Glycobiology Advance Access originally published online on May 1, 2008
Glycobiology 2008 18(7):559-565; doi:10.1093/glycob/cwn038
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Published by Oxford University Press 2008.
Adjuvant potential of archaeal synthetic glycolipid mimetics critically depends on the glyco head group structure
3 National Research Council of Canada, Institute for Biological Sciences, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada
1 To whom correspondence should be addressed: Tel: +1-613-998-7891; Fax: +1-613-952-9092; e-mail: dennis.sprott{at}nrc-cnrc.gc.ca
Received on February 28, 2008; revised on April 4, 2008; accepted on April 28, 2008
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Abstract |
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 Top Abstract IntroductionResults and discussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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Subunit vaccines capable of providing protective immunity against the intracellular pathogens and cancers that kill millions of people annually require an adjuvant capable of directing a sufficiently potent cytotoxic T lymphocyte response to purified antigens, without toxicity issues. Archaeosome lipid vesicles, prepared from isoprenoid lipids extracted from archaea, are one such adjuvant in development. Here, the stability of an archaeal core lipid 2,3-di-O-phytanyl-sn-glycerol (archaeol) is used to advantage to synthesize a series of disaccharide archaeols and show that subtle variations in the carbohydrate head group alters the type and potency of immune responses mounted in a mammal. Critically, a glycosylarchaeol was required to elicit high cytotoxic CD8+ T cell activity, with highest responses to the antigen entrapped in archaeosomes containing disaccharides of glucose in β- or
1–6 linkage (β-gentiobiose, β-isomaltose), or of β-lactose. This first study on synthetic archaeal lipid adjuvants reveals potential for this class of regulatory friendly, easily scalable, inexpensive, and potent glyco-adjuvant. Key words: adjuvant / archaea / disaccharides / glycosylarchaeols / synthesis
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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The causative agents for many of the most devastating human diseases, including malaria, tuberculosis, and HIV, are classified as intracellular pathogens. Considerable literature suggests that vaccine development for these agents, and for cancers, is likely to require a strong cell-mediated, particularly cytotoxic CD8+ T cell (CTL), response (Rappuoli 2007
). As vaccinology advances from the use of whole attenuated pathogens toward safer, defined subunit vaccines, an adjuvant system becomes critical to direct and elevate the immune response to co-delivered purified antigens (Petrovsky and Aguilar 2004). Alum, while currently approved for human vaccines, is primarily an antibody rather than a cell-mediated adjuvant. One of several adjuvant systems in development to address the need for a safe effective CTL adjuvant (Aguilar and Rodriguez 2007) is archaeosomes.
Polar lipids that are novel to the domain of life Archaea are characterized as isoprenoid glyco- and phospho-ether lipids with stereochemistry (sn-2,3) opposite to that in glycerolipids of Bacteria or Eucarya (Kates 1992). Natural archaeal total polar lipid (TPL) extracts can be hydrated to form extremely stable, nanosized vesicles (archaeosomes) that target entrapped antigens to antigen-presenting cells (Tolson et al. 1996) and upregulate the expression of their co-stimulatory proteins (Krishnan et al. 2001), without any associated toxicity observed in mice (Patel et al. 2002). Adjuvant studies using TPL archaeosomes have determined that the polar lipids extracted from Methanobrevibacter smithii are especially promising. These membrane lipids consist of 60 wt% archaeols and 40 wt% membrane-spanning bipolar caldarchaeols with head groups of primarily phosphoserine and gentiobiose, and lesser amounts of phosphoinositol and phosphate (Sprott et al. 1999). The scheme in Figure 1 summarizes what is known regarding the mechanism for M. smithii archaeosome adjuvants. As described in detail in a recent review (Krishnan and Sprott 2008), M. smithii archaeosomes promote both MHC class I and class II responses to an entrapped antigen, where the latter MHC class II response is both cell mediated (Th1) and antibody (Th2) directed. Archaeosome TPL vaccines with striking protective ability have been demonstrated for the intracellular pathogen Listeria monocytogenes (Conlan et al. 2001) as well as for solid and metastatic tumors (Krishnan et al. 2003).
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Archaea are nonpathogenic microbes (Cavicchioli et al. 2003
) and presumably would not have pathogen-associated molecular patterns to serve as danger signals that activate the innate immune system (Pulendran et al. 2001). Indeed toll-like receptors 2 and 4 do not appear to be activated by our lead archaeosomes composed of the polar lipids extracted from the commensal human gut methanogen, M. smithii (Krishnan et al. 2007). Despite the recent push toward the development of toll-like receptor agonists as adjuvants, questions remain regarding safety and efficacy (van Duin et al. 2006), and the question whether a toll-free adjuvant would lead to better vaccines remains unanswered (Ishii and Akira 2007). Archaeosomes appear to be quite novel among adjuvants by apparent inability to activate toll-like receptors, yet exhibiting long-lasting (Krishnan et al. 2007) vaccine efficacy.
Many of the mechanistic details for immune activation have yet to be determined (Medzhitov 2007) which complicates establishing structure-activity relationships for the design of directed adjuvant activity. Here we describe immunological results based on a series of novel synthetic archaeal glycolipids where some of these details can be ascertained. The presence and structural detail of the carbohydrate head group of a glycosylarchaeal lipid mimetic is shown herein to be important in the adjuvanting process.
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Results and discussion |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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Use of archaeal total polar lipid natural mixtures as an adjuvant system limits the adjuvant composition to only those lipids, and in the proportions, that are extracted from archaeal species. Our initial assumption was that natural mixtures of archaeal polar lipids in an extract would consist of immunostimulatory, immunotargeting, immunoinactive, and immunodepressing species; the latter partially based on downregulation of the adaptive immune response associated with liposomes containing phosphatidylserine (Hoffmann et al. 2005
). Theoretically then, more potent archaeal lipid adjuvants than found in extracts could be designed and synthesized chemically as simplified and defined archaeosome compositions. Our synthetic approach was to first obtain the archaeol lipid core by methanolic-HCl hydrolysis of the polar lipids extracted from Halobacterium salinarum, chosen because this archaeon has only one core lipid, namely fully saturated archaeol. Upon head group hydrolysis the mixture of natural polar lipids is converted to a single lipid, namely archaeol that is easily recovered in high yields. This approach preserves all of the archaeal lipid features including methyl 3, 7, 11 chiral centers in R-configuration and saturated C-20 chains ether-linked to glycerol in the archaeal sn-2,3-stereochemistry. Carbohydrate polar head groups of desired type, number, linkage, and configuration were then chemically coupled to the free sn-1 hydroxyl of archaeol, using to advantage the stability of the ether lipid to harsh protection/de-protection steps required in the synthesis. In fact, all the oligosaccharide structures used in this work are readily available and the target molecules were readily assembled, purified, de-protected, and characterized in a minimum of synthetic steps. All processes should be readily scalable to industrial quantities and pharmaceutical purities. Figure 2 shows some representative structures for which we present immunological data.
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Archaeosome formation from synthetic glycosylarchaeols required the presence of a negatively charged phospholipid to promote hydration and to prevent subsequent aggregation in phosphate-buffered saline, and of a stabilizing lipid such as cholesterol to prevent eventual conversion of these archaeosomes to needle-shaped crystals. Ovalbumin, used as a test protein antigen in which the dominant T cell epitope (SIINFEKL) is known (Rotzschke et al. 1991
), was entrapped within archaeosomes made from a synthetic glycosylarchaeol combined with synthetic phosphatidylglycerol (PG) and cholesterol. PG chains were dipalmitoyl to mimic the C-16 (plus 4 methyl branches) of the C-20 phytanyl chains. Upon subcutaneous injection at 0 and 3 weeks, measuring antigen-specific CD8+ CTL activity in splenic cells and titrating anti-OVA (ovalbumin) antibody in mouse sera assessed adjuvant activity for both MHC class I and class II (Th2) pathways. Results are compared with archaeosomes consisting of total polar lipid extracts from M. smithii, as these are known to promote high CTL activity to an entrapped antigen (Krishnan et al. 2003). Other adjuvant comparisons are not shown because M. smithii archaeosomes have out-performed Alum, Freund's complete adjuvant, liposomes, and live Listeria recombinant for both antibody and CTL responses (Sprott et al. 1997; Krishnan, Dicaire, et al. 2000; Krishnan, Sad, et al. 2000; Krishnan et al. 2007). PG/cholesterol liposomes or the archaeal analog of PG, archaetidylglycerol, combined with cholesterol had very low adjuvant activity (Figure 3A and B). Clearly then, the mere presence of the archaeal isoprenoid lipid, archaetidylglycerol, was insufficient to promote strong immune responses of either MHC class I or class II, and indicates that this phospholipid may be classified as immunoinactive. Critically, we observed a dramatic increase in both CTL activity and antibody titers upon addition of glycosylarchaeols to PG/cholesterol documenting the importance for a glycosylarchaeol in the adjuvant process (Figure 3A and B). Since we are unaware of a specific diglucose-binding lectin on antigen-presenting dendritic or macrophage cells (APCs) it was surprising to us that 45 mol% -tetraMannosylarchaeol (Figure 3C) and shorter variations of it (not shown) designed to target the mannose receptors on APCs (East and Isacke 2002) were less effective CTL adjuvants than 15–45 mol% β-gentiobiosylarchaeol. In fact maximal CTL activity was required in excess of 15 mol% β-gentiobiosylarchaeol, but this activity declined when the β-gentiobiosylarchaeol was increased beyond 45 mol% to 60 mol% (Figure 3C). This drop in activity was explained by instability at 60 mol% β-gentiobiosylarchaeol, as aggregation and settling of the archaeosomes occurred. This result also explains a previous erroneous conclusion that β-gentiobiosylarchaeol purified from a methanogen had little adjuvant potential (Sprott et al. 1999), as in that study the archaeosomes tested contained about 60 mol% β-gentiobiosylarchaeol. CTL adjuvant effects were antigen-specific as little response occurred for the nonspecific EL-4 target cells (Figure 3D).
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We proceeded by synthesizing a series of the diglycosylarchaeols to test if linkage configuration and sugar type were important for adjuvant activity (Figure 2). Disaccharides were chosen rather than longer oligosaccharides to minimize both the cost of synthesis and of any possibility of raising an anti-glycolipid antibody response. For all disaccharide lipids synthesized, archaeosomes could be formed readily using 25 mol% diglycosylarchaeol mixed with DPPG/cholesterol in mol% ratio of 55/20. Because archaeosomes formed less well at higher mol% ratios (35% or more) for certain of the diglycosylarchaeols to be compared, all formulations of diglycosylarchaeols with DPPG/cholesterol were tested only at 25/55/20 mol%. These preparations entrapped OVA similarly at close to 40 µg mg–1 archaeosomes, were comparable in size within the range of 50–150 nm diameter, and remained stable as judged by phase-contrast microscopy prior to each injection. CTL assays using splenic cells from immunized mice revealed a striking difference in antigen-specific adjuvant activity of archaeosomes dependent on the structural details of the disaccharide head group used (Figure 4A). Diglucosylarchaeols with either an
- or β-1,4 linkage were relatively inactive compared to 1,6 linkages. β-Gentiobiosylarchaeol consistently gave best activities, as shown by highest % lysis of target cells at a low effector/target ratio, but a change of the β-1,6 linkage between glucose residues to -1,6 (isomaltose) or change to a Gal-Glc disaccharide-linked β-1,4 (lactose) resulted in similarly high CTL adjuvant activity. Configuration of the linkage was important in the case of Gal-Glc-archaeols as the -1,6 linkage of meliobiose was less active than β-1,4 of lactose. A similar pattern of antigen-specific responses is seen by the Elispot analysis of the same splenic cell populations (Figure 4B).
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Dectin-1 binds exclusively to β-1,3 oligomers of glucose where the minimum number of residues for activity is a 10- or 11-mer (Palma et al. 2006
). Thus, the known specificity of this recently discovered β-glucan receptor excludes it as an explanation for the boost in adaptive immune responses by β-gentiobiosylarchaeol. Other known receptors will require careful scrutiny. However, the presence of novel undiscovered receptors on APCs that recognize the diglycosylarchaeols reported herein are likely.
Since the β-linkage of the monosaccharide 6-deoxyquinovose to a lipid core results in a more tightly packed bilayer membrane than its -isomer, with potential for accompanying changes in biological activity (Matsumoto et al. 2005), we tested whether this feature would influence CTL adjuvanting. Both - and β-anomers of gentiobiosylarchaeol were synthesized and used to generate antigen-loaded archaeosomes for the assessment of adjuvant activity. Significantly better activity was found for the β-anomer (Figure 4C), supporting the role of the disaccharide to engage receptors by better recognition of a more extended orientation from the surface of the archaeosome, as reported for packing of other β-glycolipids (Jarrell et al. 1987). Similar data for antigen-specific CD8+ T cell frequencies were obtained by the Elispot assay of splenic cells, where the β-gentiobiosylarchaeol adjuvant resulted in significantly higher frequencies than -gentiobiosylarchaeol (P = 0.0037) (Figure 4B).
Perhaps indicative of a selective MHC class I glycolipid adjuvant mechanism, there were no significant differences among the means of anti-OVA antibody titers in blood for any of the diglycosylarchaeols (Figure 4D), suggesting that all proceed by a similar MHC class II antigen processing pathway.
In conclusion, we have shown that a weak CTL adjuvant consisting of DPPG/cholesterol liposomes can be converted to a strong adjuvant by the addition of a synthetic diglycosylarchaeol. CTL adjuvant activity is high with gentiobiose, with linkage to the archaeol in a preferred β rather than configuration. We conclude that β-gentiobiosylarchaeol and β-gentiobiosylcaldarchaeol lipids (Sprott et al. 1999) account, at least in part, for the adjuvant activity of M. smithii TPL archaeosomes. However, strong activity also noted with β-lactosylarchaeol is encouraging for vaccine development in view of the low cost of lactose to serve as a readily available synthetic precursor. Also encouraging is the relatively simple synthesis reported herein using biosynthetic archaeol and carbohydrate precursors, compared to the multistep synthesis of other adjuvants such as glycosphingolipids (Long et al. 2007), QS-21A (Wang et al. 2005), or lipidA mimetics (Bazin et al. 2006). β-Lactosylarchaeol, for example, is synthesized from archaeol and lactose precursors in four synthetic steps.
We anticipate future design of a synthetic archaeosome adjuvant to include one or combination of glycosylarchaeol(s) combined with a synthetic anionic archaeal lipid. As a further design feature, the stability of the archaeosome may be altered as required to achieve directional preference toward either MHC class I or II responses by the addition of a caldarchaeol membrane-spanning lipid (Choquet et al. 1996), and avoid using stabilizing cholesterol that may promote membrane peroxidation (Schnitzer et al. 2007). Although the structural possibilities of archaeal glycolipids for synthesis and adjuvant testing are numerous and therefore somewhat daunting, the reward may be to develop a new class of synthetic adjuvant designed to achieve the type of immune response required for protection against specific human diseases.
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Materials and methods |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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Preparation of archaeol
Halobacterium salinarum ATCC 33170 was grown aerobically and the biomass extracted with chloroform/methanol/water ratios to obtain the total lipids. Neutral lipids were removed by precipitating the polar lipids with acetone. 3 g of polar lipids in a 500-mL round bottom flask were refluxed in 150 mL of 2.5% methanolic-HCL at 64–65°C for 2 h, while stirring magnetically. Archaeol in the methanolic-HCl was partitioned into petroleum ether by mixing methanolic HCl/water/petroleum ether (30–65°C fraction) in the ratio 93 mL/9.3 mL/93 mL. The ether was evaporated to yield the archaeol oil, further purified by passing through a silica gel G (EMD Chemicals, Inc., Gibbstown, NJ) column (bed 20 cm x 1.8 cm). Any traces of neutral lipids present were eluted with hexane prior to recovering pure archaeol by elution with hexane/ethylacetate = 9:1 (v/v) with an overall yield of 41–45% of the starting polar lipids (wt basis).
Glycosylarchaeol synthesis
Details of the chemical synthesis will be presented in a separate communication. All oligosaccharides were synthesized from commercially available monosaccharide and disaccharide precursors. For example, lactose was acetylated with acetic anhydride and sodium acetate and purified by crystallization. The resulting peracetate was converted to its known thiophenol glycoside (Tropper et al. 1991) by the action of BF3.OEt2/PhSH. This was in turn activated by N-iodosuccinimide and silver trifluoromethanesulfonate in the presence of archaeol to yield the glycoside which after purification was deacetylated to yield the β-lactosylarchaeol. All compounds were characterized by 1H and 13C NMR as well as positive ion MALDI MS of lipid-containing species which gave the expected molecular ions, typically (M+Na)+. Representative data for β-lactosylarchaeol are provided in Supplementary Figures 1A–C. Archaetidylglycerol was purified from Haloferax volcanii (Sprott, Larocque, et al. 2003).
Archaeosome preparation and characterization
Liposomes were prepared by hydrating 20–30 mg lipids at 40°C in 2 mL of PBS buffer (10 mM sodium phosphate, 160 mM NaCl, pH 7.1) with the test antigen OVA dissolved at 10 mg mL–1. In some cases cholesterol (Sigma) or DPPG (Sigma) were mixed in chloroform/methanol with the synthetic glycosylarchaeols. These were dried to remove all traces of solvent and hydrated in PBS, as above. The size of the vesicles in the preparations was decreased by sonication in a sonic water bath (Fisher Scientific) at 40°C. Antigen not entrapped was removed by centrifugation at 200,000 x g (Rmax) and pellets washed thrice with 12-mL volumes of PBS. In the case of TPL archaeosomes 82 ± 7% of protein antigen was protected from protease action, indicating entrapment within the vesicles (Krishnan, Dicaire, et al. 2000). Quantification of antigen loading by SDS polyacrylamide gel electrophoresis was as described (Sprott, Patel, et al. 2003). Antigen loading was based on salt-corrected dry weights. Average diameters were determined by the particle-size analysis using a 5 mW He/Ne laser (Nicomp 370).
Lipid vesicle vaccines were prepared and diluted just prior to each injection solely as a precaution to ensure stability. Storage of the OVA-archaeosomes shown in Figure 4 (various diglycosyl-archaeols/DPPG/cholesterol) for 16 months at 4°C resulted in no change detected upon microscopic examination or by average diameter measurements.
Animal usage
To determine adjuvant activity, OVA entrapped in archaeosomes (OVA-archaeosomes) were used to immunize female C57BL/6 mice on days 0 and 21 (8 weeks old on first injection). Injections were subcutaneous at the tail base with 0.1 mL PBS containing 15 µg OVA entrapped in 0.2–0.63 mg lipids. Blood samples were collected on week 6 from the tail vein for anti-OVA antibody titration done by ELISA (Krishnan, Dicaire, et al. 2000). Spleens were collected from duplicate mice to determine CTL activity using the Cr51-assay done in triplicate with specific and nonspecific targets EG.7 and EL-4, respectively (Krishnan, Sad, et al. 2000). In addition to CTL measurements, Elispot assays performed as previously described (Sprott et al. 2004) in triplicate on the same splenic cells used for CTL assays determined antigen-specific CD8+ T cell frequencies. All protocols were approved by the Institutional Animal Care Committee and were in compliance with guidelines set by the Canadian Council on Animal Care.
Statistical analysis
Means are reported as means ± s.e.m. and significance between means (P < 0.05) compared by the two-tailed t test.
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Supplementary data |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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Supplementary data for this article is available online at http://glycob.oxfordjournals.org/
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Conflict of interest statement |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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The authors thank Mr. John Shelvey for growing Halobacterium salinarum biomass in our cell culture facility. Mrs. Eva Eichler expertly synthesized
-tetraMannosylarchaeol in the laboratory of D.M.W. This article is publication 42524 of the National Research Council of Canada. |
Footnotes |
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2 Present address: Université de Montréal, Faculté de Médecine Vétérinaire, St. Hyacinthe, 3200 Sicotte, Québec J2S 7C6, Canada.
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Abbreviations |
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APC, antigen-presenting dendritic or macrophage cell; CTL, cytotoxic T lymphocyte; OVA, ovalbumin; PG, phosphatidylglycerol; TPL, total polar lipid
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