Liposome-Coupled Antigens Are Internalized by Antigen-Presenting Cells via Pinocytosis and Cross-Presented to CD8+ T Cells

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen-presenting cells (APCs) to CD8+ T cells, and that this process resulted in the induction of antigen-specific cytotoxic T lymphocytes. In the present study, the mechanism by which the liposome-coupled antigens were cross-presented to CD8+ T cells by APCs was investigated. Confocal laser scanning microscopic analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-based liposomes received processing at both MHC class I and class II compartments, while most of the antigens coupled to the surface of saturated-fatty-acid-based liposomes received processing at the class II compartment. In addition, flow cytometric analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-liposomes were taken up by APCs even in a 4°C environment; this was not true of saturated-fatty-acid-liposomes. When two kinds of inhibitors, dimethylamiloride (DMA) and cytochalasin B, which inhibit pinocytosis and phagocytosis by APCs, respectively, were added to the culture of APCs prior to the antigen pulse, DMA but not cytochalasin B significantly reduced uptake of liposome-coupled antigens. Further analysis of intracellular trafficking of liposomal antigens using confocal laser scanning microscopy revealed that a portion of liposome-coupled antigens taken up by APCs were delivered to the lysosome compartment. In agreement with the reduction of antigen uptake by APCs, antigen presentation by APCs was significantly inhibited by DMA, and resulted in the reduction of IFN-γ production by antigen-specific CD8+ T cells. These results suggest that antigens coupled to the surface of liposomes consisting of unsaturated fatty acids might be pinocytosed by APCs, loaded onto the class I MHC processing pathway, and presented to CD8+ T cells. Thus, these liposome-coupled antigens are expected to be applicable for the development of vaccines that induce cellular immunity

Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine

Liposomes are versatile (sub)micron-sized membrane vesicles that can be used for a variety of applications, including drug delivery and in vivo imaging but they also represent excellent models for artificial membranes or cells. Several studies have demonstrated that in vitro transcription and translation can take place inside liposomes to obtain compartmentalized production of functional proteins within the liposomes (Kita et al. in Chembiochem 9(15):2403–2410, 2008; Moritani et al.in FEBS J, 2010; Kuruma et al. in Methods Mol Biol 607:161–171, 2010; Murtas et al. in Biochem Biophys Res Commun 363(1):12–17, 2007; Sunami et al. in Anal Biochem 357(1):128–136, 2006; Ishikawa et al. in FEBS Lett 576(3):387–390, 2004; Oberholzer et al. in Biochem Biophys Res Commun 261(2):238–241, 1999). Such a minimal artificial cell-based model is ideal for synthetic biology based applications. In this study, we propose the use of liposomes as artificial microbes for vaccination. These artificial microbes can be genetically programmed to produce specific antigens at will. To show proof-of-concept for this artificial cell-based platform, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen β-galactosidase were entrapped inside multilamellar liposomes. Vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (AnExILs) elicited higher specific humoral immune responses against the produced antigen (β-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal β-galactosidase or pDNA encoding β-galactosidase). In conclusion, AnExILs present a new platform for DNA-based vaccines which combines antigen production, adjuvanticity and delivery in one system and which offer several advantages over existing vaccine formulations

Time-dependent failure in load-bearing polymers: a potential hazard in structural applications of polylactides

With their excellent biocompatibility and relatively high mechanical strength, polylactides are attractive candidates for application in load-bearing, resorbable implants. Pre-clinical studies provided a proof of principle for polylactide cages as temporary constructs to facilitate spinal fusion, and several cages already made it to the market. However, also failures have been reported: clinical studies reported considerable amounts of subsidence with lumbar spinal fusion cages, and in an in vivo goat study, polylactide spinal cages failed after only three months of implantation, although mechanical testing had predicted sufficient strength for at least eight months. The failures appear to be related to the long-term performance of polylactides under static loading conditions, a phenomenon which is common to all glassy polymers and finds its origin in stress-activated molecular mobility leading to plastic flow. This paper reviews the mechanical properties and deformation kinetics of amorphous polylactides. Compression tests were performed with various strain rates, and static stress experiments were done to determine time-to failure. Pure PLLA appeared to have a higher yield strength than its co-polymers with d-lactide, but the kinetic behaviour of the polymers was the same: an excellent short-term strength at higher loading rates, but lifetime under static stress is rather poor. As spinal implants need to maintain mechanical integrity for a period of at least six months, this has serious implications for the clinical application of amorphous polylactides in load bearing situations. It is recommended that standards for mechanical testing of implants made of polymers be revised in order to consider this typical time-dependent behaviour

Cross-Protective Peptide Vaccine against Influenza A Viruses Developed in HLA-A*2402 Human Immunity Model

Background: The virus-specific cytotoxic T lymphocyte (CTL) induction is an important target for the development of a broadly protective human influenza vaccine, since most CTL epitopes are found on internal viral proteins and relatively conserved. In this study, the possibility of developing a strain/subtype-independent human influenza vaccine was explored by taking a bioinformatics approach to establish an immunogenic HLA-A24 restricted CTL epitope screening system in HLAtransgenic mice. Methodology/Principal Findings: HLA-A24 restricted CTL epitope peptides derived from internal proteins of the H5N1 highly pathogenic avian influenza A virus were predicted by CTL epitope peptide prediction programs. Of 35 predicted peptides, six peptides exhibited remarkable cytotoxic activity in vivo. More than half of the mice which were subcutaneously vaccinated with the three most immunogenic and highly conserved epitopes among three different influenza A virus subtypes (H1N1, H3N2 and H5N1) survived lethal influenza virus challenge during both effector and memory CTL phases. Furthermore, mice that were intranasally vaccinated with these peptides remained free of clinical signs after lethal virus challenge during the effector phase. Conclusions/Significance: This CTL epitope peptide selection system can be used as an effective tool for the development of a cross-protective human influenza vaccine. Furthermore this vaccine strategy can be applicable to the development o

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 IFNγ 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