The B-cell response to a primary and booster course of MenACWY-CRM₁₉₇ vaccine administered at 2, 4 and 12 months of age

Article

Reference

The B-cell response to a primary and booster course of

MenACWY-CRM₁₉₇ vaccine administered at 2, 4 and 12 months of age

BLANCHARD ROHNER, Géraldine, et al .

Abstract

A quadrivalent meningococcal vaccine conjugated to CRM197 (MenACWY-CRM197) is immunogenic in young infants. We assessed the memory B-cell and antibody responses after a primary and booster course of MenACWY-CRM197 in children. At 5 months of age, following primary immunisation, serogroup-specific memory B-cells were detectable in fewer than 25% of children, although protective antibody titres (hSBA ≥ 4) were detectable in 69% of children against serogroup A and more than 95% against the other serogroups. At 12 months, before booster immunisation the percentages with hSBA ≥ 4 were 5% for serogroup A, and between 44 and 70% for the other serogroups. One month after booster immunisation with MenACWY-CRM197 over 50% of children had detectable memory B-cells, and 91% had hSBA ≥ 4 against serogroup A and more than 99% against the other serogroups. These data show that few antigen-specific anticapsular memory B-cells can be detected after two-doses priming with MenACWY-CRM197. For MenC and CRM197, the antigens with the highest number of B-cells at 5 months, there was a definite (p ≤0 .02) but weak correlation with [...]

BLANCHARD ROHNER, Géraldine, et al . The B-cell response to a primary and booster course of MenACWY-CRM₁₉₇ vaccine administered at 2, 4 and 12 months of age. Vaccine , 2013, vol.

31, no. 20, p. 2441-2448

DOI : 10.1016/j.vaccine.2013.03.036 PMID : 23566945

Available at:

http://archive-ouverte.unige.ch/unige:149072

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

1 / 1

ContentslistsavailableatSciVerseScienceDirect

Vaccine

j o ur na l h o me pa g e : w w w . e l s e v i e r . c o m / l o c a te / v a c c i n e

The B-cell response to a primary and booster course of MenACWY-CRM ₁₉₇ vaccine administered at 2, 4 and 12 months of age

Geraldine Blanchard-Rohner

, Matthew D. Snape

, Dominic F. Kelly

, Daniel O’Connor

, Tessa John

, Elizabeth A. Clutterbuck

, Brigitte Ohene-Kena

, Chaam L. Klinger

,

Tatjana Odrljin

, Andrew J. Pollard

aOxfordVaccineGroup,DepartmentofPaediatrics,UniversityofOxford,Oxford,UK

bDepartmentofPaediatrics,Children’sHospitalofGeneva,Geneva,Switzerland

cNIHROxfordBiomedicalResearchCentre,Oxford,UK

dNovartisVaccinesandDiagnosticsSrl,Cambridge,MA,UnitedStates

a r t i c l e i n f o

Articlehistory:

Received4October2012

Receivedinrevisedform19February2013 Accepted22March2013

Available online 6 April 2013

Keywords:

Human B-cells Memory

Meningococcalvaccines

a b s t r a c t

AquadrivalentmeningococcalvaccineconjugatedtoCRM197 (MenACWY-CRM197)isimmunogenicin younginfants.WeassessedthememoryB-cellandantibodyresponsesafteraprimaryandbooster courseofMenACWY-CRM197inchildren.At5monthsofage,followingprimaryimmunisation,serogroup- specificmemoryB-cellsweredetectableinfewerthan25%ofchildren,althoughprotectiveantibody titres(hSBA≥4)weredetectablein69%ofchildrenagainstserogroupAandmorethan95%againstthe otherserogroups.At12months,beforeboosterimmunisationthepercentageswithhSBA≥4were5%

forserogroupA,andbetween44and70%fortheotherserogroups.Onemonthafterboosterimmunisa- tionwithMenACWY-CRM197over50%ofchildrenhaddetectablememoryB-cells,and91%hadhSBA≥4 againstserogroupAandmorethan99%againsttheotherserogroups.Thesedatashowthatfewantigen- specificanticapsularmemoryB-cellscanbedetectedaftertwo-dosesprimingwithMenACWY-CRM197. ForMenCandCRM197,theantigenswiththehighestnumberofB-cellsat5months,therewasadefi- nite(p≤0.02)butweakcorrelationwithantibodypersistenceat12months.Althoughpreviousstudies suggestthatmeasuringmemoryB-cellresponsesafterprimingimmunisationsininfancycanbeusedto predictantibodypersistenceandmemoryresponses,thismaynotbesuitableforallantigensinyoung children.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

AtetravalentserogroupsA,C,WandYmeningococcalprotein polysaccharidevaccineconjugatedtoCRM₁₉₇(MenACWY-CRM₁₉₇) is licensed in theUS in persons2–55 years of age and EU for use in persons above 2 years of age. The MenACWY-CRM₁₉₇ vaccine is immunogenic in young infants, inducing antibody

Abbreviations:CRM197,crossreactivematerial(CRM197:mutantpeptiderelated todiphtheriatoxoid);DTaP-Hib-IPV,combineddiphtheriatoxoid,tetanustoxoid, acellularpertussis,Haemophilusinfluenzaetypeb,andinactivatedpoliovaccine;

GMC,geometricmeanconcentration;GMT,geometricmeantitre;MenACWY- CRM197,tetravalentserogroupsA,C,WandYmeningococcalproteinpolysaccharide vaccineconjugatedtoCRM197;PCV,pneumococcalconjugatevaccine(PCV-7:7- valentpneumococcalconjugatevaccine);SBA,serumbactericidalactivity(hSBA withhumancomplement).

∗Correspondingauthorat:CenterforClinicalVaccinologyandTropicalMedicine, ChurchillHospital,Oxford,OX37LJ,UK.

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

responses against the four serogroups in more than 80% of young children [1,2]. Similar to serogroup C meningococcal glycoconjugate vaccine (MenCV), following MenACWY-CRM₁₉₇ priminginearlyinfancy, adecline inprotective antibodieswas observedlessthanoneyearafterimmunisationdespitetheinduc- tionofimmunologicmemoryasmeasuredbytheboosterresponse [2].Long-termprotectionagainstinvasivemeningococcaldisease reliesonthepersistenceofbactericidalantibodiesinserum[3,4].

StudiesofB-cellresponsestoimmunisationinanimalmodelssug- gestthatthegenerationofmemoryB-cellsandlong-livedplasma cellsduringprimaryimmunisationmaybetheprincipaldetermi- nantoflong-termmaintenanceofantibody,asthesecellshavebeen showntopersistandtocontributetoantibodysecretion[5,6].How- ever,inhumanstheorgansofformationandresidenceofthesecells arenoteasilyaccessible,andB-cellscanonlybereadilyassessed in peripheralblood [7].In this study,memory B-celland anti- body responsesspecifictothefourserogroupsofmeningococci weredeterminedonemonthafteratwo-doseprimarycourseof MenACWY-CRM₁₉₇ vaccine,andbeforeandafteraboosterdose at 12 months of age. The primary objective of this study was 0264-410X/$–seefrontmatter© 2013 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.vaccine.2013.03.036

2442 G.Blanchard-Rohneretal./Vaccine31 (2013) 2441–2448

toassesswhetherthefrequencyofpolysaccharide-specificmem- ory B-cells, measured at 5 months of age, after priming with MenACWY-CRM₁₉₇ vaccine,predictsthehSBAtitersagainstthe fourserogroupsofmeningococciat12monthsofage.Acorrelation betweenmemoryB-cellsat5monthsandantibodypersistenceat 12monthswouldconfirmthemajorroleofmemoryB-cellsindriv- inglong-termantibodypersistenceofimmunity.Ifthisrelationship isreproducible,theassessmentofmemoryB-cellresponsesinaddi- tiontoantibodyresponsesshouldbeusedinevaluationofvaccines toprovideabetterindicationoflong-termprotection.

2. Materialsandmethods 2.1. Studypopulation

AphaseII,singlecentre,open-label,randomisedstudywascon- ductedinOxford,UK,betweenMay2007andDecember2009,to assesstheserogroup-specificB-cellresponsestoa primary and booster course of MenACWY-CRM₁₉₇ vaccine (Menveo, Novar- tis Vaccines and Diagnostics Srl, Siena). Potential participants wererecruitedbyinformationletterintheThamesValleyregion, UK.Exclusion criteria includedprevious meningococcaldisease orhouseholdcontactwithmeningococcaldisease,immunedys- function,recentreceiptofantibioticsorcorticosteroids.Written informedconsentwasobtainedfromthemothersofallenrolled infants. Ethical approval was obtained from the Oxfordshire Research Ethics Committees (approval number B07/Q1605/41;

Eudractnumber2006-003476-35).Thetrialwasregisteredwith clinicaltrials.gov(identifierNCT00488683).Participatinginfants wererandomisedtooneofthreegroupsina2:1:1ratiotoallow assessmentofimmunogenicityatvarioustime-pointswhilstmin- imizingthenumberofbloodsamplesobtainedfromeachchild.

2.2. Immunisationprocedures

One dose of 0.5ml of the MenACWY-CRM₁₉₇ vaccine was administered by intramuscular injection into the right antero- lateralthighat2,4and12monthsofage.The0.5mldoseconsisted ofN.meningitidisserogroupA,C,Yand Wcapsularsaccharides (10␮gofserogroupA;5␮geachoftheotherserogroups)individ- uallyconjugatedtoCRM197,withoutadjuvant.Atthesametime,as pertheroutineUKimmunisationschedule,concomitantvaccines wereadministeredintotheleftthigh;thecombineddiphtheriatox- oid,tetanustoxoid,acellularpertussis,Haemophilusinfluenzaetype b(Hib),and inactivatedpoliovaccine(Pediacel,Aventis-Pasteur MSDLtd)at2,3and4monthsofage,andthe7-valentpneumococ- calconjugatevaccine(PCV-7,Prevenar,WyethVaccines)at2and 4monthsofage(Table1).

Inaddition groupIreceivedthePCV-7 at13 months ofage andgroupsIIandIIIreceivedthePCV-7concomitantlywiththe MenACWY-CRM197at12monthsofage.Furthermorethecombined measles,mumpsandrubellavaccine(MMR)andaboosterdoseof Hibwereofferedonthecompletionofthestudyat13monthsof ageaccordingtotheroutineUKimmunisationscheduleatthetime (Table1).

2.3. Samplingprocedures

Forallchildren,bloodsamplesweretakenat5months,follow- ingprimingwithMenACWY-CRM197at2and4monthsofage(visit 4),immediatelybeforethethirdimmunisationwithMenACWY- CRM₁₉₇at12months(visit5),1monthfollowingthe12-months immunisation(visit6).Inaddition,participantshadoneadditional blooddrawatatimewhichwasdeterminedbytheirgroup:group Ichildren weredividedinto6subgroupsforthekineticsstudy, whichinvolvedablooddraweitherbeforeoratvariousdaysafter

theseconddoseofMenACWY-CRM₁₉₇at4months;groupIIhadan additionalblooddrawatthetimeofenrolment(2months);group IIIhadanadditionalblooddraw6–7daysfollowingthe12-months doseofMenACWY-CRM₁₉₇(Table1).

2.4. PreparationofPBMCs

Up to 5ml of heparinised blood was diluted 1:2 with RPMI-1640medium(Sigma–Aldrich,England)towhichpenicillin- streptomycinsolution(Sigma–Aldrich,England)andl-glutamine 200mM(Sigma–Aldrich,England)hadbeenaddedatadilutionof 1:100(completemedium).Theperipheralbloodmononuclearcells (PBMCs)werethenseparatedbydensitygradientcentrifugation overLymphoprep(Axis-Shield,Diagnostics,England).PBMCswere washedonceincompletemediumpriortofurtherpreparationfor ELISpotorcellculture.

2.5. PreparationofELISpotplates

ELISpotplates(96wellPVDFmembrane)(Millipore,England) werecoatedwitheitherserogroupsA,C,W-135andYmeningococ- calpolysaccharide(5␮g/ml)(NIBSC,UK)conjugatedtomethylated humanalbumin(5␮g/ml)(NIBSC,UK),or10␮g/mlCRM₁₉₇(Novar- tisVaccines,Siena,Italy),or10␮g/mlgoatanti-humanIg(Caltag laboratories,Burlingame,USA)insterilephosphatebufferedsaline (PBS).PBSalonewasaddedtotheantigenblankwells.TheELISpot plateswerestoredat4degreescentigradeuntiluse.

2.6. DetectionofmemoryB-cells

PBMCspreparedfromperipheralbloodwerere-suspendedin completemediumwith10%foetalcalfserumatafinalconcentra- tionof2×10⁶PBMCs/mland100␮lwasaddedperwellofa96-well round bottomed culture plate (Greiner One-Bio Ltd, England).

The cells were stimulated with medium, containing 1/5000 of StaphylococcusaureusCowanstrainsuspension(SAC),(Calbiochem, England), 83ng/ml Pokeweed mitogen (PWM) (Sigma–Aldrich, England)and2.5␮g/mlCpGoligonucleotide(ODN-2006)(Source Bioscience,England).Thecellswereincubatedat37^◦Cin5%CO₂ for 5.5days beforebeing re-suspendedand washed 4times in PBS-EDTAwith0.5%newbornbovineserumandresuspendedin completemediumwith10%foetalcalfserum.Theculturedcells wereplatedontopre-coatedELISpotplatesthathadbeenprevi- ouslyblockedwithcompletemediumwith10%foetalcalfserum at2×10⁵cells/well.TheELISpotplatescontainingthecellswere thenincubatedovernightat37^◦C in5%CO2 and95%humidity.

The wells were washed withPBS-Tween and bound IgG anti- bodiesdetectedusinga1:5000dilutionofgoatanti-humanIgG

␥-chainspecificalkalinephosphataseconjugate(Calbiochem,UK) incompletemediumwith10%foetalcalfserumfor4h.Boundalka- linephosphataseconjugatewasdetectedusingthesubstratekit (5-bromo-4-chloro-3-indolylphosphateinnitrobluetetrazolium dissolvedin aqueousdimethylformamide, Bio-RadLaboratories, England).Thereactionwasstoppedwithsteriledistilled water, 200␮l/well.

2.7. ELISpotcounting

SpotswerecountedusinganAIDELISpotReaderELR02(AID) andELISpotsoftware,version3.2.3(CadamaMedicalLtd,Stour- bridge,UK).Spot-formingcells werecountedandconfirmedby visualinspection.Identicalsettingswereusedforallplates but differentsettingswereusedforthedifferentantigens(polysaccha- rideantigensversusproteinantigens).Theoperatorwasblindedto whichsamplewasbeingcounted.

Table1

Studygroupsandnumberofsubjectsforeachgroup.

Visit 1 2 3 4(AllGroups)4(Kinetics)

(Group1only)

5(Group1and2) 5a(Group3)

5b(Group3only) 6 Age/Group

(no.subjects)

2months 3months 4months 5months(Group1 variable)

12months 6/7dayspostV5a 13months

1(108) Maternal venepuncture MenACWY DTaP-Hib-IPV PCV

DTaP-Hib-IPV MenACWY DTaP-Hib-IPV PCV

Venepuncture(day30 postV3)

Venepuncture MenACWY

VenepunctureHib*

MMR*PCV

Venepuncture(kinetics) (visittimingaccordingto subgroupallocation)

2(54) Maternal

venepuncture Venepuncture MenACWY DTaP-Hib-IPV PCV

DTaP-Hib-IPV MenACWY DTaP-Hib-IPV PCV

Venepuncture(day30 postV3)

Venepuncture MenACWYPCV

VenepunctureHib*

MMR*

3(54) Maternal

venepuncture MenACWY DTaP-Hib-IPV PCV

DTaP-Hib-IPV MenACWY DTaP-Hib-IPV PCV

Venepuncture(day30 postV3)

Venepuncture MenACWYPCV

Venepuncture VenepunctureHib*

MMR*

MenACWY=studyvaccineMenACWY-CRM197,DTaP-Hib-IPV=combineddiphtheriatoxoid,tetanustoxoid,acellularpertussis,Hibandinactivated poliovaccinePediacel,PCV=7-valentpneumococcalconjugatevaccinePrevenar

2.8. Anti-N.meningitidisserogroupsA,C,WandY-specific bactericidalactivityasmeasuredbyhSBA

Serumbactericidalantibody(SBA)assays usinghumancom- plement (hSBA) for meningococcal serogroups A, C, Y and W wereperformedatthelaboratoriesofNovartisVaccines,Marburg, Germanyaccording tostandard protocols[8,9]. Briefly,twofold dilutionsof heat-inactivated serawere incubated withsuspen- sionsofmeningococcalstrainsA,C,WandYandfreshlythawed humancomplement.Thelastdilutionproducinga≥50%reduction incolonies(killing)compared tocontrolwells,containingcom- plementandbacteria,wastakenastheendpointhSBAtitre.The referencestrainsusedfortherelevantserogroupswereserogroup A,F8238;C,C11;W,M01-240070;andY,860800.

2.9. Anti-N.meningitidisserogroupsA,C,YandWantibody concentrationasmeasuredbyELISA

TheconcentrationofIgGspecificformeningococcalserogroups A, C, W and Y was determined by ELISA following a previ- ouslydescribedmethod[10].Briefly,immulon-2microtiterplates (ThermoElectronCorporation,England)werecoatedwitheither serogroupA,C,WandYmeningococcalpolysaccharide(5␮g/ml) (NIBSC,England)conjugatedto5␮g/mlmethylatedhumanalbu- min (NIBSC, England) in sterile PBS. Following blocking, eight twofolddilutionsofthereferenceserum(CDC1992,NIBSC99/706);

startingdilution1:400forserogroupAand1:150forserogroupsC, WandY)andtestsera(startingdilution1:25)weremadedirectly inthemicrotiterplatebywell-to-welltransferwithamultichan- nelpipette.Thereferenceserumwasassayedintriplicate,andtest serawereassayedin duplicate.Additionally aninternal quality control(ananti-meningococcaladultimmuneserum)wasdiluted toyieldopticaldensitiesapproximatelyonthehigh,middle,and lowportionsofthereferencecurve.Afterovernightincubationat 4^◦C,microtiterplatesweredevelopedwithmonoclonal-PANanti- humanFc␥peroxidaseantibody(dilutedinserum/conjugate[S/C]

buffer)(StratechScientificLtd.,England)for2.5hatroomtempera- ture,followedbythechromogenicsubstratetetramethylbenzidine dihydrochloridemonohydrate(Sigma–Aldrich,England),andthe reactionwasstopped after30minwith2MH₂SO₄.Theoptical densityofeachwellwasthenreadat450nm.Asigmoidlogistic

plotofreferenceseradilutionswasusedtocalculatetestseraanti- bodyconcentrations.Theantibodyconcentrationswerecalculated inng/mlfromthesigmoidlogisticplotusingRevelationsoftwear (ThermalLabsInc.,Basingstoke,UK).Allthedatawerethensubse- quentlyconvertedtomcg/mlforanalysis.

2.10. Anti-CRM₁₉₇IgGconcentration

TheCRM197IgGconcentrationwasdeterminedusinganinter- nallyvalidatedELISA.Inbrief,NuncImmunoMaxisorpmicrotitre plates (Thermo Fisher Scientific) were coated with 5.0␮g/ml of diphtheria toxin mutant CRM197 (Novartis, Siena). Follow- ing blockingand washing steps,serial dilutionsof sample sera andareferencediphtheriaantitoxinserumNIBSC(00/496),were made in duplicate. Following two hours of incubation, plates werewashedandgoatanti-humanIgG-Fcspecificalkalinephos- phataseconjugateantibodies(Sigma–Aldrich)wereaddedforone hour.Plateswereoncemorewashedandp-nitrophenylphosphate (Sigma–Aldrich)added,after20mintheenzymaticreactionwas terminatedwith3MNaOH.Theopticaldensityofeachwellwas readat405nmandresultswerereportedin InternationalUnits (IU)perml.

2.11. Statisticalanalysis

Intentiontotreatanalysiswasperformed.Forthepurposesof analysis,ELISpotassaysinwhichfewerthan4spotsweredetected weretreatedasthoughnoASCsweredetected.Normally,eight replicatewellswereusedforeachmeningococcalserogroupand CRM₁₉₇,andsothemaximalsensitivityoftheassaywas2.5cellsper millionPBMCs.However,whenthetotalnumberofPBMCsavail- ablewaslimited,atleast8replicatewellswereusedforserogroup Cand4replicatewellsforCRM₁₉₇,andtherestofthecellswere usedtoassesstheotherserogroups.AmemoryBcellfrequencyof lessthan2.5cells/millionculturedlymphocyteswasassignedthe value“0”.hSBAtitreandELISAIgGconcentrationsweresummari- sedusinggeometricmeans.B-cellnumbersweresummarisedby medians.ThepercentageofparticipantswithhSBAtitre≥4were calculated.Stata(version9.1,StataCorp,USA)wasusedforthesta- tisticalanalysis.

2444 G.Blanchard-Rohneretal./Vaccine31 (2013) 2441–2448 3. Results

3.1. Population

Atotalof216childrenwereenrolledinthreegroups(108in groupI,54in groupIIand54in groupIII),ofwhom196(91%) completedthestudy(Table1).

3.2. MemoryB-cellresponses

At 5 months of age, the proportion of children who had detectablememoryB-cells(i.e.≥2.5memoryB-cellspermillion culturedlymphocytes)was25%forserogroupCandvariedbetween

12–16%fortheotherpolysaccharideserogroups.Incontrast,54%

childrenhad detectableCRM197-specificmemoryB-cells.Forall serogroupsthemedianfrequencyof–memoryB-cellswas0as comparedto3cellspermillionculturedlymphocytesforCRM₁₉₇- memoryB-cells.After5months,theproportionofchildrenwith detectablememoryB-cellswassimilaracrossalltime-pointsup toandincluding12monthsofage.At12monthstheproportionof childrenwhohaddetectablememoryB-cellswas24%forserogroup C,variedbetween13–17%fortheotherpolysaccharideserogroups, andwas80%forCRM₁₉₇.FollowingtheboosterdoseofMenACWY- CRM197at12monthsofage,theproportionofchildrenwhohad detectablememoryB-cellsincreasedandby13monthswas66%

forMenA,70%forMenC,andaround50%forMenWandMenY,and

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 1

10 100

Days after immunisation MenA

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 4 14 9 116 12 8 12 128 2 127

Median 0 0 0 0 0 0 0 0 19.8 4.1

18/116 22/128 84/127

16 17 66

No (%) with detectable cells

MenA-specific ASCs/ 106 cultured lymphocytes

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 1

10 100

Days after immunisation MenC

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 6 17 14 170 16 16 15 163 7 169

Median 0 0 0 0 0 0 0 0 32.2 5.3

42/170 39/163 118/169

25 24 70

No (%) with detectable cells

MenC-specific ASCs/ 106 cultured lymphocytes

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 1

10 100

Days after immunisation MenW

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 4 15 14 146 15 15 15 161 5 167

Median 0 0 0 0 0 0 0 0 55.3 2.5

17/146 21/161 86/167

12 13 52

No (%) with detectable cells

MenW-specific ASCs/ 106 cultured lymphocytes

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 1

10 100

Days after immunisation MenY

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 3 15 9 122 13 12 14 147 5 146

Median 0 0 0 0 0 0 0 0 63.8 2.5

16/122 23/147 74/146

13 16 51

No (%) with detectable cells

MenY-specific ASCs/ 106 cultured lymphocytes

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 1

10 100

Days after immunisation CRM

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 6 17 14 168 16 16 15 163 7 169

Median 0 10.9 5.5 3 4.1 7.7 4.7 6.1 28.4 22.5

98/168 130/163 161/169

54 80 95

No (%) with detectable cells

CRM-specific ASCs/ 106 cultured lymphocytes

Fig.1.ThememoryB-cellresponsespecifictomeningococcal-serogroupA,C,W,YandCRM197wasmeasuredfollowingprimingwith2dosesofMenACWY-CRM197at2and 4monthsofage,andfollowingboostingat12monthsofage.Theminimumsensitivityoftheassayisplottedasabrokenline.Thezerovalueshavebeengiventhearbitrary valueof1forillustrativepurposes.ThehorizontalbarsrepresentthemediannumberofspecificmemoryB-cellsateachtimepointforthewholesamplesize.Thetables beloweachgraphreporttheage,thedaysfollowingthe4-monthsimmunisation,thenumberofchildrentested,themedianfrequencyofmemoryB-cells,andthenumberof childrenwithdetectablememoryB-cells(i.e.≥2.5polysaccharideorCRM197specificmemoryB-cellspermillionculturedlymphocytes)/totalnumberofchildren,withthe percentage.

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 0.1

1 10 100 1000 10000 100000 1000000

Days after immunisation MenA

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 43 7 18 16 179 16 18 16 182 43 187

GMT 2.3 2 13.4 25.6 10.7 9.5 2.6 4.5 2.1 96.6 59.5

124/179 9/172 171/187

69 5 91

No (%) with seroprotection

MenA-specific hSBA titre

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 0.1

1 10 100 1000 10000 100000 1000000

Days after immunisation MenC

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 38 5 16 12 165 16 18 16 177 41 176

GMT 3.6 7.2 137.8 180.2 101.7 104.4 30.7 15.3 5.4 656.9 282.2

159/165 78/177 174/176

96 44 99

No (%) with seroprotection

MenC-specific hSBA titre

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 0.1

1 10 100 1000 10000 100000 1000000

Days after immunisation MenW

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 29 5 12 9 116 11 15 14 150 31 146

GMT 5.1 4.4 99.2 143.9 62.6 35.3 19.6 12.2 9.5 1063.2 741.1

115/116 108/150 146/146

99 72 100

No (%) with seroprotection

MenW-specific hSBA titre

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 0.1

1 10 100 1000 10000 100000 1000000

Days after immunisation MenY

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 21 1 8 4 39 8 9 8 93 16 76

GMT 2.9 2 31.5 95.1 22.9 33.3 11.5 8.2 8.9 435.1 457.1

37/39 64/93 76/76

95 69 100

No (%) with seroprotection

MenY-specific hSBA

Fig.2.ThefunctionalantibodyresponsemeasuredbyserumbactericidalassayusinghumancomplementspecifictoserogroupA,C,WandYwasmeasuredfollowingsecond dose-primingwithMenACWY-CRM197at4monthsofage,andfollowingboostingat12monthsofage.ThebrokenlinerepresentsthecorrelateofprotectionhSBA≥4.The horizontalbarsrepresentthegeometricmeantitre(GMT)ateachtimepointforthewholesamplesize.Thetablesbeloweachgraphreporttheage,thedaysfollowingthe 4-monthsimmunisation,thenumberofchildrentested,theGMT,andthenumberofchildrenwithseroprotection(hSBA≥1:8)/totalnumberofchildren,withthepercentage.

95%forCRM₁₉₇.Themedianfrequencyofpolysaccharide-specific memoryB-cellsat30dayspost-boostervariedbetween2and5 permillionculturedlymphocytesandwas23permillioncultured lymphocytesforCRM₁₉₇-memoryB-cells(Fig.1).

3.3. Functionalantibodyresponses(hSBA)

At 5 months of age, one month after 2 dose-priming with MenACWY-CRM₁₉₇vaccine;theproportionofchildrenwhohad hSBAtitres≥4was69%forMenA,andbetween95and99%forthe otherserogroups.Between5and12monthsofage,polysaccharide- specificfunctionalantibodytitresdecreasedslowly.At12months, theproportionofchildrenwhohadserogroupspecifichSBAtitres

≥4wasaround70%forserogroupsWandY,44%forserogroupCand only5%forserogroupA.FollowingtheboosterdoseofMenACWY- CRM197at12monthsofage,thefunctionalantibodytitreincreased rapidlyandat13monthstheproportionofchildrenwhohadSBA titres≥4was91%forserogroupAandmorethan99%fortheother serogroups(Fig.2).

3.4. Antibodyresponses(IgG)

Followingthesecond primingdoseof MenACWY-CRM197 at 4 monthsthere wasa rapid increase inpolysaccharide-specific IgG-antibody.Then, theGMCfor allserogroupsdecreasedfrom 5 monthsuntil 12 months of age.Following a booster doseof MenACWY-CRM₁₉₇at12months,theconcentrationofserogroup specific-antibodyincreasedrapidly.FortheCRM₁₉₇-IgGconcen- trationtherewasaclearrisefrombaselinefollowingthefirstdose

ofMenACWY-CRM₁₉₇at2monthsofage,butnofurtherriseafter theseconddoseofMenACWY-CRM₁₉₇ at4months.Theconcen- trationofCRM197-IgGremainedsimilarfrom4monthsuntil5.5 monthsandthendecreasedslowlyto12monthsofage.Following theboosterdoseofMenACWY-CRM₁₉₇at12monthsofage,the concentrationofCRM197-IgGincreasedrapidly(Fig.3).

3.5. AssociationbetweenthememoryB-cellfrequenciesat5 monthsofageandthehSBAtiter/IgGconcentrationat12months and13monthsofage

Therewasaweakinconsistentpositivecorrelation(Spearman’s correlationcoefficientbetween0.2and0.4)betweenmemoryB- cellfrequencymeasuredinperipheralbloodat5monthsofage andtheantibodymeasuredbyhSBAtitreandIgGconcentrationat 12monthsand13monthsofageforsomeserogroupsofmeningo- cocci(inparticularMenCandMenW)(seeTable2).Incomparison, there wasa Spearman’scorrelation coefficientof 0.5 (p<0.001, n=133) for the correlation between the frequency of CRM₁₉₇- specific memory B-cells detected at 5 months of age and the CRM197-IgGconcentrationmeasuredat12monthsofage.Further- more,therewasaSpearmancorrelationcoefficientof0.3(p=0.003, n=134)forthecorrelationbetweenCRM₁₉₇-specificmemoryB- cellsat5monthsandtheIgGconcentrationat13months.

4. Discussion

Aspreviouslyreported,thepresentstudyshowthatthereisa goodantibodyresponsefollowingMenACWY-CRM197fromearly

2446 G.Blanchard-Rohneretal./Vaccine31 (2013) 2441–2448

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 0.01

0.1 1 10 100 1000 10000

Days after immunisation

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 51 3 10 9 96 5 6 2 105 37 155

GMC 0.7 1 2.3 2.4 1.6 1.4 0.7 0.8 0.4 8.6 4.3

MenA

MenA-specific IgG concentration (μμμ μμ

g/ml)

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 0.01

0.1 1 10 100 1000 10000

Days after immunisation

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 51 3 11 9 84 6 9 3 114 36 137

GMC 0.2 0.4 3.3 4.1 1.9 2.6 0.8 0.6 0.3 7.1 5.5

MenW

MenW-specific IgG concentration (g/ml)

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 0.01

0.1 1 10 100 1000 10000

Days after immunisation

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 51 4 15 10 128 6 7 6 116 36 147

GMC 0.3 0.9 1.4 1.6 1.1 1.1 0.6 0.8 0.2 4 2

MenC

MenC-specific IgG concentration (g/ml)

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 0.01

0.1 1 10 100 1000 10000

Days after immunisation

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 44 3 14 11 124 11 12 9 162 37 166

GMC 0 1.4 1.2 1.3 1.3 1.3 0.7 0.7 0.3 3.2 22.5

CRM

CRM197-specific IgG concentration (g/ml)

2m d0 d7 d14 d30 d49 d90 d120 d240 d247 d270 0.01

0.1 1 10 100 1000 10000

Days after immunisation

Age 2m 4m 5m 7m 8m 12m 13m

Day 0 7 14 30 49 90 120 240 247 270

Number 51 3 5 4 71 3 2 102 36 136

GMC 0.3 0.8 1.5 5.5 1.8 2.5 0.6 0.4 10.7 6.1

MenY

MenY-specific IgG concentration (g/ml)

Fig.3. TheIgG-antibodyresponsespecifictoserogroupA,C,W,YandCRM197wasmeasuredfollowingseconddose-primingwithMenACWY-CRM197at4monthsofage, andfollowingboostingat12monthsofage.Thehorizontalbarsrepresentthegeometricmeanconcentration(GMC)ateachtimepointforthewholesamplesize.Thetables beloweachgraphreporttheage,thedaysfollowingthe4-monthsimmunisation,thenumberofchildrentestedandtheGMC.

infancy.However,asitisthecasewithotherconjugatevaccines, thisresponseisassociatedwithrapidwaningofantibodyinchil- drenvaccinated beforeone year of age,although the response to booster immunisation is excellent [2,11–14]. There appears to be a smaller decline in antibody for children primed with theMenACWY-CRM₁₉₇ atolderages[15].Therateofdeclinein polysaccharide-IgGconcentrationfrom5monthsto12monthsof agefollowingtwo-doses-primingwithMenACWY-CRM197,issim- ilartotherateofdeclineofserogroupC-IgGfrom5monthsto12

monthsofage,followingprimingwithtwoorthree-dosesofMenCV ininfancy[14,16],anddoesnotappeartodependonlyonthebio- logicalhalf-lifeofantibodyof3weeks[17]asthelevelat12months ofagewashigherthanwouldbepredictedsolelybythehalf-lifeof antibody.Therefore,thelevelofpersistingantibodyby12months ofageisnotonlydeterminedbythelevelreachedafterprimary immunisationat5monthsofage,butalsobyotherdeterminants suchas,presumably,theproductionoflonglivedplasmacellsand memoryB-cells.However,itappearsthatthesemechanismsarenot

Table2

CorrelationbetweenpolysaccharideandCRM197-specificmemoryB-cellsat5monthsofagewiththeantibodymeasuredbyhSBAtitreandIgGconcentrationat12months and13monthsofage.^a

hSBAtitreat12m hSBAtitreat13m IgGat12m IgGat13m

p p n p p n r p n r p n

MenA MBcfrequencyat5m 0.09 0.3 104 0.4 0.001 105 −0.08 0.6 56 0.3 0.02 89

MenC MBcfrequencyat5m 0.3 0.0003 147 0.2 0.02 143 0.3 0.02 86 0.002 1 115

MenW MBcfrequencyat5m 0.3 0.0005 112 0.3 0.004 104 0.05 0.7 74 0.2 0.02 91

MenY MBcfrequencyat5m 0.02 0.9 59 0.02 0.9 44 −0.2 0.08 57 0.3 0.03 76

CRM MBcfrequencyat5m 0.5 <0.001 133 0.3 0.003 134

aPearman’srankcorrelationcoefficient(r)withthepvalues(p)andthenumberofsubjecttestedforeachassociation(n).