【作者】 赵静；

【导师】 徐沪济；

【作者基本信息】 第二军医大学 ， 风湿免疫学， 2011， 硕士

【摘要】 一、研究背景鼠伤寒沙门氏菌是周毛菌，菌体周身遍布鞭毛，鞭毛是由基础小体、钩状体和丝状体三部分构成，它不仅是沙门氏菌的运动器官，而且同时还具有特殊的抗原性，称为H抗原，H抗原是沙门氏菌鉴定分型的重要依据，而且可以激发先天性免疫系统产生应答。随着对鞭毛主要成份——鞭毛蛋白免疫作用机理的不断深入研究，逐渐发现沙门氏菌鞭毛蛋白可以通过自身激发先天性免疫系统产生应答，以启动、增强特异性免疫系统对其他与鞭毛蛋白相连接的抗原产生特异性的应答，发挥较强的免疫佐剂作用，而且可以在粘膜表面诱导以Th2型免疫反应并且伴有IgA的分泌。其强大的免疫佐剂功效随后在多种病原体疫苗的研究中获得应用，如应用于鼠疫杆菌、变异链球菌、单核细胞增生李斯特菌、甲型流感病毒、西尼罗河病毒、和间日疟原虫等多种病原体的表位多肽和抗原蛋白，甚至用于增强甲型H1N1流感病毒灭活株的免疫应答。而且在各项实验中并没有发现各种疫苗制剂因加入沙门氏菌鞭毛蛋白引起的过敏反应或其他不良反应，因此鞭毛蛋白作为疫苗佐剂是较为安全的。先天性免疫不仅仅是抵抗病原体侵袭的第一道屏障，而且在识别和区分病原体、启动获得性免疫中起了关键的作用，抗原递呈细胞尤其是树突状细胞在其中担负了非常重要的角色。抗原递呈细胞具有所谓的模式识别受体（pattern recognition receptor，PRR），可以识别病原体相关分子模式（pathogen associated molecular pattern，PAMP）。鞭毛蛋白是一种蛋白性的PAMP，可以被PRR主要是细胞表面Toll样受体5（Toll-likereceptor 5，TLR5）识别和区分，两者特异性结合后鞭毛蛋白即可作用于表达有TLR5的细胞，如上皮细胞、内皮细胞、粒细胞、单核细胞、T淋巴细胞、NK细胞、树突细胞等，通过激活TLR5，活化MyD88依赖的信号转导通路，调控TNF-α、IL-6和IL-12等促炎细胞因子的合成，上调协同刺激分子CD80、CD86以及MHCⅡ分子，从而使这些主要的抗原递呈细胞激活和成熟，摄取抗原能力增强，向引流淋巴结迁移能力增强，从而招引、激活和分化T辅助细胞，最终启动获得性免疫应答，实现免疫佐剂功效。而对于细胞胞质内部的鞭毛蛋白则主要依赖表达在细胞胞质中的NOD样受体(Nucleotide oligomerization domain (NOD)-like receptor, NLR）家族中的IPAF（IL-1β-converting enzyme (ICE) protease activating factor）和NAIP5（neuronal apoptosisinhibitory protein 5）受体，其中IPAF受体起主要的识别作用，而NAIP5则可与IPAF联合作用，起到增强细胞对胞质内鞭毛蛋白的识别作用。目前许多疾病的疫苗候选抗原都面临在人体内低免疫原性的问题，例如恶性疟原虫裂殖子顶端膜抗原1(apical membrane antigen 1,AMA-1)蛋白，临床试验表明使用铝佐剂或Montanide ISA720佐剂都不能使AMA1在绝大多数受试者中激发出有效水平的抗体滴度，当在铝佐剂中添加了Toll样受体9（TLR9）的激动剂CpG ODN后，在受试者中抗AMA1的抗体水平得到了极大提高，并且高滴度的免疫血清在疟原虫体外抑制试验中的抑制率可达到90%以上，最高者达到96%。然而，由于在临床试验中发现1例由CpG ODN产生的副反应，所以CpG ODN的安全性已成为这种AMA1疫苗制剂在临床试验中进一步推进的重要障碍。本研究工作将以作为TLR5激动剂的纯化沙门氏菌鞭毛蛋白（flagella filamentprotein，FliC）替代作为TLR9激动剂的CpG ODN1826，加入到模型抗原——卵清蛋白（Ovalbumin，OVA）和氢氧化铝佐剂的疫苗制剂中，免疫实验小鼠，观察和比较添加鞭毛蛋白的疫苗制剂所激发的免疫应答，为鞭毛蛋白作为佐剂增强剂应用于以各种低免疫原性候选抗原的疫苗提供实验数据。二、研究目的本实验拟通过大样本随机对照的研究方法，在BALB/c小鼠中，研究鼠伤寒沙门氏菌鞭毛蛋白增强抗原特异性抗体应答的佐剂增强剂功效及其机制，同时与CpGODN进行比较，观察两者佐剂增强剂功效的差别，以及采用不同免疫方式对鞭毛蛋白作为佐剂增强剂的影响。三、研究方法1、采用酶联免疫吸附法（Enzyme Linked Immunosorbent Assay，ELISA）测定血浆中特异性抗OVA的IgG抗体滴度，以及各种IgG分型抗体（IgG1、IgG2a、IgG2b、IgG3）的滴度。2、采用ELISA法测定小鼠脾细胞体外刺激上清中干扰素gamma（Interferonγ,IFN-γ）和白介素4（Interleukin-4，IL-4）的含量。3、采用TurboFect体外转染试剂构建稳定表达pUNO-hTLR5质粒的小鼠单核巨噬细胞RAW264.7细胞株。四、研究结果1、采用肌肉注射免疫方式联合应用鞭毛蛋白的卵清蛋白/氢氧化铝佐剂（OVA+Alhydrogel+Flagellin）组诱导产生特异性抗OVA抗体滴度显著高于OVA+ Alhydrogel组，低于联合应用CpG ODN组，但随着免疫时间的延长，联合应用鞭毛蛋白组特异性抗体滴度逐渐高于联合应用CpG ODN组。整个免疫过程中，鞭毛蛋白显示出与CpG ODN相当的免疫佐剂功效。2、肌肉免疫方式中添加鞭毛蛋白组特异性抗体亚型中IgG2a和IgG2b比例较单纯铝佐剂组升高。体外刺激添加鞭毛蛋白组小鼠脾脏细胞培养基上清中检测到较单纯铝佐剂组高水平的IFN-γ和低水平的IL-4，考虑由鼠沙门氏菌鞭毛蛋白诱导的免疫应答是Th1/Th2混合型，但以Th1型为主，且可通过经典途径活化补体系统，全面提高机体对外源性抗原的特异性体液免疫反应及细胞免疫反应。3、采用皮下注射免疫和腹腔注射免疫方式，整个免疫过程中添加鞭毛蛋白或者CpGODN组特异性抗体滴度都显著高于单纯应用铝佐剂组，且添加CpG ODN组升高更为明显。4、皮下注射免疫和腹腔注射免疫方式中鞭毛蛋白诱导的免疫应答机制与肌肉免疫方式中一致，只是采用皮下免疫后特异性抗体IgG2b比例未见明显升高，考虑此种免疫方式下鞭毛蛋白不能有效活化补体系统。5、成功构建稳定表达pUNO-hTLR5质粒的小鼠单核巨噬细胞RAW264.7转染细胞株。五、研究结论本研究首次采用铝佐剂的吸附作用，选取新型免疫佐剂鞭毛蛋白，体外将鼠伤寒沙门氏菌鞭毛蛋白和OVA的氢氧化铝胶制剂（Alhydrogel）直接混合构成复合体，采用肌肉注射免疫方式，并诱导以Th1为主的Th1/2混合型免疫应答，同时有一定的诱导补体系统活化功能，在BALB/c小鼠体内全面显著增强OVA特异性的体液及细胞免疫应答，表现出CpG ODN相当的佐剂增强剂功效，为新型人用疫苗的研发提供更安全有效的佐剂选择。更多还原

【Abstract】 1. Research BackgroundAs one kind of peritrichate, the flagella of Salmonella typhimurium, which is composed ofa basal body, hook, motor, and filament, plays a major role in its motility and specialantigenicity[1, 2] . The special antigen, H antigen, is not only the systematical standards, butalso a potent inducer of innate immunity. Flagellin is a highly conserved bacterial proteinwhich is the most important part of the flagella and elicits TLR5 dependent inflammatoryresponses in both animals and plants [3, 4] and plays an significant role in bacterialpathogenicity by facilitating motility and adhesion to host mucosal tissues [5-8]. Recentstudies have shown that flagellin is associated with strong adaptive immune responses,mainly Th2-type, with the production of mucosal [9-12] and systemic IgA.Flagellin caninduce the antigen-special response which is combined with flagellin through the innateimmunology induced by it. Accordingly they have the profound significance as adjuvants,and recombinant flagellin in a soluble or fused form has been reported to play an importantrole as adjuvant for novel vaccines against epitope-based influenza vaccines [13], Yersiniapestis [8, 14], Plasmodium falciparum [15] , Tetanus toxoid [10], West Nile virus (WNV) [16]and inactivated influenza virus (A/PR/8/34, HIN1 subtype) [17] . The safety of flagellin usedas adjuvant is without doublts, because there are no seriously adverse reactions during allof the experiments.In view of the established role of the innate immune response in the development ofadaptive immunity, the strong innate response to flagellin may be the reason why flagellinpresents as an effective adjuvant. The innate immue is the firstline defense againstmicrobial pathogen. It can recognize and discriminate the different pathogen and as theinitiator of the events that are essential for the developent of an adaptive immune response,in which antigen presenting cells play the important role. Pattern recognition receptorexpressed on antigen presenting cells can recognize the corresponding pathogen associatedmolecular pattern expressed on pathogen [18-20]. Flagellin is a proteinic pathogen associatedmolecular pattern which can be recognized by pattern recognition receptor, Toll-likereceptor 5 mainly [21-23]. The direct binding of flagellin to the TLR5 expressed on cells suchas endothelial and epithelial cells, monocytes, macrophages, and immature dendritic cellsinitiates the MyD88 depended signal passway, products proinflammatory cytokines such as TNF-α、IL-6 and IL-12, and up-regulate some costimulatory molecule, CD80, CD86, MHCet al [24], then stimulate cells maturation, enhance the ability of presenting antigen, andmigrate to secondary lymphoid sites . Accordingly Th cells are attracted, stimulated,differentiated to another typical cell and finally initiate the adaptive immunity. Meanwhile,the flagellin within the cytosol of macrophages is detected through the Nod-like receptot(NLR) IPAF and NAIP5. IPAF is the mainly cytosol receptor, however, NAIP5 justenhance the detection of the flagellin with IPAF.Failing to enhance the immunogenicities of antigens to obtaina protective and sustainedresponse in humans is, so far, still a formidable problem with which many vaccines todayhave confronted including the blood-stage antigen apical membrane antigen 1 (AMA1)which is the leading malarial vaccine candidate of Plasmodium falciparum. [25,26] Despite ofthe conjugation of Alhydrogel or Montanide ISA720, the special antibody in human bodyto AMA1 still remained a low level. The addition of the TLR9 agonist CpG ODN to theAMA1/Alhydrogel formulation greatly increased the functional antibody responses inhuman, and the GIA can averagely reach 90%, in which 96% is the highest. However,along with the side effect associated with CpG ODN in an adult phase III trail, thepotential safety hazard became the biggest obstacle to the application of CpG ODN.This research focused on the immunopotention of flagellin which is conjugated withOVA/Alhydrogel formulation instead of CpG ODN. All the results has provided theevidence that the Salmonella flagellin is a potent immunopotentiator of specific immunityin mice immunized with ovalbumin .2. ObjectivesThis study tries to research the ability of flagellin as a immunopotentiator among BALB/cmice, through comparing the special antibodies in the serum during a randomizedcontrolled trial.3. Methods3.1 Analyzing the quantity of antibody to OVA from immunized BALB/c mice, includingIgG/IgG2a/IgG2b/IgG3, by EILSA. 3.2 Analyzing the concentration of IL-4 and IFN-γin supernatant of splenocytes whichwere isolated from immunized mice sacrificed after 14 days post-boost and stimulated byOVA and PMA.3.3 Contract TLR5-specific bioactivity assay: transfect the RAW264.7 cell strain withplasmid pUNO-hTLR5 expressing the human TLR5 through the TurboFect agent.4. Results4.1 The vaccine formulations of OVA+Alhydrogel+Flagellin administered intramuscularlyinduced significantly higher antibody titers comparing to OVA+Alhydrogel, meanwhile nostatistically significant difference in serum IgG titers were detected between mice injectedwith flagellin and CpG ODN. Moreover, the period of higer antibody titers is longer in thegroup co-administered with flagellin than those with CpG ODN. In the whole experiment,the immunological enhancement of flagellin is comparable to CpG ODN’s.4.2 The flagellin coadminisered intramuscularly induced higher IgG2a and IgG2b ratio ofspecial antibody, and produced higer IFN-γand less IL-4 than the other group. Based onthe above results detected with the doses tested, we found that flagellin enhanced both theTh1 and Th2 responses, Th1-biased. On the other hand the increased IgG2b levels activatecomplement via the classical pathway. It has been suggested that the classical pathway isthe most effective immune defense against microorganisms, and flagellin may thereforehave some advantages as an adjuvant for enhancing the humoral immunity againstinfection.4.3 When Vaccine formulations were administered subcutaneously and intraperitoneally,the group coadministered with flagellin induced significantly higher special antibody titersthan the group injected only with Alhydrogel and significantly lower than the ones withCpG ODN.4.4 The immune response generanted in mice with each vaccine formulationadministratered subcutaneously and intraperitoneally is the same as the results of groupsimmunized intramuscularly. Howerve, there is no higer ratio of IgG2b in groupsimmunized subcutaneously, It supposed that the administrer’s way is not suitable foractivating complement system.4.5 Get the RAW264.7 cell line which expresses TLR5 consistently. 5. ConclusionsFlagellin is a promising candidate immunopotentiator which can induce the comparableimmunological enhancement as CpG ODN does. Co-administration of flagellin with OVAand Alhydrogel induces enhanced humoral and cellular immune responses, and induces themix Th1/Th2 humoral respons, Th1 mainly.更多还原