Deconstructing the Antiviral Neutralizing-Antibody Response

22 Pages·2016·3.1 MB·English

crossmark
Deconstructing the Antiviral Neutralizing-Antibody Response:
Implications for Vaccine Development and Immunity
LauraA.VanBlargan,LeslieGoo,TheodoreC.Pierson
ViralPathogenesisSection,LaboratoryofViralDiseases,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,Bethesda,Maryland,USA
SUMMARY..................................................................................................................................................989
INTRODUCTION............................................................................................................................................989
FLAVIVIRUSESANDHIV-1:CLINICALLYSIGNIFICANTCONTEXTSFORSTUDYINGTHEHUMORALIMMUNERESPONSE................................990
Flaviviruses...............................................................................................................................................990
HumanImmunodeficiencyVirusType1.................................................................................................................992
D
HUMORALIMMUNERESPONSE............................................................................................................................993
o
DiversityoftheHumoralImmuneResponse.............................................................................................................993
w
Germinalcenterformationandaffinitymaturation....................................................................................................993 n
EffectorFunctionsofAntibodies.........................................................................................................................993 lo
WhereDoAntiviralAntibodiesComefrom?..............................................................................................................994 a
ANTIBODY-MEDIATEDNEUTRALIZATION.................................................................................................................994 d
e
NeutralizationbytheNumbers...........................................................................................................................995 d
Epitopeoccupancyandneutralization................................................................................................................995
f
MechanismsofNeutralization............................................................................................................................995 ro
VIRUSEVASIONOFANTIBODY-MEDIATEDNEUTRALIZATION ...........................................................................................996 m
SequenceVariationandAntigenicDiversity..............................................................................................................996
h
ConformationalMaskingofConservedRegions..........................................................................................................997 t
t
Regulationofepitopeaccessibilitybyflaviviruses.....................................................................................................997 p
LowDensityofSurfaceGlycoproteins....................................................................................................................997 :/
/
GlycanShields............................................................................................................................................997 m
IDENTIFYINGTHEFUNCTIONALCOMPONENTSOFTHEPOLYCLONALANTIBODYRESPONSE..........................................................998 m
NeutralizingAntibodiesAreaRareComponentoftheHumoralResponseagainstViralInfection.......................................................998 b
NeutralizingAntibodiesTargetaLimitedNumberofSpecificities.......................................................................................998 r.
a
EpitopeSpecificitiesofNeutralizingAntibodiesInformVaccineDesign.................................................................................999 s
Accessibleepitopes:easytargets......................................................................................................................999 m
Crypticepitopes:dynamictargets.....................................................................................................................999 .
o
Quaternaryepitopes:complextargets...............................................................................................................1000 r
g
CONCLUDINGREMARKS..................................................................................................................................1000
/
ACKNOWLEDGMENTS....................................................................................................................................1001 o
REFERENCES...............................................................................................................................................1001 n

A
p
r
il
SUMMARY INTRODUCTION 7
,

The antibody response plays a key role in protection against viral In the evolutionary arms race between viruses and their hosts, 20
infections.Whileantiviralantibodiesmayreducetheviralburdenvia virusesexploitcomparativelyhighmutationrates,shortgener- 1
9
severalmechanisms,theabilitytodirectlyinhibit(neutralize)infec- ation times, and large population sizes to adapt to antiviral de-
b
tion of cells has been extensively studied. Eliciting a neutralizing- fenses. In jawed vertebrates, the immune response to infection y

antibodyresponseisagoalofmanyvaccinedevelopmentprograms includestheproductionofantibodies(Abs)capableofrecogniz- g
u
andcommonlycorrelateswithprotectionfromdisease.Considerable inganextremelydiversearrayofantigens,includingproteinsand e
s
insightsintothemechanismsofneutralizationhavebeengainedfrom carbohydratesthatdecoratevirusparticles.Antibodiesareglyco- t
studiesofmonoclonalantibodies,yettheindividualcontributions proteinsofthehumoralimmunesystemwithanantigenrecogni-
anddynamicsoftherepertoireofcirculatingantibodyspecificities tion surface typically composed of two polyprotein chains en-
elicitedbyinfectionandvaccinationarepoorlyunderstoodonthe coded by a complex and dynamic array of gene segments. The
functional and molecular levels. Neutralizing antibodies with the extraordinarily broad and adaptive binding specificities of anti-
mostprotectivefunctionalitiesmaybeararecomponentofapoly- bodiesareachievedthroughallelic,combinatorial,andjunctional
clonal,pathogen-specificantibodyresponse,furthercomplicatingef-
fortstoidentifytheelementsofaprotectiveimmuneresponse.This
reviewdiscussesadvancesindeconstructingpolyclonalantibodyre- Published26October2016
sponsestoflavivirusinfectionorvaccination.Ourdiscussionsdraw CitationVanBlarganLA,GooL,PiersonTC.2016.Deconstructingtheantiviral
neutralizing-antibodyresponse:implicationsforvaccinedevelopmentand
comparisonstoHIV-1,aviruswithadistinctstructureandreplica-
immunity.MicrobiolMolBiolRev80:989–1010.doi:10.1128/MMBR.00024-15.
tioncycleforwhichtheantibodyresponsehasbeenextensivelyinves-
AddresscorrespondencetoLeslieGoo,[email protected],or
tigated.Progresstowarddeconstructingandunderstandingthecom- TheodoreC.Pierson,[email protected].
ponentsofpolyclonalantibodyresponsesidentifiesnewtargetsand Copyright©2016,AmericanSocietyforMicrobiology.AllRightsReserved.
challengesforvaccinationstrategies.
December2016 Volume80 Number4 MicrobiologyandMolecularBiologyReviews mmbr.asm.org 989
VanBlarganetal.
diversity of antibody gene segments (reviewed in reference 1). theflavivirusgenus.Toidentifygeneralconcepts,selectedinsights
Additionally,followinginfectionorvaccination,iterativerounds into the anti-HIV-1 antibody response are presented; excellent
ofsomaticmutationofantibodygenesandanaffinity-basedse- detailed reviews of the HIV-1 antibody response were reported
lectionprocessproduceantibodiesthatbindwithhighaffinityto previously(10,13–15).Bothgroupsofviruseshaveaglobalim-
viralantigens. pactonpublichealthandvaryconsiderablyinstructureandrep-
Antibody-mediatedneutralizationofvirusesisthedirectinhi- licationstrategies.
bitionofviralinfectivityresultingfromantibodydockingtovirus
particles(reviewedinreference2).Theelicitationofaneutraliz- Flaviviruses
ing-antibody(NAb)responseisacorrelateofprotectionformany TheFlavivirusgenusofthefamilyFlaviviridaeconsistsofadiverse
vaccines and contributes to long-lived protection against many groupofpositive-strandedRNAvirusestransmittedtovertebrate
viralinfections(3).Apotentantiviralresponsemayselectforvari- hostsprincipallybymosquitoortickvectors.Thisgenusof(cid:2)70
antsthatallowescapefromantibodyneutralizationand/oreffec- species includes several viruses of considerable clinical impor-
torfunctions.Neutralizationescapemechanismsarediverseand tance,includingdenguevirus(DENV),yellowfevervirus(YFV),
includetheselectionofaminoacidvariationinantibodyepitopes WestNilevirus(WNV),Zikavirus(ZIKV),Japaneseencephalitis
directlyaswellasthemodulationofstructuralfeaturestoprevent virus (JEV), and tick-borne encephalitis virus (TBEV). An esti- D
o
antibody binding. Several exciting experimental approaches to mated390millionhumansareinfectedeachyearwiththefour
w
overcomethesechallengeswererecentlydescribedforseveralviral serotypesofDENValone(16).Flavivirusesarecapableofrapid n
systems (reviewed in reference 4). Defining the specificities of emergenceandspreadinnonimmunepopulations,asillustrated lo
a
NAbselicitedbyinfectionorvaccinationallowedtheidentifica- by the extremely rapid spread of ZIKV through South America d
tionofBcelllineagesthatleadtotheproductionofpotentanti- followingitsintroductionin2015(17).Therelativelyrecentin- e
d
bodies (reviewed in reference 5). Additionally, careful study of troduction and spread of WNV throughout the Western hemi-
f
r
anti-humanimmunodeficiencyvirustype1(HIV-1)antibodies spherearewelldocumented(18).Flaviviruseswereinitiallyclas- o
m
withanabilitytorecognizeabroadrangeofdiverseviralisolates sified by using serological studies, which were subsequently

identifiedconservedstructuresoftheviralenvelopeproteintar- confirmedandextendedviaphylogeneticanalyses(19,20).Viral h
t
t
getedbyhumanNAbs(6).Insightsintoepitopesboundbyfunc- specieswithintheflavivirusgenusgenerallyshareover84%nu- p
:
tionallydesirableantibodiesmayguidethedesignofimmunogens cleotidesequenceidentity(20).However,althoughthefoursero- //
m
thatcanelicittheseresponses(7). typesofdenguevirus(DENV1to-4)shareaspeciesclassification,
m
Significantprogresstowardunderstandingthemolecularand theydifferby~25%to~40%intheiraminoacidsequences(Fig.
b
structural basis for antibody-mediated neutralization has been 1A)(21). r
.
a
made for several clinically important viruses through studies of Flavivirus virions are small, spherical, enveloped particles s
monoclonal antibodies (MAbs) (4, 8–11). How antibodies with roughly50nmindiameterthatarecomposedofthreestructural m
.
different functional properties act in concert in human sera is proteins:capsid(C),premembrane(prM),andenvelope(E).The o
r
poorly understood. Despite the fundamental and translational assemblyofnewvirionsisdirectedbyprMandEproteinsatthe g
/
importanceofcharacterizingpolyclonalantibodies,manyimpor- endoplasmicreticulum(22–24).Onimmaturevirions,thesegly- o
tantquestionsremain:(i)howmanyepitopesonavirusaretar- coproteinsareincorporatedas60icosahedrallyarrangedhetero- n

A
getedbytheneutralizing-antibodyresponse,(ii)howmanyBcell trimericspikesofthreeprM-Edimers(25–28).TheroleofprMon
p
clonal lineages are expanded during infection and vaccination, immaturevirionsistopreventlow-pH-triggeredconformational r
and(iii)howdoesthecomplexityofpolyclonalantibodymixtures changesintheEproteinsthatdrivethefusionofviralandcellular il 7
correlatewithprotectionagainstdiverseandrapidlymutatingvi- membranes(29).Asimmaturevirionstrafficthroughthesecre- ,
2
ruses?Inthisreview,wefocusonadvancesineffortstotranslate torypathway,theyundergoamaturationprocessdefinedbythe 0
1
reductionistconceptsarisingfromstudiesofMAbstowarddecon- cleavage of prM by cellular furin-like proteases (30–32). This 9
structingthepolyclonalneutralizing-antibodyresponsetoflavivi- cleavage results in an (cid:2)75-amino-acid M peptide that remains b
ruses. associatedwiththematurevirionandan(cid:2)90-amino-acid,solu- y
g
ble“pr”portionthatdisassociatesfromvirusparticlesuponre- u
FLAVIVIRUSESANDHIV-1:CLINICALLYSIGNIFICANT leasefromcells(25).ThefunctionofMonthemature,infectious e
s
CONTEXTSFORSTUDYINGTHEHUMORALIMMUNE virionisunknown.Onmaturevirions,Eproteinsexistinadense t
RESPONSE herringbonearrangementofantiparallelEproteinhomodimers
Thevirusestargetedbyantibodiescomeinmanyshapesandsizes (Fig.1BandC)(33–38).Inthisconfiguration,Eproteinslieflat
and replicate within the host via numerous strategies. In many againstthesurfaceoftheviralmembrane,incontrasttoHIV-1
respects,thesedifferencesarereflectedinthedetailsofantibody- andmanyotherviruses,whoseenvelopeproteinsexistasspikes
virusinteractionsormechanismsofinhibition.Forexample,the thatprojectawayfromthevirionsurface(Fig.2C).Critically,the
neutralizationpotencyofantibodiesagainstsomenonenveloped flavivirusmaturationprocessisnotefficient,resultinginthepro-
virusesisenhancedbyinteractionswithTRIM21inthecytoplasm ductionofaheterogeneouspopulationofvirions.Thus,inaddi-
ofinfectedcells(12).Incontrast,thismechanismofinhibitionis tiontoinfectious,fullymaturevirions(noprM)andnoninfec-
unlikelytoplayasignificantroleinpotentiatingantibodyfunc- tious, immature virions (180 uncleaved prM molecules), cells
tionforviruseswithalipidenvelopeonwhichtheprincipaltargets producepartiallymaturevirusesthatretainstructuralfeaturesof
ofNAbsaretopologicallyinaccessibletoTRIM21.While exciting bothmatureandimmaturevirusparticles(39).Partiallymature
advanceshavebeenmadetowardunderstandinghowantibodies virions can be infectious, although the extent of prM cleavage
protect against infection by many different viruses, this review requiredforinfectivityisnotknown(reviewedinreference39).
focusesontheneutralizing-antibodyresponseagainstmembersof The efficiency of virion maturation has the potential to impact
990 mmbr.asm.org MicrobiologyandMolecularBiologyReviews December2016 Volume80 Number4
DissectingAntiviralNeutralizing-AntibodyResponses
D
o
w
n
lo
a
d
e
FIG1Structureanddiversityofthesurfaceglycoproteinsofflaviviruses.(A)DendrogramdepictingtherelatednessofselectedflavivirusEproteins(thebar d

represents0.1aminoacidsubstitutionspersite).JEV,Japaneseencephalitisvirus;MVEV,MurrayValleyencephalitisvirus;WNV,WestNilevirus;SLEV,Saint fr
Louisencephalitisvirus;TBEV,tick-borneencephalitisvirus;POWV,Powassanvirus;YFV,yellowfevervirus;DENV,denguevirus.(B)Structureofthe o
m
ectodomainoftheflavivirusEproteindimer(PDBaccessionnumber1OAN)fromasideview(top)andtopview(bottom).DomainsI,II,andIIIareshownin

red,yellow,andblue,respectively.ThefusionloopindomainIIisshowningreen.(C)Structureofamatureflavivirusvirion(PDBaccessionnumber4CCT). h
TheEproteinisarrangedasantiparallelhomodimersthatdenselycoatthevirionsurface.(D)ThetwopossibleglycansontheEproteinarehighlightedinred tt
p
intheEproteindimer(PDBaccessionnumber1OAN)fromasideview(top)andtopview(bottom). :
/
/
m
virus binding, environmental conditions required to trigger protein is composed of three domains (domain I [DI] to DIII) m
membranefusion,cellulartropism,andsensitivitytoantibody- connectedtotheviralmembranebyahelicalstemandtwotrans- b
r
mediatedneutralization(40–44). membrane domains (Fig. 1B ) (46). A highly conserved, hydro- .a
TheEproteinisthemaintargetofNAbs(45).Thiselongated phobicfusionloopcomposedof13aminoacidsislocatedatthe s
m
.
o
r
g
/

o
n

A
p
r
il
7
,

2
0
1
9

b
y

g
u
e
s
t
FIG2StructureanddiversityofthesurfaceglycoproteinsofHIV-1.(A)DendrogramdepictingtherelatednessofEnvfromHIV-1groupsM,N,O,andPandSIVcpz
(CPZ)(thebarrepresents0.1aminoacidsubstitutionspersite).(B)StructureoftheectodomainoftheHIV-1Envtrimericspike(PDBaccessionnumber4TVP),
consistingoftrimersofgp120/gp41heterodimers,fromasideview(left)andtopview(right).gp120andgp41areshowningrayandcyan,respectively.Ongp120,
variableloopsareshowningreen,whiletheCD4-bindingsiteisinpurple.(C)SchematicoftheHIV-1virion.Envexistsastrimericspikesthatsparinglypopulatethe
virionsurface(showninredandgray).Avarietyofcellularproteinsareincorporatedintothevirion(showninblue)(91).(D)TheHIVglycanshieldishighlightedin
redontheEnvtrimer(PDBaccessionnumber4TVP)fromasideview(left)andtopview(right).ThemodelwasgeneratedbyusingGlyProt(298).
December2016 Volume80 Number4 MicrobiologyandMolecularBiologyReviews mmbr.asm.org 991
VanBlarganetal.
tipofDII(DII-FL).Onmaturevirusparticles,thefusionloopis (91),thesoleviralproteincomplexonthesurfaceisEnv,which
buriedinafoldcomposedofDIandDIIIoftheopposingEpro- mediatesentryintohostcellsandisthetargetofNAbs.Following
teininthedimer(47).EproteinsmaycontainuptotwoN-linked translationintheendoplasmicreticulum,thegp160polyprotein
glycosylationsites(eitheronDIonlyoronbothDIandDII)(Fig. precursorofEnvisprocessedbyafurin-likeproteaseinthetrans-
1D);somestrainsofWNVarenonglycosylated(48,49).Neutral- Golgi network, resulting in the gp120 surface subunit and the
izingantibodieshavebeenmappedtoallthreeEproteindomains gp41transmembranesubunitthatremainnoncovalentlyattached
and,inmanyinstances,bindepitopescomposedofresiduesfrom toeachother(92).Onthevirionsurface,Envexistsastrimeric
multiple domains (50–61). Antibodies that recognize prM have spikes of gp120 and gp41 heterodimers (Fig. 2B). As discussed
alsobeenidentified,buttheyhavelimitedneutralizationpotential below,boththeweakassociationofgp120withgp41andthelow
(53,62–64). densityofEnvspikesonthevirionsurfaceimpactantibodyrec-
TheEproteinsorchestratetheentryofflavivirusesintotarget ognition.
cells,whichoccursthroughreceptor-mediatedendocytosis,fol- gp120 consists of five conserved regions (C1 to C5) and five
lowedbypH-dependentfusionthattypicallyoccursinlateendo- highlyvariableregions(V1toV5)andisstructurallydefinedby
somes,althoughtherelativelyhighpHthresholdof(cid:2)6.6suggests twodomains:oneorientedtowardthecenterofthetrimer(inner
thatfusionmayalreadyoccurinearlyendosomes(65–68).Cellu- domain)andtheotherorientedtowardtheperiphery(outerdo- D
o
lar factors that mediate virus entry are not completely defined, main)(93–98).Thenativestructureofgp41hasnotbeensolvedat w
althoughattachmentfactorsthatenhancevirionbindingtocells highresolution(95,96,99),butthissubunitisfunctionallyde- n
havebeenidentified,includingtheC-typelectinsDC-SIGNand lo
finedbysixmajorregions:thefusionpeptide,heptadrepeatre- a
DC-SIGNR, mannose receptor, heparin sulfate, and phosphati- gion 1 (HR1), HR2, the membrane-proximal external region d
dylserinereceptorsoftheTIM(Tcell/transmembrane,immuno- e
(MPER),thetransmembraneanchor,andthecytoplasmictail.On d
globulin,andmucin)andTAM(TYRO3,AXL,andMER)protein
gp120,variableregionsformheavilyglycosylateddisulfide-linked fr
families(40,69–75).Therolethatthesemoleculesplayinthecell o
loopsthatshieldmoreconservedsurfaces(Fig.2D)(100).Indeed, m
biologyofvirusentryisincompletelyunderstoodandmayextend
ahigh-resolutioncrystalstructureoftheHIV-1Envtrimersug-
beyondsimplyfacilitatingvirusattachment.Todate,cellularpro- h
geststhatduetothedensearrayofglycansthatdecorateEnv,only t
t
teins absolutely required for the low-pH-mediated conforma- p
3%ofthepeptidicsurfaceisaccessibletoantibodies,comparedto :
tionalchangeinthevirushavenotbeenidentified;flavivirusesare 14%and48%oftheglycoproteinsofinfluenzavirusandrespira- //m
capableoffusingdirectlywithsyntheticmembranepreparations
torysyncytialvirus,respectively(99).Nevertheless,NAbsagainst m
(66,76).Single-particletrackingstudiessuggestthatvirusentry
HIV-1havebeenmappedtobothgp120andgp41,andremark- b
occurswithin17minofstableattachmentofthevirion(67).In r
ably, glycans themselves are also important targets for many .a
vitrostudiesclearlydemonstratethatviralfusionoccursveryrap- s
idly(withinseconds)afterexposuretomildlyacidicconditions broadlyneutralizingantibodies(98,101).Despitereachinghigh m
titers, the initial wave of NAbs elicited by infection is typically .
(58,66,76). o
strainspecific,targetingexposedvariableloopsingp120andse- r
g
HumanImmunodeficiencyVirusType1 lectingformutationsthatleadtoNAbescape(102–107).Todate, /
o
HIV-1,alentivirusintheRetroviridaefamily,isthecausativeagent NAbstargetinggp41arelesscommonthanthosetargetinggp120, n
possiblyduetosterichindranceaffordedbythecloseproximityof A
ofAIDS.DevelopinganeffectivevaccineagainstHIV-1hasbeen
gp41totheviralmembrane(108),althoughNAbsthatbindgp41, p
challengingbecauseoftheextensivediversityofthevirus,which r
resultsfromtherapidturnoverofalargenumberofinfectedcells includingthefusionpeptide,andthegp41/gp120interfacewere il 7
(77,78),anerror-proneviralreversetranscriptase(79),andahigh characterizedrecently(109–114).ThelatterNAbsareanalogous ,
2
to antiflavivirus NAbs that target quaternary epitopes spanning
frequencyofrecombinationowingtotemplateswitchingbetween 0
multipleEproteinmonomers(describedbelow). 1
twocopackagedRNAgenomesduringreversetranscription(80). 9
HIV-1originatedfromsimianimmunodeficiencyvirusinchim- HIV-1primarilyinfectsCD4(cid:3)Tcellsandcellsofthemonocyte/ b
macrophagelineageinvivo.EntryispHindependentandoccurs y
panzees(SIVcpz)andisdividedintoseveralgroups(groupsM,N,
O, and P), each of which resulted from an independent cross- mainlyattheplasmamembranefollowingEnv-receptorinterac- gu
species transmission event (81). Group M, which is responsible tions.Otherentrypathways,includingendocytosisormacropi- e
s
fortheAIDSpandemic,isfurtherdividedinto9subtypes(sub- nocytosis,mayalsobeinvolved(reviewedinreference115).Bind- t
typesAtoD,FtoH,J,andK)andanumberofcirculatingrecom- ingofgp120toCD4isthoughttotriggerconformationalchanges
binantforms(82).Thesesubtypesdifferingeographicaldistribu- thatenhancetheaffinityofgp120foritscoreceptor(92),although
tion(83)andcanbedistinguishedbysequencevariationinthe arecentstudyfromtheMotheslaboratorysuggeststhattheen-
envelope gene, which differs by (cid:2)20% at the amino acid level sembleofstructuressampledbygp120includesthosesimilarto
withinsubtypesandupto35%betweensubtypes(Fig.2A)(82, theCD4-triggeredstate(116).Interactionwithacoreceptor(G
84).Remarkably,itisestimatedthatthediversityofHIV-1strains protein-coupledreceptorseven-transmembranedomainprotein
foundwithinasingleindividualatonetimepointcanrivalthatof CCR5orCXCR4)triggersfurtherconformationalchanges,lead-
worldwideinfluenzaviruseswithinayear(85). ingtoafusogenicstateinwhichgp120dissociates,allowingthe
HIV-1virionsaresphericalparticlesthatare(cid:2)120nmindiam- fusionpeptideofgp41toextendoutwardintothetargetcellmem-
eteranddisplayalowdensityofenvelope(Env)spikesontheir brane.Next,rearrangementsinHR1andHR2facilitatetherefold-
surface ((cid:2)14 Env trimers/virion) (Fig. 2B and C) (86–88). Env ingofgp41intoasix-helixbundlethatbringstogetherthecellular
incorporationintovirionsisregulatedbyinteractionswithother andviralmembranes,creatingaporethatallowstheviralcoreto
viralproteinsandhostfactors(reviewedinreferences89and90). entercells(117).ThenumberoffunctionalEnvtrimersneededto
WhileHIV-1virionsincorporatealargenumberofhostproteins interactwithtargetcellreceptorsandmediatevirusentryhasnot
992 mmbr.asm.org MicrobiologyandMolecularBiologyReviews December2016 Volume80 Number4
DissectingAntiviralNeutralizing-AntibodyResponses
beenpreciselydeterminedandmayvarybystrainbuthasbeen memoryBcells(MBCs)(discussedfurtherbelow)(125),although
estimatedtobebetweenoneandeight(88,118–121). memoryBcellsmayalsoarisefromGC-independentmechanisms
(126).
HUMORALIMMUNERESPONSE InGCs,Bcellsrefinetheantibodyresponseviatheprocessof
Aprincipalcomponentofthehumoralimmuneresponseisthe affinitymaturation.Thisprocessoccursthroughiterativerounds
repertoireofantibodymoleculessecretedbyBlymphocytes.An- ofsomatichypermutation,duringwhichpointmutationsarein-
tibodies are Y-shaped glycoproteins composed of two identical troducedintotheantibodyVregions,followedbyT cell-based
FH
heavy chain/light chain heterodimers linked by disulfide bonds selectionofcloneswiththehighestantibodyaffinity.Onestudy
(reviewedinreference122).Thearmsofantibodymolecules(or estimatedthattheaveragenumberofmutationsintheV region
H
Fabs) are connected to the remainder of the protein by flexible ofIgGamongmemoryBcellsandgerminalcentercellswas14,
hinges,whichdiversifytheangleswithwhichantibodiesmaybind with88%ofthesequencedV genesencodingbetween3and29
H
antigens.Thedistalendofeacharmformstheantigen-binding mutations(127).GCsaresitesofcompetitionamongBcellclones
siteofthemolecule,calledthevariable(V)region.Bothheavyand forTcellhelp.Aftermultipleroundsofaffinity-basedselection,
light chains contain three hypervariable loops (called comple- GCsmayundergo“monoclonalization,”withonehigh-affinityB
mentarity-determining regions [CDRs]) that form the V struc- cellclonebeginningtodominateanygivenmatureGC(reviewed D
o
turalregion.Theconstant(Fc)portionoftheantibodyismodified inreference128).InGCs,TFHcellsalsosignalBcellstoinitiate w
by an N-linked oligosaccharide that contributes to interactions classswitching(129–131). n
withmoleculesandcellsoftheimmunesystemtomediatearange lo
EffectorFunctionsofAntibodies a
ofeffectorfunctions(discussedbelow). d
While the Fab region of an antibody defines its specificity, the e
d
DiversityoftheHumoralImmuneResponse invariant Fc portion of the heavy chain determines its effector
f
r
Thehumoralresponseiscapableofproducinganincrediblydi- function.Theantibodyclass-switchingmechanismofBcellshas o
m
verserepertoireofantibodymoleculeswithuniqueantigen-bind- thepotentialtogenerateantibodieswithasimilarspecificityca-

ing properties. In part, this diversity is encoded directly by the pableoforchestratingdiverseimmuneresponses.TheFcregions h
t
t
germline.Genesencodingthevariableheavychain(V )andvari- of antibody heavy chains interact with Fc receptors (FcRs) on p
H :
ablelightchain(VL)existasmultiplegenesegments.Theheavy immuneeffectorcellsorsolubleimmunemoleculessuchasthose //m
chainisencodedbymultiplevariable(V),joining(J),anddiver- inthecomplementsystem(122).Thestrengthsoftheseinterac-
m
sity (D) gene segments. Two chromosomes encode the V and J tionsvaryamongantibodyclassesandcanbeinfluencedbythe
b
genesegmentsthatformthelightchain(the(cid:4)and(cid:5)loci).Full- particular carbohydrate modification on the antibody molecule r
.
a
length antibody molecules are assembled from these gene seg- (132,133).AllIgGsubclassesencodeanN-linkedglycosylation s
ments in developing B lymphocytes by a process called V(D)J siteatresidue297oftheheavychain.Whilethepositionofthe m
.
recombination(reviewedinreference1).Thismechanismcreates glycosylationsiteisconserved,thecompositionandstructureof o
r
combinatorialdiversitythroughtherandompairingofVDJgene theoligosaccharideaddedtotheantibodyareinfluencedbythe g
/
segments(attheVHloci)orVJgenesegments(attheV(cid:4)orV(cid:5)loci) hostimmuneactivationstate(134,135).Variationintheantibody o
duringsomaticgenerearrangement.Additionaldiversityduring glycoform provides a mechanism by which antibody effector n

A
V(D)J recombination arises by the introduction or deletion of function can be fine-tuned beyond class switch recombination.
p
nucleotidesatthejunctionofsegmentsastheyarelinkedtogether. Forexample,theIgGrepertoireofHIV-1-infectedindividualsis r
Therecombinedvariableregionoftheheavychainisthenjoined modifiedbysugarswithanagalactosylated,proinflammatorygly- il 7
to(cid:6)and(cid:7)constantgenesegments(tomakeIgMorIgDantibody can profile compared to those of uninfected individuals; this is ,
2
classes,respectively).Randompairingofheavyandlightchains particularlypronouncedinelitecontrollersofHIV-1(136).The 0
1
resultsintheformationofanintactantibodymolecule(andBcell skewingtowardagalactosylatedantibodiesininfectedindividuals 9
receptor[BCR]).Theprocessofallelicexclusionensuresthateach andelitecontrollerswascorrelatedwithincreasedantibody-de- b
y
lymphocyteproducesonlyasingleantibodymolecule(reviewed pendentcellularviralinhibition(ADCVI)activityinvitro.
g
inreference123). Nonneutralizingantibodieselicitedbyimmunizationorinfec- u
Germinalcenterformationandaffinitymaturation.Thean- tionmayofferprotectionfromviralinfectionthroughFc-medi- e
s
tibodyrepertoireproducedbyBlymphocytesisrefinedanddiver- ated effector functions, including antibody-dependent cellular t
sifiedfurtheruponexposuretoanantigen.WithappropriateT cytotoxicity (ADCC), opsonization, mast cell activation, and
cellhelp,Bcellrecognitionofanantigenresultsincellularactiva- complement activation. Studies using murine models of both
tion, extensive proliferation, and potentially Ig class switching, WNVandHIV-1infectionhavedemonstratedtheimportanceof
during which the Fc portion of the antibody gene can be ex- theFceffectorfunctionsofantibodiesinmediatingprotectionin
changed for another with different functional properties (124). vivo(137–140).Forexample,anti-WNVMAbswithpoorinvitro
Antigen-primedBcellscandevelopintoshort-livedplasmacells neutralizingabilitiescanmediateinvivoprotectioninmiceina
(PCs),whichareterminallydifferentiatedcellscharacterizedby manner dependent on IgG binding to complement component
high-levelantibodysecretionandlow-levelBCRexpression.PCs C1q and Fc(cid:8) receptors via the N-linked glycan at N297 (137).
and their proliferating precursors, plasmablasts, are responsible Conversely,theinabilityofstronglyneutralizingMAbswithmu-
fortheproductionoftheearlyantibodyresponse(Fig.3).Anti- tationsatN297tointeractwithFc(cid:8)receptorsmaybeexploitedfor
gen-primedBcellscanalsoparticipateintheformationofgermi- thedevelopmentofDENVtherapeutics,asthesenonglycosylated
nalcenters(GCs)inlymphnodes,alongwithfollicularhelperT NAbsdonotsupport(andmaycompetitivelyinhibit)theanti-
(T )cellsandfolliculardendriticcells(FDCs).GCreactionsre- body-dependentenhancementprocessesthoughttocontributeto
FH
sult in the production of long-lived plasma cells (LLPCs) and severediseaseoutcomes(141,142).
December2016 Volume80 Number4 MicrobiologyandMolecularBiologyReviews mmbr.asm.org 993
VanBlarganetal.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
m
b
r
.
a
FIG3Sourcesofantibodies.UponnaiveBcellrecognitionofanantigenandactivationbyacognateTcell,activatedBcellsarecharacterizedbyextensive s
proliferation.ActivatedBcellscanthenfollowoneofseveralpaths:(i)theymayterminallydifferentiateintoshort-livedplasmacells(PCs),whichhavelow m
surfaceIglevelsandhighIgsecretionrates;(ii)theymaydifferentiateintomemoryBcells(MBCs),whichretainBCRexpressionbutdonotconstitutivelysecrete .
o
antibody;and(iii)theycanparticipateintheformationofgerminalcenters(GCs),alongwithfollicularhelperT(TFH)cellsandfolliculardendriticcells(FDC). rg
InGCs,Bcellsundergorapidproliferation,furtherdiversificationoftheirantibodygenethroughsomatichypermutation,andclassswitchrecombination(CSR),
/
duringwhichtheFcregionoftheIggenemaybeexchangedforanothertodetermineantibodyeffectorfunction.SelectedGCBcellsreceivesignalstodifferentiate o
intoPCsorMBCs;otherGCBcellsundergoapoptosis.PCsmaybeshort-livedandremaininthelymphoidorgansorbecomelong-livedplasmacells(LLPCs) n

andmigratetothebonemarrow,wheretheycontinuetosecreteantibodyindependentofthepresenceoftheantigen.LLPCsaremostlikelyresponsibleforthe A
long-lived,pathogen-specificantibodytitersinserumthatcanlastyearsordecadesfollowinginfectionorvaccination.DistinctfromLLPCs,MBCsarelong-lived p
r
cellsthatremainincirculationandperipherallymphoidtissue.ThroughexpressionoftheirBCR,theycanbereactivatedbyanantigen.Uponrestimulation,they il
maysetupgerminalcenters,undergofurthersomatichypermutationandclassswitching,anddifferentiateintoantibody-secretingplasmablastsandPCs. 7
,

2
0
WhereDoAntiviralAntibodiesComefrom? maturationoftheantibodiesthattheyexpress(147).OnlyBcells 1
9
Long-termhumoralimmunityresultsfromatleasttwodistinct capableofproducingantibodieswithahighaffinityfortheanti- b
cell populations (Fig. 3). Terminally differentiated LLPCs pre- genareselectedforLLPCformationandpersistentantibodypro- y
g
dominantlyresideinthebonebarrowandconstitutivelysecrete duction,whereasBcellsthatproduceantibodieswithaloweraf- u
antibodyindependentlyofthepresenceofanantigen.LLPCsdo finityfortheantigenmaysurviveasMBCs(147,148).Antibodies e
s
not possess antigen receptors, are not reactivated upon antigen from these two compartments may differentially contribute to t
reexposure, and are most likely responsible for the long-lived, protectionfrominfection.Purthaetal.(149)demonstratedthat
pathogen-specificserumantibodiesthatcanlastyearsordecades following infection of mice with WNV, antibodies from MBCs
followinginfectionorvaccination(143,144).Incontrast,MBCs wereabletorecognizenotonlytheinfectingstrainofWNVbut
remainincirculationandperipherallymphoidtissue,wherethey alsoavariantencodingamutationinaknownneutralizing-anti-
mayreencounterantigen(145).MBCsexpressBCRontheirsur- bodyepitope.Incontrast,theLLPCantibodyresponsewasspe-
facebutdonotconstitutivelysecreteantibody.Uponrestimula- cificfortheinfectingstrain.Thus,whiletheLLPC-derivedanti-
tionbyantigen,MBCsmaydifferentiateintoantibody-secreting bodyresponsewasofahigheraffinityandcapableofconferring
plasmacellsandmayformgerminalcenterstoundergofurther immediate protection against homotypic viral reinfection, the
affinitymaturationandclassswitching(146).MBCsarerespon- MBCcompartmentmaybecriticalfortherecognitionofagenet-
siblefortheanamnesticantibodyresponsethatoccursuponsec- icallydiversesecondarychallenge.
ondaryexposuretoanantigen,respondingmorerapidlyandata
greatermagnitudetoantigenicstimulationthantheirnaivepre- ANTIBODY-MEDIATEDNEUTRALIZATION
decessors. Howantibodiesblockinfectionhasbeenstudiedextensivelyfor
LLPCsandMBCsdifferwithrespecttotheextentoftheaffinity decades(reviewedinreferences150and151).BecauseNAbshave
994 mmbr.asm.org MicrobiologyandMolecularBiologyReviews December2016 Volume80 Number4
DissectingAntiviralNeutralizing-AntibodyResponses
greatpotentialasantiviraltherapeutics,andNAbtitersoftencor- tiousvirionprovidesthe“denominator”forthisrelationship,as
relate with protection following vaccination (3), structural and epitopeaccessibilityultimatelygovernsthenumberofantibody
molecularinsightsintomechanismsofneutralizationhavecon- moleculescapableofbindingthevirusparticleatsaturation.An-
siderabletranslationalvalue.Recenttechnicaladvancesthaten- tibodiesmaybindepitopesdefinedbyaminoacidcontactsfroma
able the isolation and characterization of human monoclonal singleviralstructuralprotein(167)orengageresiduesfoundon
antibodies have not only accelerated the development of thera- adjacentproteinsorcarbohydrates(61,110–113,168–172).Com-
peuticsanddiagnosticsbutalsodirectedmechanisticstudiesto- plexorquaternaryepitopeshavebeendefinedformanyviruses,
ward antibodies with the most relevant specificities in vivo (4, includingflaviviruses(seebelow).
152). Thefractionofepitopesthatantibodiesmustoccupyinorder
toexceedastoichiometricneutralizationthreshold(thefractional
NeutralizationbytheNumbers “occupancy”) may differ among antibodies as a function of the
Howmanyantibodiesarerequiredtoneutralizeavirus?Thestoi- numberoraccessibilityofepitopesonthevirusparticle.Forfla-
chiometryofantibody-mediatedneutralizationhasbeenintensely viviruses,epitopeaccessibilitymayvaryasafunctionoftheposi-
debated(reviewedinreferences2and153).A“single-hit”model tionofaparticularEproteinonthepseudoicosahedralviruspar-
suggeststhatantibodybindingtoavirusparticleintherightloca- ticle (167, 173). All else being equal, antibodies that bind D
o
tionissufficienttoinactivatethevirion.Thishypothesiswassup- abundanttargetsneedtobindasmallerfractionoftheseepitopes
w
ported largely by kinetic neutralization studies of poliovirus, inordertoinhibitinfection;theseantibodieshavearelativelylow n
Westernequineencephalitisvirus,andinfluenzavirus(154,155). occupancyrequirementforneutralization.Forexample,antibod- lo
a
However,therearesignificantlimitationsofthismodel,andthey ies that bind the exposed lateral ridge of WNV E protein DIII d
havebeenreviewedindetail(151).Analternative“multiple-hit” neutralizeinfectionmoreefficientlythanantibodiesthatbindless e
d
modelproposesthattheneutralizationofanindividualviruspar- accessiblestructureswithanequivalentaffinity(160).Incontrast,
f
r
ticlerequiresengagementbynumerousantibodymolecules(153). antibodiesthatbindinfrequentorinaccessibledeterminantsare o
m
Aninterestingextensionofthismodelsuggeststhatthenumberof characterizedbyhighfractionaloccupancyrequirementsforneu-

antibodiesnecessaryforneutralizationiscorrelatedwiththesize tralization.Notallepitopesareequallyaccessibleforbinding,and h
t
t
of the virion, which reflects a requirement to fully occlude its many factors with the potential to impact epitope accessibility p
:
surface(reviewedinreference2).Estimatesofthestoichiometry havebeendescribed(forflaviviruses,seereference165).Reduced //
m
ofneutralizationhavebeendeterminedformultipleviruses,in- epitope exposure imposes a requirement for a higher fractional
m
cluding phage MS2 (156), poliovirus (157–159), WNV (160), occupancyoftheremainingexposedepitopesinordertoexceed
b
papillomavirus (161), influenza virus A (155), and rabies virus thestoichiometricthresholdandthushavethepotentialtocon- r
.
a
(162,163).Whileinmanycases,theneutralizationthresholdfor tribute to immune evasion and antibody-dependent enhance- s
structurally distinct groups of viruses correlates positively with mentofinfection(Fig.4,inset). m
.
virionsize,inagreementwiththe“coatingtheory”(2),factorsthat Critically,therelationshipsbetweenantibodyoccupancyand o
r
determinethenumberofantibodiesrequiredforneutralization neutralizationmaybecomplicatedbyfactorsbeyondthenumber g
/
mayvaryamongviruseswithdifferentstructures,compositions, ofepitopesdisplayedbythevirion.Forexample,thebindingofa o
andentrymechanisms.Forexample,thesmallnumberoffunc- singleantibodytooneprotomerofanHIV-1envelopetrimermay n

A
tionaltrimersontheHIV-1surfaceallowsneutralizationwitha besufficienttorenderthatcomplexincapableofpromotingvirus
p
stoichiometrymuchlowerthanthatpredictedforavirionofthis entry(119,121),andthus,thecontributionofadditionaleventsof r
size(119,121).Relationshipsbetweenthenumberandoligomeric bindingtothatsametrimermaybediminished.Antibodybinding il 7
stateoffunctionalviralproteincomplexesrequiredtodrivevirus itselfmaymodulatetheaccessibilityofsurroundingepitopesin ,
2
entry and the stoichiometric requirements of neutralization are unpredictablewaysthroughdirectstericmechanisms(174)orby 0
1
notwellunderstood(86–88,118,119,121). trappingalternativeantigenicstatesofthevirion(175). 9
ForWNV,experimentswithMAbsthatbindthelateralridgeof b
DIIIindicatethatbindingof(cid:2)30MAbstothevirionisrequired MechanismsofNeutralization y
g
forneutralization(Fig.4,inset)(160,164).Whetherthestoichio- Antibodieshavetheabilitytoblockviralinfectionatanynumber u
metric requirements for the neutralization of flaviviruses vary ofstepsintheprocessofvirusentry(reviewedinreference151). e
s
amongantibodiesthatbinddifferentepitopes,engagethevirion Thesestepsincludevirusattachmenttothecellsurface,virusin- t
bivalently, or inhibit infection via distinct mechanisms is un- teractions with receptors or coreceptors, fusion with the host
known(165).Insupportofthesecaveats,thenumbersofantibod- membrane(forenvelopedviruses),membranepenetrationorge-
iesrequiredtoneutralizeflavivirusinfectionbyblockingattach- nomeinjection(fornonenvelopedviruses),orviralgenomeun-
mentormembranefusionappeartodiffer(61,166). coating(Fig.4).Blockingofcellsurfaceattachmentorreceptor
Epitopeoccupancyandneutralization.Atleasttwofactorsde- engagementthroughsterichindrancemaybeacommonmecha-
terminethenumberofantibodiesboundtoavirusparticleatany nism and has been suggested to explain the activity of DENV-
givenconcentrationofantibody(reviewedinreference165).An- immunesera(176).However,manyantibodiesarealsocapableof
tibodyaffinitydefinesthefractionofviralepitopesboundbyan- blockinginfectionatapostattachmentstep(51,59,167,177–179).
tibodyundersteady-stateconditions,asexpressedbytheequation TheseNAbsmayinhibitconformationalchangesofaviralprotein
fractionofepitopesbound(cid:9)[Ab]/([Ab](cid:3)K )(whereK isthe requiredtomediatevirusentry.Forexample,structuralanalyses
d d
dissociation constant). Thus, high-affinity antibodies bind a revealedthatWNV-specificMAbE16trapsEproteinsinaradially
largerfractionoftheirepitopesthandolower-affinityantibodies extendedintermediateatlowpH(180).LikeE16,MAbCR4354
ofthesamespecificityatadefinedantibodyconcentration.More inhibitsWNVatapostattachmentstepandcaninhibitvirusfu-
critically,thenumberofaccessibleepitopesontheintactinfec- sion with synthetic liposomes (59, 170). The structure of the
December2016 Volume80 Number4 MicrobiologyandMolecularBiologyReviews mmbr.asm.org 995
VanBlarganetal.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
m
FIG4Flavivirusentryandmechanismsofantibody-mediatedneutralization.(A)Flavivirusentryoccursfollowingvirusinteractionwithattachment b
r
factorssuchastheC-typelectinsDC-SIGNandDC-SIGNR,mannosereceptor,glycosaminoglycans(GAGS),andphosphatidylserinereceptorsofthe .
a
TIMandTAMproteinfamilies.(B)Followingvirusattachment,flavivirusesundergoclathrin-mediatedendocytosis.(C)Flavivirusescanthenentercells s
bypH-dependentfusion,typicallyinthelateendosomefordengueviruses(67).Antibody-mediatedneutralizationofflavivirusesmaybeachievedby m
inhibitingvirusinfectivityatanumberofvirusentrystepssuchas(i)preventingvirusattachmenttothecellsurface,(ii)promotingvirusdetachment .o
fromcells,and(iii)inhibitingvirusfusionwithendosomalmembranes.(Inset)Neutralizationoccurswhenantibodiesbindflaviviruseswithastoichi- r
g
ometrythatexceedsaparticularthreshold(160).Antibody-dependentenhancementofinfection(ADE)canoccurifthenumberofantibodiesboundto /

theviriondoesnotreachthestoichiometricthresholdforneutralization.Thenumberofantibodiesboundpervirionismodulatedbyantibodyaffinity o
aswellasbyepitopeaccessibility.Therefore,antibodiesthatbindcrypticepitopesthatarepoorlyaccessibleforantibodyrecognitionmaynotbeableto n

achieveastoichiometrysufficienttoexceedthethresholdrequirementsforneutralizationdespitehighaffinityfortheepitope.Incontrast,antibodiesthat A
bindhighlyaccessibleepitopescanexceedthestoichiometricthresholdforneutralizationatlowoccupancy. p
r
il
7
,

2
CR4354FabboundtoWNVrevealedadiscontinuousepitopethat processesthatoccurduringvirusentry(184,185).Forexample, 0
1
spanned neighboring E proteins, suggesting that this MAb and the orientation of the Fc region of virus-bound antibodies has 9
othersthatbindcomplexquaternaryepitopesmightblockfusion beenhypothesizedtocontrolthenumberofWNVDIII-reactive b
y
bycross-linkingEproteinsonthevirion(61,170–172).Whilethe MAbE33moleculesdockedontovirusesindifferentmaturation
g
multiple-hithypothesisassumesthatneutralizationisareversible states(174).Thus,thelargesizeoftheantibodymoleculehasthe u
process(181),insomecases,antibodybindingresultsinanirre- potential to influence the accessibility of surrounding epitopes e
s
versiblechangeinvirioninfectivitythatpersistsuponthereversal and thereby impact conditions that support antibody-mediated t
ofbinding.Someanti-HIV-1MAbs,suchasthosethatbindthe neutralization.
MPERongp41andtheCD4-bindingsiteongp120,inducethe
sheddingofviralglycoproteingp120fromthevirion,whichren-
VIRUSEVASIONOFANTIBODY-MEDIATEDNEUTRALIZATION
dersvirionsnoninfectiousevenwhentheantibodydisassociates
fromthevirusparticle(182). SequenceVariationandAntigenicDiversity
WhileFabfragmentsofneutralizingantibodiescanblockinfec- Viralgenomesmutateatarelativelyhighrate,allowingtheselec-
tion(150),theFcregionofantibodiesalsoplaysaroleinneutral- tion of mutations within antibody epitopes (186, 187). Viruses
ization.Althoughthesurfaceareaburiedbyanepitope/paratope thatcantoleratealargenumberofmutationsintheirstructural
interactionisrelativelysmall,generally(cid:10)900Å2(183),theintact proteins,suchasHIV-1,hepatitisCvirus(HCV),influenzavirus,
antibodymoleculemayoccupyalargerareaduetoitsstructural andnoroviruses,undergorapidandsubstantialantigenicdriftin
flexibility. The antiviral activity of antibodies likely reflects the thepresenceofimmunepressure(4,188–191).Thisisevidentin
contributionoftheentiremolecule,whichhasthepotentialnot theyearlyrequirementforareformulationoftheseasonalinflu-
onlytoinfluencethenumberofantibodymoleculesdockedonthe enzavaccine,asinfluenzavirusesareextremelyadeptatacquiring
virion(relativetoFabfragments)butalsotointerferewithmany antibody escape mutations (192, 193). For viruses that cause
996 mmbr.asm.org MicrobiologyandMolecularBiologyReviews December2016 Volume80 Number4
DissectingAntiviralNeutralizing-AntibodyResponses
chronic infections, intrahost generation of antigenic diversity is stratedincreaseddynamicsandtransitionedmorefrequentlybe-
observed over time as new viral variants emerge to escape the tweenclosedandopenconformationalstatesthantheclinically
NAbsgeneratedearlyintheimmuneresponse.Astheantibody isolated,neutralization-resistantstrain(116).
response evolves to recognize poorly neutralized variants, new
viralvariantsemergetoescapetheseantibodies.Thisiterativese-
LowDensityofSurfaceGlycoproteins
lectionofneutralizationescapevariantsconstantlyelicitsthepro-
ductionofnewantibodies(103,104,194).Rapidantigenicevolu- ThedensearrangementofflavivirusEproteinsmayimposesteric
tionisnotapparentforallviruses,evenamongRNAviruseswith constraintsonantibodyepitopeaccessibility(57,160,180,206)
error-proneRNApolymerases.Forexample,duetoalackofsig- (see Fig. 6). While flaviviruses incorporate a fixed number of E
nificantantigenicdrift,therehasnotbeenaneedtoreformulate proteinsintothevirusparticle,othervirusesmaylimitthenum-
the monovalent YFV 17D vaccine, even after 60(cid:3) years of use berofepitopesavailableforantibodybindingbydirectlyreducing
(195).Manyfactorsmaymodulateviralantigenicevolution,in- thenumberofsurfaceproteinsincorporatedintovirionsthatare
cluding differences in virus host range (196) and the inherent targetedbyNAbs,thuspreventingantibodyengagementfromex-
mutationaltoleranceofvirussurfaceglycoproteins(197,198). ceedingthestoichiometricthresholdrequiredforneutralization.
Forexample,humancytomegalovirus(HCMV)hasbeenshown D
ConformationalMaskingofConservedRegions toreducetheincorporationofthesurfaceglycoproteingHunder o
w
Conservedstructuralfeaturesthatplayimportantrolesintheviral selectivepressurebyantibodiesinvitro,resultinginresistanceto n
lifecyclemaybelesstolerantofsequencevariationandthusrep- neutralizationbyMAbstargetinggH(213). lo
a
resent sites of vulnerability to antibodies. Many viruses have Arelativelylowdensityofstructuralproteinsonthesurfaceof d
therefore evolved mechanisms to conceal conserved regions of virionsmayalsopreventbivalentengagementofthevirusparticle e
d
their structural proteins. For example, the “canyon hypothesis” bytheantibody,whichinturnlimitsantibodyavidityandimpacts
f
r
speculatesthatthereceptor-bindingdomain(RBD)ofsomepi- antibodyoccupancyatanygivenconcentrationofantibody.The o
m
cornavirusesisburiedinacanyononthesurfaceoftheviralcapsid lowdensityofEnvonHIV-1virionshasbeensuggestedtolimit

toevadeimmunerecognition(199),althoughthisstrategyisim- theneutralizingactivityofsomeHIV-1-reactiveMAbs(reviewed h
t
t
perfect(200).Likewise,theHIV-1gp120RBDsarepositionedin in reference 214). Biochemical and structural studies have esti- p
:
recessedpocketsoftheenvelopespikethatarenotaccessibleto matedthattheaverageHIV-1virionhasasmallnumberofEnv //
m
most antibodies (100, 201). DII-FL of flaviviruses is critical for spikesonitssurface((cid:2)14)(86–88).Comparisonsoftheneutral-
m
mediatingfusionduringvirusentry,ishighlyconservedamong izationpotenciesofIgGandFabfragmentsofanti-HIV-1anti-
b
distantlyrelatedflaviviruses,andisfrequentlytargetedbythean- bodiesrevealedsimilarpotencies,suggestingthatbivalentrecog- r
.
a
tibodyresponse(discussedbelow)(202–204).However,antibod- nition is uncommon (214). However, an elegant study by s
ieswiththisspecificityaretypicallycharacterizedbylimitedneu- Galimidietal.usingengineeredantibody-basedmoleculescapa- m
.
tralizingactivitybecausethefusionloopispoorlyaccessibledueto bleofintraspikebivalentbindingdemonstratesgreatlyincreased o
r
theproximitytoresiduesoftheopposingEproteinontheantipa- neutralizationpotenciesforantibodieswithcertainepitopespec- g
/
ralleldimer(57,205).Numerousotherexamplesofcrypticflavi- ificities (215). While the densely packed, pseudoicosahedral ar- o
virusepitopeshavebeenreported(166,175,206,207). rangementofEproteinsonflavivirusvirionsmayallowbivalent n

A
Regulationofepitopeaccessibilitybyflaviviruses.Atleasttwo engagementbyantibodies,onlyoneDENVEproteinDIII-reac-
p
factorshavethepotentialtomodulateflavivirusepitopeaccessi- tiveantiflavivirusMAbthatrequiresbivalentbindingforitsneu- r
bility.Whilestericconstraintslimitantibodyaccessibilityonthe tralizingactivityhasbeencharacterizedtodate(216). il 7
maturevirion,inefficientmaturationofflavivirusesresultsinthe ,
2
releaseofpartiallymaturevirionsonwhichepitopeaccessibility 0
GlycanShields 1
may differ (41, 42, 44, 205). Decreasing the efficiency of virion 9
maturationresultsinanincreaseinsensitivitytoneutralizationby AlthoughflavivirusEproteinscontainonlyalimitednumberof b
manyantibodies(41).TheconformationaldynamicsofEproteins glycans(1,2),manyviralstructuralproteinscontainmultiplegly- y
g
incorporatedintothevirionalsomodulateepitopeaccessibility. cosylationsites,whichcanmediateimmuneevasion(reviewedin u
Viral “breathing” has been shown in several systems to impact reference 217). The presence of N- and O-linked glycans may e
s
neutralizationsensitivity(166,208–211).Antibodieshavethepo- decreasetheimmunogenicityofparticularregionsofviralglyco- t
tentialtotraptransientlyexposedepitopes,asflavivirusessample proteins,ascarbohydratestructuresonvirionsmayberecognized
anensembleofstructuresatsteadystate.Recentstudieshavedem- as“self”bytheimmunesystem(100).Severalviruses,including
onstratedthatthereversibleexposureofcrypticepitopesbyWNV HIV-1,Ebolavirus,andHCV,utilizea“glycanshield”toavoid
andDENVcontributestotime-dependentpatternsofneutraliza- antibodyrecognition(103,218).Forexample,theHCVenvelope
tion(166,212).Intheseexperiments,anincreaseoftheincuba- glycoproteinsE1andE2togetherhaveupto16N-linkedglycosy-
tiontimeofantibody-virusimmunecomplexespriortoinfection lation sites, most of which are highly conserved. Several of the
of cells results in corresponding increases in neutralization po- glycansontheE2glycoproteinhavebeenshowntoinfluencethe
tency.Similarobservationshavebeenmadewithantibodiestar- susceptibilityofHCVvirionstoneutralization(218).Addition-
getinginternalcomponentsofthecapsidofsomepicornaviruses ally,alterationsinthenumber,placement,andtypeofglycanson
(208).ConformationaldynamicsofEnvmayalsoimpactepitope HIV-1andsimianimmunodeficiencyvirusgp120occurinorder
exposureandneutralizationsensitivityofHIV-1.Whenthecon- to mask NAb epitopes (98, 103, 219, 220). How the E protein
formational dynamics of two HIV-1 strains were compared by glycosylationstatusofflavivirusesaffectsantibodyrecognitionis
single-moleculefluorescenceresonanceenergytransfer(smFRET), notwellunderstood,althoughantibodiesthatmakecontactswith
the laboratory-adapted, neutralization-sensitive strain demon- bothglycansonDENVhavebeenreported(169).
December2016 Volume80 Number4 MicrobiologyandMolecularBiologyReviews mmbr.asm.org 997
VanBlarganetal.
IDENTIFYINGTHEFUNCTIONALCOMPONENTSOFTHE ity against diverse HIV-1 strains identified only 3 somatically
POLYCLONALANTIBODYRESPONSE relatedMAbstargetingtheCD4-bindingsitethatrecapitulated
Thehumoralresponsetoviralinfectionhasthepotentialtoyield the broad and potent neutralizing activity of the polyclonal
large numbers of B cells that produce antibodies with various serum(230).
specificities,antigenaffinities,andfunctionalproperties.Consid- PartofthechallengeofelicitingeffectiveNAbsagainststructur-
erableinsightintohowantibodiesprotectagainstviralinfection, ally complex viruses may be a requirement to engage a specific
viadirectneutralizationofinfectionorantibodyeffectorfunction, subset of the B cell repertoire. In support of this hypothesis,
hascomefromstudiesofMAbs(60,137,160,167,170,171,177, bNAbsagainsttheHIV-1CD4-bindingsiteisolatedfrommultiple
207,221,222).However,howantibodiesfunctioninconcertas donorsappeartoarisefromacommon,limitedsubsetofgerm
part of a polyclonal response is not well understood, nor is the line genes that remarkably converge in sequence and structure
breadthofthefunctionallysignificantcomponents.Forexample, duringtheaffinitymaturationprocess(231,239–242).Antibodies
itisunclearwhyinfectionorvaccinationmayelicitantibodiesthat elicitedfollowingacuteDENVinfectionorinfluenzavaccination
displayneutralizingactivityinvitrobutcontributeonlymodestly, among unrelated individuals also display convergent sequence
ifatall,toprotectioninthehost.Twocomplementaryapproaches signatures(243,244).
have been used successfully to deconstruct the functional com- D
NeutralizingAntibodiesTargetaLimitedNumberof o
plexity of the polyclonal response. First, large panels of human w
Specificities
MAbshavebeencreatedformanypathogens,allowingadetailed n
analysisinvitroandinvivooftheindividualcomponentsofan To complement studies of human MAb specificities, several lo
a
antibody response. For flaviviruses, this approach has provided groupshavedevelopedgeneticandbiochemicalmethodstoinves- d
insightsintohowantibodiesengagethemanysurfacesofthevi- tigatethecontributionofparticularepitopestotheNAbresponse ed
rion(53,62,63,223).Second,severalgroupshavedevelopedmo- toflavivirusinfectionorvaccination.Oneapproachistocompare f
r
lecular and biochemical approaches to identify epitopes recog- theneutralizingactivityofserapreincubatedwithsolublerecom- o
m
nizedbyNAbswithinsera(57,224–228). binantantigensrepresentingvariousEproteindomainstothatof

untreatedsera.SuchserumdepletionstudieshaveshownthatDIII h
t
t
is not a major target of human NAbs following vaccination or p
NeutralizingAntibodiesAreaRareComponentofthe :
naturalinfectionwithDENV(227),WNV(236),YFV(245),and //
HumoralResponseagainstViralInfection m
TBEV(246)despitebeingthetargetofverypotentmurineneu-
m
Virus-specifichumanMAbsareidentifiedbyscreeningBcells tralizingMAbs(50–52,55,59,247,248).Thesefindingsaresup-
b
fortheirabilitytoproduceantibodieswithdesiredfunctional ported by genetic approaches demonstrating that mutations in r
.
a
properties.Inmanyinstances,candidateBcells,eitherMBCs DIIIepitopesdonotresultinasignificantreductionintheneu- s
orLLPCs,arescreenedfortheabilitytobindrecombinantviral tralizationpotencyofflavivirusimmunehumansera(227,236). m
.
proteins(53,229–231),virus-likeparticles(232,233),orintact MostoftheserumneutralizingactivityfollowingYFVorDENV o
r
virions(60,234)andthenimmortalized.Theuseofrelatively infectionorvaccinationisnotaffectedbythedepletionbysoluble g
/
high-throughput neutralization assays has allowed functional Eprotein,suggestingthatquaternaryepitopesmaybeimportant o
screenstoidentifyBcellclonesthatproducepotentNAbsdi- targetsforNAbs(227,245),asdiscussedbelow.Ingeneral,most n

A
rectly(168).Thecomplexityoftheantigenicsurfaceofviruses studies characterizing antiflavivirus NAb specificities in human
p
suggests that the nature of the antigen and screening strategy polyclonalserahavelargelyruledouttheimportanceofparticular r
usedwillalmostcertainlyimpacttherepertoireofMAbsiden- epitopes,suchasthoseinDIII(227,236,249). il 7
tified(62,168,223). Only two studies have identified residues on the E protein ,
2
Inmostinstances,analysesofhumanantiflavivirusMAbssug- targetedbyNAbsinpolyclonalDENV-reactivesera(228,250). 0
1
gestthatantibodieswithlimitedinvitroneutralizingactivitycom- In one study, we analyzed the ability of serum samples from 9
prisealargefractionofthevirus-specificantibodyresponse(53, recipients of a monovalent DENV1 vaccine candidate (251– b
60,62,63,235).Forexample,(cid:10)5%oftheantibodiesisolatedfrom 254)toneutralizeacomprehensivepanelofDENV1variantsin y
g
flavivirus-infectedorvaccinatedhumansdisplayedpotent(50% which surface-accessible residues of the E protein were re- u
effectiveconcentration[EC ]of(cid:10)0.5 (cid:6)g/ml)neutralizingactiv- placedwithcorrespondingsequencesofaDENV2strain(228). e
50 s
ity(204,235).Asignificantportion((cid:2)45to60%)ofthetotalE This panel was screened to identify mutations that reduced t
protein-specificresponseappearstobedirectedagainstthehighly sensitivity to neutralization by DENV1-immune, but not
conservedfusionloopindomainII(DII-FL)(204,236).Antibod- DENV2-immune, sera. Our analysis identified mutations at
iesthattargetprMarealsofrequentlyisolated(53,62,63,235). justtworesiduesinDIandDIIthat,whencombined,ablated
However,themajorityofbothDII-FL-andprM-reactiveanti- theDENV1serotype-specificresponsetovaccination(Fig.5A
bodies inhibit infection only weakly (53, 203, 204, 206, 235). andB)(228).Theobservationthattheantiflavivirusimmune
Theseantibodiesbindepitopesthatareeitherpoorlyaccessible responseisfocusedonjustafewspecificitiesalignswithdata
forantibodyrecognition(57)ornotpresentonvirionswitha from recent studies on HIV-1, in which some (255–258) but
stoichiometrysufficienttoexceedthethresholdrequirements notall(259–262)analysesofpolyclonalserawithbroadlyneu-
forneutralization(41,57,160).Similarly,althoughbroadand tralizing activity suggest that just one to two specificities can
potent NAbs against HIV-1 have been isolated from infected recapitulatethebreadthandpotencyoftheoverallserumneu-
individuals(6,114,237,238),theseNAbsrepresentararecom- tralizingactivity.Follow-upstudieswithmultiplestrainsfrom
ponentoftheoverallhumoralimmuneresponse.Forexample, eachDENVserotypewillbeneededtoexplorethefinespeci-
ascreenof25millionperipheralbloodmononuclearcells(PB- ficityofserotype-specificNAbs.
MCs)fromanindividualwhoseserumdisplayedcross-reactiv- Another recent study investigated the epitope specificity of
998 mmbr.asm.org MicrobiologyandMolecularBiologyReviews December2016 Volume80 Number4