本文選題：H5N1疫苗佐劑 + 殼聚糖水凝膠　； 參考：《中國科學(xué)院研究生院(過程工程研究所)》2016年博士論文

【摘要】：H5N1禽流感嚴重威脅著人類生命健康,目前,商品化的H5N1疫苗多為安全性較好的滅活疫苗。然而,在鼻黏膜免疫中,滅活疫苗難以克服鼻腔黏膜屏障。在注射免疫中,H5Nl滅活疫苗也存在不能誘導(dǎo)理想的免疫反應(yīng)、需要很高抗原用量以及現(xiàn)有商品化鋁鹽佐劑效果不佳等問題。針對此現(xiàn)狀,本文選用兩種生物可降解高分子聚合物材料,殼聚糖和聚乳酸-乙醇酸共聚物(PLGA),分別制備溫敏水凝膠與均一的非球形PLGA顆粒用作黏膜免疫與注射免疫佐劑,以增強H5Nl滅活疫苗的免疫效果。針對溫敏水凝膠,考察凝膠的物化特性對免疫效果的影響,闡述其作用機制,并開展安全性與穩(wěn)定性研究,推進其臨床前研究。針對PLGA顆粒,開發(fā)簡單有效的制備均一變形顆粒的方法,并比較球形與非球形顆粒的免疫效果,探討非球形顆粒應(yīng)用于H5Nl滅活疫苗的可行性。具體研究內(nèi)容分為以下幾個部分：(1)采用放大工藝合成殼聚糖季銨鹽(HTCC),通過與甘油磷酸鈉(GP)離子交聯(lián)制得HTCC溫敏性水凝膠并用于H5N1疫苗的黏膜免疫佐劑。通過調(diào)控HTCC季銨取代度來調(diào)節(jié)凝膠的陽離子性和流變學(xué)特性,考察其物化特性對免疫效果的影響。結(jié)果表明,季銨取代度居中的41%的HTCC水凝膠既具有合適的黏度和溫敏性能延長抗原在鼻腔的停留,又具有一定陽離子性能增強與黏膜的吸附,并打開上皮細胞間通道以促進抗原的滲透,最終更好地實現(xiàn)了體液免疫效果的提升。動物實驗表明,41%凝膠組血清IgG抗體滴度是純抗原滴鼻組的5.5倍,并接近純抗原注射組的水平(0.7倍),黏膜IgA抗體滴度達到注射組的170倍。(2)針對第一章中優(yōu)化的水凝膠,系統(tǒng)考察了該凝膠佐劑的安全性。通過急毒、長毒、異常毒性、過敏反應(yīng)、熱原、溶血等實驗驗證了所開發(fā)的HTCC溫敏水凝膠是一種經(jīng)鼻應(yīng)用安全、毒副作用很低的黏膜免疫佐劑。(3)進一步采用在FDA處有備案的殼聚糖谷氨酸鹽(CS-Glu)制備水凝膠佐劑,通過正交實驗同時優(yōu)化凝膠的有效性與穩(wěn)定性。結(jié)果表明,凝膠疫苗制劑中,抗原劑量對免疫效果的影響作用最大,CS-Glu與GP的影響作用較接近。最后,進一步配方優(yōu)化后的水凝膠制劑能顯著提升純抗原的體液免疫效果(2.4倍),HI平均滴度可達120,黏膜免疫IgA抗體為注射組的178倍。該制劑在25±l。C環(huán)境下能穩(wěn)定6天,2～8℃下已經(jīng)穩(wěn)定存放兩個月,具有臨床應(yīng)用潛力。(4)針對非球形顆粒佐劑制備困難,導(dǎo)致對其形狀特性考察不足的問題,采用磷酸緩沖鹽(PBS)作為變形引發(fā)劑,利用微孔膜乳化技術(shù)成功制得不同形狀的均PLGA顆粒。該方法能同時實現(xiàn)對形狀、形貌和粒徑的三重控制,并可以實現(xiàn)規(guī)�；苽�,為其臨床應(yīng)用奠定基礎(chǔ)。通過將非球形顆粒用作H5N1裂解疫苗的注射免疫佐劑,初步闡釋了形狀對免疫效果的影響。其中球形顆粒能更好地提升體液免疫效果,而桿狀顆粒則能促進T細胞的活化以及T細胞免疫記憶的產(chǎn)生。本論文采用殼聚糖和PLGA兩種高分子聚合物材料,開發(fā)了H5N1疫苗黏膜免疫和注射免疫佐劑,探討了佐劑物化特性、流變特性以及形狀與免疫效果的關(guān)系,并開展了疫苗制劑的安全性和穩(wěn)定性評價,為實現(xiàn)臨床應(yīng)用奠定基礎(chǔ)。
[Abstract]:H5N1 avian influenza is a serious threat to human life and health. Currently, commercialized H5N1 vaccines are mostly inactivated vaccine with better safety. However, inactivated vaccine is difficult to overcome nasal mucosal barrier in nasal mucosal immunity. In injection immunization, the inactivated vaccine of H5Nl also can not induce the immune response. In this situation, two biodegradable polymer materials, chitosan and polylactic acid glycolic acid copolymer (PLGA) were used to prepare thermosensitive hydrogels and homogeneous non spherical PLGA particles as mucosal immune and injection immune adjuvant in order to enhance the immune effect of the H5Nl inactivated vaccine. According to the temperature sensitive hydrogel, the effect of the physicochemical properties of the gel on the immune effect was investigated, the mechanism of its action was expounded, the safety and stability were studied, and the pre clinical study was carried out. A simple and effective method for preparing the homogeneous deformed particles was developed for PLGA particles, and the immune effects of spherical and non spherical particles were compared, and the non sphericity was discussed. The feasibility of particles applied to H5Nl inactivated vaccine is divided into the following parts: (1) the synthesis of chitosan quaternary ammonium salt (HTCC) by amplification process, HTCC Wen Min hydrogel by crosslinking with sodium glycerol phosphate (GP), and the mucosal immune adjuvant of H5N1 vaccine, are used to regulate the gel by regulating the degree of HTCC quaternary ammonium substitution. The effects of the cationic and rheological properties on the immune effect were investigated. The results showed that the 41% HTCC hydrogel in the middle of the quaternary ammonium substitution had the appropriate viscosity and temperature sensitivity to prolong the retention of the antigen in the nasal cavity, as well as to enhance the cation performance and the adsorption of the mucous membrane, and to open the intercellular channel to promote the epithelial cells. In animal experiments, the titer of serum IgG antibody in the 41% gel group was 5.5 times that of the pure antigen intranasal group, which was close to the level of the pure antigen injection group (0.7 times) and the IgA antibody titer of the mucous membrane reached 170 times of the injection group. (2) the system was investigated systematically for the optimized hydrogel in the first chapter. The safety of the gel adjuvant. Through the experiments of acute toxicity, long toxicity, abnormal toxicity, allergic reaction, pyrogen, hemolysis and other experiments, the developed HTCC thermosensitive hydrogel is a kind of mucosal immune adjuvant with safe nasal application and low toxic side effects. (3) the preparation of hydrogel adjuvant in the preparation of chitosan glutamate (CS-Glu) in FDA is further adopted. The effectiveness and stability of the gel were optimized by over orthogonal experiments. The results showed that the effect of antigen dose on the immune effect was the greatest in the gel vaccine preparation, and the effect of CS-Glu and GP was close. Finally, the optimized hydrogel could significantly improve the humoral immune effect of the pure antigen (2.4 times), and the average HI titer of the gel vaccine could be improved. Up to 120, the mucosal immune IgA antibody was 178 times as high as that of the injection group. The preparation was stable for 6 days in the 25 + l.C environment and stable storage for two months at 2~8 centigrade. (4) the preparation of the non spherical particle adjuvant was difficult, which led to the problem of the deficiency of its shape characteristics, and the use of phosphoric acid buffer salt (PBS) as a deformable initiator. Microporous membrane emulsification has been successfully made to produce PLGA particles of different shapes. This method can simultaneously achieve three control of shape, morphology and particle size, and can be used to make a large scale preparation and lay the foundation for its clinical application. By using non spherical particles as an injection free adjuvant of H5N1 lysis vaccine, the shape of the immune effect is preliminarily explained. The spherical particles can improve the humoral immune effect, and the rod like particles can promote the activation of T cells and the production of T cell immune memory. In this paper, two kinds of polymer materials of chitosan and PLGA were used to develop the mucosal immunization and immuno adjuvant of H5N1 vaccine. The physicochemical properties, rheological properties and the rheological properties of the adjuvant were discussed. The relationship between shape and immune effect, and the safety and stability evaluation of vaccine preparations were carried out to lay the foundation for clinical application.
【學(xué)位授予單位】：中國科學(xué)院研究生院(過程工程研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R392-33
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本文編號：1901110