CpG寡去氧核醣核酸誘導石斑魚免疫反應及其在發展虹彩病毒疫苗上的應用

Abstract

先天性免疫之誘發有賴於免疫細胞能夠有效辨識病原體上稱為「病原相關分子模板」(pathogen associated molecular patterns, PAMPs )的特殊結構；主要藉由一類特殊受體PRR ( pattern-recognition receptor )來辨認PAMPs，進而激發宿主產生免疫反應。點帶石斑魚的類鐸受體9 (TLR9) 具有兩種亞型 (isofrom)，分別為gTLR9A以及經由選擇性剪切(alternative splicing)後，在C端TIR domain 缺少box3結構而形成的gTLR9B。研究結果發現CpG ODN能夠經由TLR9的訊息傳遞途徑，誘導IL-1β的產生，而gTLR9A與CpG ODN結合後，會再與其轉接子gMyD88結合，隨後招募下游的IRAK4與TRAF6，而gTLR9B亦會與CpG ODN結合，但在與轉接子gMyD88結合後，則無法招募下游的IRAK4、TRAF6，且經由CpG ODN (CpG寡去氧核醣核酸) 刺激後，gTLR9A 及 gTLR9B會隨處理時間增加而有不同趨勢的表現特性，研究結果也推測gTLR9B應是扮演negative regulate的功能。 CpG ODN 被TLR9 辨識後會啟動先天性免疫反應，而class A ODN主要是刺激漿細胞樣樹突細胞成熟和誘導IFNα分泌，本研究藉由改變class A ODN的組成結構後，再探討其所造成之影響，在體外實驗結果證實若改變central palindromic序列、phosphorothioate 修飾或3端poly-G尾巴的結構後，最終均會影響IL-1β的表現量，而改變class A ODN的組成結構會影響巨噬細胞吞噬活性與細胞內超氧陰離子的作用。本研究也發現gTLR9A及gTLR21 (點帶石斑魚類鐸受體21) 與CpG ODN、GpG ODN均能產生專一性結合，此點與哺乳動物相較之下，可知gTLR9A及gTLR21兩者對於辨識不同ODN motifs的管控並不嚴謹，而在活體實驗部分，也發現以class A ODN1966誘導IL-1β表現的效果最佳，且class A ODN 1966無論是於本研究in vitro 或 in vivo的實驗結果，均發現其對誘導IL-1β的表現有最佳效果。 進一步以人工合成CpG ODN作為佐劑，並探討其在點帶石斑魚的免疫作用功效與作用機制後發現，不活化石斑魚虹彩病毒 (iGIV) 疫苗搭配佐劑ODN 1966一同免疫點帶石斑魚後，再以不同病毒劑量進行攻毒實驗之結果顯示，注射iGIV疫苗或將其搭配不同劑量之ODN佐劑的組別，與PBS組相較之下，均可顯著降低虹彩病毒感染後所造成的死亡率，且呈現dose dependent的效果，若分析石斑魚虹彩病毒MCP 基因及genomic DNA表現量，也發現是呈現dose dependent降低的情況，顯示搭配ODN佐劑再免疫點帶石斑魚後，不僅能夠顯著降低虹彩病毒感染所造成的死亡率，更能降低魚體內所含的病毒量。另外，也發現iGIV疫苗本身即能提高石斑魚免疫後的抗體力價，若再添加低劑量之ODN佐劑使用，更能提升其力價，但過量則會呈現相反效果，推測可能與ODN在高濃度時會誘發其他免疫反應有關，因此本研究進一步分析免疫相關基因表現，發現除了在免疫後第一天即能開始誘導先天性免疫基因gTLR9A、gIL-1β、gTNF-α及gMx外，亦能在免疫後第七天開始誘導抗體的產生，gCD4和gCD8在iGIV則是在疫苗免疫後第十四天有較高的表現量，而gT-bet和gGATA-3在iGIV疫苗免疫後第七天及第十四天的基因表現則呈現相反結果，且傾向誘發Th1的免疫反應途徑。 研究結果發現iGIV疫苗搭配適量的ODN佐劑使用，確實能夠顯著降低虹彩病毒感染後所造成的死亡率，及有效提高不活化病毒疫苗在免疫早期的抗病毒保護功效，同時也可顯著降低魚體內的病毒含量與提升其專一性抗體力價，再者，iGIV疫苗除了能夠誘導先天性免疫基因之表現，也能在免疫後誘發毒殺性細胞和抗體參與抗病毒反應，本研究結果證實iGIV疫苗的抗病毒效果以及將其搭配CpG ODN佐劑免疫點帶石斑魚之可行性。

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.