改進慢性B型肝炎治療性疫苗的策略

Strategies to Improve Efficacy of Therapeutic Vaccines for Chronic Hepatitis B Infection

曾友瑜(Yo-Yu Tseng)

指導教授 ： 陶秘華

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.02901

摘要

在慢性B型肝炎病人當中，通常病人無法經由免疫反應來清除體內的病毒。因此，如何有效地引發慢性B型肝炎病人體內的免疫反應來對抗受到HBV感染的肝臟細胞以達到清除HBV的策略正被積極的發展中。
治療性疫苗具有免疫調節的能力，並且幾乎沒有副作用。然而在帶有慢性B型肝炎病人中使用疫苗卻無法表現抗病毒的能力，引起的免疫反應也很低落。不過在病毒量降低的情況下，治療性疫苗不但可以引發免疫反應，並且其效果比起在病毒量較高的時候還要好。因此我們想要測試若是在帶有HBV的基因轉殖鼠上降低HBV病毒量之後再使用疫苗免疫，其疫苗效果是否比起未降低病毒量時還要來的有效。
在我們實驗室2007年發表的文獻指出使用雙股腺相關病毒-8作為載體(double-stranded adenoassociated virus 8-pseudotyped vector; dsAAV2/8)攜帶針對HBV的shRNA，發現帶有HBV-S1的shRNA之AAV(dsAAV2/8/HBV-S1)可以有效的長期抑制HBV基因轉殖鼠體內的hepatitis B surface antigen (HBsAg)以及HBV DNA量。除此之外，dsAAV2/8/HBV-S1也幾乎完全清除了基因轉殖鼠肝臟細胞中的hepatitis B core Ag (HBcAg)。
因此在降低病毒量之後，我們使用可以表現HBcAg的DNA疫苗以及腺病毒疫苗以DNA prime-adenovirus boost的策略免疫老鼠，發現可以有效地引起基因轉殖鼠體內的CD8+ T cell分泌IFN-γ。並且我們降低病毒量之後選擇了不同時間點進行免疫，發現超過8週之後再注射疫苗，就不容易引起CD8+ T cell分泌IFN-γ。
除了降低基因轉殖鼠體內的HBV病毒量可以增強疫苗的效果，我們也嘗試使用佐劑(adjuvant)來加強疫苗的效果。Granulocyte/monocyte colony-stimulating factor (GM-CSF)目前被公認為是促使樹突細胞(dendritic cells; DCs)成熟非常重要的一個細胞激素(cytokine)。CD40 ligand (CD40L)，主要表現在已活化T細胞表面的輔助刺激分子(costimulatory molecule)。因此我們使用可以製造出CD40L/GM-CSF融合蛋白的質體作為我們疫苗的佐劑。
我們發現若是以cardiotoxin預處理過後的老鼠，使用CD40L/GM-CSF融合蛋白的質體佐劑可以有效地加強疫苗引起的抗體反應，並且也可以增強T cell的增生能力。
總而言之，使用疫苗之前先降低病毒量是可值得繼續研究的方法。另外結合兩種佐劑成為一種融合性蛋白來加強疫苗效果，或許是個值得期待的研究方向。

關鍵字

B型肝炎病毒 ； 治療性疫苗 ； shRNA ； 佐劑 ； 顆粒/單核球群落刺激生長因子 ； CD40L ； 融合性蛋白

並列摘要

In chronic hepatitis B virus infection, patients usually cannot eliminate virus by immune response themselves. Therefore, developing the stratrgy to trigger the immune response of chronic hepatitis B patients against HBV infected liver cells is actively persuaded.
Therapeutic vaccine exhibits potent immune modulatory potentials and almost no side effects, but possesses little antiviral capacity and immune response in patients with chronic hepatitis B. However, reducing virus titer can enhance therapeutic vaccine efficacy, and acquire better immune response than in higher virus titer condition as well. Therefore, we tried to test whether performing the therapeutic vaccine after reducing virus titer in HBV transgenic mice can get more efficient response than those mice didn’t reduce virus titer.
In our previously study published in 2007, using double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) cantaining shRNA specific to HBV, especially HBV-S1 (dsAAV2/8/HBV-S1), can long-term inhibit the hepatitis B surface antigen (HBsAg) and HBV DNA in HBV transgenic mice. And the inhibition can be lasting for 120 days. Besides, dsAAV2/8/HBV-S1 almost clears the hepatitis B core Ag (HBcAg) of hepatocyte in transgenic mice.
After inhibiting virus titer in transgenic mice, we used DNA vaccine and adenovirus vaccine which encode HBcAg to immunize mice with DNA prime-adenovirus boost strategy. We found out this strategy can efficiently induce CD8+ T cell secreting IFN-γ. Meanwhile, we also choose several time points of immunization after HBV titer reduction. As vaccine administration was longer than 8 weeks after inhibiting virus titer, it showed no IFN-γ secreting response.
Despite of reducing HBV titer in transgenic mice in order to enhance vaccine efficacy, we also tried to improve vaccine ability by using adjuvants. Granulocyte/monocyte colony-stimulating factor (GM-CSF) is now considered as an important cytokine to make dendritic cell maturation. CD40 ligand (CD40L) is a costimulatory molecule major expressing on activated T cell. Therefore, we construted the expression vector that encodes CD40L/GM-CSF fusion protein as our vaccine adjuvant.
We found that using plasmid encoding CD40L/GM-CSF fusion protein in cardiotoxin pretreated mice can efficiently enforce vaccine-induced antibody response. Additionly, it can improve T cell proliferation.
In conclusion, reducing virus titer before vaccination will be a strategy worth studying. Meanwhile, combination of two adjuvants as a fusion protein might be an inspiring method to improve vaccine efficacy.

並列關鍵字

Hepatitis B virus ； therapeutic vaccine ； shRNA ； adjuvant ； granulocyte/monocyte colony-stimulating factor (GM-CSF) ； CD40 ligand (CD40L) ； fusion protein

參考文獻

1. Ganem, D. and A.M. Prince, Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med, 2004. 350(11): p. 1118-29.

2. Chisari, F.V. and C. Ferrari, Hepatitis B virus immunopathogenesis. Annu Rev Immunol, 1995. 13: p. 29-60.

3. Rehermann, B., et al., Cytotoxic T lymphocyte responsiveness after resolution of chronic hepatitis B virus infection. J Clin Invest, 1996. 97(7): p. 1655-65.

4. Dienstag, J.L., et al., Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med, 1999. 341(17): p. 1256-63.

5. Boni, C., et al., Lamivudine treatment can overcome cytotoxic T-cell hyporesponsiveness in chronic hepatitis B: new perspectives for immune therapy. Hepatology, 2001. 33(4): p. 963-71.