c o m / l o c a t e / v a c c i n e Dang Duc Anha,∗, Nguyen Van Tranga, Vu Dinh Thiema, Nguyen Thi Hien Anha, Nguyen Duc Maob, Yuhuan Wangc, Baoming Jiangc,∗∗, Nguyen Dang Hiend,1, Le Th
Trang 1Vaccine
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
Dang Duc Anha,∗, Nguyen Van Tranga, Vu Dinh Thiema, Nguyen Thi Hien Anha, Nguyen Duc Maob, Yuhuan Wangc, Baoming Jiangc,∗∗, Nguyen Dang Hiend,1, Le Thi Luand,1, the Rotavin-M1 Vaccine
Trial Group2
a The National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam
b Preventive Medicine Center, Phu Tho Province, Viet Nam
c Centers for Disease Control and Prevention, CDC, Atlanta, GA, USA
d Center for Research and Production of Vaccines and Biologicals, Hanoi, Viet Nam
Article history:
Received 13 May 2011
Received in revised form 14 July 2011
Accepted 25 July 2011
Keywords:
Rotavirus vaccine
Rotavin-M1
Vietnam
Safety
Immunogenicity
Vaccine dose
Rotarix TM
© 2011 Elsevier Ltd All rights reserved
夽 Presented in part: 9th International Rotavirus Symposium, June 2010,
Johannes-burg, South Africa.
夽夽 Disclaimer: The findings and conclusions in this report are those of the authors
and do not necessarily represent the views of the Centers for Disease Control and
Prevention.
∗ Corresponding author Tel.: +84 439712989; fax: +84 438212660.
∗∗ Corresponding author Tel.: +1 404 639 2861; fax: +1 404 639 3645.
E-mail addresses: ducanh@nihe.org.vn , ducanhnihe@hn.vnn.vn (D.D Anh),
bxj4@cdc.gov (B Jiang).
1 Drs Hien and Luan are members of the Board of Directors, POLYVAC, Vietnam.
2 See Appendix A
1 Introduction Rotavirus (RV) is the most important cause of acute gas-troenteritisin children worldwide In Vietnamrotavirus causes
an estimated 122,000–140,000 hospitalizations and 2900–5400 deathsperyearamongchildrenunder5yearsofage[1].Overthe past13years,sentinelhospitalsurveillanceidentifiedrotavirusin 44–62%ofchildrenadmittedforthetreatmentofacutediarrheain Vietnam[2–4].Suchahighburdenofdiseasejustifiedaccelerated developmentofanewandlocallymanufacturedvaccineagainst rotavirusinVietnam.Itisestimatedthatifavaccinewas intro-ducedinthecurrentchildhoodimmunizationschedule,itcould 0264-410X/$ – see front matter © 2011 Elsevier Ltd All rights reserved.
Trang 2vaccineefficaciesandcoverage[5]
TheGovernmentofVietnamhaspursuedapolicyto
encour-agelocalvaccineproductionsothecountrycouldbeself-reliant
with affordable vaccines for its population [6] Over the past
decades, several locally produced vaccines for poliomyelitis,
cholera,Japaneseencephalitis,andDiphtheria–Pertussis–Tetanus
have contributed to the reduction in the prevalence of these
diseasesandtothestatusofpoliomyelitis-free.Whiletwo
com-mercialrotavirusvaccines,RotarixTM(GSK,Belgium)andRotaTeq®
(Merck),havebothbeentestedinVietnam,onlyRotarixTMis
cur-rentlyavailableinprivatemarket.TheliquidformulaofRotarix
when tested in two schedules, 1-month and 2-month interval
betweendosescomparedwithplacebocontrolin375childrenhad
aseroconversionrateof63.3%and81.5%,respectively[7].RotaTeq
showedaseroconversionrateof87.8%andanoverallefficacyof
63.9%(72.3%inthefirstyearand64.6%inthe2ndyear
following-up)inaphase3efficacytrialinVietnam[8].However,neitherof
thetwovaccinesiscurrentlyavailableatanaffordablepricefor
thenationalprogram(e.g.RotarixintheprivatemarketcostsUS
$35perdose).Therefore,thecandidatevaccine,Rotavin-M1,was
developedinordertofillthisneedforamoreaffordablevaccinefor
Vietnamesechildren[6].ThisvaccineissimilartoRotarixTM,and
wasdevelopedbyselectingacommonG1P[8]strainand
attenu-atingitthroughserialpassagesandplaquepurificationinqualified
VerocellsunderGLPconditions
Inthisstudy,wesoughttoevaluatethesafetyand
immuno-genicityofRotavin-M1producedbytheCenterforResearchand
ProductionofVaccinesandBiologicals(POLYVAC)inadult
volun-teersandininfantsinVietnam.Inaddition,weevaluateddifferent
dosagesandschedulestodeterminethebestregimentotestina
clinicaltrial
2 Materialsandmethods
2.1 Vaccine
Thevaccine,Rotavin-M1,manufacturedbyPOLYVAC-Vietnam,
wasdevelopedfromaG1P[8]strainrecoveredin2003fromachild
hospitalizedforthetreatmentofacutegastroenteritisinNhaTrang
city(KH0118-2003)[6].Themasterandworkingseedsofthis
vac-cinewereproducedunderGLPconditionsusingqualifiedVerocells
andreagentsattheUSCentersforDiseaseControlandPrevention
(CDC).Pilotvaccinelot,passage48,wasproducedbyonepassage
inVerocellsfromtheworkingseed,whichwasprovidedbythe
JapanesePolioResearch Instituteandapprovedforvaccine
pro-ductionbyWHO.Thesecellshavebeenusedfororalpoliomyelitis
vaccineproductionatPOLYVAC.Themastervirusseedfor
Rotavin-M1wastestedforporcinecircovirususingreal-timeRT-PCRatthe
USCDCandappearedtobefreeofporcinecircovirusDNA.Thetest
forporcinecircovirusinpilotvaccinelotwasnotdone
2.2 Participantsandstudydesign
Thetrials wereplannedin two stages, thefirst –a Phase 1
trial for safety in adult volunteers of a high titer preparation
of thevaccine (106.3FFU/dose) Whenresults of this trialwere
evaluatedbytheDataSafetyandMonitoringCommitteeandthe
vaccine was deemed to be safe for further study in infants, a
Phase1and2adaptivetrialwasconducted.Thistrialassessedthe
safetyandimmunogenicityoftwodifferentpreparationsof
vac-cine,oneoflowtiter(106.0FFU/dose)and thesecondwithhigh
titer(106.3FFU/dose) that wasadministered in either a 2 vs 3
doseschedulestoinfants6–12weeksofage.Acomparisongroup
wasincludedof infantswhoreceivedthelyophilizedRotarixTM
vaccine,anestablishedrotavirusvaccineofGSKthatwaslicensed
tobeusedinVietnam.ThestudywasconductedaccordingtoGood ClinicalPracticeandinaccordancewiththeDeclarationofHelsinki,
asamendedinSomersetWest,RepublicofSouthAfrica,inOctober
1996.Theprotocolandconsentformwasreviewedandapproved
bytheEthicalandScientificCommitteesoftheNationalInstitute
ofHygieneandEpidemiology(NIHE)andoftheMinistryofHealth, GovernmentofVietnam,priortoinitiatingthestudy
2.2.1 Phase1study ThePhase1studywasconductedinaCareerTrainingSchool, ThanhSondistrict,PhuThoprovincewithatotalof29healthyadult volunteers18–49yearsofage.Followingreceiptofinformed con-sent,eachofthevolunteerswasscreenedbyaphysiciantoensure theywerehealthywithnoactivemedicalproblemsandaskedto provideabloodspecimentotestforbloodcountsandlevelsofblood ureanitrogen(BUN)andtransaminase.Thevolunteerstheneach received2dosesofthehightitervaccine,106.3focus-formingunits [FFU],at1-monthinterval.Afteradministrationofeachdoseofthe vaccine,thevolunteerswerefolloweddailyfor10daysforadverse eventsandforfecalsamplecollection.Duringthenext20days,the volunteerswerefollowedbyphonetoensuretheyhadnosequelae (e.g.diarrhea,vomitingandintussusception).Serumsamplestaken beforeand30daysafterthe1stand2nddosesweretestedfor3 bloodcounts,BUNconcentrationandserumtransaminaselevels DataonthevolunteerswerereviewedbytheDataSafety Monitor-ingBoard(DSMB).Noadverseeventsorchangesinbloodcounts, BUNortransaminasewerereported.TheDSMBjudgedthevaccine
tobesafepermittingthestudiestocontinueininfants
2.2.2 Phase2study Phase2wasadoseandschedulerangingstudy,conductedat
12medicalcentersinThanhSondistrict,PhuThoprovincesfrom November2009throughApril 2010.Infants6–12weeksofage wereeligibleforinclusioninthestudyiftheywerebornatfull term(38weeks)andwerefreeofobvioushealthproblem.Infants wereexcludediftheywereimmunocompromised,hadahistoryof allergicreactiontoanyvaccinecomponentsorhadreceived vac-cinesagainstrotavirusorwereinvolvedinanyothervaccinetrials
atthesametime.Infants(n=200)wererandomlyassigned to5 groups(40infants/group)(Fig.1).Twogroupsreceived2oraldoses
ofRotavin-M1in1of2titers–106.0or106.3FFUat6–12weeks
ofage(forthefirstdose)and2monthslaterfortheseconddose (groups2Land2H),respectively.These2vaccinetiterswerealso giventoinfantsona3-doseschedule,beginning at6–12weeks
ofageforthefirstdoseand1monthand2monthslaterforthe 2ndand3rddoses(groups3Land3H,respectively).RotarixTMwas usedasthevaccinecontrolandwasgivento40infantsat6–12 weeksofageand 1monthlater(GroupRotarixTM).GSK recom-mendsthatthefirstdoseofRotarixTMbestartedbetween6and14 weeksofageandthattheseconddosebeseparatedbyatleast1 month.Thevaccinerecipients,theparents/guardians,the labora-torystaff,thefieldteamsandworkingdoctorsdidnotknowthe codingassignmentofthesegroups.Othervaccines(BCG,oralpolio vaccine,Diphtheria–Tetanus–PertussisandhepatitisB)usedinthe country’sExpandedProgramofImmunization(EPI)were adminis-terednormallytotheseinfantsondifferentdays(10–20daysbefore
orafterrotavirusvaccinewasadministered)
2.3 Assessmentofimmunogenicity Serumsampleswereobtainedfortestinglevelsofanti-rotavirus IgAand IgGantibodyonthedaythatthefirstdosewas admin-isteredand1month afterthesecondorthirddose.Inaddition, serum samples were also obtained from groups that received
3 dosesof vaccine (groups3L and 3H) immediatelybeforethe
Trang 3Fig 1 Study design including vaccination and sampling schedules for different
vac-cination groups Arrows in figure indicate times for each vaccination dose (wide
arrows) and for collection of samples of blood (thin solid arrows) and stool (dotted
arrows) In 3-dose regimen (a), groups 3L and 3H received 3 doses of Rotavin-M1
10 6.0 FFU/dose and 10 6.3 FFU/dose, respectively In 2-dose regimen (b), groups 2L and
2H received 2 doses of Rotavin-M1, 10 6.0 FFU/dose and 10 6.3 FFU/dose, respectively
(n = 40/group) Group Rotarix TM received 2 doses of Rotarix TM and only had serum
samples 1 and 2 according to schedule (c).
3rddose(Fig.1).Eachbloodsample froma childwascollected
in2tubes,onewithanti-coagulant(EDTA)(wholeblood)andone
withoutanti-coagulant(serum).Serumandwholebloodsamples
wereimmediatelytransferredtotheprovincialhospitalfor
anal-ysisofblood cellcounts(redblood cells,white bloodcells and
platelet),transaminaselevels(aspartateaminotransferase,ASTand
alanineaminotransferase,ALT)andBUNwithin4haftercollection
Aliquotsofserumsamplesforantibodydetectionwerestoredat
−20◦CattheDistrictPreventiveMedicineCenter,ThanhSon
dis-trict,PhuThoProvinceuntiltheywereshippedtothelaboratoryat
NIHE
Theimmunogenicityofthevaccinewasevaluatedatthe
Vac-cineImmunologyLaboratory,NIHE,bymeasuringseroconversion
ofrotavirus IgAantibody,usingan end-pointELISA[9].Briefly,
96-wellmicrotiterplates(NUNC,Langenselbold,Germany)were
coatedwithrabbit-antiRRVhyperimmuneserum(obtainedfrom
Dr BaomingJiang, CDC) Virus (RRV)and mock-infected
super-natantwereaddedtotheplatesinalternatewells.Serumsamples
in 2-fold serial dilutions starting at 1:10 were added tothese
virus/mockwells.Biotinylatedanti-humanIgA(␣)(Kirkegaardand
PerryLaboratory,Gaithersburg,Maryland)andperoxidaselabelled
extravidin(Sigma–Aldrich,Inc,St.Louis,MO)wereaddedforthe
detectionofRVspecificIgAantibody.Positiveandnegativecontrol
seraweretestedinthesamemanner.Antibodytitersinserumwere
calculatedasthereciprocalofthehighestdilutionthatgaveamean
opticaldensitygreaterthanthecut-offvalue(mean+3standard
deviationsofthenegativecontrolandblottowells).AnIgAtiterof
20orhigherwasconsideredpositive.Seroconversionwasdefined
asariseinanti-rotavirusIgAtiterfromundetectable(≤10)in
pre-vaccinationserumto≥20inpost-vaccinationserumora≥4-fold
risefrompre-vaccinationtopost-vaccinationserum.Forquality
assurance,ananonymizedsubsetofserumspecimens(52samples) werealsoshippedandtestedatCDC.Agreementbetweentwo lab-oratories(antibodytiterswithin2-folddilutionofthesamples)was
>90%
2.4 Assessmentofreactogenicityandsafety For30daysfollowingeachvaccineadministration,parentsor guardianswereaskedtonotegeneralsymptoms(cough,running nose,diarrhea,irritability,lossofappetite,feverandvomiting)ona dailydiarycard.Dailytemperaturewasrecordedandatemperature
>38◦Cwasconsideredasfever.Anysevereunsolicitedsymptoms andseriousadverseeventswerereportedthroughoutthestudy period(90daysforeachchild).Aliquotsofbloodfromeachchild
ateachtimepointwerealsoassayedforserumtransaminaseand BUN
2.5 Viralsheddingandstraincharacterization
Weattemptedtocollectdailystoolsamplesduringthe7days followingeachdosetoassessvirusshedding.Inaddition,stool sam-pleswerealsocollectedateveryepisodeofdiarrheaduringthe studyperiodandtestedforrotavirusantigenbyELISA(ProSpecT, Oxoid,UK).AllrotaviruspositivespecimenswereGandP-typed
by RT-PCR[3,10].To distinguishvaccinefrom wildviruses,we sequencedthe VP7 gene of theG1P [8]samples fromdiarrhea casesandselectedG1P[8]samplescollectedwithin7daysof vac-cineadministration(non-diarrhealsamples),usinganABIPrism BigDyeTerminatorCycleSequencing(AppliedBiosystems,Foster City,CA)andcomparedthesequenceswiththecorrespondinggene sequencesofRotavin-M1andRotarixTM
2.6 Statisticalanalysis DatawasmanagedusingMicrosoftVisualFoxpro7.0software (Microsoft)andanalysedusingtheStata11.1program.Themean bloodcellcountsandconcentrationsofBUNandtransaminasewere comparedbetweengroupswithaPairedStudent’st-Test.Between groups,thepercentagesofchildrenwithadverseeventswere com-paredusingFisher’sExactTest
Theanalysisforreactogenicitywasperformedonthe intention-to-treatpopulation(includingallchildrenwhoreceivedatleast1 doseofvaccine).Thenumberofchildrenwithgeneralsymptoms wasdeterminedforeachgroupafteradministrationofeachvaccine doseandcomparedbetweengroups
Theanalysisofimmunogenicitywasalsoperformedforboththe perprotocolandintention-to-treatpopulations(atleast2dosesof vaccinewererequired).TheIgAseroconversionrate(with95%CI) wascalculatedforeachgrouptoevaluatetheimmuneresponses inducedbythevaccinesandgeometricmeanantibodytiters(GMT) were calculated for those individuals who seroconverted.Viral sheddingwascalculatedasthepercentage ofchildrenshedding viruseachdaypost-vaccinationwhenstoolsampleswereavailable
Inaddition,thepercentofchildrenwhoshedatleastonceduring the7-dayobservationperiodaftereachdosewasalsocalculated
3 Results 3.1 Safetyinadultvolunteers
We firsttestedthesafety of2doses ofthehighertiter vac-cine (106.3FFU/dose) in 29 adult volunteers aged 18–40 years Duringthe30dayspost-vaccinationofeachdose,nodiarrheaor severeadverse reactionwasreported byanyof thevolunteers One month aftereach dose, neitherblood cell counts norBUN concentrationincreased.Serumtransaminaselevelsstayedbelow
Trang 410%ofvolunteersafter2dosesofvaccination.Oneindividualhad
elevatedlevelsofbothSGOTandSGPT(71and48IU/ml,
respec-tively)beforevaccinationandthelevelsremainedinthis range
after2dosesofvaccine.Nosheddingofthevaccinevirusoccurred
in these adults following vaccination Thus the Ethical Review
Committeesallowedthevaccinetobetestedfurtherin healthy
infants
3.2 Infantstudy
Atotalof200subjects(119boysand81girls)wereenrolledin
theinfantstudy.Theirmeanage(±SD)was8.7±1.6weeksatthe
timetheyreceivedthefirstdoseand17.2(±1.6)weeksatthetime
of2nddoseforgroups2Land2H.Forgroups3Land3H(the3-dose
group),themeanagewas13(±1.6)weeksatthetimeof2nddose
and17.9(±1.6)atthe3rddose.Aftereachvaccinedose,the
chil-drengainedweightandheightnormallyandwefoundnodifference
betweenvaccinationgroups.Thebloodcellcounts,serum
transam-inaselevelsandBUNwerenormalandnosignificantincreasewas
observedovertherangeofnormalhealthyinfantsafter
adminis-trationofeachvaccinedose
3.3 Reactogenicityandsafetyofinfantstudy
Duringthe entireobservationperiod (90days afterthefirst
dose), no serious adverse events that required hospitalization
and no cases of intussusception were recorded We observed
all infants for acute reactions for 30min following
vaccina-tion and parents observed their infants for 30 days aftereach
dosetonoteadverseeventsincludingdiarrhea,fever,vomiting,
loss of appetite, cough, allergy, abdominal pain and
irritabil-ity These symptoms following vaccination were grouped into
3 time periods: immediate reactions(i.e within 30min), short
term reactions (within 7 days post-vaccination) and longer
term reactions (from 8 through 30 days post-vaccination)
(Table1)
Aftereachdose,noimmediatereactionswereobserved.After
anydosefewerchildren reportedanysymptomswithin7 days
comparedtothe3-weekperiodfrom8to30dayspast
vaccina-tion.Fewerchildrenreportedanysymptomsafterdose2anddose
3,comparedwithdose1.Irritabilityand feverwerethe2most
frequentlyreportedsymptomsfollowingadministrationanydose
ofRotarixTMorRotavin-M1butnoneofthedifferencesbetween groupsreachedsignificance
Ofspecialnotes, within7daysafterreceivingthefirstdose,
3 children from group 3L (7.5%), 3 from group 2H (7.5%), 1 fromgroup3H(2.5%)and1fromgroupRotarixTM(2.5%) exhib-itedmilddiarrhea.Giventhesmallnumbers,thisdifferencewas notstatisticallysignificantandsuggestedthat thevaccinevirus hadbeenadequatelyattenuated(Table1).Rotavirusantigenwas isolated in fecal specimens from 1 case in each of the groups RotarixTM,3Hand2H duringthisperiod.Fromdays8–30, diar-rheaepisodes were reported only in groups RotarixTM and 3H (1 and 4 cases, respectively),of which only one case in group 3H was positive for rotavirus While a few infants had mild diarrhea after administration of dose 2 or 3, only 1 case in group3H (within 7 days afterdose2) and 1 case in group 3L (within7daysafterdose3)wereidentifiedasrotavirusG1P[8] SequencesofVP7geneofthesesamplesrevealedthattheywere 100%homologouswiththesequenceofRotavin-M1orRotarixTM
(inrespectivegroups).Ofnote,RotarixTM andRotavin-M1share 93.6%homologyinthe793nucleotidesequenceofVP7geneand 94.7%homologyinthe263aminoacidsequenceoftheencoded protein
3.4 Immunogenicity SerumsampleswereanalysedatNIHEandanonymizedresults were confirmed at CDC Most infants (94.5%) did not have detectableRV-IgAbeforevaccinationandallchildrenwithone pre-vaccinationserumandatleastonepost-vaccinationserumsamples wereincludedin theanalysisof immunogenicity Oneof the2 childrenwhowasseropositivebeforevaccinationseroconverted (group3H,datanotshown).Onemonthafterthe2nddoseof vac-cine,therateofseroconversiontoRotavin-M1vaccinewas61% (95%CI(45%,76%))forgroup2L(106.0FFU)and73%(95%CI(58%, 88%))forgroup2H(106.3FFU)(Table2).TheIgA-GMT,rangingfrom
76(group2H)to89(group2L),didnotdifferbetweenthesetwo groups.Forgroupsreceiving3dosesofvaccines(groups3Land3H), anti-RV-IgAseroconversionratesat1monthafter2dosesof vac-cinewere51%(95%CI(36%,67%))forgroup3L(106.0FFU)and61% (95%CI(45%,77%))forgroup3H(106.3FFU).TheIgAseroconversion rates1monthafterdose3increasedto56%(95%CI(39%,71%))in group3Land63%(95%CI(46%,79%))ingroup3H.TheIgA-GMTdid notincreasesignificantlyingroup3H(from61postdose2to83
Table 1
Number of infants with adverse events after vaccination with Rotavin-M1 or Rotarix TM during 30 days after each dose Diarrhea is defined as having more than 3 loose stools per day Diarrhea in these children is not accompanied with vomiting or fever (>37.5 ◦ C) In the majority of cases, diarrhea lasted 1–4 days Difference between groups was not significant *Figure in bracket indicates the number of samples positive for Rotavin-M1 or Rotarix vaccine.
Trang 5Table 2
Anti-RV-IgA antibody responses after administration of Rotavin-M1 vaccine (groups 2L, 2H, 3L and 3H, compared to Rotarix TM ).
Note: a Number of subjects for immunogenicity analysis (intention-to-treat) b Number of subject for immunogenicity analysis (per protocol) c Percentage of subjects sero-converted for RV-IgA d 95%CI, exact 95% confidence interval e GMT: geometric mean titers (and 95%CI) calculated for seropositive samples only.
Forchildrenreceiving3dosesofvaccine(groups3Land3H),
serumsampleswerecollected1monthafterdose2and1month
afterdose3todeterminewhetherathirddosemightimproved
theseroresponse.The3rddoseinducedseroconversionin5and3
morechildreningroup3Land3H,respectively,whohadfailedto
seroconvertafterthefirst2doses.Themajorityofchildren(14in
group3Land16ingroup3H)convertedafterseconddoseanddid
notfurtherconvertafterthethirddose.Threechildren(7.5%)from
eachgroup(3Land3H)seroconvertedafterbothdose2anddose
3
3.5 Viralsheddingaftereachdose
Weexaminedthekineticsofrotavirussheddinginvaccinated
children(Figs.2and3).Theprevalenceofchildrensheddingvirus
wasgreaterinthegroupofchildrenwhoreceivedRotarixTM(65%
afterthe1stdose)vs.anygroupthatreceivedRotavin-M1(44–48%
afterthe1stdose)(Fig.2).Furthermore,afterthefirstdose,
shed-ding of RotarixTM peaked 1 or 2 days earlier than shedding of
Rotavin-M1(Fig.3).Nonetheless,weobservedlittledifferencein
Fig 2 Dynamics of viral shedding within 7 days post-vaccination after each dose.
Percent of children shed virus in stool per day after each vaccination dose was
illus-trated The ratio was calculated per number of stool available each day because not
all children could produce stool every day for 7 days Stool samples were
consid-ered positive for RV if it is positive in ELISA (ProSpecT) and confirmed by G and P
genotyping All the samples collected during 7 days after each dose are typed as
G1P [8] Asterisk: group Rotarix TM ; opened triangle: Group 2L, 2 doses, 10 6.0 FFU;
opened circle: Group 2H: 2 doses, 10 6.3 FFU/dose; closed triangle: Group 3L, 3 doses,
0%
10%
20%
30%
40%
50%
60%
1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
2L 3L Rotarix 3H 2H
Days after dose 1 Days after dose 2 Days after dose 3 Fig 3 Shedding of vaccine virus after each dose Percent of infant shed virus at any time during 7 days after each dose was illustrated Asterisk: group Rotarix TM ; opened triangle: Group 2L, 2 doses, 10 6.0 FFU; opened circle: Group 2H: 2 doses,
10 6.3 FFU/dose; closed triangle: Group 3L, 3 doses, 10 6.0 FFU/dose; and closed circle: Group 3H, 3 doses, 10 6.3 FFU/dose.
thepatternofsheddingbetweenthe4groupsreceived Rotavin-M1.Viralsheddingreducedsignificantlyinanygroupafterdose2 (6–20%)(Fig.2).Interestinglyafterdose3,30–37%ofchildrenshed thevirusatday3post-vaccinationinboth3Land3Hgroups
4 Discussion ThisreportdocumentsthefirstPhase1and Phase2 clinical studiesofanewcandidaterotavirusvaccinedevelopedin Viet-nam,Rotavin-M1.Theliveoralvaccine,whichhasbeendescribed previously,isderivedfromthemostcommonstrainofRotavirus, genotypeG1P[8],obtainedfromaVietnamesechildwithdiarrhea, attenuatedbycellpassage(>30×),plaquepurification,and pre-paredinVerocellsforhumanstudies[6].APhase1trialin29adult volunteersdemonstratedthatthevaccineadministeredorallyina titerof106.3FFU/dosewasnotassociatedwithsymptoms,adverse eventsorlaboratorychangesinblood countsorselected chem-istryandlittlevirusshedding,similartothatreportedforRotarixTM [11].TheDSMBreviewedthedataandapprovedthecontinuation
ofstudiesininfants
InthePhase1–2adaptivestudy,thecandidatevaccine admin-istered in eithera low (106.0FFU/dose)or high(106.3FFU/dose) titerona2-or3-dosescheduletoinfants6–12weeksofagedid notcausesignificantormorediarrheathanthatassociatedwith thelicensedvaccine,RotarixTM,demonstratingthatthecandidate strainhadbeensuccessfullyattenuated.Inthisstudy,thenumberof childrenwithdiarrheaepisodeswaslow,mainlyafterthe1stdose andtherewasnodifferencebetweenRotavin-M1andRotarixTM Thesediarrheaepisodesweremildsincetheywerenot accompa-niedbyvomitingandfever.Howeverhighernumbersofdiarrhea casesoccurredinthegroupreceiving106.3FFU/doseeventhough yetvaccineviruswasonlyfoundin3diarrheacasescumulatively
Trang 6suggestingthatdietorbacterialandprotozoalinfectionsmightbe
thecauseofdiarrheainthesechildren.InanotherRotarixTMtrialin
Vietnam,thepercentageofchildrenwithdiarrheaaftereach
vac-cinationdosewas3.1–6.1%,equivalenttowhatwasfoundinthis
study[7].RotarixTMat105.6–106.8CCIDalsocaused8.5–11%
diar-rheacaseamongchildrenintheUSandCanada[12].Thedetection
ofvaccinevirusindiarrheacasesisnotanuncommonphenomenon
intrialsusingattenuatedvaccine.Inadose-escalationstudyof116E
rotavirusvaccineinIndia,virusvaccinewasalsoisolatedin2outof
19diarrheacasesand2outof17diarrheacasesafterthe1stdose
of104FFUand105FFU,respectively[13].Thus,therateofdiarrhea
observedinourstudyiscomparabletosimilarstudiesofRotarixTM
andotherliveattenuatedrotavirusvaccinesanditisunlikelythat
thevaccinecausessignificantnumbersofdiarrheacasesinour
chil-dren.Nonetheless,furtherinvestigationisinprogressinalarger
groupofinfantstodetermineifthe106.3FFUdosecancausean
increaseindiarrheacasesamongvaccinees
ThesafetyprofileofRotavin-M1isalsofeaturedinthatthe160
infantswhoreceivedthevaccineineitherofthe2or3dosesdid
nothaveanysevereadverseevents,anysignificantexcessof
symp-tomsofdiarrhea,vomiting,feverorirritability,oralterations in
bloodcountorselectedbloodchemistriescomparedtothegroup
thatreceivedthelicensedvaccine.Adverseeffectsmainlyoccurred
afterthe1stdoseanddecreasedconsiderablyafterthe2ndand3rd
doses,similartoadverseeventsobservedduringinRotarixTMtrials
inVietnamorinothercountries[7].Asacomparison,whenthe
liquidformRotarixTMwastested,approximately50–65%children
developedfeverduringtheobservationperiod[7].InSingapore,
feverrateaftervaccinationreached25–30%aftereachdoseofthis
licensedvaccine[14]
Oncesafetywasestablished,thePhase2studyexaminedthe
immuneresponseandsheddingfrombothalowandahightiter
formulationofthevaccineandbotha2-dose(8and16weeks)and
a3-dose(8,12and16weeks)schedule.Theseresultswere
com-paredwithagroupthatreceivedthelicensedvaccine,RotarixTM,in
itsstandard2-doseschedule.Overall,theimmuneresponse
mea-suredasa 4-foldriseinIgAtiterstorotavirusrangedfrom51%
to73%,arangesurroundingtheresponseobservedforRotarixTM
(58%).Whilethehighertiterformulationperformedslightlybetter
thanthelowtiterpreparation,theadditionofathirddosetothe
schedule(i.e.8,12and16weeks)didnotdramaticallyimprovethe
immuneresponsetothevaccine.Thismightbeexplainedbythe
observationthathightitersoftheremainingtransplacental
anti-bodyagainstrotaviruscaninhibittheimmuneresponsetothe2nd
doseofvaccineinthe8-12-16-weekschedule.Steelefoundthat
2dosesofRotarixTMgivenat10and14weeksperformedaswell
as3dosesgivenat6,10,and14weeksbutbetterthan2doses
givenat6and10weeks[15].Inotherwords,theoldertheinfant
waswhenhereceivedthevaccine,thelowerwastheinitialtiterof
transplacentalantibodyandthebettertheimmuneresponsetothe
vaccine[16].Inboththe2andthe3doseschedulesinourstudy,
lastdosewasadministeredwhentheinfantwasthesameage,i.e
18weeks(95%CI(16.6–19.2)),unlikestudieswiththeRotarixTM
vaccinewhereathirddosewasaddedtothescheduleat14weeks
Therefore,theimmuneresponseto2dosesofthehightiter
Rotavin-M1vaccineat2-monthintervalyieldedthemostrobustimmune
response
Ofthesamenotes,anintervalof2monthsbetweendoseswas
more efficientin inducing immune response compared toa
1-monthintervalinbothlowandhighertiterformulation.Similar
observationsweredocumentedwhentheliquidformRotarixTM
wastestedin Vietnamesechildren[7].In thatstudy,2dosesof
RotarixTM, delivered1 month apart gave a seroconversion rate
of 63.3% at 1 month after the 2nd vaccine dose The same 2
dosevaccine however, when delivered 2 months apart gave a
seroconversionrateof81.5%.Applicationofthis2-monthinterval between2dosesofRotarixTMinEuropeancountriessuchasSpain, ItalyandFinlandledtohighseroconversionratesof92.3–94.6% [17].Thusagain,thehigherimmuneresponsewiththis2-month schedulemightbeassociatedwiththeslightlyolderchildrenwho areimmunologicallymorematurecomparedtothosewiththe 1-monthschedule[7]
TheimmuneresponsesinducedbyRotavin-M1are compara-bletothose seenin theRotarixTM groupinthis studyandin a previousstudythatemployedtheliquidformofthevaccinewith
asimilarschedule(58–63.3%)[7].It isnotedthatthepatternof IgAresponsetorotavirusvaccineinVietnamseemstofollowthe trendofdevelopingcountries.Inparticular,theIgAresponsesto RotarixTMinBrazil,Mexico,VenezuelaandVietnamwerereported
at61–65%,whicharelowerthanthoseinUSA,Canada,Europeand Singapore(78.2–88.3%)[18–21]andhigherthanthoseinMalawi andSouthAfrica[22].Inparticular,whenRotarixTMisintroducedin theexpandedimmunizationprogramofEuropeancountriessuch
asFrance,Germany,SpainandCzechrepublic,theIgAresponse rateswereveryhigh,82–94.6%[17].InSingaporetheresponsewas 76–91%dependingonthevaccinetiters[23,24].ThusinVietnam,
asinotherdevelopingcountries,thevaccineisfacingthepossible influenceofhighrotavirusdiseaseburden,malnutrition, concur-rentinfections,damagedgutmucosallayers,maternalantibodies andageoffirstadministrationaspreviouslydescribed[9,25,26] Theageatwhichthechildrenwasadministeredthefirstdose mightplayanimportantroleindeterminingseroconversionrates
InthisstudyandthestudywithRotarixTMinVietnamtheaverage ageoffirstdoseadministrationwas8weeks.Incomparison,the averageageforthefirstdoseintheUSis9–11weeksand11–17 weeksinSingapore[23,24].InFinlandandItaly,vaccinehasbeen usedatevenolderage(3months)[17].Itisgenerallybelievedthat vaccinationatolderageinducesbetterimmuneresponsespossibly duetoamorematureimmunesystemofthechildanddeclining maternalantibodytitersinbreastmilkorfromplacental transmis-sion.ThisnotionisalsosupportedbyastudyofRotarixTMinthe Philippinesinwhichchildrenwere5.5weeksofageatthefirst doseandtheseroconversionratewaslowercomparedtothatin Vietnamesechildren
Asvaccines, Rotavin-M1is very similartoRotarixTM in that botharederivedfromcommonG1P[8]strainsattenuatedbyserial passageandpreparedinVerocells.LikeRotarixTM,themajority
ofchildren shedafterthe1st doseof Rotavin-M1,whereas this proportiondeclinedconsiderablyafter2nddose,similartoother studies [24] Shedding of RotarixTM in different studies world-wideis35–80%,correspondingtothesheddingrateofthisvaccine foundinourstudy[27].Oneinteresting differencebetweenthe behaviorofthetwovaccinesistheincreasedsheddingobserved forRotarixTM (65%)comparedtoRotavin-M1(44–48%)afterthe 1st dose although this was not accompanied by an increased immuneresponse.Anotherdifferencebetweenthetwovaccines
isthatRotavin-M1vaccine,atthedosageof106.0FFUor106.3FFU causeddelayedinvirussheddingcomparedtoRotarixTMatdoses
of106CCID50(correspondingto105.5FFU/dose).Thesedifferences betweenthetwovaccinessuggestthatfurtherresearchonvaccine formulation,improvingtheyieldofvirussothathighertiter candi-datescouldbeavailablewhichhelpsadvancethedevelopmentof thislocallymanufacturedvaccinethroughefficacytrials
Inthisstudy,theRotavin-M1wasadministeredseparatelyfrom theoralpoliovirusvaccine(OPV)(10–20daysfromtheEPI sched-ule),thusthestudywasnotdesignedtoinvestigatetheeffectof othervaccines,inparticularOPVonRotavin-M1.Whilethe coad-ministration ofRotarixor RotaTeqwithOPVseemed toreduce seroconversionrates,antibodytitersandvaccinetakecompared
torotavirusvaccineswithoutOPV,thereductionswerenot statis-ticallysignificant[28,29].Thusfurtherstudyshouldbedesignedto
Trang 7immuno-genicityduetoconcomitantusageofOPVandRotavin-M1
This study has several limitations which will need to be
addressed asdevelopment of this vaccine progresses First, we
electedtouseRotarixTMinsteadofaplaceboforthecomparison
groupsowecouldwellmissimportantdifferencesinbackground
ratesofnaturalinfectionoverthecourseofthestudythatmight
havealteredourresults.TheaiminincludingRotarixisto
inves-tigateif Rotavinin anyscheduleor doseshows non-inferiority
toRotarix.Inaddition,sinceRotarix(lyophilizedform)hasbeen
licensedforuseinVietnamin2007,itisofethicalconsideration
forchildren participatingin thestudytobenefitfromthis
vac-cine.While theplacebo groupis important,this backgroundof
naturalinfectioncouldbederivedfromthepreviousstudywith
theliquidformofRotarixinVietnam[7].Inaddition,theinfants
wererandomizedsothiswouldlikelyhaveaffectedtheimmune
responsesintheRotarixTMgroupaswell.Moreimportantisthat
whileweattemptedtoexaminetwodifferenttiteredformulations,
106.0FFU/doseand106.3FFU/dose,thedifferenceinthese
prepa-rationsisnotgreat,perhapsnotevenwithinthevariabilityofour
titrationmethods.Consequently,whilewebelievethatthehigher
titermightbesuperior,wereallyhavenotexaminedthefullrange
oftiterstoseeifbysignificantlyraisingthetiter,wemightimprove
theimmuneresponse.Thisdecisionismorebasedupontheability
toraisethetiterofthevaccineduringproductionwhichwellcould
bethelimitingstep.Finally,whilewetesteda2-vs.3-dose
sched-ule,wemightwellimprovetheimmuneresponsetothevaccine
substantiallyifweweretoadministerthethirddoseatanolder
age,say20or28weeks,whentransplacentalantibodyhaswaned
Atthesametime,RotarixTMprovidedsubstantialefficacyin
Viet-nameseinfantsonasimilarscheduleandiftheimmuneresponse
isatallapredictorofefficacy,Rotavin-M1mightbeexpectedto
performcomparablyinaclinicaltrial
In conclusion,theVietnamese rotavirusvaccine,Rotavin-M1
hassafetyandimmunogenicityprofileinchildren,comparableto
RotarixTM.Amulti-centerstudyisinprogresstofurtherevaluate
thisvaccinationregimeninalargernumberofchildren
Acknowledgements
Wethankallthemedicalstaffs,thevolunteersandthechildren
inThanhSon,PhuThofortheirparticipationinthisstudy
WedeeplythankDrRogerI.Glass(FogartyInternational
Cen-ter,NationalInstitutesofHealth),DrTetsuYamashiro(Nagazaki
University),DrDuncanA.Steele(PATH)andDr.JonR.Gentsch(US
CDC)forcriticalreadingofthismanuscript
Conflictofinterest:DrsAnh,Trang,Thiem,Hien-Anh,Mao,Wang
andJianghavenoconflictofinterest.Financialsupport:The
Min-istryofScienceandTechnology,KC.10.33/06-10,Governmentof
Vietnam.Ethicalapproval:Thestudyandprotocol(No
962/CN-BYT-September29,2009)wereapprovedbytheEthicsCommitteesof
theNationalInstituteofHygieneandEpidemiologyandthe
Min-istryofHealth,GovernmentofVietnam
AppendixA
MembersoftheRotavin-M1VaccineTrialGroupinclude:NIHE
– VuThiBichHau,LeThiKimAnh,LeThiHongNhung,andLeHuy
Hoang;POLYVAC– NguyenThuyHuong,NgoThuHuong,Nguyen
Thi MaiHuong, TranBich Hanh,and DangNgan Ha; Centerof
PreventiveMedicine– PhuTho:NguyenThienAn;andCenterof
PreventiveMedicine,ThanhSon– PhuTho:NguyenThiLyandLe
ThiNgan
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