Aim: Subunit vaccines have received increasing attention due to their good safety profile. However, subunit vaccines feature low immunogenicity, and soluble antigen is largely ignored by the immune system due to its lack of danger signals. To stimulate an appropriate immune response, subunit antigen vaccines require the addition of an adjuvant and multiple administrations. This study aimed to formulate biodegradable lipid implants, containing a suitable adjuvant, which delivers antigen in a sustained manner. The physico-chemical characteristics of the implants and their ability to stimulate immune responses towards a model antigen in vivo were investigated.
Methods: Lipid implants were prepared from phospholipid and cholesterol. Different adjuvants were added, and their potential to induce an immune response to the model antigen ovalbumin (OVA) was investigated. The adjuvants and immunomodulators assessed were Quil-A (QA), imiquimod, and an α-Galactosylceramide (α-GalCer) analogue. Liposomal dispersions were prepared using the lipid film hydration method. These were freeze-dried, and the powder compressed into matrices (diameter of 2 mm). Physico-chemical characterisation was undertaken by transmission electron microscopy (TEM) to investigate the release of colloidal structures (liposomes, immunostimulating complexes [ISCOMs]) upon hydration with release media. Surface changes of the implant matrices were analysed using scanning electron microscopy (SEM). The release of the fluorescently-labelled antigen ovalbumin (FITC-OVA) and its entrapment into the colloidal particles was investigated using spectrofluorophotometry. Additionally, incorporation of the cationic cholesterol derivative DC-cholesterol (DCCHOL) into implants to allow for charge-charge interactions with the negatively-charged OVA, and replacement of the phospholipid with a phospholipid having a higher transition temperature to facilitate the manufacturing process, were attempted and assessed. The immune response stimulated towards OVA released from the implants was analysed in vivo using a C57Bl/6 mouse model. Expansion of CD8⁺ T cells and CD8 T cells specific for the CD8 epitope of OVA (SIINFEKL), as well as expansion of CD4⁺ T cells, were assessed. The ability of implants to stimulate T cell proliferation and interferon-γ production after in vitro restimulation with OVA was analysed. Serum samples were analysed for OVA-specific IgG antibodies.
Results: Lipid implants containing Quil-A released colloidal structures upon hydration with buffer. The type of colloids observed by TEM depended on the ratio of QA:cholesterol:phospholipid. Release of OVA was sustained over ten days in implants prepared with egg yolk PC. However, the release kinetics depended strongly on the choice of phospholipid. In vivo, lipid implants containing Quil-A evoked expansion of CD8⁺ T cells. The immune response to one implant was comparable to that obtained by two equivalent injection immunisations. Therefore, the implants obviated the need for multiple immunisations in the vaccination regime tested here. Expansion of CD8⁺ T cells towards the Quil-A-containing implant was greater than that achieved by the immunomodulators imiquimod and the α-GalCer analogue. Quil-A-containing implants produced OVA-specific IgG antibodies to a greater extent than the implants containing imiquimod or α-GalCer. Incorporation of the cationic DCCHOL did not increase the entrapment efficiency of OVA into liposomes. However, the in vivo investigation of DCCHOL-containirig implants showed an adjuvant effect of DCCHOL on antibody responses, but not on cell-mediated immunity.
Conclusion: Lipid implants offer great potential as sustained release vaccine delivery systems. The lipid components in the implant formulation were well-tolerated and biodegradable. Lipid implants combine the advantages of sustained release of antigen and particulate delivery by the formation of colloidal particles.
| Identifer | oai:union.ndltd.org:ADTP/197597 |
| Date | January 2008 |
| Creators | Myschik, Julia, n/a |
| Publisher | University of Otago. School of Pharmacy |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Julia Myschik |
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