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INFECTIOUSDISEASE(MSPASTERNACK,SECTIONEDITOR)HostandViralFactorsinRespiratorySyncytialVirusInfectionPeterMastrangeloRichardG.
HegelePublishedonline:15May2013SpringerScience+BusinessMediaNewYork2013AbstractRespiratorysyncytialvirus(RSV)isamajorworldwidepathogenforwhichthereisstillnoeffectivevaccineorantiviraltreatmentavailable,andimmunopro-phylaxiswithRSV-specicantibodies(e.
g.
,palivizumab)isusedinlimitedclinicalsettings.
Inthisreview,wedis-cussvirus–hostinteractionsrelevanttoRSVpathobiologyandhowadvancesincellandsystemsbiologyhaveacceleratedknowledgeinthisarea.
WealsohighlightrecentadvancesinunderstandingtherelationshipbetweenRSVbronchiolitisandsequelaeofrecurrentwheezingandasthma,newndingsintoanintriguinginteractionbetweenRSVandairpollution,andexcitingdevelopmentstowardthegoalofrealizingasafeandeffectiveRSVvaccine.
KeywordsRespiratorysyncytialvirusViralpathogenesisVirus–hostinteractionsViruspersistenceAsthmaAllergySystemsbiologyNucleolinAirpollutionVaccinologyIntroductionRespiratorysyncytialvirus(RSV)isasingle-stranded,negative-polarityPneumovirusthatinfectspeopleofallagesworldwide[1].
RSVisafrequentcauseofupperrespiratorytractinfectionsandotitismedia,andalsocausesseriouslowerrespiratorytractinfectionssuchasbronchi-olitisandpneumonia,particularlyininfantsandyoungchildren,immunocompromisedindividuals,andtheelderly.
RSVinfectionsinearlypostnatallifehavealsobeenassociatedwiththeonsetofallergicsensitizationandrecurrentwheezing/asthma.
Despitealmost60yearshav-ingpassedsincethediscoveryofRSV,thereisstillnolicensedvaccineavailable,andprophylaxiswithimmu-noglobulin(palivizumab)isusedonlyforasubsetof''high-risk''children(e.
g.
,pretermbirths,andinfantswithcongenitalheartdisease,congenitalimmunodeciency,and/orbronchopulmonarydysplasia).
AlthoughanumberofpharmacologicalagentshavebeenusedinRSVtherapy,theirefcacyandeffectivenessarepoor[2].
Here,wewillbrieyreviewsomeviralandhostfactorsimplicatedinthepathobiologyofRSVinfections,focusingonhowthediversityofthesefactorsmightberelatedtothevariabilityseeninclinicaldisease.
OurdiscussionwillalsoincludecomparingandcontrastingcharacteristicsofRSVinfectionsinvitroversusinvivo;currentperspectivesontherelationshipbetweenRSVinfections,pediatricallergy,andasthma;recentworkthatdescribesanovelrelationshipbetweenRSVandparticulatematter(airpollution);andprospectsforthedevelopmentandimplementationofasafe,effectiveRSVvaccine.
ViralandHostFactorsinRSVInfection:TowardanIntegratedPerspectiveRSVStructureHumanRSVisalamentousenvelopedvirusinwhichtheviralnucleicacidandstructuralandinternalproteinsaresurroundedbyaproteinaceousoutercoat[3].
AsanP.
MastrangeloR.
G.
Hegele(&)DepartmentofLaboratoryMedicineandPathobiology,UniversityofToronto,6231-1King'sCollegeCircle,Toronto,ONM5S1A8,Canadae-mail:richard.
hegele@utoronto.
caR.
G.
HegeleDepartmentofPaediatricLaboratoryMedicine,TheHospitalforSickChildren,Toronto,ON,Canada123CurrPediatrRep(2013)1:149–157DOI10.
1007/s40124-013-0019-3envelopedvirus,RSV'sgeneralstructurehassimilaritiestothatofinuenzaandparainuenzaviruses,metapneum-oviruses,andcoronaviruses,andcontrastswiththatofothercommonrespiratorypathogenssuchasrhinovirusesandadenoviruses,whicharenonenveloped.
RSVhasthreeenvelopeproteins:G(glycoprotein),F(fusion),andSH(smallhydrophobic)[1].
HeterogeneityintheGglyco-proteindenesthetwomainsubgroupsofRSV,AandB[4].
SomeliteraturesuggeststhatRSVAstrainsproducemoreclinicallysevereinfectionsthanRSVBstrains[5],butthiscontentionhasprovendifculttoreproduceamongdifferentgroupsofinvestigators[6].
OnestrainofRSVA,so-calledline19[7],appearstobeofparticularinterestregardingmucusproductionandsecretionofIL-13,aTh2cytokineassociatedwithallergic-typehostresponses(seebelow),andtherebycontributinginparttotheheteroge-neityandseverityofclinicalmanifestations.
Inaddition,RSVGhasasecretedformthatbindshostantibodiesandtherebyservesasanimmunological''decoy''thatfacili-tatescontinuedviralreplication[8].
RSVGisalsocon-sideredtobeakeyattachmentproteinforthevirustobindtothecellsurfacetoinitiateinfection;however,withthediscoveryandengineeringofRSVstrainsdecientinG(so-calledRSVDG)thatareinfectious,albeitatwithmuchlowerefciencythanvirusexpressingG,RSVGisneithersufcientnornecessarytocauseinfection[9].
Bycontrast,RSVFiscrucialforfusionoftheviralenvelopetothehostcellsurface,sinceRSVDFcannotentercellsonitsownbutratherrequiresahelpervirustoinitiateinfection[10].
Onfreevirus,RSVFexistsina''prefusion''formthatundergoesanelegantlycharacterizedconforma-tionalchangeduringthefusionprocess[11,12].
IncontrasttoRSVG,RSVFishighlyconservedbetweenthetwomajorsubgroupsofthevirus[13].
RSVFandRSVGmayhaveanoverall''yin-yang''effectonhostadaptiveimmuneresponsesconcerningTh1–Th2balance.
Inbrief,Th1responsesinvolveasubsetofCD4lymphocytesproducingcytokinessuchasinterferon(IFN)-candinterleukin(IL)-2consideredimportantindelayedhypersensitivityandanti-viralimmunity,whereasTh2responsesinvolveproductionofinammatorymediatorssuchasIL-4,IL-5,andIL-13byadifferentsubsetofCD4lymphocytesandareimplicatedinallergy.
RSVFappearstostimulateTh1cells,whereasRSVGappearstostimulateTh2responses,andclinicalmanifestationsarebelievedtoreectwhethertheoverallresponseisshiftedtowardTh1orTh2,orremainsinbalancewithregardtocytokineproduction[14].
TheRSVSHprotein,byanalogytosimilarproteinsexpressedinotherviruses,mayhavearoleinenhancingthepermeabilityofthecellsurfacetomoleculesandions[15].
TheimportanceofthisputativefunctionofRSVSHisunclear,sincegeneticallyengineeredstrainsofRSVinwhichSHisdeletedappeartobeessentiallyasefcientaswild-typevirusincausingcellularinfections[16].
ThereisincreasinginterestinSHasapotentialviralantigentargetinvaccinedevelopment(seelater).
OtherRSVcompo-nents,includingnucleocapsid(N),polymerase(L),non-structuralcomponents(NS1,NS2),phosphoprotein(P),andmatrix(M,M2-1,M2-2)havebeenimplicatedinstimulatinghostantiviralresponses(e.
g.
,IFN-aandIFN-b)througheffectsinhostcellsignaltransductionpathwaysandimportantnucleartranscriptionfactors(e.
g.
,NF-jB,AP-1).
FurtherdetailsoftheroleofnonenvelopedproteinsinRSVreplicationandhostresponsesareprovidedinanexcellentreviewbyCollinsandMelero[1].
RSVInfectionsInVitroVersusInVivoOneofthebiggestenigmassurroundingRSVpathobiologyisthestrongtropismthevirusshowsforinfectingthehumanrespiratorytractinvivo;invitro,RSVestablishesproductiveinfectionofvirtuallyallmammaliancelltypesstudied,includingawidevarietyofprimaryandcontinuouscellslinesofepithelialcells[17],macrophages[18],otherleukocytes[19],broblasts[20],andevenneurons[21].
Infact,ndingacelltypethatisresistanttoRSVinfectioninvitroischallenging.
Aswillbediscussedin''NucleolinasaFusionReceptorforRSV:FromDiscoverytoDrugTargeting''inthecontextofthediscoveryofacellularfusionreceptorforRSV,Sf9cellsfromthemothSpo-dopterafrugiperdaaredifculttoinfectwithRSV[22].
InadenitiveautopsystudyofthelungpathologyofuntreatedacuteRSVinfections,Johnsonetal.
[23]repor-tedthatRSVantigenswereobservedwithinairwayandalveolarepithelialcells,aswellaspulmonarymacro-phages.
Similarobservationshavebeenmadeinexperi-mentalanimals.
InaguineapigmodelofexperimentalRSVinfection,ourgrouphasdocumentedtheexpressionofviralantigensviaimmunohistochemistrywithinlungepithelialcells,alveolarmacrophages,interstitialcells(presumablydendriticcells),andcellswithinbronchus-associatedlymphoidtissue,foratleast100daysafterRSVinoculation[24].
ArecentstudyhasreportedthatRSVcanbedetectedinapproximately15%ofhumantonsilsofchildrenwithchronicadenotonsillardisease[25].
GiventheabundantliteratureconcerningtheeffectsofRSVinfectiononthesecretionofvariousinammatorymedia-tors,responsesofcellsignalingpathways,andotherhostresponsesobservedinvitro,onemustexercisecautioninextrapolatingthesignicanceofobservationsmadeinvitrototheinvivostate,andwhethertheyrepresentopportu-nitiesforpotentialclinicalinterventions.
OneonlyneedstoreectonthehistoricallydisappointingresultsseenwithattemptstotreatRSVinfectionsofexperimentalanimalsorpatientswithantiviraloranti-inammatoryagentstoappreciatethispoint[2,26].
150CurrPediatrRep(2013)1:149–157123Incontrasttoothervirusessuchasinuenzavirusandadenovirus,whereextensivecytotoxicityandcelldeathisthenormwithinfectioninvitro,RSVinfectiondoesnotelicitmuchinthewayofcelldamage[17].
Thereisevi-dencethatRSVcanactuallydecreasetherateofapoptosisinvitro[27].
Takenfurther,RSVhasclearlybeenshowntocausepersistent,noncytolyticinfectionofepithelialcellsinvitro[28].
RSVpersistence,characterizedbycontinu-ous,low-levelreplicationofinfectiousvirus,hasbeendescribedinvivo,rstinamodelinwhichcottonratswereimmunosuppressedwithcyclophosphamide[29],andsub-sequentlyinimmunocompetentguineapigs[30]andinmice[31,32].
ThereisalsoevidenceconsistentwithRSVpersistenceinhumans,bestcharacterizedinthesettingofadultpatientswithchronicobstructivepulmonarydisease[33].
ApossibleroleforRSVpersistenceinthepathobi-ologyofpostbronchiolitiswheezing/asthmaisdescribedin''RSVandPediatricAsthma:HaveWeFoundtheMissingLink''RSV–HostInteractions:MovingEverMoreProximallyFormanyyears,investigationofRSV–hostinteractionsfocusedoncharacterizingtheinammatorymediatorresponsesofinfectedcells,animals,andhumansubjectswiththehopethatanunderstandingofthevirus-specichostresponsestoRSVinfectionscouldinformpossibleinterventionsdesignedtomodulaterelevantinammatorymediatorsandtherebyleadtoimprovedclinicaloutcomes[34].
Theapparentheterogeneityinvirus-specicimmu-nityobservedinhostadaptiveimmuneresponsestoRSV(bothcell-mediatedandhumoral),dependingonthepop-ulationstudied,complicatestheabilitytomakeclinicallyrelevantadvances.
ExtensiveinquirieshavefocusedoninnateimmunemechanismsintheearlyhostresponsetoRSV[35,36].
Onerationaleforthisapproachisthatbyunderstandingtheearly,''proximal''eventsthatoccurinRSVinfections,itmaybeeasiertodevelopeffectiveclinicalinterventionsthatcouldbeusedeffectivelyamongdiversehostswhohavedifferentadaptiveimmuneresponsestothevirus[15,37].
Thisrationalehasbeenrecentlyquestioned,sinceapparentdifferencesininnateimmuneresponseshavebeenobservedbetweenRSVAandRSVBinfections[38].
Nevertheless,researchintotheroleofinnateimmunemechanismsinRSVpathobiologyledtoanumberofpro-foundinsights,oneofwhichwastheabilityofRSVtoincreasethelevelofpattern-recognitionreceptors(e.
g.
,Toll-likereceptors)expressedonthecellsurface[39].
Theseobservationswereinuentialtotheeldforanumberofreasons:forexample,thenatureofcellsurfacemoleculesinvolvedininnateimmunitythatcanbeaffectedbyRSVwaspoorlyunderstood,andpattern-recognitionreceptors(e.
g.
,Toll-likereceptor4andothers)stimulatecellsig-nalingpathwaysthatareamenabletopharmacologicalmanipulation[40],eveniftheyarenotactualreceptorsforthevirus[41].
OnecellsignalingmoleculerelatedtoToll-likereceptorinteractionwithRSVisp38mitogen-activatedproteinkinase,asrevealedbyascreenofculturedcellswithapanelofpharmacologicalkinaseinhibitors.
Inhibitionofp38mitogen-activatedproteinkinasewithSB203580(acompoundoriginallydevelopedfortreatmentofarthritisandcancer)wasassociatedwithmarkedinhibitionofRSVreplicationinculturedcells,withoutinducingsignicantdrug-relatedcytotoxicity[42].
Thistypeofexperimentisanexampleofso-calleddrugrepurposing,inwhichacom-pounddevelopedforanunrelatedclinicalindicationcanbegivenonthebasisofpathobiologydenedinanotherclinicalsetting[43].
Itremainstobeseenwhethersuchanapproachtodrugrepurposingwillbeusefulintheman-agementofRSVinfectionsinvivoinanimalmodels,andultimatelyinhumans.
NucleolinasaFusionReceptorforRSV:FromDiscoverytoDrugTargetingOurgroupidentiedcellsurfacenucleolinasamoleculethatfulllsthecriteriaofafunctionalreceptorforRSV[44].
Oneofthekeychallengestoovercomeinthisdis-coverywasndingacelltyperesistanttoRSVinfectioninvitrowhichcouldbecomepermissiveafterforcedexpressionofthehumannucleolingeneandectopicexpressionofhumannucleolinproteinonthecellsurface.
ThiswasachievedbyuseofinsectSf9cells[22].
Vali-dationofnucleolinasacellularreceptorinvivowasaccomplishedinamousemodelbysmallinterferingRNAknockdownofnucleolinpriortoRSVchallenge,whichwasassociatedwithasignicantdecreaseinRSVtitersasmeasuredbyquantitativeplaqueassaysoflunghomoge-nates.
WeshowedthatRSVF,notRSVG,interactswithnucleolin,andwecoinedtheterm''fusionreceptor''todenotethisfact.
NucleolinisanespeciallychallengingpotentialdrugtargetforRSV.
Nucleolinhasaveryshorthalf-lifewhenexpressedonthecellsurface,andconstantlyundergoesrecyclingbetweenthecytoplasmicandnuclearcompart-mentsofthecell[45].
Thus,approachesthatinterferewithcellsurfacenucleolinsuchasuseofnucleolin-specicblockingantibodies,resultintheantibody–nucleolincomplexbeinginternalizedintothecellandnew(''fresh'')unboundnucleolinmovingtothesurface[46].
Thisphe-nomenoncouldexplainwhyinourantibodyneutralizationexperimentsattemptingtovalidatenucleolinasanRSVreceptorwewereabletoachieveonlyontheorderapproximately70–80%decreasedinfectionofcellsthathadbeenpreincubatedwithnucleolin-specicantibodyCurrPediatrRep(2013)1:149–157151123priortoRSVchallenge.
Secondly,nucleolinisanotori-ously''sticky''moleculethatcanbindallsortsofmole-cules,includingproteinsexpressedbyotherviruses(e.
g.
,parainuenzavirustype3,HIV),certaindrugs(e.
g.
,mid-kine,lactoferrin,pseudopeptideHB-19),andevenDNAmoieties(reviewedin[47]).
HowtotranslatethissituationintoascenariothatisRSV-specicneedstobeaddressed.
Moreover,nucleolindoesnotexistinisolationonthecellsurface;rather,itispartofamultiproteincomplexofmolecularmassofapproximately500kDa[48].
Toaddfurthercontext,intranuclearnucleolinisessentialtocellsurvivalandreplication:inhibitingnucleolininanunde-siredcellularcompartmentcouldelicitseriousoff-targeteffects[49].
Overall,theidenticationofnucleolinasafusionreceptorforRSVposesanumberofprofoundchallengestobeovercomebeforeitmayserveasabonadedrugtarget.
ARenaissanceofRSVCellBiology,andtheGrowingRoleofSystemsBiologyOneareaofincreasingresearchactivityistheintracellularcompartmentalizationofRSVconstituentsduringthevirallifecycle.
CellbiologistsinvestigatingRSVreplicationkineticsdemonstratedthatthevirusdoesnotnecessarilyfollowastereotypedreplicativecycle,butrathercanbepreferentiallydirectedintointracellularcompartmentsthatfavororinhibitreplication[50,51].
Forexample,cyto-plasmicstressgranules[52],caveolae[53],hostcytoskel-eton[54],andlipidrafts[55]havebeenreportedtointeractwithRSV,andthesearepotentialnoveltargetsforphar-macologicalintervention.
Increasingly,theuseofhigh-throughput''systemsbiology''approachesdesignedtointerrogateRSV-infectedcellsinanunbiasedmannerhasledtoidenticationofnovelcandidatemoleculesthatcouldpotentiallybetargetedpharmacologically[56,57].
SystemsbiologypermitstheapplicationofinformaticsrelatedtopathwaymodelingrelevanttoRSVinfection.
Bydeningso-calledinteractomes[58]inwhichthevirusaffectscellfunction,investigatorsaimtoachieveacomprehensiveunderstandingofRSV–hostinteractionstoinformthedevelopmentofnovelstrategiesintreatmentandprophy-laxis.
Oneofthecurrentlimitationsofinformaticsapproachesisthatthebiologicalpathwaysbeingmodelingarethemselvesincompletelycharacterized,someaningfulinterpretationswillrequireongoingiterativeactivity[59].
RSVandPediatricAsthma:HaveWeFoundtheMissingLinkWithinafewyearsofthediscoveryofRSV,inaseminalarticle,Wittigetal.
[60]reportedthatasizeablepercentage(32%)ofinfantsandyoungchildrenhospitalizedforRSVbronchiolitisdevelopedsequelaeofrecurrentwheezingandasthmasymptoms.
Thesendingshavebeenreplicatedonanumberofoccasionsoverthelastfewdecades,althoughthereissomeongoingcontroversyoverwhetherpatientscan''outgrow''theirasthmabythetimetheyreachado-lescence,orwhethertheirconditioncanpersistintoadulthood[61–63].
OnepossibilityisthatRSVbronchi-olitisisa''marker''ofchildrenwhoaregeneticallypre-disposedtodevelopasthma,asevidencedbyaclassicstudyinwhichRSV-specicIgEwasdocumentedinchildrenwhohadrecurrentwheezingasasequelaofRSVinfection[64].
Further,inahighlyinuentialarticle,Fricketal.
[65]reportedatemporalrelationshipbetweenviralinfection(particularlyRSVandparainuenzavirus)andtriggeringallergicsensitizationin14childrenborninto''high-risk''families(i.
e.
,whererst-degreerelativeshadaprovenhistoryofallergy).
Thislatterassociationhasalsobeenconrmedinlargerstudies,includingtheCanadianAsthmaPrimaryPreventionStudyofapproximately400''high-risk''childrenandtheirfamilies[66].
Toexplorepotentialmechanismsofpostbronchiolitiswheezing/asthmaandallergy,ourgroupdevelopedagui-neapigmodelofexperimentalRSVlunginfection[67].
Guineapigswerechosenbecausetheywerealreadyamodelofhumanparainuenzavirustype3infection[68],andcouldreadilybesensitizedtoallergen(aerosolizedovalbumin)[69].
AfterinitialworkestablishingthatRSVproteinandgenomecouldpersistintheguineapiglungforupleast60daysafterRSVinoculation[70],subsequentstudiesshowedthatoutbredCamHartleyguineapigscouldharborreplicatingvirusforatleast60–100daysafterinoculation,inassociationwithnonspecicairwayhyper-responsiveness(AHR)andchronicairwayinammation(physiologicalandpathologicalhallmarksofasthma)[24,30].
However,insubsequentwork,wenotedthatviralpersistenceonitsownisnotsufcienttoproduceasthma-likechangesinthelungs,asinbred,''allergy-resistant''strain2guineapigscanalsodeveloppersistentRSVlunginfectionsbuttheseanimalsdonotgetAHRorchronicairwayinammationinassociationwithviruspersistence[71].
ConcerningthepossibilitythatRSVinfectionisa''marker''ofgeneticpredispositiontoallergy,weobservedthatRSVinfectionofCamHartleyguineapigsenhancedallergicsensitizationtoaerosolizedovalbumin,consistentwiththishypothesis[72].
Interestingly,theextentofchronicAHR/airwayinammationandvirus-enhancedovalbuminsensitizationcouldbeamelioratedbyimmu-noprophylaxisofanimalswithCpG-oligodinucleotides(CpG-ODN),apotentTh1stimulus[73].
Takentogether,theseresultssuggestthatthecombinationofRSVandaeroallergenproducesadditiveeffectsonAHRandairway152CurrPediatrRep(2013)1:149–157123inammationinan''allergy-susceptible''hostandthatCpG-ODNimmunoprophylaxisappearstotargettheRSV-associated,nottheallergen-associated,component.
Alargenumberofhumanstudieshavecharacterizeddifferencesinthetypesofcytokinesandotherchemicalmediatorsofinammation—andtheirpotentialgeneticdeterminants—indifferentsubsetsofRSV-infectedpatients(reviewedinZengetal.
[74]).
ThislargebodyofworkprovidesvaluableinsightsintopossibledifferencesinhostresponsestoRSVinfectionandmayberelevanttoexplainingwhysomeinfectedindividualsdevelopmild,upperrespiratorytractinfectionswhereasotherindividualsdevelopbronchiolitisorpneumonia,orgoontodeveloprecurrentwheezing/asthma.
Unfortunately,therehasbeenarecurrentdifcultyinvalidatingthelargenumberofcan-didatemolecules/genesbyreplicationinothercohorts,andtheoverallpictureisfarfromclear[1].
Theuseofasys-temsbiologyapproach(see''ARenaissanceofRSVCellBiology,andtheGrowingRoleofSystemsBiology'')promisestoaccelerateknowledgeofidenticationofcan-didatemoleculesandgenesforvalidation.
Onerecentstudy[75]hasreportedanexcitingdevelopmentthatmaypro-videacluetothepossiblebiological''missinglink''betweensevereRSVinfectionsandthesubsequentdevel-opmentofpediatricasthma.
Inaseriesofexperimentsthatbeganwithunbiasedinterrogationofcellstoidentifycan-didategenes,followedbyvalidationinexperimentalani-malsandanalysisofhumancohorts,investigatorshavecharacterizedgeneticpolymorphismsinthepromoterregiontoNFKBIAashavingbothfunctionalandclinicalsignicanceinthecontextofRSVbronchiolitisandpedi-atricasthma.
NFKBIAisthegenewhichencodesIjBa,themajornegativeregulatorofNF-jB.
NF-jBisanimportantnucleartranscriptionfactorthathasacentralroleinregu-latingmultiplehostimmuneandinammatoryresponseelements.
NFKBIAmayendupastheunifyingconceptforbronchiolitisandallergy,asimplicatedbytheresultsofthisstudy,andthisopensupexcitingnewresearchdirectionsforpatientidentication,stratication,andtargetedtherapy.
RSVandParticulateMatter:AnEvenTighterBondThanPreviouslyThoughtEveryday,humansarebombardedwithnumerousinhaledagentsfromtheindoorandoutdoorenvironment.
Particu-latematterrelatedtoairpollutionisbutoneexampleofsuchanagent.
PreviousworkthatstudiedinteractionsbetweenRSVandparticulatematterfocusedoneffectsonthecellularinammatoryresponseinvitro[76–79]andonalargerscaleondeningtemporalrelationshipsbetweenairpollutionexposureandvirusinfectionsandtheireffectsonrespiratoryhealth[80,81].
Fromthesestudies,itisapparentthatairpollutionshouldbeconsideredasapotentialcon-founderoftheeffectivenessofRSVinterventions.
ArecentstudyhasprovidedanevenmoredramaticrelationshipbetweenRSVandparticulatematter[82].
Inthiswork,''designerparticles''ofspecicchemicalcom-positionwereconjugatedtohumanRSV,andcellcultureswereinoculatedwiththesecomplexes,withstatisticallysignicantresponsesobservedwithrespecttocytokineproductionbycells.
Interestingly,thesamestudyshowedthatRSVconjugatedtotheseparticlescouldremaininfectiousatambientroomtemperatureforatleast2monthsafterpreparation,withouttheneedforcellcul-ture,orfreezingstockstolowtemperature(-80°Corbelow).
TheseobservationsopenupanentirelynewpotentialmechanismforRSVsurvivaloutsidelivingorganisms,andsuggestamodeofenvironmentaltrans-missionofRSV,separatefromclosecontactofindividualstovirus-containingdropletsorfomites.
ObtainingimprovedunderstandingoftheimplicationsofRSVconjugatedtoparticulatematterrequiresfurtherresearch.
ProgressTowardanRSVVaccine:CloserthanEverSincethediscoveryofRSVin1955–1957[83–85],vaccinedevelopmenthasbeenamajorpriority,yetthereareanumberoflogisticalconsiderationsrelatedtoRSVinfec-tionsaffectingprogressinthisarea,asCrowe[86]sum-marizedoveradecadeago(Table1).
Thetragedythatoccurredinthe1960swithacandidateRSVvaccinethatwaspreparedbyformalininactivationofwholevirusthatunderwenteldtrialsiswelldocumented[87].
Briey,vaccinerecipientsparadoxicallydevelopedsevererclinicaldiseaseonRSVexposure,andtherewereseveraldeathsinthevaccinatedgroup.
Themechanismsresponsibleforvaccine-enhancedillnesswereultimatelydetermineddecadeslaterbyuseofanimalmodels.
ThroughapplicationoftheTh1–Th2paradigmofCD4lympho-cytes,investigatorsdeterminedthatexposureofexperi-mentalanimalstoformalin-inactivatedRSVvaccineresultedinanexaggeratedhostTh2responseaftersub-sequentexposuretoinfectiousvirus.
Thishasstimulatedconsiderableresearchactivitywiththeaimtodesignvac-cinesthatshifttheTh1–Th2balancetowardTh1[88,89].
However,thismaybeanoverlysimplisticapproach,asarecentstudyinmicehasreportedthatshiftingthehostimmuneresponsetowardTh1actuallydecreasesthepro-ductionofRSV-neutralizingantibodies,whichwouldhavetheeffectoflimitingtheeffectivenessofavaccine[90].
InadditiontoTh1andTh2lymphocytes,thesubpopulationofTh17lymphocyteshasbeenimplicatedinRSVinfection,furtheraddingtothecomplexityofthehostimmuneresponse[91].
CurrPediatrRep(2013)1:149–157153123Inrecentyears,anumberofinnovativeandcleverapproachesforvaccineengineeringhavebeendeveloped,includingtheproductionofvirus-likeparticles[92],chi-mericlivevirusvectors[93],gene-basedvectors[94],andtheuseofnoveladjuvants[95].
Atpresent,weareawareofavarietyofcandidateRSVvaccines(ontheorderoftento20differentproductsatvariousstagesofdevelopment)poisedtoenterhumanclinicaltrials,whichwillbeupdatedattheRSVVaccineConference(Porto,Portugal,October2013:http://www.
meetingsmanagement.
com/rsvvw_2013).
Itisdifculttopredictwhich,ifany,oftheseproductscouldendupasaneffectivevaccineforwidespreaduseinhumans.
Currently,thegoalisfortheretobeasimulta-neousconcertedeffortincontinuedscienticvaccinologyandinthecarefuldesignofhumanclinicaltrialprotocols,includingthepossibilityofimmunizingpregnantwomenasaformofprenatalvaccination,inanefforttodenitivelysolvethelong-standingproblemofdeterminingandimplementingasafe,effectiveRSVvaccine.
Achievingthisoutcomewouldrepresentagloriousaccomplishmentforhumankindandhaveenormousandlastingimpactaroundtheglobe.
ConclusionsWearelivinginaneraofacceleratedactivityandprogressinourunderstandingofviralandhostfactorsinRSVinfection,andarepoisedtotranslatediscoveriesmadeinthisrealmintotheclinictoachieveimprovedhealthout-comesforacuteRSVinfectionsandtheirsequelae.
ResearchhasevolvedfromcharacterizingRSV–hostinteractionstotheidenticationofnewtargetsforvaccineandpharmaceuticaldevelopment.
Advancesinsystemsbiology,informatics,cellandstructuralbiology,andbio-logicalengineeringhaveprovidednewandpowerfulapproachestostudyRSVinfections,andtheinuenceofotherenvironmentalfactorssuchasallergensandparticulatematter.
Wearecloserthanevertoachievingthedreamofasafe,effectiveRSVvaccine,andthefuturepromisestobeexcitingandrewarding.
DisclosurePeterMastrangeloandRichardG.
Hegeledeclarethattheyhavenoconictsofinterest.
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CurrPediatrRep(2013)1:149–157157123
HegelePublishedonline:15May2013SpringerScience+BusinessMediaNewYork2013AbstractRespiratorysyncytialvirus(RSV)isamajorworldwidepathogenforwhichthereisstillnoeffectivevaccineorantiviraltreatmentavailable,andimmunopro-phylaxiswithRSV-specicantibodies(e.
g.
,palivizumab)isusedinlimitedclinicalsettings.
Inthisreview,wedis-cussvirus–hostinteractionsrelevanttoRSVpathobiologyandhowadvancesincellandsystemsbiologyhaveacceleratedknowledgeinthisarea.
WealsohighlightrecentadvancesinunderstandingtherelationshipbetweenRSVbronchiolitisandsequelaeofrecurrentwheezingandasthma,newndingsintoanintriguinginteractionbetweenRSVandairpollution,andexcitingdevelopmentstowardthegoalofrealizingasafeandeffectiveRSVvaccine.
KeywordsRespiratorysyncytialvirusViralpathogenesisVirus–hostinteractionsViruspersistenceAsthmaAllergySystemsbiologyNucleolinAirpollutionVaccinologyIntroductionRespiratorysyncytialvirus(RSV)isasingle-stranded,negative-polarityPneumovirusthatinfectspeopleofallagesworldwide[1].
RSVisafrequentcauseofupperrespiratorytractinfectionsandotitismedia,andalsocausesseriouslowerrespiratorytractinfectionssuchasbronchi-olitisandpneumonia,particularlyininfantsandyoungchildren,immunocompromisedindividuals,andtheelderly.
RSVinfectionsinearlypostnatallifehavealsobeenassociatedwiththeonsetofallergicsensitizationandrecurrentwheezing/asthma.
Despitealmost60yearshav-ingpassedsincethediscoveryofRSV,thereisstillnolicensedvaccineavailable,andprophylaxiswithimmu-noglobulin(palivizumab)isusedonlyforasubsetof''high-risk''children(e.
g.
,pretermbirths,andinfantswithcongenitalheartdisease,congenitalimmunodeciency,and/orbronchopulmonarydysplasia).
AlthoughanumberofpharmacologicalagentshavebeenusedinRSVtherapy,theirefcacyandeffectivenessarepoor[2].
Here,wewillbrieyreviewsomeviralandhostfactorsimplicatedinthepathobiologyofRSVinfections,focusingonhowthediversityofthesefactorsmightberelatedtothevariabilityseeninclinicaldisease.
OurdiscussionwillalsoincludecomparingandcontrastingcharacteristicsofRSVinfectionsinvitroversusinvivo;currentperspectivesontherelationshipbetweenRSVinfections,pediatricallergy,andasthma;recentworkthatdescribesanovelrelationshipbetweenRSVandparticulatematter(airpollution);andprospectsforthedevelopmentandimplementationofasafe,effectiveRSVvaccine.
ViralandHostFactorsinRSVInfection:TowardanIntegratedPerspectiveRSVStructureHumanRSVisalamentousenvelopedvirusinwhichtheviralnucleicacidandstructuralandinternalproteinsaresurroundedbyaproteinaceousoutercoat[3].
AsanP.
MastrangeloR.
G.
Hegele(&)DepartmentofLaboratoryMedicineandPathobiology,UniversityofToronto,6231-1King'sCollegeCircle,Toronto,ONM5S1A8,Canadae-mail:richard.
hegele@utoronto.
caR.
G.
HegeleDepartmentofPaediatricLaboratoryMedicine,TheHospitalforSickChildren,Toronto,ON,Canada123CurrPediatrRep(2013)1:149–157DOI10.
1007/s40124-013-0019-3envelopedvirus,RSV'sgeneralstructurehassimilaritiestothatofinuenzaandparainuenzaviruses,metapneum-oviruses,andcoronaviruses,andcontrastswiththatofothercommonrespiratorypathogenssuchasrhinovirusesandadenoviruses,whicharenonenveloped.
RSVhasthreeenvelopeproteins:G(glycoprotein),F(fusion),andSH(smallhydrophobic)[1].
HeterogeneityintheGglyco-proteindenesthetwomainsubgroupsofRSV,AandB[4].
SomeliteraturesuggeststhatRSVAstrainsproducemoreclinicallysevereinfectionsthanRSVBstrains[5],butthiscontentionhasprovendifculttoreproduceamongdifferentgroupsofinvestigators[6].
OnestrainofRSVA,so-calledline19[7],appearstobeofparticularinterestregardingmucusproductionandsecretionofIL-13,aTh2cytokineassociatedwithallergic-typehostresponses(seebelow),andtherebycontributinginparttotheheteroge-neityandseverityofclinicalmanifestations.
Inaddition,RSVGhasasecretedformthatbindshostantibodiesandtherebyservesasanimmunological''decoy''thatfacili-tatescontinuedviralreplication[8].
RSVGisalsocon-sideredtobeakeyattachmentproteinforthevirustobindtothecellsurfacetoinitiateinfection;however,withthediscoveryandengineeringofRSVstrainsdecientinG(so-calledRSVDG)thatareinfectious,albeitatwithmuchlowerefciencythanvirusexpressingG,RSVGisneithersufcientnornecessarytocauseinfection[9].
Bycontrast,RSVFiscrucialforfusionoftheviralenvelopetothehostcellsurface,sinceRSVDFcannotentercellsonitsownbutratherrequiresahelpervirustoinitiateinfection[10].
Onfreevirus,RSVFexistsina''prefusion''formthatundergoesanelegantlycharacterizedconforma-tionalchangeduringthefusionprocess[11,12].
IncontrasttoRSVG,RSVFishighlyconservedbetweenthetwomajorsubgroupsofthevirus[13].
RSVFandRSVGmayhaveanoverall''yin-yang''effectonhostadaptiveimmuneresponsesconcerningTh1–Th2balance.
Inbrief,Th1responsesinvolveasubsetofCD4lymphocytesproducingcytokinessuchasinterferon(IFN)-candinterleukin(IL)-2consideredimportantindelayedhypersensitivityandanti-viralimmunity,whereasTh2responsesinvolveproductionofinammatorymediatorssuchasIL-4,IL-5,andIL-13byadifferentsubsetofCD4lymphocytesandareimplicatedinallergy.
RSVFappearstostimulateTh1cells,whereasRSVGappearstostimulateTh2responses,andclinicalmanifestationsarebelievedtoreectwhethertheoverallresponseisshiftedtowardTh1orTh2,orremainsinbalancewithregardtocytokineproduction[14].
TheRSVSHprotein,byanalogytosimilarproteinsexpressedinotherviruses,mayhavearoleinenhancingthepermeabilityofthecellsurfacetomoleculesandions[15].
TheimportanceofthisputativefunctionofRSVSHisunclear,sincegeneticallyengineeredstrainsofRSVinwhichSHisdeletedappeartobeessentiallyasefcientaswild-typevirusincausingcellularinfections[16].
ThereisincreasinginterestinSHasapotentialviralantigentargetinvaccinedevelopment(seelater).
OtherRSVcompo-nents,includingnucleocapsid(N),polymerase(L),non-structuralcomponents(NS1,NS2),phosphoprotein(P),andmatrix(M,M2-1,M2-2)havebeenimplicatedinstimulatinghostantiviralresponses(e.
g.
,IFN-aandIFN-b)througheffectsinhostcellsignaltransductionpathwaysandimportantnucleartranscriptionfactors(e.
g.
,NF-jB,AP-1).
FurtherdetailsoftheroleofnonenvelopedproteinsinRSVreplicationandhostresponsesareprovidedinanexcellentreviewbyCollinsandMelero[1].
RSVInfectionsInVitroVersusInVivoOneofthebiggestenigmassurroundingRSVpathobiologyisthestrongtropismthevirusshowsforinfectingthehumanrespiratorytractinvivo;invitro,RSVestablishesproductiveinfectionofvirtuallyallmammaliancelltypesstudied,includingawidevarietyofprimaryandcontinuouscellslinesofepithelialcells[17],macrophages[18],otherleukocytes[19],broblasts[20],andevenneurons[21].
Infact,ndingacelltypethatisresistanttoRSVinfectioninvitroischallenging.
Aswillbediscussedin''NucleolinasaFusionReceptorforRSV:FromDiscoverytoDrugTargeting''inthecontextofthediscoveryofacellularfusionreceptorforRSV,Sf9cellsfromthemothSpo-dopterafrugiperdaaredifculttoinfectwithRSV[22].
InadenitiveautopsystudyofthelungpathologyofuntreatedacuteRSVinfections,Johnsonetal.
[23]repor-tedthatRSVantigenswereobservedwithinairwayandalveolarepithelialcells,aswellaspulmonarymacro-phages.
Similarobservationshavebeenmadeinexperi-mentalanimals.
InaguineapigmodelofexperimentalRSVinfection,ourgrouphasdocumentedtheexpressionofviralantigensviaimmunohistochemistrywithinlungepithelialcells,alveolarmacrophages,interstitialcells(presumablydendriticcells),andcellswithinbronchus-associatedlymphoidtissue,foratleast100daysafterRSVinoculation[24].
ArecentstudyhasreportedthatRSVcanbedetectedinapproximately15%ofhumantonsilsofchildrenwithchronicadenotonsillardisease[25].
GiventheabundantliteratureconcerningtheeffectsofRSVinfectiononthesecretionofvariousinammatorymedia-tors,responsesofcellsignalingpathways,andotherhostresponsesobservedinvitro,onemustexercisecautioninextrapolatingthesignicanceofobservationsmadeinvitrototheinvivostate,andwhethertheyrepresentopportu-nitiesforpotentialclinicalinterventions.
OneonlyneedstoreectonthehistoricallydisappointingresultsseenwithattemptstotreatRSVinfectionsofexperimentalanimalsorpatientswithantiviraloranti-inammatoryagentstoappreciatethispoint[2,26].
150CurrPediatrRep(2013)1:149–157123Incontrasttoothervirusessuchasinuenzavirusandadenovirus,whereextensivecytotoxicityandcelldeathisthenormwithinfectioninvitro,RSVinfectiondoesnotelicitmuchinthewayofcelldamage[17].
Thereisevi-dencethatRSVcanactuallydecreasetherateofapoptosisinvitro[27].
Takenfurther,RSVhasclearlybeenshowntocausepersistent,noncytolyticinfectionofepithelialcellsinvitro[28].
RSVpersistence,characterizedbycontinu-ous,low-levelreplicationofinfectiousvirus,hasbeendescribedinvivo,rstinamodelinwhichcottonratswereimmunosuppressedwithcyclophosphamide[29],andsub-sequentlyinimmunocompetentguineapigs[30]andinmice[31,32].
ThereisalsoevidenceconsistentwithRSVpersistenceinhumans,bestcharacterizedinthesettingofadultpatientswithchronicobstructivepulmonarydisease[33].
ApossibleroleforRSVpersistenceinthepathobi-ologyofpostbronchiolitiswheezing/asthmaisdescribedin''RSVandPediatricAsthma:HaveWeFoundtheMissingLink''RSV–HostInteractions:MovingEverMoreProximallyFormanyyears,investigationofRSV–hostinteractionsfocusedoncharacterizingtheinammatorymediatorresponsesofinfectedcells,animals,andhumansubjectswiththehopethatanunderstandingofthevirus-specichostresponsestoRSVinfectionscouldinformpossibleinterventionsdesignedtomodulaterelevantinammatorymediatorsandtherebyleadtoimprovedclinicaloutcomes[34].
Theapparentheterogeneityinvirus-specicimmu-nityobservedinhostadaptiveimmuneresponsestoRSV(bothcell-mediatedandhumoral),dependingonthepop-ulationstudied,complicatestheabilitytomakeclinicallyrelevantadvances.
ExtensiveinquirieshavefocusedoninnateimmunemechanismsintheearlyhostresponsetoRSV[35,36].
Onerationaleforthisapproachisthatbyunderstandingtheearly,''proximal''eventsthatoccurinRSVinfections,itmaybeeasiertodevelopeffectiveclinicalinterventionsthatcouldbeusedeffectivelyamongdiversehostswhohavedifferentadaptiveimmuneresponsestothevirus[15,37].
Thisrationalehasbeenrecentlyquestioned,sinceapparentdifferencesininnateimmuneresponseshavebeenobservedbetweenRSVAandRSVBinfections[38].
Nevertheless,researchintotheroleofinnateimmunemechanismsinRSVpathobiologyledtoanumberofpro-foundinsights,oneofwhichwastheabilityofRSVtoincreasethelevelofpattern-recognitionreceptors(e.
g.
,Toll-likereceptors)expressedonthecellsurface[39].
Theseobservationswereinuentialtotheeldforanumberofreasons:forexample,thenatureofcellsurfacemoleculesinvolvedininnateimmunitythatcanbeaffectedbyRSVwaspoorlyunderstood,andpattern-recognitionreceptors(e.
g.
,Toll-likereceptor4andothers)stimulatecellsig-nalingpathwaysthatareamenabletopharmacologicalmanipulation[40],eveniftheyarenotactualreceptorsforthevirus[41].
OnecellsignalingmoleculerelatedtoToll-likereceptorinteractionwithRSVisp38mitogen-activatedproteinkinase,asrevealedbyascreenofculturedcellswithapanelofpharmacologicalkinaseinhibitors.
Inhibitionofp38mitogen-activatedproteinkinasewithSB203580(acompoundoriginallydevelopedfortreatmentofarthritisandcancer)wasassociatedwithmarkedinhibitionofRSVreplicationinculturedcells,withoutinducingsignicantdrug-relatedcytotoxicity[42].
Thistypeofexperimentisanexampleofso-calleddrugrepurposing,inwhichacom-pounddevelopedforanunrelatedclinicalindicationcanbegivenonthebasisofpathobiologydenedinanotherclinicalsetting[43].
Itremainstobeseenwhethersuchanapproachtodrugrepurposingwillbeusefulintheman-agementofRSVinfectionsinvivoinanimalmodels,andultimatelyinhumans.
NucleolinasaFusionReceptorforRSV:FromDiscoverytoDrugTargetingOurgroupidentiedcellsurfacenucleolinasamoleculethatfulllsthecriteriaofafunctionalreceptorforRSV[44].
Oneofthekeychallengestoovercomeinthisdis-coverywasndingacelltyperesistanttoRSVinfectioninvitrowhichcouldbecomepermissiveafterforcedexpressionofthehumannucleolingeneandectopicexpressionofhumannucleolinproteinonthecellsurface.
ThiswasachievedbyuseofinsectSf9cells[22].
Vali-dationofnucleolinasacellularreceptorinvivowasaccomplishedinamousemodelbysmallinterferingRNAknockdownofnucleolinpriortoRSVchallenge,whichwasassociatedwithasignicantdecreaseinRSVtitersasmeasuredbyquantitativeplaqueassaysoflunghomoge-nates.
WeshowedthatRSVF,notRSVG,interactswithnucleolin,andwecoinedtheterm''fusionreceptor''todenotethisfact.
NucleolinisanespeciallychallengingpotentialdrugtargetforRSV.
Nucleolinhasaveryshorthalf-lifewhenexpressedonthecellsurface,andconstantlyundergoesrecyclingbetweenthecytoplasmicandnuclearcompart-mentsofthecell[45].
Thus,approachesthatinterferewithcellsurfacenucleolinsuchasuseofnucleolin-specicblockingantibodies,resultintheantibody–nucleolincomplexbeinginternalizedintothecellandnew(''fresh'')unboundnucleolinmovingtothesurface[46].
Thisphe-nomenoncouldexplainwhyinourantibodyneutralizationexperimentsattemptingtovalidatenucleolinasanRSVreceptorwewereabletoachieveonlyontheorderapproximately70–80%decreasedinfectionofcellsthathadbeenpreincubatedwithnucleolin-specicantibodyCurrPediatrRep(2013)1:149–157151123priortoRSVchallenge.
Secondly,nucleolinisanotori-ously''sticky''moleculethatcanbindallsortsofmole-cules,includingproteinsexpressedbyotherviruses(e.
g.
,parainuenzavirustype3,HIV),certaindrugs(e.
g.
,mid-kine,lactoferrin,pseudopeptideHB-19),andevenDNAmoieties(reviewedin[47]).
HowtotranslatethissituationintoascenariothatisRSV-specicneedstobeaddressed.
Moreover,nucleolindoesnotexistinisolationonthecellsurface;rather,itispartofamultiproteincomplexofmolecularmassofapproximately500kDa[48].
Toaddfurthercontext,intranuclearnucleolinisessentialtocellsurvivalandreplication:inhibitingnucleolininanunde-siredcellularcompartmentcouldelicitseriousoff-targeteffects[49].
Overall,theidenticationofnucleolinasafusionreceptorforRSVposesanumberofprofoundchallengestobeovercomebeforeitmayserveasabonadedrugtarget.
ARenaissanceofRSVCellBiology,andtheGrowingRoleofSystemsBiologyOneareaofincreasingresearchactivityistheintracellularcompartmentalizationofRSVconstituentsduringthevirallifecycle.
CellbiologistsinvestigatingRSVreplicationkineticsdemonstratedthatthevirusdoesnotnecessarilyfollowastereotypedreplicativecycle,butrathercanbepreferentiallydirectedintointracellularcompartmentsthatfavororinhibitreplication[50,51].
Forexample,cyto-plasmicstressgranules[52],caveolae[53],hostcytoskel-eton[54],andlipidrafts[55]havebeenreportedtointeractwithRSV,andthesearepotentialnoveltargetsforphar-macologicalintervention.
Increasingly,theuseofhigh-throughput''systemsbiology''approachesdesignedtointerrogateRSV-infectedcellsinanunbiasedmannerhasledtoidenticationofnovelcandidatemoleculesthatcouldpotentiallybetargetedpharmacologically[56,57].
SystemsbiologypermitstheapplicationofinformaticsrelatedtopathwaymodelingrelevanttoRSVinfection.
Bydeningso-calledinteractomes[58]inwhichthevirusaffectscellfunction,investigatorsaimtoachieveacomprehensiveunderstandingofRSV–hostinteractionstoinformthedevelopmentofnovelstrategiesintreatmentandprophy-laxis.
Oneofthecurrentlimitationsofinformaticsapproachesisthatthebiologicalpathwaysbeingmodelingarethemselvesincompletelycharacterized,someaningfulinterpretationswillrequireongoingiterativeactivity[59].
RSVandPediatricAsthma:HaveWeFoundtheMissingLinkWithinafewyearsofthediscoveryofRSV,inaseminalarticle,Wittigetal.
[60]reportedthatasizeablepercentage(32%)ofinfantsandyoungchildrenhospitalizedforRSVbronchiolitisdevelopedsequelaeofrecurrentwheezingandasthmasymptoms.
Thesendingshavebeenreplicatedonanumberofoccasionsoverthelastfewdecades,althoughthereissomeongoingcontroversyoverwhetherpatientscan''outgrow''theirasthmabythetimetheyreachado-lescence,orwhethertheirconditioncanpersistintoadulthood[61–63].
OnepossibilityisthatRSVbronchi-olitisisa''marker''ofchildrenwhoaregeneticallypre-disposedtodevelopasthma,asevidencedbyaclassicstudyinwhichRSV-specicIgEwasdocumentedinchildrenwhohadrecurrentwheezingasasequelaofRSVinfection[64].
Further,inahighlyinuentialarticle,Fricketal.
[65]reportedatemporalrelationshipbetweenviralinfection(particularlyRSVandparainuenzavirus)andtriggeringallergicsensitizationin14childrenborninto''high-risk''families(i.
e.
,whererst-degreerelativeshadaprovenhistoryofallergy).
Thislatterassociationhasalsobeenconrmedinlargerstudies,includingtheCanadianAsthmaPrimaryPreventionStudyofapproximately400''high-risk''childrenandtheirfamilies[66].
Toexplorepotentialmechanismsofpostbronchiolitiswheezing/asthmaandallergy,ourgroupdevelopedagui-neapigmodelofexperimentalRSVlunginfection[67].
Guineapigswerechosenbecausetheywerealreadyamodelofhumanparainuenzavirustype3infection[68],andcouldreadilybesensitizedtoallergen(aerosolizedovalbumin)[69].
AfterinitialworkestablishingthatRSVproteinandgenomecouldpersistintheguineapiglungforupleast60daysafterRSVinoculation[70],subsequentstudiesshowedthatoutbredCamHartleyguineapigscouldharborreplicatingvirusforatleast60–100daysafterinoculation,inassociationwithnonspecicairwayhyper-responsiveness(AHR)andchronicairwayinammation(physiologicalandpathologicalhallmarksofasthma)[24,30].
However,insubsequentwork,wenotedthatviralpersistenceonitsownisnotsufcienttoproduceasthma-likechangesinthelungs,asinbred,''allergy-resistant''strain2guineapigscanalsodeveloppersistentRSVlunginfectionsbuttheseanimalsdonotgetAHRorchronicairwayinammationinassociationwithviruspersistence[71].
ConcerningthepossibilitythatRSVinfectionisa''marker''ofgeneticpredispositiontoallergy,weobservedthatRSVinfectionofCamHartleyguineapigsenhancedallergicsensitizationtoaerosolizedovalbumin,consistentwiththishypothesis[72].
Interestingly,theextentofchronicAHR/airwayinammationandvirus-enhancedovalbuminsensitizationcouldbeamelioratedbyimmu-noprophylaxisofanimalswithCpG-oligodinucleotides(CpG-ODN),apotentTh1stimulus[73].
Takentogether,theseresultssuggestthatthecombinationofRSVandaeroallergenproducesadditiveeffectsonAHRandairway152CurrPediatrRep(2013)1:149–157123inammationinan''allergy-susceptible''hostandthatCpG-ODNimmunoprophylaxisappearstotargettheRSV-associated,nottheallergen-associated,component.
Alargenumberofhumanstudieshavecharacterizeddifferencesinthetypesofcytokinesandotherchemicalmediatorsofinammation—andtheirpotentialgeneticdeterminants—indifferentsubsetsofRSV-infectedpatients(reviewedinZengetal.
[74]).
ThislargebodyofworkprovidesvaluableinsightsintopossibledifferencesinhostresponsestoRSVinfectionandmayberelevanttoexplainingwhysomeinfectedindividualsdevelopmild,upperrespiratorytractinfectionswhereasotherindividualsdevelopbronchiolitisorpneumonia,orgoontodeveloprecurrentwheezing/asthma.
Unfortunately,therehasbeenarecurrentdifcultyinvalidatingthelargenumberofcan-didatemolecules/genesbyreplicationinothercohorts,andtheoverallpictureisfarfromclear[1].
Theuseofasys-temsbiologyapproach(see''ARenaissanceofRSVCellBiology,andtheGrowingRoleofSystemsBiology'')promisestoaccelerateknowledgeofidenticationofcan-didatemoleculesandgenesforvalidation.
Onerecentstudy[75]hasreportedanexcitingdevelopmentthatmaypro-videacluetothepossiblebiological''missinglink''betweensevereRSVinfectionsandthesubsequentdevel-opmentofpediatricasthma.
Inaseriesofexperimentsthatbeganwithunbiasedinterrogationofcellstoidentifycan-didategenes,followedbyvalidationinexperimentalani-malsandanalysisofhumancohorts,investigatorshavecharacterizedgeneticpolymorphismsinthepromoterregiontoNFKBIAashavingbothfunctionalandclinicalsignicanceinthecontextofRSVbronchiolitisandpedi-atricasthma.
NFKBIAisthegenewhichencodesIjBa,themajornegativeregulatorofNF-jB.
NF-jBisanimportantnucleartranscriptionfactorthathasacentralroleinregu-latingmultiplehostimmuneandinammatoryresponseelements.
NFKBIAmayendupastheunifyingconceptforbronchiolitisandallergy,asimplicatedbytheresultsofthisstudy,andthisopensupexcitingnewresearchdirectionsforpatientidentication,stratication,andtargetedtherapy.
RSVandParticulateMatter:AnEvenTighterBondThanPreviouslyThoughtEveryday,humansarebombardedwithnumerousinhaledagentsfromtheindoorandoutdoorenvironment.
Particu-latematterrelatedtoairpollutionisbutoneexampleofsuchanagent.
PreviousworkthatstudiedinteractionsbetweenRSVandparticulatematterfocusedoneffectsonthecellularinammatoryresponseinvitro[76–79]andonalargerscaleondeningtemporalrelationshipsbetweenairpollutionexposureandvirusinfectionsandtheireffectsonrespiratoryhealth[80,81].
Fromthesestudies,itisapparentthatairpollutionshouldbeconsideredasapotentialcon-founderoftheeffectivenessofRSVinterventions.
ArecentstudyhasprovidedanevenmoredramaticrelationshipbetweenRSVandparticulatematter[82].
Inthiswork,''designerparticles''ofspecicchemicalcom-positionwereconjugatedtohumanRSV,andcellcultureswereinoculatedwiththesecomplexes,withstatisticallysignicantresponsesobservedwithrespecttocytokineproductionbycells.
Interestingly,thesamestudyshowedthatRSVconjugatedtotheseparticlescouldremaininfectiousatambientroomtemperatureforatleast2monthsafterpreparation,withouttheneedforcellcul-ture,orfreezingstockstolowtemperature(-80°Corbelow).
TheseobservationsopenupanentirelynewpotentialmechanismforRSVsurvivaloutsidelivingorganisms,andsuggestamodeofenvironmentaltrans-missionofRSV,separatefromclosecontactofindividualstovirus-containingdropletsorfomites.
ObtainingimprovedunderstandingoftheimplicationsofRSVconjugatedtoparticulatematterrequiresfurtherresearch.
ProgressTowardanRSVVaccine:CloserthanEverSincethediscoveryofRSVin1955–1957[83–85],vaccinedevelopmenthasbeenamajorpriority,yetthereareanumberoflogisticalconsiderationsrelatedtoRSVinfec-tionsaffectingprogressinthisarea,asCrowe[86]sum-marizedoveradecadeago(Table1).
Thetragedythatoccurredinthe1960swithacandidateRSVvaccinethatwaspreparedbyformalininactivationofwholevirusthatunderwenteldtrialsiswelldocumented[87].
Briey,vaccinerecipientsparadoxicallydevelopedsevererclinicaldiseaseonRSVexposure,andtherewereseveraldeathsinthevaccinatedgroup.
Themechanismsresponsibleforvaccine-enhancedillnesswereultimatelydetermineddecadeslaterbyuseofanimalmodels.
ThroughapplicationoftheTh1–Th2paradigmofCD4lympho-cytes,investigatorsdeterminedthatexposureofexperi-mentalanimalstoformalin-inactivatedRSVvaccineresultedinanexaggeratedhostTh2responseaftersub-sequentexposuretoinfectiousvirus.
Thishasstimulatedconsiderableresearchactivitywiththeaimtodesignvac-cinesthatshifttheTh1–Th2balancetowardTh1[88,89].
However,thismaybeanoverlysimplisticapproach,asarecentstudyinmicehasreportedthatshiftingthehostimmuneresponsetowardTh1actuallydecreasesthepro-ductionofRSV-neutralizingantibodies,whichwouldhavetheeffectoflimitingtheeffectivenessofavaccine[90].
InadditiontoTh1andTh2lymphocytes,thesubpopulationofTh17lymphocyteshasbeenimplicatedinRSVinfection,furtheraddingtothecomplexityofthehostimmuneresponse[91].
CurrPediatrRep(2013)1:149–157153123Inrecentyears,anumberofinnovativeandcleverapproachesforvaccineengineeringhavebeendeveloped,includingtheproductionofvirus-likeparticles[92],chi-mericlivevirusvectors[93],gene-basedvectors[94],andtheuseofnoveladjuvants[95].
Atpresent,weareawareofavarietyofcandidateRSVvaccines(ontheorderoftento20differentproductsatvariousstagesofdevelopment)poisedtoenterhumanclinicaltrials,whichwillbeupdatedattheRSVVaccineConference(Porto,Portugal,October2013:http://www.
meetingsmanagement.
com/rsvvw_2013).
Itisdifculttopredictwhich,ifany,oftheseproductscouldendupasaneffectivevaccineforwidespreaduseinhumans.
Currently,thegoalisfortheretobeasimulta-neousconcertedeffortincontinuedscienticvaccinologyandinthecarefuldesignofhumanclinicaltrialprotocols,includingthepossibilityofimmunizingpregnantwomenasaformofprenatalvaccination,inanefforttodenitivelysolvethelong-standingproblemofdeterminingandimplementingasafe,effectiveRSVvaccine.
Achievingthisoutcomewouldrepresentagloriousaccomplishmentforhumankindandhaveenormousandlastingimpactaroundtheglobe.
ConclusionsWearelivinginaneraofacceleratedactivityandprogressinourunderstandingofviralandhostfactorsinRSVinfection,andarepoisedtotranslatediscoveriesmadeinthisrealmintotheclinictoachieveimprovedhealthout-comesforacuteRSVinfectionsandtheirsequelae.
ResearchhasevolvedfromcharacterizingRSV–hostinteractionstotheidenticationofnewtargetsforvaccineandpharmaceuticaldevelopment.
Advancesinsystemsbiology,informatics,cellandstructuralbiology,andbio-logicalengineeringhaveprovidednewandpowerfulapproachestostudyRSVinfections,andtheinuenceofotherenvironmentalfactorssuchasallergensandparticulatematter.
Wearecloserthanevertoachievingthedreamofasafe,effectiveRSVvaccine,andthefuturepromisestobeexcitingandrewarding.
DisclosurePeterMastrangeloandRichardG.
Hegeledeclarethattheyhavenoconictsofinterest.
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