Bordetellae are Gram-negative bacilli that cause respiratory tract infections in mammals and birds. B. pertussis, B. parapertussis, and B. bronchiseptica are clinically important. B. pertussis vaccines have succeeded in preventing pertussis in infants and children. Veterinary vaccines against B. bronchiseptica are available, but their efficacy and mode of action have not been 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-SP obtained from B. bronchiseptica and B. parapertussis by different degradation procedures. Based on their non-reducing end saccharide structure, we identified one type of B. parapertussis O-SP and two types of B. bronchiseptica O-SP with no cross-reaction between the later two. Competitive inhibition assays showed the immunodominance of the non-reducing end of these O-SPs. We prepared conjugates of B. bronchiseptica and B. parapertussis O-SPs using either the Kdo residue exposed by mild acid hydrolysis of the LPS or the core glucosamine residue exposed by deamination of the LPS. Both coupling methods were done at a neutral pH, at room temperature and in a short time. The conjugates injected into mice as saline solutions at a fraction of an estimated human dose induced antibodies to the homologous O-SP. These methods are applicable to the preparation of LPS-based vaccines against other Gram-negative bacteria. Probably because of the absence of a direct bactericidal effect of anti-toxin antibodies, the protection afforded by licensed subunit pertussis vaccines is incomplete on an individual basis. However, herd immunity which occurs with wide vaccine usage, provides almost complete protection. Nonetheless, an additional vaccine component inducing bactericidal antibodies such as anti-LPS could increase vaccine efficacy on an individual basis. To that end, we isolated and analyzed B. pertussis and B. bronchiseptica LPS. B. pertussis expresses only the core region saccharide (OS), composed of 12 sugars. B. bronchiseptica, easier to cultivate and producing higher yields, expresses LPS with a core structure almost identical to that of B. pertussis, although we observed small variations;(1) the methylation of Fuc4Nme, 100 percent in B. pertussisis is only 50 percent in B. bronchiseptica, and (2) Heptose is phosphorylated about 30 percent in B. bronchiseptica but not in B. pertussis. B. bronchiseptica LPS is mostly further substituted by O-specific chains. For this study, we used only a free core fraction with no O-SP. We prepared conjugates of both B. pertussis OS and B. bronchiseptica core by reacting their reducing end Kdo moieties with an amino-oxy 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. Conjugates were also prepared of oligosaccharides of B. bronchiseptica mutants, and their immunogenicity is being evaluated.
