Vaccines In Aquaculture Research Articles

Distinct response of immune gene expression in peripheral blood leucocytes modulated by bacterin vaccine candidates in rainbow trout Oncorhynchus mykiss: A potential in vitro screening and batch testing system for vaccine development in aquaculture

Fish aquaculture is the world's fastest growing food production industry and infectious diseases are a major limiting factor. Vaccination is the most appropriate method for controlling infectious diseases and a key reason for the success of salmonid cultivation and has reduced the use of antibiotics. The development of fish vaccines requires the use of a great number of experimental animals that are challenged with virulent pathogens. In vitro cell culture systems have the potential to replace in vivo pathogen exposure for initial screening and testing of novel vaccine candidates/preparations, and for batch potency and safety tests. PBL contain major immune cells that enable the detection of both innate and adaptive immune responses in vitro. Fish PBL can be easily prepared using a hypotonic method and is the only way to obtain large numbers of immune cells non-lethally. Distinct gene expression profiles of innate and adaptive immunity have been observed between bacterins prepared from different bacterial species, as well as from different strains or culturing conditions of the same bacterial species. Distinct immune pathways are activated by pathogens or vaccines in vivo that can be detected in PBL in vitro. Immune gene expression in PBL after stimulation with vaccine candidates may shed light on the immune pathways involved that lead to vaccine-mediated protection. This study suggests that PBL are a suitable platform for initial screening of vaccine candidates, for evaluation of vaccine-induced immune responses, and a cheap alternative for potency testing to reduce animal use in aquaculture vaccine development.

Bacterial membrane vesicles as vaccines in aquaculture

Metalloenzymes are frequently employed by diverse microorganisms to catalyze difficult transformations during the biosynthesis of natural products. Among these, members of the versatile and prevalent radical S-adenosylmethionine (RaS) enzyme superfamily are noteworthy, as they have recently been shown to mediate an array of unusual crosslinking and cyclization reactions. Herein, we summarize the roles of RaS enzymes in the biosynthesis of ribosomally-synthesized and post-translationally modified peptides (RiPPs), an ascendant class of natural products with diverse structures and functions. We focus on cases in which the metalloenzymes have been both chemically and structurally characterized. We evaluate proposed mechanisms in each case and compare and contrast reported structural features for representative RaS enzymes. Themes that have emerged from these studies are discussed along with unresolved questions, which will provide impetus for further investigations into this rich research area at the nexus of metalloenzymology and natural product biosynthesis.

Long-term, proteome-scale analysis of rainbow trout immune proteins: Implications for aquaculture vaccine development

Long-term, proteome-scale analysis of rainbow trout immune proteins: Implications for aquaculture vaccine development

Promiscuous T cell epitopes boosts specific IgM immune response against a P0 peptide antigen from sea lice in different teleost species

The development of vaccines employing conserved protein antigens, for instance ribosomal protein P0, has as disadvantage the high degree of identity between pathogen and host proteins due to possible induction of tolerance or auto antibodies in the host organism. To overcome this drawback, peptide-based vaccines have been designed with a proved high efficacy. The use of defined peptides as antigens has the problem that they are generally poor immunogenic unless coupled to a carrier protein. Several studies have established the potential for promiscuous T cell epitopes incorporated into chimeric peptides to enhance the immunogenicity in mammals. On the contrary, studies about the role of these epitopes on teleost immune system are scarce. Therefore, the main objective of our present study was to evaluate the potential of promiscuous T cell epitopes to boost specific IgM immune response in teleost fish against a peptide antigen. With this aim, we used a peptide of 35 amino acids from the ribosomal P0 protein of Lepeophtheirus salmonis, an important parasite in salmon aquaculture. We fused this peptide to the C-terminal of T cell epitopes from tetanus toxin and measles virus and produced the chimeric protein in Escherichia coli. Following vaccination, IgM antibody production was monitored in different immunization schemes in Tilapia, African catfish and Atlantic salmon. The results demonstrated for first time that the addition of T cell epitopes at the N-terminal of a target peptide increased IgM specific response in different teleost species, revealing the potential of this approach to develop peptide-based vaccines for aquaculture. The results are also of great importance in the context of vaccine development against sea lice using ribosomal protein P0 as antigen taking into account the key role of P0 in protein synthesis and other essential physiological processes.

Attenuated Listeria monocytogenes protecting zebrafish (Danio rerio) against Vibrio species challenge

Live attenuated bacteria is a promising candidate vector for the delivery of vaccines in clinic trials. In the field of aquaculture industry, live vector vaccine also could provide long-term and effective protection against fish bacterial diseases. In our previous work, we demonstrated attenuated Listeria monocytogenes (Lm) had the potential to be an aquaculture vaccine vector in cellular level and zebrafish model. To further investigate the potential application of attenuated Lm in aquaculture vaccines, the outer membrane protein K (OmpK) from Vibrio parahaemolyticus (V. parahaemolyticus), as a conservative protective antigen, was fused to a new antigen-delivery system, and introduced into double-gene attenuated Lm strain (EGDe-ΔactA/inlB, Lmdd) to get live-vector vaccine strain Lmdd-OmpK. The strain Lmdd-OmpK showed the stable secrete efficacy of OmpK and was tested the cross-protective immunity against Vibrio species. After intraperitoneal administration in zebrafish, Lmdd and Lmdd-OmpK strain both improved the survival rates of zebrafish infected by V. parahaemolyticus, Vibrio alginolyticus (V. alginolyticus) and Vibrio anguillarum (V. anguillarum), respectively. In summary, attenuated Lm is able to protect zebrafish against Vibrio species challenge, illustrating its potential value for further aquaculture vaccines development.

Oral immunization with recombinant Lactobacillus casei expressing flaB confers protection against Aeromonas veronii challenge in common carp, Cyprinus carpio

Aeromonas veronii is an important type of gram-negative pathogen of human-livestock-aquatic animal and causes great economic losses in the aquaculture industry. Vaccination is an effective method of defence against A. veronii. There are many factors that restrict the use of vaccination, and the development of new oral vaccines is urgently needed. The selection of suitable antigens is of great significance for the development of aquaculture vaccines. Bacterial flagellin can specifically bind to TLR5 and induce the release of cytokines from the organism, which could be used in the development of vaccines. In this study, we constructed two recombinant Lactobacillus casei (L. casei) (surface-displayed or secretory) expressing the flaB of A. veronii and evaluated the effect of immune responses in common carp. The flaB gene (900 bp) of A. veronii was subcloned into the L. casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secretory). Western blot and immunofluorescence assays confirmed the expression of the recombinant flaB protein. Common carp immunized with Lc-pPG-1-flaB and Lc-pPG-2-flaB via oral administration route exhibited induction of antibody expression and innate immune responses. The results indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB can induce high levels of IgM, ACP, AKP, LZM and SOD activity in organisms, and Lc-pPG-1-flaB can induce even higher levels. The recombinant L. casei may effectively induce humoral immunity and increase the serum immunological index. Furthermore, leukocytes phagocytosis percentage and index of the recombinant L. casei were enhanced. The results of qRT-PCR showed that recombinant L. casei can significantly increase the expression of IL-10, IL-β, IFN-γ and TNF-α in the tissues of immunized common carp, compared with control groups. Viable recombinant L. casei strains, which were delivered directly survived throughout the intestinal tract. Common carp that received Lc-pPG-1-flaB (66.7%) and Lc-pPG-2-flaB (53.3%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our work indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB had beneficial effects on immune response and enhanced the disease resistance of common carp against A. veronii infection. The combination of flaB delivery and the Lactic acid bacteria (LAB) approach may be a promising method for the development of oral vaccines for treating A. veronii. In future research, we will focus on the colonization ability of LAB in the intestines and on the impact of these bacteria on intestinal flora.

Oral yeast-based DNA vaccine confers effective protection from Aeromonas hydrophila infection on Carassius auratus

Our previous study has demonstrated that recombinant yeast can induce specific immune responses in Carassius auratus and may serve as a potential carrier for oral DNA vaccines in aquaculture. In this study, we further developed an effective yeast-based oral DNA vaccine against the bacteria Aeromonas hydrophila, which was expected to provide protection from the motile aeromonad septicemia (MAS). First, two candidate antigen genes, ompG and omp48, were cloned from the Aeromonas hydrophila genome DNA. Then, relative yeast-eukaryote shuttle vectors were constructed and their expression in eukaryotes was validated. Next, crucian carps were orally administered with ompG or omp48 recombinant yeast, and the expression of the genes in the intestinal mucosa was confirmed by immunohistochemistry (IHC). The specific immune responses were further detected by Western blot and enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the production of the OVA-specific antibody in the OVA-ompG group was significantly higher than that of the OVA-omp48 group, indicating that the OVA-ompG group elicited obviously stronger immune response than OVA-omp48. Finally, the challenge experiment against Aeromonas hydrophila infection demonstrated decreased fish mortality rate after the oral administration of the OVA-ompG yeast vaccine. In conclusion, our work provided a framework for the further development of oral yeast-based fishery vaccines.

Adjuvant effects of medicinal herbs and probiotics for fish vaccines

AbstractThe most prominent limiting factor in aquaculture nowadays is infectious diseases as they lead to massive economic losses. These diseases are often partly controlled by vaccination, herbs or probiotics. Despite the fact that vaccination is the most effective prophylactic approach to disease prevention, researchers have started to use plant‐derived products and probiotics as adjuvants to boost the efficacy of fish vaccines. This review describes the efficacy and potency of plant extracts/essences and probiotics as adjuvants for various vaccines in aquaculture.

Immunogenicity study of an expressed outer membrane protein U of Vibrio vulnificus in Japanese eel (Anguilla japonica).

Vibrio vulnificus is a common bacterial pathogen causing haemorrhagic septicaemia in eel farming. This study investigates the immunogenicity of an outer membrane protein U (OmpU) of V. vulnificus and the feasibility of the protein as a new subunit vaccine against V. vulnificus. Partial gene sequence of the OmpU of V. vulnificus was cloned, and then the OmpU was expressed and purified. Three groups of Japanese eels (Anguilla japonica) were intraperitoneally (i.p) injected with bovine serum albumin (BSA group), formalin-killed whole cell of V. vulnificus (FKC group) or the expressed OmpU of V. vulnificus (OMP group). On 14, 21, 28 and 42days postimmunization (dpi), the whole blood cells were collected to evaluate the stimulation index (SI) and bactericidal activity. The serum was obtained to assess the titres of specific antibody, lysozyme activity, complement activity and bactericidal activity. The lysozyme activities in the suspension of kidney, skin mucus and liver in eels were also ascertained. The results showed that the SI and the titres of anti-V. vulnificus antibody in theOMP group was significantly increased on 28 dpi; lysozyme activity in the kidney and skin mucus of OMP group on 42 and 14 dpi were both significantly higher than BSA group; eels in OMP group showed strong bactericidal capacity on 21 and 28days; and the relative percent survival of OMP vs BSA group after challenged by V. vulnificus on 28 dpi was 80%. These results showed that the expressed OmpU of V. vulnificus could significantly improve the immune function of Japanese eel and the resistance of eels to the infection of V. vulnificus. This study offered an alternative preliminary strategy of making aquaculture vaccines against V. vulnificus for eel farming.

Exploration of the bacterial invasion capacity of Listeria monocytogenes in ZF4 cells

Despite the results from zebrafish challenged model have demonstrated that Listeria monocytogenes (Lm) has strong adjuvant effects when this attenuated pathogenic bacteria is viewed as aquaculture vaccine vector, the underlying mechanism is not clear and extensive investigations are required. To further explore the potential of Lm in the field of aquaculture vaccine, zebrafish embryonic fibroblast cell line (ZF4) was used to evaluate the invasion ability of Lm. The data from cellular level showed that Lm had the lower invasion tendentiousness in ZF4 cells while bacterial invasion capacity was compared between zebrafish embryos cell line and human intestinal epithelial cell line. In ZF4 cells, there is no significant difference in bacterial invasion capacity between wild strain EGD-e and double-deleted strain ΔactA/inlB, which suggested that this attenuated effect was not showed in zebrafish cells. In addition, translation analysis indicated that the expressions of CD4 and CD8a in ZF4 cells increased after 2-h infection of the two Lm strains. These results further demonstrated that Lm presented multiple advantages including lower pathogenicity and antigen presentation when attenuated stain was viewed as aquaculture vaccine vector.

Evaluation of an outer membrane protein as a vaccine against Edwardsiella anguillarum in Japanese eels (Anguilla japonica)

Edwardsiella anguillarum is a novel bacterial pathogen causing hemorrhagic septicemia in Japanese eels (Anguilla japonica) and results in significant economic loss to eels farming. Currently, there is no applied vaccine against E. anguillarum. In this study, the outer membrane protein A (OmpA) of E. anguillarum was expressed and purified, then Japanese eels (Anguilla japonica) were intraperitoneally injected (i.p) with bovine serum albumin (BSA group), formalin killed-whole-cell of E. anguillarum (FKC group) or the expressed OmpA of E. anguillarum (OMP group) respectively. On 14, 21, 28 and 42 d post immunization (dpi) respectively, the whole blood cells were collected to evaluate the stimulation index and bactericidal activity; the serum were obtained to assess the titers of specific antibody, lysozyme activity, complement activity and bactericidal activity; lysozyme activities in the suspension of kidney, skin mucus and liver in eels were ascertained. The results showed that the stimulation index of the whole blood cells in OMP group was significantly enhanced compared with the BSA group on 28 dpi; the titers of specific antibody in OMP group were significantly increased compared with FKC and BSA group on 28 d and 42 dpi respectively; lysozyme activity in serum of the OMP group was significantly higher than that of BSA group; complement activity of the OMP group was significantly higher than that of the BSA group; the OMP group showed strong bactericidal capacity on 21 and 28 dpi. Simultaneously, the relative percent survival (RPS) of Omp vs BSA group was 77.7% after eels were challenged by E. anguillarum on 28 dpi. These results showed that the expressed rOmpA was a good antigen of E. anguillarum and offered an alternative strategy for making applied aquaculture vaccines.

Correlates of protective immunity for fish vaccines

Vaccination is one of the most effective disease control strategies that has contributed to the significant reduction of disease outbreaks and antibiotics usage in salmonid aquaculture. To date, licensing of fish vaccines is to a limited extent based on in vitro correlates of protection, as done for many mammalian vaccines. This is because the immunological mechanisms of vaccine protection have not been clearly elucidated for most fish vaccines. Herein, we provide an overview of the different steps required to establish correlates of protective immunity required to serve as benchmarks in optimizing vaccine production in aquaculture. We highlight the importance of optimizing challenge models needed to generate consistent results used during vaccine development as a basis for establishing immune correlates of protection. Data generated this far shows that antibodies are potentially the most reliable correlates of protective immunity for fish vaccines. Our findings also show that antigen dose can be optimized to serve as a correlate of protection for fish vaccines. Further, there is need to establish signatures of T-cell protective immunity when antibodies fail to serve as proxies of immune protection, particularly for vaccines against intracellular pathogens. We can anticipate that documentation of efficacy for future vaccines in aquaculture, particularly batch testing will be based on in vitro correlates of protective immunity.

Dynamic distribution of formalin-inactivated Edwardsiella tarda in olive flounder (Paralichthys olivaceus) post intramuscular injection

Intramuscular (i.m.) injection is one of the common delivery methods of vaccination in aquaculture, which could induce an ideal immune protection to fish. In the present study, the olive flounders (Paralichthys olivaceus) were injected intramuscularly with 200 μl of three concentrations of formalin-inactivated Edwardsiella tarda bacterin (107, 108, 109 CFU ml−1) to investigate the transportation and dynamic distribution of antigen uptake in tissues by absolute real-time quantitative PCR (qPCR). The amount of uptaken antigen increased firstly, and then decreased. The peak occurred first in the blood at 6–9 h after i.m. injection, and in the spleen and head kidney at 9–15 h, then in the liver, gill and muscle at 15–24 h, finally in the skin and intestine at 36 h. The amount of uptaken antigen was highest in the head kidney, followed by in the spleen, blood, gill, and liver, and lowest in the muscle, skin and intestine. Among the three dose groups, the amount of uptaken antigen in all tested tissues became higher with the increasing dose of injected bacterin. Moreover, the tissue distribution of antigen uptake was investigated by indirect immunofluorescence assay (IIFA) at 15 h after i.m. injection with 200 μl of 108 CFU ml−1E. tarda bacterin. The distribution of antigen was mainly observed in the head kidney, then in the spleen, blood, liver, gill and muscle, and least in the skin and intestine, which correlated with the results of absolute qPCR detection. Furthermore, the expression levels of MHC Iα, MHC IIα, CD4-1 and CD8α were detected by RT-qPCR. The expression of these four genes peaked highest in the head kidney, followed by in the spleen, liver, blood and gill, and lowest in the muscle, skin and intestine, and the levels increased in parallel with the increasing dose of injected vaccine. All these results provided an important insight into the dynamic transportation of antigen uptake, and also deepened the understanding of immune response to the i.m. injection.

Effectivity of oral recombinant DNA vaccine against Streptococcus agalactiae in Nile tilapia

The effects of oral vaccination by two immune routes (gavage and mixed fodder administration) using a recombinant DNA vaccine SL7207-pVAX1-sip against Streptococcus agalactiae were assessed. SL7207-pVAX1-sip significantly enhanced various innate immune responses of tilapia, such as total serum protein, superoxide dismutase activity, lysozyme activity, complement C3 concentration in serum, serum antibacterial activity, and interleukin 1β and tumor necrosis factor-α. Vaccinated fish had higher relative percent survival values (57% and 63% for gavage group and mix fodder administration, respectively) at 10 weeks after S. agalactiae infection. When administered mixed fodder, the DNA vaccine SL7207-pVAX1-sip against S. agalactiae may produce more effective protection. These findings can promote the application and development of DNA vaccines in aquaculture.

Edwardsiella piscicida lacking the cyclic AMP receptor protein (Crp) is avirulent and immunogenic in fish

Edwardsiella piscicida is a Gram-negative pathogen that generally causes lethal septicemia in marine and freshwater fish. We generated a E. piscicida CK216 Δcrp mutant to investigate various biological roles related to this organism, including pathogenesis. Lack of Crp in CK216 was demonstrated by immunoblotting using a Crp-specific antibody. Compared to the parental strain, the mutant exhibited changes in three biochemical phenotypes, including ornithine decarboxylation, citrate utilization, and H2S production. Complementation of crp deletion in trans rescued the phenotype of the parental strain. This study proved that hemolytic activity in E. piscicida is controlled by Crp. In addition, significantly reduced motility of E. piscicida CK216 was observed, which resulted from a lack of flagella synthesis. To examine the virulence in fish, E. piscicida cells were injected into the goldfish (Carassius auratus) via intraperitoneal route. The LD50 of CK216 was 9.25 × 108 CFU, while that of the CK108 parental strain was 9.24 × 105 CFU, attenuated 1000 fold in goldfish. Fish immunized with CK216 elicited IgM responses. Moreover, 80% of goldfish immunized with 1 × 106 CFU survived after administration of a lethal dose (1 × 107 CFU) of virulent E. piscicida CK41, suggesting the potential for E. piscicida CK216 to serve as a live attenuated vaccine in aquaculture.

Immunomodulatory properties of Concholepas concholepas hemocyanin against francisellosis in a zebrafish model

The development of vaccines for aquaculture has been an important milestone in providing a continuous and sustainable production. Most of the vaccines currently on the market for aquaculture include oil as adjuvant. Nevertheless, several studies reported an occurrence of side effects after their use in farmed fish. As a result, there is a need for new and improved adjuvants that can stimulate the immune system while causing as few side-effects as possible. Hemocyanins are versatile macromolecules with strong immunogenic and immunomodulatory properties. Due to these characteristics, hemocyanin from Concholepas concholepas (CCH) has been biochemically characterized and evaluated as vaccine adjuvant in mice and humans. Francisellosis is a chronic granulomatous disease, which can result in high mortality depending on the host. The disease is caused by the facultative intracellular Gram-negative bacteria Francisella noatunensis, which remains an unsolved problem for the aquaculture, as no efficient vaccines are available. The aim of the present work was to investigate the immunoregulatory properties of CCH against francisellosis in an experimental zebrafish model. When immunized with CCH, zebrafish were protected from subsequent challenge with a lethal dose of Francisella noatunensis subsp. orientalis. Subsequently the mRNA expression levels of several immune-related genes were studied, including mhcii, il12a, tnfα and ifng1-1. Taken together, the data report the immunoregulatory properties of CCH and its potential use as a vaccine adjuvant for aquaculture.

Computational modeling of immune system of the fish for a more effective vaccination in aquaculture.

A computational model equipped with the main immunological features of the sea bass (Dicentrarchus labrax L.) immune system was used to predict more effective vaccination in fish. The performance of the model was evaluated by using the results of two in vivo vaccinations trials against L. anguillarum and P. damselae. Tests were performed to select the appropriate doses of vaccine and infectious bacteria to set up the model. Simulation outputs were compared with the specific antibody production and the expression of BcR and TcR gene transcripts in spleen. The model has shown a good ability to be used in sea bass and could be implemented for different routes of vaccine administration even with more than two pathogens. The model confirms the suitability of in silico methods to optimize vaccine doses and the immune response to them. This model could be applied to other species to optimize the design of new vaccination treatments of fish in aquaculture. The method is available at http://www.iac.cnr.it/∼filippo/c-immsim/. nromano@unitus.it. Supplementary data are available at Bioinformatics online.

A potential aquaculture vaccine vector: Evaluation of a double-gene attenuated Listeria monocytogenes in zebrafish (Danio rerio)

Although engineered Lm has strong adjuvant effects such as the delivery of antigens in antigen-presenting cells (APCs), it has not been evaluated if attenuated Lm has a potential to be used as an aquaculture vaccines vector. In this study, a double-gene attenuated Lm strain (Lm-actA/inlB) was constructed to evaluate the potential that using this attenuated strain to provide a delivery platform for aquatic vaccines. The attenuated Lm strain Lm-actA/inlB showed decreased invasion and apoptosis of HepG2 and Caco-2 host cells in in vitro infection models. Interestingly, the expression levels of the hly and plcB genes were significantly increased in Lm-actA/inlB compared with wild-type EGDe. The higher expression levels of the hly and plcB genes are indicative of greater advantages in terms of antigen presentation. To further explore the innate immune and adaptive immune response elicited by Lm-actA/inlB and its feasibility for use as a non-injection vaccine vector, we evaluated the expression of Toll-like receptor (TLR) signalling pathway-related gene markers, antigen presentation pathway-related gene markers and IgM via Lm-actA/inlB intraperitoneal injection and bath delivery. Transcription analysis indicated that the TLR signalling pathway might play a major role in the innate immune response and the major histocompatibility complex (MHC)-I and MHC-II antigen presentation pathways might also both exist in vertebrate fish. In conclusion, the live-attenuated Lm strain Lm-actA/inlB may be a potential platform for delivering aquatic vaccines against certain fish bacterial diseases commonly seen in the aquatic farm industry.

CD4-Transgenic Zebrafish Reveal Tissue-Resident Th2- and Regulatory T Cell-like Populations and Diverse Mononuclear Phagocytes.

CD4+ T cells are at the nexus of the innate and adaptive arms of the immune system. However, little is known about the evolutionary history of CD4+ T cells, and it is unclear whether their differentiation into specialized subsets is conserved in early vertebrates. In this study, we have created transgenic zebrafish with vibrantly labeled CD4+ cells allowing us to scrutinize the development and specialization of teleost CD4+ leukocytes in vivo. We provide further evidence that CD4+ macrophages have an ancient origin and had already emerged in bony fish. We demonstrate the utility of this zebrafish resource for interrogating the complex behavior of immune cells at cellular resolution by the imaging of intimate contacts between teleost CD4+ T cells and mononuclear phagocytes. Most importantly, we reveal the conserved subspecialization of teleost CD4+ T cells in vivo. We demonstrate that the ancient and specialized tissues of the gills contain a resident population of il-4/13b–expressing Th2-like cells, which do not coexpress il-4/13a. Additionally, we identify a contrasting population of regulatory T cell–like cells resident in the zebrafish gut mucosa, in marked similarity to that found in the intestine of mammals. Finally, we show that, as in mammals, zebrafish CD4+ T cells will infiltrate melanoma tumors and obtain a phenotype consistent with a type 2 immune microenvironment. We anticipate that this unique resource will prove invaluable for future investigation of T cell function in biomedical research, the development of vaccination and health management in aquaculture, and for further research into the evolution of adaptive immunity.

Design and evaluation of an Edwardsiella tarda DNA vaccine co-encoding antigenic and adjuvant peptide

Edwardsiella tarda is associated with edwardsiellosis in cultured fish, resulting in heavy losses in aquaculture. So far, different types of vaccine have been attempted against E. tarda. In this study, an optimized eukaryotic expression plasmid was developed and an optimized DNA vaccine co-encoding antigenic and adjuvant peptide using a bicistronic expression system was designed. As a result, a modified plasmid harbored cytomegalovirus (CMV) promoter attached with R region of long terminal repeat from human T-cell leukemia virus type 1 (CMV/R) and woodchuck hepatitis virus post-transcriptional response element (WPRE) component showed an increased antigenic gene expression compared with unmodified plasmid. Moreover, the designed system based on bicistronic system exhibited a stronger ability to express antigenic gene and the RPS achieved 87.3% compared with plasmid encoding antigentic gene. Finally, immunological analysis showed that the DNA vaccine induced both innate and adaptive immune responses. These results suggest that co-encoding antigenic and adjuvant proteins might be an efficient strategy to develop DNA vaccines in aquaculture in the future.