Safety of live, attenuated oral vaccines in HIV-infected Zambian adults Oral vaccines in HIV

ContentslistsavailableatSciVerseScienceDirect

Vaccine

jo u r n al h om ep a ge : 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

Safety

of

live,

attenuated

oral

vaccines

in

HIV-infected

Zambian

adults

Oral

vaccines

in

HIV

Rose

Banda

_,

_Vera

_Yambayamba

_,

_Bwalya

_Daka

_Lalusha

_,

_Edford

_Sinkala

_,

Melissa

Chola

Kapulu

_,

_Paul

_Kelly

a_{TropicalGastroenterology&NutritionGroup,DepartmentofMedicine,UniversityofZambiaSchoolofMedicine,UniversityTeachingHospital,Lusaka,Zambia} b_{BlizardInstituteofCellandMolecularScience,Barts&TheLondonSchoolofMedicineandDentistry,QueenMaryUniversityofLondon,TurnerStreet,LondonE12AD,UK}

a

r

t

i

c

l

e

i

n

f

o

Received29February2012

Receivedinrevisedform15June2012 Accepted28June2012

Available online 10 July 2012 Keywords: HIV Oralvaccines Rotavirusvaccine Typhoidvaccine ETECvaccine Phase1study

a

b

s

t

r

a

c

t

Background:CurrentrecommendationsarethatHIV-infectedpersonsshouldnotbegivenlivevaccines.

Wesetouttoassesspotentialtoxicityofthreelive,attenuatedoralvaccines(againstrotavirus,typhoid andETEC)inaphase1study.

Methods:Twocommerciallyavailableoralvaccinesagainstrotavirus(Rotarix)andtyphoid(Vivotif)and

onecandidatevaccineagainstEnterotoxigenicEscherichiacoli(ACAM2017)weregiventoHIVseropositive (n=42)andHIVseronegative(n=59)adults.Gastrointestinalsymptomsweresoughtactivelybyweekly interviewupto1monthofvaccination.Inrotavirusvaccinerecipients,intestinalbiopsieswerecollected byendoscopyandevaluatedforexpressionofIL-8andpro-inflammatorycytokines.

Results:NodifferencewasobservedbetweensymptomsinHIVinfectedandHIVuninfectedvaccinees,

exceptfordiarrhoeareportedmorethan7daysafterthelastdoseofvaccine.Ifonlydiarrhoeaepisodes within7daysofvaccinationareincluded,diarrhoeawasnotmorefrequentinHIVseropositivethan inHIVseronegativevaccinees(OR6.7,95%CI1.2–67;P=0.09).However,iflaterepisodesofdiarrhoea areincluded,asignificantincreaseindiarrhoeawasdemonstrated(OR5.3,95%CI0.98–53;P=0.04). Allepisodesweremildandtransient.IL-8wasslowlyup-regulatedovertheweekfollowingvaccination (P=0.02),butIL-␤,IFN␥orTNF␣werenot.

Conclusions:Noevidencewasfoundofadverseeventsfollowingadministrationofthesethreevaccines,

exceptforlateepisodesofdiarrhoeawhichmaynotbeattributabletovaccination.Ourdatadonot supporttheneedforaprohibitiononoraladministrationoflive,attenuatedvaccinestoallHIVinfected adults,thoughfurtherworkonseverelyimmunocompromisedadultsandchildrenarerequired.

© 2012 Elsevier Ltd.

1. Introduction

Diarrhoealdiseaseremains oneof thecommonest causesof deathinchildren,especiallyinthemalnourished.Upto2million childrendieofdiarrhoeaeachyear,anddiarrhoeahaseffectson long-termdevelopmentandgrowth[1,2].InZambia,the preva-lenceofdiarrhoeainchildrenunder5years ofageisveryhigh, with21.2%ofmothersreportingdiarrhoeaina2-weekperiod[3]. Diarrhoeainchildrenismostlyattributedtorotavirusand Entero-toxigenicEscherichiacoli(ETEC).Theprospectsforglobalprovision ofadequatequantitiesofcleanwaterareasdistantasever,with probablyover1billionpeopleunabletoaccesssafedrinkingwater. Vaccinesagainstrotavirus,choleraandtyphoidareavailable,but

∗_{Correspondingauthorat:BlizardInstituteofCellandMolecularScience,Barts} &TheLondonSchoolofMedicineandDentistry,QueenMaryUniversityofLondon, TurnerStreet,LondonE12AD,UK.Tel.:+442078827191;fax:+442078827192.

E-mailaddress:m.p.kelly@qmul.ac.uk(P.Kelly).

somearelive,attenuatedvaccineswhichwouldneedtobeused inpopulationswithhighHIVprevalence.Itwouldalsobe desir-abletoofferprotectionagainstdiarrhoea-causingpathogenstoHIV infectedadultsandchildren,soitisimperativetodetermineifthese vaccinesaresafeinHIVinfectedindividuals.

Recent highly authoritative guidelines [4] on prevention of opportunistic infection in HIV-infected children recognise the potential risk of giving live,inactivated vaccines to potentially immunocompromisedchildren but make it clearthat few data areavailableonthemagnitudeofthat risk.Arecentanalysisof rotavirusinrelationtoHIV,andtheexperienceofatrialinSouth AfricainwhichHIVinfectedchildrenweregivenarotavirus vac-cine,dosuggestthatitissafe[5].Theorallive,attenuatedcholera vaccineCVD103HgRwasfoundtobesafeinHIV-infectedadults inMali[6],andthereisevidencethatoralpoliovaccineissafein HIVinfectedchildren[7].However,uncertaintyremainsduetothe paucityofdatainAfricanpopulations[8,9].

In order toaddress theseconcerns we analysed our experi-enceofgivinganyofthreelive,attenuatedvaccinestoZambian 0264-410X© 2012 Elsevier Ltd.

http://dx.doi.org/10.1016/j.vaccine.2012.06.079

Open access under CC BY license.

Table1

PrimersusedforquantitativePCRforintestinalcytokineexpression.

Forward(5to3) Reverse(3to5)

IL-1␤ CGGCCACATTTGCTAAGA AGGGAAGCGGTTGCTCATC

TNF␣ CCCAGGCAGTCAGATCATCTTC GCTTGAGGGTTTGCTACAACATG

IFN␥ ACTGACTTGAATGTCCAACGCA ATCTGACTCCTTTTTCGCTTCC

IL-8 TGTGTGTAAACATGACTTCCAAGCT GCAAAACTGCACCTTCACACAG

GAPDH CCAGCCGAGCCACATCGCTC ATGAGCCCCAGCCTTCTCCAT

adults.Boththebacterialvaccinesare knowntobesensitiveto ciprofloxacinandsowewereconfidentthatthisevaluationwas safeinthecarefullymonitoredsettinginwhichtheyweregiven. Intheevent,noneoftherecipientsneededanymedicalsupport orantibiotictreatment.Asrotavirusvaccinationprogrammesare rolledoutacrosssub-SaharanAfrica,itisimportanttoassessthe potentialtoxicityofthis vaccinein HIVinfection,soasubsetof participantsreceivingtherotavirusvaccineunderwentintestinal biopsytoevaluateexpressionofIL-8,IL-␤,IFN␥andTNF␣. 2. Methods

2.1. Participants

ThestudywasconductedinLusaka,Zambia,betweenFebruary 2008andOctober2009.ParticipantsweredrawnfromtheMisisi cohort,amixedcohortofHIVseropositiveandseronegativeadults, whichisdefinedonlybyresidenceinadefinedarea[10]. Poten-tialparticipantswerenotgivenvaccinesiftheywerepregnantor lactating,hadexperienceddiarrhoeawithin1monthbeforetheir plannedparticipation,hadtakenantibioticsornon-steroidal anti-inflammatorydrugsinthesameperiod,hadbeenvaccinatedwith anyothervaccinewithin6months,orwerefoundtohave infec-tionwithanintestinalhelminthbyexaminationof3stoolsamples takenovera3–5-dayperiod.Ethicalapprovalwasobtainedfrom theUniversityofZambiaResearchEthicsCommittee(007-10-07) andallparticipantsgavewritteninformedconsent.

2.2. Vaccineadministration

Rotarix(Glaxo SmithKline, Brentford,UK)is derivedfrom a humanrotaviruswhichwasattenuatedbyrepeatedpassageand issafeinchildren[11].Thesecondvaccine,ACAM2017(Acambis plc,Cambridge,UK),wasderivedfromaspontaneousLT-negative ETECisolatewhichhasdeletionsofthechorismatesynthasegene aroC,themembraneproteinsompCandompF,andthetoxingenes forLT,STandEAST.ThegeneforCS1[12]hasbeenaddedandit inducesspecificmucosalIgAagainstcolisurface(CS)antigensCS1, CS2andCS3[13].Thethirdvaccine,Vivotif(Ty21avaccine;Berna Biotech,Bern,Switzerland),istheonlylicensedoraltyphoid vac-cine[14].ThegenegalEisinactivatedanditisunabletoexpress thepathogenicityfactorVi;ithasanexcellentsafetyrecord. Vivo-tifisimmunogenicinthehostbuthaslowenvironmentalsurvival. Vivotifinducesanti-LPSsystemicIgGandIgA,antibodysecreting cellsinperipheralblood,andcellmediatedimmunity(CMI). Immu-nityconferredbyTy21alastsupto7yearsbutuniquelyrequires 3–4dosesgivenonly1–2daysapart[15].Rotarixwas adminis-teredasasingledosein1.3mlliquidcarbonatebufferaccording tothemanufacturer’sinstructions.ACAM2017wasadministered asasuspensionofbacteriapreparedbyAcambisplcaspreviously described[13].Thedoseofviableorganismsinthevaccinevials (3×1010_{)wasconfirmedinthelaboratoryinthreetestvialswhich} werediscardedinordertoavoidcontaminationofthevialsused forvaccination.Thedosecontainedineachvialwasadministered asonedoseandthevialwasthendiscarded.Vivotifwas adminis-teredasacapsule,initiallyasasingledosein23participants,then

2doses(ondays1and3)in5participants,then3doses(thefull dosingscheduleondays1,3,and5)inafurther5participants,then thefullschedulefortheremainderofthestudywhensafetyand acceptabilityconcernshadbeenallayed.Altogether,81participants receivedVivotif.

2.3. Adverseeventrecording

Eachparticipantwasinterviewedat7,14,21and28daysafter vaccine administration. Direct questions were asked aboutthe experienceofthesymptomslistedinTable2.Fullbloodcountdata collectedpriortovaccineadministrationandeither7or14days afterwardswerealsocompared.

2.4. Intestinalcytokineexpression

Jejunalbiopsieswerecollectedendoscopicallyusingan Olym-pus SIF-10 endoscope under diazepam sedation as previously described[16–18].Biopsieswereobtained1daypriortovaccine administrationandat1(n=4),2(n=6),4(n=6),or7(n=5)days afterthefirstvaccinedose.Eachparticipantunderwenttwo endo-scopiesandthesebiopsieswereevaluatedasabefore/afterpair. Biopsieswerecollectedinto200␮lTriReagent(Sigma,Poole,UK) andsnap-frozeninliquidnitrogenfollowedbystorageat−80◦C. Biopsieswereusedwithin3months,andRNAisolatedaspreviously described[18].Followingreversetranscription,realtime quantita-tivepolymerasechainreaction(RT-qPCR)wascarriedoutusing SYBRGreenenzymebuffer(Qiagen)withprimersshowninTable1 for thefollowing cytokines: interleukin (IL)-8, IL-1␤,interferon (IFN)-␥,andtumournecrosisfactor(TNF)-␣.

2.5. Dataanalysis

DiarrhoeaorotherAEsattributabletovaccinewereconsidered iftheonsetwaswithin7daysofthelastdoseofvaccine.AllAEs werecomparedinHIVseropositiveversusHIVseronegative partic-ipantsandproportionsanalysedusingFisher’sexacttest.Cytokine mRNAmeasurementswerenormalisedtoGAPDHandexpressed as-foldchangefrombaselinetopost-vaccinationsample,and sta-tisticalsignificanceevaluatedusingtheWilcoxonsigned-ranktest todetermineiftherewasasignificantchangeingeneexpression followingvaccination.ForIL-8mRNA,whereatemporaltrendwas apparentfollowingvaccination,linearregressionofmRNAandtime wasusedtoconfirmstatisticalsignificance.

3. Results

Between February2008 and October 2009,100 participants betweentheagesof18and60yearswererandomlyallocatedto receiveoneof thethree vaccines:Rotarix(n=24),ETEC (n=21) orVivotif(n=81),ortoactascontrolswhoreceivednovaccine (n=21).Forty-sevenoftheseparticipantswhowereavailablewere subsequentlyinvitedtoparticipateonasecondoccasion,eitheras vaccineeorcontrol,attimepointsseparatedbyintervalsofatleast 1year.Novaccineereceivedthesamevaccinetwice.Demographic andclinicalcharacteristicsoftheparticipantsareshowninTable2.

Table2

Demographicandclinicalcharacteristicsofvaccinerecipients.

Rotavirusvaccine(n=24) ETECvaccine(n=21) Typhoidvaccine(n=81) Control(n=21) P

Sex(M:F) 10:14 11:10 29:52 7:14 0.52

Age(yrs;median,IQR) 35(29–41) 42(35–55) 39(29–47) 42(30–50) 0.37

HIVinfected(n,%) 9(38%) 7(33%) 39(48%) 11(52%) 0.36

BMI(kg/m2_{)(median,IQR) 22.3(19.3–26.9) 21.6(19–24.6) 23.0(19.26.3) 20.4(19.2–24.6) 0.66}

CD4counta_{(median,IQR) 417(294–576) 380(292–402)}b _{374(245–545)}c _{406(294–541) 0.29}

CD4count≤200cells/␮l 0 0 6 1

Pvaluesrefertoresultsofstatisticaltestingacrossallgroups.

a_{CD4countexpressedascells/␮l.} b_{Dataonlyavailablefor6samples.} c _{Dataonlyavailablefor38samples.}

Altogether,34HIVseropositiveadultsreceived58coursesoflive, attenuatedvaccinesorallyatonetimepointoranother.Vaccinees andcontrolswerewellmatchedforsex,age,bodymassindex,and (intheHIVseropositives)CD4count(Table2).

3.1. Clinicaladverseevents

Diarrhoeawasreportedwithin7daysofthelastdoseofvaccine by6participants,allofwhomhadreceived3dosesofVivotifand 5ofwhomwereHIVseropositive(ORforHIVseropositivity6.3, 95%CI0.67–303;P=0.09).Theintervalsafterwhich thesewere experiencedwere3,4,4,8,10,and13daysafterthefirstdose. Noneofthesehaddiarrhoeawhichtheyjudgedtohavebeen seri-ousenoughtoseektreatmentbuttwohadtakenthedayoffwork. TheCD4countsofthoseHIVseropositiveparticipantswho experi-enceddiarrhoeawithin7daysoflastvaccineadministrationwere (inascendingorder)175,179,351,670,and845cells/␮l.

Iftheperiodofattributionisextendedto28daysafterthefirst doseofvaccine,11episodesofdiarrhoeawerereportedby10 vac-cinees.Ofthese,3werewithin7days,5between8and14days, 2between15and21days,and1between22and28days.Ofthe 10vaccineeswhoexperienceddiarrhoea,8wereHIVseropositive (Table3).ThetwoHIVseronegativevaccineesreporteddiarrhoea 13daysafterVivotifand21daysafterACAM2017.Includingthese laterepisodesofdiarrhoeachangestheOddsRatioforHIV seropos-itivityto5.3(95%CI0.98–53;P=0.04).

Abdominalpainwasreportedby3vaccinerecipients.Intwo oftheseinstances,painoccurredduringdiarrhoealillnesses,with onset4and10daysafterthefirstdosesofVivotif.Oneparticipant reportedpainwithoutdiarrhoea5daysafterthefirstdoseofVivotif. Fever(subjective,notconfirmed)wasreportedbyoneHIV neg-ative man the dayafter rotavirusvaccination, and by two HIV positivemen13and16daysafterETECvaccination,respectively. Noneoftheseparticipantssoughtmedicalcare.

Loss of appetite (scoring 1 onanalogue scale of 1–10) was reported only by one HIV seronegative participant within 24h of receiving ACAM2017. Three other HIV positive participants reportedlossofappetite,butallover3weeksafterthevaccinedose anddesignatednotattributable.OnlyoneHIVseronegative partici-pantreportednauseaorvomiting,andthatwas12daysafteradose ofVivotif.

3.2. Intestinalcytokineexpression

Allrecipientsoftherotavirusvaccineunderwententeroscopy forjejunalbiopsycollection,butRT-PCRresultsdidnotmeetquality standardsfortwosamples,andoneparticipantdidnotreturnonthe scheduledday,sodataareavailablefor21participants.ByRT-qPCR, mRNAofIL-8showedanimmediatedown-regulationfollowedby aslowup-regulationwhichwasstatisticallysignificant(P=0.02) in a regression model against time (Fig. 1). Therewas no dis-cernibleeffectofvaccinationonIL-1␤(Fig.2)orIFN␥(Fig.3).TNF␣ expressionwasundetectableinaconsiderablenumberofsamples:

in6casestherewasnodetectableexpressionbeforeorafter vacci-nation;in5casesmRNAwasdetectedonlybeforevaccination,and in5casesonlyaftervaccination.Intheremaining5cases,there wasamodestdown-regulation,butthiswasnotstatistically sig-nificantinviewofthesmallnumber ofdatapairs.HIV-infected participantsdidnotdifferfromHIV-uninfectedparticipantswith respecttochangesincytokineexpressionfollowingvaccination, andthosebiopsiesinwhichTNF␣expressionwasnotdetectable werenotmorelikelytocomefromHIV-infectedparticipants(data notshown).

4. Discussion

Thesafetyoflive,attenuatedvaccinesinHIVinfectedpeople is of paramountimportance ifvaccines are to play anyrole in reducingtheburdenofcommondiseasesintropicalpopulations. Inthis studywefoundthatin34 HIVseropositiveadultsgiven

Fig.1. ChangesinIL-8mRNAexpression1,2,4,or7daysafterrotavirusvaccine administration.(A)Eachpointrepresentsthe-foldchangeinmRNAtranscript abun-dance(relativetoGAPDH)fromthebaselinevaluetothevalueobtainedinthebiopsy takenatthattimepoint.Eachindividualparticipantwasbiopsiedtwice(baseline andatthetimepointshown)soeachpointrepresentsalltheinformationobtained fromoneparticipant.Theseindividualchangesareshowninpanel(B).Thechange overtimeinIL-8mRNAshowsastatisticallysignificanttrend(P=0.02)using lin-earregression.CirclesrepresentHIVseronegativeindividualsanddiamondsHIV seropositiveindividuals.

Table 3 Reported adverse events in Zambian adults given live, attenuated oral vaccines. Rotavirus ETEC Vivotif Total vaccinated P HIV+ ( n = 9) HIV − ( n = 15) HIV+ ( n = 7) HIV − ( n = 14) HIV+ ( n = 42) HIV − ( n = 39) HIV+ ( n = 58) HIV − ( n = 68) Diarrhoea (within 7 days of last dose of vaccine) 0 0 0 0 5 1 5 1 0.09 Diarrhoea (within 28 days of first dose of vaccine) 0 0 3 1 6 a 1 9 2 0.04 Abdominal pain 0 0 0 0 3 0 3 0 0.09 Fever 0 1 0 0 0 0 0 1 1.00 Loss of appetite 0 0 0 1 0 0 0 1 1.00 Nausea/vomiting 0 0 0 0 0 0 0 0 – Fall in Hb > 1 g/dl 3 2 0 3 4 3 7 8 1.00 Fall in WBC >1 × 10 9/l 0 3 1 0 7 7 8 10 1.00 The P value shown relates to HIV seropositive vs. seronegative participants (i.e. not including the second episode of diarrhoea in that vaccinee who experienced two episodes) in the total group of those vaccinated, using Fisher’s exact test. a Two of these episodes were in the same vaccinee.

Fig.2.ChangesinIL-1␤mRNAexpression1,2,4,or7daysafterrotavirus vac-cineadministration.(A)Eachpointrepresentsthe-foldchangeinmRNAtranscript abundance(relativetoGAPDH)fromthebaselinevaluetothevalueobtainedin thebiopsytakenatthattimepoint.Eachindividualparticipantwasbiopsiedtwice (baselineandatthetimepointshown)soeachpointrepresentsalltheinformation obtainedfromoneparticipant.Theseindividualchangesareshowninpanel(B). atotalof58coursesofthreelive,attenuatedoralvaccinesthere wasnoevidenceofseriousadverseevents:nohospitalisations,no episodesofdiarrhoea requiringtreatment, nosignificantfebrile illnesses,andnoincreaseinsymptomssuchasabdominalpain, nauseaorlossofappetite.Therewasnoevidenceofhaematological toxicity.Ifweacceptthatoralvaccinesdonotcausediarrhoeaafter

Fig.3.Changesininterferon-␥mRNAexpression1,2,4,or7daysafterrotavirus vaccineadministration.(A)Eachpointrepresentsthe-foldchangeinmRNA tran-scriptabundance(relativetoGAPDH)fromthebaselinevaluetothevalueobtained inthebiopsytakenatthattimepoint.Eachindividualparticipantwasbiopsiedtwice (baselineandatthetimepointshown)soeachpointrepresentsalltheinformation obtainedfromoneparticipant.Theseindividualchangesareshowninpanel(B).

7dayshaveelapsedbeyondthefinaldoseofvaccine,therewasno increaseindiarrhoea.Theinterpretationofdiarrhoeadatainthis settingisdifficultifweuseHIVseronegativeadultsasthe compar-isongroup,asatanygivenpointintimeHIVinfectedadultshavea higherincidencerateofdiarrhoealdisease[19].Webelievethatthis explainsthehigherdiarrhoeaincidenceafter7daysfollowing vac-cination.Ourdataarecompatiblewiththehypothesisthatthese vaccinesleadtoamodestincreaseinmild,transientepisodesof diarrhoeabeyond1weekinHIVinfectedadults.Theyarealso expli-cablewiththerebeingaconsistentlyincreasedriskofdiarrhoeain HIVthroughouttheperiodofobservation.

Wefoundnoevidencethatvaccinesinduceintestinal inflamma-tion.IL-8isachemokineexpressedbyepithelialcellsoncontact withpotentiallyinvasive bacteria.Theother,pro-inflammatory, cytokinesshowednochangeinexpressionovertheweekfollowing vaccination.Whilethesedatadonotruleoutapathogeniceffectof thesevaccines,theyofferconsiderablereassurancethatrotavirus vaccinedoesnotinduceinflammation.Wearenotawareofother studieswhichhaveexaminedintestinaltissuefollowing vaccina-tionagainstwhichwecancompareourdata.Wild-typerotavirus infectionleadstosignificantmucosalinflammationandalthough thisinflammatoryresponseisnotfullycharacterisedinhumans, thereisevidencethatatleastinterferon-␥isimplicatedinthe sys-temicresponse[20].Incellculturemodelsusingratandhuman cells,TNF␣,IFN-␤andIL-6wereinducedbyrotavirusdsRNA[21]. Inanimalmodels,anearlyIL-8responseisseen[22].Ourdataare surprisinginasmuchastheIL-8responsewasdelayed,appearing torisefromaninitialdown-regulation,forupto7days.

Theparticipantsweenrolledweredrawnfromacommunity cohortstudywheremostHIVinfectedadultshavebeenoffered, and agree to,monitoringin anHIV treatmentprogramme, and takeHAARTwherenecessary.Only6ofourparticipantshadCD4 countsbelow200cells/␮l,allofwhomhadexperiencedarapid dropinCD4countfromtheirpreviousclinicvisit.Thuswe can-notbeconfidentthatthesevaccinesaresafeinadultswithsevere immunodeficiency(althoughthebacterialstrainsaresensitiveto ciprofloxacinandcouldbeeasilytreatedifsymptomsdevelop).For certaininfections,parenteralvaccinesareavailable(suchastheVi polysaccharidevaccinefortyphoid)ororalkilledvaccines(such asthekilledwhole-cellcholeravaccinewhichhasbeenshownto besafeinanoutbreakinMozambique[23]).However,oral admin-istrationoflive,attenuatedvaccinescombinestheadvantageof easeofadministrationonalargescalewithgoodimmunogenicity, atleastover2–3years,andthesevaccinesremainattractivefor furtherdevelopment.Whileourfindingsneedtobeconfirmedin largerstudies,theydosuggestthatsafetymaynotbeanobstacle toexploitingthepotentialfororalvaccinationinsouthernAfrica, andwedonotsupporttheview[9]thatliveoralvaccinesshouldbe withheldfromallHIV-infectedadults.However,furtherstudiesare neededofvaccinesafetyinseverelyimmunocompromisedadults andchildren.

Conflictsofinterest

Theauthorshavenocommercialorotherassociationswhich mightposeaconflictofinterest.Thefundingagencyplayednopart inthecollectionofdata,analysis,orpreparationofthemanuscript. Acknowledgements

The authors are grateful to Webby Mbuzi and Michelo Simuyandiforlaboratorywork,andtotheothermembersofthe

clinicalteam forvaccineadministration and followup: Stayner MwanamakondoandRoseSoko.

Financialsupport:Financialsupportwasobtainedfromthe Well-comeTrust,UK[grantnumber067948].

References

[1]PetriWA,MillerM,BinderHJ,LevineMM,DillinghamR,GuerrantRL.Enteric infections,diarrhea,andtheirimpactonfunctionanddevelopment.JClinInvest 2008;118:1277–90.

[2]CheckleyW,BuckleyG,GilmanRH,AssisAMO,GuerrantRL,MorrisSS,etal. Multi-countryanalysisoftheeffectsofdiarrhoeaonchildhoodstunting.IntJ Epidemiol2008;37:816–30.

[3] CentralStatisticalOffice(Zambia),CentralBoardofHealth(Zambia)andORC Macro.ZambiaDemographicandHealthSurvey2001–2002.Calverton, Mary-land,USA:CSO/CBH/ORCMacro;2003.

[4]MofensonLM, BradyMT, Danner SP, DominguezKL, Hazra R, Handels-manE,etal.Guidelinesforthepreventionandtreatmentofopportunistic infectionsamongHIV-exposedandHIV-infectedchildren:recommendations fromCDC,theNationalInstitutesofHealth,theHIVMedicineAssociation oftheInfectiousDiseasesSocietyofAmerica,thePediatricInfectious Dis-easesSociety,andtheAmericanAcademyofPediatrics.MMWRRecommRep 2009;58(September):1–166.

[5]SteeleAD,CunliffeN,TumboJ,MadhiSA,DeVosB,BouckenoogheA.Areview ofrotavirusinfectioninavaccinationofHIV-infectedchildren.JInfectDis 2009;200(Suppl.1):S57–62.

[6]PerryRT,PloweCV,KoumareB,BougoudogoF,KotloffKL,LosonskyGA,etal. AsingledoseofliveoralcholeravaccineCVD103-HgRissafeand immuno-genicinHIV-infectedandHIV-noninfectedadultsinMali.BullWHO1998;76: 63–71.

[7]MossWJ,ClementsCJ,HalseyNA.Immunizationofchildrenatriskofinfection withHIV.BullWHO2003;81:61–70.

[8]ObaroSK,PugatchD,LuzuriagaK.Immunogenicityandefficacyofchildhood vaccinesinHIV-1-infectedchildren.LancetInfectDis2004;4:510–8. [9]RivasP,HerreroMD,PuenteS,Ramirez-OlivenciaG,SorianoV.Immunizations

inHIV-infectedadults.AIDSRev2007;9:173–87.

[10]KellyP,ZuluI,AmadiB,MunkantaM,BandaJ,RodriguesLC,etal.Morbidity andnutritionalimpairmentinrelationtoCD4countinaZambianpopulation withhighHIVprevalence.ActaTrop2002;83:151–8.

[11] Ruiz-PalaciosGM,Perez-SchaelI,VelazquezFR,AbateH,BreuerT,Clemens SAC,etal.Safetyandefficacyofanattenuatedvaccineagainstsevererotavirus gastroenteritis.NewEnglJMed2006;354:11–22.

[12]TurnerAK,BeavisJC,StephensJC,GreenwoodJ,GewertC,ThomasN,etal. ConstructionandphaseIclinicalevaluationofthesafetyand immunogenic-ityofacandidateEnterotoxigenicEscherichiacolivaccinestrainexpressing colonisationfactorantigenCFA/I.InfectImmun2006;74:1062–71.

[13]DaleyAC,RandallR,DarsleyM,ChoudhryN,ThomasN,SandersonIR,etal. GeneticallymodifiedEnterotoxigenicEscherichiacolivaccinesinducemucosal immuneresponseswithoutinflammation.Gut2007;56:1550–6.

[14]GirardM.P.SteeleD,ChaignatC-L,KienyMP.Areviewofvaccineresearchand development:humanentericinfections.Vaccine2006;24:2732–50. [15] Levine MM,FerreccioC,Black RE,GermanierR. Largescalefieldtrialof

Ty21aliveoraltyphoidvaccineinenteric-coatedcapsuleformulation.Lancet 1987;i:1049–52.

[16]KellyP,Bajaj-ElliottM,KatubulushiM,ZuluI,PoulsomR,FeldmanRA,etal. Reducedgeneexpressionofintestinal␣-defensinspredictsdiarrhoeaina cohortofZambianadults.JInfectDis2006;193:1464–70.

[17]Dhaliwal W, Shawa T, Khanam M, Jagatiya P, Simuyandi M, Ndulo N, etal. Intestinal antimicrobial gene expression:impact of micronutrients inmalnourishedadultsduring arandomisedtrial.JInfectDis2010;202: 971–8.

[18]KapuluMC,SimuyandiM,SianongoS,MutaleM,KatubulushiM,KellyP. Differ-entialchangesinexpressionofintestinalantimicrobialpeptidegenesduring AscarislumbricoidesinfectioninZambianadultsdonotrespondtohelminth eradication.JInfectDis2011;203:1464–73.

[19]KellyP,ToddJ,SianongoS,MwansaJCL,SinsungweH,KatubulushiM,etal. SusceptibilitytointestinalinfectionanddiarrhoeainZambianadultsinrelation toHIVstatusandCD4count.BMCGastroenterol2009;9:7.

[20]JiangB,Snipes-MagaldiL,DennehyP,KeyserlingH,HolmanRC,BreseeJ,etal. Cytokinesasmediatorsfor,oreffectorsagainst,rotavirusdiseaseinchildren. ClinDiagnLabImmunol2003;10:995–1001.

[21]SatoA,IizukaM,NakagomiO,SuzukiM,HorieY,KonnoS,etal.Rotavirus double-strandedRNAinducesapoptosisanddiminisheswoundrepairinrat intestinalepithelialcells.JGastroenterolHepatol2006;21:521–30. [22]RolloEE,KumarKP,ReichNC,CohenJ,AngelJ,GreenbergHB,etal.Theepithelial

responsetorotavirusinfection.JImmunol1999;163:4442–52.

[23]LucasMES,DeenJL,vonSeidleinL,WangXY,AmpueroJ,PuriM,etal. Effective-nessofmassoralcholeravaccinationinBeira,Mozambique.NewEnglJMed 2005;352:757–67.