以Sindbis病毒攜帶腫瘤抗原基因對肝腫瘤進行免疫治療研究

Abstract

肝癌是遍及世界且是最致命的疾病之一，每年世界上約有一百二十五萬人因為罹患肝癌而死亡，大部分的肝細胞癌發展自因為B型、C型肝炎病毒慢性感染或酒精濫用所造成的慢性肝炎與肝硬化。肝臟移植是目前治療肝癌較理想的選擇但不是一個普及的治療方法，因為器官來源有限且只限於符合移植條件的少數病人且無法解決肝癌的復發，外科手術可切除腫瘤但也不能解決肝癌復發的問題。另外酒精注射或是動脈栓塞手術只有緩解病情的效果。加上傳統化療與放射線療法治療效果不佳，所以肝癌存活率很低，當前需要發展出治療肝癌的新策略。 在這個實驗中，我打算建立Sindbis viral vector攜帶肝腫瘤之抗原基因，甲型胎兒蛋白（AFP）基因，對帶有多發性肝腫瘤的老鼠進行免疫治療。該病毒載體已證實比傳統DNA載體疫苗更能打破對自體抗原的免疫耐受性與活化更多腫瘤相關抗原的免疫反應。利用CMV啟動子所調控的replicon載體攜帶非結構基因與吾人有趣的基因與另一個helper replicon攜帶結構基因同時以磷酸鈣沈澱法將其送入293T細胞，製造出高量的病毒顆粒。接下來將攜帶CRT、rAFP與CRT-rAFP基因的病毒顆粒感染BHK細胞後，以西方墨點法證明其在感染細胞中可表現CRT、rAFP與CRT-rAFP基因。另外學長建構的GM-CSF與IL-12載體產生的病毒感染BHK細胞後，也可以ELISA證明其表現能力。 在老鼠模式部分，我們於飲水中加入DENA可誘發Wistar大鼠產生多發性肝癌。首先，以RT-PCR證明此肝癌會表現AFP。隨後，我們以攜帶CRT-rAFP的Sindbis病毒並合併攜帶細胞激素GM-CSF或IL-12的Sindbis病毒，以肌肉內注射來治療此肝癌。以每隻大鼠的MTBI值或是腫瘤種體積（total tumor volume）來量化腫瘤的生長與衡量治療的效果。結果顯示CRT病毒治療效果與CRT-rAFP或CRT-rAFP合併GM-CSF或CRT-rAFP合併IL-12的治療效果差不多。儘管以免疫組織染色在CRT-rAFP合併GM-CSF或CRT-rAFP合併IL-12治療組可以看到較多的腫瘤滲入淋巴球（tumor infiltrate lymphocyte），但是推測活化出的免疫反應太弱以致於無法在CRT病毒產生之療效外再產生加成性的效果。另外以ELISA測量大鼠血清內IL-12濃度來分析IL-12載體病毒在活體內的表現，發現施打疫苗第二天之後表現迅速大幅下降，一週後即使再施打一次病毒，IL-12表現量遠不如第一次施打後的表現量。而只有在CRT-rAFP+IL-12治療組有看到誘發出微量的IFN-γ。推測施打第一次Sindbis病毒後，老鼠會對Sindbis病毒產生免疫力因而限制了下次病毒施打的治療成效。未來可改變治療的策略。例如以病毒DNA載體合併CRT-rAFP、GM-CSF和IL-12在腫瘤內做重複的施打，另外發現腫瘤內有免疫抑制因子包括FoxP3與TGF-β的表現，將來除了更加強正向的活化免疫反應的力道之外，另外也需注意並想辦法減少腫瘤內或腫瘤附近的免疫抑制因子，來達到最好的治療效果。

Hepatocellular carcinoma is one of the most common malignancies worldwide. The annual number of mortalities from HCC worldwide is estimated at 1,250,000. Most of the cases, this malignant tumor develops on a background of chronic hepatitis and cirrhosis caused by chronic hepatitis B and C viral infection or alcohol abuse. Liver transplantation epitomizes a radical treatment option, but it is not applicable universally because limited organ donations and the occurrence of relapse and only when patients meet stringent specific criteria. Surgical resection removes large tumors, which is associated with high risk of relapse. Ethanol injection, arterial embolization are mainly performed with palliative intent. Conventional chemotherapy or radiotherapy is ineffective for HCC. The survival rate of individuals with HCC is low. There is an urgent need for creating new effective therapeutic strategies for HCC. In this study, we attempted to treat a multifocal liver tumor model with a Sindbis viral vaccine which expresses the liver tumor antigen, α-fetoprotein(AFP). Previously, our laboratory has constructed a DNA-based Sindbis viral vector system. The system consists of a replicon vector, encoding the viral nonstructural genes and the gene of interest and a helper vector, encoding the viral structural genes, both of which were driven by the CMV promoter. The two DNA vectors were co-transfected into 293T cells by calcium phosphate method to obtain high titer of viral particles, which can infect cells once. Next, the recombinant viral particles were used to infect BHK cells. Western blot analysis indicated that these viral particles, including CRT, rAFP, and CRT-rAFP dsin/c, could express the encoded genes respectively in the infected cells. ELISA was also performed to prove the expression of GM-CSF and IL-12 from the viral vector in infected BHK cells. Expression of AFP in the DENA induced hepatocellular carcinoma was proven by RT-PCR. Next, we applied the Sindbis virus carrying an AFP gene conjugated with a calreticulin gene in combination with GM-CSF or IL-12-carrying Sindbis virus for the treatment of DENA-induced spontaneous hepatocellular carcinoma in Wistar rat by intramuscular administration. The tumor growth for each rat was evaluated by modified tumor burden index (MTBI) and total tumor volume to indicate the therapeutic effect. The results show that CRT-dsin/c virus and CRT-rAFP+cytokine viruses generated almost the same therapeutic effect, notwithstanding that CRT-rAFP+cytokine dsin/c co-administration could recruit more tumor infiltrating lymphocytes, examined by immunohistochemistry. The data suggested that the anti-tumor immunity elicited by CRT-AFP or CRT-AFP+cytokine might be too weak to augment the therapeutic effect of CRT dsin/c virus. In vivo expression of the transduced gene was analyzed by ELISA of serum IL-12 levels. It is found that in vivo expression of Sindbis virus decreased enormously 2 days after the first immunization, and that the expression level of the second round immunization taking place 7 days later was far below the first immunization. A low level of serum IFN-γ was induced only in the CRT-rAFP+IL-12 group. Therefore, we predicted that the animals might induce immunity to resist the second Sindbis virus infection thus limiting the therapeutic effect of the second immunization. To prolong the effects, one may need to modify the vaccination protocol by repeated injecting Sindbis “DNA” vector in combination with CRT-rAFP, GM-CSF, or IL-12-expressing vector DNA to elicit more effective immunity against the carcinogen-induced multifocal tumor. Furthermore, expression of immunosuppressive factors, such as FoxP3 and TGF-β, was also observed in this tumor model. As a result, in addition to enhance the anti-tumor immunity, the counter activity of the immunosuppressive factors should also be taken into consideration in order to achieve the maximal therapeutic effects.