Antiviral Functions of Monoclonal Antibodies against Chikungunya Virus

17 Pages·2017·2.9 MB·English

viruses
Review
Antiviral Functions of Monoclonal Antibodies against
Chikungunya Virus
JingJin1,2,* andGrahamSimmons1,2,*
1 VitalantResearchInstitute,SanFrancisco,CA94118,USA
2 DepartmentofPathologyandLaboratoryMedicine,UniversityofCalifornia,SanFrancisco,CA94143,USA
* Correspondence:[email protected](J.J.);[email protected](G.S.)
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:7March2019;Accepted:27March2019;Published:28March2019
Abstract: Chikungunyavirus(CHIKV)isthemostcommonalphavirusinfectinghumansworldwide.
Antibodiesplaypivotalrolesintheimmuneresponsetoinfection.Increasingly,therapeuticantibodies
are becoming important for protection from pathogen infection for which neither vaccine nor
treatmentisavailable,suchasCHIKVinfection. Thenewgenerationofultra-potentand/orbroadly
cross-reactivemonoclonalantibodies(mAbs)providesnewopportunitiesforintervention. Inthe
pastdecade,severalpotenthumanandmouseanti-CHIKVmAbswereisolatedanddemonstratedto
beprotectiveinvivo. MechanisticstudiesofthesemAbssuggestthatmAbsexertmultiplemodes
ofactioncooperatively. BetterunderstandingoftheseantiviralmechanismsformAbswillhelpto
optimizemAbtherapies.
Keywords: Chikungunya;antibodies;neutralization;budding;effectorfunctions
1. Introduction
Chikungunyavirus(CHIKV)isanemergingalphavirus. Spreadingrapidlyfromendemicareas
ofAfricaandAsiatoEuropeandtheAmericas,CHIKVisnowthemostcommonalphavirusinfecting
humansglobally[1]. CHIKVistransmittedtohumansbyAedesspeciesmosquitoesandcausesan
acute febrile illness accompanied by severe arthralgia, with relapses lasting up to several months
andrecurringforseveralyearsinupto40%ofpatients[2]. Ina2007outbreakinIndia,themedian
workdayslostduetoCHIKVinfectionwas35[3].ThisisonereasonwhyCHIKVisclassifiedbyNIAID
asabiodefenseprioritypathogen,despitelowfatalityrates. Inaretrospectivecohortstudyofthe
2005–2006LaRéunionislandoutbreak,CHIKVwasfoundtobeasignificantcauseofcentralnervous
systemdisease[4]. Currently,therearenovaccinesortreatmentsforCHIKVinfection. Inthepast
decade,severalpotenthumanandmouseanti-CHIKVmonoclonalantibodies(mAbs)wereisolated
anddemonstratedtobeprotectiveinvivo[5–13]. Inthisreview,wewilldiscussourunderstandingof
themultipleantiviralmechanismsthroughwhichthesemAbsexertviralinhibition.
2. AlphavirusStructureandLifeCycle
CHIKV is an enveloped, positive-stranded RNA virus belonging to the Alphavirus genus in
theTogaviridaefamily. TheCHIKVstructuralproteins,capsid(CP)andthreeenvelopeglycoproteins
(GPs)(E1,E2andE3),aresynthesizedasapolyproteinthatisproteolyticallyprocessed. Cryo-electron
microscopy(cryoEM)structureofmaturealphavirusparticlesandvirus-likeparticles(VLPs)revealed
that virions are composed of two icosahedral layers: the outer envelope layer and the inner
nucleocapsid (NC) core, both with T = 4 quasi-icosahedral symmetry. The envelope comprises
80 membrane embedded spikes, with each spike formed by a trimer of the E1–E2 heterodimer
(Figure1A)[13–16]. EachCPinteractswiththecytosolicdomainofE2,and240copiesofCPforman
icosahedralNCcoreenclosingtheviralgenomicRNAinside(Figure1B,C).E1isatypeIImembrane
Viruses2019,11,305;doi:10.3390/v11040305 www.mdpi.com/journal/viruses
VViriurusesse s22001199, ,1111, ,x3 F0O5R PEER REVIEW 2 2ofo f171 7
fusion protein and sits at the base of the trimeric spike, with E2 positioned on top of E1. E1 consists
offu tshiroene pdroomteaininasn: ddosmitsaaint tIh leinbkass ediosftathl deotrmimaienr iIcI sapnidk em,wemithbrEa2nep opsriotixoinmeadl odnomtoapino fIIEI.1 A. E f1ucsoionns ilsotospo f
ist hloreceatdeodm ata itnhse: ddiosmtala iennIdl oinfk Es1d disotmaladino mIIa. iCnrIyIsatanld stmruecmtubrreasn oefp CroHxIiKmVa lEd1oEm2 ahienteIIrIo.dAimfuesri orenvleoaolpedi s
thlorceaet eddomatatihnes dinis tEa2l e[1n7d]:o dfoEm1adionm Aa iins IlIo.cCatreydst ainl stthreu ccteunrteesr ooff CthHeI KspVikEe1 sEu2rhfaectee,r owdhimilee rdroemveaainle dB tahnrdee
Cd aorme aloincsatiendE a2t [t1h7e] :ddisotmala einndA ainsdlo tchaet emdeimnbthreancee nptreorxoimftahle esnpdik oef sEu2r,f arecesp,wechtiivleeldyo (mFiagiunrBe a1nDd). CThaere
alpoecxa toefd daotmthaeind iAst aalnedn ad paanrdt tohf edmomemaibnr aBn peopsrsoexssim thael epnudtaotifvEe2 b,irnedspinegc tsiviteel yfo(rF irgeucerpet1oDr )[.1T3,h1e8,a1p9]e,x
wohfidcho misa incoAnfairnmdead pbayrt tohfed oremceanint Bidpeonstsifeiscsattihoen pouft aat ivsehabriendd irnegcespitteofro, rMrexcreap8t,o rfo[r1 3a,1 8n,u1m9],bwerh iocfh
airsthcrointofigremneicd ablpyhtahveirruecseesn tinidcleundtiinfigca CtiHonIKoVf,a Os’hnayroendgr encyeopntgo rv,iMruxsr (aO8,NfoNrVa),n Mumaybaerroo vfiraurtsh (rMitoAgYeVn)ic
aanldp hRaovsisr uRsievseirn cvliurudsin (gRCRVH)I K[2V0,]O. A’nty noenugtnraylo pnHg,v Eir2u sd(oOmNaNinV B) ,pMroatyeacrtos vthireu fsu(sMioAnY lVoo)pan adt Rthoes sdRisitvael r
evnidru osf (ER1R fVro)m[2 0ac].tiAvattnioenu t[r1a7l,2p1H,2,2E]. 2Cdryoom-EaiMn Bstrpurcottuercetss othf esefvuesrioaln allopohpavaitruthseesd hisatvael sehnodwonf Eth1aftr Eo2m
daocmtivaianti oBn h[a1s7 ,a2 1lo,2w2]e.rC erleycot-rEoMn dsternuscittuy rceosmofpsaerveedr atol aoltphhearv diroumseasinhsa, vseusghgoewstinntgh tahtaEt2 Ed2o dmoaminaiBn hBa sisa
flleoxwibelree [l1e3c,t1r5o,n16d]e. nAsti tlyowco pmHp,a Ere2d dtoomoathine rBd ios mdiasionrsd,esruegdg easntdin tgheth fautsEio2nd loomopa iant Bthies tflipex oibf lEe1[ 1d3o,m15a,1in6 ].
IIA its leoxwpopsHed,E i2n dthoem Xa-irnaBy cisrydsitsaolr dsterruecdtuarned otfh eenfvueslioopnel oporoptaetintsh efrtoipmo tfhEe1 redloamtedai nalIpIhisaveixrpuoss, eSdinidnbtihse
[1X8-]r.a ycrystalstructureofenvelopeproteinsfromtherelatedalphavirus,Sindbis[18].

Figure1. StructureofChikungunyavirus(CHIKV)andenvelopeglycoproteins. CryoEMdensity
Fmigaupre( E1.M StDru-5c5tu7r7e) ooff C(Ah)ikCuHngIKunvyirau vsi-rluikse (CpHarItKicVle)s a(nVdL ePn)v,e(Blo)pCeH glIyKcoVpLrPotveiinews. eCdryaotEthMe dcreonsssi-tsye mctiaopn ,
(EaMndD(-C5)57C7H) IoKf (nAu)c lCeoHcIaKp svidirucos-rleik,ceo ploarretidclaecsc (oVrdLiPn)g, (tBo)t hCeHrIaKd iValLdPi svtaienwceedfr oamt ththee ccreonstse-sreocftitohne, vainruds ;
(C(D) )CSHtrIuKc ntuurceleoofctahpespid6 2c-oEr1e,h ceotleorroeddi macecro(rPdDinBg:3 tNo 4th0e). radial distance from the center of the virus; (D)
Structure of the p62-E1 heterodimer (PDB:3N40).
Inacidifiedendosomes,wherealphavirusesaretransportedafterendocytosisintotargetcells,
lowIpn Hactirdiigfgieedr sencodnofsoormmeast,i ownhaelrree aarlprahnagveirmuesenst sarine ttrhaenespnvoretleodp eafGtePr setnodeoxcpytoosseist hinetofu tsairognetl ocoepllsa, t
lothwe pdHis ttarilgegnedrs ocfonEf1o.rmUaptoionnraelm reoavrraalnogfeEm2efnrtosm in tthhee ceennvteelropoef vGiPrasl tosp eixkpeo,sEe1 thfoer fmussioanh loomopo tarti mtheer ,
dfiustrathl eerndex opfo Es1in. gUtphoenf uresmioonvlaolo opfs Eo2f efraocmh mthoen coemnteerr aotf tvhieraeln sdpiokfet,h Ee1t rfoimrmersi ca choommpolterximfoerr,i nfusertrhtieorn
eixnptooshinogs tthmee fmusbiorann leoo[2p3s] .ofM eaecmhb mraonneofmuesiro ant btheetw eneden ovf itrhues trainmderhioc sctocmelpllaelxl ofwors inthseerptieonne itnratoti ohnosot f
mneumclberoacnaep si[d23s](. NMCse)minbtroanthe efcuystioosno lbaentdwreaepni dvdiriusas ssaenmdb lyh.osVti racellnl onalsltorwucst utrhael pproetneeitnrsatairoen thoefn
nturaclnesolcaatepdsidfrso m(NtChse) iinnctoom thineg cyvtioraslogl eannodm raicpRidN dAisiansstehmebclyyt.o Vsoilraaln ndofnosrtmrucretuprlaicla ptiroontecionms aprlee xtehsento
translated from the incoming viral genomic RNA in the cytosol and form replication complexes to
Viruses2019,11,305 3of17
synthesizenewviralRNA[24]. Viralstructuralproteinsaretranslatedfromthenewlysynthesized
viralsubgenomicRNAinthecytosol. ThepositivelychargedresiduesattheN-terminaldomainof
CPinteractwithviralgenomicRNAtonucleatetheassemblyofNCs[25,26]. GPsaresynthesizedas
apolyprotein(p62-E1)containingE3E2(p62)andE1. Afterco-translationalproteolysis,thep62-E1
heterodimerthenassemblesintotrimericspikesintheendoplasmicreticulumandisdeliveredtothe
plasmamembranethroughintracellularmembranetrafficking. Furinandfurin-likehostproteases
cleaveE3duringthetransportthroughthetrans-Golginetworktotheplasmamembrane,primingthe
spikesforfusogenicactivation.
Attheplasmamembrane,thecytosolicdomainofE2bindstoahydrophobicpocketinCPon
thesurfaceofNC[27,28]. Theicosahedralglycoproteinshellthenassemblesinsynchronywiththe
icosahedralsymmetryoftheNC,finallyresultinginbuddingofnascentvirions. CPscanassembleinto
icosahedralNCsindependentlyofGPsinsituandinvitro[29,30]. NCscanbepreparedbyinvitro
assembly[30],isolationfromvirionsstrippedofenvelope[31],orpurificationfrominfectedcells[29].
OnlyasmallsubpopulationoftheseNCswereicosahedrallysymmetricwhenvisualizedinvitroby
cryoEMsingleparticleanalysis(SPA).TheorganizationofCPsintheisolatedNCsdifferedfromthat
insidematurevirions[29–31]. AllthesesuggestthatNCsarestructurallyheterogeneousandlessstable
thanmaturevirionsandundergoGP-drivenand/orlipid-drivenstructuralchangesuponenvelopment.
CPs that are deficient in NC assembly were able to assemble and bud into wild-type-like Semliki
Forest virus (SFV) virions driven by horizontal spike–spike interactions and vertical spike–capsid
interactions[32,33],indicatingadirectroleforviralspikesinorganizingtheicosahedralconformation
ofalphavirus.InsituelectrontomographyofchemicallyfixedSFV-infectedcellssuggestedthattrimeric
GPspikesmightalreadyassembleintohexagonallatticesincellularmembranecompartmentspriorto
beingdeliveredtotheplasmamembrane,wheretheyarerequiredforvirusbudding[34]. Mutationsin
theD-loopoftheSFVE2protein,thatdisruptthecontactbetweentheviralE1andE2proteins,inhibit
SFV budding, further suggesting that the native organization of the GP lattice is critical for virus
budding[35]. Therefore,horizontalinteractionsbetweenGPsandverticalinteractionsbetweenGPs
withCPscoordinatelydrivetheformationoftheicosahedralGPshellandinducemembranecurvature
formationrequiredforvirusbudding,similartoCOPIandCOPIIcomplex-drivenmembranevesicle
buddingforintracellularproteintransport[36].
3. EntryNeutralizationbymAbs
Antiviralantibodiesaretraditionallyscreenedfortheirabilitytoinhibitvirusentryintotarget
cells. EntryneutralizationofenvelopevirusbymAbscanbemediatedviatwomajormechanisms:
inhibition of receptor binding and prevention of conformational changes within the viral fusion
proteinspost-receptorbinding. Foralphaviruses,membranefusionoccursinacidifiedendosomes
aftervirusentersthecells. Inthisreview,neutralizingantibodies(NAbs)specificallyreferstomAbs
thatinhibitvirusentryintosusceptiblecells. Theanti-CHIKVmAbsdiscussedbelowaresummarized
inTable1.
Table1.CharacteristicsofmAbsagainstCHIKV.
mAb Species AntigenicSites CHIKV-LREC50 DemonstratedModeof Reference
ng/mL(CI) Action
2H1 human E2-DA 5.6(4.9–6.3) entryneutralization [10]
8G18 human E2-DA 7.3(6.3–8.4) entryneutralization [10]
3E23 human E2-DA 18.7(16.3–21.5) entryneutralization [10]
1O6 human E2-DA 57.5(48.8–68.1) entryneutralization [10]
4J21 human E2-DA,DB,arch 7.4(6.6–8.3) entryneutralization [10,37]
5M16 human E2-DA,DB,arch 5.9(5.0–6.8) entryneutralization [10,37]
CHK-152 mouse E2-DA,DB,arch 5(5–7) entryneutralization,ADCP [8,12,13,38–40]
CHK-9 mouse E2-DA 36(31–43) entryneutralization [8,13]
Viruses2019,11,305 4of17
Table1.Cont.
mAb Species AntigenicSites CHIKV-LREC50 DemonstratedModeof Reference
ng/mL(CI) Action
CHK-166 mouse E1-DII 154(116–205) entryneutralization,ADCP [8,12,38–40]
entryneutralization,
CHK-265 mouse E2-DA,DB 8(7–9) [6,8]
viralegressinhibition
entryneutralization,
CHK-187 mouse E2-DB 11(9–12) [6,8]
viralegressinhibition
entryneutralization,
C9 human E2-DA,DB,arch 51* [7,9,41]
buddinginhibition,ADCC
entryneutralization,
IM-CKV063 human E2-DA,DB,arch 7.4* [5,7,41]
buddinginhibition,ADCC
m242 human E2-DA 300 entryneutralization [13]
m10 human E2-DB 7 entryneutralization [13]
entryneutralization,
8B10 human E2-DA,DB 46# [12,39,40]
buddinginhibition
entryneutralization,
5F10 human E2-DB 62# [12,39,40]
buddinginhibition
CK47 mouse E1-DIII no viralegressinhibition [42]
NeutralizingactivitywasdeterminedbyFRNTonVerocellswithserialdilutionsofmAbsand100FFUofthe
CHIKV-LRstrain. *neutralizationofreporterHIVpseudotypedwithCHIKVS27E1/E2. # neutralizationof
CHIKV11strainbyPRNTassay.
3.1. BlockVirusBindingtoReceptors
AlaninescanningmutagenesisofE2revealedthebindingsiteofCHIKVreceptor,thecelladhesion
moleculeMxra8,onviralspikes: asolvent-accessibleepitopespanningthetopofE2domainAand
domainB[20]. AnumberofhumanmAbstargetingepitopesinE2domainA(2H1,8G18,3E23and
1O6) and human mAb 4J21 that binds epitopes spanning E2 domain A and domain B are able to
inhibitbindingofMxra8toCHIKV[10,20]. AmongthesemAbs,4J21bindingtoCHIKVVLPswas
resolved by cryoEM. The footprint of 4J21 Fab covers primarily the β-ribbon connector that links
E2 domain A and domain B together, as well as small regions on E2 domain A and E2 domain B
(Figure2)[37]. Cryo-EMstructuresofCHIKVVLPincomplexwithFabfragmentsofhumanmAbs
m242andm10,aswellasmousemAbCHK-9,suggesttheseE2domainAordomainBtargetingmAbs
blockreceptorbindingtoCHIKV(Figure2)[13,43],althoughexperimentsareneededtodemonstrate
these mAbs prevent CHIKV from binding to Mxra8 and susceptible cells. In addition to blocking
receptorbinding[20],4J21isalsoabletoinhibitlowpHactivatedmembranefusionattheplasma
membraneofsusceptiblecellsinfusion-from-without(FFW)experiments,wherebyafterallowing
virus to attach to cells at 4 ◦C, the environment is acidified in order to trigger viral and cellular
membranefusionatthecellsurface[10]. Incontrast,CHIKVVLPsincomplexwithFab8B10aggregate
atpH6,suggestingthathumanmAb8B10isunabletopreventlowpHactivatedmembranefusion
whilethecryo-EMstructureofthecomplexsuggeststhat8B10preventsvirusbindingtoreceptor[43].
OneoftheessentialresiduesonE2domainAforMxra8binding,W64,isasharedepitoperesidue
for a number of human NAbs [7,10,20]. Escape mutants at this residue result in attenuated viral
pathogenesis[7]. HumanNAbs,C9andIM-CKV063,targetW64onE2domainAbutdonotprevent
CHIKVfrombindingtoVerocells[7]. OnepossibleexplanationisthatonVerocellsCHIKVcanuse
alternativereceptors/attachmentfactorsthatareindependentofbindingtoW64. Alternatively,the
240receptorbindingsitesonCHIKVarenotsufficientlyoccupiedbyC9orIM-CKV063inorderto
blockcellattachment,whichwewilldiscussbelow.
Viruses2019,11,305 5of17
Viruses 2019, 11, x FOR PEER REVIEW 5 of 17

FFiigguurree 22.. FFiitt ooff FFaabb ssttrruuccttuurreess iinnttoo tthhee ccrryyooEEMM ddeennssitityy“ “ddififfefererenncece””m mapapss(g (rgeryeym mesehshsu srufarfcaec)eb)o buonudntdo
tEo1 E-E1-2Eh2e hteetreordoidmimere.rB. iBnidnidnigngo fo4f J42J121a anndd5 5MM1166F Faabbsst tooE E11 ((ggrreeeenn))--EE22 ((bblluuee)) hheetteerrooddiimmeerr wwaass aaddaapptteedd
ffrroomm [[3377]].. TThhee FFaabb ssttrruuccttuurreess aarree sshhoowwnn iinn rreedd aanndd ffiitttteedd iinnttoo tthhee ccrryyoo--EEMM ddeennssiittyy.. EE11 iiss sshhoowwnn iinn
ggrreeeenn,, aanndd EE22 iiss sshhoowwnn iinn bblluuee.. BBiinnddiningg ooff CCHHKK--115522, ,CCHHKK--99, ,mm224422 aanndd mm1100 FFaabbss ttoo EE11 ((rreedd))--EE22
((ggrreeeenn)) hheetteerrooddiimmeerr wwaass aaddaapptteedd ffrroomm [[1133]].. TThhee FFaabb ssttrruuccttuurreess aarree sshhoowwnn iinn bblluuee aanndd ffiitttteedd iinnttoo tthhee
ccrryyoo--EEMM ddeennssiittyy..
3.2. PreventActivationofMembraneFusion
3.2. Prevent Activation of Membrane Fusion
Topreventmembranefusion,NAbsneedtokeepE2domainBinpositiontopreventexposure
To prevent membrane fusion, NAbs need to keep E2 domain B in position to prevent exposure
offusionloopatlowpH.InthecryoEMstructuresofCHIKVVLPincomplexwithFabfragmentsof
of fusion loop at low pH. In the cryoEM structures of CHIKV VLP in complex with Fab fragments of
mousemAb,CHK-152[13]andhumanmAb,5M16[37],bothmAbsspanE2domainAanddomain
mouse mAb, CHK-152 [13] and human mAb, 5M16 [37], both mAbs span E2 domain A and domain
Baswellastheβ-ribbonconnectorconnectingthetwodomainswithinoneE2molecule(Figure2).
B as well as the β-ribbon connector connecting the two domains within one E2 molecule (Figure 2).
Both CHK-152 [8] and 5M16 [10] efficiently block CHIKV fusion at the plasma membrane in the
Both CHK-152 [8] and 5M16 [10] efficiently block CHIKV fusion at the plasma membrane in the FFW
experiments. They may do so by cross-linking the flexible domain B to the rest of the virus surface
Viruses2019,11,305 6of17
FFW experiments. They may do so by cross-linking the flexible domain B to the rest of the virus
surfacetherebykeepingthefusionloopunexposedbeneathdomainB.Infact,thedensityheightofE2
domainBissimilartoalltheotherdomainsinCHIKVVLP-Fab5M16[37]. Incontrast,inthecryoEM
structureofCHIKVVLPaloneE2domainBhaslowerdensityheightcomparedtootherdomains[13].
ThisindicatesstabilizationofE2domainBuponbindingof5M16toCHIKV.
HumanmAbs,C9andIM-CKV063,isolatedandcharacterizedbyourgroupandcollaborators[5,9]
neutralizeCHIKVentrybyinhibitinglowpH-activatedmembranefusion[7]. BothC9andIM-CKV063
spandomainAofoneE2moleculeanddomainBoftheneighboringE2moleculewithinonetrimeric
spike(Figure3A,B),thereforestabilizingdomainBtokeepthefusionloopprotectedatlowpH[7].
ThisissimilartothefootprintofFab3B4C-4onVenezuelanequineencephalitisvirus(VEEV)[40].
Asdiscussedabove,althoughC9andIM-CKV063bindtoW64onE2domainAthatisessentialfor
Mxra8binding,theyneutralizeCHIKVentrybyinhibitingmembranefusionnotpreventingvirus
bindingtothetargetcell. ItislikelythatbindingofonlyoneFabpertrimericspikeissufficientto
preventtheconformationalchangesinGPrequiredforactivationofmembranefusionandhencelead
toinhibitionofentry. Incontrast,topreventreceptorbinding,allthreereceptorbindingsitesineach
trimericspikemayneedtobeoccupied. ProbablysterichindrancepreventsC9andIM-CKV063from
occupyingallthreebindingsiteswithinonespike. ThismaypartiallyexplainhowC9andIM-CKV063
potentlyneutralizevirusentrywithoutinhibitingvirusbindingtothesusceptiblecell.
Broadly neutralizing antibodies (bNAbs) against HIV-1 [44], influenza virus [45], hepatitis
C virus [46], Ebola virus [47], and dengue virus [48,49] have been isolated. These bNAbs are
able to neutralize highly variable viruses by binding to conserved regions. CHIKV is relatively
conservedgenetically;withtheenvelopeGPssharing95to99.9%identityacrossthreemajorlineages
(West African; East, Central, and South African; Asian). This explains neutralization of different
strainsacrossCHIKVlineagesbyNAbsisolatedfromrecoveredpatients[5,10,12]orCHIKV-infected
animals[8,50].Interestingly,apanelofhumanandmousebNAbsthatneutralizemultiplealphaviruses
including CHIKV, MAYV, ONNV, RRV and SFV were identified [6]. Prophylactic treatment with
twoofthesebNAbsprotectedmiceagainstinfectionbyCHIKV,MAYVandONNV.ThesebNAbs
targetanepitopeinE2domainBthatisconservedacrossmultipleanddistantlyrelatedarthritogenic
alphaviruses. In the cryoEM structure of Fab fragment of one bNAb, CHK-265 in complex with
CHIKV (Figure 3C), the Fab binds to E2 domain B and pushes it further down over the fusion
loop. Concurrently,E2domainAismovedaroundE1domainIItothespacebetweenneighboring
spikes (Figure 3D) [6]. This allows CHK-265 to crosslink E2 domain B with E2 domain A of the
neighboring spike [6]. The electron density of E2 domain B is as high as the other GP domains,
indicatingthestabilizationofE2domainBexplainingthemechanismforinhibitionofmembrane
fusionbybNAbCHK-265.
HumanmAb5F10alsoinhibitslowpH-activatedconformationalchangesbyrigidlyfixingE2
domainB,butwithoutcrosslinkingdomainBwithstabledomainA.InsteadFab5F10bindstodomain
BofeachE2moleculeviaCDRsandextendstangentiallytothevirussurface,leadingtotightpacking
ofFabsboundonneighboringspikes[43]. Asaresult,theaveragedensityofE2domainBareslightly
higherthanthedensitiesofotherdomainsandthereisnomeasurabledifferencebetweenthestructures
at pH 7.0 and pH 6.0, indicating the stabilization of domain B and inhibition of low pH activated
conformationalchanges. LockingE2domainBinpositionviapackingFabsboundondomainBof
neighboringspikeswasalsoreportedforNAb3B4C-4againstVEEV[40].
Epitopes for above-described anti-CHIKV NAbs are mostly complex quaternary epitopes,
especiallyforultra-potentmulti-domaincross-linkingNAbs,like5M16,CHK-152andIM-CKV063.
ThisisreminiscentofpotentNAbsagainstflavivirusesthattargetanepitopespanningthreeseparate
Eproteinmonomers[51]oranepitopespanningbothEproteinsofanEproteindimeronmature
virus[48]. ItwasreportedthatearlyneutralizingIgGresponsestoCHIKVinpatientsmostlytargeteda
singlelinearepitope(E2EP3)attheN-terminusofE2[52]. E2EP3conjugatedtothelarge,multisubunit
carrierproteinKeyholeLimpethemocyaninwasreportedtoinducemildneutralizingactivityand
Viruses 2019, 11, x FOR PEER REVIEW 6 of 17
thereby keeping the fusion loop unexposed beneath domain B. In fact, the density height of E2
domain B is similar to all the other domains in CHIKV VLP-Fab 5M16 [37]. In contrast, in the cryoEM
structure of CHIKV VLP alone E2 domain B has lower density height compared to other domains
[13]. This indicates stabilization of E2 domain B upon binding of 5M16 to CHIKV.
Human mAbs, C9 and IM-CKV063, isolated and characterized by our group and collaborators
[5,9] neutralize CHIKV entry by inhibiting low pH-activated membrane fusion [7]. Both C9 and IM-
CKV063 span domain A of one E2 molecule and domain B of the neighboring E2 molecule within one
trimeric spike (Figures 3A,B), therefore stabilizing domain B to keep the fusion loop protected at low
pH [7]. This is similar to the footprint of Fab 3B4C-4 on Venezuelan equine encephalitis virus (VEEV)
[41]. As discussed above, although C9 and IM-CKV063 bind to W64 on E2 domain A that is essential
Virfuosre sM20x1r9a,81 1b,i3n0d5ing, they neutralize CHIKV entry by inhibiting membrane fusion not preventing vir7uosf 17
binding to the target cell. It is likely that binding of only one Fab per trimeric spike is sufficient to
prevent the conformational changes in GP required for activation of membrane fusion and hence lead
partialprotectioninvivo[52]. However,immunizationwithapentameroftheE2EP3peptidefailed
to inhibition of entry. In contrast, to prevent receptor binding, all three receptor binding sites in each
toinduceNAbsandinvivoprotectioninmice[53]. Incontrast,immunizationwithlivewild-typeor
trimeric spike may need to be occupied. Probably steric hindrance prevents C9 and IM-CKV063 from
attenuatedCHIKV[8,54–57],inactivatedCHIKV[50],orVLPs[58],inducesstrongNAbsthatprovide
occupying all three binding sites within one spike. This may partially explain how C9 and IM-
invivoprotectioninanimals,probablyduetobetterpresentationofcomplexquaternaryepitopes.
CKV063 potently neutralize virus entry without inhibiting virus binding to the susceptible cell.

Figure3.BindingofIM-CKV063andCHK-265toCHIKV.(A)CryoEMreconstructionofCHIKVLPin
Figure 3. Binding of IM-CKV063 and CHK-265 to CHIKV. (A) CryoEM reconstruction of CHIK VLP
complexwithIM-CKV063Fabfragment(EMD-6457);(B)Structureofthetrimericspikeboundwith
in complex with IM-CKV063 Fab fragment (EMD-6457); (B) Structure of the trimeric spike bound with
IM-CKV063Fabasobservedin(A),showingIM-CKV063crosslinkstwoneighboringE2molecules
IM-CKV063 Fab as observed in (A), showing IM-CKV063 crosslinks two neighboring E2 molecules
withinonetrimericspike.E1,E2andFabareshowninred,greenandblue,respectively.(C)CryoEM
within one trimeric spike. E1, E2 and Fab are shown in red, green and blue, respectively. (C) CryoEM
reconstructionofCHIKvaccinestrain181/25incomplexwithCHIK-265Fabfragment; (D)(Left)
reconstruction of CHIK vaccine strain 181/25 in complex with CHIK-265 Fab fragment; (D) (Left)
StructureoftheE1-E2heterodimer(PDB:3N42);(Right)StructureoftheE1-E2heterodimerbound

withCHIK-265Fabasobservedin(C),showingtherepositioningofE2domainAuponCHIK-265
bindingtoE2domainB.TheE2domainAanddomainBarecircled,showingthedifferenceintheir
conformationswithandwithoutFabCHIK-265binding(adaptedfrom[6]withpermission).
4. BuddingInhibitionbymAbs
Inadditiontotheircanonicalinhibitionofvirusentryintocells,humanNAbsC9andIM-CKV063
and mouse bNAbs CHK-265 and CHK-187 are able to inhibit virus release from CHIKV-infected
cells[6,7]. Fab fragments of these NAbs do not inhibit virus release but retain the ability to bind
theirepitopestoneutralizevirusentry[6,7]. Thus,theinhibitionofvirusreleasedependsonbivalent
bindingofNAbstoCHIKVGPs. ResistanceofescapemutantstoNAb-mediatedentryneutralization
andbuddinginhibitionindicatesthesameepitoperesiduesareutilizedbyNAbstoneutralizemature
virionsandtoinhibitvirusbuddingonthecellsurface[7]. ItisunknownifNAbsbindtothesame
Viruses2019,11,305 8of17
Viruses 2019, 11, x FOR PEER REVIEW 8 of 17
epitopeinGPspresentedonmaturevirionsandonthesurfaceofinfectedcells.Futurestudiestoreveal
Future studies to reveal the detailed structure of NAb crosslinking of GPs on cell surface are needed
thedetailedstructureofNAbcrosslinkingofGPsoncellsurfaceareneededtoaddressthisquestion.
to address this question. Visualization of CHIKV-infected cells cultured in the presence of NAbs with
VisualizationofCHIKV-infectedcellsculturedinthepresenceofNAbswithSTEDsuper-resolution
STED super-resolution microscopy revealed large patches of CHIKV GPs bound with NAbs on the
microscopyrevealedlargepatchesofCHIKVGPsboundwithNAbsontheplasmamembrane[41],
plasma membrane [42], suggesting that NAbs crosslink GPs and induce their coalescence into large
suggestingthatNAbscrosslinkGPsandinducetheircoalescenceintolargepatchesontheplasma
patches on the plasma membrane. Thin-section transmission electron microscopy (TEM) imaging
membrane. Thin-sectiontransmissionelectronmicroscopy(TEM)imagingrevealedadensearrayof
revealed a dense array of NAbs bound to GPs on the outer surface of CHIKV-infected cells and the
NAbsboundtoGPsontheoutersurfaceofCHIKV-infectedcellsandtheinhibitionofmembrane
inhibition of membrane curvature around NCs required for virus budding (Figure 4) [42]. In the
curvaturearoundNCsrequiredforvirusbudding(Figure4)[41]. Inthecytosol,budding-arrested
cytosol, budding-arrested NC-like particles (NCLPs) varied in size and structure with no icosahedral
NC-like particles (NCLPs) varied in size and structure with no icosahedral symmetry detected
symmetry detected by subtomogram averaging (STA) analysis of cryo-electron tomography (cryoET)
by subtomogram averaging (STA) analysis of cryo-electron tomography (cryoET) of NAb-treated
of NAb-treated CHIKV-infected cells [42]. This suggests that inhibiting surface budding arrests
CHIKV-infectedcells[41]. ThissuggeststhatinhibitingsurfacebuddingarrestsintracellularNCsat
intracellular NCs at assembly intermediates or forces NCs to disassemble. STA of membrane patches
assemblyintermediatesorforcesNCstodisassemble. STAofmembranepatchesofNAb-induced
of NAb-induced coalesced GPs did not converge to any structure with symmetric organization of
coalescedGPsdidnotconvergetoanystructurewithsymmetricorganizationofGPs,suggestingthat
GPs, suggesting that they were conformationally and/or compositionally heterogeneous [42].
theywereconformationallyand/orcompositionallyheterogeneous[41].Therefore,C9andIM-CKV063
Therefore, C9 and IM-CKV063 inhibit CHIKV budding by disrupting assembly of GP spikes into an
inhibitCHIKVbuddingbydisruptingassemblyofGPspikesintoanicosahedrallatticeviacrosslinking
icosahedral lattice via crosslinking and coalescing viral GP spikes. Other than C9 and IM-CKV063,
andcoalescingviralGPspikes. OtherthanC9andIM-CKV063,twoabove-mentionedhumanNAbs,
two above-mentioned human NAbs, 5F10 and 8B10, were also reported to strongly reduce
5F10and8B10,werealsoreportedtostronglyreduceextracellularvirusreleasedfromCHIKV-infected
extracellular virus released from CHIKV-infected cells [12], although study is needed to demonstrate
cells[12],althoughstudyisneededtodemonstrateiftheyinhibitvirusreleaseatthebuddingstep.
if they inhibit virus release at the budding step. The budding inhibition mechanism of antiviral
ThebuddinginhibitionmechanismofantiviralfunctionofmAbsisprobablyindependentoftheability
function of mAbs is probably independent of the ability to neutralize virus entry by mAbs. A mAb
toneutralizevirusentrybymAbs. AmAbCK47targetingCHIKVE1domainIIIisabletoinhibitvirus
CK47 targeting CHIKV E1 domain III is able to inhibit virus release but not entry [43]. One mAb
releasebutnotentry[42]. OnemAbtargetingE3ofVEEVwasreportedtoinhibitvirusreleasefrom
targeting E3 of VEEV was reported to inhibit virus release from VEEV-infected cells but not virus
VEEV-infectedcellsbutnotvirusentryintotargetcells[59]. Furtherstudiesareneededtounderstand
entry into target cells [60]. Further studies are needed to understand the mechanism behind the
themechanismbehindtheinhibitionofvirusreleasebythesenon-neutralizingmAbs. Asmostofthe
inhibition of virus release by these non-neutralizing mAbs. As most of the mAbs screening focused
mAbsscreeningfocusedonentryneutralization,unbiasedscreeningofmAbsforbuddinginhibition
on entry neutralization, unbiased screening of mAbs for budding inhibition function is required to
functionisrequiredtoisolatebuddinginhibitorymAbsandtesttheirinvivoprotectionefficiencies.
isolate budding inhibitory mAbs and test their in vivo protection efficiencies. Studies of budding
StudiesofbuddinginhibitorymAbstargetingdifferentepitopeswillhelpusbetterunderstandthe
inhibitory mAbs targeting different epitopes will help us better understand the mechanism behind
mechanismbehindmAb-mediatedinhibitionofalphavirusbudding.
mAb-mediated inhibition of alphavirus budding.

Figure4. NeutralizingantibodiesactontheouterleafletoftheplasmamembranetoblockCHIKV
Figure 4. Neutralizing antibodies act on the outer leaflet of the plasma membrane to block CHIKV
budding. CHIKV-infected cells treated with C9 were labeled with gold-conjugated anti-human
budding. CHIKV-infected cells treated with C9 were labeled with gold-conjugated anti-human
antibodies before visualized by thin-section TEM. Budding arrested NCLPs are indicated with a
antibodies before visualized by thin-section TEM. Budding arrested NCLPs are indicated with a red
redarrow.Scalebar:100nm(adaptedfrom[41]withpermission).
arrow. Scale bar: 100 nm (adapted from [42] with permission).
The phenotype that mAbs inhibit virus release was observed for viruses from a number of
The phenotype that mAbs inhibit virus release was observed for viruses from a number of
familiesincludinginfluenzavirus(IAV)[60,61],bovineleukemiavirus[62],herpesvirus[63],vaccinia
families including influenza virus (IAV) [61,62], bovine leukemia virus [63], herpes virus [64],
virus[64],rubellavirus[65],andMarburgvirus(MARV)[66]. MostofthosestudiesutilizedmAbs
vaccinia virus [65], rubella virus [66], and Marburg virus (MARV) [67]. Most of those studies utilized
or anti-sera that do not independently neutralize virus entry and did not resolve the mechanisms
mAbs or anti-sera that do not independently neutralize virus entry and did not resolve the
underlyingvirusreleaseinhibition. Thin-sectionTEMstudieswithtwonon-NAbsagainstMARV[66]
mechanisms underlying virus release inhibition. Thin-section TEM studies with two non-NAbs
and a broadly reactive mAb against IAV [61] suggested that those mAbs tether budded virions to
against MARV [67] and a broadly reactive mAb against IAV [62] suggested that those mAbs tether
viralGPsonthecellsurface. Thebuddinginhibitionfunctionofanti-CHIKVmAbsdescribedinthis
budded virions to viral GPs on the cell surface. The budding inhibition function of anti-CHIKV mAbs
sectionisclearlydistinctfromthetetheringmechanismformAbsagainstMARVandIAV.Itisprobably
described in this section is clearly distinct from the tethering mechanism for mAbs against MARV
and IAV. It is probably unique for alphavirus as lateral organization of viral spikes into an icosahedral
lattice drives alphavirus assembly/budding on the cell surface; therefore, alphavirus budding is
targeted by humoral immunity.
Viruses2019,11,305 9of17
uniqueforalphavirusaslateralorganizationofviralspikesintoanicosahedrallatticedrivesalphavirus
assembly/buddingonthecellsurface;therefore,alphavirusbuddingistargetedbyhumoralimmunity.
5. Antibody-ActivatedEffectorFunctions
ThroughinteractionwithFcreceptorsexpressedonallinnateimmunecells,mAbsmediatean
arrayofeffectorfunctionsthatareimportantcontributorstoprotectiveimmunity[67]. Theseinnate
immuneeffectorfunctionsincludeantibody-dependent,cell-mediatedphagocytosis(ADCP)ofvirions
and infected cells by monocytes, macrophages and neutrophils; direct killing of infected cells via
antibody-dependent, cellular cytotoxicity (ADCC) by NK cells and complement activation [68].
MountingevidencesuggeststhatFc-effectorfunctionselicitedbymAbsplayacriticalroleinprotection
againstinfection[69–72]. HumanshavesixclassicalFcreceptors(FcγRI,FcγRIIa,FcγRIIb,FcγRIIc,
FcγRIIIa and FcγRIIIb) expressed in varied combinations on all innate immune cells at different
levels[73]. OfthesixFcγRs,FcγRIIbisthesoleinhibitoryreceptor. ThecellularoutcomeofIgG-FcγR
interactionsisdeterminedbytheaffinityoftheFcforthespecificreceptorandtheexpressionpattern
of FcγRs on effector cells [74]. Classical FcγRs bind close to the hinge region of IgG [75–77] and
activatingreceptorssignalthroughanimmunoreceptortyrosine-basedactivationmotif(ITAM)either
inthecytoplasmicdomainoronthecommongammachainthatclustersduringactivation,initiating
signalingcascadesthatactivateimmunecellstoinduceeffectorfunctions[78]. ExceptforFcγRI,all
the other FcγRs exhibit low affinity for IgG and therefore are unable to bind monomeric IgG [67].
IgG-antigencomplexesareabletosufficientlyengageandcrosslinklow-affinityFcγRsviamulti-valent,
highavidityinteractionsleadingtosignalingfromoligomerizedFcγRs[79,80]. Asdescribedabove,
NAbsC9andIM-CKV063inducedcoalescenceofviralGP-NAbcomplexesontheplasmamembrane
of CHIKV-infected cells leading to inhibition of virus budding [41]. The dense layer of coalesced
NAb-GPcomplexesonthesurfaceofCHIKV-infectedcellscouldpresentmulti-valentFctoFcγRs
andpotentlyactivateADCCfromeffectorcellsthatwereengineeredtoexpresstheactivatingFcγR,
FcγRIIIa[41]. ThisissupportedbythefindingthatbivalentbindingofNAbstoviralGPsisrequired
forthepotentactivationofADCC(dataunpublished). Overalltheseresultssuggestthatmotilityof
antigenontheplasmamembraneinducedbybivalentbindingofIgGscontributestopotentADCC
activation. An assay targeting virus-infected cells is better suited to detect this ADCC activation
mechanismcomparedtoassaysbasedonantigenimmobilizedonplates[72,81–83].
Very recently, Fox et al reported that optimal therapeutic activity of mAbs against CHIKV
requires Fc-FcγR interaction on monocytes [38]. Humanized CHK-152 and CHK-166 of human
IgG1 subclass, but not their N297Q mutants that do not interact with FcγR and C1q, were able to
promoteimmunecellinfiltration,viralclearanceandreducemousefootswellingwhenadministrated
at 3 days post-infection (about the time of peak viremia) [38]. Similar protection by these mAbs
was observed in mice lacking C1q as in wild type mice, but not in mice lacking the Fc receptor
commonγchainthatabrogatesexpressionofactivatingFcγRs,suggestingthatFc-FcγRinteractions,
ratherthancomplement,mediatetheclinicalandvirologicalprotection[38]. Exvivophagocytosis
experimentswithCHIKVp62-E1-coatedbeadsdemonstratedADCPbyprimarymurinemonocytes
andneutrophils[38]. ForGP-icosahedralalphaviruses,thedensityandquaternarystructureofGPson
virionsandthesurfaceofinfectedcellsmaybedifferentfromthatoftherecombinantproteincoated
beads. ItwillbeinterestingtotestADCPofnativevirionsandvirus-infectedcells. Veryimportantly,in
thisstudy,themAb-mediatedprotectionwasdemonstratedtodependonmonocytes,notneutrophilsor
NKcells,byexperimentswithmicedepletedofindividualcelltypes[38].BecausetheNKcellistheonly
innateimmunecellsolelyexpressingactivatingFcγR,RcγIIIa,dogmahasdictatedthatNKcellsare
themajormediatorsofADCCinhumans[84]. UsingFcγR-humanizedmicethatexpressthefullarray
ofhFcγRsonabackgroundlackingallmurineFcγRs[85],DiLilloandRavetchhavedemonstratedthat
macrophagesmediateADCCofantibody-coatedtargetcellsinvivointhecontextofhuIgG1antibody
andthehumanFcγRsystem[86].Itwillbeinterestingtotestprimarymonocyte/macrophagemediated
ADCCofmAb-coatedCHIKV-infectedcellsanddetermineFcγRsexpressedonmonocyte/macrophage
Viruses2019,11,305 10of17
that signal the effector functions. Optimizing the interactions between Fc and specific FcγRs will
furtherimproveinvivoprotectionbymAbs[87–89].
6. NeutralizingIgMmAbs
Theanti-CHIKVmAbsdescribedsofarareallIgGsthatrepresentapproximately75%ofserum
antibodiesinhumans. BeforetheIgGresponse,inductionofIgMconfersprotectionagainstinfectionof
WestNilevirus[90],rabiesvirus[91],influenzavirus[92],smallpoxvirus[93]andrelatedalphaviruses
SFV [94] and Sindbis [95]. A potent neutralizing IgM against CHIKV, 3E7b, was reported [11].
3E7b targets N218 on E2 domain B and blocks virus binding to susceptible cells [11]. It potently
neutralizesCHIKVentrywithanIC of4.5ng/mL[11],comparablewiththatofthestrongesthuman
50
neutralizingIgG,5M16,3.4ng/mL[37]. ThepentamericstructureofIgMmayrenderitpotentfor
crosslinkingandcoalescingGPsonCHIKV-infectedcells,thereforestronglyinhibitvirusbudding.
Inaddition,IgMisoftenconsideredtobethemostpotentimmunoglobulinincomplementactivation
throughtheclassicalpathway[96]. ItwillbeinterestingtotestifneutralizingIgMscaninhibitCHIKV
buddingandactivatethecomplementresponsetoclearvirus-infectedcells.
7. DevelopmentofAntibodyTherapeuticsforCHIKV-Infection
Treatment with a number of mAbs in immunocomprised animal models, neonatal mice [9],
Ifnαr−/− mice [5,8,10], or AG129 mice [39], protects against lethal CHIKV challenge when
administrated24hoursbefore,orupto60hourspost-infection. Whenadministeredaspost-exposure
therapy,anumberofmAbslimitedviralspreadtojointsandmusclesandreducedtissueinflammation
in wild-type mice [7,8,38,97] and in rhesus macaques [8,97,98]. Acute CHIKV infection in mice or
rhesusmacaquesinducesviremiathatlastsforapproximately3–4days,withpeakviraltitersdetected
at 2- or 3-days post-infection [50,99,100]. In humans the acute viremic phase of CHIKV-infection
lasts4–12dayspost-symptomonset[101]. HighCHIKVviremiaisassociatedwithincreasedseverity
of illness [99,102]. Treatment with anti-CHIKV mAbs in animal models during the viremic phase
successfully reduces virus dissemination to the distal joints and tissues [38,97,98]. However, the
treatmentwindowisnarrow. Futurestudiesareneededtotestifso-faridentifiedprotectivemAbsare
abletoreducetissueinflammationorpreventCHIKVpersistencewhenadministratedpost-viremia.
Developingbetteranimalmodelsthatfullyreproducethechronicrheumatoidsyndromefollowing
CHIKFisurgentlyneeded.
ThehighmutationrateofalphviruseshelpsvirustoescapefrommAbtherapeutics,especially
when administrated during the acute phase because the high viral load increases the chance for
pre-existing or selected resistant variants to emerge. Combination of mAbs that target different
antigenicsites,eg. CHK-152andCHK-166,limitstheselectionofescapemutantsandprovidesefficient
protection [8,98]. Alternatively, if mAbs target an epitope that is critical for virus growth, fitness
andviralpathogenesis,escapingmutationwillbelethalforthevirusorattenuateviralpathogenesis.
Undertheseconditions,singlemAbtreatmentisabletoprotectthehosteffectively. Forexample,the
escapemutanttoIM-CKV063hasasingleWtoGmutationataminoacid64ofE2domainAthatis
criticalforreceptorMxra8binding[7]. W64GmutantCHIKVfailstocausedeathinneonatalmice
andresultsinlessseverejointdiseasecomparedtowild-typeCHIKV[7]. IM-CKV063administrated
post-infection provides superior protection against CHIKV-induced arthritis in 3-weeks-old mice
comparedtoapanelofNAbsincludingCHK-152andCHK-166[7].
In recent years, technological advances in high-throughput B cell isolation, transgenic
animal strains hosting human immunoglobulin sequences, antibody display platforms and
antibody engineering have drastically accelerated the discovery and optimization of antiviral
mAbs. Many excellent reviews on these new technologies have been published recently [103,104].
Withadvancedtechnologies,morehighlypotentandbroadlycross-reactivehumanmAbswillsoonbe
addedtoourcurrentarsenaltofightCHIKVandotheralphaviruses.