【作者】 谢灿；

【导师】 何金生；

【作者基本信息】 安徽医科大学 ， 免疫学， 2008， 硕士

【摘要】 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)广泛分布于世界各地,是导致婴幼儿严重下呼吸道感染的最重要的病毒病原。目前尚无特异性防治方法,世界卫生组织将发展RSV疫苗列为最优先发展的疫苗项目之一。RSV的11种蛋白中,融合蛋白(Fusion Protein,F)是中和抗原,免疫动物后,可以产生具有免疫保护作用的中和抗体,为新型疫苗候选抗原。本实验中我们用减毒鼠伤寒沙门菌(Salmonella typhimurium aroA strain SL7207,SL7207)为载体携带能够表达F蛋白的DNA疫苗,经黏膜途径,开展体内免疫效果和免疫保护作用研究,以期更好了解该候选疫苗免疫生物学特征。方法:本研究中我们将能够表达RSV F蛋白的真核表达载体pcDNA3.1/F转化到SL7207,构建重组的减毒鼠伤寒沙门菌疫苗SL7207/pcDNA3.1/F,同时用pcDNA3.1空载体做对照。分别于0、2、6周三次口服免疫小鼠,通过体液免疫(IgG)、黏膜免疫(sIgA)、细胞免疫(CTL、T淋巴细胞增殖实验)等指标分析免疫后小鼠RSV特异性免疫应答产生情况。最后,利用RSV病毒经鼻腔攻击小鼠,并分别于攻毒后3、5和7天处死小鼠进行肺组织病毒含量及肺组织病理学动态分析了解免疫保护作用的效果。结果:取已转化pcDNA3.1/F的SL7207并提取质粒,经限制性内切酶分析与预期一致。经口服免疫BALB/c小鼠,与对照组相比,实验组小鼠血清及肺泡灌洗液(bronchoalveolar lavage,BAL)中分别产生了有效的血清IgG及sIgA(p<0.01),血清中抗体亚类分析显示IgG1和IgG2a的比值为0.96。实验组CTL反应(p<0.01,效靶比值为100:1和50:1)和T淋巴细胞增殖实验(p<0.05)均明显增强。攻毒实验中,病毒攻击后3天和5天的小鼠肺部病毒载量,实验组比对照组明显降低(p<0.05),组织病理学分析结果也显示在炎性细胞浸润和肺泡壁厚度方面两组有显著性差异(p<0.01)。结论:获得的重组减毒鼠伤寒沙门菌疫苗SL7207/pcDNA3.1/F,能够刺激机体产生有效的体液免疫和细胞免疫及局部免疫,抗体亚类分析显示诱导产生了Th1和Th2平衡的免疫应答,没有产生疾病增强作用,对RSV感染具有一定的免疫保护作用。因此我们认为以减毒伤寒沙门菌为载体的口服DNA疫苗兼具黏膜免疫及DNA疫苗的双重优点,是RSV等通过黏膜途径感染的病毒疫苗研究的重要方向,为今后RSV疫苗免疫策略等研究奠定了坚实基础。更多还原

【Abstract】 Objective: Human respiratory syncytial virus (RSV) is the most important viral etiologic agent of pediatric respiratory disease worldwide. No effective vaccine is currently available for prophylaxis. The World Health Organization has affirmed RSV vaccines as one of the highest priority vaccine development projects. Among the encoded 11 viral proteins by RSV genome, fusion protein (F) is one of the neutralization antigens, which can induce protective immunity against RSV infection, and a good candidate antigen for the genetic engineering vaccine such as DNA vaccine, subunit vaccine, etc. In this research, we use the Attenuated Salmonella aroA strains SL7207 (SL7202) as the carrier to deliver DNA vaccine expressing RSV F protein orally, in order to make clear the elicited protective immunity against RSV and its mechanism in vivo.Method: The pcDNA3.1/F expressing RSV F protein was transformed into the SL7207, and the resultant recombinant attenuated Salmonella strains was named as SL7207/pcDNA3.1/F. As a control, the empty pcDNA3.1 transformed SL7207 (SL7207/ pcDNA3.1) was prepared simultaneously. Then the mice were grouped and administered orally by the above two recombinant Salmonella strains on weeks 0, 2, and 6, respectively, the induced RSV-specific immune responses, including humoral, mucosal and cellular immune responses, were investigated in detail. In the end, the mice were challenged by RSV nasally, and then sacrificed on days 3, 5 and 7 postchallenge for lung RSV load and lung histopathology assays to confirm the protective immunity.Results: The plasmids extracted from SL7207/pcDNA3.1/F and SL7207/pcDNA3.1 were confirmed by restriction endonuclease assay. SL7207/pcDNA3.1/F administered mice showed significant elevations of serum anti-RSV IgG and bronchoalveolar lavage secretory (BAL) sIgA compared with the negative control group (p<0.01). The ratio of IgG1 and IgG2a was 0.96. The experimental group also showed a stronger cytotoxic T cell response (p<0.01 at effector:target ratios of 100:1 and 50:1) and a higher stimulation index value of T cell proliferation (p<0.05) than the respective control group. RSV titers in the lung homogenates of the experimental group on day 3 and day 5 postchallenge were lower than in the control group (p<0.05). Histopathological analysis showed obvious differences in infiltration of inflammatory cells and pulmonary alveolar wall thickness (p<0.01) between the two groups.Conclusion: SL7207/pcDNA3.1/F have been constructed successfully. This candidate vaccine could induce humoral, mucosal and cellular immune response which exhibit protective immunity against the RSV challenge. Moreover, no disease enhancement was observed. The IgG subclasses analysis showes balanced Th1 and Th2 immune response has been induced. Therefore, we think this vaccine strategy delivering DNA vaccine by Attenuated Salmonella Typhimurium has the combined merit of mucosal immunity and DNA vaccine, and is of great importance for vaccine against mucosally transmissible viruses such as RSV, rotavirus, etc.更多还原