NMR characterisation of group b streptococcus capsular polysaccharide repeating units

Keresztesi, Maximillian Ludwig

NMR characterisation of group b streptococcus capsular polysaccharide repeating units

Master Thesis

2022

Abstract

Group B Streptococcus (Streptococcus agalactiae) is a Gram-positive β-haemolytic bacterium and the leading cause of neonatal mortality by sepsis, pneumonia and meningitis. To date, ten serotypes of Group B Streptococcus (GBS) have been recognised (Ia, Ib, II - IX), each identified and differentiated by their sialic acid-containing capsular polysaccharide. Capsular polysaccharides are the virulence factor for bacterial pathogens and the target for vaccine development, with multivalent polysaccharide-protein conjugate vaccines licenced against bacteria such as Neisseria meningitidis and Streptococcus pneumoniae. Nuclear magnetic resonance (NMR) spectroscopy has been established as an extremely useful and robust method for tracking the manufacturing process of carbohydrate vaccines from polysaccharide antigen through to conjugate vaccines. The 1D proton profiles of most of the GBS antigens have been published, however, the identity spectra were recorded at 298 K, resulting in broad peaks and overlap of the large water signal with diagnostic GBS signals in the anomeric region. This study attempts to aid the development of GBS glycoconjugate vaccines by fully characterising the repeating units of the six most common GBS serotypes (Ia, Ib, II - V) by NMR recorded at a higher temperature of 343 K to serve as a database of reference GBS NMR spectra and chemical shift assignments. Full NMR characterisation of the repeating unit of each serotype was achieved by use of an array of 1D and 2D NMR experiments including proton, carbon, proton-proton scalar and dipolar correlation experiments and proton-carbon heteronuclear single-quantum and multiple bond correlation experiments. The assignments of all six serotypes largely agree with NMR data published for these serotypes. The exception to this was GBS V, where data presented in this study shows that the assignments of the anomeric peaks of GlcNAc and the backbone β-Glucose are reversed relative to their assignments in the current literature. The 1D and 2D NMR spectra presented in this study can be used for identity, integrity and purity testing of polysaccharide batches. They allow identification of each serotype by its diagnostic anomeric peaks, can confirm the structural integrity of the polysaccharide both before and after conjugation and can detect the presence of impurities such as residuals. Ultimately, they represent a powerful reference resource for use in the development, preparation and control testing of future GBS glycoconjugate vaccines.

Keywords

Chemistry

Reference:

Collections

Masters

Full item page