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基于化学修饰的脑膜炎多糖结合疫苗和结核分枝杆菌亚单位疫苗
黄庆瑞
学位类型博士
导师胡涛
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
关键词多糖结合疫苗 聚乙二醇 佐剂 聚肌胞 阿拉伯半乳聚糖
摘要

近年来由于抗生素的滥用,细菌的耐药性问题越来越严重。针对各种致病菌的疫苗研发备受关注。因安全性好、化学成分明确且易于大规模生产,亚单位疫苗成为新型细菌疫苗研究的热点。通过将佐剂与抗原或者不同的佐剂连接成一个统一的整体,化学偶联技术可显著增强亚单位疫苗的免疫保护效果。在脑膜炎球菌多糖结合疫苗中,应用化学偶联技术制备三种连接桥的不同多糖结合疫苗,研究了PEG连接桥对多糖结合疫苗免疫原性的影响;在结核分枝杆菌亚单位疫苗中,通过将聚肌胞(poly(I:C))与阿拉伯半乳聚糖(AG)化学偶联,制备出新型免疫佐剂AG-P,并将结核分枝杆菌抗原Ag85B-HspX(AH)与AG-P采用化学偶联的递送方式,得到具有高免疫原性的抗原-佐剂共递送疫苗AH-AG-P。具体研究内容如下:(1)脑膜炎奈瑟球菌是一种可引发脑膜炎及其它一些病症的病原菌。脑膜炎奈瑟球菌的荚膜多糖是一种重要的致病因子,可作为免疫原来开发疫苗。与载体蛋白化学偶联后,荚膜多糖(PS)可由T细胞非依赖性抗原转化为T细胞依赖性抗原,其免疫原性得到大幅提升,并可诱导免疫记忆,从而能够为婴幼儿提供免疫保护。然而,作为生物大分子的载体蛋白可能会对荚膜多糖的一些重要抗原表位形成空间屏蔽效应在一定程度上降低多糖的免疫原性。在本论文中,我们使用一种异型双功能聚乙二醇(PEG)来连接Y型脑膜炎球菌荚膜多糖(PS)与破伤风类毒素(TT)。异型双功能PEG可以避免PS与TT的自身交联,并提高多糖结合疫苗中的多糖蛋白比。在偶联过程中,PS与TT的结构均未发生显著改变。与不含PEG的多糖结合疫苗相比,以PEG为连接桥的多糖结合疫苗诱导的PS特异性IgG抗体滴度增加了3倍,并且诱导的免疫持效性延长。PEG作为一种无免疫原性的亲水性聚合物,已被广泛应用于蛋白质药物的化学修饰。尽管PEG修饰可以提高药物的循环半衰期和降低药物的免疫原性,但是在多糖结合疫苗中PEG可以通过抑制TT的免疫原性和降低TT对PS抗原表位的空间屏蔽效应来提高多糖的免疫原性。PEG还可延长疫苗的免疫持效性。因此,作为连接桥的PEG可用于开发预防效果更好的多糖结合疫苗。(2)结核分枝杆菌(Mtb)是一种可在世界范围内引起肺结核的主要致病菌。因安全性好、化学成分明确且易于大规模生产,蛋白质亚单位疫苗有望替代完善卡介苗(BCG)。但是,蛋白质亚单位疫苗需要高效的免疫佐剂以及更好的递送系统,才能为机体提供有效的免疫保护。聚肌胞(poly(I:C))是一种人工合成的由聚肌苷酸和聚胞苷酸通过碱基配对形成的病毒双链RNA类似物。通过与免疫细胞TLR3受体的相互作用,poly(I:C)可增强机体对胞内感染菌Mtb的免疫应答。AG是一种具良好生物相容性的多糖佐剂,可刺激淋巴细胞的增殖、激活巨噬细胞、并促进IFN-γ及TNF- α等细胞因子的释放。通过AG与poly(I:C)的协同效应,AG-P表现出更强的免疫刺激作用。Ag85B与HspX是Mtb分别于活跃复制期和潜伏期表达的重要免疫原蛋白,通过对它们进行重组融合表达,可得到融合蛋白Ag85B-HspX(AH)。为确保融合蛋白与佐剂可同步到达和刺激抗原呈递细胞,我们通过将AH与AG-P化学偶联制备出一种抗原-佐剂共递送疫苗AH-AG-P。免疫学评价表明:AH-AG-P可诱导产生高滴度的AH特异性IgG抗体(以IgG2c亚型为主)并刺激淋巴细胞的增殖;AH-AG-P还可促进抗原的交叉呈递进而增加脾脏淋巴细胞中CD8+ T细胞的比例,并提高Th1细胞因子(IFN-γ、TNF- α和IL-2)的表达水平。药代动力学研究表明:与AG-P的偶联延长了AH在血浆中的循环半衰期,使其可持续地刺激免疫系统。药效动力学研究表明:AH-AG-P可快速诱导生成高滴度的AH特异性IgG抗体。因此,将AH 与AG-P共价偶联是一种开发抗Mtb感染疫苗的有效策略。

其他摘要

With the extensive abuse of antibiotics, bacterial resistance becomes very serious. Vaccine developments against various pathogenic bacteria have attracted much attention. Subunit vaccine has become a focus in the research of new bacteria vaccines for its safety, specificity and easy production. By conjugation of adjuvant to antigen or different adjuvant into a unified whole, chemical conjugation technology can enhance the immune protective effect of subunit vaccine. In Neisseria meningitidis polysaccharide conjugate vaccine, we prepared conjugate vaccines containing different spacer arms. Then, the effect of PEG as a spacer arm on immunogenicity of conjugate vaccine was investigated. In Mycobacterium tuberculosis subunit vaccine research, we prepared new adjuvant AG-P by coupling poly(I:C) to arabinogalactan (AG). In application of a co-delivery system by conjugating Mtb Ag85B-HspX (AH) fusion protein with AG-P, we developed a highly efficient vaccine (AH-AG-P). The detailed research conclusions are as follows:(1) Neisseria meningitidis is a life-threatening pathogen that causes meningitis and other clinical manifestations. As a key virulence determinant, meningococcal capsular polysaccharide (PS) can be used as an immunogen for vaccine development. Conjugation to a carrier protein can turn PS from a T cell independent antigen into a T cell dependent antigen, significantly improve its immunogenicity, induce memory response and as a result can provide immune protection for infants and young children. However, as a biomacromolecule carrier protein may cause steric shielding effect on important antigenic PS epitopes leading to lower the immunogenicity of PS. Here, a heterobifunctional polyethylene glycol (PEG) was used as a spacer arm to conjugate meningococcal group Y capsular PS with tetanus toxoid (TT). Heterobifunctional PEG can avoid self-crosslink of PS and increase the PS/TT ratio of the vaccine. Significant structural change in TT and PS was not observed upon conjugation. As compared to the vaccine without PEG, immunization with the vaccine using PEG as the spacer arm led to a 3.0-fold increase in the PS-specific IgG titers and a prolonged immune persistence. As a non-immunogenic hydrophilic polymer, PEG has been widely used to couple therapeutic protein for prolonging its circulatory time and decreasing its immunogenicity. But in polysaccharide conjugate vaccine PEG as a spacer arm can improve the immunogenicity of PS by fully decreasing the steric shielding effect of TT on antigenic epitopes of PS and suppressing the immunogenicity of TT. In addition, PEG can prolong the immune persistence of the conjugate vaccine. Thus, PEG can be used as a spacer arm to develop effective polysaccharide conjugate vaccine. (2) Mycobacterium tuberculosis (Mtb) is one of the main pathogens that can cause pulmonary tuberculosis in the world. Protein-based vaccine is promising to improve or replace BCG vaccine for its specificity, safety and easy production. However, protein- based vaccine calls for potent adjuvants and improved delivery systems to provide effective immune protection for human beings. Polyinsinic-polycytidylic acid (poly(I:C)) is a synthetic analog of viral double-stranded RNA produced by polycy-tidylic acid and polycytidylic acid through base pairing. Poly(I:C) can strengthen human immune response against intracellular Mtb by signaling via TLR3. Arabinogalactan (AG) is a biocompatible polysaccharide that can increase splenocyte proliferation, stimulate macrophages and improve the secretion of cytokines such as IFN-γ and TNF-α. The AG- poly(I:C) conjugate (AG-P) could play a potent immunostimulatory activity through a synergistic adjuvant interaction of AG and poly(I:C). Ag85B and HspX are two important virulent protein antigens expressed by Mtb at actively replicating and latent stages, respectively. Ag85B-HspX fusion protein (AH) was prepared by recombinant fusion expression of the two antigen genes. An antigen-adjuvant co-delivery system (AH-AG-P) was developed by conjugation of AH with AG-P to insure that both AH and AG-P reach and stimulate the antigen presenting cells simultaneously. Immunological evaluation revealed that AH-AG-P could elicit high AH-specific IgG titers mostly in the form of IgG2c and stimulate lympho- cyte proliferation. AH-AG-P could also increase high splenic CD8+ T cell proportion by promoting cross-presentation of antigen and provoked the secretion of Th1-type cytokines (IFN-γ, TNF-α and IL-2). Pharmacokinetic analysis revealed that conjugation with AG-P could prolong half-life of AH in serum and persistently stimulate the immune system. Pharmacodynamic analysis suggested that AH-AG-P led to a rapid and intense production of AH-specific IgG. Thus, co-delivery of AH with a new potent adjuvant AG-P is an effective strategy to develop an efficacious and protective vaccine against Mtb infection. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22909
专题研究所(批量导入)
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黄庆瑞. 基于化学修饰的脑膜炎多糖结合疫苗和结核分枝杆菌亚单位疫苗[D]. 北京. 中国科学院研究生院,2016.
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