Compared to naked
DNA immunisation, entrapment of plasmid-based
DNA vaccines into
liposomes by the
dehydration-
rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded
antigens administered by a variety of routes. In this paper, we have investigated the application of
liposome-entrapped
DNA and their cationic
lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the
nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the
dehydration-
rehydration method into
liposomes composed of 16 micromol egg
phosphatidylcholine (PC), 8 micromoles
dioleoyl phosphatidylethanolamine (DOPE) or
cholesterol (Chol) and either the cationic
lipid 1,2-diodeoyl-3-(trimethylammonium)
propane (
DOTAP) or cholesteryl 3-N-(dimethyl amino ethyl)
carbamate (
DC-Chol). This method, entailing mixing of small
unilamellar vesicles (SUV) with
DNA, followed by
dehydration and
rehydration, yielded incorporation values of 90-94% of the
DNA used. Mixing or
rehydration of preformed cationic
liposomes with 100 microg plasmid
DNA also led to similarly high complexation values (92-94%). In an attempt to establish differences in the nature of
DNA association with these various
liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the
anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal
DNA by the
dehydration-
rehydration generated submicron size
liposomes incorporating most of the
DNA in a manner that prevents
DNA displacement through
anion competition. The bilayer composition of these
dehydration-
rehydration vesicles (DRV(
DNA)) can also further influence these physico-chemical characteristics with the presence of DOPE within the
liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous
liposome-mediated
DNA immunisation employing two DRV(
DNA) formulations as well as naked
DNA revealed that humoural responses (
immunoglobulin total
IgG, and subclasses
IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 microg
liposome-entrapped
DNA compared to naked
DNA. At all time points measured, mice immunised with naked
DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with
DNA entrapped in DRV
liposomes containing
DOTAP compared to the control group and mice immunised with naked
DNA. By day 56, all total
IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing
DC-Chol dropped to significantly lower levels that those measured in mice which received the
DOTAP formulation. Similar results were found when the
IgG1 and
IgG2a subclass responses were determined. These results suggest that, not only can
DNA be effectively entrapped within
liposomes using the DRV method but that such DRV
liposomes containing
DNA may be a useful system for subcutaneous delivery of
DNA vaccines.