Evaluation on the Efficacy and Immunogenicity of Recombinant DNA Plasmids Expressing Spike Genes from Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus
et al. (2013) Evaluation on the Efficacy and Immunogenicity of Recombinant DNA Plasmids Expressing Spike Genes
from Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus. PLoS ONE 8(3): e57468. doi:10.1371/journal.pone.0057468
Evaluation on the Efficacy and Immunogenicity of Recombinant DNA Plasmids Expressing Spike Genes from Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus
Fandan Meng 0
Yudong Ren 0
Siqingaowa Suo 0
Xuejiao Sun 0
Xunliang Li 0
Pengchong Li 0
Wei Yang 0
Guangxing Li 0
Lu Li 0
Christel Schwegmann-Wessels 0
Georg Herrler 0
Xiaofeng Ren 0
Odir A. Dellagostin, Federal University of Pelotas, Brazil
0 1 College of Veterinary Medicine, Northeast Agricultural University , Harbin , China , 2 College of Life Sciences, Northeast Agricultural University , Harbin , China , 3 Institute of Virology, University of Veterinary Medicine , Hannover , Germany
Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PDEV) can cause severe diarrhea in pigs. Development of effective vaccines against TGEV and PEDV is one of important prevention measures. The spike (S) protein is the surface glycoprotein of TGEV and PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In this study, the open reading frames of the TGEV S1 protein and in addition of the S or S1 proteins of PEDV were inserted into the eukaryotic expression vector, pIRES, resulting in recombinant plasmids, pIRES(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S). Subsequently, 6-8 weeks old Kunming mice were inoculated with both DNA plasmids. Lymphocyte proliferation assay, virus neutralization assay, IFN-c assay and CTL activity assay were performed. TGEV/PEDV specific antibody responses as well as kinetic changes of T lymphocyte subgroups of the immunized mice were analyzed. The results showed that the recombinant DNA plasmids increased the proliferation of T lymphocytes and the number of CD4+ and CD8+ T lymphocyte subgroups. In addition, the DNA vaccines induced a high level of IFN-c in the immunized mice. The specific CTL activity in the pIRES-(TGEV-S1-PEDV-S) group became significant at 42 days postimmunization. At 35 days post-immunization, the recombinant DNA plasmids bearing full-length S genes of TGEV and PEDV stimulated higher levels of specific antibodies and neutralizing antibodies in immunized mice.
Funding: The authors acknowledge National Natural Science Foundation of China (31270187), The Program for New Century Excellent Talents in University of
Ministry of Education of P.R. China (NCET-10-0144), Sponsored by Chang Jiang Scholar Candidates Programme for Provincial Universities in Heilongjiang, Research
Team Program on Scientific and Technological Innovation in Heilongjiang Provincial University (2011TD001), Innovation Talent Project (Excellent discipline leader)
of the Harbin Science and Technology Bureau (RC2012XK002003). 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.
Transmissible gastroenteritis (TGE) and porcine epidemic
diarrhea (PED) are both severe enteric diseases in newborn piglets
which are characterized by extremely high mortality, as well as by
devastating economic consequences for swine industry [3,32,35].
The etiologic agents responsible for these diseases are
coronaviruses, TGEV and PEDV, respectively. TGEV was isolated for the
first time in 1946 . Japan and England reported the disease in
1956 and 1957 [12,31]. The virus replicates in the cytoplasm of
mature absorptive epithelial cells present on the tips of the villi in
the small intestine. The functions of the coronavirus spike (S)
protein are both attachment to the cell surface and fusion of the
viral membrane with the cellular membrane [7,36]. The S protein
is the major inducer of TGEV-neutralizing antibodies [11,15,19].
Therefore, it is an excellent target protein candidate for vaccine
development. The relevant epitopes for neutralization were
mapped to the N-terminal domain of S protein, and four antigenic
sites (A to D) were identified within the first 543 of the 1447
residues of the S protein [13,20]. The first 37% of the polypeptide
chain of the S protein appear to be more immunogenic than the
rest of the sequence. This region would be located in the globular
part of the peplomer, which is more exposed than the fibrillar,
Cterminal portion of the S protein . Previous reports show that
the immunogenicity of the DNA vaccine comprising the main
antigenic sites is superior to a vaccine containing the total length S
PEDV is related to TGEV and bears similarities in its structure
as well as in the clinical disease and lesions induced [1,9]. PEDV
was first separated in Belgium and the United Kingdom in 1978
[2,28,47]. The disease is characterized by severe diarrhea,
vomiting, dehydration, and death, and has a mortality rate of up
to 90% . Since 1978, the disease has frequently broken out in
many swine-raising countries and has resulted in severe economic
losses in Asia, notably in China, Japan and Korea [6,14,18]. In
1996, PED outbreaks have been reported to be responsible for the
death of more than 39,000 piglets in Japan . PED caused not
only the death of neonatal piglets, but also the weight loss in
fattening pigs due to PEDV-induced diarrhea. Therefore, it is
important to develop an effective vaccine preventing PEDV
The PEDV S protein also plays an important role in induction
of neutralizing antibodies, specific receptor binding and cell
membrane fusion . The S protein is not cleaved into S1 and
S2 subunits by furin-like proteases, due to the lack of appropriate
cleavage sites. The S1 domain (residues 1789) and the S2 domain
are artificially defined on the S protein (residues 7901.383)
[10,34]. Previous reports have shown that the main neutralizing
epitopes are located on the S1 domain that is thought to form the
globular part of S protein [34,39]. Sun et al. (2007) reported that
the epitope region designated S1D (aa 636,789) on the S1
domain of PEDV S protein is highly conserved across PEDV
isolates and that this region has the capacity to induce the
production of virus neutralization antibodies. Moreover, the
immune serum against S1D showed the binding ability to the
native S protein of PEDV. The S1D5 (aa 744759) and S1D6 (aa
756771) are two linear epitope domains. Furthermore, the SS2
(748 YSNIGVCK 755-) and SS6 (-764 LQDGQVKI 771-) are two
core epitope domains on S1D5 and S1D6, respectively, located on
the S protein of PEDV . According to the sequence
information for the neutralizing epitope of the transmissible
gastroenteritis virus (TGEV), the neutralizing epitope domain
COE (aa 499638) that is responsible for inducing virus
neutralizing antibodies on the S1 domain of PEDV S protein
was identified. It is highly conserved in PEDV strains [17,41]. A
novel antigenic domain -1368GPRLQPY1347- motif has excellent
immunogenicity has been identified on the C-terminal portion of
the S protein, which elicited a strong antibody response and
induced neutralizing antibodies .
For the protection of neonatal and older animals from disease,
vaccination is an effective prevention measure. Although, there are
many commercial vaccines, the traditional inactivated vaccines
have many deficiencies. Therefore, the two diseases are still major
problems in the swine industry [3,16,27]. The S protein is
considered to be a primary target antigen for developing an
effective vaccine against coronaviruses. There are no reports
comparing the efficiency of S and S1-based vaccines against
PEDV; therefore, we evaluated a combined vaccination approach
based on the plasmid-driven expression of two proteins, the
TGEV S protein and the PEDV S or S1 proteins using a mouse
model. This investigation is a first step for development of bivalent
vaccines against TGE and PED.
Materials and Methods
All animal experiments are approved by the Ethics Committee
of Northeast Agricultural University within the contract frame
from Program for New Century Excellent Talents in University of
Ministry of Education of P.R. China (NCET-10-0144).
Construction of plasmids containing genes encoding
TGEV-S1, PEDV-S1 and PEDV-S
The plasmid pcDNA3.1-TGEV-S containing the full-length S
gene of TGEV strain Purdue was constructed in our laboratory by
common cloning techniques and used as a template for PCR
amplification. A sense primer P1 (5
GGGGGCTAGCCCATGAAAAAACTATTTG 3) and an antisense primer P2 (5
CCCCGAATTCTTAGTTTGTCTAATAA 3) which contain
NheI (P1) and EcoRI (P2) restriction enzyme sites (underlined), were
used for PCR. The plasmid Easy-T-S containing the full-length S
gene of PEDV strain CV777 was constructed in our laboratory by
common cloning techniques and used as a template for PCR
amplification. A sense primer P3 (5
GGGGGTCGACATGGATGTCACTAGGTGCC 3), two antisense primers P4
(5CCCCGCGGCCGCTCAAATACTCATACTAAA 3) and
P5 (5 CCCCGCGGCCGCTCATCTCTGCACGTGGAC 3)
which contain SalI (P3) and NotI (P4 and P5) restriction enzyme
sites (underlined), were used for PCR.
The amplification profile for the TGEV S1 gene (primers P1/
P2) was as follows: 95uC for 5 min followed by 30 cycles of 94uC
for 1 min, 54.4uC for 1 min, and 72uC for 3 min. A final
extension of 72uC for 10 min was performed at the end of the
cycling period. The amplification profile for PEDV-S1 gene
(primers P3/P4) and PEDV-S gene (primers P3/P5) were the same
as for the TGEV-S1 gene except that the annealing temperature
was raised to 56.8uC and 56.4uC, respectively. All PCR products
were purified prior to cloning into the eukaryotic expression vector
pIRES (TaKaRa, Japan). Briefly, TGEV-S1 was digested with
NheI and EcoRI and then inserted into the multiple clone site
(MCS) A of the pIRES vector, resulting in a recombinant plasmid
pIRES-TGEV-S1; PEDV-S1 and PEDV-S were digested with SalI
and NotI and then inserted into MCS B of pIRES-TGEV-S1,
respectively. The resulting recombinant plasmids were designated
as pIRES-(TGEV-S1-PEDV-S1) and
pIRES-(TGEV-S1-PEDVS), respectively. The cloning steps are shown in Figure 1. After
transformation, clones were picked and validated by DNA
TGEV S1 and PEDV S1 proteins were expressed in E.coli,
respectively for lymphocyte stimulation assays. In brief, two
recombinant plasmids pGEX-S1 comprising the 59-end half of the
TGEV S gene  and pET30a-S1 comprising 59-end of PEDV S
gene (97 bp-1776 bp) were constructed and transformed into
E.coli BL21 (DE3) pLysS (Novagen, Germany). Expression and
purification of TGEV S1 protein were detailed in a recent
reference  and PEDV S1 protein was also obtained according
to a similar protocol. The expressed fused proteins were named
TGEV-S1and PEDV-S1, respectively,
Preparation of plasmid DNA and Western blot of
transformed BHK-21 cells
Plasmid DNAs pIRES-(TGEV-S1-PEDV-S1),
pIRES-(TGEVS1-PEDV-S) and pIRES were chromatographically purified from
bacterial lysates (Qiagen, Germany), precipitated, and then
washed with ethanol. All plasmids were dissolved in 0.1 M PBS
to a final concentration of 1 mg/ml. For immunofluorescence
assays, BHK-21 cells from American Type Culture Collection
(ATCC) were cultured to 90% confluency in 6-well plates at 37uC
(approx 24 h) then transformed with 3 mg of
pIRES-(TGEV-S1PEDV-S1) and pIRES-(TGEV-S1-PEDV-S), or pIRES using
lipofectamine 2000. The next day, indirect immunofluorescence
assays were performed with modifications as described [22,24,37].
After fixation by formalin, the cells were incubated for 20 min
with 0.2% Triton X-100. Followed by incubation for 1 h with
polyclonal antisera (1:200 dilution in 1% BSA) against PEDV
(prepared in our laboratory) or TGEV (prepared in our
laboratory). After washing with PBS, the cells were incubated in
the dark with FITC-labeled goat anti-rabbit IgG (1:500) for 1 h.
The green fluorescence signals were analyzed by fluorescence
microscopy (Leica, Germany). For Western blot, 6 mg of each
plasmid were transformed into BHK-21 cells in 6-well plates.
BHK-21 cells transfected with empty vector were used as control.
After 24 hours, cells were treated with 1% Triton X-100 and
centrifuged at 12,000 rpm for 5 minutes, respectively. Then the
supernatants were subjected to 10% SDS-PAGE. The proteins
were electronically transferred to a nitrocellulose (NC) membrane.
The NC membrane was blocked overnight at 4uC using 5%
nonfat dry milk in PBS-0.05% Tween-20 (PBST) followed by
incubation with the anti-TGEV or anti-PEDV serum (1:300
dilution in PBST) at 37uC for 1 h. The membrane was incubated
with horseradish peroxidase (HRP)-conjugated goat anti-rabbit
IgG (1:2000 dilution in PBST) at 37uC for 1 h, after complete
washing with PBST. The protein bands were visualized using
chemiluminescence reagents (Roche, Switzerland).
Immunization of mice
All animal studies were preapproved by the Animal Ethics
Committee of Northeast Agricultural University, China (approval
ID 1155-NCET-005). Prior to DNA vaccination, six-week-old
Kunming mice (Harbin Veterinary Research Institute) were
separated into 8 groups (Table 1) and injected in the medial
vastus muscle with lidocaine hydrochloride (27 ga needle; 50 ml of
0.8% v/v in PBS). After 15 min, the mice received by similar
injection, 100 mg of pIRES-(TGEV-S1), pIRES-(PEDV-S1),
pIRES-(PEDV-S) pIRES-(TGEV-S1-PEDV-S1) and
pIRES(TGEV-S1-PEDV-S), attenuated vaccine for TGEV and PEDV,
pIRES or PBS (100 mg each). The mice were boosted twice, each
at 2-week intervals as shown in Table 1.
Antibody detection and T lymphocyte analysis
Peripheral blood was collected by orbital bleeds at 3 h and then
at 7, 14, 21, 28, 35 and 42 days post-immunization (dpi); serum
and T lymphocytes were subsequently prepared. For detecting
TGEV and PEDV specific antibodies, recombinant TGEV-S1
and PEDV-S1 protein purified from pGEX-(TGEV-S1) and
pET(PEDV-S1) transformed bacterial cells was diluted to 50 mg/ml in
0.05 M NaHCO3. An ELISA was performed according to
published protocols with modifications [22,24]. To assess antibody
binding, ELISA wells were incubated with o-phenylenediamine
dihydrochloride substrate for 15 min. The reactions were
terminated with 50 ml 2M H2SO4 and the wells read at 490 nm.
Peripheral blood lymphocytes were purified using lymphocyte
separation solution (Invitrogen, USA) according to the
manufacturers instructions. Cells were suspended to 16107 cells/ml in
RPMI 1640 medium containing 10% serum and further prepared
as described . Prepared cells were then incubated at 4uC with
Attenuated TGEV/PEDV vaccine
Table 1. Experimental parameters and Cytokine levels in the blood of immunized mice (2XA22XB 6s).
Mice were immunized with PBS (A), pIRES (B), pIRES-(TGEV-S1-PEDV-S1) (C), pIRES-(TGEV-S1-PEDV-S) (D), pIRES-(PEDV-S1) (E), pIRES-(PEDV-S) (F), pIRES-(TGEV-S1) (G) or
Attenuated TGEV/PEDV vaccine (H). The number of mice, DNA dosage, and number of immunizations are indicated. The levels of IFN-c and IL-4 in the serum of
immunized mice were analyzed at 42 dpi using commercially available ELISA kits. w: p,0.01 (highly significant), compared with PBS, pIRES groups.
FITC-conjugated anti-CD4+ T cell antibody or PE-conjugated
anti-CD8+ T cell antibody (1:1000 dilutions) (Zhongshan, China)
for 30 min. After incubation, the cells were washed with cold PBS
(3X), suspended in PBS and subjected to flow cytometry. Splenic
lymphocytes were similarly prepared and analyzed.
Proliferation of T lymphocytes from immunized mice
Splenocytes from immunized mice were prepared for
lymphocyte proliferation assays as described . Prepared cells (50 ml of
26106 cells/ml) were suspended in RPMI1640 containing 10%
serum then transferred to 96-well, flat-bottom plates. To each well,
50 ml of medium containing either 20 mg/ml purified recombinant
TGEV-S1 and PEDV-S1 proteins or Concanavalin A (Sigma) was
added; all treatments were performed in triplicate. Plates were
incubated for 72 h, supplemented with 10 ml/well of
then incubated for an additional 4 h. Reactions were terminated
by adding an equal volume of DMSO and incubating the plates at
room temperature for 10 min. Proliferation was measured by
OD490 values. Proliferation of T lymphocytes in PBMCs from
vaccinated and control mice was similarly monitored.
Neutralization of PEDV and TGEV with immune mouse
To determine if mice generated TGEV and PEDV neutralizing
antibodies, sera (1:201:320 dilution, 300 ml) from
DNA-vaccinated mice were mixed with an equal volume of PEDV or TGEV
(105 pfu/ml) at 37uC. After 1 h incubation, the treated viruses
were used to infect cultured Swine testis (ST) Cells and African
green monkey kidney (Vero) cells in 24-well plates respectively
followed by overlaying with 1% methylcellulose. The plates were
incubated at 37uC in a 5% CO2 atmosphere and examined daily
for 3 days for TGEV and PEDV specific cytopathic effects (CPE).
Indirect detection of IFN-c
Serum IFN-c levels were analyzed with an IFN-c detection kit
(Excell Bio., China) according to the manufacturers instructions.
A standard curve was generated using control IFN-c diluted in
PBS at different concentrations beginning with 10,000 pg/ml
followed by two-fold serial dilutions between 2000 pg/ml and
61.25 pg/ml. Dilutions were subsequently coated onto ELISA
plates overnight at 37uC. Sera (1:100) from 42 dpi mice were also
coated onto ELISA plates and used as primary antibodies in a
parallel experiment to evaluate the virus-derived IFN-c response.
HRP-conjugated goat-anti mouse (1:2000) was used as secondary
antibody in both the control experiment and in the analysis of the
mouse sera. The OD490 values and therefore pg/ml of IFN-c in
immunized mice were determined relative to the IFN-c standard
Serum IL-4 levels were similarly analyzed using an IL-4
detection kit (Excell Bio. China). Control IL-4 was serially-diluted
two-fold in PBS between 500 pg/ml and 7.8 pg/ml then coated
onto ELISA plates at 37uC overnight. The ELISA was performed
as above and OD490 values (pg/ml) were determined relative to an
IL-4 standard curve.
Cytotoxicity (CTL) assay
Cytotoxicity was analyzed using a lactate dehydrogenase (LDH)
release assay kit according to the manufacturers instructions
(Jiancheng, Nanjing, China) using splenic lymphocytes and blood
from 42 dpi mice. Lymphocytes, suspended in complete RPMI
1640 medium were used as effector cells. Simultaneously, target
ST and Vero cells were infected with TGEV and PEDV,
respectively, at a titer of 100TCID50 for 36 h at 37uC under 5%
CO2. The effector cells were mixed with the sensitized target cells
at 25:1, added to each well of a 96-well round-bottom microplate,
and incubated for 6 h at 37uC. After centrifugation at 1500 rpm
for 10 min, 100 ml of supernatant was collected and transferred to
a fresh 96-well flat-bottomed plate, followed by the addition of
100 ml/well of LDH assay reagent. The mixture was allowed to
incubate for 15 min at 37uC after which the OD490 was measured.
Spontaneous release of LDH was determined using samples
prepared from target cells cultured in medium alone; maximum
LDH release was measured using samples prepared by lysis of
target cells in medium containing 1% (v/v) Triton X-100. The
CTL was calculated using the following equation: [(experimental
release-spontaneous release)/(maximum release-spontaneous
release)] x 100. All experiments were performed in triplicate.
Statistical analysis of the data was performed using SPSS 11.5
software; p,0.05 and p,0.01 were defined as statistically
significant and statistically very significant, respectively.
In vitro expression of pIRES-(TGEV-S1-PEDV-S1) and
Sequence analysis of pIRES-(TGEV-S1-PEDV-S1) and
pIRES(TGEV-S1-PEDV-S) showed 100% identity of the S sequences
compared to those of GenBank accession numbers DQ811789.1
(TGEV-S1) and AF353511.1 (PEDV-S1 and S), respectively.
Analysis by immunofluorescence microscopy showed that
transfection of BHK-21 cells with pIRES-(TGEV-S1-PEDV-S1) and
pIRES-(TGEV-S1-PEDV-S) plasmids resulted in the expression of
the S or S1 proteins, respectively, of TGEV and PEDV (Figure 2).
Western blot further confirmed the in vitro expression of these
genes of interest (Figure 3).
T lymphocyte proliferation in spleen and blood
To determine the immune response of mice to the DNA
vaccination, plasmids were applied by intramuscular injection. At
different times after vaccination, animals were sacrificed and
analyzed for various parameters of the immune reaction. The
proliferation of spleen T lymphocytes upon stimulation with
purified S1 protein from either TGEV or PEDV was analyzed by
MTT assays. As shown in Figure 4A, when stimulated with
PEDV-S1 protein at 28 dpi, the proliferation levels of spleen T
lymphocytes from mice injected with
pIRES-(TGEV-S1-PEDVS1) and pIRES-(PEDV-S1) were increased compared to those of
control cells and the increase was highly significant (p,0.01). By
42 dpi, the proliferation levels of cells from mice injected with
pIRES-(TGEV-S1-PEDV-S1) or pIRES-(PEDV-S1) were
somewhat lower but still were significantly increased when compared to
the control cells (p,0.01). By contrast, the proliferation levels of
Figure 3. Western blot of cells transfected with recombinant plasmids. BHK-21 cells were transfected with pIRES-(TGEV-S1-PEDV-S1) or
pIRES-(TGEV-S1-PEDV-S). Transient expression of proteins within the transfected cells was detected by Western blot with anti-TGEV antibodies (Panel
A) and anti-PEDV antibody (Panel B), respectively. Panel A; (1): cell control; (2): pIRES vector control. (3): Cells transfected with
pIRES-(TGEV-S1-PEDVS1) and (4): Cells transfected with pIRES-(TGEV-S1-PEDV-S). Panel B; (1): cell control; (2): pIRES vector control. (3): Cells transfected with
pIRES-(TGEVS1-PEDV-S1) and (4): Cells transfected with pIRES-(TGEV-S1-PEDV-S).
lymphocytes from animals immunized with
pIRES-(TGEV-S1PEDV-S) or pIRES-(PEDV-S) were highest at 42 dpi (p,0.01); no
significant differences (p.0.05) were observed between the groups
of pIRES-(TGEV-S1-PEDV-S) and pIRES-(PEDV-S). The
proliferation values upon stimulation by TGEV-S1 protein are shown
in Figure 4B. By 28 dpi the proliferation levels of spleen T
lymphocytes from animals injected with
pIRES-(TGEV-S1PEDV-S1), pIRES-(TGEV-S1-PEDV-S) or pIRES-(TGEV-S1)
were all significantly increased (p,0.01) relative to the PBS or
pIRES controls. Furthermore, the values of the
pIRES-(TGEVS1-PEDV-S1) group were somewhat higher than those of the
pIRES-(TGEV-S1-PEDV-S) group (p,0.05). By 42 dpi, the
changes in the proliferation levels relative to controls remained
highly significant (p,0.01) irrespective of the plasmid used for
vaccination, pIRES-(TGEV-S1-PEDV-S1) or
pIRES-(TGEV-S1PEDV-S). No significant differences (p.0.05) were observed
between the 28 dpi and 42 dpi values. Assays using PBMC to
determine the proliferation of peripheral blood lymphocytes upon
stimulation by PEDV-S1 protein were performed. At 14 dpi the
proliferation of blood lymphocytes from mice immunized with
pIRES-(TGEV-S1-PEDV-S1) or pIRES-(TGEV-S1-PEDV-S)
was significantly increased (p,0.05) relative to the PBS and
pIRES controls (Figure 4C) and this difference remained
significant (p,0.01) when cells were analyzed at 42 dpi. The
values of the two time points were similar in the case of the mice
immunized with pIRES-(TGEV-S1-PEDV-S). In the
pIRES(TGEV-S1-PEDV-S1) group, the proliferation values at 42 dpi
were slightly but significantly lower (p,0.05). As shown in
Figure 4D, when stimulated with TGEV-S1 protein, the
proliferation of PBMC was increased at 28 and 42 dpi in mice
immunized with pIRES-(TGEV-S1-PEDV-S1) and
pIRES(TGEV-S1-PEDV-S) (p,0.01) relative to PBS and pIRES
controls. In the group pIRES-(TGEV-S1-PEDV-S), the
proliferation significantly increased from 28 to 42 dpi whereas the
proliferation of PBMC from mice immunized with
pIRES(TGEV-S1-PEDV-S1) was similar at both time points.
Changes in CD4+ and CD8+ T lymphocytes
Flow cytometry data showed that the levels of CD4+ and CD8+ T
lymphocytes in peripheral blood gradually increased from 14 dpi to
42 dpi (Figure 3). The levels of CD4+ cells (Figure 5A) in mice
immunized with pIRES-(TGEV-S1-PEDV-S) was significantly
(p,0.01) increased already at 28 dpi. By 42 dpi the number of
CD4+ T cells from mice immunized with
pIRES-(TGEV-S1-PEDVS1) was also increased but the value for mice immunized with
pIRES(TGEV-S1-PEDV-S) was higher. In the case of CD8+ cells, cell
numbers were increased at 28 dpi in mice immunized with
pIRES(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S) to a similar
extent (Figure 5B). A further increase by 42 dpi was observed in the
pIRES-(TGEV-S1-PEDV-S) group (p,0.05) whereas in the
pIRES(TGEV-S1-PEDV-S1) group at 42 dpi only a slight increase was
determined. When spleen CD4+ lymphocyte numbers were examined
by flow cytometry, the results again showed a gradual increase from
14 dpi to 42 dpi (Figure 6A), and the highest value was determined for
the pIRES-(TGEV-S1-PEDV-S) group at 42 dpi. Analyses of
spleenderived CD8+ T cells (Figure 6B) in animals immunized with
pIRES(TGEV-S1-PEDV-S1) or pIRES-(TGEV-S1-PEDV-S) showed a
clear increase of the cell numbers by 28 and 42 dpi.
Antibody detection in immunized mice
Serum antibody levels against PEDV and TGEV were examined
in mice immunized with plasmid DNA using an indirect ELISA. In
general, between 14-42 dpi, the levels of PEDV antibodies in mice
immunized with pIRES-(TGEV-S1-PEDV-S1) and
pIRES(TGEV-S1-PEDV-S) were similar and they were significantly
(p,0.01) higher than those from animals injected with PBS or
empty vector (Figure 7A). As shown in Figure 7B, the level of TGEV
antibodies began to increase at 28 dpi and reached the peak at
35 dpi. Between 3542 dpi, the antibody levels decreased
significantly (p,0.01) in the pIRES-(TGEV-S1-PEDV-S1) group, but not
in the pIRES-(TGEV-S1-PEDV-S) group.
Assays for neutralizing anibodies against PEDV (Figure 8A)
showed a concentration-dependent decrease of the inhibitory
activity in sera of mice immunized by pIRES-(PEDV-S1),
pIRES(PEDV-S), pIRES-(TGEV-S1-PEDV-S1) and
pIRES-(TGEV-S1PEDV-S); higher titers were determined in sera from mice
immunized with pIRES-(PEDV-S) and
pIRES-(TGEV-S1-PEDVS) constructs as compared to animals injected with
pIRES-(PEDVFigure 4. Changes in T lymphocyte numbers in the spleens and peripheral blood of immunized mice. The proliferation of T lymphocytes
in mouse spleens (A and B) and peripheral blood (C and D) was analyzed by conventional MTT assays using recombinant PEDV-S1 (A and C), TGEV-S1
(B and D) protein and Con A as stimulating agents. The y-axis represents the lymphocyte proliferation index (OD490) in the spleen or peripheral blood.
w: p,0.01 (highly significant) compared with PBS group and pIRES group.
Figure 5. Changes in CD4+ and CD8+ T lymphocytes in the peripheral blood of immunized mice. Lymphocytes from the peripheral blood
of mice treated with recombinant plasmids were collected and subjected to flow cytometry to assess the numbers of CD4+ T lymphocytes (A) and
CD8+ T lymphocytes (B). w: p,0.01 (highly significant), compared with PBS group and pIRES group.
S1) and pIRES-(TGEV-S1-PEDV-S1) constructs. Neutralizing
antibodies against TGEV (Figure 8B) were also detected in mice
immunized by pIRES-(TGEV-S1), pIRES-(TGEV-S1-PEDV-S1)
and pIRES-(TGEV-S1-PEDV-S). Here, there were no pronounced
differences detected between the three groups of animals.
Changes in the levels of serum IFN-c and IL- 4 in
Changes in serum IFN-c and IL-4 levels in immunized mice
were analyzed using ELISA. The results showed that the levels of
IFN-c in mice treated with pIRES-(PEDV-S1), pIRES-(PEDV-S),
pIRES(TGEV-S1-PEDV-S) and Vaccine were significantly higher than
in mice treated with PBS or pIRES. The highest level of IFN-c
was found to be induced by pIRES-(TGEV-S1-PEDV-S) (p,0.05)
(Table 1). However, the levels of IL-4 in all treatment groups were
fluctuant, especially, in pIRES-(TGEV-S1-PEDV-S1) and
pIRES(PEDV-S1) group which did not induce any IL4 production and
the IL-4 level even much lower (P,0.05) than PBS and pIRES
control. The pIRES-(TGEV-S1) group was higher (P,0.05) than
Figure 6. Changes in CD4+ and CD8+ T lymphocytes in the spleens of immunized mice. Spleen cells from mice immunized with
recombinant plasmids were collected and subjected to flow cytometry to assess the numbers of CD4+ T lymphocytes (A) and CD8+ T lymphocytes (B).
w: p,0.01 (highly significant), compared with the PBS and pIRES groups.
pIRES-(TGEV-S1-PEDV-S), and both are highly significant
(P,0.01) than PBS and pIRES control.
Activity of CTL in spleen and blood
Cytotoxicity was analyzed using the LDH release assay. The
results showed that CTL function in the peripheral blood of the
mice treated with pIRES-(TGEV-S1-PEDV-S1) was higher
(P,0.05) than in control cells, but significantly lower than the
values determined in the pIRES-(PEDV-S1) and
pIRES-(TGEVS1-PEDV-S) groups (p,0.01). In addition, the CTL function of
pIRES-(TGEV-S1-PEDV-S) group was higher (P,0.05) than that
of the pIRES-(PEDV-S) group, (Figure 9A). In a similar way, the
CTL function of spleen cells was determined for animals
immunized by pIRES-(PEDV-S1), pIRES-(PEDV-S),
pIRES-(TGEV-S1PEDV-S) and Vaccine. In all cases the values were significantly
increased compared to the control cells (Figure 9B).
Figure 7. Antibody levels in mice treated with pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S). Anti-PEDV serum antibodies (A)
and anti-TGEV serum antibodies (B) in pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S) immunized mice were detected by indirect ELISA at
different time points following the injection of the plasmid DNAs. The OD490 was monitored as a function of time over a period of 42 days. w: p,0.01
(highly significant), compared with PBS, pIRES treatment groups.
Prevalence of TGE and PED may lead to severe economic
losses in swine-raising countries. So far, the most effective
prevention measure is vaccination. DNA vaccines can induce
complete immune responses, provide heterologous cross
protection and can be easily prepared as polyvalent vaccines . It is
documented that the S1domain of TGEV/PEDV contains the
main neutralizing epitopes [13,20,34,39]. One report indicated
that the immunogenicity of DNA plasmid bearing TGEV S1 gene
is superior to another version containing the full-length S gene
. In contrast, there are no reports regarding comparative
analysis on the efficiency of S and S1-based DNA plasmids.
Therefore, this study aimed at evaluating immune response
induced by DNA plasmids encoding the TGEV S protein and
the PEDV S or S1 proteins using a mouse model. In pIRES
vector, there is an internal ribosomal entry site (IRES), which is
derived from the encephalomyocarditis virus (ECMV); two
multiple cloning sites (MCS) A and B, allow each product of
transcription to be translated independently with the participation
of ribosome at the same time. In addition, pIRES vector has a
CMV promoter for high expression of foreign genes and a SV40
enhancer/promoter that allows the enhancement of gene
expression in many hosts . Therefore, the genes of interest in this
Figure 8. Levels of neutralizing antibodies in mice treated with pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S). Anti-PEDV
and anti-TGEV neutralizing antibodies in immunized mice were detected by plaque reduction assay with different serum dilutions from blood
samples taken at 28 days after the injection of the plasmid DNAs.
study were cloned into the pIRES vector. Both
immunufluorescence assays and Western blot indicated that the in vitro expression
of TGEV S, PEDV S or PEDV S1 proteins were successful.
It is clear that the level of neutralizing antibodies is an important
indicator to evaluate the effect of the vaccine. In this study, IgG
levels of PEDV antibodies in pIRES-(TGEV-S1-PEDV-S1) and
pIRES-(TGEV-S1-PEDV-S) groups increased between 21
42 dpi, and the differences between both groups were not
significant. In addition, the values of the
pIRES-(TGEV-S1PEDV-S1) and pIRES-(TGEV-S1-PEDV-S) groups peaked at
35 dpi and decreased thereafter, but the differences between these
two groups were also not significant. The IgG antibody levels
against TGEV of these two groups began to increase at 28 dpi and
reached the peak at 35 dpi. Between 3542 dpi, the antibody
levels decreased dramatically (p,0.01) in
pIRES-(TGEV-S1PEDV-S1) group, whereas no significant changes was observed
(p.0.05) in the pIRES-(TGEV-S1-PEDV-S) group. This result
showed that the recombinant plasmid
pIRES-(TGEV-S1-PEDVS) containing the full-length S protein of PDEV was better in
eliciting immune responses to PEDV than is
pIRES-(TGEV-S1PEDV-S1) encoding only the S1 portion of the S protein. The IgG
antibody levels and virus neutralizing assays showed that both
pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S)
groups could elicit humoral immune responses against PEDV
and TGEV S proteins, respectively. Plasmid
pIRES-(TGEV-S1PEDV-S) was more efficient in inducing neutralizing antibodies
T cells are important effector cells of immune responses. Their
activation causes the secretion of cytokines that advance cellular
and humoral immune responses and mediate CTL activity. As
such, monitoring CTL activity provides a good readout for
immune stimulation. In this study, spleen and blood-derived
lymphocytes from immunized mice clearly showed that
immunization with pIRES-(TGEV-S1-PEDV-S1) and
pIRES-(TGEV-S1PEDV-S) significantly induced T cell proliferation. When
stimulated with purified recombinant PEDV-S1 protein, there was
higher T lymphocyte proliferation in the
pIRES-(TGEV-S1PEDV-S) group than in the pIRES-(TGEV-S1-PEDV-S1) group.
Most exogenous proteins and even inactivated pathogens are
internalized and processed via the endolysosomal pathway; the
resulting degradation of proteins yields peptides that are associated
with MHC-II molecules. They not only may stimulate antibody
responses but also the production of helper T cells, which are
necessary for the specificity of antibodies and CTLs production.
Activated CD4+ T cells can be classified into at least two
subgroups, Th1 and Th2. The Th1-like phenotype is
predominantly associated with interleukin-2 (IL-2) and interferon-c (IFN-c)
[30,4346]; The Th2-like phenotype, characterized by increased
levels of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6
(IL-6), is associated with improving humoral response and mucosal
immune response [21,38]. In this study, the number of CD4+ T
lymphocytes in peripheral blood and spleen reached the peak at
42 dpi and the number of CD4+ T cells from mice immunized
with pIRES-(TGEV-S1-PEDV-S) was higher (p,0.05) than
pIRES-(TGEV-S1-PEDV-S1) group. Changes in serum IFN-c
and IL-4 levels in immunized mice were analyzed. The results
showed that the levels of IFN-c in mice treated with DNA vaccine
were significantly higher. In addition, the levels of IFN-c induced
by pIRES-(TGEV-S1-PEDV-S) were higher than other groups
(p,0.05). However, the levels of IL-4 in all treatment groups were
fluctuant, especially, in pIRES-(TGEV-S1-PEDV-S1) and
pIRES(PEDV-S1) group the IL-4 level even much lower (P,0.05) than
PBS and pIRES control. This result may be explained by the high
level of IFN-c promoting the Th1 cell differentiation, at the same
time, restraining the Th2 cell differentiation by down-regulating
IL-4 and humoral immune response. This tendency is consistent
with the IgG antibody changes in 42 dpi. Several reports also
suggested that the level of antigen presentation can affect Th1/
Th2 profiles .
Following an increased understanding of the mechanisms of
antigen processing and presentation for the generation of MHC-I
restricted cytolytic T-lymphocyte (CTL) responses, the importance
of cellular immunity became significant. The reasons for seeking to
specifically generate CTLs include not just their activity of directly
killing infected cells (versus directly killing the virus or bacteria) but
also the fact that CTLs can focus on antigens that are not
accessible to antibodies . To elicit CTL responses, the antigen
needs to be present in the cytoplasm of antigen-presenting cells
(APCs). Then some of the newly synthesized proteins are
processed in proteosomes with certain of the resultant peptides
then binding to nascent MHC-I molecules for export via the Golgi
to the cell surface . Therefore, specific CTL activity could
reflect the activity of the CD8+ T lymphocytes. In this study, the
number of CD8+ T lymphocytes in peripheral blood and spleen
reached the peak at 42 dpi and the number of CD8+ T cells from
mice immunized with pIRES-(TGEV-S1-PEDV-S) was higher
(p,0.05) than in the pIRES-(TGEV-S1-PEDV-S1) group, similar
to the CD4+ T lymphocytes changes. The activity determined by
the CTL assay showed that CTL function in the peripheral blood
of the mice treated with pIRES-(TGEV-S1-PEDV-S1) was higher
(P,0.05) than that of the PBS control, but significantly lower than
in the pIRES-(TGEV-S1-PEDV-S) group (p,0.01). As with other
studies, CTL functions in spleen cells were mirrored by a higher
efficiency of the pIRES-(TGEV-S1-PEDV-S) group compared to
the pIRES-(TGEV-S1-PEDV-S1) group.
In summary, to the best of our knowledge, this is the first time
that the immunological efficacies triggered by PEDV S protein
and the TGEV S protein were compared using a mouse model.
Both the recombinant plasmids pIRES-(TGEV-S1-PEDV-S1) and
pIRES-(TGEV-S1-PEDV-S) could elicit in mice cellular
immunity, T-cell response as well as humoral response generating CTLs,
helper T cells, particularly of Th1 cells, as well as specific
neutralizing antibodies against PEDV and TGEV, respectively. It
has been reported that an antigenic domain in the S gene that
could elicit a strong antibody response and induce neutralizing
antibodies has excellent immunogenicity on the C-terminal
portion of the S protein . Our results confirmed that the
fulllength PEDV S gene induces a better immune response than the
N-terminal half alone.
As mice are not susceptible to infection by TGEV or PEDV we
cannot use this animal model to perform challenge experiments
and evaluate the protection rate of the DNA constructs. However,
we did observe good immune responses of these DNA plasmids in
this species. On the basis of this study, our further studies will
include usage of pigs to optimize immunization procedures as well
as to evaluate host immunity and protection induced by these
DNA plasmids to develop effective DNA vaccines for controlling
TGE and PED.
Conceived and designed the experiments: XR. Performed the experiments:
FM YR SS XS XL PL WY GL LL XR. Analyzed the data: FM YR CSW
GH XR. Contributed reagents/materials/analysis tools: XR. Wrote the
paper: FM CSW GH XR.
1. Bridgen A , Duarte M , Tobler K , Laude H , Ackermann M ( 1993 ) Sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229E and porcine transmissible gastroenteritis virus . J Gen Virol 74 : 1795 - 1804 .
2. Chasey D , Cartwright SF ( 1978 ) Virus-like particles associated with porcine epidemic diarrhea . Res Vet 25 : 255 - 256 .
3. Cheng QH , Niu XY ( 1992 ) Investigation on the porcine epidemic diarrhea prevalent on Qinhai . Vet Sci 22 : 22 - 23 .
4. Claerebout E , Vercauteren I , Geldhof P , Olbrechts A , Zarlenga DS , et al. ( 2005 ) Cytokine responses in immunized and non-immunized calves after Ostertagia ostertagi infection . Parasite Immunol 27 : 325 - 331 .
5. Cruz DJ , Kim CJ , Shin HJ ( 2008 ) The GPRLQPY motif located at the carboxyterminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus . Virus Res 132 : 192 - 196 .
6. DeBouck P , Callebaut P , Pensaert M ( 1982 ) Prevalence of the porcine epidemic diarrhea (PED) virus in the pig population of different countries . Vet Soc 7 : 53 .
7. Delmas B , Gelfi J , Laude H ( 1986 ) Antigenic Structure of Transmissible Gastroenteritis Virus. II Domains in the Peplomer Glycoprotein . Virol 67 : 1405 - 1418 .
8. Doyle LP , Hutchings LM ( 1946 ) A transmissible gastroenteritis in pigs . Vet Med 108 : 257 - 259 .
9. Duarte M , Laude H ( 1994 ) Sequence of the spike protein of the porcine epidemic diarrhea virus . Gen Virol 75 : 1195 - 1200 .
10. Duarte M , Tobler K , Bridgen A , Rasschaert D , Ackermann M , et al. ( 1994 ) Sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic ORF . Virology 198 : 466 - 476 .
11. Garwes DJ , Lucas MH , Higgins DA ( 1978 /1979) Antigenicity of structural components from porcine transmissible gastroenteritis virus . Vet Microbiol 3 : 179 - 190 .
12. Goodwin RF , Jelmings AR ( 1958 ). A highly infeetious gastroenieritis of pigs . Vet Rec 70 : 271 - 272 .
13. Isabel C , Gebauer F , Bullido MJ , Su ne C, Baay MF , et al. ( 1990 ) Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus . J Gen Virol 71 : 271 - 279 .
14. Jimenez G , Castro JM , Pozzo M ( 1985 ) Identification of a coronavirus inducing porcine gastroenteritis in Spain . Pig Vet Soc Congress 9 : 186 .
15. Jimenez G , Correa I , Melgosa MP , Bullido MJ , Enjuanes L ( 1986 ) Critical epitopes in transmissible gastroenteritis virus neutralization . J Virol 60 : 131 - 139 .
16. Johnson M , Fitzgerald G , Welter MW ( 1992 ) Six most common pathogens responsible for diarrhea in newborn pigs . Vet Med 87 : 382 - 386 .
17. Jung-Eun P , Deu JM , Cruz HJS ( 2010 ) Trypsin-induced hemagglutination activity of porcine epidemic diarrhea virus . Arch Virol 155 : 595 - 599 .
18. Kweon CH , Kwon BJ , Jung TS ( 1993 ) Isolation of porcine epidemic diarrhea virus (PEDV) in Korea . Korean Vet Res 33 : 249 - 254 .
19. Laude H , Chapsal JM , Gelfi J , Labiau S , Grosclaude J ( 1986 ) Antigenic structure of transmissible gastroenteritis virus . I. Properties of monoclonal antibodies directed against virion proteins . J Gen Virol 67 : 119 - 130 .
20. Laviada MD , Videgain SP , Moreno L , Alonso F , Enjuanes L , et al. ( 1990 ) Expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infeeted cells: epitopes externally exposed . Virus Res 16 : 247 - 254 .
21. Lekutis C , Shiver JW , Liu MA , Letvin NL ( 1997 ) HIV-1 env DNA vaccine administered to rhesus monkeys elicits MHC class II-restricted CD4+ T helper cells that secrete IFN-gamma and TNF-alpha . J Immunol 158 : 4471 - 4477 .
22. Li G , Shi N , Suo S , Cui J , Zarlenga D , et al. ( 2012 ) Vaccination of Mice with ORF5 Plasmid DNA of PRRSV; Enhanced Effects by Co-immunizing with Porcine IL-15 . Immunol. Invest 41 : 231 - 248 .
23. Liu MA ( 2003 ) DNA vaccines: a review . J Intern Med 253 : 402 - 410 .
24. Liu B , Li G , Sui X , Yin J , Wang H , et al. ( 2009 ) Expression and functional analysis of porcine aminopeptidase N produced in prokaryotic expression system . J. Biotechnol 141 : 91 - 96 .
25. Margaret AL ( 2011 ) DNA vaccines: an historical perspective and view to the future . Immunological Reviews 239 : 62 - 84 .
26. Meng F , Yin J , Li X , Yang W , Li G , et al. ( 2010 ) Production and characterization of a monoclonal antibody against spike protein of transmissible gastroenteritis virus . Hybridoma 29 : 345 - 350 .
27. Moxley RA , Olson LD ( 1989 ) Clinical evaluation of transmissible gastroenteritis virus vaccines and vaccination procedures for inducing lactogenic immunity in sows . Vet Res 50 : 111 - 118 .
28. Pensaert M , Debouck P ( 1978 ) A new coronavirus-like particle associated with diarrhea in swine . Virol 58 : 243 - 247 .
29. Ren XF , Yin JC , Si W , Li YJ , Liu BQ ( 2006 ) Construction of nucleic acid vaccines containing S gene from TGEV isolate TH-98 and their immune effect in mice . Veterinary Science in China 3: 203 - 206 (in Chinese).
30. Robinson HL , Hunt LA , Webster RG ( 1993 ) Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA . Vaccine 11 : 957 - 960 .
31. Sasahara J , Harada K , Hayashi S ( 1958 ) Studies on transmissible gastroenieritis in pig In Japan . Vet Sci 20: l-6.
32. Sestak K , Lanza I , Park SK , Weilnau PA , Saif LJ ( 1996 ) Contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs . J Vet. Res 57 : 664 - 671 .
33. Shuojie L , Jizhong C , Chengwu T , Yanbin M , Shuyu W , et al. ( 2007 ) Construction and Expression of DNA Vaccine pIRES-Sj97-Sj14-Sj26 and Its Immunogenicity in Mice . Med Sci 27 : 625 - 629 .
34. Spaan W , Cavanagh D ( 1988 ) Coronavirus: structure and genome expression . Gen. Virol 69 : 29 - 52 .
35. Straw BE , Allaire SD , Mengeling WL ( 2000 ) Disease of Swine 8th ed . Beijing: The Chinese Agriculture University Press. 181 - 187 .
36. Sturman LS , Riehard CS , Murine KV ( 1985 ) Proteolytic Cleavage of the E2 Glycoprotein of Murine Coronavirus by Trysin and Separation of Two Different 90K Cleavage Fragments . Virol 56 : 904 - 911 .
37. Sui X , Yin J , Ren X ( 2010 ) Antiviral effect of diammonium glycyrrhizinate and lithium chloride on cell infection by pseudorabies herpesvirus . Antiviral Res 85 : 346 - 353 .
38. Sumi T , Fukushima A , Fukuda K , Kumagai N , Nishida T , et al. ( 2007 ) Differential contributions of B7-1and B7-2 to the development of murine experimental allergic conjunctivitis . Immunology Letters 108 : 62 - 67 .
39. Sun DB , Feng L , Shi HY , Chen JF , Liu SW , et al. ( 2007 ) Spike protein region of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies . Acta Virol 51 : 149 - 156 .
40. Sun D , Feng L , Shi H , Chen J , Cui X , et al. ( 2008 ) Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein . Vet Microbiol 131 : 73 - 81 .
41. Sun-Hwa C , Bae JL , Kang TJ , Kim J , Chung GH , et al. ( 2002 ) Identification of the Epitope Region Capable of Inducing Neutralizing Antibodies against the Porcine Epidemic Diarrhea Virus . Mol Cells Vol 14 : 295 - 299 .
42. Tsuda T ( 1997 ) Porcine epidemic diarrhea: Its diagnosis and control . Vet Soc 31 : 21 - 28 .
43. Ulmer JB , Donnelly JJ , Parker SE , Rhodes GH , Felgner PL , et al. ( 1993 ) Heterologous protection against influenza by injection of DNA encoding a viral protein . Science 259 : 1745 - 1749 .
44. Ulmer JB , Deck RR , DeWitt CM , Donnelly JJ , Friedman A , et al. ( 1997 ) Induction of immunity by DNA vaccination: application to influenza and tuberculosis . Behring Inst Mitt 98 : 79 - 86 .
45. Ulmer JB , Fu TM , Deck RR , Friedman A , Guan L , et al. ( 1998 ) Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA . J Virol 72 : 5648 - 5653 .
46. Wang B , Ugen KE , Srikantan V , Agadjanyan MG , Dang K , et al. ( 1993 ) Gene inoculation generates immune responses against human immunodeficiency virus type 1 . Proc Natl Acad Sci USA 90 : 4156 - 4160 .
47. Wood EN ( 1977 ) An apparently new syndrome of porcine epidemic diarrhoea . Vet Rec 100 : 243 - 244 .