Immunostimulatory Effect of CpG Oligodeoxynucleotide in Grouper and its Application in the Development of Anti-iridovirus Vaccine

The induction of the innate immunity depends on whether the immune cells can effectively recognize specific structures called pathogen-associated molecular patterns (PAMPs) on the pathogens. The immune cells mainly employ a specific receptor, pattern-recognition receptor (PRR), to recognize the PAMPs and stimulate the host immune response. The orange-spotted grouper (Epinephelus coioides) has two TLR9 isoforms, namely gTLR9A and gTLR9B, which are formed via alternative splicing. The main difference between them is that in gTLR9B, the box3 structure is absent in the TIR domain of the C-terminus. Studies have found that CpG oligodeoxynucleotides (ODNs) can induce the production of IL-1β via the TLR9 signaling pathway. The binding of gTLR9A with a CpG ODN was followed by co-localization with the adaptor protein, gMyD88, and subsequent recruitment of the downstream IRAK4 and TRAF6. In contrast, gTLR9B binds to a CpG ODN but cannot recruit the downstream IRAK4 and TRAF6 after binding gMyD88. A further study also found that gTlr9A and gTlr9B possessed a differential expression profiles in a time-dependent manner after stimulated by CpG ODN. Therefore, it was speculated that gTLR9B played the role of a negative regulator. The innate immune response is triggered after the recognition of CpG ODN by TLR9, and a class A ODN can stimulate the maturation of plasmacytoid dendritic cells and induce the secretion of IFNα. Hence, this study aimed at modifying the structure of a class A ODN to investigate the corresponding effects. The results from the in vitro experiments have confirmed that the modification of the central palindromic sequence, phosphorothioate, and 3’-end poly-G tail structure, affected the expression level of IL-1β. The structural changes of a class A ODN can also affect the phagocytic activity of macrophages and the action of the intracellular superoxide anion. Besides, this study also found that both gTLR9A and gTLR21 could specifically bind to a CpG ODN or GpG ODN. When compared with mammals, gTLR9A and gTLR21 were less stringent in recognizing different ODN motifs. The in vivo experiments indicated that class A ODN 1966 was most effective in inducing the expression of IL-1β. Furthermore, both in vitro and in vivo experiments showed that class A ODN 1966 was the most effective in inducing the expression of IL-1β. In this study, a synthetic CpG ODN was used as an adjuvant to further explore its effect and mechanism in the immune function of the orange-spotted grouper. Orange-spotted groupers were re-immunized with the inactivated Grouper Iridovirus (iGIV) vaccines in combination with the immunoadjuvant ODN 1966. The toxicity test using different doses of virus showed that after injecting the iGIV vaccine alone or the iGIV vaccine in combination with different doses of the ODN adjuvant, the mortality rate in the Grouper Iridovirus group was significantly reduced compared with that of the PBS group, in a dose-dependent manner. The analysis of the expression levels of the MCP gene and genomic DNA of the Grouper Iridovirus also showed a decrease in a dose-dependent manner, indicating that the co-immunization of orange-spotted groupers with the vaccine in combination with the ODN adjuvant significantly reduced mortality (a result of Grouper Iridovirus infection) as well as the viral load in the fish. The results in this study also showed that the iGIV vaccine could enhance the antibody titers after immunization of the groupers while the supplementation of ODN adjuvant at a low dose could further enhance the antibody titers of iGIV vaccine. However, excessive dosage of the ODN adjuvant would result in opposite effects, which might be related to the ODN-mediated induction of other immune responses at high concentrations. A further analysis of the expression of immune-related genes revealed that in addition to the induction of innate immune genes, such as gTLR9A, gIL-1β, gTNF-α, and gMX, on the first day after immunization, antibodies were produced on the seventh day after immunization. Additionally, the gCD4 and gCD8 genes were highly expressed on the 14th day after immunization with the iGIV vaccine, while an opposite result was obtained for gT-bet and gGATA3 genes on the seventh and 14th day after immunization in the tendency to induce the Th1 response pathway. Experimental results showed that the supplementing the iGIV vaccine with the ODN adjuvant in an appropriate amount could significantly reduce the mortality caused by the viral infection and improve the immune efficacy via early immunization with the inactivated virus vaccine. It could also effectively reduce the viral load in the fish and further enhance specific antibody titers. Furthermore, iGIV vaccine was found to induce the expression of innate immunity genes, and it might also induce cell apoptosis and involve antibodies in the antiviral responses after immunization. The above-mentioned points support the anti-viral efficacy of iGIV vaccines used in this study and the feasibility of supplementing the iGIV vaccine with a CpG ODN adjuvant for the immunization of the orange-spotted grouper.