Immunogenicity profile of a 3.75-μg hemagglutinin pandemic rH5N1 split virion AS03A-adjuvanted vaccine in elderly persons: a randomized trial.

Abstract

Elderly persons often experience a reduced immune response to influenza vaccination. We evaluated the usual dose of AS03(A)-adjuvanted H5N1 pandemic vaccine (3.75 μg hemagglutinin of A/Vietnam/1194/2004-like strain) compared with a double dose in an elderly population.
This phase 2, open-label study (NCT00397215; http://www.clinicaltrials.gov) randomized participants (age, ≥61 years) to receive, on days 0 and 21: (1) a single dose of AS03(A)-adjuvanted vaccine (n=152), (2) a single dose of nonadjuvanted vaccine (n=54), (3) a double dose of AS03(A)-adjuvanted vaccine (n=145), or (4) a double dose of nonadjuvanted vaccine (n=44). The primary end point was hemagglutination inhibition (HI) and neutralizing antibody response against vaccine antigen (according-to-protocol cohort).
Day 42 geometric mean titers for HI antibodies were 126.8 and 237.3 for single and double doses of the AS03(A)-adjuvanted vaccine, respectively. Corresponding values for neutralizing antibodies were 447.3 and 595.8. Although the immune response was higher with the double dose, European Committee for Human Medicinal Products criteria for seroconversion and seroprotection rates were achieved in both AS03(A)-adjuvanted groups. Antigen-specific CD4 T cell responses were elicited. Immune response persistence at 6 months was high. Immune response in the non-adjuvanted groups was considerably less.
The AS03(A)-adjuvanted H5N1 vaccine can be administered elderly persons at the same dose and schedule as in younger adults.

Full text

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M A J O R A R T I C L E
Immunogenicity Profile of a 3.75-lg
Hemagglutinin Pandemic rH5N1 Split Virion
AS03A-Adjuvanted Vaccine in Elderly Persons:
A Randomized Trial
Ste ´phane Heijmans,1Marc De Meulemeester,1Paul Reynders,1,aDidier Giet,1Etienne Demanet,1
Pierre-Yves Devresse,1Giancarlo Icardi,3Mamadou Drame ´,2Franc xois Roman,2and Paul Gillard2
1ResearchLink, Department Clinical Trial Network, Linkebeek, Flemish Brabant,2GlaxoSmithKline Biologicals, Wavre, Walloon Brabant, Belgium;
3Department of Health Sciences and San Martino Teaching Hospital, University of Genoa, Genoa, Italy
Background.
evaluated the usual dose of AS03A-adjuvanted H5N1 pandemic vaccine (3.75 lg hemagglutinin of A/Vietnam/1194/
2004-like strain) compared with a double dose in an elderly population.
Methods.This phase 2, open-label study (NCT00397215; http://www.clinicaltrials.gov) randomized partic-
ipants (age, >61 years) to receive, on days 0 and 21: (1) a single dose of AS03A-adjuvanted vaccine (n 5 152), (2)
a single dose of nonadjuvanted vaccine (n 5 54), (3) a double dose of AS03A-adjuvanted vaccine (n 5 145), or (4)
a double dose of nonadjuvanted vaccine (n 5 44). The primary end point was hemagglutination inhibition (HI) and
neutralizing antibody response against vaccine antigen (according-to-protocol cohort).
Results. Day 42 geometric mean titers for HI antibodies were 126.8 and 237.3 for single and double doses of the
AS03A-adjuvanted vaccine, respectively. Corresponding values for neutralizing antibodies were 447.3 and 595.8.
Although the immune response was higher with the double dose, European Committee for Human Medicinal
Products criteria for seroconversion and seroprotection rates were achieved in both AS03A-adjuvanted groups.
Antigen-specific CD4 T cell responses were elicited. Immune response persistence at 6 months was high. Immune
response in the non-adjuvanted groups was considerably less.
Conclusions.The AS03A-adjuvanted H5N1 vaccine can be administered elderly persons at the same dose and
schedule as in younger adults.
Elderly persons often experience a reduced immune response to influenza vaccination. We
The past decade has seen unprecedented preparation for
a human influenza pandemic. Until the 2009 outbreak
of human cases of (swine) influenza A (H1N1), the
avian influenza H5N1 virus was considered a potential
cause of a human influenza pandemic. The H5N1 virus
remains a considerable threat to human health, and it is
important that pandemic preparedness planning for an
outbreak continues. Moreover, vaccine development
strategies for a potential H5N1 pandemic can guide
development of vaccines to combat the 2009/10 H1N1
pandemic andotherfuture
pandemics.
Development of an influenza vaccine that induces
a cross-reactive immune response is a key component of
a pandemic preparedness strategy. Such a vaccine is
likely to prime the immune system to mount a rapid
response to vaccination with a drifted strain and/or to
potentialinfluenza
Received 15 June 2010; accepted 13 October 2010.
Potential conflicts of interest: G.I. has been an investigator of studies sponsored
by GlaxoSmithKline (GSK) and has participated in symposia funded by GSK
Biologicals, Italy; his institution has received grant support from GSK Biologicals
for conducting this study. S. H. has received compensation from GSK Biologicals for
travel and accommodation expenses to present data from this study at a congress
in 2008. M.D.M. has received investigator fees from GSK Biologicals for
conducting this study. P.-Y.D. has received grant support and D. G.'s and E.D.'s
institutions have received honoraria via ResearchLink for conducting this and other
vaccine studies. M.D., F.R., and P.G. are employees of GSK Biologicals. F.R. and P.G.
report ownership of stock options.
Presented in part: The 2nd Vaccine Congress, Boston, Massachusetts, 7-9
December 2008. Abstract AS03.
aDeceased.
Correspondence: Ste ´phane Heijmans, MD, Rue de la Station 78, 1630
Linkebeek, Belgium (stephane.heijmans@researchlink.be).
The Journal of Infectious Diseases
? The Author 2011. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved.
This is an Open Access article distributed under the terms of the Creative Commons
Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/
2.5), which permits unrestricted non-commercial use, distribution, and reproduction
in any medium, provided the original work is properly cited.
1537-6613/2011/2038-0001$15.00
DOI: 10.1093/infdis/jiq174
2011;203:1054–62
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infection and may be used before the onset of a pandemic or in
its early stages. Even partial cross-protection may have a con-
siderable impact on infection rates during early pandemic stages
[1, 2]. The vaccine must provide a high and long-lasting im-
mune response at a relatively low antigen dose, because the need
for a high dose would exhaust the limited global production
capacity for influenza antigen. Formulation of pandemic vac-
cines using adjuvants to stimulate a robust immune response is
an important approach to reducing the antigen dose and elic-
iting a cross-reactive response [3].
An H5N1 pandemic vaccine based on the A/Vietnam/1194/
2004 clade 1 strain is licensed to be used in the event of an
imminent H5N1 pandemic. The vaccine is adjuvanted with
AS03A,anoveltocopheroloil-in-wateremulsion-basedadjuvant
system(11.86mgoftocopherol).Itislicensedtobe deliveredvia
a schedule of 2 injections administered 3 weeks apart [4]. In
a study in adults aged 18-60 years, the lowest dose investigated
(3.75 lg hemagglutinin [HA]) elicited immune responses
against the vaccine strain that met all US Center for Biologics
Evaluation and Research and European Committee for Human
Medicinal Products (CHMP) immunologic licensure criteria
[5]. Much higher doses were needed to induce a moderate im-
mune response with a nonadjuvanted H5N1 split-virus vaccine
[6], indicating that adjuvantation with AS03Aallows successful
dose sparing. In addition, the vaccine has been shown to induce
a cross-reactive immune response (hemagglutination inhibition
[HI]) and neutralizing antibodies and CD4 T cell–mediated
immuneresponse)againstdriftedclade2H5N1strains[5,7–10]
An acceptable safety and reactogenicity profile has been dem-
onstrated [5, 8, 11].
Studies with seasonal influenza vaccines suggest that the re-
duced immune response to vaccination in elderly populations is
due – at least in part – to immunosenescence [12]. It is possible
that elderly people may therefore need additional or higher
doses. This study aimed to assess the immune response in an
elderly population to 2 single or 2 double doses of the AS03A-
adjuvanted H5N1 pandemic vaccine, administered 21 days
apart, compared with the same dosage and schedule without the
adjuvant system.
METHODS
The primary objectives of the study were to evaluate the im-
munogenicity of the H5N1 vaccine 21 days after receipt of the
first dose of vaccine (day 21) and 21 days after receipt of
the second dose (day 42) and to assess persistence of the im-
mune response for up to 2 years. Safety was also assessed. This
article reports immunogenicity data up to 180 days; safety data
are presented in a supplement.
The study was conducted in accordance with the current ver-
sion of the Declaration of Helsinki and the International Con-
ferenceonHarmonisationGoodClinicalPracticeguidelines.The
protocol was approved by the independent ethics committee or
institutional review board of each study center, and written
informed consent was obtained from each participant.
Study Vaccines
The H5N1 inactivated, split-virion recombinant vaccine was
manufactured by GlaxoSmithKline (GSK) Biologicals, as de-
scribed elsewhere [5, 7]. The vaccine contained 3.75 lg HA
of the A/Vietnam/1194/2004-like NIBRG-14 clade 1 strain
(National Institute for Biological Standards and Control). An
adjuvantedandanonadjuvantedvaccinewereusedinthisstudy.
The adjuvanted vaccine (Prepandrix a trademark of the GSK
group of companies) contained AS03A, an oil-in-water emul-
sion-based adjuvant system containing DL-a-tocopherol (11.86
mg) [5]. The vaccines (0.5 mL per single dose) were adminis-
tered intramuscularly into the deltoid region of the non-
dominant arm for the single dose and into the deltoid region of
each arm for the double dose.
Study Design
This
(NCT00397215). The study was conducted in 2 phases, with
the primary phase taking place between days 0 and 51, and the
follow-up phase taking place between days 51 and the end of
the study. The primary phase ended 30 days after the receipt of
the last vaccine dose, the period during which participants re-
corded all adverse events. Participants were enrolled in-
dependently in each phase and did not have to complete the
primary phase (ie, to comply with telephone contact at day 51)
to enroll in the follow-up phase.
Male and female participants aged >61 years, who were in
good healthor who had well-controlled underlying disease, were
enrolled in the study. Participants were randomized at a ratio of
3:1:3:1 to 1 of 4 vaccine study groups: (1) a single dose of the
AS03A-adjuvanted vaccine (1 3 H5N1-AS), (2) a single dose of
the nonadjuvantedvaccine (13H5N1),(3)adouble doseofthe
AS03A-adjuvantedvaccine (2 3H5N1-AS),or (4) a doubledose
of the nonadjuvanted vaccine (2 3 H5N1). Participants in the
single-dose groups received one vaccine dose on day 0 and an-
other on day 21. Participants in thedouble-dose groupsreceived
2 doses on day 0 and 2 doses on day 21. Participants who had
not received an influenza vaccine for the 2006-2007 season were
vaccinated with FluarixNorthern hemisphere 2006/2007 (GSK
Biologicals) at least 3 weeks before administration of the H5N1
vaccine.
A randomization blocking scheme was used. The randomi-
zation list was generated at GSK Biologicals using standard
SAS software. Participants ineachgroupwerestratified ataratio
of 1:1:1 by age group (61-65, 66-70, and .70 years).
wasa phase2, randomized,open-label study
Immunogenicity Evaluation
Blood samples were taken on days 0, 21, 42, and 180. Additional
samples will be taken from Belgian participants at month 12 and
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month 24. The primary end points were humoral immune re-
sponses in terms of anti-HA and neutralizing antibodies against
the vaccine antigen. The cell-mediated immune response was
also evaluated.
Anti-HA antibody titers were measured using an HI
assay which corresponded to international standards [13,14].
Each serum sample was tested in duplicate. The following
parameters with 95% confidence intervals (CIs) were de-
termined: (1) geometric mean titer (GMT) on days 0, 21, 42,
and 180; (2) seroconversion rate (SCR) on days 21, 42, and
180; (3) and seroprotection rate (SPR) on days 0, 21, 42, and
180. SCR was defined as the percentage of participants
with either (1) a prevaccination titer ,1:10 and a post-
vaccination titer >1:40, or (2) a prevaccination titer >1:10
anda
>4-foldincrease in
was defined as the percentage of participants with an HI titer
>1:40.
Virus neutralization by serum antibodies was determined by
a microneutralization assay on thawed frozen serum samples
that had been subjected to heat inactivation for 30 min at 56?C.
Each serum sample was tested in triplicate. A standard amount
of virus was mixed with serial dilutions of serum and incubated
to allow antibody binding to the virus. A cell suspension con-
taining a defined number of Madin-Darby canine kidney cells-
was then added to the mixture of virus and antiserum and
incubated for 7 days at 37?C. After the incubation period,
virus replication was visualized by hemagglutination of
chicken red blood cells. The 50% neutralization titer of the se-
rum was calculated according to the method of Reed
and Muench [15]. Neutralizing antibodies were evaluated in
a subset of participants in the AS03A-adjuvanted vaccine
groups using the following parameters with 95% CIs: (1) GMT
on days 0, 42, and 180 and (2) SCR on days 42 and 180. Sero-
conversion was defined as the percentage of participants with
a >4-fold increase in postvaccination neutralizing antibody
titer.
The cell-mediated immune response was assessed using
an adaptation of the method described by Maecker et al
[16]. Peripheral blood mononuclear cells were restimulated
in vitro using the H5N1 split antigen from the A/Vietnam/
1194/2004-like NIBRG-14 vaccine strain. The cells were
labeled using conventional immunofluorescence labeling of
cellular phenotype markers and intracellular cytokines and
analyzed by flow cytometry. Results were expressed as frequency
of CD41or CD81T cells responding to the antigen and
expressing at least 2 markers (of CD40L, interferon-c,
interleukin-2, and tumor necrosis factor–a) per million CD41
or CD81T cells in total which is deemed to give an adequate
estimate of the CD4 T cell specific response against the
A/influenza antigen, on the basis of previous published ob-
servations [17–19]. Response was measured on days 0, 21, 42,
and 180.
postvaccinationtiter.SPR
Statistical Analysis
The primary immunogenicity analysis was performed on the
according-to-protocol (ATP) cohort, including all participants
who met all eligibility criteria, complied with protocol proce-
dures, and had antibody assay results available. The immuno-
genicity analysis at day 180 was performed on the ATP
persistence cohort. A subset of 90 participants in each AS03A-
adjuvanted vaccine group enrolled in Belgium was randomly
selected for measurement of neutralizing antibodies.
The HI antibody response was analyzed in terms of CHMP
criteria for approval of an influenza vaccine in adults aged >60
years (ie, the SCR and SPR must be .30% and .60%, re-
spectively). No correlate of protection for neutralizing anti-
bodies hasbeenestablished,
postvaccination 4-fold increase in titer seems a reasonable
standard.
The target sample size was 480 participants—180 in each
AS03A-adjuvanted vaccine group and 60 in each nonadjuvanted
vaccine group—to achieve 456 evaluable participants, assuming
a 5%drop-outrate.The co-primary GMT endpoint wasusedto
estimate sample size. A sample size of 170 evaluable participants
per group was estimated to have 86% power to detect a 1.7-fold
difference between the 2 AS03A-adjuvanted vaccine groups us-
ing a 2 sample t test with a .05 2-sided significance level, as-
suming that the standard deviations were 0.738 and 0.656 (log10
unit) for the 2 3 H5N1-AS group and the 1 3 H5N1-AS group,
respectively, as previously observed [5].
Antibody titers below the assay cut-off value were given an
arbitrary value of one-half the cut-off for the GMT calculation.
A seronegative participant was defined as having an antibody
titer less than the cut-off value; a seropositive participant was-
defined as having a titer greater than or equal to the cut-off
value. Proc StatXact 5.0 was used to calculate 95% CIs for GMT,
SCR, and SPR. The single-dose and double-dose groups
were compared for HI antibodies in terms of GMT ratios
and difference in SCRs. The 95% CIs of the GMT ratios (double
dose/single dose and AS03A-adjuvanted/nonadjuvanted) were
computed using an analysis of covariance model (ANCOVA)
with the vaccine group as a fixed effect and the pre-vaccination
titer as regressor. For the difference in SCR (double-dose minus
single-dose and AS03A-adjuvanted minus nonadjuvanted), the
asymptotic standardized 95% CIs were computed. Results for
neutralizing antibodies and cell-mediated immunity were re-
ported descriptively.
buttheconventional
RESULTS
The study took place during the period from November 2006
and March 2008 (up to the point of the present analysis at Day
180) in seven centers in Belgium and five centers in Italy. A total
of 437 participants were enrolled in the primary study phase,
which 415 completed (Figure 1a). Although the target number
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of evaluable participants was not quite reached, satisfactory
statistical power was achieved. A total of 431 participants con-
tinuedinthefollow-upphase, which 421 completed (Figure 1b).
The ATP cohort for immunogenicity (primary phase) and
ATP persistence cohort (follow-up phase) included 395 and 376
participants, respectively (Figure 1a and b). Demographic
characteristics were similar across the study groups. The mean
age (6 standard deviation) in the ATP cohort for immunoge-
nicity was 69.7 6 6.5 years (range, 61–89 years); 45% of subjects
were women, and 97% were of Caucasian/European heritage.
The number of participants in different age groups is shown in
Table 1. Characteristics were similar in the ATP persistence
cohort (data not shown) and the total vaccinated cohort (data
shown in safety supplement).
Figure 1.
single dose of the nonadjuvanted vaccine; 2 3 H5N1-AS, double dose of the AS03A-adjuvanted vaccine; 2 3 H5N1, double dose of the nonadjuvanted
vaccine. AE, adverse event; ATP, according-to-protocol; SAE, serious adverse event; TVC, total vaccinated cohort.
Disposition of study participants. Vaccination groups are as follows: 1 3 H5N1-AS, single dose of the AS03A-adjuvanted vaccine; 1 3 H5N1,
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HI Antibody Response
In both AS03A-adjuvanted vaccine groups, GMTs rose sub-
stantially <3 weeks after receipt of the first dose of vaccine (day
21) (Table 2; total ATP cohort). Values increased further within
3 weeks after receipt of the second dose of vaccine (day 42)
(Table 2). Although there was a good immune response in the
13H5N1-ASgroup,itwasgreaterinthe23xH5N1-ASgroup,
with a GMT ratio (double dose/single dose) on day 42 of 1.87
(95% CI, 1.36–2.59; P 5, .001).
GMTs were considerably lower in the nonadjuvanted vaccine
groups (Table 2). The GMT ratio (AS03A-adjuvanted/non-
adjuvanted) was 5.58 (95% CI, 3.48–8.95; P , .001) for the
single-dose groups and 9.38 (95% CI, 5.93–14.83; P , .001) for
the double-dose groups.
Results for SCR and SPR mirrored those for GMT (Table 2).
For SCR and SPR, CHMP criteria were met for both AS03A-
adjuvanted vaccine dose groups (1 3 H5N1-AS and 2 3 H5N1-
AS) at days 21 and 42 but were not met at all for the
nonadjuvanted groups (1 3 H5N1 and 2 3 H5N1). All CHMP
requirements were met for both adjuvanted vaccine dose groups
inthe differentage strata(61–65,66–70, and.70years;datanot
shown). Comparing the double dose of the AS03A-adjuvanted
vaccine with the single dose, the difference in SCR (double dose
minus single dose) was 15.91% (95% CI, 7.00%–24.79%; P ,
.001), although a high SCR was achieved with the single dose.
Comparing the AS03A-adjuvanted and nonadjuvanted vaccines,
the differencein SCR(AS03A-adjuvanted
adjuvanted) was 50.15% (95% CI, 35.59%–61.73%; P , .001)
for the single-dose groups and 65.55% (95% CI, 50.30%–
76.76%; P , .001) for the double-dose groups.
Remarkably, 38% of participants were seropositive for HI
antibodies against the vaccine antigen before vaccination, and
the proportion of seropositive subjects tended to increase with
age. An exploratory analysis was therefore undertaken in par-
ticipants who were seronegative at baseline. There was a high
immune response in this population, although GMTs were
somewhat lower than in the ATP cohort as a whole (Table 2).
minusnon-
However, all CHMP criteria were met in both the single-dose
and double-dose AS03A-adjuvanted vaccine groups in seroneg-
ative participants, except for SPR at day 21 (Table 2).
There was a decrease in GMT levels between days 42 and 180;
levels remained considerably higher in the AS03A-adjuvanted
vaccine groups (Table 2). SCR was above the postvaccination
licensure criterion in both AS03A-adjuvanted vaccine groups at
day 180, and SPR was above the criterion in the double-dose
group (Table 2).
Neutralizing Antibody Response
Most participants (.90%) were seropositive for neutralizing
antibodies before vaccination. Nevertheless, there was a sub-
stantial increase in GMT levels after vaccination and a high SCR
in both AS03A-adjuvanted vaccine groups (Table 3). The im-
mune response was somewhat higher in the double-dose group
(2 3 H5N1-AS), compared with the singe-dose group (1 3
H5N1-AS). As seen with HI antibodies, GMTs and SCRs de-
creased between day 42 and Day 180, although GMTs remained
above pre-vaccination level (Table 3).
Cell-Mediated Immunity
Antigen-specific CD4 T cell responses were elicited in all study
groups but were markedly higher in the AS03A-adjuvanted
vaccine groups, particularly the double-dose group (Figure 2).
As with humoral immunity, there was a good response after the
first vaccination, with a further increase after the second vacci-
nation. Responses decreased somewhat by day 180, although
persistence was higher in the AS03A-adjuvanted vaccine groups
than in the nonadjuvanted groups (Figure 2). No CD8 T cell
responses were observed.
Safety
Safety and reactogenicity data are presented in the safety sup-
plement. No vaccine-related serious adverse events were re-
ported and no safety concerns were identified.
DISCUSSION
A high immune response in terms of HI antibodies, neutralizing
antibodies, and cell-mediated immunity was induced by 2 in-
jections of a single dose of 3.75 lg HA AS03A-adjuvanted vac-
cine given 3 weeks apart to participants aged .60 years. All
CHMP requirements for influenza vaccines in the elderly pop-
ulation were met for this regimen. The more stringent CHMP
criteria for vaccination of younger adults (.40% for SCR and
.70% for SPR) were also achieved. The benefit of the AS03A
adjuvant system was demonstrated since the nonadjuvanted
vaccine groups showed markedly lower immune responses that
failed to meet regulatory acceptance criteria. Persistence of the
immune response was also high 6 months after vaccination. The
SCR was still above the postvaccination licensure criterion with
both adjuvanted formulations, and the SPR was above the
Table 1.
Cohort)
Age Range of Participants (According to Protocol
No. of participants in each age group
Age, years13H5N1-AS13H5N123H5N1-AS23H5N1
61-654515 4612
66-70 4619 4614
71-75 309277
76-80
>81
186 165
135 106
NOTE. Vaccination groups are as follows: 1 3 H5N1-AS, single dose of the
AS03A-adjuvanted vaccine; 1 3 H5N1, single dose of the nonadjuvanted
vaccine; 2 3 H5N1-AS, double dose of the AS03A-adjuvanted vaccine; 2 3
H5N1, double dose of the nonadjuvanted vaccine.
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Table 2. Geometric Mean Titer (GMT), Seroconversion Rate (SCR), and Seroprotection Rate (SPR) for H5N1 HI Antibodies Against A/Vietnam/1194/2004
Parameter
ATP cohort (total)ATP cohort (seronegative at baseline)
13H5N1-AS
152
13H5N1
54
23H5N1-AS
145
23H5N1
44
13H5N1-AS
90
13H5N1
33
23H5N1-AS
93
23H5N1
28 No. of subjectsTime
GMT (95% CI)Day 0 11.3 (9.2–13.9)9.7 (7.3–13.0) 10.2 (8.4–12.5)8.8 (6.6–11.8)5.0 (5.0–5.0)5.0 (5.0–5.0)5.0 (5.0–5.0)5.0 (5.0–5.0)
Day 21
Day 42
50.0 (38.1–65.6)
126.8 (99.4–161.7)
16.8 (11.7–24.0)
22.7 (15.1–34.1)
69.4 (52.1–92.3)
237.3 (191.9–293.6)
20.8 (13.0–33.3)
25.3 (16.0–40.1)
25.3 (18.5–34.6)
80.0 (58.3–109.8)
9.7 (6.4–14.7)
13.4 (8.0–22.5)
40.2 (29.5–54.8)
198.5 (157.5–250.2)
10.5 (6.5–16.9)
13.1 (8.1–21.3)
SCR, % (95% CI)Day 21
Day 42
45.4 (37.3–53.7)
72.4 (64.5–79.3)
14.8 (6.6–27.1)
22.2 (12.0–35.6)
52.4 (44.0–60.8)
88.3 (81.9–93.0)
18.2 (8.2–32.7)
22.7 (11.5–37.8)
44.4 (34.0–55.3)
73.3 (63.0–82.1)
18.2 (7.0–35.5)
21.2 (9.0–38.9)
50.5 (40.0–61.1)
94.6 (87.9–98.2)
14.3 (4.0–32.7)
17.9 (6.1–36.9)
SPR, % (95% CI)Day 0
Day 21
18.4 (12.6–25.5)
61.2 (53.0–69.0)
13.0 (5.4–24.9)
27.8 (16.5–41.6)
15.9 (10.3–22.8)
62.1 (53.6–70.0)
4.5 (0.6–15.5)
34.1 (20.5–49.9)
0.0 (0.0–4.0)
44.4 (34.0–55.3)
0.0 (0.0–10.6)
18.2 (7.0–35.5)
0.0 (0.0–3.9)
50.5 (40.0–61.1)
0.0 (0.0–12.3)
14.3 (4.0–32.7)
Day 4283.6 (76.7–89.1)35.2 (22.7–49.4)95.9 (91.2–98.5)38.6 (24.4–54.5)73.3 (63.0–82.1) 21.2 (9.0–38.9)94.6 (87.9–98.2) 17.9 (6.1–36.9)
ATP persistence cohort
No. of subjects142 54 13644
GMT (95% CI) Day 18038.5 (30.0–49.5) 16.3 (11.0–24.3)53.5 (41.9–68.4)13.1 (8.9–19.2)ND NDND ND
SCR, % (95% CI) Day 18037.1 (29.1–45.7) 12.5 (4.7–25.2)53.8 (44.9–62.6) 14.3 (5.4–28.5) NDNDND ND
SPR, % (95% CI)Day 180 52.9 (44.2–61.3)26.0 (14.6–40.3)69.5 (60.8–77.2) 20.5 (9.8–35.3)NDND NDND
NOTE. Vaccination groups are as follows: 1 3 H5N1-AS, single dose of the AS03A-adjuvanted vaccine; 1 3 H5N1, single dose of the nonadjuvanted vaccine; 2 3 H5N1-AS, double dose of the AS03A-adjuvanted vaccine;
2 3 H5N1, double dose of the nonadjuvanted vaccine. European Committee for Human Medicinal Products (CHMP) criteria in adults aged >60 years were an SCR .30% and an SPR .60%.
SCR was defined as the percentage of participants with either (1) a prevaccination titer ,1:10 and a postvaccination titer >1:40 or (2) a prevaccination titer >1:10 and a >4-fold increase in postvaccination
titer; only (1) applies to initially seronegative participants. SPR: was defined as the percentage of participants with a hemagglutination inhibition titer >1:40 ATP, according to protocol; CI, confidence interval; HI,; ND,
not done.
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criterion for the double-dose adjuvanted formulation. Although
the specified sample size was not reached, adequate statistical
power was achieved. Originally, a sample size of 170 subjects per
group was estimated to have 86% power to detect a 1.7-fold
difference between GMTs in the 2 AS03-adjuvanted groups. In
fact, there was a 1.87-fold difference between GMTs in the 2
groups, and the reestimated power based on this difference was
calculated to be 94.1%.
Assessment of a double-dose vaccine regimen was motivated
by the well-recognized lower immune response in elderly per-
sons [12]. Accordingly, the single-dose regimen induced lower
immune responses after 2 vaccinations than usually observed in
adults aged 18-60 years [5, 8, 9]. However, although the double
dose of the AS03A-adjuvanted vaccine produced a higher im-
mune response, licensing criteria were easily met with the single
vaccine dose following a 2-vaccination administration schedule.
This supports the use of the single dose in both the elderly
population and among adults aged 18-60 years, as has been
recognized by regulatory authorities with the recent extension of
the vaccine licence to all adults aged .18 years [4].
When the overall study population was considered, only 1
dose of AS03A-adjuvanted vaccine (either single or double)
seemed sufficient to reach both CHMP SCR and SPR criteria.
However, further exploratory analysis showed that this effect
was mainly driven by the HI immune response in initially se-
ropositive individuals, who represented 38% of the ATP cohort
for immunogenicity. In initially seronegative individuals, the
response was lower, and all CHMP criteria were only met after 2
vaccinations with both the single- and double-dose regimens,
indicating that a majority of elderly subjects will benefit from
a 2-vaccination administration schedule, with doses given 21
days apart. The proportion of initially seropositive participants
was higher than previously observed in younger adult pop-
ulations [5, 8, 9] and may have occurred as a result of previous
seasonal influenza vaccination or natural infection, as several
studies have demonstrated cross-reactivity between avian and
human strains for cell-mediated immunity and neutralizing
antibody response [20–22]. More recently, cross-reactivity for
an HA-specific antibody response was demonstrated in a study
in which a seasonal influenza vaccine containing H1 and H3
antigens produced antibodies that cross-reacted with H5 HA
[23]. Alternatively, the high seropositivity may reflect false-
positive assay results, and it is possible that the micro-
neutralization assay may have captured some N1 response.
However, the HI assay used corresponds to international
standards [14], and we are confident that the antibody activity
measured is specific to H5N1. Thus, we believe that cross-
reactivity between seasonal and avian influenza strains is a more
likely explanation of the high levels of prevaccination seropos-
itivity. Antibody titers may have limitations as the sole indicator
of influenza vaccine efficacy in elderly persons, and it has been
suggested that measures of the cell-mediated immune response
should be included in evaluations of vaccines in this population
[24–27]. Measurement of cell-mediated immunity was therefore
included in the present study to gain a better understanding of
the overall immune response induced by the vaccine and, thus,
a better evaluation of its protective potential. The study showed
that the pattern of the immune response to the AS03A-ad-
juvanted H5N1 vaccine was similar in terms of HI antibodies,
neutralizing antibodies, and cell-mediated immunity (CD4 T
cell responses). No CD8 T cell responses were observed. This
may be due to the assay method or the protein content of the
vaccine,althoughthepossibilitythatthevaccinedoesnotinduce
CD8 T cell responses cannot be excluded.
It is interesting to note that the kinetics of the immune re-
sponse to the AS03A-adjuvanted H5N1 vaccine in elderly sub-
jects seemed different from those observed in younger adults:
3 weeks after the first dose of vaccine was administered, HI
antibody titers against the vaccine strain in the present study
were higher than those in younger adults [5, 8, 9]. In contrast,
3 weeks after the second dose of vaccine was administered, titers
were higherintheyoungerindividuals.Althoughtherewas some
Table 3.
AS03A-Adjuvanted Groups
Geometric Mean Titer (GMT) and Seroconversion Rate (SCR) for Neutralizing Antibodies Against A/Vietnam/1194/2004 in the
Parameter
Subset of ATP cohortTime13H5N1-AS23H5N1-AS
No. of subjects8782
GMT (95% CI)Day 0
Day 42
121.1 (94.6–154.9)
447.3 (359.3–557.0)
112.5 (90.7–139.5)
595.8 (487.7–727.8)
SCR, % (95% CI)Day 42 44.8 (34.1–55.9)56.1 (44.7–67.0)
Subset of ATP persistence cohort
No. of subjects76 73
GMT (95% CI) Day 180 218.2 (172.4–276.2) 260.9 (207.7–327.8)
SCR, % (95% CI) Day 18021.1 (12.5–31.9)28.8 (18.8–40.6)
NOTE. Vaccine groups are as follows: 13H5N1-AS, single dose of the AS03A-adjuvanted vaccine; 2 3 H5N1-AS, double dose of the AS03A-adjuvanted vaccine.
ATP: according to protocol; CI, confidence interval.
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decline, theimmune response persisted up to 6monthsafterfirst
vaccination, consistent with results seen in younger adults [8].
The safety and reactogenicity profile of the AS03A-adjuvanted
H5N1 vaccine in elderly subjects was similar to that in younger
adults (data shown in safety supplement), and no safety con-
cerns were raised. Although there were more local and general
symptoms reported with the adjuvanted vaccine than the non-
adjuvanted vaccine, they were mainly mild-to-moderate and
transient in nature, and reactogenicity was clinically acceptable.
In conclusion, 2 single 3.75-lg HA doses of the AS03A-
adjuvanted H5N1 vaccine administered 21 days apart induced
a high immune response in an elderly population. The study
indicates that elderly persons do not require a higher vaccine
dose than younger adults and the vaccine can be administered
according to thesameschedule. This will support a dose-sparing
strategy, because elderly persons are an important target for
vaccination in a pandemic situation.
Supplementary Data
Supplementary data are available at http://www.oxfordjournals.
org/our_journals/jid/online.
Funding
The study was funded by GSK Biologicals. Funding to pay the Open
Access publication charges for this article was provided by GlaxoSmithKline
Biologicals.
Acknowledgments
We are indebted to the participating study volunteers, clinicians, nurses,
and laboratory technicians at the study site and the sponsor’s project staff
for their support and contributions throughout the study, in particular to
Adriano Lazzarin, Giuseppe Ferrera, and Rosa Cristina Coppola as in-
vestigators, to Koen Ceulemans and Beata De Vos (GSK Biologicals,
Belgium), Maria-Primula Leone and Miriam Vighini (GSK Biologicals,
Italy) for study coordination, to Erick Rosas for preparation of the study
protocol and related study documentation and to Joe ¨lle Thonnard, safety
physician. We are grateful to Pascal Ge ´rard and his team and to Elizabeth
Neumeier, who performed the serological laboratory work, and to Phil-
ippe Moris and his team for performing the CMI analysis. We are also
grateful to the National Institute for Biological Standards and Control for
providing the vaccine virus strain for the assays and reference standards
and also to the Centers for Disease Control and Prevention (Centers for
Disease Control and Prevention) for supplying the recombinant A/In-
donesia/5/2005 strain. Finally we thank Mary Greenacre (An Sgriobha-
dair) who provided medical writing services and Isabelle Gautherot (GSK
Biologicals, Belgium) and Geraldine Verplancke (Keyrus Biopharma, on
behalf of GSK Biologicals Belgium) for coordination, funded by GSK
Biologicals.
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