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The Journal of Immunology, 2001, 167: 3324-3328.
Copyright © 2001 by The American Association of Immunologists

Sterically Stabilized Cationic Liposomes Improve the Uptake and Immunostimulatory Activity of CpG Oligonucleotides1

Ihsan Gursel, Mayda Gursel, Ken J. Ishii and Dennis M. Klinman2

Section of Retroviral Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunostimulatory CpG oligonucleotides (ODN) show promise as immune adjuvants, anti-allergens, and immunoprotective agents. Increasing the bioavailability and duration of action of CpG ODN should improve their therapeutic utility. Encapsulating ODN in sterically stabilized cationic liposomes provides protection from serum nucleases while facilitating uptake by B cells, dendritic cells, and macrophages. In a pathogen challenge model, sterically stabilized cationic liposomes encapsulation doubled the duration of CpG ODN-induced immune protection. In an immunization model, coencapsulation of CpG ODN with protein Ag (OVA) magnified the resultant Ag-specific IFN-{gamma} and IgG responses by 15- to 40-fold compared with Ag plus CpG ODN alone. These findings support the use of sterically stabilized cationic liposomes to significantly enhance the therapeutic efficacy of CpG ODN.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacterial DNA contains unmethylated CpG motifs that strongly stimulate the mammalian immune system. Synthetic oligonucleotides (ODN)3 expressing CpG motifs patterned after those found in bacteria trigger cells of the immune system to proliferate, mature, and produce cytokines, chemokines, and Ig (1, 2, 3, 4).

These immunostimulatory activities are being harnessed therapeutically. CpG ODN show promise as immune adjuvants, significantly improving the immune response to coadministered Ags (5, 6, 7, 8, 9). The strong Th1 response elicited by CpG ODN down-regulates Th2-mediated IgE and cytokine production, thereby interfering with allergic asthma (10, 11, 12). Finally, by preactivating the innate immune system, CpG ODN can protect naive animals against a variety of microbial and parasitic pathogens (13, 14, 15, 16, 17) .

Prolonging the bioavailability and duration of action of CpG ODN may improve their therapeutic efficacy. Unfortunately, phosphorothioate CpG ODN used in vivo are rapidly eliminated from the circulation due to adsorption onto serum proteins and degradation by serum nucleases (18, 19, 20). One potential method for protecting ODN from degradation while increasing their uptake by cells of the immune system involves liposome encapsulation (5, 20, 21, 22). A series of first and second generation lipid vesicles was synthesized that varied in composition, size, and charge. Sterically stabilized cationic liposomes (SSCL) contain positively charged and hydrophilic elements that efficiently encapsulate CpG ODN and increase their uptake by cells of the immune system. The immunostimulatory activity of SSCL-encapsulated ODN significantly exceeded that of free ODN in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Specific pathogen-free BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were housed in sterile microisolator cages.

Oligonucleotides

Immunostimulatory ODN1555 (GCTAGACGTTAGCGT) and ODN1466 (TCAACGTTGA), and control ODNs in which the CpG motif was methylated or inverted, were synthesized by the Center for Biologics Evaluation and Research core facility. All ODN were free of endotoxin and protein contamination.

Liposome preparation

Cholesterol and various phospholipids (Avanti Polar Lipids, Alabaster, AL) were combined in different ratios to form liposomes that varied in charge, stability, lamellarity, and fluidity (Table IGo). Lipid stocks were prepared in chloroform and were stored at 10 mg/ml under argon at -20°C until use.


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  		Table I. Efficiency of ODN encapsulation by different types of liposomes1

 
Liposomes were generated by evaporating phospholipid mixtures in a round-bottom flask using a rotary evaporator (Büchi, Flawil, Switzerland). The solvent-free dry lipid film was purged with argon to eliminate residual chloroform and oxygen, thereby preventing lipid peroxidation. To generate empty multilamellar vesicles, 1 ml PBS was added to each 20 µmol dried lipid film. These were sonicated five times for 30 s each time at 4°C using a Vibra Cell Sonicator (Sonics and Materials, Danbury, CT). The small unilamellar vesicles were then mixed with 1 mg/ml ODN, frozen on dry ice, and freeze-dried overnight (Flexi-Dry; Kinetics Group, Santa Clara, CA) (22).

ODN encapsulation was achieved during rehydration. Sterile dH2O (100 µl) was added to the dehydrated liposome/ODN powder and vortexed for 15 s every 5 min for 30 min at room temperature. PBS (900 µl) was added to the mixture, yielding a final liposome concentration of 20 µM lipid/mg DNA. Vesicles <150 nm in diameter were produced by 20–30 cycles of extrusion through polycarbonate filters using a Liposofast extruder (Avestin, Ottawa, Canada) (23). Liposome formulations were stored at 4°C until use.

Assays

RAW 264.7 mouse macrophages were transiently transfected with an IL-12 p40 promoter-luciferase gene vector (pGL3 basic vector) as previously described (24). Cells (106) were transfected with 5 µg plasmid DNA. After 24 h cells were pooled, washed, and cultured for 18 h with 1 µM ODN in 12-well macroplates. Cells were harvested and monitored for luciferase activity as recommended by the manufacturer (Promega, Madison, WI).

BALB/c spleen cells were prepared in RPMI 1640 supplemented with 5% FCS, 50 mg/ml penicillin/streptomycin, 2 mM L-glutamine, 10 mM HEPES, 0.11 mg/ml sodium pyruvate, and 0.5 mM 2-ME. The cells were cultured at 37oC in a 5% CO2 incubator. Single-cell suspensions were serially diluted in flat-bottom 96-well microtiter plates that had been precoated with anti-cytokine Abs as previously described (25). Cells were incubated with 1 µM ODN (unless otherwise stated) at 37°C for 8 h, and their secretion of cytokine was detected colorimetrically (26). ELISAs for quantifying cytokines were performed as described above, except supernatant rather than cells were added to anti-cytokine-coated plates.

In vivo CpG ODN binding and uptake

BALB/c mice were injected i.p. with 50 µg free or liposome-encapsulated FITC-labeled CpG ODN. Mice were sacrificed 2–48 h later, and single spleen cell suspensions (2 x 106/ml) were immediately prepared. One aliquot of cells was fixed and analyzed for FITC-ODN content by FACS (BD Biosciences, San Jose, CA). Another aliquot was stained with PE-labeled phenotype-specific Abs (BD PharMingen, San Diego, CA). These included CD11c for dendritic cells, CD11b for macrophages, and B220 for B cells. The autofluorescence of untreated cells was used to establish assay background.

Ag-specific immune responses

Mice were immunized i.p. with liposome (0.2 µM lipid/mouse) containing OVA (2 µg; Sigma, St. Louis, MO) and/or CpG ODN (10 µg/mouse) on days 0 and 14. Serum was collected on days 14 and 28. IgG, IgG1, and IgG2a anti-OVA titers in serum were determined by ELISA (25). Mice were sacrificed on day 28, and a single spleen cell suspension (2 x 105 cells/well) was prepared in RPMI 1640 supplemented with complete medium. Cells were stimulated in vitro with 5 µg OVA for 36 h. IFN-{gamma} levels in culture supernatants were monitored by ELISA (25).

Bacteria and growth conditions

Listeria monocytogenes strain EGD (ATCC 15313, American Type Culture Collection, Manassas, VA) was grown in modified Mueller-Hinton broth (Difco, Detroit, MI) (14). Mice treated with 50 µg ODN (alone or liposome incorporated) were challenged i.p. 2 or 4 wk later with 103 LD50 of L. monocytogenes in 500 µl PBS. Survival was monitored for 3 wk.

Statistical analysis

All cytokine and Ig assays were conducted at least twice on at least three independently studied mice per group. All immunization and bacterial challenge experiments were performed on a minimum of 5–10 mice/group. Statistical significance was evaluated using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of liposome formulation on CpG ODN incorporation

Initial experiments evaluated the efficiency with which 32P-labeled CpG ODN were incorporated into various types of lipid vesicle. Anionic liposomes interacted poorly with CpG ODN, attaining encapsulation efficiencies of <40% (Table IGo). Neutral and cationic liposomes showed progressively higher levels of encapsulation. SSCL composed of a positively charged derivative of cholesterol, a hydrophilic derivative of phosphatidylethanolamine, and the neutral lipid dioleylphosphatidylethanolamine were the most successful in incorporating anionic CpG ODN (Table IGo). Incorporation efficiency was influenced by the charge, but not by the size or sequence of the ODNs studied (data not shown).

In vitro activity of liposome-encapsulated CpG ODN

The ability of CpG ODN to stimulate cells and up-regulate cytokine expression was monitored by transiently transfecting RAW264.7 murine macrophages with an IL-12 p40/luciferase construct. It was previously shown that CpG ODN increase IL-12 p40 promoter activity in this cell line (24). As shown in Fig. 1Go, RAW264.7 cells treated with SSCL-encapsulated CpG ODN (hereafter (CpG ODN)SSCL) expressed significantly more luciferase activity than cells treated with an equal amount of free ODN (or ODN in other types of liposome; p < 0.01). Moreover, stimulation by (CpG ODN)SSCL persisted at concentrations below the effective range of free CpG ODN (p < 0.001; Fig. 2Go). The stimulation observed in these experiments was CpG motif dependent, since empty SSCL or SSCL containing control ODN did not increase luciferase expression (Fig. 2Go).



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  		FIGURE 1. IL-12 p40 expression is induced by CpG ODN-encapsulated liposomes. RAW 264.7 cells were transfected with an IL-12 promoter-luciferase gene vector (pGL3 basic vector). Transfected cells (106) were stimulated for 24 h with 0.5 µM free or liposome-encapsulated CpG ODN and monitored for luciferase activity. Data show the fold increase in luciferase activity at 24 h compared with that with medium alone and represent the mean ± SD of three independent experiments. *, p < 0.01 when compared with free CpG ODN.

 


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  		FIGURE 2. Effect of CpG ODN dose on IL-12 p40 expression. RAW 264.7 cells were transfected as described in Fig. 1Go and stimulated with 0.01–1.0 µM free or SSCL-encapsulated CpG ODN. Data show the fold increase in luciferase activity at 24 h compared with that with medium alone and represent the mean ± SD of three independent experiments. *, p < 0.01 compared with free CpG ODN.

 
The activity of (CpG ODN)SSCL was confirmed using freshly isolated BALB/c spleen cells. Consistent with previous reports (3, 27), free CpG ODN elicited a dose-dependent increase in IFN-{gamma} production by normal murine spleen cells (Fig. 3Go). (CpG ODN)SSCL stimulated significantly more IFN-{gamma} production at all concentrations examined (p < 0.001). This stimulation was CpG specific, since control ODN did not induce IFN-{gamma} (Fig. 3Go). CpG ODN encapsulated in other types of liposomes were less stimulatory (data not shown).



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  		FIGURE 3. Effect of liposome encapsulation on CpG ODN-mediated spleen cell activation. BALB/c splenocytes (2 x 105/well) were stimulated with 0.1–3.0 µM ODN. Culture supernatants were collected after 36 h and assayed for IFN-{gamma} by ELISA. Results represent the average ± SD of three independent experiments, with each assay performed in triplicate in each experiment. *, p < 0.01 compared with free CpG ODN.

 
Uptake of (CpG ODN)SSCL in vivo

Normal BALB/c mice were injected i.p. with 50 µg free or SSCL-encapsulated FITC-ODN. Uptake of these ODN by spleen and peritoneal exudate cells (PEC) 2 h postinjection was significantly higher in mice treated with (CpG ODN)SSCL than in free ODN (p < 0.05 for both sites; Table IIGo). This difference persisted at 24 h, with 3- to 6-fold more cells incorporating label (p < 0.001).


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  		Table II. SSCL encapsulation enhances CpG ODN uptake in vivo1

 
To determine the phenotype of the cells that bound and internalized (CpG ODN)SSCL, spleen cells from these mice were treated with PE-labeled phenotype-specific mAbs. The results indicate that (CpG ODN)SSCL was taken up by significantly more CD11b+ macrophages (1 vs 4%; p < 0.01), CD11c+ dendritic cells (0.5 vs 3.0%; p < 0.01), and B220+ B cells (1 vs 6%; p < 0.01) than free CpG ODN (Fig. 4Go).



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  		FIGURE 4. Uptake of (CpG ODN)SSCL by spleen cells. Mice (three per group) were injected i.p. with 50 µg FITC-labeled free or SSCL-encapsulated CpG ODN. After 48 h, spleen cells were isolated and stained with PE-labeled phenotype-specific mAbs. Data show the percentage of phenotype-positive ODN-labeled cells identified by FACS. Note that the uptake of (CpG ODN)SSCL by CD11b+ macrophages, CD11c+ dendritic cells, and B220+ B cells was significantly higher than that of free CpG ODN for all cell types. *, p < 0.01)

 
In vivo effects of (CpG ODN)SSCL: immune cell activation

Immune activation manifest by increased cytokine production and the up-regulation of CD40 expression was examined in BALB/c mice injected i.p. with 50 µg free or SSCL-encapsulated CpG ODN. Results show that spleen cells from (CpG ODN)SSCL-treated mice produced significantly more IL-6, IL-12, and IFN-{gamma} (p < 0.001; Table IIIGo) and expressed higher levels of CD40 (p < 0.001; Table IIIGo), than those from mice treated with free CpG ODN. This enhanced immune activation persisted for at least 48 h (Table IIIGo; p < 0.001 for all cytokines). Similar findings were obtained when lymph node (LN) cells and peritoneal exudate cells (PEC) from these animals were analyzed (data not shown). These findings indicate that liposome encapsulation increased both the magnitude and duration of CpG-induced immune activation in vivo.


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  		Table III. Liposome-encapsulated CpG ODN enhance cytokine production in vivo1

 
In vivo effects of (CpG ODN)SSCL: adjuvant activity

CpG ODN can act as immune adjuvants, boosting the immune response to coadministered protein Ags (16, 21). For example, coadministering free CpG ODN with OVA increases the resultant IgG2a anti-OVA response of BALB/c mice by 6-fold (p < 0.01) and IFN-{gamma} production by 1.5-fold (Table IVGo).


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  		Table IV. Coencapsulation of OVA plus CpG ODN in SSCL improves immunogenicity1

 
If CpG ODN plus OVA were coencapsulated in SSCL, a 40-fold increase in the IgG2a anti-OVA response was obtained (p < 0.01). This treatment also increased Ag-specific IFN-{gamma} production by 10-fold (p < 0.01; Table IVGo).

Optimal immunogenicity was observed when OVA and CpG ODN were encapsulated in the same liposome. Administering free OVA with encapsulated (CpG ODN)SSCL elicited immune responses of significantly lower magnitude (Table IVGo). These findings are consistent with the results of previous studies showing that optimal immunogenicity requires that the CpG ODN be kept in close physical proximity to Ag (6, 16).

In vivo effects of (CpG ODN)SSCL: immunoprotective activity

CpG ODN stimulate an innate immune response that promotes host survival following pathogen challenge (14, 28). While this response protects the host from a broad array of pathogens, it persists for only a short period (<2 wk) (>14, 16, 28). Since SSCL encapsulation increases the magnitude and duration of CpG-induced cellular activation, its ability to extend the duration of CpG-induced protection was examined.

BALB/c mice were injected i.p. with 50 µg CpG ODN or (CpG ODN)SSCL. Two weeks after treatment all animals survived infection by 103 LD50 of L. monocytogenes (data not shown). However, when challenge was delayed until 30 days, only those animals treated with encapsulated ODN survived (p < 0.0001; Fig. 5Go).



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  		FIGURE 5. (CpG ODN)SSCL provide protection against lethal pathogen challenge. BALB/c mice were injected i.p. with 50 µg free or (CpG ODN)SSCL. Four weeks later animals were challenged i.p. with 103 LD50 of L. monocytogenes and monitored for survival. Results represent the total experience from two experiments involving 10 mice/group/experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CpG ODN stimulate an innate immune response characterized by the activation of lymphocytes and professional APC. Clinical trials are underway to examine the ability of these molecules to act as immune adjuvants, anti-allergens, and immunoprotective agents (29). Yet, rapid in vivo clearance limits the uptake and shortens the therapeutic half-life of free ODN. Encapsulation in sterically stabilized cationic liposomes improves the uptake of CpG ODN by cells of the immune system, concomitantly increasing the magnitude and duration of their in vivo activity.

Multiple different liposome formulations were tested. SSCL were the most efficient at encapsulating CpG ODN. This is consistent with earlier studies showing that sterically stabilized liposomes protect and improve the uptake of antisense ODN and plasmid DNA during cancer chemotherapy (22, 30, 31, 32, 33). SSCL are composed of three distinct phospholipid elements. Dimethylaminoethane-carbamol-cholesterol increase liposome membrane stability while improving the uptake and encapsulation of DNA (34, 35). Dioleylphosphatidylethanolamine is a pH-sensitive neutral lipid that improves the cytosolic delivery of ODN following internalization (36, 37). Polyethylene glycol-PE stabilizes the liposome and also facilitates cellular uptake (37, 38, 39). Preliminary experiments showed that this three-component combination was more effective than multiple other combinations at ODN uptake and delivery (Table IGo and Fig. 1Go, and data not shown).

The uptake of CpG ODN by spleen, PEC, and LN cells was significantly enhanced by SSCL encapsulation (Table IIGo). In addition, the magnitude and duration of immune cell activation induced by (CpG ODN)SSCL significantly exceeded those of free ODN, as measured by cytokine production and the up-regulation of CD40 expression (Table IIIGo). This translated into improved in vivo activity. Consistent with this finding, Ignatius et al. (37) recently demonstrated that dendritic cells more effectively take up and present protein encapsulated in a polyethylene glycol-stabilized liposome.

Normal BALB/c mice immunized with CpG ODN coencapsulated with OVA generated Ag-specific IgG responses far greater than mice immunized with Ag alone or Ag plus nonencapsulated ODN (Table IVGo). In particular, coencapsulation increased Ag-specific IFN-{gamma} production (10-fold) and IFN-{gamma}-dependent IgG2a anti-OVA Ab production (40-fold), consistent with the preferential induction of a Th1-biased immune response. It is well established that optimal immunogenicity is achieved by maintaining Ag and ODN in close physical proximity (16, 40, 41). Liposome coencapsulation provides an effective method for ensuring the simultaneous presentation of Ag plus adjuvant to relevant APC, while providing protection against serum proteases and nucleases and eliminating the need to covalently couple ODN to Ag.

Immunoprotection experiments provided additional evidence of the in vivo activity of (CpG ODN)SSCL. Previous studies showed that the innate immune response elicited by CpG ODN protected normal mice from a variety of bacterial, viral, and parasitic pathogens for approximately 2 wk (13, 14, 15). When (CpG ODN)SSCL were administered, the duration of protection doubled, with 100% of mice surviving 103 LD50 of L. monocytogenes 4 wk post-treatment (Fig. 5Go). Thus, by facilitating the uptake of CpG ODN, SSCL encapsulation increases the magnitude and duration of their in vivo activity. These findings suggest that the immunotherapeutic potential of CpG ODN may be enhanced by SSCL incorporation.


   Footnotes
 
1 This work was supported in part by Military Interdepartmental Purchase Request MM8926. The assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Food and Drug Administration at large. Back

2 Address correspondence and reprint requests to Dr. Dennis M. Klinman, Building 29A, Room 3 D 10, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892. E-mail address: klinman{at}cber.fda.gov Back

3 Abbreviations used in this paper: ODN, oligonucleotides; SSCL, sterically stabilized cationic liposomes; PEC, peritoneal exudate cells; LN, lymph node. Back

Received for publication April 10, 2001. Accepted for publication July 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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