Consecutive CT in vivo lung imaging as
quantitative parameter of influenza vaccine
efficacy in the ferret model
Edwin J. B. Veldhuis Kroeze, Koert J. Stittelaar, Vera J. Teeuwsen, Marcel L Dijkshoorn,
Geert van Amerongen, Leon de Waal, Thijs Kuiken, Gabriel P. Krestin, Jorma Hinkula and
Albert D. M. E Osterhaus
Linköping University Post Print
N.B.: When citing this work, cite the original article.
Original Publication:
Edwin J. B. Veldhuis Kroeze, Koert J. Stittelaar, Vera J. Teeuwsen, Marcel L Dijkshoorn,
Geert van Amerongen, Leon de Waal, Thijs Kuiken, Gabriel P. Krestin, Jorma Hinkula and
Albert D. M. E Osterhaus, Consecutive CT in vivo lung imaging as quantitative parameter of
influenza vaccine efficacy in the ferret model, 2012, Vaccine, (30), 51, 7391-7394.
http://dx.doi.org/10.1016/j.vaccine.2012.10.008
Copyright: Elsevier.
Under a Creative
Commons license
http://www.elsevier.com/
Postprint available at: Linköping University Electronic Press
ContentslistsavailableatSciVerseScienceDirect
Vaccine
j o ur na l ho me p ag e : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e
Consecutive
CT
in
vivo
lung
imaging
as
quantitative
parameter
of
influenza
vaccine
efficacy
in
the
ferret
model
Edwin
J.B.
Veldhuis
Kroeze
a,b,
Koert
J.
Stittelaar
a,
Vera
J.
Teeuwsen
a,
Marcel
L.
Dijkshoorn
c,
Geert
van
Amerongen
a,
Leon
de
Waal
a,
Thijs
Kuiken
b,
Gabriel
P.
Krestin
c,
Jorma
Hinkula
d,e,
Albert
D.M.E.
Osterhaus
a,b,∗aViroClinicsBiosciencesB.V.,3000DRRotterdam,TheNetherlands
bDepartmentofVirology,ErasmusMedicalCenter,3000DRRotterdam,TheNetherlands cDepartmentofRadiology,ErasmusMedicalCenter,3000DRRotterdam,TheNetherlands dEurocineVaccinesAB,KarolinskaInstitutetSciencePark,17165Solna,Sweden eDivisionofMolecularVirology,IKE,LinkopingUniversity,58183Linköping,Sweden
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received18June2012 Accepted4October2012 Available online 17 October 2012 Keywords:
Influenza
Preclinicalvaccineefficacy Ferret
Invivoimaging CTscan Pathology
a
b
s
t
r
a
c
t
Preclinicalvaccineefficacystudiesaregenerallylimitedtocertainreadoutparameterssuchasassessment ofvirustitersinswabsandorgans,clinicalsigns,serumantibodytiters,andpathologicalchanges.These parametersarenotalwaysroutinelyappliedandnotalwaysscheduledinalogicalstandardizedway.We usedcomputedtomography(CT)imagingasadditionalandnovelreadoutparameterinavaccineefficacy studybyquantifyingalterationsinaeratedlungvolumesinferretschallengedwiththe2009pandemic A/H1N1influenzavirus.
Vaccinationprotectedfrommarkedvariationsinaeratedlungvolumescomparedtonaivecontrols. Thevaccinatedgroupshowedadailygradualmeanreductionwithamaximumof7.8%,whereasthe controlsshowedamaximumof14.3%reduction.ThepulmonaryopacitiesevidentonCTimageswere mostpronouncedintheplacebo-treatedcontrols,andcorrespondedtosignificantlyincreasedrelative lungweightsatnecropsy.
ThisstudyshowsthatconsecutiveinvivoCTimagingallowsforadaytodayreadoutofvaccineefficacy byquantificationofalteredaeratedlungvolumes.
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Thefieldofinfluenzavirusresearchisinparticularanareaof newemergingvirusesthatrequiresrapiddevelopmentofanimal modelsneededforpathogenicitystudiesandassessmentof ade-quatevaccinecandidatesandantiviraltherapies.Thiswasrecently illustrated by the emergence of the 2009 pandemic A/H1N1 influenza virus (pH1N1) [1,2]. Ferrets are being implemented extensivelyinhumaninfluenzavirusresearch.However,influenza virusresearchisconductedinmultipleseparatelaboratoriesall withtheiruniqueapproachhowtoevaluatevaccinecandidates within the ferret challenge model. Substantial differences can befoundinallstages andaspectsof challengeprotocols,study set-upsandread-outparameters.Aspectrumofrecentlypublished
Abbreviations:ALV,aeratedlungvolume;RLW,relativelungweight. ∗ Correspondingauthorat:DepartmentofVirology,ErasmusMedicalCenter,PO Box2040,3000CARotterdam,TheNetherlands.Tel.:+31107044066,
fax:+31107044760.
E-mailaddress:a.osterhaus@erasmusmc.nl(A.D.M.E.Osterhaus).
[1,3–12] infection/challenge protocols showingthis diversity is listedincomparisoninTable1.In addition,obviously, different influenza strains are used as challenge virusinstigated by the antigenicnatureofthevaccine,oralternativelytoevaluateefficacy toaheterologousinfluenzaviruschallenge.Theroutesofinfection beingintranasal,intratrachealorthroughvirustransmissionfrom experimentallyinfectedandshedding ferretsshowconsiderable differencesinimplementationandoutcomes[13].Differentviral challengedosesareused,whetherornotestablishedinpreceding dose-finding studies.However, the challenge doses are pivotal intheinterpretationofachallengeoutcome.Since,atoorobust challengemayprove,falsenegatively,apoorefficacyofahuman vaccinecandidateintheferretmodel,andviceversa.Furthermore, thedurationofthechallengereadoutperiodvaries,aswellasthe typesofsamplescollectedandfrequencyofsampling.Oftenthe designofachallengeprotocolisbasedonpredefinedendpoints andreadouts,ormayrelyonresultsfromhistoricalexperiments.
Becauseofthesevariationsintheassessmentofvaccine effi-cacy,thecomparisonoftheoutcomesofvaccinestudiesmaybe hampered,thereforeacertainwayofstandardizationcouldprove usefulbyprovidingclarity.
0264-410X/$–seefrontmatter © 2012 Elsevier Ltd. All rights reserved.
7392 E.J.B.VeldhuisKroezeetal./Vaccine30 (2012) 7391–7394
Table1
Recently,wereportedthatCT-scanningallowsquantification andcharacterisationofinfluenza-inducedpulmonarylesionsin liv-inganimals [11]. We showedthat thepulmonary ground-glass opacitiesobservedbyCTscanningcorrespondedmainlytoareas ofalveolaroedema,whichisamajorhistologicallesioninearly influenza-inducedpneumoniaandcanbeusedtoquantifythe aer-atedlungvolume(ALV).
Thepresentstudywasperformedtoevaluatethe immunogenic-ityandprotectiveefficacyofanadjuvantedinactivatedinfluenza pH1N1vaccineforintranasaluseintheferretmodel.Agroupof sixferretswasintranasallyimmunisedwiththis vaccine candi-dateandcomparedtoasecondgroupofsixferretsthatreceived intranasallyadministeredPBSasplacebo.Theseadministrations wereperformedonstudydays0,21and42.Allanimalswere subse-quentlyintratracheallychallengedwith106mediantissueculture
infectiousdose(TCID50)H1N1A/TheNetherlands/602/2009virus
onstudyday70.Theanimalsweremonitoredforvaccineinduced serologicalandimmunologicalresponsesandforinfectionrelated clinicaland virological responses (datawill be presented else-where).AsnovelreadoutparameterCT-scanningwasperformed6 daysprior,anddailyafter,virusinoculationonalltwelveferretsto monitorinfluenzainducedlungdamagebyquantifyingalterations intheALVs.Theanimalsweresacrificedat4dayspost-inoculation (dpi)toevaluatepathologicalandvirologicalparameters.
2. Materialandmethods
2.1. Animals
Theferrets(Mustelaputoriusfuro)werefemalesof8months of age, seronegative for antibodies against current circulating influenzaviruses,andAleutiandiseasevirus.Housingand hand-ling was performed under biosafety level (BSL)-3+ conditions
in negatively pressurized and high efficiency particulate air (HEPA)-filtered biocontainment isolator units, approved by an independent institutional laboratory animal ethics and welfare committee.Generalinjectionanaesthesia(ketamine8mg/kgand medetomidine-HCl 7.5g/kg body weight) was applied during handlingandscanning.
2.2. Immunisation
Theanimals(n=6)wereimmunisedthreetimeswitha3week interval withan adjuvanted inactivated vaccine.200l of vac-cinewasintranasallyadministeredanddividedequallyoverboth nostrils.Thecontrols(n=6)weresimilarlyshamimmunisedwith 200lPBSintranasally(referredtoasplacebo).
2.3. Challengevirus
Allanimalswerechallenged, 4 weeksafterthe last immun-isation, intratracheally with 106 median tissue culture
infec-tious dose (TCID50) of the 2009 pandemic influenza virus
A/Netherlands/602/2009 (pH1N1) in 3ml PBS, as described previously [2,12,14]. The virus was routinely propagated in MDCKcellculturesandinfectiousdosedeterminedasdescribed previously[15],andtitrescalculated accordingtothemethodof Spearman-Karber[16].
2.4. CT-scanning
Allanimalswerescannedon−6,1,2,3,and4d.p.i.(seealso Table1).Adual-sourceultrafastCT-system(SomatomDefinition Flash,SiemensHealthcare)wasused(temporalresolution:0.075s, spatialresolutionis0.33mm,tablespeedof458mm/s:ferret tho-raxacquisitiontime≈0.22s;enablesaccuratescanningofliving ferretswithoutthenecessityofbreath-holding, respiratory gat-ing,orelectrocardiogram(ECG)-triggering)aspreviouslydescribed [11].Briefly,duringscanningtheferretswereindorsalrecumbency inapurposelybuilt(Tecnilab-BMI)perspexbiosafetycontainerof 8.3Lcapacity.Thepost-infectiousreductionsinaeratedlung vol-umesweremeasuredfrom3-dimensionalCTreconstructsusing lowerandupperthresholdsinsubstancedensitiesof−870to−430 Hounsfieldunits(HU).
2.5. Pathology
Followingeuthanasiabyexsanguinationallanimalswere sub-mittedfor necropsy.Thelung lobeswereinspectedand lesions
wereassessedwhilethelung wasinflated.Thetracheawascut atthelevelofthebifurcationand thelungswereweighed.The relative lung weight was calculated as proportion of the body weightondayofdeath(lungweight/bodyweight×100).
Fig. 2. Changes in aerated lung volume after infection with H1N1 A/Netherlands/602/2009.Theaeratedlungvolumewascalculatedusinglowerand upperthresholdsinsubstancedensitiesof−870to−430Hounsfieldunits(HU)for theanalysisof3D-reconstructionsofthelung.Thepercentagechangeofaerated lungvolumewascalculatedusingtheindividualbaselineaeratedlungvolumesof day6againsttheaeratedlungvolumesofthedifferentdaysafterinfection.These dataareexpressedasmean±SEM.Animalswereintratracheallychallengedwith 106TCID
50H1N1A/TheNetherlands/602/2009onday0.
3. Resultsanddiscussion
Allanimals fromboth groups were scanned6 days prior to virusinoculationtodefinetheuninfectedbase-linestatusoftheir respiratorysystem.ConsecutiveinvivoimagingwithCTscanning showedthatferretsintranasallyimmunisedwiththevaccine can-didatewerelargelyprotectedagainsttheappearanceofpulmonary ground-glass opacities,as isshown by meansoftransversal CT imagesinFig.1.TheALVsmeasuredfrom3DCTreconstructs like-wiseshowedthattheimmunisedferretswereprotectedagainst majoralterationsinALV(groupmeanALVrangingfrom0.95to −7.8%) and didnot show a temporal increase in ALVon 1dpi, whichwasobservedintheplacebogroup(groupmeanALV ran-gingfrom17.3to−14.3%)(Fig.2).Thissuddenandshortincrease of17.3%(Mann–Whitneytest,two-tailed,P=0.035)inthe unpro-tectedplacebo-treatedanimalsmayresultfromavirally-induced acute respiratory depression withcompensatoryhyperinflation. A compensatoryincrease in respiratory tidal volumeby means ofhyperinflationisapathophysiologicalphenomenonknownto occurinrespiratoryviralinfections[17,18].However,CTscanning couldnotdiscernpossibleemphysemaduetorupturedalveolias causeofALVincrease.TherelativechangeofALVsondays2,3 andespecially4afterinfectiondidnotshowsignificantdifferences betweenthetwogroups.Onepossibleexplanationisthat over-expansionofthethoraxand lungsallowsforincreasedalveolar floodinginexcessofbaselineaerationresultinginapproximately unalteredALVsbetweenthetwogroups.Anotherexplanationis thattheinflamedandoedematousareaswereaeratedlessthan nor-mal,butbecausetheunaffectedareasoflungwereaeratedmore
Fig. 1. Consecutive transversal lung CT images after infection with H1N1 A/Netherlands/602/2009.Twocolumnsofconsecutive(toptobottom)transversal lungCTimagesofonerepresentativeimmunisedferret(left)andone represen-tativeplacebo-treatedferretrecordedinvivocomparedwiththeirgrossaspectat necropsy(bottom).Atday6beforeinfection,thelungsshowedtheclearaerated baselinecondition,from1dpiwiththenewpandemicH1N1influenzavirusonwards markedalmostdiffuseground-glassopacitiesappearthatshowagradualincrease withaplateauon3–4dpi.Thetwophotographstakenatnecropsyon4dpidepict theventralaspectofthelungs,withtheheartsremoved.Thelungsofthe placebo-treatedanimal(bottomright)showdiffusereddishconsolidationindicativeofacute inflammationthatessentiallymatchtheopacitiesontheCTimagestakenjustbefore necropsy;non-affectedaeratedlungtissuefromtheimmunisedanimalislightpink incolour(bottomleft).
7394 E.J.B.VeldhuisKroezeetal./Vaccine30 (2012) 7391–7394
Fig.3.Relativelungweights.Meanrelativelungweights(RLWs,relatedtobody weight;±SEM)atnecropsy(4dpi)fortheimmunisedgroupversusthe placebo-treatedcontrolgroupafterinfectionwithH1N1A/Netherlands/602/2009.
thannormal(hyperinflationoremphysema),theoverallALVvalues remainedapproximatelyunaltered.
Nevertheless,theseALVprofilesprovidemoredetailed knowl-edgeabouttheinfluenza-inducedrespiratorydiseasedevelopment thanconfineddata obtainedfrom asinglepredefined read out. Moreover,survivalandrecoveryfromchallengeinfectioncanbe includedinthisset-upandwiththeopportunitytostillmeasurethe developmentofserumantibodyresponsesuponchallenge infec-tion.
Upon necropsy, the relative lung weights (RLWs) of the intranasally immunised ferrets was about 2-fold lower (Mann–Whitney,two-tailed,P<0.0047)ascompared tothoseof theplacebo-treatedanimals(Fig.3),whichisinagreementwith theabsenceofpulmonaryground-glassopacities.Usually,more severely affected and inflamed lungs with increased amounts offluid are heaviercompared tonormal or less affectedlungs. Thistranslateswithintheferret modelin influenzaresearchto RLWs≤1.0associatedwithnon-tominimallyaffectedlungsand RLWs>1.0associatedwithseverepulmonaryinflammationwith oedema[12,19,20].
Inconclusion,theimplementationofconsecutiveCT imaging enablesrepeatedinvivomeasurementsoflungaerationas parame-tertoevaluatevaccineefficacyinpreclinicalprotocols.Consecutive daytodayimagingovercomesthelimitationsentailedbynecropsy atapredefinedtimepointafterinfection,andthelungcapacitycan berepeatedlyquantifiedinreal-time.
Acknowledgements
WearegratefultoWillemvanAert,RonaldBoom,Cindyvan Hagen,RobvanLavierenfromViroClinicsBiosciencesB.V.,Peter vanRunfromthe Department ofVirology Erasmus MC Rotter-dam,andDennisdeMeulderfromtheErasmusLaboratoryAnimal ScienceCenterRotterdamfortheirexcellenttechnicalassistance andanalyses.
Conflictofinterest:TheauthorsEVK,VT,KS,GvA,LdW,andAO areaffiliatedwithErasmusMCspin-offcompanyViroClinics Bio-SciencesB.V.TheauthorJHisaffiliatedwithKarolinskaInstitutet spin-offcompanyEurocineVaccinesAB.
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