乳劑型疫苗佐劑之免疫機轉研究

Studies on the immune mechanisms of emulsion vaccine adjuvants

沈珊珊(Shan-Shan Shen)

指導教授 ： 楊雅雯

國立臺灣大學/醫學院/藥學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.03030

摘要

抗原本身所引起的免疫原性較弱，加入適當的佐劑可以加強抗原之免疫原性和免疫保護的效果。從七十多年前在疫苗研製過程中開始使用免疫佐劑，其中以氫氧化鋁和磷酸鋁為代表的鋁礬 (alum) 至今仍是唯一被批准合法使用在人體的疫苗佐劑，因此佐劑的開發是值得研究的課題。本論文利用非離子性界面活性劑 (non-ionic surfactants)，包括 Tweens (polyoxyethylene sorbitans) 及 Spans (sorbitans)，製作出六種具不同 HLB (hydrophile-lipophile balance) 值與不同物化性質的乳劑型疫苗佐劑，以探討其造成疫苗免疫效應之機轉研究。
為了探討所製作出的乳劑型疫苗佐劑之物化性質，我們分析乳劑的電導度、黏度與蛋白質從佐劑中釋出的速率。為了要觀察六種不同乳劑型疫苗佐劑所引起的免疫反應，我們以 ovalbumin (OVA) 當作抗原，將抗原與乳劑型疫苗佐劑混合後打入雄性 C57BL/6J 小鼠，於不同時間點收集血清測量 anti-OVA 的效價 (titer) 與 IgG subtyping。為了觀察乳劑型疫苗佐劑是否會引起細胞死亡，我們分別在活體外用 EL4 細胞來觀察，並以 annexin V/propidium iodide 雙染色。我們同時也在活體內將小鼠注射部位組織取下來切片，以 H&E (hematoxylin & eosin)、TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling) 兩種方法染色，觀察乳劑型疫苗佐劑在活體內所引起之發炎反應與細胞死亡。
為了探討佐劑與死亡細胞對抗原呈獻細胞吞噬抗原的影響，我們利用小鼠的巨噬細胞株 J774A.1 當作抗原呈獻細胞來進行細胞實驗，並以 BSA-FITC 及 OVA 當作抗原。為了探討抗原呈獻細胞吞噬佐劑之抗原的機轉，我們加入三種抑制劑來觀察，其中 amiloride 可抑制 macropinocytosis、brefeldin A 可抑制 endocytosis、cytochalasin B 可抑制 phagocytosis。同時我們以 2',7'-dichlorofluorescin diacetate (DCFH-DA) 與 hydroethidine (HE) 探測乳劑型疫苗佐劑是否會產生活性氧物系以及氧化壓力。為了探討佐劑所含抗原在抗原呈獻細胞內處理的過程，我們將 J774A.1 細胞分別染上不同胞器來觀察。
由於佐劑所造成的細胞死亡在疫苗引起之免疫反應上扮演一個重要的角色，因此我們將佐劑處理過的 EL4 細胞與樹突狀細胞共同培養，以探討樹突狀細胞吞噬死亡細胞的情形；另外，也一併探討樹突狀細胞吞噬死亡細胞後，樹突狀細胞表面共刺激分子 (costimulatory molecules)，包括 MHC class II、CD40、CD80、CD86 的表現情形為何，來觀察樹突狀細胞是否進入成熟期。
樹突狀細胞在未成熟期可吞噬抗原，吞噬抗原後則進入成熟期，成熟期的樹突狀細胞可將抗原呈獻給下游的淋巴細胞。為了分析打入乳劑型疫苗佐劑後對小鼠淋巴結細胞的影響，我們首先將注射七天後的小鼠淋巴結取出，再用 methyl-[3H] thymidine 標定，以觀察淋巴結 T 細胞的增生情形；同時以 ELISPOT (enzyme-linked immunospot) 的方法來觀察經乳劑型疫苗佐劑處理後的小鼠淋巴結 B 細胞是否會產生 OVA 專一性抗體。
為了探討乳劑型疫苗佐劑是否會引起細胞免疫反應 (cell-mediated immune responses)，我們利用毒殺細胞毒殺標的細胞時會產生 granzyme B 的原理來觀察小鼠脾臟 T 細胞的細胞毒殺情形。同時也利用一種會對 OVA 引起 MHC class I 反應的 B3Z T 細胞，在活體外觀察乳劑型疫苗佐劑是否會引起細胞免疫。
由實驗結果發現，當佐劑黏度愈大時、蛋白質愈不容易自佐劑中釋出，但所製作出不同物化性質的佐劑在動物體所產生的 anti-OVA 效價並無明顯差異，且主要是引起 IgG1 subtype 的產生。乳劑型疫苗佐劑會在注射部位同時引起發炎反應及細胞死亡的情形，在體外也可以同時引起細胞凋亡與細胞壞死，且佐劑所造成的死亡細胞可被樹突狀細胞吞噬，並造成樹突狀細胞成熟。佐劑與死亡細胞可以增加 J774A.1 細胞吞噬抗原的量，吞噬抗原後會經由 lysosome、endothelium reticulum (ER)、Golgi apparatus、MHC class II 等胞器將抗原分解成胜肽片段，而吞噬抗原的步驟會被 amiloride、brefeldin A 及 cytochalasin B 等抑制劑所抑制。佐劑與死亡細胞也會造成 J774A.1 細胞產生活性氧化物以及產生氧化壓力。打入乳劑型疫苗佐劑會造成小鼠淋巴結 T 細胞增生，並造成淋巴結 B 細胞產生 OVA 專一性抗體。在脾臟細胞的細胞毒殺試驗中，除了 L121-adj. 外，其他乳劑型疫苗佐劑並無產生明顯的細胞毒殺作用，在體外實驗中，也沒有造成 B3Z T 細胞的活化。
總而言之，本研究證實了乳劑型疫苗佐劑會同時造成細胞凋亡與細胞壞死、且可增加抗原被抗原呈獻細胞所吞噬。抗原經由 macropinocytosis、phagocytosis 等方式進入抗原呈獻細胞後，會經由 lysosome、ER、Golgi apparatus、MHC class II 分解成胜肽片段。佐劑同時也會造成樹突狀細胞成熟，進而將抗原傳遞給下游的 T 細胞與 B 細胞，造成 T 細胞的增生，並使 B 細胞產生抗原專一性的抗體免疫反應。除了 L121-adj. 外，含 Tweens 與 Spans 之乳劑型疫苗佐劑並不會引起顯著的細胞免疫反應 (cell-mediated immune responses)。

關鍵字

疫苗 ； 佐劑 ； 乳劑

並列摘要

Adjuvants are used to improve the immune response to weak vaccine antigens. Aluminum adjuvant has used in human and veterinary vaccines for over seventy years, and it is the only adjuvant used in human. The development of new vaccine adjuvants is crucial. In the present study, emulsion vaccine adjuvants containing the non-ionic surfactants, including Tweens (polyoxyethylene sorbitan fatty acid esters) and Spans (sorbitan fatty acid esters) with various HLB (hydrophile-lipophile balance) values were prepared and used for studying the immunogenic mechanisms of emulsion vaccine adjuvants.
The emulsion adjuvants were prepared by mixing squalane, PBS and 2% (w/w) surfactants, and their physicochemical properties, including conductivity, viscosity, and protein release rates were determined. To examine the immunogenic response, C57BL/6J mice were immunized with various vaccine adjuvants employing ovalbumin (OVA) as a model antigen. The blood samples were collected periodically and analyzed by enzyme-linked immunosorbent assay (ELISA). To examine cell death induced by the emulsion adjuvants in vitro, EL4 cells were treated with various formulations, stained with annexin V and propidium iodide, followed by flow cytometric analysis. Cell death induced by the adjuvants in vivo was examined by histochemical staining of the subcutaneous tissues at the injection sites, followed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP- digoxigenin nick-end labeling) assay.
To investigate the effect of adjuvants on the uptake of antigen and the adjuvant-induced dead cells, murine macrophages J774A.1 cells were pretreated with macropinocytosis and phagocytosis inhibitors, including amiloride, brefeldin A and cytochalasin B, and pulsed with adjuvants containing the adjuvant-induced dead cells, followed by flow cytometric analysis and fluorescence microscopic examination. The production of reactive oxygen species (ROS) in the cells after treatment with the emulsion vaccine adjuvants was also determined. To examine antigen processing in the antigen-presenting cells, J774A.1 cells were stained with different fluorescent organelle markers, including LAMP1, cathepsin D, MHC class II, calnexin and GM130 antibodies, and examined by fluorescence microscope.
Adjuvant-induced cell death plays an important role in the initiation of immune responses. To investigate phagocytosis of adjuvant-induced dead cells by dendritic cells, the most powerful antigen-presenting cells, adjuvant-treated EL4 cells were co-cultured with dendritic cells for 6 hours. The effect of cell death induced by the adjuvants on the expressions of costimulatory molecules of DCs, including MHC class II, CD40, CD80 and CD86, was examined by flow cytometry.
To investigate the immune response induced by the emulsion vaccine adjuvants, cells in the draining lymph nodes near the injection sites were removed after immunization and analyzed by flow cytometry. To examine the effect of adjuvants on T cell proliferation in the draining lymph nodes, lymphocytes were labeled with methyl-[3H] thymidine, and the radioactivity was determined by a β-counter. The OVA-specific antibody response of B cells in the lymph nodes after immunization with various adjuvants was examined by enzyme-linked immunospot (ELISPOT) assay. To investigate cell-mediated immune responses induced by the emulsion vaccine adjuvants, splenocytes were examined at Day 10 after immunization, and the cytotoxicity assay was performed by measurement of the granzyme B activity. Primed DCs were assessed by the in vitro activation of B3Z OVA-specific CD8+ T cells.
Results obtained in this study showed that protein release from emulsion vaccine adjuvants decreased with the viscosity of the emulsions. The anti-OVA titers in the animals immunized with the emulsion vaccine adjuvants were approximately the same among six emulsion adjuvants, with IgG1 subtype being dominant, suggesting a type II immune response. Emulsion vaccine adjuvants induced inflammation and cell death, including apoptosis and necrosis, both in vitro and in vivo. Adjuvant-induced dead cells were phagocytosed by the dendritic cells, and stimulated the expression of costimulatory molecules, including CD40, CD80, and CD86. Uptake of dead cells induced by the adjuvants enhanced uptake of soluble antigens by J774A.1 macrophages, leading to generation of reactive oxygen species (ROS). The acquired antigen was processed through lysosome, endothelium reticulum (ER), Golgi apparatus, and MHC class II, the antigen uptake was inhibited by amiloride, brefeldin A, and cytochalasin B. Emulsion vaccine adjuvants were shown to induce T cell proliferation and production of OVA-specific B cells in the draining lymph nodes. However, most emulsion vaccine adjuvants, except L121-adjuvant, did not induce significant CTL effect or B3Z OVA-specific T cell activation.
In summary, we have demonstrated in this study that emulsion vaccine adjuvants stimulated humoral responses and induced cell death, resulting in inflammation at the injection sites of the animals. The antigen-presenting cells acquired antigen by macropinocytosis and phagocytosis, and the antigens were processed through lysosome, ER, Golgi apparatus, and loaded onto MHC class II molecules. The uptake of antigens by the antigen-presenting cells can be partially inhibited by amiloride, brefeldin A and cytochalasin B. The emulsion vaccine adjuvants induced antigen-specific T cell proliferation and B cell response in the draining lymph nodes.

並列關鍵字

vaccine ； adjuvant ； emulsion

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