Safety and persistence of immunological response 6 months after intramuscular vaccination with an AS03-adjuvanted H1N1 2009 influenza vaccine

Abstract

This study evaluated the long-term persistence of immune response and safety of two doses of an A/California/7/2009 H1N1 pandemic influenza vaccine adjuvanted with AS03 (an α-tocopherol oil-in-water emulsion-based Adjuvant System) in Japanese children (NCT01001169). Sixty healthy subjects aged 6 mo−17 y were enrolled (1:1) into two study groups to receive 21 d apart, two doses of 1.9µg haemagglutinin [HA]+AS03_B (5.93mg α-tocopherol) vaccine (6 mo−9 y) and 3.75µg HA+AS03_A (11.86mg α-tocopherol) vaccine (10–17 y), respectively. Immunogenicity data (by haemagglutination inhibition [HI] and microneutralisation assays) to six months after the first vaccine dose are reported here. It was observed that following Dose 2, the HI immune response against the vaccine homologous strain induced by the two different dosages of the AS03-adjuvanted vaccine met and exceeded the US and European regulatory guidance criteria for pandemic influenza vaccines (seroprotection rate[SPR]/seroconversion rate[SCR]: 100%/100%; geometric mean fold rise GMFR: 146.8/57.1). Further, the immune response persisted for at least six months after the first vaccine dose wherein these regulatory criteria were still met (SPR: 100%/100%; SCR: 96.4%/89.7%; GMFR: 25.3/23.5). The neutralising antibody response was comparable to the HI immune response at Day 42 (vaccine response rate [VRR]: 100%/100%) and at Day 182 (VRR: 96.4%/82.8%). Overall, both vaccine dosages had a clinically acceptable safety profile. Thus, two doses of a 1.9µg or 3.75µg HA AS03-adjuvanted H1N1 2009 pandemic influenza vaccine in children aged 6 mo−17 y induced strong immune responses against the vaccine homologous strain that persisted for at least six months after the first vaccine dose.

Keywords: :

• adjuvant
• H1N1
• influenza
• Japanese children
• pandemic

Introduction

The emergence of a novel, swine-origin influenza A virus (H1N1 2009) that caused the first influenza pandemic of the 21st century re-affirmed the unpredictability of influenza viruses.¹ The H1N1 2009 pandemic spread rapidly across the globe leading to over 18,449 deaths in more than 214 countries.² The highest attack and hospitalisation rates for the H1N1 2009 pandemic virus were reported in children aged < 5 y, particularly those in the first year of life, presumably due to the degree of immunological naivety of this population toward this novel strain.^1,3-5

The first case of H1N1 2009 pandemic influenza in Japan was confirmed on May 09, 2009 and by February 05, 2010, the cumulative number of confirmed H1N1 2009 cases was estimated to have reached 20 million.⁶ Significantly, a small number of deaths due to the pandemic were reported (202 deaths as of August 10, 2010).⁷ As observed in other regions,^8,9 in Japan most of the H1N1 2009 pandemic influenza infections and associated hospitalisations were reported in children and adolescents.¹⁰

Immunisation is considered to be the most efficient method of mitigating influenza pandemic related morbidity and mortality.^11,12 In this context, the immunological naivety/lack of priming of young children to the novel H1N1 2009 strain coupled with their role in indigenous transmission of the virus made them a priority group for pandemic influenza vaccination.¹³

Based on previous experience of developing a prepandemic dose-sparing H5N1 influenza vaccine (3.75µg haemagglutinin [HA] with AS03 [an α-tocopherol oil-in-water emulsion-based Adjuvant System]),^14-17 an AS03-adjuvanted H1N1 2009 pandemic vaccine with 3.75µg HA content was developed for the 2009 influenza pandemic. This H1N1 2009 vaccine has been proven to be highly immunogenic (fulfilling the US and European regulatory guidance criteria for pandemic influenza vaccines) with a clinically acceptable safety profile in different populations^18,19 including adults in Japan.²⁰

In October 27, 2009, a phase II, open-label study (ClinicalTrials.gov Identifier: NCT01001169) in Japanese children was initiated at the National Center for Child Health and Development, Tokyo, Japan. Healthy children aged 6 mo to 17 y received two doses of either 1.9µg HA with AS03_B (6 mo−9 y) or 3.75µg HA with AS03_A (10–17 y) H1N1 2009 vaccine intramuscularly, 21 d apart. The co-primary objectives of this study were to assess whether vaccination with two doses of the AS03-adjuvanted 1.9µg HA or 3.75µg HA H1N1 2009 vaccines induced an immune response against the vaccine homologous strain 21 d after the second vaccine dose (Day 42) that met and exceeded the US and European regulatory guidance criteria for pandemic influenza vaccines. The preliminary immunogenicity and reactogenicity results following the first vaccine dose (Day 21) have been published earlier.²¹ This manuscript presents the immunogenicity and safety results from the six month follow-up phase of this study. The objectives for the follow-up phase were as follows: a) to assess whether two doses of the study vaccine induced persistence of immunological response at Day 182 that met the US and European regulatory guidance criteria for pandemic influenza vaccines, b) to describe homologous HI and neutralising antibody response 21 d after the second vaccine dose and at Day 182, and c) to evaluate the safety profile of the vaccine that was administered in this pediatric Japanese population through the intramuscular route.

Results

Study population

The six month follow-up phase of this study (through Day 182) was completed on May 17, 2010.

A total of 60 subjects were enrolled to be vaccinated (Group 1.9µgHA: 30 subjects [6−35 mo: 10; 3−9 y: 20]; Group 3.75µgHA: 30 subjects [10–17 y]), of which 57 subjects completed the study at Day 182. The ATP cohort for immunogenicity at Day 42 and Day 182 included 58 and 57 subjects, respectively (Fig. 1).

Figure 1. Study design diagram. Total vaccinated cohort (TVC): all subjects with at least one documented vaccine dose with available immunogenicity results. According-to-protocol (ATP) cohort for immunogenicity: all evaluable subjects (i.e., those meeting all eligibility criteria, with no elimination criteria during the relevant analysis interval), who received two vaccine doses and for whom assay results were available at Day 42 and Day 182. Group 1.9µgHA: Subjects aged 6 mo-9 y received two doses of 1.9µg HA+AS03_B (5.93mg α-tocopherol) vaccine, 21 d apart. Group 3.75µgHA: Subjects aged 10–17 y received two doses of 3.75µg HA+AS03_A (11.86mg α-tocopherol) vaccine, 21 d apart.

The mean age of subjects in the TVC at the time of the first vaccine dose was 4.1 y (range: 7 mo−104 mo) in Group 1.9µgHA and 13.6 y (range: 10−17.9 y) in Group 3.75µgHA. The overall male to female ratio was 43.3%:56.7% and all subjects were of Japanese heritage.

Immunogenicity

HI immune response

Prior to receiving vaccination, 17.2% of subjects aged 6 mo−9 y and 60% of subjects aged 10−17 y had detectable levels of HI antibodies against the H1N1 2009 strain. The second dose of the AS03-adjuvanted vaccine elicited a strong HI immune response in both age groups that met and exceeded the CHMP guidance criteria and more stringent CBER guidance criteria for pandemic influenza vaccines at Day 42. In the 6−35 mo age stratum the sample size was small (n = 10), as a consequence the lower limit of the 95% CI for SPR was not above 70%, despite a point estimate of 100% and a high GMT value (1279.9) (Table 1).

Table 1. Immune response in terms of haemagglutination inhibition antibodies against vaccine homologous A/California/7/2009 strain [CBER^c/CHMP^d criteria] (ATP cohort for immunogenicity)

Measure	Time point	Group 1.9µgHA 6 mo−9 y						Group 3.75µgHA 10–17 y
		N^a	Overall	Age sub-strata				N	Overall
				N	6–35 mo	N	3–9 y
		Value or Point estimate (95% CI^b)
Seroprotection rates	PRE^#^	29	3.4% (0.1–17.8)	10	0.0% (0.0–30.8)	19	5.3% (0.1–26.0)	30	26.7% (12.3–45.9)
	Day 21^	29	100% (88.1–100)	10	100% (69.2-100)	19	100% (82.4–100)	30	96.7% (82.8–99.9)
	Day 42	28	100% (85.5–100)*	9	100% (66.4-100)	19	100% (82.4–100)	30	100% (86.4–100)*
	Day 182	28	100% (87.7–100)	9	100% (66.4-100)	19	100% (82.4–100)	29	100% (88.1–100)
Seroconversion rates	Day 21^	29	100% (88.1–100)	10	100% (69.2–100)	19	100% (82.4–100)	30	90% (73.5–97.9)
	Day 42^	28	100% (85.5–100)*	9	100% (66.4–100)	19	100% (82.4–100)	30	100% (86.4–100)*
	Day 182	28	96.4% (81.7–99.9)	9	100% (66.4–100)	19	94.7% (74.0–99.9)	29	89.7% (72.6–97.8)
Geometric Mean Fold Rise	Day 21^	29	27.1 (20.4–36.1)	10	23.5 (14.4–38.2)	19	29.3 (19.9–42.9)	30	22.1 (13.6–35.9)
	Day 42	28	146.8 (99.6–216.4)*	9	256.0 (161.4–406.1)	19	112.8 (74.6–170.5)	30	57.1 (33.5–97.3)*
	Day 182	28	25.3 (18.4–34.7)	9	30.8 (20.1–47.3)	19	23.1 (14.9–35.8)	29	23.5 (14.9–37.1)
Geometric Mean Titers	PRE^#^	29	6.3 (5.0–8.1)	10	5.0 (5.0–5.0)	19	7.2 (5.0–10.3)	30	15.3 (9.5–24.6)
	Day 21^	29	172 (130.1–227.6)	10	117.3 (72.2–190.8)	19	210.4 (150.4–294.5)	30	339 (238.8–481.2)
	Day 42	28	939.3 (722.9–1220.6)*	9	1279.9 (806.9–2030.4)	19	811.3 (628.4–1047.4)	30	874.3 (717.4–1065.4)*
	Day 182	28	161.9 (133.7–196.1)	9	154.0 (100.3–236.5)	19	165.8 (131.9–208.5)	29	347.9 (254.0–476.5)
Seropositivity rates	PRE^#^	29	17.2% (5.8–35.8)	10	0.0 (0.0–30.8)	19	26.3% (9.1–51.2)	30	60.0% (40.6–77.3)
	Day 21^	29	100% (88.1–100)	10	100% (69.2–100)	19	100% (82.4–100)	30	100% (88.4–100)
	Day 42	28	100% (85.5–100)*	9	100% (66.4–100)	19	100% (82.4–100)	30	100% (86.4–100)*
	Day 182	28	100% (87.7–100)	9	100% (66.4–100)	19	100% (82.4–100)	29	100% (88.1–100)

Group 1.9µgHA: Subjects aged 6 mo-9 y received two doses of 1.9µg HA+AS03_B (5.93mg α-tocopherol) vaccine, 21 d apart. Group 3.75µgHA: Subjects aged 10–17 y received two doses of 3.75µg HA+AS03_A (11.86mg α-tocopherol) vaccine, 21 d apart. ^aN: Number of subjects with available results. ^bCI: Confidence Interval. ^cCBER: Center for Biologics Evaluation and Research [Lower limit of 95% CI: SPR: ≥ 70%; SCR: ≥ 40%]. ^dCHMP: Committee for Medicinal Products for Human Use [Point estimate: SPR: > 70%; SCR: > 40%; GMFR: > 2.5].^#PRE: Pre-vaccination antibody titers for ATP cohort for immunogenicity at Day 21.*As per pre-defined primary co-objectives of this study, Day 42 immune response was calculated with 97.5% CI as per the CBER criteria for Group A (age 6 mo to 9 y), Group B (age 10–17 y), while at the other time points 95% CI was used. BOLD: Values of SPR and Geometric mean fold rise that did not meet the pre-specified criteria. ^PRE and Day 21 data have been presented previously in the primary manuscript [ref. 21: Saitoh A, et al. J Japan Pediatr Soc 2011]

Six months after the first vaccine dose (Day 182), the HI immune response against the H1N1 2009 strain still met the CHMP and CBER criteria in subjects aged 6 mo−9 y and 10−17 y; similar to the Day 42 immune response in the 6−35 mo age stratum, the lower limit of the 95% CI for SPR at Day 182 was not above 70%, despite a point estimate of 100% (with a GMT value of 154) (Table 1).

It is to be noted that the HI assays for the sequential time points Day 0, Day 21 and Day 42 were tested together. The Day 182 samples were tested separately without an assessment of variability from earlier time points. Due to potential assay variability, a comparative interpretation of the HI response at Day 182 with earlier time points should be done with caution.

Microneutralisation assay

Prior to receiving vaccination, 10.3% of subjects aged 6 mo−9 y and 46.7% of subjects aged 10−17 y had detectable levels of neutralising antibodies against the A/Netherlands/602/2009 strain which is antigenically similar to A/California/7/2009 strain. Twenty one days after the second vaccine dose (Day 42), 100% of subjects in both age groups were seropositive for antibodies against the A/Netherlands/602/2009 strain; corresponding GMTs were 551.1 and 702.4, respectively. The VRR was 100% in both age groups (Table 2). Six months after the first vaccine dose, all subjects were still seropositive for antibodies against the A/Netherlands/602/2009 strain; corresponding GMTs were 149.6 and 213.3. The VRR was 96.4% and 82.4% in the two age groups, respectively (Table 2).

Table 2. Immune response in terms of neutralising antibodies against the A/Netherlands/602/2009 strain (ATP immunogenicity cohort)

Measure	Time point		Group 1.9µgHA 6 mo−9 y						Group 3.75µgHA 10–17 y
		N^a	Overall	Age sub-strata				N	Overall
				N	6–35 mo	N	3–9 y
			Value or Point estimate (95% CI^b)
Seropositivity rates	PRE^#	29	10.3% (2.2–27.4)	10	10.0% (0.3–44.5)	19	10.5% (1.3–33.1)	30	46.7% (28.3–65.7)
	Day 21	29	96.6% (82.2–99.9)	10	100% (69.2–100)	19	94.7% (74–99.9)	29	96.6 (82.2–99.9)
	Day 42	28	100% (87.7–100)	9	100% (66.4–100)	19	100% (82.4–100)	30	100% (88.4–100)
	Day 182	28	100% (87.7–100)	9	100% (66.4–100)	19	100% (82.4–100)	29	100% (88.1–100)
Geometric Mean Titers	PRE^#	29	4.9 (3.7–6.5)	10	4.7 (3.3–6.6)	19	5.0 (3.3–7.5)	30	11.3 (6.8–18.9)
	Day 21	29	53.8 (31.2–92.7)	10	42.5 (24.4–73.9)	19	60.8 (27–137.2)	29	121.2 (68.4–214.9)
	Day 42	28	551.1 (417–728.5)	9	623.4 (369.4–1052.1)	19	519.9 (362.3–745.5)	30	702.4 (433.5–1138)
	Day 182	28	149.6 (104.7–213.6)	9	161 (85.5–303.2)	19	144.4 (90–231.7)	29	213.3 (141.7–321)
Vaccine Response Rate	Day 21	29	51.7% (32.5–70.6)	10	60% (26.2–87.8)	19	47.4% (24.4–71.1)	29	58.6% (38.9–76.5)
	Day 42	28	100% (87.7–100)	9	100% (66.4–100)	19	100% (82.4–100)	30	100% (88.4–100)
	Day 182	28	96.4% (81.7–99.9)	9	100% (66.4–100)	19	94.7% (74–99.9)	29	82.8% (64.2–94.2)

Group 1.9µgHA: Subjects aged 6 mo-9 y received two doses of 1.9µg HA+AS03_B (5.93mg α-tocopherol) vaccine, 21 d apart. Group 3.75µgHA: Subjects aged 10–17 y received two doses of 3.75µg HA+AS03_A (11.86mg α-tocopherol) vaccine, 21 d apart. ^aN: Number of subjects with available results. ^bCI: Confidence interval. ^#PRE: Pre-vaccination antibody titers for ATP cohort for immunogenicity at Day 21. Vaccine response rate defined as percentage of subjects with either a pre-vaccination titer < 1:8 and a post-vaccination titer ≥ 1:32, or a pre-vaccination titer ≥ 1:8 and at least a 4-fold increase in post-vaccination titer.

Safety and Reactogenicity

Overall, at least one solicited or unsolicited local symptom was reported for 70−100% of subjects in Group 1.9µgHA (6−35 mo, 3−5 y, 6−9 y) and 100% of subjects in Group 3.75µgHA (10−17 y); at least one solicited or unsolicited general symptom was reported for 42.9−83.3% of subjects in Group 1.9µgHA (6−35 mo, 3−5 y, 6−9 y) and 80% of subjects in Group 3.75µgHA (10−17 y). At least one MAE was reported in 58.3% of subjects aged 6 mo−5 y and 33.3% of subjects aged 6−9 y and 10−17 y.

Tables 3 and 4 present the percentage of subjects reporting solicited local and general symptoms overall and by age strata. Pain at injection site was the most frequently reported solicited local symptom across all age groups (overall 60%, 92.9%, 100% and 100% for subjects aged 6−35 mo, 3−5 y, 6−9 y and 10−17 y, respectively). The occurrence of pain was transient in most cases with the mean number of days being 1.8 d, 2.6 d, 2.7 d and 3.9 d for subjects aged 6−35 mo, 3−5 y, 6−9 y and 10−17 y, respectively. Overall, the occurrence of Grade 3 solicited local symptoms were infrequent; Grade 3 injection site pain was reported for two subjects aged 3−5 y and five subjects aged 10−17 y, and Grade 3 injection site swelling for two subjects aged 10−17 y. The reporting of solicited local symptoms after each of the two doses was comparable.

Table 3. Solicited local symptoms reported during the 7 d post-vaccination follow-up period after each vaccine dose (Total vaccinated cohort)

		Group 1.9µgHA						Group 3.75µgHA
		6–35 mo		3–5 y		6–9 y		10–17 y
		Dose 1	Dose 2	Dose 1	Dose 2	Dose 1	Dose 2	Dose 1	Dose 2
		N^a = 10	n = 10	n = 14	n = 13	n = 6	n = 6	n = 30	n = 30
		Point estimate (95% CI^b)
Pain	Any	60.0 (26.2–87.8)	50.0 (18.7–81.3)	92.9 (66.1–99.8)	84.6 (54.6–98.1)	83.3 (35.9–99.6)	83.3 (35.9–99.6)	100 (88.4–100)	100 (88.4–100)
	Grade 3	0 (0–30.8)	0 (0–30.8)	7.1 (0.2–33.9)	7.7 (0.2–36.0)	0 (0–45.9)	0 (0–45.9)	10.0 (2.1–26.5)	6.7 (0.8–22.1)
Redness	Any	0 (0–30.8)	10.0 (0.3–44.5)	0 (0–23.2)	7.7 (0.2–36.0)	16.7 (0.4–64.1)	0 (0–45.9)	23.3 (9.9–42.3)	16.7 (5.6–34.7)
	Grade 3	0 (0–30.8)	0 (0–30.8)	0 (0–23.2)	0 (0–24.7)	0 (0–45.9)	0 (0–45.9)	0 (0–11.6)	0 (0–11.6)
Swelling	Any	30.0 (6.7–65.2)	10.0 (0.3–44.5)	14.3 (1.8–42.8)	23.1 (5.0–53.8)	33.3 (4.3–77.7)	33.3 (4.3–77.7)	46.7 (28.3–65.7)	50.0 (31.3–68.7)
	Grade 3	0 (0–30.8)	0 (0–30.8)	0 (0–23.2)	0 (0–24.7)	0 (0–45.9)	0 (0–45.9)	3.3 (0.1–17.2)	3.3 (0.1–17.2)

Group 1.9µgHA: Subjects aged 6 mo-9 y received two doses of 1.9µg HA+AS03_B (5.93mg α-tocopherol) vaccine, 21 d apart. Group 3.75µgHA: Subjects aged 10–17 y received two doses of 3.75µg HA+AS03_A (11.86mg α-tocopherol) vaccine, 21 d apart. ^aN: Number of subjects with available results. ^bCI: Confidence interval

Table 4. Solicited general symptoms reported during the 7-d post-vaccination follow-up period after each vaccine dose (Total vaccinated cohort)

					Group 1.9µgHA				Group 3.75µgHA
			6–35 mo		3–5 y		6–9 y		10–17 y
			Dose 1	Dose 2	Dose 1	Dose 2	Dose 1	Dose 2	Dose 1	Dose 2
			N^a = 10	n = 10	n = 14	n = 13	n = 6	n = 6	n = 30	n = 30
			Point estimate (95% CI^b)
Drowsiness		Any	10.0 (0.3–44.5)	30.0 (6.7–65.2)	28.6 (8.4–58.1)	30.8 (9.1–61.4)	–	–	–	–
		Grade 3	0 (0–30.8)	0 (0–30.8)	0 (0–23.2)	0 (0–24.7)	–	–	–	–
Irritability		Any	30.0 (6.7–65.2)	40.0 (12.2–73.8)	21.4 (4.7–50.8)	15.4 (1.9–45.4)	–	–	–	–
		Grade 3	10.0 (0.3–44.5)	0 (0–30.8)	0 (0–23.2)	0 (0–24.7)	–	–	–	–
Loss of appetite		Any	10.0 (0.3–44.5)	30.0 (6.7–65.2)	28.6 (8.4–58.1)	15.4 (1.9–45.4)	–	–	–	–
		Grade 3	0 (0–30.8)	10.0 (0.3–44.5)	0 (0–23.2)	0 (0–24.7)	–	–	–	–
Fever		Any	0 (0–30.8)	40.0 (12.2–73.8)	21.4 (4.7–50.8)	15.4 (1.9–45.4)	33.3 (4.3–77.7)	0 (0–45.9)	13.3 (3.8–30.7)	23.3 (9.9–42.3)
		Grade 3	0 (0–30.8)	20.0 (2.5–55.6)	0 (0–23.2)	0 (0–24.7)	0 (0–45.9)	0 (0–45.9)	6.7 (0.8–22.1)	0 (0–11.6)
Fatigue		Any	–	–	–	–	16.7 (0.4–64.1)	16.7 (0.4–64.1)	36.7 (19.9–56.1)	36.7 (19.9–56.1)
		Grade 3	–	–	–	–	0 (0–45.9)	0 (0–45.9)	3.3 (0.1–17.2)	3.3 (0.1–17.2)
Gastrointestinal		Any	–	–	–	–	16.7 (0.4–64.1)	16.7 (0.4–64.1)	10.0 (2.1–26.5)	10.0 (2.1–26.5)
		Grade 3	–	–	–	–	0 (0–45.9)	0 (0–45.9)	0 (0–11.6)	0 (0–11.6)
Headache		Any	–	–	–	–	33.3 (4.3–77.7)	16.7 (0.4–64.1)	40.0 (22.7–59.4)	33.3 (17.3–52.8)
		Grade 3	–	–	–	–	0 (0–45.9)	0 (0–45.9)	0 (0–11.6)	6.7 (0.8–22.1)
Joint pain at other location		Any	–	–	–	–	0 (0–45.9)	0 (0–45.9)	16.7 (5.6–34.7)	23.3 (9.9–42.3)
		Grade 3	–	–	–	–	0 (0–45.9)	0 (0–45.9)	0 (0–11.6)	0 (0–11.6)
Muscle aches		Any	–	–	–	–	0 (0–45.9)	0 (0–45.9)	23.3 (9.9–42.3)	30.0 (14.7–49.4)
		Grade 3	–	–	–	–	0 (0–45.9)	0 (0–45.9)	0 (0–11.6)	0 (0–11.6)
Shivering		Any	–	–	–	–	16.7 (0.4–64.1)	0 (0–45.9)	23.3 (9.9–42.3)	26.7 (12.3–45.9)
		Grade 3	–	–	–	–	0 (0–45.9)	0 (0–45.9)	0 (0–11.6)	0 (0–11.6)
Sweating		Any	–	–	–	–	33.3 (4.3–77.7)	0 (0–45.9)	6.7 (0.8–22.1)	10.0 (2.1–26.5)
		Grade 3	–	–	–	–	0 (0–45.9)	0 (0–45.9)	3.3 (0.1–17.2)	0 (0–11.6)

Group 1.9µgHA: Subjects aged 6 mo-9 y received two doses of 1.9µg HA+AS03_B (5.93mg α-tocopherol) vaccine, 21 d apart. Group 3.75µgHA: Subjects aged 10–17 y received two doses of 3.75µg HA+AS03_A (11.86mg α-tocopherol) vaccine, 21 d apart. ^aN: Number of subjects with available results. ^bCI: Confidence interval

The most frequently reported solicited general symptoms varied across the different age groups; irritability (50% of subjects aged 6−35 mo), drowsiness (35.7% of subjects aged 3−5 y) and headache (50% of subjects aged 6−9 y and 66.7% of subjects aged 10−17 y). No fever was reported for subjects aged 6−35 mo following the first vaccine dose. However, following the second vaccine dose, four subjects (out of 10) developed fever, of which two cases of fever were of Grade 3 intensity (≥ 39°C). Overall, among subjects aged 6−35 mo, severe loss of appetite and irritability were reported for one subject each and Grade 3 fever for two subjects and among subjects aged 10−17 y, severe sweating was reported for one subject, Grade 3 fatigue and Grade 3 headache for two subjects each and Grade 3 fever for one subject, while no Grade 3 symptoms were reported among subjects aged 3−9 y.

Overall, 40 subjects reported at least one unsolicited adverse event upto Day 84: 17 (70.8%) in subjects aged 6 mo–5 y, 2 (33.3%) in subjects aged 6−9 y, and 21 (70%) in subjects aged 10−17 y. For subjects aged 6 mo−5 y, rhinorrhoea (7 subjects), upper respiratory tract infection and cough (6 subjects each) were the most commonly reported symptoms, while for subjects aged 6−9 y, pyrexia (2 subjects) was most frequently reported. Among subjects aged 10−17 y, there were no clear predominance of any unsolicited symptoms; axillary pain, pharyngitis, headache, cough, influenza, rhinitic allergy and acne were reported for two subjects each.

Two SAEs were reported during the entire study period. One subject in the 6−35 mo age strata presented severe febrile convulsion approximately five months after receiving the second vaccine dose which resolved in one day; the other subject in the 10−17 y age strata had a fracture in the foot approximately three months after the second vaccine dose which resolved in 72 d; none of the two SAEs were considered by the investigators to be vaccination-related. No pIMDs or fatalities were reported during the study period.

Discussion

This is the first study to report the persistence of the immunological response against the A/California/07/2009 strain in children, six months after vaccination with an AS03-adjuvanted H1N1 2009 pandemic influenza vaccine.

In this study, the 1.9µg HA AS03_B-adjuvanted H1N1 2009 pandemic influenza vaccine induced a strong HI immune response in subjects aged 6 mo−9 y as evident from the high SPR/SCR (100%) following the second vaccine dose. In previous studies, similar formulations of the AS03-adjuvanted H1N1 2009 vaccine have been shown to be optimally immunogenic in subjects aged 6−35 mo and 6 mo−12 y.^19,22 Thus, the data in Japanese children conforms to the strong immunogenicity profile of the vaccine observed in other pediatric populations.

A single dose of AS03-adjuvanted 1.9µg HA H1N1 2009 vaccine in Canadian children aged 36 mo to 9 y has been found to have a protective effectiveness of 100%, 14 d following a single vaccine dose (statistically significant difference with the control group).²³ Although this value was 96% when effectiveness was assessed 10 d after vaccination, it remained at 100% in subjects aged < 36 mo.²³ These observations are in agreement with the preliminary results obtained from this study in Japanese children, which reported a strong HI antibody immune response against the vaccine homologous strain (SPR and SCR 100%) after just one dose of the AS03-adjuvanted H1N1 2009 vaccine.²¹

The 3.75µg HA AS03-adjuvanted H1N1 2009 vaccine also induced a strong HI immune response - SPR/SCR of 96.7%/90% following the first dose and 100%, following the second vaccine dose in subjects aged 10−17 y. Considering the above mentioned protective effectiveness reported in younger Canadian children (36 mo to 9 y old),²³ these immunogenicity results obtained in subjects aged 10−17 y in this study suggested that a single dose of 3.75µg HA of AS03-adjuvanted H1N1 2009 vaccine induced a substantial protection against H1N1 2009 pandemic influenza virus.

Six months after the first vaccine dose, the HI immune response against the vaccine homologous strain were well maintained (high SPRs of 100% and SCRs of 96.4% and 89.7%, respectively), in subjects aged 6 mo−9 y and 10−17 y. Data on the long-term persistence of the immune response following pandemic influenza vaccination in children is limited. However, the observations from this study is in agreement with available data from studies in adults that the immune response induced by two doses of the 3.75µg HA AS03-adjuvanted H1N1 2009 vaccine persists for at least six months after vaccination.^24,25

The HI immune response against the A/California/7/2009 strain induced by the 1.9µg and 3.75µg HA dosages of the AS03-adjuvanted study vaccine was further corroborated when the CHMP guidance criteria²⁶ and the more stringent CBER guidance criteria²⁷ for pandemic influenza vaccines were met and exceeded, following the second vaccine dose and also six months after the first vaccine dose. The neutralising antibody titers parallel the HI immune response following each of the two vaccine doses and six months after the first vaccine dose.

Overall, the two vaccine dosages had clinically acceptable safety profiles in the respective study groups. Most solicited local and general symptoms were transient, and mild or moderate in intensity. Four cases of fever of which two were of grade 3 intensity were reported in subjects aged 6−35 mo following the second vaccine dose. Post-hoc assessments indicated that these cases may be associated with the strong increase of the humoral immune response [data not shown]. Three out of these four subjects had the highest HI antibody titers (1810 and 2560) among children aged 6−35 mo; the remaining subject did not return for visit at Day 42. A similar observation was also made in a previous study and a possible association with increase in humoral immune response was made.¹⁹ However, considering that the number of subjects in both studies is limited, further evaluation on a larger number of subjects would be required to ascertain the plausible reason for this observation.

When compared with the safety profile of a non-adjuvanted, trivalent seasonal influenza vaccine in a pediatric population aged between 6 mo and < 18 y, the AS03-adjuvanted vaccine similar to the one used in the present study demonstrated increased frequency of solicited local symptoms, though more similar for general symptoms as well as MAEs and SAEs. However, the trend of slightly lower frequencies of solicited symptoms reported following the second dose as compared that after the first dose in the referenced study was reversed in the present study.²⁸ In another pediatric study with a non-adjuvanted, trivalent seasonal influenza vaccine in subjects aged 6–9 y and 10–13 y, a similar trend of comparatively higher reactogenicity after the second vaccine dose was observed.²⁹ A recent report from six studies in children showed that the frequency of solicited local and general symptoms following vaccination with a MF59-adjuvanted seasonal influenza vaccine was comparatively (although not significantly) higher than that following vaccination with a non-adjuvanted seasonal influenza vaccine.³⁰

In Japan, the AS03-adjuvanted H1N1 2009 pandemic influena vaccine was approved as a 1.9µg HA dose in children aged 6 mo−9 y and as a 3.75µg HA dose in children aged 10 y and older, making the age-specific immunogenicity data obtained from this study particularly relevant. Also, the fact that a microneutralisation assay was used in parallel with the conventional HI assay for immunological assessments makes the findings pertinent as while HI assays are largely restricted to measuring the receptor-binding blocking activity of antibodies, theoretically, neutralisation assays can capture a broad range of anti-influenza antibody activities able to interrupt several steps of the infectious life cycle of the virus.^31,32 Further, the AS03-adjuvanted H1N1 2009 vaccines used in this study allowed dose-sparing, a property that could be beneficial in meeting the requirement for a large number of vaccine doses at the time of an influenza pandemic.

This study was restricted in drawing comparative conclusions on the persistence of the immune response following vaccination with other adjuvanted or non-adjuvanted H1N1 2009 vaccines, as it is difficult to reliably compare HI results across studies.

In conclusion, the data from this study conducted with an AS03-adjuvanted H1N1 2009 pandemic influenza vaccine establishes that, following two doses of a 1.9µg or 3.75µg HA in children aged 6 mo−17 y, the immune response against the vaccine homologous A/California/7/2009 strain persists for at least six months after the first vaccine dose and the US and European guidance criteria for pandemic influenza vaccines were still met. The safety data from this study added to the existing repertoire of safety data in published literature on the safety of this H1N1 2009 vaccine. In addition, it may contribute to a better understanding of the safety of intramuscular vaccination in Japan. Intramuscular injection has not been allowed in Japan since 1970s after more than three thousands cases of muscular contracture being reported after intramuscular injection of antibiotics and antipyretics, but not vaccines.³³ This issue needs to be clarified urgently given that new combination vaccines and adjuvanted vaccines are expected to be introduced in Japan in the near future.

Materials and methods

Study design and subjects

The primary phase of the study in Japan (NCT01001169) enrolled healthy children aged between 6 mo and 17 y before study start, without history of clinically-confirmed influenza infection or previous vaccination with a novel H1N1 vaccine or with any seasonal influenza vaccine within two weeks before study start. The subjects aged 6 mo to 9 y were further stratified by age (stratification ratio: 1:2) into 6−35 mo and 3−9 y age strata by the study personnel using GlaxoSmithKline (GSK) Biologicals’ internet-based central randomization system (SBIR). Subjects aged 6 mo−9 y received 21 d apart, two 0.25ml doses of the 1.9µg HA/AS03_B vaccine (Group 1.9µgHA) and subjects aged 10−17 y received two 0.5ml doses of the 3.75µg HA/AS03_A vaccine (Group 3.75µgHA). All subjects received the first vaccine dose between Oct 27, 2009 and Nov 06, 2009, and the subjects aged 6 mo−9 y received the second vaccine dose by Nov 30, 2009. The treatment and vial lists were generated at GSK Biologicals using SAS® (Cary, NC, USA) to assign treatments to subjects.

Written informed consent was obtained from the parents/guardians of all subjects prior to conducting any study-related procedures. Wherever deemed necessary, informed assent was collected from the subjects. The study was conducted in accordance with the Good Clinical Practice guidelines, the Declaration of Helsinki and local regulations. All study-related documents were approved by an Institutional Review Board.

Study vaccine

The study vaccine was developed and manufactured by GSK Biologicals. The H1N1 2009 pandemic influenza vaccine was a monovalent, inactivated, split-virion antigen with an oil-in-water emulsion-based Adjuvant System AS03 (Arepanrix™, a trademark of GlaxoSmithKline group of companies, Belgium). The H1N1 viral seed for the vaccine was prepared from the reassortant virus NYMC X-179A (New York Medical College, New York) generated from the A/California/07/2009 strain, as recommended by the World Health Organization (WHO).³⁴

The AS03-adjuvanted H1N1 2009 pandemic influenza vaccine was prepared prior to administration by mixing the antigen suspension and adjuvant emulsion (1:1), both of which were available in separate multi-dose vials. Group 1.9µgHA received AS03_B - an Adjuvant System containing 5.93mg α-tocopherol with 1.9µg HA (0.25ml injection dose) and Group 3.75µgHA received AS03_A - an Adjuvant System containing 11.86mg α-tocopherol with 3.75μg of HA (0.5 ml injection dose).³⁵

The first dose of the study vaccine was intramuscularly administered on Day 0 either into the anterolateral region of the thigh in children aged < 12 mo or into the deltoid of the non-dominant arm in subjects aged 12 mo or more. On Day 21, the second dose of study vaccine was administered on the opposite side.

Immunogenicity assessments

Serum samples were collected before vaccination (Day 0), 21 d after each of the two vaccine doses (Day 21 and Day 42) and six months after the first vaccine dose (Day 182).

Haemagglutination inhibition (HI) assay [cut-off: ≥ 1:10] using chicken erythrocytes as previously described³⁶ was performed at GSK Biologicals’ central laboratory. The samples from Day 0, Day 21 and Day 42 were tested at the same time point, while the Day 182 samples were tested at a later time point.

The viral microneutralisation assay was performed at Viroclinics Biosciences BV. The sera were used after heat treatment at 56°C for 30 min. Each serum was tested in triplicate. The assay used a constant amount of A/Netherlands/602/2009 pandemic H1N1 Influenza virus (a A/California/07/2009-like virus) mixed with serial 2-fold dilutions of serum samples. The mixture of virus and antiserum was added to Madin-Darby Canine Kidney (MDCK) cell cultures and incubated for one hour at 33–35°C. Then virus-antibody mixture was removed from the wells, cells were fed with fresh culture medium and further incubated for 6 d at 33–35°C. After the incubation period, virus replication was visualized by haemagglutination of red blood cells. The 50% neutralisation titer of a serum was calculated by the method of Reed and Muench.³⁷ The cut-off value of the assay was 1:8.

The assessment of the immune response was based on the seroconversion rate (SCR: percentage of subjects with pre-vaccination titer < 1:10 and post-vaccination titer ≥ 1:40, or pre-vaccination titer > 1:10 and at least 4-fold increase in post-vaccination titer), seroprotection rate (SPR: percentage of subjects with a post-vaccination titer ≥ 1:40) and geometric mean fold rise (GMFR: post-vaccination fold increase in geometric mean titers [GMTs]) in terms of HI antibodies against the vaccine homologous strain and on the the Vaccine Response Rates (VRRs: percentage of subjects with either a pre-vaccination titer < 1:8 and a post-vaccination titer ≥ 1:32, or a pre-vaccination titer ≥ 1:8 and at least a 4-fold increase in post-vaccination titer) in terms of neutralising antibodies against a strain antigenically similar to the vaccine strain.

The outcome measures of the immune response included evaluation based on the immunogenicity criteria for pandemic influenza vaccines in adults as required by the Committee for Medicinal Products for Human Use (CHMP; point estimates for HI antibody SCR: > 40%, SPR: > 70% and GMFR: > 2.5) and Center for Biologics Evaluation and Research (CBER; lower bound of 95% confidence interval [CI] for HI antibody for SCR: ≥ 40% and SPR: ≥ 70%).^26,27 In consideration of multiplicity of statistical analysis caused by co-primary endpoints of the study, 97.5% confidence intervals (CIs) were applied instead of 95% CIs (requirement of CBER guidance) for evaluation of the primary endpoints at Day 42.

Safety and reactogenicity assessments

Diary cards were used by parents/guardians to record solicited local and general adverse events up to seven days following each vaccine dose; unsolicited adverse events were recorded up to 84 d following the first vaccine dose; medically-attended events (MAEs), potential immune-mediated diseases (pIMD) and serious adverse events (SAEs) occurring during the entire study period were recorded.

Intensity of solicited symptoms was graded on a standard scale of (0–3), where Grade 1 symptoms were defined as those that were noticeable but did not interfere with normal activities and Grade 3 symptoms were defined as those that prevented normal activities (Grade 3 redness and swelling: diameter > 100mm; Grade 3 fever: temperature ≥ 39°C [≥ 102.2°F]). SAEs and pIMDs (subset of adverse events that include both autoimmune diseases and other inflammatory and/or neurologic disorders which may or may not have an autoimmune etiology) occurring throughout the study period were also recorded. Clinical laboratory parameters were assessed at all seven visits up to Day 182.

Statistical analyses

The sample size was calculated based on the co-primary objectives of the study using the results from the most recent studies with the H1N1 2009 vaccine as a reference. A population of 60 subjects (30 subjects in each study group) accounting for ≤ 10% dropout was estimated to provide a power of 84.9% to achieve the co-primary objectives, assuming log (standard deviation) for GMT to be 0.6.

The analyses of immunogenicity were performed on the According-To-Protocol (ATP) cohort that included subjects who received both vaccine doses as per protocol, complied with all protocol-defined procedures and for whom the assay results were available at the given time points (at Day 42 and Day 182). Seropositivity was defined as antibody titers greater than or equal to the cut-off value of each assay. For the purpose of GMT calculations, antibody titers below the cut-off value of each assay were substituted by half of the cut-off value.

The analyses of safety were performed on the Total Vaccinated Cohort (TVC) which included all subjects who received at least one documented vaccine dose.

Abbreviations:
ATP	=	According-To-Protocol
CBER	=	Center for Biologics Evaluation & Research
CHMP	=	Committee for Medicinal Products for Human Use
CI	=	Confidence Interval
GMFR	=	Geometric Mean Fold Rise
GMT	=	Geometric Mean Titre
HA	=	Hemagglutinin
HI	=	Hemagglutination Inhibition
MAE	=	Medically-Attended Event
pIMD	=	potential Immune-Mediated Disease
SAE	=	Serious Adverse Event
SCR	=	Seroconversion Rate
SPR	=	Seroprotection Rate
TVC	=	Total Vaccinated Cohort
VRR	=	Vaccine Response Rate
WHO	=	World Health Organization

Acknowledgments

All authors participated in the implementation of the study including substantial contributions to conception and design, the gathering of the data, or analysis and interpretation of the data. All authors were involved in the drafting of the article or revising it critically for important intellectual content, and final approval of the manuscript.

We are grateful to the New York Medical College, New York for providing the vaccine virus reassortant and to the National Institute for Biological Standards and Control (NIBSC, UK) and Therapeutic Goods Administration (TGA) from the Australian Government for providing the reference standards. The authors are indebted to the participating study volunteers and their parents, clinicians, nurses and laboratory technicians at the study sites as well as to the sponsor’s project staff for their support and contributions throughout the study. We are grateful to all teams of GSK Biologicals for their contribution to this study, especially Shinobu Tamura, Hiroshi Tamura and Kazunori Yagi for clinical study management and site monitoring, and Roger Bernhard and Urban Lundberg from the clinical and serological laboratory teams, Dorothy Slavin (Clinical Safety Representative) and Edith Lepine for project management. Finally the authors thank Dr. Karl Walravens for critical review of the manuscript, Avishek Pal (GSK Biologicals) for providing medical writing services and Dr. Wendy Van Doorslaer (XPE Pharma and Science, on behalf of GSK Biologicals) for editorial assistance and manuscript coordination.

Disclosure of Potential Conflicts of Interest

Dr. A. Nagai was the principal investigator, Dr. A. Saitoh and Dr. T. Kato contributed as a supervisor in this study funded by GlaxoSmithKline. All participating institutions received compensation for study involvement. Drs. K. Tenjinbaru, D. Vaughn, F. Roman and P. Li are employees of GlaxoSmithKline Biologicals. D. Vaughn and F. Roman report ownership of stock options.

Financial disclosure

GlaxoSmithKline Biologicals was the funding source and was involved in all stages of the study conduct and analysis (ClinicalTrials.gov Identifier: NCT01001169). GlaxoSmithKline Biologicals also took in charge all costs associated with the development and the publishing of the present manuscript. All authors had full access to the data and the corresponding author had final responsibility to submit for publication.

Trademark statement

Arepanrix is a trade mark of the GlaxoSmithKline group of companies, Belgium.