【作者】 谭磊；

【导师】 金宁一；

【作者基本信息】 吉林大学 ， 预防兽医学， 2011， 博士

【摘要】 流行性感冒(Influenza flu)是由流感病毒(Influenza Virus)引起的能够在人群中广泛传播的急性呼吸道传染病。按病毒内部核蛋白(NP)的抗原性差异分为A、B、C三型。根据病毒表面结构蛋白血凝素(Hemagglutinin, HA)和神经氨酸酶(Neuraminidase, NA)不同可分为不同的血清亚型,HA包括16个亚型,NA包括9个亚型,其中感染人类的以A型H1N1、H3N2、H2N2亚型和B型流感病毒为主,流感又分为季节性流感(Seasonal influenza)和大流行性流感(Pandemic influenza),大流行性流感病毒可以在全世界传播,造成的死亡率极高。感染禽类的主要包括A型H5/H7/H9等亚型,其中H5N1、H7N7等亚型开始频繁突破种间屏障感染人类并且引起死亡,有可能引发下一次的流感大流行。预防接种是控制流感病毒的有效手段,目前广泛采用的是传统流感灭活疫苗,但是传统疫苗存在生产周期长,生产工序多,高度依赖鸡胚,成本较高,而且只能针对特定几种亚型提供免疫保护的缺点。流感病毒多亚型共存,不同亚型之间交叉保护性不好,主要保护性抗原基因HA差异较大。研制一种可同时预防多种亚型流感病毒且制备工艺简单,无需鸡胚即可大量生产的新型疫苗显得日趋重要。目前新型流感疫苗主要集中在基因工程疫苗的研究,包括重组亚单位疫苗、病毒颗粒样疫苗、核酸疫苗、重组病毒载体疫苗等。其中核酸疫苗和重组腺病毒疫苗发展前景较好,核酸疫苗也称DNA疫苗,它具有制备过程相对简单,安全稳定,无载体免疫,可以重复多次免疫,能够刺激机体产生细胞免疫和体液免疫反应,疫苗可稳定长期保存等优点使其成为疫苗研究的热点。核酸疫苗在免疫原性上同亚单位疫苗、重组病毒载体疫苗等相比较弱,可以通过添加不同类型佐剂、辅助电穿孔免疫、基因枪等方法提高疫苗的免疫效果。腺病毒载体具有安全性好、宿主细胞广泛、感染病毒滴度高、细胞转导效率高、可容纳大片段外源基因、自身稳定等特点,可以刺激机体产生较强的细胞免疫和体液免疫反应。多表位(Multi-epitope)流感疫苗是通过对流感病毒不同亚型主要保护性抗原基因表位进行筛选,将多种不同抗原表位间连接特定的Linker以构成多表位表达盒,将其单独或者串联其他基因克隆入相应的表达载体构建的疫苗。本研究选用真核表达载体pVAX1和复制缺陷型人5型腺病毒载体,设计构建了以季节性流感H3亚型和大流行性流感H1亚型HA (HA1)基因为主,辅助以已经突破种间屏障感染人类的禽流感H5、H7、H9亚型HA抗原Th和B细胞表位盒(EHA)的多表位核酸疫苗和重组腺病毒疫苗,并分别构建了单表达对照组。通过RT-PCR、间接免疫荧光、Western Blot等方法检测各组分抗原的转录和抗原性,结果表明,构建的候选核酸疫苗和重组腺病毒疫苗均可以有效表达组分抗原,抗原性良好。选用辅助电穿孔免疫的方法对pV-H3-EHA-H1HA1 new, pV-H1HA new, pV-H1HA1 new, pV-H3HA, pV-EHA等候选核酸疫苗进行了小鼠免疫实验,分别对各免疫组进行细胞免疫和体液免疫水平检测。体液免疫结果表明,在特异性抗原刺激下,辅助电穿孔的多表位免疫组与单表达组水平相当(P>0.05),但显著高于未使用电穿孔组(P<0.05)。多表位免疫组在表位相关的H5/H7/H9亚型特异性多肽抗原刺激时产生的IgG抗体水平较未添加表位免疫组大约提高两倍。IL-2和IL-4细胞因子水平检测结果表明,多表位辅助电穿孔免疫组显著高于未使用电穿孔组和单表达辅助电穿孔免疫组(P<0.05)。选用10 LD50 H5亚型流感病毒对各免疫组小鼠进行攻毒保护实验,多表位辅助电穿孔免疫组能够产生较好的免疫保护(90%),显著高于单表达组和未使用辅助电穿孔的免疫组(P<0.05)。利用小鼠动物模型免疫候选流感重组腺病毒疫苗pacAd5-H3HA、pacAd5-H1HA1、pacAd5-H1HA1 new、pacAd5-H3-H1HA1和pacAd5-H3-EHA-H1HA1 new,结果显示,免疫组小鼠均可以产生特异性的体液免疫和细胞免疫反应,其中以多表位重组腺病毒组免疫应答水平最高。单表达H3HA的重组腺病毒设计单次免疫组和两次免疫组作为对照,结果表明,单次免疫组诱生了与两次免疫组相当的体液和细胞免疫水平(P>0.05)。加强免疫后14d,选用100 LD50 H3亚型流感病毒对各免疫组小鼠进行攻毒保护实验,结果多表位重组腺病毒组与共表达H3-H1HA1组、单表达H3HA组小鼠可以完全保护小鼠(100%),显著高于H1亚型单表达组和对照组(P<0.05),且多表位重组腺病毒组在小鼠攻毒后体重变化率较小并能够迅速恢复。根据Prime-Boost免疫策略对构建的候选核酸疫苗和重组腺病毒疫苗进行了联合免疫研究,利用核酸疫苗首免,重组腺病毒疫苗加强免疫,结果表明,多表位联合免疫组和单表达联合免疫组在抗原相关的特异性IgG抗体水平上差异不显著(P>0.05),但是联合免疫组均显著高于核酸疫苗和重组腺病毒疫苗单独免疫组(P<0.05);同时,多表位联合免疫组可以产生强烈的细胞免疫应答反应,显著高于单表达联合免疫组和单独免疫组(P<0.05)。选用H3/H5/H1亚型流感病毒攻毒保护实验结果表明,多表位联合免疫组对三种亚型病毒保护率均为最高,不但可以完全保护主要抗原成分H3亚型及表位相关抗原H5亚型致死剂量的流感病毒的攻击,对异源季节性H1N1亚型流感病毒攻击也产生了较强的交叉免疫保护(90%),显著高于单表达联合免疫组和单独免疫组(P<0.05)。综合分析,候选多表位核酸疫苗和重组腺病毒疫苗展示了较强的激发小鼠体液免疫和细胞免疫的能力,通过H5/H7/H9亚型表位盒的添加,增强了对禽流感亚型病毒跨种感染的免疫保护,且多表位疫苗联合免疫能够对异源亚型攻毒提供较好保护。多表位核酸疫苗和重组腺病毒疫苗在流感通用疫苗研究中展示了良好的应用前景。更多还原

【Abstract】 The influenza virus, the causative agent of flu and other acute respiratory infections, can spread in human populations. Based on the viral internal nucleoprotein (NP) structure, influenza viruses are divided into three different antigenic types, designated as A, B, and C. They are further divided into different subtypes based on two virus surface proteins called hemagglutinin (HA) and neuraminidase (NA). There are 16 HA subtypes and 9 NA subtypes. Three subtypes of influenza A, namely H1N1, H3N2, and H2N2, as well as influenza B mainly infect humans. Influenza may also be categorized as seasonal influenza and pandemic influenza. Pandemic influenza strain can spread worldwide and has a very high mortality rate. Avian influenza viruses mainly include subtypes H5, H7, and H9, of which H5N1, H7N7, and other avian influenza virus subtypes have crossed the species barrier, causing human infections and several deaths. This phenomenon could trigger the next influenza pandemic.Vaccination is an effective method for controlling the influenza virus. At present, the traditional influenza vaccine is widely used, but it has several limitations, including a long production period, a complicated production processes, high dependence on embryonated eggs, costly, and limited strain-specific protection. Multiple subtypes of influenza virus coexist but the vaccines provide weak cross-protection. Furthermore, sequences of the major protective antigen, HA, are quite different from each other. The development of a new influenza vaccine that can protect against different influenza virus subtypes and can be simply mass-produced independent of embryonated eggs is increasingly important. Currently, vaccine research mainly focuses on genetic engineering of new influenza vaccines, including recombinant subunit vaccines, virus-like particle vaccines, DNA vaccines, and recombinant viral vector vaccines.DNA vaccine and adenovirus vaccine offer good prospects for developing new influenza vaccine. Nucleic acid vaccines, also known as DNA vaccines, are relatively easy to prepare, safe, and stable. It also has long-term storage stability and provides non-carrier immunity and repeated immunity. DNA vaccines stimulate cellular and humoral immune responses. These advantages made DNA vaccines the focus of research. Compared with subunit vaccines and recombinant viral vector vaccines, DNA vaccines elicit a weaker immunogenic response. Vaccine efficacy can be enhanced by adding different types of adjuvants, electroporation-assisted vaccination, gene gun, and other methods. Adenoviruses have an excellent safety profile, a wide range of host cells, high viral titers, and high cell transduction efficiency, which can accommodate large segments of foreign genes. Furthermore, it is stable and can stimulate the body to produce strong cellular and humoral immune responses.A multi-epitope influenza vaccine against different influenza virus subtypes was prepared by screening the epitopes of major protective antigen genes. Different specific epitopes are connected by a linker to form the multi-epitope expression cassette, which is inserted alone or combined with other genes into the corresponding expression vector.In this study, the eukaryotic expression vector pVAX1 and the replication-defective human adenovirus type 5 vector constructs were used to design a multi-epitope DNA vaccine and adenovirus vaccine specifically against seasonal influenza H3 subtype and pandemic influenza H1 subtype HA (HA1). The genes of these subtypes were combine with the HA antigen Th and B cell epitopes cassette (EHA) of avian influenza subtypes such as H5, H7. and H9, which have crossed species barrier to infect humans, and single expression control groups were constructed. The components of antigen transcription and antigenicity were determined by reverse transcription polymerase chain reaction, indirect immunofluorescence, and western blot method. The results show that the constructed potential DNA vaccines and recombinant adenovirus vaccines effectively expressed components of antigen, thus, these are good vaccines.Potential DNA vaccines, which include pV-H3-EHA-H1HA1 new, pV-H1HA new, pV-H1HA1 new, pV-H3HA, and pV-EHA, were used to immunize mice through electroporation-assisted immunization. The levels of cellular and humoral immunity of each immunized group were determined. The results show that upon specific antigen stimulation, the group treated with the multiple epitope vaccine through electroporation-assisted immunization, had the same levels of humoral immunity as the single-expression immunized group, and had significantly higher levels than that of the group without electroporation-assisted immunization (P<0.05). In addition, the IgG antibody levels of the multi-epitope immunized group were approximately twice than that of the single-expression group upon antigen stimulation with the H5/H7/H9 epitope peptides (P<0.05). Moreover, groups treated with the multiple epitope vaccine through electroporation-assisted immunization had significantly higher IL-2 and IL-4 levels than that of the single-expression group and the non-electroporation groups (P<0.05). The multi-epitope immunization with electroporation also induced better immune protection (90%), which was significantly higher than that of the single-expression and the non-electroporation groups (P<0.05) based on the lethal dose challenge (10 LD50) with the H5 subtype influenza virus on immunized mice.Mice were immunized using potential recombinant influenza adenovirus vaccine: pacAd5-H3HA, pacAd5-H1HA1, pacAd5-H1HA1 new, pacAd5-H3-H1HA1, and pacAd5-H3-EHA-H1HA1 new. The results show that all immunized mice produced specific humoral and cellular immune response, in which the multi-epitope recombinant group had the highest level of immune response. Single and double immunization with the expressed H3HA recombinant adenovirus was used as the control. Based on the results, the single-immunized group induced considerable levels of immunity compared with the twice-immunized group. Immunized mice were challenged with a lethal dose of the H3 subtype of influenza virus 14 days after the booster immunization. The results show that the mice in the multi-epitope recombinant adenovirus group, the co-expressed H3-H1HA1 group, and the single expression group acquired complete protection, which were significantly higher than that produced in the H1 subtype single expression group and the control group (P<0.05). The body weight changes were relatively mild and quickly recovered among the mice in the multi-epitope recombinant adenovirus-immunized group.Combined immunity was achieved using DNA vaccine and recombinant vaccine based on the Prime-Boost Immunization Strategy. Prime immunization with the DNA vaccine and boost immunization with recombinant adenovirus vaccine were performed. The results show that the level of antibodies induced in the multi-epitope combined immunization group and the single combined immune-related group was not significantly different (P>0.05), but both were significantly higher than that of the single immunized with DNA vaccine and adenovirus vaccine (P<0.05). Furthermore, the multi-epitope combined immunized group exhibited a strong immune response, which was significantly higher than that of the single combined immunization group and the single-expression immunized group (P<0.05). Based on the H3/H5/H1 subtype influenza virus challenge experiment, the multi-epitope combined immunization group had the highest immune protection rates. The group was not only fully protected against the major antigenic components associated with the epitopes of the H3 and H5 subtypes, but also elicited strong cross-protective immunity against heterologous subtypes of seasonal H1N1 influenza viruses (90%) that were significantly higher than that in the combined immunization group and the single-expression immunized groups (P<0.05).In summary, the potential multi-epitope DNA vaccine and the recombinant adenovirus vaccine conferred strong humoral and cellular immunity in mice. The epitopes of the H5/H7/H9 subtypes provided enhanced immune protection against avian influenza virus subtypes that can cause cross-species infection. Combined immunization with multi-epitope vaccines effectively protects mice against heterologous subtype virus challenge. Multi-epitope DNA vaccine and adenovirus vaccine show good prospects in the study of universal influenza vaccine.更多还原