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Cutting Edge: CpG DNA Is a Potent Enhancer of Systemic and Mucosal Immune Responses Against Hepatitis B Surface Antigen with Intranasal Administration to Mice¹

Michael J. McCluskie^*, and Heather L. Davis^2,*,,,§

^* Loeb Research Institute, Ottawa, Canada; Departments of Cellular and Molecular Medicine and Microbiology, Immunology, and Biochemistry, Faculty of Medicine, and ^§ School of Rehabilitation Sciences, Faculty of Health Sciences, University of Ottawa, Ottawa, Canada

	Abstract

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Mucosal immunity is difficult to induce with subunit vaccines unless such vaccines are administered with a mucosal adjuvant such as cholera toxin (CT); however, CT is toxic in humans. Synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG) are potent adjuvants for the induction of Th1-like systemic immune responses against parenterally delivered proteins. Here, we show in mice that intranasal delivery of hepatitis B surface Ag, which alone has no effect, elicits good immune responses when given with CpG oligodeoxynucleotides and/or CT. Overall, CpG is superior to CT for the induction of humoral and cell-mediated systemic immunity as well as mucosal immune responses (IgA) at local (lung) and distant (feces) sites. Furthermore, CpG and CT act synergistically, giving stronger responses than those observed with 10 times more of either adjuvant alone. Ab isotypes were predominantly IgG1 (Th2-like) with CT, mixed IgG1/IgG2a (Th0) with CpG, and predominantly IgG2a (Th1-like) with CpG and CT together.

	Introduction

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The combined mucosal surface area is >200 times greater than that of the skin and is the primary site of transmission of the majority of infectious diseases. In general, parenteral Ag delivery induces only systemic immunity, whereas mucosal delivery can trigger both mucosal (i.e., secretory IgA Abs) immunity at local and distant sites as well as systemic responses (1, 2). Live-attenuated organisms are effective for mucosal immunization, but results have been disappointing with subunit vaccines, largely because of the lack of a safe and effective adjuvant. Cholera toxin (CT)³ is an effective mucosal adjuvant in animal models, but it is highly toxic, especially in humans. Genetically detoxified mutants of CT or the related Escherichia coli heat-labile enterotoxin have been developed using site-directed mutagenesis. These mutants appear to be nontoxic, yet retain adjuvanticity in animal models (3, 4).

A new class of adjuvant is bacterial DNA or synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs. CpG DNA triggers most (>95%) B cells to proliferate; to secrete Ig, IL-6, and IL-12; and to be protected from apoptosis (5, 6, 7). In addition, CpG DNA also directly activates monocytes, macrophages, and dendritic cells to secrete IFN-ß, IL-6, IL-12, granulocyte-macrophage CSF, chemokines, and TNF- (7, 8). These cytokines stimulate NK cells to secrete IFN- and have increased lytic activity (7, 9, 10). Overall, CpG induces a Th1-like pattern of cytokine production that is dominated by IL-12 and IFN- with little secretion of Th2 cytokines (7).

The utility of CpG as a vaccine adjuvant was suggested by 1) the strong activating effects of CpG on B cells (Ag nonspecific) (5), 2) the strong synergy between the B cell-signaling pathways triggered through the B cell AgR and by CpG (Ag specific) (11), and 3) the induction of cytokines that could have indirect effects on B and T cells via Th pathways (7). Indeed, we have shown that for systemic immunization, CpG ODN has potent adjuvant effects on the humoral and cellular responses against recombinant hepatitis B surface Ag (HBsAg) administered by i.m. injection in mice (12). The immune responses with CpG were clearly Th1-like, with predominantly IgG2a Abs and strong CTLs.

Although CpG ODN has been shown to be a potent adjuvant for the induction of systemic immune responses against a variety of other Ags (12, 13, 14, 15, 16), only one study has shown the use of CpG as a mucosal adjuvant. This study used inactivated virus, which is capable of inducing immune responses when delivered on its own to a mucosal surface (17). Here, we report that CpG ODN is a highly effective mucosal adjuvant for HBsAg, which by itself does not induce an immune response.

	Materials and Methods

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Immunization of mice against HBsAg

Groups (n = 5–15) of female BALB/c mice (6–8 wk, Charles River, Montreal, Canada) were immunized by intranasal (i.n.) inhalation (drops were applied to external nares under light anesthesia with Halothane (Halocarbon Laboratories, River Edge, NJ)) of 1 or 10 µg of HBsAg (ad subtype, Genzyme Diagnostics, San Carlos, CA) alone or in combination with 1 or 10 µg of CT (purified from Vibrio cholerae; Sigma, St. Louis, MO) and/or CpG ODN (5'-TCCATGACGTTCCTGACGTT-3') or non-CpG control ODN (5'-TCCAGGACTTCTCTCAGGTT-3') in a total volume of 150 µl.

We have shown previously that i.n. inhalation is as efficient as direct intratracheal instillation for the delivery of solutions to the lungs (with minimal oral delivery); 150 µl is the optimal volume (18). Both ODNs had a nuclease-resistant phosphorothioate backbone (12). Some mice were boosted in an identical manner at 8 wk postprime.

Collection of samples

Plasma and fecal pellets were collected at 1, 2, 4, and 8 wk postprime and at 1, 2, and 4 wk postboost; lung washes were conducted at 4 wk postprime or postboost. Plasma was obtained by retroorbital puncture. Fecal pellets were recovered from cages in which mice had been isolated without bedding for 24 h. Fecal material (0.1 mg) was rehydrated for 30 min at room temperature in 1 ml of Tris-buffered saline (TBS) (0.05 M Tris-HCl and 0.15 M NaCl (pH 7.5)) with 0.1 µg sodium azide (Sigma); next, supernatants were collected after centrifugation at 6000 rpm for 15 min. Lung washes were conducted on mice immediately after anesthetic overdose by rinsing with 1 ml of TBS using polyethylene tubing (polyethylene-20, inner diameter = 0.38 mm, Becton Dickinson, Franklin Lakes, NJ); the tubing was attached to the needle of a 1-ml insulin syringe (Becton Dickinson), inserted into the trachea via a small incision, and anchored with vascular microclamps (Fine Science Tools, North Vancouver, Canada) above and below the incision. The TBS solution was slowly instilled and withdrawn three times with 80% final recovery. Lung wash samples were centrifuged at 13,000 rpm for 7 min, and supernatants were collected. All plasma, fecal, and lung wash samples were stored at -20°C until assayed by ELISA.

Evaluation of immune responses

HBsAg-specific Abs (anti-HBs) in plasma were detected by ELISA on individual samples using HBsAg-coated plates (12). Endpoint dilution titers for total IgG as well as IgG1 and IgG2a isotypes were defined as the highest plasma dilution that resulted in an absorbance value (OD 450) of two times greater than that of nonimmune plasma, with a cutoff value of 0.05. Anti-HBs titers of responding mice (titers of >10) were expressed as means ± SEM of individual animal values, which were themselves the average of triplicate assays. Titers of HBsAg-specific IgA were detected as described for IgG, except samples were incubated on coated plates for 2 h at 37°C and captured Abs were detected with horseradish peroxidase-conjugated goat anti-mouse IgA (1:1000 in PBS-Tween, 10% PBS, 100 µl/well; Southern Biotechnology Associates, Birmingham, AL). IgA in lung washes and fecal extracts were expressed as endpoint dilution titers and OD 450 above background, respectively. Nonimmune plasma, fecal extracts, or lung wash solutions were used as negative controls.

The CTL activity of splenocytes taken 4 wk postprime or postboost was detected as described previously (12) except: 1) media used for incubating splenocytes was supplemented with 5 x 10^-5 M 2-ME (Sigma) and 3% EL-4 supernatant as a source of IL-2, and 2) splenocytes (3 x 10⁷) were cocultured with 1 x 10⁶ irradiated stimulator cells. CTL activity was expressed as group means ± SEM of individual animal values, which were themselves the average of triplicate assays.

Statistical analysis

Data were analyzed using the GraphPad InStat program (GraphPad Software, San Diego, CA). The statistical significance of the difference between two groups was determined by the two-tailed Student t test; the difference between three or more groups was determined by one-factor ANOVA followed by Tukey’s test. Differences were considered to be not significant with p > 0.05.

	Results

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i.n. inhalation of HBsAg (1 µg) alone failed to induce detectable anti-HBs IgG Abs in the plasma of any mice (0 of 15) (Fig. 1), even after boost (0 of 5) (data not shown). With the higher dose of Ag (10 µg), most mice (12 of 15) failed to seroconvert, a few (2 of 15) had extremely low titers (<40), and only one had a reasonable anti-HBs titer (1000) (Fig. 1). In contrast, high titers of plasma anti-HBs IgG were induced in all mice when adjuvant was added to the HBsAg, and surprisingly, CpG was equal to CT when used at a 1-µg dose (p = 0.73 and 0.13 with 1 and 10 µg of HBsAg, respectively) (Fig. 1) or a 10 µg dose (p = 0.08 with 10 µg of HBsAg) (data not shown). CT was only superior (by sevenfold) to CpG when a high dose of adjuvant (10 µg) and a low dose of Ag (1 µg) were used (p = 0.01) (data not shown). Nevertheless, upon boost, titers with CpG rose nearly 500-fold, whereas those with CT were increased by <100-fold (data not shown). A synergistic effect appeared with CpG and CT together (CpG/CT), since titers of plasma IgG were 5 to 10 times higher than with either adjuvant alone (Fig. 1). Indeed, 1 µg of each adjuvant together gave a better systemic humoral response against a low dose of Ag (1 µg) than did 10 µg of CpG alone (p = 0.01) and was equal to that with 10 µg of CT alone (p = 0.22) (Fig. 1). Furthermore, with a high dose of Ag (10 µg), the low dose CpG/CT combination (1 µg each) induced anti-HBs IgG titers as high as those seen with 10 times as much of either adjuvant alone (CT, p = 0.27; CpG, p = 0.09) (data not shown). Results were due to the CpG motif rather than to a nonspecific effect of the ODN backbone, since mice immunized with 1 µg of HBsAg plus 10 µg of non-CpG ODNs had no (7 of 10) or very low (3 of 10) titers of anti-HBs IgG Abs.

View larger version (77K): [in this window] [in a new window]   FIGURE 1. BALB/c mice were immunized by i.n. inhalation of 1 µg (white bars) or 10 µg (gray bars) of HBsAg alone (none) or in combination with CT and/or CpG-containing ODNs (CpG) as adjuvants (1 µg each). Each bar represents the group mean (± SEM) of the ELISA endpoint dilution titer for anti-HBs (total IgG) in plasma taken 4 wk postimmunization. Titers were defined as the highest plasma dilution resulting in an absorbance value of two times that seen for nonimmune plasma, with a cutoff value of 0.05.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. BALB/c mice were immunized at 0 and 8 wk by i.n. inhalation of HBsAg alone (none) or in combination with CT and/or CpG-containing ODNs (CpG) as adjuvants. The dose of Ag and each adjuvant was 1 µg. Each bar represents the group mean of the ELISA endpoint dilution titer for anti-HBs of IgG1 (gray bars) or IgG2a (black bars) isotypes in plasma taken 8 wk postprime (left panel) or 4 wk postboost (right panel). Titers were defined as the highest plasma dilution resulting in an absorbance value of two times that seen for nonimmune plasma, with a cutoff value of 0.05. The numbers above each bar indicate the IgG2a to IgG1 ratio, with a value >1 indicating a more Th-1-like response.

No IgA was detected in the lung washes of mice that had been given a low dose (1 µg) of HBsAg, even when 10 µg of either CpG or CT was added (data not shown). However, low IgA titers were detected with CpG/CT (1 µg each), and these titers increased 100-fold after boost (Fig. 3). In contrast, no IgA was detected in the lung washes with 1-µg doses of non-CpG ODN/CT (data not shown). With the 10-µg Ag dose, IgA was detected with the addition of CT and CpG, either alone or in combination (Fig. 3). A synergy was also noted, with lung wash IgA being significantly higher with 1 µg each of CpG/CT than with 10 µg of either adjuvant alone (p < 0.0003) (data not shown).

View larger version (54K): [in this window] [in a new window]   FIGURE 3. BALB/c mice were immunized at 0 and 8 wk by i.n. inhalation of 1 µg (white bars) or 10 µg (gray bars) of HBsAg alone or in combination with low doses (1 µg) of CT and/or CpG-containing ODNs (CpG) as adjuvants. Each bar represents the group mean of the ELISA endpoint dilution titer for anti-HBs of IgA in lung washes performed 4 wk postprime or postboost. Titers were defined as the highest sample dilution resulting in an absorbance value of two times that seen for nonimmune lung wash, with a cutoff value of 0.05.

	Discussion

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Here, we show that CpG DNA can act as a potent mucosal adjuvant for both humoral and cell-mediated systemic responses as well as both local (lung) and distant (digestive tract) mucosal immunity. Surprisingly, CpG was as good as CT and, in some aspects (e.g., IgA at distant site), was even better.

The hallmark of mucosal immunity is secretory IgA Abs, which neutralize pathogens in or adjacent to mucosal epithelial cells (19). Activated IgA cell precursors can also migrate to other mucosal sites and differentiate into IgA-secreting plasma cells (20). IgA responses depend upon T cell help, with IL-4 and TGF-ß promoting isotype switching and IL-5, IL-6, and IL-10 being required for terminal differentiation and proliferation (20, 21).

CpG and CT both activate B cells but appear to act by different mechanisms as they induce different IgG isotypes. This difference may be cytokine related, as CT induces primarily IL-4, IL-5, IL-6, and IL-10 (22, 23) and CpG induces predominantly IL-6, IL-12, and IFN- (7, 8). Notably, both induce IL-6, which is required for the development of IgA (24). Despite the predominance of IgG1 Abs with CT, its effects cannot be considered purely Th2, since CTLs are detected. Nevertheless, CpG as a mucosal adjuvant can induce IgA Abs in the context of a more pure Th1 response. This has also been reported using CpG with whole-killed influenza virus (17) and tetanus toxoid with IL-12, a Th1-like cytokine (25, 26). A Th2-like response in the lung may be undesirable because of an association with asthma (27, 28). Interestingly, CpG has recently been shown in mice to prevent allergen-induced asthmatic responses, including airway eosinophilia, Th2 cytokine induction, IgE production, and bronchial hyperactivity (29).

Further support for the different mechanisms of CpG and CT is provided by their strong synergistic effects on both humoral (systemic and mucosal) and cell-mediated responses. This finding also indicates that it may be possible to use much lower doses of mucosal toxins with CpG to obtain better immune responses with less toxicity.

CpG appears to be a safe yet effective mucosal adjuvant. Even delivery of very high doses (500 µg) to the lungs of mice was well tolerated; no more short-term distress was observed than was seen with HBsAg alone, and mice displayed a rapid, complete recovery. In contrast, mice receiving high doses of CT (>10 µg) show signs of toxicity, such as ruffling of fur and diarrhea. CT is even more toxic in humans, where a dose as low as 1 to 5 µg can cause diarrhea (30). This is the first demonstration of CpG as a mucosal adjuvant with a purified protein and of the synergistic actions of CpG and CT together. Like CT, CpG will likely be effective with other Ags such as recombinant proteins, synthetic peptides, and inactivated whole pathogens (31, 32). CpG might also be used as a mucosal adjuvant for Ags delivered to other mucosal surfaces, although the i.n. route is highly desirable for mass immunization since it is noninvasive, fast, easy, and also induces mucosal responses at distant sites (i.e., the lower digestive tract).

	Acknowledgments

 
We thank Lu Zhang, Lacrimioara Comanita, and Amanda Boyd for excellent technical assistance.

	Footnotes

 
¹ This research was supported by operating grants from the World Health Organization Global Programme for Vaccines and Immunization and the Medical Research Council of Canada (to H.L.D.). H.L.D. is also the recipient of a Career Scientist Award from the Ontario Ministry of Health. M.J.M. is the recipient of an Ontario Graduate Scholarship from the Ontario Ministry of Education and Training.

² Address correspondence and reprint requests to Dr. Heather L. Davis, Loeb Research Institute, 725 Parkdale Avenue, Ottawa, K1Y 4E9, Canada. E-mail address:

³ Abbreviations used in this paper: CT, cholera toxin; ODN, oligodeoxynucleotide; HBsAg, hepatitis B surface Ag; i.n., intranasal; anti-HBs, HBsAg-specific Abs.

Received for publication July 15, 1998. Accepted for publication August 18, 1998.

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