The avian A/H5N1 influenza virus is an infectious pathogen that has been responsible for serious human disease and death worldwide.1 Four hundred fifty-four of the 860 (~53%) confirmed human cases of A/H5N1 influenza reported to the World Health Organization between 2003 and 2018 have been fatal.2 Children are the most vulnerable population, with high rates of morbidity and hospitalization due to avian influenza and its complications.3,4 Of all the reported cases of A/H5N1 influenza, half have occurred in the pediatric population.5 Children also play a major role in seasonal influenza transmission within communities.6
The potential threat for avian H5N1 influenza to become a pandemic is based on continued circulation of the virus in birds and absence of preexisting immunity in humans.1 Advance planning and preparedness are essential in reducing the impact of a pandemic.7 The availability of safe and immunogenic vaccines against avian influenza that can be produced rapidly and efficiently is critical.7,8 Modern cell culture-derived influenza vaccine production techniques minimize dependency on egg supply and can produce vaccine as demand requires.9,10 Nonadjuvanted, cell culture-derived influenza vaccine has demonstrated immunogenicity in pediatric populations and is suitable for use in children.11–13
The ability of adjuvant to enhance immunogenicity and reduce antigen dose [eg, 7.5 μg hemagglutinin (HA) vs. 15 μg per strain in seasonal formulations] is well recognized and recommended to elicit desirable protective effects.9,14 MF59 (Novartis International AG, Basal, Switzerland) is an oil-in-water emulsion adjuvant shown to enhance immune responses to influenza vaccines in adult15 and elderly subjects.16–19 Several influenza vaccines licensed for human use contain MF59. A recent pediatric study showed that MF59 increased the immunogenicity of A/H1N1 influenza vaccine in 6-month to 17-year-old children without increasing side effects.20 MF59 has a well-established safety profile16,20,21 and promotes the production of cross-reactive antibodies exhibiting specificity for nonvaccine antigen (heterologous) strains.22,23
We present the results of a phase 2 trial, conducted in healthy pediatric subjects to evaluate the safety and immunogenicity of an MF59-adjuvanted, cell culture-derived H5N1 subunit influenza virus vaccine (aH5N1c) at 2 different doses. The study aimed to identify the optimal antigen and adjuvant dose in a pediatric population.
MATERIALS AND METHODS
This Phase 2, randomized, observer-blind, multicenter trial was conducted in the United States and Thailand. The study protocol and associated amendments, and informed consent forms were approved by the Ethics Review Committees of the participating centers. The trial was conducted in compliance with Good Clinical Practice guidelines and the Declaration of Helsinki. Before enrollment, written informed consent was obtained from the parents or legal guardians of each subject. Healthy pediatric subjects underwent stratified randomization within 3 age categories (6–35 months; 3–8 years and 9–17 years) to receive (1:1) either: 7.5 μg H5N1 HA combined with 0.25 mL MF59 (full-dose formulation), or 3.75 μg H5N1 HA combined with 0.125 mL MF59 (half-dose formulation; Fig. 1). Vaccines were administered as 2 individual intramuscular injections 3 weeks apart. The primary safety endpoint was safety and tolerability. The primary immunogenicity endpoint was antibody responses on day 43, as measured by hemagglutination inhibition (HI) assay and expressed as percentages of subjects achieving HI titers ≥1:40 and seroconversion. The trial did not include a placebo or nonadjuvanted control arm, somewhat limiting the extent of the safety analysis.
A total of 662 healthy male and female subjects 6 months–17 years of age were enrolled [73% (n = 480) and 27% (n = 182) from sites in Thailand and USA, respectively]. Female subjects of child bearing potential had to commit to use of an acceptable birth control method from day 1 (prevaccination) to day 43 of the study. Exclusion criteria were serious chronic or progressive diseases; medically significant cancer; history of impaired immune function; any progressive or severe neurologic disorder; allergy to latex or any vaccine component; receipt of any other investigational product within 30 days before study day 1; prior receipt of H5N1 vaccine; receipt of any other influenza vaccine 60 days before enrollment; body temperature ≥38.0°C and/or acute illness within 3 days of study vaccination; pregnancy or breast-feeding; body mass index ≥35 kg/m2; cognitive impairment or psychiatric diseases; history of drug or alcohol abuse and planned surgery during the study period.
The study vaccine was an MF59-adjuvanted, cell culture-derived, monovalent, inactivated, subunit formulation containing HA antigen derived from the A/turkey/Turkey/1/2005 (H5N1) NIBRG-23 strain of influenza virus (aH5N1c; f/k/a Novartis Influenza Vaccines GmbH, Marburg, Germany). The adjuvant, MF59, contained 9.75 mg squalene, 1.18 mg polysorbate 80, and 1.18 mg sorbitan trioleate per 0.25 mL. aH5N1c was administered intramuscularly on days 1 and 22. One 0.5 mL dose of aH5N1c contained 7.5 μg HA and 0.25 mL MF59 (full dose); one 0.25 mL dose contained 3.75 μg HA and 0.125 mL MF59 (half dose). Vaccines were administered by designated site staff who did not participate in any assessment of outcomes. The subjects, investigators, and site personnel who evaluated adverse events (AEs) remained blinded to treatment group assignment.
After each vaccination, subjects were monitored for 30 minutes for any immediate reactions. Solicited local reactions and systemic AEs were recorded by the subjects themselves, or the subjects’ parents or legal guardians on diary cards for 7 days after each vaccination. Solicited local reactions in subjects <6 years old included injection site induration, erythema, ecchymosis and tenderness; in subjects 6–17 years old, induration, erythema, ecchymosis and pain. Solicited systemic AEs in <6-year olds included altered eating habits, sleepiness, irritability and fever (≥38.0°C); in subjects 6–17 years old, nausea, myalgia, arthralgia, headache, fatigue, loss of appetite, malaise and fever (≥38.0°C). Severity of local reactions and systemic AEs were categorized as mild (transient with no limitation of normal daily activities), moderate (some limitation of normal daily activities) or severe (unable to perform normal daily activities). Unsolicited AEs were recorded for 3 weeks postvaccination. Serious adverse events (SAEs), new onset of chronic diseases (NOCD), medically attended AEs, AEs of special interest (AESIs), AEs leading to study withdrawal, and concomitant medications associated with these events were collected throughout the study (days 1–387). The causal relationship of AEs to study vaccine was assessed by the investigators as either nonrelated, possibly related or probably related.
Sera (stored at −18°C) were obtained for immunogenicity analyses immediately before vaccination on day 1 (baseline) and day 22 and on days 43 and 387. Immunogenicity was assessed by HI assay against the H5N1 vaccine strain according to standard methods,24 and expressed as geometric mean titers (GMTs), geometric mean ratios (GMRs; day 22/day 1, day 43/day 1 and day 387/day 1), and the percentages of subjects achieving HI titers ≥1:40 and seroconversion. Seroconversion was defined in subjects seronegative (HI titer < 1:10) at baseline as a postvaccination titer ≥1:40, and in subjects seropositive (titer ≥1:10) at baseline as a ≥ 4-fold rise in postvaccination antibody titer.
Analyses of vaccine reactogenicity and safety were performed in subjects who had received at least one study vaccination and provided either postvaccination AE or reactogenicity data (safety dataset). All safety analyses were descriptive and performed by vaccine and age groups.
Immunogenicity analyses were performed on full analysis set data, which included all subjects who received at least one vaccine dose and provided at least one evaluable serum sample at both pre and postvaccination time points. The percentages of subjects achieving titers ≥1:40 and seroconversion along with the associated 97.5% Clopper-Pearson CIs were calculated as log10-transformed values using analysis of covariance with factors for dose group, baseline titer and study site. GMTs, GMRs and associated 2-sided 95% CIs were calculated as log10-transformed values using analysis of covariance with factors for age, race, gender and site.
Primary immunogenicity endpoints (day 43 data) were based on pediatric Center for Biologics Evaluation Research and Review (CBER) licensure criteria for pandemic influenza vaccines25: (1) lower limit (LL) of 2-sided 97.5% CI for percentage of subjects achieving seroconversion should be ≥40%; (2) the LL of CI for percentage of subjects achieving titer ≥1:40 should be ≥70%. Although the CBER licensure criteria define LLs of 95% CIs, 97.5% CIs were calculated because 2 vaccine formulations were assessed, and the 0.05 alpha was distributed across tests. Secondary immunogenicity endpoints (day 43 data) were based on pandemic influenza vaccine licensure criteria established by the Committee for Medicinal Products for Human Use (CHMP).26 Adult CHMP criteria were applied as pediatric criteria do not exist: (1) percentage of subjects achieving seroconversion in HI antibody titers should be >40%; (2) percentage of subjects achieving titer ≥1:40 should be >70%; (3) GMR should be >2.5.
Ninety-nine percent (658 of 662) of enrolled subjects received at least one vaccine dose. Overall, 94% of subjects (315 of 329, full-dose group; 307 of 329, half-dose group) completed the study on day 387 (Fig. 1). Approximately 18% of subjects were excluded from the immunogenicity full analysis set data at day 387 due to missing serology results. Subject demographics and baseline characteristics were well balanced between both dose groups (Table, Supplemental Digital Content, http://links.lww.com/INF/D472). Equal percentages of male and female subjects (50%) were enrolled in the half-dose group, while more male subjects (54%) were enrolled in the full-dose group.
Percentages of children <6 years of age experiencing solicited AEs from day 1 to day 7 following any vaccination were similar in the full (70%) and half-dose (72%) groups and likewise for children 6–17 years of age (75% and 76%, respectively). Fewer solicited AEs were reported after the second dose in full- and half-dose groups across both age cohorts (Table 1). The most commonly reported solicited local reactions in both vaccine groups were injection site tenderness in children <6 years old and injection site pain in children 6–17 years old. The most commonly reported solicited systemic AE in subjects <6 years old was irritability; the most common systemic AEs in 6–17-year olds were myalgia and fatigue (Table 1).
Percentages of subjects reporting unsolicited AEs for 21 days following any vaccination were similar in the full (26%) and half-dose (29%) groups. Frequencies of unsolicited AEs were lower after the second vaccination compared with the first in both the full dose (11% vs. 20%, respectively) and half dose (14% vs. 18%, respectively) groups. In both vaccine groups, 4% of subjects experienced vaccine-related AEs. One subject withdrew from the study (nonvaccine-related gastroenteritis and rash; full-dose group). From day 1 to day 387, 2% of subjects in the full-dose group, and 3% of subjects in the half-dose group reported SAEs, none of which were vaccine related (Table 2). Three subjects (1%) in the half-dose group were diagnosed with nonvaccine-related NOCD (dyspepsia, bone cyst, attention-deficit/hyperactivity disorder). No deaths occurred during the study.
At day 43, 96% (97.5% CI: 93%–98%) and 86% (97.5% CI: 81%–90%) of subjects in the full-dose and half-dose groups achieved seroconversion, respectively (Table 3). The highest percentages of seroconversion were observed in the 6–35 months age group (full dose 99%; half dose 94%), the lowest in the 9–17 years age group (full dose 92%; half dose 79%). Three weeks after a second vaccination (day 43), CBER and CHMP criteria for seroconversion were met for both dose groups across all 3 age categories. Three weeks after a single dose (day 22), rates of seroconversion were substantially lower in all age categories and were dose related. Seroconversion rates decreased by day 387 (full dose 47%; half dose 31%), with only the full-dose group continuing to meet CBER and CHMP seroconversion criteria 1 year after the second vaccination.
The percentages of subjects with HI titers ≥1:40 increased from day 1 to day 43, then decreased by day 387 (Table 3). At day 43, 96% (95% CI: 93%–98%) and 86% (95% CI: 81%–90%) of subjects in the full-dose and half-dose groups achieved HI titers ≥1:40, respectively. The highest percentages of subjects achieving titers ≥1:40 were observed in the 6–35 months age group (full dose 98%; half dose 94%), the lowest in the 9–17 years age group (full dose 92%; half dose 79%). At day 43, CBER and CHMP criteria for HI titers ≥1:40 were met for both dose groups across all 3 age categories. At day 387, 47% (95% CI: 41%–53%) and 31% (95% CI: 26%–37%) of subjects in the full- and half-dose groups had HI titers ≥1:40, respectively; therefore, CBER and CHMP criteria were not met 1 year postvaccination. On day 43, both CBER licensure criteria were met in response to high- and low-dose formulations, regardless of whether or not subjects had received influenza vaccine 12 months before the start of the study.
GMTs were low at baseline (full-dose GMT 5.23; half dose 5.15), rose to 64 and 34 at day 22 and were maximal on day 43 at 1356 and 431 in the full- and half-dose groups, respectively (Fig. 2). At day 43, the highest GMTs were observed in subjects 6–35 months old (full-dose GMT 1842; half dose 674), followed by children 3–8 years old (full dose 1244; half dose 363), then children 9–17 years old (full dose 961; half dose 300). Day 43/day 1 GMRs for the full and half-dose groups were 262 and 84, respectively; far exceeding the CHMP criterion of >2.5 (Table 3). Day 387/day 1 GMRs also exceeded the CHMP licensure criterion (full dose GMR 12; half dose 5.6). Reverse cumulative distribution data on day 387 showed levels of antibody persistence to be higher in response to the full dose than the half-dose vaccine formulation for all 3 age categories (Figure, Supplemental Digital Content, http://links.lww.com/INF/D472).
The primary results show that both full- and half-dose formulations of aH5N1c vaccine were well tolerated and immunogenic in children 6 months to 17 years of age. No deaths, or vaccine-related SAEs, NOCDs, or study withdrawals occurred throughout the 1-year study. Solicited AE profiles were similar for the full- and half-dose groups. In both vaccine groups, frequencies of any reaction tended to be lower after the second vaccination than the first across all ages. The safety data from this study are in agreement with that of previous trials of MF59-adjuvanted A/H5N1 and A/H1N1 vaccines conducted in subjects 6 months to 17 years old3 and 3–17 years old,20 respectively. Both full- and half-dose aH5N1c formulations are immunogenic after first and second vaccinations; antibody titers in response to the first vaccination (day 22) were further increased following the second vaccination. CBER criteria for both seroconversion and HI titers ≥1:40 were met at day 43 (3 weeks after second vaccination) in both full- and half-dose groups. Similarly, all 3 CHMP criteria (seroconversion, HI titers ≥1:40 and GMR) were met in both dose groups at day 43.
In both the full- and half-dose groups, antibody titers increased from baseline following the first vaccination, reached their peak at day 43, and then declined by day 387. The full-dose group had higher overall antibody responses (seroconversion, 47%–96%; HI titers ≥1:40, 47%–96%; GMR, 12–262) than the half-dose group (seroconversion, 31%–86%; HI titers ≥1:40, 31%–86%; GMR, 5.6–84) throughout the 1-year study. In both full- and half-dose groups, sub-analysis by age showed children 6–35 months old to generate the highest antibody titers at day 43, followed by 3–8-year olds, the lowest titers were observed in 9–17-year olds. Similar comparative trends were observed at day 387: in the 6–35 months age category, 73% and 61% of full- and half-dose group subjects achieved seroconversion at day 387, compared with 45% and 22% of 3–8-year olds, and 29% and 16% of 9–17-year olds, respectively.
Immunogenicity data from this study are consistent with a recent trial which showed that MF59-adjuvanted, A/H5N1, prepandemic vaccine was highly immunogenic in pediatric subjects, with antibodies persisting up to 1 year postvaccination.27 A recent review of A/H5N1 vaccines reported acceptable safety data and robust immune responses to an MF59-adjuvanted A/H5N1 vaccine containing 7.5 μg HA per dose in children 6 months to 17 years old.28 A study of MF59-adjuvanted, cell culture-derived, A/H1N1 pandemic vaccine in 6-months to 18-year olds Japanese children observed HI titers ≥1:40 and seroconversion rates of 100% 3 weeks after the administration of second vaccine doses containing either 7.5 μg or 3.75 μg antigen; these data provide further evidence of the acceptable immunogenicity and safety profiles of MF59-adjuvanted, cell culture-derived, pandemic influenza vaccines.29
The present study was uniquely designed to provide an accurate assessment of immune responses to 2 vaccine formulations differing in quantity of antigen per dose, within a large, broad, age-stratified pediatric population (N = 662). Conducted in United States and Thailand, this study supports the suitability of aH5N1c across different geographical settings, particularly North America and Asia. Study limitations include the absence of an active or placebo control group, making interpretation of safety data somewhat limited. However, the safety profile of the full-dose formulation conforms with that reported from a recent study of MF59-adjuvanted, A/H5N1 vaccine in which an MF59-adjuvanted seasonal influenza vaccine was used for a control arm3; and also with that reported from an adult study of MF59-adjuvanted A/H5N1 vaccine in which placebo, and nonadjuvanted and aluminum hydroxide-adjuvanted H5N1 vaccine control arms were included.30 Evaluation of long-term antibody persistence beyond the 1-year time point was not planned for this study. Nor was the study designed to include the administration of a booster dose; however, a 1-year booster dose of MF59-adjuvated, A/H5N1, prepandemic vaccine has been shown to be highly immunogenic, inducing both homologous and cross-reactive A/H5N1 antibody responses in pediatric subjects.27
In conclusion, both half- and full-dose aH5N1c formulations were well tolerated and induced robust immune responses in an immunologically naive (ie, H5N1 vaccine naive), susceptible pediatric population. The safety profile of each formulation showed acceptable reactogenicity without associated severe or SAEs, suggesting benefit, especially in light of the high risk for morbidity and mortality associated with avian A/H5N1 influenza in children. Both aH5N1c formulations met CBER and CHMP licensure criteria 3 weeks after the administration of a second dose. Though both formulations could be considered for pediatric use, the full-dose formulation induced slightly higher short- and long-term antibody responses, with similar reactogenicity. During a pandemic outbreak where the production and supply of A/H5N1 vaccine may be limited, a half-dose aH5N1c formulation could prove effective in meeting the global demand for pediatric vaccine and potentially curtailing ongoing H5N1 infections in this population.
The authors would like to thank all of the study participants and investigators; Zenaida Morajes, MD, MPH for her contributions in conducting the study and data acquisition; Claudia Kittel, MSc for statistical expertise; David Lee, MD, MBA; David Hering, MBA; Heather Clouting, MSc and Robin Wallace, BSN for their operational support in conducting the study. The authors also acknowledge D Das PhD (Novartis Healthcare Pvt. Ltd., Hyderabad, India), Parexel International (IRL) Ltd., Dublin, Ireland, and J Stirling PhD (OLC Bioscience Ltd., London, UK) for providing support in the preparation, revision and editing of this manuscript. MF59 is a registered trademark of Novartis AG.
1. World Health Organization. Avian influenza fact sheet 2014. November 2018. Available at: http://www.who.int/mediacentre/factsheets/avian_influenza/en/
. Accessed April 15, 2015.
2. World Health Organization. Cumulative number of confirmed human cases for avian influenza A(H5N1) reported to WHO, 2003–2015. March 2, 2018. Available at: https://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm091985.pdf
. Accessed April 19, 2018.
3. Vesikari T, Karvonen A, Tilman S, et al. Immunogenicity and safety of MF59-adjuvanted H5N1 influenza vaccine from infancy to adolescence. Pediatrics. 2010;126:e762–e770.
4. American Academy of Pediatrics Committee on Infectious D. Prevention of influenza: recommendations for influenza immunization of children, 2008–2009. Pediatrics. 2008;122:1135–1141.
5. Oner AF, Dogan N, Gasimov V, et al. H5N1 avian influenza in children. Clin Infect Dis. 2012;55:26–32.
6. Weycker D, Edelsberg J, Halloran ME, et al. Population-wide benefits of routine vaccination of children against influenza. Vaccine. 2005;23:1284–1293.
7. Rappuoli R, Dormitzer PR. Influenza: options to improve pandemic preparation. Science. 2012;336:1531–1533.
8. World Health Organization. Pandemic influenza risk management WHO interim guidance June 2013. June 2013. Available at: http://www.who.int/influenza/preparedness/pandemic/GIP_PandemicInfluenzaRiskManagementInterimGuidance_Jun2013.pdf?ua=1
. Accessed April 15, 2015.
9. Hatz C, Cramer JP, Vertruyen A, et al. A randomised, single-blind, dose-range study to assess the immunogenicity and safety of a cell-culture-derived A/H1N1 influenza vaccine in adult and elderly populations. Vaccine. 2012;30:4820–4827.
10. Keitel W, Groth N, Lattanzi M, et al. Dose ranging of adjuvant and antigen in a cell culture H5N1 influenza vaccine: safety and immunogenicity of a phase ½ clinical trial. Vaccine. 2010;28:840–848.
11. van der Velden MV, Fritz R, Pöllabauer EM, et al. Safety and immunogenicity of a vero cell culture-derived whole-virus influenza A(H5N1) vaccine in a pediatric
population. J Infect Dis. 2014;209:12–23.
12. Vesikari T, Block SL, Guerra F, et al. Immunogenicity, safety and reactogenicity of a mammalian cell-culture-derived influenza vaccine in healthy children and adolescents three to seventeen years of age. Pediatr Infect Dis J. 2012;31:494–500.
13. Grohskopf LA, Sokolow LZ, Broder KR, et al. Prevention and control of seasonal influenza with vaccines. MMWR Recomm Rep. 2016;65:1–54.
14. World Health Organization. WHO recommendations on pandemic (H1N1) 2009 vaccines. July 2009. Available at: http://www.who.int/csr/disease/swineflu/notes/h1n1_vaccine_20090713/en/
. Accessed April 15, 2015.
15. Reisinger KS, Holmes SJ, Pedotti P, et al. A dose-ranging study of MF59(®)-adjuvanted and non-adjuvanted A/H1N1 pandemic influenza vaccine in young to middle-aged and older adult populations to assess safety, immunogenicity, and antibody persistence one year after vaccination. Hum Vaccin Immunother. 2014;10:2395–2407.
16. O’Hagan DT. MF59 is a safe and potent vaccine adjuvant that enhances protection against influenza virus infection. Expert Rev Vaccines. 2007;6:699–710.
17. Puig Barberà J, González Vidal D. MF59-adjuvanted subunit influenza vaccine: an improved interpandemic influenza vaccine for vulnerable populations. Expert Rev Vaccines. 2007;6:659–665.
18. Gasparini R, Pozzi T, Montomoli E, et al. Increased immunogenicity of the MF59-adjuvanted influenza vaccine compared to a conventional subunit vaccine in elderly subjects. Eur J Epidemiol. 2001;17:135–140.
19. De Donato S, Granoff D, Minutello M, et al. Safety and immunogenicity of MF59-adjuvanted influenza vaccine in the elderly. Vaccine. 1999;17:3094–3101.
20. Knuf M, Leroux-Roels G, Rümke HC, et al. Safety and immunogenicity of an MF59-adjuvanted A/H1N1 pandemic influenza vaccine in children from three to seventeen years of age. Vaccine. 2015;33:174–181.
21. Pellegrini M, Nicolay U, Lindert K, et al. MF59-adjuvanted versus non-adjuvanted influenza vaccines: integrated analysis from a large safety database. Vaccine. 2009;27:6959–6965.
22. Banzhoff A, Gasparini R, Laghi-Pasini F, et al. MF59-adjuvanted H5N1 vaccine induces immunologic memory and heterotypic antibody responses in non-elderly and elderly adults. PLoS One. 2009;4:e4384.
23. Galli G, Hancock K, Hoschler K, et al. Fast rise of broadly cross-reactive antibodies after boosting long-lived human memory B cells primed by an MF59 adjuvanted prepandemic vaccine. Proc Natl Acad Sci USA. 2009;106:7962–7967.
24. Belshe RB, Frey SE, Graham IL, et al.; National Institute of Allergy and Infectious Diseases–Funded Vaccine and Treatment Evaluation Units. Immunogenicity of avian influenza A/Anhui/01/2005(H5N1) vaccine with MF59 adjuvant: a randomized clinical trial. JAMA. 2014;312:1420–1428.
25. Center for Biologics Evaluation and Research (CBER). Food and Drug Administration (FDA). Guidance for industry: clinical data needed to support the licensure of pandemic influenza vaccines. May 2007 Available at: http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm074786.htm
. Accessed April 20, 2015.
26. European Committee for Proprietary Medicinal Products. Note for guidance on harmonisation of requirements for influenza vaccines (CPMP/BWP/214/96). March 1997. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003945.pdf
. Accessed April 20, 2015.
27. Vesikari T, Forstén A, Borkowski A, et al. Homologous and heterologous antibody responses to a one-year booster dose of an MF59(®) adjuvanted A/H5N1 pre-pandemic influenza vaccine in pediatric
subjects. Hum Vaccin Immunother. 2012;8:921–928.
28. Baz M, Luke CJ, Cheng X, et al. H5N1 vaccines in humans. Virus Res. 2013;178:78–98.
29. Fukase H, Furuie H, Yasuda Y, et al. Assessment of the immunogenicity and safety of varying doses of an MF59®-adjuvanted cell culture-derived A/H1N1 pandemic influenza vaccine in Japanese paediatric, adult and elderly subjects. Vaccine. 2012;30:5030–5037.
30. Bernstein DI, Edwards KM, Dekker CL, et al. Effects of adjuvants on the safety and immunogenicity of an avian influenza H5N1 vaccine in adults. J Infect Dis. 2008;197:667–675.