CMV:AnUpdate

JayA.Fishman,M.D.Professor ofMedicine,HarvardMedicalSchool

Director,TransplantInfectiousDiseaseandCompromised HostProgram,MassachusettsGeneralHospital

AssociateDirector,MGHTransplantCenter,Boston,MA,USA

Virusesmaybedangerous....

Cytomegalovirus

• Betaherpesvirinae subfamilyoftheHerpesviridae• Thestructure:

– Nucleuscontainingtheviralgenome(lineardouble-strandedDNA)– Icosahedral proteincapsid– >200geneswithsignificantvariation– Thetegumentproteinmatrix(e.g.,pp65):

• Proteinswithstructuralroles• Proteinswhichmodulatestheimmunehostcellresponse

– Anouterenvelopederivedfromthehostcellnuclearmembrane.• GlycoproteingB - involved incellattachmentandpenetration• GlycoproteingH- involved inthefusionoftheviralenvelopewiththehostcellmembrane

Pérez-Sola,M.J.etal.EIMC2008;26(1):38-47

CroughTetal.ClinMicrobRev,Jan2009,76-98

PathogenicConsiderations

• Longerreplicationcyclethanotherherpesviruses• Reactivation ofviralreplicationisrelatedtohostimmunityand

inflammation• “CMVInfection”isnotuniform

– ManygeneticvariantstrainsofCMVwithmultipletargetcells:Monocytes,macrophages,endothelialcells,epithelialcells,parenchymalcells,immaturedendriticcells,lymphocytes,CD34+cells

– “Latency”inmultiplecelltypes– Biologicaleffectsvarybyviralstrain:

• Cellulartropism,penetrationandtransfertothecellnucleus

• Viralgeneexpressioniscelltype-specific• Replicationandspreadtoothercells(virulence)• Immuneresponsestoinfectionbycelltype

DisseminatedCMV

Liver

LungKidney

Colon

RiskfactorsforCMVdiseaseinsolid-organtransplantpatients

§ Primaryinfection(D+/R−)§ Transplantedorgans,cells§ Bloodproducts

§ FactorsfavoringCMVreactivation§ Inflammation/Fever (cytokines)§ Surgery/Trauma§ Intraoperative hypothermia§ Sepsisorseverebacterial

infections§ T-celldepletion§ Co-infectionswithotherviruses

§ Herpesvirus6or7(HHV6or7)

Factorsfavoringprogressiontoinvasivedisease• Immunosuppression

• T-celldepletion• Mycophenolate,azathioprine• Methylprednisoloneboluses• Alemtuzumab• Highviralload

• Immunomodulation• Herpesvirus6(HHV6)orHHV7

• Geneticfactors• MutationsinTLR2andTLR4genes• Deficiencyofmannose-binding lectin orgenotypeassociatedwithlowproduction ofMBL

ModifiedfromGESITRA-SEIMC/REIPIrecommendationsforthemanagementofCMVinfectioninsolid-organ transplantpatients,Enferm InfeccMicrobiol Clin.2011

CMVreplicationandrelease

PathwaysforCMVreactivation

ReinkePetal.TransplantInfectDis 1999;1:157-64.

TNF-α

NF-κBStress

catechols

Pro-inflammatoryprostaglandins

cAMP

EICMV

Anti-T-cellantibodies

InmunecontrolofHCMVbyinnateandadaptativeimmunity

TaniaCrough.ClinMicrobRev.,Jan.2009,p.76–98

Replicationinmucosalepithelium

Limitedimmunerecognitionbyeffectorcells;latentreservoir

Reactivationfromlatency

ProcessingbyAPC;presentationtoAg-

specificcellsDC/MQà cytokines/chemokinesàactivateinnateimmunecells

B-cellsactivatedbyAPCsà AbsCMV-infectedMѲ à stimulateAg-specificT/NKcellsà IFNs,

TNF,cytolysis

1. CMVinhibitsdifferentiationofmyeloidantigen-presentingcellsfrommonocyticprecursors(transientblockinthecytokine-induceddifferentiationofmonocytesintofunctionallyactiveCD1a-positivedendritic cells)

2. CMValterstheimmunostimulatory propertiesofdifferent subsetsofmonocytesandDC,rendering themlesscapableofdeveloping intoprofessionalAPCs.

3. VirallyencodedIL-10(cmvIL-10)blunts theantiviralpropertiesofAPCs.4. HCMVhampersantigen-presentingcelltraffickingandphagocytic capacity.

– DecreasedAPCmigrationtositesofinflammationandtodraining lymphnodesinresponsetochemotactic stimuli(RANTES,MIP-1,andMIP-3)

• HCMV-infectedPlasmacytoid DCshavereducedsecretionofcytokinesincluding IFN-γ,reducedallostimulation,decreasedCD4andCD8T-cells

5. Transitorybutsubstantialimmunosuppressionthatinhibits theimmuneresponseagainstthevirusandunrelatedpathogens,mainlyinsubjectswithprimaryinfection

• Reactivationofotherviralinfectionsduring acuteHCMVinfection• Enhancespre-existingimmunosuppression insolid-organ transplantand

alloSCT recipients, increasingtheirriskforinvasivebacterialandfungalinfections

CMVaffectsmanyfunctionsofantigen-presentingcells

CMVRetinitis:LungTransplantRecipient

CMVcecalulcerationinpatientwithnegativeantigenemiaandPCRassaysforCMV

DirectEffects:InvasiveInfectionbyCMVInvasiveColitisAfterLiverTransplant

EffectsofViralInfectioninTransplantation

• “DIRECTEFFECTS”-- CAUSATIONOFINFECTIOUSDISEASESYNDROMES– Feverandneutropenia,hepatitis– Colitis,Retinitis,Nephritis,Pancreatitis

• “INDIRECT”orIMMUNOMODULATORYEFFECTS– SystemicImmuneSuppressionà OI’s– GraftRejection,GVHD– AbrogationOfTolerance

• Oncogenesis/CellularProliferation– HepatitisB:hepatocellular carcinoma– EpsteinBarrVirus:B-celllymphoma(PTLD)– HepatitisC:splenic lymphoma(villouslymphocytes)– Papillomavirus:Squamous cell&anogenital cancer– HHV8(KSHV):Kaposi’ssarcoma,effusionlymphoma– Acceleratedatherogenesis,BK-uretericobstruction

CMVSyndromeFever

WeaknessMyalgiaArthralgia

Myelosuppression

EndOrganDiseaseNephritisHepatitisCarditisColitis

PneumonitisRetinitis

Encephalitis

CMVdisease

LatentCMVinfection

ActiveCMVinfection(viremiaandintissue)

ALG,Fever,TNFα,Sepsis,Suppression

AtherosclerosisBronchiolitisobliterans

Vanishingbileductsyndrome

Opportunisticinfection

Systemicimmunesuppression

Acute Chronic Acute

Cellulareffects:antigenandcytokine

expression

EBV-associatedPTLD

Allograftinjury1

Allograftrejection1

FishmanJA&RubinRHNEngl JMed.1998;338:1741

HCMVProtein Function

FCReceptorhomologueTRL11/IRL11,UL118/119

Blocksantibody-dependent cytotoxicity;binding nonspecificantibodycoatingagainstCD8andNKcells

Pp65matrix Phosphorylates IE-1proteintoinhibitMHCclassI-restrictedantigenpresentation

US3,US6,US10,US11 BlockgenerationandexportofMHCclassIpeptidesUS3,US6,US10,US11 ReducedexpressionofMHCclassIpeptidesUS2 ReducedantigenpresentationinMHCclassIIpathwayMHC-Ihomologue UL40,UL122miRNA,UL142,UL141

BlocksNKcellactivation(also:UL16,pp65)

UL18 MHCclass1homologue; reducedimmunesurveillanceUL20 T-cellreceptorhomologue; reducedantigenpresentationIE86 Inactivatesp53;increasesmoothmuscleproliferationUL33,UL33,UL78,US27,

US28Transmembraneproteinschemokinereceptors;reduced

interferonandchemokineeffects;reducedinflammation, increasedviraldissemination

IL-10homologue UL111a;IL8CXC-1UL146,UL147

Immunosuppression; reducedMHCclassI/IIexpressionandlymphocyteproliferation; increasedneutrophilchemotaxis;reduceddendritic cellandmonocytechemotaxisandfunction

UL144 TNFreceptorhomologueUL36,UL37 Anti-apoptosis forinfectedcells

CMVinterfaceswithinnateandadaptiveimmunesystems

CroughandKhanna,ClinMicrobiol Rev2009

Areasofinvestigation:

- Migrationandchemotaxis

- Cellulardifferentiation- Cellsurvivaland

apoptosis- Determinantsof

reactivationandlatency

- Pathogenrecognition- Globaltranscriptional

andfunctionalchanges

CMV“IndirectEffects”:PossibleMechanisms• UpregulationofMHCclassIIantigensandhomologybetweenCMVIEantigen

andMHCclass-I(HLA-DRβ)1-2

• BlockofCD8+(MHCclassI)recognition• BlocksCMVantigenprocessinganddisplay(immediateearlyAgmodification,

poorCTLresponse)• IncreasedICAM-1,VCAM,cellularmyc&fos(adhesion)• InversionofCD4/CD8ratio3-4

• Increasedcytokines:IL-1β,TNFα,IFNγ,IL-10,IL-4,IL-8,IL-2/IL-2R,C-X-Cchemokines,andIL-85-6

• IncreasedcytotoxicIgM7

• Stimulationofalloimmuneresponsebyviralproteins1-2

• IncreasedPDGF,TGFβ;autoantibodies• IncreasedgranzymeBCD8+T-cells,γδ-T-cells

ICAM-1=Intercellularadhesionmolecule-1PDGF=Platelet-derivedgrowthfactorVCAM=Vascularcelladhesionmolecule

1.Fujinami RS,etal.JVirol. 1988;62:100-105.2.BeckS.Nature. 1988;331:269-272.3.SchooleyRT,etal.NEnglJMed.1983;308:307-313.4.Fishman JA,etal.DiagnImmunol.1983;1:261-265.5.KernF,etal.JAmSocNephrol.1996;7:2476-2482.6.TongCY,etal.JMedVirol.2001;64:29-34.7.BaldwinWM3rd,etal.BrMed(ClinResEd).1983;287:1332-1334.

Mechanisms– ShortVersion– ⇑ Adhesionmolecules(VCAM,ICAM,LFA-1,VLA-4)

– ⇑ Pro-inflammatorycytokines– ⇑ HLA-DRandMHCClassImimic– ⇑ Anti-endothelialAbs?– ⇓ Antigenpresentation(dendriticcellmaturation)

– ⇓ Leukocytemobilization

OpportunisticInfectionsPromotedbyCMVInfectioninTransplantPatients

• Pneumocystiscarinii• Fungalinfections(esp.intra-abdominaltransplants):• Candidemia andintra-abdominal infection inOLTx;patientswithinitialpoorgraftfunction

• Aspergillusspp.RoleofCMVinpromotingfulminant HCVhepatitisratherthandirecteffect

• Bacteremia:Listeria monocytogenes• Epstein-Barrvirusinfection(RCWalkeretal,CID,1995,20:1346-55),HHV6,HHV8/KSHV?

• HCV:riskforcirrhosis,retransplantation,mortality

IndirectEffectsofCMV:Organ-specific• Renal:Decreasedearlygraftfunctionandsomechronicdysfunction(increased

byHHV6andHHV7)– AcutebutpossiblynotchronicallograftrejectionisreducedbyCMV

prophylaxis

• Liver:CMVassociatedwithcirrhosis,graftfailure,needforretransplantation&death– MoreaggressiveHCVrecurrenceandfibrosisafterOLTx (partially

attributedtoHHV6)– CMVdiseaseispreventable

• Heart:cardiacallograftvasculopathy– Reducedbyganciclovir +/- CMVIg

• Lungs:CMVandD+/R- associatedwithBronchiolitisObliteransSyndrome,infection,death– Reducedbyivganciclovir +/- CMVIg

• Pancreas:Notstudied(noCMVyetinislets)

CMVandHSCTOpportunisticInfections

MarretalBlood2002 Nichols etalJID2002

Therapeutics- Terminology• Whatareyoumeasuring?

– Antigenemia– measuringCMVspecificantigeninPBMCs,forexamplepp65assay

– Viremia– culture-basedonly(requiresreplicatingvirus)– DNAemia – measuringCMVDNAbyQNAT (wholebloodor

plasma)QNATcalibratedtotheWHOstandardispreferredfordiagnosis,decisionsregardingpreemptiveantiviraltreatment,andmonitoringresponsetotherapy.

– TissueorBAL– mustbenormalizedtohousekeepinggene,urea,unitvolume,unitmass.

– Viralassaysandcultureofblood,urine,ororalsecretionsarediscouragedinadultsfordiagnosisofactiveinfectionordisease.

• Serology– PositiveIgGisamarkerofpreviousexposure(positive

serologydoesnotdefine“activeinfection”)

Recommendations:Diagnostics• EitherplasmaorwholebloodisanacceptablespecimenforQNAT.Specimentypeorassayshouldnotbechangedwhenmonitoringpatients.

• DespitereportinginIU/ml,viralloadvaluescannotbedirectlycomparedacrosscentersand/orlaboratoriesunlessidenticaltestingreagentsandprocedurescanbeassured.

• Changesinviralloadexceeding0.5log10IU/ml(3-fold)areconsideredtorepresentclinicallysignificantdifferencesinDNAemia

Terminology2

• Surveillance – patientisatrisk,butnoevidenceofanevent(infection)orbiomarker(NAT)

• Monitoring – Patienthaseventorbiomarker(previouslyknownpositiveassay)

• Hybridapproach– universalprophylaxisfollowedbypreemptivetherapy(surveillanceafterprophylaxis)

DoweknowhowtoPreventCMVInfection?

Universalvs.Pre-emptivetherapy

Prophylaxisvs PreemptiveTherapy

• ProphylaxismaybepreferredinD+/R-,withD+orR+&antilymphocyte therapy,potentimmunosuppression including desensitization (plasmapheresis orimmunoadsorption) andABOincompatibleprotocols,orwithrituximab,bortezomib,eculizumab.Rolewithbelatacept hasnotbeenstudied.

• Treatmentofacuterejectionwithantilymphocyte antibodies inat-riskrecipientsshould resultinreinitiation ofprophylaxisorpreemptivetherapyfor1to3months

• CMVDNAemia iscommonaftercessationofprophylaxis,notablyinhighriskpatients

Prophylaxis PreEmptiveTherapy

EarlyCMVDNAemia Rare CommonPreventionofCMVdisease Goodefficacy Goodefficacy*

(*less optimalinhighriskpopulations)

LateCMV(infection/disease) Common RareResistance Uncommon UncommonEaseofimplementation Relativelyeasy MoredifficultOtherherpesviruses PreventsHSV,VZV DoesnotpreventOtherOpportunisticinfections Mayprevent UnknownCost Drugcosts Monitoringcosts

Safety Drugsideeffects Lessdrugtoxicity

Preventionofrejection Mayprevent Unknown

Graftsurvival Mayimprove Mayimprove

Time(days)

DoublingTime=ln2/a

y=y0eax

AverageDT=1-2days

Log 1

0ViralLoa

d

detectionthreshold

Preemptive inD+/R-

LisboaLF,etal.Abstractpresentedat:AmericanTransplantCongress;May30-June 3,2009;Boston,Massachusetts.Abstract388. DT=Doublingtime

CMVdiseaseinD+/R- renalrecipients:Meta-analysis(allagents)

• UniversalandPre-emptiveprophylaxissignificantlyreducetheriskofCMVdisease

-81%

-64%

-100%

-80%

-60%

-40%

-20%

0%Universal Pre-emptive

CM

V di

seas

e ris

k re

duct

ion

(%)

p=ns

Kalil AC et al. Ann Intern Med 2005; 143: 870

Anti-CMVProphylaxisIsAssociatedWithIncreasedRenalGraftSurvivalat4Years(P=0.0425)

1009080706050

0

Oralganciclovirprophylaxis

IVpreemptivetherapy

Freedo

mfrom

graftlo

ss;

uncensored

ford

eath(%

)

1 2 3 4Timeaftertransplantation(years)

KliemV,etal.AmJTransplant. 2008;8:975-983. (B)Khoury JA,etal.AmJTransplant.2006;6:2134-2143. (VGCV)(B)ReischigT,etal.AmJTransplant. 2008;8:69-77. (VACV)(B)

Pvalue(Logranktest)=0.0425

ProphylaxisreducedCMVinfectionby65%(P<0.0001)

Effectofanti-CMVprophylaxisonconcomitantinfections

0.31

0.65

0.27

0.0

0.2

0.4

0.6

0.8

1.0

Placebo/notreatment

Herp. Simplex,Varic. Zoster

Bacterialinfections

Protozoalinfections

Rela

tive

risk

-73% -69%-35%

Hodson EM et al. Lancet 2005; 365: 2105

FungalInfections

l Statisticallysignificantriskreductionofmortalitywithuniversalprophylaxis(Kaliletal)andallcausemortality(Hodsonetal).

Mortality:universalprophylaxisvs.pre-emptivetherapy

-38%

-6%

-60%

-50%

-40%

-30%

-20%

-10%

0%Prophylaxis Pre-emptive

Mor

talit

y: ri

sk re

duct

ion

(%)

Kalil AC et al. Ann Intern Med 2005; 143: 870Hodson EM et al. Lancet 2005; 365: 2105

p=0.032

p=ns

àD+/R+recipientsonpreemptivetherapyhadhighestrateofCMVdisease(19.2%vs.4.4%,P0.003).àNochgGFR,rejection,graftloss(5yrf/u)

11%

39%

D+R−onPre-emptiveTherapy:DirecteffectsofCMVandanti-CMVtreatment

Prophylactic(n=32)

Preemptive(n=80)

PValue

CMVinfection 11(34%) 40(60%) 0.02CMVdisease 5(16%) 21(26%) 0.3Late-onsetinfection 9(28%) 6(8%) 0.003Peakviralload(mean,log10copies/mL)

4.2±1.1 5.0±1.0 0.06

Anti-CMVdrugresistance 1(3%) 13(16%) 0.05RecurrentCMV 3(9%) 18(23%) 0.1

Couzietal,High IncidenceofAnticytomegalovirusDrugResistanceAmongD+R−KidneyTransplantRecipientsReceivingPreemptiveTherapy,AJT,2011

Prophylaxis,Pre-emptiveTherapy,andHybridCare

1 32 765Transplant Months

Pre-emptivemonitoringperiod(onceweeklyfor12-16weeks);ifCMVdetected(viralloadorpp65Ag),treatuntilcleared

indicatesCMVsurveillancebyviralload(orpp65Ag)

AmericanSocietyofTransplantation.AmJTransplant2004;4:51–8

Prophylaxisperiod(commonly90–100days)

HYBRID:Prophylaxis+Monitoring

Long-TermOutcomesofPreemptiveValganciclovirComparedwithValacyclovirProphylaxisforPreventionofCMVinRenal

Transplantation

JournaloftheAmericanSocietyofNephrologybyAmericanSocietyofNephrologyReproducedwithpermissionofAMERICANSOCIETYOFNEPHROLOGYintheformatRepublishincontinuingeducationmaterialsviaCopyrightClearanceCenter. 35

Cooking upahybridstrategybyDavide Abate

Hybrid Approaches Vary

• Inpre-emptivetherapy:• Surveillanceonceweeklyfor3- 4months• NAT-DNAemia atpositivethresholdà treatmentdose+/- reductionin

immunuosuppression forminimumof2weeksoftreatment.• Afterresolution,discontinueantiviralandcontinueweeklysurveillance.• SpecificthresholdsfordefiningoptimalPEThavenotyetbeendefined

• Useofsurveillanceafterprophylaxismaybeconsideredinpatientsconsideredatincreasedriskforpost-prophylaxisCMVdisease(e.g.,weeklyfor~8-12weeks).

• Specialgroups:• D+/R- patientsafterliver,heart,andpancreas3-6monthsdependingonthedegreeof

immunosuppression, includingantilymphocyte antibodiesforinduction.• D+/R- lungtransplantrecipients:6-12months.R+lungtransplantrecipients6months

prophylaxiswhileD+/R+areathigherriskfordevelopingCMVdisease.

• Limitedreportsdonotsupporttheroutineuseofsecondaryprophylaxis.

• ProspectivecohortstudyofconsecutiveCMV+SOTpatientsundergoingkidney,liver,liver–kidney)andhearttransplantation

• Lowrisk,R+,noanti-lymphocytedepletiontherapy• Testedevery2weeks(first100days)&every4weeks(day100-

180),~10-11testseach• Aviralloadof3983IU/ml*(2600copies/ml)wasestablishedas

theoptimalcut-off;99.6%NPV,sensitivity90%,specificity89%• AmajorityofpatientswillnotdevelopCMVdiseasewithout

specificantiviraltherapy(18of393incohort;15beforeday100)

• Limitations:can’tapplyifhigherrisk,ATGuse,differentISregimen 39

SpecialCases• Transplantrecipientsonmammaliantargetofrapamycin

(mTOR)inhibitorssuchassirolimus andeverolimusmayhavelowerratesofCMV;whetherthisshouldaltertheirpreventionstrategyisunknown.Effectisdose-dependent.

• TherearelimiteddatatosupporttheuseofCMVimmunoglobulin(CMVIg)forprophylaxiswhenappropriateantivirals aregiven.

Molecularmechanismsofanti-CMVeffectsofmTORinhibitors

• CMVeventsaftersolidorgantransplantationoccurredsignificantlymoreoftenunderCNIs(RR=2.27)thanwithrapamycin.

• mTOR-I+CNIvs.CNIalonein15trialsofkidney,heart,andlivertransplantationàhigherCMVincidencewhenpatientsreceivedanmTOR-Ifreeimmunosuppression(RR=2.45).

AllCMVIgG+@transplant;switched@6mos

prednisolone/MMFprednisolone/everolimus

prednisolone/CyA

mTOR Effectisdosedependent

AJT,11:2453–2462,November2011

CookingTreatmentRecommendations

TherapeuticMastersforCMV

Agent Route Target Toxicity Availability

Ganciclovir(GCV)

IV>>PO UL54,DNApolymerase

Marrow Yes

Valganciclovir(VGCV)

PO UL54,DNApolymerase

Marrow Yes

Foscarnet IV UL54,DNApolymerase

Renal,metabolic

Yes

Cidofovir(CDV)

IV UL54,DNApolymerase

Renal Yes

Brincidofovir(CMX001)

PO UL54,DNApolymerase

Less renalthanCDV

NotforCMV

Letermovir(AIC246)

PO/IV UL56, DNApackaging

Limited Experimental

Maribavir(MBV)

PO UL97, egress Limited Experimental

Anti-CMVAgents

NEWAGENTSWILLNOTCOVERHSV-VZVASWELLAS

VALGANCICLOVIR.

Therapy• WiththeuseofhighlysensitiveQNAT(LLOQ<200IU/ml),considerdiscontinuingtherapyafteroneresultislessthantheLLOQ.Confirmatorytestingshouldbedoneoneweekafterdiscontinuingtherapy.

• Iftheassayisnothighlysensitivethen2consecutiveundetectable(negative)resultsareneededtodiscontinuetherapy

• Drugresistanceshouldbesuspectedinpatientswithclinicaltreatmentfailuredespitegreaterthantwoweeksofantiviraltreatment

UL97Mutations

FoldchangeinganciclovirEC50a

Genotypefrequency

5-15x 2-5x <2x

Mostcommon M460V/I,H520Q,A594V,L595S,C603W

C592G

Lesscommonatcodons460,590-

607

M460T,A594G,595delb,L595F/W,E596Y,597del2b,

599del,K599T,600del,601del,601del2,C603R,C607Y,del(≥3)c

A591V,A594E/T,E596G,C603S,596delb,600del2,C607F

E596D,N597D,K599E/R,L600I,T601M,D605Ed

Atypicalloci F342Se,K355Me,V356Ge,V466Ge,C480Re,C518Y,P521Le

L405P,I610T,A613V M615V,Y617H,A619V,L634Q,E655K,A674T

(a)Moderateresistance(5-15x),low-graderesistance(2-5x),orinsignificantresistance(<2x)

(b)=inframedeletionofcodon

(c)Inframedeletionof≥3codons inthe590-607rangecanbeassumedtoconfermoderateganciclovirresistance(8- to15-fold).Deletionoflessthan3codonsmayconfervaryingdegreesofganciclovirresistance(4- to10-fold).

(d)D605E isabaselinesequencepolymorphismcommonineastAsia,unrelatedtodrugresistance

(e)Maribavircross-resistancedocumented,allexceptF342Saremarkedlygrowth-inhibited

AlgorithmforSuspectedCMVResistance

Kotton CN et al. UpdatedInternational Consensus Guidelines ontheManagementofCytomegalovirusinSolid-OrganTransplantation.Transplantation. 2013;96(4):333-360.

Genomesequence ofUL54andUL97

Caveats• Notallgenesaresequenced• Notallmutationsareknown• Notallmutationsareequal• Somemutationsassociatedwithdecreasedviralfitness• Polyclonalinfectionsarecommon

Kotton 2013

CMVUL54DNAPolymeraseMutations

Exo andregionVmutationsconferGCV-CDVcross-resistanceFOS-Rmutationsmayconferlow-gradeGCV±CDVcross-resistance

CrossResistance

Lurain NS,Chou S.Antiviraldrugresistanceofhuman cytomegalovirus.Clin Microbiol Rev.2010;23(4):689-712.

Ratio=IC50 ofmutant/IC50 ofwildtype

“IfatypicalUL97mutationthatconfersa5- to10-foldincreaseinGCVresistanceisdetected,aswitchtoFOSisindicated,especiallyinthepresenceofongoing diseaseorhighandincreasingviralloads.”

Immunology• Serostatus isusefulbutreflectshighlyvariablepopulations

intermsofpredictionofriskforCMVinfection.• IgG hypogammaglobulinemia isassociatedwithanincreased

riskofCMVdiseaseaftertransplantation.Measurementoftotalimmunoglobulins issuggestedinsituationswhereCMVisdifficulttocontrol.

• CMV-specificcellularimmunemonitoringhasbeenshowntopredictCMVinfectioninthepre-transplantandpost-transplantsettingsinprospectiveobservational,multi-centerstudies.InterventionalstudiestodeterminepreciseclinicalutilityofCMIareongoing.

• CMVvaccinesareinpreclinical,phaseIandphaseIItrials.TheprimarygoalofaCMVvaccineshouldbetopreventormodulateCMVreplicationand/orCMVdisease.

• T-celltherapiesshouldbefurtherevaluatedforresistant/refractoryCMV.

PredictiveMarkers?• Serostatus basedriskstratification(i.e.D+ /R−,D+ /R+,D−/R+,andD−/R−)hidesasubstantialheterogeneityoftheindividualrisksforCMVreplicationandprogressiontoCMVdisease.• TheriskofCMVdiseaseamongD+ /R−patientsinthefirstyearposttransplant rangefrom6.4to58.3%whentheorganrecipientswereregroupedaccordingtoCMV-specificcell-mediatedimmunitytestingresultsattheendofantiviralprophylaxis.• Testsandmarkerscapableofindividualizing theriskofCMVreplicationmaysignificantlyimproveCMVpreventative strategies.

Cell-mediatedimmunitytopredictcytomegalovirusdiseaseinhigh-risksolidorgantransplantrecipients:LateonsetCMV.

• UtilityoftestingCD8+T-cellresponseagainstCMVasapredictoroflate-onsetCMVdiseaseafterastandardcourseofantiviralprophylaxis.108evaluablepatients(D+/R+n=39;D-/R+n=34;D+/R- n=35)ofwhom18(16.7%)developedsymptomaticCMVdisease.

• TestingusingtheQuantiFERON-CMVassayatbaseline,1,2and3monthsposttransplant (21-peptidepool).

• CMIwasdetectablein38/108(35.2%)patients(cutoff0.1IU/mLinterferon-gamma).

• CMVdiseaseoccurredin2/38(5.3%)patientswithadetectableinterferon-gammaresponseversus16/70(22.9%)patientswithanegativeresponse;p=0.038.

• InthesubgroupofD+/R- patients,CMVdiseaseoccurredin1/10(10.0%)patientswithadetectableinterferon-gammaresponse(cutoff0.1IU/mL)versus10/25(40.0%)patientswithanegativeCMI,p=0.12.

• MonitoringofCMImaybeusefulforpredictinglate-onsetCMVdisease KumarDetal.AmJTransplant.2009May;9(5):1214-22.

IFN-γ andCMVDisease

P=0.02

DetectableIFN-γ CD8+response

NoDetectableIFN-γ CD8+response

KumarD,etal.AmJTransplant. 2009;9:1214-1222.(B)

0 10 20 30 40 50 60 70 80 90 10050

60

70

80

90

100

Timeafterstoppingprophylaxis(days)

Free

domfrom

CMVdisease(%

)

Chemokines andImmuneControlofCytomegalovirusinOrganTransplantRecipients.

• CMVviremic organtransplantrecipients,chemokineexpression—specificallythechemokineCCL8(AUC0.84995%CI0.721–0.978;p = 0.003)andtheinterferon-γ inducedchemokineCXCL10(AUC0.841,95%CI0.707–0.974;p = 0.004)—wereassociatedwithcontrolofviralreplication.

• HomozygousTTpolymorphismintheCCL8promoter(SNPrs3138035)ofD+/R- transplantrecipientsconferredanincreasedriskofviralreplicationafterdiscontinuationofantiviralprophylaxis(hazardratio3.6;95%CI2.077–51.88).

• TheprimarycelltypeproducingCCL8inresponsetoCMVpeptidestimulationwasthemonocyte fractionwhereCCL8productionisassociatedwithspontaneousviralclearanceinpatientswithCMVviremia. Thereisdose-dependentreductioninCCL8productionwithimmunosuppression.Lisboa,L.F.etal.(2015),AmericanJournalofTransplantation.doi: 10.1111/ajt.13207

Virus-SpecificTcellAdoptiveTransfer

Ridell etalScience1992

Leen etalBlood2013Papadopoulou etal.Sci TransMed2014

• PBMCsfromHLAhomozygous,seropositivedonors• Generatedtrivalent(CMV,EBV,AdV)CD8Tcells• CreatingabankofreadytousevirusspecificCTLs

• Identified50patientswithrefractoryviralinfectionsafterSCT

• MatchedHLAofvirusspecificTcellswithpatients• AdoptivetransferofvirusspecificTcellsweekly• Subsequently havedevelopedpentavalent Tcells(CMV,

EBV,HHV6,AdV,BKV)

Valganciclovir

Valganciclovir ->Ganciclovir

HighdoseGanciclovir+Foscarnet

HighdoseFoscarnet

CMVTcells

UL54K513N

UL54A834P

CMVdiseaseafterSCT

MajorChanges• Biology:MechanismofCMVeffectonmacrophagefunctionappearstobeviainhibitionofinflammatorypathways.

• QNAT:increasedsensitivityà positiveinGIdiseaseandsinglenegativeassaymaybesufficientforstoppingtherapy

• Prophylaxisroutinein:– AllD+R-– D+orR+heartandlungs.

• Multiplenewdrugsà soon?• UseofCMV-specificT-celltherapymightaddressresistance.