De las infracciones

JamieWhitehorn^a,b,CameronP.Simmons^a,c,∗

aOxfordUniversityClinicalResearchUnit,HospitalforTropicalDiseases,HoChiMinhCity,VietNam

bLondonSchoolofHygieneandTropicalMedicine,UnitedKingdom

cCenterforTropicalMedicine,NuffieldDepartmentofClinicalMedicine,UniversityofOxford,UnitedKingdom

a rt i c l e i nf o

Articlehistory:

Available online 21 July 2011 Keywords:

Dengue Flavivirus Pathogenesis

a b s t ra c t

DengueisanimportantcauseofchildhoodandadultmorbidityinAsianandLatinAmericancountries anditsgeographicfootprintisgrowing.Theclinicalmanifestationsofdenguearetheexpressionofa con-stellationofhostandviralfactors,someacquired,othersintrinsictotheindividual.Thevirulenceofthe virusplustheflavivirusinfectionhistory,age,genderandgenotypeofthehostallappeartohelpshape theseverityofinfection.Similarly,thecharacteristicsoftheinnateandacquiredhostimmuneresponse subsequenttoinfectionarealsolikelydeterminantsofoutcome.Thisreviewsummarisesrecent develop-mentsintheunderstandingofdenguepathogenesisandtheirrelevancetodenguevaccinedevelopment.

© 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction. ......................................................................................................................................... 7221 1.1. Clinicalsigns,symptomsandmanagement.................................................................................................. 7222 2. Immunopathogenesisofdengue. .................................................................................................................... 7222 2.1. Thehumoralimmuneresponseandantibody-dependentenhancement................................................................... 7222 2.2. Thecellularimmuneresponse. .............................................................................................................. 7223 2.3. Cytokines..................................................................................................................................... 7223 2.4. Complement. ................................................................................................................................. 7223 2.5. Movingbeyonddescriptivestudiesinimmunopathogenesis............................................................................... 7224 3. Hostdeterminantsofdiseaseseverity............................................................................................................... 7224 3.1. Ageandgender............................................................................................................................... 7224 3.2. Geneticdeterminantsofdenguesusceptibility.............................................................................................. 7224 3.3. Pre-existingcomorbiditiesandpredispositiontoseveredengue........................................................................... 7225 4. Thrombocytopeniaandcoagulopathyindenguepathogenesis..................................................................................... 7225 4.1. Thrombocytopenia........................................................................................................................... 7225 4.2. Coagulopathy................................................................................................................................. 7225 5. Viralfactorsinpathogenesis......................................................................................................................... 7225 5.1. Virusepidemiologyandvirulence........................................................................................................... 7225 5.2. CellularandtissuetargetsofDENvirusinfection........................................................................................... 7226 6. Conclusions.......................................................................................................................................... 7226 References........................................................................................................................................... 7226

∗ Correspondingauthorat:OxfordUniversityClinicalResearchUnit,Hospitalfor TropicalDiseases,HoChiMinhCity,VietNam.

E-mailaddress:[email protected](C.P.Simmons).

1. Introduction

Dengueisagloballyimportantarboviralinfectiontransmitted byAedesmosquitoesthatendangersanestimated2.5billion peo-pleandrepresentsarapidlygrowingpublichealth problem[1].

Therearebetween50and100millioninfectionseachyear,with approximately500,000casesadmittedtohospitalwithsevereand potentiallylife-threateningdisease[2–4].Dengueisanicosahedral, 0264-410X/$–seefrontmatter © 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.vaccine.2011.07.022

envelopedviruswithasingle-strandedpositivesensegenome;itis amemberoftheflaviviridaefamilyandhas4antigenicallydistinct serotypes(DENV1-4)[3,5,6].Afterinfectionofasusceptiblehost, anacute,self-limitingfebrilesystemicsyndromeensues. Resolu-tionofinfectionoccurswithin4–7daysandisassociatedwitha robustinnateandadaptiveimmuneresponse.Atpresent diagno-sisislargelyclinical,treatmentissupportiveanddiseasecontrolis limitedtotacklingthevector[1].Developmentofavaccinewould beamajoradvanceindiseasecontrolbuteffortshavebeen ham-peredbythelackofananimalmodelofthediseaseandconcerns abouttheroleoftheimmunesystemindiseasepathogenesis[6].

Thelackofananimalmodelfurtherlimitsourunderstandingof immunopathogenesis.

Dengueisasyndromeanditspathogenesisaninterplaybetween virusandhostfactorsthatremainsincompletelyunderstood[1,7].

Explainingtheheterogeneitybetweenclinicalpresentationswithin a similarcross-section ofthepopulationremainsa vitalareaof research.Abetterunderstandingofdiseasepathogenesiswould greatlyaidvaccinedevelopmentbyaddressingspecificconcerns aboutvaccinesafetyandefficacyinlightoftheimmunesystem’s role inthedevelopmentofclinicaldisease[8].Thisreview will examinethedifferentcomponentsofourcurrentunderstanding ofdenguepathogenesisandthenconsidertheirimplicationsfor denguevaccinedevelopment.

1.1. Clinicalsigns,symptomsandmanagement

Clinicallyapparentdengue(DEN)virusinfectionisassociated witharangeofsyndromes.Classicaldenguefeverisobservedmore frequentlyin adultsandoccurs afteranincubationof 4–7days [9,10].TheclinicalmanifestationsofchildrenwithDENcan dif-ferfromadults–cough,vomitingandabdominalpainappearto bemorecommon[11].Themortalityrateinyoungchildrenwith DENissignificantlyhigherthaninolderchildrenoradults[12].The incidenceofseverediseaseisperhapshighestininfants,whichmay reflectincreasedcapillaryfragilityandlowercompensatoryreserve inthisagegroup[13].InclassicalDENfeverthereisanabruptonset offeveroftenassociatedwithmyalgia,headacheandsometimes severeretro-orbital pain.Earlyin theillnesstheskinisflushed withpetechiaeappearinginthe“critical”phase;a macularrash isobservedinconvalescence.Thecriticalphaseoccursaroundthe timeofdefervescence,typicallyondays3–7,andisassociatedwith anincreased propensityforcapillary leakageand haemorrhage.

Insomeindividuals,capillarypermeabilitymanifestsasarisein haematocrit,pleuraleffusionsorascites[14].Thisisthestagewhen life-threateningclinicalcomplicationssuchascirculatoryshockare typicallyobserved[14].Disablingfatigueanddepressionmay com-plicaterecovery[15].Thrombocytopeniaisalmostuniversaland minorbleedingmayoccurinmildinfections–thiscanbesevere inthosewithpepticulcerdisease[16].Atypicalclinicalfeatures ofDENarebeingmorefrequentlyreportedbutareprobablystill underappreciated [17]. These include encephalitis, myocarditis, hepatitis,pancreatitis,retinitisandtheacuterespiratorydistress syndrome(ARDS)[8,10,18,19].Theseatypicalpresentationslikely reflectpathologyatdifferentendothelialsurfaces[17].Recognizing thewarningsignsthatmayindicateprogressiontoseveredisease isessentialforsuccessfulcasemanagement–thesesignsinclude abdominalpain,hepatomegaly,evidenceoffluidaccumulationand arisinghaematocrit(orconverselyafallinghaematocrit sugges-tiveofhaemorrhage)[3].ThecurrentWHOguidelinesrecognize dengueasaclinicalcontinuumfromdenguetoseveredengue[3].

Carefulfluidresuscitationislife-savinginDEN[3].Ringer’slactate hasbeenshowntobeefficaciousinmoderatelysevereDEN,and starchordextranhavebeensuggestedformoreseverecases[20].

Plateletsareoftengivenasprophylaxistopreventhaemorrhage;

howeverthisisacontroversialareawithoutaclearevidencebase

[21,22].InmanysettingsDENisdiagnosedclinically;howeverthe featuresofearlyinfectionarenon-specificandmimicthoseofother febrileillnesses[23–25].Anearlydiagnosiswouldassistinpatient triageandwillhaveanincreasinglyimportantroleastherapeutic drugs,e.g.anti-virals,becomeavailableforDEN[26].

2. Immunopathogenesisofdengue

Theseverephenotypesofdengueareobservednotatthetime whentheviralburdenisatitshighestinvivo,butparadoxically whenthevirusisbeingrapidlyclearedfromhosttissuesbythe innateandadaptiveimmuneresponse.Thishasledtothe sugges-tionthatthepathogenesisofclinicallyimportantcomplicationsis closelylinkedtothehostimmunereponse[27].

2.1. Thehumoralimmuneresponseandantibody-dependent enhancement

Althoughnocorrelateofimmunitytodenguehasbeendefined, it’shypothesizedthatthehumoralimmuneresponseisvitalfor controllingDENvirusinfectionandfortheexpressionofacquired immunity.Inpart,thebeliefthatimmunitytodengueis antibody-mediatedstemsfromobservationsinothermedicallyimportant flavivirus infections,e.g. Japaneseencephalitis virusandYellow Fever,wheretheacceptedvaccine-elicitedcorrelateofimmunityis virus-neutralizingantibody[28–31].Confirmationthatvirus neu-tralizingantibodyisacorrelateofdengueimmunityismostlikely tocomefromexpandedphaseIIandphaseIIItrialsoftheSanofi PasteurdevelopedChimeriVaxvaccinethatarecurrently under-wayinAsiaandLatinAmerica[32].(SeeGuyetal.articleinthis SpecialIssue).

Sabin demonstrated in the post-warera how infectionwith oneserotypegavelong-lastingprotectiontothatspecificserotype (homotypicimmunity)andshort-livedprotectionagainsttheother serotypes (heterotypicimmunity)[33].Thebasis forhomotypic immunityisbelievedtobevirus-neutralizingantibodies.The tran-sientnatureofheterotypicimmunityislikelyduetocross-reactive E-proteinspecificantibodiesthatwhenaboveacertain concentra-tionthresholdareprotective.However,overtime,theseantibody concentrations declineand theindividualisthen susceptibleto infectionwithotherDENvirusserotypes.

Multiple prospectivecohort studiesin Asiaand Latin Amer-icahaveidentifiedsecondaryinfectionasanepidemiologicalrisk factorforseveredengue[34–38].Third,orevenfourthinfections also probably occurin endemic settings, but hospital dataand the age-related burden of dengue (children) suggests the vast majority ofthesetertiary orquaternary infectionsareclinically silentorverymild[39].Theleadingexplanationforincreasedrisk ofdiseaseinsecondaryinfectionisthatnon-neutralising, cross-reactiveantibodieselicitedbyaprimaryinfectionbindthevirus which then havegreater potentialtoinfectFc-receptor bearing cells.Thisphenomenon,calledantibody-dependentenhancement (ADE),potentiallyincreasestherisksofdevelopingseveredisease byvirtueofincreasingthenumberofvirusinfectedcellsand there-foretheviralbiomassinvivo[40,41].Someevidencealsosuggests cellsinfectedviaanADEprocessareimmunologicallymodulated suchthatthelocalenvironmentbecomesmorepermissiveforvirus replication[42].Cellsofthemonocyte–macrophagelineagein par-ticulararebelievedtobeamajorsiteofdenguereplicationunder conditionsofADE[27,43].TheconceptofADEwasinitially pro-posedfordenguein1977andwassupportedbyepidemiological observationsinCuba[44,45].DatafromCubademonstratedthat DHFwasmorefrequentlyobservedinthosepatientswhohad evi-denceofpreviousinfectionwithadifferentserotype.Recentwork hasprovidedfurthersupportfortheconceptofADE.Thisresearch

J.Whitehorn,C.P.Simmons/Vaccine29 (2011) 7221–7228 7223 hasdemonstratedthatantibodiestodenguestructural

precursor-membraneprotein(prM),acomponentofthehumoralresponseto infection,arehighlycross-reactivebetweenDENvirusserotypes [46].Invitrostudiesindicatethatevenwhenanti-prMantibodies areathighconcentrations,theyarenon-neutralisingbutpotently mediateADEinFcreceptorbearingcells[46,47].Theproposedbasis forprM-mediatedADEisthatonaproportionofvirusparticlesprM is onlypartiallycleavedfromthevirussurface duringthevirus maturationprocess.Inthisscenario,such“immature”virus parti-clesthatwouldotherwisebenon-orless-infectious,arerendered infectiousinanenvironmentwhereanti-prMantibodiescan medi-ateADE[46].Theseinvitroobservationshaveledtothesuggestion thatavaccinecandidateshouldbedesignedinawaythatwould minimisetheanti-prMresponse[46].

AsecondepidemiologicalsettingthatimplicatesaroleforADEis primaryinfectionofinfantsborntodengue-immunemothers. Pri-maryinfectionsininfantsagedbetween4and12monthsofagecan resultinseveredengue,anoutcomethatisepidemiologicallyless commoninyoungerinfantsandchildrenagedbetween1and2yrs ofage.Attheageof3–4months,maternallyderivedvirus neutral-izingantibodieshavegenerallydeclinedbelowmeasurablelevels ininfants,howevernon-neutralisingantibodies,whichrepresenta muchgreaterfractionofthevirion-bindingantibodypopulation, remain present [48–50]. These virion-binding, non-neutralising antibodiesarethoughttoenhancetheriskofclinicallyapparent andseveredenguethroughaprocessofADE[51–54].Insupportof thishypothesis,neatplasmafrom6-montholdhealthyVietnamese infantsenhancestheinfectivityofDENV-2inFcreceptorbearing cellssignificantlymorethanplasmacollectedatthetimeofbirth orat1-yrofage[52].

ConcernsaboutADEhaveledtofearsthatavaccinecould con-tribute toincreased disease severitydue tore-infection in the presenceofincompleteprotectiontooneormoreserotypes.This hypothetical concernneeds to addressedby careful, long-term follow-upofvaccinerecipients.Itisdifficulthowevertoenvisagea scenariowherebyvaccinationactuallyincreasestheriskofdengue orseveredenguetoalevelwhereitexceedsthe“naturalhistory”

ofdengueepidemiologyinendemicregions.

2.2. Thecellularimmuneresponse

Cellular immuneresponsesare alsosuggestedtoplay arole in clearing virusinfection and potentially triggeringthe devel-opmentofseveredisease.ActivatedmemoryTcellsrecognizing bothconservedandalteredpeptideligandepitopes,aresuggested tobeinvolved inthedevelopmentof plasmaleakage[55].Itis proposedthattheexpressionofviralepitopesonthesurfaceof infectedcellstriggertheproliferationofmemoryTcellsandthe production ofpro-inflammatory cytokinesthat haveanindirect effectonvascularendothelialcellsresultinginplasmaleak.The levelofTcellresponseisthoughttocorrelatetodiseaseseverity [56,57].MongkolsapayaandcolleaguesinThailandshowedthatT cellsinsevereinfectionhavearelativelylowaffinityforthe cur-rentinfectingserotypebutahighaffinityforapastinfectionwitha differentserotype,i.e.theydisplaycharacteristicsoforiginal anti-genic sin[57].Moreover,Tcellresponsesinseverepatientsare mainlymono-functionalinthattheyproduceIFN-!and/orTNF-"

onlyandrarelyCD107a,amarkerofcytotoxicdegranulation. Con-versely,inpatientswithuncomplicated dengue,relativelymore CD8⁺TcellsexpressedCD107aandonlyafewexpressedonlyIFN-!

and/orTNF-"[58].Thisissuggestedtodelayviralclearance,andvia cytokine-mediatedeffects,potentiallyincreasetheriskofsevere manifestationsofdisease.However,itremainstobeshown,ineven atemporalway,thatTcellscontributetocapillaryleakageinvivo.

RecentdatainVietnamesechildrensuggeststheemergenceof acti-vatedTcellsinbloodofchildrenwithdengueisnotsynchronous

withcommencementofcapillarypermeability[62].One possibil-ityisthatactivatedTcellsaresequesteredintissuesduringacute dengueandarethereforedifficulttodetectatthetimecapillary permeabilitybecomesapparent.TheroleofTregulatorycellsisnot clearindengue.Theirroleinchronicinfectiousdiseaseshasbeen studiedbuttheirroleinacuteviralinfectionsisnotwell estab-lished[63].HoweverLuhnandcolleaguesdemonstratedthatthey arefunctionalandexpandinacutedengueinfection[64].Theymay havearoleinsuppressionoftheproductionofvasoactivecytokines –perhapstheregulatoryresponseisinadequateinseveredisease?

Theabsenceofgoodanimalmodelsofdisease(asopposedto infection)isahurdletounderstandingtheroleofmemoryTcells inimmunityandimmunopathogenesis.For thisreason,insights intotheroleofTcellsinimmunitywillderivefromprospective cohortstudies,orvaccinetrials[65].Adetailedcommentaryon thechallengesofusingcellularimmuneparametersascorrelates ofimmunitytodenguehasrecentlybeenpublished[66].

2.3. Cytokines

It isthoughtthat in someindividuals withsecondary infec-tion,highviralburdenstriggerexpressionofawaveofcytokine and other inflammatory molecules from innate and activated, cross-reactiveTcells.Thisinflammatorymileauishypothesizedto mediatepermeabilityinthevascularendothelium,allowingwater andsmallmoleculestoleakfromtheintravascularspaceandin somecasesleadingtotheseveremanifestationsofdengue[67].

Increasedlevelsofmanydifferentcytokineshavebeenobserved indengue infection[68]. Inparticular, higherconcentrations of cytokinessuch as IFN-!, TNF-" and IL-10 have been observed in the sera of patients with severe dengue in Vietnam, Cuba andIndia[69–71].Reducedlevelsofnitricoxide(NO)associated withincreased levels of IL-10 have been described in patients withseveredengue[72].NOcontributestoimmuneregulation– lowerlevelsofNOmayresultintheincreasedexpressionof pro-inflammatorycytokines[73].Inadditionitisknownthatincreased IL-10levelscorrelatetoreducedlevelsofplateletsand reduced plateletfunction[74].Thisphenomenoncould,inpart,contribute tothe development of the bleeding complications observed in severe disease. Elevated levels of IL-6 have been observed in childrenwithascites[75].TNF-"promotesincreasedendothelial permeabilityanditisplausiblethatincreasedlevelsofthiscytokine couldresultinamoreseverediseasecourse[76].Itislikelythat cytokinesplayamutuallysynergisticroleattheendothelialsurface contributingtothedevelopmentofthetransientplasmaleak, how-evertheexactdynamicofthisinteractionhasyettobeelucidated.

It isworth notingthat otherinfectious diseasesand inflamma-torydisordersresultinelevatedcytokineswithouttheattendant increasedvascularpermeabilityseeninseveredengue.Indeed,one ofthechallengesindengueistodissectthoseelementsofthehost immuneresponsethatarecausallylinkedtocapillarypermeability fromthosethatsimplyreflectthenormalhostimmuneresponse toapathogen.Thischallengeismademoredifficultbytheabsence ofgoodanimalmodelsofdisease.

2.4. Complement

Reductioninthelevelsofcomplementcomponentshavebeen describedin patientswithseveredengue, suggestingthat com-plementactivationmayhavearoleinthepathogenesisofsevere disease[77,78].Inparticularithasbeensuggestedthatexcessive complementactivationatendothelialsurfacescontributestothe vascularleakobservedinseveredisease[79].NS1,anon-structural viralproteinthatissecretedfrominfectedcellsandpresentinblood inconcentrationsexceeding1#g/mlinsomepatients, couldbe animportantmodulatorofthecomplementpathway.DENvirus

NS1attenuatesclassicalandlectinpathwayactivationof comple-mentbydirectlyinteractingwithC4[80].NS1promotesefficient degradationofC4toC4bandbythismechanism,NS1issuggested toprotect DENV fromcomplement-dependentneutralizationin solution[80].Thesignificanceoftheseobservationswiththe inde-pendentobservationthatearlyNS1concentrations inbloodare positivelyassociatedwithdiseaseseverityarenotyetclear,but areconsistentwitharoleforNS1andcomplementinpathogenesis [81,82].Finally,itisplausiblethatthelowlevelsofcomplement observedinseveredenguearemerelyamarkerofasevere sys-temic diseaserather than anindicatorof theirrolein capillary permeability.

2.5. Movingbeyonddescriptivestudiesinimmunopathogenesis

Muchoftheliteraturedescribingtheimmunopathogenesisof denguehasbeencorrelativeinnature,e.g.temporalassociations between elevated cytokine concentrations in the febrile phase of dengue is often interpreted as being causally linked to the importantclinicaleventsofplasmaleakageorbleeding manifes-tations.Thechallengeinthisareaofresearchistomovebeyond descriptivestudiesandbegintoidentifycausalimmunopathogenic mechanisms.Therapeuticrandomizedcontrolledintervention tri-als,witheitheranti-viraldrugsorimmunomodulatoryagents,e.g.

corticosteroids,perhapswillofferinsightsintopathogenesisina moredirect fashion.Such trialsareunderway(ClinicalTrials.gov identifier NCT01096576 and ISRCTN39575233). Alternatively, improvementsinanimalmodelsofdenguehaveoccurredsuchthat a productive,sometimesfulminant,acutevirusinfectioncanbe established[83].Asyethowever,nosmallanimalmodelmimics thevirologicalandpathophysiologicaleventsthatoccurinachild withseveredengue,inparticulartherelativelyslowlyevolving cap-illarypermeabilitythatclinicallymanifestsbetweenday3and6of illnesscoupledwithafastdecliningviralburden.Clearlythenthere isscopeforfurtherresearchintoanimalmodelsandstrong justifi-cationforstudyingthehostresponseduringrandomizedcontrolled treatmenttrialsindenguepatients.

3. Hostdeterminantsofdiseaseseverity

OnlyasmallproportionofindividualswithsecondaryDENvirus infections (andan even smallerproportionwithprimary infec-tion)develop severedisease[1].Therefore itfollowsthat other variables, besidespre-existingimmunity,shape theoutcomeof infection.Thissectionwillconsidertheroleofage,gender,genetic susceptibilitydeterminantsandpre-existingmedicalconditionsin determiningtheclinicalphenotype.

3.1. Ageandgender

Ageisariskfactorforseveredengueanddeath.Forexample,the

Ageisariskfactorforseveredengueanddeath.Forexample,the