Antimicrobial Carbohydrate Vaccines: Development of Burkholderia pseudomallei immunogens

Donaldson, Matthew (2013) Antimicrobial Carbohydrate Vaccines: Development of Burkholderia pseudomallei immunogens. Doctoral thesis, University of East Anglia.

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Abstract

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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Chemistry
Depositing User: Mia Reeves
Date Deposited: 12 Mar 2014 09:43
Last Modified: 12 Mar 2014 09:43
URI: https://ueaeprints.uea.ac.uk/id/eprint/48103
DOI:

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