Bordetellae and Haemophilus ducreyi

引起哺乳动物和鸟类呼吸道感染的博德特氏菌、革兰氏阴性杆菌包括B。百日咳，B. B.副百日咳和B.支气管炎许可的百日咳疫苗在个体的基础上赋予不完全的效力，可能是因为百日咳毒素抗体不直接杀死生物体，然而群体免疫有助于广泛使用疫苗的几乎完全保护。杀菌抗体的存在将在个体基础上增加疫苗的有效性。基于IgG抗LPS提供对非包膜革兰氏阴性菌的免疫力的概念，我们研究了B的LPS衍生物的化学、血清学和免疫学性质。百日咳和B.支气管败血症，-报告共享相同的LPS核心-，通过不同的降解程序和它们的蛋白缀合物获得。 B。百日咳LPS由与脂质A结合的支链十二糖核心组成。B。支气管败血症LPS核心在结构上相同，但进一步被O-特异性多糖（O-SP）取代：1，4-连接的2，3-二乙酰氨基-2，3-二脱氧-α-半乳糖醛酸的线性聚合物。两种类型的B。支气管败血症O-SP的非还原末端糖的鉴定结果表明，两种O-SP之间无交叉反应，全细胞诱导抗血清的竞争抑制试验表明，95%的抗体针对O-SP的非还原末端。支气管败血症O-SP通过两种方法制备：使用通过LPS的温和酸水解暴露的Kdo残基或通过LPS的脱氨基暴露的核心葡糖胺残基，用于结合氨氧基化蛋白。两种偶联方法均在中性pH、室温和短时间内进行。所有的共轭物，作为盐溶液注射的一部分，估计人类剂量，诱导小鼠抗体的同源O-SP，但不核心。一个孤立的B。支气管败血症核心级分不含其O-SP，并进行ESI-MS和NMR分析，证实其结构与B相似。百日咳核心发现了小的变化：核心Fuc4 NMe在B中甲基化50%。支气管败血症，B组100%。而核心Hep在B中约有30%磷酸化。支气管败血症，B中未磷酸化。百日咳。 都是B。百日咳和B.支气管败血症核心通过其末端Kdo与氨氧基化BSA缀合。注射到小鼠中，两种缀合物诱导相似的IgG抗B。百日咳LPS水平，明显高于结合物B。支气管败血症核心+O-SP.因为B.支气管败血症比B生长快。百日咳疫苗，产量高，在简单的培养基上，进一步研究了作为潜在的百日咳疫苗来源。使用O-SP产生缺陷的突变体：1. RB50 δ（RB50衍生的突变体，具有跨越wbmB、wbmC、wbmD和wbmE基因的缺失-该菌株缺乏O-SP，但其核心结构与亲本菌株相同，2. RBA2b（RB50衍生的wbmA突变体，其产生LPS，不具有O-SP，但具有重复数次的三个非还原性末端核心酶解）。我们制备了B的馏分。支气管败血症核心具有1至4个重复的该末端三糖，并以不同的密度将它们结合到BSA。所有缀合物在小鼠中均具有免疫原性，通过每个蛋白质含有10 - 15个糖链且具有一个末端三糖重复的缀合物获得最高抗体水平。结合物诱导的血清对B具有杀菌作用。百日咳，它们的滴度与ELISA测量的IgG抗LPS水平大致相关。

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.