novel!coronavirus,!the!severe!acute!respiratory! syndrome!coronavirus!2!(SARS^CoV^2),!belonging!to!Sarbecovirus!subgenus!(genus! Betacoronavirus,! family! Coronaviridae)! and! showing! 79.6%! and! 96.2%! sequence! identity! in! nucleotide! to! SARS^CoV! and! a! bat! coronavirus! (BatCoV! RaTG13),! respectively 2^4 .!Like!SARS^CoV!infection,!a!substantial!fraction!of!COVID^19!patients! exhibits!severe!respiratory!symptoms!and!has!to!be!hospitalized!in!intensive!care!unit! (ICU) 5^8 .! Although! the! mortality! rate! of! COVID^19! is! significantly! lower! than! that! of! SARS^CoV! infection,! SARS^CoV^2! shows! much! higher! human^to^human! transmission!rate,!rapidly!leading!to!a!global!pandemic!declared!by!WHO!on!March! 11 th ,!2020 9 .! ! Currently,!there!are!no!approved!prophylactic!vaccines!or!therapeutic!drugs!that!are! specific! to! COVID^19.! Blocking! monoclonal! antibodies! (mAbs),! due! to! their! extraordinary!antigen!specificity,!are!one!of!the!best!candidates!for!neutralizing!virus! infection 10,!11 .! Therefore,! identifying! and! cloning! blocking! mAbs! that! can! specifically! target! surface! viral! proteins! to! block! the! viral! entry! to! host! cells! is! a! very! attractive! approach!for!preventing!and!treating!COVID^19,!in!particular!when!effective!vaccines! and!therapeutics!are!unavailable!in!the!outbreak!of!the!COVID^19!pandemic.!We!then! sought! to! identify! and! clone! blocking! mAbs! from! the! memory! B! cell! repertoire! of! recently!recovered!COVID^19!patients!to!prevent!the!entry!of!COVID^19!virus!to!the! host!cells.! Similar! to! SARS^CoV,! SARS^CoV^2! also! utilizes! highly! glycosylated! homotrimeric! spike! (S)! protein! for! receptor! binding! and! virus! entry 3,! 12^15 .! The! S! protein! of! SARS^CoV^2! consists! of! two! subunits,! S1! and! S2.! To! engage! host! cell! receptor! All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi. org/10.1101 org/10. /2020 human! angiotensin^converting! enzyme! 2! (hACE2),! shared! by! both! SARS^CoV! and! SARS^CoV^2,! S! protein! undergoes! dramatic! conformational! changes! to! expose! the! RBD! and! key! residues! for! receptor! binding.! S! protein! is! metastable,! and! binding! of! RBD! to! hACE2! receptor! likely! leads! to! the! shedding! of! S1! protein! from! S2! protein,! thus! promoting! S2^mediated! virus^host! membrane! fusion! and! virus! entry 16^18 .! Given! the! critical! role! of! the! RBD! in! initiating! invasion! of! SARS^CoV^2! into! host! cells,! it! becomes! a! vulnerable! target! for! neutralizing! antibodies.! Thus! far,! the! human! mAbs! specifically!target!the!SARS^CoV^2!RBD^hACE2!interaction!have!not!been!reported,! and! a! monoclonal! antibody! targeting! S1! made! from! immunized! transgenic! mice! expressing! human! Ig! variable! heavy! and! light! chains! has! been! recently! shown! to! neutralize! both! SARS^CoV^2! and! SARS^CoV! infection,! but! by! an! unknown! mechanism!that!is!independent!of!the!blockade!of!RBD^hACE2!interaction 19 .! Prior!to!cloning!SARS^CoV^2!RBD!specific!human!mAbs,!we!first!examined!whether! patients! recently! recovered! from! COVID^19! had! mounted! anti^SARS^CoV^2! S1! protein! IgG! antibodies! in! sera.! Among! 26! recovered! COVID^19! patients,! we! found! that! the! majority! of! these! recruited! patients! were! able! to! produce! high! titers! of! SARS^CoV^2!S1^specific!IgG!antibodies!and!only!3!patients!mounted!relatively!lower! anti^S1! IgG! responses,! by! enzyme^linked! immunosorbent! assay! (ELISA)! ( Fig.! 1a) .! Consistently,! we! also! found! that! SARS^CoV^2! RBD! specific! IgG! antibodies! were! present! in! sera! of! all! patients! by! ELISA! (Fig.! 1b) .! Next,! we! sought! to! investigate! whether! RBD^specific! antibodies! in! patient! serum! can! block! the! binding! of! SARS^CoV^2!RBD!to!hACE2.!To!this!end,!we!set!up!an!ELISA^based!inhibition!assay! to!examine!the!blocking!function!of!these!antibodies.!We!noted!that!there!were!only!3! out!of!26!patients!showed!effective!blockade!of!SARS^CoV^2!RBD!binding!to!hACE2! ( Fig.!1c) .!Taken!together,!these!results!suggested!that!while!all!recovered!COVID^19! patients! can! generate! anti^S1! and! anti^RBD! antibodies,! there! were! only! a! small! fraction! of! these! antibodies! can! block! the! binding! of! RBD! to! hACE2! receptor.! This! observation! may! be! explained! by! transient! and! dynamic! perfusion! conformational! All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.06.20055475 doi: medRxiv preprint states!of!S!protein!that!provide!very!limited!window!for!the!immunogenic!epitopes!of! RBD!exposure!to!specific!B!cells 20 .! Next,!we!set!out!to!clone!human!monoclonal!antibodies!using!the!blood!samples!from! three! COVID^19! recovered! patients,! of! which! their! sera! showed! potent! hACE2! receptor! binding! inhibition.! To! this! end,! we! sorted! each! SARS^CoV^2! RBD! specific,! IgG! class^switched! memory! B! cell! into! a! single! well! of! the! 96^well! microplates.! Subsequently,!we!used!reverse!transcription!polymerase!chain!reaction!(RT^PCR)!to! amplify!IgG!variable!heavy!chain!(VH)!and!light!chain!(VL)!from!each!single!memory!B! cell.! After! cloning! both! VH! and! VL,! we! inserted! both! sequences! into! expression! plasmids!that!encoding!constant!regions!of!human!IgG1!heavy!chain!and!light!chain! suggesting! the! specificity! of! our! sorting! strategy.! The! representative! RT^PCR! and! cloning!of!IgG1!heavy!chains!and!light!chains!to!expression!plasmids!were!shown!in! Figure! 2c! and! 2d.! After! antibody! cloning,! we! acquired! 3! pairs! of! IgG! VHs! and! VLs! inserted!expression!plasmids!and!the!CDR3!sequences!of!heavy!chains!were!shown! in! Figure the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.06.20055475 doi: medRxiv preprint 32D4^mediated! inhibition! of! RBD^hACE2! interaction! was! also! evidenced! by! flow! cytometry!analysis! (Fig.!3c^e) .!Furthermore,!we!determined!the!neutralization!of!these! three! mAbs! using! a! SARS^CoV^2! S! pseudotyped! lentiviral! particle 22 .! In! line! with! ELISA^! and! flow! cytometry^based! blockade! results,! both! 311mab^31B5! and! 311mab^32D4! effectively! neutralized! pseudovirus! entry! to! host! cells! ectopically! expressing! hACE2! (IC 50 =0.0338,! and! 0.0698! μg/ml,! respectively).! As! expected,! 311mab^31B9!clone!failed!to!show!any!neutralization!activities! (Fig.!3f) the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.06.20055475 doi: medRxiv preprint wrote!the!paper!with!X.C.,!R.L.,!P.L.,!Y.C.,!Z.Q.,!X.Z.,!Y.W.,!K.D.!and!Z.Z.o!and!L.Y.,! Y.K.!and!Z.Q.!supervised!the!study.! ! The!authors!declare!no!competing!interests.! All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is Biological,!10108^H08H)!in!100!μl!PBS!per!well!was!coated!on!ELISA!plates!overnight! at!4 o C.!Then,!the!ELISA!plates!were!blocked!for!1!hour!with!blocking!buffer!(5%!FBS! plus! 0.05%! Tween! 20)o! meanwhile,! three^fold! serial! dilutions! of! mAbs! or! ten^fold! diluted!patient!sera!were!incubated!with!optimal!dose!(based!on!EC 50 )!of!SARS^Cov2! RBD!protein!(Sino!Biological,!40592^V05H)!for!1!hour.!Then,!the!incubated!mixtures! were! added! to! ELISA! plates! and! allowed! to! develop! for! 30! min,! followed! by! PBST! washing!and!anti^mouse!Fc!HRP!antibody!(Thermo!Fisher!Scientific).!Next,!the!ELISA! plates! were! washed! with! PBST,! then! PBS! and! added! with! TMB! (Beyotime).! After! 5! min,! the! ELISA! plates! were! stopped! and! determined! at! 450! nm.! The! half! maximal! inhibitory! concentration! (IC 50 )! was! determined! by! using! 4^parameter! logistic! regression.& & Flow& cytometry7based& receptor7binding& inhibition& assay.& 311mab! mAbs! or! isotype! were! incubated! with! optimal! dose! (based! on! EC 50 )! of! SARS^Cov2! RBD! protein! (Sino! Biological,! 40592^V05H)! for! 1! hour! at! RT.! Then,! the! mixtures! were! incubated!with!10,000!hACE2^plasmid!transiently!transfected!293T!cells!for!40!min!on! ice,! followed! by! stained! with! Alexa! Fluor! 647^conjugated! goat! anti^mouse! IgG! (Biolegend)!and!APC^Cy7^conjugated!LIVE/DEAD!dye!(Life!Technologies).& & Pseudovirus&neutralization&assay.!Spike!protein!of!SARS^Cov^2!typed!pseudovirus! was!produced!as!previously!described 22 .!Concisely,!HEK^293T!cells!were!transfected! with! psPAX2,! pLenti^GFP! and! 2019^nCov! S! plasmids! by! using! TransIT^293! All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10. 1101 /2020 are!representative!of!two!independent!experiments!with!three!replicates!per!group!(a,! b,!d^fo!error!bars!in!a,!b,!d^f!indicate!the!SD).! All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.06.20055475 doi: medRxiv preprint Isotype 31B5 32D4 31B9 !A4new4coronavirus4associated4with4human4respiratory4disease4in4 China.!Nature,!2020 !A4pneumonia4outbreak4associated4with4a4new4coronavirus4of4 probable4bat4origin.!Nature,!2020.!579(7798):!p.!270^273.! 4.! Zhu Clinical4course4and4outcomes4of4critically4ill4patients4with4 SARSKCoVK24pneumonia4in4Wuhan,4China:4a4singleKcentered 4China:4a4descriptive4study.! Lancet,!2020.! !Monoclonal4antibodyKbased4 therapies4for4microbial4diseases.!Vaccine,!2009.!27&Suppl&6:!p.!G38^46.! All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is ):!p.!237^261.! 14 !Immunogenicity4and4structures4of4a4rationally4designed4 prefusion4MERSKCoV4spike4antigen No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is