Influenza Vaccines for the Future (Birkhäuser Advances in Infectious Diseases)

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Author: Rino Rappuoli | Giuseppe Del Giudice

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

3AMIRA-UBAREKAAND0ETER0ALESE )NmUENZAVIRUS4HEBIOLOGYOFACHANGINGVIRUS

*OHN/XFORD 2OBERT,AMBKIN 7ILLIAMSAND!NTHONY'ILBERT )NmUENZAVACCINESHAVEASHORTBUTILLUSTRIOUSHISTORY ,ONE3IMONSEN #ÏCILE6IBOUD 2OBERT*4AYLORAND-ARK!-ILLER A 4HEEPIDEMIOLOGYOFINmUENZAANDITSCONTROL +ATHRYN-%DWARDS )NmUENZAANDINmUENZAVACCINATIONINCHILDREN

*USTINE$-INTERN #AROLE'UILLONNEAU 3TEPHEN *4URNER 4 AND0ETER#$OHERTY 4HEIMMUNERESPONSETOINmUENZA!VIRUSES %MANUELE-ONTOMOLI #ORRELATESOFPROTECTIONAGAINSTINmUENZA #ATHERINE*,UKEAND+ANTA3UBBARAO 4HEROLEOFANIMALMODELSININmUENZAVACCINERESEARCH (ARRY'REENBERGAND'EORGE+EMBLE ,IVEATTENUATEDINmUENZAVACCINE $EREK4/(AGANAND!UDINO0ODDA -&!SAFEANDPOTENTOILINWATEREMULSIONADJUVANT FORINmUENZAVACCINES WHICHINDUCESENHANCEDPROTECTION AGAINSTVIRUSCHALLENGE

VI

#ONTENTS

-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

VIII

,ISTOFCONTRIBUTORS

%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

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

)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


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


!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


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


)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

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TRANSMISSION OF AVIAN INFLUENZA STRAINS IN THESE SPECIES ;= 3PECIFICITY OF VIRAL(!BINDINGISIMPARTEDBYTHERECEPTORBINDINGPOCKETON THESURFACE OF THE (! MOLECULE &IG 4HE (! IS A ROD SHAPED TRIMER ANCHORED IN THE VIRIONS ENVELOPE AND CONTAINS THREE PRIMARY LIGAND BINDING SITES ON AGLOBULARHEAD;= ;=3PECIFICITYOFBINDINGHASBEENLINKEDTOCERTAIN AMINO ACID RESIDUES IN THE (! RECEPTOR BINDING DOMAIN )N ( SUBTYPES AMINO ACID IS ONE SUCH RESIDUE WHERE THE PRESENCE OF LEUCINE ALLOWS BINDINGOF3!_ 'AL WHEREASTHEPRESENCEOFGLUTAMINEATTHISPOSITION PERMITS BINDING OF 3!_ 'AL !MINO ACID CHANGES IN THE (! OF OTHER SUBTYPES SUCHAS(VIRUSESINCLUDINGTHE(.VIRUSRESPONSIBLEFORTHE PANDEMIC HAVEBEENASSOCIATEDWITHADAPTATIONSINRECEPTORBINDING SPECIFICITY TRANSLATINGINTOASWITCHINHOSTSPECIFICITYWITHDISASTROUSCONSE QUENCES; =


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3EVERALPOSSIBLEPATHWAYSFORTHEENTRYOFINFLUENZAVIRUSESINTOHOSTCELLS HAVEBEENPOSTULATEDANDRECENTLYREVIEWED;=%NDOCYTOSISIS AMULTI STEP PROCESSCONSISTINGOFSURFACERECEPTOR MEDIATEDBINDING INTERNALIZATION AND INTRACELLULARTRAFFICKING#LATHRIN MEDIATEDANDCLATHRIN INDEPENDENTINTER NALIZATION VIA CAVEOLAE AS WELL AS CAVEOLAE INDEPENDENT ENDOCYTOSIS HAVE BEENDEMONSTRATED; =!NINITIALACIDIFICATIONSTEPINEARLYENDOSOMES ISFOLLOWEDBYTRAFFICKINGTOLOW P(LATEENDOSOMES APROCESS MEDIATEDBY MEMBERS OF THE 2AB HOST PROTEIN FAMILY &USION OF INFLUENZA VIRUS TO THE ENDOSOME IS TRIGGERED BY LOW P( CONDITIONS AND MEDIATED BY THE FUSION PEPTIDE OF (! AFTER CLEAVAGE OF (! CREATING A PORE IN THE ENDOSOME THROUGHFUSIONOFVIRALANDENDOSOMALMEMBRANES&IG ;= 3UBSEQUENT STEPS IN THE UNCOATING PROCESS INVOLVE THE INFLUENZA VIRUS TETRAMERIC-PROTEIN WHICHISINVOLVEDINTHERELEASEOF2.0 INTOTHEHOST


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;=


2.0 COMPLEXES SUBSEQUENTLY ASSOCIATE WITH - AT ITS # TERMINAL DOMAIN ANDAGGREGATIONOFTHISCOMPLEXLEADSTOINHIBITIONOFTRANSCRIPTION ;=-ALSOINTERACTSWITH.%0ATITS# TERMINALDOMAIN; =.%0 IN TURN ASSOCIATES WITH HOST NUCLEAR EXPORT RECEPTOR #RM VIA THE .%0 . TERMINALDOMAIN;= THUSORCHESTRATINGTHEEXPORTOFVIRAL2.0FROMTHE NUCLEUS

6IRALASSEMBLY BUDDING ANDRELEASE 0OST TRANSLATIONAL MODIFICATION OF THE (! CONSISTS OF GLYCOSYLATION IN THE 'OLGI APPARATUS ;= !LONG WITH VIRAL 2.0 PROTEIN COMPONENTS OF THE VIRIONARECOORDINATELYTRAFFICKEDTOTHEAPICALSURFACEOFTHE HOSTCELLFOR ASSEMBLYINTOPROGENYVIRUS 4WOMODELSFORTHEPACKAGINGOFVIRAL2.!SEGMENTSEXIST ANDINCLUDE THEhRANDOMINCORPORATIONv; =ANDTHEtSELECTIVEINCORPORATIONMOD ELS; =4HELATTERIMPLIESTHATEACH2.!SEGMENTPOSSESSESAPACKAGING SIGNAL RESULTINGINVIRIONSWITHEXACTLYEIGHTSEGMENTS0UTATIVEPACKAGING SIGNALSINCODINGREGIONSOFPOLYMERASEGENES SPIKEGLYCOPROTEINGENES AND THE.3GENEHAVEBEENIDENTIFIED; n= 6IRALASSEMBLYISCOORDINATEDBYTHE-PROTEIN WHICHASSOCIATESWITH THECYTOPLASMICTAILSOFTHEVIRALGLYCOPROTEINS; = ASWELLAS2.0AND .%0 ASDESCRIBEDABOVE,IPIDRAFTSNAVIGATEVIRALMEMBRANEGLYCOPROTEINS TOTHEAPICALSURFACEOFTHEHOSTCELL; =)NADDITION THEREISEVIDENCE THAT TARGETING OF .0 AND POLYMERASE PROTEINS TO THE APICAL SURFACE ALSO INVOLVESLIPIDRAFTS;= 'ENOMICPACKAGINGANDVIRALASSEMBLYOCCURSATTHEAPICALMEMBRANE ANDISASSOCIATEDWITHACCUMULATIONOF-ANDTHEFORMATIONOFLIPIDRAFTS 4HE - PROTEIN HAS ALSO BEEN IMPLICATED IN VIRAL MORPHOLOGY ; = "ECAUSE THE (! BINDS CELL SURFACE SIALIC ACID RECEPTORS VIRIONS MUST BE RELEASED4HE.!FUNCTIONSASASIALIDASEANDCLEAVESSIALICACIDSFROMTHE HOST CELL AND VIRAL GLYCOPROTEINS TO MINIMIZE VIRAL AGGREGATION AT THE CELL SURFACE;="ALANCEBETWEENTHE(!AND.!ISTHUSREQUIREDFOROPTIMAL RECEPTORBINDINGANDDESTRUCTION; =)NADDITIONTOITSRECEPTOR DESTROY INGACTIVITY .!ISAVIRALSPIKEGLYCOPROTEINANDIMPORTANTSURFACEANTIGEN ;=

%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


&IGURE)NFLUENZA!VIRUSSUBTYPESINHUMANS 4HREE PANDEMICS OCCURRED DURING THE TH CENTURY INCLUDING THE h3PANISHv INFLUENZA PAN DEMICOF THEh!SIANvPANDEMICIN ANDTHEh(ONG+ONGvPANDEMICIN(. VIRUSESRE EMERGEDINANDCONTINUETOCIRCULATEINTHEHUMANPOPULATION ALONGWITHTHE (. SUBTYPE )N ADDITION (. VIRUSES HAVE BEEN REPORTED TO INFECT HUMANS THROUGHOUT !SIA AND!FRICA 3EVERAL OTHER AVIAN VIRUSES HAVE ALSO RECENTLY CAUSED SPORADIC INFECTION IN HUMANS!DAPTEDFROM;=

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


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


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

#LINICALMANIFESTATIONS PATHOGENESIS ANDVIRULENCE #LINICALMANIFESTATIONS 5NCOMPLICATEDINFLUENZAINHUMANSISANUPPERRESPIRATORYTRACTINFECTION CHARACTERIZEDBYCOUGH HEADACHE MALAISE ANDFEVERINFLUENZA LIKEILLNESS 4HESE SYMPTOMS ARE NONSPECIFIC AND ARE NOT PREDICTIVE OF INFLUENZA VIRUS INFECTION PARTICULARLY IN INDIVIDUALS YEARS OLD ;= 0ULMONARY AND EXTRA PULMONARYCOMPLICATIONSMAYARISE4HELATTERCONSISTOFCENTRALNER VOUS SYSTEM INVOLVEMENT ENCEPHALITIS ACUTE NECROTIZING ENCEPHALOPATHY 2EYESSYNDROME ANDMYELITIS ;= MYOSITISRHABDOMYOSITIS;= MYOCARDI TIS; = INCREASEDCARDIOVASCULAREVENTS;= DISSEMINATEDINTRAVASCULAR COAGULATION ;= AND TOXIC AND SEPTIC SHOCK BACTERIAL AND NON BACTERIAL ; =0ULMONARYCOMPLICATIONSINCLUDEPRIMARYVIRALPNEUMONIA SEC ONDARYBACTERIALPNEUMONIA ANDEXACERBATIONOFCHRONICLUNGDISEASE; =!CUTELUNGINJURY !,) ACUTERESPIRATORYDISTRESSSYNDROME!2$3 MULTI ORGAN FAILURE PROFOUND LYMPHOPENIA AND HEMOPHAGOCYTOSIS HAVE BEENASSOCIATEDWITH(.INFECTIONANDCARRYHIGHMORTALITYRATES; n=


0ATHOGENESIS &EW HUMAN HISTOPATHOLOGICAL STUDIES OF UNCOMPLICATED INFLUENZA EXIST 0ATHOLOGICALFINDINGSFROMPOST MORTEMEXAMINATIONOFFATAL PEDIATRIC INFLUENZA!CASESINCLUDEDMAJORAIRWAYCONGESTION INFLAMMATION ANDNECROSIS ;= ,OWERAIRWAYPATHOLOGYINCLUDED HYALINE MEMBRANES INTERSTITIALCELLULARINFILTRATES ANDDIFFUSEALVEO LARDAMAGE$!$ 3ECONDARYPNEUMONIA INTRA ALVEOLARHEMORRHAGE AND VIRALPNEUMONITISWERENOTEDINAQUARTEROFCASES;=&ULMINANT$!$ WITH ACUTE ALVEOLAR HEMORRHAGE AND NECROSIS FOLLOWED BY PAUCICELLULAR FIBROSIS AND HYALINE MEMBRANE FORMATION IS OBSERVED IN (. INFECTED HUMAN LUNGS ;= %XTRA PULMONARY PATHOLOGY INCLUDES REACTIVE HEMO PHAGOCYTOSIS IN THE HILAR LYMPH NODES BONE MARROW LIVER AND SPLEEN ;= WHITE MATTER DEMYELINATION ;= AND CEREBRAL NECROSIS ;= AND ACUTETUBULARNECROSISOFTHEKIDNEYS;=$ESPITETHEPRESENCEOFDIAR RHEAAND(.VIRUSREPLICATIONINTHEGASTROINTESTINALTRACTOFHUMANS NO PATHOLOGICAL LESIONS HAVE BEEN DESCRIBED IN THE BOWEL ; = )MMUNE DYSREGULATIONHASBEENIMPLICATEDINTHEPATHOGENESISOF!2$3ANDREAC TIVEHEMOPHAGOCYTOSIS%LEVATEDLEVELSOFNEUTROPHIL MONOCYTE ANDMAC ROPHAGECHEMOATTRACTANTS), )0 -)' AND-#0 ANDPRO INFLAM MATORYCYTOKINES), ), AND)&. a AREOBSERVEDIN(. INFECTED HUMANS;=)NADDITION INCREASEDLEVELSOF), INAHUMAN CASE ;= AND2!.4%3INPRIMARYHUMANALVEOLARANDBRONCHIALEPITHELIALCELLS ;= HAVE ALSO BEEN REPORTED #ONTRIBUTION OF PRO INFLAMMATORY MEDIA TORSTOLUNGPATHOLOGYHASALSOBEENDEMONSTRATEDUSINGA4OLL LIKERECEP TOR KNOCKOUT MOUSE INFECTED WITH MOUSE ADAPTED 73. INFLUENZA ! VIRUS 4HESE MICE DEMONSTRATED ENHANCED SURVIVAL DESPITE HIGHER VIRUS REPLICATIONANDLOWERLEVELSOF2!.4%3 ), AND), PP COMPARED TOWILD TYPEMICE;=

6IROLOGICALDETERMINANTSOFVIRULENCE 4HE (! 0! 0" 0" 0" & .! AND .3 GENE PRODUCTS HAVE BEEN IMPLICATED IN VIRULENCE6IRULENCE DETERMINANTS HAVE BEEN EXPLORED USING THEREVERSEGENETICSYSTEMFOR INFLUENZAVIRUSESANDMAMMALIANFERRETAND MOUSE MODELSFORINFLUENZAVIRUSPATHOGENICITY 4HEPOLYMERASEGENECOMPLEX CONSISTINGOF0! 0" AND0"GENESARE INVOLVEDINREPLICATIONANDTRANSCRIPTIONALACTIVITY!SINGLEGENEREASSORTANT CONTAINING THE 0" FROM !(ONG +ONG (. WHICH IS FATAL IN MICE INABACKGROUNDOF!(ONG+ONG(. CAUSINGMILDRESPI RATORY INFECTION IN MICE DEMONSTRATED A LETHAL PHENOTYPE IN THIS ANIMAL MODEL;=)NADDITION REASSORTANTSCONTAININGPOLYMERASECOMPLEXGENES


FROM !CHICKEN6IETNAM# (. A NON LETHAL VIRUS IN THE BACK GROUNDOF!6IETNAM (. INFLUENZAVIRUSISOLATEDFROMAFATAL HUMANCASEWEREATTENUATEDINANANIMALMODEL;=7HENASINGLEPOINT MUTATION+%INTHE0"GENEWASGENERATEDIN!6IETNAM;= ANDIN!(ONG+ONG;= VIRULENCEWASREDUCEDINMICE ALTHOUGH IN OTHER STUDIES THIS SUBSTITUTION DID NOT REDUCE VIRULENCE SUBSTANTIALLY ;=4HEMOLECULARMECHANISMS RESPONSIBLEFORVIRULENCEHAVEYETTOBE ELUCIDATED 0" &ISTHEGENEPRODUCTARISINGFROMASECONDREADINGFRAMEOFTHE 0" GENE AND HAS BEEN IMPLICATED IN IMMUNE CELL APOPTOSIS THROUGH THE 6$!#AND!.4MITOCHONDRIAL PATHWAYS;=+NOCKOUTOF0" & DID NOTALTERVIRALREPLICATION BUTENHANCEDCLEARANCEOFTHEVIRUSANDREDUCED LETHALITYINMICEWASDEMONSTRATED SUGGESTINGTHAT0" &MAY PLAYAROLE INVIRALPATHOGENESIS;=%NHANCEDPATHOGENICITYWASOBSERVEDINMICE INFECTEDWITHRECOMBINANTINFLUENZAVIRUSCONTAININGTHE0" & GENEFROM AHIGHLYPATHOGENIC(.VIRUSISOLATEDFROMAFATALHUMANCASEIN(ONG +ONGIN;= %VASION OF THE HOST IMMUNE RESPONSE IS A KEY VIRULENCE DETERMINANT PERMITTING VIRUSES TO ESTABLISH SUSTAINABLE INFECTION 4HE INNATE IMMUNE SYSTEMISTHEFIRSTLINEOFHOSTDEFENSEANDTHEINFLUENZAVIRUSPOSSESSESTHE ABILITY TO INTERFERE WITH THIS RESPONSE4YPE ) )&.S )&. _` ARE CENTRAL TO ESTABLISHING AN ANTIVIRAL STATE IN HOST CELLS )&. ANTAGONISM HAS BEEN PRIMARILYATTRIBUTEDTOTHE.3PROTEINOFINFLUENZAVIRUS WHICHSEQUESTERS DOUBLE STRANDED2.! THUSPREVENTINGTHEACTIVATIONOF)&.TRANSCRIPTION FACTORSFORREVIEW SEE;= 4HEEFFECTOFAVIANINFLUENZAVIRUS .3ON)&.PRODUCTIONHASALSOBEEN EXPLORED!GOOSE'UANGDONG WHICHDIFFERSBYONEAMINOACIDFROM !GOOSE'UANGDONGATPOSITION ISLETHALINCHICKENSANDANTAGO NIZES)&. _`;=)NADDITION THE#TERMINUSOFTHE.3PROTEINCONTAINS A0$:LIGANDDOMAIN CAPABLEOFBINDING0$:PROTEININTERACTIONDOMAINS OFHOSTPROTEINS THUSPOTENTIALLYDISRUPTINGHOSTCELLULARPATHWAYS6IRUSES CAUSINGPATHOGENICINFECTIONINHUMANSFROMANDCONTAINEDAVIAN MOTIFSATTHE.30$:LIGAND BINDINGSITE4HESEANDTHEMOTIFFOUNDINTHE INFLUENZAVIRUS.3HADSTRONGERBINDINGAFFINITIESTO0$: DOMAINSOF HUMAN CELLULAR PROTEINS COMPARED TO LOW PATHOGENICITY INFLUENZA VIRUSES ;= .EUROVIRULENCEHASBEENASSOCIATEDWITHGLYCOSYLATIONOFTHE.! GLYCO PROTEIN;=4HE(!GLYCOPROTEINHASALSOBEENIMPLICATEDIN VIRULENCE !LTHOUGHCLEAVABILITYOFTHE(!PROTEINHASBEENPRIMARILYIMPLICATEDIN PATHOGENICITY IN CHICKENS LETHALITY HAS ALSO BEEN DEMONSTRATED IN MICE 2EPLACEMENTOFTHEMULTIBASICCLEAVAGESITEINAHIGHPATHOGENICITY(. VIRUSFROM(ONG+ONG(+ WITHANAMINOACIDSEQUENCETYPICALOFLOW PATHOGENICITYVIRUSESRESULTEDINATTENUATION;=


&IGURE#LOSERANGETRANSMISSIONOFHUMANINFLUENZA!AMONGST GUINEAPIGS )NOCULATED ANIMALS PLACED IN PROXIMITY TO UNINOCULATED ANIMALS WITHOUT DIRECT CONTACT SPREAD0ANTOALLEXPOSEDANIMALS!DAPTEDFROM;=

3EASONALITYANDTRANSMISSION )NFLUENZA!AND"VIRUSESEXHIBITMARKEDSEASONALITYANDTHISPATTERNDIC TATES THE ANNUAL VACCINATION SCHEDULE 3EVERAL THEORIES WITH RESPECT TO THE MECHANISMS DICTATINGTHISSEASONALPATTERNHAVEBEENPROPOSED FORREVIEW SEE;= 9EAR ROUNDHUMANINFLUENZAVIRUSACTIVITYINEQUATORIALREGIONS MAYBEARESERVOIRFORANNUALOUTBREAKSINTHENORTHERNANDSOUTHERNHEMI SPHERES!SRESEARCHPROGRESSESINTHISAREA FACTORSDETERMININGSEASONALITY MAYBEEXPLOITEDFORTHECONTROLOFTHESPREADOFINFLUENZAVIRUS;= 4RANSMISSION OF INFLUENZA VIRUS AMONGST HUMANS IS POORLY UNDERSTOOD AND THE MODES OF SPREAD ARE CURRENTLY UNDER DEBATE ; = )T IS WIDELYACCEPTEDTHATINFLUENZAVIRUSISTRANSMITTEDBYTHERESPIRATORYROUTE INHUMANS ALTHOUGHTHECONTRIBUTIONOFSMALLPARTICLEAEROSOLS RELATIVETO LARGE RESPIRATORY DROPLETS IS UNKNOWN )N ADDITION THE ROLE OFF FOMITES IS QUESTIONABLE5NTILRECENTLY FERRETSHAVESERVEDASTHEONLYANIMALMODEL FOR THE STUDY OF INFLUENZA VIRUS TRANSMISSION! NOVEL MAMMALIAN MODEL USINGTHEGUINEAPIGHASRECENTLYBEENDEVELOPEDTOOVERCOMETHELIMITA TIONS IMPOSED BY THE FERRET MODEL 'UINEA PIGS ARE HIGHLY SUSCEPTIBLE TO INFECTIONWITHANUNADAPTEDHUMAN(.!0ANAMA OR 0AN INFLUENZAVIRUS WITHAINFECTIOUSDOSEOF0&5 ANDTHIS VIRUSGROWSTO HIGHTITERSINTHEUPPERRESPIRATORYTRACTANDTOMODERATETITERSINTHELUNGS 4RANSMISSIONOF0ANBYDIRECTCONTACTANDAEROSOLINTHISSYSTEMIS &IG ;=#ONTROLOFINFLUENZAVIRUSSPREADDURINGINTERPANDEMICAND PANDEMICPERIODSTHROUGHPHYSICALMEANSWILLBEPARAMOUNTTOABROGATING PERSON TO PERSONTRANSMISSIONANDISPARTICULARLYCRUCIALWHERE VIRUSESARE RESISTANTTOCURRENTLYAVAILABLEANTIVIRALS


0ERSPECTIVES %FFECTIVEANDTIMELYVACCINEDEVELOPMENTDEPENDSONIN DEPTHUNDERSTAND INGOFINFLUENZAVIRUSBIOLOGY!LTHOUGHRECENTADVANCESHAVEBEENMADE ONGOING RESEARCH WILL BE REQUIRED TO FULFILL THIS GOAL )DENTIFICATION F AND CHARACTERIZATIONOFTHEMOLECULARSIGNATURESREQUIREDFORTRANSMISSIONWILL BE OF UTMOST IMPORTANCE TO PREVENTING FURTHER INFLUENZA VIRUS PANDEMICS 'LOBALIZATION OF (. INFECTION IN HUMANS REQUIRES PARALLEL EFFORTS ON BEHALFOFVIROLOGISTSINCONJUNCTIONWITHEPIDEMIOLOGISTSANDOTHERMEMBERS OFTHEPUBLICHEALTHCOMMUNITYTOTRANSLATETHEGROWINGBODYOFF KNOWLEDGE INTOMEANSBYWHICHINFLUENZASPREADCANBECONTROLLED

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2EFERENCES

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)NTRODUCTION 7HENTHEINFLUENZA!VIRUSFIRSTEMERGEDFROMAPRESUMEDAVIANRESERVOIR ATTHEENDOFTHEICEAGEORSOYEARSAGO THEREWASADISTINCTDIFFICULTY INFINDINGNEWHUMANVICTIMS&OREXAMPLE ATTHATTIME ONLYA FEWHUNDRED SETTLERSWEREINTHE,ONDONREGIONNEARTHE2OYAL,ONDON(OSPITAL NOW ACOMMUNITYOFFOURMILLIONPEOPLE!TTHATTIMEATRAVELERWOULDHAVETO WALKAMILESTOFINDANOTHERSMALLSETTLEMENT PERHAPSAT3TONEHENGE NEAR3ALISBURY .OWADAYSWEHAVEATRULYGLOBALCOMMUNITYOFSIXBILLIONPEOPLE LINKED SO THAT TWO MILLION PEOPLE ARE MOVING EACH DAY BY PLANE WHILE PERHAPS MILLION ARE JOURNEYING IN THEIR HOMELANDS )NFLUENZA LIKE ALL VIRUSES IS OPPORTUNISTIC )N IT HAD THE UNPRECEDENTED OPPORTUNITY TO SPREAD AT THEENDOFTHEFIRSTGLOBALWAR4ENMILLIONSOLDIERSBEGANTHE MOVEHOME

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*OHN/XFORDETAL

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*OHN/XFORDETAL

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*OHN/XFORDETAL

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*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


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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


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


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 SUCHAS#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 POSITIVEVIRUSTHATPRODUCEDPLAQUESAT#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 WASHINGSINTISSUECULTURESAT# -AASSAB


; = 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 TOOBTAINAVIRUSINDIRECTLYATTENUATEDBYCULTIVATIONAT#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


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

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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


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

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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


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


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

*OHN/XFORDETAL

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!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

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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

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&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=

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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


ACTIVITY)NTHE53BETWEENAND 4HOMPSONETAL; =ESTIMATED THAT SEASONAL INFLUENZA EPIDEMICS WERE ASSOCIATED WITH DEATHS PER ONAVERAGERANGEnPER DEPENDINGONTHESEVERITYOFTHE CIRCULATING STRAINS 2EASSURINGLY DIFFERENT MODEL APPROACHES YIELD SIMILAR AVERAGEESTIMATESOFTHEINFLUENZAMORTALITYBURDENINTHE53; = WHILEESTIMATESFROM%UROPEAND#ANADAARESIMILARTOTHOSEFROMTHE53 ; =4HEINTEGRATIONOFHOSPITALIZATIONORDEATHINDICATORSANDVIRAL SURVEILLANCEDATAISPARTICULARLYUSEFULFORTHESTUDYOFINFLUENZAINTROPICAL AREASWERETURNTOTHISLATER

!GEANDTIMEVARIABILITYININFLUENZA RELATEDMORTALITYINTEMPERATE CLIMATES )NFLUENZA RELATED DEATHS CONTRIBUTE ^ RANGE n OF ALL WINTER MORTALITYINSENIORSOVERINTHE53 WITHSIMILARPROPORTIONIN)TALYAND #ANADA; =3EASONSDOMINATEDBYTHEINFLUENZA!(. SUBTYPE ARETYPICALLYASSOCIATEDWITHn FOLDHIGHERMORTALITYTHANSEASONSDOMI NATEDBYINFLUENZA!(.ANDINFLUENZA"VIRUSES"UTTHEPATTERNISNOT ALWAYS CLEAR THERE HAVE BEEN INFLUENZA!(. DOMINATED SEASONS WITH LITTLE EXCESS MORTALITY EG n .ORTHERN HEMISPHERE SEASON 4HE AGE SPECIFIC RISK OF INFLUENZA RELATED EXCESS MORTALITY RATES RISES SHARPLY PAST AGE &IG 0EOPLE AGED * YEARS ARE AT APPROXIMATELY FOLD HIGHERRISKTHANPEOPLEAGEDnYEARS-OREOVER INRECENTDECADESABOUT OFALLINFLUENZA RELATEDDEATHSOCCURREDAMONGSENIORS * YEARS OCCURREDAMONGSENIORSAGED * YEARS ANDOCCURREDAMONGSENIORS OVER YEARS ;= !S THE POPULATION IN THE 53 AND OTHER DEVELOPED COUNTRIES HAS AGED SUBSTANTIALLY OVER THE LAST DECADES THE CRUDE NUMBER OF INFLUENZA RELATED DEATHS HAS BEEN RISING "ECAUSE THE RISK OF INFLUENZA RELATEDDEATHINCREASESEXPONENTIALLYWITHAGEINTHELATERDECADESOFLIFE IT IS ESSENTIAL TO STANDARDIZE FOR AGE WHEN COMPARING MORTALITY IMPACT IN DIFFERENTCOUNTRIESANDOVERTIME; =

"URDENANDCIRCULATIONPATTERNSOFINFLUENZAINTHETROPICS "ECAUSE MOST SEASONAL INFLUENZA MODELS h3ERFLING APPROACHESv DEPEND ONWINTERSEASONALITYINTHEDATA THEYARENOTGENERALLYUSEFULFORTROPICAL COUNTRIES(OWEVER SUCHMODELSCANBEUSEDFORUNUSUALLYSEVEREEPIDEM ICS AND PANDEMICS WHERE THE EXCESS DISEASE BURDEN IS MANY FOLD GREATER THANINAVERAGEYEARS;=)NTEGRATIONOFVIRALSURVEILLANCEDATAWITHDEATH OR HOSPITALIZATION INDICATORS IS THE MOST USEFUL APPROACH IN TROPICAL SET TINGS ALTHOUGH LONG TERM HISTORICAL SURVEILLANCE DATA IS USUALLY LACKING ;=! SERIES OF STUDIES IN (ONG +ONG AND 3INGAPORE RECENTLY FOUND THAT ANNUAL INFLUENZA RELATED HOSPITALIZATION AND MORTALITY RATES IN WEALTHY

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&IGURE!VERAGEAGE SPECIFICRATESOFINFLUENZA RELATEDEXCESSDEATHSANDHOSPITALIZATIONSFOR TENSEASONSDURINGnINTHE53ESTIMATEDFROM3ERFLINGREGRESSIONMODELS .OTE THE CHARACTERISTIC 5 SHAPE OF SEVERE DISEASE BURDEN BY AGE THAT CHARACTERIZES SEASONAL INFLUENZA$ATASOURCE6ITAL3TATISTICSFROMTHE.ATIONAL#ENTERFOR(EALTH3TATISTICS.#(3 ANDHOSPITALDISCHARGEDATAFROM!GENCYFOR(EALTH#ARE2ESEARCHAND1UALITY!(21

SUB TROPICALLOCATIONSARESIMILARTOTHOSEINTEMPERATECOUNTRIES;n= )N(ONG+ONG ASINMANYOTHERCOUNTRIES THEIMPACTOFINFLUENZAISSEEN NOTONLYONPNEUMONIAANDINFLUENZAOUTCOMES BUTALSOONAWIDERANGEOF CHRONICHEALTHCONDITIONSSUCHASDIABETESANDCARDIOVASCULARDISEASES;= )NADDITION INFLUENZA RELATEDHOSPITALIZATIONRATESIN(ONG +ONGVARYWITH AGEASA5 SHAPEDCURVE;= INWHICHYOUNGINFANTSANDELDERLYPEOPLEARE ATHIGHESTRISKOFSEVEREOUTCOMES REMINISCENTOFTHEAGEPATTERNOFINTER PANDEMICINFLUENZAINTHE53ANDOTHERTEMPERATECOUNTRIES 4HESPREADOFINFLUENZAINTHETROPICSHASALSOPROVENTOBEANENIGMA )NFLUENZA SEASONALITY IN THE SOUTHERNMOST TEMPERATE REGIONS IS MONTHS OUT OF PHASE WITH THE .ORTHERN HEMISPHERE -OST RECENTLY A STUDY FROM "RAZILFOUNDSEASONALINFLUENZAACTIVITYSTARTINGEARLYINREMOTE LESSDENSELY POPULATEDEQUATORIALREGIONSOFTHE.ORTH-ARCH !PRIL ANDTRAVELINGIN ^MONTHSTOTHEMORETEMPERATEAREASOFTHE3OUTHDURINGTHEIR WINTERSEA SON*UNEn*ULY ;=4HISFINDINGWASCONTRARYTOWHATWASEXPECTED GIVEN THATTHELARGER WELL CONNECTED DENSELYPOPULATEDCITIESARELOCATEDINTHE 3OUTH)FPOPULATIONMOVEMENTSWEREADRIVINGFACTORLIKEINTHE53;= THENTHEOPPOSITETRAVELINGWAVEWOULDHAVEBEENEXPECTED4HISSTUDYHAS INSPIRED FURTHER STUDIES TO INVESTIGATE THE CIRCULATION OF SPECIFIC INFLUENZA VIRUS SUBTYPES DURING A SEASON BASED ON ANALYSIS OF VIRAL GENOMICS DATA


&INDING FIRM EVIDENCE OF THIS UNUSUAL CIRCULATION PATTERN SUGGESTED FROM ANALYSIS OF REGIONAL MORTALITY DATA ALSO BEARS ON CONSIDERATIONS OF USE OF 3OUTHERNOR.ORTHERNHEMISPHEREVACCINEFORMULATIONANDTIMING "ECAUSE OFTHISSTUDY THE"RAZILIAN MINISTRYOFHEALTHISCURRENTLYCONSIDERINGCHANG ING THE TIMING OF VACCINATION IN THE .ORTH OF "RAZIL TO ACCOMMODATE THE EARLYOCCURRENCEOFINFLUENZAINTHATAREA

4HEBURDENOFINFLUENZAININFANTSANDYOUNGCHILDREN &ORAGEGROUPSOTHERTHANTHOSEOVERYEARSOFAGE ITCANBE DIFFICULTTO MEASURE THE RELATIVELY LOW SEASONAL IMPACT OF INFLUENZA MORTALITY ABOVE THE EXPECTED BASELINE "UT FOR OCCASIONAL SEVERE SEASONS A SURGE IN 0) DEATHS CAN OFTEN BE SEEN IN CHILDREN AND YOUNG ADULTS &OR EXAMPLE THE n SEASON WAS DOMINATED BY A NEW ANTIGENIC VARIANT OF!(. VIRUSES!&UJIAN ANDWASUNUSUALLYSEVEREINTHE53 CHILDREN WITHDOCUMENTEDINFLUENZAINFECTIONSDIEDOFPRIMARYORSECONDARYPNEU MONIA AND SEPSIS ;= 3URPRISINGLY OF THE CHILDREN WHO DIED HAD NO KNOWNUNDERLYINGRISKCONDITIONS4HEREASONFORTHISUNUSUALEPIDEMICOF PEDIATRIC DEATHS HAS NOT BEEN RESOLVED!S A RESULT OF THIS EXPERIENCE THE #$#ENHANCEDTHEIRINFLUENZASURVEILLANCESYSTEMWITHAREPORTINGSYSTEM FORCHILDRENHOSPITALIZEDWITHLABORATORY CONFIRMEDINFLUENZAHTTPWWW CDCGOVFLUACCESSED/CTOBER

4HEIMPACTOFINFLUENZAONMORBIDITY 6ERYFEWQUANTITATIVEDATAONMILDINFLUENZAMORBIDITYWITHKNOWNPOPULA TIONDENOMINATORSAREAVAILABLE4HEMOSTCAREFULSTUDIESUSINGTHELONGEST EXISTINGTIMESERIESCOMEFROMTHE2OYAL.ETWORKOF'ENERAL0RACTITIONERS IN THE 5+ WHICH HAS REPORTED INFLUENZA LIKE ILLNESSES ON A WEEKLY BASIS SINCE; =3UCHLONG TERMMORBIDITYRECORDSAREUNIQUEANDHAVE ALLOWED THE STUDY OF THE n INFLUENZA PANDEMIC TRANSMISSION PAT TERNSBASEDONCASEDATA;=)NADDITIONTOTHE5+ SEVERALCOUNTRIESHAVE NATIONAL SENTINEL SURVEILLANCE SYSTEMS IN PLACE 53 &RANCE .ETHERLANDS !USTRALIA AND .EW :EALAND ARE EXAMPLES 4HESE ARE USED TO DETECT THE ONSETANDPEAKTIMINGOFINFLUENZAEPIDEMICS ASWELLASTHEMAGNITUDEOF MORBIDITY IMPACT RELATIVE TO SURROUNDING SEASONS )N THE 53 EMERGENCY ROOMVISITTIMESERIESARENOWBEINGANALYZEDINTHECONTEXTOFF BIODEFENSE RESEARCHANDHAVESHEDLIGHTONINTER ANNUALANDAGE SPECIFICVARIABILITYIN INFLUENZAIMPACT; = )N CONTRAST QUANTITATIVE BURDEN STUDIES USING SAMPLES OF NATIONAL HOS PITAL DISCHARGE DATA AND ESTIMATION APPROACHES SIMILAR TO THOSE USED FOR EXCESSMORTALITYAREMOREWIDELYAVAILABLE INPARTICULARSINCE THES; =4HEPATTERNSOFEXCESSHOSPITALIZATIONSAREQUITESIMILARTOTHOSE

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OFEXCESSMORTALITY WITHA5 SHAPEDINCIDENCEREFLECTINGTHEHIGHESTVALUES INYOUNGCHILDRENANDSENIORS&IG

4HERELATIVECONTRIBUTIONOFINFLUENZAAND236 /NE CONTROVERSY IN THE LITERATURE CONCERNS THE RELATIVE CONTRIBUTIONS OF INFLUENZA AND 236 TO THE WINTER INCREASE IN RESPIRATORY HOSPITALIZATIONS ANDDEATHS ESPECIALLYAMONGSENIORS4HECURRENT#$#MODELING APPROACH SIMULTANEOUSLYESTIMATESTHEINFLUENZAAND236BURDENBYCORRELATINGPERI ODSOFEXCESSMORTALITYWITHTHEIRRESPECTIVEPERIODANDMAGNITUDEOFVIRAL ACTIVITY ;= )N THE OVERALL 53 POPULATION THE #$# INVESTIGATORS ESTIMATE THEAVERAGESEASONAL236BURDENISAPPROXIMATELYONE THIRDTHATOFINFLU ENZAFORALLSEASONSDURINGTHES(OWEVER THERELATIVECONTRIBUTIONOF 236ANDINFLUENZAVARIESGREATLYWITHAGE &OR53INFANTSOFMONTHSOFAGE THE236CONTRIBUTIONTOMORTAL ITYISMORETHAN FOLDGREATERTHANTHATOFINFLUENZAVERSUSDEATHS PER BASEDONTHE#$#MODEL;=0ASTAGE THEINFLUENZABURDEN BECOMESPREDOMINANTINTHE53DATA INAGREEMENTWITHTHEAGEPATTERNOF RESPIRATORYDEATHSINTHE5+;=&ORSENIORSOVERAGE THE #$#MODEL PUTS THE AVERAGE SEASONAL 236 BURDEN AT ^ DEATHS WHICH IS ABOUT FOLD LOWER THAN THE ESTIMATED DEATHS ATTRIBUTED TO INFLUENZA OVER THE SAME PERIOD "UT OTHERS DISAGREE SEVERAL OBSERVATIONAL STUDIES SET IN THE 5+BY&LEMINGANDCOLLEAGUES; =HAVEARGUEDTHAT236HASREPLACED INFLUENZA AS THE MAJOR CAUSE OF RESPIRATORY MORTALITY AND HOSPITALIZATION INPARTICULARINTHEELDERLY&URTHER INARECENTLABORATORY BASEDSTUDYSET IN A LARGE COHORT OF SENIORS HOSPITALIZED WITH PNEUMONIA TWICE AS MANY HOSPITALIZATIONSWEREATTRIBUTEDTO236ASINFLUENZA;="UTBECAUSEINFLU ENZA RELATEDPNEUMONIAISMOSTOFTENDUETOSECONDARYBACTERIAL INFECTIONS QUITE DISTINCT FROM PRIMARY 236 PNEUMONIA THAT OCCUR LONG AFTER THE TRIGGERINGINFLUENZAINFECTIONHASBEENCLEARED ITISPOSSIBLETHATTHISSTUDY SUBSTANTIALLYUNDERESTIMATEDTHEINFLUENZABURDEN;= 4WORECENTSTUDIESCAREFULLYDELINEATEDTHERELATIVEBURDENOFINFLUENZA AND236INCHILDREN USINGSEASONALITYINPEDIATRICRESPIRATORYHOSPITALIZA TIONS AND FOCUSING THE ANALYSIS ON SEASONS WHEN THE INFLUENZA AND 236 EPIDEMICSOCCURREDATDIFFERENTTIMES; =4HEAUTHORSSUBTRACTEDHOS PITALIZATIONRATESDURINGPERIODSOFHIGHINFLUENZACIRCULATIONFROMBASELINE hPERI INFLUENZAvWINTERPERIODSWHENNEITHERINFLUENZANOR236 WASCIRCU LATING&IG 5SINGTHISAPPROACH THEAUTHORSATTRIBUTEDASIMILARNUMBER OFHOSPITALIZATIONSTO236ANDINFLUENZAINCHILDRENUNDERIN THE53;= )N A PARALLEL STUDY FROM (ONG +ONG INVESTIGATORS ATTEMPTED TO TEASE OUT THEBURDENOF236 INFLUENZAANDOTHERRESPIRATORYPATHOGENSINVARIOUSAGE GROUPSINTHISSUB TROPICALSETTINGWITHLESSCLEARSEASONALITY ;=!LTHOUGH INFLUENZABURDENESTIMATESIN(ONG +ONGWERESIMILARTOTHOSEOFTHE53 INMOSTAGEGROUPS;= THESPECIFICSUBGROUPOFCHILDRENUNDERAPPEARED


&IGURE!NANALYTICAPPROACHTOESTIMATEINFLUENZA RELATEDHOSPITALIZATIONRATESIN53AND (ONG +ONGCHILDREN; = 4HIS METHOD RELIES ON IDENTIFYING THE PRECISE PERIODS OF INFLUENZA AND 236 VIRAL CIRCULATION FOR EACH SEASON STUDIED )NFLUENZA RELATED EXCESS RATES WERE CALCULATED AS THE DIFFERENCE IN OBSERVEDRATESBETWEENPERIODSOFINFLUENZAAND236CIRCULATIONANDTHOSEWITHLOWCIRCULA TIONOFBOTHINFLUENZAAND236

TOHAVE^ FOLDHIGHERRATESOFHOSPITALIZATIONIN(ONG +ONGTHANINTHE 53;=3UCHLARGEDISCREPANCIESMAYREFLECTTRUEGEOGRAPHICAL DIFFERENCES IN INFLUENZA TRANSMISSION AND IMPACT ALTHOUGH THEY PERHAPS MORE LIKELY RESULTFROMDIFFERENCESINACCESSTOHOSPITALCARE)NDEED YOUNGCHILDRENIN (ONG +ONG TEND TO BE RUSHED TO THE HOSPITAL WHEN THEY HAVE RESPIRATORY SYMPTOMS-ALIK0EIRIS PERSONALCOMMUNICATION &INALLY THERE IS A LARGE BODY OF LITERATURE ON RESPIRATORY VIRUS ISOLATES FROM CHILDREN HOSPITALIZED WITH RESPIRATORY SYMPTOMS IN TROPICAL AND SUBTROPICAL SETTINGS4HESE PAPERS WHICH HAVE BEEN NICELY SUMMARIZED BY 7EBERETAL;= ATTRIBUTEASUBSTANTIALPROPORTIONOFPEDIATRICRESPIRATORY HOSPITALIZATIONSTOINFLUENZA!AND"VIRUSES5NFORTUNATELY ITISDIFFICULTTO COMPARE FINDINGS ACROSS STUDIES BECAUSE THEY ARE OFTEN DONE USING DIFFER ENTLABORATORYTECHNIQUESANDARESETINDIFFERENTSTUDYYEARS SEASONS AND SETTINGSHOSPITAL OUTPATIENTCLINIC ETC 4HESESTUDIESFREQUENTLYPRESENTA SYSTEMATICAGEPATTERNTHATSUGGESTSTHAT236ISMOREIMPORTANTININFANCY WITHAGRADUALSHIFTTOINFLUENZABYABOUTAGEASTHEPATHOGENMORELIKELY TOCAUSESEVERERESPIRATORYILLNESS

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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


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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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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


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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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


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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


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

)ZURIETA (3 4HOMPSON 77 +RAMARZ 0 3HAY $+ $AVIS 2, $E3TEFANO & "LACK3 3HINEFIELD( &UKUDA+ )NFLUENZAANDTHERATES OFHOSPITALIZA TIONFORRESPIRATORYDISEASEAMONGINFANTSANDYOUNGCHILDREN .%NGL*-ED n .EUZIL+- -ELLEN"' 7RIGHT0& -ITCHEL%&*R 'RIFFIN-2 4HEEFFECT OFINFLUENZAONHOSPITALIZATIONS OUTPATIENTVISITS ANDCOURSESOFANTIBIOTICSIN CHILDREN .%NGL*-EDn )WANE -+ %DWARDS +- 3ZILAGYI 0' 7ALKER &* 'RIFFIN -2 7EINBERG '! #OULEN # 0OEHLING +! 3HONE ,0 "ALTER 3 ET AL .EW 6ACCINE 3URVEILLANCE.ETWORK0OPULATION BASEDSURVEILLANCEFORHOSPITALIZATIONSASSO CIATEDWITHRESPIRATORYSYNCYTIALVIRUS INFLUENZAVIRUS ANDPARAINFLUENZAVIRUSES AMONGYOUNGCHILDREN0EDIATRICSn 'RIFFIN -2 7ALKER &* )WANE -+ 7EINBERG '! 3TAAT -! %RDMAN $$ %PIDEMIOLOGY OF RESPIRATORY INFECTIONS IN YOUNG CHILDREN )NSIGHTS FROM THE .EW6ACCINE 3URVEILLANCE .ETWORK 0EDIATR )NFECT $IS ** 3UPPL 3n3 0OEHLING +! %DWARDS +- 7EINBERG '! 3ZILAGYI 0 3TAAT -! )WANE -+ "RIDGES #" 'RIJALVA #' :HU 9 "ERNSTEIN $) ET AL .EW 6ACCINE 6 3URVEILLANCE .ETWORK 4HE UNDER RECOGNIZED BURDEN OF INFLUENZA IN YOUNG CHILDREN .%NGL*-EDn 7EINBERG '! %RDMAN $$ %DWARDS +- (ALL #" 7ALKER &* 'RIFFIN F -2 3CHWARTZ " .EW 6ACCINE 3URVEILLANCE .ETWORK 3TUDY 'ROUP 3UPERIORITYOFREVERSE TRANSCRIPTION POLYMERASECHAINREACTIONTOCONVENTIONAL VIRALCULTUREINTHEDIAGNOSISOFACUTERESPIRATORYTRACTINFECTIONSINCHILDREN * )NFECT$ISn 0OEHLING+! 'RIFFIN-2 $ITTUS23 4ANG97 (OLLAND+ ,I( %DWARDS+ "EDSIDEDIAGNOSISOFINFLUENZAVIRUSINFECTIONSINHOSPITALIZEDCHILDREN 0EDIATRICSn "ONNER!" -ONROE+7 4ALLEY,) +LASNER!% +IMBERLIN$7 )MPACT OF THE RAPID DIAGNOSIS OF INFLUENZA ON PHYSICIAN DECISION MAKING AND PATIENT MANAGEMENTINTHEPEDIATRICEMERGENCYDEPARTMENT2ESULTSOFARANDOMIZED PROSPECTIVE CONTROLLEDTRIAL0EDIATRICSn 3HARMA6 $OWD-$ 3LAUGHTER !* 3IMON3$ %FFECTOFRAPIDDIAGNOSIS OFINFLUENZAVIRUSTYPE!ONTHEEMERGENCYDEPARTMENTMANAGEMENTOFFEBRILE INFANTSANDTODDLERS !RCH0EDIATR!DOLESC-ED n 3CHRAG3* 3HAY$+ 'ERSHMAN+ 4HOMAS! #RAIG!3 3CHAFFNER7 (ARRISON ,( 6UGIA$ #LOGHER0 ,YNFIELD2ETAL %MERGING)NFECTIONS0ROGRAM


2ESPIRATORY $ISEASES !CTIVITY -ULTISTATE SURVEILLANCE FOR LABORATORY CON FIRMED INFLUENZA ASSOCIATED HOSPITALIZATIONS IN CHILDREN n 0EDIATR )NFECT$IS*n * 'RIJALVA #' #RAIG !3 $UPONT 7$ "RIDGES #" 3CHRAG 3* )WANE -+ 3CHAFFNER7 %DWARDS+- 'RIFFIN-2 %STIMATINGINFLUENZAHOSPITAL IZATIONSAMONGCHILDREN %MERG)NFECT$ISn -ULLOOLY*0 "ARKER7( )MPACTOFTYPE!INFLUENZAON CHILDREN!RET ROSPECTIVESTUDY !M*0UBLIC(EALTHn 4HOMPSON 77 3HAY $+ 7EINTRAUB % "RAMMER , "RIDGES #" #OX .* &UKUDA + )NFLUENZA ASSOCIATED HOSPITALIZATIONS IN THE 5NITED 3TATES *!-!n /"RIEN -! 5YEKI 4- 3HAY $+ 4HOMPSON 77 +LEINMAN + -C!DAM ! 9U 8* 0LATT 2 ,IEU4! )NCIDENCE OF OUTPATIENT VISITS AND HOSPI TALIZATIONS RELATED TO INFLUENZA IN INFANTS AND YOUNG CHILDREN 0EDIATRICS n .EUZIL +- :HU 9 'RIFFIN -2 %DWARDS +- 4HOMPSON *- 4OLLEFSON 3* 7RIGHT0& "URDENOFINTERPANDEMICINFLUENZAINCHILDREN YOUNGERTHAN YEARS! YEARPROSPECTIVESTUDY *)NFECT$IS n 'LEZEN70 'REENBERG 3" !TMAR 2, 0IEDRA 0! #OUCH 2" )MPACT OFRESPIRATORYVIRUSINFECTIONSONPERSONSWITHCHRONICUNDERLYINGCONDITIONS *!-!n -ONTES - 6ICENTE $ 0ÏREZ 9ARZA %' #ILLA ' 0ÏREZ 4RALLERO % )NFLUENZA RELATEDHOSPITALISATIONSAMONGCHILDRENAGEDLESSTHANYEARSOLDIN THE"ASQUE#OUNTRY 3PAIN! YEARSTUDY*ULYn*UNE 6ACCINE n #HIU33 ,AU9, #HAN+( 7ONG7( 0EIRIS*3 )NFLUENZA RELATEDHOS PITALIZATIONSAMONGCHILDRENIN(ONG+ONG .%NGL*-EDn "HAT . 7RIGHT *' "RODER +2 -URRAY %, 'REENBERG -% 'LOVER -* ,IKOS!- 0OSEY$, +LIMOV! ,INDSTROM3%ETAL )NFLUENZA3PECIAL )NVESTIGATIONS4EAM)NFLUENZA ASSOCIATEDDEATHSAMONGCHILDRENINTHE5NITED 3TATES n .%NGL*-EDn -ORISHIMA4 4OGASHI4 9OKOTA3 /KUNO9 -IYAZAKI# 4ASHIRO- /KABE. #OLLABORATIVE3TUDY'ROUPON)NFLUENZA !SSOCIATED%NCEPHALOPATHYIN *APAN%NCEPHALITISANDENCEPHALOPATHYASSOCIATEDWITHANINFLUENZAEPIDEMIC IN*APAN #LIN)NFECT$ISn 3URTEES2 $E3OUSA# )NFLUENZAVIRUSASSOCIATEDENCEPHALOPATHY !RCH $IS#HILDn &RANK !, 4ABER ,( 7ELLS #2 7ELLS *- 'LEZEN 70 0AREDES ! 0ATTERNSOFSHEDDINGOFMYXOVIRUSESANDPARAMYXOVIRUSESINCHILDREN *)NFECT $IS n .EUZIL +- (OHLBEIN # :HU9 )LLNESS AMONG SCHOOLCHILDREN DURING INFLUENZA SEASON %FFECT ON SCHOOL ABSENTEEISM PARENTAL ABSENTEEISM FROM WORK AND SECONDARY ILLNESS IN FAMILIES !RCH 0EDIATR!DOLESC -ED n 'LEZEN70 #OUCH 2" )NTERPANDEMIC INFLUENZA IN THE (OUSTON AREA n .%NGL*-EDn

+ATHRYN-%DWARDS

2UBEN &, )NACTIVATED INFLUENZA VIRUS VACCINES IN CHILDREN #LIN )NFECT $ISn :ANGWILL +- "ELSHE 2" 3AFETY AND EFFICACY OF TRIVALENT INACTIVATED INFLUENZA VACCINE IN YOUNG CHILDREN ! SUMMARY FOR THE NEW ERA OF ROUTINE VACCINATION 0EDIATR)NFECT$IS** n *EFFERSON 4 3MITH 3 $EMICHELI 6 (ARNDEN ! 2IVETTI ! $I 0IETRANTONJ # !SSESSMENT OF THE EFFICACY AND EFFECTIVENESS OF INFLUENZA VACCINES IN T HEALTHYCHILDREN3YSTEMATICREVIEW,ANCETn .EGRI% #OLOMBO# 'IORDANO, 'ROTH. !POLONE' ,A6ECCHIA# )NFLUENZAVACCINEINHEALTHYCHILDREN!META ANALYSIS6ACCINEn -ANZOLI , 3CHIOPPA & "OCCIA ! 6ILLARI 0 4HE EFFICACY OF INFLUENZA VACCINE FOR HEALTHY CHILDREN! META ANALYSIS EVALUATING POTENTIAL SOURCES OF VARIATIONINEFFICACYESTIMATESINCLUDINGSTUDYQUALITY 0EDIATR)NFECT$IS** n .EUZIL +- $UPONT7$ 7RIGHT 0& %DWARDS +- %FFICACY OF INACTI VATED AND COLD ADAPTED VACCINES AGAINST INFLUENZA! INFECTION TO 4HEPEDIATRICEXPERIENCE 0EDIATR)NFECT$IS** n (OBERMAN! 'REENBERG$0 0ARADISE*, 2OCKETTE(% ,AVE*2 +EARNEY$( #OLBORN$+ +URS ,ASKY- (ARALAM-! "YERS#*ETAL %FFECTIVENESS OFINACTIVATEDINFLUENZAVACCINEINPREVENTINGACUTEOTITISMEDIAINYOUNGCHIL DREN!RANDOMIZEDCONTROLLEDTRIAL*!-!n #LEMENTS $! ,ANGDON , "LAND # 7ALTER % )NFLUENZA ! VACCINE DECREASES THE INCIDENCE OF OTITIS MEDIA IN TO MONTH OLD CHILDREN IN DAY CARE!RCH0EDIATR!DOLESC-ED n %DWARDS+- $UPONT7$ 7ESTRICH-+ 0LUMMER7$*R 0ALMER03 7RIGHT 0& !RANDOMIZEDCONTROLLEDTRIALOFCOLD ADAPTEDANDINACTIVATEDVAC CINESFORTHEPREVENTIONOFINFLUENZA!DISEASE*)NFECT$ISn (AMBIDGE3* 'LANZ*- &RANCE%+ -C#LURE$ 8U3 9AMASAKI + *ACKSON , -ULLOOLY*0 :ANGWILL+- -ARCY3-ETAL 3AFETYOFTRIVALENTINACTI VATEDINFLUENZAVACCINEINCHILDRENTOMONTHSOLD*!-! n &RANCE%+ 'LANZ*- 8U3 $AVIS2, "LACK3" 3HINEFIELD(2 :ANGWILL+- -ARCY 3- -ULLOOLY *0 *ACKSON ,! #HEN 2 3AFETY OF THE TRIVALENT INACTIVATEDINFLUENZAVACCINEAMONGCHILDREN!POPULATION BASEDSTUDY!RCH 0EDIATR!DOLESC-EDn "ELSHE2" -ENDELMAN0- 4REANOR* +ING* 'RUBER7# 0IEDRA0 "ERNSTEIN $) (AYDEN&' +OTLOFF+ :ANGWILL+ETAL 4HEEFFICACYOFLIVEATTENU ATED COLD ADAPTED TRIVALENT INTRANASAL INFLUENZAVIRUS VACCINE IN CHILDREN . %NGL*-ED n "ERGEN2 "LACK3 3HINEFIELD( ,EWIS% 2AY0 (ANSEN* 7ALKER2 (ESSEL 7 # #ORDOVA * -ENDELMAN 0- 3AFETY OF COLD ADAPTED LIVE ATTENUATED INFLUENZAVACCINEINALARGECOHORTOFCHILDRENANDADOLESCENTS0EDIATR)NFECT $IS** n !SHKENAZI 3 6ERTRUYEN ! !RÓSTEGUI * %SPOSITO 3 -C+EITH $$ +LEMOLA 4 "IOLEK * +àHR * "UJNOWSKI 4 $ESGRANDCHAMPS $ ET AL 3UPERIOR RELATIVEEFFICACYOFLIVEATTENUATEDINFLUENZAVACCINECOMPAREDWITHINACTIVATED INFLUENZAVACCINEINYOUNGCHILDRENWITHRECURRENTRESPIRATORY TRACTINFECTIONS 0EDIATR)NFECT$IS*n *


&LEMING$- #ROVARI0 7AHN5 +LEMOLA4 3CHLESINGER9 ,ANGUSSIS! YMAR + 'ARCIA -, +RYGIER ! #OSTA ( ET AL #OMPARISON OF THE EFFICACY AND SAFETY OF LIVE ATTENUATED COLD ADAPTED INFLUENZA VACCINE TRIVALENT WITH TRIVALENT INACTIVATED INFLUENZA VIRUS VACCINE IN CHILDREN AND ADOLESCENTS WITH ASTHMA 0EDIATR)NFECT$IS*n * 4AM *3 #APEDING -2 ,UM ,# #HOTPITAYASUNONDH4 *IANG : (UANG ,- ,EE"7 1IAN9 3AMAKOSES2 ,OLEKHA3ETAL %FFICACYANDSAFETYOFA LIVE ATTENUATED COLD ADAPTED INFLUENZA VACCINE TRIVALENT AGAINST CULTURE CON FIRMEDINFLUENZAINYOUNGCHILDRENIN!SIA 0EDIATR)NFECT$IS** n "ELSHE 2" %DWARDS +- 6ESIKARI 4 "LACK 36 7ALKER 2% (ULTQUIST - +EMBLE ' #ONNOR %- #!)6 4 #OMPARATIVE %FFICACY 3TUDY 'ROUP ,IVEATTENUATED VERSUS INACTIVATEDINFLUENZAVACCINEININFANTSANDYOUNGCHIL DREN .%NGL*-EDn 0IEDRA0! 'AGLANI-* 2IGGS- (ERSCHLER' &EWLASS# 7ATTS- +OZINETZ# (ESSEL# 'LEZEN70 ,IVEATTENUATEDINFLUENZAVACCINE TRIVALENT ISSAFE INHEALTHYCHILDRENMONTHSTOYEARS TOYEARS AND TOYEARSOFAGEIN ACOMMUNITY BASED NONRANDOMIZED OPEN LABELTRIAL0EDIATRICSEn 2EICHERT4! 3UGAYA. &EDSON$3 'LEZEN70 3IMONSEN, 4ASHIRO- 4HE*APANESEEXPERIENCEWITHVACCINATINGSCHOOLCHILDRENAGAINSTINFLUENZA . %NGL*-EDn +ING*#*R 3TODDARD** 'AGLANI-* -OORE+! -AGDER, -C#LURE% 2UBIN *$ %NGLUND*! .EUZIL+ +ING*#ETAL %FFECTIVENESSOFF SCHOOL BASED INFLUENZAVACCINATION .%NGL*-EDn HTTPWWWCDCGOVMMWRPREVIEWMMWRHTMLMMAHTM *ACKSON,! .EUZIL+- "AGGS* $AVIS2, "LACK3 9AMASAKI +- "ELONGIA % :ANGWILL+- -ULLOOLY* .ORDIN*ETAL #OMPLIANCEWITHTHERECOM MENDATIONS FOR DOSES OF TRIVALENT INACTIVATED INFLUENZA VACCINE IN CHILDREN LESSTHANYEARSOFAGERECEIVINGINFLUENZAVACCINEFORTHEFIRSTTIME!6ACCINE 3AFETY$ATALINKSTUDY0EDIATRICSn #ARPENTER ,2 ,OTT * ,AWSON "- (ALL 3 #RAIG!3 3CHAFFNER7 *ONES4& -ASSDISTRIBUTIONOFFREE INTRANASALLYADMINISTEREDINFLUENZAVACCINEIN F APUBLICSCHOOLSYSTEM0EDIATRICSEn


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

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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!


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;=

4IMMUNITYANDTHEINFLUENZA!VIRUSES $ENDRITICCELLS $#S ENABLE PATHOGEN DERIVED ANTIGENS TO BE PRESENTED IN A CONTEXT THAT FACILITATES SUCCESSFUL4 CELL IMMUNITY ;= 3PECIALIZED IN ANTIGEN PRESENTA TION THE $#S FACILITATE I THE ACQUISITION OF ANTIGEN II PROCESSING AND PRESENTATIONOFANTIGENICPEPTIDESINTHECONTEXTOFHOSTMAJOR HISTOCOMPAT IBILITYCOMPLEX-(# MOLECULESANDIII THEPROVISIONOFCOSTIMULATORYSIG NALS)MMUNITYTOINFLUENZA!VIRUSINFECTIONREQUIRES$#FORBOTHPRIMARY ;=ANDSECONDARY4CELLRESPONSES; =$#CANCONTROLTHEMAGNITUDE OFINFLUENZA!VIRUS SPECIFIC4CELLIMMUNITYVIA&ASLIGAND&AS, MEDIATED APOPTOSIS ;= )N THE RESPIRATORY TRACT AN EXTENSIVE NETWORK OF $# POPU LATIONS IS PRESENT BOTH IN THE LUNG ;= AND THE DRAINING LYMPH NODE ;= &URTHERMORE PULMONARY INFECTION RECRUITS ADDITIONAL $# POPULATIONS INTO THE LUNG ;n=4O ACQUIRE INFLUENZA! VIRUS ANTIGEN $# MAY SIMPLY BE DIRECTLYINFECTEDWITHTHEVIRUS)NFECTIONINDUCESTHEMATURATIONALCHANGES UP REGULATIONOFCOSTIMULATORYMOLECULESAND-(#CLASS)) THATARENEC ESSARY FOR $# STIMULATION OF 4 CELLS ;n= )NFECTION CAN RESULT IN THE EXPRESSIONOFINFLUENZA.!ATTHE$#SURFACE WITH.! MEDIATEDREMOVAL OFSIALICACIDSSERVINGTOBOTHENHANCE ANDINHIBIT $#FUNCTIONDEPENDING ON THE MULTIPLICITY OF INFECTION ; = $#S CAN ALSO ACQUIRE INFLUENZA ! VIRUS DERIVED ANTIGEN RELEASED FOLLOWING THE APOPTOTIC LYSIS OF INFECTED RESPIRATORYCELLS; =/NCEANTIGENISACQUIRED LUNG$#SMIGRATETOTHE LYMPHNODETHATDRAINSTHERESPIRATORYTRACT; =-IGRATIONOCCURS EARLYAFTERINFECTIONnH AFTERWHICHTHE$#DISPLAYAREFRACTORYSTATE TOFURTHERINFLAMMATORYSTIMULI;=4HELYMPHNODEALSOCONTAINSARESIDENT $#SETTHATHASNODIRECTACCESSTOTHEAIRWAYS$ESPITETHIS THESERESIDENT $#S CAN ALSO PRESENT INFLUENZA ! VIRUS DERIVED ANTIGEN ;= 4HEREFORE 4 ANTIGENTRANSFERBETWEENTHERESIDENTANDMIGRATORYLUNG$#SUBSETSMUST OCCUR; =-OSTEXPERIMENTSINDICATETHAT-(#CLASS)PRESENTATION


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


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;=

#$4CELLS 6IRUS SPECIFIC#$4CELLSAREIMPORTANTPARTICIPANTSININFLUENZAIMMU NITY ; =!LTHOUGH ACTING ALONE THESE CELLS DO NOT NORMALLY ELIMI NATE VIRUS ;= THEY EXERT DISTINCT ROLES IN BOTH HUMORAL IMMUNITY AS DISCUSSED AND#$4CELLRESPONSES!VIGOROUS HETEROGENEOUS#$ 4 CELLRESPONSEISELICITEDFOLLOWINGINFLUENZA!VIRUSINFECTION ;=!GAIN THEPROCESSOFCLONALEXPANSIONANDDIFFERENTIATIONSISINITIATEDINTHELUNG DRAINING LYMPH NODE WITH THE PEAK RESPONSE IN THE RESPIRATORY AIRWAYS OCCURRINGnDAYSFOLLOWINGINFECTION;=4HISISDOMINATED BYPRODUC ERSOFTHE4HCYTOKINES SUCHAS), )&. a AND4.& _ WHILE4HCELLSDO NOT PROVIDE ANY PROTECTION ;= &OLLOWING INFLUENZA! VIRUS CLEARANCE #$ 4 CELLS DEMONSTRATE INCREASED CONTRACTION IN THE RESPIRATORY TRACT COMPARED TO INFLUENZA! VIRUS SPECIFIC #$ 4 CELLS ; =! MAJOR ROLE FOR #$ 4 CELLS IS THE PROVISION OF @HELP FOR OPTIMAL #$ 4 CELL IMMUNITY!LTHOUGH#$4CELLSARENOTREQUIREDFORPRIMARYINFLUENZA SPECIFIC#$ 4CELLRESPONSES PRESUMABLYDUETOTHEDIRECTACTIVATIONOF $#BYVIRALINFECTION;n= THEYARECRITICALFORTHEOPTIMALESTABLISH MENTOF#$4CELLMEMORY4HEABSENCEOF#$4CELLSDURINGPRIMARY INFLUENZA!VIRUSINFECTIONSLEADSTOASIGNIFICANTREDUCTIONINTHESIZEAND MAGNITUDE OF THE SECONDARY RESPONSE AND IMPAIRED VIRAL CLEARANCE ; =!CTIVATIONOF#$4CELLSREQUIRESANTIGEN SPECIFICSIGNALING VIA4#2 RECOGNITIONOFANTIGENSPRESENTEDINTHECONTEXTOF-(#CLASS))MOLECULES 5NTILRECENTLY THESPECTRUMOFINFLUENZA!VIRUS#$ 4CELLEPITOPESWAS MUCH LESS WELL CHARACTERIZED THAN THE PANEL KNOWN FOR THE #$ SUBSET 2ECENTLY HOWEVER nPEPTIDESWEREIDENTIFIEDFORTHEINFLUENZA SPECIFIC #$ 4 CELL RESPONSE IN #", MICE WITH THE MAJORITY BEING DERIVED FROMTHE.0AND(!PROTEINS;=4HEREISSOMEEVIDENCETHAT INFLUENZA -(#CLASS))EPITOPESPERSISTFORASUBSTANTIALINTERVALAFTERTHEVIRUSHAS BEENCLEAREDFROMTHEHOST;=/VERALL THEADAPTIVEIMMUNE RESPONSETO THEINFLUENZA!VIRUSESINVOLVESCOMPLEXINTERACTIONSBETWEENASPECTRUM OFFUNCTIONALLYDIFFERENTCELLTYPES

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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; =


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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*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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&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


&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 ;=


&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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4ABLE3ENSITIVITIESANDSPECIFICITIESOFSEROLOGICALASSAYS FORDETECTIONOFANTIBODIESTO(. VIRUS !GEGROUPA

0ARAMETERB

6ALUES )NDIVIDUALSEROLOGICALTESTSC

#HILD

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#OMBINATIONOFTESTSD

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3ENSITIVITY N

3PECIFICITY N

3ENSITIVITY N

3PECIFICITY N

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A3ERUMSAMPLESFROMINDIVIDUALSnYEARSOFAGE#HILD ORFROMINDIVIDUALSnYEARSOF

AGE!DULT B3ENSITIVITY NUMBEROF(.VIRUS INFECTEDPATIENTSTESTINGPOSITIVEFORANTIBODYDIVIDEDBY

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 ;=

ASSAYHASNOCORRELATEOFPROTECTION)F(!ISPRODUCEDUSINGA BACULOVIRUS EXPRESSIONSYSTEM FALSEPOSITIVEREACTIVITYCANBEFOUNDDUETOCONTAMINAT INGBACULOVIRUSINSECTCELLPROTEINS7HENCOMBINEDWITH7ESTERNBLOTTING 7" %,)3!SHOWSIMPROVEDSPECIFICITYANDRETAINSIMPROVEDSENSITIVITY COMPAREDWITHTHE-.ASSAYAND7"COMBINATION; = 7"ISUSEFULONLYFORCONFIRMATION4HISTECHNIQUEISTOOLABOR INTENSIVE TOBECONSIDEREDADIAGNOSTICTESTFORSCREENINGSEVERALTHOUSANDSERA AND ISGENERALLYUSEDASASECONDARYSEROLOGICALTESTTOCONFIRMOTHER%-%! ACCEPTEDSEROLOGICALTESTS IE -.ASSAYOR%,)3! 4HESENSITIVITIESOF7"AND%,)3!AREGENERALLYHIGHERTHAN-. AND SPECIFICITIES OF -. ARE HIGHER THAN7" AND %,)3! PARTICULARLY FOR PAN DEMICSTRAINS4AB 4ODETERMINEWHETHERTHE-.ASSAYANDOR%,)3! COULD BE USED TO DETECT ( SPECIFIC ANTIBODY IN SINGLE SERUM SAMPLES THE RELATIVESENSITIVITIESANDSPECIFICITIESOFTHEASSAYSWERECOMPARED4ABLE 4HETESTUSINGTHE-.ASSAYWASNOTABLYSUPERIORTOTHATOF%,)3!7HEN COMBINED WITH7" EACH TEST IMPROVED IN SPECIFICITY HOWEVER MAXIMUM


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


&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;=

ANTIBODYTOINTERNALVIRUSANTIGENSINADDITIONTOTHOSEANTIBODIESDIRECTED AGAINSTSURFACEGLYCOPROTEINS ANDMAYLACKSPECIFICITYFORTHEDETECTIONOF ANTIBODIESTO(!(OWEVER INTERPRETATIONOFRESULTSISCOMPLICATEDASTHE RELATIONSHIP BETWEEN (!) TITER AND THE HEMOLYTIC AREA OBTAINED MAY NOT BEEASYTOREAD

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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

2EFERENCES

4REANOR* )NFLUENZAVACCINEn/UTMANEUVERINGANTIGENIC SHIFTANDDRIFT .%NGL*-EDn 7EBBY2* 7EBSTER2' !REWEREADYFORPANDEMICINFLUENZA 3CIENCE n "ARKER 7( -ULLOOLY *0 )MPACT OF EPIDEMIC TYPE ! INFLUENZA IN A DEFINEDADULTPOPULATION !M*%PIDEMIOLL n "ARKER7( %XCESSPNEUMONIAANDINFLUENZA ASSOCIATEDHOSPITALIZATION DURINGINFLUENZAEPIDEMICSINTHE5NITED3TATES n !M*0UBLIC(EALTH n $ORRELL , (ASSAN ) -ARSHALL 3 #HAKRAVERTY 0 /NG % #LINICAL AND SEROLOGICAL RESPONSES TO AN INACTIVATED INFLUENZA VACCINE IN ADULTS WITH ()6 INFECTION DIABETES OBSTRUCTIVEAIRWAYSDISEASE ELDERLYADULTSANDHEALTHYVOL UNTEERS )NT*34$!)$3n +àNZEL7 'LATHE( %NGELMANN( 6AN(OECKE# +INETICSOFHUMORAL ANTIBODY RESPONSE TO TRIVALENT INACTIVATED SPLIT INFLUENZA VACCINE IN SUBJECTS PREVIOUSLYVACCINATEDORVACCINATEDFORTHEFIRSTTIME6ACCINEn 3TEPHENSON) .ICHOLSON+' )NFLUENZA6ACCINATIONANDTREATMENT%UR 2ESPIR*n #OUCH 2" +ASEL *! 'LEZEN 70 #ATE 42 3IX (2 4ABER ,( &RANK !, 'REENBERG3" :AHRADNIK*- +EITEL7! )NFLUENZA)TSCONTROLINPER SONSANDPOPULATIONS*)NFECT$ISn


"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

%MANUELE-ONTOMOLI

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


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


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


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


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;=


4ABLE4HEUSEOFTHEMOUSEMODELFORTHEEVALUATIONOFVACCINESAGAINSTINFLUENZA )NFLUENZAVIRUS SUBTYPESTESTED

&INDINGS

2EFS

(UMAN

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

#LINICALSIGNSINCLUDERUFFLEDFUR HUNCHING LABOREDBREATHING UNSTEADYGAIT HYPOTHERMIAANDWEIGHTLOSS

)NFLAMMATIONISOBSERVEDINTHERESPIRATORYTRACT

; =

2ECONSTRUCTED #AUSESILLNESSINMICEANDREPLICATESEFFICIENTLYINTHE (. RESPIRATORYTRACTWITHOUTPRIORADAPTATION PANDEMICVIRUS 5PTOLOSSOFBODYWEIGHTISOBSERVED

,ETHALTOMICEWITHAN-$4OFDAYS

.OEXTRAPULMONARYSPREADOBSERVED

.ECROTIZINGBRONCHITISANDBRONCHIOLITIS ANDMODERATETO SEVEREPERIBRONCHIALANDALVEOLAREDEMAPRESENT

;=

(0!)(.

-OSTISOLATESCAUSESEVEREILLNESSANDDEATHWITHOUTPRIOR ;n ADAPTATION

2EPLICATEEFFICIENTLYINTHERESPIRATORYTRACTWITHOUTPRIOR = ADAPTATION

#AUSESIGNIFICANTWEIGHTLOSS

-OSTISOLATESARELETHALINMICEWITHA-$4OFnDAYS

3OMEISOLATESAREDETECTEDINEXTRAPULMONARYSITESINCLUDING THEBRAIN

6ARIABLEVIRULENCEINMICEISOBSERVEDWITHISOLATESFROM(ONG +ONGFROM ANDn ANDVIRUSESISOLATEDFROM %UROPEAND3OUTH!MERICA

(

(0AND,0ISOLATESREPLICATEEFFICIENTLYINRESPIRATORY TRACT OFMICEWITHOUTPRIORADAPTATION WITHSOMEVIRUSESCAUSING WEIGHTLOSSANDDEATH

%XTRAPULMONARYSPREADTOTHEBRAINANDSPLEENOBSERVED FOLLOWINGINTRANASALINFECTIONWITHSOMEISOLATES

(ISTOPATHOLOGICALOBSERVATIONSFOLLOWINGINTRANASALINFEC TIONSWITHHUMANISOLATESINCLUDENECROSISANDINFLAMMATION THROUGHOUTTHERESPIRATORYTRACT BUTNOLESIONSINTHEBRAIN HEART SPLEEN LIVERORKIDNEYS

(ISTOPATHOLOGICALLESIONSAREOBSERVEDFOLLOWINGINTRANASAL INFECTIONWITH(0AVIANISOLATES

(.

2EPLICATEEFFICIENTLYINLUNGSOFMICEWITHOUTPRIOR ADAPTATION ;

#ONFLICTINGREPORTSOFLETHALITYINMICE n

!DAPTATIONBYPASSAGEINMOUSELUNGSRESULTSININCREASED = VIRULENCE

2EPLICATIONINBRAINREPORTEDFOLLOWINGINTRANASALINFECTION WITHNON ADAPTEDANDMOUSE ADAPTEDVIRUSES

(.

!TEAL7(+(. REPLICATESEFFICIENTLYWITHOUTPRIOR ;= ADAPTATIONANDISLETHALFORMICEWHENADMINISTEREDATHIGH TITERS

3IGNIFICANTWEIGHTLOSSAVERAGE ISOBSERVEDININFECTED MICE

!DAPTATIONTOMICERESULTSININCREASEDVIRULENCEANDSPREAD TOBRAIN

;n =

-$4 MEAN TIME TO DEATH (0!) HIGHLY PATHOGENIC AVIAN INFLUENZA (0 HIGHLY PATHOGENIC ,0 LOWPATHOGENICITY


4ABLE4HEUSEOFTHEFERRETMODELFORTHEEVALUATIONOFVACCINESAGAINSTINFLUENZA )NFLUENZAVIRUS SUBTYPESTESTED

&INDINGS

(UMANINFLUENZA (. (. (. VIRUSES

%FFICIENTREPLICATIONOFNON ADAPTEDISOLATESIN ; RESPIRATORYTRACT =

)SOLATEDREPORTOFTHEPRESENCEOFAN(.HUMAN INFLUENZAVIRUSINTHEBRAIN

3IGNSOFILLNESSINCLUDEFEVER SNEEZING RHINORRHEA AND WEIGHTLOSS

-ILDINFLAMMATORYCHANGESAREOBSERVEDUPONHIS TOPATHOLOGICALEXAMINATIONOFLUNGSOFINFECTEDANIMALS

2ECONSTRUCTED 2EPLICATIONTOHIGHTITERSINRESPIRATORYTRACT (.PANDEMICVIRUS 3EVEREDISEASEOBSERVEDINCLUDINGLETHARGY ANOREXIA SEVEREWEIGHTLOSSANDHIGHFEVER

)NFECTIONISLETHALINOFINOCULATEDANIMALSDEATH OCCURSBYDAY

6IRUSISNOTDETECTEDINBRAINORHEART

.ECROTIZINGBRONCHIOLITIS ANDMODERATETOSEVERE ALVEOLITISWITHEDEMAOBSERVEDUPONHISTOPATHOLOGICAL EXAMINATION (0!)(.

2EFS

;=

%FFICIENTREPLICATIONINRESPIRATORYTRACTANDEVIDENCEOF ; EXTRAPULMONARYSPREADTOBRAIN SPLEENANDINTESTINES =

-OSTISOLATESCAUSESEVEREDISEASE INCLUDINGFEVER RHINITIS SNEEZING SEVERELETHARGY HINDLIMBPARESISAND DIARRHEA

-ANYISOLATESCAUSELETHALINFECTIONINFERRETS

(ISTOPATHOLOGICALOBSERVATIONSINCLUDEINFLAMMATORY CHANGESINTHELUNGSBRONCHIOLOITIS BRONCHITIS INTER STITIALPNEUMONIA ANDINFLAMMATIONINTHEBRAIN

!)SUBTYPES(.

%FFICIENTREPLICATIONINTHEUPPERRESPIRATORYTRACT (. (. (. .OSIGNSOFILLNESSWITHANYOFTHESEISOLATES (. (. (. (. (. SEAL (.ISOLATE

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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

3TRAIN SPECIFICIMMUNITYDIRECTEDAGAINSTTHE(! )T IS WELL ESTABLISHED THAT THE PRIMARY CORRELATE OF PROTECTION FOR INACTI VATEDWHOLE VIRUSORSUBUNITINFLUENZAVACCINESADMINISTEREDPARENTERALLYIS SERUMANTIBODYDIRECTEDAGAINSTTHE(!PROTEIN-OSTSTUDIESTHATARECON DUCTEDTOEVALUATEIMMUNERESPONSESTOINFLUENZAVACCINESARECONDUCTED INMICEANDFERRETS-EASUREMENTOFANTIBODYRESPONSESINANIMALMODELS ISVERYSTRAIGHTFORWARD SINCE(!)ANDNEUTRALIZINGANTIBODYASSAYSDONOT REQUIRE SPECIES SPECIFIC REAGENTS ,IMITED STUDIES HAVE BEEN CONDUCTED TO EVALUATE THE GUINEA PIG AS A MODEL TO STUDY IMMUNITY TO INFLUENZA VIRUS 0HAIR AND COLLEAGUES ;= DEMONSTRATED THAT INFECTION OF GUINEA PIGS WITH UNADAPTEDHUMANINFLUENZAVIRUSESRESULTEDINRESISTANCETOCHALLENGEWITH HOMOLOGOUSVIRUS ANDTHATPASSIVETRANSFEROFHYPERIMMUNESERUMTONAIVE GUINEAPIGSALSOCONFERREDPROTECTIONAGAINSTINFECTION(OWEVER THELEVELS OF (!) ANTIBODY DETECTED IN SERUM FOLLOWING INFECTION WERE LOWER THAN THOSEOBSERVEDINFERRETSORHAMSTERS ANDINFECTEDGUINEAPIGSDIDNOTPRO DUCEDETECTABLELEVELSOFLOCALANTIBODYINNASALSECRETIONS)NADDITION HIGH LEVELSOFNONSPECIFICINHIBITORSOFHEMAGGLUTINATIONWEREPRESENTINGUINEA


PIGSERA MAKINGMEASUREMENTOFSPECIFIC(!)ANTIBODIESPROBLEMATIC;= 0HAIRETALDID HOWEVER DEMONSTRATETHATGUINEAPIGSEXHIBITEDADELAYED TYPE HYPERSENSITIVITY RESPONSE TO INFLUENZA INFECTION THAT RESEMBLED THAT SEEN IN HUMANS ALTHOUGH THIS RESPONSE DID NOT APPEAR TO BE INVOLVED IN RESISTANCETOINFECTION (UMORALIMMUNERESPONSESTOTHE(!OFHUMANINFLUENZAVIRUSESAND VACCINESHAVEBEENSTUDIEDEXTENSIVELYINFERRETS%ARLYSTUDIESDETERMINED THATNAIVEFERRETSWERENOTPROTECTEDAGAINSTINFLUENZAINFECTIONBYVACCINA TIONWITHKILLEDVIRUS;=4HESEOBSERVATIONSWERECONFIRMED INLATERSTUD IESUSINGFORMALIN INACTIVATEDVACCINES;=(OWEVER KILLEDVACCINEADMIN ISTERED WITH ADJUVANT TO NAIVE FERRETS PROVIDED PARTIAL PROTECTION AGAINST INFECTION ;= 4HUS IMMUNE RESPONSES IN THE FERRET TO VACCINATION WITH INACTIVATEDVIRUSVACCINESAGAINSTHUMANINFLUENZAVIRUSESDONOTAPPEARTO BEIDENTICALTOTHOSESEENINHUMANS SINCEHUMANSDONOTGENERALLYREQUIRE ADJUVANTTOACHIEVEPROTECTIVELEVELSOF(!)ANTIBODIES)NCONTRASTTOTHE FINDINGSWITHINACTIVATEDINFLUENZAVIRUSES IMMUNIZATIONWITH LIVEINFLUENZA VIRUSRESULTEDINPROTECTIONAGAINSTSUBSEQUENTCHALLENGE;= !NEXPLANA TIONFORTHISDIFFERENCEMAYBETHATINFERRETS INFLUENZAINFECTIONISPRIMARILY ANUPPERRESPIRATORYTRACTINFECTION ANDADJUVANTISREQUIRED TOELICITHIGHER LEVELSOFSERUMANTIBODYNEEDEDTORESTRICTREPLICATIONOFVIRUSINTHEUPPER RESPIRATORY TRACT 3EVERAL STUDIES HAVE DEMONSTRATED THAT HIGHER LEVELS OF SERUM ANTIBODY ARE REQUIRED TO PROVIDE PROTECTION AGAINST RESPIRATORY VIRUSESINTHENOSEOFANIMALSTHANINTHELUNGS;n=

(ETEROSUBTYPICIMMUNITY )NRECENTYEARS ANDPARTICULARLYSINCETHEEMERGENCEOFTHEHIGHLYPATHO GENIC(.VIRUSESIN!SIAIN ANDTHECHALLENGESINDEVELOPING(. VACCINES THEREHASBEENARESURGENCEOFINTERESTINHETEROSUBTYPICIMMUNITY nTHEABILITYOFANIMMUNERESPONSEELICITEDBYAPARTICULARINFLUENZA!VIRUS TOPROTECTAGAINSTANINFLUENZA!VIRUSOFADIFFERENTSUBTYPE(ETEROSUBTYPIC IMMUNITYAGAINSTINFLUENZAHASBEENDEMONSTRATEDINANUMBEROFF STUDIES IN MICE BUT THE PRECISE MECHANISM OF THIS IMMUNITY IS NOT CLEAR ;n= 0REVIOUSLY ITWASTHOUGHTTHATTHISPHENOMENONWASMEDIATEDBYCELLULAR IMMUNERESPONSES BUTRECENTSTUDIESSUGGESTTHATANTIBODYISTHEPRIMARY MECHANISM OF HETEROSUBTYPIC IMMUNITY ;= AND THAT THE DIVERSITY OF THE ANTIBODYREPERTOIREISIMPORTANT;= (ETEROSUBTYPIC IMMUNITY HAS ALSO BEEN OBSERVED IN FERRETS ; = ALTHOUGHTHEREWASSOMEDEBATEASTOTHELENGTHOFTIMETHATSUCHIMMUNITY PERSISTS-C,ARENAND0OTTER;=REPORTEDTHATITDIDNOTPERSISTBEYOND WEEKS AFTER VACCINATION BUT IN ANOTHER STUDY PROTECTION AGAINST INFECTION WITH A HETEROSUBTYPIC VIRUS WAS OBSERVED MONTHS FOLLOWING IMMUNIZA TION;=)NBOTHCASES HETEROSUBTYPICIMMUNITYDIDNOTPREVENTINFECTION BUTLIMITEDVIRUSREPLICATIONFOLLOWINGCHALLENGE


4HEUTILITYOFTHECOTTONRATMODELTOADDRESSTHEQUESTIONOFHETEROSUB TYPICIMMUNITYWASEXPLORED;=4HEENDPOINTSINTHISSTUDYWERERESPIRA TORYRATE VIRUSREPLICATIONINLUNGSANDNASALTISSUES ANDPULMONARYHISTOPA THOLOGY!STATISTICALLYSIGNIFICANTREDUCTIONINRESPIRATORYRATEWASSEENFOL LOWINGCHALLENGEWITH!7UHAN(. INCOTTONRATSTHATHADBEEN IMMUNIZED WITH EITHER THE HOMOLOGOUS VIRUS OR WITH A VIRUS OF A DIFFERENT SUBTYPE !02 (. WEEKS EARLIER COMPARED TO NON IMMUNIZED ANIMALS4HISREDUCTIONINRESPIRATORYRATECORRELATEDWITHASTATISTICALLYSIG NIFICANTREDUCTIONINVIRUSTITERSINTHELUNGSANDNASALTISSUESINIMMUNIZED ANIMALS#OTTONRATSTHATWEREIMMUNIZEDWITHTHEHETEROSUBTYPIC!02 (. VIRUS HAD THE SAME EXTENT OF ALVEOLITIS INTERSTITIAL PNEUMONIA AND AIRWAYDEBRISASNON IMMUNE INFECTEDANIMALS AND LIKETHECOTTONRATSTHAT WERE IMMUNIZED WITH HOMOLOGOUS VIRUS THEY HAD MORE SEVERE EARLY PERI BRONCHIOLITIS THAN WAS OBSERVED IN PRIMARY INFECTION4HIS PERIBRONCHIOLITIS COULD BE INDICATIVE OF A MEMORY RESPONSE IN THE HETEROSUBTYPIC IMMUNE ANIMALS (OWEVER HETEROSUBTYPIC IMMUNE COTTON RATS HAD LESS BRONCHIOLAR EPITHELIALDAMAGETHANTHOSEANIMALSIMMUNIZEDWITHHOMOLOGOUS VIRUS 4HE ROLE OF HETEROSUBTYPIC IMMUNITY THROUGH PRIOR EXPOSURE OR VAC CINATIONINMAN ALTHOUGHINFERREDFROMRETROSPECTIVEANALYSISOFDATAFROM INFLUENZA PANDEMICS ;= IS EXTREMELY COMPLEX AND CANNOT BE READILY DETERMINED 3TUDIES IN YOUNG INFANTS AND CHILDREN IN WHICH THE EFFECT OF PRE EXISTINGIMMUNITYONREPLICATIONANDIMMUNOGENICITYOFHETEROSUBTYPIC ATTENUATED INFLUENZA VIRUSES SUGGESTED THAT HETEROSUBTYPIC IMMUNITY IN HUMANSISWEAK;=

)MMUNERESPONSESTOOTHERINFLUENZAPROTEINS !NAPPROACHTHATISBEINGEXPLOREDINTHEDEVELOPMENTOFNOVEL VACCINES FORINFLUENZAISTHATOFUNIVERSALINFLUENZAVACCINESTHATTARGETTHECONSERVED PROTEINS OF THE VIRUS n .0 - AND -! NUMBER OF MODALITIES SUCH AS .0AND-$.!VACCINES;n= BACULOVIRUS EXPRESSEDRECOMBINANT- PROTEIN ;= - PEPTIDES ;= AND RECOMBINANT - PROTEIN INCORPORATED INTOHEPATITIS"COREANTIGEN;n=HAVEBEENTESTEDINMICE ANDPREVENT DEATH BUT NOT ILLNESS FOLLOWING CHALLENGE WITH HETEROLOGOUS VIRUS )N THE CASEOFCANDIDATEUNIVERSALVACCINESFORINFLUENZA NEWANIMALMODELSAND ASSAYSARENEEDEDINWHICHANTIBODYANDCELLULARRESPONSESTOVIRALANTIGENS OTHERTHANTHE(!AND.!CANBEMEASURED3INCETHEIMMUNERESPONSES TOTHESECONSERVEDANTIGENSARENOTWELLCHARACTERIZEDINHUMANS ATPRES ENTITISNOTCLEARWHETHERTHESERESPONSESAREACCURATELYREFLECTEDINANIMAL MODELS5NDOUBTEDLYMOREINFORMATIONWILLBEOBTAINEDINTHISAREAINTHE FUTUREASCANDIDATEUNIVERSALVACCINESAREEVALUATEDINCLINICALTRIALS 4HEREHASALSOBEENRECENTINTERESTINTHEROLEOFIMMUNERESPONSESTO THE.!COMPONENTOFSEASONALVACCINESINPROTECTIONAGAINSTRELATEDSUB TYPESOFINFLUENZA INCLUDINGPOTENTIALPANDEMICSTRAINS;= !NTIBODIESTO


THE.!PROTEINCANMODULATETHESEVERITYOFINFLUENZAILLNESS;=BUTTHE .!CONTENTOFINACTIVATEDINFLUENZAVIRUSVACCINESISNOTSTANDARDIZED

%FFICACY !NIMALMODELSAREALSOUSEDTOEVALUATETHEEFFICACYOFNEWCANDIDATEINFLU ENZAVACCINES4HEMOSTCOMMONLYUSEDANIMALMODELSFORSUCHSTUDIESARE MICEANDFERRETS)NMICEANDFERRETSITHASBEENESTABLISHEDTHATANTIBODY AGAINSTTHE(!CANPREVENTINFECTIONORAMELIORATEDISEASEFOLLOWINGCHAL LENGEWITHINFLUENZAVIRUS2EDUCTIONINVIRUSTITERINTHELOWERRESPIRATORY TRACT FOLLOWING CHALLENGE CORRELATES WITH PROTECTION SO QUANTITATIVE VIROL OGYISTHEMOSTRELEVANTMEASUREOFVACCINEEFFICACYFORVACCINESDESIGNED TO GENERATE ANTIBODY RESPONSES TO THE (!!DDITIONAL ENDPOINTS SUCH AS MORBIDITY MORTALITY AND PATHOLOGICAL FINDINGS MAY PROVIDE SUPPORTING EVIDENCEOFPROTECTIONFROMINFECTIONANDDISEASE!LTHOUGHDEMONSTRATION OF VACCINE EFFICACY IN AN ANIMAL MODEL IS NOT AN ABSOLUTE REQUIREMENT IN PRE CLINICALEVALUATIONOFAVACCINECANDIDATEFROMAREGULATORYSTANDPOINT ITPROVIDESEVIDENCETHATIMMUNERESPONSESTOTHEVACCINEAREBIOLOGICALLY RELEVANT

6ACCINESFORPANDEMICINFLUENZA 4HE DIRECT TRANSMISSION OF (0!) (. (. AND LOW PATHOGENICITY!) ,0!) (.VIRUSESFROMBIRDSTOHUMANS ASSOCIATEDINMANYCASESWITH SEVEREMORBIDITYANDMORTALITY HASRAISEDCONCERNSABOUTTHEEMERGENCEOF ANEWPANDEMICVIRUSANDHASPROMPTEDEFFORTSTODEVELOPVACCINESAGAINST !)VIRUSESOFPANDEMICPOTENTIAL%VALUATIONANDCHARACTERIZATIONOFASUIT ABLEANIMALMODELFORTHESEOTHERINFLUENZAVIRUSSUBTYPESISA CRITICALSTEP INTHEDEVELOPMENTOFSUCHVACCINES

!NIMALMODELS )NTHEFOLLOWINGSECTIONWEDESCRIBETHEFEATURESOFTHEANIMALMODELSTHAT HAVE BEEN DEVELOPED TO STUDY !) VIRUSES AND THEIR CONTRIBUTIONS TO THE EVALUATIONOFPANDEMICVACCINES

-ICE -ICEHAVEBEENUSEDINPRE CLINICALSTUDIESOFINACTIVATEDANDLIVEATTENU ATEDPANDEMICINFLUENZAVIRUSVACCINES-OSTREPORTSINTHELITERATURETHAT DESCRIBETHECHARACTERIZATIONOFREPLICATION PATHOGENICITYAND THEIMMUNE


RESPONSEOF!)VIRUSESINMICEFOCUSONVIRUSESOFTHE( (AND(SUB TYPES

(.VIRUSESANDVACCINES 3EVERALSTUDIESDEMONSTRATEDTHATTHE(.VIRUSESISOLATEDFROMHUMAN CASESIN(ONG+ONGINCAUSEDISEASEANDDEATHINMICEWITHOUTPRIOR ADAPTATION;n=4HE(ONG+ONG(.VIRUSESISOLATEDFROM HUMANS INVARIEDINTHEIRABILITYTOCAUSEDISEASEANDDEATHIN"!,"CMICE ANDGENERALLYFELLINTOTWODISTINCTGROUPSnTHOSETHATWEREHIGHLYVIRULENT ANDTHOSEOFLOWVIRULENCEFORMICEnANDONEVIRUS!(+ WASOF INTERMEDIATEVIRULENCEINTWOOFTHESTUDIES; = BUT'AOETAL;= FOUNDTHISISOLATETOBEONEOFTHEMOSTHIGHLYVIRULENTINTHISMODEL4HE LETHALDOSEOF(.VIRUSESTHATWEREHIGHLYVIRULENTFORMICEWERE nTIMESLOWERTHANTHOSEOFLOWVIRULENCE THEYREPLICATEDTOTITERSUP TOTIMESHIGHERINTHELUNGSOFMICEEARLYINTHECOURSEOFINFECTION AND THEY REPLICATED IN EXTRAPULMONARY SITES INCLUDING THE BRAIN6IRAL ANTIGEN WASOBSERVEDBYIMMUNOHISTOCHEMISTRYINTHELUNGSOFMICEINFECTEDWITH !(+(. AHIGHLYVIRULENTSTRAIN AND!(+(. ALESSVIRULENTSTRAIN ANDWASASSOCIATEDWITHNECROTICBRONCHI6IRALANTI GENWASALSOOBSERVEDINBOTHGLIALCELLSANDNEURONSINTHEBRAINOFMICE INFECTED WITH THE HIGHLY VIRULENT INFLUENZA!(+ (. VIRUS A FINDINGALSOREPORTEDBY'AOETAL;=)NADDITION 'AOET ALREPORTED THEPRESENCEOFVIRALANTIGENIN THECARDIACMYOFIBERSINMICEINFECTEDWITH THEHIGHLYVIRULENTINFLUENZA!(+(. VIRUS4HEABILITYOFTHE (.VIRUSESTOREPLICATEANDCAUSEDISEASEANDDEATHINMICEDIDNOTCOR RELATEWITHTHEIRABILITYTOKILLCHICKENS;= ANDTHERELEVANCEOFREPLICA TIONOFTHESEVIRUSESINEXTRAPULMONARYSITESINMICETOTHEHUMANDISEASE ISNOTCLEAR ALTHOUGHAGENERALCORRELATIONBETWEENTHELEVELOFVIRULENCEIN MICEANDTHESEVERITYANDOUTCOMEOFDISEASEINHUMANSWASOBSERVEDWITH OFVIRUSESEVALUATED;=$YBINGANDCOLLEAGUES;=REPORTEDTHAT INFECTIONOFMICEWITHHIGHLYPATHOGENIC(!)VIRUSESTHATWEREISOLATED FROM3COTLAND;INFLUENZA!CK3COTLAND(. = )TALY;INFLUENZA!CK )TALY (. = 1UERETERO ;INFLUENZA !CK1UERETERO (. = AND %NGLAND ;INFLUENZA!TK%NGLAND (. = CAUSED LITTLEORNODISEASEIN"!,"CMICE(0!)(.INFLUENZAVIRUSESISOLATED FROMHUMANSIN!SIAINCAUSEDWEIGHTLOSS RUFFLEDFUR LISTLESSNESSAND PRONOUNCEDLEUKOPENIA ANDWERELETHALINMICEWITHOUTPRIORADAPTATION ANDREPLICATEDOUTSIDETHERESPIRATORYTRACT;=)NTHESAME STUDY (0!) (. VIRUSES ISOLATED FROM BIRDS AND A SINGLE HUMAN ISOLATE WERE LESS VIRULENTFORMICE ,U ET AL ;= USED THE "!,"C MOUSE MODEL TO EVALUATE THE IMMU NOGENICITYANDEFFICACYOFAVACCINEFOR(.INFLUENZABASEDONANANTI GENICALLY RELATED NON PATHOGENIC!) VIRUS !DUCK3INGAPORE 1&


(. 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


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 ;=


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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;=


)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


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


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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,IVEATTENUATEDINFLUENZAVACCINE (ARRY'REENBERG AND'EORGE+EMBLE $EPARTMENTSOF-EDICINEAND-ICROBIOLOGYAND)MMUNOLOGY 3TANFORD5NIVERSITY3CHOOL

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

!BSTRACT $EVELOPMENT OF THE LIVE ATTENUATED INFLUENZA VACCINE &LU-IST 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-IST WAS

,IVEATTENUATEDINFLUENZAVACCINE

LICENSEDINTHE5NITED3TATESFORTHEACTIVEIMMUNIZATIONAGAINSTINFLUENZA LIKEILLNESSINHEALTHYCHILDRENANDADULTS nYEARSOFAGE &LU-ISTISCUR RENTLY MANUFACTURED IN SPECIFIC PATHOGEN FREE EMBRYONATED CHICKEN EGGS 4HEVACCINEISATRIVALENTBLENDOFTHREE,!)6VACCINESTRAINS !(. ! (.AND" EACHRECOMMENDEDANNUALLYBYTHE530UBLIC(EALTH3ERVICE TO ANTIGENICALLY MATCH THE STRAINS EXPECTED TO CIRCULATE IN THE UPCOMING INFLUENZASEASON4HEMATERIALISBLENDEDSUCHTHATTHEDOSEOFEACHSTRAINIS F APPROXIMATELYLOG OFINFECTIOUSPARTICLESANDISFILLEDINTOSPRAYERDEVICES THATPRODUCEAMISTUPONACTUATION4HEORIGINALLICENSEDVACCINEFORMULA TIONWASSTOREDFROZENUNTILIMMEDIATELY PRIORTOUSE WHILETHECURRENTVAC CINEISSTOREDATREFRIGERATORTEMPERATURE nª#4HEFIRSTTWODECADESOF ,!)6CLINICALSTUDIES MANYSPONSOREDBYTHE.)( WEREPERFORMEDUSING MONOVALENT AND BIVALENT FORMULATIONS OF THE VACCINE DELIVERED BY NASAL DROPRATHERTHANSPRAYANDHAVEBEENEXTENSIVELYREVIEWEDPREVIOUSLY;= 4HISCHAPTERDESCRIBESTHEKEYSTUDIESDURINGTHEDEVELOPMENTANDCHARAC TERIZATIONOFTHETRIVALENTFORMULATIONOF&LU-IST

$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


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-IST 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


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


TIONCYCLEOFTHEVIRUS4HEBASICBIOCHEMICALNATUREOFTHESEPHENOTYPESIS UNCLEARANDCONTINUESTOBEINVESTIGATED

'ENETICSTABILITYOFTHEVACCINEINMANUFACTURING )NFLUENZA VIRUS LIKE OTHER 2.! VIRUSES HAS AN 2.! DEPENDENT 2.! POLYMERASE THAT LACKS A PROOFREADING FUNCTION 0ICKING AND SEQUENCING INDIVIDUAL PLAQUE ISOLATES DEMONSTRATED AN OBSERVED MUTATION FREQUENCY OFONECHANGEPERNUCLEOTIDESRESULTINGINARATEOF = n MUTA TIONS PER REPLICATION CYCLE ;= "ECAUSE OF THIS INHERENT CAPACITY OF INFLU ENZAVIRUSGENESEGMENTSTOCHANGE THEGENETICSTABILITYOFTHEVACCINEWAS CHARACTERIZEDBOTHWITHINTHECONTEXTOFTHEMANUFACTURINGPROCESSASWELL AS FOLLOWING INTRANASAL ADMINISTRATION )N GENERAL MANUFACTURING OF THE BULKVACCINEONLYREQUIRESTHESEEDMATERIALTOBEPASSAGEDONCEORTWICE INEMBRYONATEDEGGS4OEVALUATETHESTABILITYOFTHEGENETICELEMENTSDUR INGMANUFACTURE THEGENOMICSEQUENCESOFBULKVACCINEANDITS PROGENITOR SEEDMATERIALSWEREANALYZED REPRESENTEDBYOVERNINEDIFFERENTSTRAINSDIS TRIBUTEDAMONGNINEINDEPENDENTSEEDMATERIALSANDOVERBULK VACCINE STOCKS #OMPARISONS OF THE GENOMIC SEQUENCES OF THESE MATERIALS DEMON STRATEDTHATINALLCASESTHEBULKVACCINEWASIDENTICALTOTHESEEDMATERIAL 4HESE DATA DEMONSTRATED THAT THE VACCINES GENETIC COMPOSITION IS STABLE ANDUNCHANGEDWITHINTHEPARAMETERSUSEDTOMANUFACTURETHEVACCINEAT LARGESCALE;=

'ENETICSTABILITYOFTHEVIRUSINTHERESPIRATORYTRACTANDTRANSMISSION &OLLOWINGINTRANASALADMINISTRATION THEVACCINEVIRUSINFECTS ANDREPLICATES IN EPITHELIAL CELLS OF THE UPPER RESPIRATORY TRACT RESULTING IN AN IMMUNE RESPONSE #HARACTERIZING THE GENETIC STABILITY OF THE VACCINE IN HUMANS IS ANIMPORTANTELEMENTOFUNDERSTANDINGTHEPROPERTIESOFTHEVACCINE/VER THECOURSEOFMULTIPLEDECADESSTUDYINGMONOVALENT BIVALENTANDTRIVALENT FORMULATIONSOFTHISVACCINEINCLINICALSTUDIES NOREVERTANTSOFTHEVACCINE HAVEBEENIDENTIFIED;=4OEVALUATETHESTABILITYOFTHEVACCINEFOLLOWING REPLICATIONINTHEUPPERRESPIRATORYTRACTOFHUMANS APROSPECTIVESHEDDING ANDTRANSMISSIBILITYSTUDYWASDESIGNED9OUNGCHILDRENWERESELECTEDDUE TOTHERELATIVELYLONGERDURATIONANDGREATERLEVELOFSHEDDINGOFTHEVAC CINE FOLLOWING ADMINISTRATION4HEREFORE THIS POPULATION WAS EXPECTED TO REPRESENTTHEMOSTPERMISSIVESETTINGFORDETECTINGREVERTANTS IFTHEYWERE TOARISE)NTHESTUDYOFGENETICSTABILITY CHILDRENnMONTHSOFAGEWERE VACCINATEDWITH&LU-ISTANDNASALSWABSAMPLESWERETAKENATFREQUENTAND REGULARINTERVALS/FTHECHILDRENINTHESTUDY SHEDATLEASTONEOFTHE THREESTRAINSINTHEVACCINEWITHPEAKTITERSRANGINGFROMTO DAYSPOST VACCINATION AND THE LAST ISOLATE SHED DAYS POST VACCINATION4HE CA AND


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


AMOUNTSTHANCHILDREN)NASTUDYDESIGNEDTOCHARACTERIZETHE SHEDDINGOF &LU-ISTINADULTS VACCINEWASRECOVEREDFROMNASALSWABSOFONLYOF INDIVIDUALS DAYS AFTER VACCINATION AND BY DAYS AFTER VACCINATION ONLY OFINDIVIDUALSHADVACCINEDETECTABLEINTHEIRNASALSWAB;=4HISLOW PROBABILITY OF TRANSMISSION COMBINED WITH THE VACCINES GENETIC STABILITY GIVEADDITIONALCONFIDENCEINTHEUSEOF&LU-ISTINCHILDREN

"ASISOFTHEIMMUNERESPONSE )NFECTIONWITHWILD TYPEINFLUENZAVIRUSLEAVESTHEINDIVIDUAL WITHASTRONG IMMUNOLOGICALMEMORYTHATWILLPREVENTTHESAMEORANTIGENICALLYSIMILAR VARIANTFROMCAUSINGDISEASEAGAININTHESAMEINDIVIDUALFORDECADES4HIS IMMUNOLOGICAL MEMORY CAN BE DETECTED IN MANY DIFFERENT COMPARTMENTS INCLUDINGLOCALMUCOSALIMMUNITY SERUMANTIBODYAND4CELLS4HEIMMUNE 4 RESPONSETOVACCINATIONWITH,!)6HASBEENSTUDIEDINMULTIPLEDIFFERENT SETTINGSANDTHEIMMUNERESPONSEISQUALITATIVELYSIMILARBUTQUANTITATIVELY LESS THAN THAT ELICITED BY NATURAL INFECTION IMMUNITY CAN BE DOCUMENTED BY MUCOSAL )G! SERUM (!) AND NEUTRALIZING ANTIBODIES AND CELLULAR RESPONSES4HIS OBSERVATION IS NOT SURPRISING GIVEN THAT THE VACCINE STIMU LATESIMMUNITYBYREPLICATIONINTHEUPPERRESPIRATORYTRACTSIMILARTOTHATOF THEWILD TYPEVIRUS$ESPITEFINDINGEVIDENCEFORVACCINE INDUCEDIMMUNITY ATBOTHLOCALANDSYSTEMICCOMPARTMENTS THESPECIFICFUNCTIONALROLEOFANY PARTICULAR IMMUNE RESPONSE AND VALIDATED CORRELATES OF PROTECTION FROM INFLUENZADISEASEINVACCINATEDINDIVIDUALSREMAINSUNPROVEN ,!)6ELICITSAROBUSTIMMUNERESPONSEINYOUNGCHILDREN PARTICULARLY THOSETHATARESERONEGATIVEFORINFLUENZAATTHETIMEOFVACCINATION;n= 3EROCONVERSIONRATES MEASUREDBYTHEPRESENCEOFHEMAGGLUTINATIONINHIBI TIONANTIBODYINTHESERUM AREFREQUENTLYnORMOREAFTERTWODOSES OFVACCINE2ATESTYPICALLYARELOWERFORCHILDRENTHATHAVEPREEXISTINGANTI BODYATTHETIMEOFVACCINATION4HISISNOTSURPRISINGGIVENTHATTHEVACCINE MUSTREPLICATEATTHEMUCOSALSURFACESTOBEEFFECTIVE4HEPRESENCEOFANTI BODYATTHETIMEOFIMMUNIZATIONMAYBOTHLIMITTHEEXTENTOFREPLICATION OFTHEVACCINEASWELLASMASKTHEBOOSTINGOFTHEIMMUNERESPONSEUSING RELATIVELYCRUDEMEASURESOFIMMUNOGENICITYSUCHAS(!)ANTIBODYINTHE SERUM/THERIMMUNERESPONSESTO,!)6HAVEBEENDOCUMENTEDINCHIL DRENINCLUDINGSECRETORY)G!INNASALSECRETIONS#ELLULARIMMUNERESPONSES HAVEBEENEVALUATEDINOLDERCHILDRENRECEIVING&LU-IST&OLLOWINGIMMU NIZATIONOFCHILDRENAGEDnYEARS BLOODWASCOLLECTEDATANDDAYS POSTVACCINATIONANDSTIMULATEDWITHTHE!(.STRAINEXVIVO"OTHTHE #$ AND #$ INFLUENZA SPECIFIC4 CELLS WERE INCREASED IN THESE CHILDREN COMPAREDTOTHEIRPRE VACCINATIONVALUESADDITIONALLY THESEINCREASESWERE GREATERTHANTHOSEOBSERVEDFOR4)6 IMMUNIZEDCHILDRENINTHESAMESTUDY ;=)NTHISSAMEPOPULATION ANTIBODY SECRETING"CELLSWEREALSODETECTED INTHEPERIPHERYWITHINnDAYSPOSTVACCINATION;=


)MMUNOLOGICALMARKERSINADULTSHAVEBEENMOREDIFFICULTTOMEASURE -OST ADULTS HAVE HAD MULTIPLE ENCOUNTERS WITH WILD TYPE INFLUENZA AND INFLUENZA VACCINATION DURING THEIR LIFETIME AND HAVE READILY MEASURABLE LEVELSOFINFLUENZAANTIBODYINTHEIRSERUM)NCONTRASTTOSTUDIESINYOUNG CHILDREN VACCINATION OF ADULTS WITH &LU-IST INFREQUENTLY PRODUCES A MEA SURABLE INCREASE IN SERUM (!) ANTIBODY TITERS 2ECENT STUDIES ON 4 CELL IMMUNITYFOLLOWINGVACCINATIONHADSIMILARRESULTS4HEREWERENODEMON STRABLEINCREASESIN#$ #$OR.+ACTIVITYWHENCELLSWERESTIMULATEDEX VIVOEITHERORDAYSFOLLOWINGVACCINATION;=4HISMAYAGAINBEDUE TO THE A HIGHER LEVEL OF INFLUENZA IMMUNITY PRIOR TO VACCINATION IN ADULTS 4HISSAMESTUDYDEMONSTRATEDTHATTHELEVELSOFPRE VACCINATION INFLUENZA SPECIFIC #$ AND #$ CELLS INCREASED WITH AGE OF THE SUBJECTS AND THAT ADULTS HAD SIGNIFICANTLY HIGHER BASELINE QUANTITIES THAN CHILDREN )N CON TRASTTOTHE4CELLAND(!)RESPONSES ADULTSWEREGENERALLYSHOWNTOHAVE INCREASED INFLUENZA SPECIFIC ANTIBODY SECRETING " CELLS IN THE BLOOD n DAYSPOST,!)6VACCINATION7HILEONLYOFTHEADULTSHADASEROLOGI CALRESPONSEMEASUREDBYAFOURFOLDORGREATERINCREASEOF(!)ANTIBODY FOLLOWINGIMMUNIZATION APPROXIMATELYOFTHESUBJECTSHADA MEASUR ABLE INCREASE IN THE NUMBER OF INFLUENZA SPECIFIC )G' SECRETING ANTIBODY SECRETINGCELLSINTHEPERIPHERY;=4HESEDATACLEARLYDEMONSTRATEDTHAT ,!)6ELICITEDAREADILYDETECTABLE"CELLRESPONSEINMOSTADULTS WHICHIS CONSISTENTWITHTHECLINICALEXPERIENCETHAT&LU-ISTISHIGHLY EFFICACIOUSIN ANADULTPOPULATION; =4HECURRENTIMMUNOLOGICALMARKERSTYPICALLY USEDTOASSESSTHEFUNCTIONOFINFLUENZAVACCINES SUCHAS(!) INTHESERUM MAYNOTBESUFFICIENTLYSENSITIVENORMONITORTHEAPPROPRIATECOMPARTMENT TODETECTAFUNCTIONALIMMUNERESPONSETO,!)6 6ACCINE STUDIES OFTEN RELY ON CORRELATE MARKERS TO DEMONSTRATE THAT THE VACCINE WILL PERFORM AS EXPECTED UNDER THE CONDITIONS BEING STUDIED ! ROBUST CORRELATE OF PROTECTION IS AN IMMUNOLOGICAL MARKER THAT WHEN PRESENTCOINCIDESWITHPROTECTIONFROMDISEASEUPONSUBSEQUENTEXPOSURE TOTHEWILD TYPEVIRUSANDTHELACKOFWHICHCORRELATESWITHSUSCEPTIBILITYTO ILLNESS$UETOHIGHRATESOFEFFICACYDEMONSTRATEDFOR,!)6COMBINEDWITH THEDIFFICULTYINUSINGTRADITIONALSERUM BASEDINFLUENZAASSAYSTOMEASURE ANIMMUNERESPONSEINADULTS THESEMARKERSHAVEBEENDIFFICULTTOIDENTIFY FOR,!)6)NCHILDREN PARTICULARLYYOUNGCHILDRENWHOAREIMMUNOLOGICALLY NAIVETOINFLUENZA VACCINATIONWITH,!)6ELICITSAROBUSTIMMUNERESPONSE THATCANBEDETECTEDINMULTIPLECOMPARTMENTS/NESTUDYEVALUATEDPOTEN TIALCORRELATESOFPROTECTIONBYINOCULATINGCHILDRENWITHEITHERVACCINEOR PLACEBO FOLLOWEDBYARELATIVELYLONGINTERVALATWHICHPOINT SAMPLESWERE TAKENTOMEASUREHUMORALIMMUNERESPONSES ANDTHENTHESECHILDRENWERE GIVENANOTHERDOSEOFAMONOVALENTVACCINESTRAIN"YCORRELATINGTHELEVEL OF IMMUNE RESPONSE USING A VARIETY OF IMMUNOLOGICAL ASSAY SYSTEMS WITH SHEDDING OF THE CHALLENGE VACCINE VIRUS THE UTILITY OF THE VARIOUS ASSAYS FORPREDICTINGPROTECTIVEIMMUNITYWASASSESSED;=&IRST THEPRESENCEOF SERUM(!)ANTIBODYSTRONGLYCORRELATEDWITHTHEABSENCEOFSHEDCHALLENGE


VIRUS4HESEDATADEMONSTRATEDTHATATLEASTINCHILDREN APOSITIVECORRELA TION EXISTED FOR THIS MARKER (OWEVER WHEN THE TWO GROUPS OF CHILDREN WHOWERESERONEGATIVEATTHETIMEOFCHALLENGEWERECOMPARED THEREWASA SIGNIFICANTINCREASEINTHENUMBEROFCHILDRENWHOSHEDVACCINEINTHEPLA CEBOGROUPCOMPAREDTOTHE,!)6 VACCINATEDGROUP4HESEDATALEDTOTHE CONCLUSIONTHATTHEABSENCEOFSERUM(!)DIDNOTCORRELATECOMPLETELYWITH SUSCEPTIBILITY 4HE SAME TRENDS WERE OBSERVED WHEN SECRETORY NASAL )G! WASUSEDASTHEMARKER4HEOBSERVATIONTHATTHE 4 PRESENCEOFTHESEMARKERS CORRELATES WITH PROTECTION HELPS IDENTIFY POTENTIALLY USEFUL IMMUNOLOGICAL MEASURES HOWEVER THE OBSERVATION THAT ABSENCE DOES NOT CORRELATE WITH SUSCEPTIBILITY ARGUES THAT OTHER IMPORTANT IMMUNE MECHANISMS MAY BE OVERLOOKEDWHENONLYSERUM(!)ORSECRETORY)G!AREEVALUATED 3TUDIES HAVE DEMONSTRATED THAT ,!)6 ELICITS BOTH HUMORAL AND CELLULAR IMMUNE RESPONSESANDTHOSERESPONSESCANBEFOUNDATBOTHMUCOSALANDPERIPHERAL SITES (OWEVER THE FUNCTIONAL IMMUNE MEDIATORS THAT GOVERN PROTECTION FROMDISEASEHAVENOTYETBEENELUCIDATED MAYBEMULTI FACTORIALANDMAY DIFFERAMONGPOPULATIONSASWELL&URTHERSTUDYWILLBENEEDED TOIDENTIFY PRACTICAL CORRELATES FOR VACCINE EFFICACY AS WELL AS DETAILED IMMUNOLOGICAL PROFILINGTOUNDERSTANDTHEFUNCTIONALCOMPONENTSOFANEFFECTIVEIMMUNE RESPONSEANDHOWITCONTROLSDISEASE

0ERFORMANCEOFTHEVACCINEINCLINICALSTUDIES 6ACCINESDERIVEDFROM CA !!NN!RBORANDCA "!NN!RBORHAVE BEEN EXTENSIVELY CHARACTERIZED IN CLINICAL STUDIES 0RIOR TO THE MID S MONOVALENT AND BIVALENT FORMS OF THESE VACCINES WERE EVALUATED IN OVER SUBJECTS IN A NUMBER OF DIFFERENT CLINICAL STUDIES MANY OF WHICH WERESPONSOREDBYTHE.)(;=-ORERECENTLY STUDIESFOCUSEDONBOTHTHE FROZENANDREFRIGERATORSTABLETRIVALENTFORMULATIONSOF&LU-IST HAVEBEEN CONDUCTEDINAWIDERANGEOFSETTINGSININDIVIDUALSFROMMONTHSTOOVER YEARSOFAGE 4HEEFFICACYOFTHETRIVALENTFORMOFTHEVACCINEHASBEENEVALUATEDIN ANUMBEROFSETTINGSINDIFFERENTAGECOHORTSTHROUGHOUTTHEWORLD4ABLE LISTSTHESTUDYPOPULATIONS CONTROLGROUPSANDOVERALLEFFICACYAGAINSTCUL TURE CONFIRMEDINFLUENZA LIKEILLNESSFORSTUDIESINCHILDREN 4HEVACCINEHAS REPRODUCIBLYBEENSHOWNTOPREVENTINFLUENZA LIKEILLNESS),) CAUSEDBY ALLTHREEINFLUENZATYPESINCLUDING !(. !(.AND")NTHEMAJORITY OF WELL POWERED PLACEBO CONTROLLED STUDIES VACCINE EFFICACY RANGED FROM TOINCHILDRENWITHRESPECTTOCULTURE CONFIRMEDINFLUENZAILLNESS WHENCOMPAREDTOPLACEBO;n=)NTERESTINGLY FOURSTUDIESWERECONDUCT EDINWHICH&LU-ISTWASCOMPAREDTO4)6 VACCINATEDSUBJECTS)N THELARGEST OFTHESESTUDIES WHICHINCLUDEDOVERCHILDRENANDAPPROXIMATELY ISOLATESFROMCHILDRENWHOHADMODIFIED#$# ),)&LU-ISTWASSHOWNTO REDUCETHEBURDENOFILLNESSBYNEARLYCOMPAREDTO4)6/FNOTE ALL


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THE!(.STRAINSCIRCULATINGINTHISSTUDYWEREANTIGENICALLY MISMATCHED TOTHETWOVACCINESANDTHECHILDRENVACCINATEDWITH&LU-ISTHADFEWER CASESOFMODIFIED#$# ),)COMPAREDTOTHE4)6GROUP;=)NTWOOTHER STUDIES ONECONDUCTEDINCHILDRENWITHRECURRENTRESPIRATORYILLNESSANDTHE OTHER IN OLDER CHILDREN WITH ASTHMA &LU-IST WAS ALSO SHOWN TO BE MORE EFFICACIOUSTHAN4)6; =4HEFOURTHTRIAL CONDUCTEDINADULTS CONTAINED THREE COHORTS PLACEBO 4)6 VACCINATED OR &LU-IST VACCINATED7HILE ONLY ISOLATESWEREAVAILABLEFORANALYSIS &LU-ISTWASSHOWNTOBEEFFICACIOUS COMPAREDTOPLACEBOBUTNOTMOREEFFICACIOUSTHAN4)6;=4HERESULTSOF THISSTUDYCOMPAREDTOTHETHREESTUDIESINCHILDRENMAYREFLECTTHEINTERAC TION OF &LU-IST WITH THE IMMUNE SYSTEM OF THE ADULT HOST OR MAY SIMPLY REFLECTANORMALVARIATIONDUETOARELATIVELYSMALLERSAMPLESIZE


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


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


,!)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


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

2EFERENCES

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)NTRODUCTION -&ISASAFEANDPOTENTEMULSION BASEDVACCINEADJUVANTTHATHASBEEN LICENSEDINMORETHANCOUNTRIES&LUAD FORMORETHANYEARS FORUSE IN AN INFLUENZA VACCINE FOCUSED ON ELDERLY SUBJECTS4HE SAFETY PROFILE OF -&ISWELLESTABLISHEDCLINICALLYTHROUGHALARGESAFETYDATABASE SUBJECTS ANDTHROUGHPHARMACOVIGILANCEEVALUATIONSOFGREATER THANMIL LIONDOSESTHATHAVEBEENDISTRIBUTED4HE-&ADJUVANTHASASIGNIFICANT

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ANEFFECTIVEADJUVANTINOLDMICE WHICHHADPREVIOUSLYBEENINFECTEDWITH INFLUENZA4AB ASITUATIONMORESIMILARTOTHATFOUNDINHUMANS WHOARE OFTENRE INFECTEDANNUALLYWITHCIRCULATING FLUSTRAINS;=4HESEPRE CLINICAL 4 STUDIESHIGHLIGHTEDTHEHUGEPOTENTIALOF-&TOBEUSEDASAN ADJUVANT FOR AN IMPROVED FLU VACCINE POTENTIALLY ALLOWING ANTIGEN DOSE REDUCTION WHILEENHANCINGPROTECTIVEANTIBODYAND4CELLRESPONSES FOREXTENDEDTIME PERIODS4HEABILITYOF-&ADJUVANTTOOFFERASIGNIFICANTREDUCTIONINTHE PROTECTIVEDOSEFORFLUVACCINESHASSUBSEQUENTLYBECOMEVERYIMPORTANTIN THEPANDEMICFLUVACCINESETTING 4HE SMALL DROPLET SIZE OF -& ADJUVANT EMULSION GENERATED THROUGH THE USE OF A MICROFLUIDIZER IN THE PREPARATION PROCESS IS CRUCIAL TO THE POTENCY OF THE ADJUVANT BUT ALSO ENHANCES EMULSION STABILITY AND ALLOWS THEFORMULATIONTOBESTERILEFILTEREDFORCLINICALUSE/VERALL OUREARLYCLINI CALEXPERIENCEWITHOWEMULSIONSSERVESTOHIGHLIGHTTHENEEDFORCAREFUL SELECTIONOFIMMUNEPOTENTIATORS TOBEINCLUDEDINADJUVANTFORMULATIONS SHOULDTHEYPROVENECESSARY ORDESIRABLE.EVERTHELESS THEEXPERIENCEWITH -&ALSOSHOWSTHATOWEMULSIONSCANBEHIGHLYEFFECTIVEADJUVANTS WITH AN ACCEPTABLE SAFETY PROFILE WHICH MAY NOT NEED THE ADDITION OF IMMUNE POTENTIATORS

4HEMECHANISMOFACTIONOF-&ADJUVANT %ARLY STUDIES DESIGNED TO DETERMINE THE MECHANISM OF ACTION OF -& FOCUSEDONTHEPOSSIBILITYOFTHECREATIONOFA@DEPOTEFFECT FORCO ADMINIS TEREDANTIGEN SINCETHEREHADBEENSUGGESTIONSTHATEMULSIONS MAYRETAIN ANTIGEN AT THE INJECTION SITE (OWEVER EARLY WORK SHOWED THAT AN ANTIGEN DEPOT WAS NOT ESTABLISHED AT THE INJECTION SITE AND THAT THE EMULSION WAS CLEARED RAPIDLY ;= 4HE LACK OF AN ANTIGEN DEPOT WITH -& WAS CON FIRMEDINLATERSTUDIES;= WHICHALSOESTABLISHEDTHAT-& ANDANTIGEN WERECLEAREDINDEPENDENTLY3UBSEQUENTLY ITWASTHOUGHTTHATPERHAPSTHE EMULSION ACTED AS A@DELIVERY SYSTEM AND WAS RESPONSIBLE FOR PROMOTING THEUPTAKEOFANTIGENINTOANTIGEN PRESENTINGCELLS!0# 4HISTHEORYWAS LINKED TO EARLIER OBSERVATIONS WITH 3!& WHICH CONTAINED A PLURONIC SUR FACTANTTHATWASTHOUGHTTOBECAPABLEOFBINDINGANTIGENTOTHEEMULSION DROPLETSTOPROMOTEANTIGENUPTAKE;=(OWEVER STUDIESWITHRECOMBINANT ANTIGENSSHOWEDTHAT-&WASANEFFECTIVEADJUVANT DESPITENO EVIDENCE OFBINDINGOFTHEANTIGENSTOTHEOILDROPLETS-OREOVER ANADJUVANTEFFECT D WASSTILLOBSERVEDIF-&WASINJECTEDUPTOHBEFORETHEANTIGENANDUP TOHAFTER CONFIRMINGTHATDIRECTASSOCIATIONWASNOTREQUIREDFORANADJU VANTEFFECT;=.EVERTHELESS ADMINISTRATIONOF-&HAFTERTHEANTIGEN RESULTED IN A MUCH REDUCED ADJUVANT EFFECT SUGGESTING THAT THE EMULSION WAS ACTIVATING IMMUNE CELLS WHICH WERE THEN ABLE TO BETTER PROCESS AND PRESENT THE CO ADMINISTERED ANTIGEN! DIRECT EFFECT OF -& ON CYTOKINE LEVELS IN VIVO WAS ALSO OBSERVED IN SEPARATE STUDIES SUGGESTING THAT THE


DELIVERYMETHODALONEWASTOOSIMPLISTICANEXPLANATION;=4OGAINABET 4 TERUNDERSTANDINGOFTHEMECHANISMOFACTIONOF-& WERECENTLYSTUDIED THEEARLYSTEPSOFTHEIMMUNERESPONSEONHUMANCELLS INVITROANDINMOUSE MUSCLE IN VIVO7E HAVE SHOWN THAT THERE ARE AT LEAST TWO HUMAN TARGET CELLSFOR-& MONOCYTESANDGRANULOCYTES ANDTHAT-&HASARANGEOF EFFECTS INCLUDINGINCREASEDANTIGENUPTAKE THERELEASEOFCHEMOATTRACTANTS AND CELL DIFFERENTIATION4HE OBSERVATION OF INCREASED ANTIGEN UPTAKE IS IN LINEWITHPREVIOUSFINDINGSINMICE;=4HEMOSTREADILYINDUCEDCHEMOAT TRACTANT WAS THE CHEMOKINE ##, WHICH IS INVOLVED IN CELL RECRUITMENT 0REVIOUS WORK HAD SHOWN A REDUCTION OF -& INDUCED CELL RECRUITMENT INTO THE MUSCLE IN ##2 DEFICIENT MICE ;= WHICH IS CONSISTENT WITH OUR OBSERVATIONS ON HUMAN CELLS -OREOVER ONGOING EXPERIMENTS ON GENE EXPRESSIONPROFILESATTHEINJECTIONSITEAREALSOCONSISTENTWITHTHEKEYROLE OFCHEMOKINES)NADDITION ##,WASALSOFOUNDINSERUMAFTERINJECTION OF-&INTOMOUSEMUSCLE PROVIDINGFURTHERCONSISTENCYBETWEEN INVITRO ANDINVIVOOBSERVATIONS-&ALSOINDUCESPHENOTYPICCHANGESONHUMAN MONOCYTESTHATARECONSISTENTWITHAMATURATIONPROCESSTOWARDSIMMATURE DENDRITIC CELLS $#S 4HESE OBSERVATIONS WILL BE REPORTED IN DETAIL SEPA RATELY;= BUTSOFARTHEREAPPEARSTOBEANIMPRESSIVECONSISTENCYBETWEEN DATAOBTAINEDINVITRO WITHHUMANCELLS ANDTHEINVIVODATAFROMMOUSE 4HESE OBSERVATIONS SUGGEST THAT -& INDUCES A LOCAL PRO INFLAMMATORY ENVIRONMENT WITHIN THE MUSCLE WHICH PROMOTES THE INDUCTION OF POTENT IMMUNERESPONSESTOCO ADMINISTEREDVACCINES (ENCE WE CONCLUDE THAT DURING VACCINATION ADJUVANTS LIKE -& AUG MENTTHEIMMUNERESPONSEATARANGEOFINTERVENTIONPOINTS4HROUGHINDUC TIONOFCHEMOKINES THEYINCREASERECRUITMENTOFIMMUNECELLSTOTHEINJEC TION SITE THEY AUGMENT!G UPTAKE BY MONOCYTES AT THE INJECTION SITE AND THEY ENHANCE DIFFERENTIATION OF MONOCYTES INTO $#S WHICH REPRESENT THE GOLD STANDARDCELLTYPEFORPRIMINGNAIVE4CELLS!PARTICULARLYIMPORTANT FEATUREOF-&ISTHATITSTRONGLYINDUCESTHEHOMINGRECEPTOR##2ON MATURING $#S THUS FACILITATING THEIR MIGRATION INTO DRAINING LYMPH NODES WHERE THEY CAN TRIGGER THE ADAPTIVE IMMUNE RESPONSE SPECIFIC TO THE VAC CINEANTIGEN.EVERTHELESS FURTHERSTUDIESARENECESSARYTOBETTERDEFINETHE PRECISEMECHANISMOFACTIONOF-&ANDTHESESTUDIESAREONGOING

4HECOMPOSITIONOF-& -& IS A LOW OIL CONTENT OW EMULSION4HE OIL USED FOR -& IS SQUA LENE WHICHISANATURALLYOCCURRINGSUBSTANCEFOUNDINPLANTSANDINTHE LIVERS OF A RANGE OF SPECIES INCLUDING HUMANS 3QUALENE IS AN INTERMEDI ATE IN THE HUMAN STEROID HORMONE BIOSYNTHETIC PATHWAY AND IS A DIRECT SYNTHETIC PRECURSOR TO CHOLESTEROL 4HEREFORE SQUALENE IS BIODEGRADABLE ANDBIOCOMPATIBLE SINCEITISNATURALLYOCCURRING3HARKLIVEROILCOMPRISES SQUALENEANDSHARKLIVERPROVIDESTHENATURALSOURCEOFTHESQUALENE


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-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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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


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 &LUAD INELDERLYSUBJECTS &LUAD WHICHWASINITIALLYLICENSEDASANINTERPANDEMICINFLUENZAVACCINEIN )TALYINANDISNOWLICENSEDINMORETHANCOUNTRIES CONTAINS-& WHICHWASTHEFIRSTNOVELADJUVANTACCEPTEDFORHUMANUSEAFTER THEREGIS TRATIONOFINSOLUBLEALUMSALTSINTHEFIRSTPARTOFTHETHCENTURY4HEREG ISTRATIONOF&LUADWASBASEDONTHERESULTSOFALARGECLINICALDEVELOPMENT 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&LUADWEREPERFORMEDINELDERLYSUBJECTS)NTHISPOPULA TION THEADJUVANTEDVACCINECONSISTENTLYINDUCEDENHANCEDGEOMETRICMEAN TITERS SEROCONVERSIONRATESANDSEROPROTECTIONRATES4HEINCREASEDIMMUNO GENICITYOF&LUADWASPARTICULARLYIMPORTANTINSUBSETSOFTHEELDERLYPOPU 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 &LUAD PARTICULARLYPOPULATIONSATHIGHRISKOFINFLUENZA RELATEDCOMPLICA TIONS SUCH AS ()6 POSITIVE SUBJECTS &IG AND THOSE AFFECTED BY CHRONIC DISEASES&IG ; =)MPORTANTLY THEADDITIONOF-&TOTHEINFLUENZA ANTIGENSDIDNOTAFFECTTHESAFETYPROFILEOFTHEVACCINE WHICHWASVERYWELL


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

&IGURE&LUAD 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 &LUAD 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


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&LUADISSUP PORTEDBYEXTENSIVEPHARMACOVIGILANCEDATASHOWINGTHATVACCINATIONWITH &LUAD ISASSOCIATEDWITHAVERYLOWFREQUENCYOFADVERSEREACTIONS; = )NFACT THESAFETYPROFILEOF&LUADISBROADLYSIMILARTOTHEPROFILEFORNON ADJUVANTEDFLUVACCINES;=-OREOVER ITHASBEENSHOWNTHATTHERATEOF 'UILLAIN "ARRE SYNDROME AFTER &LUAD 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;=

&LUAD 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 &LUAD 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&LUADVACCINEOFFEREDSIGNIFICANTLY ENHANCEDPROTECTIONAGAINSTHETEROVARIANTSTRAINSNOTINCLUDEDINTHEVAC CINE INTHISVULNERABLEPOPULATION0ELLEGRINIETAL -&ADJUVANTENHANCES D CROSSPROTECTIVEANTIBODYRESPONSESIN YOUNGCHILDRENAGAINSTHETEROVARIANT INFLUENZASTRAINSINPREPARATION )MPORTANTLY &LUAD 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; =


&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-&&OCETRIAAND!FLUNOV 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 -&


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&OCETRIA IN-AYINALLMEMBERSTATESOF THE%UROPEAN5NION ANDTOSUBMITFORAPPROVALASAPRE PANDEMICVACCINE !FLUNOV &OCETRIA WILLBEMANUFACTUREDTOCONTAINTHEINFLUENZASTRAIN DECLARED BY THE7ORLD (EALTH /RGANIZATION AS A PANDEMIC STRAIN 2ECENT DATAHAVESHOWNTHATTHEADDITIONOF-&TOAPOTENTIALPRE PANDEMICFLU VACCINE!FLUNOV 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


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


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&OCETRIA IN-AYINALLMEMBERSTATESOFTHE %UROPEAN5NION&OCETRIA 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 -&


HAD BEEN SHOWN TO ENHANCE IMMUNE RESPONSES TO PROTEIN POLYSACCHARIDE CONJUGATE VACCINES INCLUDING AGAINST .EISSERIA MENINGITIDES SEROGROUP # ;=(ENCE -&HASBROADPOTENTIALTOBEUSEDASAVACCINEADJUVANTFORA RANGEOFVACCINES INDIVERSEAGEGROUPS INCLUDINGINFANTS!LTHOUGH-HAS BEENMAINLYUSEDINELDERLYADULTSTODATE RECENTDATAHAVEHIGHLIGHTEDTHE SIGNIFICANTPOTENTIALOF-&TOBEUSEDINANIMPROVEDINFLUENZAVACCINEFOR YOUNGCHILDREN7ECONFIDENTLYEXPECTTHATTHEADJUVANTWILLGAINAPPROVAL FORWIDERUSEINDIVERSEPOPULATIONGROUPSINTHEFORTHCOMINGYEARS

2EFERENCES

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!BSTRACT -ANKIND HAS BEEN AFFECTED BY INFLUENZA EPIDEMICS AND PANDEMICS FOR MANY CENTURIES !S FOR MANY OTHERS INFECTIOUS DISEASES MEASURES TO REDUCE THE HEAVY BURDEN OF THIS DISEASETHROUGHPREVENTIONWEREFIRSTDEVELOPEDDURINGTHETH CENTURY4HEPURPOSEOF THISCHAPTERISTODESCRIBETHENON RECENTHISTORYOFINFLUENZA INCLUDINGTHEORIGINOFTHE DISEASE THEPANDEMICSANDTHEEPIDEMICS-OREOVER WEALSODISCUSSTHEFIRSTATTEMPTSTO DEVELOPSAFEANDEFFICACIOUSVACCINES INCLUDINGTHENEGATIVEEXPERIENCEWITHTHESWINE INFLUENZA VACCINATION AND THE NUMEROUS ATTEMPTS TO IMPROVE THE EFFICACY OF INFLUENZA VACCINESUSINGADJUVANTSCARRIEDOUTSINCETHES

)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

-ARIA,ATTANZI

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


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


&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


'"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

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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


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) = )

!LUMINUMPHOSPHATE!L0/

!MORPHOUSALUMINUMHYDROXYPHOSPHATENEGATIVELY CHARGEDATPHYSIOLOGICALP(P)) n

!LUM+!L3/ q(/

!LUMINUMHYDROXIDETHATCONTAINSSOMESULFATEANIONS ASWELLASANIONSTHATAREUSEDINTHEBUFFER

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

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

3PLITVIRUS

"EGRIVAC"EHRING -ARBURG 'ERMANY )NFLUENZAVACCINE#ANTACUZINO)NSTITUTE 2OMANIA

3UBUNITVIRUS

&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


#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


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


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


&IGURE%VOLUTIONANDNOMENCLATUREOFTHE(.VIRUSISOLATES"OLDFACEINDICATESTHEMOST COMMONLYSTUDIEDVIRUSES"OLDNUMBERSONTHERIGHTHANDSIDEINDICATETHECLADESANDSUB CLADES

THE VIRUS HAD ESCAPED ANY CONTROL AND WAS SO WIDESPREAD THAT SINCE THEN CULLING HUNDREDS OF MILLION OF CHICKENS IN THE AREAS OF OUTBREAK HAS PRO VIDEDONLYATEMPORARYRELIEF BUTHASNEVERBEENABLETOCONTROLTHESPREAD OFTHEVIRUS4HEVIRUS INFACT WASSPREADINGGLOBALLYUSING MIGRATORYBIRDS ASVECTORSANDSOONAPPEAREDINTHERESTOF!SIA 2USSIA 4URKEY %GYPT AND .IGERIA4ODAY THE (. VIRUS IS ENDEMIC IN THE BIRD POPULATION IN!SIA %UROPE AND!FRICA7HILESPREADINGGEOGRAPHICALLY THEVIRUSHASALSOBEEN EVOLVING AND DRIFTING ANTIGENICALLY SO THAT TODAY WE HAVE MANY GENETI CALLYDISTINCTISOLATESOFTHE(.VIRUSTHATCANBECLASSIFIED INTOSEVERAL CLADESANDSUBCLADESSEE&IG 5PTO&EBRUARY THEVIRUSCAUSED

7AITINGFORAPANDEMIC

&IGURE9EARLYCASESANDDEATHSCAUSEDBYTHE(.VIRUSFROMTO

4ABLE#ASESANDDEATHSCAUSEDBYTHE(.VIRUSINDIFFERENTCOUNTRIES7(/WEBSITE &EBRUARY #OUNTRY !ZERBAIJAN #AMBODIA #HINA $JIBOUTI %GYPT )NDONESIA )RAQ ,AOS0EOPLES$EMOCRATIC2EPUBLIC -YANMAR .IGERIA 0AKISTAN 4HAILAND 4URKEY 6IET.AM 4OTAL

4OTAL CASES

DEATHS

4OTAL NUMBER OF CASES INCLUDES NUMBER OF DEATHS7(/ REPORTS ONLY LABORATORY CONFIRMED CASES

REPORTED CASES AND DEATHS WITH AN OVERALL MORTALITY RATE OF SEE&IGAND4AB !LLCASESDERIVEDFROMCLOSECONTACTSWITHPOULTRY ALTHOUGHINAFEWCASESCLOSECONTACTSBETWEENPEOPLEMAYHAVECAUSEDTHE INFECTION4HEVIRUSCONTINUESTOEVOLVE BUTISNOTYETABLETOBEEFFICIENTLY TRANSMITTEDFROMHUMANTOHUMAN ANDTHEREFORETHEISOLATEDVIRUSESDONOT SEEMTOBEANIMMEDIATETHREAT


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n n n n n

n n n n n n

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)NTHEMEANTIMEALLPREDICTIONSHAVEBEENMADE3OMEPREDICTTHATTHE PANDEMIC WILL COME SOON OTHERS SAY THAT (. WILL NEVER CAUSE A PAN DEMICBECAUSETHEGENETICBACKGROUNDOFTHEVIRUSDOESNOTFIT APANDEMIC STRAIN /THERS SAY THAT WE MAY EXPECT A PANDEMIC FROM A VIRUS DIFFERENT FROM(.7ESHOULDALSOCONSIDERTHEPOSSIBILITYTHATGIVENTHEINTENSE VACCINATION OF THE ELDERLY POPULATION THE EVOLVED HEALTHCARE SYSTEM THE INCREASEDGLOBALSURVEILLANCE THESTCENTURYMAYBETHEFIRSTPERIODTHAT WILL NOT SEE A PANDEMIC INFLUENZA 5NFORTUNATELY THIS SCENARIO IS UNLIKELY TOHAPPENINFACT THEINFLUENZAVIRUSISABLETOINFECTANDEVOLVEINSEVERAL ANIMAL SPECIES SEE 4AB AND CONSIDERING THAT SO FAR ONLY OF THE KNOWNHEMAGGLUTININ(! TYPESAREPRESENTINHUMANISOLATES THEVIRUS SEEMSTOHAVEENOUGHFUELTOCONTINUETOBEATHREATFORMANKINDFORTHE NEXTSEVERALCENTURIES )NCONCLUSION ALTHOUGHWEHAVENOCERTAINTYABOUTWHENAPANDEMICIS GOINGTOSTRIKE THESEFACTSSUGGESTTHATTHERISKOFAPANDEMICSHOULDBETAKEN INTOSERIOUSCONSIDERATION ANDTHATWESHOULDHAVEAPLANTOBEPREPAREDFOR IT#ONSIDERINGTHATEVENAMILDPANDEMICISPREDICTEDTOCOSTTOTHE53! ECONOMYALONEMORETHANBILLIONDOLLARSANDGLOBALLYINEXCESSOF BILLION AGLOBALINVESTMENTOFBILLIONOFTHEPOTENTIALECONOMICDAM AGE SEEMSAREASONABLEINVESTMENTINSURANCE AGAINSTTHISRISK

0RECLINICALSTUDIESWITHVACCINESAGAINSTAVIANINFLUENZA $URINGTHELASTFEWYEARS MANYACADEMICLABORATORIES BIOTECHCOMPANIES ANDVACCINEMANUFACTURERSHAVEPRODUCEDAPLETHORAOFVACCINES POTENTIALLY ACTIVEAGAINSTAVIANINFLUENZA4HEMOSTWIDELYUSEDARETHOSEMANUFACTURED INEGGSUSINGTHESAMETECHNOLOGYUSEDTOMANUFACTURESEASONAL VACCINES 4HESEVACCINESMAYBECOMPOSEDOFWHOLEINACTIVATEDVIRUS DETERGENT SPLIT VIRUS OR PURIFIED (! AND NEURAMINIDASE .! SUBUNITS 4HESE VACCINES HAVEALREADYBEENTESTEDINCLINICALTRIALSANDAREDESCRIBEDLATERONINTHIS CHAPTER(EREWEDESCRIBETHEVACCINEAPPROACHESFORWHICHRESULTSOFCLINI CALTRIALSARENOTYETAVAILABLEORTHATHAVENOTYETBEENTESTEDINHUMANS

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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6IRUS LIKEPARTICLESPRODUCEDINBACULOVIRUS 6IRUS LIKE PARTICLES 6,0S EXPRESSING THE (! .! AND - ANTIGENS OF (.VIRUSHAVEBEENPRODUCEDININSECTCELLCULTURE USINGABACULOVIRUS VECTOR4HE6,0SWEREIMMUNOGENICINMICE2ECENTLY TESTINGIN ACLINICAL TRIALINHUMANSOFAN!)NDONESIA(.VACCINEWASREPORTED;= 4WODOSESOFAND+GWEREREPORTEDTOINDUCEAFOURFOLDRISEINNEUTRAL IZINGANTIBODIESOFOFSUBJECTSWITHTHEHIGHESTDOSE

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ABSENCEOFCORRELATESOFPROTECTIONFORVACCINESBASEDON- ITISUNLIKELY THATITWILLBEPOSSIBLETOSEEAFASTDEVELOPMENTOFTHESEVACCINESBECAUSE INTHEABSENCEOFANEFFICACYTRIALITISVIRTUALLYIMPOSSIBLE TOLICENSETHESE VACCINES )T IS POSSIBLE HOWEVER THAT IN THE LONG TERM THESE COMPONENTS MAY FIND SPACE FIRST IN IMPROVED SEASONAL VACCINES AND LATER IN PANDEMIC VACCINES4HE SAME MAY APPLY TO VACCINE CONSTRUCTS BASED ON THE INTERNAL CONSERVEDNUCLEOPROTEIN.0 ; =

#LINICALSTUDIESWITHVACCINESAGAINSTAVIANINFLUENZA -ANY CLINICAL STUDIES HAVE BEEN PERFORMED DURING THE LAST YEARS WITH VACCINES AGAINST AVIAN INFLUENZA "ELOW WE DESCRIBE MOSTLY THOSE STUDIES THAT HAVE BEEN PUBLISHED ALTHOUGH IN SOME CASES WE MENTION ALSO DATA REPORTED FROM MEETINGS FOR WHICH PRELIMINARY INFORMATION IS AVAILABLE 5NLESSOTHERWISESTATEDINTHETEXTORIN4ABLE IMMUNOGENICITYWASTESTED BYHEMAGGLUTINININHIBITION() USINGHORSEREDBLOODCELLS BYMICRONEU TRALIZATION -. USING THE 2'!6IETNAMX!02 INFLUENZA VIRUS STRAIN OR BY SERUM RADIAL HEMOLYSIS 32( USING TURKEY RED BLOOD CELLS COATED WITH ANTIGENS DERIVED FROM THE TESTED VIRUS SEE THE CHAPTER BY -ONTOMOLI 3EROCONVERSION WAS CONSIDERED POSITIVE WHEN () OR -. TITERSWEREBEFOREIMMUNIZATIONANDAFTERIMMUNIZATIONORWHEN AFOURFOLDINCREASEINTITERWASOBSERVEDINTHOSESUBJECTSTHATHADAPRE IMMUNIZATIONTITER4HEOVERALLDATAAREREPORTEDIN4ABLE32(WAS CONSIDEREDPOSITIVEWHENITWASNEGATIVEBEFOREIMMUNIZATIONANDHADAN AREAOFHEMOLYSISMMAFTERIMMUNIZATION,OOKINGATTHETABLE ITIS IMPORTANTTOKEEPINMINDTHATTHEDATAARENOTDIRECTLYCOMPARABLESINCE THEY HAVE BEEN PRODUCED AT DIFFERENT TIMES IN DIFFERENT LABORATORIES () STUDIESINPARTICULARHAVEBEENPERFORMEDWITHHORSEERYTHROCYTESUSINGAN ASSAY THAT HAS NEVER BEEN VALIDATED ACROSS LABORATORIES AND FOR WHICH NO STANDARDSEXIST SOTHATLARGEDIFFERENCESHAVEBEENREPORTED!LTHOUGH-. ALSOSHOWSVARIABILITYINDIFFERENTLABORATORIES ITISPROBABLYABETTERASSAY FORCOMPARINGRESULTSBECAUSEITMEASURESTHEABILITYOFANTIBODIESTONEU TRALIZEVIRALINFECTION ANDTHISACTIVITYISBIOLOGICALLYIMPORTANT

4HEEXPERIENCEWITH-& ADJUVANTED(.VACCINE#HIRON .OVARTIS 4HEFIRSTATTEMPTSTODEVELOPVACCINESAGAINST(.FOLLOWEDTHEOUT BREAKIN(ONG+ONG!TTHATTIMEREVERSEGENETICSWASNOTAVAILABLE (. COULDNOTBEGROWNINEGGS REASSORTANTSCOULDNOTBEMADEANDTHEREFORE ONLY TWO ATTEMPTS TO MAKE VACCINES WERE MADE /NE WAS A RECOMBINANT (SUBUNITVACCINEEXPRESSEDINBACULOVIRUSPRODUCEDBY0ROTEIN 3CIENCES DESCRIBEDBELOW THESECONDONEWASASUBUNITVACCINEPREPAREDUSINGA

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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2ECOMBINANT(!0ROTEIN3CIENCES 4HERECOMBINANT(!CLONEDFROMTHE(.STRAINISOLATEDIN(ONG +ONG IN WAS PRODUCED IN BACULOVIRUS INFECTED CELLS AND PURIFIED 0LACEBO OR +GPROTEINWEREUSEDTOIMMUNIZESUBJECTS4WODOSESWERE GIVEN ATDAYANDOR!NADDITIONALBLOODSAMPLEWASTAKENATDAY 2ESPECTIVELY AND OF THE SUBJECTS HAD A -. AFTER THE SECOND IMMUNIZATION 4AB 4HE TRIAL SHOWED THAT ALTHOUGH SOME

7AITINGFORAPANDEMIC

IMMUNITYTO(.COULDBEINDUCED LARGEAMOUNTSOFANTIGENWERENEC ESSARY -ORE RECENTLY THESE SUBJECTS WERE BOOSTED WITH AN (. VACCINE FROM3ANOFI0ASTEURWITHOUTADJUVANT0RELIMINARYAVAILABLEDATASUGGEST THE +G OF ADJUVANT FREE VACCINE CAN BOOST THE ANTIBODY RESPONSE BUT WITH A STRENGTH LOWER AND A KINETIC SLOWER THAN THAT PROVIDED BY -& ADJUVANTED VACCINE .O DATA ON CROSS NEUTRALIZING ANTIBODIES HAVE BEEN REPORTED;=

.ON ADJUVANTED(.SPLITVACCINE3ANOFI0ASTEUR 4HEREALRACETODEVELOP(.VACCINESSTARTEDINANDWHEN AFTER THEQUIETPERIODBETWEENAND (.STARTEDTOCAUSENEWHUMAN CASES IN6IETNAM AND4HAILAND SEE &IG 4HIS TIME IT BECAME CLEAR THAT (. NOT ONLY HAD NOT GONE AWAY BUT IT WAS THERE TO STAY AND THE GLOBAL COMMUNITYSTARTEDTOPANICFEARINGTHATTHEPANDEMICCOULDCOMEBEFORETHE WORLD HAD THE TIME TO BE PREPARED4HE FIRST EGG BASED (. VACCINE WAS PRODUCED BY 3ANOFI 0ASTEUR4HIS WAS A SPLIT VACCINE4HE VIRUS STRAIN WAS GENERATED BY REVERSE GENETICS CONTAINING THE (! AND .! GENE SEGMENTS FROM THE INFLUENZA!6IETNAM AND ALL THE OTHER GENES FROM THE STRAIN!02THATGROWSWELLINEMBRYONATEDEGGSANDISNORMALLYUSED TOGENERATEVACCINESTRAINS4HE(!GENEWASFURTHERMODIFIEDTOREPLACETHE SIXBASICAMINOACIDSATTHECLEAVAGESITEBETWEENDOMAINANDOFTHE(! WHICHAREKNOWNTOBEASSOCIATEDWITHINCREASEDVIRULENCE4HANKSTOTHESE MODIFICATIONSTHENEWVIRUSCARRIEDTHE(!AND.!GENESOFTHE PATHOGENIC (.VIRUSBUTWASNON VIRULENTANDCOULDBEGROWNINABIOSAFETYLEVEL LABORATORY!FTERGROWTHINEGGS THEVIRUSWASPURIFIEDBYULTRACENTRIFUGATION INACTIVATEDWITHFORMALDEHYDEANDSPLITBYADDITIONOF4RITON8 4HEVAC CINEWASFINALLYREADYAFTERSTERILEFILTRATIONANDADDITIONAL STEPSOFPURIFICA TION#LINICALTRIALSSTARTEDIN!PRIL;=SUBJECTS WITHAMEDIANAGE OFYEARS WEREENROLLEDINARANDOMIZED PLACEBO CONTROLLED DOUBLE BLIND MULTICENTERTRIAL'ROUPSOFAPPROXIMATELYPEOPLERECEIVED OR +GVACCINEANTIGEN PEOPLERECEIVEDAPLACEBOINTRAMUSCULARLYIM AT DAYSAND!BLOODSAMPLEWASALSOTAKENDAYSAFTERTHESECONDDOSE 3AFETYOFTHEVACCINEWASFINEOVERALL WITHLOCALPAINANDTENDERNESSBEING THEMOSTFREQUENTLYREPORTEDSYMPTOMS4HESESYMPTOMSOCCURRED AFTERBOTH DOSES WERE DOSE DEPENDENT AND WERE REPORTED BY n OF THE SUBJECTS THAT RECEIVED THE HIGHEST VACCINE DOSES 4HE IMMUNOGENICITY RESULTS WERE CLEARLYDISAPPOINTINGANDCOMPLETELYALIGNEDWITHTHENON ADJUVANTEDGROUP OF THE (. VACCINE STUDY ;= AND WITH THOSE FROM THE 0ROTEIN 3CIENCES STUDYWITHRECOMBINANT(.;=3EROCONVERSIONUSING()AND-.WERE OBSERVED ONLY AFTER THE SECOND DOSE IN APPROXIMATELY OF THE SUBJECTS THATRECEIVEDTHEHIGHDOSEOFAND +GSEE4AB #LEARLYTHESERESULTS INDICATEDTHAT WHILEAVACCINEAGAINST(.WASPOSSIBLE WITHOUTANADJU VANT UP TO MG PER SUBJECT WERE REQUIRED7ITH THIS VACCINE THE GLOBAL


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


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


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

$ENG' ,I: 4IAN' ,I9 *IAO0 :HANG, ,IU: 7EBSTER2' 9U+ 4HEEVOLUTIONOF(.INFLUENZAVIRUSESINDUCKSINSOUTHERN#HINA 0ROC.ATL !CAD3CI53!n +ISTNER/ (OWARD-+ 3PRUTH- 7ODAL7 "RàHL0 'ERENCER- #ROWE"!


3AVIDIS $ACHO ( ,IVEY ) 2EITER - ET AL #ELL CULTURE 6ERO 6 DERIVED WHOLEVIRUS(. VACCINEBASEDONWILD TYPEVIRUSSTRAININDUCESCROSS PRO TECTIVEIMMUNERESPONSES6ACCINEn ,UKE#* 3UBBARAO+ 6ACCINESFORPANDEMICINFLUENZA %MERG)NFECT$IS n 3UGUITAN !,*R -C!ULIFFE* -ILLS+, *IN( $UKE' ,U" ,UKE#* -URPHY " 3WAYNE $% +EMBLE ' 3UBBARAO + ,IVE ATTENUATED INFLUENZA! (. CANDIDATE VACCINES PROVIDE BROAD CROSS PROTECTION IN MICE AND FERRETS 0,O3-EDE .OVAVAX )NC 0RESS RELEASE .OVAVAX ANNOUNCES PRECLINICAL RESULTS FOR SEASONALINFLUENZAVACCINEPROGRAM $E&ILETTE- -IN*OU7 "IRKETT! ,YONS+ 3CHULTZ" 4ONKYRO! 2ESCH3 &IERS7 5NIVERSALINFLUENZA!VACCINE/PTIMIZATIONOF- BASEDCON STRUCTS6IROLOGYn $E&ILETTE- &IERS7 -ARTENS7 "IRKETT! 2AMNE! ,ÚWENADLER" ,YCKE . *OU7- 3AELENS 8 )MPROVED DESIGN AND INTRANASAL DELIVERY OF AN -E BASEDHUMANINFLUENZA!VACCINE6ACCINE n *IMENEZ'3 0LANCHON2 7EI1 2USALOV$ 'EALL! %NAS* ,ALOR0 ,EAMY6 6AHLE2 ,UKE#*ETAL 6AXFECTIN FORMULATEDINFLUENZA$.!VACCINES ENCODING.0AND- VIRALPROTEINSPROTECTMICEAGAINSTLETHALVIRALCHALLENGE (UM6ACCINES n 4OMPKINS 3- :HAO :3 ,O #9 -ISPLON *! ,IU4 9E : (OGAN 2* 7U : "ENTON+! 4UMPEY4- %PSTEIN3, -ATRIXPROTEINVACCINATIONAND PROTECTIONAGAINSTINFLUENZAVIRUSES INCLUDINGSUBTYPE(. %MERG)NFECT$IS n ,IVINGSTON"$ (IGGINS$ 6AN.EST' %VOLVINGSTRATEGIESFORTHEPRE VENTIONOFINFLUENZAINFECTIONPOTENTIALFORMULTISTRAINTARGETING"IO$RUGS n .ICHOLSON+' #OLGATE!% 0ODDA! 3TEPHENSON) 7OOD* 9PMA9 :AMBON -# 3AFETY AND ANTIGENICITY OF NON ADJUVANTED AND -& ADJUVANTED D INFLUENZA !$UCK3INGAPORE (. VACCINE ! RANDOMISED TRIAL OF TWO POTENTIALVACCINESAGAINST(.INFLUENZA,ANCETT n 3TEPHENSON) .ICHOLSON+' #OLEGATE! 0ODDA! 7OOD* 9PMA% :AMBON - "OOSTINGIMMUNITYTOINFLUENZA(.WITH-& ADJUVANTED(. !$UCK3INGAPORE VACCINE IN A PRIMED HUMAN POPULATION 6ACCINE n 3TEPHENSON ) "UGARINI 2 .ICHOLSON +' 0ODDA! 7OOD *- :AMBON -# +ATZ *- #ROSS REACTIVITY TO HIGHLY PATHOGENIC AVIAN INFLUENZA (. VIRUSESAFTERVACCINATIONWITHNONADJUVANTEDAND-& ADJUVANTEDINFLUENZA !$UCK3INGAPORE (. VACCINE ! POTENTIAL PRIMING STRATEGY * )NFECT $IS n 3CHWATZ" 'ELLIN" 6ACCINATIONSTRATEGIESFORANINFLUENZAPANDEMIC F * )NFECT$IS n :ANGWILL+ #AMPBELL* .OAH$ 4REANOR* %VALUATION OFATHIRDDOSE OF SUBVIRION (. INFLUENZA VACCINE RG!6IETNAMX02" IN HEALTHY ADULTS)N/PTIONSFORTHE#ONTROLOF)NFLUENZA6)4ORONTO *UNEn ) !BSTRACTNO0

7AITINGFORAPANDEMIC

4REANOR ** #AMPBELL *$ :ANGWILL +- 2OWE4 7OLFF - 3AFETY AND IMMUNOGENICITY OF AN INACTIVATED SUBVIRION INFLUENZA ! (. VACCINE . %NGL*-EDn 4REANOR** 7ILKINSON"% -ASSEOUD& (U 0RIMMER* "ATTAGLIA2 /"RIEN$ 7OLFF- 2ABINOVICH' "LACKWELDER7 +ATZ*- 3AFETYANDIMMUNO GENICITYOFARECOMBINANTHEMAGGLUTININVACCINEFOR(INFLUENZAINHUMANS 6ACCINEn "RESSON*, 0ERRONNE, ,EUNAY/ 'ERDIL# 3AVILLE- 7OOD * (OSCHLER+ :AMBON-# 3AFETYANDIMMUNOGENICITYOFANINACTIVATEDSPLIT VIRION INFLUENZA !6IETNAM (. VACCINE 0HASE ) RANDOMIZED TRIAL ,ANCETn T ,EROUX 2OELS ) "ORKOWSKI! 6ANWOLLEGHEM4 $RAMÏ - #LEMENT & (ONS % $EVASTER *- ,EROUX 2OELS ' !NTIGEN SPARING AND CROSS REACTIVE IMMUNITYWITHANADJUVANTEDR(.PROTOTYPEPANDEMICINFLUENZA VACCINE! T RANDOMISEDCONTROLLEDTRIAL,ANCETn "ANZHOFF! !NTIGEN SPARING EFFECT OF -& IN &LUAD (. )N 4HIRD 7(/MEETINGONEVALUATIONOFPANDEMICINFLUENZAPROTOTYPEVACCINESINCLINI CALTRIALS n&EBRUARY 7(/ 'ENEVA ,IN* :HANG* $ONG8 &ANG( #HEN* 3U. 'AO1 :HANG: ,IU9 7ANG :ETAL 3AFETYANDIMMUNOGENICITYOFANINACTIVATEDADJUVANTEDWHOLE VIRION INFLUENZA ! (. VACCINE ! 0HASE ) RANDOMISED CONTROLLED TRIAL ,ANCETn T 6AJO: +OSA, 6ISONTAY) *ANKOVICS- *ANKOVICS) )NACTIVATEDWHOLE VIRUSINFLUENZA!(. VACCINE %MERG)NFECT$ISn !TMAR2, +EITEL7! 0ATEL3- +ATZ*- 3HE$ %L3AHLY( 0OMPEY* #ATE 42 #OUCH 2" 3AFETY AND IMMUNOGENICITY OF NONADJUVANTED AND -& ADJUVANTEDINFLUENZA!(.VACCINEPREPARATIONS #LIN)NFECT$IS n &ERGUSON .- #UMMINGS $! #AUCHEMEZ 3 &RASER # 2ILEY 3 -EEYAI ! )AMSIRITHAWORN3 "URKE$3 3TRATEGIESFORCONTAININGAN EMERGINGINFLU ENZAPANDEMICIN3OUTHEAST!SIA.ATUREn 'ERMANN4# +ADAU+ ,ONGINI)-*R -ACKEN#! -ITIGATIONSTRATE GIESFORPANDEMICINFLUENZAINTHE5NITED3TATES 0ROC.ATL!CAD3CI53! n &LAHAULT! 6ERGU% #OUDEVILLE, 'RAIS2& 3TRATEGIESFORCONTAININGA GLOBALINFLUENZAPANDEMIC6ACCINEn


-ODELINGINFLUENZAPANDEMICANDINTERVENTIONS #ATERINA2IZZO AND-ARTA,UISA#IOFIDEGLI!TTI .ATIONAL#ENTEROF%PIDEMIOLOGY 3URVEILLANCE AND(EALTHPROMOTION )NFECTIOUS$ISEASE5NIT

)STITUTO3UPERIOREDI3ANITÌ 2OMA )TALY$EPARTMENTOF0HARMACO "IOLOGY 5NIVERSITYOF"ARI )TALY

!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

#ATERINA2IZZOAND-ARTA,UISA#IOFIDEGLI!TTI

@(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

-ODELINGINFLUENZAPANDEMICANDINTERVENTIONS

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%PIDEMIOLOGICALPARAMETERS 0ARAMETERSUSEDINTHEMODELSINFLUENCETHETIMEESTIMATEDFORTHEPAN DEMICTOEVOLVE ALTHOUGHTHEESTIMATESARESIMILARCONSIDERINGTHATSTUDIES BASEDONDETERMINISTIC3%)2MODELSONAGLOBAL;=ORLOCAL;=SCALEOR INDIVIDUAL BASEDMODELS; =)NFACT MOSTOFTHEMODELINGSTUDIES ARECALIBRATEDUSINGPARAMETERSDERIVEDFROMTHEOBSERVEDRATES OFSERO CONVERSIONANDILLNESSDUETOTHEPANDEMICSTRAINSTHATCIRCULATEDDURING THE TH CENTURY PARTICULARLY TO SCALE INFECTIVITY )N DURING THE FIRST PANDEMICWAVE THEATTACKRATEOFCLINICALINFLUENZAWASHIGHESTINCHILDREN AND THEN FELL GRADUALLY WITH AGE REACHING IN PEOPLE AGED YEARSORMORE!NAVERAGEATTACKRATEOFWASREPORTEDDURINGTHE PANDEMIC WITHANAGEDISTRIBUTIONSIMILARTOTHATOBSERVEDDURINGTHEFIRST PANDEMICWAVEOF;=4HEAGEDISTRIBUTIONOFATTACKRATESDURINGTHE PANDEMICWASSIMILARINCHILDREN RANGINGBETWEENAND BUT DECREASED LESS MARKEDLY WITH AGE REMAINING ABOVE IN ALL OTHER AGE GROUPS;= 4HETHREEBASICPARAMETERSUSEDTODESCRIBETHENATURALHISTORYOFINFEC TIONAREI PERSON TO PERSONTRANSMISSIONRATE WHICHISASSUMEDTOVARYBY AGEOFSUSCEPTIBLEANDINFECTIOUSINDIVIDUALSANDWITHTHETIME SINCEINFEC TIONII THELENGTHOFTHELATENTPERIODTIMEELAPSEDFROMINFECTIONTOTHE ONSETOFINFECTIVITY ANDIII THELENGTHOFTHEINFECTIOUSPERIOD 0ASTMODELINGSTUDIESHAVELARGELYASSUMEDDISTRIBUTIONSOFTHEINCU BATION AND INFECTIOUS PERIODS REPORTED IN TWO HISTORICAL PAPERS ; = (OWEVER DATAFORTHESEESTIMATES USEDTOESTIMATETHEDURATIONOFINFEC TIOUSNESS ARE LACKING )N FACT RESEARCHERS HAVE ASSUMED CONSTANT INFEC TIOUSNESS FROM THE END OF INFECTION LATENCY TO RECOVERY FROM INFECTION 2ECENTLY SOMEAUTHORS; =HAVEDERIVEDNEWESTIMATESOFTHEINCUBA TIONPERIODANDDEVELOPMENTOFINFECTIOUSNESSOVERTIMEFORHUMANINFLU ENZABASEDONDATAFROMEXPERIMENTALSTUDIESINWHICHVIRALSHEDDINGWAS MEASUREDINVOLUNTEERSCHALLENGEDWITHWILD TYPEINFLUENZAVIRUSES;= 4HEPROFILESOBTAINEDWERECONSISTENTWITHTHOSEOFAPROSPECTIVEHOUSE HOLD CONTACTS SURVEY CONDUCTED IN &RANCE ; = WITH PEAK INFECTIVITY BETWEENTHEDAYSnAFTERINFECTION ANDINFECTIVITYLASTINGAMAXIMUM OFDAYS; =


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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


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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!SNON PHARMACEUTICALMEASURES THENATIONWIDECLOSINGOFALLSCHOOLS FOR WEEKS PUBLIC OFFICES FOR WEEKS AND PUBLIC GATHERING PLACES EG RESTAURANTS CINEMAS AND CHURCHES FOR WEEKS WERE CONSIDERED 4HESE MEASURES WOULD BE INTRODUCED SIMULTANEOUSLY WEEKS AFTER THE START OF THEPANDEMIC INTHECOUNTRY)NTHEMODEL SCHOOLCLOSUREWOULDREDUCETHE CONTACTSAMONGCHILDRENANDTEENAGERS THESCHOOLCOMPONENTOFTHETRANS MISSIONRATE BYWORKPLACECLOSUREWOULDREDUCETHEJOBCOMPONENT OFTHETRANSMISSIONRATEBYCLOSUREOFPUBLICGATHERINGPLACESWOULD REDUCETHERANDOMCOMPONENTOFTHETRANSMISSIONRATEBY

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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


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


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 !mUNOV 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 &LUAD &LU-IST &OCETRIA 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

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