This was a phase III open, randomized trial with two groups conducted in five centres in France from September 2009 to April 2010. The primary objective was to demonstrate that vaccination with one dose of A/H1N1/2009 vaccine containing 1.9 μg of HA adjuvanted with AS03_A results in a haemagglutination-inhibition (HI) immune response that meets or exceeds European Medicines Agency (EMA) Committee for Medicinal Products for Human Use (CHMP) criteria for pandemic vaccines 21 days post vaccination 18 . Secondary objectives included the assessment of immunogenicity following administration of a second dose at Day 21 or Day 182, assessment of persistence of the antibody response at Day 182 and Day 364, and assessment of reactogenicity and safety.

Eligible participants enrolled by the responsible on-site personnel were clinically healthy non-pregnant adults over 18 years of age at the time of the first vaccination who provided written informed consent. Subjects with a clinical history suggestive of an influenza infection within 6 months preceding the study start were excluded. The enrolment was age-stratified, with two age strata (18–60 years and >60 years, in 1:1 ratio) with a minimum of 40% of subjects aged 18–40 years and a minimum of 40% of subjects aged 41–60 years, in the 18–60 years stratum; and a minimum of 40% of subjects aged 61–70 years and a minimum of 20% of subjects aged above 70, in the >60 years stratum.

In the initial study design, all subjects were to be vaccinated on Day 0 and Day 21, however, the protocol was amended soon after the enrolment of the first subjects (and before Day 21 was reached for these subjects) to include a Day 0 and Day 182 immunization schedule. This amendment provided the opportunity to investigate the immune response 6 months after a single A/H1N1/2009 vaccine injection and to assess the flexibility of the vaccination schedule. The list of vaccinated subject numbers which was tracked using the central randomisation system on Internet (SBIR) and was randomised (1:1) by the sponsor using SAS version 9.2 to receive a second dose at either Day 21 (Group A) or Day 182 (Group B) taking into account the minimisation procedure for centre and age. At Day 21, the subjects were asked by the responsible on-site personnel to consent to either receive a second vaccination at Day 21 as initially planned or to be randomly assigned to a Day 21 or Day 182 second dose administration.

The protocol, its amendments and other relevant study documentation were approved by the appropriate Ethics Committee (CPP 1, Ile de France) and the study was conducted in accordance with good clinical practice guidelines, the Declaration of Helsinki and all applicable regulatory requirements.

The monovalent influenza A/H1N1/2009 inactivated, split-virion vaccine was manufactured by GlaxoSmithKline (GSK) Vaccines from a vaccine seed virus prepared from the reassortant virus NYMC X-179A (New York Medical College, New York) generated from the A/California/7/2009 strain as recommended by the World Health Organization (WHO) 19 . The seed virus was propagated in embryonated hen eggs and the vaccine was produced using the licensed manufacturing process for Pandemrix™ (a trade mark of the GlaxoSmithKline group of companies). AS03_A is an Adjuvant System containing α-tocopherol and squalene in an oil-in-water emulsion (11.86 mg α-tocopherol) manufactured by GSK Vaccines. The study vaccine was formulated to contain 1.9 μg of HA antigen and AS03_A [note that the standard dose registered for adults contains 3.75 μg of HA and AS03_A while the paediatric dose contains 1.9 μg of HA and AS03_B (5.93 mg α-tocopherol)].

A dose of AS03_A-adjuvanted vaccine (0.38 ml injection volume in this trial) was administered intramuscularly in the deltoid region on Day 0 and either on Day 21 or Day 182 (according to subject consent to be randomly assigned or not on Day 21 to one of the two schedules). Blood samples were collected on Day 0 (prior to vaccination), Day 21, Day 42 and Day 182, Day 364 and additionally for Group B only, on Day 203. All samples were tested under blind conditions n a validated micro-titre HI test using chicken erythrocytes as previously described 20 with the A/California/7/2009 strain used as antigen.

Data on solicited symptoms experienced within the first 7 days following each vaccination were recorded on diary cards by participants as previously described 21 . Data were also collected on the occurrence of any unsolicited adverse events occurring within 21 days after the first vaccination and either 63 days after the second vaccination (for the 21 days interval schedule group) or 30 days after the second vaccination (for the 6 months interval schedule group). The intensities of symptoms and adverse events were recorded according to a standard three-grade scale as previously described 21 . An assessment of causality was made by the investigator for solicited systemic symptoms and unsolicited adverse events. Data on serious adverse events, adverse events of specific interest (AESIs) also called potential Immune-Mediated Diseases (pIMDs) and adverse events of special interest (convulsion and anaphylaxis) are planned to be collected up to Day 546. This paper presents data up until Day 364.

The analysis was descriptive. The immune response at Day 21 addressing the primary objective was analysed according to the standard EMA CHMP HI endpoints [with 95% confidence intervals (CI)] used for evaluation of influenza vaccines 18 . The sample size of 135 evaluable subjects per age strata (270 in total) was calculated to give a power of at least 95% to fulfil the CHMP criteria for these endpoints 18 following one dose of vaccine. The immune response after the second dose was also assessed for the same endpoints.

The safety endpoints (percentage of subjects and 95% CI) were solicited adverse events and spontaneously reported adverse events. All serious adverse events occurring up to Day 364 were described. The statistical analysis was performed using SAS Version 9.2.

Safety was assessed in the Total Vaccinated Cohort including all subjects who received at least one dose of vaccine. Immunogenicity was assessed in the per protocol population including subjects without protocol violation and with serological data at each immunogenicity time point (Day 21, Day 42, Day 182, Day 203 (Group B), and Day 364).

A total of 306 subjects were vaccinated with AS03_A-adjuvanted A/H1N1/2009 vaccine on Day 0. Of these, 177 subjects received a 2^nd dose on Day 21 (group A) and 106 subjects received a 2^nd dose on Day 182 (group B). The stratification according to age led to the initial vaccination of 163 subjects aged 18 to 60 years and of 143 subjects aged over 60 years. Figure 1 shows the trial profile and Additional file 1: Table S1 provides the cohorts and rationale for exclusions from the immunogenicity assessment at each timepoint. The demographic profiles for the per-protocol analysis of immunogenicity for each group were similar with respect to age at first dose vaccination (38.9 and 41.2 years and 66.6 and 66.3 years for the two age strata for groups A and B, respectively) gender distribution (approximately 1:1) and geographic ancestry (mainly white Caucasian).

The HI antibody responses following both schedules in adults aged 18 to 60 years and above 60 years are presented in Table 1 and Figure 2.

*Seroconversion rate for haemagglutination-inhibition antibody response is defined as the percentage of vaccinees who have a prevaccination titre <1:10 and a post-vaccination titre ≥1:40, or a significant increase in antibody titre defined as the percentage of vaccinees who have a pre-vaccination titre ≥ 1:10 and at least a fourfold increase in post-vaccination titre. **Geometric mean fold rise is defined as geometric mean of the within-subject ratios of the post-vaccination reciprocal haemagglutination-inhibition titre to the Day 0 reciprocal agglutination-inhibition titre.

European Medicines Agency Committee for Medicinal Products for Human Use (CHMP) criteria for haemagglutination-inhibition antibody response in 18–60 year olds are: seroprotection rate >70%, seroconversion rate >40%, and geometric mean fold rise >2·5 and in adults >60 years are seroprotection rate >60%, seroconversion rate >30%, and geometric mean fold rise >2·0.

United States Center for Biologics Evaluation and Research (CBER) criteria for hemagglutination-inhibition antibody response in adults <65 years are: lower limit (LL) of 95% CI for seroprotection ≥70% and LL of 95% CI for seroconversion ≥40% and in adults ≥65 years are LL of 95% CI for seroprotection ≥60% and LL of 95% CI for seroconversion ≥30%.

Prior to vaccination the percentages of subjects with pre-existing HI antibody levels ≥1:40 (considered to be seroprotective) were 14.4% for subjects aged <18–60 years and 5.1% for subjects aged >60 years older adults and pre-vaccination Geometric Mean Titres (GMTs) in seropositive subjects were low in both age groups (28.1 and 16.3).

The HI response in both age strata on Day 21 following the first dose of vaccine met and exceeded all European CHMP regulatory acceptance criteria (as well as CBER criteria) for influenza vaccines and so the primary objective of the study was achieved. The results suggested a lower immune response in the older age stratum (GMTs of 459.8 for 18–60 year olds and 168.2 for above 60 year olds for pooled groups). Further stratification by age suggested slightly lower GMTs with advancing age at all time-points (data not shown).

In group A, a second dose administered at Day 21 to 177 subjects induced a further increase in HI GMTs at Day 42 in both age strata (from 459.8 after the first vaccination to 771.8 for the 18–60 years group and from 168.2 after the first vaccination to 400.9 for the >60 years group). In group B, at 42 days after a single vaccine dose, GMT values had decreased only marginally compared to the previous time point.

HI antibodies still persisted at 6 months (Day 182) with GMTs of 240.5 (18 to 60 years) and 97.8 (>60 years) in the group that had received two doses (group A) and 124.1 (18–60 years) and 48.6 (>60 years) in the group that had only received a single dose up to that time-point (group B). Despite the decline in GMTs, the percentages of subjects with putatively seroprotective antibody levels at 6 months following administration of either one (85.7% and 63.3% of the 18–60 years and the >60 years groups, respectively) or two vaccine doses (97.6% and 90.4% of the 18–60 years and >60 years groups, respectively) still complied with European regulatory criteria (also for seroconversion rates and geometric mean fold rises) and except for the older age stratum who received only one dose, still also complied with US regulatory criteria.

A second dose administered at 6 months (Day 182) interval to 106 subjects induced a further increase of GMTs 21 days later (on Day 203) in both age strata (708.3 for 18–60 years and 512.1 for >60 years). The GMTs induced by a second dose were similar whether administered at Day 21 or at 6 months interval.

HI antibody persistence at study month 12 (Day 364) tended to be higher for the 6 month interval group B (GMTs and seroprotection rates were respectively 155.5 and 93.3% for 18–60 years and 68.8 and 80.4% for >60 years) than for the 21 day interval group A (GMTs and seroprotection rates were respectively 107.8 and 82.3% for 18–60 years and 35.8 and 50.0% for >60 years).

The incidence of solicited local (injection site) and systemic symptoms reported per group within 7 days after each vaccination are presented in Tables 2 and 3 respectively.

*Grade 3 was defined as significant injection site pain at rest which prevents normal activities (inability to attend/do work).

*Grade 3 events were defined as events that prevent normal everyday activities (inability to attend/do work), or that require intervention of a physician/healthcare provider.

The profile and frequencies of solicited symptoms were similar in both study groups. No difference in profile or increase in the frequencies of solicited symptoms could be distinguished with the administration of a second dose in either vaccination schedule. The observed incidences of certain solicited symptoms were slightly numerically higher in the subjects aged 18–60 years.

Pain at the injection site was the most common solicited symptom reported following vaccination in both younger adults (94.6% and 95.7% after a first dose and 87.9% and 90% after a second dose in group A (Day 0 - Day 21 vaccinations) and group B (Day 0 - Day 182 vaccinations, respectively) and among adults aged >60 years (78.9% and 67.3% after a first dose and 72.9% and 57.8% after a second dose in groups A and B, respectively). Most injection site related symptoms were mild to moderate with a maximum of three subjects per age stratum reporting grade 3 pain or redness or swelling greater than 100 mm after any vaccination. The most frequently reported solicited systemic symptoms were fatigue (maximum 53.8% after a second dose at Day 21, 18–60 years stratum), muscle aches (maximum 51.7% after a second dose at Day 182, 18–60 years stratum) and headache (maximum 45.1% after a second dose at Day 21, 18–60 years stratum). Grade 3 systemic symptoms were rare (0 to 4 subjects for any one of these symptoms after every dose).

In group A the percentage of subjects reporting unsolicited adverse events (data not shown) within 21 days after the first vaccination and 63 days after the second vaccination was 55.9% of the 18–60 year age group (12.9% of these events were considered as related to vaccination) and 58.2% of the >60 year age group (16.5% considered as related to vaccination). In group B the percentage of subjects reporting unsolicited adverse events (data not shown) within 21 days after the first vaccination and 30 days after the second vaccination was 50.0% in the 18–60 year age group (18.6% considered as related to vaccination) and 30.8% in the >60 year age group (9.6% considered as related to vaccination). The most frequently reported adverse events were rhinitis, nasopharyngitis and bronchitis. Grade 3 adverse events were reported by 10.8% of subjects aged 18–60 years (none considered as related to vaccination) and 9.9% of subjects aged >60 years (1.1% considered as related to vaccination) in group A and by 8.6% of subjects aged 18–60 years (4.3% considered as related to vaccination) and 3.8% of subjects aged >60 years (1.9% considered as related to vaccination) in group B.

Over the Day 0 to Day 364 study period reported here, nineteen serious adverse events were reported of which two were considered by the investigator as related to the vaccination. A 63 year-old female experienced hepatic enzyme increase at 21 days after the first vaccination; this was resolved on Day 42. The second event was a Herpes zoster infection reported 18 days following the second dose and resolved 22 days later.

There was one withdrawal from the study due to a non-serious adverse event. Up to Day 364, two pIMDs were reported (ankylosing spondylitis 157 days after the 2^nd dose and multiple sclerosis 37 days after the 2^nd dose). Neither event was considered by the investigator to be related to vaccination. There were two events of special interest reported: multiple sclerosis and urticaria. The urticaria event, reported on day 1 after the 1^st dose, was considered by the investigator to be related to vaccination and resolved 15 days later.