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A novel peptide-based pan-influenza A vaccine: A double blind, randomised clinical trial of immunogenicity and safety

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FP-01.1 is a novel synthetic influenza A vaccine consisting of six fluorocarbon-modified 35-mer peptides that encapsulate multiple CD4+ and CD8+ T-cell epitopes and is designed to induce an immune response across a broad population. FP-01.1 was
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  Pleasecitethisarticlein   pressas:FrancisJN,etal.Anovelpeptide-basedpan-influenzaAvaccine:Adoubleblind,randomisedclinicaltrialofimmunogenicityandsafety.Vaccine(2014),http://dx.doi.org/10.1016/j.vaccine.2014.06.006 ARTICLE IN PRESS G Model  JVAC-15457;No.of    Pages7Vaccinexxx(2014)xxx–xxx ContentslistsavailableatScienceDirect Vaccine  j   ournal   homepage:www.elsevier.com/locate/vaccine A   novel   peptide-based   pan-influenza   Avaccine:   A   double   blind,randomised   clinical   trial   of    immunogenicity   and   safety   James   N.   Francis a ,Campbell    J.Bunce a , ∗ ,Claire   Horlock a ,    Jeannette   M.   Watson a ,Steven    J.   Warrington b ,   Bertrand   Georges a , 1 ,CarltonB.   Brown a , 1 a ImmuneTargetingSystemsLtd.,London,NW1   0NH,UK  b HammersmithMedicinesResearchLtd.,London,NW107EW,UK  a   r   t   i   c   l   e   i   nf   o  Articlehistory: Received27January2014Receivedinrevisedform8May2014Accepted2June2014Availableonlinexxx Keywords: InfluenzaVaccinePeptidesTcellsImmunogenicitySafetyFluoropeptides a   b   s   t   ra   ct Background: FP-01.1   is   anovel   synthetic   influenza   Avaccineconsisting   of    six   fluorocarbon-modified   35-merpeptides   that   encapsulate   multipleCD4+andCD8+   T-cell   epitopes   and   is   designed   toinduceanimmune   response   acrossabroad   population. Methods: FP-01.1was   evaluated   for   safetyandimmunogenicity   inarandomised,   double-blind,   placebo-controlled,   dose-escalation,phaseI   clinical   study   inhealthy   adult   volunteers( n =   49).IFN  ELISpot   assaysand   multicolourflowcytometry   were   usedto   characterisetheimmuneresponse. Results:   FP-01.1   wassafe   andwell   tolerated   atalldoses   testedwith   a   similar   adverse   event   pro-file   in   activelyvaccinated   subjects   compared   with   controls.   Maximum   immunogenicity   was   inthe150    g/peptide   dosegroupwhere   arobust   response   (243spots/million   PBMC)   wasdemonstrated   in75%   subjectscompared   with   0%   inplacebo   controls.   Allsix   peptides   were   immunogenic.   FP-01.1   induceddual   CD4+   and   CD8+Tcellresponses   and   vaccine-specific   Tcells   cross-recognise   divergent   influenzastrains. Conclusions:   Thisfirst-in-human   study   showed   thatFP-01.1has   anacceptablesafetyand   tolerabilityprofile   and   generatedrobust   anti-viral   Tcellresponses   inahigh   proportion   of    subjectstested.   Theresultssupport   the   further   clinical   testing   of    FP-01.1   prior   to   clinical,proof-of-concept,   liveviral   challengestudies.©   2014PublishedbyElsevierLtd. 1.Introduction Influenzaepidemicsareassociatedwithsignificantmorbidityandmortality,particularlyinat-riskgroupssuchastheelderly[1].InfluenzaAcanbesubjectedtomajorantigenicshiftinoneorbothsurfaceantigenspresentinga   majorriskof    worldwidepandemicwithasignificantimpactonhumanhealthandtheeconomy[2,3].Currentinactivatedtrivalentinfluenzavaccine(TIV)strategiesrelyontheinductionof    anantibody-mediatedimmuneresponsespecificfortheantigenicallyvaryinghemagglutinin(HA)surfaceantigen.TIVhaveonlymoderateefficacyoreffectiveness[4–5]andisparticularlyimpededwhenthecirculatingstrainhasdriftedsig-nificantlyfromthevaccinestrain.Arecentmeta-analysisof    TIVstudiesfoundthatanti-HAantibodiesareonlypartialcorrelatesof   Clinicaltrialsregistration.NCT01265914. ∗ Correspondingauthorat:ImmuneTargetingSystems,2RoyalCollegeStreet,London,NW1   0NH,UK.Tel.:+44(0)   2076914908;fax:+44(0)   2076819129. E-mailaddress: campbell.bunce@its-innovation.com(C.J.Bunce). 1 Equalcontributionasnon-clinicalprincipalinvestigators. protectioninthegeneralpopulationandpoorcorrelatesof    protec-tionintheelderly[4].Thereis   a   growingbodyof    evidencesupportingthepro-tectivecontributionofcell-mediatedimmune(CMI)responsestoinfluenza,intheabsenceofa   specificantibodyresponse,whicharepoorlyinducedbyTIVs[6–8].CytotoxicTlympho-cyteactivitywas   associatedwithreducedvirussheddinginacohortofexperimentallyinfectedvolunteerswithnodetectableantibodyresponsesagainsttheexperimentalstrain[9].Clini-calstudiesdemonstratedthatTcellresponses,asmeasuredbytheantiviralmediatorgranzymeB,   weredirectlycorrelatedwithprotectionagainstinfluenzaintheelderly[10,11].Tworecentstudieshaveestablisheda   directcorrelationbetweenpre-existingT   cellresponsesandreducedseverityofinfluenzadisease[12,13].Amajoradvantageof    cell-mediatedoverhumoralimmunityisthatTcellscanrecogniseepitopesthoughouttheviruspro-teome,includingfrominternalantigenswhichareconservedacrossa   rangeof    influenzasubtypes,andthusprovidebroadcross-protectiveimmunityto   both   seasonalstrainsandpandemics[14,15]. http://dx.doi.org/10.1016/j.vaccine.2014.06.0060264-410X/©2014PublishedbyElsevierLtd.  Pleasecitethisarticleinpressas:FrancisJN,etal.Anovelpeptide-basedpan-influenzaAvaccine:Adoubleblind,randomisedclinicaltrialofimmunogenicityandsafety.Vaccine(2014),http://dx.doi.org/10.1016/j.vaccine.2014.06.006 ARTICLE IN PRESS G Model  JVAC-15457;No.of    Pages72    J.N.Francisetal./Vaccinexxx   (2014)xxx–xxx We   havedevelopeda   novelinfluenzavaccinestrategybasedonlinkingafluorocarbonmoietyto35-merpeptidesspecificforconserved,internalinfluenzaantigens.Thisvaccine,calledFP-01.1(Flunisyn TM ),   isa   chemicallysynthesized,freeze-driedpreparationofsixdifferentsyntheticpeptidesconjugatedtoaninertfluoro-carbonchain.Linkinga   fluorocarbonchainto   peptidespromotestheirhalf-life invivo ,therebyputativelyallowingmoreprolongedexposureofthepeptidestotheimmunesystemandenhancingimmunogenicity.We   reporttheresultsofafirst-in-humanphaseI   clinicalstudyinhealthyadultvolunteersassessingthesafety,tolerabilityandimmunogenicityofanovel,pan-influenzaA   fluorocarbon-modifiedpeptidevaccinedesignedtoinduceoramplifypre-existingandcross-protectiveTcellresponses. 2.Materialsandmethods  2.1.Vaccinedesign VaccineFP-01.1comprisessixdifferentsynthetic35-merpeptides,eachconjugatedtothefluorocarbonmoietyC 8 F 17 (CH 2 ) 2 -COOHandarederivedfrominfluenzaAinternalproteins;nucleoprotein,matrixproteinandpolymerasebasic1andpoly-merasebasic2   proteins:P44,HMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADK;P220,VAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQG;P1100a,YITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFE;P1116b,APIMFSNKMARLGKGYMFESKRMKLRTQIPAEMLA;P3071,DQVRESRNPGNAEIEDLIFLARSALILRGSVAHKS;P3845,DLEALMEWLKTRPILSPLTKGILGFVFTLTVPSER.Eachpeptidesequencewasselectedonthebasisof    a   highlevelofconservationacrossH1–H9influenzaA   strainsisolatedfromhumans,birdsandpigs.Forexample,thereis   99.0%and97.1%identitybetweenselectedpeptidesandcorrespondingsequencesfromavianH5N1andswineH1N1(2009)respectively.Thebioin-formaticsprocessalsoselectedsequencesthat   containpredictedHLAbindingmotifsacrossthemostgloballyprevalentHLAclassIImolecules( n =   13)andHLAclassImolecules( n =35)in   ordertoachieveahighpopulationcoverage.Inaddition,peptideselectionavoidedanticipatedlarge-scalemanufacturingconstraints.EachpeptidewasmanufacturedundercurrentGoodMan-ufacturingPractice(cGMP)conditionsusingsolidphaseFmocchemistry.FP-01.1(consideredastheInvestigationalManufac-turedProduct,IMP)wasproducedinaccordancewithcGMPbyCarbogenAmcis,Franceandmanufacturedasa   freeze-driedproductcontaining350  gofeachpeptideand31.5mg   manni-tol.IMP   wasreconstitutedincGMP28mM   l -histidinebuffer,tocreateahomogeneoussolutionata   concentrationof    500  g/mL perpeptidewithcloseto   neutralpHandanosmolalityof 300mOsm/L.  2.2.Subjectsandstudydesign A   randomised,double-blind,placebo-controlled,ascendingdosestudytoassessthesafety,tolerabilityandimmunogenicityof repeatedintramuscularadministrationof    FP-01.1inhealthyvol-unteerswasperformedatHammersmithMedicinesResearchLtd.,London(Fig.1).ThestudywasconductedinaccordancewithEUDirective2001/20/ECandICHGCP.Theprotocolwas   approvedbythe   MedicinesandHealthcareProductsRegulatoryAgencyin   theUKandtheNationalResearchEthicsServiceCommitteeLondon(Brent).Writteninformedconsentwasobtainedfrom49healthymaleandfemalesubjectsagedbetween22and55yearsthatwereenrolled(Table1).Detailedinclusion/exclusioncriteriaaredescribedinclinicaltrials.govunderidentifierNCT01265914.Theprimarysafetyendpointsweretolerability,incidenceoftreatmentemergentadverseevents(TEAEs),clinicallysignificantchangesin   laboratorysafetytests,12-leadelectrocardiograms,vitalsignsandphysicalexaminationfindings.SubjectswereprovidedwithdiarycardstoreportAEswhennotattheclinicalsite.Randomisa-tioncodeswerecomputer-generatedbyanunblindedstatisticianandsubjectsweresequentialassignedanunusedrandomisationnumber.Withtheexceptionof    thepharmacistandstatistician,allstaff,subjectsandemployeesof    ITSremainedblindeduntiltheformalunblindingof    thestudy.Threeascendingdosecohorts(50,150and500  g/peptide)of16subjects(12active:4placebo)administeredtestvaccineonDay1,29(mainstudyphase)and99(followupphase).Eachcohortstartedwitha   sentinelgroupoffoursubjectsandsafetydatawasreviewedbeforeprogressiontotheremainingsubjects.Placebosubjectsreceived45mg/mLmannitolin   l -histidinebuffer.Adverseeventswererecordedandpresentedfromday7today113.Noformalsamplesizecalculationswereper-formed.Thestudywas   conductedbetweenAugust2010andMarch2011(uptoday113).  2.3.Peptidesforinvitrotesting  35-merpeptides,at ≥ 95%purity,correspondingto   theantigensinFP-01.1(butwithoutthefluorocarbonchain)wereusedas invitro recallantigensinELISpotassays.Amixtureofthesixpeptides(LPMIX6)andamixtureof    90putativeCTL(cytotoxicT   lymphocyte)targetpeptides(SPMIX;9-merpeptidespredictedusingSYFPEITHI(www.syfpeithi.de))derivedfromthe35-merpeptidesequenceswereusedasantigensforflowcytometricanalysisandformultiplexcytokineassays.  2.4.PBMCisolationandcryopreservation Within8hof    collection,PBMCwereseparatedfromheparinisedbloodbydensitygradientcentrifugationon   Histopaque-1077(Sigma).PBMCwerecryopreservedat1 × 10 7 cells/mLin   10%dimethylsulfoxide/foetalcalfserumandstoredin   liquidnitrogen.Samplesweretransferredbetweensitesusinga   liquidnitrogendryshipper.Onlycellsampleswith>80%viability(asassessedbypropidiumiodidestainingandflowcytometry)wereana-lysed.Mediancellviabilityandrecoverywere96%and79%respectively.  2.5.ELISpotassay An   IFN    enzyme-linkedimmunosorbentspot(ELISpot)assaywasutilisedto   measuretheprimaryimmunogenicityendpointfromsamplescollecteduptoday43andwas   conductedbyImmuneHealth(Belgium)followingGoodClinicalLaboratoryPracticeguidelines.Samplesfromeachindividualsubjectweretestedinparallelonthesamedaytominimiseassayvariability.PVDFplates(Millipore)werecoatedovernightat4 ◦ Cwithanti-humanIFN    antibody(R&Dsystems)andblockedfor>1   h(1%BSA(PAA),5%sucrose(Fisher),Dublecco’s-PBS(Invitrogen)).Plateswerewashedwithcompletemedia(5%humanserum(Sigma),RPMIGlutamax(Invitrogen)andgentamicin(Invitrogen))priorto   use.2 × 10 5 PBMCwerestimulatedwithindividual35-merpeptides(10  g/peptide/mL).Negative(completemedium)andpositive(phytohaemagglutininandCEF(peptidesfromcytomegalovirus,EBV,andinfluenza))controlswereincluded.After18hofculture,plateswerewashedandincubatedwithbiotinylatedanti-humanIFN    (R&Dsystems)followedbystreptavidin-HRP.ProductionofIFN  wasdetectedusingtheELISpotbluecolourmodule(R&DSystems)aspermanufacturer’sinstructions.Plateswere  Pleasecitethisarticlein   pressas:FrancisJN,etal.Anovelpeptide-basedpan-influenzaAvaccine:Adoubleblind,randomisedclinicaltrialofimmunogenicityandsafety.Vaccine(2014),http://dx.doi.org/10.1016/j.vaccine.2014.06.006 ARTICLE IN PRESS G Model  JVAC-15457;No.of    Pages7  J.N.Francisetal./Vaccinexxx(2014)xxx–xxx 3 Excluded (n= 19) Not meeting inclusion criteria (n= 12 ) Declined to participate (n= 6 ) Otherreasonsn=1  Assessed for eligibility (n= 68) Placebo (n = 12) Randomised (n= 49) FP-01.1 150 µg/peptide (n = 12) FP-01.1 50 µg/peptide (n = 12) FP-01.1 500 µg/peptide (n = 13) Lost to follow (n=0) Discontinued intervention (n=0) Lost to follow up (n=0) Discontinued intervention (n=0) Lost to follow up (n=0) Discontinued intervention (n=0) Lost to follow up (n=0) Discontinued intervention due to protocol violation (n=1)  Analysed for immunogenicity (n=12)  Analysed for safety (n=12)  Analysed for immunogenicity (n=12)  Analysed for safety (n=12)  Analysed for immunogenicity (n=12)  Analysed for safety (n=12)  Analysed for immunogenicity (n=12)  Analysed for safety (n=13) Fig.1. CONSORTdiagram. scannedandcountedusinganautomatedELISpotreadersys-tem(AID).ResultsareexpressedasSpotFormingCells(SFC)/10 6 PBMC.  2.6.Flowcytometricanalysisusingintracellularcytokinestaining  PBMCwerestimulatedwitheithercompletemediumalone(negativecontrol)ora   mixtureof    LPMIX6(5  g/mL/peptide)andSPMIX6(finalconcentrationof5  g/mL)for20h,   ora   mixtureof    Phorbol12-Myristate13-Acetate(10ng/mL)andionomycin(1  g/mL)for3hwasusedasapositivecontrol.Golgiplug(BD)wasaddedforthefinal3hofcellculture.Cellswerestainedwiththe   followingantibodiesfromBDBiosciences:CD3-APC-H7(cloneSK7),CD4-APC(cloneSK3),CD8-PE-Cy7(cloneSK1)andIFN  -PE(cloneB27).SamplesandappropriatecontrolswerecollectedonaBDFACSCantoII   andanalysedusingFACSDivav6.1.2.FP-01.1-specificcytokineproductionwasrecordedasexpressionabovebackground.  2.7.CytokineandgranzymeBmeasurementsinculturesupernatants 2 × 10 5 PBMCwereincubatedwithcompletemedia(nega-tivecontrol)orLPMIX6at5  g/peptide/mLfor48hat37 ◦ C,5%CO 2 .Supernatantswerefrozenat − 20 ◦ Cuntilanalysisusingacytometricbeadarray(BDBiosciences)aspermanufacturer’sinstructions.  2.8.Cross-reactivityassay InfluenzaA/California/7/2009,A/HongKong/8/68andA/PuertoRico/8/34werefromAdvancedBiotechnologiesInc.,whileA/Brisbane/59/2007,A/Panama/2007/99andA/Wisconsin/67/2005wereakindgiftfromRetroscreenVirologyLtd.A549cells(ATCC;HLA-A2restricted)wereseededat1.5   × 10 4 cells/wellandviruseswereaddedat3–10MOI.Afterovernightculture,A549cellswerewashedandwereco-culturedwith2.25 × 10 5 PBMC/wellataratioof    1:15for24h.PBMCwereselectedfromHLA-A2+vaccinerespon-ders( n =3).Negativecontrolsof    PBMCaloneandPBMC/A549cellswithoutviruswereassessedinparallel.Cellculturesupernatantswerecollectedandfrozenat − 20 ◦ Cbeforeanalysisof    IFN    andGranzymeBbycytometricbeadarray.  2.9.Statisticalanalysis Intheprimaryimmunologicalanalysis,anELISpotresultwasconsideredpositiveifthenumberofSFCwas   greaterthan25SFC/10 6 PBMCandgreaterorequaltotheaveragenegative  Table1 Demographiccharacteristicsofstudyparticipants.Placebo( n   =12)FP-01.150  g/peptide( n =   12)FP-01.1150  g/peptide( n =   12)FP-01.1500  g/peptide( n   =   13)Allsubjects( n =49)Gender( n ) Male/Female10/210/212/011/243/6Ethnicity( n )Europid798933Afro-Caribbean30025Asian/Indian234211Age   (  y )Median[range]27   [22–54]36[25–55]36   [26–50]39   [22–52]36[22–55]BMI   (kg/m 2 )Median[range]25   [20–31]26[22–32]26   [22–31]25   [22–32]25[20–32]  Pleasecitethisarticleinpressas:FrancisJN,etal.Anovelpeptide-basedpan-influenzaAvaccine:Adoubleblind,randomisedclinicaltrialofimmunogenicityandsafety.Vaccine(2014),http://dx.doi.org/10.1016/j.vaccine.2014.06.006 ARTICLE IN PRESS G Model  JVAC-15457;No.of    Pages74    J.N.Francisetal./Vaccinexxx   (2014)xxx–xxx controlplustwostandarddeviationsofthenegativecontrol.ThevalueofthenegativecontrolwassubtractedfromallELISpotdata.Vaccineresponderthresholdsweresetafterthestudywascom-pletedusingELISpotdatafromtheplacebogroup( n =   12)sothatnosubjectsintheplacebogroupweredefinedasvaccineresponders.AresponderwasdefinedasasubjectwithaFP-01.1-specificpos-itiveELISpotresponseabovea   thresholdof>125%ofthebaselineresponseandanELISpotscoregreaterthanthebaselineresponseplus63SFCtothesumofindividualpeptideresponsesmeasuredataminimumofonetimepointaftervaccination.Foranalysisof    thebreadthofresponse,a   thresholdof>125%of    thebaselineresponseandgreaterthanthebaselineresponseplus25SFCtoindividualpeptideresponseswasapplied. 3.Results  3.1.Vaccinetolerabilityandsafety FP-01.1demonstratedanacceptablesafetyprofileatalldoselevelstested(Table2).Acrossthedurationofthestudy(127days)atotalof46subjectsreported186TEAEs.Themostcom-mon   adverseeventswererespiratorydisordersandheadache:32subjects(65.3%)hadrespiratorydisorders(oropharyngealpain,rhinorrhoea,cough,nasalcongestion,andsneezing);and24sub- jects(49.0%)reportedheadache.Headachewasmorecommonafteractivetreatmentthanafterplacebo,buttheincidenceof    respiratorydisorderswassimilarinalltreatmentgroups.Therelativelyhighincidenceofthesesymptomsmay   beattributedtothedailymoni-toringofsymptomsfor127daysandthatthestudywasconductedduringtheUKwinter.Mostof    theseTEAEsweremildinseverityandclassedbytheinvestigatorasunlikelytoberelatedtoimmun-isationwithFP-01.1.Only12eventswereconsideredrelatedtoFP-01.1treatmentandthoseeventswerepredominantlyinjectionsitereactions.Twosevereadverseeventswerereported(Table2)andwereunrelatedvaccineadministration.Noneofthelaboratoryvaluesshowedchangesthatwereclini-callysignificantorcouldreasonablybeattributedtoadministrationofFP-01.1.Theresultsof    allphysicalexaminationswerenormal.AllvitalsignsandECGrecordingswereconsideredto   bewithinnormallimits,or,ifoutsidethereferencerange,of    noclinicalsignificance.Injectionsitetolerabilitywasfavourable,foursubjectshadmildlocalreactionsandthoseoccurredonlyafterthefirstinjectionof FP-01.1.Noitchingwasreported.Rednesswasthemostfrequentlyreportedlocalreaction,observedonlyafteractivetreatment,andmostcommonat24hafterthesecondinjection.Nosubjecthadaseverelocalreactionto   thestudymedication.Overall,FP-01.1waswelltolerateduptoandincludingthemaximumdoseof 500  g/peptideFP-01.1administeredonthreeoccasions.  3.2.Immunologicalanalysis Theprimaryimmunologicalanalysismeasuredthefrequencyofvaccine-specificTcellsinPBMCcollecteduptostudyday43usinganIFN  ELISpotassay(Fig.2).At   baselinethemedianfre-quencyofTcellsspecificforFP-01.1rangedbetween34and60SFC/10 6 PBMC.Subjectsimmunisedwith150  g/peptideshowedthegreatestincreasein   Tcellfrequenciesandmaximumresponseswereat7daysafterthesecondinjection,witha   medianresponseof243SFC/10 6 PBMC(medianincreaseof198SFC/10 6 PBMCabovebaseline; P  <   0.05).Responderfrequencieswere0%,50%,75%and83%fortheplacebo,50,150and500  g/peptidegroupsrespectively.Analysisofsubjectsdefinedasrespondersshowedamedianbreadthofresponseof    fouroutof    thesixpeptidesincludedinthevaccine.AllFP-01.1peptideswereimmunogenicin   thestudypopulation(SupplementaryFig.   1)andanalysisofsubjectHLA-restrictionshowednodifferencebetweenvaccinerespondersandnon-responders(SupplementaryTable1).  3.3.CharacterisationoftheTcellresponse Thephenotypeof    vaccine-inducedIFN  productionwasassessedin   a   subsetofsubjectsdefinedasvaccinerespon-ders.AnalysisbyflowcytometryshowssignificantincreasesinCD3+   CD4+IFN  +andCD3+   CD8+IFN  +   cellswereobserved(Fig.3)aftervaccinationwithFP-01.1.AnalysisofPBMCculturesupernatantsaftera48hincubationwithFP-0.1.1peptidesshowedpost-vaccinationincreasesinIFN  ,IL-2andGranzymeBsuggestingananti-viralphenotype.  3.4.Cross-recognitionofnaturallyprocessedandpresentedviralepitopesby   FP-01.1-specificT    cells PBMCfromFP-01.1-vaccinatedsubjectsweretestedfortheirabilitytorecogniseandrespondtoinfluenzaantigens.TargetcellswerepreparedbyincubatingdifferentinfluenzaviruseswithA549cells,a   HLA-A2-expressinghumanlungepithelialcelllinepermissivetoinfluenzainfectionandreplication.Theabilityof vaccine-specificT   cells(fromHLA-A2+subjects)tocross-recognisetargetcellsinfectedbydifferentviruseswas   establishedbycom-paringresponsesfromPBMCsamplesobtainedbeforeandaftervaccination.PBMCisolatedpost-vaccinationproducedmoreIFN  andgranzymeBinresponseto   targetcellsinfectedwithdifferentH1N1andH3N2influenzastrains(Fig4). 4.Discussion In   thisfirst-in-humanstudyofthefluorocarbon-modifiedpep-tidevaccineFP-01.1,wefoundthatintramuscularadministrationoftwodosescouldinducea   robustimmuneresponse(Fig.4).ImmunisationwithFP-01.1was   welltolerated:therewas   nodose-dependentincidenceofTEAEs,laboratoryabnormalities,orinjectionsitereactions.Nosubjectexhibitedanymarkedlocalorsystemicreactiontovaccinationoneitherthefirst,secondorthirdexposureinanyofthedosingcohorts.Administrationof 150  g/peptideinducedtheoptimumimmuneresponsemeasuredusinganIFN  ELISpotassayspecificforvaccinepeptides.AllsixpeptidesincludedinFP-01.1wereimmunogenicanda   medianbreadthofresponseof    fouroutsixpeptideswas   recorded.TheresponseinducedbyFP-01.1was   seentobecross-reactive,recog-nisingantigensprocessedandpresentedbyvirus-infectedcells.FP-01.1was   designedtoinducenewandenhanceexistinganti-viralCMI   responsesto   conserved,internalinfluenzaantigens.Mobilisinganarmof    theimmuneresponsethattargetsconservedregionsof    thevirusproteomeallowsthedevelopmentof    a   vac-cinethatinducesa   broadcross-reactiveimmuneresponse[16].WeshowthatFP-01.1-specificTcellshavethepotentialtocross-reactwithdifferentstrainsof    influenzaA.TcellsinducedbyFP-01.1vac-cinationcouldrecognisenaturallyprocessedandpresentedviralepitopesfrommultiplesubtypesof    disparateinfluenzavirusstrainsH1N1andH3N2.Thecross-reactivepotentialforFP-01.1-specificTcellssupportsthenotionthatthevaccinewouldconferbroadprotection,possiblyagainstallinfluenzaAstrains.ThemagnitudeoftheFP-01.1-specificTcellresponsemeasuredusingtheIFN  ELISpotassaywas   highestinthe150  g/peptidegroupandmaximumresponsesweremeasured7daysafterthesecondinjection.ThenumberofT   cellsrequiredtoprovideclinicalbenefitagainstinfluenzadiseasehasyettobeestablishedanditisdifficulttocompareresultsof    differentstudiesusingdivergentre-stimulationantigens.Onestudy,usinginactivatedinfluenzavirusasanantigen,suggestedthata   frequencyof    100virus-specificT   cellspermillionPBMCisassociatedwithprotectionagainst  Pleasecitethisarticlein   pressas:FrancisJN,etal.Anovelpeptide-basedpan-influenzaAvaccine:Adoubleblind,randomisedclinicaltrialofimmunogenicityandsafety.Vaccine(2014),http://dx.doi.org/10.1016/j.vaccine.2014.06.006 ARTICLE IN PRESS G Model  JVAC-15457;No.of    Pages7  J.N.Francisetal./Vaccinexxx(2014)xxx–xxx 5  Table2 Frequency(%)oftreatmentemergentadverseeventsandinjectionsitereactionsin ≥ 2subjects.Placebo( n =12)FP-01.150  g/peptide( n =12)FP-01.1150  g/peptide( n   =   12)FP-01.1500  g/peptide( n =   13)SystemicreactionsALLGrade3ALLGrade3   ALLGrade3ALLGrade3Nervoussystemdisorders(e.g.headache)25.00.066.70.075.00.053.80.0Respiratory,thoracicandmediastinaldisorders 75.0 0.0 75.00.050.0   0.061.50.0Generaldisordersandadministrationsiteconditions16.70.08.30.033.30.038.50.0Musculoskeletalandconnectivetissuedisorders33.30.016.70.033.30.023.10.0Gastrointestinalsymptoms8.30.00.00.08.30.030.00.0Infectionsandinfestations16.70.016.70.016.70.015.40.0Eye   disorders16.70.00.00.08.30.07.70.0OTHER    33.3 8.3 * 25.0 0.0 41.78.3 ˆ 53.80.0Injectionsitereactions ALLGrade2andabove ALLGrade2   andaboveALLGrade2andaboveALLGrade2andaboveBruising16.7 0.00.00.08.30.00.00.0Induration8.30.023.10.00.00.016.70.0Redness0.00.061.58.3 ** 50.00.075.00.0Pain   (VASscore) 16.7 0.0 25.00.083.30.068.20.0Note:Gradedescriptionis   0   =   absent,1=mild,2=moderateand3   =   severe.Thetwosevereadverseeventsarealsoconsideredseriousbutwerenotreportable. * Epididymo-orchitis. ˆ Wristfracture. ** Moderate. Fig.2. Magnitudeandkineticsofvaccine-inducedresponses.PBMCfromeachtreatmentgroup( n   =   12/group;all   subjectstested)werestimulatedwithpeptidesfor18hin   anIFN  ELISpotassay.A.The   graphrepresentsvaccine-specificIFN  responseatbaseline(averageresponseatday-7andday1)   andthemaximalresponserecordedpost-vaccinationforeachsubjectandfromeachtreatmentgroup.Resultsareshownasthesumofresponsesto   thesixpeptidesinthevaccineandthelinerepresentsthe   medianresponsepostvaccination.Wilcoxonmatched-pairstestswereusedforstatisticalcomparisons.Medianbackgroundresponseswere<10   SFC/10 6 PBMCandnodifferencesinbackgroundresponsesbetweenrandomisationgroupsorpre-andpost-vaccinationwereobserved.B.   IFN  responsesmeasuredatintervalsthroughthe   studyare   shownfromtheplaceboand150  g/peptidegroupsonly.Datais   shownasmedianandinterquartileranges. Fig.3. Phenotypeof    FP-01.1-specificcells.PBMCsamples( n =   11;respondersonly)wereselectedfromsubjectsdefinedasvaccinerespondersat   eitherbaseline(day-7or   day1;pre)orpost-vaccinationata   timepointwheremaximalIFN  responsesweredetectedusingthe   primaryELISpotassay.A.ThefrequenciesCD3 + CD4 + IFN  + andCD3 + CD8 + IFN  + areshownasmeanplusstandarderrorof    themeanafterbackgroundsubtractionasmeasuredby   flowcytometry.AlsorefertosupplementaryTableS1.B.   PBMCwereculturedwithFP-01.1-peptidesfor48handcellculturesupernatantswereanalysedforIL-2,IFN  andGranzymeBusinga   cytometricbeadarray.Wilcoxonmatched-pairstestswereusedforstatisticalcomparisons.
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