Antibody persistence and serological protection among seasonal 2007 influenza A(H1N1) infected subjects: Results from the FLUREC cohort study

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seasonal 2007 influenza A(H1N1) infected subjects:

Results from the FLUREC cohort study

Rosemary Markovic Delabre, Nicolas Salez, Magali Lemaitre, Marianne

Leruez-Ville, Xavier de Lamballerie, Fabrice Carrat

To cite this version:

Rosemary Markovic Delabre, Nicolas Salez, Magali Lemaitre, Marianne Leruez-Ville, Xavier de

Lam-ballerie, et al..

Antibody persistence and serological protection among seasonal 2007 influenza

A(H1N1) infected subjects: Results from the FLUREC cohort study. Vaccine, Elsevier, 2015, 33

(49), pp.7015-7021. �10.1016/j.vaccine.2015.09.016�. �hal-01225063�

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ContentslistsavailableatScienceDirect

Vaccine

jo u rn al h om ep a g 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

Antibody

persistence

and

serological

protection

among

seasonal

2007

inﬂuenza

A(H1N1)

infected

subjects:

Results

from

the

FLUREC

cohort

study

Rosemary

Markovic

Delabre

a,∗

_,

_Nicolas

_Salez

b

_,

_Magali

_Lemaitre

c

_,

Marianne

Leruez-Ville

d,e

_,

_Xavier

_de

_Lamballerie

b,f,g

_,

_Fabrice

_Carrat

a,h

a_Sorbonne_{Universités,}_UPMC_Univ_Paris_06,_INSERM,_Institut_Pierre_Louis_{d’épidémiologie}_et_de_Santé_Publique_(IPLESP_UMRS_1136),_Paris,_France b_Emergence_des_Pathologies_Virales,_UMR_D_190,_{Aix-Marseille}_Université_and_Institut_de_Recherche_pour_le_{Développement,}_Marseille,_France c_National_Agency_for_the_Safety_of_Medicine_and_Health_Products,_St_Denis,_France

d_Université_Paris_Descartes,_Sorbonne_Paris_Cité,_EA₇₃₂₈_Paris,_France e_Laboratoire_de_Virologie,_Hôpital_Necker,_AP-HP,_Paris,_France

f_IHU_{Méditerranée}_Infection,_Assistance_{Publique-Hôpitaux}_de_Marseille,_Marseille,_France g_Ecole_des_Hautes_Etudes_en_Santé_Publique,_Rennes,_France

h_Unité_de_Santé_Publique,_Hôpital_{Saint-Antoine,}_Assistance_{Publique-Hôpitaux}_de_Paris,_Paris,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Availableonlinexxx Keywords: Antibody Cohortstudies Correlate Immunity

InﬂuenzaAvirus,H1N1subtype

a

b

s

t

r

a

c

t

Introduction: Haemagglutination-inhibition (HI) antibody titer is a correlate of protection against inﬂuenza;itspersistenceafterinfectionorvaccinationisimportanttodeterminingsusceptibilityto subsequentinfection.Fewstudies,however,havereportedlongitudinaldataregardingthemagnitude anddurationofHIprotectionfollowingnaturalseasonalinﬂuenzaAinfection.

Methods:Using French influenza cohortstudydata collected from 2008 to2010, we investigated persistence of serological protection among subjects according to influenza-like illness (ILI) and laboratory-confirmedseasonal2007influenzaA(H1N1)infectionstatusatinclusionin2008(ILI-A(H1N1) positive,ILI-A(H1N1)negative,orno-ILI).Antibodytitersagainstseasonal2007A(H1N1)were deter-minedusingtheHItechniqueforsera.Regressionmodelsforinterval-censoreddatawereusedtoestimate geometricmeantiters(GMT)forHIassays.Alogisticregressionmodeladjustedforagegroup(subjects <30,30–50and>50yearsold)wasusedtoquantifytheassociationbetweenHItiterandprotection againstinfection.

Results:Basedon310totalsubjects,influenzaA(H1N1)infectionwasconfirmedin39of115ILIsubjectsat inclusion.GMTassociatedwith50%probabilityofprotectionamongILIsubjectsdecreasedwithagegroup (subjects<30yo:GMTof40.8wasassociatedwith50%[95CI:29.3%;70.7%]probabilityofprotection, subjects30–50yo:26.8[95CI:34.4%;65.6%]andsubjects>50yo:8.9[95CI:15.3%;84.7%]).GMTdeclined afterthefirstannualstudyvisitamongILI-A(H1N1)positivesubjectsbutremainedhighercompared toinclusionatthe2010studyvisit(41.5[95CI:34.8;49.5],p=0.0157).GMTremainedstableamong ILI-A(H1N1)negativesubjects(p=0.7502),butdecreasedamongno-ILIsubjects(p<0.0001).

Conclusion:OurresultsconfirmthepositiverelationshipbetweenHItiterandprobabilityof protec-tionamongnaturallyinfectedsubjects,andprovidesevidencethatprotectionassociatedwithHItiter varieswithage.ThislongitudinalanalysissuggeststheriseinHItitersfollowingseasonal2007influenza A(H1N1)infectionmaypersistintosubsequentinfluenzaseasons.

1. Introduction

Inﬂuenzarepresentsanimportantpublichealthburden;a life-time of exposure to numerous inﬂuenza viruses results in an

∗ Correspondingauthor.Tel.:+33144738451.

E-mailaddress:rosemary.delabre@iplesp.upmc.fr(R.M.Delabre).

estimated 10 infections if never vaccinated [1]. Cohort studies haveprovidedvaluableinformationconcerningtheepidemiology ofinﬂuenza[2–7],however,speciﬁcknowledgeregardingthe per-sistenceoftheimmuneresponseislimited[8].

Exposuretotheinﬂuenzavirusactivatesanimmuneresponse that includes the production of virus-speciﬁc antibodies. The concentrationofantibodiestargetingtwovirusproteins, haemag-glutinin(HA)andneuraminidase(NA),providesanindicationof

http://dx.doi.org/10.1016/j.vaccine.2015.09.016

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2 R.M.Delabreetal./Vaccinexxx(2015)xxx–xxx

thelevelofprotectionanindividualhasagainstinfection,andare thereforeconsidered“correlatesofprotection”[9,10].Inthe1970s, itwasdemonstratedthathaemagglutination-inhibiting(HI) anti-bodytitersof40wereassociatedwith50%clinicalprotectioninthe eventofinﬂuenzaA2orBexposure[11].Althoughwidelyaccepted andusedasamajorendpointinvaccineefﬁcacystudies[12,13], fewstudieshavebeenperformedtovalidatethisthresholdamong naturallyinfectedsubjects[14]orconsideredage-related differ-encesinprotection.Furthertothis,littleisknownregardinghow protectionchangesovertime.

InthisstudyweaimedtoconﬁrmtheassociationbetweenHI titerandprotectionagainstnaturalinﬂuenzainfection,investigate age-relateddifferencesinprotection,andtostudythepersistence oftheantibodyresponse.EvaluationofHItiterdurabilityfollowing infectionisof interesttodiscerningsusceptibilityof individuals torecurrentinfections,andmayhaveimportantimplicationson populationimmunity.

2. Methods

2.1. Patientrecruitment/participation

DatafromtheFLURECstudy,aFrenchcohortdesignedtostudy recurrentinfluenza infection,wasusedin this study.Thirty-six Frenchmetropolitangeneralpractitioners(GP)participatinginthe Sentinellesnetwork recruited subjectsduringGPvisits. Subject recruitmentwasstratifiedbyreasonformedicalvisit (influenza-like illness (ILI) or no-ILI related illness) and age (10-year age groups).An ILI wasdefined as a suddenonset of fever (38◦C) accompaniedbymusclesorenessandcough.Subjectsperformed aninclusionvisitandatotalof3annualstudyvisitsthroughoutthe follow-upperiod.Moredetailsregardingstudydesignandsubject recruitmentcanbefoundelsewhere[15].

2.2. Datacollection

Studydatawascollectedandenteredonelectroniccasereport formsbyGPs.Dataregardingmedicalhistory(includingchronic conditions,inﬂuenzaepisodesand/orinﬂuenzavaccinationinthe pasttwoyears)andsociodemographicinformationwerecollected atinclusion forall subjects.Symptomsdata andduration were collectedforsubjectsreportinganILI-relatedillnessatinclusion. Duringannualstudyvisits,informationregardingclinicalhistory (ILI-relatedillnesses,vaccinationstatus)wasdocumented.Nasal swabs(VIROCULT®,KITVIA,LabartheInard,France)werecollected atinclusionforILIsubjects.Serologicalbloodsampleswere col-lectedforILIsubjectsatinclusion,andforallstudysubjectsatthe annualvisits.Nasalswabsandbloodsampleswerecollectedinthe eventofanILIepisodeduringstudyfollow-upatadedicatedstudy visit.

2.3. Surveillancedata

Influenza A/Brisbane/59/2007(H1N1) (62%), A/Brisbane/ 10/2007(H3N2) (2%) and influenza B/Florida/4/2006 (36%) cir-culatedduringthe2007–2008seasonalepidemicwhich started 7 January and ended on 9 March 2008 [16]. The 2008–2009 influenzaepidemic began on15 December2008 andended on 22 February 2009, and was characterized by the predominant circulationoftheinfluenzaA/Brisbane/10/2007(H3N2)(85–88%) compared toinfluenza B(10–12%) (B/Victoria/2/87 lineage and B/Yamagata/16/88lineageweredetectedinEurope)[17,18].The 2009–2010A(H1N1)pandemicbeganinFranceon5October2009 and ended on 11 January 2010; influenza A/California/7/2009 (H1N1pdm09)represented95%ofthevirusescirculatingduring thisseason[19].

The 2007–2008 seasonal inﬂuenza vaccine was composed of the A/Solomon Islands/3/2006 (H1N1), A/Wisconsin/67/2005 (H3N2)andB/Malaysia/2506/2004strains[20].Seasonalvaccines for2008–2009 and2009–2010inﬂuenza seasonscontainedthe A/Brisbane/59/2007(H1N1)strain.

2.4. Laboratoryprocedures

2.4.1. RT-PCR

Nasopharyngealswabscollectedatinclusionweretestedforthe presenceoftheseasonal2007A(H1N1)virususingonestepreal timeRT-PCR;procedureshavebeenpreviouslydescribed[15].

2.4.2. Haemagglutinationinhibition(HI)titer

HI titers against seasonal A(H1N1) were determined using thehaemagglutination-inhibitingtechnique forserological sam-ples collected at inclusion and annual study visits using an A/Paris/6/2007 (H1N1)-like strain. Serial two-fold dilutions (1/10–1/1280)ofheat-inactivatedserawereusedinthese exper-iments.SerumHItiterwasdeterminedtobethereciprocalofthe highestserialdilutionatwhichcompleteinhibitionof haemagglu-tinationoccurredintwoindependentreadings[21].Twocontrols werepresentoneachplate;serumHItiterreadingswereadjusted accordingtocontrolresults.SamplesforwhichHItiterwas unde-tectableorhighlyelevatedweredoubleveriﬁed.Intralaboratory variationassessmentswereperformedtoevaluatetheproportion ofserumsamplesinwhicha>2-foldand>4-folddifferencewas identiﬁedinreplicateassays[22]; allsampleswerefoundtobe withina2-foldrange.

2.5. Statisticalmethods

Infectionwasdefinedaslaboratory-confirmedseasonal2007 A(H1N1)onanasopharyngealswabcollectedatinclusion.Three groupsweredefinedaccordingtopresenceofILIandinfection sta-tusatinclusion:ILI-A(H1N1)positive,ILI-A(H1N1)negative,and no-ILI.Analyseswererestrictedtosubjectswhoseinfection sta-tuscouldbedeterminedatinclusionand samplesforwhichall serologicaland vaccinationdata wascomplete. We selectedILI subjectswithaninclusionbloodsamplecollectedbeforetheend of the2007–2008seasonalepidemic and, tolimit the possibil-ityofelevatedtitersassociatedwithinfectionamongILIsubjects, within5days oftheinclusionvisit. Subjectsreportingseasonal (2008–2009,2009–2010)orA(H1N1)pdm09(pandemic) vaccina-tionduringstudyfollow-upwerecensoredatthegivenstudyvisit date.pValues<0.05weredeterminedtobesignificant.

Differencesin baselinecharacteristicsbetweenILIand no-ILI subjectswerestudiedusingtheWilcoxonrank-sumtestfor contin-uouscovariatesandtheFisherexacttestforcategoricalcovariates. Regressionmodelsforinterval-censoreddatawereusedto esti-mate geometric mean titers (GMT) for HI assays [23] and to compareGMTbetweengroups.EvolutionofGMTovertimewas evaluatedusingthesemodels(accountingforrepeateddata).The associationbetween(log2-transformed)HIestimates(GMTofthe upperandlowerboundsoftheintervalcensoredHImeasures)at inclusionandprotectionagainstinfectionwasmodeledusing logis-ticregressionamongallILIsubjectsandadjustedforageatinclusion usingthreeagegroups(subjects<30years,30–50yearsand>50 yearsold).Keepingwithpreviouslypublishedwork[24,25],we deﬁneprotectioninthisanalysisasanegativeresultbyPCRfor inﬂuenzainfection.

StatisticalanalyseswereperformedwiththeRstatistical pro-gram(v2.15.2).

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Fig.1.Studybloodsampleﬂowchart.Totalnumberofsubjectspergroup,accordingtoILIandseasonal2007A(H1N1)infectionstatus(PCRforILIsubjectsonly)atinclusion, andnumberofserologicalsamplesobtainedatinclusionandannualstudyvisits.*Includesmissingvaccinestatus:ILI-A(H1N1)positive(n=2);ILI-A(H1N1)negative(n=3); no-ILI(n=2).

2.6. Ethics

ThisstudyreceivedFrenchethicscommittee(Comitéde Protec-tiondesPersonnesIle-de-FranceV(no.07715))andDataProtection Authorityapproval(no.1261460)andallstudyparticipants pro-videdwritteninformedconsent.

3. Results

Outofa totalof382subjectsincludedin thestudyin 2008, 357providedatleastoneserologicalsample(Fig.1).ILIsubjects withincompleteinclusionvisitdata(n=4),whoseinclusion sam-plewasobtained>5daysafter inclusionvisit (n=30) andafter theseasonal2007epidemic(n=12)wereexcluded.Oneadditional subjectwasnotincludedduetoseasonalvaccinationduringﬁrst studyvisitin2009.Seasonalvaccinationwasreported(ormissing) duringthe2009(n=107)and2010studyvisits(n=9).Fiveother subjectsreportedpandemicvaccination.Thisanalysisistherefore basedon716samplesfrom310subjects.Sixty-ninepercentof sub-jects(n=213)providedatleasttwobloodsamples,48%(n=148) providedatleastthreebloodsamplesand15%(n=45)provided fourbloodsamples.Regardingthetimingoftheannualstudyvisits, 99%,86%,and100%wereconductedbeforethestartoftheseasonal epidemicperiodforthe2008,2009and2010annualstudyvisits, respectively(Fig.2).

Studysubjectswere18-85yearsoldatinclusion;No-ILIsubjects wereolderthanILI-subjects(Median:53(IQR:36;66)vsMedian: 44(IQR:35;57),p=0.0206)andweremorelikelytohavehad sea-sonal2007vaccination(No-ILI:76(39%)vs24(21%);p=0.0010).ILI andno-ILIsubjectsalsodifferedatbaselinewithregardto house-holdsize(No-ILI:Median2(IQR:2;4)vsILIsubjects:Median1 (IQR:1;3);p<0.0001),historyofhypercholesteremia(No-ILI:35 (18%)vsILIsubjects:8(7%);p=0.0064),andcardiovascularillness (No-ILI:62(32%)vsILIsubjects:24(21%);p=0.0485)(Table1).

Ofthe115ILIsubjectsincludedintheprotectionanalysis,39 werefoundtohavelaboratoryconﬁrmedA(H1N1)infection. Pro-portionofinfectiondecreasedwithincreasingagegroup,withthe highestproportion(52%)ofinfectionamongthesubjects<30years old(p<0.0001).ILI–A(H1N1)positivesubjectshadlowerGMTat inclusioncomparedtoILI–A(H1N1)negativesubjects(Fig.3)(33.8 [95CI:29.8;38.4]vs43.5[95CI:39.0;48.6],p=0.0056).

Over study follow-up, 3 subjects were diagnosed with sea-sonalA(H3N2)infection(2008–2009season)and1subjectwith A(H1N1)pdm09infection(2009–2010);allinfectedsubjectswere enrolledintheno-ILIgroupatinclusion.

3.1. HIprotectioncurves

Serologicaltiter againstseasonal 2007A(H1N1) at inclusion among ILI subjects (ILI-A(H1N1) positive, n=39; ILI-A(H1N1)

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Fig.2.Weeklyincidenceofinﬂuenza-likeillnesses(ILIs)inFranceaccordingtoFrenchSentinelnetworkandmonthlystudybloodcollectionfromJanuary2008toNovember 2010.Dottedline(leftscale)ILIweeklyincidenceper100,000;monthlystudybloodcollection(rightscale),graybarsrepresentILIinclusionvisitsamplesandblackbars representannualstudyvisitsamples.

Table1

ComparisonofbaselinecharacteristicsbetweenILI(n=115)andno-ILIsubjects(n=195)atinclusion.

No-ILI(n=195) ILI(n=115) p

Covariate

Age(yrs)—median(IQR) 53(36;66) 44(35;57) 0.0206

Numberofhouseholdmembers—median(IQR) 2(2;3.5) 1(1;3) <0.0001

Sex(Female)—N(%) 111(57) 68(59) 0.7225 Asthma—N(%) 8(4) 6(5) 0.7782 ILIseason2006–2007*_—N_(%) ₂₇₍₁₄₎ ₁₃₍₁₂₎ _0.7246 Smoker†_—N_(%) ₄₁₍₂₁₎ ₂₉₍₂₇₎ _0.2595 ChronicIllness—N(%) 100(51) 47(41) 0.0790 Hypercholesterolemia—N(%) 35(18) 8(7) 0.0064 Cardiovascular—N(%) 62(32) 24(21) 0.0485 Respiratory—N(%) 11(6) 10(9) 0.3515 Other—N(%) 19(10) 5(4) 0.1222 2007–2008seasonalvaccination—N(%) 76(39) 24(21) 0.0010

* _Indicate_unspeciﬁed_values_(ILI_season_2006–2007,_n₌_9).

† _Indicate_covariate_missing_values_(Smoker,_n₌_8).

negative,n=76)waspositivelyassociatedwithprobabilityof pro-tectionagainstA(H1N1)infection.Wefoundsigniﬁcantdifferences betweenolder(>50yearsold)and younger subjects(<30 years old)(p=0.0091);nosigniﬁcantdifferenceswerefoundbetween subjects30–50yearsoldandsubjects<30yearsold(p=0.4271). GMTassociatedwith50%probabilityofprotectiondecreasedwith increasingage:wepredictedaGMTof40.8atinclusionwas associ-atedwith50%[95CI:29.3%;70.7%]probabilityofprotectionamong subjects<30 years old; GMT 26.8for 50% [95CI: 34.4%;65.6%] amongsubjects30–50yearsold;GMT8.9for50%[95CI:15.3%;

Fig.3.Cumulativedistributioncurvesfor2007H1N1titersatinclusionamongILI subjects(n=115)accordingtoseasonal2007A(H1N1)infectionstatusbyPCR.

84.7%]amongsubjects>50yearsold(Fig.4).Overall,increasing serologicaltiterwasassociatedwithasteadyincreasein proba-bilityofprotection,however,theincreaseinprotectionwasmuch smallerforHItiters>80.

3.2. Evolutionofantibodytiter

AmongILI–A(H1N1)positivesubjectshighestGMT(54.0[95CI: 45.3;64.4])wasobservedattheﬁrstannualstudyvisit,conducted 3–12monthspost-infection(April2008andJanuary2009)(Fig.5).

Fig.4.HIprotectioncurvebyagegroup,basedonpredictionsfromthelogistic modelamongILIsubjectsatinclusion(ILI-A(H1N1)negative:subjects<30years old(n=10),30–50yearsold(n=26),>50yearsold(n=40);ILI-A(H1N1)positive: subjects<30yearsold(n=11),30–50yearsold(n=20),>50yearsold(n=8)).

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Fig.5.EvolutionofGMTaccordingtoILIandA(H1N1)infectionstatusatinclusion; errorbarsrepresent95%CI.

HItitersdeclinedthereaftertoGMT41.5[95CI:34.8;49.5]atthe time of the2010 study visit but remained signiﬁcantly higher comparedtoGMTatinclusion(p=0.0157).WhereasILI-A(H1N1) negativesubjectsmaintainedastableGMTthroughoutthestudy period(p=0.7502),GMTamongno-ILIsubjectsdecreasedsteadily (p<0.0001). GMT was signiﬁcantly differentbetweenthe three ILIgroupsatthe2009(p=0.0460)and 2010(p=0.0048)annual studyvisits.GMTamongno-ILIsubjectsdifferedfromILI-A(H1N1) positivesubjectsatthe2009studyvisit(p=0.0230)andfrom ILI-A(H1N1)negativesubjectsatthe2010studyvisit(p=0.0118).

4. Discussion

WestudiedtherelationshipbetweenHItiterandprobability ofprotectionagainstseasonal2007influenzaA(H1N1)infectionin threeagegroupsandexploredhowHItiterevolvesoverthreeyears (2008to2010)accordingtoILIandinfectionstatusatinclusion. Ouranalysissupports,amongnaturallyinfectedsubjects,previous findingswithregardtotheHIprotectioncurveandidentifies dif-ferencesaccordingtoage.Wefoundevidencetosuggestelevated HItitersassociatedwithseasonal2007A(H1N1)infectiondecrease post-infection,butpersistintosubsequentinfluenzaseasons.

Previousstudiesdemonstratingapositiveassociationbetween HItiterandclinicalprotectionhave reliedprimarilyonvaccine studydata[11,24].Hobsonetal.[11]cautionedthattheestimated HItiter(18–36)associatedwitha50%protectionmaynotbe gen-eralizabletonaturallyinfectedsubjects,howeverthis remainsa prominentoutcomeor endpointinserologicaland vaccine effi-cacystudiesregardlessoftypeofinfluenzaexposure.Wearethe first,toourknowledge,toidentifydifferencesintheHIprotection curveaccordingtoage;age-relateddifferencesinsusceptibilityto influenzainfectionarewellestablished[3,5,26,27].Furthermore, ourresultsindicate thatlow antibody titer,particularlyamong oldersubjects,mayconferasignificantlevelofprotectioninthe context ofnatural infection.Our estimates of GMTat inclusion associatedwith50%probabilityofprotection,rangingfrom8.9to 40.8accordingtoagegroup,arecomparabletoarecentestimate byCoudevilleetal.(HItiter17(CredibleInterval:10;29))[24]as wellasotherpublishedwork[11,28,29].However,onehousehold studyfoundthat50%protectionwasassociatedwithHItiter255 (CI:1:62to1:917);thehigherestimateattributedtotheintensity ofexposuresintheconfinedsettingofthehome[30].

OurmodeloftherelationshipbetweenHItiterandprobability ofprotectiondidnotsupportthedesignationofasingle thresh-oldlevel[11]andisthereforedepictedbyacurve[24].Wefound thatprobabilityofprotectionincreasedsteadilywithincreasingHI titerforHItiters≤80;higherHItiterswereassociatedwithsmaller

increasesinprobabilityofprotection.Thisphenomenonhasbeen observedpreviouslyforHItiters≥150[24,28].

Afteraninitialpeak followinginfection orvaccination, anti-bodytitersdeclinebeforereachingastabilizedlevelmaintained overseveralyears[31,32].GMTamongILI-A(H1N1)positive sub-jectspeakedatthetimeofthefirstannualstudyvisit.However, duetothetimingofthisstudyvisit(conducted3–12months post-infection)GMTatthistimepointlikelycapturesthedeclineofHI titers ratherthan thepeakGMT estimatedtooccuronemonth post-infection[33–35].Otherstudieshavealsodocumentedrapid GMTdeclineafterreachingpeaklevelspost-infection[33,35,36], however,ourresultsalsosupportrecentfindingsshowing persis-tenceofelevatedHItitersbeyond12monthspost-infection[8]. Severalstudies[37–39],includingonebasedonasubsetofthis cohort[15],suggestedthatpreviousseasonalA(H1N1)infection mayhavebeenprotectiveagainstA(H1N1)pdm09infectionduring the2009H1N1pandemicviaacross-reactiveantibodyresponse.It isunlikely,however,thatantibodypersistenceamongILI-A(H1N1) infectedsubjectsisduetoboostingduringstudyfollow-up.There isnoknowninteractionbetweenA(H3N2)(circulatingduringthe 2008–2009season)andA(H1N1)viruses.Regardingthepandemic season,noneofthesubjectswithlaboratoryconfirmed(n=1)or suspectedinfection(seroconversion,n=6)wereintheILI-A(H1N1) positivegroup.

GMTdecline amongno-ILIsubjectswasnotsurprising; anti-bodydeclineovertimehasbeendemonstrated[7,8,35]andmay explainrecurrentseasonalinfluenzaepidemics.Incontrast,GMT amongILI-A(H1N1)negativesubjectsremainedstablethroughout thestudy.Twenty-sevenofthe76ILI-A(H1N1)negativesubjects were foundto have laboratory-confirmedinfluenza Bat inclu-sion,although,cross-reactiveresponsesbetweeninfluenzaAand Bviruseshavenotbeendemonstrated[40].StableGMTmay sug-gesteffectsfromundetectedrespiratoryvirusesorasymptomatic infectionspriortostudyenrolment.

4.1. Strengths/limitations

Thisstudyisamongfewlongitudinalstudiesinvestigating dura-bilityoftheimmuneresponse[8,14].Recentstudieshaveprimarily focusedonthe2009A(H1N1)pandemicand/ordurationof vaccine-inducedantibodyresponse[33–35];thoseinvestigatingseasonal influenzainfectionwereconductedalmost30yearsago[2–4,41]. Finally,whileinfectedsubjectsmayhavealsobeenidentifiedby seroconversion, this study relied on a virological definition of infection,thus avoiding thesensitivity issues associated witha serologicaldefinition[42].

Ourresultsmaynotbegeneralizabletochildrenasthisstudyis basedonadultsubjects.HoweverourestimateofHItiterassociated with50%clinicalprotectionagainstinﬂuenzaamongsubjects<30 yearsoldiscomparabletorecentﬁndingsfromarandomized con-troltrialinvestigatingseasonalvaccinationamongchildren[29]. Anotherlimitationofourstudyisthetimingoftheserological sam-ples.InclusionsamplesamongILIsubjectswererestrictedtothose collectedwithin5 daysoftheinclusionvisit,limiting thedelay betweenstartofsymptomsandbloodcollection.Nonetheless,we cannotassurethatbloodcollectionwasperformedbeforethestart ofHItiterincreaseforallILIsubjects.

TheHIprotectioncurvesarebasedonILIsubjectsseeking medi-calcare,however,itispossiblethattheirbaselineriskforinfection maybedifferentfromthegeneralpopulation.Furthermore,our studycapturessymptomaticinfections amongILIsubjectsonly; infectedsubjectswithmildsymptoms[3]thatdidnotmeetour deﬁnitionofsymptomaticillnessand/orthosewithasymptomatic infections[43]werenotidentiﬁed.Wearethereforeunabletoinfer howprotectionandantibodypersistencechangesovertimeamong asymptomaticsubjects.

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AlthoughdeterminationofHItiterbytheHIassayisthegold standard,itsvariabilityiswellknownandthisisanotherlimitation forthisstudy[42,44].HItiteristheprimarycorrelateofprotection againstinﬂuenzainfection,howevertheassociationbetweenHI titerandprotectiondoesnotestablisha directmechanisticlink. Recentresearchhasexploredthepotentialprotectiverolesoflocal responsesintherespiratorytract[8,45,46].Furthermore,thelow titersthatwe,andotherstudies[11,14,24],havefoundtocorrelate withprotectionmayindicatethatantibodyresponsesmaywork inconjunctionwithotherimmuneresponses[30,47,48]toprotect againstinfection.ThismaypartlyexplainthedifferenceinHI pro-tectioncurvesbetweenolderandyoungersubjects.Temporaldata regardingcellularresponsesmayfurtherunderstandingofclinical protectionagainstinﬂuenzainfection,howeverthiswasoutside thescopeofthisseroepidemiologicalstudy.

5. Conclusion

WefoundapositiveassociationbetweenHItiterand probabil-ityofprotectionamongnaturallyinfectedsubjectsandidentiﬁed differencesaccordingtoage.Thisanalysisprovidesararelookinto thepersistenceoftheantibodyresponsefollowingnaturalseasonal A(H1N1)infection.Futurestudies shouldincorporate longitudi-naldataregardingotherpotentialcorrelatesofimmunity,suchas cellularimmuneresponses,tobetterunderstandthedurabilityof protectionfollowingseasonalinﬂuenzainfection.

Fundingsources

ThecohortstudywassupportedbyanAgenceNationaledela RechercheandRegionIle-de-Francegrant.TheUniversitéPierre etMarieCurie(Paris6)providedfundingforthecohortandthis researchstudy.

Authorcontributions

Participatedintheacquisitionofdata:M.LemaitreandF.Carrat. Participatedinthelaboratoryanalyses:X.deLamballerie,N.Salez, M.Leruez-Ville.Studyconceptanddesign:R.DelabreandF.Carrat. Analysisandinterpretationofdata:R.Delabre,M.Lemaitre,X.de Lamballerie,N.Salez,M.Leruez-Ville,andF.Carrat.R.Delabreand F.Carratdraftedthemanuscriptandallauthorsreviseditcritically. Allauthorsgaveﬁnalapprovalofthisversionofthemanuscript.

Conﬂictofintereststatement

F.CarratreceivedhonorariafromAstraZeneca,BoironandGSK foradvisingoninﬂuenzaepidemiology.Otherauthorshaveno con-ﬂictofinteresttodeclare.

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