针对H5N1流感疫苗的新型佐剂的设计与研究

发布时间：2018-05-17 11:00

  本文选题：H5N1疫苗佐剂 + 壳聚糖水凝胶　； 参考：《中国科学院研究生院(过程工程研究所)》2016年博士论文

【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,H5Nl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H5Nl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H5Nl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±l。C环境下能稳定6天,2～8℃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。
[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.
【学位授予单位】：中国科学院研究生院(过程工程研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392-33
，

本文编号：1901110