Archaeobacterial Ether Lipid Liposomes as Vaccine Adjuvants

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[11] Archaeobacterial Ether Lipid Liposomes as Vaccine Adjuvants By G. Dennis Sprott, Girishchandra B. Patel, and Lakshmi Krishnan Introduction

Archaeosomes, liposomes composed of the polar lipids unique to the domain Archaea, have unexpected adjuvant properties, first reported in 1997 for the humoral response in mice.1 Mixtures of total polar lipids extracted from each of several archaea were tested, and, although all formed archaeosomes that promoted an enhanced humoral response to the associated protein antigen, there were differences noted in the degrees of adjuvant activity. These differences undoubtedly reflect subtle differences in the structural properties of the lipids. The polar lipids of all Archaea share the constant-length phytanyl chains, usually fully saturated, and bonded by ether linkages to a glycerol backbone in a mirror image sn-2,3-configuration to the glycerolipids of the domains Bacteria and Eukarya.2 However, different archaea express their own unique pattern of polar lipids, which is well conserved within the genus level of classification, caused by variations in both the polar head groups and, in some cases, to the core lipid structure itself. In the case of core lipids, the sn-2,3-diphytanylglycerol is ubiquitous to all strains, but some have the ability to form additional lipid cores (Fig. 1). Those polar head groups exposed to the outer surface of archaeosomes have the potential to interact with mammalian receptors, whereas the type and proportion of lipid cores markedly influence the stability3 and permeability4 of the archaeosome structure. In vitro studies indicate that a controlling factor for the stability of archaeosomes is the proportion of caldarchaeol membrane–spanning core lipids.3,5 In vivo stability studies comparing various archaeosome types have yet to be reported. Archaeosomes are mixed adjuvants capable of promoting strong humoral (antibody and Th2),1,6 cell-mediated (Th1),6 and cytotoxic T-cell 1

G. D. Sprott, D. L. Tolson, and G. B. Patel, FEMS Microbiol. Lett. 154, 17 (1997). M. Kates, Biochem. Soc. Symp. 58, 51 (1992). 3 C. G. Choquet, G. B. Patel, and G. D. Sprott, Can. J. Microbiol. 42, 183 (1996). 4 J. C. Mathai, G. D. Sprott, and M. L. Zeidel, J. Biol. Chem. 276, 27266 (2001). 5 C. G. Choquet, G. B. Patel, T. J. Beveridge, and G. D. Sprott, Appl. Microbiol. Biotechnol. 42, 375 (1994). 6 L. Krishnan, C. J. Dicaire, G. B. Patel, and G. D. Sprott, Infect. Immun. 68, 54 (2000). 2

METHODS IN ENZYMOLOGY, VOL. 373

Copyright 2003 ß by National Research Council of Canada (NRC) All rights reserved. 0076-6879/03 $35.00

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Fig. 1. Structures of the most commonly found polar lipid cores synthesized by various members of the Archaea. Symbols X and Y represent the various headgroups of polar lipids or protons in the case of the lipid cores. Standard caldarchaeol (Cs) is 2,20 ,3,30 -tetra-Odibiphytanyl-sn-diglycerol; standard archaeol (As) is 2,3-di-O-phytanyl-sn-glycerol; hydroxyarchaeol is (AOH), and archaeol with an additional C5 unit to form a sesterterpanyl chain (A20,25 or A25,25). M. smithii total polar lipids are caldarchaeols, archaeols, and minor amounts of hydroxyarchaeols.18

responses7 to entrapped protein antigens. Physical association of the protein antigen with the archaeosome seems to be important for the induction of a strong humoral response, and all major antibody isotypes (IgG1, IgG2a, IgG2b) are induced.6 Immunization of mice with archaeosomeencapsulated bovine serum albumin, ovalbumin (OVA), or lysozyme results in strong cell-mediated immune responses as well, measured as antigen-dependent proliferation of splenic cells, and substantial production by these cells of gamma interferon (a Th1 cytokine) and interleukin-4 (a Th2 cytokine).6 In addition to major histocompatibility complex (MHC) class II presentation, archaeosomes induce a strong MHC class I presentation of associated protein antigens by antigen presenting cells (APCs), both in vitro and in vivo.7 The induced cytotoxic T-cell response is CD8þ T–cell dependent, because killing of target cells does not occur on removal of 7

L. Krishnan, S. Sad, G. B. Patel, and G. D. Sprott, J. Immunol. 165, 5177 (2000).

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effector CD8þ T cells and is primarily perforin mediated, because killing was not induced in perforin-deficient mice.7 Processing of antigen is Brefeldin A sensitive, suggesting that archaeosome-antigen presentation is through the classical cytosolic pathway with peptides being transported through the endoplasmic reticulum and presented by MHC class I molecules.7 Finally, adjuvant activity is sufficiently potent to bypass a requirement for CD4þ T-cell help.7 Comparisons with conventional liposomes and alum were made throughout all these adjuvant activity studies and indicate clearly the superior adjuvant activities of archaeosomes.6,7 Evidently, archaeosome-antigen is taken up and released into both cytosolic and phagolysosome compartments for processing, although the exact mechanism of antigen delivery has yet to be determined. To date, total polar lipid extracts from all archaea tested form archaeosomes capable of antigen delivery and subsequent presentation by both MHC pathways. Initially, we believed that the mechanism of adjuvant activity with archaeosomes was primarily attributable to a superior antigen-carrier property, featured by enhanced phagocytosis of archaeosomes by APCs compared with other liposome types.8 Discovery of immune modulating activity by archaeosomes soon changed this view.9 The addition of archaeosomes to cultures of J774A.1 macrophages resulted in up-regulation of costimulatory molecules B7.1 and B7.2, and MHC class II molecules on the cell surface. Similar results were found for mouse bone marrow–derived dendritic cells. Importantly, no such up-regulation of genes occurred in response to liposomes made from dimyristoylphosphatidylcholine(DMPC), dimyristoylphosphatidylglycerol(DMPG), and cholesterol. Furthermore, archaeosomes enhanced both the recruitment and activation of antigen presenting cells (APC) (dendritic cells and macrophages) to the injection site in mice. Finally, an enhanced production of tumor necrosis factor by APCs exposed to archaeosomes was observed. The ability of archaeosomes to activate APCs correlated with an enhanced ability of archaeosome-treated APCs to stimulate allogenic T-cell proliferation.9 Induction of immunological memory can be striking following a primary archaeosome-antigen adjuvanted immune response.6 However, different archaeosome lipid types seem to give varied levels of memory response, and these aspects critical for vaccine development are under active investigation. Recently we demonstrated that an archaeosomal peptide vaccine conferred long-term protection against infection of mice by the intracellular pathogen Listeria monocytogenes.10 Protection against 8

D. L. Tolson, R. K. Latta, G. B. Patel, and G. D. Sprott, J. Liposome Res. 6, 755 (1996) L. Krishnan, S. Sad, G. B. Patel, and G. D. Sprott, J. Immunol. 166, 1885 (2001). 10 J. W. Conlan, L. Krishnan, G. E. Willick, G. B. Patel, and G. D. Sprott, Vaccine 19, 3509 (2001). 9

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this pathogen is known to require an effective cytotoxic T-cell response, implicating archaeosomes as an effective means of conferring CD8þ T-cell memory and corroborating earlier cytotoxic T-cell assays performed up to 5 months subsequent to vaccination.7 Published reports of archaeosome action have been limited to our own laboratory11 and feature the polar lipids from Methanobrevibacter smithii, Thermoplasma acidophilum, and Halobacterium salinarum. Because much of our data is for M. smithii archaeosomes, we restrict the protocols herein to demonstrate the production of antigen (OVA) loaded M. smithii archaeosomes and to assessment of their capability to promote Th1, Th2, and cytotoxic T-cell (CTL) responses in mice. Growth of Methanobrevibacter smithii

M. smithii ALI (DSM 2375) is a mesophilic, methanogenic archaeon isolated from human feces. M. smithii derives energy for growth by using 4 moles of hydrogen to reduce 1 mole of carbon dioxide, resulting in the production of 1 mole of methane gas. Hydrogen is the preferred electron donor, with formate as a less than optimal substitute. Methanogenic bacteria are fastidiously anaerobic, requiring an initial growth Eh of below 115 mV at neutral pH. To achieve such low Eh, specialized oxygen-free gas mixtures are required, along with devices to scrub the gases of any contaminating oxygen. Special gassing stations are used to flush vessels free of oxygen, to perform the anaerobic techniques required to reduce the redox potential of the medium, and to protect the culture from exposure to oxygen during culture transfer.12 The use of sealed culture vessels that are pressurized with a H2/CO2 (4:1) gas mixture and use of sulfide require the exercise of extreme caution and safety procedures.13 These safety precautions must be followed rigorously to avoid injury and damage. By use of the safety measures outlined,12,13 we have grown methanogens successfully in 58-l batches of media contained in a 75-l fermenter for more than a decade without adverse incident. M. smithii ALI is grown in a medium modified from Balch et al.14 of the following composition (mg/l): NH4Cl, 1000; K2HPO4, 200; CH3COONa3H2O, 2500; Bacto yeast extract, 2000; Bacto tryptone, 2000; NaHCO3, 11

G. B. Patel and G. D. Sprott, Critical Rev. Biotechnol. 19, 317 (1999). K. R. Sowers and K. M. Noll, in ‘‘Methanogens’’ (K. R. Sowers and H. J. Schreier, eds.), p. 15, in ‘‘Archaea—A Laboratory Manual’’ (F. T. Robb, ed.-in chief). Cold Spring Harbor Laboratory Press, Plainview, NY, 1995. 13 L. Daniels, in ‘‘Methanogens’’ (K. R. Sowers and H. J. Schreier, eds.), p. 63, in ‘‘Archaea— A Laboratory Manual’’ (F. T. Robb, ed.-in chief). Cold Spring Harbor Laboratory Press, Plainview, NY, 1995. 12

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2500; KH2PO4, 300; (NH4)2SO4, 300; NaCl, 610; MgSO47H2O, 160; CaCl22H2O, 8; nitrilotriacetic acid, 15; MnSO4H2O, 5; FeSO47H2O, 3; CoCl26H2O, 1; ZnSO47H2O, 1; CuSO45H2O, 0.1; AlK(SO4)212H2O, 0.1; H3BO3, 0.1; Na2MoO42H2O, 0.1; resazurin, 0.2; pyridoxine-HCl, 0.1; thiamine-HCl, 0.05; riboflavin, 0.05; nicotinic acid, 0.05; p-aminobenzoic acid, 0.05; lipoic acid, 0.05; biotin, 0.02; folic acid, 0.02; vitamin B12, 0.005; 2-mercaptoethanolsulfonic acid-Na salt (HS-CoM-Na salt), 0.008; Na2S9H2O, 500; l-cysteine-HCl, 500; and the following volatile fatty acids (ml/l medium): isobutyric acid, 0.49; 2-methylbutyric acid, 0.55; n-valeric acid, 0.55; and isovaleric acid, 0.55. All ingredients are mixed, except the cysteine-HCl and Na2S9H2O, and the pH is adjusted to 7.6. The medium is boiled briefly, the headspace is flushed with H2/CO2, and cysteine-Na2S solution is added to reduce the medium (resazurin must turn colorless). The reduced medium is dispensed into glass serum vials under H2/CO2 gas phase, and the vials are capped with butyl rubber stoppers crimped in  place by aluminum seals.12 The medium is autoclaved (121 , 20 min) in sealed vials contained within a stainless steel metal container with 8-mm diameter holes in the bottom to allow steam entry, and a lid secured by metal clasps. After autoclaving, the medium is allowed to cool to room temperature and is removed from the metal container. The postautoclave pH of this medium after equilibration at room temperature for 24 h is 6.9 0.1. This medium is used to maintain working stock cultures and to prepare inoculum for the fermenter vessels. The organism is inoculated by transferring aseptically and anaerobically using syringe techniques,12 or  modifications thereof, and grown at 35 with agitation. The same medium is used for fermenters, except that the initial amount of cysteine-HCl and Na2S is decreased to 60 mg/l of each. All ingredients except the vitamins, volatile fatty acids, HS-CoM-Na salt, FeSO47H2O, and cysteine-Na2S are mixed and sterilized in situ in the fermenter (the fermenter is steam sterilized, in place), with the vessel fully vented to the atmosphere (under aerobic conditions) as is the normal practice. After sterilization, the fermenter is sparged with 80% H2:20% CO2 to maintain a positive pressure (5–10 psi) as the vessel is cooling (50 l medium in a  75 l Chemap AG fermenter). When the temperature is 35 , the remaining ingredients are added as separately autoclaved solutions, and the pH is adjusted to 6.9. The pH during growth is maintained using sterile 5 N NaOH or 5 N HCl (kept under N2). Just before inoculation with a midlogarithmic phase culture (5%–10%, v/v), the medium is supplemented

14

W. E. Balch, G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe, Microbiol. Rev. 43, 260 (1979).

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with 16% Na2S solution (autoclaved, under N2) to achieve a dissolved sulfide concentration of 0.1–0.2 mM. The sulfide is maintained in that range during growth by further additions, as needed. The 80% H2:20% CO2 sparge rate is adjusted upward during growth in response to the increase in biomass. The culture is harvested in mid to late logarithmic growth phase (A660 of > 2.0 between 48 and 72 h) using a tangential flow cell separator, and the concentrated cells are centrifuged. In addition to the safety precautions described earlier, additional steps must be adhered to as specified for fermenter-growth of methanogens on H2/CO2.13 Lipid Extraction

Total polar lipids are prepared from frozen and thawed pastes of M. smithii by the method of Bligh and Dyer,15 as described previously.16 Thirty grams (dry weight) of cell paste are thawed by adding water to a volume of 420 ml. One to 2 mg of DNase I is included, and the mixture stirred magnetically until cells are dispersed. Solvents are added to obtain a volume of 2 l in a ratio of CH3OH/CHCl3/H2O (v/v) of 2:1:0.8, and  the mixture is stirred for 16 h at 23 . The suspension is centrifuged at 4080g for 15 min in 150-ml glass centrifuge bottles. The cell pellet is extracted twice more with 1-l volumes of solvent, and the supernatants containing the lipids are combined. The total volume of extract is divided by 3.8 to obtain the volume of CHCl3 and water needed to achieve phase separation. The bottom CHCl3 phase is removed and combined with a CHCl3 wash of the methanol/water phase to obtain the total lipid extract. The volume of CHCl3 is depleted to dryness by rotary evaporation. The lipid residue is dissolved into CHCl3 adding the minimum volume of methanol required, and the polar lipids are precipitated by adding 20 volumes of ice-cold acetone. After storage at 20 for several hours, the white precipitate of total polar lipids is collected by centrifugation. The precipitate is washed once by redissolving in CHCl3, and acetoneprecipitated as before. Any acetone remaining in the pellet is dried with a stream of N2, and the total polar lipids are dissolved in CHCl3. Lipid  extracts dissolved in CHCl3 are stable indefinitely but are placed at 4 to minimize solvent evaporation over long storage periods and the resultant precipitation of lipids from concentrated extracts.

15 16

E. G. Bligh and W. J. Dyer, Can. J. Biochem. 37, 911 (1959). G. D. Sprott, C. G. Choquet, and G. B. Patel, in ‘‘Methanogens’’ (K. R. Sowers and H. J. Schreier, eds.), p. 329, in ‘‘Archaea—A Laboratory Manual’’ (F. T. Robb, ed.-in chief). Cold Spring Harbor Laboratory Press, Plainview, NY, 1995.

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Fast atom bombardment mass spectrometry (FAB MS), a technique in which molecular fragmentation is minimal, and thin-layer chromatography, combined with color development for glyco, amino, phospho, and total lipids,17 provide convenient checks on batch-to-batch consistency of lipid composition. In our laboratory, this variation has been minimal when using the following protocols, but quality control checks are still recommended. An FAB MS spectrum of a typical polar lipid extract from M. smithii18 is shown in Fig. 2. Note that signal height is not necessarily indicative of relative abundance, as shown in Table I. Indeed, the mol% of caldarchaeol lipids is much higher than predicted from the FAB MS spectrum and accounts for about 40 mol% of the polar lipids, as can be seen from the assignments and quantitative data shown in the Table. The remaining 60 mol% are primarily archaeol lipids, in particular archaetidylserine (30 mol%),
-Glcp-(1,6)-
-Glcp-(1,1)-archaeol (12 mol%), archaetidic acid (8 mol%), and archaetidylinositol (<8 mol%).

Fig. 2. Negative ion FAB MS analysis of total polar lipids from M. smithii. Data18 are shown with permission from Elsevier Science, Inc. 17

M. Kates, ‘‘Techniques of Lipidology. Isolation, Analysis and Identification of Lipids’’ (R. H. Burdon and P. H. van Knippenberg, eds.). Elsevier, New York, 1986. 18 G. D. Sprott, J.-R. Brisson, C. J. Dicaire, A. K. Pelletier, L. A. Deschatelets, L. Krishnan, and G. B. Patel, Biochim. Biophys. Acta 1440, 275 (1999).

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liposomes in immunology TABLE I Quantification of M. SMITHII Polar Lipids

Structure of main lipidsa

[M] m/zb

(Glc)2-Cs-P (Glc)2-Cs-PSc AOH-PG Unknown As-PI As-PS Glc-Cs-P As-(Glc)2 As-P

1705 1791 821 1705 893 818 1542 975 731

Mol%b 34

2

2 3 8 30 3 12 8

0.3 0.05 0.6 2 0.2 0.4 0.6

a

Cs and As represent caldarchaeol and archaeol lipid cores; P, phosphate; S, serine; G, glycerol; and I, inositol. b The [14C] total polar lipids were separated by two-dimensional thin-layer chromatography. Lipid spots were removed and counted. FAB MS was performed on unlabeled lipid spots obtained from duplicate plates. Some spots contained minor ether lipids not shown here. Further details for these data may be found in Sprott et al.18 c Presence of (Glc)2-Cs-PS is deduced from the Rf value and the ninhydrin positive nature of this spot, which also contains lipid (Glc)2-Cs-P. Data are shown with permission from Elsevier Science, Inc.

Preparation of Antigen (OVA)-Loaded Archaeosomes

Archaeosomes may be prepared from the total polar lipids of M. smithii by simple hydration in the presence of antigen, size reduction, lyophilization, and rehydration. Archaeal lipids are in a liquid crystalline-like state at ambient temperature and, hence, vesicles may be prepared at a range of temperatures including ambient. All glassware are prebaked for 6 h at  180 to render them pyrogen-free and sterilized by autoclaving. Pyrogenfree water is used to prepare autoclaved water and phosphate-buffered saline (PBS contains 160 mM NaCl and 10 mM potassium phosphate, pH 7.1). When possible, manipulations are performed aseptically in a biohood. Conventional liposomes are prepared from DMPC:DMPG:cholesterol (1.8: 0.2:1.5, molar ratio; Sigma Chemical Co., St. Louis, MO) by the same procedure described in the following for archaeosomes. 1. Thirty milligrams of total polar lipids are transferred with a glasstipped pipette into a 50-ml round-bottomed flask. A 20-ml scintillation vial may be used instead. Plastic tips should not be used when pipeting chloroform or plasticizer will be transferred. 2. The lipid is dried with an N2 stream. If solvent is trapped, the lipid film is pierced with a needle. Alternatively, to obtain a thin uniform film of

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dried lipid, the solvent is removed by rotary evaporation. The dried lipid is then placed under vacuum on a lyophilizer for 1 h. It is essential that all traces of solvent be removed. 3. Three milliliters of water and 10 glass beads (3-mm diameter) are added, then the antigen. To achieve high loading, OVA (stock 10 mg/ml water) is used at a ratio of 5 mg/10 mg lipid). The mixture is vortexed and left to hydrate on the bench for 2–3 h, and shaken/vortexed a few times during that time to aid in hydration. The hydrated lipid is then sonicated  for 2 min in a sonic bath (Branson 3200) and incubated in a 35 shaker for at least an hour. 4. The average vesicle diameter is reduced to about 100 nm. This may be achieved with a Sonic Dismembrator 550 (Fisher) at setting #4 for at least 5 min. Pressure extrusion through 100-nm filters is also effective (sequentially using 800-, 400-, and 100-nm pore filters). The average diameter is assessed with a particle sizer such as a 5-mW He/Ne laser (Nicomp 350, Santa Barbara, CA). 5. Lipid vesicles are frozen in the round-bottomed flask by immersing in dry ice–alcohol, rotating to obtain a thin layer, and dried under vacuum. 6. The dried vesides are rehydrated by adding 0.5 ml of water. The flask is rotated gently to humidify all powder and incubated for 20–30 min. Half a milliliter more of water is added, and the vesicles are sonicated at low energy for 2–5 min in a Branson 3200 bath, or equivalent, to ensure homogeneity. An additional 1.0 ml of water is added, and the vesicles are  incubated in a 35 shaker for 1–2 h.  7. The archaeosomes are annealed by incubating at 4 overnight. 8. The archaeosomes are transferred to sterile 15-ml conical tubes and centrifuged at 400g for 5 min in a clinical centrifuge. The small white pellet is discarded. 9. Nonentrapped OVA is removed by centrifugation for 1 h at 327,000g Rmax. For this step, archaeosomes are transferred to alcoholsterilized Ti 65.31 plastic tubes and the volume brought up to about 7 ml with water. After centrifugation, the archaeosome pellet is resuspended, into water by vortex mixing and washed twice. Finally, the pellet is re-suspended in 1–2 ml of water. 10. The preparation is filtered using a 0.45-m, 25-mm diameter, syringe-driven sterilizing filter (Millex-HV, nonpyrogenic, low-protein binding). The archaeosomes are combined with a 0.5-ml water rinse also passed through the filter. 11. The size distribution and dry weight of OVA-loaded archaeosomes are determined. The suspension is diluted to achieve 15 g OVA/0.1 ml PBS. Typically, the average diameter is 150 100 nm, and lipid yields are 18 to 20 mg.

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Quantification of Antigen Loading

The amount of protein antigen encapsulated in archaeosome formulations is determined by an sodiumdodecyl sulfate (SDS)-Lowry colorimetric assay.19 Interfering color from amino lipids is avoided by lipid depletion before the assay according to Wessel and Flugge,20 with modifications. Methanol (0.4 ml) is added to 0.1 ml of the archaeosomal formulation contained in a 1.5-ml microcentrifuge tube, the mixture is vortexed, and 0.2 ml CHCl3 is added. The mixture is vortexed again, 0.3-ml deionized water is added, followed by vigorous vortexing and centrifugation at 9000g for 3 min. The upper phase is removed with a Pasteur pipet and discarded, taking care not to disturb the precipitated protein at the interface. Methanol (0.3 ml), is added to the tube, and the mixture is vortexed and centrifuged for 3 min. The liquid is removed carefully, making sure not to displace any of the precipitated protein pellet. The protein pellet is dried under a stream of nitrogen and resuspended in 0.1 ml of 10% SDS for assay by the SDS-Lowry method.19 Typical loading is 80–100 g/mg archaeosomes. Filter-sterilized M. smithii archaeosomes loaded with OVA may  be stored at 4 in PBS for at least 12 months without loss of immunological responses. Antigen loading and stability may be assessed also by SDS-polyacrylamide gel electrophoresis (PAGE). Results typical for OVA entrapped in archaeosomes and conventional liposomes reveal some minor interference in band migration by the presence of lipids (Fig. 3). Immune Responses in Mice

Inbred C57BL/6 female mice, 6–8 wk of age, may be purchased from Charles River Breeding Laboratories (St. Constant, Quebec) or The Jackson Laboratory (Bar Harbor, ME). A minimum of five mice per group are immunized at 0 and 3 wk by subcutaneous injection at the base of the tail with 0.1-ml volumes of PBS containing 15 g OVA (no adjuvant) or 15 g OVA entrapped in lipid vesicles. For comparison, groups of mice are also inoculated with 15 g OVA either entrapped in conventional liposomes or mixed according to the manufacturer’s instructions with ImjectÕ Alum (Pierce, Rockford, IL). A naı¨ve group receives no injections. The animals are test-bled periodically from the tail vein to measure the systemic antibody titers. Spleens from euthanized mice are removed aseptically in a biohood and prepared for proliferative and cytotoxic T-cell assays as described in the following: 19 20

G. L. Peterson, Anal. Biochem. 83, 346 (1977). D. Wessel and U. I. Flugge, Anal. Biochem. 138, 141 (1984).

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Fig. 3. SDS-PAGE analysis showing that SDS Lowry provides a valid, comparable estimate of OVA entrapment in M. smithii archaeosomes and conventional liposomes. SDS Lowry analysis was performed on OVA entrapped in archaeosome and conventional liposome preparations. On the basis of these SDS Lowry data, lanes were loaded with 0.7 g OVA (lane 1), 0.7 g OVA entrapped in 37 g of archaeosomes (lane 2), or 0.7 g OVA entrapped in 23 g of conventional liposomes (lane 3). Data from Krishnan et al.7 are shown with permission from The Journal of Immunology.

1. The spleens are removed from duplicate mice and placed in a Petri dish, with about 10–15 ml RPMI medium (Gibco-BRL, Life Technologies Inc., Grand Island, NY), and a single cell suspension is produced by grinding between the frosted ends of two sterile glass slides. 2. The cell suspension is filtered into a 15-ml centrifuge tube, using a 70-m nylon membrane filter (both from Falcon, Becton Dickinson, Franklin Lakes, NJ), and the cells are harvested in a refrigerated  centrifuge (4 , 10 min at 470g). 3. The supernatant is discarded, and the cell pellet is tapped gently to loosen any clumps. Five milliliter of TRIS-buffered ammonium chloride (RBC lysing buffer, Sigma Chemical Co.) is added to resuspend the pellet by drawing the mixture into and out of a 10-ml pipette for about 1 min. When the cell suspension turns slightly yellow, indicating RBC lysis, 10 ml of RPMI 1640 containing 8% FBS (HyClone, Logan, Utah) is added and mixed thoroughly. The cells are harvested by centrifugation at 470g.

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4. The spleen cells are washed once or twice with RPMI 1640. Each wash will include gently tapping the cell pellet and resuspending the cells in RPMI 1640 medium and spinning at 470g to harvest the cells. After the final wash, the pellet is resuspended in 5 ml of RPMI þ 8% FBS medium. 5. The cells are counted in a hemocytometer. The recommended dilution is 10 l of spleen cell solution diluted with 90 l of RPMI (dilution 1). Ten microliters of dilution 1 are removed and 10 l of trypan blue (0.4%, Sigma Chemical Co., St. Louis, Mo.) is added. The number of cells in the 4 sets of 16 corner squares (WBC chamber) of the hemocytometer are counted No of cell=ml ¼ No of cells per 16 squares  20  5  104 6. The cells are resuspended to 5  106/ml in RPMI þ 8% FBS. Anti-Ovalbumin Antibody Titrations

Solutions To prepare PBS for ELISA, NaCl (8.0 g), KH2PO4 (0.2 g), and KCl (0.2 g) are mixed, and distilled water is added to 1 l. PBS-Tween is PBS for ELISA containing 0.5-ml Tween 20/l. Skim milk powder (Difco, Detroit, MI.) at 0.5% and 3% (wt/vol) is dissolved in PBS for ELISA. The blood is allowed to clot at 4 in Microtainer serum separator tubes (Becton Dickinson) and centrifuged at 10,000g for 5 min to obtain the serum. Antibody titers are determined by an indirect antigen-specific ELISA. Enzyme immunoassay microtitration plates (96 wells, flat bottomed, from ICN Biomedicals, Inc., Aurora, OH) are coated with antigen in water  (10 g/ml) by adding 0.1 ml/well and incubating at 37 until dry. The wells are washed four times with PBS-Tween, and all liquid is removed. To block nonspecific binding sites on the plastic 0.2 ml of 3% skim milk is added to each well, and incubated for 1 h at 37 . The wells are washed four times with PBS-Tween. Sera from individual mice are diluted 1:100 in 0.5% skim milk and further diluted serially (2x) in duplicate as the test antibody. The  plate is incubated for 1 h at 37 and then washed four times. An appropriate dilution of horseradish peroxidase–conjugated goat anti-mouse immunoglobulin-revealing antibody (Caltag, San Francisco, CA) is made in 0.5% skim milk to measure total antibody titers. The incubation and washes are repeated. Color development is initiated with the ABTS Microwell peroxidase system (Kirkegaard and Perry Laboratories, Gaithersburg,  MD), and the absorbance is determined at 415 nm after 15 min at 23 .

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12,500

Anti-OVA Ab titers

10,000 OVA Conventional-OVA M. smithii-OVA 7500

5000

2500

0

30.0

58.0 Days

150.0

Fig. 4. Comparison in anti-OVA antibody titers in sera of C57BL/6 mice immunized with 15 g of OVA (OVA, no adjuvant), M. smithii-OVA archaeosomes (15 g OVA in 0.46 mg archaeosomes), and conventional-OVA liposomes (15 g in 1.44 mg liposomes). Mice were bled at the times shown after first injections.

Titers are calculated as the end point dilutions exhibiting an optical density of 0.3 U above background. Results shown in Fig. 4 reveal higher anti-OVA antibody titers in sera of mice immunized with M. smithii OVA-archaeosomes compared with conventional OVA-liposomes. Proliferative Responses of Splenic Cells

Antigen-induced proliferation assays are performed in 96-well roundbottom tissue culture plates. Spleen cells (0.1 ml, 5  105/well) are cultured, including 0.1 ml OVA to achieve 0, 5, 25, and 100 g/ml (final concentrations) in triplicate wells, in RPMI 1640 þ 8% FBS. Dilutions for 24-, 48-, and 72-h incubations are set up in different plates. After incubation at 37 for the appropriate length of time, the supernatant (150 l) is collected without disturbing the cell pellet. If needed, the plates are centrifuged before collecting supernatants. Supernatants are stored at  70 for cytokine assays. The recommended length of cell culture time

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before collection of supernatants is 48–72 h for assays of IFN- and IL-4. Fresh RPMI 1640 (50 l) þ 8 % FBS medium is added to the cell pellet, and then 50 l [3H]thymidine (1 Ci/well) is added. The cells are incubated  for an additional 18–20 h at 37 and then harvested onto 90  120 mm glass fiber filters (Wallac, Oy Turku, Finland), and the [3H]thymidine incorporated is quantified by scintillation counting. Typical proliferative

A kCPM

60 40 20

B

IFN-γ ng/ml

6

3

0

IL-4 pg/ml

100

50

0 0.0

25.0 100.0 Ovalbumin in vitro ( m g/ml) Ova alone

Conventional

M. smithii

Alum

Fig. 5. OVA-specific spleen cell proliferation (A) and induction of both Th1 and Th2 cytokines (B) by M. smithii archaeosomes after immunization. Groups of female BALB/c mice were immunized twice at 0 and 3 weeks with 15 g OVA either without an adjuvant, adsorbed to alum, entrapped in archaeosomes, or entrapped in conventional liposomes. Spleens were harvested on day 28. Cytokines were quantified in 72-h culture supernatants. Data from Krishnan et al.6 are shown with permission from the American Society for Microbiology.

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responses of splenocytes are similar for OVA-archaeosome and OVAalum adjuvanted mice, and lesser for OVA-conventional liposome immunized animals (Fig. 5A). Cytokine Sandwich Enzyme-Linked Immunosorbent Assay

The following solutions are required to quantify cytokines in the culture supernatants prepared previously.21 Solutions 25  PBS: 33 g NaH2PO4, 188 g K2HPO4, and 1000 g NaCl taken to 5-l with distilled water, pH 7–7.5. Sample diluent: 8% FBS in RPMI 1640 medium. Secondary antibody and conjugate diluent: To prepare 1% BSA in PBS-Tween, 20 ml of 25  PBS, 0.25 ml Tween 20, and 5 g BSA are mixed and diluted to 400 ml with H2O. This is stirred until BSA dissolves, and the volume adjusted to 500 ml with distilled H2O. Na2HPO4: 26.8 g in 500 ml H2O. Citric acid: 10.5 g in 500 ml H2O (make sure citric acid is high purity, Fisher brand recommended, and check to see that the pH is strongly acidic). ABTS substrate in a 50 ml total volume: 50 mg ABTS, 11 ml Na2HPO4, 14 ml citric acid, 25 ml H2O, and 5 l H2O2. All final reagents are filter-sterilized through 0.2-m filters and stored  at 4 for long-term use. Enzyme immunoassay microtitration plates (96 wells, flexible, U-bottom, from Falcon, Beckton and Dickinson, Franklin Lakes, N. J.)  are coated for 1 h at room temperature (23 ) with 50 l/well of first antibody diluted in PBS. The wells are washed twice with PBS-Tween. All liquid is discarded and the plates are patted dry. Fifty microliters of sample are added and standards/well diluted in RPMI containing 8% FBS and incubated at room temperature for 1 h, followed by two washes in PBSTween. Usually two standard curves composed of several doubling dilutions, running in opposite directions along both edges, are included on each plate. Samples are usually tested at 1:2 dilution for cell culture supernatants from antigen-induced stimulations. The sample step is followed by 50-l/ well biotinylated-second antibody diluted in PBS-Tween, incubated for 30 min at room temperature, and washed twice. Next, 50 l/well of appropriately diluted streptavidin-HRPO conjugate is added (Jackson 21

T. R. Mosmann and T. A. Fong, J. Immunol. Methods 116, 151 (1989).

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ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS-Tween. This is incubated for 30 min at room temperature and washed twice. Color development is initiated with 100 l/well of fresh ABTS substrate prepared as described previously, or the ABTS Microwell peroxidase system as described for evaluation of antibody titers. Color development occurs optimally when incubations are done in the dark. The absorbance at 415 nm is determined when the color development reaches optimum for the standard curve ( 15–20 min). Antibody pairs used for these assays include RA-6A2 (ATCC HB170) þ XMG1.2-biotin22 for IFN-, and 11B1123 þ BVD6-24G2-biotin (Pharmingen Canada Inc, Mississauga, Canada) for IL-4. Each pair of first and second antibody is titrated, using a range of standards from 20 ng onward, to establish appropriate concentrations. IFN- and IL-4 standards may be purchased from ID Labs (London, Canada). We suggest that samples that are to be compared against each other always be tested in the same assay. Results shown in Fig. 5B reveal substantial IFN- production only from splenic cultures of M. smithii archaeosome-OVA–immunized mice. On the other hand, both archaeosomes and alum induce IL-4 production. Cytoytic T-Cell Assay

The CTL assay to follow requires that antigen-specific effector cells be restimulated in vitro to increase populations to easily measure specific killing of a target cell presenting a target peptide(s). Direct measurement of numbers of antigen-specific effectors in splenic populations requires other techniques, such as Elispot, not described here. 1. Spleen cell suspensions are prepared as described for assay of the proliferative response in the previous section. In a 25-cm2 tissue culture flask (Falcon) 30  106 spleen cells and 5  105 irradiated target cells (10,000 rad) are cultured. The target cells are EG.7, a subclone of EL-4 (T cell lymphoma) stably transfected with the gene encoding OVA (ATCC TIB 39). Interleukin-2 (0.1 ng/ml) (ID labs, London, Ont.) is added 10 l/flask from a 100 ng/ml solution. The total volume in the flask is made up to 10 ml with RPMI þ 8% FBS medium. The flask is placed upright in  the incubator at 37 , 8% CO2, and 95% humidity, and incubated for 5 days. 2. 51Cr labeled targets are prepared. EG.7 target cells (5  106), and EL-4 control cells not expressing OVA are centrifuged in 15-ml tubes at 470g for 10 min. As much of the supernatant as possible is removed, and 22

H. M. Cherwinski, J. H. Schumacher, K. D. Brown, and T. R. Mosmann, J. Exp. Med. 166, 1229 (1987). 23 J. Ohara and W. E. Paul, Nature 315, 333 (1985).

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the cell pellet is tapped gently. 51Cr (75 Ci) is added to the tube and  mixed well with a pipette tip. The targets are inculated at 37 for 45 min. The cap of the 15-ml tube is loosened during incubation. RPMI medium (10 ml) is added to the 51Cr-loaded cells and spun at 470g, 10 min. All the supernatant is discarded carefully without disturbing the cell pellet. The cell pellet is tapped to loosen the cells and 10 ml of fresh RPMI medium is  added. The cells are reincubated at 37 for 10 min. The cells are centrifuged and again the supernatant is discarded as before. The cells are resuspended in 5 ml of RPMI þ 8% FBS medium. 3. Effector cells are prepared. The cells are retrieved from the restimulation flasks into a 50-ml tube. The cells are counted using a hemocytometer as described previously. The effector cells are spun at 470g for 10 min and resuspended to obtain a final concentration of 100  106/ml. 4. The CTL assay is performed in a 96-well, round-bottomed tissue culture plate (Falcon). The killing potential of each splenic sample is tested at various effector/target ratios, in triplicate. RPMI þ 8% FBS medium (25 l) is added to wells A3–A12, numbered from right to left. In wells A1–A3 37.5 l of effector cells is added from the original stock of 100  106 cells/ml. 12.5 l is removed from wells A1–A3, transferred to wells A4–A6 and aspirated well using the same set of tips. Again 12.5 l is transfered from wells A4–A6 into wells A7–A9. This procedure is repeated for the last set of three wells of rows A9–A12. Here, after aspirating, the extra 12.5 l is discarded, and 25 l of labeled target cells (from stock of 106/ml) is added to all the wells. Thus, effector/target ratios of 100:1, 33.3:1, 11.1:1 and 3.7:1 are achieved. Finally, 50 l of RPMI þ 8% FBS medium is added to all wells to achieve a volume of 100 l. In triplicate wells measure spontaneous release (SR) by including 25 l of labeled targets and 75 l of medium, and maximal release (MR) by including 25 l of labeled targets, 25 l of medium, and 50 l of 1% NP40 (Sigma Chemical Co., St. Louis,  MO). The plates are incubated for 4 h at 37 . As a cautionary note, while plating one must ensure the cells are dropped to the bottom and not the sides of the well. If in doubt, the plate is spun for 5 min at 375g before incubating. 5. The release of 51Cr is counted from labeled EL-4 and EG.7 cells. After 4 h, the cells are sedimented by centrifuging the plate at 375g for 5 min, and 70 l of the supernatant is transferred (without disturbing the cell pellet) to counting tubes. The tip is left in the tube and counted in a gamma counter. The percent specific lysis is calculated using the formula: [(Cpm experimental  cpm spontaneous) / (cpm total  cpm spontaneous)]  100. Cytotoxic T cell data may be expressed also as lytic units defined as the number of effector cells per 106 spleen cells that yield a specific percentage killing (e.g., 20%) of a defined number (e.g., 2.5  104) of target cells.

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EG-7

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60

40

20

0

101

101

102

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E:T ratio M. smithii

CD8 depleted M. smithii

No adjuvant

Fig. 6. Abrogation of archaeosome-mediated CTL activity by elimination of CD8þ T-cell effectors. C57BL/6 mice were given intraperitoneal injections on days 0 and 21 with 15 g OVA in PBS (no adjuvant), or entrapped in M. smithii archaeosomes. Spleens were harvested on day 28. CD8þ T cells were eliminated from an aliquot of M. smithii effectors (CD8 depleted—M. smithii) on day 5 of restimulation with addition of anti-CD8 antibody and rabbit complement. Data are shown from Krishnan et al.7 with permission from The Journal of Immunology.

A typical CTL experiment for immunization by either intraperitoneal or subcutaneous routes (Fig. 6) indicates little, to no, killing of OVAnegative EL-4 cells. Furthermore, OVA-specific effectors are essentially absent in splenic cells from mice immunized with nonadjuvanted OVA. However, substantial killing of targets is observed for stimulated splenic cells from mice immunized with M. smithii OVA-archaeosomes. Because killing activity may be abolished by removing CD8þ T cells from the splenic population, it is clear that killing is mediated by OVA-specific CD8þ T cells arising in vivo from the OVA-archaeosome immunizations. Acknowledgments We wish to thank members of the archaeosome team, Mr B.J. Agnew, Ms. Lise Deschatelets, Ms. Chantal Dicaire, and Mr. P. Fleming for excellent technical expertise over the past decade. This is NRC publication 42480.