IgG abnormalities in HIV-positive Malawian women initiating antiretroviral therapy during pregnancy persist after 24 months of treatment

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IgG

abnormalities

in

HIV-positive

Malawian

women

initiating

antiretroviral

therapy

during

pregnancy

persist

after

24 months

of

treatment

Silvia

Baroncelli

a,

*

,

Clementina

Maria

Galluzzo

a

,

Giuseppe

Liotta

b

,

Stefano

Orlando

b

,

Fausto

Ciccacci

b

,

Mauro

Andreotti

a

,

Robert

Mpwhere

c

,

Richard

Luhanga

c

,

Jean

Baptiste

Sagno

c

,

Roberta

Amici

a

,

Maria

Cristina

Marazzi

d

,

Marina

Giuliano

a a

NationalCentreforGlobalHealth,IstitutoSuperiorediSanità,VialeReginaElena,299,00161Rome,Italy

b

DepartmentofBiomedicineandPrevention,UniversityofRomeTorVergata,ViaMontpellier,1,00133Rome,Italy

c_DREAM_Program,_Community_of_S._Egidio,_PO_Box_30355,_Blantyre,_Malawi d

DepartmentofHumanSciences,LUMSAUniversity,ViaTraspontina21,00193Rome,Italy

ARTICLE INFO

Articlehistory: Received24June2019

Receivedinrevisedform27August2019 Accepted3September2019

CorrespondingEditor:EskildPetersen, Aar-hus,Denmark Keywords: HIV Africa Pregnancy Hypergammaglobulinemia IgGsubclasses ABSTRACT

Objectives:HypergammaglobulinemiaandanomaliesintheIgGsubclassdistributionarecommonin HIV-infectedindividualsandpersistevenaftermanyyearsofantiretroviraltherapy(ART).Theaimofthis studywastoinvestigatetheIgGproﬁleanddynamicsinpregnantHIV-infectedMalawianwomeninthe OptionBera.

Methods:Thirty-seventreatment-naivewomenreceivedARTfromthethirdtrimesterofpregnancyto6 months postdelivery(endofthebreastfeedingperiod).ARTcontinuation(groupC)orinterruption (groupI)wasthendecidedonthebasisoftheCD4+cellcountatenrolment(>350or350/ml).TotalIgG andIgGsubclassesweredeterminedinmaternalserumusinganephelometricassayatbaselineandat6 and24monthspostpartum.

Results:Atenrolment,36/37womenhadIgGlevels>15g/landtherewasapredominanceoftheIgG1 isotype(morethan90%)inparallelwithunderrepresentationofIgG2(5.0%).After6monthsofART,both groupsshowedasignificantmediandecreaseintotalIgG( 3.1g/lingroupI, 3.5g/lingroupC)andin IgG1( 4.0g/land 3.6g/l,respectively),butonlyamodestrecoveryinIgG2levels(+0.16ingroupI,+0.14 g/lingroupC).Atmonth24,hypergammaglobulinemiawasstillpresentin73.7%ofwomeningroupC, althoughasignificantreductionwasobservedintotalIgGlevelandinIgG1andIgG3subclasses(p <0.0001inallcases).IgG2levelsdidnotshowanysignificantchange.IngroupIat24months,totalIgG andIgGsubclasseshadreturnedtolevelscomparabletothoseatbaseline.

Conclusions:Thebeneﬁcialeffectsof24monthsofARTappeartobelimitedintheB-cellcompartment, withanincompletereductionoftotalIgGlevelsandnorecoveryofIgG2depletion.AshortARTperioddid nothavesigniﬁcanteffectsonIgGabnormalitiesinwomenwhointerruptedtreatment.

http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

AnomaliesintheB-cellcompartmenthavebeendescribedinall thephasesofHIVinfection,withhypergammaglobulinemiaand defects in memory B-cells being the two most consistent observations(Pensierosoetal.,2013;MoirandFauci,2017).The mechanisms inducing immunoglobulin dysregulation are only partially known and are probably a consequence of systemic

immuneactivationandtheunbalancedproductionofproin ﬂam-matorycytokines,reﬂectingtheT-celldysfunction(DeMilitoetal., 2004;Titanjietal.,2006;MoirandFauci,2008).

The bene_ﬁcial effects of antiretroviral therapy (ART) in reversingtheimmunologicalT-celldysfunctionarealsoextended totheimprovementofB-cellcompartmentdisorders(Amuetal., 2014),buttherearecontrastingresultsregardingthetimingand quality of this normalization. Although a reduction in the hypergammaglobulinemiaafterARTinitiationiswidelydescribed (Notermansetal.,2001;Serpaetal.,2010),mostofthestudieshave reportedthepersistenceofachronicstateofB-cellhyperactivation (Regidoretal.,2011; Pogliaghiet al.,2015), consistentwiththe

* Correspondingauthor.

E-mailaddress:silvia.baroncelli@iss.it(S.Baroncelli).

https://doi.org/10.1016/j.ijid.2019.09.001

1201-9712/©2019TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

International

Journal

of

Infectious

Diseases

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incompleterecoveryoftheimmunologicalfunctionobservedin theT-cellcompartment.

EvenaftermanyyearsofART,B-cellcompartmentanomalies have been observed in aviremic patients (Chong et al., 2004; d’Orsognaetal.,2007;Huetal.,2015;Pogliaghietal.,2015)with IgGlevelsexceedingreferencelimits.Inparticular,thepolyclonal B-cellactivationcauses anunbalancedIgGsubclassproﬁlewith signiﬁcantelevationofIgG1andIgG3levelsand underrepresenta-tionoftheIgG2isotype(Rauxetal.,2000;McGowanetal.,2006). The combination of these abnormalities in IgG levels and distributionhasbeenassociated witha poorresponsiveness to vaccinationsinadultsandchildren(Cagigietal.,2010)andtoa higher incidence of B-cell malignancies observed in HIV ART-treatedpatients(Bohliusetal.,2009;HentrichandBarta,2016).

InHIVpregnancy,thedysregulationofB-cells,andinparticular maternalhypergammaglobulinemia, hasa signiﬁcant impacton IgGtransferfrommothertofoetus(Palmeiraetal.,2012),causinga saturationofFcRNreceptors,whichhavea keyrolein transpla-cental passage (de Moraes-Pinto et al., 1998). A reduced IgG concentrationinthecordofneonatesfrommotherswithIgGlevels of>15g/landinadequatelevelsofspeciﬁcIgGofmaternalorigin hasbeenextensively reportedinHIV-exposedinfants( Cumber-landetal.,2007;Babakhanyanetal.,2016).

Inthelastdecade,ARTadministrationtoHIV-positivewomen duringpregnancyandthebreastfeedingperiodhasresultedina strongreductionintherateofverticaltransmission(JointUnited NationsHIV/AIDSProgramme,2017);this in turnhasledtoan increasingrate of HIV-exposeduninfected (HEU) children, who neverthelesshave a signiﬁcantly higher rate of morbidity and mortalityin theﬁrstyearsof lifecompared totheirunexposed counterparts (Brennan et al., 2016). Efforts made towards understandingthehighervulnerability ofHEUchildrenarestill inconclusive.

Inthiscontext,amaternalIgGdisordercanhaveacriticalrole consideringtheimportanceofmaternalimmunologicalprotection duringtheﬁrst6monthsoflife,whenthehumoralresponsesto infectionsaremostlysustainedbyIgGofmaternalorigin(Simon etal.,2015).

InthisstudyinvolvingMalawianHIV-positivepregnantwomen intheOptionBera,theaimswere(1)tomonitorthemagnitude andthetimingoftotalIgGandIgGsubclassnormalization(ifany) after24monthsofART,and(2)todeterminethedurabilityofthe beneﬁcialeffectsofARTonplasmaIgGlevelsandthedistribution ofthesubclassesinwomenwhointerruptedARTafter6months andwerefollowedupto24months.

Materialsandmethods Studypopulation

Thisstudyformspartofalargerobservationalstudy(enrolment duringFebruary2008toFebruary2009)aimedatassessingthe safetyandefﬁcacyofARTadministrationinMalawianHIV-positive pregnantwomen(Safe MilkforAfrican Children(SMAC)study) (Giuliano et al., 2013). The study was conducted in Malawi (BlantyreandLilongwesites)withintheDREAM(DrugResource Enhancement against AIDS and Malnutrition) Program of the CommunityofS.Egidio,anItalianfaith-basednon-governmental organization.

In thisstudy, treatment-naiveHIV-infectedpregnantwomen startedARTduringpregnancy:inthepresenceofaCD4+cellcount <350cells/

m

l, theyreceiveda combination ofstavudine (30mg twicedaily),lamivudine(150mgtwicedaily),andnevirapine(200 mgtwicedaily)assoonaspossibleaftertheﬁrsttrimesterand continueditindeﬁnitely(groupC,continuoustherapy);or,inthe caseofaCD4+count>350/

m

l,theyreceivedzidovudine(300mg

twicedaily),lamivudine,andnevirapinefromweek26ofgestation until6monthspostpartum(groupI,interruptionof treatment). Womenwereinstructedtoexclusivelybreastfeedupto6months. Afterdelivery,themotherswerevisitedmonthlyforclinicaland laboratory assessment up to24 months postpartum. Haemato-chemistryandviro-immunologicalanalyseswereperformedatthe localDREAMlaboratoriesinMalawi.Womenwereincludedinthis sub-study if they breastfed their infants for the scheduled 6 monthsandhadplasma samplesavailable atenrolment(before ART)andat6and24monthspostdelivery.

PlasmaIgGlevelsandIgGsubclasses

MaternaltotalIgGandsubclassplasmalevelsweredetermined using IgG total, IgG1, IgG2, IgG3, and IgG4 reagents(Siements; SiemensHealthcareDiagnostics)andreadbyautomated nephe-lometry (BN ProSpec System Analyzer; Siemens Healthcare Diagnostics).

Statisticalanalysis

IBMSPSSStatisticsversion25.0(IBMCorp.,Armonk,NY,USA) wasusedforthestatisticalanalyses.Resultsarepresentedasthe median values with interquartile range (IQR) or percentages. DifferenceswereevaluatedusingtheChi-squaretestorFisher’s exacttest,asappropriate,forcategoricalvariables,andbyMann– WhitneyU-testforquantitativevariables.Longitudinaldifferences weredetectedusingtheWilcoxontest,andSpearman’scorrelation coefﬁcient was used for the correlation analysis between quantitative variables. Differences were considered statistically signiﬁcantwhenp<0.05.

Ethicalconsiderations

Ethical approval was obtained from the National Health Research Committee in Malawi (approval number #486), and informedconsent was obtainedfrom allindividualparticipants includedinthestudy.

Results

The characteristics of the two groups of women (group I, interruption of treatment,n=18; groupC, continuoustherapy, n=19) are reportedin Table 1.Group C women wereolder (p =0.022),withhigherHIV-RNAlevels(p=0.020),andwithamore immunocompromised proﬁle. The median time of ART during pregnancywas 9.0weeks, withaslightdifference betweenthe groups(groupI:7.0weeks,IQR4.5–13.0weeks;groupC:10weeks, IQR8.0–15.0weeks;p=0.051).Aftervaginaldelivery,allwomen continuedthetherapyfor6themonthsofexclusivebreastfeeding. IgGlevelsandimmunologicalparametersbeforeARTinitiation

Atenrolment,thetotalIgGconcentrationwas_>15g/lforall butonewoman,with medianlevelsof 24.1g/l ingroupIand 27.8g/l in group C, and no statistically significant difference betweenthegroups(p=0.221).ThecompositionofIgGsubclasses showedapredominanceoftheIgG1isotype,whichaccountedfor morethan90%inbothgroups,aswellasanunderrepresentation ofIgG2(5.2%)(Table1).Althoughnosignificantdifferenceswere observed in IgG levels between the groups, a strong inverse correlationwasfoundbetweentotalIgGlevelsandtheCD4+cell count(r= 0.426,p=0.009;Figure1).TheassociationwithCD4+ cells was also confirmed for IgG1 and IgG3 plasma levels (r= 0.419, p=0.010;r= 0.340,p=0.039,respectively), but not forIgG2orIgG4levels.

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ChangesinIgGproﬁleafter6monthsofART,attheendofthe breastfeedingperiod

At6months,signiﬁcantimprovementsin the viro-immuno-logicalparameters wereobservedin bothgroups, andHIV-RNA plasmalevelsbecameundetectablein mostwomen (groupI:n =15,83.3%;groupC:n=16,84.3%).TheCD4+cellcountincreased signiﬁcantlyfrombaselineinbothgroups(groupI:+204cells/

m

l; groupC:+182cells/

m

l),andtheCD4+countremainedbelow350 cells/

m

linonlysevenofthe37women.

However,hypergammaglobulinemiawasstillpresentin34of the37 women, even thougha signiﬁcant decreasein total IgG levelswasobservedinbothgroups(groupI: 6.5g/l,IQR 3.1to +0.05,p=0.010;groupC: 8.0g/l,IQR 3.5 to 0.50,p=0.006;

Table2,Figure2).Regardingthesubclasscomposition,thetrendin changes was similar in the two groups, with some difference: whilea significant decreasein thecytophilicisotype(IgG1and IgG3) levels was observed in group I (IgG1, p=0.004; IgG3, p=0.016) and in group C (IgG1, p<0.0001; IgG3, p<0.0001), IgG2 levels showed a modest increase that reached statistical significanceonlyforthewomeningroupI(p=0.003).IgG4levels decreased minimally (statistically significant only in group I, p=0.031).

The6–24-monthperiod

According tothe Option Bstrategy, onlygroup C, including women with a more severe viro-immunological proﬁle at enrolment,continuedARTafterthebreastfeedingperiod.Inthis group,all womenreached HIV-RNA <50copies/ml anda CD4+ count>350cells/

m

l(median560cells/

m

l,IQR 420–730cells/

m

l) after24 monthsoftreatment. Thelevelsof totalIgG decreased between 6 and 24 months, but after 2 years,14of 19 women (73.7%) still had hypergammaglobulinemia, with a median IgG levelof19.0g/l(IQR14.3–23.7g/l).ThedecreaseintotalIgGwas paralleled by a signiﬁcant decrease in IgG1 levels ( 2.3g/l, p<0.0001)andalesspronounceddecreaseinIgG3( 0.23g/l,p =0.453)andIgG4( 0.04g/l,p=0.004).IgG2levelsdidnotshow anysigniﬁcantchange,andafter24monthsofARTtreatmentno recoverywasobservedinplasmaandtheIgG2isotyperemained largelyunderrepresented.

Asexpected,15of18womeningroupIwereviremicatmonth 24(HIV-RNAmedian3.42logcopies/ml),whiletheCD4+cellcount remainedstablewithamedianof629cells/

m

l(+66cells/

m

l,IQR 21.3to+307cells/l;p=0.05)withrespecttopre-ARTvalues.All womeninthisgrouphadanIgGconcentration>15g/lat2years postpartum.ThemedianlevelofIgGreturnedtovaluessimilarto thosepre-ART(24.9g/lvs.24.1g/l;p=0.616),withnovariationin subclassdistribution.

Discussion

TheseresultsshowthatinHIV-positivepregnantwomen,24 months of ART can improve the IgG abnormalities, but is not sufﬁcienttonormalizetheIgGhyperproductionortorestorethe normal subclassdistribution.Overall,24monthsofARThadno impactontheIgG2levels,whichremainedstrongly underrepre-sented.ItwasalsofoundthatashortperiodofART(about7–10 months),althoughassociatedwithatemporaryimprovementin IgGanomalies,wasnotsufﬁcienttomaintainthesechangesover2 yearspostpartum.

Polyclonalhypergammaglobulinemiaoccurs inthesetting of HIVinfection(Cagigietal.,2008),causedbyT-celldysregulation and bya direct interactionbetweenmatureB-cells andvirions (MoirandFauci,2008).ManystudieshavereportedthatARTcan partially reversesome immunological dysfunctionof theB-cell compartment(Chongetal.,2004;Redgraveetal.,2005;Abudulai etal.,2016),decreasingtheIgGhyperproductionoveravariable intervaloftime,dependingmostlyonthetimesinceHIVprimary

Figure1.CorrelationbetweentheCD4+cellcount(cells/ml)andtotalIgGlevels(g/ l)inHIV-positivepregnantMalawianwomenbeforeARTinitiation(r= 0.426, p=0.009).GroupI:whitedots;groupC:blackdots.

Table1

Patientcharacteristicsatenrolment;valuesareexpressedasthemedian(interquartilerange),orpercentage.

All GroupI GroupC p-Value

Number 37 18 19

Age(years) 27.0(23.0–32.0) 23.5(20.8–30.0) 30.0(25.0–33.0) 0.022 Weight(kg) 57.0(49.0–62.7) 56.9(50.6–60.8) 57.3(47.4–64.0) 0.855 Haemoglobin(g/dl) 10.3(9.5–11.1) 10.6(9.8–11.5) 9.8(9.1–10.0) 0.142 WHOclassiﬁcation,I/II/III(%) 78.4/13.5/8.1 94.4/5.6/0.0 63.1/21.1/15.8 0.060 ARTduringpregnancy(weeks) 9.0(7.0–13.0) 7.0(4.5–13.0) 10(8.0–15.0) 0.051 CD4+(cells/ml) 340(214–503) 503(401–776) 223(152–302) <0.0001 HIVRNA(logcopies/ml) 4.17(3.49–4.69) 3.74(3.51–4.46) 4.54(3.78–4.75) 0.020 IgGtotal(g/l) 25.1(21.2–29.3) 24.1(19.5–27.7) 27.8(21.3–30.9) 0.221 IgG1(g/l) 23.5(18.6–28.4) 22.2(16.8–26.3) 25.3(18.9–28.3) 0.245

ProportionoftotalIgG(%) 91.8(87.5–93.9) 92.3(87.4–93.6) 91.6(87.6–94.6)

IgG2(g/l) 1.44(0.96–1.82) 1.38(0.93–1.81) 1.57(0.94–1.85) 0.753 ProportionoftotalIgG(%) 5.2(3.9–7.8) 5.4(4.2–8.0) 5.8(3.4–7.6)

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infection(Pensierosoetal.2009;Moiretal.,2010).Althoughthe majority of these studies involved patients from developed countries, where better sanitary conditions, a lack of chronic parasite infections, and the wider availability of different therapeuticapproachescanhaveanimportantimpact,analogous effectsofARTonthememoryB-cellsandactivationofB-specific markers have been observed in the few studies performed in African cohorts, reporting incomplete recovery of the B-cell anomaliesinducedbyHIV(Longweetal.,2010;Tankoetal.,2017). ThisstudyfocusedontheIgG profile,since hypergammaglo-bulinemia in pregnancy has been associated with severe im-pairment of transplacental passage and can have important consequencesfor theclinicaloutcomeof theinfants(Abu-Raya etal.,2016).In apreviousstudyinvolvingwomen intheSMAC study,withARTinitiatedatweek26ofgestation,wecorrelatedthe highIgGlevelsofmothersatdeliverytothereducedIgGlevels observedintheir1-montholdinfants(Baroncellietal.,2018).We concluded that the short period of ART exposure may not be sufficientformaternalIgGnormalization.Hereweextendedthe study to 24 months of observation in an attempt to better understandthedynamicsofIgGlevelsanddistributioninAfrican HIV-positive pregnant women receiving ART. The inclusion of women who interrupted therapy according to the Option B strategygaveustheopportunitytoexplorethedurabilityofthe beneficialeffectsofARTontheIgGlevels.

Atbaseline,HIV-positive,treatment-naïvewomenhadmedian levelsofIgGashighas25.1g/l,conﬁrmingthat hypergammaglo-bulinemiaiscommonintheHIV-infectedAfricanpopulation,as alreadyreportedbyusandbyothers(deMoraes-Pintoetal.,1998; Baroncellietal.,2018).Itwasalsofoundthatthehighlevelsoftotal IgGandIgG1werenotassociatedwiththeviralburden,butrather withtheCD4+ cellcount,suggestingthattheT-celldysfunction couldbetheprimaryforceleadingtothegeneraldysregulationof theimmunesystem(Malaspinaetal.,2003).Theseresultsarein agreementwiththose of previous studies that havefound IgG abnormalitiesassociatedwithmarkersofimmunoactivationand to the decline in T-cell functionality (Abudulai et al., 2016 D’Orsogna et al., 2007; Tanko et al., 2017). The IgG isotype distributionwasdominatedbyIgG1,whichrepresentedmorethan 90%oftotalIgG,followedbyIgG3(5.3%),whiletheIgG2isotype, whichaccountsfor20–30%oftotalIgGinthenormalpopulation (Vidarssonetal.,2014;Puissant-Lubranoetal.,2015),accounted foronly5.2%.Thepolarizationofimmunoglobulinstowardsthe IgG1andIgG3subclassesisacharacteristicofHIV-inducedB-cell dysregulation(Rauxetal.,2000),butwecannotexcludethatthe predominanceofthecytophilicisotypeinwomencouldalsobea

consequenceofexposuretomalariainfection,whichinMalawihas a prevalenceofaround24%in thegeneral population(National Malaria Control Programme (NMCP) and ICF, 2018). Cytophilic antibodiesmediateparasite-killingresponses(Scopeletal.,2006), and their increase in populations living in areas endemic for Plasmodiumfalciparumhasbeenwidelyreported(Rouhanietal., 2015;Dobañoetal.,2019).

ThelongitudinalmonitoringoftheIgGproﬁleshowedasigniﬁcant reductionintotalIgG,IgG1,IgG3,andIgG4levelsinthewomeninboth groupsafter6monthsofART.Nevertheless,onlyasmallpercentageof women(8.1%)reachedIgGlevelsbelowtheclinicalcut-offindicating hypergammaglobulinemia,whilemostofthem reachedviralload suppressionandaCD4+cellrecoveryof>350cell/

m

l.Thesignificant trendinreductionofIgGandisotypesIgG1,IgG3,andIgG4(butnot IgG2)wassimilarinthetwogroups,indicatingthebeneficialeffectsof 6monthsofARTalsoinwomenstartingwithamorecompromised viro-immunologicalprofile.However,6monthsofARThadnoeffect onIgG2levelsingroupCwomen,andwasassociatedwithamodest increaseingroupIwomenthat,althoughstatisticallysignificant,did notseemtobeofclinicalrelevance.

In women on continuous therapy, a slow but statistically significantprogressivedecreaseintotalIgGandIgG1continued, although73.7%ofwomenstill hadIgGlevelsabove15g/lat24 months postpartum, and no significant improvement was ob-servedintermsofsubclassproportions.AgainIgG2levelsdidnot change with ART, remaining at the pre-therapy levels. In agreement with others (Tanko et al., 2017), it was found that thepersistenceofIgGdisorderswasnotassociatedwiththeviral load(undetectableinallwomen).Indexesofimmuneactivation werenotmeasuredinthestudywomen,butitislikelythatthe persistenceofin_flammatorycytokines,ahallmarkofHIV-induced immune activationstatus (Brenchleyetal.,2006;Mehrajet al., 2019), couldhave affectedIgGisotype switching(Tangyeet al., 2002)andsubclasssynthesis(Kawanoetal.,1994).

ItisinterestingtonotethatthebeneﬁcialeffectofARTonIgG levelsingroupIwomenwasonlytransitory,and18monthsafter theinterruptionofARTallwomenhadpre-ARTIgGvalues,while theCD4+ cellcountsremainedhigher withrespecttobaseline. Althoughfunctionaltestswerenotperformed,thestudyresults conﬁrmthat thehumoraldysfunctionsin patientstreatedwith ARThavea differentialtiminginrecoverywithrespecttoCD4+ cells,probablydependingonthepatient’sinitialimmunological condition(d’Orsogna et al.,2007; Moir et al.,2010; Pensieroso etal.,2009).

Thisstudyhasseverallimitations.Thepatientsamplesizewas limited, but thelow degreeof variabilityin IgG measurements

Table2

Patientviro-immunologicalcharacteristicsat6and24monthspostpartum;valuesarereportedasthemedian(interquartilerange),orpercentage.

6Months 24Months

GroupI(n=18) GroupC(n=19) GroupI(n=18) GroupC(n=19)

ARTregimen AZT3TCNVP d4T3TCNVP None d4T3TCNVP

CD4+(cells/ml) 733(585–940) 402(333–539) 629(492–875) 560(420–734) CD4+increase(cells/ml) +204(50–366) +182(116–271) 35( 191to+47.5) +197(91–258) HIVRNA>50copies/ml,n(%) 3(16.7%) 3(15.7%) 15(83.3%) 0(0%) IgGtotal(g/l) 20.5(17.9–22.5) 21.1(18.8–28.2) 24.9(21.4–27.8) 19.0(14.3–23.7) PatientswithIgG>15g/l(%) 15/18(83.3%) 19/19(100%) 18/18(100%) 14/19(73.7%)

IgG1(g/l) 17.5(15.7–20.3) 19.4(15.9–25.6) 23.6(19.2–27.4) 16.4(12.8–21.6) IgG1change(g/l) 4.0( 8.6to+0.2) 3.6( 8.3to 1.4) +3.7(+1.5to+8.4) 2.3( 5.4to 0.7) IgG2(g/l) 1.47(1.25–1.89) 1.59(1.15–2.20) 1.36(1.13–1.77) 1.43(1.20–2.13) IgG2change(g/l) +0.16(+0.05to+0.27) +0.14( 0.04to+0.40) 0.11( 0.46to+0.07) 0.20( 0.17to+0.05) IgG3(g/l 1.02(0.61–1.31) 1.10(0.79–1.41) 1.10(0.83–150) 0.97(0.8–1.34) IgG3change(g/l) 0.25( 0.52to+0.03) 0.23( 0.50to 0.09) +0.03( 0.10to+0.33) 0.23( 0.50to 0.09) IgG4(g/l) 0.19(0.12–0.32) 0.27(0.14–0.34) 0.23(0.13–0.33) 0.21(0.12–0.34) IgG4change(g/l) 0.02( 0.19to+0.11) 0.04( 0.12to+0.02) +0.02( 0.46to+0.03) 0.04( 0.10to+0.00) ART,antiretroviraltherapy;AZT,zidovudine;3TC,lamivudine;NVP,nevirapine;d4T,stavudine.

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allowsustobequiteconfidentregardingthevalidityofthedata. Unfortunatelyitwasnotpossibletoperformfunctionaltestsonthe B-cellcompartment,whichwouldhaveallowedamorecomplete analysisofthehumoraldysfunction.Further,IgGreferenceranges basedmostlyonWesternpopulationvalueswereused,whichcan differ from those of African populations (Pieters et al., 1997; Bénigueletal.,2004)andcouldmaketheevaluationoftheIgG abnormalitiesinapopulationalreadyexposedtoenvironmental factorsthathaveanimpactonthereconstitutionoftheimmune profileduringARTdifficult.

InconclusionitwasfoundthatinMalawianwomenreceiving ART,thetrendofIgGrecoveryisprogressivebutslowandisstill incomplete after 24 months in terms of total IgG levels and proportions.

Considering the physiological importance of IgG acquisition throughtransplacentalpassageforinfanthealth,thereductionof hypergammaglobulinemia in HIV-positive pregnant women should bemonitored under the current Option B+approach to assessthebeneﬁtsofthestrategyinreducingtheriskofinfection inHIV-exposedinfants.

Figure2.LongitudinalchangesintotalIgGandsubclasses.Valuesareexpressedasthemedian+standarderror.(A)TrendoftotalIgGlevels;groupIgreydots,groupCwhite dots.GraphsB–EshowtheIgGsubclasslevels.Greybars:T1(pre-ART);whitebars:month6(endofbreastfeedingperiod);dottedbars:month24(endofstudyperiod).

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Funding

ThisworkwassupportedbyagrantfromtheMinistryofHealth, Rome,Italy (grantnumber 3C04/1), by Esther-Italy,Ministryof Health(grantnumber9M34).

Conﬂictofinterest

Theauthorsdeclarethattheyhavenoconﬂictofinterest. Acknowledgements

WethankAlessandraMatteiandStefaniaDonniniforproviding secretarialhelp,MarcoMirra,MassimilianoDiGregorio,Stefano Lucattini,andLucaFuciliforITsupport,andFerdinandoCostaand PatriziaCocco fortechnicalsupport.WearegratefultoRiccardo d’AmicofortheprecioustechnicalsupportwiththeBNProSpec SystemAnalyzer,andtoallthemotherswhoparticipatedinour study.

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