Liposome-based subunit and DNA hepatitis B vaccines

It has been shown that liposomes have immunological adjuvant properties and earlier evidence suggested that liposomes could play a role in vaccination against hepatitis B. The objective of this thesis is to optimize the liposomal formulations for hepatitis B vaccines and to investigate the effect of these formulations on antibody induction by hepatitis B surface antigen (HBsAg). In the present studies, the work on liposomes as a carrier for a hepatitis B vaccine was carried out and promising formulations were selected for further research. The HBsAg and plasmid pRc/CMV HBS DNA were entrapped into liposomes and these formulations were evaluated in terms of preparation procedures, characteristics, stability upon storage and in vitro release behaviours. Recombinant HBsAg and a plasmid DNA (pRc/CMV HBS) encoding for HBsAg (S region) were entrapped into liposomes by the dehydration-rehydration method yielding dehydration-rehydration vesicles (DRV). The effect of lipid composition, vesicle size, protein characteristics, routes of administration and direct or indirect immunisation were studied. To investigate the adjuvant effect of liposomes, the mice were immunised with a variety of formulations. HBsAg-containing DRVs composed of DSPC/Chol (1:1 molar ratio) elicited stronger IgG responses in mice than liposomes composed of PC/Chol (1:1 molar ratio). Furthermore, microfluidised HBsAg-containing DRVs (smaller vesicle size) did not influence the IgG responses. However, HBsAg-containing DRVs prepared in the presence of sucrose, which have a similar size to microfluidised DRVs, were found to enhance the IgG responses to HBsAg. In addition, mannosylated HBsAg changes the adjuvant effect of liposomes. Cholesterol- containing DRVs did not enhance the IgG responses to mannosylated HBsAg, but DSPC/DOPE DRVs induced stronger IgG responses in mice immunized with mannosylated HBsAg. There was improvement of immunological adjuvant properties of liposomes by the oral route by conjugating cholera toxin B (CTB) to HBsAg-containing DRVs. Furthermore, a novel method was produced to prepare the liposomal formulations of DNA by entrapping calcium phosphate-DNA complexes into the liposomes (capisomes). Capisomes incorporating pRc/CMV HBS plasmid were evaluated in mice in terms of humoral immunity to the encoding antigen. From these studies, liposomes were shown to be versatile as immunological adjuvants for protein or DNA vaccines, or as a carrier for a combination of the two types of vaccines to develop single-dose vaccines against hepatitis B. HBsAg-containing DRVs prepared in the presence of sucrose can enhance the antibody responses to HBsAg. CTB-conjugated DRVs provide an opportunity to deliver antigen orally and capisomes offer another delivery system for DNA vaccination against hepatitis B. Further studies for these formulations are recommended