A structural comparison of the total polar lipids from the human archaea Methanobrevibacter smithii and Methanosphaera stadtmanae and its relevance to the adjuvant activities of their liposomes.

Mice were immunized with bovine serum albumin (BSA) entrapped within archaeosomes (i.e. liposomes) composed of the total polar lipids (TPL) from the two methanogenic archaea common to the human digestive tract. Methanobrevibacter smithii archaeosomes boosted serum anti-BSA antibody to titers comparable to those achieved with Freund's adjuvant, whereas Methanosphaera stadtmanae archaeosomes were relatively poor adjuvants. An explanation for this difference was sought by analysis of the polar lipid composition of each archaeobacterium. Fast atom bombardment mass spectrometry and NMR analyses of the purified lipids revealed a remarkable similarity in the ether lipid structures present in each TPL extract. However, the relative amounts of each lipid species varied dramatically. The phospholipid fraction in M. stadtmanae TPL was dominated by archaetidylinositol (50 mol% of TPL) and the glycolipid fraction by beta-Glcp-(1,6)-beta-Glcp-(1,1)-archaeol (36 mol%), whereas in M. smithii extracts, both caldarchaeol and archaeol lipids containing a phosphoserine head group were relatively abundant. Liposomes prepared from purified archaetidylinositol and from M. stadtmanae TPL supplemented with increasing amounts of phosphatidylserine elicited poor humoral responses to encapsulated BSA. A dramatic loss in the adjuvanticity of M. smithii archaeosomes was seen upon incorporation of 36 mol% of the uncharged lipid diglucosyl archaeol and, to a lesser extent, of 50 mol% of archaetidylinositol. Interestingly, the relative rates of uptake of M. smithii and M. stadtmanae archaeosomes by phagocytic cultures in vitro were similar. Thus, the lipid composition may influence archaeosome adjuvanticity, particularly a high diglucosyl archaeol and/or archaetidyl inositol content, resulting in a low adjuvant activity.     

In vitro evaluation of archaeosome vehicles for transdermal vaccine delivery

Abstract

Archaeosomes composed of archaeal total polar lipids (TPL) or semi-synthetic analog vesicles have been used as vaccine adjuvants and delivery systems in animal models for many years. Typically administered by intramuscular or subcutaneous injections, archaeosomes can induce robust, long-lasting humoral and cell-mediated immune responses against entrapped antigens and provide protection in murine models of infectious disease and cancer. Herein, we evaluated various archaeosomes for transdermal delivery, since this route may help eliminate needle-stick injuries and needle re-use, and therefore increase patient compliance. Archaeosomes composed of TPL from different archaea (Halobacterium salinarum, Methanobrevibacter smithii, Haloferax volcanii) and various semi-synthetic glycolipid combinations were evaluated for their ability to diffuse across the skin barrier using an ex vivo pig skin model and the results were compared to conventional synthetic ester liposomes. Physicochemical characteristics were determined for selected formulations including vesicle size, size distribution, zeta potential, fluidity, antigen (ovalbumin) incorporation efficiency and release. Archaeosomes, in particular those composed of M. smithii TPL or the synthetic glycolipid sulfated S-lactosylarchaeol (SLA) mixed with uncharged glycolipid lactosyl archaeol (LA), appeared to be effective carriers for ovalbumin, achieving much better antigen distribution and vesicle accumulation in the skin epidermis than conventional liposomes. The enhanced skin permeation of archaeosomes may be attributed to their chemical structure and physicochemical properties such as particle size, surface charge, stability, and fluidity of their lipid bilayer.     

Archaeosomes as adjuvants for combination vaccines

The present study evaluated the potential of archaesomes, prepared from the total polar lipids extracted from Methanobrevibacter smithii, as adjuvants for combination (multivalent) vaccines. Groups of Balb/c mice were immunized subcutaneously at day 0 and 21 with one of the following vaccines: trivalent vaccine formulated by the simultaneous co-encapsulation of bovine serum albumine (BSA), ovalbumin (OVA) and hen egg lysozyme (HEL) into archaeosomes (CEC vaccine); an univalent archaeosome vaccine (UVE vaccine) containing either BSA, OVA or HEL; or an admixture vaccine (AMC vaccine) consisting of the three UVE vaccines. Serum specific antibody (IgG + M) responses were determined at day 32, 112 and 203, and specific IgG1 and IgG2a responses were determined at day 112. Mice immunized with the CEC of AMC vaccine developed strong and sustained specific antibody responses to all three antigens at a magnitude similar to those seen in control mice immunized with UVE vaccines. Moreover, the serum BSA-, OVA-, and HEL-specific IgG1 and IgG2a levels in the CEC and AMC immunized mice were overall comparable to those of the UVE immunized control mice. Boosting CEC and AMC vaccinated mice with antigens alone at day 203 elicited strong antibody memory responses, comparable to those in the UVE vaccinated groups. These results show that archaeosomes could be used as adjuvants in developing combination vaccines.     

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, D011.10 (CD4(+) cells expressing OVA(323-339) TCR) or OT1 (>90% CD8(+) OVA(257-264) cells), indicating both MHC class I and II presentations. In vivo, when naive (Thy1.2(+)) CFSE-labeled OT1 cells were transferred into OVA-archaeosome-immunized Thy 1.1(+) recipient mice, there was profound accumulation and cycling of donor-specific cells, and differentiation of H-2K(b)Ova(257-264) CD8(+) T cells into CD44(high)CD62L(low) effectors. Both macrophages and dendritic cells (DCs) efficiently cross-presented OVA-archaeosomes on MHC class I. Blocking phagocytosis by phosphatidylserine-specific receptor agonists strongly inhibited MHC class I presentation of OVA-archaeosomes, whereas blocking mannose receptors or FcRs lacked effect, indicating specific recognition of the archaetidylserine head group of M. smithii lipids by APCs. In addition, inhibitors of endosomal acidification blocked MHC class I processing of OVA-archaeosomes, whereas endosomal protease inhibitors lacked effect, suggesting acidification-dependent phagosome-to-cytosol diversion. Proteasomal inhibitors blocked OVA-archaeosome MHC class I presentation, confirming cytosolic processing. Both in vitro and in vivo, OVA-archaeosome MHC class I presentation required TAP. Ag-free archaeosomes also activated DC costimulation and cytokine production, without overt inflammation. Phosphatidylserine-specific receptor-mediated endocytosis is a mechanism of apoptotic cell clearance and DCs cross-present Ags sampled from apoptotic cells. Our results reveal the novel ability of archaeosomes to exploit this mechanism for cytosolic MHC class I Ag processing, and provide an effective particulate vaccination strategy.     

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. 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 of archaeosomes 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.     

Interleukin-2/liposomes potentiate immune responses to a soluble protein cancer vaccine in mice

A critical element in improving the potency of cancer vaccines, especially pure protein or peptide antigens, is to develop procedures that can strongly but safely increase their ability to induce immune responses. Here, we describe that encapsulation of a pure protein antigen and interleukin-2 (IL-2) together into liposomes significantly improves immune responses and tumor protection. Groups of C57Bl/6 mice were immunized weekly ×4 with –0.1 mg of ovalbumin (OVA) injected subcutaneously in PBS or encapsulated in liposomes with or without human recombinant IL-2. Control groups included mice immunized to irradiated E.G7-OVA cells (that express ovalbumin), or to PBS. Sera were collected and pooled by immunization group at baseline and at weeks 2 and 4 to measure antibody responses to OVA by ELISA. Splenocytes obtained at week 4 were tested for anti-OVA cellular responses by ELISPOT. Mice were then challenged to a lethal dose of E.G7-OVA cells to measure tumor-protective immunity. IL-2 liposomes caused no detectable toxicity. Antibody, CD8⁺ T cell, and tumor-protective immune responses were markedly enhanced in mice immunized to OVA + IL-2 in liposomes compared to mice immunized to OVA, either alone or encapsulated into liposomes without IL-2. These results indicate that IL-2 liposomes enhance antibody, cellular, and tumor-protective immune responses to immunization with a soluble protein. This may provide a simple, safe, and effective way to enhance the immunogenicity of vaccines that consist of pure protein antigens. Supported by grant CA096804 (DJ)     

Stability of Liposomes Prepared from the Total Polar Lipids of Methanosarcina Mazei is Affected by the Specific Salt form of the Lipids

Abstract

The total polar lipids (TPL) extracted from the archaeobacterium Metha-nosarcina mazei were predominantly in the form of sodium and potassium salts. Upon passage of this natural salt form of TPL (ns-TPL) through a silica gel G column (s-TPL), the molar ratio of the monovalent cations, sodium plus potassium, to that of the divalent cations, magnesium plus calcium, decreased from about 11:1 to 2:1. Liposomes (archaeosomes) made from ns-TPL were unable to efficiently retain low molecular weight aqueous markers such as 5(6)-carboxyfluorescein (CF). In phosphate buffered saline (PBS, pH 7.14), between 60-90% and 90-100% of entrapped CF leaked out during 21 h storage at room temperature (20-22°) and 3 h at 50°, respectively. However, archaeosomes made with the s-TPL, as well as from TPL converted to the salt forms of predominantly sodium, potassium, calcium or magnesium, were significantly less leaky. The archaeosomes made with s-TPL were the most stable, showing less than 7% leakage after 21 h at room temperature and only 14% leakage of CF after 3 h at 50°. The stability of s-TPL archaeosomes (21 h, 20-22°) was not affected when Tris-HCI buffer (pH 7.4) was used instead of PBS. However, the inclusion of 1 or 10 mM EGTA in the Tris-HCI buffer increased the amount of CF leaked, to about 25% and 100%, respectively. Except for some differences in phosphatidylserine and phosphati-dylglycerol, the lipid compositions of ns-TPL, s-TPL, and the magnesium form of TPL were similar, as determined by thin layer chromatography of labeled lipids. Archaeosomes prepared from s-TPL and ns-TPL had -“P NMR spectra that were similar to each other, but distinct from those of vesicles prepared from the ester lipid dimyristoylphosphatidylcholine. The types and relative proportions of cations associated with the lipids of M. mazei prior to their hydration and vesicle formation have a major influence, although other factors such as lipid composition may have an effect, on the permeability of the bilayer to low molecular weight compounds.     

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). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.     

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers — OS) or completely (dense shell glycodendrimers — DS) modified with maltose residues. As a model membrane, two types of 100 nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D₂O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.     

Structural characterization of archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) formulations prepared by different protocols and their efficacy upon intranasal immunization of mice

Intranasal administration of ovalbumin (OVA) formulated in an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system prepared by the addition of CaCl2 to small unilamellar archaeosomes (liposomes made from archaeal polar lipids) containing encapsulated OVA, was recently shown to elicit strong and sustained OVA-specific mucosal and systemic immune responses. In this study, we show that the centrifugation/washing and antigen quantization steps required in the standard protocol for obtaining OVA/AMVAD model vaccine formulations can be eliminated by using simpler protocols such as admixing OVA with preformed empty archaeosomes, or by changing the starting ratio (w/w) of archaeal lipid to antigen at the archaeosome preparation stage, prior to the addition of CaCl2 to convert to the AMVAD structures. Irrespective of the vaccine preparation protocol, the AMVAD particle typically comprised of larger spherical structures that had aggregated like a bunch of grapes, and it contained aqueous compartment(s). The anti-OVA IgA antibody responses in vaginal wash, nasal wash, serum, and bile samples, and the anti-OVA IgG antibody responses in sera, in mice intranasally immunized with the OVA/AMVAD formulations prepared by the simplified or the standard protocols, were comparable.     

Derivative spectrophotometry as a tool for the determination of drug partition coefficients in water/dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) liposomes.

The partition coefficients (K(p)) between lipid bilayers of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) unilamellar liposomes and water were determined using derivative spectrophotometry for chlordiazepoxide (benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and dibucaine (local anaesthetic). A comparison of the K(p) values in water/DMPG with those in water/DMPC (dimyristoyl-L-alpha-phosphatidylcholine) revealed that for chlordiazepoxide and isoniazid, neutral drugs at physiological pH, the partition coefficients are similar in anionic (DMPG) and zwitterionic (DMPC) liposomes. However, for ionised drugs at physiological pH, the electrostatic interactions are different with DMPG and DMPC, with the cationic dibucaine having a stronger interaction with DMPG, and the anionic rifampicin having a much larger K(p) in zwitterionic DMPC. These results show that liposomes are a better model membrane than an isotropic two-phase solvent system, such as water-octanol, to predict drug-membrane partition coefficients, as they mimic better the hydrophobic part and the outer polar charged surface of the phospholipids of natural membranes.     

Solubilization of Multilamellar Liposomes in the Presence of Non-ionized Drug

The solubilization of multilamellar liposomes by metoprolol tartrate (MPL) has been studied as a function of pH, [MPL], [dimyristoylphosphatidylcholine (DMPC)], temperature and lipid composition. The solubilization of liposomes at 37° C by 7.3 mM MPL occurred at different rates at different pH values. MPL completely solubilized by 7.2 mM DMPC liposomes after about 17 hat pH 12, but only a partial solubilization occurred at pH 10 and 11. Between pH 7 and 9 no change in turbidity was observed after 1 week. Addition of cholesterol (CHOL) to DMPC (2:1 mol) had very little effect on solubilization after 24 h, however with DMPC:CHOL (5:1 mol) the decrease in turbidity was observed after 24 h, even though solubilization was much less compared with that of DMPC alone. The rate of solubilizaiton was decreased when dipalmitoylphosphatidylcholine liposomes were employed. Addition of dicetylphosphate (DCP) to DMPC liposomes reduced the rate of solubilization significantly. The solubilization of liposomes by 7.3 mM MPL as a function of [DMPC], indicated that the lower the liposome concentration the greater the effect on solubilization. It is concluded that MPL in the non-ionized form has a solubilizing effect on liposomes, and addition of CHOL or DCP to DMPC has a stabilizing effect against solubilization.     

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. A starch-derived trimer was also synthesized as a control. To promote hydration and form stable archaeosomes, an archaeal anionic lipid archaetidylglycerol (AG) was included in a 4:1 molar ratio. Archaeosomes prepared from Manp(1-2)-AG were recovered at only 34-37%, whereas Manp(3-4)-AG recoveries were 72-77%. Lipid recovery following hydration of Manp(5)-AG archaeosomes declined to 34%, indicating an optimum of 3-4 Manp units for bilayer formation. The CD8(+) T cell response in mice immunized with Manp(3-5) archaeosomes containing ovalbumin was highest for Manp(4) and declined for Manp(3) and Manp(5), revealing an optimum length of four unbranched units. The starch-derived trimer was more active than the Manp oligomers, suggesting the involvement of either a general binding lectin on antigen-presenting cells with highest affinity for triglucose or multiple lectin receptors.     

Serum albumin-lipid membrane interaction influencing the uptake of porphyrins

It is frequently observed in pharmaceutical practice that entrapped substances are lost rapidly when liposomes are used as carriers to introduce substances into cells. The reason for the loss is the interaction of serum components with liposomes. To elucidate the mechanism of this phenomenon the partition of mesoporphyrin (MP) was systematically studied in model systems composed of various lipids and human serum albumin (HSA). As surface charge is an important factor in the interaction, neutral (1, 2-dimyristoyl-sn-glycero-3-phosphatidylcoline, DMPC) and negatively charged (1,2-dimyristoyl-sn-glycero-3-phosphatidylcoline/1, 2-dimyristoyl-sn-glycero-3-phosphatidylglycerol, DMPC/DMPG = 19/1 w/w) lipids were compared. The liposome/apomyoglobin system was the negative control. The size distribution of sonicated samples was carefully analyzed by dynamic light scattering. Constants of association of MP to the proteins and to the liposomes were determined: K(p,1) = (2.5 +/- 0.7) x 10(7) M(-1), K(p,2) = (1.0 +/- 0.7) x 10(8) M(-1), K(L,1) = (1.3 +/- 0.3) x 10(5) M(-1), and K(L,2) = (3.2 +/- 0.6) x 10(4) M(-1) for HSA, apomyoglobin, DMPC, and DMPC/DMPG liposomes, respectively. These data were used to evaluate the partition experiments. The transfer of MP from the liposomes to the proteins was followed by fluorescence spectroscopy. In the case of apomyoglobin, the experimental points could be interpreted by ruling out the protein-liposome interaction. In the case of HSA, the efflux of MP from the liposomes was strongly inhibited above a critical HSA concentration range for negatively charged vesicles. This effect was interpreted as the result of HSA coat formation on the liposome surface. This direct interaction is significant for small liposomes. The interpretation is fully supported by differential scanning calorimetry experiments.     

Molecular Dynamics Simulations of Mixed Acidic/Zwitterionic Phospholipid Bilayers

Anionic lipids are key components in the cell membranes. Many cell-regulatory and signaling mechanisms depend upon a complicated interplay between them and membrane-bound proteins. Phospholipid bilayers are commonly used as model systems in experimental or theoretical studies to gain insight into the structure and dynamics of biological membranes. We report here 200-ns-long MD simulations of pure (DMPC and DMPG) and mixed equimolar (DMPC/DMPG, DMPC/DMPS, and DMPC/DMPA) bilayers that each contain 256 lipids. The intra- and intermolecular interaction patterns in pure and mixed bilayers are analyzed and compared. The effect of monovalent ions (Na⁺) on the formation of salt-bridges is investigated. In particular, the number of Na⁺-mediated clusters in the presence of DMPS is higher than with DMPG and DMPA. We observe a preferential clustering of DMPS (and to some extent DMPA) lipids together rather than with DMPC molecules, which can explain the phase separation observed experimentally for DMPC/DMPS and DMPC/DMPA bilayers.     

Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies

Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG.     

Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Gramicidin A was incorporated at a peptide/lipid ratio of 1:10 mol/mol in aligned bilayers of dimyristoyl phosphatidylcholine (DMPC), phosphatidylserine (DMPS), phosphatidylglycerol (DMPG), and phosphatidylethanolamine (DMPE), from trifluoroethanol. Orientations of the peptide and lipid chains were determined by polarized attenuated total reflection infrared spectroscopy. Lipid-peptide interactions with gramicidin A in DMPC bilayers were studied with different spin-labeled lipid species by using electron spin resonance spectroscopy. In DMPC membranes, the orientation of the lipid chains is comparable to that in the absence of peptide, in both gel and fluid phases. In gel-phase DMPC, the effective tilt of the peptide exceeds that of the lipid chains, but in the fluid phase both are similar. For gramicidin A in DMPS, DMPG, and DMPE, the degree of orientation of the peptide and lipid chains is less than in DMPC. In the fluid phase of DMPS, DMPG, and DMPE, gramicidin A is also less well oriented than are the lipid chains. In DMPE especially, gramicidin A is largely disordered. In DMPC membranes, three to four lipids per monomer experience direct motional restriction on interaction with gramicidin A. This is approximately half the number of lipids expected to contact the intramembranous perimeter of the gramicidin A monomer. A selectivity for certain negatively charged lipids is found in the interaction with gramicidin A in DMPC. These results are discussed in terms of the integration of gramicidin A channels in lipid bilayers, and of the interactions of lipids with integral membrane proteins.     

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). In this study, we evaluated the priming, phenotype, and functionality of the CD8(+) T cells induced after immunization of mice with OVA-Methanobrevibacter smithii archaeosomes (MS-OVA). A single injection of MS-OVA evoked a profound primary response but the numbers of H-2K(b)OVA(257-264)-specific CD8(+) T cells declined by 14-21 days, and <1% of primarily central phenotype (CD44(high)CD62L(high)) cells persisted. A booster injection of MS-OVA at 3-11 wk promoted massive clonal expansion and a peak effector response of approximately 20% splenic/blood OVA(257-264)-specific CD8(+) T cells. Furthermore, contraction was protracted and the memory pool (IL-7Ralpha(high)) of approximately 5% included effector (CD44(high)CD62L(low)) and central (CD44(high)CD62L(high)) phenotype cells. Recall response was observed even at >300 days. CFSE-labeled naive OT-1 (OVA(257-264) TCR transgenic) cells transferred into MS-OVA-immunized recipients cycled profoundly (>90%) within the first week of immunization indicating potent Ag presentation. Moreover, approximately 25% cycling of Ag-specific cells was seen for >50 days, suggesting an Ag depot. In vivo, CD8(+) T cells evoked by MS-OVA killed >80% of specific targets, even at day 180. MS-OVA induced responses similar in magnitude to Listeria monocytogenes-OVA, a potent live vector. Furthermore, protective CD8(+) T cells were induced in TLR2-deficient mice, suggesting nonengagement of TLR2 by archaeal lipids. Thus, an archaeosome adjuvant vaccine represents an alternative to live vectors for inducing CD8(+) T cell memory.     

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. Archaeosomes contrasted to the liposome adjuvants by exhibiting higher amounts of surface amino groups and inducing superior shorter and, especially, longer-term CTL responses. The incorporation of dipalmitoyl lipids into archaeosomes induced instability and prevented long-term, but not short-term, CTL responses in mice. The importance of glycero-lipid cores (isopranoid versus dipalmitoyl) to the longevity of the CTL response achieved was shown further by incorporating dipalmitoyl phosphatidylethanolamine (DPPE) or equivalent amounts of synthetic archaetidylethanolamine (AE) into archaeosome adjuvants. Both DPPE and AE at equivalent (5 mol%) concentrations enhanced the rapidity of CTL responses in mice, indicating the importance of the head group in the short term. In the longer term, 5% of DPPE (but not 5% of AE) was detrimental. In addition to head-group effects critical to the potency of short-term CTL responses, the longer term CTL adjuvant properties of archaeosomes may be ascribed to stability imparted by the archaeal isopranoid core lipids.     

Response of the phosphatidylcholine headgroup to membrane surface charge in ternary mixtures of neutral, cationic, and anionic lipids: a deuterium NMR study.

Deuterium nuclear magnetic resonance (2H NMR) spectroscopy was used to investigate the response of the phosphatidylcholine headgroup of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) to changes in surface electrostatic charge in membranes consisting of ternary mixtures of lipids. DMPC was deuterated at the choline alpha- and beta-methylene segments. The membrane surface charge was manipulated by the simultaneous addition of cationic didodecyldimethylammonium bromide (DDAB) and anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) to neutral DMPC. Addition of increasing amounts of DDAB caused a progressive decrease (increase) in the 2H NMR quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Addition of increasing amounts of DMPG caused a progressive increase (decrease) in the quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Qualitatively, the 2H NMR quadrupole splitting charge response exhibited the same main features for ternary mixtures of DDAB/DMPG/DMPC and binary mixtures of DDAB/DMPC or DMPG/DMPC. Quantitatively, however, the 2H NMR quadrupole splittings obtained from ternary mixtures did not coincide with those obtained from binary mixtures of nominally identical surface charge densities. Hence, the quadrupole splitting did not respond directly to the net membrane surface charge. Instead, the quadrupole splitting measured for a given ternary lipid composition could be reproduced by summing the individual effects of the charged lipids in binary mixtures, weighted according to their appropriate mole fractions.