Bordetellae, Brucellae and Haemophilus ducreyi

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提供几乎完全的保护。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。

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, likely 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 has the same structure 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 their non-reducing end saccharide; no cross-reaction between the 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 carried out at a neutral pH, room temperature, and in a short time. Injected as saline solutions into mice, these conjugates, induced antibodies 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 and non phosphorylated in B. pertussis. 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 with intact 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. B. bronchiseptica core with 1 to 4 repeats of this terminal trisaccharide were prepared and bound 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. Previously, we developed Bordetella bronchiseptica strain TY-178 that produces a genetically inactivated analog of Bordetella pertussis toxin for use as in acellular pertussis vaccines. During the past year we have focused on increasing the yield and stability of the toxin analog of the TY-178 strain. To this end we designed specific protocols that met our research goals. The bacterial strains generated by these protocols are consistent with use in vaccine manufacturing process. Brucellosis is a zoonosis caused by various Brucella species affecting sheep, goats, cattle, deer, elk, pigs and more. Humans become infected by contact with animals or animal products contaminated with the organism. Symptoms in animals depend on the animal and Brucella species. Symptoms in humans may include fever, sweats, headaches, back pains, and weakness. Severe infections of the CNS, heart, or GI tract may occur. Brucellosis can also cause chronic symptoms that include recurrent fevers, joint pain, and fatigue. Brucella are categorized as group B agents. Ten species are recognized within the genius Brucella with the main species pathogenic for humans worldwide being B. abortus, B. melitensis and B. suis. The lipopolysaccharide (LPS) of Brucella is considered a major virulence factor and potentially a protective antigen. The 3 Brucella species share an O-SP composed of a non-branched homopolymer of 4,6-dideoxy-4-formamido--D-mannopyranose though variations may exist. Conjugates of O-SPs isolated from the 3 species and aminooxylated BSA were prepared. The ratios of protein to saccharide were: 1:0.23 for B. abortus, 1:0.24 for B. melitensis, and 1:0.16 for B. suis. The lower amount of saccharide in the last conjugate is explained by the lower molecular size of B. suis O-SP, visualized by silver staining (SDS-PAGE). The B. abortus O-SP conjugate was injected into mice; it induced IgG anti O-SP antibodies, with the booster responses, and reacted with both B. melitensis and B. suis by ELISA. Chancroid, a sexually transmitted genital ulcer disease is caused by infection with Haemophilus ducreyi, gram negative bacteria producing a tri component cytolethal toxin; cdtA, cdtB, and cdtC, responsible for the genital ulcers of chacroid. CdtA and cdtC comprise the binding toxin component to host cell receptors and translocating cdtB, the catalytic toxin component, into host cell nuclei. Three plasmids carrying modified forms of the cdtABC genes were obtained, sequenced and the reading frames of each identified. Using the DNA information, we produced PCR primers designed to amplify portions of the cdtABC genes encoding the mature cdtA and cdtC subunits less the signal sequence. Using these primers, we successfully amplified and cloned the modified genes into a protein expression plasmid. These plasmids were transformed into host E. coli DH5-alpha for seed stocks and E. coli BL21(DE3) for expression. The recombinant cdtA and cdtC protein subunits were purified from inclusion bodies using 6 M urea and Ni-ion affinity chromatography. The proteins were characterized by sequence analysis, SDS-PAGE and by western blots.