Preservation of Influenza Virosome Structure and Function During Freeze-Drying and Storage

Virosomes derived from influenza virus are reconstituted viral envelopes, which retain the receptor-binding and cell entry properties of the native virus, but lack the viral genetic material. These virosomes are of interest because of their potential use as vaccines or cellular delivery systems. However, in aqueous dispersion influenza virosomes have a relatively poor stability. Although freeze-drying of the virosomes could improve their stability, a lyoprotectant is required to preserve the structure and function of the virosomes during the lyophilization process as well as during subsequent storage of the dry powder formulation. In this study, inulin, a medium-chain oligosaccharide, was identified as an effective stabilizer of influenza virosomes. When inulin was added to an aqueous virosomal dispersion, the vesicular structure of the virosomes, with spike proteins protruding from the virosomal surface, as well as their membrane fusion activity were completely preserved during freeze-drying. When the freeze-drying process was performed from dispersions lacking a lyoprotectant, both structure and fusogenic properties of the virosomes were lost. Moreover, it was shown that the immunogenicity of inulin-stabilized virosomes was preserved. For example, dry powder formulations of virosomes retained HA potency for at least 12 weeks at 20°C. Virosomes with encapsulated pDNA encoding for the eGFP reporter gene were also found to be stabilized by inulin. The fusion capacity and the transfection efficacy (determined in BHK-21 cells) could be preserved for 12 weeks during storage at 4°C. It is concluded that freeze-drying in the presence of inulin as a lyoprotectant completely preserves the structure and function of influenza virosomes.