評估水禽雷氏桿菌不同類型疫苗以不同免疫策略之免疫調節效力

水禽雷氏桿菌(Riemerella anatipestifer)為造成台灣水禽業之重要傳染性疾病，以雛鵝和雛鴨最易受感染且死亡率高，因而造成嚴重的經濟損失。水禽雷氏桿菌是革蘭氏陰性桿菌，目前至少有21多種血清型。主要引起敗血症以及傳染性漿膜炎(infectious serositis)。感染的病禽會有打噴嚏、咳嗽、排綠色下痢便及頭頸震顫等病徵。現今使用之市售疫苗，為不活化三價菌苗，無法提供其他血清型有效的交叉保護效果。研究指出雷氏桿菌之外膜蛋白(Outer membrane protein A; OmpA)是重要的共同抗原，可應用於跨越血清型之疫苗開發。本實驗室已經成功建立原核系統表現外膜蛋白OmpA (rOmpA)，及將OmpA基因選殖於pTCY載體上，結合CpG ODN佐劑能有效引起免疫反應。本研究目的將DNA疫苗、次單位疫苗，和市售不活化菌苗進行prime-boost免疫策略於鴨隻，以ELISA抗體力價、細胞激素表現量及T細胞含量，探討最適合用於prime-boost免疫策略之組合，及新型疫苗與傳統疫苗免疫反應之差異。在抗體力價結果，再補強免疫後所有免疫組接顯著高於控制組(p<0.05)，而且Subunit + Inactivated組與Inactivated + Inactivated能夠誘導長期抗體反應直至12週。在CD4+/CD8+ T細胞數結果上，CD4+在初次免疫後，所有免疫組皆顯著高於控制組；CD8+在DNA+DNA，Subunit + Subunit，Subunit + Inactivated和DNA + DNA皆顯著高於Inactivated + Inactivated組。在細胞激素結果上，IFN-γ、IL-6與IL-12 mRNA表現量在免疫後四週結果發現，DNA + Subunit組相較其他免疫組有顯著性差異(p < 0.05)。在交叉反應試驗結果，Subunit + Subunit組及DNA+DNA組證明我們所構築之Subunit疫苗及DNA疫苗是具交叉反應的。結論，統整免疫分析後結果，我們發現不同疫苗免疫組合相較於相同疫苗免疫組合是具有較好的結果，尤其是有免疫Subunit的組別，在免疫分析結果上是較好的。

關鍵字

水禽雷氏桿菌；次單位疫苗； DNA vaccine ； CpG ODN

並列摘要

Goslings and ducklings are susceptible to Riemerella anatipestifer (Ra) infection with high mortality. Ra is a Gram-negative bacillus, and at least 21 serotypes exist. Ra causes septicemia and infectious serositis. The infection of Ra causes sneezing, cough, greenish diarrhea, and trembling of the head and neck. Nowadays, commercial inactivated trivalent consisting of vaccines do not provide an effective cross-protection for other serotypes. Previous research indicates that outer membrane protein A ( OmpA ) of Ra is the most cross-reactive antigen and cross-serotype of vaccine development. We have expressed recombinant OmpA (rOmpA) protein by the prokaryotic expression system cloned the OmpA gene into the pTCY vector and combined with the CpG ODN adjuvant. They effectively elicited immune responses. The objective of this study was to use DNA vaccine, subunit vaccine with CpG ODN, and commercially inactivated vaccines for the prime-boost immunization in duck. We detection antibody titer by ELISA, cytokine expression and percentages of CD4+/CD8+ cells to explore the most suitable combination for prime-boost immunization. In the antibody titer results, that after boosting the antibody titer of all vaccinated groups were significantly higher control group (p < 0.05); and Subunit + Inactivated and DNA + DNA were able to induce effective immune long-term antibodies responses till 12 weeks. In the percentage of CD4+/CD8+ T cells results, that after prime immunization the CD4+ of all immunized groups were significantly higher than control group; the CD8+ of DNA + Subunit, Subunit+ Subunit, Subunit + Inactivated and DNA + DNA were significantly higher than Inactivated + Inactivated. In the cytokine expression results, the IFN-γ, IL-6 and IL-12 mRNA expression of DNA + Subunit was significantly higher than other immunized groups (p<0.05). In the cross-reactive test results, the Subunit + Subunit and DNA + DNA are demonstrated our constructed Subunit vaccines and DNA vaccines are cross-reactive. Conclusions, we found immunization strategies that heterologous vaccine have better results than homologous vaccine, especially those with immune Subunit of the group, in the immunoassay results are better.