Bordetellae and Haemophilus ducreyi

博氏杆菌和杜克雷嗜血杆菌

• 批准号: 8149365
• 负责人: Rachel Schneerson
• 金额: $ 35.53万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149365
• 关键词: Hemophilus ducreyi

Bordetellae, Gram-negative bacilli causing respiratory tract infections of mammals and birds include B. pertussis, B. parapertussis and B. bronchiseptica. The licensed pertussis vaccines confer incomplete efficacy on an individual basis, probably because pertussis toxin antibodies do not kill the organism directly, however herd immunity contributes to the almost complete protection with wide vaccine usage. The presence of bactericidal antibodies would increase vaccine effectiveness on an individual basis. Based on the concept that IgG anti-LPS provides immunity to non-capsulated Gram-negative bacteria we studied chemical, serological and immunological properties of LPS-derived saccharides of B. pertussis and B. bronchiseptica, -reported to share the same LPS core-, obtained by different degradation procedures and their protein conjugates. B. pertussis LPS is composed of a branched dodecasaccharide core bound to Lipid A. B. bronchiseptica LPS core is structurally the same but is further substituted by the O-specific polysaccharide (O-SP): a linear polymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-alpha-galacturonic acid. Two types of B. bronchiseptica O-SPs were identified based on the identity of their non-reducing end saccharide; no cross-reaction between these two types was found. Competitive inhibition assays of whole cell induced antisera showed that 95% of the antibodies were directed to the non-reducing end of these O-SP. Conjugates of B. bronchiseptica O-SPs were prepared by two methods: using the Kdo residue exposed by mild acid hydrolysis of the LPS or the core glucosamine residue exposed by deamination of the LPS, for binding to an aminooxylated protein. Both coupling methods were carried out at a neutral pH, room temperature, and in a short time. All conjugates, injected as saline solutions at a fraction of an estimated human dose, induced antibodies in mice to the homologous O-SP but not to the core. An isolated B. bronchiseptica core fraction without its O-SP and subjected to ESI-MS and NMR analysis confirmed its structural similarity to that of the B. pertussis core. Small variations were found: the core Fuc4NMe was 50% methylated in B. bronchiseptica, 100% in B. pertussis and the core Hep was about 30% phosphorylated in B. bronchiseptica, non phosphorylated in B. pertussis. Both B. pertussis and B. bronchiseptica cores were conjugated to aminooxylated BSA via their terminal Kdo. Injected into mice, both conjugates induced similar IgG anti B. pertussis LPS levels, significantly higher than a conjugate of B. brochiseptica core + O-SP. Because B. bronchiseptica grows faster than B. pertussis, with high yields and on simple culture media it was further investigated as a potential pertussis vaccine source. Mutants deficient in O-SP production were used: 1. RB50 delta (RB50-derived mutant, with a deletion spanning the wbmB, wbmC, wbmD and wbmE genes - this strain lacks the O-SP but its core structure is identical to that of the parent strain, 2. RBA2b (RB50-derived wbmA mutant producing LPS with no O-SP, but with the three non-reducing end core saccharides repeated several times). We prepared fractions of the B. bronchiseptica core with 1 to 4 repeats of this terminal trisaccharide and bound them to BSA at different densities. All conjugates were immunogenic in mice, the highest antibody levels were obtained by conjugates containing 10-15 saccharide chains per protein and with one repeat of the terminal trisaccharide. Conjugate-induced sera were bactericidal against B. pertussis, their titers correlated roughly with IgG anti LPS levels measured by ELISA.

博德氏杆菌是引起哺乳动物和鸟类呼吸道感染的革兰氏阴性杆菌，包括百日咳双歧杆菌、副百日咳双歧杆菌和支气管吸虫病双歧杆菌。许可的百日咳疫苗在个体基础上具有不完全的效力，可能是因为百日咳毒素抗体不会直接杀死生物体，然而群体免疫有助于广泛使用疫苗，几乎完全保护。在个体基础上，杀菌抗体的存在将提高疫苗的有效性。基于IgG抗LPS对非包膜革兰氏阴性菌提供免疫的概念，我们研究了通过不同降解程序及其蛋白偶联物获得的具有相同LPS核心的百日咳双歧杆菌和支孢双歧杆菌的LPS衍生糖的化学、血清学和免疫学特性。