Saccharide/protein conjugate vaccines for Bordetella species: preparation of saccharide, development of new conjugation procedures, and physico-chemical and immunological characterization of the conjugates

Abstract

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 the structure of their non-reducing end saccharide; 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 two methods: using the anhydro-Kdo residue exposed by mild acid hydrolysis of the LPS or the 2,5-anhydromannose residue exposed by deamination of the core glucosamine 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. These methodologies can be applied to prepare O-SP-based vaccines against other Gram-negative bacteria.

Fig. 1

Non-reducing end-group types of B. bronchiseptica O-SP.

Fig. 2

Immunodifusion assays of anti-B parapertussis (Bpp) 15989 (A) and anti-B. bronchiseptica (Bb) 10580 (B) with Bordetella LPS. The LPS from Bpp15989 forms a spur over Bb10580 LPS, both ‘ala-type’, when tested against anti-Bpp15989 serum; Bb10580 LPS forms a spur over Bpp15989 using anti-Bb10580 serum. No reaction was seen between with BbRb50 LPS (‘lac’ type) and the two antisera.

Fig. 3

Schemes of conjugation of B. bronchiseptica and B. parapertussis O-SP to protein Protein was first amiooxylated in a two step procedure and reacted with the carbonyl group on the terminal reducing end introduced into the O-SP either by deamidation of the GlcN, a first non-reducing end sugar of Band B (Panel A) or by acetic acid hydrolysis of the Kdo converting it to anhydro-Kdo (Panel B). Conjugates preserved the external nonreducing end of the O-SP. Below, the structure and the cleavage sides of B. bronchiseptica LPS.

Fig. 4

MALDI-TOF of conjugates. (A) BSA, (B) aminooxylated BSA (BSA-ONH₂), (C) BSA-ONH₂/Bb10580 Conjugate #1 (C).