- Email: [email protected]
Protective immunity of grass carp immunized with DNA vaccine against Aeromonas hydrophila by using carbon nanotubes as a carrier molecule
Accepted Manuscript Protective immunity of grass carp immunized with DNA vaccine against Aeromonas hydrophila by using carbon nanotubes as a carrier molecule Lei Liu, Yu-Xin Gong, Guang-Lu Liu, Bin Zhu, Gao-Xue Wang PII:
S1050-4648(16)30386-2
DOI:
10.1016/j.fsi.2016.06.026
Reference:
YFSIM 4026
To appear in:
Fish and Shellfish Immunology
Received Date: 8 March 2016 Revised Date:
18 June 2016
Accepted Date: 21 June 2016
Please cite this article as: Liu L, Gong Y-X, Liu G-L, Zhu B, Wang G-X, Protective immunity of grass carp immunized with DNA vaccine against Aeromonas hydrophila by using carbon nanotubes as a carrier molecule, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.06.026. 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.
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Protective immunity of grass carp immunized with DNA vaccine against Aeromonas hydrophila by
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using carbon nanotubes as a carrier molecule
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Lei Liua, Yu-Xin Gongb, Guang-Lu Liuc, Bin Zhua, Gao-Xue Wanga,*
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College of Animal Science and Technology, Northwest A&F University, Xinong Road 22nd, Yangling,
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Shaanxi 712100, China b
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College of Veterinary Medicine, Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi
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712100, China c
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College of Science, Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China
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* Corresponding author at: Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100,
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China. Tel./fax: +86 29 87092102.
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E-mail addresses: [email protected] (Gao-Xue Wang)
Keywords: Single-walled carbon nanotubes; Aeromonas hydrophila; DNA vaccine; immunological
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activity
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With the capture fishing industry declined and wild stocks diminished, the aquaculture industry has
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become an important source of food fish [1]. However, microbial pathologies are gradually primary
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constraints factors to hinder desirable production output. One of the major bacterial diseases is caused by
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a Gram-negative bacterium Aeromonas hydrophila which can cause serious damage in many animals [2],
ACCEPTED MANUSCRIPT especially fish [3,4], and is also harmful to human [5]. Generally, farmers use a wide range of antibiotics
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or chemicals to control A. hydrophila infection [6]. However, these treatments may be detrimental to
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environment and human health [7], since they pose negative such as antibiotic resistance in environment
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and fish [8]. Therefore, vaccines have been developed as a sustainable alternative against A. hydrophila.
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For example, some functional or structural proteins of A. hydrophila in recombinant form have been
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evaluated for vaccine candidates [9-13], and attempts were also made to use recombinant A. hydrophila
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vaccine via intramuscular injection to control the bacterial spread [11,14]. Currently, one of the most
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promising vaccine preparations against fish diseases is the DNA vaccine (delivered intramuscularly)
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consisting of naked plasmid DNA that will result in gene expression of pathogenic proteins in the muscle
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tissue of the vaccinated fish [15]. Although these methods have been developed for many years, there are
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still a number of limitations, such as stress on the fish, labor costs, time required and safety issues [16].
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Furthermore, intramuscular injection of naked DNA vaccine generally induces few and transient immune
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protection in fish [17]. To be more effective, DNA vaccines required effective carriers to overcome these
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obstacles.
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In the field of carrier systems, carbon nanotubes (CNTs) are potentially useful in their development
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for transporting and translocating bioactive molecules [18], because they can rapidly enter into
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antigen-presenting cells and make them especially useful as carriers of antigens/DNA [19-21]. More
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importantly, a study was found that CNTs could enhance the expression of β-galactosidase marker gene
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5~10 times higher levels than the plasmid DNA delivered alone [22]. Recently, a novel functionalized,
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single-walled carbon nanotubes (SWCNTs) as a vector for recombinant vaccines were found. They can
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produce specific antibodies to resist A. hydrophila or grass carp reovirus (GCRV) infection effectively
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[23,24]. Hence, the CNTs as a vaccine carrier could be potentially used to break through the cell barrier,
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improve vaccine and induce a protective immune response. In this study, CNTs-aerA (a virulence factor that has hemolytic and cytolytic properties) vaccine was
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prepared to enhance the efficacy of an aerA DNA vaccine against A. hydrophila in grass carp. We
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evaluated the immune efficacy of CNTs-aerA DNA vaccine in vaccinating fish after intramuscular
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injection. This study will provide a further information for future work on a wide range of CNTs-DNA
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vaccine delivery systems in fish.
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Grass carp with the total length and body weight of 3.0 ± 0.5 cm and 1.0 ± 0.1 g (mean values ± SD)
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were kindly provided by the Xinmin Aquatic Breeding Center (Heyang, Shaanxi, China) and acclimatized
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in the laboratory for two weeks before experimental manipulation. Fish were reared daily with a diet of
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commercial dry pellets (Tianlong Feed Company, China) and held in 300 L aquaria at 28°C aerated fresh
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water under laboratory conditions for two weeks, in which approximately 25% of the water was replaced
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daily. According to the study of Vazquez-Juarez et al. [25], a PCR technique was used to confirm free
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from A. hydrophila in fish. All protocols strictly adhered to the guide for care and use of laboratory
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animals, and were approved by Northwest A&F University animal protection committee.
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A. hydrophila strain XS91-4-1 used in this experiment was preserved in our laboratory with 20%
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glycerol at -80°C. During the experimental period, A. hydrophila was inoculated into liquid LB medium
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and grown overnight with constant shaking. Additionally, Escherichia coli DH5α (CWBIO, Beijing,
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China) was also cultured in LB medium.
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Cloning of aerA gene of A. hydrophila strain XS91-4-1 were performed according to our previous study
[24].
Two
oligonucleotide
primers
(F:
5'-GCAGAGCCCGTCTATCCAGA-3';
R:
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respectively. After aerA gene were ligated with pMD19T vector (Takara, Dalian, China), transformed into
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competent E. coli DH5α cells (CWBIO, Beijing, China) and sequenced by Sangon Biological Company
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(Shanghai, China), the aerA gene excised from pMD19T-aerA by digestion with Xho I/BamH I restricion
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enzyme (Takara, Dalian, China) was inserted into the pEGFP (Invitrogen, Carlsbad, CA, USA) to
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generate pEGFP-aerA. A recombinant plasmid pEGFP-aerA containing the capsid open reading frame
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was transformed into E. coli DH5a cells (CWBIO, Beijing, China) and the recombinant constructs were
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sequenced by Sangon Biological Company (Shanghai, China). The positive clone was grown in LB broth
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with ampicillin and incubated at 37
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with the Endo-free Plasmid Mini Kit (Omega, USA) following the manufacturer's instructions and the
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concentration was measured by the NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies
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Inc., Wilmington, DE) and conserved at -20
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overnight with shaking. Recombinant plasmid DNA was isolated
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The raw SWCNTs were purchased from Chengdu Organic Chemicals Co., Ltd. Chinese Academy of
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Sciences (Chengdu, China). Preparation of SWCNTs-pEGFP-aerA vaccine was performed as described
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previously [23].
For vaccination experiments, healthy grass carp were distributed randomly into eight groups
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(containing six treatment groups and two control groups, 80 fish per group). The experimental fish were
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injected intramuscularly with 0.1 mL pEGFP-aerA/SWCNTs-pEGFP-aerA (dissolve in PBS, pH = 7.4) in
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three different doses (1, 5 and 10 µg) in the right, dorsal, epaxial muscle in front of the dorsal fin after
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being anaesthetized with 20.0 g/m3 MS-222 [6]. The control groups were injected intramuscularly with 10
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µg of pEGFP diluted in 0.1 mL PBS or 0.1 mL of PBS alone. Subsequently, control and vaccinated
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groups were transferred to different tanks during the whole immunization period (6 weeks). Antiserum preparation and measurement of anti-aerA antibody responses were made by
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enzyme-linked immunosorbent assay (ELISA) according to the previous study [26], and rabbit sera
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anti-IgM was prepared following the method of Ding et al. [27]. For analyses of the presence of specific,
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neutralizing antibodies, vaccinated and control fish were sampled weekly until 6 weeks for antibody
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determination. At each time points, five samples from each control and vaccinated groups were pooled
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and prepared according the previous method [24]. Rabbit anti-IgM polyclonal antibody was used as
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primary antibody, and HRP-conjugated goat anti-rabbit IgG (CWBIO, Beijing, China) was used as
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secondary antibody. The primary and secondary antibodies were diluted 1:1000 immediately before use
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with PBS containing 3% skimmed milk. Additionally, DAB horseradish peroxidase color development kit
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(Tiangen Biotech, Beijing, China) was used for color development. The plate was read at 450 nm with a
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precision microplate reader (Molecular Devices Corp., Palo Alto, CA).
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After 21 d, 15 sampled fish from each control and vaccinated groups were euthanized through
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washrag soaked with 20.0 g/m3 MS-222 and dissected immediately with sterile scissors. The kidney was
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removed, where five fish from one pool per condition were sampled for analyzing gene expression and
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this experiment was performed in triplicate replicates. Pooled kidney tissue samples were taken from the
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fish and immediately frozen in liquid nitrogen for subsequent RNA isolation. Total RNA was extracted
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using TRIZOL reagent (CWBIO, Beijing, China) following manufacturer’s instruction. The isolated RNA
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samples were diluted in RNase-free double distilled water and treated with DNase I (Takara, Dalian,
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China) according to the manufacturer’s instructions. RNA was reverse transcribed using PrimeScript RT
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reagent Kit (Perfect Real Time) and oligo d (T) primer (Vazyme, USA). Real-time PCR (RT-PCR) was
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Green Master Mix (Vazyme, USA), with 18S RNA as the endogenous control [28]. The primers of seven
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immune-relevant genes were designed by other studies [26] and are listed in Table 1. The relative
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quantification of gene transcription was performed as described in the previous study [6]. Each individual
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sample was run in triplicate wells. Relative mRNA expression was calculated using 2-△△Ct method with
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the formula, F = 2-△△Ct, △△Ct = (Ct, target gene – Ct, reference gene) – (Ct, target gene – Ct, reference gene)control [29].
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Table 1
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At the time point of 21 days post-vaccination, each group was randomly chosen 30 fish to transfer to
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different tanks. Then fish were challenged by intraperitoneal injection with 30 µL culture suspension of A.
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hydrophila (5.0 × 106 CFU/mL of fish). Mortality rates were recorded daily, and dead fish were removed
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from the tank daily. The relative percent survival (RPS) was calculated according to Amend's method
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[30].
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Data were presented as mean ± standard deviation (SD). Differences between experimental group
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and control were generated with SPSS (version 18.0) statistical software (SPSS Inc., Chicago, IL, USA)
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using ANOVA followed by Tukey’s post hoc tests using SPSS 18.0. A P value of <0.05 or <0.01 was
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considered statistically significant.
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The amplification of the aerA gene (expected size 1332 bp) of A. hydrophila was shown in Fig. 1A.
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When the aerA gene was cloned into the cloning vector pMD19T and eukaryotic expression plasmid
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pEGFP, it was confirmed by restriction enzyme digestion (Fig. 1B and C) and sequence analysis (data not
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show). Both results confirmed that recombinant plasmid was successfully constructed.
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[Fig. 1]
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obtained from vaccinated fish during 1-6 weeks post-initiation vaccination. ELISA analysis showed that
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the antibody levels increased with the increasing doses of pEGFP-aerA or SWCNTs-pEGFP-aerA (Fig. 2).
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The highest levels of specific antibodies were observed at 4 weeks post vaccination in 10 µg pEGFP-aerA
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or SWCNTs-pEGFP-aerA groups. Generally, SWCNTs-pEGFP-aerA elicited higher levels of antibody
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compared with free pEGFP-aerA at the same immunization dose. Meanwhile, no significant difference
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was found in the fish injected with PBS and pEGFP.
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As shown in Fig. 3, both aerA and SWCNTs-aerA in highest immunization dose induced a
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significant enhancement of all immune-related genes (type I interferon (IFN-I), tumor necrosis factor α
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(TNFα), C-reactive protein (CRP), interleukin 8 (IL-8), immunoglobulin M (IgM), major
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histocompatibility complex (MHC) class I and Cluster of differentiation 8α (CD8α)) expression in the
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kidney. Furthermore, all these genes expression were induced by 10 µg SWCNTs-pEGFP-aerA to
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increase
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SWCNTs-pEGFP-aerA treatments were significantly higher than that in the pEGFP-aerA groups.
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inductions observed.
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And
the
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[Fig.3]
Grass carp were challenged by injected intramuscularly with 5.0 × 106 CFU/mL of A. hydrophila.
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The result in Fig. 4 showed that the cumulative mortality of treatment and control groups after being
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challenged with a lethal dose of A. hydrophila at 21 days post vaccination. For two control groups (PBS
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and pEGFP) and two vaccinated groups (1 and 5 µg pEGFP-aerA), the mortalities were occurred at 3-day
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after challenge and after 8-day, the cumulative mortality of two control groups was reached to 100%. For
ACCEPTED MANUSCRIPT SWCNTs-pEGFP-aerA vaccinated groups (1, 5 and 10 µg), the cumulative mortalities were reached to
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42.7%, 30.4% and 16.3%, respectively, and comparably, a lower immune protective effects were obtained
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in pEGFP-aerA vaccinated groups (1, 5 and 10 µg) (Table 2).
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[Fig. 4]
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[Table 2]
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In the past decade, DNA vaccine has been extensively applied for the prevention of aquaculture
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diseases caused by different pathogens, especially infectious virus [31-33]. Compared with live,
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attenuated vaccines, inactivated vaccines, and purified subunits of the pathogen, DNA vaccine has the
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advantage of being inexpensive, safe, stable, and inducing both humoral and cellular immunities [34,35].
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Immunization with antigen-encoding plasmid DNA can produce the target protein in vivo upon
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introduction to the host, thereby eliciting powerful and long-lasting cellular and humoral immune
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responses that is similar to that induced by natural infection with intracellular pathogens [36]. However,
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the rapid tissue clearance and on-site degradation of DNA of conventional DNA vaccination may reduce
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pDNA uptake and transgene expression [37,38]. To enhance the efficacy of DNA vaccines, in this study,
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SWCNTs vaccine delivery system was used to deliver pEGFP-aerA DNA vaccine against A. hydrophila.
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Our previous study have found that higher levels of transcription and expression of the vp7 gene of
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GCRV could be detected in muscle tissues of grass carp 28 days post-injection in SWCNTs-pcDNA-vp7
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treatment groups comparing with naked pcDNA-vp7 treatment groups [23]. In addition, the potency of
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ammonium group-functionalized multi-walled carbon nanotubes (MWCNTs) could enhance the
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transfection and expression efficiency of plasmid DNA (pEGFP-vp5) in Ctenopharyngodon idellus
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kidney (CIK) cells [39]. SWCNTs was also found to facilitate higher pDNA uptake and gene expression
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ACCEPTED MANUSCRIPT than pDNA alone in Chinese hamster ovary cells [40]. In the challenge test, the result reflected that
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pEGFP-aerA DNA vaccine could protect grass carp against A. hydrophila infection, with a lower
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percentage of mortality rates in 15 days. Further, SWCNTs- pEGFP-aerA DNA vaccine showed a better
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protection efficacy on grass carp than pEGFP-aerA, and naked plasmid vaccine did not enhance innate
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antibacterial immunity of fish as the control group. These results were in agreement with the previous
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findings that SWCNTs can improve the plasmid/antigen stability, and prolong the time of plasmid/antigen
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degradation [23].
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In the past, most research about DNA vaccines against bacterial pathogens in aquaculture have been
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reported, and these vaccine were demonstrated to be effective against challenges of the respective
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pathogens [41-44]. In our tests, the levels of specific antibody increased significantly and persisted up to
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6 weeks of post immunization. It might be that the SWCNTs-pEGFP-aerA could be delivered into the fish
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cells, and the plasmid DNA was released from the nanotubes, and thus expressed antigen. With the
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increase of amounts of plasmid DNA released from SWCNTs, more antigens were expressed and stronger
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immune response was induced.
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In the present study, we compared the expressions of immune-related genes of free pEGFP-aerA and
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SWCNTs-pEGFP-aerA DNA vaccine on grass carp via intramuscular injection immunization. The results
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indicated that fish immunization with SWCNTs-pEGFP-aerA DNA vaccine augmented the production of
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specific antibodies and exhibited a high level of survival rate compared with pEGFP-aerA alone. Herein,
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consistent with the production of specific antibodies (Fig. 2), the IgM expression was increased
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significantly in vaccinated fish kidney. It is known that IgM is a major component of the humoral immune
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system of teleost fish, regarded as the first antibody [45,46]. Some investigators have reported that the
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ACCEPTED MANUSCRIPT IgM expression would be intensively increase in many tissues and organs from the second week after
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immunization and maintained almost one month [47]. Besides, IL-8, IFN-I and TNFα, an important
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component in innate immunity and the inflammatory, were also found up-regulated by the DNA vaccine
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treatment in this study. These genes were considered to be an important component in innate immunity
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and antivirus response in fish [48-50], and especially TNFα is considered as an important
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pro-inflammatory factors to induce the inflammatory response by regulating the expression of other
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cytokines [51]. Thus, the risen gene levels may reflect the initiation of specific adaptive immune
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responses [52]. Additionally, CRP levels were observed to increase in grass carp vaccinated with
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pEGFP-aerA/SWCNTs-pEGFP-aerA DNA vaccines, while CRP usually served as the major acute phase
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reactant and played an important role in the innate immune response to an inflammatory stimulus in fish
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[53]. Since MHC I are involved in offering antigenic peptides for recognition by the TCR/CD8 complex
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of cytotoxic T lymphocytes and CD8 molecules acts as a coreceptor and lies in its active role in activating
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T cells [6,54,55], their up-regulation indicated that the protective effect of the DNA vaccines might be
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due to the induction of humoral and cellular immune response.
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Currently, it is an important consideration on safety in preparing the vaccine on fish. Even though a
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series of researches have assessed toxicity of raw carbon nanotubes in mice, no significant effect was
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found in cell culture experiments [56]. For fish embryos and larvae, Zhu et al. found that a series of
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tested index were significantly changed when the concentration of SWCNTs was above 50 mg/L by bath
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exposure [57]. However, there was no report about the toxicity of CNTs in fish by intramuscular injection,
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and the information was lack in relative action mechanisms. Therefore, toxicity assessment on aquatic
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animals of SWCNTs is also needed to make further studies.
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ACCEPTED MANUSCRIPT In conclusion, our results showed that SWCNTs loaded with DNA vaccine induced a better
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protection to juvenile grass carp against A. hydrophlia. Moreover, SWCNTs conjugated with DNA
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vaccine provided significantly protective immunity compared with free DNA vaccine. Thereby, SWCNTs
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may be considered as a potential efficient DNA vaccine carrier to enhance the immunological activity.
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This work was supported by the Scientific Innovation and Achievements Transformation Fund for Northwest A&F University (XNY2013-25).
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in orange-spotted grouper after immunization with Cryptocaryon irritans vaccine. Fish Shellfish
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Immunol. 2013 34:885–91.
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vivo detection, imaging and drug delivery. Nano Res. 2009 2:85–120. [57] Zhu B, Liu G L, Ling F, Song LS, Wang GX. Development toxicity of functionalized single-walled
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carbon nanotubes on rare minnow embryos and larvae. Nanotoxicology, 2014 9:1–12.
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ACCEPTED MANUSCRIPT Figure captions
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Fig. 1. Analysis of aerA expression. (A) RT-PCR amplification of aerA: lane M, DNA marker; lane 1,
362
aerA. (B) analysis of recombinant plasmid: lane M, DNA marker; lane 1, double enzymes digested
363
pMD19T-aerA with Xho I and BamH I; lane 2, pMD19T-aerA. (C) analysis of recombinant plasmid: lane
364
M, DNA marker; lane 1, pEGFP-aerA; lane 2, double enzymes digested pEGFP-aerA with Xho I and
365
BamH I.
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Fig. 2. Specific antibody levels of fish vaccinated with DNA vaccine were determined by ELISA. Data
367
are means for three assays and presented as the means ± SE. **P < 0.01; *P < 0.05.
368
Fig. 3. Real-time PCR analysis of the expression of immune-related genes in fish vaccinated with
369
different vaccine formulations after 21 d. (A) IL-8; (B) IFN-I; (C) TNFa; (D) CRP; (E) MHC-I; (F) IgM;
370
(G) CD8α. Data are means for three assays and presented as the means ± SE. **P < 0.01; *P < 0.05.
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Fig. 4. Cumulative mortalities of vaccinated fish. The accumulated mortalities were calculated at the end
372
of the monitored period.
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ACCEPTED MANUSCRIPT Table 1 Sequences of primer pairs used for the analysis of mRNA expression by qRT-PCR.
Product size (bp)
GenBank accession no.
Forward
ATTTCCGACACGGAGAGG
90
EU047719
Reverse
CATGGGTTTAGGATACGCTC
Forward
GGTGAAGTTTCTTGCCCTGACCTTAG
173
AB196166
Reverse
CCTTATGTGATGGCTGGTATCGGG
TNF-α
Forward
TGTGCCGCCGCTGTCTGCTTCACGCT
291
EU047718
Reverse
GATGAGGAAAGACACCTGGCTGTAGA
CRP
Forward
CTGCCTCCGCTCTCCATCTT
300
FJ547474
Reverse
TCCCTTTGGCACATACGGTTCCTGA
IL-8
Forward
AGGTCTGGGTGTAGATCCACGCTG
Reverse
TTAGTGTGAAAACTAACATGATCTCT
IgM
Forward
GCTGAGGCATCGGAGGCACAT
MHC-I
Reverse
TTGGGTCTCGCACCATTTTCTC
Forward
CCTGGCAGAAAAATGGACAAG
Reverse
CCAACAACACCAATGACAATC
Forward
GAGTCTCTGCACGGATCTAT
Reverse
GTGTAGTGTTCCGAATTTAAGT
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CD8α
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IGF-I
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EU047717
170
DQ417927
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Primer sequences (from 5' to 3')
Genes
271
AY391782
172
GQ355586
ACCEPTED MANUSCRIPT Table 2 Corresponding RPS values of vaccinated and control fish. Cumulative percentage mortality (%) and calculated relative percentage survival (RPS) following challenge with A. hydrophila in experimental
Fish injected with
Cumulative mortality (21 d)
PBS
100.0%
pEGFP 10 µg
100.0%
pEGFP-aerA 1 µg
92.1%
pEGFP-aerA 5 µg
70.7%
pEGFP-aerA 10 µg
54.9%
SWCNTs-pEGFP-aerA 1 µg
42.7%
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7.9%
29.3%
45.1%
57.3%
30.4%
69.6%
16.3%
83.7%
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SWCNTs-pEGFP-aerA 10 µg
RPS (21 d)
—
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groups of DNA vaccinated fish by intramuscular injection.
ACCEPTED MANUSCRIPT
M
1
B
M
1
2
M
1
2
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C
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A
Fig. 1.
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0.7
**
PBS
**
**
** **
**
0.5 0.4
**
**
0.3
** **
**** **
** **
**
** ** ** **
**
**
** **
**
**
** **
pEGFP-aerA 1 µg pEGFP-aerA 5 µg pEGFP-aerA 10 µg SWCNTs-pEGFP-aerA 1 µg
*
*
SWCNTs-pEGFP-aerA 5 µg
0.2
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Absorbance (OD450)
**
0.6
SWCNTs-pEGFP-aerA 10 µg
0.1
pEGFP
0 1
2
3
4
6
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Weeks post-vaccination
5
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Fig. 2.
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4
pEGFP-aerA
IL-8
**
Fold change
SWCNTs-pEGFP-aerA
3
** *
2
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1 0 PBS
pEGFP
*
*
1
5
10
Fold change
4
pEGFP-aerA
IFN-I
SWCNTs-pEGFP-aerA
3
**
2
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PBS
pEGFP-aerA
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5 4
**
*
0
Fold change
**
**
1
C
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5
B
SC
Injected dose (µg)
1
5
10
Injected dose (µg)
TNFα
**
**
SWCNTs-pEGFP-aerA
**
3
**
*
2 1 0
PBS
pEGFP
1
5 Injected dose (µg)
10
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Fold change
5
pEGFP-aerA
CRP
**
SWCNTs-pEGFP-aerA
**
4 3
**
*
2
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1 0 PBS
pEGFP
1
5
10
pEGFP-aerA
MHC-I
Fold change
SWCNTs-pEGFP-aerA
6 4
**
**
12
pEGFP
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PBS
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0
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pEGFP-aerA
1
**
**
**
2
F
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E
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Injected dose (µg)
5
10
Injected dose (µg)
IgM
**
Fold change
SWCNTs-pEGFP-aerA
9
**
6
**
** **
3 0 PBS
pEGFP
1
5 Injected dose (µg)
10
ACCEPTED MANUSCRIPT 5
Fold change
4
pEGFP-aerA
CD8α
**
SWCNTs-pEGFP-aerA
**
3
**
**
2 1 0 PBS
pEGFP
1
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G
5
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Injected dose (µg)
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Fig. 3.
10
100 90 80 70 60 50 40 30 20 10 0
PBS pEGFP 10 µg pEGFP-ae rA 1 µg pEGFP-ae rA 5 µg pEGFP-ae rA 10 µg SWC NTs-pEGFP-ae rA 1 µg SWC NTs-pEGFP-ae rA 5 µg SWC NTs-pEGFP-ae rA 10 µg
3
6
9
12
Days after challenge
18
21
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Fig. 4.
15
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Cumulative mortality (%)aaa
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ACCEPTED MANUSCRIPT Highlights: 1. SWCNTs can be used as carriers for DNA vaccine. 2. SWCNTs-DNA vaccine enhanced the immunological activity.
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3. SWCNTs-DNA vaccine induced a better protection to grass carp against A. hydrophlia.
S1050-4648(16)30386-2
DOI:
10.1016/j.fsi.2016.06.026
Reference:
YFSIM 4026
To appear in:
Fish and Shellfish Immunology
Received Date: 8 March 2016 Revised Date:
18 June 2016
Accepted Date: 21 June 2016
Please cite this article as: Liu L, Gong Y-X, Liu G-L, Zhu B, Wang G-X, Protective immunity of grass carp immunized with DNA vaccine against Aeromonas hydrophila by using carbon nanotubes as a carrier molecule, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.06.026. 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.
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Protective immunity of grass carp immunized with DNA vaccine against Aeromonas hydrophila by
2
using carbon nanotubes as a carrier molecule
3
Lei Liua, Yu-Xin Gongb, Guang-Lu Liuc, Bin Zhua, Gao-Xue Wanga,*
a
College of Animal Science and Technology, Northwest A&F University, Xinong Road 22nd, Yangling,
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Shaanxi 712100, China b
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College of Veterinary Medicine, Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi
8
712100, China c
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College of Science, Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China
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* Corresponding author at: Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100,
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China. Tel./fax: +86 29 87092102.
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E-mail addresses: [email protected] (Gao-Xue Wang)
Keywords: Single-walled carbon nanotubes; Aeromonas hydrophila; DNA vaccine; immunological
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activity
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With the capture fishing industry declined and wild stocks diminished, the aquaculture industry has
19
become an important source of food fish [1]. However, microbial pathologies are gradually primary
20
constraints factors to hinder desirable production output. One of the major bacterial diseases is caused by
21
a Gram-negative bacterium Aeromonas hydrophila which can cause serious damage in many animals [2],
ACCEPTED MANUSCRIPT especially fish [3,4], and is also harmful to human [5]. Generally, farmers use a wide range of antibiotics
23
or chemicals to control A. hydrophila infection [6]. However, these treatments may be detrimental to
24
environment and human health [7], since they pose negative such as antibiotic resistance in environment
25
and fish [8]. Therefore, vaccines have been developed as a sustainable alternative against A. hydrophila.
26
For example, some functional or structural proteins of A. hydrophila in recombinant form have been
27
evaluated for vaccine candidates [9-13], and attempts were also made to use recombinant A. hydrophila
28
vaccine via intramuscular injection to control the bacterial spread [11,14]. Currently, one of the most
29
promising vaccine preparations against fish diseases is the DNA vaccine (delivered intramuscularly)
30
consisting of naked plasmid DNA that will result in gene expression of pathogenic proteins in the muscle
31
tissue of the vaccinated fish [15]. Although these methods have been developed for many years, there are
32
still a number of limitations, such as stress on the fish, labor costs, time required and safety issues [16].
33
Furthermore, intramuscular injection of naked DNA vaccine generally induces few and transient immune
34
protection in fish [17]. To be more effective, DNA vaccines required effective carriers to overcome these
35
obstacles.
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In the field of carrier systems, carbon nanotubes (CNTs) are potentially useful in their development
37
for transporting and translocating bioactive molecules [18], because they can rapidly enter into
38
antigen-presenting cells and make them especially useful as carriers of antigens/DNA [19-21]. More
39
importantly, a study was found that CNTs could enhance the expression of β-galactosidase marker gene
40
5~10 times higher levels than the plasmid DNA delivered alone [22]. Recently, a novel functionalized,
41
single-walled carbon nanotubes (SWCNTs) as a vector for recombinant vaccines were found. They can
42
produce specific antibodies to resist A. hydrophila or grass carp reovirus (GCRV) infection effectively
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[23,24]. Hence, the CNTs as a vaccine carrier could be potentially used to break through the cell barrier,
44
improve vaccine and induce a protective immune response. In this study, CNTs-aerA (a virulence factor that has hemolytic and cytolytic properties) vaccine was
46
prepared to enhance the efficacy of an aerA DNA vaccine against A. hydrophila in grass carp. We
47
evaluated the immune efficacy of CNTs-aerA DNA vaccine in vaccinating fish after intramuscular
48
injection. This study will provide a further information for future work on a wide range of CNTs-DNA
49
vaccine delivery systems in fish.
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Grass carp with the total length and body weight of 3.0 ± 0.5 cm and 1.0 ± 0.1 g (mean values ± SD)
51
were kindly provided by the Xinmin Aquatic Breeding Center (Heyang, Shaanxi, China) and acclimatized
52
in the laboratory for two weeks before experimental manipulation. Fish were reared daily with a diet of
53
commercial dry pellets (Tianlong Feed Company, China) and held in 300 L aquaria at 28°C aerated fresh
54
water under laboratory conditions for two weeks, in which approximately 25% of the water was replaced
55
daily. According to the study of Vazquez-Juarez et al. [25], a PCR technique was used to confirm free
56
from A. hydrophila in fish. All protocols strictly adhered to the guide for care and use of laboratory
57
animals, and were approved by Northwest A&F University animal protection committee.
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A. hydrophila strain XS91-4-1 used in this experiment was preserved in our laboratory with 20%
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glycerol at -80°C. During the experimental period, A. hydrophila was inoculated into liquid LB medium
60
and grown overnight with constant shaking. Additionally, Escherichia coli DH5α (CWBIO, Beijing,
61
China) was also cultured in LB medium.
62 63
Cloning of aerA gene of A. hydrophila strain XS91-4-1 were performed according to our previous study
[24].
Two
oligonucleotide
primers
(F:
5'-GCAGAGCCCGTCTATCCAGA-3';
R:
ACCEPTED MANUSCRIPT 5'-TCACTCCAGCCTCAGGCCTTG-3') consists of Xho I and BamH I restriction enzyme sites,
65
respectively. After aerA gene were ligated with pMD19T vector (Takara, Dalian, China), transformed into
66
competent E. coli DH5α cells (CWBIO, Beijing, China) and sequenced by Sangon Biological Company
67
(Shanghai, China), the aerA gene excised from pMD19T-aerA by digestion with Xho I/BamH I restricion
68
enzyme (Takara, Dalian, China) was inserted into the pEGFP (Invitrogen, Carlsbad, CA, USA) to
69
generate pEGFP-aerA. A recombinant plasmid pEGFP-aerA containing the capsid open reading frame
70
was transformed into E. coli DH5a cells (CWBIO, Beijing, China) and the recombinant constructs were
71
sequenced by Sangon Biological Company (Shanghai, China). The positive clone was grown in LB broth
72
with ampicillin and incubated at 37
73
with the Endo-free Plasmid Mini Kit (Omega, USA) following the manufacturer's instructions and the
74
concentration was measured by the NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies
75
Inc., Wilmington, DE) and conserved at -20
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overnight with shaking. Recombinant plasmid DNA was isolated
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Sciences (Chengdu, China). Preparation of SWCNTs-pEGFP-aerA vaccine was performed as described
78
previously [23].
For vaccination experiments, healthy grass carp were distributed randomly into eight groups
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(containing six treatment groups and two control groups, 80 fish per group). The experimental fish were
81
injected intramuscularly with 0.1 mL pEGFP-aerA/SWCNTs-pEGFP-aerA (dissolve in PBS, pH = 7.4) in
82
three different doses (1, 5 and 10 µg) in the right, dorsal, epaxial muscle in front of the dorsal fin after
83
being anaesthetized with 20.0 g/m3 MS-222 [6]. The control groups were injected intramuscularly with 10
84
µg of pEGFP diluted in 0.1 mL PBS or 0.1 mL of PBS alone. Subsequently, control and vaccinated
ACCEPTED MANUSCRIPT 85
groups were transferred to different tanks during the whole immunization period (6 weeks). Antiserum preparation and measurement of anti-aerA antibody responses were made by
87
enzyme-linked immunosorbent assay (ELISA) according to the previous study [26], and rabbit sera
88
anti-IgM was prepared following the method of Ding et al. [27]. For analyses of the presence of specific,
89
neutralizing antibodies, vaccinated and control fish were sampled weekly until 6 weeks for antibody
90
determination. At each time points, five samples from each control and vaccinated groups were pooled
91
and prepared according the previous method [24]. Rabbit anti-IgM polyclonal antibody was used as
92
primary antibody, and HRP-conjugated goat anti-rabbit IgG (CWBIO, Beijing, China) was used as
93
secondary antibody. The primary and secondary antibodies were diluted 1:1000 immediately before use
94
with PBS containing 3% skimmed milk. Additionally, DAB horseradish peroxidase color development kit
95
(Tiangen Biotech, Beijing, China) was used for color development. The plate was read at 450 nm with a
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precision microplate reader (Molecular Devices Corp., Palo Alto, CA).
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After 21 d, 15 sampled fish from each control and vaccinated groups were euthanized through
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washrag soaked with 20.0 g/m3 MS-222 and dissected immediately with sterile scissors. The kidney was
99
removed, where five fish from one pool per condition were sampled for analyzing gene expression and
100
this experiment was performed in triplicate replicates. Pooled kidney tissue samples were taken from the
101
fish and immediately frozen in liquid nitrogen for subsequent RNA isolation. Total RNA was extracted
102
using TRIZOL reagent (CWBIO, Beijing, China) following manufacturer’s instruction. The isolated RNA
103
samples were diluted in RNase-free double distilled water and treated with DNase I (Takara, Dalian,
104
China) according to the manufacturer’s instructions. RNA was reverse transcribed using PrimeScript RT
105
reagent Kit (Perfect Real Time) and oligo d (T) primer (Vazyme, USA). Real-time PCR (RT-PCR) was
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107
Green Master Mix (Vazyme, USA), with 18S RNA as the endogenous control [28]. The primers of seven
108
immune-relevant genes were designed by other studies [26] and are listed in Table 1. The relative
109
quantification of gene transcription was performed as described in the previous study [6]. Each individual
110
sample was run in triplicate wells. Relative mRNA expression was calculated using 2-△△Ct method with
111
the formula, F = 2-△△Ct, △△Ct = (Ct, target gene – Ct, reference gene) – (Ct, target gene – Ct, reference gene)control [29].
112
Table 1
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At the time point of 21 days post-vaccination, each group was randomly chosen 30 fish to transfer to
114
different tanks. Then fish were challenged by intraperitoneal injection with 30 µL culture suspension of A.
115
hydrophila (5.0 × 106 CFU/mL of fish). Mortality rates were recorded daily, and dead fish were removed
116
from the tank daily. The relative percent survival (RPS) was calculated according to Amend's method
117
[30].
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Data were presented as mean ± standard deviation (SD). Differences between experimental group
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and control were generated with SPSS (version 18.0) statistical software (SPSS Inc., Chicago, IL, USA)
120
using ANOVA followed by Tukey’s post hoc tests using SPSS 18.0. A P value of <0.05 or <0.01 was
121
considered statistically significant.
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The amplification of the aerA gene (expected size 1332 bp) of A. hydrophila was shown in Fig. 1A.
123
When the aerA gene was cloned into the cloning vector pMD19T and eukaryotic expression plasmid
124
pEGFP, it was confirmed by restriction enzyme digestion (Fig. 1B and C) and sequence analysis (data not
125
show). Both results confirmed that recombinant plasmid was successfully constructed.
126
[Fig. 1]
ACCEPTED MANUSCRIPT To compare the humoral immune response, we evaluated the neutralizing activity of sera samples
128
obtained from vaccinated fish during 1-6 weeks post-initiation vaccination. ELISA analysis showed that
129
the antibody levels increased with the increasing doses of pEGFP-aerA or SWCNTs-pEGFP-aerA (Fig. 2).
130
The highest levels of specific antibodies were observed at 4 weeks post vaccination in 10 µg pEGFP-aerA
131
or SWCNTs-pEGFP-aerA groups. Generally, SWCNTs-pEGFP-aerA elicited higher levels of antibody
132
compared with free pEGFP-aerA at the same immunization dose. Meanwhile, no significant difference
133
was found in the fish injected with PBS and pEGFP.
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As shown in Fig. 3, both aerA and SWCNTs-aerA in highest immunization dose induced a
136
significant enhancement of all immune-related genes (type I interferon (IFN-I), tumor necrosis factor α
137
(TNFα), C-reactive protein (CRP), interleukin 8 (IL-8), immunoglobulin M (IgM), major
138
histocompatibility complex (MHC) class I and Cluster of differentiation 8α (CD8α)) expression in the
139
kidney. Furthermore, all these genes expression were induced by 10 µg SWCNTs-pEGFP-aerA to
140
increase
141
SWCNTs-pEGFP-aerA treatments were significantly higher than that in the pEGFP-aerA groups.
143
inductions observed.
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greater
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And
the
relative
expression levels in
[Fig.3]
Grass carp were challenged by injected intramuscularly with 5.0 × 106 CFU/mL of A. hydrophila.
144
The result in Fig. 4 showed that the cumulative mortality of treatment and control groups after being
145
challenged with a lethal dose of A. hydrophila at 21 days post vaccination. For two control groups (PBS
146
and pEGFP) and two vaccinated groups (1 and 5 µg pEGFP-aerA), the mortalities were occurred at 3-day
147
after challenge and after 8-day, the cumulative mortality of two control groups was reached to 100%. For
ACCEPTED MANUSCRIPT SWCNTs-pEGFP-aerA vaccinated groups (1, 5 and 10 µg), the cumulative mortalities were reached to
149
42.7%, 30.4% and 16.3%, respectively, and comparably, a lower immune protective effects were obtained
150
in pEGFP-aerA vaccinated groups (1, 5 and 10 µg) (Table 2).
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[Fig. 4]
152
[Table 2]
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In the past decade, DNA vaccine has been extensively applied for the prevention of aquaculture
154
diseases caused by different pathogens, especially infectious virus [31-33]. Compared with live,
155
attenuated vaccines, inactivated vaccines, and purified subunits of the pathogen, DNA vaccine has the
156
advantage of being inexpensive, safe, stable, and inducing both humoral and cellular immunities [34,35].
157
Immunization with antigen-encoding plasmid DNA can produce the target protein in vivo upon
158
introduction to the host, thereby eliciting powerful and long-lasting cellular and humoral immune
159
responses that is similar to that induced by natural infection with intracellular pathogens [36]. However,
160
the rapid tissue clearance and on-site degradation of DNA of conventional DNA vaccination may reduce
161
pDNA uptake and transgene expression [37,38]. To enhance the efficacy of DNA vaccines, in this study,
162
SWCNTs vaccine delivery system was used to deliver pEGFP-aerA DNA vaccine against A. hydrophila.
163
Our previous study have found that higher levels of transcription and expression of the vp7 gene of
164
GCRV could be detected in muscle tissues of grass carp 28 days post-injection in SWCNTs-pcDNA-vp7
165
treatment groups comparing with naked pcDNA-vp7 treatment groups [23]. In addition, the potency of
166
ammonium group-functionalized multi-walled carbon nanotubes (MWCNTs) could enhance the
167
transfection and expression efficiency of plasmid DNA (pEGFP-vp5) in Ctenopharyngodon idellus
168
kidney (CIK) cells [39]. SWCNTs was also found to facilitate higher pDNA uptake and gene expression
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ACCEPTED MANUSCRIPT than pDNA alone in Chinese hamster ovary cells [40]. In the challenge test, the result reflected that
170
pEGFP-aerA DNA vaccine could protect grass carp against A. hydrophila infection, with a lower
171
percentage of mortality rates in 15 days. Further, SWCNTs- pEGFP-aerA DNA vaccine showed a better
172
protection efficacy on grass carp than pEGFP-aerA, and naked plasmid vaccine did not enhance innate
173
antibacterial immunity of fish as the control group. These results were in agreement with the previous
174
findings that SWCNTs can improve the plasmid/antigen stability, and prolong the time of plasmid/antigen
175
degradation [23].
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In the past, most research about DNA vaccines against bacterial pathogens in aquaculture have been
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reported, and these vaccine were demonstrated to be effective against challenges of the respective
178
pathogens [41-44]. In our tests, the levels of specific antibody increased significantly and persisted up to
179
6 weeks of post immunization. It might be that the SWCNTs-pEGFP-aerA could be delivered into the fish
180
cells, and the plasmid DNA was released from the nanotubes, and thus expressed antigen. With the
181
increase of amounts of plasmid DNA released from SWCNTs, more antigens were expressed and stronger
182
immune response was induced.
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In the present study, we compared the expressions of immune-related genes of free pEGFP-aerA and
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SWCNTs-pEGFP-aerA DNA vaccine on grass carp via intramuscular injection immunization. The results
185
indicated that fish immunization with SWCNTs-pEGFP-aerA DNA vaccine augmented the production of
186
specific antibodies and exhibited a high level of survival rate compared with pEGFP-aerA alone. Herein,
187
consistent with the production of specific antibodies (Fig. 2), the IgM expression was increased
188
significantly in vaccinated fish kidney. It is known that IgM is a major component of the humoral immune
189
system of teleost fish, regarded as the first antibody [45,46]. Some investigators have reported that the
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ACCEPTED MANUSCRIPT IgM expression would be intensively increase in many tissues and organs from the second week after
191
immunization and maintained almost one month [47]. Besides, IL-8, IFN-I and TNFα, an important
192
component in innate immunity and the inflammatory, were also found up-regulated by the DNA vaccine
193
treatment in this study. These genes were considered to be an important component in innate immunity
194
and antivirus response in fish [48-50], and especially TNFα is considered as an important
195
pro-inflammatory factors to induce the inflammatory response by regulating the expression of other
196
cytokines [51]. Thus, the risen gene levels may reflect the initiation of specific adaptive immune
197
responses [52]. Additionally, CRP levels were observed to increase in grass carp vaccinated with
198
pEGFP-aerA/SWCNTs-pEGFP-aerA DNA vaccines, while CRP usually served as the major acute phase
199
reactant and played an important role in the innate immune response to an inflammatory stimulus in fish
200
[53]. Since MHC I are involved in offering antigenic peptides for recognition by the TCR/CD8 complex
201
of cytotoxic T lymphocytes and CD8 molecules acts as a coreceptor and lies in its active role in activating
202
T cells [6,54,55], their up-regulation indicated that the protective effect of the DNA vaccines might be
203
due to the induction of humoral and cellular immune response.
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Currently, it is an important consideration on safety in preparing the vaccine on fish. Even though a
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series of researches have assessed toxicity of raw carbon nanotubes in mice, no significant effect was
206
found in cell culture experiments [56]. For fish embryos and larvae, Zhu et al. found that a series of
207
tested index were significantly changed when the concentration of SWCNTs was above 50 mg/L by bath
208
exposure [57]. However, there was no report about the toxicity of CNTs in fish by intramuscular injection,
209
and the information was lack in relative action mechanisms. Therefore, toxicity assessment on aquatic
210
animals of SWCNTs is also needed to make further studies.
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ACCEPTED MANUSCRIPT In conclusion, our results showed that SWCNTs loaded with DNA vaccine induced a better
212
protection to juvenile grass carp against A. hydrophlia. Moreover, SWCNTs conjugated with DNA
213
vaccine provided significantly protective immunity compared with free DNA vaccine. Thereby, SWCNTs
214
may be considered as a potential efficient DNA vaccine carrier to enhance the immunological activity.
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This work was supported by the Scientific Innovation and Achievements Transformation Fund for Northwest A&F University (XNY2013-25).
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ACCEPTED MANUSCRIPT Figure captions
361
Fig. 1. Analysis of aerA expression. (A) RT-PCR amplification of aerA: lane M, DNA marker; lane 1,
362
aerA. (B) analysis of recombinant plasmid: lane M, DNA marker; lane 1, double enzymes digested
363
pMD19T-aerA with Xho I and BamH I; lane 2, pMD19T-aerA. (C) analysis of recombinant plasmid: lane
364
M, DNA marker; lane 1, pEGFP-aerA; lane 2, double enzymes digested pEGFP-aerA with Xho I and
365
BamH I.
366
Fig. 2. Specific antibody levels of fish vaccinated with DNA vaccine were determined by ELISA. Data
367
are means for three assays and presented as the means ± SE. **P < 0.01; *P < 0.05.
368
Fig. 3. Real-time PCR analysis of the expression of immune-related genes in fish vaccinated with
369
different vaccine formulations after 21 d. (A) IL-8; (B) IFN-I; (C) TNFa; (D) CRP; (E) MHC-I; (F) IgM;
370
(G) CD8α. Data are means for three assays and presented as the means ± SE. **P < 0.01; *P < 0.05.
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Fig. 4. Cumulative mortalities of vaccinated fish. The accumulated mortalities were calculated at the end
372
of the monitored period.
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ACCEPTED MANUSCRIPT Table 1 Sequences of primer pairs used for the analysis of mRNA expression by qRT-PCR.
Product size (bp)
GenBank accession no.
Forward
ATTTCCGACACGGAGAGG
90
EU047719
Reverse
CATGGGTTTAGGATACGCTC
Forward
GGTGAAGTTTCTTGCCCTGACCTTAG
173
AB196166
Reverse
CCTTATGTGATGGCTGGTATCGGG
TNF-α
Forward
TGTGCCGCCGCTGTCTGCTTCACGCT
291
EU047718
Reverse
GATGAGGAAAGACACCTGGCTGTAGA
CRP
Forward
CTGCCTCCGCTCTCCATCTT
300
FJ547474
Reverse
TCCCTTTGGCACATACGGTTCCTGA
IL-8
Forward
AGGTCTGGGTGTAGATCCACGCTG
Reverse
TTAGTGTGAAAACTAACATGATCTCT
IgM
Forward
GCTGAGGCATCGGAGGCACAT
MHC-I
Reverse
TTGGGTCTCGCACCATTTTCTC
Forward
CCTGGCAGAAAAATGGACAAG
Reverse
CCAACAACACCAATGACAATC
Forward
GAGTCTCTGCACGGATCTAT
Reverse
GTGTAGTGTTCCGAATTTAAGT
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CD8α
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IGF-I
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EU047717
170
DQ417927
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Primer sequences (from 5' to 3')
Genes
271
AY391782
172
GQ355586
ACCEPTED MANUSCRIPT Table 2 Corresponding RPS values of vaccinated and control fish. Cumulative percentage mortality (%) and calculated relative percentage survival (RPS) following challenge with A. hydrophila in experimental
Fish injected with
Cumulative mortality (21 d)
PBS
100.0%
pEGFP 10 µg
100.0%
pEGFP-aerA 1 µg
92.1%
pEGFP-aerA 5 µg
70.7%
pEGFP-aerA 10 µg
54.9%
SWCNTs-pEGFP-aerA 1 µg
42.7%
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7.9%
29.3%
45.1%
57.3%
30.4%
69.6%
16.3%
83.7%
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RPS (21 d)
—
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groups of DNA vaccinated fish by intramuscular injection.
ACCEPTED MANUSCRIPT
M
1
B
M
1
2
M
1
2
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A
Fig. 1.
ACCEPTED MANUSCRIPT
0.7
**
PBS
**
**
** **
**
0.5 0.4
**
**
0.3
** **
**** **
** **
**
** ** ** **
**
**
** **
**
**
** **
pEGFP-aerA 1 µg pEGFP-aerA 5 µg pEGFP-aerA 10 µg SWCNTs-pEGFP-aerA 1 µg
*
*
SWCNTs-pEGFP-aerA 5 µg
0.2
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Absorbance (OD450)
**
0.6
SWCNTs-pEGFP-aerA 10 µg
0.1
pEGFP
0 1
2
3
4
6
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Weeks post-vaccination
5
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Fig. 2.
ACCEPTED MANUSCRIPT A
4
pEGFP-aerA
IL-8
**
Fold change
SWCNTs-pEGFP-aerA
3
** *
2
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1 0 PBS
pEGFP
*
*
1
5
10
Fold change
4
pEGFP-aerA
IFN-I
SWCNTs-pEGFP-aerA
3
**
2
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PBS
pEGFP-aerA
AC C
5 4
**
*
0
Fold change
**
**
1
C
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5
B
SC
Injected dose (µg)
1
5
10
Injected dose (µg)
TNFα
**
**
SWCNTs-pEGFP-aerA
**
3
**
*
2 1 0
PBS
pEGFP
1
5 Injected dose (µg)
10
ACCEPTED MANUSCRIPT 6
Fold change
5
pEGFP-aerA
CRP
**
SWCNTs-pEGFP-aerA
**
4 3
**
*
2
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1 0 PBS
pEGFP
1
5
10
pEGFP-aerA
MHC-I
Fold change
SWCNTs-pEGFP-aerA
6 4
**
**
12
pEGFP
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PBS
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AC C
pEGFP-aerA
1
**
**
**
2
F
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E
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Injected dose (µg)
5
10
Injected dose (µg)
IgM
**
Fold change
SWCNTs-pEGFP-aerA
9
**
6
**
** **
3 0 PBS
pEGFP
1
5 Injected dose (µg)
10
ACCEPTED MANUSCRIPT 5
Fold change
4
pEGFP-aerA
CD8α
**
SWCNTs-pEGFP-aerA
**
3
**
**
2 1 0 PBS
pEGFP
1
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G
5
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Injected dose (µg)
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Fig. 3.
10
100 90 80 70 60 50 40 30 20 10 0
PBS pEGFP 10 µg pEGFP-ae rA 1 µg pEGFP-ae rA 5 µg pEGFP-ae rA 10 µg SWC NTs-pEGFP-ae rA 1 µg SWC NTs-pEGFP-ae rA 5 µg SWC NTs-pEGFP-ae rA 10 µg
3
6
9
12
Days after challenge
18
21
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Fig. 4.
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Cumulative mortality (%)aaa
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ACCEPTED MANUSCRIPT Highlights: 1. SWCNTs can be used as carriers for DNA vaccine. 2. SWCNTs-DNA vaccine enhanced the immunological activity.
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3. SWCNTs-DNA vaccine induced a better protection to grass carp against A. hydrophlia.