以CBA 小鼠研究單株B型肝炎表面抗原抗體對於小鼠體內所帶之B型肝炎病毒的影響

The effect of anti-HBs monoclonal antibody on intrahepatic HBV in the hydrodynamic injection CBA mouse model

羅際勝(Ji-Sheng Lo)

指導教授 ： 陳培哲

國立臺灣大學/醫學院/微生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10831

摘要

B型肝炎是世界主要流行的傳染疾病之一，目前世界上約有三分之一的人口可能感染B型肝炎，而其中約有三點五億人是B型肝炎的帶原者。慢性B型肝炎的成因有許多，比如宿主的免疫系統、性別、年齡，病毒本身的基因表型，基因的表現能力，宿主所在的環境因素影響。其中宿主免疫系統是相當重要的。在慢性B型肝炎患者中，NK cell活性，IFN-γ，CD4、CD8 T cell活性均較急性感染的患者來得低。表示B型肝炎患者無法有效的清除病毒。為了能夠有效地治療甚至根除慢性B型肝炎，強化宿主的免疫反應以達此一目的是近年來思考的方向之一。近來研究指出了免疫蛋白除了直接作用在抗原的功能外，也可能扮演了調整免疫系統的角色，因其可和許多免疫細胞，如巨噬細胞、樹突細胞等結合而抑制了發炎作用。但以B型肝炎免疫蛋白作為治療慢性B肝感染並沒有顯著的效果。
實驗合作團隊製造了特定的B型病毒表面抗原抗體─E6F6，其所針對的抗原表現位置是第119至125的氨基酸序列，並且在B型肝炎基因轉殖小鼠研究中有明顯抑制表面抗原的作用。但基因轉殖小鼠並不適合做為研究抗體是否會誘發宿主免疫反應而達到抑制病毒表現此一假說。因此本篇論文將聚焦於E6F6對於B型肝炎清除能力弱的小鼠─CBA/Caj的影響，並長時間的觀察。初步觀察的結果中發現，給予E6F6的小鼠其血清中的B型肝炎表面抗原在一天之內就很明顯地下降，且在九個星期的觀察期內，表面抗原完全沒有回升的跡象。並在第六個星期測其血液中anti-HBs 抗體的量，發現雖然小鼠沒有自主的產生抗體，但也沒有偵測到E6F6的存在。小鼠肝組織中surface protein表現量並沒有完全受到E6F6抑制，仍有少部分表現，而core protein也沒受到顯著地抑制。但HBV RNA的表現量完全不受到影響。為了研究E6F6早期對於小鼠中HBV的影響，我們在七天內觀察其血清中HBsAg的變化以及在細胞中蛋白質及RNA的表現。血清中HBsAg下降的十分迅速，但細胞中core protein 受到的影響較少，但surface protein便可清楚發現施打前與施打後七天有所差異。但只有一劑E6F6的給予似乎無法完全地抑制血液中HBsAg。
綜合以上結果，我們提出了一個可能性：在施打E6F6初期，可能因抗體中和反應使得在血液中的HBsAg和部分細胞中surface protein受到抑制。但在後期，數周後，E6F6逐漸降解後，可能改由其他由抗體所引發的機制繼續抑制HBsAg分泌至細胞外。而此一機制初期可能需要高劑量的antibody所誘發。

關鍵字

B型肝炎病毒 ； B型肝炎表面抗原 ； 慢性B型肝炎 ； B型肝炎免疫蛋白 ； 靜脈給予免疫蛋白

並列摘要

Hepatitis B virus (HBV) is a common infectious disease in the world. About one-third people in the world may be infected, and 350 million people are chronic infected. There are many factor involve in HBV pathogenesis, host immunity, gender, age, viral genome type, gene expression, and environmental factor. The immunity response is important for viral clearing. The NK cell activity, IFN-γproduction, CD4 and CD8 T cell activity are relatively weak than acute infection in the patients with HBV chronic infection. In order to control, even eliminate, the viral infection, the activation of dysfunctional immunity is a new way. In recent studies think the intravenous immunoglobulin (IVIG) is as an immune-modulator because IVIG can interact with immunity effector, DC cell and macrophage. However in the treatment with hepatitis B immunoglobulin (HBIG) to chronic viral infection is not useful.
E6F6 is the monoclonal anti-HBs antibody against the a.a.119-125 of HBV surface protein, and dramatically suppress the HBsAg in transgenic mouse model. However the transgenic mouse is a good model to identification the immunoglobulin to suppress HBsAg via induction of immunity. This thesis will focus on the E6F6 effects on the HBV expression in the long-term HBV carried CBA/Caj mouse. In the nine weeks of observation, the serum HBsAg is fully suppressed by E6F6. And the HBsAg never recover in the period. In the 6th weeks after antibody injection, the ani-HBs Ab is negative that means the amount of host antibody production and E6F6 are low. Mice were sacrifice in 9th weeks, the core and surface protein in the liver is not fully inhibited by E6F6. And the RNA expression also does not interfere by E6F6. To study the E6F6 effect on the early time after administration, we observe the serum HBsAg in a week. And the Western blots show that the core protein is relatively weaker difference between day0 and day 7 than surface protein. RNA expression does not interfere by E6F6. However, only one dose administration of antibody cannot suppress the serum HBsAg for long time.
The possibility is that the inhibition of serum HBsAg in the early time may due to the antibody nebulization, either extracellular or intracellular. In several weeks, the E6F6 degradation, the other mechanism induction by high dose antibody to suppress the serum HBsAg.

並列關鍵字

HBV ； HBsAg ； chronic hepatitis B ； hepatitis B immunoglobulin (HBIG) ； intravenous immunoglobulin (IVIG)

參考文獻

1. Barker, L.F., et al., Transmission of Type B Viral Hepatitis to Chimpanzees. Journal of Infectious Diseases, 1973. 127(6): p. 648-662.

2. Wang, Q., et al., Experimental chronic hepatitis B infection of neonatal tree shrews (Tupaia belangeri chinensis): A model to study molecular causes for susceptibility and disease progression to chronic hepatitis in humans. Virology Journal, 2012. 9(1): p. 170.

3. Yan, H., et al., Molecular Determinants of Hepatitis B and D Virus Entry Restriction in Mouse Sodium Taurocholate Cotransporting Polypeptide. Journal of Virology, 2013. 87(14): p. 7977-7991.

4. Zhong, G., et al., Sodium Taurocholate Cotransporting Polypeptide Mediates Woolly Monkey Hepatitis B Virus Infection of Tupaia Hepatocytes. Journal of Virology, 2013. 87(12): p. 7176-7184.

5. Araki, K., et al., Expression and replication of hepatitis B virus genome in transgenic mice. Proceedings of the National Academy of Sciences, 1989. 86(1): p. 207-211.