Emerging Antiviral Strategies to Interfere with Influenza Virus Entry

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Emerging Antiviral Strategies to Interfere
with Influenza Virus Entry
EvelienVanderlindenandLieveNaesens
RegaInstituteforMedicalResearch,KULeuven,Leuven,Belgium
PublishedonlineinWileyOnlineLibrary(wileyonlinelibrary.com).
DOI10.1002/med.21289
(cid:2)
Abstract: InfluenzaAandBvirusesarehighlycontagiousrespiratorypathogenswithaconsiderable
medicalandsocioeconomicalburdenandknownpandemicpotential.Currentinfluenzavaccinesrequire
annual updating and provide only partial protection in some risk groups. Due to the global spread of
viruseswithresistancetotheM2protonchannelinhibitoramantadineortheneuraminidaseinhibitor
oseltamivir,novelantiviralagentswithanoriginalmodeofactionareurgentlyneeded.Weherefocuson
emergingoptionstointerferewiththeinfluenzavirusentryprocess,whichconsistsofthefollowingsteps:
attachment of the viral hemagglutinin to the sialylated host cell receptors, endocytosis, M2-mediated
uncoating,lowpH-inducedmembranefusion,and,finally,importoftheviralribonucleoproteinintothe
nucleus.Wereviewthecurrentfunctionalandstructuralinsightsintheviralandcellularcomponentsof
this entry process, and the diverse antiviral strategies that are being explored. This encompasses small
molecule inhibitors as well as macromolecules such as therapeutic antibodies. There is optimism that
atleastsomeoftheseinnovativeconceptstoblockinfluenzavirusentrywillproceedfromtheproofof
concepttoamoreadvancedstage.Specialattentionisthereforegiventothechallengingissuesofinfluenza
virus(sub)type-dependentactivityorpotentialdrugresistance. (cid:2)C 2013WileyPeriodicals,Inc.Med.Res.Rev.,
00,No.0,1–39,2013
Keywords: influenzavirus;antiviral;hemagglutinin;M2channel;nucleoprotein
1. INTRODUCTION
HumaninfluenzaAandBvirusescausesignificantmorbidityandmortality,particularlyinin-
fantsandelderlypeople,orthosesufferingfrompreexistingpathologyorimmunodeficiency.1,2
The United States Centers for Disease Control and Prevention estimated that, from 1976 to
2000,seasonalinfluenzaepidemicswereresponsiblefor>200,000annualhospitalizationsand
anannualaverageof>30,000influenza-associateddeathsintheUSA.3Approximately90%of
theinfluenza-associateddeathsoccuramongadultsaged≥65years.4
To evade the immune response, the circulating influenza H3N2, H1N1, and B viruses
continuously change their antigens, and this explains why current influenza vaccines require
Correspondenceto:LieveNaesens,RegaInstituteforMedicalResearch,KULeuven,Minderbroedersstraat10,
B-3000Leuven,Belgium.E-mail:[email protected]
MedicinalResearchReviews,00,No.0,1–39,2013
(cid:2)C 2013WileyPeriodicals,Inc.
(cid:2)
2 VANDERLINDENANDNAESENS
Figure1. Overviewoftheinfluenzavirusentryandreplicationprocess.Intheinsetontheright,thedifferent
virioncomponentsarespecified.(a)AfterbindingoftheviralHAtosialylatedglycansonthehostcellsurface,
thevirusisinternalizedbyendocytosis.(b)AcidificationoftheendosomeleadstoactivationoftheM2proton
channelandvirionacidification,resultinginvirusuncoating(i.e.,dissociationofthevRNPsfromtheM1capsid
protein).ThelowpHinsidetheendosomealsotriggersaconformationalchangeintheHA,leadingtofusion
oftheviralandendosomalmembranes.AftervRNPreleaseinthecytoplasmanddissociationofresidualM1,
nuclearlocalizationsignalsinNPdirectthetransportofthevRNPsintothenucleus.(c)Inthenucleus,theviral
polymerasestartsmRNAsynthesisbycleavingoff5(cid:4)-cappedRNAfragmentsfromhostcellpre-mRNAs.Then,
viralmRNAtranscriptionisinitiatedfromthe3(cid:4)endofthecleavedRNAcap.(d)ViralmRNAsaretransportedto
thecytoplasmfortranslationintoviralproteins.HA,M2,andNAareprocessedintheendoplasmicreticulumand
theGolgiapparatus,andsubsequentlytransportedtothecellmembrane.(e)BesidesviralmRNAsynthesis,
theviralpolymeraseperformstheunprimedreplicationofvRNAs.ThevRNAsarefirsttranscribedintopositive-
strandedcRNAs,whichthenfunctionasthetemplateforthesynthesisofnewvRNAs.Duringtheirsynthesis,
vRNAsandcRNAsareencapsidatedbyNPs.ExportofthenewlyformedvRNPsintothecytoplasmismediated
byanM1-NS2complexthatisboundtothevRNPs.(f)Astheyreachthecellmembrane,thevRNPsassociate
withviralglycoproteinsattheplasmamembranefromwhichnewvirionsbudoff.Finally,theNAcleavesthesialic
acidterminionviralandcellmembraneglycoproteins,therebyreleasingtheprogenyvirionsfromthehostcell.
annual updating. These vaccines provide inadequate protection in some target populations
(particularlytheelderly).5Besides,thereisconcernthatthewidelyspreadandhighlypathogenic
avian H5N1 influenza virus may acquire human transmissibility and become a potentially
disastrouspandemicvirus.Thehumancase-fatalityrateofthisavianH5N1isreportedtobe
59%,6 although some investigators have raised the possibility that subclinical cases of H5N1
infectionsinhumansmayremainunnoticed.7Forcomparison,thecase-fatalityrateofthe1918
influenzaviruswasestimated>2.5%.8
AsshowninFigure1,theinfluenzavirusreplicationcyclecontainsseveralstepsamenable
toantiviralintervention.Thisreviewfocusesontheviralentrypathway,which,giventheacute
onset of influenza virus infection and the inflammation associated with it, is a particularly
attractive process to interfere with. We describe the current insights into the structure and
functionsoftheviralandcellularcomponentsinvolvedinthisentryprocess,andtheantiviral
strategiesthatarebeingexplored(anoverviewofthedescribedcompoundsisgiveninTableI).
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EMERGINGOPTIONSTOINTERFEREWITHINFLUENZAVIRUSENTRY 3
s
e
Referenc 79,808283,84858688,8990,92,939496,97101,102103104105106,107109112,114 124 126–128 129 135135137–139107,131
2
1 c
cspectrum H3,H9,HH13,H16 ype-specifi
ctivity 11,H2,1,H2, AsubtandBandBandB andB andB
A HHHBBHAAA A A
b
y
Demonstratedactivitorclinicalstatus nvitronvivonvivonvitronvivonvivonvitro/Invivonvitro/Invivonvitro/Invivonvitronvivonvitronvivonvitronvitronvivo/PhaseII nvitro nvivoNaturalmedicine nvitro nvitronvitroNotactiveinhumansnvitro
IIIIIIIIIIIIIIII I I I II I
ors aProposedmodeofaction BindingclosetoHARBSIdemIdemIdemIdemIdemIdemIdemIdemBindingtoHARBSIdemIdemIdemNotpreciselyknownBindingtosialicacidReceptordestruction MembranefluiditymodulatorInhibitsvirusinternalization;immunomodulatorIntercalatesintoviralmembranesV-ATPaseinhibitorIdemRaisesendosomalpHIntra-cytoplasmicvirustrapping
bit
hi
n
i b y
y a d
r F o e
OverviewofreportedinfluenzavirusentTableI. CompoundcodeorclassGeneralstructure InhibitorsoftheHA-receptorinteractionCH65ImmunoglobulinorC05IdemS139/1Idem3A2and10C4IdemCR8033andCR8071IdemNanobodiesSingle-domainantibSurfactantproteinDLectinCL-43LectinCyanovirin-NLectinSPGandGM3SialylatedgangliosidLSTc-bearingliposomesSialylatedliposomePentadecapeptidesPeptideA22AptamerNMSO3Sulfatedsialyllipidc01andc03AcylatedpeptidesDAS181Sialidaseenzyme InhibitorsofviralendocytosisFattiviracinNeutralglycolipid GlycyrrhizinTriterpeneglycoside LJ001Smallmolecule BafilomycinA1SmallmoleculeDiphyllinSmallmoleculeChloroquine4-AminoquinolineSA-19Aglycoristocetinlipoglycopeptide
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References 146,155,159148161,166–169 55,175 176177,182179180178184,187–189194196,197,199 206–208 20921086 236–239 245 o(i.e.animal wasnot(yet)
v
cActivityspectrum AAA H3-specific H3-specificH1-andH2-specificH1-andH2-specificH1-andH2-specificH1-andH2-specificA AandB Group1HAs Group2HAsAllAHAsAandB A incellculture),invi heactivityspectrum
b (i.e. ce,t
DemonstratedactivityaProposedmodeofactionorclinicalstatus BlocktheM2channelApprovedIdemApprovedIdemInvitro BindstoHAstemandinhibitsInvitroHArefoldingIdemInvitroIdemInvitroIdemInvitroIdemInvitroIdemInvitroInhibitsmembranemixing?InvitroImmobilizessurfaceglycoproteinsInvitroInhibitorofHArefolding;Approved(RussiaandmembranefluiditymodulatorChina)BindtoconservedHA-stemInvivoepitope;inhibitHArefoldingIdemInvivoIdemInvivoIdemInvivo AggregatesNP;alsoinhibitsInvivovRNPactivity BroadproteinkinaseCinhibitorInvitro hemodeofactionrelevantforinfluenzavirusisgiven.nfluenzavirusactivityisgiven,inthesequence:invitro oanumberofinfluenzaAorBvirusestested,andheneclearlydefinedactivityspectrum.
ContinuedTableI. CompoundcodeorclassGeneralstructure InhibitorsofM2-mediatedprotontransportAmantadineAdamantaneRimantadineAdamantane+analoguesAdamantane InhibitorsofHA-mediatedfusionatlowpHTBHQSmallmolecule 4cSmallmoleculeBMY-27709SmallmoleculeCL61917SmallmoleculeStachyflinSmallmoleculeRO5464466SmallmoleculeDextransulfateSulfatedpolysaccharideRetrocyclin-2CircularpeptideArbidolSmallmolecule F10;CR6261ImmunoglobulinorFab CR8020ImmunoglobulinorFabFI6v3ImmunoglobulinorFabCR9114ImmunoglobulinorFab InhibitorsofNP-mediatedviralnuclearimport+NucleozinanaloguesSmallmolecule Inhibitorsofentry-relatedcellularproteinkinasesBisindolylmaleimideISmallmolecule aForcompoundswithbroaderantiviralactivity,onlytbThemostadvancedlevelfordemonstrationofanti-istudies),andinhumans.cFormostcompounds,literaturereportsarelimitedtspecified.Theinformationinthiscolumnindicatesth
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For antiviral approaches affecting other stages in the viral life cycle, the reader is referred to
otherrecentreviewarticles.9–12
2. CURRENTLYAVAILABLEANTI-INFLUENZAVIRUSDRUGS
Effectiveantiviraldrugstopreventortreatinfluenzainfectionsshouldatalltimesbeavailable.
Today, two classes of anti-influenza virus drugs exist: the M2 proton channel blockers (i.e.,
theadamantanecompounds,amantadineandrimantadine),andtheneuraminidaseinhibitors
(NAIs)(oseltamivirandzanamivir).13 Thefirsttwocompoundshavelimitedutility,sincethey
areassociatedwithneurologicalsideeffects,havenoactivityagainstinfluenzaBvirus,andthe
vastmajorityofcirculatingstrainsareadamantane-resistant.13 Adetaileddescriptionoftheir
mode of action and resistance mechanisms will be given below. The obviously superior class
of anti-influenza virus drugs are the NAIs oseltamivir and zanamivir that are active against
all influenza A and B viruses. These structural analogues of sialic acid bind to the catalytic
pocketoftheviralNAandinhibititsfunctioninreleasingthenewlyproducedvirusfromthe
host cells.14,15 Thereisa criticaldifferenceintheNAbinding modeofoseltamivir compared
to that of zanamivir, which explains their significantly different resistance profile. Due to its
largerhydrophobicsidechain,oseltamivirrequiresrotationofthenoncatalyticGlu276residue
withinNAtocreateabindingspaceforoseltamivir.16Bycontrast,thesmallersizeofzanamivir
enablesdirectbindingofthiscompoundtoNA.InamutantN1NAcontainingaHistoTyr
substitution at position 274, this rotation can no longer occur, rendering the NA resistant to
oseltamivir binding. During the 2008–2009 season, oseltamivir-resistant H1N1 viruses were
isolated all over the globe, even from untreated patients.17,18 In a Japanese study in 2004,
nine out of 50 children treated with oseltamivir carried oseltamivir-resistant H3N2 viruses.19
Fortunately, oseltamivir-resistant viruses are still sensitive to zanamivir, for which resistance
hasonlyscarcelybeenreported.20,21 Ontheotherhand,thepatient-unfriendlyadministration
routeforzanamivir(i.e.,bypowderinhalationdevice)explainswhyoseltamivir(whichisgiven
byoralcapsules)isgenerallypreferredintheclinicalsetting.Inhalationofzanamivirisapriori
excludedinpatientssufferingfromsevereinfluenzasymptomswithacuterespiratorydistress,
such as patients infected with the highly pathogenic avian H5N1 virus, or severe cases of the
2009pandemicH1N1virus.Toaddressthisissue,anintravenousformulationofzanamiviris
under consideration.22,23 Besides, new NAIs are being developed. Peramivir, which has to be
administeredintravenously,hasbeenlicensedinJapanandSouthKorea,while,intheUnited
States,itsusewastemporarilyallowedduringthe2009H1N1pandemic.24 Unfortunately,the
widespreadoseltamivir-resistantH1N1His274Tyrmutantsshowintermediatecross-resistance
to peramivir.25 Another NAI, laninamivir (CS-8958), was approved in Japan in 2010 and is
currentlyinPhaseIIItrialsintheUnitedStates.26,27 Thispromisingcompoundrequiresonly
onesingleintranasaladministration(basedonitslonghalf-life),andhasasimilarNAbinding
mode and favorable resistance profile as zanamivir.28 Finally, novel NAIs with a sialic acid-
relatedorunrelatedstructurehavebeendevelopedbyrationaldesign,butarestillintheearly
experimentalstage.29–31
To face the emerging resistance to NAIs (in particular, oseltamivir), entirely novel anti-
influenzavirusdrugsareurgentlyneeded.Thetwoproductsthataremostadvancedinclinical
developmentarethenucleobaseanalogueT-705(favipiravir)andthereceptordestroyingpro-
tein DAS181. For T-705, Phase III trials in the United States are pending. Its active ribose-
triphosphate metabolite is recognized by the influenza virus polymerase, causing competitive
inhibitionofviralRNAsynthesisand/orlethalviralmutagenesis.32T-705hasbroadanti-RNA
virus activity beyond influenza virus and is presumed (based on cell culture data) to have a
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high barrier for viral resistance.33 The second agent, DAS181, is currently in Phase II trials.
Thisrecombinantproteinisasialidasethatcleavestheinfluenzavirusreceptorsintheairway
epithelia.MoredetailsonDAS181areprovidedinSection3.
3. INHIBITORSOFTHEHEMAGGLUTININ-RECEPTORINTERACTION
A. StructureoftheViralHemagglutinin
Within the influenza virus particle, the single-stranded, negative-oriented RNA genome is
dividedovereightviralribonucleoprotein(vRNP)segments,whichareprotectedbythecapsid
shell formed by the M1 protein, further surrounded by the viral envelope. Two viral spike
proteinsprotrudefromthevirion:thehemagglutinin(HA)andNA,whichhavealeadingrole
in viral entry and release, respectively. The HA and NA glycoproteins are the main antigens
against which the host immune response is raised. In the case of influenza A virus, 17 HA
and 10 NA subtypes are known, which are all present in aquatic birds, the natural reservoir
for influenza A viruses.34 The only exception is H17, which was isolated only recently from
bats.35,36 Theemergenceofanewpandemicvirusisexplainedbythereassortmentofgenome
segments,whichoccasionallyoccursupondualinfectionofananimalspecies(suchasapig)
thatcarriestheavian-aswellasthehuman-typeinfluenzavirusreceptors.37
The influenza virus HA (Fig. 2A) is a homotrimeric type 1 membrane glycoprotein. Its
membrane-distal globular head domain contains the receptor binding site (RBS), whereas
the HA stem structure (which contains the fusion peptide) is responsible for intraendosomal
membrane fusion.34 In influenza virus-infected cells, HA is first synthesized as its precursor
proteinHA0,whichassemblesintoanoncovalentlylinkedhomotrimer38andiscleavedintotwo
polypeptides(HA1andHA2containing,inthecaseofH3,328and221aminoacids,respec-
tively),whichremaincovalentlyattachedbyadisulfidebond.39 FormostHAs,HA0cleavage
occursatasinglearginineresidueandisperformedbyamembrane-boundorsecretedserine
proteasethatisrestrictedtobronchiolarepithelium,suchastryptaseClara,thehumanairway
trypsin-likeproteaseorTMPRSS2.40,41TheHAsfromhighlypathogenicavianvirusescontain
aseriesofbasicresiduesattheircleavagesite,42allowingrecognitionbyfurin-likeintracellular
proteasesthatarewidelydistributedinaviantissues,thusexplainingtheirsystemicspreadand
high virulence.43 Inhibition of the cellular proteases performing HA0 cleavage is an original
antiviralstrategy,andpeptidomimeticfurininhibitorshaveproventoinhibitthereplicationof
an avian influenza virus in cell culture.44 After HA0 cleavage, minor rearrangements lead to
insertion of the fusion peptide (located at the N-terminus of HA2) into a negatively charged
cavity,thusprimingtheHAforpH-dependentfusion.40PosttranslationalmodificationsofHA
comprise the addition of acyl chains to the short cytoplasmic tail,45 and N-glycosylation at
several asparagine residues in the ectodomain.39 Besides masking the antigenic epitopes by
stericallyhinderingantibodyrecognition,46,47theN-linkedglycansalsofunctioninthecorrect
foldingofHAintheendoplasmicreticulum,48,49modulationofreceptorbinding,50controlling
HA0 cleavage,51 and maintaining the HA in its metastable conformation required for fusion
activity.52 TheN-glycansthataremostconservedamongvariousinfluenzaHAsarelocatedat
theN-terminusofHA0(oraftercleavage,HA1)48andintheHAstemregion.53
The 17 influenza HA subtypes are classified into two phylogenetic groups (Fig. 2B). The
H1andH5HAsbelongtothesamecladewithingroup1,whereasH3HAbelongstogroup
2.35,54,55AlthoughthisphylogeneticclassificationwasprimarilybasedonHAproteinsequence,
comparisonofavailableHAcrystalstructuresindicatesthattheregionsinvolvedinmembrane
fusionshowstrikingsimilaritiesonagroup-specificbasis.54
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Figure2. StructureandclassificationofinfluenzaAHAs.(A)Structureoftheviralhemagglutinin,showingthe
bindingsiteforsialicacid(violet)intheglobularheaddomain(blueribbonstructure),aswellasthebinding
pocketsintheHAstemstructureforfusioninhibitorsreportedtopreventtheHAconformationalchange,thatis,
thesmall-moleculeinhibitorTBHQ(orange)andthebroad-actingantibodiesF10(pink)andCR6261(yellow).
TwoHAsubunitsarerepresentedbytheircombinedmolecularsurface,whilethethirdoneisshowninaribbon
diagram.[ReprintedbypermissionfromMacmillanPublishersLtd:NatureStructural&MolecularBiologyRef.
Dasetal.10 (cid:2)C (2010).](B)PhylogenetictreeofinfluenzaAHAs.Group1(cyan)canbesubdividedintothree
clades (H8, H9, and H12; H1, H2, H5, and H6; H11, H13, and H16). Group 2 (green) is subdivided in two
clades(H3,H4,andH14;H7,H10,andH15).ThenewlyidentifiedH17isclassifiedintheH1cladeofgroup
1.35 [TakenfromRusselletal.,55 Copyright(2008)NationalAcademyofSciences,USA.](C)DetailoftheHA
RBSindicatingthebindingmodeoftheCDR-H3loop(heavy-chaincomplementaritydeterminingregion3)of
antibodyCH65,whichactsasasialicacidmimic.TheHARBSiscoloredpinkandtheCDR-H3loopisshown
inblue.Theresiduesrelevantfortheantibody-HAinteractionarelabeled;someoftheseareconservedHA1
residuesinvolvedinsialicacidbinding(Ser1361,Trp1531,andLeu1941).[Taken,withpermission,fromWhittle
etal.79](D)CartoonofthestructuralchangesinHAduringtheHA-mediatedmembranefusionprocess.[a]The
HARBSbindstothesialylatedcellreceptor(ingreen).[b]TheacidicpHintheendosomeinducesHArefolding,
whichleadstotheexposureofthefusionpeptide(inred)anditsinsertionintheendosomalmembrane.[c]As
aresultoffurtherconformationalchangesinHA,theviralandendosomalmembranesarepulledtogether.[d]
Mixingoftheoutermembraneleafletsgeneratestheprefusionstalkintermediate.Thedashedlinesseparatethe
innerandoutermembraneleaflets.[TakenfromHamiltonetal.,251withpermission]
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B. Species-SpecificVirusBindingtoSialylatedGlycanReceptors
In the first step of the infection cycle, the HA attaches, via the RBS in its globular head, to
sialylatedglycoproteinsorglycolipidsonthehostepithelialcells.56ThisHA-receptorinteraction
ishighlyspecificforsialylglycoconjugatesandplaysanessentialpartinthespeciesrecognition
of avian versus human influenza viruses.57 The HAs from human-adapted viruses, including
thepandemicvirusesoftheH1N1,H2N2,orH3N2subtype,preferentiallybindtocell-surface
glycansterminatinginα2-6-linkedsialyl-galactosylresidues[Neu5Ac(α2-6)Gal],whereasavian
influenza A viruses have a preference for α2-3-linked sialyl-galactosyl termini.58–62 The HAs
ofinfluenzaBviruseswhich,innature,areonlydetectedinhumansandseals,showabinding
preference for α2-6-linked glycans.63–65 Thus, it is important to underline that the species
specificityoftheHA–glycaninteractionisnotbasedonrecognitionoftheterminalsialicacid
itself, but, rather, its linkage to the vicinal galactose and the sugars beyond galactose.66,67 A
correlation between glycan topology and species specificity was established from HA–glycan
cocrystal structures as well as glycan array data.58 With regard to the HA residues that are
directlyinvolvedinsialicacidbinding,thesearehighlyconservedacrossdifferentHAsubtypes.
Theseaminoacids(Tyr98 ,Ser136 ,Trp153 ,His183 ,Leu194 )[aminoacidnumberingbased
1 1 1 1 1
on the H3 HA sequence; the suffixes 1 and 2 denote location in the HA1 and HA2 subunit,
respectively]leadtoafixedorientationofthesialicacidrelativetotheHARBS.68
Althoughsialicacidisgenerallyconsideredtobetheprimaryattachmentreceptor,influenza
virusisabletobindandenter(thoughconsiderablylessefficiently)intocellsofwhichallsurface
sialicacids,whetherattachedtoglycolipidsorglycoproteins,wereremovedbytreatmentwith
exogenousMicromonosporaviridifacienssialidase.69 Hence,ithasbeenproposedthat,besides
sialic acid, other receptors may be involved in influenza virus entry, which can work either
independentlyorviaamultistepprocess.69,70
Which specific amino acid residues in HA govern its avian versus human receptor pref-
erence, varies among the different HAs, and is still incompletely understood, although α2-6
tropismisgenerallylinkedtoresiduesAsp190 andAsp225 inH1andLeu226 inH2andH3
1 1 1
HAs.71 To cross the avian–human species barrier, acquisition of the human receptor binding
preference is not sufficient, since additional amino acid changes are required, particularly in
theinfluenzaviruspolymerasecomplex.71InarecentstudyinwhichtheavianH5N1viruswas
passedinferrets,fourmutationsintheheaddomainofH5HA,combinedwiththeGlu627Lys
hallmarkmutationinthePB2subunitofthepolymerasecomplex,wereabletoleadtoairborne
transmissionofthisvirusinferrets.72AsimilarstudywithareassortantviruscarryingtheHA
ofavianH5N1alsoconcludedthatitsavian-to-mammalianadaptationrequiresacombination
ofHAmutationstonotonlyswitchitsreceptorpreferencefromα2-3toα2-6,butalsoincrease
thestabilityoftheHAprotein.73
C. AntiviralStrategiestoInterferewithHA-ReceptorBinding
When considering the HA-receptor binding as an antiviral target, the multivalent nature of
this interaction may present as a challenge. This binding is highly dynamic and involves an
ensembleofsialylatedglycansmakingcontactwithmultipleHAtrimers.74Inthismanner,the
avidityeffectsofthemultivalentinteractioncompensatefortheintrinsicallylowglycanbinding
affinity for a single binding site on HA [with a dissociation constant (Kd) in the millimolar
range].75
Thus, to develop inhibitors that block the receptor binding of HA, at least three factors
needtobetakenintoaccount:largesequencevariationamongHAsubtypesandantigenicdrift
of HA; avian versus human-specific receptor use; and multivalent nature of the HA-receptor
interaction. An ideal inhibitor would be species- and HA subtype-independent. There are
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threeconceivablestrategiesforinhibitingattachmentofinfluenzavirustoitstargetcell:(i)an
antiviralcompoundbindingtotheHARBS;(ii)aninhibitorblockingthesialicacid-containing
receptorsontheepithelialcellmembrane;or(iii)areceptor-destroyingagent.
1. HA-BindingAgents
Virus-neutralizingantibodies
Thefirstandnaturaltypesofbindinginhibitorsarethevirus-neutralizingantibodiesraiseddur-
ingthecourseofaninfluenzavirusinfection.Theseneutralizingantibodiesarepredominantly
directed toward the surface of the membrane-distal globular head domain of HA.76 During
the 1918 pandemic, some patients were treated with human blood products from recovering
influenza patients.77 Eight controlled studies reported between 1918 and 1925 were recently
reviewed,anditwasconcludedthattheoverallcase-fatalityratewasreducedfrom37%among
control patients to 16% among treated patients. Treatment was most effective when initiated
early(i.e.,lessthan4daysafterpneumoniabecameapparent).77 Thesehistoricaldatademon-
stratethatpassiveimmunizationwithanti-HAantibodiescanbeconsideredincaseapandemic
occurs.Obviously,safetyconsiderationsabouttheuseofpatient-derivedmaterialsneedtobe
addressed.AnelegantmethodfortheisolationofhumanantibodieswasreportedbySimmons
etal.,78 whopreparedH5N1neutralizingmonoclonalantibodiesfromthememoryB-cellsof
patients recovered from an H5N1 infection. Two monoclonal antibodies were effective in a
mouseinfluenzamodelwhenadministerednolaterthan72hrafterinfection.78 Anattractive
new concept is the development of monoclonal antibodies that bind to the conserved RBS
of HA and, hence, are endowed with heterosubtypic HA neutralizing activity. A first human
monoclonalantibodydirectedagainstH1HA,encodedCH65,wasderivedfromplasmacells
of a person immunized with the 2007 trivalent influenza vaccine. Cocrystallization of its Fab
fragment with H1 HA revealed that this antibody acts as a sialic acid mimic since the tip of
itsheavy-chaincomplementaritydeterminingregion3(HCDR3)insertsintheRBSofH1HA
(Fig.2C).79SinceCH65wasshowntoneutralize31outof36H1N1isolatescoveringaperiod
of more than 30 years, and to interact with the conserved RBS itself, resistance selection by
CH65 may be expected to be rare, unless associated with reduced viral fitness.79,80 It should
however be noted that the RBS of HA is smaller than the interaction site of an antibody81
and,therefore,CH65formsadditionalinteractionswithRBSsurroundingresiduesthatareless
conservedamongthedifferentHAs.ThemorebroadlyactingmonoclonalantibodyC05binds
toH1,H2,H3,H9,andH12HAsandwasisolatedfromaphage-displaylibraryconstructed
frombonemarrowdonatedafterseasonalinfluenzainfection.Cocrystallizationstudiesdemon-
stratedthattheHCDR3partofC05formsaloopthatinsertsintotheconservedRBSofHA,
whileitsHCDR1regionmakesonlyminimalcontactwithRBSsurroundingandmorevariable
residues.82 Athirdcross-reactivemonoclonalantibody,S139/1,neutralizesH1,H2,H3,H13,
andH16virusstrains.83TheHCDR2regionofS139/1wasshowntoformmultiplehydropho-
bicinteractionswithintheRBSofH3HA.Theratherlowaffinityofthisbindinginteraction
iscompensatedinthebivalentIgGmolecule,andthisavidityeffectisrequiredtobroadenthe
neutralizing activity of S139/1 to strains of the H1, H2, H13, and H16 subtypes.84 Regard-
ing influenza B viruses, the human monoclonal antibodies 3A2 and 10C4, reactive against B
viruses of the Yamagata lineage, recognize the 190-helix (residues 190–198 in HA1) near the
RBS.85ThehumanmonoclonalantibodiesCR8033andCR8071wereshowntoneutralizeboth
YamagataandVictorialineageBvirusesandprotectmiceafterchallengewithalethaldoseof
influenzavirus.86Althoughthetherapeuticuseofananti-influenzaantibodymayappearcom-
plicated,someparallelcanbeseenwiththepalivizumabantibodythatisalreadyinuseforthe
prophylaxisofanotherrespiratoryvirus,thatis,respiratorysyncytialvirus(RSV).87 Aninno-
vativestrategytoimprovethepharmacokineticsandreducetheproductioncostoftherapeutic
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10 VANDERLINDENANDNAESENS
antibodiesconsistsofsingle-domainantibodyfragments(alsoreferredtoasNanobodies)de-
rivedfromcamelidimmunoglobulins.88 ANanobodydirectedtotheglobularheadofH5HA
wasshowntobeeffectiveinH5N1-infectedmice.TheactivityofthemonovalentNanobodywas
increasedbyafactor60whenusingabivalentformat,consistingoftwoparatopecontaining
domainsconnectedbyaflexiblelinker.89
Lectins
AnothertypeofimmuneproteinscapableofcatchingvirusesisthecollagenousC-typelectins
(referredtoascollectins)suchasthelungsurfactantproteins.TheroleofsurfactantproteinD
(SP-D)intheinnateimmuneresponsetoinfluenzavirusisexplainedbyitscapacitytocause
virus particle aggregation, thereby preventing virus attachment to the host cells.90 Besides,
SP-Dhasvariousimmunologicaleffectsthataccountforitsabilitytolimitlunginflammation
byrespiratorypathogens.91 Regardingpotentialantiviraluse,designofmodifiedformsofthe
porcineSP-Dlectin(whichhashigheranti-influenzavirusactivitythanitshumancounterpart)
is aided by the growing insight into how its carbohydrate recognition domain (CRD) pre-
ciselyinteractswiththehigh-mannoseglycansattachedneartheRBSofHA.92,93 Inaddition,
N-linkedsialoglycansattachedtotheCRDofSP-Dareconsideredimportant,sincetheymay
cause additional interactions between the SP-D and the HA RBS and enhance the antiviral
effect.90Asimilaractionprinciple,thatis,bindingtohigh-mannosecarbohydratesontheviral
HA,accountsfortheanti-influenzavirusactivityofthebovineserumlectinCL-43.94Likewise,
cyanovirin-N,alectinisolatedfromEscherichiacoli,recognizeshigh-mannoseoligosaccharide
structuresondiverseviralglycoproteins,explainingitsbroadactivityagainstunrelatedviruses
such as influenza virus and HIV.95 Cyanovirin-N was shown to inhibit influenza virus repli-
cation in cell culture as well as mouse and ferret infection models.96,97 Although SP-D and
cyanovirin-N manifest broad anti-influenza A and B virus activity, some virus strains (such
as the A/PR/8/34 H1N1 strain) are known to be insensitive, due to the lack of particular
Asn-linked oligosaccharides on the head of their HA.98 The location and number of glycans
attached to the head of HA is quite variable, since acquisition of epitope shielding oligosac-
charides is part of the viral immune escape.46 In contrast, the glycans attached to the HA
stemhaveastructuralfunctioninproteinrefolding,andthecorrespondingglycosylationsites
arethereforemoreconserved.52,53 Thisimpliesthatantiviraluseoflectincompoundsdirected
towardHAheadglycansmightleadtoescapemutantsdevoidofspecificglycans,althoughthe
newlyexposedantigenicsitesmightalsorenderthemutatedvirussusceptibletoimmunological
control.99
Sialyl-containingmacromoleculesandsialomimetics
An alternative approach to block the HA RBS makes use of receptor mimics, such as
sialyl-containing macromolecules. The gangliosides sialylparagloboside (SPG) and GM3
(Neu5Acα2-3Galβ1-4Glcβ1-1(cid:4)ceramide) were proven to bind to HA and inhibit the virus-
induced cytopathic effect,100–102 and their antiviral activity correlated with their HA binding
affinities.101ThehydrophobicceramidemoietyofSPGandGM3wasfoundessential,sincethe
uncoupledtrisaccharides3(cid:4)-sialyllactosamineand3(cid:4)-sialyllactose(whichconstitutethetermini
ofSPGandGM3,respectively)producednoeffect.Micelleformationofthesegangliosidesin
aqueous solution likely causes protrusion of their sialic acid parts toward the outside of the
micelles,resultinginhighsialicaciddensityand,hence,amultivalentbindinginteractionwith
HA.101
In a recent report, Hendricks et al. described that liposomes bearing sialylneolacto-N-
tetraose c (LSTc) can form multivalent interactions with influenza virus.103 In contrast to
MedicinalResearchReviewsDOI10.1002/med