牛流行熱病毒(bovine ephemeral fever virus, BEFV)為子彈型病毒科(Rhabdoviridae)一員，經由庫蠓等節肢動物為媒介，會感染牛隻造成急性發熱，精神抑鬱、食慾不振、呼吸症狀、流產、產乳量下降等症狀。其外套膜G醣蛋白(envelope glycoprotein G)具中和性抗原決定部位，可以刺激牛隻產生保護性免疫反應，但目前疫苗效力仍不佳。VP22為第一型單純疱疹病毒的內殼蛋白，具有特殊的細胞間散佈能力，可由被感染或轉染的原發細胞進入周邊未感染或轉染的細胞，已被利用為蛋白質傳輸的運載工具。
先前，本實驗室沈雅玲學姊已成功利用BEFV-G重組載體合併使用小鼠細胞素IL-2和GM-CSF的DNA載體為混合DNA疫苗，但所產生的免疫效果不夠顯著。本研究擬改進之，另行構築一結合牛流行熱病毒醣蛋白G基因和單純疱疹病毒VP22基因的重組融合載體(pBEFV-G/VP22)為DNA疫苗，使其在細胞內形成融合蛋白，並藉由VP22的細胞間運輸能力，將標的G抗原散佈到周邊鄰近細胞，以增加抗原呈現機率，提高免疫反應。動物實驗方面，我們以BALB/c小鼠為模式，肌肉注射pBEFV-G/VP22、pBEFV-G、pVP22、或不活化的BEFV，每十天一劑共三次，並於每次接種完的48小時後施打CpG核苷佐劑(ODN1628、ODN1628 control)。結果顯示，三次接種完成21天後(即第一次免疫接種後第42天)，控制組及免疫接種pBEFV-G、pBEFV-G/VP22或不活化BEFV的組別，在搭配注射ODN1628後，血清與病毒結合之抗體效價分別為≦1：2、1：32、1：64、1：128；病毒中和抗體效價亦同。顯示VP22確實可以提升二倍抗體免疫反應。未添加ODN1628(即使用ODN1628 control或以PBS取代ODN1628)之實驗中，控制組及免疫接種pBEFV-G、pBEFV-G/VP22或不活化BEFV的組別，病毒中和抗體效價分別為≦1：2、1：8、1：16、1：64，皆比添加ODN1628低，顯示ODN1628佐劑可以提高二至四倍免疫反應。

Bovine ephemeral fever virus (BEFV), a member of the rhabdoviridae, is an arthropod-borne viral disease. It can cause a sudden onset of fever, depression, lameness, respiration symptoms, increase in abortions, and reduce milk production in cattle. The BEFV envelope G glycoprotein contains type-specific and neutralizing antigenic sites and can induce protective immunity in cattle. VP22 is a tegument protein of herpes simplex virus type 1. It has the specific capability of intercellular spread, where by the VP22 protein exits infected or transfected cells and enters neighboring cells. VP22 has been utilized as a vehicle for trafficking other proteins.
Previously, we had explored the use of pBEFV-G DNA vector combined with IL-2 and/or GM-CSF gene DNA vectors as adjuvants. The antibody responses were not satisfactory. This study was aimed to construct a new plasmid pBEFV-G/VP22, i.e., G gene fused to VP22, as vaccine exploiting the intercellular trafficking capacity of VP22 to help disseminating G protein to neighboring cells and enhancing immune response. The BALB/c mice were passively immunized by intramuscular inoculation with pBEFV-G/VP22, pBEFV-G, pVP22, or inactivated BEFV, CpG ODN adjuvant (ODN1628 and ODN1628 control) was administered 48h later. Three immunizations were performed with 10-days interval. After 21 days of the last immunization, mice sera were collected and antibody titers were determined. The virus-binding antibody titers for mice groups of control, pBEFV-G, pBEFV-G/VP22, or inactivated BEFV immunized in combination with ODN1628 adjuvant were 1:2, 1:32, 1:64, and 1:128, respectively. The virus-neutralizing antibody titers were same as above. The VP22 can thus enhance antibody response by two-fold. The virus-neutralizing antibody titers for mice groups of control, pBEFV-G, pBEFV-G/VP22, or inactivated BEFV immunized without ODN1628 (i.e. with ODN1628 control or PBS control) were ≦1:2, 1:8, 1:16, 1:64, respectively. The ODN1628 adjuvant thus shows two- to four-fold increase of immune responses.

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