Attenuated Vibrio Anguillarum Research Articles

Maternal transfer and protection role in zebrafish (Danio rerio) offspring following vaccination of the brood stock with a live attenuatedVibrio anguillarumvaccine

In this study, expressions of some immune parameters among embryos during different development stages from immunized and mock-immunized female fish were compared at day 14 post immunization of the zebrafish brood stock vaccinated with the live attenuated Vibrio anguillarum vaccine MVAV6203. It was found that transcriptional levels of innate immune cell gene markers including spi.1, l-plastin, mpx and gata-1 as well as the myeloid transcription factor c/ebpβ were up-regulated and expressed earlier in embryos of immunized female zebrafish than naive embryos during early development stage. Investigation through ELISA revealed an increase in antibody level in egg cytosol of the female zebrafish following immunization prior to breeding, which was transferred to larvae through eggs. Rise of specific antibody against wild-type V. anguillarumMVM425 was detected at hour 24 post fertilization (p.f.) eggs from immunized brood stock. Extracts of the newly fertilized eggs from immunized zebrafish also showed a higher capability of killing V. anguillarum MVM425. Moreover, larvae from immunized zebrafish also showed a higher capability of resisting the V. anguillarum MVM425 from proliferation after immersion challenge. Interestingly, the higher content of total IgM and specific antibody in eggs and larvae at 24, 48 and 72 h p.f. were positively correlated with their higher antibacterial activity against V. anguillarum MVAV425. Our results showed that, the development of immune cells was enhanced and the maternally derived antibody could protect early embryos and larvae from attack of specific pathogens via vaccination with a live attenuated vaccine.

A controllable bacterial lysis system to enhance biological safety of live attenuated Vibrio anguillarum vaccine.

Bacterial strains used as backbone for the generation of vaccine prototypes should exhibit an adequate and stable safety profile. Given the fact that live attenuated vaccines often contain some potential risks in virulence recovery and spread infections, new approaches are greatly needed to improve their biological safety. Here, a critically iron-regulated promoter PviuA was screened from Vibrio anguillarum, which was demonstrated to respond to iron-limitation signal both in vitro and in vivo. By using PviuA as a regulatory switch to control the expression of phage P22 lysis cassette 13-19-15, a novel in vivo inducible bacterial lysis system was established in V. anguillarum. This system was proved to be activated by iron-limitation signals and then effectively lyse V. anguillarum both in vitro and in vivo. Further, this controllable bacterial lysis system, after being transformed into a live attenuated V. anguillarum vaccine strain MVAV6203, was confirmed to significantly improve biological safety of the live attenuated vaccine without impairing its immune protection efficacy.

Profiling immune response in zebrafish intestine, skin, spleen and kidney bath-vaccinated with a live attenuated Vibrio anguillarum vaccine

Profiling immune response in zebrafish intestine, skin, spleen and kidney bath-vaccinated with a live attenuated Vibrio anguillarum vaccine

Notable mucosal immune responses induced in the intestine of zebrafish (Danio rerio) bath-vaccinated with a live attenuated Vibrio anguillarum vaccine

Live attenuated vaccine is one of the efficient vaccine candidates in aquaculture, which can be easily delivered to fish via bath-vaccination. An outstanding advantage of bath-vaccination is that vaccine delivery is through the same route as that utilized by many fish pathogens, generating specific mucosal immune responses. In this work, we investigated the mucosal immune responses induced by a live attenuated Vibrio anguillarum vaccine in zebrafish via bath-vaccination. Bacteria proliferated rapidly in 3 h after vaccination and maintained at a high level until 6 h in the intestine. Besides, bacteria persisted in the intestine for a longer time whereas decreased rapidly in the skin and gills. Moreover, a significant up-regulation of TLR5 triggering a MyD88-dependent signaling pathway was observed in the intestine, which implied that flagella were the crucial antigenic component of the live attenuated vaccine. And macrophages and neutrophils showed active responses participating in antigen recognition and sampling after vaccination. Furthermore, an inflammation was observed with plenty of lymphocytes in the intestine at 24 h post vaccination but eliminated within 7 days. In conclusion, the live attenuated V. anguillarum vaccine induced notable mucosal immune responses in the intestine which could be used as a mucosal vaccine vector in the future.

Th17-like immune response in fish mucosal tissues after administration of live attenuated Vibrio anguillarum via different vaccination routes

This study aimed to investigate the protective mucosal immunity elicited by live attenuated Vibrio anguillarum in fish. Zebrafish were immunized by bath or injection way, and undertook bath challenge at 28 days post vaccination. The results implied that bath vaccination was the better delivery route for inducing the protective immunity against bath challenge in zebrafish. The expressions of genes related to Th1, Th2 and Th17 cells were measured in the mucosal tissues of vaccinated and challenged zebrafish. Gene expression profiles showed that Th17-like responses were induced in mucosal immune system by vaccination via bath and injection routes while Th1 and Th2-like responses were not remarkable. Compared to injection vaccination, bath vaccination elicited the intense Th17-like immune responses in the gut tissue of zebrafish. Additionally, in gills and skin, Th17-like mucosal immunity elicited by injection vaccination occurred later than that by bath vaccination. Our results proved the immunological importance of gut in bath vaccination and the presence of two-compartmental model for immune response in zebrafish. In conclusion, bath vaccination more efficiently elicited protective Th17-like immunity than injection vaccination in mucosal tissues of vaccinated zebrafish. In turbot, effective immune protection against wild-type V. anguillarum was obtained by bath-vaccinated and the Th17-like responses were found in mucosal and systemic tissues.

Booster vaccination with live attenuated Vibrio anguillarum elicits strong protection despite weak specific antibody response in zebrafish

Summary A live attenuated Vibrio anguillarum vaccine was recently established in the laboratory that induced immunoprotection against vibriosis in zebrafish, Danio rerio. To improve immunogenicity, the effects of different booster vaccination regimens were investigated using bath-vaccination in a zebrafish model. Zebrafish receiving booster doses at 2 weeks or at both 2 and 4 weeks after primary vaccination were better protected in comparison to fish that received a single vaccination. In addition, the booster vaccination induced a prolonged specific antibody response. No correlation between a weak specific antibody response and a strong protection was observed, indicating that the booster vaccination could enhance the affinity of Immunoglobulin M (IgM) rather than the amount. Moreover, changes in the immune-related gene expression of the booster-vaccinated group suggested that the booster enhanced the adaptive immune responses.

Transcriptome Profiling Reveals Th17-Like Immune Responses Induced in Zebrafish Bath-Vaccinated with a Live Attenuated Vibrio anguillarum

BackgroundA candidate vaccine, live attenuated Vibrio anguillarum developed in our laboratory could prevent vibriosis of fish resulted from V. anguillarum and V. alginolyticus. To elucidate the molecular mechanisms underlying the vaccine protection, we used microarray technology to compare the spleen transcriptomes of bath-vaccinated and unvaccinated zebrafish at 28 days post vaccination.Principal FindingsA total of 2164 genes and transcripts were differentially expressed, accounting for 4.9% of all genes represented on the chip. In addition to iron metabolism related to the innate immunity and the signaling pathways, these differentially expressed genes also involved in the adaptive immunity, mainly including the genes associated with B and T cells activation, proliferation and expansion. Transcription profiles of Th17-related transcription factors, cytokines and cytokine receptors during 35 days post-vaccination implied that Th17 cells be activated in bath-vaccinated zebrafish.Conclusion/SignificanceThe transcriptome profiling with microarray revealed the Th17-like immune response to bath-vaccination with the live attenuated V. anguillarum in zebrafish.

Immune responses evoked by infection with Vibrio anguillarum in zebrafish bath-vaccinated with a live attenuated strain

Live attenuated vaccines are a promising application to control bacterial fish diseases. A live attenuated Vibrio anguillarum vaccine candidate was established in our laboratory to protect fish against vibriosis. To elucidate the mechanism of immunoprotection, it is necessary to compare the different immune responses to infection between vaccinated and non-vaccinated fish. In this study, the expression levels of pathogen-specific antibodies and immune-related genes upon challenge at 28 days post-vaccination were compared between vaccinated and non-vaccinated zebrafish. In the results, the specific antibody levels against virulent V. anguillarum in the vaccinated group did not rise significantly following infection, which suggested that high-affinity antibodies were induced by the vaccine. In the non-vaccinated group, the specific IgM response was triggered at 3 days post-infection and showed a delayed antibody response. Meanwhile, the transcription levels of the genes encoding the pro-inflammatory cytokine IL-1β and the chemokine IL-8 were more highly up-regulated in non-vaccinated fish than in vaccinated fish. This suggests that the overwhelming inflammatory response trigged by infection in non-vaccinated zebrafish was controlled in vaccinated zebrafish. Interestingly, the expression levels of adaptive immune-related genes were increased in vaccinated fish after challenge, compared to the non-vaccinated fish. These results suggest that inoculation with the live attenuated vaccine triggered protection by curbing inflammation and strengthening the adaptive immune response.

FERMENTATION PREPARATION OF RECOMBINANT Vibrio anguillarum VACCINE WITH HETEROGENEOUS ANTIGEN DISPLAY

In the design of recombinant bacterial vector vaccine, heterogeneous antigen is displayed on the outer membrane of the vector strain to evoke polyvalent immunological protection. Thus, the expression of heterogeneous antigen in cells and its display on the outer membrane are of great concern for vaccine preparation. In our previous work, a multivalent bacterial vector vaccine MVAV6203A-1 was constructed by displaying the protective antigen GAPDH from Aeromonas hydrophila on the surface of an attenuated Vibrio anguillarum MVAV6203. In this work, a new fermentation medium was designed by a four-step method to improve the cell growth and antigen display of V. anguillarum MVAV6203A-1. First, suitable carbon and nitrogen sources were selected by a component swapping method. Second, the initial concentrations of carbon and nitrogen sources were determined by orthogonal design. Then three main factors to significantly affect cell growth and antigen expression were screened by a Plackett–Burman design. Finally, the three main factors were meticulously optimized by response surface methodology. Based on this medium, a fed-batch fermentation process was established in a 5-L bioreactor, and the dry cell weight, the antigen expression in cells, and its display on outer membrane reached 5.98 g/L, 2.82 mg/g DCW, and 0.119 mg/g DCW, respectively.

C312M: an attenuated Vibrio anguillarum strain that induces immunoprotection as an oral and immersion vaccine

Vibrio anguillarum, a Gram-negative bacterial pathogen, is the causative agent of vibriosis that affects a wide range of aquatic animals. In this study, we obtained a mutant V. anguillarum, C312M, derived from the pathogenic V. anguillarum C312 by selection of rifampicin resistance. C312M was slower in growth than the wild type C312, particularly under conditions of iron depletion. Compared to C312, C312M was altered in protein production profile and exhibited a dramatically increased median lethal dose. Safety analysis showed that C312M was stable in virulence in the absence of selective pressure. To examine the potential of C312M as a live attenuated vaccine, Japanese flounder Paralichthys olivaceus were vaccinated with C312M via oral, immersion, and oral plus immersion routes. Microbiological analysis showed that C312M was recovered from the gut, liver, kidney, and spleen of the vaccinated fish in 1 to 14 d post-vaccination. When the fish were challenged with C312 at 1 mo post-vaccination, C312M-vaccinated fish exhibited relative percent survival rates of 60 to 84%. Comparable protection was observed when the fish were challenged with a heterologous V. anguillarum strain. Further analysis showed that C312M-vaccinated fish produced specific serum antibodies which enhanced serum bactericidal activity in a manner that is probably complement-dependent. These results indicate that C312M is highly attenuated in virulence but still retains residual infectivity, and that C312M is an effective vaccine when delivered alive via immersion and oral feeding.

Different approaches to expressing Edwardsiella tarda antigen GAPDH in attenuated Vibrio anguillarum for multivalent fish vaccines

With the development of gene technology, expressing heterologous antigens in attenuated bacteria has become an important strategy to design multivalent vaccines. In our previous work, an attenuated Vibrio anguillarum named MVAV6203 was developed and proven to be an efficient live vaccine candidate. In this research, we aimed to express protective antigen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda in attenuated Vibrio anguillarum to establish a multivalent V.anguillarum vector vaccine. Several strategies were compared between low- vs. high-copy plasmid-mediated antigen expression, in vivo-inducible vs. constitutive antigen expression and intracellular vs. surface-displaying antigen expression. Zebrafish, Danio rerio (Hamilton), was applied as the fish model to evaluate the immune protection of the V.anguillarum vector vaccine candidates. Our results demonstrated that V.anguillarum MVAV6203 (pUTatLNG40), which harbours a low-copy plasmid-loaded antigen surface display system under the control of a constitutive promoter, presented the best protective efficacy against the infection of Vibrio anguillarum (relative per cent survival, RPS = 85%) and Edwardsiella tarda (RPS = 70%).

Immune responses of zebrafish (Danio rerio) induced by bath-vaccination with a live attenuated Vibrio anguillarum vaccine candidate

A fish vaccine candidate, live attenuated Vibrio anguillarum, which can protect fish from vibriosis, was established in our laboratory. In this study, the protective immunological mechanism of live attenuated V. anguillarum was investigated in zebrafish as a model animal. After bath-vaccinated with the live attenuated strain, zebrafish were challenged with wild pathogenic strain to test the immunoprotection of the live attenuated strain. As the results, specific antibody response of fish against V. anguillarum was found to gradually increase during 28 days post-vaccination, and remarkable protection was showed with a high relative protection survival (RPS) of about 90%. Moreover, the vaccination changed the expressions of several immune-related genes in the spleens and livers of zebrafish. Among them, the expressions of pro-inflammatory factors such as IL-1 and IL-8 were tenderly up-regulated with about 3–4 fold in 1–7 days post-vaccination, while MHC II rose to a peak level of 4-fold in 7th day post-vaccination. These results gave some important messages about the mechanism of specific protection induced by live attenuated V. anguillarum and showed the availability of zebrafish model in the evaluation of the vaccine candidate.

A stable plasmid system for heterologous antigen expression in attenuated Vibrio anguillarum

To stably synthesize heterologous protein in an attenuated Vibrio anguillarum strain (MVAV6203) for potential multivalent live vaccine application, plasmids with different replicons were used to construct protein expression systems in this work. The gfp fragment under control of a strict low-iron-regulated promoter P viua was inserted into seven plasmids with varied replicons derived from pAT153, pACYC184, pBBR1, pEC, pMW118, pRK2, and pSC101, to generate seven corresponding plasmids. Our results revealed that the plasmid pUTat with the replicon from pAT153 was retained by 100% of the host cells and mediated stable expression of heterologous protein in antibiotic-free medium within 250 generations. Further analyses in animal model (zebrafish larvae) demonstrated that the constructed plasmid pUTat was well retained by bacteria and continuously expressed GFP in vivo in zebrafish. The gapA40 gene, encoding Glyceraldehyde-3-phosphate dehydrogenase from the fish pathogen Edwardsiella tarda, was introduced into the pUTat-based protein expression system, and transformed into V. anguillarum MVAV6203. The resultant recombinant vector vaccine 6203/pUTatgap was evaluated in turbot ( Scophtalmus maximus). After 30 days post vaccination, the fish showed an increased survival ratio by 80% and 67% under the challenge of wild V. anguillarum and E. tarda, respectively. Our results suggested that the pUTat-based antigen expression system had great potential with its efficiency and stability in the design of bacterial vector vaccine.

Surface Display of Aeromonas hydrophila GAPDH in Attenuated Vibrio anguillarum to Develop a Noval Multivalent Vector Vaccine

Displaying foreign antigens on the surface of attenuated or avirulent bacteria is an important strategy to develop live multivalent vector vaccines. In our previous work, several efficient surface display systems have been established based on outer membrane anchoring elements, which could successfully display heterologous proteins in attenuated Vibrio anguillarum. In this work, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from pathogenic Aeromonas hydrophila LSA34 was fused to seven display systems and introduced into attenuated V. anguillarum strain MVAV6203 (AV) to get seven GAPDH-display strains. The strain AV/pN-gapA showed the best display efficacy of GAPDH and was tested as the multivalent vaccine candidate. Further immune protection evaluation of AV/pN-gapA in turbot (Scophtalmus maximus) demonstrated that the attenuated V. anguillarum with surface-displayed GAPDH of A. hydrophila LSA34 effectively protected turbot from the infections of A. hydrophila and V. anguillarum and showed potential value for further multivalent vaccine development.

Differential gene expression in Japanese flounder (Paralichthys olivaceus) induced by live attenuated Vibrio anguillarum

Using suppression subtractive hybridization technique, a purpose-designed cDNA library was established to investigate the changes in gene expression and isolate the immune-related genes induced or activated by live attenuated Vibrio anguillarum. Forty-eight genes were obtained in flounder immune tissues, including cytokines, cytokine receptors, cell surface molecules, transcription factors, complement components, antimicrobial peptides and cell surface molecules. Among them, seven genes were found for the first time in flounder, named mannose-binding lectin-associated serine protease (MASP) gene, mannose-binding lectin, QM gene, tumour necrosis factor superfamily member 13b (BAFF), natural killer-lysin, chemotaxin and nuclear factor κB (NF-κB). The expression of four selected genes encoding MASP, QM, BAFF and NF-κB was further studied in immune tissues of the flounder. An obviously up-regulated expression was observed in live and spleen between challenged and control fish. These results could be beneficial for a better understanding of the immune mechanism in flounder.

A novel multivalent vaccine based on secretary antigen-delivery induces protective immunity against Vibrio anguillarum and Aeromonas hydrophila

In our previous work, four secretory antigen-delivery systems based on different signal peptides (SPs) (SP hlyA , SP rtxA , SP vah3 and SP empA ) have been successfully established in attenuated Vibrio anguillarum. To investigate the potential application of the antigen-delivery systems in V. anguillarum multivalent vector vaccine, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from pathogenic Aeromonas hydrophila, as a putative protective antigen, was fused to the four delivery systems, respectively, and introduced into attenuated V. anguillarum strain MVAV6203 to get four GAPDH-delivery strains in this work. Immunodetection of GAPDH indicated that among the four constructs, the SP empA -mediated GAPDH-delivery strain AV(pGap-empA) showed the highest level of GAPDH expression and secretion, and was determined as the multivalent vaccine candidate. Further immune protection evaluation of AV(pGap-empA) in turbot ( Scophtalmus maximus) demonstrated that the vaccine candidate could induce efficient protection against V. anguillarum and A. hydrophila, suggesting that this vector vaccine had great potential in serving as a valuable multivalent live vaccine.

Secretory delivery of heterologous proteins in attenuated Vibrio anguillarum for potential use in vaccine design

To synthesize and secrete heterologous proteins in an attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different antigen-delivery systems based on bacterial-originated secretion signal peptides (SPs) were designed and identified in this work. Four SPs were derived from hemolysin of Escherichia coli, RTX protein of V. cholerae, hemolysin of V. anguillarum, zinc-metalloprotease of V. anguillarum, respectively, and their abilities to support secretion of green fluorescent protein (GFP) in an attenuated V. anguillarum strain MVAV6203 were assayed. Immunodetection of GFP showed that the capability of the tested signal leaders to direct secretion of GFP varied greatly. Although all the four signal peptide-fused GFPs could be expressed correctly and trapped intracellularly in recombinant strains, only the EmpA signal peptide could confer efficient secretion to GFP. For the investigation of its potential application in live bacteria carrier vaccines, a heterologous protein EseB of Edwardsiella tarda was fused to the SP(empA) antigen-delivery system and introduced into the strain MVAV6203. Further analysis of EseB demonstrated that the constructed SP(empA) antigen-delivery system could be used to secrete foreign protein in attenuated V. anguillarum and be available for carrier vaccines development.

Surface Display of GFP by Pseudomonas Syringae Truncated Ice Nucleation Protein in Attenuated Vibrio Anguillarum Strain

Microbial cell surface display of foreign proteins has been widely developed for many potential applications in live vaccine construction, whole-cell biocatalysts, and bioadsorption. To investigate the feasibility of displaying heterologous proteins on the surface of attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different display systems were built upon a truncated ice nucleation protein (INP) from Pseudomonas syringae ICMP3023 whose N- and C-terminal domains were considered to be the putative membrane-anchoring motifs. Green fluorescent protein (GFP), as a reporter, was fused with the display systems in different forms of N-GFP, NC-GFP, and N-GFP-C. Analysis of the total expression level and surface localization of GFP demonstrated that the truncated P. syringae INP could be used to display foreign protein in V. anguillarum, while the system of N-GFP showed the higher levels of total expression and surface display based on unit cell density among the three and might be available for further carrier vaccine development.

Freeze-drying of live attenuated Vibrio anguillarum mutant for vaccine preparation

Vibrio anguillarum MVAV6203 is a mutant strain as a candidate of live attenuated vaccine. In vaccine preparation, the freeze-drying conditions of the strain were investigated to improve the survival after freeze-drying, including the protectant, rehydration medium, freezing temperature, and initial cell concentration. Vibrio anguillarum MVAV6203 is sensitive to freeze-drying and the viability was only 0.03% in the absence of protectant. Of the tested protectants, 5% trehalose with 15% skimmed milk gave the highest viability of 34.2%. Higher cell survival was obtained by quick freezing at −80 °C than slow freezing at −20 °C. Initial cell concentration was another important factor, preferable for 1–3 × 10 10 CFU/ml. The supplementation of 10% skimmed milk in rehydration medium improved obviously freeze-drying viability. The combination of the optimal conditions achieved 51.4% cell viability after freeze-drying.