Glycans Function as Anchors for Antibodies and Help Drive HIV Broadly Neutralizing Antibody

29 Pages·2017·15.33 MB·English

by  Raiees Andrabi

Article
Glycans Function as Anchors for Antibodies and Help
Drive HIV Broadly Neutralizing Antibody
Development
Graphical Abstract Authors
RaieesAndrabi,Ching-YaoSu,
Chi-HuiLiang,...,Chung-YiWu,
Chi-HueyWong,DennisR.Burton
Correspondence
[email protected]
In Brief
Understandingthemolecularbasisfor
thedevelopmentofHIVEnv-specific
bnAbsiskeyforvaccinedesign.Andrabi
etal.findthatCAP256V2apexlineage
Absaffinitymaturewithsialicacid
containingcomplex-typeglycansonHIV
envelopetrimer.Thisglycanrecognition
enablesthedevelopmentintobnAbsby
resistingvirusescape.
Highlights
d CAP256V2apexbnAbsaffinitymaturewithsialicacid(SIA)-
bearingHIVEnvglycans
d Bothgermlineandsomatic-mutatedHC-Abresidues
contributetoSIArecognition
d SIA-specificmaturationpatternisconservedintwo
geneticallyrelatedbnAblineages
d SIA-specificaffinitymaturationcountersvirusescapeand
helpsbnAbdevelopment
Andrabietal.,2017,Immunity47,524–537
September19,2017ª2017ElsevierInc.
http://dx.doi.org/10.1016/j.immuni.2017.08.006
Immunity
Article
Glycans Function as Anchors for Antibodies
and Help Drive HIV
Broadly Neutralizing Antibody Development
RaieesAndrabi,1,2,3,6Ching-YaoSu,1,2,3,6Chi-HuiLiang,1,2,3SachinS.Shivatare,4BryanBriney,1,2,3JamesE.Voss,1,2,3
SalarKhanNawazi,1,2Chung-YiWu,4Chi-HueyWong,4andDennisR.Burton1,2,3,5,7,*
1DepartmentofImmunologyandMicrobiology,TheScrippsResearchInstitute,LaJolla,CA92037,USA
2InternationalAIDSVaccineInitiative,NeutralizingAntibodyCenter,TheScrippsResearchInstitute,LaJolla,CA92037,USA
3CenterforHIV/AIDSVaccineImmunologyandImmunogenDiscovery,TheScrippsResearchInstitute,LaJolla,CA92037,USA
4GenomicsResearchCenter,AcademiaSinica,Nankang,Taipei115,Taiwan
5RagonInstituteofMassachusettsGeneralHospital,MassachusettsInstituteofTechnology,andHarvard,Cambridge,MA02114,USA
6Theseauthorscontributedequally
7Leadcontact
*Correspondence:[email protected]
http://dx.doi.org/10.1016/j.immuni.2017.08.006
SUMMARY broad spectrum of native Env molecules (Andrabi et al., 2015;
Bhimanetal.,2015;Dimitrov,2010;Doria-Roseetal.,2014;Gor-
Apex broadly neutralizing HIV antibodies (bnAbs) manetal.,2016;Jardineetal.,2013;Liaoetal.,2013;McGuire
recognize glycans and protein surface close to the etal.,2016;Steichenetal.,2016;Xiaoetal.,2009).Oneofthe
3-fold axis of the envelope (Env) trimer and are targetsofbnAbsthathasattractedagreatdealofattentionis
among the most potent and broad Abs described. theV2apexregionofEnv(Andrabi etal.,2015;Bhimanetal.,
The evolution of apex bnAbs from one donor 2015; Doria-Rose et al., 2014; Sok et al., 2014; Walker et al.,
2009, 2011). bnAbs to this region are induced in a substantial
(CAP256) has been studied in detail and many
proportion of the individuals who make such Abs (Georgiev
Abs at different stages of maturation have been
et al., 2013; Landais et al., 2016; Walker et al., 2010) and the
described. Using diverse engineering tools, we
V2apexresponsetendstoappearearlierinnaturalHIVinfection
investigated the involvement of glycan recognition
than other bnAb responses (Doria-Rose et al., 2014; Wibmer
inthedevelopmentoftheCAP256.VRC26Ablineage.
etal.,2013),bothfactorsthatfavoritasavaccinetarget.
We found that sialic acid-bearing glycans were TheV2apexrepresentsacomplexantigenicregion.Broadly,a
recognized by germline-encoded and somatically lysine-richsetofresiduesonV2cometogetherinspacearound
mutatedresiduesontheAbheavychain.Thisrecog- the3-foldaxisatthe‘‘top’’ofthespiketoconstituteabasicre-
nition provided an ‘‘anchor’’ for the Abs as the core gionthatisrecognizedbyacidicresidues(aspartateandsulfated
proteinepitopevaries,preventedcompleteneutrali- tyrosines)atthetipofalongHCDR3(heavychaincomplemen-
zation escape, and eventually led to broadening tarity-determiningregion3oftheheavychain)ofthebnAb.Inor-
dertoaccessthebasicregion,theHCDR3,andtosomeextent
of the response. These findings illustrate how
theother CDRs,of thebnAb mustpenetrate theglycan shield
glycan-specific maturation enables a human Ab to
of Env and particularly interact with and/or accommodate a
cope with pathogen escape mechanisms and will
conserved glycan at N160 and, to a lesser extent, a second
aid in optimization of immunization strategies to
nearbyglycanatN156orrarelyN173(Aminetal.,2013;Gorman
induceV2apexbnAbresponses.
etal.,2016;Leeetal.,2017;McLellanetal.,2011;Panceraetal.,
2013; Walker et al., 2009). Detailed studies have combined
structure,nuclearmagneticresonance(NMR),andmutagenesis
INTRODUCTION analyses to characterize glycan recognition by the V2 apex
bnAbs PG9 and PG16 (McLellan et al., 2011; Pancera et al.,
ApromisingtrackforHIVvaccinedesignistotakelessonsfrom 2013). These studies identified PG9 and PG16 as interacting
broadly neutralizing antibodies (bnAbs) and their interaction withaMan GlcNAc structureatN160andasialicacidcontain-
5 2
withEnvelopetrimer(Env)tocraftpotentialimmunogensinan inghybrid-typeglycanatN173orN156.Thestudiesemployed
approach described as ‘‘Reverse Vaccinology 2.0’’ (Burton, V1V2loopsonscaffoldsratherthannativeEnvandthisremains
2002;Rappuolietal.,2016).Ithasbecomeapparentthatmulti- acaveattothisanalysis.Particularfocuswasplacedonatermi-
ple immunogens will likely be required to guide the antibody nalsialicacidontheglycanatN173orN156interactingwitha
response from their unmutated ancestors to bnAbs (Briney tripeptide(RSH)motifinthelightchainofPG16.
etal.,2016a;Dosenovicetal.,2015;Escolanoetal.,2016;Jar- The molecular information provided by these studies is
dine et al., 2015; Tian et al., 2016), including immunogens to extremelyvaluablebutmoreinsightisneededpriortoembarking
trigger unmutated precursor B cells, to shepherd them along uponasystematicmultistagevaccineefforttotargettheV2apex
favorable pathways, and to maximize their interaction with a region.OneapproachistocomparedifferentV2apexbnAbsand
524 Immunity47,524–537,September19,2017ª2017ElsevierInc.
identifycommonfeaturesthatcouldbeimportantinconsidering prototype is partially dependent on both N156 or N173 and
immunogendesign.Thisapproachhasbeenvaluableinidenti- N160 glycans in an isolate- and context-dependent manner
fyingpotentialV2apexbnAbprecursor-targeting immunogens (Doria-Roseetal.,2014,2015).
(Andrabietal.,2015;Gormanetal.,2016;Sandersetal.,2015) To assess the dependency of CAP256 lineage Abs during
that are currently under study. Four prototype V2 apex bnAb maturation on Env glycans and glycan character in detail, we
lineages have been described and two of them, PG9 and employedthreeapproaches.Wecarriedoutourstudiesinthe
CAP256-VRC26, are derived from variable heavy chain (VH) contextoftheCRF250isolatetrimerasthisisolatebindstoin-
genesegmentsthatshow99%sequenceidentity andusethe ferred-unmutated ancestor versions of multiple V2 apex bnAb
sameD-genethatprovidescriticalanionicresiduesforprotein prototypesandrepresentsapotentialBcellprecursortargeting
epitope recognition (Andrabi et al., 2015; Bonsignori et al., immunogen(Andrabietal.,2015).
2011;Doria-Roseetal.,2014;Walkeretal.,2009,2011).Asec- First, we generated CRF250 Env virus variants that lacked
ondapproachistoinvestigatetheco-evolutionofbnAbandvirus glycans at positions N156 and N160 and investigated neutral-
duringnaturalinfectiontoidentifycriticalstagesinthematura- ization byCAP256 lineage members (Figures1A,1B, and S1).
tion of the response and attempt to extract information that The wild-type (WT) CRF250 virus grown in 293T cells was
can aid in immunogen design (Doria-Rose et al., 2014; Liao neutralizedbyallCAP256Ablineagemembers,withtheexcep-
etal.,2013;MacLeodetal.,2016).TheCAP256lineage(referred tionofAbCAP256.20.Thispatternwassimilartothatseenfor
to as CAP256.01-33) has been extensively studied and first theCAP256superinfecting virus(CAP256.SU), highlighting the
appeared after superinfection of the donor. This lineage has antigenic resemblance of CRF250 Env and the CAP256.SU
a particularly long HCDR3, which is 35 amino acids (Kabat Env,which,oraclosedescendantthereof,isbelievedtohave
numbering convention) in the unmutated common ancestor led to initiation of the CAP256 Ab lineage (Figures 1B and S1;
(UCA) and increases by insertions to 37 amino acids in the Bhiman et al., 2015; Doria-Rose et al., 2015). Both N156 and
most potent mature bnAbs (Doria-Rose et al., 2014, 2015). N160 glycan-eliminated variants were less well neutralized by
One detailed study of co-evolution in the CAP256 lineage CAP256 bnAbs, the effect being more prominent with N156
showeddifferentfatesforAbsoriginatingintheUCA.Onetrack glycan elimination (N156A; 882-fold drop) than N160 glycan
led to ‘‘dead-end’’ Abs that did not evolve as they could not removal(N160A;80-folddropinaverageIC comparedtothe
50
respondtoviraldiversity.Asecondtrackcontinuedtoevolve,re- WT CRF250 virus) (Figures 1B and S1). Effects were seen
sultinginhighlysomaticallymutatedAbs,somethatwereableto throughout the lineage although the most potent neutralizing
neutralizemanyviralvariantsaswellas‘‘off-track’’Absthatwere Abs to CRF250, CAP256.03 and .25, tolerated the loss of the
limitedintheirneutralizationbreadth(Bhimanetal.,2015). N160glycanverywell(FigureS1).
Here,wesoughttotakeadvantage oftheavailabilityofAbs AsasecondapproachtoglycandependenceofAbbinding,
fromtheCAP256lineagetostudytheroleofglycaninteractions wemodifiedtheglycancompositionontheCRF250Envtrimer
intheevolutionofthelineage.Wecomparedanumberoffea- by growing virus in the presence of the glycosidase inhibitors
turesoftheinteractionsofCAP256Abswithglycansandinves- kifunensine (ER a-mannosidase I inhibitor) and swainsonine
tigatedthe role ofglycan recognition in resisting neutralization (Golgi a-mannosidase II inhibitor) and in 293S cells, which
escape. We concluded that recognition of sialic acid-bearing lack an N-acetyl glucosaminyl transferase enzyme. Kifunen-
glycanswascriticalintheevolutionofCAP256familyantibodies sine-treated cells were expected to yield viruses with mostly
anddependedupongermline-encodedandsomaticallymutated high-mannose (Man GlcNAc ) glycans; swainsonine-treated
8-9 2
residues in CDRH2, and that acquisition of these somatic cellstoyieldviruseswithhigh-mannoseorhybrid-typeglycans
mutationsearlyinAbmaturationcouldhelpanchorAbstotheir with a Man GlcNAc glycan core and a branch that may
5 2
epitopeeveninthefaceofmutationsinthecoreproteinpartof potentiallyterminateinasialicacid(DooresandBurton,2010;
theepitope. Pancera et al., 2013); and 293S cell-grown viruses to have
Man GlcNAc glycans without any hybrid or complex-type
5-9 2
RESULTS glycan character. Neutralization sensitivity was substantially
reducedbykifunensinetreatment(a58-foldincreaseinaverage
CAP256LineageAbsEvolveinTermsofGlycan IC titers),implyingthatbindingofCAP256Abswasdisfavored
50
RecognitionduringAffinityMaturationwithPossible byhigh-mannose-onlyglycans(Man GlcNAc )atoneorboth
8-9 2
ContributionfromTerminalSialicAcids oftheN156andN160positions.Incontrast,neutralizationsensi-
The four V2 apex bnAb prototype lineages—PG9, CH01, tivity was increased by swainsonine treatment as compared
PGT145,andCAP256.09—typicallydependonglycansatposi- to the WT CRF250 virus, suggesting a preference for hybrid
tionsN160andN156,orlessoftenN173(McLellanetal.,2011; glycans at either position. The 293S cell-treated virus overall
Panceraetal.,2013;Walkeretal.,2009),forneutralizationand showedadecreaseinneutralizationcomparedto293T-WTvirus
bindingtotheEnvtrimer.TheN173glycansiteisrelativelyclose (a28-folddropinaverageIC titers);however,thisdropinIC
50 50
totheN156siteanditappearsthattheN173glycancanactto titersappearedtobemainlycontributedbythelaterAblineage
replacetheN156glycaninthecontextofcertainisolatesinthe members(Figures1BandS1).Thislatterobservationwasagain
binding of V2 apex bnAbs (McLellan et al., 2011; Pancera consistentwithanapparentpreferenceforhybridglycans,espe-
et al., 2013). The first three Ab prototypes show an absolute ciallyasthelineageprogresses.
dependenceontheN160glycansiteforneutralizationwhereas TogiveinsightintotheglycansrecognizedattheN156andN160
the dependence is variable with respect to glycan N156 or positionsbytheCAP256bnAbs,wecombinedtheCRF250Env
N173.Incontrast,theneutralizingactivityoftheCAP256.09Ab glycan eliminations at N156 and N160 with glycan-modifying
Immunity47,524–537,September19,2017 525
Figure1. CAP256LineageAbsShowVaryingGlycanSpecificityandSialicAcidReactivityasMeasuredbyNeutralization,GlycanArray,and
EnvTrimerBinding
(A)Aphylogenetictreebasedonthevariableheavy(VH)chainregionofCAP256lineagemonoclonalantibodies(CAP256.01-32,I1,andI2).Thetreeoriginates
fromtheVH3-30*18germlinegenepresentinCAP256UCA.TheAbsarecoloredbasedontimeofsampling(wpi:weekspostinfection).
(B)HeatmaprepresentationoftheIC neutralizationtitersoftheCAP256AbswithCRF250wild-typevirus(293T).FoldchangesintheIC neutralizationtitersof
50 50
theCAP256AbsforCRF250glycaneliminatedvariantsatpositionsN156(N156A)andN160(N160A)andviralglycovariantsproducedinpresenceofglycosidase
inhibitors(kifunensine[Kif]andswainsonine[Swain])orina293Scellline,whichlacksanN-acetylglucosaminyltransferaseenzyme.ThemAbsweretestedina
TZM-blcellbasedinfectivityassay(Abconcentrationrange:IC <0.00001or>10mg/mL)andtheneutralizationIC foldchangesarerepresentedinheatmap
50 50
formatasindicatedinthekey.AnIC foldchangevalueof1.0indicatesthatIC neutralizationtiterisunchangedbetweentheWTvirus(293T)andthevirus
50 50
variant.
(C)ReactivityoftheCAP256Abstoglycansonglycanmicroarrays.Bindingisrepresentedasaveragefluorescenceintensity(a.u.)witherrorbarsshowing
standarddeviationfromthemean.Thesymbolsforeachmonosaccharideareindicated.
(D)OctetbindingresponseoftheCAP256AbstoCRF250WTtrimeranditsthreedesialylatedforms(a2,3ora2,6aloneora2,3,6,8,9cocktailsialidase-treated
trimers).ThemAbswereimmobilizedontoananti-humanIgG-FcsensorfollowedbydippingthesebiosensorsintothetrimersolutionandtheAb-trimer
interactionisrepresentedasbindingcurvesshowingtheassociation(120s;180–300)anddissociation(240s;300–540).
SeealsoFiguresS1andS2.
reagents to produce homogeneous virus glycovariants. One S1A and S1D). These results suggest that the CAP256 bnAbs
caveat of this analysis was that the modifications could affect preferhigh-mannoseglycans(Man GlcNAc )attheN160glycan
8-9 2
glycanprocessingthatmay,inturn,influenceproteinfoldingin positionbutthatthenativesugaratthispositionseemstoinclude
theV2apexregionandthusAbaccessibilitytotheregion.Never- some proportion of hybrid or complex-type. In contrast, the
theless,weconsidereditusefultostudytheeffectsofmodifica- CRF250N160Aviruswithkifunensinehadanoveralloppositeef-
tionsononeglycanintheabsenceoftheother.Weobservedan fectonneutralizationsensitivity(FiguresS1AandS1E),suggesting
increaseinneutralizationsensitivitytotheCAP256bnAbs,when thatasmallerhighmannoseoronewithhybridorcomplexglycan
theCRF250N156Aviruswasproducedinthepresenceofkifunen- charactermaybepreferredattheN156position.Again,theabove
sinewhileswainsoninetreatmenthadlittletonoeffect(Figures caveatshouldbenoted.
526 Immunity47,524–537,September19,2017
AsathirdapproachtoglycandependenceofAbbinding,we against CRF250 virus and for binding to soluble CRF250 and
tested CAP256 mAbs binding to a variety of human glycans BG505SOSIP.664trimersbyOctet(FigureS3B).Withtheexcep-
onglycanmicroarrays.Thisapproachrevealedastrongbinding tion of K100S-A Ab variant, whose neutralizing activity with
ofsomeofthemorematureCAP256Ablineagememberstobi-, CRF250viruswasslightlyenhanced,theneutralizingandbinding
tri-,andtetra-antennaryglycanswitha2,6-linkedsialicacidres- activitiesofalltheCAP256.09Abvariantswasreducedsubstan-
idueasaterminalsugar(Figures1C,S2A,andS2B).Asforthe tiallyascomparedtotheWTAb,indicatingthatthesubstituted
CAP256 Ab members here, V3-glycan PGT121 bnAb lineage residuescontributetoneutralizationandtrimerbinding.
membershavebeenpreviouslyshowntobindcomplex-typegly- To specifically identify residues in the HC most critical for
cansterminatingwitha2,6-linkedsialicacidresidues(Mouquet glycanrecognition,wefirstassessedthebindingofCAP256.09
etal.,2012).TheearlyCAP256Abmembersshowednodetect- WT Ab and nine variants to glycans on a microarray. The
ableinteraction—thisdoesnotmeanthatthereisnocontactof WTAb reactedstronglytoa2,6-linked sialic acidresidue-con-
these early Abs with sialic acid. Indeed, data presented in the taining glycanswhile theCDRH2variants (D53A,K57A, H59A,
nextsectionsuggestedthereiscontact.However,suchcontact and W64A) Ab variants, in addition to a single CDRH3 variant
mustpresumablyinvolveaweakerinteraction.Afailuretodetect (A100X-G),lostbindingtothetheseglycans(Figure2A).Asimilar
glycan binding on arrays despite interaction being shown be- glycanbindingpatternwasobservedforamorepotentCAP256
tweenAbandglycanonanativeEnvstructurehasbeenprevi- Ablineagemember,CAP256.25,anditscorrespondingCDRH2
ouslydescribed(Julienetal.,2013). Abvariants(Figure2B),thussuggestingaroleforthesegermline
Tospecificallyprobetheimportanceofsialicacidrecognition andsomaticallymutatedresiduesinrecognitionofa2,6-linked
bytheCAP256AblineageonthenativeEnvtrimer,weinvesti- sialicacidonglycans.
gatedtheeffectsofsialidasetreatmentofCRF250SOSIPtrimer Previously, we showed that the VH-germline gene families
onthebindingofCAP256AbsbyBio-LayerInterferometry(BLI) of the CAP256 and PG9 or PG16 Ab lineages share amino
orOctet.WetreatedtheCRF250SOSIP.664trimerwitha2,3-or acidsequencesimilarities(Andrabietal.,2015).Analysisofthe
a2,6-sialidasesaloneorwithacocktailofa2,3-,2,6-,2,8-,and matureVH-geneoftheCAP256andPG9Ablineagesrevealed
2,9-sialidasestoremoveterminalsialicacid-specificglycosidic a common maturation pattern in the CDRH2 residues of the
linkages. We assessed the binding of the CAP256 Ab lineage two lineages (Y59 to H and K64 to W) (Figure 2C). The data
members to the WT CRF250 trimer and its three desialylated suggestthatthispatternmaybeassociatedwiththecriticalna-
forms. Octet revealed that the binding of CAP256 Abs to tureoftheseresiduesforglycanrecognition.Indeed,functional
a2,3-sialidase-treated CRF250trimerremainedmostlyuniform analysisoftheCDRH2substitutionsinthecontextofPG9and
throughoutthelineageandonlyasmallfractionofAbsshowed PG16 bnAbs showed a similar dependence on the glycans as
small changes in binding to the a2,3-desialylated protein as CAP256 Ab lineage members (Figures S3C–S3F). Examination
comparedtotheWTtrimer(Figures1DandS2C).However,for ofthecrystalstructureofPG9andCAP256.09bnAbprototypes
a2,6-sialidase-treated trimer, a number of later more mature showsthatthefourresiduesdiscussedaboveareclosetoeach
Absshowedatrendforsomewhatweakerbindingascompared otherandformacage-likestructurewithK57andH59residues
toWTtrimer,consistentwitharraydata(Figures1DandS2C). atthecenter(Figure2D).
The effect was most pronounced for the CAP256.21 mAb. Overall, therefore, we observed two genetically related Ab
In contrast to the later Abs, the earlier Abs did not show any lineages following a conserved affinity maturation pattern that
noticeablea2,6-sialidaseeffectsinlinewiththeirlackofbinding helps them to recognize sialic acid-containing glycans on HIV
toarraysexpressinga2,6-sialicacid. Envtrimer.
Overall, the findings suggest that the CAP256 lineage Abs
haveevolvedintermsofglycanrecognitionduringaffinitymatu- BothGermlineandSomaticallyMutatedCAP256CDRH2
ration and there is an indication that binding of terminal sialic ResiduesContributetoEnvBindingandNeutralization
acidscontributes. buttheLatterAreMostImportant
WenextsoughttoassesshowtheCDRH2germlineandsomat-
CriticalCAP256AntibodyResiduesforSialicAcid icallymutatedresiduesidentifiedabovecontributedtoV2apex
RecognitionAreLocatedintheCDRH2 bnAb binding to Env and neutralization, in particular in the
Tobetterunderstandhowsialicacidrecognitionevolvesduring contextofthecriticalglycansatN160andN156.Previously,a
CAP256 Ab lineage development, we sought to identify the number of structural and analytical studies have argued the
most critical CAP256 Ab residues for glycan recognition and presence of both high-mannose and complex-type glycans at
investigatehowtheseresiduesweremaintainedorevolveddur- glycan positions N160 and N156 (or occasionally N173) on
ingmaturationoftheresponse.Wefocusedontheheavychain diverseisolates(Aminetal.,2013;Behrensetal.,2016;Gristick
(HC)oftheCAP256Abssincethischaindominatestheactivity etal.,2016;McLellanetal.,2011;Panceraetal.,2013).Further,
of these Abs. Analysis of the HC sequences revealed nine structural studies have shown that PG9 and PG16 Abs make
amino acid positions, including four positions in CDRH2 and contactwithN160andN156orN173glycansonEnvV2scaf-
five positions in CDRH3, that could potentially differentiate folds through both germline and somatically mutated residues
glycan-reactive Ab sub-lineage members from non-reactive (McLellanetal.,2011;Panceraetal.,2013).
ones (Figure S3A). We chose CAP256.09 as a prototype Ab To investigate, we generated a series of N156 or N173 and
from the glycan-reactive Ab sub-lineage and generated single N160 glycan-eliminated variants on a panel of diverse env
aminoacidsubstitutionsattheaboveninepositionsintheHC. backgrounds.Wetestedthesevirusesandtheglycanmutants
TheWTAbanditsvariantsweretestedforneutralizingactivity in neutralization against two WT V2 apex bnAbs, PG16 and
Immunity47,524–537,September19,2017 527
Figure 2. CDRH2GermlineEncoded andSomatically Mutated ResiduesofCAP256.09andCAP256.25Abs AreImportantinSialicAcid
RecognitiononGlycanArrays
(A)ReactivityonglycanmicroarraysofCAP256.09wild-type(WT)antibodyandninevariants.ThenineaminoacidsubstitutionsintheCAP256.09heavychain
variableregionincludefourinCDRH2andfiveinCDRH3.
(B)BindingofCAP256.25WT,CDRH2Abvariantstoglycansontheglycanmicroarray.
(C)AminoacidsequencealignmentoftheCDRH2andpartsoftheFRH2andFRH3regionofCAP256.09andPG9V2apexbnAbprototypeswiththeirrespective
germlineVHgenes.Kabatnumberingisindicated.ThealignmentshowsthatbothV2apexbnAbprototypesretainthegermline-encodedresiduesatCDRH2
positionsD53andK57andaccumulatetwocommonsomaticmutations(Y59HandK64W).
(D)StructuralalignmentofCAP256UCA(wheat),CAP256.25(lightpink),PG9(cyan),andPG16(green)bnAbshighlightingfouraminoacidpositionsintheCDRH2
regionwithsidechainsassticks.
SeealsoFigureS3.
CAP256.25,andtheircorrespondingCDRH2variants(Figure3A). affected by eliminating glycans (Doria-Rose et al., 2015), we
For PG16 Ab and its Ab variants, neutralization was invariably notedadependenceofneutralizationontheglycansN156and
completelyeliminatedonallN160glycan-removedenvmutants. N160 in an isolate- and context-specific manner, the loss of
This feature rendered it impossible to separate effects of the neutralizing activity being more common for the N160 glycan
CDRH2substitutionsonN160andN156orN173glycaninter- elimination,i.e.,whenN156wastheremainingglycan(Figure3A).
action. However, CAP256 Ab lineage member retained some Considering next neutralization of the WT viruses by the two
activityinmanycasesinN160-andN156-orN173-eliminated germline CDRH2 Ab variants (D53A and K57A), we showed
mutations,allowingthecorrespondingglycan-deficientviruses limitedreduction(upto18-fold)intheneutralizingactivities as
to be used to explore the behavior of the glycans individually compared to the WT CAP256.25 Ab. However, substitutions
asdescribedearlier,althoughnowinthecontextofthecontribu- involving two of the CDRH2 somatic mutations (H59A and
tionoftheCDRH2substitutionstobindingtoeachglycan.Again, W64A), the WT virus-neutralizing activities of the Ab variants
wenotethecaveatthatthelossofoneglycanmayimpactthe weresignificantlyreducedwiththeeffectbeinggenerallymore
processingoftheother. pronounced for the W64residue substitution (>1,000-fold IC
50
Focusing on WT CAP256 Ab lineage member, CAP256.25, change) (Figure3A).Focusing thenon the CDRH2variant Abs
whose neutralizing activity was previously shown to be less (D53A and K57A) neutralizing the N160A and N156A viruses,
528 Immunity47,524–537,September19,2017
Figure3. NeutralizationofaPanelofVirusesandGlycovariantVirusesSuggeststhatCAP256.25AbCDRH2ResiduesInteractwithSialicAcid
LikelyonN156Glycan
(A)NeutralizationofapanelofvirusesincludingN156or173AandN160AvariantsbyCAP256.25andPG16AbsandtheirCDRH2variants.Neutralization
heatmapsbasedontheIC titersshowtheeffectofglycanremovalsonantibodyneutralizingactivity.PGT128(anN332-V3Ab)andPGT151(agp120-41
50
interfaceAb)wereusedascontrols.
(B)NeutralizationtitrationofCAP256.25WTAbanditsCDRH2substitutedvariantsagainsttworepresentativeviruses,CAP45_G3andDu156.12,andtheir
glycan-eliminatedN156A,N160Avariants.
Immunity47,524–537,September19,2017 529
we observed a similar sensitivity pattern as that of the WT glycan. The binding of CAP256.25 bnAb to WT CRF250
CAP256.25Ab,suggestingthatthegermlineresiduescontribute trimerandglycan-modifiedvariantsshowedreducedbindingre-
inalimitedwaytoneutralizationthroughtheirinteractionswith sponses upon a2,6-sialidase treatment, except for the N156A
the Env glycans. In contrast, the CDRH2 somatically mutated variant, to which the relatively low binding response remained
substitutedAbvariants,particularlyinvolvingtheH59Asubstitu- largely unchanged upon desialylation (Figures 4B and S4B).
tion,showedsomeverymarkeddifferenceswithN160glycan- Overall, for WT, N135A, and N141A CRF250 trimer variants,
eliminatedvirusesfromWTCAP256.25Ab.Figure3Aillustrates theeffectsofsialidaseweremodestformostAbs(FiguresS4A
considerabledecreasesorevencompletelossofneutralizingac- and S4B). For the N160A variant, the effects of sialidase were
tivityinsomecases(Figure3A).Forexample,Figure3Bshows somewhat greater (Figures S4Aand S4B).Therefore, although
that WT CAP256.25Ab neutralizes two representative viruses, the earlier data provide evidence as to the interaction of
CAP45_G3andDu156_12,andtheirN160AandN156Avariants CAP256 Abs with sialic acid, the glycan elimination studies
butthattheH59AvariantAbisfarlesseffectiveagainsttheN160A here suggest possible involvement of N156 but they do not
viruses.FarlessdramaticeffectsareseenfortheN156Avirus, unambiguouslydeterminetheglycaninvolved.
implyingthatinteractionoftheCDRH2somaticmutatedresidues Inearlierstudies,structuralanalysisofthePG16AbwithaV1V2
withtheN156glycan(theremainingkeyglycanintheabsenceof scaffold showed that the terminal sialic acid residue at glycan
N160)ismostimportantfortheeffectsobserved. N173 makes one and two hydrogen bonds with the CDRH2
WealsoincludedinthisanalysisaCAP256Ablineagemem- germline-encodedresidueK57andthesomaticallymutatedres-
ber,CAP256.12,thathadaccumulatedsomaticmutationscom- idueH59,respectively,inadditiontothreehydrogenbondswith
parabletotheotherCAP256bnAbmembers,includingthetwo PG16Ablightchainresidues(Figure4C;Panceraetal.,2013).Of
glycan-specific CDRH2 somatic mutations, but failed to show note,however,thePG16Abshowedanenrichmentofonly2%of
broadreactivitytotheviruses(an‘‘off-trackAb’’inthenomencla- thetotal ZM109FV1V2on scaffold thatpossessed theappro-
ture of Bhiman et al., 2015). The results revealed that the priateglycoforms,suggestinganinherentglycanheterogeneity
CAP256.12 Ab could efficiently neutralize certain isolates only atthissiteandastrongpreferenceofPG16tobinda2,6-linked
when the glycan at position N160 was eliminated, suggesting sialicacid-containingglycoforms.Recentstudiesusingchroma-
thatthisAbmayhavefailedtoaccommodateoraffinitymature tography coupled mass spectrometry analysis of soluble Env
withrespecttotheN160glycan(Figure3A). proteins suggested that the N156 glycan position is occupied
Overall,theresultsdemonstratethatboththegermlineandso- largelybyahigh-mannoseglycan(Behrensetal.,2016;Panico
matic mutated CDRH2 key heavy chain residues contribute to et al., 2016). Another study (Cao et al., 2017) indicated that
neutralization by CAP256.25 and PG16 but that somatically N156 is occupied either by high-mannose or high-mannose
mutatedresiduesarelikelymostimportant.Further,N156may with some proportion of complex glycans for several soluble
harbortheinteractingsialicacids. SOSIP.664 trimers, including the CRF250 trimer used in this
study.Furtherstudiesarerequiredtofullyunderstandthecom-
N156GlycansMayBeartheTerminalSialicAcidsthat positionsoftheglycansintheV2apexregionondifferentisolates
ContacttheKeyHeavyChainResiduesonCAP256and andexpressedfromdifferentcelltypes.Itshouldalsobenoted
PG9FamilyAntibodies thatonlyasub-populationofN156orN173glycanmaybesialy-
Wewishedtoobtainfurtherevidencefortheglycanorglycans latedbutthismaybesufficienttodrivesialicacid-specificaffinity
thatbearthesialicacidtargetedbyCAP256Abs.Weincorpo- maturationintwoindependentV2apexbnAblineages.
ratedsubstitutionsontheCRF250SOSIPbackbonetoindividu- Altogether, these results are consistent with the importance
allyeliminateeachofthefourV1V2apexglycans(N135,N141, of a sialic acid residue, possibly on the glycan N156 or N173
N156, and N160) in turn, reasoning that treatment with a2,6- at the V2 apex site, in driving the affinity maturation of the
sialidaseandcomparisontotheeffectonWTtrimermightreveal CDRH2residues,Y59toHandK64toW,inCAP256andPG9
whichoftheglycanswasmostlikelytoharborthecriticalsialic V2apexbnAbprototypes.
acid(s) (Figure 4A). Due to the inability of PGT145 Ab to bind
N160glycan-eliminatedtrimers(Andrabietal.,2015),wechose ImprovedSialicAcidBindingIsAssociatedwith
2G12AbtopurifytheWTCRF250trimeranditssingleglycan DevelopmentofbnAbs;FailuretoMatureSuchBinding
variants prior to a2,6-sialidase treatment. Octet was used LeadstoNon-bnAbs
to investigate the binding of a selection of CAP256 mAbs to Togainabetterunderstandingoftheimportanceofsialicacid
theglycovariantsuntreatedortreatedwitha2,6-sialidase. recognition and the Ab mutations described above on bnAb
Theeffectsofsialidasetreatmentwereparticularlymarkedfor development,weassessedtheeffectsofsialidasetreatmentof
mAbCAP256.21,producinggenerallygreatlyreducedbinding. CRF250trimeronbindingbythecompletesetofCAP256Abs.
TheN156Avariantshowedlowerbindingoverallandalesseref- WerankedthesetofAbsaccordingtothepercentagechange
fect of sialidase treatment, implying that this Ab is dependent in octet binding (Figures 5A and 5B). The CAP256 Abs that
upon asialic acid at N156.Of note, CAP256.21 Ab bound the displayed the most significant loss of binding (red color) upon
N160A variant trimer with a higher affinity than even the WT removal of a2,6-linked sialic acid (CAP256.21, CAP256.30,
CRF250trimer,presumablyinvolvinganincreaseddependence CAP256.31,CAP256.12,andCAP256.13)hadeitherthegerm-
ontheN156glycanintheabsenceoftheN160glycan(Figure4B). line CDRH2 residue, D53, mutated to E or had both CDRH2
However,upona2,6-sialidasetreatment,bindingofCAP256.21 germlineresiduesD53andK57mutatedtoAandY,respectively
was dramatically lost, indicating that the sialic acid-specific (Figures 5A and 5B). All fiveof these Abs became ‘‘off target’’
interaction of this Ab at least is mediated through the N156 Abs,suggestingthatan‘‘over-dependence’’onsialicacidmight
530 Immunity47,524–537,September19,2017
Figure4. BindingofSelectCAP256AbstoCRF250TrimerV2ApexGlycanVariantsShowsVaryingSensitivitytoa2,6-SialidaseTreatment
(A)SchematicrepresentationoftheV2apexindividualglycansubstitutedvariantsonCRF250SOSIP.664trimerbackbone.
(B)Octetbindingcurves(association:120s[180–300]anddissociation:240s[300–540])ofCAP256.21andCAP256.25AbswithCRF250trimerglycovariants.
ThebindingresponseofCAP256.25bnAbwiththeCRF250N156Avariantisfixedto0.2nanometer(nm)forclarity.
(C)RibbonrepresentationofPG16antibody(green)incomplexwithisolateZM109FV1V2loop(cyan)onascaffold(modifiedfromPanceraetal.,2013).The
glycansatN160(orange;shownaslines)andN173(magenta;shownaslines)positionsandthesialicacidresidue(SIA;red)arelabeled.ThePG16heavychain
CDRH2residues(D53,K57,H59,andW64)arehighlighted(blue).TheresiduesK57andH59ofthePG16heavychainshowhydrogenbonding(yellowdottedline)
withaterminalsialicacidresidueofN173glycan.
SeealsoFigureS4.
bedetrimentaltothedevelopmentofneutralizationbreadth.In InthemiddlepartofFigure5A(lightred)areAbsthatshowed
contrast,CAP256Abmembersthatretainedthegermlineresi- very modest changes upon removal of a2,6-linked sialic acid.
dues Y59 and K64, or mutated at these positions to residues These Abs typically have D53-K57-H59-W64 and show the
other than H at 59 and W at 64, generally showed enhanced greatest breadth of neutralization. For example, the broadest
binding to the a2,6-linked sialic acid-trimmed CRF250 trimer neutralizing Ab member, CAP256.25, exhibited very modest
(Figures5Aand5B,yellowin5B).Thus,theunmutatedancestor dependenceonthea2,6-linkedsialicacid.Thus,thedatasug-
version of CAP256 Abs may actually bind better to trimers gestthatitmaybevitalforadevelopingbnAbto initiatesialic
lackingsialicacid. acidrecognitionbutthenkeepafinebalanceandnotbecome
Immunity47,524–537,September19,2017 531
Figure5. BindingoftheFull-RangeofCAP256LineageAbstoCRF250SOSIP.664ShowsaGradationofSensitivitytoa2,6-SialidaseTreat-
mentDependentuponKeyHCDR2Residues
(A)OctetbindingofCAP256-derivedmAbstoPGT145Abaffinity-purifiedCRF250SOSIP.664trimer(WT)anda2,6-desialylatedversion.TheCAP256mAbswere
capturedonananti-humanIgG-FcsensorandthentheCRF250trimerforms(100nM)flowedover.Thebindingresponse(nanometers[nm])toWTandthea2,6-
desialylatedtrimersisshownascolor-codednumericalvaluesandtheCAP256mAbsarearrangedwithrespecttotheirbindingsensitivitytoa2,6-desialylated
CRF250trimer.TheaminoacidsatfourpositionsintheCDRH2(withrespecttogermlinepositionsD53,K57,Y59,andK64)areindicatedforeachAb.
(B)Percent(%)changeintheoctetbindingresponsesareshownasheatmapforthedesialylatedtrimerformandiscalculatedas(Abbindingresponsetothe
a2,6-desialylatedCRF250trimer)/(bindingresponsetotheCRF250WTtrimer).
(C)PhylogenetictreeshowingthedevelopmentoftheCAP256bnAblineagefromtheVH3-30*18germlinegene.Thebranchesinthetreearecolor-codedand
demonstratetheevolutionofaminoacidresiduesattheabove-mentionedfourpositionsinCDRH2.Thegermlineresidues(DKYK)andthemostevolvedresidues
(DKHW)inglycan-reactiveAbsareindicatedingreenandred,respectively.
SeealsoFigureS2C.
over focused on sialic acid. Subsequently, this ability of the previously(Doria-Roseetal.,2014,2015).Thephylogenetictree
most mature CAP256 bnAb members to recognize a broad showed a strong bias toward the development of CAP256 Ab
diversity of features within the V2 apex epitope, including sub-lineagemembersthathadaccumulatedsialicacid-specific
a reduced dependence on sialic acid-bearing glycans, may CDRH2somaticmutations(Figure5C).
increase their effectiveness against a wide range of viruses. Overall,theseresultssuggestthataffinitymaturationinasub-
The bnAbs may not only neutralize viruses with more varied setofCAP256Ablineagememberstoimprovesialicacidbind-
protein epitopes but also tolerate glycoform heterogeneity at ingprovidesthemwithaselectiveadvantageintheprogression
thissite. towardabnAbphenotype.Anothersubsetoflineagemembers
To further examine whether the sialic acid-specific affinity becomes over-dependent on sialic acid binding, loses some
maturation of the glycan-specific CAP256Ab sub-lineage was interaction flexibility, and shows moderate neutralization
beneficial for the overall development of broad and potent Ab breadth.Athirdsubsetfailstoacquirethecrucialmutationsto
sub-lineagemembers,weanalyzednext-generationsequencing effectivelyinteractwithsialicacidandshowslittleornobreadth
(NGS)dataofBcelltranscriptsfromtheCAP256donorreported ofneutralization.
532 Immunity47,524–537,September19,2017