Baseline polysaccharide-specific antibodies may not consistently inhibit booster antibody responses in infants to a serogroup C meningococcal protein-polysaccharide conjugate vaccine

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Article

Reference

Baseline polysaccharide-specific antibodies may not consistently inhibit booster antibody responses in infants to a serogroup C

meningococcal protein-polysaccharide conjugate vaccine

BLANCHARD ROHNER, Géraldine, et al .

Abstract

Negative correlations between baseline antibody concentrations and increases in antibody concentrations (after booster doses of vaccines) have been reported previously. Such correlation coefficients are widely reported by statisticians to be subject to mathematical coupling. Negative correlations may be attributable partly or wholly to the combination of mathematical coupling and measurement error (or other short term fluctuations in measurements) and therefore not clinically interpretable. In this study we re-analysed the serum antibody responses from five clinical trials of serogroup C meningococcal conjugate vaccine (MenCV) given to infants for priming followed by boosting with MenCV or a meningococcal A/C polysaccharide vaccine (MenA/C) at 12 months of age. Using Pearson's correlation method to assess the effect of pre-booster MenC-IgG concentration on the relative increase in MenC-IgG concentration post-booster, a significant negative correlation was observed for all the groups, indicating that high pre-booster antibody was associated with a smaller rise in antibody post-booster. We tested two additional statistical [...]

BLANCHARD ROHNER, Géraldine, et al . Baseline polysaccharide-specific antibodies may not consistently inhibit booster antibody responses in infants to a serogroup C meningococcal protein-polysaccharide conjugate vaccine. Vaccine , 2012, vol. 30, no. 28, p. 4153-4159

PMID : 22554466

DOI : 10.1016/j.vaccine.2012.04.063

Available at:

http://archive-ouverte.unige.ch/unige:149071

Disclaimer: layout of this document may differ from the published version.

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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

Baseline polysaccharide-speciﬁc antibodies may not consistently inhibit booster antibody responses in infants to a serogroup C meningococcal

protein–polysaccharide conjugate vaccine

Geraldine Blanchard-Rohner

^a,b,∗

, Hilary Watt

^c

, Dominic F. Kelly

^a

, Ly-Mee Yu

^c

, Matthew D. Snape

^a

, Andrew J. Pollard

^a

aOxfordVaccineGroupandtheNIHROxfordBiomedicalResearchCentre,UniversityofOxford,CentreforClinicalVaccinologyandTropicalMedicine,Oxford,UK

bDepartmentofPaediatrics,Children’sHospitalofGeneva,UniversityHospitalofGeneva,Switzerland

cCentreforStatisticsinMedicine,UniversityofOxford,Oxford,UK

a r t i c l e i n f o

Articlehistory:

Received25October2010

Receivedinrevisedform12April2012 Accepted19April2012

Available online 1 May 2012

Keywords:

MenC

Baselineantibody Responsetobooster

Protein–polysaccharideconjugatevaccine

a b s t r a c t

Negativecorrelationsbetweenbaselineantibodyconcentrationsandincreasesinantibodyconcentra- tions(afterboosterdosesofvaccines)havebeenreportedpreviously.Suchcorrelationcoefficientsare widelyreportedbystatisticianstobesubjecttomathematicalcoupling.Negativecorrelationsmaybe attributablepartlyorwhollytothecombinationofmathematicalcouplingandmeasurementerror(or othershorttermfluctuationsinmeasurements)andthereforenotclinicallyinterpretable.Inthisstudy were-analysedtheserumantibodyresponsesfromfiveclinicaltrialsofserogroupCmeningococcalcon- jugatevaccine(MenCV)giventoinfantsforprimingfollowedbyboostingwithMenCVorameningococcal A/Cpolysaccharidevaccine(MenA/C)at12monthsofage.UsingPearson’scorrelationmethodtoassess theeffectofpre-boosterMenC-IgGconcentrationontherelativeincreaseinMenC-IgGconcentration post-booster,asignificantnegativecorrelationwasobservedforallthegroups,indicatingthathighpre- boosterantibodywasassociatedwithasmallerriseinantibodypost-booster.Wetestedtwoadditional statisticalmethodsthataccountformathematicalcoupling.UsingBlomqvistmethodofadjustmentto assesstheplausibleextentofbias,correlationcoefficientswerestillnegativeprovidingerrorvariance waslow.Theothermethod,amultilevelmodellingspecificationofOldham’smethodappearednottobe appropriate.Incontrast,usingPearson’scorrelationmethodaconsistentnegativecorrelationbetween carrierprotein-specificbaselineantibodyconcentrationandtheincreaseinMenC-specificantibodycon- centrationwasonlyobservedfollowingboosterimmunisationwiththeprotein-polysaccharideconjugate vaccinebutnotfollowingtheMenA/Cplainpolysaccharidevaccine.Thesefindingssuggestthatanalysis oftheinhibitoryeffectofbaselineantibodyontheresponsetoboosterimmunisationischallengingand shouldaccountforthepossibilityofmathematicalcouplingandmeasurementerror.Thataninhibitory effectofbaselineantibodycannotbeassumedaprioriissupportedbyobservationsinanimalmodels, whichshowthatbaselineantibodycanbothsuppressorenhancetheantibodyresponsetoaspecific antigen.

Abbreviations: CRM197,amutantofdiphtheriatoxoidusedascarrierprotein indiverseproteinpolysaccharideconjugatevaccines;GMC,geometricmeancon- centration;Hib,Haemophilusinﬂuenzaetypeb;MenC,meningococcalserogroupC polysaccharide;MenA/C,meningococcalAandCpolysaccharidevaccine;MenCV, serogroup C meningococcal conjugate vaccine; MMR, measles, mumps and rubellavaccine;9vPnC-MenCV,combined9-valentpneumococcal/meningococcal serogroupCconjugatevaccine;DT,diphtheriatoxoid;SD,standarddeviation;TT, tetanustoxoid.

∗Correspondingauthorat:CentreforClinicalVaccinologyandTropicalMedicine, ChurchillHospital,Oxford,OX37LJ,UK.Tel.:+441865857444;

fax:+441865857420.

E-mailaddress:geraldine.blanchardrohner@paediatrics.ox.ac.uk (G.Blanchard-Rohner).

1. Introduction

Vaccinesarefrequentlyadministeredtoindividualswithpre- existingserumantibodyspeciﬁcforthevaccineantigen.Thisoccurs becauseofeither(i)presenceoftransplacentallyacquiredmater- nal antibodyin infants, (ii)the existence of naturallyacquired antibodyfollowingexposuretotheorganismfromwhichthevac- cineisderived (orrelatedorganisms)throughcarriage,(iii) the administration of prior doses of vaccine, or (iv) in the case of polysaccharideconjugatevaccines,thepriorand/orconcomitant administrationofvaccinescontainingtheproteincarrier(i.e.rou- tine diphtheriatoxoid,tetanustoxoidandpertussisvaccinesin, forexample,Hib-TT,MenC-TT/-CRM₁₉₇andPnC-TT/-DT/-CRM₁₉₇ 0264-410X/$–seefrontmatter© 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.vaccine.2012.04.063

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4154 G.Blanchard-Rohneretal./Vaccine30 (2012) 4153–4159

antigens)thatisalsopresentinthevaccine.Variousstudiesinani- malshavereportedthatthepresenceofantibodyatbaselinecan eithersuppressorenhancetheantibodyresponsestothespecific antigentowhichtheyaretargeted[1–3].Possiblemechanismsfor enhancementofantibodyresponsebybaselineantibodyinclude (1)thecaptureofsmallquantitiesofantigenbypre-existingIgG (especiallysubclassesIgG1,IgG2aandIgG2b)whichthentargets theantigentoantigen-presenting-cells,leadingtomoreefficient activationofTcells,asIgG–antigenimmunecomplexesaretaken upmore efficiently than antigenalone via Fc␥R+expressedon antigen-presenting-cells;(2)IgM and IgG3enhancement ofthe antibodyresponsesthroughtheactivationofcomplement,which willthenlowerthethresholdforB-cellactivation[3].Mechanisms proposedforsuppressionofantibodyresponsesmaybe(1)forma- tionofIgG/antigenimmunecomplexeswhichmaybeeliminated byphagocyticcellsbeforetheycanactivatespecificBcells;(2)epi- topemaskingbyIgGwhichmaypreventB-cellsfromrecognising andrespondingtotheantigen[1–3].Theeffectofbaselineanti- bodyontheactivationofBcellsisnotwellunderstoodandyetis likelytohaveimportantimplicationsforthedesignofschedules forre-immunisation.

Whilsttheinhibitoryeffectofmaternalantibodyiswelldoc- umented[4,5]thesituation isless certainfor individuals given boosterdoses of vaccines following immunisation at an earlier time-point,becauseofconcernswithstatisticalmethodsandtheir interpretationinpublishedstudies.Manystudiesinhumanshave usedcorrelationorlinearregressiontoinvestigatehowtheabso- lute or relative increase in antibody concentration following a boosterdoseofvaccinerelatestopre-boosterantibodyconcentration.Thesestudieshavereportedanegativecorrelationbetween baselineantibodyconcentrationandtheincreaseinantibodycon- centrationafteraboosterdoseofvaccine[6–9].Otherstudieshave investigatedtheratiobetweenpre-andpost-boosterantibodyconcentration (foldincrease) in individuals withdifferent baseline antibodyconcentrations(usinganarbitrarycut-off)andcommonly reportedhigherfoldincreasesinthegroupswithlowerpre-booster antibodytitre[6,7,10].

However,theuseofsimplecorrelationorlinearregressionto assesstherelationshipbetweenbaseline valuesand changes in valuehasbeencriticisedbystatisticiansformanyyears,sinceitis pronetobias[11,12].Theissueofconcernismathematicalcoupling betweenthetwovariables,i.e.thefactthatthechangescore(which iscorrelatedwiththebaselinevalue)isderivedfromasimpleequa- tionwhichincludesthebaselinevalueitself(changeiscalculated aspostvalueminusbaselinevalue).Thisisanimportantissue,for example,ifmeasurementerrorover-estimatestheobservedvalue ofpre-boosterantibodyconcentration(i.e.thereading ishigher thanitstruevalue),thenthereportedchangeinantibodyconcen- trationwillbelowerthanitstruevalue(sincethehighervalueof pre-boosterantibodyissubtractedincalculatingtheincreasein antibodyconcentration)[12].Ifmeasurementerrordecreasesthe observedvalueofpre-boosterantibodyconcentration,thentheval- uesofthepre-boosterantibodyconcentrationwillbelowerthanit shouldbeandthevaluesofthechangeinantibodyconcentration willbehigher.Thus,anegativecorrelationisaconsistentresult arisingsolelyfromthecombinationofmathematicalcouplingand measurementerrors[13].Measurementerrorhereisdeemedto includeshortterm ﬂuctuationsin truevalues oftheantibodies beingmeasured,andanyrandomerrorsinherentinhandlingand storageofserumsamples,aswellastruemeasurementerrorofthe antibodylevels.Systematicerrorsattributabletostorageorhan- dlingmaynotaffectsresults.Forinstance,aconstantpercentage deteriorationinantibodycountsisequivalenttoaconstantvalue beingsubtractedfromallvaluesonthelogarithmicscale,sowill notaffectreportedassociationsbetweenbaselineandchangeson thelogscale(whichweareusinginthispaper).Themathematical

issuesapplysimilarlytocomparingthestrengthsofassociationsin sub-setswithdifferentdegreesofprotectionatbaseline.

Inresponsetotheseissues,statisticianshavedevelopedalterna- tivemethods[11–13,21],toassesstherelationshipbetweenchange andbaselinevalues,namelyOldham’smethodandBlomqvist’sfor- mula,whichhavedifferentmerits.Blomqvist’sformulaisbasedon knowledgeofwithinpersonﬂuctuationsinmeasurements(“error variance”).Thismaybeobtainedfromaseparatedataset,con- tainingrepeatedmeasurementsofantibodyconcentrationstaken overa shortperiod oftime. Intheabsenceofsuchdata,sensi- tivity analysesmaybeundertaken todeterminetheeffectof a plausiblerangeofestimatesofwithinpersonerrorvariances[11].

Oldham’smethodcorrelatesthechangebetweenpre-andpost- treatmentvalueswiththemeanofthepre-andpost-treatment values.Thismethodissaidtoreducetheeffectofmathematical couplingandbelesspronetobias.Undertherelativelyplausible assumptionsthatthemeasurementerror/shorttermﬂuctuations termsofpre-andpost-treatmentvaluesareindependentofeach other,and thatboth havethesame variance,then thismethod reducestheproblemsarisingfrommathematicalcoupling.These assumptionsareplausibleincomparisontotheassumptionofno measurementerror/shorttermﬂuctuationsrequired forvalidity of thestandard correlationor linearregression methods. How- ever,Oldham’smethodislikelytobesomewhatbiasedtowards apositiveassociationbetweenthechangeandmean(ofmedium termaveragesofbaselineand offollow-upconcentrations)values. Sinceboth changeand meaninvolveadditionof follow-up values,anymeasurementerrorinthesevaluesinducesapositive bias.

Oldham’stechniquecanbeextendedtoallowconsiderationof additionalcovariatesorconfounders(e.g.,ageandgenderofstudy participants,variation inthe doseofvaccine antigens,studyin meta-analyses), ortoallowindividuals tobemeasuredvarying numbersoftimes.Thisrequiresuseofmulti-levelmodelling(MLM) techniques [12]. With correct model speciﬁcation and without additionalcovariates,theresultsareequivalenttothoseobtained fromOldham’smethod.

Inthepresentstudy,we usedstandardcorrelationmethods, BlomqvistadjustmentmethodandanMLMversionofOldham’s methodtoassesstheeffectofpre-boosterMenC-IgGconcentra- tionontherelativeincreaseinMenC-IgGconcentrationfollowing booster immunisation in ﬁve studieswe previously conducted.

Since estimates of within person ﬂuctuations in measurement (“error variance”) are not available, Blomqvist adjustment was basedonarangeofplausibleestimatesforthesevalues.

2. Materialsandmethods

Datawereobtainedfromﬁvestudiespreviouslyconductedin Oxford,UK[10,14–20].Inthesestudieschildrenwereprimedat2, 3,4monthsofagewitheitherserogroupCmeningococcalconju- gatevaccine(MenCV),oracombinedHaemophilusinﬂuenzaetype bandserogroupCNeisseriameningitidistetanustoxoidconjugate vaccine(Hib-MenC-TT), ora combined 9-valentpneumococcal- groupCmeningococcalconjugatevaccine(9vPnc-MenCV).They wereboostedat12monthsofagewitheitherMenCVormeningo- coccalA/Cpolysaccharidevaccine(MenA/C),or Hib-MenC-TT.A totalof6differentvaccinescheduleswereusedacrossthe5stud- ies(seeTable1)andthechildrenwereseparatedinto6groups accordingtotheirvaccineschedule.EachdoseofMenCVcontained 10␮gofN.meningitidisgroupColigosaccharide(MenC),however, theycontainedbetween12.5and25mcgofCRM197 [10,14–20].

TheyweremanufacturedeitherbyNovartisVaccinesandDiagnos- tics(studies1and3)[10MacLennan,2000#36,16],orbyWyeth Vaccines(studies2,4and5)[14,17,19].

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Table1

Summarydescribingthestudiesfromwhichthedataweretakenforthepresentanalysis.

Studyno Studyname Groupofvaccineschedule Primingat2,3,4months Boostingat12months References

1 NovartisM14P5E1 1 MenCV MenCV [10]

2 WyethCombostudy 1 MenCV MenCV [14,15]

2 MenCV MenA/C

5 Pnc9-MenC MenCV

6 Pnc9-MenC MenA/C

3 NovartisMenCstudy 1 MenCV MenCV [16]

2 MenCV MenA/C

7^a HepB MenCV

8^a HepB MenA/C

4 WyethMenCstudy 1 MenCV MenCV [17,18]

2 MenCV MenA/C

7^a HepB MenCV

8^a HepB MenA/C

5 GSKHib-MenCstudy 4 Hib-MenC Hib-MenC [19,20]

3 MenCV Hib-MenC

aVaccineschedulenotusedinthepresentstudy.

EachdoseofMenA/Ccontained50␮gofMenC,however,only 1/5thoftheMenA/Cwasadministeredinstudies2and4[14,17], andthefulldoseofMenA/Cwasusedinstudy3[16].

ThecombinedHib-MenC-TTvaccinecontained5␮gofMenC, the combined Pnc9-MenC contained 10␮g of MenC. All the protein–polysaccharideconjugatevaccinesusedineachofthe6 studieshad CRM₁₉₇ (a mutantof diphtheriatoxoid)as thecar- rierprotein,exceptthecombinedHib-MenC-TT,whichusedthe tetanustoxoidascarrierprotein.ThePnC9-MenCvaccinecontained 38.5mcgofCRM197[14].TheHib-MenC-TTcontained12.5mcgof tetanustoxoid[19].

TheconcentrationofIgGspecifictoMenCwasavailablebefore theboosterat12monthsofageandaftertheboosterat13months ofageforthechildrenparticipatinginallthestudies.Theconcen- trationsofIgGspecifictodiphtheriatoxoid(DT)beforethebooster wereavailableforallthestudiesexceptstudy4,andtheconcen- trationsoftetanustoxoid(TT)beforetheboosterwereavailablefor allthestudiesexceptstudies2and4.Foranyparticularstudy,the concentrationofeitherMenC,diphtheriaortetanus-specificIgG wasmeasuredinoneoffourlaboratories(OxfordVaccineGroup, UK;Novartis Vaccines(ex-ChironVaccines), Emeryville,Califor- nia;Wyeth,Rochester,NY;GlaxoSmithklineBiologicals,Belgium).

Theresultswerepooledandanalysedtoassesstheeffectofpre- boosterMenC-specificIgGconcentrationontherelativeincrease inMenC-IgGconcentrationafterthebooster.Theanalysiswasper- formedfirstusingthePearsons’scorrelationmethod,thenusing Blomqvist’s adjustment method to assess the plausible extend of bias to the standard correlation method, and finally using theMLMspecification ofOldham’s method[21]for eachgroup across 5 studies receiving 1 of 6 different vaccine schedules (seeTables1and2).

2.1. Blomqvistmethod

Foreachgroup,andindeedforeachstudywithingroups1and 2,Blomqvistadjustmentwasused.Thisadjuststheregressioncoef- ficient ofchanges onbaseline score (theunadjusted values are obtaineddirectlyfromourdatasets).Theadjustedregressioncoef- ficientsareconvertedtocorrelationcoefficientsusingthestandard formula;correlationofbaselineandchanges=(regressioncoeffi- cientofchangesonbaseline)SD(baseline)/SD (changes),where SD=standarddeviation.Thebaselineerrorvariancesusedwereas follows:theerrorvariancewasassumedtobe10%,and50%ofthe totalbaselinevarianceinthespecificgroupandstudyofinterest.

Theerrorvarianceatfollow-upwasassumedtobeﬁrstlythesame asthatatbaseline,thenhalfthatatbaseline,andthenjust10%

ofthatatbaseline.Errorvariance inthechangescoresiscalcu- latedasthesumoferrorvariancesatbaselineandatfollow-up(by independenceoftheseerrorterms).Itisplausiblethattheerror varianceatfollow-upwillbesmallerthanatbaseline,sincethe meanvaluesaresubstantiallyhigher,soitmaybelesslikelythat theywillbeoutbyafactoroftwo(oranyotherchosenfactor), anditisthesefactordifferencesthatarerelevantwhenworking, ashere,onthelogarithmicscale.Forgroupswithmorethanone study,apooled correlationcoefﬁcientwasderived bymeansof meta-analysis(inversevariancemethodonFishertransformation ofcorrelationcoefﬁcients).

2.2. MLMversionofOldham’stechnique

For each group a MLM model was fitted using multilevel software(MLwiNversion2.0.01;http://www.cmm.bristol.ac.uk), where theoutcome ofthemodel wasthelogof theMenC-IgG concentration.Pre-andpost-treatment measureswerefittedas repeatedmeasureswithinsubject.Forgroupswithmorethanone study,a3-levelmodelwasfittedwithstudyatlevel3,subjects atlevel2,andtime(i.e.pre-andpost-boostercodedas-1and+1 respectively)atlevel1.A2-levelmodelwasusedforgroupswith onestudy,withsubjectsatlevel2andtime atlevel 1.Thecor- relationcoefficientwasderivedfromthecovariancebetweenthe randomslopeandrandominterceptofthemodel[21].Thisgives thecorrelationbetweenmean(ofpre-andpost-booster)valuesand changes.

Furthermore, in order to assess the role of carrier-specific antibodies onthe MenC-antibodies response tobooster immunisation, we assessed the correlations between the change in MenC-IgG concentration and the pre-booster diphtheria toxoid/tetanus toxoid-IgG concentration (depending of the carrier proteincontainedinthevaccineusedforbooster)usingPearson’s correlationmethod,for thestudieswhere pre-boosterdiphthe- riatoxoid/tetanustoxoidIgG concentrationswereavailable.For groups with more than one study,a pooled correlation coeffi- cient wasderived bymeansof meta-analysis (inverse variance methodonFishertransformationofcorrelationcoefficients).Note thatthereisnomathematicalcouplingwhenlookingatcorrelation betweenbaselineprotein-carrierspecific-IgGconcentrationswith changesinMenC-IgGconcentrations,asthebaselinevariableisnot thesameasthevariableusedforthe“change”.

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Table2 Analysisoftheeffectofpre-boosterMenCspecificIgGconcentration,andtherelativeincreaseinMenCspecificIgGconcentrationafterthebooster,usingunadjustedPearson’scorrelationcoefficients,theBlomqvistadjustment onthesecorrelations,andthenusingtheMLMtechnique. GroupPriming2,3,4months Boosting12monthsPre-boostMenC-IgG GMC(␮g/ml) (95%CI) Post-boost MenC-IgGGMC (␮g/ml) (95%CI) Men-CIgG GMR (95%CI) UnadjustedPearson’s correlation(preand change) (95%CI) Estimatedtruecorrelation(preandchange),usingBlomqvistmethodMLMcorrelation betweenmean(ofpre andpost)andchange (95%CI)10%errorvar, sameatf/u50%error var,same atf/u

10%errorvar, halfatf/u50%error var,halfat f/u

10%errorvar, 10%thatatf/u50%error var,10% thatatf/u 1(n=202)MenCVMenCV1.81(1.59,2.06)36(32,40)20(17,23)−0.58*(−0.48,−0.67)−0.57NA−0.55NA−0.53−0.08−0.15*(−0.29,−0.02) 2(n=130)MenCVMenA/C1.87(1.61,2.17)11(8.5,13)5.7(4.5,7.3)−0.5*(−0.36,−0.62)−0.51NA−0.48NA−0.46NA0.21*(0.04,0.39) 3(n=41)MenCVHib-MenC-TT1.11(0.8,1.55)3.7(2.9,4.7)3.3(2.6,4.3)−0.68*(−0.47,−0.82)−0.71NA−0.67NA−0.64−0.2−0.35*(−0.67,−0.03) 4(n=134)Hib-MenC-TT Hib-MenC-TT0.73(0.62,0.85)10(8.5,12)14(11,16)−0.58*(−0.46,−0.68)−0.56−0.42−0.55−0.3−0.54−0.25−0.02(−0.19,0.15) 5(n=44)9vPnc-MenCVMenCV0.51(0.41,0.63)6.5(4.6,9.2)13(8.9,19)−0.44*(−0.17,−0.65)−0.42−0.28−0.42−0.26−0.41−0.250.45*(0.13,0.76) 6(n=44)9VPnc- MenCVMenA/C0.4(0.32,0.49)2.2(1.7,2.9)5.6(4.4,7.3)−0.48*(−0.21,−0.68)−0.45−0.16−0.44−0.13−0.43−0.110.09(−0.19,0.38) ErrorvariancesforBlomqvistadjustmentaretakentobe10%and50%ofthetotalbaselinevariance,andatfollow-uparetakentoequaltheerrorvarianceatbaseline,ortobehalfofit,ortobe10%ofbaselineerrorvariance.NA, notavailable,missingvaluesforBlomqvistadjustedcorrelationsarearesultofthefollow-upvariancebeingtoosmalltosupportsuchlargeerrorvariancesasassumed. *Correlationthatarestatisticallysigniﬁcant.

AnalysiswascarriedoutusingStata(version10,StataCorp,USA) andMLwiN(version2.0.01,UniversityofBristol,UK).Dataofall analyseswerelogtransformedpriortoanalysestomeetthenormal distributionassumption.

3. Results

MenC-IgG antibody data were available from 646 children, whohadbeenimmunisedaccordingto1of6differentschedules (Table 1). The children were primed witheither MenCV, com- binedHib-MenC-TT,orcombined9vPnc-MenCV.Boostingwaswith either MenCV, MenA/C,or Hib-MenC-TT. Allgroups had a pre- booster MenC-geometric mean concentration(GMC) below the postulatedcorrelateofprotectionof2␮g/ml[22,23].However,the GMCofMenC-antibodyofgroups1,2,and3primedwithamono- valentMenCVwas≥1␮g/ml,incontrasttotheothergroupsprimed withacombinedvaccinecontainingtheMenCV.Followingbooster immunisation,allgroupshadasigniﬁcantincreaseinMenC-IgG concentration,andallthegroupshadaMenC-GMCabove2␮g/ml (Table2).

Using Pearson’s correlation method to assess the effect of pre-boosterMenC-IgGconcentrationontherelative increase in MenC-IgGconcentrationpost-booster,asignificantnegativecor- relation was observed for all the groups (Table 2). As already discussed,measurementerrorandshorttermfluctuationsinpre- boosterlevelscouldcause,orcontributetosuchassociations.The Blomqvistadjustmentshowshowmuchofaninfluencethisbias mayplausiblyhave,byshowingestimatedcorrelationcoefficients betweenunderlyingtruevaluesatbaselineandchanges(i.e.taken frombaselineandfollow-upvaluesaveragedovershorttermfluc- tuationswithoutmeasurementserror,Table2).Blomqvistadjusted correlationcoefficientsremainnegativeinmostcasesexceptwhen theerrorvarianceishigh.Forexampleforgroup6,primedwith 9vPnc-MenCVandboostedwithMenA/C,wheretheadjustedcor- relationis−0.16,when50%ofthebaselinevarianceisattributable toerror,evenassumingthesameerrorvarianceatfollow-up.

UsingtheMLMtechniquetoassesstherelationshipbetween mean(ofpre-andpost-booster)MenC-IgGconcentrationandthe relative increase in MenC-IgG concentration post-booster, con- trastingresultswereobservedwitheitherapositive,negativeor nocorrelation(Table2).

Toassesstherelationshipbetweencarrier-proteinspecificanti- bodyconcentrationsandtheincrease inMenC-specificantibody concentrationsPearsons’scorrelationcoefficientwasused.Note thatinthiscasetheuseofPearsons’scorrelationisvalidsincethe variablesconcernedarenotmathematicallycoupled.Anegative correlationwasobservedwhena protein–polysaccharideconjugatevaccine(MenCVorHib-MenC-TT)wasusedforbooster.This wassignificantfor3outofthe4suchgroups(Table3).Incontrast, therewasnocorrelationwhentheplain-polysaccharideMenA/C vaccinewasusedasbooster(Table2).

4. Discussion

Theeffectofpre-existingantigen-speciﬁcserumantibodyon responsetosubsequentdosesofvaccineisnotwellunderstood.

Studying this phenomenon in humans, outside the settings of transplacentalantibodytransferandclinicalsituationswhereanti- bodiesareadministeredpassively(e.g.hepatitisBimmunoglobulin inindividualsexposedtoaninfectioussource),islimitedtoobser- vational studies usingcorrelation or regression analysis.In the currentstudyusingstandardcorrelationmethodstheincreasein MenC-antibodyconcentrationwasconsistentlynegativelycorre- latedwithpre-boosterMenC-antibodyconcentrationaspreviously described [6–9]. Negative correlations, in other disease areas,

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Table3

CorrelationbetweenthechangeinMenC-IgGconcentrationandthepre-boostercarrierprotein-IgGconcentrationusingPearson’scorrelationmethod.

GroupPrimingat2,3,4months Boostingat12months Studyno. No.ofparti-cipants Correlationbetweenpre-boost carrierprotein^aIgGandchangein MenCIgGonboosting(95%CI)

p-Value

1 MenCV MenCV 1,2,3 110 −0.43(−0.58,−0.26) <0.0001

2 MenCV MenA/C 2,3 97 0.06(−0.14,0.26) 0.57

3 MenCV Hib-MenC-TT 5 41 −0.21(−0.49,0.1) 0.19

4 Hib-MenC-TT Hib-MenC-TT 5 132 −0.18(−0.34,0) 0.04

5 9vPnC-MenCV MenCV 2 35 −0.47(−0.7,−0.17) 0.004

6 9vPnC-MenCV MenA/C 2 38 −0.04(−0.36,0.28) 0.8

aDiphtheriatoxoid-IgGwasusedforallthegroupsexceptgroups3and4wheretetanustoxoidwasusedbecausetheHib-MenC-TTcontainstetanustoxoidascarrier protein.

betweenbaselineandchangeshavebeenshowntoresultfromthe combinedeffectofmathematicalcouplingandmeasurementerror [24,25],andsocannotbeinterpretableinaclinicallymeaningful way.UseofBlomqvistadjustment,onarangeofplausibleerror variances,suggeststhatthetrueunderlyingcorrelationbetween baselineandchangesremainsnegativeinthecasesofsmallerror variance,asitcanbeexpectedassumingthatthereisasmallcoef- ficientofvariationintheELISAassayused[26,27]andassuming thatthereislittledaytodayfluctuationinMenC-IgGconcentra- tions[10].In contrast,usingtheMLMtechnique,toaccountfor themathematicalcouplingofthepre-andpost-boosterantibody concentration,aconsistentnegativecorrelationwasnotobserved betweenthemeanMenC-specificantibodyconcentrationandthe MenC-specificantibodyincreasepost-booster.However,theuseof theMLMmethodappearsnottobeappropriateasitmayintroduce apositivebias,sincethefollow-upvaluesareaddedincalculating bothbaselineandchanges,soanymeasurementerrorinfollow-up terminducesapositivebias.Furthermore,itdoesnotdirectlymea- surethecorrelationbetweenbaselineandchange(ratherituses meanandchange).Thesedatasuggestthattheinhibitoryeffect ofbaselineantibodyontheimmuneresponsetoboostermaybe accurateassumingthatthereisalowerrorvariance(duetosmall measurementerrorduetotheELISAassayand littledaytoday fluctuationsinmeasurements)[10,26,27].

TheuseofOldham’smethodassociatedwithMLMtoaccount formathematicalcouplingbetweenbaselinevaluesandchangesis supportedbyTuetal.fromaseriesofpublicationsmostlyonden- talliterature.However,thismethodhasalsobeencriticisedasitis basedonseveralassumptions(suchassameerrorvarianceatbase- lineandatfollow-up)whichcannotbeveriﬁed.Furthermore,this methoddoesnotdirectlymeasurethecorrelationbetweenbase- lineandchanges,whereasthatisthecorrelationwhichisclinically relevant(ratherthantheOldham’scorrelationbetweenmeanand changes).OtherssupporttheuseofBlomqvist[11],althoughaddi- tionaldata(repeatedmeasurementstakenshortintervalsapart)are neededtoapplythistechniquedirectly,whichisnotreallyfeasi- blewhendoingvaccinetrialsinyoungchildrenwithlimitedblood draws.However,we haveusedthemethodsuggestedbyHayes etal.(1988)amongothers,whichiseffectivelyundertakingasen- sitivityanalysis,toestimateerrorvariance,accordingtoarangeof plausibleestimates.Asdiscussedearlier,onlyalowerrorvariance islikelytooccurinthestudiesreportedinthepresentmanuscript.

Ourstudyislimitedbytheheterogeneityofthedifferentstud- iesused.Indeed,sinceavarietyofotherfactorsvariedamongstthe infantsstudied[e.g.thedoseofMenCusedinprimingandboosting, theroleofcombinationversusmonovalentvaccineinprimingor boosting,thegenerallevelofpre-boostantibody(highervslower)]

itwasnotpossibletoinferwhichoneswereindependentlyimpor- tantduetothenumberofgroupsavailableforstudyinrelationto thenumberofpossiblevariables.Also,theantigen-speciﬁcIgGcon- centrationsweremeasuredindifferentlaboratories,andalthough laboratoriesderivedtheiranti-polysaccharideIgGprotocolsfrom twopublications[26,28],theassayspooled,analysedandcompared inthisstudyhavenotbeencomparedtoeachotherdirectly,which shouldbeacknowledgedasa limitationofthis study.However, themethodusedaccountsforthisheterogeneity(causedbyassay variationsorinfantselection)betweenthedifferentstudiesused.

There are a number of factors that might explain why pre-immunisation antibody concentrations may have a weaker inhibitory effect on vaccine immunogenicity than previously reported.First, thestatistical methodsused inprevious studies didnotaccountfortheproblemsofcouplingwhichareinherent in studieswherea variableis measuredinthesame individual pre-andpost-intervention(asdiscussedabove).Second,antibody mediatedinhibitionmaynotbeasignificantimmunologicalphe- nomenoninthecontextofboosterimmunisationofinfantsorat leastitmayonlybesignificantathigherconcentrationsofpre- immunisationantibodythantheonesobservedintheinfantsof thepresentstudy.Inseveralreviewsofanimaldata,ithasbeen reportedthattheantibody-feedbackregulationonBcellresponses toimmunisationcouldbeeitherpositiveornegative[1–3].Finally, analternativepossibilityisthatevenifantibodymediatedinhibi- tionoccurs,thepre-immunisationantibodycorrelateswithsome otherimmunologicalparameter(e.g.immunologicalmemory)that ismoresignificantinenhancingtheimmuneresponse thanthe antibodyisatdirectlyinhibitingit.Itisthoughtthatserumanti- bodyismaintainedbythedifferentiationofmemoryBcellsinto plasmacells.Iftheantibodylevelsatbaselinereflectthesizeofthe memoryBcellpoolavailable,thenonemightexpectgreatersec- ondaryantibodyresponsestobeassociatedwithhigherlevelsof pre-existingantibody.

Astrikingfindingof thecurrentstudywasaconsistentneg- ative correlation between the carrier protein-specific baseline antibodyconcentrationandtheincreaseinMenC-specificantibody concentrationfollowingboosterimmunisationwiththeprotein- polysaccharideconjugatevaccine,whichwassignificantin3out of the4 groups tested. Note that theonly group (group 3)for whichthecorrelationwasnotsignificantwasagroupwitharela- tivelysmallsamplesize.Incontrast,therewasnocorrelationwhen theplain-polysaccharideMenA/Cvaccinewasusedasbooster.For variableswhichaprioriareunrelatedstandardcorrelationmeth- odscanbeused,sincethereisnomathematicalcouplingpresent.

“Carrierinducedepitopesuppression”describestheeffectofpre- existing immunitytoa carrier-protein, onresponse toa linked

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haptenicantigen(inthiscasethepolysaccharide)[14,29].Avariety ofmechanismshavebeendescribedinvolvingbothcarrier-protein specificBandT-cellsinadditiontopre-existingserumantibody [30]. Preformed Bcells and T cells specific for the carrierpro- teinscouldcompeteforantigencapturewithBcellsspecificfor thepolysaccharide,thusinhibitingtheimmuneresponse tothe polysaccharidecomponentoftheconjugatevaccine[30–32].These findingssuggestthatthelevelofcarrier-specificantibodymight correlatewiththelevelofcarrier-specificBcells,whichmaycom- petewith polysaccharide-specificBcells for thecaptureof the MenCV. An alternative explanationfor this could bethat anti- bodytothecarrierproteinprovidesmoreeffectivesterichindrance oftheinteractionofvaccineantigenandantigen-specificB-cells thantheanti-polysaccharideantibody[30].Thiscouldbedueto eitheroftheantigenqualityandquantityorthatoftheantibody.

Anintriguingpossibilityisthatpre-existingcarrier-specificantibody may bea more potent inhibitor of T-cell responses than B-cells.Fromthispointofviewitwouldbeusefultolookatanti- bodymediatedinhibitionduetoavaccineantigencontainingonly T-dependentconstituent antigens. However, another studyhas reportedthat high pre-booster concentrationof carrier-specific antibodycorrelatedpositivelywiththeincreaseinantibodyspe- cificforthepolysaccharideantigenfollowingimmunisationwith a H. influenzae type b conjugate vaccine in adults [33]. It was suggestedbytheinvestigatorsof thisstudythatcarrier-specific antibodiescorrelatedwiththenumbersofcarrier-specifichelperT cells,whichinturncouldincreasetheBcellresponseagainstthe polysaccharidecomponentoftheconjugatevaccine.Therefore,the phenomenonofcarrier-inducedepitopesuppressionmaydepend onseveralfactors. Forexample, thedoseof thecarriermaybe important.Itwasobservedinamurinestudythatlow-dosecarrier primingimprovedsubsequentresponsetopolysaccharidescon- jugatedtothesamecarrier,whilehighdoseprimingsuppressed theresponse [34].Similarly,it wasreportedthat thekey factor for the inhibitory effect of maternal antibodies on infant vac- cineresponseswastheratioofantibodytoimmunisingantigen [4].Inthepresentstudy,theconcentrationofCRM197wasquite highforall thevaccines (MenCV and9vPnC-MenCV), however, thedoseoftetanustoxoidusedintheHib-MenC-TTvaccinewas quitelow.

Arecommendationforfurtherresearchisthat themeasure- mentsofMenC-IgGandprotein-carrierspecific-IgGaretakenat leasttwiceatashorttimeintervalapart,certainlyaroundbaseline andpreferablyalsoatfollow-up,sothatthevarianceofthemea- surementerror/shorttermfluctuationsinthesemeasurementscan befounddirectly.ThiswouldallowBlomqvist’smethodtobeused directlytoadjustthecorrelationsbetweenpre-andpost-MenC-IgG measurements,andgivefurtherinsightintotheresultspresented here.Itwouldalsoallowforadjustmentstobemadetothecorrela- tioncoefficientsbetweenbaselineprotein-carrierspecific-IgGand changesinMenC-IgG.Itwouldalsobedesirableinfuturevaccine trialsingeneraltobuildrepeatedmeasurementsofantibodiesinto theprotocol,atleastforasubsetofpatients,sothattheseissues canbemorefullyassessed.

Inconclusion,wehavedemonstratedthatunderstandingthe effectof pre-existent antibody onresponse toimmunisation is dependentonusing statisticalmethods that account for mathematical coupling.Using Blomqvist method of adjustment and assumingthat thereis low errorvariance,we haveshown that theinhibitoryeffectofpre-existingMenCantibodyconcentration ontheMenC-antibodyresponsetoboosterimmunisationreported previouslymightbeaccurate.Incontrast,therewasaconsistent inhibitionoftheproteincarrier-speciﬁcantibodypre-boosteron theMenC-antibodyincrease post-boosterwiththeMenCV sug- gestiveofthefactthateither(i)serumantibodyisinhibitorythe effectofimmunological memoryis excludedor(ii)that carrier

proteinantibodiesaremorelikelytomediateinhibitionthrough mechanismssuchasdiversionoralterationsinT-cellhelp.These ﬁndingsshouldbefurtherexploredastheyhaveimplicationsfor theoptimaltimingofdosesofconjugatevaccinesandtheirrela- tionshipto boosterdosesof vaccines containingcarrier-protein epitopes.

Conﬂictofinterests

AJPhasconductedclinicaltrialsonbehalfofOxford Univer- sity, sponsored by Wyeth Vaccines, GlaxoSmithKline Vaccines, SanoﬁPasteur,SanoﬁPasteurMSDandNovartisVaccines,butdoes notaccept anypersonalpayments fromvaccinemanufacturers.

Industry-sourcedhonorariaforlecturing,writingor consultancy andtravelexpensesforattendanceatscientiﬁcmeetingsarepaidto aneducational/administrativefundheldbytheDepartmentofPae- diatrics,UniversityofOxford.MDShasreceivedassistancetoattend scientiﬁcmeetingsfromWyethVaccines,NovartisVaccinesand GlaxoSmithKlineVaccinesandhashadtravelandaccommodation expensespaidbyNovartisVaccineswhileworkingincollaboration withNovartisVaccinesinSiena,Italy.

Acknowledgements

Wewouldliketothankthefollowingcompanies(Novartis,GSK and Wyeth)for allowingustousetheirdata,and inparticular GSKforveryusefulcommentsonthemanuscript.G.B.-R.wassup- portedbytheSwissNationalScienceFoundation.DFKandMDS arefundedbytheOxfordPartnershipComprehensiveBiomedical Research CentrewithfundingfromtheDepartment of Health’s NIHR Biomedical Research Centres funding scheme. The views expressedinthispublicationarethoseoftheauthorsandnotnec- essarilythoseoftheDepartmentofHealth.A.J.P.isaJennerInstitute InvestigatorandJamesMartinFellow.

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