title: Structural perturbation of diphtheria toxoid upon adsorption to aluminium hydroxide adjuvant.
abstract: Aluminium-containing adjuvants are often used to enhance the potency of vaccines. In the present work we studied whether adsorption of diphtheria toxoid to colloidal aluminium hydroxide induces conformational changes of the antigen. Diphtheria toxoid has a high affinity for the aluminium hydroxide particles based on a high adsorption degree, adsorption rate and adsorptive capacity. The conformation and stability of diphtheria toxoid in solution and adsorbed to aluminium hydroxide adjuvant were characterized using five physicochemical techniques: intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, infrared spectroscopy and differential scanning calorimetry. Diphtheria toxoid adsorbed to aluminium hydroxide resulted in a minimal shift of the tryptophan fluorescence spectrum, whereas a large increase in the emission of the Bis-ANS probe was observed, indicating that hydrophobic sites of the protein became accessible due to adsorption. In addition, circular dichroism and infrared spectroscopy revealed that adsorption to aluminium hydroxide caused an increase of β-sheet content and a decrease of α-helix content in diphtheria toxoid. Differential scanning calorimetry demonstrated a major decrease in the enthalpy of denaturation upon adsorption. In conclusion, the adsorption of diphtheria toxoid to aluminium hydroxide adjuvant leads to substantial conformational changes in the antigen. Since physicochemical methods can be used to monitor these conformational changes, these analytical methods might offer a tool in regulatory required vaccine quality control by demonstrating consistency in production.
authors.affiliation: Unit Vaccinology, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
authors.forename: Marie
authors.initials: M
authors.lastname: Régnier
authors.forename: Bernard
authors.initials: B
authors.lastname: Metz
authors.forename: Wichard
authors.initials: W
authors.lastname: Tilstra
authors.forename: Coenraad
authors.initials: C
authors.lastname: Hendriksen
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
authors.forename: Willem
authors.initials: W
authors.lastname: Norde
authors.forename: Gideon
authors.initials: G
authors.lastname: Kersten
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry*; D000327:Adsorption; D000536:Aluminum Hydroxide / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004168:Diphtheria Toxoid / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared
chemical_list: D000276:Adjuvants, Immunologic; D004168:Diphtheria Toxoid; D000536:Aluminum Hydroxide