A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate

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.

Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein

“…The focus of this study has been sedimentation velocity in the analytical ultracentrifuge, together with sedimentation equilibrium and viscosity to examine the molecular integrity of the samples in terms of heterogeneity, molar mass distribution and conformational flexibility of purified native, micro-fluidized and activated (chemically modified to facilitate conjugation) capsular polysaccharides from N. meningitidis serogroups as well as meningococcal-tetanus toxoid (TT) conjugates. It follows our recent studies on (i) S. pneumoniae polysaccharides24; (ii) the tetanus toxoid protein25 and (iii) on the Hib-TT system26 the latter study clearly demonstrating it was the carbohydrate polymer component rather than the protein which dictated the solution properties rather than the protein component for that glycoconjugate vaccine system. We now seek to establish if this is true also for the Men-TT system.…”

“…Consistent with the behaviour of the sedimentation coefficients, the intrinsic viscosity results for the polysaccharides are decreasing with the size reduction and activation of native polysaccharide. Intriguingly they are generally lower for the glycoconjugates even though the sedimentation coefficients and molar masses are much higher, although still an order of magnitude higher than for compact globular particles (including the tetanus toxoid protein2526 and large molar mass spherical viruses28). Nonetheless they are lower than for the Hib- glycoconjugates26 indicating greater compaction.…”

“…Analysis of the sedimentation coefficient distributions g* (s) vs s profiles in phosphate-chloride buffer (pH = 6.8, I = 0.10) showed that, in common with Hib polysaccharides26 the native Men polysaccharides are bimodal systems with the high molar mass components ranging from 5% by mass in native Men Y to 20% in native Men A (for both Na + and Ca 2+ salts). The high molar mass component was relatively less significant at low concentrations.…”

“…However, the conformation of the conjugates has become more flexible than native, micro-fluidized and activated polysaccharides and that the conjugates are highly flexible chains or highly flexible random coil structures. The lower intrinsic viscosities compared with the native or activated polysaccharides is indicative of some degree of wrapping of the polysaccharide chains around the tetanus toxoid protein core, although, like with glyco-vaccines based on Hib 26 it seems it is the carbohydrate polymer component which dictates the hydrodynamic properties of these substances.…”

“…The experiment was performed at different reclining angles of 70° ( n  = 4 times), 60° ( n  = 4 times) and 50° ( n  = 6 times) under precise temperature control (20.00 ± 0.01) °C. Huggins56 and Kraemer57 extrapolations forms were performed to obtain the intrinsic viscosity, as described in ref 26. Intrinsic viscosities were also estimated using the Solomon – Ciutâ relation which represents a combination of the opposite trends of the Huggins and Kraemer relations allowing estimation of [ η ] without extrapolation5859: Nonetheless as a check on consistency of data such estimates are still registered at different concentrations.…”