Synthetic archaeosome vaccines containing triglycosylarchaeols can provide additive and long-lasting immune responses that are enhanced by archaetidylserine.

The relation between archaeal lipid structures and their activity as adjuvants may be defined and explored by synthesizing novel head groups covalently linked to archaeol (2,3-diphytanyl-sn-glycerol). Saturated archaeol, that is suitably stable as a precursor for chemical synthesis, was obtained in high yield from Halobacterium salinarum. Archaeosomes consisting of the various combinations of synthesized lipids, with antigen entrapped, were used to immunize mice and subsequently determine CD8(+) and CD4(+)-T cell immune responses.

The olsA gene mediates the synthesis of an ornithine lipid in Pseudomonas aeruginosa during growth under phosphate-limiting conditions, but is not involved in antimicrobial peptide susceptibility.

Pseudomonas aeruginosa responds to phosphate limitation by inducing the expression of phosphate transport systems, phosphatases, hemolysins and a DNase, many of which are important for virulence. Here we report that under phosphate-limiting conditions, P. aeruginosa produces a phosphate-free ornithine lipid (OL) as the primary membrane lipid.

Archaeosome adjuvant overcomes tolerance to tumor-associated melanoma antigens inducing protective CD8 T cell responses.

Vesicles comprised of the ether glycerolipids of the archaeon Methanobrevibacter smithii (archaeosomes) are potent adjuvants for evoking CD8(+) T cell responses. We therefore explored the ability of archaeosomes to overcome immunologic tolerance to self-antigens. Priming and boosting of mice with archaeosome-antigen evoked comparable CD8(+) T cell response and tumor protection to an alternate boosting strategy utilizing live bacterial vectors for antigen delivery.

Isopranoid- and dipalmitoyl-aminophospholipid adjuvants impact differently on longevity of CTL immune responses.

The success of lipid membranes as cytotoxic T-cell (CTL) adjuvants requires targeted uptake by antigen-presenting cells (APCs) and delivery of the antigen cargo to the cytosol for processing. To target the phosphatidylserine (PS) receptor of APCs, we prepared antigen-loaded liposomes containing dipalmitoylphosphatidylserine and archaeal lipid liposomes (archaeosomes), containing an equivalent amount of archaetidylserine, and compared their ability to promote short and long-term CTL activity in animals. CTL responses were enhanced by the incorporation of PS into phosphatidylcholine/cholesterol liposomes and, to a lesser extent, into phosphatidylglycerol/cholesterol liposomes, that correlated to the amount of surface amino groups reactive with trinitrobenzoyl sulfonate.

Development of new glycosylation methodologies for the synthesis of archaeal-derived glycolipid adjuvants.

To commercialize the production of glycolipid adjuvants, their synthesis needs to be both robust and inexpensive. Herein we describe a semi-synthetic approach where the lipid acceptor is derived from the biomass of the archaeon Halobacterium salinarum, and the glycosyl donors are chemically synthesized. This work presents some preliminary results using the promoter system N-iodosuccinimide (NIS) and a stable 0.

Glycosidase-induced fusion of isoprenoid gentiobiosyl lipid membranes at acidic pH.

A difficulty in explaining the mechanism whereby archaeal lipid membrane vesicles (archaeosomes) deliver entrapped protein antigens to the MHC class I cytosolic pathway from phagolysosomes of antigen-presenting cells has been the observation that they tend not to fuse. Here, we determine that archaeosomes, composed of archaeal isoprenoid mixtures of glyco and phospholipids, can be highly fusogenic when exposed to the pH and enzymes found in late phagolysosomes. Fusions were strictly dependent on acidic pH and the presence of alpha- or beta-glucosidase.

Synthesis of archaeal glycolipid adjuvants-what is the optimum number of sugars?

As part of a programme to optimize the use of archaeal-lipid liposomes (archaeosomes) as vaccine adjuvants, we present the synthesis and immunological testing of an oligomeric series of mannose glycolipids (Manp(1-5)). To generate the parent archaeol alcohol precursor, the polar lipids extracted from the archaeon Halobacterium salinarum were hydrolyzed to remove polar head groups, and the archaeol so generated partitioned into diethyl ether. This alcohol was then iteratively glycosylated with the donor 2-O-acetyl-3,4,6-tri-O-benzyl-alpha/beta-d-mannopyranosyl trichloroacetimidate to yield alpha-Manp-(1-->2) oligomers.

Archaeosome adjuvants: immunological capabilities and mechanism(s) of action.

Archaeosomes (liposomes comprised of glycerolipids of Archaea) constitute potent adjuvants for the induction of Th1, Th2 and CD8(+) T cell responses to the entrapped soluble antigen. Archaeal lipids are uniquely constituted of ether-linked isoprenoid phytanyl cores conferring stability to the membranes. Additionally, varied head groups displayed on the glycerol-lipid cores facilitate unique immunostimulating interactions with mammalian antigen-presenting cells (APCs).

Rapid clonal expansion and prolonged maintenance of memory CD8+ T cells of the effector (CD44highCD62Llow) and central (CD44highCD62Lhigh) phenotype by an archaeosome adjuvant independent of TLR2.

Vaccines capable of eliciting long-term T cell immunity are required for combating many diseases. Live vectors can be unsafe whereas subunit vaccines often lack potency. We previously reported induction of CD8(+) T cells to Ag entrapped in archaeal glycerolipid vesicles (archaeosomes).

Identification of sulfoquinovosyl diacylglycerol as a major polar lipid in Marinococcus halophilus and Salinicoccus hispanicus and substitution with phosphatidylglycerol.

The sulfonolipid sulfoquinovosyl diacylglycerol normally associated with photosynthetic membranes was identified as a major lipid in Marinococcus halophilus, Salinicoccus hispanicus ("Marinococcus hispanicus"), and Marinococcus sp. H8 (Planococcus sp. H8).

Salt tolerance of archaeal extremely halophilic lipid membranes.

The membranes of extremely halophilic Archaea are characterized by the abundance of a diacidic phospholipid, archaetidylglycerol methylphosphate (PGP-Me), which accounts for 50-80 mol% of the polar lipids, and by the absence of phospholipids with choline, ethanolamine, inositol, and serine head groups. These membranes are stable in concentrated 3-5 m NaCl solutions, whereas membranes of non-halophilic Archaea, which do not contain PGP-Me, are unstable and leaky under such conditions. By x-ray diffraction and vesicle permeability measurements, we demonstrate that PGP-Me contributes in an essential way to membrane stability in hypersaline environments.

Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen.

Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M.

Activation of dendritic cells by liposomes prepared from phosphatidylinositol mannosides from Mycobacterium bovis bacillus Calmette-Guerin and adjuvant activity in vivo.

Liposome vesicles could be formed at 65 degrees C from the chloroform-soluble, total polar lipids (TPL) extracted from Mycobacterium bovis bacillus Calmette-Guérin (BCG). Mice immunized with ovalbumin (OVA) entrapped in TPL liposomes produced both anti-OVA antibody and cytotoxic T lymphocyte responses. Murine bone marrow-derived dendritic cells were activated to secrete interleukin-6 (IL-6), IL-12, and tumor necrosis factor upon exposure to antigen-free TPL liposomes.

Phosphatidylserine receptor-mediated recognition of archaeosome adjuvant promotes endocytosis and MHC class I cross-presentation of the entrapped antigen by phagosome-to-cytosol transport and classical processing.

Archaeal isopranoid glycerolipid vesicles (archaeosomes) serve as strong adjuvants for cell-mediated responses to entrapped Ag. We analyzed the processing pathway of OVA entrapped in archaeosomes composed of Methanobrevibacter smithii lipids, high in archaetidylserine (OVA-archaeosomes). In vitro, OVA-archaeosomes stimulated spleen cells from OVA-TCR-transgenic mice, D011.

Liposome adjuvants prepared from the total polar lipids of Haloferax volcanii, Planococcus spp. and Bacillus firmus differ in ability to elicit and sustain immune responses.

Immune stimulating activity was compared for lipid vesicles consisting of the total polar lipids of an archaeon Haloferax volcanii, and the eubacteria Planococcus spp. and Bacillus firmus. Each total polar lipid extract readily formed liposomes of similar size, within which the protein antigen ovalbumin was entrapped, with comparable loading and internalization.

Novel polar lipids of halophilic eubacterium Planococcus H8 and archaeon Haloferax volcanii.

As part of a study to identify novel lipids with immune adjuvant activity, a structural comparison was made between the polar lipids from two halophiles, an archaeon Haloferax volcanii and a eubacterium Planococcus H8. H. volcanii polar lipid extracts consisted of 44% archaetidylglycerol methylphosphate, 35% archaetidylglycerol, 4.

Archaeosomes induce enhanced cytotoxic T lymphocyte responses to entrapped soluble protein in the absence of interleukin 12 and protect against tumor challenge.

Archaeosome adjuvants formulated as archaeal ether glycerolipid vesicles induce strong CD4(+) as well as CD8(+) CTL responses to entrapped soluble antigens. Immunization of mice with ovalbumin (OVA) entrapped in archaeosomes composed of the total polar lipids of Methanobrevibacter smithii resulted in a potent OVA-specific CD8(+) T-cell response, and subsequently, the mice dramatically resisted solid tumor growth of OVA-expressing EG.7 cells and lung metastasis of B16OVA melanoma cells.

Safety of archaeosome adjuvants evaluated in a mouse model.

Archaeosomes, liposomes prepared from the polar ether lipids extracted from Archaea, demonstrate great potential as immunomodulating carriers of soluble antigens, promoting humoral and cell mediated immunity in the vaccinated host. The safety of unilamellar archaeosomes prepared from the total polar lipids (TPL) of Halobacterium salinarum, Methanobrevibacter smithii or Thermoplasma acidophilum was evaluated in female BALB/c mice using ovalbumin (OVA) as the model antigen. Groups of 6-8 mice were injected (0.

Molecular mechanisms of water and solute transport across archaebacterial lipid membranes.

Archaebacteria thrive in environments characterized by anaeobiosis, saturated salt, and both high and low extremes of temperature and pH. The bulk of their membrane lipids are polar, characterized by the archaeal structural features typified by ether linkage of the glycerol backbone to isoprenoid chains of constant length, often fully saturated, and with sn-2,3 stereochemistry opposite that of glycerolipids of Bacteria and Eukarya. Also unique to these bacteria are macrocyclic archaeol and membrane spanning caldarchaeol lipids that are found in some extreme thermophiles and methanogens.

Immunization of mice with lipopeptide antigens encapsulated in novel liposomes prepared from the polar lipids of various Archaeobacteria elicits rapid and prolonged specific protective immunity against infection with the facultative intracellular pathogen, Listeria monocytogenes.

Protective immunity to intracellular bacterial pathogens usually requires the participation of specific CD8+ T cells. Natural exposure of the host to sublethal infection, or vaccination with attenuated live vaccines are the most effective means of eliciting prolonged protective cell-mediated immunity against this class of pathogens. The ability to replace these immunization strategies with defined sub-unit vaccines would represent a major advance for clinical vaccinology.

The potent adjuvant activity of archaeosomes correlates to the recruitment and activation of macrophages and dendritic cells in vivo.

The unique glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with potent adjuvant activity. We studied the effect of archaeosomes on APCs to elucidate the mechanism(s) of adjuvant action. Exposure of J774A.

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help.

The unique ether glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264).

Archaeobacterial ether lipid liposomes (archaeosomes) as novel vaccine and drug delivery systems.

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents.