Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses.

Abstract

Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus (CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA.
In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P < 0.05) higher S-specific IgG1 in the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those in mice challenged with pci-S alone, the number of B220+ cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-Ad) were increased on CD11c+ dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice.
These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.

Full text

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RESEARCH ARTICLEOpen Access
Intranasal immunization with plasmid DNA
encoding spike protein of SARS-coronavirus/
polyethylenimine nanoparticles elicits antigen-
specific humoral and cellular immune responses
Byoung-Shik Shim1,3†, Sung-Moo Park2,3†, Ji-Shan Quan1,4†, Dhananjay Jere1, Hyuk Chu5, Man Ki Song3,
Dong Wook Kim3, Yong-Suk Jang6, Moon-Sik Yang6, Seung Hyun Han3,7, Yong-Ho Park2, Chong-Su Cho1,
Cheol-Heui Yun1,8*
Abstract
Background: Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus
(CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV.
Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA.
Results: In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with
SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles
appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro.
The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P < 0.05) higher S-specific IgG1 in the
sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those
in mice challenged with pci-S alone, the number of B220+cells found in PEI/pci-S vaccinated mice was elevated.
Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-Ad) were
increased on CD11c+dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The
percentage of IFN-g-, TNF-a- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control
mice.
Conclusion: These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific
humoral and cellular immune responses.
Background
Severe acute respiratory syndrome (SARS) is an emer-
ging infectious disease [1]. In contrast to most other
coronaviruses, which cause mild infection, the new
SARS-CoV has a high mortality rate. Because the
re-emergence of SARS is possible due to existence of
SARS-CoV like strains in animal reservoir, development
of safe and effective vaccines is highly desired. The
SARS-CoV genome is composed of single positive
stranded RNA and encodes four main structural pro-
teins including spike protein (S), membrane protein
(M), envelope protein (E) and nucleocapsid protein (N)
[2]. The S protein is involved in not only receptor
recognition but also in virus attachment and its entry
into target cells [3].
In attempts to develop vaccines against various patho-
gens, research on DNA vaccine has been widely carried
out. Using DNA vaccines, both humoral and cellular
immune responses are induced [4]. A few studies
demonstrated that DNA-based vaccines can induce pro-
tective immune response against several viruses [5,6].
However, one of the problems with DNA-based vaccines
is that they are incapable of inducing immune response
* Correspondence: cyun@snu.ac.kr
† Contributed equally
1Department of Agricultural Biotechnology and Research Institute for
Agriculture and Life Sciences, Seoul National University, Seoul 151-921,
Republic of Korea
Full list of author information is available at the end of the article
Shim et al. BMC Immunology 2010, 11:65
http://www.biomedcentral.com/1471-2172/11/65
© 2010 Shim et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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in mice when injected through the intranasal (i.n.) route
[7]. In light of the fact that the entry of most respiratory
diseases is through the mucosal surface, it is obvious
and ideal that a vaccine should induce both systemic
and mucosal immune responses. Secretory IgA plays a
major role in mediating mucosal immunity [8]. Mucosal
immune responses take an important role as a first line
of immune defense system against influenza virus infec-
tion although parenteral immunization is not enough to
provoke protective immunity [9].
Polyethylenimine (PEI) has been widely used as the non-
viral vector in vitro and in vivo due to high transfection
efficiency and buffering capacity [10]. It has been shown
that mucosal administration with PEI could function as a
potent mucosal immunostimulator [11]. It has been
revealed that PEI is a very effective gene delivery vehicle
for lung transfection producing high antibody titers
against the encoded protein [12]. In the present study, the
immune responses in BALB/c mice immunized with
SARS DNA vaccine via i.n. route have been examined.
Results
Characterization of PEI/pci-S complexes
It is well known that transfection efficiency of gene car-
rier depends upon its ability to condense DNA into
nano-sized particles [13]. As expected, PEI condensed
DNA into nano-sized particles, suggesting their endocy-
tosis potential (Figure 1A).
The formation of PEI/pci-S nanoparticles was further
confirmed by morphology observation. Representative
energy-filtering transmission electron microscopy
(EF-TEM) images of the PEI/pci-S nanoparticles at N/P
ratio 10 are shown in Figure 1B. The nanoparticles were
observed as spherical shape with around 200 nm size,
which are similar to those measured by dynamic light
scattering. It is notable that cytotoxicity of PEI was mea-
sured in RAW 264.7 cells after transfection with PEI/
pci-S complexes by using MTT assay. The cell viabilities
decreased slightly when the N/P ratios of PEI/pci-S
complexes increased. When the N/P ratio was 10, the
cell viability was 87.5 ± 7.3% (data not shown). In order
to confirm PEI/pci-S uptaken by RAW264.7 cells, rho-
damine labeled pci-S DNA was used to form the nano-
particles with the PEI and the complex was visualized
by confocal microscopy. As shown in Figure 1C, the
labeled nanoparticles can be seen in the cells, near to
the nucleus. RT-PCR analysis showed both pci-S DNA
and PEI/pci-S nanoparticles can transfect the cells. In
fact, the PEI/pci-S nanoparticles induced much stronger
S mRNA expression than that of naked DNA (Figure
1D). Western blot analysis further confirmed that the
cells treated with PEI/pci-S nanoparticles can induce the
expression of detectable S protein whereas no protein
was detected when pci-S was used alone (Figure 1E).
SARS-CoV S-specific antibody responses and B cell
proliferation capacity
To evaluate the influence of PEI on adaptive immunity
to SARS-CoV S protein, specific antibody responses
were examined in mice immunized i.n. with SARS-CoV
DNA vaccine. Immunization with PEI/pci-S complexes
elicited high level of SARS-CoV S-specific-serum IgG
antibody but not in mice immunized with SARS-CoV S
DNA alone (Figure 2A). To access the balance of Th1/
Th2 response, SARS S-specific IgG1 and IgG2 were
evaluated in immunized mice. SARS S-specific IgG1
antibody was significantly (P < 0.01) increased in mice
immunized with SARS-CoV S DNA vaccine plus PEI
whereas little increase was observed on SARS-specific
IgG2a antibody production (Figure 2A), indicating Th2
dominant response. To examine mucosal antibody pro-
duction, lung wash, nasal wash, fecal extracts, saliva and
vaginal wash of immunized mice were collected. The
result showed that SARS S-specific IgA antibody
response was significantly (P < 0.01) increased in lung
wash from mice immunized with PEI/pci-S complexes
(Figure 2B).
To assess B lymphocyte proliferation against SARS-
CoV spike protein, the notion that antibody responses
enhanced was further confirmed by proliferation ability
of B220+cells at 1 week after the last vaccination. B220+
cells from mice immunized with PEI/pci-S complexes
were highly proliferated after in vitro re-stimulation
with SARS-CoV S protein (Figure 2C).
Expression of cell surface molecules and maturation of
dendritic cells (DCs) from the mice stimulated with PEI/
pci-S complexes
The maturation of DCs is accompanied with enhanced
expression of surface markers, including co-stimulatory
and MHC class molecules. To examine the effect of
DNA vaccination on DC maturation in vivo, mice were
immunized i.n. with PEI/pci-S complexes. The surface
expression of CD80 and CD86 co-stimulatory molecules
were significantly (P < 0.05) higher on DCs from mice
treated with PEI/pci-S complexes than those from
SARS-CoV DNA S vaccine alone (Figure 3). MHC class
II, I-Ad, expression was also up-regulated significantly (P
< 0.05) in PEI/pci-S complexes group as compared with
that of SARS-CoV DNA alone (Figure 3).
Improvement of SARS-CoV S-specific T cell responses by
PEI/pci-S complexes
To examine T cell immunity to SARS-CoV S DNA
vaccine, cytokine profiles were examined by using intra-
cellular cytokine assays. The cells were harvested from
the lung at 6 days after the immunization. It has been
suggested that T cells producing IFN-g, IL-2, IL-17, and
TNF-a are especially effective in protective immunity
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[14]. Amount of IFN-g-producing cells were increased in
CD4+and CD8+T cells from mice immunized with PEI/
pci-S whereas IL-17-producing cells were increased only
in CD4+, not CD8+T cells. It is notable that IFN-g+,
IL-2+and IL-17+cells were not detected in CD8+
T cells from the mice immunized with PEI/pci-S
complexes (Figure 4A and 4B). Re-stimulation with
SARS-CoV S peptide induced the activation of cytokine-
producing CD4+and CD8+T cells with a predominant
production of TNF-a as well as TNF-a and IL-2 double
cytokine-producing T cells in mice immunized with
PEI/pci-S complexes (Figure 4A and 4B). Furthermore,
PBS
S gene
GAPDH
pci-S PEI/pci-S
mock
pci-S PEI/pci-S
S protein
β-actin
AB
Average 194.7 ? ? 99.3
C
D
E
Figure 1 Characterization of SARS-CoV S DNA vaccine (pci-S)-PEI complexes. (A) Transmission electron micrographic image of PEI/pci-S
complexes at N/P ratio 10. Scale bar represents 0.5 μm. (B) Size distribution of complexes prepared at N/P ratio 10. (C) The cell uptake of PEI/
pci-S complexes was observed by confocal laser scanning microscope. Upper left, intracellular distribution of rhodamine-labeled PEI/pci-S (red).
Upper right, cell nuclei by DAPI staining. Lower left, differential interference images of RAW 264.7 cells. Lower right, overlapping image of nuclei
and rhodamine-labeled PEI/pci-S. (D) Expression of S mRNA in RAW 264.7 cells transfected with PEI/pci-S complexes was detected by RT-PCR. (E)
S protein in RAW 264.7 cells with PEI/pci-S complexes was detected by Western blot.
Shim et al. BMC Immunology 2010, 11:65
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IFN-g and IL-17 double cytokine-producing cells were
found more in the PEI/pci-S complexes group while it
was not detectable in pci-S group. It is to note that IL-
4-producing cells were detectable neither in the lung
nor spleen (data not shown).
Discussion
In the twenty first century, SARS was the first emerging
infectious disease that has seriously threatened public
health and the economy throughout the world [1]. Over
8,000 people from 26 countries were infected with SARS-
coronavirus, resulting 774 deaths [15]. It has been shown
that SARS-CoV spike protein (S) plays an important role
in receptor recognition, virus attachment and its entry
[16]. It represented one of the most important targets for
the development of SARS vaccine [6]. To prevent and con-
trol SARS outbreaks, several vaccine studies based on the
spike protein of SARS have been done including S protein
vaccine, fragment DNA vaccine, full-length DNA vaccines
and receptor binding domain [17]. DNA vaccine encoding
full-length S protein has shown to induce humoral, cellu-
lar and protective immune responses against SARS-CoV
[6]. In the current study, we evaluated the immunogenicity
of a PEI/pci-S in mice through intranasal immunization.
There have been several reports that PEI/DNA com-
plexes enhance transfection efficiency in mammalian
cells and augment immunogenicity [18]. In the present
study, we have adapted this PEI/pci-S complex for
mucosal DNA vaccination. The size of PEI/pci-S com-
plexes appeared to be about 200 nm. The effect of PEI/
DNA complexes on transfection, and gene and protein
expression in RAW 264.7 cells were evaluated by
measuring the expression of the mRNA and protein,
respectively. This result, thus, let us move ahead to in
vivo studies for mouse immunization via intranasal
route using PEI/pci-S complexes.
A number of studies attempted to develop SARS DNA
vaccines, exclusively via systemic routes including
Vaginal wash
Fecal
Saliva
Nasal wash
Lung wash
pci-Mock
pci-Spci-S + PEI
B
**
IgG
IgG1
IgG2a
Absorbance at 450nm
pci-Mock
pci-Spci-S + PEI
A
**
**
*
pci-Mock
pci-Spci-S+PEI
CFSE
Cell NumberCell Number
2.57
8.23
19.7
C
Figure 2 SARS-CoV S protein-specific humoral and mucosal immune responses in BALB/c mice intranasally immunized with PEI/pci-S
complexes. The samples were collected on day 7 after the last immunization. Induction of (A) S-specific IgG subclasses in serum and (B) S-
specific IgA in various mucosal samples were determined by ELISA. The results were expressed as means ± SEM for the group (n = 5 to 8).
Significant differences compared with pci-S group were expressed as *P < 0.05 and **P < 0.01, respectively. (C) Proliferation activity of B220+
cells from spleen of mice immunized with PEI/pci-S complexes. Bar and number in each panel present the percentage of proliferated cells.
Shim et al. BMC Immunology 2010, 11:65
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intramuscular injection [19]. As SARS is a respiratory
pathogen, among the SARS vaccine candidates, targeting
intranasal immunization was likely to be more effective
to induce protective immune responses against infection
when compared with other delivery routes. Intranasal
immunization of PEI/pci-S complexes induced higher
antigen-specific serum IgG responses than pci-S alone.
Coincidently, antigen-specific IgG1 was also dramatically
increased when compared to IgG2a, suggesting Th2
dominant response. Also the immunization enhanced
antigen-specific IgA response in bronchoalveolar lavage
fluid and B cell proliferation after in vitro re-stimulation
with the spike protein. In Garzon’s study, antigen-speci-
fic antibody and T cell responses have been dose-depen-
dently increased in mice immunized with DNA vaccine
up to 100 μg [20]. However, in our study each mouse
was immunized with only 20 μg of DNA. Despite the
small amount of DNA, it has induced not only systemic
but also mucosal immune responses.
DCs play a pivotal role for the effective induction of
antigen-specific immune responses. DCs are found
throughout the body and are considered as one of the
first-line sentinel cells [21]. When DCs recognize
pathogen-associated molecular patterns from microor-
ganisms, DCs became mature and acquired capacity
for the antigen presentation, concomitantly augmented
the expression of MHC proteins [22], cytokines [23]
and number of co-stimulatory molecules including
CD80, CD83, and CD86 [24]. Thus, the maturation of
DCs is essential for appropriate initiation of the subse-
quent adaptive immune responses [22]. In the present
study, we demonstrated that the PEI/pci-S complexes
increase the co-stimulatory and MHC class II mole-
cules on DCs from cervical lymph nodes after intrana-
sal immunization.
The cellular immune responses are mediated by both
CD4+and CD8+T cells. Functional study indicated that
antigen-specific T cells produce cytokines including
IFN-g, TNF-a, IL-2, and IL-17 after in vitro re-stimula-
tion with SARS spike peptides. IFN-g is an effector cyto-
kine, critical for activating macrophages and DCs and
inhibiting viral infection [25]. TNF-a is a cytokine that
probably regulates immune cells and inhibits viral repli-
cation [26]. IL-2 mediates the expansion of T cells and
maintains memory T cells [27]. IL-17 mediates the pro-
duction of antimicrobial peptide and immunoglobulin
for neutralizing viral infection [28]. In the current study,
we have shown that antigen-specific CD4+and CD8+T
cells secreted IFN-g, TNF-a, IL-2, and IL-17 in non-
lymphoid tissues such as lung. Furthermore multiple-
cytokine producing cells are increased in mice immu-
nized i.n. with PEI/pci-S. It is probable that these cells
are likely responsible for the protection when host is
infected with SARS-CoV after the vaccination. In fact, it
has been suggested that multi-cytokine producing anti-
gen-specific CD4+T cells are functionally superior on
protection to single-cytokine producing cells [29]. There
are several reports that multi-cytokine producing T cells
have shown to correlate with protection against Leish-
mania major infection [30]. In the current study, the
PEI/pci-S complexes induced a high frequency of TNF-
a+IL-2+CD4+T cells and IFN-g+IL17+CD4+T cells,
and TNF-a+IL-2+CD8+T cells when assessed by SARS
peptide recall responses. Taken together, our results
support the hypothesis that intranasal immunization
with PEI/pci-S complexes induces ideal cellular
responses for the protection.
Conclusion
PEI is effective in delivering DNA onto the mucosal sur-
face, in maturation of dendritic cells and in improving
the immunogenicity of the DNA vaccine. Our results
indicated that PEI can be used as a vector for the muco-
sal delivery of DNA vaccine and play an important role
in B cell and T cell immunities.
Methods
Construction of plasmid expressing SARS-CoV S protein
The gene encoding SARS-CoV spike (S) protein without
transmembrane domain (amino acids 14-1154) was
synthesized. The sequence was codon optimized for
mammalian cell expression and the natural signal
CD80CD83CD86CCR7I-Ad
Mock44.0 ? ? 3.4 38.9 ? ? 2.349.9 ? ? 3.435.4 ? ? 2.51379.0 ? ? 25.2
pci-S35.2 ? ? 0.119.0 ? ? 0.551.4 ? ? 0.137.3 ? ? 5.41379.1 ? ? 0.6
pci-S+PEI61.3 ? ? 3.7 *20.2 ? ? 0.777.2 ? ? 3.7 *41.3 ? ? 3.81791.2 ? ? 40.9 *
A
pci-Mock
pci-S
pci-S+PEI
B
Cell Number
I-Ad
Figure 3 Expression of cell surface molecules, CD80, CD83,
CD86, CCR7 and MHC class II, on CD11c+cells in cervical
lymph nodes from mice immunized with PEI/pci-S complexes.
BALB/c mice were immunized with PBS, pci-mock, pci-S, or PEI/pci-S
complexes and then cell surface molecules on CD11c+cells from
cervical lymph nodes were analyzed on day 3 after the last
immunization. (A) The expressions of major cell surface molecules,
CD80, CD83, CD86, CCR7, and MHC class II (I-Ad) on CD11c+DC
were determined by flow cytometry. Data were expressed as the
mean value of mean fluorescence intensity ± SD. Significant
differences compared with pci-S group were expressed as *P < 0.05.
(B) Expression of MHC class II molecules was represented by
histogram.
Shim et al. BMC Immunology 2010, 11:65
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