| 摘要: | 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?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. |
