Research Paper Immunogenicity of Liposomes Containing Lipid Core Peptides and the Adjuvant Quil A Karen White, 1 Thomas Rades, 1 Philip Kearns, 2 Istvan Toth, 2,3 and Sarah Hook 1,4 Received July 23, 2005; accepted February 21, 2006 Purpose. The purpose of this study was to investigate the immunogenicity of liposomes containing mannosylated lipid core peptide (manLCP) constructs, both in vitro and in vivo, with or without the addition of the immune stimulating adjuvant Quil A. Methods. Mouse bone marrow dendritic cells (BMDC) were cultured with liposome formulations for 48 h, and the resulting level of BMDC activation was determined by flow cytometry. BMDC pulsed with liposome formulations were incubated with 5,6-carboxyfluoroscein diacetate succinimidyl ester-labeled T cells for 72 h and the resulting T cell proliferation was determined by flow cytometry. To investigate the immunogenicity of formulations in vivo, groups of C57Bl/6J mice were immunized by subcutaneous injection, and the resulting antigen-specific cytotoxic and protective immune responses toward tumor challenge evaluated. Results. Despite being unable to demonstrate the activation of BMDC, BMDC pulsed with liposomes containing manLCP constructs were able to stimulate the proliferation of naı ¨ve T cells in vitro. However, in vivo only liposomes containing both manLCP and Quil A were able to stimulate a strong antigen- specific cytotoxic immune response. Liposomes containing manLCP and Quil A within the same particle were able to protect against the growth of tumor cells to a similar level as if the antigen was administered in alum with CD4 help. Conclusion. ManLCPs administered in liposomes are able to stimulate strong cytotoxic and protective immune responses if Quil A is also incorporated as an adjuvant. KEY WORDS: CD8 T cell; lipid core peptide; liposome; Quil A; vaccine. INTRODUCTION Synthetic lipid core peptide (LCP) constructs containing minimal CD8 + T cell epitopes of model proteins conjugated to a polylysine backbone have previously been investigated for their ability to stimulate strong MHC class II, CD4 + T-cell- mediated immune responses (1Y3). We have recently shown that these LCP constructs are also capable of stimulating MHC class I restricted, CD8 + T-cell-mediated immune re- sponses, but only if the constructs are administered in alum and with ovalbumin protein to provide CD4 help (4). The requirement for CD4 help in priming CD8 + T-cell immune responses is controversial with both CD4-dependent and CD4-independent CD8 + T cell activation being described (5 Y9). In general, it seems that weakly immunogenic antigens stimulate stronger CD8 cytotoxic immune responses if CD4 help is provided. Previous experiments investigating the immunogenicity of peptide and lipopeptide antigens have found that deliver- ing the antigen in a particulate delivery system is one means of increasing the resulting immune response (10,11). In gen- eral, particulate delivery systems, such as immune stimulating complexes (ISCOMs) and liposomes, more closely resemble the size of the pathogens phagocytic cells have evolved to rec- ognize, thereby enhancing antigen phagocytosis and presen- tation by specialized antigen presenting cells. In addition, ISCOMs contain the immune stimulating adjuvant Quil A or its derivatives, which have been demonstrated to be potent stimulators of both antibody- and cell-mediated immune re- sponses (12,13). Liposomes can also be modified to include additional immune stimulating molecules such as cytokines and targeting molecules that are capable of increasing the im- mune response toward entrapped protein and peptide anti- gens (14 Y18). Although ISCOMs have been shown to be capable of stimulating effective immune responses and indeed have been licensed for use in several veterinary vaccines, their potential toxicity and lipid matrix structure, which means only antigens with hydrophobic domains can be incorporated, 1473 0724-8741/06/0700-1473/0 # 2006 Springer Science + Business Media, Inc. Pharmaceutical Research, Vol. 23, No. 7, July 2006 ( # 2006) DOI: 10.1007/s11095-006-0272-z 1 School of Pharmacy, University of Otago, PO Box 913 Dunedin, New Zealand. 2 School of Pharmacy, University of Queensland, Brisbane, Australia. 3 School of Molecular and Microbial Sciences, University of Queens- land, Brisbane, Australia. 4 To whom correspondence should be addressed. (e-mail: sarah.hook@ stonebow.otago.ac.nz) ABBREVIATIONS: BMDC, bone marrow dendritic cell; DC, dendritic cell; HEPES, N-2-hydroxyethylpiperazine- N-2- ethanesulfonic acid; ISCOMs, immune stimulating complexes; LCP, lipid core peptide; manLCP, mannose-conjugated LCP; PBS, phos- phate-buffered saline; PC, L-a-phosphatidylcholine; PRR, pattern re- cognition receptor; TEM, transmission electron microscopy.