| Summary: | 碩士 === 國立臺灣大學 === 免疫學研究所 === 86 === C型肝炎，一個造成全世界公共健康問題的疾病，正肆虐著我們。以台灣而言，一般
人口中C型肝炎感染率平均約有百分之二左右，估計在台灣約有30萬以上C型肝炎感
染者，造成流行病學上的恐慌。更甚者，由於C型肝炎的變異性大，常常造成感染者
無法有效地清除病原菌，造成慢性肝炎，經年累月轉變成肝硬化，甚至肝癌。而至
目前為止，仍無有效的疫苗被使用。
DNA疫苗，一種可以有效地引發宿主產生體液性 (humoral) 與細胞性 (cell-mediated)
免疫反應的新技術產物，給了我們一道曙光。尤其，DNA疫苗在純化與製備上，
相當簡單、快速、花費又少；又因為相當穩定，可耐熱，故在保存上極為方便。
而且，所引起的免疫反應較類似於病原菌疫苗：成效高、免疫性強、且能廣泛地
預防類似病原之傳染。
本論文即是基於上述二者，應用C型肝炎病毒核心蛋白來探討DNA疫苗的免疫反應
與機制。我們選定C型肝炎最具有高度保留性的核心蛋白為主角，經由肌肉注射的
方式，將質體DNA送進老鼠體內，以期能了解其免疫反應的發生。本實驗設計主要
是針對：(1) DNA疫苗的免疫機制探討；(2) DNA疫苗結合淋巴介質佐劑是否能強
化免疫反應的發生；(3) DNA疫苗經由多部位 (multiple sites) 與單一部位
(single site) 多次注射 (multiple boost) 後，其免疫反應是否有差異；(4)
DNA疫苗與傳統蛋白質疫苗所引起的免疫反應是否有不同。而由實驗結果顯示：
1. 以報告基因所得的結果顯示，合併淋巴介質載體注射後，可以使得蛋白質的
表現量增多；同時，也引發較多的浸潤性細胞出現在肌肉組織周圍；而且，在
近端的淋巴結中，於施打後24小時，可以偵測到抗原的活性；
2. 在合併了淋巴介質的質體DNA共同注射至老鼠肌肉後，於第四週即可測得HCV
專一性抗體，但是，在無淋巴介質的共同施打下，則無法偵測到抗體反應。此外
，在淋巴介質的幫助下，也引發了較好的細胞性反應；
3. 利用多部位多次注射的結果，可以使得老鼠的免疫反應增強，尤其在結合了
淋巴介質後，效果可以更好；
4. 傳統蛋白質疫苗確實在抗體的效價上比DNA疫苗高出許多，且產生時間相對快
很多，約只需2∼3週即可；至於細胞性免疫反應也可產生，但是反應的程度較
DNA疫苗低。
Hepatitis C virus (HCV) infection has become an important public health
problem worldwide. In Taiwan area, it has been estimated that more than
300,000 people, 2% of general population, are found to have HCV
infection. Furthermore, the high mutation rate of HCV results in the
chronicity of viral infection and subsequent development of
hepatocellular carcinoma. Both the epidemiological and clinical features
indicate that control of HCV infection demands more effectively
preventive regimens, an efficient vaccine is among one of them.
DNA vaccine can effectively induce humoral and cellular immune responses.
Compared to traditional protein vaccines, generation and purification
of DNA vaccine is relatively simple and inexpensive, which is especially
of benefit for vaccine design in developing countries. Most importantly,
DNA vaccines have been shown to evoke a strong cell-mediated immune
response which is more close to natural infection of pathogens.
In this study, mice were injected with HCV core protein-encoding DNA
plasmid into skeletal muscle to induce immune response and explore
the mechanisms of DNA vaccine. The core protein of HCV has been noted
with highly conserved sequence and strong immunogenecity. The aims of
this study are to explore the mechanisms of DNA vaccination and
possibly enhance immune response with cytokine adjuvant or multiple
boost of DNA immunization. Furthermore, the difference between immune
responses triggered by protein and DNA immunization was also investigated.
The data demonstrated that: 1) Mice immunized with HCV core plasmid
DNA and adjuvant cytokine gene can induce a strong cellular immunity
and HCV core-specific antibody titers as early as 4 weeks after
injection; 2) The increased expression level of protein and
infiltrating cells around muscle tissue were noted when mice were
coinjected with reporter gene and cytokine gene plasmid. Moreover,
the protein can be detected in inguinal node 24 hours after
injection; 3) With multiple sites and multiple boost immunization,
both humoral and cellular immunity were noted even without cytokine
plasmid DNA; 4) The antibody titer induced by protein immunization
was higher than that of DNA immunization, however, the relatively
lower cellular immune response was noted.
In the future, more experiments are needed to clarify the
mechanisms and the novel strategy of delivering naked DNA to
induce effective immune response. Furthermore, the feasibility
of gene delivery as a potential vaccine for HCV infection will
also be investigated.
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