Current status and progress of prepandemic and pandemic influenza vaccine development

401

Review

www.expert-reviews.com ISSN 1476-0584© 2009 Expert Reviews Ltd 10.1586/ERV.09.15

In the 20th Century, humanity was struck by three major influenza pandemics. The most recent outbreak dates back from 1968, when an influenza A/H3N2 virus, known as the Hong Kong influenza, killed an estimated 2 million people. In 1957, an influenza A/H2N2 virus, known as the Asian influenza, caused an esti-mated 1 million deaths. No pandemic in recent history beats the devastating influenza outbreak of 1918, caused by an exceptionally pathogenic H1N1 influenza virus, known as the Spanish influenza, which killed 40–50 million people worldwide [1]. Over the past decade, we have been constantly reminded of the threat of an influenza pandemic and this has generated concern among politicians, policy makers, healthcare profession-als and the general public. In 2005, the WHO prepared a global influenza preparedness plan to assist WHO member states and those responsi-ble for public health to respond to threats and occurrences of pandemic influenza [101].

The influenza virus that has raised most con-cern and is considered the prime candidate to cause the next pandemic is the highly pathogenic

H5N1 avian inf luenza virus. The incessant spread and evolution of highly pathogenic H5N1 influenza in birds worldwide [102] and the con-tinuing infections of humans, limited in number but alarmingly fatal, have focused most attention and vaccine development efforts to this strain. From 2003 until 18 February 2009, 408 human cases of documented H5N1 infections have been reported, 254 (62%) of which were fatal [103]. Since no sustained human–human spread of the virus has been demonstrated, the WHO considers that we are now in phase 3 (of 6) of a pandemic alert [104].

Nonpharmaceutical interventions, such as quarantine, isolation, school closure and com-munity social distancing, represent a first layer of defense in the fight against a pandemic out-break. Combining these societal measures with targeted antiviral prophylaxis and treatment is expected to delay the course of a pandemic and lower the illness attack rate [2–5]. Vaccines, pre-pandemic and pandemic, are considered to be the most powerful tools to mitigate the effects of an influenza pandemic [2,3,6–8].

Isabel Leroux-Roels and Geert Leroux-Roels†

†Author for correspondenceCenter for Vaccinology, Ghent University and Hospital, De Pintelaan 185, 9000, Ghent, Belgium Tel.: +32 9332 3422 Fax: +32 9332 6311 [email protected]

H5N1 viruses are widely considered to be a probable cause of the next influenza pandemic. Influenza vaccines are considered to form the main prophylactic measure against pandemic influenza. The world’s population is expected to have no pre-existing immunity against the pandemic virus strain and will need two vaccine doses to acquire protective immunity. A pandemic outbreak will spread much faster than it will take for pandemic vaccines to be produced and distributed. Therefore, increasing efforts are being made to develop prepandemic vaccines that can induce broad cross-protective responses and that can be administered as soon as a pandemic is declared or even before, in order to successfully prime the immune system and allow for a rapid and protective antibody response with one dose of the pandemic vaccine. Several vaccine manufacturers have developed candidate pandemic and prepandemic vaccines, predominantly based on reverse-genetics reference strains and have improved the immunogenicity by formulating these vaccines with different adjuvants. Clinical studies with inactivated split-virion or whole-virion vaccines based on H5N1 indicate that two immunizations appear necessary to elicit the level of immunity required to meet licensure criteria. A detailed overview is given of the most successful candidate vaccines developed by seven vaccine manufacturers.

Keywords: adjuvant • cross-neutralization • dose sparing • H5N1 • influenza • pandemic • prepandemic • preparedness plan • vaccine

Current status and progress of prepandemic and pandemic influenza vaccine developmentExpert Rev. Vaccines 8(4), 401–423 (2009)

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Expert Rev. Vaccines 8(4), (2009)402

Review Leroux-Roels & Leroux-Roels

Being prepared for a pandemic means that the world needs sufficient vaccines, in time and against the right influenza A virus strain. In this review, we will elaborate on these enormous challenges and give an overview of the major achievements that have been accomplished to date. It must be stressed that the pandemic threat and the intensive vaccine development efforts that have been made in the past decade have been the subject of a number of recent, interesting and most informative editorials, commentaries and review papers [8–19].

Challenges of producing a pandemic influenza vaccineThere will be an unprecedented need for influenza vaccine doses. A pandemic outbreak will be caused by a new and potentially, highly pathogenic influenza virus. Most, if not all, of the world’s population will lack pre-existing immunity to this virus and will probably require two doses of vaccine, administered 3–4 weeks apart, to acquire adequate levels of immune protection. With the global population estimated at 6.7 billion, this represents a need for over 13 billion vaccine doses that will have to be produced and distributed in a limited time frame. To date, most trivalent seasonal influenza vaccines contain fragments of influenza viruses that were grown in embryonated chicken eggs. Today’s global production capacity, largely based on this technology, is approxi-mately 350 million doses per year. The International Federation of Pharmaceutical Manufacturers (IFPMA) estimates that the global capacity to make seasonal influenza vaccine will rise from 350 mil-lion to 1 billion doses by 2010. This increased production capacity combined with an intensified use of production plants (continuous production will double production volume) and a dose-sparing strategy (see below) would allow the production of sufficient vac-cines for the global population in 12 months in 2010; whereas, this would have theoretically taken 21 months in 2007 [20].

To exert its mitigating effect on the progression and attack rate of an influenza outbreak, a ‘pandemic vaccine’ needs to be avail-able in huge quantities at the start of the pandemic or immediately thereafter. There is still an alarming contrast between the speed with which the virus will spread around the world and the time needed to produce influenza vaccines today. The production of the seasonal influenza vaccine is a complex undertaking that is highly time constrained. From the WHO strain recommendation to the release of the first vaccine batches takes approximately 20–24 weeks and this will not be different in the case of a pandemic outbreak. Mathematical modeling suggests that it will take approximately 8 weeks for an outbreak starting in Thailand to reach the UK or the USA and that the epidemic will peak 50–65 days after the first case in the UK, and approximately 60–80 days after the first case for the USA [4]. Osterhaus stated that a pandemic vaccine based on the pandemic-causing virus isolate will become available after the start of the pandemic or, rather, as a postpandemic vaccine. Stockpiling a prepandemic vaccine, in the format of separately packaged adjuvant and rapidly replaceable viral antigen, appears to be today’s best strategy to prepare for a pandemic [8].

Since the nature of the influenza strain that will ultimately cause the next pandemic is unknown, the selection of the influ-enza strain to be used in a prepandemic vaccine poses a problem.

The highly pathogenic avian influenza H5N1 virus is still con-sidered to be the leading contender for the next human influenza pandemic [21,22,105]. The development of influenza A (H5N1) vaccines has been put forward by the WHO as one of the stra-tegic actions in its pandemic-preparedness plan. Numerous vac-cine companies have responded to this signal and have developed and produced such vaccines and performed preclinical and clini-cal tests of these products. H5N1 viruses isolated from human victims since 2005 belonged principally to clade 2. Subclades of clade 2 have been distinguished, three of which (subclades 2.1, 2.2 and 2.3) also differ in geographical distribution and have been largely responsible for the human cases in Indonesia, countries in the Middle East, Europe and Africa, and China, respectively. Considering the increasing importance of clade 2 strains, the human Indonesia isolate (A/Indonesia/5/2005) is also being used either as a priming or heterologous boost vaccine strain. Throughout the years, the H5N1 virus has been evolving constantly, resulting in many antigenic drift variants that have been classified into ten clades and various subclades [106].

Most studies executed and reported to date used prototype vaccines that were generated from human isolates belonging to the clade 1 viruses (A/Vietnam/1194/2004, NIBRG-14 and A/Vietnam/1203/2004, CDCRG-1 and SJRG-161052) or the clade 2 virus (A/Indonesia/5/2005, IBCDC-RG2) [23].

Considering the diversity and continuing evolution of H5N1 viruses, a prepandemic vaccine will need to induce crossreactive antibodies that are able to, ideally, neutralize all, or at least as many as possible, H5N1 drift strains.

The immunogenicity of candidate vaccines is evaluated by serological assays that measure the influenza-induced antibody responses. The most commonly used assays are the hemag-glutination inhibition (HI), single-radial hemolysis (SRH) and the microneutralization (MN) test. Since the HI assay was found to significantly underestimate responses to H5 [24–26], the more sensitive SRH and MN assays are being used more frequently today, despite the fact that these assays are more complicated and labor intensive. To increase the sensitivity of the HI assay, horse erythrocytes are used as substrate instead of fowl red blood cells [27]. The variability of the assay systems and a lack of stand-ardization preclude a direct comparison of serology results from different trials. A uniform serum standard was shown to improve the reproducibility of both HI and MN, but this standard is currently not yet applied [28].

To evaluate pandemic influenza vaccine immunogenicity, the European Committee for Medicinal Products for Human Use (CHMP) [107] and the US FDA [108] developed guidelines that are based on experience and data from interpandemic (seasonal) influ-enza studies. These criteria are shown in Box 1. Whether fulfillment of these surrogate criteria results in protection against pandemic influenza remains unknown, although challenge experiments in animal models can provide indicative results. Although MN results are not strictly required to obtain CHMP approval, the EMEA recommends the quantification of neutralizing antibodies, which confirm the presence of biologically functional antibodies that can inhibit viral attachment, entry and the release of new virions.

www.expert-reviews.com 403

ReviewPrepandemic & pandemic influenza vaccine development

Overall, an improved understanding of the correlates of pro-tection against influenza is urgently needed and the criteria to assess and approve candidate pandemic vaccines need to be reconsidered [29].

Challenges of implementing prepandemic & pandemic vaccinesIn case of a pandemic outbreak, the first batches of the pandemic vaccine will become available after a lead time of no less than 4 months. Moreover, vaccine supply will only gradually become available and the large populations that need it will receive the vaccine over a long period of time. This fact, together with recent data on the cross-neutralizing qualities of some prototype vaccines and the qualities of the booster responses they elicit (discussed further for each of the candidate vaccines), has changed our view on the deployment of prepandemic vaccines.

Using the influenza A/H5N1 strain that is considered the most likely candidate for causing the next outbreak, a prepandemic vaccine can be developed and produced today. This vaccine can be produced in a timely manner and stockpiled until an outbreak is declared. Eventually, one or two doses of the vaccine can be administered to high-risk populations. Governments may even consider to prime their entire population or particularly those at high risk such as healthcare workers, with one dose of the pre-pandemic vaccine so that cross-clade antibodies can be generated rapidly by the administration of a second dose of this vaccine (in case the pandemic virus is related to the vaccine virus) or by a single dose of the pandemic vaccine or by natural exposure to the pandemic virus once the outbreak (with a more distant virus strain) has started.

In January 2006, 28 vaccine trials against avian or pandemic studies were registered at the IFPMA, of which 19 vaccines were against the H5N1 subtype [109]. In the most recent update (26 May, 2008) of this document, 72 studies are tabulated, of which 57 were against H5N1 [110]. In the following paragraphs, we will present a limited selection of these candidate (pre)pandemic vaccines based on their advanced development stage. We will describe the developmental efforts of seven vaccine manufactur-ers and summarize the safety and immunogenicity data of their most advanced vaccine candidates. Dose-sparing qualities and crossreactivity of the induced responses will be highlighted as well as the protective efficacy in animal models whenever such data are available. An overview of these critical qualities of the most promising candidates is shown in TaBles 1–11. IFPMA and the WHO provide extensive overviews of all vaccine candidates under development [110,111].

Baxter (Czech Republic/Austria)The majority of the vaccine-producing companies that are devel-oping H5N1 influenza vaccines are producing these vaccines using conventional egg-based manufacturing methods, based on modified, attenuated reassortant viruses produced using reverse genetics. Baxter has devised a strategy that differs in both pro-duction aspects, since this company uses a wild-type virus and grows it in Vero cells. The advantages of this approach are a reduction of the lead time to produce the vaccine, since there is no need to generate attenuated, reassortant virus and the independ-ence of embryonated chicken eggs, which are a limiting factor in today’s production that may become even more pronounced in a pandemic situation. A drawback of this novel approach is the

Box 1. Criteria for the annual update of human seasonal influenza vaccines have also been applied to ‘mock-up’ pandemic vaccines.

The European Committee for Medicinal Products for Human Use uses three criteria to assess the antibody response to influenza vaccines:

• Seroconversion rate: the percentage of subjects (sera) with negative prevaccination hemagglutination inhibition (HI) titer and postvaccination titer of at least 1:40 or, for sera with positive prevaccination titer, at least a fourfold increase in HI titer. In single-radial hemolysis (SRH) tests, seroconversion corresponds to negative prevaccination serum and postvaccination values of at least 25 mm2, or for sera with positive prevaccination SRH tests, at least a 50% increase in area.

• Seroprotection rate: the percentage of subjects achieving a postvaccination HI titer of at least 1:40 or SRH area of at least 25 mm2.

• Mean geometric titer ratio: the mean geometric increase in titer.

Parameter Adults 18–60 years Adults ≥60 years

Seroconversion rate ≥40% ≥30%

Seroprotection rate ≥70% ≥60%

Geometric mean titer ratio ≥2.5 ≥2.0

For ‘mock-up’ pandemic vaccines and prepandemic vaccines, it is expected that all three criteria will be met.The US FDA uses the same criteria but requires:

• For adults under 65 years of age and for the pediatric population; that the lower bound of the two-sided 95% CI for the percent of subjects achieving seroconversion for HI antibody should meet or exceed 40% and that the lower bound of the two-sided 95% CI for the percent of subjects achieving seroprotection should meet or exceed 70%.

• For adults of 65 years of age or older; that the lower bound of the two-sided 95% confidence interval (CI) for the percent of subjects achieving seroconversion for HI antibody should meet or exceed 30% and that the lower bound of the two-sided 95% CI for the percent of subjects achieving seroprotection should meet or exceed 60%.



requirement for the use of enhanced biosafety level 3 (BSL-3+) facilities, the biohazard these facilities represent and the costs involved in their exploitation.

Candidate vaccines based on clade 1 (A/Vietnam/1203/2004) and clade 2 (A/Indonesia/5/2005) influenza H5N1 strains were developed and shown to be highly immunogenic in guinea pigs and mice [30]. The vaccines induced cross-neutralizing anti bodies, highly cross-reactive T-cell responses and were protective in a mouse challenge model not only against the homologous virus but also against H5N1 strains from other clades (see later) [30]. These preclinical data led to the development of a candidate whole-virus vaccine for human use. On 18 December 2008, the EMEA issued a positive opinion for the marketing authorization of Celvapan®, the nonadjuvanted 7.5 µg hemagglutinin (HA) formulation. This is the first cell culture-based H5N1 pandemic vaccine approved in the EU [112].

Pharmaceutical properties & formulation of the vaccineBaxter’s monovalent avian influenza H5N1 whole-virus vaccine is produced using the wild-type strain A/Vietnam/1203/2004, obtained from the US CDC. The virus is grown on Vero cells in a BSL-3+ facility [31]. The vaccines that are being examined in ongoing and planned Phase III studies contain 7.5 or 3.75 µg of HA (A/Vietnam/1203/2004) in a nonadjuvanted formula-tion. The vaccination schedule consists of two doses of 0.5 ml administered intramuscularly (to the deltoid muscle) with an interval of 3 weeks.

Safety & tolerability of the vaccineThe most relevant information on the safety and immuno genicity of the Baxter vaccine is taken from the dose-finding study wherein 275 subjects between the ages of 18 and 45 years received the first dose of vaccine and 257 received the second dose [32]. Subjects were administered a H5N1 whole-virus formulation containing 3.75, 7.5, 15 or 30 µg of HA antigen with a 0.2% alum adjuvant or 7.5 and 15 µg of HA antigen without adjuvant. The most com-monly reported local solicited adverse event was injection site pain (in 9–27% of subjects). The most commonly reported solicited general adverse event was headache (in 6–31% of participants). There were no significant differences between the vaccine formu-lations with respect to local or general solicited adverse effects. No serious, vaccine-related adverse effects were recorded.

Immunogenicity The immunogenicity against the influenza virus strain used in the vaccine (A/Vietnam/1203/2004) was tested 21 days after the first and the second dose with HI, MN and SRH assays [32]. Since the HI assay may be less sensitive for the detection of anti-H5 antibodies, the immunogenicity analyses focused on MN antibody responses. Virus-neutralizing titers of 1:20 or greater, seroconversion rates (fourfold increase of the titer) and geometric mean increase of the titer were all higher in the 7.5 and 15 µg formulations without adjuvant than in those with alum [32]. For example, seroconversion rates in vaccinees that received two doses of 7.5 and 15 µg HA without adjuvant were 69.0 Ta

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ReviewPrepandemic & pandemic influenza vaccine developmentTa

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.



(95% CI: 52.9–82.4) and 68.3% (95% CI: 51.9–81.9), respec-tively. Seroconversion rates induced with 7.5 and 15 µg HA of adjuvanted vaccine were 51.3 (95% CI: 34.8–67.6) and 46.3% (95% CI: 30.7–62.6), respectively. The superior immuno-genicity of the nonadjuvanted formulation was also observed when responses were measured with HI and SRH assays [32]. Cross-neutralizing capacity of the antibodies induced with the A/Vietnam vaccine was tested against a clade 0 strain (A/Hong Kong/156/1997, HK156) and a clade 2 strain (A/Indonesia/5/2005). The 7.5 and 15 µg formulations without adjuvant showed high levels of cross reactivity against the HK156 strain (76.2 and 78.0%, respectively, with a neutralizing titer of ≥1:20 after two doses). The responses against the clade 2 strain were lower with neutralizing titer of ≥1:20 observed in 45.2 and 36.6% of those receiving 7.5 and 15 µg without adjuvant, respec-tively. Formulations without adjuvant were consistently better than adjuvanted ones [32]. This study showed that the whole-virus clade 1-based vaccine can induce strong immune responses against the vaccine strain as well as against heterologous clade 0 and 2 strains.

Phase III studies to assess the safety and tolerability of and the immune response to the nonadjuvanted, whole-virus influenza vaccine (A/Vietnam/1203/2004) in adult and elderly popula-tions and in special risk groups are ongoing and/or planned. Further studies will assess the need for booster vaccination and the appropriate interval (6 or 12 months) between the priming and the booster dose. In addition, the effect of two-dose prim-ing with a clade 1-based vaccine (A/Vietnam/1203/2004) and boosting with a clade 2-based vaccine (A/Indonesia/5/2005) will be explored [113].

Challenge studies in a mouse modelOutbred mice (CD1 strain) were subcutaneously immunized twice, 3 weeks apart, with doses ranging from 0.001 to 3.75 µg HA antigen (A/Vietnam/1203/2004) using nonadjuvanted material and vaccine adjuvanted with 0.2% Al(OH)

3 [30]. Control groups

received buffer or adjuvanted buffer. A total of 3 weeks after the second dose, all animals were challenged intranasally with 105 50% tissue culture infectious dose (TCID

50) of the A/Vietnam

virus strain and survivors were counted 14 days postchallenge. The nonadjuvanted vaccine protected 100% of immunized mice with a dosage as low as 30 ng HA antigen; whereas, 750 ng of the alum-adjuvanted vaccine was required to provide full protection against lethal infection. The whole-virus vaccine was highly effec-tive in inducing protective immunity in CD1 mice and complete protection correlated with positive neutralization results. The nonadjuvanted vaccine formulation appeared more potent than the alum-formulated material.

To evaluate the crossprotective potential against antigenically different H5N1 strains, CD1 mice were immunized as described above with A/Vietnam vaccine in a dose range of 0.006–0.750 µg with the nonadjuvanted material. A total of 3 weeks after the sec-ond immunization, the animals were challenged with either the homologous clade 1 A/Vietnam strain, the heterologous clade 0 HK156 strain or the heterologous clade 2 A/Indonesia strain. Ta

ble

4. O

verv

iew

of

pre

pan

dem

ic a

nd

pan

dem

ic v

acci

ne

can

did

ates

: Gla

xoSm

ith

Klin

e B

iolo

gic

als:

pre

pan

dem

ic v

acci

ne.

Typ

e o

f va

ccin

eIn

flu

enza

vi

rus

stra

in

Sub

stra

teA

dju

van

t (c

om

po

nen

ts

in m

g/d

ose

)

An

tig

en

do

se

(HA

µg

/d

ose

)

Pro

du

ct

nam

e(l

icen

se)

Vac

cin

atio

n

Sch

edu

leH

I res

po

nse

MN

re

spo

nse

%

wit

h

fou

rfo

ld

incr

ease

(9

5% C

I)

Cro

ss-

neu

tral

izat

ion

% w

ith

fo

urf

old

in

crea

se

In v

ivo

p

rote

ctio

nR

ef.

SCR

(%

)(9

5% C

I)SP

R (

%)

(95%

CI)

GM

TR

(95%

CI)

Inac

tiva

ted

split

-viri

onRe

asso

rtan

t A

/V

ietn

am/

1194

/20

04

NIB

RG-1

4

Eggs

AS0

3 (s

qual

ene:

10

.68

mg

; d

l-α

-t

oco

pher

ol:

11.8

6 m

g,

po

lyso

rbat

e 8

0: 4

.85

mg

)

3.8

Prep

andr

ix

(lice

nsed

in

EU

, 20

08

)

Two

do

ses

im.,

3 w

eeks

ap

art

82

(68

.6–

91.4

)

84

(70.

9–

92.8

)

27.9

(1

7.2–

45.2

)

86

(72.

8–9

4.1

)A

/Ind

ones

ia/

5/0

5: 7

5;

A/t

urke

y/Tu

rkey

/1/0

5:

85;

A/A

nhui

/1/0

5:

75

Full

prot

ecti

on

achi

eved

w

ith

two

3.8

µg

do

ses

in

ferr

et

chal

leng

e m

od

el

[40,46

,53]

Ant

igen

do

se: s

ho

ws

the

HA

do

se o

f th

e fi

nal v

acci

ne

form

ula

tio

n (w

hen

defi

ned

) or

the

HA

do

se t

hat

elic

its

the

hig

hes

t H

I tit

ers,

mea

sure

d 3

(or

4) w

eeks

aft

er t

he

seco

nd

vacc

ine

do

se.

CI:

Co

nfi

den

ce in

terv

al; G

MTR

: Geo

met

ric

mea

n ti

ter

rati

o; H

A: H

emag

glu

tini

n; H

I: H

emag

glu

tina

tio

n in

hib

itio

n; i

m.:

Intr

amu

scu

lar;

MN

: Mic

ron

eutr

aliz

atio

n; N

A: N

ot a

vaila

ble

; SC

R: S

ero

conv

ersi

on

rate

; SP

R: S

ero

pro

tect

ion

rate

.



Challenge with the homologous A/Vietnam strain resulted in full protection against lethal infection with doses greater than 30 ng. Challenges with A/Indonesia and HK156 showed good dose-dependent cross-protection, reaching 95–100% at a dose of 750 ng. Next, the reciprocal experiment was carried out with mice immunized with the clade 2 A/Indonesia/5/2005 strain vaccine and challenged with the homologous A/Indonesia or the heter-ologous clade 1 A/Vietnam strain. The study confirmed the high level of cross-protection that can be induced by immunization with the whole-virus vaccine and suggests that the virus strain used for vaccination need not fully match the challenge virus to achieve high levels of protection against lethal infection.

The protective efficacy of the vaccine at doses as low as 3.75 µg has also been confirmed in ferret challenge studies [33,34].

CSL Biotherapies Limited (Australia)CSL Biotherapies, Australia’s leading biopharmaceutical com-pany, has developed a pandemic influenza vaccine that was recently (17 June 2008) approved by Australia’s Therapeutic Goods Administration. The vaccine, named Panvax®, can only be used when the Australian government officially declares that a influenza pandemic is underway.

Pharmaceutical properties & formulation of the vaccinePanvax is a monovalent, A/H5N1, inactivated split-vir-ion inf luenza vaccine. The vaccine seed virus was the A/Vietnam/1194/2004 NIBRG-14 reference strain [23]. The vac-cine was produced on embryonated chicken’s eggs using standard techniques. In a Phase I study, 7.5 and 15 µg H5 HA with or without 0.5 mg AlPO

4 were administered in 0.5 ml [35]. In a

Phase II trial, vaccines (0.5 ml) containing 30 and 45 µg H5 HA with AlPO

4 were evaluated [35]. In a pediatric study, vaccines

containing 30 and 45 µg H5 HA with adjuvant (0.5 mg AlPO4)

were evaluated [36]. All vaccines contained 50 µg of thiomersal. The vaccination scheme consisted of two doses administered 3 weeks apart in the deltoid muscle (in adults and children aged >12 months) or in the antero–lateral side of the thigh (in children aged ≤12 months).

Safety & tolerability of the vaccineThe safety profile has been studied in adults aged 18–45 years (Phase I), 18–64 years (Phase II) [35] and in healthy infants and children (≥6 months to <9 years) [36]. All vaccine formulations were well tolerated by adults. The most frequently reported injec-tion site reactions were pain and redness (varying between 53–83 and 24–41.7%, respectively) and these tended to be more pro-nounced in the adjuvanted vaccine recipients than in the alum-free vaccinees. The most frequently reported systemic adverse events were headache and fatigue (varying between 45.0–47.0 and 35.0–44.2%, respectively). Adjuvantation had no effect on systemic adverse events. Most solicited local and general adverse events were mild and did not increase with successive doses. Of all unsolicited adverse events reported, 25.9% (301 out of 1160) were considered to be related to the study vaccine; 54.3% (631 out of 1160) were rated as mild and 8.7% (101 out of 1160) as severe.Ta

ble

5. O

verv

iew

of

pre

pan

dem

ic a

nd

pan

dem

ic v

acci

ne

can

did

ates

: No

vart

is: p

and

emic

vac

cin

e.

Typ

e o

f va

ccin

eIn

flu

enza

vi

rus

stra

in

Sub

stra

teA

dju

van

t (c

om

po

nen

ts

in m

g/d

ose

)

An

tig

en

do

se

(HA

µg

/d

ose

)

Pro

du

ct

nam

e (l

icen

se)

Vac

cin

atio

n

sch

edu

leH

I res

po

nse

MN

re

spo

nse

% t

iter

≥1

:40

(95%

CI)

Cro

ss-

neu

tral

izat

ion

In v

ivo

p

rote

ctio

nR

ef.

SCR

(%

)(9

5% C

I)SP

R (

%)

(95%

CI)

GM

TR(9

5% C

I)

Inac

tivat

ed

subu

nit

Reas

sort

ant

A/V

ietn

am/

119

4/2

00

4N

IBRG

-14

Eggs

MF5

9C

.1

(squ

alen

e:

9.75

mg

; p

oly

sorb

ate

80

: 1.1

75 m

g so

rbit

an

trio

leat

e:

1.17

5 m

g)

7.5

Foce

tria

®

(lice

nced

in

EU

, 20

07)

Two

do

ses,

im

., 3

wee

ks

apar

t

Adu

lts

(18

–6

0 ye

ars)

: 85

(7

9–9

1)

Eld

erly

(≥

61 y

ears

):

71 (

60

–81)

Adu

lts

(18

–6

0 ye

ars)

: 8

6 (7

9–9

1)

Eld

erly

(≥

61 y

ears

):

81 (

71–8

9)

Adu

lts

(18

–6

0 ye

ars)

: 7.

85

(6.7

–9.2

)

Eld

erly

(≥

61 y

ears

):

5.02

(3

.91–

6.4

5)

Adu

lts

(18

–6

0 ye

ars)

: 85

(78

–90

)

Eld

erl y

(≥

61 y

ears

):

79 (

68

–87)

NA

NA

[117]

Ant

igen

do

se: s

ho

ws

the

HA

do

se o

f th

e fi

nal v

acci

ne

form

ula

tio

n (w

hen

defi

ned

) or

the

HA

do

se t

hat

elic

its

the

hig

hes

t H

I tit

ers,

mea

sure

d 3

(or

4) w

eeks

aft

er t

he

seco

nd

vacc

ine

do

se.

CI:

Co

nfi

den

ce in

terv

al; G

MTR

: Geo

met

ric

mea

n ti

ter

rati

o; H

A: H

emag

glu

tini

n; H

I: H

emag

glu

tina

tio

n in

hib

itio

n; i

m.:

Intr

amu

scu

lar;

MN

: Mic

ron

eutr

aliz

atio

n; N

A: N

ot a

vaila

ble

; SC

R: S

ero

conv

ersi

on

rate

; SP

R: S

ero

pro

tect

ion

rate

.


Review Leroux-Roels & Leroux-RoelsTa

ble

6. O

verv

iew

of

pre

pan

dem

ic a

nd

pan

dem

ic v

acci

ne

can

did

ates

: No

vart

is: p

rep

and

emic

vac

cin

e.

Typ

e o

f va

ccin

eIn

flu

enza

vi

rus

stra

in

Sub

stra

teA

dju

van

t (c

om

po

nen

ts

in m

g/d

ose

)

An

tig

en

do

se

(HA

µg

/d

ose

)

Pro

du

ct

nam

e (l

icen

se)

Vac

cin

atio

n

sch

edu

leH

I res

po

nse

MN

re

spo

nse

% t

iter

≥1

:40

(95%

CI)

Cro

ss-

neu

tral

izat

ion

%

tit

er ≥

1:4

0 (9

5% C

I)

In v

ivo

p

rote

ctio

nR

ef.

SCR

(%

)(9

5% C

I)SP

R (

%)

(95%

CI)

GM

TR(9

5% C

I)

Inac

tiva

ted

subu

nit

Reas

sort

ant

A/

Vie

tnam

/ 11

94

/20

04

NIB

RG-1

4

Eggs

MF5

9C

.1

(squ

alen

e 9.

75 m

g;

po

lyso

rbat

e 8

0: 1

.175

mg

sorb

itan

tr

iole

ate:

1.

175

mg

)

7.5

Aflu

nov®

(m

arke

ting

appl

icat

ion

wit

hdra

wn

)

Two

do

ses

im.,

3 w

eeks

ap

art

Adu

lts

(18

–6

0 ye

ars)

: 73

(65

–80

)

Adu

lts

(18

–6

0 ye

ars)

: 73

(65

–80

)

Adu

lts

(18

–6

0 ye

ars)

: 16

(12–

21)

Adu

lts

(18

–6

0 ye

ars)

: 85

(78

–90

)

A/t

urke

y/Tu

rkey

/1/2

005

(N

IBRG

-23

);

Adu

lts

(18

–6

0 ye

ars)

: 27

(17–

39);

NA

[66]

Eld

erly

(≥

61

year

s):

67

(55

–77

)

Eld

erly

(≥

61

year

s):

75

(64

–84

)

Eld

erly

(≥

61ye

ars)

: 9.

52

(6.6

–11

.4)

Eld

erly

(≥

61 y

ears

):

79 (

68

–87

)

Eld

erly

(≥

61 y

ears

):

11 (3

–25

)

Ant

igen

do

se: s

ho

ws

the

HA

do

se o

f th

e fi

nal v

acci

ne

form

ula

tio

n (w

hen

defi

ned

) or

the

HA

do

se t

hat

elic

its

the

hig

hes

t H

I tit

ers,

mea

sure

d 3

(or

4) w

eeks

aft

er t

he

seco

nd

vacc

ine

do

se.

CI:

Co

nfi

den

ce in

terv

al; G

MTR

: Geo

met

ric

mea

n ti

ter

rati

o; H

A: H

emag

glu

tini

n; H

I: H

emag

glu

tina

tio

n in

hib

itio

n; i

m.:

Intr

amu

scu

lar;

MN

: Mic

ron

eutr

aliz

atio

n; N

A: N

ot a

vaila

ble

; SC

R: S

ero

conv

ersi

on

rate

; SP

R: S

ero

pro

tect

ion

rate

.



Tab

le 8

. Ove

rvie

w o

f p

rep

and

emic

an

d p

and

emic

vac

cin

e ca

nd

idat

es: S

ano

fi P

aste

ur

USA

: pan

dem

ic v

acci

ne.

Typ

e o

f va

ccin

eIn

flu

enza

vi

rus

stra

in

Sub

stra

teA

dju

van

tA

nti

gen

d

ose

(H

A µ

g/

do

se)

Lice

nse

Vac

cin

atio

n

sch

edu

leH

I res

po

nse

MN

re

spo

nse

% w

ith

fo

urf

old

in

crea

se

Cro

ss-

neu

tral

izat

ion

In v

ivo

p

rote

ctio

nR

ef.

SCR

(%

)(9

5% C

I)SP

R (

%)

(95%

CI)

GM

TR

Inac

tivat

ed

split

-viri

onRe

asso

rtan

t A

/Vie

tnam

/ 12

03/2

00

4C

DC

RG-1

Eggs

Non

e9

0 A

ppr

oved

in

USA

, 20

07

Two

do

ses,

im

., 4

wee

ks

apar

t

57 (

46

–67

)57

(4

6–

67)

5.4

53 (

42–

63)

NA

NA

[67]

Ant

igen

do

se: s

ho

ws

the

HA

do

se o

f th

e fi

nal v

acci

ne

form

ula

tio

n (w

hen

defi

ned

) or

the

HA

do

se t

hat

elic

its

the

hig

hes

t H

I tit

ers,

mea

sure

d 3

(or

4) w

eeks

aft

er t

he

seco

nd

vacc

ine

do

se.

CI:

Co

nfi

den

ce in

terv

al; G

MTR

: Geo

met

ric

mea

n ti

ter

rati

o; H

A: H

emag

glu

tini

n; H

I: H

emag

glu

tina

tio

n in

hib

itio

n; i

m.:

Intr

amu

scu

lar;

MN

: Mic

ron

eutr

aliz

atio

n; N

A: N

ot a

vaila

ble

; SC

R: S

ero

conv

ersi

on

rate

; SP

R: S

ero

pro

tect

ion

rate

.

Tab

le 7

. Ove

rvie

w o

f p

rep

and

emic

an

d p

and

emic

vac

cin

e ca

nd

idat

es: O

mn

inve

st.

Typ

e o

f va

ccin

eIn

flu

enza

vi

rus

stra

in

Sub

stra

teA

dju

van

tA

nti

gen

d

ose

(H

A µ

g/

do

se)

Pro

du

ct

nam

e(l

icen

se)

Vac

cin

atio

n

sch

edu

leH

I res

po

nse

MN

re

spo

nse

%

wit

h

fou

rfo

ld

incr

ease

Cro

ss-

neu

tral

izat

ion

%

wit

h f

ou

rfo

ld

incr

ease

In v

ivo

p

rote

ctio

nR

ef.

SCR

(%

)SP

R (

%)

GM

TR

Inac

tiva

ted

who

le-

viri

on

Reas

sort

ant

A/V

ietn

am/

119

4/2

00

4N

IBRG

-14

Eggs

AlP

O4

(0.3

1 m

g al

um)

6 Fl

uva

l™

(lice

nsed

in

H

unga

ry,

2007

)

On

e d

ose

, im

.A

dult

s (1

8–

60

year

s):

63.7

Adu

lts

(18

–6

0 ye

ars)

: 63

.7

Adu

lts

(18

–6

0 ye

ars)

: 5.

6

Adu

lts

(18

–6

0 ye

ars)

: 5

4.5

; El

der

ly

(≥61

yea

rs):

47

.7;

A/A

nhui

/1/0

5 A

dult

s (1

8–

60

year

s): 4

5.5

Eld

erly

(≥6

1 ye

ars)

: 3

4.1

NA

[54– 56]

Chi

ldre

n (3

–18

year

s):

75

Chi

ldre

n (3

–18

year

s):

75

Chi

ldre

n (3

–18

year

s):

16.9

5

Chi

ldre

n (3

–18

year

s):

66

.7

[55]

Ant

igen

do

se: s

ho

ws

the

HA

do

se o

f th

e fi

nal v

acci

ne

form

ula

tio

n (w

hen

defi

ned

) or

the

HA

do

se t

hat

elic

its

the

hig

hes

t H

I tit

ers,

mea

sure

d 3

(or

4) w

eeks

aft

er t

he

seco

nd

vacc

ine

do

se.

CI:

Co

nfi

den

ce in

terv

al; G

MTR

: Geo

met

ric

mea

n ti

ter

rati

o; H

A: H

emag

glu

tini

n; H

I: H

emag

glu

tina

tio

n in

hib

itio

n; i

m.:

Intr

amu

scu

lar;

MN

: Mic

ron

eutr

aliz

atio

n; N

A: N

ot a

vaila

ble

; SC

R: S

ero

conv

ersi

on

rate

; SP

R: S

ero

pro

tect

ion

rate

.



Tab

le 1

0. O

verv

iew

of

pre

pan

dem

ic a

nd

pan

dem

ic v

acci

ne

can

did

ates

: San

ofi

Pas

teu

r: p

rep

and

emic

vac

cin

e.

Typ

e o

f va

ccin

eIn

flu

enza

vi

rus

stra

inSu

bst

rate

Ad

juva

nt

An

tig

en

do

se

(HA

µg

/d

ose

)

Vac

cin

atio

n

sch

edu

leH

I res

po

nse

MN

res

po

nse

%

wit

h

fou

rfo

ld

incr

ease

(9

5% C

I)

Cro

ss-n

eutr

a%

MN

tit

er

≥1:2

0 (9

5% C

I)

agai

nst

A

/In

do

nes

ia

/5/2

005

RG

2

In v

ivo

p

rote

ctio

nR

ef.

SCR

(%

)(9

5% C

I)SP

R (

%)

(95%

CI)

GM

TR

(95%

CI)

Inac

tiva

ted

split

-viri

onRe

asso

rtan

t A

/Vie

tnam

/ 11

94

/20

04

NIB

RG-1

4

Eggs

AF0

3 (5

%

squa

len

e-in

-wat

er

emul

sion

)

3.8

Two

do

ses

im.,

3 w

eeks

ap

art

81

(67.

4–9

1.1)

81 (

67.4

–91.

1)15

.5

(11.

3–2

1.4

)8

8 (7

5.2–

95.4

)28

(16

.0–4

3.5

)Pr

otec

tion

in

m

acaq

ues

[73]

and

fe

rret

s [74]

[69]

Ant

igen

do

se: s

ho

ws

the

HA

do

se o

f th

e fi

nal v

acci

ne

form

ula

tio

n (w

hen

defi

ned

) or

the

HA

do

se t

hat

elic

its

the

hig

hes

t H

I tit

ers,

mea

sure

d 3

(or

4) w

eeks

aft

er t

he

seco

nd

vacc

ine

do

se.

CI:

Co

nfi

den

ce in

terv

al; G

MTR

: Geo

met

ric

mea

n ti

ter

rati

o; H

A: H

emag

glu

tini

n; H

I: H

emag

glu

tina

tio

n in

hib

itio

n; i

m.:

Intr

amu

scu

lar;

MN

: Mic

ron

eutr

aliz

atio

n; n

eutr

a: N

eutr

aliz

atio

n; S

CR

: Ser

oco

nver

sio

n ra

te;

SPR

: Ser

op

rote

ctio

n ra

te.

Tab

le 9

. Ove

rvie

w o

f p

rep

and

emic

an

d p

and

emic

vac

cin

e ca

nd

idat

es: S

ano

fi P

aste

ur

Fran

ce: p

and

emic

vac

cin

e.

Typ

e o

f va

ccin

eIn

flu

enza

vi

rus

stra

inSu

bst

rate

Ad

juva

nt

An

tig

en

do

se

(HA

µg

/d

ose

Lice

nse

Vac

cin

atio

n

sch

edu

leH

I res

po

nse

MN

re

spo

nse

% w

ith

fo

urf

old

in

crea

se

(95%

CI)

Cro

ss-n

eutr

a%

wit

h t

iter

≥1

:20

amo

ng

se

ra w

ith

h

om

olo

go

us

MN

tit

er ≥

1:20

In v

ivo

p

rote

ctio

nR

ef.

SCR

(%

)(9

5% C

I)

SPR

(%

) (9

5% C

I)

GM

TR

(95%

CI)

Inac

tivat

ed

split

-viri

onRe

asso

rtan

t A

/V

ietn

am/

119

4/2

00

4N

IBRG

-14

Eggs

Al(

OH

) 33

0A

pplic

atio

n su

bmit

ted

to E

MEA

Two

do

ses

im.,

3 w

eeks

ap

art

66

(51–

79)

67

(52–

79)

11.6

(6

.7–2

0.0

)41

(28

–56

)A

/Ind

o/5

/05

CD

C-RG

2: 9

A

/Ind

o/5

/05

wild

-typ

e: 8

2 A

/tk/

Tk/1

/05

NIB

RG23

: 42

A/t

k/Tk

/1/0

5 w

ild-t

ype:

90

NA

[68,71]

Ant

igen

do

se: s

ho

ws

the

HA

do

se o

f th

e fi

nal v

acci

ne

form

ula

tio

n (w

hen

defi

ned

) or

the

HA

do

se t

hat

elic

its

the

hig

hes

t H

I tit

ers,

mea

sure

d 3

(or

4) w

eeks

aft

er t

he

seco

nd

vacc

ine

do

se.

CI:

Co

nfi

den

ce in

terv

al; G

MTR

: Geo

met

ric

mea

n ti

ter

rati

o; H

A: H

emag

glu

tini

n; H

I: H

emag

glu

tina

tio

n in

hib

itio

n; i

m.:

Intr

amu

scu

lar;

MN

: Mic

ron

eutr

aliz

atio

n; N

A: N

ot a

vaila

ble

; neu

tra:

Neu

tral

izat

ion

; SC

R: S

ero

conv

ersi

on

rate

; SPR

: Ser

op

rote

ctio

n ra

te.



ImmunogenicityThe immunogenicity of the different vaccine formulations was tested with HI and MN assays. In two trials in which adult par-ticipants (n = 100 per group) were given either 7.5 or 15 µg H5 HA with or without AlPO

4 (Phase I) and 30 or 45 µg HA with

AlPO4 (n = 200 per group, Phase II), all subjects displayed at least

a 2.5-fold increase in the HI titer, but the CHMP criterion for over 70% seroprotection (HI ≥ 1:32) was not met in any of the study groups [35]. Only 58% of the subjects receiving the 45-µg dose (with AlPO

4) reached a HI titer of at least 1:32. After two

vaccine doses MN titers of at least 1:20 were measured in 37% (7.5 µg without alum) to 73% (30 and 45 µg with alum) of the participants. Adjuvantation of the vaccine had a positive effect on the MN titers. Vaccines containing 7.5 and 15 µg HA without adjuvant induced MN titers of at least 1:40 in 19 and 30% of subjects, respectively, while this was 34–41% with adjuvant [35]. This may explain why in the Phase II study only an adjuvan-ted vaccine was used. The 6-month persistence of the antibody responses in the Phase I study was low; HI titers of at least 1:32 were measured in less than 12% of participants and MN titers of at least 1:20 in less than 10%. This drop of antibodies was less prominent in the Phase II study. At month 6, 60–62% of subjects had retained MN titers of at least 1:20, while these were 73% 21 days post dose two [35].

The booster vaccination administered at 6 months in the Phase I trial induced a marked response to all formulations [35]. However, the postbooster HI titers were slightly lower than those observed 21 days after the second dose of the primary vaccination cycle [35]. By contrast, postbooster MN titers were higher than those achieved after the second dose of 7.5- or 15-µg formulations and were comparable to the responses induced with two doses of the 30 and 45 µg adjuvanted formulations in the Phase II trial [35]. Vaccinees were more likely to achieve MN titers of at least 1:20 to booster vaccinations with the adjuvanted formulations rather than with the nonadjuvanted formulations [35].

Antibody responses to variant clade 2 strains (A/Turkey/ Turkey/1/2005 NIBRG23 and A/Indonesia/5/2005 IBCDC-RG2) were lower than against the vaccine strain (A/Vietnam /1194/2004 NIBRG-14). Both HI and MN responses were somewhat higher against the NIBRG23 strain than against the IBCDC-RG2 strain [35].

In a study targeting a pediatric population, vaccines con-taining 30- and 45-µg H5 HA with adjuvant (0.5 mg AlPO

4)

were evaluated [36]. While the first vaccine dose induced rather modest responses, especially when measured by the MN assay, the second dose elicited strong immune responses by both the MN and HI assay. While the proportion of all children with MN titers of at least 1:20 was 26 and 31% after the first dose for the 30- and 45-µg HA formulations, respectively, this was 99% for both formulations after the second dose [36]. Following the second dose of vaccine, the proportion of children with HI titers of at least 1:32 was 100% in all children receiving the 45-µg formulation. In the children receiving the 30-µg formulation, the overall proportion of those with HI titers of at least 1:32 was 95%, with 88% in the youngest ones (≥6 months to <3 years) Ta

ble

11.

Ove

rvie

w o

f p

rep

and

emic

an

d p

and

emic

vac

cin

e ca

nd

idat

es: S

ino

vac.

Typ

e o

f va

ccin

eIn

flu

enza

vi

rus

stra

in

Sub

stra

teA

dju

van

tA

nti

gen

d

ose

(H

A µ

g/

do

se

Pro

du

ct

nam

e (l

icen

se)

Vac

cin

atio

n

sch

edu

leH

I res

po

nse

MN

re

spo

nse

%

tite

r ≥1

:40

(95%

CI)

Cro

ss-

neu

tral

izat

ion

In v

ivo

p

rote

ctio

nR

ef.

SCR

(%

) (9

5% C

I)SP

R (

%)

(95%

CI)

GM

TR

(95%

CI)

Inac

tivat

ed

who

le-

viri

on

Reas

sort

ant

A/V

ietn

am/

119

4/2

00

4N

IBRG

-14

Eggs

Al(

OH

) 3

(0.5

mg

/ml

alum

)

10 µ

gPa

nflu

(ap

prov

ed b

y th

e C

hina

St

ate

Foo

d an

d D

rug

Adm

inis

trat

ion

)

Two

do

ses,

im

., 4

wee

ks

apar

t

64

(4

1–8

3)

64

(41–

83

)10

.3

(9.9

–10

.7)

50

(28

–72)

NA

NA

[75]

Ant

igen

do

se: s

ho

ws

the

HA

do

se o

f th

e fi

nal v

acci

ne

form

ula

tio

n (w

hen

defi

ned

) or

the

HA

do

se t

hat

elic

its

the

hig

hes

t H

I tit

ers,

mea

sure

d 3

(or

4) w

eeks

aft

er t

he

seco

nd

vacc

ine

do

se.

CI:

Co

nfi

den

ce in

terv

al; G

MTR

: Geo

met

ric

mea

n ti

ter

rati

o; H

A: H

emag

glu

tini

n; H

I: H

emag

glu

tina

tio

n in

hib

itio

n; i

m.:

Intr

amu

scu

lar;

MN

: Mic

ron

eutr

aliz

atio

n; N

A: N

ot a

vaila

ble

; SC

R: S

ero

conv

ersi

on

rate

; SP

R: S

ero

pro

tect

ion

rate

;



and 100% in the older group (≥3 years to <9 years) [36]. Neutralizing antibodies persisted substantially for 6 months following the primary course. The proportion of all children maintaining MN titers of at least 1:20 was 85 and 87% for the 30- and 45-µg HA formulations, respectively [36]. The responses to the heterologous influenza variant (IBCDC-RG2, clade 2.1) were lower than against the vaccine strain (NIBRG-14). After the primary two-dose vaccine course, the crossreactivity to IBCDC-RG2 by HI assay (HI ≥1:32) was demonstrated in 80 and 86% of infants and children receiving the 30- and 45-µg formulations, respectively.

GlaxoSmithKline Biologicals (Belgium)Alerted by the 1997 H5N1 outbreak in Hong Kong, GlaxoSmithKline (GSK) started to explore the safety and immunogenicity of adjuvant use and varying doses of whole-virus H2N2 and H9N2 pandemic candidate vaccines. These studies demonstrated that alum-adjuvanted whole-virus vac-cine with low HA content (up to eightfold less than the sea-sonal influenza vaccine) can raise protective antibody levels after two vaccine doses. The need for a two-dose schedule in unprimed populations was shown and neither the use of alumi-num adjuvant nor whole virus had a significant effect on general reactions [37,38]. This experience led to the development of an alum-adjuvanted, whole-virus influenza A/Vietnam/1194/2004 (H5N1) vaccine. Two injections of this vaccine, even at doses as low as 3.8 µg, raised HI responses that complied with two out of three CHMP criteria (seroconversion rate [SCR] and geometric mean titer ratio [GMTR]). However, in order to meet all three criteria, at least a 15-µg dose (with or without adjuvant) was needed [39]. In March 2007, this vaccine, known as Daronrix®, received a marketing authorization valid throughout the EU for the indication of ‘prophylaxis of influenza in an officially declared pandemic situation’ [114]. Subsequent developments focused on the dose-sparing capacity and the persistence and broadness of the immune response induced by the candidate H5N1 vaccine and examined the effects of adjuvantation on these qualities of the vaccine.

Pharmaceutical properties & formulation of the vaccineGlaxoSmithKline’s prepandemic vaccine is a monovalent, A/H5N1, inactivated split-virion inf luenza vaccine. The vaccine seed virus was a H5N1 reassortant reference virus (A/Vietnam/1194/2004 NIBRG-14) derived by reverse genetics from the highly pathogenic avian strain A/Vietnam/1194/2004 by the UK National Institute for Biological Standards and Control [23] and recommended as suitable for use as a prototype by the CHMP [107]. The vaccine was produced on fertilized hen’s eggs according to the licensed manufacturing and testing process for the interpandemic trivalent split-virion influenza vaccine Fluarix®. The vaccine was formulated in GSK’s proprie-tary adjuvant system 03 (AS03). This adjuvant consists of a 10% (by volume) oil-in-water-based emulsion. The oil phase con-tained 5% dl-α-tocopherol and squalene, and the aqueous phase contained 2% non-ionic detergent polysorbate 80 (Tween 80).

This vaccine, with the trade name Prepandrix™, is approved in the EU for use as an active immunization against H5N1 subtype influenza virus in adults aged 18–60 years. The rec-ommended schedule in this population consists of two doses of 0.5 ml containing 3.8-µg H5 HA, administered 21 days apart. The vaccine presentation consists of a multidose vial (ten doses) with the antigen and a multidose vial (ten doses) containing the adjuvant. The content of both vials has to be mixed prior to administration. This two-vial format allows for the replace-ment of the antigen in case viral evolution creates this need. The stabilities of the antigen and adjuvant are estimated to be at least 3 years and studies are ongoing to evaluate whether longer storage periods can be achieved.

Safety & tolerability of the vaccineIn a dose-finding study [40] and in subsequent studies [41–44], pain at the injection site was the most frequently reported solic-ited local adverse event. The incidence of pain was significantly higher in those who received adjuvanted vaccine than in those who received nonadjuvanted H5N1 vaccine or the (nonadjuvan-ted) seasonal influenza vaccine. Local pain was rarely severe (in 0–3.7% of doses administered to adults between 18 and 60 years of age and in 0.5% of doses administered to persons aged over 60 years). Local swelling and redness at the injection site were also reported, but at a lower frequency. Fatigue, myalgia and headache were the most frequently occurring solicited general adverse events. Most local and general side effects were of mild or moderate severity and resolved within 48 h. Overall, the vaccine was well tolerated and its side effects were considered clinically acceptable [45].

ImmunogenicityIn an initial dose-finding study, two doses of vaccine contain-ing varying amounts (3.8, 7.5, 15 and 30 µg) of H5 HA for-mulated with and without AS03 were administered 3 weeks apart to eight groups of healthy adult volunteers aged between 18 and 60 years [40]. The adjuvanted formulations were sig-nificantly more immunogenic than the nonadjuvanted ones at all antigen doses. At the lowest antigen doses of 3.8-µg H5 HA, immune responses for the adjuvanted vaccine, measured as HI titers against the recombinant homologous vaccine strain (A/Vietnam/1194/2004 NIBRG-14, clade 1), met or exceeded all FDA and CHMP licensure criteria. None of the FDA criteria and only two out of three CHMP criteria were met with the nonadjuvanted vaccine and only at the highest dose of 30 µg H5 HA. Neutralizing antibody seroconversion (defined as a four-fold increase in neutralizing antibodies) against the homologous strain occurred in 86 and 22% of the vaccinees administered 3.8 µg of the AS03-adjuvanted and nonadjuvanted vaccine, respectively. Neutralizing antibodies against hetero logous strains were also measured to estimate the cross-protective potential of the candidate vaccine. Vaccination with two doses of 3.8 µg of the AS03-adjuvanted vaccine induced neutralizing seroconversion rates in 75, 85 and 75% of the subjects against A/Indonesia/5/2005 (subclade 2.1), A/turkey/Turkey/1/2005



(subclade 2.2) and A/Anhui/1/2005 (subclade 2.3), respec-tively [46]. Based on these results, the dose of 3.8 µg was selected for further development of the vaccine.

Subjects participating in the dose-finding study were followed up until 6 months after the first vaccination (day 180). The HI seroprotection rates against the homologous A/Vietnam strain for the adjuvanted 7.5- and 3.8-µg groups were 64.0 and 54.0%, respectively, which is lower than at day 42, but markedly higher than in the nonadjuvanted vaccine groups (14.3 and 4.0%) [47].

Several studies have been performed in larger adult popula-tions in Europe and Asia [41,42] and in pediatric populations in Europe [43,44]. The results of these trials, published as full papers or presented at recent conferences (abstracts), confirm the safety and immunogenicity data found in the dose-finding study.

During the dose-finding study, peripheral blood mononuclear cells were collected before vaccination and 3 weeks after the administration of the second dose, in order to study the cell-mediated immune (CMI) response to 3.8 and 7.5 µg of AS03-adjuvanted and nonadjuvanted vaccines. The frequency and functionality of H5-specific CD4+ T-cells were determined by intracellular cytokine staining after an overnight in vitro stimu-lation with a pool of peptides covering the entire H5 sequence or with the H5N1 split-vaccine preparation. Before vaccination, frequencies of H5-specific CD4+ T cells (expressed per million of CD4+ T cells) were not different between the groups. No antigen dose effect was detected on the CMI response in terms of influenza-specific CD4+ T cells at any time point. After two vaccine doses the frequencies were significantly higher in the adjuvanted 3.8-µg vaccine group (3050; 95% CI: 2340–4010) than in the nonadjuvanted one (1650; 95% CI: 1080–2160) [48]. The frequencies of CD4+ T cells specific for the conserved sequences between the clade 1 (A/Vietnam/1194/2004) and the clade 2.1 (A/Indonesia/5/2005) viruses were also higher in the adjuvanted group than in the nonadjuvanted group (430; 95% CI: 220–667 vs 150; 95% CI: 89–266) [48]. The anti-gen-specific CD4+ lymphocytes predominantly expressed IL-2 and CD40L, a marker profile consistent with that of memory T cells [48]. Vaccine strain-specific CD4+ T-cell responses per-sisted 6 months after the first vaccination and the difference between the adjuvanted and nonadjuvanted groups remained significant. The frequency of memory B cells, enumerated by B-cell ELISPOT method, was also significantly higher in the group that received the adjuvanted vaccine (p < 0.001) [48].

Challenge studies in the ferret modelThe protective efficacy of a prepandemic vaccine in human populations will only be demonstrated during a pandemic out-break. The best alternative to this undesired and unplanned experiment of nature is a challenge test in an appropriate ani-mal model. Ferrets are considered to be one of the best animal models to study infections with H5N1 influenza viruses [49–51]. Therefore, challenge tests were performed in ferrets that were vaccinated with different vaccine doses or controls [52,53]. In a first experiment designed to test protection against homologous

challenge, four groups of six ferrets were immunized intra-muscularly with two doses of 15-, 5-, 1.7- and 0.6-µg HA of inactivated split A/Vietnam/1194/04 NIBRG-14 vaccine adju-vanted with AS03 [52]. Control groups consisted of six ferrets treated with either phosphate-buffered saline (PBS) or AS03 only. Animals were vaccinated on days 0 and 21 and challenged intratracheally on day 49 with a lethal dose of the homologous virus (105 TCID

50). Most animals (20 out of 23) immunized

with adjuvanted vaccine survived to the end of the observation period (5 days after the lethal challenge); whereas, most animals in the control groups (ten out of 11 ferrets) died prematurely as a consequence of the infection. All animals receiving PBS or adjuvant alone exhibited high viral load (>105 TCID

50/g of lung

tissue) and shed high levels of virus in the pharynx throughout the course of the infection. Administration of adjuvanted vac-cine reduced the viral load in the majority of the animals below the threshold of 102 TCID

50/g lung tissue or per ml of pharyn-

geal fluid. A vaccine dose effect was observed in the levels of HI titers induced that correlated with the level of protection [52].

A second experiment was set up to assess whether the AS03-adjuvanted, split A/Vietnam/1194/04 NIBRG-14 (clade 1) vaccine also induced cross-clade protection against the A/Indonesia/5/2005 (clade 2.1) strain [53]. Four groups of six ferrets were immunized intramuscularly with two doses of 15-, 7.5-, 3.8- or 1.7-µg HA of A/Vietnam vaccine adjuvanted with AS03. The two control groups each included six ferrets administered with either AS03 alone or the nonadjuvanted vac-cine containing 15-µg HA. Animals were vaccinated on days 0 and 21 and challenged intratracheally on day 49 with a lethal dose of wild-type A/Indonesia/5/2005 virus (105 TCID

50). All

animals that received two doses of at least 3.8 µg of the adjuvan-ted vaccines survived the lethal heterologous challenge. All but one animal that received the lowest 1.7-µg dose of adjuvanted vaccine survived the challenge. By contrast, all control animals (12 out of 12) died or were moribund and were euthanized on days 3 or 4 [53]. Serological assays showed that the adjuvanted A/Vietnam vaccine formulations induced neutralizing anti-body responses (response defined as titers ≥ 1:28) against the homologous A/Vietnam strain in 17 out of 23 ferrets (74%) and against the heterologous A/Indonesia strain in 14 out of 23 ani-mals (61%). Neutralizing responses against the homologous as well as the heterologous virus were absent in the control groups. All animals without a detectable neutralizing antibody response to A/Vietnam or A/Indonesia exhibited a viral load of greater than 8 × 103 TCID

50/g tissue; whereas, 94% (16 out of 17) of

ferrets with anti-A/Vietnam neutralizing anti bodies and 93% (13 out of 14) with anti-A/Indonesia responses showed virus loads in the lungs below the detection limit of 102 TCID

50/g

tissue. A total of 17 out of 22 animals (77%) that survived until the termination of the experiment had a neutralizing antibody response. Four of the five nonresponder animals that were pro-tected from mortality had lower viral loads in the lung than unvaccinated control animals, suggesting a possible role for vaccine-induced cellular immune responses in the control of viral replication [53].



Omninvest (Hungary)Pharmaceutical properties & formulation of the vaccineOmninvest produced a vaccine using the A/Vietnam/1194/2004 NIBRG-14 reference strain. The vaccine was made by essen-tially the same method as the yearly interpandemic influenza vaccine Fluval AB used in Hungary for the past 11 years. The egg-grown virus was inactivated by formaldehyde treatment. One vaccine dose consisted of a 0.5-ml ampoule containing 6-µg HA formulated with 0.31-mg AlPO

4 as adjuvant and

methiolate at 0.1 mg/ml as preservative. This product received a final marketing authorization from the National Institute of Pharmacy (Hungary) in September 2007.

Safety & tolerabilityIn a first clinical study, in which 146 healthy adults with a mean age of 42 years were given a single dose of the candi-date pandemic vaccine (6-µg HA), 15.7% of the participants reported adverse reactions in the form of local pain at the injec-tion site that occurred within the first 48 h [54]. These reactions disappeared within 1 day and were not different from those observed upon administration of Omninvest’s interpandemic vaccine. In 12 healthy children (mean age ± standard deviation: 12.73 ± 2.77 years) that were administered one dose of the 6-µg HA vaccine no side effects were detected [55].

ImmunogenicityThe immunogenicity of Fluval was measured using HI and MN assays. In the adult population the following values were reached 21 days after the administration of a single vaccine dose: sero-conversion and seroprotection rates of 63.7%; and geometric mean titer ratio of 5.6 [54]. In the pediatric population a single vaccine dose elicited the following responses: seroconversion and seroprotection rates of 75%; and geometric mean titer ratio of 16.95 [55]. These studies demonstrate that the administration of a single dose of Omninvest’s inactivated whole-virus vaccine induces responses that meet two out of three CHMP criteria in adults [54] and all three criteria in children [55].

Cross-reactivity of antibodies induced with a single dose (6 µg) of the H5N1 (NIBRG-14) vaccine in adults (18–65 years of age; n = 44) and elderly (>65 years of age; n = 44) was tested against the following H5N1 strains: rg A/Anhui/1/2005-PR8-IBCDC-RG5, rg A/Bar headed goose/Qinghai/1A/05 and A/Swan/Nagybaracska/01/2006-like A/PR8/34 reassor-tant; and the non-H5N1 influenza virus strain A/Solomon Island/13/2006 (H1N1)-like IVR-145 reassortant [56] . Antibody titers were measured by HI and MN assays. A total of 3 weeks after the administration of a single vaccine dose in the adult group, the HI seroconversion rates against Anhui, Qinghai, Nagybaracska and Solomon were 56.8, 31.8, 40.9 and 18.2%, respectively. In the elderly group, these HI sero-conversion rates were 50.0, 45.5, 61.4 and 56.8%, respectively. HI GMT ratios against these four virus strains were 7.28, 4.19, 3.31 and 2.78 in adults and 5.07, 5.48, 6.22 and 6.73 in elderly individuals. Neither in adults nor in the elderly, the required criterion for seropositivity rate was met for any of these four

strains. The MN seroconversion rates against Anhui, Qinghai, Nagybaracska were 45.5, 27.3 and 36.4, respectively in adults and 34.1, 31.8 and 13.6, respectively, in elderly individuals [56].

The effects of a second dose of vaccine on the magnitude and the persistence of the response have not been assessed. To our knowledge, no protection studies in animals have been performed.

Novartis V&D (Italy)Chiron Corporation, acquired by Novartis International AG in 2006, was one of the first companies to start the development of candidate vaccines against highly pathogenic avian influenza viruses. Their first candidate vaccine used the influenza strain A/Duck/Singapore-Q/F119–3/97 (H5N3) that was nonpatho-genic to birds and antigenically similar enough to human and avian A/Hong Kong/97 (H5N1) to be tested. In June 1999, a Phase I trial was undertaken to examine the safety and immuno-genicity of two injections (3 weeks apart) of escalating doses of a monovalent surface antigen A/Duck/Singapore/97 vaccine, administered without and with the adjuvant MF59 to 65 healthy adults aged between 18 and 40 years [24]. A few years prior to this, Martin demonstrated that MF59 adjuvant enhanced the immune response to influenza vaccine in the elderly [57]. This H5 vaccine study showed that nonadjuvanted surface antigen A/Duck/Singapore/97 vaccines at doses of 7.5, 15 and 30 µg were poorly immunogenic and unlikely to convey protection against A/Hong Kong/97. Adjuvantation with MF59 enhanced the immune response against the vaccine, measured by HI, MN and SRH. Two doses of 7.5 µg with MF59 induced the highest SCR and the antibody titers measured against the heter ologous virus A/Hong Kong/97 were approximately half of those against the vaccine strain [24]. MF59-adjuvanted vaccines were well tolerated but induced more frequent moderate and severe pain reactions at the injection site than nonadjuvanted vaccines [24]. A total of 16 months later, a follow-up study was performed to assess the effect of H5N3 revaccination on a primed immune system [26]. Subjects (26 of the original 65) were administered one injection of the same vaccine formulation and dose that they had origi-nally received and serum was examined for anti bodies against A/Duck/Singapore/97 (HI, MN and SRH) before and 21 days after the booster dose. A total of 16 months after the two-dose priming, GMT of HI, MN, and SRH antibodies had dropped to prevaccination titers. A total of 21 days after revaccination with MF59-adjuvanted vaccines, strong anamnestic responses were observed, with antibody titers that largely exceeded those measured on day 42 (21 days after the second dose of the prim-ing series). MN and SRH titers were distinctively higher than HI titers. SRH titers for the heterologous H5N1 were approxi-mately half of those for the homologous H5N3. Nonadjuvanted vaccines induced very weak anamnestic responses in MN and SRH assays only. This study confirmed the immune enhancing and potentially dose-sparing effects of MF59 adjuvant in com-bination with a surface antigen influenza vaccine [26]. Serum samples from the 26 subjects that had received three doses of A/Duck/Singapore/97 (H5N3) vaccine (15 subjects received



MF59-adjuvanted vaccine and 11 subjects received nonadjuvan-ted vaccine) were examined by virus MN assay against highly pathogenic avian influenza H5N1 viruses isolated from humans between 1997 and 2004 [58]. Three doses of nonadjuvanted A/Duck/Singapore/97 surface antigen vaccine were poorly immunogenic even against the closely related HK/156/97-like strains. Adjuvantation of the vaccine with MF59 significantly enhanced the immune responses and induced broadly cross-reactive antibodies able to neutralize antigenically distinct HPAI H5N1 viruses. SCR (defined as MN titer of ≥1:80) to A/Hong Kong/156/97, A/Hong Kong/213/03, A/Thailand/16/2004 and A/Vietnam/1203/2004 were 100, 100, 71 and 43% in 14 sub-jects that received MF59-adjuvanted vaccine, respectively, com-pared with 27, 27, 0 and 0% in 11 individuals who received nonadjuvanted vaccine [58].

MF59 adjuvant has also been combined with a subunit influenza A/H9N2 vaccine [59]. To examine the safety and immunogenicity of a two-dose schedule (administered intramuscularly, 4 weeks apart) of four dose levels (3.75-, 7.5-, 15- and 30-µg HA) of inac-tivated influenza A/chicken/Hong Kong/G9/97 (H9N2) vaccine with and without MF59, 96 healthy adults (aged 18–34 years) were enrolled and randomly assigned to one of the eight study groups [59]. Geometric mean titers (GMT) for HI and MN anti-bodies to the vaccine strain for all vaccine groups were similar prior to vaccination but were significantly higher on days 28 and 56 for the MF59-adjuvanted vaccine groups than for those receiving the nonadjuvanted vaccine. There was no evidence of an antigen dose effect. In addition, the HI and MN GMT after administration of a single dose of MF59-adjuvanted vaccine were similar to those measured after two doses of nonadjuvanted vaccine [59]. Although this study showed that two injections of 3.75-µg HA induced a satisfactory humoral immune response, the formulation of the final candidate vaccine does not use such low antigen content.

In 2006, a multicenter, randomized, double-blind, placebo-controlled trial was conducted in 394 healthy adults to evalu-ate the safety and immunogenicity of varying doses of nonad-juvanted, Al(OH)

3-adjuvanted and MF59-adjuvanted influenza

A/Vietnam/1203/2004 (H5N1) vaccine [60]. MF59 increased the HI and MN responses, whereas Al(OH)

3 did not. The highest

antibody responses were measured in the group that received 15 µg of vaccine with MF59, in which 63% of subjects reached a HI titer of at least 1:40 after the second dose. In the groups that received 45-µg nonadjuvanted and 30-µg Al(OH)

3-adjuvanted vaccine, 29

and 14%, respectively, achieved a HI titer of at least 1:40. The vaccine formulations were well tolerated but local adverse effects were common and increased in a dose-dependent manner and by the addition of adjuvant.

Recently, Stephenson and colleagues administered two doses of 7.5-µg HA of clade 1 influenza A/Vietnam/1194/2004 NIBRG-14 adjuvanted with MF59, intramuscularly, 3 weeks apart, to subjects who had been primed with clade 0 H5 vaccine at least 6 years ear-lier [61]. These 24 subjects had received two doses of either MF59-adjuvanted or nonadjuvanted (plain) A/Duck/Singapore/97 (H5N3) vaccine containing 7.5–30-µg HA in studies summa-rized above [24,26]. Some subjects had also received a booster dose

16 months after the primary immunization [26]. A total of 30 sub-jects were unprimed. Serum samples were obtained immediately before each of the two vaccine administrations (days 0 and 21) and on days 7, 14, and 42 after vaccination. Antibody responses were measured with HI and MN assays using the homologous clade 1 NIBRG-14 and the heterologous clade 2.2 NIBRG-23 vaccine-reference strains. On each postvaccination timepoint and with each assay, GMT of antibodies to NIBRG-14 and NIBRG-23 were significantly higher among the primed subjects than among the unprimed. From day 14 onward, and for both assays, titers of antibodies were significantly higher in the MF59-primed group than in the plain primed group. The highest titers were measured on day 14 in the MF59-primed group, with GMT of HI anti-bodies to NIBRG-14 and NIBRG-23 of 378 and 347, respectively, and GMT of MN antibodies of 1754 and 2128, respectively. The number of preceeding H5N3 vaccine doses or the antigen content of these vaccines had an effect on the postvaccination titers. This study clearly shows that priming subjects with MF59-adjuvanted H5 HA antigen induces a long-lasting immune memory that can be rapidly mobilized and effectively boosted by the administration of a low-dose, antigenically distinct vaccine. It supports the idea of administering prepandemic vaccines to selected high-risk popula-tions (healthcare workers) or less-selected populations to prime their immune systems in order to evoke more rapid, protective immune responses in case of a pandemic outbreak.

Pharmaceutical properties & formulation of the vaccineBased on the data summarized above and results of clinical trials that have not yet been published as full papers, Novartis has devel-oped a pandemic mock-up vaccine, named Focetria®, and a pre-pandemic vaccine, named Aflunov®. The former was approved by CHMP and granted a marketing authorization under exceptional circumstances in the EU in May 2007. This mock-up vaccine is based on the studies using the afore mentioned H5N3, H9N2 and H5N1 vaccines. The CHMP has judged that the application of Aflunov for use in the prophylaxis of H5N1 avian influenza in adults and elderly was not approvable and a recommendation of a marketing authorization could not be granted because of a major objection related to the conduct of a pivotal clinical trial [115]. Each 0.5-ml dose of Focetria as well as Aflunov contains 7.5-µg HA of the NIBRG-14 influenza strain, adjuvanted with MF59C.1. The vaccine virus was grown on embryonated eggs using a process similar to that of seasonal vaccine production. MF59C.1 is an oil-in-water emulsion that contains squalene, polysorbate 80, sorbitan trioleate, citric acid monohydrate, and sodium citrate dehydrate in water [62]. This proprietary adjuvant has a proven safety and tolerability since it is used in the commercially available seasonal influenza vaccine Fluad of which more than 40 million doses have been distributed worldwide since 1997 [63,64].

ImmunogenicityThe results of the early dose-finding studies with H5N3 and H9N2 pilot vaccines (described above) were the basis for the design of Aflunov [24,26,58,59]. More recently, three studies have been conducted with the final candidate H5N1 vaccine [115].



A small Phase II study (V87P2) in adults aged below 60 years investigated the cellular immune responses induced by two injec-tions of 7.5 µg (n = 14) and 15 µg (n = 13) of MF59-adjuvanted H5N1 vaccine and 15-µg nonadjuvanted comparator (n = 13). In each group, a booster dose was administered 6 months later. The frequency and functionality of H5-specific CD4+ T cells was determined by intracellular cytokine staining after a short in vitro pulse, with a pool of peptides covering the entire H5 sequence or with the H5N1 antigen preparation [65]. MF59-adjuvanted H5N1 vaccines enhanced both neutralizing antibody and CD4+ T-cell responses to H5N1. Both MF59-adjuvanted vaccines primed H5-specific CD4+ T cells with a Th1 effector/memory phenotype. The rise in H5-specific CD4+ T-cells could already be detected after the first vaccine dose and remained above pre-immune levels at day 223. These cells were further expanded after the booster dose administered at 6 months [65].

Recently, data have been published from a study that encom-passed 313 adults aged between 18 and 60 years and 173 adults over 60 years of age [66]. These participants were randomized (1:1) to receive two primary and one booster injection of 7.5 or 15 µg of a subunit MF59-adjuvanted H5N1 (A/Vietnam/1194/2004), clade 1) vaccine. The humoral responses, measured as HI, SRH and MN titers, to 7.5- and 15-µg doses were comparable. The rates of seroprotection (HI ≥ 1:40, SRH ≥ 25 mm2) and MN of at least 1:40 ranged between 72 and 85%. A total of 6 months after primary vaccination with the 7.5-µg dose, 27 and 54% of the nonelderly and elderly adults were seroprotected (HI); HI rates increased to 83 and 92%, respectively, after the booster vaccination. In the 15-µg group, 6 months after primary vac-cination, the HI seroprotection rates among the nonelderly and elderly adults increased from 34 and 62% to 76 and 96% after booster vaccination, respectively. After the second dose of the pri-mary vaccination and the booster dose, a heterologous immune response against H5N1 A/turkey/Turkey/1/05 (NIBRG-23) was detectable, indicating the crossreactivity of the antibodies induced with the A/Vietnam-based vaccine. The CHMP criteria for vaccine licensure were met with the lower (7.5 µg) dose tested.

Challenge studies in ferretsAlthough two ferret challenge studies with an influenza virus homologous to the vaccine virus have been performed, no data are yet available [115].

Safety The studies discussed above have provided safety data in indi-viduals below 60 years and subjects older than 60 years of age. Overall, the reactogenicity of the MF59-adjuvanted H5N1 vac-cine is comparable to that of Fluad. After the first vaccine dose, more frequent reactions are observed in younger adults than in older ones (68 vs 50%, respectively). Most solicited reactions were mild or moderate and rarely lasted longer than a few days. Severe reactions were less frequent with the adjuvanted H5N1 vaccine than with Fluad (3 vs 7%, respectively; p < 0.001) after the first dose. Severe reactions occurred less frequently (1–2%) after the second dose and no difference was noted between

younger and older vaccinees. For both age groups, local reac-tions were reported more frequently than systemic reactions, as well as for the adjuvanted H5N1 vaccine or Fluad. No severe adverse events related to the study vaccines were reported [66,115].

Sanofi Pasteur (France & USA)Sanofi Pasteur has been committed to global pandemic prepared-ness and has responded quickly to the guidelines for the fast-track licensing of pandemic influenza vaccines developed by the CHMP. These recommended the development of a mock-up pan-demic vaccine to be submitted for regulatory approval in the form of a core pandemic dossier during the interpandemic period [107].

Pharmaceutical properties & formulation of the vaccineIn the past 5 years, Sanofi Pasteur has developed candidate pan-demic vaccines that were evaluated in a series of consecutive clini-cal trials. Initially the immunogenicity was tested of a two-dose regimen of nonadjuvanted subvirion influenza A vaccine generated from the human H5N1 isolate A/Vietnam/1203/2004. Although this vaccine induced modest responses even at the highest dose tested (90 µg), it has been approved by the FDA for use in case of a pandemic [67]. Another clinical trial revealed that adjuvanta-tion with Al(OH)

3 of a candidate H5N1 influenza vaccine based

on the human isolate A/Vietnam/1194/2004 had only a limited immune-enhancing effect [68]. The adjuvanted 30-µg formula-tion induced the greatest response and alum-adjuvantation did not improve the response to the lower doses (7.5 and 15 µg) [68]. Aware of the need for antigen dose-sparing and the shortcomings of its alum-adjuvanted formulations, Sanofi Pasteur developed a novel oil-in-water adjuvant (adjuvant formulation 03 or AF03) and evaluated its effect on the immune responses induced by varying doses of vaccine [69]. The investigational vaccines were monovalent, inactivated split-virion vaccines using the NIBRG-14 reassortant H5N1 reference strain [23]. This virus was propagated in embryo-nated chicken’s eggs, using the licenced manufacturing process for the interpandemic vaccine Vaxigrip® [70]. The adjuvant produced by Sanofi Pasteur was a 5% squalene-in-water emulsion stabilized by two nonionic surfactants. This emulsion was prepared to have a fine (mean particular diameter < 100 nm) and monodispersed emulsion, with a narrow particle size distribution. Vaccine doses were prepared just before injection by mixing vaccine antigen from multidose vials and adjuvant from monodose vials [69].

Safety & tolerability of the vaccinesThe nonadjuvanted influenza A/H5N1 vaccine induced more local side effects than a placebo and these increased with increas-ing doses. Pain and tenderness at the injection site were most common and generally mild to moderate. Systemic adverse events were dose independent and occurred less frequently than local ones. Headache was the most frequently reported systemic adverse event. No laboratory abnormalities were observed [67]. Addition of Al(OH)

3 increased the incidence of local but not systemic adverse

reactions. Pain at the injection site and headache were most com-mon and generally mild to moderate. No vaccine-related serious adverse events and no fever of at least 38.0°C were reported [68].



Formulation of the H5N1 vaccine with an oil-in-water adjuvant induced injection site pain in almost all (≥94%) subjects after the first vaccination and in 20–48% this pain was reported as moderate (interfering with normal daily activity) [69]. Local pain was reported by 48% of the subjects in the nonadjuvanted control group [69], a frequency similar to that observed in the nonadju-vanted vaccine study [67]. Injection site erythema, swelling and induration, but not ecchymosis, were also more frequent with adjuvant than without [69]. Local reactions were less frequent after the second dose. The most frequently reported general symptom was headache, which occurred in approximately 40 and 30% of the vaccinees after the first and the second dose, respectively. There was no trend for higher reactions with higher antigen dose and the majority (86.4%) of solicited local and general reactions lasted 1–3 days. No vaccine-related serious adverse events occurred [69].

ImmunogenicityTwo 90-µg doses of a nonadjuvanted inf luenza A/Vietnam/1203/2004 (H5N1) vaccine induced neutralizing antibody titers of at least 1:40 in 53% of the vaccinees and HI titers of at least 1:40 in 57%. Neutralizing antibody titers of at least 1:40 were observed in 41, 20 and 7% of the subjects receiving two doses of 45, 15 and 7.5 µg, respectively [67]. These responses did not meet FDA or CHMP criteria defined for sea-sonal influenza vaccines. Adding Al(OH)

3 to an investigational

influenza A/Vietnam/1194/2004 (H5N1) vaccine improved the immune responses to the 30- and 45-µg doses but had no effect on the lower doses (7.5 and 15 µg) [68]. A first dose of the vaccine induced HI titers of at least 1:32 in 6% (7.5 µg plus Al) to 34% (30 µg) of the vaccine recipients and these responses rose to 28% (7.5 µg plus Al) and 67% (30 µg plus Al) 21 days after the second dose [68]. The highest responses were observed after the second vaccination with adjuvanted 30-µg HA. Only this formulation induced a HI SCR in excess of 60% after two vaccinations. In this study, the 1:40 dilution of sera was not actually tested and the proportion of participants that might have reached a titer of at least 1:40 was calculated. These calculated numbers were slightly lower than those effectively measured for titers of at least 1:32 and were, for example, 63% (instead of 67%) in the adjuvanted 30 µg group and 39% (instead of 43%) in the non adjuvanted 7.5 µg group. Neutralizing antibody responses followed a similar pattern to those of HI. On day 42 (21 days following the second dose), the proportion of vaccinees in the nonadjuvanted 7.5-, 15-, and 30-µg groups with MN titers of at least 1:40 were 20, 22 and 27%, respectively. In the adjuvanted groups, these numbers were 16, 18 and 41%. As with HI, the neutralizing response elicited by the non adjuvanted 7.5 µg formulation was similar to that elicited by the two 15-µg formulations and was higher than that elicited by the adjuvanted 7.5-µg vaccine [68].

Cross-neutralizing capacity of antibodies elicited during this study was tested on postvaccination sera in MN assays against highly pathogenic, wild-type clade 2 H5N1 strains isolated from human cases and their corresponding reverse-genetics-derived strains [71]. This survey led to some interesting findings. When tested against the homologous strain A/Vietnam/1194/2004,

MN antibody titers were higher against the wild-type virus than against the reverse genetics NIBRG14 strain and within the 63 NIBRG14-seronegative samples tested against the wild-type virus, 65% were also able to neutralize the virus. Of the 127 NIBRG14-seropositive samples, 35% were also able to cross-neutralize the clade 2.2 NIBRG23 strain and 80% of these samples neutralized the parental wild-type A/turkey/Turkey/1/2005 strain. Of the 45 NIBRG14-seropositive samples tested, three were able to neutral-ize the reverse-genetics clade 2.1 A/Indonesia/5/2005 CDCRG2 strain, whereas 29 out of 45 were able to neutralize the parental wild-type A/Indonesia/5/2005 strain. Antibodies induced by alum-adjuvanted (and to a lesser extent nonadjuvanted) clade 1, NIBRG14-derived HA are able to cross-neutralize clade 2.1 and, even more so, clade 2.2 strains (wild-type virus better than reverse-genetics strains). These data suggest that the use of reverse genet-ics viruses in neutralization assays may underestimate the extent of crossprotective antibodies present in vaccine-induced sera. It further emphasizes the need for further studies to extend our knowledge on correlates of immune protection and the need for standardization of assays and international standard reagents [71].

The safety, humoral and CMI responses of two formulations of an inactivated, split-virion influenza A/H5N1 vaccine were also tested in children [72]. In a Phase II open, randomized, multi-center trial, 180 children aged 6 months–17 years received two doses, 3 weeks apart, of a vaccine containing either 30-µg HA with adjuvant (30 µg plus Al) or 7.5-µg HA without adjuvant. An additional 60 children aged 6–35 months were administered two half-dose injections (i.e., 15 µg plus Al or 3.8 µg). Antibody responses were measured 21 days after each dose. The 30 µg plus Al formulation was more immunogenic than 7.5 µg in all age groups and in the 6–35-month-olds, the full doses were more immunogenic than their half-dose equivalents. Vaccination induced a predominant Th2 response against H5 HA [72].

The results of a clinical trial evaluating Sanofi Pasteur’s emulsion-adjuvanted pandemic influenza vaccine were published recently [69]. In this multicenter, randomized, blind-observer Phase I trial, groups of 50 healthy adult (18–40-year-old) volunteers received two doses, 21 days apart, of influenza A/Vietnam/1194/2004 NIBRG-14 (H5N1) vaccine containing 1.9, 3.8, 7.5 or 15 µg of HA with oil-in-water adjuvant or 7.5 µg of HA without adjuvant. One vaccination, with or without adjuvant, induced significant (seroconversion or fourfold rise) HI and neutralizing responses in 22–40% of sub-jects. A second vaccination with adjuvant substantially increased the response in all four groups. The 1.9-µg formulation was the least immunogenic but even this low dose induced HI responses that met the three CHMP criteria (SPR: 72%, SCR: 72% and GMTR: 11.0) and MN response (≥fourfold increase from day 0 to 42) in 92% of subjects [69]. Adjuvanted 3.8-, 7.5- and 15-µg formulations induced stronger responses, with HI SPRs and SCRs of 81, 89 and 86%, respectively, and significant MN responses (≥fourfold increase from day 0 to 42) in 88, 92 and 96%, respectively. Immunogenicity of adjuvanted vaccines increased from 1.9 to 7.5 µg, but increasing the dose to 15 µg did not further enhance the immunogenicity [69]. Crossreactivity of the induced anti bodies was tested in HI and MN assays using the clade 2 strain A/Indonesia/5/2005 RG2. HI titers of



at least 1:32 against the heterologous clade 2 strain were observed in 4–23% of the recipients of the adjuvanted vaccines versus 72–89% against the vaccine strain. For MN titers of at least 1:20, these values were 18–43 and 88–96%, respectively [69]. The persistence of the antibodies beyond day 42 is currently being evaluated.

Challenge study in macaquesMacaques were vaccinated with adjuvanted, formaldehyde-inacti-vated split-virion vaccines against influenza A/Vietnam/1194/2004 (H5N1) to investigate, in the first place, whether this vaccine might induce more severe disease in animals upon subsequent natural homologous infection, in a manner analogous to that observed with aluminum-adjuvanted RSV investigational vaccines, and to also examine whether these H5N1 vaccines could protect against infection [73]. Groups of eight cynomolgus monkeys received a monovalent H5N1 vaccine containing 30 µg per dose of HA of the influenza virus A/Vietnam/1194/2004 NIBRG-14 reassortant vaccine strain, without adjuvant, with Al(OH)

3 or with the oil-

in-water adjuvant AF03. A fourth group received PBS control. Two doses were injected in the quadriceps muscle, 27 days apart. A total of 3 months later, the animals were challenged with 106 TCID

50 of the homologous wild-type virus. Four animals per

group were sacrificed 5 and 15 days after challenge. No signs of vaccine-induced disease exacerbation were noted. The adjuvanted vaccines induced HI antibodies against the vaccine strain and a heterologous clade 2 strain (A/Indonesia/5/2005 CDCRG-2) and protected the lungs and upper respiratory tract. Histopathological signs of pneumonia were less severe and lung tissue and pharyngeal swabs contained less virus. Without adjuvant, partial protection was induced. Best results were obtained with the oil-in-water adju-vant [73]. In another challenge experiment, the ability of an adju-vanted A/Vietnam/1194/04 NIBRG14 H5N1 vaccine to protect ferrets against mortality and disease was evaluated [74]. Four groups of six ferrets received two intramuscular vaccinations, 4 weeks apart, with either 1.9-, 3.8-, 7.5- or 15-µg HA adjuvanted with the AF03 oil-in-water emulsion. Four more groups of six ferrets received 3.8-, 7.5-, 15- or 30-µg HA without adjuvant. Six control animals received saline or adjuvant alone. A total of 2 months later, ferrets received an intranasal challenge of 103 TCID

50 of wild-type

influenza A/Vietnam/1194/04 strain and surviving animals were sacrificed 14 or 21 days later. The adjuvanted vaccine formulations induced high HI titers against the vaccine strain, which strongly cross-reacted against the clade 2 Indonesia/5/05 strain. In the con-trol group, five out of six ferrets died within 6 days of wild-type viral challenge; in contrast, all vaccinated animals survived the challenge. As compared with animals receiving nonadjuvanted product, ferrets vaccinated with adjuvanted vaccine, even at very low dose (1.9-µg HA) showed fewer clinical signs of disease, shed less virus in nasal samples and had fewer and milder lesions in the lungs upon histopathological examination [74].

Sinovac (China)Sinovac Biotech Ltd, a China-based biopharmaceutical company that focuses on R&D, manufacturing and commercialization of vaccines against hepatitis A virus, combined hepatitis A and B

virus, influenza, pandemic influenza and Japanese encephalitis, has developed an inactivated whole-virion influenza (H5N1) vaccine adjuvanted with Al(OH)

3.

Pharmaceutical properties & formulation of the vaccineThe vaccine strain was the NIBRG14 strain prepared by the NIBSC [23]. The vaccine was produced on embryonated chicken’s eggs and then concentrated and purified by chromatography. Based on pre-clinical tests in mice and rats that showed the need for adjuvanta-tion, the vaccine was formulated with Al(OH)

3 to contain a final

concentration of aluminum of 0.5 mg/ml [75]. A Phase I clinical trial has been completed and published and a Phase II trial aiming at the evaluation of the safety of different doses and schedules has been executed but no final dosage had yet been defined [75].

Safety & tolerability of the vaccinesThe safety of the Sinovac candidate H5N1 vaccine was evaluated via a Phase I, monocentric, stratified, placebo-controlled, double-blind clinical trial encompassing 120 healthy individuals aged between 18 and 60 years [75]. Groups of 24 subjects were ran-domly assigned to receive two intramuscular injections, 28 days apart, of 1.25-, 2.5-, 5- and 10-µg HA or placebo. All vaccine formulations were well tolerated and no serious adverse events were reported between day 0 and 56 (end of follow-up). Most of the local (pain and swelling at the injection site) and systemic (fever, headache, myalgia and nausea) symptoms were mild and recovered within 72 h. No antigen dose effect was apparent [75].

ImmunogenicityThe immune response to the vaccine was measured with HI and MN assays. With the HI assay, antibody responses against H5N1 were seen in all four vaccine groups 14 days after the first dose. The highest antibody response after two doses was measured in the 10 µg group where 78% of subjects had HI titers of at least 1:40. Lower doses induced lower antibody responses in a clear dose-dependent manner. Responses that met the CHMP criteria for seasonal influ-enza vaccines were measured only in the 10-µg group at day 42 (2 weeks following the second dose) [75]. Serum from 88 subjects that had participated at this study were collected 6 months later and serum from 57 of these 88 were collected at 12 months [76]. These 57 subjects were then administered a third dose of the same vaccine they had received for priming. Serum samples were col-lected 15 and 30 days after the third dose to be tested by HI and MN assays against the homologous NIBRG-14 vaccine strain. A total of 6 and 12 months after the two-dose priming with alum-adjuvanted H5N1 inactivated whole-virion vaccine the HI antibody titers had declined substantially. A total of 12 months after the prim-ing both HI and MN GMT were statistically similar in the four groups and compared with the GMT measured 4 weeks after the 2-dose priming had declined 18.1–52.8% for HI and 25.8–67.1% for MN (of the starting value). Combining the four groups, 14.8% (13 out of 88) and 8.8% (five out of 57) had seropositive HI titers 6 and 12 months after priming, respectively. The third dose boosted immune responses in a dose-dependent manner in the four groups. A total of 15 days after the third dose, HI GMT was 12.8–65 and



30 days after the booster HI GMT of 13.9–52.8 was reached. Both the 5- and 10-µg groups met all three CHMP criteria at 15 and 30 days after boosting. MN responses showed a similar response pattern and MN GMT were generally higher than HI GMT. A total of 15 and 30 days after the third dose, 41.2–100% and 52.9–100% of the participants of the four groups had MN titers of at least 1:40, respectively [76]. This study shows that antibody titers induced by two doses of Sinovac’s candidate vaccine are not very durable and require a third dose to return to seroprotective levels.

A Phase II trial to evaluate the safety and immunogenicity of varying doses (5-, 10- and 15-µg HA) of Sinovac’s inactivated whole-virion H5N1 vaccine, administered via a 0-, 4- and a 0-, 2-week scheme, is ongoing in 402 healthy volunteers aged between 18 and 60 years. The vaccine was approved by the Chinese authori-ties in April 2008. Cross-reactivity data and results from challenge studies in experimental animal models are not available.

We realize that the present overview provides only a limited selection of the countless and intense efforts that are ongoing to develop pandemic and prepandemic vaccines and strategies to prevent or contain the next pandemic outbreak. Our selection has been based on the availability of published data and the advanced stage of the products discussed. We are well aware that numerous vaccine-development efforts have not been given the attention they deserve. We did, for example, not mention the baculovirus-recombinant HA antigen vaccines that, similar to the cell-cul-ture vaccines, hold promise for large-scale production [77,78]. In addition, we did not discuss the vaccines that target other viral

antigens, such as matrix proteins, and other projects in search of a universal influenza vaccine [79,80]. We hope, however, that these projects may mature successfully and soon become useful tools in the global fight against influenza in general and the pandemic threat in particular.

Expert commentary & five-year view

• Within 5 years, a pandemic may have struck the world and this experience will teach us whether the preparedness plans have been successful and whether the developed vaccines and vaccination strategies have been efficacious;

• As immunogenicity of vaccines increases and production capac-ity grows, one can be more confident that sufficient vaccines can be produced rapidly enough to protect the global population;

• Today’s vaccine development and clinical evaluation has mainly focused on the adult population aged between 18 and 60 years. Efforts are ongoing and will be pursued to evaluate the safety and efficacy of (pre)pandemic vaccines in the most vulnerable populations, namely the very young (6–24-month-old indi-viduals), the elderly (≥60 years of age) and patients with chronic diseases;

• The urgent calls to prepare for the next pandemic have fueled all aspects of influenza virus research and vaccine development, in particular, and will most likely continue to do so for at least another decade;

Key issues

• The WHO has designed a preparedness plan to respond to the threats and occurrence of pandemic influenza.

• The highly pathogenic influenza A (H5N1) virus is considered the most likely agent to cause the next pandemic. However, other subtypes, such as H2, H7 and H9 should not be left out of sight.

• The development of (pre)pandemic vaccines and strategies to produce, stockpile, distribute and administer these vaccines have been considered as high-priority issues.

• To facilitate the approval of pandemic vaccines, the EMEA has developed a novel approach that allows a mock-up vaccine to be authorized in advance of an actual pandemic outbreak.

• The development and production of (pre)pandemic vaccines are immensely challenging because: – The ultimate causative agent is not known

– The timing of the pandemic outbreak cannot be predicted

– The world needs 13 billion vaccine doses

• Ideally, (pre)pandemic vaccines are safe and well tolerated and induce strongly protective immune responses that are broadly cross-reactive, highly persistent and can be elicited with a small amount (a fraction of the standard 15 µg) of antigen.

• Adjuvants are needed to convey these unique qualities to split and subunit influenza vaccines. Whole-virus vaccines appear to be endowed with these qualities without the use of adjuvants.

• Oil-in-water emulsions provide the best results when added to subunit and split influenza vaccines and convey increased immunogenicity, better persistence of antibody response, dose-sparing capacity and cross-reactivity.

• Whole-virus vaccines are more immunogenic than nonadjuvanted split or subunit vaccines.

• The mock-up vaccine exercise has led to the development of so-called prepandemic vaccines that combine a series of unique qualities that make these appropriate for usage in earlier phases, before an actual pandemic has struck.

• The lack of standardization of the assays to measure the magnitude and quality of the humoral immune response to influenza antigens makes comparisons between different vaccine candidates and clinical trials impossible.

• Administration of one or two doses of prepandemic vaccines during the pandemic alert period (phases 3–5) is considered in order to prime selected groups, such as healthcare workers and poultry farmers, or entire populations, for an improved and more rapid response to a single booster dose with the pandemic vaccine.



• Alternative methods to produce (non-egg-based) and deliver (DNA and viral vectors) influenza vaccines will be explored further and may become increasingly successful;

• Efforts to improve seasonal vaccines and the search for uni-versal influenza vaccines has never been as intense as today and will undoubtedly lead to major improvements in our approach to cope with the consequences of influenza virus drift and shift.

Financial & competing interests disclosureGeert Leroux-Roels was principal investigator of clinical studies of seasonal and (pre)pandemic influenza vaccines for the following manufacturers: Baxter, GlaxoSmithKline Biologicals, Novartis and Sanofi Pasteur. The

Ghent University and University Hospital received sponsoring for the con-duct of these studies. Geert Leroux-Roels also performed consulting services for the following manufacturers: GlaxoSmithKline Biologicals and Novartis. Isabel Leroux-Roels assisted in the conduct of clinical studies of seasonal and (pre)pandemic influenza vaccines for the following manufac-turers: GlaxoSmithKline Biologicals and Sanofi Pasteur. Isabel Leroux-Roels also received travel and speaker fees from the following manufacturers: GlaxoSmithKline Biologicals and Sanofi Pasteur. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

ReferencesPapers of special note have been highlighted as:•ofinterest••ofconsiderableinterest

1 Taubenberger JK, Morens DM. 1918 Influenza: the mother of all pandemics. Emerg. Infect. Dis. 12(1), 15–22 (2006).

2 Longini IM Jr, Nizam A, Xu S et al. Containing pandemic influenza at the source. Science 309(5737), 1083–1087 (2005).

3 Ferguson NM, Cummings DA, Cauchemez S et al. Strategies for containing an emerging influenza pandemic in Southeast Asia. Nature 437(7056), 209–214 (2005).

4 Ferguson NM, Cummings DA, Fraser C, Cajka JC, Cooley PC, Burke DS. Strategies for mitigating an influenza pandemic. Nature 442(7101), 448–452 (2006).

•• Simulationstudyestimatingtheeffectofvariousmitigationstrategies,suchastheadvancestockpilingofprepandemicvaccine.

5 Halloran ME, Ferguson NM, Eubank S et al. Modeling targeted layered containment of an influenza pandemic in the United States. Proc. Natl Acad. Sci. USA 105(12), 4639–4644 (2008).

•• Anothermodelingstudythatpredictsthattherapidproductionanddistributionofvaccines,evenifpoorlymatched,couldsignificantlyslowpandemicspread,particularlyifchildrenarevaccinated.

6 Webster RG, Govorkova EA. H5N1 influenza – continuing evolution and spread. N. Engl. J. Med. 355(21), 2174–2177 (2006).

7 Subbarao K, Joseph T. Scientific barriers to developing vaccines against avian influenza viruses. Nat. Rev. Immunol. 7(4), 267–278 (2007).

8 Osterhaus AD. Pre- or post-pandemic influenza vaccine? Vaccine 25(27), 4983–4984 (2007).

9 Wood JM. Selection of influenza vaccine strains and developing pandemic vaccines. Vaccine 20(Suppl. 5), B40–B44 (2002).

10 Wood JM, Nicholson KG, Stephenson I et al. Experience with the clinical development of influenza vaccines for potential pandemics. Med. Microbiol. Immunol. 191(3–4), 197–201 (2002).

11 Stephenson I, Gust I, Pervikov Y, Kieny MP. Development of vaccines against influenza H5. Lancet Infect. Dis. 6(8), 458–460 (2006).

12 Subbarao K, Murphy BR, Fauci AS. Development of effective vaccines against pandemic influenza. Immunity 24(1), 5–9 (2006).

13 Stephenson I. H5N1 vaccines: how prepared are we for a pandemic? Lancet 368(9540), 965–966 (2006).

14 Fedson DS. Vaccine development for an imminent pandemic: why we should worry, what we must do. Hum. Vaccin. 2(1), 38–42 (2006).

15 Poland GA, Jacobson RM, Targonski PV. Avian and pandemic influenza: an overview. Vaccine 25(16), 3057–3061 (2007).

16 El Sahly HM, Keitel WA. Pandemic H5N1 influenza vaccine development: an update. Expert Rev. Vaccines 7(2), 241–247 (2008).

17 Poland GA, Sambhara S. Vaccines against influenza A (H5N1): evidence of progress. J. Infect. Dis. 198(5), 629–631 (2008).

18 Subbarao K, Luke C. H5N1 viruses and vaccines. PLoS Pathog. 3(3), e40 (2007).

19 Jennings LC, Monto AS, Chan PK, Szucs TD, Nicholson KG. Stockpiling prepandemic influenza vaccines: a new cornerstone of pandemic preparedness plans. Lancet Infect. Dis. 8(10), 650–658 (2008).

•• Interestingreviewon(pre)pandemicvaccine-deploymentstrategies.

20 Kieny MP, Subbarao K. The pandemic influenza vaccine challenge. Vaccine 26S(Suppl. 4), D3–D4 (2008).

21 Gambotto A, Barratt-Boyes SM, de Jong MD, Neumann G, Kawaoka Y. Human infection with highly pathogenic H5N1 influenza virus. Lancet 371(9622), 1464–1475 (2008).

• Interestingseminaronclinicalfeatures,diagnosis,hostrestrictionandpathogenesisofhumanH5N1infection.

22 Peiris JS, de Jong MD, Guan Y. Avian influenza virus (H5N1): a threat to human health. Clin. Microbiol. Rev. 20(2), 243–267 (2007).

23 Wood JM, Robertson JS. From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza. Nat. Rev. Microbiol. 2(10), 842–847 (2004).

24 Nicholson KG, Colegate AE, Podda A et al. Safety and antigenicity of non-adjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza. Lancet 357(9272), 1937–1943 (2001).

25 Rowe T, Abernathy RA, Hu-Primmer J et al. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J. Clin. Microbiol. 37(4), 937–943 (1999).



26 Stephenson I, Nicholson KG, Colegate A et al. Boosting immunity to influenza H5N1 with MF59-adjuvanted H5N3 A/Duck/Singapore/97 vaccine in a primed human population. Vaccine 21(15), 1687–1693 (2003).

27 Stephenson I, Wood JM, Nicholson KG, Charlett A, Zambon MC. Detection of anti-H5 responses in human sera by HI using horse erythrocytes following MF59-adjuvanted influenza A/Duck/Singapore/97 vaccine. Virus Res. 103(1–2), 91–95 (2004).

28 Stephenson I, Das RG, Wood JM, Katz JM. Comparison of neutralising antibody assays for detection of antibody to influenza A/H3N2 viruses: an international collaborative study. Vaccine 25(20), 4056–4063 (2007).

29 Girard MP, Osterhaus A, Pervikov Y, Palkonyay L, Kieny MP. Report of the third meeting on “influenza vaccines that induce broad spectrum and long-lasting immune responses”, World Health Organization, Geneva, Switzerland, 3–4 December 2007. Vaccine 26(20), 2443–2450 (2008).

30 Kistner O, Howard MK, Spruth M et al. Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses. Vaccine 25(32), 6028–6036 (2007).

31 Kistner O, Barrett PN, Mundt W, Reiter M, Schober-Bendixen S, Dorner F. Development of a mammalian cell (Vero) derived candidate influenza virus vaccine. Vaccine 16(9–10), 960–968 (1998).

32 Ehrlich HJ, Muller M, Oh HM et al. A clinical trial of a whole-virus H5N1 vaccine derived from cell culture. N. Engl. J. Med. 358(24), 2573–2584 (2008).

• Keypaperpresentingverypromisingresultswithanonadjuvantedwhole-virionvaccinederivedfromVerocellculture.

33 Barrett N. Vero cell technology and H5N1 influenza vaccines – a unique combination. Presented at: 13th International Congress on Infectious Diseases. Kuala Lumpur, Malaysia, 19–22 June 2008.

34 Kistner O, Howard MK, Sabarth N et al. Induction of cross-clade anti-H5N1 immune responses in mice, guinea pigs and ferrets by Vero cell-derived H5N1 whole virus candidate vaccine. Presented at: Third European Influenza Conference – ESWI. Vilamoura, Portugal, 14–17 September 2008.

35 Nolan TM, Richmond PC, Skeljo MV et al. Phase I and II randomised trials of the safety and immunogenicity of a prototype adjuvanted inactivated split-virus influenza A (H5N1) vaccine in healthy adults. Vaccine 26(33), 4160–4167 (2008).

36 Nolan T, Richmond PC, Formica NT et al. Safety and immunogenicity of a prototype adjuvanted inactivated split-virus influenza A (H5N1) vaccine in infants and children. Vaccine 26(50), 6383–6391 (2008).

37 Hehme N, Engelmann H, Kuenzel W, Neumeier E, Saenger R. Immunogenicity of a monovalent, aluminum-adjuvanted influenza whole virus vaccine for pandemic use. Virus Res. 103(1–2), 163–171 (2004).

38 Hehme N, Engelmann H, Kunzel W, Neumeier E, Sanger R. Pandemic preparedness: lessons learnt from H2N2 and H9N2 candidate vaccines. Med. Microbiol. Immunol. 191(3–4), 203–208 (2002).

39 Hehme N, Kuhn A, Mueller M et al. Whole virus alum-adjuvanted pandemic vaccine: safety and immunogenicity data on a vaccine formulated with H5N1. Presented at: International Conference on Influenza Vaccines for the World – IVW2006. Vienna, Austria, 18–20 October 2006.

40 Leroux-Roels I, Borkowski A, Vanwolleghem T et al. Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised controlled trial. Lancet 370(9587), 580–589 (2007).

• Firststudytodemonstratesignificantantigen-sparingthroughadjuvantation.

41 Rumke HC, Bayas JM, de Juanes JR et al. Safety and reactogenicity profile of an adjuvanted H5N1 pandemic candidate vaccine in adults within a Phase III safety trial. Vaccine 26(19), 2378–2388 (2008).

42 Chu D-S, Dramé M, Hwang S-J. Safety and immunogenicity of an AS adjuvanted H5N1 pepandemic influenza vaccine: a Phase III study in a large population of Asian adults. Presented at: Xth International Symposium on Respiratory Viral Infections. Singapore, 28 February–1 March 2008 (Abstract).

43 Ballester A, Garcés-Sanches M, Planelles Cantinaro M. Pediatric safety evaluation of an AS-adjuvanted H5N1 prepandemic candidate vaccine in children aged

6–9 years. A Phase II study. Presented at: 26th Annual Meeting of the European Society for Pediatric Infectious Diseases. Graz, Austria, 13–17 May 2008.

44 Ballester A, Garcés-Sanches M, Planelles Cantinaro M et al. Pediatric safety evalation of an AS-adjuvanted H5N1 prepandemic candidate vaccine in children aged 3–9 years. A Phase II study. Presented at: 13th International Congress of Infectious Diseases. Kuala Lumpur, Malaysia, 19–22 June 2008.

45 Carter NJ, Plosker GL. Prepandemic influenza vaccine H5N1 (split virion, inactivated, adjuvanted) [Prepandrix]: a review of its use as an active immunization against influenza A subtype H5N1 virus. BioDrugs 22(5), 279–292 (2008).

46 Leroux-Roels I, Bernhard R, Gerard P, Drame M, Hanon E, Leroux-Roels G. Broad clade 2 cross-reactive immunity induced by an adjuvanted clade 1 rH5N1 pandemic influenza vaccine. PLoS ONE 3(2), e1665 (2008).

47 Leroux-Roels I, Moris P, Dramé M et al. Persistent immune response against clade 1 and 2 H5N1 strains induced by an AS adjuvanted H5N1 clade 1 candidate prepandemic influenza vaccine. Presented at: International Conference on Avian Influenza. Bangkok, Thailand, 23–25 January 2008.

48 Moris P, Leroux-Roels I, Dramé M et al. Heterologous cell-mediated immunity priming using adjuvanted H5N1 vaccine. Presented at: IXth International Symposium on Respiratory Viral Infections. Hong Kong, China, 3–6 March 2007.

49 Zitzow LA, Rowe T, Morken T, Shieh WJ, Zaki S, Katz JM. Pathogenesis of avian influenza A (H5N1) viruses in ferrets. J. Virol. 76(9), 4420–4429 (2002).

50 Govorkova EA, Rehg JE, Krauss S et al. Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004. J. Virol. 79(4), 2191–2198 (2005).

51 Hampson AW. Ferrets and the challenges of H5N1 vaccine formulation. J. Infect. Dis. 194(2), 143–145 (2006).

52 Baras B, Stittelaar K, Simon JH. Immunization with low dose adjuvanted split H5N1 pandemic vaccine protects ferrets against homologous challenge. Presented at: IXth International Symposium on Respiratory Viral Infections. Hong Kong, China, 3–6 March, 2007.



53 Baras B, Stittelaar KJ, Simon JH et al. Cross-protection against lethal H5N1 challenge in ferrets with an adjuvanted pandemic influenza vaccine. PLoS ONE 3(1), e1401 (2008).

• Challengestudyinferretsdemonstratingtheaddedvalueofvaccineadjuvantationonprotectiveefficacyandviralshedding.

54 Vajo Z, Kosa L, Visontay I, Jankovics M, Jankovics I. Inactivated whole virus influenza A (H5N1) vaccine. Emerg. Infect. Dis. 13(5), 807–808 (2007).

55 Vajo Z, Kosa L, Szilvasy I et al. Safety and immunogenicity of a prepandemic influenza A (H5N1) vaccine in children. Pediatr. Infect. Dis. J. 27(12), 1052–1056 (2008).

56 Fazekas G, Martosne-Mendi R, Jankovics I, Szilvasy I, Vajo Z. Cross-reactive immunity in adult and elderly patients against clade 2 influenza A H5N1 virus strains induced by Fluval, a reverse genetic-derived adjuvanted H5N1 clade 1 prototype pandemic influenza vaccine. Clin. Vaccine Immunol. (Epub ahead of print) (2008).

57 Martin JT. Development of an adjuvant to enhance the immune response to influenza vaccine in the elderly. Biologicals 25(2), 209–213 (1997).

58 Stephenson I, Bugarini R, Nicholson KG et al. Cross-reactivity to highly pathogenic avian influenza H5N1 viruses after vaccination with nonadjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a potential priming strategy. J. Infect. Dis. 191(8), 1210–1215 (2005).

59 Atmar RL, Keitel WA, Patel SM et al. Safety and immunogenicity of nonadjuvanted and MF59-adjuvanted influenza A/H9N2 vaccine preparations. Clin. Infect. Dis. 43(9), 1135–1142 (2006).

60 Bernstein DI, Edwards KM, Dekker CL et al. Effects of adjuvants on the safety and immunogenicity of an avian influenza H5N1 vaccine in adults. J. Infect. Dis. 197(5), 667–675 (2008).

61 Stephenson I, Nicholson KG, Hoschler K et al. Antigenically distinct MF59-adjuvanted vaccine to boost immunity to H5N1. N. Engl. J. Med. 359(15), 1631–1633 (2008).

• Shortreportshowingstrongandrapidrecallresponseswithanadjuvantedvaccineinaheterologousprime–boostregimen.

62 Podda A, Del Giudice G. MF59-adjuvanted vaccines: increased immunogenicity with an optimal safety profile. Expert Rev. Vaccines 2(2), 197–203 (2003).

63 O’Hagan DT. MF59 is a safe and potent vaccine adjuvant that enhances protection against influenza virus infection. Expert Rev. Vaccines 6(5), 699–710 (2007).

64 Barbera J P, Vidal DG. MF59-adjuvanted subunit influenza vaccine: an improved interpandemic influenza vaccine for vulnerable populations. Expert Rev. Vaccines 6(5), 659–665 (2007).

65 Borgogni E, Castellino F, Galli G et al. MF59-adjuvanted H5N1 subunit vaccine induces a high frequency of Th1 effector/memory CD4 T-cells which persist over time. Presented at: 3rd European Influenza Conference. Vilamoura, Portugal, 14–17 September 2008.

66 Banzhoff A, Gasparini R, Laghi-Pasini F et al. MF59-adjuvanted H5N1 vaccine induces immunologic memory and heterotypic antibody responses in non-elderly and elderly adults. PLoS ONE 4(2), e4384 (2009).

67 Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M. Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N. Engl. J. Med. 354(13), 1343–1351 (2006).

68 Bresson JL, Perronne C, Launay O et al. Safety and immunogenicity of an inactivated split-virion influenza A/Vietnam/1194/2004 (H5N1) vaccine: Phase I randomised trial. Lancet 367(9523), 1657–1664 (2006).

69 Levie K, Leroux-Roels I, Hoppenbrouwers K et al. An adjuvanted, low-dose, pandemic influenza A (H5N1) vaccine candidate is safe, immunogenic, and induces cross-reactive immune responses in healthy adults. J. Infect. Dis. 198(5), 642–649 (2008).

70 Lina B, Fletcher MA, Valette M, Saliou P, Aymard M. A TritonX-100-split virion influenza vaccine is safe and fulfills the committee for proprietary medicinal products (CPMP) recommendations for the European community for immunogenicity, in children, adults and the elderly. Biologicals 28(2), 95–103 (2000).

71 Höschler K, Gopal R, Andrews N et al. Cross-neutralisaton of antibodies elicited by an inactivated split-virion influenza A/Vietnam/1194/2004 (H5N1) vaccine in healty adults against H5N1 clade 2 strains. Influenza Other Respir. Viruses 1(5–6), 199–206 (2008).

72 Chotpitayasunondh T, Thisyakorn U, Pancharoen C, Pepin S, Nougarede N. Safety, humoral and cell mediated immune

responses to two formulations of an inactivated, split virion influenza A/H5N1 vaccine in children. PLoS ONE 3(12), e4028 (2008).

73 Ruat C, Caillet C, Bidaut A, Simon J, Osterhaus AD. Vaccination of macaques with adjuvanted formalin-inactivated influenza A virus (H5N1) vaccines: protection against H5N1 challenge without disease enhancement. J. Virol. 82(5), 2565–2569 (2008).

74 Bigger J, Ruat C, Vasconcelos D, Legastelois I, Stark G, Caillet C. A new adjuvanted low-dose candidate pandemic influenza A(H5N1) vaccine protects ferrets against homologous clade 1 viral challenge and induces clade 2 cross-reactive antibodies. Presented at: International Conference on Avian Influenza. Bangkok, Thailand, 23–25 January 2008.

75 Lin J, Zhang J, Dong X et al. Safety and immunogenicity of an inactivated adjuvanted whole-virion influenza A (H5N1) vaccine: a Phase I randomised controlled trial. Lancet 368(9540), 991–997 (2006).

76 Lin JT, Li CG, Wang X et al. Antibody persistence after 2-dose priming and booster response to a third dose of an inactivated, adjuvanted, whole-virion H5N1 vaccine. J. Infect. Dis. 199(2), 184–187 (2009).

77 Jin R, Lv Z, Chen Q et al. Safety and immunogenicity of H5N1 influenza vaccine based on baculovirus surface display system of Bombyx mori. PLoS ONE 3(12), e3933 (2008).

78 Treanor JJ, Schiff GM, Hayden FG et al. Safety and immunogenicity of a baculovirus-expressed hemagglutinin influenza vaccine: a randomized controlled trial. JAMA 297(14), 1577–1582 (2007).

79 Gerhard W, Mozdzanowska K, Zharikova D. Prospects for universal influenza virus vaccine. Emerg. Infect. Dis. 12(4), 569–574 (2006).

80 Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers W. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat. Med. 5(10), 1157–1163 (1999).

Websites

101 WHO. Responding to the avian influenza pandemic threat: recommended strategic actions www.who.int/csr/resources/publications/influenza/who_cds_csr_gip_05_8-en.pdf



102 WHO. Toward a unified nomenclature system for highly pathogenic avian influenza virus (H5N1; conference summary) www.cdc.gov/eid/content/14/7/e1.htm

103 WHO. Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to WHO www.who.int/csr/disease/avian_influenza/country/cases_table_2009_02_18/en/index.html

104 WHO. WHO global influenza preparedness plan: the role of WHO and recommendations for national measures before and during pandemics www.who.int/csr/resources/publications/influenza/who_cds_csr_gip_2005_5/

105 WHO. Epidemic and pandemic alert and response www.who.int/csr/disease/avian_ influenza/en/

106 WHO. Antigenic and genetic characteristics of H5N1 viruses and candidate H5N1 vaccine viruses developed for potential use as human vaccines www.who.int/csr/disease/avian_influenza/guidelines/h5n1virus/ en/index.html

107 EMEA. Guideline on dossier structure and content for pandemic influenza vaccine marketing authorisation application (CPMP/VEG/4717/03) www.emea.europa.eu/pdfs/human/vwp/471703en.pdf

108 US FDA. Guidance for industry. Clinical data needed to support the licensure of pandemic influenza vaccines www.fda.gov/cber/gdlns/panfluvac.htm

109 International Federation of Pharmaceutical Manufacturers. R&D for avian/pandemic influenza vaccines by IFPMA Influenza Vaccine Supply International Task Force (IVS ITF) members www.ifpma.org/Influenza/content/pdfs/table_avian_pandemic_influenza_vaccine_rnd_24jan06.pdf

110 International Federation of Pharmaceutical Manufacturers. About clinical trials conducted by the members of the IFPMA & IFPMA Influenza Vaccine Supply International Task Force (IVS ITF) www.ifpma.org/influenza/content/pdfs/table_H5N1_pandemic_influenza_rnd_26may08.pdf

111 WHO. Tables on the clinical trials of pandemic influenza prototype vaccines www.who.int/vaccine_research/diseases/influenza/flu_trials_tables/en/

112 European Medicines Agency. Committee for medicinal products for human use. Summary of positive opinion for celvapan www.emea.europa.eu/pdfs/human/opinion/celvapan_65700508en.pdf

113 US NIH. ClinicalTrials.gov database www.clinicaltrials.gov/ct2/results?term=baxter+and+H5N1+vaccine

114 European Medicines Agency. European Medicines Agency adopts first positive opinion for mock-up pandemic influenza vaccine www.emea.europa.eu/pdfs/general/direct/pr/50287306en.pdf

115 European Medicines Agency. Withdrawal assessment report for aflunov www.emea.europa.eu/humandocs/pdfs/epar/aflunov/H-804-AR-en.pdf

116 European Medicines Agency. European public assessment report for daronrix www.emea.europa.eu/humandocs/humans/epar/daronrix/daronrix.htm

117 European Medicines Agency. European public assessment report for focetria www.emea.europa.eu/humandocs/humans/epar/focetria/focetria.htm

Affiliations

• Isabel Leroux-Roels Center for Vaccinology, Ghent University and Hospital, De Pintelaan 185, 9000, Ghent, Belgium Tel.: +32 9332 3422 Fax: +32 9332 6311 [email protected]

• Geert Leroux-Roels Center for Vaccinology, Ghent University and Hospital, De Pintelaan 185, 9000, Ghent, Belgium Tel.: +32 9332 3422 Fax: +32 9332 6311 [email protected]

Documents

Current status and progress of prepandemic and pandemic influenza vaccine development