Bureau GGO

GEKOMEN 2 1 FE

tfli9

Intervet International bv Wim de Körverstraat 35 P0. Box 31 5830 AA Boxmeer

The Netherlands msd-animal-nealth.com

RIVM / SEC / Bureau GGO Postbus 1 3720 BA BILTHOVEN

.) MSD

Anima! Health

Boxmeer, 17 februari 2012 Betreft: nieuwe aanvraag

Geachte mevrouw, heer,

Hierbij doet Intervet International bv een verzoek tot een aanvraag van voorgenomen ingeperkt gebruik van genetisch gemodificeerde organismen, als bedoeld in paragraaf 2 van het Besluit Genetisch Gemodificeerde Organismen. De aanvraag betreft productie van Yellow Fever-West Nile chimeer virus t.b.v. vaccinproductie. Bijgevoegd zult u het aanvraagformulier met 3 bijlagen aantreffen.

Intervet International bv

Cc:

Trade register No. 16028015

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AANVRAAGFORMULIER INGEPERKT GEBRUIKGROOTSCHALIG: HANDELINGEN IN PROCESINSTALLATIES EN BIOREACTOREN MET GENETISCH GEMODIFICEERDE ORGANISMEN Indien u vragen heeft kunt u contact opnemen met Bureau GGO (email: bggo(rivm.nl, telefoon: 030-2742793). Alle gevraagde gegevens op dit formulier zijn openbaar. Vertrouwelijke informatie dient in een aparte bijlage meegezonden te worden. Het aanvraagformulier omvat vragen die mogelijk niet van toepassing zijn voor uw aanvraag. U wordt vriendelijk verzocht de onderdelen die geen betrekking hebben op de aan te vragen werkzaamheden NIET in uw aanvraag op te nemen. LEES VOOR HET INVULLEN EERST DE TOELICHTING! De toelichting vindt u aan het einde van formulier.

INHOUDSOPGAVE A

ALGEMENE GEGEVENS

8

TABEL VAN GGO’s

C

Beschrijving van het GGO

D

Handelingen met GGP’s in procesinstallaties of bioreactoren

INTERNET

www.vrom.nl/ggo-vergunninqverleninq

AFKORTINGEN Regeling ggo

Regeling genetisch gemodificeerde organismen Genetisch Gemodificeerd Organisme

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ALGEMENE GEGEVENS Vergunningaanvraag titel Antwoord: Productie in roller botties van YelIow Fever-West Nile chirneer virus A.1 .2 geef een gedetailleerde beschrijving van de werkzaamheden (zie toelichting) Antwoord: Het doel van de werkzaamheden is het op corn merciele schaal maken van antigeen voor een vaccin tegen West Nile virus in paarden. Hiervoor wordt een chimeer virus gebruikt, dat gebaseerd is op de Yellow Fevervaccinstam 17D. In de chimeer zijnde prM en E genen van Yellow Fever virus vervangen door de prM en E genen van West Nile virus. Het YeIlow Fever-West Nile (YF-WN) chirneer virus wordt vermenigvuldigd in Vero cellen. De Vero-cellen worden gekweekt in roller bottles. Nadat de cellen in de roller bottles de gewenste celdichtheid hebben bereikt, worden de roller bottles leeggegoten en wordt er medium met een lage hoeveelheid YF-WN chimeer virus toegevoegd. Na een vastgesteld aantal dagen worden alle roller bottles leeggegoten in een steriel vat met een grootte van niet meer dan 750L. Dit vat is het eerste inactiveringsvat, waarin het virus chemisch wordt geïnactiveerd. De inhoud uit het eerste inactiveringsvat wordt na begin van de inactivering naar een tweede vat overgepompt om zeker te zijn dat de gehele inhoud met het chemische inactiveringsmiddel in contact is geweest. De inactivering wordt gedurende de vastgestelde tijd bij de juiste temperatuur en pH gehouden, waarna de inactivatie chemisch wordt gestopt. Vervolgens wordt het antigeen 5-1 Ox geconcentreerd door middel van ultrafiltratie. Het geconcentreerde antigeen wordt afgevuld en ingevroren. Alle aseptische handelingen, inclusief monstername, vinden plaats in een klasse A cleanroom (met Ml-(ll classificatie).

A.1.1

A.1 .3

wordt er gebruik gemaakt van ggo’s die onder een andere vergunning binnen Nederland zijn vervaardigd? zo ja, geef de projectnummer(s) Antwoord: De GGO is al sinds 2003 in gebruik onder beschikking nummer lG 03-089. De GGO is vervaardigd door Acambis lnc. Cambridge, MA, USA.

Vergunningaanvrager A.2.1 instellinglbedrijf (zie toelichting) Antwoord: Intervet International bv A.2.2 afdelinglfaculteit Antwoord: Tissue Culture Boxmeer A.2.3 correspondentieadres Antwoord: Postbus 31, 5830 AA Boxmeer: Plaats van uitvoering A.3.1 plaats van uitvoering (bezoekadres) Antwoord: Wim de Korverstraat 35, 5831 AN Boxmeer A.3.2 naam vergunningverlenende instantie Wet milieubeheer Antwoord: Gemeente Boxmeer A.3.3 nummer Wet milieubeheervergunning en datum van afgifte Antwoord: R-VLR/201 1/4493, juli 2011 A.3.4 inperkingniveaus van de ruimten waarvoor Wet milieubeheer voor activiteiten met ggos is afgegeven Antwoord: Ml III voor productiewerkzaamheden en maximaal ML III voor researchwerkzaamheden. Verantwoordelijk medewerker verantwoordelijk medewerker (zie toelichting) (titel, voorlefter(s), achternaam)

A.4.1

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A.4.2 A.4.3 A.4.4 A.4.5

Antwoord: instellinglbedrijf Antwoord: Intervet International bv afdeling/faculteit en vakgroep Antwoord: correspondentieadres Antwoord: Postbus 31 5830 AA Boxmeer telefoon/faxnummer Antwoord e-mail: Antwoord: -

A.4.6

Biologischeveiligheidsfunctionaris (B VF) A.5.1

biologischeveiligheidsfunctionaris (titel, voorletter(s), achternaam)

A.5.3

Antwoord: correspondentieadres Antwoord: Postbus 31 5830 AA Boxmeer telefoonnummer

A.5.4

e-mail

A.5.2

Verklaring van de BVF en de verantwoordelijk medewerker

De BVF en de verantwoordelijk medewerker verklaren kennis te hebben genomen van de inhoud van deze aanvraag Datum:

cj

Handtekening BVF:

Datum: Handtekening verantwoordelijk medewerker:

3

B.1 .2

B.1 .1

1

-,

T-1, T-2 of T-3)

Yellow Fever virus stam 17D

Alle vectoren te gebruiken in cornbinatie met vermelde gastheersoort

T= Toxiciteitsklasse(

nvt

T

West Nile virus genen coderend voor het envelop (E) proteine en het pre rnembraan (pr-M) proteine

Alle te kloneren donor Genen/Sequenties

West Nile virus

Donororganismen

3

PG

nvt

T

Aanvraagformulier

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Ingeperkt gebruik

4

Betreft het commerciële activiteiten of onderzoeks & ontwikkelings (R&D) activiteiten (zie toelichting)? Antwoord: Commerciele productie

liter.

Met welke volumina worden er handelingen uitgevoerd ? (zie toelichting), per nummer uit tabel B specificeren Antwoord: Het volume van de productie unit (roller bottles) is 700 ml met een maximum van 1 liter. Deze worden gepoold tot een maximum batchgrootte van 750

PG= Klasse van pathogeniteit (1 2, 3 of 4)

African green monkey kidney cellen (Verocellen)

1

PG

(ELEMENTEN GEBRUIKT VOOR DE VERVAARDIGING VAN DE GGO’S DIE IN DEZE KENNISGEVING WORDEN TOEGEPAST)

Gastheersoort

Tabel van GGO’s

{PR IVA TE }N r.

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Aanvraagformulier

Beschrijving van het GGO Plantaardige en dierlijke cellen als gastheer

C.6.1 C.6.2 C.6.3

C.6.4

C.6.5

Gastheer nummer in de tabel van GGOs Antwoord: 1 geef de naam van de cellijn/cellen (zie toelichting) Antwoord: Vero cellen (African green monkey kidney cellen) geef een beschrijving van de cellijnlcellen met name met betrekking tot de wijze van vervaardiging (bv. immortalisatie) en opsomming van eventueel aanwezige virale sequenties (welke en uit welk virus) Antwoord: Verocellen zijn zeer algemeen gebruikte cellen in zowel onderzoek als productie van virale vaccins, waaronder levende vaccins voor mensen (oa polio vaccin) en dieren. De cellijn is op 27 maart 1962 geisoleerd uit een nier van een gezonde volwassen Cercopithecus aethiops door Y. Yasumura and Y. Kawakita aan de Chiba University in Chiba, Japan. Het is een continue, aneuploide cellijn, die in principe ‘oneindig” in cultuur kan worden gehouden. De cellijn is ontwikkeld door langdurige passages in kweek, er is dus geen immortalisatie of vergelijkbare techniek op toegepast. De cellijn vertoont geen retrovirale reverse transcriptase activiteit. Voor zover bekend, bevat de cellijn geen virale sequenties. De in gebruik zijnde Vero-cellen worden regelmatig op de aanwezigheid van “extraneous agents” getest. zijn de gastheercellen genetisch gemodificeerd? zo ja, geef een beschrijving van de genetische mod ificatie Antwoord: NEE is de gastheer geschikt voor de vervaardiging van groep IAB ggo’s en is dit bevestigd in een erkenningsbrief (geef datum en kenmerk)? Antwoord: NEE

Indien het een gastheercelljn betreft die niet IAB erkend is dienen de vragen onder C. 7 beantwoord te worden. Deze gegevens hebben betrekking op de eigenschappen van het aan te melden organisme en moeten onderbouwd worden met onderzoeks- of literatuurgegevens. C.7.1 C.7.2 C.7.3

C.7.4

klasse c.q beschrijving van pathogeniteit van de gastheer

Antwoord: 1 produceert de gastheer agentia die potentieel schadelijk zijn? Zo ja, welke? Antwoord: NEE bevat de gastheer helperfuncties die, in combinatie met een virale vector, tot de vorming van autonoom replicerend, infectieus virus kunnen leiden? Antwoord: Het doel van de gastheer in deze aanvraag is het produceren van infectieus virus. Echter, dit virus kan niet autonoom repliceren; het zal daar altijd delende (gastheer)cellen voor nodig hebben. is de gastheer veilig gebleken bij langdurig gebruik onder condities van fysische inperking die het Ml-l niveau niet te boven gaan?

Antwoord: Vero-cellen zijn zeer algemeen gebruikte cellen in zowel onderzoek als productie van virale vaccins, waaronder levende vaccins voor mensen (oa polio vaccin) en dieren.

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heeft de gastheer een biologische inperking waardoor de overlevings- of reproductiekansen in het milieu beperkt zijn?

Antwoord: Vero-cellen kunnen alleen overleven en reproduceren bij zeer strikte condities: de omgevingstemperatuur moet rond de 37°C liggen en er moet celkweekmedium met verschillende voedingsstoffen aanwezig zijn in een gesloten systeem onder aseptische condities. Deze condities zijn zodanig dat de kans op overleving en reproductie in het milieu nihil zijn.

C.8.1

C.8.2

C.8.3

C.8.4

C.8.5

C.8.6 C.8.7

C.8.8 C.8.9

C.8.1O

Vectoren staat de vector vermeld op de lijst van vectoren die geschikt zijn voor de vervaardiging van groep IA ggo’s (bijlage 2.1.2 van de Regeling)? Antwoord: NEE indien nee: voeg een plasmidekaart toe waarop duidelijk de grootte en de verschillende onderdelen aangegeven zijn (antibiotica resistentie genen, virale sequenties, promotoren, enhancers, merkers, etc). Antwoord: De methode van vervaardiging van de ggo is beschreven in bijlage 1 (Arroyo et al, 2001). Virale vectoren beschrijf per vector alle virale onderdelen (functie en herkomst, zie toelichting) Antwoord: Voor het chimere construct is de virusstam gebruikt van het gele koorts vaccinvirus, bekend als YF-1 7D. Deze vaccinstam is een zeer effectief en veilig levend geattenueerd vaccin tegen gele koorts. De stam wordt sinds 1936 gebruikt als vaccin; er zijn wereldwijd meer dan 400 miljoen doseringen van dit vaccin aan mensen toegediend. wat is de klasse van pathogeniteit van het virus waarvan de vectoren zijn afgeleid: 2/3/4 Antwoord: Wildtype Yellow fever is klasse 3, vaccinstam YF-17 D is klasse 2 ingeschaald (zie COGEMadvies CGM/070724-01) op welke wijze wordt het virus overgedragen? Antwoord: Het virus waar de vector van is afgeleid: het gele koorts virus, wordt verspreid door muggen. Er zijn twee vormen te onderscheiden: stedelijke gele koorts en jungle gele koorts. De stedelijke gele koorts wordt verspreid door de mug Aedes aegypti. De jungle variant wordt verspreid door verschillende muskieten soorten die enkel voorkomen in de jungles van tropisch Afrika en zuid/centraal Amerika. De Aedes aegypti mug komt van nature niet voor in Nederland. zijn de virale vectoren infectieus en replicatiecompetent? Antwoord: JA zijn de vectoren in omvang beperkt tot de voor het beoogde doel noodzakelijke sequenties? Antwoord: JA. Er wordt een chimeer virus gemaakt van Yellow Fever en West Nile fever virus. De vector sequenties aanwezig in het construct zijn noodzakelijk om een antigeen te produceren dat voldoende immunogeen is om het als vaccin te kunnen gebruiken. zijn de vectoren vrij van schadelijke sequenties? Antwoord: JA. De vector bevat geen sequenties die leiden tot de productie van schadelijke stoffen. beschrijf de toepassing (integratie, suïcide vector etc.) van de vectoren Antwoord: De virale vector vermenigvuldigt zich in de gastheer (Vero-cellen), zonder dat daarbij integratie van het genoom van de virale vector in dat van de gastheer plaats vindt. Uiteindelijk zal de cellulaire gastheer hierdoor lysis ondergaan. Als de gastheercellen gelyseerd zijn, zal de vermenigvuldiging van de gastheer stoppen. Doel is antigeenproductie t.b.v. geïnactiveerd vaccin worden bepaalde vectoren (obligaat) in combinatie gebruikt (zie ook toelichting)?

Antwoord: NEE

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C.9.1 C.9.2

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Inserties Gekarakteriseerde sequenties/genen wat is de grootte van het insert? Antwoord: 1998 bp wat is de functie van de elementen van het insert (genen, regulatiesignalen etc.)? Op welke wijze zijn deze elementen gekarakteriseerd? Antwoord: De genen prM en E, die het insert vormen, coderen voor structurele eiwitten van West Nile virus. Het envelop eiwit is het oppervlakte eiwit van het virion. E is daardoor het immunodominante eiwit en belangrijk voor het opwekken van een goede immuunrespons. Het M eiwit (prM is de precursorvorm) is het eiwit dat zorgt voor de verbinding van het E eiwit met het nucleokapsied en is dus nodig voor de vorming van een functioneel virusdeeltje. codeert het insert voor een toxine? welk toxine wordt er gevormd; wat is de werking van het toxine; tot welke klasse behoort dit toxine (TIIT2IT3); op welke wijze is de LD5O bepaald; is de bepalingswijze relevant voor de gastheer; wat is de blootstelling aan het toxine bij gebruik in de gastheer Antwoord: het insert codeert niet voor een toxine, de subvragen zijn niet van toepassing. zijn (gedeelten van) transposons aanwezig? Indien ja, geef een beschrijving van de aanwezige sequenties en hun functionaliteit Antwoord: NEE codeert het insert voor een schadelijk product anders dan een toxine? Zo ja, waarvoor zijn de genproducten schadelijk? Antwoord: NEE is de insertie vrij van sequenties waarvan bekend is dat zij bijdragen tot de mobiliseerbaarheid van vector en insertie? Antwoord:JA kan het insert in combinatie met de vector en de gastheer tot de vorming van infectieus, replicatiecompetent virus leiden? Zo nee, toelichting

-

-

-

-

-

-

C.9.4

C.9.5

C.9.6

C.9.7

C.9.8 C.9.9

Antwoord: JA Zo ja, is er een verandering van het tropisme te verwachten (geef een motivatie)? Antwoord: NEE, zie vraag C 9.9 worden er chimeren gemaakt (zie toelichting)? Indien ja, geef aan welke delen van de verschillende virussen afkomstig zijn. Is er sprake van een kans op tropismeverandering (geef een motivatie)? Antwoord: JA er worden chimeren gemaakt. De genen coderend voor het pre-membrane eiwit (prM) en het envelop eiwit (E) zijn afkomstig van West Nile virus, alle overige genen van het virus zijn afkomstig van YF-17D. Uitgebreid intern en extern onderzoek heeft uitgewezen dat het recombinant YF-WNV een replicatiekinetiek en een weefseltropisme vertoont die vergelijkbaar zijn met dat van de vaccinstam YF 17D, en verschillend van WNV. Deze onderzoeken zijn uitgebreid besproken in het verzoek tot wijziging van de beschikking IG 03-089 dd 21 maart 2007. Over deze wijziging van de beschikking is advies uitgebracht door de COGEM (kenmerk CGM/070724-01)

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verhoogt de insertie de overlevingskans van het ggo in het milieu? Antwoord: NEE GGO Is het ggo erkend als een ggo waarmee zowel handelingen van categorie A als B mogen worden uitgevoerd door middel van een erkenningsbrief (geef datum en kenmerk)? Antwoord: NEE

C.11.2

wat is de klasse van pathogeniteit van het ggo Antwoord: 2

Ci1.3

is het GGO in de installatie en het productieproces net zo veilig als de gastheer of ouderstammen? Antwoord: Verschillende onderzoeken hebben aangetoond dat het YF-WN chimeer virus zeker net zo veilig is als het YF-1 7D virus, de vaccinstam van het gele koorts vaccin (zie vraag C 9.9). De virulentie van het YF WN chimeer virus is ook lager dan de West Nile virus donor van prM en E. Hieruit mag men concluderen dat het GGO in de installatie en het productieproces net zo veilig is als de gastheer of ouderstam men. Zie ook vraag 011.4.

C.11.4

heeft het GGO ten opzicht van een natuurlijk isolaat van dezelfde soort een beperkte overlevingskans buiten het fysisch inperkend systeem? Antwoord: Virussen kunnen enkel overleven in delende cellen. De overlevingskans buiten het inperkend systeem hangt mede af van de capaciteit om cellen te infecteren en er in te repliceren. Studies hebben aangetoond dat het YF-WN chimeer virus ongeveer 1 OOx slechter in staat is om muskietencellen te infecteren dan de YF-17D stam, die in dit geval beschouwd kan worden als het natuurlijke isolaat van dezelfde soort (zie Bijlage 2)

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D D.1

D.2

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Handelingen met GGP’s in procesinstallaties of bioreactoren Bijproducten Kan of worden er andere producten dan het bedoelde geproduceerd tijdens het gebruik o.i.v. het ingebrachte genetisch materiaal? Zo ja, welke? Antwoord: NEE Inrichting beschrijf de toegepaste technische procédés Antwoord: Vero-cellen worden gekweekt in roller botties met een kweekvolume van 700 ml. Als er voldoende cellen zijn gekweekt, wordt een preculture van maximaal 100 roller botties aangelegd met deze cellen. Nadat de cellen in de roller bottles de gewenste celdichtheid hebben bereikt, worden de roller bottles geleegd en wordt er eenzelfde hoeveelheid virushoudend medium toegevoegd. De virusproductie vindt plaats voor de voorgeschreven tijdsduur en daarna worden alle roller bottles geleegd. De oogst wordt verzameld in een steriel vat en heeft een batchgrootte van niet meer dan 750L. Dit vat is het eerste inactiveringsvat, waarin het virus chemisch met binair ethylenimine (BEl) wordt geïnactiveerd. De inhoud uit het eerste inactiveringsvat wordt enkele uren na begin van de inactivering naar een tweede vat overgepompt om zeker te zijn dat de gehele inhoud met BEl in contact is geweest. Tijdens het inactiveringsproces wordt de virus-BEl suspensie continue gemengd en gedurende de vastgestelde tijd bij de juiste temperatuur en pH gehouden, waarna de inactivatie chemisch wordt gestopt (mbv natrium thiosulfaat). Vervolgens wordt het antigeen 5-lOx geconcentreerd door middel van ultrafiltratie. Het geconcentreerde antigeen wordt afgevuld in flacons en opgeslagen. Alle aseptische handelingen, inclusief monstername, vinden plaats in een MI-lIl ruimte (klasse A cleanroom). Flowschema zie bijlage 3 geef een beschrijving van het (downstream)proces; specificeer de stappen waarbij de afdoding van het ggo plaatsvindt Antwoord: 1. Inactivatie, hierbij vindt het “afdoden” van het ggo plaats: De virusoogst wordt verzameld in een steriel vat. Dit vat is het eerste inactiveringsvat, waarin het virus chemisch met BEl wordt geïnactiveerd. De inhoud uit het eerste inactiveringsvat wordt enkele uren na begin van de inactivering naar een tweede vat overgepompt om zeker te zijn dat de gehele inhoud met BEl in contact is geweest. Tijdens het inactiveringsproces wordt de virus-BEl suspensie continu gemengd en gedurende de vastgestelde tijd bij de juiste temperatuur en pH gehouden. Na het bereiken van de vereiste inactivatieduur wordt de inactivatie chemisch gestopt (met natrium thiosulfaat) en wordt het antigeen gekoeld tot een temperatuur van 2-8°C. 2. Concentratie van het afgedode ggo: Het antigeen (geïnactiveerde virussuspensie) wordt geconcentreerd afgevuld en opgeslagen.

0.4

geef een beschrijving van de methode van afdoding van de GGOs en de validatiegegevens Antwoord: De virusoogst wordt verzameld in een steriel vat. Dit vat is het eerste inactiveringsvat, waarin het virus chemisch met BEl wordt geïnactiveerd. Aan de virusoogst wordt 2-bromo ethylamine hydrobromide (BEA) toegevoegd tot de gewenste eindconcentratie. BEA wordt omgezet tot de actieve vorm BEl door de pH boven de 7.5 te brengen. De inhoud uit het eerste inactiveringsvat wordt 2 uur na begin van de inactivering naar een tweede vat overgepompt om zeker te zijn dat de gehele inhoud met BEl in contact is geweest. Tijdens het inactiveringsproces wordt de virus-BEl suspensie continu gemengd en gedurende ± 18 -24 uur bij de juiste temperatuur en pH gehouden. Validatiestudies hebben laten zien dat inactivatie van een virus oogst met een normaal te verwachten concentratie virus maximaal 4 uur duurt. Inactivatie van een (artificieel, door concentratie verkregen) virus oogst met een virusconcentratie die 1 Ox boven de normaal te verwachten concentratie lag, duurde

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maximaal 8 uur. De duur van het inactiveringsproces van de YF-WN chimeer is voor praktische redenen ingesteld op 18-24 uur, dat overschrijdt dus ruim de vereiste duur om virus met een concentratie van 1 Ox de normale opbrengst af te doden. Na het bereiken van de vereiste inactivatieduur wordt de inactivatie chemisch gestopt (met natrium thiosulfaat) en wordt de virussuspensie gekoeld tot 2-8°C. D.5

D.6

geef het maximale volume in het downstreamproces per run en maximaal te produceren volume Antwoord: De maximale batchgrootte in het downstream proces is 750L per run (hoeveelheid vôôr concentreren). Er zullen per cleanroom niet meer dan 2 batches tegelijkertijd aanwezig zijn. Ongevallen geef een analyse van de risicobronnen en omstandigheden waaronder ongelukken kunnen gebeuren Antwoord: Voor het productiegebouw is een calamiteitenprocedure opgesteld, waarin onder andere is opgenomen hoe te handelen in het geval van morsen van levend virus. De ruimten waar open handelingen plaatsvinden zijn voorzien van onderdruk. Elke cleanroom is voorzien van een afzonderlijke luchtbehandeling met dubbel HEPA-filtersysteem. De viruskweek zelf vindt plaats in roller bottles met een volume van circa 700 ml elk. Bij het oogsten wordt het antigeen in een vat verzameld, waarbij mogelijk een groter volume virus vrij kan komen in de ruimte. De opzet van de cleanroom is zo, dat de totale inhoud van het systeem altijd binnen het ingeperkte gebied wordt opgevangen. Indien er door een onvoorziene situatie virus in de cleanroom vrijkomt, wordt de spillprocedure gevolgd. Het materiaal zal worden ingedamd, chemisch gedesinfecteerd en opgeruimd. Medewerkers dragen cleanroomkleding en handschoenen zodat ze beschermd zijn tegen blootstelling en infectie door niet intacte huid. Verspreiding is via een vector (mug) en dit zal niet optreden bij een dergelijke spill. Buiten de cleanrooms (ingeperkt gebied) zijn alleen virusseed (1 ml afvulling in glazen 10 ml flacon afgesloten met rubber stopper en aluminium cap) en kwaliteitscontrolemonsters (enkele ml, verpakt in onbreekbare plastic flacons) gedurende korte tijd aanwezig voor opslag of transport. Het materiaal is buiten inperking verpakt in dubbele, lekdichte verpakking. Indien er toch een kleine hoeveelheid virus vrijkomt door een onvoorziene situatie tijdens transport of opslag buiten inperking gemorst, wordt het materiaal gedesinfecteerd en opgeruimd.

D.7

geef de aan de werknemers verstrekte informatie over ongevallenpreventie en rampenplan Antwoord: Er staat een algemene instructie in het bioveiligheidshandboek op intranet. Alle medewerkers zijn getraind in de spill procedure, een SOP die beschrijft wat te doen bij bijvoorbeeld een lekkage waarbij virussen vrijkomen. Medewerkers zijn op de hoogte dat bij een ongeval/incident met GGO’s altijd een BVF moet worden geïnformeerd.

D.8

geef informatie waarmee de bevoegde autoriteiten een rampenplan voor gebruik buiten de installatie kunnen opstellen Antwoord: De maatregelen in geval van calamiteiten zijn onderdeel van het noodplan dat is opgesteld voor Intervet International bv. Hierin zijn ook de afspraken met brandweer en locale hulpverleners vastgelegd. Er worden periodiek gezamenlijke oefeningen gehouden.

D.9

Afvalstoffen beschrijf de aard, hoeveelheid en potentiële gevaren van de geproduceerde afvalstoffen Antwoord: Vast afval dat (mogelijk) met virus in contact is geweest, wordt thermisch gedestrueerd (autoclaaf) Vloeibaar afval dat (mogelijk) met virus in contact is geweest, wordt door middel van een chemisch destructieproces behandeld (Chloramine T).

10

0

VROM 15november2005

Ingeperkt gebruik Grootschalig Aanvraagformulier

Herbruikbare materialen en apparaten die (mogelijk) met virus in contact zijn geweest en die niet door middel van één van bovenstaande methoden kunnen worden gedecontamineerd, worden gedecontamineerd d.m.v. begassing (H -gas). Deze techniek wordt ook routinematig gebruikt voor 0 2 desinfectie van de cleanrooms waarin met dit virus is gewerkt. De mobiele vaten die gebruikt zijn zullen van binnen gedesinfecteerd worden d.m.v. een stoomdesinfectie (steaming in place). D.1O

Inschaling van de handelingen samenvatting van de relevante eigenschappen van de ggo’s

Antwoord: Het GGO is een chimeer virus, dat gebaseerd is op de Yellow Fever vaccinstam 1 7D. In de chimeer zijn de prM en E genen van Yellow Fever virus vervangen door de prM en E genen van West Nile virus. Het Yellow Fever-West Nile (YF-WN) chimeer virus wordt vermenigvuldigd in Vero cellen. DII

geef de inperkingscategorie(en) waaronder deze werkzaamheden kunnen worden uitgevoerd, en het artikel uit de Regeling/Richtlijnen waarop deze inschaling is gebaseerd

Antwoord: Deze werkzaamheden kunnen worden uitgevoerd op inperking MI-lIl (bijlage 5, 5.7.3.a) D.1 2

geef aan welke aanvullende inrichtingsvoorzieningen of werkvoorschriften noodzakelijk zijn

Antwoord: geen

c

11

L’j —

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71 Hu, J. etal. (1998) Hypoxia regulates expression of the endothelin-1 gene through a proximal hypoxia-inducible factor-1 binding site on the antisense strand. Biochem. Biophys. Res. Commun. 245, 894-899 72 Jiang, B-H. etal. (1996) Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.]. Bio). Chem. 271, 17771—17778 73 Lee, P.J. etal. (1997) Hypoxia-inducible factor 1 rnediates transcriptional activation of the heme oxygenase- 1 gene in response to hypoxia. J Bio). Chem. 272, 5375—5381 74 Feidser, D. eta]. (1999) Reciprocalpositive regulation of hypoxia-inducible factor lcz and insulin-like growth factor 2. CancerRes. 59, 3915—3918 75 Pairner. LA. eta). (1998) Hypoxia induces type II NOS gene expression in pulmonary artery endothelial ceils via HIF-1. Am. 1. Physiol. 274, L212—L219 76 Bruick, RK. (2000) Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc. Nat). Acad. Sd. U S. A. 97, 9082-9087 77 Bhattacharya, S. eta). (1999) Functional role of p35srj, a novel p300ICBP binding protein, during transactivation byHIF-1. Genes Dcv 13, 64—75

O

78 Kietzmann,T. ctal. (1999) Induction of the plasminogen activator inhibitor-1 gene expression hy mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes. B)ood94, 4177—4185 79 Takahashi, Y. eta], (2000) 1-lypoxic induction of prolyl 4-hydroxylase tc(I) in cultured tells. 1 Bio). Chem. 275, 14139-14146 80 Rolfs,A. etal. (1997) Oxygen-regulated transferrin expression is mediated by hypoxia inducible factor 1.]. Bio). Chem. 272, 20055—20062 81 Lok, C.N. and Ponka, P. (1999) Identification of a hypoxia response element in the transferrin receptorgene. .1. Bio). Chem. 274, 24147—24152 82 Tacchini, L. eta). (1999) Transferrin receptor induction by hypoxia: HIF-1-mediated transcriptional activation and cell-specific post-transcriptional regulation. 1. Bio). Chom. 274, 24142—24146 83 Gerber, H-P. eta). (1997) Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes: Flt-1, but not Flk-1IKDR, is upregulated by hypoxia. J. Bio), C’hem. 272,23659-23667 84 Carrero, P. eta). (2000) Redox-regulated recruitment of the transcriptional coactivators

CREB-binding protein and SRC-1 to hypoxia-inducible factor la. Mol. Ce))Bio). 20, 402 -415 85 Erna, M. eta). (1999) Molecular rnechanisrns of transcription activation by HLF and HIF-la in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. EMBOJ. 18,1905—1914 86 Zhang,W. eta). (1999) Transitional change between HIF-1 and HNF-4 in response to hypoxia. J. Hum. Genet. 44, 293-299 87 Gradin, K. eta). (1996) Functional interference between hypoxia and dioxin signal transduction pathways: cornpetition for recruitrnent of the Amt transcription factor. Afo). Ce)) Bio). 16, 5221—5231 88 Hogenesch.J.B. cta).(1997)Characterizationofa subset of the basic-helix-loop-helix-PAS superfarnily that interacts with cornponents of the dioxin signaling pathway. J. Bio). Chem. 272, 8581—8593 89 An, W.G. etal. (1998) Stabilization of wild-type p53 by hypoxia.inducible factor lcc. Nature 392, 405—408 90 Arany,Z. ctal.(1996)Anessentialrolefor p300ICBP in the cellular response to hypoxia. Proc. Nat). Acad. Sci. U S. A. 93, 12969—12973

Yellow fever vector live-virus vaccines: West Nile virus vaccine development Juan Arroyo, Charles A. Miller, John Catalan and Thomas P Monath By combining molecular-biological techniques with our increased understanding of the effect of gene sequerice modification on viral function, yellow fever 17D, a positive-strand RNA virus vaccine, has been manipulated to Iduce a protective immune response against viruses of the same family \e.g. Japanese encephalitis and dengue viruses). Triggered by the emergerice of West Nile virus infections in the New World afflicting humans, horses and birds, the success of this recombinant technology has prompted the rapid development of a live-virus attenuated candidate vaccine against West Nile virus.

r

Juan Arroyo Charles A. Miller John Catalan Thomas R Monath Acambis mc. 38 Sidney Street. Cambriclgo, MA 02319. USA. ‘e-maii:juan.arroyo@

acarnbis.com

Yellow fever (YF) virus is a positive-strand RNA virus widely used as an attenuated live-virus vaccine. Other RNA live-virus vaccines inciude measles, mumps, rubella and poliovirus, all of which have been in use for many years with remarkable , although this has been 2 safety and efficacy profiles” challenged by recent reports linking measles virus or measles vaccines with the occurrence ofjuvenile . 5 3 CR0HX’s DISEASE (see Glossary) and autism Recently, some of these RNAviruses have been explored as vectors to deliver foreign genes. A promising example is influenza virus, a negative strand RNA virus commercialized as a nonliving virus vaccine, first used in 1989 as a vaccine vector. Influenza vector technolog based on delivering defined EPITOPES by substitution of surface residues of http://tmm.trends.com

1471-4914/01/s

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the iniluenza spike proteins, exploits influenza TROPISM to target generation oftiucosAL IMMLINJTY in , as well as 6 the upper and lower respiratory tracts systemic responses, which result in immunity in genital and intestinal tracts . Poliovirus, a positive 7 strand RNA virus like YF, has been taken a step forward in preclinical studies and tested as a vaccine vector genetically modified to induce immunogenicity against tetanus toxin and simian . Most advances regarding 89 immunodeficiency virus the use of RNA viruses as delivery systems rely on the significant progress of RT-PCR, REVERSE GENETICS, plasmid vectors and in vitro transcription systems. The first infectious animal RNA virus done to be recovered from a full-length cDNA molecule was the 7.5-kb poliovirus in 198110; only last year, a 27-kb porcine coronavirus, the longest viral RNA genome known, was successfully cloned usirig a bacterial artificial chromosome (BAC), a low-copy-number plasmid”. The instability of bacterial vectors carrying viral cDNA was a gigantic hurdie for this technology, as experienced during the YF virus 2 were the first to generate YF cloning. Rice et al.’ virus RNA from a pair of cDNA clones ligated in vitro before RNA transcription. The same technology was

see front matter © 2001 Elsevier Science Ltd. All rights reserved. Pil: S1471.4914(01l02048-2

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later used by Thomas J. Chambers to create a chimeric virus where the sequence of YF envelope genes was substituted with the sequence from Japanese encephalitis (JE), a member of the same group of viruses known as flaviviruses’ . Chambers’ 3 chimeric concept originated from work in 1992 where another pair of flaviviruses (i.e. tick-borne encephalitis and dengue) was used to create a chimeric live virus with vaccine potential’ . Pletnev’s 4 dengue chimera encodes the structural genes of a virulent tick-borne encephalitis strain, while retaining ?‘EURO\’IRuLENCE in a mouse model. Attenuation was achieved owing to loss of peripheral invasiveness, thus creating a conceptual method for attenuating flaviviruses. Following these principles, the chimeric YF vaccine technology has evolved to create a platform, now delivering structural genes encoding sufficient protective antigens to produce live-attenuated candidate vaccines against JE, t5 and more recently, West Nile (WN). dengue Vellow lever live-attenuated vaccine

YF 17D vaccine strain was developed 65 years ago by empirical methods, which included a substantial passage history’ . The vaccine is used for wide-scale 6 immunization of children in tropical areas, travelers and military personnel. This long experience in >350 million people has provided assurances of safety and efficacy, making yellow fever an ideal vector for foreign genes. However, there are disadvantages to this practice. As with any other positive-strand virus, the high rate of genetic variation caused by viral replication by polymerases without proofreading enzymes and mutations involved in adaptation to different host ceils could lead to unexpected surprises. Replacing the structural genes of YF 17D with those of other flaviviruses might alter tropism, and the potential of replication in unanticipated tissues will have to be examined individually. On the other hand, a significant advantage of alive vaccine inciudes the development of rapid and durable HUMORAL and ceil-mediated immune responses that closely mimic those directed against the wild-type virus. Chimeric vaccine construct technology

YF and WN are members of the genus Flavivirus enveloped, positive-strand RNA viruses of approximately 11 kb, which are transmitted by arthropods, like ticks and mosquitoes’ . WN virus 7 was first isolated from a febrile adult woman in the West Nile district of Uganda in 193718 °. The virus was not recognized as a cause of severe meningoencephalitis until an outbreak in Israel in 195720. After appearing over subsequent years in Europe, Asia and Australia, where it afflicted humans and horses, WN made its appearance in North America in 199918, Since the first recorded outbreak in the western hemisphere, WN has become part of the prevention and control mandate of several —

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Glossary Crohn’s disease: A chronic infiammatory disease. primarily involving the small and large intestine, butwhich can affect other parts of the digestive system as weTI. CytotoxicT celi: T celi that can kilI other cells. Most cytotoxic T ceils are MHC class 1-restricted CD8T cdle, but CD4T ceils can also kili in some cases. Cytotoxic Tcells are important in host clefense against cytosolic pathogens. Epitopes: Refers to a site 0fl an antigen recognized by an antibody; epitopes are also called antigenic determinants. A T-cell epitope is a short peptide derived from a protein antigen. It binds toen MHC molecule and is recognized by a particularTcell. Humoral immuriity: Specific immunity mediated by antibodies made ina humoral immune response. Mucosal immunity: Resistance to infection across the mucous membranes, Mucosal immunity depends on immune cdle and antibodies present in the linings of reproductive tract, gastrointestinal tract and other moist surfaces of the body exposed to the outside world, Neuroinvasive: A virus capable of invading the central nervous system upon peripheral inoculation. Neurovirulence: The ability of a virus to replicate in bram tissue and cause encephalitis. Reverse genetics: A strategy for studying gene structure and function by site-directed mutagenesis. The modified nucleic acid can then be introduced into an organism to study the effect of the mutation. RT-PCR (reverse transcriptase polymerase chain reaction): PCR in which the stareing template is RNA, implying the need for an initial reverse transcriptase step to make a DNA template, followed by separate conventional PCR. Tropism: Tissues or host cclie in wh ich a virus can replicate. Virion: A virus particle existing freely outside a host ccli,

government agencies. The availability of a technology (ChimeriVax) based on the use of YF 1 7D as a vector to deliver effective immunity against JE virus, a close relative of WN virus, prompted the development of a WN vaccine. A formalin-inactivated JE vaccine (JE-VAX®, Aventis Pasteur) is available in the USA and marketed to travelers. Although immunity to JE might provide cross protection against WN in macaques there are insufficient data to recommend the use of the JE-VAX® to protect humans and horses. The development of the WN vaccine followed the path drawn by the development of a live-attenuated YF—JE chimeric vaccine (ChimeriVax-JE) 13,22• The same two-plasmid system used to develop ChimeriVax-JE was utilized by replacing the sequences for the JE prMand Egenes with those of the WN NY-99 strain virus (Fig. 1). The resulting VIRION has the envelope of WN, containing structures involved in virus—ceil attachment and virus internalization, all antigenic determinants for neutralization, and epitopes for T-cell mediated immunity. The capsid protein, nonstructural proteins and non-translated termini (UTR) responsible for virus replication remain those of YF 17D vaccine. Like ChimeriVax-JE, the chimeric YF/WN virus replicates in tissue culture to titers in excess of 7 1og 10 plaque-forming units (PFU)ml-’. The chimeric virus is expected to replicate efficiently in the host as well as provide protective immunity against WN virus. ,

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prM

Na,1

WN E

Eag I/Bsp El digestion Isolation of fragments In vitro ligation Xho 1 linearize

Sp6

BspEl 5 WN YF

XhoI

YF Sp6 transcription

RNA TF?ENDS in Molecuiar Medic,ne

Fig. 1. Two-plasmid system encoding the YF—WN chimeric vaccirle. Plasmid VF5’3’IV WN preMembrane and envelope protein genes (prME) encodes the 5’ UTR, yellow fever capsid (VFC),

West Nile virus prM (gray) and 5’ end of E (blue), and the 3’ end of yellow fever NS5 and UTR. Plasmid YFM5.2 WN encodes the second half of E (blue) and the non-structural genes of yellow fever NS1 NS2A, NS2B, NS3, NS4A, NS4B NS5. West Nile prMEgene fragments were amplified by RT-PCR and subcloned into the two-plasmid system by overlap-extension PCR. Silent Eag land Bsp El sites ware introduced for in vitro ligation steps necessary to create a full-length cDNA before in vitro transcription. Naked RNA initiates productive infections after transfection ofa Vero cell line.

Therefore, relative to peripheral natural infection, PFU is a severe test of 10 TO inoculation with 5 1og protection. All ChimeraVax-JE immunized rhesus developed high titers of neutralizing antibodies after subcutaneous vaccination. Following IC challenge with a wild-type JE virus, vaccinated monkeys were 100% protected against viremia and clinical encephalitis providing dear evidence of . 26 vaccine efficacy Because the neurotropic nature of these viruses might lead to encephalitis in humans, the safety of live JE and WN vaccines has to be carefully addressed. Flavi virus genome replication lacks proofreading activity, resulting in a significant rate of mutations. For example, one single codon alteration from GAA (glutamic acid) t0AAA (lysine) in the envelope protein gene markedly reduced the . To study the genetic 28 virulence of a JE virus isolate stability of a ChimeriVax vaccine construct, the virus was passed six times in bram tissue of mice and up to 18 times in ceil culture. For ChimeriVax JE, the vaccine genome and attenuated phenotype . The molecular 24 were shown to be stable on passage basis of attenuation of ChimeriVax-JE vaccine was . Neurovirulent 22 studied by systematic mutagenesis JE strains and attenuated JE strains were compared by sequence analysis, and amino acid residues in the prMEsequence of the vaccine that were implicated in virulence were reverted. The elucidation of multiple attenuation determinants in the Egene provided a rationale for the development of a chimeric WN vaccine.

ChimeriVax-J E vaccine: immunological basis for protection, efficacy and safety

Molecular basis of ChimeriVax-JE vaccine attenuation and the ratioriale for WN vaccine development

Neutralizing antibodies are the first line of defense against flaviviruses. In a live vaccine, virus replication induces a fast and durable response. CYTOTOXIC T CELLS eliminate viruses that were able to establish intracellular infections. This defense mechanism recognizes the E protein as well as nonstructural proteins, making infected celis a target . Exposure to the virus by natural 23 for ceil kifling infection post-vaccination would lead to a rapid secondary response, stronger than the primary response, and increased cross-reactivity with other members of the Flavivirusgenus encoding similar 2526 were 24 and monkeys epitopes. Studies in mice conducted to determine the ability of ChimeriVax-JE vaccine to protect animals against challenge. Challenge of both immunized and control monkeys with JE virus was perfornted by an intracerebral (IC) 10 PFU. Natural JE infection inoculation of 5 1og occurs by peripheral inoculation of small doses of virus in mosquito saliva; it was estimated by in vitro 10 methods that a female mosquito releases about 2 1og . Typically, rhesus monkeys 27 PFU while blood feeding do not develop bram infection and encephalitis after peripheral JE virus inoculation. The IC inoculation disease model is thus used in vaccine efficacy tests,

In principle, the attenuated phenotype and safety profile of the ChimeriVax-JE virus are based on the derivation of all its genome from proven vaccine strains (i.e. YF 17D and JE 5A14-14-2). The live-attenuated JE SA14-14-2 vaccine is used only in . 29 China and possesses an excellent safety record The basis of attenuation lies within the genome sequence of these RNA viruses. Sequence changes affect the phenotype by altering the function of the gene products, the secondary structure integrity of the RNA molecules, or both. The effect of the sequerice of the prMEgenes of JE on its virulence phenotype was revealed following mouse studies that compared ChimeriVax-JE with a corresponding neurovirulent YF/JE Nakayama . The study showed lack of neurovirulence 3 construct’ for ChimeriVax-JE relative to the Nakayama construct or to a YF 1 7D vaccine control. The E proteins of wild-type and SA14-14-2 (attenuated) JE strains differ in ten amino acid residues (Table 1), A rational sequence reversion study to understand the basis of ChimeriVax-JE attenuation provided additional insight. Envelope amino acid residue E138 had a dominant effect, being required for reconstitution of neurovirulence ina mouse model,

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Table 1. Mutagenesis target residues for West Mle vaccine attenuation Virus

Amino acid 107

138

176

177

227

244

264

279

315

439

West NiIeNY S 9

L

E

Y

T

S

E

Q

K

A

K

JE wild type

L

E

1

T

3 P

E

Q

K

A

K

14142 strain F JEEA

K

V

A

S

G

H

M

V

R

aE227 was found to ho a proline (P) in Japanose encephalitis (JE) Nakayama (wild-type) strain.

whereas E107, 176, 279, 264 and 227 residues, in that order, only contributed to measurable increases in neurovirulence . 22

WN virus is a member of the JE antigenic complex, a subgroup of very closely related flaviviruses defined by cross-neutralization . The sequence similarity of 30 the E protein of WN and JE is shown in Fig. 2. Because of the high degree of similarity, it is hypothesized that the introduction of mutations linked to the attenuation ofJE SA14-14-2 will also attenuate ChimeriVax-WN. For ChimeriVax-WN, the approach will be to systematically mutate the sequence of the wild-type WN NY99 strain E protein to assess the effect of single mutations at the equivalent of residues E138, 107, 176 and 279. Additional mutagenesis targets could he considered from the outcome of other F]avivfrus studies. Mutations in the vicinity of E3 15 are associated with altered virus tropism and changes in . 33 3t virulence Position E244 might not play a significant role as it is either a glycine (G) or glutamic acid (E) in several virulent JE strains analyzed . Position E439 3436 EN NY99 JE EA14142

1 1

EN NY99 JE EA14142

43 63

EN N199 JE 3514142

125 125

na=rirntant__

EN NYS9 JE 4514142

197 197

c

EN NY99 JE 4514142

249 249

EN NY99 JE 4514142

311 310

372 371

EN NYS9 JE 0514142

373 372

434 433

EN NY99 JE 0A14142

435 434

EN NY99 JE EA14142

457 t 496

42 62 107)

flîIrr 139)

176,177Jj_________

2271

244

2791

186 186

1

—4 :zz4ri 2641

124 124

:

249 248

315 309

3191

4391 496 490 541 50E 1 EOI9IJN LE MOI4CL1I0 M6CICIE4

Fig. 2. sequence alignment of the E protein of WN NV-99 strain and theJE 5A14-14-2 vaccine strain. Identical and conserved residues are shown in blue and green, respectively. A 77% identity was predicted by CLU5TAL W alignment. Red arrows map the location of ten amino acid residues that distinguish virulentJE Nakayama strain E protein (see Table 1).

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represents a conserved K—R substitution in the transmembrane region of the E protein with very littie chance of any major effect. A tyrosine (Y) at position E176 (Table 1) might contribute to the attenuation of ChimeriVax-WN in its present configuration. The hypothesis will be tested by targeted mutation of El 76 to isoleucine and testing for an increase in neurovirulence. Eventually, combinatorial mutation studies will define an attenuated vaccine. Surprisingly, the chimeric ChimeriVax-WN construct containing wild-type \VN prME was found to be significantly Iess neurovirulent than YF 1 7D vaccine. Young adult mice (five per group) inoculated IC with graded doses between 2 and 6 log 10 PFU resulted in scattered deaths (ranging from 20—60% mortality) without a dear dose effect, perhaps owing to the susceptibility of the model used. By contrast, YF 17D was 100% lethal at doses >1 1og 10 PFU in th same mouse model. In addition, ChimeriVax-WN ha 3 lost the NEUROINVA5IVE property typical of wild-type WN. Virus replication in bram tissue or viremia levels in morihund animals was not measured. However, the average survival times (nine days) were similar for both viruses. The data suggest that the sequence unmodified chimera might he a vaccine candidate. Ultimately, the safety of a live attenuated ChimeriVax-WN will rely on sequence stability of the genome particularly at the E protein amino acid positions identified to play a role in attenuation. Additional WN vaccine approaches

The hiotech company Baxter/Immuno (Austria) has initiated efforts to develop a formalin-inactivated human vaccine. Fort DodgeAnimal Health (USA) has initiated development of botha formalin-inactivated and a DNA plasmid vaccine for horses. The DNA technology was developed at the Centers for Disease Control and Prevention, Ft. Collins (CC, USA) and study in horses at Colorado State University demonstrated protection against virus challenge . 35 Lustig er al. produced alive attenuated WN virus isolate derived from empirical passage of a wild-type strain in Aedesaegyprimosquito cells. One dose of the attenuated virus showed 100% protection in both mice and geese IC inoculation disease models challenged with a homologous wild-type WN (ReL 37). A formalin-inactivated WN vaccine should work as effectively as JE-VAX® against JE. 1-lowever, requiring multiple doses for efficacious protection, this vaccine would not be beneficial in an immediate threat of epidemic disease. A DNA vaccine for expression of WN prMEgenes only might be protective for horses but is likely to require multiple doses. Considering the possibility of a spontaneous virulent reversion owing to the relatively high mutation rate of RNA viruses, alive attenuated full length WN vaccine is not as safe as the WN prME DNA vaccine. Furthermore, the WN prMEDNA vaccine will produce antibodies only against the WN

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membrane proteins encoded in the plasmid thus the resporise to nonstructural viral gefleS, typical of natural WN infections, will be absent. This will facilitate screening of traded horses by the existing antibody detection methods. A chimeric live vaccine (ChimeriVax-WN) encoding the nonstructural genes of YF 17D might be as effective in horses as the DNA vaccine. However, it has to be determined whether the vaccine will replicate and prove effective, Conciusions

C

(

Acknowledgements We would like to thank Thomas Ermak (Acambis mc., Cambridge, MA, USA) and Farrukh Rizvi (Aventis Pasteur, Toronto, Canada) for theircritical review of this manuscript.

The need for a WN virus vaccine will be defined by the progression of WN outbreaks. The most promising candidates for a human vaccine include an ‘old technology’ formalin-inactivated virus vaccine, and a live chimeric vaccine where a rational approach is used to create a safe recombinant vaccine. A live vaccine that rapidly elicits immunity after a single dose would be much preferred over a multi-dose

References 1 Corter, H. and Campbell, H. (1993) Rational use of measles, mumps and rubella (MMR) vaccine. Drugs45, 677-683 2 Guillot, S. ctal. (2000) Natural genetic exchanges between vaccine and wild poliovirus strains in humans. 1. Viro?. 74, 8434-8443 3 Petrovic, M. etal. (2001) Second dose of measles, mumps, and rubella varcine: questionnaire survey of health professionals. Br ,fcd. 1322, 82—85 4 Afzal, M.A. c’tal. (2000) Clinical safetyissuesof measles, mumps and rubella vaccines. Bull. l4drldHcalth Organ. 78, 199-204 5 Kawashima, H. etal. (2000) Detection and sequencing of measles virus from peripheral mononuclear ceils from patients wild inilammatory bowel disease and autism. Dig. Dis. Sri. 45, 723—729 6 Hewson, R. (2000) RNA viruses: emerging vectors for vaccination and gene therapy. Mol. Mcd. Today6, 28-35 7 Muster, T. cta]. (1995) Mucosal model of immunization against human immunodeilciency virus type 1 withachimericinfluenzavirus. J. l’irol. 69,6678 6686 8 Porter, D.C. etal. (1997) Immunization of mice with poliovirus replicons expressing the C fragment of tetanus toxin protects against lethal challenge with tetanus toxin. Vaccine 15, 257—264 9 Tang, S. etal. (1997) Toward apoliovirus-based simian immunodeficiency virus vaccine: correlation between genetic stability and immunogenirity. 1. Virol. 71, 784 1—7850 10 Raraniello, VR, and Baltimore, D. (1981) Cloned poliovirus complernentary DNA is infectious in marnmalian cells. Science 214,916—919 11 Almazan, F. et al. (2000) Engineering the largest RNA virus genome as an infectious bacterial artificial chrornosome. Proc. Nat]. Acad. Sci. U S. A. 97,5516-5521 12 Rice, CM. etal. (1989) Transcriptionofinfectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation. NewBiol. 1,285—296 13 Chambers, T.J. etal. (1999) Yellow Fever/Japanese encephalitis chimeric viruses: construction and biological properties. J. l’irol. 73. 3095 -3101 http://tmm.trends.conl

product for use in an impending epidemic, as surveillance of birds and mosquitoes provides only a brief warning of virus activity before the risk of human disease. The ecology of WN virus in the western hemisphere is stili evolving. At least five species of mosquitoes were found to be competent vectors in experimental transmission studies with the WN NY99 strain’ . Further understanding of the 8 basic transmission cycle of WN in birds and mosquitoes is needed to implement proper control mechanisms. Ultimately, dear definition of the risks associated with WN virus epidemics might help create sufficient preventive measures to avoid the use of a vaccine. In the meantime, for as long as outbreaks continue to occur predominaritly in urban areas where media attention is so ubiquitous, and with a system that relies on sightings of dead crows to forecast a potential WN epidemic, the need to develop a vaccine will continue.

14 Pletnev, A.G. etal. (1992) Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses. Proc. Nat?. Acad. Sci, U S. A. 89, 10532—10536 15 Guirakhoo, F. et al. (2000) Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates. 1. Virol. 74. 5477—5485 16 Monath, T.P. (1999) in K’llowFever(Plotkin, S. and Orenstein,X’fA.,eds), pp. 815—879.WBSaunders and Company 17 Monath, T.P. and Heinz, F.X. (1996) Flaviviruses. in Fields Virology (Fields, B.N. etal. eds), pp. 961—1034. Lippincott-Raven 18 Komar, N. (2000) WestNileviralencephalitis. Rei: sci. tech. 0ff int. Epiz 19. 166—176 19 Smithburn, K. etal. (1940)Aneurotropic virus isolated from the bloed of a native of Uganda. Am. 1. Lop. Mcd. Hyg. 20,471-492 20 Jordan, 1. etal. (2000) Discovery and molecular characterization of West Nile virus NY 1999. Viral Immunol. 13, 435—446 21 Goverdhan, M.K. etal. (1992) Two.way cross-protection between West Nile and Japanese encephalitis viruses in bonnet macaques. Acta Drol. 36,277—283 22 Arroyo,J. etal. (2001) Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE). J. Virol. 75,934—942 23 Aihara, H. eta]. (1998) Establishment and characterization ofJapanese encephalitis virus specific, human CD4(+) T-ceIl clones: flavivirus cross-reactivity. protein recognition, and cytotoxic activity. 1. Virol. 72,8032-8036 24 Guirakhoo, F. eta?. (1999) Immunogenicity. genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-Japanese encephalitis virus (ChimeriVax-JE) as alive, attenuated vacrine candidate against Japanese encephalitis. lîrology257, 363—372 25 Monath,T.P. etal. (1999) Recombinant, chimaeric live, attenuated vaccine (ChimeriVax) incorporating the envelope genes of Japanese encephalitis (SA14-14-2) virus and the capsid and nonstructuralgenesofyellowfever (17D) virus is safe. immunogenic and protective in non-human prinlates. tccine17, 1869—1882

26 Monath,T.P. eta]. (2000) Chimericyellowfever virus 1 7D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys. J. Virol. 74, 1742—1751 27 Reisen, W.K. ctal. (2000) Method of infection does not alter response of chicks and house finches to western equine encephalomyelitis and St. Louis encephalitis viruses. 1. Mcd. Entornol. 37, 250-258 28 Sumiyoshi, H. etal. (1995) Characterization of a highly attenuated Japanese encephalitis virus generated from molecularly cloned cDNA. 1 In[eet. Dis. 171, 1144—1151 29 Tsai, T. etal. (1999) JapaneseEncephalitis 1dccines, WB Saunders and Company 30 Calisher, CH. wal. (1989) Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera, J. Gen. Virol. 70,37—43 31 Jennings, AD. wal. (1994) Analysis ofa yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis. J. In[eet. Dis. 169, 512—518 32 Ni, H. and Barrett, AD. (1998) Attenuation of Japanese encephalitis virus by selection of its mouse bram rnembrane receptor preparation escapevariants. Drvlogv24l. 30—36 33 Ryman, K.D. etal, (1998) Mutation ina 17D-204 vaccine substrain-specific envelope protein epitope alters the pathogenesis of yellow fever virus in mice. Virology244, 59-65 34 Ni, H. wal. (1995) Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14.i Gen. Virol.76, 409—413 35 Nitayaphan, S. wal. (1990) Nucleotide sequence of the virulent SA- 14 strain of Japanese encephalitis virus and its attenuated vaccine derivative. SA-14-14-2. Virologyl77, 541—552 36 Davis,B.S. etal. (2001) West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a non-infectious recombinant antigen that can be used in enzyme-linked immunosorbent assays. J. Virol, 75, 4040-4047 37 Lustig, S. etal. (2000) Alive attenuated West Nile virus Strain as a potential veterinary vaccine. Virallmmunol 13, 401-410

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jfr2

Short title: Growth characteristics of ChimeriVaxTMWN vaccine virus in mosquitoes

Growth charaeteristics of the veterinary vaceine candidate ChimeriVaxTWest Nile (WN) virus in Aedes aegypti, Aedes albopictus, and Culex xnosquitoes

BW Johnson,* TV Chambers,* ME Crabtree,* J Arroyo, T Monath,t and BR Miller*

*Djvjsjon of Vector-Bome Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado; Acambis mc., Cambridge, Massachusetts

Correspondence: Barbara W. Johnson, Centers for Disease Control and Prevention, Division of Vector-Borne Tnfectious Diseases, Arbovirus Diseases Branch, Rampart Road, Foothilis Campus, Fort Collins, Colorado 80521, U.S.A. E-mail: bfj9cdc,gov

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Abstract: The chimeric virus ChimeriVaxTMWest Nile (WN) (trademark Acambis mc., Cambridge, MA) is being developed for use as a vaccine to protect against WN infetion.

The veterinary vaccine virus ChimeriVax&WN(vet) contains the premembrane (prM) and envelope (B) genes from a wild-type WN virus, isolated from a flarningo in the New

York 1999 WN epidemic (WN NY99), in the yellow foyer (YF) 1 7D vaccine virus backbone. A vaccine canclidate for human use containing three specific animo acid

mutations in the WN insert is also under developnient. Replication ldnetics of -WN(vet) virus in rnosquito ceil culture and in vivo in the WN mosquito TM ChinieriVax

vectors Culex tritaeniorhynchus Giles (Diptera: Culicidae), Cx. nigripalpus Theobald (Diptera: Culicidae), and Cx. quinquefasciatus Say (Diptera: Culicidae), as well as the YF vectors Aedes aegypti (Linneaus) (Diptera: Culicidae)and 4e. albopictus (Skuse) (Diptera: Culicidae) were evaluated to determine the potential of mosquitoes to become infected through feeding on a viremic vaccinee and to transmit the virus. Growth of ChimeriVaxTMWN(vet) virus was restricted compared to WN virus in 4e. albopictus -WN(vet) and YF 17D viruses did not replicate in IT TM C6136 celis. CbiineriVax

inoculated Culex tritaeniorhynchus or Cx. nigrzpalpus, and replication was restricted in Cx. cjuinquefasciatus, 4e. aegypti, and 4e. albopictus compared to wt WN virus. None of the Culex mosquitoes were infected orally with CbimeriVaxTMWN(vet) virus; one 4e. albopictus and 10% of the 4e. aegypti became infected, but the titer was very low and virus did not disseminate to head tissue. ChimeriVaxTM.WN(vet) virus had a replication

profile siinilar to that of the attenuated vaccine virus YF 17D, which is not transmitted by mosquitoes. These resuits suggest that the natural mosquito vectors of WN and YF

2

viruses which may ineidentally take a blood meal from a vaccinated host, will not become infected with ChimeriVax’-WN(vet) virus. Key words. Aedes aegypti, Aedes albopictus, ChimeriVaxTMWN(vet) virus, Culex nigripalpus, Culex quinquefasciatus, Culex tritaeniorhynchus, West Nile vaccine, West Nile virus

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Introduction

West Nile (WN) virus is positive-strand RNA flavivirus, which is maintained in an enzoonotic cycle between birds and primarily Culex mosquitoes (Hayes, 1989). During periods of high density of mosquito populations, hijmans and equines are infected incidentally through the bite of an infected mosquito, but do not play a role in amplification or Iransmission of the virus (Bmming et al., 2001; Bunning ei al., 2002).

-WN virus (Acambis mc., Cambridge, MA) is being developed as a TM ChimeriVax potential vaccine against WN virus (Arroyo et al., 2001). ChimeriVax-WN contains

the preinembrane (prM) and envelope (E) genes from WN virus in the YF 1 7D vaccine -WN(vet), contains the TM virus backbone. The veterinary vaccine candidate, ChimeriVax prM and E genes from wild-type (wt) WN virus, which was isolated from a flamingo in

the New York 1999.WN epideniic (WN NY99) (Lanciotti et al., 1999). The human ChimeriVaxTMWN vaccine construct contains three specific animo acid mutations in the WN insert. It is currently under development and will be the subject of future research. The YF 17D vaccine is an attenuated, live virus wbich has been used sucessful1y and safely for over 65 years (Monath, 1991; Robertson ei al., 1996). Aedes aegypti, the principal mosquito vector species of YF virus, is poorly infected by the attenuated YF

3

17D vaccine strain and does not transmit the virus (Whitman, 1939). However, the introduction of heterologous flavivirus genes into the YF 17D genome may affect the

susceptibility of mosquitoes to infection by the chimerie virus. Therefore, infection and transmission studies of ChimeriVaxTMWN(vet) virus were eonducted in five mosquito species based on their importance as veetors of WN or YF virus, or because of their preference for human or mammalian hosts. West Nile virus is endemic in the Middie East, Afriea, and parts of Burope. Culex tritaeniorhynchus Giles mosquitoes are major vectors of Japanese encephalitis (JE) virus in Asia and of WN virus in India and Pakistan (Hayes et al., 1980; Ilkal et al., 1997). Although Cx. tritaeniorhynchus prefers large mammalian hosts, such as cattie, to birds and humans, it has been shown to be a competent vector species in the laboratory (Hayes et al., 1980; Hayes, 1989). In addition, the high population density reached by this mosquito in nature compensates for a low transmission rate, resulting in maintenance of active transmission (Hayes, 1989). West Nile virus was introduced into North America in the summer of 1999, and as a consequence of the presence of competent mosquito vectors, it distribution has expanded each sunimer since 1999 south and north along bird migration routes, and westward (Bernard & Kramer, 2001). Culex quinquefasciatus Say has become the primary enzootic veotor in the southern United States (Bernard & Kramr, 2001). In addition, Cx. quinquefasciatus feeds on a variety of hosts, which inoreases the risk of humans and equines becoming infected with WN virus (Niebylski & Meek, 1992; Bernard & Kramer, 2001). Culex nigripalpus Theobald may serve as bridge vector in the WN virus transmission cycle, as this species has been shown to prefer bird hosts but also feeds readily on mammals (Bernard & Kramer, 2001; Zyzak et al., 2002).

4

Aedes aegypti (Linneaus) is found throughout the fropics, and although not considered a competent vector species for WN virus, it is the principal vector of urban YP (Monath, 1989). The arbovirus transmission potential of Aedes albopictus (Skuse) has been a concern in the United States since its introduction in the 1980s, as it feeds on a variety of hosts, including manimals and birds, and is a competent vector of both dengue and YF viruses (Miller & Ballinger, 1988; Savage ei al., 1993; Niebylski ei al., 1994; Miteheli, 1995; Moore & Mitchell, 1997). West Nile virus RNA has also been isolated from field collectedAe. albopictus in the United States (Bernard ei al., 2001). Replication of ChimeriVax -WN(vet) virus was assesseci in Ac. albopictus C6/36 ceil TM culture, and the ability of ChirneriVaxTMWN(vet) virus to infect and be transmitted by mosquitoes was evaluated in vivo. Mosquitoes were either intrathoracically inoculated

with virus, to preclude the effect of the niidgut infection barrier, or fed an artificial, virusladen blood meal, and growth ldnetics of ChimeriVaxTM.WN(vet) virus were compared

to those of wt WN (NY99) and YF 17D vaccine viruses. Virus irifection was deterrnined by plaque titration over a 10-day time course and after a 14-day extrinsic incubation

period. Disseminated virus infection into head tissue was used as an indicator of transmission potential. Materials and Methods

Mosquiloes Culex quinquefasciatus and Cx. nigripalpus mosquitoes from the laboratory colonies of the Florida Medlical Entomology Laboratory (University of Florida, Vero Beach, FL) were provided by Dr. Jai Nayar. Culex tritaeniorhynchus and Ae, albopictus were from the laboratory colonies maintained at Centers for Disease Control and Prevention (CDC),

5

Division of Vector-Borne Infectious Diseases, Fort Collins, CD. Aedes aegypti collected

in the Republic of Vanuatu (F2), an island in the Pacific Ocean, were provided by Dr. Thomas Burkot (CDC). F4 and F5 generations from these mosquitoes were used in this study. Mosquitoes were maintained as previously deseribed (Bhatt et al., 2000; 3ohnson

et al., 2002). Viruses ChimeriVax-WN(vet) virus was obtained from Acambis Tno. at Vero ceil (green monkey kidney) passage 3. West Nile virus from the CDC Diagnostic and Reference Section, Division of Vector-Bome Diseases, Fort Collins, CD, was isolated from a flaniingo at the Broux Zoo in New York in 1999 (passed once in Vero celis, once in

C6136 ceils) (Lanciotti et al., 1999). Yellow fever 17D vaccine virus (Aventis-Pasteur, lot VA433AA) was used unpassaged. Stook virus titers were as follows: ChimeriVaxTM WN(vet), 7.7 Iog PFU!mL; WN NY99, 7.2 logo PFU!rnL; YF 17D, 6 log PFUIrnL. Virus titration Virus titers were assayed by plaque titration. Jnclividual mosquitoes were triturated by pestie in 1 .7-mL tubes (Kontes Glasa Co., Vineland, Nl) in 1 rnL BA-l diluent (1X M 199 medium withHank’s saits, 0.05M Tris, pH 7.6, 1% bovine albun’iin, 0.35 g/L of sodium bicarbonate, 100 ofU/mLpenicillin, 100 g/mL streptomycin, 1 L/mL of fungizone). Moaquito slispensions were clarified by centrifugation at 8000 rpm for 10 min. Virus titer was determined by double-overlay plaque assay in Vero celis as previously described (Muller et al., 1989). Second overlays were applied after a 4-day -WN(vet) viruses, and after 2 days for TM inoubation period for YF 17D and ChimeriVax wt WN virus.

6

Growth curves

Growth curves were conducted for each of the ChimeriVax -WN(vet), WN NY99, and TM YF 17D viruses in both Vero and 06/36 celis. Celi monolayers were infected at a multiplicity of infection (m.o.i.) of 0.01 plaque-forming units (PFU) per cefl. Following adsorption of 2 hr in C6136 ceils and 1 hr in Vero celis, monolayers were washed and celis were maintained in Dulbecco’s modified minimal essential medium containing gentamycin and 5% FBS. Samples were removed every other day for 10 days and titers were determined by plaque titration as described above. Int rathoracic inoculation of Inosquitoes Mosquitoes that had eclosed 1 to 2 days earlier were cold-anesthetized and inoculated intrathoracically (IT) using a ruicrocapillary needie that bad been pulled to a point with a Narishige (Tokyo) needile puller. Approximately 0.34 itL of virus standardized to 6.0 o PFIJ/mL was injected into each mosquito (3.5 1ogo PFU/mosquito)(Rosen & 1 log Gubler, 1974). Inoculated mosquitoes were maintained in cartons at 27°C and 85% humidity with 5% sugar water. Three mosquitoes were collected at 48-hr intervals for 10 days and the remainder were collected at 14 days postinoculation, and then were frozen at -70°C until assayed. Oral infection ofrnosquitoes

Blood meals were prepared from fresh virus grown in Vero ceils as described above. Virus supematant was collected 2-6 days after infeofion (76% CPE) and clarified by centrifugation at 8000 rpm, 4°C for 20 min. Defibrinated sheep’s blood (Colorado Serum Co., Denver, CO) was washed with ice-cokl phosphate-buffered saline (PBS) three times. Two parts fresh virus (7-8 logw PFU/mL) was rnixed with two parts defibrinated sheep’s

7

blood and one part 10% sucrose in fetal bovine serum (FBS). The artificial blood rneal was quickly heated to 37°C, then offered in hanging droplets to 7- to 10-day-old mosquitoes that had been starved for 24 hours (Gubler & Rosen, 1976). Mosquitoes weie allowed to feed for 30 min, then were cold anesthetized and sorted. Fully engorged mosquitoes were collected and incubated at 27°C and 85% humidity on sugar water as deseribed previously. Three mosquitoes were removeci at 48-hr intervals; the rest were collected at 14 days. Mosquitoes were stored at -70°C until they were assayed. Amplification and sequencing of C7zimeriVaxTMJVN(’ve) virus isolatedfrom inoculated inosquitoes Genomic sequences of the WN prM and E region in ChimeriVaxTMWN(vet) were obtained from individual Cx. quinquefasciatus, Ae. aegypti, and Ae, albopictus mosquitoes that were positive for virus infection at 14 days after IT inoculation, RNA was extracted from titurated mosquito tissue in 1 mL BA-1 diluent using the QIAamp viral RNA kit (QIAGEN, Valencia, CA). One hundred microliters of mosquito suspension from the titurated Cx. quinquefasciatus was used to inoculate a T-25 cm 2 flask of Vero ceils as described above. Virus supernatant was collected at 6 days, 70% CPE,

and RNA was extracted using the QIAamp viral RNA kit. The WN prM and E genes and surrounding YF 1 7D gene region were amplified using the Titan One Tube RT-PCR Kit

and primers designed from the ChimeriVax”-WN(vet) virus sequence (forward position 413: 5 ‘-ACGCCGTTCCCATGATGTTCTGAC-3’; reverse position 2557: 5’CTTGTTCAGCCAGTCATCAGAGTC-3’). The resultant 2400-bp DNA product was

purified by gel electrophoresis followed by extraction from the gel using the QlAquick gel extraction kit (Qiagen) and sequenced using CEQ2000 Dye Teniiinator Cycle

8

Sequencing with Quick Start kit (Beckman Coulter, Fuilerton, CA). Sequencing reactions were analyzed on a Beckman Coulter CEQS000 Genetic Analysis System. Resuits In vitro virus replication

Replication of ChimeriVaxTMWN(vet) and YF 170 viruses in Ae. albopictus C6136 ceils was restricted compared to WN virus (Figure 1A). Peak mean virus titers of 10 PFU/mL) et) (5.2 logio PFU/mL) at 6 days and YF 170 (6.5 1og ChinieriVax -WN(v TM at 14 days were 4.0 and 2.7 logs lower, respectively, than the 9.2 logo PFU/mL peak mean titer of WN virus at day 10. All viruses grew well in Vero celis (Figure 1B). The peak mean titer of WN virus in Vero ceils was 8.7 logio PFU/mL at 2 days (Figure 1B). Aithough supematant was collected throughout the 1 0-day time interval, extensive cell death (>70% CPB) was observed by day 4, which resulted in a decrease in viral titer. On day 4 ChimeriVax-WN(vet) reached a titer of 8.4 logio PFTJ/rnL and at 6 days YF 7D grew to 7.8 logio PFU/mL, after whioh ceil death corresponded to decreased viral titer. Growth and dissemi,uzfion in IT-inocutated mosquitoes

Figure 2 illustrates growth of WN, ChimeriVax-WN(vet), andYF 170 viruses in IT inoculated mosquitoes over a 10-day time course. Presence and dissemination of viruses in mosquitoes at 14 days postinoculation is shown in Table 1. Mosquitoes were assayecl individually for infection by plaque titration of mosquito bodies. Disseminated infection was determined by plaque assay of individual mosquito heads. No Cx. tritaeniorhynchus IT inoculated with ChimeriVax-WN(vet) or YF 170 virus were infected at 14 days (Table 1). Two mosquitoes were infected with ChimeriVax 10 PFU!mosquito) and one at 8 days (1.1 logjo WN(vet) virus, one at 4 days (2.1 1og

9

PFU/mosquito) postinoculation (Figure 2A). In contrast, the titer of WN virus increased to 6.7 logio PFU/mosquito at 10 days followirig inoculation, and at 14 days 21 of the 22 WN-inoculated mosquitoes were infected, with a niean titer of 6.5 logio PFU!mosquito. WN virus disseminated to heads in all WN infected Cx. fritaeniorhynchus (Table 1). Replication of CbimeriVaxTMWN(vet) and YF 170 viruses was similarly restricted in IT-inoculated Cx. nigripalpus. No mosquitoes were infected with ChimeriVax’-WN(vet) virus after the 14-day extrinsic incubation period; at 4 days postinoculation, one mosquito (1.6 log o PFU/mosquito) was infected (Figure 2B). At 14 1 days postinoculation, 2 of 24 Cx. nigripalpus 1T-inocu1âted with YF 170 virus were stil infected; however, the mean virus titer was very low (0.7 1ogo PFU!mosquito) (Table 1). West Nile virus replicated in all tE’ inoculated Cx. nigripal,pus, reaching a mean titer of 6 logo PFU!mosquito at 4 days; at 14 days the niean titer was 6.1 logo PFU/mosquito. In Cx. quinquefasciaius mosquitoes, ChimeriVax”’-WN(vet) virus replicated at a low level tbroughout the 10-day time course (Figure 2C) and was present in 22 of 24 mosquitoes 14 days following inoculation (mean titer 1.9 logio PFU/mosquito) (Table 1). ChimeriVaxTMWN(vet) virus disseminated to the head in 11 of 24 Cx. quinquefasciatus (mean titer of positives 1.0 logo PFU/ mosquito). Similarly, Cx. quinquefasciatus were infected with YF 170 virus throughout the 10-day time cdurse but virus liter was very low (Figure 2C). YF 170 virus was detectedin 7 of 8 mosquitoes at 14 days postinoculation (mean liter of positives 2.0 logio PFU/mosquito); dissemination occurred o PFU/ mosquito). In contrast, WN 1 in 3 of 8 mosquitoes (mean liter ofpositives 1.0 log virus grew in all IT-inoculated Cx, quinquefasciatus. The peak mean titer occurred at 6 o 1 days (7.3 legio PFU/mosquito) (Figure 2C); at 14 days the mean titer was 7.1 log

10

PFU/mosquito (Table 1). Virus disseminated to heads in all WN-inoculated mosquitoes, reaching a mean titer of 5.3 logio PFU/mosquito. et) and YF 170 viruses IT inoculated into Ae. aegypti reached ChirneriVax -WN(v TM o PFU/mosquito, respectively, at 6 days postinoculation (Figure 1 titers of 6.3 and 4.7 log 20). At 14 days, all Ae. aegypti inoculated with ChimeriVaxTMWN(vet) and YF 170 viruses were infected; mean titers were 4.9 and 4.2 logio PFU/mosquito, respectively. Virus disseniinated to heads, with mean titers of 3.8 logio PFU/mosquito in ChimeriVaxTMWN(vet) infected mosquitoes and 3.7 logio PFU/mosquito in YF 170 infected mosquitoes (Table 1). Four days following IT inoculation with WN virus titer 10 PFU/mosquito at 14 days. reached 8.3 logio PFUfmosquito, with a mean titer of 7.5 log Mean virus titer of WN virus inAe. aegypti heads was 6.2 logio PFU/mosquito. All Ae. albopictus were infected by the three viruses and virus disseminated to head tissues in all specirnens. ChimeriVax”&WN(vet) and YF 170 viruses replicated to peak mean titers of 5.8 and 4 logio PFLT/mosquito, respectively, at 4 days (Figure 2E). 10 PFU/mosquito at day 8. Mean 14-day titers WN virus reached apeak titer of 7.6 1og were 6.7 logio PFU/mosquito for WN virus, and 4.0 logio PFU/mosquito for both ChimeriVax’-WN(vet) and YF 170 viruses (Table 1). Oral iizfection of mosqriitoes

Mosquitoes were fed an artificial blood meal containing 8,1 log PFU/mL of WN virus, -WN(vet) virus, or 7.3 logio PFU/mL YF 170 virus TM 7.7 logio PF[J/rnL of ChimeriVax (2.5-4 logio PFU/mosquito). Mosquitoes were collected at 48-hr intervals for 10 days (Figure 3), and after a 14-day extrinsic incubation period (Table 2). Mosquitoes were

ii

assayed individually for infection and dissemination by plaque titration of mosquito bodies and heads, respectively. -WN(vet) and YF 170 viruses did not infect Cx. tritaeniorhynchus or TM ChimeriVax

Cx. quinquefasciatus by the oral route (Figure 3A and B; Table 2). In contrast, WN virus infected and disseminated to head fissues in 100% of Cx; tritaenior]zynchus and Cx. quinquefasciatus mosquitoes tested, with mean titers of 6.1 and 6.7 logjo PFU/mosquito, respectively, at day 14 (Table 2). One of three 4e. aegypti collected at 2 and at 6 days was infected with ChimeriVax WN(vet) virus (Figure 3C), and at 14 days 2 of 20 were infeoted (Table 2). YF 170 virus was foimd in 2 of 20 Ae. aegypti at 14 days. However, virus titers were very low -WN(vet) 1.8 logo PFU/mosquito; YF 1 7D 1.6 logo PFU/Inosquito) and TM (ChimeriVax virus did not clisseminate to head tissue (Table 2). Seven of 11 4e. aegypti becanie infected with WN virus, which replicated to a mean titer of 6.6 log PFU/mosquito and and disseminated to head tissue in all infected specimens. One 4e. albopictus was orally infected with ChimeriVaxTMWN(vet) virus at 8 days

(Figure 30); none were irifected at 14 days (Table 2). YF 170 virus infected 2 of 24 4e. albopictus at 14 days, but no dissemination occurred (Figure 30; Table 2). Aedes albopictus were inefficiently infected orally with WN virus (Figure 30). At 14 days 19 of

244e. albopictus were infected with WN virus (mean liter of positives 5.5 log o 1 PFU/mosquito), although there was a wide range of titer between individual mosquitoes o PFU/mosquito, data not shown). WN virus disseminated to head tissue in 1 (2.0-7.6 log 12 of 24 mosquitoes.

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Sequencing ofprM and E genes of C’hiineriVax -WN’vet,) viruses isolatedfroin Ae. TM aeypti, Ae. albopictus, and Cx. quinquefasciatus ?nosquitoes

-WN(vet) RNA was extracted from 14-day IT inoculated Ae. aegypti and TM ChimeriVax Ac. albopictus mosquitoes. Virus titer from a single Cx. quinquefasciatus 14 days following IT inoculation with ChimeriVaxTM..WN(vet) virus was low, and we were unable to isolate enough RNA clirectly from the mosquito for sequencing. Therefore, supernatant from a triturated ChirneriVaxTMWN(vet) infected Cx. quinquefasciatus mosquito was used to infect Vero celis and amplify the virus liter, and virus was recovered and extracted after a single Vero celi culture passage. The prM and E regions of these three virus isolates were sequenced and the sequences were compared to the prM and E gene sequence of the ChimeriVax-WN(vet) seed virus. The nucleotide sequences of CbimeriVax-WN viruses isolated from these mosquitoes were identical to the parent ChimeriVax-WN virus. Discussion -WN(vet) virus fed to mosquitoes in an artificial blood meal did not infect TM ChimeriVax any of the Culex mosquitoes tested. One Ae. albopictus was orally infected by

(E

ChimeriVaxTMWN(vet) at 8 days, but none of the Ae. albopictus remained infected after

the 14-day extrinsic incubation period. In Ae. aegypti, ChimeriVaxTMWN(vet) virus infeeted 10% of the mosquitoes at 14 days, but virus titer was very low and virus did not disseminate to the mosquito head. Introduced direetly into mosquitoes via IT inoculation, the chimeric virus failed to replicate to 14 days in Cx. tritaeniorhchus and Cx.

nigpalpus mosquitoes, and grew poorly in IT-inoculated Cx. quinquefasciatus. InAe. -WN(vet) virus, TM aegypti and Ae. albopictus mosquitoes IT inoculated with ChimeriVax

13

replication was restricted compared to wt WN virus, and was similar to that of the YF 17D virus. In contrast, WN virus efficiently infected the Culex and Aedes mosquitoes tested, both by IT inoculation and artificial blood meaL We conciude therefore that the poor infectivity of ChimeriVaxTMWN(vet) virus in the mosquito vectors of WN and YF viruses makes it unlikely that mosquitoes, which may bite a mammalian host vaccinated with ChimeriVaxTMWN(vet) virus, would become infected. Hence there is very littie potential for the tiansmission of ChimeriVaxTMWN(vet) virus by mosquitoes. These studies have also provided us with the opportunity to further characterize flaiviviral genes and analyze how they contribute to tissue specificity in the mosquito. Both WN and YF viruses are in the virus faniily Flavividae, and as such their genome organization and replication activities are siniilar (Lindenbach & Rice, 2001). However, they have very different ecology. Although the trarismission cycle of both viruses involves mosquitoes, they lack common mosquito vectors and vertebrate hosts. WN

virus cycles primarily between birds and Culex mosquitoes. Mammals, inciuding humans and horses, are incidental bosts and viremia does not reach high enough liters to infect mosquitoes (Hayes, 1989; Bunning etal., 2001; Bunning ei aL, 2002). Culexmosquitoes

are not important vectors of YF virus. Further, Cx. tritaeniorhynchus and Cx. nigripalpus are resistant to irifection by the attenuated YF 1 7D virus, oth by the oral route and by IT inoculation; Cx. quinquefasciatus mosquitioes are not infected by YF 17D virus via an

infectious blood meal, and are infected poorly by IT inoculation. Humans are the primary host in the urban YF cycle, and Ae. aegypti mosquitoes are an important urban YF vector species (Monath, 1999). However, Ae. aegypti has been shown to be refractory to oral infection with YF17D previously and in this study, and

14

virus replication is restricted coinpared to that of wt YF virus upon IT inoculation (Whitman, 1939; Bhatt et al., 2000; Johnson et al., 2002). The vaccine has been.safely used for over 60 yr with no reports of YF 17D transmission byAe. aegypti. Aedes aegypti

can become infected with WN virus, as shown in this study, but are not

considered important vectors because of their anthropophilicity. The mosquito midgut is an important factor in vector competence. The ability of a virus to infect the midgut following sri infectious blood meal and Uien to replicate and clisseminate tbxoughout the mosquito deterniines whether or not the virus will be transmitted. In this and in previous studies, the chimeric virus as well as the attenuated YF1 7D vaccine virus failed to infect mosquitoes ‘via a blood meal, probably due to attenuating mutations, primarily in the structural gene region (Bhatt et al., 2000; Johnson

et al., 2002). However, it has been shown that a virus that may not be able to iufect a mosquito orally, because of the midgut infection barrier, can. nevertheless replicate in a mes quito species which is not a competent vector of the virus, when introdueed into the mosquito directly by intrathoracic inoculation (Gubler & Rosen, 1976). The flavivirus envelope protein is involved in ceil attachment, receptor binding, and celi membrane fusion (Lindenbach & Rice, 2001). The viral envelope also contain epitopes against which host antibodies are produced. Thus host spcificity and tissue tropism are largely determined by virus E protein. Culex tritaeniorynchus, Cx. quinquefasciatus, and

Cx. nigripalpus are competent vectors of WN virus (Hayes et al., 1980; Bernard & Kramer, 2001). As expected, in these studies WN virus infected and replicated efficiently in these mosquitoes both by IT inoculation and via

sri

artificial blood meal. Therefore, as

the prM and E genes inserted into ChimeriVaxTMWN(vet) virus were cloned from a wt

15

WN (NY99) isolate, ChimeriVaxTM_WN(vet) virus might be expeeted to infect these tbree Culex species, particularly upon IT inoculation, which bypasses the midgut infection barrier. However, ChimeriVaxTMWN(vet) virus did not grow in either Cx.

tritaeniorhynchus or Cx. nigrzpalpus, and replication was restricted in Cx. quinquefasciatus, as virus titer remained below the inoeulum dose. These resuits correspond to those of a previous study with a ChimeriVax virus containing prM and E genes from Tapanese encephalitis (IR) virus. Culex tritaeniorhynchus is the primary mosquito vector in the IE transmission cycle, yet ChimeriVax-IE did not repilcate in IT

inoculated or orally fed Cx. tritaeniorhynehus (Bbatt et al., 2000). Clearly, the YF 171) nonstructural proteins contribute to the restricted replication of the chimeric virus. The nonstructural region of the flavivirus genome enco des proteins required for replication of the viral genonie, cleavage processing of the viral polyprotein, and virus

assembly and packaging (Schiesinger et al., 1996). Host ceil factors also are involved in virus replication. Lindenbach and Rice showed that the insertion of a heterologous

flavivirus NS 1 gene into wt YF virus rendered the virus incapable of negative strand production and thus replication in BHK-21 ceils (Lindenbach & Rice, 1999). Our resuits are in agreement with this observation. Hence the YF 171) nonstructural genes in ChimeriVax-WN(vet) presumably were responsible for the failure of the chimeric virus to replicate in WN virus susceptible Culex mosquitoes.

16

Acknowledgnients

This study was partiafly supported by grant A148297 from the National Institute of Allergy & Infectious Diseases. B.W. Johnson was supported by an American Society for Microbiology/ National Center for Infectious Diseases (ASM/NCID) postdoctoral training fellowship.

17

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WNV antigen production flowchart

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