AS03 A ‐Adjuvanted Influenza A (H1N1) 2009 Vaccine for Adults up to 85 Years of Age

668 • CID 2010:51 (15 September) • Roman et al

M A J O R A R T I C L E

AS03A-Adjuvanted Influenza A (H1N1) 2009 Vaccinefor Adults up to 85 Years of Age

Francois Roman,1 Tejaswini Vaman,1 Froukje Kafeja,2 Emmanuel Hanon,1 and Pierre Van Damme2

1GlaxoSmithKline Biologicals, Wavre, and 2Vaccine and Infectious Disease Institute, Centre for the Evaluation of Vaccination,University of Antwerp, Antwerp, Belgium

Background. Vaccination of high-risk groups was started shortly after the emergence of the influenza A (H1N1)2009 pandemic virus.

Methods. Healthy adults were enrolled into 2 age strata: 18–60 years and 160 years, and received monovalentinfluenza vaccine containing 3.75 mg of A/California/2009 (H1N1) hemagglutinin antigen, adjuvanted with AS03A.Hemagglutination inhibition assay–based antibody titers against H1N1 vaccine were assessed after 1 vaccine dose(primary endpoint), after which subjects were randomized 1:1 to receive no further vaccination or a second dose.Immunogenicity endpoints were European licensure criteria for influenza vaccines. Exploratory analyses assessedthe effect of previous seasonal influenza vaccination on responses to the H1N1 vaccine.

Results. Licensure criteria for immunogenicity were fulfilled after 1 dose of H1N1 vaccine ( ). ForN p 240subjects 18–60 years of age, previous vaccination against seasonal influenza within the preceding 2 seasons resultedin significantly lower geometric mean titers (adjusted for baseline antibody titer) after 1 or 2 doses of H1N1vaccine ( and , respectively). Transient mild or moderate injection-site pain was reported by 87.5%P ! .001 P p .003and 65.0% of subjects 18–60 years of age and 160 years of age, respectively, after the first dose, and in 63% ofsubjects overall after the second dose.

Conclusions. A single dose of 3.75 mg hemagglutinin antigen, AS03A-adjuvanted H1N1 2009 vaccine wasimmunogenic and well tolerated in adults. In exploratory analyses (of subjects 18–60 years of age), postvaccinationantibody titers were lower in subjects who had previously received seasonal influenza vaccination, compared withthose who had not. This phenomenon warrants further investigation.

Clinical trials registration. NCT00968526.

The swine-origin influenza A H1N1v strain was first

identified in April 2009, and following the rapid world-

wide spread of the virus, on 11 June 2009, the World

Health Organization (WHO) declared a phase 6 pan-

demic alert [1, 2]. As of 3 April 2010, the WHO stated

that laboratory-confirmed cases of H1N1 2009 pan-

demic influenza had been reported in 214 countries

and were associated with 117,798 deaths [3].

Disease control and prevention agencies recom-

mended that vaccination of high-risk groups should

begin as soon as vaccine against the novel H1N1 virus

Received 22 February 2010; accepted 6 June 2010; electronically published 4August 2010.

Reprints or correspondence: Dr Pierre Van Damme, Vaccine and Infectious DiseaseInstitute, Centre for the Evaluation of Vaccination, University of Antwerp, Campus 3Eiken, Universiteitsplein 1, 2610 Antwerpen, Belgium ([email protected]).

Clinical Infectious Diseases 2010; 51(6):668–677� 2010 by the Infectious Diseases Society of America. All rights reserved.1058-4838/2010/5106-0005$15.00DOI: 10.1086/655830

became available [4, 5]. However, meeting the global

demand for vaccine within the constraints of the ex-

isting vaccine-manufacturing infrastructure involves re-

ducing the amount of antigen required per dose (an-

tigen sparing), which can be achieved by formulating

the vaccine with an adjuvant [6, 7]. Experience with

H5N1 pandemic vaccines shows that formulation with

oil-in-water adjuvant systems allows for antigen spar-

ing [8–10].

Recent experience has shown that robust immune

responses are obtained with single doses of H1N1 2009

vaccine formulated with or without adjuvants [11–16].

Furthermore, in recent studies with H1N1 2009 vac-

cines, the concomitant administration of H1N1 2009

vaccine and Northern Hemisphere trivalent seasonal

influenza vaccine had no effect on the immunogenicity

of either vaccine [8, 13, 16, 17]. These findings support

a coadministration strategy, which is favorable for the

administration of vaccines to high-risk individuals for

by guest on October 14, 2011

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AS03A-Adjuvanted Pandemic H1N1 Vaccine • CID 2010:51 (15 September) • 669

Figure 1. Subject enrollment in study to evaluate immunogenicity and safety of an investigational influenza vaccine (H1N1) in adults (8–16 September2009).

whom both pandemic and seasonal influenza vaccines are

indicated.

However, recent trial data regarding H5N1 pandemic influ-

enza vaccines have shown that previous receipt of seasonal

influenza vaccination may lower immunogenicity to subse-

quent pandemic influenza vaccination [18, 19]. Moreover, al-

though giving pandemic and seasonal influenza vaccination

together does not appear to influence immunogenicity, the pre-

vious receipt of Northern Hemisphere trivalent influenza vac-

cine for any season between 2004–2005 and 2007–2008 was

recently reported to reduce geometric mean titers (GMTs)

against subsequent doses of nonadjuvanted H1N1 2009 vaccine

[13]. As mass vaccination programs cover populations with

widely heterogeneous seasonal vaccination histories, further

characterization of the influence of exposure to previous sea-

sonal influenza vaccination on immunogenicity against H1N1

2009 vaccine is needed.

We describe hemagglutination inhibition (HI) antibody re-

sponses in healthy adults 18–85 years of age after 1 or 2 doses

of 3.75 mg A/California/2009 (H1N1) hemagglutinin antigen,

with an oil-in-water adjuvant (AS03A). We conducted explor-

atory analyses to assess the effect of previous trivalent seasonal

influenza vaccination history on immune responses to AS03A-

H1N1 2009 vaccine for subjects 18–60 years of age.

METHODS

This phase III, open-label study assessed the immunogenicity,

reactogenicity, and safety of 1 or 2 doses of 3.75 mg hemag-

glutinin antigen, A/California/2009 (H1N1)–like candidate vac-

cine adjuvanted with AS03A in healthy subjects 18–60 years of

age ( ) and in healthy subjects 160 years of age (n p 120 n p

). The primary objective was to demonstrate that the im-120

mune response after 1 dose of H1N1 vaccine fulfilled the im-

munogenicity criteria recommended by European guidelines

for the evaluation of candidate influenza vaccines (European

Medicines Agency, Committee for Human Medicinal Products

[CHMP]) [20].

The study was conducted at the Vaccine and Infectious Dis-

ease Institute, Centre for the Evaluation of Vaccination (WHO

Collaborating Centre), University of Antwerp, Belgium. The

protocol was approved by an independent ethics committee,

and the study was conducted in accordance with the Inter-

national Conference on Harmonization guidelines of Good

Clinical Practice and with the Declaration of Helsinki. All sub-

jects provided informed written consent (ClinicalTrials.gov Iden-

tifier: NCT00968526).

Vaccines. The AS03A-adjuvanted, monovalent, split-virion

H1N1 vaccine was manufactured by GlaxoSmithKline (GSK)

Biologicals. The candidate vaccine was developed from the reas-

sortant reference virus generated from the A/California/7/2009

(H1N1)v–like strain (NYMC X-179A; New York Medical Col-

lege), distributed by the US Centers for Disease Control and

Prevention. The vaccine contained 3.75 mg of hemagglutinin

antigen and was adjuvanted with AS03A, which contains 11.86

mg tocopherol, 10.69 mg squalene, and 4.86 mg polysorbate

80 [21]. The vaccine was produced according to licensed man-

ufacturing and testing processes for seasonal trivalent influenza

vaccine and H5N1 pandemic vaccines [22].

Subjects and study schedule. Men and women �18 years

of age who had not received an investigational drug or any



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Table 1. Baseline Characteristics of Study Subjects, by Age Group (Total Vaccinated Cohort)

CharacteristicSubjects 18–60 years of age

( )n p 120Subjects 160 years of age

( )n p 120

Age, yearsMean � SD 39.7 � 13.90 69.1 � 6.12Median 42.5 68.0Range 19–60 61–85

SexFemale 65 (54.2) 52 (43.3)Male 55 (45.8) 68 (56.7)

RaceWhite (European heritage) 120 (100) 120 (100)

Receipt of seasonal influenza vaccinationAt least 1 season 64 (53.3) 105 (87.5)2007–2008 season 57 (47.5) 93 (77.5)2008–2009 season 49 (40.8) 95 (79.2)2009–2010 season 0 (0) 0 (0)

NOTE. Data are no. (%) of subjects, unless otherwise indicated. SD, standard deviation.

vaccines within 30 days of the study were eligible for inclusion.

Eligible participants were either healthy or had controlled

chronic illness. Women of child-bearing age gave a history of

reliable contraceptive practices. The Higher Health Council

guidelines in Belgium recommend that individuals 150 years

of age and healthcare workers be vaccinated against seasonal

influenza; subjects in these risk groups were eligible for the

study if they fulfilled the inclusion criteria. All subjects were

systematically questioned about previous seasonal influenza

vaccination, and the investigator relied on the subject’s recall

for the information. Subjects were recruited from the University

Hospital and by using the study center database.

An internet-based algorithm was used to ensure that an equal

number of subjects were enrolled in the 2 age strata (18–60

years and 160 years). In the 18–60 years of age stratum, at least

40% of subjects were 18–40 years of age, and at least 40% were

41–60 years of age. In the 160 years of age stratum, at least

40% of subjects were 60–70 years of age, and at least 20% were

170 years of age. Following reports that 1 dose of H1N1 vaccine

may meet the immunogenicity endpoints, the protocol was

amended, and on day 21, subjects were randomized 1:1 to

receive no further vaccination or a second dose. Subjects pro-

vided a new informed written consent before entry into the

second-dose phase of the study.

Subjects received H1N1 vaccine administered intramuscu-

larly (deltoid of nondominant arm). A blood sample was taken

for serologic assessment before vaccination (day 0) and 21 and

42 days after the first vaccination.

Immunogenicity. The immunogenicity outcomes were (1)

the proportion of subjects with antibody titers of �1:40 (sero-

protection rate); (2) the proportion of subjects who were se-

ronegative at baseline with postvaccination antibody titers of

�1:40, or the proportion who were seropositive at baseline

with �4-fold increase in antibody titer postvaccination (se-

roconversion rate); and (3) the geometric mean of the within-

subject ratios of pre- and postvaccination reciprocal antibody

titers (geometric mean ratio). The primary immunogenicity

endpoint was day-21 antibody titers in the day-21 immuno-

genicity cohort; the secondary endpoints were day-42 responses

in subjects who received 1 or 2 doses.

In each age stratum, exploratory analyses were performed to

investigate the immunogenicity endpoints according to sea-

sonal influenza vaccination history, including the current in-

fluenza season (2009–2010) and any of the preceding 2 influ-

enza seasons (2007–2008 and 2008–2009). The immunogenicity

of the H1N1 vaccine was assessed by use of HI assays to measure

antibody titers against the A/California/2009 (H1N1) hemag-

glutinin antigen, according to an established method [23].

Hemagglutinin antibody titers were measured in thawed frozen

samples by use of a standardized and validated micromethod

that used 4 hemagglutination-inhibiting units of the antigen

and a 0.45% chicken erythrocyte suspension. The assays were

performed at the central GSK laboratories.

Reactogenicity and safety. The safety endpoints were to

describe solicited and spontaneously reported adverse events.

Solicited local and general events were recorded by subjects

using diary cards for 7 days after the vaccination. Severity grad-

ing was based on a standard scale: the diameters of injection-

site redness and swelling were 120–50 mm (grade 1), 150–100

mm (grade 2), and 1100 mm (grade 3), and the intensity of

other symptoms were graded as follows: “easily tolerated” (“on

touch” for injection-site pain [grade 1]), “interferes with nor-

mal activity” (“when limb is moved” for injection-site pain

[grade 2]), and “prevents normal activity” (“significant pain at



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Table 2. Hemagglutination Inhibition Assay–Based Immune Responses against A/California/2009 (H1N1)Hemagglutinin Antigen after 1 Dose of AS03A-H1N1 2009 Vaccine in the Day-21 Immunogenicity Cohort

ImmunogenicitySubjects 18–60 years of age

( )n p 118Subjects 160 years of age

( )n p 119

At baseline% of subjects with titers �1:40 (95% CI) 8.5 (4.1–15.0) 9.2 (4.7–15.9)% of subjects with titers �1:10 (95% CI) 37.3 (28.6–46.7) 42.0 (33.0–51.4)Geometric mean titer (95% CI) 8.6 (7.4–10.0) 10.0 (8.2–12.2)

At day 21Seroprotection ratea (95% CI) 97.5 (92.9–99.5) 87.4 (80.1–92.8)Seroconversion rateb (95% CI) 94.9 (89.3–98.1) 79.0 (70.6–85.9)Geometric mean titer (95% CI) 358.9 (285.8–450.7) 137.4 (111.2–169.9)Geometric mean ratioc (95% CI) 41.73 (32.98–52.81) 13.76 (10.95–17.29)

NOTE. To fulfill CHMP licensure criteria for immunogenicity, the point estimates in subjects 18–60 years of age and insubjects �60 years of age needed to be as follows: a seroconversion rate of �40% and �30%, respectively; a seroprotectionrate of �70% and �60%, respectively; and a geometric mean ratio of �2.5 and �2.0, respectively [20]. CHMP, Committeefor Human Medicinal Products; CI, confidence interval.

a Proportion of subjects with antibody titers of �1:40.b Proportion of subjects who were seronegative at baseline with postvaccination antibody titers of �1:40, or the proportion

of subjects who were seropositive at baseline with �4-fold increase in antibody titer postvaccination.c Geometric mean of the within-subject ratios of pre- and postvaccination reciprocal antibody titers.

rest” for injection-site pain [grade 3]). All local solicited events

were considered H1N1 vaccine related, and the investigator

provided causality assessments for solicited general events. As-

sessment of spontaneously reported adverse events was planned

from day 0 to day 84, and serious adverse events, and events

of special interest, were to be assessed prospectively from day

0 to 1 year postvaccination. Spontaneously reported adverse

events up to day 42 are provided in this report.

Statistics. On basis of recent experience with AS03A-ad-

juvanted pandemic influenza vaccines, it was estimated that a

sample size of 120 subjects per age stratum would provide

�95% power to demonstrate that 1 dose of the H1N1 vaccine

fulfills influenza vaccine licensure criteria for immunogenicity

overall [14, 24]. The safety population included all subjects

who received the H1N1 vaccine. The primary immunogenicity

assessment included subjects in the safety population with eval-

uable serum samples at day 0 and day 21 (day-21 immuno-

genicity cohort). The day-42 immunogenicity assessment was

performed on subjects with serum samples available at day 21

and day 42 (day-42 immunogenicity cohort).

To fulfill CHMP licensure criteria for immunogenicity, the

point estimates in subjects 18–60 years of age and in subjects

�60 years of age needed to be as follows: a seroconversion rate

of �40% and �30%, respectively; a seroprotection rate of

�70% and �60%, respectively; and a geometric mean ratio of

�2.5 and �2.0, respectively [20]. Immunogenicity and reac-

togenicity results were described as point estimates with 95%

confidence intervals (CIs). In the exploratory analysis, the GMT

ratio and the 95% CI between the groups with or without

receipt of previous seasonal influenza vaccination in the pre-

vious 2 years were obtained by use of analysis of covariance

for logarithm-transformed titers with log-prevaccination results

as the covariate.

RESULTS

Subjects. A total of 240 subjects were enrolled during the

period from 8 September to 16 September 2009 (Figure 1).

The mean age was 39.7 years (range, 19–60 years) in the youn-

ger group and 69.1 years (range, 61–85 years) in the older

group. No subject had received trivalent influenza vaccine for

the 2009–2010 season. In the age group of 18–60 years, 53.3%

of subjects had received seasonal influenza vaccine in the pre-

ceding 2 influenza seasons; in the age group of 160 years, 87.5%

of subjects had received seasonal influenza vaccine in the pre-

ceding 2 influenza seasons (Table 1).

Of the 120 subjects 18–60 years of age, 10 (8.3% [95% CI,

4.1%–14.8%]) had baseline antibody titers of �1:40. Of the

120 subjects 160 years of age, 11 (9.2% [95% CI, 4.7%–15.8%])

had baseline antibody titers of �1:40 (Table 2). There was no

clear relationship between seasonal influenza vaccine history

and baseline antibody titers against the H1N1 2009 vaccine.

Immunogenicity. After 1 dose (day 21), all immunogenicity

outcomes fulfilled the licensure criteria for candidate influenza

vaccines (Table 2). The day-21 seroprotection rate was 97.5%

(95% CI, 92.9%–99.5%) in subjects 18–60 years of age and

87.4% (95% CI, 80.1%–92.8%) in subjects 160 years of age.

The immune responses persisted at day 42 among subjects who

received only 1 dose (Table 3). At day 42 in the 2-doses group,

the seroprotection rate was 100% (95% CI, 94.6%–100%) in

subjects 18–60 years of age and 98.5% (95% CI, 92.0%–100%)

in subjects 160 years of age (Table 3).



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Table 3. Hemagglutination Inhibition Assay–Based Immune Responses against A/California/2009 (H1N1) Hemagglutinin Antigen after 1or 2 Doses of AS03A-H1N1 2009 Vaccine in the Day-42 Immunogenicity Cohort

Immunogenicity

Subjects who received first dose Subjects who received second dose

18–60 years of age( )n p 50

160 years of age( )n p 49

18–60 years of age( )n p 66

160 years of age( )n p 67

At baseline% of subjects with antibody titers �1:40 (95% CI) 8.0 (2.2–19.2) 10.2 (3.4–22.2) 9.1 (3.4–18.7) 9.0 (3.4–18.5)% of subjects with antibody titer �1:10 (95% CI) 40.0 (26.4–54.8) 42.9 (28.8–57.8) 36.4 (24.9–49.1) 41.8 (29.8–54.5)Geometric mean titer (95% CI) 8.6 (6.8–10.8) 9.7 (7.3–13.0) 8.8 (7.0–10.9) 10.3 (7.7–13.7)

At day 21Seroprotection ratea (95% CI) 96.0 (86.3–99.5) 85.7 (72.8–94.1) 98.5 (91.8–100) 88.1 (77.8–94.7)Seroconversion rateb (95% CI) 94.0 (83.5–98.7) 77.6 (63.4–88.2) 95.5 (87.3–99.1) 79.1 (67.4–88.1)Geometric mean titer (95% CI) 388.6 (262.0–576.2) 129.3 (89.9–186.0) 339.1 (258.6–444.8) 142.7 (108.5–187.8)Geometric mean ratioc (95% CI) 45.30 (30.32–67.68) 13.32 (9.04–19.61) 38.71 (29.10–51.51) 13.86 (10.29–18.65)

At day 42Seroprotection ratea (95% CI) 94.0 (83.5–98.7) 87.8 (75.2–95.4) 100 (94.6–100) 98.5 (92.0–100)Seroconversion rateb (95% CI) 92.0 (80.8–97.8) 79.6 (65.7–89.8) 98.5 (91.8–100) 94.0 (85.4–98.3)Geometric mean titer (95% CI) 331.2 (220.7–497.1) 117.1 (82.4–166.4) 610.6 (507.9–734.0) 345.8 (278.0–430.1)Geometric mean ratioc (95% CI) 38.62 (25.96–57.45) 12.06 (8.39–17.33) 69.70 (53.79–90.32) 33.57 (24.87–45.31)

NOTE. To fulfill CHMP licensure criteria for immunogenicity, the point estimates in subjects 18–60 years of age and in subjects �60 years of age neededto be as follows: a seroconversion rate of �40% and �30%, respectively; a seroprotection rate of �70% and �60%, respectively; and a geometric mean ratioof �2.5 and �2.0, respectively [20]. CHMP, Committee for Human Medicinal Products; CI, confidence interval.

a Proportion of subjects with antibody titers of �1:40.b Proportion of subjects who were seronegative at baseline with postvaccination antibody titers of �1:40, or the proportion of subjects who were seropositive

at baseline with �4-fold increase in antibody titer postvaccination.c Geometric mean of the within-subject ratios of pre- and postvaccination reciprocal antibody titers.

At day 21 in subjects 18–60 years of age, the GMT adjusted

for baseline antibody titer was significantly lower in subjects

who had received seasonal influenza vaccine within the pre-

ceding 2 influenza seasons, compared with subjects who had

not (Table 4). The adjusted GMT ratio at day 21 was 0.42 (95%

CI, 0.28–0.64; ). The effect was also observed at day 42P ! .001

in subjects 18–60 years of age who had received a second dose,

with an adjusted GMT ratio of 0.60 (95% CI, 0.41–0.85;

). A similar trend was observed in the age group ofP p .003

160 years, but too few subjects had not received previous sea-

sonal influenza vaccination ( ), compared with those whon p 15

had ( ), to allow a statistical comparison (Table 4). Inn p 105

the age group of 18–60 years, day-21 and day-42 GMTs were

numerically lower in subjects with previous vaccination, relative

to those with no previous vaccination, regardless of baseline

antibody titer (ie, !1:10 and �1:10) (Table 4 and Figure 2).

Reactogenicity and safety. Solicited adverse events during

the initial 7-day postvaccination period are shown in Table 5.

In the total vaccinated cohort after the first dose ( ),N p 240

local reactions (solicited and unsolicited) were reported more

frequently in subjects 18–60 years of age (87.5% [95% CI,

80.2%–92.8%]) than in subjects 160 years of age (68.3% [95%

CI, 59.2%–76.5%]). Injection-site pain was the most frequent

local reaction during the 7-day postvaccination period, with a

mean duration of 3.1 days in subjects 18–60 years of age and

2.8 days in subjects 160 years of age. The incidence of redness

and swelling was low in both age groups. There were no grade-

3 local reactions after the first dose. Local reactions after the

second dose ( ) were similar to those after the first dose,n p 138

but with 2 reports of severe pain and 1 report of severe swelling.

In the total vaccinated cohort after 1 dose, 64.2% (95% CI,

54.9%–72.7%) of subjects 18–60 years of age and 44.2% (95%

CI, 35.1%–53.5%) of subjects 160 years of age experienced

general reactions (solicited and unsolicited) during the 7-day

postvaccination period. After 1 dose in subjects 18–60 years of

age and in subjects 160 years of age, general reactions were

most frequently fatigue (35.8% and 21.7%, respectively), head-

ache (36.7% and 18.3%, respectively), and muscle aches (24.2%

and 20.8%, respectively). The incidences of solicited general

reactions after the first and second doses were similar.

Up to day 42, spontaneously reported adverse events oc-

curred in 46 subjects (45.1%) who received 1 dose and in 61

subjects (44.2%) who received 2 doses. The most common

spontaneously reported adverse events in subjects who re-

ceived 1 or 2 doses were nasopharyngitis (12 subjects who

received 1 dose and 14 subjects who received 2 doses) and

headache (5 subjects who received 1 dose and 6 subjects who

received 2 doses). Grade-3 unsolicited adverse events occurred

in 5 subjects (4.9%) who received 1 dose and in 12 subjects

(8.7%) who received 2 doses.

With regard to serious adverse events, in subjects who re-

ceived 1 dose, there was 1 case of headache, and in those who



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Table 4. Hemagglutination Inhibition Assay–Based Immune Responses against A/California/2009 (H1N1) Hemagglutinin An-tigen after 1 Dose (Day-21 Immunogenicity Cohort) or 2 Doses (Day-42 Immunogenicity Cohort) of AS03A-H1N1 2009 Vaccine,According to Age Group, Seasonal Influenza Vaccination History, and Antibody Titers

Seasonal influenza vaccination history,a immunogenicity

Antibody titer

All subjects!1:10 at baseline �1:10 at baseline

No. of subjects 18–60 years of age with a history 44 19 63

At baseline

GMT (95% CI) 5.0 (5.0–5.0) 17.6 (13.7–22.5) 7.3 (6.2–8.6)

% of subjects with titers �1:40 (95% CI) 0.0 (0.0–8.0) 10.5 (1.3–33.1) 3.2 (0.4–11.0)

At day 21

GMT (95% CI) 157.6 (108.3–229.2) 533.3 (353.7–804.1) 227.6 (165.8–312.4)


No. of subjects 18–60 years of age with a history 27 11 38

At day 42

GMT (95% CI) 408.5 (289.7–576.1) 726.0 (548.2–961.3) 482.5 (370.4–628.6)


No. of subjects 18–60 years of age without a history 30 25 55

At baseline

GMT (95% CI) 5.0 (5.0–5.0) 24.9 (17.3–35.9) 10.4 (7.9–13.6)


At day 21

GMT (95% CI) 496.3 (330.6–745.2) 766.3 (528.3–1111.7) 604.7 (458.7–797.1)


No. of subjects 18–60 years of age without a history 15 13 28

At day 42

GMT (95% CI) 844.6 (642.6–1110.0) 835.6 (586.4–1190.5) 840.4 (684.8–1031.3)


No. of subjects 160 years of age with a history 58 46 104

At baseline

GMT (95% CI) 5.0 (5.0–5.0) 22.5 (17.1–29.6) 9.7 (8.1–11.7)


At day 21

GMT (95% CI) 95.6 (68.9–132.7) 202.1 (153.8–265.5) 133.1 (106.1–167.2)


No. of subjects 160 years of age with a history 33 27 60

At day 42

GMT (95% CI) 285.0 (213.7–380.1) 424.4 (294.7–611.3) 341.0 (271.7–427.9)


No. of subjects 160 years of age without a history 11 4 15

At baseline

GMT (95% CI) 5.0 (5.0–5.0) 134.5 (5.8–3107.1) 12.0 (4.5–31.9)


At day 21

GMT (95% CI) 132.3 (63.1–277.2) 348.7 (53.3–2280.2) 171.3 (89.8–326.9)


No. of subjects 160 years of age without a history 6 1 7

At day 42

GMT (95% CI) 302.0 (112.5–810.6) 1810.0 390.0 (141.9–1072.3)


NOTE. CI, confidence interval; GMT, geometric mean titer.a Subjects who did or did not receive the 2007–2008 and/or 2008–2009 seasonal influenza vaccine.



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Figure 2. Hemagglutination inhibition assay–based, day-21 reverse cu-mulative distribution curves in subjects 18–60 years of age after 1 doseof A/California/2009 (H1N1) hemagglutinin antigen, by baseline serostatusand seasonal influenza vaccination history in the day-21 immunogenicitycohort.

received 2 doses, there was 1 case of atrial fibrillation and 1

case of intervertebral disc disorder. The serious adverse events

were not considered by the investigators to be vaccine related.

There were no adverse events of special interest.

DISCUSSION

A single 3.75-mg dose of AS03A-adjuvanted H1N1 2009 vaccine

elicited immune responses that fulfilled the European licensure

immunogenicity criteria for immunogenicity of influenza vac-

cines [20]. At day 21, HI antibody titers of �1:40 were achieved

by 97.5% of subjects 18–60 years of age and by 87.4% of sub-

jects 160 years of age. Similar immune responses were observed

at day 42 in subjects who received a second dose of vaccine,

and immune responses persisted at day 42 in subjects who did

not receive a second dose.

Mild or moderate injection-site pain was the most common

solicited postvaccination event, and the incidence of redness

and swelling was low. Three subjects reported a grade-3 local

reaction after the second dose. The most frequent general re-

actions were fatigue, headache, and muscle aches. These find-

ings are consistent with previous experience with avian-origin

pandemic influenza vaccines, in which formulation with AS03A

increased the incidence of postvaccination reactions, compared

with nonadjuvanted vaccine [22, 25]. Prospective assessments

are ongoing to establish the long-term safety profile of the

H1N1 2009 vaccine.

On the basis of numerous clinical studies, it is widely ac-

cepted that vaccination history does not negatively influence

protection associated with subsequent seasonal influenza vac-

cination [26, 27]. Nonetheless, reduced HI antibody responses

in subjects with a history of influenza vaccination, compared

with those without, have been repeatedly observed in clinical

studies of trivalent seasonal influenza vaccines [28–30]. Fur-

thermore, recent experience with AS03A-adjuvanted avian-or-

igin H5N1 influenza vaccine in adults showed that vaccination

with nonadjuvanted pandemic influenza vaccine was associated

with reduced immune responses to booster doses of AS03A-

H5N1 vaccine, compared with responses in subjects who re-

ceived the AS03A-H5N1 vaccine for the first time [18]. And in

a recent pediatric study, postvaccination neutralizing antibody

titers after 2 doses of alum-adjuvanted H5N1 vaccine were

significantly higher in children who had not previously received

nonadjuvanted seasonal influenza vaccination, compared with

those who had [19].

To investigate this phenomenon, we conducted explorato-

ry analyses on the immunogenicity of the AS03A-adjuvanted

H1N1 2009 vaccine in subjects with and without a history of

seasonal influenza vaccination. Although all immunogenicity

endpoints were met, we found that, among subjects 18–60 years

of age, the adjusted GMTs at day 21 and day 42 were signifi-

cantly lower among subjects who had previously received at

least one seasonal influenza vaccination during the preceding

two seasons, compared with those who had not. It should be

noted, however, that the sample was relatively small at day 42

(38 subject with previous vaccination and 28 subjects without).

A relationship between baseline serostatus and postvaccina-

tion antibody titers has been previously described, with reduced

postvaccination responses observed in subjects with higher pre-

vaccination titers of antibodies derived from natural influenza

infection or previous influenza vaccination [31]. Contrary to

this, in our study, among subjects who had previously received

trivalent influenza vaccination, antibody titers after H1N1 2009

vaccine were numerically lower in subjects who were seroneg-

ative at baseline, compared with subjects who were seropositive.

This suggests that the observed effect of previous seasonal in-

fluenza vaccination on subsequent responses to H1N1 2009

vaccination is not conditioned by the existence of HI antibodies

at baseline.

Various studies have shown that subjects who have previously

received seasonal influenza vaccine may have reduced antibody

titers following subsequent pandemic influenza vaccination, yet

the mechanisms underlying this phenomenon remain unknown

[13, 18, 19]. It can be speculated that previous nonadjuvanted

seasonal influenza vaccination led to a “skewing” of the T cell

and/or B cell repertoires toward epitopes specific for those vac-

cines, making it more difficult to generate or select highly H1N1

pandemic-specific T and B cells after subsequent vaccination

with the H1N1 pandemic vaccine, and translating into lower

HI titers. Indeed, it has been shown that seasonal influenza

vaccination induces a profound selection of antigen-specific B

cells, with major changes in the B cell repertoire as a result

[32].

Another hypothesis, not necessarily exclusive from the for-

mer, could be that previous seasonal influenza vaccination pre-

vented natural seasonal H1N1 infection and thereby altered the

development of heterosubtypic immunity against the H1N1

2009 strain. Under the assumption that virally induced im-

munity induces optimal cross-reactive responses, this would

eventually result in a lower responsiveness state to subsequent

H1N1 2009 pandemic vaccination in subjects who were pre-

viously vaccinated against seasonal influenza, compared with

subjects who were not. This hypothesis has been verified in a

mouse model, in which the heterosubtypic immunity against



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Table 5. Data on Solicited Local and General Adverse Events Occurring within 7 Days after Receipt of AS03A-H1N1 2009Vaccine, According to Age Group in the Total Vaccinated Cohort after the First Dose and in the Vaccinated Cohort after theSecond Dose

Type of adverse event

First dose Second dose

Subjects18–60 years of age

( )n p 120

Subjects160 years of age

( )n p 120

Subjects18–60 years of age

( )n p 68

Subjects160 years of age

( )n p 70

No. % (95% CI) No. % (95% CI) No. % (95% CI) No. % (95% CI)

Local adverse eventsPain

Mild 64 53.3 (44.0–62.5) 65 54.2 (44.8–63.3) 26 38.2 (26.7–50.8) 37 52.9 (40.6–64.9)Moderate 41 34.2 (25.8–43.4) 13 10.8 (5.9–17.8) 23 33.8 (22.8–46.3) 5 7.1 (2.4–15.9)Severe 0 0 2 2.9 (0.4–10.2) 0

Redness120–50 mm 1 0.8 (0.0–4.6) 7 5.8 (2.4–11.6) 1 1.5 (0.0–7.9) 4 5.7 (1.6–14.0)150–100 mm 0 2 1.7 (0.2–5.9) 0 1 1.4 (0.0–7.7)1100 mm 0 0 0 0

Swelling120–50 mm 5 4.2 (1.4–9.5) 8 6.7 (2.9–12.7) 3 4.4 (0.9–12.4) 0150–100 mm 6 5.0 (1.9–10.6) 4 3.3 (0.9–8.3) 0 2 2.9 (0.3–9.9)1100 mm 0 0 0 1 1.4 (0.0–7.7)

General adverse eventsFatigue

Grade 1 28 23.3 (16.1–31.9) 20 16.7 (10.5–24.6) 10 14.7 (7.3–25.4) 13 18.6 (10.3–29.7)Grade 2 13 10.8 (5.9–17.8) 5 4.2 (1.4–9.5) 7 10.3 (4.2–20.1) 2 2.9 (0.3–9.9)Grade 3 2 1.7 (0.2–5.9) 1 0.8 (0.0–4.6) 2 2.9 (0.4–10.2) 0

HeadacheGrade 1 33 27.5 (19.7–36.4) 16 13.3 (7.8–20.7) 12 17.6 (9.5–28.8) 10 14.3 (7.1–24.7)Grade 2 10 8.3 (4.1–14.8) 6 5.0 (1.9–10.6) 2 2.9 (0.4–10.2) 2 2.9 (0.3–9.9)Grade 3 1 0.8 (0.0–4.6) 0 1 1.5 (0.0–7.9) 0

Muscle achesGrade 1 15 12.5 (7.2–19.8) 18 15.0 (9.1–22.7) 5 7.4 (2.4–16.3) 8 11.4 (5.1–21.3)Grade 2 12 10.0 (5.3–16.8) 6 5.0 (1.9–10.6) 9 13.2 (6.2–23.6) 5 7.1 (2.4–15.9)Grade 3 2 1.7 (0.2–5.9) 1 0.8 (0.0–4.6) 1 1.5 (0.0–7.9) 0

ShiveringGrade 1 14 11.7 (6.5–18.8) 6 5.0 (1.9–10.6) 9 13.2 (6.2–23.6) 3 4.3 (0.9–12.0)Grade 2 9 7.5 (3.5–13.8) 1 0.8 (0.0–4.6) 7 10.3 (4.2–20.1) 1 1.4 (0.0–7.7)Grade 3 0 0 2 2.9 (0.4–10.2) 1 1.4 (0.0–7.7)

SweatingGrade 1 14 11.7 (6.5–18.8) 6 5.0 (1.9–10.6) 9 13.2 (6.2–23.6) 4 5.7 (1.6–14.0)Grade 2 4 3.3 (0.9–8.3) 0 1 1.5 (0.0–7.9) 1 1.4 (0.0–7.7)Grade 3 1 0.8 (0.0–4.6) 0 1 1.5 (0.0–7.9) 0

Joint painGrade 1 10 8.3 (4.1–14.8) 10 8.3 (4.1–14.8) 7 10.3 (4.2–20.1) 11 15.7 (8.1–26.4)Grade 2 9 7.5 (3.5–13.8) 6 5.0 (1.9–10.6) 3 4.4 (0.9–12.4) 2 2.9 (0.3–9.9)Grade 3 0 1 0.8 (0.0–4.6) 1 1.5 (0.0–7.9) 0

Increased temperature38�C–38.5�C 0 2 1.7 (0.2–5.9) 1 1.5 (0.0–7.9) 038.5�C–39.0�C 1 0.8 (0.0–4.6) 0 1 1.5 (0.0–7.9) 0139.0�C 0 0 0 0

NOTE. Adverse events were graded from 1 to 3 for severity: the diameters of injection-site redness and swelling were graded as follows: 120–50 mm (grade 1), 150–100 mm (grade 2), and 1100 mm (grade 3), and the intensity of other symptoms were graded as follows: “easily tolerated”(“on touch” for injection-site pain [grade 1]), “interferes with normal activity” (“when limb is moved” for injection-site pain [grade 2]), and “preventsnormal activity” (“significant pain at rest” for injection-site pain [grade 3]).



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a pandemic H5N1 strain was blunted after prevention of H3N2

infection through subunit vaccination [33]. Explanations for

the effect of previous seasonal influenza vaccine on immune

responses to pandemic influenza vaccine remain speculative,

and further investigation is warranted.

To conclude, a single dose of AS03A-adjuvanted H1N1 2009

vaccine (3.75 mg hemagglutinin antigen) elicited immune re-

sponses that fulfilled the European licensure criteria for im-

munogenicity of influenza vaccines in adults. Because immu-

nogenicity criteria were reached after 1 dose, a second dose

may not be necessary. Exploratory analyses in subjects 18–60

years of age showed that seasonal influenza vaccination within

the previous 2 seasons was associated with reduced responses

to the H1N1 2009 vaccine.

Acknowledgments

We are grateful to the New York Medical College for providing thevaccine virus strain. We are indebted to the participating study volunteers,study doctors, trial nurses, and laboratory technicians at the study site aswell as to the sponsor’s project staff for their support and contributionsthroughout the study. We are grateful to all teams of GSK Biologicals fortheir contribution to this study, Karl Walravens and the clinical and se-rological laboratory teams, Julie De Wever for preparation of the studyprotocol and related study documentation, Anaelle Delhage for global studymanagement, Dorothy Slavin (clinical safety representative), Carine Mag-getto (clinical data coordinator), Walthere Dewe, Olivier Ghys, and VinodBambure for input on statistical analysis, and Edith Lepine for projectmanagement. We would like to address special thanks to Paul Gillard andRobbert Van der Most for their critical reading of the manuscript and fortheir helpful suggestions. Finally, we thank Annick Moon (of Moon MedicalCommunications, Oxford, UK), who provided medical writing services,and Isabelle Gautherot for editorial assistance and manuscript coordination.

Potential conflicts of interest. P.V.D. acts as chief and principal in-vestigator for vaccine trials conducted on behalf of the University of Ant-werp, for which the university obtains research grants from several vaccinemanufacturers. F.R., E.H., and T.V. report that they are employees of GSKBiologicals. E.H. reports that he owns equity or stock options. GSK Bio-logicals sponsored the study and, together with the authors, was involvedin all stages of the study, including analysis of data. GSK Biologicals alsopaid the costs associated with the development and publication of thepresent study.

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AS03 A ‐Adjuvanted Influenza A (H1N1) 2009 Vaccine for Adults up to 85 Years of Age

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