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In mammals, CD83 is a surface marker on mature dendritic cells and vital to lymphocyte activation. In teleost, studies on the function of CD83 are very limited. In this study, we examined the potential involvement of turbot ( Scophthalmus maximus ) CD83, SmCD83, in vaccine-induced immunity. For this purpose, turbot were immunized with pORF75, a DNA vaccine against megalocytivirus, in the presence or absence of pSmCD83, a plasmid that constitutively expresses SmCD83. Immune response and protection analysis showed that the presence of pSmCD83 significantly (i) enhanced the activation of head kidney macrophages (HKM) and immune gene expression, (ii) inhibited viral replication in fish tissues following megalocytivirus challenge and increased the survival of the vaccinated fish, and (iii) stimulated production of specific serum antibody and the cytotoxicity of peripheral blood leukocytes. To further examine the effect of SmCD83, pORF75 was administered into turbot in which SmCD83 was knocked down. Subsequent analysis showed that in fish with SmCD83 knockdown, vaccine-induced HKM activation and antibody production were severely reduced, and, consistently, the protectivity of pORF75 was drastically decreased. Taken together, these results indicate for the first time that teleost CD83 is required for the induction of protective immune response by DNA vaccine. [ABSTRACT FROM AUTHOR]

Highlights: [•] Three CpG ODNs that inhibited megalocytivirus replication in flounder were selected. [•] ODN C7 was able to promote leukocyte and phagocyte activation. [•] Knockdown of toll-like receptor 9 expression blocked C7-mediated immune response. [•] C7 exhibited adjuvant property and augmented the protection of a DNA vaccine. [•] C7 enhanced the expression of vaccine-induced genes of innate and adaptive immunity. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. [Copyright &y& Elsevier]

Abstract: Megalocytivirus is an important fish pathogen with a broad host range that includes turbot. In this study, proteomic analysis was conducted to examine turbot proteins modulated in expression by megalocytivirus infection. Thirty five proteins from spleen were identified to be differentially expressed at 2days post-viral infection (dpi) and 7dpi. Three upregulated proteins, i.e. heat shock protein 70 (Hsp70), Mx protein, and natural killer enhancing factor (NKEF), were further analyzed for potential antiviral effect. For this purpose, turbot were administered separately with the plasmids pHsp70, pMx, and pNKEF, which express Hsp70, Mx, and NKEF respectively, before megalocytivirus infection. Viral dissemination and propagation in spleen were subsequently determined. The results showed that the viral loads in fish administered with pNKEF were significantly reduced. To examine the potential of Hsp70, Mx, and NKEF as immunological adjuvant, turbot were immunized with a DNA vaccine in the presence of pHsp70, pMx, or pNKEF. Subsequent analysis showed that the presence of pNKEF and pHsp70, but not pMx, significantly reduced viral infection and enhanced fish survival. Taken together, these results indicate that NKEF exhibits antiviral property against megalocytivirus, and that both NKEF and Hsp70 may be used in DNA vaccine-based control of megalocytivirus infection. Biological significance: This study provides the first proteomic picture of turbot in response to megalocytivirus infection. We demonstrated that megalocytivirus infection modulates the expression of turbot proteins associated with various cellular functions, and that one of the upregulated proteins, NKEF, exhibits antiviral effect when overexpressed in vivo, while another upregulated protein, Hsp70, exhibits adjuvant effect when co-immunized with a DNA vaccine. These results add molecular insights into turbot immune response induced by megalocytivirus and provide candidate proteins with application potentials in the control of megalocytivirus-associated disease. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda is a Gram-negative bacterium that is reckoned one of the most severe fish pathogens. In this study, we analyzed the biological properties of the E. tarda major fimbrial subunit protein, FimA. We found that mutation of fimA resulted in defective biofilm growth, attenuated infectivity against host cells, and impaired ability to disseminate into and colonize host tissues following experimental infection. When used as a subunit vaccine, recombinant FimA (rFimA) elicited a high level of protection in turbot (Scophthalmus maximus) against lethal E. tarda challenge. Immunological analysis showed that rFimA vaccination induced production of specific serum antibodies that bound to live E. tarda via interaction with the FimA on bacterial cells, and that antibody–E. tarda interaction blocked bacterial infection. Furthermore, passive immunization of turbot with anti-rFimA serum before E. tarda infection reduced bacterial loads in fish tissues to significant extents. To examine the adjuvant potential of FimA, turbot were vaccinated with rVhhP2, a protective Vibrio harveyi antigen, in the presence or absence of rFimA. Subsequent analysis showed that the presence of rFimA significantly augmented the protectivity of rVhhP2. ELISA and quantitative real time RT-PCR showed that rFimA significantly increased rVhhP2-specific serum antibody production and enhanced the expression of immune relevant genes. Taken together, these results indicate that FimA is a virulence-associated protein that possesses vaccine as well as adjuvant potentials, and that the immunoprotectivity of FimA is most likely due to its ability to induce specific immune response that inhibits E. tarda infection. [Copyright &y& Elsevier]

Abstract: Vibrio anguillarum is the etiological agent of vibriosis, an aquaculture disease that affects a wide range of farmed fish. The genome of V. anguillarum contains five flagellin genes, i.e. flaA, flaB, flaC, flaD, and flaE. In this study, we analyzed the vaccine potential and adjuvanticity of FlaA, FlaB, FlaD, and FlaE in a model of Japanese flounder (Paralichthys olivaceus). For this purpose, recombinant FlaA, FlaB, FlaD, and FlaE were expressed in and purified from Escherichia coli. In vivo immunogenicity analysis showed that antibodies against rFlaA, rFlaB, rFlaD, and rFlaE were detected in rat antiserum raised against live V. anguillarum, with the highest antibody level being that against rFlaB. When administered into flounder via intraperitoneal injection, rFlaA, rFlaD, and rFlaE induced comparable relative percent survival (RPS) rates, which were significantly lower than that induced by rFlaB. Specific serum antibodies were induced by all flagellins, however, the antibody level induced by rFlaB was significantly higher than those induced by other three flagellins. Compared to sera from fish vaccinated with rFlaA, rFlaD, and rFlaE, serum from fish vaccinated with rFlaB significantly reduced the infectivity of V. anguillarum against host cells. To examine the potential adjuvant effect of the flagellins, flounder were immunized with rEsa1, a D15-like surface antigen that induces protective immunity as a subunit vaccine, in the presence or absence of rFlaA, rFlaB, rFlaD, and rFlaE respectively. The results showed that rFlaE, but not other three flagellins, significantly increased the RPS of rEsa1. Compared to fish vaccinated with rEsa1, fish vaccinated with rEsa1 plus rFlaE exhibited a significantly higher level of serum antibodies and enhanced expression of the genes involved in innate and adaptive immunity. Taken together, these results indicate that FlaA, FlaB, FlaD, and FlaE have different immunological properties and, as a result, differ in vaccine and adjuvant potentials. [Copyright &y& Elsevier]

Abstract: Heat shock protein (Hsp) 70 is a molecular chaperone that plays an important role in protein folding and transport. In addition, Hsp70 is also involved in regulation of innate and adaptive immune response. In this study, we examined the biological activity and the immunomodulatory property of an Hsp70 homologue, PoHsp70, from Japanese flounder (Paralichthys olivaceus). Recombinant PoHsp70 purified from Escherichia coli exhibits apparent ATPase activity; however, a mutant PoHsp70, PoHsp70M, that bears mutation of the ATPase-associated domain, was completely abolished in activity. Expression of PoHsp70 was upregulated in a time-dependent manner by vaccination of flounder with a DNA vaccine, pSia10, that expresses a Streptococcus iniae antigen, Sia10. To examine whether PoHsp70 possessed any adjuvant potential, the DNA vaccine plasmids pSia10Hsp70 and pSia10Hsp70M were constructed. pSia10Hsp70 co-expresses Sia10 and PoHsp70, while pSia10Hsp70M co-expresses Sia10 and PoHsp70M. Following vaccination of flounder, production of Sia10 plus PoHsp70 and Sia10 plus PoHsp70M was detected in pSia10Hsp70- and pSia10Hsp70M-vaccinated fish respectively. At one month post-vaccination, comparable levels of serum antibodies were detected in fish vaccinated with pSia10Hsp70, pSia10Hsp70M, and pSia10. Subsequent protection analysis showed that, following S. iniae challenge, pSia10Hsp70 induced a survival rate that was significantly higher than that induced by pSia10, while pSia10Hsp70M induced a survival rate similar to that induced by pSia10. These results indicate that PoHsp70 is an effective adjuvant and that the adjuvanticity of PoHsp70 requires the intrinsic ATPase activity. [Copyright &y& Elsevier]

Abstract: Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class IIα, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda is a severe aquaculture pathogen that can infect many different fish species cultured worldwide. Et49 is a major E. tarda antigen with weak immunoprotective potential. In this study, using Et49 as an example vaccine, the adjuvanticity of Freund''s incomplete adjuvant (FIA), aluminum hydroxide, and aluminum phosphate adjuvant were evaluated in a Japanese flounder model. The results showed that the presence of FIA, aluminum hydroxide, and aluminum phosphate adjuvant increased the relative percent of survival of Et49-vaccinated fish by 47%, 19%, and 35%, respectively. Fish vaccinated with FIA-adjuvanted Et49 exhibited longer persistence of vaccine at the injection site and more severe intra-abdominal lesions than fish vaccinated with aluminum-adjuvanted Et49. Both aluminum adjuvants and, to a lesser degree, FIA augmented the production of specific serum antibodies, which reached the highest levels at 6 and 7 weeks post-vaccination. Passive immunization of Japanese flounder with sera from fish vaccinated with aluminum- and FIA-adjuvanted Et49 induced no protection against lethal E. tarda challenge. Examination of the transcription profile of immune-related genes showed that vaccination with aluminum-adjuvanted Et49 significantly enhanced the expression of the genes that are associated mainly with humoral immunity, whereas vaccination with FIA-adjuvanted Et49 induced the expression of a much broader spectrum of genes that are likely to be involved in humoral and innate cellular immunity. These results provide new insights to the action mechanisms of FIA and aluminum adjuvants in Japanese flounder and may be useful for the selection of adjuvant for vaccine formulations intended for Japanese flounder. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunoprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish. [Copyright &y& Elsevier]