Atlantic halibut (Hippoglossus hippoglossus L.) T-cell and cytokine response after vaccination and challenge with nodavirus.

Viral encephalopathy and retinopathy (VER), caused by nodavirus, is one of the major infectious diseases affecting the marine fish farming industry, yet no effective vaccine is available. In this study, we examined the halibut immune response following administration of an experimental vaccine comprising a recombinant nodavirus capsid protein in combination with an oil adjuvant (OA). Four groups of halibut were injected with either: PBS alone, PBS plus OA, 10µg recCP plus OA, or 50µg recCP plus OA. 15 weeks later, half the fish in each group were challenged with nodavirus and the immune response investigated by analysis of: serum levels of recCP-specific halibut immunoglobulins (Igs), and mRNA transcript levels of several T-cell markers (CD3ɛ, Lck, CD4, CD4-2, CD8α and CD8ß) and cytokines (IL-1ß, IL-6, IL-12ßc and IFNγ). Additionally, the presence of nodaviral RNA2 transcripts in the brains of infected halibut was analysed. After vaccination, the level of IL-6 was consistently elevated in the spleens of fish given injections containing the OA. The combination of recCP and OA increased the expression of IL-1ß and IFNγ, as well as the level of recCP-specific Igs in blood plasma. Following challenge with nodavirus, IL-1ß and IFNγ were elevated in halibut spleens after 24h in all groups that had received OA with or without recCP antigen. In brain, a general increase in the expression levels of all T-cell markers and IFNγ was observed following challenge with nodavirus. The viral load at 8 weeks post-challenge was lower in the fish that received 50µg recCP, with 5 out of 8 individuals being negative for nodavirus. Additionally, a better correlation between these markers (apart from the CD8 markers), and the viral RNA2 was also observed in this group, suggesting that the activation of CD4+T-cells might be important in reducing the viral load. In conclusion, this study identifies recCP as a promising candidate antigen for the future development of a vaccine against nodavirus.

Characterisation and expression analysis of the Atlantic halibut (Hippoglossus hippoglossus L.) cytokines: IL-1ß, IL-6, IL-11, IL-12ß and IFNγ.

Genes encoding the five Atlantic halibut (Hippoglossus hippoglossus L.) cytokines; interleukin (IL)-1ß, IL-6, IL-11b, IL-12ßc, and interferon (IFN) γ, were cloned and characterised at a molecular level. The genomic organisation of the halibut cytokine genes was similar to that seen in mammals and/or other fish species. Several mRNA instability motifs were found within the 3'-untranslated region (UTR) of all cytokine cDNA sequences. The putative cytokine protein sequences showed a low sequence identity with the corresponding homologues in mammals, avian and other fish species. Nevertheless, important structural features were presumably conserved such as the presence, or absence in the case of IL-1ß, of a signal peptide, secondary structure and family signature motifs. The relative expression pattern of the cytokine genes was analyzed in several halibut organs, revealing a constitutive expression in both lymphoid and non-lymphoid organs. Interestingly, the gills showed a relatively high expression of IL-1ß, IL-12ßc and IFNγ. The real time RT-PCR data also showed that the mRNA level of IL-1ß, IL-6, IL-12ßc and IFNγ was high in the thymus, while IL-11b was relatively highly expressed in the posterior kidney and posterior gut. Moreover, the halibut brain showed a relatively high level of IL-6 transcripts. Anterior kidney leucocytes in vitro stimulated with imiquimod showed a significant increase in mRNA level of the five halibut cytokine genes. The sequence and characterisation data presented here will be useful for further investigation of both innate and adaptive immune responses in halibut, and be helpful in the design of vaccines for the control of various infectious diseases.

Cloning, characterization, and expression pattern of Atlantic halibut (Hippoglossus hippoglossus L.) CD4-2, Lck, and ZAP-70.

As known from mammalia, the co-receptors CD4 or CD8 associate with a lymphocyte cell-specific kinase (Lck) upon T-cell activation. Lck phosphorylates tyrosine residues within the CD3 chains, providing docking sites for a 70 kDa zeta-associated-protein (ZAP-70), a tyrosine protein kinase important for T-cell signaling. The sequences of a CD4-like gene (CD4-2), Lck, and ZAP-70 were cloned, characterized, and the relative expression pattern was explored in several organs of Atlantic halibut (Hippoglossus hippoglossus L.). Important structural features, as a signal peptide, two Ig-like domains followed by a connecting peptide, a transmembrane region, and a CxC motif within the cytoplasmic tail were conserved within the predicted halibut CD4-2 protein. The deduced halibut Lck protein sequence was found to be composed of a N-terminal Src homology (SH) 4 domain, required for membrane attachment and CD4/CD8 binding, SH3 and SH2 adapter domains, and a SH1 domain followed by a regulatory C-terminal tail (COOH-domain). Tyrosine residues important in Lck activation were conserved within the SH1 and COOH-domain. Structural features of ZAP-70 as tandem SH2 domains and a C-terminal SH1 domain were predicted within the halibut ZAP-70 sequence, having the highest level of conservation within these regions. Several important phosphorylation sites found to play a critical role in T-cell antigen receptor signaling in mammalian were conserved. The overall expression pattern of the three genes was highly similar, showing the highest mRNA level of all three genes in thymus. Some expression was seen in spleen, anterior and posterior kidney, gills, and fin, as seen for other halibut T-cell markers. This study will enable further experiments on halibut T-cell signaling and activation, and enhance understanding about the development of immunological memory T-cells of halibut.

Cloning and expression analysis of Atlantic halibut (Hippoglossus hippoglossus) CD3 genes.

The CD3 complex is in higher vertebrates shown to be important for the activation of T-cells. The T-cell system in fish is believed to be similar to that in higher vertebrates, and the CD3 chains could therefore be an important marker for identification of T-cells in fish. Here, we report the cDNA and corresponding gene sequence of Atlantic halibut (Hippoglossus hippoglossus) CD3gammadelta, CD3varepsilon, and CD3zeta chains, and the tissue-specific expression pattern of CD3 and T- cell receptor (TCR) genes. Important structural characteristics defining the CD3 genes seemed to be conserved in the halibut CD3 chains, such as a signal peptide, an extracellular region, a transmembrane helix having a negatively charged residue, and an ITAM bearing cytoplasmic tail. The extracellular domain of halibut CD3gammadelta and CD3varepsilon included two cysteines presumably involved in Ig-fold stabilisation and the CxxCxE motif important for dimerization. A spliced variant of CD3varepsilon was identified, lacking the Ig-fold, but with the CxxCxE motif intact. The real time RT-PCR analysis revealed a highly similar expression pattern of the CD3 genes and the TCRalpha and TCRbeta genes, indicating that the functional relationship between the TCR and the CD3 genes are preserved in teleosts.