ChrisParkerMSThesis

EffectofaCodonOptimizedDNAPrimeonInductionofAnti-InfluenzaProtectiveAntibodiesAThesisSubmittedtotheFacultyoftheWORCESTERPOLYTECHNICINSTITUTEInpartialfulfillmentoftherequirementsfortheDegreeofMasterofScienceinBiologyandBiotechnologyBy______________________ChristopherParkerApril9,2007APPROVED:____________________________________________________________ShanLu,M.D.,Ph.D.DavidAdams,Ph.D.SamuelPolitzPh.D.DepartmentofMedicineDeptofBiologyandBiotechDeptofBiologyandBiotechUMassMedicalSchoolWPIWPIMajorAdvisorCommitteeMemberCommitteeMember2ABSTRACTAneffectiveantibodyresponseisessentialforimmunityagainstinfluenzavirusinfectionandistheprimarygoalforvaccinedevelopment.Inthisstudy,codonoptimizedandwildtypeDNAvaccinesexpressinghemagglutinin(HA)antigensofhumanfluvirusesA/H1N1/NewCal/20/99(H1serotype)werecomparedtotesttheantigenicdifferencesoftheconstructsinmammaliansystems.Furthermore,todetermineifaprime-boostimmunizationstrategywasmoreeffectiveinelicitingagreaterimmuneresponse,acodonoptimizedHAvaccinewasadministeredasaprimeinconjunctionwiththetrivalentinactivatedvaccine(TIV),Fluzone,asaboostandimmuneresponsesweremeasured.WefoundthatproteinexpressionandantibodyresponselevelsofHAantigenswereincreasedwiththecodonoptimizedconstructwhencomparedtothewildtypeHAgeneconstruct.Prime-boostvaccinationofNZWrabbitswasabletoelicitagreaterimmuneresponsewhencomparedtoTIValoneasmeasuredbyenzyme-linkedimmunosorbentassay(ELISA),hemagglutinininhibition(HI)andneutralizingantibody(NAb)assays.Together,thesestudiesindicatethatoptimalHADNAvaccineformulationsshouldbecodonoptimizedandcanbeusedaspartofaprime-boostvaccinationstrategy.3TABLEOFCONTENTSSignaturePage…………………………………………………....………….1Abstract……………………………………………………………………...2TableofContents……………………………………………………………3Acknowledgements………………………………………………………….4Background………………………………………………………………….5ProjectPurpose……………………………………………………………....19Methods………………………………………………………………………20Results…………………………………………………………………….....30Discussion…………………………………………………………..…...…..47References……..……………………………………………………..….......514ACKNOWLEDGEMENTSFirstandforemost,IwanttoexpressmydeepgratitudeandrespecttoShanLu,M.D.,Ph.D.,DirectoroftheLaboratoryofNucleicAcidVaccinesattheUniversityofMassachusettsMedicalSchool.Dr.Lufacilitatedvaluableandexcitinglearningexperiencesforme.HiscuttingedgeinstructionhasgivenmeauniqueandsolidfoundationinthenascenttechnologyofDNAvaccineresearch.IalsoextendthankstoeveryscientistattheLaboratoryofNucleicAcidVaccines,especiallyShixiaWang,HongCao,ScottColey,SiyuanShen,AnthonyHackett,TomLingandJillGrimes.Theyextendedtheirprofessionalexpertise,resourcesandsupporttome.ThankyoutothelabofDr.AdolfoGarcia-Sastreforconductingprotectiveantibodyassaysandtechnicalexpertisewhenneeded.Inaddition,IextendsincereappreciationtomyWPIfacultyadvisors,ProfessorDaveAdamsandProfessorSamPolitz,forguidancewiththisprojectandthesispreparation.5BACKGROUNDInfluenzarelatedsicknesseshavebecomeaglobalcommonalityduringthewintermonths.Theinfluenzavirusishighlycontagious,attackstheupperrespiratorytract,andhasplaguedtheworldsincenearlythebeginningofwrittenhistory.The“flu,”asitiscommonlyknown,primarilycausesachymuscles,feveranddigestivesystemsymptoms,andcancausedeathinpeoplewithweakenedimmunesystems(LangleyandFaughnan,2004).Somestrains,however,causeglobalpandemicsofcatastrophicproportionssuchastheSpanishinfluenzaof1918-1919duringwhichupto50millionpeoplewerekilledworldwide,regardlessofthestatusoftheirimmunesystem(PaleseandGarcia-Sastre,2002).AccordingtotheWorldHealthOrganization(WHO),wearedueforanotherpandemicwithahumanadaptedformoftheavianfluastheleadingpotentialstrain.Globalpandemicsofthepasthavereachedallcontinentswithin6-9monthsbutwitheasiertravel,thenextpandemiccouldinfectallcontinentsinlessthan3months(WHO,2005).VirologicalFeaturesofInfluenzaVirusTypesofInfluenzaVirusTheinfluenzavirusbelongstothefamilyOrthomyxoviridaewhichareenvelopedviruseswithasegmentedsingle-strandedRNAgenome(PaleseandGarcia-Sastre,2002).Therearethreetypesofinfluenzacurrentlyinexistence,influenzaA,BandC.InfluenzaAandBaremorphologicallyindistinguishable,whileinfluenzaCisdifferentiatedbyitsglycoproteinspikes.InfluenzaAcanspreadamongbothhumansandanimals,influenza6Baffectsonlyhumans,andwhileinfluenzaCaffectspredominantlyhumansitrecentlyhasinfectedswineinChina(LambandKrug,1996).TheinfluenzaAvirionischaracterizedbythe16subtypesofhemagglutinin(HAprotein)andbythe9subtypesofneuraminidase(NAprotein)bothofwhichareproteinspikesonthesurfaceofthevirus(LambandKrug,1996;MurphyandWebster,1996).Strainsarenamedbasedonthesetwoproteins.Forexample,thecurrentavianflustraindesignatedH5N1,whichcurrentlyhasnotestablishedhuman-to-humantransmission,isaninfluenzaAviruswithasubtype5HAproteinandasubtype1NAprotein.Newinfluenzavirusstrainsresultprimarilyfromantigenicchangesofthehemagglutininandneuraminidaseproteins.Antigenicdriftresultsfrompointmutationsthatoccurduringviralreplicationwithinamajorserotype,whileantigenicshiftoccurswhengenesfromanimalinfluenzavirusesarecapturedbythehumanvirusviareassortmentusuallyres