Archaeosome Vaccine Adjuvants Induce Strong Humoral, Cell-Mediated, and Memory Responses: Comparison to Conventional Liposomes and Alum^†

Growth of archaea and extraction of lipids.

Halobacterium salinarum (“Halobacterium cutirubrum”) (ATCC 33170), Methanobacterium espanolae GP9 (DSM 5982), Methanobrevibacter smithii ALI (DSM 2375), Methanosphaera stadtmanae MCB-3 (DSM 3091), Thermoplasma acidophilum 122-1B3 (ATCC 27658), Methanosarcina mazei S-6 (DSM 2053), and Natronobacterium magadii MS3 (ATCC 43099) were cultivated in 75- to 250-liter fermentors as described earlier (10). Total lipids were extracted from frozen cell pastes, and the TPL were collected as the acetone-insoluble fraction (10).

Preparation and characterization of archaeosomes and conventional liposomes.

Archaeosomes were composed of the TPL from the different archaea mentioned above except for PGP-0-CH₃ archaeosomes. These were prepared from PGP-0-CH₃ (phosphatidylglyceromethylphosphate diether analog [20]) isolated from H. salinarum with a purity of at least 79%, determined by negative-ion fast atom bombardment-mass spectrometry. l-α-Dimyristoylphosphatidylcholine (DMPC), l-α-dimyristoylphosphatidylglycerol (DMPG), and cholesterol (CHOL) were purchased from Sigma Chemical Co., St. Louis, Mo., for the preparation of conventional liposomes, defined herein as DMPC-DMPG-CHOL (1.8:0.2:1.5 molar ratio) unless otherwise stated. Vesicles were prepared by pressure extrusion at 23°C with 400-nm-pore-size filters (9). Briefly, 20 mg of dried lipid was hydrated in 1 ml of phosphate-buffered saline (PBS) containing the protein Ag (10 mg/ml). Ag that was not associated with the vesicles was removed by ultracentrifugation (200,000 × g_max for 30 min) three times from 7-ml volumes of PBS. Mean vesicle diameters were determined by number-weighted Gaussian size distributions with a Nicomp Particle sizer (model 370; Nicomp, Santa Barbara, Calif.). The amount of protein incorporated into the vesicles was estimated, after lipid removal (45), by the sodium dodecyl sulfate (SDS)-Lowry method and comparison with standard curves constructed for the relevant protein. The ratio of protein to lipid (micrograms per milligram) is based on the salt-free dry weights of the vesicles.

Encapsulation versus surface localization of Ag in lipid vesicles.

Archaeosomes and conventional liposomes containing BSA were incubated with protease type IV from Streptomyces griseus (Sigma Chemical Co.). The assay consisted of incubating 50 μl of BSA-vesicles with, or without, 0.028 U of protease for 2 h at 35°C. Protease inhibitors, phenylmethylsulfonyl fluoride and leupeptin (Sigma), were then added from ethanolic solutions to achieve 50 μM each. After 0.5 h at ambient temperature, 5 μg of hen egg lysozyme (HEL) (Sigma) was added just prior to vesicle lysis with SDS sample buffer, and samples of 50 μl were placed immediately in a boiling water bath for 3 min. SDS-polyacrylamide gel electrophoresis and quantitative densitometry on bands stained with Coomassie brilliant blue R-250 (Bio-Rad, Richmond, Calif.) were done as described elsewhere (42). Controls were included within each experiment to verify that the protease inhibitors were effective and to confirm digestion of surface-bound Ag. For the latter, BSA was surface bound by incubating empty H. salinarum archaeosomes overnight at 4°C with 10 mg of BSA/ml of PBS, followed by one wash. Interfering bands were absent from all vesicle types lacking Ag.

Mice, immunization, and Ags.

Inbred, 6- to 8-week-old female BALB/c and C57BL/6 mice were purchased from Charles River Laboratories (St. Constant, Canada), and C3H/HeJ mice were from The Jackson Laboratory (Bar Harbor, Maine). Mice were maintained in the animal facility of the Institute for Biological Sciences, National Research Council, in accordance with guidelines of the Canadian Council on Animal Care.

Groups of four to six mice received two immunizations of the Ag in PBS (no adjuvant), in alum or Freund's adjuvant (FA), or entrapped in archaeosomes or conventional liposomes, as specified in the figure legends. Immunization volume was 100 to 200 μl, Ag dose was 8 to 15 μg/injection, and lipid concentration was 0.2 to 1.7 mg/injection. For alum immunizations, the Ag was adsorbed onto Imject alum (Pierce, Rockford, Ill.) according to the manufacturer's protocol. When FA (Sigma Chemical Co.) was used, Ag in PBS was emulsified with 62.5% complete FA in the first injection and incomplete FA for the second. Immunization routes used included intraperitoneal (i.p.), intramuscular (i.m.), and subcutaneous (s.c.) injection at the base of the tail. Ags tested included fatty-acid-free BSA, HEL, and ovalbumin (OVA), all purchased from Sigma Chemical Co.

Evaluation of antibody titers.

Mice were bled either from the tail veins or by cardiac puncture, and blood was collected in Microtainer serum separator tubes (Becton Dickinson, Franklin Lakes, N.J.). After the blood was allowed to clot (1 h at 4°C), the serum was separated by centrifugation and frozen at −20°C until assayed. The antibody levels were determined by indirect Ag-specific enzyme-linked immunosorbent assay (ELISA) (42). Briefly, ELISA plates (enzyme immunoassay microtitration plates, 96 wells and flat bottom; ICN Biomedicals, Inc., Aurora, Ohio) were coated with Ag in PBS (10 μg/ml), and serial twofold dilutions of serum (from individual mice) were assayed in duplicate. Horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Ig; IgG plus IgM) revealing antibody (Caltag, San Francisco, Calif.) was used to determine total antibody titers of sera. For isotyping of the sera, horseradish peroxidase-conjugated sheep anti-mouse isotype-specific (IgM-, IgG1-, IgG2a-, IgG2b-, or IgG3-specific) revealing antibodies (Serotech Inc.; distributed by Cedarlane Laboratories, Hornby, Canada) were used. The reactions were developed with the ABTS Microwell peroxidase system (Kirkegaard and Perry Laboratories, Gaithersburg, Md.), and absorbance was determined at 415 nm after 15 min. Antibody titers are represented as endpoint dilutions exhibiting an optical density of 0.3 U above background. Samples pertaining to each experiment were evaluated in the same assay.

Ag-specific proliferation of splenocytes.

Splenocytes were obtained on day 28 from individual mice immunized on days 0 and 21. Selective lysis of erythrocytes was performed with Tris-buffered ammonium chloride, pH 7.2 (Sigma Chemical Co.). Cells (5 × 10⁶/ml) were washed twice, and triplicate cultures were set up (72 h, 7% CO₂) in RPMI 1640 medium (Gibco-BRL, Life Technologies Inc., Grand Island, N.Y.) in 96-well round-bottom microtiter plates (Falcon; Becton Dickinson) in the presence or absence of Ag. The serum supplement used for culture included either defined equine serum (2%, for BSA-stimulated cultures) or fetal bovine serum (FBS; 8%, for HEL- and OVA-stimulated cultures), obtained from HyClone Laboratories Inc., Logan, Utah. Wells were pulsed with 1 μCi of [³H]thymidine (ICN Pharmaceuticals Canada, Montreal, Quebec, Canada) for 18 h, cultures were harvested onto glass-fiber filters, and the radioactivity incorporated was determined by liquid scintillation counting.

Cytokine assays.

Cytokines produced in supernatants of Ag-stimulated cultures were measured by a sandwich ELISA (29). Antibody pairs used included RA-6A2 (ATCC HB170) and XMG1.2-biotin (8) for IFN-γ, and 11B11 (31) and BVD6-24G2-biotin (Pharmingen Canada Inc., Mississauga, Canada) for IL-4. IFN-γ and IL-4 standards were purchased from ID Labs, London, Canada. Duplicate standard curves were included on each plate. The sensitivities of the ELISAs were as follows: IFN-γ, >100 pg/ml, and IL-4, >15 pg/ml. Regardless of thresholds, samples from each experiment were tested in the same assay.

Cell cycle analysis.

Cell cycle analysis was performed by quantitating the incorporation of DNA-binding dye (propidium iodide) by flow cytometry. Briefly, splenocytes (10⁶) were stained with fluorescein isothiocyanate-conjugated anti-CD4 antibodies (Pharmingen Canada Inc.) for 30 min on ice in 50 μl of RPMI medium containing 8% FBS. Cells were then washed and fixed overnight at 4°C in 1 ml of 70% ice-cold ethanol. Fixed cells were stained with propidium iodide (Calbiochem, La Jolla, Calif.) in the presence of RNase A (100 U/ml; Boehringer Mannheim, Laval, Canada) and analyzed by flow cytometry (EPICS XL; Coulter Corp., Hialeah, Fla.). DNA content histograms gated on CD4⁺ T cells were obtained with EXPO software (Coulter Corp.). As the ploidy of the DNA increases as cells enter synthetic and mitotic phases, each cell incorporates greater amounts of propidium iodide. Thus, the percentages of cells in various phases of the cell cycle (apoptotic, G₁ [resting], S [synthetic], and G₂/M [mitotic]) were calculated based on DNA content.

Statistical analyses.

Student's t test was done to determine statistical significance between different immunization groups for the various experiments, whereas analysis of variance (ANOVA) was used for long-term memory data to determine differences within groups (over time). P < 0.05 was considered statistically significant.

Choice of archaeosome types.

The archaeosome types tested were chosen to represent divergent sources of TPL, based on their lipid structures. TPL with high caldarchaeol (tetraether lipids) content (T. acidophilum, >90%; M. espanolae, 65%) are known to form the most stable archaeosomes (40). M. smithii was chosen because it represents the major archaeon resident in the human gut (28) and has a caldarchaeol content of 40 mol%. On the other hand, M. stadtmanae was included occasionally as an example of a human isolate with relatively low humoral, adjuvant properties. The phospholipids and glycolipids of H. salinarum and their sulfated forms are unique and have been characterized extensively (20). M. mazei was of interest as a representative TPL composed almost exclusively of phosphoarchaeols with simple phospho-head groups of inositol, glycerol, serine, and ethanolamine (41). Overall, we attempted to include at least one of the three most promising candidate archaeosomes (M. smithii, T. acidophilum, or H. salinarum) in each experiment.

BSA-archaeosomes induce strong humoral responses in mice of varying genetic backgrounds.

In earlier studies, we have demonstrated the ability of several archaeosome types to induce a strong antibody response to the entrapped protein Ag (42). The three most potent archaeosome adjuvants were tested in the current study for their ability to induce antibody responses in mice of different genetic backgrounds. Inbred mouse strains BALB/c, C57BL/6, and C3H/HeJ were immunized (i.p.) with BSA entrapped in archaeosomes comprised of TPL of different archaea or conventional liposomes or in conjunction with traditional adjuvants such as alum and FA, and anti-BSA antibodies were evaluated in the sera. Entrapment of BSA in archaeosomes resulted in elevated anti-BSA antibody titers (total IgG plus IgM) in all three mouse strains (Fig. 1). The antibody titers achieved by entrapping BSA in archaeosomes were often comparable to that found with the potent FA and superior to those with alum and conventional liposomes. A low Ag dose (10 μg/injection) was used for these experiments, demonstrating the potency of archaeosomes in facilitating a strong humoral response. Immunization with bare Ag (BSA in PBS) failed to evoke an anti-BSA antibody response, reiterating the poor immunogenicity of BSA. Thus, adjuvant activity of archaeosomes is not restricted by major histocompatibility complex haplotype of the mouse strains.

Isotype distribution.

Sera collected from immunized BALB/c mice were analyzed for isotype distribution. Figure 2 indicates the serum distribution of Ag-specific IgG1, IgG2a, and IgG2b antibodies. The overall magnitude of anti-BSA-specific IgG1 antibody titers was higher than that of IgG2a and IgG2b. Nevertheless, titers of all three antibodies were significantly enhanced by BSA-archaeosomes in comparison to those induced after BSA-alum immunization (P < 0.05). The induction of both IgG1 and IgG2a antibodies by archaeosomes suggests efficient major histocompatibility complex class II presentation of the Ag leading to both a humoral (IgG1) and a cell-mediated (IgG2a; complement-fixing antibodies) response. In all cases, very little anti-BSA IgM antibodies were detected, indicating strong class switching, whereas IgG3 antibodies were undetectable (data not shown).

Internalization of BSA within archaeosomes.

Because of the lack of information on the stability-degradability of archaeosomes in vivo, it was especially relevant to determine whether the protein Ag had first to be released from the internal space of the archaeosome or whether only surface-bound Ag could be presented from these relatively stable vesicles. Data showed that any BSA which was surface bound was susceptible to protease IV treatment and that surface localization versus internalization of the protein varied with the composition of the vesicle (Table 1). However, the equivalent ability to evoke an antibody response by archaeosome types with >80% internalized Ag (M. smithii and T. acidophilum), compared to those where substantial amounts of the antigen were surface located (H. salinarum), suggested that efficient vesicle degradation had occurred in vivo.

BSA must be archaeosome associated to induce strong anti-BSA antibody titers.

To test the effect of Ag encapsulation on the adjuvant action of archaeosomes, mice were immunized with BSA entrapped in archaeosomes or given separate injections of the respective weights-concentrations of empty archaeosomes and BSA. Archaeosome types were chosen to represent a high caldarchaeol (M. espanolae and T. acidophilum), a moderate caldarchaeol (M. smithii), or a simple phosphoarchaeol (M. mazei) composition. Data in Table 2 indicate that association of archaeosomes and Ag, either internalized or surface bound, is required to achieve high antibody titers, by both i.p. and i.m. routes. A significant drop in the titers was observed (for all archaeosome types irrespective of lipid composition) when empty archaeosomes and BSA were administered separately. The TPL of M. espanolae are largely uncharacterized structurally and were excluded from further studies. Similarly, M. mazei archaeosomes were excluded upon discovering that the permeability of these vesicles is dependent upon the salt form of the TPL used in their construction (32).

Archaeosomal lipids induce superior humoral responses compared to conventional liposomal lipids.

Liposomes composed of synthetic ester-lipids (conventional liposomes) have been used in the past to facilitate humoral responses to entrapped Ags (1, 14). In earlier studies (26, 42), as well as the current one (Fig. 1), we have observed that archaeosome-facilitated humoral responses are significantly superior to that of conventional liposomes. The adjuvant action of conventional liposomes was directly compared to that of archaeosomes by immunizing mice with BSA entrapped in M. smithii archaeosomes, conventional DMPC-DMPG liposomes, or vesicles composed of mixtures of archaeal and ester lipid types (Table 3). A potent anti-BSA antibody response was observed when Ag was delivered in vesicles made with 100% M. smithii TPL. This response dropped significantly (P < 0.05) with increasing proportions of DMPC-DMPG in the vesicle composition, although entrapment of Ag in 100% DMPC-DMPG vesicles yielded a moderately higher antibody titer than did bare antigen. Day 35 titers were evaluated after three (injections on days 0, 14, and 31) instead of the usual two immunizations. Even under these conditions, antibody titers induced by conventional liposomes failed to equal that of archaeosomes.

Ag/lipid ratio influences the magnitude of humoral response.

One factor that may influence the adjuvant action of archaeosomes is the ratio of Ag to TPL. To specifically address this issue, mice were immunized with archaeosomes containing varying Ag loading to achieve a constant entrapped-Ag dose of 8 μg/injection within a range of 0.15 to 3.1 mg (dry weight) of BSA-archaeosomes. Data in Fig. 3 demonstrate the dose-dependent effect of H. salinarum lipids on the antibody titers to the entrapped Ag. Interestingly, both very low and high doses of lipids resulted in lowering of the antibody titer, whereas intermediate doses (0.4 to 0.7 mg of lipid/injection) yielded optimum titers. In most experiments herein, archaeosome preparations contained lipid doses of <1 mg/injection.

Archaeosomes facilitate humoral responses to other entrapped protein Ags.

In the studies described above, BSA was used as the model Ag for entrapment in archaeosomes and assessment of humoral responses. Potential vaccine adjuvants must possess the capacity to facilitate immune responses to a wide array of proteins irrespective of antigenic properties. Therefore, the immune response to other Ags entrapped within archaeosomes was studied. Figure 4 illustrates the antibody response in mice, obtained after immunization with two other model Ags (HEL and OVA) entrapped within M. smithii and M. stadtmanae archaeosomes. As with BSA, M. smithii archaeosomes induced markedly enhanced antibody responses in comparison to bare Ag (no adjuvant) and conventional liposomes. Interestingly, s.c. immunizations with M. smithii archaeosome-Ag resulted in very strong titers, with a dramatic enhancement (∼2 log) over that of alum (Fig. 4b). Antibody titers induced by M. stadtmanae were relatively lower than that of M. smithii, as has been observed by us in other studies as well (39).

Induction of cell-mediated immunity: archaeosomes facilitate Ag-specific proliferation of spleen cells.

The induction of a cell-mediated response against Ags is often critical for sustained and effective immunity, particularly against intracellular pathogens. One of the first indicators of an effective cell-mediated immune response is the ability of lymphocyte populations of immunized mice to respond to Ag in vitro. Spleen cells from immunized mice were cultured in vitro with varying doses of Ag, and at 72 h the proliferative response was assessed by [³H]thymidine incorporation. Figures 5a and b indicate the BSA-specific proliferation of spleen cells after i.p. immunization. Immunization with BSA precluded the use of FBS-supplemented medium for in vitro Ag stimulation. Therefore, the overall low levels of proliferation noted may be attributed to the inability of horse serum supplement to support strong growth. Interestingly, even under such suboptimal stimulation conditions, a dose-dependent proliferation to BSA was seen with spleen cells of mice immunized with BSA entrapped in archaeosomes. Furthermore, the proliferative response of spleen cells following archaeosome immunization was seen in both BALB/c and C3H/HeJ (lipopolysaccharide [LPS]-hyporesponsive) mice. On the other hand, conventional liposomes as well as alum failed to induce BSA-specific proliferation. Ag-specific proliferation was more pronounced with other Ags (HEL and OVA). Both HEL-archaeosomes and OVA-archaeosomes induced strong Ag-specific proliferation, as did alum (Fig. 5c and d). Immunization by the s.c. route also resulted in similar responses. However, even with robust Ag stimulation, conventional liposomes failed to equal M. smithii archaeosomes in their ability to induce proliferation.

Archaeosomes induce Th1 and Th2 cytokine production by Ag-stimulated spleen cells.

Ag-specific lymphocyte proliferation results in activation of specific T helper responses and cytokine production. While Th2 (IL-4) responses are important for aiding antibody production by B cells, Th1 (IFN-γ) responses are important for induction of effective cell-mediated immunity. Th1 and Th2 responses may be dichotomous, and Ags may themselves skew immunity toward one or the other (22). A superior adjuvant, on the other hand, may possess the desirable capacity of inducing both Th1 and Th2 responses irrespective of antigenic nature.

Data in Fig. 6a indicate Ag-dependent cytokine production by cultured spleen cells following i.p. immunization with HEL-archaeosomes. Substantial IFN-γ production was seen only in cultures of cells from M. smithii HEL-archaeosome-immunized mice. The IFN-γ levels showed a clear dose-dependent production in response to stimulation by Ag. Alum failed to evoke any IFN-γ production. On the other hand, both archaeosomes and alum induced IL-4 production. M. stadtmanae archaeosomes and conventional liposomes induced neither IFN-γ nor IL-4, as might have been expected from their poor adjuvanticity. The ability of M. smithii archaeosomes to induce Th1 (IFN-γ) as well as Th2 (IL-4) responses to entrapped Ags was further corroborated with another Ag system. Immunization of mice with OVA-archaeosomes by the s.c. route also resulted in production of both IFN-γ and IL-4 by spleen cells (Fig. 6b). After s.c. immunization with archaeosomes, cytokine production was detectable even in popliteal lymph node cell cultures (data not shown). Interestingly, even the potency of s.c. immunization failed to evoke IFN-γ production by alum. Also, conventional liposomes induced neither IL-4 nor IFN-γ.

T. acidophilum archaeosomes induce a long-term memory response.

The success of vaccines often depends on the induction of a strong persistent memory response. It was reasoned that archaeosomes, because of their stability properties (40), may sustain Ag in vivo for longer periods than other adjuvant formulations, aiding memory responses.

Mice were immunized on days 0 and 14 with BSA-archaeosomes or other adjuvant-BSA preparations. The antibody titers were monitored in groups of mice at regular intervals, and memory responses were assessed after boosting with bare Ag in the absence of adjuvant (Fig. 7). Figure 7a indicates that T. acidophilum archaeosomes induced a moderately enhanced primary antibody response that was sustained for nearly 200 days (P < 0.02 by ANOVA, for all time points compared to no-adjuvant immunization). Following an Ag-alone boost on day 210, a remarkable memory antibody response was seen (∼2-log enhancement, P = 0.0004) that was sustained to at least 300 days. On the other hand, alum, despite inducing an enhanced primary response (days 35 and 70, P < 0.01 compared to no-adjuvant immunization), showed a subsequent drop in antibody titers and failed to evoke significant memory response (following Ag boost). BSA-archaeosomes prepared from the TPL of M. stadtmanae (1.1 mg/injection) resulted in low initial titers of antibody and little indication of a memory response (data not shown).

In contrast to the T. acidophilum BSA-archaeosome response noted above, BSA-archaeosomes composed of the TPL of M. mazei (0.53 mg/injection), N. magadii (0.34 mg/injection) (data not shown), or H. salinarum (Fig. 7b) failed to evoke a strong memory response despite a substantially enhanced primary response. Vesicles composed of purified PGP-0-CH₃ (the major lipid in H. salinarum TPL) showed a near-identical response to TPL of H. salinarum. FA was similar to H. salinarum vesicles by inducing a strong and sustained primary response but failing to evoke an enhanced memory response.

Induction of T-cell memory by T. acidophilum archaeosomes: increased cell cycling of CD4⁺ cells.

As antibody response to protein Ags is often T cell dependent, we tested if the strong memory antibody response induced by T. acidophilum archaeosomes was accompanied by stimulation of T helper (CD4⁺) subsets. Thus, the groups of mice used for experimentation in Fig. 7a were challenged again with Ag (25 μg of BSA in PBS, without adjuvant) on day 334. Seven days later, the propensity of cell cycling (based on propidium iodide staining) by splenic CD4⁺ T cells was analyzed. The frequency of CD4⁺ staining of spleen cell populations obtained from control (no-adjuvant) or alum- or T. acidophilum archaeosome-immunized mice is shown in Fig. 8a. The total CD4⁺ cell number was increased from 29% in the controls to 37% in the archaeosome-immunized group. Analysis of cell cycling profiles of the CD4⁺ cells revealed that antigenic challenge of T. acidophilum archaeosome-immunized mice resulted in a dramatic increase in the numbers of cells in the S (synthetic) and G₂/M (mitotic) phase and a concomitant decrease of cells in the G₁ (resting) phase (Fig. 8b). In contrast, alum immunization evoked no change in the percentages of cells in the various phases compared to the no-adjuvant control. Thus, archaeosomes induced the development and persistence of memory CD4⁺ T cells against the entrapped protein Ag.