Patel, Brijesh Prakash; (2004) Polymerised liposomes as intranasal vaccine adjuvants. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The purpose of this thesis was to establish whether polymerised liposomes possessed adjuvant activity following intranasal administration and whether the inclusion of poloxamers or chitosan with such a formulation further potentiated the immune response when using tetanus toxoid (TT) as a model soluble protein antigen. Early studies were based on the preparation, optimisation and characterisation of polymerised liposomes of a size appropriate for mucosal vaccination (with respect to attaining a high systemic and mucosal immune response), which was hypothesised to be between 0.3-1 ?m in diameter. Such particle sizes were achieved through use of a freeze-thaw and extrusion procedure. Liposomes were prepared from a polymerisable phospholipid that was polymerised using UV light (254 nm), the extent of which was followed by measuring the decrease in UV absorption by the conjugated diene bond at 254 nm. Polymerised liposomes were shown to possess a less negative zeta potential and to be more hydrophobic (determined using the hydrophobic dye Rose Bengal) than non-polymerised liposomes. A modified dehydration-rehydration vesicle (DRV) method was used for the post-polymerisation association of bovine serum albumin and TT to liposomes and hence avoided the exposure of the antigen to formulation processes such as UV light, which may have resulted in decreased protein immunogenicity. The effect of the association of TT to polymerised and non- polymerised liposomes using the modified DRV method was compared to polymerised liposomes with surface adsorbed antigen using in vitro release studies and also following intramuscular and intranasal administration to BALB/c mice. Polymerised liposomes were further modified by surface adsorption of poloxamer 331 (L101) and 401 (L121), chitosan chloride or chitosan glutamate for further investigation of intranasal adjuvanticity. The systematic addition of poloxamer or chitosan to liposomes was conducted to minimise vesicle aggregation and the levels of association were quantified using spectroscopic assays. Animals immunised intramuscularly with TT associated to non-polymerised DRVs or polymerised liposomes with surface adsorbed TT produced a higher mean antigen-specific serum IgG antibody response compared to animals given free protein in solution. Unexpectedly, administration of TT associated with polymerised DRVs resulted in the lowest mean antigen-specific serum IgG antibody levels. In comparison, intranasal administration of all liposomal formulations resulted in lower levels of TT-specific serum IgG titres for all formulations investigated. However, consistent with intramuscular administration studies, animals immunised with polymerised liposomes with surface adsorbed TT elicited the highest mean TT-specific serum IgG response amongst all liposomal formulations. In contrast, nasal administration of TT associated with non- polymerised DRVs showed a similar antigen-specific serum IgG response to that elicited by free TT. Further modification of polymerised liposomes by coating with L101 or L121 before surface adsorption of TT resulted in an enhancement of antigen-specific serum IgG antibody levels. Surprisingly, chitosan-coated polymerised liposomes did not enhance the systemic immune response markedly in comparison to non-coated polymerised liposomes. The high immune response elicited by L101-coated polymerised liposomes was also confirmed on analysis of cytokine production after antigen stimulation of mixed spleen cells of immunised mice, which secreted significantly higher levels (p<0.05) of IL-2, IFN-γ, IL-4, IL-6 and TNF-α than mice given L121-coated, chitosan-coated or non-coated polymerised liposomes. The results reported in this thesis further emphasise the importance of the route of administration, which affects the type of immune response elicited to a particular vaccine, possibly due to differences in antigen uptake and subsequent processing by APCs. The overall findings indicated that the surface adsorption of TT to polymerised liposomes led to the highest immune response amongst liposomal formulations following intranasal administration and was further enhanced with the addition of L101 andL121.

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