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Vaccine 29 (2011) 1836–1843

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

he development of vaccine viruses against pandemic A(H1N1) influenza

ames S. Robertsona,∗, Carolyn Nicolsona, Ruth Harveya, Rachel Johnsona, Diane Majora,ate Guilfoylea, Sarah Rosebya, Robert Newmana, Rebecca Collina, Chantal Wallisa,thmar G. Engelhardta, John M. Wooda, Jianhua Leb, Ramanunninair Manojkumarb,arbara A. Pokornyb, Jeanmarie Silvermanb, Rene Devisb, Doris Bucherb, Erin Verityc,atherine Agiusc, Sarina Camugliac, Chi Ongc, Steven Rockmanc, Anne Curtis c, Peter Schoofsc,lga Zouevad, Hang Xied, Xing Lid, Zhengshi Lind, Zhiping Yed, Li-Mei Chene,1, Eduardo O’Neill e,1,manda Balishe , Aleksandr S. Lipatove , Zhu Guoe , Irina Isakovae , Charles T. Davise , Pierre Rivaillere ,ortney M. Gustine, Jessica A. Belsere, Taronna R. Mainese, Terrence M. Tumpeye, Xiyan Xue, Jacqueline. Katze, Alexander Klimove, Nancy J. Coxe, Ruben O. Donise

Division of Virology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar EN6 3QG, UKDepartment of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USACSL Limited, AustraliaCenter for Biologics Evaluation and Research, 8800 Rockville, Bethesda, MD 20892, USAInfluenza Division, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA

r t i c l e i n f o

rticle history:eceived 26 July 2010eceived in revised form6 November 2010ccepted 14 December 2010

a b s t r a c t

Wild type human influenza viruses do not usually grow well in embryonated hens’ eggs, the substrateof choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developedspecifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be createdwith utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratoriesbegan a race against time to develop suitable candidate vaccine viruses. Two approaches were followed,

vailable online 1 January 2011

eywords:nfluenza vaccinesandidate vaccine viruseseverse genetics

the classical reassortment approach and the more recent reverse genetics approach. This report describesthe development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

© 2010 Elsevier Ltd. All rights reserved.

igh growth reassortantsA antigen yield

. Introduction

A novel influenza A(H1N1) virus was identified in the USA on5 April 2009 and shown to be genetically related to recent swine

nfluenza viruses. This virus was later shown to be the cause of res-iratory disease prevalent in Mexico in March 2009. In subsequenteeks sustained human-to-human transmission of the virus was

eported in various parts of the world and as a result a pandemic

f influenza was declared on June 11, 2009 by the World Healthrganisation (WHO) [1].

There are two principal pharmaceutical approaches for combat-ng influenza; use of anti-viral drugs or vaccines. While influenza

∗ Corresponding author. Tel.: +44 1707 641304; fax: +44 1707 641050.E-mail address: jim.robertson@nibsc.hpa.org.uk (J.S. Robertson).

1 Contributed equally to the work at CDC.

264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2010.12.044

antiviral drugs are broadly reactive against all strains of influenza,vaccines have to be tailor-made to individual viruses. Conse-quently, immediately upon recognition that this novel 2009 H1N1virus was spreading from person-to-person, a network of laborato-ries associated with the WHO Global Influenza Programme starteddevelopment of viruses suitable for production of vaccine againstthis influenza virus. These laboratories were experienced in thedevelopment of seasonal and pandemic vaccine viruses and workedcollaboratively towards the common goal of deriving an effectivevaccine virus.

The most widely used influenza vaccines consist of chemicallyor physically inactivated virus. For decades, this has been pre-

pared from virus grown in embryonated hens’ eggs and remains theprincipal mode of influenza vaccine manufacture. Other methodsinvolve growth of virus in cell culture or, alternatively, productionof live, attenuated vaccine (also currently prepared in eggs). Iso-lates of human influenza generally grow very poorly in eggs (and

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he novel H1N1 virus is no exception). Consequently, the devel-pment of a vaccine virus involves adapting the virus to grow inggs and improving its antigen yield. The ideal influenza vaccineeed virus grows to high titre in eggs, has good purification prop-rties, provides a high yield of antigen (the surface glycoproteinsA and NA) and is of low pathogenicity, being safe to work with inmanufacturing setting.

The initial stage of egg-adaptation of a virus from a clinical spec-men can be problematic and typically involves blind passage ofespiratory tract secretions in eggs and selection of a variant capa-le of replication within the egg. Once this is achieved, usually byne of the WHO Collaborating Centres (WHO-CCs), efforts are thenade by specialist laboratories to increase virus/antigen yield by

eassorting the egg-adapted wild type virus with a high-yieldingaboratory virus [2].

Currently there are two techniques by which to generate theesired reassortant; one is classical reassortment and the other ishe more recently developed reverse genetics approach. Classicaleassortment of an influenza A virus involves the co-infection ofggs with: (1) the wild type virus against which a vaccine is soughtnd (2) a high yielding donor virus (PR8 [A/Puerto Rico/8/34] orderivative). Following a series of steps involving selection for

igh growth and the use of antiserum directed against the surfacelycoproteins of the high yielding donor, a high growth reassor-ant (HGR) can be derived that grows to high titre and has theppropriate antigenic characteristics of the wild type virus. Thushe HGR needs to contain both the haemagglutinin (HA) and neu-aminidase (NA) genome segments from the wild type virus whilehe remaining segments can derive from either parent althoughhe PR8M gene is generally required for high yield; achievementf the appropriate antigenic and good growth characteristics arehe principal requirements. There is no guarantee that an HGRill be obtained from such a procedure, or that it will have theesired growth properties. Nonetheless, the classical reassortmentpproach has served the influenza vaccine community exceedinglyell since it was first used in an influenza vaccine in 1971 by Kil-

ourne [3,4].Reverse genetics, in comparison to classical reassortment, is an

pproach that produces a virus with a predefined genetic constella-ion [5,6]. The desired genome segments of the wild type virus, i.e.hose encoding the HA and the NA, are cloned into ‘rescue’ plasmidsnder the control of a human Pol I promoter and transfected intosuitable mammalian cell line such as Vero cells, along with cor-

esponding plasmids encoding the remaining six genes from PR8.ith the help of plasmids expressing influenza polymerase activ-

ty, infectious virus (in this case with a 6:2 genetic constellation, i.e.genome segments from the high growth parent and 2 from theild type) can be readily reconstituted (or rescued). Advantages of

he reverse genetics approach are the predictability of the resultingirus genotype and the robustness of the process. One potential dis-dvantage might be that there is no selection for a virus with a geneonstellation that provides the highest growth; however in com-arative analyses, 6:2 reassortants produced by reverse geneticsnd compared with antigenically equivalent HGRs can have similarrowth characteristics [7,8].

Whereas the classical reassorting approach has been used suc-essfully now for several decades in the development of influenza Aeasonal vaccine viruses, experience with the use of reverse genet-cs in developing vaccine viruses is limited to the development of5N1 vaccine viruses. In the case of H5N1, reverse genetics was

he approach of choice because of the need to delete basic amino

cids at the cleavage site of the HA gene in order to attenuate theirulence of the highly pathogenic H5N1 avian influenza viruses9]. However, the pandemic H1N1 virus HA does not contain a

ulti-basic cleavage site and consequently, reverse genetics doesot have to be employed to remove it.

29 (2011) 1836–1843 1837

This report describes the immediate response that was madeby a network of laboratories in the early stages of the currentpandemic in developing influenza A(H1N1) pandemic candidatevaccine strains and the outcome of these efforts.

2. Methods

The precise details involved for both classical reassortment andreverse genetics vary between laboratories and so only an outlineof the process is described here, with references provided.

2.1. Classical reassortment

Classical reassortment involves the co-infection of eggs withthe wild type strain and a high yielding virus, typically PR8, anH1N1 subtype [3,4]. The virus harvest is passaged sequentially 3–4times at low dilution with treatment of the harvested virus betweenpasses with rabbit anti-PR8 antiserum in order to suppress viruseswith the HA and NA surface proteins of the PR8 donor virus. Finally,virus is passaged a minimum of 2 times at limiting dilution, ascloning steps, followed by a passage to prepare adequate stocksof the virus.

For the development of HGRs for H1N1 wild type strains, it isuseful to have a high yielding donor virus of a subtype other thanH1N1, e.g. H3N2, to permit elimination of the donor HA and NAgenes by antiserum selection after reassortment; such an alterna-tive donor should contain the remaining six PR8 genome segments.For example, X-157 virus (used in developing a pandemic H1N1high growth reassortant – see below) is a cross between A/NewYork/55/2004 × A/PR/8/34 and contains genes for the H3N2 surfaceantigens of the New York isolate and the remaining six genome seg-ments from PR8. Selection against reassortants containing H3 or N2genes is achieved by treatment with antisera raised against purifiedHA and NA of X-157 and successful HGRs will have the H1 and N1wild type antigens and up to six of the remaining genome segmentsof PR8.

Details of the classical reassortment method employed at NewYork Medical College (NYMC) are as follows; the methods usedat other laboratories were essentially similar and are not statedto avoid repetition. The development of the X-179A pandemicH1N1 high growth reassortant at NYMC (see Section 3.2.4) pro-ceeded in eight steps, with each step requiring 42 h incubation at35 ◦C followed by harvest of allantoic fluid and passage to the nextstep. Specified pathogen free 10–11 day embryonated hen’s eggs(Charles River, CT, USA) were used throughout. Eggs were initiallyco-infected with A/California/07/2009 and X-157 at 10−2 and 10−3

dilution, respectively, regardless of virus titre. Harvested allantoicfluid was treated with purified polyclonal rabbit antibodies to theH3N2 X-157 HA and NA and further passaged at 10−1 dilution. Anti-body treatment and passage was repeated two additional times at10−1 and 10−3 dilution, respectively, followed by a further passageat 10−4 dilution. Two further passages were performed at limitingdilution (10−8) as cloning steps, following which virus was pas-saged at 10−5 to prepare stocks of the high growth reassortant viruswhich was designated NYMC X-179A.

2.2. Reverse genetics

Reverse genetics is a well established process whereby infec-tious influenza virus is reconstituted from plasmid DNAs encoding

the genome segments [5,6,10]. The plasmids are transfected intoqualified Vero cells obtained either from a vaccine manufacturer orderived in-house and viral RNA corresponding to the genome seg-ments is transcribed from the Pol I promoter within the plasmids.Helper plasmids expressing influenza NP and polymerase function

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o kick-start a cycle of replication of the transcribed RNAs and infec-ious virus is produced. The fine details of this process vary betweenaboratories and are as described in Nicolson et al. [7], O’Neill andonis [11] and Liu and Ye [12].

.3. Viral protein yield assessment

Evaluation of total protein yield of a candidate vaccine virus iserformed by growth of virus in 100–300 eggs per virus under opti-al conditions. Harvested virus is pelleted by ultracentrifugation

nd purified on a continuous sucrose density gradient. The virusand is collected, pelleted by ultracentrifugation and resuspended

n a small volume of buffer. The total protein content is deter-ined using a standard recognised protein assay (such as Lowry

r Bradford) and expressed as protein per 100 eggs.

.4. Safety studies in ferrets

In all studies, ferrets were handled in compliance with all localequirements regarding their health and well-being. Prior to use,ll animals were determined to be serologically negative for cur-ently circulating influenza viruses. Typically in safety studies ofpandemic candidate vaccine virus (in vivo safety studies are noterformed for seasonal inactivated vaccine), the virus was analysed

n parallel with the parental wild type virus. Animals were infectedntranasally with approximately 106–107 50% egg infectious dosesEID50) and assessed for clinical signs of infection including tem-erature, weight loss, lethargy and respiratory signs for 14 daysnd for virus replication by titration of nasal washes collectedn alternate days over the first 7 days of infection. Some ani-als were euthanised for necropsy and quantification of infectious

irus in lungs and other organs was performed (see Ref. [13] andupplementary Online Material for further methodology details).

. Results

.1. Attempts to rescue vaccine viruses from cell-derived/California/04/2009, without egg-adaptive mutations

The first virus isolates became available in late April and thoseere distributed by the Centers for Disease Control and Prevention

CDC), USA. Initially egg-isolates were not obtained and one of therst prototype pandemic A(H1N1) viruses, A/California/04/2009,as isolated in MDCK cell cultures. The initial absence of an egg

solate probably reflected a requirement for the wild type virus tondergo an egg-adaptation mutation. Such a feature is an estab-

ished phenomenon for seasonal human isolates and typicallynvolves the selection by egg passage of a variant with an aminocid substitution in the HA in the vicinity of the receptor bind-ng site [14]. The successful selection of such a variant is a chancevent dependent upon their existence in the small quantity of virusnoculated into the egg from a clinical specimen.

Viruses isolated on mammalian cell cultures in research andiagnostic laboratories are usually not suitable for taking forward

nto the classical reassorting technique for derivation of a vaccineirus due to the cells not being approved for human vaccine pro-uction. In contrast, viruses isolated in embryonated hen’s eggs or

n primary cells derived from eggs are deemed suitable [13]. In thisegard, viruses derived from cells or eggs are equally acceptable asstarting material for cloning the HA and NA for reverse genet-

cs [7]. Thus, in the absence of a direct egg-isolate CDC began the

erivation of a vaccine virus using reverse genetics possessing theA and NA of A/California/04/2009, the cell-derived isolate, alongith the six ‘internal’ genome segments of PR8. The procedure

nvolved rescue of virus in qualified Vero cells and amplification ofescued virus in eggs. No virus was recovered from embryonated

29 (2011) 1836–1843

eggs presumably because the small quantity of virus obtained fromtransfected Vero cells did not contain an appropriate variant thatwould enable replication in eggs. These results were immediatelycommunicated to the WHO network laboratories to recommendalternative approaches for rescuing H1N1 viruses isolated in cellculture (see Section 3.2.1).

3.2. Vaccine viruses derived from A/California/07/2009 andA/Texas/05/2009 containing egg-adaptive mutations

The first direct egg-isolates of this new pandemic H1N1 virus,e.g. A/California/07/2009, became available in late April and meantthat vaccine development by classical reassorting and by reversegenetics could commence from these isolates. A/California/07/2009was dispatched from CDC to various centres for vaccine virus devel-opment including New York Medical College which specialises inhigh growth reassortant derivation in eggs, to NIBSC (UK), whichhas the capability to derive both classical egg reassortants and areverse genetics virus, and to CSL Limited (Australia), also capable ofderiving a virus by both technologies. CDC reported the sequence ofthe HA and NA for the A/California/07/2009 isolate, noting that theHA had a mixed sequence at codons encoding HA1 residues 222 and223 which would result in 222D/G and 223Q/R (singly or in combi-nation). Such a mixed population was potentially the result of theselection of more than one variant from the clinical specimen dur-ing egg passage. Interestingly, previous studies showed selectionof D222G during egg-adaptation of seasonal H1N1 viruses [14].

3.2.1. CDCCDC selected the A/Texas/05/09 cell isolate as an alternative to

A/California/04/2009. The cloned A/Texas/05/2009 HA cDNA wasengineered to include the two amino acid substitutions detectedin the A/California/07/2009 egg isolate; D222G and Q223R, eithersingly or in combination. The three viruses rescued containingthe modified HA and the NA of A/Texas/05/09 could be readilyamplified in eggs and were termed IDCDC-RG14 (HA mutation:D222G), RG15 (HA mutation: Q223R) and RG16 (HA mutation:D222G, Q223R). The A/Texas/05/2009 virus HA was cloned on 4thMay, 2009 and IDCDC-RG14, 15 and 16 E2 reassortant virus stocksverified by sequence analysis were ready for antigenic analysis on22nd May giving a preparation time of 18 days.

3.2.2. CSL limitedA high growth reassortant (IVR-153) was generated from a

mixed infection between A/California/07/2009 and the high yield-ing donor IVR-6 (which itself is a 5:3 reassortant containing 5internal genes from PR8 and the HA, NA and PB1 genome segmentsfrom A/Texas/1/77, an H3N2 virus). Using real time PCR with minorgroove binding probes for the 6 internal genes of PR8 and HA andNA probes for PR8 and A/California/07/2009, it was demonstratedthat IVR-153 is a 5:2:1 reassortant with the five internal genomesegments of PR8, the PB1 from A/Texas/1/77 (via IVR-6) and theHA and NA from A/California/07/2009. The HA sequence of IVR-153 was confirmed by the WHO Collaborating Centre for InfluenzaResearch (WHO-CC) (Melbourne) and showed a Q223R substitutionfrom the wild type sequence.

The A/California/07/2009 virus was received from CDC on 4thMay and the high growth reassortant IVR-153 was supplied tothe WHO-CC Melbourne on 26th May for genetic and antigeniccharacterisation giving a preparation time for IVR-153 of 23 days.Following confirmation of antigenic and genetic suitability at WHO-

CC Melbourne, IVR-153 was distributed globally on 29th May, 26days following receipt of the wild type virus.

The introduction of a Q223R or K209T, or both, mutations intosynthetic HAs of A/California/04/2009 and A/California/07/2009resulted in the successful rescue of 6:2 reassortants. In each case the

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irus with the double mutation demonstrated increased growth asetermined by HA titre (data not shown). These viruses howeverere derived experimentally and were not suitable to progress as

andidate vaccine viruses.

.2.3. NIBSCAt NIBSC, both reverse genetics and classical reassorting were

sed to derive a vaccine virus from the A/California/07/2009 eggsolate. During cloning of the HA for the reverse genetics approach,lternative clones were identified having an HA sequence of G/Q,/R or D/Q at HA1 positions 222/223; 6:2 viruses were rescued

uccessfully with all three HA clones. All viruses grew to similarA titres (circa 320–640); however previous studies had shown

hat a D222G mutation associated with egg-adaptation of seasonal1N1 HA did not affect the antigenic nature of the virus whereas no

uch data was available for a virus with a Q223R substitution [14].onsequently, the virus containing the G/Q (222/223) HA sequenceas taken forward as the candidate vaccine virus; this was namedIBRG-121.

Full antigenic characterisation of candidate vaccine viruses pre-ared at NIBSC was undertaken at the WHO CC located at theational Institute for Medical Research, London.

Viruses derived from a mixed infection between/California/07/2009 and PR8 appeared to have increased growthut were antigenically distinct from the wild type virus withutations in the HA which could be responsible for the altered

ntigenicity. Furthermore, they were not reassortants as assessedy RT-PCR and RFLP analysis.

The A/California/07/2009 virus was received from CDC on 30thpril and the second egg passage of NIBRG-121 which was prepareds the virus stock was harvested on 22nd May; thus the preparationime for NIBRG-121 was 22 days.

.2.4. NYMCThe laboratory at NYMC generated two candidate high growth

eassortants from A/California/07/2009 and the high yielding donor-157 (a 6:2 reassortant containing the six internal genome seg-ents from PR8 and the HA and NA of A/New York/55/2004,

n H3N2 virus). The reassortants were sent to the WHO-CC atDC for antigenic analysis and sequencing. One high growth reas-ortant, X-179A, was determined serologically to be identical to/California/07/2009 by HI analysis. A second reassortant, X-179,as antigenically altered from the parental wild type with a G155Eutation in the HA potentially responsible for these changes.

he PB1 and NA genes of X-179A were also determined to be/California/07/2009-like by RT-PCR and RFLP analysis while theemaining five genome segments were identified as PR8 (donatedy X-157). Thus, X-179A is a 5:3 reassortant with the PB1, HAnd NA segments derived from the wild type A/California/07/2009

able 1ntigenic analysis of candidate vaccine viruses by haemagglutination inhibition.a

Virus Ferret antiserum

CA/07 X-179A X-181

A/California/07/2009 2560 1280 2560NYMC X-179A 5120 2560 2560NYMC X-181 2560 1280 2560IVR-153 5120 2560 ndIDCDC-RG-20 2560 1280 ndA/Texas/05/2009 5120 2560 ndIDCDC-RG15 2560 1280 ndA/Brisbane/59/2007b 5 5 5

a Performed at CDC.b Seasonal H1N1 virus.d: not tested.he values in boldface/underlined represent the homologous titres of the antigens with c

29 (2011) 1836–1843 1839

virus. Furthermore, sequence analysis of the HA segment revealedQ223R as well as a K209T mutation.

X-179A had a haemagglutination titre of 2048 as assayed withchicken erythrocytes. This represented a 32-fold increase over theoriginal A/California/07/2009 isolate, which had an HA titre of 64.The A/California/07/2009 virus was received at NYMC on 28th April2009 and X-179A was shipped to the CDC on 21st May for geneticand antigenic characterisation giving a preparation time for X-179Aof 23 days.

3.3. WHO pandemic virus recommendation

At the April 27, 2009 conference call led by the WHO, anA/California/7/2009-like virus isolated at CDC was selected as vac-cine candidate for laboratories preparing high growth reassortants.Pandemic A(H1N1) viruses isolated during May 2009 were deter-mined by antigenic and genetic analyses to be highly homogeneousand closely related to A/California/07/2009. [15]. It is essential thatthe candidate vaccine viruses remain A/California/07/2009-like andso they are assessed by HI using relevant ferret antisera for thispurpose. Table 1 shows the HI analyses of candidate viruses X-179A, X-181 (see below, section 3.6.3), IVR-153, IDCDC-RG15 andIDCDC-RG20 (see below, section 3.6.1) and confirms that they areA/California/07/2009-like.

On 27th May, the WHO announced the availability of the firstcandidate vaccine viruses IDCDC-RG15 (ex CDC), NIBRG-121 (exNIBSC) and X-179A (ex NYMC) and on 4th June, the availability ofIVR-153 (ex CSL) (Table 2, Fig. 1) [16–19].

3.4. Antigen yield

An important characteristic of a candidate vaccine virus is itsgrowth and antigen yield. Growth is measured directly by thehaemagglutination assay although results of such measurementsmade in separate laboratories are not always directly comparable.A parameter that provides a better indication of the yield of a can-didate virus at large scale manufacture is the amount of protein ina small scale purified preparation of the virus, typically expressedin weight of protein per 100 eggs. This approach can be subjectto variation according to which laboratory performs the assay,and possibly also variation between operators, but in any singleexperiment performed, in which alternative candidate viruses areassessed in parallel, it provides a very good and robust comparisonof these alternative candidates.

At NIBSC, viruses X-179A, NIBRG-121 and IVR-153 wereassessed in this way and the amount of protein per 100 eggs deter-mined. In duplicate experiments, X-179A clearly produced moreprotein than either NIBRG-121 or IVR-153 (average 5.9 mg proteinper 100 eggs versus 3.6 and 3.4 for NIBRG-121 and IVR-153 respec-

IVR-153 RG-20 TX/05 RG-15 AS/59

1280 640 2560 320 52560 1280 5120 1280 5

nd nd nd nd 52560 1280 1280 640 51280 640 2560 160 52560 1280 5120 1280 51280 640 5120 640 5

5 5 5 5 320

onvalescent ferret serum.

1840 J.S. Robertson et al. / Vaccine 29 (2011) 1836–1843

Table 2Initial panel of pandemic H1N1 vaccine viruses distributed to vaccine manufacturers.

Virus Origin Derivation Wild type parent Gene constellation Viral protein (mg/100 eggs)a,b

IDCDC-RG15 CDC Reverse genetics A/Texas/05/2009 6:2 ndX-179A NYMC Classical reassortant A/California/07/2009 5:3 5.85NIBRG-121 NIBSC Reverse genetics A/California/07/2009 6:2 3.55IVR-153 CSL Classical reassortant A/California/07/2009 5:2:1 3.4

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ively) (Table 2). However, this value for X-179A was only 30–50%f what is typically produced for a seasonal human H1N1 virus∼10–15 mg per 100 eggs). While no specific data on protein yieldere produced by vaccine manufacturers themselves, several of

hem acknowledged similar findings, that the yield of X-179A wasetter than for other candidates but was much less than seasonal1N1 viruses (communicated via teleconference calls organised by

he International Federation of Pharmaceutical Manufacturers andssociations [IFPMA]).

Consequently, during June 2009 most manufacturers of egg-erived vaccine initiated vaccine production with X-179A. At theame time, it was made clear by industry that an alternative vaccineirus with further improved growth and antigen yield characteris-ics was highly desirable.

.5. Safety testing

During May 2009 when there was limited spread of the pan-emic H1N1 virus in the population, WHO recommended for safetyeasons that the viruses should be handled at biosafety level (BSL)

arch

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August

Sept

March–April: Outbreaks of respiratory disease in Mexico

April 15: Novel 2009 A(H1N1) iden�fied by US CDC

April 27: WHO selects A/California/07/2009-like virus as vaccine candidate

April 28: CA/07/09 egg isolate virus distributed for reassortment

May 21-26: RG and egg reassortant viruses available foran�genic characteriza�on

May 27: WHO announced availability of X-179A , IDCDC-RG15,and NIBRG-121 viruses (Table 2)

June 4: WHO announced the availability of IVR-153

June 11: WHO declared pandemic by novel 2009 A(H1N1)

July 23: WHO announced safety tes�ng results of vaccine viruses and recommended BSL2 prac�ces for manufacturing

August 6: WHO announced availability of improved yieldvaccine virus NIBRG-121xp

September 14: WHO announced availability of improved yield vaccine viruses X-181 and X-181A

2009

Fig. 1. Timeline of 2009 pandemic H1N1 vaccine virus development.

3. This was problematic for egg manufacture of vaccine becausethis level of biocontainment was not available at the majority ofvaccine manufacturing plants. It was anticipated however that avaccine virus that had much of its genetic makeup derived from PR8would be attenuated in comparison to wild type pandemic H1N1viruses since PR8 itself is fully attenuated for humans. In addition,reassortants between PR8 and wild type viruses (both seasonal andviruses of pandemic potential) were partially attenuated [20]. Thus,although prior evidence would suggest that the candidate vaccineviruses developed so far would be sufficiently attenuated, it wasrequired by WHO, and prudent, to measure this directly [13,21,22].

The ferret infection model is the most appropriate with whichto address this question and assessment of the pathogenicityof candidate vaccine viruses in comparison to wild type pan-demic H1N1 viruses was performed at CDC (on X-179A and onIDCDC-RG15), at NIBSC (on NIBRG-121) and at the AustralianAnimal Health Laboratories, Geelong (on IVR-153) [23]. A standard-ized experimental protocol, reporting criteria and template weredeveloped and used by all three laboratories. Studies were per-formed comparing the respective vaccine virus against the parentalwild type virus, A/California/07/2009, or A/California/04/2009 andA/Texas/15/2009 (both A/California/07/2009-like viruses); in thestudy for IVR-153, the high growth parent IVR-6 was also included.In the other studies, data for PR8 was derived from previous exper-iments. In each study, ferrets were inoculated intra-nasally with astandard dose of infectious virus. Animals were assessed for clini-cal signs of infection (temperature, weight loss, etc.) and for virusreplication by titration of nasal washes. Animals were euthanisedfor necropsy at day 3, or days 3 and 5, and histological analyses ofspecified organs, and titration of infectious virus in lungs and otherorgans was performed.

In each of the studies, it was concluded that the reassortant can-didate vaccine viruses were attenuated in ferrets relative to the wildtype pandemic H1N1 isolates [24,25]. Consequently, based uponthese studies, WHO recommended that vaccine production usingfully trained and competent staff in accordance with national safetyguidelines may proceed at biosafety level 2 enhanced, as describedin the WHO Technical Report Series No. 9411, Annex 5 (Fig. 1)[13,21,22]. A summary report on the assessment of NIBRG-121 ispresented in the Supplementary Online Material. Data for the otherthree candidate viruses were essentially the same as for NIBRG-121(not shown).

Indeed, as the pandemic progressed it became unnecessaryto maintain containment at BSL 2 enhanced as WHO had previ-ously announced that where there was significant infection withina region where a vaccine manufacturing facility was sited, thenin consultation with local authorities, vaccine manufacture couldproceed at BSL level 2, the standard level of biosafety for the man-ufacture of seasonal influenza vaccine (Fig. 1) [21,22].

3.6. Further development of candidate H1N1v vaccine viruses

Due to the low yield of viral protein from X-179A and the othercandidate vaccine viruses, the network of laboratories was urgedby WHO and by vaccine manufacturers to continue vaccine devel-

J.S. Robertson et al. / Vaccine 29 (2011) 1836–1843 1841

Table 3Growth and protein yield of viruses X-179A, NIBRG-121xp, X-181 and X-181A.a

Virus HA titre Viral protein (mg/100 eggs)

X-179A 640 4.9NIBRG-121xp 1280 10.9

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Table 4HA sequence features in principal pandemic H1N1 candidate vaccine viruses.a

Virus Position 222 Position 223 Others

A/California/04/2009 D QA/California/07/2009 D/G Q/RIDCDC-RG15 – RNYMC X-179A – R K209TNIBRG-121 G –IVR-153 – RIDCDC-RG18 G –IDCDC-RG20 – RIDCDC-RG22 – – S183PNIBRG-122 G – A135S, I321VNYMC X181 – R N129D, K209TNYMC X-181A – R N129D, K209T

X-181 1280 10.1X-181A 1920 12.0

a Determined at NIBSC.

pment with the hope of deriving a much higher yielding vaccineirus.

.6.1. Reverse genetics vaccine viruses derived during June/July009

The Center for Biologics Evaluation and Research (CBER), USAsed reverse genetics to derive the candidate vaccine virus CBER-G2, a 6:2 reassortant with the HA and NA genome segments

rom the cell-derived isolate A/California/04/2009. This virus hadn L191I substitution in the HA sequence and its availability wasnnounced by WHO on June 19, 2009 [26]. As this virus had a 6:2ene constellation and the HA and NA of A/California/04/2009, itas deemed that ferret safety testing would not be required as the

esults of tests on similar viruses had shown them to be attenuatedompared with the wild type virus [24,25,27].

At CDC, three further 6:2 candidate vaccine viruses wereerived as mix-and-match HA and NA reassortants between/Texas/05/2009 and A/New York/18/2009. IDCDC-RG18 contains

he HA of A/Texas/05/2009 with the D222G change and theA of A/New York/18/2009; IDCDC-RG20 contains the HA of/New York/18/2009 with the Q223R substitution and the NA of/Texas/05/2009; IDCDC-RG22 has both the HA (with an S183Pequence change) and NA of the A/New York/18/2009 isolate [28].

At NIBSC, a 6:2 candidate vaccine virus (NIBRG-122) was derivedrom a UK isolate, A/England/195/2009. The HA had the D222Ghange as well as A135S and I321V substitutions [28].

For the above reverse genetics viruses, there was no clear advan-age over X-179A in virus growth and protein yield (data nothown).

.6.2. Serial passage of NIBRG-121The in-house seed stock of NIBRG-121 is at passage level V1E2

Vero × 1, egg × 2). The virus was subjected to serial passage andt passage level V1E8, an increase in virus titre (as measured byaemagglutination assay) was observed. By passage level V1E12consistent high HA titre was obtained and a further passage to1E13 was made to prepare a stock for distribution. This new virusas named NIBRG-121xp (extended passage). The HA titre was

560 and, similar to new candidate viruses developed at NYMC (X-81 and X-181A, see section 3.6.3) protein yield analyses indicateduperior characteristics as compared to the X-179A high growtheassortant where NIBRG-112xp virus yielded 2.5 times more viralrotein per 100 eggs than X-179A (Table 3). NIBRG-121xp hadn amino acid substitution in HA compared with NIBRG-121 of119N/K (see Table 4).

.6.3. High growth reassortant vaccine viruses derived duringuly/August 2009

At NYMC by reassorting X-179A with X-157, the high yieldingonor used in the generation of X-179A, a new set of reassortantsere generated with improved growth characteristics, X-181, X-

81A and X-181B. With an HA titre of 4096, X-181 and X-181A haditres 2–4 times greater than X-179A while X-181B had no discern-ble improvement and was not studied further. The antigenic andenetic characteristics were determined at WHO-CC CDC and afternitial studies indicated them to be A/California/07/2009-like, they

NIBRG-121xp G – K119N/KCBER-RG2 – – L191I

a H1N1 numbering; all changes relative to A/California/04/2009.

were deemed suitable for distribution to manufacturers and otherpartners for further characterisation. Sequence analysis revealedan HA substitution of N129D in both viruses compared to the HA ofX-179A while full antigenic characterisation confirmed X-181 andX-181A to be A/California/07/2009-like (data not shown). Assess-ment of the yield of purified viral protein showed that both viruseshad considerably improved characteristics and yielded 10–12 mgviral protein per 100 eggs; in comparison X-179A in this experimentgave 4.9 mg protein per 100 eggs (Table 3).

3.6.4. WHO announcement of improved vaccine virusesOn 6th August, the WHO announced on their website the avail-

ability of improved vaccine virus NIBRG-121xp [29] and on 14thSeptember viruses X-181 and X-181A (Fig. 1) [30]. Informal reportsfrom industry on their assessment of viruses NIBRG-121xp, X-181and X-181A generally, but not entirely, backed-up the data on virusgrowth and protein yield provided by NIBSC. A summary of all avail-able candidate vaccine viruses for development of pandemic H1N1vaccine was produced by the WHO on 26th October 2009 [31].

4. Discussion

To develop a suitable candidate vaccine virus by classical reas-sortment, there was a requirement to use an egg-isolate as thisis the procedure approved by regulatory authorities for licensedinactivated vaccines produced in hens’ eggs. In contrast, the start-ing point for a reverse genetics reassortant virus can be a cell-or an egg-isolate. Initially, attempts to derive a vaccine virusfor pandemic H1N1 in eggs using reverse genetics with theA/California/04/2009 cell culture-derived isolate were unsuccess-ful; this was likely to be due to the absence of an egg-adaptingmutation in the HA of the virus. It was with the availability of asecond cell-isolate, A/Texas/05/2009 combined with the insertionof mutations that enabled replication in eggs, and an egg-isolate,A/California/07/2009, that derivation of candidate vaccine virusesbegan to be achieved by reverse genetics. The availability ofthe A/California/07/2009 egg-isolate also enabled vaccine virusderivation by classical reassortment. Of note, one candidate virus(CBER-RG2) was eventually derived in eggs directly from the cell-isolate A/California/04/2009 and contained an L191I substitution inits HA.

The timelines for pandemic H1N1 vaccine virus developmentare shown in Fig. 1. The generation of vaccine viruses by both clas-

sical reassortment and reverse genetics routes was rapid, takingapproximately three weeks for either approach. Indeed, the firstvaccine viruses were available prior to the WHO announcementof a pandemic on June 11, 2009. However, it took a few moremonths before vaccine viruses with improved antigen yield became

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vailable. Nonetheless, manufacturers began vaccine manufacturesing X-179A very early in the pandemic. With the availability of-181 with an improved antigen yield, several vaccine manufac-

urers switched to this virus; use was also made of NIBRG-121xp.lthough additional vaccine viruses with a further improvement

n antigen yield might have been generated and tested for vaccineanufacture, this option was not exercised because parallel devel-

pment and calibration of the necessary vaccine potency assayeagents could not have been achieved within acceptable timelines.

Table 4 shows amino acid substitutions in the HA of allrincipal candidate vaccine viruses compared to the wild type/California/04/2009 cell isolated virus. Most of the substitutionsre in the proximity of the receptor binding site of the HA moleculend one can make a reasonable assumption based upon historicalata that these mutations allow the virus to replicate successfully inggs [32,33]. Of interest, the D222G substitution has been observedreviously during egg-adaptation of seasonal H1N1 viruses [14]hile residue 223, commonly Q in H1N1 viruses, has not been pre-

iously implicated [33]. However, the corresponding residue in H3A (226) is implicated in determining the specificity of the linkageetween the sialic acid residue bound by the H3 HA and the adjacentalactose residue conferring specificity for either an �2,6 or �2,3inkage [34]. In virus CBER-RG2, the 222/223 sequence remains DQnd the virus HA has the substitution L191I. This residue is adjacento the receptor binding site and may be impacting on the ability ofhis virus to replicate readily in eggs.

In the three viruses for which growth in eggs was furthernhanced by multiple sequential passages, NIBRG-121xp, X-181nd X-181A, each had an additional HA substitution, K119N inIBRG-121xp and N129D in both X-181 and X-181A. Residue 129

s close to the RBS and thus the enhanced growth of X181/181Aay be due to more efficient receptor binding. Residue 119 is on

he surface of the HA, half way down the globular head and is notocated in the proximity of the RBS or an adjacent monomer withinhe trimeric HA structure. However, the substitution of K119 with Nreates a potential glycosylation site and if glycosylated, a carbohy-rate structure might in some way affect receptor binding despiteesidue 119 being distant from the receptor binding site.

Fortuitously, the above substitutions had no detectableffect on the antigenicity of the viruses and they remained/California/07/2009-like; consequently they remained suitable in

his respect for vaccine manufacture. The antigenicity of candidateaccine viruses was assessed at WHO Collaborating Centres at CDCUSA), NIMR (UK) and Melbourne (Australia), and at CBER (USA).

There was initial concern from industry that vaccine man-facture might have to be undertaken at BSL 3. This wouldave made vaccine manufacture difficult for some manufactur-rs and prohibitive for others. However, the inoculation of the 6:2nd 5:3 reassortants into ferrets showed that they were attenu-ted with respect to the wild type pandemic H1N1 viruses (seeupplementary Online Material). Based upon this evidence, theHO recommended that production could be undertaken at BSLenhanced [24,25]. This was still disconcerting for industry whose

arge scale production is more attuned to BSL 2; however, with thencreasing spread of pandemic H1N1 in communities worldwide,he WHO indicated that where there was widespread infection inregion where vaccine manufacture was taking place, then a BSL 2

afety level might be appropriate [13,21,22].The first batch of successfully derived candidate viruses pro-

uced only 30–50% of the level of viral protein typically foundor seasonal H1N1 viruses. This would have had a profound effect

n the worldwide supply of pandemic vaccine, and industry andHO made an appeal for improved viruses. Given that the derived

iruses already had a full or nearly full backbone of PR8 genomeegments and each HA had an egg-adapting substitution, it was notlear how further improvements in growth and viral protein yield

29 (2011) 1836–1843

could be approached. In the end it was classical virology methodsthat provided the required viruses; by sequential passaging in eggs(in the case of NIBRG-121xp) or further reassorting (in the caseof X-181/X-181A), which in itself involves extended egg passag-ing, viruses were selected that provided the desired characteristics.Note that X-181 and X-181A have the same genetic constellation asX-179A and the increased growth of these two viruses is potentiallydue to the extended egg passaging of X-179A during the reassortingprocess and the selection of a virus with the N129D HA mutation.Thus the poor antigen yield of the initial set of reassortant viruses ismost likely due to a lack of adaptation of the novel H1 HA to replica-tion in eggs. The protein yield of NIBRG-121xp, X-181 and X-181Awere all considerably improved and the values were more typicalof seasonal vaccine viruses (Table 3). In this way, the mission of thecollaborative group of laboratories was achieved.

To date the H1N1 viruses causing disease remain antigenicallyhomogeneous so that the A/California/07/2009 virus remains thebest choice for the vaccine. There is no doubt that drifted antigenicvariants will appear and that at some time in the not too distantfuture, the generation of new vaccine candidates will need to beundertaken. But for the moment, vaccine manufacturers have suit-able vaccine strains with which to produce high levels of vaccinefor the benefit of world health.

The derivation of candidate vaccine viruses is not confined to thelaboratories contributing to this report. Other laboratories such asSt Jude Children’s Research Hospital, USA, and the National Institutefor Infectious Diseases, Japan, have established the reverse geneticstechnology with which to derive vaccine viruses in a regulatory-compliant environment. Consequently, the world is well servedwith expertise to create rapidly the influenza viruses required ona continuous basis by the vaccine manufacturing industry. Whilereverse genetics has emerged as the only viable means of develop-ing vaccine viruses for highly pathogenic avian influenza and canhave some advantages for viruses that cannot be readily isolatedin eggs, the classical approach, developed many decades ago byKilbourne, has served the influenza community well and clearlycontinues to do so. There is a danger that classical virological lab-oratory skills could be lost in favour of newer technology; bothapproaches have an important role to play in the continuouslyvariable and unpredictable world of the influenza virus.

Acknowledgements

The authors are grateful to the World Health Organisation Col-laborating Centres in London and Melbourne for their antigeniccharacterisation of candidate vaccine viruses, to the WHO GlobalInfluenza Surveillance Network as a whole for the surveillance ofpandemic H1N1 viruses, and to the WHO and IFPMA for close dis-cussions throughout the period of vaccine virus development. Thefindings and conclusions in this report are those of the authors anddo not necessarily represent the views of the Centers for DiseaseControl and Prevention or the Agency for Toxic Substances andDisease Registry.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.vaccine.2010.12.044.

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