CpG-containing Oligodeoxynucleotides Research Articles

CpG‐containing oligodeoxynucleotide promotes microglial cell uptake of amyloid β 1–42 peptide by up‐regulating the expression of the G‐protein‐coupled receptor mFPR2

Human G protein-coupled formyl peptide receptor like 1 (FPRL1) and its mouse homologue murine formyl peptide receptor 2 (mFPR2) mediate the chemotactic activity of amyloid beta 1-42 (Abeta42), a key pathogenic peptide in Alzheimer's disease (AD). Since mFPR2 is up-regulated in mouse microglia by lipopolysaccharide (LPS), a Toll-like receptor 4 ligand, we investigated the capacity of CpG-containing oligodeoxynucleotide (ODN), a Toll-like receptor (TLR) 9 ligand, to regulate the expression of mFPR2 in mouse microglia. CpG ODN markedly enhanced the expression and function of mFPR2 in microglial cells, which exhibited increased chemotactic responses to mFPR2 agonists, including Abeta42. The effect of CpG ODN is dependent on activation of p38 MAPK. Further studies showed that CpG ODN-treated microglia increased their capacity to endocytose Abeta42 through mFPR2, as this process was abrogated by pertussis toxin, a Gi protein inhibitor, and W peptide, another potent mFPR2 agonist. Our results suggest that TLR9 may play an important role in promoting microglial recognition of Abeta42, thus affecting the pathogenic process of AD.

CpG-Containing Oligodeoxynucleotides Act through TLR9 to Enhance the NK Cell Cytokine Response to Antibody-Coated Tumor Cells

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcgammaRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-gamma (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1alpha was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 +/- 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-gamma compared with mice receiving either agent alone. TLR9-/- animals reconstituted with TLR9+/+ NK cells secreted IFN-gamma in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.

A CpG-containing oligodeoxynucleotide as an efficient adjuvant counterbalancing the Th1/Th2 immune response in diphtheria–tetanus–pertussis vaccine

Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responses to vaccine component(s). Diphtheria–tetanus–pertussis (DPT) vaccine contains not only aluminum hydrate (alum) to enhance the immune response to the vaccine ingredients, but also, both for that purpose and as a principal ingredient, pertussis toxin (PT). However, both adjuvants strongly promote T helper (Th) 2 type immune responses. Th1 and Th2 type immune responses are counterbalanced in vivo, and a Th2-prone immune response is not effective against intracellular infections but promotes IgE production, which is related to allergic disease. In this study, we used the CpG motif contained in oligodeoxynucleotide (CpG-ODN), which has an adjuvant effect and also induces the Th1 response, as an adjuvant to this vaccine, and we investigated its adjuvanticity and its potential to modulate immune responses to DPT vaccine. Administration of DPT vaccine with CpG-ODN (DPT-alum/ODN) to mice significantly reduced the total IgE levels and increased the anti-PT specific IgG2a titer in serum, in comparison with ordinary DPT vaccine (DPT-alum). Moreover, we investigated the antibody response to orally administrated ovalbumin (OVA) after vaccine administration. In the DPT-alum/ODN-administered group, the OVA specific IgE production in serum greatly decreased in comparison with that in the DPT-alum-administered group. These data indicate that CpG-ODN was not useful only as an efficient vaccine adjuvant but also shifted the immune responses substantially toward Th1 and modulated the Th1/Th2 immune response in DPT vaccine. These data suggested new applications of CpG-ODN as adjuvants in DPT vaccine.

CpG motif in ATCGAT hexamer improves DNA-vaccine efficiency against lethal Pseudorabies virus infection in pigs

This study evaluated the adjuvant activity of a porcine-specific type A CpG-containing oligodeoxynucleotide (CpG-ODN) in a DNA-vaccine against Pseudorabies virus (PrV) infection. This vaccine, composed of three independent plasmids encoding PrV gB, gC and gD glycoproteins, was injected alone or was co-injected with CpG-ODN or control GpC-ODN. CpG-ODN improved the PrV-specific humoral immune response and provided better clinical protection against lethal PrV-infection. The PrV-specific cell-mediated immune response seemed also to be increased.

Inhibitors of TLR-9 Act on Multiple Cell Subsets in Mouse and Man In Vitro and Prevent Death In Vivo from Systemic Inflammation

In parallel with the discovery of the immunostimulatory activities of CpG-containing oligodeoxynucleotides, several groups have reported specific DNA sequences that could inhibit activation by CpG-containing oligodeoxynucleotides in mouse models. We show that these inhibitory sequences, termed IRS, inhibit TLR-9-mediated activation in human as well as mouse cells. This inhibitory activity includes proliferation and IL-6 production by B cells, and IFN-alpha and IL-12 production by plasmacytoid dendritic cells. Our studies of multiple cell types in both mice and humans show the optimal IRS to contain a GGGG motif within the sequence, and the activity to require a phosphorothioate backbone. Although the GGGG motif readily itself leads to formation of a tetrameric oligodeoxynucleotide structure, inhibitory activity resides exclusively in the single-stranded form. When coinjected with a CpG oligodeoxynucleotide in vivo, IRS were shown to inhibit inflammation through a reduction in serum cytokine responses. IRS do not need to be injected at the same site to inhibit, demonstrating that rapid, systemic inhibition of TLR-9 can be readily achieved. IRS can also inhibit a complex pathological response to ISS, as shown by protection from death after massive systemic inflammation induced by a CpG-containing oligodeoxynucleotides.

CpG oligodeoxynucleotides promote the host protective response against infection withCryptococcus neoformansthrough induction of interferon-gamma production by CD4+ T cells

In the present study, we elucidated the effect of synthetic CpG-containing oligodeoxynucleotides (ODN) on pulmonary and disseminated infection caused by Cryptococcus neoformans. CDF-1 mice were inoculated intratracheally with a highly virulent strain of this pathogen, which resulted in massive bacterial growth in the lung, dissemination to the brain and death. Administration of CpG-ODN promoted the clearance of C. neoformans in the lungs, decreased their dissemination to brain and prolonged the survival of infected mice. These effects correlated well with the enhanced production of interleukin (IL)-12 and interferon (IFN)-gamma and attenuated secretion of IL-4 in bronchoalveolar lavage fluids (BALF) and promoted development of Th1 cells, as indicated by the increased production of IFN-gamma by paratracheal lymph node cells upon restimulation with cryptococcal antigens. The IFN-gamma synthesis in BALF was inhibited by depletion of CD8(+) and CD4(+) T cells on days 7 and 14 after infection, respectively, but not by depletion of NK and gammadelta T cells. Consistent with these data, intracellular expression of IFN-gamma was detected predominantly in CD8(+) and CD4(+) T cells in the lung on days 7 and 14, respectively. The protective effect of CpG-ODN, as shown by the prolonged survival, was completely and partially inhibited by depletion of CD4(+) or CD8(+) T cells, respectively, but not by depletion of other cells. Finally, TNF-alpha was markedly induced by CpG-ODN, and the protective effect of this agent was strongly inhibited by neutralizing anti-TNF-alpha MoAb. Our results indicate that CpG-ODN alters the Th1-Th2 cytokine balance and promotes host resistance against infection with C. neoformans.

Serum Alters the Uptake and Biologic Activity of CpG Oligodeoxynucleotides in B Cell Chronic Lymphocytic Leukemia

Immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) have a number of effects on B cells, including upregulation of immunogenic molecules, and, therefore, appear attractive as potential components of immunotherapy for B cell chronic lymphocytic leukemia (B-CLL). Previous in vitro studies investigating the effect of CpG ODN on B-CLL cells used serum-low conditions and did not account for the longer-half life of CpG ODN in vitro. The present study was designed to explore how the presence of serum and exposure time affect CpG ODN-mediated changes on B-CLL cells. The optimal concentration for CpG ODN-mediated effects in the presence of 100% serum or plasma was higher (10-20 microg/ml) than for serum-low conditions. Maximal CpG ODN-mediated effects required the presence of ODN for no longer than 3 hours. The inhibition of CpG ODN-mediated effects by serum correlated with lower uptake of ODN into B-CLL cells in the presence of serum. A threshold effect on biologic response was observed, with a given amount of ODN internalized, resulting in phenotypic changes. In conclusion, systemic short-term application of CpG ODN appears to be sufficient to induce phenotypic changes, but higher doses of CpG ODN than previously thought may be necessary because of inhibition of their uptake by serum.

CpG inhibits IgE class switch recombination through suppression of NFκB activity, but not through Id2 or Bcl6

The CpG motif in DNA plays a critical role in immunity via modulating the Th1/Th2 balance. In B cells, CpG-containing oligodeoxynucleotides (CpG ODNs) inhibit IL-4-mediated class switch recombination (CSR) to IgG1 and IgE through inhibition of the germline transcription (GLT) of these isotypes. However, the molecular mechanism of this inhibitory effect remains elusive. We showed here that Id2 and Bcl6, both of which inhibit IgE GLT and CSR, are not involved in this inhibitory pathway. We demonstrated that there is reduced activity of NFκB binding to the IgE promoter and a reduction of Irf4 protein in CpG ODN-treated B cells. These data indicate the critical role of NFκB and Irf4 in the regulation of IgE CSR through actions downstream of CpG signaling.

CpG oligodeoxynucleotides inhibit tumor growth and reverse the immunosuppression caused by the therapy with 5-fluorouracil in murine hepatoma

To investigate the effect of CpG-containing oligodeoxynucleotides (CpG ODN) alone or in combination with the chemotherapeutic agent 5-fluorouracil (5-FU) on tumor growth and whether CpG ODN can reverse the immunosuppression caused by the chemotherapy with 5-FU in murine hepatoma model. Hepatoma model was established by subcutaneous inoculation with hepatoma-22 (H(22)) cells into the right flank of BALB/c mice. Mice with tumor were treated by peritumoral injection of CpG ODN alone or in combination with subcutaneous injection of 5-FU. Tumor size was quantified regularly. Serum levels of IL-12 and IFN-gamma in mice were assayed by enzyme-linked immunosorbent assay (ELISA). The lytic capacity of splenic NK cells was tested by lactate dehydrogenase release assay. Peritumoral injection of CpG ODN alone or in combination with subcutaneous injection of 5-FU, and the treatment with 5-FU alone all led to significant inhibition of hepatoma growth. The mean tumor volumes fell by 46.66% in mice injected with CpG ODN, 68.34% in the 5-FU treated mice, and 70.23% in mice treated with the combination of CpG ODN and 5-FU than in controls. There was no significant difference in tumor size between 5-FU-treated mice and mice treated with the combination of 5-FU and CpG ODN (P>0.05). The serum levels of IL-12 and IFN-gamma of mice treated with CpG ODN alone (IL-12: 464.50+/-24.37 pg/mL; IFN-gamma: 134.20+/-25.76 pg/mL) or with the co-administration of CpG ODN and 5-FU (IL-12: 335.83+/-28.74 pg/mL; IFN-gamma: 111.00+/-5.33 pg/mL) were significantly higher than that of controls (IL-12: 237.50+/-45.31 pg/mL; IFN-gamma: 56.75+/-8.22 pg/mL). The production of IL-12 and IFN-gamma was suppressed moderately in 5-FU-treated mice (IL-12: 166.67+/-53.22 pg/mL; 53.33+/-16.98 pg/mL) compared to control mice (P>0.05), whereas the combination of CpG ODN and 5-FU significantly increased the serum levels of IL-12 and IFN-gamma compared to 5-FU alone (P<0.05). The NK cell killing activity in CpG ODN-treated mice (44.04+/-1.38%) or the mice treated with CpG ODN combined with 5-FU (30.67+/-1.28%) was significantly potentiated compared to controls (19.22+/-0.95%, P<0.05). The co-administration of CpG ODN and 5-FU also significantly enhanced the lytic activity of NK cells when compared with the treatment with 5-FU alone (12.03+/-1.42%, P<0.05). The present data suggests that CpG ODN used as single therapeutic agent triggers anti-tumor immune response to inhibit the growth of implanted hepatoma and reverses the immunosuppression caused by the chemotherapy with 5-FU.

Anti-proliferative effects of phosphodiester oligodeoxynucleotides

The immunostimulatory effects of cytosine-phosphate-guanosine (CpG)-containing oligodeoxynucleotides (ODNs) have been extensively documented. In this paper, we describe the inhibitory effects of ODNs that contain natural phosphodiester backbones (O-ODNs) on the immunostimulation caused by CpG-containing phosphorothioated ODNs (CpG-S). CpG-S stimulation of mouse splenocyte proliferation was reduced by the addition of O-ODNs that contained or lacked the CpG-motif (CpG-containing phosphodiester oligodeoxynucleotide, CpG-O or GpC-O). The total number of cultured splenocytes was up-regulated by CpG-S, whereas repetitive addition of O-ODNs to the cell cultures inhibited this effect. The frequency of T2-like B cells was found to be increased by CpG-S. The culture supernatants of CpG-S-treated splenocytes contained elevated levels of IL-10 and IL-6. However, IL-10 and IL-6 production was down-regulated significantly by the combination of CpG-S and either CpG-O or GpC-O. The O-ODN mediated inhibition of proliferation was less pronounced in IL-10 −/− mice. Thus, the O-ODNs, irrespective of CpG content, exerted inhibitory activities on the proliferation of B cells. These anti-proliferative effects appear to be mediated both by the down-regulation of IL-10 production and increased apoptosis.

Cpg Oligodeoxynucleotide-Mediated Effects on B-Cell Chronic Lymphocytic Leukemia In Vitro Are Influenced by Cytogenetic Status and the Presence of Serum.

Immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) are TLR9 agonists that mediate a number of immunologic effects in normal and malignant B cells including upregulation of immunogenic molecules. They are therefore felt to be attractive as potential components of immunotherapy for B cell chronic lymphocytic leukemia (B-CLL). The cytogenetic status of B-CLL is predictive of clinical prognosis, but little is known about how CpG ODN-mediated effects on B-CLL cells correlate with cytogenetic status. The present study was designed to explore the impact cytogenetic status has on in vitro B-CLL cell survival and immunogenicity. Since most in vitro studies to date have been performed under serum-low conditions, we also evaluated how the presence of autologous serum or plasma impacts on CpG ODN-mediated effects on B-CLL cells. B-CLL cytogenetic status by interphase FISH, as well as immunophenotype and cell survival in the absence or presence of CpG ODN was determined in 23 samples. CpG ODN decreased in vitro survival of B-CLL cells with good prognosis cytogenetics, but had little effect on cells with poor prognosis cytogenetics. In contrast, CpG ODN upregulated costimulatory and antigen-presenting molecules and enhanced allogeneic T cell response in samples with good and poor prognosis cytogenetics. The optimal concentration for CpG ODN-mediated effects in the presence of 100% autologous serum or plasma was higher (10–20 micrograms/ml ODN) than for serum-low conditions (2.5–5 micrograms/ml ODN). The observed inhibition of CpG ODN-mediated effects by serum directly correlated with a lower uptake of ODN into B-CLL cells in the presence of serum. In conclusion, CpG ODN induced changes in B-CLL consistent with enhanced immunogenicity in all samples studied, but induced apoptosis most effectively in the subset of B-CLL cells with good prognosis cytogenetics. Approximately 4-fold more CpG ODN was needed to induce these changes when serum was present as compared to low serum conditions due to decreased ODN uptake by the cells in the presence of serum. These studies suggest CpG ODN may be useful as immunotherapeutic agents for B-CLL irrespective of cytogenetic status because of their potential effects on immunogenicity. Higher systemic doses of CpG ODN than previously thought might be necessary to induce these changes because of the effect of serum proteins on ODN uptake.

Immunostimulatory CpG motifs induce CTL responses to HIV type I oligomeric gp140 envelope protein

In the present study we investigated the immunomodulatory effects of two adjuvants, liposomal lipid A [L(LA)] and CpG-containing oligodeoxynucleotides (CpG ODN), to the HIV-1 ogp140 envelope protein. Administration of each of these adjuvants separately with unencapsulated ogp140 resulted in low antibody titres. Encapsulation of ogp140 in liposomes containing lipid A resulted in a sixfold increase in anti-ogp140 antibodies. The antibody titres were further enhanced threefold by the addition of CpG ODN. Priming and boosting BALB/c mice with unencapsulated ogp140 with L(LA) or encapsulation in liposomes containing lipid A induced a mixed Th1/Th2 type of immune response. In contrast, immunization with L(ogp140 + LA) plus CpG ODN switched the immune response to a Th-1 response with elevated anti-ogp140 IgG2a antibodies and IFN-gamma levels. Both adjuvants induced excellent ogp140-specific proliferative and CTL responses. Therefore, for the induction of high titre antibodies, but not for cellular responses, the antigen and lipid A have to be present in the same liposomes. These results can have significant implications in directing the Th1 or Th2 differentiation of antigen-specific immune responses in the context of vaccine development.

CpG immunostimulatory sequences enhance contact hypersensitivity responses in mice.

Bacterial DNA and synthetic cytidine-phosphate-guanosine-oligodeoxynucleotides (CpG ODN) potently activate dendritic cells (DC) and therefore have been proposed as adjuvants for vaccination strategies. Although CpG ODN are considered as safe adjuvants this study shows that CpG ODN are responsible for enhanced antigen-specific skin inflammatory reactions. We used the murine model of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB) in which hapten-specific CD8+T cytotoxic 1 cells are effector cells. Subcutaneous injection of CpG ODN, 1 d before sensitization enhanced the CHS response to DNFB and resulted in increased recruitment of CD8+ T cells at the challenge sites, whereas control ODN injection did not have any effect. This effect was local and not systemic as it was only observed when DNFB was applied at the same site as the CpG motifs. CpG ODN-induced enhancement of CHS was due to increased antigen-presenting cell functions of DC since: (i) CpG ODN-injected skin revealed upregulated expression of major histocompatibility complex class II, CD80, and CD86 molecules and (ii) CpG ODN treatment of DNFB-derivatized DC enhanced the intensity of CHS responses after in vivo transfer. Taken together, the results show that CpG ODN may be responsible for immune side-effects such as worsening of T cell-mediated skin diseases.

Synergistic activation of innate immunity by double-stranded RNA and CpG DNA promotes enhanced antitumor activity.

Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity. To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN). Combined treatments demonstrated synergy in nitric oxide, interleukin (IL)-12, tumor necrosis factor alpha, and IL-6 production. Studies using neutralizing antibodies for type I interferons (IFNs), IFN-alpha and IFN-beta, indicated that nitric oxide synthase synergism is mediated by paracrine/autocrine effects of IFN-beta. In contrast, enhanced cytokine production occurred independent of type I IFN and was maintained in macrophages from IFN-alpha/beta receptor knockout mice. Cotransfection of human Toll-like receptors 3 and 9 (receptors for dsRNA and CpG DNA, respectively) into 293T cells supported synergistic activation of an IL-8 promoter reporter construct by pIC, indicating interaction of the signaling pathways in driving the synergy response. In vivo stimulation of mice with pIC and CpG-ODN demonstrated synergy for serum IL-6 and IL-12p40 levels that correlated with an enhanced antitumor effect against established B16-F10 experimental pulmonary metastases. Treatment of tumor-bearing mice with pIC and CpG-ODN in combination resulted in enhanced nitric oxide synthase expression in lung tissue and enhanced up-regulation of class I major histocompatibility complex on splenic dendritic cells relative to treatments with either agent alone. In conclusion, the combined detection of viral pathogen-associated molecular patterns, i.e., dsRNA and CpG DNA, may mimic definitive viral recognition, resulting in an enhanced innate immune response that could be used for tumor vaccination or immunotherapy.

Vaccination with gp120-depleted HIV-1 plus immunostimulatory CpG oligodeoxynucleotides in incomplete Freund's adjuvant stimulates cellular and humoral immunity in rhesus macaques

Whole killed human immunodeficiency virus type 1 (HIV-1) immunogens contain the more conserved epitopes of HIV-1 and therefore may provide some utility as potential HIV-1 vaccine candidates. Previous studies have shown that synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine–guanine (CpG) dinucleotides trigger rapid stimulation of both CD4+ and CD8+ T cells. Here, we investigated whether immunization of rhesus macaques with an inactivated gp120-depleted HIV-1 immunogen, emulsified in incomplete Freund's adjuvant (IFA) together with immunostimulatory CpG-containing ODN (ODN 2006), would elicit HIV-specific cellular and humoral immune responses. High titer anti-p24 antibody levels were induced in all four immunized animals that were sustained 6 weeks after the fifth and final boost at 23 months. These anti-gag antibodies mapped to linear B-cell epitopes within the matrix (MA), capsid (CA), p2, nucleocapsid (NC) and p6 proteins of HIV-1 gag. HIV-specific interferon-γ-producing CD4+ and CD8+ T-cell responses were measured before and after the fourth and fifth immunizations by both intracellular cytokine (ICC) and ELISPOT techniques; responses were detected in three of the four immunized animals. CD4+ T-cell epitopes appear to map within amino acids 261–290 and 291–320 of p24 CA protein. Immunizations were well tolerated both locally and systemically. Based on these results, further studies of this approach are warranted.

Heterogeneity in the response of pigs and mice to specific CpG oligodeoxynucleotides

AbstractDifferences in immunostimulation activity of CpG oligodeoxynucleotides (ODNs) between pigs and mice were studied. In accordance with current research data, three kinds of CpG ODN – 2006, D19 and 1826, which were identified as optimally active in humans, pigs and mice, respectively – and control ODN D48, a non CpG-containing ODN, were selected and synthesized. Results indicated that CpG ODN D19 strongly stimulated the proliferation of porcine peripheral blood mononuclear cells (PBMCs), and the amount of γ-interferon in the supernatant of activated cells increased, exceeding that of all other ODNs tested, while mouse splenocytes did not respond. CpG ODN 1826 strongly stimulated the proliferation of mouse splenocytes, but porcine PBMCs remained unresponsive. CpG ODN 2006 effectively stimulated proliferation of both porcine PBMCs and mouse splenocytes. Therefore some specific CpG ODNs, D19 and 1826, selectively stimulated animal species, while CpG ODN 2006 possessed a common stimulation effect. In addition, CpG ODN D19 predominantly induced the response of type 1 T-helper cellsin vivo.

Polymyxin B enhances ISS-mediated immune responses across multiple species

The immunostimulatory effects of bacterial DNA on mammalian cells have been localized to unmethylated CpG motifs, and synthetic CpG-containing oligodeoxynucleotides that mimic these effects are known as immunostimulatory sequences (ISS). We have found that the polycationic antibiotic, polymyxin B (PMXB), associates with ISS and serum albumin in vitro and forms microparticles that greatly increase the activity of ISS on plasmacytoid dendritic cells (PDCs). Specifically, ISS/PMXB greatly enhanced IFN-α production from PDCs and other activities downstream of IFN-α, including IFN-γ secretion, NK lytic activity, and the expression of genes dependent upon IFN-α/IFN-γ. This amplification was specific for the IFN-α pathway since other ISS activities, including B cell proliferation, B cell IL-6 secretion, and PDC maturation, were not affected by PMXB. Both the polycationic peptide and lipophilic fatty acid side chain domains of PMXB, as well as the presence of a third party stabilizing agent such as albumin or Tween 85, were required for particle formation and enhanced ISS activity. The ISS-enhancing activity of PMXB was observed across multiple species (human, primate, and mouse) and in vivo (primate, mouse). These data illustrate the usefulness of formulating ISS with a cationic lipopeptide such as PMXB, which focuses and greatly amplifies the ISS-induced pathway of IFN-α-mediated responses.

Dendritic cells (DC) activated by CpG DNA ex vivo are potent inducers of host resistance to an intracellular pathogen that is independent of IL-12 derived from the immunizing DC.

We used the model of murine leishmaniasis to evaluate the signals enabling Ag-pulsed dendritic cells (DC) to prime a protective Th1 response in vivo. Bone marrow-derived DC (BMDC) that had been activated by TNF-alpha or CD40 ligation were not able to induce protection against leishmaniasis in susceptible BALB/c mice. In contrast, all mice vaccinated with a single dose of Leishmania major Ag-pulsed BMDC stimulated by prior in vitro exposure to CpG-containing oligodeoxynucleotides (ODN) were completely protected, had a dramatic reduction in parasite burden, and developed an Ag-specific Th1 response. Importantly, systemic administration of CpG ODN was not required. Protection mediated by ex vivo CpG ODN-activated and Ag-pulsed DC was solid, as documented by resistance to reinfection with a higher parasite dose, and long-lasting, as immunized mice were still protected against L. major challenge 16 wk after vaccination. A significantly increased level of protection could also be elicited in resistant C57BL/6 mice. Surprisingly, IL-12 expression by the immunizing BMDC was not required for induction of host resistance. In contrast, the availability of IL-12 derived from recipient cells was essential for the initial triggering of protective immunity by transferred BMDC. Together, these findings demonstrate that the type of stimulatory signal is critical for activating the potential of DC to induce a Th1 response in vivo that confers complete protection against an intracellular pathogen. Moreover, they show that the impact of activated DC on the initiation of a protective Th cell response in vivo may be independent of their ability to produce IL-12.

Combination of a CpG-oligodeoxynucleotide and a topoisomerase I inhibitor in the therapy of human tumour xenografts

The study was conducted to investigate the effects of a novel therapeutic approach, i.e. the combination of chemotherapy and immunotherapy, against a human prostate carcinoma xenograft. A topoisomerase I inhibitor, topotecan, and CpG-containing oligodeoxynucleotides (CpG-ODN) were combined. Athymic mice bearing the PC-3 human prostate carcinoma were treated with the maximum tolerated dose (MTD) of topotecan (3 weekly treatments) and with repeated treatments of CpG-ODN (40 and 20 μg/mouse); tumour growth and lethal toxicity were monitored. Topotecan effect on CpG-ODN-induced production of interleukin (IL) 12, interferon (IFN)-γ and tumour necrosis factor-α was also assessed. Since topotecan pretreatment differentially influenced CpG-ODN-induced production of IL-12 and IFN-γ, the antitumour effects of the two therapies were investigated in a sequential (full topotecan regimen followed by CpG-ODN) or in an alternating sequence (starting with CpG-ODN). Topotecan inhibited PC-3 tumour growth, inducing 95% tumour volume inhibition. All combined treatments resulted in a significant delay in tumour growth, compared to the effects in topotecan-treated mice (P<0.01, by analysis of tumour growth curves). The combination regimens were well tolerated, except for the alternating sequence of 40 μg CpG-ODN and topotecan, which resulted in three out of eight toxic deaths. This alternating sequence was highly toxic even when another cytotoxic drug (doxorubicin) was used in healthy mice. In conclusion, the combination of topotecan and CpG-ODN increased antitumour effects over chemotherapy alone in the growth of a human prostate carcinoma xenograft. Administration sequence was critical to the combination toxicity: the complete regimen of the cytotoxic drug followed by repeated administrations of the immunomodulator seemed the most promising for further investigations.

Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration

DNA containing unmethylated cytidyl guanosyl (CpG) sequences, which are underrepresented in mammalian genomes but prevalent in prokaryotes, is endocytosed by cells of the innate immune system, including macrophages, monocytes and dendritic cells, and activates a pathway involving Toll-like receptor-9 (TLR9). CpG-containing oligodeoxynucleotides (CpG-ODN) are potent stimulators of innate immunity, and are currently being tested as adjuvants of antimicrobial, antiallergic, anticancer and antiprion immunotherapy. Little is known, however, about the consequences of repeated CpG-ODN administration, which is advocated for some of these applications. Here we report that daily injection of 60 microg CpG-ODN dramatically alters the morphology and functionality of mouse lymphoid organs. By day 7, lymphoid follicles were poorly defined; follicular dendritic cells (FDC) and germinal center B lymphocytes were suppressed. Accordingly, CpG-ODN treatment for > or =7 d strongly reduced primary humoral immune responses and immunoglobulin class switching. By day 20, mice developed multifocal liver necrosis and hemorrhagic ascites. All untoward effects were strictly dependent on CpG and TLR9, as neither the CpG-ODN treatment of Tlr9(-/-) mice nor the repetitive challenge of wild-type mice with nonstimulatory ODN (AT-ODN) or with the TLR3 agonist polyinosinic:cytidylic acid (polyI:C) were immunotoxic or hepatotoxic.