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针对H5N1流感疫苗的新型佐剂的设计与研究
范清泽
学位类型博士
导师马光辉
2016-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词H5n1疫苗佐剂 壳聚糖水凝胶 Plga颗粒 非球状 安全性
其他摘要

H5N1禽流感严重威胁着人类生命健康,目前,商品化的H5N1疫苗多为安全性较好的灭活疫苗。然而,在鼻黏膜免疫中,灭活疫苗难以克服鼻腔黏膜屏障。在注射免疫中,H5N1灭活疫苗也存在不能诱导理想的免疫反应、需要很高抗原用量以及现有商品化铝盐佐剂效果不佳等问题。针对此现状,本文选用两种生物可降解高分子聚合物材料,壳聚糖和聚乳酸-乙醇酸共聚物(PLGA),分别制备温敏水凝胶与均一的非球形PLGA颗粒用作黏膜免疫与注射免疫佐剂,以增强H5N1灭活疫苗的免疫效果。针对温敏水凝胶,考察凝胶的物化特性对免疫效果的影响,阐述其作用机制,并开展安全性与稳定性研究,推进其临床前研究。针对PLGA颗粒,开发简单有效的制备均一变形颗粒的方法,并比较球形与非球形颗粒的免疫效果,探讨非球形颗粒应用于H5N1灭活疫苗的可行性。具体研究内容分为以下几个部分:(1)采用放大工艺合成壳聚糖季铵盐(HTCC),通过与甘油磷酸钠(GP)离子交联制得HTCC温敏性水凝胶并用于H5N1疫苗的黏膜免疫佐剂。通过调控HTCC季铵取代度来调节凝胶的阳离子性和流变学特性,考察其物化特性对免疫效果的影响。结果表明,季铵取代度居中的41%的HTCC水凝胶既具有合适的黏度和温敏性能延长抗原在鼻腔的停留,又具有一定阳离子性能增强与黏膜的吸附,并打开上皮细胞间通道以促进抗原的渗透,最终更好地实现了体液免疫效果的提升。动物实验表明,41%凝胶组血清IgG抗体滴度是纯抗原滴鼻组的5.5倍,并接近纯抗原注射组的水平(0.7倍),黏膜IgA抗体滴度达到注射组的170倍。(2)针对第一章中优化的水凝胶,系统考察了该凝胶佐剂的安全性。通过急毒、长毒、异常毒性、过敏反应、热原、溶血等实验验证了所开发的HTCC温敏水凝胶是一种经鼻应用安全、毒副作用很低的黏膜免疫佐剂。(3)进一步采用在FDA处有备案的壳聚糖谷氨酸盐(CS-Glu)制备水凝胶佐剂,通过正交实验同时优化凝胶的有效性与稳定性。结果表明,凝胶疫苗制剂中,抗原剂量对免疫效果的影响作用最大,CS-Glu与GP的影响作用较接近。最后,进一步配方优化后的水凝胶制剂能显著提升纯抗原的体液免疫效果(2.4倍),HI平均滴度可达120,黏膜免疫IgA抗体为注射组的178倍。该制剂在25 ± 1 °C环境下能稳定6天,2~8 °C下已经稳定存放两个月,具有临床应用潜力。(4)针对非球形颗粒佐剂制备困难,导致对其形状特性考察不足的问题,采用磷酸缓冲盐(PBS)作为变形引发剂,利用微孔膜乳化技术成功制得不同形状的均一PLGA颗粒。该方法能同时实现对形状、形貌和粒径的三重控制,并可以实现规模化制备,为其临床应用奠定基础。通过将非球形颗粒用作H5N1裂解疫苗的注射免疫佐剂,初步阐释了形状对免疫效果的影响。其中球形颗粒能更好地提升体液免疫效果,而杆状颗粒则能促进T细胞的活化以及T细胞免疫记忆的产生。本论文采用壳聚糖和PLGA两种高分子聚合物材料,开发了H5N1疫苗黏膜免疫和注射免疫佐剂,探讨了佐剂物化特性、流变特性以及形状与免疫效果的关系,并开展了疫苗制剂的安全性和稳定性评价,为实现临床应用奠定基础。

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Highly pathogenic avian H5N1 influenza is a serious infectious disease. Currently, most of the commercial H5N1 vaccines are inactivated vaccines. While, in the intranasal immunization, these inactivated vaccine could not overcome the nasal mucosal barrier and usually generate relative weak immunity. Moreover, in the intramuscular immunization, these inactivated H5N1 vaccines could not induce optimal immune response, even with commercial Alum adjuvant and always require large antigen dosage. Therefore, the thesis employed two biodegradable high molecular polymer, chitosan and poly(lactic-co-glycolic) acid (PLGA), to prepare thermal-sensitive hydrogel and uniform non-spherical PLGA particles as the intranasal and intramuscular adjuvant respectively, which could enhance the immune responses. For hydrogel adjuvant, the relationship between the physiochemical properties and immune effects and the underlying mechanism were explored. Then, toxicity and stability of the hydrogel formulation were investigated. For particle adjuvant, a simple method was developed to prepare non-spherical PLGA particles and explore their shape effects. This thesis was divided into the following parts.(1) The synthesis of quaternized chitosan (HTCC) was magnified and the thermal-sensitive HTCC hydrogel was well prepared by ionic gelation with glycerol phosphate sodium (GP). By adjusting the quaternize degree (QD) of HTCC, the influence of hydrogel cationic character and rheological behavior on the corresponding immune responses was investigated. Results showed there was competition between cationic character and thermal-sensitivity. As a consequence, the hydrogel with a moderate QD of 41% had both the good cationic character and good thermal-sensitivity, resulting in the induction of the best humoral immunity. In-vivo animal experiments showed that the IgG titer induced by 41% hydrogel was 5.5 times higher than the pure antigen intranasal group (Ag IN) and comparable to the pure antigen intramuscular group (Ag IM). The nasal IgA titer of hydrogel group was 170 times higher than Ag IM.(2) The safety and toxicity of the hydrogel were systematically investigated. The tests of acute toxicity, repetitive dose toxicity, abnormal toxicity, allergy reaction, pyrogen and hemolysis were carried out in the animal models of mice, rats, rabbits and guinea pigs. Results showed that the HTCC hydrogel was well tolerated and had very low toxicity when used as an intranasal vaccine adjuvant.(3) Furthermore, we chose the import chitosan glutamine salt (CS-Glu) which has presented file to the Food and Drug Administration (FDA) to prepare the CS-Glu hydrogel. Through the orthogonal experiment, the influence of CS-Glu concentration, GP concentration and antigen dose on the immune responses were investigated and optimized along with the formulation stability. Results showed that the antigen dose had a dominant effect on the immune effects and the influences of CS-Glu and GP were comparable. At last, the CS-Glu hydrogel adjuvant was optimized, which could induce the value of 120 for HI titer and IgA titer 178 times higher than Ag IM group. This hydrogel could store at 25 ± 1 °C for 6 days and 2~8 °C for more than 2 months。(4) There is lack of researches about the influence of particle shape on the biological outcomes because of the difficulty in the preparation of non-spherical particles. In order to resolve this problem, a novel method was developed based on the premix membrane emulsification technique to prepare uniform non-spherical particles. By employing an inorganic reagent, phosphate buffer saline (PBS), as the deformation initiator, non-spherical PLGA particles were prepared. The shape, morphology and size of the particles could be well controlled in this method and it is easy to scale up the production. Then, the non-spherical particles were used as H5N1 vaccine adjuvants and the shape effects were studied. The preliminary results showed that the spherical particles could potentiate humoral immunity while the rod particles could promote the activation of antigen specific T cells and increase the antigen specific memory T cells.In summary, by using the high molecular polymers of chitosan and PLGA, two adjuvants of H5N1 inactivated vaccine were developed. The influence of the physicochemical properties, the rheological behavior and the shape on the corresponding immune responses was deeply investigated. Moreover, the safety and formulation stability were studied and optimized, which could establish a foundation for clinical application. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22889
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范清泽. 针对H5N1流感疫苗的新型佐剂的设计与研究[D]. 北京. 中国科学院研究生院,2016.
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