减毒活疫苗D39ΔCPS-TA对小鼠感染肺炎链球菌的保护效果和机制研究
发布时间：2018-08-07 10:11
【摘要】：【背景】肺炎链球菌是一种常见革兰阳性致病菌,可引起肺炎、脑膜炎、败血症、鼻窦炎及中耳炎等疾病。由于抗生素的滥用,已经出现了耐万古霉素和多重耐药性的肺炎链球菌菌株。世界卫生组织建议将肺炎链球菌结合疫苗纳入国家免疫计划,并将S.pn疫苗列为全球最急需的前3位疫苗之一。目前已上市的23价荚膜多糖疫苗和7-13价的荚膜多糖结合疫苗由于存在荚膜转换、成本高昂等种种不足,限制了它们的推广使用。因此开发新型肺炎链球菌疫苗迫在眉睫。 减毒活菌疫苗是一种免疫原性强、保护效果好、成本低廉的疫苗。由于通过传统方法不易获得减毒菌株,因此其开发有一定的困难。本课题组在基因突变实验中偶然获得了一株荚膜丢失的菌株,前期研究显示其较肺炎链球菌无感染致病能力的R6菌株的毒力还弱,因此安全性较高,有望开发成一种减毒活菌疫苗。本研究拟在全面鉴定其安全性的基础上,对其保护作用进行评价,并进行初步的分子机制研究,为该活菌疫苗的研发提供可靠的实验依据。 【方法】通过荚膜检测、荚膜磷壁酸检测、溶血活性检测、小鼠体内毒力实验、肺组织病理切片观察及缺陷菌在小鼠体内定植来评价D39△CPS-TA的安全性;候选疫苗免疫小鼠后,通过检测特异性抗体效价、疫苗对定植的保护效果、疫苗对肺炎和败血症的保护效果、疫苗对临床菌株的保护效果及疫苗的长期保护效果来评价D39△CPS-TA的有效性;最后通过检测抗体亚型、抗体的被动保护效果及细胞因子的检测来初步探讨D39△CPS-TA的保护机制。 【结果】D39△CPS-TA作为减毒活菌疫苗安全性较好。与D39相比,D39△CPS-TA的荚膜明显变薄变稀;荚膜磷壁酸的表达量显著减少;溶血活性也显著降低;毒力明显减弱;对小鼠肺组织只引起轻微的可耐受的炎症,损伤较轻;在小鼠体内的定植能力也明显减弱,24小时后完全被清除。减毒活菌疫苗D39△CPS-TA的保护效果很好。免疫小鼠后,D39△CPS-TA可以诱导高效价的特异性抗体;也能减少肺炎链球菌19F和TIGR4在小鼠体内的定植;可以对D39引起的肺炎和败血症产生很好的保护效果,特别是粘膜免疫的保护效果达100%;对临床菌株6B和3型感染的保护效果也较市面疫苗好(p0.05);粘膜免疫对小鼠长期保护效果为100%,系统免疫的保护效果为75%。抗体的被动保护效果为100%;其抗体亚型主要为IgG1、IgG2a、IgG2b;D39△CPS-TA系统免疫产生高水平的IL-10,而粘膜免疫产生高水平的IL-10、IL-17A和IFN-γ。 【结论】D39△CPS-TA作为减毒活菌疫苗安全性较高。它对小鼠的保护效果非常好,尤其是通过粘膜途径免疫,其对多种血清型肺炎链球菌感染的保护效果都可达100%,较市面上的疫苗效果好。其通过系统免疫主要诱导体液免疫反应,通过粘膜免疫既可诱导体液免疫反应也可诱导细胞免疫反应。
[Abstract]:Background Streptococcus pneumoniae is a common gram-positive pathogen, which can cause pneumonia, meningitis, septicemia, sinusitis and otitis media. Due to the abuse of antibiotics, vancomycin-resistant and multi-resistant Streptococcus pneumoniae strains have emerged. The World Health Organization recommends that streptococcus pneumoniae conjugate vaccines be included in national immunization plans and that S.pn vaccines be listed as one of the three most needed vaccines in the world. The 23 valent capsule polysaccharide vaccine and the 7-13 valent capsule polysaccharide conjugate vaccine have been put on the market at present. Because of the shortage of capsule conversion and high cost, their popularization and application are restricted. Therefore, it is urgent to develop a new type of Streptococcus pneumoniae vaccine. Attenuated live bacteria vaccine is a kind of vaccine with strong immunogenicity, good protective effect and low cost. Because it is difficult to obtain attenuated strains by traditional methods, it is difficult to develop them. In the gene mutation experiment, our team accidentally obtained a strain with lost capsule. Previous studies showed that the strain R6 was less virulent than Streptococcus pneumoniae, so it was more safe. It is expected to develop a live attenuated vaccine. On the basis of comprehensive identification of its safety, this study aims to evaluate its protective effect and to study its molecular mechanism. [methods] through capsule detection, capsule phosphoric acid detection, hemolytic activity test, virulence test in mice. The safety of D39 CPS-TA was evaluated by observing the pathological section of lung tissue and colonization of defective bacteria in mice. After immunizing mice with candidate vaccine, the specific antibody titer was detected and the protective effect of vaccine on colonization was obtained. The protective effect of vaccine on pneumonia and septicemia, the protective effect of vaccine on clinical strain and long-term protective effect of vaccine were evaluated to evaluate the effectiveness of D39 CPS-TA. The protective mechanism of D39 CPS-TA was preliminarily investigated by the passive protective effect of antibody and the detection of cytokines. [results] D39 CPS-TA was safe as a live attenuated vaccine. Compared with D39, the capsule of D39 CPS-TA was thinned and thinned, the expression of phosphatidic acid in capsule decreased significantly, the hemolytic activity also decreased, the virulence was obviously weakened, and the lung tissue of mice was only slightly tolerated inflammation, and the injury was less. The colonization ability in mice was also significantly reduced after 24 hours. The protective effect of live attenuated vaccine D39 CPS-TA was very good. After immunizing mice, D39 CPS-TA could induce specific antibodies with high titer, reduce the colonization of streptococcus pneumoniae 19F and TIGR4 in mice, and have a good protective effect on pneumonia and septicemia induced by D39. Especially, the protective effect of mucosal immunity was 100%, the protective effect of clinical isolates 6B and type 3 infection was better than that of marketable vaccine (p0.05), the long-term protective effect of mucosal immunity was 100 and the protective effect of systemic immunity was 7575%. The passive protective effect of the antibody was 100. The main subtypes of antibody were IgG1T IgG2bmD39 CPS-TA system to produce high level of IL-10, while mucosal immunity produced high levels of IL-10, IL-17A and IFN- 纬. [conclusion] D39 CPS-TA is more safe as a live attenuated vaccine. It has a very good protective effect on mice, especially through mucous membrane immunization. Its protective effect on various serotypes of Streptococcus pneumoniae infection can reach 100%, which is better than the vaccine available on the market. The humoral immune response was mainly induced by systemic immunity, and the cellular immune response was induced by mucosal immunity as well as humoral immune response.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2169703
[Abstract]:Background Streptococcus pneumoniae is a common gram-positive pathogen, which can cause pneumonia, meningitis, septicemia, sinusitis and otitis media. Due to the abuse of antibiotics, vancomycin-resistant and multi-resistant Streptococcus pneumoniae strains have emerged. The World Health Organization recommends that streptococcus pneumoniae conjugate vaccines be included in national immunization plans and that S.pn vaccines be listed as one of the three most needed vaccines in the world. The 23 valent capsule polysaccharide vaccine and the 7-13 valent capsule polysaccharide conjugate vaccine have been put on the market at present. Because of the shortage of capsule conversion and high cost, their popularization and application are restricted. Therefore, it is urgent to develop a new type of Streptococcus pneumoniae vaccine. Attenuated live bacteria vaccine is a kind of vaccine with strong immunogenicity, good protective effect and low cost. Because it is difficult to obtain attenuated strains by traditional methods, it is difficult to develop them. In the gene mutation experiment, our team accidentally obtained a strain with lost capsule. Previous studies showed that the strain R6 was less virulent than Streptococcus pneumoniae, so it was more safe. It is expected to develop a live attenuated vaccine. On the basis of comprehensive identification of its safety, this study aims to evaluate its protective effect and to study its molecular mechanism. [methods] through capsule detection, capsule phosphoric acid detection, hemolytic activity test, virulence test in mice. The safety of D39 CPS-TA was evaluated by observing the pathological section of lung tissue and colonization of defective bacteria in mice. After immunizing mice with candidate vaccine, the specific antibody titer was detected and the protective effect of vaccine on colonization was obtained. The protective effect of vaccine on pneumonia and septicemia, the protective effect of vaccine on clinical strain and long-term protective effect of vaccine were evaluated to evaluate the effectiveness of D39 CPS-TA. The protective mechanism of D39 CPS-TA was preliminarily investigated by the passive protective effect of antibody and the detection of cytokines. [results] D39 CPS-TA was safe as a live attenuated vaccine. Compared with D39, the capsule of D39 CPS-TA was thinned and thinned, the expression of phosphatidic acid in capsule decreased significantly, the hemolytic activity also decreased, the virulence was obviously weakened, and the lung tissue of mice was only slightly tolerated inflammation, and the injury was less. The colonization ability in mice was also significantly reduced after 24 hours. The protective effect of live attenuated vaccine D39 CPS-TA was very good. After immunizing mice, D39 CPS-TA could induce specific antibodies with high titer, reduce the colonization of streptococcus pneumoniae 19F and TIGR4 in mice, and have a good protective effect on pneumonia and septicemia induced by D39. Especially, the protective effect of mucosal immunity was 100%, the protective effect of clinical isolates 6B and type 3 infection was better than that of marketable vaccine (p0.05), the long-term protective effect of mucosal immunity was 100 and the protective effect of systemic immunity was 7575%. The passive protective effect of the antibody was 100. The main subtypes of antibody were IgG1T IgG2bmD39 CPS-TA system to produce high level of IL-10, while mucosal immunity produced high levels of IL-10, IL-17A and IFN- 纬. [conclusion] D39 CPS-TA is more safe as a live attenuated vaccine. It has a very good protective effect on mice, especially through mucous membrane immunization. Its protective effect on various serotypes of Streptococcus pneumoniae infection can reach 100%, which is better than the vaccine available on the market. The humoral immune response was mainly induced by systemic immunity, and the cellular immune response was induced by mucosal immunity as well as humoral immune response.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 白雪源,车凤翔;粘膜免疫进展[J];国外医学(免疫学分册);1999年05期
2 高杰英;粘膜免疫向免疫学提出了新问题[J];上海免疫学杂志;2000年05期
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