Glycoconjugate vaccines: some observations on carrier and production methods

© 2019 Informa UK Limited, trading as Taylor & Francis Group. Glycoconjugate vaccines use protein carriers to improve the immune response to polysaccharide antigens. The protein component allows the vaccine to interact with T cells, providing a stronger and longer-lasting immune response than a polysaccharide interacting with B cells alone. Whilst in theory the mere presence of a protein component in a vaccine should be sufficient to improve vaccine efficacy, the extent of improvement varies. In the present review, a comparison of the performances of vaccines developed with and without a protein carrier are presented. The usefulness of analytical tools for macromolecular integrity assays, in particular nuclear magnetic resonance, circular dichroism, analytical ultracentrifugation and SEC coupled to multi-angle light scattering (MALS) is indicated. Although we focus mainly on bacterial capsular polysaccharide-protein vaccines, some consideration is also given to research on experimental cancer vaccines using zwitterionic polysaccharides which, unusually for polysaccharides, are able to invoke T-cell responses and have been used in the development of potential all-polysaccharide-based cancer vaccines. A general trend of improved immunogenicity for glycoconjugate vaccines is described. Since the immunogenicity of a vaccine will also depend on carrier protein type and the way in which it has been linked to polysaccharide, the effects of different carrier proteins and production methods are also reviewed. We suggest that, in general, there is no single best carrier for use in glycoconjugate vaccines. This indicates that the choice of carrier protein is optimally made on a case-by-case basis, based on what generates the best immune response and can be produced safely in each individual case. Abbreviations: AUC: analytical ultracentrifugation; BSA: bovine serum albumin; CD: circular dichroism spectroscopy; CPS: capsular polysaccharide; CRM197: Cross Reactive Material 197; DT: diphtheria toxoid; Hib: Haemophilius influenzae type b; MALS: multi-angle light scattering; Men: Neisseria menigitidis; MHC-II: major histocompatibility complex class II; NMR: nuclear magnetic resonance spectroscopy; OMP: outer membrane protein; PRP: polyribosyl ribitol phosphate; PSA: Polysaccharide A1; Sa: Salmonella; St.: Streptococcus; SEC: size exclusion chromatography; Sta: Staphylococcus; TT: tetanus toxoid; ZPS: zwitterionic polysaccharide(s)

Probing the effect of aroma compounds on the hydrodynamic properties of mucin glycoproteins

© 2020, The Author(s). Aroma compounds are diverse low molecular weight organic molecules responsible for the flavour of food, medicines or cosmetics. Natural and artificial aroma compounds are manufactured and used by the industry to enhance the flavour and fragrance of products. While the low concentrations of aroma compounds present in food may leave no effect on the structural integrity of the mucosa, the effect of concentrated aroma volatiles is not well understood. At high concentrations, like those found in some flavoured products such as e-cigarettes, some aroma compounds are suggested to elicit a certain degree of change in the mucin glycoprotein network, depending on their functional group. These effects are particularly associated with carbonyl compounds such as aldehydes and ketones, but also phenols which may interact with mucin and other glycoproteins through other interaction mechanisms. This study demonstrates the formation of such interactions in vitro through the use of molecular hydrodynamics. Sedimentation velocity studies reveal that the strength of the carbonyl compound interaction is influenced by compound hydrophobicity, in which the more reactive short chain compounds show the largest increase in mucin-aroma sedimentation coefficients. By contrast, the presence of groups that increases the steric hindrance of the carbonyl group, such as ketones, produced a milder effect. The interaction effects were further demonstrated for hexanal using size exclusion chromatography light scattering (SEC-MALS) and intrinsic viscosity. In addition, phenolic aroma compounds were identified to reduce the sedimentation coefficient of mucin, which is consistent with interactions in the non-glycosylated mucin region

Submaxillary Mucin: its Effect on Aroma Release from Acidic Drinks and New Insight into the Effect of Aroma Compounds on its Macromolecular Integrity

Submaxillary mucin is a major component that defines the makeup and functionality of saliva. Understanding its structure and function during food intake is key to designing appropriate strategies for enhancing the delivery of flavour. In the present study, the hydrodynamic integrity of bovine submaxillary mucin was characterised under physiological and acidic conditions and it was shown to have a broad molecular weight distribution with species ranging from 100 kDa to over 2000 kDa, and a random coil type of conformation. A decrease in the pH of mucin appeared to result in aggregation and a broader molecular weight distribution, which was shown to correlate with a release of flavour compounds. Our study also provides indications that p-cresol may have an effect on the macromolecular integrity of mucin

Hydrodynamics and stability of Group B Streptococcus polysaccharides and CRM197 protein

Group B Streptococcus is a pathogen that chiefly infects immunocompromised individuals and neonates. To combat this infection, at-risk mothers are currently given antibiotics before birth to prevent exposure of neonates to the bacteria. A vaccine would be beneficial, as it would allow mothers to combat this infection without excessive use of antibiotics. As encapsulated bacteria, the main structure available as a target antigen is the polysaccharide capsule itself, rather than any underlying structures. For this reason, glycoconjugate vaccines are being synthesised which use surface polysaccharides conjugated to carrier proteins to improve their immunogenicity. This project studies the stability of candidate polysaccharides for use in a glycoconjugate vaccine. The polysaccharides were provided by GSK vaccines, who requested the use of specific hydrodynamic methodology. The polysaccharides were found to be generally stable, only seeing small changes in hydrodynamic properties after exposure to stress, but in many cases it is likely that the differences seen are due to minor experimental variation rather than genuine structural differences. The polysaccharides were also tested for possible association effects with blood plasma proteins: serum albumin, fibrinogen and immunoglobulin. The polysaccharides were again found to be stable in the presence of these molecules. Together, these results suggest that these molecules are suitable for further testing and development to generate a vaccine

Hydrodynamics and stability of Group B Streptococcus polysaccharides and CRM197 protein

Group B Streptococcus is a pathogen that chiefly infects immunocompromised individuals and neonates. To combat this infection, at-risk mothers are currently given antibiotics before birth to prevent exposure of neonates to the bacteria. A vaccine would be beneficial, as it would allow mothers to combat this infection without excessive use of antibiotics. As encapsulated bacteria, the main structure available as a target antigen is the polysaccharide capsule itself, rather than any underlying structures. For this reason, glycoconjugate vaccines are being synthesised which use surface polysaccharides conjugated to carrier proteins to improve their immunogenicity. This project studies the stability of candidate polysaccharides for use in a glycoconjugate vaccine. The polysaccharides were provided by GSK vaccines, who requested the use of specific hydrodynamic methodology. The polysaccharides were found to be generally stable, only seeing small changes in hydrodynamic properties after exposure to stress, but in many cases it is likely that the differences seen are due to minor experimental variation rather than genuine structural differences. The polysaccharides were also tested for possible association effects with blood plasma proteins: serum albumin, fibrinogen and immunoglobulin. The polysaccharides were again found to be stable in the presence of these molecules. Together, these results suggest that these molecules are suitable for further testing and development to generate a vaccine

Heparin in Acid and Alkaline Environments- a Study of the Correlations Between Hydrodynamic Properties and Desulfation

This work evaluated the hydrodynamic properties of heparin hydrolysed at temperatures ranging from 40 °C to 80 °C in buffered acid and alkaline environments. The correlation between hydrodynamic parameters led to the conclusion that polymer conformational changes appeared to be minimal until chain depolymerisation, initiated at pH 1 and 80 °C. However, the synergy of conformational changes, even if minimal, and sulphate loss observed at pH 1, pH 3 and pH 12 (various temperatures) resulted in a loss of the antifactor Xa activity. Therefore, the ‘contribution’ of conformational changes should be added to the generally recognized effect of desulphation towards the activity of heparin. This is of significance as the processing of medical heparin is complex, and requires adjustment of several physical and chemical factors, including pH and temperature

Comparative sedimentation equilibrium analysis of two IgG1 glycoforms: IgGCri and IgGWid.

The solution properties of two different glycoforms of IgG1 (IgG1Cri and IgG1Wid) are compared using primarily sedimentation equilibrium analysis with two complementary analysis routines: SEDFIT-MSTAR and MULTISIG. IgGCri bears diantennary complex-type glycans on its Fc domain that are fully core fucosylated and partially sialylated, whilst on IgGWid, they are non-fucosylated, partially galactosylated and non-sialylated. IgGWid is also Fab glycosylated. Despite these differences, SEDFIT-MSTAR analysis shows similar weight average molar masses Mw of ~ (150 ± 5) kDa for IgGCri and ~ (154 ± 5) kDa for IgGWid and both glycoforms show evidence of the presence of a small fraction of dimer confirmed by MULTISIG analysis and also by sedimentation coefficient distributions from supportive sedimentation velocity measurements. The closeness of the sedimentation equilibrium behaviour and sedimentation coefficient distributions with a main peak sedimentation coefficient of ~ 6.4S for both glycoforms at different concentrations suggest that the different glycosylation profiles do not significantly impact on molar mass (molecular weight) nor conformation in solution