基於HBV X蛋白之治療性疫苗於慢性B型肝炎病毒帶原小鼠之療效及相關免疫機轉研究

Efficacy and Mechanism of the HBV X protein based therapeutic vaccine in a chronic HBV mouse model

洪昭皓(Jau-Hau Horng)

指導教授 ： 陳培哲

國立臺灣大學/醫學院/微生物學研究所/博士(2020年)

https://doi.org/10.6342/NTU202004254

摘要

B型肝炎病毒是造成慢性B型肝炎感染的主因，目前在全世界約有2億5千萬的慢性B型肝炎感染者，但目前醫學上仍缺乏有效的根治方法，因此找尋有效的療法是目前的當務之急。目前開發中的療法中，慢性B型肝炎的免疫治療被寄予厚望，其中治療性疫苗已經研究超過20年，但目前仍未能在臨床試驗成功。因此，在合適的動物模式研究治療性疫苗及其相關的免疫機轉是有必要的。B型肝炎病毒的免疫治療之所以困難的主因是由於抗B型肝炎病毒T細胞的耗竭(T cell exhaustion)現象，當耗竭現象發生時，抗原特異性的T細胞的毒殺及分泌細胞激素的功能會大幅減弱，因而無法順利清除入侵的病原。目前科學上已經知道T細胞受體接受大量且持續的抗原暴露是產生耗竭現象的重要因子。由於目前研發中的B型肝炎治療性疫苗大多使用病毒的結構蛋白，例如表面或核心抗原，但這些抗原在肝臟細胞內表現量較高，慢性B型肝炎病人的表面(HBs)或核心抗原(HBc)的毒殺型T細胞在長期抗原暴露下，已經有嚴重的耗竭現象。我們的假說是X蛋白(HBx)特異性的T細胞因在宿主的表現量，對抗原的暴露量遠少於其他結構蛋白，因而能夠避開或減緩T細胞的耗竭現象，並更容易被治療性疫苗刺激重新恢復功能。
HBx目前免疫原性以及作為治療性疫苗使用的數據較少，為了解HBx的免疫原性及其作為治療性疫苗的潛力，我們選擇HBx作為主要的抗原，並且輔以強力的免疫佐劑CpG-ODN作為測試用的治療性疫苗。為了在動物模式研究此疫苗之效力，我們選用先前建立之基於高壓流體注射法的小鼠慢性B型肝炎病毒帶原模型。我們將從病人身上的B型肝炎病毒序列複製到pAAV重組質體上，再透過高壓流體注射法轉染至CBA/CaJ品系小鼠的肝臟細胞，此時B型肝炎病毒質體能在小鼠體內穩定表現超過6個月。我們選用血清表面抗原效價>500IU/mL並維持超過4周以上的小鼠來模擬人類的慢性B型肝炎，並注射前述的HBx疫苗來研究其抗HBV的效果及其免疫原性。我們以酶聯免疫吸附斑點法(ELISpot)分析脾臟內抗原特異性T細胞的產生。血液中的病毒量以HBs的酵素免疫分析法及HBV-DNA的定量即時聚合酶鏈鎖反應(Q-PCR)評估。肝臟中的病毒量以HBc的西方墨點法(Western blotting)及免疫組織化學(immunohistochemistry)分析。此外，我們也針對血液或肝臟內的免疫細胞，經分離程序後進行流式細胞儀的定量分析。
我們的研究結果顯示，在未受HBV轉染的小鼠，注射HBx疫苗3次後，脾臟能夠產生HBx特異性的T細胞，並且經過後續HBV轉染後，血清中病毒量有顯著的降低。另一方面，對HBV帶原小鼠給予3次HBx疫苗後，血清在3周後即無法偵測到病毒，實驗終點時分析肝臟內的病毒抗原亦達到完全消失，同時 HBx疫苗亦能夠在HBV帶原小鼠引發系統性的CD4及CD8 T細胞免疫力。在治療性疫苗注射後以流式細胞儀分析肝臟淋巴球組成，我們發現肝臟內有疫苗誘導的T細胞累積。治療成功的小鼠若於一個月後接受第二次HBV轉染，第二次注射後的血清病毒量與只打CpG佐劑的小鼠相比有顯著的下降，顯示HBx疫苗能產生持續的免疫力。此外，我們也發現疫苗抗原的選擇對其療效有顯著的影響:比較以HBx及HBc作為治療性疫苗的抗原時，僅HBx能清除小鼠血液中的病毒抗原並產生系統性的抗原特異性T細胞。與HBc相比，HBx疫苗注射亦能在肝臟造成更多的單核白血球(monocytes)累積。在疫苗治療的期間若以藥物移除不同的肝臟骨髓系細胞(myeloid cell)子群體，影響疫苗治療效果的程度也不同。若僅移除巨噬細胞類，包含monocyte衍生的巨噬細胞(Monocyte-derived Macrophage)以及庫式細胞(Kupffer cells)，僅能暫時抑制病毒清除的作用，但最終仍會達到清除。若連炎症性單核球(Inflammatory monocyte)一併移除，則病毒清除的作用將受到大幅度的影響，最終造成病毒清除失敗。
我們的研究展示了HBx作為慢性B型肝炎治療性疫苗的潛力以及不同肝臟內骨髓系細胞對疫苗清除病毒的重要性。適當的抗原設計以及透過適當的佐劑調節肝臟內免疫微環境可能是發展下一代慢性B型肝炎治療性疫苗的關鍵。

關鍵字

慢性B型肝炎 ； 免疫治療 ； 先天性免疫 ； 骨髓系細胞 ； HBV X蛋白

並列摘要

Background and Hypothesis: Hepatitis B virus (HBV) persistently infects about 250 million people worldwide, and a curative treatment remains an unmet medical need. Among many approaches to treat chronic hepatitis B (CHB), therapeutic vaccines have been developed for two decades, but none have yielded promising results in clinical trials. Therefore, dissection of HBV clearance mechanisms during therapeutic vaccination in appropriate models, which could give rise to new curative therapies, is urgently needed. Growing evidence indicates that prolonged and intensive exposure of antigen-specific T cells to viral antigens is a major cause of T cell exhaustion and decreases anti-HBV immunity efficacy of therapeutic vaccination. Hence, we propose that in chronic HBV carriers, antigen specific T cell targeting low-level expressing viral proteins may be less exhausted and their antiviral function could be rejuvenated through therapeutic vaccination.
Methods: HBV X protein (HBx) is expressed at low levels during natural infection, and the understanding of its immunogenicity and potential in therapeutic CHB vaccines is limited. We selected HBx as the major antigen, and a robust adjuvant CpG-ODN-1826, was added to boost antigen’s immunogenicity. To test the therapeutic potential of HBx based therapeutic vaccine in an immunocompetent animal model, HBV genome sequences from CHB patients were cloned into a pAAV plasmid backbone and transfected into immunocompetent mouse hepatocytes through hydrodynamic injection. Mice carrying >500 IU/mL serum HBV surface antigen (HBs) for more than 4 weeks were considered HBV carriers mimicking human CHB and received 3 doses of weekly HBx vaccine by subcutaneous immunization. Serum HBV clearance was evaluated by monitoring serum HBs and HBV-DNA titers. Residual HBV in the liver was evaluated by western blotting for HBV core antigen. The splenic antigen-specific T cell response was quantified by a 15-mer overlapping peptide-stimulated interferon-γ enzyme-linked immunospot assay. Blood and hepatic immune cells were quantified by flow cytometric analysis.
Results: Naïve CBA/CaJ mice immunized with HBx-based vaccine shows the induction of HBx specific T cell immunity and protected mice from HBV exposure. In HBV carrier mice, HBx vaccination results in significant HBs and HBV-DNA elimination. The vaccine induced systemic HBx-specific CD4+ and CD8+ T cell responses, and liver CD8+ T cell influx was also observed. The protective effect persisted for at least 30 days without additional booster immunization.
The antigen selection largely affects the potency of vaccine-mediated HBV clearance. Compare with HBx, HBV core antigen (HBc) serving as vaccine antigen cannot induce systemic T cell immunity or hepatic HBV clearance. HBx vaccine also induces higher hepatic monocyte infiltration than HBc, suggesting that the hepatic myeloid cell infiltration may be important to hepatic viral clearance. Different infiltrating myeloid cell subsets, each with distinctive roles during vaccine-mediated HBV clearance, were found in mouse liver after HBx vaccination. During vaccine therapy, Ly6Chigh monocyte derived macrophage (MDM) depletion resulted in sustained HBV clearance inhibition. In contrast, phagocytic monocyte-derived macrophage and Kupffer cell elimination resulted in only transient inhibition of vaccine-induced HBV clearance.
Conclusions: We report the potential of HBx protein as a major immunogen in an HBV therapeutic vaccine in an HBV-persistent animal model. Appropriate antigen selection significantly affects the efficacy of therapeutic vaccine. Among the vaccine-induced hepatic myeloid cell subsets, the Ly6Chigh MDMs may have unique functions to promote hepatic T cell immunity and viral clearance. Appropriate antigen design and the liver resident myeloid cell microenvironment may be the keys to developing next-generation therapeutic HBV vaccines.

並列關鍵字

Chronic Hepatitis B ； Immune therapy ； Hepatic innate immunity ； myeloid cell ； HBV X protein

參考文獻

1. Lok AS, Zoulim F, Dusheiko G, Ghany MG (2017) Hepatitis B cure: From discovery to regulatory approval. Journal of Hepatology 67(4):847-861.

2. Iloeje UH, et al. (2007) Risk and Predictors of Mortality Associated With Chronic Hepatitis B Infection. Clinical Gastroenterology and Hepatology 5(8):921-931.

3. Yapali S, Talaat N, Lok AS (2014) Management of hepatitis B: our practice and how it relates to the guidelines. Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association 12(1):16-26.

4. Wherry EJ (2011) T cell exhaustion. Nature Immunology 12(6):492-499.

5. Wherry EJ Kurachi M (2015) Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 15(8):486-499.