Bordetellae and Haemophilus ducreyi

波尔德泰莱： 波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。临床上重要的是百日咳杆菌、副百日咳杆菌和败血杆菌。百日咳疫苗在预防婴儿和儿童百日咳方面取得了成功。针对支气管败血杆菌的兽医疫苗是可用的，但其效力和作用模式尚未确定。目前还没有针对副咳嗽杆菌的疫苗。基于血清中抗内毒素抗体可诱导对非包膜革兰氏阴性菌免疫的观点，我们研究了不同降解方法获得的支气管败血杆菌和副咯血杆菌的O-SP的化学、血清学和免疫学性质。根据非还原末端糖结构识别出一种副咳杆菌类型和两种类型的支气管败血杆菌O-SP，两种类型的支气管败血杆菌之间没有交叉反应。竞争抑制实验显示这些O-SP的非还原末端具有免疫优势。支气管败血杆菌和副肺结核杆菌O-SP的结合物是利用脂多糖温和酸解暴露的Kdo残基或脱氨基暴露的氨基葡萄糖残基与氨基氧基化的蛋白结合而成的。这两种偶联方法都是在中性pH、室温下进行的，而且时间短。所有的结合物都作为生理盐水注射到小鼠体内，剂量是估计的人类剂量的一小部分，可以诱导出对同源O-SP的抗体。这些方法可用于制备针对其他革兰氏阴性菌的脂多糖疫苗。 经许可的亚单位百日咳疫苗提供的保护在个体基础上是不完整的，可能是由于缺乏抗毒素抗体的直接杀菌作用。然而，随着疫苗的广泛使用，群体免疫几乎提供了完全的保护。诱导杀菌抗体的附加疫苗成分，如抗内毒素，可以提高个体的疫苗效力。为此，我们分离并分析了百日咳杆菌和败血杆菌的脂多糖。百日咳杆菌只表达由12种糖组成的核心区糖(OS)。百日咳杆菌中Fuc4NMe的甲基化程度为100%，而百日咳杆菌中Hep的甲基化程度仅为50%；(2)Hep在百日咳杆菌中的磷酸化程度约为30%，而百日咳杆菌中Hep的磷酸化程度约为30%。支气管炎杆菌的内毒素主要被O-特异链(O-SP)进一步取代。在这项研究中，只使用了不含O-SP的游离核心部分。百日咳杆菌和支气管败血杆菌核心蛋白的还原端Kdo部分与BSA结合的氨氧基连接物反应，制备了它们的偶联物。这两种结合物平均每个BSA分子含有10个糖链，都与抗B抗体反应。百日咳和抗BSA血清具有相同的同源性，并且两者在诱导相似抗体水平的小鼠中具有免疫原性，通过ELISA检测。

Bordetellae: Bordetellae are Gram-negative bacilli causing respiratory tract infections of mammals and birds. Clinically important are B. pertussis, B. parapertussis and B. bronchiseptica. B. pertussis vaccines have been successful in preventing pertussis in infants and children. Veterinary vaccines against B. bronchiseptica are available, but their efficacy and mode of action are not established. There is no vaccine against B. parapertussis. Based on the concept that immunity to non-capsulated Gram-negative bacteria may be conferred by serum IgG anti-LPS we studied chemical, serological and immunological properties of the O-specific polysaccharides (O-SP) of B. bronchiseptica and B. parapertussis obtained by different degradation procedures. One type of the B. parapertussis and two types of B. bronchiseptica O-SP were recognized based on their non-reducing end saccharide structure; no cross-reaction between the two B. bronchiseptica types was observed. Competitive inhibition assays showed the immunodominance of the non-reducing end of these O-SP. Conjugates of B. bronchiseptica and B. parapertussis O-SP were prepared by 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 into mice as saline solutions, at a fraction of an estimated human dose, induced antibodies to the homologous O-SP. These methodologies can be applied to prepare LPS-based vaccines against other Gram-negative bacteria. The protection afforded by licensed subunit pertussis vaccines is incomplete on an individual basis, likely due to the lack of direct bactericidal effect of anti toxin antibodies. However herd immunity, that occurs with wide vaccine uasage, provides the almost complete protection. An additinal vaccine component inducing bactericidal antibodies such as anti LPS, could increase vaccine efficacy on an individual basis. To that end B. pertussis and B. bronchiseptica LPS were isolated and analyzed. B. pertusis expresses only the core region saccharide (OS) composed of 12 sugars. B. bronchiseptica, easier cultured and with better yields, expresses LPS with almost identical core structure to that of B. pertussis, though we observed small variations: (1) the methylation of Fuc4NMe, 100% in B. pertussis is only 50% in B. bronchiseptica; (2) Hep is phosphorylated in about 30% in B. bronchiseptica,, while in B. petrussis it is not phosphorylated. B. bronchispetica LPS is mostly further substituted by O-specific chains (O-SP). For this study only a free core fraction, with no O-SP was used. Conjugates of both B. pertussis OS and B. bronchiseptica core were prepared by reacting their reducing-end Kdo moieties with an aminooxy linker bound to BSA. Both conjugates incorporated an average of 10 saccharide chains per BSA molecule, both reacted with anti-B. pertussis and anti-BSA sera with a line of identity and both were immunogenic in mice inducing similar antibody levels, measured by ELISA.