Antimicrobial Carbohydrate Vaccines:Development of Burkholderia pseudomalleiimmunogens

The potential bio-terror threat posed by Burkholderia pseudomallei highlights the need for an effective vaccine. Immunisation and challenge experiments in mice have demonstrated that the capsular polysaccharide (CPS-1) of B. pseudomallei, which is composed of β-1,3-linked 6-deoxy-D-manno-heptopyranose residues, is a promising candidate for vaccine development. This thesis set out to explore routes to potential vaccine candidates for Burkholderia pseudomallei infection based on both bottom-up synthetic monosaccharide constructs and top-down CPS-1 conjugates. Initially this thesis focused on the development of a monosaccharide antigen based on the 6-deoxy-D-manno-heptose building block of CPS-1. The antigens were conjugated to TetHc carrier protein via an 8-(methoxycarbonyl)octyl linker using traditional acyl hydrazide conjugation chemistry. The TetHc monosaccharide conjugates were analysed by SDS-PAGE and MALDI-ToF mass spectrometry before undergoing immunisation trials in sheep. Immunological assessment of the resulting polyclonal antisera was conducted by ELISA, slot-blot and the Octet biosensor system. These studies demonstrated the generation of antibodies that were specific for the cognate monosaccharide. Preliminary investigations showed that the 6-deoxy-D-mannoheptose- derived antibodies reacted positively with the natural CPS-1. The linker was shown to be important in the specificity of the polyclonal sera, leading to cross reactivity with non-parent monosaccharide antigens. A second generation of antigens was developed using a short linker, 3-(methyl mercaptopropionate). The expression and purification of the natural B. pseudomallei CPS-1 was achieved in an avirulent strain of Burkholderia, B. thailandensis E555. Characterisation of the capsular polysaccharide highlighted the co-expression of an α-1,3-mannan polysaccharide. Mild acid hydrolysis of the CPS-1 preparation, combined with capillary electrophoresis was trialled with a view to the generation of oligosaccharide fragments of CPS-1 for conjugation and immunisation studies. While initial carbohydrate conjugates were prepared with a previously described tetanus toxoid Hc fragment, the development and in planta expression of a chemically conjugatable Hepatitis B core virus-like particle system was also achieved.