Toll-like receptor 9 (TLR9) agonists in the treatment of cancer.

Although still early in clinical development, agonists of Toll-like receptor 9 (TLR9) have demonstrated potential for the treatment of cancer. TLR9 agonists directly induce activation and maturation of plasmacytoid dendritic cells and enhance differentiation of B cells into antibody-secreting plasma cells. Preclinical and early clinical data support the use of TLR9 agonists in patients with solid tumors and hematologic malignancies. In preclinical studies, TLR9 agonists have shown activity not only as monotherapy, but also in combination with multiple other therapies, including vaccines, antibodies, cellular therapies, other immunotherapies, antiangiogenic agents, radiotherapy, cryotherapy and some chemotherapies. Phase I and II clinical trials have indicated that these agents have antitumor activity as single agents and enhance the development of antitumor T-cell responses when used as therapeutic vaccine adjuvants. The activity and safety of these novel anticancer agents are being explored in a wide range of tumor types as part of a variety of therapeutic strategies with the goal of harnessing the immune response to fight cancer.

Enhancement of infectious disease vaccines through TLR9-dependent recognition of CpG DNA.

The adaptive immune system-with its remarkable ability to generate antigen-specific antibodies and T lymphocytes against pathogens never before "seen" by an organism-is one of the marvels of evolution. However, to generate these responses, the adaptive immune system requires activation by the innate immune system. Toll-like receptors (TLRs) are perhaps the best-understood family of innate immune receptors for detecting infections and stimulating adaptive immune responses. TLR9 appears to have evolved to recognize infections by a subtle structural difference between eukaryotic and prokaryotic/viral DNA; only the former frequently methylates CpG dinucleotides. Used as vaccine adjuvants, synthetic oligodeoxynucleotide (ODN) ligands for TLR9--CpG ODN--greatly enhance the speed and strength of the immune responses to vaccination.

CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study.

Oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) act as potent Th1-like immune enhancers with many antigens in animal models. We have extended these observations to the first clinical evaluation of the safety, tolerability and immunogenicity of CPG 7909 when added to a commercial HBV vaccine. In a randomized, double-blind phase I dose escalation study, healthy volunteers aged 18-35 years were vaccinated at 0, 4 and 24 weeks by intramuscular injection with Engerix-B (GlaxoSmithKline). The regular adult dose of 20 microg recombinant hepatitis B surface antigen (HBsAg) adsorbed to alum was administered mixed with saline (control) or with CPG 7909 at one of three doses (0.125, 0.5 or 1.0 mg). HBsAg-specific antibody responses (anti-HBs) appeared significantly sooner and were significantly higher at all timepoints up to and including 24 weeks in CPG 7909 recipients compared to control subjects (p< or = 0.001). Strikingly, most CpG 7909-vaccinated subjects developed protective levels of anti-HBs IgG within just two weeks of the priming vaccine dose. A trend towards higher rates of positive cytotoxic T cell lymphocyte responses was noted in the two higher dose groups of CPG 7909 compared to controls. The most frequently reported adverse events were injection site reactions, flu-like symptoms and headache. While these were more frequent in CPG 7909 groups than in the control group (p<0.0001), most were reported to be of mild to moderate intensity regardless of group. In summary, CPG 7909 as an adjuvant to Engerix-B was well-tolerated and enhanced vaccine immunogenicity. CPG 7909 may allow the development of a two-dose prophylactic HBV vaccine.

Safety and immunogenicity of CPG 7909 injection as an adjuvant to Fluarix influenza vaccine.

CPG 7909, a 24-mer B-Class CpG oligodeoxynucleotide (ODN), was tested for safety, tolerability and its ability to augment the immunogenicity of a commercial trivalent killed split influenza vaccine (Fluarix containing A/Beijing/262/95, A/Sydney/5/97 and B/Harbin/7/94; SmithKline Beecham) in a phase Ib blinded, randomized, controlled clinical trial. Sixty healthy volunteers were recruited in two consecutive cohorts of 30 subjects, who were randomly assigned to receive Fluarix plus 1mg CPG 7909 or Fluarix plus saline control (15 subjects each). Vaccines were administered by intramuscular injection on a single occasion with subjects in the first cohort receiving a 1/10th dose of Fluarix and those in the second cohort receiving the full-dose. All safety measures including physical evaluation, laboratory blood assays, and assays for DNA autoimmunity were within normal values except for transient and clinically inconsequential decreases in total white blood cell counts in groups receiving CPG 7909. All vaccines were found to be generally well tolerated with similar frequency and intensity for most adverse reactions for groups receiving CPG 7909 as controls. Exceptions were injection site pain and headache, which were reduced in frequency in subjects receiving the 1/10th Fluarix dose without CpG, compared to the frequency in all other groups. There was a lack of pre-existing immunity, defined as hemagglutinin inhibition (HI) activity < or =20, for all subjects to the influenza strains A/Beijing/262/95 and B/Harbin/7/94 and for some subjects to A/Sydney/5/97. Post-vaccination humoral immune responses, as determined 2 and 4 weeks later by assay of HI activity and ELISA to detect antibodies against hemagglutinin (anti-HA) were similar for both full and reduced Fluarix doses but the cellular immune responses (measured as PBMC antigen-specific IFN-gamma secretion) were reduced in the 1/10th Fluarix dose group. Humoral responses were not significantly enhanced by the addition of CPG 7909, except in individuals with pre-existing immunity to A/Sydney/5/97 strain (baseline HI activity titre >20), where there was a trend to higher HI activity with CPG 7909 (P = 0.06). The addition of CPG 7909 to the 1/10th dose of Fluarix did however result in significantly higher levels of IFN-gamma secretion from peripheral blood mononuclear cells recovered at 4 weeks and restimulated ex vivo with A/Beijing/262/95 (P = 0.048) and B/Harbin/7/94 (P = 0.0057), restoring these to the level seen with full-dose vaccine. These results suggest that addition of CPG 7909 to Fluarix may allow the use of reduced vaccine doses without reduced immunogenicity.

CpG oligodeoxynucleotide enhances immunity against blood-stage malaria infection in mice parenterally immunized with a yeast-expressed 19 kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) formulated in oil-based Montanides.

The 19kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)), an analog of the leading falciparum malaria vaccine candidate, induces protective immunity to challenge infection when formulated with complete/incomplete Freund's adjuvant (CFA/IFA), an adjuvant unsuitable for use in humans. In this study, we investigate Montanide ISA51 and Montanide ISA720 as well as CpG oligodeoxynucleotide (ODN) as adjuvants for induction of immunity to MSP1(19). Mice immunized with MSP1(19) adjuvanted with Montanide ISA51 were protected even though some mice experienced low-grade parasitemia before resolving the infection. Mice immunized with MSP1(19) adjuvanted with Montanide ISA720 showed delayed patent parasitemia with all mice ultimately succumbing to infection. Interestingly, when the synthetic CpG ODN 1826 was included in either Montanide formulation, mice were completely protected with no parasites detected in the blood. MSP1(19)-specific antibodies in MSP1(19)-immunized mice adjuvanted with Montanide ISA51 or Montanide ISA720 showed predominantly IgG1 antibody and low levels of IgG2a. CpG ODN 1826 significantly enhanced both IgG1 and IgG2a antibody responses in Montanide ISA51-adjuvanted mice but significantly enhanced only the IgG2a antibody response in Montanide ISA720-adjuvanted mice. To investigate the relative roles of antibody and CD4(+) T cells in protection, MSP1(19)-immunized mice adjuvanted with Montanide ISA720 and CpG ODN 1826 were depleted of CD4(+) T cells just prior to challenge. Results showed that three of nine immunized/T cell depleted mice died following infection. These results suggest that antibody and CD4(+) T cells are critical for protection following immunization with MSP1(19) adjuvanted with Montanide and CpG ODN and that the formulation of a human malaria vaccine candidate in Montanide ISA720 or ISA51 together with human compatible CpG ODN would be useful for improving efficacy.

CpG ODN enhances uptake of bacteria by mouse macrophages.

Unmethylated CpG motif in synthetic oligodeoxynucleotide (CpG ODN) or bacterial DNA is well recognized for its role in innate immunity, including enhancing production of NO and cytokines by macrophages. In the present study, we demonstrated the effect of CpG ODN on the phagocytic uptake of bacteria by macrophages. Flow cytometric analysis of mouse macrophages (RAW 264.7) incubated with fluorescein isothiocyanate (FITC)-labelled Burkholderia pseudomallei, Salmonella enterica serovar Typhi or Escherichia coli showed that CpG ODN increased the uptake of these bacteria by mouse macrophages. The enhancement of bacterial uptake by CpG ODN was concentration-dependent. The increase of bacterial uptake by CpG ODN-activated macrophages shown above is consistent with the result of bacteria internalization study using a standard antibiotic protection assay. There was also an increase in the rate and degree of multi-nucleated giant cell formation, phenomena which have been shown previously to be unique when the cells were infected with B. pseudomallei. These observations may provide significant insights for future investigation into host cell-pathogen interaction.

Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5'-TCGTCGTT-3' motif with two 5'-GTCGTT-3' motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM(+)) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3(+)) from the PBMC population did not affect IFN-gamma secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B(+)) completely abrogated the ability of CpG-ODN to stimulate IFN-gamma secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy.

Distinct CpG oligonucleotide sequences activate human gamma delta T cells via interferon-alpha/-beta.

Oligodeoxynucleotides with CpG motifs (CpG ODN) mimic microbial DNA and activate effectors of innate immunity including NK cells. Human gamma delta T cells (Vgamma9/Vdelta2) are antigen specific "natural memory" T cells in a preactivated stage, which respond to common non-protein phosphoantigens. Among several CpG ODN tested, distinct CpG ODN sequences characterized by inducing high amounts of IFN-alpha/-beta in PBMC elicited strong gamma delta T cell and NK cell responses, as determined by CD69 expression, IFN-gamma production, perforin content and lytic activity. These CpG ODN activated gamma delta T cells and NK cells in the absence of an additional stimulus and synergistically increased responsiveness to cell-type-specific antigens like isopentenylpyrophosphate for gamma delta T cells and NK-sensitive tumor cells for NK cells. NK cells and gamma delta T cells were activated via IFN-alpha/-beta released by CpG ODN-stimulated PBMC. Purified gamma delta T cells and NK cells did not respond to CpG ODN but to recombinant IFN-alpha/-beta. In conclusion, CpG ODN sequences were identified which, based on their ability to induce high amounts of IFN-alpha/-beta, represent strong adjuvants for "natural memory" cells including responses of gamma delta T cells to non-protein antigens. Early IFN-alpha/-beta dependent stimulation of IFN-gamma synthesis in NK cells and gamma delta T cells may contribute to the CpG ODN-induced Th1 bias of an evolving immune response.

Biodegradable microspheres containing group B Streptococcus vaccine: immune response in mice.

OBJECTIVE: This study seeks to show the feasibility of producing a group B Streptococcus (GBS) vaccine, which is capable of producing both a local IgA immune response at the mucosal surface where GBS is colonized and a humoral IgG response, which is capable of transplacental passive immunization. STUDY DESIGN: Inactivated GBS antigen was microencapsulated in poly (D, L-lactic-co-glycolic acid) (PLG) with a water-in-oil-in-water double emulsion technique. Immunostimulatory synthetic oligodeoxynucleotides containing cytidine-phosphate-guanosine (CpG) motifs were coencapsulated as a potent adjuvant. The ICR strain of mouse was used in these studies. Female mice with normal immune systems were immunized with the PLG microparticles containing GBS type III polysaccharide (GBS PS) vaccine and CpG adjuvant (PLG/GBS/CpG) via the oral, vaginal, or nasal routes or by the intramuscular or intraperitoneal routes. Booster doses were administered 4 weeks after the initial immunization. Vaginal washings and blood samples were obtained 3 weeks after the booster dose and examined for both IgG and secretory IgA (sIgA) GBS antibodies with the use of an enzyme-linked immunoabsorbent assay method. RESULTS: PLG/GBS/CpG microparticles elicited a significantly higher GBS antibody response when compared with nonencapsulated GBS antigen or PLG-encapsulated GBS PS vaccine without the addition of the CpG adjuvant. IgG and secretory IgA (sIgA) antibodies to GBS antigen were documented in both the vaginal washings and blood samples. CONCLUSION: Preliminary findings indicate that this novel PLG/GBS/CpG vaccine elicited both IgA and IgG antibody responses to the GBS PS antigen studied. This antibody response may provide both protection against maternal GBS colonization and passive transplacental immunization for the fetus and neonate.

Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs.

Immune stimulatory oligodeoxynucleotides (ODN) with unmethylated CpG motifs are potent inducers of both innate and adaptive immunity. It initially appeared that a single type of optimal CpG motif would work in all applications. We now report that specific motifs of CpG ODN can vary dramatically in their ability to induce individual immune effects and that these differences impact on their antitumor activity in different tumor models. In particular, a distinct type of CpG motif, which has a chimeric backbone in combination with poly(G) tails, is a potent inducer of NK lytic activity but has little effect on cytokine secretion or B cell proliferation. One such NK-optimized CpG ODN (1585) can induce regression of established melanomas in mice. Surprisingly, no such therapeutic effects were seen with CpG ODN optimized for activation of B cells and Th1-like cytokine expression (ODN 1826). The therapeutic effects of CpG 1585 in melanoma required the presence of NK but not T or B cells and were not associated with the induction of a tumor-specific memory response. In contrast, CpG 1826, but not CpG 1585, was effective at inducing regression of the EL4 murine lymphoma; this rejection was associated with the induction of a memory response and although NK cells were necessary, they were not sufficient. These results demonstrate that selection of optimal CpG ODN for cancer immunotherapy depends upon a careful analysis of the cellular specificities of various CpG motifs and an understanding of the cellular mechanisms responsible for the antitumor activity in a particular tumor.

Lipopolysaccharide and CpG DNA synergize for tumor necrosis factor-alpha production through activation of NF-kappaB.

Unmethylated CpG motifs in bacterial DNA (CpG DNA) activate host innate immune responses synergistically with some other microbial products, such as endotoxins, and may contribute to disease pathogenesis through excessive production of proinflammatory cytokines. Because monocyte-derived tumor necrosis factor (TNF)-alpha is an important mediator of disease, we investigated whether CpG DNA and lipopolysaccharide (LPS) synergize for inducing TNF-alpha biosynthesis. CpG DNA and LPS synergistically induce TNF-alpha production in RAW264.7 cells and J774 cells through activation of NF-kappaB. Furthermore, transient transfection with a super-repressive mutant of IkappaBalpha (IkappaBalpha-AA) demonstrated that NF-kappaB plays a critical role in CpG DNA-mediated TNF-alpha expression. Like NF-kappaB activation, CpG DNA-induced activation of mitogen-activated protein kinases (MAPK) regulates TNF-alpha production. Both extracellular receptor kinase (ERK) and p38 can regulate TNF-alpha gene transcription induced by CpG DNA. Although CpG DNA at the higher concentration slightly enhanced LPS-mediated phosphorylation of ERK, it did not alter the LPS-mediated activation of c-Jun N-terminal kinase and p38. In addition, CpG DNA showed little or no enhancement of LPS-mediated AP-1 activation. These results suggest that CpG DNA- and LPS-mediated signals converge at or above the level of NF-kappaB and ERK, and that there are distinct, as well as common, signaling pathways which are utilized by both CpG DNA and LPS for activating various transcription factors and MAPK.

Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12.

Human plasmacytoid dendritic cells (DC) (PDC, CD123+) and myeloid DC (MDC, CD11c+) may be able to discriminate between distinct classes of microbial molecules based on a different pattern of Toll-like receptor (TLR) expression. TLR1-TLR9 were examined in purified PDC and MDC. TLR9, which is critically involved in the recognition of CpG motifs in mice, was present in PDC but not in MDC. TLR4, which is required for the response to LPS, was selectively expressed on MDC. Consistent with TLR expression, PDC were susceptible to stimulation by CpG oligodeoxynucleotide (ODN) but not by LPS, while MDC responded to LPS but not to CpG ODN. In PDC, CpG ODN supported survival, activation (CD80, CD86, CD40, MHC class II), chemokine production (IL-8, IP-10) and maturation (CD83). CD40 ligand (CD40L) and CpG ODN synergized to activate PDC and to stimulate the production of IFN-alpha and IL-12 including bioactive IL-12 p70. Previous incubation of PDC with IL-3 decreased the amount of CpG-induced IFN-alpha and shifted the cytokine response in favor of IL-12. CpG ODN-activated PDC showed an increased ability to stimulate proliferation of naive allogeneic CD4 T cells, butTh1 polarization of developing T cells required simultaneous activation of PDC by CD40 ligation and CpG ODN. CpG ODN-stimulated PDC expressed CCR7, which mediates homing to lymph nodes. In conclusion, our studies reveal that IL-12 p70 production by PDC is under strict control of two signals, an adequate exogenous microbial stimulus such as CpG ODN, and CD40L provided endogenously by activated T cells. Thus, CpG ODN acts as an enhancer of T cell help, while T cell-controlled restriction to foreign antigens is maintained.

CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-kappaB activation.

Bacterial DNA and CpG-oligodeoxyribonucleotides (ODN) are powerful B cell activators, inducing apoptosis protection, cell cycle entry, proliferation, costimulatory molecule expression, immunoglobulin (Ig) and interleukin-6 (IL-6) secretion. However, proximal events in B cell activation by ODN are only partially characterized, including the translocation of NF-kappaB to the nucleus. In this paper, we provide evidence that CpG-ODN-induced cell cycle entry and apoptosis protection are blocked by SN50 or gliotoxin and thus require NF-kappaB activation. NF-kappaB activation occurred within 30 minutes of stimulation of murine B cells with a phosphorothioate (S) CpG-ODN and persisted for up to 40 hours, with p50, p65, and c-Rel as the major components. Similar to other NF-kappaB inducers, CpG-ODN caused an early IkappaBalpha and IkappaBbeta degradation plus cleavage of the p50 precursor and subsequent NF-kappaB nuclear translocation. A group of closely related S-ODN, which specifically blocked CpG-induced B cell activation at submicromolar concentrations, also prevented NF-kappaB DNA binding and transcriptional activation. These inhibitory S-ODN differed from stimulatory S-ODN by having 2-3 G substitutions in the central motif. As inhibitory S-ODN did not directly interfere with the NF-kappaB DNA binding but prevented CpG-induced NF-kappaB nuclear translocation of p50, p65, and c-Rel and blocked p105, IkappaBalpha, and IkappaBbeta degradation, we concluded that their putative target must lie upstream of inhibitory kinase (IKK) activation.

Type I interferon is the primary regulator of inducible Ly-6C expression on T cells.

Ly-6C has been proposed as a marker of memory CD8+ T cells. Reports have indicated that Ly-6C is upregulated after T cell receptor (TCR) stimulation or exposure to proinflammatory cytokines. This study examined the relative roles of proinflammatory cytokines and TCR engagement in Ly-6C induction. In vitro experiments tested the effects of cytokines on Ly-6C expression and confirmed interferon-alpha (IFN-alpha) as a primary cytokine that induces Ly-6C expression on CD4+ and CD8+ T cells. The amount and duration of Ly-6C expression were examined on T cells after in vivo induction of proinflammatory cytokines (CpG oligodeoxynucleotides [ODN]) or TCR activation (staphylococcal enterotoxin B [SEB]). In vivo, proinflammatory cytokines transiently upregulated Ly-6C on T cells in the absence of TCR stimulation. TCR stimulation by SEB transiently upregulated Ly-6C expression on antigen-specific and antigen-nonspecific T cells but did not cause long-term upregulation of Ly-6C expression in either population. IFN-alpha was confirmed as a primary inducer of Ly-6C in vivo, as CpG ODN were unable to induce Ly-6C expression in IFN-alphaRI(-/-) mice. Thus, inducible Ly-6C expression on CD4+ and CD8+ T cells is largely due to IFN-alpha in the environment and appears not to be directly correlated with the development of T cell memory.

Synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides are potent stimulators of antileukemia responses in naive and bone marrow transplant recipients.

Immunostimulatory cytosine-phophate-guanosine (CpG)--containing motifs in bacterial DNA are potent immune system activators. Depending on the bases flanking the CpG motif and on the DNA backbone, CpG oligodeoxynucleotides (ODNs) can induce relatively more B-cell activation or relatively more natural killer (NK)--cell activation. To evaluate their antitumor activities, an NK-optimized ODN (1585) and 2 B-cell--optimized ODNs (1826 and 2006) were compared for their ability to protect naive mice against a lethal acute myelogenous leukemia (AML) challenge. CpG 2006, but not CpG 1585, administered 2 days before the AML challenge, allowed mice to survive more than 100 times a lethal tumor dose. Cell depletion studies showed that protection did not require T or B cells but depended on NK cells and also on an NK-independent mechanism. CpG 2006 protected against AML challenge in both syngeneic and allogeneic bone marrow transplant (BMT) recipients at both early and late time points after transplantation. Although CpG 1585 had no protective effect on its own, it showed a striking synergy with CpG 2006 to induce prolonged survival to AML challenge in allogeneic recipients of T-cell-depleted marrow grafts, exceeding the survival benefit of donor lymphocyte infusion (DLI). When combined with DLI, a synergistic effect was observed in recipients of CpG2006 or 2006 + 1585 with 88% of mice surviving long-term. These data are the first to indicate that the systemic administration of CpG ODNs is a potent means of inducing therapeutic anti-AML innate immune responses in naive and BMT recipients. (Blood. 2001;98:1217-1225)

Bacterial DNA does not increase serum corticosterone concentration or prevent increases induced by other stimuli.

Bacterial DNA containing unmethylated CpG motifs (CpG DNA) and other microbial molecules such as lipopolysaccharide (LPS) have a broad range of immune stimulatory effects, which may include many shared cell signaling pathways leading to enhanced cytokine production. Some cytokines activate the hypothalamic-pituitary-adrenal (HPA) axis, and their production is downregulated by products of the HPA axis (glucocorticoids). Because such interactions have practical implications in the clinical use of CpG DNA, the present study was done to examine the effects of CpG DNA and LPS on serum corticosterone concentrations. In contrast to LPS, administration of CpG DNA (DNA from Escherichia coli) (30-300 microg) alone did not significantly increase serum corticosterone concentrations 1 or 4 h after administration. Administration of CpG DNA to mice prior to LPS caused a synergistic increase in serum tumor necrosis factor-alpha (TNF-alpha), indicative of an immune stimulatory effect. LPS and TNF-alpha, however, induced similar levels of corticosterone with or without concomitant CpG DNA. Increasing doses of LPS caused peak corticosterone levels similar to those induced by LPS in combination with CpG DNA. Exogenous TNF-alpha administered in vivo induced comparable concentrations of corticosterone with or without CpG DNA. An alternative stressor (restraint) yielded similar levels of corticosterone with or without CpG DNA. These results indicate that CpG DNA does not induce corticosterone release or alter its release by other stimuli, indicating biologically important differences in its immune effect compared to those of LPS, and possibly reduced toxicity.

Lactoferrin binds CpG-containing oligonucleotides and inhibits their immunostimulatory effects on human B cells.

Unmethylated CpG dinucleotide motifs in bacterial DNA, as well as oligodeoxynucleotides (ODN) containing these motifs, are potent stimuli for many host immunological responses. These CpG motifs may enhance host responses to bacterial infection and are being examined as immune activators for therapeutic applications in cancer, allergy/asthma, and infectious diseases. However, little attention has been given to processes that down-modulate this response. The iron-binding protein lactoferrin is present at mucosal surfaces and at sites of infection. Since lactoferrin is known to bind DNA, we tested the hypothesis that lactoferrin will bind CpG-containing ODN and modulate their biological activity. Physiological concentrations of lactoferrin (regardless of iron content) rapidly bound CpG ODN. The related iron-binding protein transferrin lacked this capacity. ODN binding by lactoferrin did not require the presence of CpG motifs and was calcium independent. The process was inhibited by high salt, and the highly cationic N-terminal sequence of lactoferrin (lactoferricin B) was equivalent to lactoferrin in its ODN-binding ability, suggesting that ODN binding by lactoferrin occurs via charge-charge interaction. Heparin and bacterial LPS, known to bind to the lactoferricin component of lactoferrin, also inhibited ODN binding. Lactoferrin and lactoferricin B, but not transferrin, inhibited CpG ODN stimulation of CD86 expression in the human Ramos B cell line and decreased cellular uptake of ODN, a process required for CpG bioactivity. Lactoferrin binding of CpG-containing ODN may serve to modulate and terminate host response to these potent immunostimulatory molecules at mucosal surfaces and sites of bacterial infection.

Identification of methylated CpG motifs as inhibitors of the immune stimulatory CpG motifs.

The unmethylated CpG motifs within E. coli DNA (EC) cause immune stimulation. In contrast, mammalian DNA such as calf thymus (CT) DNA had been thought to be immunologically inert. In this article, we demonstrate that CT DNA unexpectedly specifically inhibits the immune activation by EC but not that by endotoxin. This inhibitory effect was mediated in the signaling pathway activated by EC since CT DNA markedly inhibited the CpG-induced nuclear translocation of the transcription factors, NF-kappaB and AP-1. In addition, CT DNA significantly inhibited the synergistic immune activation by EC and endotoxin. The mechanism of the inhibition by CT DNA probably did not involve the inhibition of the cellular uptake of EC. Using a CpG-depleted plasmid, we demonstrated that CpG methylation played an important role in the inhibition by CT DNA. Compared with unmethylated plasmid DNA, CpG-methylated DNA inhibited the immune activation by EC to the same extent as did CT DNA. Importantly, the inhibitory effect of CT DNA was also observed in vivo. Our results suggest that methylated DNA may be applied to alleviate the unwanted immune stimulation and inflammation in systemic inflammatory response syndrome and in gene therapy with plasmid DNA.

From bugs to drugs: therapeutic immunomodulation with oligodeoxynucleotides containing CpG sequences from bacterial DNA.

Several types of immune cells possess pattern recognition receptors (PRR) that can distinguish prokaryotic DNA from vertebrate DNA by detecting unmethylated CpG dinucleotides in particular base contexts (CpG motifs). Bacterial DNA or synthetic oligodeoxynucleotides containing these CpG motifs activate both innate and acquired immune responses that have evolved to protect against intracellular infections. These T helper 1 (Th1)-like immune responses include activation of B cells, dendritic cells, macrophages, and natural killer (NK) cells. CpG DNA-induced immune activation can protect against infection either alone or in combination with a vaccine and is effective in the immunotherapy of allergic diseases and cancer. Human clinical trials using such CpG DNA are currently underway.

Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells.

The immature plasmacytoid dendritic cell (PDC) is identical with the principal type I IFN-producing cell upon viral infection. Oligodeoxynucleotides which contain unmethylated CpG motifs (CpG ODN) are recognized by the vertebrate immune system. Previously, we described CpG ODN that strongly activate human B cells and human blood dendritic cells. Here we describe distinct CpG-containing oligonucleotide sequences which, in contrast to previously described CpG ODN, induced high amounts of IFN-alpha and IFN-beta in peripheral blood mononuclear cells (PBMC). Intracellular staining for IFN-alpha revealed that within PBMC CpG ODN-induced IFN-alpha is produced exclusively by PDC. Unlike IFN-alpha, TNF-alpha is up-regulated in PDC by all CpG ODN tested. Purified PDC responded to CpG ODN, demonstrating direct activation of PDC by CpG ODN. The most active sequence induced the production of up to 5 pg IFN-alpha per single PDC, resulting in more than 400 ng/ml IFN-alpha in the supernatant of PBMC enriched for PDC. The potency of CpG ODN to stimulate IFN-alpha correlated with their ability to stimulate NK cell lytic activity, while purified NK cells did not respond to CpG ODN. IFNgamma production in PBMC was dependent on CpG ODN-induced IFN-alpha/beta as demonstrated by IFN-alpha/beta blocking antibodies. IFN-alpha-inducing CpG ODN strongly supported IFN-gamma production of TCR-triggered CD4 T cells but were less active than other CpG ODN in stimulating B cells. In conclusion our results demonstrate that particular CpG ODN sequences exist which, due to high IFN-alpha/beta induction in PDC, induce a set of immune responses typical for viral infection.