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Vaccine 27 (2009) 4565–4570
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Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Short communication
Immunogenicity and protective efficacy of ApxIA and ApxIIA DNA vaccine
against Actinobacillus pleuropneumoniae lethal challenge in murine model
Chung-Hao Chiang a , Wei-Fang Huang a , Li-Pu Huang b , Shuen-Fuh Lin b , Wen-Jen Yang a,b,∗
a
b
Institute of Biotechnology, National University of Kaohsiung, Kaohsiung, 811, Taiwan
Department of Life Science, National University of Kaohsiung, Kaohsiung, 811, Taiwan
a r t i c l e
i n f o
Article history:
Received 6 February 2009
Received in revised form 13 May 2009
Accepted 21 May 2009
Available online 9 June 2009
Keywords:
Actinobacillus pleuropneumoniae
Apx toxin
DNA vaccine
a b s t r a c t
Actinobacillus pleuropneumoniae is the major etiological agent of swine pleuropneumonia that causes
critical economic losses in swine industry. The use of DNA vaccines encoding Apx exotoxin structural
proteins is a promising novel approach for immunization against A. pleuropneumoniae. The goal of this
study was to design DNA vaccines which encode the gene of ApxIA or ApxIIA, and to evaluate the elicited
immune responses and protective efficacy in mice. Significant humoral immune responses were induced
by these DNA vaccines through intramuscular immunization. The IgG subclass (IgG1 and IgG2a) analysis indicates that divalent DNA vaccine induces both Th1 and Th2 immune responses. The protective
efficacy was evaluated by the survival against lethal challenge with A. pleuropneumoniae serotype 1. The
groups of vaccination with pcDNA-apxIA or divalent (pcDNA-apxIA and pcDNA-apxIIA) DNA vaccine provided protective efficacy significantly higher than that of the negative control groups (P < 0.05). However,
pcDNA-apxIIA vaccine conferred protection was limited and not significant than that of the negative control groups (P > 0.05). These results show that the divalent DNA vaccine could confer the best protection.
This finding indicates that DNA immunization should facilitate the development of a ‘third-generation’
of vaccines and provide a novel strategy against A. pleuropneumoniae infection.
© 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Swine pleuropneumonia caused by Actinobacillus pleuropneumoniae is a severe and high mortality respiratory disease which
leads to major economic losses in the swine industry [1]. The
clinical features of infected animals range from acute, extensive
necrotizing hemorrhagic bronchopneumonia to chronic, local lung
lesions with pleuritis [2]. To date, two biotypes and 15 serotypes
of A. pleuropneumoniae have been identified based on the nicotinamide adenine dinucleotide (NAD) requirement [3] and surface
capsular polysaccharide antigens [4,5], respectively. Several possible virulence factors have been described for A. pleuropneumoniae
such as capsular polysaccharides [6], iron-regulated proteins [7],
lipopolysaccharides (LPS) [8], fimbriae [9], flagella [10], secreted
protease [11], and most importantly, four exotoxins called Apx toxins (for A. pleuropneumoniae RTX toxins). RTX toxins (repeats in the
structural toxin) are pore-forming, cytolytic protein toxins that are
produced by a wide range of pathogenic Gram-negative bacteria
[3,12]. Although the virulence determinants are multifactorial, sev-
∗ Corresponding author at: Institute of Biotechnology, National University of
Kaohsiung, 700, Kaohsiung University Road, Nanzih District, Kaohsiung, 811, Taiwan.
Tel.: +886 7 5919454; fax: +886 7 5919404.
E-mail address: [email protected] (W.-J. Yang).
0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2009.05.058
eral investigations show that the virulence of A. pleuropneumoniae
is strongly linked with the production of Apx toxins, especially in
which serovars producing ApxI and ApxII exotoxins are the most
virulent. The toxins ApxI and ApxII are encoded on polycistronic
operons apxICABD and apxIICA, respectively, and the A gene (apxIA
and apxIIA) encodes the structural protein pretoxin in the operons
[3,13].
Several researches in vaccine development against A. pleuropneumoniae have been stimulated in the past years due to the
economic impact of this disease in swine industry. The state-ofthe-art of A. pleuropneumoniae vaccines was reviewed and showed
that it is promising in vaccine research and development [14].
Many virulence factors of A. pleuropneumoniae have been studied
for their protective potential including LPS [15], outer membrane
proteins [16,17] and Apx toxins [18,19]. Most of the commercial
vaccines against A. pleuropneumoniae were composed of chemical inactivated whole-cell bacterins of various serotypes known
as ‘first-generation’ vaccines. However, the antigenicites of certain bacteria-associated virulence factors might be altered by heat
or chemical treatments during bacterin preparation and might
influence the efficacy of the vaccine [20]. Furthermore, the inactivated whole-cell bacterins, lack of Apx exotoxins which are
known to be highly immunogenic and crucial for protection, usually conferred the limited protective efficacy [21]. The so-called
‘second-generation’ commercially available A. pleuropneumoniae
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subunit vaccines almost contain Apx detoxified toxins [22,23].
However, the difficulty and cost of large-scale purification and
inactivation impede the extensive use of Apx toxoids as vaccine
components. In several approaches, the usage of recombinant
proteins as subunit vaccine candidates although showed hopeful
results [24,25]. However, it requires additional steps for protein
purification, concentration and a method to maintain the stability
of the recombinant proteins.
DNA vaccination has emerged as an exciting new approach
with great promising in vaccine development against viruses [26],
bacteria [27], parasites [28,29], and tumor [30]. DNA vaccines
may provide several crucial advantages than traditional vaccines
including the relative safe, the inherent stability of their molecular
structure, the specificity of the antigen produced, both of humoral
and cellular immune responses could be elicited, the easy to prepare in large amount with high purity, and a few of plasmid DNA
(microgram to milligram level) are enough to raise a strong immune
response [31]. To date, the use of DNA immunization strategy has
not been reported to control A. pleuropneumoniae infection. In this
study, we developed a rational DNA immunization approach utilizing ApxIA and ApxIIA genes, which encode the structural proteins
of Apx toxins, to develop ‘third-generation’ novel types of vaccines
against A. pleuropneumoniae.
2. Materials and methods
2.1. Bacteria strains, plasmids, and culture conditions
The A. pleuropneumoniae serotype 1 Denmark strain was
obtained from Animal Health Research Institute, Council of Agriculture (Tansui, Taiwan). The bacteria were cultured in brain heart
infusion medium (Bacto), supplemented with 0.1% ␤-nicotinamide
adenine dinucleotide (Sigma) at 37 ◦ C in a shaking incubator at
180 rpm. Escherichia coli TOP10 (Invitrogen) was used in the construction and screening of the transformants and was cultured in
Luria-Bertani (LB) medium. Ampicillin 50 ␮g/ml was added for the
selection of recombinant plasmid-containing strains.
2.3. ApxIA and ApxIIA proteins expression
Because of the inclusion of CMV and T7 promoter in expression vector (Fig. 1A), the cloned apxIA and apxIIA gene encoded
proteins could be expressed in prokaryotic and eukaryotic system.
The expression of these proteins was first verified in prokaryote by Western blot analysis and these proteins were used for
the enzyme-linked immunosorbent assay (ELISA) for antisera
responses detection. The expression of these proteins in eukaryote also further confirmed in porcine kidney cells LLC-PK1 (BCRC
60080, Bioresource Collection and Research Center, Taiwan) by
immunofluorescence assay.
The expression of proteins was analyzed by Western blot
following a standard protocol. Briefly, the total lysates of
cloned plasmid-containing E. coli were loaded on 10% SDS-PAGE
gels and subsequently transferred to nitrocellulose membrane.
After blocking with 5% skim milk powder in TBST, proteins
were selectively identified using 1:5000 dilution of alkaline
phosphatase-conjugated anti-V5 antibody (Invitrogen). The color
development of blot was developed and visualized with the
NBT/BCIP substrate.
Expression of the ApxIA and ApxIIA proteins in LLC-PK1 cells was
confirmed by indirect immunofluorescence assay (IFA). The cells
were transfected with 16 ␮g of plasmid DNA using the transfecting
reagent Lipofectamine (Invitrogen) according to the manufacturer’s
protocol. Eighteen hours after transfection the medium was aspirated, and the transfected cells were washed and then fixed with 5%
formalin in PBS. After washing three times with PBS/1% Triton X-100
(PBS/Triton) the cells were subsequently incubated with 3% bovine
serum albumin (BSA) in PBS at 37 ◦ C for 1 h to block non-specific
binding. After aspirating the blocking solution mouse anti-V5 antibody diluted at 1:500 in PBS was added and incubated at 37 ◦ C
for 1 h. The cells were washed and then 100 ␮l of 1:300 diluted
fluorescein-conjugated goat anti-mouse IgG antibody (KPL) was
added and incubated in the dark at 37 ◦ C for 1 h. After washing the
2.2. Cloning and construction of apxIA and apxIIA in expression
vector
The genomic DNA of A. pleuropneumoniae serotype 1 was
extracted using Blood and Tissue Genomic DNA Extraction
Miniprep System (Viogene) and used as a template for PCR amplification of the target sequences. The apxIA and apxIIA genes were
amplified with Pfu Turbo DNA polymerase (Stratagene) according to the sequences of GenBank accession numbers AF240779
and AF363362, respectively. The primer pair sequences for apxIA
and apxIIA amplification were 5 -CACCATGGCTAACTCTCAGCTCGA3 ; 5 -AGCTGCTTGTGCTAAAGAATAA-3 and 5 -CACCATGTCAAAAATCACTTTGTCA-3 ; 5 -AGCGGCTCTAGCTAATTG-3 . The underline of
the forward primer indicates that the additional four bases used
for the PCR products were directionally cloned. The amplified
fragments were cloned into pcDNA3.1 Directional TOPO vector
(Invitrogen), transformed to E. coli TOP10 and the transformants
were selected from LB-Amp medium. The brief map of recombinant plasmids was shown in Fig. 1. Recombinant plasmids were
amplified in E. coli TOP10 and isolated using High-Speed Plasmid
Mini Kit (Geneaid) according to the manufacturer’s recommendations. Restriction enzymes BamHI and XbaI or EcoRV were
used for the preliminary identification of recombinant plasmids.
Cloned apxIA and apxIIA gene sequences were further sequenced
using fluorescence-based sequencer at Seeing Bioscience Company
(Taipei, Taiwan). Sequences were analyzed through the BLAST software provided by NCBI to verify the cloned gene sequences.
Fig. 1. Map of DNA vaccines and cloning of the apxIA and apxIIA genes. (A) Schematic
map of recombinant plasmid. The inserted gene can be driven by prokaryotic or
eukaryotic promoter. PCMV , human cytomegalovirus (CMV) immediate-early promoter; T7, T7 promoter/priming site; V5, V5 epitope (GKPIPNPLLGLDST); His6,
polyhistidine; BGH pA, bovine growth hormone polyadenylation signal. (B) The
apxIA and apxIIA fragments were generated using primers indicated in Table 1
and cloned into pcDNA3.1 vector and identified with the restrict enzymes. Lane
M, molecular weight markers; lane 1, the 3070 bp of apxIA PCR fragment; lane 2,
represent the 2872 bp of apxIIA PCR fragment. Lanes 3 and 4 represent the pcDNAapxIA digested with BamHI/XbaI and the pcDNA-apxIIA digested with BamHI/EcoRV,
respectively. The inserted DNA fragment and pcDNA3.1 vector are correspondence
to the predicted fragment size.
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C.-H. Chiang et al. / Vaccine 27 (2009) 4565–4570
cells with PBS/Triton three times, the cells were examined with a
fluorescence microscope (Nikon, Japan).
2.4. Animals and immunization regimen
Six to eight weeks old female inbred specific pathogen-free
(SPF) BALB/c mice were purchased from the National Laboratory
Animal Center (Taipei, Taiwan) of the National Science Council.
The immunization experiments were performed at the Laboratory
Animal Center of National University of Kaohsiung. Animals were
allowed to stabilize for 10 days before the experiments to minimize any stress due to transportation and environmental changes
on the immune system. All experiments were performed according to protocols in accordance with institutional guidelines. The
DNA vaccines used for immunization were prepared by Winzard
Puls Maxipreps DNA Purification System (Promega) according to
the manufacturer’s instructions. Groups of ten mice were injected
intramuscularly in the hind thigh muscle with 100 ␮g of plasmids
pcDNA-apxIA, pcDNA-apxIIA, or divalent vaccines (pcDNA-apxIA
and pcDNA-apxIIA, 100 ␮g for each plasmid) dissolved in 100 ␮l
PBS. Mice immunized with 100 ␮g of pcDNA vector and 100 ␮l of
PBS served as the negative controls. One hundred microliter of a
commercial inactivated vaccine which contained serotypes 1–5 and
7 with aluminum hydroxide as adjuvant was used as positive control. Animals were boosted with the same dose on day 14th and 21st
after the first immunization. Serum samples were collected by tail
bleeding before immunization (preimmune sera), one day before
each boost and one week after 2nd boost, and stored at −20 ◦ C until
use.
2.5. Measurement of antibody response by ELISA
Two ELISA systems were performed to evaluate the antisera
responses. For DNA vaccine groups, the microtiter plates were
coated with 1:5000 diluted anti-V5 antibody (Invitrogen) and incubated at 4 ◦ C overnight. After washing three times with TBST the
wells were blocked with TBST containing 5% (w/v) skim milk at 37 ◦ C
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for 1 h. The wells were washed with TBST and then 300 ␮g of total
lysate of cloned plasmid-containing E. coli, which could express the
target protein (Fig. 2A, lanes 1 and 2), was added and incubated at
37 ◦ C for 1 h. The serum samples in duplicate were diluted with 100fold and then serial two-fold dilutions and incubated at 37 ◦ C for
1 h. The wells were washed with TBST and incubated with 1:5000
diluted goat anti-mouse IgG alkaline phosphatase-conjugated antibodies (Sigma) at 37 ◦ C for 1 h. The wells were then washed and
developed with substrate p-nitrophenyl phosphate (Amresco) at
37 ◦ C for 30 min and the plate was read at 405 nm with Multiskan EX
ELISA reader (Thermo Fisher Scientific, USA). For commercial inactivated vaccine, empty vector, and PBS groups, the microtiter plates
were coated with 100 ␮l of A. pleuropneumoniae serotype 1 (containing 103 colony-forming units) and incubated at 4 ◦ C overnight.
After blocking step, the serum samples were added and the following steps were the same as DNA vaccine groups measurement
described above. A similar protocol was followed for ELISA assessing IgG subclass analysis. The serum samples were diluted with
100-fold and assayed in duplicate against target antigen. Rabbit
anti-mouse IgG1 or IgG2a antibodies conjugated to alkaline phosphatase (Invitrogen) were used as second antibodies and measure
the results at 405 nm as above described.
2.6. Challenge test
Ten days after 2nd boost, the animals were challenged
intranasally with A. pleuropneumoniae serotype 1. The culture broth
of A. pleuropneumoniae serotype 1 grown at 37 ◦ C for 6 h to achieve
OD600 = 0.6. The culture broth estimated containing 2 × 109 CFU/ml
of A. pleuropneumoniae. The bacteria were harvested by centrifugation, washed twice with PBS and suspended in PBS containing
5 × 109 CFU/ml. One hundred microliters of bacteria saline suspension containing 5 × 108 CFU (10 times ID50 in mice) were used as
challenge dose for each mouse according to a previous description
[32]. The intranasal challenge was performed with a 26-gauge needle attached to 1 ml syringe and applied drop by drop onto both
of the nasal orifices of partially ethyl ether-anesthetized mice [33].
Fig. 2. Expression of apxIA and apxIIA in E. coli and LLC-PK1 cells. (A) The protein expression by E. coli TOP10 cells that had been transformed with pcDNA-apxIA (lane 1),
pcDNA-apxIIA (lane 2), pcDNA3.1 vector (lane 3), or non-transformed (lane 4) was determined by Western blot analysis using anti-V5 antibody as described in Section 2.
The molecular weight standards (M) are indicated. (B) Expression of DNA vaccine construct analyzed through indirect immunofluorescence (IFA) as described in Section 2.
The fluorescence microscopy picture of non-transfected LLC-PK1 cells (left panel) and pcDNA-apxIA transfected cells (right panel). The expressed ApxIA proteins in cells are
indicated by arrows.
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Table 1
The differences of nucleotide and encoded amino acid sequences of apxIA and apxIIA
gene between strain 4074 in the GenBank and the strain used in this study.
very well in the LLC-PK1 cells and the ApxIA protein expression as
shown by immunofluorescence (Fig. 2B).
Antigen gene
Nucleotide position
Nucleotide
differencea
Encoded amino
acid differencea
3.3. Antibody response analysis
apxIA
apxIIA
381
2536
2539
2577
2632
2680
2699
2771
2772
2790
2812
2833
C→T
A→G
A→G
A→G
A→G
A→G
G→T
A→G
A→G
A→G
G→A
A→G
Gly → Gly
Lys → Glu
Asn → Asp
Thr → Thr
Lys → Glu
Lys → Glu
Gly → Val
Glu → Gly
Glu → Gly
Ser → Ser
Ala → Thr
Asn → Asp
The levels of antibody response specific to these DNA vaccines
were evaluated in mice through intramuscular immunization. The
humoral immune responses of the mice immunized with either
pcDNA-apxIA or pcDNA-apxIIA vaccine were analyzed by IgG ELISA
against the recombinant protein itself as described in Section 2. The
results shown in Fig. 3A clearly reveal that antibody responses were
induced significantly by these DNA vaccines before challenge. The
responses of antibodies specific to ApxIA or ApxIIA were produced
at comparable levels in the group immunized with divalent DNA
vaccine. The antibody responses of commercial inactivated vaccine,
pcDNA3.1 vector, and PBS immunization groups were analyzed by
ELISA against the bacteria A. pleuropneumoniae serotype 1. Significant antibody response was observed with commercial inactivated
vaccine immunization compare with the negative control groups
(P < 0.05). However, no significant level of antibody was detected
in animals immunized with pcDNA3.1 vector or PBS throughout
the experiment (Fig. 3A). The IgG subclass was further analyzed for
one week after third immunization to evaluate the type of Th cell
responses associated with DNA vaccination. As shown in Fig. 3B,
both IgG1 and IgG2a responses were induced by divalent DNA vaccine, with IgG1 slightly higher than IgG2a. The levels of IgG1 and
IgG2a were also significant compared with the negative control
groups. This result reveals that the immune response induced by
divalent DNA vaccine is a mixed Th1 and Th2 type.
a
The bold type letter represents the nucleotide or encoded amino acid of the
strain used in this study.
The survival of the challenged mice was observed at 6 h intervals
within 24 h and then at 12 h intervals for five days post-challenge.
2.7. Statistical analysis
The statistical analysis was performed by using Student’s t-test
with Excel software. Differences were considered to be statistically
significant if probability values of P < 0.05 were obtained.
3. Results
3.1. Cloning and sequence analysis of cloned apxIA and apxIIA
genes
To construct the DNA vaccines, two pairs of primers were
designed to amplify apxIA and apxIIA genes from A. pleuropneumoniae genomic DNA. The amplified fragment of apxIA (3070 bp) and
apxIIA (2872 bp) genes was generated after PCR reactions (Fig. 1B).
These fragments were cloned into pcDNA3.1 vector, designed as
pcDNA-apxIA and pcDNA-apxIIA. The recombinant plasmids were
identified by restriction enzymes digestion with BamHI and XbaI or
EcoRV (Fig. 1B). According to the length of digested DNA fragments,
the correct clones were further sequenced using fluorescencebased sequencer. The sequences of cloned apxIA and apxIIA genes,
as well as the deduced protein sequences, were aligned with the
gene/protein sequences of A. pleuropneumoniae serovar 1 strain
4074 in the NCBI GenBank database. As shown in Table 1, one
nucleotide difference but encoded the same amino acid glycine in
apxIA gene was found between these two strains. However, in apxIIA
gene, there are 11 nucleotide differences and nine of them altered
A→G were found and eventually made nine encoded amino acids
are different between these two strains.
3.2. Detection of ApxIA and ApxIIA expression
In pcDNA3.1 expression vector, both of the CMV and T7 promoters were included (Fig. 1A). Western blot analysis was used to
verify whether the cloned apxIA and apxIIA gene encoded proteins
could be expressed in prokaryotic cells and be used for ELISA in
antiserum responses detection. The results of Western blot analysis clearly reveal significant expression levels of both the ApxIA
and ApxIIA proteins with an apparent molecular weight of 110 kDa.
No band was detected in control pcDNA3.1 transformed E. coli cells
and non-transformed E. coli cells after incubation with anti-V5 antibodies (Fig. 2A). The expression of these proteins in eukaryotic cells
also further confirmed their expression in porcine kidney cells LLCPK1 by immunofluorescence assay. Both of these proteins expressed
Fig. 3. Antibody response of each group before challenge. (A) The serum antibody
response of pcDNA-apxIA () immunized mice was assayed against ApxIA protein;
pcDNA-apxIIA immunized group was analyzed against ApxIIA protein (); divalent DNA vaccine group was analyzed against ApxIA () or ApxIIA () protein,
respectively. Other groups were determined against 103 CFU of A. pleuropneumoniae.
Duplicate ELISA measurements were performed for each sample. The data shows the
average absorbance at 405 nm of 10 mice serum samples and one of three different
experiments with similar observations is shown. (B) Levels of IgG subclass analysis.
The T cell response was measured by IgG1 (gray column) and IgG2a (white column)
analysis in the sera one week after third immunization. The serum samples were
diluted 1:100 and optical density read at 405 nm. Each column represents the mean
and standard deviation of OD405 from 10 mice of one experiment.
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Fig. 4. The protective efficacy of immunized mice. Survival curves for mice after
intramuscular immunization with pcDNA-apxIA (䊉), pcDNA-apxIIA (), divalent
DNA vaccine (), pcDNA3.1 vector (), commercial inactivated vaccine (), and PBS
(), followed by intranasal challenge with 5 × 108 CFU A. pleuropneumoniae serotype
1. The figure shows the survival percentage of post-challenge as a representative vaccine efficacy evaluation and difference in time period between mortality of vaccine
and control groups. Representative results from three independent experiments are
shown. An asterisk indicates a significant difference in survival compared to that of
negative control groups (P < 0.05).
3.4. DNA vaccine protective efficacy against serovar 1 lethal dose
challenge
The protective efficacy of these DNA vaccines was evaluated
in terms of survival of mice. Mice were challenged with a dose
of 5 × 108 CFU of A. pleuropneumoniae serotype 1 ten days after
2nd boost, and the survival percentage of mice was recorded for
five days after the challenge. As shown in Fig. 4, the survival for
animals immunized with divalent vaccine (70%) was better but
not significant (P > 0.05) than the groups that immunized with
pcDNA-apxIA or commercial inactivated vaccine which provided
60% protective efficacy. However, the survival of mice immunized
with divalent DNA vaccine, pcDNA-apxIA or commercial inactivated
vaccine was significantly higher than that of the negative control
groups (P < 0.05). In addition, pcDNA-apxIIA vaccine conferred limited protection only upon 20% and the survival was not significant
(P > 0.05) than that of the negative control groups. No protective
efficacy was observed for pcDNA3.1 vector immunization group and
PBS control group and all mice were died within 24 h.
4. Discussion
Several types of vaccines, including inactivated whole-cell vaccine, live attenuated vaccine, subunit vaccines have been developed
to control the infection of A. pleuropneumoniae [14]. However, the
use of inactivated whole-cell vaccine only showed the limited protective efficacy [34]. The lack of secreted Apx toxins, which are
known to be crucial for protection, might explain the reason why
the protective efficacy of inactivated whole-cell vaccine is limited
[35]. The results of our study also supported this issue (Fig. 4). DNA
immunization is a promising strategy to develop a new generation of vaccine and the licensure of three kinds of animal DNA
vaccines has provided broader potential of this technology [36].
In this work, we demonstrated that the divalent DNA vaccines
containing apxIA and apxIIA induced significant humoral immune
responses (Fig. 3) and conferred the best protection (Fig. 4) in this
study. The pcDNA-apxIA DNA vaccine provided the same protective efficacy as commercial inactivated vaccine. As expected, the
pcDNA-apxIIA DNA vaccine conferred limited protection. The possible explanation for these results is that ApxI is strongly hemolytic
activity and strongly cytotoxic for pagocytic cells, however, ApxII
is weakly hemolytic and cytotoxic activities. The humoral immune
response produced by ApxIA plays a more crucial role than ApxIIA
in protection against the highly lethal dose challenge. The highest survival was observed in the group immunized with divalent
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DNA vaccine. Similar results also obtained by oral immunization
with 10 mg each of S. cerevisiae expressing recombinant ApxIA and
ApxIIA antigens [37]. The divalent DNA vaccine produced satisfactory humoral immune responses to Apx toxins and conferred the
highest protective efficacy. These results agree with previous report
indicating that both ApxI and ApxII exotoxins were necessary for the
full virulence of A. pleuropneumoniae infection [13].
As shown in Table 1, eleven nucleotides and encoded nine amino
acids differences of cloned apxIIA gene were found between A. pleuropneumoniae serovar 1 strain 4074 and the strain used in this study.
These results were confirmed with different PCR experiments using
Pfu Turbo DNA polymerase to reduce the error producing by DNA
polymerase during PCR. It indicates that the strain difference may
cause the divergence. However, the cloned apxIIA gene still induced
significant humoral immune response and conferred limited protection. The relevant IgG subclass and Th type could be critical for
protection against a particular disease. The production of IgG1 is
representative for the Th2 response, while IgG2a is typical for the
Th1 response. In this study, the IgG1 response was slightly higher
than IgG2a in low dilution sera. It is possible without the detection
of the cytokines associated with the cellular immunity. One foot
and mouth disease virus DNA vaccine has been showed that the
similar immune response was induced [38].
Based on the protective efficacies, several antigens of A. pleuropneumoniae have been evaluated the potential as subunit vaccine
candidates. Although significant advances made in the vaccine
development, thus far, none of the commercial subunit vaccines
provide complete protection against A. pleuropneumoniae infection
[14]. The divalent DNA vaccine developed in this study without the
usage of adjuvant could provide considerably protective efficiency
and superior to a commercial inactivated whole-cell vaccine containing aluminum hydroxide as adjuvant. Therefore, it is worthy
to investigate the administration of adjuvant in DNA vaccine formulation to improve plasmid DNA immunogenicity and protective
efficacy. Furthermore, the DNA vaccine efficacy should be enhanced
through the combination of other crucial antigens and/or different
administration pathway.
In conclusion, the present work demonstrates that the DNA
vaccination approach could induce significant humoral immunity
and protective efficacy and represent a potentially novel approach
to design ‘third-generation’ vaccine against A. pleuropneumoniae.
It indicates that these DNA vaccines may be useful in modifying
current commercial inactivated vaccine to improve the protective
efficacy under lethal dose challenge. Certainly, there are several differences existing between mice and pigs. Based on the results in this
study, the protective results in mice could not infer straightly that
the same protective efficacies will occur in pigs. Fortunately, the
murine model was used to evaluate the immune responses and protective efficacy against A. pleuropneumoniae challenged in several
studies and showed the similar results in pigs [32,39]. Undoubtedly, the potential of using these DNA vaccines for the control of
porcine pleuropneumonia will be further studied in pigs based on
the present mice data.
Acknowledgments
We thank Dr. Wei-Ming Zhang for kindly providing the bacteria
of A. pleuropneumoniae serotype 1. This research was supported in
part by grants NSC-95-2317-B-390-002 from the National Science
Council, Republic of China.
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