Thedegreeofmaturationoftheavianimmunesystemisanimportantaspecttoconsiderforthe

developmentofinovovaccines.SuccessfuluseoftheinovoroutetodeliverMLVsisachieved,even
thoughonday18ofincubation(E18),embryosdonothaveafullymatureimmunesystem[6,7].In
fact,MLVforMD[8,9],avianmetapneumovirus[10,11]andIB[12]producedearlierimmunityor
betterprotectiveimmunitywhengiveninovocomparedwithdayofhatchadministration.Itis
commonforbroilerchickenstobevaccinatedforseveraldiseasespriortoleavingthehatchery.
SimultaneousinovoadministrationofMLVsroutinelyoccurswithoutinterferenceamongthemselves
[2,3,13]orwithdayofhatchvaccinesforNDandIB[14,15].
Chickenimmunesystemdevelopmentstartsearlyinembryoniclifebutisincompleteuntilseveral
weeksafterhatching.Similartomammals,birdsrespondtoantigenicstimulationbygenerating
antibodies,aswellascellularimmunity[16].Tcellmaturationoccursinthethymusasalternating
wavesduringembryogenesisandcontinuesafterhatching[17].MatureTcellsemigratefromthe
thymusandcolonizeperipheralorgans.ChickenTcellimmunityhasbeenimplicatedinresistanceto
severalpathogens,asshownbyalackofcorrelationbetweenantibodyresponsesinvaccinated
chickensanddiseaseresistancetosubsequentinfections,andcontinuedresistanceinbursectomized
vaccinatedbirds.
ThebursaofFabriciusistheprimarysiteofBcelldevelopmentrequiredforhumoralimmunity[18].
Aroundthetimeofhatching,matureBcellsbegintoemigratefromthebursatotheperipheryand
continuetodosountilsexualmaturitywhenthebursabeginsinvolution.Asthebursallumenis
directlyconnectedtothegutlumen,thisprovidesthebursawithconstantsamplingofgutderived
antigens.Withoutthissampling(e.g.,iftheductisligated),theBcellrepertoireisseverelylimited
[19].
Chickensdonothavewelldevelopedlymphnodes[20].Instead,chickensdevelopdiffuselymphoid
tissuesatantigenicallystimulatedsitesinthebody,suchasthemucosalassociatedlymphoidtissue
(MALT).Dependingupontherouteofantigenicexposure,suchasinhalationoringestion,different
tissuetypesmaybepreferentiallystimulated.Theimmunesystemassociatedwiththemucosalsurfaces
isthefirstpointofcontactbetweentheavianimmunesystemandantigen.Inovoadministrationof
vaccinesintotheamnioticfluidonE18conceivablyintroducesantigenstoMALT.
Inadditiontoadaptiveimmunity,defenseagainstmicrobialinvadersinpoultryalsoinvolvesinnate
mechanismsassociatedwithnaturalkiller(NK)cells,granulocytes,macrophagesandtheirsecreted
products,suchasnitricoxide(NO),andavarietyofcytokines[21].
Chickenmacrophagesformthefirstlineofdefensebyphagocytizingbacteriaandviralparticlesand
areasourceofendogenousenzymaticactivities,suchasnonspecificesteraseactivityandacid
phosphataseactivity[22].Cellsbelongingtothemononuclearphagocyticsystemariseinthebone
marrowandsubsequentlyenterthecirculationasbloodmonocytes.Uponmigrationtotissues,
monocytesmatureanddifferentiateintotissuemacrophagescapableofprocessingantigensand
secretingseveralimmunoregulatorycytokinesormetabolites[23].Chickenmacrophagesalsoproduce
NOwithcytostaticorcytotoxicactivityagainstviruses,bacteria,fungi,protozoa,helminthesand
tumorcells.TheantimicrobialandcytotoxicactionsofNOareenhancedbyothermacrophage
products,suchasacid,glutathione,cysteine,hydrogenperoxideorsuperoxide[24].
NKcellactivityhasbeenimplicatedasamajorlimitingfactorinavianviralandprotozoaninfections.
IthasbeenshownthatMDresistantchickenstrainshaveanelevatedlevelofNKcellsfollowing
infection,whereasdecreasedNKcellcytotoxicitywasfoundinMDsusceptiblechickenstrains
[25,26].NKcellscanbedetectedinembryonicspleenbeforehatching[27].SubpopulationsofTcells,
Bcells,macrophagesandellipsoidassociatedreticularcellscanalsobefoundinchickenspleenon
E20andthespleengraduallymaturesduringthefirstweekposthatch[6].Basedontheincomplete
structuralorganizationofsecondaryimmuneorgans,theimmunefunctionofthelatestageembryonic
chickenisnotentirelydeveloped.Thereasonwhyinovoimmunizationcanbemoreeffectivethan
posthatchvaccinationonsomeoccasionsisnotwellunderstood.

Inovoimmunizationofpoultry

Section:

Today,approximately90%ofbroilerchickensgrownintheUSAarevaccinatedinovo23daysprior
tohatchatthetimewheneggsaretransferredfromtheincubatortothehatcher.Globally,inovo
vaccinationhasbeenusedin26ofthetop30poultryproducingcountries.Theinovoroutewasfirst
usedcommerciallyin1992withthedevelopmentoftheInovojectsystem(Embrex,Inc.),an
automatedinovoinjector(Figure

1A)[28].TheInovojectsystemdeliversvaccinesviaaneedlewithin
aneedle,inwhichthepunchneedlepenetratestheshellonthebluntaircellendoftheeggandthe
smallerdiametervaccinedispenseneedlepenetratestheaircellandchorioallantoicmembranesto
depositvaccineintheamnion(Figure

1B).Theinjectionsystemvaccinatesupto70,000chickeneggs
perhourinamoreuniformandprecisemannerthanposthatchmassvaccinationmethods.Inaddition
tochickens,turkeyscanbeeffectivelyimmunizedinovousingMLV[10,29]anditisconceivablethat
otherdomesticpoultryspeciescanalsobeimmunizedinovo.
Thefirst10yearsofinovovaccinationwaslimitedtovaccinesforMD,IBDandFP.Duringthis
period,experimentalinovovaccinesweredevelopedforseveralothermajorpoultrydiseases,
includingND[3033]andIB[34].However,thesevaccineshavenotadvancedtoroutinecommercial
use.
Theprospectfornewinovovaccinesisgrowing,asevidencedbythenumberofrecombinantpoultry
vaccinesenteringthemarketorabouttodoso.Currently,theserecombinantvaccinesuseeitherFPor
theMDvaccineherpesvirusofturkeys(HVT)asthevectoragenttoexpressprotectiveantigensof
Mycoplasmagallispeticum,infectiouslaryngotracheitisvirus,AIvirus,NDvirusorIBDvirus.HVT
[31]andFP[32]vectorsaresafeandeffectivewhenadministeredinovo.In2006,anonrecombinant
liveoocystvaccinefortheparasiticdiseasecoccidiosis[35]waslicensedfortheinovorouteandis
currentlyincommercialuse.
Anumberofexperimentalvaccinesthatmayprovetobecompatiblewiththeinovorouteof
administrationareunderdevelopmentandthemostpromisingofthesearenonreplicatinghumanAd
vectoredAIvaccine[36]andalphavirusvectoredAIvaccine[37].NDvirusvectoredAIvaccineis
anotherapproachunderdevelopment[3840].ItisyettobedeterminedwhethertheNDvirusvectors
areattenuatedenoughforsafeinovoadministration.However,datafrominoculationofE10embryos
suggestthatsomeNDvirusvectorsarehighlyattenuatedandmaybeinovocompatible[38,39].A
standaloneIBvaccinedevelopedspecificallyforinovoadministrationappearspromising[41].Inovo
deliveryofaDNAvaccineforIB[42]providedpartialprotection,whereasaninovoDNApriming
vaccinationforIBDfollowedwithaposthatchFPvectoredIBDvaccinebooststimulatedcomplete
protectionwhenneithervaccinealoneprovidedsufficientimmunity[43].However,commercialuseof
DNAvaccinesinpoultryawaitsfurtheradvances[44].Itislikelythatthenumberofmajorpoultry
diseasescontrolledbyinovovaccinationwillincreasesubstantiallyduringthenext5years.
Inovovaccinationismorecomplexthaninjectinganegg.Thecomplexityisdueinparttothefive
inovocompartmentsintowhichvaccinecanbedelivered:theaircell,allantoicfluidsac,amnioticfluid
(inwhichtheembryofloats),embryobodyandyolksac(Figure

2).Vaccineeffectivenesscanvary
basedontheinovocompartmentintowhichavaccineisdeposited.Variationinvaccineeffectiveness
byinovocompartmentwasdemonstratedwhenitwasshownthatMDvaccinedeliveredtotheaircell
membraneorallantoicsacprovidedpoorprotectiveimmunity,whereasMDvaccinedeliveredtothe
amnioticfluidorembryobodywashighlyeffective[45].DuringE1719,theembryoimbibesthe
amnioticfluid.Inertparticlesofvarioussizes,viralparticlesorDNAdeliveredtotheamnioticfluidare
rapidlyfoundintheupperrespiratorytractanddeepinthelungs[42,46,47].Recentworkhasshown
thatinovoadministrationofHVTinducesthestructuralmaturationofbronchialassociatedlymphoid
tissue(BALT)[48].StructuralmaturationofposthatchavianMALTisantigendrivenasevidencedby
thedevelopmentofdiffuselymphoidtissuesatantigenstimulatedsites[49,50].Anotherimportant

componentoftheamnioticfluidrouteisvaccinedeliverytothedigestivesystem,includingthe
intestine[42,47].Theintestinefunctionsasamajorimmuneorgan[51]andincludesthemajorityof
avianMALTandthebursaofFabricius.Thus,theamnioticfluidrouteofinovovaccinationresultsin
vaccinedeliverytorespiratoryanddigestivesystemMALT,aswellasprimaryBcelllymphoid
organs.
EvidenceshowsthatantigendrivesthematurationofavianMALT[49,50]andsuggeststhatinovo
immunizationmayacceleratethematurationofembryonicBALT[48].Acceleratedmaturationwould
beexpectedtoresultinchickenswithagenerallymorematureimmunesystemathatchandwith
antigenspecificimmuneresponsesinitiatedatmucosalsites.Understandingtheontogenyofthe
embryonicimmunesystemandhowMLVs,vector,antigenandadjuvantstimulationaffectthat
ontogenyareofparamountimportanceforunderstandingthemechanismsbehindsuccessfulinovo
vaccination.
Whenconsideringinovovaccination,maternalantibodyinterferencewithvaccineefficacyiscritical
[52].Itiscommonforpoultryproducersworldwidetovaccinateand,inmanycases,hyperimmunize
theirbreederstockformanydiseases,includingMD,FP,ND,IB,IBDandAI.Maternalantibody
derivedfromtheyolkreachesthehighestlevelintheprogenyscirculationbetweendays1and3
posthatch.KowalczykandcolleaguesreportedasteepriseinyolkmaternalIgGtransfertothe
embryoniccirculationbetweenE19andE21[53].Embryonicpathogenspecificserumantibodylevels
aremuchloweronE1719thanfromE20toimmediatelyposthatch[54].Thus,theinovoroute
appearstoofferawindowofopportunityforvaccinationwhencirculatingmaternallyderivedantibody
iscomparativelylow,allowingtraditionalandrecombinantMLVtoestablishinfectionsandstimulate
immunitypriortothesteepriseincirculatingantibody[54].Vaccinesadministeredviatheamniotic
fluidroutelocateatmucosalsitesintherespiratorysystemwherematernallyderivedIgGwouldbe
expectedtobeconsiderablylowerthanthatfoundintheembryoniccirculation.Ontheotherhand,yolk
enteringtheembryosintestinallumenlateinincubationwouldbeexpectedtocontainsignificant
maternallyderivedIgG,potentiallyimpactingvaccinesthatpreferentiallylocatetointestinalmucosal
sites.Avaccinethatisnotimpactedbymaternalimmunitywouldbeexpectedtohaveabetterchance
ofstimulatingrobustimmuneresponses.Onewaytoavoidthenegativeimpactofmaternalantibodyis
touseavectoragentthatdoesnotnaturallyinfectchickens,suchasthehumanAdvector[36].Another
approachistouseHVTasavectorsincethecellassociatednatureofHVTsignificantlyreducesthe
impactofmaternalimmunitytoMD[8].

Nonreplicatinghumanadenovirusvectorasanemerging
Section:
vaccinecarrierforinovovaccination
Theperformancerequirementsofvaccinesincludetherapiditywithwhichaprotectiveimmune
responsecanbeelicited,ahighbenefitriskratio,thespeedandeaseofmanufacture,distributionand
administration;andtheinherentstabilityofthevaccinetoallowforlongtermstockpiling.The
developmentofAdvectoredvaccinesholdsgreatpromiseforcreatingamajorimpactoncommercial
vaccinesbymeetingmost,ifnotall,oftheabovecriteria.TheuseofhumanAdvectorstoexpress
foreignantigensinanimalsandhumanshasbeenmotivatedbyefficienttransductionofawidevariety
ofcelltypesbyAd[55,56],thetransientbutproductivewaveoftransgeneexpressionfromAdvectors
intransducedcells[55],rapidgenerationofnewrecombinantAdvectorsinresponsetoemergenceof
newpathogens[57,58],economicadvantagesavailablethroughmassproductionofAdvectorsina
wellcharacterizedpackagingcelllineinserumfreesuspensionbioreactors[59],andinexpensive
purificationofAdparticlesbycolumnchromatography[60].AlthoughahumanAdvectoredvaccineis
capableofelicitingimmuneresponsesinrodents[61,62],dogs[63],cattle[64],nonhumanprimates
[65]andhumans[66]withoutanyappreciablesideeffects(e.g.,adversehostresponsethatneeds
medicalattention),itspotentialtoimmunizebirdswasnotdemonstrateduntilrecently.
ThepotencyofhumanAdvectoredvaccinesinimmunizingbirdswasshownbystimulatingprotective
immuneresponsesinchickensfollowingintramuscularinjection[67]andinovoadministration[36]of

humanAdvectoredAIvaccinesinasingledoseregimen.Thesefindingsrepresentauniqueand
innovativeconceptinthefieldofvaccinology.NotonlycanAdvectoredvaccinesbegeneratedrapidly
andmassproducedbuttheyalsodonotreplicateandwillnotintroducegeneticallymodifiedorganisms
intothefoodchainortheecosystem.IncontrasttohumanAd,fowlAdhasnotbeenbioengineeredinto
areplicationincompetentvectorbecauseitsE1regionhasnotbeenidentified[68].Mass
administrationofnonreplicatinghumanAdvectoredvaccinesintoembryonatedeggsusingan
automatedinovoinjectorwouldprovideamajoradvantageoverothermethodsthatrequireindividual
handlingofbirds(e.g.,posthatchinjection)orinvolvelessuniformexposure(e.g.,sprayanddrinking
waterapplications).
AhumanAdvectormaypotentiallytransducepartofachickenembryofollowinginovo
administrationthroughbindingoftheAdfibertothecoxsackievirusandadenovirusreceptorfoundon
thesurfaceofchickencells[69].
AtleastoneofthehumanAdcomponents,hexon,ishighlyimmunogenicandconfersadjuvantactivity
toexogenousantigens[70].ThisintrinsicadjuvantassociatedwithAdvectorsmayfacilitateefficient
immunizationfollowingexpressionofasmallnumberofantigenmoleculesintheembryo.
AlthoughhumanAdgenomicmaterialdoesnotpersistinrodents[71],thefateofhumanAdvectorsin
birdsfollowinginovoadministrationremainstobedetermined.
TwocontinuouspackagingcelllinesarecommonlyusedforpropagationofE1defectivehumanAd
vectors:293,alineestablishedbytransforminghumanembryonickidneycellswiththeleftendofthe
humanAdserotype5genome[72];andPER.C6,alineestablishedbytransforminghumanembryonic
retinalcellswiththehumanAdserotype5E1region[73].Eachinfected293packagingcellcan
produceupto10,000plaqueformingunits(pfu)ofAdvectors[74].However,alowlevelof
contaminationbyreplicationcompetentAd(RCA)appearstobeanintrinsicproblemforthe293cell
line[75].RCAfreeAdvectorscanbeproducedinPER.C6packagingcells[73]andtheyieldis
comparabletothatofitscounterpartproducedin293cellsinourexperience.Advectorsareroutinely
producedbyinfectingPER.C6suspensioncellsataconcentrationof1106cells/mlasdescribed[76].
A5lcellbagcontainingAdinfectedPER.C6cellsonawavebioreactor(Figure

3)canthusproduce
51013pfuofRCAfreeAdvectorsinafewdays.A500lcellbagcapableofproducing51015pfu
ofAdvectorsatonetimeiscommerciallyavailable(WaveBiotech,LLC,NJ,USA).Specific
pathogenfreechickenswerewellprotectedagainsthighlypathogenicAIviruschallengefollowing
inovovaccinationonE18byahumanAdvectoredAIvaccineatadoseof1.5108infectiousunits
[36].AlthoughpfurepresentAdparticlesthatreplicate,whereasinfectiousunitsrepresentthosethat
expresshexoninpackagingcells,thetwoAdtiterassaysarecomparableandthelattercanbe
completedinamoretimelymannerusingtheAdenoXrapidtiterkit(BDClontech,CA,USA).A
single500lcellbagcanproduceAdvectoredvaccinesintherangeof3107doses,althoughtheyield
isexpectedtovaryfromvaccinetovaccineandfrombatchtobatch.Itisconceivablethattheeffective
doseofanAdvectoredinovovaccinecanbefurtherreducedafteroptimization(e.g.,adjuvantation
andcodonoptimizationoftransgenes).Useofdisposablecellbagsminimizescontaminationand
eliminatestherequirementstocleanandsterilizetheproductionsystem.PurificationofAdvectorsby
specificanionexchangecolumnsallowsnearly100%recovery[60].Developmentofastableliquid
formulationenablesAdvectoredvaccinestobestoredforlongperiodswithouttheneedforfreezing
andreconstitution[77].ItisexpectedthatAdvectoredinovovaccinescanbemanufacturedrapidlyat
lowcosts.Moreover,generationofRCAfreeAdvectorsinPER.C6cells[73]foregoesmanyofthe
potentialsafetyconcernsrelatedtoreplicationassociatedbiologicalhazards.
Althoughmultiplevectorsareavailableasvaccinecarriers,bioengineeredavianvirusderivedvectors
(e.g.FPvirus)haveshownreducedprotectionassociatedwithpreexistingimmunitytothevectordue
topreexposureofbirdstotheavianvirus[78].Incontrasttoavianvirusderivedvectors,thereisno
evidenceofpreexistingimmunitytohumanAdinchickens,eventhoughthebirdwasdomesticated
morethan8000yearsago[79].ThefailureofdomesticchickenstodevelopimmunitytohumanAd
maybeattributedtoinefficientinfectionofchickensbyhumanAdthroughtheintranasalroute[36,67]
andnonpermissivenessofchickencellsinsupportofreplicationofhumanAd[80].TheuseofE1
defectiveRCAfreeAdvectorasaninovovaccinecarrierisendowedwithahighcompliancerateas

thisclassofvectorsdoesnotreplicateinhumancellswithoutE1expression[73].Thevastmajorityof
broilerchickensrearedforhumanconsumptionliveforfewerthan60daysandvaccineinduced
protectiveimmunityisrequiredtolastonlyforthisshorttime.Therefore,Advectoredinovovaccine
mayemergeasacriticaltoolforcontrollingdiseaseinbroilerchickens.Itmayalsobepossibleto
immunizebroilerbreederhenswithanAdvectoredinovovaccinewithoutinterferingwiththe
effectivenessofAdvectoredvaccinesintheprogenyiftheAdvectoredvaccineisadministeredtothe
progenyonE18priortoasteepriseofmaternalantibodyinembryoniccirculation[53,54].
TheautomatedproductionandadministrationofAdvectoredvaccinesminimizelabor,timeandcosts
byallowinguniforminoculationofvaccinesinhundredsofeggsperminute.Furthermore,theAd
vectoredvaccineiscompatiblewithadifferentiationbetweeninfectedandvaccinatedanimalsstrategy
becausethevectorencodesalimitednumberofpathogenderivedantigens.Thus,markerantigens
expressedbythepathogenbutnotthevectorcanbeusedtodiscriminatenaturalinfectionfrom
vaccination.UnliketheuseofinactivatedvaccinesandMLV,Advectoredvaccinesmayrefocus
immuneresponsesawayfromnonprotectiveimmunodominantepitopestowardsmoreprotectiveones.
Therefocusingofimmuneresponsesmayenableasinglevaccinetotargetmultipleprotectiveepitopes
inonepathogenandinducepotentprotectiveimmunityagainstmultiplepathogens.Whenprotective
epitopesofmajoravianpathogensarebettercharacterized,inovoadministrationofAdvectored
vaccinesmaybringpoultryvaccinationtoahorizonwheretheabilityofanembryotorespond
effectivelytoamulticomponentvaccinewillbetheultimatelimitationofinovovaccination.

Expertcommentary

Section:

Massimmunizationandcosteffectivenessarecrucialforpoultryvaccinationprograms.Thepotency
ofahumanAdvectoredinovovaccinehasbeendemonstratedandmaybefurtherenhancedby
adjuvantationand/orcodonoptimizationoftheantigengenetomatchthetRNApoolfoundinchicken
cells.Furthermore,characterizationofprotectiveepitopesforantigensfoundinmajoravianpathogens
mayallowcoexpressionofmultipleprotectiveepitopesfromasingleAdvector.Collectively,these
approachesmaysignificantlyreducetheeffectivedoseandexpandthespectrumofvaccinesthatcanbe
coadministeredinasingledoseregimen.Automatedsimultaneousinovoadministrationofmultiple
poultryvaccinesshouldincreasetheuniformityofvaccineapplicationandreducevaccinationcosts.

Fiveyearview

Section:

Massimmunizationofpoultrybyautomatedinovoadministrationofvaccineshasbeensuccessful;
however,itisrestrictedtoalimitednumberofdiseasesatpresent.Thislimiteduseisbecauseseveral
commonlyusedposthatchvaccines(e.g.,NDandIB)arepathogenictothechickenembryo.The
demonstrationthatanRCAfreehumanAdvectoredinovoAIvaccineeffectivelyimmunizedchickens
withoutcausingharmtoembryosopensthepossibilitythatvaccinesforthemajorpoultrydiseasesmay
besimultaneouslyadministeredusinganautomatedinovoinjectorinatime,laborandcostsaving
manner.Overthenext5years,weshouldbeabletobringthepotentialandlimitationofhumanAd
vectoredpoultryvaccinesintoaclearfocus,anddeterminewhetherthisnoveltechnologywillextend
inovovaccinationtoallmajorpoultrydiseasesandtodevelopedanddevelopingcountries.

ReadMore:http://ezproxyprd.bodleian.ox.ac.uk:2497/doi/full/10.1586/14760584.6.3.457