Immune response to tetanus booster in infants aged 15 months born prematurely with very low birth weight

ContentslistsavailableatSciVerseScienceDirect

Vaccine

j o ur na l ho me p ag 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

Immune

response

to

tetanus

booster

in

infants

aged

15

months

born

prematurely

with

very

low

birth

weight

Maria

Cristina

Abrão

Aued

Perin

_,

_Carolina

_Frank

_Schlindwein

_,

_Maria

_Isabel

_de

_Moraes-Pinto

_,

Raquel

Maria

Simão-Gurge

_,

_Ana

_Flavia

_de

_Mello

_Almada

_Mimica

_,

_Ana

_Lucia

_Goulart

_,

Amélia

Miyashiro

Nunes

dos

Santos

a_{DepartmentofPediatrics,NeonatalDivisionofMedicine,FederalUniversityofSãoPaulo,SãoPaulo,SP,Brazil}

b_{DepartmentofPediatrics,DivisionofPediatricInfectiousDiseases,FederalUniversityofSãoPaulo,SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received29May2012

Receivedinrevisedform21August2012 Accepted23August2012

Available online 6 September 2012

Keywords: Prematureinfant Immunization Tetanustoxoid Humoralimmunity Cellularimmunity

a

b

s

t

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Objectives:Tocomparehumoralandcellularimmuneresponsestotetanusboostervaccinationininfants bornprematurelywiththosebornatfulltermandidentifyfactorsassociatedwiththehumoralresponse. Methods:Aprospectivestudywascarriedoutonchildrenbornprematurelyandwithabirthweight <1500gandwith infantsborn atfullterm.At15months (pre-vaccination)and 18months (post-vaccination), anti-tetanus antibodieswere measuredby ELISA;the intracellularinterferon-gamma percentagesofCD4+TandCD8+Tcellsafterinvitrostimulationwithtetanustoxoidweredeterminedby flowcytometry.Chi-squaredorFisher’sexacttestwasusedtocomparecategoricalvariables.Student’s t-testorMann–Whitneytestwasusedtocomparenumericalvariables.Regressionanalysiswas per-formedtodeterminefactorsassociatedwithhumoralimmunity.Statisticalsignificancewasconsidered ifp<0.05.

Results:Sixty-fourprematureand54full-terminfantswerestudied.Theproportionofchildrenimmune againsttetanusat15and18monthswassimilarinbothgroups.Thegeometricmeanoftheantibodies wasloweramongtheprematurechildrenat15months(p=0.025)andwassimilarinbothgroupsat 18months(p=0.852).ThepercentagesofCD4+andCD8+TcellsexpressingintracellularIFN-␥were

similarinbothgroupsat15and18months.Gestationalage<32weekswasassociatedwithareduction of−0.116IU/mLinthelevelofantibodiesat15months.Breastfeeding>6monthswasassociatedwitha 3.5-foldgreaterchanceofoptimalprotective(≥0.1IU/mL)antibodylevelagainsttetanusat15months andanincreaseof0.956IU/mLinthelevelofantibodiesat18months.

Conclusions:Humoralandcellularresponsefollowingatetanusboosterwassimilarinbothgroups. Pre-matureinfantsexhibitedlowerlevelsofanti-tetanusantibodiesat15monthsofage,withthelowest levelsinthosebornatagestationalageoflessthan32weeks.Breastfeedingwasassociatedwithgreater levelsofantibodyagainsttetanus.

© 2012 Elsevier Ltd.

1. Background

Studiessuggestthatevenpatientsvaccinatedagainsttetanus and with antibody levels considered protective may acquire tetanus,dependingontheimmunestatusofthehostandamountof tetanusneurotoxinproducedbyClostridiumtetani[1,2].Moreover, childrenbornpriortoagestationalageof37weeksmayexhibita lowerimmuneresponsetovaccinationand/orhaveanaccelerated decayofserumantibodies[3–5].

∗_{Correspondingauthorat:RuaDiogodeFaria764,CEP:04037-002,SãoPaulo,} SP,Brazil.Tel.:+551150840535;fax:+551150840535.

E-mailaddress:ameliamiyashiro@yahoo.com.br(A.M.N.dosSantos).

Factorsthatcontributetothesurvivalofprematureinfants,such astheuseof prenatalsteroidsinwomen athighrisk ofgiving prematurebirth[6] andtheuseofpostnatal corticosteroidsfor thetreatmentofbronchopulmonarydysplasia[7],mayalsoaffect theimmuneresponsetovaccinationinchildrenbornprematurely

[5,8].AccordingtoSlacketal.[5],theproductionofanti-tetanus antibodiesinprematureinfantswithagestationalageoflessthan 32 weeksis negatively associatedwiththenumber of dosesof prenatalcorticosteroids.Robinsonetal. [8]foundthat antibody levelsfollowingvaccinationfortetanus,diphtheriaandwhooping coughwerelowerinchildrenwithbronchopulmonarydysplasia treatedwithdexamethasone.Moreover,breastfeeding,less preva-lentamongprematureinfants,andnutritionalstatus,whichmaybe compromisedinthispopulation,arealsoinvolvedintheimmune responsetovaccination[9,10].

0264-410X© 2012 Elsevier Ltd.

http://dx.doi.org/10.1016/j.vaccine.2012.08.056

Open access under the Elsevier OA license.

Itisnotknownwhetherthecompromisedimmuneresponseto vaccinationinprematureinfantsisonlyrelatedtovaccines admin-isteredinthefirstsixmonthsoflife.However,Kirmanietal.[3]

reportedlowerantibodylevelsfollowingvaccinationfor diphthe-ria,tetanustoxoid,poliovirus,Haemophilusinfluenzaetypeband hepatitisBinseven-year-oldchildrenbornatagestationalageof lessthan29weeksandwithabirthweightoflessthan1000gin comparisontochildrenofthesameagebornatfullterm.

Theaimsofthepresentstudyweretocomparethehumoraland cellularimmuneresponsetoatetanusboostervaccineat15months ofagein infantsbornprematurelywiththosebornatfullterm andtoidentifyfactorsassociatedwithhumoralimmuneresponse. Specificallywithregard toimmuneresponse,theconcentration ofanti-tetanusantibodiesandpercentagesofCD4+TandCD8+T cellsexpressingintracellularinterferon-gammaafterinvitro stim-ulationwithtetanustoxoidwerecomparedbeforeandafterthe tetanusboostervaccination.

2. Methods

The present prospective study was carried out between September2007andJanuary2010andreceivedapprovalfromthe EthicsCommitteeoftheinstitution.Allparents/guardiansofthe participantssignedastatementofinformedconsent.

Theinclusioncriteria werechildrenaged 15months,having receivedthreedosesoftetanusvaccine(at2,4and6monthsof age)andnothavingyetreceivedthetetanusboostervaccine. Partic-ipantsweredividedintotwogroups.Theprematuregroupincluded childrenbornwitha gestationalageoflessthan 37weeks and birthweightoflessthan1500g(verylowbirthweightpreterm infants).Theseinfantswereassistedattheneonatalintensivecare unitoftheFederalUniversityofSãoPaulo,SP,Brazil,wherepreterm infantswithbirth weightless than1500gwerefollowed upat themultidisciplinaryprematureoutpatientclinicof the institu-tion. Afterdelivery from theneonatal unit, these infantswere routinelyfollowedupbypediatricians,neurologists, physiother-apists,ophthalmologists,nutritionists,psychologistsanddentists until20yearsofage.

Thecontrolgroupincludedchildrenbornatfullterm,adequate forgestational age,withnoneonatal complications, discharged fromthematernityunitattwotofourdaysoflifeandinfollow upatapediatricoutpatientclinic.

The exclusion criteria were: congenital malformation, chil-drenofHIV-infectedmothers,primaryimmunodeficiency,children whoreceivedplasmaorimmunoglobulintransfusionsfivemonths beforeorthreeweeksaftertheboosterdoseorreceivedthetetanus booster vaccinationprior to being invited toparticipate in the study.

Infantsincludedinthestudywerevaccinatedaccordingtothe Brazilianimmunizationrecommendations.Briefly,theroutine vac-cinescheduleinBrazilis:BCGatbirth;HepatitisBatbirth,1,2and 6monthsofage(the1-monthdose,onlyforchildrenbornwith lessthan2kg);tetanusanddiphtheriatoxoidsandpertussis(DTP) at2,4,6monthsand4–6years;H.influenzaetypeb(Hib)at2, 4and6months;oralpoliovirusat2,4,6monthsand4–6years; rotavirusat2and4months;10-valentpneumococcalconjugate vaccineat3,5,7,15months;meningococcalCconjugatevaccine (MenC)at3,5,and12months;yellowfevervaccineat9months; measles–mumps–rubellavaccineat12monthsand4–6yearsof age.

Maternaldemographic and clinicalcharacteristicsas wellas children’sdatarelatedtotheperiodofhospitalizationinthe neona-talunit and clinicalcomplications in thefirstyear oflife were collected.Gestationalagewasdeterminedeitherbythebest obstet-ricestimateorusingtheNewBallardmethod[11].Theadjustment

ofbirthweighttogestationalagewasperformedusingthecurve proposedbyAlexander etal.[12].Clinical severityscore inthe first12hoflifewasdeterminedusingtheScoreforNeonatalAcute Physiology,PerinatalExtension,VersionII(SNAPPEII)[13]. Nutri-tionalstatusatthetimeofvaccinationwasdeterminedbasedon therecommendationsoftheWorldHealthOrganization[14].

Fourmililitersofbloodwascollectedforthedeterminationof humoralandcellularimmunityagainsttetanustoxoidat15months ofage(priortotheboostervaccinedoseagainsttetanus,diphtheria andwhoopingcough)andat18monthsofage(post-vaccination). Double-antigenenzyme-linkedimmunosorbentassay(ELISA) wasusedtodeterminehumoralimmunity,asdescribedby Kris-tiansen et al. [15]. The resultswere expressedin international unitspermilliliter(IU/mL)by comparisonsofthecurvesofthe plasmasamplestestedandtheinternationalreferencestandard. Concentrations of anti-tetanus antibodies equal to or greater than0.1IU/mLwereconsideredoptimalprotectivelevelsagainst tetanus, concentrations between 0.01 and 0.1IU/mL were con-sideredminimalprotectivelevelsandconcentrationslowerthan 0.01IU/mL,susceptibilitytothedisease[1,16].

Cellularimmune responseagainst tetanustoxoidwas deter-minedbythepercentagesofCD4+TandCD8+Tcellsexpressing intracellular interferon-gamma after in vitro stimulation with tetanustoxoidbyflowcytometry.Aculturewasdoneusingfull blooddilutedto1:10inRPMI1640culturemedium(Gibco,NY, USA)supplementedwithl-glutamine,penicillinandstreptomycin.

Thediluted blood wasthendistributed in polystyrenetubes in volumesof1mL.Followingtheadditionoftheantigen,thetubes weresealedandincubatedat37◦_{Cfor72hinanatmospherewith}

5%CO2.Foralltests, onetubeofblood stimulatedwith

phyto-hemagglutininwasused asthepositive control and anotherof non-stimulatedblood wasusedasthenegativecontrol.Tetanus toxoidwas obtainedfrom Butantã Institute(São Paulo,Brazil). Inthelast4hofincubation,brefeldinAwasaddedata concen-trationof10␮g/mLtoalltubes(Sigma,St.Louis,USA).CD3-APC

andCD8-PerCPconjugatedmonoclonalantibodies(BDBiosciences) wereusedforcell-surfacestaining.Cellswerefixed,washed, resus-pendedwithpermeabilizationbuffer,andincubatedfor10minin thedarkatroomtemperature.IFN␥-FITC-conjugatedmonoclonal

antibodywasthenadded.Finally,thecellswerewashedandkept at+4◦_{Cinthedarkuntildataacquisition.Sampleacquisitionwas}

performedwithFACSCaliburCytometer(BDBiosciences)usingCell Questsoftware(BDBiosciences).Theanalysiswasperformedusing FlowJosoftware(TreeStar,Ashland,USA).Fiftythousandevents wereacquiredinthelymphocytegatebasedontheforward scat-terandsidescatterdotplot.CD3+Tcellswereselectedbasedon thesidescatterprofileandCD3-APCfluorescence.CD8+T lympho-cytesweredefinedasCD3+/CD8+;duetodownregulationofCD4 moleculesduring activation,CD4+ T lymphocytesweredefined asCD3+/CD8−. IntracellularIFN-␥production wasevaluated in

CD3+/CD8+andCD3+/CD8−cells.Thefinalvalueofpositivecellsto eachstimuluswasobtainedbysubtractingthepercentageof pos-itivecellsoftheculturewithoutstimulus(negativecontrol)from thecultureinthepresenceofstimulus.

2.1. Statisticalanalysis

Thenumericalvariableswerecomparedusingeitherthe Stu-dent’s t-test (normal distribution) or the Mann–Whitney test (non-normal distribution). The categorical variables were com-paredusingeitherthechi-squared(2)testorFisher’sexacttest.

wascarriedoutusingtheStatisticalPackageforSocialSciencesfor Windows,version17.0(SPSS,Chicago,IL,USA),withthelevelof significancesetto5%(p<0.05).

3. Results

Amongthe89prematureinfantsin followupatthe prema-tureoutpatientclinicoftheinstitutionduringthestudyperiod, 20fulfilledtheexclusioncriteria.Sixty-nineprematureinfantsand 60full-terminfantsfulfilledtheinclusioncriteria.Amongthese, 5(3.9%)prematureinfantsand6(10.0%) full-terminfantswere excludedbecausetheparentsabandonedthestudyprior tothe blood collection for the immunity analyses. Thus, data on 118 patients(64intheprematuregroupand54inthecontrolgroup) wereanalyzed(Fig.1).

Prematureinfantshadmeangestationalageof29.9±2.2weeks (variation:25.6–34.4weeks),birthweightof1185±216g (vari-ation: 714–1480g), 23 (35.9%) were small for gestational age, and48(75.0%)hadantenatalcorticosteroidsexposure.Duringthe neonatal period, 36 (56.3%), 17 (26.6%), 29 (45.3%), 36 (56.3%), and16(25.0%)hadrespiratorydistresssyndrome,patentductus arteriosus,clinicalsepsis,intraventricularhemorrhage, retinopa-thyofprematurity,respectively.Also,duringtheneonatalperiod, 40(62.5%)neonatesweresubmittedtomechanicalventilationon medianfor6days(variation:1–57days),25(39.1%)wereonneed ofoxygentherapyat28dayoflife,6(9.4%)receivedcorticosteroids duringhospitalizationintheneonatalunit,31(48.4%)receivedat leastoneredbloodcellstransfusion,2(3.1%)receivedplasmaand 4(6.3%)receivedatleastoneplatelettransfusion.

Table1summarizesthedifferencesbetweentheprematureand full-terminfants.

At the beginning of the study, the premature infants had lowerweight(8119±1122gvs.9743±1100g;p<0.001),stature (69.9±3.4cm vs. 75.0±2.8cm, p<0.001) and body mass index (BMI)(16.5±1.5vs.17.3±1.3;p=0.005),incomparisontothe full-terminfants.Fourprematureinfants(6.3%)hadaBMIbelowthe

−2z-scoreand22(34.3%)prematureinfantshadastature/agez -score<−2,whereasallfull-terminfantswerewithinthenormal rangefortheseindices.

Regarding clinical evolution following discharge from the neonatalunit,18(28.1%)prematureinfantsdevelopedpneumonia, 41 (64.1%) exhibited wheezing and 24 (37.5%) required pred-nisolone,5.7±4.5monthsbeforeboosterdoseat15months,at adoseof1mg/kg/dayforfivedays.Moreover,24(37.5%)required hospitalization,withamedianvalueof1(range:1–12) hospitaliza-tionperprematureinfanthospitalized.Onlyonechildinthecontrol groupdevelopedpneumoniaandrequiredhospitalization.

Mother’smilkwasadministeredto37(57.8%)prematureinfants and 48 (88.9%) full-term infants(p<0.001). Breastfeeding con-tinued for more than six months among 9 (14.1%) premature infantsand32(59.3%) full-terminfants(p<0.001)andformore than one yearamong 0 (0%) prematureinfants and 15 (27.8%) full-terminfants(p<0.001).Meandurationofbreastfeedingwas shorteramongtheprematureinfants(3.2±3.7monthsvs.9.1±6.3 months;p<0.001).

Prematureinfantswereolderatthetimeofthefirstdose(mean age,2.4±0.8monthsvs.2.1±0.2months;p=0.002),seconddose (4.6±0.9monthsvs.4.2±0.3months;p=0.001)andthird dose (6.9±1.2 monthsvs. 6.2±0.4 months;p<0.001) ofthetetanus vaccineincomparisontothefull-terminfants.

Thetetanusboosterdosewasadministeredatameanageof 15.2±0.3months.

The percentage of infants with optimal protective humoral immunitywassimilarinbothgroupspriortoandfollowing vacci-nation(Table2).Amonginfantswithminimalhumoralimmunity

fortetanusat15months,agreaterpercentageofthemhadbeen breastfedforlessthansixmonths(37%vs.17%;p=0.026). Geo-metric mean of the anti-tetanus antibody levels was lower in theprematureinfantsat15 months(0.147±0.2 vs.0.205±0.3; p=0.025)andsimilarinbothgroupsat18months(1.997±2.2vs. 1.867±2.5;p=0.852).

Regardingcellularimmunity,thepercentagesofCD4+ Tand CD8+Tcellsexpressingintracellularinterferon-gammawere sim-ilar in both groups at pre-booster and 3 months post-booster (Table3).

Multiple linear regression and multiple logistic regression analyseswere performed todetermine anassociation between demographic/clinical factors and humoral immune response to anti-tetanus vaccination. The following independent variables wereincorporatedintoallregressionmodels:useofatleastone cycleofantenatalcorticosteroids;gestationalage<32weeks;small forgestationalage;clinicalseverityscoreassessedbySNAPPEII; needforerythrocytetransfusions;BMI;andbreastfeedingformore than six months.After controllingfor thesevariables, thefinal linearregressionmodelshowedthathavingbeenbornata ges-tationalageoflessthan32weekswasassociatedwithareduction of−0.116IU/mL(95%CI:−0.219to−0.014;p=0.027)inthelevel of antibodies and breastfeedingfor more than six months was associatedwithanincreaseof0.956IU/mL(95%CI:0.080–1.832; p=0.033)inthelevelofantibodiesafterboosterdose. Likewise, after controlling for the same variables, the logistic regression revealedthatbreastfeedingformorethansixmonthswas asso-ciatedwitha3.455-fold(95%CI:1.271–9.395;p=0.015)greater chanceofhavingoptimalprotectiveantibodylevel(≥0.1IU/mL) againsttetanusat15monthswhencomparedtobreastfeedingfor lessthansixmonths.

4. Discussion

Inthepresentstudy,theproportionofchildrenwithminimal protective (≥0.01–≤0.09IU/mL) antibody levels and potentially susceptibletotetanuswassimilarbetweengroupsat15months ofage.However,meananti-tetanus antibodylevelswerelower amongtheprematureinfantsat15monthsofageincomparison tothefull-terminfants.Thisfindingisimportant,asdelayed vac-cinationismorecommonamonginfantsbornprematurely,when comparedtothegeneralpopulation,whichmayleadsomeofthese childrentobecomemoresusceptibletotetanus[17].

Meanageatadministrationofthefirstthreedosesofthetetanus vaccinewassignificantlygreateramongtheprematureinfantsthan thefull-terminfants,butprobablynotenoughtoconferabetter immuneresponseforprematureinfants[18].Moreover,thefact thatprematureinfantshavelowerlevelsofmaternalantibodies thanfull-terminfantsmaybeanadditionalfactorinvolvedinthe betterhumoralimmune response tovaccination[19,20].In the sameway,Baxteretal.referredthat100%and98.3%of121 pre-matureinfantsbornlessthan 32weeksofgestationdeveloped, respectively,aminimumandoptimumantibodylevelsaftera3 doseprimaryseriesoftetanusvaccine[21].However,despitethese factors,theprematureinfantsanalyzedinthepresentstudyhad lowerlevelsofantibodies.Thisfindingsuggeststheinfluenceof prematurebirthand/orpossiblefactorsassociatedwithalower immuneresponsetovaccinationorafasterdecayinantibody lev-elsresultingfromtheprimaryvaccinationinprematureinfantsin comparisontothosebornatfullterm[22].

89 Pre

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low up at the

Premature Outpa

tient

Clinic

69 in

fants born premature

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fulfil

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64 infants born

premature

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were studied

Full-term

infan

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a Pediatric

Outpa

tient

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20 in

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5 (3.9%)

premature infants

abandoned

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6 (10.0%) infan

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abandoned

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study

60 infants

born at

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Fig.1. Flowdiagramshowingtheflowofthestudiedinfantsbornprematureandatfull-term.

Table1

Demographicandclinicalcharacteristicsofthestudiedinfants,duringtheperinatalperiod,expressedbymean±sdornumberandpercentage.

Premature(n=64) Full-term(n=54) p

Motherage(years) 28 ± 8 26± 6 0.138*

Numberofgestations 2.4±1.5 1.7±1.0 0.001**

Numberofantenatalappointments 5.5±3.6 7.9±1.9 <0.001**

Hypertensivesyndromes 24(37.5%) 0(0%) <0.001***

Diabetesmellitus 5(7.8%) 0(0%) 0.062***

Chorioamnionitis 6(9.4%) 0(0%) 0.031***

Cesareansection 48(75.0%) 31(57.4%) 0.043****

Male 31(48.4%) 28(51.9%) 0.712****

1stminuteApgar 7.1±1.7 8.3±0.6 <0.001*

5thminuteApgar 8.6±0.7 9.2±0.4 <0.001*

Gestationalage(weeks) 29.9±2.2 39.0±1.0 <0.001*

Birthweight(g) 1185±216 3231±269 <0.001*

Daysofhospitalstay 61±33 3±1 <0.001**

* _p:ttest.

** _{p:Mann–Whitneytest.} ***_{p:exactFishertest.}

****_p:2_.

Table2

Humoralimmunityagainsttetanus:geometricmean(variation)ofantibodylevelsbefore(at15months)andafter(at18months)vaccinationagainsttetanus,expressedin IU/mLandnumberandpercentagesofchildren,accordingtotheirimmunitystatus.

n Premature n Full-term p

Pre-vaccination(IU/mL) 63 0.147(0.015–0.969) 54 0.205(0.012–1.080) 0.025*

Post-vaccination(IU/mL) 62 1.997(0.280–9.959) 53 1.867(0.164–12.506) 0.852*

>0.1IU/mLat15mo 63 42(66.7%) 54 41(75.9%) 0.271**

0.01–0.1IU/mLat15mo 63 21(33.3%) 54 13(24.1%) 0.271**

>0.1IU/mLat18mo 62 62(100%) 53 53(100%)

Table3

PercentagesofCD4+TandCD8+Tcellsexpressingintracellularinterferon-gammaafterinvitrostimulationwithtetanustoxoidbefore(at15months)andaftervaccination (at18months).

n Premature n Fullterm p*

CD4+Tcells-15mo 53 2.10±8.89 54 1.01±3.18 0.514

CD8+Tcells-15mo 53 2.66±9.66 54 2.06±6.02 0.254

CD4+Tcells-18mo 54 1.55±6.02 52 0.75±3.36 0.686

CD8+Tcells-18mo 54 1.97±4.78 52 1.18±3.58 0.564

IFN-␥:interferon-gamma;mo:months.Missingdataoccurredduetodifficultyvenousaccessthatledtobloodclottingorasmallbloodvolumewhichallowedonlythe assessmentofhumoralimmuneresponses.Parentsofoneterminfantabandonedthestudyafterthefirstbloodcollectionat15months.

*_{p:Mann–Whitney.}

describedintheliteraturestatingthatimmuneresponsein pre-matureinfantsmaybelowerinthefirstsixmonthsoflifedueto thelowernumberofcirculatingTandBlymphocytesandThelper cellsaswellasthelowerCD4/CD8ratioincomparisontochildren havingbeenbornatfullterm[23].

Inthepresentstudy,theprematuregroupwasrecruitedamong childrenbornprematurelywithabirthweightoflessthan1500g andthecontrolgroupwascomposedofchildrenbornatfullterm, adequateforgestationalage,withnoclinicalcomplicationsinthe neonatalperiodanddischargedfromhospitalbyfourdaysoflife. Moreover,thecontrolgrouphadchildrenwithintheidealweight range and a good breastfeeding index until six months of age, whereas77%oftheprematuregrouphadbeenbornatagestational ageoflessthan32weeks,hadahighrateofhospitalization follow-ingdischargefromtheneonatalunitandalowbreastfeedingindex. Nonetheless,thehumoralresponsetothetetanusboosterwas sim-ilarbetweengroupsandcellimmunitywassimilarbetweengroups at15 and 18months ofage.Thesefindings showthat thetwo groupshadasimilargoodmemoryresponseafteraboosterdose at15monthsafterhavingreceiveda3doseprimaryseriesvaccine againsttetanus.

Vermeulen et al.compared 48 prematureinfantswho were bornat less than31 weeks ofgestational ageaftervaccination at2, 3,and 4monthsof chronologicalagewithan acellularor awhole-celltetravalentdiphtheria–tetanus–pertussis–polio vac-cine.Whileassessingcellularimmuneresponse,theauthorsfound thatmostofthepreterminfantsdevelopedagammainterferon (IFN-␥)responsetotheBordetellapertussisantigens.Thisfinding

suggeststhatmostpreterminfantsareabletomountaspecific cellularimmuneresponse[24].

In the present study, thetime of immune evaluation, three monthsafter thebooster dose, couldbe stated asa limitation. It is possible that theantibody titers and numbers of circulat-ingtetanus-specific Tcells mayhave decayedfrom peaklevels threemonthsaftervaccination.Antibodylevelsfollowingabooster dose usually peak after 15 and 30 days. The antigen-specific IFN-producingcells mostprobablyare foundamongcirculating Peripheralbloodmononuclearcells1–2weeksaftervaccination verytransiently,thereafter,theyrapidlyreachthelymphnodes andthendecaywithtime[24–27].

Withtheincreaseinthesurvivalrateofprematureinfantsat progressivelyyoungergestationalagesandthegrowinguseof ther-apeutic resources,premature infantscurrentlyexhibit different characteristicsfromthoseofpastdecades[28,29]andfactorsother thanprematurityitselfmaybeinvolvedintheimmuneresponse. Thus,apartfromthedirectcomparisonofantibodylevelsbetween groups,linearandlogisticregressionanalyseswereperformedto controlforvariablesthatmayaffecttheresponsetovaccination.It shouldbepointedoutthatthesameindependentvariableswere incorporatedintoallmultiplelinearandlogisticregressionmodels, whichcontributestotheconsistencyofthefindings.

Breastfeedingformorethansixmonthswasassociatedwitha 3.5foldincreaseinthechanceofhavingoptimalprotective anti-bodylevelsagainsttetanusat15monthsofage,anda0.96IU/mL

(95%CI:0.08–1.83)increaseofantibodylevels3monthsafterthe boosterdose.However,giventhesignificantlylowerratesof breast-feedinginprematureinfants,theeffectobservedofbreastfeeding couldbeaconfoundingofotherfactors(e.g.gestationalage,affinity maturation,etc.)thatcouldinfluencetheantibodyresponselevels intheseinfants.However,thiseffecthasalsobeendescribedby Greenbergetal.[30],whofoundhighlevelsofantibodiesamong childrenwhoreceivedaconjugatedvaccineagainstH.influenzae typeb andtetanustoxoidand hadbeenbreastfeduntilat least sixmonthsofage.Jeppesenetal.[31]foundacorrelationbetween breastfeedingandthepopulationofTCD8+cells.Itissuggestedthat breastfeedingcontributestothestructuralandfunctional develop-mentofthethymusandthecontroloftheapoptosisofimmature thymocytes,whichsubsequentlytransformintoCD4+TandCD8+ Tcells[32].

The use of antenatal corticosteroids, nutritional status and erythrocytetransfusions were notassociated withthe humoral responsetothetetanusvaccineat15and18months,whichisin agreementwithfindingsdescribedinpreviousstudies[5,8–10,33]. Inconclusion,thelevelsofanti-tetanusantibodieswerelower amongtheprematureinfantsat15 months(pre-booster)when comparedtochildrenbornatfullterm,althoughthepercentage ofchildrenwithoptimalprotectiveantibodylevelagainsttetanus wassimilarinbothgroups.At18months,theprematureand full-terminfantshadsimilarhumoralandcellularimmuneresponses tothetetanusboostervaccine.Moreover,breastfeedingincreased theoddsofoptimalprotectiveantibodylevel againsttetanusat 15 monthsof ageand raisedlevelsof antibodiesconcentration followingthetetanusboostervaccine.

Acknowledgments

TheauthorsacknowledgeFundac¸ãodeAmparoàPesquisado Estado de São Paulo (FAPESP), Brazil, for research support (# 06/51865-8 and #09/14351-4). The authors also acknowledge Juliana Pires and Mônica Lopes for laboratorialanalysis of the patients included in the study, and Dra. Célia Cristina Pereira BortolletofromHealthSecretaryofSuzanoMunicipalityand pro-fessionals from Unidade Básica de Saúde Pref. Alberto Nunes Martins,Suzano,fortheirsupport.

Support statement: This study was funded by Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil: 06/51865-8and09/14351-4.

Conflictofinterest:Nonetodeclare.

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