The immune response of ruminant livestock to bluetongue virus:From type I interferon to antibody

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The immune response of ruminant livestock to bluetongue virus:From type I interferon to antibody

N. James Maclachlana, Christine Henderson, Isabelle Schwartz-Cornil, Stephan Zientara

To cite this version:

N. James Maclachlana, Christine Henderson, Isabelle Schwartz-Cornil, Stephan Zientara. The immune

response of ruminant livestock to bluetongue virus:From type I interferon to antibody. Virus Research,

Elsevier, 2014, 182, pp.71-77. �10.1016/j.virusres.2013.09.040�. �hal-01190011�

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ContentslistsavailableatScienceDirect

Virus Research

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / v i r u s r e s

The immune response of ruminant livestock to bluetongue virus:

From type I interferon to antibody

N. James Maclachlan

^a,∗

, Christine Henderson

^b

, Isabelle Schwartz-Cornil

^c

, Stephan Zientara

^c

aDepartmentofPathologyMicrobiologyandImmunology,SchoolofVeterinaryMedicine,UniversityofCalifornia,Davis,CA95616,USA

bMerialInc.,Athens,GA30601,USA

cANSESAlfort,UMR1161ANSES/INRA/ENVA-UPEC,23AvduGénéraldeGaulle,BP63,94703Maisons-AlfortCédex,France

a r t i c l e i n f o

Articlehistory:

Availableonline10October2013

Keywords:

Bluetonguevirus Immuneresponse Innate

Adaptive

a b s t r a c t

Infectionofruminantswithmost(butnotall)serotypesofbluetonguevirus(BTV)leadstoahighlyblood cell-associatedviremiathatmaybeprolongedbutnotpersistent.Furthermore,recoveredanimalsare resistanttoreinfectionwiththehomologousvirusserotype,whichisthebasisforvaccinationstrategies topreventBTVinfectionandtheclinicaldisease(bluetongue)thatitcausesindomesticlivestock.BTV infectionisinitiatedatthesiteofvirusinoculationandtheassociateddraininglymphnode,fromwhere thevirusisthenspreadinlymphcellstothesystemiccirculationandsecondarysitesofreplication.

ReplicationofBTVintargetcells,notablymononuclearphagocyticcells(dendriticcellsandmacrophages) andendothelium,leadstothegenerationoftheinnateandadaptiveimmuneresponsesthatmediateboth initialvirusclearanceandsubsequentresistancetoinfectionwiththehomologousvirusserotype.The goalofthisreviewistosummarizecurrentunderstandingoftheseinnateandadaptiveimmuneresponses ofanimalstoBTVinfection.

1. Introduction

Bluetongue(BT)isanarboviraldiseaseofdomesticand wild ruminantscharacterizedbyvascularinjurythatresultsintissue necrosis,hemorrhage, andedema (Hutcheon,1902; Maclachlan et al., 2009; Spreull, 1905; Verwoerd and Erasmus, 2004).The causativeagentofBTisbluetonguevirus(BTV),theprototypemem- berofthegenusOrbivirus,familyReoviridae(Bordenetal.,1971;

MatsuoandRoy,2013;Murphyetal.,1971;Schwartz-Corniletal., 2008).TheBTVvirionconsistsofadiffuseouterproteincoatand aninnericosahedralcorethatenclosesthetranscriptasecomplex (Stewartetal.,2012;Verwoerdetal.,1972).Thegenomeincludes 10 distinctgenome segmentsofdouble-stranded RNA(dsRNA).

Eachgenesegmentencodesatleast1protein,speciﬁcally7struc- tural(VP1-7)and5nonstructural(NS1-3/3A,4)proteins(Grubman etal.,1983;Mertensetal.,1984;Ratinieretal.,2011).Some26BTV serotypesarenowrecognizedglobally(Hofmannetal.,2008;Maan etal.,2012).

BTVinfectionoccursthroughouttemperateandtropicalregions oftheworldcoincidentwiththedistributionofspeciﬁcspecies ofvectorCulicoidesbitingmidges,howevertheglobaldistribution

∗Correspondingauthor.Tel.:+15307548125.

E-mailaddress:njmaclachlan@ucdavis.edu(N.J.Maclachlan).

ofBTVhasrecentlyalteredmarkedly, particularlyin thenorth- ern hemisphere (Gibbs and Greiner, 1994; Maclachlan, 2011;

MaclachlanandMayo,2013;Saegermanetal.,2008;Tabachnick, 2010).Thus,BT isviewedcurrentlyas aglobally emerging dis- ease(Gibbsetal.,2008;Guisetal.,2012;Jiminez-Clavero,2012;

Maclachlan and Guthrie,2010; Purse et al., 2008; Weaver and Reisen, 2010).OutbreaksofBTcanbeeconomicallydevastating tolivestockproductionandthepresenceofBTVinacountrycan adverselyimpactthetradeandmovementoflivestock(Maclachlan andOsburn,2006;Velthuisetal.,2010).ControlofBTVinfection andBT diseasecanbedifﬁcult,andpreventionofthediseaseis typicallyreliantonvaccinationofsusceptiblelivestock(reviewed:

MaclachlanandMayo,2013).Soundvaccinationstrategiesshould logicallybebasedonathoroughunderstandingoftheruminant immuneresponsetoBTV,whichisthefocusofthisreview.

2. Innateimmunity

Theinnateimmuneresponseconstitutestheﬁrstlineofdefense ofanimalsagainstviralinfections.Innateimmunemechanismsare alsocriticaltotransitiontoaneffectiveadaptiveimmuneresponse (humoralandcellular).NaturalBTVinfectionofruminantstypi- callyoccursfollowingthebiteofanyoneofthemultiplespecies ofhematophagousCulicoidesmidgesthatserveasbiologicalvec- torsofthevirus(Barratt-BoyesandMaclachlan,1995;Gibbsand 0168-1702/$–seefrontmatter©2013ElsevierB.V.Allrightsreserved.

http://dx.doi.org/10.1016/j.virusres.2013.09.040

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72 N.J.Maclachlanetal./VirusResearch182(2014)71–77

Greiner,1994; Melloretal., 2000;Tabachnick,2010;Verwoerd andErasmus,2004).AftercutaneousinstillationofBTVfollowing thebiteofaninfectedmidge,thevirusisquicklytranslocatedin dendriticcells totheregionallymphnodewhere initialreplica- tionoccurs(Barratt-BoyesandMaclachlan,1994;Ellisetal.,1991;

Hematietal.,2009;Pini,1976).Conventionaldendriticcellsare recruitedinlargenumberstotheskinfollowingvirusinfection, facilitatingfurtherreplicationofthevirusatthesiteofinitialinfec- tionintheskin(Hematiet al.,2009).Interruptionoflymphatic ﬂowtotheregionallymphnodeadjacenttothesiteofvirusinoc- ulationmarkedlydelaystheonsetofviremia(Barratt-Boyesand Maclachlan,1994).

InitialreplicationofBTVintheregionallymphnodelikelyoccurs indendriticcells,macrophages,endotheliumandpossiblylympho- cytes,asthesearemajorcellulartargetsofBTV(Maclachlanetal., 2009;Darpeletal.,2012).InvitroBTVinfectionofovineconven- tionaldendriticcellsfromskinlymphleadstotheproductionof proinﬂammatoryandimmunecytokines(interleukins[IL-1␤,IL-6, IL-12])aswellastoexpressionofco-stimulatorymoleculesthat promotetheproliferationofBTV-speciﬁcCD4+andCD8+Tcells (Hemati etal.,2009).Similarly,invitro BTVinfectionofbovine monocyte- derivedmacrophages leadstotheiractivationwith productionoftissuenecrosisfactor(TNF-␣),interleukins(e.g.IL- 1␤,IL-8)andinduciblenitricoxidesynthetase(Drewetal.,2010a).

ReplicationofBTVintheregionallymphnodestimulatesamarked cellularresponsecharacterizedbyincreasednumbersof Bcells withinthenodeitselfandtheappearanceofvirus-speciﬁcanti- bodiesandactivatedCD8+Tcellsinthedrainingefferentlymph (Barratt-Boyesetal.,1995).

FollowinginitialreplicationofBTVatthesiteofinoculationand withintheadjacentdraininglymphnode,thevirusisthendissem- inatedtoavarietyoftissuesthroughoutthebodywheresecondary replicationoccursinmononuclearphagocyticcells(macrophages anddendriticcells),endothelialcells,lymphocytesincluding␥␦

Tcells,andperhapsothercelltypes(Barratt-Boyes etal.,1992;

Barratt-Boyes and Maclachlan, 1994,1995; Darpel et al., 2012;

DeMaulaetal.,2001,2002;Ellisetal.,1991;Hematietal.,2009;Lee etal.,2011;Maclachlanetal.,1990,2009;MahrtandOsburn,1986;

Umeshappaetal.,2012;Whetteretal.,1989).ViralRNA(especially dsRNA)isdetectedbypatternrecognitionreceptors(PRR)inthe cytoplasmicandendosomalcompartmentsofhostcells,notably toll-likereceptors(TLRs)ormembersoftheretinoicacidinducible gene(RIG-1)-likefamilyofcytoplasmicreceptors(Levyetal.,2011).

DifferentPRRutilizedistinctsignalingpathwaystotriggerthepro- ductionof TypeIand TypeIIIIFNs and otherproinﬂammatory cytokines.

IthaslongbeenrecognizedthatBTVinfectionisastronginducer ofTypeIIFN,bothinvivoandinvitro(Fosteretal.,1991;Fulton and Pearson, 1982; Huismans, 1969; MacLachlan et al., 1984;

MaclachlanandThompson,1985;Russelletal.,1996).Whereas adultmiceareresistanttoproductiveBTVinfection,TypeI(␣/␤) IFNreceptorknockoutmice(IFNAR[−/−])arehighlysusceptible tolethalinfectionwiththevirus(Calvo-Pinillaetal.,2009a,2010;

Franceschietal.,2011).ItrecentlyhasbeenshownthatBTVinduces TypeIIFNproductioninplasmacytoiddendriticcellsviaaMYD88- dependentTLR7/8-independentsensing and signalingpathway, whereascytoplasmichelicases(RIG-1,MDA5)mediatesensingand signalinginBTV-infected epithelialcells(Chauveauetal.,2012;

Ruscanuetal.,2012).TheBTVNS3proteinspeciﬁcallyinterferes withIFNproductioninepithelialcellsbyinhibitingtheRIG1-like receptormediatedsignalingpathway(Chauveauetal.,2013).Plas- matocytoiddendriticcellsandType1IFNmayexertacentralrole in triggeringBcellresponses and promotingBTV-speciﬁc anti- bodysynthesis(humoralimmunity),ashasrecentlybeenshown forrotavirus,whichisanotherdsRNAvirusofthefamilyReoviridae (Dealetal.,2013).

Infectionofdendriticcells,macrophages,endothelialcellsand othersduringBTVinfectionofruminantsleadstotheproduction ofavarietyofproinﬂammatoryandvasoactivemediatorsinclud- ingTypeIIFNs,TNF-␣,andvariousinterleukinsandprostanoids, buttheresponse canvarydependingonthecelltypesthatare infected aswellastheirtissue location(Channappanavaretal., 2012;DeMaulaetal.,2001,2002;Drewetal.,2010a;Hematietal., 2009;Ruscanuetal.,2012,2013;Umeshappaetal.,2012).Fur- thermore,althoughthesevirus-inducedcytokineandchemokine mediatorsmayservetolimit/controltheinfectionduringitsini- tialstagesandtopromotethedevelopmentofastrongacquired immuneresponse,theymayalsocontributetothepathogenesis ofthecapillaryleakagesyndromeandcoagulopathythatcharac- terizesclinicalBTinruminants-theso-called“cytokinestorm”of hemorrhagicfevers(Fig.1)(Drewetal.,2010b;Maclachlanetal., 2008,2009;Umeshappaetal.,2012).Thus,thevirulencetosheep ofdifferentstrains/serotypesofBTViscorrelatedwiththesever- ityofthevascularlesionstheyinduceandtoserumconcentrations ofacutephase proteins,andnot toviralloads(Sanchez-Cordon etal.,2013).Similarly,DeMaulaetal.(2002)showedthatvascu- larinjuryand enhancedcoagulation,asassessedbytheratioof plasmathromboxanetoprostacyclin,weresigniﬁcantlygreaterin sheepthancattleinoculatedwiththesamestrainofBTV,consis- tentwiththesensitivityofsheepandrelativeresistanceofcattle toexpressionofclinicalBT.

3. Cellularimmunity

Cell-mediatedimmunity(CMI)typicallylimitsviralspreaddur- ingtheinitialstagesofacuteviralinfectionsbythedestruction ofvirus-infectedcells(virusfactories).AlthoughBTVinfectionof ruminantsclearlyresultsinchanges inlymphocytepopulations anddynamicsbothlocally(adjacenttothesiteofinfection)andsys- temically(Barratt-Boyesetal.,1995;Ellisetal.,1990,1991;Hemati etal.,2009;Umeshappaetal.,2012),theCMIresponseofruminants toBTVinfectionremainspoorlycharacterized.CD8+cytototoxicT lymphocytes(CTLs)havebeenidentiﬁedfollowingBTVinfectionof bothmiceandsheep,althoughtheiractivityisapparentlytransient andtheirpreciseroleinmediatingvirusclearanceispoorlydeﬁned (Andrewetal.,1995;Calvo-Pinillaetal.,2012;JeggoandWardley, 1982a,b;Jeggoetal.,1984,1986).AdoptivetransferofCTLshar- vestedfromthethoracicductofasheeppreviouslyinfectedwith BTVpartiallyprotectedrecipientsheepagainstchallengeinfection witheitherhomologousorheterologousserotypesofBTV(Jeggo etal.,1984,1986).

The BTV NS1 and VP2 proteins have been proposed to be major targets of the ovineCMI response to BTV,although the viralprotein-specificCTLresponse ofindividualsheepcanvary markedly(Andrewetal.,1995;Janardhanaetal.,1999;Takamatsu andJeggo,1989).Ovine CTLsspecificforthenon-structuralNS1 proteinarecrossreactiveamongstdifferentBTVserotypes.Simi- larly,Calvo-Pinillaetal.(2012)describedgenerationofNS1-specific CD8+TcellsandheterotypicimmunityinIFNARmicethatwere immunizedwithrecombinantBTVproteins,andJonesetal.(1996, 1997)earliershowedthattheBTVnonstructuralproteins,espe- ciallyNS2,caninducecross-reactiveCTLsinmice.Togetherthese findingsoffersomepromisethatapolyvalentvaccinestrategyto preventBTVinfectionispotentiallyfeasible,astheNS1andNS2 proteinsarerelativelyconservedamongfieldstrainsofBTVregard- lessofserotype.However,neitherhomologousnorheterologous protectionwasobtainedwitharecombinantadenovirusexpressing onlytheNS1proteinofBTV-2despitestronginductionofprotein- specificCD8+Tcellsinthevaccinatedsheep(unpublisheddata).

Similarly,althoughBTVcore-likeparticlesthatincludeonlyVP3 andVP7havebeenreportedtoinducepartialprotectionagainst

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Fig.1.PotentiallycriticalinteractionsofBTVwithmononuclearphagocytes(dendriticcells,macrophages)duringinfectionofruminants.Speciﬁcally,thesecellsmay:(1) contributetodisseminationofBTVinafferentlymphandblood,(2)produceantiviralandinﬂammatorycytokines/chemokinesthatinitallycontrolBTVinfectionandpossibly triggerthecytokinestormandassociatedcoagulopathyandhemorrhageand(3)elicitacquiredimmuneresponsesthatleadtoviralclearanceandsubsequentprotection againstre-infectionwiththehomologousBTVserotype.

bothhomologousandheterologouschallengeinsheep,complete protectionrequiresimmunizationwiththeVP2oftherespective BTVserotype(Royetal.,1994;Stewartetal.,2012).Thus,despite thepotential promise of a polyvalent BTVvaccination strategy basedonCMIresponsestodifferentBTVproteins,littleprogress hasbeenmadeinconﬁrmingtheviabilityofsuchanapproachand thepreciserole ofCMIineithermediatingvirusclearancedur- ingprimaryinfectionorconferringresistancetoreinfectionwith homologousorheterologousBTVserotypesawaitsthoroughclari- ﬁcation.

4. Antibody(humoral)immunity

BTVinfectionof ruminantsresultsinthepromptproduction ofantibodiestoboth structuralandnonstructuralviralproteins (HuismansandErasmus,1981;Maclachlanetal.,1987;Richards etal.,1988).ImmunecompetenceoffetalruminantstoBTVinfec- tionarisespriortomid-gestation(Osburnetal.,1982;MacLachlan etal.,1984).AntibodiestocoreproteinVP7arebroadlyserogroup reactiveasthisproteinisconservedamongstmostBTVserotypes andstrains,thusdetectionofantibodiestoVP7isthebasisofthe competitiveELISA(cELISA)assaysthat arenowwidelyusedfor serologicaldiagnosisofBTVinfectionoflivestock(Afshar,1994;

Anderson,1984;MaclachlanandMayo,2013).BTV-speciﬁcneu- tralizingantibodiescanconferlong-lastingresistancetoreinfection withthehomologousBTVserotype,whichisthebasisofvaccina- tionstrategiestopreventBTdisease(Huismansetal.,1987;Noad andRoy,2009;Savinietal.,2008;Spreull,1905;Stewartetal.,2012;

VerwoerdandErasmus,2004).Interestingly,althoughneutraliz- ingantibodiesconferserotype-speciﬁcresistancetoreinfection, viremiacancontinueforextendedintervalsinthecourseofprimary infectiondespitethepresenceofsuchantibodies(Barratt-Boyes andMaclachlan,1994,1995;Bonneauet al.,2002;Fosteretal., 1991;DiGialleonardoetal.,2011;Luedkeetal.,1969;Martinelle et al.,2011; Richards et al.,1988).This abilityof BTV toavoid neutralizationduring primaryinfection isattributed toitsinti- mateassociationwiththecellmembranesofcirculatingbloodcells, erythrocytesinparticular,whichalsoleadstoanextendeddura- tion(severalmonths)ofreverse-transcriptase-polymerasechain reaction(RT-PCR)positivestatusofruminantsfollowingBTVinfec- tion(Barratt-BoyesandMaclachlan,1994;BrewerandMaclachlan,

1992, 1994; Di Gialleonardo et al., 2011; Luedke et al., 1969;

Maclachlanetal.,1990,1994,2009;Martinelleetal.,2011).

TheneutralizingepitopesofBTVarelocalizedtospecificinter- activeregionsoftheVP2outercapsidprotein.Sheepinoculated withVP2thatiseitherisolatedfromviralparticlesorgeneratedby invitroexpressionproducevirusneutralizingantibodiesandare resistanttochallengeinfectionwiththehomologousBTVserotype (HuismansandErasmus,1981;Huismansetal.,1987;Royetal., 1990,1992,1994;Stewartetal.,2012).Atleastsomeneutraliza- tionepitopesarehighlyconformationallydependent,reflectingnot onlyfoldingoftheVP2proteinonitselfbutalsoitsconformational interaction withthe otherouter capsidprotein, VP5 (Appleton andLetchworth,1983;CowleyandGorman,1987;DeMaulaetal., 1993,2000;Gouldetal.,1988;GouldandEaton,1990;Heidner etal.,1990;Mertensetal.,1989).DeMaulaetal.(2000)showed thatwhereasmostvariantsthatwereresistanttoneutralization withindividual neutralizingmonoclonalantibodies (MAbs)had amino acidsubstitutionsinkey regions ofVP2, theVP2 ofone MAb-neutralizationresistantvariantviruswasunchangedbutthis virushad a substitution in VP5, confirmingunambiguously the importanceof theconformationalinteractionbetweenVP2 and VP5 in BTV neutralization. Individual neutralizing epitopes are sometimesconservedbetweenserotypesineitheraneutralizing ornon-neutralizingconformation(Maclachlanetal.,1992;Pierce etal.,1995;Ristowetal.,1988;RossittoandMaclachlan,1992;

WhiteandEaton,1990).Thepresenceofcommonneutralization epitopesbetweenBTVserotypesisfurtherconﬁrmedbyserolog- icalcrossreactivityofcertainserotypesofthevirus,andbythe factthatanimalsinoculatedorimmunizedwithoneormoreBTV serotypescandevelopneutralizingantibodiesagainstserotypesto whichtheywereneverexposed(Dunguetal.,2004;Rossittoand Maclachlan,1992;Thomas,1985).

StudieswithBTVproteinsexpressedfromrecombinant pox, herpesandbaculovirusesconﬁrmthatinductionofneutralizing antibodiesandprotectionofsheepagainstchallengeinfectionwith thehomologousBTVserotypeisbestachievedbyco-expression of both theVP2 and VP5 outercapsidproteins, includingtheir co-expressionasvirus-likeparticlesorincombinationwithBTV core proteins (Lobato et al., 1997; Ma et al., 2012; Roy et al., 1990, 1992, 1994). Thus, we have utilized the canarypox (CP) virusexpressionvectortoevaluateinductionofBTV-neutralizing antibodiesinsheepusingaCPconstructco-expressingbothVP2

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74 N.J.Maclachlanetal./VirusResearch182(2014)71–77

Table1

Effectofimmunizationofsheepwithdifferentdosesofrecombinantcanarypoxvirus co-expressingtheVP2andVP5genesofbluetonguevirus(BTV)priortochallenge infectionwithBTVserotype17.

TREATMENT SN⁺ cELISA⁺⁺ qRTPCR⁺⁺⁺ Temp.

(>103.5^◦F) Pre^* Post Pre Post

Control 0/4 3/3^* 0/4 3/3 4/4 4/4

Vaccine–low 0/4 4/4 0/4 4/4 4/4 1/4

Vaccine–med 3/4 4/4 0/4 1/4 1/4 0/4

Vaccine–high 4/4 4/4 0/4 0/4 0/4 0/4

+SN=serumneutralizationtesttoBTV-17;titer<10considerednegative.

++cELISA=competitiveELISAtoBTVcoreproteinVP7(VMRD).

+++ qRT-PCR=quantitativePCRbasedondetectionofBTVS10geneafterchallenge infection,aspreviouslydescribed(Booneetal.,2007).

* Serologicalstatusimmediatelybeforeand14daysafter(Post)BTVchallenge infection.

** 1sheepdiedinthecourseofthestudy.

andVP5ofastrainofBTV-17(CP-BTV)(Booneetal.,2007).This recombinantCP-BTVconstructconferscompleteresistanceofvac- cinatedsheeptochallengeinfectionwiththehomologousvirus serotype.Inanefforttobetterestablishtheprotectiveimmunity inducedbytherecombinantCP-BTVconstructweundertookadose titration/challengeinfection study using 16 seronegative sheep housedin insect-secure isolation facilities and methods essen- tiallyaspreviouslydescribed(Booneetal.,2007).Thesheepwere assignedindividuallytooneoffourtreatmentgroups:fourcon- trolsheepwereimmunizedwitha CP-vectoredvaccineagainst WestNilevirus(Recombitek^®,Merial,GA);fourhighdosevacci- natedsheepwereinoculatedsubcutaneouslyandintramuscularly (SC/IM)with 2×10⁸ particles of therecombinant CP-BTV construct;fourmediumdosevaccinatedsheepwereinoculatedSC/IM with2×10⁷ particlesof CP-BTV;and fourlow dosevaccinated sheepwereinoculatedSC/IMwith2×10⁶particlesofCP-BTV.All sheepwereimmunizedtwicewiththeirrespectiveinoculaata3 weekintervalandthenchallengedat3weeksafterboosterimmu- nizationbySCinoculationof approximately10^4.9 tissueculture infectiousdosesofafieldstrainofBTV-17,aspreviouslydescribed (Booneetal.,2007).Allsheepwereseronegativebygroup-specific cELISAprior to challenge(Table1), confirmingthat sheep vaccinatedwiththeCP-BTVcanbedistinguishedfromthoseprevi- ouslyinfectedwithBTV(DIVA).Whereasallcontrolandlowdose vaccinates, andone of thefourmedium dosevaccinatedsheep seroconvertedbycELISAat14daysafterchallengeinfection,the three other medium dose and all of the high dose vaccinated sheepremainedseronegativebycELISAfollowingchallenge.Fur- thermore,whereasthefourcontrolandonelowdosevaccinated sheepalldevelopedmarkedfever(>103.5^◦F)byday6–7afterchal- lengeinfection,threeofthefourlowdosevaccinatesandallsheep immunizedwitheithermediumorhighdosesoftheCP-BTVcon- structwereresistanttochallengeinfectionasdeterminedbythe absenceoffever.ViralRNAwasnotdetectedbyquantitativeRT- PCR(qRT-PCR)inthebloodanyofthehighdosevaccinatedsheep, andinonlyoneofthefourmediumdosevaccinatedsheep.Incon- trast,allofthecontrolandlowdosevaccinatedbecameinfected afterchallengeasconfirmedbyqRT-PCRanalysisoftheirblood (Table1).

Insummary,thesestudiesconﬁrmthatinductionofprotective immunityinsheepcanbeaccomplishedbyimmunizationwith onlytheBTVoutercapsidproteins,andthatthereisatitratable responsetovaccinationsuchthat immunizationwithrelatively largequantities of the CP-BTV construct can induce sterilizing immunitywhereas lowerdoses protectagainst developmentof clinicalsignsfollowing challengebut notinfection.This immu- nityissomewhatcorrelatedtoinductionofneutralizingantibody inserum,howeverthedatafurthersuggestthatanimalswitha

minimalneutralizingantibodyresponsefollowingvaccinationmay beprimedtomorequicklyandeffectivelyrespondtochallenge infection.Similarobservations of immune “priming” havebeen madewithotherrecombinantvaccineconstructsthatutilizeother expressionvectorssuchascapripoxandmyxomaviruses(Perrin etal.,2007;Topetal.,2012),andwithotherCP-vectoredanimal vaccinessuchasthattoWestNilevirus(Gardneretal.,2007).

5. Vaccinology

Athoroughunderstandoftheimmuneresponsetoanimalsto BTVinfectioniscentraltothelogicaldevelopmentofefﬁcacious vaccines,whichisimportantbecauseat-riskcountriestypicallyuse vaccinationoflivestocktocontrolanyincursionoroutbreakofBT (MaclachlanandMayo,2013).Earlyinvestigatorsrecognizedthat livestockthatrecoveredfromBTwereoftenresistanttothedisease, whichspurredinitialeffortstodevelopvaccines(Spreull,1905).

However,itwaslaterrecognizedthatvaccinationcanbeproblem- aticgiventhepluralityof BTVserotypes(currently26) coupled withserotype-speciﬁcimmunityin livestock,meaningthatvac- cinespotentiallyhavetobedevelopedtoall26currentlyrecognized BTVserotypes(reviewed:NoadandRoy, 2009;OyaAlparetal., 2009;Savinietal.,2008;Stewartetal.,2012;Zientaraetal.,2010).

Furthermore,therearefewavailablecommercialvaccineproducts forBTVotherthanthelive-attenuated(modifiedlivevirus[MLV]) vaccinesthatcontinuetobeusedtopreventBTamongcommercial sheepinseveralareasoftheworld(e.g.SouthAfrica,theUnited States of America). MLV vaccines are relatively inexpensiveto produceandtheyareeffectiveastheycaninduceprotectiveimmu- nitysimilartothatwhichfollowsnaturalinfection.Despitethese attributes,theuseofinactivatedvaccinesisincreasinglypreferred asinactivatedvaccinesenjoyseveralinherentadvantagesoverMLV vaccines.Specifically,ascompared toMLVvaccines,inactivated vaccinescannotcausediseaseorreverttovirulence,reassortgenes withfieldorMLVviruses,norcrosstheplacentatocauserepro- ductivelosses(reviewed:MaclachlanandMayo,2013;Noadand Roy,2009;Savinietal.,2008;Stewartetal.,2012;Zientaraetal., 2010).However,inactivatedvaccinessufferfromtheirrelativeslow onsetofimmunityascomparedtoMLVvaccines,andthelackof commercialproductsformostBTVserotypes.

Although not yet available commercially, replication defec- tivevaccinevirusesproducedusingreversegeneticstechnology offerthepotentialforvaccineconstructsthatcombinetheben- eﬁtsofMLVvaccineswiththesafetyofinactivatedones(Celma etal.,2013;Matsuoetal.,2011).Other“nextgeneration”products suchasbaculovirus-expressedviruslikeparticles(VLPs)andviral- vectoredrecombinantvaccines,includingtheCP-BTVrecombinant describedearlier,havebeenshowntobeeffectiveexperimentally buttheirinherentcostandlimitedmarketpotentialhaveprevented theircommercialusetodate(Booneetal.,2007;Celmaetal.,2013;

NoadandRoy,2009;Stewartetal.,2010).

TherecentavailabilityofIFNARmiceasanexperimentalmodel systemhasfurtherfacilitatedthestudyofimmunitytoBTVinfec- tion, and tothe development of potentially efﬁcaciousvaccine constructs(Calvo-Pinillaetal.,2009a,2010).IFNARmicecanbe protected againstlethalBTV challengebyneutralizing antibod- iesinducedbyimmunizationwithrecombinantVP2aloneorin combinationwithotherviralproteins(Calvo-Pinillaetal.,2009b, 2012;Franceschietal.,2011;Jabbaretal.,2013;Maetal.,2012).

ImmunizationofIFNARmicewithindividualorcocktailsofrecom- binantBTVproteinsalsocaninducevaryingdegreesofheterotypic immunityassociatedwiththeproductionofprotein-speciﬁcCD8+

Tcells(Calvo-Pinillaetal.,2012).Itistobestressed,however,that althoughtheIFNARmurinemodelprovidesausefulmodelforini- tialscreeningofpotentialvaccines,validationinruminantlivestock

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isclearlyrequiredandparticularlyinsituationswheretheTypeI IFNresponsemaybecriticaltothestimulationofcriticalacquired immuneresponses.

In summary,although ongoing studieshave repeatedly con- ﬁrmedthatarecombinantsubunitvaccinationstrategyisviable forprotectionoflivestockagainst BTVinfection,theexpression ofimmunogenicVP2canbechallenginggiventheconformational natureofindividualepitopesandtheirstabilityduringstorage,and becauseoftheapparentlyserotype-speciﬁcnatureofimmunityto BTVinfectionoflivestock(DeMaulaetal.,2000;Savinietal.,2008;

Stewartetal.,2012;Zientaraetal.,2010).Thus,currentvaccina- tionstrategiestopreventBTVinfectionoflivestockcontinuetobe reliantonhistorictechnologies,withalloftheirattendantpotential disadvantages(MaclachlanandMayo,2013;Savinietal.,2008).

Acknowledgements

TheauthorsthankDr.DamienVitourforhisconstructivereview ofthemanuscript. Portionsofthesestudiesweresupportedby USDANRICompetitiveGrant#2002-35204-12314,andfundspro- videdbytheHarrietE.PﬂegerFoundation.

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