Approaches to achieve a higher efficacy include optimising the delivery to and interaction with dendritic cells (DCs) and the addition of immune potentiators to improve the activation of these DCs. Lessons to improve the interaction with DCs can be learned from nature, as all pathogens are particulates. Particles

are better taken up by DCs and may provide an additional benefit by offering prolonged antigen delivery due to slow antigen release [2]. Liposomes are elegant and flexible nanoparticulates that have been used for a long time as JQ1 cell line drug delivery systems. Actually, when they were used for the first time in the pharmaceutical field in 1974, it was for the delivery of vaccines [3]. Since then they have been used successfully for the delivery of protein antigens [4], [5] and [6] and DNA vaccines [7] and [8]. By changing the lipid composition of liposomes, their characteristics can be varied. The usage of positively charged lipids, for instance, creates cationic liposomes. It has become clear that cationic liposomes are one of the most effective liposomal delivery systems for antigens to antigen presenting cells [9], [10], [11] and [12]. Liposomes themselves may function as an adjuvant by improving the uptake of antigens by DCs, but generally lack VE-822 order intrinsic immune-stimulatory effects [11] and [13]. By co-encapsulation

of an immune potentiator, the immunogenicity of liposomes can be improved. As classified by Schijns [14], immune potentiators until (i) interact with pattern recognition receptors (PRRs) (Signal 0) [15] and [16]; (ii) are co-stimulatory molecules necessary for activating naïve T cells (Signal 2) or (iii) act as a ‘danger-signal’ [17]. Pathogens express specific pathogen-associated molecular patterns (PAMPs) that are recognised by PRRs, of which the Toll-like receptors (TLRs) are an important subclass. All cells, but mainly antigen presenting cells such as DCs, have TLRs that recognise specific ligands. In humans 11 different TLRs have been identified, the majority of them being specific for microbial products. Most TLRs are present on

the cell surface, but TLRs that recognise nucleic acids (TLR3, 7, 8 and 9) are located intracellularly [18]. In this study we co-encapsulated a model antigen, ovalbumin (OVA) and two TLR ligands in cationic liposomes. The selected TLR ligands are Pam3CSK4, a synthetic lipoprotein consisting of a tri-palmitoyl-S-glyceryl cysteine lipopeptide with a pentapeptide SKKKK (PAM), and unmethylated CpG oligonucleotide (CpG). PAM is recognised by TLR2 in association with TLR1, both cell surface expressed receptors. CpG is a TLR9 ligand, which is expressed intracellularly. By co-encapsulation in liposomes it is ensured that both the antigen and the immune potentiator are co-delivered to the DCs, which is considered essential for induction of a strong immune response [19], [20] and [21]. To examine the effect of co-encapsulation, a comparison was made to solutions of OVA mixed with the respective TLR ligands.