α-Enolase as a novel vaccine candidate against Streptococcus dysgalactiae infection in cobia (Rachycentron canadum L.)

Journal Pre-proof α-Enolase as a novel vaccine candidate against Streptococcus dysgalactiae infection in cobia (Rachycentron canadum L.) Thuy Thi Thu Nguyen, Hai Trong Nguyen, Yi-Ting Wang, Pei-Chi Wang, Shih-Chu Chen PII:

S1050-4648(19)31093-9

DOI:

https://doi.org/10.1016/j.fsi.2019.11.050

Reference:

YFSIM 6619

To appear in:

Fish and Shellfish Immunology

Received Date: 18 June 2019 Revised Date:

6 November 2019

Accepted Date: 19 November 2019

Please cite this article as: Thu Nguyen TT, Nguyen HT, Wang Y-T, Wang P-C, Chen S-C, α-Enolase as a novel vaccine candidate against Streptococcus dysgalactiae infection in cobia (Rachycentron canadum L.), Fish and Shellfish Immunology (2019), doi: https://doi.org/10.1016/j.fsi.2019.11.050. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

α-Enolase as a novel vaccine candidate against Streptococcus dysgalactiae infection

2

in cobia (Rachycentron canadum L.)

3 4

Thuy Thi Thu Nguyena, Hai Trong Nguyena, Yi-Ting Wanga, Pei-Chi Wanga,b,c,e*, Shih-Chu Chena,b,c,d,e*

5

a

6

Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan, ROC

7 8 9

b

10 11

c

12 13

d

14

e

15

International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan, ROC Southern Taiwan Fish Disease Centre, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan, ROC Research Center for Animal Biologics, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan, ROC Research Center for Fish Vaccines and Diseases, College of Veterinary Medicine, National Pingtung University of Science and Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan, ROC

16 17

* Corresponding author:

18

Department of Veterinary Medicine, National Pingtung University of Science and

19

Technology, No. 1, Shuefu Road, Neipu, Pingtung 91201, Taiwan

20

Tel.: +886 8 7703202 (ext. 5076 or 5095), Fax: +886 8 7700447

21

E-mail: [email protected], [email protected]

22

E-mail address: [email protected] (T.T.T. Nguyen); [email protected]

23

(H.T. Nguyen); [email protected] (Y.-T. Wang); [email protected] (P.-Y. Wang);

24

[email protected] (S.-C. Chen)..

25

Abstract

26

Streptococcus dysgalactiae is an important pathogenic bacterium that has caused economic loss for the

27

cobia industry in Taiwan, ROC. This study presents a highly effective subunit vaccine composed of a

28

moonlight protein, α-enolase, for the prevention of S. dysgalactiae infection. First, α-enolase was

29

cloned, transformed, and expressed in E. coli for production of recombinant protein. Then, the

30

protective efficacies of α-enolase recombinant protein were evaluated in combination with either a pro-

31

inflammatory cytokine, TNF-α, or an oil adjuvant, ISA 763 AVG. The results showed that the

32

combination of α-enolase and ISA 763 AVG was highly protective (RPS = 88.89%), while a negative

33

effect was found in the group immunised with α-enolase adjuvanted with TNF-α (RPS = 22.22%). A

34

further study was conducted with double dose of ISA 763 AVG, which led to an increased RPS value

35

of 97.37%. Moreover, immunised cobia exhibited significantly greater lysozyme activity, antibody

36

responses, and expression of certain immune-related genes post-challenge. Altogether, our results

37

demonstrated that a combination of α-enolase recombinant protein with ISA 763 AVG adjuvant is a

38

promising vaccine that can be employed for protection of cobia against S. dysgalactiae infection.

39

Keywords: α-enolase, Streptococcus dysgalactiae, cobia, ISA 763 AVG, subunit vaccine

40

1 Introduction

41

Cobia (Rachycentron canadum) is an attractive fish species for marine aquaculture in Asian

42

countries [1, 2]. In 2003, nearly 4,000 metric tons of cobia was produced in Taiwan, ROC; however,

43

this value decreased to 884 metric tons in 2012 [3]. Disease, especially Streptococcosis and

44

Photobacteriosis, is the major reason for this decline in cobia production [2]. Streptococcosis caused by

45

Streptococcus dysgalactiae, a Gram-positive cocci bacterium, is the recent concern of cobia farmers in

46

Taiwan, ROC [4-6]. This disease usually occurs during the hot season (from July to October), causing

47

fish death and negatively impacting the market size, which results in economic losses for the cobia

48

industry [4]. In addition, S. dysgalactiae is known as causative agent of streptococcosis in a wide range

49

of farmed fish species from diverse habitats worldwide: 1) In sea-water fish, e.g. amberjack (Seriola

50

dumerili) and yellowtail (Seriola quinqueradiata) in Japan [5, 7, 8], pompano (Trachinotus blochii) in

51

Taiwan, ROC and Malaysia, and white-spotted snapper (Lutjanus stellatus) in Malaysia [8]; 2) In

52

brackish-water fish such as soiny mullet (Liza haematocheila) and grey mullet (Mugil cephalus) in

53

Taiwan, ROC [4, 9]; 3) In fresh-water fish such as Nile tilapia (Oreochromis niloticus) in Brazil,

54

hybrid red tilapia (Oreochromis sp.) in Indonesia [8]. Therefore, prevention and control of this

55

infectious disease are crucial for the sustainable development of these potential farmed fish species.

56

Vaccination has had a great impact on disease reduction and has been a key reason for the

57

success of salmon cultivation [10]. Inactivated vaccines derived from whole pathogens were previously

58

the most common strategy for the prevention of aquatic animal diseases. However, currently, the

59

recombinant subunit approach is more attractive for the development of new vaccines owing to its

60

effectiveness, safety, and serotype independence [11]. In most cases, potential protein candidates were

61

identified based on either surface exposed/secreted proteins, or polysaccharide capsules [12-14]. These

62

components can reduce toxicity as well as achieve cross-protection due to the use of conserved

63

antigens [12, 13]. Of note, there are a number of reports on the protective potentials of some

64

cytoplasmic

glycolytic

enzymes

such

as

α-enolase

[15,

16],

glyceraldehyde-3-phosphate

65

dehydrogenase (GAPDH) [17-19], and fructose-1, 6-bisphosphate aldolase (FBA) [20] in pathogenic

66

streptococci.

67

α-Enolase is a highly conserved and prevalent protein among pathogenic S. dysgalactiae [5]. It

68

is a glycolytic enzyme that presents on the surface of most streptococcus species as a multifunctional

69

protein [21]. In the cytoplasm, α-enolase catalyses the conversion of phosphoglycerate into

70

phosphoenolpyruvate. However, when α-enolase is exported to the cell-surface, it becomes involved in

71

the pathogenicity by binding the plasminogen and cell adhesion/invasion of several infectious

72

streptococcus species [22-25]. Moreover, α-enolase was identified as an important antigen for the

73

development of subunit vaccines against S. iniae in tilapia [15], catfish [26], against Streptococcus

74

agalactiae in tilapia [27], and against Candida albicans [28], Ascaris suum [29], and Streptococcus

75

suis [25] in mice. Therefore, α-enolase is a promising protein candidate that needs to be explored in an

76

emerging pathogen, S. dysgalactiae.

77

Although recombinant subunit proteins provide many advantages for future vaccine

78

development, they generally have poor immunogenicity when administered alone [30]. Thus, adjuvants

79

are required to hold antigens in a stable form, slowing the antigen processing and prolonging both

80

tissue retention and the period of immune stimulation [31, 32]. Various adjuvants are used in fish

81

vaccines, including oil-based adjuvants along with immunostimulatory adjuvants such as cytokines

82

(TNF-α, IL-8, IL-6) [33-36], cytosine phosphate-guanine (CpG) [37], and lipopolysaccharide (LPS)

83

[38]. To date, Montanide™ ISA 763 AVG (Seppic, Puteaux, France), a non-mineral oil emulsion

84

adjuvant, is effective and often used in fish vaccines [39-41]. Moreover, mature TNF-α recombinant

85

protein was also used as an oral adjuvant to enhance disease resistance against Vibrio anguillarum for a

86

commercial vaccine in seabass [35]. However, the adjuvanticity of TNF- α by intraperitoneal injection

87

has not been investigated in fish. Therefore, in the present study, the protective efficacies of a novel

88

immunogenic protein of S. dysgalactiae, α-enolase, adjuvanted with ISA 763 AVG or TNF-α

89

recombinant protein were investigated to identify a good vaccine formulation for cobia immunization.

90

2 Materials and Methods

91

2.1 Fish husbandry and bacterial preparation

92

Healthy cobia (117.3 ± 13.16 g; 28.29 ± 2.1 cm) purchased from a cobia farm in Pingtung

93

County, Taiwan, ROC, were reared in 3500 L aquaria with a circulatory system and fed twice daily

94

with commercial floating pellets. Fish were kept at 28°C for acclimatization to the laboratory

95

conditions for one week. Fish were confirmed to be free of S. dysgalactiae infection and antigens by

96

bacterial isolation and sera agglutination tests, respectively, from 5 fish before conducting experiments.

97

Feeding was stopped 24 h before and after vaccination, and fish were anesthetized using 2-

98

phenoxythanol when handled.

99

For challenge studies, the S. dysgalactiae strain SD12 was revived from -80°C stock and grown

100

on blood agar containing 1.5% NaCl at 25°C for 36 h. Bacterial cells were harvested in phosphate-

101

buffered saline (7 mM Na2HPO4.12H2O, 5.6 mM NaH2PO4.2H2O, and 150 mM NaCl, pH 7.2) (PBS)

102

prior to injection.

103

2.2 Cloning, expression and sequence analysis of recombinant α-enolase protein (rEnolase)

104

α-enolase was amplified from the total DNA of S. dysgalactiae strain SD12 using the pair of

105

primers EnoE1 and EnoE2 (Table 1), designed based on the nucleotide sequence on the National

106

Center for Biotechnology Information (NCBI; accession no.: AB758245). PCR amplification was

107

carried out under the follow thermal parameters: a denaturation step at 95°C for 5 min; 30 cycles of

108

denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and elongation at 72°C for 1 min 30 s; and a

109

final elongation step at 72 °C for 10 min. The PCR product was then detected on agarose gel and

110

purified using a Gel/PCR Purification Kit (Viogene, Taiwan) according to the manufacturer’s

111

instruction.

112

Table 1. The sequences and product sizes of primers used in this study Target gene

Primer name

Sequence (5′-3′)

PCR products size (bp)

α-enolase

TNF-α

β-Actin

18S rRNA

TNF-α

IL-1β

IL-8

EnoE1

CACCATGTCAATTATTACTGATG

EnoE2

CGCCGTCATTTTTTTAAGTTATAG

cTNF-F

CACCATGAGCAAAGCCAAGGCAG

cTNF-R

GTCGACTCAAAGTGCAAACACACC

β-Actin RC-F

CAGACTGTTCCTCCTCCCC

β-Actin RC-R

AAAATCCTGAGTCAAGCGCC

18S rRNA-F

CGTATTGTGCCGCTAGAGGTG

18S rRNA-R

GTTGGCATCGTT TATGGTCG

cTNF-qF

GAGGACCGGACAGTGATTGTGG

cTNF-qR

GCTTCAGTGTGTAGTGGGGCTC

RCIL-1βF1

CAGGCAGAACAACCACTGAC

RCIL-1βR2

TTCCAAGTCCAGTCCTTTGG

cIL8-qF

GTCATTGTCATTGCTGTGGTGGTGC

cIL8-qR

CCTCGCAATGAGAATTGGCAGGAATC

1308

510

160

153

214

170

171

113 114

The purified PCR product was cloned into the pET151D/TOPO vector (Invitrogen), and then

115

transformed into E. coli DH5α competent cells (RBCBioscience, Taiwan). Transformed E. coli cells

116

were plated on LB-ampicillin (100 µg/mL) agar and PCR with T7 primers were used to identify

117

positive transformants. Plasmids were extracted from positive colonies and then transformed into E.

118

coli BL21 (DE3). Positive transformants in E. coli BL21 were determined as described above and then

119

sub-cultured in 2 mL LB-ampicillin broth medium at 37°C till an OD600 of 0.5–0.6 was reached.

120

Then, 1 mM isopropyl thiogalactoside (IPTG) (Amresco, Solon, OH) was supplied to the cultured

121

suspension for protein expression at 37°C for 4 h. Cell pellets were then harvested by centrifugation at

122

13000 ×g for 10 min and mixed with 5X SDS-PAGE sample-loading buffer. The mixture was heated at

123

100°C for 5 min and subjected to SDS-PAGE gel for detection of protein expression level.

124

The nucleotide sequence of α-enolase cloned from S. dysgalactiae was blasted on the GenBank

125

database (available on NCBI website) to search for homologous sequences. The amino acid (aa)

126

translation and peptide molecular weight prediction were performed using the ExPASy tools

127

(https://web.expasy.org/compute_pi/ and https://web.expasy.org/translate/). The multiple aa sequence

128

alignments were conducted using the CLUSTAL method of MEGALIGN (DNAstar 5.0, DNASTAR

129

Inc., Madison, WI). B cell epitope prediction was conducted using BepiPred, version 2.0

130

(http://www.cbs.dtu.dk/services/BepiPred) to analyse the possible strong immunogenicity fragments of

131

S. dysgalactiae α-enolase. The binding sites of S. iniae DGX07 α-enolase protein were used as a

132

reference to S. dysgalactiae α-enolase.

133

2.3 Cloning and expression of mature TNF-α recombinant protein (rTNF-α)

134

Total RNA was extracted from cobia liver after 12 h of S. dysgalactiae infection using the Total

135

RNA Extraction Kit (Viogene, Taiwan). One microgram of total RNA was used to synthesise cDNA

136

using iScript™ cDNA Synthesis Kit (Bio-Rad, Laboratories, Inc.) as described previously [6]. The

137

TNF-α gene (excluding the transmembrane domain) was amplified using the specific primer pairs

138

cTNF-F3 and cTNF-R (Table 1), which were designed based on a previous published sequence [6].

139

cDNA derived from cobia liver was used as template for PCR amplification. The PCR thermal

140

parameters were as follows: 95°C for 5 min; 32 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30

141

s; and 5 min at 72°C. The PCR product with an expected size of about 510 bp was purified and cloned,

142

and mature TNF-α recombinant protein was expressed in E. coli using the routine protocol as described

143

above.

144

2.4 Purification of recombinant proteins

145

E. coli BL21 (DE3) harbouring recombinant α-enolase and TNF-α proteins were revived from -

146

80°C stock on a LB-ampicillin plate and then sub-cultured in 1 L LB-ampicillin broth medium for

147

expression of protein as described above. The cell pellets were collected by centrifugation at 6,000 ×g

148

for 15 min. rEnolase and rTNF-α proteins were purified and dialysed, and the endotoxin level was

149

determined as described by Nguyen et al. [19].

150

2.5 Preliminary screening with two adjuvant combinations

151

For the preliminary screening, α-enolase recombinant protein was mixed with either the

152

adjuvant ISA 763 AVG at a ratio of 3:7, rTNF-α (1 µg/g fish), or PBS to obtained 1 µg/g fish. The

153

cobia were randomly divided into four groups with 32 fish each and kept separately in a 3.5 m3 tank

154

with a circulation system as described above. Fish were anesthetized and injected intraperitoneally with

155

0.1 mL of PBS, rEnolase + PBS, rEnolase + TNF-α, or rEnolase + ISA (ISA 763 AVG). The booster

156

was performed 2 weeks after the first immunization using the same doses. Blood samples were

157

collected from vaccinated and control fish, 10 fish/group, at 0, 2, and 6 week(s). Six weeks post-

158

primary immunization, fish were infected by intraperitoneal injection with live S. dysgalactiae at a

159

concentration of 7 × 107 CFU/fish. Then, fish from each group were divided into two sub-groups (16

160

fish each) which maintained separately in two different tanks for 2 weeks. Mortalities were observed

161

daily over two weeks period and all dead fish were collected for bacterial re-isolation. The relative

162

percentage of survival (RPS) was calculated using the following formula:

163 164

RPS = [1 – (% mortality of vaccine group/% mortality of control group)] × 100 2.6 Second immunization of α-enolase delivered with double the dose of ISA 763 AVG

165

In order to enhance the protective effectiveness of rEnolase adjuvanted with ISA763 AVG,

166

rEnolase was mixed with the adjuvant ISA763 AVG at the same ratio but using double the dose of

167

adjuvant. Hence, rEnolase (diluted in PBS to obtain 2 mg/mL) was mixed with the adjuvant ISA763

168

AVG at a ratio of 3:7. A total of 159 cobia were randomly divided into three groups with 53 fish each

169

and then each group were randomly separated into two sub-groups (26 and 27 fish each) which were

170

reared in two different tanks. Fish were intraperitoneally injected with 0.2 mL of PBS, PBS + ISA763

171

AVG, or rEnolase + ISA763 AVG to obtain approximately 1 µg protein/g fish. The booster was carried

172

out using the same method and dosage at 2 weeks after the first immunization. Bleedings were

173

performed at 0, 1, 2, and 6 week(s) post-primary immunization from 10 fish/group and sera were

174

divided into five samples of two fish each. The non-vaccinated and vaccinated fish were challenged by

175

intraperitoneal injection with live S. dysgalactiae at a concentration of 1.45 × 108 CFU/fish (2 LD50) 4

176

weeks after the booster. Mortality was recorded and RPS values were determined as described above

177

after 14 days of observation. Moreover, five fish from each group were randomly collected for head-

178

kidney sampling at 0 h, 12 h, and 24 h post challenge (PC) for cytokine expression studies.

179

2.7 SDS-PAGE and western blot

180

Protein samples were mixed with 5X SDS-PAGE sample loading buffer, heated at 100°C for 5

181

min and then subjected to 12% (w/v) SDS-PAGE gel as described previously [42, 43]. Following

182

electrophoresis, the gel was stained with Coomassie brilliant blue or directly used for western blot.

183

Western blotting was carried out as described by Mahmood and Yang (2012) [44] with some

184

modifications. Proteins from SDS-PAGE gels were transferred to polyvinylidene difluoride membranes

185

(Invitrogen). Following blocking at 4°C with 5% skim milk in PBST (PBS with 0.1% Tween 20), the

186

membranes were incubated with either mouse anti-6-Histidine antiserum (diluted 1:2000 in PBST) or

187

anti-S. dysgalactiae cobia antisera (prepared as described by Nguyen et al. [6]; diluted 1:500 in PBST)

188

for 2 h at room temperature (RT). The membrane incubated with anti-S. dysgalactiae cobia antisera

189

was then bound to mouse anti-cobia IgG1 antibody (Aquatic Diagnostic Ltd., Scotland, diluted 1:2000

190

in PBST) for 1 h at RT with gentle shaking. Then, both membranes were incubated with the HRP-

191

conjugated goat anti-mouse IgG antibody (diluted 1:3000 in PBST) for 1 h. The membranes were

192

washed five times with PBST for 5 min each between each reaction. Finally, Western Lightning™

193

Plus-ECL (PerkinElmer Inc., USA) was added to recognise the expressed proteins from recombinant E.

194

coli cell lysates using the Luminescence Fluorescence Imaging System (Syngene).

195

2.8 Serum lysozyme assay The lysozyme assay was performed based on the method described by Parry et al. [45] and

196 197

Nguyen et al. [46].

198

2.9 Evaluation of specific antibody

199

An indirect ELISA with α-enolase as the coating antigen was employed to quantify the specific

200

antibodies from cobia sera post-vaccination (PV). The detailed protocol was well documented by

201

Nguyen et al. [19]. 500 ng/well of rEnolase was used as the coating antigen and cobia sera were diluted

202

1:50 in PBST.

203

2.10

Gene expression analysis

204

Total RNA was extracted from the tissues using Trizol® (Invitrogen, USA) and cDNA was

205

synthesised using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.) as described

206

previously by Nguyen et al. [6].

207

The expression of immune-related genes such as IL-1β [47], TNF-α, and IL-8 [6] in the head

208

kidney was evaluated by qRT-PCR. Two reference genes, β-actin and 18S rRNA, were used as

209

housekeeping genes for normalisation [6, 47]. The primer pairs of each gene and their sequences are

210

presented in table 1. qRT-PCR was performed using the iQSYBR Green Supermix (Bio-Rad

211

Laboratories, Inc.) following the protocols described in the cited references. Each sample was analysed

212

in triplicate. The relative expression levels were determined by the 2(-∆∆Ct) method described by Livak

213

et al. [48] with ∆∆Ct values described by Nguyen et al. [19].

214

2.11

Statistical analysis

215

SPSS 22 software was used for statistical analysis. The differences in antibody titres, lysozyme

216

activity, and gene expression levels at each time point were analysed by one-way ANOVA with

217

Duncan’s test. A P value < 0.05 was considered to indicate statistical significance.

218

3 Results

219

3.1 Production of rEnolase

220

The α-enolase gene (1308 bp) amplified from S. dysgalactiae was cloned into the

221

pET151/TOPO vector and transformed into E. coli DH5α (Fig. 1A). The generated 6x His-tagged α-

222

enolase was then transformed and expressed in E. coli BL21 (DE3). An expected band of about 51 kDa

223

(Fig. 1B), close to the predicted molecular weight, was observed on an SDS-PAGE gel after

224

overexpression. Western blot analysis revealed that the 51 kDa recombinant protein, rEnolase, reacted

225

specifically with the mouse anti-His and cobia anti-Streptococcus dysgalactiae antisera (Fig. 1B).

226

Purification of recombinant protein using Ni–NTA affinity chromatography exhibited a unique protein

227

of this size (Fig. 1B).

228

(A) 1

M

bp (B)

229

kDa M 1500

230

1000

1

2

3

4

5

90 72

231 500

232

55

233

43

234

34

235

Figure 1. PCR amplification of the α-enolase gene; expression, western blotting, and purification of α-

236

enolase recombinant protein (51 kDa). (A) PCR amplicons of α-enolase. Lane M: 100 bp DNA ladder

237

(NEB); lane 1: the PCR product of α-enolase (1308 bp). (B) Expression, western blotting, and

238

purification of α-enolase recombinant protein. Lane M: Protein ladder; lane 1: non-induced E. coli

239

BL21 (pET151. α-enolase); lane 2: induced E. coli BL21 (pET151. α-enolase); lane 3: western blot

240

analysis using anti-His antibody; Lane 4: western blot analysis using S. dysgalactiae-cobia antiserum;

241

lane 5: purified rEnolase.

242

3.2 α-enolase sequence analysis

243

The full-length sequence of the α-enolase gene consisted of 1308 base pairs (bp) with an open

244

reading frame (ORF) encoding 435 amino acids (aa) (Fig. 2). The predicted molecular weight of the

245

deduced aa sequence was approximately 47 kDa (pI = 4.64). The α-enolase aa sequences derived from

246

cobia S. dysgalactiae strains SD12 and SD49 shared 100% identity with each other. The sequences also

247

exhibited highly similarity with S. dysgalactiae subsp. dysgalactiae (SDSD) isolated from kingfish

248

(AB758245, 99.3%), S. dysgalactiae subsp. equisimilis (SDSE, AP012976) (99.8%), Streptococcus

249

pyogene (CP007241, 99.8%), Streptococcus iniae DGX07 (KF460454, 97.5%), Streptococcus

250

agalactiae (AF439649, 96.6%), and Streptococcus suis (FJ895346, 96.1%).

251

Analysis of aa sequences revealed putative active sites found in S. iniae DGX07, e.g. one

252

plasminogen-binding site (249-257 FYDKELGVY), seven substrate-binding sites (155 H, 164 E, 292

253

E, 319 D, 344 K, 371-374 SHRS, 395 K), two enzyme active sites (205 E, 344 K), and three metal-

254

binding sites (243 D, 292 E, 319 D; magnesium). In the immunological characteristic aspect, 14 linear

255

B-cell epitopes were found in this protein (Fig. 2). Neither signal peptide cleavage sites nor

256

transmembrane domains were found in the aa sequences.

257

EEEEEE

EEEEEEEEEEEEEEE

258 259 260

EEEEEEEEE

EEEEEEEEE

EEEEEEE

EEEE

261 262 EEEEEEEEEEE EEEEEEEEEEEE

263

EEEEE

264 265 266

EEEEEEEEEEEEEEEEEEEEEEEEEEEE

EEEEE

267 268 269

EE

EEEE

270 271 EEEEEEEEEEEE

272 273 274 275

Figure 2. Multiple amino acid sequence alignments of α-enolase from cobia S. dysgalactiae with those

276

from other Streptococcus species. Dots (.) indicate similar residues, and different letters represent

277

different residues at the same position. A residue annotated with a red “E” was predicted as being part

278

of a linear B-cell epitope. The putative plasminogen-binding regions (249-257 FYDKELGVY),

279

substrate-binding sites (155 H, 164 E, 292 E, 319 D, 344 K, 371-374 SHRS, 395 K), enzyme active

280

sites (205 E, 344 K), Mg2+-binding sites (243 D, 292 E, 319 D) were in blue, green, yellow and black

281

boxes, respectively. The numbers on amino acid and in the left hand site displayed the numbers of

282

amino acid and the derived nucleotide sequences, respectively.

283

3.3 Production of rTNF-α

284

To determine the role of cobia TNF-α in immune regulation, the mature form of TNF-α

285

(excluding the transmembrane domain) was successfully amplified by PCR (Fig. 3A), cloned into the

286

pET151/TOPO vector, and transformed into E. coli DH5α. The plasmid harbouring the TNF-α gene

287

was isolated and transformed into E. coli BL21 (DE3). The product of positive transformants in E. coli

288

BL21 (DE3) produced an expressed protein band of approximately 23 kDa in an SDS-PAGE gel (Fig.

289

3B). This result suggested that the mature form of TNF-α is approximately 19 kDa, similar to the

290

predicted molecular weight using the ExPASy tool. The western blot using anti-His antibody showed a

291

strong band with the same size as the expressed protein (Fig. 3B). As expected, the mature form of

292

cobia TNF-α was soluble and could be purified using Ni–NTA affinity chromatography (Fig. 3B).

293

(B)

(A) 1

294

M

bp

295 1000 296 500

kDa M 55 43

1

2

3

4

34 26

297 300

15

298 299 300

Figure 3. PCR amplification of the TNF-α gene; expression, western blotting, and purification of

301

rTNF-α protein (23 kDa). (A) PCR amplification of TNF-α. Lane M: 100 bp DNA ladder (NEB); lane

302

1: PCR product of TNF-α (510 bp). (B) Expression, western blotting, and purification of rTNF-α. Lane

303

M: Protein ladder; lane 1: non-induced E. coli BL21 (pET151. TNF-α); lane 2: induced BL21 (pET151.

304

TNF-α); lane 3: western blot analysis of rTNF-α protein using anti-His antibody; lane 4: purified rTNF-

305

α.

306

3.4 Effectiveness of α-enolase recombinant protein in combination with ISA 763AVG and TNF- α

307

The first screening experiment was conducted with 4 different groups (32 fish/group): the

308

control (PBS), rEnolase, rEnolase + ISA, and rEnolase + TNF-α. Sera samples were collected from 5

309

fish in each group before immunization and challenged to check the antibody responses. ELISA using

310

rEnolase as the coating antigen revealed that antibody titres were remarkably upregulated in three

311

vaccinated groups at 2- and 6-week PV. Of note, the sera antibody levels in cobia injected with

312

rEnolase only and rEnolase + ISA at 6 weeks (1.07 and 1.49, respectively) were greater than at 2 weeks

313

(0.59 and 1.16, respectively). In contrast, antibody levels in rEnolase + TNF-α immunised fish

314

displayed a slightly decrease at 6 weeks (0.52) in comparison with 2 weeks (0.6).

315

6 weeks PV, vaccinated and non-vaccinated fish were challenged by S. dysgalactiae using a low

316

dose of approximately 7 × 107 CFU/fish (close to the LD50 dose). Following challenge, the first

317

mortality occurred on day 2 in three groups – control (PBS), rEnolase, and rEnolase + TNF-α – and

318

continued until day 10 (Fig. 4). Two fish from the rEnolase + ISA group died on day 3 PC, after which

319

no more fish died. After 14 days of observation, the cumulative mortality in the control group reached

320

56.25%, while the mortalities in the rEnolase, rEnolase + ISA, and rEnolase + TNF-α groups were

321

28.13%, 6.25%, and 43.37%, respectively (Fig. 4A). Many pure S. dysgalactiae colonies could be re-

322

isolated from the kidneys, livers, spleens, and brains of the dead fish, while only a few colonies were

323

recovered from ~10% of surviving fish on day 15 PC. At 6 weeks PV, the average RPS values in the

324

rEnolase, rEnolase + ISA, and rEnolase + TNF-α groups were 50%, 88.89%, and 22.22%, respectively.

325

These results indicate that ISA 763 AVG could enhance the protective efficacy of rEnolase, whereas

326

TNF-α exerted negative effects on vaccinated fish.

327

(A)

328

1.6

56.25

60

(B)

d

331 332 333

50

43.75

40

28.13 30 20 10

6.25

b

c 1.2 1 0.8

b

c

b

0.6 0.4

a a

0.2 0

0

334

Absorbance at 405 nm

330

Cumulative mortality (%)

1.4

329

1

2

3

4

5

6

7

8

9 10 11 12 13 14

0

2 Week(s) post vaccination

6

Day(s) post challenge

335

PBS

ENO

ENO+ISA

ENO+TNF-α

PBS

ENO

ENO + TNF-α

ENO + ISA

336 337

Figure 4. Cumulative mortalities PC and antibody responses PV. (A) Cumulative mortalities of

338

vaccinated fish during the 14 days after S. dysgalactiae challenge. (B) Antibody titres of vaccinated

339

cobia sera at 0, 2, and 6 week(s) PV. Sera were collected from 5 fish/group at each time point.

340

Different letters indicate significantly different data at each time point (p < 0.05) using Duncan’s test in

341

one-way ANOVA analysis.

342

3.5 Effectiveness of α-enolase delivered with a double dose of ISA 763 AVG

343

3.5.1

Antibody response

344

The second immunisation was carried out with two control groups (PBS and ISA alone) and

345

one vaccinated group, rEnolase + ISA, with a double dose (0.2 mL/fish). However, the protein

346

concentration was administered as a same dose (1 µg/g fish) as with the first screening test. In this

347

experiment, sera were collected at 1 week, 2 weeks, and 6 weeks PV. One week post vaccination,

348

ELISA assay indicated that cobia immunised with rEnolase + ISA 763 AVG (OD405 = 1.26) induced

349

significantly higher antibody titres than two control groups (OD405 = 0.3 in PBS and OD405 = 0.66 in

350

ISA groups). While the antibody levels from the two control groups remained the same, the antibody

351

levels in vaccinated fish were remarkably increased at 2 (OD405 = 1.5) and 6 weeks (OD405 = 2.29) PV

352

(Fig. 5).

353 2.5

Absorbance at 405 nm

354 355 356 357 358

b

2

b

b

1.5 PBS 1

a

a

a

PBS + ISA rEnolase + ISA

a

0.5

a a

359

0 0

360

1

2

6

Week(s) after immunization

361

Figure 5. Antibody titres of vaccinated cobia PV measured using ELISA. Sera were collected from 10

362

fish and divided into five samples of two fish each. Data are presented as means ± SD (n = 5). One-way

363

ANOVA and Duncan’s test were used to identify differences among groups. The data of different

364

groups at the same time point differ significantly (p < 0.05) were displayed in different letters.

365

3.5.2

Lysozyme activity after immunization and challenge

366

Cobia sera were sampled for a lysozyme assay at 1, 2, and 6 week(s) PV, as well as 12 h and 24

367

h PC. Lysozyme activity was determined by a turbidimetric assay, revealing greater lysozyme activity

368

in rEnolase + ISA (281 units/mL) than in PBS (178 units/mL) and ISA (177 units/mL) groups 1 week

369

PV. No significant differences in lysozyme activities among groups were observed at 2 and 6 weeks

370

PV, 12 and 24 h PC (Fig. 6).

500

372

450

373 374 375 376 377

Lysozyme activity (units ml-1)

371

400

b

350 300

PBS

a

250

PBS + ISA

a

200

rEnolase + ISA

150 100 50

378

0 379

0 day

1 week

2 weeks

6 weeks

AC-12H AC-24H

380

Figure 6. Lysozyme activity of vaccinated fish following immunization and challenge (AC). Blood

381

was collected from 10 fish/group and pooled into 5 samples of 2 fish each. Data are presented as means

382

± SD (n=5). Different letters mean significantly different data at the same time point among groups

383

using one-way ANOVA and Duncan’s test (p < 0.05).

384

3.5.3

Gene expression after challenge

385

The mRNA expression levels of IL-1β, IL-8, and TNF-α in the kidneys of two control groups

386

(PBS and PBS + ISA) were upregulated, reaching to highest levels 12 h PC, and decreasing 24 h PC. In

387

contrast, the expression levels of these genes in the rEnolase + ISA immunised group increased

388

gradually and peaked at 24 h PC. Of note, the mRNA expression levels of IL-1β and IL-8 in the

389

vaccinated fish (557-fold and 1.87-fold, respectively) at 24 h PC were greater than in PBS (57-fold and

390

0.7-fold, respectively) and PBS + ISA (58-fold and 0.5-fold, respectively) injected fish. Additionally,

391

although mRNA expression of TNF-α was upregulated PC, no significant difference was observed

392

among groups.

393 IL-1β

394

IL8

700

395

398

150

500 400 300

a a

200

Fold change

397

a

600 Fold change

396

200

b

100

b ab 50

100 0

399

b

a a

12

24

a ab b 0 0

12

24

0

TNF-α

400 30

401 25

403 404

Fold change

402

20 PBS

15

PBS+ISA

10

rEnolase+ISA 5

405 0

406

0

12

24

Hour(s) post challenge

407 408

Figure 7. mRNA expression levels of IL-1β, IL-8, and TNF-α in the kidneys of control and vaccinated

409

fish 0, 12, and 24 h PC. Expression levels of target genes are compared with the expression level in the

410

PBS group at 0 h (calibrate to equal 1). Data are displayed as means ± SD (n=5). Different letters

411

indicate significantly different expression at the same time point.

412

3.5.4

Disease resistance efficacy

413

The second immunization and challenge with higher doses in duplicates showed similar patterns

414

to the first screening test, in which major mortalities occurred from days 2 to 8 PC (Fig. 8). However,

415

100% mortality was observed in the two control groups, while 2.63% of fish died in the vaccinated

416

group (rEnolase + ISA) in two replicates of 19 fish each. The RPS value was increased to 97.37% in

417

this immunization application (Table 2). Bacteria could be re-isolated from all dead fish and no special

418

cross sign was found in the surviving fish.

420 421 422 423 424

Cumulative mortality (%)

419

100 80 60 PBS 40

PBS + ISA

20

rEnolase + ISA

0 1

425

2

3 4

5 6 7 8 9 10 11 12 13 14 Day(s) post challenge

426

Figure 8. Cumulative mortality of vaccinated and non-vaccinated cobia up to 14 days PC with S.

427

dysgalactiae.

428

4 Discussion

429

Lancefield group C Streptococcus dysgalactiae is one of leading causes of the decline in cobia

430

production in Taiwan, ROC in recent years. The screening and identification of protective antigens

431

from S. dysgalactiae are ongoing to prevent or develop therapies against this bacterial infection. So far,

432

the antigenicity of α-enolase, has been explored for protection against various pathogenic

433

microorganisms [16, 25, 28, 29, 49-52]. Here, we cloned α-enolase recombinant protein from a virulent

434

S. dysgalactiae strain, expressed it using E. coli as an expression system, and immunized cobia.

435

Purified α-enolase was preliminarily examined in cobia using two adjuvants: an oil adjuvant (ISA763

436

AVG) and recombinant mature TNF-α. The results indicated that α-enolase provided the highest

437

protection against S. dysgalactiae infection in cobia when it was formulated with the oil adjuvant

438

ISA763 AVG.

439

α-enolase is a moonlighting protein serving as a protective antigen owing to its bacterial surface

440

exposure and interaction with the host plasminogen system. α-Enolase is exposed on the surface of

441

various microorganisms, even though it lacks cell wall anchoring motifs, transmembrane domains, and

442

a signal peptide cleavage site. This non-classical surface-associated protein was shown to be produced

443

in the cytoplasm and then exported to the cell surface depending on covalent binding to the substrate

444

[21]. It has been confirmed in many microorganisms that α-enolase is secreted and attaches to the cell

445

surface in a complex with plasminogen (Plg) [23, 53-55], laminin, or fibronectin to assist with

446

microbial invasion and dissemination in hosts. Amino acid sequences analysis in our study revealed

447

that α-enolase from S. dysgalactiae also possesses a plasminogen-binding site at the position from aa

448

249 to 257 (FYDKELGVY), which may play an important role in interacting with host plasminogen

449

system. Notably, multiple predicted B-cell epitopes found in this protein have shown the potential of α-

450

enolase as a protective antigen for vaccine development.

451

His-enolase recombinant protein with an expected size of 51 kDa was confirmed using western

452

blotting with anti-His antiserum. Of note, the recombinant protein reacted strongly with anti-S.

453

dysgalactiae cobia antisera. The deduced amino acid sequence of the α-enolase gene in S. dysgalactiae

454

isolated from cobia is highly conserved, displaying 100% similarity with each other and >99% with

455

other Streptococcus species, such as S. dysgalactiae subsp. dysgalactiae from Kingfish, S. dysgalactiae

456

subsp. equisimilis, and S. pyogene. Additionally, α-enolase presented with high prevalence (100%)

457

among S. dysgalactiae isolates in our previous study [5]. Therefore, α-enolase is a potential vaccine

458

candidate that has a high possibility of cross-protection and is worth investigating.

459

First, we attempted to screen for the protective efficacy of recombinant protein in combination

460

with two adjuvants, ISA 763AVG and TNF-α. ISA 763 AVG, a non-mineral oil adjuvant, is a

461

metabolisable and injectable adjuvant. This adjuvant has been proven to be non-toxic and can enhance

462

the protective effectiveness of different vaccines used in aquaculture [40, 56]. TNF-α is a pro-

463

inflammatory cytokine that was shown to exhibit adjuvanticity in combination with an oral commercial

464

vaccine against V. anguillarum infection in sea bass [35]. Additionally, TNF-α plays an important role

465

in controlling diseases as the natural induction of TNF-α can protect the host against bacterial,

466

parasitic, and viral infection [57]. In our previous study, we successfully identified this cytokine in

467

cobia [6]. Next, we tried to produce a recombinant protein from the secreted form and screened its

468

efficacy as an adjuvant. We hypothesised that recombinant TNF-α could help to enhance the vaccine

469

efficacy of α-enolase recombinant protein. However, based on our preliminary results, intraperitoneal

470

supplementation with TNF-α at a dose of 1µ g/g fish produced negative side effects in S.

471

dysgalactiae-infected fish. Although the cobia looked normal after immunization, the results

472

revealed that TNF-α made the disease more serious, resulting in more fish deaths in the TNF-α-

473

adjuvanted group compared to α-enolase alone. However, a variety of administered doses should be

474

considered in later studies to characterize the dose effects of this pro-inflammatory cytokine as a

475

vaccine adjuvant.

476

α-Enolase exhibited strong immunogenicity with highly protective efficacy in cobia.

477

Immunization with α-enolase alone can protect 50% of cobia from S. dysgalactiae infection. This

478

effectiveness was improved when α-enolase was adjuvanted with ISA 763 AVG (RPS = 88.89%).

479

Furthermore, protection could reach 97.37% RPS when the dose was increased to 0.2 mL per fish with

480

a double dose of adjuvant. Although no protection was observed when it was administered alone, ISA

481

763 AVG adjuvant enhanced the protective efficacy when combined with α-enolase. Therefore, the

482

effects of this combination were further studied to understand the immune responses during vaccination

483

and infection.

484

Serum antibody response is a key part of adaptive immunity and plays a significant role in

485

combating an extracellular pathogen. In line with previous studies, the protective effectiveness of a

486

vaccine for the prevention of extracellular pathogen infection usually increases with increasing

487

antibody titre [39, 40, 46]. Inducing antibody production can help eliminate invading pathogens by

488

neutralization, opsonization, or activation of the complement system to lyse their cell walls. In this

489

study, vaccinated fish induced significantly greater specific serum antibody titres at early time points

490

(just 1 week PV), and induced higher antibody levels along with the higher RPS value obtained at 6

491

weeks PV. These results again suggest a correlation between increasing antibody levels and protective

492

effectiveness against S. dysgalactiae.

493

Innate immunity is fast acting but plays an important role in bacterial defence. Lysozyme is an

494

important component of innate immunity due to its ability in lysis of peptidoglycans in gram-positive

495

bacterial cell walls [58]. Lysozyme splits the 1,4-beta-linkages between N-acetylmuramic acid and N-

496

acetyl-D-glucosamine residues in the peptidoglycans of Gram-positive bacteria cell walls, leading to

497

lysis of bacterial cells and preventing further invasion [59]. Lysozyme is also known as an opsonin and

498

as an activator of the complement system and phagocytic activity [60]. Lysozyme is widely distributed

499

in teleosts – on their body surface, gill, skin, intestinal tract, and in serum – as a protective factor

500

against bacterial infection. Cobia immunized with an α-enolase recombinant subunit vaccine induced

501

higher lysozyme activity 1-week PV. Upregulation of lysozyme was observed in immunized fish in the

502

present work, suggesting that lysozyme is involved in cobia immune responses and plays a role in the

503

elimination of the pathogenic S. dysgalactiae.

504

Furthermore, expression levels of immune-related genes are important when studying vaccines.

505

Good vaccine candidates are found to stimulate upregulation of some pro-inflammatory cytokines after

506

immunization. Two available cytokines in cobia, TNF-α and IL-1β, are key mediators of the

507

inflammatory response to microbial infection. TNF-α and IL-1β are produced and secreted by various

508

immune cells – especially macrophages, monocytes, and dendritic cells – to induce inflammation as

509

well as the secretion of other cytokines and chemokines [61]. Additionally, IL-8 is an early release

510

chemokine whose main role is the recruitment of other immune cells, particularly neutrophils, to the

511

infected sites to kill pathogens [62]. Therefore, these are important cytokines for the establishment of

512

inflammation and antimicrobial activities. In this study, vaccinated fish induced significantly higher

513

expression levels of IL-1β and IL-8 in the kidney 24 h PC. Although stronger expression levels of these

514

genes were found in control groups 12 h PC, these decreased and presented at lower levels in

515

comparison with the vaccinated fish 24 h PC. These results demonstrate that vaccinated fish induced

516

and maintained antigen-specific cell-mediated immune responses, resulting in the better antimicrobial

517

defence.

518

In conclusion, a novel protein candidate from S. dysgalactiae, α-enolase, is a potential vaccine

519

in combination with adjuvant ISA 763 AVG to protect cobia against this pathogen. An α-enolase

520

subunit vaccine induced both humoral and cellular immune responses with enhanced serum antibody

521

titres, lysozyme activity, and expression of pro-inflammatory cytokines in vaccinated fish. Importantly,

522

the vaccine elicited a highly protective effectiveness, with an RPS value of up to 97.37% with the

523

highly challenged dose. Although α-enolase recombinant protein in combination with oil adjuvant ISA

524

763 AVG is a good vaccine candidate that could be used for prevention of S. dysgalactiae infection in

525

the future, further studies on one-shot immunization regime and reducing the vaccinated dose maybe

526

worth investigations to reduce stress for fish and vaccination costs. Furthermore, in order to enhance

527

the applicable capacity of vaccine, other administrated route of vaccines such as immersion or oral

528

immunization should be considered in future studies.

529 530 531 532

Funding This work was supported by the Ministry of Science and Technology, Taiwan, ROC under Grant nos. MOST 104-2313-B-020-009-MY3 and NSC 101-2313-B-020-015-MY3.

533 534

Conflicts of interest

535

The authors declare no conflicts of interest.

536 537

References

538

[1] U.R. Estrada, F.A. Yasumaru, A.G.J. Tacon, D. Lemos, Cobia (Rachycentron canadum): A

539

Selected Annotated Bibliography on Aquaculture, General Biology and Fisheries 1967–2015, Reviews

540

in Fisheries Science & Aquaculture 24(1) (2015) 1-97.

541

[2] I.C. Liao, Huang, T.S., Tsai, W.S., Hsueh, C.M., Chang, S.L., Leaño, E.M.,, Cobia culture in

542

Taiwan: current status and problems, Aquaculture 237(1-4) (2004) 155-165.

543

[3] H.J. Kaiser JB, © FAO 2007-2016. Cultured Aquatic Species Information Programme.

544

Rachycentron canadum, In: FAO Fisheries and Aquaculture Department [online]

545

http://www.fao.org/fishery/culturedspecies/Rachycentron_canadum/en#tcNA00FE.

546

[4] M.A. Tsai, P.C. Wang, T. Yoshida, S.C. Chen, Genetic characteristics of Streptococcus

547

dysgalactiae isolated from cage cultured cobia, Rachycentron canadum (L.), J Fish Dis 38(12) (2015)

548

1037-46.

(2016)

549

[5] T.T.T. Nguyen, H.T. Nguyen, M.-A. Tsai, O. Byadgi, P.-C. Wang, T. Yoshida, S.-C. Chen, Genetic

550

diversity, virulence genes, and antimicrobial resistance of Streptococcus dysgalactiae isolates from

551

different aquatic animal sources, Aquaculture 479 (2017) 256-264.

552

[6] T.T.T. Nguyen, H.T. Nguyen, P.C. Wang, S.C. Chen, Identification and expression analysis of two

553

pro-inflammatory cytokines, TNF-alpha and IL-8, in cobia (Rachycentron canadum L.) in response to

554

Streptococcus dysgalactiae infection, Fish & shellfish immunology 67 (2017) 159-171.

555

[7] R. Nomoto, Munasinghe, L.I., Jin, D.H., Shimahara, Y., Yasuda, H., Nakamura, A.,, N. Misawa,

556

Itami, T., Yoshida, T., Lancefield group C Streptococcus dysgalactiae infection responsible for fish

557

mortalities in Japan, Journal of fish diseases 27 (2004) 679-686.

558

[8] M. Abdelsalam, S.C. Chen, T. Yoshida, Phenotypic and genetic characterizations of Streptococcus

559

dysgalactiae strains isolated from fish collected in Japan and other Asian countries, FEMS Microbiol

560

Lett 302(1) (2010) 32-8.

561

[9] Z.T. Qi, Tian, J.Y., Zhang, Q.H., Shao, R., Qiu, M., Wang, Z.S., Wei, Q.J., Huang, J.T.,

562

Susceptibility of Soiny Mullet (Liza haematocheila) to Streptococcus dysgalactiae and physiological

563

response to formalin inactivated S. dysgalactiae, Pakistan Veterinary Journal 33 (2012) 234-237.

564

[10] Ø. Evensen, Development in fish vaccinology with focus on delivery methodologies, adjuvants

565

and formulations, Options Méditerranéennes 86(177-186) (2009).

566

[11] W. Yajun, G. Yi, W. Erlong, C. Defang, H. Yang, H. Xiaoli, W. Kaiyu, O. Ping, Y. Qian, L.

567

Weimin, W. Jun, S. Cunbin, Identification and screening of effective protective antigens for channel

568

catfish against Streptococcus iniae, Oncotarget 8(19) (2017) 30793-30804.

569

[12] B. Brodeur, M. Boyer, I. Charlebois, J. Hamel, F. Couture, C. Rioux, D. Martin, Identification of

570

group B streptococcal Sip protein, which elicits cross-protective immunity, Infect Immun 68(10)

571

(2000) 5610-5618.

572

[13] X. Li, H. Wu, M. Zhang, S. Liang, J. Xiao, Q. Wang, Q. Liu, Y. Zhang, Secreted glyceraldehyde-

573

3-phosphate dehydrogenase as a broad spectrum vaccine candidate against microbial infection in

574

aquaculture, Letters in applied microbiology 54(1) (2012) 1-9.

575

[14] Y.N. Mizrachi, K. Blau, T. Kushnir, M. Shagan, M. Portnoi, A. Cohen, S. Azriel, I. Malka, A.

576

Adawi, D. Kafka, S. Dotan, G. Guterman, S. Troib, T. Fishilevich, J.M. Gershoni, A. Braiman, A.M.

577

Mitchell, T.J. Mitchell, N. Porat, I. Goliand, V. Chalifa Caspi, E. Swiatlo, M. Tal, R. Ellis, N. Elia, R.

578

Dagan,

579

Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and

580

Evaluation of Its Potential as a Vaccine, PloS one 11(3) (2016) e0150320.

581

[15] P. Kayansamruaj, H.T. Dong, N. Pirarat, D. Nilubol, C. Rodkhum, Efficacy of α-enolase-based

582

DNA vaccine against pathogenic Streptococcus iniae in Nile tilapia (Oreochromis niloticus),

583

Aquaculture 468 (2017) 102-106.

584

[16] A. Zhang, B. Chen, X. Mu, R. Li, P. Zheng, Y. Zhao, H. Chen, M. Jin, Identification and

585

characterization of a novel protective antigen, Enolase of Streptococcus suis serotype 2, Vaccine 27(9)

586

(2009) 1348-53.

587

[17] J. Perez-Casal, A.A. Potter, Glyceradehyde-3-phosphate dehydrogenase as a suitable vaccine

588

candidate for protection against bacterial and parasitic diseases, Vaccine 34(8) (2016) 1012-7.

589

[18] Z. Zhang, A. Yu, J. Lan, H. Zhang, M. Hu, J. Cheng, L. Zhao, L. Lin, S. Wei, GapA, a potential

590

vaccine candidate antigen against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus), Fish

591

& shellfish immunology 63 (2017) 255-260.

592

[19] T.T. Thu Nguyen, H.T. Nguyen, H. Vu-Khac, P.C. Wang, S.C. Chen, Identification of protective

593

protein antigens for vaccination against Streptococcus dysgalactiae in cobia (Rachycentron canadum),

594

Fish & shellfish immunology 80 (2018) 88-96.

595

[20] E.S. Goldman, S. Dotan, A. Talias, A. Lilo, S. Azriel, I. Malka, M. Portnoi, A. Ohayon, D. Kafka,

596

R. Ellis, M. Elkabets, A. Porgador, D. Levin, R. Azhari, E. Swiatlo, E. Ling, G. Feldman, M. Tal, R.

Streptococcus

pneumoniae

Cell-Wall-Localized

Phosphoenolpyruvate

Protein

597

Dagan, Y.N. Mizrachi, Streptococcus pneumoniae fructose-1,6-bisphosphate aldolase, a protein

598

vaccine candidate, elicits Th1/Th2/Th17-type cytokine responses in mice, International journal of

599

molecular medicine 37(4) (2016) 1127-38.

600

[21] V. Pancholi, Multifunctional α-enolase: Its role in diseases, Cell. Mol. Life Sci. 58 (2001) 902-

601

920.

602

[22] A. Diaz-Ramos, A. Roig-Borrellas, A. Garcia-Melero, R. Lopez-Alemany, alpha-Enolase, a

603

multifunctional protein: its role on pathophysiological situations, Journal of biomedicine &

604

biotechnology 2012 (2012) 156795.

605

[23] S. Agarwal, P. Kulshreshtha, D. Bambah Mukku, R. Bhatnagar, alpha-Enolase binds to human

606

plasminogen on the surface of Bacillus anthracis, Biochimica et biophysica acta 1784(7-8) (2008) 986-

607

94.

608

[24] J. Wang, K. Wang, D. Chen, Y. Geng, X. Huang, Y. He, L. Ji, T. Liu, E. Wang, Q. Yang, W. Lai,

609

Cloning and Characterization of Surface-Localized alpha-Enolase of Streptococcus iniae, an Effective

610

Protective Antigen in Mice, International journal of molecular sciences 16(7) (2015) 14490-510.

611

[25] Y. Feng, X. Pan, W. Sun, C. Wang, H. Zhang, X. Li, Y. Ma, Z. Shao, J. Ge, F. Zheng, G.F. Gao, J.

612

Tang, Streptococcus suis enolase functions as a protective antigen displayed on the bacterial cell

613

surface, The Journal of infectious diseases 200(10) (2009) 1583-92.

614

[26] W.J. Wang E, Long B, Wang K, He Y, Yang Q, Chen D, Geng Y, Huang X, Ouyang P, Lai W,

615

Molecular cloning, expression and the adjuvant effects of interleukin-8 of channel catfish (Ictalurus

616

Punctatus) against Streptococcus iniae, Scientific reports 6 (2016) 29310.

617

[27] T. Yi, Y.-W. Li, L. Liu, X.-X. Xiao, A.-X. Li, Protection of Nile tilapia ( Oreochromis niloticus

618

L.) against Streptococcus agalactiae following immunization with recombinant FbsA and α-enolase,

619

Aquaculture 428-429 (2014) 35-40.

620

[28] W. Li, X. Hu, X. Zhang, Y. Ge, S. Zhao, Y. Hu, R.B. Ashman, Immunisation with the glycolytic

621

enzyme enolase confers effective protection against Candida albicans infection in mice, Vaccine 29(33)

622

(2011) 5526-33.

623

[29] N. Chen, Z.G. Yuan, M.J. Xu, D.H. Zhou, X.X. Zhang, Y.Z. Zhang, X.W. Wang, C. Yan, R.Q.

624

Lin, X.Q. Zhu, Ascaris suum enolase is a potential vaccine candidate against ascariasis, Vaccine 30(23)

625

(2012) 3478-82.

626

[30] H.T. Nguyen, T.T.T. Nguyen, Y.C. Chen, H. Vu-Khac, P.C. Wang, S.C. Chen, Enhanced immune

627

responses and effectiveness of refined outer membrane protein vaccines against Vibrio harveyi in

628

orange-spotted grouper (Epinephelus coioides), J Fish Dis 41(9) (2018) 1349-1358.

629

[31] D.P. Anderson, G. Jeney, Immunostimulants added to injected Aeromonas salmonicida bacterin

630

enhance the defense mechanisms and protection in rainbow trout (Oncorhynchus mykiss). , Vet

631

Immunol Immunopathol 34 (1992) 379-389.

632

[32] O. Evensen, B. Brudeseth, S. Mutoloki, The vaccine formulation and its role in inflammatory

633

processes in fish-effects and adverse effects. , Dev Biol (Basel) 121 (2005) 117-125.

634

[33] E. Wang, J. Wang, B. Long, K. Wang, Y. He, Q. Yang, D. Chen, Y. Geng, X. Huang, P. Ouyang,

635

W. Lai, Molecular cloning, expression and the adjuvant effects of interleukin-8 of channel catfish

636

(Ictalurus Punctatus) against Streptococcus iniae, Scientific reports (2016) 29311-29322.

637

[34] E. Wang, Q. Yang, B. Long, T. Liu, K. Wang, D. Chen, J. Wang, Y. Geng, H. Yang, X. Huang, P.

638

Ouyang, W. Lai, Interleukin-8 holds promise to serve as a molecular adjuvant in DNA vaccination

639

model against Streptococcus iniae infection in fish, Oncotarget (2016) 83938-83950.

640

[35] J. Galindo-Villegas, I. Mulero, A. Garcia-Alcazar, I. Munoz, M. Penalver-Mellado, S.

641

Streitenberger, G. Scapigliati, J. Meseguer, V. Mulero, Recombinant TNFalpha as oral vaccine

642

adjuvant protects European sea bass against vibriosis: insights into the role of the CCL25/CCR9 axis,

643

Fish & shellfish immunology 35(4) (2013) 1260-71.

644

[36] M. Guo, X. Tang, X. Sheng, J. Xing, W. Zhan, The Immune Adjuvant Effects of Flounder

645

(Paralichthys olivaceus) Interleukin-6 on E. tarda Subunit Vaccine OmpV, International journal of

646

molecular sciences 18(7) (2017).

647

[37] H.T. Nguyen, T.T.T. Nguyen, Y.T. Wang, P.C. Wang, S.C. Chen, Effectiveness of formalin-killed

648

vaccines containing CpG oligodeoxynucleotide 1668 adjuvants against Vibrio harveyi in orange-

649

spotted grouper, Fish & shellfish immunology 68 (2017) 124-131.

650

[38] H. Diao, H.-B. Ye, X.-Q. Yu, Y. Fan, L. Xu, T.-B. Li, Y.-Q. Wang, Adjuvant and

651

immunostimulatory effects of LPS and β-glucan on immune response in Japanese flounder,

652

Paralichthys olivaceus, Veterinary Immunology and Immunopathology 156(3-4) (2013) 167-175.

653

[39] H.Y. Huang, Y.C. Chen, P.C. Wang, M.A. Tsai, S.C. Yeh, H.J. Liang, S.C. Chen, Efficacy of a

654

formalin-inactivated vaccine against Streptococcus iniae infection in the farmed grouper Epinephelus

655

coioides by intraperitoneal immunization, Vaccine 32(51) (2014) 7014-20.

656

[40] H.T. Nguyen, T.T. Thu Nguyen, M.A. Tsai, E. Ya-Zhen, P.C. Wang, S.C. Chen, A formalin-

657

inactivated vaccine provides good protection against Vibrio harveyi infection in orange-spotted grouper

658

(Epinephelus coioides), Fish & shellfish immunology (2017).

659

[41] R.M. Jaafar, J.K. Chettri, I. Dalsgaard, A. Al-Jubury, P.W. Kania, J. Skov, K. Buchmann, Effects

660

of adjuvant Montanide ISA 763 A VG in rainbow trout injection vaccinated against Yersinia ruckeri,

661

Fish & shellfish immunology 47(2) (2015) 797-806.

662

[42] B.D. Hames, D. Rickwood, Gel Electrophoresis of Proteins: A Practical Approach, 2nd ed.; IRL

663

Press Limited: Oxford, UK., (1999) 383.

664

[43] J.L. Brunelle, Green R., One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-

665

PAGE), Methods in enzymology 541 (2014) 151-9.

666

[44] T. Mahmood, P.C. Yang, Western blot: technique, theory, and trouble shooting, North American

667

journal of medical sciences 4(9) (2012) 429-34.

668

[45] R.M. Parry, R.C. Chandan, K.M. Shahani, A rapid and sensitive assay of muramidase, Proc Soc

669

Exp Biol Med (1965) 119:384.

670

[46] T.T.T. Nguyen, H.T. Nguyen, Y.-C. Chen, H.H. Hoang, P.-C. Wang, S.-C. Chen, Effectiveness of

671

a divalent Streptococcus dysgalactiae inactivated vaccine in cobia (Rachycentron canadum L.),

672

Aquaculture 495 (2018) 130-135.

673

[47] O. Byadgi, D. Puteri, Y.H. Lee, J.W. Lee, T.C. Cheng, Identification and expression analysis of

674

cobia (Rachycentron canadum) Toll-like receptor 9 gene, Fish & shellfish immunology 36(2) (2014)

675

417-27.

676

[48] Livak KJ, Schmittgen TD, Analysis of relative gene expression data using real-time quantitative

677

PCR and the 2(-Delta Delta C(T)) Method, Methods 25(4) (2001) 402-8.

678

[49] V. Mundodi, A.S. Kucknoor, J.F. Alderete, Immunogenic and plasminogen-binding surface-

679

associated alpha-enolase of Trichomonas vaginalis, Infect Immun 76(2) (2008) 523-31.

680

[50] W. Gan, G. Zhao, H. Xu, W. Wu, W. Du, J. Huang, X. Yu, X. Hu, Reverse vaccinology approach

681

identify an Echinococcus granulosus tegumental membrane protein enolase as vaccine candidate,

682

Parasitol Res 106(4) (2010) 873-82.

683

[51] J. Sha, T.E. Erova, R.A. Alyea, S. Wang, J.P. Olano, V. Pancholi, A.K. Chopra, Surface-expressed

684

enolase contributes to the pathogenesis of clinical isolate SSU of Aeromonas hydrophila, J Bacteriol

685

191(9) (2009) 3095-107.

686

[52] I. Pal-Bhowmick, M. Mehta, I. Coppens, S. Sharma, G.K. Jarori, Protective properties and surface

687

localization of Plasmodium falciparum enolase, Infect Immun 75(11) (2007) 5500-8.

688

[53] W. Jiang, X. Han, Q. Wang, X. Li, L. Yi, Y. Liu, C. Ding, Vibrio parahaemolyticus enolase is an

689

adhesion-related factor that binds plasminogen and functions as a protective antigen, Applied

690

microbiology and biotechnology 98(11) (2014) 4937-48.

691

[54] A.M. Floden, J.A. Watt, C.A. Brissette, Borrelia burgdorferi enolase is a surface-exposed

692

plasminogen binding protein, PloS one 6(11) (2011) e27502.

693

[55] C.M. Marcos, J. de Fatima da Silva, H.C. de Oliveira, R.A. Moraes da Silva, M.J. Mendes-

694

Giannini, A.M. Fusco-Almeida, Surface-expressed enolase contributes to the adhesion of

695

Paracoccidioides brasiliensis to host cells, FEMS yeast research 12(5) (2012) 557-70.

696

[56] Jaafara R.M., Chettri J.K., I. Dalsgaard, A. Al-Jubury, W.P. Kania, J. Skov, K. Buchmann, Effects

697

of adjuvant Montanide™ ISA 763 A VG in rainbow trout injection vaccinated against Yersinia ruckeri,

698

Fish & shellfish immunology 47(2) (2015) 797-806.

699

[57] H. Clay, H.E. Volkman, L. Ramakrishnan, Tumor necrosis factor signaling mediates resistance to

700

mycobacteria by inhibiting bacterial growth and macrophage death, Immunity 29(2) (2008) 283-94.

701

[58] S. Saurabh, P.K. Sahoo, Lysozyme: an important defence molecule of fish innate immune system,

702

Aquaculture Research 39(3) (2008) 223-239.

703

[59] G.P. Manchenko, Lysozyme, Handbook of Detection of Enzymes on Electrophoretic Gels. Boca

704

Raton, Fla.: CRC Press (1994) 223.

705

[60] B. Grinde, Lysozyme from rainbow trout, Salmo gairdneri Richardson, as an antibacterial agent

706

against fish pathogens., J Fish Dis 12 (1989) 95-104.

707

[61] G. Arango Duque, A. Descoteaux, Macrophage cytokines: involvement in immunity and

708

infectious diseases, Front Immunol 5 (2014) 491.

709

[62] S. de Oliveira, C.C. Reyes-Aldasoro, S. Candel, S.A. Renshaw, V. Mulero, A. Calado, Cxcl8 (IL-

710

8) mediates neutrophil recruitment and behavior in the zebrafish inflammatory response, J Immunol

711

190(8) (2013) 4349-59.

Highlights Recombinant α-enolase (rEnolase) derived from Streptococcus dysgalactiae was produced in Escherichia coli and formulated with ISA763 AVG adjuvant or cobia TNFα. Vaccination of cobia with rEnolase and ISA763 AVG significantly induced greater serum antibody response, lysozyme activity and immune-related genes expression. Vaccinated fish was highly protected from a virulent Streptococcus dysgalactiae challenge with the relative percent survival of 97.37%.