Liposome-mediated antigen delivery: formulation and optimisation
Live conventional vaccines are generally effective at provoking protective immunity against the infectious
agent. However, there are many disadvantages regarding their adverse side effects and overall safety profile.
Alternative vaccine strategies such as subunit and plasmid-based vaccines, using recombinant technology, are
much safer, yet less effective. Therefore, the immunogenicity of such vaccines could be enhanced by utilising
delivery and/or adjuvant systems, to provoke the appropriate immune responses.
The role of liposomal systems within plasmid-based delivery was examined, looking at the effects of varying
liposomal composition and method of preparation on the physical characteristics, transfection efficiency in
vitro and immunogenicity in vivo. Compared to naked DNA alone, entrapment of plasmid DNA within
liposomal vesicles results in complete protection from degradation by intracellular enzymes, with the DNA
maintaining full structure and function. For liposome-mediated gene delivery small cationic lipids have been
shown to be potent candidates, acquiring a net positive charge, which effectively interact with the anionic
charges of the DNA to generate high incorporation values. In contrast, neutral liposomes are much larger
aggregated structures with lower incorporation of DNA. The method of preparation was shown to effect the
spatial location of plasmid DNA to liposomal systems. The dehydration-rehydration procedure (DRV) carried
out in the presence of DNA effectively entraps the plasmid with little effect on liposome size and surface
charge. Alternatively, upon addition of plasmid DNA the measured vesicle size of small unilamellar vesicles
(SUV) or 'empty' (water containing) DRV increases due to the formation of SUV-DNA or DRV-DNA
complexes, with the majority of the DNA localised on the surface of the liposomes. When applied in vitro,
transfection efficiency of SUV-DNA complexes was greater than DRV(DNA). Transfection efficiency of SUVDNA
complexes varied depending on the cationic lipid present within the lipid bilayer, with DC-Chol showing
most efficiency. Furthermore, these DC-Chol cationic liposomes were formulated in combination with two
different 'helper' lipids, the fusogenic lipid dioleoyl phosphatidylethanolamine (DOPE) or the stabilising lipid
Cho!. The manner in which complexes form, the resultant structure and their transfection efficiency in vitro
varied depending on the combining effects of both the type of 'helper' lipid incorporated within the lipid
bilayer and total lipid to DNA charge ratio, with the overall structural size playing a significant role in
promoting transfection. Transfection efficiency in vitro was significantly reduced when complexes were
stabilised by the inclusion of phosphatidylcholines, with both the phospholipid head group and the alkyl-chain
length influencing transfection efficiency. The production of DRV vesicles incorporating DNA were also
produced in the range of IOO-200nm by the addition of a disaccharide (i.e. sucrose), prior to freeze-drying
during the dehydration-rehydration procedure. In this instance, with an increase in sucrose/lipid mass ratio, the
z-average diameter of liposomes decreased, while the percentage plasmid DNA, pRc/CMV HBS, entrapment
remained relatively high (92%). Despite this, these small DRV(DNA) were found to be poor transfecting
agents in vitro.
After an initial screening process in vitro, a select few liposomal systems were subcutaneously administered in
vivo. For all the liposomal formulations tested there was no induction of a humoral immune response, as no
antibody titres were detected against the encoded antigen. However, SUV-DNA complexes composed of
PC:Choi:DC-Chol (16:8:4 Ilmole/ml) and the production of small modified DRV(DNA) by the addition of
sucrose generated sufficiently high levels of cell-mediated immunity.
With regard to protein antigen delivery and adjuvanticity, the association with liposomal systems significantly
enhances the immunogenicity of the fusion protein, Ag8SB-ESAT -6, a promising tuberculosis vaccine antigen.
Several factors were shown to influence adjuvanticity of these liposomal systems. For example, the inclusion of
the immunomodulator, TDB, effectively enhanced immunity against tuberculosis by increasing the
adjuvanticity of these liposomal systems. Such immune responses were prolonged and most effective when
these liposomal systems were either neutral or possessed a net positive charge rather than a negative charge and
when the protein antigen was entrapped within these vesicles rather than surface complexed. Therefore, the
overall protection against infection by tuberculosis was enhanced, presumably as a result of these liposomes
forming depots, whereby the protein antigen is released slowly and at a controlled rate, maintaining therapeutic
levels of the antigen in vivo to exert its therapeutic effect.