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Construction and analysis of the immune effect of Vibrio harveyi subunit vaccine and DNA vaccine encoding TssJ antigen
Journal Pre-proof Construction and analysis of the immune effect of Vibrio harveyi subunit vaccine and DNA vaccine encoding TssJ antigen Yun Sun, Susu Ding, Mingwang He, Anzhu Liu, Hao Long, Weiliang Guo, Zhenjie Cao, Zhenyu Xie, Yongcan Zhou PII:
S1050-4648(19)31218-5
DOI:
https://doi.org/10.1016/j.fsi.2019.12.079
Reference:
YFSIM 6721
To appear in:
Fish and Shellfish Immunology
Received Date: 11 October 2019 Revised Date:
24 December 2019
Accepted Date: 26 December 2019
Please cite this article as: Sun Y, Ding S, He M, Liu A, Long H, Guo W, Cao Z, Xie Z, Zhou Y, Construction and analysis of the immune effect of Vibrio harveyi subunit vaccine and DNA vaccine encoding TssJ antigen, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/ j.fsi.2019.12.079. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Construction and analysis of the immune effect of Vibrio
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harveyi subunit vaccine and DNA vaccine encoding TssJ
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antigen
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Yun Suna,b,1, Susu Dinga,b,1, Mingwang Hea,b, Anzhu Liub, Hao Longa, Weiliang Guoa,b,
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Zhenjie Caoa,b, Zhenyu Xie a,b,*, Yongcan Zhoua,b,* *
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a
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P.R. China
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b
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Marine Science, Hainan University, P.R. China
State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University,
Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of
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1
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* To whom correspondence should be addressed
These authors contributed equally to this work.
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Mailing address:
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College of Marine Sciences
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Hainan University
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58 Renmin Avenue
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Haikou 570228
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PR ChinaPhone and Fax: 86-898-66256125
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Email: [email protected] (Z.Y. Xie) [email protected] (Y.C. Zhou)
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Abstract
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Vibrio harveyi, a severe pathogen infects different kinds of sea animals, causes huge
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economic loss in aquaculture industry. In order to control the Vibriosis disease caused mainly
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by V. harveyi and other Vibrio spp., the best solution lies in developing corresponding
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efficient vaccines. In this study, we have cloned and analyzed a putative antigen TssJ from the
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T6SS of V. harveyi, which has the potential as a vaccine against infection. The sequence
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analysis and western blotting experiments indicated that TssJ anchored in outer membrane
38
and there were several antigenic determinants existed on its extracellular region. Two forms
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of universal vaccines, subunit vaccine and DNA vaccine, were developed based on TssJ and
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applied in Trachinotus ovatus. The results showed that both of the two vaccines could
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generate a moderate protection in fish against V. harveyi. The relative percentage survival
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(RPS) of subunit vaccine and DNA vaccine were 52.39% and 69.11%, respectively.
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Immunological analysis showed both subunit vaccine and DNA vaccine enhanced acid
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phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme activities. Specific
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serum antibodies against TssJ in the fish vaccinated with subunit vaccine was much higher
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than that in the DNA vaccine group. Several immune-related genes, i.e., IL10, C3, MHC Iα,
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MHC IIα, and IgM, were induced both by the two forms of vaccines. TNFα and Mx were
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only upregulated in the DNA vaccine group. However, the induction levels of these genes
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induced by DNA vaccine were higher than subunit vaccine. All these findings suggested that
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TssJ from V. harveyi had a potential application value in vaccine industry.
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Keywords: TssJ, DNA vaccine, subunit vaccine, RPS
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1. Introduction
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Bacterial pathogens are one of the major causes of aquaculture diseases. Among the
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pathogens, Vibrio harveyi, a Gram-negative halophilic bacterium, is recognized as a severe
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pathogen in aquaculture. Vibrio harveyi infects many commercially farmed marine
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invertebrate and vertebrate, including thunberg (Crassostrea gigas), pearl oyster (Pinctada
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maxima), tiger prawn (Penaeus monodon), short sunfish (Mola mola), greasy grouper
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(Epinephelus tauvina), brill (Colistium guntheri), Japanese flounder (Paralichthys olivaceus),
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seabass (Lates calcarifer), et al [1-7]. The disease caused by Vibrio spp. and termed as
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Vibriosis is often fatal and can lead to heavy economic losses under adverse conditions such
63
as high temperature and heavy stocking density. In China, Vibriosis has become a major
64
constraint to fisheries production [8-11]. In our previous studies, we isolated one V. harveyi
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strain QT520 from a golden pompano (Trachinotus ovatus)-disease breakout and sequenced
66
and analyzed its genome [12].
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In aquaculture, vaccine is an effective preventive tool used in a health management
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strategy of aquatic organism for controlling infectious diseases [13]. To date, many vaccines
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against V. harveyi were developed. For example, an inactivated vaccine using formalin-killed
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cells of V. harveyi with adjuvant Montanide™ ISA 763 AVG provided good protection against
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V. harveyi infection in turbot [14]. As well, subunit vaccines of conserved antigens, such as
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the outer membrane proteins K (OmpK) and U (OmpU), contributed to protective efficacy in
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fishes [15,16]. In addition, a DNA vaccine was constructed to express glutathione peroxidase
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and showed effective immune protection to orange-spotted grouper [17]. Different types of
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vaccines have been prepared and tested [17-19]. Among these vaccines, there are few reports
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on the development of vaccine against V. harveyi in T. ovatus.
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Since T6SS secretion system was found in V. cholerae in 2006, many studies have
78
focused on it [20,21]. It’s reported that there are 13 genes required to assemble a functional
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T6SS, among which, TssJ, TssK, TssL, and TssM were co-occur in most clusters and only
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TssJ was anchored in outer membrane (OM) [22,23]. In this study, the full-length sequence of
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TssJ was obtained from the V. harveyi strain QT520 and was predicted as a new candidate
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antigen.
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For V. harveyi, several purified recombinant subunit vaccines and DNA vaccines have
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been reported; however, there is no comparative study on these two forms of vaccines. In this
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study, we first identified a vaccine candidate, TssJ, from a pathogenic V. harveyi strain.
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Meanwhile, we first examined the immune effect of TssJ in two different forms, i.e., as a
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purified recombinant subunit vaccine and as a DNA vaccine. The protective effects and some
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immune parameters such as antibody titer, acid phosphatase, alkaline phosphatase, superoxide
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dismutase, and lysozyme activities in sera from vaccinated fish with two vaccine forms were
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studied. The aim of our study was to ascertain the immune responses induced by different
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vaccination conditions in T. ovatus.
92 93 94
2. Materials and methods
95 96
2.1. Bacterial strains and growth conditions
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Vibrio harveyi QT520 is a fish pathogen reported previously [12]. Escherichia coli DH5a
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and BL21 (DE3) were purchased from Takara, Dalian, China. Vibrio harveyi QT520 was
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cultured in Luria-Bertani broth (LB) medium at 28 °C and E. coli was cultured in LB medium
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at 37 °C.
101 102
2.2. Plasmid construction and preparation
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According to the genome of QT520 in our previous study, the coding sequence of the
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TssJ outside domain (residues 27–151), which was predicted by TMHMM, was amplified
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with the primer pair of TSSJ-F1/TSSJ-R1 (Table S1). The PCR product was ligated into the
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pEASY®-T1 Simple Cloning Vector (Transgen, Beijing, China). To construct pETTssJ, the
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recombined plasmid above was digested by restriction enzyme SmaI and then the fragment
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was inserted into pET32a [24]. And then, the correct recombinant plasmid pETTssJ was
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transformed to BL21 (DE3). The positive colony verified by PCR was cultured in LB broth at
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37 °C to an OD of approximately 0.7; subsequently, IPTG was added to the medium at a final
111
concentration of 0.5mM for culturing an additional 6 h. The cells were harvested by
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centrifugation. Proteins were purified, analyzed, and measured as reported previously [25].
113
To construct pCTssJ, the fragment digested by restriction enzyme SmaI from pEASY-T1
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recombined plasmid above was inserted into pCN3 [26] at the SmaI site. Thus, pCTssJ, which
115
expresses a His-tag, was constructed. The plasmids of pCTssJ were prepared by the EndoFree
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plasmid Kit (Tiangen, Dalian, China) and the purity was measured by spectrophotometer
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using the A260/280 and A260/230 absorbances.
118
[Table S1]
119 120
2.3. Western blot analysis of the subcellular localization
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Vibrio harveyi QT520 were grown in LB medium to an OD600 of 1 and harvested by
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centrifugation at 4 °C. Protein preparation of the outer membrane, periplasm, and cytoplasm
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were carried out as reported previously [27]. And then the samples obtained above were
124
subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose
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membranes (Amersham, Cambridge, UK). Immunoblotting was performed as described
126
previously [27] using anti-TssJ antibodies.
127 128
2.4. Agglutination assay
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Vibrio harveyi QT520 was first labeled with FITC. Briefly, QT520 was cultured at 30 °C
130
in LB medium and then diluted with 1mM FITC solution by anhydrous DMSO. The
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FITC-labeled QT520 was suspended in PBS, and then antisera against rTssJ (from
132
rTssJ-vaccinated mouse) and control sera (from PBS-injected mouse) were added to cell
133
suspension and incubated at 28 °C for 3 h, separately. After that, the mixtures were diluted
134
with PBS and were observed by microscopy.
135 136
2.5. Immunohistochemical analysis
137
To detect the TssJ protein produced from the DNA plasmids, immunohistochemistry was
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performed to examine the TssJ vaccine at the protein level. Briefly, muscle tissues were taken
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from fish which were vaccinated with pCTssJ at 7 days and the tissue samples were treated as
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reported previously [28]. And then the sections were incubated firstly with mouse anti-His
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monoclonal antibody and then were incubated with gold-labeled goat anti-mouse IgG (Bios,
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Beijing, P.R. China). After that, the sections were stained with uranyl acetate and lead citrate,
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and then were observed with a transmission electron microscope (GEM-1200, GEOL, Japan).
144 145
2.6. Vaccination
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Golden pompano (average weight, 18.5 g ± 3.2 g) were purchased from a commercial
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fish farm in Chengmai (Hainan province, China) and were acclimatized in fresh seawater for
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one week. The fish was randomly divided into five groups (110 fish/group). For vaccination
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with subunit vaccine, the protein was mixed with aluminum hydroxide and diluted with PBS
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to 200 µg/ml and injected intraperitoneally (i.p.) with 100 µl rTssJ or rTrx, respectively. For
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vaccination with DNA vaccine, the plasmids were diluted with PBS to 200 µg/ml and injected
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intramuscularly (i.m.) with 100 µl pCTssJ and pCN3, respectively. And fish injected with
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PBS were used as control group. At one month post-vaccination (p.v.), 75 fish were taken out
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from each group and challenged by i.p. injection with 100 µl of V. harveyi QT520
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in three parallel (25 fish/parallel), respectively. V. harveyi QT520 was cultured in LB medium
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to an OD600 of 0.7 and resuspended in PBS to 1 × 106 CFU/ml. For all vaccinations, the
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mortality rate of the fish was monitored for two weeks. Relative percent survival (RPS) was
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determined as follows: RPS = 100*(1 - (% mortality in vaccinated fish /% mortality in control
159
fish)). RPS values are reported here as the mean mortality results.
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2.7. Innate immune responses analysis After 1, 2, 3, and 4 weeks vaccination, three fish were selected randomly from each
163
group. Blood was sampled from each fish and centrifuged at 800 × g for 15 min at 4 °C to
164
collect serum. The non-specific immune responses analysis of acid phosphatase (ACP),
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alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) were
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performed by the kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).
167
168
2.8. Enzyme-linked immunosorbent assay (ELISA)
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To detect antibody titres, sera were collected at 3, 4, 5, 6, 7, and 8 weeks p.v. from three
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fish each point which were vaccinated with rTssJ, pCTssJ, rTrx, pCN3, or PBS. Briefly, a
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96-well ELISA plate was coated with rTssJ and placed at 4 °C overnight. The samples were
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washed by PBST for 3 times and then seal with skimmed milk powder at 37 °C for 2 hours.
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After washed by PBST, the sera were diluted 50-fold in PBST (0.1% Tween-20 in PBS) and
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added to the wells in triplicate and incubated at 37 °C for an hour. And then the wells were
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washed by PBST and incubated with diluted Goat anti mouse (IgG) antibody at 37 °C for an
176
hour. After colour reaction, the absorbance were read at 450nm by a microreader as described
177
previously [29].
178
179
2.9. qRT-PCR
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At 4 weeks, spleen was taken from the fish which was immune by rTssJ or pCTssJ and
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challenged with V. harveyi for 24 h. Total RNA extraction and cDNA synthesis were
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performed using Eastep® Super and Eastep® RT Master Mix Kit according to the
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manufacturer's instructions. qRT-PCR was carried out in an ABI 7300 Real-time Detection
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System (Applied Biosystems, Foster City, CA, USA) using SYBR ExScript qRT-PCR Kit
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(Takara, Dalian, China) as described previously [30]. Each assay was performed in triplicate
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with B2M as the control. The primers used for qRT-PCR were in Table S1. All data are given
187
in terms of relative mRNA, expressed as means plus or minus standard deviation of the means
188
(SD).
189 190
2.10. Statistical analysis
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All statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago,
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IL, USA). Differences in mortality rate, non-specific immune responses analysis, antibody
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titer, and transcription levels were analysed with ANOVA. In all cases, the significance level
194
was defined as P < 0.05 or P < 0.01.
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3. Results
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3.1. Sequence characterization of TssJ
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TssJ was cloned from the pathogenic V. harveyi QT520. The open reading frame (ORF)
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of TssJ is 456 bp and encodes a putative protein of 151 amino acid residues (GenBank
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accession no. MN510468). BLAST analyses showed that TssJ of QT520 shares 88.74%,
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86.75%, 78.15%, and 77.48% overall sequence identities with the type VI secretion system
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lipoprotein TssJ of V. alginolyticus, V. parahaemolyticus, V. hyugaensis, and V. jasicida
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(GenBank accession no. WP_005375933.1, WP_054576750.1, WP_045399867.1, and
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WP_104028255.1, respectively). TMHMM predicted that TssJ has an inside part (residues
206
1–6), a TM helix structure (residues 7–26), and an outside part (residues 27–151).
207
Consistently, a signal peptide consisting of the N-terminal 22 amino acid residues and a
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transmembrane region from residues 7 to 26 were identified in TssJ. Meanwhile, some
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probable antigenic epitopes were also predicted by prediction server.
210 211
3.2. Subcellular localization of TssJ
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The recombined proteins (rTssJ) were purified (Fig. S1) and the polyclonal antibody
213
were prepared. To examine the subcellular localization of TssJ, QT520 was cultured in LB
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medium to an OD600 of about 0.8, and then proteins from the outer membrane, periplasm, and
215
cytoplasm of bacteria cells were prepared. Western blot analysis was used for the subcellular
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localization. The results showed that TssJ was found in the preparations of the outer
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membrane and periplasm proteins but not in that of the cytoplasm (Fig. 1). To further examine
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whether TssJ was expressed on the outer membrane, QT520 was incubated with rat anti-rTssJ
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serum or preimmune serum, and was examined by microscope. Consistent with the western
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blot results, QT520 became agglutinated in the presence of anti-rTssJ antibodies (Fig. 2 B and
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D). In contrast, QT520 treated with preimmune serum showed no agglutination (Fig. 2 A and
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C). These results suggested that TssJ could locate and display on cell surface.
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[Fig. S1] [Fig. 1] [Fig. 2]
224 225 226
3.3. Expression of TssJ in fish tissues following vaccination Golden pompano were vaccinated with pCTssJ, pCN3, and PBS, respectively. To
227
examine the expression of TssJ protein in the vaccinated fish, immunocolloidal gold electron
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microscopy analysis was performed, which showed that production of TssJ protein was
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detected in the muscle tissue of pCTssJ-vaccinated fish, but not in that of pCN3-vaccinated
230
fish (Fig. 3) and PBS fish (data not show). [Fig. 3]
231 232 233
3.4. Immunoprotection induce by rTssJ and pCTssJ against V. harveyi challenge
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At one-month p.v., the fish vaccinated with rTssJ or pCTssJ were challenged with V.
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harveyi QT520, and the mortality was monitored for 14 days. The results showed the mean
236
survival rates for rTssJ-, pCTssJ-, rTrx-, pCN3-, and PBS-vaccinated fish were 73.33%,
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82.67%, 45.33%, 46.67%, and 40%, respectively (Fig. 4). Hence, the mean protective efficacy
238
of rTssJ and pCTssJ, in terms of RPS, were 52.39% and 69.11% with PBS as control. The
239
results showed an extremely significantly higher (P < 0.01) protection induced by pCTssJ than
240
rTssJ. [Fig. 4]
241 242 243
3.5. Innate immune responses analysis
244
Compared with the control group, significantly increased activity of ACP (P < 0.05) in
245
pCTssJ-vaccinated fish was observed at 2 weeks post-vaccination, as well as an
246
extremely significant increase in rTssJ-vaccinated fish (P < 0.01) was found. In addition, fish
247
vaccinated rTssJ exhibited significantly higher (P < 0.05) and pCTssJ extremely significantly
248
higher (P < 0.01) AKP activity at 1 week post-vaccination compared with the control group,
249
respectively. Moreover, the LZM activity of rTssJ-vaccinated fish was extremely significantly
250
higher (P < 0.01) than those of the control group at 2 and 3 weeks post-vaccination. Fish
251
vaccinated pCTssJ can extremely improve the LZM activity compare to that of the control
252
group at 1, 2, and 3 weeks post-vaccination (P < 0.01). Furthermore, at 1 week
253
post-vaccination, fish vaccinated with rTssJ and pCTssJ both exhibited significantly higher
254
SOD activity (P < 0.05) than that fish vaccinated PBS (Fig. 5). [Fig. 5]
255 256 257
3.6. Serum antibody production
258
ELISA analysis showed that both rTssJ and pCTssJ induced the production of specific
259
serum antibodies at 3 to 8-week p.v., which indicated that the two forms of vaccines could
260
elicit the adaptive immunity. The highest antibody level induced by rTssJ occurred at 5-week
261
p.v., but occurred at 4-week induced by pCTssJ (Fig. 6). Meanwhile, the antibody level
262
inducted by rTssJ was much higher than that in pCTssJ-vaccinated fish at each of the
263
examined time point. [Fig. 6]
264 265 266
3.7. Expression of immune-related genes
267
To examine the expression of immune genes induced by rTssJ and pCTssJ, qRT-PCR
268
analysis was carried out. The transcription of the genes encoding interleukin 10 (IL10), tumor
269
necrosis factor-alpha (TNFα), interferoninduced Mx protein (Mx), complement C3 (C3),
270
major histocompatibility complex (MHC) class Iα and class IIα, immunoglobulin M (IgM)
271
and D (IgD), and CD8α were examined. The results showed that in rTssJ-vaccinated fish,
272
IL10, C3, MHC Iα, MHC IIα, and IgM were induced to significant (P < 0.01 or P < 0.05)
273
extents (Fig. 7). In pCTssJ -vaccinated fish, all genes except IgD and CD8α were significantly
274
(P < 0.01 or P < 0.05) induced, and the induction levels of seven of these genes, i.e., IL10,
275
TNFα, Mx, C3, MHC Iα, MHC IIα, and IgM, were higher than that were induced by rTssJ
276
(Fig. 7). [Fig. 7]
277 278 279 280
4. Discussion
281
Vibrio harveyi is a severe aquaculture pathogen with a broad host range and has become
282
a major constraint to fisheries production. As we all know, vaccination is an effective
283
preventive tool which serves as a health management strategy in aquaculture, among which
284
subunit vaccines and DNA vaccines are two important forms of vaccine [13]. In this study, we
285
described the identification of a new V. harveyi antigen TssJ and constructed subunit vaccine
286
and DNA vaccine based on it. We examined and comparatively analysed the vaccine potential
287
and the immune effect induced by the two different forms.
288
In Gram-negative bacteria, the type VI secretion system (T6SS) is discovered as a new
289
type of secretion system for translocation proteins and is essential for virulence [21,23,31].
290
T6SS has been reported in many bacteria, such as, Escherichia coli, Edwarsiella tarda, Vibrio
291
cholerae, Agrobacterium tumefaciens, Pseudomonas aeruginosa, et al [20,21,32]. Previous
292
studies reported that T6SS contains 13 genes which are required to play its roles [23]. Among
293
these 13 genes, TssJ, TssK, TssL and TssM were co-occur in most clusters, with the exception
294
of TssK that locates in the cytoplasm, TssJ, TssL and TssM fractionate with the membranes
295
[23]. In enteroaggregative E. coli (EAEC), TssJ was identified as an outer membrane protein
296
that bears a typical lipobox sorting motif [33-35]. Likewise, TssJ protein was solved to be
297
anchored in outer membrane in Serratia marcescens and P. aeruginosa [31,36]. Similar to the
298
current study, our results of western immunoblot analysis showed that TssJ in V. harveyi
299
QT520 was found in the preparations of the outer membrane and periplasm proteins.
300
Consistent with the western blot results, QT520 became agglutinated in the presence of
301
anti-rTssJ antibodies, suggesting that TssJ in V. harveyi may tether to the outer membrane.
302
These results indicate that TssJ from different bacterial species may be conserved at certain
303
structural and can be used as a novel antigenic candidate for vaccines.
304
Though no magic ways can avoid fish infection of V. harveyi, several control methods
305
including chemotherapy, preventive measures, and vaccines were performed to prevent or
306
reduce the infection [13,37,38] . Studies in previous studies showed that subunit vaccines
307
have to be co-formulated with adjuvants to recall pre-existing immunity and to elicit a
308
balanced immune response [39,40]. In the present study, we vaccinated fish with rTssJ which
309
was mixed with aluminum hydroxide. After a one-month vaccination period, the fish were
310
challenged with V. harveyi QT520 to examine the protective effects as a subunit vaccine.
311
Compared with the PBS group, fish vaccinated with rTssJ exhibited RPS of 52.39%. To
312
investigate whether rTssJ could induce an effective immunity, we analysed some serum
313
parameters, which are usually used to monitor the innate immunity of fish [41]. AKP and ACP
314
are important components of animal lysosomal enzymes, which play important roles in the
315
immune response. In the vertebrate, the increase of AKP and ACP activities indicates
316
increased immunity and is often regarded as a marker for macrophage activation of immune
317
system [42]. LZM is also an important member of lysosomal enzymes, which can lyse and
318
digest pathogenic microorganism. SOD is one of the key antioxidant enzymes for scavenging
319
free radicals, which plays a vital role in the body's oxidation and antioxidant balance [43]. In
320
this study, comparing to control fish, fish vaccinated with rTssJ showed stronger AKP, ACP,
321
LZM, and SOD activities, suggesting that rTssJ could enhance the activation of lysosomal and
322
antioxidant enzymes. Subunit vaccines have been found to provoke not only innate immunity
323
but also adapt immunity in mammals and fish [44-48]. In flounders, outer membrane protein
324
(Omp) K, OmpR, and VAA have been identified with good immunogenicity from V.
325
anguillarum, and their recombinant proteins could induce the proliferation of CD3+ T or sIg+
326
B lymphocytes and the specific antibodies [49-51]. For example, in OmpK group, antibody
327
titer was significantly increased at 2-weeks, and then enhanced steadily and peaked at
328
5-weeks immunization [49]. Similarly, our results showed the specific antibody induced by
329
rTssJ from 3- to 8-weeks vaccination and the highest antibody level occurred at 5-week p.v.,
330
demonstrating that rTssJ could elicit the adapt immunity of fish. In addition, our qRT-PCR
331
analysis revealed an enhanced range of gene expression in fish vaccinated with rTssJ,
332
including IL10, C3, MHC Iα, MHC IIα, and IgM. All of these results suggested that rTssJ
333
could stimulate the innate and adaptive immunity of fish.
334
In case of pCTssJ, the RPS was 69.11%, which was extremely significantly higher than
335
that induced by rTssJ. Like the subunit vaccine (rTssJ), fish injected with pCTssJ also showed
336
stronger AKP, ACP, LZM, and SOD activities when comparing to the control fish. But the
337
results showed no significance between pCTssJ- and rTssJ- immunitied fish, suggesting that
338
pCTssJ and rTssJ elicited similar innate immune responses. It’s well known that lots of V.
339
harveyi vaccines can induce specific serum antibody production [19,49]. In previous studies,
340
Hu et al. found that pDegQ and pVhp1 exhibited respectively, 64.1% and 56.5% relative
341
percent survival rates following lethal V. harveyi challenge, and antibody production was
342
detected [19]. In this study, we found that both rTssJ and pCTssJ could induce serum antibody
343
production. However, they have different patterns of serum antibody production, which in
344
pCTssJ-vaccinated and rTssJ-vaccinated fish peaked at approximately 4-week p.v. and 5-week
345
p.v., respectively. And the actual antibody levels were much higher in rTssJ-vaccinated fish
346
than in pCTssJ-vaccinated fish. Compared to other types of vaccines, DNA vaccine possesses
347
the advantage of being able to elicit systemic immune response of both the humoral and the
348
cellular arms [52-54]. In Japanese flounder, pDegQ and pVhp1 induced higher expression of
349
IgM, MHC Iα, MHC IIα, CD8α, C3, IL-1β, IFN, TNFα, and Mx [19]. In line with this, our
350
qRT-PCR results showed after 4-week p.v. and challenged with V. harveyi, with the exception
351
of IgD and CD8α, whose expression was not affected, all other examined immune genes i.e.,
352
IL10, TNFα, Mx, C3, MHC Iα, MHC IIα, and IgM, were upregulated by pCTssJ. However,
353
the induction folds induced by pCTssJ were higher than those induced by rTssJ, which
354
probably accounts for the much stronger protective effect observed in pCTssJ than rTssJ. The
355
expression of immune factors, which included those participating in specific and nonspecific
356
immunities, indicated that DNA vaccines might induce a wider range of quick and efficient
357
immune response.
358
In conclusion, we constructed and analysed the immune effect of a V. harveyi subunit
359
vaccine and a DNA vaccine encoding TssJ antigen, demonstrating that TssJ is a protective
360
immunogen that could induce different immune responses as both forms of vaccines. DNA
361
vaccine plasmids administered via i.m. injection are transported to internal tissues, thus it
362
might be easier to induce the humoral and the cellular immune response compared to subunit
363
vaccine. However, the specific antibodies elicited by purified recombinant TssJ are much
364
stronger than that induced by TssJ as a DNA vaccine. These results suggest a stable and
365
efficient immune response is important for protection and TssJ may be an effective candidate
366
antigen against V. harveyi infection in aquaculture.
367 368
Acknowledgements
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This research was supported financially by the National Natural Science Foundation of
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China (No. 31702379 and No. 31660744), the Key Research and Development Projects of
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Hainan Province (No. ZDYF2018109), and the Nanhai Famous Youth Project.
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Figure legends
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Fig. 1. Western immunoblot analysis of the subcellular localization of TssJ in Vibrio harveyi.
545
Proteins of the outer membrane (lane 1), periplasm (lane 2), and cytoplasm (lane 3) were
546
resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-TssJ
547
antibodies. Lane 1, protein markers.
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Fig. 2. Effect of rTssJ on agglutination of Vibrio harveyi. Vibrio harveyi QT520 was first
551
labeled with FITC. The cells were incubated with antisera against PBS (A and C) or TssJ (B
552
and D). Agglutination was observed by light fields (A and B) and fluorescence fields (C and
553
D). Bar = 10 µm.
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Fig. 3. Production of TssJ protein in pCTssJ-vaccinated fish. Muscle tissues were taken from
557
golden pompano vaccinated with pCN3 (A) and pCTssJ (B) at 7 days post-vaccination and
558
used for immunohistochemistry with gold-labeled antibodies. Arrows indicate gold particles.
559
Bar = 200 nm.
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Fig. 4. Survival of vaccinated fish. Golden pompano vaccinated with rTssJ, pCTssJ, rTrx,
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pCN3 or PBS (control) were challenged with Vibrio harveyi and monitored daily for survival.
566
The results are one representative vaccination trial. *P < 0.05, **P < 0.01.
567 568 569
Fig. 5. Effect of rTssJ and pCTssJ on innate immune responses analysis in golden pompano.
570
Serum acid phosphatase (ACP) activity (A), alkaline phosphatase (AKP) activity (B), serum
571
lysozyme (LZM) activity (C), serum superoxide dismutase (SOD) activity (D).
572
Data are presented as means ± standard deviation (SD, N= 3). *P < 0.05, **P < 0.01.
573 574 575
Fig. 6. Serum antibody production in vaccinated fish. Golden pompano were vaccinated with
576
rTssJ, pCTssJ or PBS (control). Sera were collected from the fish at 3-8 weeks
577
post-vaccination, and serum antibodies against TssJ were determined by ELISA. Data are
578
presented as means ± standard deviation (SD, N= 3). *P < 0.05, **P < 0.01.
579 580 581
Fig. 7. qRT-PCR analysis of the expression of immune genes in vaccinated fish. Golden
582
pompano were vaccinated with rTssJ, pCTssJ or PBS (control) and challenged with V. harveyi.
583
Total RNA was extracted from spleen at 24 h post-challenge and used for qRT-PCR. For each
584
gene, the mRNA level of the fish vaccinated with PBS was set as 1. Data are presented as
585
means ± standard deviation (SD, N= 3). *P < 0.05, **P < 0.01.
586 587 588 589 590 591 592 593 594 595 596 597 598
599 600
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Fig. 4.
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Fig. 5.
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Fig. 6.
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Fig. 7.
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Supplementary data
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Table S1
709
Primers used in this study. Primer name
Primer sequence (5′-3′)
Amplification target
TssJ-F1 TssJ-R1 B2M-F B2M-R IL10-F IL10-R TNFα-F TNFα-R
CCCGGGATGGACCCATATTCAGATTTAG CCCGGGTTTTACGACCAAAGACAGAT AAGTCAGTCCACCCAAGGTTCA GGGATTTCCATTCCGTTCTTCATG GATATCGCCACCATGTCTGCGGCTGTGAAT GATATCGACACACATGTCTACAATGC GGCGTCGTTCAGAGTCTCCT TCCTCCTGGGCAGTGGTTT CCAGGAGCATGAGGAAGAGAT AGGTCAATGAGCGTCAGGTCT CGCCGCTCCCTCTACATCA GACAGCCACCCAACCATTACG ACTGGACCTTTCATCAGTCAAACCTG GGACGCTTGGCACTCTTCTTTCT GGCGTGAAGCCATGAAGGACAT AGACTTTGACGGGAGCAGGGTAGA AGAAATGGCTGACTCCTCAATGA CCCGCCTTCCTTTCATAGAG GCAAAGCACCGATACTCTAACC AATATCCAGAAACTAGGCAAGTAAGT CCCGCAGAGTTCGACAGCA CAGGTTGGGTTTGACGGTGAT
Outside part cloning Outside part cloning qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR
Mx-F Mx-R C3-F C3-R MHC Iα-F MHC Iα-R MHC IIα-F MHC IIα-R IgM-F IgM-R IgD-F IgD-R CD8α-F CD8α-R 710 711 712 713 714 715 716 717
Notes: The underlined bases indicate the SmaI restriction enzyme sites that were added to the 5’ end of primers.
718 719 720
Fig. S1. SDS-PAGE analysis of rTssJ and rTrx. Protein were analyzed by SDS-PAGE and
721
viewed after staining with Coomassie brilliant blue R-250. M: protein molecular weight
722
marker; lane 1, Induced rTssJ; lane 2, Purified rTssJ; lane 3, Purified rTrx.
723
Highlights: 1. Subunit vaccine and DNA vaccine were constructed based on the TssJ gene of Vibrio harveyi for Trachinotus ovatus against V. harveyi. 2. Both innate and adaptive immune responses were induced in Trachinotus ovatus after vaccination with subunit vaccine and DNA vaccine. 3. Both of subunit vaccine and DNA vaccine could induce high levels of protection against Vibrio harveyi for Trachinotus ovatus.
S1050-4648(19)31218-5
DOI:
https://doi.org/10.1016/j.fsi.2019.12.079
Reference:
YFSIM 6721
To appear in:
Fish and Shellfish Immunology
Received Date: 11 October 2019 Revised Date:
24 December 2019
Accepted Date: 26 December 2019
Please cite this article as: Sun Y, Ding S, He M, Liu A, Long H, Guo W, Cao Z, Xie Z, Zhou Y, Construction and analysis of the immune effect of Vibrio harveyi subunit vaccine and DNA vaccine encoding TssJ antigen, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/ j.fsi.2019.12.079. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Construction and analysis of the immune effect of Vibrio
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harveyi subunit vaccine and DNA vaccine encoding TssJ
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antigen
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Yun Suna,b,1, Susu Dinga,b,1, Mingwang Hea,b, Anzhu Liub, Hao Longa, Weiliang Guoa,b,
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Zhenjie Caoa,b, Zhenyu Xie a,b,*, Yongcan Zhoua,b,* *
6
a
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P.R. China
8
b
9
Marine Science, Hainan University, P.R. China
State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University,
Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of
10 11
1
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* To whom correspondence should be addressed
These authors contributed equally to this work.
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Mailing address:
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College of Marine Sciences
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Hainan University
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58 Renmin Avenue
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Haikou 570228
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PR ChinaPhone and Fax: 86-898-66256125
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Email: [email protected] (Z.Y. Xie) [email protected] (Y.C. Zhou)
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Abstract
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Vibrio harveyi, a severe pathogen infects different kinds of sea animals, causes huge
33
economic loss in aquaculture industry. In order to control the Vibriosis disease caused mainly
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by V. harveyi and other Vibrio spp., the best solution lies in developing corresponding
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efficient vaccines. In this study, we have cloned and analyzed a putative antigen TssJ from the
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T6SS of V. harveyi, which has the potential as a vaccine against infection. The sequence
37
analysis and western blotting experiments indicated that TssJ anchored in outer membrane
38
and there were several antigenic determinants existed on its extracellular region. Two forms
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of universal vaccines, subunit vaccine and DNA vaccine, were developed based on TssJ and
40
applied in Trachinotus ovatus. The results showed that both of the two vaccines could
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generate a moderate protection in fish against V. harveyi. The relative percentage survival
42
(RPS) of subunit vaccine and DNA vaccine were 52.39% and 69.11%, respectively.
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Immunological analysis showed both subunit vaccine and DNA vaccine enhanced acid
44
phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme activities. Specific
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serum antibodies against TssJ in the fish vaccinated with subunit vaccine was much higher
46
than that in the DNA vaccine group. Several immune-related genes, i.e., IL10, C3, MHC Iα,
47
MHC IIα, and IgM, were induced both by the two forms of vaccines. TNFα and Mx were
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only upregulated in the DNA vaccine group. However, the induction levels of these genes
49
induced by DNA vaccine were higher than subunit vaccine. All these findings suggested that
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TssJ from V. harveyi had a potential application value in vaccine industry.
51 52
Keywords: TssJ, DNA vaccine, subunit vaccine, RPS
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1. Introduction
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Bacterial pathogens are one of the major causes of aquaculture diseases. Among the
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pathogens, Vibrio harveyi, a Gram-negative halophilic bacterium, is recognized as a severe
57
pathogen in aquaculture. Vibrio harveyi infects many commercially farmed marine
58
invertebrate and vertebrate, including thunberg (Crassostrea gigas), pearl oyster (Pinctada
59
maxima), tiger prawn (Penaeus monodon), short sunfish (Mola mola), greasy grouper
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(Epinephelus tauvina), brill (Colistium guntheri), Japanese flounder (Paralichthys olivaceus),
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seabass (Lates calcarifer), et al [1-7]. The disease caused by Vibrio spp. and termed as
62
Vibriosis is often fatal and can lead to heavy economic losses under adverse conditions such
63
as high temperature and heavy stocking density. In China, Vibriosis has become a major
64
constraint to fisheries production [8-11]. In our previous studies, we isolated one V. harveyi
65
strain QT520 from a golden pompano (Trachinotus ovatus)-disease breakout and sequenced
66
and analyzed its genome [12].
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In aquaculture, vaccine is an effective preventive tool used in a health management
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strategy of aquatic organism for controlling infectious diseases [13]. To date, many vaccines
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against V. harveyi were developed. For example, an inactivated vaccine using formalin-killed
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cells of V. harveyi with adjuvant Montanide™ ISA 763 AVG provided good protection against
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V. harveyi infection in turbot [14]. As well, subunit vaccines of conserved antigens, such as
72
the outer membrane proteins K (OmpK) and U (OmpU), contributed to protective efficacy in
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fishes [15,16]. In addition, a DNA vaccine was constructed to express glutathione peroxidase
74
and showed effective immune protection to orange-spotted grouper [17]. Different types of
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vaccines have been prepared and tested [17-19]. Among these vaccines, there are few reports
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on the development of vaccine against V. harveyi in T. ovatus.
77
Since T6SS secretion system was found in V. cholerae in 2006, many studies have
78
focused on it [20,21]. It’s reported that there are 13 genes required to assemble a functional
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T6SS, among which, TssJ, TssK, TssL, and TssM were co-occur in most clusters and only
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TssJ was anchored in outer membrane (OM) [22,23]. In this study, the full-length sequence of
81
TssJ was obtained from the V. harveyi strain QT520 and was predicted as a new candidate
82
antigen.
83
For V. harveyi, several purified recombinant subunit vaccines and DNA vaccines have
84
been reported; however, there is no comparative study on these two forms of vaccines. In this
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study, we first identified a vaccine candidate, TssJ, from a pathogenic V. harveyi strain.
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Meanwhile, we first examined the immune effect of TssJ in two different forms, i.e., as a
87
purified recombinant subunit vaccine and as a DNA vaccine. The protective effects and some
88
immune parameters such as antibody titer, acid phosphatase, alkaline phosphatase, superoxide
89
dismutase, and lysozyme activities in sera from vaccinated fish with two vaccine forms were
90
studied. The aim of our study was to ascertain the immune responses induced by different
91
vaccination conditions in T. ovatus.
92 93 94
2. Materials and methods
95 96
2.1. Bacterial strains and growth conditions
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Vibrio harveyi QT520 is a fish pathogen reported previously [12]. Escherichia coli DH5a
98
and BL21 (DE3) were purchased from Takara, Dalian, China. Vibrio harveyi QT520 was
99
cultured in Luria-Bertani broth (LB) medium at 28 °C and E. coli was cultured in LB medium
100
at 37 °C.
101 102
2.2. Plasmid construction and preparation
103
According to the genome of QT520 in our previous study, the coding sequence of the
104
TssJ outside domain (residues 27–151), which was predicted by TMHMM, was amplified
105
with the primer pair of TSSJ-F1/TSSJ-R1 (Table S1). The PCR product was ligated into the
106
pEASY®-T1 Simple Cloning Vector (Transgen, Beijing, China). To construct pETTssJ, the
107
recombined plasmid above was digested by restriction enzyme SmaI and then the fragment
108
was inserted into pET32a [24]. And then, the correct recombinant plasmid pETTssJ was
109
transformed to BL21 (DE3). The positive colony verified by PCR was cultured in LB broth at
110
37 °C to an OD of approximately 0.7; subsequently, IPTG was added to the medium at a final
111
concentration of 0.5mM for culturing an additional 6 h. The cells were harvested by
112
centrifugation. Proteins were purified, analyzed, and measured as reported previously [25].
113
To construct pCTssJ, the fragment digested by restriction enzyme SmaI from pEASY-T1
114
recombined plasmid above was inserted into pCN3 [26] at the SmaI site. Thus, pCTssJ, which
115
expresses a His-tag, was constructed. The plasmids of pCTssJ were prepared by the EndoFree
116
plasmid Kit (Tiangen, Dalian, China) and the purity was measured by spectrophotometer
117
using the A260/280 and A260/230 absorbances.
118
[Table S1]
119 120
2.3. Western blot analysis of the subcellular localization
121
Vibrio harveyi QT520 were grown in LB medium to an OD600 of 1 and harvested by
122
centrifugation at 4 °C. Protein preparation of the outer membrane, periplasm, and cytoplasm
123
were carried out as reported previously [27]. And then the samples obtained above were
124
subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose
125
membranes (Amersham, Cambridge, UK). Immunoblotting was performed as described
126
previously [27] using anti-TssJ antibodies.
127 128
2.4. Agglutination assay
129
Vibrio harveyi QT520 was first labeled with FITC. Briefly, QT520 was cultured at 30 °C
130
in LB medium and then diluted with 1mM FITC solution by anhydrous DMSO. The
131
FITC-labeled QT520 was suspended in PBS, and then antisera against rTssJ (from
132
rTssJ-vaccinated mouse) and control sera (from PBS-injected mouse) were added to cell
133
suspension and incubated at 28 °C for 3 h, separately. After that, the mixtures were diluted
134
with PBS and were observed by microscopy.
135 136
2.5. Immunohistochemical analysis
137
To detect the TssJ protein produced from the DNA plasmids, immunohistochemistry was
138
performed to examine the TssJ vaccine at the protein level. Briefly, muscle tissues were taken
139
from fish which were vaccinated with pCTssJ at 7 days and the tissue samples were treated as
140
reported previously [28]. And then the sections were incubated firstly with mouse anti-His
141
monoclonal antibody and then were incubated with gold-labeled goat anti-mouse IgG (Bios,
142
Beijing, P.R. China). After that, the sections were stained with uranyl acetate and lead citrate,
143
and then were observed with a transmission electron microscope (GEM-1200, GEOL, Japan).
144 145
2.6. Vaccination
146
Golden pompano (average weight, 18.5 g ± 3.2 g) were purchased from a commercial
147
fish farm in Chengmai (Hainan province, China) and were acclimatized in fresh seawater for
148
one week. The fish was randomly divided into five groups (110 fish/group). For vaccination
149
with subunit vaccine, the protein was mixed with aluminum hydroxide and diluted with PBS
150
to 200 µg/ml and injected intraperitoneally (i.p.) with 100 µl rTssJ or rTrx, respectively. For
151
vaccination with DNA vaccine, the plasmids were diluted with PBS to 200 µg/ml and injected
152
intramuscularly (i.m.) with 100 µl pCTssJ and pCN3, respectively. And fish injected with
153
PBS were used as control group. At one month post-vaccination (p.v.), 75 fish were taken out
154
from each group and challenged by i.p. injection with 100 µl of V. harveyi QT520
155
in three parallel (25 fish/parallel), respectively. V. harveyi QT520 was cultured in LB medium
156
to an OD600 of 0.7 and resuspended in PBS to 1 × 106 CFU/ml. For all vaccinations, the
157
mortality rate of the fish was monitored for two weeks. Relative percent survival (RPS) was
158
determined as follows: RPS = 100*(1 - (% mortality in vaccinated fish /% mortality in control
159
fish)). RPS values are reported here as the mean mortality results.
160 161 162
2.7. Innate immune responses analysis After 1, 2, 3, and 4 weeks vaccination, three fish were selected randomly from each
163
group. Blood was sampled from each fish and centrifuged at 800 × g for 15 min at 4 °C to
164
collect serum. The non-specific immune responses analysis of acid phosphatase (ACP),
165
alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) were
166
performed by the kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).
167
168
2.8. Enzyme-linked immunosorbent assay (ELISA)
169
To detect antibody titres, sera were collected at 3, 4, 5, 6, 7, and 8 weeks p.v. from three
170
fish each point which were vaccinated with rTssJ, pCTssJ, rTrx, pCN3, or PBS. Briefly, a
171
96-well ELISA plate was coated with rTssJ and placed at 4 °C overnight. The samples were
172
washed by PBST for 3 times and then seal with skimmed milk powder at 37 °C for 2 hours.
173
After washed by PBST, the sera were diluted 50-fold in PBST (0.1% Tween-20 in PBS) and
174
added to the wells in triplicate and incubated at 37 °C for an hour. And then the wells were
175
washed by PBST and incubated with diluted Goat anti mouse (IgG) antibody at 37 °C for an
176
hour. After colour reaction, the absorbance were read at 450nm by a microreader as described
177
previously [29].
178
179
2.9. qRT-PCR
180
At 4 weeks, spleen was taken from the fish which was immune by rTssJ or pCTssJ and
181
challenged with V. harveyi for 24 h. Total RNA extraction and cDNA synthesis were
182
performed using Eastep® Super and Eastep® RT Master Mix Kit according to the
183
manufacturer's instructions. qRT-PCR was carried out in an ABI 7300 Real-time Detection
184
System (Applied Biosystems, Foster City, CA, USA) using SYBR ExScript qRT-PCR Kit
185
(Takara, Dalian, China) as described previously [30]. Each assay was performed in triplicate
186
with B2M as the control. The primers used for qRT-PCR were in Table S1. All data are given
187
in terms of relative mRNA, expressed as means plus or minus standard deviation of the means
188
(SD).
189 190
2.10. Statistical analysis
191
All statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago,
192
IL, USA). Differences in mortality rate, non-specific immune responses analysis, antibody
193
titer, and transcription levels were analysed with ANOVA. In all cases, the significance level
194
was defined as P < 0.05 or P < 0.01.
195 196 197
3. Results
198
3.1. Sequence characterization of TssJ
199
TssJ was cloned from the pathogenic V. harveyi QT520. The open reading frame (ORF)
200
of TssJ is 456 bp and encodes a putative protein of 151 amino acid residues (GenBank
201
accession no. MN510468). BLAST analyses showed that TssJ of QT520 shares 88.74%,
202
86.75%, 78.15%, and 77.48% overall sequence identities with the type VI secretion system
203
lipoprotein TssJ of V. alginolyticus, V. parahaemolyticus, V. hyugaensis, and V. jasicida
204
(GenBank accession no. WP_005375933.1, WP_054576750.1, WP_045399867.1, and
205
WP_104028255.1, respectively). TMHMM predicted that TssJ has an inside part (residues
206
1–6), a TM helix structure (residues 7–26), and an outside part (residues 27–151).
207
Consistently, a signal peptide consisting of the N-terminal 22 amino acid residues and a
208
transmembrane region from residues 7 to 26 were identified in TssJ. Meanwhile, some
209
probable antigenic epitopes were also predicted by prediction server.
210 211
3.2. Subcellular localization of TssJ
212
The recombined proteins (rTssJ) were purified (Fig. S1) and the polyclonal antibody
213
were prepared. To examine the subcellular localization of TssJ, QT520 was cultured in LB
214
medium to an OD600 of about 0.8, and then proteins from the outer membrane, periplasm, and
215
cytoplasm of bacteria cells were prepared. Western blot analysis was used for the subcellular
216
localization. The results showed that TssJ was found in the preparations of the outer
217
membrane and periplasm proteins but not in that of the cytoplasm (Fig. 1). To further examine
218
whether TssJ was expressed on the outer membrane, QT520 was incubated with rat anti-rTssJ
219
serum or preimmune serum, and was examined by microscope. Consistent with the western
220
blot results, QT520 became agglutinated in the presence of anti-rTssJ antibodies (Fig. 2 B and
221
D). In contrast, QT520 treated with preimmune serum showed no agglutination (Fig. 2 A and
222
C). These results suggested that TssJ could locate and display on cell surface.
223
[Fig. S1] [Fig. 1] [Fig. 2]
224 225 226
3.3. Expression of TssJ in fish tissues following vaccination Golden pompano were vaccinated with pCTssJ, pCN3, and PBS, respectively. To
227
examine the expression of TssJ protein in the vaccinated fish, immunocolloidal gold electron
228
microscopy analysis was performed, which showed that production of TssJ protein was
229
detected in the muscle tissue of pCTssJ-vaccinated fish, but not in that of pCN3-vaccinated
230
fish (Fig. 3) and PBS fish (data not show). [Fig. 3]
231 232 233
3.4. Immunoprotection induce by rTssJ and pCTssJ against V. harveyi challenge
234
At one-month p.v., the fish vaccinated with rTssJ or pCTssJ were challenged with V.
235
harveyi QT520, and the mortality was monitored for 14 days. The results showed the mean
236
survival rates for rTssJ-, pCTssJ-, rTrx-, pCN3-, and PBS-vaccinated fish were 73.33%,
237
82.67%, 45.33%, 46.67%, and 40%, respectively (Fig. 4). Hence, the mean protective efficacy
238
of rTssJ and pCTssJ, in terms of RPS, were 52.39% and 69.11% with PBS as control. The
239
results showed an extremely significantly higher (P < 0.01) protection induced by pCTssJ than
240
rTssJ. [Fig. 4]
241 242 243
3.5. Innate immune responses analysis
244
Compared with the control group, significantly increased activity of ACP (P < 0.05) in
245
pCTssJ-vaccinated fish was observed at 2 weeks post-vaccination, as well as an
246
extremely significant increase in rTssJ-vaccinated fish (P < 0.01) was found. In addition, fish
247
vaccinated rTssJ exhibited significantly higher (P < 0.05) and pCTssJ extremely significantly
248
higher (P < 0.01) AKP activity at 1 week post-vaccination compared with the control group,
249
respectively. Moreover, the LZM activity of rTssJ-vaccinated fish was extremely significantly
250
higher (P < 0.01) than those of the control group at 2 and 3 weeks post-vaccination. Fish
251
vaccinated pCTssJ can extremely improve the LZM activity compare to that of the control
252
group at 1, 2, and 3 weeks post-vaccination (P < 0.01). Furthermore, at 1 week
253
post-vaccination, fish vaccinated with rTssJ and pCTssJ both exhibited significantly higher
254
SOD activity (P < 0.05) than that fish vaccinated PBS (Fig. 5). [Fig. 5]
255 256 257
3.6. Serum antibody production
258
ELISA analysis showed that both rTssJ and pCTssJ induced the production of specific
259
serum antibodies at 3 to 8-week p.v., which indicated that the two forms of vaccines could
260
elicit the adaptive immunity. The highest antibody level induced by rTssJ occurred at 5-week
261
p.v., but occurred at 4-week induced by pCTssJ (Fig. 6). Meanwhile, the antibody level
262
inducted by rTssJ was much higher than that in pCTssJ-vaccinated fish at each of the
263
examined time point. [Fig. 6]
264 265 266
3.7. Expression of immune-related genes
267
To examine the expression of immune genes induced by rTssJ and pCTssJ, qRT-PCR
268
analysis was carried out. The transcription of the genes encoding interleukin 10 (IL10), tumor
269
necrosis factor-alpha (TNFα), interferoninduced Mx protein (Mx), complement C3 (C3),
270
major histocompatibility complex (MHC) class Iα and class IIα, immunoglobulin M (IgM)
271
and D (IgD), and CD8α were examined. The results showed that in rTssJ-vaccinated fish,
272
IL10, C3, MHC Iα, MHC IIα, and IgM were induced to significant (P < 0.01 or P < 0.05)
273
extents (Fig. 7). In pCTssJ -vaccinated fish, all genes except IgD and CD8α were significantly
274
(P < 0.01 or P < 0.05) induced, and the induction levels of seven of these genes, i.e., IL10,
275
TNFα, Mx, C3, MHC Iα, MHC IIα, and IgM, were higher than that were induced by rTssJ
276
(Fig. 7). [Fig. 7]
277 278 279 280
4. Discussion
281
Vibrio harveyi is a severe aquaculture pathogen with a broad host range and has become
282
a major constraint to fisheries production. As we all know, vaccination is an effective
283
preventive tool which serves as a health management strategy in aquaculture, among which
284
subunit vaccines and DNA vaccines are two important forms of vaccine [13]. In this study, we
285
described the identification of a new V. harveyi antigen TssJ and constructed subunit vaccine
286
and DNA vaccine based on it. We examined and comparatively analysed the vaccine potential
287
and the immune effect induced by the two different forms.
288
In Gram-negative bacteria, the type VI secretion system (T6SS) is discovered as a new
289
type of secretion system for translocation proteins and is essential for virulence [21,23,31].
290
T6SS has been reported in many bacteria, such as, Escherichia coli, Edwarsiella tarda, Vibrio
291
cholerae, Agrobacterium tumefaciens, Pseudomonas aeruginosa, et al [20,21,32]. Previous
292
studies reported that T6SS contains 13 genes which are required to play its roles [23]. Among
293
these 13 genes, TssJ, TssK, TssL and TssM were co-occur in most clusters, with the exception
294
of TssK that locates in the cytoplasm, TssJ, TssL and TssM fractionate with the membranes
295
[23]. In enteroaggregative E. coli (EAEC), TssJ was identified as an outer membrane protein
296
that bears a typical lipobox sorting motif [33-35]. Likewise, TssJ protein was solved to be
297
anchored in outer membrane in Serratia marcescens and P. aeruginosa [31,36]. Similar to the
298
current study, our results of western immunoblot analysis showed that TssJ in V. harveyi
299
QT520 was found in the preparations of the outer membrane and periplasm proteins.
300
Consistent with the western blot results, QT520 became agglutinated in the presence of
301
anti-rTssJ antibodies, suggesting that TssJ in V. harveyi may tether to the outer membrane.
302
These results indicate that TssJ from different bacterial species may be conserved at certain
303
structural and can be used as a novel antigenic candidate for vaccines.
304
Though no magic ways can avoid fish infection of V. harveyi, several control methods
305
including chemotherapy, preventive measures, and vaccines were performed to prevent or
306
reduce the infection [13,37,38] . Studies in previous studies showed that subunit vaccines
307
have to be co-formulated with adjuvants to recall pre-existing immunity and to elicit a
308
balanced immune response [39,40]. In the present study, we vaccinated fish with rTssJ which
309
was mixed with aluminum hydroxide. After a one-month vaccination period, the fish were
310
challenged with V. harveyi QT520 to examine the protective effects as a subunit vaccine.
311
Compared with the PBS group, fish vaccinated with rTssJ exhibited RPS of 52.39%. To
312
investigate whether rTssJ could induce an effective immunity, we analysed some serum
313
parameters, which are usually used to monitor the innate immunity of fish [41]. AKP and ACP
314
are important components of animal lysosomal enzymes, which play important roles in the
315
immune response. In the vertebrate, the increase of AKP and ACP activities indicates
316
increased immunity and is often regarded as a marker for macrophage activation of immune
317
system [42]. LZM is also an important member of lysosomal enzymes, which can lyse and
318
digest pathogenic microorganism. SOD is one of the key antioxidant enzymes for scavenging
319
free radicals, which plays a vital role in the body's oxidation and antioxidant balance [43]. In
320
this study, comparing to control fish, fish vaccinated with rTssJ showed stronger AKP, ACP,
321
LZM, and SOD activities, suggesting that rTssJ could enhance the activation of lysosomal and
322
antioxidant enzymes. Subunit vaccines have been found to provoke not only innate immunity
323
but also adapt immunity in mammals and fish [44-48]. In flounders, outer membrane protein
324
(Omp) K, OmpR, and VAA have been identified with good immunogenicity from V.
325
anguillarum, and their recombinant proteins could induce the proliferation of CD3+ T or sIg+
326
B lymphocytes and the specific antibodies [49-51]. For example, in OmpK group, antibody
327
titer was significantly increased at 2-weeks, and then enhanced steadily and peaked at
328
5-weeks immunization [49]. Similarly, our results showed the specific antibody induced by
329
rTssJ from 3- to 8-weeks vaccination and the highest antibody level occurred at 5-week p.v.,
330
demonstrating that rTssJ could elicit the adapt immunity of fish. In addition, our qRT-PCR
331
analysis revealed an enhanced range of gene expression in fish vaccinated with rTssJ,
332
including IL10, C3, MHC Iα, MHC IIα, and IgM. All of these results suggested that rTssJ
333
could stimulate the innate and adaptive immunity of fish.
334
In case of pCTssJ, the RPS was 69.11%, which was extremely significantly higher than
335
that induced by rTssJ. Like the subunit vaccine (rTssJ), fish injected with pCTssJ also showed
336
stronger AKP, ACP, LZM, and SOD activities when comparing to the control fish. But the
337
results showed no significance between pCTssJ- and rTssJ- immunitied fish, suggesting that
338
pCTssJ and rTssJ elicited similar innate immune responses. It’s well known that lots of V.
339
harveyi vaccines can induce specific serum antibody production [19,49]. In previous studies,
340
Hu et al. found that pDegQ and pVhp1 exhibited respectively, 64.1% and 56.5% relative
341
percent survival rates following lethal V. harveyi challenge, and antibody production was
342
detected [19]. In this study, we found that both rTssJ and pCTssJ could induce serum antibody
343
production. However, they have different patterns of serum antibody production, which in
344
pCTssJ-vaccinated and rTssJ-vaccinated fish peaked at approximately 4-week p.v. and 5-week
345
p.v., respectively. And the actual antibody levels were much higher in rTssJ-vaccinated fish
346
than in pCTssJ-vaccinated fish. Compared to other types of vaccines, DNA vaccine possesses
347
the advantage of being able to elicit systemic immune response of both the humoral and the
348
cellular arms [52-54]. In Japanese flounder, pDegQ and pVhp1 induced higher expression of
349
IgM, MHC Iα, MHC IIα, CD8α, C3, IL-1β, IFN, TNFα, and Mx [19]. In line with this, our
350
qRT-PCR results showed after 4-week p.v. and challenged with V. harveyi, with the exception
351
of IgD and CD8α, whose expression was not affected, all other examined immune genes i.e.,
352
IL10, TNFα, Mx, C3, MHC Iα, MHC IIα, and IgM, were upregulated by pCTssJ. However,
353
the induction folds induced by pCTssJ were higher than those induced by rTssJ, which
354
probably accounts for the much stronger protective effect observed in pCTssJ than rTssJ. The
355
expression of immune factors, which included those participating in specific and nonspecific
356
immunities, indicated that DNA vaccines might induce a wider range of quick and efficient
357
immune response.
358
In conclusion, we constructed and analysed the immune effect of a V. harveyi subunit
359
vaccine and a DNA vaccine encoding TssJ antigen, demonstrating that TssJ is a protective
360
immunogen that could induce different immune responses as both forms of vaccines. DNA
361
vaccine plasmids administered via i.m. injection are transported to internal tissues, thus it
362
might be easier to induce the humoral and the cellular immune response compared to subunit
363
vaccine. However, the specific antibodies elicited by purified recombinant TssJ are much
364
stronger than that induced by TssJ as a DNA vaccine. These results suggest a stable and
365
efficient immune response is important for protection and TssJ may be an effective candidate
366
antigen against V. harveyi infection in aquaculture.
367 368
Acknowledgements
369
This research was supported financially by the National Natural Science Foundation of
370
China (No. 31702379 and No. 31660744), the Key Research and Development Projects of
371
Hainan Province (No. ZDYF2018109), and the Nanhai Famous Youth Project.
372 373 374
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375 376 377 378 379 380 381 382 383 384 385 386 387 388 389
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Figure legends
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Fig. 1. Western immunoblot analysis of the subcellular localization of TssJ in Vibrio harveyi.
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Proteins of the outer membrane (lane 1), periplasm (lane 2), and cytoplasm (lane 3) were
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resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-TssJ
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antibodies. Lane 1, protein markers.
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Fig. 2. Effect of rTssJ on agglutination of Vibrio harveyi. Vibrio harveyi QT520 was first
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labeled with FITC. The cells were incubated with antisera against PBS (A and C) or TssJ (B
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and D). Agglutination was observed by light fields (A and B) and fluorescence fields (C and
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D). Bar = 10 µm.
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Fig. 3. Production of TssJ protein in pCTssJ-vaccinated fish. Muscle tissues were taken from
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golden pompano vaccinated with pCN3 (A) and pCTssJ (B) at 7 days post-vaccination and
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used for immunohistochemistry with gold-labeled antibodies. Arrows indicate gold particles.
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Bar = 200 nm.
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Fig. 4. Survival of vaccinated fish. Golden pompano vaccinated with rTssJ, pCTssJ, rTrx,
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pCN3 or PBS (control) were challenged with Vibrio harveyi and monitored daily for survival.
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The results are one representative vaccination trial. *P < 0.05, **P < 0.01.
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Fig. 5. Effect of rTssJ and pCTssJ on innate immune responses analysis in golden pompano.
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Serum acid phosphatase (ACP) activity (A), alkaline phosphatase (AKP) activity (B), serum
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lysozyme (LZM) activity (C), serum superoxide dismutase (SOD) activity (D).
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Data are presented as means ± standard deviation (SD, N= 3). *P < 0.05, **P < 0.01.
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Fig. 6. Serum antibody production in vaccinated fish. Golden pompano were vaccinated with
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rTssJ, pCTssJ or PBS (control). Sera were collected from the fish at 3-8 weeks
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post-vaccination, and serum antibodies against TssJ were determined by ELISA. Data are
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presented as means ± standard deviation (SD, N= 3). *P < 0.05, **P < 0.01.
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Fig. 7. qRT-PCR analysis of the expression of immune genes in vaccinated fish. Golden
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pompano were vaccinated with rTssJ, pCTssJ or PBS (control) and challenged with V. harveyi.
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Total RNA was extracted from spleen at 24 h post-challenge and used for qRT-PCR. For each
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gene, the mRNA level of the fish vaccinated with PBS was set as 1. Data are presented as
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means ± standard deviation (SD, N= 3). *P < 0.05, **P < 0.01.
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Fig. 4.
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Fig. 5.
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Fig. 6.
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Fig. 7.
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Supplementary data
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Table S1
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Primers used in this study. Primer name
Primer sequence (5′-3′)
Amplification target
TssJ-F1 TssJ-R1 B2M-F B2M-R IL10-F IL10-R TNFα-F TNFα-R
CCCGGGATGGACCCATATTCAGATTTAG CCCGGGTTTTACGACCAAAGACAGAT AAGTCAGTCCACCCAAGGTTCA GGGATTTCCATTCCGTTCTTCATG GATATCGCCACCATGTCTGCGGCTGTGAAT GATATCGACACACATGTCTACAATGC GGCGTCGTTCAGAGTCTCCT TCCTCCTGGGCAGTGGTTT CCAGGAGCATGAGGAAGAGAT AGGTCAATGAGCGTCAGGTCT CGCCGCTCCCTCTACATCA GACAGCCACCCAACCATTACG ACTGGACCTTTCATCAGTCAAACCTG GGACGCTTGGCACTCTTCTTTCT GGCGTGAAGCCATGAAGGACAT AGACTTTGACGGGAGCAGGGTAGA AGAAATGGCTGACTCCTCAATGA CCCGCCTTCCTTTCATAGAG GCAAAGCACCGATACTCTAACC AATATCCAGAAACTAGGCAAGTAAGT CCCGCAGAGTTCGACAGCA CAGGTTGGGTTTGACGGTGAT
Outside part cloning Outside part cloning qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR qRT-PCR
Mx-F Mx-R C3-F C3-R MHC Iα-F MHC Iα-R MHC IIα-F MHC IIα-R IgM-F IgM-R IgD-F IgD-R CD8α-F CD8α-R 710 711 712 713 714 715 716 717
Notes: The underlined bases indicate the SmaI restriction enzyme sites that were added to the 5’ end of primers.
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Fig. S1. SDS-PAGE analysis of rTssJ and rTrx. Protein were analyzed by SDS-PAGE and
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viewed after staining with Coomassie brilliant blue R-250. M: protein molecular weight
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marker; lane 1, Induced rTssJ; lane 2, Purified rTssJ; lane 3, Purified rTrx.
723
Highlights: 1. Subunit vaccine and DNA vaccine were constructed based on the TssJ gene of Vibrio harveyi for Trachinotus ovatus against V. harveyi. 2. Both innate and adaptive immune responses were induced in Trachinotus ovatus after vaccination with subunit vaccine and DNA vaccine. 3. Both of subunit vaccine and DNA vaccine could induce high levels of protection against Vibrio harveyi for Trachinotus ovatus.