Conjugation of Lipid and CpG-Containing Oligonucleotide Yields an Efficient Method for Liposome Incorporation

For optimal stimulation of T cells, protein-based vaccines must deliver protein antigens to antigen-presenting cells while simultaneously providing immunostimulatory signals. Listeriolysin O (LLO)-containing liposomes have been utilized to efficiently deliver protein antigens to the cytosolic pathway for antigen processing and major histocompatibility complex class I-dependent presentation while codelivering immunostimulatory CpG-oligodeoxyribonuceotides (ODNs). In this report, we describe the synthesis of lipid-CpG-ODN conjugates utilizing maleimide-phosphatidylethanolamine (PE) lipids and 5'-sulfhdryl-containing CpG-ODNs as a method for facile incorporation of CpG-ODNs in liposomal vaccine carriers, an alternative to co-encapsulation inside liposomes and as a means to enhance delivery of CpG-ODNs to their major receptor, Toll-like receptor 9 (TLR9), in the endosome. The characterization and biological evaluation of the vaccine delivery system made of liposomes, which contain the lipid-CpG-ODN conjugates inserted in the liposomal membrane, is described. We demonstrate in vitro in bone marrow derived macrophages that the lipid-CpG-ODN conjugates incorporated onto the liposome bilayers interact with their receptor TLR9 as readily as liposome-encapsulated ODNs and exert their immunostimulatory capabilities. The liposomal vaccine delivery systems were evaluated in mice using ovalbumin (OVA) as a model antigen, and the results indicate equally robust OVA-specific cytotoxic T lymphocyte responses and similar Th1 immune skewing capabilities between liposomes containing lipid-conjugated or encapsulated CpG-ODNs. Overall, this work indicates that conjugating PE lipids and CpG-ODNs results in an efficient method that allows facile incorporation of CpG-ODNs into a liposome-based delivery platform while retaining the immune-stimulating capabilities of CpG-ODNs.