2015 Age-dependent immune responses and immune protection after avian coronavirus vaccination

2015·1.23 MB·English

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ARTICLE IN PRESS
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Vaccinexxx(2015)xxx–xxx

ContentslistsavailableatScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Age-dependent immune responses and immune protection after

avian coronavirus vaccination

FrederikW.vanGinkel∗,JustinPadgett,GiselaMartinez-Romero,MatthewS.Miller,

KellyeS.Joiner,StephenL.Gulley

DepartmentofPathobiology,CollegeofVeterinaryMedicine,AuburnUniversity,217ScottRitcheyResearchCenter,Auburn,AL36849,USA

a r t i c l e i n f o a b s t r a c t

Articlehistory: Infectiousbronchitisvirus(IBV)isanendemicdiseaseofchickensandamajorcontributortoeconomic
Receiv ed17December2014 lossesfort hepoultry indus tryde sp ite vaccinati on.Rece nt observati ons in dicated thatchicks ma yhavean
RAeccceepivteedd i1n0 r Aevpirsield2 0fo1r5m 20 March 2015 immat ure im mune s ystem im mediat ely after hatc hing w hen vaccinat ed for IBV . The refore we h ypot h-
esized that early IBV vaccination will generate an immature, poorly protective IBV-specific immune
Availableonlinexxx
responsecontributingtoimmuneescapeandpersistenceofIBV.TotestthishypothesistheIBV-specific

immuneresponseandimmuneprotectionweremeasuredinchicksvaccinatedatdifferentages.This
Keywords:
demonstratedadelayedproductionofIgGandIgAplasmaantibodiesinthe1,7and14-day-oldvaccina-
Age-dependentimmunity
tiongroupsandalsolowerIgAantibodylevelswereobservedinplasmaofthe1-day-oldgroup.Similar
Infectiousbronchitisvirus
MAnutciboosadlyi mk imneutincisty olobwseerrvaavtiidointsy winedriec emsathdaen fodra ayn2t8ibvoadciceins ainte tdeabrisr.d Isn. Tahdedidteiolany, eIgdGa nandt/iobrolodwiees rfraonmtib tohde y1r-edsapyo-onlsde gcroomubpi nheadd
Avidityi ndex withl owerIg Gavidit yind ices co incidedwit hincr ease dtrache alinfla mmat ionandd epletiono ftracheal

Aviancoronavirus epitheliacellsandgobletcellsuponIBVfieldstrainchallenge.Thelackofvaccine-mediatedprotection

wasmostpronouncedinthe1-day-oldvaccinationgroupandtoalesserextentthe7-day-oldgroup,

whilethe14-day-oldandolderchickenswereprotected.ThesedatastronglysupportIBVvaccination

afterday7posthatch.

©2015ElsevierLtd.Allrightsreserved.

1. Introduction well as antigenic variants [4] complicates vaccination programs.

Sinceimmunityinducedbyvaccinationagainstasingleserotype

IBVisendemicandcurrentlyoneofthemostimportantcauses generallyprovidesinsufficientprotectionagainstotherserotypes

ofeconomiclossesforthepoultryindustryandrepresentsacontin- [5,6].

uousthreatforthisindustry.Inthepast,itwasestimatedthatwith MucosalimmunityplaysaroleinthecontrolofIBVinchick-

thebestpossiblemanagementofflocksIBVinfectionwillreduce ensaswasdemonstratedusingIBV-resistantandIBV-susceptible

incomebyapproximately3%whencomparedtoanIBV-freeflock inbred chicken lines [7]. This combined with the finding of Gelb

[1].Thereisapproximatelya50%vaccinefailureforArkansas(Ark) et al. [8], in which ocular immunization with the Massachusetts

serotypeofIBV[2],themostprevalentvaccineserotypeusedin Connaught strain of IBV only on day 1 or on day1 plus day 14

the USA. Symptoms of IBV infection include, but are not limited followedbychallengewithMassachusetts41providedprotection

to,weteyes,swollenface,trachealandkidneylesions,respiratory of 8% and 50% of the chickens, respectively, while the same SPF

disease,reducedweightgaininbroilers,decreasingandpooregg White leghorns only ocularly immunized on day 14 were 100%

quality in layers [3,4]. The existence of various IBV serotypes as protected [8]. BSA immunization of 1, 7 and 12 day old broiler

chickens obtained very similar results [9]. This raises questions

pertaining the maturity of the immune system and in particular

themucosalimmunesystem,andtheabilityofchickstogenerate
assoAcbiabtreedvilaytmiopnhs:o Aidrkt iDssPuI,e A;rEkLaInSPsaOsT D,eelnmzyamrvea- Plionukletdryi Imndmuusntroys;p CoAt;LTH, Gco,nHjaurndcetriviaan- a pr otective immune respons e wh en vaccin ate d at a ve ry young

gland;HALT,head-associatedlymphoidtissues;HRP,horseradishperoxidase;IBV, age.

irnefceecpttiooru.s bronchitis virus; EID50, median embryo infectious dose; TLR, Toll-like Conjunctiva-associated lymphoid tissue (CALT) and Harderian
∗ glandsdonotfullymatureasalymphoidorganuntilweeksafter
Correspondingauthor.Tel.:[phone];fax:[phone].
hatching[5,6,10–12].Thiscombinedwiththepracticeofimmuniz-
(F.WE.-vmaanilG aidndkreels).ses: [e-mail], [e-mail] ingandb oostingforIB Vea rlyafterha tchin gm aysetup th eimmune

http://dx.doi.org/10.1016/j.vaccine.2015.04.026
0264-410X/©2015ElsevierLtd.Allrightsreserved.

Pleasecitethisarticleinpressas:vanGinkelFW,etal.Age-dependentimmuneresponsesandimmuneprotectionafteraviancoronavirus

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response for failure to protect. The second IBV immunization challengedocularlywith7.3×105EID oftheAL/4614/98IBVfield
50
onday14ofage,whichbyitselfisfullyprotective,doesnotcom- strain21daysaftervaccination

pletelycompensatefortheprematureprimingonday1[8].Field Samplecollection:Tearswerecollectedaspreviouslydescribed

studiesbydeWitetal.[3]demonstratedalackofprotectiveimmu- [19].Bloodsampleswereobtainedbypuncturingthebrachialvein

nitywhenbirdswereboostedbetweenday8throughday13of withasterile20GneedleintoKendallmonoject,EDTAcontaining,

age.ThepercentageofbirdsinacommercialflockpositiveforIBV- bloodcollectiontubes(TycoHealthcareGroupLP,Mansfield,MA)
spec ificI gMantibod ies wasc or re latedwithv accin eprotect ion and andin cubatedo nice. Blood samplesw erecen trif ugedat50 0×g
increase dw iththeage ofb oosting.Th isda tasuppo rtstheno tion for3 0min.Plas ma wa scollec tedand stored at−80◦Cun til test ed .

thatearlyvaccinationandboostingoftheIBVimmuneresponse IBVpropagationandpurificationforELISA:IBVwaspropagated

maylimitinductionofprotectiveimmuneresponsestoIBV.Unlike inSPFWhiteLeghornembryonatedchickeneggs(SunriseFarms,

thestudybyGelbetal.[8],thedeWitetal.[3]studycanalsobe Inc., Catskills, NY) by inoculation on day 10 of embryonation as

interpretedthatmaternalantibodiesinterferewithvaccinedeliv- previously reported [20]. Supernatants were titrated for the IBV

eryduringthefirst2weeksoflife[13]. virus using the Reed and Muench method [21]. IBV was treated
F urther evid ence t hatthe im mu neresponsemaybelimiteddur- with 0.1% (cid:3) -pro priol acton e for 30 min at 3 7◦C [ 22]. Inac tivation

ingthefirstweeksoflifecomesfromtheobservationthatIgAlevels of the virus was confirmed by injection into embryonated eggs.

areundetectableinplasmathefirstweekoflifeandIgMlevelsare TheinactivatedIBVwaspurifiedbasedonapreviouslypublished
low [14].Thisind ic atestha tim mun oglob ul inc lass swit chinga nd prot ocol[23].Th evi rusw asthen stored at − 80◦Cuntilu sed.

productionofantibodiesisverylimitedduringthefirstweekpost

hatchandthereforechicksarehighlydependentonmaternalIgY
antibo dies forprotec tionag ains tIBV,w hichdrops ∼5 0%during the 2.1. IBV-specific ELISA

firstweekoflife[14].
In order to measure IgG (IgY), IgA and IgM antibody levels
BesidesdiminishedBcellresponseaftervaccination,splenicT
in plasma and tears of chicken, an IBV-specific enzyme-linked
cellsfromoneweekoldchickensarealsolessresponsivetopoly-
immunosorbent assay (ELISA) was developed as previously
clonal activation than that of older chickens. The splenic T cells from described [20]. In Bri ef, ELISA pla tes were c oate d with (cid:3)-
1 day old chicks even produce inhibitory factors for proliferation of propriolac tonek ille d,puri fiedIBV at2(cid:2)g /mlin carbona tebu ffer.
matureTcellsinvitro[15].Furthermore,spleniclymphocytesof
The plates were blocked with PBS-BSA (1%) after which the
40dayoldchickendisplayedbetterantigenspecificproliferation
samples were loaded at two-fold dilutions. Binding of chicken
afteroralSalmonellaexposurethan10dayoldchicken[16].When
antibodieswasdetectedusingbiotinylatedanti-chicken-IgG,-IgA
measuringgeneexpressioninlungandtracheain1and4week
and -IgM monoclonal antibodies (Southern Biotechnology Asso-
old birds after avian influenza exposure a reduced expression of
ciates,Inc.,Birmingham,AL)followedbystreptavidin-horseradish
immune-related genes was shown and included innate immune peroxi dase . The plates we re devel ope d using TMB (3,3(cid:5),5,5-
responsegenesintheyoungerbirds[17].Additionalevidencethat
Tetramethylbensidine;Invitrogencorp.,Frederick,MD)substrate.
innate immune mechanisms are diminished in young chickens
Thehighestsampledilutionwithatleastanopticaldensityof0.100
wasdemonstratedbyalowerSalmonellaphagocyticindexofhet-
above background level at 450nm was defined as the endpoint-
erophilsduringthefirstfewdaysoflife[18].Thus,earlyexposureto
titer.Tentothirteenchickenswereanalyzedpergroup.Thecontrol
pathogensorvaccinesmayinducesuboptimalinnateandadaptive
groupconsistedout3chickenfromeachagegroupforIgAandIgG,
immuneresponses.
whichwerepooledinonegroupof15sincenodifferenceswere
Based on these observations we hypothesized that early IBV
observedbetweenthecontrols.ForIgMlevelsinplasmathecon-
vaccination,i.e.,withinthefirstweekafterhatching,willgener-
trols(eachgroupcontaining5chickensexceptday28whichhad3)
ateanimmature,poorlyprotectiveIBV-specificimmuneresponse
weredisplayedseparateforeachagegroup.Thiswasdonebecause
contributingtoIBVimmuneescapeandpersistence.Therefore,the
significantdifferenceswereobservedbetweencontrolIgMlevels
abilityofSPFchickensofdifferentagetoinduceanIBV-specificanti-
toIBVindifferentagegroups.
bodyresponseandprotectagainstchallengewithanIBVfieldstrain

wasmeasured.Ourdataindicatethatearlyvaccinationissubopti-
mal forinducti ono fIBV -specific imm uner esponsesan d immune 2.2. Avidityindex

protection.
The avidity index was determined as previously described

[24,25]usingtheabovedescribedIBV-specificELISA.Plasmaand
2. Materialsandmethods tears w ere di lute d 1:64 in ELISA buffer and w ere lo aded on (cid:3)-
propi olacto nekilled IBV co atedEL ISApla tes( 2(cid:2)g/ ml)[20] .Af ter
Chickens:Specific-pathogen-free(SPF)whiteleghorneggswere overnight incu bation of these s ample s at 4◦ C, 100(cid:2)l of inc reas-
obtainedfro mSunriseFarms,Inc.,C atski ll,NY, hatched and used ing conce ntrations of po tassiu m thiocy an ate ( 0.00, 0. 09 4, 0.187,
inallexp erime nts.All hatched chi ckenswe reu sedfort heb elow 0.37 5,0.75,1.50,3.0 M KSCN)wer eloadedinto thew ellsand incu-
ou tlin edexperimen ts regardles sofsex. Chicke nsw ere hou sedin bated for30 min at ro omtem pera ture.Af terw ash ingt hep lates,
cagesin BSL2facilitie sforthed ur ation oftheex perim ent.Fo od thede tect ion ofIB V- specifi cantibodiesw asac complishe das previ-
andw at erw er eprovide da dlib itum.Alle xp erim entalproce dures ous lyreporte d[ 20].Thedata werenorm aliz edtopercentin hi bition
and anima lcare wereperf orm edinco mp liancewithal lapplicable relativ etosam plesn ote xpos edto KSCN.Theco nc entratio nofKSCN
fede raland insti tution alanimalu se guidelines. Aubu rn University toinhib it5 0%ofthe rea ctivityo fth eELIS Aw asdefinedast he avid-
College of V eterinary M edicine is a n Associati on for A ssessment ity index [24,2 5] .Th eOD450re ad -ou tfort heK SCNinh ibi tion data
andAcc red itationofL aboratory An im alCare(AAA LAC )-accredited wa scurv e-fitted usin g3-orderpolyn omi alr egress ionanalys esin
inst itution. Micr osoftofficeE xcellp rogram. TheExcellp rovidedform ulafort he

IBV-vaccinationandchallenge:SPFchickenswereocularlyvac- inhibitioncurvewasusedtodeterminethexvaluesofy=0.5,which
cinatedwith3×10 550 %embryo infe ctiousdo ses(E ID )ofa live arethecon centr ation sof KS CNinhibiti ng5 0 %ofth eE L IS Are activ-
attenua tedA rk DP IIBVvac cinestra in(Zoetis, NewY ork,N50Y) in 5 0(cid:2)l ity, rep resentingtheav id ityind icesofth oses am ple s.All samples
PBS,which wasex pan dedino urlab oratory .Chic kens wer ev ac ci- wer eanalyzedin tri plicates and4– 5s ample swerea naly zedper
nate d1day ofa geand1, 2, 3or 4weeksof age.Allg roups were group .

Pleasecitethisarticleinpressas:vanGinkelFW,etal.Age-dependentimmuneresponsesandimmuneprotectionafteraviancoronavirus

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2.3. Histomorphometricsandhistopathology scoringoftheseparametersisprovidedinthesupplementaldata

(supplementalFigs.1–3).

The cranial 1/3 of tracheae was collected 4 days after IBV Statisticalanalysis:Datawereanalyzedusingaone-wayANOVA
challen ge with 7.3 ×1 05 EID of A L/4614/98 I BV fie ld st rain. testwithNew man–Ke ulsm ulti-c ompariso ntest o rthet-te stusing
50

The tracheae were formalin-fixed and embedded in paraffin. GraphPadPrism5software.Groupswereconsideredsignificantly
Long itudinal 5(cid:2)m sections were m ade and were hem atoxylin differentw henP< 0.05.

and eosin (H&E) stained and analyzed for mucosal thickness

usingAperioScanScopeandtheImageJmorphometryprogram 3. Results

(rsb.info.nih.gov/ij/download.html).Tomeasurethemucosalthick-

ness 5 measurements were made at regular intervals on one To determine whether age of IBV vaccination affected the

trachealring(seeSupplementalFig.1). humoralimmuneresponse,plasmasampleswerecollected14and
Assta ted abov e,histopathol ogy wasanalyzedinH&Estained 21daysa ndtears 14daysaf tervacc inationw ith3 ×105EID ofa
50
trachealslides4daysafterIBVchallenge.Besidesmucosalthick- live-attenuatedArkDPIIBVvaccinestrainon1,714,21or28days

ness(seesupplementalFig.1),deciliation(seesupplementalFig. ofage.TheIBV-specificIgGendpointtitersinplasma14daysafter

2),gobletcells(seesupplementalFig.3)andlymphocytesscores vaccinationaresignificantlylowerforday1vaccinatedbirdswith
(se esupp lemen talF ig.1)ofthetr ach eal muc osawereeva luated a mean of 7 .8± 1.0 when co mpar ed t o da y 14, 21 and 28 v acci-

blindlyandscored1through5basedonseverity(i.e.,normal,mild, natedbirds,whichmeansvarybetween10.2–11.4.The7-day-old

moderate,marked,severe).Fivechickenswereusedaspositiveand groupdoesnotdiffersignificantlyfromday1orlatervaccinated

negative controls, i.e., one of each age group, and 10–13 chick- bird(Fig.1A).TheIgGIBV-specificplasmalevels21daysaftervacci-

ens were analyzed for each age group. A visual depiction of the nationdemonstrate,thattheday21and28oldvaccinationgroups

Fig.1. TheIBV-specificIgG,IgAandIgMresponseinplasma.EndpointtitersofIBV-specificIgGonday14(A)and21(C)aswellasIgAonday14(B)and21(D)andIgMon

day7postIBVvaccination(squares)andincontrols(circles)(E)weremeasuredbyELISA.Chickenswerevaccinatedatday1,7,14,21or28ofage.Unvaccinatedchickensof

thedifferentagegroupsservedasnegativecontrol.ThedatawasanalyzedbyonewayANOVAwiththeNewman–Keulspost-test.Thecontrolgroupcontains3datapoints

foreachagegroup,whichwerepooledinonegroup(n=15).Allvaccinatedagegroupscontainedbetween10and13chickensforIgGandIgA.Asignificantdifferenceis

observedatP<0.05andisindicatedbydifferentletters.IgMlevelsinIBVvaccinatedbirdsaredepictedbysquares(n=5)andthecontrolsbycircles(n=5,d28groupn=3).

FortheIgMcontrolsthedifferentagegroupsareshownseparatelysincesignificantdifferenceswereobservedbetweenthem.Asignificantincrease(P<0.05)ofIgMlevels

inIBVvaccinatedbirdsovertheircontrolgroupisindicatedbya(*).

Pleasecitethisarticleinpressas:vanGinkelFW,etal.Age-dependentimmuneresponsesandimmuneprotectionafteraviancoronavirus

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stayed the same, while IgG antibody titers in groups vaccinated significantlylowerthanthoseintheday28vaccinationgroupbut

onday1,7and14stillincreased(Fig.1C).Thisshowsthatearly notcomparedtotheotheragegroups.

vaccinationcausesadelayintheIBV-specificIgGantibodykinetics. IntearstheIBV-specificIgGresponseissignificantlyhigherin

TheIBV-specificIgAplasmaantibodytitersarenotsignificantly theday21and28vaccinatedgroupsthanintheday1and7vacci-

different between groups, although the day 1 vaccinated group natedchickensforday14oftheimmuneresponse(Fig.2A).TheIgG

meanantibodytiteristhelowestofallgroups(Fig.1B)andatleast endpointtiterintheday1immunizedgroupisevensignificantly

3-foldlowerthanthenextlowestgroup.UnliketheIgGantibody lowerthantheday7immunizedgroup,whiletheday14immu-

titersonlytheday7vaccinationgroupincreasesinmeanIgAplasma nizedgroupisintermediatebetweentheday7groupandchickens

titeronday21postvaccination,whileday14groupstaysthesame vaccinatedatanolderage.Thus,acorrelationbetweenageofvacci-

andtheday21,28and1groupsdeclineinmeanIgAtiter.Thus,only nationandthemagnitudeoftheIBV-specificIgGresponseintears

theday7groupincreasesantibodytitersonday21whencompared isobservedonday14oftheIBV-specificimmuneresponse.

toolderbirds.Thus,theday7groupdisplaysadelayedresponse Theday14and21groupshavesignificantlyhigherIgAanti-IBV

inantibodyproductioncomparedtoolderbirds.Theday7andday responsesintearsonday14aftervaccinationcomparedtotheday

14groupshaveplasmaIgAtiterstoIBVthatarecomparableto,or 1group.Theday1groupisnotsignificantlydifferentfromtheday

higherthan,theday21and28groupsonday21oftheresponse. 7and28vaccinationgroups.Theday28groupisalsosignificantly

Unliketheday7andday14vaccinationgroupstheday21and28 lowerthantheday14and21groups(Fig.2B).Thisislikelydueto

groupsaredecliningonday21oftheresponsecomparedtothe theday28groupdisplayingfasterkineticsforIgAantibodylevelsin

immuneresponseonday14.Thisindicatesthattheyarepasttheir tearsaftervaccination,ratherthanalowerresponse[20].Theday

peakresponseonday21andpossiblyevenonday14basedonpre- 14groupIgAresponseissignificantlyhigherthantheday1and

viousobservations[20].Thesedataareconsistentwithadelayin day7groupsconsistentwithadelayordeficiencyinthemucosal

theIgAplasmaresponsetoIBVinbirdsvaccinatedatayoungerage antibodyresponsewhenvaccinatedatanearlierage.

andanon-significantdeclineinmeanIgAtitersinthe1-day-old Ourdataindicatethereisadelayinantibodyproductionwhen

group. vaccinated at a younger age. The day 1 vaccination group not

IBV-specificIgMantibodytitersweremeasuredinplasma.The onlydisplaysadelaybutalsolowerlevelsofantibodyproduction.

plasma samples analyzed were collected on day 7 post vaccina- Todeterminewhethertherearenotonlyquantitativedifferences

tion.Thistimepointwasselectedbasedontheliteratureinwhich betweenantibodiesproducedwhenvaccinatedonday1butalso

thepeakIgMresponsewasobservedbetween5–9daysaftervirus qualitativedifferenceswecomparedavidityindicesforIgGandIgA

challengeorlivevirusvaccination[26–28].Duetothevariability antibodiesfromplasmaandtearsgeneratedin1dayoldversusfully

ofIBV-reactiveIgMinthecontrolsbetweendifferentagegroups, matured28dayoldbirds14daysafterIBVvaccination.Asisillus-

independentcontrolswereincludedforeachagegroup.TheIgM tratedinFig.3A,Basignificantlyhigheravidityindexisobserved
levels in the controls decre ased cons ide rably by ∼2 wee k of age forIgG p lasm aant ib odiesforthe day28 vaccin ationg ro upwhen

after(7daysaftertheday7oldchickvaccination)whichincreased comparedtotheday1vaccinatedbirds,whilenosignificantdiffer-

oneweeklaterandstabilizedinolderagecontrolgroups(Fig.1E). enceisobservedforIgAplasmaantibodies.Thesameobservations

The day 14 through day 28 control IgM levels were significantly arealsomadefortearIgGandIgAantibodies(Fig.3C,D).

higherthanintheday1andday7agegroupcontrols.Andtheday CiliatedcellsandgobletcellsaretheprimarytargetofIBVinthe

7controlwassignificantlylowerthantheday1controlIgMlevels. respiratorytract[29].Fig.4displaysthedeciliation(Fig.4A)and

AllIgMtitersfromtheIBVvaccinatedagegroupsweresignificantly gobletcell(Fig.4B)scores4daysafterIBVchallenge.IBVchallenge

higherwhencomparedtotheircontrols(P<0.05)withexception decreasedciliatedepithelialcellsandgobletcellscore.Ciliatedcells

oftheday21agegroups(P=0.08).Thiswasduetohighercontrol werefullyprotectedwhenvaccinatedonday7ofageorlaterbut
va lues ,wh ich we re∼1.6 fol d highe rtha nin thea ge 14or 28days notw hen vaccinated onda y1ofage (Fig .4A ). Th ep rot ection of

oldgroups.Thismayreflectaninitialpeakofnaturalantibodies gobletcellsincreaseswiththeageofvaccinationandwerefully

inducedtoIBVbeforestabilizing.TheIBVvaccinatedagegroupsdid protectedwhenvaccinatedonday21ofageorolder(Fig.4B).

notdiffersignificantlyinIgMantibodylevelstoIBVwithexception Thelymphocytesscoreandmucosalthicknesswerealsomea-
oft heday 1IBVvaccin at edg roup,wh ichme an ±S Ewa s8.7±0.6 suredin trachealsam pleso nda y4postA L/4614/98 IBVfi eld strain

hadsignificantlylowerIBV-specificIgMlevelsinplasmathanthe challengeasindicatorsofinflammation.AsisillustratedinFig.5A,a
day 14(10.2±0.4 )day2 1(10.6±0.7 )and day2 8( 11.2±0 .5)va cci- significan td ecreasein lym phocytescore w as observedi nt hed ay 7
nate db irdsb u tdid not dif fersig n ifica ntly from th eday 7 (9.2 ±0.5) and14vac cinationg ro upswhenco mpar edto theday1 g roup .Th e

vaccinatedbirds(Fig.1E).Theday7IgMantibodytiterswerealso day1vaccinationgroupdidnotsignificantlydifferfromtheday21

Fig.2. TheIBV-specificIgGandIgAresponseintears.EndpointtitersofIBV-specificIgG(A)andIgA(B)onday14oftheimmuneresponseweremeasuredbyELISA.Chickens

werevaccinatedonday1,7,14,21or28ofage.Unvaccinatedchickensofthedifferentagegroupsservedasnegativecontrol.Depictedarethemeansandstandarderror

ofeachagegroupandthecontrolgroup,anddifferentagegroupsareasdescribedinFig.1(n=10–14pergroup).ThedatawasanalyzedbyonewayANOVAwiththe

Newman–Keulspost-test.DifferenceswereconsideredsignificantatP<0.05andareindicatedbydifferentletters.

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Fig.3. AvidityindicesofIBV-specificIgGandIgAantibodiesinplasmaandtearsfromchickensvaccinatedat1and28daysofage.Avidityindicesweredeterminedusing

KSCNinhibitionoftheIBVELISA.DepictedarethemeansandstandarderrorofplasmaIgG(A)andIgA(B)andtearIgG(C)andIgA(D)ofn=4–5observationspergroup.The

datawereanalyzedusingtheStudentt-test.DifferenceswereconsideredsignificantatP<0.05.

Fig.4. TrachealdeciliationandgobletcelldepletionafterIBVchallenge.TomeasurethedegreeofprotectionagainstIBVchallengeaftervaccinationtrachealdeciliationand

gobletcelldepletionweremeasured.Chickenswerevaccinatedatday1,7,14,21or28ofageandchallenged21dayslater.Unvaccinated/unchallengedchickensofallage

groupsservedasnegativecontrolandunvaccinated/IBVchallengechickensaspositivecontrol.Tracheawerecollected4dayspostchallenge.Depictedarethemeanand

onestandarderror.Forthenegativeandpositivecontrolsn=5forthedifferentagegroupsn=10–13.ThedatawereanalyzedbyonewayANOVAwiththeNewman–Keuls

post-test.SignificantdifferencewasobservedatP<0.05andareindicatedbydifferentletters.

and28vaccinationgroupsduetoasmallincreaseinlymphocyte response,isconfirmed.IBVvaccinationonday1ofage,whichis

scoreinthelattertwogroups(Fig.5A).Theday1groupdisplayed routinelyperformedinthepoultryindustry,willnotbefullypro-

thehighestlymphocytesscorefromallvaccinationgroups,which tectiveandasaconsequencethechicksremainvulnerabletoIBV

isconsistentwiththehighestinflammatoryresponsetoIBVchal- exposure.Thus,earlyvaccinationperpetuatestheIBVproblemsand

lenge.Thiswasalsosupportedbyasignificantincreaseinmucosal isafactorintheestimated$48millionormoreannuallosstothe

thicknessintheday1vaccinatedgroupwhencomparedwiththe poultryindustryduetoIBVinfection[30].Ourmeasurementsof

day14,21and28groupsbutnotday7group,whichwasinter- mucosalandsystemicantibodylevelsdemonstratesadelayedpro-

mediatebetweentheday1andgroupsvaccinatedatanolderage ductionofIgGandIgAplasmaantibodiesintheday1,day7and14

(Fig.5B). ofagevaccinationgroups.IgAantibodylevelsintheday1group,

unlikeIgGantibodies,donotrecoverlaterintheresponse.Besides

4. Discussion delayedIgGkinetics,theday1groupdisplaysalsoaloweravid-

ityindexthantheday28vaccinatedgroup.Loweravidityindex

Based on our data, the hypothesis that early IBV vaccination isnotobservedinIgAantibodies.Thedelayedand/orloweranti-

willgenerateanimmature,poorlyprotectiveIBV-specificimmune bodyresponseandlowerIgGavidityindextranslatedinincreased

Pleasecitethisarticleinpressas:vanGinkelFW,etal.Age-dependentimmuneresponsesandimmuneprotectionafteraviancoronavirus

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6 F.W.vanGinkeletal./Vaccinexxx(2015)xxx–xxx

Fig.5. TracheallymphocyteinfiltrationandmucosalthicknessafterIBVchallenge.TomeasurethedegreeofinflammationinducedbyIBVchallengetracheallymphocyte

infiltrationandmucosalthicknessweremeasured.Chickenswerevaccinatedatday1,7,14,21or28ofageandchallenged21dayslater.Unvaccinated/unchallengedchickens

ofallagegroupsservedasnegativecontrolandunvaccinated/IBVchallengedchickensaspositivecontrol.Tracheawerecollected4dayspostchallenge.Depictedarethe

meanandonestandarderrorinpanelAandthemean,95–5%interval(boxes)andthemaximumandminimum(whiskers)inpanelB.Thedatawereanalyzedbyoneway

ANOVAwiththeNewman–Keulspost-test.SignificantdifferencewasobservedatP<0.05andareindicatedbydifferentletters.Forthenegativeandpositivecontrolsn=5

forthedifferentagegroupsn=10–13.

trachealinflammationanddepletionoftrachealepitheliacellsand maturation in 1-day-old chicks. Neither monomeric plasma nor

goblet cells upon IBV challenge when compared to chicks vacci- dimerictears-derivedIgA[37]displaysthisdropinavidityindex

natedlaterinlife.Alackofvaccine-mediatedprotectionismost fortheday1vaccinatedgroup(Fig.3B,D).Althoughthereisalower

noticeable in the 1 day of age vaccination group and to a lesser level of IgA antibodies produced by the day 1 vaccinated birds

extend the day 7 vaccination group, while the day 14 and older compared to the older groups, which is consistent with a delay

vaccinatedchickensareprotected. inclass-switchingintheday1oldgroup,itisnotclearwhythe

TheIgMantibodiesspecificforIBVweresignificantlyelevated lackofamaturemucosalimmunesysteminthe1-day-oldgroup

above controls on days 7 of the IBV immune response in all age [5,6,11–13]didnotresultinloweraffinitymaturationofmucosal

groupsexcepttheday21group.Theday21groupwasnotquite IgAantibodiescomparedtothe4-week-oldgroup.

significant (P=0.08) because of higher IgM levels in the control Another factor influencing early vaccination is the level of

group.ThiscouldbeduetoaninitialsurgeofnaturalIgMantibod- maternal antibodies, an issue not addressed in this study. There

iestoIBVinthisagegroup.Asignificantdeclineinthe7dayold existsalinearrelationshipwiththehens’plasmaantibodylevels

groupisobservedwhencomparingtheIgMantibodylevelstoIBV andtransferofIgYtothechicks’circulation[14].Chickswereover

inthecontrolday1group.Thiswouldbeconsistentwithadropof 95%protectedagainstIBVchallengeonday1iftheyhadhighlevels

presumablynaturalmaternalIBV-specificIgMantibodiesinthese of maternal antibodies but less than 30% protected when chal-

SPFchickensintheday7controlagegroup.TheseIgMantibodies lengedonday7.Thisprotectioncorrelatedwithlocalrespiratory

rapidlyincreasesintheday14groupafterwhichtheystabilizein antibodies and not serum antibodies [38]. IBV-specific maternal

theolderagegroups.Thisindicatesthataconsiderableportionof antibodiesdecreasedtheinductionofneutralizingantibodiesfol-

IgMantibodiesinplasmafromtheoldervaccinationgroupsreacts lowingboosting[38].Despitethisinhibitionbymaternalantibodies

withIBVwithoutseeingthevirus,indicatingthesearenaturalanti- of the memory response, low or erratic maternal antibody titers

bodies to IBV, which only increase after day 14 of age. Bacterial toIBVinbroilerflocksareassociatedwithIBV-inducedeconomic

colonizationoftheintestinaltractofchickensisestablisheddur- losses[39].Thisfurthersupportsthatprotectionbymaternalanti-

ingthefirsttwoweekspost-hatch[31].This,combinedwiththe bodies,whicharepredominantlyoftheIgGisotype,isimportant

observationthatprobioticsenhancenaturalantibodiesinchicken topreventactivationofanimmatureimmunesystemthatisnot

[32],indicatesthatIBV-specificnaturalIgMantibodiestoIBVare capablegeneratingafullyprotectiveimmuneresponseearlyinlife.

possiblygeneratedfollowingintestinalcolonizationpresumablyby InseveralstudiesIBVvaccinationwaseffectiveagainstIBVchal-

stimulatingB1cells,whicharethemainproducersofnaturalIgM lenge in both SPF chickens and commercial broilers when the

antibodiesinseraofmammals[33].IntheIBVvaccinatedgroups initialvaccinationwasperformedonday1[40–42].However,in

weseeasteadilyinclineofIgMIBV-specificantibodiesinplasma thesestudiesday1vaccinationwasfollowedwithasecondvac-

withageashasbeenreportedbyDeWitetal.[3].Onlytheday cinationtwoweekslaterforoptimalprotectionagainstchallenge,

1agegroupdisplayssignificantlylowerIgMantibodylevelswhen whichwouldhavemaskedtherelativepoorIBV-specificimmune

comparedwiththeday14–28agegroupsbutnotwiththeday7 responsesaftertheday1immunization.Extensiveimmunization

agegroup.Thisisconsistentwithanearlyinlifedeficiencyordelay on the day of hatch containing three different live attenuated

intheIgMresponse. viruses,whichcausedseverevaccinesymptoms,providedsimilar

The lower avidity index for the IgG antibodies in 1-day-old protectionastwosingleliveattenuatedIBVvirusvaccinesonday

chicks is an important factor contributing to decreased protec- ofhatchandday14ofagewhenchallengedwithaheterologous

tion to IBV challenge. Increased antibody affinity maturation to virus[42].Thisseemstoindicatethatinductionofcross-protective

virusvaccinesstronglycorrelatedwithbetterprotection[34,35]. immunitymaybelessimpairedwhenvaccinatedearlyinlife.How-

A lack of antibody affinity maturation observed following vacci- ever,nodirectcomparisonofthesamevaccinationprotocolwas

nationagainstrespiratorysyncytialviruswasduetoalackofTLR analyzedbetweenthesetwochallengegroups,whichmakesthis

stimulation[35].Thisindicatesthat1-day-oldbirdsmaybedefi- datahardertointerpretinthecontextofage-dependentimmune

cientinTLRexpression.Evidencethatthisisthecasecomesfroma responses.

recentpublication[36]demonstrating,thatsignificantlylowerlev- A decreased humoral immune response to vaccines early in

elsofTLR7expressioninspleenandsmallintestineswereobserved lifeasseeninchickensisalsoobservedinhumans.Neonatesare

whencomparing1-day-oldchickswith4-to5-week-oldchickens. highly dependent upon passively acquired maternal antibodies,

InclusionofTLRactivatingadjuvantscouldalleviatetheproblems sincetheirhumoralimmunesystemremainsunderdeveloped[43].

ofearlyvaccinationbyboostingantibodyproductionandaffinity Thesepassivelyobtainedantibodiesininfantscanalterhumoral

Pleasecitethisarticleinpressas:vanGinkelFW,etal.Age-dependentimmuneresponsesandimmuneprotectionafteraviancoronavirus

vaccination.Vaccine(2015),http://dx.doi.org/10.1016/j.vaccine.2015.04.026
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ARTICLE IN PRESS
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F.W.vanGinkeletal./Vaccinexxx(2015)xxx–xxx 7

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Pleasecitethisarticleinpressas:vanGinkelFW,etal.Age-dependentimmuneresponsesandimmuneprotectionafteraviancoronavirus

vaccination.Vaccine(2015),http://dx.doi.org/10.1016/j.vaccine.2015.04.026