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Evaluation of recombinant protein superoxide dismutase of
Haemophilus parasuis strain SH0165 as vaccine candidate
in a mouse model
Journal: Canadian Journal of Microbiology
Manuscript ID cjm-2016-0671.R2
Manuscript Type: Article
Date Submitted by the Author: 12-Dec-2016
Complete List of Authors: Guo, Ling; Hubei Key Laboratory of Animal Nutrition and Feed Science,
Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety Xu, Lei; Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety Wu, Tao; Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety Fu, Shulin; Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety Qiu, Yinsheng; Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety Hu, Chien-An Andy ; Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety; Biochemistry and Molecular Biology, University of New Mexico
School of Medicine, Albuquerque, New Mexico 87131, USA Ren, Xinglong; College of Science,Huazhong Agricultural University ,Wuhan 430070,PR China Liu, Rongrong; School of Animal Science and Nutritional Engineering, Wuhan Polytechnic University, Wuhan 430023, PR China Ye, Mengdie; School of Animal Science and Nutritional Engineering, Wuhan Polytechnic University, Wuhan 430023, PR China
Keyword: Haemophilus parasuis; Glässer's disease; superoxide dismutase; vaccine
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Evaluation of recombinant protein superoxide dismutase of
Haemophilus parasuis strain SH0165 as vaccine candidate in a mouse
model
Ling Guo 1†
, Lei Xu 1†
, Tao Wu 1, Shulin Fu
1, *, Yinsheng Qiu
1, *, Chien-An Andy
Hu 1, 2
, Xinglong Ren 3, Rongrong Liu
4, Mengdie Ye
4
1. Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative
Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic
University, Wuhan 430023, PR China
2. Biochemistry and Molecular Biology, University of New Mexico School of Medicine,
Albuquerque, New Mexico 87131, USA
3. College of Science,Huazhong Agricultural University ,Wuhan 430070,PR China
4. School of Animal Science and Nutritional Engineering, Wuhan Polytechnic
University, Wuhan 430023, PR China
† These authors contributed equally to the work.
* Corresponding author:
Shulin Fu, Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei
Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan
Polytechnic University, Wuhan 430023, PR China.
E-mail address: fushulin2016@126.com
Yinsheng Qiu, Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei
Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan
Polytechnic University, Wuhan 430023, PR China.
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E-mail address: qiuyinsheng6405@aliyun.com
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Abstract
Haemophilus parasuis, can cause a severe membrane inflammation disorder. It
has been documented that superoxide dismutase is a potential target to treat systemic
inflammatory diseases. Therefore, we constructed an experimental H. parasuis
subunit vaccine SOD and determined the protective efficacy of SOD using a lethal
dose challenge against H. parasuis serovar 4 strain MD0322 and serovar 5 strain
SH0165 in a mouse model. The results demonstrated that SOD could induce a strong
humoral immune response in mice and provide significant immunoprotection efficacy
against a lethal dose of H. parasuis serovar 4 strain MD0322 or serovar 5 strain
SH0165 challenge. IgG subtype analysis indicated SOD protein could trigger a bias
toward a Th1-type immune response and induce the proliferation of splenocytes and
secretion of IL-2 and IFN-γ of splenocytes. In addition, serum in mice from SOD
immunized group could inhibit the growth of strain MD0322 and strain SH0165 in the
whole-blood killing bacteria assay. It was the first time reported that immunization of
mice with SOD protein could provide protective effect against a lethal dose of H.
parasuis serovar 4 and serovar 5 challenge in mice, which may provide a novel
approach against heterogenous serovar infection of H. parasuis in future.
Keywords: Haemophilus parasuis; Glässer's disease; superoxide dismutase; vaccine
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Introduction
Haemophilus parasuis (H. parasuis), is the causative agent of the Glässer's
disease in swine with phenotypic presentation of polyserositis, meningitis and arthritis
(Oliveira and Pijoan 2004). In recent years it has become one of the most important
respiratory bacterial pathogens of livestock worldwide and can cause gross economic
losses (Rapp-Gabrielson et al. 1997). To date, fifteen serovars of H. parasuis have
been identified, however, up to 20% isolates could not be sero-typed in some
countries (Kielstein and Rapp-Gabrielson 1992). Serovar 4 and serovar 5 are the most
frequently detected in most countries (Rúbies et al. 1999; Cai et al. 2005). Although
vaccine immunization is thought to be the best method to control and prevent
infectious disease (Rappuoli et al. 2002), currently no good vaccine is available which
can provide protection against the challenge of all serovars of pathogenic H. parasuis.
The pathogenesis of H. parasuis infection is poorly understood - only a few
number of virulence - related factors have been linked to the pathogenicity of
Glässer's disease. ClpP plays an essential role in stress tolerance, and negatively
regulates biofilm formation by H. parasuis (Huang et al. 2016). cheY plays a crucial
role in growth, colonization, biofilm formation and autoagglutination of H. parasuis
(He et al. 2016). Deletion of rfaE gene can attenuate resistance, adhesiom and
invasion of H. parasuis to PUVEC and PK-15 cells (Zhang et al. 2014). capD gene is
a novel pathogenicity-associated determinant and involves in serum-resistance ability
of H. parasuis (Wang et al. 2013). Previous reports demonstrated that immunization
against virulence - related proteins, which mostly were outer membrane proteins
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(OMPs), could confer protection against the infection of microbe (Tavares er al. 2003;
Vilanova et al. 2004). Importantly, some protective or immunogenic antigens have
been identified that potentially can be used as vaccine candidates, for example,
Omp26, VacJ, HAPS-0742 (Li et al. 2016), PflA, Gcp, Ndk, HsdS, RnfC, HAPS-0017
(Li et al. 2015), TbpB (Frandoloso et al. 2015), rGAPDH, rOapA, rHPS-0675 (Fu et
al. 2013a) and 6PGD (Fu et al. 2012a). Even though some potential vaccine
candidates have been studied, the mechanism of immune protection triggered by
vaccines is poorly understood.
The genomic sequencing of H. parasuis SH0165 strain has been completed (Yue
et al. 2009). We have found superoxide dismutase (SOD) gene present in H. parasuis
genome. SOD is a family of antioxidant enzymes that function as a frontline defense
against superoxide anion radicals (.O2
-) which were produced in all aerobic cells
(Fridovich 1998). It has been documented that SOD is very useful to treat systemic
inflammatory diseases (Shafey et al. 2010). To date, using H. parasuis SOD protein as
a vaccine candidate has not been reported previously.
In this study, we constructed an experimental H. parasuis SOD vaccine and
determined the protective efficacy of the H. parasuis SOD using a lethal dose
challenge of H. parasuis serovar 4 and serovar 5 in a mouse model.
Materials and methods
Bacteria strains and growth conditions
H. parasuis (serovar 4, strain MD0322; serovar 5, strain SH0165) (Cai et al. 2005)
were grown in tryptic soy broth (TSB) (Difco, USA) or tryptic soy agar (TSA) (Difco,
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USA) supplemented with 10 µg/ml NAD (Sigma, St. Louis, MO) and 10% newborn
calf serum (Gibco, USA). Escherichia coli was grown in Luria-Bertani (LB) broth or
agar (Difco, USA) at 37 °C in the presence of 25 µg/mL kanamycin (Sigma, USA)
when necessary. All bacterial strains were grown at 37 °C.
Purification of recombinant SOD protein
sodA gene was amplified by PCR using the following primers: sodAF
(5’-GCGGATCCATGGCATACACATTAC-3’; underlined, BamHI site) and sodAR
(5’-GCAAGCTTTTATGCTTGGGATTCA-3’; underlined, HindIII site). Then the
PCR products were sub-cloned into the pET28a expression vector.
E. coli BL21 (DE3) (OD600=0.6) were induced with 1mM IPTG (Sigma, USA). A
pilot experiment was performed and the molecular mass of the SOD protein was
assessed by SDS-PAGE. The SOD protein was purified, as previously described with
some minor modifications (Fu et al. 2013a). Briefly, the cell pellet was suspended in
20 mL of buffer solution (50 mM Tris-HCl at pH 8.0, 500 mM NaCl and 1 mM PMSF)
and disrupted in a French pressure cell. After centrifugation at 12,000 rpm for 20 min
at 4 ℃, the supernatant was mixed with 5 mL of Ni2+
-NTA agarose (Qiagen, Germany)
in a column. Proteins not bound to Ni-NTA agarose were removed using 150 mL
binding buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 5 mM imidazole). The
target protein was eluted by 5 mL elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM
NaCl, and 500 mM imidazole) and stored at -80 ℃ for further study.
Animals and vaccination
This study was carried out in strict accordance with the recommendations in the
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China Regulations for the Administration of Affairs Concerning Experimental
Animals 1988 and the Hubei Regulations for the Administration of Affairs
Concerning Experimental Animals 2005. The protocol was approved by China Hubei
Province Science and Technology Department (Permit Number: SYXK(ER)
2010-0029). The animals were euthanized at the end of the experiments or when
moribund during the experiments.
Before immunization, the purified SOD protein was mixed with Marcol 52
(ESSO). 99 numbers of four to five weeks old female BALB/c mice were divided
randomly into three groups. Mice were immunized subcutaneously with 50 µg of
SOD protein (100 µL, 50 µg) mixed with a 1.5-fold volume of Marcol 52 adjuvant
(immunized group). The other two groups were injected PBS (100 µL) emulsified in
the same adjuvant as negative control (negative group) or PBS only as blank control
(blank group). Mice were injected a vaccination boost of the same dose on day 14
following the first immunization. The blood samples were collected for immunologic
assays by tail bleeding on day 14 after the booster immunization.
Determination of antibody titers
Serum for IgG titers against SOD protein was analyzed by enzyme linked
immunosorbent assay (ELISA), as described previously (Fu et al. 2012b). Briefly,
96-well microtiter plates were coated with purified SOD protein and serially-diluted
mouse serum was added and incubated for 45 min at 37 ℃. Hence goat anti-mouse
IgG-HRP (CST, USA) was added and the samples were incubated for 30 min at 37 ℃.
To determine IgG subclass, coated plates were incubated with dilutions of mice sera
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and added 100 µL of goat anti-mouse IgG1-HRP or IgG2a-HRP (Santa Cruz
Biotechnology, USA) diluted 1:5000. The color was developed by adding activated
substrate solution (sodium citrate buffer containing 1 mg/mL of 3, 3, 5,
5-tetramethylbenzidine and 0.03% H2O2) and the reaction was stopped by adding
0.25% hydrofluoric acid to each well. The plates were read at an absorbance of 630
nm.
Lymphocyte proliferation
Lymphoproliferation assays were determined, as previously described with some
minor modifications (Khan et al. 2006; Fu et al. 2013a). Three mice from immunized
group, the negative control group and the blank control group, were sacrificed on the
14th day following the boost immunization, and the spleens from the mice were
isolated as recorded previously (Silva and Benitez 2005). Briefly, spleens were
aseptically harvested and processed by gentle disruption with sterile stainless steel
sieve and glass pestle and suspension of splenocytes in RPMI incomplete medium
(Gibco, USA). Cell suspensions were centrifuged for 20 min at 190 × g. Erythrocytes
were lysed by treatment with 0.84% ammonium sulfate for 15 min on ice. Then the
cells were gently washed three times with Hank’s Balanced Salt Solution (HBSS)
(Hyclone, USA), then resuspended in complete RPMI medium (Gibco, USA). 200 µL
of cells were cultured (1 × 105 cells/mL) in 96-well culture plates at 37 ℃. The cells
were stimulated with the SOD protein and concanavalin A (4 µg/well) (WAKO, Japan)
in vitro and incubated for 72 h at 37 ℃ in a 5% CO2 incubator. Lymphoproliferation
assays were determined using
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3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra
zolium (MTS) reagent with a cell proliferation kit (Promega, USA), according to the
manufacturer’s instructions. The lymphocytes were co-cultured in 96-well culture
plates with MTS reagent for 4 h, and the absorbance was read at 490 nm wavelength
using ELISA reader.
Determination of cytokines by ELISA
The supernatants from splenocytes cultures were collected after 72 h and stored at
-80 ℃. The production of IL-2, IL-4, and IFN-γ from culture supernatants were
measured using ELISA kits (R&D, USA), according to the manufacturer’s
instructions.
Whole blood killing bacteria assay
The bactericidal assay was determined as described previously (Furano and
Campagnari 2003; Fu et al. 2013b), with some minor modifications. Briefly, live
bacteria of H. parasuis strain SH0165 or strain MD0322, were washed and diluted in
sterile physiological saline. Samples of diluted bacteria (20 µL; 106 cells) were
mixed with 180 µL inactivated test sera which were inactivated in a 56 ℃ water bath
for half an hour diluted 2-fold with physiological saline, or diluted bacteria were
mixed with saline alone. After incubation at 30 min under 37 ℃ and then 45 min on
ice, nonimmune whole heparinized (10 U/mL) mouse blood (100 µL) was added,
and the mixture was incubated at 37 ℃ with shaking for 1 h. Bacteria were then
plated on TSA containing 10% newborn calf serum and 10 µg/mL NAD. Bacterial
colony counts (CFU) were recorded after 36 h. Results were expressed as percent
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killing according to the following formula: (CFU after 1 h of growth with control
sera - CFU after 1 h of growth with immune sera)/CFU after 1 h of growth with
control sera × 100. All assays were performed in triplicate and repeated three times.
Challenge experiments in mice
On day 14 following the booster immunization, mice immunized with SOD
protein were randomly divided into groups 1 and 2 (15 mice/group) (immunized
groups), mice immunized with Marcol 52 adjuvant were randomly divided into group
3 and 4 (15 mice/group) (negative control groups), mice immunized with PBS were
randomly into 5 and 6 (15 mice/group) (blank control groups), respectively. Hence,
the mice from groups 1, 3, and 5 were challenged intraperitoneally against a lethal
dose of 6.0 × 109 CFU of H. parasuis SH0165 strain and the mice of groups 2, 4, and
6 were challenged intraperitoneally against a lethal dose of 6.0×109
CFU of H.
parasuis MD0322 infection, respectively. All mice were monitored regularly for 7
days following the challenge, and morbidity and mortality were recorded.
Histopathology
Lung tissues of mice from immunized group, negative control group and blank
control group were fixed by immersion in 10% neutral buffered formalin and
embedded in paraffin. And then 4 µm tissue sections were cut and stained with
hematoxylin and eosin (H&E) according to a standard protocol and examined under
light microscopy.
Passive immunization
Passive immunization experiments were conducted as previously described (Sun
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et al. 2011; Fu et al. 2012b). Briefly, the newly purchased 40 numbers four to five
weeks old female BALB/c mice were randomly divided into four groups which were
named groups 7-10. The groups 7 and 9 (ten mice per group) were intraperitoneally
injected with 100 µL of serum from immunized mice and groups 8 and 10 (ten mice
per group) were intraperitoneally injected with 100 µL of serum from
adjuvant-immunized mice (negative group). At 24 h post-immunization, groups 7 and
8 were challenged against a lethal dose of 3.0 × 109 CFU of SH0165 strain of serovar
5, and groups 9 and 10 were challenged against a lethal dose of 3.0 × 109 CFU of
MD0322 strain of serovar 4. All mice were monitored as described above and
morbidity and mortality were recorded.
Statistical analysis
The experimental data were expressed as mean ± SD. The difference between two
groups was analyzed using Student’s t-test and the difference among three groups was
analyzed using the ANOVA and survival analysis was used the Logrank test. p values
of <0.05 were considered significant. *p < 0.05; **p < 0.01 and ***p < 0.001.
Results
Expression and purification of the recombinant SOD
The recombinant SOD protein was successfully cloned and expressed as HIS
fusion protein which corresponded to its predicted size (Fig. 1A), and the His-tagged
SOD protein was purified by Ni+NTA affinity chromatography. We showed that the
purified recombinant SOD migrated as a single band in SDS-PAGE (Fig. 1B).
Antibody response to SOD recombinant protein in mice
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The results demonstrated that the mice immunized with the purified SOD protein
exhibited significant antibody immune responses (p < 0.01) (Fig. 2A). However, the
antibody production was not observed in mice from negative control group or blank
control group (Fig. 2A).
To further determine the type of immune response, the subclass of IgG1 and
IgG2a were investigated. We showed that the levels of antibody isotypes were higher
in the SOD-immunized group compared to the negative control group and blank
control group (p < 0.001) (Fig. 2B). Furthermore, IgG2a immune responses
predominated over IgG1 immune responses (p < 0.01) (Fig. 2B).
Determination of cytokine production
We showed that a significant proliferative T-cell immune response was displayed
in the mice from immunized with SOD protein (p < 0.001) (Fig. 3A). However, the
blank control group and the negative control group could not induce a proliferative
T-cell immune response (Fig. 3A). In addition, a strong T cell proliferative response
was elicited by concanavalin A (Con A) (p < 0.001) (Fig. 3A).
The levels of IL-2 and IFN-γfrom the culture supernatants of splenocytes
isolated from SOD-immunized group mice were significantly higher than that from
the negative control group and the blank control group (p < 0.01) (Fig. 3B, 3C). These
results strongly suggested that immunization with SOD protein in mice could elicite a
Th1-type immune response.
Mouse whole-blood killing bacteria assay
The results demonstrated that serum from the negative control group and the blank
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control group could not inhibit the growth of either SH0165 strain or MD0322 strain
(Fig. 4). However, the serum from SOD immunized group displayed a clearly
reduction of the viable titers of SH0165 strain and MD0322 strain (p < 0.01) (Fig. 4),
which demonstrated that the antibodies induced by the recombinant SOD protein
potentially provide a certain immunoprotection against the challenge of H. parasuis.
Protective efficacy against H. parasuis challenge in mice
The results of immunization and challenge experiments showed that the survival
of mice from immunized with SOD protein against MD0322 strain challenge was
significantly better than that group immunized with SOD protein against SH0165
strain challenge, 80% and 73.3%, respectively (p < 0.05) (Fig. 5A and 5B). In addition,
the immunoprotective efficacy in groups immunized with SOD protein were markedly
greater compared with the blank control groups and the negative control groups (p <
0.01) (Fig. 5A), in which no immunoprotective efficacy was observed, because all
mice in the blank control group died within 2 days after challenge (Fig. 5B).
The significance of antibody response to immunoprotection effect
The results showed that the percentages of surviving mice immunized with the
serum from SOD protein against SH0165 strain or MD0322 strain challenge were
37.5% and 50%, respectively, which were significantly higher than that from the
negative control group (p < 0.01) (Fig. 6A, 6B).
Histopathologic analysis
All of the mice from the blank control groups and from the negative control
groups displayed severe tissue damage in the lung. However, all survival mice from
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SOD protein immunized groups did not appear obvious pathological damage. Lung
tissues from blank control groups or negative control groups challenged by either H.
parasuis MD0322 strain or SH0165 strain exhibited extensive edema with massive
proliferation of fibroblasts and connective tissue formation. The bronchioles were
suffused with cellular exudates composed of neutrophils (Fig. 7C, 7D, 7F, 7G).
However, only minor pathological damages were detected in the tissue of lung from
mice immunized with SOD protein (Fig. 7E, 7H). These results indicated that
immunization with SOD protein could inhibit tissue pathological damage following H.
parasuis challenge, which also confirm that SOD protein could confer
immunoprotection efficacy against infection of H. parasuis.
Discussion
The current studies were carried out to explore the role of superoxide dismutase
(SOD) of H. parasuis in triggering immunoprotection efficacy against a lethal
challenge in mice. In this study the sodA gene of H. parasuis was cloned and
expressed and its immunoprotection effect was determined. To our best knowledge,
this is the first report documenting that SOD protein of H. parasuis has immunogenic
characteristic and can trigger humoral and cell mediated immune responses in the
SOD-immunized mice. In addition, immunization of mice with SOD protein could
provide protective effect against the lethal dose of H. parasuis serovar 4 and serovar 5
challenge in mice.
Previous studies have reported that SOD can control the levels of reactive oxygen
species (ROS) and reduce neutrophil recruitment to the lung and dampen
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inflammatory responses during noninfectious insult (Bowler et al. 2004; Yao et al.
2010). Extracellular SOD has a high affinity in binding to heparin sulfate and collagen
in the extracellular matrix (ECM) and has important effects on protecting tissues from
oxidative damage and inflammation (Petersen et al. 2004; Gao et al. 2008).
Extracellular SOD also plays a detrimental effect during L. monocytogenes infection
which decreased host survival, bacterial clearance, TNF-α and peroxynitrite
production, and neutrophil function (Break et al. 2012). SOD has been used in many
pharmaceutical compositions for treatment of diseases. To evaluate the immune
responses and immunoprotection effect against H. parasuis challenges, a murine
model has been developed in the past (Zhou et al. 2009; Fu et al. 2012a). Again, we
used this well - developed murine model and demonstrated that the SOD protein can
protect against lethal dose of H. parasuis infection, which provides a novel approach
for the development of a H. parasuis SOD subunit vaccine.
Previous research has been showed that the subtype of IgG and the type of Th
immune responses are important for protective immunity against certain disease
(Chiang et al. 2009; Fu et al. 2013b). The production of IgG1 subclass is
representative of T helper 2 (Th2) immune response, whereas the IgG2a is the marker
of Th1 immune response. In this study, we showed that SOD protein of H. parasuis
can trigger the production of both subclasses of IgG1 and IgG2a. Furthermore, the
higher levels of IgG2a demonstrated the dominance of Th1 immune response over
Th2 immune response. To further verify the types of immune response, the
productions of cytokine from the splenocytes were measured. Our results showed that
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SOD protein of H. parasuis also induced the secretion of IL-2 and IFN-γ of
splenocytes, but not the production of IL-4. Th1 immune cells mainly produce IL-2
and IFN-γ and Th2 immune cells mainly produce IL-4 (Gagliani and Huber, 2017).
These results, together with the data of passive immunization assay and whole-blood
killing bacteria assay, indicate that the effect of SOD protein of H. parasuis is likely
related to cellular immune response which elicits strong immunoprotection function
against H. parasuis infection.
It has been documented that serum resistance is thought to be an important
virulence pathological mechanism in H. parasuis leading to pig systemic disease and
virulent strain were mainly more resistant to the bactericidal effect of the serum than
the non-virulent strains (Cerdà-Cuéllar and Aragon 2008). Serovars are commonly
considered as a marker of virulence in H. parasuis (Oliveira and Pijoan 2004). In
general, serovar 5 was thought to be highly virulent, whereas serovar 4 was
considered to be moderately virulent (Amano et al. 1994). In this study, we elected to
use the Chinese local isolates, MD0322 (serovar 4) strain and SH0165 (serovar 5)
strain, as the challenge strains. These virulence strains allow us to simulate a natural
situation of infection. In addition, we showed that both highly virulent SH0165 strain
and moderately virulent MD0322 strain were killed by the antiserum induced by SOD
protein with different degrees and serovar 4 MD0322 strain is more sensitive to
bactericidal effect of the antiserum. This new finding is somehow contradictory to our
previous finding (Fu et al. 2013a). To address this discrepancy, we are in the process
of designed new experiments to dissect the underlying mechanism. Furthermore, the
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result of blood killing bacteria assay also suggests that the serovar 5 is more virulent
than serovar 4, which implies that antibody responses seems to play an important role
in SOD-triggered immunoprotection.
Our results showed that SOD protein could provide immnoprotection effect in the
female BALB/c mice against lethal dose challenge of serovar 4 and serovar 5 of H.
parasuis.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(grant no. 31402225, 31572572, 31302089, 31601922), the Natural Science
Foundation of Hubei Province, China (grant no. 2014CFB345, 2014CFC1022,
2012FFBO4804) and Youth Science and Technology of Wuhan Morning program
(grant no. 2015070404010194).
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Legends to figures
Fig.1. Expression of SOD protein in E. coli BL21 (DE3). The E. coli BL21 (DE3)
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cells were grown to log-phase and induced with 1mM IPTG for 3 h at 37 ℃. The
target protein was eluted using 500 mM of imidazole. (A) (1) (2) (3) induced with
IPTG; M: molecular weight marker. (B) (1): elution; M: molecular weight marker.
Fig.2. (A) Antibody titers in mice from immunized group, the negative control
group and the blank control group. Blood samples were collected on the 14 day
after the booster immunization, and the antibody response was determined by ELISA;
optical density readings of > 0.3 were scored as positive. (B) The levels of IgG1
and IgG2a in mice from immunized group, the negative control group and the
blank control group. Results are expressed as means ± SD. **, significance at a p
value of < 0.01; ***, significance at a p value of < 0.001. △, versus the negative
control group and the blank control group.
Fig.3. (A) Lymphocyte proliferation assay. On the 14th day following the boost
immunization, 200 µL of splenocytes from immunized group, the negative control
group and the blank control group were cultured (1 × 105 cells/mL) were stimulated
with the SOD protein (4 µg/well) and concanavalin A (4 µg/well) and incubated for 72
h at 37 in a 5% CO℃ 2 incubator. (B) and (C) Levels of IL-2 and IFN-γ secretion in
cultured splenocytes of mice from immunized group, the negative control group
and the blank control group. Results are means ± SD; ***, significance at a p value
of < 0.001. △, versus the negative control group and the blank control group.
Fig.4. The whole blood bactericidal activity of SOD immunized, negative control
and blank control mice. The results are expressed as the percent killing bacteria
activity according to the percentage of reduction of bacteria growth in the presence of
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immune blood serum. The results were performed from three independent assays. “*”
indicates significance at p < 0.05 and “**” indicates significance at p < 0.01. △,
versus the negative control and the blank control.
Fig.5. (A) Survival of mice from SOD immunized group, negative control group and
blank control group following challenge against H. parasuis MD0322 strain. (B)
Survival of mice from SOD immunized group, negative control group and blank
control group following challenge against H. parasuis SH0165 strain.
Fig.6. (A) Survival of mice immunized with serum from immunized group and
negative control group following challenge against H. parasuis MD0322 strain. (B)
Survival of mice immunized with serum from immunized group and negative control
group following challenge against H. parasuis SH0165 strain.
Fig.7. Representative lung sections of mice in different treatment groups
(immunized groups, the negative control groups and the blank control groups).
(A) Negative control groups, blank control groups and SOD-immunized groups
challenged against H. parasuis MD0322 strain. (B) Negative control groups, blank
control groups and SOD-immunized groups challenged against H. parasuis SH0165
strain. Negative control group: C, F; Blank control group: D, G; SOD protein
immunized group: E, H.
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Fig.1. Expression of SOD protein in E. coli BL21 (DE3). The E. coli BL21 (DE3) cells were grown to log-phase and induced with 1mM IPTG for 3 h at 37 ℃. The target protein was eluted using 500 mM of imidazole. (A)
(1) (2) (3) induced with IPTG; M: molecular weight marker. (B) (1): elution; M: molecular weight marker. black and white
75x41mm (300 x 300 DPI)
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Fig.2. (A) Antibody titers in mice from immunized group, the negative control group and the blank control group. Blood samples were collected on the 14 day after the booster immunization, and the antibody
response was determined by ELISA; optical density readings of > 0.3 were scored as positive. (B) The levels
of IgG1 and IgG2a in mice from immunized group, the negative control group and the blank control group. Results are expressed as means ± SD. **, significance at a p value of < 0.01; ***, significance at a p value
of < 0.001. △, versus the negative control group and the blank control group.
black and white
98x49mm (300 x 300 DPI)
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Fig.3. (A) Lymphocyte proliferation assay. On the 14th day following the boost immunization, 200 µL of splenocytes from immunized group, the negative control group and the blank control group were cultured (1
× 105 cells/mL) were stimulated with the SOD protein (4 µg/well) and concanavalin A (4 µg/well) and incubated for 72 h at 37 ℃ in a 5% CO2 incubator. (B) and (C) Levels of IL-2 and IFN-γ secretion in cultured
splenocytes of mice from immunized group, the negative control group and the blank control group. Results are means ± SD; ***, significance at a p value of < 0.001. △, versus the negative control group and the
blank control group. black and white
98x34mm (300 x 300 DPI)
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Fig.4. The whole blood bactericidal activity of SOD immunized, negative control and blank control mice. The results are expressed as the percent killing bacteria activity according to the percentage of reduction of
bacteria growth in the presence of immune blood serum. The results were performed from three independent assays. “*” indicates significance at p < 0.05 and “**” indicates significance at p < 0.01. △,
versus the negative control and the blank control. black and white
79x81mm (300 x 300 DPI)
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Fig.5. (A) Survival of mice from SOD immunized group, negative control group and blank control group following challenge against H. parasuis MD0322 strain. (B) Survival of mice from SOD immunized group, negative control group and blank control group following challenge against H. parasuis SH0165 strain.
black and white 98x42mm (300 x 300 DPI)
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Fig.6. (A) Survival of mice immunized with serum from immunized group and negative control group following challenge against H. parasuis MD0322 strain. (B) Survival of mice immunized with serum from immunized group and negative control group following challenge against H. parasuis SH0165 strain.
black and white 98x36mm (300 x 300 DPI)
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Fig.7. Representative lung sections of mice in different treatment groups (immunized groups, the negative control groups and the blank control groups). (A) Negative control groups, blank control groups and SOD-immunized groups challenged against H. parasuis MD0322 strain. (B) Negative control groups, blank control groups and SOD-immunized groups challenged against H. parasuis SH0165 strain. Negative control group:
C, F; Blank control group: D, G; SOD protein immunized group: E, H. black and white
155x85mm (300 x 300 DPI)
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