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.

Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4 roles for cd14, lps-binding protein, and md2 as targets for specificity of inhibition

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysac-charide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor- production, IB degradation, p38 MAPK phosphorylation, and NF-B-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I·C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from 30 µM. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.

Ascorbate and α-tocopherol differentially modulate reactive oxygen species generation by neutrophils in response to FcγR and TLR agonists

Periodontitis, a ubiquitous chronic inflammatory disease, is associated with reduced antioxidant defences and neutrophil hyperactivity in terms of reactive oxygen species (ROS) generation. Its phenotype is thus characterized by oxidative stress. We have determined the effect of antioxidant micronutrients ascorbate and α-tocopherol on neutrophil ROS generation. Peripheral neutrophils from periodontally-healthy individuals (n = 20) were challenged with phorbol myristate acetate, IgG-opsonised Staphylococcus aureus, Fusobacterium nucleatum or PBS in the presence and absence of micronutrients (50 μM). Total and extracellular ROS were measured by luminol and isoluminol chemiluminescence respectively. Total and extracellular unstimulated, baseline ROS generation was unaffected by α-tocopherol, but inhibited by ascorbate and a combination of both micronutrients. Fcγ-receptor (Fcγ-R)-stimulated total or extracellular ROS generation was not affected by the presence of individual micronutrients. However, the combination significantly reduced extracellular FcγR-stimulated ROS release. Neither micronutrient inhibited TLR-stimulated total ROS, but the combination caused inhibition. Ascorbate and the micronutrient combination, but not α-tocopherol, inhibited extracellular ROS release by TLR-stimulated cells. Such micronutrient effects in vivo could be beneficial in reducing collateral tissue damage in chronic inflammatory diseases, such as periodontitis, while retaining immune-mediated neutrophil function. © The Author(s) 2012 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

The role of the innate immune system in the clearance of apoptotic cells

Rapid clearance of dying cells is a vital feature of apoptosis throughout development, tissue homeostasis and resolution of inflammation. The phagocytic removal of apoptotic cells is mediated by both professional and amateur phagocytes, armed with a series of pattern recognition receptors that participate in host defence and apoptotic cell clearance. CD14 is one such molecule. It is involved in apoptotic cell clearance (known to be immunosuppressive and anti-inflammatory) and binding of the pathogen-associated molecular pattern, lipopolysaccharides (a pro-inflammatory event). Thus CD14 is involved in the assembly of two distinct ligand-dependent macrophage responses. This project sought to characterise the involvement of the innate immune system, particularly CD14, in the removal of apoptotic cells. The role of non-myeloid CD14 was also considered and the data suggests that the expression of CD14 by phagocytes may define their professional status as phagocytes. To assess if differential CD14 ligation causes the ligand-dependent divergence in macrophage responses, a series of CD14 point mutants were used to map the binding of apoptotic cells and lipopolysaccharides. Monoclonal antibodies, 61D3 and MEM18, known to interfere with ligand-binding and responses, were also mapped. Data suggests that residue 11 of CD14, is key for the binding of 61D3 (but not MEM18), LPS and apoptotic cells, indicating lipopolysaccharides and apoptotic cells bind to similar residues. Furthermore using an NF-kB reporter, results show lipopolysaccharides but not apoptotic cells stimulate NF-kB. Taken together these data suggests ligand-dependent CD14 responses occur via a mechanism that occurs downstream of CD14 ligation but upstream of NF-?B activation. Alternatively apoptotic cell ligation of CD14 may not result in any signalling event, possibly by exclusion of TLR-4, suggesting that engulfment receptors, (e.g. TIM-4, BAI1 and Stablin-2) are required to mediate the uptake of apoptotic cells and the associated anti-inflammatory response.

The effect of ageing on macrophage Toll-like receptor-mediated responses in the fight against pathogens

The cellular changes during ageing are incompletely understood yet immune system dysfunction is implicated in the age-related decline in health. The acquired immune system shows a functional decline in ability to respond to new pathogens whereas serum levels of cytokines are elevated with age. Despite these age-associated increases in circulating cytokines, the function of aged macrophages is decreased. Pathogen-associated molecular pattern receptors such as Toll-like receptors (TLRs) are vital in the response of macrophages to pathological stimuli. Here we review the evidence for defective TLR signalling in normal ageing. Gene transcription, protein expression and cell surface expression of members of the TLR family of receptors and co-effector molecules do not show a consistent age-dependent change across model systems. However, there is evidence for impaired downstream signalling events, including inhibition of positive and activation of negative modulators of TLR induced signalling events. In this paper we hypothesize that despite a poor inflammatory response via TLR activation, the ineffective clearance of pathogens by macrophages increases the duration of their activation and contributes to perpetuation of inflammatory responses and ageing.

Liposomes:Formulation and characterisation as contrast agents and as vaccine delivery systems

Liposome systems are well reported for their activity as vaccine adjuvants; however novel lipid-based microbubbles have also been reported to enhance the targeting of antigens into dendritic cells (DCs) in cancer immunotherapy (Suzuki et al 2009). This research initially focused on the formulation of gas-filled lipid coated microbubbles and their potential activation of macrophages using in vitro models. Further studies in the thesis concentrated on aqueous-filled liposomes as vaccine delivery systems. Initial work involved formulating and characterising four different methods of producing lipid-coated microbubbles (sometimes referred to as gas-filled liposomes), by homogenisation, sonication, a gas-releasing chemical reaction and agitation/pressurisation in terms of stability and physico-chemical characteristics. Two of the preparations were tested as pressure probes in MRI studies. The first preparation composed of a standard phospholipid (DSPC) filled with air or nitrogen (N2), whilst in the second method the microbubbles were composed of a fluorinated phospholipid (F-GPC) filled with a fluorocarbon saturated gas. The studies showed that whilst maintaining high sensitivity, a novel contrast agent which allows stable MRI measurements of fluid pressure over time, could be produced using lipid-coated microbubbles. The F-GPC microbubbles were found to withstand pressures up to 2.6 bar with minimal damage as opposed to the DSPC microbubbles, which were damaged at above 1.3 bar. However, it was also found that DSPC-filled with N2 microbubbles were also extremely robust to pressure and their performance was similar to that of F-GPC based microbubbles. Following on from the MRI studies, the DSPC-air and N2 filled lipid-based microbubbles were assessed for their potential activation of macrophages using in vitro models and compared to equivalent aqueous-filled liposomes. The microbubble formulations did not stimulate macrophage uptake, so studies thereafter focused on aqueous-filled liposomes. Further studies concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyldioctadecylammonium (DDA) and immunomodulatory trehalose dibehenate (TDB) with the addition of polyethylene glycol (PEG). One of the proposed hypotheses for the mechanism behind the immunostimulatory effect obtained with DDA:TDB is the ‘depot effect’ in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. The depot effect has been suggested to be primarily due to their cationic nature. Results reported within this thesis demonstrate that higher levels of PEG i.e. 25 % were able to significantly inhibit the formation of a liposome depot at the injection site and also severely limit the retention of antigen at the site. This therefore resulted in a faster drainage of the liposomes from the site of injection. The versatility of cationic liposomes based on DDA:TDB in combination with different immunostimulatory ligands including, polyinosinic-polycytidylic acid (poly (I:C), TLR 3 ligand), and CpG (TLR 9 ligand) either entrapped within the vesicles or adsorbed onto the liposome surface was investigated for immunogenic capacity as vaccine adjuvants. Small unilamellar (SUV) DDA:TDB vesicles (20-100 nm native size) with protein antigen adsorbed to the vesicle surface were the most potent in inducing both T cell (7-fold increase) and antibody (up to 2 log increase) antigen specific responses. The addition of TLR agonists poly(I:C) and CpG to SUV liposomes had small or no effect on their adjuvanticity. Finally, threitol ceramide (ThrCer), a new mmunostimulatory agent, was incorporated into the bilayers of liposomes composed of DDA or DSPC to investigate the uptake of ThrCer, by dendritic cells (DCs), and presentation on CD1d molecules to invariant natural killer T cells. These systems were prepared both as multilamellar vesicles (MLV) and Small unilamellar (SUV). It was demonstrated that the IFN-g secretion was higher for DDA SUV liposome formulation (p<0.05), suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

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.

Nanotechnology for the delivery of vaccines

Liposomes offer an ideal platform for the delivery of subunit vaccines, due to their versatility and flexibility, which allows for antigen as well as immunostimulatory lipids and TLR agonists to become associated with these bilayered vesicles. Liposomes have the ability to protect vaccine antigen, as well as enhance delivery to antigen presenting cells, whilst the importance of cationic surface charge for delivery of TB subunit vaccines and formation of an ‘antigen depot’ may play a key role in boosting cell-mediated immunity and Th1 immune responses. The rational design of vaccine adjuvants requires the thorough investigation into the physicochemical characteristics that dictate the function of a liposomal adjuvant. Within this thesis, physicochemical characteristics were investigated in order to show any effects on the biodistribution profiles and the ensuing immune responses of these formulations. Initially the role of liposome charge within the formulation was investigated and subsequently their efficacy as vaccine adjuvants in combination with their biodistribution was measured to allow the role of formulation in vaccine function to be considered. These results showed that cationic surface charge, in combination with high loading of H56 vaccine antigen through electrostatic binding, was crucial in the promotion of the ‘depot-effect’ at the injection site which increases the initiation of Th1 cell-mediated immune responses that are required to offer protection against tuberculosis. To further investigate this, different methods of liposome production were also investigated where antigen incorporation within the vesicles as well as surface adsorption were adopted. Using the dehydration-rehydration (DRV) method (where liposomes are freeze-dried in the presence of antigen to promote antigen encapsulation) and the double emulsion (DE) method, a range of liposomes entrapping antigen were formulated. Variation in the liposome preparation method can lead to antigen entrapment within the delivery system which has been shown to be greater for DRV-formulated liposomes compared to their DE-counterparts. This resulted in no significant effect on the vaccine biodistribution profile, as well as not significantly altering the efficacy of cationic liposomal adjuvants. To further enhance the efficacy of these systems, the addition of TLR agonists either at the vesicle surface as well as within the delivery system has been displayed through variation in the preparation method. Anionic liposomal adjuvants have been formulated, which displayed rapid drainage from the injection site to the draining lymph nodes and displayed a reduction in measured Th1 immune responses. However, variation in the preparation method can alter the immune response profile for anionic liposomal adjuvants with a bias in immune response to Th2 responses being noted. Through the use of high shear mixing and stepwise incorporation, the efficient loading of TLR agonist within liposomes has been shown. However, interestingly the conjugation between lipid and non-electrostatically bound TLR agonist, followed by insertion into the bilayer of DDA/TDB resulted in localised agonist retention at the injection site and further stimulation of the Th1 immune response at the SOI, spleen and draining lymphatics as well as enhanced antibody titres.

25-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6 signalling in human macrophages

Recent studies have shown that Toll-like receptor (TLR)- signalling contributes significantly to the inflammatory events of atherosclerosis. As products of cholesterol oxidation (oxysterols) accumulate within atherosclerotic plaque and have been proposed to contribute to inflammatory signalling in the diseased artery, we investigated the potential of 7-ketocholesterol (7-KC), 7β-hydroxycholesterol (7β-HC) and 25-hydroxycholesterol (25-HC) to stimulate inflammatory signalling via the lipid-recognising TLRs 1, 2, 4 and 6. Each oxysterol stimulated secretion of the inflammatory chemokine interleukin-8 (IL-8), but not I?B degradation or tumour necrosis factor- release from monocytic THP-1 cells. Transfection of TLR-deficient HEK-293 cells with TLRs 1, 2, 4 or 6 did not increase sensitivity to the tested oxysterols. Moreover, blockade of TLR2 or TLR4 with specific inhibitors did not reduce 25-hydroxycholesterol (25-HC) induced IL-8 release from THP-1 cells. We conclude that although the oxysterols examined in this study may contribute to increased expression of certain inflammatory genes, this occurs by mechanisms independent of TLR signalling.

Oxidised phospholipid regulation of toll-like receptor signalling

Toll-like receptors (TLRs) serve to initiate inflammatory signalling in response to the detection of conserved microbial molecules or products of host tissue damage. Recent evidence suggests that TLR-signalling plays a considerable role in a number of inflammatory diseases, including atherosclerosis and arthritis. Agents which modulate TLR-signalling are, therefore, receiving interest in terms of their potential to modify inflammatory disease processes. One such family of molecules, the oxidised phospholipids (OxPLs), which are formed as a result of inflammatory events and accumulate at sites of chronic inflammation, have been shown to modulate TLR-signalling in both in vitro and in vivo systems. As the interaction between OxPLs and TLRs may play a significant role in chronic inflammatory disease processes, consideration is given in this review to the potential role of OxPLs in the regulation of TLR-signalling.

Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via toll-like receptor 2

To determine whether non-enterobacterial endotoxins, which are likely to constitute the majority of the circulating endotoxin pool, may stimulate coronary artery endothelial cell activation. Interleukin-8 secretion, monocyte adhesion, and E-selectin expression were measured in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) challenged in vitro with highly purified endotoxins of common host colonisers Escherichia coli, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Bacteroides fragilis. HCAECs but not HUVECs expressed Toll-like receptor (TLR)-2 and were responsive to non-enterobacterial endotoxins. Transfection of TLR-deficient HEK-293 cells with TLR2 or TLR4/MD2 revealed that while E. coli endotoxin utilised solely TLR4 to signal, the endotoxins, deglycosylated endotoxins (lipid-A), and whole heat-killed bacteria of the other species stimulated TLR2-but not TLR4-dependent cell-signalling. Blockade of TLR2 with neutralizing antibody prevented HCAEC activation by non-enterobacterial endotoxins. Comparison of each endotoxin with E. coli endotoxin in limulus amoebocyte lysate assay revealed that the non-enterobacterial endotoxins are greatly underestimated by this assay, which has been used in all previous studies to estimate plasma endotoxin concentrations. Circulating non-enterobacterial endotoxins may be an underestimated contributor to endothelial activation and atherosclerosis in individuals at risk of increased plasma endotoxin burden.

Toll-like receptor 4 signalling is neither sufficient nor required for oxidised phospholipids mediated induction of interleukin-8 expression

Toll-like receptor (TLR)-4 signalling has been shown to accelerate atherosclerosis. As oxidised phospholipids are present in atherosclerotic plaque and have been shown to modulate TLR4 signalling, we investigated the role of oxidised 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC) in the regulation of TLR 1, 2, 4 and 6 signalling. Unlike established TLR agonists, OxPAPC did not induce NF-?B-dependent gene expression in monocytic THP-1 cells, human aortic endothelial cells or TLR-deficient HEK-293 cells transfected with TLRs 1, 2, 4 or 6. OxPAPC induction of IL-8 was not blocked by the TLR4 specific antagonist Rhodobacter sphaeroides LPS in human aortic endothelial cells, though OxPAPC potently inhibited TLR4 mediated IL-8 induction in these cells. OxPAPC upregulated IL-8 production in TLR4 deficient HEK-293 cells and this was not increased following TLR4 overexpression. Lipids extracted from carotid atherectomy samples did not stimulate TLR 1, 2, 4 or 6 signalling in a HEK-293 transfection assay. TLR4 signalling does not contribute to OxPAPC induced IL-8 expression in human epithelial HEK-293, monocytic THP-1 or aortic endothelial cells. As lipids extracted from diseased human artery also induced no TLR signalling, it is likely that the TLR-activating materials contributing to atherosclerosis are not of endogenous lipid origin.

Phosphatidylserine-containing liposomes:modulators of rhinovirus induced inflammatory responses

Background: Human rhinoviral infections are major contributors to the healthcare burden associated with acute exacerbations of asthma. We, and others have recently demonstrated that rhinovirus (RV)-induced inflammatory responses are mediated by multiple signalling mechanisms, such as IL-1/MyD88 (1) and TLR3/RIGI (2). We have also previously published work showing that TLR signalling is effectively inhibited by phosphatidylserine-containing liposomes (SAPS), through the disruption of membrane microdomains (3). Evidence has also suggested that membrane microdomains may influence infections with RV. In this study, we explored the ability of SAPS to modulate responses to the natural viral pathogens, RV-1B and RV-16. Method: The immortalized bronchial epithelial cell line, BEAS-2B or primary bronchial epithelial cells were infected with RV-1B or RV-16 at a TCID50/ml of 19107 for 1 h. Immediately following infection, various concentrations of SAPS were added and changes in cytokine release were measured at 24 h. SAPS remained present throughout. Type I and III interferon (IFN) expression and rates of viral replication were measured by quantitative PCR. Virus quantification was also performed using a viral CPE assay, and IFN signalling was measured by western blot. Liposome stability was characterised and intracellular trafficking of fluorescently labelled SAPS in BEAS-2B cells was investigated using confocal microscopy. For in vivo studies, female wt Balb/c mice were pre-treated with SAPS for 2 h prior to infection with RV as previously described and changes in BAL cell number, BAL cytokine production and viral replication were quantified (4). Results: Characterisation of SAPS liposomes by mass spectrometry showed no obvious signs of oxidation over the time period tested, and liposome size remained constant. Preliminary confocal studies revealed that SAPS was rapidly internalised within the cell and was found to associate with intracellular compartments such as the early endosome and golgi. Viral infected BEAS-2B cells co-incubated with SAPS, showed notably impaired responses to RV as assessed by release of CXCL8 and CCL5. SAPS also reduced RV-induced IFNb production and STAT-1 phosphorylation, without significantly influencing viral replication rates. Modest increases in viral particle production were only observed at 48 and 72 h time points. Suppression of viral-induced cytokine production was also observed in primary bronchial epithelial cells and pilot in vivo studies showed that SAPS results in reduced KC production at 24 h post viral infection, and this was associated with reduced neutrophil numbers within the BAL fluid. Conclusion: Our data demonstrates a potential means of modulating inflammatory responses induced by human rhinovirus.