Background: There is a pressing need for antibodies and vaccines of improved efficacy, and improved methods for their production. This project is aimed at creating a new enabling technology for controlled synthesis of a carbohydrate-protein vaccine conjugate in pursuit of a broad-spectrum antibacterial vaccine. PNAG is a polysaccharide conserved on the surface of multiple bacterial, pathogens, including E. coli O157 and multidrug resistant S. aureus and A. baumannii, and is therefore a tantalizing antigenic target for developing a broad-spectrum vaccine for resistant bacteria (PNAS, 2013, 110, E2209). However access to pure PNAG polysaccharides of defined composition and degree of polymerization (dp), is a significant challenge with purification from bacteria yielding impure glycans and different dp. These glycans are therefore of limited utility for the construction of carbohydrate-protein conjugate vaccines, which are necessary for weak antigens like polysaccharides to increase the immune response.
Objectives: We aim to utilise a multidisciplinary approach for the synthesis of complex PNAG oligosaccharides, uniting the capability of UK’s first automated oligosaccharide synthesiser the ‘Glyconeer’, based in the UoY, with a novel strategy for enzyme mediated on-protein polymerization, following bioconjugation using technology developed in our lab (Chem Sci., 2018, 9(25), 5593). The resulting carbohydrate-protein conjugates will then be evaluated as components of broad-spectrum antibacterial vaccines
Experimental Methodology: As synthesis of pure PNAG polysaccharides (>20 monosacchride units) of defined composition and dp is challenging, we aim to utilise the Glyconeer to access ‘primer’ oligosaccharide glycans by automated glycan assembly (AGA) (Org. Biomol. Chem., 2018, 17(7), 1821), prior to conjugation to protein platforms and on-protein polymerisation using mutant endoglycosynthase enzymes (Chem. Soc. Rev., 2017, 46, 5128). In order to control the degree of polymerization we will also deploy PNAG terminator which will lack the hydroxyl required for enzymatic elongation, therefore conceivably controlling dp through altering the ratio of glycan to terminator in the reaction.
The project’s focus will be to wholly unite both automated and enzymatic assembly of oligosaccharides for the first time, taking advantage of the ‘Glyconeer’, the only automated oligosaccharide synthesis platform in the UK, and one of 5 around the world. We will also seek to develop novel endoglycosynthase enzymes through rational genetic engineering for on-protein glycan polymerisation.
Scientific training activities in this project being flexible and continuous over the 3 years. Primarily, training in Synthetic carbohydrate chemistry will be provided in the Fascione group, including in the use of the Glyconeer. Training in synthetic organic chemistry will also be provided to ensure the student has an established organic chemistry knowledge of the proposed transformations/methodology at the molecular level. On a practical level, the student will be trained in solid phase oligosaccharide synthesis and novel bio-orthogonal methodologies that have been developed and patented within the group, in addition to the synthesis of other small molecules and the associated practical techniques all organic chemists need to master i.e. anhydrous technique, chromatographic purification, compound characterisation etc. The student will also be trained in the NMR characterisation of small molecules, specifically carbohydrates, which often have unique and complex spectra. As the project progresses, the student will be taught how to express and purify proteins using existing constructs in E. coli. They will also learn how to characterise proteins and their modifications in water/buffer i.e. simple SDS-polyacrylamide electrophoresis gels, and mass spec characterisation within the Davies group. As a member of both groups, the student will attend weekly group meetings and journal clubs that will highlight core technologies and advancements in this field.
All Chemistry research students have access to our innovative Doctoral Training in Chemistry (iDTC): cohort-based training to support the development of scientific, transferable and employability skills: https://www.york.ac.uk/chemistry/postgraduate/idtc/
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. This PhD project is available to study full-time or part-time (50%).
This PhD will formally start on 1 October 2020. Induction activities will start on 28 September.