Characterization of the structural modifications accompanying the loss of HBsAg particle immunogenicity.
The aim of this work was to further understand the relationship between the immunogenicity and the structure of Hepatitis B surface antigen (HBsAg) particles used in Hepatitis B vaccines. To reach this aim, we compared by using a large range of techniques, the structure and properties of untreated particles with those of particles stored for 3 weeks at +60°C, a treatment which resulted in a loss of HBsAg antigenicity (toward RF-1 mAb) and immunogenicity (in mice). While untreated particles imaged by electron microscopy and atomic force microscopy appeared as isolated nanoparticles of ∼ 20nm, heated particles appeared as long chains of particle aggregates with a partial loss of their protein protrusions. Moreover, infrared spectroscopy and circular dichroism revealed that the secondary structure of the S proteins was significantly affected, with a loss of 10% of their α-helix content. Steady-state and time-resolved fluorescence data further revealed strong modifications of the most emitting Trp residues at the particle surface, confirming significant changes in the conformation of the S proteins. Moreover, modifications in the organization of both the lipid core and lipid membrane surface of the heated particles were evidenced by environment-sensitive 3-hydroxyflavone probes. Taken together, our data evidenced a clear relationship between the bona fide S protein structure and lipid organization notably at the particle surface and the particle immunogenicity.
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France.
Vanille J
VJ
Greiner
Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Étoile, France.
Catherine
C
Manin
Laboratoire d'Enzymologie et Biochimie Structurales (LEBS) UPR 3082 du CNRS, Campus CNRS, Bâtiment 34, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France.
Eric
E
Larquet
Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Étoile, France.
Nabila
N
Ikhelef
Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Étoile, France.
Frédéric
F
Gréco
Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Étoile, France.
Sophie
S
Naville
Centre de Biochimie Structurale, CNRS UMR 5048-Inserm U1054, Université de Montpellier, 29 rue de Navacelles, 34090 Montpellier Cedex, France.
Pierre-Emmanuel
PE
Milhiet
Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Étoile, France.
Frédéric
F
Ronzon
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France.
Andrey
A
Klymchenko
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France. Electronic address: yves.mely@unistra.fr.
Yves
Y
Mély
D000818:Animals; D002942:Circular Dichroism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D006514:Hepatitis B Surface Antigens / Q000737:chemistry* / Q000276:immunology; D006358:Hot Temperature; D051379:Mice; D008807:Mice, Inbred BALB C; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D017433:Protein Structure, Secondary; D013055:Spectrophotometry, Infrared
D000939:Epitopes; D006514:Hepatitis B Surface Antigens
Atomic force microscopy; Electron microscopy; Fluorescence spectroscopy; Fourier transform infrared spectroscopy; Hepatitis B surface antigen; Immunogenicity