Reconstruction of H3N2 influenza virus based virosome in-vitro

Background and Objectives: Virosomes are Virus Like Particles (VLP) assembled in-vitro. Influenza virosomes maintain the cell binding and membrane fusion activity of the wild type virus but are devoid of viral genetic material or internal proteins. Influenza virosomes mimic the natural antigen presentation route of the influenza virus. Materials and Methods: Virosomes were prepared by membrane solubilization and reconstitution. Briefly, the Madine- Darby Canine kidney (MDCK) cell line was cultivated on microcarrier beads inoculated with influenza virus strain A/X-47 (H3N2). The culture medium was harvested and clarified. Subsequently, virus was concentrated and purified by ultrafiltration and ultracentrifugation. The purified viral membrane was dissolved by adding 375 μl of 200 mM 1, 2-dicaproyl-sn-glycero- 3-phosphocholine (DCPC) in HEPES-buffered saline (HBS). Nucleocapsid was removed by ultracentrifugation. The supernatant consisting of phospholipids and glycoproteins of the influenza virus was reconstituted by removal of DCPC using overnight dialysis against Hank’s Buffered Saline (HBS) solution at 4°C. After dialysis, crude virosome preparation was layered over a discontinuous sucrose gradient in order to separate non-incorporated material from the reconstituted virus membranes. Results: The virosome harvested from the boundary of the two sucrose layers successfully was identified by the Hemagglutination assay and western blotting. Conclusion: Use of a dialyzable short-chain phospholipid (DCPC) is an efficient procedure for solubilization and reconstitution of influenza virus virosomes and has not caused structural changes in a major envelope glycoprotein (hemagglutinin protein) on the surface of virosome.