DefenseAgainstViruses/AIDS

ShomysehSanjabi,PhDShomyseh.Sanjabi@gladstone.ucsf.edu

Micro204,11/30/18

RecommendedReading:Textbook:• Janeway chapter13.22-13.38.Prof.V.Racaniello onlinematerial(ColumbiaUniversityvirologycourse)• http://www.virology.ws/course/Reviews:• Paludan SR.InnateantiviraldefensesindependentofinducibleIFNα/βproduction.(2016)

TrendsinImmunology 37:588• Deeks etal.InternationalAIDSSocietyglobalscientificstrategy:towardsanHIVcure.

(2016)NatureMedicine22:839.• Sengupta andSiliciano.TargetingtheLatentReservoirforHIV-1.(2018)Immunity 48:872.• Elong Ngono andShresta.ImmuneResponsetoDengueandZika.(2018)AnnulReviewof

Immunology 36:279.Primaryliterature:• Yuanetal.AsinglemutationintheprM proteinofZika viruscontributestofetal

microcephaly.(2017)Science 358:933.• Estesetal.Definingtotal-bodyAIDS-virusburdenwithimplicationsforcurativestrategies.

(2017)NatureMedicine23:1271.• Buggert etal.IdentificationandcharacterizationofHIV-specificresidentmemoryCD8+T

cellsinhumanlymphoidtissue.(2018)ScienceImmunology3,eaar4526

Whatisavirus?

• Aninfectious,obligateintracellularparasitecomprisinggeneticmaterialsurroundedbyaproteincoatand/oranenvelopederivedfromahostcellmembrane.

• Theyareclassifiedbasedon:– Natureandsequenceofnucleicacidinvirion (RNA/DNA)– Symmetryofproteinshell(capsid)– Presenceorabsenceoflipidmembrane(+/- envelope)– Dimensionsofvirion andcapsid

Envelope

Capsid

Nucleicacid(DNAorRNA)

Spikes(forattachingtospecificcellsurfaces)

GenericViralLifeCycle1. Attachmentandentry

(cellularreceptors)2. Convertgenometo

mRNA3. TranslatemRNAto

protein4. Replicategenome5. Assembleproteins

aroundgenome6. Viralreleasefrom

infectedcell

Cytopathic effect(CPE):Morphologicalalterationsininfectedcells

causedbyviralinvasion.

BaltimoreSystemtoDescribeViralGenomes

I. dsDNAII. ssDNAIII. dsRNAIV. ss (+)RNAV. ss (-)RNAVI. ss (+)RNAwith

DNAintermediateVII. GappeddsDNA

Theyallhaveto:1. MakemRNA2. Usehostribosomesto

translatemRNAàprotein

Retrovirus

PoliovirusZika virus

Influenzavirus

Reovirus

HepatitisBvirus

AdenovirusHerpessimplexvirus

Parvovirus

• Replicatingvirusesproducelargenumbersofmutantgenomes!• MostDNAvirusesevolveslowerthanRNAvirusesastheirgenomereplicationisnotaserrorproneasRNAviruses.

FundamentalQuestionsofViralPathogenesis

• Howdoesavirusparticleenterthehost?(transmission)• Whatistheinitialhostresponse?(innatesensing)• Wheredoesprimaryreplicationoccur?(tropism)• Howdoestheinfectionspreadinthehost?(viral

dissemination)• Whatorgansandtissuesareinfected?(localvs.

systemicinfection)• Istheinfectionclearedfromthehostorisapersistent

infectionestablished?(acutevs.chronic)• Howisthevirustransmittedtootherhosts?(viral

shedding)

ViralTropismisDeterminedbySusceptibility,Permissivity,AccessibilityandDefense

• Asusceptible cellhasafunctionalreceptorforagivenvirus- thecellmayormaynotbeabletosupportviralreplication

• Aresistantcellhasnoreceptor- itmayormaynotbecompetenttosupportviralreplication

• Apermissivecellhasthecapacitytoreplicatevirus-itmayormaynotbesusceptible

• AsusceptibleANDpermissivecellistheonlycellthatcantakeupavirusparticleandreplicateit

TwoComponentstoViralPathogenesis

1.Effectsofviralreplicationonthehostà Cytopathic vs.non-cytopathic effect

2.Effectsofhostresponseonvirusandhostà Immuneprotectionvs.Immunopathology

Viralvirulenceisinfluencedby:• Viraldose• Routeofinfection• Species• Age• Gender• Susceptibilityofhost

3Requirementsforsuccessfulinfection:• Enoughvirus• Cellsaccessible,susceptible,

andpermissive• Localantiviraldefenseis

absentorovercome

ImmuneDefenseBeginsbyViralSensing

Melchjorsen 2013Viruses

SpecificityofInnateImmuneReceptorsforViralNucleicAcid

Aoshi etal.2011CurrentOpinioninVirology

IFNstimulatedgenes(ISGs=IRF7,RIG-I,MDA5)

ViralSensingInitiatesTypeIIFNResponse

Paludan 2016,TrendsinImmunology

LargequantitiesofIFNhavedramaticphysiologicalconsequences:fever,chills,nausea,malaise

à ‘flu-like’symptoms

MostofourcellshaveIFNreceptorsà Antiviralactivitytoblockinfection!

MacMicking 2012NatRevImmunol

Cell-autonomousMechanismsUsedbyIFN-InducedProteinsAgainstViruses

• Viralentryanduncoating:IFITMs,TRIM5α

• Nucleocapsid transport:MXs

• InhibitionofRNAreversetranscription,proteintranslationandstabilityAPOBEC3,SAMHD1,ADAR1,NOS2,OAS,RNaseL,ISG20,PKR,andISG15

• PreventviralassemblyandreleaseViperin andTetherin

ViralCountermeasuresAgainstInnateandAdaptiveImmunity

• Sensing• IFNproduction• IFNsignaltransduction• Cytokineproduction• Chemokineproduction• NKcellactivation• DCantigen

presentation• Complementinhibition

http://viralzone.expasy.org/all_by_protein/886.html

Flaviviral InhibitionoftheInterferonSystem

Elong Ngono andShresta 2018Annu RevImmunol

ManyVirusesInterferewithAntigenPresentation(Reasoncross-presentationisimportant!)

MechanismstoSubverttheHostImmuneSystem(Reasonwestudyviralvirulencefactors!)

TakeHomeMessagesI:

• Virustransmitsthroughaccessible(routematters),susceptible(cellularreceptors)andpermissive(cellhastherightmachinery)cells.

• Virusencountershostinnatesensors.• TypeIIFNisproduced.• ISGshaveantiviralandinflammatoryactivity.• Viruscounteractsinnateandadaptiveimmunity.

LocalizedControlversusSystemicDisseminationofViruses

• Ifimmunesystemprevails,viruseswillremainlocalized.

• Ifphysicalandimmunebarriersarebreached,viruswilldisseminate byspreadingbeyondtheprimarysiteofinfection.

• Ifmanyorgansareinfected,virushasgonesystemic.

• Ifvirusentersbloodsystem,itwillhematogenously spreadtootherorgans.

• Importanttostudyviralpathogenesisvianaturalroutesoftransmission!

Acutevs ChronicViremia

Viralsetpointduringchronicviremia

GeneralPatternsofViralInfection

Whataresomeofthereasonsforthesevariouspatternsofviralinfection?

*SIV-infectedrhesusmacaques

*SIV-infectedsootymangabey

*LCMV(Armstrong)

*LCMV(Clone13)

Cytopathic andNon-cytopathic VirusesElicitDifferentImmuneResponses

Cytopathic viruses• Causehighinflammationduetocellandtissuedamage

– ActivatetheinnateresponseNon-cytopathic viruses• Typicallycauselowinflammationduetolackofcellular

damage,apoptosis/necrosis– Loworineffectiveinnateimmuneresponse– Donoteffectivelyactivateadaptiveimmuneresponse

• Dramaticallydifferentinteractionswiththehostimmunesystem– Persistentinfections:rarelyorinefficientlycleared

• Diseaseisusuallyaconsequenceoftheimmuneresponse– Immunopathology

Persistent(Chronic)Infections

• Occurwhenprimaryinfectionisnotclearedbyimmuneresponse

• Virions,protein,genomescontinuetobeproduced• Viralgenomesmayremainafterproteinsarenotdetected

• Nosinglemechanism:– Whencytopathic effectsareabsentandhostdefensesarereduced,persistentinfectionislikely

– Reducedimmunesurveillance inimmuneprivilegedsites(CNS,eye…)

– Viralimmunemodulationà immunosuppression– Latentinfectionsà poorrecognitionbyimmunesystem

ImmunopathologyCausedbyAntiviralImmunity

ViralInfectioncanbeBlockedbyNeutralizingAbs

Antibody-dependentEnhancement(ADE)ofDengueInfection

• Primarydenguevirus(DENV)infectiongeneratesAbs.• Subsequentinfectionwithadifferentserotypewillbindthesenon-

neutralizingAbs.• Instead,theAb–viruscomplexattachestoreceptorscalledFcγ receptors

(FcγR)oncirculatingmonocytes.• Theantibodieshelpthevirusinfectmonocytesmoreefficiently.• Theoutcomeisanincreaseintheoverallreplicationofthevirus,more

inflammation,andahigherriskofseveredenguefever.

TCellOriginalAntigenicSin

Elong Ngono andShresta 2018Annu RevImmunol

presentationofcross-reactiveviralantigen

ViralEvolution:TheConstantChangeofaViralPopulationintheFaceofSelectionPressures

• Ashostpopulationsgrowandadapt,viruspopulationsareselectedthatcaninfectthem– Newviralpopulationsemergeeveryday

• Italsoworkstheotherway– Viralpopulationscanbesignificantselectiveforcesintheevolutionof

hostpopulations• Ifahostpopulationcannotadapttoalethalvirusinfection,

thepopulationmaybeexterminated

• Examplesofviralevolution:Influenza,Zika virus,andHIV

NotethattheseareallRNAviruses!

AntigenicDriftversusAntigenicShift

• Drift- diversityarisingfromcopyingerrorsandimmuneselection.– Mayoccureachtimea

genomereplicates– Causeofepidemics

• Shift - diversityarisingafterrecombinationorreassortment ofsegmentedgenomes.– Isrelativelyrare– Causeofpandemics

Reassortment EventsGiveRisetoPandemicInfluenza:TheEvolutionofthe2009InfluenzaA(H1N1)Virus

• InfluenzaAvirusesareclassifiedbyantigeniccomposition,byserologictestingofHAandNA.

• H1-17caninfectbirds;H1-3caninfectandtransmitbetweenhumans.

• The2009H1N1pandemicstrainisareassortment ofavian,human,andswineinfluenzaviruses!

Trifonov etal.2009NEJM

Zika Virus:TheEvolvingEpidemic

• Zika viruswasdiscoveredin1947inZika ForestinUganda.• ItwasisolatedfromRhesusMacaques.• Arbovirus ofthegenusFlavivirus (YellowFever,Dengue,WestNile,

Japaneseencephalitis)• Firsthumancasewasdetectedin1952.• SporadicinfectionsreportedintropicalAfrica,SoutheastAsia,andthe

PacificIslandspriorto2014.

Zika CanCauseMicrocephalyandOtherSevereBrainDefectsinUnbornBabies

Petersen2016NEJM

LinkBetweenZika andMicrocephaly

Neurovirulence PhenotypesoftheContemporaryZIKVStrainsandTheirAncestralAsianStrain

The Serine 139 àAsparagine mutant causes more severe microcephaly.

Yuanetal.(2017)Science 358:933.

MutationThatShoweduparound2014inZIKVFormsaReceptorforNeuralProgenitorCells?

Screaton andMongkolsapaya 2017Science358:863

PhylogeneticOriginsofHIV-1andHIV-2• HIV-1showsmarkedgenetic

variability.• Classifiedonthebasisofgenomic

sequenceintofourmajorgroups:– M(main),– O(outlier),– N(non-M,non-O),– P(non-M,non-N,non-O),

• Thesearefurtherdiversifiedintosubtypes,orclades,thataredesignatedbythelettersAtoK.

• Indifferentpartsoftheworld,differentsubtypespredominate.

HIVisaMemberoftheLentivirusFamilyofRetroviruses

• Lentiviruses caninfectnondividing cells

• Replicationdrivenfromlongterminalrepeats

• RTmakesalotofmistakes!

HIVTropismDeterminedbyReceptor/Co-receptor

CXCR4isexpressedprimarilybynaiveandcentralmemoryCD4Tcells.

CCR5ispredominantlyexpressedonsubsetsofeffectormemoryCD4Tcells(speciallyinthemucosa!),dendriticcells,andmacrophages.

DendriticCellsTransportHIVfromMucosalSurfacestoCD4TCellsinLymphoidTissue

• HIVisasexuallytransmittedpathogenthatmustcrossmucosalbarriers.• Abrasioncausedbysexualcontactcanpromotetransmission.• DirectlypickedupbyDCsthathaveprotrudedbetweenepithelialcellsto

sampletheexternalworld.• HIVvirions aretranslocated backtothecellsurfaceandtransferredtoT

cellsinsecondarylymphoidtissue.

Transmission:R5Tropicvirus(CCR5)

Mutates

Chronicphase:X4Tropicvirus(CXCR4)

• Structuralgenes- gag,pol,env

• Regulatorygenes- tat,rev

• Accessorygenes- vif,vpr,vpu,nef

ThegenomicorganizationofHIV• Genomeisreadinthreeframes,

allowingthevirustoencodemanyproteinsinasmallgenome.

• Geneproductsofgag,pol,andenv togetherwithviralRNAarepresentinthematureviralparticle.

• ThemRNAsforTat,Rev,andNefproteinsareproducedbysplicingofviraltranscripts,sotheirgenesaresplitintheviralgenome.(BasisofusingmultiplysplicedHIVRNA(msRNA)fortat/revgenomicregioninPCRassaystodetectviralreplication.)

• Gp120bindsCD4andundergoesaconformationalchange,exposingahigh-affinitysitethatisboundbytheco-receptor(CCR5/CXCR4).

• gp41unfoldsandinsertsaportionofitsstructureintotheplasmamembrane.

• Fusionofviralenvelopewiththecell’splasmamembraneoccurs.

HIVLifeCycle

• Viralnucleocapsid,composedoftheviralgenomeandassociatedviralproteins,enterthehost-cellcytoplasm.

• RTtranscribestheviralRNAintocDNA,encoding9genes.

• Viralintegrase theninegrates viralcDNAintohostgenome,creatingprovirus.

• InactivatedCD4Tcells,NF-κBandNFATbindtoLTRandinitiatetranscriptionoftheHIVgenome.

• Regulatoryproteins,TatandRev,areproduceduponextensiveprocessing.

• TatbothenhancestranscriptionfromtheprovirusandbindstotheRNAtranscripts,stabilizingtheminaformthatcanbetranslated.

• RevbindstheRNAtranscriptsandtransportsthemtothecytosol.

• Thesinglysplicedandunspliced transcriptsencodethestructuralproteinsofthevirus.

• Unspliced transcripts,whicharealsothenewviralgenomes,arepackagedwiththeseproteinstoformmanynewvirusparticles.

HIVCounteractsIntrinsicAntiviralFactors

YanandChen2012NatureImmunology

TypicalCourseofUntreatedInfectionwithHIV

• Acuteflu-likedisease,seroconversion disease,withhightitersofvirusintheblood.• Adaptiveimmunityà controlsacuteillness,largelyrestoresCD4Tcells,butdoesnot

eradicatethevirusà asymptomaticphase(5–10years)• CD4Tcellnumbersfallà opportunisticinfectionsoccurà symptomaticphase• CD4T-cellcountsbelow200cells/μl ,thepatientisconsideredtohaveAIDS.

TheImmuneResponsetoHIV

• Asymptomaticphaseà lowlevelofvirusinblood,butviruscontinuestoreplicateinlymphoidtissues.

• CD4T-cellcountsgraduallydecline,althoughantibodiesandCD8CTLsremainhigh.• OnceAb andCTLsdecline,infectiousvirusincreasesinblood.

MassiveDamagetotheGIAssociatedLymphoidTissueFollowingAcuteInfection

Microbialtranslocation

Systemicinflammation

Absenceoflymphoidcellaggregatesinterminalileum

Brenchley etal.2004JEM

PathogenesisofInflammation-AssociatedDiseaseinHIV-InfectedAdults

Deeks etal.2013Immunity

TheVariableCourseofHIV-1Infection

Elitecontrollers:IndividualswhomaintainnormalCD4countsandundetectableviralloads(<50copiesHIVRNA/mlofplasma)for>10yearsintheabsenceofantiretroviraltherapy.Virusnotdefective.

Mechanism?

HostGenesthatcanAlterHIV-1Progression

*TheseHLAsarealsoassociatedwithautoimmunity!

ResidentMemoryCD8+TCellsinLymphoidTissuesProvideContinuousProtectionAgainstHIVinECs

Roan, Sci. Immunol. (2018)Buggert et al., Sci. Immunol. 3, eaar4526 (2018)

HostGenesthatcanAlterHIV-1Progression

• CCR5-delta32mutationispresentin4-16%ofEuropeandescent.• StemcelltherapycuredGermanAIDSpatient– onlypersoncuredsofar!• Possibletodisruptccr5 withnucleases,CRISPR/Cas9technology?

BasicResearchLedtoDevelopmentofDrugTargetsthatInterferewithHIVLifeCycle

• Duetohighrateofviralmutations,combinationtherapy(HAART)ismuchmoreeffectivethanusingasingledrug.

AdventofHighlyActiveAntiretroviralTherapy(HAART)

HIVEstablishesLatencyinRestingMemoryCD4+TCells

Murrayetal.JImmunol,2016

HAARTDoesNotCurethePatients!

Latentvirushidesintissues!

• LatentlyinfectedCD4memoryTcellshaveahalf-lifeof~44months.• HAARTneedstobeadministeredforover70yearstocompletely

clearthevirus.

OnlyOnePersonsofarHasBeenCured

Deeks etal.2016NatureMedicine

HowcanwepreventoreliminateHIV?

PreventingorEliminatingReservoirstoCureHIV-1

Siliciano 2014NatureMedicine

• ARTandneutralizingantibodiescanstopnewcellsfrombecominginfected,butlatentcellsremain.

• Preexistingvaccine-inducedCTLscanlyseinfectedcellsbeforetheycantransitiontoastateoflatentinfection.

How do we make latent cells visible to the immune system?

LatencyistheMajorBarriertoHIVCure

• “Shock and Kill”- Reactivate virus via immune activation and use of latency reversing agents, followed by CTL killing of virus-producing cells.

Deeks Nature 2012; Archin et al. Nature 2012

Rejuvenated CTLs?

Latently infected cells?

Latency reversal?

Where are the latent cells located?

HIVRemainsintheGutEvenAfterAntiretroviralTherapy

Estesetal.,2017,NatureMedicine

Beforetherapy Aftertherapy

Despitehighlevelsofproviral DNA,lowlevelsofHIVtranscriptionalactivityisobservedinthelargebowel,suggestingalargefractionoflatentHIVresidesinthegut.(Yukl etal.2010&2013JInfectDis)

How can we activate the latent reservoir?

StrategiestoDisruptHIVLatencybyUsingLatency-ReversingAgents(LRAs)

Archin 2014NatRevMicrobio

Telwatte et al 2018 PLOS Pathogens

BlockstoHIVTranscriptionDifferintheGutandBlood

How can we generate and retain HIV-specific CTLs in the gut?

Different LRAs for the gut?

WhyDon’tWeHaveanHIVVaccine?

1. Escapevariants/alteredpeptideligands- virusoperatesnearmutationalthreshold

2. Neutralizingantibodieslow-affinity,ariselate

3. LossofCD4helprequiredforCD8,antibodyresponses

4. ImmuneexhaustionwithPD-1expressiononCD4andCD8anti-HIVTcells

5. Prolongedtimerequiredtodevelopbroadlyneutralizingprotectiveantibodies(bnAbs)

6. NeedtolearnhowtogenerateprotectiveTRMsthroughvaccination.

IfwecurepeopleoftheirHIV,willtheybeprotectedagainstre-infection?

MethodstoMeasureViralTiters

• Plaqueassay• FocusFormingAssay(FFA)• Endpointdilutionassay• Enzyme-LinkedImmunosorbent Assay(ELISA)• QuantitativePolymeraseChainReaction(qPCR)/ddPCR

• ViralOutgrowthAssay(VOA)

PlaqueAssay/FocusFormingAssay

1. Serialdilutionofvirusismade.2. Aconfluentmonolayerofhost

(susceptibleandpermissive)cellsisinfectedwithvaryingdilutionofthevirus.

3. Cellsarecoveredwithasemi-solidmedium,suchasagar.

4. APlaqueisformedwhenavirusinfectsacellwithinthefixedcellmonolayer,lysesthatcellandinfectsneighboringcells.Infection-to-lysis cycleisrepeated.

5. Theplaquesareseenvisually(withhelpofdeadcellstaining)orwithanopticalmicroscope.

6. ForFFA,plaquesarevisualizedusingimmunostaining ofviralantigens.

EndpointDilutionAssay

TCID50=50%TissueCultureInfectiveDose

Quantifiestheamountofvirusrequiredtokill50%ofinfectedhostsortoproduceacytopathic effectin50%ofinoculatedtissueculturecells.

Enzyme-LinkedImmunosorbent Assay(ELISA)

TodetectAb(Seroconversion)

TodetectAg(viralproteins)

TodetectAg

Quantitative/DropletDigitalPCR

PCRreactionmixtureispartitionedintothousandsofindividualdropletssuchthateachcontainsasinglecopyofthetarget,whichfacilitatespreciseendpointquantification.

PCR-basedassayscannotdistinguishdefectivevirusfromintactreplication-competentones!

Archin 2014NatRevMicrobio

ViralOutgrowthversusPCR-basedAssaystoMeasureLatentHIV

• Culture-basedassaysdetectinducedreplication-competentprovirusesonly,whilePCR-basedassaysdetectalltypesofproviruses.

• Onlyinducedreplication-competentprovirusesandintact,noninduced proviruses(INPs)poseabarriertoanHIV-1cure.

Bruneretal.2015TrendsinMicrobiology