Draft · 2017-03-28 · Draft 1 Evaluation of recombinant protein superoxide dismutase of Haemophilus parasuis strain SH0165 as vaccine candidate in a mouse model Ling Guo 1†, Lei

Draft

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

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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1

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: [email protected]

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: [email protected]

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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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Draft · 2017-03-28 · Draft 1 Evaluation of recombinant protein superoxide dismutase of Haemophilus parasuis strain SH0165 as vaccine candidate in a mouse model Ling Guo 1†, Lei