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Vaccine
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Immunogenicity and safety of AS03A-adjuvanted H5N1 influenza oossMark
vaccine prepared from bulk antigen after stockpiling for 4 years
Olivier Godeauxa*, Patricia Izurietaa, Miguel Madariagab, Mamadou Draméc, Ping Lic, David W. Vaughn d
a GlaxoSmithKline Vaccines, Wavre, Belgium b Naples Community Hospital, Naples, FL, USA c GlaxoSmithKline Vaccines, King of Prussia, PA, USA d GlaxoSmithKline Vaccines, Rixensart, Belgium
ARTICLE INFO
ABSTRACT
Article history: Received 3 February 2014 Received in revised form 2July 2014 Accepted 17 July 2014 Available online 30 July 2014
Keywords: AS03
H5N1 vaccine Pandemic influenza Stockpile Bulk antigen
Background: Stockpiling vaccine for deployment in the event of an influenza pandemic is an important mitigation strategy. A necessary aspect of stockpiling is to determine the shelf-life of the stored vaccine. Methods: In this Phase II, open-label study we assessed the immunogenicity and safety of H5N1 A/Indonesia/5/2005 vaccine adjuvanted with AS03a. The AS03a-H5N1 vaccine was prepared from bulk antigen that had been stored for 4 years, and adjuvant that had been stored for 2.5 years. Both the antigen and adjuvant were filled in separate multi-dose vials within 4 months of use, and on the day of vaccination, the contents of antigen and adjuvant vials were mixed. Seventy-eight adults aged 18-64 years were scheduled to receive two doses of hemagglutinin-antigen (3.75 ^g) given 21 days apart. Antibody responses were assessed by hemagglutination-inhibition (HI) assay according to age (18-30 years, 31-40 years, 41-50 years, and 51-64 years). Reactogenicity was assessed for 7 days after each vaccination, and safety was assessed for 385 days post-vaccination (NCT01416571).
Results: The vaccine was immunogenic. Twenty-one days after the second dose of vaccine in the overall population, the HI seroconversion rate and seroprotection rate (SPR; titer >1:40) was 96.0% and 98.7%, respectively. At Day 182 after vaccination, the SPR was 76.7% in the overall population. Injection site pain was the most frequent solicited adverse event (91.0%), and no safety concerns were raised. Conclusion: The immunogenicity and safety observed with AS03A-H5N1 vaccine formulated with bulk antigen which had been stockpiled before vialing and administration was consistent with that previously observed with newly manufactured AS03a-H5N1 vaccine. This suggests that stockpiling bulk antigen for 4 years does not compromise the immunogenicity or reactogenicity of the vaccine.
© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
The development of vaccines against influenza viruses with pandemic-potential is a public health priority. Among influenza viruses, avian-origin A/H5N1 remains a threat, and as such, the
Abbreviations: AEs, adverse events; CBER, US Center for Biologics Evaluation and Research; CHMP, Committee for Medicinal Products for Human Use; CIs, confidence intervals; GMTs, geometric mean titers; HA, hemagglutinin antigen; HI, hemagglutination-inhibition; MAEs, medically attended adverse events; MGI, mean geometric increase; pIMDs, potentially immune-mediated diseases; SAEs, serious adverse events; SCR, seroconversion rate; SPR, seroprotection rate; WHO, World Health Organization.
* Corresponding author. Tel.: +32 473893771.
E-mail address: olivier.o.godeaux@gsk.com (O. Godeaux).
World Health Organization (WHO) recommends vaccines are produced against clade 1 or clade 2 H5N1 strains [1].
Meeting demand for vaccines at the start of a pandemic will depend upon the ability to provide sufficient doses within the existing worldwide vaccine manufacturing infrastructure. As it typically takes 4-6 months to release the first doses of vaccine using egg-derived antigens, producing a vaccine matched to the novel emerging strain in time to mitigate the spread of virus is a major challenge [2]. Strategies proposed to overcome these issues include: the formulation of vaccines with an adjuvant to decrease the amount of antigen needed per dose ('antigen sparing'), thus increasing the number of doses available early in the response; and stockpiling subtype-matched but not necessarily strain-matched H5N1 vaccine to be deployed during a pandemic alert or at the start of a pandemic to prime a population in advance of a booster dose of vaccine matched to the novel pandemic strain [3,4].
http://dx.doi.org/10.1016/j.vaccine.2014.07.062
0264-410X/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
There are two H5N1 vaccines currently licensed for the national stockpile in the United States, which are manufactured by Sanofi-Pasteur Inc. and GlaxoSmithKline (GSK) Vaccines, and various pandemic influenza vaccines are licensed in Europe, including Prepandrix™, Adjupanrix™, and Pumarix™ which are manufactured by GSK Vaccines. The vaccines produced by GSK Vaccines are formulated with an oil-in-water Adjuvant System 03 (AS03), and have been shown to elicit strong, durable, cross-clade immune responses in adults [5-8]. The bulk antigen and AS03 adjuvant are stored separately, and then presented in two vials to be mixed at the time of administration. An important aspect of stockpiling is to determine the shelf-life of the vaccine.
In this study we evaluated the immunogenicity, reactogenicity, and safety profile of H5N1 vaccine antigen which had been stored for 4 years, and AS03a adjuvant which had been stored for 2.5 years. The antigen and adjuvant were filled in separate multi-dose vials within 4 months of the study, to be mixed on the day of administration. The aim of the study was to assess the feasibility of stockpiling adjuvanted pandemic influenza vaccines.
2. Methods
This Phase II, open-label study of adults 18-64 years of age, was conducted to assess the immunogenicity and safety of AS03A-adjuvantedH5N1 (A/lndonesia/5/2005) pandemic influenza vaccine in which the antigen had been stockpiled for 4 years.
Adults who were in stable health were eligible to participate. Women of child bearing potential were required to practice adequate contraception for 30 days before and 2 months after study vaccination, and had a negative pregnancy test on the day of each vaccine dose. Exclusion criteria included: previously received any H5N1 vaccine; received any investigational or non-registered drug or vaccine within 30 days; received seasonal influenza vaccine within 14 days before, or any other vaccine within 30 days; significant acute or chronic, uncontrolled medical or psychiatric illness; cancer diagnosis or treatment within 3 years; temperature of >38 °C (>100.4 °F) at baseline assessment; immunosuppressive or immunodeficient condition; systemic glucocorticoids within 1 month, immunoglobulins or any blood products within 3 months; suspected or known allergy to any constituent of influenza vaccines; neurological disorder or history of Guillain-Barré syndrome.
The study was performed at a single center in the United States. All protocols and study documentation were approved by independent/local ethics committees in accordance with Good Clinical Practice, the Declaration of Helsinki, and regulatory requirements (ClinicalTrials.gov NCT01416571). All subjects provided informed written consent.
2.1. Vaccines and enrolment
The study vaccine was an H5N1 inactivated, split-virion influenza vaccine manufactured by GSK Vaccines in Québec, Canada. AS03 was manufactured by GSK Vaccines in Rixensart, Belgium.
The vaccine contained 3.75 |ig H5N1 A/lndonesia/5/2005 hemagglutinin antigen (HA), which was adjuvanted with AS03A containing 11.86 mg tocopherol. The bulk antigen was manufactured in January 2007. The adjuvant was manufactured and stored in February 2009. The bulk antigen and adjuvant were formulated and presented in separate multi-dose vials in April 2011 for use in the study. The vials were stored at +2-8 °C and were mixed by study personnel prior to administration. Mixing of 1 vial of antigen (lot number DFLPA659A) and 1 vial of adjuvant (lot number AA03A208A) provided 8x 0.5 mL doses of AS03a-H5N1 containing 3.75 | g HA.
Stratification was performed by the study sponsor using MATEX in SAS (Cary, NC, USA), and vaccine allocation at study sites was performed using a central internet-based system. Subjects were stratified 1:1:1:1 according to age groups: 18-30 years, 31-40 years, 41-50 years, and 51-64 years. Subjects received two doses of vaccine 21 days apart administered intramuscularly in the deltoid muscle of the non-dominant followed by the dominant arm, using a 25 mm (1 in.), 22-25 gauge needle. The study was open-label.
2.2. Immunogenicity assessments
Blood samples were taken for the evaluation of immune responses on Days 0, 42, and 182. All serological testing was performed at GSK Vaccines (Dresden, Germany) using validated procedures. Antibody responses against the vaccine strain (A/lndonesia/05/2005) were assessed using hemagglutination-inhibition (HI) assays, which were performed using an established HI method, modified for equine rather than avian ethrocytes [9-12]. Serum antibody titers were calculated as the geometrical mean values following duplicate testing.
HI antibody parameters were geometric mean titer (GMT), sero-protection rate (SPR; percentage of subjects with HI titers >1:40 post-vaccination), seroconversion rate (SCR; percentage of subjects achieving an increase in HI titers from <1:10 to >1:40 or at least a fourfold post-vaccination increase in Hl titer from a pre-vaccination titer of >1:10), and mean geometric increase (MGl; geometric mean of the ratio between post-vaccination and pre-vaccination reciprocal HI titers). Subjects with HI antibody titers of >1:10 were considered to be seropositive.
2.3. Reactogenicity and safety assessment
Solicited injection site and general symptoms were assessed during the 7-day post-vaccination period after each dose of vaccine. Subjects recorded the occurrence and severity of symptoms using diary cards, including injection site symptoms (pain, redness, and swelling), and general symptoms (fatigue, fever, gastrointestinal symptoms, headache, joint pain, muscle aches, shivering, sweating). Fever was defined as a temperature of >38.0 °C (>100.4°F) by any route or method. The severity of solicited symptoms was graded from 1 to 3 defined as: (1) mild and does not prevent daily activities; (2) moderate and interferes with daily activities; (3) severe and prevents daily activities. For redness and swelling, severity was defined in terms of the diameter of the affected area: (1) >20-50 mm; (2) >50-100 mm; (3) >100 mm. Fever was defined as a temperature of >38.0 °C (>100.4°F) by any route or method. Grade 3 fever was defined as a temperature of >39.0 °C (there was no grade 4 fever, >40.0°C). All solicited injection site events were considered to be vaccine-related, and investigators provided causality assessments for solicited general events.
Unsolicited adverse events (AEs) were recorded from Day 0 to Day 84. Medically attended AEs (MAEs), serious AEs (SAEs), and potentially immune-mediated diseases (plMDs) were recorded from Day 0 to Day 385. Potential lMDs were included in the protocol in accordance with regulatory requirements and are a subset of AEs including autoimmune diseases and neurologic disorders, such as neurological/demyelinating events (including narcolepsy), rheumatic and connective diseases, inflammatory bowel diseases, autoimmune endocrine diseases (including autoimmune hepatitis), autoimmune blood disorders, and inflammatory skin disorders.
Smoking history was recorded whenever an SAE was reported. Unsolicited events were coded using the Medical Dictionary for Regulatory Activities and investigators provided causality assessments for unsolicited AEs, MAEs, SAEs, and plMDs. Serum chemistries and hematology were assessed on Days 0 and 42.
2.4. Objectives
The primary objective was to evaluate vaccine-homologous HI antibody titers 21 days after the second vaccination (Day 42) to assess whether licensure criteria for immunogenicity were fulfilled according to the US Center for Biologics Evaluation and Research (CBER) immunogenicity criteria and the European Committee for Medicinal Products for Human Use (CHMP) licensure criteria [13,14]. Fulfillment of the CBER licensure criteria was defined as: (1) lower limit of the 95% Confidence Intervals (CIs) for SCR >40%; (2) lower limit of 95% CIs for SPR >70%. The fulfillment of the CHMP licensure criteria was defined as: (1) the point estimate for SCR >40%; (2) the point estimate for SPR >70%; (3) the point estimate for MGI >2.5. Collectively these five measures represented the primary endpoint.
Further immunogenicity objectives were to describe vaccine-homologous HI antibody responses at Days 0 and 42, and the persistence of antibody responses at Day 182. HI antibody parameters were seropositivity rates, GMTs, SCR, SPR, and MGI in the population overall, and stratified by age (18-30 years, 31-40 years, 41-50 years, and 51-64 years).
Safety objectives were to assess solicited local and general symptoms during the 7-day post-vaccination period after each dose of vaccine, unsolicited AEs 21 days after each dose of vaccine and from Day 0 to Day 84, MAEs, SAEs and pIMDs from Day 0 to Day 385, and laboratory parameters on Days 0 and 42.
2.5. Statistical analyses
Based on a previous study of AS03a-H5N1 vaccine [15], it was estimated that 78 subjects assuming a drop-out rate of 10% would provide 71 evaluable subjects which would have 98% power to fulfill the licensure criteria after two doses of vaccine [13,14].
Immunogenicity, reactogenicity, and safety data were summarized with 95% CIs. Reactogenicity and safety were assessed in the total vaccinated cohort including all subjects who received at least one dose of vaccine. Immunogenicity was assessed in the per-protocol immunogenicity cohort (Day 42) or the per-protocol immunogenicity persistence cohort (Day 182) including subjects in the total vaccinated cohort without protocol violation and with a serology sample available at the assessment time-point.
It should be noted that the safety assessment was descriptive and the study was not powered to detect rare adverse events (i.e., pIMDs), nor was the study sample sufficient to allow conclusions to be drawn about the safety of stockpiled vaccine.
3. Results
A total of 77 subjects received both doses of vaccine and 1 subject received 1 dose of vaccine. Three subjects were excluded from the per-protocol immunogenicity analysis at Day 42 for the following reasons: administration of vaccine(s) forbidden in the protocol (n = 1), non-compliance with vaccination schedule (n = 1), and noncompliance with blood sampling schedule (n = 1); and 1 further subject was excluded from the Day 182 analysis for receiving a medication forbidden by the protocol. No subject withdrew from the study due to an AE. The first and last subjects were enrolled on the 12th and 26th of August 2011, respectively, meaning that vials were stored for 4-4.5 months. The last study contact (Day 385) was in September 2012.
The mean age of subjects at the first vaccination in the total vaccinated cohort was 40.6 years (±13.05 years), 52.6% were women, the mean body mass index was 31.5 kg/m2 (±7.63 kg/m2), and 96.2% were of Caucasian/European heritage (Table 1). A total of 64 subjects (82.1%) had received an influenza vaccine (including A(H1N1)pdm09 vaccine) within the preceding 3 seasons.
Table 1
Demographic characteristics (total vaccinated cohort).
Characteristic
N = 78
Age, years Mean (standard deviation) Median (range) Men, n (%) Women, n (%) Body mass index (kg/m2) Mean (standard deviation) Median (range) Geographic ancestry, n (%) African heritage
Native Hawaiian or pacific Islander White, Arabic/north African heritage White, Caucasian/European heritage
40.6(13.05) 40.0 (l8-64) 37(47.4) 4l(52.6)
31.5(7.63) 30.0(l9.9-57.6)
1(1.3) 1(1.3) 1(1.3)
75(96.2)
3.1. Immunogenicity
At Day 0,34.7% of subjects overall were seropositive for the vaccine strain, which increased to 100% at Day 42. By age strata, the seropositivity rate at Day 0 in the 18-30 years stratum was 20.0%, in the 31-40 years stratum was 29.4%, in the 41-50 years stratum was 42.1%, and in the 51-64 years stratum was 47.4%.
GMTs at Days 0 and 42 overall and by age strata are shown in Table 2. In the overall population, HI antibody responses at Day 42 exceeded CBER and CHMP criteria. The SCR was 96.0% (95% CIs: 88.8-99.2%), the SPR was 98.7% (95% CIs: 92.8-100%), and the MGI was 41.7 (Fig. 1). HI antibody responses at Day 42 exceeded CBER and CHMP criteria for each of the age strata (Fig. 1). In the 18-30 years stratum, the SCR and SPR were each 100%, in the 31-40 years stratum the SCR and SPR were each 100%, in the 41-50 years stratum were 89.5% and 100%, respectively, and in the 51-64 years stratum, the SCR and SPR were each 94.7%.
At Day 182, all subjects were seropositive. GMTs overall and by age strata at Day 182 are shown in Table 2. In the overall population, at Day 182, the SCR was 65.8% (95% CIs: 53.7-76.5%), and the SPR was 76.7% (95% CIs: 65.4-85.8%). At Day 182, in the 18-30 years stratum, the SCR and SPR were each 94.7%, in the 31-40 years stratum the SCR and SPR were 83.3% and 94.4%, respectively, in the 41-50 years stratum were 50.0% and 77.8%, respectively, and in the 51-64 years stratum, the SCR and SPR were 33.3% and 38.9%, respectively (Fig. 2).
Table 2
Hemagglutination-inhibition GMTs overall and by age stratum for the per-protocol immunogenicity cohort (Days 0 and 42) and the per-protocol immunogenicity persistence cohort (Day 182).
Age group Day N GMT value (95% CIs)
0 75 7.5 (6.5-8.6)
Overall 42 75 311.3(249.2-389.0)
182 73 56.3 (47.0-67.5)
0 20 6.0 (5.0-7.3)
18-30 years 42 20 629.1 (475.1-833.1)
182 19 75.8 (55.5-103.4)
0 17 6.6 (5.1-8.6)
31-40 years 42 17 400.4 (288.7-555.4)
182 18 71.4 (54.8-93.0)
0 19 9.6 (6.4-14.6)
40-50 years 42 19 276.8(182.4-420.1)
182 18 51.4 (35.2-75.0)
0 19 8.0 (6.1-10.6)
51-64 years 42 19 133.3 (82.8-214.9)
182 18 35.6 (22.4-56.5)
GMT, geometric mean titer.
100 90 80 70 60 50 40 30 20 10 0
□ Day 42
Licensure threshold
18-30 years 31-40 years 41-50 years 51-64 years
100 Ü 90 g 80
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70 60 50 40 30 20 10 0
□ Day 0
□ Day 42
Licensure threshold
18-30 years 31-40 years 41-50 years 51-64 years
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4= 120-
ä 100-
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□ Day 42
18-30 years 31-40 years 41-50 years 51-64 years
Licensure threshold
Fig. 1. Hemagglutination-inhibition antibody seroconversion rates (A), seroprotection rates (B), and mean geometric increase (C) after two doses of AS03A-H5N1 vaccine, overall and by age stratum in the per-protocol immunogenicity cohort. Seroconversion rate defined as proportion of subjects with a pre-vaccination HI antibody titer <1:10 and post-vaccination HI antibody titer >1:40, or subjects with at least a fourfold increase in the post-vaccination HI antibody titer; licensure thresholds defined as a lower limit of 95% CI >40% (CBER) and point estimate >40% (CHMP). Seroprotection rate defined as proportion of subjects with HI antibody titers >1:40; licensure threshold defined as lower limit of 95% CI >70% (CBER) and point estimate >70% (CHMP). Mean geometric increase defined as the geometric mean of the within subject ratios of reciprocal HI antibody titers for post-vaccination versus pre-vaccination; No CBER threshold, CHMP threshold defined as >2.5 increase. CBER, the US Center for Biologics Evaluation and Research; CHMP, European Committee for Medicinal Products for Human Use [13,14]; CI, confidence interval.
At Day 42, MGl values exceeded the target threshold (>2.5) for all groups, although age-related declines were observed from 104.0 in the 18-30 years stratum to 16.6 in the 51-64 years stratum (Fig. 1). MGls decreased at Day 182, yet the target threshold was fulfilled for all age groups from 12.4 in the 18-30 years stratum to 4.3 in the 51-64 years stratum.
3.2. Reactogenicity
Reactogenicity assessments included all 78 subjects, and are reported as overall rate by subject (rate for both doses combined). The most common injection site symptom was pain which was reported by 91.0% (n = 71) of subjects, including 7.7% (n = 6) of
_ 100 u
Ï 70 S? 60 -
•i= 50 2
c 40 ^ (3
i- 30 -
□ Day 182
18-30 years 31-40 years 41-50 years 51-64 years
SS 90 m ® 80
èS 60
ra 50 ^
••p
2 30 o
□ Day 0
□ Day 182
18-30 years 31-40 years 41-50 years 51-64 years
Fig. 2. Hemagglutination-inhibition antibody seroconversion rates (A), and seroprotection rates (B), after two doses of AS03a-H5N1 vaccine in the per-protocol immuno-genicity persistence cohort. Seroconversion rate defined as proportion of subjects with a pre-vaccination HI antibody titer <1:10 and post-vaccination HI antibody titer >1:40, or subjects with at least a fourfold increase in the post-vaccination HI antibody titer; seroprotection rate defined as proportion of subjects with HI antibody titers >1:40.
subjects reporting Grade 3 pain. Swelling and redness were reported by 15.4% (n = 12) and 9.0% (n = 7) of subjects, respectively. The most common general solicited symptoms were fatigue, muscle aches, and headache, which were reported by 44.9% (n = 35), 39.7% (n = 31), and 35.9% (n = 28) of subjects, respectively. The rate of Grade 3 general events was 0.0-3.8%. There was a trend for a decreased frequency of injection site symptoms after the second compared with the first dose of vaccine (pain after the first and second doses: 89.9% and 77.9%, respectively). There was no trend for differences in general adverse events between the first and second doses of vaccine.
3.3. Safety
Safety assessments included all 78 subjects. A total of 38 subjects (48.7%) reported at least one unsolicited AE up to Day 21 after either dose, including 5 subjects (6.4%) with a Grade 3 event. The most frequently report AEs were cough (6.4%; n = 5) and oropharyngeal pain (5.1%; n = 4). A total of 7 subjects (9.0%) reported 8 AEs which were considered by the investigator to be related to vaccination, including injection site dryness (n = 1), injection site hematoma (n = 2), injection site pruritus (n = 2), dysgeusia (n = 1), pruritus (n = 1), and pruritic rash (n = 1). From Day 0 to 84, 46 subjects (59.0%) reported at least 1 AE, including 7 (9.0%) who reported a Grade 3 event. The most frequent AEs reported up to Day 84 were nasopharyngitis (12.8%; n = 10), cough (6.4%; n = 5), and neck and oropharyngeal pain (5.1%; n = 4). None of the AEs reported after
Day 21 were considered by the investigator to be related to vaccination.
From Day 0 to 385,49 subjects (62.8%) reported at least one MAE, which were most frequently upper respiratory tract infection (6.4%; n = 5) and sinusitis (7.7%; n = 6). From Day 0 to 385, three subjects (3.8%) reported at least one SAE, which were obstructive hernia, pneumonia, and small cell lung cancer. The SAEs were not fatal or considered by the investigator to be related to vaccination. There were no pIMDs reported from Day 0 to 385. No safety concerns were identified based on serum chemistries and hematology (data not shown).
4. Discussion
This Phase II, open-label study of adults showed that the storage of bulk H5N1 antigen for 4 years and AS03A adjuvant for 2.5 years, before vialing and administration, does not appear to compromise immunogenicity and reactogenicity of the vaccine. The AS03A-H5N1 vaccine elicited HI antibody responses that fulfilled the immunogenicity threshold values recommended by European and US regulatory authorities. The antibody responses and the reactogenicity were consistent with previous studies using AS03A-H5N1 vaccine which had been recently manufactured before administration [6,16].
The availability of influenza vaccines may mitigate the severity of a pandemic, yet there are many challenges that must be overcome for the successful global deployment of vaccine. Major
issues include producing enough doses to provide appropriate coverage and delivering those doses in time to prevent the spread of infection. Developments in vaccine technology over the past decade mean that 'pre-pandemic' vaccination strategies are now feasible, which may offset to a degree the challenges posed by manufacturing and logistical constraints. For example, a pre-pandemic approach involves the stockpiling of vaccine to be deployed during a pandemic alert (phase 4 or 5) to prime a population, in advance of the manufacture and distribution of a second dose of vaccine matched to the novel emerging strain during the pandemic period (phase 6) [4]. Formulation of H5N1 vaccines with adjuvants, such as AS03, enhances the immune response therefore reducing the amount of antigen needed per dose, which increases the number of doses available within the existing manufacturing infrastructure [16]. In addition, AS03A-adjuvanted H5N1 vaccine has been shown to elicit durable cross-clade reactivity, which is desirable to allow flexible prime-boost vaccination schedules [7].
The stockpiling of pandemic vaccines against prevalent 'bird-flu' strains based on international surveillance is now the key pandemic preparedness vaccination strategy recommended by WHO, and the approach has been adopted by various national public health agencies [4,17]. An important aspect of stockpiling is to determine the shelf-life of the stored vaccine. Holding the antigen in bulk form may slightly increase deployment time compared with filled doses; although no data are available comparing shelf-lives of vaccines according to different storage methods, the shelf-life for bulk antigen is expected to be greater than for filled doses. In this study, the bulk antigen had been stored for 4 years, and the AS03a adjuvant had been stored for 2.5 years. Both the antigen and adjuvant were filled in separate multi-dose vials within 4 months of supply to study centers, and on the day of vaccination, the contents of antigen and adjuvant vials were mixed providing a vial containing 8 vaccine doses of 0.5 mL.
We showed that two doses of AS03A-H5N1 vaccine which had been stockpiled for 4 years were immunogenic in adults and fulfilled European and US thresholds for immunogenicity. After vaccination, there were no differences in SCRs and SPRs in the population stratified by age at Day 42, although GMTs appeared to decrease with increasing age (18-30 and 51-64 years, 629.1 and 133.3, respectively). At Day 182, antibody levels remained above pre-vaccination levels, and the age-related effect on GMTs was less pronounced than at Day 42. Before vaccination, 34.7% of subjects were seropositive (titer >1:10) for the vaccine HA, and seropos-itivity rates were higher in the oldest (47.4%) than the youngest (20.0%) group; in addition, two subjects in the 41-50 years stratum had titers of >1:40 at baseline. The immunogenicity observed at baseline was similar to that in previous studies of H5N1 vaccines. For example, in a study of AS03A-H5N1 vaccine including 437 elderly adults, 38% of subjects were seropositive at baseline and 4.5-18.4% of subjects had baseline titers of >1:40; the authors suggested that this may have resulted from previous seasonal influenza vaccination or natural infection [18]. The subjects in our study with baseline titers of >1:40 had the lowest SCR, although the SCR (89.5%; 95% CIs: 66.9, 98.7) in the 41-50 years group was similar to SCRs in the other age groups based on overlapping CIs, and fulfilled the pre-defined threshold for this outcome. In previous studies, pre-vaccination titers were found to be associated with HI responses to vaccination [18]; in our study however, no trends were observed based on GMTs, and given the small sample size, it is not possible to draw conclusions about the effect of pre-vaccination titers.
The lack of a study arm in which subjects received recently manufactured vaccine is a limitation of our study. Nonetheless, the HI antibody responses we observed were similar to those in previous studies of AS03A-adjuvanted H5N1 vaccines including the A/Indonesia/5/2005 hemagglutinin antigen, in which both the adjuvant and antigen had been recently manufactured before
administration [6,15]. In a previous Phase II/III study of AS03A-H5N1 vaccine, Day 42 HI antibody SCRs in adults aged 18-64 years were 97.2% (95% CIs: 93.0, 99.2) [6], and in a Phase III study of AS03A-H5N1 vaccine in adults aged >18 years, Day 42 HI antibody SCRs were 90.8% (95% CIs, 89.3-92.2) in subjects aged 18-64 years and were 74.0% (95% CIs, 69.4-78.2) in subjects aged >65 years [15]. Both of the previous studies showed cross-reactive responses against vaccine-heterologous strains, which is an important property of a vaccine manufactured prior to the identification of the pandemic strain. A further limitation of our study was the lack of data regarding cross-reactive responses. Despite the small study sample, and the above-mentioned limitations, our findings are consistent with the results of previous studies of recently manufactured vaccines, suggesting that stockpiling AS03A-H5N1 vaccine is feasible without compromising immunogenicity.
The reactogenicity profile of the stockpiled vaccine in our study was consistent with that observed in previous studies of newly manufactured AS03A-H5N1 vaccine in adults [19].
In summary, a vaccine composed of H5N1 antigen that had been stockpiled as bulk antigen for 4 years, adjuvanted with AS03A that had been stored for 2.5 years, elicited HI antibody responses that fulfilled licensure criteria for immunogenicity, and had a reactogenicity consistent with previous assessments using newly produced materials. This study suggests that stockpiling of AS03A-H5N1 vaccine does not compromise the immunogenicity and reactogenicity of the vaccine, therefore supporting the pre-pandemic approach to pandemic preparedness.
Trademarks
Prepandrix, Adjupanrix, and Pumarix are trademarks of Glaxo-SmithKline group of companies.
Conflict of interest
OG, PI, PL, and DV are employees of GSK group of companies and own restricted shares in GSK. MD is an employee of GSK. MM was an employee of GSK at the time of the study.
Financial disclosure
The study was funded by the US Department of Health and Human Services (HHS), Assistant Secretary for Preparedness and Response (ASPR), Biomedical Advanced Research and Development Authority (BARDA) and GlaxoSmithKline Biologicals SA. Glaxo-SmithKline Biologicals SA was involved in all stages of the study conduct and analysis, and also took charge of all costs associated with the development and publishing of the manuscript. All authors had full access to the data, and the lead author had the final responsibility to submit the manuscript for publication.
Acknowledgements
The authors are indebted to the study volunteers, clinicians, nurses, and laboratory technicians at the study site. The authors are grateful to principal investigator Gerald Shockey. The authors also thank all teams of the GlaxoSmithKline (GSK) group of companies including Global Study Managers Catena Lauria, Cherie Barreca and Sandra Fenstermacher, Study Manager Dawn Hall, Clinical Data Coordinator Rosalia Calamera, Clinical Immunology Representative Karl Walravens, Clinical Safety Representative Dorrie Slavin, and Local Study Manager Amie Blanchfield. The authors also thank Janine Linden for writing the protocol and Manavi Narendra for writing the study report (both GSK), and Domenica Majorino (XPE Pharma&Science on behalf of GSK) for writing the annex report.
Thanks also to Annick Moon (Moon Medical Communications Ltd, UK on behalf of GSK) for providing medical writing services, and Shirin Khalili (XPE Pharma&Science on behalf of GSK) for editorial assistance and manuscript co-ordination.
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