Millar, Benjamin M. G.; (1997) Studies of membrane fusion by influenza haemagglutinins, and their application to liposome delivery systems in gene therapy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of the work presented in this thesis was to investigate the membrane fusion activity of influenza haemagglutinin (HA), particularly with regard to the contribution of receptor binding to the efficiency of the membrane fusion process. Since the membrane fusion potential of HA could provide an efficient mechanism to deliver molecules to the cell cytoplasm the ability of HA—containing liposomes to fuse with membranes was also established. The role of receptor binding by HA with regard to the efficiency of membrane fusion has been extensively investigated. Liposome coupled anti-HA monoclonal Fab' fragments with various specificities towards the HA molecule were used as surrogate receptors for HA. Electron microscopy (EM) studies of HA-receptor complexes using liposome coupled Fab' fragments as surrogate receptors are reported. On the basis of results from EM, membrane fusion experiments were done between HA containing lipid vesicles (virosomes) and anti HA Fab' coupled liposomes. The virosomes used contained two antigenically distinct strains of HA, which underwent their acid induced conformational change at a significantly different pH from each other. Anti HA Fab' fragments which recognised one of the two HA strains were used as surrogate receptors. These experiments concluded that HA bound to receptor can be more efficient at causing membrane fusion than a HA molecule held close to but not directly bound by the target membrane. These results have implications for the design of a HA based delivery vector which are discussed. Various procedures were investigated for the reconstitution of HA containing lipid vesicles (virosomes), using purified HA and purified lipids. Using the detergent octaethylene glycol monododecyl ether virosomes with a lipid composition of phosphatidylcholine and cholesterol (2:1 molar ratio) were made. These virosomes could participate in HA mediated membrane fusion. Virosomes made using the detergent octylglucoside were not fusion - active. The fusion efficiency of reconstituted vesicles containing HA was shown to be lower than that of influenza virus. Initial experiments using virosomes as vehicles for DNA delivery are also presented.

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