Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli

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DOI: 10.1371/journal.pone.0052488 · Source: PubMed
Abstract
The 23-amino acid extracellular domain of matrix 2 protein (M2e) and the internal nucleoprotein (NP) of influenza are highly conserved among viruses and thus are promising candidate antigens for the development of a universal influenza vaccine. Various M2e- or NP-based DNA or viral vector vaccines have been shown to have high immunogenicity; however, high cost, complicated immunization procedures, and vector-specific antibody responses have restricted their applications. Immunization with an NP-M2e fusion protein expressed in Escherichia coli may represent an alternative strategy for the development of a universal influenza vaccine. cDNA encoding M2e was fused to the 3' end of NP cDNA from influenza virus A/Beijing/30/95 (H3N2). The fusion protein (NM2e) was expressed in E. coli and isolated with 90% purity. Mice were immunized with recombinant NM2e protein along with aluminum hydroxide gel and/or CpG as adjuvant. NM2e plus aluminum hydroxide gel almost completely protected the mice against a lethal (20 LD(50)) challenge of heterologous influenza virus A/PR/8/34. The NM2e fusion protein expressed in E. coli was highly immunogenic in mice. Immunization with NM2e formulated with aluminum hydroxide gel protected mice against a lethal dose of a heterologous influenza virus. Vaccination with recombinant NM2e fusion protein is a promising strategy for the development of a universal influenza vaccine.
Robust Immunity and Heterologous Protection against
Influenza in Mice Elicited by a Novel Recombinant NP-
M2e Fusion Protein Expressed in
E. coli
Wenling Wang
.
, Baoying Huang
.
, Tao Jiang, Xiuping Wang, Xiangrong Qi, Yingying Gao, Wenjie Tan,
Li Ruan*
National Institute for Viral Disease Control & Prevention, Chinese Center for Disease Control and Prevention, China CDC, Beijing, People’s Republic of China
Abstract
Background:
The 23-amino acid extracellular domain of matrix 2 protein (M2e) and the internal nucleoprotein (NP) of
influenza are highly conserved among viruses and thus are promising candidate antigens for the development of a universal
influenza vaccine. Various M2e- or NP-based DNA or viral vector vaccines have been shown to have high immunogenicity;
however, high cost, complicated immunization procedures, and vector-specific antibody responses have restricted their
applications. Immunization with an NP–M2e fusion protein expressed in Escherichia coli may represent an alternative
strategy for the development of a universal influenza vaccine.
Methodology/Principal Findings:
cDNA encoding M2e was fused to the 39 end of NP cDNA from influenza virus A/Beijing/
30/95 (H3N2). The fusion protein (NM2e) was expressed in E. coli and isolated with 90% purity. Mice were immunized with
recombinant NM2e protein along with aluminum hydroxide gel and/or CpG as adjuvant. NM2e plus aluminum hydroxide
gel almost completely protected the mice against a lethal (20 LD
50
) challenge of heterologous influenza virus A/PR/8/34.
Conclusions/Significance:
The NM2e fusion protein expressed in E. coli was highly immunogenic in mice. Immunization
with NM2e formulated with aluminum hydroxide gel protected mice against a lethal dose of a heterologous influenza virus.
Vaccination with recombinant NM2e fusion protein is a promising strategy for the development of a universal influenza
vaccine.
Citation: Wang W, Huang B, Jiang T, Wang X, Qi X, et al. (2012) Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel
Recombinant NP-M2e Fusion Protein Expressed in E. coli. PLoS ONE 7(12): e52488. doi:10.1371/journal.pone.0052488
Editor: Gourapura J. Renukaradhya, The Ohio State University, United States of America
Received August 14, 2012; Accepted November 13, 2012; Published December 21, 2012
Copyright: ß 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the National High Technology Research and Development Program of China (863 Program) (grant number
2006AA02A203). The funders had no role in study design, data collection and analysis, decision to publish, or preperation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ruanl@bbn.cn
. These authors contributed equally to this work.
Introduction
Currently, vaccination is the most effective method for
prevention of influenza [1,2]. However, conventional flu
vaccines based on hemagglutinin (HA) and neuraminidase
(NA) have failed to induce heterosubtypic protection owing to
the high variability of these two antigens [3–5]. To afford
intrasubtypic and heterosubtypic cross-protection, a universal
influenza vaccine based on the more conserved antigens of
influenza viruses is desirable, as conserved antigens are
consistent across strains and do not exhibit frequent variation
[2,6,7]. Matrix 2 protein (M2) and nucleoprotein (NP) are
conserved antigens of influenza A virus and thus are promising
candidate antigens for the development of a universal influenza
vaccine [8,9]. Recent studies have investigated the potential of
M2 (mainly M2e) [10–19] or NP [20–24] as alternative antigens
in preventing seasonal and pandemic flu outbreaks. In these
cases, M2e was fused genetically or linked chemically with large
carriers such as hepatitis B virus core (HBVc), flagellin, phage
Qb, Neisseria meningitides outer membrane complex (OMPC). M2
and NP have also been used together, because the combination
of multiple antigens is often superior to a single antigen in terms
of eliciting an immune response. In previous studies, injections
of vaccines based on NP and M2 recombinant DNA and/or
adenovirus have conferred protection to mice against a lethal
virus challenge, and it showed that the protection induced by
the combination of NP and M2 was superior to the sole one
[25–31]. However, the poor immunogenicity of DNA-based
vaccines may restrict their wide application [32], and vector-
based vaccines have the potential to elicit anti-vector antibodies
which may interfere with immunization [33].
A prokaryotic system may be the simplest and fastest method for
expression and purification of large quantities of a single antigenic
protein for the production of a new influenza vaccine [34,35]. The
influenza A virus NP protein [36] and M2 protein with residues
26–55 deleted [37] have been expressed fom E. coli successfully
and they induced broad protective immunity against influenza. A
fusion protein consisting of NP and M2 expressed in a prokaryotic
system is a promising candidate antigen for a universal influenza
vaccine, and the new construct may contain the character of the
PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e52488
two antigens. Protein vaccines are superior to attenuated live
vaccines and inactivated virus vaccines with respect to safety.
However, due to the poor immunogenicity of protein vaccines, an
appropriate adjuvant must be used to induce effective and long-
term protection [38]. The development of more effective vaccine
and adjuvant formulations, as well as procedures to enhance the
immunogenicity of influenza virus proteins and peptides, could
result in improved humoral and cell-mediated immunity [15,39–
43].
In this study, NP and the 23-amino acid extracellular domain
of M2 (M2e), which are highly conserved among viruses, were
selected as candidate antigens for a universal influenza A
vaccine. The cDNA encoding M2e was fused to the 39 end of
the full-length cDNA for NP from influenza virus A/Beijing/
30/95 (H3N2). The resultant fusion protein (NM2e) was
expressed in Escherichia coli and isolated with 90% purity.
Recombinant NM2e fusion protein was highly immunogenic in
mice, and NM2e formulated with aluminum hydroxide gel as
an adjuvant almost completely protected the mice against
challenge with a lethal dose of heterologous influenza virus A/
Puerto Rico/8/34. Thus, NM2e fusion protein expressed in E.
coli is a promising candidate antigen for the development of
a universal influenza vaccine.
Materials and Me thods
Ethics Statement
This mouse study was conducted in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the Chinese Center for Disease control
and prevention. The protocol was approved by the Committee on
the Ethics of Animal Experiments of the Institute for Occupational
Health and Poison Control (Permit Number: EAWE-2010-029).
Serum was obtained by orbital sinus puncture. In the ELISPOT
assay mice were sacrificed by cervical dislocation. Challenge
experiment was performed under sodium pentobarbital anesthe-
sia, and all efforts were made to minimize suffering. After
influenza a virus PR8 challenge, mice were monitored closely
for approximately 21 days for signs of illness. Any animals in
a moribund condition were euthanized.
Construction, Expression, and Purification of NM2e
Fusion Protein
Genes of influenza A virus A/Beijing/30/95 (H3N2) strain
(supplied by Department of Influenza Virus, National Institute for
Viral Disease Control and Prevention, China CDC) were used as
templates for cloning NP and M2e. The cDNA encoding M2e
(amino acid sequence: SLLTEVETPIRNEWGCRCNDSSD) was
fused to the 39 end of the full-length cDNA for NP (498 amino
acids) without any linker sequence to form the fusion construct
NM2e (Fig. 1). The optimized NM2e cDNA was synthesized and
ligated into the pET-30a(+) vector (Merck-Novagen, Darmstadt,
Germany) between the NdeI and EcoRI sites to form pET30a-
NM2e, which was used to transform E. coli BL21(DE3) (Merck-
Novagen). The expressed NM2e protein was purified by ion
exchange chromatography (DEAE-Sepharose Fast Flow, Amer-
sham Biosciences Corp., New Jersey, USA), followed by gel
chromatography (Superdex 200, Amersham Biosciences Corp,
New Jersey, USA). The purified NM2e was concentrated,
quantified using the bicinchonic acid protein assay (PIERCE
Biotechnology, Rockford, Illinois, USA), and stored at 270uC.
Endotoxin levels were determined using the Tachypleas Amebo-
cyte Lysate assay (Chinese Horseshoe Crab Reagen Manufactory,
Xiamen, China) as directed by the manufacturer. The endotoxin
level of the NM2e protein was about 2000 EU/mg. Furthermore,
the recombinant NM2e was analyzed by Western blot assay.
Samples were electrophoresed in a 12% polyacrylamide gel under
reducing conditions, transferred to nitrocellulose, probed with
influenza A virus NP-immunized murine serum and a monoclonal
antibody (14C2; Abcam, Cambridge, UK) against influenza A
virus M2e, and incubated with HRP-conjugated goat anti-mouse
IgG (1:10000) (Sigma-Aldrich, St. Louis, Mousa, USA). Immuno-
reactivity was detected with 3, 39-diaminobenzidine (DAB)
substrate.
Immunogenicity and Protective Efficacy of NM2e
Immunization in mice
The immunogenic potential of NM2e fusion protein was
evaluated in six groups of 4- to 6-week-old BALB/c (H-2
d
)
mice. Mice were purchased from the Institute of Laboratory
Animals, Chinese Academy of Medical Sciences, and raised in
cages in the Institute for Occupational Health and Poison
Control, Chinese center for Disease Control and Prevention.
Aluminum hydroxide gel (Al[OH]
3
, Alhydrogel; Brenntag
Biosector, DK-3600 Frederikssund, Denmark) and/or CpG
1826 (59-TCCATGACGTTCCTGACGTT-39 ) were used as
adjuvants. The mice in each group (n = 33) were injected
intramuscularly in the right posterior gastrocnemius as follows:
group 1, 0.9% NaCl solution (normal saline, NS); group 2, NS
plus Al(OH)
3
and CpG 1826; group 3, 10 mg of NM2e; group
4, 10 mg of NM2e plus CpG 1826; group 5, 10 mg of NM2e
plus Al(OH)
3
; and group 6, 10 mg NM2e plus Al(OH)
3
and
CpG 1826 (Table 1). Immunization was performed three times
at 2-week intervals. Blood samples were collected by orbital
sinus puncture from six mice in each group at the times
indicated in Fig. 2, and mice were sacrificed by cervical
dislocation, then spleen were removed and grinded up sterilely.
Spleen mononuclear cells (SMNCs) were obtained after the red
blood cells in the spleen cell suspension were lysed. Ten days
after the third immunization, the protective potential of
recombinant NM2e fusion protein was evaluated in the
vaccinated mice. Mice were anesthetized intraperitoneally with
sodium pentobarbital (10 mg/ml) at a dose of 60 mg/kg body
weight and infected with 50 ml of influenza A/PR/8/34 (H1N1)
(abbreviated PR8), containing 20-fold the LD
50
. The daily body
weight loss and mortality were monitored for 3 weeks after the
challenge.
ELISA Protocol
Specific antibodies from serum samples of NM2e-immunized
mice were determined by ELISA. Microtiter plates were coated
with 2 mg/ml recombinant NP [44] or M2e peptide (synthesized
in Beijing Scilight Biotechnology Ltd. Co., Beijing, China)
overnight at 4uC. Washed wells were blocked by incubation with
PBS containing 2% BSA (Amresco, Solon, Ohio, USA) for 2 h at
37uC. Serum samples were serially diluted in PBS containing 1%
BSA, added to the wells, and incubated for 1.5 h at 37uC. After
repeated washes, bound total IgG and IgG1 and IgG2a isotypes
were detected by incubation with HRP-conjugated goat anti-
mouse IgG (Sigma) (1:10000 dilution), IgG1 (Southern Biotech,
Birmingham, Alabama, USA) (1:5000 dilution), and IgG2a
(Southern Biotech) (1:5000 dilution), respectively, for 1.5 h at
37uC. After washing, 100 ml of TMB substrate solution was added
to each well, and plates were incubated for 5 min at room
temperature in darkness. The reaction was stopped by addition of
50 mlof1MH
2
SO
4
to each well. The color produced by the
enzymatic reaction was determined by measuring the absorbance
at 450 nm.
Protection Elicited by NP and M2e Fusion Protein
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Protection Elicited by NP and M2e Fusion Protein
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ELISPOT Protocol
Murine ELISPOT kits (BD Biosciences, Franklin Lakes, New
Jersey, USA) were used to detect the numbers of IFN-c-, IL-4-,
and IL-10-secreting SMNCs separated from blood samples of mice
immunized with NM2e protein. ELISPOT plates were coated
with anti-murine IFN-c, IL-4, and IL-10 antibodies overnight at
4uC. RPMI-1640 medium containing 10% FBS (R-10; GIBCO,
Langley, Oklahoma, USA) was added to block nonspecific sites for
2 h at room temperature. SMNCs were aseptically isolated, and 5
6 10
5
SMNCs suspended in 100 ml of R-10 containing 10 mg/ml
NP
55–69
(RLIQNSLTIERMVLS; H-2d-restricted Th epitope),
NP
147–155
(TYQRTRALV; H-2d-restricted CTL epitope), or M2e
peptide pool (peptides of residues 1–15, 5–19, and 9–23) were
added to each well. After incubation for 40 h in a 5% CO
2
incubator at 37uC, 100 ml of detection antibody was added to each
well and incubated for 2 h at room temperature. Then, 100 mlof
enzyme complex solution was added, followed by incubation for
1 h at room temperature. AEC substrate solution (100 ml) was
added to each well and the reaction was allowed to proceed for
20 min at room temperature in darkness. To terminate the
reaction, the ELISPOT plate was rinsed with flowing water. An
ELISPOT image analyzer (Bioreader 4000; Bio-Sys, Karben,
Germany) was used to determine the number of spot-forming cells
(SFCs).
Statistical Analysis
Statistical analysis was performed using SPSS, version 17.0, and
Prism, version 5.0a, software. Log conversion was performed for
antibody titers. Differences in antibody titers and ELISPOT
results among groups were analyzed by one-way ANOVA. The
paired t-test and log-rank test were used to analyze differences in
body weight change curves and survival rate curves, respectively.
Differences with p-values #0.05 were considered significant.
Results
Design, Expression, Purification, and Identification of
NM2e Fusion Protein
The constructed cDNA encoding a fusion protein containing
full-length NP (498 amino acids) with the extracellular domain of
M2 (M2e, 23 amino acids) at its C-terminus, with no linker
sequence, was expressed in E. coli as a protein of 521 amino acids,
and designated NM2e (Fig. 1A). Native sequences from influenza
A virus A/Beijing/30/95 (H3N2) were used as templates for
cloning the target genes. The codons of the NM2e cDNA
sequence were optimized for expression in E. coli, and the
synthesized cDNA was cloned in the prokaryotic expression vector
pET-30a(+). The protein was expressed in E. coli BL21(DE3) with
high efficiency (Fig. 1B). Most of the recombinant protein was
produced as insoluble inclusion bodies with induction at 37uC, but
as soluble protein with induction at 25uC. The optimal conditions
for producing the maximal amount of soluble NM2e protein were
induction with 0.1 mM IPTG for 12 h at 25uC. Under these
culture conditions, the target protein accounted for 20–30% of the
total soluble E. coli protein. The NM2e protein yield of the gene-
codon optimized cDNA was two- to three-fold that of the original
cDNA (data not presented).
The fusion protein was purified by ion-exchange chromatog-
raphy followed by gel filtration chromatography, with a yield of
3.20 mg of NM2e protein per liter of culture medium. SDS-
PAGE with Coomassie Blue staining showed that the fusion
protein was 58 kDa and about 90% pure (Fig. 1C). On Western
blots, the expressed NM2e protein was recognized by serum
from NP-immunized BALB/c mice and by a monoclonal
antibody against M2e (Fig. 1C). These results demonstrate the
Figure 1. Construction, expression, and characterization of NM2e fusion protein. (A) Schematic of influenza A virus NP and M2, and
diagram for the construction of recombinant NM2e fusion protein. The entire sequence of NM2e is shown. The cDNA sequences encoding residues
1–498 of NP and residues 2–24 of M2 (extracellular domain of M2, M2e) from influenza A virus A/Beijing/30/95 (H3N2) were fused directly, with no
linker, and this was cloned as pET30a-NM2e for expression in Escherichia coli. (B) Protein profile of cell lysates from induction experiments in E. coli
BL21 (DE3) transformed with pET30a-NM2e at 25 and 37uC. Lane 1, Mid-range protein molecular weight marker; lane 2, whole-cell lysate of
transformed E. coli before induction; lanes 3–5, whole-cell lysate, soluble supernatant, and insoluble fraction after 4-h induction with 0.1 mM IPTG at
25uC; lanes 6–8, whole-cell lysate, soluble supernatant, and insoluble fraction after 4-h induction with 0.1 mM IPTG at 37uC; lane 9, whole-cell lysate
after 4 h of cultivation without IPTG at 37uC. The arrow indicates the 58-kDa band corresponding to NM2e. (C) SDS-PAGE (left) showing the purified
NM2e fusion protein, NP of influenza A virus A/Beijing/30/95(H3N2), and lysates of E. coli transformed with pET30a(+). NM2e fusion protein was
detected on Western blots probed with NP-immunized mouse serum (middle) and mouse anti-M2e monoclonal antibody (right). Lanes 1–3, purified
influenza A virus NP, recombinant NM2e fusion protein expressed in E. coli, cell lysates of E. coli transformed with pET30a(+). M, protein molecular
weight marker.
doi:10.1371/journal.pone.0052488.g001
Figure 2. NM2e protein immunization schedule. The indicated
mice were immunized intramuscularly with NM2e protein with or
without adjuvant, three times at 2-week intervals. Blood was collected
on days 14, 28, and 38, respectively. The immunized mice were
challenged with influenza A virus PR8 at 20-fold the LD
50
on day 38.
Body weight and survival were monitored for 3 weeks, until day 59.
doi:10.1371/journal.pone.0052488.g002
Table 1. Summary of mouse groups immunized with NM2e.
Group NM2e/dose
a
Adjuvant/dose
b
Volume
G1 (NS/triple dose) 100 ml
G2 (NS/triple dose) Al(OH)
3
/100 mg+CpG/10 mg100ml
G3 Triple dose/10 mg– 100ml
G4 Triple dose/10 mg CpG/10 mg100ml
G5 Triple dose/10 mg Al(OH)
3
/100 mg100ml
G6 Triple dose/10 mg Al(OH)
3
/100 mg+CpG/10 mg100ml
NS, normal saline.
a
mice in G1 and G2 were mock-immunized with NS instead of NM2e protein.
b
mice in G1 and G3 were immunized without adjuvant.
doi:10.1371/journal.pone.0052488.t001
Protection Elicited by NP and M2e Fusion Protein
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successful in vitro expression of NM2e protein and its isolation
with 90% purity.
An Immune Response in Mice is Elicited by NM2e
Immunization
To determine the immunogenicity of recombinant NM2e
protein, it was used to immunize groups of BALB/c mice three
times at 2-week intervals, as described in Fig. 2. Immunogen and
adjuvant doses used in each group are presented in Table 1.
In group 3, the priming vaccination with NM2e alone induced
a high titer of anti-NP IgG (9.6 6 10
3
) (Fig. 3A, left), but a low titer
of anti-M2e IgG (40) (Fig. 3A, right). The second immunization
significantly improved the serum anti-NP IgG titer (8.5 6 10
5
;
p,0.001), but not that of anti-M2e IgG (82; p.0.05). The third
immunization had no additional effect on either titer.
To test whether an adjuvant could increase the immunogenicity
of NM2e, the protein was formulated with aluminum hydroxide
gel (Al[OH]
3
) alone (group 5). The inclusion of Al(OH)
3
with
NM2e induced higher levels of anti-NP IgG (3.4 6 10
4
) and anti-
M2e IgG (126) after the prime immunization, which was
significantly improved after the second immunization (4 6 10
6
,
p,0.001 and 2 6 10
4
, p,0.001) (Fig. 3A). Based on IgG titers after
the third immunization, the inclusion of Al(OH)
3
significantly
improved the anti-M2e (5 6 10
4
, p,0.001) and anti-NP IgG
(5610
6
, p,0.01) titers compared with immunization using NM2e
protein alone (Fig. 3B). Long term monitoring data showed that
when mice were immunized with NM2e formulated with Al(OH)
3
three times at 2-week intervals, the high anti-NP and anti-M2e
IgG levels were maintained for at least 7 months, while single
immunization with the same formulation induced only low-level
anti-NP and anti-M2e IgG, which was reduced further 3 months
after the immunization (Fig. 3C). We can infer from the long-term
data that NM2e formulated with Al(OH)
3
induces stronger and
longer-lasting anti-NP and anti-M2e IgG responses than does
NM2e protein alone. Furthermore, we identified the anti-NP and
anti-M2e antibody isotypes by ELISA (Fig. 4). NM2e immuniza-
tion elicited high levels of anti-NP IgG1 (2 6 10
5
) and IgG2a (4 6
10
5
) (Fig. 4, left) after three immunizations. When Al(OH)
3
was
included in the formulation with NM2e, both the anti-NP and
anti-M2e IgG1 and IgG2a titers were markedly increased (Fig. 4
A, B). To further characterize the cellular immune responses
elicited by NM2e protein in mice, IFN-c-, IL-4-, and IL-10-
secreting SMNCs were quantified by ELISPOT assays (Fig. 5).
The priming and second immunizations elicited only a few IFN-c-,
IL-4-, or IL-10-secreting SMNCs (data not shown), and the third
immunization elicited a limited number of IL-4- or IL-10-secreting
SMNCs (Fig. 5B, C) in each group. Therefore, only the IFN-c-
specific ELISPOT results were analyzed in detail. Immunization
with NM2e alone did not elicit a clear cellular response, however,
Al(OH)
3
significantly enhanced the NM2e-induced cellular
immune response when NP55-69 (p,0.01) (Fig. 5A, middle) and
the M2e peptide pool (p,0.001) (Fig. 5A, right) were used as
stimuli.
CpG oligodeoxynucleotide alone was used to increase the
immunogenicity of NM2e in group 4 mice. The data showed
that CpG improved the immune response elicited by NM2e,
although it displayed poorer efficacy than Al(OH)
3
. The
inclusion of CpG with NM2e induced higher levels of anti-NP
IgG titer (2.3 6 10
4
) and low level of anti-M2e IgG titer (66)
after the prime immunization, the anti-NP IgG was significantly
improved after the second immunization (1.1 6 10
6
, p,0.001)
(Fig. 3A, left), whereas the anti-M2e IgG titer was improved
markedly till the third immunization (252, p,0.05) (Fig. 3A,
right). Based on IgG titers after the third immunization, the
inclusion of CpG significantly improved the anti-M2e IgG titer
(252, p,0.01), but not the anti-NP IgG titer (2.3 6 10
4
,
p.0.05) compared with immunization using NM2e protein
alone (Fig. 3A, B, right). The anti-NP and anti-M2e IgG titers
were significantly lower in group 4 than in group 5 (p,0.01 and
p,0.001, respectively) (Fig. 3B). When antibody subtype was
considered, the inclusion of CpG in NM2e significantly
increased the NP-specific IgG2a titer (2 6 10
6
, p,0.001) and
decreased the IgG1 titer (7 6 10
4
, p, 0.05) compared with
NM2e immunization alone. This resulted in a lower anti-NP
IgG1/IgG2a ratio indicative of a potent Th1 response, which is
different from the IgG1/IgG2a pattern in groups 3 and 5
(Fig. 4, left). Anti-M2e antibody subtype data showed that
inclusion of CpG with NM2e improved the anti-M2e IgG1 titer
(89), but not the IgG2a titer (178) (Fig. 4, right). Meanwhile,
ELISPOT results suggested that the inclusion of CpG had no
clear effect on the cellular immune response induced by NM2e
alone (Fig. 5A).
Al(OH)
3
and CpG were also used together to improve the
immunogenicity of NM2e protein (group 6). Based on IgG titers
after the third immunization, anti-NP and anti-M2e IgG titers,
regardless of antibody subtype, of group 6 were not markedly
higher than those of group 5 (Fig 3, 4). Meanwhile, the inclusion of
Al(OH)
3
plus CpG with NM2e failed to show a stronger cellular
immune response than Al(OH)
3
alone (Fig. 5).
These results indicate that the NM2e fusion protein expressed in
E. coli is immunogenic in mice. Al(OH)
3
markedly improved the
immune response of NM2e in mice. However, NM2e formulated
with CpG induced poorer immune responses than with Al(OH)
3
,
although CpG also improved the immunogenicity of NM2e.
Moreover, Al(OH)
3
and CpG showed no clear synergistic effect
when combined in the NM2e formulation.
NM2e Ind uces Protection against Influenza A virus PR8
Challenge in Mice
We investigated the potential for immunization with the
NM2e fusion protein to induce cross protection. All mice in
control groups 1 and 2 showed marked weight loss and died
from lethal infection upon heterologous virus challenge (Fig. 6).
Mice immunized with NM2e alone showed severe body weight
loss, as much as 30%, and only 27% of the mice survived the
lethal virus challenge. Although weight loss was similar between
the NM2e-only (group 3) and NM2e/CpG (group 4) immunized
groups, 40% of the NM2e/CpG immunized mice survived the
lethal challenge. Compared with groups 3 and 4, mice
immunized with NM2e/Al(OH)
3
(group 5) exhibited significant-
ly lesser weight loss (19%, p,0.001) and significantly higher
survival (93%, p ,0.001). Although the transient weight loss in
mice immunized with NM2e formulated with Al(OH)
3
plus
CpG (group 6) was slight (12%), the survival percentage was not
significantly different from that of group 5 (p.0.05). These
results indicate that the inclusion of Al(OH)
3
alone for NM2e
immunization may significantly improve the protective efficacy
of NM2e, whereas the inclusion of CpG only results in no such
effect. Moreover, Al(OH)
3
and CpG did not show a synergistic
effect on mortality.
NM2e induced stronger immune response and higher pro-
tection efficacy than NP only in mice.
To test whether NM2e was able to induce stronger immune
response than NP, 10 mg of NM2e (group 2, g2) and NP
protein (group 3, g3) formulated with Al(OH)
3
were used to
immune BALB/c mice three times respectively according to the
time schedule in Fig. 2. Mice immunized with Al(OH)
3
were
treated as negative control (group 1, g1). Ab analysis result
Protection Elicited by NP and M2e Fusion Protein
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showed that after the third immunization NM2e formulated
with Al(OH)
3
induced comparable anti-NP IgG and IgG1,
IgG2a with NP formulated with Al(OH)
3
(Fig. 7A, left).
Different from NP, NM2e did induce anti-M2e Ab (Fig. 7A,
Figure 3. Antibody response trend and long-term humoral immune response induced by NM2e protein in mice. (A and B) Mice were
immunized intramuscularly with 10 mg of NM2e protein three times at 2-week intervals. Al(OH)
3
and/or CpG 1826 were used as adjuvants. Mice
immunized with normal saline (NS) or adjuvant alone was used as negative controls. Serum was obtained from each mouse on days 14, 28, and 38,
respectively, and analyzed for the presence of IgG antibodies specific for NP (left) or M2e (right), in an ELISA, as described in the Materials and
Methods. Antibody response trends after three immunizations are presented in A, and the comparison of results on day 38 are presented in B.
Columns show geometric mean antibody titers, and bars indicate the 95% confidence interval in each group. Plots in B show the NP- and M2e-
specific IgG titers of all of the mice in each treatment group on day 38, and bars indicate the geometric mean antibody titers of each treatment group
(n = 6 mice per experimental group, except n = 5 mice in the NS group). Lines above two or more groups indicate that they have the same
comparative results. *, p#0.05; **, p#0.01; ***, p#0.001 by one-way ANOVA. (C) Mice were immunized intramuscularly with 10 mg of NM2e protein
formulated with Al(OH)
3
three times at 2-week intervals or immunized with a single dose of 10 mg of NM2e formulated with Al(OH)
3
. Serum was
prepared from each mouse at the indicated times, and NP- and M2e-specific IgG antibodies were analyzed by ELISA, as described in the Materials and
Methods.
doi:10.1371/journal.pone.0052488.g003
Protection Elicited by NP and M2e Fusion Protein
PLOS ONE | www.plosone.org 6 December 2012 | Volume 7 | Issue 12 | e52488
right). ELISPOT result showed NM2e induced comparable
NP
147–155
and NP
55–69
specific cellular immune response with
NP, more importantly it succeeded in inducing cellular response
against M2e (Fig. 7B), whereas NP didn’t. Morbidity data
showed NP immunized mice experienced significantly greater
weight loss (p,0.01) than mice in group 2, the survival mice in
group 3 began to recover their body weight on day 10, 4 days
later than NM2e immunized mice (Fig. 7C, left). NP
immunization did induce protective immunity, however the
Figure 4. IgG1 and IgG2a isotypes in serum from NM2e-immunized mice. Mice were treated as described in Fig. 3. NP- and M2e-specific IgG
isotypes in mouse serum were analyzed by ELISA. The plots show the (A) NP- (left) and M2e-specific (right) IgG1 isotypes and (B) the respective IgG2a
isotypes. The scatter dot plots show the results for every mouse in each group, and the bars show the geometric mean of each group. The plots in (C)
present the NP-(left) and M2e-specific (right) IgG1/IgG2a ratios, and the bars show the means with SD. Lines above two or more groups indicate that
they have the same comparative results. *, p#0.05; **, p#0.01; ***, p#0.001 by one-way ANOVA.
doi:10.1371/journal.pone.0052488.g004
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PLOS ONE | www.plosone.org 7 December 2012 | Volume 7 | Issue 12 | e52488
survival rate of mice group 3 was significantly lower than that
in group 2 ( p ,0.01) (Fig. 7C, right).
Protective Efficacy is Correlated with Humoral and
Cellular Immune Responses
To better understand the relationship between protective
efficiency and the humoral and cellular immune responses,
correlation coefficients were analyzed. With respect to humoral
immune responses, the survival percentage induced by NM2e
immunization was highly related to NP- and M2e-specific total
IgG, IgG1, and IgG2a antibody levels (Fig. 8A). Moreover, the
survival percentage was markedly related to NP55-69- and M2e-
specific cellular immune responses, but not to NP147-155-specific
cellular immune responses (Fig. 8 B). Thus, the protective efficacy
of NM2e immunization was related to both the humoral and
cellular immune responses.
Discussion
A universal influenza vaccine capable of inducing cross-
protection among heterosubtypic influenza strains is critically
needed to prevent seasonal and pandemic flu outbreaks. The
highly conserved NP and M2 of influenza A virus have been used
as target antigens in the development of universal influenza
vaccines. In mice, previous studies have shown that gene- and/or
vector-based NP+M2 vaccines induced strong humoral and
cellular responses, and protected against lethal influenza virus
challenges, the combination of NP and M2 is superior to the sole
one when the immune response and protection efficacy were
considered [25–31]. Here, we describe a vaccine based on a fusion
protein of NP and M2e expressed in E. coli. In mice, the NM2e
fusion protein elicited robust antibody responses and T cell
responses against NP and M2e. More importantly, mice immu-
nized with NM2e formulated with Al(OH)
3
adjuvant were
protected against lethal challenge with high doses of influenza A
Figure 5. Cellular immune response in NM2e-immunized mice. SMNCs secreting IFN-c, IL-4, or IL-10 upon stimulation were detected by
ELISPOT assay. Groups of six mice were immunized intramuscularly three times at 2-week intervals using 10 mg of NM2e protein in normal saline (NS)
or 10 mg of NM2e formulated with Al(OH)
3
, CpG 1826, or Al(OH)
3
plus CpG 1826. Mice immunized with NS or Al(OH)
3
plus CpG 1826 were treated as
negative controls. All mice in each treatment group were sacrificed on day 38. SMNCs were separated from mouse spleen samples, and 5 mg/ml
NP
147–155
,NP
55–69
, and M2e peptide pool were used as stimulants in the ELISPOT assays. The numbers of SMNCs producing IFN-c (A), IL-4 (B), or IL-10
(C) after stimulation for 40 h with NP
147–155
(left), NP
55–69
(middle), or M2e peptides (right) are presented as spot-forming cells (SFCs)/10
6
SMNCs.
Columns show the average SFCs/10
6
SMNCs, and bars indicate the standard deviation of each group. Lines above two or more groups indicate that
they have the same comparative results. *, p#0.05; **, p#0.01; ***, p#0.001 by one-way ANOVA.
doi:10.1371/journal.pone.0052488.g005
Protection Elicited by NP and M2e Fusion Protein
PLOS ONE | www.plosone.org 8 December 2012 | Volume 7 | Issue 12 | e52488
virus PR8 (20LD50) compared with the relatively low challenge
dose (1 LD90 [12,13] or 4 LD50 [11,17]) in other studies with M2-
based vaccines. It is noticeable here that NM2e did contain the
character of both NP and M2e and induced stronger protective
immune response than NP protein. The present results suggest
that E. coli-expressed NM2e fusion protein is immunogenic in mice
and may be a suitable candidate for a universal influenza vaccine.
It is necessary to understand the immune correlates of
protection for new vaccine types. Here, NM2e immunization
induced a substantial antibody response to M2e in mice, and an
analysis showed that the protection was closely correlated with the
anti-M2e antibody titer. Previous experiments have shown similar
results [16,43,45]. Anti-M2e antibody is unable to neutralize the
virus to prevent infectivity, but it is able to disrupt the viral life
cycle and kill infected cells by the mechanism of antibody-
dependent cell-mediated cytotoxicity (ADCC) [46]. M2e-specific
IgG2a isotype has been proposed as an effective inducer of the
ADCC response [47]. Jegerlehner et al. [46] suggested that IgG2a
levels were correlated with protection against influenza infection in
mice. However, Denis et al. [45] reported that low levels of anti-
M2e IgG2a induced by PapMV-CP-M2e immunization did not
efficiently protect mice against a challenge with 4 LD
50
of
influenza A virus; thus, they considered that anti-M2e IgG1 may
also play a role in M2e-mediated protection. Our results
demonstrated that protection was highly correlated with not only
IgG2a but also IgG1. The M2e peptide contains an MHC class II-
restricted epitope [11], and studies have documented that
influenza-specific CD4+ T cells are involved in immune protection
[11,48]. In the present study, M2e-specific IFN-c-, IL-4-, and IL-
10-secreting SMNCs (mainly CD4+ T cells; data not shown) were
significantly correlated with protection. It was reported that
following influenza infection, antigen-presenting cells secrete IL-
10, which contributes to the differentiation of Th0 cells into Th2
cells; subsequently, Th2 cells secrete IL-4, IL-5, and IL-6, which
help to preferentially drive IgG1, IgA, and IgE antibody pro-
duction by antibody-secreting plasma cells [49]. Th1 cells secrete
IFN-c, which helps to produce IgG2a antibodies [50]. Thus, our
data indicate that both M2e-specific antibodies and CD4+ T cells
contribute to the protection induced by NM2e protein.
Several studies have reported that NP plays a role in the
elimination of influenza virus-infected cells via specific CD8+ killer
T cells [22,26,27,51]. However, recent studies have suggested that
antibodies against NP are necessary for NP immunization to
confer protection and that NP-immune serum can transfer
protection [36,52,53]. Although NP-specific IgG antibodies had
no effect on neutralization and failed to block viral infection of
cells, antibodies to NP may nevertheless provide an unexpected yet
important mechanism of protection against influenza [11,18]. In
the present study, protection against virus challenge was closely
correlated with the presence of anti-NP antibodies, including IgG1
and IgG2a isotypes. Furthermore, the protective effect was
significantly correlated with SMNCs specific for NP55–69 (H-2
d
-
restricted Th epitope), but not SMNCs specific for NP147–155 (H-
2
d
-restricted CTL epitope). It is not surprising that NP-specific
CD8+ T cells were not elicited in response to NM2e immunization
because although gene- and vector-based NP vaccines easily
induce CD8+ T cells, protein vaccines might not, even with CpG
as adjuvant. Further research is needed to identify a suitable
adjuvant that favors the induction of CD8+ T cells in response to
NM2e protein vaccination. Meanwhile, it is essential to identify
the mechanism of action of NM2e immunity and to establish
in vitro assays for measuring immunity.
Adjuvant is required for protein subunit vaccines to elicit
effective and long-lasting immune protection [38]. In this study,
inclusion of the Al(OH)
3
adjuvant, which is widely and safely used
in human vaccines [54], enhanced both humoral and cellular
immune responses elicited by NM2e. However, it should be noted
that alum boosts mainly the Th2 immune response, while it
inhibits the Th1 and CTL response elicited by many antigens
Figure 6. Protec tive efficacy of immunization with NM2e
formulated with Al(OH)
3
and CpG in mice. Groups of 15 mice
were immunized with NM2e protein or NM2e formulated with adjuvant
and were challenged with 20 LD
50
of influenza virus PR8. Mice
immunized with normal saline or adjuvant alone were challenged as
negative controls. Mice were monitored daily for 21 days after PR8
challenge. Mice were weighed daily to detect morbidity (A). Average
weights in each treatment group were followed for the duration of the
study, and the percentage of the original body weight was calculated
based on the average starting weight for each group at day 0. Survival
rates (B) following the challenge within each experimental group were
calculated. Tables above the graph compare the results for groups 3, 4,
5, and 6. *, p#0.05; **, p#0.01; ***, p#0.001; ns, not significant.
doi:10.1371/journal.pone.0052488.g006
Protection Elicited by NP and M2e Fusion Protein
PLOS ONE | www.plosone.org 9 December 2012 | Volume 7 | Issue 12 | e52488
Figure 7. Comparison of the immunogenicity and protection efficacy induced by NM2e and NP. Mice were immunized intramuscularly
with 10 mg of NM2e (g2)or NP protein (g3) formulated with Al(OH)
3
according to the time schedule in Fig. 2. Mice immunized with adjuvant alone
were used as negative controls (g1). Serum, SMNC was prepared from each mouse and analyzed at the indicated times as described in the Materials
and Methods. A), Ab and subtypes against NP (left) and M2e (right) on day 38. Columns show geometric mean antibody titers, and bars indicate the
95% confidence interval in each group (n = 6 mice per experimental group). B), SMNCs secreting IFN-c, IL-4, or IL-10 upon stimulation were detected
by ELISPOT assay. Six mice in each treatment group were sacrificed on day 38. The numbers of SMNCs producing IFN-c (left), IL-4 (middle), or IL-10
(right) after stimulation for 40 h with NP
147–155
,NP
55–69
, or M2e peptides are presented as spot-forming cells (SFCs)/10
6
SMNCs. Columns show the
average SFCs/10
6
SMNCs, and bars indicate the standard deviation of each group. *, p#0.05; **, p#0.01; ***, p#0.001 by one-way ANOVA. C),
Protective efficacy of immunization with NM2e or NP formulated with Al(OH)
3
in mice. Three mice group were challenged with 20 LD
50
of influenza
virus PR8 (n=15). Mice were monitored daily to detect morbidity (left) and mortality (right). *, p#0.05; **, p#0.01; ***, p#0.001.
doi:10.1371/journal.pone.0052488.g007
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[38,54], which was also shown in this study. Different from the
immune mechanism of alum adjuvant, CpG efficiently promotes
Th1 and CTL responses [55–57]. In the present study, inclusion of
CpG alone considerably enhanced the humoral immune response
elicited by NM2e, however it did not enhance the CTL response
markedly. The inclusion of CpG with NM2e improved the anti-
M2e IgG1 titer, but not the IgG2a titer (Fig. 4, right), which was
different from the NP-specific IgG1/IgG2a pattern (anti-NP IgG1
titer decreased and anti-NP IgG2a titer increased). McCluskie MJ
et al [58] once reported CpG together with the E. coli heat-labile
enterotoxin (LT) strengthened the humoral response against some
antigen, while others not, so adjuvanticity may have also depended
on the particular antigen. NP is one of the internal protein of
influenza virus, while M2e is the extracellular domain of the
membrane protein 2, thus we infer CpG showed different
adjuvanticity when two different antigens were used. In addition,
Shim B-S [37] once prepared the construct expressing three
tandem copies of M2e conjugated to C-terminus sequence of M2
protein (3M2eC), their research showed that immunization with
3M2eC induced predominantly IgG1 as compared to IgG2a
subclass, similar to the result in this study.
CpG was expected to complement the immune effect induced
by Al(OH)
3
when used together in the formulation of NM2e
protein. However, CpG plus Al(OH)
3
did not show a clear
synergistic effect on the immunogenicity of NM2e. It is possible
that remnant endotoxin (about 2000 EU/mg) in the NM2e
formulation expressed in E. coli might have interfered with the
effect of CpG. Our recent data indicated that once remnant
endotoxin was removed further from the NM2e protein formu-
lation, CpG and Al(OH)
3
used together did show a clear
synergistic effect on the immune response (Fig. S1) and protective
efficacy (Fig. S2) of NM2e, and inclusion of Al(OH)
3
alone still
markedly improved the immune efficacy of the NM2e vaccine.
In conclusion, we have described a potential universal influenza
vaccine that provides cross-protective immunity against influenza
A virus PR8 in mice. The vaccine is based on the recombinant
fusion protein NM2e expressed in E. coli, which consists of the 23-
amino acid external domain of M2 protein attached to the C-
terminus of NP. When administered with Al(OH)
3
as adjuvant,
NM2e provided almost complete protection against a lethal-dose
challenge of A/PR8 in mice. The combination of Al(OH)
3
and
NM2e offers promising prospects for further vaccine development.
Supporting Information
Figure S1 The study of the immunogenicity of NM2e in pre-
production in mice. The pre-production fermentation and
purification of NM2e were finished in SINOVAC BIOTECH
CO.,LTD., Beijing, China. The concentration of endotoxin in
NM2e protein was decreased to 250 EU/mg after the purification.
Mice were immunized with 10 mg of NM2e (G3), 10 mg of NM2e
formulated with Al(OH)
3
(G5), 10 mg of NM2e formulated with
Al(OH)
3
plus CpG (G6) according to the procedure in Fig. 2. Mice
immunized with NS (G1), Al(OH)
3
plus CpG (G2) were treated as
negative controls. A), Ab and subtypes against NP (left) and M2e
(right) on day 38 were analyzed by ELISA. Columns show
geometric mean antibody titers, and bars indicate the 95%
Figure 8. Correlations between survival percentage and immune responses in mice. A, Correlation analysis was conducted to determine
the relationships of the survival percentage data from Fig. 6 with the NP-, M2e-specific IgG (left) ELISA data from Fig. 3, IgG1 (middle) and IgG2a
(right) ELISA data in Fig. 4. Log conversion was performed for the murine serum antibody titers. B, Correlation analysis was conducted to determine
the relationships of the survival percentage data in Fig. 6 with the IFN-c- (left) IL-4- (middle), and IL-10-secreting (right) SMNCs stimulated with NP147-
155, NP55-69, or M2e peptide pool based on the ELISPOT data in Fig. 5.
doi:10.1371/journal.pone.0052488.g008
Protection Elicited by NP and M2e Fusion Protein
PLOS ONE | www.plosone.org 11 December 2012 | Volume 7 | Issue 12 | e52488
confidence interval in each group (n = 6 mice per experimental
group). B), SMNCs secreting IFN-c, IL-4, or IL-10 upon
stimulation were detected by ELISPOT assay. Six mice in each
treatment group were sacrificed on day 38. The numbers of
SMNCs producing IFN-c (left), IL-4 (middle), or IL-10 (right) after
stimulation for 40 h with NP
147–155
,NP
55–69
, or M2e peptides are
presented as spot-forming cells (SFCs)/10
6
SMNCs. Columns
show the average SFCs/10
6
SMNCs, and bars indicate the
standard deviation of each group. *, p#0.05; **, p#0.01; ***,
p#0.001 by one-way ANOVA.
(TIF)
Figure S2 Protective efficacy of immunization with the formu-
lation of NM2e in pre-production in mice. Immunized mice
(n=15) were challenged with 30 LD
50
of influenza virus A/
Brisbane/59/2007(H1N1)-like (MA) on day 38, then they were
monitored daily to detect morbidity (left) and mortality (right). *,
p#0.05; **, p#0.01; ***, p#0.001.
(TIF)
Author Contributions
Conceived and designed the experiments: WW BH LR. Performed the
experiments: WW BH TJ XW XQ YG. Analyzed the data: WW BH LR.
Contributed reagents/materials/analysis tools: XW. Wrote the paper: WW
LR. Revised the article critically: WW WT LR. Final approval of the
version of the article to be published: LR.
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Protection Elicited by NP and M2e Fusion Protein
PLOS ONE | www.plosone.org 13 December 2012 | Volume 7 | Issue 12 | e52488
    • "Several strategies have been proposed to expand the breadth of immunity against divergent strains of influenza. The use of conserved genes such as the nucleoprotein (NP) and matrix (M) have shown the potential to induce cross-reactive T cell immunity against divergent influenza strains5678. Studies have shown that antibodies directed to the stalk region of HA induce broadly reactive and neutralizing antibodies in humans and mice91011. "
    [Show abstract] [Hide abstract] ABSTRACT: With the exception of the live attenuated influenza vaccine there have been no substantial changes in influenza vaccine strategies since the 1940's. Here we report an alternative vaccine approach that uses Adenovirus-vectored centralized hemagglutinin (HA) genes as vaccine antigens. Consensus H1-Con, H3-Con and H5-Con HA genes were computationally derived. Mice were immunized with Ad vaccines expressing the centralized genes individually. Groups of mice were vaccinated with 1 X 1010, 5 X 107 and 1 X 107 virus particles per mouse to represent high, intermediate and low doses, respectively. 100% of the mice that were vaccinated with the high dose vaccine were protected from heterologous lethal challenges within each subtype. In addition to 100% survival, there were no signs of weight loss and disease in 7 out of 8 groups of high dose vaccinated mice. Lower doses of vaccine showed a reduction of protection in a dose-dependent manner. However, even the lowest dose of vaccine provided significant levels of protection against the divergent influenza strains, especially considering the stringency of the challenge virus. In addition, we found that all doses of H5-Con vaccine were capable of providing complete protection against mortality when challenged with lethal doses of all 3 H5N1 influenza strains. This data demonstrates that centralized H1-Con, H3-Con and H5-Con genes can be effectively used to completely protect mice against many diverse strains of influenza. Therefore, we believe that these Ad-vectored centralized genes could be easily translated into new human vaccines.
    Full-text · Article · Oct 2015
    • "We used CpG and Al(OH) 3 to improve the immunogenicity of the NM2e fusion protein of the influenza virus in a previous report; the adjuvant showed potential efficacy, strengthened the immune response and elicited cross protection after NM2e immunization in mice. A combination of Al(OH) 3 and CpG in the formulation of NM2e induced the highest protection in mice (Wang et al., 2012). In this study, CpG and Al(OH) 3 were employed separately or in combination with 10 mg NP expressed in E. coli to improve its Fig. 3. IgG1 and IgG2a isotypes of serum from NP-immunized mice. "
    [Show abstract] [Hide abstract] ABSTRACT: The highly conserved internal nucleoprotein (NP) is a promising antigen to develop a universal influenza A virus vaccine. In this study, mice were injected intramuscularly with Escherichia coli-derived NP protein alone or in combination with adjuvant alum (Al(OH)3), CpG or both. The results showed that the NP protein formulated with adjuvant was effective in inducing a protective immune response. Additionally, the adjuvant efficacy of Al(OH)3 was stronger than that of CpG. Optimal immune responses were observed in BALB/c mice immunized with a combination of NP protein plus Al(OH)3 and CpG. These mice also showed maximal resistance following challenge with influenza A virus PR8 strain. Most importantly, 10µg NP formulated with Al(OH)3 and CpG induced higher protection than did 90µg NP. These findings indicated that a combination of Al(OH)3 and CpG may be an efficient adjuvant in the NP formulation.
    Full-text · Article · Sep 2014
    • "Thus, the development of vaccines able to trigger strong CD4+ responses could be central for the induction of memory responses capable of combating divergent influenza viruses through multiple pathways. In this context, there is experimental evidence on NP-based vaccines which promote CD4+ T cell responses contributing towards protective immunity [22,66]. This phenomenon is not restricted only to influenza, since numerous infectious models have demonstrated the importance of CD4+ T cells in cellular mediated protection [67,68]. "
    [Show abstract] [Hide abstract] ABSTRACT: There is a critical need for new influenza vaccines able to protect against constantly emerging divergent virus strains. This will be sustained by the induction of vigorous cellular responses and humoral immunity capable of acting at the portal of entry of this pathogen. In this study we evaluate the protective efficacy of intranasal vaccination with recombinant influenza nucleoprotein (rNP) co-administrated with bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) as adjuvant. Immunization of BALB/c mice with two doses of the formulation stimulates high titers of NP-specific IgG in serum and secretory IgA at mucosal sites. This formulation also promotes a strong Th1 response characterized by high secretion of INF-γ and IL-2. The immune response elicited promotes efficient protection against virus challenge. These results suggest that c-di-AMP is a potent mucosal adjuvant which may significantly contribute towards the development of innovative mucosal vaccines against influenza.
    Full-text · Article · Aug 2014
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