﻿Single HA2 Mutation Increases the Infectivity and
Immunogenicity of a Live Attenuated H5N1 Intranasal
Influenza Vaccine Candidate Lacking NS1
Brigitte M. Krenn1.
, Andrej Egorov1.
*, Ekaterina Romanovskaya-Romanko2
, Markus Wolschek1
,
Sabine Nakowitsch1
, Tanja Ruthsatz1
, Bettina Kiefmann1
, Alexander Morokutti1
, Johannes Humer1
,
Janina Geiler5
, Jindrich Cinatl5
, Martin Michaelis5
, Nina Wressnigg1
, Sanda Sturlan1
, Boris Ferko1
, Oleg V.
Batishchev3
, Andrey V. Indenbom3
, Rong Zhu4
, Markus Kastner4
, Peter Hinterdorfer4
, Oleg Kiselev2
,
Thomas Muster1
, Julia Romanova1
1 Avir Green Hills Biotechnology AG, Vienna, Austria, 2 Influenza Research Institute, Russian Academy of Medical Sciences, St. Petersburg, Russia, 3 A.N. Frumkin Institute
of Physical Chemistry and Electrochemistry of Russian Academy of Sciences (RAS), Moscow, Russia, 4 Christian Doppler Laboratory of Nanoscopic Methods in Biophysics,
Institute for Biophysics, Johannes Kepler University Linz, Linz, Austria, 5 Institute for Medical Virology, Johann Wolfgang Goethe University, Frankfurt, Germany
Abstract
Background: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to
seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza
viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian
influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts
to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the
acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for
mammals.
Methodology/Principal Findings: We demonstrate that original human influenza viruses infect primary human nasal
epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH#5.6. The HA of A/Vietnam/1203/04
was modified by introducing the single substitution HA2 58KRI, decreasing the pH of the HA conformational change. The
H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature
treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by
immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the
reassortants keeping 58KRI mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced
superior systemic and local antibody response after the intranasal immunization of mice.
Conclusion/Significance: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may
require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal
epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a
substantial role in the infectivity of HPAIVs for mammals.
Citation: Krenn BM, Egorov A, Romanovskaya-Romanko E, Wolschek M, Nakowitsch S, et al. (2011) Single HA2 Mutation Increases the Infectivity and
Immunogenicity of a Live Attenuated H5N1 Intranasal Influenza Vaccine Candidate Lacking NS1. PLoS ONE 6(4): e18577. doi:10.1371/journal.pone.0018577
Editor: Andrew Pekosz, Johns Hopkins University - Bloomberg School of Public Health, United States of America
Received November 17, 2010; Accepted March 6, 2011; Published April 7, 2011
Copyright: ß 2011 Krenn 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 has been partially funded by the European Commission's 6th Framework Program projects ``Intranasal H5 Vaccine'' (SP5B-CT-2007-044512)
and ``FLUVACC'' (LSHB-CT-2005-518281). Costs not covered by the European Grants are funded by AVIR Green Hills Biotechnology AG (AGHB) and in part by
Russian Foundation for Basic Researches (project #08-03-00971), Program of Presidium of RAS ``Molecular and Cell Biology,'' Federal Task Program ``Scientific and
Scientific-Pedagogic Personnel of Innovative Russia'' for 2009-2013 (state contract #P337) and Austrian Institute for Biophysics, Christian Doppler Laboratory of
Nanoscopic Methods in Biophysics (Johannes Kepler University Linz). No additional external funding was received for this study. AGHB performed the study
design, data collection and analysis, decision to publish, and preparation of the manuscript.
Competing Interests: AVIR Green Hills Biotechnology (AGHB) is a privately held biopharmaceutical company based in Vienna (Austria) that draws on its own
knowledge in virology to develop and commercialize an intranasal live attenuated vaccine targeting pandemic influenza disease among other products in the
pipeline. BMK, AE, MW, SN, TR, BK, AM, NW, SS, BF, JH, TM, and RJ were employees of AGHB at the time of study. IPR rights related to this manuscript are covered
by the following IP rights: DelNs1 mutants are covered by patents from AGHB and Mount Sinai School of Medicine. Patent applications covering the technology
platform of the influenza virus and virus amplification procedure are fully owned by AGHB. In adherence to the PLoS guidelines, AGHB will make freely available
any materials and information described in the publication that are reasonably requested by others for the purpose of academic, non-commercial research.
* E-mail: a.egorov@greenhillsbiotech.com
. These authors contributed equally to this work.
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Introduction
An unprecedented spread of highly pathogenic avian influenza
viruses (HPAIV) of the H5N1 subtype was observed among wild
and domestic birds throughout the last decade. Hundreds of cases
of the direct transmission of avian viruses to humans with a case
fatality rate exceeding 50% raised great concerns of a possible new
pandemic.
Numerous clinical studies with vaccines produced from H5N1
viruses have demonstrated that the inactivated vaccines produced
from the H5 hemagglutinin (HA) appeared to be poorly
immunogenic compared to seasonal influenza strains [1,2]. A
broader and longer lasting immunity might be induced by live
attenuated influenza vaccines, which are believed to be superior to
inactivated vaccines [3,4]. However, H5N1 cold adapted vaccine
strains comprising surface antigens derived from A/Vietnam/
1203/04 (VN1203) or A/Hong Kong/213/03 lacked replication
in the human nasal mucosa, correlating with the observed poor
immunological outcome [5].
The effectiveness of intranasal live attenuated influenza vaccines
is substantially dependent on the efficient virus uptake and
subsequent replication in the cells of the upper respiratory tract.
Human influenza viruses are known to attach predominantly to
the surface of ciliated epithelial cells in the human trachea,
bronchi, and bronchioles while avian H5N1 viruses prefer the
lower respiratory tract, in turn binding more abundantly to the
alveoli [6]. This could be explained by the preferential affinity of
H5 HA to sialic acid receptors with an a2,3 galactose (a2,3Gal)
linkage dominating on the cells of the lower respiratory tract, but
not to the a2,6Gal type, which is abundantly present in the human
trachea [7,8]. However, in spite of the difference in the receptor
specificity, it was demonstrated that H5N1 viruses are able to
infect ex vivo cultures of human nasopharyngeal, adenoid, and
tonsillar tissues [9]. Consistently, another live attenuated H5
vaccine candidate comprising the HA of the low pathogenic avian
influenza virus (LPAIV) A/duck/Potsdam/86/92 (H5N3) was
shown to replicate efficiently in the human upper respiratory tract
(for at least 11 days) [10,11]. Therefore, the receptor specificity
properties of influenza surface glycoprotein might not be the only
responsible reason for the low infectivity of avian viruses in
humans.
Unlike isolated epithelial cells in vitro, the human airway luminal
surface in vivo presents a significant extracellular barrier to
influenza infection. It includes the mucociliary clearance system,
viscous fluids, and macrophages interfering with the virus access to
the cell surface. In addition, the measurement of the pH of the
human nasal cavity revealed that the overall pH range at the
anterior and posterior sites is 5.2­8.0 [12­15]. Acidic pH, heat, or
chemical denaturants are known to promote the conformational
change of HA into its fusogenic form, which is responsible for the
complete viral inactivation [16]. Therefore, in order to overcome
the human mucosal barrier, influenza viruses require a certain
level of HA stability towards inactivating factors.
It was described that human and LPAIVs are relatively
resistant to an acidic pH environment because they perform the
pH dependent conformational change of the HA at a pH range of
5.1 to 5.4 [17]. In contrast, the HAs of H5 and H7 HPAIVs are
much more sensitive undergoing conformational modification
already at a pH of 5.6 to 6.0 [18]. By the mutagenesis of viral
HA, Reed et al. succeeded in increasing the environmental
stability of an HPAIV H5N1 strain (A/Chicken/Vietnam/C58/
04) through the reduction of the pH of HA activation [19]. The
obtained mutant demonstrated reduced virulence and transmis-
sibility in wild ducks.
We hypothesized that the poor infectivity of HPAIVs for
mammals and, as a consequence, the insufficient efficacy of live
attenuated H5 vaccines is related to the low stability of their HA
protein. In order to prove our hypothesis, we constructed two
H5N1 live attenuated vaccine candidates lacking NS1 based on
the VN1203 virus, which differ in a single amino acid substitution
58KRI in the HA2 subunit [20]. Reed et al. described this
mutation as decreasing the threshold pH of the activation of the
H5 HA protein of approx. 0.5 pH units [21]. The obtained viruses
were compared for their stability in vitro and for the infectivity and
immunogenicity after intranasal application in vivo in mice.
We demonstrated that the stabilization of the HA molecule
towards acidic pH and high temperature promotes superior
vaccine virus infectivity in the respiratory tract and, consequently,
increased immunogenicity in mice.
Results
HPAIVs but not human seasonal viruses lose their
infectious activity at an acidic pH in primary Human
Nasal Epithelial cells (HNEpC)
The acidification machinery of the human nasal passageway is
known to release protons in response to irritation or inflammation
[22] acidifying the mucosal surface (down to pH of 5.2).
Therefore, viruses must possess a certain level of stability to low
pH in order to infect the upper respiratory tract. We analyzed
whether human seasonal virus isolates differ from HPAIVs in
infectious activity at an acidic pH. Human viruses A/Brisbane/
59/2007 (H1N1) (BN/59/07), A/Vienna/25/07 (H3N2) (VI/25/
07), A/St. Petersburg/14/10 (H1N1v) (SP/14/10) and HPAIVs
VN1203 (H5N1) and A/Thailand/01/04 (H5N1) (TH/01/04) of
an early passage level adjusted to a similar multiplicity of infection
(moi) were admixed with acidic (pH 5.4 or/and 5.6) or neutral
(pH 7.4) buffer followed by the inoculation of the HNEpCs. After
removing the inoculum, the cells were incubated at a neutral pH
for 5 hours without trypsin. The effectiveness of the infection was
visualized by influenza nucleoprotein (NP) staining (Fig. 1). All
human influenza viruses, including the recently appeared
pandemic H1N1v virus, were capable of infecting cells at acidic
conditions (Fig. 1A), while both highly pathogenic H5N1 viruses
infected HNEpCs only at a neutral pH (Fig. 1B). A similar pH
dependence of infection was observed in Vero cells (results are not
shown).
Generation of H5N1 viruses with 58KRI substitution in
the HA2 subunit
To investigate whether the infectivity and, consequently, the
immunogenicity of H5N1 viruses might be improved by
decreasing the threshold pH of HA fusion to the range described
for human influenza viruses, we modified the HA protein by
implementing the 58KRI substitution in the HA2 subunit of the
VN1203 virus, according to Reed et al. [21]. This mutation
located in the HA2 coiled-coil domain was shown to decrease the
pH of the activation of the H5 HA protein. An additional attempt
failed to rescue a corresponding virus comprising HA2 23GRC
modification, which is described to decrease the pH of the fusion
of H7N7 viruses [23].
Increased resistance of influenza HA to an acidic pH is known
to be linked to a decreased isoelectric point (pI) of the HA2 protein
subunit, the pH value at which the protein charge is neutral [24].
The calculated value pI = 4.59 for the mutated HA2 subunit was
lower than that of the natural VN1203 HA2 subunit (pI = 4.67),
supporting the assumption of the enhanced resistance of the
mutated HA to acidic pH (http://isoelectric.ovh.org/EMBOSS).
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The reassortant virus containing mutation HA2 58KRI and the
deletion of the NS1 ORF was generated by the eight-plasmid
rescue method in Vero cells analogously to the previously
described VN1203DNS1 virus [20] and was named
VN1203DNS1-K58I. Both viruses comprised an HA with a
polybasic cleavage site modified in a trypsin dependent manner
[25]. In addition, a corresponding pair of reassortants with a
complete NS segment was generated in the same way and named
VN1203wtNS and VN1203wtNS-K58I, respectively.
Mutation HA2 58KRI decreases the pH threshold of HA
fusion and increases the virus resistance to acidic pH and
high temperature
To prove the impact of the introduced mutation on the pH
optimum of HA fusion activity, we analyzed the reassortants
VN1203DNS1 and VN1203DNS1-K58I in a fusion assay. The
original virus had the capacity to trigger a fusion at pH
values#5.6, whereas the same fusion rate for the mutant virus
was observed at pH#5.3 (Fig. 2). It is noteworthy that the mutant
H5N1 virus VN1203DNS1-K58I showed the same activation
profile of HA as observed for the human seasonal virus A/
Solomon Island/03/06 (H1N1) (SL/03/06).
The result of the HA fusion assay was confirmed by the atomic
force microscopy (AFM) technique allowing visualization of viral
Figure 1. Infectivity of human and highly pathogenic avian viruses at an acidic pH in HNEpCs. Primary Human Nasal Epithelial cells were
infected with human (A) epidemic BN/59/07 (H1N1), VI/25/07 (H3N2), SP/14/10 (H1N1v) or avian (B) highly pathogenic viruses VN/1203 (H5N1) and
TH/01/04 (H5N1) at the indicated pH values. Influenza NP protein was visualized by immunostaining after incubating for 5 h.
doi:10.1371/journal.pone.0018577.g001
Figure 2. Hemolytic activity of influenza viruses as a function
of pH. Viruses VN1203DNS1, VN1203DNS1-K58I, and seasonal A/SL/03/
06 (H1N1) standardized to 128 HA units were mixed with 1%
suspension of human erythrocytes at 0uC and pH 7.4 for 1 h and then
the buffer was replaced with MES buffer at various pH values. After
incubation at 37uC for 1 h hemolysis representing the fusion activity
was determined spectrophotometrically at 405 nm. Error bars represent
the standard deviation from triplicate experiments.
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interaction with the lipid membrane. Unlike the electron
microscopy, this technique enables the imaging of biological
samples in a natural fluid environment with atomic resolution.
Virus particles were adsorbed on a supported bilayer lipid
membrane (sBLM) containing GD1a ganglioside, which served
as a receptor for the influenza virus. The conversion of the
conformation of HA at the threshold pH value leading to the
activation of the fusion of the virus envelope with sBLM was
registered by changing the domain structure of the lipid bilayer.
We found that the HA conformational change of VN1203DNS1
occurred already at pH 5.8, whereas the VN1203DNS1-K58I
mutant was stable under these conditions. The image confirming
the conformational change of the mutant virus was observed at
pH 5.0 (detailed data are presented as supporting information,
Text S1, Fig.S1).
Next, we investigated whether the virus VN1203DNS1-K58I,
which was more resistant to an acidic pH, was also more stable at
an increased temperature. VN1203DNS1 and VN1203DNS1-
K58I viruses were incubated at a temperature ranging from 46uC
to 58uC for 30 min with the subsequent measurement of the
remaining infectious and hemagglutination titers. We found that
the infectivity of the VN1203DNS1 virus was impaired already at
50uC, as indicated by a drop in the infectious titer more than 100
times. At the same time, this temperature did not affect the
infectious titer of the mutant virus (Fig. 3). After incubation at
52uC, the VN1203DNS1 virus completely lost the infectious titer,
whereas the titer of the mutant virus VN1203DNS1-K58I
decreased by 1.0 log10 TCID50/ml. Analogous results were
obtained by assessing HA titers indicating improved temperature
stability of mutated surface glycoprotein.
Mutation HA2 58KRI preserves virus infectivity at an
acidic pH in cell culture
The infectivity of reassortants VN1203DNS1 and VN1203DNS1-
K58I under acidic conditions was compared in Vero cells at pH 5.4,
5.6, 5.8, or 7.4. The human BN/59/07 virus was taken as a control
sample resembling a pH stable phenotype. As is shown in Fig. 4A, the
mutant virus VN1203DNS1-K58I gained a resistance to low pH
infecting Vero cells at pH 5.6, whereas the limit of infection was at
pH 5.8 for the original virus VN1203DNS1. These results were
confirmed on MDCK cells estimated by the flow cytometry analysis
under similar conditions (Fig. 4B). As expected, the human BN/59/
07 (H1N1) virus was infectious on Vero cells at pH 5.4 and infected
32% of the MDCK cells even at pH 5.2 (Fig. 4C).
Mutation HA2 58KRI increases virus sensitivity to
lysosomotropic agents
Avian influenza viruses in contrast to human strains are more
resistant to the prevention of endosomal acidification mediated by
lysosomotropic agents, such as chloroquine and NH4Cl [24].
Therefore, we compared the infectivity of both viruses in the
presence of chloroquine or NH4Cl (Fig. 5). As a control, the
seasonal influenza strain BN/59/07 (H1N1) was included in the
study. The infection of Vero cells with VN1203DNS1-K58I was
decreased by 10 mM chloroquine and completely inhibited by
50 mM. In contrast, the growth of the original virus was hardly
affected even at 50 mM chloroquine. BN/59/07 (H1N1) was most
sensitive to chloroquine as the infection was reduced by 1 mM.
These results were confirmed by another lysosomotropic agent
(1 mM NH4Cl) when the infectivity of the VN1203DNS1-K58I or
BN/59/07 (H1N1) viruses was almost lost, whereas the
VN1203DNS1 virus was not affected. Thus, the mutation HA2
58KRI converted the phenotype of the avian influenza virus to be
more similar to that of the human isolate.
Mutation HA2 58KRI decreases the replication efficiency
of H5N1 viruses in cell culture
To elucidate whether the changed stability of HA to an acidic
pH has any influence on the replication efficiency in various cell
lines, we compared the viral growth capacity in Vero, MDCK,
A549 (human lung carcinoma cell), and in HBE (human bronchial
epithelial) cells. As the DNS1 viruses are replication deficient in
interferon competent cell lines (all mentioned above except Vero),
two similar viruses expressing functional NS1 protein
VN1203wtNS and VN1203wtNS-K58I were used in these
experiments. Similar results of stability towards an acidic pH
and elevated temperature were obtained for these viruses (results
are not shown). Growth curves made for each cell line revealed
that the introduced mutation HA2 58KRI impaired the yield of
the mutant virus VN1203wtNS-K58I in HBE cells of about 150
Figure 3. Effect of elevated temperature on the viral HA and infectious titer. Virus preparations were incubated at indicated temperatures
for 30 min. HA titers (bars, left Y-axis) and infectious titers measured as log10 TCID50 (graphs, right Y-axis) were determined for VN1203DNS1 (black
bars and lines) and VN1203DNS1-K58I (gray bars and lines) viruses. The lower limit of detection for the TCID50 titer is 1.5 log10 TCID50/ml indicated by
the horizontal dashed line.
doi:10.1371/journal.pone.0018577.g003
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times. A similar tendency was observed in Vero, MDCK, and
A549 cells (Fig. 6). Therefore, a mutation decreasing the pH
threshold of the HA conformational change may affect the growth
of H5N1 viruses in continuous cell lines.
Mutation HA2 58KRI increases virus infectivity in mice
To assess the relevance of the in vitro findings in animals, we
compared the virus infectivity in mice. First, we measured the
MID50 of viruses with mutant or original HA. The animals were
infected with VN1203wtNS or VN1203wtNS-K58I viruses in a
dose ranging from 2.5 to 4.5 log10 TCID50/animal. Nasal tissues
were collected 60 h after i.n. administration and the tissue
homogenates were investigated for the presence of infectious virus
by TCID50 assay. The mutant virus VN1203wtNS-K58I had an
approximately 25-fold lower MID50 (MID50 2.9 log10) compared
to that found for the original virus (MID50 4.3 log10) (Fig. 7). This
result was consistent in two independent experiments.
To compare the extent of the uptake of the original and mutated
viruses, the animals were infected i.n. with live attenuated
replication deficient VN1203DNS1 or VN1203DNS1-K58I vaccine
candidates to ensure single round infection at a dose of 6 log10
TCID50/animal. A qualitative assessment of infected foci was
carried out by the immunohistochemistry of the sections obtained
from the nasal mucosa, trachea, and lung tissues stained with anti-
influenza NP primary antibody. The mutant VN1203DNS1-K58I
virus increased the extent of the infection of the alveolar regions of
lungs, trachea and nasal mucosa, where more foci of the infection
were seen for all 5 immunized animals when compared to the
original VN1203DNS1 virus (Fig. 8). Thus, the introduced mutation
promoted more intensive viral uptake in the mouse respiratory tract.
Figure 4. Infectivity of viruses at different pH values in tissue culture. (A) Virus VN1203DNS1 or VN1203DNS1-K58I was mixed with MES
buffer at indicated pH values and used for infection of Vero cells at moi 2. Influenza NP was visualized by immunostaining after incubating for 5 h. (B)
MDCK cells were infected with VN1203DNS1 or VN1203DNS1-K58I viruses at indicated pH values. The number of infected cells was determined after
the immunostaining of viral NP (5 h p.i.) by flow cytometry. The indicated numbers show the percentage of infected cells. (C) Vero or MDCK cells were
infected with seasonal human influenza virus BN/59/07 (H1N1) at indicated pH values. Virus infectivity was analyzed analogously by immunostaining
or by flow cytometry.
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Figure 5. Viral sensitivity to the lysosomotropic agents chloroquine or NH4Cl. Pretreated Vero cells were infected with VN1203DNS1,
VN1203DNS1-K58I, or seasonal BN/59/07 (H1N1) virus at moi 5 in a medium supplemented with chloroquine or NH4Cl at indicated concentrations.
The infected cells were visualized by the immunostaining of influenza NP (5 h p.i.).
doi:10.1371/journal.pone.0018577.g005
Figure 6. Growth curves on different cell lines. Vero (A), MDCK (B), A549 (C), or HBE (D) cells were infected with VN1203wtNS or VN1203wtNS-
K58I virus at moi 0.001. Supernatants were collected at indicated time points and virus infectious titers were determined by TCID50 assay on Vero
cells.
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Mutation HA2 58KRI contributes to increased virus
immunogenicity in mice
In a previous study, we noticed that vaccine candidate
VN1203DNS1 did not induce any HAI antibodies in mice in
contrast to macaques and ferrets, although still providing
protection against challenge viruses [20]. Therefore, we were
interested in whether modified VN1203DNS1-K58I virus is able
to evoke functional HAI or neutralizing antibodies in a mouse
model. A single i.n. vaccination with the VN1203DNS1 virus
induced detectable HAI titers only in 2 out of 6 animals
Figure 7. Viral growth capacity in the mouse upper respiratory tract. Mice were infected i.n. at doses of 2.5, 3.5, or 4.5 log10 TCID50/animal of
VN1203wtNS or VN1203wtNS-K58I virus. The presence of the virus was determined in 10% w/v nasal tissue homogenates by TCID50 assay 60 h p.i.
The lower limit of detection for the TCID50 titer is 1.5 log10 TCID50/ml indicated by the horizontal dashed line. The number of animals infected/total
number is indicated above the bars. Error bars represent the standard deviation.
doi:10.1371/journal.pone.0018577.g007
Figure 8. Viral infectivity in the mouse respiratory tract. Mice were infected i.n. with 6 log10 TCID50/animal of VN1203DNS1 or VN1203DNS1-
K58I virus or mock-infected with PBS. Nasal, tracheal, and lung tissues were examined 12 h p.i. for the presence of virus infected cells by the
immunostaining of influenza NP.
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(GMT = 6.3), whereas the administration of the mutant virus
VN1203DNS1-K58I induced a response in 5 animals (GMT = 25.4)
(Fig. 9A). An MNA test also revealed higher titers of neutralizing
antibodies induced by VN1203DNS1-K58I (GMT = 57) virus than
that induced by VN1203DNS1 (GMT = 27.9) (Fig. 9B). The
significant superior induction of specific antibodies by the mutated
vaccine virus was confirmed in an ELISA, where the IgG GMT
evoked by VN1203DNS1 was 3225 and by VN1203DNS1-K58I
was 8127 (Fig. 9C). The measurement of specific IgA antibodies in
pooled nasal washes of immunized mice indicated fourfold more
antibodies after immunization with the mutant virus (Fig. 9D).
Thus, a single immunization of mice with the VN1203DNS1-K58I
virus, containing the stability improved HA, induced a significantly
higher immune response as reflected by increased serum and
mucosal antibody titers.
Despite the observed differences in immunogenicity, both
VN1203DNS1 and VN1203DNS1-K58I viruses completely pre-
vented the replication of the homologous challenge virus
VN1203wtNS in the lungs of 3/5 and 4/5 animals, respectively,
taken 3 days post immunization, (data not shown) showing no
statistically significant difference.
Discussion
The effective spread of influenza viruses in the human
population may require a certain level of environmental stability
of the virus particles depending on a pathway of their transmission.
Three modes of infection with influenza viruses in people have
been postulated, including aerosol transmission, infection with
large droplets, and self-inoculation of the nasal mucosa by
contaminated hands [26]. Although, these routes of infection are
not mutually exclusive, effective infection via the nasal route could
be dependent on the pH stability of the virus particles due to the
mechanisms of the fast acidification of human nasal mucosal
surfaces [22].
Similar to the previous observation [27], we found that human
epidemic influenza viruses, irrespective of their subtype (H1, H1v,
H3), are more resistant to physical factors such as low pH (5.4­5.6)
or elevated temperature exposure than highly pathogenic H5N1
influenza strains. It should be mentioned that the pH stability
phenotype might be abrogated upon passaging in tissue culture
[28]. Therefore, the virus isolates of the early passage level were
used in this study. Contrary to human strains, HPAIVs and their
derivates with a modified HA cleavage site were not infectious at
pH#5.6.
We hypothesize that this relatively low pH stability of HPAIVs
viruses might be a limiting factor for infectivity in the mammalian
upper respiratory tract particularly in the nasal compartment. To
prove the hypothesis we introduced a known stabilizing mutation
58KRI into HA2 subunit of the VN1203 H5N1 virus and
compared the properties of the obtained viruses in vitro and in vivo.
We revealed that the in vitro parameters, such as the pH threshold
Figure 9. Antibody titers after single immunization of mice. Mice were vaccinated i.n. with 6 log10 TCID50/animal of VN1203DNS1 or
VN1203DNS1-K58I virus. Sera (collected 4 weeks p.i.) were analyzed for HAI (A), neutralizing (B) and for total IgG (C) antibodies measured by ELISA.
The titers of the individual animals (symbols) and the geometric mean titers (horizontal lines) are indicated. An undetectable antibody titer was
assigned a value of 4 (HAI; A) or 8 (MNA; B). (D) Pools of nasal washes (collected 4 weeks p.i.) were examined for the presence of homologous IgA
antibodies. * indicates p,0.05 determined by a t-Students test.
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of the HA conformational change, thermo-stability of HA protein,
and virus sensitivity to drugs preventing the endosomal acidifica-
tion of mutated virus, correspond (although not to the full extent)
to those of the human influenza isolates. Moreover, by applying
the FMA technique, we could reveal that the conformational
change of the original, but not the mutated HA, occurs already at
pH 5.8.
Interestingly, the introduction of the 58KRI substitution
impaired the viral growth of the pH stable mutant in HBE cells.
A similar finding was made for the HA2 G23RC mutant of A/
Nederlands/219/03 (H7N2) virus with a decreased pH of HA
activation showing a reduced replication in MDCK cells [23].
Thus, decreasing the pH value of HA activation to the level of
human viruses might not be beneficial for growth in tissue culture.
These data are consistent with the previous observation showing
that the adaptation of human influenza viruses to high growth in
MDCK cells was accompanied with the appearance of mutations
that elevate the pH of HA membrane fusion [28].
In contrast to the tissue culture results, the intranasal infection
of mice revealed that the pH stable variant was more infectious in
the mouse respiratory tract, including nasal mucosa, trachea, and
lungs, as demonstrated by immunohistochemistry. Such a
difference could not be explained by the superior replication
capacity of the mutant virus, since NS1 protein deletion abrogated
the multicycle replication. [20,29].
For an analogous pair of viruses comprising a functional NS1,
the HA2 modification diminished the MID50 25 times compared
to the non-mutated strain. These results are in agreement with the
data of Ilyushina et al., showing that equivalent modifications of
the HA of an H7N7 virus decreased the mouse lethal dose 50 by 3
log10 units [23]. Reed et al. demonstrated, for a highly pathogenic
H5N1 strain, that the reduction of the pH of HA activation
increased the viral shedding from the trachea but not from the
cloaca of infected birds, indicating that the threshold pH of HA
conformational change could be important for host and/or tissue
tropism [19]. Consistently for better viral uptake, the
VN1203DNS1-K58I virus appeared to be more immunogenic in
mice, inducing significantly higher HAI and neutralizing antibody
titers as well as mucosal IgA antibodies. We observed a similar
regularity for seasonal influenza viruses in ferrets where the stable
virus induced an enhanced antibody response when compared to
the mutant having HA destabilizing mutation [30].
The acidification of the mucosal surfaces is one of the oldest
primary innate defense mechanisms in mammals against a variety
of pathogens. The degree of acidification depends on the
distribution of the submucosal glands responsible for the liquid
secretion and, therefore, might differ in various animal species
[31]. There is evidence that mouse epithelium cell composition
and ion transport traits are similar to that of the human
conducting airways [32,33]. The nasal epithelia of humans and
mice are abundantly supplied with qualitatively similar glands
[34]. The pH of the human nasal mucosa in healthy adults is
known to be slightly acidic [12­15]. Moreover, the human nasal
airway epithelium starts to release acid in reaction to organic dust
or inflammation [22,34]. In combination with the receptor
specificity factor, the human nasal compartment might be
suboptimal for an efficient infection by HPAI viruses, hence
requiring a low respiratory tract delivery by aerosols (reviewed in
[26]). In this regard, mutations decreasing the pH of HA
conformational change and, therefore, stabilizing the HA of H5
HPAIVs might be a prerequisite for their efficient spread in
humans via a large droplet mechanism infecting the upper
respiratory tract (nose). A relatively high pH of HA activation
(ranging from 5.6 to 5.9) was described as being essential for
infectivity, shedding, and transmission in wild ducks for HPAIVs
[19]. However, for the efficient infection of mice and probably
other mammals including humans, we found that a lower pH
optimum of the HA activation might be required.
Interestingly, the mutation HA2 K58RI was first described as
stabilizing the HA towards a low pH and thereby conferring
resistance to the antiviral substance amantadine in a variant of the
HPAI virus Rostock (H7N1) [35]. Amantadine, besides for
inhibiting virus uncoating during the entry step, also prevents
the release of infectious influenza virus particles comprising an HA
with a polybasic cleavage site [36]. As our results indicate that the
mutation HA2 K58RI increases the infectivity of H5 viruses for
mice, the use of amantadine in clinics, or especially in farming,
could support the appearance of H5 HPAIVs more infectious for
mammals [37].
It is noteworthy that LPAIVs (H5N1) are known to have a pH
range of HA activation similar to that of human influenza viruses
and, therefore, are extremely stable at a low pH [18]. This
property is essential for the efficient replication of viruses in the
low-pH intestinal tract, which is the main site of replication for
these viruses. In addition, LPAIVs circulate in wild birds and
transmit through the fecal-oral route, requiring virus stability in
fresh water, usually with a pH of around 6.0 [38]. In contrast,
HPAIVs circulate mostly in domestic poultry and are excreted
mainly from the trachea (upper respiratory tract), rather than the
cloaca, and they do not persist very long in water [39].
Theoretically, stable LPAIVs might be potentially more infectious
for the human upper respiratory tract. However, other factors
such as the incompatibility of the avian virus polymerase complex
with mammalian host factors or the temperature optimum of the
polymerase activity were shown to restrict their efficient
replication in mammals [40­45]. In this regard, it seems logical
that a live influenza H5N1vaccine reassortant virus comprising the
HA of the LPAIV A/Potsdam/1402-6/86 (H5N3) in combination
with the polymerase complex of a human cold-adapted influenza
virus was shown to replicate in the upper respiratory tract of
humans [11], whereas analogous vaccine candidates comprising
the HAs of VN1203 or A/Hong Kong/213/03 did not.
Intranasal vaccination with live attenuated influenza vaccines
implies the infection of the nasal mucosa with the large droplet
mode. Our findings suggest that an efficient intranasal vaccination
may require a certain level of pH and the temperature stability of
HA in order to achieve an optimal uptake of the attenuated virus
by the nasal epithelial cells. In this context, any mutation
destabilizing the HA appearing during vaccine construction or
production can severely affect vaccine immunogenicity. In
addition, since the nasal mucosa serves as the first target for large
droplet settling, we believe that the HA mutations involved in
modulating the pH and temperature stability, might be also
important for the virus transmissibility besides the mutations
regulating receptor binding specificity.
Materials and Methods
Cell culture
A Vero cell line was obtained from the European Collection of
Cell Cultures and was adapted and further cultivated at 37uC and
5% CO2 in a serum-free cultivation medium (SFM; Opti-pro
medium supplemented with 4 mM L-glutamine; Invitrogen).
Madin-Darby canine kidney (MDCK; ATCC CCL-34) cells were
cultivated at 37uC and 5% CO2 in DMEM medium (Invitrogen)
comprising 2% Fetal Bovine Serum (FBS, Invitrogen) and 2 mM
L-glutamine. Human bronchial epithelial 16HBE14o­
(HBE) cells
(obtained from J. Seipelt, Austria) were grown in MEM
Increased Infectivity of H5N1 Influenza Virus
PLoS ONE | www.plosone.org 9 April 2011 | Volume 6 | Issue 4 | e18577
(Invitrogen) supplemented with 10% FBS and 2 mM L-glutamine.
For the latter the dishes, were coated with 10 mg/ml BSA (Sigma),
30 mg/ml bovine collagen type I (Promocell), and 10 mg/ml human
fibronectin (BD Pharmingen) in Ham's F12 medium (HyClone).
The carcinoma human alveolar basal epithelial A549 cells (obtained
from J. Seipelt, Austria) were maintained in MEM supplemented
with 10% FBS and 2 mM L-glutamine. Human Nasal Epithelial
Cells (HNEpCs) were provided by PromoCell (Germany) and
cultivated according to the manufacture's procedure.
Viruses
The human influenza viruses A/Solomon Island/3/2006
(H1N1; NIBSC) (SL/03/06) and A/Brisbane/59/2007 (H1N1;
NIBSC) (BN/59/07) were propagated in the allantoic cavity of 9-
to 11-days old embryonated hen's eggs at 37uC. Allantoic fluids
were collected 48 h post infection (p.i.) and clarified by
centrifugation. The primary influenza isolate A/Vienna/25/07
(H3N2) (VI/25/07) (MDCK - derived, A/Wisconsin/67/05-like,
HA and NA GenBank accession numbers are JF340081 and
JF340082 respectively) was provided by the Austrian National
Reference Centre for Influenza (Institute of Virology, Vienna,
Austria). The primary influenza isolate A/St.Petersburg/14/10
(H1N1v) (SP/14/10, MDCK ­ derived, HA and NA GenBank
accession numbers are JF340083 and JF340084) was provided by
Influenza Research Institute (Saint Petersburg, Russia). Avian
influenza viruses A/Vietnam/1203/04 (H5N1) (VN1203)
(MDCK - derived, passage 3) and A/Thailand/01/04 (H5N1)
(TH/01/04) (MDCK - derived, passage 2) were provided by the
Institute for Medicinal Virology, Johann Wolfgang Goethe
University Frankfurt (Frankfurt, Germany). Viral stocks were
produced in MDCK cells in SFM supplemented with 5 mg/ml
porcine trypsin (Sigma) cultivated at 37uC and 5% CO2.
The generation of the reassortant virus VN1203DNS1 was
described previously [20]. Briefly, the 5:3 reassortant viruses
contain HA, NA, and M from VN1203 (H5N1) and the internal
protein genes from the WHO influenza vaccine strain IVR-116
(H1N1). The HA polybasic cleavage site as well as the NS1 open
reading frame were deleted. The virus VN1203DNS1-K58I differs
from VN1203DNS1 by a single mutation in the HA2 58KRI
introduced by site directed mutagenesis (Stratagene).
In addition, a corresponding couple of viruses was constructed
as 5:3 reassortants deriving the HA with a modified cleavage site as
described above, the M and the NA of influenza virus VN1203 in
combination with all other genes including the complete NS
segment of the IVR-116 strain and named VN1203wtNS or
VN1203wtNS-K58I (comprising the HA2 58KRI mutation).
For virus stocks, Vero cells were inoculated at a moi of 0.001 in
SFM or MES buffer (100 mM MES, 150 mM NaCl, 0.9 mM
CaCl2, 0.5 mM MgCl2; pH 5.6) and cultivated at 37uC and 5%
CO2 in SFM supplemented with 0.25 mg/ml Amphotericin B
(Amph B; Bristol-Myers Squibb) and 5 mg/ml porcine trypsin
(Sigma). Infectious virus titers were determined in Vero cells by
calculating the 50% tissue culture infectious doses per ml
(TCID50/ml) according to Reed and Muench [46].
Fusion assay
Viruses standardized to 128 HA units (HAU; determined
following standard procedure) per 50 ml were diluted 1:4 in a 1%
suspension of human erythrocytes (Siemens) and incubated on ice
for 1 h to allow virus binding. Then, the mixtures were pelleted at
72 g and the supernatants were removed. 100 ml of MES buffer at
various pH values (from 5.0 to 6.0) were added, followed by
incubating at 37uC for 1 h. After centrifugation (72 g), 50 ml of
supernatant were transferred to 96-well plates and the amount of
hemoglobin released by virus-cell fusion induced hemolysis was
determined by the measurement of optical density at 405 nm.
Reported results are the means 6 standard deviations (SD) of 3
replicates at the indicated pH value.
Effect of various pH values or lysosomotropic reagents
on virus infectivity in vitro
Semi-confluent Vero cells were infected with virus at moi 2.
Virus inoculum was prepared in MES buffer (0.25 mg/ml Amph
B) at the indicated pH value or in SFM supplemented with
0.25 mg/ml Amph B and chloroquine (Sigma) or NH4Cl at
indicated concentrations. It was applied to the cells at 37uC and
5% CO2 for 30 min. Then, the inoculum was replaced with SFM
and 5 h p.i. cells were fixed with 4% para-formaldehyde,
permeabilized with 1% Triton X100 (in PBS) and blocked (PBS
+ 1% BSA). Infected cells were stained with influenza anti-
nucleoprotein (NP) monoclonal antibody (Millipore, 1:5000 in
PBS + 1% BSA) followed by Alexa Fluor 488 conjugated anti-
mouse antibody (Invitrogen, 1:1000 in PBS + 1% BSA). Images
were taken on an Olympus CKX41 Fluorescence Microscope with
connected Olympus camera system E330.
Flow cytometry
Confluent MDCK cells were infected with virus at moi 2 in
MES buffer (0.25 mg/ml Amph B) at the indicated pH values or
mock-infected. 5 h p.i. cells were detached with 0.2% trypsin­
EDTA (Cellgro), fixed with Fix/Perm buffer (BD Cytofix/
Cytoperm Plus Kit), blocked in PBS (+1% FBS) overnight (o.n.),
permeabilized and stained with the FITC-labelled mAb against
NP (Imagen, UK). The percentage of NP-positive cells was
determined by flow cytometry using an Epics Elite XL-MCL flow
cytometer and EXPO 32 software (Coulter Immunotech, F).
Virus growth kinetics
To determine the growth kinetics, four different cells lines
(Vero, MDCK, A549, and HBE cells) were infected with
VN1203wtNS or VN1203wtNS-K58I at moi 0.001 in SFM (+
0.25 mg/ml Amph B). After incubating at 37uC and 5% CO2 for
30 min, the inoculum was removed and the cells were maintained
at 37uC 5% CO2 in SFM supplemented with 4 mM L-glutamine,
0.25 mg/ml Amph B, and 5 mg/ml porcine trypsin (Sigma).
Supernatants were collected 16, 24, 48, and 72 h p.i. and virus
titers were determined by TCID50 assay on Vero cells.
Infectivity in mice
Animal experiments were conducted in accordance with the
Declaration of Helsinki and approved by National Authorities:
One mouse study was approved by the Austrian regulatory
authorities (MA58/000351/2009/13). The other two studies were
done under the Russian Influenza Research Institutes Ethics
Committee that is registered at the US Department of Health and
Human Services under the IORG Number IORG0004322 Rsch
inst of Influenza IRB # 1.
Groups of 6 to 8 weeks old out bred female mice were infected
i.n. without narcosis with 20 ml of preparations of VN1203wtNS or
VN1203wtNS-K58I virus at a dose of 4.5, 3.5, or 2.5 log10
TCID50/animal. 60 h p.i. nasal tissues were collected and the
presence of infectious virus in 10% w/v tissue homogenates [in
SFM supplemented with 1% antibiotic-mix (Invitrogen), 25 mg/ml
gentamicin (Invitrogen)] was determined by TCID50 assay on
Vero cells. Mouse infectious dose 50 (MID50) titers were calculated
by using the method of Kerber and expressed as the TCID50 value
corresponding to 1 MID50 [47].
Increased Infectivity of H5N1 Influenza Virus
PLoS ONE | www.plosone.org 10 April 2011 | Volume 6 | Issue 4 | e18577
Histopathology
Groups of five 6 to 8 weeks old female BALB/c mice (Charles
River) were intranasally (i.n.) infected under ether narcosis with
50 ml of VN1203DNS1 or VN1203DNS1-K58I viruses at a dose of
6 log10 TCID50/animal. An additional group of four mice was
mock treated i.n. with 50 ml of PBS. 12 h p.i. mice were sacrificed
and tissues of the respiratory tract were fixed in 7% formalin and
paraffin embedded. Sections of the nasal epithelium, the trachea
and lungs were prepared and influenza infected cells were detected
with influenza anti-NP monoclonal antibody (1:5000 in PBS;
Millipore) followed by immunoperoxidase visualization (standard
protocol). Sections were counterstained with hemalum. This
animal study was approved by the Austrian regulatory authorities
(MA58/000351/2009/13).
Immunogenicity and protective efficacy in mice
Groups of 6 to 8 week old out bred female mice were
immunized i.n. under narcosis once with 50 ml of VN1203DNS1
or VN1203DNS1-K58I virus at a dose of 6 log10 TCID50/animal.
The control group was treated with PBS. 28 days post treatment,
sera and nasal washes were taken and analyzed for the presence of
vaccine strain specific antibodies by HAI assay, MNA, or ELISA.
To obtain nasal secretions, salivation was induced by i.p. injection
of 0.1 mg pilocarpine-HCl (Sigma). Concurrent small amounts of
nasal secretions in the nostrils were immediately absorbed with the
aid of sterile wicks and eluted in 50 ml of ice-cold sterile PBS for
4 h. Group-specific pools were made and stored at 220uC.
Treated animals were challenged on day 28 p.i. with 50 ml of
VN1203wtNS (4 log10 TCID50/animal) virus under ether narcosis.
On day 3 post challenge, 3 mice from each group were euthanized;
the lung tissues were collected. The viral load was determined in a
10% w/v tissue homogenate by TCID50 assay on Vero cells.
Detection of serum antibody titer by a hemagglutination
inhibition assay (HAI)
Sera from the mice immunized with VN1203DNS1 or
VN1203DNS1-K58I or mock-treated were diluted 1:4 with
Receptor Destroying Enzyme (RDE; Denka Seiken, Japan) and
incubated at 37uC o.n. Thereafter, samples were inactivated at
56uC for 30 min and serial twofold dilutions were prepared.
25 ml/well of the standardized antigen (4 HAU/25 ml) were
added. After incubating 1 h at RT, 50 ml of 1% horse erythrocytes
(+ 1% BSA, Sigma) were added and the plates were incubated at
RT for 1 h.
Detection of neutralizing antibody titer by a
microneutralization assay (MNA)
MNA was performed as previously described [20]. Briefly, serial
twofold dilutions of RDE-pre-treated sera were combined with
50 ml of a standardized viral suspension (100 TCID50/50 ml) and
incubated for 2 h at 37uC. Vero cells were added and incubated
for 20 h, washed, and acetone fixed. An influenza A virus NP-
specific monoclonal antibody conjugated with HRP (107L-Px,
4 mg/ml, Dr. E. Vareckova, Slovak Academy of Sciences) diluted
in a blocking buffer (PBS containing 0.5% I-Block and 0.1%
Tween-20) was added for 1 h. After adding the substrate (TMB,
KPL), the average absorption at 450 nm (A450) was determined for
the control wells of virus-infected (VC) and uninfected (CC) cells
and the neutralizing endpoint (NEP) was determined by using a
50% specific signal calculation.
NEP ~
average A450 of VC wells
ð Þ { average A450 of CC wells
ð Þ
½ 
2
z average A450 of CC wells
ð Þ
The endpoint titer was expressed as the reciprocal of the highest
dilution of serum with an A450 value less than NEP.
Detection of vaccine specific serum IgG and mucosal IgA
by ELISA
A modified enzyme-linked immunosorbent assay (ELISA)
protocol was performed as described previously [48,49]. Briefly,
UV-inactivated VN1203DNS1 virus (adjusted to 40 HAU/well in
carbonate buffer; pH 9.6) was used as a coating antigen. After
washing and blocking serially twofold diluted samples (serum for
IgG ELISA or group specific pools of nasal washes for mucosal
IgA ELISA) were added and incubated at RT for 1.5 h. H5-
specific IgG or IgA antibodies were detected with goat anti-mouse
IgG conjugated to HRP (0.25 mg/ml; KPL) or goat anti-mouse
IgA conjugated with AP (0.25 mg/ml; Rockland Immunochemi-
cals). Amount of IgG was determined by using TMB (KPL)
substrate and level of IgA by Lumi-Phos Plus substrate (Aureon
Biosystems). The cutoff values were defined as the mean value of
the negative control samples plus 3 standard deviations. H5-
specific IgG as well as IgA were presented in log2 titer.
Supporting Information
Figure S1 Virus stability to low pH determined by
atomic force microscopy (AFM). (A) Supported bilayer lipid
membrane (sBLM) on mica: phase and topography image.
Topography image of VN1203DNS1-K58I virus adsorbed on
sBLM at pH 6.5. (B) Phase images of VN1203DNS1 and
VN1203DNS1-K58I viruses on sBLM at pH 6.5­5.8­5.0. The
bright regions in the phase images correspond to the liquid
disordered domains in the lipid bilayer. (C) Change of the area
ratio of the bright and dark regions on the phase images of the two
different viruses. The error bars represent the standard deviation
from five measurements.
(TIF)
Text S1 The stability of the viruses VN1203DNS1 and
VN1203DNS1-K58I to low pH analyzed by AFM.
(DOC)
Acknowledgments
We would like to thank Nicole Ferstl and Andrea Triendl for their excellent
technical assistance. We would also like to thank Dr. Horst Fischer (CHORI,
Oakland) for his helpful discussions and reviewing the manuscript.
Moreover, we would like to acknowledge Dr. E. Vareckova of the Slovak
Academy of Sciences for providing the influenza A anti-NP antibody.
Author Contributions
Conceived and designed the experiments: BMK AE MW SN RZ PH OK
TM JC MM JR. Performed the experiments: BMK ER TR BK AM NW
SS BF OB AI MK JH JG. Analyzed the data: BMK AE RZ PH TM JR.
Contributed reagents/materials/analysis tools: ER TR SS RZ PH. Wrote
the paper: BMK AE JR OB AI.
Increased Infectivity of H5N1 Influenza Virus
PLoS ONE | www.plosone.org 11 April 2011 | Volume 6 | Issue 4 | e18577
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