減毒活疫苗D39ΔCPS-TA對小鼠感染肺炎鏈球菌的保護(hù)效果和機(jī)制研究
發(fā)布時間：2018-08-07 10:11
【摘要】：【背景】肺炎鏈球菌是一種常見革蘭陽性致病菌,可引起肺炎、腦膜炎、敗血癥、鼻竇炎及中耳炎等疾病。由于抗生素的濫用,已經(jīng)出現(xiàn)了耐萬古霉素和多重耐藥性的肺炎鏈球菌菌株。世界衛(wèi)生組織建議將肺炎鏈球菌結(jié)合疫苗納入國家免疫計(jì)劃,并將S.pn疫苗列為全球最急需的前3位疫苗之一。目前已上市的23價莢膜多糖疫苗和7-13價的莢膜多糖結(jié)合疫苗由于存在莢膜轉(zhuǎn)換、成本高昂等種種不足,限制了它們的推廣使用。因此開發(fā)新型肺炎鏈球菌疫苗迫在眉睫。 減毒活菌疫苗是一種免疫原性強(qiáng)、保護(hù)效果好、成本低廉的疫苗。由于通過傳統(tǒng)方法不易獲得減毒菌株,因此其開發(fā)有一定的困難。本課題組在基因突變實(shí)驗(yàn)中偶然獲得了一株莢膜丟失的菌株,前期研究顯示其較肺炎鏈球菌無感染致病能力的R6菌株的毒力還弱,因此安全性較高,有望開發(fā)成一種減毒活菌疫苗。本研究擬在全面鑒定其安全性的基礎(chǔ)上,對其保護(hù)作用進(jìn)行評價,并進(jìn)行初步的分子機(jī)制研究,為該活菌疫苗的研發(fā)提供可靠的實(shí)驗(yàn)依據(jù)。 【方法】通過莢膜檢測、莢膜磷壁酸檢測、溶血活性檢測、小鼠體內(nèi)毒力實(shí)驗(yàn)、肺組織病理切片觀察及缺陷菌在小鼠體內(nèi)定植來評價D39△CPS-TA的安全性;候選疫苗免疫小鼠后,通過檢測特異性抗體效價、疫苗對定植的保護(hù)效果、疫苗對肺炎和敗血癥的保護(hù)效果、疫苗對臨床菌株的保護(hù)效果及疫苗的長期保護(hù)效果來評價D39△CPS-TA的有效性;最后通過檢測抗體亞型、抗體的被動保護(hù)效果及細(xì)胞因子的檢測來初步探討D39△CPS-TA的保護(hù)機(jī)制。 【結(jié)果】D39△CPS-TA作為減毒活菌疫苗安全性較好。與D39相比,D39△CPS-TA的莢膜明顯變薄變稀;莢膜磷壁酸的表達(dá)量顯著減少;溶血活性也顯著降低;毒力明顯減弱;對小鼠肺組織只引起輕微的可耐受的炎癥,損傷較輕;在小鼠體內(nèi)的定植能力也明顯減弱,24小時后完全被清除。減毒活菌疫苗D39△CPS-TA的保護(hù)效果很好。免疫小鼠后,D39△CPS-TA可以誘導(dǎo)高效價的特異性抗體;也能減少肺炎鏈球菌19F和TIGR4在小鼠體內(nèi)的定植;可以對D39引起的肺炎和敗血癥產(chǎn)生很好的保護(hù)效果,特別是粘膜免疫的保護(hù)效果達(dá)100%;對臨床菌株6B和3型感染的保護(hù)效果也較市面疫苗好(p0.05);粘膜免疫對小鼠長期保護(hù)效果為100%,系統(tǒng)免疫的保護(hù)效果為75%�？贵w的被動保護(hù)效果為100%;其抗體亞型主要為IgG1、IgG2a、IgG2b;D39△CPS-TA系統(tǒng)免疫產(chǎn)生高水平的IL-10,而粘膜免疫產(chǎn)生高水平的IL-10、IL-17A和IFN-γ。 【結(jié)論】D39△CPS-TA作為減毒活菌疫苗安全性較高。它對小鼠的保護(hù)效果非常好,尤其是通過粘膜途徑免疫,其對多種血清型肺炎鏈球菌感染的保護(hù)效果都可達(dá)100%,較市面上的疫苗效果好。其通過系統(tǒng)免疫主要誘導(dǎo)體液免疫反應(yīng),通過粘膜免疫既可誘導(dǎo)體液免疫反應(yīng)也可誘導(dǎo)細(xì)胞免疫反應(yīng)。
[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- 緯. [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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392
本文編號：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- 緯. [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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 白雪源,車鳳翔;粘膜免疫進(jìn)展[J];國外醫(yī)學(xué)(免疫學(xué)分冊);1999年05期
2 高杰英;粘膜免疫向免疫學(xué)提出了新問題[J];上海免疫學(xué)雜志;2000年05期
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