Vaccination against Human Influenza A/H3N2 Virus
Prevents the Induction of Heterosubtypic Immunity
against Lethal Infection with Avian Influenza A/H5N1
Rogier Bodewes, Joost H. C. M. Kreijtz, Chantal Baas, Martina M. Geelhoed-Mieras, Gerrie de Mutsert,
Geert van Amerongen, Judith M. A. van den Brand, Ron A. M. Fouchier, Albert D. M. E. Osterhaus,
Guus F. Rimmelzwaan*
Department of Virology, Erasmus Medical Center, Rotterdam, The Netherlands
Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for
complications resulting from infection with these viruses. Recently it was recommended in a number of countries including
the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically
naı ¨ve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially
pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.
Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/
H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were
no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice
continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and
more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.
The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection.
These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal
influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.
Citation: Bodewes R, Kreijtz JHCM, Baas C, Geelhoed-Mieras MM, de Mutsert G, et al. (2009) Vaccination against Human Influenza A/H3N2 Virus Prevents the
Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus. PLoS ONE 4(5): e5538. doi:10.1371/journal.pone.0005538
Editor: Wanda Markotter, University of Pretoria, South Africa
Received January 5, 2009; Accepted April 21, 2009; Published May 14, 2009
Copyright: ? 2009 Bodewes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was financially supported in part by EU Grant FluVac (grant SP5B-CT-2007-044407). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Since 2003, more than 380 human cases of infection with highly
pathogenic avian influenza A virus (IAV) of the H5N1 subtype
have been reported to the World Health Organization (WHO) of
which more than 60% were fatal . Because of the continuous
spread of these viruses among domestic birds, the frequent
introduction into wild birds and the increasing number of human
cases, a pandemic outbreak caused by influenza A/H5N1 viruses
is feared [2–4].
It has been demonstrated in animal models that prior exposure
to an IAV can induce heterosubtypic immunity to infection with
an IAV of an unrelated subtype (for review see ). Also in
humans there is evidence that infection with IAV can induce
heterosubtypic immunity . Individuals that had experienced an
infection with an H1N1 IAV before 1957 less likely developed
influenza during the H2N2 pandemic of 1957 . In particular,
the induction of cell-mediated immune responses after infection
contributes to protective immunity against infection with hetero-
subtypic IAVs. The presence of cross-reactive cytotoxic T
lymphocytes (CTL) in humans inversely correlated with the
amount of viral shedding in the absence of antibodies directed
against the virus used for experimental infection . It is well
documented that seasonal human IAVs and avian IAVs share
CTL epitopes located in the internal viral proteins like the
nucleoprotein [8–10]. Thus, cell-mediated immunity induced by
natural infection with seasonal IAVs may confer protection against
heterosubtypic pandemic influenza viruses. In this respect, the
disproportional age distribution of severe human H5N1 cases is of
interest . Especially younger individuals are at risk and
although other confounding factors cannot be excluded, it is
tempting to speculate that young subjects have been infected with
seasonal influenza viruses less frequently and therefore have not
developed protective heterosubtypic immune responses against
infection with the highly pathogenic avian A/H5N1 viruses.
Since seasonal IAVs of the H3N2 and H1N1 subtypes cause
epidemic outbreaks annually associated with excess morbidity and
mortality mainly among infants, the elderly, immuno-compro-
mised and other high-risk patients, influenza vaccination is
recommended for these high-risk groups. In general, the influenza
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vaccines most frequently used are inactivated vaccines, including
subunit preparations that consist of the viral hemagglutinin (HA)
and neuraminidase (NA). Due to the higher risk of complications
and hospitalizations secondary to influenza in children [12,13],
annual vaccination of all healthy children 6 to 59 months of age
was recommended in various countries including the United States
since 2007 .
However, annual vaccination may prevent the induction of
heterosubtypic immunity by infection with seasonal influenza virus
strains. In addition, it is unlikely that seasonal inactivated influenza
vaccines, unlike live attenuated vaccines, induce heterosubtypic
immunity since they induce cross-reactive CTL responses
Thus, we hypothesized that vaccination against seasonal flu
prevents the induction of cross-protective cell-mediated immunity,
which consequently may lead to more severe clinical outcome of
infection with a future pandemic virus. Here we show in a mouse
model that protective immunity against lethal infection with H5N1
IAV Indonesia/5/05 (IND/05) was induced by infection with
H3N2 IAV HongKong/2/68 (HK/68), which was prevented by
effective vaccination against the A/H3N2 virus. The lack of
protection against IAV IND/05 correlated with reduced virus-
specific CTL responses.
Antibody responses against IAV HK/68 (H3N2) after
Mice were vaccinated with subunit vaccine with or without
Alum or were ‘mock’ vaccinated (table 1). HI antibody titers were
detected 28 days after the first vaccination with subunit and Alum
(groups 2 and 5) and in 3 out of 26 mice vaccinated with
unadjuvanted subunit vaccine (group 6). Four weeks after the
second vaccination, geometric mean titers (GMTs) increased to
244 and 218 in mice from group 2 and group 5, respectively. Four
mice of group 6 developed detectable HI-antibody responses with
a GMT of 48, the other mice of this group did not seroconvert
(figure 1A). Sera of mice were also analysed for the presence of
virus neutralizing (VN) antibodies. Four weeks after the second
vaccination, mice vaccinated with adjuvanted subunit vaccine
developed VN antibodies with a GMT of 38 and 29 in group 2
and group 5 respectively, while only two mice of group 6
developed detectable VN antibody titers (figure 1B).
Outcome of infection with IAV HK/68 (H3N2)
Mice that developed HI-antibodies against IAV HK/68 (all
mice of group 2 and four of group 6) were protected from weight
loss after infection with IAV HK/68, while mice of other groups
lost weight until day seven post infection (p.i.) and showed mild
clinical symptoms for 2–3 days (figure 2A). Clinical signs and
weight loss after infection correlated well with virus titers in the
Table 1. Experimental groups and design of the study.
Mice were divided over seven groups and were either vaccinated twice with
subunit vaccine with or without adjuvant (Alum), PBS, or adjuvant only as
indicated. Four weeks after the second vaccination, mice were infected with IAV
HK/68 (H3N2) or mock-infected. Twenty-nine days after the infection with IAV
HK/68, mice were challenged with IAV IND/05 (H5N1).
Figure 1. Induction of serum antibodies against IAV HK/68 (H3N2) by vaccination. Serum antibody levels were determined before and at
the indicated time points after vaccination of mice with PBS (groups 1, 3 and 4; #), subunit vaccine with alum (groups 2 and 5; m), subunit vaccine
only (group 6; &) and alum only (group 7; 6) by HI assay (A) and VN assay (B).
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lungs of infected mice 4 days p.i.. No virus was detected in lungs of
mice vaccinated with adjuvanted subunit vaccine, while the
average lung virus titer of mock-vaccinated mice was 108.1
TCID50/gram lung. Similar titers were observed for the mice in
groups 6 and 7 with the exception of one mouse in group 6 with a
HI antibody titer of 40 induced by vaccination with unadjuvanted
subunits that had a lung virus titer of 105.7TCID50/gram lung
(figure 2B). The virus titers detected on day 4 p.i. correlated with
the absence or presence of virus infected cells in the lungs detected
by immunohistochemistry (data not shown).
Virus-specific CTL and antibody responses after infection
with IAV HK/68 (H3N2)
Four days p.i. with IAV HK/68 the frequency of splenic CD8+
T lymphocytes specific for the NP366–374epitope of IAV HK/68
Figure 2. Outcome of infection with IAV HK/68 (H3N2). Mice were inoculated with IAV HK/68 (groups 2 (m), 3 (#), 6 (&) and 7 (6)) or PBS
(groups 1 (
), 4 (,) and 5 (e)). (A) Body weight after infection was determined daily and expressed as the percentage of the original body weight
before infection. (B) Lung virus titers measured on day 4 p.i. in mice from the indicated experimental groups. Horizontal bars represent the average
titers of five mice. The dotted line represents the cut-off value for obtaining a positive result. *This mouse from group 6 had before infection an HI
antibody titer of 40. (C) Vaccination prevented the induction of iBALT after infection. Twenty-eight days post infection with IAV HK/68 iBALT was
detected in mice from group 3, but not in mice from group 2. Lung tissue sections were stained with HE. (D) Virus-specific CD8+ T cell responses
detected 28 days post infection. Splenocytes of mice from the indicated experimental groups were tested for the presence of CD8+ T cells that bound
the H2-Db NPHKTetramer. Horizontal bars represent the average of 2–4 mice. The difference in %CD8+ Tm+ T cells between groups 2 and 3 was
statistically significant (P=0.030).
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(CD8+ TmHK+ T-cells) as determined by tetramer staining
remained at background levels in all groups (data not shown).
In all infected mice a raise in the frequency of CD8+ TmHK+ T-
cells was detected twelve days p.i.. No statistically significant
differences were observed between the experimental groups.
Essentially the same results were observed using intracellular
IFN-c staining after re-stimulation with peptides representing the
NP366–274 and PA224–233 epitopes of IAV HK/68 (NPHK and
PAHK). The NPHKand PAHKspecific CTL induced by infection
with IAV HK/68 cross-reacted to various extents with their
counterparts derived from IAV IND/05 (NPINDand PAIND). The
cross-reactive nature of a proportion of the NP366–374specific CTL
was confirmed by double staining with TmHKand TmIND(data
By day 28 p.i. with IAV HK/68, just before challenge infection
with IAV IND/05, the frequency of virus-specific CTL in the
spleen had declined and virus-specific CTL were not detectable by
intracellular IFN-c staining. However, TmHKand TmINDpositive
cells were detected in mice that were mock vaccinated prior to
infection (group 3). Strikingly, the frequency of TmHKpositive
CD8+ T lymphocytes was significantly lower in mice of group 2
that were effectively vaccinated against infection with IAV HK/68
(p=0.030) (figure 2D).
Vaccination prevents induction of iBALT after IAV HK/68
Following infection with IAV HK/68, no significant lesions
were found in lungs of mice vaccinated with adjuvanted subunit
vaccine (group 2), whereas mice that were mock-vaccinated or
vaccinated with Alum or subunit preparation only (mice of groups
3, 6 and 7) developed a multifocal mild subacute necrotizing
bronchopneumonia four days after infection, which on day 12 p.i.
progressed into a multifocal moderate chronic necrotizing
bronchopneumonia. On day 28 p.i., these mice had developed
perivascular moderate proliferation of inducible Bronchus Asso-
ciated Lymphoid Tissue (iBALT), consisting mainly of mononu-
clear cells, which was absent in mice effectively vaccinated against
infection with IAV HK/68 (figure 2C).
Effective vaccination prevents heterosubtypic immunity
against IAV IND/05 (H5N1)
After infection with IAV IND/05, all mice developed clinical
signs (weight loss, ruffled fur, lethargy) from day two p.i. onwards.
Mice that developed clinical signs p.i. with IAV HK/68 (groups 3,
6 and 7) lost weight until day 6–7 after infection with IAV IND/05
and then started to gain weight and fully recovered, while mice of
other groups, not previously infected with IAV HK/68 (groups 4
and 5) and more strikingly, those effectively vaccinated against
infection with IAV HK/68 (group 2) lost significantly more weight
(group 2 versus group 3: p=0.0001) on day 7 p.i. with IAV IND/
05 and showed more severe clinical signs (lethargy, ruffled fur,
hunched posture) than mice of the other groups (figure 3A).
Moribund animals were euthanised when they reached pre-fixed
criteria regarding weight loss (.20%) and clinical signs, which was
used to determine mortality rates. One mouse out of 10 (10%) of
group 2 survived lethal challenge, while all mice but one (91%) of
group 3 survived lethal challenge (n=11). This difference in
survival rate was statistically significant (p=0.0003) as was
calculated with the Logrank test (figure 3B). All other mice not
previously exposed to IAV HK/68 became moribund, whereas all
mice not adequately vaccinated against IAV HK/68 (groups 6 and
Replication of IAV IND/05 (H5N1) in the lungs
The lung virus titers at days four and seven p.i. were compared
between groups of IAV IND/05 infected mice. Four days p.i. no
significant differences were found between mice of different
groups. The average virus titer in mice of group 3 was 107.7
TCID50/gram lung, which was similar to that observed in mice
from group 2 that were effectively vaccinated against IAV HK/68
(107.6TCID50/gram lung). In contrast, there were significant
differences in lung viral titers between mice of the different groups
seven days p.i. (figure 3C). Group 3 mice, not vaccinated against
infection with IAV HK/68, had virus titers of 104.8TCID50/gram
lung while mice of group 2, vaccinated with adjuvanted subunits,
had significantly higher virus titers with an average of 106.5
(p=0.025), which was similar to that observed in naı ¨ve mice
infected with IAV IND/05 virus (group 4) or those that were
vaccinated against, but not infected with IAV HK/68 virus (group
5). Mice unsuccessfully vaccinated against IAV HK/68 infection
with adjuvant or subunits only also displayed lower lung viral titers
(groups 6 and 7).
Induction of CD8+ T cell responses p.i. with IAV IND/05
Four and seven days p.i. infection with IAV IND/05,
splenocytes were stained for intracellular IFN-c after incubation
with peptides NPINDand PAIND. Four days p.i., no virus-specific
CD8+ T cell responses were detected in any of the IAV IND/05
infected mice. However, seven days p.i, anamnestic NPINDand
PAINDspecific IFN-c+CD8+ T-cell responses were observed in
mice from group 3, which were significantly lower in mice
effectively vaccinated against IAV HK/68 (group 2) (p=0.038 and
p=0.002 respectively) (figure 3D)
Histopathology and detection of infected cells after
infection with IAV IND/05 (H5N1)
On day four p.i. with IAV IND/05, mice developed a
multifocal severe subacute necrotizing bronchopneumonia, of
which the severity was similar for all experimental groups.
However, seven days p.i. there were marked differences between
the groups. The mock-vaccinated mice or those vaccinated with
adjuvant only prior to infection with IAV HK/68 had a multifocal
moderate chronic necrotizing bronchopneumonia characterized
by a perivascular core of lymphocytes and plasma cells,
proliferation of bronchiolar epithelium and hyperplasia of
pneumocytes with a type II appearance. In contrast, mice of
groups 4, 5 and especially group 2 had more severe lung pathology
characterized by a multifocal to coalescing severe subacute
In general, the extent of lung histopathology and the lung virus
titers after infection with IAV IND/05 correlated with the
presence of virus-infected cells in the lungs as determined by
immunohistochemistry. Four days p.i., virus-infected cells were
detected in all IAV IND/05 infected mice. In contrast, seven days
p.i., antigen positive cells were found sporadically in lungs of mice
of groups 3 (figures 4C–D) and 7 (figures 4I–J), whereas in the
lungs of mice from group 2 (figures 4A–B), 4 (figures 4E–F) and
5 (figures 4G–H) virus-infected cells were still abundantly
Here we demonstrate that successful vaccination of mice against
human IAV HK/68 (H3N2) prevented the induction of hetero-
subtypic immunity against a lethal challenge with IAV IND/05
(H5N1). As a result, H3N2 vaccinated mice had a fatal clinical
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outcome of infection with IAV IND/05, associated with higher
virus titers and more severe histopathological lesions in the lungs
seven days p.i. and reduced virus-specific CD8+ T cell responses
compared to mice that experienced a productive, self-limiting
infection with IAV HK/68.
It has been well established that infection with IAV can induce a
certain degree of protective immunity against infection with an
heterosubtypic strain of IAV, which was already recognized more
than 40 years ago . This so-called heterosubtypic immunity
was not only demonstrated in animal models [17–20] but there is
Figure 3. Outcome of infection with IAV IND/05 (H5N1). Mice were inoculated with IAV IND/05 (groups 2 (m), 3 (#), 4 (,), 5 (e), 6 (&) and 7
(6)) or PBS (group 1 (
). (A) Body weight after infection was determined daily and expressed as the percentage of the original body weight before
infection. (B) Survival rates after infection with IAV IND/05. The proportion of mice from the indicated groups that survived infection is shown in a
Kaplan-Meier plot. Moribund animals were euthanized when they reached pre-fixed criteria regarding weight loss (.20%) and disease severity score,
which was used to determine mortality rates. (C) Lung virus titers measured on 7 days p.i. in mice from the indicated groups. Horizontal bars
represent the average of 2–6 mice. The difference in virus titers between mice of group 2 and group 3 was statistically significant (p=0.025). N.S.: not
significant. (D) Virus-specific CD8+ T cell responses on day 7 p.i.. The frequency of CD3+ CD8+ splenocytes specific for peptide NP366–374and PA224–233
derived from IAV IND/05 was determined by intracellular IFN-c staining. The horizontal bars represent the average frequency of IFN-c+ cells in the
CD8+ T cell population of 2–7 mice in the indicated groups. Differences between group 2 and group 3 were statistically significant for both peptides.
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also direct and indirect evidence that it exists in humans [6,7] and
that cell-mediated immune responses contribute to this type of
immunity (for review see ).
Of special interest in this respect is that there is a dispropor-
tionate age distribution of human cases . Especially younger
subjects are at risk for severe A/H5N1 disease and fatal outcome,
which may inversely correlate with the history of infections with
seasonal influenza viruses and the cross-reactive CTL responses
 associated with these infections. Also the results from the
Cleveland study indicate that a prior infection with seasonal
influenza virus strains induced protective immunity against a new
heterosubtypic pandemic strain . Nevertheless, severe A/H5N1
infections with fatal outcome do occur. However, little is known
about the history of previous infections of these patients. Although
most adults must have experienced an infection with seasonal
influenza viruses, it is possible that individual cases did not develop
adequate heterosubtypic immunity against A/H5N1 strains.
To test the hypothesis that successful immunization against
seasonal influenza could interfere with the induction of hetero-
subtypic immunity, mice were vaccinated with an Alum-adjuvanted
subunit vaccine. The use of an adjuvant was necessary since
vaccination with subunit alone induced detectable antibody
responses in a small proportion of mice only and would not provide
a useful model for successful vaccination against seasonal influenza.
Indeed, all mice vaccinated with Alum alone and most mice
vaccinated with subunits alone were not protected against infection
with A/H3N2 virus. In contrast, all mice vaccinated with
adjuvanted subunits, were fully protected against infection with
IAV HK/68. This prevented the induction of heterosubtypic
immunity against infection with IAV IND/05 normally seen in
mice that had experienced a productive IAV HK/68 infection. The
severity of the clinical signs and histopathological lesions, the extent
of weight loss, lung virus titers and mortality rates of these mice was
comparable of those that were immunologically naı ¨ve prior to
infection with IAV IND/05 (group 4) or that were vaccinated
against IAV HK/68 virus, but not subsequently infected with IAV
HK/68 virus (group 5). It could be argued that in the present study
the vaccine matched the A/H3N2 virus perfectly, while under field
conditions the match may not always be optimal allowing sub-
clinical infections to occur, which may induce heterosubtypic
immunity despite vaccination. However, also in our mouse model
there is indication that in vaccinated mice sub-clinical infection with
influenza virus A/HK/2/68 took place, since weak, short-lived
virus-specific CTL responses were observed, which did not protect
against challenge infection with the A/H5N1 strain.
Four weeks after infection with IAV HK/68 virus, the number
of virus-specific CD8+ T cells in the spleen was significantly lower
in mice vaccinated against IAV HK/68 than in unvaccinated
mice. The differences were not observed at earlier time points p.i..
Further evaluation of the CD8+ TmHK+ T cells indicated that the
numbers of CD62Lhigh and CD127+ cells were higher in
unvaccinated mice than in vaccinated mice on day 28 p.i. (data
not shown). This may indicate that the control of IAV HK/68
replication in the lungs had prevented the efficient induction of
virus-specific central and effector memory CD8+ T cell responses.
These results resemble those found in a mouse model for Listeria
monocytogenes infection, in which shortening of the duration of the
infectious period did not impact the size of the primary CD8+ T
cell response, but diminished the memory population of CD8+ T
cells . The analysis of the CD8+ T cells responses seven days
after challenge infection with IAV IND/05 further indicated that
indeed prior vaccination against HK/68 (H3N2) prevented the
efficient induction of memory CTL responses. Both the secondary
response to the NPIND and the PAIND epitope were reduced
compared to the responses observed in un-vaccinated mice.
Although it has been described that the NP366–374 is more
immunodominant than the PA224–233epitope in secondary CTL
responses , a stronger response was observed against the
PA224–233epitope after infection with IAV IND/05. This could be
Figure 4. Histopathological analysis and immunohistochemis-
try of the lungs of mice infected with IAV IND/05. Mouse lung
sections were stained for influenza A virus nucleoprotein. Cytoplasm of
infected cells stain red, the nuclei of infected cells stain deep red. In the
groups without a history of productive A/H3N2 infection, including
group 2 (A,B), infection with IAV IND/05 led to severe histopathological
changes and to viral antigen expression in cells of the bronchiolar walls
and in the alveoli (group 4: E,F and group 5: G,H). In mice of groups 3
(C,D) and 7 (I,J) that had experienced a productive infection with IAV
HK/68 only moderate histopathological changes were observed and
virus infected cells were detected sporadically (see insert in panel D).
For more information please see text.
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explained by the lower cross-reactivity of CTL directed to the
NP366–374epitope derived from IAV HK/68 (ASNENMDAM)
with that derived from IAV IND/05 virus (ASNENMEVM)
compared to the cross-reactivity of CTL specific for the PA224–233
epitope as was observed after the analysis of the CTL measured by
tetramerstaining p.i. with IAV HK/68 and IND/05 (data not
shown). Apart from systemic CTL responses measured in the
spleen also local CTL responses may contribute to protective
immune responses, such as in the draining lymph nodes and in the
lung tissue itself [24,25]. Since the frequency of virus-specific
CD8+ T cells in the spleen reflected that in the lymph nodes
[26,27], we analyzed CTL responses in the spleen only. It was of
interest to note that infection with IAV HK/68 resulted in the
formation of iBALT structures. Prior vaccination against IAV
HK/68 infection prevented the formation of iBALT completely.
iBALT consists mainly of B cells, T cells and dendritic cells and it
has been shown that mice with iBALT but without peripheral
lymphoid organs can clear virus infection . Also in humans, T
cells specific for viral respiratory pathogens have been detected in
lung tissue and may play a protective role against subsequent
infections in this species as well . Although no IAV IND/05
cross-reactive antibodies were detected by VN or HI assay on the
day of challenge infection, it is possible that infection with IAV
HK/68 induced M2 specific antibodies that potentially cross-
reacted with the M2 protein of IAV IND/05. However it is
unlikely that these antibodies accounted for the heterosubtypic
immunity induced by primary infection with IAV HK/68 [30,31].
Thus prior infection with seasonal influenza viruses, which
generally results in a self-limiting upper respiratory tract infection,
may afford at least partial protection against potentially pandemic
heterosubtypic influenza virus strains. At present vaccination
against seasonal influenza is recommended for all healthy children
6–59 months of age in a number of countries, including the USA
. Also in Europe vaccination of children is currently
considered and a number of countries already decided to
recommend vaccination of healthy children . Although
vaccination is (cost-) effective in this age group [33–37], it may
interfere with the induction of heterosubtypic immunity against
potentially pandemic strains of a novel subtype, e.g. H5N1, by
creating an immunological ‘‘blind spot’’. Furthermore, the use of
adjuvants is considered to increase vaccine efficacy in young
children . Thus during a next pandemic, especially children
that received the annual flu-shot would be at higher risk to develop
severe illness and a fatal outcome of the disease than those that
experienced an infection with a seasonal IAV strain. This of
course, would be of great concern and is supported by the data
obtained in our mouse model. Ideally, seasonal influenza vaccines
are used that also induce heterosubtypic immunity [16,39]. More
research is required in this field to define vaccine preparations that
not only induce protective immunity against seasonal influenza,
but also induce heterosubtypic immunity. With the current
pandemic threat caused by A/H5N1 viruses this would be highly
Materials and Methods
Virus stocks of influenza viruses A/Hong Kong/2/68 (IAV
HK/68) and A/Indonesia/5/05 (H5N1) (IAV IND/05) were
prepared by infecting confluent Madin-Darby-Canine-Kidney
(MDCK) cells. After cytopathologic changes were complete,
culture supernatants were cleared by low speed centrifugation
and stored at 270uC. Infectious virus titers were determined in
MDCK cells as described previously .
Influenza subunit antigen derived from IAV X-31 (H3N2) was
essentially prepared as described previously . X-31 is a
reassortant vaccine strain of A/Aichi/2/68 and A/PR/8/34, of
which the HA and NA resemble that of IAV HK/68 closely. The
purity of the subunit preparations was tested by SDS-polyacryl-
amide gel electrophoresis and the absence of the nucleoprotein
and matrix protein of the subunit preparations was tested by
western blotting using monoclonal antibodies against the influenza
A nucleoprotein and the influenza A matrix protein. The protein
concentration was determined using a BCA Protein Assay Kit
(Pierce, Rockford, USA).
Immunization and infection of mice
Female specified pathogens free 6–8 weeks old C57BL/6J (H-2b)
micewerepurchasedfrom CharlesRiver(Sulzfeld, Germany).Mice
were immunized twice with an interval of four weeks intramuscu-
larly (i.m.) in both hind legs in a total volume of 100 ml. Mice
(n=19–40 per group) received PBS (phosphate buffered saline)
(Groups 1,3 and 4), 15 mg subunit vaccine with (Groups 2 and 5) or
without (Group 6) 1 mg Aluminum hydroxide gel (Alum) (Sigma-
Aldrich, Zwijndrecht, The Netherlands) or Alum only (Group 7).
Eight days after the second vaccination, four mice of each group
were bled and spleens were resected. Four weeks after the second
vaccination, mice of groups 2, 3, 6 and 7 were infected intranasally
with 56102TCID50IAV HK/68 in a volume of 50 ml. Four and
twelve days post infection (p.i.), 5–7 mice were bled and lungs and
spleens were resected. Four weeks after infection with IAV HK/68,
all mice except mice of group 1 were challenged with 26102
TCID50IAV IND/05. A dose of 26102TCID50was used because
this was the minimal dose resulting in a lethal infection in .90%
mice reproducibly. The day before challenge with IAV IND/05,
mice of each group (n=2–4) were euthanized and lungs and spleens
were resected as well as on day four (n=4–6), seven (n=2–9) and
fourteen (n=3–8) days after challenge. Vaccinations, intranasal
infections, orbital punctures and euthanasia were performed under
anesthesia with isoflurane in O2. After infection with IAV HK/68
and IAV IND/05, mice were monitored for the presence of clinical
signs,includingweight loss. Allexperimentswith IAVIND/05 were
performed under Biosafety Level 3 conditions. An independent
animal ethics committee (DEC consult) approved the experimental
protocol before the start of the experiments.
Serum samples of mice were collected at various time points
during the experiment and tested for the presence of HA-specific
antibodies against IAV HK/68 and IAV IND/05 using the
hemagglutination inhibition (HI) assay  and virus neutralizing
(VN) antibodies using the VN assay .To determine the titer of
antibodies against IAV IND/05 before infection with IAV IND/
05, a reverse genetics virus was produced from which the basic
cleavage site was removed. Antibody titers obtained with this
reverse genetics virus was comparable with that against the wild-
type strains (data not shown). Positive control serum specific for
IAV HK/68 was obtained by injecting a rabbit with sucrose
gradient purified virus . Hyper-immune serum obtained from
a swan immunized twice with inactivated H5N2 influenza virus
A/Duck/Potsdam/1402/86 (Intervet, Boxmeer, the Netherlands)
was used as a positive control against IAV IND/05 .
Lung virus titers
Lungs of mice were snap frozen on dry ice with ethanol and
stored at 270uC. Lungs were homogenized with a FastPrep-24
Flushot Prevents H5N1 Immunity
PLoS ONE | www.plosone.org7 May 2009 | Volume 4 | Issue 5 | e5538
(MP Biomedicals, Eindhoven, The Netherlands) in medium
consisting of Hank’s balanced salt solution containing 0.5%
lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 mg/ml
streptomycin, 100 U/ml polymyxin B sulfate, 250 mg/ml genta-
mycin, and 50 U/ml nystatin (ICN Pharmaceuticals, Zoetermeer,
The Netherlands) and centrifuged briefly. Quintuplicate 10-fold
serial dilutions of these samples were used to infect MDCK cells as
described previously . HA activity of the culture supernatants
collected 5 days post inoculation was used as indicator of infection.
The titers were calculated according Spearman-Karber .
Flow cytometry of virus-specific CD8+ T cells
Peptides and intracellular IFN-c staining.
suspensions of spleens were prepared as described previously .
CD8+ T cell responses after infection were measured by incubation
with peptides representing two immunodominant epitopes of IAVs in
C57BL/6J mice (H2-b), PA224–233 and NP366–374 [23,49]. The
peptides of the PA224–233 epitope of influenza A virus were
manufactured at Eurogentec (Seraing, Belgium), while peptides of
the NP366–374 epitope were manufactured at Sanquin Research
(Amsterdam, The Netherlands). Four hundred thousand splenocytes
were cultured for 6 h at 37uC in the presence of 5 mM of either the
NP366–374 ASNENMDAM (NPHK), PA224–233 SCLENFRAYV
(PAHK) peptides derived from IAV HK/68 or the NP366–374
ASNENMEVM (NPIND) or SSLENFRAYV (PAIND) peptides
(derived from IAV IND/05) in IMDM (Lonza, Breda, The
Netherlands) with 5% FCS and Golgistop (BD). After incubation,
cells were o/n stored at 4uC, stained with monoclonal antibody
permeabilized with Cytofix and Cytoperm and stained with
Pharmingen, Alphen a/d Rijn, The Netherlands). Data were
acquired using a FACSCalibur and analyzed with Cellquest Pro
Splenocytes were washed and stained
with mAbs CD3e-PerCP, CD8b.2-FITC (BD Pharmingen,
for IFN-c-PE (allfromBD
immunodominant NP366–374 epitope derived from IAV X-31
ASNENMETM (TmX-31) or IAV HK/68 ASNENMDAM
(TmHK) or the APC labeled tetramer derived from IAV IND/05
NP366–374ASNENMEVM (TmIND). All tetramers were purchased
from Sanquin Research, Amsterdam, The Netherlands. Following
incubation with tetramers and mAbs for 20 minutes, cells were
washed twice and analysed by flow cytometry using a FACSCanto
in combination with FACS Diva software (BD).
Histopathology and immunohistochemistry
After euthanasia, lungs of mice were inflated with 10% neutral
buffered formalin. After fixation and embedding in paraffin, lungs
were sectioned at 4 mm and tissue sections were examined by
staining for hematoxylin and eosin (HE). Using an immunoperox-
idase method, sequential slides were also stained with a monoclonal
antibody directed against the nucleoprotein of IAV .
Data for weight loss after infection, viral load in the lungs,
tetramerstaining, and peptide pulsing were analyzed statistically
using the two-sided student’s T test. Survival was analyzed using
the Logrank test. Differences were considered significant at
The authors wish to thank Theo Bestebroer, Peter van Run and Pascal
Lexmond for excellent technical assistance.
Conceived and designed the experiments: RB GFR. Performed the
experiments: RB JK CB MMGM GdM GvA. Analyzed the data: RB
JMAvdB GFR. Wrote the paper: RB RAMF ADMEO GFR.
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