新型流感通用疫苗研究

发布时间：2019-06-06 07:51

【摘要】：流感病毒是一种重要的传染源。可以引起流感爆发流行。通用流感疫苗已经研制多年。目前已经发现一些保守抗原靶位，如HA2, M2和NP蛋白。血凝素C端的HA2形成茎部结构锚定在病毒囊膜中,引起病毒囊膜与细胞膜的融合和病毒粒子核衣壳的释放。血凝素C端的HA2形成茎部结构锚定在病毒囊膜中,引起病毒囊膜与细胞膜的融合和病毒粒子核衣壳的释放。以往的研究表明，HA2是一个重要的保守性抗原，可能用于通用的研究。目的：本研究旨在通过选择流感病毒保守抗原片段，获得可对各种流感病毒具有保护作用的通用疫苗，利用优化编码病毒抗原密码子策略，同时采用CD40L作为分子佐剂，获得可显著提高免疫效果的流感疫苗。方法：本研究拟将CD40L作为佐剂，采用病毒载体在体内真核表达HA2基因与CD40L基因重组表达融合蛋白的策略。 CD40L序列的加入可以使融合蛋白通过CD40L与免疫效应细胞APC和B细胞，直接激活免疫系统，。CD40L可与APC表面的CD40结合，从而诱导细胞毒性T淋巴细胞（CTLs）的活化，释放IFN-γ，使CTLs直接杀伤感染细胞。另一方面，CD40L与B细胞表面的CD40结合，可以使刺激B细胞活化的抗原阈值降低，诱导B细胞针对其所携带抗原HA2的活化和增殖、促进HA2特异性的免疫球蛋白类型转换、抗体合成和分泌。此外，SHAF2CD40L复合物也可诱导DC细胞分泌一系列重要的细胞因子和趋化因子，进一步上调T细胞表面CD40L的表达。本研究首先通过PCR拼接及酶切连接的方法重组出HA2、SHA2、SHA2FCD40L、SFCD40L、HA2FCD40L、SFCD40L及S2HA2FCD40L共6个基因重组片段，其中S2HA2FCD40L为优化密码子的重组质粒。将这6个重组基因通过穿梭质粒插入腺病毒载体。选取SHA2、SHA2FCD40L、S2HA2FCD40L及空载体对照组进行动物实验。对于每种腺病毒，小鼠体内融合蛋白功能实验均包括2个部分：病毒攻击保护实验和免疫效果检测。同时为了对各组腺病毒免疫保护效果进行全面观察，每种腺病毒分为1×10~7PFU/只、1×10~8PFU/只、1×10~9PFU/只三个剂量组进行免疫。为了观察疫苗组小鼠抵抗感病毒的保护能力，分别用5LD50H1N1（PR8株）和H9N2流感病毒进行攻毒，观察小鼠在不同剂量组（10~7PFU、10~8PFU、10~9PFU）的生存率、体重下降、肺部及鼻腔病毒滴度等指标。结果：Western－blot结果表明，构建的6种腺病毒在Hela细胞内均可表达出目的大小的融合蛋白。用HA抗体和CD40L抗体分别检测的结果分别证实了6种融合蛋白大小及抗原性均正确。免疫荧光的结果表明，所有6种腺病毒均可以在细胞膜上检出，说明这6种融合蛋白具有接近天然的生物活性。小鼠体内免疫应答检测结果表明，在第一次免疫两周后，腺病毒载体SHA2FCD40L、S2HA2FCD40L疫苗3个剂量组都能够检测到分泌TNF-α，IFN-γ及IL-4的T细胞。此外，细胞免疫应答的水平与疫苗的免疫剂量呈现出正相关的关系，即随着免疫剂量的增加，诱导机体的细胞免疫应答的水平也随之上升。二次免疫后，SHA2FCD40L,S2HA2FCD40L和SHA2组刺激产生的细胞因子明显增多。其中Ad-S2HA2FCD40L诱导的细胞免疫应答较强。与初次免疫相比，，二次加强免疫能够显著性地增强机体的细胞免疫应答水平，并且细胞免疫应答水平与免疫剂量呈正相关。虽然流式细胞检测结果表明腺病毒空载体可以刺激机体产生免疫应答,但综合ELISPOT结果分析不是针对HA2蛋白产生特异性的免疫应答。体液免疫检测结果表明，AD-SHA2、AD-SHA2FCD40L和AD-S2HA2FCD40L三种腺病毒载体疫苗组都能够诱导机体产生体液免疫应答。体液免疫应答的水平与疫苗的免疫剂量呈正相关，其中后2组有更高的IgG和IgA抗体分泌，动物保护实验结果表明，10~9PFU的AD-SHA2腺病毒载体疫苗对PR8和H9N2有一定的交叉保护作用，其体内的病毒滴度水平也相对较低。小鼠肺部和鼻腔中流感病毒滴度在有佐剂CD40L的组别SHA2FCD40L感染滴度更低，存活率明显升高（P0.05）。优化密码子的S2HA2SFCD40L保护效果更好。结论：可以表达SHA2与CD40L融合蛋白的腺病毒载体，可以引起小鼠细胞及体液免疫应答，保护小鼠抵抗不同亚型流感病毒的攻击，为新型流感疫苗的研制打下了基础。
[Abstract]:Influenza virus is an important source of infection. The outbreak of the flu can be caused. The universal influenza vaccine has been developed for many years. Some conserved antigen targets, such as HA2, M2 and NP proteins, have been found. The HA2-forming stem structure at the C-terminus of the hemagglutinin is anchored in the viral envelope, resulting in the fusion of the viral envelope to the cell membrane and the release of the viral particle nucleocapsid. The HA2-forming stem structure at the C-terminus of the hemagglutinin is anchored in the viral envelope, resulting in the fusion of the viral envelope to the cell membrane and the release of the viral particle nucleocapsid. Previous studies have shown that HA2 is an important conservative antigen and may be used in general research. Objective: The purpose of this study was to obtain a universal vaccine with a protective effect on various influenza viruses by selecting the conservative antigen fragment of influenza virus. the agent is used for obtaining the influenza epidemic disease which can remarkably improve the immune effect, Methods: CD40L was used as an adjuvant, and the expression of the fusion protein was expressed by the recombinant expression of the HA2 gene and the CD40L gene in the body by using the viral vector. The addition of CD40L sequence can enable the fusion protein to directly activate the immune system through the CD40L and the immune effector cells APC and B cells. CD40L can be combined with CD40 on the APC surface to induce activation of cytotoxic T lymphocytes (CTLs), release of IFN-1, and direct the direct killing of CTLs. The CD40L, on the other hand, binds to the CD40 on the surface of the B cell, which reduces the antigen threshold that stimulates the activation of the B-cell, induces the activation and proliferation of the B-cell against the antigen HA2, promotes the type conversion of the HA2-specific immunoglobulin, the synthesis of the antibody, and the separation. In addition, the SHAF2CD40L complex can also induce a series of important cytokines and chemokines in the DC cell, and further up-regulate the surface of the T-cell surface CD40L. In this study,6 gene recombinant fragments of HA2, SHA2, SHA2FCD40L, SFCD40L, HA2FCD40L, SFCD40L and S2HA2FCD40L were recombined by PCR and enzyme digestion. The 6 recombinant genes were inserted into the adenosis through the shuttle plasmid. Toxic carrier. SHA2, SHA2FCD40L, S2HA2FCD40L and empty vector control group were selected. Animal experiments were performed. For each of the adenoviruses, the mouse in vivo fusion protein function test consisted of two parts: virus attack protection experiment and maintenance-free in order to make a comprehensive observation on the effect of the immune protection of each group, each adenovirus is divided into 1-10-7 PFU/ only,1-10-8 PFU/ only,1-10-9 PFU/ only three doses The mice were challenged with 5LD50H1N1 (PR8 strain) and H9N2 influenza virus, and the survival rate, weight loss, lung and nasal cavity of the mice were observed in different dose groups (10-7PFU,10-8PFU,10-9PFU). Results: The results of Western-blot show that the six adenoviruses can be expressed in Hela cells. The size of the fusion protein was confirmed by the results of the detection by the HA antibody and the CD40L antibody, respectively. The results of the immunofluorescence showed that all six kinds of adenoviruses can be detected on the cell membrane, indicating that the six fusion proteins are close to each other. The results of immune response in mice showed that, after the first immunization for two weeks, the recombinant adenovirus vector SHA2FCD40L and S2HA2FCD40L vaccine can detect the secretion of TNF-1, IFN-1, And the level of the cellular immune response is positively correlated with the immune dose of the vaccine, that is, the cellular immunity of the induction body should be induced with the increase of the immune dose. The level of response of SHA2FCD40L, S2HA2FCD40L and SHA2 was also increased after secondary immunization. The cytokines of Ad-S2HA2FCD40L induced by Ad-S2HA2FCD40L were significantly increased. The cellular immune response of the cell was stronger than that of the primary immunization, and the level of cellular immune response of the body was significantly enhanced and the cellular immune response level was increased. The results of flow cytometry show that the adenovirus empty vector can stimulate the immune response of the body, but the analysis of the comprehensive ELISPOT results is not for the production of the HA2 protein. The results of humoral immunity test showed that the three adenoviral vector vaccine groups of AD-SHA2, AD-SHA2FCD40L and AD-S2HA2FCD40L could be induced The level of humoral immune response is positively related to the immune dose of the vaccine, and the latter group is higher. IgG and IgA antibody secretion, animal protection experiment results show that the AD-SHA2 adenovirus vector vaccine of 10-9PFU is used for PR8 and H 9N2 has a certain cross-protection effect, The level of virus titer in mouse lung and nasal cavity was also relatively low. The titer of influenza virus in mouse lung and nasal cavity was lower than that of group SHA2FCD40L with adjuvant CD40L. Obvious increase (P0.05). The optimized codon of S2HA2 Conclusion: The adenovirus vector of SHA2 and CD40L fusion protein can be expressed, and the immune response of mouse and humoral immune response can be induced.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.1

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