牛流行熱病毒( bovine ephemeral fever virus, BEFV )為子彈型病毒科( Rhabdoviridae )的暫時熱病毒屬( Ephemerovirus )，經由糠蠓等節肢動物作為媒介，會感染牛隻造成急性發熱、呼吸症狀、流產、產乳量下降和死亡等症狀，影響畜牧業經濟損失。現行使用之疫苗為民國73年所分離之病毒株經福馬林不活化後，加入氫氧化鋁膠作為佐劑製成不活化疫苗，保護力仍不理想。本實驗室相繼投入了針對牛流行熱外套膜醣蛋白G基因 pBEFV-G核酸疫苗之研發與改良，前後曾搭配細胞激素IL-2或GM-CSF基因，或是改良G基因本身結構，融合在細胞間具移動能力之VP22基因，或給予CpG核苷佐劑，或搭配CC型趨化素CCL5基因與核輸出訊號NES基因作為免疫佐劑改進核酸疫苗，雖然有較好的免疫效力產生，但中和抗體效價仍不夠理想。
本研究擬利用CXC型趨化素CXCL10基因與調控造血相關之細胞激素Flt-3L基因作為免疫佐劑，期望能藉由趨化免疫細胞的能力和促進樹突細胞聚集累積的能力，增加核酸疫苗施打處的局部免疫反應並提升抗原呈現細胞移動至抗原呈現處的機會，進而提高G基因抗原之呈現，提升核酸疫苗之效力。首先在細胞培養實驗中發現，共同轉染G基因與pCXCL10的組別具有較好的G基因表現量，另外預先轉染pCXCL10也可以提升後續感染病毒後病毒G基因之表現量，而從病毒效價(TCID50)測定發現預先轉染pBEFV-G、pCXCL10、pFlt-3L、pNES或mock pcDNA3.1，各為10-4.84 、10-5.30、10-5.25、10-5.37及10-5.37，與對照組(10-5.50)比較並無明顯差異，僅感染pBEFV-G組病毒效價略微降低。
在動物實驗方面，將各類重組DNA載體注射於BALB/c小鼠肌肉組織中，每隔兩週注射一次，總共施打三劑，並固定每週眼窩採血，利用ELISA測試抗病毒抗體效價及中和抗體效價，發現在第一次注射後42天相較於單獨注射pBEFV-G所引發病毒抗體效價 (1：32)，pBEFV-G搭配pCXCL10或pFlt-3L基因組別，其效價皆明顯提升 (1：128和1：256)，而中和抗體方面 (1：64和1：128) 相較於單獨注射pBEFV-G (1：32) 亦有二到四倍之提升，另外pBEFV-G合併使用pCXCL10和pFlt-3L基因組別，所引發中和抗體效價為1：256，而抗病毒抗體效價更可達1：1024，免疫反應最佳。

Bovine ephemeral fever virus (BEFV) is classified as the genus Ephemerovirus in Rhabdoviridae family. Arthropods, like Culicoides, are the media of the BEFV. The BEFV can cause a sudden onset of fever, depression, lameness, respiration symptoms, increasing in abortions, reducing milk production and death in cattle. It can cause significant economic impact in livestock industry. The vaccine currently used was formaldehyde-inactivated virus of 1984 isolated in Taiwan and added Al(OH)3 as adjuvant for inactivated vaccine. But its protection is not effective enough. We have tried to improve DNA vaccine of BEFV before. The glycoprotein G gene of BEFV was tested as a target gene combined with cytokine genes, e.g. IL-2 and GM-CSF, or combined with CC type chemokine CCL5 and nuclear export signal (NES). The G gene was also fused with VP22 to help spreading G gene. All of these studies showed better immune responses, but the neutralizing antibody responses were not very satisfactory.

This study was aimed to further improve G gene DNA vaccine. CXCL10 , which is a CXC type chemokine, was reported to have ability of attracting leukocytes; while Flt-3L, which is a cytokine related to hematopoiesis, was reported to have ability of accumulating dendritic cells. CXCL10 and Flt-3L were expected to improve local immune response by improving the chance of APC aggregation to the antigen site, that increasing efficiency of DNA vaccination. In cell culture experiments, we found that co-transfection of G gene with pCXCL10 gene could increase G gene expression. Pre-transfection of pCXCL10 before virus infection could also elevate the expression of G gene of virus. However, in virus growth experiment, results (TCID50) of pre-transfection with pBEFV-G, pCXCL10, pFlt-3L, pNES and mock pcDNA3.1 were 10-4.84 , 10-5.30, 10-5.25, 10-5.37 and 10-5.37, respectively. All showed similar viral titers as compared with control group (10-5.50). Only pBEFV-G group showed slightly reducing titer (10-4.84).

The female BALB/c mice were inoculated by i.m. route with several combinations of the above vectors and three immunizations were performed with two-week intervals. Blood samples were weekly collected by orbital bleeding. Sera antibody titers for virus were determined by ELISA and viral neutralization antibody titers were also determined in cell culture. The results showed that mice immunized with pBEFV-G combined with pCXCL10 or pFlt-3L had better anti-virus antibody titers (1:128 and 1:256, respectively) than those with pBEFV-G alone (1:32) 42 days after first immunization. Viral neutralizing antibody titers of the above two groups (1:64 and 1:128, respectively) were also higher than pBEFV-G alone (1:32). Viral antibody titers of the group injected with pBEFV-G, pCXCL10 and pFlt-3L altogether showed highest responses of anti-virus antibody titers and viral neutralizing antibody responses , 1:1024 and 1:256, respectively.

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