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; 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.

bordetelae，革兰氏阴性杆菌引起哺乳动物和鸟类的呼吸道感染，包括百日咳芽孢杆菌，B。parapertussis和B.b。bronchiseptica。获得许可的百日咳疫苗以个人为基础赋予不完整的功效，这可能是因为百日咳毒素抗体不会直接杀死生物体，但是牛群的免疫力几乎可以通过广泛的疫苗使用来造成几乎完全的保护。杀菌抗体的存在将在单独的基础上提高疫苗有效性。基于IgG抗LPS对非封装的革兰氏阴性细菌的免疫力，我们研究了B. t. b. b. t. b. b. t. b. b. b. b. b. tussiss and B. b. b.b。bronchiseptica的化学，血清学和免疫学特性，并通过不同的LPS核心报告，通过不同的LPS核心，通过不同的DENGRACTATION PROCTARADATION PROTICATION PROTICATION PROTINATION和PROTEIN CONTERATES和他们的蛋白质可获得。 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.根据其非还原末端糖的身份鉴定了两种类型的支气管杆菌O-SP。没有发现这两种类型之间的交叉反应。全细胞诱导的抗血清的竞争抑制测定表明，有95％的抗体针对这些O-SP的非还原端。支气管杆菌O-SP的偶联物是通过两种方法制备的：使用LPS的温和酸水解暴露的KDO残基或通过LPS脱氨基暴露于LPS暴露的核心葡萄糖胺残基，与氨基二氧化蛋白结合。两种耦合方法均在中性pH，室温和短时间内进行。所有共轭物，在估计的人剂量的一部分处注射为盐水溶液，诱导小鼠对同源O-SP的抗体，但对核心不诱导。一个孤立的支气管杆菌核心分数没有其O-SP并进行ESI-MS，NMR分析证实了其与芽孢杆菌核心核心的结构相似性。发现小变化：在支气管杆菌中，核心FUC4NME甲基化50％甲基化，百日咳芽孢杆菌为100％，核心HEP在支气管杆菌中大约30％磷酸化，在B. tellussis中未磷酸化。 百日咳芽孢杆菌和支气管杆菌的核心均通过其末端KDO连接到氨基化的BSA。两种结合物被注射到小鼠中，诱导了类似的IgG抗B.百日咳LPS水平，显着高于B. brochiseptica core + O-Sp的偶联物。由于支气管杆菌的生长速度比百日咳B.百日咳更快，因此，在简单的培养基上，它作为潜在的百日咳疫苗来源进一步研究。 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糖的重复几次是针对百日咳芽孢杆菌的杀菌性，它们的滴度与ELISA测量的IgG抗LPS水平大致相关。