A recombinant vaccine
against the H5N1
influenza virus
Presented by: Steven Mitchell
Background
 Influenza is an infectious disease that arises within
mammals and birds.
 The influenza virus is categorized into three groups:
 Influenza A: infects birds, swine, and humans
 Influenza B: infects seals and humans
 Influenza C: infects swine and humans
 Influenza is named using the system H_N_
H for
hemagglutinin; N for neuraminidase
 Traditional vaccinations cover only H3N2, Influenza B,
and H1N1 strain variants
Step 1
Health officials globally surveys year-round which viral
strains will be most prominent for a year
Step 2
Officials analyze and target the most dominant strains
and are submitted to the FDA, who will choose the three
most worthy candidates
Step 3
Each virus is separately created then combined to form
the final vaccine. Millions of chicken eggs are specially
designed to incubate one strand per egg
Step 4
Viral fluid from the eggs is purified and the virus is
inactivated. The virus is cleaved, and fragments are
combined to form the vaccine
Step 5
The vaccine is packaged into vials and syringes after
each batch, or lot, is approved individually by the FDA
Problems with traditional
vaccines
 Not all infectious agents can be grown in culture
 Production of animal/human viruses require animal cell
culture, which is expensive
 Yield and production rate of viruses is relatively low
 Batches of vaccine may not be killed, leading to inadvertent
disease transmission. Attenuated strains may revert, and so
constant testing is needed
 Most vaccines have a limited shelf life and require
refrigeration
Recombinant vaccine to
prevent H5N1
 HPAI-H5N1 (bird flu) is an influenza A subtype virus
that is highly pathogenic and has a high mortality rate
 Current efforts toward a vaccine have been
unsuccessful due to traditional methods unable to
handle the rapid mutation of hemagglutinin (HA), a
diverse surface protein present on all influenza viruses
 Recombinant DNA technology may provide a
preventative treatment for this pathogen
Diagram of an influenza virus
Principles of H5N1
recombinant vaccine
 The Fc portion of IgG is an important fusion tag for
expressing several viral proteins, such as SARS and
HIV
 The Fc portion helps folding of the viral protein to
enhance binding of antigen-presenting cells. The Fd
(foldon sequence of T4 phage fibritin) sequence was
also expressed to further promote proper folding
 Two vaccines were created by hybridizing the HA
protein to either the Fc sequence of IgG, or Fc plus Fd
HA1 Vaccine Induced Cross-Protection against H5N1
Structure of HA protein
ch as matrix protein 2 (M2), HA, and nucleoprotein (NP)
and
recombinant
Fc and
4,17], updating
vaccine
delivery systems[18], or combining
viral
oteins with other components
[19]. The above findings
Fdc
protein
present a significant advancement towards the development of
ore efficacious H5N1 influenza vaccines.
Fc of IgG is considered an important fusion tag for coTheseveral
HA protein
of H5N1
consists of
pression with
viral proteins,
such as receptor-binding
a signal
HA1,
HA2,coronavirus
and a
main (RBD)
of severe peptide,
acute respiratory
syndrome
ARS-CoV),protease
in order tocleavage
facilitate purification
sites and subsequent
munogenicity of the proteins [20,21]. For example, fusion of Fc
was replaced
HIV-1 The
proteinSP
hassequence
also been shown
to increase by
the
IL2ss
sequence
theto Fc
to
munogenicity
of fusion
proteinsinand
elicitvector
neutralizing
tibody responses
[22,23].
This isprotein
explained by the ability of Fc
create
HA1-Fc
promote correct folding of the fusion proteins following
HA1-Fdc
was
byhelp
attaching
pression and
the possibility
thatcreated
IgG Fc may
to enhance
HA1 to the Fd
C-terminus,
followed
nding to antigen-presenting
cells(APCs)
and cell linesexpressing
receptorsby
(FcR)the
[22,24].
foldon (Fd) sequence derived from
Fc The
vector
tive T4 phage fibritin has been typically incorporated at the
terminus of collagen-like protein molecules to facilitate
bilization of protein trimer or oligomer [25,26], indicating that
terminal Fd is essential for correct trimerization and folding of
e protein [27].
In this study, we designed two recombinant influenza vaccines
sed on the recombinant proteinsencoding HA1 of a H5N1 virus
sed to either Fc of pFUSE-hIgG1-Fc2 (human IgG Fc, thereafter
med Fc) or Fd plus Fc. We then tested their immune responses
Figure 1. Structure of HA protein of AH/1 and construction of
recombinant HA1-Fc and HA1-Fdc proteins. (A) Structure of HA
protein of H5N1 AH/1 strain. The HA protein of AH/1 H5N1 virus
consists of signal peptide (SP, residues 1–18), HA1 (residues 19–345,
corresponding to +3-329aa) and HA2 (residues 346–567, corresponding
to +330-551aa) fragments spanned by a protease cleavage site RERRRKR
between HA1 and HA2. Amino acid 19 of HA1 of AH/1 corresponds to
Results
 Transfected HEK 293T cells were used as expression
vectors for HA1-Fc and HA1-Fdc proteins
 293T cells were used because of their ability to amplify
transfected plasmids. Other cell lines could be used for
substitution (such as CHO cells)
 Proteins were extracted from the supernatant medium,
purified, then analyzed by SDS-PAGE and a western
blot
Schematic of a subunit
vaccine
of mice vaccinated with the fusion proteins,
he
-Fdc, reacted strongly with this HA1 protein,
point titer of 1:1.36 107, demonstrating that the
sie
es induced by the expressed proteins were mainly
icprotein. It was further shown that the induced
As
1at
re
be
ot
y
Western blot for HA1 fusion
protein
The purified HA1-FC and
HA1-Fdc proteins were
analyzed using a western blot
with an anti-HA mAb. Purified
proteins were separated by
10-20% Tricine gels and
transferred to nitrocellulose.
Proteins were blocked
overnight, then incubated with
anti-HA mAb for 1 hour. Blots
were incubated with HRPconjugated goat anti-mouse
IgG for 1 hour.
is of t he expression of HA1-Fc and HA1-Fdc
pression of HA1-Fc and HA1-Fdc proteins were
PAGE and Coomassie blue staining (A), and Western
-specific mAb. The protein molecular weight marker
on the left.
Figure 2. Analysis of t he expression
.pone.0016555.g002
th
As
es
he
of HA1-Fc and HA1-Fd
prot eins. The expression of HA1-Fc and HA1-Fdc proteins we
performed by SDS-PAGE and Coomassie blue staining (A), and Weste
Results
 Mice injected with the HA1-Fc and HA1-Fdc
recombinant proteins were analyzed via ELISA for
antibody responses
 Both proteins induced IgG antibody responses specific
to the proteins
 Subsequent injections with different clade’s HA proteins
showed an antibody response (pseudoviruses)
IgG antibody response to HA1-Fc and HA1Fdc vaccine treatments
HA1 Vaccine Induced Cross-Protection against H5N1
Left: Reactivity of IgG antibody with HA1-Fc and HA1-Fdc proteins. Time
scale was 10 days between first vaccination and boosters
Right: The ability of IgG to bind to the proteins and the control
Results
 The antibodies produced were found to reduce the viruses
pathogenic qualities such as reproductive capabilities
 The antibodies were effective for a wide array of live H5N1 strains,
as well as H5N1 pseudoviruses, including:
 HK/156
 VN/1194
 SZ/406H
 HK-HA
 1194-HA
 QH-HA
 XJ-HA
 AH-HA
 These three avian flu strands are highly pathogenic in humans
Antibodies present from
heterologous strains of H5N1
Graphs show various strains of
live influenza being introduced
into mice after being vaccinated.
The neutralizing antibodies,
NAb, block biological effects the
virus has on it’s host cells.
Hemagglutination antibodies, HI
Ab, disable the binding ability of
the influenza virus.
elicit strong HA1-specific antibody responses and to neutralize
divergent strains of homologous and heterogeneous H5N1
antibodies
with neutralizing
and/ orlive
protective
immunity
pseudoviruses,
as well asactivity
heterologous
virus. Importantly,
against
virus proteins
[31,32], could
and elicit
it has
a protective
greater
bothHAPI
HA1-FcH5N1
and HA1-Fdc
potent
immunitythan
in the
immunized
mice against
of atM2,
least
contribution
other
viral proteins,
such aschallenges
NA, NP and
three heterogeneous strains of H5N1 viruses.
to the induction of neutralizing antibodies and/ or protection
In addition to Fc, another component, Fd, could also be
[31,33].
HA-based
have been
shown
to elicitimmunogenicity
higher titers
considered
as avaccines
fusion partner
to help
increasing
of neutralizing
antibodies
prevent
influenza
virus
infectionmotif,
in
of recombinant
vaccines.toThis
is because
that Fd,
a trimeric
Viral challenge in vaccinated
mice
Discussion
Antibody responses provide essential immunity against infection
of IAVs [30]. As the main surface protein of the virus, HA of
H5N1 virus serves as an important target for induction of specific
tested animals [14,18,34]. In addition, human clinical trials of the
HA-containing H5N1 vaccines have revealed the production of
strong and broad antibody responses after vaccine injection
[35,36]. However, current influenza vaccines have failed to
provide sufficient protection against infections of rapidly mutated
influenza viruses. Thus, novel strategies are urgently needed to
develop vaccines potentially inducing cross-clade protection
against multiple strains of influenza viruses.
Fc fragment of human IgG has been previously used asa fusion
compartment to improve immunogenicity of the proposed
proteins [20,21,23]. The underlying mechanism of Fc fusion
protein-based vaccines may be partially due to that proteins with
Fc could bind to cells with FcR, while the latter plays important
roles in the clearance of virus infections and protection against
Figure 6. Cross-clade p rotect ion of HA1-Fc- or HA1-Fd cvaccinated mice against lethal H5N1 virus challenge. HA1-Fc- virus challenge via FcR-mediated phagocytosis [37].
and HA1-Fdc-vaccinated mice were challenged with 10 LD50 of three Indeed,
Figure our
7. Detectio
of viral
RNA copies
by quantitative
reversestudy nhas
demonstrated
that
both Fc-containing
clades (clade 0: HK/156, clade 1: VN/1194, and clade 2.3.4: SZ/406H) of
transcript ion PCR (Q-RT-PCR) in lung tissues of H5N1 virusvaccinemice.
candidates,
and HA1-Fdc,
wereH5N1
ablevirus
to
H5N1 virus. PBS was used as the negative control. Groups of 10 mice influenza
challenged
Titers of HA1-Fc
HK/156, VN/1194
and SZ/406H
Left: Lethal
H5N1
virus
challenge
in
vaccinated
mice.
Three
different
clades
were observed for survival for 21 days post-virus challenge, and elicitwere
determined
in lung antibody
tissues of the
mice vaccinated
with HA1-Fc
strong
HA1-specific
responses
and to neutralize
corresponding survival rates were calculated. Survival rate (%) of mice
and HA1-Fdc proteins, respectively. Mice vaccinated with PBS were
divergent
strains of homologous
and heterogeneous
H5N1
were tested
forHK/156
21 (A),
days.
the difference
in
proteins
challenged with
VN/1194Note
(B), and SZ/406H
(C) H5N1 virus
usedsurvival
as the negativerate
control.between
The data are expressed
as mean 6 SD of
was shown.
pseudoviruses,
as lung
well tissues
as heterologous
live
RNA copies per
from five mice
pervirus.
group. Importantly,
HA1-Fc
and HA1-Fdc
doi:10.1371/journal.pone.0016555.g006
doi:10.1371/journal.pone.0016555.g007
both HA1-Fc and HA1-Fdc proteins could elicit potent protective
Right: Detection of viral RNA copies by quantitative
PCR
immunity in thereverse-transcription
immunized mice against challenges
of at least
PLoS ONE
www.plosone.org
6
2011 viruses.
| Volume 6 | Issue 1 | e16555
three
heterogeneous
of H5N1
(Q-RT-PCR)
in | lung
tissues of H5N1 challenged
mice strainsJanuary
In addition to Fc, another component, Fd, could also be
considered as a fusion partner to help increasing immunogenicity
of recombinant vaccines. This isbecause that Fd, a trimeric motif,
Histopathological changes in
lung tissue
Mat e
Ethic
The
the re
Labor
Unive
standa
(BSLthe U
Unive
Lung tissue samples were
collected five days after all mice
were sacrificed. All sections were
stained with hematoxylin and
eosin and observed under light
microscope. Tissues are fixed in
paraffin wax.
Cons
Reco
Figure 8. Evaluatio n of histopath ological changes in lung
tissues of H5N1 virus-ch allenged mice. HA1-Fc- and HA1-Fdcvaccinated mice were challenged with 10 LD50 of three clades (clade 0:
HK/156, clade 1: VN/1194, and clade 2.3.4: SZ/406H) of H5N1, and lung
tissues were collected at day 5 post-challenge. Lung tissues from mice
The
using
ABD2
vector
The F
to the
prime
encod
and H
with s
binan
Mana
calciu
fresh
later,
Overview
 Targeting HA, the main surface protein of the virus,
would provide a feasible means of producing an
effective virus
 The HA gene was fused with the Fc of IgG antibodies,
or with Fc and Fd (foldon) to promote trimeric folding
 Both subunit vaccines were shown to be effective for a
wide variety of avian influenza strains. However, the
HA1-Fdc vaccine proved to be the most effective
Methods and Materials

Construction, expression, and purification of recombinant HA1-Fc and HA1-Fdc proteins
 Genes encoding HA1 of H5N1 were amplified by PCR using full-length HA as the template and inserted
into the Fc expression vector. The Fd sequence derived from bacteriophage T4 fibritin was fused to the Cterminus of HA1 sequences by PCR with overlapping primers. The recombinant proteins were expressed
in 293T cells using calcium phosphate method. The recombinant proteins were purified by protein A affinity
chromatography.

Western Blot
 The purified proteins were analyzed by SDS-PAGE and western blot using an anti-HA mAb. The proteins
were transferred to nitrocellulose membranes. After blocking, blots were incubated with HA specific mAb
for 1 hour. Blots were incubated with HRP-conjugated goat anti-mouse IgG for 1 hour. Signals were
visualized with ECL reagents.

ELISA
 The antibody response was evaluated by ELISA in collected mouse sera. 96-well ELISA plates were
coated with recombinant HA1-Fc and HA1-Fdc fusion proteins, HA1 protein without Fd and Fc, and
inactivated H5N1 virus and blocked overnight with non-fat milk. Bound antibodies were incubated with
HRP-conjugated goat anti-mouse IgG for 1 hour. The reaction was visualized by TMP and stopped by 1N
H2SO4.
Further information
 http://www.ted.com/talks/seth_berkley_hiv_and_flu_the
_vaccine_strategy.html
 http://www.who.int/csr/disease/avian_influenza/country/
cases_table_2010_12_09/en/index.html
 Barry, John. The Great Influenza. New York: Penguin
Publishing, 2005. Print.
 Shanks, Dennis, and John Brundage. "Pathogenic
Responses among young adults during the 1918
Influenza Pandemic." Emerging Infectious Diseases.
18.2 (2012): 201-207. Print. <www.cdc.gov/eid>.
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