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
Stability
of
a
formalin-inactivated
Rift
Valley
fever
vaccine:
Evaluation
of
a
vaccination
campaign
for
cattle
in
Mozambique
N.
Lagerqvist
a,b,∗,1,
B.
Moiane
b,c,1,
G.
Bucht
d,
J.
Fafetine
c,
J.T.
Paweska
e,
Å.
Lundkvist
a,b,
K.I.
Falk
a,baSwedishInstituteforCommunicableDiseaseControl,SE-17182Solna,Sweden
bDepartmentofMicrobiology,TumorandCellBiology,KarolinskaInstitutet,SE-17177Stockholm,Sweden cVeterinaryFaculty,EduardoMondlaneUniversity,C.Postal257,Maputo,Mozambique
dSwedishDefenseResearchAgency,DepartmentofCBRNDefenseandSecurity,SE-90182Umeå,Sweden
eNationalInstituteforCommunicableDiseasesoftheNationalHealthLaboratoryService,Sandrigham-Johannesburg,SouthAfrica
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received3May2012
Receivedinrevisedform18July2012 Accepted21August2012
Available online 2 September 2012 Keywords:
RiftValleyfever Virus
Formalin-inactivatedvaccine Bovine
Mozambique
a
b
s
t
r
a
c
t
InAfricaandtheArabianPeninsula,outbreaksofRiftValleyfever(RVF)arecharacterizedbyabortions
ingestatinganimalsandhighmortalityratesamongdomesticruminants.Animmunizationprogram
usingaformalin-inactivatedvaccinewasinitiatedinMozambiquein2002tocontrolRVFincattle.Inthis
intervention,thevaccinemustbetransportedformorethanaweekwithinthecountrybeforeitcanbe
administeredtotheanimals,anditispracticallyimpossibletomaintainlowstoragetemperaturesduring
thattime.Here,weevaluatedtheinfluenceoftransportationconditionsontheefficacyofthevaccine.
Sixty-threepreviouslyunvaccinatedandRVFvirusseronegativecattleweredividedintofourgroups,
whichweregivenvaccinethathadbeenstoredfor1weekat4◦C(n=9,groupA),at25◦C(n=8,group B),oralternatingbetween4and25◦C(n=8,groupC),orunderthetemperatureconditionsordinarily
occurringduringtransportationwithinMozambique(n=38,groupD).Theantibodyresponsesinduced
weremonitoredfor6–9monthsandinsomeanimalsupto21months.Twoimmunizations(3weeks
apart)withtheformalin-inactivatedvaccineinducedalong-lastingneutralizingantibodyresponsethat wasstilldetectableupto21monthslater.Theantibodytitersintheanimalsdidnotdiffersignificantly
betweenthetemperature-assignedvaccinegroupsA,B,andC,whereastheyweresignificantlyhigherin
groupD.Theseresultsshowthattheformalin-inactivatedRVFvirusvaccineisstable,and,importantly, itisnotadverselyaffectedbythevariationintemperaturethatordinarilyoccursduringtransportwithin Mozambique.
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ThefirstreportofRiftValleyfever(RVF)amongsheepinKenya waspublishedin1930[1],andsincethenthediseasehasbecome widespreadandendemicinSub-SaharanAfricaandEgypt,andlater alsointheArabianPeninsula.ThecausativeagentistheRVFvirus (RVFV),whichbelongstothegenusPhlebovirusofthefamily Bun-yaviridae.Thetripartite RNAgenome(S,M,and Lsegments) is ofnegativeorambisense(Ssegment)polarityandencodesfour structuralproteins[2].Thehighlyimmunogenicnucleocapsid(N)
∗ Correspondingauthorat:Nobelsväg18,SE-17182Solna,Sweden. Tel.:+4684572472;fax:+468302566.
E-mailaddresses:nina.lagerqvist@smi.se(N.Lagerqvist),belisario.moiane@ki.se (B.Moiane),goran.bucht@foi.se(G.Bucht),jfafetine@yahoo.com(J.Fafetine), januszp@nicd.ac.za(J.T.Paweska),ake.lundkvist@smi.se(Å.Lundkvist), kerstin.falk@smi.se(K.I.Falk).
1 Theseauthorscontributedequallytothiswork.
protein,whichisencodedbytheSsegment,isincreasinglybeing used as the antigen of choice in serological assays [3,4]. The membrane-associatedGnandGcglycoproteinsareencodedbythe Msegment,andconstitutethetargetsforvirus-neutralizing anti-bodies[5].
RVFVcaninfectabroadrangeofanimalhosts,andoutbreaks of this diseaseoften occurafter heavyrains and flooding have generatedfavorablebreedingconditionsforthemosquitovectors [6,7].Viralinfectionsinhumansareusuallymanifestedasmild, self-limitingfebrileillness,althoughinsomecasestherearemore severesymptoms,suchasencephalitis,lossofvision,and hem-orrhagicfever [8]. In animals infected withRVFV, spontaneous abortionsarefrequentlyobservedamonggestatingdomestic rumi-nants,andthemortalityratecanreach100%amongnewbornand youngindividuals.RVFcanbesubclinicalinolderanimals,but clin-icalmanifestationsarealsooftensevereinadults,withmortality ratesrangingfrom10%to70%,dependingontheage,species,and breedoftheinfectedhost[9].EpizooticsofRVFhaveresultedin rigorousrestrictionsonthetradeofanimalsandanimalproducts, 0264-410X/$–seefrontmatter © 2012 Elsevier Ltd. All rights reserved.
whichinturnhavehadaconsiderablesocio-economicimpact,not onlyonlivestock keepersand foodproducers, buton the non-agriculturalsectoraswell[10].
Vaccinationoflivestockisthemainmeasureusedtocontrol RVF in endemic areas. Many promising vaccine candidates are underevaluation[11–13],although animalsarecurrentlybeing immunized with the live-attenuated Smithburn strain [14] or formalin-inactivatedRVFVvaccines[15].Unfortunately,the Smith-burnstrain hasretained teratogeniccharacteristics,asnoted in studiesofpregnantcowsandgoats[16,17].Comparedtothe live-attenuatedstrain,theformalin-inactivatedvaccinesaresaferfor useingestatinganimals,buttheyarealsolessimmunogenic,and annual boosters are required to maintainprotective immunity [18].
OutbreaksofRVFarereportedregularlyinseveralofthe Sub-Saharancountries[19,20], althoughthesituationregarding this diseaseinMozambiqueisunclear.StrongevidenceofRVFV trans-missioninMozambiquewasobtainedasearlyasthe1960s[21], andthiswasconfirmedinthe1980s,whenvirus-specific antibod-iesweredetectedin2%ofpregnantwomeninthecountry[22]. Furthermore,surveysofcattleinZambeziaProvince(thecentral regionofMozambique)revealedhighseropositivity,whichledto introductionofanimmunizationprogramin2002usinga formalin-inactivatedvaccine[23].
Inthepresentstudy,weinvestigatedwhetherdifferentstorage andtransportconditionsaffecttheabilityofformalin-inactivated RVFV vaccine to induce immune responses in cattle. For that purpose, a commercialvaccinewasstored under fourdifferent conditions:at4◦C,at25◦C,alternatingbetween4◦Cand25◦C,or accordingtothestandardproceduresstipulatedbythe Mozambi-canDirectorateofNationalVeterinaryServices(seeSection2.3). Theantibodyresponsesinducedbythevaccineweremonitored byELISAandplaquereductionneutralizationtests(PRNTs)for6–9 months,andinsomeanimalsupto21months.
2. Materialsandmethods
2.1. Cellsandviruses
Verocells(ATCCno.CCL-81)weregrowninMedium199(Gibco, LifeTechnologiesCorporation)supplementedwith100U/mL peni-cillin, 100g/mL streptomycin, and 5% heat-inactivated fetal bovineserum(FBS).Thecellswereculturedat37◦Cina humid-ifiedatmospherecontaining5%CO2.TheRVFVstrainZH548[24],
waspropagatedand titrated in Verocells beforebeingused in immunofluorescenceassays(IFAs)andPRNTs.
2.2. Animals
The experimental procedures using animals were approved bytheMozambicanBoardofAgriculture(ethicalpermissionno. 555/DNSV/2008,Maputo).Thirtymixedbreedcattlewereraised inafree-rangefeedingsystemintheNamaachaDistrict,Maputo Province(Fig.1), withthemaindiet basedongrass andhayto whichmineralsaltsandfoodsupplementswereaddedduringthe dryseason(March–August).Theseanimalsweredividedintothree groups(designatedA,B,andC)initiallywith10animalsineach. Anadditional41 headof mixed-breedcattle (designatedgroup D)were raisedintheAltoMolocue District,ZambeziaProvince (Fig.1),underconditionssimilartothosedescribedforgroupsA–C, butwithoutfoodsupplementsduringthedryseason.Twomore animalswerekeptasnegativecontrolsintheNamaachaDistrict, MaputoProvince.Noneofthecattlehadpreviouslybeen vacci-natedagainstRVFV. Characteristicsof the animalsare given in Table1.
Fig.1. MapofMozambiqueandneighboringcountries.Themapshowsthecapital cityofMaputo(italics)andtheprovincesreferredtointhearticle(boldcapital let-ters),aswellasthecityofQuelimaneandtheapproximatelocationsoftheNamaacha andAltoMolocueDistricts.
2.3. Vaccinationandsamplecollection
InMaputoProvince,vialscontainingformalin-inactivatedRVFV vaccineandaluminumhydroxidegeladjuvant(Onderstepoort Bio-logical Products,batchno.398) werestored for1 weekat4◦C (accordingtothemanufacturer’sinstructions),at25◦C,or alternat-ingbetween4and25◦C(at12-hintervals);thereafter,thevaccine wasadministeredtothecattleingroupsA,B,andC,respectively. ForgroupDinZambeziaProvince,vaccineinvialswastransported ascurrentlystipulatedbytheDirectorateofNationalVeterinary Servicesbeforeitwasadministeredtothecattle.Moreprecisely, thevialsweresentbyairfromMaputotoQuelimaneand there-afterbycartotheAltoMolocueDistrict(Fig.1),andthevaccine wassubsequentlygiventotheanimals.Thedeliverytook approx-imately1week,andthevialswerestoredoniceorat4◦Cwhen possible.
Theanimalswereinoculatedsubcutaneously(16G½in.needle) lateralontheneckwith2mLofvaccineonday0andboostedon day21,accordingtothemanufacturer’sinstructions.Bloodsamples werecollectedbyvenipunctureofthecoccygealveinonthedayof primaryvaccinationandthereafterfor287–630daysingroupsA–C andfor147daysingroupD.Samplescollectedondays45and48 wereanalyzedtogetherandarereferredtobelowashavingbeen takenonday45.Thebloodsampleswerestoredat4◦Candlater centrifugedat1500×gtoobtainsera,whichwerestoredat−20◦C
pendinganalysis.
2.4. Immunofluorescenceassay
Bovineserumsamplesdiluted1:40inPBSwereincubatedwith monolayersofacetone-fixedRVFV-infectedVerocellsfor30minat
Table1
Characteristicsofthecattle.
Group Province Animala Age(yr) Sex Weightb(kg) Samplingdaysc
0 10 20 30 45 87 117 147 207 267 630 A(4◦C) Maputo A1 5 F 300 • • • • • • • • • • x A2 4 F 260 • • • • • • • • • • • A3 3 F 250 • • • • • • • • • • • A5 8 F 400 • • • • • • • • • + + A6 8 F 380 • • • • • • • • • • • A7 8 F 380 • • • • • † A8 8 F 360 • • • • • • • • x A9 8 F 360 • • • • • • • • • • • A10 8 F 300 • • • • • • • • • • • B(25◦C) Maputo B2 5 F 320 • • • • • • • • x B4 4 F 300 • • • • • • • x B5 5 M 340 • • • • • • • • • • • B6 6 F 320 • • • • • • • • † B7 6 F 300 • • • • • • + • • • • B8 8 F 350 • • • • • • • • • • • B9 8 F 250 • • • • • • • • • • x B10 8 F 300 • • • • • • • • • • • C(4/25◦C) Maputo C1 8 M 280 • • • • • • • • • • • C2 4 F 260 • • • • • • • • • • • C3 6 F 340 • • • • • • • • • • • C5 5 F 350 • • • • • • • • • • • C6 5 F 300 • • • • • • • • • • • C7 8 F 260 • • • • • • • • x C8 8 F 250 • • • • • • • • • • x C10 8 F 400 • • • • • • • • • • • D (customary)d
Zambezia D1–D5 4–8 n/a n/a • • • • • • • •
D7 4–8 n/a n/a • • • • • • • + D8–D29 4–8 n/a n/a • • • • • • • • D30 4–8 n/a n/a • • • • • • • + D31 4–8 n/a n/a • • • • • • • • D33–D39 4–8 n/a n/a • • • • • • • • D41 4–8 n/a n/a • • • • • • • • Maputo ctrl1 6 F 300 • • • • • • • • • • • ctrl2 5 F 300 • • • • • • • • • • •
aAnimalsA4,B1,B3,C4,C9,D6,D32,andD40wereexcludedduetopre-exposuretoRiftValleyfevervirus. bWeightwasestimatedusingatapemeasure.
c Symbolsindicatesamplesthatcouldnotbecollected(+)orwerenotavailablebecauseananimaldied(†)orwasstolenorsold(x).
d VaccinewastransportedaccordingtothecustomaryproceduresoftheMozambicanDirectorateofNationalVeterinaryServices(seeSection2.3).F,female;M,male;n/a,
notavailable;ctrl,control.
37◦C.Next,thecellswerewashed3× 10minin0.9%NaClsolution, andthenfluorescein-conjugatedanti-bovineIgGantibody(Cappel, MPBiomedicalsInc.)wasaddedtoafinaldilutionof1:50. There-after,thesampleswereincubatedfor 30minat 37◦C and then washedasaboveandmounted.Thepresenceofanti-RVFV anti-bodieswasdeterminedbyfluorescencemicroscopy,andallserum sampleswereanalyzedinduplicate.
2.5. ELISA
Full-length RVFV N protein (GenBank ID: AF134534) [25] was produced from a prokaryotic expression vector pET14b (Novagen),andtherecombinantNproteinantigenwaspurified fromEscherichiacolicelllysatesundernativeconditionsusing Ni-NTAagarose(Qiagen),asdescribedelsewhere[26].IndirectELISA wasperformedasreportedbyotherauthors[27]withtheseminor changes: the plates were coated with 3g/mL recombinant N protein,andtheantigen-antibodycomplexeswerevisualizedby useofperoxidase-conjugatedanti-bovine IgGantibody(Jackson ImmunoResearchLaboratories)diluted1:5000andTMBsubstrate (3,3,5,5-tetramethylbenzidine,SigmaAldrich).Thereactionwas stoppedwith50Lof1MH2SO4,andtheabsorbancewas
mea-suredat450nm.TheELISAcut-offvaluewascalculatedusingthe resultsfor25negativebovineseraandtheStudentt-distribution
test with99%confidence intervals,asdescribedin detailin the literature[28].Negativeandpositivebovineserumcontrol sam-ples wereincludedineach plate.Allsampleswereanalyzedin duplicate,andthetitersweredeterminedasthereciprocalofthe highestserumdilutionthatresultedinareadingabovethecut-off value.
2.6. Plaquereductionneutralizationtest
Heat-inactivatedserumsampleswereseriallydilutedtwofoldin MinimalEssentialMedium(MEM,Invitrogen)supplementedwith 3%FBS,100U/mLpenicillin,and100g/mLstreptomycin,andthen incubatedwith60 PFUof RVFVfor30minat37◦C. Adsorption oftheserum-virusmixturetoVerocellswasallowedtoproceed for1.5hat37◦C,andthenthecellswerewashed,overlaidwith MEMmediumcontaining0.75%carboxymethylcellulose(Sigma Aldrich),andincubatedfor4daysat37◦Cunder5%CO2.Thecells
weresubsequentlyfixedin4%formaldehyde,andplaques were visualizedbycounterstainingwithcrystalviolet.The neutraliza-tiontitersweredeterminedasthereciprocalofthehighestserum dilutionthat reducedvirusinfectivityby80%.Atiterof20 was consideredpositiveafterevaluatingtheassayusing25negative bovinesera.AllproceduresusingviableRVFVwerecarriedoutina biosafetylevel3containmentlaboratory.
Fig.2.Percentageofseropositivecattleineachofthefourvaccinegroups.Formalin-inactivatedRVFVvaccinewasstoredfor1weekat4◦C,at25◦C,alternatingbetween4◦C
and25◦C,orinvialstransportedinthemannercustomaryinMozambiqueandthenadministeredtwice(3-weekinterval,day0andday21)toanimalsingroupsdesignated
A,B,C,andD,respectively.Thepercentageofseropositiveanimalswasdeterminedontheindicateddaysbyplaquereductionneutralizationtests(A)andindirectELISA againstNprotein(B).Arrowsalongthex-axisindicateimmunizationdays.NodatawereavailablefortheanimalsingroupDonthedaysmarkedwithasuperscript‘x’.
2.7. Statisticalmethods
Statisticalcorrelations wereanalyzedusing Spearman’srank test. TheKruskal–Wallis test wasappliedto achieve theinitial comparisonsoftheantibodyresponsesexhibitedbythefour vac-cination groups, and, if systematic variations were found, the Mann–WhitneyUtestwascarried outtocomparethe vaccine-inducedtitersoftheanimalsintheindividualgroups.Statistical analyseswereperformedwithStatisticaTM10(StatSoftInc.),and
graphswerevisualizedusingGraphPadTMPrism4(GraphPad
Soft-wareInc.).
3. Resultsanddiscussion
ThesporadicreportsofoutbreaksofRVFinneighboring coun-triesillustrate that there is a continuous risk to Mozambique, becauseitisoneofthemostfrequentlyfloodedcountriesinthe southernpartofAfrica[29].Notably,Mozambiquehasbeenspared fromRVFinhumansorlivestock,withtheexceptionofthe out-breaksthatoccurredin1969and1994,whichcausedabortionsand deathsincattleandwaterbuffalo[9,21,30].Nevertheless, surveil-lanceof cattle in ZambeziaProvince (Fig. 1)in 1996and 2001 revealedseroprevalenceof37%and53%,respectively[23]. Accord-ingly,in2002,avaccinationprogramwasinitiatedindistrictsin thatprovincetopreventRVFVinfectionsin cattle.Inasmuchas therewasconsideredtobeahighriskofRVFafterthefloodingthat hadaffectedthesouthernpartofthecountryin2000andthecentral regionin2001,thevaccinationprogramwasextendedinlate2002 toincludetheprovincesofGazaandManica(Fig.1)[31].However, after2004,vaccinationoflivestockwasonceagainlimitedtoonly afewdistrictsinZambeziaProvince.
Theantibodystatusofthecattletobeusedinourstudywas initially unknown, and thus, to start with, we assessed RVFV seronegativitybyIFA,andtheresultswerelaterconfirmedbyPRNT
(datanotshown).Aseropositivityrateof7%wasfoundamongthe 41animalskeptinZambeziaProvince(groupD),whichwasa rela-tivelylowlevelcomparedtothesero-surveillancedatapreviously recordedinthatprovince[23].Ontheotherhand,17%ofthe30 animalsinMaputoProvince(groupsA,B,andC)werepositivefor anti-RVFVantibodies,whichwasasurprisinglyhighrate.It was inthisregionthatthelastsmalloutbreaksofRVFincattlewere reportedin1969[21].TheseresultsindicatethatRVFVis circulat-inginMaputoProvince,butitiseithernotcausingmanifestclinical diseaseornotbeingdiagnosed.
CattlethatwerefoundtohaveelevatedRVFVantibodytiters beforetheprimaryimmunizationwerenotincludedinthestudy (Table 1). Furthermore, none of the negative control animals showedseroconversionovertheexperimentalperiod,analyzedas thepresenceofantibodiesagainsttheNproteinorbyvirus neu-tralizationtests(datanotshown).
TheabilityofthebovineantiseratoneutralizeRVFVinvitro wasanalyzedbyPRNT.Our datashowthat themajorityofthe animals in all four groups (78%, 88%, 88%, and 74% in A, B, C, and D, respectively) seroconverted rapidly in response to the primary vaccination (Fig. 2A). After the second immunization, allanimalsingroupsA,B,andChadelevatedPRNTtiters(≥80) (Figs.2Aand3A).However,fouroftheanimalsingroupD(n=38) failed to seroconvert 9 days after receiving the booster dose; threeofthosefourseroconvertedbyday45,whereasthefourth animaldidnotdevelopdetectableanti-Nproteinorneutralizing antibodiesovertheentireexperimentalperiod.Ingeneral, neu-tralizingantibodytitersshowedsmallpeaksondays30–45and thereafter declined relatively rapidlyin groups A–C but not in groupD(Fig.3A).Interestingly,theantibodytitersof thecattle ingroupDpeaked onday45 andremainedhighuntilday147 (Fig.3A).Furthermore,two-thirdsoftheanimalsingroupsA,B, andCstillhaddetectablelevelsofneutralizingantibodies267days aftervaccination(Fig.2A).It canbementionedthatcomparable
Fig.3. Barchartsshowingthemedianantibodytitersinthevaccinatedcattle.Fourgroupsofcattlewereimmunizedwithformalin-inactivatedRVFVvaccinethathadbeen storedat4◦C(groupA),25◦C(groupB),oralternatingbetween4◦Cand25◦C(groupC),orhadbeentransportedwithinMozambiqueaccordingtothepracticestipulatedin
thatcountry.Twodoseswereadministered(primaryvaccinationonday0andboosteronday21),andthevaccine-inducedneutralizingantibodyresponses(A)andanti-N antibodyresponses(B)weredeterminedbyaplaquereductionneutralizationtestandindirectELISA,respectively.Serumsampleswerecollectedfromtheanimalsonthe indicateddays.NodatawereavailableforgroupDonthedaysmarkedwithasuperscript“x”.Arrowsalongthex-axisindicateimmunizationdays.Bracketsandasterisks indicatevaluesfoundtobesignificantlydifferentbytheMann–WhitneyUtest(*p<0.05;**p≤0.01;***p≤0.001).
long-lastingtitersofneutralizingantibodieshadpreviouslybeen observed by other researchers in a study using a comparable formalin-inactivatedRVFVvaccine[32].Wealsonotedthat73% oftheanimalsinourinvestigationstillhadneutralizingantibody titersintherangeof40–320(median40)(Figs.2Aand3A)630days aftertheinitialvaccination.Theextentanddurationofthe pro-tectiveimmunityinducedbyformalin-inactivatedRVFVvaccines are considered to be “short termed”, and consequently annual boosterimmunizationsaregiventocattletomaintainimmunity. However,ourobservationsindicatethattheneutralizingantibody responsesinducedbytheformalin-inactivatedRVFVvaccinesmay bemorerobustandlong-lastingthanexpected.
IndirectELISAbasedonrecombinantNproteinhasthe poten-tialforuseinroutinediagnosticsandepidemiologicalstudiesof RVF[33].Therefore,wedecidedtomonitorthelevelsof anti-N-proteinantibodyinserumsamplesofthevaccinatedcattle.Anti-N antibodyresponsesweredetectedinaboutone-thirdoftheanimals aftertheprimaryvaccination,andnearly80%oftheanimalswere seropositiveninedaysaftertheboosterimmunization,regardless oftreatmentgroup(Fig.2B).Incontrasttotheneutralizingantibody responses,circulatinganti-Nantibodiescouldonlybedetectedfor ashortperiodoftime.Onday147aftertheprimary immuniza-tion(126daysafterbooster),themajority(74%)oftheanimalsin groupsA–Cwerenegativeforantibodiesagainsttherecombinant Nprotein(Fig.2B),whereassuchseronegativitywasobservedin only14%oftheanimalsingroupDatthattime(Fig.2B).Bythe endofthestudyperiod,allbutfouroftheinvestigatedanimals
hadexhibitedanti-Nantibodyresponsesafterthetwo immuniza-tions.
Wealsonotedthat,afterreceivingtheimmunizations,the ani-malsthathadlowvaccinationanti-Nproteintiters(≤100)showed neutralizingantibodytiterssimilartothoseseenintheanimalsthat hadstrongervaccinationanti-Nantibodyresponses.TheELISAused inthisstudymeasuresantibodiesdirectedtowardtheNprotein, whilethePRNTmainlymeasuresneutralizingantibodiesdirected towardtheglycoproteins.Thusthelevelofanti-Nantibodiesmay notberelevantasanindicatorofvaccinationstatus. Itis possi-blethatthelackofarelationshipbetweenanti-Nandneutralizing antibodytiters(groupA,Spearman’sranktest)canbeexplained bydifferencesinassaysensitivity,howeverouranalysesindicate thatserologybasedontheresponsetoNproteinmaynotbethe methodofchoiceforevaluatingtheefficacyofimmunizationwith formalin-inactivatedRVFVvaccines.
Formalin-inactivatedvaccinesareingeneralmorestablethan attenuatedvaccines[34].Effortshavebeenmadetoincreasethe stability of formalin-inactivated RVFV vaccines even furtherby lyophilization[35],butthepreparationsusedinourstudywere in fluidform,and,toourknowledge,noevaluationshave been performed to determine the efficacy of that type of formalin-inactivatedRVFVformulaafterstorageortransportationatambient temperatures.
In our investigation, analysis of the impact of the differ-ent vaccine storage conditions on neutralizing antibody titers measured after immunization (Fig. 3A) revealed no significant
differences betweenthe four experimental groups ondays 10, 20, and 45, or between groups A, B, and C on days 207, 267 and630(Kruskal–Wallistest).However,theneutralizingantibody responsesdifferedsignificantlybetweenthefourgroupsondays 30,87,117, and147afterthefirstvaccination(p=0.023,0.004, 0.000,and0.001,respectively;Kruskal–Wallistest).Accordingly, theindividualgroupsofanimalsweresubjectedtofurtheranalysis usingtheMann–WhitneyUtest.Onday30,neutralizingantibody titerswerehigherin groupBthan ingroupD,which mightbe explainedbytheearlierantibodypeak(Fig.3A).However,from day45andonward,theresultswerereversed,thatis,thetitersin groupDwereelevated(albeitonlyslightly)comparedtothosein groupB(Fig.3A).Furthermore,somewhatsurprisingly,the anti-bodytitersobservedondays87,117,and147werehigheringroup DthaningroupsAandC(Fig.3A).
Asimilarpatternwasobservedconcerningthelevelsofanti-N proteinantibody(Fig.3B),whichdifferedbetweenthegroupson days10,20,87,117,and147(p=0.035,0.006,0.001,0.000,and 0.000,respectively;Kruskal–Wallis).Comparisonsofthe individ-ualgroupsregardingtheresultsfortheindicatedsamplingdays revealedthatthetitersmeasuredingroupDwerehigherthanthose ingroupsAandC,andalsohigherthanthoseingroupBonsomeof thedays(Mann–WhitneyUtest)(Fig.3B).Asfortheneutralizing antibodytiters,theanti-Nproteinantibodyresponsesdidnot dif-fersignificantlybetweengroupsA,B,andCondays207,267,and 630aftervaccination.Severalunrelatedfactors,suchasthebreed andthenutritionalstatusoftheanimalsmayinfluence vaccine-inducedimmuneresponses[36],whichmightexplainthehigher antibodytitersweobservedintheanimalsingroupD.Moreover, ingeneral,thevaccinefromvialsstoredattherecommended tem-perature(4◦C)inducedequivalentorslightlylowerantibodylevels comparedtothevaccinestoredatambienttemperatures (alternat-ingbetween4and25◦C)orat25◦C.Theseeffectswerenotedin cattlebelongingtothesameherd(groupsA,BandC),which sug-geststhatstoragefor1weekunderadverseconditionsdoesnot impairtheabilityofthevaccinetoinducethehumoralantibody responses.
Insummary, oftheanimalsincludedin thisstudy,only one failedtoacquireadetectableantibodyresponseaftertwo vacci-nations.Furthermore,theantibodyresponseswerestilldetectable up to 21 months after the immunizations. Based on the data presentedhere,weconcludethatthestorageandtransport con-ditionsused in Mozambique do not have anadverse effecton theantibodyresponsesinducedbytheformalin-inactivatedRVFV vaccine.
Acknowledgements
WegratefullyacknowledgetheSwedishInternational Devel-opment Cooperation Agency (SIDA) and the Swedish Civil Contingencies Agency (MSB) for their support. We also thank Dr.LarsEriksson(KarolinskaInstitutet,Sweden)forvaluable sta-tistical advice, LouiseTreiberg Berndtsson (National Veterinary Institute,Sweden)forprovidingbovineserum,andtheDirectorate ofNationalVeterinaryServices(DNSV,Mozambique)forfurnishing thevaccine.
Conflictofinterest: Thefunders hadno rolein study design, datacollectionandanalysis,orpreparationofthemanuscript. Con-tributions: K.F. initiated the study concept and design. N.L. did theserologicalanalysisandshecarriedoutworksrelatedtothe interpretationofdataandthepreparationofthemanuscriptwith thehelpofK.F.WhilehelpingK.F.andN.L.forthepreparationof themanuscript,Å.L.effectedsomevaluablescientificdiscussions. Besides,B.M.endeavouredvaccinationandsamplecollection,and J.F.wasresponsibleforinitialprojectdesign.ThoughG.B.prepared
thecontent,hewashelpedbyJ.P.forthecriticalrevisionofthe manuscript.
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