A Rapid Flp-In System for Expression of Secreted H5N1
Influenza Hemagglutinin Vaccine Immunogen in
Hanxin Lu1, Surender Khurana1, Nitin Verma1, Jody Manischewitz1, Lisa King1, John H. Beigel2,
1Division of Viral Products, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Bethesda, Maryland, United States of America,
2Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, SAIC-Frederick, NCI-Frederick, Frederick,
Maryland, United States of America
Background: Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for
a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic
hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation
of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled
Methodology/Principal Findings: We have developed a Flp-In based 293 stable cell lines through targeted site-specific
recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular
domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both
proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal
antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon
rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/
2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/
2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine.
Conclusions/Significance: Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA
immunogens in mammalian cells from emerging influenza strains.
Citation: Lu H, Khurana S, Verma N, Manischewitz J, King L, et al. (2011) A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine
Immunogen in Mammalian Cells. PLoS ONE 6(2): e17297. doi:10.1371/journal.pone.0017297
Editor: Ralph Tripp, University of Georgia, United States of America
Received November 9, 2010; Accepted January 28, 2011; Published February 28, 2011
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This project was funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract no.
HHSN261200800001E and in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health. 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
The recent global spread of swine-origin H1N1 highlighted the
need for rapid development of effective vaccines against pandemic
influenza viruses. Much of our recent knowledge was derived from
studies with the highly pathogenic (HP) H5N1 avian influenza A
viruses (AIV) . The H5N1 viruses still cause severe human
disease, and may undergo adaptation for human-to-human
transmission. As of October 18, 2010 there have been 507 human
cases of H5N1 resulting in 302 deaths (fatality rate=59%) (http://
Production of hemagglutinin using recombinant technology
could overcome the constraints of traditional inactivated influenza
vaccine manufacturing that require several months for generation
of vaccine viruses using reassortment/reverse genetics, and
adaptation for high growth in eggs. Most of the influenza
protective antigenic sites are conformation dependent and map
primarily to HA1 globular head . In previous reports, codon-
optimized HA ectodomain with mutated cleavage site (to prevent
processing of HA1-HA2) and an added exogenous foldon
sequence at the C-terminus was expressed transiently in 293 cells
in order to produce stable oligomers [3,4,5]. Technologies that can
be easily translated into well controlled large scale manufacturing
process will have a great advantage. Thus far, various influenza
vaccine prototypes produced in a baculovirus-insect cell expression
system have undergone pre-clinical and clinical development
[6,7], but it is not well understood if the baculovirus produced HA
products are identical in terms of antigenicity and immunogenicity
to the egg grown or mammalian cells based vaccines.
Recombinant proteins produced from mammalian cells are
expected to have the same extent of posttranslational modifications
as egg grown influenza viruses. Transient transfection of
mammalian cells (often HEK293T cells) followed by selection of
stable tranfectants, usually result in random integration, clone-to-
PLoS ONE | www.plosone.org1 February 2011 | Volume 6 | Issue 2 | e17297
clone variability and upredicatable level of expression due to
position effects based on the site of integration in the host genome.
Therefore, one of the important parameters of recombinant
protein production for manufacturing is the ability to derive stable
mammalian cell lines with defined integration site(s) and
reproducible level of protein expression.
Previously, the Flp-In system has been used to express proteins
for basic research purposes and in one instance, to produce
monoclonal antibodies in CHO cells [8,9]. In the current study,
we have constructed the first vector system for HA protein
expression in mammalian cells using the FRT/FLP strategy to
overcome position effects and for rapid derivation of stable cell
lines expressing HA. As a proof-of-concept, we have cloned the
HA0 and HA1 proteins from the highly pathogenic H5N1, A/
Vietnam/1203/2004. No codon optimization or modifications to
the polybasic cleavage site were made in order to exactly match
the HA sequence of the transmitted avian influenza strain .
We provide data demonstrating that both proteins are properly
folded and can elicit homologous and heterologous H5N1
neutralizing antibodies in rabbits. Importantly, in the face of
impending pandemic, cloning, transfection, and stable cell line
generation can be done within 2–3 weeks.
Cloning of HA1 and HA0 from H5N1 A/Vietnam/1203/
2004 in 293Flp-In system
The Flp-In system, which offers a single targeted integration site
was found to be more efficient than the conventional transient
transfection/selection protocols. Based on a site-specific recombi-
nation strategy, a modified Flp-In system was successfully
developed to generate stably expressing cell lines. The recombi-
nation occurs at a single site and eliminates clonal variability.
Expression from the same integration site resulted in reproducible
stable levels of insert expression and protein secretion [10,11].
Therefore such a system is amenable to controlled manufacturing
with built in quality assurance steps.
To enhance HA protein expression and protein secretion from
the FRT-CMV vector, a RNA splicing sequence from HTLV-I
gene and a cassette containing NotI and PacI cloning sites was
introduced after the CMV promoter using Topo cloning system,
followed by a secretory signal peptide from IgG kappa chain
(Fig. 1A). The H5N1 A/Vietnam HA sequences coding for either
HA1 (1-330) or HA0 (1-500) were inserted into the vector as NotI-
PacI inserts. In the case of HA0, the polybasic cleavage site
between HA1 and HA2 was not removed or modified, to ensure
proper cleavage and folding of the secreted HA0 protein (Fig. 1B).
Stable cell clones were selected by growing in DMEM with
150 mg/ml of hygromycin for 10–14 days followed by expansion of
individual clones. The selected stable integrants were 100%
homogeneous, an important attribute of the Flp-in system. All 40
of selected single–cell clones expressed HA protein at similar
levels. For HA protein production, individual cell clones
expressing either HA1 globular domain (1-330) or HA0 (1-500)
were expanded in T175 flask in serum-free medium for 24 hours.
Expression of HA proteins from 293 Flp-In cell lysates (C) and
secreted proteins in supernatants (S) were resolved on SDS-PAGE
under reducing conditions and detected by Western blot using
anti-V5 MAb (Fig. 1C). As expected, under reducing conditions, a
significant portion of the cleaved HA0 was separated into HA1
and HA2, only the later reacted with the anti-V5 tag (in the C-
terminus of HA2) (Fig. 1C lanes 3, 4). Supernatants were collected
at 24, 48, 72 and 96 hours post culture splitting and were analyzed
in SDS PAGE followed by western blot with anti-HA1 polyclonal
sera. In both HA1 and HA0 expressing cells, the highest level of
HA secretion was observed at 24 and 48 hours following cell
splitting (Fig. 1D).
In subsequent experiments, H5N1 A/Vietnam/1203/2004
HA1 (1-330) protein and HA0 (1-500) proteins were affinity
purified from the 293 Flp-In cell culture supernatant collected
24 hours after culture in serum-free medium using HisTrap
Characterization of purified HA proteins from 293 Flp-In
Proper protein folding is critical for preservation of HA
antigenicity and immunogenicity. Since the majority of influenza
neutralizing antibodies recognizes conformational epitopes, we
used a panel of human H5N1 neutralizing antibodies generated
from B cells of H5N1 A/Vietnam/1203/2004 recovered individ-
uals, that recognized conformational dependent epitopes in HA1
domain of H5N1 A/Vietnam virus [12,13]. Steady-state binding
equilibrium analysis with conformation-dependent human H5N1
neutralizing MAb demonstrated that 293 Flp-In secreted H5N1
HA1 and HA0 proteins were properly folded (Fig. 2A). The
licensed inactivated subunit H5N1 vaccine (Sanofi Pasteur) was
used as positive controls (Fig. 2A). Similar binding was measured
with additional two human MAbs (data not shown).
To determine if the secreted HA1 and HA0 proteins purified
from Flp-In cells form higher order quaternary forms, they were
analyzed in SDS-PAGE under reducing vs. non-reducing
conditions. For comparison, subunit H5N1 vaccine (Sanofi
Pasteur) was also subjected to analysis. The Flp-In derived HA0
ran as a single band under non-reducing conditions, but was also
present in cleaved form that separated into HA1 and HA2 under
reducing conditions (Fig. 2B and 2C, lane 2). Unexpectedly, the
Flp-In derived HA1 (1-330) contained both a monomer band and
higher MW species under non-reducing conditions, (Fig. 2C, lane
1). Western blot confirmed that both bands are reactive with
rabbit anti HA sera. Subunit H5N1 HA vaccine ran primarily as
oligomers in non-reducing gel, which dissociated into HA1 and
HA2 forms under reducing conditions (Fig. 2B and 2C, lane 3).
To better decipher the quaternary forms in the Flp-In derived
HA1 and HA0, we subjected the purified HA proteins to Superdex
S-200 gel filtration chromatography. While the HA0 (1-500)
protein was predominantly monomeric (Fig. 3B), the HA1 (1-330)
protein contained monomers and higher MW oligomers (Fig. 3A).
In comparison, the subunit H5N1 HA vaccine contained
predominantly oligomers (Fig. 3C).
Flp-In derived proteins elicit H5N1-specific broadly
neutralizing antibodies in Rabbits
New Zealand rabbits were immunized thrice intra-muscularly at
21-days interval with 100 mg of purified HA1 and HA0 proteins
based on the currently recommended human dose of 90 mg HA
for the licensed unadjuvanted subunit H5N1 vaccine for pre-
pandemic use in US . Total binding to either HA1 or HA0 was
measured after each vaccination using SPR. At least two doses of
the recombinant HA proteins were required to elicit antibody
response, as previously described for the inactivated H5N1 vaccine
. Post-second and third vaccination sera from rabbits
immunized with either HA1 or HA0 derived from the 293 Flp-
In system bound with high avidity to both HA1 and HA0 proteins
(Fig. 4 A–D). In the microneutralization assays, the rabbit sera
after two immunizations with the H5N1 HA0 protein elicited very
significant neutralizing antibody titers against both homologous
(A/Vietnam/1203/2004, clade 1) and heterologous strains (A/
H5N1 HA Secretion from Stable Flp-In Cells
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Turkey/1/05, clade 2.2 and A/Anhui/1/05, clade 2.3.4). After
the third vaccination these titers further increased and cross
neutralization of H5N1 A/Indonesia/5/05 (clade 2.1) was also
observed (Fig. 4E). Interestingly, the HA1 globular domain protein
generated similar titers of neutralizing antibodies with the same
profile of cross-clade neutralizing activity as was observed for the
intact HA0 protein (two-fold difference in titers falls within assay
variability). Similar results were obtained with the licensed
inactivated H5N1 subunit vaccine administered to rabbits at
100 mg dose mixed with the TiterMax adjuvant. After the second
and third vaccination a titer of 2560 against A/Vitenam/1203/04
was observed with modest cross reactivity against A/Indonesia/
05/2005 (clade 2.1), A/Turkey/1/05 (clade 2.2), and A/Anhui/
1/05 (clade 2.3.4).
For comparison we measured neutralizing titers in the plasma of
humans enrolled in a clinical trial, using the monovalent
inactivated subunit A/Vietnam/1203/04 H5N1 vaccine (Sanofi
Pasteur) that has subsequently been licensed. As previously
described, increasing doses of vaccine (90, 120, 180 mg of HA),
were administered to study participants on days 0, 28, 56, and 84,
for a total of four vaccinations . We have expanded the
previously published microneutralization assays to include several
heterologous H5N1 strains from clade 2 sub lineages (Table 1).
Even after three immunizations the neutralization titers in humans
against the homologous strain were lower than what was observed
with the rabbit sera. In addition, minimal if any cross
neutralization of heterologous strains was measured irrespective
of the vaccine dose even after three immunizations (Table 1). The
differences in the vaccination outcome between rabbits and
humans with the inactivated H5N1 subunit vaccine may reflect
the lower body mass and/or the addition of the TiterMax (water
in oil) adjuvant. Several clinical trials demonstrated that oil-in-
water adjuvants such as MF59 and AS03 greatly enhanced the
immunogenicity of inactivated H5N1 vaccines in human popula-
These data confirmed that the 293 Flp-In derived proteins were
properly folded, presented relevant conformational epitopes, and
were immunogenic in rabbits. The ability to elicit cross
neutralizing antibodies against clade 2 viruses, including A/
Indonesia/5/2005 (clade 2.1) is very important, since it is
Fig. 1. Construction of expression vector for constitutively H5N1 HA secretion in Flp-In based mammalian expression system. (A) To
enhance HA protein expression from the FRT-CMV vector (Invitrogen), an RNA splicing sequence from HTLV-I gene and a cassette containing NotI and
PacI cloning sites was introduced after CMV promoter using Topo cloning system. (B) Schematic of HA1 (1-330) & HA0 (1-500) V5-His6tagged fusion
proteins expressed in Flp-In system. The H5N1/Vietnam HA sequence coding for either HA1 (1-330) or HA0 (1-500) was inserted into the vector as a
NotI-PacI insert. (C–D) Expression and purification of H5N1 A/Vietnam/1203/2004 hemagglutinin proteins from 293 Flp-In stable cell lines. (C)
Expression of HA proteins secreted into supernatant from 293 Flp-In cells [S], and in the cell lysates [C] was analyzed by western blot using anti-V5
MAb. (D) HA protein level from supernatant of 293 Flp-In cell culture in serum free medium, collected at different time points: 24, 48, 72 and 96 hours
post culture splitting and was analyzed in SDS PAGE followed by western blot with anti-HA1 polyclonal sera.
H5N1 HA Secretion from Stable Flp-In Cells
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unknown which H5N1 strain will adapt to bind better to the
human a2-6 sialic acid receptor, resulting in human to human
Recombinant protein technology may provide a rapid response
to emerging influenza infections with pandemic potential. The
current vaccine campaign is based on a traditional process that
depends on the derivation of reassortant viruses in mammalian
cells followed by adaptation to high growth in eggs, which may
result in mutations in the key antigenic sites. In addition, vaccine
potency reagents require repeat vaccinations of sheep with
bromelain cleaved HA from egg-grown vaccine strains. Therefore,
recombinant technology can alleviate some of the bottlenecks and
assist in production of early vaccines or potency reagents for
vaccine lot release. Transient expression or random integration
based cell lines are widely used for protein expression, but these
technologies have several significant shortcomings including time-
consuming, unpredictable and unreproducible levels of gene
expression primarily due to lack of control over the position of
integration. To that end, it will be important to use a certified
mammalian cell line with a built in genomic site specific
recombination cloning site and an optimized expression vector
that can be used within short time frame for cloning and selection
of stable integrants secreting properly folded HA proteins .
In the current study we provide the first data supportive of the
use of 293 Flp-In based stable mammalian system for influenza
hemagglutinin expression. The main findings were (a) Both HA1
and HA0 proteins from H5N1 HP A/Vietnam/1203/2004 were
successfully expressed in 293 Flp-in cells and stable integrants were
selected within 10-14 days; (b) the secreted HA1 and HA0 were
purified from the supernatants of cells growing in serum free
conditions and were shown to be properly folded as determined by
binding to conformation dependent human MAbs; (c) the HA0
with intact polybasic cleavage site contained cleaved (HA1 and
HA2) as well as uncleaved molecules (similar to HA on virions); (d)
rabbit vaccination with the HA1 and HA0 proteins resulted in
homologous virus-neutralizing antibodies with a significant cross
neutralizationof heterologous strains representing
transmitted H5N1 clades; and (e) sera from humans vaccinated
three times with the licensed H5N1 A/Vietnam/1203/2004
inactivated vaccine had lower titers against the homologous virus
and negligible neutralizing antibody titers against heterologous
H5N1 strains from multiple clades.
The use of site-directed recombinant technology for HA
production in stable mammalian expression cell lines eliminates
the handling of live highly pathogenic H5N1 viruses. It also
circumvents the need for generation of egg-adapted high growth
virus in the face of impending pandemic. High-level protein
expressing mammalian cell lines could be reproducibly construct-
ed based on the exchange of HA expression cassettes in a pre-
tagged desired locus of the engineered cell line by site-specific
Our data supports and extend the findings of Wei et al.  and
Bosch et al.  who used transient expression systems for
expression of HA proteins. In this study, both secreted HA1 and
HA0 expressed in the Flp-In system in the same integration site
and were purified from the culture supernatants. The proteins
were properly folded and were recognized by conformation
dependent H5N1 neutralizing human MAbs.
Following vaccination, the kinetics of HA-binding antibody
development (measured by SPR) paralleled the appearance of
potent broadly neutralizing antibodies confirming the immunoge-
nicity of Flp-in derived secreted HA1 and HA0 proteins. From the
rabbit studies, the Flp-In derived HA1 (1-330) protein seems as
immunogenic as intact HA0 (1-500) in generation of broadly
H5N1 neutralizing antibodies and might be a promising vaccine
Importantly, the neutralization titers and breadth of cross clade
neutralization observed in the current study using recombinant
HA proteins, were higher than the neutralization titers measured
in the sera of human volunteers vaccinated with escalating doses of
the currently licensed H5N1 inactivated vaccine. Suguitan et al.
previously demonstrated that restoring the polybasic cleavage site
in the H5 HA of the vaccine virus improved its immunogenicity
and efficacy, suggesting that removal of polybasic cleavage site due
Fig. 2. Characterization of purified HA proteins from 293 Flp-In
cell. (A) Proper protein folding as demonstrated by steady-state
binding equilibrium analysis of conformational dependent human
H5N1 neutralizing MAb FLA5.10 (10 mg/ml) to purified Flp-In expressed
H5N1 HA1 proteins immobilized on a sensor chip through the free
amine group, and onto a blank flow cell, free of peptide. Purified
mammalian cell derived H5N1 HA1 or the HA0 proteins obtained from
Immune Technology Corp were also analyzed. Binding was recorded
using ProteOn system surface plasmon resonance biosensor instrument
(BioRad Labs, Hercules, CA). (B–C) Analysis of purified H5N1 protein
from Flp-In cells in SDS-PAGE under reducing conditions (B) and non-
reducing conditions (C). Purified HA protein from Flp-In cell has higher
order protein structure as analyzed by coomassie staining of the
reducing SDS PAGE (B) and by coomassie stained non-reducing SDS
PAGE (C). Subunit H5N1 vaccine (Sanofi Pasteur) was run as comparator.
Western blot analysis of non-reducing SDS PAG using an anti-H5N1 HA1
antibody confirmed the identity of bands observed in coomassie
stained gel in Fig. 1C.
H5N1 HA Secretion from Stable Flp-In Cells
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to safety concerns, had a deleterious effect on the immunogenicity
and efficacy of the H5N1 HA . Therefore, it is plausible that
the HA0 with intact polybasic cleavage site expressed in Flp-In
system, which was cleaved into HA1 and HA2 forms provided
better immunogenicity than what has been earlier reported for HA
with mutated cleavage site .
In the face of impending pandemic, generation of vaccine in a
timely manner is critical. While adjuvants were shown to improve
the immunogenicity of inactivated vaccines against H5N1 and
SOIV-H1N1 [13,15,16,17], there is still a considerable time gap
between virus isolation and generation of reassorted vaccine seed,
as was evident during the recent SOIV-H1N1 pandemic.
Therefore, recombinant technologies may provide an important
early vaccine response alternative. The Flp-In system provides an
advantage over the transient transfection approach.
Based on these data it will be prudent to derive a 293 Flp-In
system that can be used for production of HA proteins for vaccine
generation as well as reagents that can be used for quality
assurance and vaccine release purposes. In addition to human
vaccines, poultry vaccination may become an important early
approach for curtailing the spread of newly emerging HP viruses
and their transmissionto susceptible
Materials and Methods
Cloning and protein expression
A pSecRTag vector was modified to incorporate a HTVL-I
splice site (R) using Topo cloning system (Invitrogen) into the
pSecTag vector (Invitrogen). This vector further modified that
incorporated the NotI and PacI cloning site between the splice site
and the V5-His6tag for cloning of desired genes (Fig. 1A). A
cDNA corresponding to residues HA1-330 or HA1-500 of the
hemagglutinin (HA) from A/Vietnam/1203/2004 (GenBank
accession no. GI:50296053) was cloned into the pSecRTag vector
using NotI and PacI cloning site. The expressed fusion protein
contains V5-tag and His6-tag residues at the C terminus
(GKPIPNPLLGLDSTRTGRTGHHHHHH), and IgK-chain se-
cretion signal peptide at the N terminus (Fig. 1B).
Protein expressing and purification
Plasmids expressing a secretory HA and a Flp recombinase were
transfected into the human embryonic kidney cell line 293 Flp-In
cell (Invitrogen, Carlsbad, CA) using lipofectamine 2000 reagent
(Invitrogen, Carlsbad, CA). At the day of transfection, DMEM
medium was replaced with fresh medium. One mg of HA
expression plasmid pSecRHA and 9 mg of pOG44 were co-
transfected using lipofectamine 2000 reagent. Twenty-four hours
after transfection, cells were grown in the fresh DMEM medium
containing 150 mg/ml of hygromycin. The medium was replaced
every three days. Ten days after selection, about 40 cell clones
were selected for protein expression analysis. For control, mock
transfected cells were all killed by hygromycin. Individual clones
were expanded and maintained in DMEM with hygromycin.
For HA protein production, cells expressing either HA(1-330) or
HA(1-500) were grown in T175 flask in serum-free medium and
incubated for 24 hours. Then HA was purified from the cell
supernatant by metal affinity chromatography using HisTrap
columns (GE Healthcare, Piscataway, NJ). Fractions containing
Fig. 3. Analysis of HA protein by gel filtration chromatography.
Superdex S-200 gel filtration chromatography of Flp-In derived H5N1
HA proteins. Purified H5N1 HA1 (1-330) protein (A) or the HA0 (1-500)
protein (B) were subjected to gel filtration. The panels present
superimposed elution profiles of purified HA proteins (blue line)
overlaid with calibration standards (grey line). The elution volumes of
protein species are shown in parenthesis. While purified HA 1-330
protein was presented in monomer, trimer and oligomer form (A), the
HA1-500 protein was observed primarily in a monomeric form (B). Gel
filtration profile of Subunit H5N1 vaccine (Sanofi Pasteur) (C) show
predominance of oligomers.
H5N1 HA Secretion from Stable Flp-In Cells
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Fig. 4. Development of neutralizing and anti-HA binding antibodies in rabbits following vaccination with H5N1 HA1 and HA0
proteins produced using Flp-In system. (A–D) Antibody kinetics following H5N1 HA1 & HA0 vaccination in rabbits. Steady-state binding
equilibrium analysis of pre & post-H5N1 HA1 immune sera (rabbit K8) in (A–B) or pre- & post-H5N1 HA0 immune sera (rabbit K9) in (C–D) to Flp-In
derived H5N1 HA1 and HA0 proteins were measured using SPR. Ten-fold diluted individual post-vaccinated sera from each time point, were injected
simultaneously onto recombinant Flp-In H5N1 HA1 in (A and C) and H5N1 HA0 in (B and D), immobilized on a sensor chip through the free amine
group, and onto a blank flow cell, free of peptide. Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument
H5N1 HA Secretion from Stable Flp-In Cells
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HA proteins were combined and desalted using PD10 column (GE
Healthcare, Piscataway, NJ). The expression of the HA proteins
was confirmed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under reducing and non-reducing
conditions and western blotting using a mouse monoclonal anti-
V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1
Gel filtration chromatography
Proteins at a concentration of 5 mg/ml were analyzed on
Superdex S200 XK 16/60 column (GE Healthcare, Piscataway,
NJ) pre-equilibrated with PBS, and the protein elution was
monitored at 280 nm. Protein molecular weight marker standards
(GE healthcare) were used for column calibration and generation
of standard curve to identify the molecular weights of the test
Protein conformation analysis by surface plasmon
Steady-state equilibrium binding of conformation dependent
human H5N1 neutralizing MAb FLA5.10 was monitored at 25uC
using a ProteOn surface plasmon resonance biosensor (XPR36,
BioRad Labs). The HA proteins were coupled to a GLC sensor
chip using amine coupling with 100 resonance units (RU) in the
test flow cells. Samples of freshly prepared FLA5.10 human
monoclonal antibody at various concentrations were injected at a
flow rate of 30 ml/min (120-s contact time). Flow was directed over
a mock surface to which no protein was bound, followed by the
HA proteins coupled surface. Responses from the peptide surface
were corrected for the response from the mock surface and for
responses from a separate, buffer only, injection. MAb 2D7 (anti-
CCR5) was used as a negative control antibody in various binding
experiments. Binding Kinetics for the MAbs and the data analysis
was performed using BioRad ProteON manager software (version
New Zealand rabbits were immunized thrice intra-muscularly at
21-days interval with 100 mg of purified HA proteins mixed with
Viral-neutralizing activity was analyzed in a microneutralization
assay based on the methods of the pandemic influenza reference
laboratories of the Center for Disease Control and Prevention
(CDC). Low pathogenicity H5N1 viruses, generated by reverse
genetics, were obtained from CDC: A/Vietnam/1203/2004
(SJCRH, clade 1), A/Indonesia/5/2005 (PR8-IBCDC-RG2;
clade 2.1), A/Turkey/1/05 (NIBRG-23; clade 2.2), A/Anhui/1/
05 (IBCDC-RG5, clade 2.3.4). The experiments were conducted
with three replicates for each serum sample and performed at least
Microneutralization titers in human sera vaccinated with
the licensed inactivated H5N1 vaccine
This study was conducted by the NIAID, NIH. Inactivated
subvirion H5N1 vaccine (rgA/Vietnam/1203/04 X A/PR/8/34)
that contains 90 mg/ml HA was used as a vaccine (Sanofi Pasteur). A
single-center dose escalating unblinded clinical trial was conducted
to determine if it is possible to increase the HAI and neutralizing
(BioRad Labs, Hercules, CA). (E) Flp-In HA1 and HA0 elicit high cross-neutralizing antibody titers against homologous and heterologous H5N1 viruses.
Animals were immunized with 100 mg proteins mixed every three weeks. Sera was collected at 8thday after each vaccination and analyzed in a
microneutralization assay against various H5N1 virus strains. Data is representative of three experiments.
Table 1. Mean Reciprocal Neutralizing Titers of Post-H5n1 Human Sera.
(CLADE 1)(CLADE 2.2) (CLADE 2.3.4)(CLADE 2.1)
90 mg H5N1 Vaccine
120 mg H5N1 Vaccine
180 mg H5N1 Vaccine
2 8020 40
3 8040 40 20
4 4020 20
*End-point titers (mean of three replicates) using human sera after two vaccinations with inactivated H5N1 vaccine in a microneutralization assay performed with
performed with rgH5N1 x PR8.
H5N1 HA Secretion from Stable Flp-In Cells
PLoS ONE | www.plosone.org7 February 2011 | Volume 6 | Issue 2 | e17297
antibody titers against avian influenza by additional boosts and/or Download full-text
increasing the vaccine dose beyond 90 mg HA . The study was
conducted under NIH IRB approval (Clinical Trials.gov identifier
NCT00383071). Post vaccination sera samples were evaluated in
the microneutralization assays (under FDA/NIH Research
Involving Human Subjects exemption 03-118B), using multiple
low pathogenicity H5N1 viruses generated by reverse genetics as
previously described .
All procedures were in accordance with the National Research
Council (NRC) Guidelines for the Care and Use of Laboratory
Animals, the Animal Welfare Act, and the Centers for Disease
Control (CDC)/National Institutes of Health (NIH) Bio-Safety
Guidelines in Microbiological and Biomedical Laboratories and
approved by the Institutional Animal Care and Use Committee
(IACUC). The study was conducted under National Institutes of
Health (NIH) IRB approval (Clinical Trials.gov identifier
Conceived and designed the experiments: HL SK HG. Performed the
experiments: HL SK NV JM LK HG. Analyzed the data: HL SK HG.
Contributed reagents/materials/analysis tools: HL SK NV JM LK JHB
HG. Wrote the paper: HL SK HG.
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H5N1 HA Secretion from Stable Flp-In Cells
PLoS ONE | www.plosone.org8 February 2011 | Volume 6 | Issue 2 | e17297