title: Real time monitoring of antigenicity development of HBsAg virus-like particles (VLPs) during heat- and redox-treatment.
abstract: The Hepatitis B virus major surface antigen (HBsAg) is a cysteine-rich, membrane-bound protein which self-assembles into 22-nm spherical virus-like particles (VLPs). While this VLP based human vaccine has been demonstrated to be safe and efficacious since 1986, the structural and exact molecular basis for its antigenic determinants has not been elucidated. Maturation of the yeast-derived purified VLPs was characterized for the changes in 37 their biophysical properties. Using rapid and label-free surface plasmon resonance technique with a neutralizing monoclonal antibody - A1.2, the epitope evolution kinetics of purified VLPs was monitored in real time. Evidence supporting the mechanism that the correct disulfide bond pairing is the molecular basis for shaping up the native virion-like epitopes was obtained. At least 10-fold enhancement in antigenicity probed by A1.2 of the VLPs was achieved by heat-treatment (t(1/2) ∼ 6-10 h), and another 2- to 3-fold enhancement was obtained when they were treated with redox buffer. This antigenicity development, presumably via disulfide formation/isomerization, was shown to be inhibited by a free radical scavenger and facilitated in the presence of light. Relative antigenicity determination with surface plasmon resonance was shown to be a valuable tool for process characterization in the kinetic monitoring mode or for final VLP product assessment in the end point antigenicity testing mode.
authors.affiliation: Merck Research Laboratories, West Point, PA 19486, USA. qinjian_zhao@merck.com
authors.forename: Qinjian
authors.initials: Q
authors.lastname: Zhao
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Dicky
authors.initials: D
authors.lastname: Abraham
authors.forename: Victoria
authors.initials: V
authors.lastname: Towne
authors.forename: Ronald
authors.initials: R
authors.lastname: Kennedy
authors.forename: Robert D
authors.initials: RD
authors.lastname: Sitrin
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D003545:Cysteine / Q000737:chemistry / Q000276:immunology; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D016166:Free Radical Scavengers / Q000737:chemistry; D006509:Hepatitis B / Q000276:immunology*; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000276:immunology*; D017325:Hepatitis B Vaccines / Q000737:chemistry / Q000276:immunology*; D006358:Hot Temperature; D006801:Humans; D010084:Oxidation-Reduction; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors; D014771:Virion / Q000737:chemistry / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D016166:Free Radical Scavengers; D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D003545:Cysteine