Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene (vp7) of grass carp reovirus using bacterial ghost as delivery vehicles

Accepted Manuscript Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene (vp7) of grass carp reovirus using bacterial ghost as delivery vehicles Kai Hao, Xiao-Hui Chen, Xiao-Zhou Qi, Xiao-Bo Yu, En-Qi Du, Fei Ling, Bin Zhu, Gao-Xue Wang PII:

S1050-4648(17)30140-7

DOI:

10.1016/j.fsi.2017.03.021

Reference:

YFSIM 4490

To appear in:

Fish and Shellfish Immunology

Received Date: 4 December 2016 Revised Date:

16 February 2017

Accepted Date: 10 March 2017

Please cite this article as: Hao K, Chen X-H, Qi X-Z, Yu X-B, Du E-Q, Ling F, Zhu B, Wang G-X, Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene (vp7) of grass carp reovirus using bacterial ghost as delivery vehicles, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.03.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT 1

Protective immunity of grass carp induced by DNA vaccine encoding

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capsid protein gene (vp7) of grass carp reovirus using bacterial ghost

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as delivery vehicles

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Kai Haoa, Xiao-Hui Chena, Xiao-Zhou Qia, Xiao-Bo Yua, En-Qi Dub, Fei Linga, Bin

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Zhua*, Gao-Xue Wanga*

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College of Animal Science and Technology, Northwest A&F University, Xinong

Road 22nd, Yangling, Shaanxi 712100, China

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Yangling, Shaanxi 712100, China

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College of Veterinary Medicine, Northwest A&F University, Xinong Road 22nd,

*Corresponding author. Phone (Office): +86 029 87092102

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FAX (Office): +86 02987092164, Phone (Home) No. +86 02987091516

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E-mail address: [email protected]; [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Grass carp reovirus (GCRV) is one of the most pathogenic aquareovirus and can cause

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lethal hemorrhagic disease in grass carp (Ctenopharyngodon idella). However,

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management of GCRV infection remains a challenge. Therefore, it is necessary to find

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effective means for the control of its infection. The uses of bacterial ghost (BG,

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non-living bacteria) as carriers for DNA delivery have received considerable

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attentions in veterinary and human vaccines studies. Nevertheless, there is still no

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report about intramuscular administration of bacterial ghost-based DNA vaccines in

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fish. In the current study, a novel vaccine based on Escherichia coli DH5α bacterial

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ghost (DH5α-BG)，delivering a major capsid protein gene (vp7) of grass carp reovirus

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encoded DNA vaccine was developed to enhance the efficacy of a vp7 DNA vaccine

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against GCRV in grass carp. The grass carp was injected intramuscularly by different

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treatments -i) naked pcDNA-vp7 (containing plasmid 1, 2.5 and 5 µg，respectively), ii)

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DH5α-BG/pcDNA-vp7 (containing plasmid 1, 2.5 and 5 µg，respectively) and iii)

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naked pcDNA, DH5α-BG or phosphate buffered saline. The immune responses and

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disease resistance of grass carp were assessed in different groups, and results

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indicated that the antibody levels, serum total antioxidant capacity (T-AOC),

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superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity and alkaline

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phosphatase (AKP) activity and immune-related genes were significantly enhanced in

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fish immunized with DH5α-BG/pcDNA-vp7 vaccine (DNA dose ranged from

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2.5-5µg). In addition, the relative percentage survival were significantly enhanced in

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fish immunized with DH5α-BG/pcDNA-vp7 vaccine and the relative percentage

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ACCEPTED MANUSCRIPT survival reached to 90% in DH5α-BG/pcDNA-vp7 group than that of naked

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pcDNA-vp7 (42.22%) at the highest DNA dose (5 µg) after 14 days of post infection.

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Moreover, the level of pcDNA-vp7 plasmid was higher in DH5α-BG/pcDNA-vp7

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groups than naked pcDNA-vp7 groups in muscle and kidneys tissues after 21 days.

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Overall, those results suggested that DH5α bacterial ghost based DNA vaccine might

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be used as a promising vaccine for aquatic animals to fight against GCRV infection.

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Key words: Grass carp, Grass carp reovirus, Bacterial ghost, Vaccine, Innate immunity

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1. Introduction Aquaculture is regarded as one of the fastest growing and expanding industries in

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the world and significantly contributes to the world economy. Grass carp

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(Ctenopharyngodon idella) is an important freshwater economic fish, occupying a

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vital position in aquaculture of China [1]. However, hemorrhagic disease caused by

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grass carp reovirus (GCRV) has a serious threat to the grass carp cultivation industry.

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Over the past decades, to propose the effective prevention or therapeutic strategy for

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hemorrhagic disease, a series of antibiotics and chemotherapeutants have been

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developed and partially solve the problem [2]. Nevertheless, long term use of the

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antibiotics and chemotherapeutants lead to many negative impacts such as antibiotics

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residues and drug resistance, which drive us to find effective alternative means to

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control the GCRV viral infection [3].

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Nucleic acid vaccination has emerged as powerful technology, which can be

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applied for development of either prophylactic or therapeutic vaccines [4]. It has been

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extensively studied and a variety of nucleic acid vaccines using naked DNA have

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undergone clinical trials in both human and veterinary practices [5, 6]. The first

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demonstration of the efficacy of a DNA vaccine in fish was in rainbow trout

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immunized against infectious hematopoietic necrosis virus [7]. Subsequently, various

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DNA vaccines were wildly studied e.g., viral hemorrhagic septicemia [8], viral

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haemorrhagic septicemia viruses [9], infectious pancreatic necrosis viruses [10] and

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spring viremia of carp viruses [11]. However, treatment with naked DNA vaccine

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generally induces weak immune protection in fish [12]. Therefore, DNA vaccines

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require the more effective carrier to improve the protection of host, which becomes a

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vital issue now. Recently, a great attempt focused on the studies of vaccine carrier systems,

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because they could efficiently deliver antigen/DNA and transport them to the specific

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cells/tissues. For example, our lab utilized carbon nanotubes (CNTs) as vehicle to

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deliver DNA, which could significantly induce immune protection in fish [2, 13].

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Now, bacterial ghost (BG, non-living bacteria) is a novel vaccination technology

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platform produced by controlled expression of lysis genes which can lead to the

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formation of a transmembrane tunnel through the bacterial cellular envelope [14]. In

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recent years, the use of BGs as carrier to deliver DNA is an attractive vaccine

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development strategy because of its safety to organism, excellent loading capacity and

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adjuvant properties [15, 16], making it a good candidate for use in DNA vaccine

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development in fish.

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In this study, gene E came from bacteriophage PhiX 174, which could lead to the

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formation of bacterial ghosts; Smap-29 (sheep myeloid antimicrobial peptide-29) gene

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referring to the peptide of 29 amino acids was also used to inactivate the bacteria

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along with lysis E gene [17]. Moreover, during BG production, nucleic acid

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degradation could be used to eliminate danger related to nucleic acid e.g., horizontal

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genes transfer of either pathogenic or antibiotic-resistance genes. To avoid that,

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Staphylococcus aureus nucleic acid enzyme A (SNA) was expressed along with lysis

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E gene to degrade the host DNA [18].

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VP7 is encoded by the S10 gene fragment and is an important outer capsid

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protein of GCRV [28]. It had been expressed in Escherichia coli which indicated the

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recombinant VP7 could be used as a potential subunit vaccine against GCRV infection

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by our previous study. Taking into account all these previous considerations, we prepared E. coli

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DH5α-BG/vp7 DNA vaccine and evaluated immune responses in immunized grass

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carp and immune protection elicited in grass carp by E. coli DH5α-BG/vp7 DNA

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vaccine against GCRV infection.

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2. Materials and methods

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2.1. Fish and virus

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Grass carp (average weight 1.5-1.8 g) used in the experiment was provided by a

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fish farm in Heyang (Shanxi, China). Prior to the initiation of the experiment, the fish

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were acclimatized to laboratory conditions for one week in 300 L aerated aquaria at

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28 °C, fed twice daily. Possible virus contamination in fish was evaluated by reverse

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transcription quantitative real-time PCR (RT-qPCR) [3]. The GCRV strain used as a

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challenge pathogen in this work isolated from the infected grass carp in fish farm

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located in Rougu (Shaanxi, China) and stored in our laboratory [2]. The viruses were

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cultured in Ctenopharyngodon idellus kidney (CIK) cell. The CIK cell culture

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methods and 50% tissue culture infective doses (TCID50) of the virus were performed

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according to established protocols [19]. Care of animals was in compliance with the

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guidelines of the Animal Experiment Committee, Northwest A&F University.

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2.2 Bacterial strains and plasmids

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Escherichia coli (E. coli) DH5α was purchased from Invitrogen (Life

ACCEPTED MANUSCRIPT Technologies, NY, USA). The pCIts857/pR/pL, which contains whole temperature

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regulatory expression cassette (promoter, multiple clone site and terminator) and

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Pmd-E/SNA/Smap29 (it contains lysis E, Staphylococcus aureus nucleic acid enzyme

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A and sheep myeloid antimicrobial peptide-29) vectors were kindly provided by Dr

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Enqi Du Northwest A&F University (China). pCAT vector carrying chloramphenicol

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and pcDNA-vp7 containing GCRV vp7 antigen gene were constructed by our

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laboratory [2].

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2.3 Construction of the pCAT-lysisE/SNA/Smap29 plasmid

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The E/SNA/Smap29 gene was obtained by PCR amplification from

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pmd-E/SNA/Smap29 vector with primer pair consisting of lysis E forward primer

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(5'CGGAATTCATGAATCACAAAGTGATGGTACGCT3'

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restrictive endonuclease site; as underlined) and the lysis E reverse primer,

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(5'CGCGGATCCTTAACCAGCGATACGGATGATA3'

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restrictive endonuclease site; as underlined). The PCR parameters consisted of one

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cycle of 5 min at 95 °C, followed by 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and

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72 °C for 30 s, with a final extension step of 5 min at 72 °C using a CFX96 Real-Time

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PCR Detection System (Bio-Rad, USA). The products were visualized on 1% agarose

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gels stained with ethidium bromide, and purified with a Gel midi Purification Kit

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(Tiangen, Beijing, China). The DNA fragment by digestion with EcoRI/BamHI

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restrictive endonucleases (Takara, Dalian, China) was cloned into the pCIts857/pR/pL

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to obtain pCIts857/ E/SNA/Smap29. The construct was then transformed into E. coli

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DH5α cells (Invitrogen, USA) and sequenced by Sangong Biological Company

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BamHI

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ACCEPTED MANUSCRIPT (Shanghai, China). The fragment CIts857/pR/pL-lysisE/SNA/Smap29 was amplified

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from the construct pCIts857/E/SNA/Smap29 with CIts857 forward primer

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(5'GCTCTAGAAACCTCAAGCCAGAATGC3', the underline show XbaI site) and

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CIts857 reverse primer (R5' TCCCCCGGGTTGTAGAAACGCAAAAAGGCCATCC

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3, the underline show SmaI site). The PCR reaction consisted of one cycle of 3 min at

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95 °C, followed by 30 cycles at 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 1 min,

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with a final extension step of 10 min at 72 °C. The DNA fragment was purified and

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cloned into pCAT, after digestion with XbaI/SmaI restrictive endonucleases (Takara,

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Dalian, China) to generate pCAT-lysisE/SNA/Smap29 recombination plasmid. The

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recombinant cassette pCAT-lysisE/SNA/Smap29 was transformed into E. coli DH5α

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cells and identified by restriction enzyme digestion, PCR amplification and DNA

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sequencing.

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2.4 Production of bacterial ghosts

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Bacterial ghosts from E. coli DH5α were produced by the regulated expression

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of the lysis E gene, SNA gene and Smap29 gene as described elsewhere [20-22].

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Briefly，the positive recombinant DH5α strain containing pCAT-lysisE/SNA/Smap29

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vector was named DH5α-E and grown in Luria broth (LB) liquid medium

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supplemented with chloramphenicol (34 µg mL-1, Sigma) at 28 °C with agitation of

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200 rpm to reach the optical density of 0.3–0.4 at 600 nm (OD600). The expression of

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the lysis E gene, SNA gene and Smap29 gene was induced by a temperature upshift

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from 28 to 42 °C. Meanwhile, 1 Mm MgCl2 and 10 mM CaCl2 (at final concentrations)

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were added post-induction to stimulate nuclease activity of the SNA. The number of

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ACCEPTED MANUSCRIPT OD600 was monitored and measured in the course of lysis (0, 0.5, 1. 1.5, 2, 2.5, 3, 3.5

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and 4 h). The efficiency of lysis was determined by viable cell counts prior to (0 h)

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and at the end of the lysis process (4 h). Total DNA was extracted to confirm the

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complete degradation of DNA molecules by nuclease activity prior to (0 h) and at the

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end of the lysis process (4 h). Extraction of the total DNA was performed using

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TIANamp Bacteria DNA kit (Tiangen, Beijing, China) according to the

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manufacturer’s instructions. Degradation of DNA was analyzed on 1% agarose gel.

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The bacterial ghosts were centrifuged, washed three time with PBS (sterile

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phosphate buffered saline, pH 7.4), lyophilized, and stored at -80 °C until use. The

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DH5α-E ghosts were characterized by field emission scanning electron microscopy

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(FE-SEM, S-4800, Hitachi Ltd., Tokyo, Japan).

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2.5 Loading of DH5α-BG with pcDNA-vp7

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GCRV recombinant DNA vaccine, pcDNA-vp7 was constructed by our

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laboratory previously [2]. The plasmid pcDNA-vp7 was prepared by large-scale using

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LB broth and isolated with the Endo-free Maxi Kit (Omega, USA) following the

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manufacturer's instructions. The absorbance at 260 and 280 nm was measured to

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determine its concentration using the NanoDrop spectrophotometer (ND-1000,

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NanoDrop Technologies Inc., Wilmington, DE). The plasmid pcDNA-vp7 was

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lyophilized and conserved at -20 °C until use.

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DH5α-BG were loaded with pcDNA-vp7 by diffusion of plasmid DNA through

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the lysis holes into the ghosts with a similar protocol as described by S. Paukner [20]

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and was named as DH5α-BG/pcDNA-vp7. Briefly, The lyophilized DH5α-BG were

ACCEPTED MANUSCRIPT resuspended in HBS (100 mM NaCl, 10 mM sodium acetate, 10 mM Hepes, pH 7.0)

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containing pcDNA-vp7 (680–700 ng µl-1) at a ratio of 2 mg lyophilized DH5α-BG per

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200µl DNA solution. Subsequently, CaCl2 (final concentration 25 mM) was added

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into the suspension and the mixed suspension was incubated for 1 h at 24 °C with

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agitation. Afterward, The DH5α-BG were separated by centrifugation (12,000 g) and

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washed twice with HBS. The DH5α-BG/pcDNA-vp7 were stored at -80 °C until use.

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The qualitation, as well quantitation of loading efficiency were analyzed by flow

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cytometry and real-time PCR respectively as the described elsewhere [20, 22].

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2.6 Vaccination and Challenge

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Healthy grass carps were randomly divided nine groups (50 fish per group) and

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immunized in each group (three control groups and six vaccinated groups). The

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vaccinated groups, fish were anaesthetized in 0.01% benzocaine and then

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intramuscular injected with 20 µL pcDNA-vp7 and DH5α-BG/pcDNA-vp7 (dissolve

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in PBS, pH 7.4) in three doses (1, 2.5, 5 µg pcDNA-vp7 per fish in pcDNA-vp7 and

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DH5α-BG/pcDNA-vp7 vaccinated groups). While, the control groups were injected

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with pcDNA (5µg), DH5α-BG (0.5 mg) or PBS, respectively. All groups were run in

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triplicate. Subsequently, the immunized fish were transferred to different tanks and

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maintained as above during the whole immunization period. At the end of 21-days

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immunization experiment, 30 fish were selected randomly from each replication for

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the challenge trial. The selected fish was challenged with 1×105 TCID50 GCRV by

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intraperitoneal injection. Mortality of the post infection fish was recorded everyday

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for up to 14 days. The relative percent survival (RPS) was calculated after 14 days of

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post infection by the following formula of Amend [23]. Relative percentage survival (RPS) = {1-[% mortality rate (treatment group)/% mortality rate (PBS control)]}×100.

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2.7 Detection of injected DNA in fish tissues

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DNA was extracted from muscle and kidneys tissues (three fish) as described

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elsewhere. Briefly, fish tissues (muscle covering the area of injection and kidneys)

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were taken from grass carp at 21 days after vaccination. The tissues were pulverized

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to powder using liquid nitrogen and dissolved in 3 mL genomic DNA isolation buffer

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(1.0% sodium dodecyl sulfate, 100 mM NaCl, 50 mM Tris-HCl, 100 mM EDTA, pH

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8.0, 20 mg mL-1 RNase). After incubated for 1 h at 37 °C, proteinase K was added into

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the suspension with a concentration of 150 mg mL-1 and then the sample was

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incubated at 60 °C overnight. The conventional phenolchloroform procedure was used

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to extract the DNA. The vp7 gene (831 bp) was amplified with specific primers (A-F

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5’-CTAGAGAACCCACTGCTTAC-3’, A-R 5’-TAGAAGGCACAGTCGAGG-3’)

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and β-actin was used as an internal gene (Table 1).

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2.8 RT-PCR detection of the expression of plasmid DNA in fish tissues

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Kidneys (three fish for each group) were taken from the fish at 21 days after

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vaccination to examine the expression of vp7. Total RNA was extracted from kidneys

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using the Trizol reagent (TaKaRa, Japan) following the manufacturer’s protocol, and

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then incubated with RNase-free DNase I (TaKaRa, Japan) to eliminate contaminated

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genomic DNA before being reversely transcribed into cDNA using random hexamer

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primers and M-MLV Reverse Transcriptase (TaKaRa, Japan). The expression of

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plasmid DNA was determined by PCR with specific primers A-F/R and β-actin was

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used as an internal gene (Table 1).

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2.9 Measurement of anti-vp7 antibody The preparation of rabbit sera anti-IgM were performed according to regular

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method as described previously [2, 24]. The titers of the antibodies were measured by

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ELISA (Enzyme-linked immunosorbent assay) as described elsewhere [2, 3, 24]. For

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analyses of the presence of specific neutralizing antibodies, serum samples (3 fish per

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group) were collected from each vaccinated and control groups on days 3, 7, 14 and

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21 post-immunization and used for antibody determination according to previous

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method [25]. Briefly, the blood collected from the caudal vein of grass carp was

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placed overnight at 4 °C and then centrifugated at 5000×g for 15 min. The supernatant

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was collected and stored at -20 °C until use. Purified recombinant VP7 protein was

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used as antigen. The Rabbit anti-IgM polyclonal were used as primary antibody, and

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HRP-conjugated goat anti-rabbit IgG (Beijing CoWin Biotech Corp., Beijing, China)

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was used as secondary antibody. The primary and secondary antibodies were diluted

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1:1000 immediately before use with PBS containing 3% skimmed milk. Color

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development was performed using DAB horseradish peroxidase color development kit

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(Tiangen Biotech, Beijing, China). The absorbance of each well at 450 nm was read

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with a precision microplate reader ((Molecular Devices Corp., Palo Alto, CA).The

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antibody response was expressed in terms of O.D.

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2.10 Non-specific immune parameters assay

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At 3, 7, 14 and 21 days post-immunization, blood samples were collected from

ACCEPTED MANUSCRIPT the caudal vein of 3 grass carp from each group. The serum was obtained from the

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blood with the same method as described before. Total antioxidant capacity (T-AOC),

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superoxide dismutase (SOD) activity, ACP activity and AKP activity were measured

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using assay kits (Nanjing Jiancheng Institute, China) according to the manufacturer’s

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instructions. Details of the procedures were described by the previous methods[26].

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2.11 Determination of immune-related genes expression

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3 fish from each group were anesthetized and killed at 3, 7, 14 and 21 days after

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immunization. The kidneys of fish were excised. Total RNA was extracted from

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kdneys using the Trizol reagent (TaKaRa, Japan) following the manufacturer’s

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instruction. RNA quality was verified by electrophoresis on ethidium bromide staining

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1.0% agarose gels. RNA concentration was determined by measuring the absorbance

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at 260 nm and its purity was assessed by the 260/280 nm ratios.

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Total RNA was reverse transcribed into cDNA using the reverse transcriptase kit

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(TaKaRa, Japan) following the manufacturer's protocol. All the RNA and cDNA

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samples were stored at – 80 °C for further use.

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The expression of five target genes (TNFα，IL-1β，MHC-I, IgD and IgM) and

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internal gene (β-actin) were quantified by real-time qPCR using a CFX96 Real-Time

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PCR Detection System (Bio-Rad, USA) and SYBR Premix Ex Taq II kit (TaKaRa).

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Specific primers were designed to amplify genes (Table 1). The amplifications were

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performed in a 96-well plate in a 12.5 µl reaction volume including 6.25 µl 1 × SYBR

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Premix Ex Taq™ (TaKaRa), 0.25 µM of each primer, and 200 ng of cDNA template.

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The PCR parameters consisted of one cycle of 3 min at 95 °C followed by 40 cycles

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with 15 s at 95 °C and 30 s at 60 °C. Each individual sample was run in triplicate

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wells. The relative quantification of gene expression among the every groups was

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analyzed by the 2-

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2.12 Statistical analysis

The data were expressed as the arithmetic mean ± standard deviation (SD) and

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were analyzed by one-way ANOVA after normalization. Differences in antibody titers,

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and transcription levels of the immune-related genes were analyzed with two-tailed

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student's t-test; the data of mortality rate and relative percentage survival were

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transformed to square-root arcsine values before performing the differences test with

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SPSS statistical software (SPSS Inc., USA). Levels of P < 0.01 and P < 0.05 were

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considered significant.

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3. Results

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3.1 Characterization of the pCAT-lysisE/SNA/Smap29 plasmid

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E/SNA/Smap29 gene (size in 915bp) was obtained by PCR amplification from

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pmd-E/SNA/Smap29 vector with primer pair and the result was shown in Fig.1(A)

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When the E/SNA/Smap29 gene was cloned into pCIts857/pR/pL, it was confirmed by

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sequence analysis (data not show). The fragment CIts857/pR/pL-lysisE/SNA/Smap29

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(size in 2199bp) was amplified from the construct pCIts857/E/SNA/Smap29. The

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result was shown in Fig. 1(B) When the CIts857/pR/pL-lysisE/SNA/Smap29 gene

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was cloned into pCAT plasmid (size in 3572bp), it was confirmed by restriction

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enzyme digestion (Fig.1(C)) and sequence analysis (data not show).

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ACCEPTED MANUSCRIPT [Fig. 1]

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3.2 Production of bacterial ghosts E. coli DH5α cells harboring lysis plasmid were utilized for the production of

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bacterial ghosts. When a temperature shifted from 28 to 42 °C, onset of lysis was

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observed 30 min after induction of lysis genes expression by a decrease of OD600 until

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the next 2.5 to 3 hours, and then finally the OD600 remained almost constant (Fig.

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2(A)). Loss of viability of the ghost preparation (colony forming unit, cfu) was

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assessed by viable cell counts. The number of E. coli expressing lysis gene decreased

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from 3.1 ×106 cfu mL-1 before induction of lysis to 4×104 cfu mL-1 at the end of the

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lysis process and the lysis efficiency was determined almost 99% (see Fig. 2(B)).

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Meanwhile, total DNA was extracted to confirm the degradation of DNA molecules

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by nuclease activity at the end of the lysis process. The results in Fig. 2(C) showed

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that the DNA in bacterial ghost was degraded by the nuclease activity of SNA.

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3.3 Characterization of DH5α-BG

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Electron microscopic analysis was performed to reveal morphology changes of

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DH5α-BG, compared with that of unlysed and intact DH5α cells. The results showed

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that the DH5α-BG envelope appeared lysis holes structure, which mainly were

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distributed in the middle of the bacterial cell or at the polar sites (Fig. 3(A) and (C),

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(white arrowheads). However, the unlysed cells grown at 28 °C had no changes in the

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morphology by the SEM observation (Fig. 3(B)). Above results indicated that

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DH5α-E can be inactivated by the lysis genes expression.

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3.4 Analysis of DH5α-BG loaded with pcDNA-vp7. We loaded the DH5α-BG cells with the plasmid pcDNA-vp7. The dye Propidium

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Iodide (PI) was used to stain pcDNA-vp7 loaded. Flow cytometry revealed that the

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DH5α-BG cells were loaded with pcDNA-vp7, as the cells showed distinct red

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fluorescence (Fig. 4(B)), whereas that of empty DH5α-BG cells(no-load plasmid) had

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almost no fluorescence after staining with PI (see Fig. 4(A)). We also investigated

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whether the lyophilized intact E.coli DH5α could be loaded with plasmid with the

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same method as described before. Flow cytometry revealed that the overlays of the

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histograms of lyophilized, intact E.coli DH5α did not have a distinct shift compared to

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empty DH5α-BG (FL2-Log ranged from 100 to 101), demonstrating that they could

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not be loaded with plasmid DNA (Fig. 4(C)). Above results also indicated DH5α-E

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can be inactivated by the lysis genes expression and the DH5α-BG cells were also

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accessible to plasmid pcDNA-vp7 through the lysis tunnel structure. Additional, the

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standard curve of pcDNA-vp7 plasmid was obtained by real-time PCR. The

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correlation coefficient and amplification efficiency (E) was 0.9935 and 114%,

347

respectively (Fig. 4(D)). According to the standard curve, it was calculated about 1

348

mg DH5α-BG carried 10 µg pcDNA-vp7.

350

SC

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EP

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349

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333

[Fig. 4]

3.5 Persistence of pcDNA-vp7 in different tissues

351

PCR was performed with pcDNA-vp7 specific primers to confirm the presence

352

of the pcDNA-vp7 plasmid in muscle covering the area of injection and kidneys

ACCEPTED MANUSCRIPT tissues at 21 days post-injection. The amplification of vp7 gene was detected both in

354

DH5α-BG/pcDNA-vp7 and naked pcDNA-vp7 groups at different tissues (muscle and

355

kidneys) (Fig. 5). In addition, the electrophoresis strips in DH5α-BG/pcDNA-vp7

356

groups were much brighter than naked pcDNA-vp7 groups. Whereas there was no

357

amplification detected for vp7 gene in the control groups (Fig. 5).

359

3.6 Transcription of pcDNA-vp7 gene in vivo

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[Fig. 5]

358

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RT-PCR reaction was performed to analyze transcription of the vp7 gene of

362

kidneys in different groups. Transcripts of the vp7 gene was detected both in

363

DH5α-BG/pcDNA-vp7 and naked pcDNA-vp7 groups at 21 days after immunization

364

(Fig. 6). The electrophoresis strips in DH5α-BG/pcDNA-vp7 groups were much

365

brighter than naked pcDNA-vp7 groups. No amplification was observed in fish

366

injected with PBS, pcDNA and DH5α-BG (Fig. 6).

369

EP

368

[Fig. 6]

3.7 Serum antibody production

AC C

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The ELISA results showed that specific antibodies were produced in fish

370

vaccinated groups. The antibody level increased as pcDNA-vp7’ concentration

371

increasing and immunized time extending. Meanwhile, the DH5α-BG/pcDNA-vp7

372

elicited higher antibody levels at the same examined time point, compared to naked

373

pcDNA-vp7 (Fig. 7). However, No specific antibody responses were observed in PBS,

374

pcDNA and DH5α-BG groups.

ACCEPTED MANUSCRIPT [Fig. 7]

375 376

3.8 Change of non-specific immune parameters assay The AKP, ACP, SOD and T-AOC activities were recorded in different groups, as

378

showed in Fig. 8. It was found that AKP activity initially increased after 7 days and

379

then it decreased as time further extended to 21 days in vaccinated groups (DH5α-BG/

380

pcDNA-vp7, pcDNA-vp7) (Fig. 8 (B)). AKP activity at the highest dose groups

381

(DH5α-BG/pcDNA-vp7 group 5.679 ± 0.167 U mL-1, pcDNA-vp7 group 4.393 ±

382

0.094 U mL-1) was significantly higher than PBS control (P < 0.01) at days 7. At days

383

21, only DH5α-BG/pcDNA-vp7 groups (containing 5 µg and 2.5 µg pcDNA-vp7,

384

respectively) had significantly higher AKP activity (5µg group 4.521 ± 0.113 U mL-1,

385

2.5µg group 3.883 ± 0.355 U mL-1), compared to PBS control (P < 0.01 and P < 0.05,

386

respectively).

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Fig.8

(A)

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377

showed

that

the

change

tendency

of

ACP

activity

in

DH5α-BG/pcDNA-vp7 groups was similar to AKP and it was significantly influenced

389

in a time-dependent and dose-dependent manner. DH5α-BG/pcDNA-vp7 groups

390

(containing 5µg and 2.5µg pcDNA-vp7, respectively) had significantly higher ACP

391

activity compared to PBS control during the whole immunization period (P < 0.05).

392

Only naked pcDNA-vp7 group with the highest dose had significantly higher ACP

393

activity compared to PBS control (P < 0.05), at days 3 (3.203 ± 0.320 U mL-1), 7

394

(3.039 ± 0.076 U mL-1), 14 (3.126 ± 0.284 U mL-1) and 21 (2.916 ± 0.275 U mL-1).

AC C

EP

388

395

The SOD activity was showed in Fig. 8(C). The peak of SOD activity appeared

396

at days 3 in each group, and then the SOD content was gradually attenuated. At the

ACCEPTED MANUSCRIPT last

time,

the

SOD

activities

at

5

of

pcDNA-vp7

in

398

DH5α-BG/pcDNA-vp7 and naked pcDNA-vp7 group (DH5α-BG/pcDNA-vp7 group

399

91.205 ± 3.465 U mL-1, pcDNA-vp7 group 90.354 ± 0.014 U mL-1) were significantly

400

higher than PBS control (P < 0.05).

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sampling

µg

397

The data in Fig. 8(D) reflected that T-AOC activity cloud be positively impact

402

via both DH5α-BG/pcDNA-vp7 and naked pcDNA-vp7 immunization and these

403

effects were also dose dependent at each of the examined time point. The naked

404

pcDNA-vp7 group with highest dose had significantly higher T-AOC activity than

405

PBS control between 7 and 14 days (P < 0.05). The T-AOC activity in all

406

DH5α-BG/pcDNA-vp7 groups, except the lowest dose, also increased significantly

407

between 7 and 21 days (P < 0.05).

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401

In addition, the control group injected with DH5α-BG had a little effect on ACP,

409

SOD and T-AOC activities at the initial 7 days and then it recovered to normal levels.

410

Meanwhile, it was also found that the DH5α-BG/pcDNA-vp7 groups stimulated

411

higher ACP, AKP, SOD and T-AOC levels at the same dose and examined time point

412

than those of pcDNA-vp7 groups.

414 415

EP

AC C

413

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408

[Fig. 8]

3.9 Expression of immune-related genes Expression of immune-related genes was examined by qRT-PCR analysis of the

416

transcription of the genes encoding tumor necrosis factor α (TNF-α), interleukin-1β

417

(IL-1β), major histocompatibility complex (MHC) class I, immunoglobulin M (IgM)

418

and immunoglobulin D (IgD), in the kidney of fish. The results showed that the

ACCEPTED MANUSCRIPT transcript levels of examined genes increased with different degree in naked

420

pcDNA-vp7 and DH5α-BG/pcDNA-vp7 groups (Fig. 9). The mRNA levels of IL-1β，

421

IgM and MHC-I were significantly increased in DH5α-BG/pcDNA-vp7 groups, with

422

greater than 6-fold inductions (except the lowest dose) from 14 to 21 days (P < 0.01),

423

whereas in naked pcDNA-vp7 groups, only the highest dose group had a 4-5 fold

424

significant increase of genes expression level (P < 0.01) between 14 and 21 days (Fig.

425

9).

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For TNF-α (Fig. 9(A)), the mRNA was significantly up regulated, reaching

427

maximal mRNA expression level at days 7 in all DH5α-BG/pcDNA-vp7 groups, and

428

the mRNA level of the highest dose reached to 5.68-fold of the PBS control (P < 0.01),

429

then the mRNA displayed a tendency of down regulation. At days 21, only in the

430

highest dose in DH5α-BG/pcDNA-vp7 group, TNF-α transcript level was

431

significantly up regulated (2.28-fold, P < 0.05). For pcDNA-vp7 group, only 5 µg

432

naked pcDNA-vp7 showed significantly higher (2.44-fold, P < 0.05) mRNA

433

expression level of TNF-α at days 7, whereas other sampling time and doses had no

434

significant difference. For IgD (Fig. 9(E)), the mRNA transcript levels increased

435

significantly only at days 14 (5.41-fold, P < 0.05) and 21 (3.80-fold, P < 0.05) in

436

DH5α-BG/pcDNA-vp7 group with the highest dose.

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EP

AC C

437

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In addition, we also found that the control group injected with DH5α-BG could

438

slightly increase mRNA expression level of examined genes, but no significant

439

difference, except MHCI at days 14.

440

[Fig. 9]

ACCEPTED MANUSCRIPT 441

3.10 Challenge test Cumulative mortalities of grass carp from all groups after being injected with 20

443

µL live GCRV were recorded everyday for up to 14 days. The results showed (Fig. 10)

444

that for PBS, pcDNA and DH5α-BG groups, the cumulative mortalities were reached

445

to 100% at 9 days after challenge. Cumulative mortality of pcDNA-vp7 group (5 µg

446

per fish) was 57.78% after 14 days. In DH5α-BG/pcDNA-vp7 group (5 µg per fish),

447

the cumulative mortalities was much lower than pcDNA-vp7 group and only reached

448

to 10% at 14-day. Meanwhile, DH5α-BG/pcDNA-vp7 group had the highest

449

protective efficacy at 14 days after challenge, and the RPS value reached to 90%

450

(Table 2). During challenge trials, dead fish showed typical clinical symptoms of

451

GCRV infection, and no pathogen other than GCRV was detected from dead fish.

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442

[Fig. 10]

[Table 2]

453 454

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452

4. Discussion

Bacterial ghost is a novel vaccination technology platform produced by

456

controlled expression of lysis genes. This leads to the formation of a transmembrane

457

tunnel through the bacterial cellular envelope (Fig. 3) [14]. BGs own excellent

458

loading capacity and could be filled with large amounts of DNA [16, 28, 29]. In recent

459

years, the use of BGs as a carrier to deliver DNA is an attractive vaccine development

460

strategy because of its safety to organism, excellent loading capacity (foreign DNA,

461

peptides, protein, or drugs) and adjuvant properties [15, 22, 30]. Their uses as

462

combination vaccines mainly focused on veterinary vaccine trials and animal studies

AC C

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ACCEPTED MANUSCRIPT for human vaccine candidates (e.g., mice, rabbits and pigs) [4], for example, Chen et

464

al. used Salmonella typhimurium BG to deliver plasmid DNA, and they found it

465

facilitated stronger humoral and cellular immune responses in immunized mice [22].

466

However, Their application in fish was not intensively studied [28]. In the present

467

study, we prepared E. coli DH5α-BG delivering pcDNA-vp7 vaccine and investigated

468

its immune responses and efficacy against GCRV challenge in grass carp for the first

469

time.

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E. coli DH5α is a simple and efficient vector system for loading foreign DNA. In

471

addition, it is also nonpathogenic and avirulent, which makes it a good candidate for

472

use in DNA vaccine development [30]. Previous study by our laboratory had

473

confirmed that the pcDNA-vp7 constructed by ourselves could effectively express vp7

474

in grass carp tissue [2]. Our result in this study showed that almost 99% E. coli DH5α

475

formed BG (Fig. 2(B)) and was loaded with pcDNA-vp7 (Fig. 4); and average per

476

milligram (dry wt) of DH5α-BG could load 10 µg DNA by real-time PCR (Fig. 4).

477

Similarly, Paukner et al. found that lyophilized E. coli ghost (dry wt, per milligram)

478

was able to carry 5 µg pEGFP-N1 and the DNA can effectively be taken up and

479

expressed by macrophages (RAW264.7 cell) [20].

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480

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470

To defense against pathogens invasion in fish, the non-specific immunity is

481

considered as the primary defense [31]. Superoxide dismutase (SOD) is a vital

482

antioxidant enzyme, which could eliminate superoxide radicals to reduce intracellular

483

oxidant stress level [32]. T-AOC is also effective antioxidant, which can reflect a

484

comprehensive situation of the defense system [33]. ACP and AKP have been used as

ACCEPTED MANUSCRIPT a symbol of macrophage activation for the ability of intracellular digestion of

486

phagocytized antigens in the immune system of invertebrates [34]. It was found that

487

innate immune parameters, e.g., superoxide dismutase, T-AOC, ACP and AKP

488

activities were significantly increased both in the DH5α-BG/pcDNA-vp7 and naked

489

pcDNA-vp7 groups with high DNA dose (Fig. 8). It’s possibly that the increased

490

numbers of cells involved in the process (e.g.,migration of head and kidney

491

leukocytes) or enhanced pathogen resistance led to the increase of innate immune

492

parameters activities [2]. We also found that the innate immune parameters activities

493

in DH5α-BG/pcDNA-vp7 groups were much higher than naked pcDNA-vp7 groups at

494

the same sample time and DNA dose. It could be due to the fact that the adjuvant

495

properties of BG resulted in the above phenomenon. In other words, perhaps, native

496

cytomembrane of BG protected the DNA from being degraded, and the pill or out

497

membrane protein in the BG makes it suitable for attachment to specific target cells

498

[15].

499

phosphatidylethanolamine, which induced stronger reaction [20]. This may partly

500

explain the improved performance of the bacterial ghost-based DNA vaccines;

501

however, the exact mechanism needs further investigation.

SC

M AN U

TE D the

DNA

escaped

from

BG

mediated

by

the

EP

Subsequently,

AC C

502

RI PT

485

Furthermore, the mRNA expression levels of immune-related genes were also

503

detected.

The

IgM

gene

expression

was

significantly

up-regulated

in

504

DH5α-BG/pcDNA-vp7 groups after 21 days (Fig. 9), which was consistent with the

505

production of specific serum antibodies (Fig. 7). Except for IgM gene, MHC

506

molecules play a vital player in adaptive immunity. MHC class I, important

ACCEPTED MANUSCRIPT constitution of MHC family, has the ability to present antigen to T-killer cells to attack

508

cells invaded by pathogens [35]. In the present study, the mRNA levels of MHCI were

509

significantly up-regulated both in DH5α-BG/pcDNA-vp7 and naked pcDNA-vp7

510

groups (DNA dose ranged from 2.5-5µg) from days 14 to 21. Recently, Jiao et al.[36]

511

study the effect of a DNA vaccine in Japanese flounder, their results demonstrated that

512

the fish injected with the DNA vaccine showed significant up-regulation in the MHCI

513

mRNA expression. Meanwhile, previous study in our group also indicated that DNA

514

carried by carbon nanotubes could significant up-regulation transcription of genes

515

encoding MHCI in grass carp [2, 13]. In addition, some studies report that the BG

516

came from pathogenic bacteria could stimulate up-regulation in the expression of

517

MHCI because of its potential antigen in pathogenic bacteria BG [4, 16, 37]. Ebensen

518

et al. observed an up-regulation in the expression of MHCI on ghost-treated DC [4].

519

In our result, it was also found the nonpathogenic E. coli DH5α-BG significant

520

up-regulation mRNA expression of MHCI at days 14. It may be explained, at least in

521

part, that ghost may improve the capacity of APC to process and present

522

MHCI-restricted Ags [4], which may also explain the improved performance of the

523

bacterial ghost-based DNA vaccines.

SC

M AN U

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EP

AC C

524

RI PT

507

TNF-α is an important pro-inflammatory factors produced by various cells (e.g.,

525

macrophages/monocytes, T/B lymphocytes and NK cells) which can regulate immune

526

functions and mediates the inflammatory responses in mammals [38]. TNF-α in fish

527

showed similar functions with their mammalian counterparts. For example, the TNF-α

528

induces chemotactic response, phagocytosis and nitric oxide production in

ACCEPTED MANUSCRIPT macrophages in goldfish [39]. In the present study, the TNF-α transcript level

530

increased significantly in DH5α-BG/pcDNA-vp7 group (pcDNA-vp7 dose ranged

531

2.5-5µg) from days 7 to 14. After 21 days, only in the highest dose in

532

DH5α-BG/pcDNA-vp7 group, TNF-α transcript level was significantly up regulated.

533

Wang et al. utilized SWCNTs to deliver plasmid DNA, they also found the TNF-α

534

expression was significantly increased in the grass carp [13]. IL-1β is the master

535

inflammatory cytokine in the IL-1 family [40]. Our data showed that E. coli

536

DH5α-BG increased mRNA expression of IL-1β, but no significant difference,

537

compared to the PBS control. Wen et al. [41] studied the effect of Salmonella typhi

538

Ty21a ghost on the expression of IL-12, which found that S. typhi Ty21a ghost could

539

not markedly increase the expression of IL-12 in RAW264.7 cells. However, Ebensen

540

et al. reported that there is a significant increment in IL-12 secretion by DC in the

541

presence of Mannheimia haemolytica ghosts[4]. The differences in bacteria type,

542

research model or immunizing dose may lead to above results. We also found IL-1β

543

was significantly up-regulated in kidney in all DH5α-BG/pcDNA-vp7, as well as in

544

naked pcDNA-vp7 (pcDNA-vp7 dose ranged from 2.5-5µg) between days 14 and 21.

545

Zhu et al. [2] utilized SWCNTs to deliver pcDNA-vp7 which can also increase IL-1β

546

expression in grass carp. Additional, there is evidence that recombinant TNF-α can

547

up-regulate the expression of IL-1β in grass carp head kidneys leukocytes in vitro [42].

548

Perhaps, the increase of TNF-α expression may also have some positive effects on the

549

IL-1β expression in grass carp.

550

AC C

EP

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SC

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529

Cumulative mortality of grass carp after the challenges with live GCRV virus

ACCEPTED MANUSCRIPT was lower in vaccinated groups compared with the control (Fig. 10), which indicated

552

that pcDNA-vp7 DNA vaccine could protect fish from GCRV infection. The results in

553

Fig. 10 also reflected sole PBS, naked pcDNA plasmid and DH5α-BG did not enhance

554

the antiviral ability in fish. Moreover, percentage of mortality rates in

555

DH5α-BG/pcDNA-vp7 group was significantly lower compared to naked pcDNA-vp7

556

group, above results were consistent with the production of specific serum antibodies

557

(Fig. 7) that the specific antibody response was significantly increased after 21 days

558

post immunization by DH5α-BG/pcDNA-vp7 DNA vaccine. Similarly, Chen et al.

559

also found that H. pylori DNA vaccine in Salmonella typhimurium bacterial ghost

560

could significantly increase IgG antibody titer and protect the mice from H. pylori

561

infection [22]. Our results also demonstrated the plasmid could be detected in muscle

562

and kidneys tissues with relatively little degradation at the latest time point tested,

563

which was 21 days after injection with DH5α-BG/pcDNA-vp7 (Fig. 5). Prolong the

564

time of plasmid degradation in bacterial ghost-based DNA vaccines may contribute to

565

the production of more specific antibody response which may induce stronger

566

immunoprotection. Besides, The adjuvant effect of ghost bacterial has been reported

567

in pathogenic bacteria [43], the out membrane protein in the DH5α-BG may make the

568

loaded DNA suitable for attachment to specific target cells and more vp7 antigens

569

were expressed resulting in stronger immune responses and immunoprotection [15].

570

5. Conclusions

AC C

EP

TE D

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SC

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551

571

To conclusion, the present study demonstrated that E. coli DH5α ghost-based

572

pcDNA-vp7 DNA vaccine can induce stronger immune responses and protect grass

ACCEPTED MANUSCRIPT carp from GCRV infection as compared to conventional naked DNA vaccine.

574

Therefore, bacteria ghost may be considered as promising and efficient DNA vaccine

575

vehicle against viral pathogens in fish and ghost-based DNA vaccine will provide

576

extensive prospects for application to vaccines for aquatic animals.

577

Acknowledgments

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573

This work was supported by the special grade of the financial support from the

579

China Postdoctoral Science Foundation (Program No. 2016T90956) and Natural

580

Science Foundation of Shaanxi Province, China (Program No. 2016JQ3016). Authors

581

sincerely thank Xiaozhou Qi, Xiao Tu, Aiguo Huang and other laboratory members

582

for fish infection and sampling.

583

587 588 589 590 591 592 593 594 595 596 597

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[24] L.Yan, H. Guo, X. Sun, L. Shao, Q. Fang, Characterization of grass carp reovirus minor core protein VP4, J. Virol. 9 (2012), 1.

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[28] A. Muhammad, J. Champeimont, U.B. Mayr, W. Lubitz, P. Kudela, Bacterial ghosts as carriers of protein subunit and DNA-encoded antigens for vaccine applications, Expert Rev. Vaccines 11 (2014) 97-116.

[29] S.R. Kwon, Y.K. Nam, S.K. Kim, K.H. Kim, Protection of tilapia (Oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts, Fish. shellfish immunol. 20 (2006)

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[32] J. Tian, J. Yu, Poly(lactic-co-glycolic acid) nanoparticles as candidate DNA vaccine carrier for oral immunization of Japanese flounder (Paralichthys olivaceus) against lymphocystis disease virus, Fish. shellfish immunol. 30 (2011) 109-117. [33] D.Q. Sun, A.W. Li, J. Li, D.G. Li, Y.X. Li, F. Hao, M.Z. Gong, Changes of lipid peroxidation in carbon disulfide-treated rat nerve tissues and serum, Chem-biol. Interac. 179 (2009) 110-117. [34] F. Yin, H. Gong, Q. Ke, A. Li, Stress, antioxidant defence and mucosal immune responses of the large yellow croaker Pseudosciaena crocea challenged with Cryptocaryon irritans, Fish. shellfish immunol. 47 (2015) 344-51. [35] F. Buonocore, E. Randelli, D. Casani, S. Costantini, A. Facchiano, G. Scapigliati, R.J.M. Stet,

ACCEPTED MANUSCRIPT Molecular cloning, differential expression and 3D structural analysis of the MHC class-II β chain from sea bass (Dicentrarchus labrax L.), Fish. shellfish immunol. 23 (2007) 853-866. [36] X.D. Jiao, M. Zhang, Y.H. Hu, L. Sun, Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens, Vaccine 27 (2009) 5195-5202. [37] U.B. Mayr, C. Haller, W. Haidinger, A. Atrasheuskaya, E. Bukin, W. Lubitz, G. Ignatyev, Bacterial ghosts as an oral vaccine: a single dose of Escherichia coli O157:H7 bacterial ghosts protects mice against lethal challenge, Infect. Immun. 73 (2005) 4810-4817. Bi. 9 (1993), 317-343.

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[38] K.J. Tracey, A. Cerami, Tumor necrosis factor, other cytokines and disease, Annu. Rev. Cell. Dev [39] L. Grayfer, J.G. Walsh, M. Belosevic, Characterization and functional analysis of goldfish (Carassius auratus L.) tumor necrosis factor-alpha, Dev. Comp. immunol. 32 (2008) 532-543.

[40] L.A. Joosten, M.G. Netea, C.A. Dinarello, Interleukin-1beta in innate inflammation, autophagy

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and immunity, Semin. Immunol. 25 (2013) 416-424.

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[42] S. Zhang, R. Zhang, T. Ma, X. Qiu, X. Wang, A. Zhang, H. Zhou, Identification and functional characterization of tumor necrosis factor receptor 1 (TNFR1) of grass carp (Ctenopharyngodon idella), Fish. shellfish immunol. 58 (2016) 24-32.

[43] K. Jalava, A. Hensel, M. Szostak, S. Resch, W. Lubitz, Bacterial ghosts as vaccine candidates for

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veterinary applications, J. Control. Release 85(2002) 17-25.

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686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708

ACCEPTED MANUSCRIPT Figure captions Fig. 1 Analysis of genes expression. (A) PCR amplification of E/SNA/Smap29: lane M, DNA marker; lane 1, E/SNA/Smap29. (B) PCR amplification of CIts857/pR/pL-lysisE/SNA/Smap29:

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lane M, DNA marker; lane 1, CIts857/pR/pL-lysisE/SNA/Smap29. (C) analysis of recombinant plasmid: lane M, DNA marker; lane 1, double enzymes digested pCAT-lysisE/SNA/Smap29 with

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XbaI and SmaI; lane 2, pCAT-lysisE/SNA/Smap29.

Fig. 2 Analysis of the production of Escherichia coli DH5α ghost. (A) Growth curves (OD600) of

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DH5α-E by temperature induction of lysis gene expression. The temperature was up-shifted from 28 to 42 °C. (B) Numbers of viable cell prior to (0 h) and at the end of the lysis process (4 h). (C) Electrophoretic analysis of DH5α-E total DNA prior to (0 h) and at the end of the lysis process (4 h): lane M, DNA marker; lane 1, total DNA at the end of the lysis process (4 h); lane 2, total DNA

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prior to the lysis process (0 h).

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Fig. 3 Characterization of DH5α-BG by SEM. (A) and (C): The lysed cells grown at 42 °C

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(arrows indicated the transmembrane holes). (B): The unlysed cells grown at 28 °C.

Fig. 4 Analysis of DH5α-BG loaded with pcDNA-vp7. (A) Flow cytometric of Empty DH5α-BG cells staining with Propidium Iodide (PI). (B) Flow cytometric of DH5α-BG cells with pcDNA-vp7 staining with PI. (C) Flow cytometric of Lyophilized, intact E.coli DH5α with pcDNA-vp7 staining with PI. The x-axis represented relative fluorescence intensity and the y-axis represented relative cell numbers. (D) Analysis of the standard curve and loading efficiency with pcDNA-vp7 plasmid by real-time PCR.

ACCEPTED MANUSCRIPT Fig. 5 Detection of vaccine DNA in fish muscle and kidneys tissue extracts by PCR. Total DNA was extracted from grass carp muscle (A) and kidneys (B) at 21 days after immunity. M, DNA marker; lane 1, PBS group; lane 2, pcDNA group; lane 3, DH5α-BG group; lane 4, pcDNA-vp7 (1

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µg) group; lane 5, pcDNA-vp7 (2.5 µg) group; lane 6, pcDNA-vp7 (5 µg) group; lane 7, DH5α-BG/pcDNA-vp7 (1 µg) group; lane 8, DH5α-BG/pcDNA-vp7 (2.5 µg) group, lane 9,

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DH5α-BG/pcDNA-vp7 (5 µg) group.

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Fig. 6 PCR detection of vp7 transcription in grass carp. Total RNA was extracted from grass carp kidneys at 21 days after immunity. M, DNA marker; lane 1, PBS group; lane 2, pcDNA group; lane 3, DH5α-BG group; lane 4, pcDNA-vp7 (1 µg) group; lane 5, pcDNA-vp7 (2.5 µg) group; lane 6, pcDNA-vp7 (5 µg) group; lane 7, DH5α-BG/pcDNA-vp7 (1 µg) group; lane 8,

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DH5α-BG/pcDNA-vp7 (2.5 µg) group, lane 9, DH5α-BG/pcDNA-vp7 (5 µg) group

Fig. 7 Specific antibody levels of fish vaccinated with pcDNA-vp7 and DH5α-BG/pcDNA-vp7.

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Serum was collected from the fish at 3, 7, 14 and 21 days post-vaccination, and serum antibodies

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against recombinant VP7 were determined by ELISA. Data are means for three assays and presented as the means ± SD. **P < 0.01; *P < 0.05.

Fig. 8 (A) acid phosphatase (ACP) activity, (B) alkaline phosphatase (AKP) activity, (C) superoxide dismutase (SOD) activity and (D) Total antioxidant capacity (T-AOC) activity of grass carp post-immunization with different VP7 formulations by intramuscular injection at 3, 7, 14 and 21 days. Data are means for three assays and represented as mean ± SD. **P < 0.01; *P < 0.05.

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Fig. 9 Quantitative expression analysis of immune genes in grass carp vaccinated with different VP7 formulations at 3, 7, 14 and 21 days. (A) TNF-α; (B) IL-1β; (C) IgM; (D) MHCI; (E) IgD.

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Data are means for three assays and presented as the means ± SD. **P < 0.01; *P < 0.05.

Fig. 10 Cumulative mortalities after artificial challenging with GCRV in vaccinated grass carp for

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PBS, pcDNA, DH5α-BG, pcDNA-vp7 and DH5α-BG/pcDNA-vp7. Data are means for three

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assays and presented as the means ± SD.

ACCEPTED MANUSCRIPT Table 1 Sequences of primer pairs used in real-time PCR Product length (bp)

GATGATGAAATTGCCGCACTG ACCGACCATGACGCCCTGATGT

M25013

135

TGTGCCGCCGCTGTCTGCTTCACGCT

EU047718

291

EU047716

448

AY391782

271

Sequence (5’– 3’)

β-actin forward β-actin reverse TNFα forward TNFα reverse IL-1β forward IL-1β reverse

GATGAGGAAAGACACCTGGCTGTAGA GGAGAATGTGATCGAAGAGCGT GCTGATAAACCATCCGGGA

MHC-I forward MHC-I reverse IgD forward IgD reverse IgM forward IgM reverse

CCTGGCAGAAAAATGGACAAG

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CCAACAACACCAATGACAATC

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Accession number GenBank

Primer

CTGGCGCAGCTCTGAATTTG

GQ429174

287

DQ417927

170

TCGGAGGATGCTCACAATGG

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GCTGAGGCATCGGAGGCACAT

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TTGGGTCTCGCACCATTTTCTC

ACCEPTED MANUSCRIPT Table 2 Mortality rate and relative percentage survival (RPS) of fish challenged with GCRV after 14 days. Fish injected

Cumulative mortality (%/14 d)

RPS (%/ 14 d)

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PBS 100 ± 0a a pcDNA 100 ± 0 a DH5α-BG 100 ± 0 c 57.78 ± 1.92 42.22 ± 1.92d pcDNA-vp7 5 µg b pcDNA-vp7 2.5 µg 76.67 ± 3.33 23.33 ± 3.33e pcDNA-vp7 1 µg 94.44 ± 1.92a 5.56 ± 1.92f DH5α-BG/pcDNA-vp7 5 µg 10 ± 0f 90 ± 0a DH5α-BG/pcDNA-vp7 2.5 µg 27.78 ± 1.92e 72.22 ± 1.92b 46.67 ± 5.77d 53.33 ± 5.77c DH5α-BG/pcDNA-vp7 1 µg Values are expressed as mean ± S.D; three replicates were set for the tests. Data with different letters are significantly different (p < 0.01)

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Fig. 1

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y = -3.0261 x + 35.1095 R2 = 0.9935 E = 114%

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Fig. 4

4

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7 8 Log Starting Quantity

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1000 bp 750 bp 500 bp

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200 bp 100 bp

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vp7

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β-actin

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Fig. 8

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(B)IL-1β

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(A)TNF-α

(C)IgM

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(E)IgD

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(D)MHCI

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Fig. 10

ACCEPTED MANUSCRIPT E. coli DH5α ghost was used as carrier to prepare a novel DNA vaccine in grass carp. Immunization with DH5α/pcDNAvp7 induced stronger immune response than naked pcDNAvp7.

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Immunization with DH5α/pcDNAvp7 enhanced disease resistance against GCRV in fish.

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Bacterial ghost based DNA vaccine had prospects for application to aquatic vaccine.