APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.     

Suppression of skin lesions by transdermal application of CpG-oligodeoxynucleotides in NC/Nga mice, a model of human atopic dermatitis

Atopic dermatitis (AD) is a pruritic inflammatory skin disease characterized by an elevation of the total IgE level in plasma, the infiltration of mast cells and eosinophils, and the expression of cytokines by Th2 cells. NC/Nga mice kept in conventional conditions are known to develop skin lesions resembling human AD. We examined in this study the alterations of immune response in NC/Nga mice kept in conventional conditions, following transdermal application of CpG-oligodeoxynucleotides (ODN), which plays a critical role in immunity via the augmentation of Th1-type and suppression of Th2-type responses. CpG-ODN remarkably changed the immune response from type Th2 to Th1 as determined from cytokine mRNA and Ab levels. The serum IgE level was decreased and the expression of IgG2a was up-regulated. The application of CpG-ODN to the skin also decreased inflammatory infiltration of mast cells, and suppression in the skin lesions was observed. Furthermore, the generation of regulatory T cells, which are considered immune suppressive T cells, was observed in the skin on treatment with CpG-ODN. These results suggested CpG-ODN is effective for immunotherapy in patients with AD, which is characterized by Th2-dominated inflammation.     

Development of atopic dermatitis-like skin lesions in STAT6-deficient NC/Nga mice

Atopic dermatitis (AD) is a pruritic inflammatory skin disease characterized by elevation of plasma levels of total IgE, infiltration of mast cells and eosinophils, and the expression of cytokines by Th2 T cells. However, the role of Th2 cells in the pathogenesis of AD is not fully understood. In this study we examined the NC/Nga (NC) mouse model of AD and established STAT6-deficient (SATA6(-/-)) NC mice to investigate the relevance of IL-4-mediated immune responses. Surprisingly, these mice elicited AD-like skin lesions at equivalent frequency and time of onset compared with normal NC littermates. Histological features of the lesion in STAT6(-/-) NC mice fulfilled the criteria for the pathogenesis of AD, although these mice fail to produce IgE and Th2 cytokines. The lymph nodes proximal to the regions of skin that developed lesions exhibited massive enlargement elicited by the accumulation of activated IFN-gamma-secreting T cells. Moreover, caspase I, IL-18, IL-12, and IFN-gamma are found to be highly expressed at the skin lesion, occurring simultaneously with elevation of eotaxin 2 and CCR3 expression. Therefore, the Th2-mediated immune response is not necessary for the development of AD-like skin disease in NC mice. The skin microenvironment that favored IFN-gamma production tightly correlates with the skin disease in NC mice through the infiltration of eosinophils.     

A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. We therefore investigated whether such a CpG-containing ODN (CpG ODN) given mucosally in the female genital tract could enhance innate immunity and protect against genital herpes infection. Groups of C57BL/6 mice were treated intravaginally with either CpG ODN or a non-CpG ODN control in the absence of any antigen either 2 days before or 4 h after an intravaginal challenge with a normally lethal dose of herpes simplex virus type 2 (HSV-2). Mice treated with CpG ODN exhibited significantly decreased titers of HSV-2 in their vaginal fluids compared with non-CpG ODN-treated mice. Furthermore, CpG ODN pretreatment significantly protected against development of disease and death compared to non-CpG ODN pretreatment. Most strikingly, CpG ODN conferred protection against disease and death even when given after the viral challenge. The CpG ODN-induced protection was associated with a rapid production of gamma interferon (IFN-gamma), interleukin-12 (IL-12), IL-18, and RANTES in the genital tract mucosa following CpG ODN treatment. The observed protection appeared to be dependent on IFN-gamma, IL-12, IL-18, and T cells, as CpG ODN pretreatment did not confer any significant protection in mice deficient in IFN-gamma, IL-12, IL-18, or T cells. Further, a complete protective immunity to reinfection was elicited in CpG ODN-treated, HSV-2-challenged mice, suggesting a role for mucosally administered CpG ODN in inducing the development of an acquired immune response in addition to its potent stimulation of innate immunity.     

Therapeutic effect of CpG motifs on the development of chronic graft-versus-host disease in mice

Transferring DBA/2 spleen cells into (C57BL/10xDBA/2) F1 (referred to as BDF1) mice induces a chronic graft-versus-host disease (GVHD), characterized by the production of Th(2) cytokines, hypergammaglobulinemia, and immune complex-mediated glomerulonephritis that resembles systemic lupus erythematosus. DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (CpG ODN) induces Th(1) cytokine production in mice. This study examines the effect of administering CpG ODN to mice undergoing chronic GVHD, based on the premise that altering Th(1)/Th(2) activity might beneficially impact on disease progression.GVHD BDF1 mice injected with DBA/2 spleen cells were treated with weekly intraperitoneal injection of 50 microg CpG ODN. This treatment significantly suppressed the production of IgG anti-DNA autoantibody and reduced the development of glomerulonephritis. Serum IgG2a titers were higher in the CpG ODN than in non-CpG control group, whereas IgG1 titers were unchanged. As predicted, IFN-gamma levels were significantly higher in the CpG ODN-treated group, while IL-4 levels were lower, resulting in a shift in the Th(1)/Th(2) cytokine ratio. Results suggest that CpG ODN administration may be of therapeutic benefit in chronic GVHD.     

Pyridone 6, a pan-JAK inhibitor, ameliorates allergic skin inflammation of NC/Nga mice via suppression of Th2 and enhancement of Th17

Atopic dermatitis (AD) is a common pruritic inflammatory disease triggered by a defective skin barrier and immunodysregulation. AD has been considered a typical example of a Th2 response associated with allergic disease. In the early phases of the disease, symptoms include IgE hyperproduction, eosinophil accumulation, and mast cell activation; in the chronic phase, a Th1-dominant immune response is also observed at the sites of AD skin lesions. The role of IL-17-producing Th (Th17) cells in AD has not been established. In the current study, we found that pyridone 6 (P6), a pan-JAK inhibitor, delayed the onset and reduced the magnitude of skin disease in an AD-like skin-disease model of NC/Nga mice. P6 reduced IFN-γ and IL-13, whereas it enhanced IL-17 and IL-22 expression. In vitro, P6 also inhibited both Th1 and Th2 development, whereas it promoted Th17 differentiation from naive T cells when present within a certain range of concentrations. This was probably because P6 strongly inhibited STAT1, STAT5, and STAT6 phosphorylation, whereas STAT3 phosphorylation was less efficiently suppressed by P6 at the same concentration. Furthermore, IL-22 protects keratinocytes from apoptosis induced by IFN-γ, and administration of IL-17 and IL-22 partially ameliorated skin diseases in NC/Nga mice. These results suggested that the JAK inhibitor P6 is therapeutic for AD by modulating the balance of Th2 and Th17.     

Formulation with CpG oligodeoxynucleotides prevents induction of pulmonary immunopathology following priming with formalin-inactivated or commercial killed bovine respiratory syncytial virus vaccine

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-gamma production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.     

Enhanced tumor-specific long-term immunity of hemagglutinating [correction of hemaggluttinating] virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides

Immunization with dendritic cells (DCs) using various Ag-loading approaches has shown promising results in tumor-specific immunotherapy and immunoprevention. Fused cells (FCs) that are generated from DCs and tumor cells are one of effective cancer vaccines because both known and unknown tumor Ags are presented on the FCs and recognized by T cells. In this study, we attempted to augment antitumor immunity by the combination of DC-tumor FC vaccination with immunostimulatory oligodeoxynucleotides containing CpG motif (CpG ODN). Murine DCs were fused with syngeneic tumor cells ex vivo using inactivated hemagglutinating virus of Japan (Sendai virus). Mice were intradermally (i.d.) immunized with FCs and/or CpG ODN. Coadministration of CpG ODN enhanced the phenotypical maturation of FCs and unfused DCs, and the production of Th1 cytokines, such as IFN-gamma and IL-12, leading to the induction of tumor-specific CTLs without falling into T cell anergy. In addition, immunization with FCs + CpG ODN provided significant protection against lethal s.c. tumor challenge and spontaneous lung metastasis compared with that with either FCs or CpG ODN alone. Furthermore, among mice that rejected tumor challenge, the mice immunized with FCs + CpG ODN, but not the mice immunized with FCs or CpG ODN alone, completely rejected tumor rechallenge, indicating that CpG ODN provided long-term maintenance of tumor-specific immunity induced by FCs. Thus, the combination of DC-tumor FCs and CpG ODN is an effective and feasible cancer vaccine to prevent the generation and recurrence of cancers.     

In vivo role of p38 mitogen-activated protein kinase in mediating the anti-inflammatory effects of CpG oligodeoxynucleotide in murine asthma

DNA containing unmethylated CpG motifs is intrinsically immunostimulatory, inducing the production of a variety of cytokines and chemokines by immune cells. The strong Th1 response triggered by CpG oligodeoxynucleotide (ODN) inhibits the development of Th2-mediated allergic asthma in mice. This work documents that CpG ODN-induced IL-12 production plays a critical role in this process, because intrapulmonary CpG ODN inhibits allergic inflammation in wild-type but not IL-12(-/-) mice. CpG ODN rapidly localized to alveolar macrophages (AM), thereby triggering the phosphorylation of p38 mitogen-activated protein kinase (MAP kinase). AM cultured with CpG but not control ODN up-regulated IL-12 p40 expression and release, and these effects were blocked by the highly specific p38 MAP kinase inhibitor SB202190. Intrapulmonary administration of this inhibitor blocked the ability of CpG ODN to produce IL-12 in the lungs and reversed the anti-inflammatory effects of CpG ODN on allergic lung inflammation. These findings indicate that IL-12 production by AM is stimulated by intrapulmonary CpG ODN administration through a p38 MAP kinase-dependent process, and IL-12 is a key cytokine that mediates CpG ODN-induced protection against allergic lung inflammation.     

Effects of a hexameric deoxyriboguanosine run conjugation into CpG oligodeoxynucleotides on their immunostimulatory potentials

CpG oligodeoxynucleotides (ODNs) are promising immunomodulatory agents for treating human diseases and vaccine development. Phosphodiester CpG ODNs were demonstrated to have poor immunostimulatory potentials for cytokine production. However, the conjugation of consecutive deoxyriboguanosine residues, called a dG run, at the 3' terminus of phosphodiester CpG ODNs significantly enhanced TNF-alpha and IL-12 production from mouse splenic dendritic cells (DCs). The optimal induction of cytokine production was achieved by the addition of a hexameric dG (dG6) run. In contrast, the existence of a dG6 run either at the 5' terminus of phosphodiester CpG ODNs or at the 3' terminus of phosphorothioate CpG ODNs diminished CpG-mediated cytokine induction, suggesting that the effects of a dG run depend on its location and the chemical property of the ODN backbone, respectively. In addition, we provided the evidence that the conjugation of a dG6 run caused the structural transformation of CpG ODNs, which facilitates their targeting into mouse APCs such as splenic DCs, B cells, and peritoneal macrophages with a scavenger receptor type A ligand specificity. Among primary APCs, DCs were the most potent for CpG ODN-mediated IL-12 production. Furthermore, we demonstrated that the conjugation of a dG6 run into the 3' terminus of phosphodiester CpG ODNs was crucial for their ability to generate Th1 immunity in vivo. Thus, the conjugation of a dG6 run into phosphodiester CpG ODNs would be an alternative way to optimize their immunostimulatory potentials in vitro and in vivo.     

Stimulation of innate immune responses by CpG oligodeoxynucleotide in newborn lambs can reduce bovine herpesvirus-1 shedding

Stimulation of the innate immune system is potentially very important in neonates who have an immature adaptive immune system and vaccination cannot be used to reduce the risk of infection. CpG oligodeoxynucleotide (ODN) can stimulate innate immune responses in newborn chickens and mice, but similar studies are lacking in other mammalian species. We have shown previously that CpG ODN can both stimulate an acute-phase immune response and induce the antiviral effector molecule, 2'5'-A synthetase, in adult sheep. Therefore, the immunostimulatory activity of A class and B class CpG ODN was evaluated in newborn lambs, and the capacity of CpG ODN-induced responses to reduce viral shedding was evaluated following aerosol challenge with the respiratory pathogen, bovine herpesvirus-1 (BHV-1). In vitro CpG ODN stimulation of peripheral blood mononuclear cells (PBMC) isolated from newborn lambs (3-5 days old) and adult sheep induced equivalent CpG-specific proliferative responses and interferon-alpha (IFN-alpha) secretion. CpG ODN-induced IFN-alpha secretion by neonatal PBMCs was, however, significantly (p < 0.01) enhanced 6 days after subcutaneous (s.c.) injection of 100 microg/kg CpG ODN 2007. Newborn lambs injected s.c. with B class CpG ODN 2007 or the inverted GpC control ODN formulated in 30% Emulsigen (MVP Laboratories, Ralston, NE) displayed CpG ODN-specific increases in body temperature (p < 0.0001), serum 2'5'-A synthetase activity (p = 0.0015), and serum haptoglobin (p = 0.07). CpG ODN-treated lambs also displayed a transient reduction in viral shedding on day 2 postinfection (p < 0.05), which correlated (p < 0.03) with serum 2'5'-A synthetase levels on the day of viral challenge. These observations confirmed that CpG ODNs effectively activate innate immune responses in newborn lambs and CpG ODN-induced antiviral responses correlated with a reduction in viral shedding.     

Intranasal mite allergen induces allergic asthma-like responses in NC/Nga mice

Airway responses induced by intranasal administration of mite allergen without adjuvant were studied in NC/Nga mice. A crude extract of Dermatophagoides farinae (Df) was administered for 5 consecutive days and a single intranasal challenge booster dose was given 1 week after the last sensitization. 24 h after the single challenge, the airway hyperresponsiveness (AHR) was measured and the bronchoalveolar lavage fluid (BALF) was analyzed for numbers of eosinophils and neutrophils, and both cytokine and chemokine levels. There were marked increases in number of eosinophils in the BALF, AHR, Th2 cytokines (IL-5 and IL-13), and chemokine (eotaxin-1 and eotaxin-2) levels in the BALF following Df exposure. C57BL/6N, A/J, BALB/c, and CBA/JN mouse strains were also exposed to Df crude extract, but all of the measured responses were strongest in NC/Nga mice. Furthermore, Df-exposed NC/Nga mice showed the goblet cell hyperplasia, pulmonary eosinophilic inflammation, and increases in both total serum IgE and Df-specific IgG1. After intranasal exposure of NC/Nga mice to crude extract of Dermatophagoides pteronyssinus, the BALF eosinophilia and AHR were similar to responses induced by Df. None of the study parameters were increased in response to intranasal exposure to ovalbumin. These data demonstrated that NC/Nga mice developed allergic asthma-like responses after intranasal exposure to mite allergens.     

The mechanism of a defective IFN-gamma response to bacterial toxins in an atopic dermatitis model, NC/Nga mice, and the therapeutic effect of IFN-gamma, IL-12, or IL-18 on dermatitis

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-gamma levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-gamma response to staphylococcal enterotoxin B was correlated to the lack of regular Vbeta8(+) T cells and Vbeta8(+) NK T cells, and the low IFN-gamma response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-gamma, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.     

Inability of IL-12 to down-regulate IgE synthesis due to defective production of IFN-gamma in atopic NC/Nga mice.

NC/Nga mice raised in nonsterile circumstances spontaneously suffer from atopic dermatitis-like skin lesions with IgE hyperproduction. We investigated effects of rIL-12 on the IgE production in NC/Nga mice. rIL-12 administration was successful to suppress the increase of IgE levels in BALB/c mice immunized with OVA and aluminum hydroxide, but failed to abrogate that in NC/Nga mice. Both in vivo and in vitro IFN-gamma production induced by rIL-12 was less in NC/Nga mice than in BALB/c mice. Addition of rIFN-gamma to rIL-4 and LPS completely abrogated IgE production by B cells of BALB/c mice, but was insufficient to suppress it by B cells of NC/Nga mice. In splenic cells pretreated with Con A, STAT4 was phosphorylated at the tyrosine residue by addition of rIL-12, which was more weakly inducible in NC/Nga mice than in BALB/c mice. Finally, we examined the preventive ability of rIL-12 on the clinical aspects of atopic dermatitis in NC/Nga mice. rIL-12 administration resulted in exacerbation of development of the skin lesions and IgE production in NC/Nga mice raised in nonsterile circumstances. These results suggest that defective production of IFN-gamma by T cells less sensitive to IL-12 and low responsiveness of B cells to IFN-gamma may contribute to IgE hyperproduction in NC/Nga mice, and that IL-12 may have no ability to improve the clinical aspects of NC/Nga mice.     

Immunostimulatory CpG oligonucleotides abrogate allergic susceptibility in a murine model of maternal asthma transmission

We tested the potential of CpG oligodeoxynucleotides (ODN) to reverse the increased susceptibility to allergic airways disease in neonatal mice in a model of maternal transmission of asthma risk. Offspring of OVA-sensitized and challenged BALB/c mother mice were subjected to an intentionally suboptimal sensitization protocol that has minimal effects on normal mice, but results in airway hyperresponsiveness (AHR) and airway inflammation (AI) in babies of asthmatic mother mice. We evaluated pulmonary function and AI in CpG- or control ODN-treated offspring. CpG treatment of neonates on day 4 of life prevents the AHR otherwise seen in this model (enhanced pause at 100 mg/ml methacholine: CpG, 0.9 +/- 0.1; ODN control, 3.8 +/- 0.6; n = 62; p < 0.005). It also prevented the development of AI, as evident in decreased bronchoalveolar lavage eosinophilia (CpG, 1.2 +/- 0.3%; ODN, 31.4 +/- 4.1%; n = 56; p < 0.005), diminished the severity of AI on histopathology, and resulted in lower IL-5 levels in bronchoalveolar lavage fluid. The effect of CpG persisted for at least 4-6 wk and was allergen independent. Treatment with CpG just before OVA aerosol challenge also prevented allergic responses. The data support the potential for immunomodulatory therapy with CpG in early life to reduce susceptibility to asthma.     

CpG oligonucleotides can prophylactically immunize against Th2-mediated schistosome egg-induced pathology by an IL-12-independent mechanism

Using a Schistosoma mansoni egg-induced granuloma model, we examined the ability of CpG oligodeoxynucleotides (ODN) to suppress Th2-type cytokine expression and to prophylactically immunize against Th2-dependent pulmonary pathology. The mechanism was examined by studying Th2 response regulation in cytokine-deficient mice. Surprisingly, our findings revealed several functions of CpG DNA that were completely IL-12 independent. Most striking was the marked suppression in Th2 cytokine expression and granulomatous inflammation observed in egg/CpG-sensitized IL-12-deficient mice. Immune deviation was not dependent on NK or B cells. However, a role for IL-10, B7.1, and CD40 expression in Th2 response inhibition was suggested. Indeed, CpG ODN up-regulated all three elements in both wild-type and IL-12-deficient mice. The role of IL-10 was demonstrated in mice exhibiting combined deficiencies in IL-12 and IL-10. Here, a marked increase in egg-specific IL-4/IL-5-producing cells confirmed a role for both cytokines in Th2 response inhibition. Nevertheless, the frequency of Th2-producing cells was again reduced by CpG ODN. However, in marked contrast to IL-12-deficient animals, a significant increase in IFN-gamma-producing cells likely explains the reduced Th2 response in IL-10/IL-12-deficient mice. Thus, a novel IL-12-independent type 1-inducing pathway was revealed in the combined absence of IL-12 and IL-10. Together, these data demonstrate 1) that the Th1-promoting activity of CpG DNA is controlled by IL-12 and IL-10, and 2) that Th2 response inhibition by CpG ODN involves IL-12-independent changes in IL-10 and costimulatory molecule expression. These findings illustrate the utility of CpG DNA as adjuvants for vaccines designed to prevent Th2-dependent immunopathology.     

Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA

Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein antigens administered by parenteral or mucosal routes to BALB/c mice. To date, there have been no studies using combined parenteral/mucosal approaches with CpG DNA as adjuvant. In this study we evaluated different parenteral prime-mucosal boost and mucosal prime-parenteral boost strategies using hepatitis B surface antigen (HBsAg) alone or with different adjuvants: aluminum hydroxide (alum), cholera toxin (CT), CpG ODN. In addition, since CpG ODN has previously been shown to act synergistically with other adjuvants after parenteral or mucosal delivery, we also evaluated adjuvant combinations: alum+CpG ODN and CT+CpG ODN. The effects of adjuvant and administration strategy on systemic and mucosal humoral responses were measured, as well as cell-mediated immune responses (cytotoxic T lymphocyte activity). These results were compared to parenteral only or mucosal only strategies. Our findings demonstrate that parenteral immunization can prime for mucosal responses even when different lymph nodes were being targeted. HBsAg-specific immune responses (IgG in plasma, cytotoxic T lymphocytes) induced by parenteral prime could all be significantly enhanced by mucosal boosting and despite the fact that intramuscular immunization alone could not induce mucosal IgA, it could prime for a subsequent mucosal boost. In addition, the presence of adjuvant at time of boosting could influence the nature of subsequent immune responses (Th1 vs. Th2). Mice primed intranasally could have their systemic immune responses boosted with a parenteral administration and it was also possible to enhance mucosal responses induced by intranasal prime with an intramuscular boost.     

Intranasal immunization of mice with CpG DNA induces strong systemic and mucosal responses that are influenced by other mucosal adjuvants and antigen distribution

BACKGROUND: Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory cytosine-guanine phosphate-linked dinucleotide (CpG) motifs are potent systemic and mucosal adjuvants in mice that have synergistic action with numerous other adjuvants, including alum and cholera toxin (CT). Herein, we evaluate CpG ODN with intranasal (IN) delivery of purified hepatitis B surface antigen (HBsAg), relative to and in combination with CT, Escherichia coli heat labile enterotoxin (LT), the B subunit of CT (CTB), and a nontoxic derivative of LT (LTK63). MATERIALS AND METHODS: BALB/c mice were immunized by IN administration of HBsAg, alone or combined with CT, LT, CTB, or LTK63, and/or CpG ODN, or non-CpG control ODN. In addition, the effect of low-or high-volume administration was assessed, in order to target upper respiratory or entire respiratory tract, respectively. HBsAg-specific systemic (immunoglobulins: IgG, IgG1, IgG2a in plasma) and mucosal (IgA in fecal, lung, vaginal, saliva, and gut samples) humoral responses, as well as cell-mediated immune responses including T-cell proliferation and cytokines (interleukins: IL-4, IL-5; interferon: IFN-gamma) were evaluated. RESULTS: CpG ODN, CT, and LT augmented anti-HBs titers equally, and more so than did CTB or LTK63. CpG ODN acted synergistically with CT and LT, but not CTB or LTK63 to enhance anti-HBs titers. Nevertheless, CpG ODN induced a more Th1-like response for all combinations, compared with the same formulation without CpG. Strength of induced systemic and mucosal immune responses was better with IN delivery of a large volume. A small volume required multiple administrations and higher doses of antigen and adjuvant for equal results. This suggests that delivery of antigen to the lung and/or diges-tive system is superior to delivery to the nasal cavity. CONCLUSIONS: Our results suggest that the synergy between CpG ODN and native toxins (CT, LT) may depend on their enzymatic activity and that the lack of synergy with nontoxic derivatives (LTB, LTK63) arises, since they do not have enzymatic activity. Because both CT and LT are too toxic for use in humans, it is possible that CpG ODN may be combined with bacterial toxin mutants that retain some enzymatic activity to optimize immune augmentation.