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ARTICLE IN PRESS Model
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
ARTICLE IN PRESSG Model
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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;
wg bloo
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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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