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Preprint:Pleasenotethatthisarticlehasnotcompletedpeerreview.

ASARS-CoV-2surrogatevirusneutralizationtest(sVNT)basedonantibody-mediatedblockageofACE2-spike(RBD)protein-proteininteractionCURRENTSTATUS:UNDERREVIEW

CheeWahTanDuke-NUSMedicalSchool

WanNiChiaDuke-NUSMedicalSchool

MarkI-CChenNationalCentreforInfectiousDiseases

ZhiliangHuNanjingUniversityofChineseMedicine

BarnabyE.YoungNationalCentreforInfectiousDiseases

Yee-JooTanNationalUniversityofSingapore

YongxiangYiNanjingUniversityofChineseMedicine

DavidC.LyeNationalCentreforInfectiousDiseases

DanielleE.AndersonDuke-NUSMedicalSchool

Danielle.anderson@duke-nus.edu.sgCorrespondingAuthor

Lin-FaWangDuke-NUSMedicalSchool

linfa.wang@duke-nus.edu.sgCorrespondingAuthor

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DOI:10.21203/rs.3.rs-24574/v1

SUBJECTAREASLaboratoryDiagnostics

KEYWORDSCOVID-19,serologicaltest,detectionofneutralizingantibodies,SARS-CoV-2surrogatevirusneutralizationtest

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AbstractAtthiscriticalmomentoftheinternationalresponsetotheCOVID-19outbreak,thereisanurgent

needforarobustserologicaltesttodetectneutralizingantibodiestoSARS-CoV-2.Suchatestisnot

onlyimportantforcontacttracing,butfordetermininginfectionrate,herdimmunityandpredicted

humoralprotection.Thecurrentgoldstandardisavirusneuralizationtest(VNT)requiringlivevirus

andabiosafetylevel3(BSL3)laboratory.Ontheotherhand,theELISA-orlateralflow-basedassays

areforthedetectionofbindingantibodies,whichdoesnotdirectlycorrelatewiththeirneutralizing

ability.HerewereportaSARS-CoV-2surrogatevirusneutralizationtest(sVNT)thatisdesignedto

detecttotalneutralizingantibodiesinanisotype-andspecies-independentmanner.Oursimpleand

rapidtestisbasedonantibody-mediatedblockageofvirus-hostinteractionbetweentheACE2

receptorproteinandthereceptorbindingdomain(RBD)oftheviralspikeprotein.Thetesthasbeen

validatedwithtwoCOVID-19patientcohortsintwodifferentcountries,achieving100%specificityand

95-100%sensitivityandiscapableofdifferentiatingantibodyresponsesfromotherknownhuman

coronaviruses.Importantly,thesVNTdoesnotrequireBSL3containment,therebymakingthetest

immediatelyaccessibletotheglobalcommunity.

Introduction

TheCOVID-19outbreakwasfirstrecognizedinDecember2019inWuhan,China1,whichhassince

spreadtoallpartsoftheworldresultinginatotal2,160,207infectionswith146,088deathsasof18

April,20202.Thecausativeagentwasidentifiedas2019-nCoV,subsequentlydesignatedSARS-CoV-

23,4,whichbelongstothespeciesSARS-relatedcoronavirus(SARSr-CoV),sameasforSARS-CoV,the

causativeagentoftheSARSoutbreak17yearsago5.

Whilemoleculardetection,suchaspolymerasechainreaction(PCR)andnextgenerationsequencing

(NGS),playedandcontinuetoplayanimportantroleinacutediagnosisandmonitoringofgenetic

changesofthevirus,thereisnowanurgentneedforareliableandversatileserologicalorantibody

test.Suchatestisneededforretrospectivecontacttracing,investigationofasymptomaticinfection

rate,accuratedeterminationofcasefatalityrate,assessmentofherdimmunityandhumoral

protectiveimmunityinrecoveredpatientsandrecipientsofvaccinecandidates,andinthesearchfor

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thenaturalreservoirhostandintermediatehost(s)6.Researchlaboratoriesandpharmaceutical

companiesareracingtoproduceantibodyteststhatcandetectCOVID-19infectionwithsufficient

specificityandsensitivity6.Therearetwotypesofantibodytestsonecanaimfor.Thefirsttypeisthe

virusneutralizationtest(VNT)whichdetectsneutralizingantibodies(NAbs)inapatient’sblood.VNT

requireshandlingliveSARS-CoV-2inaspecializedbiosafetylevel3(BSL3)containmentfacilitywhich

istediousandtimeconsuming,taking2-4daystocomplete.Pseudovirus-basedvirusneutralization

test(pVNT)issimilar,butstillrequirestheuseoflivevirusesandcellsalthoughhandledinaBSL2

laboratory7,8.Allotherassays,suchasELISAandlateralflowrapidtests,representthesecondassay

typewhichdetectonlybindingantibodies,andnotNAbs6,9-11.

Inthisstudy,weestablishedasurrogatevirusneutralizationtest(sVNT)whichdetectsNAbs,but

withouttheneedtouseanylivevirusorcellsandcanbecompletedin1-2hoursinaBSL2lab.Using

purifiedreceptorbindingdomain(RBD)proteinfromtheviralspike(S)proteinandthehostcell

receptorACE2,ourtestisdesignedtomimicthevirus-hostinteractionbydirectprotein-protein

interactioninatesttubeoranELISAplatewell.Thishighlyspecificinteractioncanthenbe

neutralized,i.e.,blockedbyhighlyspecificNAbsinpatientoranimalserainthesamemannerasina

conventionalVNT.

ResultsBiochemicalsimulationofvirus-receptorinteractionandantibody-mediatedneutralization

ImmediatelyafterSARS-CoV-2wasidentifiedasthecausativeagentoftheCOVID-19outbreak,itwas

shownthatthehumanangiotensinconvertingenzyme-2(hACE2)isthemainfunctionalreceptorfor

viralentry3.Wehypothesizedthatthevirus-receptorbindingcanbemimickedinvitroviaaprotein-

proteininteractionusingpurifiedrecombinanthACE2andtheRBDoftheSARS-CoV-2Sprotein.This

interactioncanbeblockedbyvirusNAbspresentinthetestserum,usingthesameprincipleasa

conventionalVNTconductedusinglivevirusinsideaBSL3facility(Fig.1aandb).

Inourstudy,thedirectbindingwasdemonstratedusingdifferentSARS-CoV-2proteinsconjugated

withhorseradishperoxidase(HRP).Thereisadose-dependentspecificbindingbetweenhACE2and

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RBDorS1,butnotwiththenucleocapsid(N)protein,withtheRBDproducingthebestbinding

characteristics(Fig.1c).TheHRP-RBDproteinwaschosenforsubsequentstudies.Wethen

demonstratedthatthespecificRBD-hACE2bindingcanbeblockedorneutralizedbyCOVID-19serain

adose-dependentmanner,butnotbyserafromhealthycontrols(Fig.1d).Toprovethatthesame

principleworkswiththecloselyrelatedSARS-CoV,whichalsouseshACE2astheentryreceptor12,we

repeatedthesimilarexperimentsandprovedthattheSARS-CoVRBDperformedinanalmostidentical

mannerinthisnewtestformat(Fig.1e,f),termedsurrogatevirusneutralizationtest(sVNT).

Isotype-andspecies-independentneutralization

OneoftheadvantagesofsVNTisitsabilitytodetecttotalantibodiesinpatientsera,incontrastto

mostSARS-CoV-2antibodytestspublishedormarketed,whicharealmostallisotype-specific,mostly

forIgMorIgG,withsomeforIgA9-11.Fromapanelof77COVID-19positiveserafrompatientsin

Singapore,wehavedesignatedfourgroupsbasedonIgMorIgGELISAlevels,determinedbyourin-

housecaptureELISAassays(seeMethods),presentinthepatientconvalescentsera:a)highIgM/low

IgG;b)lowIgM/highIgG;c)lowIgM/lowIgG;andd)highIgM/highIgG.Allgroupsshowedstrong

neutralizationactivityinthesVNT(Fig.2),demonstratingtheisotype-independentperformanceofthe

assay.ItisworthtonotethatforpanelcwithlowIgM/IgG,the%inhibitioninsVNTisstillsignificant

at70-75%,demonstratingitssuperiorsensitivityasthisgroupofseraweredeemednegativeor

weaklypositivewithisotype-specificcaptureELISAbasedonIgMorIgGalone.

WethentesteddifferentanimalserainthesVNTassaystodemonstratespecies-independent

performance.ResultsfromthreeindependentrabbitsimmunizedwiththeSARS-CoV-2RBDprotein,

demonstrateverypotentneutralizingactivityintheSARS-CoV-2sVNT(Fig.3a).Similarly,serafrom

ferretsinfectedwithSARS-CoV(Fig.3b)andrabbitsimmunizedwithinactivatedSARS-CoV(Fig.3c)

alsodemonstrateanefficientdose-dependentinhibitionofthehACE2-SARS-CoVRBDinteractionin

theSARS-CoVsVNT.

SpecificityagainstotherhCoVsandcomparisonofSARSseracollectedin2003vs2020

Todemonstratespecificity,wetesteddifferentpanelsofseraagainstotherknownhuman

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coronaviruses(hCoVs)andconfirmedthattheSARS-CoV-2sVNTcandifferentiateantibodyresponses

betweenCOVID-19andothercoronavirusinfections(Fig.3d).ForSARSsera,thereissomelevelof

crossreactivityasexpectedfromtheirclosegeneticalrelatednessandpreviouspublishedstudies3,7.

Butthedifferenceinneutralizationisstatisticallysignificant,andhencethesVNTcanbeusedto

differentiateCOVID-19infectionfrompastSARSinfection.Forhumanserafrompatientswith

229/NL63orOC43infectionandalpacaserafromexperimentalMERS-CoVinfection,thereisno

detectablecrossneutralization.

DuringtheinvestigationofpotentialcrossreactivitybetweenSARSseraandSARS-CoV-2virus,we

madeseveralimportantobservations.Firstly,despitethelackofcrossneutralizationbySARSsera

againsttheliveSARS-CoV-2virusinVNTobservedbyusandothergroups13,wedetectedsomelevel

ofcrossneutralizationinsVNT(Fig.3d),indicatingsVNTismoresensitivethanVNT;secondly,SARS

NAbsaredetectableforatleast17yearsinrecoveredpatients(Fig.3f);thirdly,thecross

neutralizationlevelishigherinthe2020SARSserathanthe2003samples(Fig.3d)althoughthe

homologousneutralizinglevelofthe2020SARSsera(Fig.3f)islowerthanthe2003SARSsera(Fig.

3e);lastly,wehavefoundthattheN-specificantibodylevelismuchlowerinthe2020SARSserathan

the2003samples(Fig.3g).

CorrelationbetweenlivevirusVNTandbiochemicalsVNT

ApanelofCOVID-19serawithdifferentlevelsofSARS-CoV-2NAbsasshownbysVNT(SupplFig.1)

werechosenforacomparativeandcorrelationstudybetweenthelivevirusbasedVNTandtheRBD-

hACE2basedsVNT.Theresultsdemonstrategoodoverallthecorrelationbetweenthetwoassays

(Fig.4aandSupplTable1).TheSARS-CoV-2sVNTismoresensitivethanVNT.Atthe50%inhibition

cutoff,whichisconsideredastringentcutoffasevidentfromthetitrationcurvesinSuppl.Fig.1,all

COVID-19patientsserashowedneutralizationat1:20orgreaterwiththeCOVID-19Patient13serum

reachinganeutralizationtiterequaltoorgreaterthan640(Suppl.Fig.1).

Validationwithtwocohortsofpositiveandnegativeserafromtwocountries

TovalidatetheperformanceoftheSARS-CoV-2sVNT,wetestedtwodifferentcohortsofpositiveand

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negativesera.Theassaywasperformedintwodifferentcountriesbytwoindependentgroupsto

furtherassurereliabilityandreproducibility.Forthefirstcohort,wetested77serafromPCR-

confirmedCOVID-19patientsinSingaporecollectedondays14-33aftersymptomonsetand75

healthycontrolsera.Allcontrolserawerenegative,resultingina100%specificity.Usingacutoffat

20%inhibition,theassaysensitivityisat100%.Thesensitivitydecreasesto95.6%whena40%cutoff

isused(Fig.4b).Forthesecondcohort,wetested50seraeachofhealthycontrolsandPCR-

confirmedCOVID-19patientsinNanjing,China,sampledondays27-61aftersymptomonset.The

specificityis100%.Thesensitivityis98%and96%usinga20%and40%cutoff,respectively(Fig.

4c).

DiscussionWearemorethan100daysintotheCOVID-19outbreakandattentionworldwide,bothinthescientific

communityandforpolicymakers,hasshiftedfocusfromacutediagnosticstrategyandcapacityto

theuseofserologyforthe“exitstrategy”,relyingonaccurateassessmentofinfectionprevalenceat

theindividualandpopulation(herd)level.Discussionanddebateontheroleofserologyhas

intensifiedgreatlyinthiscontext6.

WhiletherearemanyCOVID-19lab-basedorpoint-of-care(POC)antibodytestkitscommercially

available,nonearecapableofmeasuringNAbs.VNTorpVNTremaintheonlyplatformfordetectionof

NAbs.Bothrequirelivevirusandcells,highlyskilledoperators,arelesssensitiveingeneral,andtake

daystoobtainresults.VNTandpVNTarethusnotsuitableformassproductionandtesting,evenin

themostdevelopednations.

TheWorldHealthOrganization(WHO)hasrecentlycautionedthatpositiveresultsfromantibodytests

donotequaltoprotectiveimmunity14duetotwoaspectsorobstacles.Firstly,most,ifnotall,current

testingdoneatlargescaleisfordetectionofbindingantibodiesonlyanddoesnotmeasureNAbs;

secondly,thepresenceofNAbsmayormaynotcorrelatewithprotection.Whilethesecondaspect

willtakemuchmorein-depthscientificandclinicalresearchtoresolveinthespecificcontextof

COVID-19infection,pastexperienceswithviralinfectioningeneralarguethatinmostrecovered

patientsNAblevelisagoodindicatorofprotectiveimmunity,despitethefactthatsomepatientsmay

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notobeythis“ruleofthumb”15,16.Inthisstudy,wehavedevelopedanovelsVNTplatformtotackle

thefirstobstacle.

ThedatapresentedheredemonstratedthatsVNTisasspecificas,andmoresensitivethanVNT.The

resultsobtainedfromsVNTcorrelateswellwithVNT.ThemajoradvantageofsVNTisthatitcanbe

rapidlyconductedinmostresearchorclinicallabswithouttheneedtouselivebiologicalmaterials

andbiosafetycontainment.ThesVNTisalsoamenabletohighthroughputtestingand/orfully

automatedtestingafterminimaladaptation.

AnotheradvantageofsVNTisitsabilitytodetectSARS-CoV-2antibodiesinaspecies-independent

manner.AstheoriginofSARS-CoV-2andearlytransmissioneventremainelusive,thesVNTassaywill

beideallysuitedfor“virushunting”aspaststudieshaveamplydemonstratedthatserological

surveysaremoresuperiorthanmoleculardetectionasthevirus-specificantibodieslastmuchlonger

inanimalsthantheviralgeneticmaterial17-19.Samplingserumforantibodydetectionisalsomore

reliablethanothersamplingapproachesusedformoleculardetectionasthetargettissuescanvary

fromvirustovirus20-22.

Inaddition,sVNToffersakeyadvantageovermostELISAorPOCtestsinitsabilitytodetecttotal

NAbsinanisotype-independentmanner.Thiswillnotonlysimplifythetestingstrategy,butalso

furtherincreasetestsensitivity.AsshowninFig.2cfortheserumpanelofCOVID-19patientsshowing

lowIgMandIgGintheisotype-specificELISAs,thesVNTassaystilldetectedsignificantlevelofNAbs.

Althoughthemechanismneedsfurtherinvestigation,thereareatleasttwopossibilities:thepresence

ofotherIgisotypesorneutralizationsynergyorcooperativityfromthecombinationofdifferent

isotypeantibodiestargetingdifferentneutralizationcriticalepitopes,aspreviouslyobservedforHIV

andotherviruses23-25.

ResultsobtainedforthetwoSARSserumpanelsareveryinteresting.ThelonglastingNAbs17years

afterinitialinfectionisencouragingnewsforrecoveredCOVID-19patientsconsideringtheclose

relationshipofthetwoviruses.Themechanismandbiologicalsignificanceoftheincreasedcross

neutralizationtowardsSARS-COV-2coupledwiththedecrease/disappearanceofN-specificantibodies

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17yearsafterinfectionwarrantsfurtherinvestigationinthecontextofbetterunderstandingSARSr-

CoVimmuneresponsedynamics.

Insummary,wehaveaddressedthechallengeofCOVID-19serologywithanewapproachthat

enablesthedetectionofNAbsinaneasy,safe,rapidandinexpensivemannerwithenhanced

specificityandsensitivity.WhilethesVNTassaymayneverbeabletocompletelyreplacethe

conventionalVNT,ourdataindicatethattheirperformanceisgenerallywellcorrelated.Itsapplication

cancovermanyaspectsofCOVID-19investigationfromcontacttracing,sero-prevalencesurvey,

reservoir/intermediateanimaltrackingtoassessmentofherdimmunity,longevityofprotective

immunityandefficacyofdifferentvaccinecandidates.

MethodsCellsandvirus.Humanembryonickidney(HEK293T)cells(ATCC#CRL-3216)andAfricangreen

monkeykidney,cloneE6(Vero-E6)cells(ATCC#CRL-1586)weremaintainedinDulbecco’smodified

EagleMedium(DMEM)supplementedwith10%fetalbovineserum.SARS-CoV-2,isolate

BetaCoV/Singapore/2/2020(AccessionIDEPI_ISL_406973),wasusedforvirusneutralizationteston

Vero-E6cellsasdescribedpreviously26.

Panelsofhumanandanimalserausedinthisstudy.InSingapore,COVID-19patientseraused

inthisstudywasfromtheSingaporePROTECTstudyasdescribed[13].SerafromrecoveredSARS

patientsfrom2003wereaspreviouslydescribed[15].ForSARSrecallsamplingin2020,wecontacted

andthenobtainedbloodfromconsentingindividualspreviouslyadmittedforSARS(ethicsapproval

number:NHGDSRBE2020/00091).ThehCoVserumpanelincludedpost-infectionsamplesfrom

subjectsconfirmedCoV229/NL63andCoVOC43positiveusingtheSeeGeneRV12respiratory

multiplexkitinapreviousstudy(ethicsapprovalnumber:NUS-IRB11-3640)27.Negativecontrolsera

wereobtainedfromresidualserumsamplesfrompreviousunrelatedstudies.InNanjing,China,

COVID-19convalescentserawerecollectedwithwritteninformedconsentandapprovedbytheethics

committeeoftheSecondHospitalofNanjing(ethicsapprovalnumber:2020-LS-ky003).Rabbitanti-

SARS-CoV-2RBDserawerepurchasedfromGenScript.Rabbitandferretanti-SARS-CoVsera,and

alpacaanti-MERS-CoVserawereasdescribedinpreviousstudies28,29.

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DirectbindingandsVNTassay.Fordirectbinding,hACE2protein(GenScript)wascoatedat100

ng/wellin100mMcarbonate-bicarbonatecoatingbuffer(pH9.6).HRP-conjugatedSARS-CoV-2N,S1,

RBDorHRP-conjugatedSARS-CoV-RBD(allpurchasedfromGenScript)wasaddedtothehACE2

coatedplateatdifferentconcentrationinOptEIAassaydiluent(BD)for1hatroomtemperature.

UnboundHRP-conjugatedantigenswereremovedbyfivephosphatebufferedsaline,0.05%tween-20

(PBST)washes.ColorimetricsignalwasdevelopedontheenzymaticreactionofHRPwithchromogenic

substrate,3,3’,5,5’-tetramethylbenzidine(TMB)(Invitrogen).EqualvolumeofTMBstopsolution(KPL)

wasaddedtostopthereaction,andtheabsorbancereadingat450nmand570nmwereacquired

usingCytation5microplatereader(BioTek).Forthesurrogateneutralizationtest(sVNT),6ngofHRP-

RBD(fromeithervirus)waspre-incubatedwithtestserumatthefinaldilutionof1:20for1hat37°C,

followedbyhACE2incubationfor1hatroomtemperature.Inhibition(%)=(1-SampleOD

value/NegativeControlODvalue)x100.

IndirectELISA.SARS-CoV-2andSARS-CoVNproteinswereexpressedfromthepcDNA3.1SARS-CoV-

2NandpDualGCSARS-CoVNtransfectedHEK293TcellsandpurifiedusingNiSepharose(GE

healthcare).ForindirectELISA,100ngofeachproteinwascoatedontoMaxiSORPELISAplate(Nunc)

using100mMcarbonatebufferandblockedwithBDOptEIA(BD).COVID-19,SARSpatientserawere

testedatadilutionof1:50anddetectedbyGoat-anti-humanIgG-HRP(SantaCruz)at1:10,000

dilution.ThechromogenicsignalwasdevelopedusingTMBsubstrate(Invitrogen)andthereaction

wasstopwithTMBstopsolution(KPL).Absorbancereadingsat450and570nmwereobtainedusing

Cytation5microplatereader(Bio-Tek).

CaptureELISA.96-wellMaxisorpplates(Nunc)werecoatedwith10µg/mlofanti-humanIgM

(SeraCare)oranti-humanIgG(Jacksonlabs)inbicarbonatebufferovernightat4oC.Wellswere

blockedusingBDOptEIAassaydiluent(BD)for1hat37oCandheat-inactivatedseradiluted1:50

werenextaddedandincubatedfor1hat37oC.Followingextensivewashing,SARS-CoV-2-HRP

(GenScript)diluted4µg/mlwasaddedandincubatedfor30minat37oC.Chromogenicreactionwas

quantifiedfollowingtheadditionofTMBsubstrate(Invitrogen)andstopsolution(KPLSeraCare).The

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absorbanceofthesampleswasmeasuredat450nmandthebackgroundat570nm.Negative

controlsconsistedof37naïvehumansera.Resultsarepresentedasfold-changeoveraverage

readingofnegativecontrols.

Statisticalanalysis.StatisticalanalysiswasperformusingGraphPadPrismsoftwarewiththe

Kruskal-Wallistesttocomparemultiplegroups,followedbyDunn’smultiplecomparisonstest.Data

wereconsideredsignificantif*P<0.05,**P<0.01,***P<0.001,****P<0.0001.

DeclarationsOnlinecontent.Anymethods,additionalreferences,NatureResearchreportingsummaries,

supplementaryinformation,acknowledgements;detailsofauthorcontributionsandcompeting

interestsareavailableat[ArticleDOI].

Acknowledgements

WethankXijianQin,ShuangshuangTang,PeiLiuandWeihuiShaofortechnicaladviceandassistance

withassaydevelopmentandtesting;YilongPeng,CharlesTiu,AkshamalGamage,BengLeeLim,

VivianChen,WanRongSiaandXinMeiOngforassistanceinproteinpurification,sample

managementandtesting;YazidAbdadandLindaWeiLinTanforhelpwithhCoVseracollection;Viji

Vijayan,BensonNgandVelrajSivalingamoftheDuke-NUSMedicalSchoolABSL3facilityforlogistics

managementandassistance.L-FWandDEAaresupportedbygrantsfromtheSingaporeNational

ResearchFoundation(NRF2016NRF-NSFC002-013)andNationalMedicalResearchCouncil(STPRG-

FY19-001andCOVID19RF-003).

Authorcontributions

L-FWconceivedandguidedthestudy.CWT,WNCandDEAperformedlaboratoryworkincludingdata

analysis.MI-CC,ZH,BEY,Y-JT,YYandDCLprovidednecessarysamplesandcoordinationforthe

study.L-FWinitiatedthemanuscriptwritingwithinputfromallauthors.

Conflictofinterest

ApatentapplicationhasbeenfiledforthecontentdisclosedinthisstudyandaSARS-CoV-2sVNTKit

isunderdevelopmentwithanindustrialpartnerforcommercialization.

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Figures

15

Figure1

PrincipleandinitialvalidationoftheSARS-CoV-2surrogatevirusneutralizationtest(sVNT).

(a)Mechanismofconventionalvirusneutralizationtest(VNT).Anti-SARS-CoV-2neutralizing

antibodiesblockSARS-CoV-2SpikeproteinfrombindingtohACE2receptorproteinsonthe

16

hostcellsurface.(b)InthesVNTassay,anti-SARS-CoV-2neutralizingantibodiesblockHRP-

conjugatedRBDproteinfrombindingtothehACE2proteinpre-coatedonanELISAplate.(c)

BindingofHRP-conjugatedSARS-CoV-2N,S1andRBDproteinstohACE2.(d)Inhibitionof

SARS-CoV-2RBD-hACE2interactionbyCOVID-19patientsera.(e)BindingofHRP-

conjugatedSARS-CoVRBDtohACE2.(f)InhibitionofSARS-CoVRBD-hACE2interactionby

SARSpatientsera

17

Figure2

Isotype-independentneutralizationbyhumanserawithdifferentlevelsofIgMandIgG

antibodies.(a)HighIgM/LowIgG(n=5);(b)LowIgM/HighIgG(n=3);(c)LowIgM/LowIgG

(n=9);(d)HighIgM/HighIgG(n=5).TheIgMandIgGlevelsweredeterminedbyisotype-

specificcaptureELISAdetailedinMethods.

18

Figure3

Species-independentandvirus-specificneutralization.(a)Rabbitanti-SARS-CoV-2RBDsera

fromimmunization(n=3).(b)Ferretanti-SARS-CoVserafrominfection(n=2);(c)Rabbit

anti-SARS-CoVserafromimmunization(n=2).(d)SARS-CoV-2sVNTusingdifferent

coronavirussera:humanCOVID-19sera(n=10),humanSARSserasampledin2003(n=7,

<1year),humanSARS-CoVserasampledin2020(n=10,>17years),humanOC43sera(n

=8),human229E/NL63sera(n=10),MERS-CoVserafromexperimentallyinfectedalpaca

(n=4).(e)ComparativeanalysisofhomologousandheterologousNAblevelsforthe2003

SARSserumpanel.(f)ComparativeanalysisofhomologousandheterologousNAblevelsfor

the2020SARSserumpanel.(g)ComparativeanalysisofhomologousN-specificantibodies

inthethreeserumcohorts.SARS-CoV-2NproteinindirectELISAforCOVID-19seraand

SARS-CoVNproteinindirectELISAforthetwoSARSserumpanels.

19

Figure4

CorrelationbetweensVNTandVNTandsVNTtestingwithtwoCOVID-19patientcohorts

fromtwodifferentnations.(a)Correlationanalysisfor13COVID-19serawithdifferentlevels

ofSARS-CoV-2antibodiesbyVNTandsVNTat70%inhibition.Testingofhealthycontroland

COVID-19serumcohortsinSingapore(b)(COVID-19n=77,controln=75)andNanjing(c),

China(COVID-19n=50,controln=50).

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