基于不同黏膜佐剂的艰难梭菌类毒素疫苗微针经皮免疫效应研究

Clostridium difficile infection(CDI) has been identified as the leading cause of nosocomial infection. With the emergence of hypervirulent strains and the strains resistant to metronidazole or vancomycin in recent years, the prevalence and severity of CDI has increased in the world. As C. difficile toxins A and B play important roles in infection as well as in gut inflammation, the highly effective mucosal toxoid vaccines are necessary for CDI control. According to the immunological principle and technology of vaccine preparations, first of all, the C.difficile are cultured anaerobically by the dialysis bag methods, the toxin A and toxin B will be purified by precipitation with (NH4)2SO4 followed by ion-exchange chromatography. C. difficile toxoids vaccine are prepared with formaldehyde-inactivated the C. difficile toxins A,toxin B and toxin A+B). Secondly, the female BALB/C mice will be transcutaneous immunized with or without cholera toxin(CT) and E. coli heat-labile enterotoxin (LT) as mucosal adjuvant respectively by microneedle patches. And then, in order to detect the systemic and mcosal immune responses, the specific serum immunoglobulin (IgG) and secretory IgA (S-IgA) in stool against to the C. difficile toxins will be measured by enzyme-linked immunosorbent assay. Finally, the immunogenicity and protective activity of the vaccine will be examined in the mice C. difficile challenge model, Serum from individual animals was tested for neutralizing activity against native C. difficile toxin A and toxin B in the Vero cell based neutralization assay. By analysis of the immune effects of different adjuvants used, not only the most effective mucosal adjuvants, optimal antigen ratio of C. difficile toxoid vaccine could be established, and the role played by intestinal mucosal immunity in the occurrence and change of the CDI will be revealed, but also the study will offer technology and biological data for C.difficile toxoid vaccine candidate for human use.

艰难梭菌是医院内感染的主要病原菌之一，近年来，随着高致病株的出现和菌株耐药性的增加，艰难梭菌的感染在全球呈流行趋势，由于毒素是其主要的感染因子，且病变部位是肠道，因此，研制粘膜免疫效果良好的类毒素疫苗是防治艰难梭菌感染的关键。本项目利用疫苗免疫原理和技术，首先通过艰难梭菌透析培养、盐析、离子交换层析等方法纯化艰难梭菌A、B毒素，经脱毒后分别制备艰难梭菌疫苗(类毒素A，类毒素B和类毒素A+B)，与粘膜佐剂(CT/LT)配伍后，按特定的免疫程序，采用微针技术经皮免疫BALB小鼠，定期检测特异性血清IgG和粪便中S-IgA的效价水平变化，最后采用艰难梭菌动物感染模型和细胞检测来评价疫苗的实际免疫保护效果，通过分析不同佐剂对免疫效果的影响，确立艰难梭菌疫苗最有效的粘膜免疫佐剂、最佳类毒素抗原种类和配比，揭示肠道粘膜免疫水平在细菌感染中的作用，为艰难梭菌类毒素疫苗的研制提供技术和理论参考。

艰难梭菌是医院内感染的主要病原菌之一，近年来，随着高致病株的出现和菌株耐药性的增加，艰难梭菌的感染在全球呈流行趋势，由于毒素是其主要的感染因子，且病变部位是肠道，因此，探索基于不同粘膜佐剂的微针经皮免疫的艰难梭菌类毒素疫苗的效果是防治艰难梭菌感染的关键。. 本项目利用疫苗免疫原理和技术，首先通过艰难梭菌透析培养、盐析、离子交换层析等方法纯化艰难梭菌A、B毒素，经甲醛脱毒（甲醛终浓度0.4%）。经动物免疫效果证实，该艰难梭菌类毒素A毒素和类毒素B疫苗适合动物免疫。采用微针技术经皮免疫BALB/C小鼠，在免疫后0d、7d、14d、24d、42d检测特异性血清IgG和粪便中S-IgA的效价水平变化，通过艰难梭菌人工感染后的小鼠组织学切片病理观察和细胞中和实验来评价疫苗的实际免疫效果。结果表明：（1）用艰难梭菌类毒素A而言，单独微针免疫小鼠，随着免疫剂量的增加，血清抗体IgG效价逐步增高，艰难梭菌类毒素A使用5μg免疫7d后效果组好。对于肠道粘膜免疫效果而言，使用10μg免疫14d后效果组好；合用LT佐剂（25μg），艰难梭菌类毒素A 5μg免疫7d后效果组好，而最佳剂量为25μg。对于粪便中IgA效价，使用10μg免疫14d后效果组好。（2）用艰难梭菌类毒素B而言，使用10μg微针免疫后血清抗体IgG效价最好，加入LT佐剂，5μg免疫后效果较好；对于肠道粘膜免疫而言，则使用10μg微针免疫效果最好。（3）无论艰难梭菌类毒素A还是艰难梭菌类毒素B，分别加入LT佐剂，CBT佐剂微针免疫小鼠，无论血清抗体IgG效价还是粪便中IgA效价，结果提示LT比CBT佐剂效应明显，具有明显统计学差异。（4）用艰难梭菌类毒素A和艰难梭菌类毒素B混合，互为佐剂免疫小鼠，使用剂量为50μg免疫效果最好。两种类毒素佐剂效应不明显。实验结果提示艰难梭菌类毒素疫苗BALB/C小鼠微针免疫具有较好的免疫效果，无论是血清抗体IgG效价还是粪便中IgA效价，都具有较好的免疫效果，为艰难梭菌类毒素疫苗的研制提供重要的技术和理论参考。

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