CLINICAL AND VACCINE IMMUNOLOGY, Nov. 2010, p. 1817–1819
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 17, No. 11
A Heterologous MF59-Adjuvanted H5N1 Prepandemic Influenza
Booster Vaccine Induces a Robust, Cross-Reactive Immune
Response in Adults and the Elderly?
Elena Fragapane,1* Roberto Gasparini,2Francesco Schioppa,3Franco Laghi-Pasini,4
Emanuele Montomoli,5and Angelika Banzhoff6
Novartis Vaccines and Diagnostics, Siena, Italy1; Department of Health Sciences, Section of Hygiene and Preventive Medicine,
University of Genoa, Genoa, Italy2; University of Annunzio, Chieti, Italy3; Department of Allergology and Clinical Immunology,
Le Scotte University Hospital, University of Siena, Siena, Italy4; Department of Pathophysiology, Experimental Medicine and
Public Health, Laboratory of Molecular Epidemiology, University of Siena, Siena, Italy5; and
Novartis Vaccines and Diagnostics, Marburg, Germany6
Received 9 November 2009/Returned for modification 3 January 2010/Accepted 14 August 2010
Immunogenicity and safety of a booster dose of an MF59-adjuvanted H5N1 vaccine containing 7.5 ?g
A/turkey/Turkey/1/2005-like (clade 2.2) H5N1 hemagglutinin, given approximately 18 months after primary
vaccination with a heterologous strain, were evaluated. The booster vaccine was well tolerated and induced a
robust, cross-reactive immune response.
Immunization against pandemic virus strains, such as H5N1,
is a keystone of pandemic preparedness plans (5, 7, 12). In
addition, due to the need to rapidly produce many doses,
vaccine hemagglutinin (HA) content may be a limiting factor,
which may be countered by the inclusion of an adjuvant, such
as MF59. Considering the unpredictable emergence and rapid
spread of pandemic influenza together with the time needed to
produce and distribute a pandemic influenza vaccine, proactive
prepandemic vaccination presents a valuable opportunity to
reduce the impact of pandemic influenza disease. In addition
to having an excellent safety profile, a prepandemic vaccine
should offer broad, robust immunity that can be easily boosted
with a flexible dosing schedule (5).
(This work was presented in part at Influenza Vaccines for
the World [IVW 2009], 27 to 30 April 2009, Cannes, France.)
An H5N1 vaccine containing the MF59 adjuvant (Aflunov;
Novartis Vaccines and Diagnostics) was developed and admin-
istered to healthy volunteers in a clinical trial setting. The
present study was an extension of a trial (NCT00311480) in
which 486 subjects over 18 years of age received two primary
doses of the MF59-adjuvanted H5N1 vaccine, formulated with
7.5 ?g or 15 ?g HA per dose of the A/Vietnam/1194/2004
(clade 1), at an interval of 3 weeks; a subset of 223 subjects
received a homologous booster dose at 6 months (2). Those
who did not receive the booster dose at 6 months were eligible
for inclusion in this extension study (NCT 00561184), which
evaluated the safety and immunogenicity of one 0.5-ml dose of
MF59-adjuvanted H5N1 vaccine, containing 7.5 ?g of HA
from the A/turkey/Turkey/1/2005-like strain (clade 2), approx-
imately 18 months after primary vaccination. The inclusion and
exclusion criteria and laboratory and safety surveillance meth-
ods used in this extension study were similar to those of the
initial study (2). There was no statistical null hypothesis for the
immunogenicity assessments, which were based on European
Committee for Medicinal Products for Human Use (CHMP)
criteria (4), and the calculations of all statistical parameters
and confidence intervals are descriptive.
Following completion of the primary vaccination course in
the initial study using the MF59-adjuvanted H5N1 vaccine
formulated with A/Vietnam/1194/2004 (clade 1), all CHMP
criteria were met (2). Following the primary course, hemag-
glutination inhibition (HI) antibody for the priming strain,
A/Vietnam/1194/2004 (clade 1), declined to low levels by the
time of the booster dose (Table 1). Antibody levels increased
1 week following the booster vaccination for both the booster
(A/turkey/Turkey/1/2005-like [clade 2.2]) and heterologous
priming (A/Vietnam/1194/2004 [clade 1]) strains and remained
high 3 weeks postbooster (Table 1). The CHMP criterion for
the seroprotection rate by HI was met 3 weeks following the
booster vaccination for the A/turkey/Turkey/1/2005-like (clade
2.2) and A/Vietnam/1194/2004 (clade 1) strains in elderly sub-
jects and for the A/Vietnam/1194/2004 (clade 1) strain in non-
elderly subjects. The seroprotection rates 3 weeks after booster
vaccination were comparable to those reached after comple-
tion of the primary vaccination course (2). The CHMP crite-
rion for the seroconversion rate by HI was met for both strains
1 week after the booster dose in the nonelderly subjects. Sero-
conversion rate criteria were met for both strains for elderly
and nonelderly subjects 3 weeks after the booster dose. Immu-
nogenicity, when measured using the SRH and MN assays,
showed similar trends (data not shown). Overall, the results
from this study compare favorably with those in other clinical
* Corresponding author. Mailing address: Novartis Vaccines and
Diagnostics, Via Fiorentina, 1, 53100 Siena, Italy. Phone: 39 0577
245114. E-mail: email@example.com.
?Published ahead of print on 1 September 2010.
trials, with similar immune responses after primary vaccination
and booster doses (6, 9). These results suggest that subjects
were effectively primed, which facilitated a rapid immune re-
sponse to the heterologous A/turkey/Turkey/1/2005-like (clade
2.2) strain after a single dose.
The incidence of solicited reactions reported within 7 days of
booster administration was 72% (22/29) in nonelderly subjects
and 39% (7/18) in elderly subjects. The most frequently re-
ported local reactions for all subjects were pain and induration
(Fig. 1). The most frequently reported solicited systemic reac-
tions were myalgia and headache for nonelderly subjects and
myalgia and fatigue for elderly subjects (Fig. 1). No subject
reported fever. All reactions were transient (?2 days) and
were considered mild to moderate in intensity. No unsolicited
AEs and SAEs were considered to be vaccine related. The
incidence of AEs compares favorably with the results from the
initial study (2). Overall, the safety assessments confirmed that
the A/turkey/Turkey/1/2005-like (clade 2.2) booster was well
tolerated when administered after primary vaccination with
A/Vietnam/1194/2004 (clade 1), supporting the safety profile
of MF59-adjuvanted vaccines (8, 10).
Several studies have demonstrated that the inclusion of
MF59 in a seasonal or pandemic influenza vaccine increases
both the homologous and heterologous immune responses (1,
3, 9–11) even at low antigen doses. The findings from this
extension study provide further support for both the immuno-
potentiating capabilities of MF59 and the potential for adop-
tion of antigen-sparing strategies in a prepandemic context.
This study further illustrates how prepandemic vaccination
may prime a population, providing initial protection against an
influenza pandemic that can be boosted with a different strain.
This prime-boost strategy is likely to be the most effective way
to protect populations against future influenza pandemics.
A. Banzhoff had full access to all data in the study and takes re-
sponsibility for the integrity of the data and the accuracy of the data
analysis. R. Gasparini was the principal investigator. All authors par-
ticipated in the analysis and interpretation of the data and/or were
involved in drafting and revising the manuscript for important intel-
Novartis Vaccines and Diagnostics sponsored this study.
E. Fragapane and A. Banzhoff are employees of Novartis Vaccines
and Diagnostics, the sponsor of the study; R. Gasparini has no conflict
of interest; F. Schioppa received funding from Novartis Vaccines and
Diagnostics to perform the study; E. Montomoli received funding from
Novartis Vaccines and Diagnostics to perform single radial hemolysis
TABLE 1. Hemagglutination inhibition response by MF59-adjuvanted H5N1 subunit influenza vaccine formulation and age cohorta
Value for age cohortb
Seroprotection rate, %
1 week after boosterGMT
Seroprotection rate, %
Seroconversion rate, %
3 weeks after boosterGMT
Seroprotection rate, %
Seroconversion rate, %
aPrimary vaccination with H5N1 A/Vietnam, 7.5 or 15 ?g on study days 1 and 22; booster with H5N1 A/turkey/Turkey, 7.5 or 15 ?g on study day 382. GMT, geometric
mean titer; GMR, geometric mean ratio. Two-sided 95% confidence intervals are shown in parentheses. CHMP criteria: nonelderly, GMR ? 2.5; seroprotection ? 70%;
seroconversion ? 40%; elderly, GMR ? 2.0; seroprotection ? 60%; seroconversion ? 30%.
bNonelderly cohort (n ? 29), 18 to 60 years of age; elderly cohort (n ? 17), ?60 years of age.
FIG. 1. Reported incidences of local and systemic reactions for
nonelderly adults (18 to 60 years of age; white bars) and elderly adults
(?60 years of age; black bars).
1818 NOTESCLIN. VACCINE IMMUNOL.
analysis; F. Laghi-Pasini has received a travel grant from Novartis
Vaccines and Diagnostics.
1. Atmar, R. L., W. A. Keitel, S. M. Patel, et al. 2006. Safety and immunoge-
nicity of nonadjuvanted and MF59-adjuvanted influenza A/H9N2 vaccine
preparations. Clin. Infect. Dis. 43:1135–1142.
2. Banzhoff, A., R. Gasparini, F. Laghi-Pasini, et al. 2009. Pre-pandemic influ-
enza immunization: MF59-adjuvanted H5N1 vaccine induces immunogenic
memory and heterotypic antibody response in non-elderly and elderly adults.
PLoS One 4:e4384.
3. Banzhoff, A., M. Pellegrini, G. Del Giudice, E. Fragapane, N. Groth, and A.
Podda, 2008. MF59-adjuvanted vaccines for seasonal and pandemic influ-
enza prophylaxis. Influenza Other Respir. Viruses 2:243–249.
4. Committee for Proprietary Medicinal Products. 1997. Note for guidance on
harmonization of requirements for influenza vaccines CPMP/BWP/214/96.
European Agency for the Evaluation of Medicinal Products, London, United
5. Jennings, L. C., A. S. Monto, P. K. Chan, T. D. Szucs, and K. G. Nicholson.
2008. Stockpiling prepandemic influenza vaccines: a new cornerstone of
pandemic preparedness plans. Lancet Infect. Dis. 8:650–658.
6. Lin, J. T., C. G. Li, X. Wang, et al. 2009. Antibody persistence after 2-dose
priming and booster response to a third dose of an inactivated, adjuvanted,
whole-virion H5N1 vaccine. J. Infect. Dis. 199:184–187.
7. Monto, A. S. 2009. The risk of seasonal and pandemic influenza: prospects
for control. Clin. Infect. Dis. 48(Suppl. 1):S20–S25.
8. Nicholson, K. G., A. E. Colegate, A. Podda, et al. 2001. Safety and antige-
nicity of non-adjuvanted and MF59-adjuvanted influenza A/Duck/Singa-
pore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against
H5N1 influenza. Lancet 357:1937–1943.
9. Nolan, T., P. C. Richmond, M. V. Skeljo, et al. 2008. Phase I and II ran-
domised trials of the safety and immunogenicity of a prototype adjuvanted
inactivated split-virus influenza A (H5N1) vaccine in healthy adults. Vaccine
10. Stephenson, I., R. Bugarini, K. G. Nicholson, et al. 2005. Cross-reactivity to
highly pathogenic avian influenza H5N1 viruses after vaccination with non-
adjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3)
vaccine: a potential priming strategy. J. Infect. Dis. 191:1210–1215.
11. Stephenson, I., K. G. Nicholson, A. Colegate, et al. 2003. Boosting immunity
to influenza H5N1 with MF59-adjuvanted H5N3 A/Duck/Singapore/97 vac-
cine in a primed human population. Vaccine 21:1687–1693.
12. World Health Organization. Accessed 17 March 2008. Responding to the
avian influenza pandemic threat: recommended strategic actions. http://www
.pdf. World Health Organization, Geneva, Switzerland.
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