Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes--including size, antigen association and addition of TLR agonists--to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFN? responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.

Vascular cell responsiveness to toll-like receptor ligands in carotid atheroma

Background Atherosclerosis is potentiated by stimulation of Toll-like receptors (TLRs), which serve to detect pathogen associated molecular patterns (PAMPs). However little is known of which PAMPs may be present in atheroma, or capable of stimulating inflammatory signalling in vascular cells. Materials and Methods DNA extracted from human carotid atheroma samples was amplified and sequenced using broad-range 16S gene specific primers to establish historical exposure to bacterial PAMPs. Responsiveness of primary human arterial and venous endothelial and smooth muscle cells to PAMPs specific for each of the TLRs was assessed by measurement of interleukin-8 secretion and E-selectin expression. Results Extracts of atheromatous tissue stimulated little or no signalling in TLR-transfected HEK-293 cells. However, sequencing of bacterial DNA amplified from carotid atheroma revealed the presence of DNA from 17 different bacterial genera, suggesting historical exposure to bacterial lipopeptide, lipopolysaccharide and flagellin. All cells examined were responsive to the ligands of TLR3 and TLR4, poly inosine:cytosine and lipopolysaccharide. Arterial cells were responsive to a wider range of PAMPs than venous cells, being additionally responsive to bacterial flagellin and unmethylated cytosine-phosphate-guanosine DNA motifs, the ligands of TLR5 and TLR9, respectively. Cells were generally unresponsive towards the ligands of human TLR7 and TLR8, loxoribine and single stranded RNA. Only coronary artery endothelial cells expressed TLR2 mRNA and responded to the TLR2 ligand Pam3CSK4. Conclusions Vascular cells are responsive to a relatively diverse range of TLR ligands and may be exposed, at least transiently, to ligands of TLR2, TLR4, TLR5 and TLR9 during the development of carotid atheroma.