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1.  Synthetic Archaeosome Vaccines Containing Triglycosylarchaeols Can Provide Additive and Long-Lasting Immune Responses That Are Enhanced by Archaetidylserine 
Archaea  2012;2012:513231.
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. Addition of 45 mol% of the glycolipids gentiotriosylarchaeol, mannotriosylarchaeol or maltotriosylarchaeol to an archaetidylglycerophosphate-O-methyl archaeosome, significantly enhanced the CD8+ T cell response to antigen, but diminished the antibody titres in peripheral blood. Archaeosomes consisting of all three triglycosyl archaeols combined with archaetidylglycerophosphate-O-methyl (15/15/15/55 mol%) resulted in approximately additive CD8+ T cell responses and also an antibody response not significantly different from the archaetidylglycerophosphate-O-methyl alone. Synthetic archaetidylserine played a role to further enhance the CD8+ T cell response where the optimum content was 20–30 mol%. Vaccines giving best protection against solid tumor growth corresponded to the archaeosome adjuvant composition that gave highest immune activity in immunized mice.
PMCID: PMC3465877  PMID: 23055819
2.  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. Vaccination with melanoma antigenic peptides TRP181-189 and Gp10025-33 delivered in archaeosomes resulted in IFN-γ producing antigen-specific CD8+ T cells with strong cytolytic capability and protection against subcutaneous B16 melanoma. Targeting responses against multiple antigens afforded prolonged median survival against melanoma challenge. Entrapment of multiple peptides within the same vesicle or admixed formulations were both effective at evoking CD8+ T cells against each antigen. Melanoma-antigen archaeosome formulations also afforded therapeutic protection against established B16 tumors when combined with depletion of T-regulatory cells. Overall, we demonstrate that archaeosome adjuvants constitute an effective choice for formulating cancer vaccines.
PMCID: PMC3034908  PMID: 21318177
3.  Activation of Dendritic Cells by Liposomes Prepared from Phosphatidylinositol Mannosides from Mycobacterium bovis Bacillus Calmette-Guérin and Adjuvant Activity In Vivo†  
Infection and Immunity  2004;72(9):5235-5246.
Liposome vesicles could be formed at 65°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. Three phosphoglycolipids and three phospholipids comprising 96% of TPL were identified as phosphatidylinositol dimannoside, palmitoyl-phosphatidylinositol dimannoside, dipalmitoyl-phosphatidylinositol dimannoside, phosphatidylinositol, phosphatidylethanolamine, and cardiolipin. The activation of dendritic cells by liposomes prepared from each purified lipid component of TPL was evaluated in vitro. A basal activity of phosphatidylinositol liposomes to activate proinflammatory cytokine production appeared to be attributable to the tuberculosteric fatty acyl 19:0 chain characteristic of mycobacterial glycerolipids, as similar lipids lacking tuberculosteric chains showed little activity. Phosphatidylinositol dimannoside was identified as the primary lipid that activated dendritic cells to produce amounts of proinflammatory cytokines several times higher than the basal level, indicating the importance of mannose residues. Although the activity of phosphatidylinositol dimannoside was little influenced by palmitoylation of mannose at C-6, a further palmitoylation at inositol C-3 diminished the induction levels of IL-6 and IL-12. Further, OVA entrapped in palmitoyl-phosphatidylinositol dimannoside liposomes was delivered to dendritic cells for major histocompatibility complex class I presentation more effectively than TPL OVA-liposomes. BCG liposomes containing mannose lipids caused up-regulation of costimulatory molecules and CD40. Thus, the inclusion of pure phosphatidylinositol mannosides of BCG in lipid vesicle vaccines represents a simple and efficient option for targeting antigen delivery and providing immune stimulation.
PMCID: PMC517455  PMID: 15322018
4.  Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen 
Archaea  2003;1(3):151-164.
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. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis ofarchaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.
PMCID: PMC2685569  PMID: 15803661
antibody; archaea; cytotoxic T lymphocyte; liposomes; phagocytosis; phosphatidylserine
5.  Archaeosome Vaccine Adjuvants Induce Strong Humoral, Cell-Mediated, and Memory Responses: Comparison to Conventional Liposomes and Alum† 
Infection and Immunity  2000;68(1):54-63.
Ether glycerolipids extracted from various archaeobacteria were formulated into liposomes (archaeosomes) possessing strong adjuvant properties. Mice of varying genetic backgrounds, immunized by different parenteral routes with bovine serum albumin (BSA) entrapped in archaeosomes (∼200-nm vesicles), demonstrated markedly enhanced serum anti-BSA antibody titers. These titers were often comparable to those achieved with Freund's adjuvant and considerably more than those with alum or conventional liposomes (phosphatidylcholine-phosphatidylglycerol-cholesterol, 1.8:0.2:1.5 molar ratio). Furthermore, antigen-specific immunoglobulin G1 (IgG1), IgG2a, and IgG2b isotype antibodies were all induced. Association of BSA with the lipid vesicles was required for induction of a strong response, and >80% of the protein was internalized within most archaeosome types, suggesting efficient release of antigen in vivo. Encapsulation of ovalbumin and hen egg lysozyme within archaeosomes showed similar immune responses. Antigen-archaeosome immunizations also induced a strong cell-mediated immune response: antigen-dependent proliferation and substantial production of cytokines gamma interferon (Th1) and interleukin-4 (IL-4) (Th2) by spleen cells in vitro. In contrast, conventional liposomes induced little cell-mediated immunity, whereas alum stimulated only an IL-4 response. In contrast to alum and Freund's adjuvant, archaeosomes composed of Thermoplasma acidophilum lipids evoked a dramatic memory antibody response to the encapsulated protein (at ∼300 days) after only two initial immunizations (days 0 and 14). This correlated with increased antigen-specific cell cycling of CD4+ T cells: increase in synthetic (S) and mitotic (G2/M) and decrease in resting (G1) phases. Thus, archaeosomes may be potent vaccine carriers capable of facilitating strong primary and memory humoral, and cell-mediated immune responses to the entrapped antigen.
PMCID: PMC97101  PMID: 10603368
6.  Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts † 
The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per μg of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per μg of vector DNA. However, a 380-fold improvement (3,417 transformants per μg of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.
PMCID: PMC201408  PMID: 16349218
7.  Hydroxydiether Lipid Structures in Methanosarcina spp. and Methanococcus voltae† 
Hydroxylated diether lipids are the most abundant lipids in Methanosarcina acetivorans, Methanosarcina thermophila, and Methanosarcina barkeri MS and Fusaro, regardless of the substrate used for growth. Structural analysis of the lipid moiety freed of polar head groups revealed that the hydroxydiether lipids of all the Methanosarcina strains were hydroxylated at position 3 of sn-2 phytanyl chains. The finding that Methanosarcina strains synthesize the same hydroxydiether structure suggests that this is a taxonomic characteristic of the genus. Methanococcus voltae produced minor amounts of the 3-hydroxydiether characteristic of Methanosarcina spp. and also the 3′-hydroxydiether described previously for Methanosaeta concilii.
PMCID: PMC202207  PMID: 16348899
8.  Metabolic Pathways in Methanococcus jannaschii and Other Methanogenic Bacteria † 
Eleven strains of methanogenic bacteria were divided into two groups on the basis of the directionality (oxidative or reductive) of their citric acid pathways. These pathways were readily identified for most methanogens from the patterns of carbon atom labeling in glutamate, following growth in the presence of [2-13C]acetate. All used noncyclic pathways, but members of the family Methanosarcinaceae were the only methanogens found to use the oxidative direction. Methanococcus jannaschii failed to incorporate carbon from acetate despite transmembrane equilibration comparable to other weak acids. This organism was devoid of detectable activities of the acetate-incorporating enzymes acetyl coenzyme A synthetase, acetate kinase, and phosphotransacetylase. However, incorporation of [1-13C]-, [2-13C]-, or [3-13C]pyruvate during the growth of M. jannaschii was possible and resulted in labeling patterns indicative of a noncyclic citric acid pathway operating in the reductive direction to synthesize amino acids. Carbohydrates were labeled consistent with glucogenesis from pyruvate. Leucine, isoleucine, phenylalanine, lysine, formate, glycerol, and mevalonate were incorporated when supplied to the growth medium. Lysine was preferentially incorporated into the lipid fraction, suggesting a role as a phytanyl chain precursor.
PMCID: PMC202243  PMID: 16348909
9.  Nature of the Specificity of Alcohol Coupling to l-Alanine Transport into Isolated Membrane Vesicles of a Marine Pseudomonad 
Journal of Bacteriology  1974;117(3):1043-1054.
Ethanol stimulated the uptake of l-alanine into isolated membrane vesicles of a marine pseudomonad at a rate and to an extent comparable with that obtained with reduced nicotinamide adenine dinucleotide (NADH) or the artificial electron donor ascorbate-N, N, N′, N′-tetramethyl-p-phenylenediamine (ascorbate-TMPD). Methanol and branched-chain alcohols had little or no capacity to energize transport. No quantitative relationship was found between the ability of a compound to induce oxygen uptake and to energize transport, since with ethanol initial rates of oxygen uptake were approximately 4% of that obtained with NADH or ascorbate-TMPD. Cytochrome analysis revealed that NADH and ethanol reduced cytochromes b and c, whereas ascorbate-TMPD coupled primarily at the level of cytochrome c. Approximately 25% of the cytochromes reduced by dithionite were reducible by ethanol. Ethanol reduction of both cytochromes b and c was prevented by 2-heptyl-4-hydroxyquinoline-N-oxide, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. The ethanol- and NADH-energized transport systems for l-alanine were subject to quantitatively similar inhibition by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, 2, 4-dinitrophenol, and the sulfhydryl reagents p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. In contrast, for ascorbate-TMPD-driven transport, only cyanide and 2, 4-dinitrophenol remained fully effective as inhibitors, p-chloromercuribenzoate was only half as effective, and the other compounds stimulated transport. Inhibition of ethanol oxidation strikingly paralleled the inhibition of ethanol-driven transport for each of the inhibitors, including 2, 4-dinitrophenol. Marked differences between inhibition of oxygen uptake and inhibition of transport were observed when NADH or ascorbate-TMPD were the electron donors. The data indicate that only a small proportion of the respiratory chain complexes in the membrane vesicles are involved in transport and these are efficiently coupled to ethanol oxidation. The results also suggest that when 2, 4-dinitrophenol inhibits transport it is not acting as an uncoupling agent.
PMCID: PMC246583  PMID: 4360536

Results 1-9 (9)