新型疫苗锌佐剂的制法优化及ATP与铝、锌复合佐剂免疫增强效果研究
发布时间：2018-08-28 08:56
【摘要】：疫苗佐剂的发展直接影响到新型疫苗的开发及应用,为了开发出更安全、高效、经济以及更具有靶向性的佐剂,国内外研究人员进行了大量的研究。目前,铝佐剂仍然是唯一在世界范围内被批准用于人用疫苗的佐剂,它主要依赖储存库效应和免疫刺激效应的作用机制,能够显著提高疫苗在体内的效果,经过了几十年的应用,虽然出现部分轻微过敏反应,但总体来说其安全性和有效性均得到了证实。 锌是人体必需微量元素之一,人体生长发育离不开锌,体内锌水平的高低会对神经、生殖和免疫系统产生影响。利用锌与机体免疫系统之间的复杂关系,锌疗法被越来越广泛地应用于一些疾病的治疗中。氢氧化锌与氢氧化铝理化性质相近,在一定条件下,二者均能以稳定的胶体溶液形式存在,由于铝佐剂在临床应用中取得了良好的效果,因此将氢氧化锌作为疫苗佐剂进行的研究也非常具有潜力,在前期研究中,也初步证实了氢氧化锌佐剂的安全性和有效性。在本研究中,参照铝佐剂的制备方法,首先将可溶性锌盐葡萄糖酸锌作为研究对象,利用其与低浓度氢氧化钠溶液发生反应,制备出性质稳定质地均匀的氢氧化锌胶体溶液,之后再针对疫苗佐剂的要求对其理化性质进行改进,最终制得可以作为疫苗佐剂使用的氢氧化锌胶体溶液。 三磷酸腺苷二钠(ATP)是一种辅酶,有改善机体代谢的作用,同时又是体内能量的主要来源。ATP能在某些免疫反应中扮演重要角色,实验表明,ATP在与MgCl2复合使用后,可以对体内IgG、IgA、IgM表达水平产生一定的影响,同时,ATP是体内一类重要的内源性危险信号因子,在缺氧、组织损伤、病变发生时,多会伴随周围组织体液中ATP水平的变化,利用这一特点,改变体内ATP的水平在 一定条件下也能有效激活免疫系统,并影响其效应过程。因此,ATP也作为一类潜在疫苗佐剂得到进一步的开发和研究。 复合佐剂是近年来佐剂研究的新热点,由于一些疫苗佐剂单独作用效果并不理想,而将多种疫苗佐剂复合使用以后却能达到让人满意的效果,因此关于复合佐剂的研究也不断深入。本研究中,分别使用已经得到广泛运用的铝佐剂以及实验中制备的氢氧化锌胶体佐剂,与ATP制备得到复合佐剂,再进一步研究复合佐剂与铝、锌佐剂单独作用的效果差异。实验中,以人用狂犬病疫苗作为抗原,分别设置不添加佐剂的抗原对照组、仅添加铝、锌佐剂的单独佐剂组和分别添加了ATP-氢氧化铝佐剂和ATP-氢氧化锌佐剂的复合佐剂组,对ICR小鼠进行免疫,在初次免疫后不同时间段通过间接ELISA法检测小鼠血清中抗-狂犬病病毒IgG的滴度进行免疫效果评价。 体液免疫效果评价结果表明,Zn(OH)2胶体与铝佐剂类似,其作为佐剂单独使用或与ATP联合使用均能一定程度上增强机体针对狂犬病毒抗原的免疫应答,虽然佐剂1次免疫组尚未达到抗原5次免疫组(抗原对照Ⅳ组)效果(P0.05),但抗体水平已经基本高于抗原1-3次免疫组(抗原对照Ⅰ、Ⅱ、Ⅲ组)(P0.05),提示Zn(OH)2胶体佐剂单独使用以及与ATP联合使用均能增强狂犬病疫苗特异性体液免疫应答,有一定的免疫增强作用。通过横向对比可以发现,氢氧化锌胶体作为佐剂使用的效果已经接近甚至超过铝佐剂,加之锌元素的毒副作用要远远低于铝元素,锌佐剂的开发应用也有望克服铝佐剂的应用瓶颈,达到一个新的高度。
[Abstract]:The development of vaccine adjuvants has a direct impact on the development and application of new vaccines. In order to develop safer, more efficient, more economical and more targeted adjuvants, researchers at home and abroad have done a lot of research. The mechanism of response and immune stimulation can significantly improve the efficacy of the vaccine in vivo. After decades of application, although some slight allergic reactions have occurred, its safety and effectiveness have been confirmed in general.
Zinc is one of the essential trace elements in human body. Zinc can not be separated from the growth and development of human body. The level of zinc in the body will affect nervous, reproductive and immune system. Using the complex relationship between zinc and the immune system, zinc therapy is more and more widely used in the treatment of some diseases. Recently, both of them can exist in a stable colloidal solution under certain conditions. As aluminum adjuvant has achieved good results in clinical application, the study of zinc hydroxide as vaccine adjuvant has great potential. In the previous study, the safety and effectiveness of zinc hydroxide adjuvant were preliminarily confirmed. According to the preparation method of aluminium adjuvant, zinc gluconate, a soluble zinc salt, was used as the research object to prepare zinc hydroxide colloidal solution with stable and homogeneous properties by reacting with low concentration of sodium hydroxide solution. Then the physicochemical properties of zinc hydroxide colloidal solution were improved according to the requirements of vaccine adjuvant. Zinc hydroxide colloid solution for vaccine adjuvants.
ATP is a coenzyme that can improve metabolism and is also the main source of energy in the body. ATP can play an important role in some immune responses. The experiment shows that ATP combined with MgCl2 can affect the expression of IgG, IgA and IgM in vivo, and ATP is an important class in vivo. Endogenous risk factors, when hypoxia, tissue damage, lesions occur, are often associated with changes in the levels of ATP in the surrounding tissues and body fluids, using this feature to change the level of ATP in the body.
Under certain conditions, ATP can activate the immune system effectively and affect its effect process. Therefore, ATP has been further developed and studied as a potential vaccine adjuvant.
Complex adjuvant is a new hotspot in adjuvant research in recent years, because some vaccine adjuvants alone effect is not ideal, but after the use of multiple vaccine adjuvants can achieve satisfactory results, so the study of complex adjuvant is also in-depth. In this study, the use of aluminum adjuvant and solid adjuvant has been widely used. In the experiment, human rabies vaccine was used as antigen, and the antigen control group without adjuvant was set up, and the adjuvant group with aluminum and zinc adjuvant and the adjuvant group with ATP were added respectively. The ICR mice were immunized with the combined adjuvant of - aluminium hydroxide adjuvant and ATP-zinc hydroxide adjuvant. The titers of anti-rabies virus IgG in the serum of the mice were detected by indirect ELISA at different time after the first immunization.
The results of humoral immunity evaluation showed that Zn (OH) 2 colloid was similar to aluminum adjuvant, and its adjuvant alone or combined with ATP could enhance the immune response to rabies virus antigen to a certain extent. Although the adjuvant once immunized group had not reached the effect of antigen five times immunization group (antigen control group IV) (P 0.05), the antibody level had already been increased. The results showed that Zn (OH) 2 colloidal adjuvant alone and combined with ATP could enhance the specific humoral immune response of rabies vaccine, and had a certain immune enhancement effect. The development and application of zinc adjuvant is expected to overcome the bottleneck of the application of aluminum adjuvant and reach a new height.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
[Abstract]:The development of vaccine adjuvants has a direct impact on the development and application of new vaccines. In order to develop safer, more efficient, more economical and more targeted adjuvants, researchers at home and abroad have done a lot of research. The mechanism of response and immune stimulation can significantly improve the efficacy of the vaccine in vivo. After decades of application, although some slight allergic reactions have occurred, its safety and effectiveness have been confirmed in general.
Zinc is one of the essential trace elements in human body. Zinc can not be separated from the growth and development of human body. The level of zinc in the body will affect nervous, reproductive and immune system. Using the complex relationship between zinc and the immune system, zinc therapy is more and more widely used in the treatment of some diseases. Recently, both of them can exist in a stable colloidal solution under certain conditions. As aluminum adjuvant has achieved good results in clinical application, the study of zinc hydroxide as vaccine adjuvant has great potential. In the previous study, the safety and effectiveness of zinc hydroxide adjuvant were preliminarily confirmed. According to the preparation method of aluminium adjuvant, zinc gluconate, a soluble zinc salt, was used as the research object to prepare zinc hydroxide colloidal solution with stable and homogeneous properties by reacting with low concentration of sodium hydroxide solution. Then the physicochemical properties of zinc hydroxide colloidal solution were improved according to the requirements of vaccine adjuvant. Zinc hydroxide colloid solution for vaccine adjuvants.
ATP is a coenzyme that can improve metabolism and is also the main source of energy in the body. ATP can play an important role in some immune responses. The experiment shows that ATP combined with MgCl2 can affect the expression of IgG, IgA and IgM in vivo, and ATP is an important class in vivo. Endogenous risk factors, when hypoxia, tissue damage, lesions occur, are often associated with changes in the levels of ATP in the surrounding tissues and body fluids, using this feature to change the level of ATP in the body.
Under certain conditions, ATP can activate the immune system effectively and affect its effect process. Therefore, ATP has been further developed and studied as a potential vaccine adjuvant.
Complex adjuvant is a new hotspot in adjuvant research in recent years, because some vaccine adjuvants alone effect is not ideal, but after the use of multiple vaccine adjuvants can achieve satisfactory results, so the study of complex adjuvant is also in-depth. In this study, the use of aluminum adjuvant and solid adjuvant has been widely used. In the experiment, human rabies vaccine was used as antigen, and the antigen control group without adjuvant was set up, and the adjuvant group with aluminum and zinc adjuvant and the adjuvant group with ATP were added respectively. The ICR mice were immunized with the combined adjuvant of - aluminium hydroxide adjuvant and ATP-zinc hydroxide adjuvant. The titers of anti-rabies virus IgG in the serum of the mice were detected by indirect ELISA at different time after the first immunization.
The results of humoral immunity evaluation showed that Zn (OH) 2 colloid was similar to aluminum adjuvant, and its adjuvant alone or combined with ATP could enhance the immune response to rabies virus antigen to a certain extent. Although the adjuvant once immunized group had not reached the effect of antigen five times immunization group (antigen control group IV) (P 0.05), the antibody level had already been increased. The results showed that Zn (OH) 2 colloidal adjuvant alone and combined with ATP could enhance the specific humoral immune response of rabies vaccine, and had a certain immune enhancement effect. The development and application of zinc adjuvant is expected to overcome the bottleneck of the application of aluminum adjuvant and reach a new height.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
【参考文献】
相关期刊论文 前4条
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2 李德娟;何巍;张睿;付琳娜;赵丽剑;王宇;金立杰;;氢氧化铝佐剂配制工艺的优化[J];中国生物制品学杂志;2010年10期
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