title: Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes.
abstract: Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.
authors.affiliation: Department of Medical Microbiology, Molecular Virology Section, University of Groningen, University Medical Center Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Jørgen
authors.initials: J
authors.lastname: de Jonge
authors.forename: Pieter
authors.initials: P
authors.lastname: Schoen
authors.forename: Wouter
authors.initials: W
authors.lastname: ter Veer
authors.forename: Toon
authors.initials: T
authors.lastname: Stegmann
authors.forename: Jan
authors.initials: J
authors.lastname: Wilschut
authors.forename: Anke
authors.initials: A
authors.lastname: Huckriede
mesh_terms: D003956:Dialysis; D007700:Kinetics; D009975:Orthomyxoviridae / Q000502:physiology / Q000648:ultrastructure*; D010713:Phosphatidylcholines / Q000737:chemistry*; D012995:Solubility; D014759:Viral Envelope Proteins / Q000737:chemistry*; D038301:Virus Inactivation
chemical_list: D010713:Phosphatidylcholines; D014759:Viral Envelope Proteins; C017251:1,2-oleoylphosphatidylcholine