Comparative Hepatitis (Birkhäuser Advances in Infectious Diseases) 376438557X, 9783764385576

Acute and chronic hepatitis and hepatitis-related diseases such as liver failure, liver cirrhosis and hepatocellular car

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Comparative Hepatitis (Birkhäuser Advances in Infectious Diseases)
 376438557X, 9783764385576

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#ONTENTS ,ISTOFCONTRIBUTORS  VII 2INO2APPUOLIAND'IUSEPPE$EL'IUDICE )NTRODUCTION 



3AMIRA-UBAREKAAND0ETER0ALESE )NmUENZAVIRUS4HEBIOLOGYOFACHANGINGVIRUS 



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-ARIA,ATTANZI .ON RECENTHISTORYOFINmUENZAPANDEMICS VACCINESANDADJUVANTS   2INO2APPUOLIAND'IUSEPPE$EL'IUDICE 7AITINGFORAPANDEMIC    #ATERINA2IZZOAND-ARTA,UISA#IOlDEGLI!TTI -ODELINGINmUENZAPANDEMICANDINTERVENTIONS   )NDEX   

,ISTOFCONTRIBUTORS -ARTA,UISA#IOlDEGLI!TTI .ATIONAL#ENTREOF%PIDEMIOLOGY 3URVEILLANCE AND(EALTHPROMOTION )NFECTIOUS$ISEASE5NIT )STITUTO3UPERIOREDI3AN ITÌ 2OMA )TALY 'IUSEPPE$EL'IUDICE .OVARTIS6ACCINESAND$IAGNOSTICS 6IA&IORENTINA 3IENA )TALYE MAILGIUSEPPEDEL?GIUDICE NOVARTISCOM 0ETER#$OHERTY $EPARTMENTOF-ICROBIOLOGYAND)MMUNOLOGY 5NIVERSITY OF-ELBOURNE 0ARKVILLE 6ICTORIA !USTRALIA$EPARTMENTOF)MMU NOLOGY 3T*UDE#HILDRENS2ESEARCH(OSPITAL -EMPHIS 4. 53! E MAIL0ETER$OHERTY 34*5$%/2' +ATHRYN - %DWARDS 6ANDERBILT 5NIVERSITY 3CHOOL OF -EDICINE $EPART MENT OF 0EDIATRICS 0EDIATRIC #LINICAL 2ESEARCH /FlCE .ASHVILLE 4.  53!E MAILKATHRYNEDWARDS VANDERBILTEDU !NTHONY'ILBERT 2ETROSCREEN6IROLOGY,TD 3T"ARTSANDTHE2OYAL,ON DON(OSPITAL 2OYAL#OLLEGE3TREET ,ONDON.7/.( 5+ (ARRY'REENBERG $EPARTMENTSOF-EDICINEAND-ICROBIOLOGYAND)MMU NOLOGY 3TANFORD5NIVERSITY3CHOOLOF-EDICINE 3TANFORD #!AND 6ETERANS!FFAIRS 0ALO!LTO (EALTH #ARE 3YSTEM 0ALO!LTO #!  53!E MAILHARRYGREENBERG STANFORDEDU #AROLE 'UILLONNEAU $EPARTMENT OF -ICROBIOLOGY AND )MMUNOLOGY 5NI VERSITYOF-ELBOURNE 0ARKVILLE 6ICTORIA !USTRALIA 'EORGE +EMBLE -ED)MMUNE 6ACCINES )NC -OUNTAIN 6IEW #!  53!E MAILKEMBLEG MEDIMMUNECOM 2OBERT,AMBKIN 7ILLIAMS 2ETROSCREEN6IROLOGY,TD 3T"ARTSANDTHE2OY AL,ONDON(OSPITAL 2OYAL#OLLEGE3TREET ,ONDON.7/.( 5+ -ARIA,ATTANZI .OVARTIS6ACCINESAND$IAGNOSTICS 6IA&IORENTINA  3IENA )TALYE MAILMARIALATTANZI NOVARTISCOM #ATHERINE*,UKE ,ABORATORYOF)NFECTIOUS$ISEASES .ATIONAL )NSTITUTEOF !LLERGYAND)NFECTIOUS$ISEASES .ATIONAL)NSTITUTESOF(EALTH "ETHESDA -$ 53!E MAILCLUKE NIAIDNIHGOV -ARK!-ILLER &OGARTY)NTERNATIONAL#ENTER .ATIONAL)NSTITUTESOF(EALTH "ETHESDA-ARYLAND 53!E MAILMILLEMAR MAILNIHGOV *USTINE$-INTERN $EPARTMENTOF-ICROBIOLOGYAND)MMUNOLOGY 5NIVER SITYOF-ELBOURNE 0ARKVILLE 6ICTORIA !USTRALIA

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%MANUELE-ONTOMOLI $EPARTMENTOF0HYSIOPATHOLOGY %XPERIMENTAL-ED ICINEAND0UBLIC(EALTH ,ABORATORYOF-OLECULAR%PIDEMIOLOGY 5NIVER SITYOF3IENA 6IA!LDO-ORO 3IENA )TALY E MAILMONTOMOLI UNISIIT 3AMIRA -UBAREKA $EPARTMENT OF -ICROBIOLOGY -OUNT 3INAI 3CHOOL OF -EDICINE .EW9ORK .9 53! $EREK 4 /(AGAN .OVARTIS 6ACCINES AND $IAGNOSTICS 6IA &IORENTINA  3IENA )TALYE MAILDEREKOHAGAN NOVARTISCOM *OHN /XFORD 2ETROSCREEN6IROLOGY ,TD 3T "ARTS AND THE 2OYAL ,ONDON (OSPITAL 2OYAL#OLLEGE3TREET ,ONDON.7/.( 5+ E MAILJOXFORD RETROSCREENCOM 0ETER0ALESE $EPARTMENTOF-ICROBIOLOGY -OUNT3INAI3CHOOLOFF -EDICINE .EW9ORK .9 53!E MAILPETERPALESE MSSMEDU !UDINO0ODDA .OVARTIS6ACCINESAND$IAGNOSTICS 6IA&IORENTINA   3IENA )TALYE MAILAUDINOPODDA NOVARTISCOM 2INO2APPUOLI .OVARTIS6ACCINESAND$IAGNOSTICS 6IA&IORENTINA  3IENA )TALYE MAILRINORAPPUOLI NOVARTISCOM #ATERINA2IZZO .ATIONAL#ENTREOF%PIDEMIOLOGY 3URVEILLANCE AND(EALTH PROMOTION )NFECTIOUS$ISEASE5NIT )STITUTO3UPERIOREDI3ANITÌ 2OMA AND$EPARTMENTOF0HARMACO "IOLOGY 5NIVERSITYOF"ARI )TALYE MAIL E MAILCATERINARIZZO ISSIT ,ONE3IMONSEN '753CHOOLOF0UBLIC(EALTHAND(EALTH3ERVICES 7ASH INGTON$# 53!E MAILLONE GWUEDU +ANTA 3UBBARAO ,ABORATORY OF )NFECTIOUS $ISEASES .ATIONAL )NSTITUTE OF !LLERGYAND)NFECTIOUS$ISEASES .ATIONAL)NSTITUTESOF(EALTH "ETHESDA -$ 53!E MAILKSUBBARAO NIAIDNIHGOV 2OBERT*4AYLOR 3!'%!NALYTICA ,,# "ETHESDA -ARYLAND 53! E MAIL RTAYLOR SAGEANALYTICACOM 3TEPHEN*4URNER $EPARTMENTOF-ICROBIOLOGYAND)MMUNOLOGY 5NIVER SITY OF -ELBOURNE -ELBOURNE 6ICTORIA  !USTRALIA $EPARTMENT OF )MMUNOLOGY 3T*UDE#HILDRENS2ESEARCH(OSPITAL -EMPHIS 4. 53! #ÏCILE6IBOUD &OGARTY)NTERNATIONAL#ENTER .ATIONAL)NSTITUTESOF(EALTH "ETHESDA-ARYLAND 53!E MAILVIBOUDC MAILNIHGOV

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)NTRODUCTION 2INO2APPUOLIAND'IUSEPPE$EL'IUDICE .OVARTIS6ACCINESAND$IAGNOSTICS 6IA&IORENTINA 3IENA )TALY

7E HAVE BEEN LIVING WITH INFLUENZA AS LONG AS HISTORY CAN REMEMBER AND WEARESOUSEDTOTHEFACTTHATTHISDISEASEISPARTOFLIFETHATWEUSUALLYDO NOTPAYMUCHATTENTIONTOIT%VENTHESCIENCEOFINFLUENZAVIRUSHASBEEN DORMANT FOR MORE THAN HALF A CENTURY!FTER THE EXCITEMENT OF THE INITIAL DISCOVERYOFTHEVIRUSINANDTHEDEVELOPMENTOFTHEFIRSTVACCINESBY GROWINGTHEVIRUSINTHEALLANTOICCAVITYOFEMBRYONATEDHENS EGGSINTHE S RELATIVELY LITTLE HAS HAPPENED IN THE FIELD DEVELOPMENT OF THE FIRST SEROLOGICAL ASSAYS DEFINITION OF SOME SEROLOGICAL CORRELATES OF PROTECTION AND LITTLE MORE 4HERE WAS NO REAL BREAKTHROUGH UNTIL THE APPLICATION OF REVERSE GENETICS AND OF NOVEL ADJUVANT TECHNOLOGY TO THE FIELD 3OMEHOW PEOPLE THOUGHT THAT THE INFLUENZA PROBLEM HAD BEEN SOLVED OR WAS NOT A PROBLEM VERY LITTLE MONEY WAS AVAILABLE FOR RESEARCH 7ORLDWIDE ONLY A FEW LABORATORIES CONTINUED TO PERFORM RESEARCH ON INFLUENZA 6ACCINE MANUFACTURERS HAD NO INCENTIVE TO INVEST IN IMPROVED INFLUENZA VACCINES BECAUSE THE LOW PRICE AND THE LIMITED MARKET DID NOT JUSTIFY INVESTMENTS INNEWTECHNOLOGIES7HENTHESTCENTURYARRIVED WITHEVERY SINGLEFIELD WAS CELEBRATING TECHNOLOGICAL QUANTUM JUMPS AND THE EXCITEMENTS OF THE HUMANGENOMEPERMEATINGTHEGLOBE INFLUENZAWASSTILLHAPPYTOUSETHE TECHNOLOGIESOFS COMPLACENTWITHTHESTATUSQUO 4ODAY FORANUMBEROFCONCOMITANTREASONS THEINFLUENZAFIELDISABOUT TOMAKETHEFIRSTIMPORTANTCHANGESAFTERMORETHANHALFACENTURY ANDIT ISLIKELYTOTAKELEADERSHIPININTRODUCINGTECHNOLOGICALINNOVATIONS3INCE THEFIELDISNOWMOVINGFASTANDTHEREAREMANYNEWCOMERS THEREISARISK TORE DISCOVEREVERYTHING TOFORGETTHEPASTEXPERIENCE ANDTOREPEATTHE MISTAKESALREADYDONEINTHEPAST&ORHISREASON THISBOOKISDESIGNEDTO PROVIDETHEBACKGROUNDINFORMATIONNECESSARYTOUNDERSTANDTHE STATE OF THE ART OF THE INFLUENZA FIELD )N THE BOOK THERE ARE CHAPTERS DEALING WITH



2INO2APPUOLIAND'IUSEPPE$EL'IUDICE

THE BASIC BIOLOGY AND MOLECULAR BIOLOGY OF THE VIRUS CHAPTERS DESCRIBING THENON RECENTANDRECENTHISTORYOFINFLUENZAANDINFLUENZAVACCINES CHAP TERSONEPIDEMIOLOGY ANIMALMODELS CORRELATESOFPROTECTION THEIMMUNE RESPONSETOINFLUENZAINFECTIONANDVACCINES ADJUVANTS LIVE ATTENUATEDVAC CINES AVIANVIRUSES ANDPANDEMICVACCINES/VERALLTHEMESSAGESTHATCOME OUTOFTHECHAPTERSCANBESUMMARIZEDASFOLLOWS

)NFLUENZAISAGLOBALUNDERESTIMATEDKILLER 4HE 7ORLD (EALTH /RGANIZATION REPORTS THAT INFLUENZA INFECTS ANNUALLY nOFTHEPOPULATION CAUSESnMILLIONCASESOFSEVEREILLNESS ANDREG ISTERSUPTO DEATHS2EADINGTHECHAPTERBY3IMONSENET AL ITISCLEAR THAT WHILEINDEVELOPEDCOUNTRIESTHEEPIDEMIOLOGYOFINFLUENZAISDIFFICULT ANDMORTALITYDATAARECONFOUNDED BYTHEMANYSECONDARYDISEASESTHATARE THE ULTIMATE CAUSE OF DEATH FOLLOWING AN INFLUENZA INFECTION IN DEVELOP INGCOUNTRIESTHEREARENODATAATALL0OSSIBLY ALOTOFTHECHILDMORTALITY RECENTLY CALCULATED FOR PNEUMOCOCCUS MORE THAN  MILLION ANNUALLY IS A CONSEQUENCEOFAPRIMARYINFLUENZAINFECTION%VENINARICHCOUNTRYSUCH AS THE 53! INFLUENZA CAUSES   DEATHS PER YEAR! SIMPLE CALCULATION APPLYINGTHEANNUALDEATHRATEOFTHE53!TOTHEGLOBALPOPULATIONWOULD PREDICTANANNUALGLOBALINFLUENZAMORTALITYOF 4HESECALCULATIONS AREOBVIOUSLYINAPPROPRIATEBECAUSETHEPOPULATIONAGEISDIFFERENTANDTHE SOCIO ECONOMICCONDITIONSVARY(OWEVER THEYCANBEUSEFULJUSTTOSUGGEST THATTHEMORTALITYOFINFLUENZAANDTHEIMPACTONTHEGLOBALPOPULATIONARE UNDERESTIMATED ANDTHATTHEREISANURGENTNEEDTOLEARNMORE ABOUTINFLU ENZA ESPECIALLYINDEVELOPINGCOUNTRIESANDTROPICALAREAS7EBELIEVETHAT WHEN APPROPRIATE STUDIES ARE PERFORMED WE WILL FIND OUT THAT THE GLOBAL MORTALITYDUETOINFLUENZAISINEXCESSOFMILLION

)NFLUENZAISANIMPORTANTDISEASEFORCHILDREN 4HECHAPTERSOF%DWARDSAND3IMONSENETALBOTHREPORTTHATTHEMAJORITY OFTHEINFLUENZADISEASECASESOCCURININFANTS CHILDREN ANDTHEELDERLY)N THE 53! HOSPITALIZATION RATES CAUSED BY INFLUENZA ARE  PER  CHIL DREN OF LESS THAN  MONTHS OF AGE AND DECREASE TO  TO  PER  WITH INCREASINGAGE-ORTALITY UPTOCASESINn ISALSOPRESENTIN SPITEOFTHEEXTREMELYSOPHISTICATEDHEALTHCARESYSTEM(OWEVER WHILETHE ELDERLYPOPULATION ATLEASTINTHEWESTERNWORLD ISCOVEREDBYVACCINATION PROGRAMS VACCINATIONOFINFANTSANDCHILDRENHASONLYBEENRECENTLYRECOM MENDEDINTHE53!ANDISNOTPERFORMEDELSEWHERE4HEFIRSTRESULTSOFTHE 53! VACCINATION OF SCHOOLCHILDREN SUGGEST THAT IN ADDITION TO PROTECTING CHILDREN VACCINATIONPROVIDESAHERDIMMUNITYANDEXTENDSTHE PROTECTION TONON IMMUNIZEDHOUSEHOLDS4HEOBSERVATIONTHATVACCINATIONOFCHILDREN

)NTRODUCTION



PROTECTS THE ELDERLY IS VERY ENCOURAGING AND SUGGESTS THAT POLICY MAKERS SHOULD URGENTLY INTRODUCE INFANT AND CHILDREN VACCINATION AS A COMMON PRACTICE)NSPITEOFTHEAVAILABILITYOFTHERECENTLYINTRODUCEDLIVE ATTENUAT ED COLD ADAPTEDVACCINE,!)6 ANDTHECONVENTIONALTRIVALENTINACTIVATED VACCINE4)6 THEREISTHENEEDFORIMPROVEDVACCINESFORCHILDREN)NFACT THEEFFICACYOF4)6ISNOTOPTIMAL ANDTHE,!)6CANNOTBEUSEDINCHILDREN BELOWYEARSOFAGE!GAIN NODATAONINFLUENZAININFANTSANDCHILDRENARE AVAILABLEFROMDEVELOPINGCOUNTRIESHOWEVER ITISEASYTOIMAGINETHAT IN THEABSENCEOFAGOODHEALTHCARESYSTEM INFLUENZACANBEDEVASTATING7E BELIEVETHATTHEREISANURGENTNEEDTOGENERATEDATAANDTHEINCLUSIONOF INFLUENZAAMONGTHEVACCINEPRIORITIESOF'!6)FORCHILDRENANDPREGNANT WOMENMAYBEONEOFTHELOWHANGINGFRUITSTHATWOULDMAKEAGREATCON TRIBUTIONTOGLOBALHEALTH

#ORRELATESOFPROTECTIONRELYONSERUMANTIBODIES BUTLIVE ATTENUATEDVACCINESUSEALSOSOMETHINGELSE 4HELICENSUREOFINFLUENZAVACCINES DESCRIBEDINTHECHAPTERSBY-ONTOMOLI AND/XFORDETAL ISBASEDONTHEINDUCTIONOFSERUMANTIBODIES4HESEARE MEASUREDBYEITHERTHECAPACITYOFSERATOINHIBITTHEBINDING OFTHEVIRUS TO THE RECEPTORS PRESENT OF THE SURFACE OF THE RED BLOOD CELLS AND CAUSE AGGLUTINATION OF THE CELLS () OR ON THE ABILITY OF THE ANTIBODIES TO FORM ACOMPLEXONREDBLOODCELLSCOATEDWITHTHEINFLUENZAANTIGEN ANDCAUSE A COMPLEMENT MEDIATED HEMOLYSIS OF THE CELLS SINGLE RADIAL HEMOLYSIS OR 32(  "OTH METHODS USE CONVENIENT NON SOPHISTICATED READ OUTS WHERE THECOLOROFTHEREDBLOODCELLSISAMARKERTHATCANBESCOREDBYTHEEYE !LTHOUGHNOTSOPHISTICATED THESEMETHODSARERELIABLEANDWELLVALIDATED BYTHEEXPERIENCEOFDECADES ANDWEKNOWTHATAN()TITEROFCORRELATES WITH PROTECTIVE EFFICACY IN HUMANS (OWEVER THESE VERY OLD TECHNOLOGIES START TO SHOW A LOT OF SHORTCOMINGS &OR INSTANCE () EXHIBITS A VERY HIGH DEGREEOFVARIABILITYFROMONELABORATORYTOANOTHER4HESITUATIONISEVEN WORSEFORAVIANVIRUSESSUCHAS(.VIRUSSTRAINS)NDEED TO MEASURE() TITERSAGAINST(.VIRUSES THEREDBLOODCELLSOFCHICKENTHATWEROUTINELY USEFORCONVENTIONALVACCINESCOULDNOTBEUSEDBECAUSETHEAVIANVIRUSREC OGNIZES MOSTLY AVIAN OLIGOSACCHARIDE RECEPTORS CONTAINING . ACETYL NEUR AMINIC ACID LINKED BY _   GALACTOSE WHICH ARE NOT ABUNDANT IN CHICKEN REDBLOODCELLSTHATCONTAINMOSTLYHUMANOLIGOSACCHARIDERECEPTORSLINKED BY AN _   GALACTOSE BOND 4HUS TO DETERMINE THE () TITERS AGAINST THE (.VIRUS WEHADTOSWITCHTOHORSEREDBLOODCELLS WHICHCONTAINMORE _   GALACTOSE BONDS (OWEVER IN DOING SO WE MOVED AWAY FROM WELL STANDARDIZED ALTHOUGHVARIABLE ASSAYS ANDTODAYTHE()TITERSREPORTEDFOR (.AREEVENLESSREPRODUCIBLEANDEVENMOREVARIABLEFROMLABORATORY TOLABORATORYINTHEABSENCEOFSTANDARDIZEDCONTROLS.EUTRALIZATIONOFVIRAL INFECTION IS A NEW POSSIBLY MORE RELIABLE METHOD THAT CAN BE BIOLOGICALLY



2INO2APPUOLIAND'IUSEPPE$EL'IUDICE

MORE MEANINGFUL THAN () AND 32( (OWEVER OUR EXPERIENCE WITH THIS NEWMETHODISLIMITEDANDTHEREISNOTYETATITERTHATWECANCORRELATEWITH EFFICACYINHUMANS4HELIMITOFTHENEUTRALIZATIONISOFTENTHEAVAILABILITY OFWILD TYPEVIRUSESTHATREQUIREHIGHCONTAINMENTTOPERFORMTHEASSAYS)N THIS REGARD THE PSEUDOTYPE ASSAYS THAT CAN USE HIGH THROUGHPUT METHODS USINGANYVIRALCONSTRUCTWITHOUTTHENEEDOFANYCONTAINMENTISAPROMISING ASSAYFORTHEFUTURE &INALLY THE MOST IMPORTANT QUESTION THAT ARISES FROM THE DEVELOPMENT ANDLICENSUREOFLIVEATTENUATEDVACCINES WELLDESCRIBEDBY%DWARDSANDBY 'REENBERGAND+EMBLE ISTHAT THE,!)6VACCINEINCHILDRENISMOREEFFICA CIOUS THAN THE4)6 INACTIVATED VACCINE IN SPITE OF THE FACT THAT IT INDUCES MUCH LOWER LEVELS OF ANTIBODIES4HE QUESTION RAISED BY THIS OBSERVATION WHICH IS OF FUNDAMENTAL IMPORTANCE FOR THE UNDERSTANDING OF THE MECHA NISMOFIMMUNITYTOINFLUENZAANDALSOTOMANYOTHERVACCINES ISDISCUSSED INTHECHAPTERBY-INTERNETALDEALINGWITHTHEIMMUNERESPONSETOINFLU ENZA4HE CONCLUSION IS THAT BY MECHANISMS OTHER THAN SERUM ANTIBODIES MAYBEMUCOSALANTIBODIESOR4CELLS CLEARLYCONTRIBUTETOTHE PREVENTION OF INFLUENZA HOWEVER WE HAVE NO IDEA OF WHAT THEY ARE HOW TO MEASURE THEM AND HOW TO CORRELATE THEM WITH EFFICACY IN HUMANS 5NDERSTANDING THESEMECHANISMSOFPROTECTIONWILLBEOFPARAMOUNTIMPORTANCE TOUNDER STANDTHEVACCINESOFTHEFUTURE

#ELLCULTURE&ORTHEFIRSTTIMEWEHAVEANALTERNATIVETOEGGS FOR PRODUCTIONOFINFLUENZAVACCINES )N FORTHEFIRSTTIME THE%UROPEANAUTHORITYAPPROVEDAVACCINEPRO DUCEDINMAMMALIANCELLS-$#+ OFCANINEORIGIN 4HISSTEPIS THEFIRST MEANINGFULCHANGESINCEWHENTHEFIRSTVACCINEPRODUCEDINEMBRYO NATEDEGGSOFHENSWASAPPROVED/THERVACCINESPRODUCEDINMAMMALIAN CELLS USING NON HUMAN PRIMATE 6ERO OR HUMAN 0ER#  CELLS ARE IN DEVELOPMENT AND ARE COVERED MOSTLY IN THE CHAPTER BY /XFORD ET AL4HE AVAILABILITY OF A CELL CULTURE PRODUCTION TECHNOLOGY THAT CAN SIMPLIFY THE MANUFACTURINGPROCESS PROVIDESVACCINESOFINCREASEDPURITY ANDPRODUCES FASTERWITHAMORERELIABLEMETHOD ISATREMENDOUSSTEPFORWARDINITSELF .EVERTHELESS POSSIBLYTHEREALVALUEOFTHISTECHNOLOGYISTHEFACTTHATTHIS PROVIDES A PLATFORM THAT ALLOWS THE DEVELOPMENT OF THE VACCINES OF THE FUTURE4ODAY FORINSTANCE MOSTOFINFLUENZAPRIMARYISOLATESDONOTEASILY GROWINEGGSANDTHEREFORECANNOTBEUSEDTOMAKEVACCINES SO THATEVERY YEAR THE VACCINE COMPOSITION REFLECTS A COMPROMISE BETWEEN THE MEDI CAL NEED AND WHAT CAN BE GROWN IN EGGS! TYPICAL EXAMPLE IS THE SEASON n WHEN THE (.!&UJAN VIRUS STRAIN DID NOT GROW IN EGGS AND THE VACCINE CONTAINED A STRAIN THAT DID NOT COVER THE HIGHLY PATHOGENIC STRAINCIRCULATINGTHATYEAR)SOLATINGTHEVIRUSDIRECTLYINMAMMALIANCELLS

)NTRODUCTION



WILLALLOWTHESELECTIONOFTHEMOSTAPPROPRIATEVACCINECOMPOSITIONEVERY YEAR)NADDITION ITISWELLKNOWNTHATPASSAGEINEGGSSELECTSFORMUTATIONS INTHEVIRALGENOMETHATMAYCOMPROMISESOMEOFTHEIMPORTANTEPITOPES NECESSARYFORPROTECTION4HESECHANGESWILLNOLONGERHAPPENWHENVACCINE SEEDSAREISOLATEDDIRECTLYINMAMMALIANCELLS

2EVERSEGENETICSALLOWSNAVIGATINGNEWHORIZONS 4HEBIOLOGYOFTHEINFLUENZAVIRUSHASSEENAQUANTUMLEAPSINCETHEEND OFTHECENTURY4HECHAPTERSBY-UBAREKAAND0ALESEANDBY/XFORDETAL PROVIDEACOMPREHENSIVEANALYSISOFTHEBIOLOGYOFTHEVIRUSANDTHEIMPACT OFTHENEWDISCOVERIES 5PTOVERYRECENTLY THEONLYWAYTOSTUDYTHEGENETICSOFINFLUENZAAND GENERATENEWSTRAINSOFVIRUSESHASBEENTHECO INFECTIONOFCHICKENEMBRYO CELLSWITHTWOVIRUSESANDTHESELECTIONOFREASSORTANTSEACHCONTAININGTHE DESIREDCOMBINATIONOFTHEEIGHT2.!FRAGMENTSTHATCONSTITUTETHEINFLU ENZAGENOME4HISTECHNOLOGYHASBEENANDSTILLISVERYUSEFUL ANDCONTINUES TOALLOWTHEGENERATIONOFTHESEEDVIRUSESTHATAREUSEDEVERY YEARTOMANU FACTURETHEVACCINE)NTHISPROCEDURE THEDESIREDVACCINEVIRUSISUSEDINA CO INFECTIONWITHTHEHIGHYIELDSTRAIN!02 ANDREASSORTANTSCONTAIN INGTHE(!AND.!SEGMENTSOFTHEVACCINEVIRUSANDTHEOTHERGENESFROM THEHIGHYIELD02VIRUSARESELECTED4HEABILITYTOGENERATENEWVIRUSESBY TRANSFECTING CELL WITH PLASMIDS CONTAINING THE EIGHT FRAGMENTS OF THE VIRAL GENOMEFIRSTREPORTEDINISONEOFTHEMOSTIMPORTANTMILESTONESINTHE HISTORYOFINFLUENZA ANDHASALREADYCAUSEDAREVOLUTIONINTHEWAYRESEARCH ISPERFORMEDANDVACCINESAREDESIGNED.OWEVERYSINGLEGENEOFINFLUENZA CANBEMANIPULATEDATWILLANDVIRUSESTHATCONTAINTHEDESIRED 2.!FRAG MENTSCANBEGENERATEDWITHOUTTHECO INFECTIONANDSELECTIONMETHODUSED SOFARTOGENERATEREASSORTANTS4HEFIRSTIMPORTANTCONTRIBUTIONOFREVERSE GENETICS HAS BEEN THE ABILITY TO GENERATE SEEDS FOR THE (. VACCINES )N FACT THESEREASSORTANTSCOULDNOTBEGENERATEDBYTHECLASSICALMETHODSINCE THE(.VIRUSISPATHOGENICFORAVIANCELLSANDKILLSTHECHICKENEMBRYO CELLSTHATAREUSUALLYUSEDTOGENERATETHESEEDVIRUSESFORVACCINEPRODUC TION7HILE REVERSE GENETICS HAS ALREADY BEEN OF FUNDAMENTAL IMPORTANCE BYENABLINGTHEPRODUCTIONOFVACCINESAGAINST(.VIRUSES THEPOTENTIAL OFTHISTECHNOLOGYISJUSTATTHEBEGINNING3OMETIME NOTINTHETOODISTANT FUTURE WECANIMAGINETHATALLSEEDVIRUSESWILLBESYNTHESIZEDINTHELABO RATORY ENGINEEREDANDOPTIMIZEDATWILL ANDTHATHEALTHAUTHORITIESWILLNO LONGER PROVIDE SEED VIRUSES BUT FILES OF SEQUENCES TO BE INCORPORATED IN VACCINES!NOTHERIMPORTANTCONTRIBUTIONOFREVERSEGENETICSHASBEENTHE ABILITY TO GENERATE VIRUSES NO LONGER EXISTING SUCH AS THE ONE THAT CAUSED THE  PANDEMIC TO GENERATE NOVEL LIVE ATTENUATED VIRUSES CONTAINING WELL DEFINED GENETIC DEFECTS AND TO ADDRESS FUNDAMENTAL QUESTIONS ABOUT THEBIOLOGYOFTHEVIRUS



2INO2APPUOLIAND'IUSEPPE$EL'IUDICE

!DJUVANTSWILLBEANESSENTIALCOMPONENTOFINACTIVATED INFLUENZAVACCINES 4HE CHAPTER BY ,ATTANZI REPORTS THAT THE NEED TO IMPROVE4)6 VACCINES WAS ALREADY RECOGNIZED IN  WHEN AN INACTIVATED VACCINE ADJUVANTED WITHMINERALOILWASGIVENTOPEOPLE WHOWEREFOLLOWEDFORSAFETY FORTWODECADES4HELONG TERMSAFETYTURNEDOUTTOBEVERYSATISFACTORY ANDTHEYWEREABLETODEMONSTRATETHATTHEADJUVANTINTHELONG TERMDID NOT HAVE ANY EFFECT ON MORTALITY MALIGNANT DISEASES OR ALLERGY4HIS Y WAS ANIMPORTANTFINDINGBECAUSEATTHATTIMEPEOPLEWEREWORRIEDTHATADJU VANTSMAYHAVEINCREASEDTHEFREQUENCYOFCANCER#ONVERSELY THESHORT TERMSAFETYWASDISAPPOINTING BECAUSEUPTOOFVACCINESDEVELOPEDA DELAYEDLOCALCYSTICREACTIONATTHESITEOFINJECTIONTHATREQUIREDSURGICAL INTERVENTION 7HILE THE VACCINE ADJUVANTED WITH MINERAL OIL WAS NEVER LICENSEDBECAUSEOFTHELOCALREACTIONS THEFINDINGSOFTHETRIAL REPORTED IN4ABLEOFTHECHAPTERBY,ATTANZI WEREIDENTICALTOTHOSEREPORTED YEARSLATERWITHTHENEWGENERATIONOFADJUVANTS)N (ENNESSYAND $AVENPORTWROTETHATh-INERALOILADJUVANTVACCINEISREMARKABLYEFFEC TIVE FOR STIMULATING HIGH BROAD UNIFORM AND PERSISTENT ANTIBODY LEVELS AGAINSTPROTOTYPESTRAINSOFINFLUENZA!vANDCANBEHELPFULTO SPARETHE VACCINEDOSE hAPHENOMENALECONOMYCANBEAFFECTEDINTHEREQUIREMENT OFANTIGENv;= 4HE NEED TO IMPROVE INFLUENZA VACCINES BY ADDING ADJUVANTS WAS PER CEIVEDALSOINTHESANDS ANDVACCINESWERELICENSEDFORAWHILE USING ALUMINUM SALTS (OWEVER THESE VACCINES WERE EVENTUALLY REMOVED BECAUSETHEALUMINUMSALTSDIDNOTINCREASEIMMUNOGENICITYBUT INCREASED REACTOGENICITY)TISINTERESTINGINTHISCONTEXTTOREMEMBERTHATDURINGTHE LASTYEARSABIGEFFORTHASBEENDONETOADJUVANT(.VACCINESWITHALUM TOREDISCOVERTHATTHISADJUVANTISNOTUSEFULFOR4)6VACCINES THESAMECON CLUSIONALREADYACHIEVEDYEARSAGO 4HE NEED FOR IMPROVED4)6 VACCINES CONTINUED DURING THE S AND SWHENFINALLYASAFE ADJUVANTEDVACCINEWASLICENSEDFOR HUMANUSE IN4HECHAPTERBY/(AGANAND0ODDADESCRIBESTHEHISTORY ANDTHE PROPERTIESOFTHENEWADJUVANTNAMED-&THATREPRESENTEDAFUNDAMEN TALMILESTONEINTHEHISTORYOFINFLUENZAVACCINESANDMARKEDTHEBEGINNING OFANEWERA4HEADJUVANTISANOILINWATEREMULSIONCONTAININGABIODE GRADABLEOILSQUALENE WHICHISPRESENTINEVERYEUKARYOTICORGANISM)N THEEMULSIONTHEOILISSURROUNDEDBYDROPLETSOFWATERTHATARESTABILIZED AROUNDTHEOILBYDETERGENTSSUCHAS4WEENAND3PAN4HEBIODEGRADABLE OILINSTEADOFTHENON BIODEGRADABLEMINERALOIL ANDTHEEMULSION WHICH PREVENTSDIRECTCONTACTOFTHEOILWITHTHEBIOLOGICALFLUIDSANDCELLMEM BRANES SOLVEDALLTHESAFETYPROBLEMSOBSERVEDWITHMINERALOILINTHES ANDALLOWEDTHELICENSUREOFSAFEADJUVANTEDINFLUENZAVACCINES$URINGTHE LAST  YEARS MORE THAN  MILLION DOSES OF THE ADJUVANTED VACCINE HAVE BEEN ADMINISTERED AND NOW THE SAFETY ON A NEW ADJUVANT IS WELL ESTAB

)NTRODUCTION



LISHED)NGENERAL THEADJUVANTEDVACCINESHOWSINCREASEDIMMUNOGENICITY ANDBROADERIMMUNITYAGAINSTNON VACCINESTRAINS SIMILARLYTOTHEVACCINES ADJUVANTEDWITHMINERALOIL WHICHWERETESTEDINTHES4HEEFFECTSOF THEADJUVANTEDVACCINESAREMOSTLYVISIBLEINCHILDREN THEELDERLY PEOPLE WITHCHRONICDISEASES ANDWHENPEOPLEARENAIVETOANINFLUENZAVIRUSAS ISTHECASEOF(.)NTHECASEOF(. VACCINEDEVELOPMENTWASIMPOS SIBLEWITHOUTTHEPRESENCEOFOILINWATEREMULSIONSASADJUVANTS)NFACT IN DEVELOPING VACCINES AGAINST AVIAN INFLUENZA NON ADJUVANTED VACCINES WEREFOUNDTOBENON IMMUNOGENICATMEANINGFULDOSES ALUM ADJUVANTED VACCINES WERE FOUND TO BE USELESS WITH WHOLE VIRUS INACTIVATED VACCINES ALTHOUGHIMMUNOGENIC THEYNEEDTOTAKEINTOACCOUNTTHATTHESE VACCINES WERE ABANDONED IN THE S AND S BECAUSE OF THE NON ACCEPTABLE SYSTEMICREACTOGENICITYESPECIALLYINCHILDRENANDNON IMMUNEPEOPLESEE THECHAPTERBY/XFORDETAL  !GAINST(. THE-&ADJUVANTEDVACCINE WAS SHOWN TO INDUCE A VERY HIGH IMMUNE RESPONSE AT VERY LOW ANTIGEN CONTENTANDTOINDUCEABROADIMMUNERESPONSEABLETONEUTRALIZEVIRUSES THATWERENOTCONTAINEDINTHEVACCINE/THEROILINWATEREMULSIONSCON TAININGSQUALENESIMILARTO-&HAVE BEENRECENTLYTESTEDINCLINICALTRIALS WITH ENCOURAGING RESULTS /NE OF THEM DEVELOPED BY 'LAXO 3MITH+LINE ANDNAMED!3 ISLIKELYTOBELICENSEDINTHENEARFUTURE!NOTHERONE REFERREDTOAS!&ANDDEVELOPED BY3ANOFI 0ASTEUR HASBEENANNOUNCED RECENTLY #LEARLY IMPROVED INACTIVATED INFLUENZA VACCINES WILL MAKE USE OF ADJUVANTS WHICHWEANTICIPATEWILLBECOMEMORESOPHISTICATEDINTHENEXT DECADEBYTHEINTRODUCTIONOF4OLL LIKERECEPTORAGONISTSOROTHERIMMUNO MODULATORYAGENTS

!NIMALMODELSANDHUMANCHALLENGEARENECESSARYBUTNOTPERFECT )T IS NOT POSSIBLE TO STUDY THE VIRULENCE TRANSMISSIBILITY PATHOGENESIS IMMUNE RESPONSE AND VACCINE POTENCY OF INFLUENZA WITHOUT THE USE OF APPROPRIATE ANIMAL MODELS4HE CHAPTER BY ,UKE AND 3UBBARAO DESCRIBES THEDIFFERENTANIMALMODELSAVAILABLEFORINFLUENZASTUDIES4HESEAREMOSTLY MICE FERRETS ANDGUINEAPIGS!LTHOUGHEACHOFTHESEMODELSISIMPORTANT NONEOFTHEMFULLYREPRODUCESTHEHUMANINFECTION(UMANCHALLENGETRI ALSCANBEPERFORMEDSEECHAPTERBY/XFORDETAL BUTTHESEARENOTHIGH THROUGHPUT AND CANNOT BE CARRIED OUT WITH HIGHLY VIRULENT STRAINS SUCH AS THE (. AND THE (. WHICH CAUSED THE  PANDEMIC )MPROVEMENT OFINFLUENZAVACCINESWILLREQUIREASMARTUSEOFEACHOFTHESE MODELS FOR INSTANCEMICEFORHIGH THROUGHPUTSTUDIES FERRETSFORPROTECTIONFROMINFEC TION ANDGUINEAPIGSFORTRANSMISSIBILITYSTUDIES4HEHUMANCHALLENGECAN BEUSEDTOACHIEVEPROOFOFPRINCIPLEINMAN)NTHEFUTUREWE HOPETHATTHE ANIMALMODELSWILLIMPROVEANDWEWILLHAVEMICETHATCANBETTERSUSTAIN THE INFLUENZA INFECTION4HIS WILL PROBABLY REQUIRE UNDERSTANDING THE SPE



2INO2APPUOLIAND'IUSEPPE$EL'IUDICE

CIES SPECIFIC INNATE IMMUNITY WHICH PREVENTS MICE FROM BEING INFECTED BY HUMAN VIRUSES AND THE DEVELOPMENT OF TRANSGENIC MICE SUSCEPTIBLE TO HUMANVIRUSES

2EFERENCES 

(ENNESSY!6 $AVENPORT&- 2ELATIVEMERITSOFAQUEOUSANDADJUVANT INFLUENZA VACCINES WHEN USED IN A TWO DOSE SCHEDULE 0UBLIC (EALTH 2EP  n

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)NFLUENZAVIRUS4HEBIOLOGYOFACHANGINGVIRUS 3AMIRA-UBAREKAAND0ETER0ALESE $EPARTMENTOF-ICROBIOLOGY -OUNT3INAI3CHOOLOF-EDICINE .EW 9ORK .9 53!

!BSTRACT 2ECENTDISCOVERIESINTHEFIELDOFINFLUENZAVIROLOGYARETHEFOCUSOFTHISCHAPTER)NFLUENZA VIRUSESAREMEMBERSOFTHEFAMILY/RTHOMYXOVIRIDAEANDINCLUDEINFLUENZAVIRUSTYPES! " AND#4HISINTRODUCTIONPROVIDESADETAILEDOVERVIEWOFINFLUENZAVIRUSCLASSIFICATION F STRUCTURE ANDLIFECYCLE7EALSOINCLUDEABRIEFREVIEWOFTHECLINICALMANIFESTATIONSOF INFLUENZAANDTHEMOLECULARDETERMINANTSFORVIRULENCE4HEGENETICDIVERSITYOFINFLUENZA !VIRUSESANDTHEIRCAPABILITYTOSUCCESSFULLYINFECTANARRAY OFHOSTS INCLUDINGAVIANAND MAMMALIAN SPECIES IS HIGHLIGHTED IN A DISCUSSION ABOUT HOST RANGE AND EVOLUTION4HE IMPORTANCE OF VIRAL RECEPTOR BINDING HEMAGGLUTININS AND HOST SIALIC ACID DISTRIBUTION IN SPECIES RESTRICTED BINDING OF VIRUSES IS UNDERSCORED &INALLY RECENT ADVANCES IN OUR UNDERSTANDINGOFTHESEASONALITYANDTRANSMISSIONOFINFLUENZA VIRUSESAREDESCRIBEDAND THEIRIMPORTANCEFORTHECONTROLOFTHESPREADOFTHESEVIRUSES DISCUSSED

)NTRODUCTION )NFLUENZA HAS HAD SIGNIFICANT HISTORICAL IMPACT AND CONTINUES TO POSE A CONSIDERABLE THREAT TO PUBLIC HEALTH /UR UNDERSTANDING OF THE BIOLOGY OF INFLUENZAVIRUSANDITSEFFECTONTHEHOSTHASADVANCEDCONSIDERABLYINTHE LASTYEARS2ECENTEVENTSININFLUENZAVIRUSRESEARCHHAVECONTRIBUTEDTO THISPROGRESS;=4HESEINCLUDETHEDEVELOPMENTOFPLASMID BASEDREVERSE GENETICSSYSTEMS; = THEGENERATIONOFTHEPANDEMIC(.INFLUENZA VIRUS ;= IMPROVED ACCESS TO BIOSAFETY LEVEL  FACILITIES AND THE ESTABLISH MENT OF INTERNATIONAL INFLUENZA VIRUS SEQUENCE DATABASES ; =!DVANCES HAVE ALSO LED TO THE PRODUCTION OF &$! APPROVED ANTIVIRALS FOR INFLUENZA ANDNOVELVACCINEAPPROACHESSUCHASTHEUSEOFLIVEATTENUATEDVACCINES #ONTINUEDACCELERATIONOFINFLUENZAVIRUSRESEARCHHASDIRECTIMPLICATIONS FOR THE DEVELOPMENT OF IMPROVED VACCINES AND INFLUENZA CONTROL DURING PANDEMICANDINTERPANDEMICPERIODS



3AMIRA-UBAREKAAND0ETER0ALESE

/VERVIEWANDCLASSIFICATION )NFLUENZAVIRUSESAREMEMBERSOFTHEFAMILY/RTHOMYXOVIRIDAEANDINCLUDE INFLUENZAVIRUSTYPES! " AND#)NFLUENZAVIRUSESPOSSESSSEVENINFLUENZA # OREIGHTINFLUENZA!AND" GENOMESEGMENTSCOMPOSEDOFNEGATIVE SENSESINGLE STRANDED2.!4HESETYPESDIFFERINVARIOUSASPECTS THEMOST IMPORTANT OF WHICH INCLUDE ANTIGENICITY HOST RANGE PATHOGENICITY TRANS MISSION AND SEASONALITY 3TANDARD NOMENCLATURES FOR HUMAN INFLUENZA VIRUSES INCLUDE TYPE GEOGRAPHIC LOCATION OF ISOLATION ISOLATE NUMBER AND YEAROFISOLATION&OREXAMPLE ANINFLUENZA!VIRUSISOLATEDIN0ANAMAIN  WOULD BE REFERRED TO AS!0ANAMA 3UBTYPES OF INFLUENZA !VIRUSESAREDESCRIBEDBYHEMAGGLUTININ(! ANDNEURAMINIDASE.! DESIGNATIONS4ODATE (!AND.!SUBTYPESHAVEBEENDESCRIBED )NFLUENZA!VIRUSESAREMOSTLYRESPONSIBLEFORSEASONALEPIDEMICSAND GLOBALPANDEMICS ANDSUSTAINTHEBURDENOFDISEASEATTRIBUTABLETOINFLU ENZA#LINICALDISEASEISAPPARENTANDINCLUDESPRIMARILYRESPIRATORYMANI FESTATIONS ANDMAYBECOMPLICATEDBYCENTRALNERVOUSSYSTEMINVOLVEMENT AND RARELY TOXIC SHOCK AND MULTI ORGAN SYSTEM FAILURE ; = #IRCULATING STRAINSOFINFLUENZA!VIRUSESARETARGETSFORANNUALVACCINATIONTOMITIGATE MORBIDITYANDMORTALITYIMPARTEDBYTHESEVIRUSES)NADDITIONTOINFECTING HUMANS INFLUENZA! VIRUSES CIRCULATE IN OTHER MAMMALS INCLUDING SWINE ANDHORSES7ATERFOWLHARBORSEVERALLINEAGESOFINFLUENZA!VIRUSES AND SERVE AS A RESERVOIR 4RANSMISSION AMONGST WILD AND DOMESTIC FOWL AND MAMMALIANSPECIESISANIMPORTANTCHARACTERISTICOFINFLUENZA! ENABLING VIRALREASSORTMENTANDEMERGENCEOFNOVELSUBTYPESINSUSCEPTIBLEHUMAN POPULATIONS )N CONTRAST INFLUENZA " VIRUS HAS A RESTRICTED HOST RANGE CIRCULATING ONLYINHUMANS ALTHOUGHTHEVIRUSHASBEENISOLATEDINSEALS;=)NFLUENZA " VIRUS DEMONSTRATES SEASONALITY AND IS RESPONSIBLE FOR HUMAN DISEASE ALTHOUGHTHECLINICALMANIFESTATIONSAREGENERALLYLESSSEVERECOMPAREDTO INFLUENZA!VIRUS ASSOCIATEDILLNESS.ONETHELESS RARECASESOFENCEPHALITIS AND SEPTIC SHOCK HAVE BEEN DESCRIBED IN CHILDREN ; =!T PRESENT THE TWOMAJORLINEAGESAREREPRESENTEDBYINFLUENZA"6ICTORIAAND" 9AMAGATAVIRUSES;=2E EMERGENCEOFTHE6ICTORIALINEAGEAFTER ADECADEOFABSENCEWASASSOCIATEDWITHANOUTBREAKDURINGTHEn INFLUENZA SEASON AFFECTING HEALTHY IMMUNOLOGICALLY NAIVE CHILDREN ;= )NFLUENZA " VIRUS IS INCLUDED IN INACTIVATED AND LIVE ATTENUATED ANNUAL INFLUENZAVACCINES 5NLIKEINFLUENZA!AND"VIRUSES INFLUENZA#VIRUSESLACKTHE .!GENE AND CODE FOR A SINGLE SURFACE HEMAGGLUTININ ESTERASE FUSION (%& GLY COPROTEIN4HIS VIRUS DOES NOT DEMONSTRATE MARKED SEASONALITY AND IS NOT INCLUDEDINTHEANNUALINFLUENZAVACCINE ALTHOUGHITHASBEENRESPONSIBLE FOROCCASIONALOUTBREAKS PREDOMINANTLYINCHILDREN;=)LLNESSINHUMANSIS GENERALLYMILDANDCONSISTSOFANUPPERRESPIRATORYTRACTINFECTION)NFLUENZA

)NFLUENZAVIRUS4HEBIOLOGYOFACHANGINGVIRUS



&IGURE3CHEMATICSTRUCTUREANDELECTRONMICROGRAPHOFINFLUENZAVIRUS! ! 4HEVIRALENVELOPANCHORSTHEHEMAGGLUTININ(! ANDNEURAMINIDASE.! GLYCOPROTEINS ANDMATRIX- PROTEINANDISDERIVEDFROMTHEHOSTCELLDURINGTHEPROCESSOFBUDDING- LIES BENEATH THE VIRAL ENVELOPE .UCLEAR EXPORT AND NONSTRUCTURAL PROTEIN .%0.3 AND THECOREOFTHEVIRIONARECONTAINEDWITHIN4HECORECONSISTSOFEIGHTSEGMENTSOFVIRAL2.! ASSOCIATEDWITHTHEPOLYMERASECOMPLEX0" 0" AND0! ANDNUCLEOPROTEIN.0 !DAPTED FROM ;= AND KINDLY PROVIDED BY - , 3HAW " .EGATIVELY STAINED ELECTRON MICROGRAPH OF MOUSE ADAPTED INFLUENZA ! 73. 'LYCOPROTEIN SPIKES ARE VISIBLE ON THE SURFACE OF THE VIRION+INDLYPROVIDEDBY-,3HAW

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CELL CYTOPLASM THROUGH ION CHANNEL ACTIVITY ; = 6IRAL 2.! V2.! SYNTHESISOCCURSINTHENUCLEUS ANDVIRAL2.0SMUSTTHEREFOREBEIMPORTED 4HISPROCESSISPRIMARILYMEDIATEDBYVIRAL.0 WHICHCOATSVIRAL2.!AND POSSESSES NUCLEAR LOCALIZATION SIGNALS .,3 INCLUDING AN UNCONVENTIONAL .,3WHICHBINDSHOSTKARYOPHERIN ALPHAANDISESSENTIALFORENERGY DEPEN DENT2.0NUCLEARIMPORT; =

4RANSCRIPTION REPLICATION ANDNUCLEAREXPORT 6IRAL 2.! SERVES AS A TEMPLATE FOR THE PRODUCTION OF MESSENGER 2.! M2.! AND SUBSEQUENT TRANSCRIPTION AS WELL AS FOR THE GENERATION OF COMPLEMENTARY 2.! C2.! WHICH IS POSITIVE SENSE AND FUNCTIONS AS A TEMPLATE FOR THE GENERATION OF MORE V2.! VIRAL REPLICATION  2.! SEGMENTS ARE COATED BY .0 THROUGH NONSPECIFIC INTERACTIONS BETWEEN THEARGININE RICHPOSITIVELYCHARGED.0ANDTHENEGATIVELYCHARGED2.! PHOSPHATEBACKBONE;=4HEVIRALPOLYMERASECOMPLEXCONSISTSOFTIGHTLY ASSOCIATED0" 0" AND0!ANDASSOCIATESWITH.0 COATED2.!WITHOUT DISRUPTING THIS INTERACTION ;= 0" IS AN ENDONUCLEASE INVOLVED IN BOTH REPLICATION AND TRANSCRIPTION AND BINDS THE PROMOTER REGION OFF 2.! SEGMENTS ;= )T FUNCTIONS AS AN 2.! DEPENDENT 2.! POLYMERASE AND CATALYZES 2.! CHAIN ELONGATION )NTERACTION WITH 0! IS REQUIRED FOR THIS FUNCTIONANDVIRALREPLICATION;=0"BINDSBOTH.0AND0" VIA SEPARATE BINDINGSITES;=)NITIATIONOFTRANSCRIPTIONISRELIANTON0" WHICHBINDS THECAPONHOSTPRE M2.! ANDTHISCAPSERVESASAPRIMERFORTRANSCRIPTION ; =)NADDITION INTERACTIONSBETWEEN0"ANDHOSTPROTEINSMAYALSO BE SPECIES SPECIFIC AND MAY PLAY A ROLE IN RESTRICTING HOST RANGE ;= 0! ISACOMPONENTOFTHEPOLYMERASEHETEROTRIMER ISCO TRANSPORTEDINTOTHE NUCLEUSWITH0" ANDISTHUSIMPORTANTINTHEFORMATIONOFTHISCOMPLEX ; = 3YNTHESISOFM2.!BEGINSWITHAHOSTCELLCAPPEDPRIMER GENERATED BYHOSTCELL2.! POLYMERASE))ANDOBTAINEDFROM HOSTPRE M2.! ;= 4RANSCRIPTIONISTHUSINITIATEDANDSYNTHESISONTHETEMPLATEOCCURSINA TODIRECTION!POLYADENYLATIONSIGNALCONSISTINGOFFIVETO SEVENURIDINES ATTHEENDOFV2.!PREMATURELYTERMINATESTRANSCRIPTIONAFTERINDUCING STUTTERINGOFTHEVIRALPOLYMERASE;n=4HEGENERATIONOF.0AND.3 TENDSTOOCCUREARLIERAFTERINFECTIONCOMPAREDTOTHEGENERATIONOFSURFACE GLYCOPROTEIN AND - M2.!S ;= -ECHANISMS FOR THE REGULATION OFF GENE EXPRESSIONREMAINEVASIVE ALTHOUGH.0HASBEENIMPLICATEDINTHECONTROL OFGENEEXPRESSION;= 6IRAL REPLICATION REQUIRES THE SYNTHESIS OF V2.! WHICH IS PRIMER INDEPENDENT AND OCCURS THROUGH A C2.! INTERMEDIATE .ASCENT C2.! IS THEREFORE NOT CAPPED OR POLYADENYLATED UPON TERMINATION 4HE NOTION THAT C2.! SYNTHESIS IS INITIATED AFTER A SWITCH FROM M2.! SYNTHESIS HAS RECENTLYBEENCHALLENGED;=



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%VOLUTION !MONGST THE INFLUENZA VIRUS TYPES INFLUENZA ! DEMONSTRATES THE MOST GENETICDIVERSITYANDISCAPABLEOFSUCCESSFULLYINFECTINGANARRAYOFHOSTS INCLUDING AVIAN AND MAMMALIAN SPECIES )NFLUENZA ! VIRUSES EXHIBIT AN EVOLUTIONARY PATTERN THAT IS COMPLEX AND CONSISTS OF ANTIGENIC DRIFT AND SHIFT$RIFTOCCURSONANANNUALBASISANDHASBEENATTRIBUTEDTOLOWFIDELITY

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OF THE 2.! POLYMERASE AND SUBSEQUENT SELECTION FROM IMMUNE PRESSURE EXERTEDBYTHEHOST;=4HISRESULTSINANTIGENICDIVERSITYOFTHE(!AND .!GLYCOPROTEINS ANDISONEOFTHEMAJORCHALLENGESTOVACCINE PRODUCTION REQUIRINGANNUALCHANGESTOVACCINECOMPONENTS4HE(!DOMAINCONTAINS SEVERALEPITOPES ANDISTHEMOSTDYNAMICASACONSEQUENCE DEMONSTRATING CLUSTERSOFANTIGENICVARIANCEOVERTIME;=!NTIGENICSHIFTRESULTSAFTERA VIRALREASSORTMENTEVENTWHEREEXCHANGEOFONEORMOREOFTHEVIRALSEG MENTSWITHTHATOFANOTHERSTRAINMAYRESULTINANOVELSEROTYPE POTENTIALLY DIVERSIFYING THE HOST RANGE OF THE VIRUS )T IS IN THIS SETTING THAT PANDEMIC STRAINS HAVE EMERGED IN IMMUNOLOGICALLY NAIVE POPULATIONS IN THE PAST INCLUDINGTHE(.WITHNEW(! .! AND0"SEGMENT SUBTYPEIN AND THE (. INFLUENZA VIRUS WITH NEW (! AND 0" SEGMENTS WHICH CAUSEDAPANDEMICIN&IG 4OTACKLETHECHALLENGEOFUNDERSTANDING THEEVOLUTIONOFINFLUENZAVIRUS LARGE SCALECOLLABORATIVEEFFORTSSUCHASTHE )NFLUENZA'ENOME3EQUENCING0ROJECTHAVEBEENUNDERTAKEN4HEPRESENCE OFSEVERALCO CIRCULATINGCLADESINTHEHUMANPOPULATIONHASBEENDESCRIBED ACCOUNTINGFORREASSORTMENT4HISCANRESULTINLIMITEDVACCINE EFFECTIVENESS ASSEENWITH!&UJIAN LIKEVIRUSDURINGTHEnSEASON;= 'ENETICEVOLUTIONAPPEARSTOBE ARELATIVELYGRADUALPROCESSHOWEVER ANTI GENICCHANGESINTHE(!DOMAINTENDTOCLUSTER;=/NGOINGCHANGESOF



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THE(HEMAGGLUTINININTHEHUMANPOPULATIONRESULTFROMSELECTIVEPRES SUREEXERTEDBYTHEHOSTIMMUNESYSTEM)NCONTRAST THE(LINEAGEINBIRDS HAS REMAINED RELATIVELY STABLE ;=4HE RATE OF CHANGE OF THE ( SUBTYPE IS GREATER WHEN COMPARED TO ( VIRUSES AND INFLUENZA " WITH ESTIMATED NUCLEOTIDE CHANGES PER SITE PER YEAR OF  FOR (  FOR ( AND FORINFLUENZA";=!SGREATERNUMBERSOFINFLUENZAVIRUSGENOME SEQUENCESBECOMEAVAILABLEANDWEGAININSIGHTINTOANTIGENICPATTERNSOF CHANGE THIS KNOWLEDGE MAY BE APPLIED TO ANNUAL VACCINE DEVELOPMENT 0REDICTION OF FUTURE INFLUENZA SEQUENCES COULD LEAD TO THE MORE TIMELY DEVELOPMENT OF EFFECTIVE VACCINES ;= ALTHOUGH MODELING METHODS HAVE YETTOBEVALIDATED 2ECENTLY SEVERALAVIANINFLUENZAVIRUSES INCLUDING(. (. (. (. (. AND (. SUBTYPES HAVE INFECTED HUMANS ;= ALTHOUGH LIMITEDEVIDENCEFORPERSON TO PERSONSPREADEXISTS;=,ACKOFTRANSMIS SIONAMONGSTHUMANSREMAINSABARRIERTOPANDEMICSPREADOFTHESEVIRUSES 4HE (. SUBTYPE ISOLATED FROM AVIAN SPECIES HAS UNDERGONE GENETIC REASSORTMENT ANDSEVERALGENOTYPESEXIST'ENOTYPES:AND6ARELARGELY RESPONSIBLE FOR OUTBREAKS OF HIGHLY PATHOGENIC INFLUENZA VIRUSES (0!) IN DOMESTIC BIRDS IN 3OUTHEAST!SIA BEGINNING IN  ;= (. VIRUSES MAYALSOBEDIVIDEDINTOCLADESANDBASEDUPONGENOMICANALYSISOFTHE (!GENES ANDCLADEISFURTHERDIVIDEDINTOSUBCLADES;=,ESSTHAN DIVERGENCE FROM AVIAN ISOLATES HAS BEEN REPORTED IN VIRUSES ISOLATED FROM HUMANSIN!SIA;=

(OSTRANGE )NFLUENZA ! VIRUS IS A ZOONOTIC PATHOGEN CAPABLE OF INFECTING BIRDS WATERFOWLANDCHICKENS SWINE HORSES FELINES ANDOTHERSPECIES(OST RANGE RESTRICTION OF DIFFERENT TYPES OF INFLUENZA VIRUSES IS OBSERVED 3PECIES RESTRICTED BINDING OF VIRUSES IS MEDIATED BY DIFFERENT TYPES OF RECEPTOR BINDING HEMAGGLUTININS ;n= 4HE DISTRIBUTION OF DIFFERENT TYPES OF 3! LINKAGES HAS RECENTLY BEEN ELUCIDATED IN HUMANS ALTHOUGH THE TYPE OF CELL INFECTED CILIATED VS NON CILIATED IS CURRENTLY UNDER DEBATE; =3!WITH_ 'ALLINKAGEPREDOMINATESONEPITHELIALCELLS OFTHEUPPERAIRWAY INCLUDINGNASALMUCOSA SINUSES BRONCHI ANDBRON CHIOLES ;= )N HUMAN TRACHEOBRONCHIAL EPITHELIAL (4"% CELLS OLIGO SACCHARIDESWITH3!WITH _ 'ALLINKAGEPREDOMINATEDONNONCILIATED EPITHELIALCELLS;= ALTHOUGHTHESEOLIGOSACCHARIDESHAVEBEENDESCRIBED ONCILIATEDANDGOBLETCELLSINTHEHUMANAIRWAY;=,OWERAIRWAYSALSO CONTAIN3!WITHMOSTLY _ 'ALLINKAGE INADDITIONTO3!WITH _ 'AL LINKAGE; = (OST RESTRICTION IS NOT ABSOLUTE AND HUMAN INFECTIONS WITH AVIAN INFLUENZA VIRUSES INCLUDING (. (. AND (. VIRUSES HAVE BEEN

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EXTENSIVELYDESCRIBED;n=(.BINDSTYPE))PNEUMOCYTESANDMACRO PHAGESOFTHELOWERRESPIRATORYTRACTINHUMANS;  =(.INFECTION OF CILIATED CELLS IN (4"% CELL CULTURE WITH LIMITED CELL TO CELL SPREAD ;= ANDOFHUMANNASOPHARYNGEAL ADENOID ANDTONSILLAR EXVIVOCELLCULTURES HAVE BEEN SHOWN ;= "INDING OF (. VIRUSES TO SACCHARIDES TERMINAT INGIN_ 'AL3!LINKAGEHASBEENACHIEVEDBYMUTATING(!AMINOACIDS RESIDUESATPOSITIONSAND SUGGESTINGPOTENTIALFORADAPTATIONTOTHE HUMANHOST;= $IFFERENCES IN INFLUENZA VIRUS RECEPTORS AMONGST AVIAN SPECIES HAVE BEENDESCRIBED ANDISREFLECTEDINBINDINGOFDIFFERENTTYPESOFAVIANINFLU ENZA VIRUSES !LTHOUGH CHICKEN AND DUCK INFLUENZA VIRUSES PREFERENTIALLY BIND_ 'AL LINKED3! VIRUSESFROMCHICKENSHADGREATERAFFINITYFOR3! WHERE THE THIRD SUGAR MOIETY WAS A `  'LC.!C CONTAINING SYNTHETIC SIALYLGLYCOPOLYMER $UCK VIRUSES PREFERRED `  'AL.!C SUGAR MOIETIES INTHETHIRDPOSITION;=$ISTRIBUTIONOFINFLUENZAVIRUSRECEPTORSREFLECTS THESITESOFREPLICATION)NCHICKENSANDWATERFOWL 3!WITH _ 'ALLINKAGE ISFOUNDINTHEUPPERRESPIRATORYTRACTANDINTESTINES3OMESPECIESDEMON STRATETHEABILITYTOSUPPORTREPLICATIONOFBOTHHUMANANDAVIANINFLUENZA VIRUSES4HE RESPIRATORY TRACT AND INTESTINES OF QUAIL CONTAIN BOTH _ 'AL AND_ 'AL LINKEDTERMINALSIALICACIDS;=)NSWINE OLIGOSACCHARIDESWITH BOTH TYPES OF LINKAGES MAY BE FOUND AND SUGGEST THIS SPECIES SERVES AS A MIXINGVESSELWHEREHUMAN AVIAN ANDSWINEINFLUENZAVIRUSESCANREASSORT ; = (OWEVER NO IN VIVO EVIDENCE HAS BEEN PROVIDED THAT PANDEMIC STRAINSHAVEEMERGEDFROMTHISSOURCE

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OBSERVERSREPORTEDRESULTS WHICHULTIMATELYLEDTOTHERECOMMENDEDUSEIN CHILDRENOFSPLIT TYPERATHERTHANWHOLE VIRUSVACCINES3UCHRECOMMENDA TIONSCONTINUEATTHEPRESENTTIME)NADULTS TOO THEOLDERWHOLE VIRUSVAC CINESGAVEAHIGHERPROPORTIONOFFEBRILEREACTIONSTHANSPLITVIRUS;=

%ARLYPROGRESS4HESTANDARDIZATIONOFPOTENCY COMPOSITIONAND DOSAGEOFINACTIVATEDVACCINES &ORMERMETHODSFORASSAYSOFTHEPOTENCYOFINACTIVATEDVACCINEDEPENDED UPONMEASURINGTHE(!ACTIVITIESOFTHEVACCINESWITHERYTHROCYTESUSPEN SIONSUSINGTHE3ALKPATTERNTECHNIQUEOF-ILLERAND3TANLEY;=)NAMAJOR TECHNOLOGICALBREAKTHROUGH 3CHILDETAL;=PROPOSEDAMETHODOFASSAY BASED ON SINGLE RADIAL IMMUNODIFFUSION 32$ &IG   4HE (! ANTIGEN CONTENTOFVACCINESWASESTIMATEDUSING32$TESTSINAGAROSEGELSCONTAIN INGSPECIFIC()ANTIBODIES4HE32$METHODWASMODIFIEDANDREFINEDBY F 7OOD ET AL ;=4HE 32$ TECHNIQUE WAS VALID FOR BOTH WHOLE VIRUS AND SPLIT VIRUS VACCINES AND WAS QUICKLY ADOPTED FOR INTERNATIONAL USE AND IS STILL THE GOLD STANDARD )N THIS TEST VACCINE VIRUS PREPARATIONS AND REFER



*OHN/XFORDETAL

&IGURE3INGLE RADIALDIFFUSION32$ TESTTOSTANDARDIZE(! 6ACCINEANTIGENISPIPETTEDINTO MMWELLSINANAGARPLATECONTAININGSPECIFICANTI (! .! AND .0ANTIBODIES!FTERAFEWHOURSINCUBATIONAZONEOFPRECIPITATIONISQUANTIFIEDANDTHE AREAISPROPORTIONALTOTHEQUANTITYOF(!INTHEVACCINE

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(!DOSAGEOFVACCINESANDRELATIONSHIPTO()ANTIBODYRESPONSE )THASBEENKNOWNFORMANYYEARSTHATTHESEROLOGICALRESPONSETOINACTIVAT EDVACCINEDEPENDSONTHEPREVIOUSEXPERIENCEOFTHERECIPIENTTOINFECTION BYVIRUSESOFTHESAMESUBTYPEOFINFLUENZA!VIRUSASTHATPRESENTINTHE VACCINE7HEREASASINGLESUBCUTANEOUSINJECTIONOF(. VACCINEGAVEAS GOODARESPONSEASTWODOSESPRIORTO THEADVENTOFTHENEWPANDEMIC !!SIAN(. VIRUSPRODUCEDADIFFERENTEFFECT4HUS (OLLANDETAL;= DEMONSTRATEDTHATTWODOSESATANINTERVALOFORMOREWEEKSPRODUCEDA BETTERRESPONSETOONEDOSEANDINTHISREGARDTHEVACCINEINDUCEDIMMUNE

)NFLUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY



RESPONSEWASMUCHINFERIORTOTHATNOTEDBEFORETHECHANGEINVIRUSSUBTYPE 3UCHANEXPERIENCEWASAGAINNOTEDDURINGTHEFIRSTYEAROFCIRCULATIONOF !(ONG+ONG(. VIRUSANDALSOWHENTHE!.EW*ERSEY(SW. VACCINEWASUSEDINCHILDRENANDYOUNGADULTS!LSO INTHECIRCUMSTANCESOF n WHENMOSTPERSONSUNDERYEARSOFAGEHADNOPREVIOUSANTI BODYTOTHERE CIRCULATING(.VIRUS ATWO DOSEREGIMENFORCHILDRENAND YOUNGADULTSPRODUCEDAMORESATISFACTORYRESPONSETHANASINGLEINJECTION ;=4HECONTRASTBETWEENTHEEFFECTSOFASINGLEDOSEOFVACCINEINPERSONS INFECTEDWITH(.VIRUSESATLEASTYEARSEARLIERWASVERYSTRIKING4HESE DATAHAVEIMMEDIATERELEVANCETODAYINTERMSOF(.VACCINE 3EVERAL FACTORS ARE OF IMPORTANCE IN THE DETERMINATION OF THE QUANTITY ANDTHEPRECISECOMPOSITIONOFTHEANTIBODYRESPONSETOTHESURFACEANTIGENS OFTHEVIRUSPRESENTININACTIVATEDVACCINE&IRSTANDFOREMOST THEQUANTI TIESOF()AND.)ANTIBODIESINDUCEDBYVACCINEAREBROADLYRELATEDTOTHE QUANTITYOFANTIGENPRESENTINASINGLEDOSE3ECONDLY THEPRECISECOMPOSI TIONOFTHEANTIBODIESFORMEDINRESPONSETOINFLUENZA!VIRUS ISIMPORTANT 4HUS REINFORCEMENTOFPREVIOUSLYACQUIREDANTIBODIESBYTHEORIENTATIONOF THE"LYMPHOCYTERESPONSETOTHEFIRSTINFECTIONBYTHEPARTICULARSUBTYPE OF VIRUS EXPERIENCED IN CHILDHOOD OR LATER MAY TAKE PRECEDENCE OVER THE STRAIN SPECIFIC ANTIBODY RESPONSE TO THE VACCINE VIRUS4HIRDLY Y THE PRECISE RESPONSEISINFLUENCEDBY THEROUTEBYWHICHTHEVACCINEISPRESENTEDTOTHE BODYSIMMUNESYSTEM &IRSTTHEN SEVERALEARLIERSTUDIESREPORTEDAGRADEDRELATIONSHIPBETWEEN THE QUANTITY OF ANTIGEN INOCULATED AND THE ANTIBODY RESPONSE THAT RESULTS 4HISWASSOINTHESTUDYOF-OSTOWETAL;= WHOGAVEINCREASINGDOSESOF VACCINEINASINGLEINJECTIONCONTAININGnCHICKCELLAGGLUTINATIONUNITS ##! CONTAINING!*APAN(. VIRUSGROUPSOFVOLUNTEERS 4HESERUM ()RESPONSEWASTESTEDWITHFOURDIFFERENT(.VIRUSESISOLATEDn ANDALSOTHEHOMOLOGOUSVIRUS7ITHMORETHANA FOLDINCREASEIN(!FROM THE LEAST TO THE HIGHEST DOSE THE GEOMETRIC MEAN TITER '-4 OF ANTIBODY INCREASEDONLY FOLD3IMILARRESULTSWEREOBTAINEDBY0OTTERETAL;= WHO INOCULATEDSTUDENTVOLUNTEERSWITHVACCINESRANGINGINDOSAGEFROMTO F )NTERNATIONAL 5NITS AND CONTAINING !0ORT #HALMERS (. VIRUS 4HE VACCINE WAS A SURFACE ANTIGEN DETERGENT TREATED MATERIAL ;= ADSORBED TO ALUMINUMHYDROXIDEGEL'-4()SERUMTITERSINCREASEDAGAINSTHOMOLOGOUS VIRUSFROM TO FOLDWITHTHEINCREASEINDOSEOFVACCINE(!4HREEOTHER (.STRAINSAND!3INGAPORE(. VIRUSWEREALSOTESTEDANDALLTHREE (.VIRUSESSHOWEDGRADED()ANTIBODYRESPONSESPROPORTIONALINMAGNI TUDETOINCREASEINANTIGENDOSE ASDIDTHEHOMOLOGOUSVIRUS 4HE 0ANDEMIC 7ORKING 'ROUP OF THE -2# #OMMITTEE ON )NFLUENZA 6ACCINE ;= GAVE GRADED DOSES OF WHOLE VIRUS VACCINE CONTAINING THE! .EW*ERSERY(SW. STRAINTOGROUPSOFVOLUNTEERSIN4HOSELESS THANYEARSOFAGE WHODIDNOTPOSSESSSIGNIFICANTSERUM() ANTIBODYTO THE VIRUS BEFORE IMMUNIZATION SHOWED A POST VACCINATION ANTIBODY TITER RANGINGFROMTO'-4WITH ANEARLY FOLDINCREASEINDOSEFROMTO



*OHN/XFORDETAL

+G(!!BOVETHISAGE INTHOSETOWITHPRE EXISTING(SW ANTIBODY THERE WAS AN INCREASE IN ANTIBODY TITER FROM  TO  TIMES '-4 WITH A CHANGEIN(!FROMTO+G4HUS THEEFFECTOFINCREASINGTHEPOTENCYOF THISVACCINEONTHEANTIBODYRESPONSEWASMUCHGREATERINTHOSE SERA WHICH INDICATEDTHATTHEYHADBEENEXPOSEDTOTHEANTIGEN PRESUMABLY BYINFEC TIONWITHARELATEDVIRUS THANINTHOSEWITHNOSUCHEXPOSURE"OTHWHOLE ANDDETERGENT SPLIT VIRUSVACCINESSHOWEDARELATIVELYPOOR()RESPONSEIN VOLUNTEERSLESSTHANYEARSOFAGEWHOSEINITIALSERUMHADNO SIGNIFICANT AMOUNT OF PRE VACCINATION OR POST INFECTION () ANTIBODY )N THIS GROUP OF SUBJECTSTWODOSESOFVACCINEGAVEABETTERANTIBODYRESPONSETHANDIDONE BUTTHERESULTANTPOST VACCINATION'-4WASHALFTHATOBTAINEDWITHASINGLE DOSEINTHEVACCINEESOVERYEARSOFAGE 4HESE EXAMPLES UNDERLINE THE PRACTICAL IMPORTANCE OF A CONSIDERABLE DEGREEOFANTIGENICDRIFTWITHINASUBTYPECOMPRISING()ANTIBODYRESPONSE !LSO THE RECALL OF ANTIBODIES INDUCED BY PREVIOUS INFECTION ILLUSTRATES THE GENERAL RULE THAT AN UP TO DATE MONOVALENT VACCINE REINFORCES ANTIBODIES AGAINST FORMER MEMBERS OF THE SUBTYPE WHILE ALSO INDUCING SPECIFIC ANTI BODIES TO THE VACCINE VIRUS4HIS WAS CLEARLY SHOWN BY DIRECT COMPARISON OF MONOVALENT AND POLYVALENT VACCINES SUCH AS THE -2# #OMMITTEE ON )NFLUENZA6ACCINESTRIALS;n= 4HE QUANTITATIVE DOSE RESPONSE ALREADY DESCRIBED FOR () IS ALSO FOUND WITH .) ANTIBODY BUT IS LESS CONSISTENT4HUS 0OTTER ET AL ;= NOTED THAT THERE WAS A  TO  FOLD INCREASE IN .) ANTIBODY AS VACCINE POTENCY WAS INCREASEDFROMTO)5(!9ETTHETRIALOF!.EW*ERSEY (SW. VACCINECONDUCTEDBYTHE0ANDEMIC7ORKING'ROUPOFTHE-2#)NFLUENZA 6ACCINE #OMMITTEE ;= FOUND ONLY A SLIGHT INCREASE IN .) ANTIBODY AFTER AN INCREASED DOSE FROM  TO  )5 USING  )5 (! IN THE VACCINE .ICHOLSONETAL;=GAVEAWHOLE VIRUSVACCINEOFTHE!5332(. VIRUS WHICHRANGEDINPOTENCYUPTO FOLD ANDFOUND INTHOSEUNDER A  FOLD INCREASE IN .) ANTIBODY (OWEVER IN THOSE OVER  YEARS OF AGE AN INCREASE IN DOSE OF VACCINE HAD A LESS CONSTANT EFFECT ON .) ANTIBODY FORMATION /NE POSSIBLE REASON FOR THE VARIATION IN THE EFFECT OF DIFFERENT VACCINES ON THE .) ANTIBODY IS THE LACK OF CONSISTENCY IN THE .! CONTENT ;=HOWEVER ANOTHERPOSSIBILITYMAYBETHATIMMUNOLOGICALPRIMINGTOTHE (!INTHEVACCINECANINSOMEWAYSUPPRESSTHEIMMUNOGENICITYOFTHE.! ANTIGEN WHICHMAYBEPHYSICALLYASSOCIATEDWITHTHE(! 4HE SECOND IMPORTANT VARIABLE IN THE IMMUNE RESPONSE TO INACTIVATED VACCINEARISESFROMTHERELATIVEAMOUNTSOFCROSS REACTIVEANDSTRAIN SPECIFIC ANTIBODIESTHATAREGENERATED4HEDIFFERENTIATIONOFTHESEREQUIRESSPECIAL TECHNIQUESSUCHAS32$ANDTHEADSORPTIONSTUDIES7EBSTERETAL;=COM PARED INADULTS THERESPONSETOAN!0ORT#HALMERS(. SUBUNITVAC CINETOHOMOLOGOUSANDHETEROLOGOUS(.VIRUSES-OSTOFTHEANTIBODY WAS CROSS REACTIVE WITH !(ONG +ONG VIRUS BUT WHEN HIGHER DOSES OF THE VACCINES WERE EMPLOYED STRAIN SPECIFIC!0ORT #HALMERS ANTIBODY WAS PRODUCED IN ADDITION TO THAT AGAINST HETEROLOGOUS VIRUS /XFORD ET AL

)NFLUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY



; =COMPAREDWHOLE ANDSPLIT VIRUSVACCINESCONTAINING!6ICTORIA OR!3COTLANDVIRUSESANDUSINGSINGLE RADIAL HEMOLYSISAND ADSORPTION TECHNIQUESSHOWEDTHATINANIMMUNIZEDADULT CROSS REACTIVEANTIBODYWAS INDUCEDMUCHMOREFREQUENTLYTHANSPECIFICANTIBODYAGAINSTHOMOLOGOUS VIRUS4HEY SHOWED THE SAME PHENOMENON IN ADULTS DURING INFECTION WITH !0ORT#HALMERSVIRUS WHOFREQUENTLYALSODEVELOPEDANTIBODYRISESTO !(ONG+ONGANTIGENSFROM/XFORDETAL;=USEDSIMILAR TECHNIQUES TO ANALYZE SERA FROM CHILDREN AGED n YEARS IMMUNIZED WITH A SURFACE ANTIGEN VACCINE CONTAINING!6ICTORIA (. ANTIGENS -OST CHILDREN PRODUCEDASTRAIN SPECIFICSERUMANTIBODYTOTHEVACCINEANTIGENS WHEREAS ADULTSSIMILARLYVACCINATEDTENDEDTOPRODUCEANTIBODYCROSS REACTINGWITH ALLVARIANTSOFTHE(.SUBTYPETESTED0OST EPIDEMICSERAFROMTHOSEOF VARIOUSAGESRECENTLYINFECTEDBY!4EXASnLIKESTRAINSHOWEDCROSS REAC TIVE ANTIBODY IN ADULTS BUT IN CONTRAST MOSTLY STRAIN SPECIFIC RESPONSES IN CHILDREN3TRAIN SPECIFICANTIBODYISCONSIDEREDTOBEMOREPROTECTIVE

4HEROUTEOFVACCINATION 4HEINFLUENCEOFTHEROUTEOFIMMUNIZATIONWITHINACTIVATEDVACCINEHASBEEN STUDIEDINTHEPASTBYMANYOBSERVERS4HECHIEFALTERNATIVETO THESUBCUTA NEOUSnINTRAMUSCULARROUTEISINTRADERMALINJECTIONUSINGAREDUCEDAMOUNT OF VACCINE 4HE ADVANTAGES OF THIS ROUTE ARE ECONOMY AND THE AVOIDANCE OFFEBRILEREACTION4HEPRINCIPALDISADVANTAGEISTHEFACTTHATTHEANTIBODY RESPONSEISLESSCONSISTENT)TWASSHOWNBY!PPLEBYETAL;=THATTHE'-4 AFTERINTRADERMALVACCINEWASLESSTHANHALFTHATOBTAINEDWITH SUBCUTANEOUS VACCINE ANDTHISSEEMEDLOGICALINTHATONLYONE TENTHOFTHE VACCINEDOSE WAS GIVEN INTRADERMALLY -C#ARROLL AND +ILBOURNE ;= FOUND LITTLE DIFFER ENCEINTHEANTIBODYRESPONSESTOINTRADERMALANDSUBCUTANEOUS VACCINESIN EQUIVALENTDOSES4AURASOETAL;=REINVESTIGATEDTHEQUESTIONUSINGATWO DOSEREGIMEBEFORETHEARRIVALOFTHE!(ONG+ONG(. EPIDEMIC)N THE EQUIVALENT AMOUNT OF  ML VACCINE ANTIBODIES FORMED IN HIGHER TITER AFTERINTRADERMALTHANSUBCUTANEOUSVACCINE(OWEVER THETITERSAFTERML VACCINESUBCUTANEOUSLYWERELITTLEDIFFERENTTHANAFTERINTRADERMALINJECTION OFML)TISCONSIDEREDADVISABLE HOWEVER INPRACTICETOLIMITINTRADERMAL VACCINATIONTOWHENTHEVACCINEISINSHORTSUPPLYORWHEN IN CHILDRENORTHE AGED REACTIONSAFTERSUBCUTANEOUSVACCINEMIGHTPOSEPROBLEMS 4HENASALROUTEOFINOCULATIONEITHERBYINSTILLATIONOFDROPS ORBYSPRAY WASFIRSTSTUDIEDINDETAILBY7ALDMANETAL;=#OMPAREDWITHTHESUB CUTANEOUS VACCINE IN A DOSE OF  ML ANTIBODIES CAPABLE OF NEUTRALIZING THEVIRUS!4AIWAN(. INCREASEDTOAGREATEREXTENTINSPUTUMAND NASALSECRETIONSAFTERREPEATEDNASALINOCULATIONWITHATOTAL VOLUMEOF MLVACCINE)NCONTRAST THEINTRANASALVACCINEPRODUCEDAMUCH LOWERRISE INSERUMANTIBODY THE'-4BEINGONLYONE SIXTHTHATAFTERSUBCUTANEOUS VACCINE 7ALDMAN ET AL ;= USING AN AEROSOL SPRAY FOUND THAT A BETTER



*OHN/XFORDETAL

SERUM ANTIBODY RESPONSE OCCURRED WITH A SMALL SIZED PARTICLE SPRAY THAN ALARGERONE BUTTHENASALANTIBODYRESPONSEWASBETTERAFTER THELATTEROR WITH NASAL DROPS!BSORPTION STUDIES SHOWED THAT A MAJORITY OF THE SECRE TORY ANTIBODY )G! RESPONSE IN NASAL SECRETION WAS CROSS REACTIVE WITH HETEROLOGOUSVIRUSES!(ONG+ONG(. 0HILLIPSETAL;=COMPARED SUBCUTANEOUSORINTRADERMALVACCINEINNURSESWITHVACCINEDROPPEDINTRA NASALLY 4HE SUBCUTANEOUS ROUTE PRODUCED THE BEST SERUM ANTIBODY RISES AND INTRADERMAL VACCINE WAS SUPERIOR TO THE INTRANASAL ROUTE IN TERMS OF ANTIBODY RESPONSE4HE NASAL ANTIBODY TITERS AFTER IMMUNIZATION BY EITHER SUBCUTANEOUSORRESPIRATORYROUTESPARALLELEDTHOSEINSERUM 4HE FACT THAT NASAL ANTIBODIES INCREASE AFTER SUBCUTANEOUS VACCINE ; =ISIMPORTANTBECAUSETHELACKOFAGOODRESPONSEINSERUMANTIBODYIN THOSE GIVEN THE SAME VACCINE INTRANASALLY IS A LIMITATION HARDLY OFFSET BY LOCAL NASAL SECRETORY CHANGES #HALLENGE OF IMMUNIZED GROUPS OFF PERSONS BYLIVEATTENUATEDVIRUSALSOSUPPORTSTHEVIEWTHATNASALANTIBODIESPLAYA SUPPLEMENTARYROLETOSERUM()ANTIBODY;=

%ARLYQUANTIFICATIONOFSIDEREACTIONSTOVACCINES7HOLEVIRUS VERSUSSPLITANDSUBUNIT 4HEFIELDTRIALSOFINACTIVATEDVACCINESINANDADDED TOKNOWLEDGE CONCERNINGTHEREACTOGENICITYOFDIFFERENTPREPARATIONS4HESPLIT VIRUSTYPE OFVACCINETHENUSEDUNQUESTIONABLYCAUSEDFEWERSYSTEMICFEBRILERESPONSES INBOTHCHILDRENANDADULTS4HEFACTTHATREACTIONSWITHWHOLE VIRUSVACCINES USEDATTHETIMEWEREUNPLEASANTLYSEVEREFORTHOSEWITHOUTSERUMANTIBOD IESTOTHEVACCINEVIRUSBEFOREINOCULATIONHADNOTBEENFULLYAPPRECIATED )NTHECASEOFCHILDRENAGEDnINTHE!MERICANTRIALSOF!.EW*ERSEY (SW. VIRUS THEMOSTPOTENTVACCINESCAUSEDFEVERINUPTO OFVAC CINEES)NTHE5+ THE0ANDEMIC7ORKING'ROUPOFTHE-2##OMMITTEEON )NFLUENZA6ACCINEFOUNDTHATADOSEOF+G(!)5 OFWHOLE VIRUS VACCINEWITHTHESAME(SW.STRAINPRODUCED INADULTS LOCAL REACTIONSIN ANDSYSTEMICEFFECTSINOVEROFVOLUNTEERS%VENTHELOWERDOSES OFn +G(!CAUSEDLOCALREACTIONSINANDSYSTEMICEFFECTSIN 4HE!5332(. VIRUSVACCINETRIALININ"RITAINSHOWEDTHAT ADSORBEDORAQUEOUSSPLIT VIRUSVACCINEPRODUCEDFEWERREACTIONSTHANDID WHOLEVIRUS;=!FTERASECONDDOSEOFTHESAMEVACCINE FEWERVOLUNTEERS EXPERIENCED REACTIONS THAN SEEN AFTER THE FIRST DOSE ,ATER STUDIES OF THE ENDOTOXINCONTENTOFVARIOUSPOOLSOFINACTIVATEDTYPE!OR"VACCINESUSING THELIMULUSLYSATETESTGAVENOHINTOFAPARALLELBETWEENTHE OCCURRENCEOF GENERALREACTIONSANDTHEENDOTOXINCONTENT;= .EUROLOGICALILLNESSISARECOGNIZEDSEQUELTOIMMUNIZATIONWITHAVARI ETY OF VACCINES BUT HAD NOT PREVIOUSLY BEEN OBSERVED WITH ANY FREQUENCY F AFTERINFLUENZAVIRUSVACCINES7ELLS;=NOTED 7 THERAREINSTANCEOF'UILLAIN "ARRÏ 3YNDROME '"3 WHICH APPEARED IN EXCESS AMONG THE PERSONS

)NFLUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY



RECENTLY VACCINATED WITH !3WINE VACCINE COMPARED WITH THE NUMBERS IN UNVACCINATED INDIVIDUALS /F  PERSONS WITH '"3 REPORTED FROM  /CTOBERTOTHE*ANUARY HADRECEIVEDVACCINEPRIORTOTHE ONSETOFNEUROLOGICALSYMPTOMS4HEOVERALLRISKOF'"3WASCALCULATEDAS TENCASESPERMILLIONVACCINATED4HERATEOFOCCURRENCEDURING THE WEEK SWINEVACCINEPERIODWASFIVETOSIXTIMESGREATERTHANINUNVACCINATEDPER SONS(OWEVER THEEXCESSINNUMBERWASGREATERINTHENDANDRDWEEKS AFTER INOCULATION THAN EITHER THE ST OR SUBSEQUENT WEEKS!S REPORTED BY ,ANGMUIR ;= '"3 WAS NOT ASSOCIATED WITH A PARTICULAR VARIETY OF VAC CINE OR AGE GROUP (OWEVER THAT NUMBERS WERE SLIGHTLY GREATER IN THOSE AGEDnTHANINMIDDLEAGEDORELDERLYPERSONSAPPEARSTORULEOUTTHE POSSIBILITY THAT THE SYNDROME WAS IN SOME WAY RELATED TO THE ABSENCE OF ANTIBODIESTOTHESWINEVIRUSBEFOREIMMUNIZATION FORMOSTOFTHOSEOVER WOULDHAVEBEENEXPOSEDTOANTIGENSOFTHISVIRUSMANYYEARSBEFORE!FTER THESWINEINFLUENZACAMPAIGNWASTERMINATED SURVEILLANCEWASCONTINUED ANDDURINGTHEPERIODn WHENMILLIONDOSESOFORDINARYINAC TIVATEDVACCINEWEREESTIMATEDTOHAVEBEENUSED THERELATEDRISKOF'"3 WASTIMESTHEINCIDENCEINUNVACCINATEDPERSONS4HISRISK WASREGARDED ASNOTSIGNIFICANT;=.OCLUETOTHECAUSEOFTHEMARGINALLYINCREASEDRISK OF'"3INIMMUNIZEDPERSONSINHASYETBEENOBTAINED BUTCOULDBE VIRUSSTRAINRELATED

!DVENTOFTHEPANDEMICVIRUSANDUSEOFINACTIVATEDVACCINES !T THE TIME WHEN!(ONG +ONG  (. VIRUS WAS SPREADING IN!SIA PLANSWEREMADEBYTHE-2##OMMITTEEON)NFLUENZA6ACCINETOPROTECT CHILDREN IN RESIDENTIAL SCHOOLS AND OTHER GROUPS IN A CONTROLLED MANNER )NACTIVATEDPOLYVALENTVACCINECONTAININGTWO(.VIRUSES!%NGLAND AND!%NGLAND ANDA"STRAINWERECOMPAREDWITHAN(.!(ONG +ONG WHOLE OR DEOXYCHOLATE TREATED VIRUS VACCINE IN INITIAL SEROLOGICAL TRIALS !NTIBODY FORMATION EVEN IN THOSE WITHOUT DETECTABLE SERUM () ANTIBODY GAVE '-4S OVER  IN THOSE RECEIVING !(ONG +ONG VACCINE INTRAMUSCULARLY(OWEVER CONTROLLEDTRIALSINTWOBOARDINGSCHOOLSSHOWED NOCONVINCINGEVIDENCEOFPROTECTION)NUNCONTROLLEDTRIALSIN OTHERSCHOOLS EITHER THE POLYVALENT OR THE!(ONG +ONG VACCINE WERE GIVEN OR NO VAC CINE AT ALL4HERE WERE  SCHOOLS WHERE EPIDEMICS OF INFLUENZA OCCURRED IN*ANUARYAND&EBRUARYBUTNOEVIDENCEOFPROTECTIONWASFOUNDIN THOSERECEIVING!(+VACCINES4HEONLYCLUEOBTAINEDCONCERNINGTHEVAC CINEFAILUREWASFIRSTTHATONLYONEDOSEOFVACCINEHADBEENGIVEN ANDTHIS ISKNOWNTOBEINADEQUATETOGIVEASATISFACTORYANTIBODYRESPONSEINPRE VIOUSLYSERONEGATIVEPERSONS ANDSECONDLY THEREWASANINTERVALBETWEEN VACCINE ADMINISTRATION AND INFECTION OF n MONTHS 4HESE TWO FACTORS MAY HAVE COMBINED TO EXPLAIN THE ABSENCE OF PROTECTION BECAUSE OF THE INADEQUACYOFTHEANTIBODYRESPONSEATTHETIMEOFCHALLENGE)TWOULDBE



*OHN/XFORDETAL

FAIRTOADDTHATOTHERS; =DIDOBTAINPROTECTIONFROM!(ONG+ONG WHOLE VIRUSVACCINEDURINGTHEFIRSTOUTBREAKOFINFLUENZADUETOTHISVIRUS INTHE53!4HEUSEOFMODERNADJUVANTED(.VACCINEINTWODOSESIS ANTICIPATEDTOGIVEPROTECTIVEEFFECTS

&IRSTSTUDIESWITHLIVEINFLUENZAVACCINES 4HE USE OF LIVING BUT ATTENUATED VIRUS AS AN IMMUNIZING AGENT DEVELOPED SLOWLY FROM THE INITIAL STUDIES OF -AWSON AND 3WAN ;= IN!USTRALIA AND THE53324HEMAJORDIFFICULTYOFTHELACKOFALABORATORYTESTTOINDICATE THATCULTUREDVIRUSHADLOSTITSPATHOGENICITY WHILERETAININGINFECTIVITYFOR MAN MEANT THAT DELIBERATE INTRANASAL INOCULATION OF VOLUNTEERS FURNISHED THEONLYWAYTOSELECTASUITABLESTRAINFORINFECTIONWITHOUT CAUSINGCLINICAL REACTION )N SPITE OF THE WIDESPREAD ADOPTION OF LIVE VACCINES SELECTED BY THISMETHODANDGIVENASANINTRANASALSPRAYINTHE5332 LITTLEINTERESTWAS EXHIBITEDINMOSTOTHERCOUNTRIES&ROMONWARDS TRIALSTOOKPLACEIN VOLUNTEERSIN%NGLANDAND7ALESTOPROVIDEEVIDENCEOFSAFETYANDIMMU NOGENICITY OF CULTURED VIRUSES AND THE DRAWBACK OF A REDUCED INFECTIVITY OFWELL ATTENUATEDVIRUSESHANDICAPPEDPROGRESS4HENECESSITY TOOBSERVE A MATCH BETWEEN THE ANTIGENS OF EPIDEMIC VIRUSES AND THOSE PRESENT IN THEVACCINEWASAFURTHERDRAWBACKUNTILTHETECHNIQUEOFREASSORTMENTOF CHARACTERS BETWEEN TWO STRAINS ONE OF WHICH WAS OF PROVEN ATTENUATION WAS UTILIZED TO YIELD SEED VIRUSES WITH THE DESIRABLE CLINICAL AND ANTIGENIC PROPERTIES/THERDISADVANTAGESOFLIVEVIRUSESAPPEAREDDURINGTHEINTEN SIVE RESEARCHES OF THE S PARTICULARLY IN THE 53! AND IN %NGLAND ; =)TCANNOTYETBECLAIMEDTHATTHEIDEALLIVEATTENUATEDVIRUSVACCINEHAS BEENFORMULATED BUTREVERSEGENETICSANDINCREASEDKNOWLEDGEOFVIRULENCE GENESHAVENOWLEADTOARESURGENCEOFINTEREST )NTHES GENETICSTUDIESWEREINTENSIVELYPURSUEDINATTEMPTSFIRSTTO DEFINETHEPARTICULARGENEORCOMBINATIONOFGENES DONATEDBY THEATTENU ATED VIRUS THAT CONFERS THE PROPERTY OF ATTENUATION UPON THE REASSORTANT STRAIN)TWASFOUNDTHATTHEBIOLOGICALPROPERTIESOFEXCRETEDVIRUSMAYBE ALTERED COMPARED WITH THOSE OF THE ORIGINAL VIRUS IN THE VACCINE AND THE MANNEROFTHISALTERATIONWASALSOSTUDIEDGENETICALLY3UCHWORKISESSENTIAL INACHIEVINGTHEGOALOFANEFFECTIVEANDSAFEVACCINEVIRUSFORHUMANUSE %XPERIMENTALINOCULATIONSWERECARRIEDOUTINITIALLYINSMALL SCALETESTSIN VOLUNTEERSUNDERSEMI ISOLATIONTOPERMITCLOSEOBSERVATIONSEEBELOW 

(OST RANGEVIRUSMUTANTSASLIVEVACCINES -ULTIPLE CULTIVATION AND PASSAGE OF VIRUSES EITHER IN ANIMAL HOSTS SUCH AS FERRETSANDMICE ORINDEVELOPINGCHICKEMBRYOSORTISSUECULTURESHADBEEN PRACTICEDEVENBEFORETHEUSEOFTEMPERATURE SENSITIVETS ORCOLD ADAPTED

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CA MUTANTSWASSUGGESTED%ARLYWORKERSIN"RITAINUSEDTHE02VIRUS ASAHOSTRANGEMUTANT WHICH ALTHOUGHNONINFECTIVEFORMAN HASRETAINED ANIMALPATHOGENICITYEVENAFTERMANYPASSAGESINEGGS!SADONORPARENT WITHGOODPOWERSOFMULTIPLICATIONINTHELABORATORY 02WASMATEDWITH VARIOUSSTRAINSOFWILD TYPEINFLUENZA!VIRUSESTOOBTAINRECOMBINANTSWITH UP TO DATESURFACE(!AND.!ANTIGENS4HISMETHODWASPREFERABLETOSIM PLELABORATORYCULTIVATIONBECAUSESOMEVIRUSESFAILEDTOALTERINPATHOGE NICITYAFTERASMANYASSERIALPASSESINCULTURES;= ALTHOUGHOTHERVIRUS STRAINSAPPEAREDTOBECOMEATTENUATEDWITHONLYAFEWPASSAGESINEGGS 02VIRUSWASCHOSENALSOBYWORKERSIN"ELGIUMWHOPREPAREDREASSOR TANTSFROMANUMBEROFVIRUSES SOMEOFWHICHWERELICENSEDFOR HUMANUSE ;=4OSELECTRECOMBINANTSWITHASHIGHPROPORTIONOF2.!COMPONENTS ASPOSSIBLEDERIVEDFROMTHEHOSTRANGEMUTANT02 &LORENTETAL;=USED 2.! 2.!HYBRIDIZATIONTOIDENTIFYGENEORIGINS,ATERTHEGENE CONSTELLA TIONOFFOUROFTHECANDIDATEVACCINEVIRUSESWEREDETERMINEDAND&LORENT ;=FOUNDTHATSOMECLONESOF"EAREAND(ALLS;=RECOMBINANTSOF02 AND!%NGLANND(. CONTAININGFIVEGENESFROM02WERESATISFAC TORILYATTENUATED(OWEVER ONE CLONETHOUGHCONTAININGSIX02 GENESWAS NEVERTHELESSCLINICALLYVIRULENTTOVOLUNTEERS!FURTHERGENETICSTUDYOF02 HOSTRANGERECOMBINANTSUSINGVIRUSESTESTEDCLINICALLYBY"EAREAND2EED ;=WASMADEBY/XFORDETAL;=)TWASAGAINFOUNDTHATRECOMBINANTS FROM02AND!%NGLANDVIRUSES COULDCONTAINONLYTHESURFACE(!AND .!GENESFROMWILD TYPEVIRUSANDYETRETAINVIRULENCEFORMAN !DDITIONAL ATTEMPTS TO STABILIZE THE ATTENUATION OF CANDIDATE VIRUSES WEREMADEBOTHBY"EAREATTHE-EDICAL2ESEARCH#OUNCILSLABORATORIES AT 3ALISBURY AND THE 2)4 WORKERS BY RENDERING THE VIRUS RESISTANT TO AN INHIBITORPRESENTINNORMALHORSESERUM4HISPROPERTYWASPRESENTINTHE 2)4SERIESOFRECOMBINANTS)TSEEMSSTRANGETHATSTABILIZATIONHASNOTBEEN PURSUEDSINCE NORHASCULTIVATIONOFHOSTRANGEMUTANTVIRUSES SUCHAS02 ATABNORMALLYLOWTEMPERATURES SUCHASŽ#4HISMETHODWASFOUNDBY 3ABIN;=TOBEPREFERABLETONORMALTEMPERATURESWHENATTENUATINGPOLIO VIRUSES ANDITWASEXPLOITEDBYBOTHWORKERSINTHE53!AND5332 -ARKER TESTS WHICH CAN BE EQUATED WITH ATTENUATION OF VIRULENCE FOR MAN WERESOUGHTWITHRELATIVELYVARIABLERESULTS/NESUCHTESTUSEDWEAN LING RATS THAT WERE INOCULATED INTRANASALLY FIRST WITH VIRUS AND LATER WITH CULTURESOF(AEMOPHILUSINFLUENZAE6IRULENTVIRUSINDUCESBACTEREMIAAND MENINGITISANDUSINGTHISMETHOD*ENNINGSETAL;=SUCCESSFULLYSEPARATED ANUMBEROFREASSORTANTVIRUSESANDOBTAINEDSOMECORRELATIONWITHCLINICAL VIRULENCE9ETTHEHOSTRANGEMUTANTPARENT02AND2)4 WHICHARE BOTHATTENUATEDINMAN WERECLASSEDASVIRULENTBYTHERAT ! NEW APPROACH AT THAT TIME USED AN AVIAN DUCK VIRUS WHICH WAS FOUNDTOHAVEONLYLOWPATHOGENICITYFORSQUIRRELMONKEYSINOCULATEDINTRA NASALLY AND WAS PROPOSED AS A DONOR OF ATTENUATION! REASSORTANT WITH A VIRULENTHUMAN!5DORN(. VIRUSBEHAVEDASDIDTHEAVIAN PARENT INTHESQUIRRELMONKEY ALTHOUGHIMMUNIZINGTHELATTERAGAINST THEVIRULENT



*OHN/XFORDETAL

PARENT #LINICAL TRIALS HAVE SUGGESTED THAT THIS VIRUS IS ATTENUATED FOR MAN ANDISIMMUNOGENIC BUTHASNOTBEENINVESTIGATEDSINCE;=

4EMPERATURE SENSITIVEVIRUSMUTANTSASLIVEVACCINES -OST WORK ON THE DEVELOPMENT OF VIRUSES WITH RESTRICTED MULTIPLICATION AT TEMPERATURES ABOVE THE NORMAL RANGE FOR CULTIVATION HAS BEEN AFFECTED BY #HANOCK -URPHY AND ASSOCIATES AT THE .ATIONAL )NSTITUTES OFF (EALTH "ETHESDA ;= 4HE TECHNIQUE EMPLOYED CHEMICALLY PRODUCED MUTATION IN VIRUS2.!BYCULTIVATIONINTHEPRESENCEOFTHEMUTAGENICAGENT FLUOROURA CIL!FTERCULTIVATIONANDPLAQUINGATª ªANDª# MUTANTVIRUSESWITH THEREQUISITETEMPERATURESENSITIVITYWEREOBTAINED)NTRANASAL INOCULATIONOF HAMSTERSCONFIRMEDTEMPERATURERESTRICTION INTHATMUCHLOWER TITERSOFVIRUS WEREFOUNDINTHEHOTTERLUNGSTHANINTHECOOLERUPPERRESPIRATORYTRACT 3PREADFROMINOCULATEDVOLUNTEERSTOADULTSINCONTACTWASNOT OBSERVED AND NO EVIDENCE OF A CHANGE IN VIRULENCE WAS FOUND IN VIRUSES RECOVERED FROM ADULT RECIPIENTS OF VACCINE ;= "UT IN SERONEGATIVE CHILDREN THE! (ONG+ONG TS L;%=VIRUSBOTHPRODUCEDMILDFEBRILEREACTIONSANDAVIRUS THATHADLOSTITSPROPERTIESWASRECOVEREDFROMSOMEWHOWEREINFECTED !SECONDSERIESOFTS AWASTHENDEVELOPEDBYCOMBININGTWODEFECTIVE TSVIRUSES EACHOFWHICHBELONGEDTOADIFFERENTCOMPLEMENTATIONGROUPIN RESPECT OF THE GENETIC DEFECT4HE PROGENY EXHIBITED GREATER TEMPERATURE RESTRICTIONTHANTHETS ;%=LINEOFVIRUSES)TWASTERMED!5DORN TS ! ANDITWASRECOMBINEDWITHTHREEFURTHERVIRUSESWILD TYPE!6ICTORIA 6 !!LASKA (. AND ALSO !(ONG +ONG (.  4HESE TS ! VIRUSESWEREHIGHLYIMMUNOGENICANDEXHIBITEDTEMPERATURERESTRICTIONOF MULTIPLICATIONINCELLCULTURESANDREDUCEDREPLICATIONINTHEHAMSTERLUNG 4HE !6ICTORIA TS ! RECOMBINANT RETAINED ITS TS PROPERTIES AFTER INOCULATION INTO DOUBLY SERONEGATIVE CHILDREN 5NFORTUNATELY WHEN THE !!LASKA TS ! VIRUS WAS SIMILARLY TESTED IN A SINGLE CHILD AFTER TESTS IN ADULTS HAD SHOWN GENETIC STABILITY THE NASAL SECRETIONS OF THE VACCINEE YIELDEDATS POSITIVEVIRUSTHATPRODUCEDPLAQUESATŽ#EVEN THOUGHTHE CHILDHADSHOWNNOSYMPTOMSORFEVER4HERECOMBINANT!VIRUS WITH! (ONG+ONG(. PARENTEXHIBITEDACAPACITYTOINFECTOFF DOUBLY SERONEGATIVEADULTSANDWASATTENUATEDCOMPAREDWITHTHEWILD TYPEPARENT .EVERTHELESS ITAPPEAREDPOSSIBLETHATAVIRUSSUCHASTHE!!LASKA TS ! MIGHT IFTRANSFERREDTOCONTACTSFROMANINOCULATEDCHILD RESULTINCLINICAL ILLNESSANDCLINICALSTUDIESWITHTHISPARTICULARVIRUSWERENOTPURSUED

#OLD ADAPTEDVIRUSMUTANTSASLIVEVACCINES "EGINNINGWITHASTRAINOF(.VIRUSRECOVEREDIN!NN!RBOR -ICHIGAN INBYCULTIVATIONOFTHROAT WASHINGSINTISSUECULTURESATŽ# -AASSAB

)NFLUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY



; = EVOLVED A VIRUS !!NN !RBOR (. WHICH HAS ACTED AS A DONOR OF ATTENUATION TO OTHER VIRUSES BY GENETIC REASSORTMENT %ARLIER PASSAGESWEREMADEINCHICKKIDNEYTISSUECULTURESFOLLOWEDBYINTRANASAL PASSAGES IN MICE AND THEN A GRADUAL ADAPTATION TO LOWER TEMPERATURES IN TISSUECULTURESANDINDEVELOPINGHENSEGGSINOCULATEDALLANTOICALLY LEDTOA VIRUSWITHGOODPOWERSOFMULTIPLICATIONATª#4HECAVARIANTWASFOUND TORETAINTHEINFECTIVITYOFTHEORIGINALSTRAINFORBOTHTHEMOUSEANDFERRET ALTHOUGH IT PRODUCED NO DEATHS IN MICE AND NO FEVER OR TURBINATE LESIONS IN FERRETS WHEREAS THE ORIGINAL VIRUS WAS PATHOGENIC FOR BOTH SPECIES4HE VIRUS PROVED TO BE TEMPERATURE SENSITIVE WITH A SHUT OFF TEMPERATURE OF ª# ;= 2ECOMBINANTS WITH WILD TYPE VIRUSES OF BOTH (. AND (. SUBTYPES WERE PREPARED STUDIED IN THE LABORATORY AND IN VOLUNTEERS AND ANALYZEDGENETICALLY4HEORIGINAL!!NN!RBOR(. VIRUSWASNOT HOWEVER TESTEDINFULLYSUSCEPTIBLEPERSONSPRESUMABLYBECAUSEOFTHEDIF FICULTYINTHATPERIODOFFINDINGSERONEGATIVEADULTS!FEWPERSONSWITHLOW TITERSOFSERUMNEUTRALIZINGANTIBODIESTO WEREINOCULATEDANDAS JUDGEDBYANTIBODYRESPONSES BECAMEINFECTEDWITHOUTUNDERGOINGCLINICAL ILLNESSES -ORE RIGOROUS CLINICAL STUDIES HAVE BEEN PURSUED WITH RECOMBI NANTS INPARTICULARTHOSEWITH(.ANTIGENS ANDDETAILSOFTHERESULTSHAVE BEEN BOUGHT TOGETHER AND EARLIER DATA SUMMARIZED BY +ENDAL ;= 4HE DONORCAPARENTHASBEENMORERECENTLYREASSORTEDWITH(.GENES )T IS CLEAR THAT INFECTIVITY AND IMMUNOGENICITY WERE FULLY RETAINED FOR SERONEGATIVE ADULTS OF WHOM  RECEIVED (. RECOMBINANTS !MONG THOSERECEIVINGTHREEOFFOURRECOMBINANTS CLINICALREACTIONSWEREMINIMAL ORNEGLIGIBLEBUTWITHTHEFOURTH DERIVEDFROMTHE!3COTLANDPARENT IN OFVOLUNTEERSRECEIVING ANDINRECEIVING 4#)$ THEREWERE CLINICALILLNESSES6IRUSESRE ISOLATEDFROMTHEVACCINEESRETAINEDTS PROPER TIESANDSODIDTHOSEGIVENRECOMBINANTSOF!6ICTORIA(. AND! !LASKA(. (OWEVER SOMELOSSOFCARESTRICTIONWASFOUNDINVIRUS RE ISOLATEDFROMVOLUNTEERSGIVENTHE!3COTLANDRECOMBINANT #OLD ADAPTED RECOMBINANTS WITH !5332 (. LIKE VIRUS HAVE ALSOBEENSTUDIEDINADULTVOLUNTEERSANDFOUNDTOBELESSIMMUNOGENICAS JUDGEDBY()ANTIBODYRESPONSES!BETTERRESPONSEWASOBTAINEDBY7RIGHT ETAL;=INCHILDRENIN.ASHVILLEGIVEN 4#)$OFSTRAIN#2(. ANDNONEOFCHILDRENDEVELOPEDADVERSECLINICALREACTIONSEVENTHOUGH BECAMEINFECTED!LLRE ISOLATEDVIRUSESRETAINEDTHETSPHENOTYPE4HEFAIL URETOELICITSERUMANTIBODYRESPONSEINADULTSGIVENTHISSAME VIRUSRECOM BINANT IS PUZZLING 5SING THE %,)3! ENZYME LINKED ASSAY -URPHY AND OTHERS;=FOUNDTHATBYTHISMORESENSITIVEMETHODANTIBODYRISESCOULD BEDEMONSTRATEDANDTHERESULTSTALLIEDBETTERWITHTHEABILITYTORE ISOLATE VIRUSESFROMTHEINOCULATEDVOLUNTEERSTHANDIDTHESERUM()RESPONSES 4HE,ENINGRADGROUPOFWORKERSLEDBY3MORODINSTEV;=WERETHEFIRST TOOBTAINAVIRUSINDIRECTLYATTENUATEDBYCULTIVATIONATŽ#4HEGROUPUSED STRAINSSELECTEDBYINOCULATINGVOLUNTEERSWITHSEVERALVIRUSES DERIVEDFROM CULTURES REPEATEDLY INCUBATED AT nª# TO SPEED UP ATTENUATION 3OME



*OHN/XFORDETAL

nMONTHSWEREREQUIREDFORTHEPREPARATIONANDPRODUCTIONOFNEWSTRAINS EVENUSINGGENETICRECOMBINATIONTOINCORPORATENEWSURFACE(! AND.! ANTIGENS!LTHOUGH!LEXIEVAETAL;=FOUNDTHATCOLDCULTIVATIONWASNOT SUCCESSFULINPRODUCINGRELIABLYATTENUATEDVIRUSESFORUSEINCHILDREN THE TECHNIQUE WAS ADOPTED FOR GENERAL USE 'ENETIC STUDIES OF THE ,ENINGRAD VIRUSESAREDESCRIBEDBRIEFLYBY+ENDALETAL;=ANDTHESEPARENTCAVIRUSES ARECURRENTLYTHECENTEROFNEWINTERESTFORATTENUATED(.VACCINES 5SUALLY PRELIMINARY STUDIES WERE MADE IN THE 5332 IN n YEAR OLD SERONEGATIVE ADULTS WHO RECEIVE VIRUS TWICE AT INTERVALS OFF n DAYS ADMINISTRATEDBYNASALSPRAY6IRUSESWEREATTENUATEDBYPASSAGEFORVARY INGPERIODSATª#ANDBOTHDONORVIRUSESANDRECOMBINANTSPROVEDTEM PERATURESENSITIVE)Nn WHEN(.VIRUSESWERECIRCULATING  CHILDREN AGED FROM  TO  YEARS RECEIVED THE CA !,ENINGRAD (. VIRUS3OMEFEBRILEREACTIONSOCCURREDBUTONLYINLESSTHANOFTHECHIL DREN&URTHERSTUDIESOFRECOMBINANTSWITH(.OR(.ANTIGENSANDTHE SAME,ENINGRAD(.PARENTAFTERSERIALPASSAGESUNDERCOLDCONDITIONS OF CULTIVATION ª# WERE CONDUCTED IN CHILDREN HALF OF WHOM HAD NO DETECTABLESERUMANTIBODYTOTHEVACCINESTRAIN.OREACTIONSOCCURREDAND OVER  OF THE CHILDREN RESPONDED WITH ANTIBODY PRODUCTION )T IS CLEAR FROMTHEEARLIERPAPERSBY!LEXIEVAETAL; =THATINTRANASALADMINISTRA TION OF CHILDREN AGED n YEARS WERE TOO REACTOGENIC AND THAT THIS IS THE REASONWHYTHEPERORALROUTEHASBEENCHOSENFORROUTINEADMINISTRATIONIN THE5332 ! *APANESE VIRUS RECOVERED IN  !/KUDA(. WAS FOUND TO BE ATTENUATED FOR CHILDREN AND SERVED AS A DONOR OF ATTENUATION BOTH IN *APAN AND IN %NGLAND *APANESE WORKERS :HILOVA ET AL ;= DEVELOPED A RECOMBINANT VIRUS +/  FROM ULTRAVIOLET IRRADIATED !/KUDA AND WILD TYPE!+UMAMOTO(. 3ERIALPASSAGINGINEGGS IN THEPRES ENCEOFNORMALHORSESERUMWASFOLLOWEDBYPLAQUEPURIFICATION ANDLATER CLINICALTESTSINAFEWCHILDREN4HE-MEMBRANE GENEWASFOUNDTOHAVE BEEN DONATED BY THE /KUDA PARENT &ROM REASSORTANTS WITH OTHER HUMAN VIRUSES ACANDIDATE72,VIRUSWASSELECTEDANDUNDERWENTCLINICALTRI ALSWITHOUTHARMFULCLINICALEFFECTS;= BUTHASBEENLITTLEINVESTIGATEDSINCE THATTIME

-AMMALIANCELLCULTUREVACCINES #ULTIVATION OF INFLUENZA VIRUSES IN MAMMALIAN CELLS RATHER THAN EGGS INI TIALLYENCOURAGEDTWOMANUFACTURESTOINVESTINCELLCULTUREFERMENTERSFOR VACCINEPRODUCTION; =-ANYMOREGROUPSARENOWRESEARCHINGTHESE TECHNOLOGIES#APACITYCANBEINCREASEDTOCOPEWITHASURGEINDEMANDFOR A PANDEMIC VIRUS VACCINE -OREOVER THE FINAL VACCINE HAS THE THEORETICAL ADVANTAGEOFTHEABSENCEOFEGGPROTEINS4HECELLCULTUREVACCINEVIRUSIS ALSO EASIER TO PURIFY7HERE CLINICAL ISOLATES OF INFLUENZA VIRUSES ARE CULTI

)NFLUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY



VATEDINMAMMALIANCELLSANDEGGSINPARALLEL DIFFERENTANTIGENICVARIANTS MAYBESELECTED;=4HEBIOLOGICALVARIANTSHAVEAMINOACIDSUBSTITUTIONS INTHERECEPTORBINDINGSITEINPROXIMITYTOANANTIGENICSITEONTHE(! AND ANAMINOACIDCHANGEINTHISREGIONCANALTERANTIGENICITY/FTHETWOVIRUS SUBPOPULATIONSTHATCANBESELECTED THEVIRUSWHICHISGROWNON-$#+OR 6ERO CELLSRATHERTHANINEGGSAPPEARSMORECLOSELYRELATEDTOTHEWILD TYPE CLINICALVIRUS4HEREISSOMEINDICATIONTHATCELL GROWNVIRUS VACCINESOFFER GREATERPROTECTIONINANIMALMODELSTHANTHECORRESPONDINGEGG GROWNVAC CINE4HESE ARE ALL POWERFUL ARGUMENTS IN FAVOR OF THE NEW GENERATION OF INFLUENZAVACCINESBEINGCULTIVATEDCURRENTLYIN6ERO;=OR-$#+;=OR 0ERCELLS

5NLIKEHISTORICALVACCINESCOULDNEWLYDEVELOPEDSTCENTURY VACCINESINDUCEPROTECTIONACROSSTHEDIFFERENTVIRUSSUBTYPES 4HERE ARE  KNOWN SUBTYPES OF THE (! OF INFLUENZA! VIRUS /NLY THREE SUBTYPESHAVECAUSEDPANDEMICSINHUMANS ( (AND( WHILE ( ( AND( PREDOMINANTLYCIRCULATINGINBIRDSHAVECROSSEDTHESPECIESBARRIER INTOHUMANSANDCAUSEDHUMANOUTBREAKS7EDONOTKNOWWHETHER THESE LATTERTHREESUBTYPESCOULDMUTATEINTOHUMAN TO HUMANTRANSMITTERSAND THEREBYACQUIREPANDEMICPOTENTIAL!TPRESENT(.ISCAUSING CONSIDER ABLE CONCERN IN 3% !SIA !N IMPORTANT QUESTION THEREFORE IS WHETHER A VACCINECOULDBEENGINEEREDTOGIVESO CALLEDHETEROTYPICORCROSS SUBTYPE IMMUNITYTOPROTECTAGAINSTALLTHESEPOTENTIALLYPANDEMICVIRUSES)TISWELL KNOWNTHATTHEINTERNALPROTEINSOFINFLUENZA!VIRUSSUCHAS- -AND .0ARESHAREDBYALLINFLUENZA!VIRUSES4HESEINTERNALLYSITUATEDPROTEINS ARECERTAINLYIMMUNOGENICPARTICULAR.0 BUTCOULDTHEIMMUNITYINDUCED EITHER4CELLORANTIBODY BEBROADLYREACTING 4O BACK UP THE CENTRAL CORE OF THIS APPROACH IT HAS BEEN KNOWN FOR YEARSTHATMICEINFECTEDWITHANINFLUENZA!(. VIRUSWOULDLATER RESISTALETHALCHALLENGEFROMANINFLUENZA!(. VIRUS'IVENTHELACK OF GENETIC AND ANTIGENIC RELATEDNESS BETWEEN THE ( AND ( PROTEINS OR INDEED THE CORRESPONDING . AND . PROTEINS THIS STRONG CROSS IMMUNITY WASATTRIBUTEDTOANINTERNALPROTEINSUCHAS.0OR-(OWEVER ITHASBEEN DIFFICULTTOCONSTRUCTASOLIDDATABASEANDTHEREHASBEENALINGERINGDOUBT ABOUT THIS SO CALLED CROSS PROTECTIVE IMMUNITY -OST VIROLOGISTS DEDUCED VIRTUALLY BY ELIMINATION THAT A CROSS REACTIVE PORTION OF THE (! (! COULDHAVEPROVIDEDTHECROSSPROTECTION&URTHERMORE THISCROSSPROTECTION ISPARTICULARLYSEENINTHEMOUSEMODEL LEADINGSOMETOCONCLUDETHATTHE MOUSERECOGNIZEDCROSSPROTECTIONEPITOPESTHATPERHAPSHUMANS DIDNOT &UNDAMENTAL STUDIES TO CORRELATE THE GENETICS AND IMMUNOLOGY OFF .0 AND-ESTABLISHEDTHECYTOTOXIC4CELLRESPONSETOPORTIONSOFTHESEPROTEINS (OWEVER THEWORKCLEARLYSHOWEDTHAT-COULDBEACROSS REACTIVEIMMU NOGEN ALTHOUGH A RELATIVELY WEAK ONE ;=4HE - PROTEIN IS AN INTEGRAL



*OHN/XFORDETAL

MEMBRANE PROTEIN OF INFLUENZA! VIRUSES THAT IS EXPRESSED AT THE PLASMA MEMBRANE OF VIRUS INFECTED CELLS AND IS ALSO PRESENT IN SMALL AMOUNTS ON VIRIONS 4HE IMPORTANT EXTRACELLULAR DOMAIN POTENTIALLY TARGETED BY ANTI BODIESAND4CELLS ISCONSERVEDBYVIRTUALLYALLINFLUENZA!VIRUSES%VENTHE PANDEMICVIRUSDIFFERSONLY INONEAMINOACID4HEFIRSTINDICATIONTHAT THE - WAS IMMUNOLOGICALLY ACTIVE WAS THE OBSERVATION THAT AN ANTI - MONOCLONALANTIBODYREDUCEDTHESPREADOFVIRUSCELLCULTURE.OTUNEXPECT EDLY THEANTIBODYREACTEDWITHTHEEXTRACELLULARDOMAINOF- %VENMORE EXCITINGLY THE ANTIBODY REDUCED THE REPLICATION OF VIRUS IN MOUSE LUNGS )MMUNIZATIONSTUDIESWITH-CONSTRUCTS HOWEVER HAVEGIVENMOREMIXED RESULTS)MMUNIZATIONOFMICEWITH$.!PLASMIDOF-AND-GENEGAVE PROTECTIONMAINLYVIA4HELPERCELLACTIVITY!NALTERNATIVEAPPROACHUTILIZED A HEPATITIS " CORE AND - FUSION PROTEIN4HE CROSS PROTECTION RESIDED IN ANTIBODIES ALTHOUGH- SPECIFICANTIBODIESDIDNOTNEUTRALIZE THEVIRUSIN VITRO0RESUMABLY PROTECTIONWASMEDIATEDBYANINDIRECTMECHANISM SUCH AS COMPLEMENT MEDIATED CYTOTOXICITY OR ANTIBODY DEPENDANT CYTOTOXICITY (OWEVER THEPROTECTIONINDUCEDINTHEMOUSEMODELWASCONSIDERABLYLESS THANTHATINDUCEDBYACONVENTIONALSUBUNIT(!.!VACCINE )T COULD BE ARGUED THAT WEAK HETEROTYPIC IMMUNITY MAY BE PRESENT ALREADYINTHECOMMUNITYANDTHATTHISISHELPINGTOPREVENTTHEEMERGENCE OFCHICKENINFLUENZA!(. IN3%!SIA;=#ERTAINLYWITHEVIDENCEOF TENS OF MILLIONS OF DOMESTIC BIRDS INFECTED SINCE LATE  IN  COUNTRIES IN 3%!SIA WITH ONLY A HANDFUL OF HUMAN INFECTIONS AND ONLY HUMAN TO HUMANTRANSMISSIONINFAMILYGROUPS THEREISAPOSSIBILITYTHATTHEUNIQUE CO CIRCULATIONSINCEOFTWOINFLUENZA!VIRUSES(.AND(. MAY HAVE ENHANCED HETEROTYPIC IMMUNITY IN MOST COMMUNITIES WHICH IN TURN ABROGATES THE EMERGENCE OF CHICKEN INFLUENZA! (. INTO HUMANS )T WOULDBEFOOLHARDY THOUGH TOTAKETHISARGUMENTTOAFULLERCONCLUSIONAND RELAXPREPARATIONSFORANEWPANDEMICINFLUENZA!VIRUS

4HEHISTORICALUSEOFVOLUNTEERSTOSTUDYINFLUENZAANDVACCINES !T PRESENT WITH THE UNPRECEDENTED RESEARCH INVESTMENT INTO INFLUENZA F VACCINES THERE ARE NEW DISCOVERIES OF ADJUVANTS AND VACCINE FORMULATIONS TO BE TESTED AS WELL AS FUNDAMENTALS OF VIRUS TRANSMISSION INFECTIOUSNESS ANDPATHOGENICITY4HEULTIMATETESTISININFLUENZA INFECTEDVOLUNTEERS4HIS SPECIALIZEDWORKWASINITIATEDOVERYEARSAGO $URINGTHEGREATPANDEMICOF WHENTHEPRECISENATUREOFTHECAUS ATIVE MICROBE OF THE 3PANISH INFLUENZA HAD NOT BEEN ESTABLISHED A GROUP OF!MERICANSCIENTISTSASKEDFORYOUNGVOLUNTEERSFROMTHEARMY ANDNAVY 4HEQUESTWASTOPROBETHENATUREOFTHEMICROBETHATWASALREADYCAUSING DEVASTATIONINTHEIROWNCOUNTRYANDWHERE BY  YOUNGPEOPLE WERETODIE(OWEVER THISWASNOTTHEFIRSTSTUDYINTOTHEPRECISENATUREOF THEMICROBE4HEINFECTIONHADFIRSTBEENDOCUMENTEDAYEAREARLIERASAHER

)NFLUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY



ALDWAVEINTHEGREATCITY SIZEDMILITARYBASEANDENCAMPMENTOF%TAPLES; =(ERETHE"RITISH!RMYCONSTRUCTEDTHELARGESTESTABLISHMENT;=INITS HISTORY WHERE NEWLYRECRUITEDSOLDIERSEACHDAYINTERMINGLEDWITH THOUSANDSOFWOUNDEDSOLDIERS PIGSAND INTHENEARBYVILLAGES ANDMARKETS WITHDUCKS DOMESTICCHICKENSANDGEESE4HESEARENOWRECOGNIZEDASTHE NECESSARYBIOLOGICALFEATURESOFANEPICENTERFORTHECREATIONOFAPANDEMIC VIRUS7ESURMISE INRETROSPECT THATANAVIANVIRUSFROMASILENTLYINFECTED GOOSE OR DUCK COULD HAVE CROSSED SPECIES EITHER TO A PIG OR TO A SOLDIER ALREADYINFECTEDWITHAHUMANSTRAINOFINFLUENZA4HISISTHE MIXINGBOWL HYPOTHESIS)NDEED COMMONEPIDEMICINFLUENZAWASKNOWNTOBECIRCULAT INGINTHEWINTEROFnIN%TAPLES!NOTHERFACTORIN%TAPLESCOULD HAVE BEEN THE HUNDREDS OF TONS OF GAS OF  VARIETIES CONTAMINATING THE LANDSCAPEOFTHENEARBY3OMMEBATTLEFIELD ASWELLASMANYOFTHEWOUNDED SOLDIERSBROUGHTBYTHENIGHTTRAINSINTOTHEHOSPITALSONSITEANDCAUS ING RESPIRATORY DISTRESS ! GROUP OF PATHOLOGISTS THERE AND AT !BBEVILLE LEDBY''IBSON RAISEDTHEQUESTIONOFTHENATUREOFTHEMICROBE#OULD IT BE A 'RAM NEGATIVE BACTERIUM SUCH AS ( INFLUENZAE ALREADY DESCRIBED BY 0FEIFFER AS THE CAUSE OF THE PREVIOUS INFLUENZA PANDEMIC OF  /R COULDITBEAVIRUS6IRUSESWERERATHERUNKNOWNENTITIESATTHATTIMEBUT HAD BEEN IDENTIFIED BY THEIR FILTER PASSING NATURE 3O 'IBSONS EXPERIMENT WASQUITESIMPLYTOTAKESPUTUMFROMASOLDIERVICTIMANDFILTERITTHROUGH A"ERKEFIELDCANDLEFILTER WHICHWOULDHOLDBACKANYKNOWNBACTERIUMBUT ALLOWTHEPASSAGEOFTHEMUCHSMALLERULTRAFILTERABLEVIRUS"UTWHATTHEN 'IBSONHADNOTEVENCONSIDEREDTHATAHUMANVOLUNTEERWOULDRECEIVETHE FILTRATE)NFACT HEGAVEITTOASERIESOFMACAQUESAND INADVERTENTLY TOHIM SELF(EDIEDANDTHEMACAQUESBECAMEILL(ISPREMATUREDISCOVERYOFNEW VIRUSINFLUENZAHASLAINUNDISCOVEREDANDHITHERTOUNQUOTEDINTHEARCHIVES OFTHE&IRST7ORLD7AR;= -EANWHILE INTHE53! AMOREVIGOROUSDECISIONHADBEENTAKEN AND ARMYANDNAVYVOLUNTEERSWEREINFECTEDINTRANASALLYWITHFILTEREDMATERIAL FROM 3PANISH INFLUENZA VICTIMS 3OME VOLUNTEERS WERE PLACED  M FROM DYINGSERVICEMEN WHOCOUGHEDINTHEIRFACES4HEINCREDIBLERESULTOFTHIS HEROICENDEAVORISTHATNOTASINGLEVOLUNTEERBECAMEILL WHEREASALLAROUND THE 53! THEIR COMPANIONS WERE DYING )T IS MORE THAN POSSIBLE THAT THE VOLUNTEERSHADALREADYBEENSUBCLINICALLYINFECTEDINTHEEARLYSUMMEROUT BREAKOF WHICHWASLESSVIRULENTTHANTHEAUTUMNVIRUSANDWOULDBE EXPECTEDTOGIVECROSSIMMUNITY

4HE-2#COMMONCOLDANDINFLUENZAQUARANTINEUNITIN3ALISBURY 5+ !SSOONASTHE3ECOND7ORLD7ARWASOVER THE-EDICAL2ESEARCH #OUNCIL INTHE5+ESTABLISHEDTHE#OMMON#OLD5NITIN3ALISBURYATTHE (ARVARD (OSPITAL4HEHOSPITALWASADONATIONFROMTHE53!TOCOPEWITHEXPECTED



*OHN/XFORDETAL

BOMBCASUALTIESFROM,ONDON)NTHEEVENT THISFULLYEQUIPPED MULTI BUILD ING FACILITY WAS USED AS AN ACUTE SURGICAL HOSPITAL FOR SERVICEMEN 7ITH #HRISTOPHER!NDREWES AS ITS FIRST CHIEF SCIENTIST THE UNIT RECRUITED VOLUN TEERSTOUNRAVELTHEVIROLOGICALMYSTERIESOFRESPIRATORYDISEASE&ORTHENEXT YEARS ASMALLTEAMOFVIROLOGISTSANDCLINICIANSINFECTEDVOLUNTEERSAND DISCOVERED THE FIRST HUMAN CORONAVIRUS THE COMMON COLD VIRUS AND WERE THE FIRST TO DESCRIBE THE CLINICAL EFFECTS OF INTERFERONS %SSENTIALLY SIMILAR UNITSWERESETUPINTHE53!AND2USSIA

%STIMATESOFVACCINEPROTECTIONOBTAINEDINTHEPASTBYDELIBERATE CHALLENGEINQUARANTINEUNITS 4HE CONSIDERABLE DIFFICULTIES ENCOUNTERED IN MOUNTING FIELD TRIALS LED TO EXPERIMENTS IN WHICH IMMUNIZED VOLUNTEERS WERE SUBJECTED TO DELIBERATE INOCULATIONWITHLIVEVIRUSEITHERINTHEFORMOFATTENUATEDSTRAINORMODI FIEDWILD TYPESTRAIN4HISPROTOCOLWASSUGGESTEDBY(ENLEET AL;= WHO IMMUNIZEDAGROUPOFCHILDRENWITHINACTIVATEDINFLUENZA!(. VIRUS VACCINE AND THEN INOCULATED THEM WITH EGG CULTURED VIRUS OF THE SAME SUBTYPE BUT RECENTLY ISOLATED BY INHALATION OF AN AEROSOL (IGH RATES OF INFECTION  WERE PRODUCED IN  UNIMMUNIZED CHILDREN OF WHOM  BECAMEILL4HOSERECEIVINGVACCINEEITHERESCAPEDSUBSEQUENTINFECTIONOR DEVELOPED SEROLOGICAL CHANGES ONLY  CHILD OF THE  CHILDREN THUS CHAL LENGEDBECAMEILL!LTHOUGHTHISSTUDYILLUSTRATEDTHEOUTSTANDINGSUCCESSOF THEIMMUNIZEDPROTOCOL THEREAREPROBABLYFEWOBSERVERSTODAY WHOWOULD BEPREPAREDTOSUBMITTHEIRCHILDRENTOASIMILARRISKOFDELIBERATELYINDUCED ILLNESS 3UCH A RISK IS OF COURSE EXPERIENCED DURING EPIDEMICSAND "ELL ET AL;=UNDERTOOKASIMILAREXPERIMENTINADULTVOLUNTEERSSOMEOFWHOM WEREIMMUNIZEDWITHASINGLEDOSEOFINACTIVATED!*APAN(. VIRUSVACCINESOONAFTERTHE!!SIANEPIDEMICBEGAN4HEVOLUNTEERSWERE ISOLATED BEFORE BEING GIVEN INTRANASALLY POOLED NASOPHARYNGEAL WASHINGS FROMPATIENTSWITHINFLUENZAANDTHISCAUSEDCLINICALILLNESSINOFVOL UNTEERS PREVIOUSLY GIVEN A PLACEBO!S  OF THE VACCINATED VOLUNTEERS DEVELOPED FEVER AFTER CHALLENGE IN THIS EXPERIMENT THE SINGLE INJECTION OF INACTIVATEDVACCINEPROVEDRELATIVELYINEFFECTIVE PRESUMABLYBECAUSEOFITS INADEQUATEIMMUNOGENICITY 4HEINFORMATIONOBTAINEDBYDELIBERATECHALLENGEOFIMMUNIZEDVOLUN TEERSHASBEENEXPLOREDINTHEPASTUSINGMODIFIEDATTENUATEDVIRUSSTRAINS "EAREETAL;=DIDTHISINTHEIRCOMPARISONOFINACTIVATEDORLIVEINFLUENZA " VACCINES IN WHICH A CHALLENGE FROM THE LIVE VIRUS " STRAIN WAS USED TO ASSESS THE COMPARATIVE EFFICACY OF THE TWO VACCINES 2E INOCULATION WITH LIVEVIRUSWASRESISTEDBETTERBYTHOSERECEIVINGTHESAMEMATERIALAMONTH PREVIOUSLYTHANBYTHOSEINJECTEDWITHINACTIVATEDVACCINE #OUCH ;= HAS REPORTED A NUMBER OF TRIALS IN VOLUNTEERS AFTER INAC TIVATED VACCINE USING A LOW DOSE OF AN ESSENTIALLY UNMODIFIED (. VIRUS

)NFLUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY



THATHADRECEIVEDONEORTWOPASSAGESINHUMANEMBRYONICKIDNEYCULTURE )TWASFIRSTESTABLISHEDBY'REENBERGETAL;=THATPREVIOUSINFECTIONBY HOMOTYPIC(.VIRUSGAVEPROTECTIONAGAINSTDELIBERATEEXPOSUREFORUP TO  YEARS AFTER THE ORIGINAL INFECTION #OMPARISON OF INACTIVATED VACCINE !(ONG+ONG(. GIVENINTRANASALLYORSUBCUTANEOUSLYSHOWEDTHAT FOLLOWINGCHALLENGEWITHLIVEVIRUSONLYTHOSEWHOHADDEVELOPEDASERUM ANTIBODYRESPONSEAFTERVACCINEBYEITHERROUTERESISTEDINFECTION )N A FURTHER TRIAL OF AN ANTI .! INACTIVATED VACCINE MADE FROM AN (EQ. VIRUS IT WAS SHOWN THAT A REDUCED FREQUENCY OF ILLNESS AND A REDUCEDTITEROFVIRUSINNASALWASHSPECIMENSRESULTEDFOLLOWINGLIVE(. VIRUSCHALLENGECOMPAREDWITHTHEFINDINGSINCONTROLSUBJECTS 4HENUMBER OFTHOSEWHOCONTRACTEDINFECTIONWASALSOREDUCEDSOMEWHATBY THEINAC TIVATED.!VACCINE THUSSUPPORTINGTHESUGGESTIONOF3CHULMAN ETAL;= THAT.!ANTIBODY ALTHOUGHINCAPABLEOFNEUTRALIZINGVIRALINFECTIVITY COULD LIMIT THE EXTENT OF VIRAL REPLICATION "EUTNER ET AL ;= ALSO IMMUNIZED CHILDRENWITHAN.! SPECIFICVACCINEANDNOTEDTHATANTIBODYTO .!HADA ROLEPROTECTINGAGAINSTILLNESSRATHERTHANAGAINSTINFECTION3LEPUSHKINETAL ;=AND-ONTOAND+ENDAL;=CAMETOSIMILARCONCLUSIONSWITHREGARD TO.!VACCINEANDTHECLINICALEVIDENCEOFPROTECTIONFROMILLNESS ! SERIES OF EXPERIMENTS ON VOLUNTEERS DESIGNED TO OBTAIN EVIDENCE OF PROTECTIONFROMVACCINESCONTAININGVIRUSESTHATWEREHOMOTYPIC ORHETER OLOGOUSTOTHECHALLENGEVIRUS ISIMPORTANTINRELATIONTOTHE DETERMINATION OF THE BEST COMPOSITION OF INACTIVATED VACCINE 0OTTER ET AL ;= GAVE ONE OFFOURINACTIVATEDMONOVALENT(.VIRUSVACCINESTOGROUPSOFF STUDENTS MEASUREDTHEIRPRE ANDPOST IMMUNIZATIONANTIBODIESBY()AND.)TESTS ANDLATERCHALLENGEDALLTHEGROUPSWITHALIVEINTRANASAL(.VIRUS72,  4HIS VIRUS WAS ANTIGENICALLY NEAREST TO THE!0ORT #HALMERS VIRUS AND VACCINE FROM THIS LATTER STRAIN AND ALSO THAT CONTAINING!3COTLAND VIRUSGAVEBETTERPROTECTIONAGAINSTINFECTIONTHANEARLIER(.VIRUSVAC CINESTHERESULTTHUSCORRELATEDWITHTHEINDUCED()ANTIBODY TITERS ,ARSONANDOTHERS;=ALSOCHALLENGEDTHEIMMUNITYPRODUCEDBYINAC TIVATEDVACCINEMADEFROM!0ORT#HALMERS(. VIRUSWITH THATFROM A STRAIN DEVELOPED BY THE 0ASTEUR )NSTITUTE ;=4HIS VIRUS C WITH AN ANTIGENCLOSELYSIMILARTO!%NGLAND(. WASSELECTEDINTHELABORA TORYBYAMETHODANALOGOUSTONATURALSELECTIONBYANTIGENICDRIFT ANDTHUS REPRESENTSTHEFIRSTHUMANATTEMPTTOANTICIPATEANTIGENVARIATIONINNATURE #HALLENGEOFTHOSEIMMUNIZEDWITHONEORTHEOTHERVACCINESSHOWEDTHAT PROTECTIONBYTHEHETEROLOGOUSCVIRUSWASABOUTONE QUARTERASEFFECTIVE ASTHATPRODUCEDBYTHEHOMOLOGOUS!0ORT#HALMERSVIRUS %XPERIENCESRELATEDBY#OUCHALSOCONFIRM;=THATANTIBODYEFFECTIVE AGAINSTTHEHOMOLOGOUS(!OFTHECHALLENGINGVIRUSISMOREPROTECTIVETHAN THAT FORMED BY HETEROLOGOUS ANTIGEN 0ROTECTION WAS ALSO COMPARED AFTER INACTIVATEDVACCINEBYINTRANASALORSUBCUTANEOUSROUTES WHICHSHOWEDTHAT THEIMPORTANTMEDIATOROFIMMUNITYWASTHESERUM)G'CONTENTOFF ANTI (! RATHERTHANTHERESPIRATORYSECRETIONCONTENTOFSPECIFIC)G!



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&IGURE  ! VOLUNTEER ROOM AT THE #OMMON #OLD AND )NFLUENZA 5NIT (ARVARD (OSPITAL 3ALISBURY IN THE S6OLUNTEERS WOULD STAY FOR  WEEKS IN THIS COUNTRY PLACED UNIT TO BE INFECTEDANDCAREFULLYSTUDIEDFORCLINICALSYMPTOMS

!NEWQUARANTINEUNITIN,ONDON 7EHAVEESTABLISHEDANEWQUARANTINEUNIT BASEDIN,ONDONWWWRETRO SCREENCOM BUTVERYMUCHCENTEREDUPONTHEEXPERIENCEANDETHOSOFTHE #OMMON #OLD 5NIT OF THE PAST ;= )N A SERIES OF EXPERIMENTS OVER THE PASTYEARS WEHAVEINFECTEDOVERYOUNGVOLUNTEERSWITHINFLUENZA! (. INFLUENZA"ANDINFLUENZA (. VIRUSANDMORERECENTLYRESPIRA TORYSYNCYTIALVIRUS ANDNOWHAVEFULLYCHARACTERIZEDVIRUSPOOLS;=)N THE 53! A QUARANTINE UNIT HAD ALREADY BEEN ESTABLISHED IN6IRGINIA AND ALSOAT"AYLOR ANDPIONEEREDWORKINTOTHENEW.!INHIBITORSOFINFLUENZA USING AN INFLUENZA! VIRUS ISOLATED IN  ;= 3O FAR OUR OWN UNIT HAS FOCUSED ON EVALUATING NEW INFLUENZA VACCINES ;=7E USE GROUPS OF  YOUNG VOLUNTEERS AND QUARANTINE THEM IN A STUDENT HOSTEL OR HOTEL ALONG WITH CLINICIANS AND SCIENTISTS &IG  4HE -2# #OMMON #OLD 5NIT WAS ROOTEDSTRONGLYINTHEPOST WARERAWITHDECKCHAIRS FREERUNRABBITS COUN

)NFLUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY



TRYWALKS AFTERNOONCREAMTEASANDTWO COURSE%NGLISHMEALS/URNEWUNIT REFLECTS A MORE DIVERSE COMMUNITY SO CHICKEN TIKKA IS AS COMMON ON THE MENUASROASTLAMBANDBAKEDPOTATOES BUTTHEWISHOFMANYOFTHEVOLUN TEERSISTHESAMETOCONTRIBUTETOKNOWLEDGE

#ONCLUSION )NFLUENZA!VIRUSHASAPROVENRECORDASAhBIOTERRORISTvVIRUSBUTDRIVENNOT IN#HURCHILLSWORDSBYTHEhEVILFORCESOFPERVERTEDSCIENCEv BUTBYTHEVAST UNFATHOMABLE LAWS OF NATURE AND EMERGENCE RE EMERGENCE AND RESURGENCE OF NATURAL DISEASE )NFORMATION FROM THE HUMAN GENOME PROJECT WHEREBY A SIGNIFICANT PROPORTION OF THE   ACTIVE GENES ARE ALREADY KNOWN TO BE INVOLVED IN INNATE AND ACQUIRED IMMUNITY PROVIDES REASSURANCE THAT THE IMMUNESYSTEMWILLCONTINUETOPROVIDESOMEPROTECTIONAGAINST NEWVIRUSES 'AUGUIN IN HIS LAST GREAT PAINTINGh7HO ARE WE WHERE HAVE WE COME FROM WHERE ARE WE GOINGv ASKS CRUCIAL QUESTIONS ABOUT THE FUTURE OF HUMANKIND"UTITWASTHEMEDIEVALPAINTER"REUGELWHOASKEDTHEMAJOR QUESTION YET TO BE ANSWERED IN THE ST CENTURY (IS MEDIEVAL PAINTING h4HE 4RIUMPH OF $EATHv SHOWS A HORSEMAN ON A WHITE CHARGER SCYTHING AT RANDOM AND GATHERING SOULS DURING AN OUTBREAK OF 0ASTEURELLA PESTIS IN MEDIEVALTIMES4HEQUESTIONHAUNTINGTHEPAINTINGIShWHYDOSOMEPERSONS SURVIVEWHILEOTHERSDIEv%VENININMOSTCOMMUNITIESOFPERSONS INFECTEDWITHTHEVIRUSSURVIVED"UTWHYDIDSOMEDIEANDEXACTLYHOWWERE THEY KILLED BY SUCH A MINUTE AND FRAGILE FORM OF LIFE THAT WE KNOW AS THE ORTHOMYXOVIRUSINFLUENZA7ASTHEIMMUNEREACTIONANDENSUINGCYTOKINE STORMOVERWHELMING ORWASVIRUSREPLICATIONINTHEENDOTHELIALCELLSOFTHE AIRSACSMOREIMPORTANT !NEXTRAORDINARYCLEARMESSAGEISEMERGING WHICHTELLSUSTOBUILDOUR PUBLICHEALTHINFRASTRUCTUREANDCONTINUEANDEXPANDOUREPIDEMIOLOGICAL VIGILANCEANDSURVEILLANCEAGAINSTALLTHESEINFECTIOUSVIRUSES ANDBACTERIA &ORPANDEMICINFLUENZA EVERYCOUNTRYNEEDSADETAILEDANDPRACTICALPLAN ANDASUPPLYOFANTIVIRALDRUGSANDNEWVACCINESATHAND7EWOULDTHENBE hATTHEENDOFTHEBEGINNINGvASREGARDSPROTECTIONOFALLCITIZENS)NFLUENZA WASTHETHCENTURYSWEAPONOFMASSDESTRUCTION.ATUREISTHEGREATEST BIOTERRORISTOFOURWORLDANDEMERGINGVIRUSESCOULDDOFORUS ALL ASEASILY ANDASQUICKLY OREVENMORESO THANTHE'REAT)NFLUENZAOF EXCEPTFOR THEFACTTHATWENOWHAVETHEAMMUNITIONTOFIGHTBACKKNOWLEDGEOFVIRUS TRANSMISSIONANDEFFECTIVEANTIVIRALSANDVACCINES

!CKNOWLEDGMENTS 7EAREPLEASEDTORECEIVEGRANTINCOMEFROMTHE%5TODEVELOPNEWINFLU ENZAVACCINES



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)DENTIFICATIONOFTHEHAEMAGGLUTININANDNEURAMINIDASEGENES 0ROC.ATL!CAD 3CI53!n (OFFMAN% .EUMANN' +AWAOKA9 (OBORN' 7EBSTER2' ! $.! TRANSFECTION SYSTEM FOR GENERATION OF INFLUENZA! VIRUS FROM EIGHT PLASMIDS 0ROC.ATL!CAD3CI53!n 3CHICKLI *( &LANDORFER ! .AKAYA 4 -ARTINEZ 3OBRIDO , 'ARCIA 3ASTRE ! 0ALESE 0  0LASMID ONLY RESCUE OF INFLUENZA! VIRUS VACCINE CANDIDATES 0HILOS4RANS23OC,ONDON"n +ISTNER / "ARRETT 0. -UNDT 7 2EITER - 3CHOBER "ENDIXEN 3 $ORNER &  $EVELOPMENTOFAMAMMALIANCELL6ERO DERIVEDCANDIDATEINFLUENZA VIRUSVACCINE6ACCINEn 0ALACHE!- "RANDS2 VAN3CHARRENBURG' )MMUNOGENICITYANDREAC TOGENICITYOFINFLUENZASUBUNITVACCINESPRODUCEDIN-$#+CELLS ORFERTILISED CHICKENEGGS*)NFECT$IS3UPPL 3n3 &RANCIS4*R .AGILL40 )MMUNOLOGICALSTUDIESWITHTHEVIRUSOFINFLUENZA *%XP-ED  !NDREWES #( 3MITH7  )NFLUENZA &URTHER EXPERIMENTS ON THE ACTIVE IMMUNISATIONOFMICE"R*%XP0ATHOLL  #OMMISSIONON)NFLUENZA "OARDOF)NFLUENZAANDOTHEREPIDEMICDISEASESIN THE!RMY  ! CLINICAL EVALUATION OF VACCINATION AGAINST INFLUENZA *!M -ED!SSOC  $AVENPORT&- (ENNESSY!6 "RANDON&- 7EBSTER2' "ARRETT#$*R ,EASE '/ #OMPARISONSOFSEROLOGICALANDFEBRILERESPONSESINHUMANSTOVAC CINATION WITH INFLUENZA VIRUSES OR THEIR HAEMAGGLUTININS * ,AB #LIN -ED  n "RANDON &" #OX & ,EASE '/ 4IMM %! 1UINN % -C,EAN )7 *R  2ESPIRATORYVIRUSVACCINES)))3OMEBIOLOGICALPROPERTIESOFSEPHADEX PURIFIED ETHER EXTRACTEDINFLUENZAVIRUSANTIGENS*)MMUNOLL n $UXBURY!% (AMPSON!7 3IEVERS*'- !NTIBODYRESPONSEINHUMANS TODEOXYCHOLATE TREATEDINFLUENZAVIRUSVACCINES*)MMUNOLL n &RANCIS4*R 3ALK*% 1UILLIGAN***R %XPERIENCEWITHVACCINATIONAGAINST INFLUENZAINTHE3PRINGOF!M*0UBLIC(EALTH n ,OOSLI #' 3CHOENBERGER * "ARNETT '  2ESULTS OF VACCINATION AGAINST INFLUENZADURINGTHEEPIDEMICOF*,AB#LIN-ED  +ILBOURNE %$  &UTURE INFLUENZA VACCINES AND USE OF GENETIC RECOMBI NANTS"ULL7ORLD(EALTH/RGAN n 2EIMER #" "AKER 23 VAN &RANK 2- .EWLIN4% #LINE '" !NDERSON .'  0URIFICATION OF LARGE QUANTITIES OF INFLUENZA VIRUS BY DENSITY GRADIENT CENTRIFUGATION*6IROLL n 'LEZEN 70 ,ODA &! $ENNY &7  ! FIELD EVALUATION OF INACTIVATED ZONAL CENTRIFUGEDINFLUENZAVACCINESINCHILDRENIN#HAPEL(ILL .ORTH#AROLINA n"ULL7ORLD(EALTH/RGAN n 3YMPOSIUMOFINFLUENZA!(.  *)NFECT$IS 3UPPL L  3ALK *%  2EACTIONS TO CONCENTRATED INFLUENZA VACCINES * )MMUNOL  0OTTER#7 *ENNINGS2 #LARK! 4HEANTIBODYRESPONSEANDIMMUNITYTO







 









    





*OHN/XFORDETAL

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 /XFORD*3 3CHILD'# 0OTTER# *ENNINGS2 4HESPECIFICITYOFTHEANTI F HAEMAGLUTTININANTIBODYRESPONSEINDUCEDINMANBYINACTIVATED VACCINESAND BYNATURALINFECTION *(YG#AMB n  /XFORD*3 (AAHEIM,2 3LEPUSHKIN! 7ERNER* +UWERT% 3CHILD'# 3TRAIN SPECIFICITY OF SERUM ANTIBODY TO THE HAEMAGGLUTININ OF INFLUENZA ! (. VIRUSESINCHILDRENFOLLOWINGIMMUNISATIONORNATURALINFECTION *(YG #AMB n  !PPLEBY*# (IMMELWEIT& 3TUART (ARRIS#( )NFLUENZA VIRUS!VACCINES #OMPARISONOFINTRADERMALANDSUBCUTANEOUSROUTES,ANCETn T  -C#ARROLL*2 +ILBOURNE%$ )MMUNISATIONWITH!SIANSTRAININFLUENZA VACCINE n %QUIVALENCE OF THE SUBCUTANEOUS AND INTRADERMAL ROUTES . %NGL * -EDn  4AURASO .- 'LECKMAN 2 0EDREIRA &! 3ABBAJ * 9AHWAK 2 -ADOFF -!  %FFECT OF DOSAGE AND ROUTE OF INOCULATION UPON ANTIGENICITY OF INACTI VATED INFLUENZA VIRUS VACCINE (ONG +ONG STRAIN IN MAN "ULL7ORLD (EALTH /RGANn  7ALDMAN2( #ASE*! &ULK26 4OGO9 (ORNICK2" (EINER'' $AWKIN*UN !4 -ANN** )NFLUENZAANTIBODYINHUMANRESPIRATORYSECRETIONSAFTER SUBCUTANEOUS OR RESPIRATORY IMMUNISATION WITH INACTIVATED VIRUS .ATURE  n  7ALDMAN2( 7IGLEY&- 3MALL0!*R 3PECIFICITYOFRESPIRATORYSECRE TIONANTIBODYAGAINSTINFLUENZAVIRUS*)MMUNOLn L  0HILLIPS#! &ORSYTHE"2 #HRISTMAS7! 'UMP$7 7HORTON%" 2OGERS) 2UDIN! 0URIFIEDINFLUENZAVACCINECLINICALANDSEROLOGICALRESPONSETO VARYINGDOSESANDDIFFERENTROUTESOFIMMUNISATION*)NFECT$IS n  0OTTER #7 3TUART (ARRIS #( -C#LAREN #  !NTIBODY IN RESPIRATORY SECRETIONSFOLLOWINGIMMUNISATIONWITHINFLUENZAVIRUSVACCINES)N &40ERKINS 2(32EGAMEYEDS )NTERNATIONAL3YMPOSIUM3ERIES)MMUNOLOGICALSTANDAR R DISATION +ARGER "ASEL   2UBEN&, 0OTTER#7 3TUART (ARRIS#( (UMORALANDSECRETORYANTI BODYRESPONSESTOIMMUNISATIONWITHLOWANDHIGHDOSAGESPLITINFLUENZAVIRUS VACCINES!RCH6IROLL n  $OWNIE *# 3TUART (ARRIS #(  4HE PRODUCTION OF NEUTRALISING ACTIVITY INSERUMANDNASALSECRETIONSFOLLOWINGIMMUNISATIONWITHINFLUENZA"VIRUS* (YG#AMB n  %NNIS &! $OWDLE 72 "ARRY $7 (OCHSTEIN ($ 7RIGHT 0& +ARZON $4 -ARINE7- -EYER(-*R %NDOTOXINCONTENTANDCLINICALREACTIVITYTO INFLUENZAVACCINES*"IOL3TAND n  7ELLS #%#  ! NEUROLOGICAL NOTE ON VACCINATIONS AGAINST INFLUENZA "R -ED** n  ,ANGMUIR !$  'UILLAIN "ARRÏ SYNDROME 4HE SWINE INFLUENZA VIRUS VACCINE INCIDENT IN THE 5NITED 3TATES OF !MERICA n *2 3OC -ED  n  (URWITZ%3 3CHONBERGER," .ELSON$" (OLMAN2# 'UILLAIN "ARRÏ SYNDROMEANDTHEnINFLUENZAVACCINE.%NGL*-EDn  -OGABGAB7* ,IEDERMAN% )MMUNOGENICITYOFPOLYVENTAND (ONG+ONGINFLUENZAVACCINES *!M-ED!SSOCn



*OHN/XFORDETAL

 +NIGHT6 #OUCH2" $OUGLAS2' 4AURASO.- 3EROLOGICALRESPONSES AND RESULTS OF NATURAL INFECTIOUS CHALLENGE OF RECIPIENTS OF ZONAL ULTRACENTRI FUGEDINFLUENZA!!)#()VACCINE "ULL7ORLD(EALTH/RGANn  -AWSON* 3WAN# )NTRANASALVACCINATIONOFHUMANSWITHLIVINGATTENU ATEDINFLUENZAVIRUSSTRAINS -ED*!UST T  3TUART (ARRIS#( 0RESENTSTATUSOFLIVEINFLUENZAVIRUSVACCINE *)NFECT $IS  +ENDAL!0 -AASAB(& !LEXANDROVA') 'HENDON9: $EVELOPMENT OFCOLD ADAPTEDRECOMBINANTLIVEATTENUATEDINFLUENZA!VACCINESINTHE53! AND5332 !NTIVIRAL2ES  "EARE !3 "YNOE -, 4YRRELL $!*  )NVESTIGATION INTO ATTENUATION OF INFLUENZAVIRUSESBYSERIALPASSAGE "R-ED** n  (UYGELEN# 0ETERMANS* 6ASCOBOINIC% "ERGE% #OLINET' ,IVEATTEN UATED INFLUENZA VIRUS VACCINE IN VITRO AND IN VIVO PROPERTIES )N &4 0ERKINS 2(32EGAMEYEDS )NTERNATIONAL3YMPOSIUMON)NFLUENZA6ACCINESFOR-AN AND(ORSES3ERIES)MMUNOBIOLOGICAL3TANDARDS VOL+ARGER "ASEL   &LORENT' ,OBMANN- "EARE!3 :YGRAICH. 2.!SOFINFLUENZAVIRUS RECOMBINANTSDERIVEDFROMPARENTSOFKNOWNVIRULENCEFORMAN!RCH6IROL L n  &LORENT' 'ENECONSTELLATIONOFLIVEINFLUENZA!VACCINES !RCH6IROLL  n  "EARE!3 (ALL43 2ECOMBINANTINFLUENZA!VIRUSESAS LIVEVACCINEFOR MAN,ANCETT ))n  "EARE!3 2EED3 4HESTUDYOFANTIVIRALCOMPOUNDSIN VOLUNTEERS)N*3 /XFORD ED  #HEMOPROPHYLAXIS AND6IRAL )NFECTIONS OF THE 2ESPIRATORY4RACT VOL#2#0RESS #LEVELAND   /XFORD*3 -C'EOCH$* 3CHILD'# "EARE!3 !NALYSISOFVIRION2.! SEGMENTSANDPOLYPEPTIDESOFINFLUENZA!VIRUSRECOMBINANTSOFDEFINEDVIRU LENCE.ATURE n  0OLIOMYELITIS#ONGRESSESn 0APERSANDDISCUSSIONSATST ND RD TH ANDTH)NTERNATIONAL0OLIOMYELITIS#ONGRESSES   AND ,IPPINCOTT 0HILADELPHIA  *ENNINGS2 0OTTER#7 4EH#: -AHMUD-) 4HEREPLICATIONOF4YPE! INFLUENZAVIRUSESINTHEINFANTRAT!MARKERFORVIRUSATTENUATION*'EN6IROL n  -URPHY"2 #LEMENTS-, -AASAB(& "UCKLER 7HITE!* 4IAN3 & ,ONDON 74 #HANOCK 2-  4HE BASIS OF ATTENUATION OF VIRULENCE OF INFLUENZA VIRUSFORMAN)N#(3TUART (ARRIS #70OTTEREDS -OLECULAR6IROLOGYAND %PIDEMIOLOGYOF)NFLUENZA!CADEMIC0RESS ,ONDON   #HANOCK2- -URPHY"2 'ENETICAPPROACHESTOCONTROLOFINFLUENZA 0ERSPECT"IOL-ED 3  2ICHMAN $$ -URPHY "2 #HANOCK 2- 'WALTNEY *- *R $OUGLAS 2' "ETTS2& "LACKLOW.2 2OSE&" 0ARRINO4! ,EVINE-- #APLAN%3 4EMPERATURE SENSITIVE MUTANTS OF INFLUENZA ! VIRUS 8)) 3AFETY ANTIGENIC ITY TRANSMISSIBILITY AND EFFICACY OF INFLUENZA!5DORN TS ;%= RECOMBINANT VIRUSESINHUMANADULTS *)NFECT$ISn

)NFLUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY



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*OHN/XFORDETAL

 !RTIFICIALLY INDUCED !SIAN INFLUENZA IN VACCINATED AND UNVACCINATED VOLUNTEERS *!M-ED!SSOCn #OUCH 2"  !SSESSMENT OF IMMUNITY TO INFLUENZA VIRUS USING ARTIFICIAL CHALLENGE OF NORMAL VOLUNTEERS WITH INFLUENZA VIRUS $EV "IOL 3TAND  n  'REENBERG 3" #OUCH 2" +ASEL *!  $URATION OF IMMUNITY TO TYPE! INFLUENZA #LIN2ES 3CHULMAN*, +HAKPOUR- +ILBOURNE%$ 0ROTECTIVEEFFECTSOFSPECIFIC IMMUNITYTOVIRALNEURAMINIDASEONINFLUENZAVIRUSINFECTIONOFF MICE *6IROLL  n "EUTNER +2 #HOW 4 2UBI 5 3TRUSSENBERG * #LEMENT * /GRA 0,  %VALUATION OF A NEURAMINIDASE SPECIFIC INFLUENZA! VIRUS VACCINE IN CHILDREN !NTIBODYRESPONSESANDEFFECTSONTWOSUCCESSIVEOUTBREAKSOFNATURALINFEC TION *)NFECT$ISn 3LEPUSHKIN!. 3CHILD'# "EARE!3 #HINN3 4YRRELL$!* .EURAMINIDASE ANDRESISTANCETOVACCINATIONWITHLIVEINFLUENZA!(ONG+ONG VACCINE *(YG #AMB n -ONTO!3 +ENDAL!0 %FFECTOFNEURAMINIDASEANTIBODYON(ONG+ONG INFLUENZA,ANCET)n T ,ARSON(% 4YRRELL$!* "OWKER#( 0OTTER#7 3CHILD'# )MMUNITY TO CHALLENGE IN VOLUNTEERS VACCINATED WITH AN INACTIVATED CURRENT OR EARLIER STRAINOFINFLUENZA!(.  *(YG#AMB n &AZEKAS DE 3T 'ROTH 3 (ANNOUN #  3ÏLECTION PAR PRESSION IMMU NOLOGIQUE DE MUTANTS DOMINANTS DU VIRUS DE LA GRIPPE! (ONG +ONG  # 2 !CAD3CIDE0ARIS$ 4YRRELL $ &IELDER -  #OLD7ARS4HE &IGHT!GAINST THE #OMMON #OLD /XFORD5NIVERSITY0RESS /XFORD &RIES , ,AMBKIN 7ILLIAMS 2 'ELDER # 7HITE ' "URT $ ,OWELL ' /XFORD *  &LU)NSURE4- AN INACTIVATED TRIVALENT INFLUENZA VACCINE FOR INTRANA SAL ADMINISTRATION IS PROTECTIVE IN HUMAN CHALLENGE WITH !0ANAMA (. VIRUS )N 9 +AWAOKA ED  /PTIONS FOR THE #ONTROL OF )NFLUENZA 6 )NTERNATIONAL#ONGRESS3ERIES%LSEVIER ,ONDON  n 4REANOR** (AYDEN&' 6OLUNTEERCHALLENGESTUDIES)N+'.ICHOLSON 2'7EBSTER !*(AYEDS  4EXTBOOKOF)NFLUENZA"LACKWELL3CIENCE /XFORD *ONES3 %VANS+ -C%LWAINE *OHN( 3HARPE- /XFORD* ,AMBKIN 7ILLIAMS 2 -ANT4 .OLAN! :AMBON- $.!VACCINATIONPROTECTSAGAINSTAN INFLUENZACHALLENGEINAPHASEBDOUBLEBLINDRANDOMISEDPLACEBOCONTROLLED CLINICALTRIALSUBMITTED

)NFLUENZA6ACCINESFORTHE&UTURE EDBY22APPUOLIAND'$EL'IUDICE ¥"IRKHËUSER6ERLAG"ASEL3WITZERLAND



4HEEPIDEMIOLOGYOFINFLUENZAANDITSCONTROL ,ONE3IMONSEN #ÏCILE6IBOUD 2OBERT*4AYLORAND-ARK!-ILLER 'EORGE7ASHINGTON5NIVERSITY3CHOOLOF0UBLIC(EALTHAND(EALTH3ERVICES 7ASHINGTON $#

53!&OGARTY)NTERNATIONAL#ENTER .ATIONAL)NSTITUTESOF(EALTH "ETHESDA -ARYLAND 53! 3!'%!NALYTICA ,,# "ETHESDA -ARYLAND 53!

!BSTRACT )NTHISCHAPTERWEHIGHLIGHTHOWRECENTADVANCESININFLUENZAEPIDEMIOLOGYCANINFORM EXISTING STRATEGIES FOR DISEASE CONTROL!S A FIELD INFLUENZA EPIDEMIOLOGY HAS BENEFITED GREATLY FROM ANALYSIS OF LARGE DATA SETS REGARDING HOSPITALIZATION MORTALITY AND OUTPA TIENT VISITS4HESE DATA HAVE ALLOWED COMPARISON OF THE IMPACT OF INFLUENZA IN VARIOUS CLIMATESANDTHEEVALUATIONOFTHEDIRECTANDINDIRECTBENEFITSOFVACCINATION THELATTER THROUGHTHEVACCINATIONOFhTRANSMITTERPOPULATIONSvSUCHASSCHOOLCHILDREN TOACHIEVE HERDIMMUNITY-OREOVER THERESOLUTIONOFINFLUENZAEPIDEMIOLOGYHASUNDERGONEALEAP TO THE MOLECULAR LEVEL DUE TO THE INTEGRATION OF NEW ANTIGENIC AND VIRAL GENOMIC DATA WITHCLASSICALEPIDEMIOLOGICALINDICATORS&INALLY THENEWDATAHAVELEDTOANINFUSIONOF QUANTITATIVE STUDIES FROM THE FIELDS OF EVOLUTIONARY AND MOLECULAR BIOLOGY POPULATION GENETICSANDMATHEMATICS 4HEPROGRESSCANBESEENINMANYFORMS4HEEMERGINGFIELDOFMOLECULARINFLUENZA EPIDEMIOLOGY IS PROVIDING DEEPER INSIGHT INTO GLOBAL PATTERNS OF VIRAL EMERGENCE THE IMPORTANTROLEOFREASSORTMENTINGENERATINGGENETICNOVELTY ANDGLOBALDIFFUSIONOFVIRUS VARIANTSnINCLUDINGTHEMYSTERIOUSBUTCRUCIALROLE OF THE4ROPICS ESPECIALLY 3OUTHEAST !SIA AS A SOURCE OF NEW VARIANTS $EEPER STRATIFICATION OF CONTEMPORARY AND HISTORIC EPIDEMIOLOGICALDATAISPROVIDINGAMOREDETAILEDPICTUREOFTHEEFFECTOFAGEANDOTHER HOST CHARACTERISTICS ON OUTCOMES AS WELL AS BETTER ESTIMATES OF THE TRANSMISSIBILITY OF PANDEMIC AND SEASONAL INFLUENZA VIRUSES 2E EXAMINATION OF OBSERVATIONAL STUDIES OF VACCINEEFFECTIVENESSINSENIORSISLEADINGTORECONSIDERATIONOFSEASONALANDPANDEMIC VACCINEPRIORITIES WHILEMATHEMATICALMODELERSHAVEDEVELOPEDTOOLSTOEXPLOREOPTIMAL STRATEGIES FOR MITIGATING A FUTURE PANDEMIC 4HE FIELD OF INFLUENZA EPIDEMIOLOGY HAS RAPIDLYPROGRESSEDINTHEPASTDECADEANDBECOMETRULYMULTIDISCIPLINARY0ROGRESSCOULD BESUSTAINEDINTHENEXTDECADEBYEVENCLOSERTIESWITHVIROLOGY EVOLUTIONARYBIOLOGY IMMUNOLOGY ANDGENETICS

)NTRODUCTION )NFLUENZA VIRUSES EVOLVE CONTINUOUSLY CHALLENGING MAMMALIAN AND AVIAN HOSTSWITHNEWVARIANTSANDCAUSINGCOMPLEXEPIDEMICPATTERNSWITHREGARD



,ONE3IMONSENETAL

TO AGE PLACE AND TIME (UMAN INFLUENZA VIRUSES CAUSE DISEASE THROUGH A VARIETYOFDIRECTANDINDIRECTPATHOLOGICALEFFECTS4HEDIRECTEFFECTSINCLUDE DESTRUCTIONOFINFECTEDCELLS DAMAGETORESPIRATORYEPITHELIUM ANDIMMU NOLOGICAL RESPONSES THAT CAUSE GENERAL MALAISE AND PNEUMONIA )NDIRECT CONSEQUENCESOFINFECTIONINCLUDESECONDARYBACTERIALINFECTIONPATHOGENS AS A RESULT OF TISSUE DAMAGE AND EXACERBATION OF UNDERLYING CO MORBID CONDITIONSSUCHASCARDIOVASCULARDISEASE RENALDISEASE DIABETESORCHRONIC PULMONARYDISEASE; =-ORBIDITYANDMORTALITYASSOCIATEDWITHINFLUENZA ISFREQUENTLYCITEDASTHATWHICHFALLSWITHINBROADDISEASECATEGORIES SUCH AS PNEUMONIA AND INFLUENZA 0) RESPIRATORY ILLNESS OR ALL CAUSE !# MORTALITY)NTHELATTERCASE INFLUENZAINFECTIONISUSUALLYNOTLABORATORYCON FIRMEDBUTITSHEALTHIMPACTCANBEDETERMINEDTHROUGHSTATISTICALINFERENCE BASED ON SEASONAL COINCIDENCE OF VIRUS CIRCULATION AND DISEASE OUTCOMES ;n= 'IVENTHEDIFFICULTYOFDIRECTLYMEASURINGINFLUENZAMORBIDITY ANDMOR TALITY TIME SERIESMODELSHAVEBEENDEVELOPEDTOELUCIDATEPATTERNSOFDIS EASEWITHINVARIOUSAGEGROUPSANDPOPULATIONS;n=3UCHMODELSALLOW FOR QUANTIFICATION OF DISEASE BURDEN BY SEASON AND SEVERITY OF CIRCULATING STRAINS;=(ISTORICALARCHIVEDDATAHAVEALSOELUCIDATEDTHE LINKSBETWEEN INFLUENZA TRANSMISSION ACROSS GEOGRAPHIC REGIONS AND POPULATION MOVE MENTS ;= AND ALLOWED COMPARISON OF THE IMPACT AND TRANSMISSIBILITY OF PASTPANDEMICSANDEPIDEMICSINMULTIPLECOUNTRIES;n=3IMILARMODELS APPLIEDTOPROSPECTIVESYNDROMICSURVEILLANCEDATAHAVEALLOWEDTHESTUDY OF THE EPIDEMIOLOGICAL SIGNATURE OF RECURRING AND REEMERGING STRAINS OF INFLUENZAONPOPULATIONS;=-ATHEMATICALMODELINGANDSTATISTICALANALY SESOFINFLUENZAACTIVITYINTROPICALCOUNTRIESHAVEREKINDLEDINTERESTINTHE OLD MYSTERY OF THE SEASONAL DRIVERS OF INFLUENZA AND OFFERED NEW INSIGHTS INTOTHECIRCULATIONPATTERNSOFTHISVIRUSATTHEGLOBALANDREGIONALSCALES ;n=&IG  4HEFIELDOFINFLUENZAEPIDEMIOLOGYHASRECENTLYUNDERGONEAQUANTUM LEAP IN RESOLUTION DUE TO THE INCREASED AVAILABILITY OF ANTIGENIC AND VIRAL GENOMICDATAANDTHEINTEGRATIONOFTHESEDATAWITHCLASSICALEPIDEMIOLOGI CALINDICATORS;n=4HEEMERGINGFIELDOFMOLECULARINFLUENZAEPIDEMI OLOGYALSOKNOWNAShPHYLODYNAMICSv;= HASALREADYPROVIDEDAMUCH CLEARER PICTURE OF THE COMPLEX DYNAMICS OF GLOBAL INFLUENZA VIRUS CIRCULA TIONANDREASSORTMENTPATTERNS4HEGROWINGNUMBEROFAVAILABLE INFLUENZA GENOMESEQUENCESFROMSPECIMENSCOLLECTEDAROUNDTHEWORLDHASSTARTED TOCREATEAMORECOHERENTPICTUREOFTHEGLOBALEPIDEMIOLOGYOFF INFLUENZA IN PARTICULAR THE INTERPLAY BETWEEN VIRUS EVOLUTION POPULATION IMMUNITY ANDIMPACT 4HROUGHOUT THIS CHAPTER OUR INTENTION IS TO HIGHLIGHT HOW INFLUENZA EPIDEMIOLOGYCANHELPREFINEEXISTINGSTRATEGIESFORINFLUENZACONTROL ESPE CIALLYVACCINATION7EBEGINBYEXAMININGTHESPATIALANDTEMPORALSPREAD OFSEASONALINFLUENZA ANDWEDISCUSSHOWOLDANDNEWANALYTICALTOOLSARE RESHAPINGQUANTITATIVETHINKINGININFLUENZAEPIDEMIOLOGYANDCONTROL7E

4HEEPIDEMIOLOGYOFINFLUENZAANDITSCONTROL



&IGURE#OMPARISONOFINFLUENZAVIRUSSEASONALPATTERNSINTEMPERATEANDTROPICALCOUNTRIES INTHE!MERICAS 0IECHARTSREPRESENTTHEPERCENTDISTRIBUTIONOFINFLUENZAVIRUSISOLATIONBYMONTHASCOMPILED FROM7(/DATABETWEENANDCOLORBAR .OTETHETRANSITIONINSEASONALPATTERNS FROM.ORTHTO3OUTH4HELATITUDEOFTHECAPITALCITYISINDICATEDFOREACHCOUNTRYINTHELEGEND !DAPTEDFROM6IBOUDETAL;=

THENMOVEONTOSEVERALASPECTSOFHISTORICALPATTERNSOFDISEASEOBSERVED DURINGTHETHREEPANDEMICSOFTHETHCENTURY ANDTOUCHUPONTHEEPIDE MIOLOGYOFTHERECENTAVIAN!(.INFLUENZATHREAT7ECONCLUDEWITHAN IN DEPTHREVIEWOFWHATISKNOWNABOUTVACCINEBENEFITSINSENIORSnTHE GROUP THAT BEARS THE GREATEST INFLUENZA RELATED MORTALITY BURDEN n AND A DISCUSSION OF THE IMPLICATIONS OF INFLUENZA EPIDEMIOLOGY FOR PANDEMIC PLANNING 2EADERS LOOKING FOR A MORE COMPREHENSIVE TREATMENT OFF THE VAST FIELD OF INFLUENZA EPIDEMIOLOGY SHOULD CONSIDER SUPPLEMENTING THIS CHAPTERWITHSOMEOFTHECLASSICALREVIEWSPUBLISHEDOVERTHELASTDECADES ; n=



,ONE3IMONSENETAL

3EASONALINFLUENZA.EWINSIGHTS 4HE HEALTH IMPACT OF ANNUAL INFLUENZA EPIDEMICS VARIES GREATLY IN TERMS OF HOSPITALIZATIONS AND DEATHS )N THE 5NITED 3TATES CLINICAL ILLNESS AFFECTS n OF THE POPULATION AND ASYMPTOMATICALLY INFECTS A LARGER NUMBER ;=)NFANTS WHOAREEXPOSEDTOINFLUENZAEPIDEMICSASANOVELANTIGENIC CHALLENGEAFTERMATERNALANTIBODIESDECLINE MAYHAVEATTACKRATESASHIGH ASnINTHEIRFIRSTYEAROFLIFE DEPENDINGONTHEFREQUENCYOFCONTACTS WITHOLDERSIBLINGS;=&ORREASONSNOTFULLYUNDERSTOOD INFLUENZAVIRUSES F CAUSESEASONALEPIDEMICSINTHE.ORTHERNAND3OUTHERN(EMISPHEREDURING THEIRRESPECTIVEWINTERS)NTHETROPICSTHETIMINGOFACTIVITYISLESSDEFINED WITHSOMETIMESYEAR ROUNDCIRCULATIONORBI SEASONALPEAKSDURINGTHEYEAR &IG ;  n=

-ETHODSUSEDTOESTIMATETHEMORTALITYBURDENOFINFLUENZA %STIMATESOFTHENUMBEROFINFLUENZA RELATEDDEATHSARETYPICALLYINFERRED THROUGHSTATISTICALANALYSIS4HESYNDROMICDIAGNOSIShINFLUENZA LIKEILLNESSv ISRARELYLABORATORYCONFIRMEDANDISOFTENCAUSEDBYNON INFLUENZARESPIRA TORYVIRUSES-OREOVER INFLUENZAISOFTENAPRECIPITATINGFACTORTHATBRINGS ABOUTDEATHFROMSECONDARYBACTERIALPNEUMONIAORANUNDERLYING CHRONIC DISORDER)NTHESECASES THEUNDERLYINGDISORDERSARETYPICALLYIDENTIFIEDAS THECAUSEOFDEATH"ECAUSEOFTHESEASCERTAINMENTPROBLEMS DETERMINING THE MAGNITUDE OF INFLUENZA RELATED DEATHS REQUIRES INDIRECT APPROACHES IN WHICHMATHEMATICALORSTATISTICALMODELSAREAPPLIEDTOBROADDEATHCATEGO RIES4HISAPPROACHWASFIRSTUSEDINBY7ILLIAM&ARRTOCHARACTERIZEAN INFLUENZAEPIDEMICIN,ONDONANDWASFURTHERDEVELOPEDANDEXTENSIVELY USED THROUGHOUT THE TH CENTURY 4HE REFINEMENTS INCLUDE 3ERFLING LIKE CYCLICAL REGRESSION MODELS ;    n= AND !RIMA MODELS ;   = WHICHAREAPPLIEDTOMONTHLYORWEEKLYTIMESERIESOF0)OR!# MORTALITY /VERALL INVESTIGATORS FROM AT LEAST  COUNTRIES HAVE USED VARI ANTS OF THESE 3ERFLING TYPE MODELS TO ESTIMATE THE MORTALITY BURDEN OF INFLUENZA3IMILARISSUESANDSTATISTICALAPPROACHESAPPLYTOTHEESTIMATION OFTHEINFLUENZABURDENONHOSPITALIZATION;  =4HEVARIOUSSTATISTICAL APPROACHESALLATTRIBUTEhEXCESSDEATHSvINWINTERMONTHSTOINFLUENZA3UCH SEASONALAPPROACHESARENOTSUITEDTOSTUDYINGDISEASEBURDENOFINFLUENZA INCOUNTRIESWITHTROPICALCLIMATESBECAUSETHEYREQUIREANANNUALSEASONAL PATTERNOFVIRALACTIVITYINTERRUPTEDBYINFLUENZA FREEPERIODS -ORE RECENTLY THE 53 #ENTERS FOR $ISEASE #ONTROL AND 0REVENTION #$# HAS USED AN APPROACH TO MEASURE HOSPITALIZATION AND MORTALITY BURDENBASEDONANEWGENERATIONOFSEASONALREGRESSIONMODELSINTEGRAT INGLABORATORYSURVEILLANCEDATAONINFLUENZAANDRESPIRATORYSYNCYTIALVIRUS 236 ; =)NSUCHMODELS WINTERSEASONALINCREASESINDEATHSORHOS PITALIZATIONSAREDIRECTLYPROPORTIONALTOTHEMAGNITUDEOFRESPIRATORYVIRUS

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&IGURE#OMPARISONOFAGEMORTALITYPATTERNSDURINGTHE PANDEMICANDASEVEREINTER PANDEMICSEASON .EW 9ORK#ITY ! )NFLUENZA SEASON ATTRIBUTABLEEXCESSDEATHSAREPLOTTEDFOR THEnINTER PANDEMIC SEASONS THEPANDEMICHERALDWAVEEPIDEMICMONTHS-ARCHAND!PRIL' ANDTHE MAINFALLPANDEMICWAVE3EPTEMBERTO!PRIL . " 2ELATIVERISKOFDEATHISPLOT TEDBYAGEGROUPONALOG SCALEFORTHEHERALDANDFALLPANDEMICWAVES RELATIVETOTHESEVERE INTER PANDEMICSEASON!DAPTEDFROM/LSONETAL;=

HYPOTHESISWOULDSUGGESTSENIORSCOULDBEATGREATRISK ASSUGGESTEDBYONE AUTHOR;=)NCONTRAST WERETHEIMMUNEPATHOLOGYHYPOTHESISCORRECT THE RELATIVE IMMUNE SENESCENCE IN SENIORS MAY MITIGATE THE FULL IMPACT OF A PANDEMICVIRUSANDTHEYOUNGADULTSCOULDBEATHIGHESTRISKOFF DYING #OMPARATIVE STUDIES OF PANDEMIC INFLUENZA IN MULTIPLE COUNTRIES HAVE REVEALEDMANYINTERESTINGINSIGHTS&OREXAMPLE ARECENTSTUDYUSEDANNUAL MORTALITYDATAFROMMULTIPLECOUNTRIESTOESTIMATETHEMORTALITYBURDENOF THEnINFLUENZAPANDEMICANDUNCOVEREDSUBSTANTIALGEOGRAPHICAL

4HEEPIDEMIOLOGYOFINFLUENZAANDITSCONTROL



DIFFERENCESININFLUENZA RELATEDMORTALITYRATES4HEPERCENTAGEOFTHEPOPU LATION THAT DIED VARIED FROM  IN 3CANDINAVIA TO  IN SOME AREAS OF )NDIA REPRESENTINGA FOLDDIFFERENCEINMORTALITYRISKINTHESESETTINGS;= 4HE UNDERLYING REASONS FOR THIS SUBSTANTIAL VARIABILITY ARE NOT WELL UNDER STOOD BUTMIGHTBEREVEALEDBYADDITIONALHISTORICALPANDEMIC STUDIES )NASECONDEXAMPLE ANALYSISOFEXCESSMORTALITYDATAFROMSEVERALCOUN TRIESPUTASURPRISINGSPINONTHEPANDEMIC;=!NUNEXPECTEDPATTERN OFAhSMOLDERINGvMORTALITYIMPACTIN%UROPEANANDSOME!SIANCOUNTRIES WASREVEALEDnARELATIVELYMILDFIRSTWAVEOFTHEEMERGINGVIRUSINTHEn SEASON FOLLOWEDBYAVERYSEVEREnSEASON4HISISDIFFERENT FROMTHECLASSICALIMPRESSIONBASED ONTHE.ORTH!MERICANEXPERIENCETHAT MOSTOFTHEIMPACTOCCURSWITHTHEFIRSTEXPOSURETOPANDEMICSTRAINS)TMAY BEMORECOMMONTHANPREVIOUSLYTHOUGHTTHATTHEFIRSTWAVEOFAPANDEMIC VIRUSRESULTSINLOWMORTALITY ONLYTOBEFOLLOWEDBYAMOREDRAMATICIMPACT A FEW MONTHS LATER )NDEED THIS INTRIGUING PATTERN WAS NOT ONLY OBSERVED INSOMECOUNTRIESDURINGTHEMILDPANDEMIC BUTITISALSOCONSISTENT WITHTHEHERALDWAVEEXPERIENCEIN.EW9ORK#ITYAND3CANDINAVIADURING THE CATASTROPHIC  PANDEMIC ; = &URTHER HISTORICAL MORTALITY DATA FROMTHELESS STUDIEDnPANDEMICIN%NGLANDALSOSUGGESTAPATTERN OFSUCCESSIVEPANDEMICWAVESWHERETHEFIRSTENCOUNTERWASNOTTHEMOST LETHAL;=4HEREASONSFORTHIShSMOLDERINGvORHERALDWAVE PATTERNARE STILL UNKNOWN AND MAY BE PARTLY RELATED TO ON GOING ADAPTATION IN NEWLY EMERGEDPANDEMICVIRUSESANDPRE EXISTINGPOPULATIONIMMUNITY

4RANSMISSIONMODELSUSEDTOPREDICTFUTUREPANDEMICSCENARIOS ,ARGE SCALE MATHEMATICAL TRANSMISSION MODELS HAVE BEEN EMPLOYED TO SIMULATEINDETAILTHEPOSSIBLESPREADOFAFUTUREPANDEMICVIRUSINASUS CEPTIBLEHUMANPOPULATIONSEEFOREXAMPLE;   = 4HESEMODELS 4 SEEKTOPREDICTTHESPATIO TEMPORALDYNAMICOFAHYPOTHETICALFUTUREPAN F DEMICVIRUSANDEFFECTIVENESSOFPOSSIBLEINTERVENTIONSTRATEGIES INCLUDING VACCINATIONBEFOREANOUTBREAKWITHAPARTIALLYMATCHED LOW EFFICACYVAC F CINE DISTRIBUTIONOFANTIVIRALSFORPROPHYLAXISORTREATMENT SCHOOLCLOSURE CASEISOLATIONORHOUSEHOLDQUARANTINE4HESEMODELSGENERALLY AGREETHATA COMBINATIONOFMEASURES IFIMPLEMENTEDEARLYANDWITHSUFFICIENTCOMPLI ANCE MIGHTBRINGABOUTAMEANINGFULLEVELOFMITIGATIONANDSUBSTANTIALLY SLOWGEOGRAPHICSPREAD3UBSEQUENTSTUDIESFOUNDTHATEARLY TARGETED AND LAYERED USE OF NON PHARMACEUTICAL INTERVENTIONS COULD GREATLY REDUCE THE OVERALL PANDEMIC ATTACK RATE PROVIDED THE INTRINSIC TRANSMISSIBILITY BASIC REPRODUCTIVE NUMBER 2O OF THE EMERGING VIRUS IS NOT GREATER THAN  ;n=-ATHEMATICALMODELSCANBEUSEFULTOESTIMATETHELEVELSOFDRUG STOCKPILESNEEDEDTOBUYTIME HOWANDWHENTOMODIFYVACCINES WHOMTO TARGETWITHVACCINESANDDRUGS ANDWHENTOENFORCEQUARANTINE MEASURES "UTTHESIMULATIONMODELSCURRENTLYUSEDFORPANDEMICPLANNING STILLNEED



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TO BE TESTED AGAINST REAL DISEASE DATA AND FOR THIS WE MUST CONTINUE TO GATHERDATAONINFLUENZAMORBIDITY MORTALITY ANDVIRALGENETICSEQUENCES NOTJUSTFOREXCEPTIONALSCENARIOSBUTALSOFORSEASONALINFLUENZA;=

0REDICTINGTHEIMPACTOFAFUTUREPANDEMICINLIGHTOFTHE!(. THREAT #URRENTLY PANDEMICCONCERNSAREFOCUSEDONTHEHIGHLYPATHOGENICVARIANT OF!(.INFLUENZATHATEMERGEDIN(ONG+ONGINANDREMERGEDIN !(.HASNOWSPREADTOAVIANPOPULATIONSINMORETHANCOUN TRIES)TISPRESENTENDEMICALLYIN3OUTH %AST!SIA CAUSINGREGULARDIE OFFS INPOULTRYANDWILDBIRDS ANDOCCASIONALLYAFFECTSHUMANS!S OF!UGUST  THE7ORLD(EALTH/RGANIZATION7(/ HADCOUNTEDLABORATORY CONFIRMED(.CASESANDNOTEDAVERYHIGHCASEFATALITYOF^HTTP WWWWHOINTTOPICSAVIAN?INFLUENZAEN 7HILE(.CONTINUESTOBE AN IMPORTANT ECONOMIC PROBLEM IN !SIA !FRICA %UROPE AND THE -IDDLE %ASTSOFARSPARINGTHE!MERICAS THECRITICALQUESTIONFORPUBLICHEALTHIS WHETHERITWILLGAINTHEABILITYTOEFFECTIVELYTRANSMITAMONGHUMANS4HIS COULDOCCURINONEOFTWOWAYS BYGRADUALMUTATIONSOFPURELYAVIAN(. VIRUSES OR BY REASSORTMENT WITH CIRCULATING HUMAN INFLUENZA ! VIRUSES (.OR(. INHUMANS OROTHERANIMALhMIXINGVESSELSv 3EVERALCOM PREHENSION DISCUSSIONS AT THE THREAT OF AN AVIAN INFLUENZA PANDEMIC HAVE BEENPUBLISHEDSEEFOREXAMPLE; n=  4HEREARESTILLMANYUNCERTAINTIESABOUTTHEPANDEMICPOTENTIALOFTHE CIRCULATINGAVIAN(.VIRUS INCLUDINGITSPOTENTIALTOEFFECTIVELYTRANSMIT BETWEENHUMANSANDTHEEVOLUTIONARYMECHANISMSTHATMAYCONCURRENTLY AFFECTITSVIRULENCE4HECLASSICALBELIEFISTHATEXTREMELYPATHOGENICVIRUSES ARE NOT WELL ADAPTED TO THEIR HOSTS n MORIBUND PATIENTS DO NOT TRANSMIT VIRUSESASEASILYASTHOSEWHOREMAINMOBILE&URTHER THEPATHOGENESISOF NOVEL PANDEMIC VIRUSES REMAINS UNCLEAR IN PARTICULAR THE PROPORTIONS OF SEVEREDISEASECAUSEDBYIMMUNE MEDIATEDPATHOLOGICALRESPONSES SECOND ARY BACTERIAL INFECTIONS FOR WHICH TREATMENTS EXIST AND EXACERBATION OF CHRONIC ILLNESSES -ODERN MEDICINE CAN MITIGATE SOME OF THE PATHOLOGICAL MECHANISMSANDCONTROLSECONDARYBACTERIALINFECTIONSTOACERTAINEXTENT HOWEVER THEREISUNDOUBTEDLYADIFFERENTPROPORTIONOFPERSONS LIVINGWITH CHRONICCO MORBIDCONDITIONSNOWTHANWASTHECASEDURINGPREVIOUSPAN DEMICS&INALLY WEDONOTKNOWTHEDEGREEOFCROSSPROTECTIONAFFORDEDBY EARLYEXPOSURETOOTHERINFLUENZAVIRUSANTIGENS;=)FONESIMPLYAPPLIES THEMORTALITYEXPERIENCETOTODAYSPOPULATION ANYWHEREFROM F TOOFACOUNTRYSPOPULATIONCOULDDIE ANDTHEHIGHESTBURDENWOULDBE SUFFEREDBYDEVELOPINGCOUNTRIES;=

4HEEPIDEMIOLOGYOFINFLUENZAANDITSCONTROL



%PIDEMIOLOGYANDTHECONTROLOFINFLUENZA )NFLUENZAVACCINESWEREORIGINALLYDEVELOPEDFORUSEBYTHEMILITARYAND HAVEBEENSHOWNTOBEHIGHLYEFFECTIVEINPREVENTINGINFECTION INHEALTHY ADULTS ;= -OST COUNTRIES THAT USE SEASONAL INFLUENZA VACCINE HAVE ADOPTED A POLICY OF TARGETING INFLUENZA VACCINATION EFFORTS TO THOSE AT hHIGHRISKvOFSEVEREOUTCOMES INCLUDINGTHOSEAGEANDOLDER PERSONS WITH CERTAIN CHRONIC DISEASES AND THEIR CLOSE CONTACTS!LTHOUGH CURRENT POLICYCONTINUESTOEMPHASIZEVACCINATIONOFSENIORS THEhGOLDSTANDARDv EVIDENCETHATTHISSTRATEGYEFFECTIVELYREDUCESINFLUENZA RELATEDMORTALITY INTHATAGEGROUPISNOTSTRONG;=)THASRECENTLYBECOMEEVIDENTTHAT INFLUENZA RELATED MORTALITY HAS NOT DECREASED IN AT LEAST SOME COUNTRIES DESPITEMAJORGAINSINVACCINATIONCOVERAGEAMONGPEOPLEATHIGHESTRISK ;   ="ECAUSEhGOLDSTANDARDvEVIDENCEFROMRANDOMIZEDCLINICAL TRIALSISSCARCE EPIDEMIOLOGICALTOOLSANDSTUDIESCONSTITUTE THEVASTMAJOR ITYOFTHEEVIDENCEBASEFORWHETHERVACCINATIONPROGRAMSAREBENEFICIAL 0ARADOXICALLY OBSERVATIONAL STUDIES HAVE CONSISTENTLY ARGUED THAT ABOUT  OF ALL WINTER DEATHS IN SENIORS ARE PREVENTABLE WITH INFLUENZA VAC CINATION DESPITE THE RELATIVELY LOW IMMUNE RESPONSE TO VACCINE IN THIS POPULATION;=

4HESCARCEEVIDENCEFROMCLINICALTRIALS ,ANGMUIR WHO ORIGINALLY FORMULATED THE POLICY OF TARGETING SENIORS AND HIGHRISKPOPULATIONFORVACCINATION QUESTIONEDWHETHERTHEVACCINEWOULD REALLY BE EFFECTIVE IN SENIORS WHO RESPOND LESS VIGOROUSLY TO THE VACCINE THAN YOUNGER ADULTS ;= /NLY A SINGLE RANDOMIZED PLACEBO CONTROLLED CLINICAL TRIAL SET IN YOUNG HEALTHY SENIORS IS AVAILABLE )T SHOWED THAT VAC CINATION EFFECTIVELY PREVENTS INFLUENZA ILLNESS IN SENIORS AGED n YEARS BUTCOULDNOTDOCUMENTSIGNIFICANTBENEFITSINSENIORS *YEARS;=4HE AUTHORSEXPRESSEDCONCERNTHATTHEIRNONSIGNIFICANTFINDINGOFEFFICACY INSENIORSYEARSOLDINDICATEDIMMUNESENESCENCEADECLINEINIMMUNE RESPONSE WITH AGE ALTHOUGH THEY ALSO NOTED LIMITATIONS ON THE STATISTICAL POWER OF THEIR STUDY TO ADDRESS THIS QUESTION !S BOTH 4 CELL AND " CELL RESPONSESAREIMPAIREDINOLDERINDIVIDUALS ITISPLAUSIBLETHATTHEVACCINE ANTIBODYRESPONSETOTHEDRIFTINGINFLUENZAVIRUSESANDVACCINECOMPONENTS IS LESS VIGOROUS IN SENIORS ;= #ONSEQUENTLY IMMUNOLOGISTS HAVE LONG PERCEIVED A NEED FOR MORE EFFECTIVE VACCINE FORMULATIONS FOR THIS POPULA TION INCLUDINGTHENEEDFORADJUVANTSANDAMOVEBACKTOWHOLE CELLVACCINE PRODUCTS4HERECENTEMERGENCEOFNOVELAVIANSTRAINSANDDEVELOPMENTOF VACCINESAGAINSTTHEMHASREOPENEDMANYOFTHEDISCUSSIONSOFIMMUNOGE NICITYANDCORRELATESOFPROTECTION



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%VIDENCEFROMOBSERVATIONALSTUDIES )NTHENEAR ABSENCEOFRANDOMIZEDCLINICALTRIALS THESECOHORTSTUDIESHAVE LONG PROVIDED THE EVIDENCE BASE THAT SUPPORTS INFLUENZA VACCINE POLICY 0ARADOXICALLY THE CONCERNS ABOUT IMMUNE SENESCENCE AND VACCINE FAILURE HAVEEXISTEDINPARALLELWITHCOHORTSTUDIESREPORTINGEXTRAORDINARILYLARGE MORTALITY BENEFITS IN VACCINATED SENIORS ;n= )N THESE STUDIES COM PARISON OF VACCINATED AND UNVACCINATED SENIORS INDICATES THAT VACCINATION COULDPREVENTFULLYOFALLDEATHSAMONGDURINGWINTERMONTHS IMPLY ING THAT INFLUENZA CAUSES HALF OF ALL WINTER DEATHS AMONG SENIORS )NSTEAD META ANALYSESCONSOLIDATEDTHEFINDINGSANDPRODUCEDESTIMATES WITHTIGHT CONFIDENCEINTERVALS"UTONLYABOUTOFALLWINTERDEATHSCANBEATTRIB UTEDTOINFLUENZAINANAVERAGESEASONACCORDINGTOEXCESSMORTALITYSTUDIES ;  =%VENINTHE!(.PANDEMICANDINMORERECENTSEASONS SUCHASn WHENTHEVACCINEWASCOMPLETELYMISMATCHEDTOTHENEW CIRCULATING VARIANT OF !(. THE PROPORTION OF ALL DEATHS ATTRIBUTED TO INFLUENZANEVEREXCEEDEDOFALLWINTERDEATHSAMONGSENIORS;= ! FEW RESEARCHERS SUBSEQUENTLY ADDRESSED THIS PARADOX DIRECTLY AND INVESTIGATED THE POSSIBILITY THAT UNRECOGNIZED BIAS HAS LED THE MAJORITY OF COHORT STUDIES TO SYSTEMATICALLY OVERESTIMATE INFLUENZA VACCINE BENEFITS )N TWOPUBLISHEDREPORTSCLEARLYDEMONSTRATEDTHATTHESENIORCOHORT STUDY FINDINGS ARE LARGELY A RESULT OF SYSTEMATIC MIS MEASUREMENTS ; = &IRST THEY SHOWED THAT THE GREATEST MORTALITY REDUCTIONS OCCURRED IN EARLY WINTER BEFORE INFLUENZA EVER CIRCULATED AND WERE NOT SPECIFICALLY ASSOCIATED WITH THE PEAK INFLUENZA PERIOD 3ECOND THEY SHOWED THAT THE ANALYTICAL ADJUSTMENT TECHNIQUES TYPICALLY USED IN COHORT STUDIES ACTUALLY MAGNIFIED THE MIS MEASUREMENT RATHER THAN REDUCING IT4HE AUTHORS CON CLUDEDTHATTHEMAGNITUDEOFTHEUNADJUSTEDBIASDETECTEDWASSUFFICIENTTO ACCOUNTENTIRELYFORTHEOBSERVEDBENEFITOFMORTALITYREDUCTIONDURING THEENTIREWINTERPERIOD4HISPROBLEMINTHEEVIDENCEBASEWAS ALSOHIGH LIGHTEDINARECENT#OCHRANEREVIEWANDANEDITORIAL; =4HESOURCE OFBIASMAYBEASUBSETOFFRAILSENIORSWHOAREUNDERVACCINATEDINTHEFALL MONTHSFORTHATSEASONANDSUBSEQUENTLYCONTRIBUTESUBSTANTIALLYTOMORTAL ITY IN THE EARLY WINTER MONTHS ;= 3TUDIES HAVE SUBSTANTIATED THAT FRAIL ELDERLYAREINDEEDVACCINATEDLESSOFTENTHANTHEIRHEALTHYPEERS; = #ONTROLLINGFORTHESEBIASESYIELDSFARMOREMODESTESTIMATESOFMORTALITY REDUCTIONS;= )NSUMMARY THEEMERGINGPICTUREISAMIXTUREOFTHATRESIDUALSELECTION BIAS COUNTER PRODUCTIVE ADJUSTMENT EFFORTS AND LOW SPECIFICITY ENDPOINTS HASLEDTOSYSTEMATICOVERESTIMATIONINVIRTUALLYALLCOHORTSTUDIESPUBLISHED OVERTHELASTDECADES!DJUSTMENTSFORSELECTIONBIASMAYBEPOSSIBLE BUT ONLY IF HIGH SPECIFICITY ENDPOINTS ARE STUDIED "EYOND THAT A COMMONLY AGREEDSETOFSTANDARDSFOR CARRYINGOUTANDREPORTINGOBSERVATIONALSTUDIES THATINCLUDESAFRAMEWORKFORDETECTIONOFBIASWOULDBEHELPFUL!LSO PRE VIOUSLY PUBLISHED OBSERVATIONAL STUDIES COULD UNDERGO RE ANALYSIS GUIDED

4HEEPIDEMIOLOGYOFINFLUENZAANDITSCONTROL



BY SUCH EXPECTATIONS AS THAT VACCINE BENEFITS SHOULD BE HIGHEST IN PEAK INFLUENZAPERIODSANDFORWELL MATCHEDINFLUENZAVACCINES7EHAVERECENTLY PROPOSEDSUCHAFRAMEWORK;=

2EVISITINGTHEEVIDENCEBASESUPPORTINGSTRATEGIESFORPROTECTING POPULATIONSWITHVACCINE )FWEDISCOUNTTHEBIASEDCOHORTSTUDIES THEREMAININGSTUDIES SUGGESTTHE VACCINEBENEFITSAREINFACTMUCHLOWERTHANPREVIOUSLYTHOUGHTnPROBABLY LOWERTHANINSENIORSYEARSOFAGE4HISASSESSMENTISBASEDONTHE hGESTALTv OF RESULTS FROM THE RANDOMIZED PLACEBO CONTROLLED CLINICAL TRIAL DESCRIBED EARLIER ;= A NESTED CASE CONTROL STUDY USING LABORATORY CON FIRMEDENDPOINTSFROMAN236STUDY; = ANDTHEEXCESSMORTALITYSTUD IESSHOWINGLITTLEDECLINEINMORTALITYASVACCINECOVERAGEROSE; =.ONE OFTHESESTUDIESARECONCLUSIVEBUTIFTHESEFINDINGSHOLDUPINFUTURESTUDIES THENTHEREISAMPLEROOMFORIMPROVEMENTOFINFLUENZAVACCINES INCLUDING BETTERVACCINEFORMULATIONS ADJUVANTS ORHIGHERDOSESORCOMBINATIONSOF LIVEANDKILLEDVACCINEDOSES;n= *APANISTHESINGLECOUNTRYTHATHASIMPLEMENTEDAPOLICYOFVACCINATING SCHOOLCHILDREN WITHASTRATEGYOFREDUCINGTRANSMISSIONINTHECOMMUNITY AND THEREBY INDIRECTLY PROTECTING HIGH RISK POPULATIONS !LTHOUGH *APAN ABANDONED THIS POLICY IN  AN EXCESS MORTALITY STUDY FOUND EVIDENCE THATITWASASSOCIATEDWITHSUBSTANTIALLYREDUCEDEXCESSMORTALITYINELDERLY PEOPLE FOR THE DECADES IT WAS IN PLACE ;= &IG   /THER STUDIES HAVE EXAMINEDTHEVALUEOFINDUCINGGREATERHERDIMMUNITYBASEDONLOCALCOM MUNITY TRIALS OR MATHEMATICAL MODELS ;n= BUT UNFORTUNATELY NONE HAVE THUS FAR PROVED CONCLUSIVE ENOUGH TO EXTEND THE POLICY OF SCHOOL CHILDRENVACCINATIONNATIONALLY4OFULLYINVESTIGATETHEINDIRECTBENEFITSOF ASCHOOLCHILDRENVACCINATIONPROGRAM ITWOULDBENECESSARYTOCONDUCTA LARGE CLUSTER RANDOMIZED STUDY ACROSS THE COUNTRY SUCH A STUDY HAS BEEN PROPOSEDBUTHASNOTYETBEENUNDERTAKEN;=

6ACCINESFORTHECONTROLOFPANDEMICINFLUENZA 0OLICYPLANNERSAREANTICIPATINGTHATANEFFECTIVEVACCINECAN BEDEVELOPED MANUFACTURED ANDDISTRIBUTEDRAPIDLYENOUGHHELPLIMITTHEIMPACTOFTHE NEXTINFLUENZAPANDEMICHTTPWWWHHSGOVNVPOPANDEMICPLANACCESSED  -ARCH  HTTPWWWHHSGOVPANDEMICFLUPLANAPPENDIXDHTML ACCESSED  $ECEMBER  HTTPWWWHHSGOVPANDEMICFLUPLANSUP HTMLACCESSED$ECEMBER !GREATDEALOFEFFORTHASALREADYBEEN EXPENDED TO DEVELOP AND CLINICALLY TEST SEVERAL TYPES OF VACCINES AGAINST (. INFLUENZA INCLUDING INACTIVATED LIVE ATTENUATED AND $.! VACCINE PREPARATIONS3EVERALCOUNTRIESHAVESTOCKPILEDMILLIONDOSESOFhPRE PAN



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&IGURE(ERDIMMUNITYANDINFLUENZAVACCINATION %NCOURAGINGEVIDENCEFROMTHE*APANESEEXPERIENCEOFVACCINATINGSCHOOLCHILDRENBETWEEN AND4HEGRAPHCOMPARESTHEDIFFERENTPHASESOFTHEVACCINATIONPROGRAMWITHBASE LINETOTALDEATHRATES RATESOFEXCESSDEATHSFROMALLCAUSESANDPNEUMONIAANDINFLUENZA IN *APAN n!DAPTEDFROM2EICHERTETAL; =

DEMICvINACTIVATEDVACCINESBASEDON(.STRAINS.ATIONALPLANNINGDOCU MENTSHAVESETFORTHPRIORITIESFORHOWTODEPLOYANEFFECTIVE VACCINEASIT BECOMESAVAILABLE ANDDETAILEDLOGISTICALPLANSHAVEBEENLAIDFORVACCINE DISTRIBUTION4HE7ORLD(EALTH/RGANIZATIONANDNATIONALPANDEMICPLANS AREREVIEWEDIN5SCHER 0INESETAL;= "UTUNCERTAINTIESABOUND ANDITISUNKNOWNHOWMUCHWARNINGWE WILL HAVE BEFORE THE FULL BLOWN IMPACT OF THE NEXT PANDEMIC .OR IS IT KNOWN WHICHAGEGROUPSWILLBEMOSTATRISKALTHOUGHASHIFTINMORTALITYTOWARD YOUNGER PEOPLE IS VERY LIKELY WHETHER THIS SHIFT WILL PUT YOUNG ADULTS AT GREATESTABSOLUTERISKASWASTHECASEINn ORRELATIVERISKASIN nANDn CANNOTBEPREDICTED7HILETHECHANCESAREGOOD THATANEFFECTIVEVACCINEWILLEVENTUALLYBEDEVELOPEDANDMANUFACTUREDIN LARGE QUANTITIES IT IS UNKNOWN HOW LONG PRODUCTION AND DISTRIBUTION WILL TAKE )N FACT THE FEAR AMONG RESOURCE POOR COUNTRIES THAT THEY WILL ONLY BE ABLE TO OBTAIN VACCINE AFTER WEALTHY COUNTRIES HAVE COVERED THEIR OWN POPULATIONS HAS ALREADY EXACERBATED TENSIONS OVER SHARING OF (. DATA ANDSAMPLES;=&ORALLTHESEREASONS ITISNOTCLEARTHAT PLANNERSHOPES THATVACCINESWILLPLAYAMAJORROLEINLIMITINGTHEGLOBALIMPACTOFTHENEXT PANDEMICWILLBEREALIZED

#ONCLUSION -ANY UNSOLVED YET INTRIGUING MYSTERIES ABOUT INFLUENZA EPIDEMIOLOGY REMAIN ; n= 3OLVING THESE RIDDLES WILL DEPEND ON THE SUCCESSFUL INTEGRATION OF MANY SEPARATE FIELDS INCLUDING IMMUNOLOGY PHYLOGENETICS

4HEEPIDEMIOLOGYOFINFLUENZAANDITSCONTROL



VIROLOGY AND CLINICAL ASCERTAINMENT %XCITING PROGRESS HAS RECENTLY BEEN MADEINAREASWHEREMATHEMATICALMODELERSANDPHYLOGENETICRESEARCHERS HAVEENTEREDTHEINFLUENZAFIELD;   =4HISCROSS FERTILIZATIONHAS FOREXAMPLE PRODUCEDUSEFULNEWFINDINGSINMOLECULARINFLUENZAEPIDEMI OLOGY WHICHMAYINTURNLEADTOIMPROVEDTOOLSFORTHESELECTIONOFVACCINE STRAINS;= 2EGARDING PANDEMIC INFLUENZA THE ENORMOUS VARIABILITY OF EXPERIENCE WITHPASTPANDEMICSnWHICHHAVE RANGEDFROMMILDTODEVASTATINGnMAKES PREDICTIONS ABOUT A FUTURE PANDEMIC UNRELIABLE7ILL IT BE THE YOUNG AND HEALTHYWHODIEATTHEHIGHESTRATEASWASTHECASEIN ORWILLITBE hBUSINESS AS USUALv SIMILAR TO SEVERE SEASONAL INFLUENZA EPIDEMICS WHERE ELDERLY PEOPLE AND OTHER HIGH RISK POPULATIONS ARE AT HIGHEST RISK7HAT WILL BE THE EFFECT OF THE PRESENCE OF LARGE NUMBERS OF IMMUNOSUPPRESSED PEOPLE INCLUDINGPEOPLEWITH()6INFECTION!NDPERHAPSMOSTIMPORTANT WILL THE NEXT PANDEMIC MERELY HAVE A MODERATE IMPACT LIKE  OR BE CATASTROPHICLIKE  4HEFACTIS WECANNOTKNOWTHEANSWERSTOTHESEQUESTIONSINADVANCE 4HE AGE DISTRIBUTIONS OF THE ^ HUMAN CASES AND DEATHS FROM!(. INFLUENZA MOST CLOSELY RESEMBLE THOSE OF THE  PANDEMIC WITH HIGHEST IMPACT AMONG THE YOUNG HTTPWWWWHOINTTOPICSAVIAN?INFLUENZAEN  n  "UT THAT COULD CHANGE CONSIDERABLY AS THE VIRUS GAINS THE ABILITYTOTRANSMITEFFECTIVELYINHUMANPOPULATIONS&URTHERMORE THENEXT PANDEMICMAYNOTBECAUSEDBYAN!(.VIRUS 7HATEVER THE SCENARIO THE EPIDEMIOLOGICAL CHARACTERISTICS OF A FUTURE PANDEMICDIRECTLYAFFECTTHEETHICALPRINCIPLESTHATSHOULDBE INVOKEDWHEN ALLOCATINGLIMITEDVACCINEDOSES; =&ORTHATREASON ITISABSOLUTELY ESSENTIALTHATREAL TIMESURVEILLANCEDATAFROMTHEEARLYPHASE OFTHENEXT PANDEMICBEFREELYSHAREDANDRAPIDLYINTERPRETED TODETERMINE WHOISAT RISK AND WHERE SCARCE RESOURCES SUCH AS PANDEMIC VACCINE AND ANTIVIRALS COULD BEST BE USED -OREOVER PANDEMIC PLANNERS SHOULD BUILD SUFFICIENT FLEXIBILITYINTOTHEIRPLANSTOALLOWRAPIDSHIFTSINPLANNEDCONTROLSTRATEGIES AS KEY EPIDEMIOLOGICAL INSIGHTS HOPEFULLY BECOME AVAILABLE IN THE EARLY PANDEMICPHASE#ONTINUEDINFLUENZASURVEILLANCEEFFORTSINTEMPERATEAND TROPICAL REGIONS COMBINED WITH INTERNATIONAL SHARING OF EPIDEMIOLOGICAL ANDVIRALSEQUENCEDATAAREOURBESTHOPEFORLIMITINGTHEIMPACTOFTHENEXT INFLUENZAPANDEMIC

!CKNOWLEDGEMENTS 7EAREENORMOUSLYGRATEFULTOOURMANYCOLLEAGUESnNATIONALLY ANDINTER NATIONALLY THROUGH THE -ULTINATIONAL )NFLUENZA 3EASONAL -ORTALITY 3TUDY NETWORKnFORTHEMANYINSPIRINGCONVERSATIONSWEHAVEHADOVER THEYEARS ABOUTTHEhMYSTERIESvOFINFLUENZAEPIDEMIOLOGY



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!BSTRACT %COLOGICAL AND ACTIVE POPULATION BASED SURVEILLANCE STUDIES HAVE CLEARLY SHOWN THE LARGEBURDENOFINFLUENZADISEASEINCHILDREN BOTHINHOSPITALANDOUTPATIENTSETTINGS -ORTALITYANDENCEPHALITISDUETOINFLUENZAHAVEALSOBEENREPORTED4WOVACCINESARE LICENSEDFORUSEINCHILDRENTRIVALENTINACTIVATEDANDLIVEATTENUATEDVACCINES"OTHHAVE BEENSHOWNTOBEEFFICACIOUSFORTHEPREVENTIONOFCLINICALLYANDLABORATORY CONFIRMED INFLUENZA)NRECENTCOMPARATIVETRIALSINYOUNGCHILDREN LIVE ATTENUATEDVACCINESWERE SHOWN TO BE MORE EFFECTIVE THAN TRIVALENT INACTIVATED VACCINES FOR THE PREVENTION OF LABORATORY CONFIRMEDINFLUENZA(OWEVER EPISODESOFWHEEZINGWEREINCREASEDINTHE YOUNGESTCHILDRENRECEIVINGLIVEATTENUATEDVACCINE4RIVALENTINACTIVATEDINFLUENZAVAC CINEHASANEXCELLENTSAFETYPROFILEANDHASBEENMAINLYASSOCIATEDWITHLOCALPAINAND TENDERNESS AT THE INJECTION SITE6ACCINE EFFICACY FOR THE INACTIVATED VACCINE HAS BEEN SHOWNTOBEGREATERINOLDERCHILDREN)NCREASEDUSEOFEITHERINFLUENZAVACCINEHASTHE POTENTIAL TO REDUCE THE DISEASE BURDEN IN CHILDREN AND TO EXTEND HERD PROTECTION TO THOSEWHOARENOTVACCINATED

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3AFETYOF4)6 4WO LARGE STUDIES HAVE ASSESSED THE SAFETY OF 4)6 IN CHILDREN AND PRO VIDEASSURANCETHATTHEVACCINEISWELLTOLERATEDINTHISAGEGROUP; = (AMBIDGEETAL;=CONDUCTEDARETROSPECTIVECHARTREVIEWOFSIGNIFICANT MEDICALLY ATTENDED EVENTS AT EIGHT MANAGED CARE ORGANIZATIONS IN THE 5NITED3TATESTHATPARTICIPATEINTHE#$# FUNDED6ACCINE3AFETY $ATALINK !LLCHILDRENINTHISCOHORTWHOWEREnMONTHSOLDANDWHORECEIVED4)6 BETWEEN*ANUARY AND-AYWEREASSESSED4HISREPRESENTED CHILDRENWHORECEIVEDATOTALOF4)6VACCINATIONS!NYMEDI



+ATHRYN-%DWARDS

CALLY ATTENDED EVENT ASSOCIATED WITH4)6 WAS EVALUATED IN FOUR RISK WIN DOWSnDAYS nDAYS nDAYS ANDnDAYSAFTERVACCINATIONAND COMPAREDWITHTWOCONTROLPERIODS ONEBEFOREVACCINATIONANDTHESECOND AFTERTHERISKWINDOW4HERESULTSOFTHISSTUDYINDICATETHATTHEREWEREVERY FEW MEDICALLY ATTENDED EVENTS NONE WERE SERIOUS AND NONE WERE SIGNIFI CANTLYASSOCIATEDWITHTHEVACCINE4HISSTUDYPROVIDESADDITIONALEVIDENCE SUPPORTINGTHESAFETYOFIMMUNIZINGCHILDRENnMONTHSOLDWITH4)6 )NANOTHERSTUDYFROM THE#$# FUNDED6ACCINE3AFETY$ATALINK &RANCE & ETAL;=EVALUATEDCHILDRENYOUNGERTHANYEARSOFAGEWHO RECEIVED4)6 -EDICAL VISITS IN THE  WEEKS AFTER4)6 VACCINATION WERE COMPARED WITH THOSEINTWOCONTROLPERIODS#HILDRENVACCINATEDFROM*ANUARYTO $ECEMBERWERERANDOMLYDIVIDEDINTOTWOEQUALGROUPS)NGROUP RISKSOFOUTPATIENT EMERGENCYDEPARTMENT ANDINPATIENTVISITS DURINGTHE DAYSAFTERVACCINATIONWERECOMPAREDWITHTHERISKSOFVISITSINTHECONTROL PERIODS 3IGNIFICANT MEDICALLY ATTENDED EVENTS IDENTIFIED IN GROUP  WERE THEN EVALUATED IN GROUP  USING THE SAME TWO CONTROL PERIODS -EDICALLY ATTENDED EVENTS SIGNIFICANT IN BOTH GROUPS WERE CONSIDERED POTENTIALLY ASSOCIATED WITH VACCINATION AND WERE ASSESSED BY MEDICAL RECORD REVIEW ! TOTAL OF   VACCINATION EPISODES WERE ASSESSED 3TUDY PARTICIPANTS INCURRED   AND  DIFFERENT DIAGNOSES DURING THE  DAYS AFTER VACCINATIONACCORDINGTOTHEOUTPATIENT EMERGENCYDEPARTMENT ANDINPA TIENTDATA RESPECTIVELY!FTERMEDICALRECORDREVIEW IMPETIGOINCHILDREN nMONTHSOFAGEWASSIGNIFICANTLYMORECOMMONAFTERVACCINATION4HE CONCLUSIONOFTHISLARGESAFETYSTUDYWASTHAT4)6WASWELLTOLERATEDINTHE ENTIREAGERANGEOFPEDIATRICVACCINATIONS

%FFICACYSAFETYOF,!)6 ! NUMBER OF STUDIES OF ,!)6 HAVE BEEN PUBLISHED USING MONOVALENT BIVALENT AND TRIVALENT EXPERIMENTAL AND MANUFACTURING LOT PREPARATIONS OF,!)6/NEOFTHELARGESTWASAMULTICENTER DOUBLE BLIND PLACEBO CON TROLLED TRIAL OF TRIVALENT ,!)6 CONDUCTED IN CHILDREN n MONTHS OLD IN THE LATE S ;= )N THIS PIVOTAL STUDY  CHILDREN WERE ASSIGNED TO RECEIVE TWO DOSES OF LIVE ATTENUATED INTRANASAL VACCINE AND  CHILDREN WEREASSIGNEDTORECEIVEONEDOSEOFEITHERLIVEATTENUATEDVACCINEORPLA CEBO4HESTRAINSINCLUDEDINTHELIVEATTENUATEDVACCINEWEREANTIGENICALLY EQUIVALENTTOTHOSEINTHECONTEMPORARYINACTIVATEDINFLUENZAVIRUSVACCINE )LLSUBJECTSWEREEVALUATEDWITHVIRALCULTURESFORINFLUENZADURINGTHESUBSE QUENTINFLUENZASEASON!CASEOFINFLUENZAWASDEFINEDASILLNESSASSOCIATED WITHISOLATIONOFWILD TYPEINFLUENZAVIRUSFROMRESPIRATORYSECRETIONS4HE INTRANASALVACCINEWASWELLTOLERATED WITHNOSERIOUSADVERSEEVENTSREPORT ED!MONGCHILDRENWHOWEREINITIALLYSERONEGATIVE FOURFOLDTITERRISESWERE NOTEDINnOFTHESUBJECTS DEPENDINGONTHEINFLUENZASTRAIN#ASES OFINFLUENZAWERESIGNIFICANTLYLESSCOMMONINTHEVACCINEGROUPTHANTHE

)NFLUENZAANDINFLUENZAVACCINATIONINCHILDREN



PLACEBOGROUPANDVACCINEEFFICACYAGAINSTCULTURE CONFIRMEDINFLUENZAILL NESS WAS   #) n  )N ADDITION THE ONE DOSE REGIMEN HAD EFFICACYAGAINSTCULTURE CONFIRMEDDISEASE 4O DETERMINE THE SAFETY OF COLD ADAPTED TRIVALENT INTRANASAL INFLUENZA VIRUS VACCINE A RANDOMIZED DOUBLE BLIND PLACEBO CONTROLLED SAFETY TRIAL WASCONDUCTEDINNEARLY HEALTHYCHILDRENAGEMONTHSTOYEARS OF AGE GIVEN LIVE VACCINE OR PLACEBO IN A  RANDOMIZATION SCHEME ;= #HILDREN YEARSOFAGERECEIVEDTWO DOSESOFEITHERVACCINEOR PLACEBO nDAYSAFTERTHEFIRSTDOSE%NROLLEDCHILDRENWERETHENFOLLOWEDFOR DAYSAFTEREACHVACCINATIONFOR ALLMEDICALLYATTENDEDEVENTS!CUTERESPIRA TORY TRACT EVENTS SYSTEMIC BACTERIAL INFECTIONS ACUTE GASTROINTESTINAL TRACT EVENTSANDRAREEVENTSPOTENTIALLYASSOCIATEDWITHWILD TYPEINFLUENZAWERE ASSESSED AND NONE WERE FOUND TO BE ASSOCIATED WITH VACCINE (OWEVER A STATISTICALLYSIGNIFICANTINCREASEINTHERELATIVERISKFORREACTIVEAIRWAYDIS EASE;#)n =WASOBSERVEDINCHILDRENn MONTHSOF AGE"ASEDONTHEHIGHEFFICACYRATESOBTAINEDINTHE"ELSHEET AL;=STUDY BUTTEMPEREDBYTHESAFETYCONCERNSASSOCIATEDWITHWHEEZINGIN THISLARGE STUDY THE,!)6WASLICENSEDFORUSEINCHILDRENOVER YEARSOFAGEWITHOUT APREVIOUSHISTORYOFWHEEZING 'IVEN CONCERNS OVER THESE REACTIVE AIRWAY FINDINGS IN THE STUDY OF "ERGENETAL;= ANOTHERSTUDYCOMPARINGTHEEFFICACYANDSAFETYOF,!)6 WITHINACTIVATEDINFLUENZAVACCINEINCHILDRENnMONTHSOFAGEWITHAHIS TORYOFRECURRENTRESPIRATORYTRACTINFECTIONSWASCONDUCTED;=#HILDREN WERERANDOMIZEDTORECEIVETWODOSESOFEITHER,!)6N ORINACTI VATEDVACCINEN BEFORETHEnINFLUENZASEASON0ARTICIPANTS WERE FOLLOWED FOR CULTURE CONFIRMED INFLUENZA ILLNESS AND VACCINE SAFETY /VERALL THEREWERE#)n FEWERCASESOFCONFIRMED INFLUENZACAUSEDBYANTIGENICALLYSIMILARSTRAINSINTHELIVEATTENUATEDTHAN INTHEINACTIVATEDVACCINEGROUPS4HEREWERENODIFFERENCESBETWEENGROUPS INTHEINCIDENCEOFWHEEZINGAFTERVACCINATION 4O FURTHER COMPARE THE SAFETY AND EFFICACY OF THE ,!)6 AND 4)6 IN ASTHMATIC CHILDREN &LEMING ET AL ;= RANDOMIZED OVER  ASTHMATIC CHILDRENnYEARSOFAGE TOEITHER4)6OR,!)6INANOPEN LABELSTUDY DURINGTHEnINFLUENZASEASON0ARTICIPANTSWEREASSESSEDFORCUL TURE CONFIRMEDINFLUENZAILLNESSANDVACCINESAFETY7HENTHEINCIDENCEOF CULTURE CONFIRMEDINFLUENZAILLNESSWASCOMPAREDBETWEENTHETWOVACCINE GROUPS THE,!)6HADSIGNIFICANTLYGREATERRELATIVEEFFICACY#) n .OSIGNIFICANTDIFFERENCESWERENOTEDBETWEENTHETWOVACCINE GROUPS IN THE INCIDENCE OF ASTHMA EXACERBATIONS MEAN PEAK EXPIRATORY FLOWRATEFINDINGS ASTHMASYMPTOMSCORES ORNIGHTTIMEAWAKENINGSCORES 2UNNYNOSEANDNASALCONGESTIONWEREMORECOMMONINTHERECIPIENTSOF THE,!)6ANDMOREINJECTIONSITEREACTIONSWERENOTEDFORTHE4)6RECIPI ENTS !NOTHERTRIALTOASSESSVACCINEEFFICACYOF,!)6WASRECENTLYCONDUCTED IN!SIA4AM ET AL ;= EVALUATED THE EFFICACY AND SAFETY OF ,!)6 AGAINST



+ATHRYN-%DWARDS

CULTURE CONFIRMEDINFLUENZAINAPLACEBO CONTROLLEDTRIALDURINGTWOINFLU ENZASEASONS)NYEAR CHILDRENnMONTHSOFAGEWERERANDOMIZED TORECEIVETWODOSESOF,!)6ORPLACEBO)NYEAR SUBJECTSWEREAGAIN RANDOMIZED TO RECEIVE ONE DOSE OF ,!)6 OR PLACEBO6ACCINE EFFICACY IN YEARWAS#)n AGAINSTANTIGENICALLYSIMILARINFLU ENZASUBTYPES AND#)  AGAINSTANYSTRAIN)NYEAR  ,!)6 WAS EFFECTIVE AGAINST ANTIGENICALLY SIMILAR   #) n  ANDANY#)n INFLUENZASTRAINS.OINCREASE INWHEEZINGEPISODESWASNOTEDINVACCINERECIPIENTSINEITHERSTUDYYEAR )N THE MOST RECENT COMPARATIVE EFFICACY STUDY "ELSHE ET AL ;= COM PAREDTHESAFETYANDEFFICACYOFINTRANASALLYADMINISTERED,!)6WITHTHAT OFINACTIVATEDVACCINEININFANTSANDYOUNGCHILDREN#HILDRENnMONTHS OFAGE WITHOUTARECENTEPISODEOFWHEEZINGILLNESSORSEVEREASTHMA WERE RANDOMLY ASSIGNED IN A  RATIO TO RECEIVE EITHER COLD ADAPTED TRIVALENT ,!)6OR4)6INADOUBLE BLINDMANNER)NFLUENZA LIKEILLNESSWASASSESSED WITH CULTURES DURING THE n INFLUENZA SEASON AND SAFETY WAS CARE FULLY MONITORED /VERALL THERE WERE  FEWER CASES OF CULTURED CON FIRMED INFLUENZA IN THE ,!)6 RECIPIENTS THAN IN THE 4)6 RECIPIENTS  VS  CASES P 4HE BETTER EFFICACY OF LIVE ATTENUATED VACCINE WAS SEENFORBOTHANTIGENICALLYWELL MATCHEDANDDRIFTEDVIRUSES!MONGPREVI OUSLYUNVACCINATEDCHILDREN WHEEZINGWITHINDAYSOFADMINISTRATIONOF DOSEONEOF,!)6WASMORECOMMONTHANWITH4)6!MONGCHILDREN n MONTHS OF AGE  MORE EPISODES OF WHEEZING WERE NOTED WITHIN  DAYS AFTER RECEIPT OF DOSE ONE AMONG RECIPIENTS OF ,!)6  THAN AMONG RECIPIENTS OF4)6  P   2ATES OF HOSPITALIZATION FOR ANY CAUSE DURINGTHEDAYSAFTERVACCINATIONWEREHIGHERAMONGTHERECIPIENTSOF ,!)6WHOWEREnMONTHSOFAGE THANAMONGTHERECIPIENTSOF 4)6 IN THIS AGE GROUP  P  "ASED ON THESE RESULTS ,!)6 IS LICENSED DOWN TO  YEARS OF AGE IN CHILDREN WITHOUT A PREVIOUS HISTORY OF WHEEZINGORASTHMA &INALLY REVIEWOFAVERYLARGEOPEN LABEL NONRANDOMIZED COMMUNITY BASEDTRIALOFA,!)6WASCONDUCTEDBY0IEDRAETAL;=ANDPROVIDESSOME OFTHEMOSTCOMPREHENSIVESAFETYDATAAVAILABLEINTHELITERATUREFORTHELIVE VACCINE-EDICALRECORDSOFALLCHILDRENWHORECEIVED,!)6WERESURVEYED FORSERIOUSADVERSEEVENTS3!%S ANDHEALTHCAREUTILIZATION WEEKSAFTER VACCINATION )N THE FOUR STUDY YEARS   DOSES OF ,!)6 WERE ADMINIS TEREDTOCHILDREN!TOTALOF  ANDDOSES OF,!)6 4 WERE ADMINISTERED TO CHILDREN WHO WERE  MONTHS TO  YEARS n YEARS ANDnYEARSOFAGE RESPECTIVELY$URINGTHEFOURSTUDYYEARS3!%S WERE IDENTIFIED BUT NONE WERE ATTRIBUTED TO ,!)6 4 #OMPARED WITH THE PREVACCINATIONPERIOD THEREWERENOINCREASESINMEDICALLYATTENDEDACUTE RESPIRATORY INFECTIONS FROM  TO  AND  TO  DAYS AFTER VACCINATION IN CHILDRENWHOWEREMONTHSTOYEARS nYEARS ANDnYEARSOFAGE! RELATIVERISKOF#)n FORASTHMAEVENTSnDAYSAFTER

)NFLUENZAANDINFLUENZAVACCINATIONINCHILDREN



VACCINATIONWASDETECTEDINCHILDRENWHOWEREMONTHSTOYEARSOFAGE DURINGONESTUDYYEAR BUTWASNOTSIGNIFICANTLYINCREASEDFORTHEOTHERTHREE VACCINEYEARS;VACCINEYEAR 22#)n VACCINEYEAR 22#)n VACCINEYEAR 22#) n = 4HEYCONCLUDEDTHAT,!)6WASSAFEINCHILDRENRECEIVINGTHEVACCINE;=

#ANHERDIMMUNITYFORINFLUENZABEACHIEVED 4HERE ARE A NUMBER OF HIGHLY CONTAGIOUS INFECTIONS SUCH AS MEASLES AND VARICELLA WHEREIMMUNIZATIONOFAPORTIONOFTHEPOPULATIONCONFERSPRO TECTION TO UNIMMUNIZED INDIVIDUALS BY DECREASING THE CIRCULATION OF THE PATHOGEN A CONCEPT CALLED HERD IMMUNITY 3EVERAL YEARS AGO A STUDY IN *APAN ASSESSED THE IMPACT OF INFLUENZA IMMUNIZATION OF SCHOOL CHILDREN ONINFLUENZAMORTALITYINELDERLYPERSONSANDOTHERSATHIGHRISK;=&ROM TO *APANESESCHOOLCHILDRENWEREMANDATEDTORECEIVE 4)6 AND MOSTWEREVACCINATEDINTHELAWSWERERELAXEDANDINTHEYWERE REPEALED4HE STUDY LOOKED AT INFLUENZA VACCINATION RATES AND DEATH RATES SPANNING THIS TIME PERIOD IN *APAN AND COMPARED THEM TO DATA FROM F THE 53&IG  !FTERTHEVACCINATIONPROGRAMFORSCHOOLCHILDRENWASINITIATEDIN*APAN EXCESSMORTALITYRATESDROPPEDFROMVALUESTHREETOFOURTIMES THOSEINTHE 53TOVALUESSIMILARTOTHOSEINTHE532OUTINEVACCINATIONOF*APANESE CHILDRENWASESTIMATEDTOHAVEPREVENTEDnDEATHSPERYEAR OR ABOUTDEATHFOREVERYCHILDRENVACCINATED!STHEVACCINATIONMANDATE WASRELAXED VACCINATIONRATESDROPPEDANDEXCESSMORTALITYRATESINCREASED )NCONTRAST EXCESSMORTALITYRATESINTHE53WERENEARLYCONSTANTOVERTHE SAMEPERIODOFTIME4HEDATAFROM*APANSUGGESTEDTHATVACCINATINGSCHOOL CHILDRENAGAINSTINFLUENZAREDUCEDINFLUENZAMORTALITYAMONGOLDERPERSONS CONFIRMINGTHATHERDIMMUNITYWASOCCURRINGWITHINFLUENZAVACCINE;= ! SIMILAR STUDY WAS RECENTLY REPORTED FROM THE 53 WHERE SCHOOL CHILDREN WERE VACCINATED WITH ,!)6 AND ITS IMPACT WAS ASSESSED IN THEIR HOUSEHOLDS AND COMMUNITY ;= %LEVEN DEMOGRAPHICALLY SIMILAR CLUSTERS OFELEMENTARYSCHOOLSINFOURSTATESWERECHOSEN7ITHINEACHCLUSTER ONE SCHOOLWASSELECTEDTORECEIVEVACCINATIONINTERVENTIONSCHOOL ANDONEOR TWO SCHOOLS IN THAT CLUSTER DID NOT PARTICIPATE CONTROL SCHOOLS  $URING A PREDICTEDWEEKOFPEAKINFLUENZAACTIVITYINEACHSTATE ALLHOUSEHOLDSWITH CHILDRENINTHEINTERVENTIONANDCONTROLSCHOOLSWEREASKEDABOUTINFLUENZA VACCINATIONANDINFLUENZA LIKEILLNESS0ERSONSLIVINGININTERVENTIONSCHOOL HOUSEHOLDSHADSIGNIFICANTLYFEWERINFLUENZA LIKESYMPTOMSAND OUTCOMES DURINGTHERECALLWEEKTHANTHOSEINCONTROLSCHOOLHOUSEHOLDS EVENTHOUGH THEYTHEMSELVESMIGHTNOTHAVEBEENIMMUNIZED4HISSUGGESTSTHATVACCI NATING CHILDREN PROTECTS THEIR UNIMMUNIZED CONTACTS THE ESSENTIAL MECHA NISMOFHERDIMMUNITY



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&IGURE%XCESSDEATHSATTRIBUTEDTOBOTHPNEUMONIAANDINFLUENZAANDALLCAUSES SPANNING THEYEARSWHENTHE*APANESESCHOOLIMMUNIZATIONPROGRAMWASDISMANTLEDFROM;= 

0RACTICALIMPLICATIONSFORINFLUENZAVACCINATIONOFCHILDREN &OR MANY YEARS ALL CHILDREN WITH HIGH RISK CONDITIONS ASSOCIATED WITH INFLUENZA HAVE BEEN RECOMMENDED TO RECEIVE ANNUAL INFLUENZA VACCINA TION4HESECONDITIONSINCLUDEASTHMAOROTHERCHRONICPULMONARYDISEASES SIGNIFICANT CARDIAC DISEASE IMMUNOSUPPRESSIVE DISORDERS HUMAN IMMU NODEFICIENCYVIRUSINFECTION SICKLECELLANEMIA LONG TERMASPIRINTHERAPY CHRONIC RENAL DISEASE CHRONIC METABOLIC DISORDERS AND CHILDREN WITH NEUROLOGICALDISORDERS"EGINNINGWITHTHEnINFLUENZA SEASONIN THE5NITED3TATES ALLCHILDRENBETWEENTHEAGESOFMONTHSANDYEARS WERE RECOMMENDED TO RECEIVE ANNUAL4)6 TO REDUCE THE BURDEN OF BOTH HOSPITALIZATION AND OUTPATIENT VISITS ASSOCIATED WITH INFLUENZA 7ITH THE UNIVERSALRECOMMENDATIONSFORINFLUENZAVACCINEINYOUNGCHILDREN STUDIES HAVEBEENCONDUCTEDTOMONITORVACCINEUPTAKE$ATAFROMTHE.ATIONAL )MMUNIZATION3URVEYMEASUREDVACCINATIONRATESINCHILDRENn MONTHS

)NFLUENZAANDINFLUENZAVACCINATIONINCHILDREN



OFAGETHEYEARAFTERTHEUNIVERSALINFLUENZARECOMMENDATIONSWEREISSUED !LTHOUGHINFLUENZAIMMUNIZATIONRATESVARIEDWIDELYAMONGTHEDIFFERENT STATES OVERALLOFCHILDRENBETWEENANDMONTHSOFAGERECEIVED ONEDOSEOFVACCINEANDRECEIVEDTWODOSES;=#ONTINUEDASSESS MENTOFINFLUENZAVACCINATIONRATESINTHISPOPULATIONISNEEDED)TISHOPED THATIMMUNIZATIONRATESWILLINCREASEWITHEACHYEARTHATPASSESAFTERANEW RECOMMENDATION #ONSIDERABLEDISCUSSIONHASSURROUNDEDTHEQUESTIONWHETHERROUTINE INFLUENZAVACCINATIONOFALLSCHOOLCHILDRENMIGHTREDUCEDISEASEINBOTH THECHILDRENANDOTHERCOMMUNITYMEMBERS ASDISCUSSEDEARLIER !STUDY CONDUCTEDTHROUGHTHE#$# FUNDED6ACCINE3AFETY$ATALINKADDRESSED THE SIMPLE QUESTION OF WHETHER TWO DOSES OF4)6 COULD BE DELIVERED TO CHILDREN  YEARS OF AGE WHO HAD NOT PREVIOUSLY RECEIVED VACCINE ;= ! TOTAL OF   CHILDREN  MONTHS TO  YEARS OF AGE WERE EVALUATED !MONG CHILDREN n MONTHS OF AGE A FAIRLY HIGH PROPORTION OF FIRST TIME VACCINATEDCHILDRENALSORECEIVEDASECONDVACCINATION WITHRATESOF  IN n  IN n AND  IN n A SEASON WITH VACCINE SHORTAGES  )N CONTRAST AMONG CHILDREN n YEARS OF AGE THECORRESPONDINGRATESWEREONLY  AND4HEFACTTHATTHE MAJORITY OF CHILDREN WHO REQUIRED TWO DOSES OF VACCINE DID NOT RECEIVE THEMHIGHLIGHTSSOMEOFTHEDIFFICULTIESTHATMAYBEENCOUNTEREDINIMPLE MENTINGUNIVERSALVACCINATIONOFALLSCHOOLCHILDRENINTHEPRIMARYCARE SETTING -IGHT SCHOOL BASED VACCINE DELIVERY CIRCUMVENT SOME OF THESE PROB LEMS! RECENT REPORT DESCRIBING ON SITE ADMINISTRATION OF ,!)6 TO ALL STUDENTSINALARGE METROPOLITANPUBLICSCHOOLSYSTEMDEMONSTRATEDTHAT LARGE NUMBERS OF SCHOOL CHILDREN COULD BE EFFECTIVELY IMMUNIZED ;= 4HEREWERE STUDENTSINTHESYSTEMOFTHEELEMENTARY SCHOOL STUDENTS OFTHEMIDDLESCHOOLSTUDENTS ANDOFTHEHIGHSCHOOL STUDENTS WERE IMMUNIZED4HIS EXPERIENCE CLEARLY HIGHLIGHTS THAT A VAC CINATION CAMPAIGN IN A LARGE PUBLIC SCHOOL SYSTEM CAN ACHIEVE RELATIVELY HIGH COVERAGE LEVELS HOWEVER CONSIDERABLE EFFORT BY THE LOCAL HEALTH DEPARTMENTWASEXPENDEDINTHEPROCESS!NALYSESOFSTUDIESASSESSINGTHE EFFECTIVENESSOFTHESCHOOL BASEDINFLUENZAIMMUNIZATIONPROGRAMSAREIN PROGRESS

#ONCLUSION 'IVENTHECLEAREVIDENCETHATBOTHLIVEANDINACTIVATEDINFLUENZAVACCINES CANPREVENTINFLUENZADISEASE INFLUENZAVACCINATIONSHOULDBE OFFEREDTOALL CHILDRENWHOWISHTOREDUCETHEIRBURDENOFINFLUENZA4HERECENTEVIDENCE OF IMPROVED VACCINE EFFICACY FOR ,!)6 IN YOUNG CHILDREN AND THE LIKELY APPROVALOFTHEVACCINEFORAYOUNGERAGEGROUP ALSOSUGGESTSTHATITMIGHT BEABETTERALTERNATIVETO4)6INYOUNGCHILDRENWITHOUTAHISTORYOFASTHMA



+ATHRYN-%DWARDS

!LSO GIVENTHATINFLUENZADISEASEISSORARELYSPECIFICALLYDIAGNOSED;=AND THAT IT CAN MIMIC OTHER RESPIRATORY VIRAL INFECTIONS IT IS IMPERATIVE THAT LABORATORY BASED SURVEILLANCE BE CONDUCTED TO ASSESS VACCINE EFFICACY F AS INFLUENZAVACCINEISUTILIZEDMOREBROADLY4HEFUTUREFORINFLUENZAPREVEN TIONISBRIGHT BUTCONTINUEDATTENTIONTOMEASURINGVACCINEEFFECTISNEEDED F TOSUSTAINTHISEFFORT

2EFERENCES 

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4HEIMMUNERESPONSETOINFLUENZA!VIRUSES *USTINE$-INTERN #AROLE'UILLONNEAU 3TEPHEN*4URNER AND0ETER#$OHERTY  $EPARTMENTOF-ICROBIOLOGYAND)MMUNOLOGY 5NIVERSITYOF-ELBOURNE 0ARKVILLE 6ICTORIA

 !USTRALIA$EPARTMENTOF)MMUNOLOGY 3T*UDE#HILDRENS2ESEARCH(OSPITAL -EMPHIS 4. 53!

!BSTRACT 4HEINFLUENZA!VIRUSESAREDANGEROUSPATHOGENSWITHTHEPOTENTIALTOPROVOKEDEVAS TATING DISEASE4HE CHALLENGE FOR THE MEDICAL RESEARCH COMMUNITY IS TO DESIGN PREVEN TIVE MEASURES AND THERAPEUTIC INTERVENTIONS THAT WILL LIMIT THE SEVERE CONSEQUENCES OF PANDEMIC INFLUENZA! VIRUS INFECTIONS6ACCINES HAVE LONG BEEN AVAILABLE BUT THERE IS CONSIDERABLESCOPEFORIMPROVEMENTASTHEYTARGETONLYTHEPREVAILINGINFLUENZA!VIRUS STRAINS DONOTGIVEBROADIMMUNITYANDWORKPOORLYINTHEELDERLY THETARGETGROUPTHAT ISMOSTATRISKOFFATALDISEASE)MPROVEDVACCINESWILLONLYEMERGEIFTHEDEVELOPMENT STRATEGYISBASEDONAFIRMUNDERSTANDINGOFTHEHOSTIMMUNERESPONSETOTHEVIRUS(ERE WESUMMARIZETHERESEARCHTODATETHATDETAILSIMMUNEMECHANISMSPARTICIPATINGINTHE CONTROLANDELIMINATIONOFINFLUENZA!VIRUSES

)NTRODUCTION 4HE INFLUENZA VIRUSES ARE /RTHOMYXOVIRUSES WITH AN EIGHT SEGMENTED NEGATIVE SENSE SINGLE STRANDED SS 2.! GENOME 4HERE ARE THREE TYPES INFLUENZA! "AND#4HEINFLUENZA!VIRUSESTHATCAUSETHEMOSTSERIOUS PROBLEMS IN HUMANS ARE THE SUBJECT OF THIS REVIEW 4HESE PATHOGENS ARE CLASSIFIEDACCORDINGTOTHEIRTWOMAJORSURFACEGLYCOPROTEINSHEMAGGLUTININ (! OR( ANDNEURAMINIDASE.!OR. )NFECTINGBOTHMAMMALIANAND AVIANSPECIES THEHIGHLYCONTAGIOUSINFLUENZA!VIRUSESARERESPONSIBLEFOR WIDESPREAD MORBIDITY AND MORTALITY ;= )N MAMMALS INFECTION IS ESTAB LISHED IN THE UPPER AND LOWER RESPIRATORY TRACTS PROVOKING AN ILLNESS THAT IS ASSOCIATED WITH FEVER MYALGIA CONGESTION PHARYNGITIS AND IN SEVERE CASES PNEUMONIA%ARLYON SOMEOFTHEVERYVIRULENTINFLUENZA!VIRUSES CANINDUCEAhCYTOKINESHOCKvSYNDROMEMEDIATED VIATHEINNATEIMMUNE RESPONSEPATHWAY&ORTUNATELY INFECTIONALSOELICITSPOTENTADAPTIVEIMMU NITY AND LONG TERM MEMORY ALTHOUGH THE VIRUS CAN MUTATE READILY ALLOW INGSTRAINSWITHVARIANT(!MOLECULESTOCAUSESUCCESSIVEPANDEMICS4HE



*USTINE$-INTERNETAL

CURRENT KILLED OR SUBUNIT VACCINES INDUCE EFFECTIVE ANTIBODY RESPONSES IN NORMAL ADULTS ALTHOUGH THEY DO NOT PROMOTE A VIRUS SPECIFIC #$ 4 CELL RESPONSEANDMEMORYANDTHEYAREPOORLYIMMUNOGENICINTHOSEWHOARE EVENMARGINALLYIMMUNOLOGICALLYCOMPROMISED4HEMAJORTASKFOR IMMU NOLOGISTSINTERESTEDINTHEPROBLEMTHATINFLUENZAVIRUSPOSES ISTODEVELOP BETTER VACCINES -OST OF OUR DETAILED KNOWLEDGE ABOUT IMMUNITY TO THE INFLUENZA!VIRUSESISDERIVEDFROMTHEMURINEMODELTHATALLOWSRIGOROUS ANALYSISDUETOTHEAVAILABILITYOFANEXTENSIVEPANELOFDEFINEDANALYTICAL REAGENTS (ERE WE PROVIDE A COMPREHENSIVE SUMMARY OF A LARGE BODY OF RESEARCH EXAMINING THE IMMUNE MECHANISMS THAT ACT TO CONTROL INFLUENZA !VIRUSINFECTION&IG 4HISINFORMATIONSHOULDPROVIDEAUSEFULBASISFOR THEINFORMEDDESIGNOFNOVEL NEXTGENERATIONINFLUENZA!VIRUSVACCINES

$ETECTIONOFINFLUENZA!VIRUS )NVADINGINFLUENZA!VIRUSESAREDETECTEDINTHEHOSTENVIRONMENTBYhPAT TERNRECOGNITIONRECEPTORSv022 ;=0REVIOUSLY THEMOLECULARTARGETWAS CONSIDERED TO BE DOUBLE STRANDED VIRAL 2.! DS2.! RECOGNIZED BY THE 022 TOLL LIKE RECEPTOR  4,2 ; =! ROLE FOR4,2 WAS QUESTIONED HOWEVER GIVENTHATTHECONCENTRATIONOFDS$.!ISUNLIKELYTOBESUFFICIENT TO SIGNAL 4,2 ;= )T IS NOW CONSIDERED THAT INFLUENZA ! VIRUS INFECTION DOES NOT GENERATE DS2.! AT ALL ;= )NSTEAD THE INFLUENZA ! VIRUS POLY MERASEGENERATESSS2.!WITHAN UNCAPPED  PHOSPHATETHATSERVESASTHE MOLECULARSIGNATUREIDENTIFIEDBYTHEIMMUNESYSTEM;=4HECYTOPLASMIC 2.!HELICASE 2)' ; = BUTNOT-$!; = ISRESPONSIBLEFORINFLU ENZA! VIRUS RECOGNITION WHICH OCCURS INDEPENDENTLY OF VIRAL REPLICATION ;= )N ADDITION TO 2)'  4,2 IS IMPLICATED IN INFLUENZA! VIRUS DETEC TION%XPRESSEDINTHEENDOSOMALCOMPARTMENTSOFPLASMACYTOIDDENDRITIC CELLS$#S AND"CELLS 4,2DETECTSINFLUENZA!VIRUSSS2.!; =4HE PARTICIPATIONOFMULTIPLE022INTHESURVEILLANCEOFINFLUENZA!VIRUSMAY REFLECT CELL TYPE SPECIFIC ROLES ;= /NCE INFLUENZA ! VIRUS IS RECOGNIZED 022 INITIATE MULTIPLE SIGNALING CASCADES THAT FACILITATE BOTH INNATE AND ADAPTIVEIMMUNITYTOENABLEVIRALERADICATION

)NNATEIMMUNITYANDTHEINFLUENZA!VIRUSES )NNATE IMMUNITY DIRECTED AGAINST INFLUENZA ! VIRUS PROVIDES AN IMMEDI ATEANDRAPIDRESPONSETOTHEPATHOGEN4HEPULMONARYINFILTRATEOFINNATE IMMUNECELLSISCOMPRISEDMAINLYOFNATURALKILLER.+ CELLS NEUTROPHILS AND MACROPHAGES4HE .+ CELL REPRESENTS THE MAJOR INNATE RESPONSE ELE MENT AND ARE DETECTED IN THE INFECTED LUNG AS EARLY AS  H FOLLOWING INFLUENZA! VIRUS INFECTION ; = 0ROTECTION IS THOUGHT TO BE MEDIATED BYBOTHCYTOKINEPRODUCTION;INTERFERON)&. aANDTUMORNECROSISFACTOR

4HEIMMUNERESPONSETOINFLUENZA!VIRUSES



&IGURE3UMMARYOFTHEHOSTIMMUNERESPONSETOINFLUENZA!VIRUS

4.& _ = AND DIRECT CYTOTOXICITY OF VIRUS INFECTED CELLS ;= )NFLUENZA ! VIRUSnINFECTED CELLS ARE RECOGNIZED BY .+P ;= AND .+P ;= INTER ACTION WITH (!4HE CRITICAL ROLE FOR THIS PATHWAY IN INFLUENZA CONTROL IS ILLUSTRATEDBYTHEFATALINFECTIONTHATOCCURSINMICETHATLACK.+P;= 4OGETHER WITH .+ CELLS NEUTROPHILS ALSO CONTRIBUTE TO INFLUENZA ! VIRUS CLEARANCETHROUGHTHESECRETIONOFANARRAYOFPRO INFLAMMATORYMOLECULES THAT SERVE TO LIMIT VIRAL REPLICATION ;n= &INALLY ALVEOLAR MACROPHAGES !- AREALSOPRESENTINTHEINNATEPULMONARYINFILTRATE ALTHOUGHINITIALLY THEYFORMONLYASMALLCONTRIBUTION BUTARERECRUITEDINLARGENUMBERSLATER BYTHE4CELLRESPONSE!-REPRESENTTHEMAJORPHAGOCYTICCELLTYPERESIDENT INTHELUNG;= ACTINGTOSCAVENGEINFLUENZA!VIRUS DERIVED ANTIGEN;=)N ADDITION !- SECRETE PRO INFLAMMATORY CYTOKINES INCLUDING4.& _ INTER LEUKIN ), ` ),  AND )&. _` ; = TOGETHER WITH THE CHEMOKINES MACROPHAGEINFLAMMATORYPROTEIN-)0 A MONOCYTECHEMOTACTIC PROTEIN -#0  2!.4%3AND)&. INDUCIBLEPROTEIN)0 ;  n=4HE !-CANALSOMODULATEADAPTIVE4CELLIMMUNITYTOINFLUENZA!VIRUSES;= 0RESENTINTHELUNGDURINGACTIVEVIRALREPLICATION !-AREFULLYSUSCEPTIBLE TO INFLUENZA! VIRUS INFECTION ;= 5NLIKE IN EPITHELIAL CELLS HOWEVER THE INFECTIONISNON PRODUCTIVEWITHLITTLE IFANY VIRIONRELEASE ; = ALTHOUGH IT DOES LEAD TO SUBSEQUENT APOPTOSIS ;= $EPLETION OF MACROPHAGES DUR INGINFLUENZA!VIRUSINFECTIONRESULTSINELEVATEDVIRALTITERSANDINCREASED MORBIDITYANDMORTALITY ILLUSTRATINGTHEIRPARTICIPATIONINTHERESPONSE;= 4HEREFORE MULTIPLE IMMUNE CELL TYPES PROVIDE IMMEDIATE INNATE DEFENSE AGAINSTTHEINFLUENZA!VIRUSES



*USTINE$-INTERNETAL

4HEPULMONARYINFILTRATERELEASESATORRENTOFINNATEIMMUNEMOLECULES THATARECONSIDEREDTOLIMITINFLUENZA!VIRUSINFECTION!LONGLISTOFCYTO KINES AND CHEMOKINES ARE POTENTIALLY INVOLVED ! MAJOR PLAYER IS TYPE ) )&. REPRESENTING THE MOST POTENT CYTOKINE ATTACK AGAINST THE VIRUS ;= 3O POTENT IS THE )&. RESPONSE THAT THE INFLUENZA! VIRUSES ENCODE A PRO TEIN.3 TODISABLETHISPATHWAYDESCRIBEDIN)NFLUENZA!6IRUS%SCAPE BELOW .ASALANDPULMONARY)&. _AND `RISERAPIDLYFOLLOWINGINFLUENZA !VIRUSINFECTION;=ANDACTTODIRECTLYLIMITVIRALREPLICATIONANDINDUCE FURTHER CYTOKINES ANDOR CHEMOKINE SECRETION THAT ENHANCES RECRUITMENT ANDACTIVATIONOFMULTIPLEIMMUNECELLTYPES4YPE))&.SERVES TOENHANCE MACROPHAGE FUNCTION PROMOTE ANTIGEN PRESENTATION BY ANTIGEN PRESENT ING CELLS !0# AND TO MODULATE ADAPTIVE IMMUNITY 4HE IMPORTANCE OF THISPATHWAYISEXEMPLIFIEDBYTHESEVEREPULMONARYDISEASETHATDEVELOPS FOLLOWING INFLUENZA ! VIRUS INFECTION OF MICE WITH DISRUPTED TYPE ) )&. SIGNALING; =0LASMACYTOID$#SARETHEMAJORPRODUCERSOFF TYPE))&. IN RESPONSE TO MANY VIRUSES INCLUDING INFLUENZA ! VIRUS ;n= /THER CYTOKINESIMPLICATEDININFLUENZA!VIRUSIMMUNITYINCLUDE4.& _ ;= ), ; = ), ;= ), ;=AND), ; =)NCONTRAST MICETHATLACK FUNCTIONAL )&. a CAN EFFICIENTLY CLEAR INFLUENZA! VIRUSES SUGGESTING ONLY AMINORORREDUNDANTROLEFOR)&. aINTHERESPONSE;n=#HEMOKINES WITHDEFINEDROLESININFLUENZA!VIRUSIMMUNITYINCLUDE-)0 _ ;=AND ##2;= ASILLUSTRATEDBYTHEELEVATEDDISEASEBURDENFOLLOWINGINFECTION OF THE CHEMOKINE DEFICIENT MICE &INALLY WHILE CYTOKINES AND CHEMOKINES AREIMPORTANTINTHEIMMUNECONTROLOFINFLUENZA!VIRUSINFECTIONS THEIR CONTRIBUTION CAN BE DETRIMENTAL AS THEY ELICIT POTENTIALLY FATAL @CYTOKINE SHOCK;=2ECENTSTUDIESDRAMATICALLYILLUSTRATETHEDEVASTATINGIMPACTOF INCREASEDINFLAMMATORYINFILTRATESUPONVIRAL INDUCEDPATHOLOGY)NANIMAL Y MODELS INFECTION WITH THE RECONSTRUCTED  INFLUENZA! VIRUS PROMOTES MASSIVEINFLAMMATORYINFILTRATESWITHSIGNIFICANTLYHIGHERLEVELSOFCYTOKINES )&. a 4.& _ ),  ),  ),  ), ANDGRANULOCYTE COLONYSTIMULATING FACTOR ANDCHEMOKINES-)0  -)0 _` -#0  ; n=4HEREFORE PARTICULARLYEARLYON POTENTINFLAMMATORYANTI VIRALACTIVITY MAYBEDANGER OUS RATHERTHANPROTECTIVE TOTHEHOSTDUETOTHEDELETERIOUSIMPACTUPON LUNGPATHOLOGY #OLLECTINS ARE COLLAGEN LIKE LECTINS THAT PARTICIPATE IN INNATE IMMUNITY TOVIRALPATHOGENS;=#OLLECTINFAMILYMEMBERS THESURFACTANTPROTEINS! 30 ! AND30 $ ARECONSTITUTIVELYPRESENTINTHEFLUIDSTHAT LINETHERESPI RATORYTRACT;=4OGETHERWITHTHEMANNANBINDINGLECTIN-", 30 !AND 30 $CONTRIBUTETOINFLUENZA!VIRUSCLEARANCEVIAANUMBEROFMECHANISMS (EMAGGLUTINATIONANDVIRALINFECTIVITYISINHIBITEDBY30 !; = 30 $ ; = AND -", ;  = )N ADDITION COMPLEMENT MEDIATED LYSIS OF INFLUENZA!VIRUS INFECTEDCELLSISENHANCEDBY-",;= WHILE30 !AND 30 $PROMOTETHEBINDINGANDUPTAKEOFINFLUENZA!VIRUSESBYNEUTROPHILS ; =AND30 !PROMOTESOPSONIZATIONANDPHAGOCYTOSISOFINFLUENZA! F VIRUS BY THE!- POPULATION ;=4HE SENSITIVITY OF DIFFERENT INFLUENZA!

4HEIMMUNERESPONSETOINFLUENZA!VIRUSES



VIRAL STRAINS TO COLLECTIN MEDIATED DEFENSE CORRELATES WITH THE DEGREE OF GLYCOSYLATIONOFTHE(!GLYCOPROTEIN; = $EFENSINS ARE CATIONIC PEPTIDES PRODUCED BY BOTH LEUKOCYTES AND EPITHELIAL CELLS $EFENSINS CAN EXERT DIRECT MICROBIAL ACTIVITY OR PROMOTE IMMUNITYBYACTINGASCHEMOTACTICAGENTS%XAMPLESOFDEFENSIN MEDIATED ANTI INFLUENZA!VIRUSACTIVITYINCLUDERETROCYCLIN e DEFENSIN ANDHUMAN `DEFENSININHIBITIONOF(! MEDIATEDMEMBRANEFUSION;=4HE HUMAN NEUTROPHIL PEPTIDE (.0  _ DEFENSIN DIRECTLY INACTIVATES INFLUENZA! VIRUSBYANUNKNOWNMECHANISM;=

(UMORALIMMUNITYANDTHEINFLUENZA!VIRUSES (UMORALIMMUNITYPROVIDESHOSTDEFENSETHROUGH"LYMPHOCYTESECRETION OF ANTIBODY 0ROTECTIVE ANTIBODIES TARGET ANTIGENIC STRUCTURES EXPOSED ON THEPATHOGENSURFACE!NTIBODY MEDIATEDIMMUNITYCONTRIBUTESTODEFENSE AGAINSTTHEINFLUENZA!VIRUSES;n= BUTISNOTALWAYSESSENTIALFOROPTIMAL VIRALCLEARANCE; =)NANYCASE THEINFLUENZA!VIRUSES ELICITADIVERSE SPECTRUM OF ANTI VIRAL ANTIBODY RESPONSES .ATURAL ANTIBODIES PRESENT THE FIRSTLINEOFANTIBODY MEDIATEDDEFENSE;=4HESEARELOW AFFINITYANTIBOD F IESTHATRESTRICTEARLYVIRUSDISSEMINATION;=ANDPROMOTETHERECRUITMENT OF VIRAL ANTIGEN TO THE SECONDARY LYMPHOID ORGANS ;= .ATURAL ANTIBOD IESREDUCETHEOVERALLLOADOFINFLUENZA!VIRUSAND ASSUCH AREREQUIRED FOR OPTIMAL SPECIFIC )G' ANTIBODY RESPONSES ; = 3ECRETION OF NATURAL ANTIBODIESREQUIRESTHETRANSCRIPTIONALREPRESSOR"LIMP MICE WITH"LIMP  DEFICIENT"CELLSAREMORESUSCEPTIBLETOINFLUENZA!VIRUSINFECTION;= !LTHOUGHNATURALANTIBODIESAREINVOLVEDINTHEPRIMARYRESPONSETOINFLU ENZA!VIRUSES THEYARENOTREQUIREDFOROPTIMALPROTECTIONFROMSECONDARY CHALLENGE;=7HILENATURALANTIBODIESCLEARLYDISPLAYANTI VIRALPROPERTIES EFFECTIVE VIRUS CLEARANCE REQUIRES THE INDUCTION OF NEUTRALIZING ANTIBODY 3UCHNEUTRALIZINGANTIBODIESCANBERAPIDLYINDUCEDANDPOSSESSHIGHAFFIN ITY OR AVIDITY FOR VIRAL ANTIGEN -OSTLY VIRUS NEUTRALIZATION IS THOUGHT TO BEOPTIMALLYACHIEVED VIA ANTIBODY MEDIATEDINTERFERENCEWITHVIRALBIND ING TO THE HOST RECEPTORS REQUIRED FOR CELL ENTRY OR EGRESS #ONSEQUENTLY THEINFLUENZAVIRUS(!ISHEAVILYTARGETEDBYNEUTRALIZINGANTIBODIES;= #RYSTALLOGRAPHICEXAMINATIONOF(!INCOMPLEXWITHNEUTRALIZINGANTIBOD IES REVEALSTHATANTIBODYBINDINGCANOCCURATTHESAMESITEASHOSTRECEPTOR BINDING ;= OR IN DISTAL REGIONS WHERE RECEPTOR BINDING IS OBSTRUCTED BY STERIC HINDRANCE ;= 3IMILAR TO (! .! IS ALSO TARGETED BY NEUTRALIZING ANTIBODIES ;= .EUTRALIZING ANTIBODIES REPRESENT THE MAJOR TARGET OF CUR RENTINFLUENZA!VIRUSVACCINESTRATEGIES7HILEMOSTNEUTRALIZINGANTIBODY STRATEGIES TARGET (! OR .! ;= THE MATRIX PROTEIN  - REPRESENTS AN INTERESTINGPOTENTIALVACCINECANDIDATE;=-ISATRANSMEMBRANEPROTEIN EXPRESSEDATTHEINFECTEDCELLSURFACE;= BUTINCONTRASTTO (!AND.! IS HIGHLYCONSERVEDAMONGSTINFLUENZA!VIRUSSTRAINS5NFORTUNATELY THUSFAR



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- TARGETEDVACCINESTRATEGIESHAVEELICITEDONLYWEAKIMMUNITYTHATDOES NOTPROTECTMICEFROMLETHALCHALLENGE;= #$ 4 HELPER CELLS CONTRIBUTE TO HUMORAL IMMUNITY BY PROMOTING " CELLDIFFERENTIATIONINTOIMMUNOGLOBULINCLASS SWITCHED ANTIBODY SECRETING CELLS )N MOST STUDIES THE PRODUCTION OF ANTI INFLUENZA! VIRUS ANTIBODY IS #$ 4 CELL DEPENDENT ; n= ALTHOUGH EXCEPTIONS ARE REPORTED ; =#LASSICALLY #$ 4CELLHELPINVOLVESI THERECOGNITIONOFVIRALANTIGEN ANDII THEDELIVERYOFANACTIVATIONSIGNALTOTHE"CELL VIATHE4.&2FAM ILYMEMBER #$-ICEDEFICIENTIN#$GENERATESIGNIFICANTLY IMPAIRED INFLUENZA ! VIRUS SPECIFIC ANTIBODY RESPONSES ; = /F INTEREST #$ 4 CELLS CAN HELP " LYMPHOCYTES BY NON COGNATE INTERACTIONS THAT DO NOT REQUIRESPECIFICINFLUENZA!VIRUSANTIGENRECOGNITION;=

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4HEIMMUNERESPONSETOINFLUENZA!VIRUSES



OFINFLUENZA!VIRUS DERIVEDANTIGENINTHELUNGDRAININGLYMPHNODECEASES BEYONDnDAYS; = ALTHOUGHRECENTLYITHASBEENSUGGESTEDTHAT ANTIGENPRESENTATIONCANOCCURFORUPTOMONTHSFOLLOWINGINFECTION;= -(#CLASS))PRESENTATIONISALSOREPORTEDTOPERSISTFORASLONGASWEEKS AFTERINFECTION;=4HISISSURPRISINGGIVENTHATINFECTIOUSVIRUSISCLEARED BYDAY;=4HEREFORE ITHASBEENPOSTULATEDTHATTHERESPIRATORYTRACT $#SCANSERVEASARESERVOIRFOR ANTIGEN WITHADEPOTBEINGMAINTAINEDWELL BEYONDTHECLEARANCEOFPATHOGENFROMTHEINFECTEDRESPIRATORYTISSUE; =4HISHOWEVER REMAINSTOINDEPENDENTLYVERIFIED

#OSTIMULATION 4HEPARTICIPATIONOF$#SINADAPTIVEIMMUNITYISCRITICALDUETOTHERICHARRAY OF COSTIMULATORY MOLECULES EXPRESSED AT THE CELL SURFACE! GROWING LIST OF COSTIMULATORYMOLECULESHASBEENIDENTIFIED MOSTOFWHICHBELONGTOEITHER THE#$";=OR4.&2;=FAMILIES#OSTIMULATIONSERVESTOENHANCE THE ANTIGEN SPECIFIC SIGNALS THAT ARE DELIVERED THROUGH THE 4 CELL RECEPTOR 4#2 !S SUCH COSTIMULATION IS REQUIRED FOR OPTIMAL4 CELL IMMUNITY IN MANY VIRAL INFECTIONS ;=4HE MAJOR PATHWAY OF COSTIMULATION IS VIA THE #$"INTERACTIONTHATPLAYSANIMPORTANTROLEININFLUENZA! VIRUSIMMU NITY4HISSIGNALCONTRIBUTESTOTHEGENERATIONOFINFLUENZA!VIRUS SPECIFIC4 CELLIMMUNITYATMULTIPLELEVELS&OR#$ 4CELLS #$"CONTRIBUTESTO EXPANSION;n= CYTOTOXICITYANDOREFFECTORCYTOKINEPRODUCTION;  = RECRUITMENT TO THE INFECTED AIRWAYS ;= AND SURVIVAL ;= )N CONTRAST THEHIERARCHYOF4CELLRESPONSEMAGNITUDETOINDIVIDUALINFLUENZA ! VIRUS DERIVED EPITOPES A PHENOMENON TERMED IMMUNODOMINANCE ; = ISNOTALTEREDINTHEABSENCEOF#$"SIGNALING;= -ICEDEFICIENT IN #$" ALSO DISPLAY IMPAIRED INFLUENZA SPECIFIC NEUTRALIZING ANTIBODY RESPONSES;=7HILE#$"PLAYSAPROMINENTPARTEARLYINRESPONSETO INFLUENZA! VIRUS INFECTION """", IS IMPORTANT FOR SUSTAINED #$ 4CELLEXPANSIONANDISCRITICALFOROPTIMALRECALLRESPONSES;  = %FFECTIVE #$ 4 CELL IMMUNITY DURING INFLUENZA ! VIRUS INFECTION ALSO REQUIRES#$";= /8/8,;=AND)#/3)#/3,;= MEDI ATEDCOSTIMULATION4HEACCUMULATIONOF4CELLSININFLUENZA!VIRUS INFECTED LUNGSDEPENDSON#$#$SIGNALING; =4HISISDUETOITSIMPACT ON4 CELL SURVIVAL ANDOR MIGRATION TO THE INFECTED RESPIRATORY TRACT ;= 4OGETHER MULTIPLE COSTIMULATORY SIGNALS ARE DELIVERED VIA THE $#S TO PRO MOTEOPTIMALADAPTIVEIMMUNITYAND INTURN INFLUENZA!VIRUSELIMINATION

#$ 4CELLS %FFECTOR#$ 4CELLS ALSOKNOWNASCYTOLYTIC4LYMPHOCYTES#4, ARE IMPORTANTINTHENORMALCLEARANCEOFINFLUENZA!VIRUSES;= -ICEDEFI



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CIENTIN#$ 4CELLSSHOWDELAYEDINFLUENZA!VIRUSCLEARANCE ALTHOUGH THEY EVENTUALLY CONTROL INFECTION WITH ALL BUT THE MOST VIRULENT VIRUSES ;=4HEINFLUENZA!VIRUS SPECIFIC#$ 4CELLRESPONSEHASBEENEXTEN SIVELYCHARACTERIZEDUTILIZINGMURINEMODELSOFINFECTION PARTICULARLYWITH THE(+X(. AND02(. INFLUENZA!VIRUSES#$ 4CELLSARE PRIMED ACTIVATEDANDEXPANDIN THELUNGDRAINING LYMPHNODESDURINGTHE FIRSTWEEKORSOAFTERPRIMARYINFECTION; =!CTIVATED#$ 4CELLS THEN TRAFFIC TO THE RESPIRATORY AIRWAYS AND THE INFECTED LUNG TO MEDIATE VIRALCLEARANCE;=4HETRAFFICKING;=ANDRETENTIONOF#$ 4CELLSIN THELUNG;= ISDEPENDENTON,&! EXPRESSION!TTHESITEOFINFECTION #$ 4CELLSTARGETVIRUS INFECTEDCELLSTHATEXPRESSPEPTIDEDERIVEDFROM INFLUENZA!VIRUSPROTEINASSOCIATEDWITH-(#CLASS)!NARRAYOFEPIT OPESISRECOGNIZEDINTHE#"," MOUSEMODEL WITHTHEDOMINANT EPITOPES IN TERMS OF RESPONSE MAGNITUDE SEEN BY #$ 4 CELLS BEING PROVIDED BY THE VIRAL POLYMERASE! 0!n ;= AND NUCLEOPROTEIN .0n ; = 3UBDOMINANT EPITOPES ARE DERIVED FROM THE BASIC POLYMERASE SUBUNIT  0"n ;= THE MITOCHONDRIAL PROTEIN 0" &n ; = NON STRUCTURAL PROTEIN  .3n ;= AND MATRIX PROTEIN  -n ;= )N THE ABSENCE OF THE DOMINANT EPITOPES SUBDOMINANT EPITOPE SPECIFIC #$ 4 CELLS ACCOUNT FOR A COMPENSATORY RESPONSE ALTHOUGHASLIGHTDELAYINVIRALCLEARANCEISOBSERVED; = $EPENDINGONTHEEXPERIMENTALMODEL nOF#$ 4CELLSRECOVERED FROMTHERESPIRATORYTRACTAREINFLUENZA!VIRUSSPECIFICATTHEPEAKOFTHE PRIMARY RESPONSE ILLUSTRATING THEIR ENRICHMENT IN THE PNEUMONIC LUNG ;   = %PITOPE SPECIFIC #$ 4 CELLS CAN BE FOUND WIDELY DISPERSEDTHROUGHOUTVARIOUSBODYORGANS INCLUDINGTHELUNG SPLEEN BONE MARROW BLOOD LIVERANDNON DRAININGLYMPHNODES; =/NCETHEIR TARGET ANTIGEN IS RECOGNIZED #$ 4 CELLS EXERT MULTIPLE EFFECTORS FUNC TIONS#YTOKINESSUCHAS)&. a 4.& _ AND), ARESECRETEDBYINFLUENZA ! VIRUS SPECIFIC #$ 4 CELLS ;= )N ADDITION #$ 4 CELLS MEDIATE DIRECTCYTOLYSISOFINFLUENZA!VIRUS INFECTEDTARGETCELLSBYTHEEXOCYTOSIS OFCYTOLYTICGRANULESTHATCONTAINPERFORINANDGRANZYMES; =ANDOR THROUGHTHEEXPRESSIONOF&AS,;n= &OLLOWING INFLUENZA ! VIRUS CLEARANCE VIRUS SPECIFIC #$ 4 CELLS DECREASE IN NUMBER UNTIL A PLATEAU IS REACHED APPROXIMATELY  MONTHS FOLLOWING INFECTION ; = !FTER PRIMARY INFECTION THE CO DOMINANT $B.0n AND $B0!n SPECIFIC #$ 4 CELL POPULATIONS CONTRACT AT THESAMERATE;=TOMEMORYPOOLSTHATAREAPPROXIMATELYEQUIVALENTIN NUMBERANDREPRESENTSOFTHEPOPULATIONATTHEPEAKOFTHERESPONSE ;=)NFLUENZA!VIRUS SPECIFIC#$ 4CELLSPERSISTASASTABLEPOPULATION FOR THE LIFE OF A LABORATORY MOUSE ;  = 2ETENTION OF MEMORY #$ 4 CELLS IN NON LYMPHOID TISSUE SUCH AS THE LUNG IS MEDIATED BY 4 CELL EXPRESSION OF6,!  ;= 3ECONDARY CHALLENGE RECRUITS THE MEMORY #$ 4CELLSTHATEXPANDINTHELYMPHNODESANDPROMOTEVIRALCLEARANCE

4HEIMMUNERESPONSETOINFLUENZA!VIRUSES



APPROXIMATELYDAYSEARLIERTHANAFTERPRIMARYINFECTION;=$URINGSEC ONDARYINFECTION THE.0n#$ 4CELLPOPULATIONISCLEARLYDOMINANT REPRESENTINGUPTOOFTHEVIRUS SPECIFIC#4,RESPONSES;   =4HISDOMINANCEISMAINTAINEDINTHEMEMORYPOPULATIONSTHATPERSIST FOLLOWING THE PEAK OF THE SECONDARY RESPONSE DAY  ;= 4HE SKEWED IMMUNODOMINANCE HIERARCHY OBSERVED IN SECONDARY VERSUS PRIMARY INFLU ENZA!VIRUSINFECTIONWASINITIALLYTHOUGHTTOBELARGELYACONSEQUENCEOF DIFFERENTIALANTIGENPRESENTATION;= ALTHOUGHITISNOWCONSIDEREDTHAT4 CELLPRECURSORFREQUENCYANDANTIGENDOSEARELIKELYTOBEIMPORTANTDETER MININGVARIABLES;=

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)NFLUENZA!VIRUSESCAPE 4HEMAJORINFLUENZA!VIRUSESCAPEMECHANISMRESTSINTHEINHERENTGENETIC VARIATIONOFTHESE2.!VIRUSES COMBINEDWITHTHESELECTIVEPRESSUREEXERT ED BY (! SPECIFIC NEUTRALIZING ANTIBODY ;n=4HIS PROCESS IS KNOWN AShANTIGENIC DRIFTv ,ACKING PROOF READING CAPACITY THE INFLUENZA! VIRUS 2.!POLYMERASEPROMOTESTHEACCUMULATIONOFNUCLEOTIDEPOINTMUTATIONS 3UCH MUTATIONS GENERATE APPROXIMATELY  AMINO ACID SUBSTITUTIONS PER YEAR;=#IRCULATINGVIRALSUBTYPESARETHENSELECTEDWHERESUBSTITUTIONS HAVE OCCURRED THAT MAINTAIN VIRAL FITNESS ;= BUT ABROGATE IMMUNE REC OGNITION&OREXAMPLE VIRUSESCAPEMUTANTSAREPOORLYRECOGNIZEDBYNEU TRALIZING ANTIBODY DUE TO I INTRODUCED STERIC INTERFERENCE WITH ANTIBODY BINDING;= II VIRUSCONFORMATIONALCHANGESTHATRENDERANTIBODYBINDING ENERGETICALLYUNFAVORABLE;= ORIII THEINTRODUCTIONOFNEWOLIGOSACCHA RIDEATTACHMENTSITESTOSURFACEGLYCOPROTEINSTHATOBSCUREANTIBODYBIND ING; =2ETENTIONOFAMINOACIDSUBSTITUTIONSATTHE(!MEMBRANE DISTALSURFACE ANAREATARGETEDBYANTIBODIES ISFAVOREDOVER THOSEBURIED WITHINTHEPROTEIN;=6IRUS SPECIFIC#4,IMMUNITYCANALSOBETARGETEDBY ANTIGENICDRIFT;=(ERE VIRUSESARESELECTEDWITHMUTATIONSTHATINTERFERE WITH EPITOPE BINDING TO -(# CLASS ) OR WITH EPITOPES THAT ARE NO LONGER RECOGNIZED BY THE 4#2 4HE .0n ; = AND .0n ; = #4,PEPTIDESHAVEBOTHSHOWNEVIDENCEOFANTIGENICDRIFT(YPERVARIABILITY WITHIN A #4, EPITOPE CORRELATES WITH THE FUNCTIONAL AVIDITY OF THE 4#2 ;= 3UCH ANTIGENIC DRIFT CAN FUNCTION TO LIMIT CROSS PROTECTIVE IMMUNITY AGAINSTMULTIPLEINFLUENZA!VIRUSSTRAINSAND ASACONSEQUENCE CONTRIBUTE TOSEASONALEPIDEMICS 7HILEANTIGENICDRIFTREPRESENTSASUBTLEMODEOFIMMUNEESCAPE INFLU ENZA!VIRUSESCANALSOUNDERGOMAJORANTIGENICVARIATIONTOOUTMANEUVER THEIMMUNESYSTEM4HISTAKESPLACEBYhANTIGENICSHIFTv WHEREINFECTIONOF THESAMECELLWITH TWODISTINCTINFLUENZA!VIRUSSTRAINSALLOWSREASSORTMENT OFTHEVIRALGENOMICSEGMENTS GENERATINGANEWHYBRIDINFLUENZA!VIRUS 2EASSORTMENTCANOCCURFOLLOWINGINFECTIONWITHDIFFERENTSPECIES ADAPTED VIRUSES&OREXAMPLE PIGSCANBEINFECTEDWITHBOTHHUMANAND AVIANINFLU ENZA ! VIRUSES 3IMULTANEOUS INFECTION MAY THEREBY GENERATE A REASSORT MENTVIRUSWHERETHEhHUMANvPATHOGENACQUIRESANhAVIANvVIRUS (!OR .!GENE)NTHISCASE FORTHE(!AND.!INPARTICULAR THEREWOULDBENO PREVAILINGIMMUNITYINTHEHUMANPOPULATIONLEADINGTOTHEPOSSIBILITYOFA HUMANPANDEMIC; =3UCHANTIGENICSHIFTINVOLVINGAVIANANDHUMAN STRAINSHASBEENIMPLICATEDINTWOOFTHEINFLUENZA!VIRUSPANDEMICSTHAT HAVEOCCURREDINTHETHCENTURYTHE(.; =AND (. ; = INFECTIONS /F INTEREST THE INFLUENZA! VIRUS THAT PROVOKED THE PANDEMICDIDNOTARISETHROUGHANTIGENICSHIFT)NSTEAD THE(. VIRUS THATWASRESPONSIBLEFORMILLIONSOFDEATHSWORLDWIDE ISBELIEVEDTO BE AN ENTIRELY AVIAN VIRAL STRAIN THAT MUTATED IN A WAY THAT ALLOWED IT TO INFECTHUMANS; =

4HEIMMUNERESPONSETOINFLUENZA!VIRUSES



4HE NONSTRUCTURAL PROTEIN  .3 ENCODED BY INFLUENZA! VIRUS PRO VIDESAMODEOFIMMUNEESCAPETHATDOESNOTREQUIREMANIPULATIONOFTHE GENOME .3 INHIBITS THE HOST CELL )&. _` RESPONSE ; = A MAJOR PATHWAY OF IMMUNE DEFENSE AGAINST THE VIRUS AS DISCUSSED 4YPE ) )&. INDUCTIONISANTAGONIZEDBY.3 MEDIATEDSUPPRESSIONOF)&. INDUCEDPRO TEINSDS2.! ACTIVATEDPROTEINKINASE   OLIGO! SYNTHETASE;n= THETRANSCRIPTIONFACTORS.&g";=ANDTHE)&.REGULATORYFACTOR ;= #ONTAININGAN2.! BINDINGDOMAINATITS.TERMINUS;= ITWASPREVIOUS LYCONSIDEREDTHAT.3SEQUESTEREDINFLUENZA!VIRUSDS2.!;=)NSTEAD .3FORMSACOMPLEXWITH2)'  THECELLULARSENSOROFINFLUENZA!VIRUS UNCAPPED SS2.! ;=4HEREFORE .3 ACTS TO DISABLE THE HOST MECHANISM FORDETECTIONOFVIRUS DERIVED2.!ANDTHEINDUCTIONOFTHE)&.RESPONSE )NFLUENZA!VIRUSESLACKINGTHE.3PROTEINAREGOODVACCINECANDIDATES AS THEABSENCEOFTHISIMMUNOMODULATORYPROTEINGREATLYENHANCESTHEIMMU NOGENICITYOFTHEVIRUS;=

(ETEROTYPICINFLUENZA!VIRUSIMMUNITY (ETEROTYPICIMMUNITYINTHISSYSTEMISDEFINEDBYCROSS REACTIVE PROTECTIVE RESPONSESBETWEENSEROLOGICALLYDIFFERENT(! DISTINCT INFLUENZA!VIRUSES )T WOULD OBVIOUSLY BE ADVANTAGEOUS IF FOR EXAMPLE PRIOR INFECTION WITH A HUMAN INFLUENZA! VIRUS COULD GENERATE IMMUNE MEMORY THAT PROVIDES AT LEASTSOMERESISTANCETOAHIGHLYPATHOGENICAVIANVIRUSTHATSUDDENLYADAPT ED TO TRANSMIT BETWEEN PEOPLE ; = #LEARLY PROMOTING HETEROTYPIC IMMUNITYISADESIRABLESTRATEGYFORINFLUENZA!VIRUSVACCINEDEVELOPMENT $ESCRIBED MANY DECADES AGO ;= HETEROTYPIC IMMUNITY HAS NOW BEEN SHOWNFORMANYINFLUENZA!VIRUSCOMBINATIONS;n=!TLEASTINMICE HETEROTYPICIMMUNITYCANBOTHBELONGLASTINGANDPROVIDEPROTECTIONAGAINST OTHERWISE LETHAL VIRUS CHALLENGE 4HE BEST UNDERSTOOD COMPONENT OF SUCH RESPONSES IS #4, IMMUNITY DIRECTED AT GENERALLY CONSERVED INTERNAL VIRAL PROTEINS;  =(OWEVER THEREISALSOEVIDENCEFORTHERETENTIONOFA MEASUREOFHETEROTYPICIMMUNITYINMICELACKING#$ 4CELLS; =)N ADDITIONTOTHE#$ 4EFFECTORS #$ 4CELLS NON NEUTRALIZING)G!ANTIBODY .+4CELLSAND ab 4CELLSHAVEALLBEENCONSIDEREDASPOSSIBLEPLAYERS;= )MMUNIZATIONWITHALOWDOSEOFACOLD ADAPTED ATTENUATEDINFLUENZA!VIRUS PROVIDES ONE VACCINATION STRATEGY THAT HAS THE POTENTIAL TO INDUCE AT LEAST SOME DEGREE OF LONG TERM HETEROTYPIC IMMUNITY ;= 4HE PROMOTION OF SUCHRESPONSESISCLEARLYAWORTHWHILEFOCUSFORFUTUREVACCINATIONSTRATEGIES

#ONCLUSION 4HE INFLUENZA! VIRUSES ARE DANGEROUS PATHOGENS WITH THE POTENTIAL TO PROVOKEDEVASTATINGDISEASE4HECHALLENGEFORTHEMEDICALRESEARCHCOM



*USTINE$-INTERNETAL

MUNITY IS TO DESIGN PREVENTIVE MEASURES AND THERAPEUTIC INTERVENTIONS THAT WILL LIMIT THE SEVERE CONSEQUENCES OF PANDEMIC INFLUENZA ! VIRUS INFECTIONS;=6ACCINESHAVELONGBEENAVAILABLE BUTTHEREISCONSIDER ABLESCOPEFORIMPROVEMENTASTHEYTARGETONLYTHEPREVAILINGINFLUENZA! VIRUSSTRAINS DONOTGIVEBROADIMMUNITYANDWORKPOORLYINTHEELDERLY THE TARGET GROUP THAT IS MOST AT RISK OF FATAL DISEASE )MPROVED VACCINES WILL ONLY EMERGE IF THE DEVELOPMENT STRATEGY IS BASED ON A FIRM UNDER STANDING OF THE HOST IMMUNE RESPONSE TO THE VIRUS -OVING BEYOND THE CURRENTLYAVAILABLEPRODUCTSWILLDEPENDONEXPLOITINGOURUNDERSTANDING OF IMMUNE DEFENSE MECHANISMS AGAINST THIS IMPORTANT AND POTENTIALLY VERY DANGEROUS GROUP OF HUMAN PATHOGENS (ERE WE HAVE BRIEFLY SUM MARIZEDACURRENTVIEWOFHOWTHESEVIRUSESARECONTROLLEDBYELEMENTS OFBOTHTHEINNATEANDADAPTIVEHOSTRESPONSE TOGETHERWITHTHEESCAPE STRATEGIES THAT INFLUENZA ! VIRUSES EXPLOIT TO SURVIVE IN NATURE AND TO MAINTAIN TRANSMISSION AT THE SPECIES LEVEL !N IDEAL VACCINE COULD BE THOUGHTTOINDUCEHIGHLEVELSOFNEUTRALIZINGANTIBODYAND#4, MEMORY 4HISMIGHTOPTIMALLYBEACHIEVEDBYPROMOTINGMOREEFFECTIVE$# VAC CINATION PERHAPSVIATHEPATHWAYOFDRIVINGTHEINNATERESPONSEINWAYS THATENHANCE4CELLIMMUNITY!NIMPORTANTCAVEATIS THOUGH THATMUCH OFOURUNDERSTANDINGOFPARTICULARLY THEINNATEAND4CELLRESPONSESTO THEINFLUENZA!VIRUSESISBASEDONMOUSEEXPERIMENTS!SWEGOFORWARD TODEVELOPVACCINECANDIDATES ITISIMPORTANTTHATTHEANALYSISOFINFLUEN ZAVIRUSCELL MEDIATEDIMMUNITY INPARTICULAR SHOULDBEGREATLYEXTENDED INHUMANSUBJECTS

2EFERENCES   









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*USTINE$-INTERNETAL

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*USTINE$-INTERNETAL

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#ORRELATESOFPROTECTIONAGAINSTINFLUENZA %MANUELE-ONTOMOLI $EPARTMENTOF0HYSIOPATHOLOGY %XPERIMENTAL-EDICINEAND0UBLIC(EALTH ,ABORATORYOF -OLECULAR%PIDEMIOLOGY 5NIVERSITYOF3IENA 6IA!LDO-ORO 3IENA )TALY

!BSTRACT #ORRELATESOFPROTECTIONFORINFLUENZAVIRUSHASNOTBEENDEFINED BUTITISWIDELYBELIEVED THAT PROTECTION AGAINST INFLUENZA CAN BE CONFERRED BY SERUM HEMAGGLUTININ (! ANTI BODIES4HEIMMUNERESPONSESTOINJECTEDINFLUENZAVACCINESARE ROUTINELYASSESSEDUSING SEROLOGICAL (! ANTIBODIES TITRATION )T IS GENERALLY ACCEPTED THAT NEUTRALIZING AND (! ANTIBODIES AS WELL AS ANTIBODIES TO NEURAMINIDASE CAN BE DETECTED IN SERUM AFTER n WEEKSPOSTPRIMARYINFECTIONORVACCINATION 3EROLOGICAL ASSAYS COMMONLY USED TO QUANTIFY ANTIBODIES SPECIFIC FOR INFLUENZA VIRUSESINCLUDEHEMAGGLUTINATIONINHIBITION(!) SINGLERADIALHEMOLYSIS32( MICRO NEUTRALIZATION-. %,)3!AND7ESTERNBLOT THEMOSTWIDELYUSEDASSAYSBEING(!) AND32(%ACHMETHODUSEDFORANTIBODIESTITRATIONHASDIFFERENTCHARACTERISTICS ANDTHE VALIDITYINDEXANDSPECIFICUSESEROEPIDEMIOLOGY SERODIAGNOSIS RESPONSETOVACCINATION ETC HAVETOBECONSIDEREDTOSELECTTHEMOSTSUITABLEONE2ECENTLY%,)3!AND-.TESTS HAVEBEENDEVELOPEDTHANKSTOTHEDISCOVERYOFTHE(!STRUCTURE7HILETHENUMBEROF DATACOLLECTEDBYCONVENTIONALASSAYS(!)AND32( HASPERMITTEDAFAIRLYGOODOPTI MIZATION SEROLOGICALMEASURESAREUSEDTOCHARACTERIZETHENUMBEROFANTIBODIESBEFORE ANDAFTERVACCINATION (!)ISTHEASSAYUSEDMOSTFREQUENTLYFORINFLUENZAANTIBODYTITRATIONHOWEVER ITHAS LOWSENSITIVITYINDETECTINGRESPONSESTOAVIANVIRUSESINMAMMALIANSERAANDALTERNATIVE SEROLOGICAL TESTS ARE NEEDED 32( UTILIZES A COMPLEMENT MEDIATED HEMOLYSIS REACTION TOMEASURETHEAMOUNTOFANTIBODY4HISTESTAPPEARSTOBEASSENSITIVEASTHE-.ASSAY (!)AND32(ASSAYSARENOTFUNCTIONALTESTSFORMEASURINGIMMUNITYTOINFLUENZAAND SUFFERSFROMSEVERALTECHNICALDRAWBACKS $EVELOPMENTOFTHESEASSAYSWILLBEAFURTHERSTEPINPREPARATIONOFNEWINFLUENZA VACCINES PARTICULARLY FOR CELL DERIVED PRODUCTS !DDITIONAL IMMUNOLOGICAL ASSESSMENTS SUCHASCELL MEDIATEDIMMUNITYANDTHEROLEOFNEURAMINIDASE NEEDTOBEEXPLOREDTO GIVEMOREINSIGHTINTOTHEOVERALLEFFECTSOFVACCINATION

)NTRODUCTION 4HE #ENTERS FOR $ISEASE #ONTROL AND 0REVENTION ESTIMATE THAT BETWEEN   AND   PERSONS ARE HOSPITALIZED EACH YEAR BECAUSE OF INFLU



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ENZA!LTHOUGHTHEEXACTTABULATIONSOFILLNESSESANDCOMPLICATIONSATTRIBUT ABLETOINFLUENZAVIRUSINJECTIONARENOTAVAILABLE THEPRECEDINGESTIMATES INDICATETHATMORBIDITYANDMORTALITYCAUSEDBYINFLUENZAAREMAJORHEALTH PROBLEMS 4HE INFLUENZA VIRUS BELONGS TO THE FAMILY OF /RTHOMYXOVIRIDAE AND IS CLASSIFIEDINTOTHREEDIFFERENTTYPES! "AND#ONTHEBASISOFDIFFERENTEPI TOPES THE ANTIGENIC DIFFERENCES IN THEIR RESPECTIVE NUCLEOCAPSID )NFLUENZA !AND"ARETHETWOTYPESOFINFLUENZAVIRUSESTHATCAUSEEPIDEMICHUMAN DISEASE )NFLUENZA ! VIRUSES ARE FURTHER CATEGORIZED INTO SUBTYPES EG (. (. AND(.ONTHEBASISOFTWOSURFACEANTIGENSHEMAGGLUTI NIN(! ANDNEURAMINIDASE.! )NFLUENZA"VIRUSESARENOTCATEGORIZED INTO SUBTYPES 3INCE  INFLUENZA! AND " VIRUSES HAVE BEEN IN GLOBAL CIRCULATION"OTHAREFURTHERSEPARATEDINTOGROUPSONTHEBASISOFANTIGENIC CHARACTERISTICS.EWINFLUENZAVIRUSVARIANTSRESULTFROMFREQUENTANTIGENIC CHANGEIEANTIGENICDRIFT RESULTINGFROMPOINTMUTATIONSTHATOCCURDUR INGVIRALREPLICATION)NFLUENZA"VIRUSESUNDERGOANTIGENICDRIFTLESSRAPIDLY THANINFLUENZA!VIRUSES )NFLUENZAISCHARACTERIZEDBYTHEOCCURRENCEOFFREQUENTUNPREDICTABLE EPIDEMICS AND MUCH LESS FREQUENT WORLDWIDE PANDEMICS %PIDEMICS ARISE BECAUSE DIFFERENT STRAINS OF INFLUENZA ARE CONSTANTLY GENERATED THROUGH ANTIGENICDRIFT ANDINDIVIDUALSARE LESSORNOTATALLPROTECTEDINSOMEYEARS !PANDEMICISRESPONSIBLEFORHIGHERMORBIDITYANDMORTALITYTHANANEPI DEMICBECAUSEITAFFECTSALARGERPROPORTIONOFTHEPOPULATION4HEBURDEN OF EPIDEMICS HOWEVER IS CUMULATIVELY GREATER THAN THAT OF PANDEMICS! WORLDWIDE PANDEMIC IS CAUSED BY THE SPREAD OF A NEW INFLUENZA SUBTYPE ARISINGFROMANTIGENICSHIFT;=7HENSUCHASUBTYPEENTERSTHEPOPULATION THERE IS NO NATURAL IMMUNITY AND THE NEW VIRUS CAN EASILY INFECT EXPOSED INDIVIDUALS !N INFLUENZA PANDEMIC OCCURS WHEN A NOVEL INFLUENZA VIRUS EMERGES AGAINSTWHICHTHEVASTMAJORITYOFTHEWORLDSPOPULATIONHASNOIMMUNITY )FSUCHAVIRUSDEMONSTRATESTHEABILITYTOTRANSMITEFFICIENTLYFROMPERSONTO PERSON THERESULTISA GLOBALOUTBREAKOFDISEASETHATAFFECTS AHIGHPERCENT AGEOFINDIVIDUALSINASHORTPERIODOFTIMEANDISLIKELYTOCAUSESUBSTAN TIALLYINCREASEDMORBIDITYANDMORTALITYINALLCOUNTRIESOFTHEWORLD/VER MILLIONPEOPLEAREESTIMATEDTOHAVESUCCUMBEDTOTHEMOSTDEVASTATING INFLUENZAPANDEMICIN THESO CALLEDh3PANISHFLUvh!SIANFLUvOF HASBEENRESPONSIBLEFORABOUTDEATHSINTHE53!ONLY 0RELIMINARYFINDINGSHAVEIDENTIFIEDTHE( ( ( (AND( SUBTYPES OFINFLUENZA!VIRUSASTHOSEMOSTLIKELYTOBETRANSMITTEDTOHUMANS THERE FOREPRESENTINGAPOTENTIALPANDEMICTHREAT;= )NFLUENZAVIRUSESCAUSEDISEASEACROSSALLAGEGROUPS2ATESOFF INFECTION AREHIGHESTAMONGYOUNGCHILDREN BUTRATESOFSERIOUSILLNESSANDDEATHARE HIGHEST AMONG PERSONS AGED * YEARS AND PERSONS OF ANY AGE WHO HAVE MEDICALCONDITIONSTHATPLACETHEM ATINCREASEDRISKFORCOMPLICATIONSFROM INFLUENZA;=3TUDIESOFMORBIDITYANDMORTALITYASSOCIATEDWITHINFLUENZA

#ORRELATESOFPROTECTIONAGAINSTINFLUENZA



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ISAMONGCHILDREN.EITHERANTIVIRALDRUGHASBEENUSEDWIDELY DUETOTHEIR SPECTRUMOFACTIVITY THERAPIDONSETOFRESISTANCE ANDTHERELATEDADVERSE EFFECTS;=:ANAMIVIRANDOSELTAMIVIRARE.!INHIBITORSWITHACTIVITYAGAINST BOTHINFLUENZA!AND"VIRUSES4HESITEOFENZYMEACTIVITYOFTHEINFLUENZA .!ISHIGHLYCONSTANTBETWEENDIFFERENTTYPES SUBTYPESANDSTRAINSOFINFLU ENZA ANDHASTHEREFOREEMERGEDASTHETARGETOFTHISNEWCLASSOFANTIVIRAL AGENTS EFFECTIVE IN PREVENTION AND TREATMENT )N THE 53 BOTH DRUGS WERE APPROVEDINFORTHETREATMENTOFUNCOMPLICATEDINFLUENZAINFECTIONS :ANAMIVIRWASAPPROVEDFORTHETREATMENTOFPATIENTSAGED* YEARS AND OSELTAMIVIR WAS APPROVED FOR TREATMENT OF PATIENTS AGED *  YEAR AND FOR THEPROPHYLAXISOFPERSONSOFAGE * YEARS4HESEANTIVIRALDRUGSAREONLY EFFECTIVEIFSTARTEDSOONAFTERTHEONSETOFDISEASE )NFLUENZA VACCINATION IS THE PRIMARY METHOD FOR PREVENTING INFLUENZA AND ITS SEVERE COMPLICATIONS6ACCINATION IS ASSOCIATED WITH A REDUCTION IN INFLUENZA RELATEDRESPIRATORYILLNESSANDPHYSICIANVISITSATALLAGES INHOSPI TALIZATIONSANDDEATHSAMONGHIGH RISKPERSONS OTITISMEDIAAMONGCHILDREN ANDWORKABSENTEEISMAMONGADULTS6ACCINATIONWITHINACTIVATEDINFLUENZA VIRUS CURRENTLY REPRESENTS THE MOST IMPORTANT MEASURE FOR REDUCING THE IMPACTOFINFLUENZA 4HETHREECURRENTTYPESOFVACCINESAREINACTIVATEDVACCINE ATTENUATED VACCINE AND VECTOR BASED VACCINES )NACTIVATED VACCINES ARE PRODUCED BY PROPAGATION OF THE VIRUS IN EMBRYONATED HENS EGGS4HE VACCINE IS AVAIL ABLE CONTAINING WHOLE SPLIT CHEMICALLY DISRUPTED AND SUBUNIT PURIFIED SURFACEGLYCOPROTEINS VIRUS$EVELOPMENTOFCELLCULTURE BASED VACCINESIS ANATTRACTIVEALTERNATIVEAPPROACHTOTHEPRODUCTIONOFINACTIVATEDVACCINE PRODUCTION ,IVEATTENUATEDVACCINETHATCANBEADMINISTEREDBYNASALSPRAY ISNEAR ING AVAILABILITY )T HAS BEEN SHOWN TO BE AS EFFICACIOUS AS INACTIVATED VAC CINE;=,IVEINFLUENZAVACCINESELICITSYSTEMICANDLOCALMUCOSALIMMUNE RESPONSES THAT INCLUDE STIMULATING SECRETORY IMMUNOGLOBULIN )G! IN THE RESPIRATORY TRACT A PORTAL FOR THE VIRUS4HEY ALSO ELICIT CELLULAR IMMUNITY WHICHMIGHTPROVIDEBETTERPROTECTIONTHANTHATGIVENBYINACTIVATEDVAC CINES;=,IVEATTENUATEDVACCINESWILLNOTBEUSEDUNTILTHE(.VIRUSHAS BECOME WIDESPREAD AMONG HUMANS SO AS NOT TO INTRODUCE NEW INFLUENZA VIRAL(!AND.!GENESINTOHUMANPOPULATION 4HESE VECTOR BASED VACCINES ARE EGG INDEPENDENT REPLICATION INCOMPE TENTHUMANADENOVIRALVECTOR BASED(INFLUENZAVACCINESANDHAVEBEEN SHOWN TO SUCCESSFULLY INDUCE (! SPECIFIC HUMORAL AND CELLULAR IMMUNE RESPONSES 4HESE VACCINES PROVIDE PROTECTIVE EFFECTS AGAINST HOMOLOGOUS ANDANTIGENICALLYDISTINCT(.STRAINSINAMOUSEMODEL;= 2APIDANDEARLYDIAGNOSISOFINFLUENZAVIRUSINFECTIONISIMPORTANTTOAID SURVEILLANCEOFCIRCULATINGSTRAINSANDENABLETHEEARLYVACCINATIONORPRO PHYLACTICTREATMENTOFHIGH RISKGROUPS,ABORATORYDIAGNOSISOFINFLUENZA IS MADE BY THE DETECTION OF VIRUS IN RESPIRATORY SECRETIONS BY SEROLOGICAL METHODS

#ORRELATESOFPROTECTIONAGAINSTINFLUENZA



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#ORRELATESOFPROTECTIONAGAINSTINFLUENZA



"VIRUS;=)TISPOSSIBLETOINCREASESENSITIVITYBYETHERTREATMENTOFTHE VIRUS BUTTHISCANALSOBEASOURCEOFVARIABILITY ANDPOTENTIALLYREDUCESTHE STRAINSPECIFICITYOFTHETEST; =!NOTHERDISADVANTAGEISTHATSERAMUST BETREATEDTOREMOVENONSPECIFICINHIBITORSBEFOREBEINGTESTED 4HE(!)ASSAYISASEROLOGICALTECHNIQUEUSEDTODETECT(!ANTIBODYIN SERUMRESULTINGFROMINFECTIONORVACCINATIONWITHINFLUENZAVIRUS AND(!) TITERSCORRELATEWITHPROTECTIONFROMINFLUENZAINHUMANS;= 4HISASSAYIS SUITABLEFORSCREENINGALARGENUMBEROFSAMPLESTHEREISAGOODCORRELATION WITH-.TESTSSEEBELOW FORSEASONAL(AND(STRAINSONLYABIOSAFETY LEVELISNEEDEDANDTHISTESTIS%-%!APPROVED !LTHOUGHTHISTESTISASTANDARDTECHNIQUEFORTHEMEASUREMENT OFANTI BODYTOINFLUENZAVIRUS ITSSENSITIVITYISMUCHTOOLOWTODETECTRESPONSES TOAVIANVIRUSESINMAMMALIANSERA SOTHATALTERNATIVESEROLOGICALTESTSARE NEEDED ;= 3TUDIES HAVE SHOWN THAT THE (!) ASSAY SENSITIVITY FOR AVIAN INFLUENZA VIRUSES CAN BE IMPROVED THROUGH THE USE OF SUBUNIT (! RATHER THANINTACTVIRUS;= 4HE (!) ANTIBODY TITER IS EXPRESSED AS THE RECIPROCAL OF THE HIGHEST SERUMDILUTIONSHOWINGCOMPLETEHEMAGGLUTINATIONUSINGFOURVIRALHEMAG GLUTINATIONUNITS!N(!)TITEROFORHIGHERISTAKENASSEROPROTECTIVE ANDAFOURFOLDINCREASEIN(!)TITERINSAMPLESTAKENAFTERANDBEFOREVAC CINATIONISTHEMINIMUMINCREASECONSIDEREDNECESSARYFORCLASSIFICATIONOF SEROCONVERSION 4HE(!)TESTHASANUMBEROFADVANTAGES)TCANBEPERFORMEDWITHA VARIETYOFERYTHROCYTESPECIESANDTHETESTISSIMPLETOPERFORMANDISHIGHLY REPRODUCIBLE4HETESTISSTRAINSPECIFIC 4HIS ASSAY UNDERESTIMATES ANTIBODY FOR PANDEMIC STRAINS BECAUSE THE EFFICIENCYOFBINDINGOFINFLUENZAVIRUSISDEPENDENTONTHESPECIFICITYOFTHE SIALICACIDCELLULARRECEPTOR4HERECEPTORSPECIFICITYOFINFLUENZAVIRUSESCOR RELATESWITHABILITYTOAGGLUTINATE2"#SFROMDIFFERENTSPECIES!NIMPOR TANTSOURCEOFVARIATIONINTHE(!)TESTISTHEDIFFERINGSENSITIVITYOF2"#S FROMINDIVIDUALANIMALSOFTHESAMESPECIES (UMAN VIRUSES PREFERENTIALLY BIND TO OLIGOSACCHARIDES CONTAINING . . ACETYLNEURAMINIC ACID _  GALACTOSE .EU!C _ 'AL WHILE AVIAN AND EQUINE INFLUENZA STRAINS BIND TO .EU!C _ 'AL -ANY ANIMAL SPECIES INCLUDINGTHEHORSEANDCOW HAVEHIGHAMOUNTOF.EU!C _ 'ALRECEPTORS BUT VIRTUALLY NO .EU!C _ 'AL RECEPTORS IN ERYTHROCYTES #HICKEN 2"#S HAVE LESS .EU!C _ 'AL AND MORE .EU!C _ 'AL TURKEY 2"#S HAVE MORE.EU!C_ 'ALTHANCHICKEN2"#S;=4HEREFORE SEASONAL(AND ( INFLUENZA VIRUSES PREFERENTIALLY AGGLUTINATE CHICKEN OR TURKEY BUT NOT HORSEORCOW2"#S WHEREASAVIANVIRUSESAGGLUTINATEPREFERENTIALLY2"#S FROMHORSEORCOW )NACCORDANCEWITHTHESEHYPOTHESES THESENSITIVITYOFTHE(!)ASSAYIS LARGELYDETERMINEDBYTHETYPEOFERYTHROCYTESUSED ANDTHEMEASUREMENT OF(!)TITERSAGAINSTAVIANVIRUSESHASBEENSIGNIFICANTLYIMPROVEDBYUSEOF HORSEERYTHROCYTES4URKEYERYTHROCYTESCOULDBERESPONSIBLEFORTHERELATIVE



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&IGURE"ASICSOFHORSE2"#(!)ASSAY #ONDITIONSARE6 BOTTOM WELLPLATES2$% TREATEDSERA STARTINGDILUTIONSERIALTWOFOLD DILUTIONSINFINALVOLUMEOF +LVIRUS(.6IETNAM"0, INACTIVATED(!5IN+L HORSE2"#SCOLLECTEDINCITRATEDEXTROSEACID!#$ SOLUTIONWASHEDANDSTANDARDIZEDTO VVIN0"3"3!ADDEDTOASSAYIN +LVOLUMEHINCUBATIONTIMEATROOMTEMPERATURE TOALLOWHORSE2"#TOSETTLE

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#ORRELATESOFPROTECTIONAGAINSTINFLUENZA



&IGURE32(REACTIONS 32(OFHUMANSERUMINAGAROSEGELIMMUNOPLATESCONTAININGGUINEAPIGCOMPLEMENTFINAL CONCENTRATION  AND CHICKEN ERYTHROCYTES  TREATED WITH!0ORT #HALMERS VIRUS 4HECLEARAREASREPRESENTZONESOFLYSEDERYTHROCYTESPRODUCEDBYANTIBODYTO(!4HEWELLS INTHETOPROWCONTAINSERIAL TWOFOLDDILUTIONSn OFAPOTENTHUMANSERUMHAVINGAN (!)TITEROFWITH!0#VIRUS4HEBOTTOMROWCONTAINSSIMILARDILUTIONSOFASERUM WITHAN(!)TITEROF;=

OF HEMOLYSIS ARE MEASURED AND AREAS ARE CALCULATED !REAS OF HEMOLYSIS EQUALORHIGHERTHANMM ARECONSIDEREDSEROPROTECTIVE)NTHECASEOF (STRAINS BETTERRESULTSWEREOBTAINEDUSINGTURKEYERYTHROCYTES 32(ISSUITABLEFORSCREENINGFORLARGENUMBEROFSAMPLES4HIS FEATURE HAS MADE THE TEST USEFUL FOR RAPID SCREENING OF ANTIBODIES AGAINST NEWLY DETECTED INFLUENZA VARIANTS MAKING IT VALUABLE FOR LARGE SCALE SERO EPIDE MIOLOGICALSTUDIES)THASGOODCORRELATIONWITH-.FORPANDEMIC(STRAINS ANDIS%-%!APPROVED 4HE 32( TEST WORKS WELL WITH INACTIVATED VIRUSES SO THAT SEROLOGY OF (.CANBESAFELYANALYZEDATABIOSAFETYLEVELCONTAINMENT !LTHOUGH THISTESTCANDETECT(.ANTIBODY ITCROSS REACTSWITHNONSPECIFICANTIBODY INHUMANANDRABBITSERA4HEREFORE PRELIMINARYSCREENFORCROSS REACTIVITY ANDCONFIRMATORYTESTSWITHANALTERNATIVETECHNIQUEARERECOMMENDED 4HISTESTALLOWSSMALLERDIFFERENCESINANTIBODYLEVELTOBEDETECTEDTHAN IS POSSIBLE BY CONVENTIONAL (!) TESTS )T IS A CONVENIENT TEST THAT CAN BE PERFORMEDWITHUNDILUTEDSERUM!NTIBODIESRESPONSESTONATURAL INFECTION ANDVACCINATIONAREREADILYDETECTEDBY32(TESTS 7HETHERCOMPARING(!)AND32(TESTSFORSEASONALSTRAINS&IG  A CLOSE CORRELATION BETWEEN THE ANTIBODIES POTENCIES MEASURED IN BOTH TEST SYSTEMSWASOBSERVED3ERUMSAMPLESWITHHIGH(!)TITERSn GAVE ZONE DIAMETER OF n MM HEMOLYSIS AREA n MM M  /F THE  SAMPLESSHOWNIN&IGURETHATWERENEGATIVEIN(!)TESTSTITER  WEREALSONEGATIVEBY32(;=#OMPLETELYDIFFERENTRESULTSWEREACHIEVED WHENCOMPARING(!)WITH32(AND-.FOR(STRAINS&IG  4HE-.ASSAYORVIRUSNEUTRALIZATIONASSAY 6. REQUIRESONLY ASTOCK OFINFECTIOUSVIRUSASTHEANTIGEN4HEADVANTAGESOFTHE-.ASSAYARETHAT ITISMORESENSITIVETHAN(!) MORESPECIFIC ANDISSUITABLEFORAUTOMATION



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&IGURE  #ORRELATION BETWEEN (!) TITER AND 32( ZONE DIAMETER FOR!0ORT #HALMERS (. VIRUSINHUMANSERUMSAMPLES;=

;=4HISASSAYDETECTSFUNCTIONALANTI (!ANTIBODY WHICHISHIGHLYSPECIFIC FORTHESUBTYPEINQUESTION-OREOVER THISASSAYCANBEDEVELOPEDQUICKLY UPON RECOGNITION OF A NOVEL VIRUS AND CAN BE USED EVEN BEFORE SUITABLE RECOMBINANT OR PURIFIED VIRAL PROTEINS BECOME AVAILABLE FOR USE IN OTHER ASSAYS )N ADDITION -. WILL ALSO MEASURE NEUTRALIZING RESPONSES AGAINST OTHERENVELOPEGLYCOPROTEINS IE .! INCONTRASTTOTHE(!) ASSAYTHATONLY MEASURESRESPONSESAGAINSTTHE(!COMPONENT4HE-.TESTSEEMS TOHAVE ADVANTAGESWHENANTIBODYLEVELSARELOW WITHNEGLIGIBLEORNEGATIVETITERS INTHE(!)TEST; = -.TESTSHAVENOTBEENUSEDWIDELYINSEROLOGICALSTUDIESBECAUSETHEY ARELENGTHYANOVERNIGHTTEST THUSTHISASSAYISNOTEASYWHENSCREENING A LARGE NUMBER OF SAMPLES ;= -OREOVER THIS ASSAY NEEDS LIVE VIRUS AND THUS AHIGHCONTAINMENTINCASEOFPANDEMICSTRAINS!TTHEFRONTLINEOFAN OUTBREAK ESPECIALLYINRESOURCE LIMITEDREGIONS BIOSAFETYLEVELLABORATORY FACILITIESORHIGHERARENOTALWAYSAVAILABLE4HEREFORE THIS ASSAYISTHEGOLD STANDARDFORCONFIRMATION &ORTHE-.ASSAY THEREISNORECOGNIZEDCORRELATEOFPROTECTIONHOW EVER AFOURFOLDINCREASEINTITERAFTERVACCINATIONHASBEENUSEDINTHELITERA TURETOASSESSIMMUNERESPONSESTO(VIRALANTIGENSBY-.; = "ASED ON THE SENSITIVITY AND SPECIFICITY OF ANALYSIS DESCRIBED THE -. ASSAY IS NOW BEING USED AS PART OF A SERO EPIDEMIOLOGICAL INVESTIGATION OF THEOUTBREAKIN(ONG +ONGTODETECT(VIRUSINSERAFROM THOUSANDS OFSUBJECTSEVALUATED"ECAUSEAVIANVIRUSESMAYALSOINDUCELOWLEVELSOF SERUMANTIBODY ANDMAYNOTHAVEBEENDETECTEDINPREVIOUSSURVEYSTHAT

#ORRELATESOFPROTECTIONAGAINSTINFLUENZA



&IGURE'EOMETRICMEANTITERSOFANTIBODYFOR-& ADJUVANTEDANDCONVENTIONALSURFACE ANTIGEN (. VACCINE BEFORE AND AFTER  AND  DOSES OF VACCINE ! (!) TEST USING (. ANTIGEN " -. USING (. ANTIGEN # 32( USING (. ANTIGEN $ 32( USING (. ANTIGEN;=



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&IGURE  2ELATIONSHIP BETWEEN (!) AND NEUTRALIZING ANTIBODY -. TITERS AGAINST ! 9AMAGATA (. ! !&UKUOKA# (. " !3HISEN (. # ".AGASAKI$ AND"/SAKA%  4HESERUMANTIBODYWASMEASUREDBY-.AND(!)TESTS7HENTITRATEDAGAINSTVACCINESTRAINS ! " $ DIFFERENCESBETWEENTHE-.AND(!)TITERSARESMALL WHENTITRATEDAGAINSTHETEROLO GOUSSTRAINSTHEDIFFERENCESARELARGE4HEPOSSIBILITYTHATNONINFECTIOUSVIRUSPARTICLESCONSUME NEUTRALIZINGANTIBODIES ANDTHUSRESULTINLOWERACTUALTITERS ASSUSPECTEDINTHENEUTRALIZATION TESTS WITH HETEROLOGOUS STRAINS SEEMS UNLIKELY BECAUSE THE RATIO BETWEEN INFECTIVITY AND THE HEMAGGLUTININTITEROF!3HISENWASNOTLOWERTHANTHATOFF OTHERSTRAINS4HEREFORE THESE OBSERVATIONSSEEMTOINDICATETHATTHENEUTRALIZATIONTESTISMORESPECIFICTHANTHE(!)TEST ;=

#ORRELATESOFPROTECTIONAGAINSTINFLUENZA



&IGURE#ORRELATIONBETWEEN-.ANDHORSE2"#S(!)TITERSUSINGSAMPLESVACCINATEDWITH ADJUVANTED!(.$UCK3INGAPOREVACCINE-&N 2CORRELATIONCOEFFICIENT ;=

RELIEDONTHE(!)ASSAY;= THE-.ASSAYMAYALSOBEAPPLIEDTOSEROSUR VEYSTODETECTEVIDENCEOFHUMANINFECTIONWITHAVIANINFLUENZA!VIRUSES OFOTHERSUBTYPES -.ASSAYSAREUSUALLYDONEIN WELLSPLATES WHERESERAHEAT INACTI VATEDFORMINATª# ARETESTEDTHROUGHMIXINGWITHANEQUALVOLUME OFINFLUENZAVIRUSAT=4#)$ML!FTER HINCUBATIONATª#UNDER #/ +LCELLS =  ISADDEDTOEACHWELL!FTERASECONDINCUBA TIONATª#FORHUNDER#/ THECELLSAREFIXEDINACETONEFOR MIN4HEPRESENCEOFVIRALPROTEINISDETECTEDBY%,)3!USINGAMONOCLONAL ANTIBODYTOTHEINFLUENZANUCLEOPROTEIN!LTERNATIVELY THEPRESENCEOFVIRUS CANBEDETECTEDINTHEMEDIUMOFCULTUREUSINGSIMPLEHEMAGGLUTINATIONOF ANIMAL2"#S;= &IGURE  SHOWS THE RELATIONSHIP BETWEEN (!) AND -. ANTIBODY TITERS AGAINSTSEASONALSTRAINS4HE-.ANTIBODYTITERSARESLIGHTLYHIGHERTHANTHE (!) TITERS AGAINST THE HOMOLOGOUS STRAINS 4HIS FIGURE ALSO DEMONSTRATES THAT CORRELATION IS DEPENDENT ON THE STRAIN ANALYZED ,ESS CORRELATION BETWEEN-.AND(!)WASALSOOBSERVEDFORDETECTIONOFANTIBODIESTHAT MAYBEPRESENTINSERAFROMINDIVIDUALSRECEIVINGTHE(.6IETNAMVAC CINE&IG 

/THERASSAYS !N%,)3!HASBEENDEVELOPEDTODETECTANTIBODIESTO(!FROMNOVELINFLU ENZAVIRUSES4HISASSAYISSUITABLEFORSCREENINGALARGENUMBEROFSAMPLES WITHTHEPREFERABLEUSEOF(!WITH(!BEINGPURIFIEDONLYIFTHISGIVES LESSCROSS REACTIVITY 4HUS THISOFFERSTHEPOSSIBILITYOFAUTOMATION BUTTHE



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THE TOTAL NUMBER OF PATIENTS WITH CONFIRMED (. INFECTIONS TESTED 3PECIFICITY NUMBER OF CONTROL AGE MATCHED SERA TESTED MINUS THE NUMBER OF CONTROL SERA TESTING POSITIVE FOR ANTI BODYDIVIDEDBYTOTALNUMBEROFCONTROLSERA C4ESTSFORDETERMINATIONOF(.VIRUSPOSITIVITY. MICRONEUTRALIZATIONTEST7 7ESTERNBLOT TING% %,)3!  D#OMBINATION OF TESTS -ICRONEUTRALIZATION TEST WITH!(ONG +ONG VIRUS FOLLOWED BY 7ESTERNBLOTCONFIRMATIONWITHR(!OF!(ONG+ONGVIRUS . 7 AND%,)3!WITH R(! OF!(ONG +ONG VIRUS FOLLOWED BY7ESTERN BLOT CONFIRMATION WITH R(! OF! (ONG+ONGVIRUS% 7  E.UMBEROFSAMPLES  F3TATISTICALANALYSISFORPOSITIVEASSOCIATIONBETWEENTESTRESULTANDKNOWNSTATUSOFSAMPLESWAS NOTSIGNIFICANT&ISHEREXACTTESTP !LLOTHERASSAYSWERESIGNIFICANTP   ;=

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#ORRELATESOFPROTECTIONAGAINSTINFLUENZA



SENSITIVITYANDSPECIFICITYWERESTILLACHIEVEDBYACOMBINATIONOFTHE-. ASSAYAND7";=%,)3!AND7"TESTDETECTEDANTIBODYOFLOWERAVIDITY ANDORQUANTITYTHANTHATREQUIREDFORDETECTIONBYTHE-.ASSAY 4HEDEVELOPMENTOFPOLYMERASECHAINREACTION0#2 ;=HASPROVIDED A HIGHLY SPECIFIC AND SENSITIVE METHOD FOR THE DETECTION OF VIRAL GENOMES 0#2USESTWOOPPOSITELYORIENTEDPRIMERFLANKINGASPECIFIC$.! REGION WHICH BY REPEATED CYCLES OF HEAT DENATURATION ANNEALING AND EXTENSION OFTHEPRIMERSWITH4AQPOLYMERASE ALLOWTHEAMPLIFICATIONOFTHESPECIFIC TARGETSEQUENCEWITHINTHEVIRUSGENOME3TUDIESHAVECARRIEDOUTREVERSE TRANSCRIPTASE24 0#2TODETECTINFLUENZAVIRUS2.!INCLINICALMATERIAL ANDCELLCULTUREFLUIDS!LTHOUGH0#2ISNOTASRAPIDAS%,)3! WHICHCAN BEPERFORMEDINAFEWHOURS ITISCONSIDERABLYMORERAPIDTHANCELLCULTURE OFVIRUS&URTHERMORE THESENSITIVELYOF0#2HASBEENDEMONSTRATEDTOBE COMPARABLETOTHATOFISOLATIONOFVIRUSBYCELLCULTURE;= 4HEREARETWOREALISTICOPTIONSFORRAPIDDEVELOPMENTOFNEUTRALIZATION ASSAYS TO MAKE THEM MORE WIDELY APPLICABLE TO USE REVERSE GENETICS TO ENGINEERASAFER ATTENUATEDVIRUSBYDELETIONOFTHEPOLYBASIC CLEAVAGESITE IN(! ASISDONEFORTHEDEVELOPMENTOFINACTIVATEDVACCINESFORPANDEMIC INFLUENZA; = ORTHECONSTRUCTIONOFVIRALPSEUDOTYPESBEARINGTHEINFLU ENZA(!GLYCOPROTEINSASSURROGATEVIRUSESFORUSEINNEUTRALIZATIONASSAYS 4HEFIRSTOPTIONHASITSINHERENTPROBLEMS IE THEISSUEOFPOSSIBLEREVERSION TOTHEWILD TYPEVIRUSVIAGENETICREASSORTMENT;= 7ITH THE PSEUDOTYPE SYSTEM HOWEVER ONLY THE (! FROM INFLUENZA IS REQUIRED WITH NO POSSIBILITY OF RECOMBINATION OR VIRUS ESCAPE 4HESE PAR TICLESUNDERGOABORTIVEREPLICATIONANDDONOTGIVERISETOREPLICATION COM PETENTPROGENY4HESEPSEUDOTYPESENCODEREPORTERGENESANDBEARFOREIGN VIRALENVELOPESOFINTEREST;=4HETRANSFEROFMARKERGENESTOTARGETCELLS DEPENDSONTHEFUNCTIONOFTHEENVELOPEPROTEINTHEREFORE THE TITEROFNEU TRALIZINGANTIBODIESAGAINSTTHEENVELOPECANBEMEASUREDBYA REDUCTIONIN MARKERGENETRANSFER&IG  2ETROVIRAL PSEUDOTYPE BASED ASSAY FACILITATES THE ACCURATE DETERMINA TIONOFNEUTRALIZINGANTIBODYBODYRESPONSESTOINFLUENZA(. WITHOUTTHE NEEDTOUSEREPLICATION COMPETENTVIRUS ANDTHUSCANBECARRIEDOUTATBIO SAFETYLEVEL4HEPSEUDOTYPEASSAYISSIGNIFICANTLYMORESENSITIVETHANTHE TURKEYERYTHROCYTE(!) ANDATLEASTASSENSITIVEASTHEHORSE ERYTHROCYTE (!)AND-.FORTHEDETECTIONOFANTIBODIESTO((!&IG 4HESAFETY 4 SPECIFICITYANDSENSITIVITYOFTHEPSEUDOTYPEASSAYSUGGESTTHATITMAYHAVEA ROLEASANALTERNATIVEORSUPPLEMENTTOCONVENTIONALASSAYS &URTHERDEVELOPMENTOF` 'AL BASEDPSEUDOTYPEASSAYSWILLALLOWWIDER APPLICATION AS AN %,)3! TYPE ASSAY IN LABORATORIES WITHOUT SPECIALIZED EQUIPMENT )N ADDITION WITH MINOR MODIFICATIONS THESE ASSAYS COULD BE USEDTOSCREEN(!AND.!INHIBITORSWITHHIGH THROUGHPUTUSAGE 4HE APPLICATION OF PSEUDOTYPES IN IMMUNOLOGICAL ASSAYS SUCH AS -. %,)3! (!) USINGSERAFROMVACCINATEDVOLUNTEERSHASBEENDEMONSTRAT ED FOR (.6IETNAM STRAIN 4HE PSEUDOTYPE BASED NEUTRALIZATION



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&IGURE(.(!RETROVIRALPSEUDOTYPES-,6(! AND()6(! PSEUDOTYPECONSTRUC TIONANDNEUTRALIZATIONASSAYFORINFLUENZA!(.;=#ONFLUENTPLATESOF4CELLSWERE PREPAREDTHEDAYBEFORETRANSFECTION%ACHPLATEWASTRANSFECTEDWITH+GGAGPOLCONSTRUCT +GOF'&0OR,UCREPORTERCONSTRUCTAND +G(!CONSTRUCTUSING&UGENE TRANSFECTION REAGENT!T H POST TRANSFECTION 5 OF EXOGENOUS NEURAMINIDASE WAS ADDED TO INDUCE THE RELEASEOF(! PSEUDOTYPEDPARTICLESFROMTHESURFACEOFTHEPRODUCERCELLS4HESUPERNATANT WASHARVESTEDHPOST TRANSFECTIONANDPASSEDTHROUGH +MFILTERS-,6VECTORTITERSWERE MEASUREDONHUMAN4 14 -$#+CELLSANDAREPRESENTEDASINFECTIOUSUNITS)5 PER MILLILITER()6VECTORTITERSWEREMEASUREDON4 14AND-$#+CELLS

ASSAYISPROMPTLYADAPTABLETOAHIGH THROUGHPUTFORMATFORTHE EVALUATION OFVACCINEEFFICACY&URTHERMORE USINGPLASMIDSENCODINGFORANYOTHER(! SEQUENCE APANELOFPSEUDOTYPESEXPRESSING(!FROMDIFFERENTVIRALSTRAINS COULDBEGENERATEDANDUSEDTOEVALUATECROSS PROTECTIVEANTIBODIES

#ONCLUSION 4RADITIONAL (!) METHOD PERFORMED USING TURKEY 2"#S OFFERS A SIMPLER ASSAY FOR DETECTION OF HUMAN ANTIBODY TO SOME INFLUENZA STRAINS PARTICU LARLY(AND(THISASSAYMAYBEUSEFULFORLARGESEROSURVEYSASANINITIAL SCREENINGTOOL$ETECTIONOFANTIBODIESTOAVIANINFLUENZAVIRUSESINMAM MALIAN SPECIES INCLUDING HUMANS USING (!) HAS GENERALLY FAILED EVEN IN CASES WHERE INFECTION WAS CONFIRMED BY VIRUS ISOLATION (!) ASSAY IS LESS DIFFICULTANDLESSTIMECONSUMINGTOPERFORMTHAN-.OR32(ASSAYS 32(ASSAYREQUIRESONLYASMALLAMOUNTOFWHOLEINACTIVATEDVIRUS THUS THEREISNONEEDTOADAPTTHEVIRUSTORAPIDGROWTH4HISFEATUREHASMADE THISTESTUSEFULFORRAPIDSCREENINGOFANTIBODIESAGAINSTNEWLYDETECTEDINFLU ENZAVARIANTS)TCANBEVALUABLEFORLARGE SCALESERO EPIDEMIOLOGICALSTUDIES OF NEW INFLUENZA VIRUS VARIANTS 32( HAS A BIG DISADVANTAGE IN DETECTING

#ORRELATESOFPROTECTIONAGAINSTINFLUENZA



&IGURE#OMPARISONOF-,6(! WITH(!)AND-.FORTHEMEASUREMENTOFANTIBODIESTO (. A !NTI ( ANTIBODY RESPONSES IN HUMAN SERA 3CATTERPLOT SHOWING THE CORRELATION OF ANTI BODY GEOMETRIC MEAN TITERS '-4 MEASURED BY HORSE (!) SQUARES AND -. DIAMONDS WITH)#'-4MEASUREDBY-,6(! PSEUDOTYPEASSAYWITHAGREENFLUORESCENTPROTEIN '&0 REPORTER(!)AND-.ASSAYSPERFORMEDAGAINST.)"2' VIRUS4OTALNUMBEROFSERA ASSAYEDISPOSITIVEFORANTIBODIESAGAINST( AND( NEGATIVECONTROLSERA ,INEAR TRENDLINESWEREFITTEDTOTHEDATABYUSING-ICROSOFT%XCELB #ORRELATIONCOEFFICIENTSOF (!)HORSE -,6(! -.-,6(! AND(!)HORSE -.FORHUMAN FERRETANDCHICKEN SERA$ATAPLOTTEDARERCORRELATIONCOEFFICIENTSDETERMINEDUSING-ICROSOFT%XCEL;=

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-.ASSAYISSPECIFIC MORESENSITIVETHANTHE(!)ASSAY ANDSUITABLEFOR AUTOMATION (OWEVER THIS ASSAY REQUIRES TIME AND LIVE AND TITRATED VIRUS THUSAHIGHCONTAINMENTAREAISNEEDEDWHENTESTINGPANDEMICSTRAINS !N%,)3!TESTSPECIFICFOR(!ANTIBODIESREQUIRESHIGHLYPURIFIEDANTI GEN WHICHCANBEDIFFICULTTOOBTAININSUFFICIENTAMOUNT 4HEPSEUDOTYPENEUTRALIZATIONASSAYISASSENSITIVEASHORSEERYTHROCYTES (!)AND-.FORTHEDETECTIONOFANTIBODIESAGAINST(.)TISSAFER AND CANBEAPPLIEDINAHIGH THROUGHPUTFORMATFORHUMANANDANIMALSURVEIL LANCEANDFORTHEEVALUATIONOFVACCINES4OACHIEVEMAXIMUMSENSITIVITYIN SEROLOGICAL ASSAYS THE SELECTION OF VIRUS ISOLATED FROM THE SAME INFLUENZA OUTBREAK OR THE USE OF AN ANTIGENICALLY EQUIVALENT STRAIN IS REQUIRED FOR OPTIMALANTIGENICMATCH#OMPETENTMOLECULARVIROLOGYLABORATORIESCOULD PRODUCE(!PSEUDOTYPEVIRUSWITHINnWEEKSOFTHEAVAILABILITYOFVIRAL 2.!&URTHERSTUDIESAREUNDERWAY MAKINGUSEOFAPANELOF(RETROVIRAL PSEUDOTYPESWITH(!COMPONENTSDERIVEDFROM(.VIRUSESINVOLVEDIN THERECENTHUMANANDAVIANOUTBREAKS $EVELOPMENTOFSUCHASSAYSWILLBEANIMPORTANTFURTHERSTEPIN PREPA RATIONOFNEWINFLUENZAVACCINES NOTONLYFORPREPANDEMICANDPANDEMIC PRODUCTS BUT ALSO FOR CELL DERIVED VACCINES !DDITIONAL IMMUNOLOGICAL ASSESSMENTSSUCHASCELL MEDIATEDIMMUNITYAND.!INHIBITIONNEEDTOBE EXPLOREDTOGIVEMOREINSIGHTINTOTHEOVERALLEFFECTSOFVACCINATION

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"EYER7% 0ALACHE!- DE*ONG*# /STERHAUS!$ #OLD ADAPTEDLIVE INFLUENZAVACCINE VERSUS INACTIVATEDVACCINE3YSTEMICVACCINEREACTIONS LOCAL ANDSYSTEMICANTIBODYRESPONSE ANDVACCINEEFFICACY!META ANALYSIS 6ACCINE n (OELSCHER-! 'ARG3 "ANGARI$3 "ELSER*! ,U8 3TEPHENSON) "RIGHT2! +ATZ *- -ITTAL 3+ 3AMBHARA 3  $EVELOPMENT OF ADENOVIRAL VECTOR BASEDPANDEMICINFLUENZAVACCINEAGAINSTANTIGENICALLYDISTINCT HUMAN(. T STRAINSINMICE,ANCETn "EYER 7% 0ALACHE !- ,àCHTERS ' .AUTA * /STERHAUS !$  3EROPROTECTIONRATE MEANFOLDINCREASE SEROCONVERSIONRATE7HICHPARAMETER ADEQUATELY EXPRESSES SERORESPONSE TO INFLUENZA VACCINATION 6IRUS 2ES  n %-%! .OTEFOR'UIDANCEONHARMONISATIONOFREQUIREMENTSFORINFLU ENZAVACCINES#0-0"70 %-%! 'UIDELINEONDOSSIERSTRUCTUREANDCONTENTFOR PANDEMICINFLU ENZAVACCINEMARKETINGAUTHORISATIONAPPLICATION#0-06%' (IRST '+  4HE AGGLUTINATION OF RED CELLS BY ALLANTOIC FLUID OF CHICK EMBRYOSINFECTEDWITHINFLUENZAVIRUS3CIENCEn 7RIGHT 0& "RYANT *$ +ARZON $4  #OMPARISON OF INFLUENZA "(ONG +ONG VIRUS INFECTIONS AMONG INFANTS CHILDREN AND YOUNG ADULTS * )NFECT $IS n -ONTO!3 -AASSAB(& %THERTREATMENTOFTYPE"INFLUENZAVIRUSANTI GENFORTHEHEMAGGLUTINATIONINHIBITIONTEST*#LIN-ICROBIOLL n +ENDAL!0 #ATE42 )NCREASEDSENSITIVITYANDREDUCEDSPECIFICITYOFHEM AGGLUTINATIONINHIBITIONTESTSWITHETHER TREATEDINFLUENZA"3INGAPORE *#LIN-ICROBIOLn 0ALMER $& #OLEMAN -4 $OWDLE72 3CHILD '#  !DVANCED LABORA TORYTECHNIQUESFORINFLUENZADIAGNOSIS)MMUNOLOGY3ERIES 0ROCEDURAL'UIDE 53$EPARTMENTOF(EALTH %DUCATION AND7ELFARE0UBLIC(EALTH3ERVICE #ENTERFOR$ISEASE#ONTROL  (INSHAW63 7EBSTER 2' %ASTERDAY "# "EAN7* *R  2EPLICATION OF AVIANINFLUENZA!VIRUSESINMAMMALS)NFECT)MMUNn 2OWE4 !BERNATHY2! (U 0RIMMER* 4HOMPSON77 ,U8 ,IM 7 &UKUDA + #OX.* +ATZ*- $ETECTIONOFANTIBODYTOAVIANINFLUENZA!(. VIRUS IN HUMAN SERUM BY USING A COMBINATION OF SEROLOGIC ASSAYS * #LIN -ICROBIOLL n 3TEPHENSON) 7OOD*- .ICHOLSON+' :AMBON-# 3IALICACIDRECEP TORSPECIFICITYONERYTHROCYTESAFFECTSDETECTIONOFANTIBODYTOAVIANINFLUENZA HAEMAGGLUTININ*-ED6IROLL n 3CHILD'# 0EREIRA-3 #HAKRAVERTY0 3INGLE RADIAL HEMOLYSIS!NEW METHODFORTHEASSAYOFANTIBODYTOINFLUENZAHAEMAGGLUTININ!PPLICATIONSFOR DIAGNOSIS AND SEROEPIDEMIOLOGIC SURVEILLANCE OF INFLUENZA "ULL7ORLD (EALTH /RGAN n -UMFORD * 7OOD *  7(//)% MEETING #ONSULTATION ON NEWLY EMERGING STRAINS OF EQUINE INFLUENZA n -AY  !NIMAL (EALTH4RUST .EWMARKET 3UFFOLK 5+6ACCINEn 7OOD *- 'AINES $AS 2% 4AYLOR * #HAKRAVERTY 0  #OMPARISON OF



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INFLUENZA SEROLOGICAL TECHNIQUES BY INTERNATIONAL COLLABORATIVE STUDY 6ACCINE n  7OOD *- -ELZACK $ .EWMAN 27 -AJOR $, :AMBON - .ICHOLSON +' 0ODDA! !SINGLERADIALHAEMOLYSISASSAYFORANTIBODYTO(HAEMAG GLUTININ )NTERNATIONAL#ONGRESS3ERIESn  'ROSS 0! "ARRY $7 $@%SOPO .  )NFLUENZA IMMUNIZATION IN CHRONIC BRONCHITIS ,OCAL AND SYSTEMIC IMMUNE RESPONSE !M 2EV 2ESPIR $IS  n  "ENNE #! (ARMSEN - DE *ONG *# +RAAIJEVELD #!   .EUTRALIZATION L ENZYMEIMMUNOASSAYFORINFLUENZAVIRUS *#LIN-ICROBIOLn  (ARMON -7 2OTA 0! 7ALLS (( +ENDAL!0  !NTIBODY RESPONSE IN HUMANS TO INFLUENZA VIRUS TYPE " HOST CELL DERIVED VARIANTS AFTER VACCINATION WITHSTANDARDEGG DERIVED VACCINEORNATURALINFECTION *#LIN-ICROBIOL n  4REANOR ** #AMPBELL *$ :ANGWILL +- 2OWE4 7OLFF -  3AFETY AND IMMUNOGENICITY OF AN INACTIVATED SUBVIRION INFLUENZA ! (. VACCINE . %NGL*-EDn  "RESSON*, 0ERRONNE# ,AUNAY/ 'ERDIL# 3AVILLE- 7OOD* (ÚSCHLER+ :AMBON-# 3AFETYANDIMMUNOGENICITYOFANINACTIVATEDSPLIT VIRION INFLUENZA !6IETNAM (. VACCINE 0HASE ) RANDOMISED TRIAL T ,ANCETn  0ROFETA-, 0ALLADINO' 3EROLOGICALEVIDENCEOFHUMANINFECTIONSWITH L AVIANINFLUENZAVIRUSES !RCH6IROLn  &RANK !, 0UCK * (UGHES "* #ATE 42  -ICRONEUTRALIZATION TEST FOR INFLUENZA!AND"ANDPARAINFLUENZAANDVIRUSESTHATUSESCONTINUOUSCELL LINESANDFRESHSERUMENHANCEMENT *#LIN-ICROBIOLL n  3TEPHENSON ) 7OOD *- .ICHOLSON +' #HARLETT ! :AMBON -#  $ETECTIONOFANTI (RESPONSESINHUMANSERABY()USINGHORSE ERYTHROCYTES FOLLOWING-& ADJUVANTEDINFLUENZA!$UCK3INGAPOREVACCINE 6IRUS2ES n  $OLLER' 3CHUY7 4JHEN+9 3TEKELER" 'ERTH(* $IRECTDETECTIONOF INFLUENZAVIRUSANTIGENINNASOPHARYNGEALSPECIMENSBYDIRECTENZYMEIMMU NOASSAY IN COMPARISON WITH QUANTITATING VIRUS SHEDDING * #LIN -ICROBIOLL  n  3AKAI2+ 'ELFAND$( 3TOFFEL3 3CHARF&* (IGUCHI2 (ORN'4 -ULLIS+" %RLICH(! 0RIMER DIRECTEDENZYMATICAMPLIFICATIONOF$.!WITHTHER MOSTABLE$.!POLYMERASE 3CIENCEn  $ONOFRIO*# #OONROD$ $AVIDSON*. "ETTS2& $ETECTIONOFINFLUENZA ! AND " IN RESPIRATORY SECRETIONS WITH THE POLYMERASE CHAIN REACTION 0#2 -ETHOD!PPLL n  7OOD *- 2OBERTSON *3  &ROM LETHAL VIRUS TO LIFE SAVING VACCINE $EVELOPINGINACTIVATEDVACCINESFORPANDEMICINFLUENZA .AT2EV-ICROBIOL n  (OFFMANN% +RAUSS3 0EREZ$ 7EBBY2 7EBSTER2' %IGHT PLASMID SYSTEMFORRAPIDGENERATIONOFINFLUENZAVIRUSVACCINES 6ACCINEn  4EMPERTON.* (OSCHLER+ -AJOR$ .ICOLSON# -ANVELL2 (IEN6- (A$1 DE*ONG- :AMBON-# 4AKEUCHI9 7EISS2! !SENSITIVERETROVIRAL

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PSEUDOTYPE ASSAY FOR INFLUENZA (. NEUTRALIZING ANTIBODIES )NFLUENZA  n  3ANDERS $!  .O FALSE START FOR NOVEL PSEUDOTYPED VECTORS #URR /PIN "IOTECHNOLn L  .ICHOLSON+' #OLEGATE!% 0ODDA! 3TEPHENSON) 7OOD* 9PMA% :AMBON -#  3AFETY AND ANTIGENICITY OF NON ADJUVANTED AND -& ADJUVANTED D INFLUENZA !$UCK3INGAPORE (. VACCINE ! RANDOMISED TRIAL OF TWO T POTENTIALVACCINESAGAINST(.INFLUENZA ,ANCETn  /KUNO9 4ANAKA+ "ABA+ -AEDA! +UNITA. 5EDA3 2APIDFOCUS REDUCTIONNEUTRALIZATIONTESTOFINFLUENZA!AND"VIRUSESINMICROTITERSYSTEM *#LIN-ICROBIOLL n

)NFLUENZA6ACCINESFORTHE&UTURE EDBY22APPUOLIAND'$EL'IUDICE ¥"IRKHËUSER6ERLAG"ASEL3WITZERLAND



4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH #ATHERINE*,UKEAND+ANTA3UBBARAO ,ABORATORYOF)NFECTIOUS$ISEASES .ATIONAL)NSTITUTEOF!LLERGYAND)NFECTIOUS$ISEASES G .ATIONAL)NSTITUTESOF(EALTH "ETHESDA -$ 53!

!BSTRACT !MAJORCHALLENGEFORRESEARCHONINFLUENZAVACCINESISTHESELECTIONOFANAPPROPRIATE ANIMALMODELTHATACCURATELYREFLECTSTHEDISEASEANDTHEPROTECTIVEIMMUNERESPONSETO INFLUENZA INFECTION IN HUMANS6ACCINES FOR SEASONAL INFLUENZA HAVE BEEN AVAILABLE FOR DECADESANDTHEREISAWEALTHOFDATAAVAILABLEONTHEIMMUNERESPONSETOTHESEVACCINES INHUMANS WITHWELL ESTABLISHEDCORRELATESOFPROTECTIONFORINACTIVATEDINFLUENZAVIRUS VACCINES-ANYOFTHESEMINALSTUDIESONVACCINESFOREPIDEMICINFLUENZAWERECONDUCTED INHUMANSUBJECTS3TUDIESINHUMANSAREPERFORMEDLESSFREQUENTLYNOWTHANTHEYWERE INTHEPAST4HEREFORE ASTHEQUESTFORIMPROVEDINFLUENZAVACCINESCONTINUES ITISIMPOR TANTTOCONSIDERTHEUSEOFANIMALMODELSFORTHEEVALUATIONOFF INFLUENZAVACCINES AND AMAJORCHALLENGEFORRESEARCHONINFLUENZAVACCINESISTHESELECTIONOFANAPPROPRIATE ANIMALMODELTHATACCURATELYREFLECTSTHEDISEASEANDTHEPROTECTIVEIMMUNERESPONSETO INFLUENZAINFECTIONINHUMANS 4HE EMERGENCE OF HIGHLY PATHOGENIC (. AVIAN INFLUENZA !) VIRUSES AND THE THREATOFAPANDEMICCAUSEDBY!)VIRUSESOFTHISORANOTHERSUBTYPEHASRESULTEDINA RESURGENCE OF INTEREST IN INFLUENZA VACCINE RESEARCH4HE DEVELOPMENT OF VACCINES FOR PANDEMICINFLUENZAPRESENTSAUNIQUESETOFOBSTACLES NOTTHE LEASTOFWHICHISTHATTHE DEMONSTRATION OF EFFICACY IN HUMANS IS NOT POSSIBLE 3INCE THE CORRELATES OF PROTECTION FROMPANDEMICINFLUENZAARENOTKNOWN WERELYONEXTRAPOLATION OFLESSONSFROMSEA SONALINFLUENZAVACCINESANDONDATAFROMTHEEVALUATIONOFPANDEMICINFLUENZAVACCINES IN ANIMAL MODELS TO GUIDE OUR DECISIONS ON VACCINES FOR USE IN HUMANS 4HE FEATURES AND CONTRIBUTIONS OF COMMONLY USED ANIMAL MODELS FOR INFLUENZA VACCINE RESEARCH ARE DISCUSSED

)NFLUENZAVIRUSES )NFLUENZA IS A NEGATIVE SENSE SINGLE STRANDED 2.! VIRUS BELONGING TO THE FAMILY /RTHOMYXOVIRIDAE /RTHOMYXOVIRIDAE CONSIST OF FOUR GENERA INFLU ENZA! INFLUENZA" INFLUENZA#AND4HOGOTOVIRUSES4HEPROTEINSOFINFLU ENZA!VIRUSESAREENCODEDBYGENESONEIGHT2.!SEGMENTS)NFLUENZA! VIRUSESAREWIDELYDISTRIBUTEDINNATUREANDCANINFECTAWIDEVARIETYOFBIRDS



#ATHERINE*,UKEAND+ANTA3UBBARAO

ANDMAMMALS INCLUDINGHUMANS)NFLUENZA!VIRUSSUBTYPESARECLASSIFIED ONTHEBASISOFTHEANTIGENICITYOFTHEIRSURFACEGLYCOPROTEINS HEMAGGLUTI NIN (! AND NEURAMINIDASE .! ; = INTO  (! SUBTYPES AND  .! SUBTYPES AND ALL OF THESE SUBTYPES HAVE BEEN FOUND TO INFECT BIRDS ; = 7ATERFOWLANDSHOREBIRDSARETHENATURALRESERVOIRSOF!)VIRUSES )NTHEIRNATURALHOSTS MOST!)INFECTIONSARENOTASSOCIATEDWITHCLINICAL DISEASE AND THE VIRUSES ARE GENERALLY THOUGHT TO BE IN EVOLUTIONARY STASIS ;=)NTHEHUMANPOPULATION RELATIVELYFEWSUBTYPESOFINFLUENZA!VIRUSES HAVE CAUSED SUSTAINED OUTBREAKS OF DISEASE VIRUSES BEARING ( ( AND ( (! AND . AND . .! GENES HAVE CIRCULATED IN THE HUMAN POPULA TIONDURINGTHETHCENTURY(.VIRUSESAPPEAREDINANDCIRCULATED UNTIL  WHEN THEY WERE REPLACED BY (. VIRUSES4HESE IN TURN WERE REPLACEDINBY(.VIRUSES WHICHCONTINUETOCIRCULATEATTHEPRES ENTTIME)N (.VIRUSESREAPPEAREDANDHAVECONTINUEDTOCO CIR CULATEWITHTHE(.VIRUSES)NFLUENZA!AND"VIRUSESCAUSEEPIDEMICSIN HUMANSEACHWINTER )N ADDITION TO THE SEASONAL INFLUENZA EPIDEMICS THE POTENTIAL ALSO EXISTS FOR AN INFLUENZA PANDEMIC AT ANY TIME ! PANDEMIC OCCURS WHEN ANINFLUENZASTRAINWITHANOVEL(!SUBTYPEWITHORWITHOUTANOVEL.! SUBTYPE APPEARS AND SPREADS IN A SUSCEPTIBLE HUMAN POPULATION )N THE TH CENTURY INFLUENZA PANDEMICS OCCURRED IN   AND  AND WEREASSOCIATEDWITHSIGNIFICANTMORBIDITYANDMORTALITY;=)TISESTIMATED THAT INTHE5NITED3TATESALONE THENEXTINFLUENZAPANDEMICCOULDCAUSE  n  DEATHS AND  n  HOSPITALIZATIONS AND TENS OF MILLIONS OF OUTPATIENT VISITS AND ILLNESSES ;= !) VIRUSES IN THEIR NATURAL RESERVOIR IN WATERFOWL AND SHOREBIRDS ARE THE SOURCE FROM WHICH NOVEL (!AND.!SUBTYPESAREINTRODUCEDINTOTHEHUMANPOPULATION! NOVEL !)(!ANDOR.!CANBEINTRODUCEDINTOTHEHUMANPOPULATIONBYDIRECT SPREAD FROM EITHER WILD BIRDS OR DOMESTIC POULTRY AS WAS SEEN WHEN AN (.!)VIRUSINFECTEDHUMANSIN;=!LTERNATIVELY HUMANANDAVIAN INFLUENZAVIRUSESCANREASSORT GENERATINGVIRUSTHATCANEFFICIENTLYSPREAD INHUMANS ASHAPPENEDINTHECASEOFTHE(.AND(.PAN DEMICVIRUSES;= )NFLUENZA! VIRUSES ALSO INFECT AND CAUSE DISEASE IN A WIDE VARIETY OF MAMMALIAN SPECIES INCLUDING SWINE HORSES FERRETS MINK DOGS SEALS AND WHALES4HE CURRENTLY CIRCULATING HIGHLY PATHOGENIC!) (. VIRUSES THAT EMERGEDIN!SIAINCANALSOINFECTANDCAUSELETHALINFECTIONINFELIDS INCLUDINGTIGERS LEOPARDSANDDOMESTICCATS; = !LTHOUGH SEVERAL ANIMAL SPECIES ARE INFECTED WITH INFLUENZA ! VIRUSES NATURALLY AND EXPERIMENTALLY AN IDEAL ANIMAL MODEL FOR STUDYING INFECTION AND IMMUNITY TO HUMAN INFLUENZA HAS NOT BEEN IDENTIFIED 3EVERAL ANIMAL SPECIESAREPERMISSIVETOINFECTIONWITHINFLUENZA!AND"VIRUSESTOVARYING DEGREES AND SOME EXHIBIT CLINICAL SIGNS OF ILLNESS AND PATHOLOGICAL CHANGES IN THE RESPIRATORY TRACT THAT ARE SIMILAR TO THOSE SEEN IN HUMAN INFLUENZA )NTHISCHAPTER WEDISCUSSTHEMAINFEATURESOFTHEANIMALMODELSUSEDFOR

4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



EVALUATION OF INFLUENZA VACCINES THEIR ADVANTAGES AND DISADVANTAGES AND THEIRCONTRIBUTIONTORESEARCHONVACCINESAGAINSTINFLUENZAIN HUMANS7E ALSODISCUSSTHEROLEOFANIMALMODELSINTHEDEVELOPMENTOFVACCINESAGAINST PANDEMICINFLUENZA6ETERINARYVACCINESFORSWINE EQUINE AVIANANDCANINE INFLUENZACANBEEVALUATEDINTHEIRNATURALHOSTSANDARENOTBEDISCUSSED

)NFLUENZAVACCINES 6ACCINESHAVEBEENAVAILABLEFOREPIDEMICOR@SEASONALINFLUENZASINCETHE S)NACTIVATEDINFLUENZAVIRUSVACCINESARELARGELYTHESAMENOWASTHEY WEREWHENFIRSTDEVELOPED4HEYARESTILLGENERALLYPRODUCEDIN EMBRYONAT EDHENSEGGS4HEREHASBEENMUCHRECENTINVESTMENTINTHEDEVELOPMENT OFCELL BASEDINFLUENZAVACCINES OFWHICHATLEASTTWOARELICENSEDIN%UROPE ANDSEVERALOTHERSAREINDEVELOPMENTIN%UROPEANDTHE5NITED3TATES)N  LIVEATTENUATEDINFLUENZAVACCINESWERELICENSEDINTHE5NITED3TATES FORANNUALUSEINHEALTHYINDIVIDUALSBETWEENANDYEARSOFAGE F ! SERUM HEMAGGLUTINATION INHIBITING (!) ANTIBODY TITER OF  OR ORGREATERISASSOCIATEDWITHPROTECTIONFROMSEASONALINFLUENZA;n= F ANDTHISISUSEDASAMEASURETOPREDICTTHEPROTECTIVEEFFICACYOFSEASONAL INACTIVATED INFLUENZA VIRUS VACCINES 4HE CORRELATES OF PROTECTION FOR LIVE ATTENUATED VACCINES ARE LESS CLEAR CUT 4HESE VACCINES ELICIT SYSTEMIC AND MUCOSAL IMMUNE RESPONSES AND MUCOSAL ANTIBODY IN THE RESPIRATORY TRACT IS BELIEVED TO PLAY A MAJOR ROLE IN PROTECTION AFFORDED BY THESE VACCINES ;n= !NTIGENICDRIFTDESCRIBESTHEGRADUALCHANGEINANTIGENICITYOFF ANINFLU ENZA VIRUS THAT ALLOWS THE VIRUS TO ESCAPE NEUTRALIZATION BY ANTIBODIES INDUCED BY INFECTION OR IMMUNIZATION WITH PREVIOUSLY CIRCULATING STRAINS !NTIGENICDRIFTRESULTSFROMPOINTMUTATIONSINANDAROUNDANTIBODY COM BININGSITESINTHE(!AND.!PROTEINS)NFLUENZAVIRUSVACCINESAREUNUSUAL INTHATONEORMOREOFTHECOMPONENTSOFTHETRIVALENTVACCINE FORMULATION MAYHAVETOBECHANGEDANNUALLYTOKEEPPACEWITHANTIGENICDRIFTOFTHE VIRUSBUTASLONGASTHELICENSEDMANUFACTURINGPROCESSISUSED THECHANGEIN COMPOSITIONOFTHEVACCINEISCONSIDEREDASTRAINCHANGEANDITISNOTTREATED ASANEWVACCINE!PPROVALOFSEASONALINFLUENZAVACCINESFORUSEINHUMANS REQUIRESLIMITEDTESTINGINANIMALS ANDANEVALUATIONOFIMMUNOGENICITYIN HUMANSISREQUIREDIN%UROPEBUTNOTINTHE5NITED3TATES )N RECENT YEARS A RESURGENCE OF INTEREST IN IMPROVEMENT OF SEASONAL INFLUENZAVACCINES ANDTHELOOMINGTHREATOFAPOSSIBLEINFLUENZAPANDEMIC HAVESPURREDEFFORTSTODEVELOPVACCINESTHATCOULDTHWARTTHE SPREADOFAN EMERGING PANDEMIC VIRUS %XTENSIVE PRE CLINICAL CHARACTERIZATION OF THESE NEW VACCINES IN ANIMALS WILL BE NECESSARY -ANY RESEARCHERS ARE ENGAGED INEFFORTSTODEVELOP@UNIVERSALINFLUENZAVACCINESTHATWILL PROTECTAGAINST BOTHEPIDEMICANDPANDEMICSTRAINSBYTARGETINGTHEMORECONSERVEDANTI GENS OF THE VIRUS SUCH AS NUCLEOPROTEIN .0 OR MATRIX PROTEIN - THUS



#ATHERINE*,UKEAND+ANTA3UBBARAO

ELIMINATINGTHENEEDFORCONSTANTUPDATINGOFTHECOMPOSITIONOFTHEANNUAL SEASONAL INFLUENZA VACCINE4HE IMMUNE RESPONSES TO CANDIDATE UNIVERSAL VACCINESAREENTIRELYDIFFERENTFROMTHOSEELICITEDBYTHECURRENTLYLICENSED SEASONALINACTIVATEDINFLUENZAVIRUSVACCINES WHEREPROTECTIVE IMMUNITYIS BASEDMAINLYONNEUTRALIZINGANTIBODIESPRODUCEDAGAINSTTHE(! PROTEIN !NIMAL MODELS ARE NEEDED IN WHICH DIFFERENT TYPES OF IMMUNE RESPONSES CANBEEVALUATED /NE OF THE MAJOR CHALLENGES IN THE DEVELOPMENT OF PANDEMIC INFLU ENZAVACCINESISTHATCORRELATESOFPROTECTIONFROM!)VIRUSESOFPANDEMIC POTENTIAL ARE NOT KNOWN %FFICACY OF THESE NOVEL INFLUENZA VIRUSES CANNOT BEESTABLISHEDINHUMANS SOASSESSMENTOFEFFICACYISBASEDONINFORMATION GLEANEDFROMCHALLENGESTUDIESINANIMALS

!NIMALMODELSFORINFLUENZA $ESPITETHEDIVERSITYOFMAMMALIANSPECIESINFECTEDBYINFLUENZAVIRUSES IN NATURE ONLY A FEW SPECIES ARE AMENABLE TO STUDY IN THE LABORATORY 4ABLESnANDTHEFOLLOWINGSECTIONSSUMMARIZETHEFEATURESOFF THEMOST COMMONLYUSEDSMALLANIMALMODELSFORTHESTUDYOFINFLUENZA ANDTHEIR RESPECTIVEUTILITIESINTHEEVALUATIONOFINFLUENZAVACCINESARESUMMARIZED IN4ABLE  #OMMONLY USED LABORATORY ANIMAL SPECIES MAY NOT BE FULLY PERMISSIVEFORINFECTIONWITHWILD TYPE NON ADAPTEDISOLATESOFINFLUENZA VIRUSES ANDCANVARYINSUSCEPTIBILITYTOINFECTIONBYSPECIFICVIRUSSTRAINS ANDSUBTYPES/THERVARIABLESTHATCANINFLUENCETHEOUTCOMEOFF INFECTION ARETHEUSEOFANESTHESIA ROUTEOFVIRUSADMINISTRATIONANDTHEVOLUMEOF INOCULUM

2ODENTMODELS 2ODENT MODELS OF INFECTIOUS DISEASES ARE ATTRACTIVE FOR A NUMBER OF SCI ENTIFIC AND PRACTICAL REASONS4HEY ARE SMALL AND RELATIVELY INEXPENSIVE TO PURCHASE AND HOUSE -ANY INBRED STRAINS ARE AVAILABLE AND A BATTERY OF IMMUNOLOGICALREAGENTSAREAVAILABLEFORSOMESPECIES

-ICE -ICEHAVEBEENUSEDFORINFLUENZAVACCINERESEARCHFROMTHEEARLIESTDAYSOF THESTUDYOFINFLUENZAVIRUSBIOLOGY3HORTLYAFTERTHEFIRSTHUMANINFLUENZA VIRUSWASISOLATEDFROMFERRETSINBY7ILSON3MITHANDCOLLEAGUESATTHE .ATIONAL)NSTITUTEFOR-EDICAL2ESEARCHIN,ONDON;= ITWASDISCOVERED THATHUMANINFLUENZAVIRUSESWOULDCAUSEDISEASEINMICEONLYIFTHEYWERE FIRSTADAPTEDTOTHESPECIESBYSERIALPASSAGESINTHELUNGS;=4HISWASSUB

4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



SEQUENTLYFOUNDTOBETRUEOFALLHUMANINFLUENZAVIRUSISOLATES/NEOFTHE MOSTCOMMONLYUSEDHUMANINFLUENZAVIRUSESINMOUSESTUDIESIS INFLUENZA !0UERTO2ICO02 AN(.VIRUSWITHACOMPLEXPASSAGEHISTORY INCLUDINGSEVERALPASSAGESINFERRETS ANDHUNDREDSOFPASSAGES INEGGSAND MICE#"3MITH #$# !TLANTA '! PERSONALCOMMUNICATION 4HISVIRUS ISWELLADAPTEDTOMICEANDCAUSESALETHALINFECTION4HENEED FORADAPTA TIONTHROUGHSERIALPASSAGEOFHUMANINFLUENZAVIRUSESISONEOFTHEMAJOR DRAWBACKS OF USING MICE IN INFLUENZA RESEARCH BECAUSE MANY MUTATIONS CANARISEDURINGADAPTATIONTOTHEMURINEHOST;n=THATCANALTERTHEIR REPLICATIONKINETICS ANDCANRESULTINTHEABILITYOFTHEVIRUSTOESCAPEINNATE IMMUNERESPONSES;= )NFLUENZA VIRUSES THAT CAUSE DISEASE AND ARE LETHAL FOR MICE PROVIDE A USEFUL ENDPOINT FOR VACCINE EFFICACY STUDIES $EPENDING ON THE STRAIN OF VIRUS USED MICE MAY BECOME LETHARGIC ANOREXIC DEVELOP RUFFLED F FUR AND MAY ALSO EXHIBIT NEUROLOGICAL SYMPTOMS OF INFECTION IN ADDITION TO WEIGHTLOSS WHICHISOFTENTHEPRIMARYOBJECTIVEMEASUREOFTHESEVERITYOF INFECTION"ODYTEMPERATUREISNOTAUSEFULMEASUREMENTINMICE BECAUSE HYPOTHERMIA CAN OCCUR FOLLOWING INFECTION WITH MOUSE ADAPTED VIRUSES )RRESPECTIVE OF WHETHER AN INFLUENZA VIRUS INDUCES MORBIDITY OR MORTAL ITYINMICE THELEVELOFREPLICATIONOFINFLUENZAVIRUSESINTHELUNGSISTHE MOSTINFORMATIVEENDPOINTFOREFFICACYSTUDIESINMICE SINCEEVENAMODEST REDUCTIONINTITEROFINFECTIOUSVIRUSINTHELUNGSCANBEASSOCIATEDWITHSUR VIVALFROMLETHALINFECTION; =-ICEIMMUNIZEDWITHINFLUENZAVIRUSES OR VACCINES DEVELOP SERUM (!) AND NEUTRALIZING ANTIBODIES THE TITERS OF WHICH CORRELATE WITH PROTECTION FROM SUBSEQUENT CHALLENGE 3TUDIES BY 6IRELIZIER ;= DEMONSTRATED THAT ANTIBODY ALONE COULD PROTECT AGAINST INFLUENZA INFECTION IN MICE 0ASSIVE TRANSFER OF IMMUNE SERUM TO NAIVE MICEREDUCEDREPLICATIONOFVIRUSINTHELUNGS ANDPROTECTEDTHERECIPIENT MICE FROM LETHAL INFLUENZA PNEUMONITIS BUT DID NOT PREVENT TRACHEITIS OR REPLICATIONOFVIRUSINTHEUPPERRESPIRATORYTRACT;=4HEOBSERVATIONTHAT PASSIVELYTRANSFERREDSERUMANTIBODIESCANREDUCEPULMONARYVIRUSREPLICA TIONBUTNOTVIRALREPLICATIONINTHEUPPERRESPIRATORYTRACTISNOTUNIQUETO INFLUENZA!3IMILAROBSERVATIONSHAVEBEENREPORTEDININFLUENZA#VIRUS ;= RESPIRATORY SYNCYTIAL VIRUS 236 ;= AND SEVERE ACUTE RESPIRATORY SYNDROME ASSOCIATEDCORONAVIRUS3!23 #O6 INFECTION;=7HEN MEA SURINGTHEAMOUNTOFVIRUSINVARIOUSTISSUESINCASESWHEREHIGHLEVELSOF SERUMANTIBODYAREPRESENT FOREXAMPLE WHENVACCINESAREADMINISTERED WITH ADJUVANT THE PRESENCE OF VIRUS SHOULD BE MEASURED BY QUANTITATIVE MOLECULAR METHODS TO RULE OUT THE POSSIBILITY OF EX VIVO NEUTRALIZATION BY SERUM ANTIBODY DURING TISSUE PREPARATION 3UCH EX VIVO NEUTRALIZATION ACCOUNTEDFORAREDUCTIONINDETECTABLEVIRUSOFUPTO FOLDINTHELUNGS OF MICE THAT HAD UNDERGONE PASSIVE TRANSFER OF IMMUNE SERUM AGAINST 3!23 #O6;=4HEUSEOFNASALANDBRONCHIOLARWASHSAMPLESINSTEADOF TISSUEHOMOGENATESFORVIRALQUANTITATIONWASALSOEMPLOYEDAS ASOLUTION TOTHISISSUE;=



#ATHERINE*,UKEAND+ANTA3UBBARAO

4ABLE4HEUSEOFTHEMOUSEMODELFORTHEEVALUATIONOFVACCINESAGAINSTINFLUENZA )NFLUENZAVIRUS SUBTYPESTESTED

&INDINGS

2EFS

(UMAN

(UMANINFLUENZAVIRUSISOLATESREQUIREADAPTATIONTOCAUSE INFLUENZA ILLNESSLETHALITY INMICE (. (. )NFECTIONUNDERANESTHESIARESULTSINVIRALPNEUMONIA (.

#LINICALSIGNSINCLUDERUFFLEDFUR HUNCHING LABOREDBREATHING UNSTEADYGAIT HYPOTHERMIAANDWEIGHTLOSS

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;n= )N THESE STUDIES SIGNS OF CLINICAL ILLNESS INCLUDING FEVER AND OTHER SYSTEMICSIGNS APPEAREDWITHINHOFADMINISTRATIONOFTHEVIRUS WHICHIS MORERAPIDINGENERALTHANTHEAPPEARANCEOFSYMPTOMSASSOCIATEDWITHPRO DUCTIVE INFLUENZA VIRUS INFECTION4HE SYSTEMIC SYMPTOMS DID NOT CORRELATE WITHTHETITEROFVIRUSSHEDINRESPIRATORYSECRETIONS ORWITH THEOCCURRENCE OFRESPIRATORYSYMPTOMS4HEOCCURRENCEOFSYSTEMICILLNESSINHUMANSFOL LOWINGADMINISTRATIONOFHIGHDOSESOFINFLUENZAVIRUSINTHEABSENCEOFHIGH LEVELSOFVIRUSREPLICATIONMAYBEEXPLAINEDBYTHEINNATEIMMUNERESPONSE TOANABORTIVEINFECTIONOFEPITHELIALCELLS 4HECURRENTPROCEDURESFORMARKETINGAPPROVALOFVACCINESFORSEASONAL INFLUENZA DO NOT INVOLVE EXTENSIVE SAFETY TESTING IN ANIMALS )N THE 53 A STANDARD GENERAL SAFETY TEST WHICH IS DESIGNED TO DETECT EXTRANEOUS TOXIC COMPONENTSINTHEVACCINEPREPARATION ISUSUALLYPERFORMEDWITHTHEFINAL DRUGPRODUCTINMICEANDGUINEAPIGS;=4HISTESTISPERFORMEDFORBOTH INACTIVATED AND LIVE ATTENUATED VIRUS VACCINES &OR INACTIVATED INFLUENZA VIRUSVACCINES VACCINECANBEADMINISTERED VIAEITHERTHESUBCUTANEOUSOR INTRAPERITONEALROUTESFORTHEGUINEAPIGTEST WHEREASONLYTHEINTRAPERITO NEALROUTECANBEUSEDFOROTHERTYPESOFVACCINE4HEVACCINE FORMULATION MUSTALSOBECERTIFIEDTOBEFREEOFENDOTOXIN .EWVACCINECANDIDATESORNOVELPREPARATIONSINCLUDINGVACCINESPRE PARED BY CURRENTLY LICENSED METHODOLOGIES THAT ARE NOW FORMULATED WITH ADJUVANT REQUIRE EXTENSIVE PRE CLINICAL SAFETY TESTING )N ADDITION TO TESTS SUCHASREPEATDOSETOXICOLOGYTESTINGANDGENERALSAFETYTESTING SOMETESTS WOULDBEAPPROPRIATEFORTHESPECIFICTYPEOFVACCINE EG DEMONSTRATIONOF ATTENUATION OF LIVE ATTENUATED VACCINES COMPARED TO THE WILD TYPE PARENT VIRUSINMORETHANONEANIMALSPECIES;  = ANDBIODISTRIBUTIONSTUD IESFORPLASMID BASEDVACCINES;n= &ERRETS HAVE BEEN USED TO ASSESS THE ATTENUATION OF COLD ADAPTED LIVE ATTENUATED VACCINES FOR INFLUENZA ;= 4HESE STUDIES SHOWED THAT COLD ADAPTED   REASSORTANT VACCINE VIRUSES GENERATED FROM HUMAN INFLUENZA VIRUSESFAILEDTOREPLICATEINTHELOWERRESPIRATORYTRACTOFFERRETS3INCEFER RETS ARE A GOOD MODEL FOR INFLUENZA INFECTION IN HUMANS THEY CAN ALSO BE USEDINTOXICOLOGICALSTUDIESOFINFLUENZAVACCINES 4HEATTENUATIONPHENOTYPEOFSEVERALLIVEATTENUATEDINFLUENZA VACCINE CANDIDATES WAS EVALUATED USING THE HAMSTER MODEL ; = &OR THE SMALL NUMBEROFTEMPERATURE SENSITIVE COLD ADAPTEDREASSORTANTINFLUENZAVIRUSES TESTED IN HAMSTERS AND LATER IN HUMANS THERE WAS GENERALLY A CORRELATION BETWEENTHELEVELOFREPLICATIONINHAMSTERSANDHUMANS(OWEVER INSTUD IESWITH!)HUMANINFLUENZAVIRUSREASSORTANTS THEFINDINGSIN HAMSTERSDID NOTACCURATELYPREDICTTHELEVELOFATTENUATIONOFTHEVIRUSESFORHUMANS;= 3UCHDATAAREIMPORTANTBECAUSETHEYDEMONSTRATETHATTHEGENETICDETERMI NANTSFORATTENUATIONOFINFLUENZAVIRUSESAREDIFFERENTINDIFFERENTSPECIES F .ON HUMANPRIMATESPECIESHAVENOTBEENUSEDEXTENSIVELYINSTUDIESOF THESAFETYOFINFLUENZAVACCINES#HIMPANZEESWEREUSEDINSEVERALSTUDIES TOEVALUATETHELEVELOFATTENUATIONANDSAFETYOFCANDIDATELIVEATTENUATED

4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



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)MMUNOGENICITY 4HE VAST MAJORITY OF STUDIES CONDUCTED IN ANIMALS IN INFLUENZA VACCINE RESEARCHARETHOSETHATEVALUATETHEIMMUNERESPONSETOCANDIDATEVACCINES !LTHOUGHITISCLEARTHATTHEIMMUNERESPONSESTOVACCINESINANIMALSARE NOT OFTEN IDENTICAL TO AND MAY NOT BE DIRECTLY PREDICTIVE OF THOSE SEEN IN HUMANS THEFIRSTSTEPINTHEPROOF OF PRINCIPLEOFANEWVACCINEISTOESTAB LISHTHEIMMUNOGENICITYOFAVACCINECANDIDATEINANIMALSBEFOREPROCEED ING TO CLINICAL EVALUATION4HE IMMUNE RESPONSES MEASURED IN THE ANIMAL MODELSHOULDBERELEVANTTOTHEDESIREDRESPONSEINHUMANS3UCHSTUDIES MAYPROVIDEUSEFULINFORMATIONREGARDINGREGIMENANDROUTESOFVACCINA TIONTOGUIDETHEDESIGNOFCLINICALTRIALS

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#ATHERINE*,UKEAND+ANTA3UBBARAO

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4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



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#ATHERINE*,UKEAND+ANTA3UBBARAO

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4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



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#ATHERINE*,UKEAND+ANTA3UBBARAO

(. 4HEYFOUNDTHATTWODOSESOFINACTIVATEDVACCINEWEREREQUIREDTO ELICIT(!)ANTIBODYRESPONSESOFAMAGNITUDETHATWOULDBEPROTECTIVEIN HUMANINFLUENZAINTHEMAJORITYOFVACCINATEDANIMALS ANDTHATTHEADDITION OFALUMADJUVANTRESULTEDINHIGHERLEVELSOF(!)ANTIBODYAND AGREATER SEROCONVERSIONRATE4HESEFINDINGSGENERALLYAGREEDWITHTHEOBSERVATIONS MADEINHUMANSWHENASIMILARVACCINEWASTESTEDINCLINICALSTUDIESTWO DOSES OF VACCINE WERE NECESSARY TO ACHIEVE ACCEPTABLE LEVELS OF F ANTIBODY ANDTHEADDITIONOFADJUVANT INTHISCASE-&INSTEADOFALUMUSEDINTHE STUDIESINMICE INCREASEDTHEMAGNITUDEOFTHEANTIBODYRESPONSEASWELL AS THE SEROCONVERSION RATE ;n= %FFICACY OF THIS VACCINE IN MICE WAS DETERMINED BY MEASUREMENT OF THE LEVEL OF VIRUS REPLICATION IN THE LUNGS ANDPROTECTIONAGAINSTLETHALCHALLENGEWITHAN(.ISOLATETHATWASHIGHLY VIRULENTINMICE 4HEEFFICACYOFSEVERALDIFFERENT(.VIRUSVACCINESHAVEBEEN EVALU ATEDINMICEANDINALLCASES THEVACCINESWEREIMMUNOGENICANDPROTECTIVE INMICEREVIEWEDIN;= 7HENTESTEDIN0HASE)STUDIESIN HUMANS INAC TIVATED(.VIRUSVACCINESWEREFOUNDTOBESUBOPTIMALLYIMMUNOGENIC REQUIRING HIGH DOSES ; = TO ELICIT NEUTRALIZING AND (!) ANTIBODY RESPONSES !DMINISTRATION OF WHOLE VIRION VACCINES AND INACTIVATED VIRUS VACCINES WITH ADJUVANT INCREASED IMMUNOGENICITY IN MICE AND IN HUMANS ; =)TISUNCLEARWHETHERDATAOBTAINEDINMICEWITHPANDEMICINFLU ENZA VACCINES ARE PREDICTIVE OF VACCINE IMMUNOGENICITY IN HUMANS SINCE PRE CLINICALDATAFORTHESPECIFICVACCINEFORMULATIONSTHATHAVEBEENTESTED INHUMANSTODATEHAVENOTBEENREPORTED #OLD ADAPTEDLIVEATTENUATEDVACCINECANDIDATESAGAINST(.!)VIRUS ESHAVEBEENDEVELOPED ANDWEREFOUNDTOBEIMMUNOGENICANDTO CONFER PROTECTIONAGAINSTCHALLENGEWITHHOMOLOGOUSANDHETEROLOGOUSWILD TYPE VIRUSESINMICE;  =3OMEOFTHESEVACCINESARECURRENTLYINCLINICAL TRIALS ANDSOTHEPREDICTIVEVALUEOFTHEINFORMATIONGAINEDFROMSTUDIESIN F MICECANNOTBEFULLYASSESSEDATTHISTIME

(VIRUSESANDVACCINES 2EPRESENTATIVE LOW PATHOGENICITY AND HIGHLY PATHOGENIC ( !) VIRUSES FROM BOTH THE %URASIAN AND .ORTH!MERICAN LINEAGES REPLICATED IN MICE WITHOUTPRIORADAPTATION;=(IGHLYPATHOGENIC(VIRUSESDEMONSTRATED EXTRAPULMONARY SPREAD TO THE SPLEEN AND BRAIN AS HAS BEEN OBSERVED WITH(0!)(.ISOLATES ALTHOUGH(VIRUSESWEREDETECTEDINTHEBRAIN EARLIERININFECTIONDAYPIFOR(ANDDAYFOR( DE7ITETAL;= 7 REPORTED THAT INTRANASAL INFECTION OF MICE WITH THE NON ADAPTED (0!) !.ETHERLANDS(.VIRUS THATWASISOLATEDFROMAFATALHUMAN CASE RESULTED IN SEVERE ILLNESS INDICATED BY WEIGHT LOSS LETHARGY RUFFLED FUR ANDLETHALITY4HERATEOFLOSSINBODYWEIGHTWASSIMILAR OVERARANGE OFDOSESOFVIRUSBETWEEN = AND = %)$4HEVIRUSWASDETECTED

4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



INTHESPLEEN LIVER KIDNEYSANDBRAIN ASWELLASINTHELUNGSOFMICE4HIS MODEL WAS USED FOR THE EVALUATION OF THE IMMUNOGENICITY AND EFFICACY F OF CANDIDATE(INFLUENZAVACCINES;=!SINGLEDOSEOFAN)3#/-VACCINE AND TWO DOSES OF A SUBUNIT VACCINE FAILED TO PROTECT MICE AGAINST LETHAL INFECTION WITH THE!., (. VIRUS WITH ONE EXCEPTION -ICE VACCINATEDWITHTWODOSESOF+GOR +G)3#/-VACCINEEXHIBITEDASMALL TEMPORARYLOSSINBODYWEIGHTBUTOTHERWISEAPPEAREDHEALTHYAFTERCHAL F LENGE6ACCINATIONWITHTWODOSESOFTHE)3#/-VACCINERESULTEDINATLEAST A  FOLD REDUCTION IN VIRUS REPLICATION IN THE LUNGS AND NEAR COMPLETE REDUCTIONOFEXTRAPULMONARYREPLICATIONOFCHALLENGEVIRUS(OWEVER INALL VACCINATEDMICE VIRUSWASSTILLPRESENTINTHELUNGSATHIGHTITERS -UNSTERETAL;=REPORTEDTHATTHEHUMAN(0!)(.VIRUSES ! .,AND!.,BOTHCAUSEDLETHALINFECTIONINMICEWHEN ADMINISTEREDINTRANASALLYATAHIGHDOSEDOSENOTSPECIFIED  !TADOSEOF  =4#)$ INFLUENZA!.,VIRUS WHICHWASISOLATEDFROMA FATALHUMANCASE RESULTEDINLOSSOFBODYWEIGHT RUFFLEDFUR LETHARGY AND RESPIRATORYPROBLEMSFROMDAYPI ANDINFECTEDMICEWEREEUTHANIZED ON DAY  PI WHEREAS MICE INFECTED INTRANASALLY WITH = 4#)$ OF INFLUENZA!.,VIRUS ISOLATEDFROMAHUMANWITHCONJUNCTIVITISIN THESAMEOUTBREAK NOSIGNSOFILLNESSORLOSSINBODYWEIGHTWEREOBSERVED UP TO DAY  PI4HE INFLUENZA!., VIRUS REPLICATED TO A TITER THAT WAS MORE THAN  FOLD HIGHER IN THE LUNGS OF INFECTED MICE THAN INFLUENZA !., VIRUS AND WAS ISOLATED FROM THE BRAIN SPLEEN LIVER AND KIDNEY OF ALL INFECTED ANIMALS )NFLUENZA !., VIRUS WAS ISOLATED FROM THE BRAIN OF ONLY ONE OUT OF THREE MICE AND WAS NOT DETECTEDFROMTHEOTHERORGANSEXAMINED(ISTOPATHOLOGICALFINDINGSFOR ALLMICEINFECTEDWITHINFLUENZA!.,VIRUSINCLUDEDNECROSISAND INFLAMMATIONTHROUGHOUTTHERESPIRATORY TRACTTHATWASPRONOUNCEDINTHE TRACHEA ANDBECAMEPROGRESSIVELYMILDERINTHEBRONCHI BRONCHIOLESAND ALVEOLI )NCONTRAST LESIONSINTHERESPIRATORYTRACTWEREONLYOBSERVEDIN ONEOUTOFFOURMICEINFECTEDWITHINFLUENZA!.,VIRUS ANDWERE CHARACTERIZEDASMILDTOMODERATECELLNECROSIS WITHNEUTROPHILINFILTRATES INTHETRACHEA BRONCHIANDBRONCHIOLES,ESIONSWERENOTOBSERVEDUPON HISTOPATHOLOGICAL EXAMINATION OF BRAIN HEART SPLEEN LIVER OR KIDNEYS OF MICEINFECTEDWITHEITHERVIRUS 6IRALANTIGENEXPRESSIONWASLIMITEDTOTHE RESPIRATORY TRACT TISSUES IN MICE INFECTED WITH EITHER VIRUS BUT WAS MORE ABUNDANTINMICEINFECTEDWITHINFLUENZA!.,VIRUS2IGONIAND COLLEAGUES;=REPORTEDTHAT(0!)(.VIRUSESISOLATEDFROM CHICKENS AND OSTRICHES COULD INFECT AND REPLICATE IN MICE WITHOUT ADAPTATION AND WEREASSOCIATEDWITHDISEASESIGNSOFVARYINGSEVERITY"RONCHITIS TRACHE ITIS ALVEOLITISANDBRAINLESIONSWEREOBSERVEDINMICEINFECTEDWITHTHREE (0!) (. INFLUENZA VIRUSES (OWEVER THE INFLUENZA!OSTRICH VIRUSCAUSEDMORESEVERELESIONSANDSPREADMORERAPIDLYINTHE LUNGSAND BRAINTHANTHEOTHERTWOVIRUSESINFLUENZA!OSTRICHANDINFLUENZA !CK ;=



#ATHERINE*,UKEAND+ANTA3UBBARAO

,OWPATHOGENICITY(VIRUSESREPLICATEDTOHIGHTITERSINTHEUPPERAND LOWER RESPIRATORY TRACT OF MICE BUT WERE NOT LETHAL EVEN AT HIGH DOSES )MMUNOGENICITYOFTHESEVIRUSESWASALSOEVALUATEDINMICE;=

(VIRUSESANDVACCINES (UMAN INFECTIONS WITH (.!) VIRUSES WERE FIRST REPORTED IN  ; = AND ALTHOUGHTHEILLNESSINTHEINFECTEDINDIVIDUALSWASRELATIVELYMILD THERE IS STILL CONCERN OVER THE PANDEMIC POTENTIAL OF ( VIRUSES BECAUSE VIRUSESOFTHISSUBTYPEAREHIGHLYPREVALENTINBIRDS;n=4HEPATHOGE NICITYOFHUMANANDAVIAN(INFLUENZAVIRUSESINMICEHASBEENSTUDIEDBY SEVERAL LABORATORIES WITH A VIEW TO THE ESTABLISHMENT OF AN ANIMAL MODEL THATCANBEUSEDTOSTUDYSTRATEGIESFORPREVENTION INCLUDING VACCINESAND ANTIVIRALDRUGS3OME(INFLUENZAVIRUSESREPLICATEINTHERESPIRATORYTRACT OF MICE WITHOUT PRIOR ADAPTATION ; n= BUT SERIAL PASSAGE OF THE !QUAIL(ONG+ONG'(. VIRUSINMICERESULTEDINANINCREASEIN VIRULENCEANDINEXTRAPULMONARYSPREADANDLETHALITYOFTHISVIRUSININTRA NASALLYINFECTEDMICE; =$ATAFROMDIFFERENTLABORATORIESUSINGTHE SAME(.VIRUSTOINFECTMICEARENOTCONSISTENT3OMEOFTHEFACTORSTHAT CAN INFLUENCE THE OUTCOME OF INFECTION ARE ANESTHESIA DOSE VOLUME AND ROUTEOFVIRUSADMINISTRATIONANDPASSAGEHISTORY)TISDIFFICULTTOCOMPARE STUDIES WHEN COMPLETE INFORMATION IS NOT PROVIDED &OR EXAMPLE IN STUD IESREPORTEDBY,UETAL;= THEHUMANINFLUENZA!(ONG+ONG (. VIRUSREPLICATEDEFFICIENTLYINTHELUNGSOFMICEBUTFAILEDTOCAUSE DEATH OR SIGNS OF DISEASE SIGNIFICANT WEIGHT LOSS OR TO SPREAD TO EXTRAPUL MONARYSITES(OWEVER ,ENEVAETAL;=REPORTEDTHATINFECTIONOFMICE WITHTHISVIRUSRESULTEDINMORTALITYANDSIGNIFICANTWEIGHTLOSSINTHE SURVIVING MICE )N THESE DISCORDANT STUDIES MICE WERE ANESTHETIZED WITH #/ ;= OR WITH METOFANE ;= WERE INFECTED BY THE SAME ROUTE USING VIRUSTHATHADBEENPROPAGATEDINEMBRYONATEDEGGS ATAPPROXIMATELYTHE SAMEDOSE %)$ BUTINOCULUMVOLUMESUSEDWERENOTSTATEDINEITHER STUDY SOITISNOTCLEARWHYTHISVIRUSWASLETHALINONESTUDYANDNOTINTHE OTHER3IMILARLY LETHALCHALLENGEOFMICEWITHTHEHUMANINFLUENZA!(ONG +ONG(. VIRUSWASREPORTEDASPARTOFASTUDYTODETERMINETHE EFFICACYOFAN-LIPOSOMEVACCINE;= ALTHOUGHTHISVIRUSDIDNOTCAUSE DISEASE OR LETHALITY IN THE HANDS OF OTHER INVESTIGATORS ;  =!LL LABORATORIESDELIVEREDVIRUSINTRANASALLYTOANESTHETIZEDMICE(OWEVER IN THESTUDYREPORTEDBY%RNSTETAL;= MICEWEREANESTHETIZEDINTRAPERITO NEALLYWITHKETAMINEXYLAZINE WHEREASINTHEOTHERTWOSTUDIES INHALATIONAL ANESTHESIAWASUSED WHICHMAYRESULTINALIGHTERSTATEOFANESTHESIA 4HEMOUSEMODELWASUSEDTOEVALUATETHELEVELOFATTENUATIONANDTHE PROTECTIVEEFFICACYOFACOLD ADAPTEDLIVEATTENUATED(.VACCINECANDI DATE BEARING THE (! AND .! FROM THE INFLUENZA!CK(+' (. VIRUSANDTHEINTERNALPROTEINGENESFROMTHEINFLUENZA!!NN!RBOR

4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



COLD ADAPTEDVIRUS;=4HE(.LIVEATTENUATEDVACCINEWAS RESTRICTED INREPLICATIONANDPROTECTEDMICEFROMCHALLENGEWITHHOMOLOGOUSANDHET EROLOGOUSWILD TYPE(.INFLUENZAVIRUSES4HISVACCINEISBEINGEVALUATED FORSAFETYANDIMMUNOGENICITYINCLINICALTRIALS

(.PANDEMICVIRUS ,IKETHEHIGHLYPATHOGENIC(.!)VIRUSES THEFULLYRECONSTRUCTEDRECOM BINANT(.PANDEMICINFLUENZAVIRUSWASHIGHLYLETHALINMICEWITH OUTPRIORADAPTATION;=4HEMEANTIMETODEATHINMICEINFECTEDINTRA NASALLYWASDAYS(OWEVER INCONTRASTTOTHEHIGHLYPATHOGENIC(. INFLUENZA VIRUSES THIS VIRUS WAS NOT DETECTED IN EXTRAPULMONARY TISSUES (ISTOPATHOLOGICALFINDINGSINCLUDEDNECROTIZINGBRONCHITISAND BRONCHIOLITIS MODERATETOSEVEREALVEOLITISANDSEVEREPERIBRONCHIALANDALVEOLAREDEMA 4HE MOUSE MODEL APPEARS TO BE POTENTIALLY USEFUL FOR THE EVALUATION OF PANDEMIC INFLUENZA VACCINES -OST !) VIRUSES STUDIED IN MICE TO DATE CAN REPLICATE WITHOUT ADAPTATION ALTHOUGH THE OUTCOME OF INFECTION WITH SOME!) VIRUSES IS CLEARLY DIFFERENT DEPENDING NOT ONLY ON THE PARTICULAR VIRUS BEING STUDIED BUT ALSO ON THE LABORATORY IN WHICH THE STUDIES WERE CONDUCTED)TISIMPORTANTTHAT!)VIRUSESCONTINUETOBEEVALUATEDINMICE USINGSTANDARDIZEDINOCULATIONPROCEDURESANDDOSES WITHMEASUREMENTOF THESAMEENDPOINTS SOTHATTHEUTILITYOFTHISMODELCANBEMAXIMIZEDFOR THEEVALUATIONOFPANDEMICINFLUENZAVACCINES

&ERRETS (.VIRUSESANDVACCINES 4HEABILITYOFALIMITEDNUMBEROF!)SUBTYPESTOREPLICATEAND CAUSEDISEASE IN FERRETS HAS BEEN INVESTIGATED AND NOT SURPRISINGLY THE BEHAVIOR OF ( SUBTYPE VIRUSES HAS BEEN STUDIED IN THE MOST DETAIL :ITZOW AND COLLEAGUES ;= DEMONSTRATED THAT TWO (. INFLUENZA VIRUSES ISOLATED FROM HUMAN CASESOFINFECTIONIN(ONG+ONGINWERECAPABLEOFREPLICATIONNOTONLY INTHERESPIRATORYTRACT BUTALSOINTHEBRAIN SPLEENANDINTESTINESOFFERRETS 6IRUSREPLICATIONWASASSOCIATEDWITHCLINICALSIGNSOFDISEASESUCHASSEVERE LETHARGY SNEEZING RHINITIS HINDLIMBPARESISAND INSOMECASES DIARRHEA AND SOME(.VIRUSESWERELETHALTOFERRETS(OWEVER THEHIERARCHYOFSEVERITY OFDISEASESEENWITHTHEDIFFERENT(.ISOLATESININFECTIONOFMICEWAS NOTOBSERVEDINFERRETSINFLUENZA!(+AND!(+WEREEQUAL LYVIRULENTAFTERINTRANASALINFECTIONOFFERRETS WHEREASTHE!(+VIRUS WASMOREVIRULENTINMICETHANTHE!(+VIRUSWASINSEVERALSTUDIES ;n =!SWITHMICE THESIGNIFICANCEFORHUMANSOFTHEDISEASESIGNS ANDEXTRAPULMONARYREPLICATIONOF(.VIRUSESINFERRETSISNOTCLEAR PAR



#ATHERINE*,UKEAND+ANTA3UBBARAO

TICULARLYSINCEINTHESAMESTUDY :ITZOWETALREPORTEDISOLATIONOFAHUMAN (.INFLUENZAVIRUSFROMTHEBRAINOFFERRETSFOLLOWINGINTRANASALINFECTION 3IMILARSTUDIESWERECONDUCTEDUSINGHUMANANDAVIAN(.VIRUSESISOLATED INn; ='OVORKOVAETAL;=EVALUATEDFOUR HUMAN(. INFLUENZAISOLATESANDNINEAVIAN(.ISOLATESFROM!SIAFROM!WIDE SPECTRUMOFINFECTIVITY SEVERITYOFDISEASEANDLETHALITYWAS OBSERVEDINFER RETSINOCULATEDWITHTHESEVIRUSES4HE(.VIRUSESISOLATEDFROMHUMANS F AND TWO OF THE AVIAN ISOLATES CAUSED SEVERE DISEASE IN FERRETS WITH SOME LETHALITY (OWEVER IT IS DIFFICULT TO DRAW GENERAL CONCLUSIONS REGARDING THE BEHAVIOR OF THESE VIRUSES IN THIS MODEL BECAUSE SMALL NUMBERS OF ANIMALS WEREUSEDONLYTWOANIMALSPERGROUPFORALLBUTONEOFTHEVIRUSESTESTED AND THERE WAS VARIABILITY IN INFECTIVITY &OR EXAMPLE ALTHOUGH THE INFLUENZA !6IETNAM VIRUS CAUSED SEVERE DISEASE IN TWO OUT OF TWO FERRETS INOCULATED IT WAS LETHAL IN ONLY ONE ANIMAL AND VIRUS WAS ONLY RECOVERED FROMTHENASALWASHES)NCONTRAST THEINFLUENZA!6IETNAMVIRUS WHICH WAS ALSO LETHAL IN ONE OUT OF TWO FERRETS INOCULATED WAS RECOVERED FROMTHELUNGS BRAIN SPLEENANDINTESTINEOFTHESEANIMALS3IMILARLY -AINES ETAL;=EVALUATED(.ISOLATESFROM!SIAFROMINTHE FERRETMODEL !LTHOUGH DIFFERENT VIRUSES WERE USED IN THIS STUDY COMPARED TO THAT CON DUCTED BY 'OVORKOVA ET AL WITH THE EXCEPTION OF !6IETNAM SIMILARFINDINGSWEREREPORTEDTHEHUMANISOLATESCAUSEDSEVEREDISEASE WITH SOMELETHALITY INFERRETS!GAIN SMALLNUMBERSOFANIMALSWEREUSEDTHREE PERGROUPFORMOSTOFTHEISOLATESTESTED ANDSOMEVARIABILITY ININFECTIVITY ANDSEVERITYOFDISEASEWASOBSERVED)NTHESTUDYCONDUCTEDBY :ITZOWETAL GROSS PATHOLOGICAL CHANGES OBSERVED IN FERRETS INFECTED WITH HIGHLY VIRULENT (0!)(.VIRUSESINCLUDEDFOCALAREASOFREDNESSINTHELUNGS CONSOLIDA TIONOFTHELUNGSANDRAREDISCOLORATIONOFTHELIVER PETECHIAEONTHELIVERAND LESIONSONTHEINTESTINESANDKIDNEYS;=-AINESETAL;=REPORTEDTHE PRESENCEOFHEMORRHAGE INTHEADIPOSETISSUESURROUNDINGTHE LIVER KIDNEY AND BLADDER IN TWO THIRDS OF INFECTED FERRETS (ISTOPATHOLOGICAL FINDINGS IN THELUNGSOFINFECTEDFERRETS INCLUDEDACUTEBRONCHIOLITIS BRONCHOPNEUMONIA INTERSTITIALPNEUMONIA WITHSUPPURATIVEEXUDATESINTHEBRONCHI BRONCHIOLES ANDADJACENTALVEOLARSPACES WITHPROMINENTEPITHELIALNECROSISANDMARKED INTRAALVEOLAR EDEMA BY DAY  PI AND BRONCHITIS BRONCHIOLITIS AND PNEU MONIAOBSERVEDONDAYSnPI;  =)NFLAMMATORYCHANGESWERE ALSOEVIDENTINTHEBRAINOFFERRETSINFECTEDWITHHIGHLYVIRULENT(0!)(. VIRUSES FROMDAYSnPI INCLUDINGGLIALNODULES PERIVASCULARINFILTRATIONOF LYMPHOCYTES AND POLYMORPHONUCLEAR LEUKOCYTES IN THE BRAIN PARENCHYMA NEURONOPHAGIA AND LYMPHOCYTIC INFILTRATES IN THE CHOROID PLEXUS ; = 6IRALANTIGENWASOBSERVEDBYIMMUNOHISTOCHEMISTRYINNEURONSINTHESAME AREASOFTHEBRAINASTHEINFLAMMATION;='OVORKOVAETAL ;=REPORTED HISTOPATHOLOGICALCHANGESINTHELIVER INCLUDINGDIFFUSEVACUOLIZATIONOFTHE HEPATOCELLULAR CYTOPLASM MONONUCLEAR INFILTRATES PERIPORTAL HEMORRHAGE ANDHEPATOCELLULARNECROSIS'ENERALLY THEVIRUSESISOLATEDFROMAVIANSPECIES CAUSEDLESSSEVEREDISEASETHANTHOSEISOLATEDFROMHUMANS

4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



4HENUMBEROFFERRETSINOCULATEDWITHEACHVIRUSWASSMALLAND FERRETS AREANOUTBREDSPECIES SOTHESIGNIFICANCEOFVARIABILITYINDATASUCHASVIRUS REPLICATIONANDCLINICALILLNESSAREDIFFICULTTOINTERPRET5NTILTHESCIENTIFIC COMMUNITYHASMOREEXPERIENCEWITHTHEBEHAVIOROF!)VIRUSESINANIMAL MODELS IT WOULD BE PRUDENT TO COMPARE NEW ISOLATES WITH WELL CHARACTER IZEDSTRAINSANDTOSTUDYTHESEPATHOGENSINMORETHANONEMODEL 4HEFERRETMODELHASBEENUSEDTOEVALUATETHEEFFICACYOFSEVERALEXPER IMENTAL INACTIVATED ; = AND LIVE ATTENUATED ;  = VACCINES AGAINST(.INFLUENZA)NACTIVATED(.VACCINESWEREIMMUNOGENICAND PROTECTIVEINTHEFERRETMODEL; =(OWEVER INACTIVATED (.VAC CINESTHATWERETESTEDINCLINICALTRIALSWERESUBOPTIMALLYIMMUNOGENIC; =4HE ATTENUATION OF COLD ADAPTED LIVE ATTENUATED (. VACCINES WAS DEMONSTRATEDINFERRETS4HESEVACCINECANDIDATESWEREALSOIMMUNOGENIC ANDPROTECTIVEAGAINSTCHALLENGEWITHHOMOLOGOUSANDHETEROLOGOUS(. WILD TYPE VIRUSES IN FERRETS ;=7HETHER THE OBSERVATIONS OF ATTENUATION AND CROSS REACTIVE IMMUNE RESPONSES IN FERRETS ARE BORNE OUT IN CLINICAL STUDIESINHUMANSREMAINSTOBESEEN#LINICALEVALUATIONOFTHESAFETYAND IMMUNOGENICITYOFTHESEVACCINESISCURRENTLYUNDERWAY 0ROTECTION FROM LETHAL (. INFECTION AND LEVEL OF REPLICATION OF THE CHALLENGE VIRUS IN THE LUNGS AND OTHER TISSUES ARE THE ENDPOINTS USED FOR EVALUATIONOFEFFICACYINTHISMODEL6AN2IELETAL;=DEMONSTRATEDTHAT THEPATTERNOFATTACHMENTOF(.INFLUENZAHUMANISOLATESINTHERESPIRA TORYTRACTOFFERRETSWASSIMILARTOTHATSEENINTHEHUMANRESPIRATORYTRACT WHEREBY VIRUS ATTACHED PREDOMINANTLY TO TYPE )) PNEUMOCYTES ALVEOLAR MACROPHAGES AND NONCILIATED CUBOIDAL EPITHELIAL CELLS OF THE TERMINAL BRONCHIOLESINTHELOWERRESPIRATORYTRACT ANDBECAMEPROGRESSIVELYRARER MOREPROXIMALLYINTHERESPIRATORYTRACT TOWARDSTHETRACHEA4HISPATTERN OF (. VIRUS ATTACHMENT PREDOMINANTLY TO THE LOWER RESPIRATORY TRACT IS THOUGHTTOBERELATEDTOTHEDISTRIBUTIONOF_  SIALICACIDRECEPTORS;= (OWEVER OTHERINVESTIGATORSFOUNDTHAT(.INFLUENZAVIRUSESWEREABLE TO INFECT EX VIVO CULTURES OF THE HUMAN UPPER RESPIRATORY TRACT IE NASO PHARYNGEAL ADENOIDANDTONSILLARTISSUES DESPITEALACKOF_  SIALICACID RECEPTORS IN THESE TISSUES ;=4HE TROPISM OF (. INFLUENZA VIRUSES IN THE RESPIRATORY TRACT OF HUMANS AND OTHER SPECIES REMAINS EQUIVOCAL AND FURTHER STUDIES IN WHICH A NUMBER OF DIFFERENT ISOLATES ARE EVALUATED IN LARGERNUMBERSOFANIMALSARENEEDED

/THER!)SUBTYPES 4HEREAREONLYISOLATEDREPORTSINTHELITERATURETHATDESCRIBETHEREPLICATION ANDCLINICALSIGNSRESULTINGFROMINFECTIONOFFERRETSWITHOTHER!)SUBTYPES (INSHAW ET AL ;= DEMONSTRATED THAT!) VIRUSES OF THE ( ( ( ( AND(SUBTYPES ASWELLASAN(.VIRUSISOLATEDFROMASEAL REPLICATED IN THE UPPER RESPIRATORY TRACT OF FERRETS BUT ELICITED LOW OR UNDETECTABLE



#ATHERINE*,UKEAND+ANTA3UBBARAO

LEVELSOFANTIBODY.ONEOFTHESE!)ISOLATESTESTEDCAUSEDSIGNSOFDISEASE ININFECTEDFERRETS *OSEPH ET AL ;= EVALUATED THE IMMUNOGENICITY OF (!) VIRUSES IN FERRETS AND DEMONSTRATED THAT THE PATTERN OF ANTIGENIC RELATEDNESS OF THE VIRUSESSTUDIEDWASSIMILARTOTHATOBSERVEDINMICE

(.PANDEMICVIRUS 4HE RECONSTRUCTED  (. INFLUENZA VIRUS REPLICATED TO HIGH TITERS IN THEUPPERRESPIRATORYTRACTOFFERRETSFOLLOWINGINTRANASALINOCULATION;= !LLINOCULATEDFERRETSEXHIBITEDSEVERESIGNSOFDISEASETHATINCLUDEDLETH ARGY ANOREXIA SNEEZING RHINORRHEA SEVEREWEIGHTLOSSANDHIGHFEVERFROM DAYPI ANDTWOOUTOFTHREEANIMALSSUCCUMBEDTOINFECTIONBYDAY 5NLIKETHEHIGHLYPATHOGENIC(.VIRUSESINFERRETS VIRALREPLICATIONWAS NOTDETECTEDINTISSUESOUTSIDETHERESPIRATORYTRACT.ECROTIZINGBRONCHIOL ITIS MODERATETOSEVEREALVEOLITISANDEDEMAWEREOBSERVEDINTHELUNGSOF INFECTEDFERRETSONDAYPI4HEPRESENCEOFVIRALANTIGENINTHEUPPERAND LOWER PORTIONS OF THE BRONCHI BRONCHIAL AND BRONCHIOLAR EPITHELIUM AND HYPERPLASICEPITHELIUMWITHINTHEALVEOLIWASOBSERVED

#ATS 4HERE ARE FEW REPORTS IN THE LITERATURE ON INFLUENZA INFECTION IN CATS )N STUDIES CONDUCTED BY 0ANIKER AND .AIR IN THE S ; = INTRANASAL INFECTION OF ANESTHETIZED CATS WITH INFLUENZA !(ONG+ONG (. VIRUS FRESHLY ISOLATED FROM HUMAN CASES OR LABORATORY AND EGG ADAPTED ISOLATESDIDNOTRESULTINCLINICALSIGNSOFINFLUENZABUTVIRUSWASRECOVERED FROMPHARYNGEALSECRETIONS ANDINFECTIONINDUCED(!)ANTIBODIESANDWAS TRANSMITTEDTOCONTACTANIMALS)NFECTEDCATSDIDNOTDISPLAYCLINICALSIGNSOF INFLUENZA (INSHAW AND COLLEAGUES ;= LATER DEMONSTRATED THAT INTRANA SALLYADMINISTERED(.AND(.!)VIRUSESREPLICATEDINTHEUPPERRESPI RATORYTRACTOFCATSWITHOUTCLINICALSIGNSOFDISEASE ANDTHECATSDEVELOPED (!)ANTIBODIESAFTERINFECTION

(.!)VIRUSES 4HERE WAS LITTLE INTEREST IN INFLUENZA INFECTION AND IMMUNITY IN CATS UNTIL THERECENTRE EMERGENCEOFHIGHLYPATHOGENICAVIAN(.VIRUSESIN!SIA WHENITWASREPORTEDTHATANUMBEROFBIGCATS NAMELYTIGERSANDLEOPARDS INZOOSIN4HAILAND BECAMEINFECTEDWITH(0!)(.VIRUSES APPARENTLY AFTERTHEYWEREFEDINFECTEDCHICKENCARCASSES;=)NFECTIONINMANYOFTHESE FELIDSWASFATAL ANDLATERANECDOTALREPORTSOF(.INFECTION INDOMESTIC

4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



CATSINAREASWHERETHEREWEREOUTBREAKSOF(.INFECTIONINAVIANPOPULA TIONSCONTRIBUTEDTOASURGEININTERESTIN(.INFLUENZAINCATS4HEPAT TERN OF ATTACHMENT OF A HUMAN (. INFLUENZA VIRUS TO RESPIRATORY TRACT TISSUESOFACATWASSIMILARTOTHATSEENWITHHUMANTISSUE;= %XPERIMENTAL INFECTION OF %UROPEAN SHORT HAIRED CATS WITH AN (. VIRUSISOLATEDFROMAHUMANIN6IETNAMINRESULTEDINCLINICALDISEASE VIRUSREPLICATIONINRESPIRATORYANDEXTRA PULMONARYTISSUES ANDPATHOLOGI CAL CHANGES CONSISTENT WITH (. INFECTIONS IN HUMANS ; = #LINICAL SIGNS INCLUDINGSIGNIFICANTELEVATIONINBODYTEMPERATURE DECREASEDACTIVITY CONJUNCTIVITIS AND LABORED BREATHING WERE SEEN IN EXPERIMENTALLY INFECTED CATS THAT WERE INFECTED INTRATRACHEALLY OR BY FEEDING ON INFECTED CHICKS ;=3IMILARDISEASESYMPTOMSWEREOBSERVEDINSENTINELCATSTHATBECAME INFECTED FROM BEING HOUSED WITH CATS THAT HAD BEEN INFECTED INTRATRACHE ALLY)LLNESSINCONTACTCATSBECAMEAPPARENTABOUTDAYSLATERTHANINTHE CATSINFECTED VIA THEINTRATRACHEALROUTE0EAKVIRALTITERSINTHROATSWABSOF INTRATRACHEALLYINFECTEDCATSWERE^ 4#)$ML WHEREASTHEPEAKTITERS OBSERVED IN NASAL SWABS RANGED FROM  TO  4#)$ML ;=6IRUS WASALSORECOVEREDFROMRECTALSWABSOFCATSINFECTEDBYFEEDINGONINFECTED CHICKS BUTTHETITERSOFVIRUSINTHESESAMPLESVARIEDWIDELY )NADDITION CATS INFECTED THROUGH FEEDING HAD LESIONS IN THE INTESTINES )N ANIMALS INFECTED INTRATRACHEALLY OR BY FEEDING VIRUS WAS ALSO RECOVERED FROM EXTRA PULMO NARY TISSUES MOST OFTEN FROM THE BRAIN LIVER KIDNEY AND HEART )NFECTED SENTINELCATSDIDNOTHAVEDETECTABLEVIRUSINTISSUESOUTSIDETHERESPIRATORY TRACT HOWEVER PATHOLOGICAL CHANGES WERE OBSERVED IN THE ADRENAL GLANDS INONEOFTHETWOSENTINELCATSINFECTEDINTHISMANNER4HESESTUDIESDEM ONSTRATEDTHAT(0!)(.VIRUSESARECAPABLEOFEXTRAPULMONARYSPREAD IN CATS AND CAN CAUSE SEVERE DISEASE AND EVEN DEATH IN ANIMALS INFECTED INTRATRACHEALLY OR BY FEEDING ON INFECTED BIRD CARCASSES4HE OBSERVATIONS ALSORAISETHEPOSSIBILITYTHAT(0!)(.INFLUENZAINCATSMAY BESPREAD FROMTHEGASTROINTESTINALTRACT +ARACAETAL;=REPORTEDSTUDIESONTHEIMMUNOGENICITYOFA FOWLPOX BASED ( VACCINE IN CATS (!) ANTIBODIES WERE DETECTED IN SERUM OF CATS FOLLOWINGASINGLESUBCUTANEOUSDOSEOFVACCINE ANDASIGNIFICANTBOOSTIN ANTIBODYTITERSWASOBSERVEDFOLLOWINGASECONDVACCINATION )TREMAINSTOBESEENWHETHERCATSWILLBEUSEDEXTENSIVELYINTHEEVALU ATIONOFPANDEMICINFLUENZAVACCINES

(AMSTERS (VIRUSESANDVACCINES 3AITO AND COLLEAGUES CONDUCTED A STUDY TO EVALUATE THE REPLICATION AND PATHOGENICITY OF INFLUENZA VIRUSES OF VARIOUS SUBTYPES IN 3YRIAN HAMSTERS ;=4HEINFLUENZA!(+(. VIRUSREPLICATEDTOHIGHTITERSIN



#ATHERINE*,UKEAND+ANTA3UBBARAO

THELUNGS BUTWASNOTLETHALTOHAMSTERSANDWASNOTDETECTED INTHEBRAIN 4HE (0!) (. INFLUENZA!(+ VIRUS THAT WAS HIGHLY VIRULENT IN MICEWASALSOLETHALINHAMSTERS WITHALLANIMALSSUCCUMBINGTOINFECTION BYDAYPI AND ASINMICE VIRUSWASRECOVEREDFROMTHEBRAINOFINFECTED HAMSTERS!VIAN (. AND (. ISOLATES REPLICATED IN THE LUNGS OF HAM STERS BUTTOLOWERTITERSTHANHUMANISOLATES4HEHUMAN(. VIRUSELICITED LOWLEVELSOFNEUTRALIZINGANTIBODYININFECTEDHAMSTERS WHEREASTHEAVIAN (.ISOLATEDIDNOTELICITDETECTABLENEUTRALIZINGANTIBODY4HEBEHAVIOROF 4 THISLIMITEDNUMBEROF!)ISOLATESINTHE3YRIANHAMSTERMODEL SUGGESTTHAT THESEVIRUSESMAYBESIMILARTOTHATOBSERVEDINMICE ANDFURTHEREVALUATION OFTHISMODELFOREVALUATINGTHEEFFICACYOFPANDEMICINFLUENZA VACCINESIS WARRANTED

.ON HUMANPRIMATES 4HEREISRENEWEDINTERESTINTHEUSEOFNON HUMANPRIMATESFORIMMUNOGE NICITYSTUDIESFORPANDEMICVACCINES BASEDONTHEPRESUMPTION THATIMMUNE RESPONSES IN THESE ANIMALS HAVING A CLOSER EVOLUTIONARY RELATIONSHIP TO HUMANS MAYBEMOREPREDICTIVEOFTHERESPONSESINHUMANSTHANSMALLER ANIMALSLIKEMICEANDFERRETS4ODATE THEREAREFEWDATAAVAILABLEONTHE SEROLOGICALRESPONSESINNON HUMANPRIMATESTO!)VIRUSVACCINES

(.!)VIRUSES 4HEUSEOFCYNOMOLGUSMACAQUESASAMODELFORINFLUENZAVIRUSINFECTION INHUMANSWASREVISITEDFOLLOWINGTHEEMERGENCEOFTHEHIGHLYPATHOGENIC (.!) VIRUSES IN  ;=4HE INITIAL HUMAN (. INFLUENZA ISOLATE !(ONG+ONG ISOLATEDFROMAFATALCASEOFINFLUENZAINACHILD;= WASINOCULATEDATMULTIPLESITES INCLUDINGTHETRACHEA TONSILSANDCONJUNC TIVA4HREEOFFOURANIMALSDEVELOPEDFEVERWITHINDAYS ANDONESHOWED SIGNS OF ANOREXIA AND ACUTE RESPIRATORY DISTRESS (IGH TITERS OF VIRUS WERE RECOVERED FROM LUNGS ON DAY  PI AND VIRUS WAS ALSO ISOLATED FROM THE TRACHEA TRACHEOBRONCHIALLYMPHNODESANDHEART6IRUSWASNOT RECOVERED FROMTHESETISSUESONDAYPI6IRUSWASALSORECOVEREDFROMBRONCHIOAL VEOLARLAVAGEFROMTWOOUTOFTWOANIMALSONDAYSANDPI FROMPHA RYNGEALSWABSFROMTWOANIMALSONDAYPI ANDFROMNASALSWABSFROM ONEANIMALONDAYSAND6IRAL2.!WASDETECTEDINTHEBRAINSOFTWO ANIMALSBY24 0#2ONDAYPI ANDINTHESPLEENOFALLFOURANIMALSTESTED ONDAYPI0ATHOLOGICALCHANGESINTHELUNGSOFINFECTEDANIMALSINCLUDED PULMONARY CONSOLIDATION NECROTIZING BRONCHO INTERSTITIAL PNEUMONIA AND FLOODING OF ALVEOLI WITH EDEMA FLUID FIBRIN ERYTHROCYTES CELL DEBRIS MAC ROPHAGESANDNEUTROPHILSANDINFLAMMATORYCHANGESWERESEENINMULTIPLE ORGANS;=

4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



)NFECTION OF 2HESUS MACAQUES WITH AVIAN (. ISOLATES REPORTED BY #HEN ET AL ;= INDICATED THAT RESULTS OF INTRANASAL INOCULATION VARIED DEPENDING ON THE INFLUENZA VIRUS ISOLATE USED #LINICAL SIGNS OF INFECTION INCLUDINGELEVATIONINBODYTEMPERATURE ANOREXIAANDINCREASED RESPIRATORY RATEWEREOBSERVEDINMACAQUESINOCULATEDWITHTHEFOLLOWING(.VIRUS ES!BAR HEADEDGOOSE1INGHAI !GREATCORMORANT1INGHAI AND!DUCK'UANGXI0ATHOLOGICALCHANGESWERESEENINTHELUNGS OFALLINFECTEDANIMALS BUTWEREMOREPRONOUNCEDINTHEMONKEYSINOCU LATEDWITHTHEDUCKISOLATE(OWEVER THEONLYVIRUSTOBERE ISOLATEDFROM INFECTED ANIMALS WAS!DUCK'UANGXI AND THIS VIRUS WAS ISOLATED FROMRESPIRATORYTRACTSECRETIONSANDTISSUESANDALSOFROMSPLEEN LIVERAND HEART

(.PANDEMICVIRUS #YNOMOLGUS MACAQUES WERE EVALUATED AS A MODEL FOR THE RECONSTRUCTED (.PANDEMICINFLUENZAVIRUS;=-ONKEYSWEREINFECTEDBYMUL TIPLEROUTESnINTRATRACHEALLY ORALLY ONTHETONSILSANDCONJUNCTIVAnBASED ON THE EARLIER STUDIES WITH (0!) (. INFLUENZA VIRUSES IN THIS SPECIES ;=!NIMALSINFECTEDWITHTHERECONSTRUCTEDVIRUSHADSEVERECLINI CALILLNESS HIGHLEVELSOFVIRUSREPLICATIONINTHERESPIRATORYTRACTANDSEVERE PATHOLOGICALCHANGESINTHELUNGS COMPAREDTOCONTROLANIMALSINFECTEDWITH ARECOMBINANTHUMAN(.INFLUENZAVIRUS !+AWASAKI;= 4HEREMAYBEAPLACEFORNON HUMANPRIMATESINTHEEVALUATIONOFPAN DEMICINFLUENZAVACCINES BUTTHECURRENTLYAVAILABLEDATAARENOTSUFFICIENT TOSUPPORTTHEUSEOFTHESEMODELSFORIMMUNOGENICITYOREFFICACYSTUDIES &URTHER STUDIES ARE NEEDED TO CHARACTERIZE!) INFECTION AND THE IMMUNE RESPONSESTO!)VIRUSESANDVACCINESINTHESESPECIES

#ORRELATESOFPROTECTIONFROM!)VIRUSESANDREGULATORYCONCERNS $ESPITETHEFACTTHATTHECORRELATESOFPROTECTIONFROM!)VIRUSINFECTIONSIN HUMANSARENOTKNOWN THECRITERIAFORLICENSINGPANDEMICINFLUENZAVACCINES AREBASEDONTHEPREVIOUSHUMANEXPERIENCEWITHVACCINESAGAINSTSEASONAL INFLUENZA)N%UROPEANDTHE5NITED3TATES REGULATORYAUTHORITIESHAVEPUB LISHEDGUIDANCEFORVACCINEMANUFACTURERSTHATATTEMPTTOBALANCETHENEED FOREXPEDITEDAPPROVALOFPANDEMICINFLUENZAVACCINESWITHTHEREQUIREMENTS OFDEMONSTRATIONOFSAFETYANDIMMUNOGENICITYOFCANDIDATEVACCINES )NTHE5NITED3TATES FOREXAMPLE AGUIDANCEFORVACCINEMANUFACTURERS WASPUBLISHEDIN;= WHICHSTATESTHATLICENSUREOFBOTHINACTIVATED ANDLIVEATTENUATEDVACCINESFORPANDEMICINFLUENZASHOULDBEBASEDONTHE PERCENTOFSUBJECTSACHIEVINGAN(!)ANTIBODYTITEROFORGREATER AND UPONTHERATEOFSEROCONVERSION WHICHISDEFINEDASAFOURFOLDORGREATERRISE



#ATHERINE*,UKEAND+ANTA3UBBARAO

IN POST VACCINATION (!) ANTIBODY TITER %FFICACY STUDIES IN ANIMAL MODELS ALTHOUGH NOT AN ABSOLUTE REQUIREMENT MAY AT LEAST PROVIDE EVIDENCE THAT BIOLOGICALLYRELEVANTIMMUNERESPONSESAREELICITEDBYCANDIDATEVACCINES 4HISGUIDANCEISINTENDEDTOALLOWFORTHERAPIDMARKETINGAPPROVALOF PANDEMIC INFLUENZA VACCINES THAT ARE PRODUCED USING MANUFACTURING PRO CESSES THAT ARE ALREADY VALIDATED FOR SEASONAL INFLUENZA VACCINES SO THAT THE LICENSURE OF THE PANDEMIC VACCINE IS ESSENTIALLY A STRAIN CHANGE 3UCH APPROVAL REQUIRES MUCH MORE LIMITED TESTING OF THE CANDIDATE VACCINES IN ANIMALMODELS)NTHE%UROPEAN5NION MANUFACTURERSAREREQUIREDTOSUB MIT INFORMATION ON THE PRODUCTION AND PRE CLINICAL TESTING OF A @MOCK UP PANDEMICVACCINE)NTHEEVENTOFAPANDEMIC AVACCINEMADEINTHESAME WAYASTHEMOCK UPVACCINE BUTBASEDONTHENASCENTPANDEMICVIRUS WILL BE PRODUCED AND WILL BE SUBJECT TO LIMITED PRE CLINICAL CHARACTERIZATION INCLUDING IMMUNOGENICITY STUDIES IN ANIMALS ON AT LEAST ONE BATCH OF THE PRODUCT;=%FFICACYSTUDIESOFTHEACTUALPANDEMICVACCINE FORMULATION INANIMALSARENOTREQUIRED(OWEVER EXTENSIVEPRE CLINICALTESTINGOFTHE VACCINECANDIDATEISREQUIREDFORNEWVACCINEMODALITIESANDFORMULATIONS INCLUDINGFORMULATIONOFAPPROVEDVACCINESWITHADJUVANTS )N THE 5NITED 3TATES A REGULATORY MECHANISM WAS INTRODUCED UNDER WHAT IS COMMONLY REFERRED TO AS THE @ANIMAL RULE ;= FOR MARKETING APPROVALOFVACCINESFORWHICHEFFICACYSTUDIESINHEALTHYHUMANVOLUNTEERS AREEITHERUNETHICALORNOTFEASIBLE4HISREGULATIONSTIPULATESTHAT INCASES WHERE EFFICACY OF VACCINES IN HUMANS CANNOT BE DEFINITIVELY DETERMINED MARKETINGAPPROVALFORAVACCINEMAYBEGRANTEDBASEDON@ADEQUATEAND WELL CONTROLLEDANIMALSTUDIESPROVIDINGTHATTHEBASISFORVACCINEEFFICACY IS REASONABLY WELL UNDERSTOOD AND THAT THE ANIMAL RESPONDS TO THE VAC CINE IN A MANNER THAT IS PREDICTIVE FOR HUMANS 3TUDIES IN MORE THAN ONE ANIMAL SPECIES WOULD TYPICALLY BE REQUIRED UNLESS A SINGLE ANIMAL MODEL IS AVAILABLE THAT FAITHFULLY PREDICTS EFFICACY IN HUMANS )T IS UNCLEAR AT THIS TIMEWHETHERTHISRULEWILLEVENTUALLYBEAPPLIEDTOVACCINESFORPANDEMIC INFLUENZA)NANYEVENT ITISCRITICALTHATTHEPREDICTIVEVALUEOFTHEAVAILABLE ANIMALMODELSFORIMMUNOGENICITYANDEFFICACYOFPANDEMICINFLUENZAVAC CINESBEDETERMINEDSYSTEMATICALLYUSINGTHESAMEVACCINEFORMULATIONSTHAT AREPROGRESSINGINTOCLINICALSTUDIES

#ONCLUSION !LTHOUGHSEVERALANIMALSPECIESSUPPORTTHEREPLICATIONOFHUMANAND!) VIRUSES A SURVEY OF THE LITERATURE LEADS TO THE CONCLUSION THAT THERE IS NO SINGLEIDEALANIMALMODELFORTHEEVALUATIONOFINFLUENZAVACCINES3OMEANI MALMODELSAREMORESUITABLETHANOTHERSTOPREDICTTHEATTENUATIONOFLIVE VIRUSVACCINES ORMORECLOSELYREFLECTTHEHUMANIMMUNERESPONSETOVAC CINES!NIMALMODELSCERTAINLYPLAYACRUCIALROLEINTHEEVALUATIONOFINFLU ENZAVACCINES BUTTHELIMITATIONSOFTHEMODELSMUSTBETAKENINTOACCOUNT

4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH



WHENDECISIONSAREMADEREGARDINGWHICHVACCINECANDIDATESSHOULDMOVE FORWARDINTOCLINICALTRIALS 4HE EVALUATION OF VACCINES FOR PANDEMIC INFLUENZA PRESENTS ADDITIONAL CHALLENGES IN THAT THE CORRELATES OF PROTECTION FROM !) VIRUSES ARE NOT KNOWN ANDSOTHEREMAYBEAGREATERNEEDFORRELIANCEONDATA FROMANI MALSTUDIESFORTHESEVACCINES)TISCRITICALTHATTHEBEHAVIOROF!)VIRUSES WITHPANDEMICPOTENTIALBECHARACTERIZEDINARANGEOFANIMALMODELS%VEN FROMLIMITEDOBSERVATIONSITISCLEARTHATREPLICATIONOF!)VIRUSESANDTHEIR ABILITYTOCAUSEDISEASEINANIMALSDEPENDSONTHEHOSTSPECIES ANDISSUB TYPEANDEVENSTRAINSPECIFIC4HEREFORE PRE CLINICALSAFETY IMMUNOGENICITY ANDEFFICACYDATAFROMANIMALSTUDIESMUSTBECAREFULLYCONSIDEREDINTHE EVALUATIONOFPANDEMICINFLUENZAVACCINES

!CKNOWLEDGEMENTS 7ETHANK"RIAN-URPHYFORCRITICALREVIEWOFTHISMANUSCRIPT4HISRESEARCHWAS 4 SUPPORTEDINPARTBYTHE)NTRAMURAL2ESEARCH0ROGRAMOFTHE.)!)$ .)(

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#ATHERINE*,UKEAND+ANTA3UBBARAO

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4HEROLEOFANIMALMODELSININFLUENZAVACCINERESEARCH





 







 





 







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OF-EDICINE 3TANFORD #!AND6ETERANS!FFAIRS0ALO!LTO(EALTH#ARE3YSTEM 0ALO!LTO #! 53!-ED)MMUNE6ACCINES -OUNTAIN6IEW #! 53!

!BSTRACT $EVELOPMENT OF THE LIVE ATTENUATED INFLUENZA VACCINE &LU-ISTš HAS SPANNED SEVERAL DECADES4HE VACCINE CONTAINS THREE VACCINE STRAINS TWO ATTENUATED INFLUENZA! STRAINS AND ONE ATTENUATED INFLUENZA " STRAIN THESE VACCINE STRAINS ARE GENETIC REASSORTANTS EACHHARBORINGTWOGENESEGMENTSFROMTHEWILDTYPEVIRUSCONFERRINGTHEAPPROPRIATE ANTIGENS EG!(. !(. OR " AND THE REMAINING  GENE SEGMENTS OF THE LIVE ATTENUATED INFLUENZA! OR INFLUENZA " DONOR VIRUS "OTH DONOR VIRUSES HAVE COMPLEX GENETICSIGNATURESTHATCONTROLTHEKEYBIOLOGICALTRAITSOFTHERESULTINGGENETICREASSOR TANTS INCLUDINGTEMPERATURE SENSITIVITYINVITROANDATTENUATIONINANANIMALMODEL AND THE OVERALL ATTENUATION OF THE VACCINE 3TUDIES IN HUMANS HAVE DEMONSTRATED THAT THE ATTENUATEDVACCINESTRAINSCANELICITHUMORALANTIBODIESASWELLASCELLULARIMMUNITYBOTH RESPONSESAREGENERALLYMOREREADILY DETECTABLEINCHILDRENTHANADULTS!NUMBEROFDIF FERENTCLINICALSTUDIESINCHILDRENANDADULTSHAVESHOWNTHAT THISVACCINECANREDUCETHE BURDENOFINFLUENZAILLNESSINVACCINATEDSUBJECTS INCLUDINGSEASONSINWHICHTHECIRCULAT INGWILDTYPESTRAINWASANTIGENICALLYDRIFTEDFROMTHEANTIGENSINCLUDEDINTHEVACCINE 4HESEATTRIBUTES ANDOTHERS OFTHELIVEVACCINEMAKEITAPOTENTIALLYUSEFULPLATFORMTO GENERATEANEFFECTIVEVACCINETOCOMBATAFUTUREINFLUENZAPANDEMIC

)NTRODUCTION /NE KEY PRINCIPLE IN DETERMINING WHETHER A VACCINATION STRATEGY WILL LIKELY BE AN EFFECTIVE MEANS OF CONTROLLING DISEASE IS THE OBSERVATION THAT NATURAL INFECTION BY A PATHOGEN RESULTS IN THE PROTECTION OF THE INDIVIDUAL FROMSUBSEQUENTILLNESSWITHTHESAMEORHIGHLYRELATEDPATHOGENS.ATURAL INFECTION WITH WILD TYPE INFLUENZA VIRUS ELICITS A HIGHLY PROTECTIVE AND LONG LASTING IMMUNE RESPONSE THAT PROTECTS THE INDIVIDUAL FROM SUFFERING INFLUENZA ILLNESS FOLLOWING RE EXPOSURE TO THE SAME OR SIMILAR STRAIN OF INFLUENZA (ISTORICAL ANALYSES HAVE SHOWN THAT INDIVIDUALS INFECTED WITH AN!(.STRAININTHEEARLYSWEREPROTECTEDFROMILLNESSWHENTHE SAMEVIRUSCIRCULATEDNEARLYYEARSLATER;=$ESPITETHISLONG LASTINGAND



(ARRY'REENBERGAND'EORGE+EMBLE

HIGHLYEFFECTIVEIMMUNITY ADULTSARESUSCEPTIBLETOINFLUENZA LIKEILLNESSON A REGULAR BASIS4HIS APPARENT PARADOX IS NOT DUE TO WANING OR INEFFECTIVE IMMUNERESPONSES BUTRATHERTOTHEFACTTHATTHEINFLUENZAVIRUSCONTINU ALLYEVOLVESINTHEHUMANPOPULATIONBYUNDERGOINGGENETICCHANGESINALL ITSGENESINCLUDINGTHOSEENCODINGTHEMAJORANTIGENSONTHEVIRIONSURFACE WHICHARETARGETSOFPROTECTIVEIMMUNITY4HESECHANGESINTHE TWOSURFACE PROTEINS THE HEMAGGLUTININ (! AND NEURAMINIDASE .! GLYCOPROTEINS LEADTOANTIGENICDRIFT!TSOMEPOINT THENEWLYEVOLVEDDRIFTEDINFLUENZA STRAINDIFFERSSUFFICIENTLYFROMITSPROGENITORSOTHATTHEIMMUNITYBUILTUP TOTHEPROGENITORINTHEHUMANPOPULATIONISNOLONGERCAPABLEOFEFFICIENTLY REACTINGWITHTHENEWINFLUENZASTRAIN AND THEDRIFTEDVARIANTISNOWPOISED TO CAUSE A NEW EPIDEMIC WAVE OF DISEASE )MMUNE RESPONSES TO INFLUENZA CAN BE MEASURED IN MANY DIFFERENT COMPARTMENTS INCLUDING )G' AND )G! INTHESERUM SECRETORY)G!INTHENASALSECRETIONS AND4 "AND.+CELLS INTHEPERIPHERYASWELLASVARIOUSLYMPHOIDTISSUES ESPECIALLYTHOSEINTHE RESPIRATORYTREE&UNCTIONALANTIBODIESTHATNEUTRALIZETHEVIRUSORPREVENTIT FROMBINDINGITSCOGNATERECEPTOR FREQUENTLYDESIGNATEDHEMAGGLUTINATION INHIBITING (!) ANTIBODIES CAN BE FOUND IN THE SERUM AND OCCASIONALLY INNASALSECRETIONSTHECELLULARIMMUNERESPONSESANDADDITIONALANTIBODY RESPONSESTARGETAVARIETYOFREGIONSONTHEVIRAL(! .!ANDOTHERPROTEINS ENCODED BY THE VIRUS PARTICULARLY - .0 AND .3 3EVERAL OF THE IMMUNE RESPONSES TO THE VIRUS HAVE BEEN CORRELATED WITH PROTECTION FROM DISEASE ESPECIALLYTHEQUANTITYOF(!)ORNEUTRALIZINGANTIBODIESINTHESERUMAND POTENTIALLY THETITEROFSERUMANTIBODYTO.!ASWELL$ESPITE THEPRESENCE OF THESE MULTIPLE MEASURES OF IMMUNOLOGICAL MEMORY AND EFFECTOR FUNC TIONS AND AVAILABILITY OF SUBSTANTIAL INFORMATION CORRELATING SOME OF THESE MEASURESWITHPROTECTION THEFUNDAMENTALROLEEACHHASINPREVENTINGILL NESS FOLLOWING RE EXPOSURE TO INFLUENZA REMAINS TO BE ELUCIDATED $UE TO THECOMPLEXITYOFTHEIMMUNERESPONSETOINFLUENZAINFECTIONANDTHELACK OFADETAILEDMECHANISTICUNDERSTANDINGOFTHESPECIFICCOMPONENTSOFTHE RESPONSETHATPROVIDEPROTECTION DESIGNINGANOPTIMALLYEFFECTIVEVACCINE THAT TARGETS ONLY A LIMITED SUBSET OF VIRAL PEPTIDES OR ANTIGENS HAS BEEN DIFFICULT! VACCINE STRATEGY THAT EFFECTIVELY MIMICS THE IMMUNE RESPONSE ELICITED BY NATURAL INFECTION WOULD BE EXPECTED TO PROVIDE AN EFFECTIVE CROSS REACTIVE ANDLONG LASTINGIMMUNITY

"ACKGROUND )NACTIVATEDINFLUENZAVACCINESWEREFIRSTPUTINTOUSEOVERYEARSAGOFOR THE MILITARY )N THE LATE S THE PROCESS OF COLD ADAPTATION WAS APPLIED TO INFLUENZA VIRUS FOR THE PURPOSE OF GENERATING A LIVE ATTENUATED VACCINE WITHTHEHOPETHATSUCHAVACCINEWOULDGENERATEBROADERANDHIGHERLEVELS OFIMMUNITY)N FOLLOWINGSEVERALDECADESOFCLINICALSTUDYANDDEVEL OPMENT A LIVE ATTENUATED INFLUENZA VACCINE ,!)6 CALLED &LU-ISTš WAS

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$EVELOPMENTOFCOLD ADAPTEDINFLUENZAVACCINESTRAINS ,IVEATTENUATEDVACCINESTHATAREDELIVEREDBYTHESAMEROUTEOFENTRYAS THE WILD TYPE PATHOGEN ARE EXPECTED TO INDUCE AN IMMUNE RESPONSE THAT IS SIMILAR TO THE NATURAL PATHOGEN WITHOUT ELICITING THE TYPICAL SIGNS OR SYMPTOMSASSOCIATEDWITHILLNESS4HISAPPROACHDOESNOTREQUIREAPREDE TERMINED KNOWLEDGE OF THE IDENTITY OR STRUCTURE OF THE CRUCIAL PROTECTIVE ANTIGENS NOR A DEFINED MECHANISTIC UNDERSTANDING OF THE IMMUNE EFFEC TOR FUNCTIONS THAT MEDIATE PROTECTION )N THE S *OHN -AASSAB AT THE 5NIVERSITYOF-ICHIGANSETOUTTOATTENUATEINFLUENZAVIRUSFORVACCINEUSE THROUGHAPROCESSDESIGNATEDCOLD ADAPTATION &ORCINGTHEVIRUS TOREPLICATE EFFICIENTLY AT LOWER THAN NORMAL TEMPERATURES WAS PREDICTED TO RESULT IN CHANGESTOITSGENETICMAKEUPMAKINGITLESSFITTOREPLICATEATNORMALAND ELEVATED BODY TEMPERATURES THEREBY ATTENUATING THE STRAIN 4HE !!NN !RBOR(. STRAINWASISOLATEDFROMAPATIENTANDSERIALLYPASSAGED ATREDUCEDTEMPERATURESINBOTHEGGSANDCHICKENCELLSALONGWITHBIOLOGI CALLYCLONINGTHEPROGENYATSEVERALINTERVALS;="IOLOGICALCHARACTERIZATION DEMONSTRATED THAT THE RESULTING VIRUS WAS COLD ADAPTED CA AS DEFINED BYITSABILITYTOREPLICATETOTITERSATª#THATWEREWITHIN LOGOFTITERS OBTAINEDATª# ANDTEMPERATURESENSITIVETS ASDEFINEDBYREPLICATION OFTHEVIRUSATª#THATWASDEBILITATEDBYATLEASTLOG COMPAREDTOITS REPLICATIONATª#;=4HESENEWLYACQUIREDPROPERTIESOFTHECOLD ADAPTED PROGENY DESIGNATED CA !!NN!RBOR DISTINGUISHEDITFROMITSPARENT ASWELLASMOSTWILD TYPEINFLUENZASTRAINS4HESPECTRUMOFTEMPERATURESAT WHICHTHE CA VIRUSREPLICATEDWELLWASLOWERTHANTHEWILD TYPEVIRUSESTHAT CAUSED DISEASE /F NOTE FURTHER CHARACTERIZATION OF CA!!NN!RBOR INTHEHIGHLYSUSCEPTIBLEFERRETMODELDEMONSTRATEDTHATITWAS ATTENUATED



(ARRY'REENBERGAND'EORGE+EMBLE

COMPARED TO WILD TYPE INFLUENZA VIRUSES )N CONTRAST TO THE PARENTAL WILD TYPE!!NN!RBOR STRAIN THE CA VIRUS WAS UNABLE TO REPLICATE IN THE LUNGTISSUESOFFERRETSORELICITSIGNSOFINFLUENZA LIKEILLNESS;=&OLLOWING THESUCCESSOFADAPTATIONOFINFLUENZA! -ASSAABANDHISCOLLEAGUESLATER ISOLATED AND COLD ADAPTED AN INFLUENZA " VIRUS IN A SIMILAR MANNER4HIS VIRUS DESIGNATED CA "!NN !RBOR HAD SIMILAR CA TS AND ATTENUATED ATT PROPERTIESASITSINFLUENZA!COUNTERPART;=4HISVIRUSWAS EVENMORE RESTRICTEDATHIGHERTEMPERATURESTHANCA !!NN!RBOR INTHATITWAS SIGNIFICANTLYRESTRICTEDINREPLICATIONATTEMPERATURESASLOW ASª#4HESE TWOSTRAINSPROVIDETHEGENETICBACKGROUNDOFALL&LU-ISTš VACCINESTRAINS IMPARTINGTHEIRCA TSAND ATTPROPERTIESTOTHEVACCINE T 4HE INFLUENZA VIRUS GENOME IS COMPRISED OF EIGHT DIFFERENT 2.!S OR GENESEGMENTS)NDIVIDUALMONOVALENT,!)6STRAINSAREDERIVEDBYCOMBIN INGTHEGENESEGMENTSENCODINGTHETWOSURFACEGLYCOPROTEINS (!AND.! OFACONTEMPORARYFIELDISOLATEOFINFLUENZAWITHTHEREMAININGSIXINTERNAL GENESEGMENTSOFTHEAPPROPRIATECAMASTERDONORVIRUS-$6 EITHER CA !!NN!RBORORCA"!NN!RBOR4HERESULTINGGENETICREAS SORTANTCOMBINESTHEATTENUATIONINHERENTTOTHE-$6SWITHTHE ANTIGENS NEEDEDTOELICITANEUTRALIZINGIMMUNERESPONSETHATSHOULDPREVENTDISEASE CAUSEDBYCURRENTLYCIRCULATINGSTRAINSOFINFLUENZA

"ASICPROPERTIESOFTHEVACCINE 'ENETICBASISOFBIOLOGICALPROPERTIESOFTHEVACCINE 3EQUENCEANALYSISANDCOMPARISONOFTHEGENOMESOFCA !!NN!RBOR AND CA "!NN !RBOR TO THEIR RESPECTIVE PARENTAL STRAINS CONFIRMED THATANUMBEROFCHANGESHADACCUMULATEDDURINGPASSAGE7HICH OFTHESE CHANGES WERE KEY TO CONTROLLING THE NEWLY ACQUIRED BIOLOGICAL PROPERTIES WASNOTIMMEDIATELYEVIDENTBYMERELYEXAMININGTHESEQUENCES4HEFIRST STUDIESTODETERMINETHEGENETICBASISOFTHECA TS AND ATTT PHENOTYPESWERE PERFORMEDBYCREATINGREASSORTANTVIRUSESVIACO INFECTIONOFACELLWITHTWO BIOLOGICALLYDISTINCTSTRAINS THE-$6ANDTYPICALLYAWILD TYPEFIELDISOLATE 4HE GENE SEGMENTS OF THESE TWO VIRUSES WOULD REASSORT AND THE RESULTING PROGENYCOULDBEISOLATEDFROMEACHOTHERANDINDEPENDENTLYCHARACTERIZED FORRETENTION LOSSORMODIFICATIONOFTHESPECIFICPHENOTYPEANDTHEIRGENET ICCOMPOSITION"ECAUSETHEREWASLITTLECONTROLOVERWHICHSEGMENTSWOULD REASSORT DISTINCTSTRAINSWEREUSEDTOFACILITATESELECTIONANDSCREENINGFOR THE DESIRED PROGENY4HESE STUDIES HELPED IDENTIFY THE CONTRIBUTION OF THE 0"GENESEGMENTTOTHETS PHENOTYPEOFCA !!NN!RBOR3OMETIMES HOWEVER THERESULTSWEREMISLEADINGINTHATTHELOSSOFABIOLOGICALPROP ERTY WAS NOT DUE TO A SPECIFIC MUTATION OR SET OF MUTATIONS BUT RATHER TO AN INCOMPATIBILITY OF GENE SEGMENTS FROM TWO DIVERSE PARENTAL INFLUENZA STRAINS ALSOKNOWNASTHECONSTELLATIONEFFECT/NEOFTHEBEST DOCUMENTED

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CONSTELLATION EFFECTS WAS OBSERVED WITH THE -$6 ! - GENE SEGMENT ! RECOMBINANTWILD TYPEVIRUSHARBORING THE-GENESEGMENTOF-$6 !WAS 6 ATTENUATED IN ANIMALS LEADING TO THE CONCLUSION THAT SPECIFIC MUTATIONS IN T THISGENESEGMENTWERERESPONSIBLEFORTHEATTPHENOTYPE;=(OWEVER LATER WORK SHOWED THAT A SIMILAR RECOMBINANT HARBORING THE - GENE SEGMENT FROMTHEPARENTALWILD TYPE!!NN!BORSTRAINWASALSOATTENUATED;= 4HEBIOLOGICALRESULTWASNOTDUETOTHEONENUCLEOTIDEDIFFERENCEINTHEGENESEGMENTSBETWEEN CAANDWILD TYPEPAIRRATHERTHEPHENOTYPEWASDUE TOTHEINABILITYOFTHE!NN!RBOR-GENESEGMENTTOINTERACTOPTIMALLYWITH THEOTHERGENESEGMENTSOFTHEDIVERGENTFIELDISOLATE 4HE INTRODUCTION OF REVERSE GENETICS ENABLED BIOLOGICAL TRAITS TO BE ASSOCIATED WITH SPECIFIC NUCLEOTIDES WITHOUT HAVING TO ACCOUNT FOR POTEN TIAL PROBLEMS CAUSED BY CONSTELLATION EFFECTS 5SING REVERSE GENETICS RECOMBINANT VIRUSES ARE DERIVED DIRECTLY FROM C$.! CLONES OF THE VIRAL GENE SEGMENTS ALLOWING RECOMBINANT VIRUSES WITH ONLY ONE OR A DEFINED SET OF CHANGES TO BE PRODUCED .O SELECTION SYSTEM OR EXTENSIVE SCREEN ING PROCEDURE IS REQUIRED TO OBTAIN THE DESIRED RECOMBINANT VIRUS THE GENOME OF THE RECOMBINANT VIRUS ACCURATELY REFLECTS THE GENETIC CONTENT OF THE C$.!S USED TO PRODUCE IT 4O MAP AND STUDY THE IMPACT OFF THE GENETICCHANGESBETWEENTHEWILD TYPEAND CAVIRUS PAIRSTWO DERIVATIVES OF!!NN!RBORAND"!NN!RBORWEREPRODUCED ONECONTAINED THE NUCLEOTIDES PRESENT IN THE -$6 THE OTHER ENCODED EITHER EIGHT OR NINE AMINO ACID CHANGES IN THE INTERNAL GENE SEGMENTS RESPECTIVELY REP RESENTINGTHEWILD TYPEPROGENITORS4HECHANGESRESULTINGINTHEWILD TYPE PROGENITORSEQUENCEWEREEXPECTEDTORESULTINNON TSANDNON ATTT PROPER TIES THESEPROPERTIESWERECONFIRMEDFOLLOWINGBIOLOGICALCHARACTERIZATION 2ECOMBINANTVIRUSESWERETHENDERIVEDBYMAKINGINDIVIDUALORGROUPED CHANGESINTHEWILD TYPEOR-$6VERSIONOFTHESTRAINANDEVALUATINGTHE RESULTING PHENOTYPE4HE CULMINATION OF THESE STUDIES DEMONSTRATED THAT FIVE NUCLEOTIDE POSITIONS DISTRIBUTED BETWEEN THE 0" 0" AND .0 GENE SEGMENTSOF!!NN!RBORCONTROLLEDBOTHTHETSANDATTT PROPERTIES;= 3TUDIES WITH "!NN!RBOR REVEALED THAT THREE POSITIONS TWO IN 0! ANDONEIN.0 CONTROLLEDTHE TSPHENOTYPE ANADDITIONALTWONUCLEOTIDES IN-CONTROLLEDTHEATTT PHENOTYPEANDANOTHERSUBSETOFTHREECHANGESIN 0! AND 0" WERE RESPONSIBLE FOR THE CA PHENOTYPE ; =4HE ROBUST NESS OF THESE GENETIC SIGNATURES WAS DEMONSTRATED BY PLACING ONLY THE MINIMALSETOFCHANGESINTODIVERGENTINFLUENZASTRAINSANDDEMONSTRATING THE ACCOMPANYING TRANSFER OF THE BIOLOGICAL TRAITS &OR EXAMPLE THE FIVE CHANGESRESPONSIBLEFORCONTROLLING TSANDATTOF-$6 !WEREINTRODUCED T INTO!02(. ANDTHERESULTINGRECOMBINANTVIRUSACQUIREDBOTH THE TS AND ATT T PHENOTYPES ;= 3IMILAR STUDIES WITH CA "!NN!RBOR AND "9AMANASHI WERE CONDUCTED WITH SIMILAR RESULTS ;= 4HE FUNDAMENTAL MECHANISM RESULTING IN THE EXPRESSION OF THESE PHENOTYPES IS A RESULT OF CHANGES IN MULTIPLE DIFFERENT VIRAL PROTEINS AND MOST LIKELY THEIMPACTOFTHESECHANGESISEXHIBITEDATMULTIPLEPOINTSOFTHEREPLICA



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TS PHENOTYPESWEREPRESERVEDINALLTHESHEDVIRUSESTESTED OFISO LATESWERETESTED ;=/FTHEISOLATES WERECHOSENATRANDOMANDTHEIR GENOMESSEQUENCEDINTHEIRENTIRETYANDCOMPAREDTOTHESEQUENCESOFTHE STRAINS USED TO VACCINATE THE CHILDREN4HESE ANALYSES REVEALED THAT SOME GENETIC CHANGES HAD OCCURRED IN A MAJORITY OF SHED ISOLATES AND IN SOME CASES THE MUTATIONS WERE SHARED BY MULTIPLE ISOLATES ;=4HESE CHANGES COULDHAVEARISENDURINGREPLICATIONINTHEUPPERRESPIRATORYTRACTORCOULD HAVEPREEXISTEDINTHEVACCINEMATERIALATALEVELNOTDETECTEDBYSEQUENCE ANALYSISOFTHEBULKMATERIAL4OADDRESSTHELATTERHYPOTHESIS SAMPLESOFTHE VACCINEMATERIALWEREOBTAINED AMPLIFIEDBY24 0#2ANDINDIVIDUALCLONES WERE SEQUENCED )NTERESTINGLY IN MOST CASES THE CHANGES EVIDENT IN THE ISOLATESHEDFROMTHECHILDWEREREPRESENTATIVEOFCHANGESTHATPREEXISTED IN THE BULK VACCINE MATERIAL $ESPITE THE PRESENCE OF THESE MUTATIONS ALL ISOLATES INVARIABLY RETAINED THEIR CHARACTERISTIC BIOLOGICAL PROPERTIES CON FIRMINGTHEEXQUISITEGENETICSTABILITYTHATHADBEENPREVIOUSLYDESCRIBEDIN OBSERVATIONALSTUDIES !COROLLARYCONCERNASSOCIATEDWITHGENETICSTABILITYANDVACCINESHED DINGISTHEPOTENTIALFORPERSON TO PERSONTRANSMISSIONOFTHEVIRUS3HEDDING OFTHEVACCINEFROMANINDIVIDUALISANECESSARYPREDECESSORFORTRANSMISSION TOANUNIMMUNIZEDCONTACTHOWEVER SHEDDINGISNOTNECESSARILYSUFFICIENT FORTRANSMISSIONTOOCCUR4HESTUDYOFTHEGENETICSTABILITYOFTHEVACCINEIN CHILDRENWASALSODESIGNEDTOASSESSTHEPROBABILITYOFTRANSMISSIONOFTHE VACCINEVIRUS9OUNGCHILDREN INADAYCARESETTINGWEREEXPECTEDTOINCREASE THELIKELIHOODOFDETECTINGATRANSMISSIONEVENTSHOULDITOCCURDUETOTHE RELATIVELY HIGH LEVEL OF SHED VIRUS IN RESPIRATORY SECRETIONS RELATIVELY HIGH LEVELOFSUSCEPTIBILITYTOVACCINETAKEANDTHEGENERALABSENCEOFHYGIENIC PRACTICES AMONG YOUNG CHILDREN THAT GENERALLY INHIBIT TRANSMISSION OF VIRUSESINADULTS)NADDITIONTOTHECHILDRENVACCINATEDWITH&LU-IST  CHILDREN RECEIVED PLACEBO4HESE CHILDREN WERE INTERMIXED AND PLACED IN COHORTSTHATPLAYEDTOGETHERINADAYCAREENVIRONMENTFORATLEASTHEVERY DAY FOR  OR MORE DAYS EACH WEEK .ASAL SWABS WERE OBTAINED AT REGULAR ANDFREQUENTINTERVALSFROMEACHCHILDANDTHEPRESENCEOFVACCINEVIRUSWAS ASSESSED6ACCINEVIRUSWASRECOVEREDFROMOFTHEVACCINATEDCHILDREN ANDINONLYONECONFIRMEDCASE FROMAPLACEBORECIPIENT;=4HEINFLUENZA 4 "VACCINEVIRUSRECOVEREDFROMTHEPLACEBORECIPIENTWASSHEDONONLYDAY ANDMATCHEDTHEGENETICSIGNATUREOFANINFLUENZA"VACCINEVIRUSSHEDBY AVACCINATEDMEMBEROFTHESAMEPLAYGROUPSEVERALDAYSEARLIER 4HETRANS MITTEDVACCINEISOLATEWASSHOWNTORETRAINITSCHARACTERISTIC CAANDTS PROP ERTIESANDEXHIBITEDTHEATTENUATIONPHENOTYPEINFERRETSADDITIONALLY THE PLACEBOCHILDHADNOSIGNSORSYMPTOMSDISTINGUISHABLEFROMOTHERCHILDREN INTHESTUDY4HESERESULTSWEREAPPLIEDTOTHE2EED &ROSTMODELTHATINDI CATEDAPROBABILITYOFVACCINETRANSMISSIONOCCURRINGFROMASINGLE CONTACTOFAVACCINATEDYOUNGCHILDWITHANUNVACCINATEDYOUNG CHILD;= 4HELIKELIHOODOFTRANSMISSIONFROMAVACCINATEDADULTWOULDBEEXPECTED TOBESUBSTANTIALLYLOWER SINCEADULTSSHEDVIRUSLESSFREQUENTLYANDINLOWER



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(ARRY'REENBERGAND'EORGE+EMBLE

4HESAFETYOF&LU-ISTHASBEENEVALUATEDINOVER SUBJECTSIN COMPLETED STUDIES ;= )N CONTROLLED STUDIES THE MOST COMMON ADVERSE EVENTS IN CHILDREN WERE RUNNY NOSE OR NASAL CONGESTION LOW GRADE FEVER DECREASED ACTIVITY AND DECREASED APPETITE )N THE YOUNGEST CHILDREN WHO RECEIVED TWO DOSES OF VACCINE NO SIGNIFICANT DIFFERENCES WERE OBSERVED FOLLOWINGTHESECONDDOSE)NADULTS THEMOSTCOMMONADVERSEEVENTSARE RUNNYNOSENASALCONGESTION COUGHANDSORETHROAT4HEREACTOGENICITYOF THE VACCINE IS CONSISTENT WITH REPLICATION OF A LIVE ATTENUATED VIRUS IN THE NASALEPITHELIUMOFTHESUBJECT)NALARGESAFETYDATABASESTUDYPERFORMED IN.ORTHERN#ALIFORNIA+AISER(OSPITALSYSTEM A FOLDINCREASEINASTHMA EVENTS WERE NOTED WITHIN  DAYS OF VACCINATION IN THE PRE SPECIFIED AGE STRATUMOFnMONTHS;=4HEOBSERVATIONWASFURTHERINVESTIGATEDIN THELARGEEFFICACYSTUDYOF&LU-ISTAND4)6INYOUNGCHILDREN )NTHISLATTER STUDY IN THE AGE STRATUM LESS THAN  MONTHS OF AGE n MONTHS THERE WERE  OF CHILDREN IN THE &LU-IST GROUP WHO HAD MEDICALLY ATTENDED WHEEZING EVENTS WITHIN  DAYS OF VACCINATION COMPARED TO  IN THE 4)6GROUP4HEREWASNOSIGNIFICANTDIFFERENCEINRATESAFTERDAYSORIN THECHILDRENMONTHSOFAGEOROLDER;=)NASTUDYOFOLDERCHILDRENWITH ASTHMA THE REACTOGENICITY AND SAFETY OF &LU-IST WAS SIMILAR TO CHILDREN RECEIVING4)6 ANDINPLACEBO CONTROLLEDSTUDIESINCHILDREN YEARSOFAGE OLDERWITHMODERATETOSEVEREASTHMA THEVACCINEWASSAFEANDGENERALLY WELL TOLERATED ; = 4HESE OBSERVATIONS OF INCREASED WHEEZING IN THE YOUNGEST AGE GROUPS REQUIRE FURTHER INVESTIGATION TO UNDERSTAND THE RELA TIONSHIPBETWEENTHESEEVENTSANDVACCINATIONWITH,!)6

,!)6PROTECTSAGAINSTANTIGENICALLYDIFFERENTSTRAINS )NFLUENZAVIRUSCONTINUALLYEVOLVESCHANGESINTHE(!MOLECULEGIVERISE TO VARIANTS THAT ARE CAPABLE OF ESCAPING FROM THE PREEXISTING IMMUNITY IN THE POPULATION 0REDICTING WHICH OF THESE VARIANT DRIFTED STRAINS WILL GIVE RISETOTHENEXTEPIDEMICWAVEOFSEASONALINFLUENZAISANANNUALCHALLENGE ADDRESSEDBYTHEGLOBALPUBLICHEALTHAUTHORITIES)NGENERAL MATCHINGTHE VACCINEANTIGENTOUPCOMINGSEASONSINFLUENZASTRAINSHOULDRESULTINTHE BEST OPPORTUNITY TO PRODUCE EFFECTIVE INFLUENZA VACCINES HOWEVER OCCA SIONALLYSTRAINSCHOSENFORINCLUSIONINTHEVACCINEDONOTMATCHWELLWITH THEEPIDEMICVIRUS)MMUNITYELICITEDBY&LU-ISTMAYPROVIDEFORALARGER MARGINOFERRORFORANTIGENICMATCHINGTHANOCCURSAFTERINACTIVATEDVACCINE ADMINISTRATION&LU-ISTHASBEENSHOWNTOPROVIDEPROTECTIONAGAINSTSIG NIFICANTLYANTIGENICALLYDRIFTEDVARIANTSINSEVERALCLINICALSETTINGS)Nn  CHILDRENWEREIMMUNIZEDWITHATRIVALENTBLENDOF&LU-ISTCONTAINING THE!7UHAN(. STRAIN4HEVIRUSTHATCIRCULATEDINTHECOMMU NITYTHATYEARWASDESIGNATED!3YDNEY(. ANDWASANTIGENICALLY QUITEDISTINCTFROMTHE(.ANTIGENCONTAINEDINTHEVACCINE $ESPITETHIS

,IVEATTENUATEDINFLUENZAVACCINE



LEVELOFMISMATCH THEVACCINECONFERREDEFFICACYGREATERTHAN AGAINST THE!3YDNEY (. VIRUS ;=4HAT SAME SEASON &LU-IST WAS ALSO SHOWNTOBEEFFECTIVEINADULTSBYMONITORINGFEBRILEUPPERRESPIRATORYTRACT INFECTIONSANDOTHERASSOCIATEDMEDICALUTILIZATIONS;=)NTHEHEADTOHEAD STUDYOF&LU-ISTAND4)6INCHILDREN &LU-ISTREDUCEDMODIFIED #$# ),) CAUSEDBYANANTIGENICALLYDRIFTED!(.STRAINBYCOMPAREDTO4)6 ;= 4HE CROSS PROTECTIVE NATURE OF &LU-IST IMMUNITY IS REFLECTED IN THE SERUMANTIBODYRESPONSETOTHEVACCINE; =9OUNGCHILDREN VACCINATED WITH&LU-ISTDEVELOPAROBUSTIMMUNERESPONSETOVACCINATIONTHATCANBE MEASUREDBYARISEINSERUM(!)ANDNEUTRALIZINGANTIBODYTITERS#HILDREN IMMUNIZEDWITH,!)6CONTAININGTHE!0ANAMASTRAINDEVELOPED HIGHLEVELSOF(!)ANDNEUTRALIZINGANTIBODYFOLLOWINGONEDOSEINCONTRAST ONLYAMINORITYOFCHILDRENRECEIVINGONEDOSEOF4)6WITHTHE SAMEANTIGEN RESPONDEDTOTHEVACCINE.OTABLY ANTIBODIESFROMCHILDRENVACCINATEDWITH ,!)6HADSIGNIFICANTREACTIVITYTOTHEDRIFTVARIANTTHATCIRCULATEDTHROUGH THE COMMUNITY THAT YEAR THE!&UJIAN LIKE (. WHEREAS THE CHILDREN RECEIVING4)6HADLITTLETONOREACTIVITYTOTHISSTRAIN;=

&IELDSTUDIESOF,!)6 )NFLUENZA VACCINATION HAS BEEN SHOWN TO HAVE INDIRECT BENEFITS TO OTHERS INTHECOMMUNITYWHOARENOTVACCINATED)N*APAN THEIMPLEMENTATIONOF MANDATORY VACCINATION OF SCHOOL AGED CHILDREN WITH INACTIVATED INFLUENZA VACCINE RESULTED IN A SIGNIFICANT DROP IN RATE OF PNEUMONIA AND INFLUENZA 0) MORTALITYINTHEELDERLY4HERATEOF0)MORTALITYREMAINEDLOWFOR THE DURATION OF MANDATORY VACCINATION PROGRAM AND RETURNED TO HIGHER BASELINELEVELSWITHINYEARSAFTERTHEPROGRAMWASABOLISHED DEMONSTRAT INGTHEPOWERFULIMPACTOFREDUCINGTHEBURDENOFILLNESSINYOUNGCHILDREN ONTHECOMMUNITYATLARGE; =4WOLARGEFIELDSTUDIESOFTHEVACCINEHAVE BEEN REPORTED IN WHICH THE IMPACT OF VACCINATION ON BOTH THE VACCINATED POPULATIONANDTHENONVACCINATEDPOPULATIONWERESTUDIED)NALARGEOPEN LABELSTUDYINTHE4EMPLE "OLTONAREAOF4EXAS ,!)6WASADMINISTEREDTO SEVERALTHOUSANDCHILDRENANDTHERATESOFMEDICALLYATTENDEDACUTERESPIRA TORYILLNESSWEREMEASUREDANDCOMPAREDTOASIMILARCONTROLCOMMUNITY ,!)6VACCINATIONSIGNIFICANTLYREDUCED ILLNESSINTHEVACCINATEDINDIVIDUALS IN THE INTERVENTION COMMUNITY EVEN IN YEARS IN WHICH A DRIFT STRAIN CIRCU LATEDTHROUGHTHECOMMUNITY; =2ECENTLY ASCHOOL BASED PROGRAMWAS CONDUCTED IN WHICH  OF THE CHILDREN IN THE INTERVENTION SCHOOLS WERE VACCINATED4HERATESOFABSENTEEISMWERELOWERINTHEVACCINATEDSUBJECTS COMPAREDTOTHEIRUNVACCINATEDSCHOOLMATESORCHILDRENINCONTROLSCHOOLS THATWERENOTACTIVELYRECRUITEDFORVACCINATION;=.OTABLY OTHERINDICA TORSOFVACCINEEFFECTIVENESSWERENOTEDINCLUDINGAREDUCTION INILLNESSIN OLDERSIBLINGSINTHESAMEHOUSEHOLDASTHEVACCINATEDSUBJECTS



(ARRY'REENBERGAND'EORGE+EMBLE

,!)6ANDPANDEMICPREPAREDNESS !PPLICATIONOFANEFFECTIVEANDWIDELYAVAILABLEVACCINEISTHEONLYLIKELY SOLUTION TO PREVENT SIGNIFICANT MORBIDITY AND MORTALITY IN THE NEXT PAN DEMIC ,!)6 HAS MANY ATTRIBUTES THAT MAKE IT A GOOD CANDIDATE FOR AN EFFECTIVEPANDEMICVACCINE3EVERAL,!)6VACCINECANDIDATESHAVEBEEN CONSTRUCTED THAT EXPRESS (. (! AND .! 4HE (! OF HIGHLY PATHO GENICWILD TYPE(.STRAINS ASWELLASHIGHLYPATHOGENICSTRAINSOFOTHER SUBTYPES HAVEASTRETCHOFBASICAMINOACIDSBETWEENTHE(!AND(! DOMAINSTHATCONTRIBUTESTOTHEVIRULENCEOFTHESTRAIN4HE,!)6(. VACCINE CANDIDATES WERE CONSTRUCTED BY FIRST DELETING THIS MULTIPLE BASIC AMINOACIDMOTIFPRIORTORESCUINGTHEVACCINESTRAIN4HERESULTINGVACCINE CANDIDATESHAVEBEENSHOWNTOEXHIBITTHECHARACTERISTICCA ANDTS INVITRO PHENOTYPES OF ALL ,!)6 STRAINS )N ADDITION THESE CANDIDATES ARE HIGHLY ATTENUATED IN CHICKENS MICE AND FERRETS YET PRODUCE IMMUNE RESPONSES THATPROTECTMICEANDFERRETSFROMCHALLENGEWITHANTIGENICALLY SIMILARAS WELL AS ANTIGENICALLY DRIFTED VARIANTS ;= 3EVERAL ASPECTS OFF A PANDEMIC ARELIKELYTOBEDIFFERENTTHANATYPICALSEASONALEPIDEMICANDTHESEUNIQUE FEATURES WILL ALTER THE NORMAL COURSE OF ACTIONS TAKEN BY PUBLIC HEALTH AUTHORITIESASWELLASVACCINEMANUFACTURERS&IRST THEPANDEMICISLIKELY TOSPREADQUICKLYANDONAGLOBALSCALE!NEFFECTIVEVACCINEWILLNEEDTO BE ADMINISTERED TO A LARGE PORTION OF THE WORLDS POPULATION #URRENTLY THEANNUALWORLDWIDEDISTRIBUTIONOFINFLUENZAVACCINESISONLY ADEQUATE FORMILLIONDOSES FARSHORTOFTHEBILLIONPEOPLEWHOWILLNEEDTHE VACCINE)NADDITION THENATUREOFTHEPANDEMICANTIGENWILLBEATYPICAL ITWILLBECOMPRISEDOFAN(!THATHASNOTCIRCULATEDPREVIOUSLYANDVAC CINE SEED STRAINS WILL NEED TO BE QUICKLY ASSEMBLED ,!)6 HAS CLINICALLY DEMONSTRATED ITS CAPACITY FOR PREVENTING DISEASE CAUSED BY ANTIGENICALLY DRIFTEDSTRAINS4HISATTRIBUTEOF,!)6MAYALLOWPREBANKEDOR STOCKPILED VACCINES THAT ARE NOT ANTIGENICALLY IDENTICAL TO THE CIRCULATING VACCINE STRAINTOBEMADEWELLINADVANCE OFTHEPANDEMICANDBEEFFECTIVEDURING THEEARLYSTAGESOFAPANDEMIC!SECONDESSENTIALFEATUREOFANEFFECTIVE PANDEMICSTRATEGYISRAPIDANDLARGE SCALEPRODUCTIONCAPACITIES4HEDOSE OFLOG INFECTIOUSPARTICLESOF,!)6ISASMALLANTIGENICMASS/NEDOSE OF,!)6REPRESENTSLESSTHANAPPROXIMATELYOFANINACTIVATED  +G DOSE OF ANTIGEN 4HIS EFFICIENCY TRANSLATES INTO THE POTENTIAL TO RAPIDLY PRODUCELARGEQUANTITIESOFBULK,!)6COMPAREDTOINACTIVATEDVACCINE 4HECAPACITYTOPRODUCELARGEAMOUNTSOF,!)6RAPIDLYCOMBINED WITHITS CROSS PROTECTIVENATURE EASEOFADMINISTRATIONANDHIGHDEGREE OFEFFICACY INIMMUNOLOGICALLYNAIVEPOPULATIONSMAKETHISAPROMISINGCANDIDATEFOR PANDEMICPREPAREDNESS

,IVEATTENUATEDINFLUENZAVACCINE



#ONCLUSION 4HEUTILIZATIONOFTHISNOVELVACCINETECHNOLOGYCONTINUESTOBEREFINEDAND IMPROVED2ECENTSTUDIESINCHILDRENSHOULDENABLEGREATERUSEOFTHISVAC CINEINTHISHIGHLYSUSCEPTIBLEPOPULATION4HECURRENTMANUFACTURINGMETH ODSUSEDTOMAKE,!)6AREBASEDONPRODUCTIONTECHNOLOGIESTHAT AREOVER YEARSOLD MOREMODERNPRODUCTIONMETHODSINCLUDINGMANUFACTURINGIN CELLCULTURESUBSTRATES AREBEINGDEVELOPED)NADDITION THE GENERATIONOF THEREASSORTANTVIRUSESUSEDTOINITIATESEEDSTRAINISBEINGREFINEDAND INTEGRATEDWITHTHEUSEOFREVERSEGENETICSTECHNOLOGY&INALLY THEATTRIBUTES Y THATMAKETHISVACCINEEFFECTIVEINYOUNGCHILDRENISBEINGFURTHEREXPLORED ANDDEVELOPEDTOAPPLYTOPANDEMICSOLUTIONS

2EFERENCES 

7RIGHT 0% .EUMANN ' +AWAOKA9  /RTHOMYXOVIRUS )N $ +NIPE 0 (OWLEYEDS  &IELDS6IROLOGY THEDN,IPPINCOTT 0HILADELPHIA  -URPHY "2 #OELINGH +  0RINCIPLES UNDERLYING THE DEVELOPMENT AND USE OF LIVE ATTENUATED COLD ADAPTED INFLUENZA ! AND " VIRUS VACCINES 6IRAL )MMUNOLn  -AASSAB(& !DAPTATIONANDGROWTHCHARACTERISTICSOFINFLUENZAVIRUSAT ª#.ATUREn  -AASSAB(& 0LAQUEFORMATIONOFINFLUENZAVIRUSATª#.ATURE n  -AASSAB(& &RANCIS4*R $AVENPORT&- (ENNESSY!6 -INUSE% !NDERSON ' ,ABORATORYANDCLINICALCHARACTERISTICSOFATTENUATED STRAINSOFINFLU ENZAVIRUS "ULL7ORLD(EALTH/RGANn  -AASSAB(& $EVELOPMENTSOFVARIANTSOFINFLUENZAVIRUS)N2$"ARRY "7*-AHYEDS 4HE"IOLOGYOF,ARGE2.!6IRUSES!CADEMIC0RESS ,ONDON n  3NYDER-( "ETTS2& $E"ORDE$ 4IERNEY%, #LEMENTS-, (ERRINGTON$ 3EARS3$ $OLIN2 -AASSAB(& -URPHY"2 &OURVIRALGENESINDEPEN DENTLY CONTRIBUTE TO ATTENUATION OF LIVE INFLUENZA!!NN!RBOR (. COLD ADAPTEDREASSORTANTVIRUSVACCINES*6IROLL n  3UBBARAO %+ 0ERKINS - 4REANOR ** -URPHY "2  4HE ATTENUATION PHENOTYPECONFERREDBYTHE-GENEOFTHEINFLUENZA!!NN!RBORCOLD ADAPTEDVIRUS(. ONTHE!+OREA(. REASSORTANTVIRUSRESULTSFROM AGENECONSTELLATIONEFFECT6IRUS2ESn  *IN( ,U" :HOU( -A# :HAO* 9ANG#& +EMBLE' 'REENBERG( -ULTIPLEAMINOACIDRESIDUESCONFERTEMPERATURESENSITIVITYTOHUMANINFLUENZA VIRUSVACCINESTRAINS&LU-IST DERIVEDFROMCOLD ADAPTED!!NN !RBOR 6IROLOGY n  #HEN: !SPELUND! +EMBLE' *IN(E 'ENETICMAPPING OFTHECOLD ADAPTED PHENOTYPE OF "!NN !RBOR THE MASTER DONOR VIRUS FOR LIVE ATTENUATEDINFLUENZAVACCINES&LU-IST 6IROLOGY n



(ARRY'REENBERGAND'EORGE+EMBLE

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(ARRY'REENBERGAND'EORGE+EMBLE

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WHICH IS USED TO PREPARE -& -& ALSO CONTAINS TWO NON IONIC SUR FACTANTS4WEENAND3PAN WHICHAREDESIGNEDTOOPTIMALLYSTABILIZE THEEMULSIONDROPLETS&IGURESHOWSTHECOMPOSITIONOF-&ADJUVANT #ITRATE BUFFER IS ALSO USED IN -& TO STABILIZE P(!LTHOUGH SINGLE VIAL FORMULATIONS CAN BE DEVELOPED WITH VACCINE ANTIGENS DISPERSED DIRECTLY IN-& -&CANALSOBEADDEDTOANTIGENSIMMEDIATELYPRIORTOTHEIR ADMINISTRATION !LTHOUGH A LESS FAVORABLE OPTION COMBINATION PRIOR TO ADMINISTRATION MAY BE NECESSARY TO ENSURE OPTIMAL ANTIGEN STABILITY FOR SOMEANTIGENS BUTNOTFORFLU

-ANUFACTURINGOF-& $ETAILS OF THE MANUFACTURING PROCESS FOR -& AT THE  LITER SCALE HAVE PREVIOUSLYBEENDESCRIBED;=4HEPROCESSINVOLVESDISPERSING3PANIN THESQUALENEPHASEAND4WEENINTHEAQUEOUSPHASE BEFOREHIGHSPEED MIXING TO FORM A COARSE EMULSION 4HE COARSE EMULSION IS THEN PASSED REPEATEDLY THROUGH A MICROFLUIDIZER TO PRODUCE AN EMULSION OF UNIFORM

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&IGURE3ERUMHEMAGGLUTINATION(! TITERSAGAINSTTHETHREESTAINSOFINFLUENZAVIRUSINCLUD EDINVACCINES(. (.AND" FORFLUCELLCULTUREVACCINE INCOMBINATIONWITHVARIOUS ADJUVANTS4HEADJUVANTSEVALUATEDINCLUDED-& ALUMINUMSALT !LUM CALCIUMPHOSPHATE #!0 POLYLACTIDECO GLYCOLIDEMICROPARTICLES0,' #P'OLIGONUCLEOTIDE#P' ANDVAC CINEALONEWITHOUTADJUVANTNIL -&WASTHEMOSTPOTENTADJUVANTFORALLTHREESTRAINS

SMALLDROPLETSIZENM WHICHCANBESTERILEFILTEREDANDFILLEDINTOVIALS -ETHODS HAVE ALSO BEEN PUBLISHED PREVIOUSLY TO ALLOW THE PREPARATION OF -&ONASMALLSCALE FORUSEINRESEARCHSTUDIES;=-&ISEXTENSIVELY CHARACTERIZEDBYVARIOUSPHYSICOCHEMICALCRITERIAAFTERPREPARATION

0RE CLINICALEXPERIENCEWITH-& 0RE CLINICAL EXPERIENCE WITH -& IS EXTENSIVE AND HAS BEEN REVIEWED ON SEVERAL OCCASIONS PREVIOUSLY ;  = -& HAS BEEN SHOWN TO BE A POTENTADJUVANTINADIVERSERANGEOFSPECIES INCOMBINATIONWITHABROAD RANGEOFVACCINEANTIGENS TOINCLUDERECOMBINANTPROTEINANTIGENS ISOLATED VIRAL MEMBRANE ANTIGENS BACTERIAL TOXOIDS PROTEIN POLYSACCHARIDE CONJU GATES PEPTIDES AND VIRUS LIKE PARTICLES -& IS PARTICULARLY EFFECTIVE FOR INDUCINGHIGHLEVELSOFANTIBODIES INCLUDINGFUNCTIONALTITERSNEUTRALIZING BACTERICIDALANDOPSONOPHAGOCYTICTITERS ANDISGENERALLYMORE POTENTTHAN !LUM )NARECENTSTUDY WEDIRECTLYCOMPARED-&AND!LUMFORSEVERALDIF FERENTVACCINESANDCONFIRMEDTHAT-&WASMOREPOTENT ALTHOUGH!LUM PERFORMEDWELLFORBACTERIALTOXOIDANTIGENS PARTICULARLYDIPHTHERIATOXOID ;= -& HAS ALSO SHOWN ENHANCED POTENCY OVER ALUM WHEN DIRECTLY COMPARED IN NON HUMAN PRIMATES WITH PROTEIN POLYSACCHARIDE CONJUGATE VACCINES;=ANDWITHARECOMBINANTVIRALANTIGEN;= )NPRE CLINICALSTUDIES -&ISTHEMOSTPOTENTADJUVANTFORFLUVACCINES F IN COMPARISON TO VARIOUS ALTERNATIVES &IG   )N A RECENT STUDY WE COM PAREDANUMBEROFADJUVANTSFORFLUVACCINEINMICE ANDSHOWED THAT-& SIGNIFICANTLY OUTPERFORMS ALTERNATIVES INCLUDING !LUM FOR BOTH ANTIBODY AND4 CELL RESPONSES ;= -OREOVER WE HAVE RECENTLY SHOWN THAT -& OFFERS ENHANCED PROTECTION AGAINST CHALLENGE WITH PANDEMIC FLU STRAINS IN MICE5NPUBLISHEDOBSERVATIONS +ANTA 3UBBARAO WHICHISCONSISTENTWITH



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OUREARLIERWORKONINTERPANDEMICSTRAINS;=)NADDITIONTO IMMUNOGENIC ITYSTUDIES EXTENSIVEPRE CLINICALTOXICOLOGYSTUDIESHAVEBEENUNDERTAKEN WITH-& INCOMBINATIONWITHARANGEOFDIFFERENTANTIGENSIN ANUMBEROF SPECIES)NTHESESTUDIES ITHASBEENSHOWNTHAT-&ISNEITHERMUTAGENIC NORTERATOGENIC ANDDIDNOTINDUCESENSITIZATIONINANESTABLISHEDGUINEAPIG MODELTOASSESSCONTACTHYPERSENSITIVITY4HEFAVORABLETOXICOLOGICALPROFILE ESTABLISHED FOR -& ALLOWED EXTENSIVE CLINICAL TESTING FOR -& WITH A NUMBER OF DIFFERENT VACCINE CANDIDATES AND THE APPROVAL OF A FLU F VACCINE CONTAINING-&IN%UROPEIN

#LINICALEXPERIENCEWITH-&ADJUVANT &LUADš INELDERLYSUBJECTS &LUADš WHICHWASINITIALLYLICENSEDASANINTERPANDEMICINFLUENZAVACCINEIN )TALYINANDISNOWLICENSEDINMORETHANCOUNTRIES CONTAINS-& WHICHWASTHEFIRSTNOVELADJUVANTACCEPTEDFORHUMANUSEAFTER THEREGIS TRATIONOFINSOLUBLEALUMSALTSINTHEFIRSTPARTOFTHETHCENTURY4HEREG ISTRATIONOF&LUADšWASBASEDONTHERESULTSOFALARGECLINICALDEVELOPMENT PLAN WHICHSHOWED INMORETHE SUBJECTS THATTHE-& ADJUVANTED VACCINEWASWELLTOLERATEDANDMOREIMMUNOGENICTHANCONVENTIONALNON ADJUVANTEDINFLUENZAVACCINES4HEADJUVANTISGENERALLYWELLTOLERATED WITH ONLY A LOW INCIDENCE OF LOCAL MILD REACTIONS FOLLOWING IMMUNIZATION AND WITH NO INCREASES IN INCIDENCE FOLLOWING SUBSEQUENT IMMUNIZATIONS ;= 4HEADJUVANTEDINFLUENZAVACCINEWASINITIALLYDEVELOPEDFORVACCINATIONOF THE ELDERLY TO FILL THE MEDICAL NEED FOR AN IMPROVED INFLUENZA VACCINE FOR THISAGEGROUP INWHICHCONVENTIONALINFLUENZAVACCINESARECOSTEFFECTIVE BUTDONOTPROVIDEOPTIMALPROTECTION;=&ORTHISREASON MOSTOFTHEEARLY CLINICALTRIALSWITH&LUADšWEREPERFORMEDINELDERLYSUBJECTS)NTHISPOPULA TION THEADJUVANTEDVACCINECONSISTENTLYINDUCEDENHANCEDGEOMETRICMEAN TITERS SEROCONVERSIONRATESANDSEROPROTECTIONRATES4HEINCREASEDIMMUNO GENICITYOF&LUADšWASPARTICULARLYIMPORTANTINSUBSETSOFTHEELDERLYPOPU LATION WHOHAVEAHIGHERRISKOFDEVELOPINGINFLUENZAANDITSMOSTSEVERE COMPLICATIONSTHISINCLUDESSUBJECTSWITHALOWPRE IMMUNIZATIONTITERAND SUBJECTS AFFECTED BY CHRONIC CARDIOVASCULAR AND RESPIRATORY DISEASES ; =!NINCREASEDRESPONSEAGAINSTHETEROVARIANTINFLUENZASTRAINS DIFFERENT FROMTHESTRAINSINCLUDEDINTHEVACCINE WASALSOCONSISTENTLYOBSERVED4HIS WILLBEPARTICULARLYBENEFICIALWHENEVERTHEVACCINEANTIGENSDONOTMATCH PERFECTLYTHOSEOFTHECIRCULATINGVIRUSES; n=-ORERECENTDATAHAVE INDICATED THAT YOUNGER ADULTS MAY ALSO BENEFIT FROM IMMUNIZATION WITH &LUADš PARTICULARLYPOPULATIONSATHIGHRISKOFINFLUENZA RELATEDCOMPLICA TIONS SUCH AS ()6 POSITIVE SUBJECTS &IG  AND THOSE AFFECTED BY CHRONIC DISEASES&IG ; =)MPORTANTLY THEADDITIONOF-&TOTHEINFLUENZA ANTIGENSDIDNOTAFFECTTHESAFETYPROFILEOFTHEVACCINE WHICHWASVERYWELL

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&IGURE  #OMPARISON OF HEMAGGLUTINATION INHIBITION ANTIBODY TITERS IN ()6  SEROPOSITIVE INDIVIDUALSIMMUNIZEDWITH-&ADJUVANTEDVACCINE&LUADš ORWITHANON ADJUVANTEDCOM PARATOR!GAINSTALLTHREESTAINSINTHEVACCINE -&INDUCED HIGHERGEOMETRICMEANTITERS A HIGHERPROPORTIONOFINDIVIDUALSSEROCONVERTEDANDAHIGHERPROPORTIONOFSUBJECTSACHIEVEDA HEMAGGLUTINATIONTITEROF

&IGURE&LUADš ISMOREIMMUNOGENICINADULTSnYEARSWITHCHRONICDISEASESTHANNON ADJUVANTEDVACCINES'EOMETRICMEANRATIOOFHEMAGGLUTINATIONINHIBITIONRESPONSEINADULTS n YEARS OF AGE WITH CHRONIC DISEASES WHO WERE IMMUNIZED WITH AN -& ADJUVANTED SUBUNIT INFLUENZA VACCINE VERSUS A SIMILAR GROUP IMMUNIZED WITH A VACCINE WITHOUT -& 'EOMETRIC MEAN RATIOS WERE HIGHER FOR THE -& ADJUVANTED GROUP &LUADš FOR ALL THREE VACCINESTRAINS

TOLERATEDINALLCLINICALTRIALSPERFORMEDINELDERLYANDNON ELDERLYSUBJECTS ;= -OST ENCOURAGINGLY CLINICAL EFFECTIVENESS DATA HAVE NOW BEGUN TO ACCUMULATE TO INDICATE THAT THE INCREASED IMMUNOGENICITY OF -& ALSO TRANSLATESINTOIMPROVEDPROTECTION0UIG "ARBERAETAL;=DESCRIBEDTHE IMPROVED EFFECTIVENESS OF -& IN PREVENTING EMERGENCY ADMISSIONS FOR PNEUMONIA WHILESUPERIORCLINICALPROTECTIONAGAINSTINFLUENZA LIKEILLNESS HAS BEEN REPORTED ;=4HE DATA SHOWING IMPROVED CLINICAL EFFECTIVENESS



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OF -& ADJUVANTED INFLUENZA VACCINE WERE RECENTLY REVIEWED ;= AND SHOWED SIGNIFICANTLY REDUCED INFLUENZA LIKE DISEASE AND REDUCED HOSPITAL IZATION RATES FOR PNEUMONIA CARDIOVASCULAR AND CEREBROVASCULAR DISEASE INTHEELDERLY)MPORTANTLY -&CANCOMPENSATEFORTHEREDUCEDEFFICACY OF VACCINES WHICH OCCASIONALLY HAPPENS DUE TO A MISMATCH BETWEEN THE ANTIGENICCOMPOSITIONANDTHECIRCULATINGINFLUENZASTRAINS;=)THASBEEN SHOWNONSEVERALOCCASIONSBOTHINTHEELDERLYANDINNON ELDERLYSUBJECTS THAT-&INDUCESASIGNIFICANTLYENHANCEDRESPONSEAGAINSTHETEROVARIANT VIRUSSTRAINSNOTINCLUDEDINTHEVACCINE; n = 7ITHMORETHANMILLIONDOSESDISTRIBUTED THESAFETYOF&LUADšISSUP PORTEDBYEXTENSIVEPHARMACOVIGILANCEDATASHOWINGTHATVACCINATIONWITH &LUADš ISASSOCIATEDWITHAVERYLOWFREQUENCYOFADVERSEREACTIONS; = )NFACT THESAFETYPROFILEOF&LUADšISBROADLYSIMILARTOTHEPROFILEFORNON ADJUVANTEDFLUVACCINES;=-OREOVER ITHASBEENSHOWNTHATTHERATEOF 'UILLAIN "ARRE SYNDROME AFTER &LUADš IMMUNIZATION IS WITHIN THE NORMAL RANGEOFRATESFOUNDINTHE53AFTERIMMUNIZATIONWITHCONVENTIONALNON ADJUVANTEDINFLUENZAVACCINES;=)NADDITION ARECENTSTUDY HIGHLIGHTED THAT IMMUNIZATION WITH -& ADJUVANT NEITHER RAISES THE LEVELS OF PRE EXISTING ANTIBODIES NOR INDUCES NEW ANTIBODY RESPONSES AGAINST SQUALENE ALTHOUGH ANTIBODIES TO SQUALENE ARE ALREADY NATURALLY OCCURRING IN MANY SUBJECTS;=

&LUADš INYOUNGCHILDREN )NARECENTCLINICALTRIAL WEEVALUATEDTHEPOTENCYOFAN-& ADJUVANTED SUBUNIT INFLUENZA VACCINE IN YOUNG CHILDREN n MONTHS OF AGE AND DIRECTLY COMPARED IT WITH A LICENSED SPLIT INFLUENZA VACCINE PRODUCT 4HE -& ADJUVANTED VACCINE &LUADš WAS SIGNIFICANTLY MORE IMMUNOGENIC THANTHECOMPARATORFORALLTHREESTRAINSINCLUDEDINTHEVACCINE-OREOVER THE-& ADJUVANTEDVACCINEMETALLTHREE%-%!#(-0CRITERIATO GAIN VACCINE APPROVAL FOR HEALTHY ADULTS SEROCONVERSION SEROPROTECTION AND MEAN GEOMETRIC INCREASE IN RESPONSE WHILE THE COMPARATOR VACCINE DID NOTUNPUBLISHEDDATA )NADDITION THE&LUADšVACCINEOFFEREDSIGNIFICANTLY ENHANCEDPROTECTIONAGAINSTHETEROVARIANTSTRAINSNOTINCLUDEDINTHEVAC CINE INTHISVULNERABLEPOPULATION0ELLEGRINIETAL -&ADJUVANTENHANCES D CROSSPROTECTIVEANTIBODYRESPONSESIN YOUNGCHILDRENAGAINSTHETEROVARIANT INFLUENZASTRAINSINPREPARATION )MPORTANTLY &LUADš WASWELLTOLERATEDIN THIS YOUNG POPULATION AND HAD A SIMILAR REACTOGENICITY PROFILE TO THE SPLIT VACCINEPRODUCT46ESIKARI PERSONALCOMMUNICATION  .EVERTHELESS DESPITETHESEENCOURAGINGDATA THESAFETYOF-&ADJU VANTNEEDSTOBECAREFULLYEVALUATEDINTHISYOUNGPOPULATION PRIORTOCON SIDERATIONFORPRODUCTAPPROVAL(OWEVER THEDATAONTHESAFETYOF-& INTODDLERS INFANTSANDNEWBORNS AREVERYENCOURAGINGANDSUGGESTTHATTHE ADJUVANTWILLHAVEANACCEPTABLESAFETYPROFILEINYOUNGCHILDREN; =

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&IGURE  3ERUM HEMAGGLUTINATION INHIBITION AND MICRONEUTRALIZATION GEOMETRIC MEAN TITERS AGAINST(.INFLUENZAATVARYINGVACCINEDOSESnMG WITHANDWITHOUT-&ADJUVANT AFTER ONE AND TWO VACCINE DOSES POST  AND POST  IN HEALTHY ADULTS n YEARS OF AGE  !LL DOSE LEVELS OF VACCINE WERE SIGNIFICANTLY MORE POTENT WITH -& THAN WITHOUT PLAIN  -OREOVER ONEDOSEOFVACCINEWITH-&ADJUVANTWASCOMPARABLETOTWODOSESOFTHEPLAIN VACCINE

0ANDEMICFLUVACCINESCONTAINING-&&OCETRIAšAND!FLUNOVš 5NPRECEDENTED EFFORTS ARE BEING UNDERTAKEN WORLDWIDE TO DEVELOP PLANS TOMITIGATETHEDIREPUBLICHEALTHCONSEQUENCESOFANINFLUENZAPANDEMIC 4HE DEVELOPMENT OF EFFECTIVE VACCINES IS OF PARAMOUNT IMPORTANCE FOR THESE PLANS 2ECENT STUDIES HAVE ESTABLISHED THAT SINCE HUMANS ARE NAIVE TO POTENTIAL PANDEMIC INFLUENZA STRAINS THEY RESPOND POORLY TO CONVEN TIONAL VACCINES AND LARGE DOSES ARE NEEDED TO INDUCE ACCEPTABLE LEVELS OF ANTIBODIES;=)NCONTRAST THERESPONSETOPOTENTIALPANDEMICFLUVACCINES ISMUCHIMPROVEDBYTHEADDITIONOF-&ADJUVANT;n=&IG  -&



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ALSO ALLOWS FOR SIGNIFICANT DOSE SPARING WHICH IS ESSENTIAL TO INCREASE THE PANDEMICVACCINEPRODUCTIONCAPACITY5NFORTUNATELY TRADITIONALADJUVANTS LIKE!LUMDONOTAPPEARTOBEPOTENTFORPANDEMICINFLUENZAVACCINES;= )NCONTRAST HEMAGGLUTININ(! ANTIGENSDERIVEDFROMSEVERALPOTENTIALLY PANDEMICSTRAINS INCLUDING(. (.AND(. HAVEBEENCOMBINED WITH-&ANDINDUCEDASIGNIFICANTENHANCEMENTOFANTIBODYTITERSCOM PAREDTONON ADJUVANTEDVACCINES;  =AND!"ANZHOFF! PERSONAL COMMUNICATION  )MPORTANTLY ADJUVANTATION WITH -& ALLOWED A SIGNIFI CANTREDUCTIONINTHEANTIGENCONCENTRATIONPERDOSE &IG 4HISFINDING HAS SIGNIFICANT IMPLICATIONS IN TERMS OF PRODUCTION CAPACITY FOR PANDEMIC VACCINES;=.OTABLY  +G-& ADJUVANTED((!AND+G-& ADJUVANTED((!WERESIGNIFICANTLYMOREIMMUNOGENICTHAN +GOFTHE RESPECTIVEPLAIN(!S; =!SALREADYSHOWNFORTHEINTERPANDEMICVAC CINE BROADER CROSS NEUTRALIZATION AGAINST HETEROVARIANT PANDEMIC STRAINS WASANADDITIONALBENEFITOFAN-& ADJUVANTEDVACCINE;=4HESEDATA 4 WEREUSEDTOGAINAPPROVALFORANEWPANDEMICINFLUENZAVACCINECONTAIN INGTHE-&ADJUVANT&OCETRIAš IN-AYINALLMEMBERSTATESOF THE%UROPEAN5NION ANDTOSUBMITFORAPPROVALASAPRE PANDEMICVACCINE !FLUNOVš &OCETRIAš WILLBEMANUFACTUREDTOCONTAINTHEINFLUENZASTRAIN DECLARED BY THE7ORLD (EALTH /RGANIZATION AS A PANDEMIC STRAIN 2ECENT DATAHAVESHOWNTHATTHEADDITIONOF-&TOAPOTENTIALPRE PANDEMICFLU VACCINE!FLUNOVš ALLOWSTHEVACCINETOMEETALLTHREE#(-0CRITERIAFOR IMMUNOGENICITY FOR INTERPANDEMIC FLU VACCINES IN ADULTS AT A  +G DOSE LEVEL-OREOVER THEVACCINEWASSHOWNTOINDUCEHIGHLEVELSOFNEUTRALIZING F ANTIBODIESINMORETHANOFSUBJECTSIMMUNIZED WHILEBEINGVERYWELL TOLERATEDINALLPOPULATIONSIMMUNIZED"ANZHOFFETAL UNPUBLISHEDDATA  -OREOVER THEADDITIONOFTHEADJUVANTINDUCEDPOTENT#$ 4CELL MEDI ATED IMMUNE RESPONSES AFTER A SINGLE IMMUNIZATION $EL 'IUDICE ET AL UNPUBLISHEDDATA 4HEUSEOFAPRE PANDEMICFLUVACCINEWOULDBELIKELYTO SIGNIFICANTLYREDUCETHECATASTROPHICPUBLICHEALTHCONSEQUENCESOFAPAN DEMIC SINCELARGESUBSETSOFTHEPOPULATIONMIGHTBEALREADYPROTECTEDOR ATLEASTPRIMEDAGAINSTTHEPANDEMICVIRUSATTHEINITIATIONOFTHEPANDEMIC F THREAT4HEDOSE SPARINGASPECTOF-&ISPARTICULARLYATTRACTIVEFORPAN DEMICFLUVACCINES GIVENTHECURRENTLIMITEDCAPACITYWORLDWIDEFORINFLUEN ZAVACCINEPRODUCTION WHICH ISNOTSUFFICIENTTODEALWITHAPANDEMIC GIVEN THEHIGH DOSEREQUIREMENTSFORUNADJUVANTEDINFLUENZAVACCINES ;= !LTHOUGHCLINICALTRIALSWITHINTERPANDEMICANDPANDEMICVACCINESREP RESENTTHEGREATMAJORITYOFTHECLINICALEXPERIENCEWITH-& SEVERALTRI ALSHAVEBEENPERFORMEDALSOWITHOTHER-& ADJUVANTEDINVESTIGATIONAL VACCINES INCLUDING ()6 (36 #-6 ("6 AND (#6 4HESE STUDIES HAVE PROVIDEDADDITIONALEVIDENCEOFTHESAFETY TOLERABILITYANDADJUVANTICITYOF D -&;n= 4HECLINICALEXPERIENCEWITHTHESEVACCINESHAS BEENPREVI OUSLYREVIEWED;=/FNOTE -&HASBEENUSEDASANADJUVANTFORPEDI ATRIC VACCINES SUCH AS THE #-6 AND ()6 VACCINES 3ERONEGATIVE TODDLERS IMMUNIZED WITH THE #-6 G" VACCINE SHOWED ANTIBODY TITERS THAT WERE

-&!SAFEANDPOTENTOILINWATEREMULSIONADJUVANTFORINFLUENZAVACCINES



HIGHERTHANTHOSEFOUNDINADULTSNATURALLYINFECTEDWITH#-6-OREOVER THE -& ADJUVANTED VACCINE WAS WELL TOLERATED IN THIS AGE GROUP ;= !DDITIONALLY AN-& ADJUVANTED()6VACCINEWASEVALUATEDINNEWBORNS BORNTO()6 POSITIVEMOTHERS;  =4HEVACCINEWASVERYWELLTOLER ATEDAND DESPITETHEPRESENCEOFMATERNALANTIBODIES INDUCEDANANTIBODY RESPONSEINOFTHEIMMUNIZEDINFANTS; =-OREOVER THE-&VAC CINEWASSIGNIFICANTLYMOREPOTENTTHANALUMFORTHEINDUCTIONOFCELL MEDI ATEDIMMUNERESPONSESPROLIFERATIVE 4CELLRESPONSES AGAINSTHOMOLOGOUS ANDHETEROLOGOUSSTRAINSOF()6;= )NSUMMARY CLINICALTESTINGOF-&ADJUVANTHASRESULTEDINTHEREGIS TRATION IN MORE THAN  COUNTRIES OF AN EFFECTIVE AND WELL TOLERATED INFLU ENZA VACCINE FOR USE IN THE ELDERLY POPULATION )N ADDITION CLINICAL TRIALS HAVEDEMONSTRATEDTHAT-&CANBESAFELYADMINISTEREDWITHARANGEOF ANTIGENS TODIVERSEAGEGROUPS INCLUDINGTHEPEDIATRICPOPULATION

#OMBINATIONOF-&WITHIMMUNOPOTENTIATORS !LTHOUGH -& IS GENERALLY A MORE POTENT ADJUVANT THAN ALUM ;= IT CANNOT BE EXPECTED TO BE A SUITABLE ADJUVANT FOR ALL VACCINES THERE IS NO hUNIVERSAL ADJUVANTv (OWEVER -& IS PARTICULARLY EFFECTIVE FOR ENHANC INGANTIBODYAND4CELLPROLIFERATIVERESPONSES; =(OWEVER ITISNOT A POTENT ADJUVANT FOR THE INDUCTION OF4H CELLULAR IMMUNE RESPONSES IN PRE CLINICAL MODELS INVOLVING NAIVE MICE ; = 0OTENT 4H RESPONSES MAYBEREQUIREDTOPROVIDEPROTECTIVEIMMUNITYAGAINSTSOMEVIRUSESAND ADDITIONALINTRACELLULARPATHOGENS4HIMMUNEPOTENTIATORS INCLUDING#P' OLIGONUCLEOTIDES;=HAVEBEENSUCCESSFULLYADDEDTO-&TOIMPROVEITS POTENCY AND TO ALTER THE KIND OF RESPONSE INDUCED ;=!LTHOUGH THE FOR MULATIONOF-&CANBEMODIFIEDTOPROMOTETHEASSOCIATIONOF#P'WITH THEOILDROPLETS;= MORERECENTSTUDIESSUGGESTTHATTHISMAYNOTBENECES SARY ANDSIMPLEADDITIONOF#P'TO-&MAYBESUFFICIENTINSOMESITUA TIONS ;=(OWEVER CAREFULCONSIDERATIONISREQUIREDINRELATIONTOWHICH IMMUNEPOTENTIATORSTOADDTO-&EMULSIONANDHOWBESTTOFORMULATE THEM!LTHOUGHPRE CLINICALSTUDIESSHOWEDTHATTHEPOTENCYOF-&WAS ENHANCEDBYTHEINCLUSIONOF-40 0%;= OUREARLYEXPERIENCE INTHECLINIC SHOWEDTHAT-40 0%ADDEDTO-&GAVEANUNACCEPTABLELEVELOFREACTO GENICITY; =5NFORTUNATELY THEANIMALMODELSAVAILABLEATTHETIMEWERE NOTABLETOPREDICTTHEPOORTOLERABILITYOF-40 0%INHUMANS &ORTUNATELY SUBSEQUENTDATASHOWEDTHATTHEINCLUSIONOF-40 0%WASNOTNECESSARYTO ENHANCETHEIMMUNOGENICITYOFANTIGENSCOMBINEDWITH-& )NADDITIONTOIMMUNEPOTENTIATORS ALTERNATIVEDELIVERYSYSTEMS INCLUD INGMICROPARTICLESCANALSOBEADDEDTO-&TOENHANCEITSPOTENCY;= (OWEVER THELEVELOFENHANCEMENTACHIEVEDWOULDNEEDTOBEHIGHLYSIG NIFICANTANDENABLINGFORVACCINEEFFICACY TOJUSTIFYDEVELOPMENTOFSUCHA COMPLEXFORMULATION



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4HEUSEOF-&INPRIMEBOOSTSETTINGS !S AN ALTERNATIVE TO THE INCLUSION OF IMMUNE POTENTIATORS IN -& TO PROMOTEA4HRESPONSE -&CANALSOBEUSEDASABOOSTERVACCINEWITH PROTEINS ONCE A4H RESPONSE HAS ALREADY BEEN ESTABLISHED BY IMMUNIZA TION WITH $.! ;= 2ECENTLY THIS STRATEGY HAS BEEN SHOWN TO BE HIGHLY PROMISINGFORTHEDEVELOPMENTOFAVACCINEAGAINST()6 SINCEALLARMSOF THE IMMUNE RESPONSE INCLUDING #4, RESPONSES 4 HELPER RESPONSES AND NEUTRALIZINGANTIBODIESWEREINDUCEDBYTHISCOMBINATIONAPPROACH;n= !SIMILARAPPROACHOF$.!PRIMEANDPROTEINBOOSTWITH-&HAS ALSO SHOWN SIGNIFICANT PROMISE IN NON HUMAN PRIMATES AS A VACCINE STRATEGY AGAINST (#6 ;= !LTERNATIVELY -& AND PROTEIN ANTIGENS CAN ALSO BE USEDTOBOOST4HRESPONSESPRIMEDBYIMMUNIZATIONWITHATTENUATEDVIRAL VECTORS4HECONCEPTOFANATTENUATEDVIRALVECTORPRIMEFOLLOWEDBY-& BOOSTHASBEENESTABLISHEDINTHECLINICUSINGCANARYPOXVECTORS ASASTRAT EGYFORBOTH()6;=AND#-6;=

#ONCLUSION )NANEXTENSIVERANGEOFPRE CLINICALANDCLINICALSTUDIES-& HASPROVEN TO BE A SAFE AND POTENT VACCINE ADJUVANT RESULTING IN THE LICENSURE OF AN -& ADJUVANTEDINFLUENZAVACCINEINMORETHANCOUNTRIES4HEABILITYOF -&TOINDUCESIGNIFICANTLYENHANCEDTITERSAGAINSTPOTENTIAL PANDEMICFLU STRAINSATLOWANTIGENDOSESAPPEARSHIGHLYPROMISING ASTOODOESTHEABIL ITYOF-&TOOFFERNEUTRALIZATIONAGAINSTHETEROLOGOUSSTRAINS4HESEDATA WEREUSEDTOGAINAPPROVALFORANEWPANDEMICINFLUENZAVACCINE CONTAINING THE-&ADJUVANT&OCETRIAš IN-AYINALLMEMBERSTATESOFTHE %UROPEAN5NION&OCETRIAš WILLBEMANUFACTUREDTOCONTAINTHEINFLUENZA STRAINDECLAREDBYTHE7ORLD(EALTH/RGANIZATIONASAPANDEMIC STRAIN4HE DOSE SPARING ASPECT OF -& IS PARTICULARLY ATTRACTIVE GIVEN THE CURRENT LIMITED CAPACITY WORLDWIDE FOR INFLUENZA VACCINE PRODUCTION WHICH IS NOT SUFFICIENT TO DEAL WITH A PANDEMIC GIVEN THE HIGH DOSE REQUIREMENTS FOR UNADJUVANTEDINFLUENZAVACCINES;= 4HEENCOURAGINGSAFETYANDTOLERABILITYPROFILEOF-& INCOMBINATION WITH IMMUNOGENICITY DATA SUGGEST THAT -& IS AN APPROPRIATE ADJUVANT FORUSEINPEDIATRICPOPULATIONS)NATRIALINNEONATES -& WASSHOWNTO BEMOREPOTENTTHANAN!LUM ADJUVANTEDCOMPARATORVACCINE WHILEBEING SIMILARLY WELL TOLERATED ;=!N -& ADJUVANTED #-6 VACCINE WAS ALSO SHOWNTOBESAFE POTENTANDWELLTOLERATEDINTODDLERS;=-OREOVER PRE CLINICAL DATA HAVE FIRMLY ESTABLISHED THAT -& IS A MORE POTENT ADJUVANT THAN!LUMFORAWIDERANGEOFVACCINES INCLUDINGRECOMBINANTPROTEINSAND PROTEINPOLYSACCHARIDECONJUGATES;=4HEPOTENTIALFOR-& 4 TOBEUSEDIN RECOMBINANTVACCINESWASRECENTLYHIGHLIGHTEDINASTUDYONANEWGENERA TION SEROGROUP " MENINGOCOCCUS VACCINE CANDIDATE ;= 0REVIOUSLY -&

-&!SAFEANDPOTENTOILINWATEREMULSIONADJUVANTFORINFLUENZAVACCINES



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)NTRODUCTION 4HE INFLUENZA VIRUS DERIVED ITS NAME IN THE THnTH #ENTURY WHEN A &LORENTINEFAMILYUSEDTHEWORDhINFLUENCEvTOSUGGESTANUNUSUALCONJUNC TION OF PLANETS AT TIMES OF EPIDEMICS OF COUGH COLDS AND FEVER4HE WORD hINFLUENZAvWASTHUSDERIVEDASADESCRIPTIONNAMEFORTHEEPIDEMICSDUE TOhINFLUENCESvhABOCCULTACOELIINFLUENTIAv;= 4HEREALCAUSEOFTHEhINFLUENZAvWASDISCOVEREDINWITHTHEFIRST ISOLATIONOFTHEFLUVIRUSBY3MITH3INCETHEN THEKNOWLEDGE OFTHEVIRUS HASINCREASEDEXPONENTIALLYANDREACHESTOTHEFINESTMOLECULARDETAILS*UST AFEWYEARSAFTERTHEISOLATIONOFTHEVIRUS THEFIRSTFLUVACCINESWEREDEVEL OPEDANDTHEIRUSEHASBECOMEANANNUALROUTINE )NTHE-IDDLE!GES THE'ERMANSTHOUGHTINFLUENZAWASCAUSEDBYEAT ING TOO MANY SOUR APPLES AND SALT FISH .OW WE KNOW MUCH MORE ABOUT THEFLUVIRUS BUTOURKNOWLEDGEONHOWTOTAKECONTROLOFPANDEMICAND EPIDEMICINFLUENZAISSTILLPRIMITIVEANDPOOR ANDTHISVIRUSCONTINUESEVEN TODAYTOBEONEOFTHEMAJORTHREATSFORHUMANHEALTHANDONEOFTHEMOST IMPORTANTPUBLICHEALTHCONCERNS;= )N ANNUAL SEASONAL INFLUENZA EPIDEMICS AS MUCH AS n OF THE POPULATIONISAFFECTEDBYUPPERRESPIRATORYTRACTINFECTIONS WHICHIMPOSEA



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CONSIDERABLEECONOMICBURDENDUETOINDIRECTANDDIRECTCOSTSDUETOHOSPI TALIZATIONS OTHERHEALTHCARECOSTSANDALOSSOFPRODUCTIVITY;= )NFLUENZA PANDEMICS CAN AFFECT UP TO  OF THE POPULATION WITH A DRAMATICINCREASEINMORBIDITYANDMORTALITYTHATCOULDLEADTO THEFAILURE OFHEALTHCARESYSTEMSASALARGEPROPORTIONOFTHEPOPULATIONISAFFECTEDAT ANYONETIME !LTHOUGHASINGLEPANDEMICLEADSTOAHIGHERTOLLTHATASINGLEEPIDEMIC THECUMULATIVEDISEASEBURDENOFEPIDEMICSOVERALLISGREATERTHANTHATOF PANDEMICS;=)NTHISCHAPTERWEFOCUSONTHENON RECENTHISTORYOFINFLU ENZA THE ORIGIN OF THE DISEASE THE PANDEMICS FOR WHICH WE HAVE RECORDS STARTINGFROM THEFIRSTVACCINE THENEGATIVEEXPERIENCE WITHTHESWINE INFLUENZAVACCINATIONANDTHENUMEROUS ATTEMPTSTOIMPROVETHEEFFICACYOF INFLUENZAVACCINESUSINGADJUVANTSTHATWERECARRIEDOUTSINCES

(ISTORYOFINFLUENZA (ISTORICAL RECORDS CARRY EVIDENCE OF THE CIRCULATION OF THE INFLUENZA F VIRUS STARTINGFROMTHEANCIENT'REEKS!LTHOUGHITISDIFFICULTTOINTERPRETOLDLIT ERATURE ITCOULDBETHAT(IPPOCRATESWRITINGSOF"#ARETHEFIRSTREPORTS OFPOSSIBLEINFLUENZA;=)NTHE-IDDLE!GESSEVERALWIDESPREADOUTBREAKS THATWEREPROBABLYDUETOINFLUENZAOCCURREDIN%UROPE(OWEVER THEFIRST REPORTOFANINFLUENZAEPIDEMIC WHERESYMPTOMSCANBEREGARDED WITHCON FIDENCEASPROBABLYDUETOINFLUENZA OCCURREDINn;= WHILETHE FIRSTINFLUENZAPANDEMICAGREEDBYALLAUTHORSOCCURREDIN4HISPAN DEMICORIGINATEDIN!SIADURINGTHESUMMEROFTHATYEAR SPREAD TO!FRICA ANDTHENTO%UROPEALONGTWOCORRIDORSFROM!SIA-INORAND.ORTH 7EST !FRICA;=4HEWHOLE%UROPEWASINFECTEDFROMSOUTHTONORTHINA MONTH PERIOD AND INFECTION SUBSEQUENTLY SPREAD TO!MERICA ; = )LLNESS RATES WEREHIGHDEATHSWEREREPORTEDFROM2OME ANDSOME3PANISHCITIES WEREDECIMATED)TWASSAIDTHATONLYONETWENTIETHOFTHEPEOPLEESCAPED THEILLNESS;= !CCORDING TO THE DICTIONARY DEFINITION A PANDEMIC IS SIMPLY A WIDE SPREADEPIDEMIC BUTWHENREFERRINGTOINFLUENZA APANDEMICNOWSIGNIFIES AWORLDWIDEEPIDEMICCAUSEDBYANEWSUBTYPEOFINFLUENZA!VIRUS4HUS ALTHOUGHONLYDURINGTHEVIROLOGICALERAWECANRECOGNIZEAPANDEMICWITH CERTAINTY AT LEAST TEN PANDEMICS HAVE BEEN AGREED BY ALL THE REVIEWERS SINCETHEFIRSTONEINANDANOTHERTHREEHAVEBEENCLASSIFIEDASPOS F SIBLE;= .UMEROUSREFERENCESTOINFLUENZAPANDEMICSANDEPIDEMICSWEREMADE FORTHETHCENTURYIN!MERICAAND%UROPE&ROMTHEBEGINNING OFTHETH CENTURY THEQUALITYANDQUANTITYOFDATAINCREASEDANDMEDICAL HISTORIANS WEREDRAWNTOCOMMENTONTHENUMBEROFINFECTEDPERSONS WHETHERTHEY WERECONSIDERINGANEPIDEMICORAPANDEMIC THECOUNTRIESINVOLVEDANDTHE POSSIBLEORIGINSOFTHEVIRUSSTRAINSINVOLVED

.ON RECENTHISTORYOFINFLUENZAPANDEMICS VACCINES ANDADJUVANTS



4HE FIRST AGREED INFLUENZA PANDEMIC OF THE TH CENTURY BEGAN IN!$ ;   =THEOUTBREAKSTARTEDIN2USSIAINTHESPRING SPREADWEST WARDSINEXPANDINGWAVESTOEMBRACEALL%UROPEWITHINA MONTH PERIOD ;= ANDENCOMPASSEDTHEWHOLEKNOWNWORLDOVERA YEARPERIODWITHHIGH DEATHRATES$ISTINCTWAVESOFINFECTIONWERERECORDEDTHELATERWEREMORE SEVERETHANTHEFIRST;n= 4HENEXTPANDEMICOCCURREDAFTERAGAPOFSOMEYEARSBETWEEN AND; =-OSTAUTHORSAGREETHATTHEOUTBREAKBEGANIN#HINAINTHE AUTUMN SPREADTO2USSIAANDFROMTHEREWESTWARDSINWIDENINGCIRCLESTO ENCOMPASSTHEWHOLEOF%UROPEINAPERIODOFMONTHS;=4HEREISEVI DENCEOFEXTENSIVESEEDINGINBOTH2USSIAAND.ORTH!MERICAIN THEEARLY MONTHSOFTHEPANDEMIC FOLLOWEDBYEXTENSIVEOUTBREAKS4HEATTACKRATE WASREPORTEDTOBEHIGH PARTICULARLYAMONGYOUNGADULTS;= ATTHEPEAK OFTHEPANDEMIC  FELLILLEACHDAYIN3T0ETERSBURGTWO THIRDSOFTHE POPULATIONOF2OMEBECAMEILLANDTHEOUTBREAKISREPORTEDTO HAVERAGED THROUGH"RITAINDURINGTHESUMMEROF 4HE FIRST PANDEMIC OF THE TH CENTURY BEGAN IN THE WINTER OF  IN #HINA FROM WHERE IT SPREAD SOUTHWARDS BY SEA TO REACH THE 0HILIPPINES )NDIAAND)NDONESIA ANDACROSS2USSIAINTO%UROPE4HECONTAGIONSPREAD INTO.ORTH!MERICATOCAUSEOUTBREAKSINn RECURREDIN %UROPEAT THESAMETIMEANDRECURREDAGAININ%UROPEINn;=!LLAUTHORS COMMENTONTHEHIGHATTACKRATEOFTHEPOPULATION BUTTHEMORTALITYRATE WASNOTEXCEPTIONALLYHIGH;= 3INCE THE PANDEMIC OF n DATA HAVE BEEN MORE RELIABLE AND MORETHOROUGHLYREVIEWED ANDSINCETHECAUSALVIRUSESWEREAVAILABLE FORANALYSIS 4HEEPIDEMICOFnSTANDSOUTNOTONLYINSEVERITY BUTALSOINITS WAVELIKEEXTENSIONINANNUALRECURRENCESINTOTHEIMMEDIATELYSUCCEEDING YEARS )N ,ONDON THE EPIDEMIC INCREASED IN SEVERITY WITH EACH SUCCESSIVE WAVESOTHATMORTALITYWASHIGHESTIN;=!NOTHERNOTABLEFEATUREOF THISEPIDEMICWASTHEOCCURRENCEINOFANINCREASEINTHEPROPORTIONOF DEATHSINTHEn YEAR OLDAGEGROUP ORDINARILYCONSIDEREDTOBEAUNIQUE CHARACTERISTICOFTHEPANDEMIC 4HE GREATEST PANDEMIC OF THE TH CENTURY WAS THE PANDEMIC OF  POPULARLY KNOWN AS h3PANISH FLUv WHICH IS BELIEVED TO BE CAUSED BY AN INFLUENZA!(.VIRUS WHICHISCLOSELYRELATEDTOTHEVIRUSLATERFOUNDIN PIGS;= ANDWHICHREMAINSANINFECTIONOFTHISSPECIESTOTHEPRESENTTIME %PIDEMIOLOGICAL EVIDENCE SUGGESTS THAT THE 3PANISH FLU ORIGINATED IN THE 53 IN -ARCH AND!PRIL  AND WAS TRANSPORTED BY!MERICAN TROOPS TO %UROPE WHENITFIRSTREACHEDEPIDEMICLEVELSIN&RANCEIN!PRIL/VER THENEXTSEVERALMONTHS INFLUENZASPREADTHROUGHOUTTHEWHOLEOF%UROPE -IDDLE %AST )NDIA %ASTAND3OUTH%AST!SIA !USTRALIAAND!FRICAINTHREE SUBSEQUENTWAVES)NTHE53 THEINFLUENZAPANDEMICKILLEDAPPROXI MATELY PEOPLEn OFTHEPOPULATION ABOUTTENTIMESMORETHAN THENUMBEROF!MERICANSWHODIEDINTHE&IRST7ORLD7AR)N%NGLANDAND



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7ALESTHEOFFICIALDEATHSNUMBERED )NAFEWPLACES SUCHAS3AMOA AND!LASKASOMEORMOREOFTHEPOPULATIONDIED.OFIGURESEXISTFOR MANY PARTS OF THE WORLD BUT THE PANDEMIC IS ESTIMATED TO HAVE INFECTED  OF THE WORLDS POPULATION  SUFFERED A CLINICAL INFECTION AND THE TOTALMORTALITYWASnMILLION4HEINFLUENZAPANDEMICKILLEDMORE PEOPLEINLESSTIMETHANANYOTHERDISEASEBEFOREORSINCE 3INCETHEPANDEMIC THEREHAVEBEENTWOMAJOROUTBREAKSOFF INFLU ENZA!INTHETHCENTURY)NINFLUENZAWASINDUCEDBYTHEh!SIANv INFLUENZAVIRUS!(. ANDIN INFLUENZAWASINDUCEDBYTHEh(ONG +ONGvINFLUENZAVIRUS!(..EITHERPANDEMICWASASBLOODTHIRSTYASIN NEVERTHELESS INTHE53ALONE AND DEATHSWEREATTRIBUTED TOTHEh!SIANvANDTOTHEh(ONG+ONGvPANDEMICS RESPECTIVELY )NADDITIONTOTHETHREEMAJORINFLUENZAPANDEMICS AFEWSMALLERINFLU ENZAEPIDEMICSASWELLASFLUhSCARESvOCCURREDINTHETHCENTURY/FTHESE THE MOST FAMOUS IS THE OUTBREAK OFh3WINE FLUv THAT HAPPENED IN  AT &ORT$IX .*4HERECOVERYOFTHISVIRUSRAISEDCONCERNSABOUT AWORLDWIDE PANDEMIC AND A MASS VACCINATION PROGRAM WAS LAUNCHED IN THE 53 SEE BELOW  4HERE IS NO PERIODICITY TO THE OCCURRENCE OF PANDEMICS AND NO BASIS FORPREDICTINGWHEREANDWHENANEWOUTBREAKMAYARISE;=3INCE PANDEMICSOFINFLUENZAHAVEOCCURREDATINTERVALSOFANDYEARS ANDINFACTSEVERALSUPPOSITIONSONTHEPOSSIBILITYOFPREDICTINGANINFLUENZA PANDEMICONTHEBASISOFCYCLICALRECURRENCEOFTHEPANDEMICSTRAINSORSOLAR ACTIVITY OR OTHER FACTORS ARE ONLY SPECULATIVE )N THE TH CENTURY (OPE 3IMPSONPUBLISHEDANARTICLE;=INWHICHHESUGGESTEDTHATANTIGENICSHIFTS OF INFLUENZA! VIRUS AND ACCORDINGLY A PANDEMIC COINCIDED WITH SUNSPOT ANDSHOWEDSOMEEXAMPLESOFTHISFROMTO"UTIN SUNSPOTS ACTIVITYTOOKPLACEBUTTHEREWASNOPANDEMIC3OWESHOULDBE PREPARED FORUNFORESEENINFLUENZAPANDEMICINANYGIVENYEAR;=

&IRSTINFLUENZAVACCINES %FFORTS TO DEVELOP INFLUENZA VIRUS VACCINES BEGAN SOON AFTER INFLUENZA! AND " VIRUSES WERE RECOGNIZED AS THE ETIOLOGICAL AGENTS OF INFLUENZA4HE FIRSTPAPERSONIMMUNOGENICITYOFINFLUENZAVACCINESWEREPUBLISHEDINTHE SECOND HALF OF S ;=4HE VACCINE TESTED WAS A CRUDE POORLY PURIFIED PREPARATIONOFTHE0UERTO2ICO02 STRAINOBTAINEDFROMTHE LUNGSOF INFECTEDMICEANDINACTIVATEDWITHFORMALIN 3UBSEQUENTLY IT WAS NOTICED THAT THE INOCULATION AND PASSAGING OF THE FLUSTRAINSONTHECHORIO ALLANTOICMEMBRANESOFDEVELOPINGCHICKEMBRYOS LEADTOANINCREASEINTHEPRODUCTIONPROCESSANDTOAMOREIMMUNOGENIC VACCINE 4HE MAIN INPUT AND INTEREST IN THE DEVELOPMENT OF INFLUENZA VACCINES CAMEFROMTHE53MILITARYDURING7ORLD7AR)) INPARTBECAUSE OFTHEDEV

.ON RECENTHISTORYOFINFLUENZAPANDEMICS VACCINES ANDADJUVANTS



ASTATION CAUSED IN BOTH MILITARY AND CIVILIAN POPULATION BY THE n INFLUENZAPANDEMIC DURINGTHELATESTAGESOF7ORLD7AR) )N VERY FEW YEARS WHOLE VIRUS INACTIVATED BIVALENT TYPE! 02 STRAIN AND TYPE " ,EE STRAIN AND TRIVALENT VACCINES TYPE ! 02 AND 7EISS STRAINSANDTYPE",EESTRAIN WEREDEVELOPEDANDTESTEDINLARGEFIELDTRIALS MAINLYAMONGHEALTHYMILITARYRECRUITSANDCOLLEGESTUDENTS; =4HESE FIELDTRIALSLEADTOTHEAPPROVALOFTHEFIRSTCOMMERCIALVACCINESINTHE53 IN 4HERESULTSOFALLTHOSEFIELDTRIALSSUGGESTEDTHATVACCINATIONWITHCON CENTRATEDINACTIVEVIRUSINDUCEDANINCREASEINANTIBODIESAGAINSTFLUANDA DISTINCTEFFECTINREDUCINGTHEINCIDENCEOFINFLUENZA )T IS VERY IMPRESSIVE TO NOTICE HOW THE VAST MAJORITY OF KNOWLEDGE ON THEEFFECTSOFINFLUENZAVACCINATIONWASALREADYDESCRIBEDINTHOSEPAPERS WRITTENINTHEEARLYS )T WAS NOTED INDEED THAT THE INCREASE IN VIRUS NEUTRALIZING CAPACITY FOLLOWING VACCINATION WAS RELATED TO THE PRE VACCINATION LEVEL )N GENERAL THELOWERTHENEUTRALIZINGCAPACITYBEFOREVACCINATIONTHEGREATERWASTHE INCREASEINANTIBODIESAFTERVACCINATION!DDITIONALLY ITWAS OBSERVEDTHAT THE MEAN NEUTRALIZING CAPACITY FOLLOWING VACCINATION WAS HIGHEST IN THE GROUP WHO POSSESSED THE HIGHEST ANTIBODY LEVELS BEFORE VACCINATION EVEN THOUGHTHEMEANINCREASEINNEUTRALIZINGCAPACITYINTHISGROUPWASLOWEST ATALL-OREOVER ITWASSHOWNTHATTHEREWASACORRELATIONBETWEENNEUTRAL IZINGANTIBODYLEVELSAGAINSTINFLUENZAANDTHEOCCURRENCEOFTHEDISEASE)T WASFOUNDTHATTHEHIGHERTHELEVELOFANTIBODIES THELESSLIKELYITWASTHAT DISEASEWOULDDEVELOP ALTHOUGHANEXACTQUANTIFICATIONOFTHIS CORRELATION WASNOTPOSSIBLEATTHATTIME;= 4HOSEFIRSTEXPERIENCESSHOWEDALSOTHAT WHILEVACCINATIONPREVENTEDTHE DEVELOPMENTOFCLINICALINFECTIONTOACONSIDERABLEDEGREE ITALSOREDUCED THESEVERITYOFTHEDISEASE;=)TISOFINTERESTTHATNOSIGNIFICANTDIFFERENCES ININCIDENCEWEREOBSERVEDBETWEENCONTROLANDVACCINATEDSUBJECTSWHEN THEDIAGNOSISOFCOMMONCOLDORLOCALRESPIRATORYINFECTIONWEREANALYZED -OREOVER THEINCIDENCEOFLOCALRESPIRATORYINFECTIONSINEITHERGROUPDID NOTVARYSIGNIFICANTLYFROMTHEPRE EPIDEMICLEVEL4HESEFACTS SUGGESTEDTHE SPECIFICITYOFACTIONOFTHEINFLUENZAVACCINE;= &URTHERMORE THESAMEAUTHORSALSOSUGGESTEDHOWTOBESTUSEVACCINA TIONINAPUBLICHEALTHSETTINGTOMAXIMIZEPOTENTIALITIESOFSUCHATOOL)TWAS PROPOSEDTOCONTROLTHEEPIDEMICRECURRENCEOFINFLUENZABYREVACCINATION ATINTERVALSSHORTERTHANTHEEPIDEMICPERIODICITY ORATINTERVALSDETERMINED BYIMMUNOLOGICALSURVEYSh4HISWOULDAPPEARAMOREPRACTICALMETHODOF ADMINISTERING VACCINE FOR PROPHYLAXIS THAN TO VACCINATE IN THE FACE OF AN OUTBREAK AFTER ITS IDENTIFICATION PARTICULARLY SINCE INFLUENZA SPREADS WITH SUCHRAPIDITYTHATTHEEPIDEMICMIGHTBEWELLUNDERWAYBEFORE ITWOULDBE POSSIBLETOVACCINATEASIGNIFICANTPROPORTIONOFTHEPOPULATIONv;= 4HEPROCESSUSEDTOMAKETHECURRENTCOMMERCIALLYAVAILABLEINACTIVATED INFLUENZA VIRUS VACCINES SHARE CERTAIN KEY FEATURES WITH THOSE PIONEER



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ING EARLY VACCINES4HE INFLUENZA VACCINE VIRUSES ARE STILL REPLICATED IN THE ALLANTOIC CAVITIES OF EMBRYONATED HENS EGGS AND THE INACTIVATION OF THE HARVESTEDINFLUENZAVIRUSESISDONEEVENNOWUSINGEITHERFORMALINORBETA PROPRIOLACTONE (OWEVER EARLYINFLUENZAVACCINESWERERELATIVELYCRUDEPREPARATIONSOF CHICKEMBRYOALLANTOICFLUIDVIRUSANDWEREFREQUENTLYCONTAMINATEDWITH BACTERIAL ENDOTOXIN WHICH QUITE OFTEN EVOKED FEBRILE REACTIONS AFTER VAC CINATIONSANDWERECONTRAINDICATEDINCHILDREN !LTHOUGHTHEEXTENSIVEFIELDTRIALSTHATESTABLISHEDINFLUENZAVACCINESAS SAFEANDREASONABLYEFFECTIVEFORPREVENTIONOFINFLUENZAINMILITARYPOPULA TIONBEGANINTHEMIDS WASTHEFIRSTYEARINWHICHTHEREWASWIDE SPREAD CIVILIAN USE OF INFLUENZA VACCINES IN 53 APPROXIMATELY  MILLION DOSES WERE DISTRIBUTED IN ANTICIPATION OF THE ONSET OF THE!SIAN INFLUENZA PANDEMIC&ROMTOTHESECONDHALFOFTHESPOLYVALENT ANDBIVALENT VACCINESINCORPORATINGEITHERMULTIPLESTRAINSOFINFLUENZA!AND"ORONLY THEMOSTCURRENTINFLUENZA!AND"STRAINHADBEENMARKETED!TTHATTIME THERATIONALEFORPOLYVALENTVACCINEUSAGEWASBASEDONTHEINABILITYTOPRE DICTEPIDEMICSTRAINSLIKELYTOBEPREVALENTANDTHEBELIEFTHATABROADBASE OFPROTECTIONWOULDRESULTFROMMULTIPLEANTIGENICSTIMULATIONS ;= 7HOLE VIRUSVACCINESPRODUCEDWITHMOREORLESSTHESAMETECHNOLOGIES ASTHOSEPIONEERVACCINESHAVEBEENUSEDSINCETHES(OWEVER ATTHAT TIME YEARLY VACCINE FORMULATION DEPENDED UPON LESS THAN ADEQUATE VIRAL SURVEILLANCE RELATIVELY CUMBERSOME METHOD OF VACCINE PRODUCTION AND OFTEN NO ESTABLISHED KNOWLEDGE OF EFFECTIVENESS &URTHERMORE THOSE VAC CINESPRODUCEDADISTURBINGLYHIGHPERCENTAGEOFSIDEREACTIONS4HEREFORE DURINGTHES INFLUENZAVACCINESNEVERENJOYEDWIDEPOPULARITYAMONG PRIVATE PHYSICIANS NOR THEY WERE PROMOTED UNIVERSALLY BY PUBLIC HEALTH OFFICIALS4HEY HAVE BEEN USED SPARINGLY IN PERHAPS n OF THE AT RISK POPULATION;= !LL THOSE REASONS BUT ESPECIALLY THE HIGH INCIDENCE OF REACTOGENICITY INDUCEDBYTHESEVACCINES PROMPTEDATTEMPTSTOPRODUCEALESS REACTOGENIC VACCINE3UBVIRIONPREPARATIONSWEREDEVELOPEDTOMEETTHISMEDICALNEED RETAININGTHEIMMUNOGENICPROPERTIESOFTHEWHOLE VIRUSPREPARATIONSBUT WITH GREATLY REDUCED REACTOGENICITY ;= 3UBVIRION OR SPLIT VACCINES ARE PREPAREDUSINGASOLVENTSUCHASETHERORADETERGENT TODISSOLVEORDISRUPT THEVIRALLIPIDENVELOPE4HEYCONTAINMEMBRANEPROTEINS THEANTIGENSHEM AGGLUTININ(! ANDNEURAMINIDASE.! ANDPARTSOFTHELIPID ENVELOPE BUT NO 2.! MOLECULES THE GENETIC MATERIAL 4HESE VACCINES HAVE BEEN SHOWNTOINDUCEFEWERSIDEEFFECTSINTHEVACCINEESBUTAREEQUALLYIMMU NOGENICASWHOLE VIRUSVACCINE3PLITVIRUSVACCINESARESUITABLEFORTHEUSE INADULTSANDCHILDREN&IG  !DDITIONALPURIFICATIONSTEPSCANBETAKENTOREDUCETHEAMOUNT OFVIRAL PROTEINSESSENTIALLYINFLUENZAMATRIXPROTEINANDNUCLEOPROTEIN TOPRODUCE ASUBUNITORPURIFIEDSURFACEANTIGENVACCINE)NTERNALPROTEINSOFTHEVIRUS CANBEREMOVEDLEAVINGONLY(!AND.! WHICHARETHEONLYTWOANTIGENS

.ON RECENTHISTORYOFINFLUENZAPANDEMICS VACCINES ANDADJUVANTS



&IGURE-ILESTONESOFINACTIVATEDINFLUENZAVACCINEDEVELOPMENT

NECESSARYTOELICITAPROTECTIVEANTIBODYRESPONSE6OLUNTEERS GIVENSUBUNIT VACCINESEXPERIENCEDFEWERADVERSEREACTIONSTHANTHOSEGIVENWHOLEVIRUS VACCINES;= $UE TO THEIR GOOD TOLERABILITY AND IMMUNOGENICITY SPLIT AND SUBUNIT VACCINESHAVEBEENTHEMOSTWIDELYUSEDINFLUENZAVACCINEDURINGTHELAST FEWDECADES

4HESWINEINFLUENZAOUTBREAK !NEGATIVEEXPERIENCEWITHINFLUENZAANDINFLUENZAVACCINATIONCOMESFROM THE 3WINE INFLUENZA! OUTBREAK THAT TOOK PLACE IN &ORT $IX .EW *ERSEY 53! ATTHEBEGINNINGOF 4HE SEVERITY OF THE  INFLUENZA! (. h3PANISHv PANDEMIC AND EVIDENCE FOR A CYCLE OF PANDEMICS AROUSED CONCERN THAT THE  DISASTER COULDRECUR)NTHEEARLYSTHEONLY!VIRUSTOCIRCULATEWASAN(. SUBTYPE4HUS WHEN IN CASESOFINFLUENZAINSOLDIERS MOSTLYRECRUITS AT&ORT$IXWEREASSOCIATEDWITHISOLATIONOFINFLUENZA!(. SEROTYPE



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WHICHINWASLABELED!.EW*ERSEY(SW. ;= THERE WASGREAT CONCERN THAT THIS MIGHT MARK THE BEGINNING OF A NEW DREADFUL PANDEMIC LIKETHE3PANISHONE 4HANKFULLY !.EW*ERSEYVIRUSWASDETECTEDONLYFROM*ANUARYTO &EBRUARY ANDDIDNOTSPREADBEYOND&ORT$IX)NTHISTIMEPERIOD UPTO SOLDIERS WERE INFECTED INCLUDING  SOLDIERS WITH SEVERE RESPIRATORY ILLNESS AND  DEATH!6ICTORIA (. SPREAD SIMULTANEOUSLY IN THE SAME SET TING ANDCAUSEDILLNESSCASESDOCUMENTEDBYVIRALISOLATION SEROLOGICAL RESPONSES OR BOTH AND PERSISTED UNTIL -ARCH $ESPITE INTENSIVE INVESTIGA TION THESOURCEOFTHEVIRUS THEEXACTTIMEOFITSINTRODUCTIONINTO&ORT$IX ANDFACTORSLIMITINGITSSPREADANDDURATIONREMAINEDUNKNOWN;n= 4HEAPPEARANCEOFANEWFLUVIRUSSUBTYPEHADALWAYSPRESAGEDTHEDIS APPEARANCEOFTHEPREVALENTSUBTYPEOFVIRUSANDRAPIDSPREADOFTHENEW STRAINCOINCIDENTWITHPANDEMICDISEASE7HENTHENEWVIRUSWASIDENTIFIED ASTHEPUTATIVEAGENTOFPANDEMIC CONCERNEVOKEDCAUTION4HECHANCE THATTHENEWVIRUSMIGHTPOSSESSTHEVIRULENCEATTRIBUTEDTOTHEVIRUS WASONEOFTHEMAINREASONSTOUNDERTAKEMASSIMMUNIZATIONSIN THE53 3UPPORTEDBYA53MILLIONAPPROPRIATIONFORMTHE#ONGRESS MANU FACTUREOFVACCINEANDINTENSIVESURVEILLANCEFORNEWSWINEINFLUENZACASES F BEGAN"ETWEEN!PRILAND/CTOBERTHEMOSTEXTENSIVETRIALS EVERPER FORMEDWITHANINFLUENZAVACCINEWERECONDUCTEDONVOLUNTEERS .ATIONAL)NFLUENZA)MMUNIZATION0ROGRAM.))0 WASDESIGNEDTO PRO VIDE!.EW*ERSEYINFLUENZAVACCINEFORALMOSTTHEENTIREADULT POPULATION INTHE53!/NLYCHILDRENATHIGHRISKOFSERIOUSILLNESSFORINFLUENZAVIRUS INFECTIONRECIEVEDTHEVACCINEDUETHEUNACCEPTABLETOXICITYOFF WHOLEVIRUS VACCINEINYOUNGERCHILDRENCONFIRMEDFROMTHECLINICALTRIALS "ETWEEN/CTOBER AND$ECEMBER MILLIONPEOPLE WERE IMMUNIZED INCLUDINGNEARLYOFTHEHIGH RISKGROUP THEGREATESTPRO TECTIONEVERAFFORDEDINTHATGROUPANDTWICETHENUMBEROFPERSONSINTHAT GROUPUSUALLYIMMUNIZED &OURVACCINEMANUFACTURERSPRODUCEDOVERMILLIONDOSESAPPROXI MATELY  MILLION DOSES OF MONOVALENT !.EW *ERSEY (SW. VAC CINE REASSORTANT VIRUS 8 A AND  MILLION DOSES OF BIVALENT !.EW *ERSEY (SW. AND !6ICTORIA (. VACCINE 7HOLE VIRUS WAS USEDINOFTHEMONOVALENTVACCINEANDINOFTHEBIVALENTVACCINE FROMTWOPRODUCERS WHILETHEREMAININGDOSESWERESPLITORSUBUNITVAC CINESFROMTHEOTHERTWOPRODUCERS  6ACCINEMANUFACTURERSANDTHEIRINSURERSHADFORESEENPOSSIBLE LIABILITY ISSUESOFTHISUNPRECEDENTEDVACCINATIONCAMPAIGN ANDWEREABLETOFORCE THE'OVERNMENTTOASSUMEPRIMARYLIABILITYRESPONSIBILITY 4HEVACCINATIONPROGRAMINCORPORATEDANATIONALWIDESURVEILLANCESYS TEMTOEVALUATEPOSSIBLEADVERSEVACCINEREACTIONS/N$ECEMBER THE APPARENT ASSOCIATION BETWEEN VACCINATION AND THE ONSET OF 'UILLAIN "ARRÏ SYNDROME '"3 AND THE CONTINUING ABSENCE OF SWINE INFLUENZA VIRUS IN MAN LED TO THE SUSPENSION OF THE .))0 AND TO THE EXPANSION OF

.ON RECENTHISTORYOFINFLUENZAPANDEMICS VACCINES ANDADJUVANTS



'"3SURVEILLANCENATIONALLYTODETERMINEASQUICKLYASPOSSIBLEWHETHERTHE INFLUENZAVACCINATIONSWERERELATEDTO'"3"Y*ANUARYOF MORETHAN CASESOF'"3HADBEENREPORTEDWITHDEATHS;= )NITIALREPORTSINDICATEDTHAT INPERSONSOLDERTHANYEARS THERISKOF GETTING'"3WASFIVETOSIXTIMESGREATERINVACCINATEDTHANIN UNVACCINATED INDIVIDUALS;=4HISREPORTWASCRITICIZEDDURINGSUBSEQUENT COURTPROCEED INGSINWHICHVICTIMSSUEDFORDAMAGESBECAUSEITDIDNOTCONTAINTHEBASIC DATAFORCALCULATIONOFINCIDENCEANDESTIMATIONOFRISK)NDEED NOCONTROL GROUP OF SIMILAR SIZE WAS FOLLOWED AS CAREFULLY AT THAT TIME 3O THE TRUE DOMINATORISNOTKNOWN ! DEFINITIVE STUDY HAS SUBSEQUENTLY BEEN MADE ;=4HIS STUDY ESSEN TIALLY CORROBORATED THE EARLIER REPORT BUT REFINED THE ANALYSIS 6ACCINE ASSOCIATEDRISKnPERMILLION WASFOUNDTOLASTFORAT LEASTWEEKS FOLLOWING VACCINATION BUT NOT BEYOND  WEEKS 3UBSEQUENT STUDIES ON SEASONAL INFLUENZA VACCINES FOUND LOW RELATIVE RISKS OF  IN n n IN n AND n AND  IN n THESE RELATIVE RISKS WERE NOT SIGNIFICANTLY DIFFERENT FORM  NO ASSOCIATION  -OREOVER A MARKEDDECLINEINTHE INCIDENCEOF'"3WAS ALSOREPORTEDFROMTHETO ; =4HEREFORE ITHASBEENPROPOSEDTHATTHEELICITATIONOF'"3IS UNIQUETOSWINEINFLUENZAVACCINE(OWEVER ITHASTOBECONSIDEREDTHATTHE REVELATIONOFINCREASEDRISKCOULDBEUNIQUETOTHEMASSIVESWINEINFLUENZA IMMUNIZATIONPROGRAMANDTOTHESUBSEQUENTINTENSIVESURVEILLANCERATHER THATTOTHEVACCINEITSELF )NCONCLUSION THE.))0FORhSWINEFLUvISANEXPERIENCETHATWENEEDTO LEARNFROMTOAVOIDTHESCIENTIFIC TECHNICAL HUMAN ANDPOLITICALPROBLEMS ENCOUNTERED AT THAT TIME AND THAT CAN PROBABLY BE AVOIDED WITH PROPER PLANNING

%ARLYADJUVANTEDINFLUENZAVACCINES 1UITESOONAFTERTHEDEVELOPMENTOFTHEFIRSTINACTIVATEDINFLUENZAVACCINES SCIENTISTS WITHTHESPONSOROFTHE53!RMY TRIEDTOFINDAWAYTOIMPROVE THEIMMUNOGENICITYOFTHOSEVACCINESTHROUGHTHEADDITIONOFADJUVANTS D 4HE MOST POWERFUL ADJUVANT USED IN RESEARCH IS THE &REUNDS COM PLETE ADJUVANT &#! A WATER IN OIL EMULSION CONTAINING  MINERAL OIL !RLACEL! A SURFACTANT AND KILLED MYCOBACTERIA &#! GENERATES SIDE EFFECTSFARTOOSEVERETOBEUSEDINHUMANVACCINEAPPLICATIONS(OWEVER THEFIRSTADJUVANTSUSEDININFLUENZAVACCINESWERESUBSTANTIALLYBASEDONA SLIGHTLYMODIFIED&#! )N THEFIRSTCLINICALTRIALSOFMINERALOIL ADJUVANTEDINFLUENZAVAC CINESWEREPERFORMED AFTERPRECLINICALTESTINGINMORETHANMONKEYSTHAT WERECAREFULLYSTUDIEDHISTOLOGICALLYANDSEROLOGICALLYFORMORETHANYEAR )NTHESUBSEQUENTYEARS EXTENSIVEFIELDCLINICALTRIALSWERESPONSOREDBYTHE #OMMISSIONON)NFLUENZA !RMED&ORCES%PIDEMIOLOGICAL"OARDAND MAN



-ARIA,ATTANZI

AGEDBY*3ALKAT&ORT$IXINn;=4HESETRIALSINCLUDED !RMYPERSONNELGIVENADJUVANTEDINFLUENZAVIRUSVACCINE GIVENSTAN DARDAQUEOUS VACCINEANDGIVENFORMALINIZEDSALINEASACONTROL 4HE VACCINES USED IN THESE STUDIES WERE PREPARED BY COMMERCIAL BIO LOGICALFIRMS6IRUSSUSPENSIONSWEREINACTIVATEDWITHFORMALIN AND MERTHIOLATE WASADDEDASAPRESERVATIVE-INERALOIL ADJUVANTED VACCINESWEREMADEBYEMULSIFYINGONEVOLUMEOFVIRUSCONCENTRATEWITHAN EQUALVOLUMEOFAMIXTURECONTAININGNINEPARTSLIGHTMEDICINALMINERALOIL $RAKEOL 62 ANDONEPARTEMULSIFIERPURIFIED!RLACEL!  2ESULTS OF THESE TRIALS SHOWED THAT EFFECTIVENESS OF INFLUENZA VACCINES COULD BE GREATLY INCREASED BY EMULSIFICATION IN LIGHT MINERAL OIL ;= BUT ALSO THAT REACTOGENICITY INCREASED 3HORT TERM RESULTS REPORTED THAT ABOUT  OF RECIPIENTS DEVELOPED DELAYED LOCAL CYSTIC REACTIONS REQUIRING SURGI CAL INTERVENTION AT THE SITE OF INOCULATION -OREOVER A HIGHER INCIDENCE OF ALLERGIC MANIFESTATIONS ALMOST ENTIRELY URTICARIA WAS REPORTED 4HOSE ALLERGICREACTIONSWEREDUETOASENSITIZATIONTOPENICILLINBYTHEADJUVANT BECAUSEPENICILLINHADBEENUSEDINITSMANUFACTUREANDNOTTOTALLYREMOVED BYPURIFICATIONPROCESSES (OWEVER THE MAJOR CONCERNS ABOUT MINERAL OIL ADJUVANTED VACCINES WERENOTRELATEDTOTHEDESCRIBEDREACTOGENICITY BUTTOTHELONG TERMSAFETY ISSUESLIKECARCINOGENICITYANDHYPERSENSITIVITY DUETOTHEFACTTHATMINERAL OIL PRODUCTS WERE CATEGORIZED AS NON METABOLIZABLE AND NON EXCRETABLE 4HUS ALTHOUGH THE EFFECTIVENESS OF THE FLU VACCINE WAS ENHANCED BY THE MINERAL OILADJUVANT DOUBTSABOUTTHELONG TERMSAFETYOFTHE OILADJUVANT PREPARATIONGENERALLYPREVENTEDITSACCEPTANCEFORROUTINEUSE ;= 4OADDRESSTHELONG TERMSAFETYISSUESOFTHEADJUVANTEDVACCINESUSED AT &ORT $IX IN n A  YEAR AND SUBSEQUENTLY AN EXTRA  YEAR FOL LOW UPWASPERFORMEDONMORTALITYAFTERVACCINATION; ="OTHSTUDIES CONCLUDED THAT THERE WAS NO SUGGESTION OF A MORTALITY EFFECT ATTRIBUTABLE TO THE ADJUVANT!LL THE FINDINGS WERE ESSENTIALLY NEGATIVE WITH RESPECT TO MALIGNANT NEOPLASMS ALLERGIC DISEASES AND COLLAGEN DISEASES )N ADDITION THEREWASNOEVIDENCETHATMENKNOWNTOHAVEHADTHECYST LIKEREACTIONAT THESITEOFINOCULATION ORTHOUGHTTOHAVEHADALLERGICREACTIONSMEDIATEDBY THEADJUVANTEDVACCINEHADEXPERIENCEDAHIGHERMORTALITYRISK 4HOSEEARLYADJUVANTEDFLUVACCINESWEREEXTENSIVELYSTUDIEDINCLINICAL TRIALSDURINGTHESANDTHESBECAUSEOFTHECARDINALADVANTAGEOVER AQUEOUSVACCINESSUCHASHIGHERANDMOREDURABLELEVELSOFBROADLYREAC TIVEANTIBODIES4AB ANDTHEREDUCTIONOFTHEANTIGENCONTENT; = ! COUPLE OF STUDIES SHOWED THAT THE PERSISTENCE OF HIGH LEVELS OF ANTI BODIESAFTERASINGLEDOSE OFMINERALOIL ADJUVANTEDVACCINEWASATLEASTn YEARS; = BUTONETRIALFOUNDA YEARPERSISTENCE;=4HEULTIMATEGOAL OFTHOSETRIALSWASTOEVALUATETHEPOSSIBILITYOFACHIEVINGADURABLEPROTEC TIONWITHOUTTHETROUBLESOMEEVENTODAY NECESSITYFORANNUALREVACCINA TION (OWEVER REGARDLESS THE POTENCY OF THE ADJUVANT THE EMERGENCE OF DRIFTEDSTRAINSHASNOTALLOWEDSKIPPINGTHEANNUALUSEOFFLU VACCINES

.ON RECENTHISTORYOFINFLUENZAPANDEMICS VACCINES ANDADJUVANTS



4ABLE#OMPARATIVEIMMUNOGENICITYOFONEANDTWODOSESOFAQUEOUSANDADJUVANTINFLUENZA VACCINESINHEALTHYADULTSSUBJECTS;= 0OLYVALENTVACCINES

4ESTSTRAINANDANTIBODYLEVELS 3WINE

02

02

!QUEOUSAQUEOUS "EFORESECONDDOSE !FTERSECONDDOSE

 

 

 

!DJUVANT ADJUVANT "EFORESECONDDOSE !FTERSECONDDOSE

 

 

 

4HE REDUCED ANTIGEN CONTENT REQUIREMENT USING IN MINERAL OIL ADJU D VANTED VACCINES COULD ALLOW MULTIPLE ANTIGENS TO BE INCLUDED IN THE SAME VACCINE TO ACHIEVE PROTECTION AGAINST MORE THAN ONE DISEASE THROUGH A SINGLEINJECTION -OREOVER THOSEINFAVOROFTHEADOPTIONOFMINERALOIL ADJUVANTEDVAC CINESSTRESSEDTHEFACTTHATTHOSEVACCINESAFFORDEDTHEGREATESTPROMISEFOR COPINGWITHANEWPANDEMICSTRAIN;=4ODAY THEFEARFORANEWINFLUENZA PANDEMICISHIGH BASEDONTHEHISTORICALRECORDSOFTHE3PANISHFLU BUTAT THATTIME IN ONLYYEARSHADPASSEDANDTHEVASTMAJORITYOFTHEKEY OPINIONLEADERSANDPOLICYMAKERSHADWITNESSEDTHEnDISASTER 4OIMPROVETHESAFETYPROFILEOFMINERALOIL ADJUVANTEDVACCINES OTHER TYPESOFADJUVANTWERETESTED LIKEVEGETABLEOILEMULSIONS(OWEVER THOSE FORMULATIONSWERENOTCONSIDEREDASSUBSTITUTESOFTHEMINERAL OILPRODUCTS BECAUSEVEGETABLEOILSEMULSIONSDIDNOTPERFORMASEFFECTIVELYASMINERAL OIL PREPARATIONS WITH RESPECT TO HEIGHT OF TITER ACHIEVED AND DURATION OF ANTIBODYATHIGHLEVELS;= !LUMINUMSALTS THEMOSTWIDELYUSEDADJUVANTFORHUMANVACCINES WERE TESTED AS A WAY TO INCREASE INFLUENZA VACCINE EFFICACY WITHOUT SAFETY CON CERNS4ABLEDESCRIBESTHEDIFFERENCESBETWEENTHEALUMINUMSALTSUSEDAS VACCINEADJUVANTS0RECLINICALOBSERVATIONSHADSHOWNTHATTHEADSORPTIONOF INFLUENZAVIRUS(!ON!L0/ ENHANCEDTHEIRANTIGENICPOTENCYINMICE;= 4HUS ACLINICALTRIALONHEALTHYMILITARYRECRUITSWASPERFORMEDTOCOMPARE IMMUNOGENICITYOFTWOINFLUENZAVACCINES WITHTHESAMECONCENTRATIONOF (! 4HE ADSORBED VACCINE CONTAINED  MG !L0/ML 2ESULTS SHOWED THATTHEFREQUENCIESOFANTIBODYINCREASEANDTHEANTIBODYLEVELSACHIEVED AFTERADMINISTRATIONOFUNADSORBEDORADSORBEDVACCINEINDICATEDTHATBOTH PREPARATIONSWEREANTIGENICALLYPOTENTWITHNOSIGNIFICANTDIFFERENCES4HESE RESULTS DEMONSTRATED THAT!L0/ FAILS TO EXERT AN ADJUVANT EFFECT ON (! VACCINESGIVENTOADULTHUMANSUBJECTS;= (OWEVER DESPITETHEMENTIONEDSCIENTIFICEVIDENCE DURINGTHESAND THESMANYINFLUENZAVACCINESCOMMERCIALLYAVAILABLEINBOTH%UROPE AND53EITHERWHOLEVIRUS SPLITVIRUSORSUBUNIT WEREALUMINUMADSORBED



-ARIA,ATTANZI

4ABLE!LUMINUMSALTSUSEDASADJUVANTINVACCINES;= #HEMICALFORMOF!L

#HARACTERISTICS

!LUMINUMHYDROXIDE!L/( 

#RYSTALLINEALUMINUMHYDROXIDEPOSITIVELYCHARGEDAT PHYSIOLOGICALP(;ISOELECTRICPOINTP) = )

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4ABLE!LUMINUMANDINFLUENZAVACCINES(ISTORICALVIEW %XAMPLESOFALUMINUM ADSORBEDINFLUENZAVACCINES COMMERCIALLYAVAILABLEDURINGTHES IN%UROPEOR53 7HOLEVIRUS

!LORBAT!34!,ABORATORIES "IELEFELD 'ERMANY !GRIPPAL)3643CLAVO 3IENA )TALY

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&LUVIRIN-EDEVA ,IVERPOOL 5+

4AB !LUMINUMSALTSWEREREMOVEDINTHEEARLYSBECAUSEAGROWING BODYOFEVIDENCESHOWEDTHATTHEADJUVANTDIDNOTINCREASEDIMMUNOGENIC ITYBUTSLIGHTLYINCREASEDREACTOGENICITY ESPECIALLYINCHILDREN;n= 4HOSEUNSUCCESSFULEARLYEXPERIENCESDIDNOTSTOPTHERESEARCHFORASAFE AND POTENT ADJUVANT TO BE USED NOT ONLY WITH INFLUENZA VACCINES BUT ALSO WITH OTHER HUMAN VACCINES $URING THE S EFFORTS TOWARD GENERATING POTENTBUTSAFEADJUVANTFORMULATIONSHAVECENTEREDONTHEDEVELOPMENTOF OIL BASEDADJUVANTS LIKE&#!WIDELYUSEDINPRECLINICALRESEARCH4OREDUCE THE TOXICITY ASSOCIATED WITH &REUNDS LIKE ADJUVANTS FORMULATIONS WERE DEVELOPEDTHATREDUCEDTHEAMOUNTOFOILFROMTOnANDREPLACED ALL OF THE &REUNDS COMPONENTS WITH SIMILAR ACTING BUT LESS TOXIC COMPO NENTS )N THE MOST SUCCESSFUL OF THESE FORMULATIONS -& THE MINERAL OIL OF&REUNDSADJUVANTWASREPLACEDWITHTHEMETABOLIZABLEOILSQUALENE AND THE !RLACEL ! SURFACTANT WAS REPLACED WITH CLEANER MORE DEFINED SURFAC TANTSSUCHAS4WEENAND3PAN4HEREDUCTIONOFTHEOILCONTENTTO RESULTS IN THE FORMATION OF OIL IN WATER EMULSIONS RATHER THAN WATER IN OIL EMULSIONS;= -& HAS BEEN EXTENSIVELY STUDIED IN PRECLINICAL AND CLINICAL MODELS WITHMANYDIFFERENTANTIGENS4HELARGESTEXPERIENCEISWITHSUBUNITINFLU ENZA ANTIGENS -& ADJUVANTED INFLUENZA SUBUNIT VACCINE WITH ENHANCED IMMUNOGENICITY HAS NOW BEEN AVAILABLE IN %UROPE FOR APPROXIMATELY  YEARS&IG 

.ON RECENTHISTORYOFINFLUENZAPANDEMICS VACCINES ANDADJUVANTS



#ONCLUSION -ANKIND HAS BEEN AFFECTED BY INFLUENZA EPIDEMICS AND PANDEMICS FOR MANYCENTURIES/VERTHECENTURIES WEHAVESEENATTEMPTSTODEVELOPSAFE ANDEFFICACIOUSVACCINES INCLUDINGTHENEGATIVEEXPERIENCEWITHTHESWINE INFLUENZAVACCINATIONANDTHENUMEROUS ATTEMPTSTOIMPROVETHEEFFICACYOF INFLUENZAVACCINESUSINGADJUVANTSCARRIEDOUTSINCETHES (OWEVER AS HASBEENTHECASEFORMANYOTHERSINFECTIOUSDISEASES ITWASONLYDURINGTHE THCENTURYTHATWEHAVESUCCEEDEDINDEVELOPINGMEASURESTOREDUCETHE HEAVYBURDENOFTHISDISEASETHROUGHPREVENTION

2EFERENCES   



    

     



'HENDON9 )NTRODUCTIONTOPANDEMICINFLUENZATHROUGHHISTORY %UR* %PIDEMIOLn L 7(/  &ACT SHEET NO  HTTPWWWWHOINTMEDIACENTREFACTSHEETS FSEN7EBSITEACCESSED3EPTEMBER &UKUDA+ ,EVANDOWSKI2! "RIDGES#" #OX.* )NACTIVATEDINFLUENZA VACCINES )N 3! 0LOTKIN 7! /RENSTEIN EDS  6ACCINES TH EDN 3AUNDERS 0HILADELPHIA n 0OTTER #7  #HRONICLE OF INFLUENZA PANDEMICS )N +' .ICHOLSON 2& 7EBSTER !* (AY EDS  4EXTBOOK OF )NFLUENZA "LACKWELL 3CIENCE /XFORD n (IRSCH !  (ANDBOOK OF 'EOGRAPHICAL AND (ISTORICAL 0ATHOLOGY .EW 3YDENHAM3OCIETY ,ONDON 0YLE'& 4HE$IFFUSIONOF)NFLUENZA0ATTERNSAND0ARADIGMS2OWAN ,ITTLEFIELD .EW*ERSEY "EVERIDGE 7)"  4HE CHRONICLE OF INFLUENZA EPIDEMICS (ISTORY 0HILOS ,IFE3CIn 0OTTER#7 !HISTORYOFINFLUENZA *!PPL-ICROBIOLn L &INKLER$ )NFLUENZAINTWENTIETHCENTURYPRACTICE)N4,3HIPMANED  !N)NTERNATIONAL%NCYCLOPAEDIAOF-ODERN-EDICAL3CIENCE3AMPSON,AW -ARSTON ,ONDON ABSTRACT n 0ATTERSON +$  0ANDEMIC )NFLUENZA n ! 3TUDY IN (ISTORICAL %PIDEMIOLOGY2OWMAN,ITTLEFIELD .EW*ERSEY "ROWN -7  %ARLY EPIDEMICS OF INFLUENZA IN!MERICA * -ED 2EC  n "EVERIDGE7)" )NFLUENZA4HE,AST'REAT0LAGUE(EINEMANN ,ONDON 0YLE'& 0ATTERSON+ )NFLUENZADIFFUSIONIN%UROPEAN HISTORY0ATTERNS ANDPARADIGMS %COL$ISn 4HOMPSON%3 )NFLUENZA0ERVICAL ,ONDON "URNET&- #LARK% )NFLUENZA!SURVEYOFTHELAST YEARSINTHELIGHT OFMODERNWORKONTHEVIRUSOFEPIDEMICINFLUENZA -ONOGR7ALTER%LIZA(ALL )NST2ES0ATHOL-ED n 3HOPE2% 3WINEINFLUENZA%XPERIMENTALTRANSMISSIONANDPATHOLOGY * %XP-EDn



-ARIA,ATTANZI

 (OPE 3IMPSON2 3UNSPOTSANDFLU!CORRELATION.ATURE  &RANCIS4*R -AGILL40 4HEANTIBODYRESPONSEOFHUMANSUBJECTSVAC CINATEDWITHTHEVIRUSOFHUMANINFLUENZA *%XP-EDn  &RANCIS4*R 4HEDEVELOPMENTOFTHEVACCINATION STUDYOFTHECOM MISSIONONINFLUENZA !M*(YGIENEn  &RANCIS4*R 3ALK*% 0EARSON(% "ROWN0. 0ROTECTIVEEFFECTOFVAC T CINATIONAGAINSTINDUCEDINFLUENZA! *#LIN)NVESTn  3ALK*% -ENKE7* &RANCIS4*R !CLINICAL EPIDEMIOLOGICALANDIMMU NOLOGICALEVALUATIONOFVACCINATIONAGAINSTEPIDEMICINFLUENZA !M*(YG G  n  'REGG-" 2ECENTADVANCESININFLUENZAVACCINES *-EDn  #ATE42 #OUCH2" 0ARKER$ "AXTER" 2ACTOGENICITY IMMUNOGENICITY AND ANTIBODY PERSISTENCE IN ADULTS GIVEN INACTIVATED INFLUENZA VIRUS VACCINES n 2EV)NFECT$ISn  3PILA !LEGIANI3 3ALMASO3 2OTA-# 4OZZI! 2ASCHETTI2 THE)TALIAN36%6! GROUP 2EACTOGENICITYINTHEELDERLYOFNINECOMMERCIALINFLUENZAVAC CINES2ESULTSFROMTHE)TALIAN36%6!STUDY6ACCINEn  +ENDAL !0 'OLDFIELD - .OBLE '2 $OWDLE 72  )DENTIFICATION AND PRELIMINARY ANTIGENIC ANALYSIS OF SWINE INFLUENZA LIKE VIRUSES ISOLATED DUR ING AN INFLUENZA OUTBREAK AT &ORT $IX .EW *ERSEY * )NFECT $IS  3UPPL  3n3  'OLDFIELD- "ARTLEY*$ 0IZZUTI7 "LACK(# !LTMAN2 (ALPERIN7% )NFLUENZAIN.EW*ERSEYIN)SOLATIONOFINFLUENZA!.EW*ERSEYVIRUS AT&ORT$IX *)NFECT$IS3UPPL 3n3  4OP &( 2USSELL 0+  3WINE INFLUENZA ! AT &ORT $IX .EW *ERSEY *ANUARYn&EBRUARY   )6 3UMMARY AND SPECULATION * )NFECT $IS  3UPPL 3n3  'AYDOS *# 4OP &( (ODDER 2! 2USSER 0+  3WINE INFLUENZA! OUT BREAK &ORT$IX .EW*ERSEY %MERG)NFECT$IS n  ,ANGMUIR!$ 'UILLAIN "ARRÒSYNDROME4HESWINEINFLUENZAVIRUSVAC CINEINCIDENTINTHE5NITED3TATESOF!MERICA nPRELIMINARYCOMMU NICATION*23OC-EDn  3CHONBERGER," "REGMAN$* 3ULLIVAN "OLYAI*: +EENLYSIDE2! :IEGLER$7 2ETAILLIAU(& %DDINS$, "RYAN*! 'UILLAIN "ARRÒSYNDROMEFOLLOW INGVACCINATIONINTHENATIONALINFLUENZAIMMUNIZATIONPROGRAM 5NITED3TATES n!M*%PIDEMIOLn  ,ANGMUIR!$ "REGMAN $* +URLAND ,4 .ATHANSON . 6ICTOR -  !N EPIDEMIOLOGIC AND CLINICAL EVALUATION OF 'UILLAIN "ARRÒ SYNDROME REPORTED IN ASSOCIATION WITH THE ADMINISTRATION OF SWINE INFLUENZA VACCINES !M * %PIDEMIOLn  ,ASKY4 4ERRACCIANO '* -AGDER , +OSKI #, "ALLESTEROS - .ASH $ #LARK 3 (ABER0 3TOLLEY0$ 3CHONBERGER," #HEN24 4HE'UILLAIN "ARRÒ SYNDROMEANDTHEnANDnINFLUENZAVACCINES .%NGL*-ED n  (ABER0 $E3TEFANO& !NGULO&* )SKANDER* 3HADOMY36 7EINTRAUB% #HEN 24  'UILLAIN "ARRÒ SYNDROME FOLLOWING INFLUENZA VACCINATION *!-! n

.ON RECENTHISTORYOFINFLUENZAPANDEMICS VACCINES ANDADJUVANTS



 3ALK *%  5SE OF ADJUVANTS IN STUDIES ON INFLUENZA IMMUNIZATION  $EGREEOFPERSISTENCEOFANTIBODYINHUMANSUBJECTSTWOYEARSAFTERVACCINA TION *!-!n  3ALK *% "AILEY -, ,AUREL !-  4HE USE OF ADJUVANTS IN STUDIES ON INFLUENZA IMMUNIZATION )) )NCREASED ANTIBODY FORMATION IN HUMAN SUBJECTS INOCULATED WITH INFLUENZA VIRUS VACCINE IN A WATER IN OIL EMULSION !M * (YG n  $AVENPORT&- 3EVENTEENYEARSEXPERIENCEWITHMINERALOILADJUVANT INFLUENZAVIRUSVACCINES !NN!LLERGYn  "EEBE'7 3IMON!( 6IVONA3 &OLLOW UPSTUDYON!RMY PERSONNEL WHORECEIVEDADJUVANTINFLUENZAVIRUSVACCINEn !M*-ED3CI n  "EEBE '7 3IMON !( 6IVONA 3  ,ONG TERM MORTALITY FOLLOW UP OF !RMYRECRUITSWHORECEIVEDADJUVANTINFLUENZAVIRUSVACCINEIN n !M *%PIDEMIOLn  (ENNESSY!6 $AVENPORT&- 2ELATIVEMERITSOFAQUEOUS ANDADJUVANT INFLUENZA VACCINES WHEN USED IN A TWO DOSE SCHEDULE 0UBLIC (EALTH 2EP  n  $AVENPORT &- (ENNESSY !6 "ELL *!  )MMUNOLOGIC ADVANTAGES OF EMULSIFIEDINFLUENZAVIRUSVACCINES -IL-EDn  $AVIS $* 0HILIP 2. "ELL *! 6OEGEL *% *ENSEN $6  %PIDEMIOLOGICAL STUDIESONINFLUENZAINFAMILIARANDGENERALPOPULATIONGROUPSn))) G ,ABORATORYOBSERVATIONS !M*(YGn  3TUART (ARRIS#( 3CIENTIFIC0UBLICATION.O 0!))/   $AVENPORT &-  !NTIGENIC ENHANCEMENT OF ETHER EXTRACTED INFLUENZA VIRUSVACCINESBY!)0/ 0ROC3OC%XP"IOL-EDn  $AVENPORT &- (ENNESSY !6 !SKIN &"  ,ACK OF ADJUVANT EFFECT OF !L0/ ON PURIFIED INFLUENZA VIRUS HEMAGGLUTININS IN MAN * )MMUNOLL  n  7ERNER * +UWERT %+ 3TEGMAIER 2 3IMBOCK (  ;,OCAL AND SYSTEMIC ANTIBODYRESPONSEAFTERVACCINATIONWITHDIFFERENTTYPESOFVACCINESAGAINST INFLUENZA)).EURAMINIDASEINHIBITINGANTIBODIESAUTHORSTRANSL = :ENTRALBL "AKTERIOL!n L  $%RRICO-- 'RASSO'- 2OMANO& -ONTANARO$ ;#OMPARISONOF ANTI INFLUENZAVACCINES7HOLEADSORBEDTRIVALENT TRIVALENTSUBUNITANDTETRA VALENTSUBUNIT="OLL)ST3IEROTER-ILANn  )ONITA % ,UPULESCU % !LEXANDRESCU 6 -ATEPIUC - #ONSTANTINESCU # #RETESCU,ETAL #OMPARATIVESTUDYOFTHEIMMUNOGENICITYOFAQUEOUS VERSUSALUMINIUMPHOSPHATEADSORBEDSPLITINFLUENZAVACCINE#) !RCH2OUM 0ATHOL%XP-ICROBIOLn L  6AN.EST'! 3TEIMER+3 (AIGWOOD., "URKE2, /TT' !DVANCED ADJUVANTFORMULATIONSFORUSEWITHRECOMBINANTSUBUNITVACCINES)N&"ROWN 2-#HANOCK (3'INSBERG 2!,ERNEREDS  6ACCINES#OLD3PRING(ARBOR ,ABORATORY0RESS #OLD3PRING(ARBOR n  "AYLOR.7 %GAN7 2ICHMAN0 !LUMINUMSALTSINVACCINESn53PER SPECTIVE6ACCINE 3n3

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7AITINGFORAPANDEMIC 2INO2APPUOLIAND'IUSEPPE$EL'IUDICE .OVARTIS6ACCINESAND$IAGNOSTICS 6IA&IORENTINA 3IENA )TALY

!BSTRACT !FTERAQUIETPERIODOFNEARLYYEARS INFLUENZASTRAINSWITH HEMAGGLUTININTYPESTHAT HAVENOTBEENSEENINHUMANSPREVIOUSLYSTARTEDTOJUMPFROMBIRDSTOMAN SUGGESTING THERISKOFANEWINFLUENZAPANDEMIC(OWEVER INCONTRASTTOTHESITUATIONWITHALLTHE OTHERINFLUENZAPANDEMICSOCCURRINGINTHETHCENTURYANDBEFORE INTHESTCENTURY WE HAVE SOPHISTICATED TECHNOLOGIES FOR DIAGNOSIS THERAPY AND PREVENTION -ODELING OF THE POSSIBLE SPREAD OF A PANDEMIC SUGGESTS THAT VACCINATION IS BY FAR THE ONLY WAY TO ELIMINATETHERISKOFANEWPANDEMIC)NTHISCHAPTERWEREVIEWTHEDEVELOPMENTOFNEW VACCINES AGAINST (. VIRUSES SHOWING THAT EFFECTIVE VACCINES ADJUVANTED WITH OIL IN WATEREMULSIONAREABOUTTOBELICENSEDANDWILLSOONBEAVAILABLE4HERACEAGAINSTAN INFLUENZAPANDEMICHASBEGUNITISABATTLEAGAINSTTIMETHAT MANKINDCANNOTAFFORDTO LOSE

)NTRODUCTION 3INCE  AT LEAST TEN INFLUENZA PANDEMICS HAVE OCCURRED WITH AN AVER AGEOFONEPANDEMICEVERYYEARS!NALYSISOFTHEMOSTRECENTANDMORE ACCURATE DATA PREDICTS ONE PANDEMIC EVERY  YEARS SEE THE CHAPTER BY ,ATTANZI 4HELASTPANDEMICWASIN YEARSAGOANDTHEREFORECOM MONSENSEANDMATHEMATICALMODELSPREDICTTHATWESHOULDBEPREPAREDFOR ANEWPANDEMIC$URINGTHELASTYEARS ALLTHEEVENTSTHATAREEXPECTEDTO HAPPENBEFOREAPANDEMICDIDHAPPEN&IRST ANEWVIRUSCARRYINGTHE( ANTIGEN THATHADNEVERBEENINHUMANSBEFOREJUMPEDFROMCHICKENSINTO MANANDKILLEDSIXPEOPLEIN(ONG+ONGIN4HISEARLYOUTBREAKWAS CONTAINED BY CULLING CHICKENS IN THE (ONG +ONG AREA4HE VIRUS MOMEN TARILYDISAPPEAREDANDWEFORGOTABOUTITFORAFEWYEARS(OWEVER THEVIRUS WASNOTDEADATALLITWASSUCCESSFULLYBREADING MULTIPLYING ANDEXPANDING INBIRDSIN3OUTH%AST!SIA;= UNTILITSUDDENLYBLEWUPAGAINWITHHUMAN CASESINANDIN6IETNAM 4HAILAND )NDONESIA AND#HINA#LEARLY



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7AITINGFORAPANDEMIC



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7AITINGFORAPANDEMIC



4HEWILD TYPEWHOLEINACTIVATEDVIRUSGROWNINCELLCULTURE"AXTER "AXTERTOOKTHEUNUSUALAPPROACHOFUSINGFULLYVIRULENTWILD TYPE(.VIRUS ESGROWNIN6EROCELLSTOMAKE(.VACCINES4HERATIONALEPROVIDEDISTHAT USINGAREVERSEGENETICPROCEDURE WHICHDELIVERSANATTENUATED NON DANGEROUS VIRUS THAT CAN BE GROWN IN BIOSAFETY LEVEL  LABORATORIES TAKES n WEEKS TO GENERATETHEINITIALVIRUSSEED WHILETHEWILD TYPEVIRUSCAN BEUSEDIMMEDI ATELYFORMANUFACTURING4HECAUTIONWITHTHISAPPROACHISTHATTHOUSANDSOF LITERSOFWILD TYPE(.VIRUSEVENIFGROWNUNDERABIOSAFETYLEVELSREPRE SENTANEXTREMELYDANGEROUSPROCEDURE ANDINCASEOFLEAKAGEMAYBEENOUGH TOCAUSEAPANDEMICINITSELF!6IETNAM !)NDONESIAAND OTHERVIRUSESWEREPRODUCEDUSINGTHISPROCEDURE;=4HEVIRUS WASFIRSTINAC TIVATEDBYADOUBLEPROCEDUREINVOLVINGFORMALDEHYDEAND56TREATMENTAND THEN PURIFIED BY SUCROSE GRADIENT ULTRACENTRIFUGATION FOLLOWED BY DIAFILTRA TION4HE VACCINE WAS IMMUNOGENIC IN PRECLINICAL STUDIES PROTECTIVE IN MICE INDUCED4 CELL RESPONSES AND WAS CROSS PROTECTIVE 0RELIMINARY CLINICAL DATA HAVEBEENREPORTEDATMEETINGSBUTNOPUBLISHEDDATAAREAVAILABLEYET

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7AITINGFORAPANDEMIC



NON VIRULENT DUCK (. VIRUS THAT COULD GROW IN CHICKEN EGGS ;=4HIS (.VACCINEWASAVERYINTERESTINGEXPERIENCETHATPROVIDEDMOSTOFTHE USEFUL INFORMATION WE HAVE TODAY AND PAVED THE WAY TO FURTHER VACCINE DEVELOPMENT4HISVACCINEWASTESTEDINHUMANADULTVOLUNTEERS WITHAND WITHOUTTHEADJUVANT-&SEETHECHAPTERBY/(AGAN0ODDA AT  AND+GPERDOSELICENSEDVACCINESCONTAIN+GDOSE 4HEDATAOFTHE TRIALREPORTEDIN&IGINTHECHAPTERBY-ONTOMOLI WEREVERYSURPRISING 4AB   &IRST THE CONVENTIONAL NON ADJUVANTED VACCINE DID NOT ELICIT A SIGNIFICANTPROTECTIVEANTIBODYRESPONSENO()RESPONSEANDONLYn -. )NRETROSPECTIVEWESHOULDNOTBETOOSURPRISEDBYTHISRESULTWHILE WEUSEONEDOSEOFSEASONALINFLUENZAVACCINEINPEOPLETHATALREADYHAVE IMMUNITYAGAINSTINFLUENZA INTHECASEOF( PEOPLEARENAIVETOTHISANTI GENANDTHEREFORETHEVACCINATIONNEEDSTOPRIMETHEIMMUNESYSTEMBEFORE ITCANINDUCEAHIGHANTIBODYRESPONSE4HEREALLYSURPRISINGFINDINGINSTEAD F WASTHATTHEVACCINEADMINISTEREDWITHTHENEWLYLICENSED-&ADJUVANT DID INDUCE A GOOD IMMUNE RESPONSE AFTER ONE DOSE AND A SEROCONVERSION BY -. AND 32( IN NEARLY ALL SUBJECTS AFTER TWO DOSES )NTERESTINGLY THE HIGHESTIMMUNERESPONSEWASACHIEVEDWITHTHE+GDOSE WHICHWASTHE LOWESTUSEDINTHETRIAL;=!TMONTHSAFTERTHEPRIMARYIMMUNIZATION THETESTPERSONSWEREGIVENABOOSTWITHTHESAMEVACCINE!GAINTHESUB JECTSVACCINATEDWITHTHE-& ADJUVANTEDVACCINERESPONDEDWITHAVERY STRONGANDLONG LASTINGIMMUNERESPONSE WHILETHOSEVACCINATEDWITHTHE NON ADJUVANTED VACCINE SHOWED STILL A VERY LOW BUT DETECTABLE PROTECTIVE ANTIBODY RESPONSE ;= 7HEN THE (. VIRUS STARTED TO CIRCULATE AGAIN IN4HAILAND AND6IETNAM IN  AND  IT WAS INTERESTING TO GO BACK TO THE SERA OBTAINED FROM THE TRIAL AND ASK WHETHER THE PEOPLE THAT HAD BEENIMMUNIZEDWITHTHE(.VIRUSHADANTIBODIESABLETONEUTRALIZETHE (.CLADEVIRUSESDERIVINGFROMANANTIGENICDRIFTINGTHATHADBEENTAK INGPLACEFORnYEARS&IGURESHOWSTHAT WHILETHESUBJECTSVACCINATED WITHTHENON ADJUVANTEDVACCINESHADNOCROSS CLADENEUTRALIZINGANTIBOD IES THEMAJORITYOFTHESUBJECTSVACCINATEDWITH-& ADJUVANTEDVACCINE HAD PROTECTIVE LEVELS OF ANTIBODIES AGAINST THE HETEROLOGOUS CLADE  VIRUS ISOLATEDIN6IETNAMAND4HAILAND; =)NSUMMARY THEDATAHADSHOWN THAT -& ADJUVANTED VACCINES COULD INDUCE PROTECTIVE IMMUNITY AGAINST VIRUSESNOTMATCHINGTHEVACCINESTRAINANDCOULDCOVERTHEANTIGENICDRIFT OFTHEVIRUSFORnYEARS&INALLY MORERECENTLY THEPEOPLE VACCINATEDWITH (.INANDBOOSTEDWITH(.IN WEREVACCINATEDINWITH ACLADEVACCINE0RELIMINARYDATASHOWTHATTHOSEWHOHADBEENPRIMED WITHTHE-& ADJUVANTED(.VACCINEPRODUCEDVERYHIGHLEVELOFANTI BODIES AGAINST THE CLADE  STRAIN BY  DAYS AFTER VACCINATION WHILE THOSE PRIMEDWITHOUTADJUVANTALSOHADARESPONSEBUTOFMUCHLOWERMAGNITUDE 0EOPLE THAT HAD NOT BEEN PRIMED RESPONDED AS EXPECTED ONLY AFTER TWO DOSESOFVACCINEANDREACHEDPROTECTIVELEVELSONLYDAYSAFTERTHEFIRST IMMUNIZATION4HISLATTERSTUDYPROVIDEDEXTREMELYIMPORTANTINFORMATION SHOWING THAT WHEN PEOPLE ARE PRIMED WITH ANY (. STRAIN ADJUVANTED

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7AITINGFORAPANDEMIC



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2INO2APPUOLIAND'IUSEPPE$EL'IUDICE

INFLUENZA VACCINE MANUFACTURING CAPACITY APPROXIMATELY  - DOSES OF TRIVALENTVACCINEYEAR WASABLETOPROVIDEAVACCINEFORNOTMORETHAN MILLIONPEOPLEYEAR4HISVACCINEHASBEENLICENSEDINTHE53! BY&$!

3PLITVACCINEADJUVANTEDWITHALUM3ANOFI0ASTEUR )N*ULY3ANOFI0ASTEURPERFORMED ATRIALIN%UROPEUSINGASIMILARSPLIT VACCINEINARANDOMIZED MULTICENTER OPENLABELTRIALINSUBJECTSAGED nYEARS;=4HESTRAINUSEDWASINFLUENZA!(.6IETNAMPRE PAREDBYREVERSEGENETICSBYTHE.ATIONAL)NSTITUTEFOR"IOLOGICAL3TANDARDS AND#ONTROL.)"3# 4HEVACCINEWASADJUVANTEDBYALUMINUMHYDROXIDE 4HREEDOSES AND+GWEREINJECTEDIMATDAYSAND AFURTHER BLOODSAMPLEWASOBTAINEDATDAY()AND-.WEREMEASUREDASINTHE PREVIOUSTRIAL4HERESULTSWEREDISAPPOINTING.EITHERTHEVACCINEWITHOUT THEADJUVANTNORTHEONEADJUVANTEDWITHALUMWEREABLETOINDUCESUBSTAN TIAL()OR-.SEROCONVERSIONS4AB 3OMEDOSE DEPENDENTIMMUNOGE NICITYWASOBSERVEDHOWEVER SEROCONVERSIONWASUSUALLYACHIEVEDINLESS THANOFTHECASESANDTHEONLYGROUPTHATHADAN()SEROCONVERSION ABOVEWASTHEONEWITH+GANDALUM(OWEVER THISGROUPHADVERY LOWSEROCONVERSIONIN-. RAISINGQUESTIONSONTHEHOWTHE()TITERSSHOULD BEINTERPRETED)NCONCLUSION THISSTUDYCONFIRMEDWHATHADALREADYBEEN WELL DESCRIBED IN THE S AND S THAT IS THAT THE ALUMINUM IS NOT A GOODADJUVANTFORTHEINFLUENZAVACCINESEETHECHAPTERBY,ATTANZI 

!3ADJUVANTED(.SPLITVACCINE'LAXO3MITH+LINE 4HESPLITVACCINEWASMADEUSINGTHESTRAIN!6IETNAM .)"2'  GROWN IN EGGS4HE STRAIN WAS GENERATED BY REVERSE GENETICS AND CON TAINEDTHE(!AND.!GENESEGMENTSFROMTHE(.6IETNAMAND THE OTHER GENE SEGMENTS FROM THE!02 STRAIN )N THIS CASE AS WELL THE(!GENEWASENGINEEREDTOREMOVETHESIXBASICAMINOACIDS SOTHAT THE STRAIN COULD BE GROWN UNDER BIOSAFETY LEVEL  CONDITIONS 4HE 4 TRIAL STARTED IN -ARCH  AND  SUBJECTS n YEARS OLD WERE ENROLLED IN ANOBSERVER BLIND RANDOMIZEDTRIAL;='ROUPSOFRECEIVED    OR +GOFVACCINEALONEORADJUVANTEDWITHTHE!3OILINWATEREMUL SION  DL _ TACOPHEROL AND  SQUALENE OIL IN WATER CONTAINING  4WEEN ACOMPOSITIONVERYSIMILARTOTHATOF-&SEETHECHAPTERBY /(AGAN0ODDA 4WOIMDOSES ATDAYSANDWEREGIVEN"LOODWAS ALSOTAKENATDAY/VERALL SAFETYWASFINE WITHLOCALPAIN BEINGTHEMOST FREQUENTLY REPORTED SYMPTOM AND WAS DOSE DEPENDENT IN UP TO  OF THENON ADJUVANTEDGROUPSANDDOSEINDEPENDENTINVIRTUALLYALL SUBJECTS n IN THE ADJUVANTED GROUPS () AND -. WERE MEASURED AGAINST THE HOMOLOGOUS VACCINE STRAIN AND ALSO AGAINST THE HETEROLOGOUS CLADE 

7AITINGFORAPANDEMIC



!)NDONESIA STRAIN !S SHOWN IN 4ABLE  DATA OBTAINED WITH THE !3 ADJUVANTEDVACCINEWEREVERYSIMILARTOTHOSEPREVIOUSLYDESCRIBED FORTHE-& ADJUVANTED(.VACCINESEROCONVERSIONMEASUREDBY -. AND()WASACHIEVEDINnANDnOFTHESUBJECTS RESPECTIVELY INDEPENDENTLYOFTHEDOSEUSED4HEDATAOBTAINEDWITHTHEVACCINEWITHOUT ADJUVANTWEREASDISAPPOINTINGASTHOSEPREVIOUSLYOBTAINEDWITHTHENON ADJUVANTED(.AND(.VACCINESSEROCONVERSIONWASDOSEDEPENDENT ANDREACHEDAMAXIMUMOFANDIN -. AND() RESPECTIVELY AT THEMAXIMUMDOSEUSED)NTERESTINGLY INTHISCASE #(-0CRITERIAOFSERO CONVERSION RATES IN AT LEAST  OF THE SUBJECTS WERE ACHIEVED ALSO AFTER THEFIRSTDOSEINTHOSESUBJECTSTHATRECEIVED +GORMOREOFADJUVANTED VACCINE4HEVACCINEWASALSOSHOWNTOINDUCESEROCONVERSIONAGAINSTTHE HETEROLOGOUS CLADE  )NDONESIA STRAIN WHICH WAS OBSERVED IN n OF THESUBJECTSWHENUSINGTHE -. BUTCOULDBEDETECTEDINONLYnOF THESUBJECTSWHENUSINGTHE()

3UBUNITVACCINEADJUVANTEDWITH-&.OVARTIS 4HE SUBUNIT VACCINE WAS PRODUCED IN EGGS USING THE STRAIN ! 6IETNAM .)"2'  AND ACCORDING TO THE PROCEDURES USED FOR THE SEASONAL INFLUENZA VACCINE ;=  AND  +G WERE USED TO IMMUNIZE ADULTSANDELDERLYSUBJECTSIM ATDAYSAND!BLOODSAMPLE WASTAKENALSOATDAY4ABLESHOWSTHATBOTHDOSESINDUCEDSEROCONVER SIONINTHEMAJORITYOFTHESUBJECTSnOFTHEADULTSAND nOF THEELDERLYSEROCONVERTEDBY-.OR32( 4HESEDATACONFIRMEDTHATTHE DATAOBTAINEDWITH(.ALSOAPPLYTO(.4HESEAREALSOTHE FIRSTDATA AVAILABLE OF THE IMMUNOGENICITY OF THE VACCINE IN THE ELDERLY POPULATION ANDTHEYINDICATETHATTHEADJUVANTEDVACCINEISSUITABLEFORTHEIMMUNIZA TIONOFYEARSANDOLDERSUBJECTS;=

7HOLEINACTIVATEDVIRUSVACCINEADJUVANTEDWITHALUMINUMHYDROXIDE 3INOVAC 4HESTRAIN!6IETNAM .)"2' GROWNINEGGS INACTIVATEDBY FORMALIN CONCENTRATED AND PURIFIED BY CHROMATOGRAPHY AND FORMULATED WITH ALUMINUM HYDROXIDE WAS USED TO IMMUNIZE  SUBJECTS ;= 4HE RANDOMIZED PLACEBO CONTROLLED DOUBLE BLINDSTUDY STARTEDIN *ULYIN nYEARSOLDSUBJECTSIN"EIJING #HINA  AND +GANTIGENPER DOSEWEREUSEDFORIMMUNIZATIONATDAYAND!BLOODSAMPLEWASALSO OBTAINEDATDAY4HEVACCINEWASREPORTEDTOBESAFEHOWEVER THENUM BEROFADVERSEEVENTSREPORTEDISSOLOWTHATITISLIKELYTHATTHESTANDARDSFOR REPORTINGINTHISTRIALWEREDIFFERENTFROMTHEOTHERTRIALS4HE()TITERSWERE 4 MEASUREDUSINGTURKEYERYTHROCYTES ANDTHEREFORECANNOTBECOMPAREDTO



2INO2APPUOLIAND'IUSEPPE$EL'IUDICE

ANY OF THE OTHER DATA )N ANY CASE A DOSE DEPENDENT SEROCONVERSION WAS OBSERVEDINnOFTHESUBJECTS4AB -.SEROCONVERSIONOCCURREDIN ONLYnOFTHESUBJECTS3OFAR THISISTHEONLYPUBLISHEDTRIALUSINGWHOLE INACTIVATEDVIRUS4HEDATAASJUDGEDFROMTHE-.SEROCONVERSION ARENOT VERY EXCITING ! SECOND WHOLE VIRUS VACCINE PRODUCED BY THE (UNGARIAN #ENTERFOR!LLERGYAND)MMUNOLOGYREPORTEDSEROCONVERSIONBY() (OWEVER EVENINTHISCASE()WASMEASUREDBYCHICKENERYTHROCYTESAND THEREFOREDATACANNOTBECOMPAREDWITHTHEOTHERS;=-ANYOTHERCLAIMS WERE MADE WITH WHOLE INACTIVATED VIRAL VACCINES BY SEVERAL INDEPENDENT GROUPS(OWEVER SOFAR NONEOFTHEMHASPROVIDEDROBUSTDATAABLETOSUP PORTTHECLAIMSMADEOFTENINPRESSRELEASES

(.VACCINEADJUVANTEDWITH-&.OVARTIS 4HEVACCINESTRAINWASAREASSORTANTCONTAININGTHE(!AND.!GENESEG MENTSFROMTHEINFLUENZA!CHICKEN(ONG+ONG'ANDTHEREMAINING GENESEGMENTSFROMTHE!02STRAIN4HEVIRUSWASGROWNINEGGSAND THE VACCINE PRODUCED ACCORDING TO THE PROCEDURES USED FOR THE SEASONAL SUBUNITVACCINE)N!PRIL SUBJECTSPERGROUP RECEIVED   OR+GVACCINEWITHORWITHOUT-&ADJUVANT)MMUNIZATIONWASIM ATDAYSAND!FURTHERBLOODSAMPLEWASTAKENATDAY 4HERESULTS REPORTEDIN4ABLE CONFIRMEDTHEDATAOBTAINEDWITHALLTHE OTHER TRIALS PERFORMED WITH (. AND (. ;n= "ASICALLY NEARLY ALL SUBJECTS IMMUNIZED WITH THE ADJUVANTED VACCINE ACHIEVED SEROCONVERSION BOTHIN()AND-. INDEPENDENTLYOFTHEDOSEUSED SHOWINGTHAT +G ARESTILLABLETOINDUCE AFULLIMMUNERESPONSE)NMARKEDCONTRAST THENON ADJUVANTEDVACCINESHOWEDAMUCHLOWER DOSE DEPENDENTRESPONSE;=

0OLICY $URING THE n PERIOD WE HAVE SEEN THE MOST DIVERSE REACTIONS OF POLICYMAKERS TOWARDS THE POTENTIAL RISK OF A PANDEMIC INFLUENZA 4HE APPROACHES HAVE BEEN FROM TOTALLY IGNORING THE PROBLEM n TO PANICn TOAMORERECENTBALANCEDRATIONALAPPROACH"ELOWARE THE POSSIBLE OPTIONS THAT POLICYMAKERS HAVE! SUMMARY OF THE DIFFERENT OPTIONSISREPORTEDIN&IGURE

/PTIONSAND6ACCINATINGPANDEMICSURVIVORS /PTIONSISBASEDONTHESTRATEGYTOWAITFORTHE7(/TODECLARETHEBEGIN NINGOFTHEPANDEMIC IDENTIFYTHESTRAINCAUSINGTHEPANDEMIC RUSHTOMANU FACTURE THE VACCINE AND THEN VACCINATE PEOPLE WITH TWO DOSES OFF VACCINE

7AITINGFORAPANDEMIC



&IGURE  /PTIONS AVAILABLE TO POLICYMAKERS TO VACCINATE AGAINST A PANDEMIC INFLUENZA VIRUS /PTIONSnAREDESCRIBEDINTHETEXT9ELLOWTRIANGLESSHOWIMMUNIZATIONSBEFORETHEBEGIN NING OF THE PANDEMIC GREEN TRIANGLES INDICATE VACCINATION AFTER THE BEGINNING OF THE PAN DEMIC

/PTIONISTOSTOCKPILEENOUGHVACCINETOADMINISTERONEDOSE WHEN7(/ DECLARESTHEPANDEMICANDTHENMANUFACTURETHEVACCINEUSINGTHEPANDEMIC STRAINANDGIVETHESECONDDOSE#LEARLYTHESEOPTIONSHAVETHEINSURMOUNT ABLE LIMIT THAT THE VACCINATION CAMPAIGN BECOMES EFFECTIVE WHEN IT IS TOO LATE ANDTHEREFOREVACCINATIONCANONLYHELPTHOSEPEOPLETHAT HAVEALREADY SURVIVED THE PANDEMIC )N FACT WHILE ALL MODELING STUDIES ;n= PREDICT THATINONLYMONTHSAPANDEMICWILLSWEEPACOUNTRY PREPARINGANDDELIV ERINGTWODOSESOFVACCINESWILLTAKEATLEASTnMONTHS INDEPENDENTLYOF THECAPACITYANDTECHNOLOGYAVAILABLEnWEEKSTOPREPARETHESEEDSTRAIN n MONTHS TO MANUFACTURE ANY SIGNIFICANT AMOUNT OF VACCINE  MONTH TO BUILDIMMUNITY 4HESETWOOPTIONSHAVEBEENVERYPOPULARBETWEEN AND  WHEN VACCINES HAD NOT YET BEEN TESTED IN CLINICAL TRIALS SAFETY DATAWERENOTYETAVAILABLE ANDCROSSPROTECTIONHADNOTBEEN CONVINCINGLY DEMONSTRATED"OTHOPTIONSAREBASEDONTHEASSUMPTIONTHATTHEVACCINES NEEDTOBEMADEUSINGTHESAMESTRAINCAUSINGTHEPANDEMICANDTHATCROSS PROTECTIONISNOTEFFECTIVE/PTIONHASTHEADDITIONALDISADVANTAGETHATIF THESTRAINCAUSINGTHEPANDEMICISOFADIFFERENTCLADEOFTHEONESTOCKPILED ITWOULDBENOTUSEFUL4ODAYWEKNOWTHAT WHILENON ADJUVANTEDORALUM ADJUVANTEDVACCINESHAVENOTBEENSHOWNTOINDUCECROSSPROTECTIONAMONG



2INO2APPUOLIAND'IUSEPPE$EL'IUDICE

DIFFERENT CLADES OF (. THE VACCINES ADJUVANTED WITH OIL IN WATER EMUL SIONSHAVEBEENSHOWNTOINDUCECROSSPROTECTION ANDTHEREFOREITISPOSSIBLE TOVACCINATEWITHOUTWAITINGFORTHESTRAINTHATCAUSESTHEPANDEMIC

/PTIONSAND0RE PANDEMICVACCINATION /PTIONISTOVACCINATEPEOPLEWITHONEDOSEOFADJUVANTEDVACCINEBEFORE THEPANDEMICSTARTSANDTHENTOGIVEABOOSTWITHTHEPANDEMICSTRAINONCE THE PANDEMIC STARTS 4HIS OPTION IS BASED ON THE OBSERVATION THAT WHILE PRIMING WITH ONE DOSE IS NOT ABLE TO PRODUCE A FULLY PROTECTIVE AND LONG LASTING IMMUNITY IT WILL BE ABLE TO PRIME THE IMMUNE SYSTEM AND INDUCE AMEMORYTHATCANBEBOOSTEDLATERWITHTHEPANDEMICSTRAIN4HISOPTION ALTHOUGHNOTIDEALWOULDHAVETHEGREATMERITTOPRIMETHEIMMUNESYSTEM AGAINSTAN(VIRUSSOTHATTHEBASICASSUMPTIONSFORPANDEMIC TOHAPPEN WHICHISTHECIRCULATIONOFTHEVIRUSINANUNPRIMEDPOPULATION WOULDBE ELIMINATED)FOPTIONWASADOPTED WESHOULDNOLONGERHAVETOFEARAPAN DEMICCAUSEDBYAN(VIRUS BECAUSETHISVIRUSWOULDCIRCULATEINAPRIMED POPULATIONANDINTHEWORSECASESCENARIOTHEVIRUSWOULDBEABLETOCAUSE ONLYABADINFLUENZASEASON /PTIONISTOINDUCEFULLYPROTECTIVEANDCROSSPROTECTIVETITERSOFANTI BODIESANDMEMORY"AND4CELLSBYVACCINATINGTHEPOPULATIONWITHTWO DOSES OF ADJUVANTED VACCINE USING ANY ( VIRUS AND STILL HAVE THE OPTION TO BOOST THE IMMUNITY WITH A PANDEMIC VACCINE WHEN THE PANDEMIC IS DECLARED4ODAY THISISPOSSIBLEUSINGTHEVACCINESADJUVANTEDWITH-&OR !3)NFACT THESEVACCINESHAVEBEENSHOWNTOINDUCEHIGHLY PROTECTIVE LEVELOFANTIBODIESAGAINSTTHEVACCINESTRAIN WHICHARECROSSREACTIVEWITH STRAINS FROM DIFFERENT CLADES AND TO INDUCE A MEMORY THAT WHEN BOOSTED BYANYDISTANTLYRELATED(VIRUSITPROVIDES INJUSTDAYS AFULLYPROTEC TIVERESPONSEAGAINSTTHENEWVIRUS4HISIMPLIESTHATREGARDLESSOFTHEVIRUS STRAINUSEDFORTHEPRE PANDEMICVACCINATION THEAPPROPRIATEIMMUNITYWILL BETHEREWHENNEEDED)NFACT THEBOOSTCANBEPROVIDEDBYANEWIMMUNI ZATIONASSHOWNIN&IGURE ORBYTHEINFECTIONWITHTHEPANDEMICSTRAINTHAT WILLBEABLETOINDUCEAPROTECTIVEIMMUNITYBEFOREITINDUCES THEDISEASE )NCONCLUSION PRE PANDEMICVACCINATIONISTHEONLYOPTIONTHATWEHAVE TOMAKESURETHATWEAREINTHEPOSITIONTOPREVENTANDCONTROLTHERISKOFA PANDEMIC)NTHISWAY THEVACCINEWOULDBLOCKTHEDISEASEORWOULDSIGNIFI CANTLYAFFECTITSMORBIDITYMORTALITY

"ARRIERSTOPRE PANDEMICVACCINATION )FPRE PANDEMICVACCINATIONHASTHEPOTENTIALTOELIMINATETHERISKOFAPAN DEMIC WHYAREPOLICYMAKERSNOTRUSHINGTOIMPLEMENTIT4HERE ARESEVERAL BARRIERSBETWEENTHEPOLICYMAKERSANDTHEIMPLEMENTATION4HEFIRSTONEIS F

7AITINGFORAPANDEMIC



THATPRE PANDEMICVACCINESHAVENOTYETBEENLICENSEDANDTHEREFORETHEY ARENOTYETAVAILABLE4HISBARRIERISABOUTTODISAPPEARBECAUSETHE.OVARTIS AND'3+VACCINESARELIKELYTOBELICENSEDIN%UROPEINTHENEARFUTUREAND WILLBESUBMITTEDFORREGISTRATIONINTHE53!SHORTLY4HESECONDBARRIERHAS BEENTHAT WHILECROSSPROTECTIONHADBEENAMPLYDEMONSTRATEDWITH(. AND IN PRE CLINICAL STUDIES HUMAN DATA ON (. WERE NOT YET AVAILABLE 4ODAY THESEDATAAREAVAILABLEANDSTRONG0ERHAPSTHESTRONGESTDATADERIVE FROMTHEBOOSTINUSINGACLADEVIRUSOFTHEPEOPLEIMMUNIZEDIN ANDWITH(.4HESEDATASHOWTHATWHENTHEAPPROPRIATEMEMORY ISPRESENT AFEWDAYSAFTERBOOSTARESUFFICIENTTOINDUCEAFULLYPROTECTIVE F RESPONSEAGAINSTADRIFTEDVIRUS4HISCANBE 4 ACHIEVEDBYVACCINATINGWITHAN ADJUVANTEDVACCINEORBYINFECTION )N CONCLUSION THE TECHNICALLY SOUND BARRIERS TO PRE PANDEMIC VAC CINATION ARE DISAPPEARING AND WE SEE NO REASON WHY THIS SHOULD NOT BE IMPLEMENTED (OWEVER THERE ARE PROBABLY EVEN MORE IMPORTANT BARRIERS THATARELIKELYTOBEINTHEWAYOFMAKINGTHERIGHTDECISIONS4HEFIRSTAND MOSTIMPORTANTISTHATADECISIONTOACTIVELYVACCINATEPEOPLEIMPLIESTAK INGARISK WHILEWAITINGISNOTRISKYFORTHEPOLICYMAKERS(OWEVER ITISA RISKFORTHEPOPULATION 4HEMAJORFEAROFTHEPOLICYMAKERSIS SAFETY7HAT HAPPENSIFTHEYRECOMMENDAVACCINATIONANDTHENWEHAVEASAFETYPROB LEMWITHTHEVACCINE4HISISREASONABLECONCERNTHATSHOULDBEADDRESSED 0OLICYMAKERSLOOKATTHESWINEFLUEXPERIENCEOFSEETHE CHAPTERBY ,ATTANZI AND THEY DO NOT WANT TO FIND THEMSELVES IN A SIMILAR SITUATION 4HEYAREABSOLUTELYRIGHTINTHISNOCOMPROMISEISACCEPTABLEFORSAFETYIN APRE PANDEMICSITUATION/NTHEOTHERHAND THEREAREANUMBEROFREAS SURING DATA THAT SHOULD MAKE POLICYMAKERS CONFIDENT &IRST THE SWINE FLU VACCINATIONWASMOSTLYDONEWITHAWHOLEINACTIVATEDVIRUS WHICHATTHAT TIMEWASKNOWNTOBENOTPUREANDTOINDUCEALOTOFSIDEEFFECTS4HATIS THEREASONWHYSPLITANDSUBUNITVACCINESWEREDEVELOPEDANDREPLACEDTHE WHOLEVIRUSINSEASONALVACCINES 4ODAY WEHAVETHEOPTIONTO USEVACCINES THATARENOTBASEDONWHOLE INACTIVATEDVIRUS3ECOND THESAFETYOFSOME OFTHEADJUVANTSHASBEENAMPLY DEMONSTRATED-&HASBEENSAFELYGIVEN TOMILLIONPEOPLE ANDPASSIVESURVEILLANCESHOWEDTHATTHEREISNORISK OF'UILLAIN"ARRÏREPORTEDCASESWERESIMILARTOTHOSEOFNON ADJUVANTED VACCINES  )N CONCLUSION ALL THE BARRIERS TO PRE PANDEMIC VACCINATION ARE DISAPPEARINGANDWEARECONFIDENTTHATOURGENERATIONWILLBETHEFIRSTONE INCENTURIESTHATWILLBEABLETOPREVENTANINFLUENZAPANDEMIC

2EFERENCES 



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7AITINGFORAPANDEMIC



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!BSTRACT -ODELING IS AN IMPORTANT ASPECT OF PANDEMIC PREPAREDNESS AND DIFFERENT APPROACHES HAVE BEEN CONDUCTED TO DATE 4HE EPIDEMIC DYNAMICS AND THE ASSESSMENT OF CONTAIN MENTORMITIGATIONSTRATEGIES SUCHASVACCINATION BORDERRESTRICTION ANTIVIRALTREATMENT OFCASESANDPROPHYLAXISOFHOUSEHOLDORSCHOOLWORKPLACECONTACTSOFINDEXCASESCAN BE PREDICTED BY EMPLOYING CLASSICAL COMPARTMENTAL MODELS ;SUSCEPTIBLE EXPOSED BUT NOTYETINFECTIOUS INFECTIOUS RECOVEREDANDNOLONGERSUSCEPTIBLE3%)2 POSSIBLYWITH AGEANDORGEOGRAPHICCOMPONENT=/THERAUTHORSHAVEIMPLEMENTEDTHEEVALUATIONOF REALISTIC INDIVIDUALLYTARGETED PUBLICHEALTHINTERVENTIONSTRATEGIESTHATREQUIRESHIGHLY DETAILED MODELS SUCH AS THE INDIVIDUAL BASED MODEL )"-  !CCORDING TO THE PREDIC TIVEMODELSUSED ANINFLUENZAPANDEMICWOULDSPREADWORLDWIDE OVERAPERIODOFn MONTHS DEPENDINGONTHEBASICREPRODUCTIVENUMBERAMEASUREONHOWMANYPEOPLE ANINFECTIOUSINDIVIDUALINFECTSON AVERAGE ANDREDUCINGTRANSMISSIONWOULDENTAILCOM BININGCONTROLMEASURES SPECIFICALLY REDUCINGCONTACTSANDPERFORMINGBOTHTHERAPEUTIC ANDPROPHYLACTICUSEOFANTIVIRALSANDVACCINATION)NTERNATIONALBORDERRESTRICTIONSARE UNLIKELY TO DELAY THE SPREAD BY MORE THAN n WEEKS UNLESS MORE THAN  EFFECTIVE 3IMILAR RESULTS WERE OBTAINED FOR SOCIAL DISTANCING MEASURES SUCH AS SCHOOL CLOSURE 4REATMENT OF CLINICAL CASES AND PROPHYLAXIS OF CLOSE CONTACTS CAN LOWER TRANSMISSION REDUCING THE CLINICAL ATTACK RATE BY n BUT IT WOULD BE LOGISTICALLY CHALLENGING REQUIRINGASTOCKPILEFORMORETHANOFTHEPOPULATION6ACCINESTOCKPILEDINADVANCE EVENIFLESSEFFICACIOUSTHANPANDEMICVACCINES COULDSIGNIFICANTLYREDUCETHECUMULATIVE ATTACKRATES)N)TALY THECOMBINATIONOFTHEDESCRIBEDMEASURESWOULDLEADTOnMIL LIONOFCASESBEINGAVOIDED DEPENDINGONVACCINEEFFECTIVENESS TAKENASOR  -ATHEMATICALMODELSARENECESSARYTOPLANANDEVALUATEINTERVENTIONSBASEDONDIFFER ENTSTRATEGIES4HEYREPRESENTARELEVANTTOOLTOASSISTPUBLIC HEALTHDECISION MAKINGIN PREPARINGTHERESPONSETOANEWINFLUENZAPANDEMIC

)NTRODUCTION 4HREE INFLUENZA PANDEMICS OCCURRED IN THE TH CENTURY4HEIR MORTALITY IMPACT RANGED FROM DEVASTATING THE  @3PANISH !(. INFLUENZA TOMODERATETHE!SIAN!(.PANDEMIC TORATHERMILDTHE



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@(ONG+ONG!(.VIRUS ; =2ECENTLY THEEMERGENCEOFTHEHIGHLY VIRULENT!(.AVIANINFLUENZASTRAIN;= WHICHHASPROVENITSABILITYTO PASS DIRECTLY FROM BIRDS TO HUMANS ;= AND COULD POTENTIALLY ACQUIRE THE CAPACITY FOR EFFICIENT PERSON TO PERSON TRANSMISSION HAS RAISED CONCERN OVER THE RISK OF A FUTURE INFLUENZA PANDEMIC ;= 4HIS VIRUS OR A CLOSELY RELATEDONEISCONSIDEREDTOBETHELEADINGCONTENDERASTHESOURCEOFTHE NEXT HUMAN INFLUENZA PANDEMIC ; = -OREOVER THE INCREASING MOBIL ITY OF THE POPULATION AT A GLOBAL LEVEL AND THE GREATER SPEED OF MEANS OF TRANSPORT WOULD MAKE THE CONTROL OF THE SPREAD OF INFECTION PARTICULARLY PROBLEMATIC

-EASURESTOBEADOPTEDINCASEOFAPANDEMIC 4OHARMONIZEINTERVENTIONSTOBEADOPTEDINCASEOFANINFLUENZAPANDEMIC COUNTRIES HAVE BEEN URGED TO STRENGTHEN THEIR PREPAREDNESS PLANS ;= FOL LOWING THE  7(/ RECOMMENDATIONS ;= &OR THIS REASONS A NUMBER OFCOUNTRIESHAVEDEVELOPEDORUPDATEDANATIONALINFLUENZAPREPAREDNESS PLAN INCLUDING MEASURES TO BE ADOPTED IN CASE OF A PANDEMIC! RANGE OF MEASURESHAVEBEENSUGGESTED INCLUDINGTHOSETHATINVOLVEPERSONALACTIONS HAND WASHINGANDMASKWEARING NON PHARMACEUTICALINTERVENTIONSEG INTERNATIONAL AIR TRAVEL RESTRICTION BORDER CLOSURE SOCIAL DISTANCING MEA SURES QUARANTINE ;= AND PHARMACEUTICAL INTERVENTIONS ANTIVIRAL DRUGS AND VACCINES ;= ! FEW OF THE MEASURES ARE RELATIVELY STRAIGHTFORWARD TO IMPLEMENT EG HAND WASHING WHILE OTHERS ARE GOING TO BE DIFFICULT TIMELY MASS USE OF ANTIVIRAL DRUGS FOR INDEX AND CLOSE CONTACTS OF CASES ANDYETOTHERSARECOSTLY ANDPOTENTIALLYHIGHLYDISRUPTIVEBORDERCLOSURES ;=(OWEVER THEAPPLICATIONOFTHESEMEASURESHASTHEOBJECTIVETOREDUCE THENUMBEROFPEOPLEWHOAREINFECTED NEEDMEDICALCAREANDDIEDURING ANEPIDEMIC"YLOWERINGTHENUMBERSOFAFFECTEDPEOPLE THEMEASURESCAN ALSOLESSENTHESECONDARYCONSEQUENCESOFEPIDEMICSSUCHASTHEIMPACTOF MASS ABSENTEEISM ON KEY FUNCTIONS EG DELIVERING HEALTHCARE FOOD SUP PLIES FUELDISTRIBUTION THEUTILITIES ETC  4HE EXPERIENCE GATHERED FROM PAST PANDEMICS COULD BE USEFUL TO ORI ENTATEPUBLICHEALTHDECISION MAKINGINTHECASEOFANINFLUENZAPANDEMIC -ODELS ARE A USEFUL TOOL TO PLAN AND EVALUATE INTERVENTIONS BASED ON DIF FERENTSTRATEGIES(OWEVER MODELSAREONLYASRELIABLEASTHE ESTIMATESOF THEBIOLOGICALANDEPIDEMIOLOGICALPARAMETERSTHEYREQUIREINTHECASEOF PANDEMICINFLUENZA ITISPOSSIBLETOUSEDATAFROMPASTPANDEMICSTOIMPLE MENT SOME IMPORTANT PARAMETERS SUCH AS THE LENGTH OF LATENT AND INFEC TIOUSPERIODOFTHENEWPANDEMICINFLUENZAVIRUS 0ASTPANDEMICS HOWEVER WIDELYDIFFERBETWEENCOUNTRIESINTERMSOFSEVERITYANDINTERMSOFGROUPS OF INDIVIDUALS MOST AFFECTED AND EXPERIENCING MOST TRANSMISSION ; = &OR INSTANCE IN )TALY AS IN OTHER %UROPEAN COUNTRIES ;= THE PANDEMIC SEASONDEVELOPEDAMOREDESTRUCTIVECONFLAGRATIONINTHESECOND SEASONOF

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!NEXAMPLEOFSIMULATIONMODEL4HECASEOF)TALY 4OESTIMATETHEEXPECTEDREDUCTIONINTHEATTACKRATEFORDIFFERENTMITIGA TION STRATEGIES IN )TALY A DETERMINISTIC MATHEMATICAL MODEL OFF INFLUENZA TRANSMISSIONWITHASTOCHASTICSIMULATIONCOMPONENTWASUSED4HEMODEL 4 USED TO PREDICT THE SPREAD OF PANDEMIC INFLUENZA IN )TALY AND TO EVALUATE THE IMPACT OF VACCINATION ANTIVIRAL FOR PROPHYLAXIS !60 AND SOCIAL DIS TANCING MEASURES IS AN 3%)2 DETERMINISTIC MODEL4HE MODEL INCLUDED A STOCHASTICCOMPONENTTHATTAKESINTOACCOUNTALLOFTHERANDOMEFFECTSTHAT



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AREIMPORTANTDURINGAPANDEMICSINITIALANDFINALSTAGES WHENTHENUMBER OFINFECTEDINDIVIDUALSWOULDBELOW0RECISELY WHENEVERTHEDETERMINISTIC PREDICTIONOFTHENUMBEROFINFECTEDINDIVIDUALSINANAGECLASSREGIONWAS BELOWTHETHRESHOLDVALUEOF ITWASREPLACEDBYA0OISSONVARIABLEWITH THESAMEMEAN)NTHISWAYALLTHESIMULATIONSWEREDIFFERENTANDINSOMEOF THEMSTOCHASTICEXTINCTIONMAYOCCURDUETOTHEFACTTHATTHETOTALNUMBER OFINFECTEDMAYDROPTOINTHEINITIALPHASESOFTHEEPIDEMICS!NALYZING THE RESULTS OF THE MODEL WE HAVE CONSIDERED ONLY RESULTS REGARDING THOSE SIMULATIONSINWHICHSUSTAINEDTRANSMISSIONISOBSERVEDANDUSEDTOEVALU ATETHEEFFECTOFPOSSIBLECONTAINMENTSTRATEGIES )NEACHSIMULATION THEPANDEMICBEGANWITHTHEINTRODUCTIONOFINFECTED F ADULTSINTHE,AZIO2EGION WHERE2OMESINTERNATIONALAIRPORTISLOCATED4HE RESULTSWEREOBTAINEDBYAVERAGINGOVERSIMULATIONSFOREACHSCENARIO 5SINGTHISTYPEOFMODEL ANALYTICALCALCULATIONOF2 ISNOTFEASIBLE;= &ORTHISREASON WEPROCEEDEDBYSIMULATION RANDOMLYCHOOSING INFECTIVE INDIVIDUAL ASDESCRIBEDABOVE ANDTHENCONTINUINGTHENUMBEROFSECONDARY INFECTIONS4HE MEAN 2 WAS  WHICH WHEN APPLYING THE CONTACT MATRIX CORRESPONDS TO A CUMULATIVE INFECTED ATTACK RATE OF  (OW THE RESULTS WOULDCHANGEDEPENDINGONDIFFERENTLEVELSOFPATHOGENTRANSMISSIBILITY WITH ARESULTING2 OFOR WASTHENEXPLOREDWITHASENSITIVITYANALYSIS

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#ATERINA2IZZOAND-ARTA,UISA#IOFIDEGLI!TTI

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4REATEDINDIVIDUALS 7ITH VACCINE

7ITH ANTIVIRAL

&IRSTVACCINEDOSEATMONTHSFORALLCATEGORIESB 3OCIAL DISTANCING MEASURES

!NTIVIRALFOR WEEKS

 n

  

  

 

!NTIVIRALFORTHE ENTIREEPIDEMIC

 n

  

  

  

&IRSTVACCINEDOSEATMONTHSFORALLCATEGORIESB 3OCIAL DISTANCING MEASURES

!NTIVIRALFOR WEEKS

 n

  

  

 

!NTIVIRALFORTHE ENTIREEPIDEMIC

 n

  

  

  

A6ALUEINPARENTHESESREPRESENTTHEnPERCENTILEVALUESOFTHEATTACKRATEESTIMATES B4HETARGETPOPULATIONWASDIVIDEDINTOFOURCATEGORIESI PERSONNELPROVIDINGESSENTIALSERV

ICESOFTHEn YEAR OLDWORKINGPOPULATION ;=II ELDERLYPERSONS* YEARS III CHILDRENANDADOLESCENTSAGEDnYEARSANDIV ADULTSnYEARS 

EVEN IF CONSIDERING THE PRE PANDEMIC VACCINE LESS EFFECTIVE THAN THE PAN DEMICVACCINE;  =

$YNAMICOFANINFLUENZAPANDEMICINABSENCEOFINTERVENTIONS $EPENDINGONTHE2VALUESCONSIDERED FROMMILDTOHIGHTRANSMISSIBILITY SCENARIOS MODELINGSTUDIESHAVEESTIMATEDTHATTHEFIRSTCASES OFINFLUENZA INAFUTUREPANDEMICWOULDBEIMPORTEDTO%UROPEWITHINnDAYSOFITS EMERGENCE ELSEWHERE IN THE WORLD ;  =4HE INCIDENCE WOULD PEAK nDAYSAFTERIMPORTATIONOFFIRSTCASES;   =!LLTHERESULTS OF THE CONSIDERED STUDIES SHOWED THAT INFECTION WOULD SPREAD SIMULTANE OUSLYTONEARLYTHEENTIRECOUNTRYCONSIDERED WITHNOCLEARSPATIALPATTERN !CCORDING WITH THESE STUDIES THE RESULTS REPORTED FOR )TALY ;= SHOWED THAT WITHAN2 VALUEOF THEINCIDENCEWOULDPEAKAFTERDAYS4HE EPIDEMIC IN)TALY WOULDBEOVERINDAYS (OWEVER ALLTHEDYNAMICSOFANINFLUENZAPANDEMICDESCRIBEDBYMATH EMATICAL MODELS DO NOT TAKE IN ACCOUNT THE FACT THAT THE PAST INFLUENZA PANDEMICSIN%UROPEAND)TALYOCCURREDOVERTWOCONSECUTIVEWINTERS WITH THEHIGHEST!2INTHESECONDWINTER;  =4HISTWO WAVEPATTERNIS PROBABLYANEFFECTOFTHECLOSINGOFSCHOOLSDURINGTHESUMMER;=4HUS MODELS PROBABLY DEPICT AhWORST CASE SCENARIOv WHICH COULD BE USEFUL IN EVALUATINGCONTROLMEASURES;=

-ODELINGINFLUENZAPANDEMICANDINTERVENTIONS



4ABLE%FFECTIVENESSOFCOMBINEDCONTROLMEASURESONTHEDYNAMICSOFANINFLUENZAPANDEMIC IN)TALYWITHAN2 OFANDANATTACKRATEOF WITHVACCINEEFFECTIVENESS6% !TTACKRATEA

)NTERVENTIONS

!VOIDED CASES

4REATEDINDIVIDUALS 7ITH VACCINE

7ITH ANTIVIRAL

&IRSTVACCINEDOSEATMONTHSFORALLCATEGORIESB 3OCIAL DISTANCING MEASURES

!NTIVIRALFOR WEEKS

 n

  

  

 

!NTIVIRALFORTHE ENTIREEPIDEMIC

 n

  

  

  

&IRSTVACCINEDOSEATMONTHSFORALLCATEGORIESB 3OCIAL DISTANCING MEASURES

!NTIVIRALFOR WEEKS

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!NTIVIRALFORTHE ENTIREEPIDEMIC

 n

  

  

  

A6ALUEINPARENTHESESREPRESENTTHEnPERCENTILEVALUESOFTHEATTACKRATEESTIMATES

B4HETARGETPOPULATIONWASDIVIDEDINTOFOURCATEGORIESI PERSONNELPROVIDINGESSENTIAL

SERVICESOFTHEn YEAR OLDWORKINGPOPULATION ;=II ELDERLYPERSONS*  YEARS III CHILDRENANDADOLESCENTSAGEDnYEARSANDIV ADULTSnYEARS 

0HARMACEUTICALINTERVENTIONS -ODELINGSTUDIESHAVESHOWNTHATVACCINATIONANDTHEUSEOFANTIVIRALDRUGS SEEMTOBETHEMOSTEFFECTIVEMEASURESFORPANDEMICCONTROL ESPECIALLYWHEN RAPIDLYIMPLEMENTED;  =)NTHE)TALIANCASE CONSIDERINGAN2VALUE OF THECUMULATIVEINFECTED!2WOULDBE BUTTHISWOULDREQUIREAN EXTREMELYHIGHNUMBEROF!60DOSES0ROVIDING!60FORWEEKS ONLYWOULD INCREASECUMULATIVEINFECTED!2TO WHICHISSIMILARTOTHATOBSERVED DURINGSEVERESEASONALEPIDEMICS;= WITHACONSIDERABLEREDUCTIONINTHE NUMBEROFDOSESPROVIDED!LLTHEMODELINGSTUDIESPUBLISHEDTODATEHAVE SHOWNASUCCESSFULCONTROLOFPANDEMICS EXCEPTTHEHIGHESTLEVELOFTRANSMIS SIBILITY USINGVACCINATION;  =(OWEVER ASREPORTED INOTHERSTUDIES ;  = INDEPENDENTLYOFTHE6% VACCINATIONOFCHILDRENANDYOUNGADULTS WOULDCONSIDERABLYREDUCETHEINCIDENCE ALSOINOTHERAGEGROUPSIE RESULT INGINhHERDIMMUNITYv PROBABLYBECAUSEOFTHEIMPORTANTROLE OFCHILDREN ANDADOLESCENTSINTHESPREADOFINFLUENZA ASALSOOBSERVEDININTER PANDEMIC PERIODS; =!LLTHEMODELINGSTUDIES PUBLISHEDTODATE SUPPORTTHEIDEA THAT DURINGAPANDEMIC VACCINATINGCHILDRENSHOULDBEAHIGHERPRIORITYTHAN VACCINATINGELDERLY;  =&ORTHEHIGHLEVELOFTRANSMISSIBILITY THETIMING OFVACCINATIONISALSOCRUCIAL/NLYTHEADMINISTRATIONOFTHEFIRSTVACCINEDOSE WITHINMONTHSFROMTHEFIRSTWORLDCASE;=WOULDCONTROLTHEPANDEMIC (OWEVER THIS WOULD BE POSSIBLE ONLY IF VACCINES AGAINST hHIGH PANDEMIC RISKv AVIAN INFLUENZA STRAINS SUCH AS!(. WERE STOCKPILED BEFORE THE



#ATERINA2IZZOAND-ARTA,UISA#IOFIDEGLI!TTI

PANDEMIC SINCE TO DEVELOP A VACCINE AGAINST A NEW POTENTIALLY PANDEMIC INFLUENZAVIRUSWOULDTAKEAPPROXIMATELYnMONTHS;= 2EGARDING ANTIVIRAL DRUGS !2 REDUCTION WERE OBTAINED WHEN TREATING CLOSECONTACTSOFINDEXCASESIE HOUSEHOLDCONTACTSANDCLOSECONTACTSIN THESCHOOLORWORKPLACE WITH!60ONLYINTHEEARLIESTSTAGESOFANOUTBREAK ; = (OWEVER TO OBTAIN A REDUCTION IN THE CUMULATIVE!2 FOR A HIGH TRANSMISSIBILITYSCENARIO2  INTERVENTIONSBASEDSOLELYON!60USEARE INADEQUATETOMITIGATEANINFLUENZAPANDEMIC EVENINTHECASE OFADEQUATE STOCKPILEOFANTIVIRALDRUGSSUPPLIES FORAPPROXIMATELYOFF THEPOPULA TION; =

.ON PHARMACEUTICALINTERVENTIONS 4HE SIMULATIONS SHOW THAT APPROPRIATE AND PROMPT MEASURES WHEN COM BINED COULD BE EFFECTIVE IN CONTAINING AN INFLUENZA PANDEMIC ;n = 4IMINGISALSOESSENTIAL ANDMEASURESTHATATFIRSTGLANCEAPPEARTOBELESS IMPORTANT SUCHASINCREASINGSOCIALDISTANCING COULDBEEXTREMELYUSEFULIN DELAYINGTHEEPIDEMICPEAKANDTHUSPROVIDINGMORETIMEFORVACCINESTOBE PRODUCED )MPLEMENTING SUCH MEASURES HOWEVER WOULD ENTAIL ORGANIZING AVARIETYOFBOTHMEDICALANDNON MEDICALRESOURCES ANDSOMEMEASURES SUCH AS THE CLOSING OF SCHOOLS WOULD ALSO HAVE A SOCIAL IMPACT!FTER THE IDENTIFICATIONOFANEWHUMAN TO HUMANTRANSMISSIBLEINFLUENZA VIRUSSTRAIN SOMEWHEREINTHEWORLD EFFORTSTOPREVENTTHESPREADTOUNAFFECTEDCOUN TRIESAREEXPECTED4HEROLEOFGLOBALAIRTRAVELRESTRICTIONHASBEENEXTEN SIVELYSTUDIED BUTITSTILLREMAINSCONTROVERSIAL2VACHEVAND,ONGINI;= DEVELOPEDADETERMINISTICMODELTOSTUDYTHEROLEOFGLOBALAIRTRAVELRESTRIC TION IN THE n INFLUENZA PANDEMIC 2ECENTLY OTHER STUDIES HAVE INDICATED MODELS TO UPDATE POPULATION LEVELS ; = INCORPORATE MORE RECENTAIRTRAVELPATTERNS; = ADJUSTSEASONALITYPARAMETERS; = ADD STOCASTICITYTOTHEMODEL;= ANDEXTENDITTOMORECITIES;=4HESEMOD ELSFOUNDTHATANINFLUENZAPANDEMICATTHEPRESENTTIME WHEN COMPARED TO  WOULD SPREAD FASTER AND THAT THE INTERNATIONAL AIR TRAVEL RESTRIC TIONS ALONE WILL NOT CONTAIN A PANDEMIC BUT CAN DELAY THE EPIDEMIC PEAK ABOUTnWEEKS ;=)NCONTRAST OTHERAUTHORS; =HAVECONCLUDED THAT INTERNATIONAL TRAVEL RESTRICTION DO LITTLE TO REDUCE THE RATE OF SPREAD GLOBALLY SLOWINGDOWNBYONLYAFEWDAYSORWEEKSTHEOVERALL EVOLUTIONOF APANDEMIC!LLTHESESTUDIESSUGGESTTHATUNLESSTHEREISALMOSTCOMPLETE CESSATIONOFINTERNATIONALTRAVELTOACOUNTRY THEATTEMPTSAT BORDERCLOSURE WILLBEUNSUCCESSFULINPREVENTINGDISEASEENTRY;= 3IMILAR RESULTS WERE OBTAINED FOR SOCIAL DISTANCING MEASURES AND QUAR ANTINE ; =4HE MODEL IMPLEMENTED IN )TALY ; = FOUND THAT SOCIAL DISTANCING MEASURES INCLUDING CLOSING OF ALL SCHOOLS FOR  WEEKS PUBLIC OFFICESFORWEEKS ANDPUBLICGATHERINGPLACEFORWEEKS WERENOTEFFEC TIVEINREDUCINGTHECUMULATIVE!2 BUTWOULDDELAYTHEEPIDEMICPEAKBY

-ODELINGINFLUENZAPANDEMICANDINTERVENTIONS



WEEKS4HESERESULTSARECONSISTENTWITHTHATREPORTEDINPREVIOUSSTUDIES WHERE SOCIAL DISTANCING MEASURES WOULD DELAY THE EPIDEMIC PEAK BY n WEEKS;=

#ONCLUSION 4HETRANSMISSIBILITYOFAFUTUREPANDEMICVIRUSISUNCERTAIN SOISIMPORTANT TOEXPLOREDIFFERENTPOSSIBLESCENARIOS-ATHEMATICALMODELINGSTUDIESHAVE SHOWNTHEIMPORTANCEOFTHECOMBINATIONOFDIFFERENTMITIGATION MEASURES FORPANDEMICCONTROL&ORTHEHIGHESTLEVELOFTRANSMISSIBILITY INPARTICULAR Y ONLY THE VERY RAPID TREATMENT WITH ANTIVIRAL DRUGS AND VACCINES CAN SIG NIFICANTLY REDUCE TRANSMISSION &OR THESE REASON PANDEMIC PREPAREDNESS ISCRUCIALALARGESTOCKPILEOFAVACCINEWITHPOTENTIALPANDEMICINFLUENZA ANTIGENS COUPLEDWITHTHECAPACITYTORAPIDLYMAKEABETTER MATCHEDVAC CINE BASED ON THE HUMAN STRAIN WOULD BE THE BEST STRATEGY TO CONTROL AN INFLUENZAPANDEMIC)N)TALY ITHASBEENSHOWN;= FOREXAMPLE THATUSING THISSTRATEGYCOMBINEDWITHANTIVIRALFORWEEKS ANDWITHSOCIALDISTANCING MEASURESWOULDAVOIDTOMILLIONOFCASES DEPENDINGON6%OR  4ABSAND  &ORALLTHESEREASONS MODELINGTOOLSCANHELPTODETERMINEANDPRIORITIZE THEMEASURESMOSTLIKELYTOMITIGATEANINFLUENZAPANDEMIC ANDCANBEVERY USEFULININFORMINGHEALTHAUTHORITIES ATTHENATIONALANDINTERNATIONALLEVELS INTHEABSENCEOFDATAFROMACTUALINTERVENTIONTRIALS WHICHWOULDBEUNFEA SIBLEOUTSIDE ANDIMPRACTICALWITHIN THECONTEXTOFANACTUAL PANDEMIC

!CKNOWLEDGEMENT 4HIS WORK WAS PARTIALLY SUPPORTED BY %PICO 0ROJECT OF THE 0ROVINCIA !UTONOMA DI4RENTO )TALY WE GRATEFULLY ACKNOWLEDGE THE %PICO WORKING GROUPFORTHEIRCONTRIBUTIONTOTHEWORK

2EFERENCES   



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)NDEX



)NDEX

!(.INmUENZA MORTALITY  !(.INmUENZA TRANSMISSION  ADJUVANT  ADJUVANT-&  ADJUVANTVACCINE  ADJUVANTEDINmUENZAVACCINE  AEROSOL  !mUNOVš   AGEMORTALITYPATTERN  ALUM  ANIMALMODELFORINmUENZA   ANIMALMODELFORTHEEVALUATIONOFINmUENZA VACCINES  ANTIGEN AMOUNTFORIMMUNIZATION  ANTIGENICDRIFT  ANTIGENICSHIFT  ANTIVIRALTREATMENTANDPROPHYLAXISUSE DURINGAPANDEMICINmUENZA  ARCHAEO EPIDEMIOLOGY  !RIMAMODEL  !3  !STRA  AVIANINmUENZA  AVIANINmUENZAVIRUS   BASICREPRODUCTIVENUMBER    "AXTER  "IAS INCOHORTSTUDIES  BIOSAFETY  BUDDINGOFINmUENZAVIRUS  #$ 4CELL  #$ 4CELL  CELLCULTURE VACCINEPRODUCTION  CELLCULTUREVACCINE  #HIRON  CLADE  CLASSIlCATIONOFINmUENZAVIRUS  CLINICALMANIFESTATION  CLINICALTRIAL   COMPLICATIONSOFINmUENZA  CORRELATESOFPROTECTION  

COSTIMULATION  CROSSPROTECTION   DENDRITICCELL  DETECTIONOFINmUENZA!VIRUS  DIFFUSEALVEOLARDAMAGE$!$  EFFECTIVEREPRODUCTIVENUMBER 2  EFlCACYOFVACCINE  EMBRYONATEDHENSEGGINOCULATION  EMULSION   EPIDEMIOLOGYOFINmUENZA   EVOLUTIONOFINmUENZAVIRUS  EXCESSDEATH  EXCESSHOSPITALIZATION  EXTRA PULMONARYPATHOLOGY  &LUADš   &LU-ISTš  &OCETRIAš   FOMITE  GENETICREASSORTANT  GENOMICS  GENOTYPE  'LAXO3MITH+LINE  'UILLAIN "ARRÏSYNDROME'"3  GUINEAPIG MODELOFTRANSMISSION  (.VIRUS  (.VIRUS  (.VACCINE  HEMAGGLUTININ      HEMAGGLUTININ RECOMBINANT  HEMAGGLUTININBINDING  HEMAGGLUTINATIONINHIBITION   HEMAGGLUTINATIONINHIBITIONANTIBODY  HEMAGGLUTINATIONINHIBITIONTEST   HERDIMMUNITY  HETEROLOGOUSVACCINESTRAIN  HETEROTYPICINmUENZA!VIRUSIMMUNITY HISTOPATHOLOGICALSTUDY 



HISTORYOFINmUENZA  HOMOLOGOUSVACCINESTRAIN  HOSTCELLULARRECEPTORS  HOSTRANGEMUTANT  HOSTRANGEOFINmUENZAVIRUS  HOST RANGEMUTANT  HUMORALIMMUNITYANDTHEINmUENZA! VIRUSES  )&.ANTAGONISM  )&. a  ), PP  ),   ),   IMMUNEDYSREGULATION  IMMUNERESPONSE  IMMUNESENESCENCE  IMMUNIZATION  IMMUNOGENICITY  INACTIVATEDINmUENZAVACCINE4,6  INmUENZA!  INmUENZA!VIRUSESCAPE  INmUENZAAND236  INmUENZA"  INmUENZABURDEN  INmUENZA#   INmUENZAEPIDEMIC  INmUENZAEPIDEMIOLOGY  INmUENZA'ENOME3EQUENCING0ROJECT  INmUENZAINTHETROPICS  INmUENZAPANDEMIC  INmUENZAVIRUS CLASSIlCATION  INmUENZAVIRUS HOSTRANGE  INmUENZAVIRUSRECEPTOR   INmUENZAVIRUSVACCINE  INmUENZAVIRUS ATTACHMENT  INmUENZAVIRUS C2.!  INmUENZAVIRUS ENDOCYTOSIS  INmUENZAVIRUS ENTRY   INmUENZAVIRUS M2.!  INmUENZAVIRUS NUCLEARIMPORT  INmUENZAVIRUS POLYADENYLATION INmUENZAVIRUS REASSORTMENT  INmUENZAVIRUS SEASONALITY   INmUENZAVIRUS TRANSMISSION  INmUENZAVIRUS V2.!  INmUENZA BURDENININFANTS  INmUENZA MORBIDITY  INmUENZA ASSOCIATEDENCEPHALOPATHY  INmUENZA LIKEILLNESS  INmUENZA RELATEDMORTALITY   INNATEIMMUNITYANDTHEINmUENZA! VIRUSES  )0  

)NDEX

LICENSURE  LIPIDRAFT  LIVEATTENUATEDINmUENZAVACCINE,!)6   LIVEATTENUATEDVACCINE   -   -  -PROTEIN  - BASEDVACCINE  MAGNITUDEOFINmUENZA RELATEDDEATHS  MANUFACTURINGCAPACITY  MATHEMATICALMODEL  MATHEMATICALTRANSMISSIONMODEL  -#0   -ED)MMUNE  -& COMPOSITION  -& ADJUVANTEDVACCINE  MICRONEUTRALIZATION  -)'  MICRONEUTRALIZATION-.  MITIGATIONMEASURESDURINGAPANDEMIC INmUENZA  MIXINGBOWLHYPOTHESIS  MODELLINGINmUENZAPANDEMIC  MOLECULAREPIDEMIOLOGYOFINmUENZA  .%0.3  NEURAMINIDASE.!     NON ADJUVANTVACCINE  NON PHARMACEUTICALINTERVENTION  NON PHARMACEUTICALINTERVENTIONSDURINGA PANDEMICINmUENZA  .OVARTIS   .0   .3   OBSERVATIONALTRANSMISSIONSTUDY  0!    PACKAGINGOFVIRAL2.!  PANDEMIC   PANDEMIC TREATMENTANDPROPHYLAXIS  PANDEMICAGESHIFT  PANDEMICINmUENZA  PANDEMICINmUENZAVACCINE  PANDEMICINmUENZAVACCINEEFFECTIVENESS  PANDEMICMORTALITY  PANDEMICMORTALITY  PANDEMICMORTALITY .EW9ORK #ITY  PANDEMICPREPAREDNESS  PANDEMICSMOLDERINGMORTALITY 

)NDEX

PANDEMICVACCINES  PATHOGENESIS  0"    0" &    0"    PHARMACEUTICALINTERVENTIONSDURINGA PANDEMICINmUENZA  POLICY  PRE PANDEMICINACTIVATEDVACCINE  PRE PANDEMICVACCINATION  PRE PANDEMICVACCINES  QUARANTINE  2AB  2!.4%3  RELEASEOFINmUENZAVIRUS  RESERVOIR  RESPIRATORYDROPLET  RETROVIRALPSEUDOTYPE BASEDASSAY  REVERSEGENETICS  RIBONUCLEOPROTEIN2.0 COMPLEX  2.!POLYMERASE  2.0COMPLEX  SAFETY  3ANOl0ASTEUR  3A_ 'AL   SEASONALINmUENZAVACCINE  SEASONALINmUENZAVACCINE INmUENZA RELATED MORTALITY  SEASONALINmUENZAVACCINE RANDOMIZED CLINICALTRIALS  SEASONALINmUENZAVACCINE OBSERVATIONAL STUDIES 



3%)2MODEL  3ERmING LIKECYCLICALREGRESSIONMODEL  SEROCONVERSION  SERO EPIDEMIOLOGY  SINGLERADIALHEMOLYSIS32(  SINGLE RADIALIMMUNODIFFUSION32$   3INOVAC  SOCIALDISTANCINGMEASURESDURINGA PANDEMICINmUENZA  SPLITVACCINE  STRUCTUREOFINmUENZAVIRUS  SUBCLADE  SUBUNITVACCINE  SURFACTANT   SWINEmU  SWINEINmUENZAVACCINATION  SYMPTOMSOFINmUENZA  SYNDROMICSURVEILLANCE  3YNTEXADJUVANTFORMULATION  TIME SERIESMODEL  TRIVALENT INACTIVATEDINmUENZAVACCINE 4)6  TYPE))&.S  VACCINATINGSCHOOLCHILDREN  VACCINEBENElT  VIRALASSEMBLYOFINmUENZAVIRUS  VIRULENCE  VIRUS LIKEPARTICLE  WATEREMULSION  WHOLEINACTIVATEDVIRUS 

4HE"!)$ 3ERIES "IRKHËUSER!DVANCESIN)NFECTIOUS$ISEASES )NFECTIOUS DISEASES REMAIN A SUBSTANTIAL DRAIN ON HUMAN WELL BEING AND ECONOMIESDESPITETHEAVAILABILITYOFMODERNDRUGS.EWPATHOGENSEMERGE AND KNOWN PATHOGENS CHANGE THEIR GEOGRAPHICAL DISTRIBUTION AND THEIR SUSCEPTIBILITYTOTHEAVAILABLEDRUGS!NUNDERSTANDINGOFTHESTRUCTUREAND FUNCTIONOFINFECTIOUSDISEASEPATHOGENSISAMAJORSCIENTIFIC CHALLENGEWITH IMPORTANTPOTENTIALAPPLICATIONS 4HIS NEW CROSS DISCIPLINARY MONOGRAPH SERIES WILL PROVIDE UP TO DATE INFORMATIONONTHELATESTDEVELOPMENTSININFECTIOUSDISEASERESEARCH4HE MULTI AUTHOREDVOLUMESWILLCOVERBASICBIOLOGYANDBIOCHEMISTRYOFPATHO GENSASWELLASAPPLIEDMEDICALASPECTSANDIMPLICATIONSFORPUBLICHEALTH ANDPOLICY 4HECONTRIBUTIONSAREWRITTENBYLEADINGINFECTIOUSDISEASERESEARCHERS ANDPHARMACEUTICALSCIENTISTSWITHAWIDERANGEOFEXPERTISE 4HEENVISAGEDREADERSHIPINCLUDESACADEMICANDINDUSTRIALRESEARCHERS INMEDICINEANDINFECTIOUSDISEASESASWELLASCLINICIANSANDOTHERSINVOLVED INDIAGNOSTICSANDDRUGDEVELOPMENT

!VAILABLEVOLUMES #ORONAVIRUSES WITH 3PECIAL %MPHASIS ON &IRST )NSIGHTS #ONCERNING 3!23 !3CHMIDT -(7OLFF /7EBER%DITORS  4HE'RAND#HALLENGEFORTHE&UTURE6ACCINESFOR0OVERTY 2ELATED$ISEASES FROM "ENCH TO &IELD 3(% +AUFMANN AND 0 ( ,AMBERT %DITORS  #OMMUNITY !CQUIRED0NEUMONIA .3UTTORP 47ELTE 2-ARRE%DITORS  0OXVIRUSES !-ERCER !3CHMIDT /7EBER%DITORS  0EDIATRIC )NFECTIOUS $ISEASES 2EVISITED ( 3CHROTEN 3 7IRTH %DITORS  #OMPARATIVE(EPATITIS /7EBER 50ROTZER%DITORS 

BIRKHÄUSER ADVANCES IN INFECTIOUS DISEASES

Comparative Hepatitis Weber, O., Bayer Healthcare AG, Wuppertal, Germany / Protzer, U., Technical University/ Helmholtz Center Munich, Germany (eds)

2008. 396 p. 59 illus., 37 in color. Hardcover ISBN 978-3-7643-8557-6

Acute and chronic hepatitis and hepatitis-related diseases such as liver failure, liver cirrhosis and hepatocellular carcinoma are among the most important causes for disabilities and death. Hepatocellular injury is due to a variety of agents including viruses, toxins, radiation, injury and drugs but also bacteria, parasites and autoimmune reactions. This volume reviews today’s knowledge about hepatitis with emphasis on comparative aspects between hepatitis in humans and animals, but also between different etiological agents. This particular viewpoint makes the book relevant for scientists from both human and veterinary medicine, gastroenterologists, pathologists, virologists and students of human and veterinary medicine.

From the contents: Hepatitis in the clinics – treatment options.- Differential diagnosis of human hepatitis.- Comparative pathology.- Hepatitis B virus: lessons learned from the virus life cycle.- Chronic hepatitis C: Portrait of a silent epidemic and the etiologic agent.- Hepatitis A infection.- Hepatitis E infection.- Bacterial infections of the liver.- Comparative hepatitis: Diseases caused by adult parasites or their distinct life cycle stages.- Autoimmune hepatitis in humans.Hepatitis in dogs.- Hepatitis in horses.- The Woodchuck model of hepadnavirus infection.- Hepadnaviruses have a narrow host range – do they?- The liver as immune escape site for pathogens.- Drug candidates for the treatment of viral hepatitis.

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BIRKHÄUSER ADVANCES IN INFECTIOUS DISEASES

Poxviruses Mercer, A., University of Otago, New Zealand / Schmidt, A., University Witten/Herdecke, Germany / Weber, O., Bayer Healthcare AG, Wuppertal, Germany (eds)

2007. XX, 441 p. 34 illus., 4 in color. Hardcover ISBN 978-3-7643-7556-0

The recent international alarm at the possible use of smallpox as weapon of bioterror has refocused the attention of the scientific fi community, public health policy makers and the public on poxviruses. In contrast to the threat posed by some of these viruses, other poxviruses show exciting benefi ficial potential including their use as valuable tools in biomedical research and in the delivery of vaccines. This monograph provides a comprehensive review of the poxvirus family with a particular emphasis on current developments. It includes the latest insights into poxviral molecular biology, diagnosis, therapy, vaccine development and the beneficial fi exploitation of these viruses in biomedical research. Each chapter is written by a leader in the fi field. The reviews present all aspects of the field, including historical perspectives, along with summaries of the latest advances. This combination makes the book relevant for graduate students, researchers new to the field fi and to experienced poxvirologists. From the contents: Historic aspects and early smallpox management approaches in the new world.- Genus Orthopoxvirus: Vaccinia virus.Genus Orthopoxvirus: Variola virus.- Genus Orthopoxvirus: Monkeypox virus.- Genus Orthopoxvirus: Cowpox virus.- Genus Molluscipoxvirus.- Genus Yatapoxvirus.- Genus Parapoxvirus.Genus Capripoxvirus.- Genus Leporipoxvirus.- Genus Suipoxvirus.Genus Avipoxvirus.- Subfamily Entomopoxvirinae.- Vaccines and vaccination.- Poxvirus diagnostics.- Therapy of poxvirus infections.- Environmental resistance, disinfection, and sterilization of poxviruses.- Early disease management strategies in case of a smallpox outbreak.- Recombinant poxviruses in biomedical research.- Immunomodulation by poxviruses.- Immunomodulation by inactivated Parapoxvirus ovis (PPVO) - therapeutic potential.

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BIRKHÄUSER ADVANCES IN INFECTIOUS DISEASES

Pediatric Infectious Diseases Revisited Schroten, H., Universitätskinderklinik Düsseldorf, Germany / Wirth, S., Helios Klinikum Wuppertal, Germany (Eds)

2007. XIV, 503 p. 35 illus. Hardcover ISBN 978-3-7643-7997-1

The successful prevention of childhood diseases like diphtheria, tetanus and pertussis has made a major contribution to the improvement of public health, and vaccines and a variety of drugs are amongst the most fruitful applications of 20th-century research. Today pediatric infectious disease research is closely interconnected with other disciplines. Understanding the biology of the causative agents and the pathogenesis of disease is an essential step in achieving control and elimination of the diseases. Starting with historical, epidemiological and sociocultural issues, the volume presents clinical as well as molecular biological aspects of pediatric infectious diseases. New insights into the pathogenesis of infection are presented and an update on diagnostics, prevention and treatment of pediatric viral, fungal and bacterial diseases provided. The role of emerging new pathogens is also highlighted. The book aims at an interdisciplinary audience of clinicians and non-clinicians: pediatricians, infectious disease researchers, virologists, microbiologists, public health politicians and university libraries. From the contents: Global control of infectious diseases by vaccination programs.Potential impact of rotavirus vaccination on the mortality of children in developing countries.- Controversially discussed indications for immunization.- Gonorrheal ophthalmia neonatorum: historic impact of Credé’s eye prophylaxis.- Malnutrition and infection in industrialized countries.- Better education through improved health and nutrition: Implications for early childhood development programs in developing countries.- Early childhood caries [ECC] and childhood periodontal diseases.- Role of the blood-brain barrier and blood-CSF barrier in the pathogenesis of bacterial meningitis.The molecular basis of paediatric malarial disease.- Epidemiology and etiology of Kawasaki disease.- Helicobacter pylori infection in children.- Human metapneumovirus infection.- Avian influenza fl viruses: a severe threat of a pandemic in children?.- Human papillomavirus infections in children.

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