牛流行熱病毒(bovine ephemeral fever virus, BEFV)為子彈型病毒科(rhabdoviridae)，具有外套膜之負股RNA病毒，牛流行熱主要影響到畜牧業，會感染乳牛、水牛、黃牛、造成急性發熱、泌乳量下降、死亡等危害，現在主要使用的疫苗乃民國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所製成不活化疫苗，保護力仍不理想。而本實驗室則相繼投入牛流行熱DNA疫苗的研發與改良，前後利用了針對牛流行熱外套膜醣蛋白G基因，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佐劑等製備DNA疫苗，雖然有較好的免疫效力產生，然中和抗體效價仍不夠理想，仍須改進之。
本研究擬結合趨化素CCL5基因，利用其趨化免疫細胞的能力，增加G基因疫苗抗原呈現的機會，增強DNA疫苗的效力。首先完成CCL5基因之選殖並構築於表現載體，藉由轉染細胞的觀察可以證明於第12個小時即開始表現，當與G基因載體共同轉染細胞時，搭配pCCL5的組別能有較好的G基因表現量，可是對於病毒感染細胞所造成的G基因表現量則無影響，顯示CCL5在DNA疫苗方面可以促進G基因載體的表現，但對於病毒複製時G基因表現無影響。此外以前探討VP22作用時，其載體有融合NES序列，故本研究另行構築了NES的表現載體，探討其作用。將上述各重組載體預先轉染BHK-21細胞，再感染病毒測其病毒效價，結果發現預先轉染pBEFV-G、pCCL5、pVP22/NES、pNES、mock，各組病毒效價（TCID50）依序各為10-4.78、10-5.48、10-5、10-5.52、10-5.14 對照組為10-5.49顯示病毒效價並無受到轉染這些載體的影響，僅感染pBEFV-G這組病毒效價略微降低。
在動物實驗方面，利用BALB/c小鼠進行重組載體的肌肉注射，每隔兩週注射一次，總共施打三劑，並固定每週眼窩採血，進行酵素連結免疫吸附試驗（ELISA）測試病毒抗體及中和抗體，發現pBEFV-G搭配pCCL5或是pNES，在第一次注射後第42天相較於單獨pBEFV-G對照組（1:32）都能產生較佳的血清抗病毒抗體效價（1:128），而中和抗體方面（1: 64）相較於對照組（1:16）亦有四倍提升的效力，然而仍無法超越免疫接種不活化病毒組之效力（1:128）。故此G 基因DNA疫苗效力仍須研究加強 。

Bovine ephemeral fever virus (BEFV) is a member of the rhabdoviridae. The BEFV virion contains single-strained, negative-sense RNA genome and envelope. The BEFV can cause acute febrile diease, depressed secretion of milk and even death in cattle and water buffalo. The current vaccine for BEFV were prepared by use of formaldehyde-inactivated virus of 1984 isolate. But its efficacy is not 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, IL-2 and GM-CSF. The G gene was also fused with VP22 to help spreading G gene. All results showed better immune responses, but the antibody responses were not satisfactory.
This study was aimed to further improve G gene DNA vaccine. The chemokine CCL5 was reported to have the ability of attracting leukocytes and dendritic cells, therefore amplifying and shaping the immune responses to DNA vaccine. The CCL5 gene was thus cloned and successfully constructed in expressing plasmid. We demonstrated that CCL5 plasmid could be expressed efficiently in BHK-21 cell. The NES gene which was previously co-constructed with VP22 was also successfully constructed alone. Several combinations of co-transfection with pBEFV-G, pCCL5, pVP22/NES, pNES, and mock vector were performed in BHK-21 cells. We found that pCCL5 could significantly promote the expression of G gene. The pre-transfections of BHK-21 with pCCL5 before virus inoculation were also done. The results showed that pCCL5 had no influence on viral G gene expression. The viral titers(TCID50) were also determined. Results of pre-transfection with pCCL5, pVP22/NES, pNES, and mock vector were 10-5.48, 10-5, 10-5.52, 10-5.14, respectively. All showed similar viral titers as compared with control group (10-5.49). Only pBEFV-G group showed slightly reduced titer of 10-4.78 .
The female BALB/c mice were inoculed by i.m. route with several combinations of the above vectors and three immunizations were performed with two-weeks intervals. Blood samples were weekly collected by orbital bleeding. Sera antibody titers for binding virus were determined by ELISA. Viral neutralizing antibody titers were performed in BHK-21 cell culture system. The results showed that mice immunized with pBEFV-G combined with pCCL5 or pNES had better antibody titer (1:128) than those with pBEFV-G alone (1:32) 42 days after first immunization. Their neutralizing antibody titers (1:64) were also higher than control group (1:16); however still not better than that immunized with inactivated virus (1:128). This modulation of BEFV G DNA vaccine still awaited further improvement.

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