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VAC 13599 1–8

Vaccine xxx (2012) xxx– xxx

Contents lists available at SciVerse ScienceDirect

Vaccine

jou rn al h om epa ge: www.elsev ier .com/ locate /vacc ine

he immunogenicity and safety of a single 0.5 mL dose of virosomal subunitnfluenza vaccine administered to unprimed children aged ≥6 to <36 months:ata from a randomized, Phase III study

usanna Espositoa,∗, Paola Marchisioa, Valentina Montinaroa, Sonia Bianchinia,errit Jan Weverlingb, Elena Parianic, Antonella Amendolac, Valentina Fabianod,e,alentina Pivettid,e, Alessandro Zanetti c, Gian Vincenzo Zuccottid,e

Pediatric Clinic 1, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,ia Commenda 9, 20122 Milan, ItalyCrucell, Biostatistics, Leiden, The NetherlandsDepartment of Biomedical Science for Health, Università degli Studi di Milano, Milan, ItalyDepartment of Pediatrics, Università degli Studi di Milano, L. Sacco Hospital, Via GB Grassi 74, 20157 Milan, ItalyDepartment of Clinical Sciences, Università degli Studi di Milano, L. Sacco Hospital, Via GB Grassi 74, 20157 Milan, Italy

r t i c l e i n f o

rticle history:eceived 21 June 2012eceived in revised form7 September 2012ccepted 26 September 2012vailable online xxx

a b s t r a c t

This study evaluated the immunogenicity, safety and tolerability of a single 0.5 mL dose of the seasonalvirosomal subunit influenza vaccine (Inflexal V, Crucell, Switzerland) in 205 healthy, unprimed childrenaged at least 6 to <36 months, evaluated at four weeks post-vaccination and seven months from baseline.Of the enrolled children, 102 received one single 0.5 mL dose and 103 received the standard two 0.25 mLdoses given four weeks apart. Both treatments evoked an immune response that satisfied the EMA/CHMPcriteria for yearly vaccine licensing for all three vaccine strains. Exploratory analyses revealed no differ-

eywords:nfluenzairosomalhildrenaccination schedule

ences between the groups at four weeks post-vaccination. Furthermore, immunogenicity was maintainedseven months after the first vaccination after both the 0.5 mL and standard two 0.25 mL doses. Adverseevents were comparable between groups and were as expected according to the safety profile of thevaccine; overall, the vaccine was well tolerated. Our results show that a single 0.5 mL dose effectivelyand safely provided long-term immunogenicity to all three influenza strains in unprimed children aged

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mmunogenicity at least 6 to <36 months.

. Introduction

Vaccination remains the leading method of preventingnfluenza-associated morbidity and mortality [1]. It has been pre-iously shown by Neuzil et al. [2] that two doses of trivalentnfluenza vaccine provide optimal immunogenicity against threetrains (H1N1, H3N2 and B strains) of influenza in children andhis is the general recommendation [3]. However, compliance with

Please cite this article in press as: Esposito S, et al. The immunogeinfluenza vaccine administered to unprimed children aged ≥6 to <36 mhttp://dx.doi.org/10.1016/j.vaccine.2012.09.069

he recommended two-dose vaccination schedule is poor, rangingrom 29 to 65% and 12 to 58% for children aged 6–23 months and 2–8ears, respectively [4,5]. Poor compliance to the two-dose schedule

∗ Corresponding author. Tel.: +39 02 55032498; fax: +39 02 50320206.E-mail addresses: susanna.esposito@unimi.it (S. Esposito),

aola.marchisio@unimi.it (P. Marchisio), vali.m85@gmail.com (V. Montinaro),ianchini.sonia@fastwebnet.it (S. Bianchini), GWeverli@its.jnj.com (G.J. Weverling),lena.pariani@unimi.it (E. Pariani), antonella.amendola@unimi.it (A. Amendola),abiano.valentina@hsacco.it (V. Fabiano), valentina.pivetti@studenti.unimi.itV. Pivetti), alessandro.zanetti@unimi.it (A. Zanetti), gianvincenzo.zuccotti@unimi.itG.V. Zuccotti).

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264-410X/$ – see front matter © 2012 Published by Elsevier Ltd.ttp://dx.doi.org/10.1016/j.vaccine.2012.09.069

© 2012 Published by Elsevier Ltd.

in children has particular bearing on the transmission of influenza,

as children are recognized as the principal vectors of influenza

transmission within the community [6]. Studies have also shownthat both the infection rate and the burden of disease in the elderly

can be reduced through proper vaccination of children [7,8].

To overcome compliance issues, highly immunogenic vaccines

(trivalent live attenuated vaccine) or modified vaccination sched-

ules have been used [9]. A recent study [10] in children aged 6–35

months randomized subjects to two 0.25 mL doses (standard regi-

men) of virosomal subunit influenza vaccine or two 0.5 mL doses.

After vaccination with a single 0.5 mL dose, the immune responsesatisfied the European Medicines Agency (EMA)/Committee for

Medicinal Products for Human Use (CHMP) criteria for immuno-

genicity. The 0.5 mL dose also induced high immunogenicity

persisting for six months in >90% of children without increasing

local or systemic adverse events (AEs). For a review on virosomal

nicity and safety of a single 0.5 mL dose of virosomal subunitonths: Data from a randomized, Phase III study. Vaccine (2012),

subunit influenza vaccine see Herzog et al. [11] 54

Our study aimed to show whether a single 0.5 mL dose of 55

virosomal subunit influenza vaccine safely provides adequate 56

immunogenicity in children. The primary objectives were to 57

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valuate the immunogenicity of a single full 0.5 mL dose and a.25 mL two-dose regimen of the virosomal subunit influenzaaccine in healthy, unprimed children aged from 6 to <36 months.s reference, the EMA/CHMP immunogenicity criteria for thee-registration of seasonal influenza vaccines in adults (aged ≥18o ≤60 years) was used [12].

Secondary objectives were immunogenicity and safety for thege ranges 6 to <21 months and 21 to <36 months; furtherbjectives were immunogenicity and safety data at seven-monthsost-baseline.

. Materials and methods

.1. Study design and population

This open, randomized, Phase III study was performed in twoenters in Milan, Italy, between 6 October 2010 and 29 July 2011.he Ethics Committees for the IRCCS Ca’ Granda Ospedale Maggioreoliclinico and the Luigi Sacco Hospital (both Milan, Italy) approvedhe study, which was performed in accordance with the Declara-ion of Helsinki and Good Clinical Practice standards and registeredith clinicaltrials.gov (NCT01229397). Written informed consent,

igned by both parents or by the legal guardian, was obtainedor all participants. Healthy, unprimed children aged ≥6 to <36

onths were eligible for inclusion. Exclusion criteria included:cute respiratory infection/other acute disease; acute febrile ill-ess (≥38 ◦C); previous influenza vaccine; laboratory-confirmed

nfluenza infection; hypersensitivity to any vaccine component;gg protein allergy or severe atopy; blood coagulation disorder;mmunodeficiency; treatment with an investigational medicinalroduct; chronic administration of immunosuppressants/other

mmune modifying drugs, or immunoglobulin/blood transfusionithin 6 months or 3 months before first vaccine dose, respectively;MR vaccination within 4 weeks prior to first or 4 weeks after last

accine dose; participation in another clinical trial, non-compliancer relationship to an investigator/site employee.

After screening, children were randomized 1:1 to Group A (one.5 mL dose) or Group B (two 0.25 mL doses), using a computer-enerated randomization sequence, with assignments enclosed inealed envelopes. Each full dose of the 2010/2011 season viroso-al subunit influenza vaccine (Inflexal V, Crucell, The Netherlands;

ot Number: 3001875 both groups) contained 15 �g eachf the hemagglutinin antigen of A/California/7/2009-H1N1-like,/Perth/16/2009-H3N2-like, and B/Brisbane/60/2008-like virus,ith solvent added to reach a volume of 0.5 mL. The 0.25 mL dose

half the 0.5 mL dose was expelled from the syringe and discardedefore vaccination) contained half the amount (7.5 �g) of hemag-lutinin antigens as in the full dose, and was administered on Days

and 29. The vaccine was administered intramuscularly (needleength: 16 mm) into Musculus deltoideus or Musculus vastus later-lis. A minimum of 0.5 mL blood for immunogenicity assessmentsas taken on Days 1 (both groups), 29 (Group A), 59 (Group B) and

12 (both groups).Reactogenicity was evaluated in all children receiving at least

ne dose of the vaccine and for whom post-baseline safetyata were available (safety population). Solicited local (pain, ery-hema, ecchymosis, and induration) and systemic (fever, shivering,rritability, and fussiness) AEs were documented by parents/legaluardian for four days post-vaccination (from Day 1 to 4 for bothroups and Day 29 to 32 for Group B) in a subject diary. If thereas no entry for an AE in the subject diary, then the default was

hat the event had occurred. Body temperature was measured in the

Please cite this article in press as: Esposito S, et al. The immunogeinfluenza vaccine administered to unprimed children aged ≥6 to <36 mhttp://dx.doi.org/10.1016/j.vaccine.2012.09.069

vening, and fever was defined as a body temperature ≥ 38 ◦C inde-endent of the route of measurement. If more than one temperatureeasurement was performed in a day, then the highest tempera-

ure was recorded in the study diary. Unsolicited AEs were observed

PRESSxxx (2012) xxx– xxx

directly by the investigator or reported by parents/legal guardian

spontaneously or in response to a direct question. Unsolicited AE

data were collected at baseline, Day 29/57 (Group B) and Day 212

(both groups). Serious AEs were reported from Day 1 to 212. The

overall tolerability of the vaccine was assessed as ‘very good’, ‘good’,

‘moderate’, or ‘bad’. Additionally, the parents/legal guardian stated

whether they would allow their child to be revaccinated.

2.2. Immunogenicity evaluation

Blood-sample analysis was performed at the Dipartimento diScienze Biomediche per la Salute, Milan, Italy. Hemagglutination-inhibiting (HI) antibodies were titrated in two independent testruns in duplicate, according to standard procedures [13]. The HItiter was the reciprocal value of the highest dilution which inhibited

hemagglutination. The titer assigned to each sample was the geo-metric mean (GM) of the two independent determinations. The

group GM was the back-transformed arithmetic mean of the log10-

transformed individual GMs. The confidence interval (CI) of GM

was calculated as the back-transformed CI of the mean of the log10-

transformed value.

Primary endpoints were the immunogenicity parameters at

Days 29 (Group A) and 57 (Group B) compared to baseline (Day

1). Further endpoints were the immunogenicity parameters at Day

212. Immunogenicity was determined by: seroprotection rate (pro-

portion of subjects with a HI antibody titer ≥ 1:40); seroconversion

rate (proportion of subjects with a ≥4-fold increase in HI antibody

titer and a titer of ≥1:40); and GMT-fold increase versus base-

line. The immunogenicity variables were analyzed according to set

criteria with at least one of the criteria having to be met for each

strain according to EMA/CHMP guidelines for adults. The criteria

specify seroprotection and seroconversion rates four weeks after

completion of the vaccination (Day 29 for Group A and Day 57 for

Group B) of ≥70% and ≥40%, respectively, and a GMT-fold increase

of >2.5-fold compared to baseline four weeks after completion ofvaccination [12].

2.3. Statistical analysis

In this proof-of-concept study, neither formal statistical hypoth-

esis testing nor sample size calculation were performed. To fulfill

the minimum requirement by EMA/CHMP [12] (for influenza vac-

cine relicensing trials, the sample size is set to 50 persons per age

group), the sample size was set to 120 children eligible for evalua-

tion per treatment group (60 children per age group per treatmentgroup) allowing a drop-out rate of up to 17%.

At seven months post-baseline, samples for serology taken at

Day 212 from both groups were analyzed in parallel to those from

Day 1/29 for Group A, and Day 1/57 for Group B. Immunogenicity

analyses were repeated for each age group separately and for those

children with baseline HI antibody titers < 1:40. Immunogenicity

analyses for HI antibody titers and all exploratory efficacy analyses

were based on the intention-to-treat (ITT) population.

Although the study was not powered to evaluate the superi-

ority or non-inferiority of the vaccination schedules, exploratorybetween-group comparisons were performed. The seroprotection

and seroconversion rates in the two groups were compared using

the Chi-squared test, and the GMTs using an analysis of variance

model for the log-transformed concentrations, at each time point,

with treatment and age category as factors. A two-sided p-value of

nicity and safety of a single 0.5 mL dose of virosomal subunitonths: Data from a randomized, Phase III study. Vaccine (2012),

<0.05 was statistically significant. 177

Safety analyses were performed on the safety population per 178

vaccination group. AEs were presented as frequencies and percent- 179

ages with exact two-sided 95% CIs. 180

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. Results

.1. Study population

Fig. 1 shows the patient disposition. More boys (63.4%) were vac-inated than girls for both groups. The median age was 23.5 (Group) and 22.5 months (Group B). At baseline, all children had a body

emperature < 38 ◦C and shivering and malaise were absent. Table 1resents other baseline demographic data. Ten children experi-nced influenza-like illness (no virological testing was performed)n the year prior to vaccination.

.2. Immunogenicity

.2.1. SeroprotectionAt baseline, the majority of children in both groups had base-

ine HI antibody titers < 1:40 against any of the three strains.n Group A, 65.7%, 94.5% and 99.0% of children had baseline HIntibody titers < 1:40 against strains A/California/7/2009-H1N1,/Perth/16/2009-H3N2 and B/Bristbane/60/2008, respectively; inroup B, these figures were 71.4%, 93.9% and 98.0%, respectively.able 2 summarizes the seroprotection rates. Immunogenicityriteria for seroprotection were fulfilled, with rates above theMA/CHMP threshold (70%) at Day 29/Day 57 and at Day 212or both groups and all strains except for B/Brisbane/60/2008 inroup B at Day 212. Those children with baseline HI antibody

iters < 1:40 had similar results for both groups. The seropro-ection rates were higher for strains A/California/7/2009-H1N1nd A/Perth/16/2009-H3N2 than for B/Brisbane/60/2008 at allime points post-baseline (data not shown). The between-groupxploratory analysis showed no statistically significant differencesor either the overall population or the children with baseline HIntibody titers < 1:40.

.2.2. SeroconversionTable 3 summarizes seroconversion rates. Immunogenicity

riteria for seroconversion were fulfilled, with rates above theMA/CHMP threshold (≥40%) at both Days 29 and 57, and at Day12 for both groups and all strains. Children with baseline antibodyiters < 1:40 had similar results; both groups were comparable (dataot shown). The between-group exploratory analysis showed notatistically significant differences for either the overall populationr the children with baseline HI antibody titers < 1:40.

.2.3. GMT-fold increaseTable 4 summarizes the GMTs of HI antibodies and GMT-fold

ncreases. The GMT-fold increase was above the EMA/CHMP thresh-ld (>2.5-fold increase) at both Days 29 and 57, and Day 212 for bothroups and all strains.

The GMT-fold increase from Day 1 to Day 29 (Group A) or to Day7 (Group B) and to Day 212 in the ITT population was higher fortrains A/California/7/2009-H1N1 and A/Perth/16/2009-H3N2 thanor B/Brisbane/60/2008 (Table 5). This trend was maintained forhildren with baseline HI antibody titers < 1:40 (data not shown).he GMT-fold increase at Day 57 was statistically significantlyigher after two 0.25 mL doses (Group B) than at Day 29 afterne 0.5 mL dose (Group A) for both A/California/7/2009-H1N1p = 0.031) and A/Perth/16/2009-H3N2 (p = 0.036). Although thereas initially a difference, at the end of the study this difference waso longer significant. However, the GMT-fold increases were still

Please cite this article in press as: Esposito S, et al. The immunogeinfluenza vaccine administered to unprimed children aged ≥6 to <36 mhttp://dx.doi.org/10.1016/j.vaccine.2012.09.069

bove the threshold of 2.5.For the subgroup populations of children aged 6 to <21 months

nd 21 to <36 months, immunogenicity results were similar to theverall population (data not shown).

PRESSxxx (2012) xxx– xxx 3

3.3. Adverse events

Table 5 summarizes solicited local and systemic AEs. Erythema

and pain were the most frequently reported solicited local AEs

for both groups. Most events were mild/moderate in intensity and

were reported on the day of vaccination, resolving within four days

of vaccination. Fever (≥38 ◦C) and malaise were the most frequently

reported solicited systemic AEs. Of the children with fever (≥38 ◦C),

high fever (≥39 ◦C) was reported for 5 children in Group A and 3 in

Group B (after first vaccination). Further, 40.0% and 25.0% of chil-

dren with fever (≥38 ◦C) had onset of fever on Day 1 in Group A and

Group B, respectively, with 36.0% in Group B having onset on Day

29 (second vaccination). Fever resolved within one day for 12/15 of

children vaccinated with 0.5 mL virosomal subunit influenza vac-

cine. For Group B, fever also resolved within one day for 4/8 children

and 4/11 children after the first and second vaccination, respec-tively.

Most unsolicited AEs were mild, and the most frequent werefever (≥38 ◦C), rhinitis and cough. The onset of fever (≥38 ◦C)

was more than eight days post-vaccination for the majority ofunsolicited fever cases, which were assessed as unrelated by the

investigator except for one case. Six unsolicited AEs in Group A

and four in Group B were assessed as related to the vaccine by the

investigator, including one event each of bronchitis, nasopharyn-

gitis, oral herpes, fever, acute otitis media, bronchospasm, cough,

erythema and swelling face.

Six serious AEs were reported during the study; all were

assessed as unrelated to the study vaccine and resolved without

sequelae. No deaths occurred during the study.

4. Discussion

This study expands our knowledge of the feasibility and the

long-term (up to seven months post-vaccination) immunogenic-

ity of a single full 0.5 mL dose of virosomal subunit influenza

vaccine given as a one-dose regimen in previously unvaccinated

children aged from 6 to 36 months. Our most important find-

ing is that the single 0.5 mL dose evoked high seroprotection

and seroconversion rates, and high GMT-fold increases, with val-

ues persisting in the protective range for the duration of the

influenza season [14]. These results were comparable with those

achieved for the standard 0.25 mL two-dose regimen. Immuno-

genicity to the influenza B virus antigen was lower than for the

two influenza A virus antigens, but was still within the protective

range (above EMA/CHMP thresholds) up to seven months post-

vaccination for those children vaccinated with the single 0.5 mL

dose. These results are similar to those seen in a previous pilot

study with a double 0.5 mL dose [10], and are expected since

young children have an unexplained decrease in immunogenicity

to B influenza viruses [9]. Since the burden of B-strain influenza

falls on young children, a good immune response to influenza B

virus antigens is particularly important and necessary to reduce

morbidity in this population [15]. Our results show that a single

0.5 mL dose of virosomal subunit influenza vaccine is sufficient

to provide a long-term protective immune response to B-strain

influenza, which would cover the duration of the influenza sea-

son [14]. Furthermore, a high level of seroprotection was evoked in

those children with low baseline titers (≤1:40), especially against

the A/Perth/16/2009 and B/Brisbane/60/2008 strains. It should,

however, be noted that satisfying the EMA/CHMP immunogenicity

criteria may not always guarantee protection against influenza dis-

nicity and safety of a single 0.5 mL dose of virosomal subunitonths: Data from a randomized, Phase III study. Vaccine (2012),

ease in vaccinated children, as the immunogenicity thresholds are 297

administrative and serve for the licensing and delivery of influenza 298

vaccines from the pharmaceutical companies to the market in 299

Europe. 300

Please cite this article in press as: Esposito S, et al. The immunogenicity and safety of a single 0.5 mL dose of virosomal subunitinfluenza vaccine administered to unprimed children aged ≥6 to <36 months: Data from a randomized, Phase III study. Vaccine (2012),http://dx.doi.org/10.1016/j.vaccine.2012.09.069

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4 S. Esposito et al. / Vaccine xxx (2012) xxx– xxx

Enrolled and rando mize d

N = 20 5

All ocated t o Group AReceived 0.5 mL virosomal subun it

influen za vacc ine

n = 10 2

All ocated t o Group BReceived 2 x 0.25 mL virosomal subun it

influen za vacc ine

n = 10 3

Withd rawn

n = 9Rea son s f or withd rawal:

• Con sent withd rawal: n = 3

• Lost to follow-up: n = 6

Withd rawn

n = 11Rea son s f or withd rawal:

• Con sent withd rawal: n = 3

• Migrated/moved: n = 1

• Lost to follow-up: n = 7

Completed stud y

n = 93

Completed stud y

n = 92

Excluded f rom ana lyses

Safety: n = 2 (miss ing sa fety da ta)ITT: n = 3 (missing pre- and post-

vaccination da ta)ATP: n = 13 (protoco l violation )

Excluded from analy ses

Safety: n = 1 (miss ing sa fety da ta)ITT: n = 5 (missing pre- and post-

vaccination da ta)ATP: n = 38 (protoco l violation )

Ana lysed

Safety popu lation: n = 10 0ITT popu lation: n = 99

ATP popu lation: n = 86

Ana lysed

Safety popu lation: n = 10 2ITT population: n = 98

ATP popu lation: n = 60

Fig. 1. Consort diagram (ITT = intention-to-treat, ATP = according-to-protocol).

Table 1Baseline demographic data (safety population).

Group A1 × 0.5 mL virosomal subunit influenza vaccine

Group B2 × 0.25 mL virosomal subunit influenza vaccine

Total

N = 100 N = 102 N = 202

Sex [n (%)] Female 37 (37.0) 37 (36.3) 74 (36.6)Male 63 (63.0) 65 (63.7) 128 (63.4)

Age [months] Median (min, max) 23.5 (6, 35) 22.5 (7, 34) 23.0 (6, 35)Mean ± SD 21.9 ± 7.33 21.5 ± 6.24 21.7 ± 6.79

Age group ≥6 to <21 months 41 (41.0) 42 (41.2) 83 (41.1)≥21 to <36 months 59 (59.0) 60 (58.8) 119 (58.9)

Weight [kg] Median (min, max) 12.5 (6.3, 18.0) 12.0 (7.2, 19.1) 12.0 (6.3, 19.1)Height [cm] Median (min, max) 86.0 (64, 100) 84.0 (67, 100) 85 (64, 100)BMI [kg/m2] Median (min, max) 17.1 (12.5, 24.3) 17.1 (13.8, 22.1) 17.1 (12.5, 24.3)

Percentages are based on N (number of subjects in specified group in safety population).Sn, number of subjects in specified category; SD, standard deviation; BMI, body mass index.

Please cite this article in press as: Esposito S, et al. The immunogenicity and safety of a single 0.5 mL dose of virosomal subunitinfluenza vaccine administered to unprimed children aged ≥6 to <36 months: Data from a randomized, Phase III study. Vaccine (2012),http://dx.doi.org/10.1016/j.vaccine.2012.09.069

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Table 2Seroprotection rates (hemagglutination-inhibiting antibody titers ≥ 1:40) in children aged ≥6 to <36 months previously unvaccinated against influenza receiving one 0.5 mLdose or two 0.25 mL doses of virosomal subunit influenza vaccine.

Group A Group B p-Value1 × 0.5 mL virosomal subunit influenza vaccineN = 99

2 × 0.25 mL virosomal subunit influenzavaccine N = 98

n/n′ % (95% CI) n/n′ 95% CI

A/California/7/2009-H1N1Baseline (Day 1) 34/99 34.3

(25.1, 44.6)28/98 28.6

(19.9, 38.6)0.383

Day 29 ± 3 days 97/99 98.0(92.9, 99.8)

– –

Day 57 ± 3 days – – 97/98 99.0(94.4, 100)

0.567

Day 212a ± 7 days 85/88 99.6(90.4, 99.3)

78/84 92.2(85.1, 97.3)

0.272

A/Perth/16/2009-H3N2Baseline (Day 1) 5/99 (5.1) 5.1

(1.7, 11.4)6/98 6.1

(2.3, 12.9)0.743

Day 29 ± 3 days 96/99 97.0(91.4, 99.4)

– –

Day 57 ± 3 days – – 97/98 99.0(94.4, 100)

0.317

Day 212a ± 7 days 78/88 88.6(80.1, 94.4)

79/84 94.0(86.7, 98.0)

0.209

B/Brisbane/60/2008Baseline (Day 1) 1/99 1.0

(0.0, 5.5)2/98 2.0

(0.2, 7.2)0.555

Day 29 ± 3 days 86/99 86.9(78.6, 92.8)

– –

Day 57 ± 3 days – – 91/98 92.9(85.8, 97.1)

0.164

Day 212a ± 7 days 66/88 75.0(64.6, 83.6)

57/84 67.9(56.8, 77.6)

0.300

Percentages for seroprotection are based on n′ = number of subjects with valid measurements in the specified group and population.CI, confidence interval; N, number of subjects in the specified group and population; n, number of subjects seroprotected.

a The ITT population for Day 212 is smaller than for Day 29/Day 57 (Group A: n = 88, Group B: n = 84) because blood samples were missing for more subjects.

Table 3Seroconversion rates (≥4-fold increase in hemagglutination-inhibiting antibody titers compared to baseline and maintenance of the protective tier of ≥1:40) in children aged≥6 to <36 months previously unvaccinated against influenza receiving one 0.5 mL dose or two 0.25 mL doses of virosomal subunit influenza vaccine.

Group A Group B p-Value1 × 0.5 mLN = 99

2 × 0.25 mLN = 98

n/n′ % (95% CI) n/n′ % (95% CI)

A/California/7/2009-H1N1Day 29 ± 3 days 97/99 98.0

(92.9, 99.8)– – 0.567

Day 57 ± 3 days – – 97/98 99.0(94.4, 100)

Day 212a 84/88 95.5(88.8, 98.7)

78/84 92.9(85.1, 97.3)

0.467

A/Perth/16/2009-H3N2Day 29 ± 3 days 96/99 97.0

(91.4, 99.4)– – 0.317

Day 57 ± 3 days – – 97/98 99.0(94.4, 100)

Day 212 a 78/88 88.6(80.1, 94.4)

79/84 94.0(86.7, 98.0)

0.209

B/Brisbane/60/2008Day 29 ± 3 days 86/99 86.9

(78.6, 92.8)– – 0.164

Day 57 ± 3 days – – 91/98 92.9(85.8, 97.1)

Day 212a 66/88 75.0(64.6, 83.6)

57/84 67.9(56.8, 77.6)

0.300

Percentages for seroconversion are based on n′ = number of subjects with valid measurements in the specified group and population.CI, confidence interval; N, number of subjects in the specified group and population; n, number of subjects seroconverted.

a The ITT population for Day 212 is smaller than for Day 29/Day 57 (Group A: n = 88, Group B: n = 84) because blood samples were missing for more subjects.

ARTICLE IN PRESSG Model

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6 S. Esposito et al. / Vaccine xxx (2012) xxx– xxx

Table 4Geometric mean titers (GMTs) of hemagglutination-inhibiting antibody and GMT-fold increase from baseline to Day 29 ± 3 days or Day 57 ± 3 days and to Day 212 ± 3 daysin children aged ≥6 to <36 months previously unvaccinated against influenza receiving one 0.5 mL dose or two 0.25 mL doses of virosomal subunit influenza vaccine.

Group A Group B p-Value1 × 0.5 mLN = 99

2 × 0.25 mLN = 98

n′ GMT GMT-fold increase (95% CI) n′ GMT GMT-fold increase (95% CI)

A/California/7/2009-H1N1Baseline (Day 1) 99 13.1 – 98 10.6 –Day 29 ± 3 days 99 257.6 19.6 (16.9, 22.7) – – – 0.031Day 57 ± 3 days – – – 98 270.0 25.5 (21.3, 30.5)Day 212a ± 7 days 88 180.1 14.4 (12.0, 17.4) 84 132.3 12.3 (10.3, 14.6) 0.110A/Perth/16/2009-H3N2Baseline (Day 1) 99 5.7 – 98 5.8 –Day 29 ± 3 days 99 140.6 24.6 (20.7, 29.3) 0.036Day 57 ± 3 days – – – 98 183.0 31.6 (26.7, 37.3)Day 212a ± 7 days 88 80.6 13.8 (11.5, 16.5) 84 89.1 16.1 (13.6, 19.1) 0.318B/Brisbane/60/2008Baseline (Day 1) 99 5.3 – 98 5.6 –Day 29 ± 3 days 99 77.0 14.7 (12.3, 17.4) – – – 0.263Day 57 ± 3 days – – – 98 71.9 12.8 (11.2, 14.6)Day 212a ± 7 days 88 41.3 7.9 (6.6, 9.3) 84 37.8 6.8 (5.8, 7.9) 0.214

G rval;

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Recently, awareness of the global influenza disease burden inhildren has increased. In 2008, an estimated 90 million new casesf influenza occurred worldwide in children aged < 5 years [14];0 million were influenza-associated lower respiratory tract infec-ions (LRTIs) [14]. The hospitalization rate in children aged < 6

onths from 2003 to 2008 was 30/10 000 cases [15], and in 2008orldwide between 28 000 and 111 500 deaths occurred in chil-ren aged < 5 years due to influenza-associated LRTIs [14]. Figuresrom the US show the majority of deaths actually occur in healthyhildren, and data from Europe further confirms the risk to healthyhildren [16]. Further economic data shows that influenza in younghildren costs annually $44–163 million in the US [17], and onetalian study estimated the cost to society of influenza in children

Please cite this article in press as: Esposito S, et al. The immunogeinfluenza vaccine administered to unprimed children aged ≥6 to <36 mhttp://dx.doi.org/10.1016/j.vaccine.2012.09.069

ged from birth to 14 years at more than D 1 billion over a five-yeareriod in Italy alone [18]. Together, these data show that there areoth health and economic reasons for vaccination of all children

able 5umber of children with solicited local and systemic adverse events (safety population).

Group A

1 × 0.5 mLN = 100 (99a)

Day 1

n (%c) Number of eve

Number of children with ≥1 solicited local AEs 17 (17.2)

Total number of AEs 30

Pain 9 (9.1) 9

Erythema 10 (10.1) 10

Eccymosis 5 (5.1) 5

Induration 6 (6.1) 6

Number of children with ≥1 solicited systemic AEs 20 (20.2)

Total number of AEs 22

Fever (≥38 ◦C) 15d (15.2) 15

Shivering 0 0

Malaise 7 (7.1) 7

E, Adverse event.a Number of children who returned the study diary.b The parents/legal guardian of one child withdrew consent before receipt of the secon

ncluded in the Day 29 ± 3 safety analysis.c Percentage is based on the number of children with at least one diary returned.d One child had missing temperature data which was considered per definition as fevee Three children had missing temperature data which was considered per definition as

N, number of subjects in the specified group and population; n′ , number of subjects

d samples.

aged > 6 months, not just those with chronic underlying medical

conditions. Despite this, there are still obstacles to the introduction

of universal pediatric influenza vaccination, such as minimizing

costs to families and health care providers and increasing aware-

ness of the disease [19,20].

To combat this high disease burden in children, 6 European

countries recommend influenza vaccination in all children aged > 6

months, with some country specific variations in the upper age

limit [21], yet this is not the case for the rest of Europe [22]. In

2010, the Advisory Committee on Immunization Practices (ACIP)

in the US updated their recommendations on influenza vacci-

nation to include all persons aged > 6 months [3], with Canada

following in 2011 [23]. However, this gives a total of only eight

nicity and safety of a single 0.5 mL dose of virosomal subunitonths: Data from a randomized, Phase III study. Vaccine (2012),

countries worldwide which in practice recommend and actively 330

vaccinate healthy children aged > 6 months against influenza [24], 331

with all other countries still only vaccinating patients with severe 332

Group B2 × 0.25 mLN = 102

Dayn = 102 (98a)

Day 29 ± 3n = 101b (93a)

nts n (%c) Number of events n (%c) Number of events

22 (22.4) 16 (17.2)36 23

11 (11.2) 11 10 (10.8) 1015 (15.3) 15 7 (7.5) 7

3 (3.1) 3 4 (4.3) 47 (7.1) 7 2 (2.2) 2

16 (16.3) 16 (17.2)18 19

8 (8.2) 8 11e (11.8) 111 (1.0) 1 0 09 (9.2) 9 8 (8.6) 8

d vaccination. This child is included in the safety population for Group B, but is not

r. fever.

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ARTICLEVAC 13599 1–8

S. Esposito et al. / Va

hronic underlying disease at risk of influenza complications andhe elderly. This is paradoxical given that the incidence of influenza-elated hospitalization is the same for children and elderly [16].

Prior to the introduction of the US ACIP recommendations, vac-ine coverage in children was particularly low. In the 2008–2009 USeason, 48.0% [24,25] of children aged 6–23 months received at leastne dose of influenza vaccine, but only 29.0% were fully vaccinated24]. Since 2010, this figure has increased with recent estimatesf vaccine coverage with one or more doses for the 2011–2012nfluenza season of 66.3% in children aged 6–23 months [26]. Inurope, the figures for influenza vaccination coverage in childrenre extremely low. Data from the Vaccine European New Inte-rated Collaboration Effort (VENICE) II report [27] revealed that,rstly, very few countries collect coverage data in children, and,econdly, of the two that provided data for the VENICE survey,rance reported influenza vaccination coverage in the 2008–2009eason of 9.9% for children aged 6 months to 4 years, and Portugalf 13% in children aged 6 months to 15 years. It should, however,e noted that neither of these two countries recommend influenzaaccination in children. In a surveillance study by Blank et al. [28],nfluenza vaccination coverage rates ranged from 4.0% to 19.0% inurope (France, Poland and Ireland 4.0%, Germany 19.0%). Theseata confirm that influenza vaccination coverage in children is par-icularly low, especially when compared with those of the elderlyopulation (∼70.0% in Europe) [16].

One of the main reasons behind the low influenza vaccinationoverage in children is non-compliance with the second dose [16].n our study, we eliminated the second dose by giving one sin-le 0.5 mL dose, a strategy which appears immunogenic and couldheoretically improve compliance and vaccination coverage rates.

e also show that the single 0.5 mL dose of virosomal subunitnfluenza vaccine was both safe and well tolerated by the children.

good and comparable safety profile was observed in both vacci-ated groups indicating a high overall tolerability of the virosomalubunit influenza vaccine, which supports safety data gathered in

previous study [10]. The incidence of AEs was within the normalange observed for seasonal influenza vaccines [29,30]. As recentvents associated with the use of influenza vaccine highlighted theisk of fever convulsions [31], we looked closer at this aspect. Noebrile convulsion was reported in this study. However, our studys limited by the relatively small sample size. Since fever rates tendo vary from year to year [32], the fever incidence observed inur study is within the variability of the fever incidences notedcross seasons. The study presented here was conducted in the010–2011 influenza season. During this season an increased fre-uency of fever cases (≥38 ◦C) was reported in other studies withn influenza trivalent inactivated vaccine (TIV) [33–35]. Further-ore, the CDC reported a safety signal triggered by the influenza

IV Fluzone of febrile convulsion [31]. To determine whether thesebservations were related to the strain composition of the vac-ine further research is needed, but it could be concluded thatn increased reactogenicity was seen overall after the use of theeasonal influenza vaccines in the 2010–2011 season [31,36]. More-ver, since the launch of Inflexal V, 68 million doses have beenistributed and there has been only one report of febrile convulsion

n association with Inflexal V occurring in 2006, in a one-year-oldhild with predisposing factors.

. Conclusions

Our results suggest a single 0.5 mL dose of virosomal subunit

Please cite this article in press as: Esposito S, et al. The immunogeinfluenza vaccine administered to unprimed children aged ≥6 to <36 mhttp://dx.doi.org/10.1016/j.vaccine.2012.09.069

accine is an immunogenic and safe measure of providing a long-erm protective immune response against influenza in children.hese findings will help to decrease the cost of influenza vaccina-ion and vaccine administration and the simplified dosing regimen

[

[

PRESSxxx (2012) xxx– xxx 7

is a potential way to improve compliance with vaccination. This,

in turn, should increase vaccination coverage in countries where

influenza vaccination in young children is recommended, ensur-

ing that a larger number of younger children, for whom an already

crowded vaccination schedule is the norm, are protected.

Role of the funding source

Crucell Switzerland AG was involved in study design, analysisand interpretation of data, writing of the report and in the decisionto submit the article for publication.

Acknowledgements

This study was sponsored by Crucell Switzerland AG. We would

like to thank Lyndsey Kostadinov (Crucell Switzerland AG) for writ-ing the manuscript.

Disclosure statement: GJW is an employee of Crucell. VM, SB,

EP, AA, VF, VP, and GVZ declare no conflicts of interest. AZ doesnot have any personal conflict of interests to declare; however,

the Dipartimento di Scienze Biomediche per la Salute, Universitàdegli Studi di Milano has received payment for assays performed

in laboratory on the serum samples collected for this study. SE and

PM have received research grants from Crucell, GlaxoSmithKline,

Medimmune, Novartis and Pfizer.

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