Adjuvant activity of fish type I interferon shown in a virus DNA vaccination model

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Vaccine

jou rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

Adjuvant activity of ﬁsh type I interferon shown in a virus DNA vaccination model

Chia-Jung Chang, Baojian Sun, Børre Robertsen

^∗

NorwegianCollegeofFisheryScience,UniversityofTromsø,9037Tromsø,Norway

a r t i c l e i n f o

Articlehistory:

Received29January2015

Receivedinrevisedform24March2015 Accepted27March2015

Availableonline9April2015

Keywords:

IFN Adjuvant DNAvaccine

Infectioussalmonanemiavirus Atlanticsalmon

Antibody

Hemagglutinin-esterase

a b s t r a c t

ThereisaneedformoreefficientvaccinestocombatviraldiseasesofAtlanticsalmonandotherfarmed fish.DNAvaccinesarehighlyeffectiveagainstsalmonidrhabdoviruses,buthaveshownlesseffectagainst otherviruses.InthepresentworkwehavestudiediftypeIIFNsmightbeusedasadjuvantsinfish DNAvaccines.ForthispurposewechoseaDNAvaccinemodelbasedonthehemagglutinin-esterase (HE)geneofinfectioussalmonanemiavirus(ISAV)asantigen.Salmonpresmoltswereinjectedwitha plasmidencodingHEaloneortogetherwithaplasmidencodingAtlanticsalmontypeIIFN(IFNa1,IFNb orIFNc).Serawereharvestedafter7–10weeksformeasurementsofantibodyagainstISAVandthefish werechallengedwithISAVtomeasureprotectiveeffectsofthevaccines.Theresultsshowedthatall threeIFNplasmidsdeliveredtogetherwithHEplasmidpotentlyenhancedprotectionofsalmonagainst ISAVmediatedmortalityandstimulatedanincreaseinIgMantibodiesagainstthevirus.Incontrast, HEplasmidalonegavelowantibodytitersandaminorprotectionagainstISAV.Thisdemonstratesthat typeIIFNsstimulateadaptiveimmuneresponsesinfish,whichmaybeabenefitalsoinotherfishDNA vaccines.QuantitativeRT-PCRstudiesshowedthatthesalmonIFNscausedanincreasedinfluxofB-cells andcytotoxicT-cellsatthemuscleinjectionsite,whichmayinpartexplaintheadjuvanteffectofthe IFNs.

1. Introduction

Thelargeincreaseinﬁshfarmingintheworldrequiresade- quatecontrolofvirusdiseases,whichisamajorprobleminthe industry[1].Theorthomyxovirusinfectioussalmonanemiavirus (ISAV)isanexampleofavirusthatcausesseriousdiseaseoffarmed AtlanticsalmoninEurope,ChileandNorthAmerica.In2007,ISAV causedanoutbreakin Chilethathad devastatingeffects onthe country’ssalmonfarmingindustry[1].Whilemostbacterialdis- easesoffarmedsalmonhavebeendefeatedbyhighlyprotective vaccines,virusvaccinesbasedonformalininactivatedvirusesor recombinantviralproteinshavebeenunabletoefﬁcientlycombat virusdiseases[2].DNAvaccinesseemedtobemorepromisingsince theyprovideahighlevelofprotectionagainsttherhabdoviruses viralhemorrhagicsepticaemiavirusandinfectioushematopoietic

Abbreviations:IFN,interferon;ISAV,infectioussalmonanemiavirus;IPNV,infec- tiouspancreaticnecrosisvirus;HE,hemagglutinin-esterase;i.m.,intramuscular;i.p., intraperitoneally;PBS,phosphate-bufferedsaline;RT-qPCR,reversetranscription quantitativePCR.

∗Correspondingauthor.Tel.:+4777644487;fax:+4777644900.

E-mailaddress:borre.robertsen@uit.no(B.Robertsen).

virusinsalmonids[3].However,DNAvaccinesagainstISAVand infectiouspancreaticnecrosisvirushaveshowninferiorprotec- tiveeffectscomparedtorhadoviralDNAvaccines[4].Inthiswork wedecidedtostudyiftypeIIFNs(IFN-I)mightbeusedasadju- vantsinDNAvaccinesagainstvirusinﬁshsinceitisknownthat IFN-Ienhancetheadaptiveimmuneresponseinmammals[5].The adjuvanteffectofIFN-Iinmammalshasbeenlinkedtodirectstim- ulationofBcellsTcellsanddendriticcells[6,7].

AtlanticsalmonpossessesatleastfourIFN-Isubtypes,named IFNa,IFNb,IFNcandIFNd,whichhaveonly22to37%aminoacid sequenceidentityandshowmajordifferencesincellularexpres- sionandantiviralactivity[8–10].IFNa1andIFNcinduceantiviral activityagainstIPNVandISAV,whileIFNbislessactiveandIFNd shownoantiviralactivity[9,11].Inthisworkwehavetestedthe adjuvanteffectofIFNa1,IFNbandIFNcinaDNAvaccinemodel, usingISAVhemagglutinin-esterase(HE)asantigen[12].Atlantic salmonpresmoltswereinjectedwithplasmidexpressingHEalone ortogetherwithaplasmidexpressingIFN,andprotectionandanti- bodylevelsagainstISAVwerethemeasured7–10weekslater.The resultsshowedthatallthreeIFNplasmidsdeliveredtogetherwith HEplasmidpotentlyenhancedprotectionandantibodyproduction againstISAV.Incontrast,HEplasmidalonegavelowantibodytiters andaminorprotectionagainstISAV.RT-qPCRofimmunegenesat http://dx.doi.org/10.1016/j.vaccine.2015.03.093

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themuscleinjectionsitesuggestedthattheIFNsattractedBcells andcytotoxicTcells.

2. Materialsandmethods 2.1. Fish

Atlantic salmon (Salmo salar L.) presmolts (30–45g) were obtained,labeledandkeptin300ltanksat10^◦Casdescribed[13].

AllhandlingofﬁshwasinaccordancewiththeNorwegian“Regu- lationonAnimalExperimentation”andallﬁshexperimentswere submittedtoand approvedby theNorwegianAnimal Research Authority(NARA)beforeinitiation.

2.2. Virus

The ISAV Glesvaer/2/90 isolate was obtained and grown as described[11].VirustiterswerecalculatedbytheTCID₅₀method [14].

2.3. Plasmidsusedforintramuscularinjection

PlasmidsencodingAtlanticsalmonIFNa1,IFNbandIFNcwere availablefromapreviousstudy[9]andweresub-clonedintothe pcDNA3.3-TOPOvector(Invitrogen)downstreamoftheCMVpro- moter.InthisworkwenamedtheseplasmidspIFNa,pIFNband pIFNc.AreligatedpcDNA3.3plasmidwithoutinsertwasusedas negativecontrol.The ISAVhemagglutinin-esterase(HE) expres- sionvectorwasobtainedfromprofessorEspenRimstad,Norwegian SchoolofVeterinaryScience,Oslo,andconsistsofISAVHEfused totheN-terminusofEGFPintheeukaryoticexpressionplasmid pEGFP-N1[12].ThepEGFP-N1plasmidwasusedasnegativecon- trol.InthepresentworkwenamedtheseplasmidspHEandpEGFP, respectively.PlasmidsweretransformedandgrowninOneShot TOP10Escherichiacoli(Invitrogen)andpuriﬁedbyEndoFreeplas- midpuriﬁcationkit(Qiagen).

2.4. DNAvaccinationandchallenge

Presmoltsalmonwereinjectedi.m.approximately1cmbelow thedorsalﬁnwithoneortwoplasmids(15␮geach)in50␮lsterile PBSatpH7.4orwithPBSonlyinthreedifferenttanksasdescribedin Fig.1A(Tank1),Fig.1B(Tank2)andFig.1C(Tank3),respectively.

InFig.1AandBtanks,theﬁshwerekeptfor7weeksandthen infectedbyi.p.injectionwith10⁴ISAV.InFig.1Ctank,theﬁshwere challenged8weekspostvaccinationbyadditionof20%“shedder”

presmolts,whichhadbeeninjectedi.p.with10⁵ISAV.Mortality wasrecordeddailyuntilday32inTank1andTank2,anduntilday 62inTank3.

2.5. Quantitationofvirusinheadkidneyofsurvivalﬁshfromthe cohabitationchallenge

ISAVwasquantitatedbyRT-qPCRofsegment8inheadkidney fromsurvivalﬁshinthecohabitationchallengetrial62daysafter additionofshedderﬁsh(experiment1C).Headkidneyswerekeptin RNAlater(Ambion)andtotalRNAwasisolatedbyQiagenRNeasy minikit(Qiagen).One␮gRNAwasusedforcDNAsynthesisby QuantiTectReverseTranscriptionKit(Qiagen).qPCRwascarried outusingISAVsegment8 primersasdescribed [15].Elongation Factor1␣B(EF1␣B)wasusedasareferencegene[16].ISAVseg- ment8clonedintothepCR-BluntIITOPOplasmid(Invitrogen)was usedforcalculatingISAVsegmentcopynumbersper0.1␮gRNAin theheadkidneysamplesbypreparingastandardcurveofplasmid copynumbersversusCT-valuesasdescribedinthemanualfrom themanufacturerofthe7500FASTReal-TimePCRsystem(Applied

Biosystems),whichwasusedforqPCR.Basedonthis,viruscopy numbersinﬁshwasplottedasacolumnusingboxandwhiskers, andsigniﬁcantdifferenceswereanalyzedbyKruskal–Wallistest andposthocDunn’smultiplecomparisonstestmethod(GraphPad Prismvision6.01forWindows,SanDiego).

2.6. MeasurementofantibodyresponsebyELISA

ISAV-speciﬁcIgMantibodiesinsalmonseraweremeasuredby ELISAusingISAVasantigen.ISAV4waspropagatedinASKcellsand puriﬁedbyultracentrifugationasdescribed[17]usinga15to65%

(wt/vol)sucrosegradient.Thevirusfractionwasharvestedandthe totalproteinconcentrationwasmeasuredwithBCAproteinassay kit(Pierce,ThermoScience).

Microtiterplates(Immuno-PlateMaxisorp,Nunc)werecoated overnightat 4^◦C withISAV correspondingto200ng proteinin 100␮l0.1Msodiumcarbonatebuffer(pH9.6)ineachwell.The plateswerethenwashedandblockedwith5%dryskimmilkin TBSTbuffer(50mMTris–HCl,150mMNaCl,0.1%Tween20, pH 7.6).Fishseraweretwo-folddilutedinTBSTbufferfrom1:50to 1:12,800folddilutionand100␮lofeachdilutionwasaddedtoeach antigen-coatedwellandincubatedovernightat4^◦C.Monoclonal mouseanti-salmonIg(H)(CloneIPA3D1,mouseIgG1,Cedarlane labs)(1:300)andsubsequentlyperoxidaseconjugatedpolyclonal goatanti-mouseIg(Invitrogen)(1:2000)wereaddedinTBSTbuffer andincubatedatroomtemperaturefor2h.Onehundredmicroliter TMBsubstrate(ThermoScientificPierce)wasaddedandreactedfor 10minwhenthereactionswerestoppedwith100␮l2MH2SO4. Opticaldensitywasmeasuredat450nm.SerafromISAV-infected fishwereusedasthepositivecontrol.Allseraweretestedindupli- catesandantibodytiterwasdeterminedasthehighestdilution whoseOD₄₅₀wasabovethemeanOD₄₅₀plus2standarddevia- tionsofthesameserumdilutionfromfishinjectedwithcontrol plasmidsorfromfishinjectedwithHEK293cellsupernatantfor oil-formulatedvaccineexperiment.Theantibodytiterofeachfish wasplottedasacolumnusingscattergraph(GraphPadPrismvision 6.01forWindows,SanDiego).Ifthetiterwaslowerthanitsstarting dilutionof1:50,thetiterwasascribedavalueof25whencalcu- latingthemeantiter.Themeanofeachgroupwasprintedabove itscolumn.Significantdifferencesofthemeanranksbetweendif- ferentcolumnswereanalysisbyKruskal–Wallistestandposthoc Dunn’smultiplecomparisonstestmethod(GraphPadPrismvision 6.01forWindows,SanDiego).

2.7. Virusneutralizingactivity

ISAVneutralizing activityin serawastested asdescribedby Lauscher[16].

2.8. Vaccinationofﬁshwithanoil-formulatedvaccinecontaining inactivatedISAVwithorwithoutrecombinantIFNc

RecombinantIFNc(rIFNc)waspreparedinHEK293asdescribed [9]. ISAVwasgrown in ASKcells and after freezingand thaw- ing,virusinthecelllysateswereinactivatedwith0.1%formalin atroomtemperaturefor3days.Afterinactivation,formalinwas removed by dialysis against PBS. The antigenic content of the inactivatedviruswasmeasuredbyhemagglutinationassay[18].

Eachvaccine doseconsistedof0.1mlcontaining 8hemaggluti- nationunitsofinactivatedISAV(WVV)aloneorcombinedwith 0.1ml HEK293medium inoiladjuvant orWVV combinedwith 3.6×10⁴unit rIFNc in oil adjuvant. Control vaccines contained HEK293mediumorrIFNcinoiladjuvant.Thevaccineswereformu- latedasawater-in-oilemulsionusingMontanide^TMISA763AVG (Seppic)withanadjuvant/antigenratioof70/30(weight/weight).

Groups of Atlantic salmon presmolts (65 ﬁsh per group,

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Fig.1. SurvivalplotsandvirusquantitationforsalmonvaccinatedbyintramuscularinjectionofISAVhemagglutininesteraseplasmid(pHE)incombinationwithIFNplasmids afterchallengewithISAV.(A)Fishchallengedbyi.p.injectionofISAVat7weeksaftervaccinationwithpHEincombinationwithpIFNa1,pIFNcorpcDNA3.3orwithpEGFP incombinationwithIFNplasmidsorpcDNA3.3(n=50).(B)Fishchallengedbyi.p.injectionofISAVat7weeksaftervaccinationwithpHEincombinationwithpIFNbor pcDNA3.3orwithpEGFPincombinationwithpIFNborpcDNA3.3(n=50).(C)FishchallengedbyadditionofISAVinfectedcohabitantfish8weeksaftervaccinationwith pHEincombinationwithpIFNa1,pIFNcorpcDNA3.3plasmidsorwithpEGFPincombinationwithIFNplasmidsorpcDNA3.3(n=50).pcDNA3.3istheplasmidcontrolfor IFNplasmidswhilepEGFPistheplasmidcontrolfortheHEplasmid.(D)QuantitationofISAVinheadkidneyofsurvivingfishfromthecohabitationchallengein(C)Atday62 afteradditionofinfectedcohabitantsinthevaccineexperimentdescribedin(C)headkidneysweresampledfrom8survivalfishineachofthevaccinegroups.ISAVlevelsin theorganswerecomparedbetweenvaccinegroupsbyRT-qPCRofISAVsegment8,whichwasconvertedtovirusparticlesasdescribedinMaterialsandmethods.Statistical significantdifferencesbetweenpIFNs+HEandpcDNA3.3+HEgroupsareindicatedwithasterisks,*p<0.05,**p<0.01,***p<0.001or****p<0.0001.

averageweight30g)wereanesthetizedwith0.005%benzocaine and taggedbyalcian bluetattooing. Each ﬁshwasinjected i.p.

withavaccinedoseof0.1ml.Allgroupswerekeptinone900l tankin fresh water of 10^◦C. Group 1 fish were injected with WVVonly,Group2 fishwereinjectedwithHEK293cellsuper- natantalone,Group3fishwereinjectedwithWVVtogetherwith HEK293supernatant,Group4fishwereinjectedwithrIFNcalone andGroup5fishwereinjectedwithWVVtogetherwithrIFNc.Eight weekspostvaccination,seraweresampledfrom15fishbefore cohabitantinfectionbyadditionof20%shedderfish,whichwere injectedi.p.with0.1mlISAV(10⁵TCID₅₀)andplacedintothesame tank.Mortalitywasrecordeddaily.Theexperimentwasendedat day62p.i.whenthemortalityofthecontrolgrouphadreached approximately70%.

2.9. ReversetranscriptionquantitativePCR(RT-qPCR)

Musclesamplesfromplasmidinjectionsiteswerecollectedin RNAlater (Ambion)and RNA isolation, cDNAsynthesis and real timePCRwereperformedasdescribedpreviously[13].Therela- tiveexpressionofeachgenewascalculatedbytheCtmethod [19]usingEF1␣Basa referencegene[16].Datawerecalculated fromtriplicatesofﬁvesamplesineachgroup,andexpressedas mean±standard errors.Theprimersusedin RT-qPCRarelisted in Table1. Gene transcriptswere compared using anunpaired Student’s t-test and considered as statistically signiﬁcant at p≤0.05.

3. Results

3.1. IFNsincreasetheprotectiveeffectofHEagainstchallenge withISAV

TotestiftheIFNa1,IFNborIFNcmightincreasetheprotective effectofHE againstISAVinfectionofsalmon,fishwereinjected withvariouscombinationsof IFNplasmids and HEplasmids or therespectivecontrolplasmids,keptfor7or8weeksandthen challengedbyISAV(Fig.1).Inthe7weekvaccinationexperiment, theeffectsofpIFNa1andpIFNcwerestudiedinonetank(Fig.1A) andtheeffectofthepIFNbinanothertank(Fig.1B).32daysafter injectionofISAV,fishinjectedwithcontrolplasmidsshowedhigh mortalityinbothtanks(<5%survival).Intank1,fishinjectedwith pHEandpIFNa1showedsignificantlyhigherprotection(64%survival)thanfishinjectedwithpHEandpcDNA3.3(28%survival)or pIFNa1andpEGFP(10%survival).Asexpected[13],pIFNcalsogave significantprotection(55%survival),butfishinjected withpHE togetherwithpIFNcobtainedevenhigherprotection(74%survival).

Intank2,survivalofﬁshinjectedwithpHEtogetherwithpIFNbwas muchhigher(76%)thanofﬁshinjectedwithpIFNbandpEGFP(20%) orwithpHEandpcDNA3.3(14%).

Inthe8weekvaccinationexperiment,theadjuvanteffectsof pIFNa1andpIFNcwerecomparedbycohabitationchallengewith ISAVinjectedﬁsh,whichissimilartonaturalinfection(Fig.1C).The mortalityofﬁshinjectedwithpIFNa1orcontrolplasmidsagain developedsimilarly(36and30%survival)whilepHEgave some

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Table1

PrimersusedforqPCRofimmunegenes.

Genename Accessionno. Forwardprimer(5–3) Reverseprimer(5–3)

IgM Y12457.1 TGAGGAGAACTGTGGGCTACACT TGTTAATGACCACTGAATGTGCAT

IgT ACX50290 CAACACTGACTGGAACAACAAGGT CGTCAGCGGTTCTGTTTTGGA

CD3epsilon NM001123622 CTCAGGGCTCGGAAGAAGTCT GGCCACGGCCTGCTGA

CD4 EU409794.1 GAGTACACCTGCGCTGTGGA GGTTGACCTCCTGACCTACAA

CD8a NM001123583.1 CGTCTACAGCTGTGCATCAATCAA GGCTGTGGTCATTGGTGTAGTC

CD83 DQ339141.1 GCACCTGTAGGAGAGCAGAACC TCCCTTTCTTCTGATTGGTCTGT

CD86 DW580717.1 TAGACCACACACAGGGAACAATG ATTGAGATGTATGTTCTTGTCGTCG

MHCII ABX44766.1 ATGGTGGAGCACATCAGCC CTCAGCCTCAGGCAGGGAC

MHCI JN561338.1 GAAGAGCACTCTGATGAGGACAG CACCATGACTCCACTGGGGTAG

Perforin1-1 ACI33854.1 CGTTGTCACCATGGAACGTAA GCCTCTGAGCCTGTCTATCCA

Perforin1-2 ACI33201.1 CTCCATCGCTCCAGTGAGTCT TGGTTCCAGCGAGCATAAACT

GranzymeA NM001141037.1 GCTAAGGGGAGTCGTGTCCTT CAATGACTTCCGCATGGTTTT

IFNgamma AY795563.1 AAGGGCTGTGATGTGTTTCTG TGTACTGAGCGGCATTACTCC

EF1a BG933853 TGCCCCTCCAGGATGTCTAC CACGGCCCACAGGTACTG

protection(60%survival).Incontrast,pIFNa1pluspHE,pIFNcplus HEorIFNcalonegavemuchhigherprotection(94%,90%and82%

survival),whichweresigniﬁcantlyhigherthaninthepHEgroup.

Viruscopynumbersinheadkidneyofsurvivalfishwasestimated byRT-qPCR(Fig.1D).FishinjectedwithcontrolplasmidsorpIFNa alonecontainedthehighestvirusnumbers,whichwasnotsignifi- cantlydifferentfromfishinjectedwithpHEalone.Asexpected,fish injectedpIFNa+pHEorpIFNc+pHEcontainedsignificantlylower virusnumbers.Takentogether,theseresultsdemonstratestrong adjuvanteffects ofIFNaandIFNbplasmidswhen administrated togetherwiththeHE plasmid.TheadjuvanteffectofpIFNcwas lessclearfromthesechallengeexperimentsbecausepIFNcalone providessuchahighlevelofprotectionagainstISAVinfection[13].

3.2. TypeIIFNsenhancetheantibodyresponseagainstISAV

TotestwhetherIFNsstimulatetheantibodyresponseofAtlantic salmonagainstISAVHEantigen,theﬁshwereinjectedi.m.with pHEtogetherwitheitherpIFNa,pIFNb,orpIFNcorwithpcDNA3.3 withoutinsert.ControlswereIFNplasmidsinjectedtogetherwith pEGFP. Tenweeks later,serawereharvested and IgMantibody responses were measuredby ELISA using whole ISAV as coat- ing antigen(Fig. 2).The resultsshowed that antibodytiters of serafromﬁsh injected withpEGFPtogetherwithIFN-plasmids

Fig.2. Anti-ISAVantibodytitersinserumoffish10weeksafteri.m.injectionof differentcombinationsofHEandIFNplasmids.Fishimmunizedbyinjectionwith pcDNA3.3,pIFNa1,pIFNborpIFNctogetherwithpHEorbyinjectionwithpIFNa1, pIFNborpIFNctogetherwithpEGFP(controlforHE)orbyinjectionwithpcDNA3.3 andpEGFP.IgMantibodyresponsesweremeasuredbyELISAusingpurifiedISAV ascoatingantigen.Antibodytiterwasdeterminedasthehighestdilutionwhose OD450wasabovethemeanOD450plus2standarddeviationsofthesameserumdilu- tionfromfishinjectedwithpcDNA3.3andpEGFP(n=8).Numbersabovecolumns showmeanantibodytiters.StatisticalsignificantdifferencebetweenpIFNs+HEand pcDNA3.3+HE(p<0.05)isindicatedwithanasterisk.

orthepcDNA3.3controlplasmidweresimilarand rangedfrom 72 to 462.The mean antibody titer of sera from fish injected withthepHEtogetherwithpcDNA3.3wasalsoonly412.Incon- trast,themeanantibodytitersoffishinjectedwithpIFNa+pHE, pIFNb+pHEorpIFNc+pHEplasmidswere9603,8803and9600, respectively,whichweresignificantlyhigherthanantibodytiters from fishinjected withpHE togetherwithpcDNA3.3 (p<0.05).

Thisshowedthat allthree IFNsenhanced theadaptive humoral immuneresponseagainstISAVHEtoasimilarextentandconﬁrmed the adjuvant activity of type I IFN in salmon. ISAV neutraliz- ingactivitiesintheseraweremeasured, butshowedlowtiters.

Therewerenosignificantdifferencesbetweenfishvaccinatedwith pcDNA3.3+pEGFPandfishvaccinatedwithpHEwithorwithoutan IFNplasmid(SupplementalTable1).

3.3. AdjuvantactivityofrecombinantIFNwithinactivatedwhole virusasvaccine

WealsotestedifrecombinantIFNc(rIFNc),mightincreasethe protectiveeffectofinactivatedISAVasvaccine(WVV)orincrease the humoral immune response against WVV. IFNc waschosen becauseitseemedtobemorestablethanIFNainvivo[13]andwas producedinHEK293cells.Groupsofsalmonpresmoltswerevacci- natedbyi.p.injectionofWVValone,HEK293mediumalone,WVV andHEK293medium,rIFNcaloneorWVVandrIFNc,allmixedin awaterinoilemulsion.Eightweeksaftervaccination,serawere harvestedandthefishwerechallengedwithISAVinacohabita- tioninfectionmodel.Thechallengeexperiment(Fig.3A)showed thatthesurvivaloffishvaccinated withWVVplusrecombinant IFNc(76%)washigherthanfishvaccinatedwithWVValone(66%) orWVVplusHEK293medium(64%).FishinjectedwithHEK293 mediumalone showed30%survival. Antibodytiters weremea- suredbyELISAusingISAVascoatingantigen(Fig.3B).Serafrom fishvaccinated withWVV aloneor WVVplus HEK293medium showeda significantincrease inantibodytiterwhen compared tofishinjectedwithHEK293mediumonly(p<0.05).Serafrom fishvaccinatedwithWVVplusrIFNcshowed3-foldhighertiters (p<0.01) than thegroups vaccinated with WVV withoutrIFNc.

Takentogether,theseresultsfurtherconﬁrmedtheadjuvantactiv- ityoftypeIIFNinsalmon.

3.4. InjectionofIFNplasmidsincreaseinﬂuxofBcellsandCD8 positivecells

TheoutcomeofDNAvaccinationislikelytodependonthetype ofimmunecellsthatareattractedtothemuscleinjectionsite.We thusmeasuredthechangeingenetranscriptsofvariousmarker genesforB-cells(IgM,IgT),T-cells(CD3,CD4,CD8)anddendritic cells (CD83,CD86,MHCII)inthemuscleattheinjectionsiteof

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Fig.3. SurvivalplotsafterchallengewithISAV(A)andanti-ISAVantibodyresponses (B)offish8weeksafteri.p.injectionwithvaccinescontaininginactivatedISAV (WVV)alone,WVVandmediumfromHEK293cells(HEK),rIFNcaloneorWVVand recombinantIFNcprotein(rIFNc).A.FishchallengedbyadditionofISAVinfected cohabitantfish8weeksafter vaccination(n=50).Statisticalsignificantdiffer- enceswereobservedbetweenthesurvivaloftheHEKgroupandtheWVV+rIFNc group(p<0.0001****),betweentheHEKgroupandWVVgroup(p<0.001***)and betweentheHEKgroupandtheWVV+HEKgroup(p<0.01**),butnotbetweenthe WVV+rIFNcgroupandtheWVVorWVV+HEKgroups.B.IgMantibodyresponses weretestedbyELISAusingpurifiedISAVascoatingantigen(n=15).Antibodytiter wasdeterminedasthehighestdilutionwhoseOD450wasabovethemeanOD450plus 2standarddeviationsofthesameserumdilutionfromfishinjectedwithHEK293 cellsupernatant.Thefigureshowsantibodytitersofindividuals.Numbersabove columnsshowmeanantibodytiters.Statisticalsignificantdifferencewasobserved betweentheWVV+rIFNcgroupandtheWVVorWVV+HEKgroups(p<0.01**).

IFNa,IFNbandIFNcplasmidscomparedtocontrolplasmidandPBS (Fig.4).ThedatashowedsigniﬁcantincreasesinIgM,IgTandCD8a forallthreeIFNplasmidscomparedtocontrolplasmid(p<0.05), whichsuggeststhatIFNexpressioncausedanincreasedinﬂuxofB- cellsandcytotoxicT-cells.Increasedtranscriptswereobservedalso forthepanT-cellantigenCD3forallthreepIFNs,whileCD4showed aminorincreaseforIFNbandIFNc,butnotforpIFNa.Increased expressionofperforin1–2andGranzymeAbytheallthreeIFN plasmidssupportthattheCD8positivecellsarecytotoxicT-cells.

IFNgtranscriptswereincreasedonlyinmuscleinjectedwithpIFNa, however.TheIFNplasmidsdidnotseemtoinﬂuencetheinﬂuxof dendriticcellssincetherewerenoorminorincreasesintranscripts ofCD83,CD86andMHCII.MHCIwasincreasedforallthreeIFN plasmids.Inaseparateexperimentweobservedthatinjectionof HEplasmiddidnotcauseincreaseintranscriptionofanyofthe genesIgM,CD8a,CD3,CD4,CD83orCD86,MHCIorMHCIIcom- paredtoinjectionofthecontrolplasmidEGFPN1(Supplemental Fig.1).

4. Discussion

We hereprovideevidencethat ﬁshIFN-Ihave animportant roleinkick-startingtheadaptiveimmuneresponsesagainstvirus.

Strong adjuvant activity of IFNa, IFNb and IFNc plasmids was observedwheninjectedi.m.togetherwithaplasmidexpressing ISAVHEasantigen,bothwithrespecttoprotectionagainstISAV infectionandIgMantibodyresponseagainstISAV.I.m.injectionof HEplasmidalonegavesomeincreaseinprotectionasdescribed inapreviouswork[12],butfarlessthanincombinationwithIFN plasmids.AdjuvanteffectwasalsoobservedforrecombinantIFNc, which increasedtheIgM antibodyresponsetoinactivatedISAV uponi.p.delivery.WhiletheadjuvantactivityofIFN-Iinmammals hasbeenknownpreviously,thepresentworkshowsthatthelink betweentypeIIFNsandtheadaptiveimmunesystemwasestab- lishedinﬁshseveralhundredmillionyearsago.Thisworksupports thatIFN-ImayplayaroleintheprotectiveeffectofDNAvaccines basedonVHSVandIHNVGproteinssincethelattervaccinesare knowntoinduceIFN-IandIFN-inducedgenesinthemuscleofthe ﬁshandtheVHSVG-proteinhasbeenshowntoinduceIFN-Iincell culture[20,21].

TheprotectionobtainedwithIFNa+HEplasmidsandIFNb+HE plasmidsmustbeduetoadaptiveimmune responsessincenei- therIFNaalone norIFNbplasmidalone provideany signiﬁcant protectionagainstISAVmediatedmortality[13].Ontheotherhand, theprotectionobtainedwithIFNc+HEplasmidsismostprobably duebothtoadaptiveandinnateimmuneresponsesbecauseinjec- tionofIFNcplasmidaloneprovidesahighlevelofprotectionagainst ISAVinfectionduetosystemicinductionofantiviralproteins[13].

TheadjuvanteffectofIFNcwas,however,conﬁrmedbyitsabilityto enhancetheantibodyresponseagainstISAV,bothwhendelivered asgeneandasrecombinantprotein.

ThefactthatIFNa,IFNbandIFNcallshowedsimilaradjuvant effectswassurprisingsince theyhavequitedifferentproperties inAtlanticsalmon.ThethreeIFNsareinducedthroughdifferent signalingpathwaysandshowdifferentexpressionincellsandtis- sues[9].RecentworkhasshownthatsalmonIFNa,IFNbandIFNc utilizedifferentreceptors[22].Theimmunecellsthatcontribute totheadjuvanteffectofIFN-IinAtlanticsalmonmustthushave receptorsforallthreeIFNsubtypessincetheyallshowsimilaradju- vantproperties.Interestingly,theIFNa1plasmidhasbeenshownto inducesantiviralgenesonlyatthemuscleinjectionsite,whilethe IFNbandIFNcplasmidsinduceantiviralgenessystemicallyinthe ﬁsh[13].ThismeansthattheadjuvanteffectoftheIFNaplasmidis causedbystimulationofimmunecellsatthemuscleinjectionsite sinceIFNadoesnotinduceantiviralgenessystemicallyinsalmon.

Accordingly,theadjuvantactivityofIFNbandIFNcisalsolikelyto occuratthemuscleinjectionsite.Mammalianstudiessuggestthat adjuvantactivityofIFN-Iispleiotropicandduetodirectstimula- toryeffectsonTcells,Bcellsaswellasdendriticcells,whichare themainantigenpresentingcells[7,23–25].IFN-Ihavebeenshown tobeimportantforclonalexpansionofCD4andCD8T-cellsand forinitiationofcross-primingofCD8Tcells[26–28].Inaddition, IFN-IhavebeenshowntopromotesurvivalofB-cellsbyinhibi- tionofapoptosis[29].TheeffectofIFN-Ionfishimmunecellsis unknown,butthepresentRT-qPCRstudiesshowedthatinjectionof allthreeIFNplasmidscausedanincreaseintranscriptsforIgMand IgT,whichsuggestsanincreasedinfluxofB-cells;andanincrease ofCD8,perforinandgranzymeAtranscripts,which suggestsan increasedinflux ofcytotoxicT-cells.Thismightbeexplainedby thefactthattheIFNsinduceseveralchemokinegenesinthemuscle tissue(unpublisheddata).IncreasedexpressionofMHCIIwasnot observedforthethreeIFNplasmids,whichsuggestnoincreased attractionofprofessionalantigenpresentingcellsbyIFNsatthe muscleinjectionsite.Antigenpresentingcellsmaystillbeinvolved intheadjuvantactivityoftheIFNssincethesecellsmaybeattracted

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Fig.4.Expressionofimmunecellmarkergenesinsalmonpresmoltsafteri.m.injectionofIFNexpressionplasmids.GroupsofpresmoltswereinjectedwithPBS,control plasmid(Cp)orplasmidsexpressingIFNa1,IFNborIFNc,respectively(n=5).GenetranscriptionwasmeasuredbyRT-qPCR7daysafterinjection,andpresentedasfold increase(mean±SD)comparedtoPBSinjectedﬁsh.Statisticalsigniﬁcantdifferences(p<0.05)betweenIFNplasmidgroupsandcontrolplasmidgroupareindicatedwithan asterisk.

bythewounding caused byinjection. The questionof whether muscle cells or professional antigen presenting cells are most importantinDNAvaccinationmechanism,isstilldebated[30,31].

ThemechanismofprotectionobtainedbyinjectionofIFNplas- midtogetherwiththeHEplasmidisuncertainsincetheantiserum fromthevaccinatedﬁshshowedlowvirusneutralizing activity.

PoorISAV-neutralizing activitywasalsoobservedby antiserum fromAtlanticsalmonimmunizedwithahighdoseofinactivated ISAVeveniftheﬁshwashighlyprotectedagainstISAVinfection [16].Non-neutralizingantibodiesmaycontributetoprotectionby increasedphagocytosisanddestructionofvirus,butthishasyet tobeshownforﬁshantibodies.TheroleofcytotoxicT-cellsinthe protectiveimmuneresponsehastobeexaminedinfuturestudies.

ThepresentdemonstrationofadjuvanteffectsofIFNexpression plasmidsprovidesanovelmethodforimprovingDNAvaccination offish.ThisisimportantsinceonlyDNAvaccinesagainstfishrhab- dovirusbasedontheG-proteinhaveuntilnowshownsatisfactory protectionagainstvirusinfection.AbenefitofDNAvaccinesisthat theyinduceboth humoral and cellmediated adaptive immune responsesbecausetheproteinantigensareproducedwithinthe hostcells[3].Moreover,theyareeasytoaccommodatetovarious virulentvirusstrainsandcanbepreparedevenagainstvirusesthat cannotbegrowninculture.DNAvaccinesarealsosafetouseand showlesssideeffectsthantraditionalfishvaccines,whichhaveto bedeliveredinoiladjuvantstogiveaprotectiveeffect.

Conﬂictofintereststatement Therearenoconﬂictsofinterest.

Acknowledgements

WethankProfessorEspenRimstad,NorwegianSchoolofVet- erinary Science,Oslo, for providing theplasmid encoding ISAV hemagglutinin-esterasefusedtoEGFP.

ThisworkwasinpartfundedbytheRegionalResearchFundfor NorthernNorway(grant212426).

AppendixA. Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/

j.vaccine.2015.03.093.

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