Towards the development of a capsular polysaccharide-protein conjugate vaccine to protect against Burkholderia pseudomallei

Abstract

A thesis by Alison Jane George entitled 'Towards the development of a capsular polysaccharide-protein conjugate vaccine to protect against Burkholderia pseudomallei' and submitted to The University of Manchester for the degree of Doctor of Philosophy.Burkholderia pseudomallei is the causative agent of melioidosis, a debilitating disease in endemic areas and a potential biowarfare agent, for which there is currently no vaccine available. Many licensed vaccines exist for protection against a range of other bacterial pathogens and many of these are based on the cell-surface polysaccharides of the respective infectious agent. The immunogenicity of bacterial polysaccharides may be improved by conjugating to a carrier protein.The aim of this thesis was to develop a method for production and conjugation of the capsular polysaccharide (CPS) from B. pseudomallei. This involved two areas of development; to improve the production and extraction of B. pseudomallei CPS and to develop a chemical-based method for conjugation of both a synthetic CPS and a native CPS to a carrier protein.To simplify production and extraction of B. pseudomallei CPS, a recombinant source was investigated. Two genomic libraries were screened for an Escherichia coli clone containing the B. pseudomallei CPS gene cluster. A clone was identified but failed to express the CPS.The extraction and purification of CPS from B. pseudomallei was optimised to produce a pure source of material. CPS was extracted from a B. pseudomallei LPS knock-out strain, DeltarmlB and purified using an affinity chromatography column labelled with a monoclonal antibody against the CPS. This purified CPS was found to be protective in a murine model of melioidosis. Mice immunized with CPS produced higher levels of IgM and IgG and showed an increased survival compared to the control groups. Furthermore, bacterial loads were lower in organs from mice immunized with CPS, compared to the controls.A method for chemical conjugation of a synthetic B. pseudomallei CPS to the tetanus Hc fragment protein was developed using EDC to activate the protein, followed by sodium borohydride to covalently attach synthetic CPS. Unfortunately, this conjugate could not be proven using analytical methods. A second approach using CDAP activation of native CPS extracted from B. pseudomallei, followed by addition of an adipic acid dihydrazide linker and EDC-mediated attachment of the carrier protein successfully produced a conjugate vaccine candidate.

Date of Award	1 Aug 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ian Roberts (Supervisor) & James Linton (Supervisor)