Abstract
Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 106 g.mol−1) compared to the native (Mw ~ 1.2 × 106 g.mol−1). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 106 g.mol−1), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution.
Highlights
Until the availability of the Hib vaccine, the type b H. influenzae was the main cause of meningitis in children between 6 months and 5 years old, older children, adolescents and adults can be infected[8]
Unlike other bacteria (e.g Streptococcus pneumoniae and Neisseria meningitidis) the type b polysaccharide capsule of H. influenzae is attractive as a vaccine antigen since invasive disease is almost exclusively restricted to only one serotype, which in turn has rendered its capsular polysaccharide a prime candidate for vaccine studies
While many other aspects of polysaccharide characterisation have been relatively thoroughly explored[26,27,28], the physical characterisation of capsular polysaccharides has been less extensively pursued. Such physical studies for capsular polysaccharides have been generally limited to low pressure chromatographic analyses calibrated with “standards” and more recently to high performance size exclusion chromatography (SEC) coupled to on-line refractive index detector (RI), multi-angle light scattering (MALS)
Summary
Until the availability of the Hib vaccine, the type b H. influenzae was the main cause of meningitis in children between 6 months and 5 years old, older children, adolescents and adults can be infected[8]. The first generation of Hib vaccine was based on the purified type b capsular polysaccharide and its ribose-ribitol phosphate repeating units[2,18,19,20]. While many other aspects of polysaccharide characterisation have been relatively thoroughly explored[26,27,28], the physical characterisation of capsular polysaccharides (molecular size and mass distribution and conformational flexibility) has been less extensively pursued. This study is designed principally to characterise the purified native and activated capsular polyribosylribitol polysaccharides (PRP) from Haemophilus influenzae type b (referred to as PRP native and PRP-ADH respectively) as well as the final PRP- TT conjugate This study is designed principally to characterise the purified native and activated capsular polyribosylribitol polysaccharides (PRP) from Haemophilus influenzae type b (referred to as PRP native and PRP-ADH respectively) as well as the final PRP- TT conjugate (with a polysaccharide: protein ratio of ca. 0.4) and to establish whether it is the protein component or carbohydrate component which principally influences the physical or hydrodynamic properties in solution This study has been designed to demonstrate the usefulness of analytical ultracentrifuge based procedures – all not requiring a separation column or matrix - in the characterisation of large glycoconjugate vaccines
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