Vaccine 29 (2011) 5785–5792

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Immunogenicity and safety of inactivated influenza vaccines in primed

populations: A systematic literature review and meta-analysis
W.E.P. Beyer a , J.J.P. Nauta b , A.M. Palache c , K.M. Giezeman d , A.D.M.E. Osterhaus a,∗
a
National Influenza Centre and Department of Virology, Erasmus Medical Centre, Rotterdam, The Netherlands
b
Abbott Established Products Division, Weesp, The Netherlands
c
Abbott Health Care Products, Weesp, The Netherlands
d
Abbott Product Operations AG, Allschwil, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Several inactivated influenza vaccine formulations for systemic administration in man are currently avail-
Received 2 December 2010 able for annual (seasonal) immunization: split virus and subunit (either plain-aqueous, or virosomal, or
Received in revised form 9 May 2011 adjuvanted by MF59). From a literature search covering the period 1978–2009, 33 articles could be iden-
Accepted 13 May 2011
tified, which described randomized clinical trials comparing at least two of the four vaccine formulations
Available online 30 May 2011
with respect to serum hemagglutination inhibition (HI) antibody response, local and systemic vaccine
reactions and serious adverse events after vaccination, and employing seasonal vaccine components and
Keywords:
doses. In total, 9121 vaccinees of all ages, either healthy or with underlying diseases, were involved. Most
Influenza
Vaccine
vaccinees were primed or had been vaccinated in previous years.
Immunogenicity For immunogenicity, homologous post-vaccination geometric mean HI titers (GMTs) were analyzed
Safety by a random effects model for continuous data. Unreported standard deviations (SD) were addressed by
Meta-analysis imputing assumed SD-values. Age and health state of the vaccinees appeared to have little influence on the
outcome. The immunogenicity of split, aqueous and virosomal subunit formulations were similar, with
geometric mean ratio values (GMR, quotient of paired GMT-values) varying around one (0.93–1.24). The
MF59-adjuvanted subunit vaccine induced, on average, larger antibody titers than the non-adjuvanted
vaccine formulations, but the absolute increase was small (GMR-values varying between 1.25 and 1.40).
Vaccine reactions were analyzed using a random effects model for binary data. Local and systemic
reactogenicity was similar among non-adjuvanted formulations. The adjuvanted subunit formulation
was more frequently associated with local reactions than the non-adjuvanted formulations (rate ratio:
2.12, significant). Systemic reactions were similar among all vaccine formulations. The original articles
emphasized the mild and transient character of the vaccine reactions and the absence of serious vaccine-
related adverse events.
This adequate amount of evidence led to the conclusion that all the currently available inactivated
influenza vaccine formulations are safe, well tolerated and similarly effective to control seasonal influenza
outbreaks across primed populations and age ranges.
© 2011 Published by Elsevier Ltd.

1. Introduction (HA) and neuraminidase (NA) are linked to liposomes (globular

Different formulations of inactivated, non-adjuvanted influenza
vaccines for intramuscular or subcutaneous use in man are
currently marketed for routine annual (seasonal) influenza immu-
nization throughout the world: the split virus vaccine [1], the
aqueous (plain) subunit (or surface antigen) vaccine [2], and the
virosomal subunit vaccine where the viral antigens hemagglutinin
∗ Corresponding author at: WHO National Influenza Centre, Institute of Virology,
Erasmus Medical Center, PO Box 1738, NL-3000 DR Rotterdam, The Netherlands.
Tel.: +31 10 7044066; fax: +31 10 7044760.
E-mail address: a.osterhaus@erasmusmc.nl (A.D.M.E. Osterhaus).
lipid membranes) [3]. In addition, an adjuvanted subunit vaccine
has been available since the 1990s: an oil-in-water emulsion of
squalene where the squalene oil droplets serve as carriers for HA
and NA (MF59-adjuvanted subunit vaccine) [4]. The classical whole
virus (WV) vaccine introduced in the 1940s [5] is not widely used
anymore.
This investigation is a systematic review and meta-analysis
of data from randomized clinical trials with any two or three
of the four current influenza formulations for seasonal use,
published during the last 30 years, with the objective to com-
pare serum hemagglutination inhibition (HI) antibody induction
(immunogenicity), the occurrence of local and systemic vac-
cine reactions (reactogenicity), and serious adverse events after

0264-410X/$ – see front matter © 2011 Published by Elsevier Ltd.

doi:10.1016/j.vaccine.2011.05.040
5786 W.E.P. Beyer et al. / Vaccine 29 (2011) 5785–5792
vaccination (safety). Live virus influenza vaccine formulations, pre-
pandemic and pandemic vaccines with avian influenza A strains
and the novel pandemic 2009 A-H1N1 strain are not part of this
review.
Two main research questions were covered: (1) How do
immunogenicity and reactogenicity of the three non-adjuvanted
formulations compare mutually? (2) Are there differences in
immunogenicity and reactogenicity between the non-adjuvanted
formulations and the adjuvanted formulation – in other words:
Is there an adjuvant effect? In this meta-analysis, we include the
MF59-adjuvanted formulation for seasonal use only although we
are aware there are also studies with MF59 subunit vaccine using
(pre-)pandemic strains (see Section 4).
In 1998, we published a meta-analysis of clinical trials com-
paring aqueous subunit vaccine with whole-virus and split virus
vaccine [6]. We found no relevant differences in immunogenic-
ity between these vaccine formulations for any age group or virus
(sub)type, except between subunit and whole-virus vaccine for
the A-H1N1 subtype in people born after 1957, in whom whole
virus vaccine was more immunogenic. Subunit vaccine was asso-
ciated with the lowest incidence of local and systemic vaccine
reactions when compared to split and whole virus vaccines. How-
ever, these reactions generally were mild and transitory. The 1998
meta-analysis had some methodological limitations. First, both
randomized and non-randomized trials had been included, and
the estimates for the two study types of trial differed, indicat-
ing possible selection bias in the non-randomized trials. In the
present meta-analysis, we therefore excluded non-randomized tri-
als. Secondly, trials had been included that used the now obsolete
‘international units’ of chick red cell agglutination (CCA) for the
assessment of vaccine potency (antigenic contents). In 1978, the
World Health Organization advised to use instead the single-radial
immunodiffusion (SRD) method [7], expressed in microgram HA.
In the present meta-analysis, only studies published from 1978
onwards and using the SRD method were selected. Thirdly, in 1998,
we had used the seroprotection rate as the primary measure of
immunogenicity. Recently, however, we have advocated that this
statistic is not an optimal predictor of protection against infection
[8]. Therefore, we now use the post-vaccination geometric mean
titer (GMT) as primary measure of immunogenicity. Many articles,
however, do not report a measure for the precision of the GMT,
such as a standard deviation or a confidence interval, which would
preclude its use in meta-analyses. This problem [9] is addressed
by imputing multiple values for missing standard deviations. For
completeness, we have also extracted and analyzed seroprotection
and seroconversion rates.
2. Materials and methods
2.1. Literature retrieval, data extraction, and data management
The literature search covered the period 1978 through Augus-
tus 2009. The primary search was carried out electronically using
ScopusTM (http://www.scopus.com) (Elsevier B.V., Amsterdam, The
Netherlands). The following combination of terms was applied:
influenza and (vaccin* or immunization or immunisation) and
(inactivated or whole virus or split or subunit or subvirion or
virosom* or MF59 or MF-59 or adjuvan*). Secondary searches
were carried out using the Cochrane Central Register of Con-
trolled Trials (http://www.cochrane.org), the Monthly Influenza
Bibliography of the National Institute for Medical Research (Medi-
cal Research Council and WHO World Influenza Centre, London, UK;
http://www.nimr.mrc.ac.uk), other sources as described in [10],
and cross references of articles already selected. There was no
restriction on the article language.
On the basis of titles and abstracts, those articles were disqual-
ified, which did not describe clinical trials comparing influenza
vaccines. The remaining articles were read in full text to select
those, which fulfilled the following criteria:
1. At least two of four inactivated vaccine formulations were com-
pared: split virus vaccine (SPL), aqueous subunit vaccine (SU),
virosomal subunit vaccine (VIR), or MF59-adjuvanted subunit
vaccine (adjSU). Comparisons with whole virus vaccines, live
vaccines, or experimental vaccines were not included.
2. Administration of vaccines was stated to be randomized. Tri-
als were regarded as not randomized when randomization was
not explicitly mentioned in the article, even when vaccines were
administered in a double-blind manner.
3. Vaccine strains belonged to the influenza virus (sub)types that
circulated seasonally between 1978 and 2009: A-H3N2, A-H1N1,
and B. Only homologous titers were regarded. Pre-pandemic and
pandemic vaccines were not considered.
4. The antigenic contents of influenza vaccines was expressed in
microgram hemagglutinin (␮g HA), and the vaccine doses given
were 7–21 ␮g HA (or half the amount in children).
5. Data on at least one of the following endpoints were retrievable:
serum hemagglutination inhibition (HI) antibody titers, local or
systemic vaccine reactions, serious adverse events.
From the original articles, data were extracted and entered
in a database by a first reviewer (WB). A second reviewer (JN)
compared the extracted data with that in the articles. The final
database, approved by both reviewers, included the following
information:
General study data: First author, year and language of publica-
tion, year and place(s) of study performance, level of blinding, ethics
committee approval.
Trial population: Numbers, age range and mean (or median),
health status, prior vaccinations.
Vaccines: Formulation, vaccine strains, HA dose per strain, single
or booster vaccinations.
Immunogenicity: Pre- and post-vaccination homologous HI geo-
metric mean titers (GMTs) with a measure of variability (if
given), threshold for seroprotection, numbers or percentages
of subjects in whom seroprotection and seroconversion were
achieved.
Reactogenicity and safety: Numbers or percentages of sub-
jects reporting local or systemic reactions, or serious adverse
events.
This meta-analysis is using summary results of randomized clin-
ical trials (RCTs) comparing two or more vaccine formulations,
obtained from journal articles. In most articles, 2-arm RCTs were
described, but there were also three articles with 3-arm RCT. In
long-term trials where a study group was vaccinated annually dur-
ing several years, only the first vaccination event was taken into
account. In most RCTs, the vaccines involved were trivalent (few
were bi- or monovalent). For immunogenicity, bi- and trivalent
RCTs had to be subdivided into two and three comparisons, respec-
tively, according to viral (sub)type.
Based on the available information in the articles and accepting
a certain overlap, three age groups were established: (predomi-
nantly) children/adolescents (<18 years of age), adults (18–59 years
of age) and the elderly (≥60 years of age). In articles where results
were reported separately for two age groups, data were treated
to form two separate, age-defined RCTs. Health state was classi-
fied as either ‘(predominantly) healthy’ or ‘(predominantly) having
chronic disease’.
In trials involving children, usually a second vaccine dose
was given, typically one month after the first dose (booster),
to overcome the poor antibody response in unprimed sub-
 W.E.P. Beyer et al. / Vaccine 29 (2011) 5785–5792
jects. In these cases, we included the antibody response after
the second dose (booster) in the meta-analysis, according
to [11].
2.2. Statistical methods
The extracted immunogenicity and reactogenicity endpoints
were either post-vaccination GMTs or rates (seroprotection and
seroconversion rates, rates of vaccine reactions). For a compari-
son between two vaccine formulations A and B, ratios A:B with
their 95% confidence intervals (CIs) were calculated, i.e. geometric
mean ratios (GMRs) from GMTs, and rate ratios (RRs) from rates.
Confidence intervals excluding 1.0 would indicate a significant dis-
tinction between A and B at the 5% level.
For overall estimates from ratios of different trials calculated by
techniques of meta-analysis, we used the term ‘meta-analytical’
[12] rather than ‘combined’ or ‘pooled’. Log-transformed post-
vaccination GMTs and their standard deviations (SDs) were
meta-analyzed using a mixed model with vaccine formulation and
trial fitted as fixed effects, and the interaction between trial and
vaccine formulation as random [13]. Thus, we assumed that the
GMRs varied randomly between studies, but around a fixed mean.
When in a comparison GMTs were reported without a measure of
variability, seven SDs were imputed: five fixed values (0.5, 1.0, 1.5,
2.0 or 2.5), one value depending on the log-transformed GMT, and
one varying randomly between 0.5 and 2.5 (this range was derived
from those articles, which did report SDs). Using PROC MIXED of
SAS® (version 9.2; The SAS Institute Inc., Cary, NC, USA), seven
log-transformed GMRs with CIs were calculated, which were then
averaged and back-transformed to obtain a final GMR estimate with
95% CI for the comparison.
Seroprotection, seroconversion and reactogenicity rates were
analyzed using the DerSimonian and Laird random effects model
for meta-analysis of binary data [14]. In the case of 3-arm RTCs
where three formulations A, B, and C were compared, data were
partitioned into three 2-arm RCTs: A–B, A–C, and B–C. Presented
are meta-analytical RRs with 95% CIs.
3. Results
3.1. Numbers and characteristics of selected articles
Primary and secondary literature searches resulted in a list of
2955 articles, due to the broad combination of search terms (see
Section 2). Of these, 2851 did not describe suitable clinical vaccine
trials, as based on titles and abstracts. The remaining 104 articles
were read in full. Of these, 33 articles (Table 1) met the selection
criteria: i.e. a randomized clinical trial comparing at least two of
the four studied seasonal formulations [15–47].
The number of articles increased from 5 in the period,
1978–1987, to 19 in the period, 2000–2009. Most articles (29) were
written in English, and one in German, Spanish, Italian, and Chi-
nese, respectively. All except one article (in German) [19] were
found by ScopusTM . Most studies were conducted in countries of
the European Union (20) and the USA (5). In 20 articles, information
of blinding was provided: 11 studies were double-blind and nine
were partially blind. Half of the articles (16) mentioned approval of
an ethics committee.
Most articles (21) did not provide any history of prior vaccina-
tions of the vaccinees. Four studies were performed in persons who
had never received an influenza vaccine before, and eight stud-
ies in populations, which had been previously vaccinated in part
(32–90%). In 18 articles, the study populations could be character-
ized as (predominantly) healthy. Seven studies were performed in
subjects with a specified chronic disease: cystic fibrosis [15–17,25],
5787
HIV infection [42,44], and asthma bronchiale [19]. The other eight
articles described persons with various or unspecified chronic, in
particular geriatric, diseases.
The total number of vaccinees was 9121, comprising all ages.
When classifying the vaccinees into age groups, overlap could not
always been avoided. In two articles [29,32], separate results were
reported for adults and elderly. The final number of age-defined
RCTs subjected to meta-analysis, was therefore 35. The number of
vaccinees in an RCT varied between 19 and 2150 (median: 172 sub-
jects). Nine trials were done predominantly in children, eight in
adults, and 18 in the elderly. In eight of the nine trials in children,
a booster dose was given three to four weeks after the first dose.
Thus, most of the vaccinees could be regarded as primed, i.e., ever
exposed to influenza antigens.
Table 1 is already arranged according to the use of adjuvant. In 20
articles, comparisons between the non-adjuvanted vaccine formu-
lations SPL, SU and VIR were given, and in 13 articles, comparisons
between non-adjuvanted and adjuvanted formulations were given.
Three articles describing 3-arm trials provided data for both sorts
of comparison.
In 32 out of 35 RCTs, post-GMT values were given as a measure
of immunogenicity. In only 11 RCTs, SD-values for log-transformed
post-GMT were also given or could be calculated (mostly from
reported confidence intervals). SD-values varied between 0.671
and 2.720 with a mean of 1.207. In all other RCTs (66%), SD-values
were imputed (see Section 2). As the number of vaccine com-
ponents varied per RCT (mostly trivalent vaccines but also one
bivalent and two monovalent vaccines), in total 109 GMRs could
be calculated; they varied between 0.42 and 5.25.
In 22 and 25 of 33 articles, respectively, local and systemic vac-
cine reactogenicity was assessed. In some articles, summary values
(e.g., ‘symptom score’) or qualitative statements were given rather
than quantitative incidences for single reactions. Incidences for sin-
gle local reactions were rarely reported, except for ‘pain on contact
(at vaccination site)’ in 13 of the articles. Results for other single
local reactions were reported in only two articles. Therefore, the
analysis of local reactions was limited to the reported rates of ‘pain
on contact’. Similarly, the most commonly reported single systemic
reaction, headache, was analyzed.
3.2. Immunogenicity of non-adjuvanted vaccine formulations
Table 2 presents the meta-analyzed GMRs and RRs of the non-
adjuvanted formulations (split, aqueous subunit, and virosomal
subunit). All ratios were around unity, and with two exceptions,
none of their CIs excluded one. We may therefore conclude that
there are no relevant overall immunogenicity differences between
these formulations. With regard to age and health state, the data
allow only limited conclusions, in part because there was a certain
overlap between the age and health state categories, in part because
there were only few trials in some sub-groups. Nevertheless, the
meta-analyzed ratios in the sub-groups were also close to one (not
shown), with CIs including one, suggesting that the overall con-
clusion of immunogenic similarity between the non-adjuvanted
formulations was robust with respect to the explored study and
population characteristics.
3.3. Immunogenicity of adjuvanted versus non-adjuvanted
vaccine formulations
The immunologic similarity among the three non-adjuvanted
formulations justified averaging, to be compared to the adjuvanted
subunit formulation (adjSU). The resulting meta-analytical ratios
can be interpreted as a measure for the adjuvant effect. Overall,
a moderate but significant adjuvant effect could be demonstrated
for all meta-analytical ratios (largest for GMRs, smallest for protec-
 5788
Table 1
Characteristics of 33 selected articles.

Article First author Year of Country Health statea Ageb Vaccine formulationsc Immunogenicity Reactogenicity
number publication
Nd GMTe Sero-protection Sero-response Local Systemic
rate rate

1 Gross [15] 1981 USA CD Ch SPL SU 76 Yes (SD−) Yes Yes

2 Gross [16] 1983 USA CD Ch SPL SU 63 Yes (SD−) Yes Yes
3 Adlard [17] 1987 United Kingdom CD Ch SPL SU 19 Yes (SD−) Yes Yes Yes Yes
4 Hall [18] 1987 United Kingdom H Ch SPL SU 34 Yes (SD−) Yes Yes Yes Yes
5 Ortwein [19] 1987 Germany CD Ch SPL SU 52 Yes (SD−)
6 Glück [20] 1994 Italy H E SU VIR 94 Yes (SD−) Yes Yes Yes
7 Bautista [21] 1995 Spain H E SPL SU Yes Yes
8 Powers [22] 1995 USA H E SPL VIR 77 Yes (SD−) Yes Yes
9 Conne [23] 1997 Switzerland CD E SU VIR 72 Yes (SD−) Yes Yes
10 Powers [24] 1997 USA H E SU VIR 77 Yes (SD−) Yes Yes Yes Yes

W.E.P. Beyer et al. / Vaccine 29 (2011) 5785–5792

11 Schaad [25] 2000 Switzerland CD Ch SU VIR 64 Yes Yes Yes Yes
12 Baldo [26] 2001 Italy CD E SPL VIR 186 Yes (SD+) Yes Yes
13 Ben Yehuda [27] 2003 Israel H A SPL SU 73 Yes (SD−) Yes Yes Yes Yes
14 Dong [28] 2003 China H Ch SPL SU 499 Yes (SD−) Yes Yes Yes
15 Morales [29] 2003 Colombia H A E SPL SU 344 Yes (SD+) Yes Yes Yes Yes
16 Kanra [30] 2004 Germany, Italy, Turkey H Ch SPL VIR 452 Yes Yes Yes Yes
17 Ruf [31] 2004 Germany H E SPL VIR 545 Yes (SD+) Yes Yes Yes Yes
18 De Bruijn [32] 2005 Not given CD A E SU VIR 382 Yes (SD+) Yes Yes Yes Yes
19 De Bruijn [33] 2006 Not given H E SU VIR 256 Yes (SD−) Yes Yes
20 Evison [34] 2009 Switzerland CD A SU VIR 304 Yes (SD+) Yes Yes Yes Yes

21 Baldo [35] 1999 Italy CD E VIR 163 Yes (SD−) Yes Yes
22 De Donato [36] 1999 Italy H E SU 211 Yes (SD+) Yes Yes Yes
23 Menegon [37] 1999 Italy CD E SPL 194 Yes (SD−) Yes Yes Yes
24 Minutello [38] 1999 Italy H E SU 92 Yes (SD−) Yes Yes Yes
(12) CD E SPL VIR 285 Yes (SD+) Yes Yes
25 Gasparini [39] 2001 Italy H E SU 308 Yes (SD+) Yes Yes Yes
26 Pregliasco [40] 2001 Italy CD E VIR 422 Yes (SD−) Yes Yes Yes Yes
27 Frey [41] 2003 USA H A SPL 301 Yes (SD−) Yes Yes Yes
adjSU
28 Iorio [42] 2003 Italy CD A SU 84 Yes (SD−) Yes Yes
29 Squarcione [43] 2003 Italy H E SPL 1186 Yes (SD−) Yes Yes Yes Yes
(17) H E SPL VIR 820 Yes (SD+) Yes Yes Yes Yes
30 Gabutti [44] 2005 Italy CD A SU 37 Yes (SD−) Yes Yes Yes Yes
31 Baldo [45] 2006 Italy H E SPL 338 Yes (SD+) Yes Yes
(19) H E SU VIR 386 Yes (SD−) Yes Yes
32 Baldo [46] 2007 Italy CD A SU 238 Yes (SD−) Yes Yes
33 Vesikari [47] 2009 Finland H Ch SPL 222 Yes (SD+) Yes Yes Yes Yes
a
H, (predominantly) healthy; CD, (predominantly) having chronic disease.
b
Ch, (predominantly) children; A, (predominantly) adults; E, (predominantly) elderly.
c
SPL, split vaccine; SU, aqueous subunit vaccine; VIR, virosomal subunit vaccine; adjSU, adjuvanted subunit vaccine.
d
Number of vaccinated subjects with immunogenicity data.
e
SD+, SD−, standard deviation given (or computable) or not.
 W.E.P. Beyer et al. / Vaccine 29 (2011) 5785–5792 5789

Table 2
Non-adjuvanted formulations: immunogenicity ratios with 95% confidence intervals.

Meta-analytical parameter Comparison A-H3N2 A-H1N1 B

SPL/SU 1.16 (0.99–1.35) 1.19 (0.93–1.52) 1.18 (0.94–1.48)

Geometric mean ratio SPL/VIR 1.08 (0.91–1.27) 1.24 (0.94–1.63) 1.15 (0.88–1.50)
SU/VIR 0.93 (0.80–1.08) 1.04 (0.82–1.31) 0.97 (0.78–1.22)

SPL/SU 1.00 (0.98–1.02) 1.00 (0.99–1.02) 1.03 (0.99–1.06)

Seroprotection rate ratio SPL/VIR 1.07 (0.97–1.17) 1.03 (0.97–1.09) 1.02 (0.99–1.05)
SU/VIR 0.97 (0.92–1.02) 1.00 (0.94–1.05) 1.0 (0.92–1.03)

SPL/SU 1.09 (1.01–1.18) 1.05 (0.90–1.24) 1.08 (1.03–1.13)

Seroconversion rate ratio SPL/VIR 1.01 (0.93–1.10) 1.01 (0.93–1.10) 1.02 (0.96–1.08)
SU/VIR 0.89 (0.75–1.05) 0.93 (0.79–1.09) 0.97 (0.82–1.16)

Table 3
Adjuvanted versus all non-adjuvanted formulations: immunogenicity ratios with 95% confidence intervals.

Meta-analytical statistic Population A-H3N2 A-H1N1 B

All 1.38 (1.22–1.58) 1.25 (1.04–1.51) 1.40 (1.17–1.68)

[GMTadj /GMTnon-adj ] [215.0/155.2] [156.1/124.6] [115.4/82.4]
Healthy 1.38 (1.13–1.69) 1.19 (0.90–1.57) 1.50 (1.09–2.07)
Geometric mean ratio Health state
Chronic disease 1.40 (1.12–1.76) 1.39 (1.08–1.80) 1.24 (1.01–1.52)
Adults 1.49 (0.76–7.25) 1.17 (0.79–1.92) 1.36 (0.67–2.79)
Agea
Elderly 1.31 (1.16–1.48) 1.23 (0.98–1.55) 1.32 (1.19–1.47)
Seroprotection rate ratio All 1.03 (1.01–1.06) 1.03 (0.99–1.07) 1.09 (1.03–1.16)
Seroconversion rate ratio All 1.12 (1.05–1.19) 1.06 (0.98–1.13) 1.30 (1.14–1.48)
a
One study in children [47] not included.

tion rate ratios), varying between 1.03 (seroprotection) and 1.40
(GMR) (Table 3). The absolute GMT-values varied between 82.4 (B-
strain) and 155.2 (H3N2-strain) for non-adjuvanted formulations,
and between 115.4 and 215.0 for the adjuvanted formulation.
When the GMRs were divided according to health state and age,
no relevant differences could be demonstrated, neither between
predominantly healthy vaccinees and those with chronic disease,
nor between adults and the elderly. We could not calculate meta-
analytical ratios for children as there was only one study in that
age group [47]. The single GMRs of this study showed a significant
adjuvant effect greatly exceeding the meta-analytical estimates in
adults and the elderly: 2.60 (A-H3N2), 2.12 (A-H1N1), and 5.25 (B).
cantly more frequent the local vaccine reaction ‘pain on contact
at the vaccination site’. With respect to the systemic reaction
‘headache’, no differences were found.
4. Discussion
The current meta-analysis is based on data from random-
ized clinical trials with inactivated influenza vaccine formulations
developed for the prevention of clinical illness from seasonal
influenza.
4.1. Immunogenicity of non-adjuvanted vaccine formulations

3.4. Safety and reactogenicity The meta-analysis methods applied here are more refined
than those in a previous publication [6]; yet the results confirm
In 30 articles with 8462 vaccinees (of the total 9121), serious the previously reported similar immunogenicity of the two most
adverse events after vaccination were assessed. Few events requir- widely used formulations: split virus and aqueous subunit vaccines,
ing major medical attention or hospitalization were described. Of regardless of age and health state. The virosomal subunit formu-
these, none was judged as related to vaccination by the clinical lation was found similarly immunogenic as the split and aqueous
investigators. All four vaccine formulations involved can there- subunit formulations. It has been suggested that virosomal vaccines
fore be regarded as safe, based on the serious adverse events may also considerably stimulate cellular immunity [48], which was
information. not assessed in the meta-analysis. It has not yet been demonstrated
Local and systemic reactogenicity rate ratios between the non- whether an elevated cellular immune response translates into a
adjuvanted formulations did not significantly differ from one larger clinical protection.
(Table 4) allowing the conclusion that these three formulations
were associated with similar incidences of local and systemic vac- 4.2. Immunogenicity of non-adjuvanted versus adjuvanted
cine reactions. vaccine formulations
Compared to vaccinees receiving a non-adjuvanted formulation,
vaccinees receiving the adjuvanted formulation reported signifi- The MF59-adjuvanted subunit vaccine showed significantly
larger HI antibody responses compared to the other three vac-
cine formulations, with meta-analytical GMRs varying between
Table 4
Reactogenicity ratios. 1.25 and 1.40 in primed populations (predominantly adults and
the elderly). These results are in good agreement with Banzhoff
Comparison Meta-analytical rate ratio
et al. [49] who found pooled GMR-values between 1.14 and 1.32.
Pain on contact (local Headache (systemic The authors had analyzed data from 13, in part unpublished, ran-
vaccine reaction) vaccine reaction) domized clinical trials in the elderly comparing adjSU with SPL
SPL/SU 0.99 (0.82–1.20) 0.73 (0.41–1.30) or SU formulations by a statistical approach different from ours:
SPL/VIR 0.91 (0.65–1.29) 1.22 (0.81–1.84) they applied an ANOVA model on the HI antibody titers from all
SU/VIR 1.03 (0.70–1.52) 1.16 (0.91–1.47) studies combined. Banzhoff et al. analyzed their data according to
adjSU/(SPL, SU, VIR) 2.12 (1.65–2.71) 1.07 (0.87–1.31)
health state as well. In contrast to our analysis they had access
5790 W.E.P. Beyer et al. / Vaccine 29 (2011) 5785–5792
to the records of the individual vaccinees and could therefore
make a better distinction between ‘healthy’ and ‘having chronic
disease’ than we could do. While we found no meaningful influ-
ence of health state in our data, Banzhoff et al. reported a slightly
larger immunogenicity of adjSU in chronically ill elderly than in
healthy elderly, compared to non-adjuvanted formulations: the
largest GMR difference was seen for the A-H3N2 subtype: 1.43
versus 1.18 (significant); for the other two (sub)types, the differ-
ence was smaller and not statistically significant. Yet, Banzhoff et al.
found their results suggestive for an increased efficacy (i.e., clinical
protection) of adjSU over non-adjuvanted formulations especially
in the elderly with chronic disease.
The pivotal question is whether an HI antibody titer improve-
ment of up to ∼1.4-fold translates into a meaningful increase in
clinical protection. We argued earlier that this is not likely [50].
Evidence should come from clinical exposure trials with persons
of known pre-exposure HI antibody titers whose susceptibility to
influenza infection is recorded, like the well-known experiments
of Hobson et al., published in 1972 [51]. A literature review of
such experiments in healthy children and adults showed indeed
that pre-exposure HI antibody titers were a suitable surrogate of
protection to predict field efficacy [52]. Nauta et al. studied the
relationship between HI antibody titers and clinical protection
from infection [8]. They found that a moderate positive difference
between two GMTs (like in our case) may translate into a moderate
positive difference in clinical protection, but also in a zero, or even
negative, difference. Knowledge of the underlying clinical protec-
tion curve is indispensable for the interpretation of antibody titers.
Estimations of such a curve by meta-analytical methods from pub-
lished clinical exposure trials have been made [53,54]. Coudeville
et al. analyzed data of nearly 6000 adults in 15 studies [54]. Their
meta-analytical clinical protection curve was remarkably robust
with respect to vaccine (sub)type and source of pre-exposure anti-
body (either naturally required or vaccine-induced). An important
feature of the curve is its high steepness for low titers and flatten-
ing of the curve for large titers: the mean protection rises from 0%
for seronegative subjects to 75% for subjects with a titer of 40; for
titers >40, the increase in protection rapidly diminishes.
We may apply Nauta’s approach and Coudeville’s protection
curve to our findings in Table 3 using the mean of reported SDs
(1.207, see Section 3): For the A-H3N2 vaccine component, the
mean titer value (GMT) of the non-adjuvanted formulations is
155.2, which translates to an already high probability of protection
of ∼89%. The corresponding GMT of the adjuvanted formulation is
1.38-fold larger, namely 215.0. This translates to a probability of
protection of ∼92%.
While MF59 has only a modest additional effect on the vac-
cine immunogenicity of previously exposed subjects in our data, its
effect in naïve, unexposed subjects may be much larger, as has been
demonstrated in a randomized trial in adults with a pre-pandemic
avian A-H5N3 strain [55]. Probably, a meaningful adjuvant effect on
antibody formation materializes only in unexposed subjects. If this
is true, then adjSU would perform better in children than in adults
or the elderly. Exactly this has been found in the only study avail-
able in children comparing adjSU with a split formulation [47]: for
example, the A-H3N2 GMT for split vaccine was 195 and for adju-
vanted vaccine 507, which results in an impressive immunogenicity
ratio of 2.60. If we may apply Coudeville’s approach, established in
adults, to antibody titers in children, then both vaccine formula-
tions would translate to a probability of protection greater than
90%.
Our conclusions are based on homologous HI titers, i.e., anti-
body titers against the actual vaccine strains. In the study of Baldo
et al. [46], also the titers against seasonal heterologous drift variants
were determined, demonstrating higher heterologous antibody
responses after adjSU vaccination than after comparator (SU) vac-
cination. This observation suggests a broader cross-reactivity of
antibodies induced by adjSU, compared to non-adjuvanted for-
mulations, and justifies the hypothesis that adjSU confers larger
protection in case of vaccine mismatch (significant discrepancy
between vaccine strain and circulating variant).
One could argue that the ultimate clinical confirmation of
these considerations should come from methodologically sound
(yet laborious) head-to-head comparative efficacy trials with viro-
logical and clinical outcome parameters [56]. We are aware of
two publications assessing vaccine effectiveness ([57,58] claim-
ing that the adjSU formulation was more protective than SPL or
SU. However, one study assessed mortality from all causes with-
out virological confirmation, and the other was not randomized,
which rules out any valid conclusion. We realize that random-
ized clinical protection studies need a large statistical power to
detect a small difference between protection rates. For example,
the head-to-head comparison study of Belshe et al. [59] between
a live attenuated and an inactivated influenza vaccine in children
involved nearly 8000 persons to detect an infection rate difference
of ∼5% in favor of the live vaccine. As the infection incidence dur-
ing an influenza season is usually smaller in the elderly than in
children, a trial in the elderly to detect a similar difference would
easily require up to 20,000 vaccinees.
All available evidence taken together, the four inactivated vac-
cine formulations induce more or less similar homologous humoral
immunity in primed populations and offer a high chance of clin-
ical protection. There may be some elevated immunogenicity of
the adjuvanted formulation, but this antibody gain is not or only
marginally translated into a gain in clinical protection. From a
public health viewpoint therefore, it might be more favorable to
improve general vaccine acceptance and actual use of existing vac-
cines, regardless whether adjuvanted or non-adjuvanted, rather
than to develop new vaccine formulations [60].
4.3. Safety and reactogenicity
Scrutiny of the few reported serious adverse events after vacci-
nation did not reveal any causal relationship with vaccination, thus
there is no safety concern for any of the vaccine formulations. Vac-
cine reactions, both local and systemic, were consistently reported
to be mild and transient, and thus of little clinical relevance. These
results are in line with the favorable safety assessment of the World
Health Organization [61].
The adjuvanted formulation was associated with an increased
level of local reactogenicity compared to SPL, SU and VIR for-
mulations: the rate ratio adjuvanted/non-adjuvanted for the local
symptom ‘pain on contact’ was 2.12 (significant). In good agree-
ment, Banzhoff et al. found a rate ratio of 2.5 [49]. These findings are
not unexpected, since the long-term experience with vaccine adju-
vants has led to the paradigm that higher vaccine immunogenicity
generally comes at the expense of elevated vaccine reactogenicity
[62]. With regard to systemic reactogenicity, Banzhoff et al. found
a significantly elevated rate ratio of 1.5, while in our data, such a
difference between adjuvanted and non-adjuvanted vaccine for-
mulations could not been demonstrated for the systemic reaction
symptom ‘headache’ (rate ratio 1.07, not significant).
4.4. Methodological aspects of meta-analysis
As main source for the identification of suitable articles to
be included in this meta-analysis, we used the ScopusTM system,
currently the largest abstract and citation database of research lit-
erature and web sources with coverage of MedLine® . Thirty-two
out of 33 articles selected were retrieved by Scopus (and only one,
non-English, article from alternative search strategies).
 W.E.P. Beyer et al. / Vaccine 29 (2011) 5785–5792
In the period of three decades covered by this review, vac-
cine immunogenicity was virtually always assessed by pre- and
post-vaccination serum hemagglutination inhibition antibody
titers in groups of persons. It is well-known that post-vaccination
titers are linearly related to pre-vaccination titers [63]. Pre-
vaccination titer, as a person’s characteristic expressing the
individual history of exposition to influenza (previous infections
and vaccinations), constitutes a prognostic baseline factor possi-
bly biasing outcomes in non-randomized comparative trials. As we
retrieved only randomized trials where baseline imbalances tend
to zero, we made no attempt to correct for pre-vaccination titers.
In the influenza literature, HI antibody response has been
expressed by a variety of measures, both quantitative (e.g., geo-
metric mean titer) and dichotomous ones (e.g., seroprotection rate,
seroconversion rate). We regard the GMT the most informative
statistic as it is formed by the absolute post-vaccination titers of
the entire group without applying cut-off thresholds and limita-
tions. For the meta-analysis of means, information on the precision
of the means (e.g., standard deviations) is required. However, in the
majority of trials, dispersion was not at all reported. Such substan-
tial amounts of missing data on dispersion form a, still debated,
challenge in meta-analysis. In 1987, Rubin discussed simulation-
based multiple imputation procedures [64]. Here, we imputed, for
each missing SD-value, seven values within a range, which was
derived from the trials with information on dispersion. Five val-
ues were fixed, one value was based on the actual log-transformed
GMT, and another was chosen at random. As an advantage of mul-
tiple imputation over single imputation, the uncertainty about the
correct value to input is represented by replacing each missing
value with a set of plausible values. Our strategy to use (partly)
fixed imputations is probably not optimal because the underlying
assumption of homogeneity of variance may not hold. However,
we compensated for this by using both small and large fixed impu-
tations. The resulting seven versions of the completed data were
combined and subjected to a mixed model procedure, allowing
also for indirect comparisons between vaccine formulations. This
procedure resulted in stable estimates for mean ratios (GMRs) and
allowed inferences.
For the purpose of completeness, we also collected and analyzed
seroprotection and seroconversion rates. While seroconversion
rate ratios showed a fair accordance with GMRs (although con-
fidence bounds were often wider), the seroprotection rate ratios
were usually close to one, possibly the result of loss of information
inherent to the seroprotection rate concept: a serum antibody titer
is regarded protecting when it exceeds a pre-set titer threshold (40
with current HI-assays). Thus, titers of, for example, 50 and 500 are
both treated the same in the seroprotection concept (as they are
both larger than 40), while they may actually be associated with
different levels of clinical protection.
4.5. Overall conclusion
In conclusion, we found the currently available inactivated
influenza vaccine formulations to be safe, well tolerated and sim-
ilarly effective in primed populations, suitable for the control of
seasonal influenza outbreaks according to existing policy recom-
mendations.
Acknowledgements
We wish to thank Mrs. Lily Lu, Shanghai, China, for the transla-
tion of a Chinese article.
Conflict of interest: W.E.P. Beyer has held consultancies with
pharmaceutical companies. A.D.M.E. Osterhaus is CSO of Viroclin-
ics Biosciences BV, a CRO that collaborates with pharmaceutical
5791
companies. This study is a cooperation between Erasmus Medical
Centre and Abbott.
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