臺灣博碩士論文加值系統

次單位疫苗的優點是因為它為非病毒基因體，不具傳染性且可簡單保存。當病毒樣顆粒疫苗利用抗原自體組裝後，可形成重複的抗原片段模仿病毒的外部結構，提高免疫原性，被抗原呈現細胞吞噬，並蓄積在淋巴結，活化免疫反應。但常見生產病毒樣顆粒的平台如細菌或酵母菌，其蛋白質轉譯後修飾與哺乳類動物會有所差異，進而影響蛋白質結構，使得此平台有所限制。本研究將開發，卵清蛋白類病毒樣聚乳酸甘醇酸奈米顆粒 (Ovalbumin virus like PLGA nanoparticles, OVA-VLPN) 的載體系統，病毒樣聚乳酸甘醇酸奈米顆粒開發為蛋白質次單位疫苗平台，以便接種宿主，可以產生更有效與特異性的中和抗體。相較於一般生產病毒樣顆粒的系統，沒有表達系統造成汙染的風險與高成本設備支出。本研究採用單乳相溶劑揮發法，利用卵清蛋白的硫醇基團與帶有馬來酰亞胺的DSPE-PEG2000-Maleimide經麥克爾加成反應 (Michael addition reaction) 反應後，製備成OVA-VLPN，其產率為37.76±0.7 %，粒徑為160.33±13.3 nm、PDI為0.164±0.04、ζ(mV)為-39.6±10.16。在 4°C 下，顆粒大小與均一性於28天內維持一定安定性，具有良好的安定性。於冷凍電子顯微鏡 (cryo-electron microscopy)下，觀察到完整球形結構與均一性的ovalbumin (OVA) 修飾於顆粒表面。在體外試驗中，OVA-VLPN能夠誘導CD80與CD86的活化，引起樹突細胞的成熟並且增強細胞吞噬能力。而在體內研究中，V25組別 (OVA-VLPN, 25 μg OVA and 5 μg Quil-A) 顯示出高度IgG與CD4+CD8+ T 細胞。具備抗體依賴性細胞介導的細胞毒性(antibody-dependent cell-mediated cytotoxicity, ADCC)與記憶型T細胞的特性，在未來可用於治療癌症以及防止癌症復發的潛力。在心肌纖維、肝細胞、脾髓細胞、肺泡或腎小球上皮細胞中沒有發現明顯的發炎現象、腫脹區域以及細胞損傷，顯示OVA-VLPN在主要器官中表現出良好的生物相容性與安全性。本研究開發的OVA-VLPN具有效增強體液性免疫反應，誘導抗體生成以及具有高度安全性。具有極大潛力應用於廣泛適用的疫苗平台。

The advantages of protein subunit vaccines are non-virus genome, non-infectious and simple conservation. Virus like particle (VLP) Utilizing the characteristic of nanoparticle, highly repetitive antigen fragments via antigen self-assembly to mimic the outer structure of the virus, improving immunogenicity, be phagocytized by antigen-presenting cells, and accumulate in lymph nodes to enhance the immune response. However, common production of virus-like particle platforms such as bacteria or yeast, Its protein post-translational modification is different from mammals and affects protein structure, which limits this platform. In this study, the carrier of Ovalbumin virus like nanoparticle (OVA-VLPN), was developed as a protein subunit vaccine platform so that the vaccinated host can produce more potent and specific neutralizing antibodies. Compared with the general production of virus-like particles (VLP), there is no possibility of contamination of the expression system and high equipment expenditure. The OVA-VLPN via thiol-maleimide conjugation reaction was prepared by a single-emulsion solvent evaporation method. The yield ratio, particle size, polydispersity index, and zeta potential of OVA-VLPN was 37.76±0.7%, 160.33±13.3 nm, 0.164±0.04, -39.6±10.16 mV. The particle size distribution and uniformity of OVA-VLPN remain stable at 4°C for one month. The spherical structure and uniform decorative ovalbumin (OVA) on the particle surface could be observed by the cryo-electron microscopy (cryo-EM). In the in vitro study, OVA-VLPN was able to induce the much higher activation of CD80 and CD86 leading to maturation of dendritic cells and enhance phagocytosis. In the in vivo study, the administration of V25 group (OVA-VLPN, 25 μg OVA and 5 μg Quil-A) shows high IgG and CD4+CD8+ T cells. Possesses the characteristics of antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity, ADCC) and memory T cells, and can be potential for treating cancer and preventing cancer recurrence in the future The tissue sections of major organs showed no inflammatory, swollen regions, , or damaged cells found in cardiac muscle fibers, hepatic cells, splenic cell , pulmonary alveoli cell , and glomerular epithelial cells, indicating that these OVA-VLPN exhibited good biocompatibility. In conclusion, the OVA-VLPN could effectively enhance humoral immune responses and reliable safety, thus, showed a well potential to be developed as a broadly applicable vaccine platform technology.

目錄

致謝 I
中文摘要 II
Abstract IV
目錄 VI
表目錄 IX
圖目錄 X
縮寫表 XVIII
第一章 緒論 1
一、 疫苗 1
1. 疫苗功能 1
2. 疫苗種類 3
3. VLP介紹 5
4. VLP的表達宿主系統 6
二、 奈米載體系統 10
1. 奈米載體種類 12
2. 奈米疫苗之被動運輸 20
第二章 實驗動機與目的 21
第三章 材料與實驗方法 24
一、 實驗材料 24
二、 實驗儀器 25
三、 Ovalbumin Virus like PLGA Nanoparticles (OVA-VLPN) 製備 27
四、 物化特性分析 29
1. Maleimide Virus like PLGA Nanoparticles (MAL-VLPN) 與Ovalbumin Virus like PLGA Nanoparticles (OVA-VLPN) 之粒徑、修飾率測定 29
2. Ovalbumin (OVA) 含量測定 29
3. 最佳化處方篩選 30
4. Ovalbumin Virus like PLGA Nanoparticles (OVA-VLPN) 結構分析 30
5. Ovalbumin Virus like PLGA Nanoparticles (OVA-VLPN) 安定性評估 30
五、 Ovalbumin Virus Like PLGA Nanoparticles (OVA-VLPN) 之體外試驗 32
1. DC 2.4 Mouse Dendritic Cell細胞培養 32
2. 細胞計數 32
3. 細胞毒性試驗 33
4. 抗原呈現細胞之活化能力評估 34
5. 細胞吞噬試驗 35
六、 Ovalbumin Virus like PLGA Nanoparticles (OVA-VLPN) 之動物試驗 37
1. 生物體內分布評估-活體成像系統(IVIS Spectrum) 39
2. 專一性抗體分析-酵素結合免疫吸附分析法 (ELISA) 40
3. 脾臟內免疫細胞分析 41
4. 組織染色切片 42
5. 免疫組織染色切片 42
6. 統計方法分析 42
第四章 實驗結果與討論 43
一、 處方設計與物化特性 43
1. 最佳化處方 43
2. MAL-VLPN之maleimide的分析方法確效 45
3. OVA-VLPN之ovalbumin的分析方法確效 47
4. MAL-VLPN之物化特性分析 49
5. OVA-VLPN之物化特性與OVA含量分析 49
6. OVA-VLPN 安定性評估 52
7. OVA-VLPN 結構分析 52
二、 OVA-VLPN細胞實驗 54
1. 細胞安全性試驗 54
2. 樹突細胞活化能力評估 55
3. 樹突細胞吞噬能力評估 56
4. 樹突細胞吞噬影像評估 57
三、 OVA-VLPN 動物實驗 60
1. 生物分佈 60
2. 抗體分析 65
3. 脾臟細胞分析 71
4. 組織切片 75
5. 安全性評估 77
第五章 結論 78
第六章 參考文獻 79

表目錄

Table 1 Commercial virus like particle vaccine. 5
Table 2 Classification, application areas, advantages, and disadvantages of polymer-based nanomaterials.[54] 15
Table 3 Classification, application areas, advantages, and disadvantages of polymer-based nanomaterials.[54] 16
Table 4 Protein and peptide decorative lipid polymer hybrid nanoparticle.[57-61] 19
Table 5 Optimal formulation of ovalbumin virus like PLGA nanoparticles (OVA-VLPN) 31
Table 6 The characteristic of optimal formulation of ovalbumin virus like PLGA nanoparticles (OVA-VLPN) 44
Table 7 Physical characteristics of maleimide virus like PLGA nanoparticles (MAL-VLPN). Data are presented as the mean ±SD (n = 3). 50
Table 8 Physical characteristics of ovalbumin virus like PLGA nanoparticles (OVA-VLPN). Data are presented as the mean ±SD (n = 3). 51
Table 9 C57BL/6 mice differentiation of B cells form spleen after 84 days post first injection. Maleimide virus like nanoparticle (MAL-VLPN) and quil-A (5 μg) group (blank), ovalbumin (OVA) and quil-A (5 μg) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) and quil-A (5 μg) group (V10, V25, V50,100). 73
Table 10 C57BL/6 mice differentiation of T cells form spleen after 84 days post first injection. Maleimide virus like nanoparticle (MAL-VLPN) and quil-A (5 μg) group (blank), ovalbumin (OVA) and quil-A (5 μg) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) and quil-A (5 μg) group (V10, V25, V50,100).. Statistical significance is indicated by **** p < 0.0001 compared to other groups. 74


圖目錄

Figure 1 The generation of an immune response to a vaccine.[1] 2
Figure 2 Advantages and limitations of different expression systems for the development of virus-like particles. [19] 6
Figure 3 Designing nanoparticles for intracellular applications. Nanoparticles can be modularly assembled from different materials composition with different physical and chemical properties and functionalized with a myriad of ligands for biological targeting. Such flexibility in design freedom enables researchers to tailor nanoparticle for specific intracellular applications as contrast agents, drug delivery vehicles, and therapeutics.[41] 11
Figure 4 Overall schematic representation of polymer-lipid hybrid nanoparticles (PLHNPs) and their applications. The hybrid system is developed from polymeric nanoparticles and liposomes covered by different types of PLHNPs. (A) Polymer core lipid shell, (B) Polymer caged liposome, (C) Monolithic PLHNPs, (D) Erythrocyte membrane-coated PLHNPs), (E) Core shell-type hollow lipid-polymer-lipid hybrid nanoparticles. The different applications of PLHNPs are also shown. (1) Delivery of various chemotherapeutic drugs. (2) Delivery of genes (siRNA, DNA). (3) Delivery of vaccines, ovalbumin (OVA), and Toll-Like Receptor (TLR) agonist for immune activation. (4) Imaging applications based on gadolinium (Gd), manganese (Mn), and gold (Au). (5) Photothermal therapy (PTT), photodynamic therapy (PDT), ultrasound, and alternative magnetic field (AMF). [56] 18
Figure 5 Schematic illustration of immunogenicity of ovalbumin virus like PLGA nanoparticle (OVA-VLPN) to enhance dendritic cell uptake and traffic to the lymph node, induce higher specific humoral immunity. 22
Figure 6 Flow chart of MAL-VLPN and OVA-VLPN preparation, physical characteristic, in vitro, in vivo study to ovalbumin virus like PLGA nanoparticles (OVA-VLPN). 23
Figure 7 The preparation of ovalbumin virus like PLGA nanoparticle (OVA-VLPN) using single emulsion solvent evaporation method. 28
Figure 8 Schematic illustration of animal protocol of ovalbumin virus like PLGA nanoparticle (OVA-VLPN) vaccine. 38
Figure 9 The H spectrum of dspe-peg020-maleimide-standard curve from 0.3125 to 10 mg /mL. The peak at (δ = 6.7 ppm) of was measured by 600H NMR and calculate the area of maleimide group .Each point is the average of three determinations. 45
Figure 10 Standard curve of dspe-peg2000-maleimide in dimethyl sulfoxide (A)Interday. (B) Intraday. The graph shows detection of dspe-peg2000-maleimide from 0.3125 to 10 mg /mL. The peak at (δ = 6.7 ppm) of was measured by 600H NMR and calculate the area of maleimide group .Each point is the average of three determinations. 46
Figure 11 Standard curve of ovalbumin by CBQCA Protein Quantitation Kit. The graph shows detection of ovalbumin from 0.03125 to 1 mg / mL. Fluorescence is detected by microplate reader with (Ex/Em：465/550 nm). Each point is the average of three determinations. (A) Interday. (B) Intraday. 48
Figure 12 The ovalbumin virus like PLGA nanoparticle (OVA-VLPN) image of cryo-EM 52
Figure 13 The stability of ovalbumin virus like PLGA nanoparticle (OVA-VLPN) after lyophilisation. (A) particle size and PDI. (B) Zeta potential(mV) at 4 °C in 28 days Data are presented as mean ± SD (n = 3). 53
Figure 14 Cell viability (%) of maleimide virus like nanoparticle (MAL-VLPN) group (blank), ovalbumin (OVA) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) group (V10, V25, V50,100). Live cells at 24 hours of exposure administration. Data are presented as mean ± SD (n = 3). 54
Figure 15 Dendritic Cell maturation at 24 hours of exposure to maleimide virus like nanoparticle (MAL-VLPN) group (blank), ovalbumin (OVA) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) group (V10, V25, V50,100). Data are presented as mean ± SD (n = 3). Statistical significance is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to other groups 55
Figure 16 The dendritic cells uptake ability after exposure to maleimide virus like nanoparticle (MAL-VLPN) group (blank), ovalbumin (OVA) and ovalbumin virus like PLGA nanoparticle (OVA-VLPN). Statistical significance is indicated by ** p < 0.01, **** p < 0.0001 compared to other groups. 56
Figure 17 Cell uptake analysis by confocal microscopy. at 6, 12 hours of ovalbumin (OVA) and ovalbumin virus like PLGA nanoparticle (OVA-VLPN) exposure. 58
Figure 18 Cell uptake analysis by confocal microscopy. at 24, 36 hours of ovalbumin (OVA) and ovalbumin virus like PLGA nanoparticle (OVA-VLPN) exposure. 59
Figure 19 The back side of in vivo imaging system of C57/6 mice(N=4) after first intramuscular administration of control group (normal saline(DiR) ), DiR labeled maleimide virus like PLGA nanoparticle (MAL-VLPN(DiR)) group (blank), DiR labeled ovalbumin group (O10, O25, O50, O100), DiR labeled ovalbumin virus like PLGA nanoparticle (OVA-VLPN(DiR)) group (V10, V25, V50, V100). 61
Figure 20 The ventral side of in vivo imaging system of C57/6 mice(N=4) after first intramuscular administration of control group (normal saline(DiR) )DiR labeled maleimide virus like PLGA nanoparticle (MAL-VLPN(DiR)) group (blank), DiR labeled ovalbumin group (O10, O25, O50, O100), DiR labeled ovalbumin virus like PLGA nanoparticle (OVA-VLPN(DiR)) group (V10, V25, V50, V100). 62
Figure 21 The site of injection in vivo imaging system of C57/6 mice(N=4) after first intramuscular administration of control group (normal saline(DiR)), DiR labeled maleimide virus like PLGA nanoparticle (MAL-VLPN(DiR)) group (blank), DiR labeled ovalbumin group (O10, O25, O50, O100), DiR labeled ovalbumin virus like PLGA nanoparticle (OVA-VLPN(DiR)) group (V10, V25, V50, V100). 63
Figure 22 Inguinal lymph nodes of in vivo imaging system of C57/6 mice(N=4) after first intramuscular administration of control group (normal saline(DiR) ). DiR labeled maleimide virus like PLGA nanoparticle (MAL-VLPN(DiR)) group (blank), DiR labeled ovalbumin group (O10, O25, O50, O100), DiR labeled ovalbumin virus like PLGA nanoparticle (OVA-VLPN(DiR)) group (V10, V25, V50, V100). 64
Figure 23 The mice immunogenicity of VLPN vaccines after three intramuscular administration (BIW), maleimide virus like nanoparticle (MAL-VLPN) and quil-A (5 μg) group (blank), ovalbumin (OVA) and quil-A (5 μg) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) and quil-A (5 μg) group (V10, V25, V50,100). The mouse anti ovalbumin immunoglobulin G (IgG) titer be analyzed by ELISA until 84 days of post-administration. (A) Line chart for analyzing booster immune response. (B) Bar chart for evaluating immune response of different group. Statistical significance is indicated by * p < 0.05, *** p < 0.001, **** p < 0.0001 compared to other groups. The IgG concentration of day1 is zero in order to be able to express the value on the log scale, it is represented by 1. N.D. is non-detect. 67
Figure 24 The mice immunogenicity of VLPN vaccines after three intramuscular administration (BIW), maleimide virus like nanoparticle (MAL-VLPN) and quil-A (5 μg) group (blank), ovalbumin (OVA) and quil-A (5 μg) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) and quil-A (5 μg) group (V10, V25, V50,100). The mouse anti ovalbumin immunoglobulin G1 (IgG1) titer be analyzed by ELISA until 84 days of post-administration. (A) Line chart for analyzing booster immune response. (B) Bar chart for evaluating immune response of different group Statistical significance is indicated by **** p < 0.0001 compared to other groups. The IgG1 concentration of day1 is zero in order to be able to express the value on the log scale, it is represented by 1. N.D. is non-detect. 68
Figure 25 The mice immunogenicity of VLPN vaccines after three intramuscular administration (BIW), maleimide virus like nanoparticle (MAL-VLPN) and quil-A (5 μg) group (blank), ovalbumin (OVA) and quil-A (5 μg) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) and quil-A (5 μg) group (V10, V25, V50,100). The mouse anti ovalbumin immunoglobulin G2a (IgG2a) titer be analyzed by ELISA until 84 days of post-administration. (A) Line chart for analyzing booster immune response. (B) Bar chart for evaluating immune response of different group. The IgG2a concentration of day1 is zero in order to be able to express the value on the log scale, it is represented by 1. N.D. is non-detect. 69
Figure 26 The mice immunogenicity of VLPN vaccines after three intramuscular administration (BIW), maleimide virus like nanoparticle (MAL-VLPN) and quil-A (5 μg) group (blank), ovalbumin (OVA) and quil-A (5 μg) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) and quil-A (5 μg) group (V10, V25, V50,100). The ratio of mouse anti ovalbumin immunoglobulin G1 (IgG1) to mouse anti ovalbumin immunoglobulin G2a (IgG2a) be analyzed by ELISA until 84 days of post-administration. (A) Line chart for analyzing booster immune response. (B) Bar chart for evaluating immune response of different group. 70
Figure 27 Flowchart of flow cytometry analysis to C57BL/6 spleen cells. (A) B cell analysis of CD45 and MHCII B cell. (B) .T cell analysis of CD4 and CD8 T cell. Maleimide virus like nanoparticle (MAL-VLPN) and quil-A (5 μg) group (blank), ovalbumin (OVA) and quil-A (5 μg) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) and quil-A (5 μg) group (V10, V25, V50,100). 72
Figure 28 Histopathology analysis of four organ (hearts, livers, lungs, kidneys) isolated form mice after three i.m. administration of maleimide virus like nanoparticle (MAL-VLPN) and quil-A (5 μg) group (blank), ovalbumin (OVA) and quil-A (5 μg) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) and quil-A (5 μg) group (V10, V25, V50,100).BIW until 84 days. Magnification: 200X 76
Figure 29 Body weight changes of C57BL/6 mice (%) after three intramuscular(i.m.) administration BIW maleimide virus like nanoparticle (MAL-VLPN) and quil-A (5 μg) group (blank), ovalbumin (OVA) and quil-A (5 μg) group (O10, O25, O50, O100), ovalbumin virus like PLGA nanoparticle (OVA-VLPN) and quil-A (5 μg) group (V10, V25, V50,100). 77

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