CpG DNA: a novel immunomodulator.
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Adjuvants, Immunologic↗
Biomedical subjects
A M Krieg
Publications and source records attributed to A M Krieg.
CpG DNA and LPS induce distinct patterns of activation in human monocytes.
A specific set of immune functions is switched on in response to DNA containing unmethylated CpG dinucleotides in particular base contexts ('CpG motifs'). Plasmids, viral vectors and antisense oligodeoxynucleotides used for DNA vaccination, gene replacement or gene blockade contain immunostimulatory CpG motifs which may have independent biological activity. Although the immune stimulatory effects of CpG motifs on murine cells are well established, the evaluation of their possible effects on human cells is complicated by the higher LPS sensitivity of human leukocytes compared with those in mice. To address this issue, we analyzed CpG- and LPS-mediated immune activation of human PBMC. The biologic activity of LPS could be detected within 4 h using intracellular TNF staining of monocytes with flow cytometry at concentrations just one-twentieth (0.0014 Eu/ml) of the lower detection limit for the routinely used LAL assay (0.03 EU/ml). In contrast to the rapid LPS response, CpG DNA-stimulated TNF and IL-6 synthesis in human monocytes was not detectable until 18 h. E. coli DNA induced IL-6 synthesis in a concentration-dependent manner (30 micrograms/ml E. coli DNA; 409 pg/ml +/- 75 pg/ml, n = 7, IL-6 ELISA), but calf thymus DNA did not (< 10 pg/ml). Likewise, the CpG oligodeoxynucleotides 1760 (phosphorothioate) and 2059 (unmodified) induced IL-6 synthesis, but the corresponding control oligonucleotides 1908 and 2077 did not CpG DNA and LPS enhanced IL-6 synthesis synergistically. ICAM-1-expression of monocytes was increased 4.6-fold by E. coli DNA, 3.5-fold by 1760 and three-fold by 2059, compared with 3.6-fold by a maximal LPS stimulus and no change with non-CpG DNA. In conclusion, CpG-motifs induce TNF, IL-6 and ICAM-1 expression in human monocytes, but the kinetics of this differ from that induced by LPS, which makes it possible to distinguish immune activation by these agents. These results have important implications for the clinical development of therapeutic DNA in humans.
Cells, Cultured↗
CpG DNA rescues B cells from apoptosis by activating NFkappaB and preventing mitochondrial membrane potential disruption via a chloroquine-sensitive pathway.
Isolated murine splenic B cells gradually undergo spontaneous apoptosis while WEHI-231 B lymphoma cells undergo activation-induced apoptosis. Unmethylated CpG dinucleotides in a particular sequence context (CpG motif) in bacterial DNA or in synthetic oligodeoxynucleotides (CpG DNA) rescue both splenic B cells and WEHI-231 cells from apoptosis, an effect which could potentially contribute to autoimmune disease. Chloroquine has been used as an effective therapeutic agent for some autoimmune diseases, although the mechanism of action is not clearly understood. Low concentrations of chloroquine (<5 microM) selectively abolished CpG DNA-mediated protection against spontaneous apoptosis of splenic B cells and against anti-IgM-induced apoptosis of WEHI-231 cells without affecting anti-apoptotic activities of anti-CD40 or lipopolsaccharide. CpG DNA effectively prevented mitochondrial membrane potential disruption through a chloroquine-sensitive pathway in splenic B cells. Apoptosis protection by CpG DNA was also associated with increased expression of several proto-oncogenes and oncoproteins directly and/or indirectly through a rapid and sustained activation of NFkappaB in splenic B cells and WEHI-231 cells. These effects were also suppressed by chloroquine. Our results suggest that despite the difference in maturation phenotype of splenic B cells and WEHI-231 cells, CpG DNA rescues both from apoptosis by similar pathway, which is blocked at an early step by chloroquine.
Animals↗
Phagocytic antigen processing and effects of microbial products on antigen processing and T-cell responses.
Processing of exogenous antigens and microbes involves contributions by multiple different endocytic and phagocytic compartments. During the processing of soluble antigens, different endocytic compartments have been demonstrated to use distinct antigen-processing mechanisms and to process distinct sets of antigenic epitopes. Processing of particulate and microbial antigens involves phagocytosis and functions contributed by phagocytic compartments. Recent data from our laboratory demonstrate that phagosomes containing antigen-conjugated latex beads are fully competent class II MHC (MHC-II) antigen-processing organelles, which generate peptide:MHC-II complexes. In addition, phagocytosed antigen enters an alternate class I MHC (MHC-I) processing pathway that results in loading of peptides derived from exogenous antigens onto MHC-I molecules, in contrast to the cytosolic antigen source utilized by the conventional MHC-I antigen-processing pathway. Antigen processing and other immune response mechanisms may be activated or inhibited by microbial components to the benefit of either the host or the pathogen. For example, antigen processing and T-cell responses (e.g. Th1 vs Th2 differentiation) are modulated by multiple distinct microbial components, including lipopolysaccharide, cholera toxin, heat labile enterotoxin of Escherichia coli, DNA containing CpG motifs (found in prokaryotic and invertebrate DNA but not mammalian DNA) and components of Mycobacterium tuberculosis.
Animals↗
Induction and regulation of Th1-inducing cytokines by bacterial DNA, lipopolysaccharide, and heat-inactivated bacteria.
Th1 immune responses, characterized by production of gamma interferon (IFN-gamma), are associated with protective immunity to viruses and intracellular bacteria. Heat-killed Brucella abortus promotes secretion of Th1-inducing cytokines such as interleukin-12 (IL-12) and IFN-gamma and has been used as a carrier to induce Th1 responses to vaccines. To explore which bacterial constituents could mediate this response and how it is regulated, murine spleen cells were cultured with B. abortus derived DNA, lipopolysaccharide (LPS), or whole killed organisms. Each constituent induced similar, substantial amounts of IL-10. However, only B. abortus and B. abortus DNA induced high levels of IFN-gamma and IL-12. B. abortus and B. abortus DNA-stimulated IL-12 production was maximal by 6 to 18 h, while IL-10 production steadily accumulated over this time period. These kinetics suggested that IL-10 may eventually downmodulate the Th1-like cytokine response to B. abortus and B. abortus DNA, which was confirmed by using neutralizing antibody. In the absence of IL-10, B. abortus LPS induced strong IFN-gamma responses, but IL-12 p70 levels were still undetectable from BALB/c spleen cells. LPS induced IL-12 if the spleen cells were primed with IFN-gamma and IL-10 was neutralized, indicating that LPS can stimulate IL-12 production under the most favorable conditions. Responses to Escherichia coli LPS and DNA mirrored the responses to B. abortus components, suggesting that immune effects observed with these constituents may be generalizable to many microbial species. In vivo experiments demonstrated the same hierarchy of responses for IL-12 production. These findings support the likelihood that microbial components, if used as carriers or adjuvants, can differ substantially in their ability to effect a Th1 response.
Animals↗
CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice.
Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1-14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0-10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.
Aging↗
Immunostimulatory CpG oligodeoxynucleotides enhance the immune response to vaccine strategies involving granulocyte-macrophage colony-stimulating factor.
Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can activate various immune cell subsets and induce production of a number of cytokines. Prior studies have demonstrated that both CpG ODN and granulocyte-macrophage colony-stimulating factor (GM-CSF) can serve as potent vaccine adjuvants. We used the 38C13 murine lymphoma system to evaluate the immune response to a combination of these two adjuvants. Immunization using antigen, CpG ODN, and soluble GM-CSF enhanced production of antigen-specific antibody and shifted production towards the IgG2a isotype, suggesting an enhanced TH1 response. This effect was most pronounced after repeat immunizations with CpG ODN and antigen/GM-CSF fusion protein. A single immunization with CpG ODN and antigen/GM-CSF fusion protein 3 days before tumor inoculation prevented tumor growth. CpG ODN enhanced the production of interleukin-12 by bone marrow-derived dendritic cells and increased expression of major histocompatibility complex class I and class II molecules, particularly when cells were pulsed with antigen/GM-CSF fusion protein. We conclude that the use of CpG ODN in combination with strategies involving GM-CSF enhances the immune response to antigen and shifts the response towards a TH1 response and that this approach deserves further evaluation in tumor immunization approaches and other conditions in which an antigen-specific TH1 response is desirable.
Adjuvants, Immunologic↗
Rapid induction of mitogen-activated protein kinases by immune stimulatory CpG DNA.
Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) rapidly activate B cells and monocyte-derived cells; however, the intracellular signaling pathways involved in this process are unclear. Here, we demonstrate that CpG DNA induces the activation of c-Jun NH2-terminal kinase and p38 but does not activate the extracellular receptor kinase in murine B and monocyte-like cell lines. CpG DNA also induces the phosphorylation of activating transcription factor-2, c-Jun, and mitogen-activated protein kinase (MAPK)-activated protein kinase 2 as well as the activation of activator protein-1 (AP-1) DNA binding. Inhibition of p38 led to the suppression of CpG DNA-induced AP-1 DNA-binding activity and cytokine production, indicating that the p38 pathway is required for mediating these immune stimulatory effects of CpG DNA. Chloroquine, an endosomal acidification inhibitor, selectively abolished CpG DNA-mediated MAPK activation. Our results indicate that CpG DNA activates the p38 and c-Jun NH2-terminal kinase MAPK and leads to the activation of AP-1 via a pathway which is sensitive to chloroquine.
Activating Transcription Factor 2↗
Cyclosporin A enhances IL-12 production by CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides.
Certain sequences of nucleotides (CpG motifs) in bacterial DNA or synthetic oligonucleotides (CpG DNA) promote the production of proinflammatory cytokines, including TNF-alpha, IFN-gamma, IL-6, and IL-12. Here we demonstrate that the immunosuppressant cyclosporin A (CsA) unexpectedly enhanced CpG DNA-induced IL-12 production in murine splenocytes. CsA did not inhibit CpG DNA-induced TNF-alpha or IL-6 production, but decreased the production of IFN-gamma by CpG DNA. Upon examining mechanisms by which CsA increases IL-12 production, we found that CpG DNA can also induce IL-10 production in B cells and that this production was sensitive to CsA. IL-10 has anti-inflammatory effects and can reduce the production of IL-12. To determine the possible role of CsA-modulated IL-10 production in mediating the increased IL-12 levels, splenocytes from IL-10 gene-disrupted mice (IL-10 -/-) and splenocytes cultured in anti-IL-10 Ab were studied. CpG DNA-stimulated IL-10 (-/-) splenocytes demonstrated no increase in IL-12 levels in the presence of CsA. Anti-IL-10 Ab treatment of normal splenocytes increased the magnitude of CpG DNA-induced IL-12 production to that seen with CsA. These results suggest that CpG DNA induces CsA-sensitive IL-10 production in B cells and that IL-10 acts as a negative feedback regulator of CpG DNA-induced IL-12 production.
Animals↗
Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs.
Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA. B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immune-stimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA. In type 12 genomes, the distribution of CpG-flanking bases is similar to that predicted by chance. However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15- to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5' side or a G on the 3' side. Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs. Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing. These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.
Adenoviridae↗
Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA.
Genetic vaccination depends at least in part on the adjuvant properties of plasmids, properties that have been ascribed to unmethylated CpG dinucleotides in bacterial DNA. Because dendritic cells (DC) participate in the T cell priming that occurs during genetic vaccination, we reasoned that CpG-containing DNA might activate DC. Thus, we assessed the effects of CpG oligodeoxynucleotides (CpG ODN) on Langerhans cell (LC)-like murine fetal skin-derived DC (FSDDC) in vitro and on LC in vivo. Treatment with CpG ODN as well as LPS induced FSDDC maturation, manifested by decreased E-cadherin-mediated adhesion, up-regulation of MHC class II and costimulator molecule expression, and acquisition of enhanced accessory cell activity. In contrast to LPS, CpG ODN stimulated FSDDC to produce large amounts of IL-12 but only small amounts of IL-6 and TNF-alpha. Injection of CpG ODN into murine dermis also led to enhanced expression of MHC class II and CD86 Ag by LC in overlying epidermis and intracytoplasmic IL-12 accumulation in a subpopulation of activated LC. We conclude that immunostimulatory CpG ODN stimulate DC in vitro and in vivo. Bacterial DNA-based vaccines may preferentially elicit Th1-predominant immune responses because they activate and mobilize DC and induce them to produce large amounts of IL-12.
Adjuvants, Immunologic↗
CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge.
Vertebrates have evolved innate immune defense mechanisms that recognize and respond to structural patterns that are specific to microbial molecules. One such pattern recognition system is based on unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs); these motifs are common in bacterial DNA but are under-represented ("CpG suppression") and methylated in vertebrate DNA. Mice that are injected with bacterial DNA or synthetic oligodeoxynucleotides (ODNs) containing CpG motifs respond with a rapid production of IL-12 and IFN-gamma. The serum levels of IL-12 were increased for at least 8 days after a single injection of CpG ODNs, but IFN-gamma levels returned to baseline within 24 h. This Th1-like cytokine response to CpG motifs induces a state of resistance to infection by Listeria monocytogenes in susceptible specific pathogen-free BALB/c mice. Resistance developed within 48 h of pretreatment with CpG ODNs, persisted for at least 2 wk, and was dependent upon IFN-gamma secretion. These data support the hypothesis that CpG DNA motifs are a "danger signal" that activates protective innate immune defenses and may have therapeutic potential.
Animals↗
CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry.
Isolated murine splenic B cells undergo spontaneous apoptosis. Motifs containing unmethylated CpG dinucleotides in bacterial DNA or in synthetic oligodeoxynucleotides (ODN) are known to activate murine B cells. Now we show that ODN that induce spleen B cell cycle entry also inhibit spontaneous apoptosis in a sequence-specific fashion. Reversal of the CG to GC abolished activity. Methylation of the central cytosine decreased activity. When CpG is preceded by a cytosine or followed by a guanine, activity was abolished. Other substitutions at the same positions had no effect. Dose-response curves for apoptosis protection and G1 entry suggested that a uniform population of ODN recognition sites controlled downstream ODN effects. A CpG ODN with a nuclease-resistant phosphorothioate backbone (S-ODN) was also active, and increased the levels of c-myc, egr-1, c-jun, bclXL, and bax mRNA and c-Myc, c-Jun, Bax, and BclXL protein in spleen B cells. Levels of c-myb, myn, c-Ki-ras, and bcl2 mRNA remained unchanged. When protein synthesis was inhibited, at 16 h ODN-induced cell cycle entry was abolished and apoptosis protection was partially preserved. Under these conditions, c-Myc was still present, but c-Jun and BclXL were not detected. Our results suggest that CpG containing ODN motifs provide signals for both survival and cell cycle entry. Single base changes determine whether this signal proceeds through a rate-limiting step governing at least two steps in apoptosis (plasma membrane transition, DNA cleavage) and two phases of the cell cycle (G1 and S phase entry). This biologic action is associated with increased c-Myc, c-Jun, and BclXL expression.
Animals↗
CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species.
B cells and monocytes endocytose DNA into an acidified intracellular compartment. If this DNA contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs), these leukocytes are rapidly activated. We now show that both B cell and monocyte-like cell line responses to DNA containing CpG motifs (CpG DNA) are sensitive to endosomal acidification inhibitors; they are completely blocked by bafilomycin A, chloroquine, and monensin. The specificity of these inhibitors is demonstrated by their failure to prevent responses to LPS, PMA, or ligation of CD40 or IgM. Acidification of endosomal CpG DNA is coupled to the rapid generation of intracellular reactive oxygen species. The CpG DNA-induced reactive oxygen species burst is linked to the degradation of IkappaB and the activation of NFkappaB, which induces leukocyte gene transcription and cytokine secretion. These studies demonstrate a novel pathway of leukocyte activation triggered by CpG motifs.
Animals↗
Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma.
Asthma has been increasing in industrialized countries. Evidence suggests that asthma is caused by a Th2 immune response to inhaled environmental Ags and that childhood infections protect against this. We have shown that bacterial DNA contains motifs, centered on unmethylated CpG dinucleotides, which induce Th1-type responses. We hypothesized that the Th1 effect of these CpG motifs may oppose the Th2 type allergic response and suggest that this may account for the protective effect of childhood infection against asthma. We examined the effects of CpG-motif oligodeoxynucleotides (CpG ODN) in a murine model of asthma. Airway eosinophilia, Th2 cytokine induction, IgE production, and bronchial hyperreactivity were prevented by coadministration of CpG ODN with the Ag. Significantly, in a previously sensitized mouse, CpG ODN can prevent allergen-induced airway inflammation. These studies suggest that exposure to CpG DNA may protect against asthma.
Animals↗
CpG DNA rescue from anti-IgM-induced WEHI-231 B lymphoma apoptosis via modulation of I kappa B alpha and I kappa B beta and sustained activation of nuclear factor-kappa B/c-Rel.
Unmethylated CpG dinucleotides in particular base contexts in oligonucleotides (CpG DNA) rescue WEHI-231 cells from anti-IgM-induced cell cycle arrest and apoptosis. Anti-IgM rapidly elevated the levels of NFkappaB p50/c-Rel heterodimers followed by a decline of p50/c-Rel heterodimers by 3 h and a concomitant increase of p50/p50 homodimers. In contrast, CpG DNA induced and maintained the levels of p50/c-Rel heterodimers in the presence or absence of anti-IgM, while control non-CpG DNA failed to induce NFkappaB activation. Anti-IgM induced IkappaB alpha degradation followed by increased IkappaB alpha protein levels. The levels of IkappaB beta were increased after anti-IgM treatment. In contrast, CpG DNA, but not non-CpG DNA, induced sustained IkappaB alpha and IkappaB beta degradation in the presence or absence of anti-IgM. Inhibition of IkappaB degradation blocked CpG DNA-induced NFkappaB activation and expression of c-myc. Prevention of NFkappaB activation by inhibiting IkappaB degradation also suppressed the ability of CpG DNA to rescue WEHI-231 cells from anti-IgM-induced apoptosis. These results indicate that CpG DNA-mediated sustained activation of NFkappaB depends on the degradation of IkappaB alpha and IkappaB beta and is required for the CpG DNA-mediated anti-apoptosis gene expression and the protection against anti-IgM-induced apoptosis of WEHI-231 cells.
Animals↗
CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen.
Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODN) cause B cell proliferation and Ig secretion, monocyte cytokine secretion, and activation of NK cell lytic activity and IFN-gamma secretion in vivo and in vitro. The potent immune activation by CpG ODN suggests possible utility for enhancing immune responses to vaccines. Mice immunized with recombinant hepatitis B virus surface Ag and a CpG ODN as an immune enhancer have titers of Abs against HBsAg (anti-HBs) that are five times higher than those of mice immunized with HBsAg and the standard adjuvant, aluminum hydroxide (alum). Ab titers in mice immunized with HBsAg and both CpG ODN plus alum were 35 times higher than the titers in mice immunized with alum alone, indicating a strong synergistic interaction between the CpG ODN and alum. ODN without CpG motifs had little or no immune-enhancing activity at the doses used herein. Alum induces a Th2 humoral response (mostly IgG1) and no CTL. In contrast, CpG ODN gives a strong Thl response with predominantly IgG2a Abs and CTL, even when mixed with alum. In vitro studies to determine possible mechanisms of CpG immune-enhancing effects show that CpG ODN induce expression of costimulatory molecules on Ag-presenting cells and drive B cell isotype switching in the appropriate cytokine milieu. These studies demonstrate that CpG ODN are promising new immune enhancers for vaccination applications.
Adjuvants, Immunologic↗
CpG DNA, a novel immune enhancer for systemic and mucosal immunization with influenza virus.
Bacterial DNA causes B cell proliferation, immunoglobulin secretion, and Th1-like cytokine secretion, due to unmethylated CpG dinucleotides in particular base contexts (CpG motifs), which are far more common in bacterial DNA than in vertebrate DNA. Synthetic oligodeoxynucleotides (ODN) containing CpG motifs also trigger immune activation, suggesting possible utility as vaccine enhancers. Mice systemically primed with formalin-inactivated influenza virus mixed with CpG ODN, generated virus-specific serum antibodies at titres approximately seven times higher than mice immunized without CpG; the titres were further increased following an identical second injection. To determine whether CpG could be absorbed through mucosae and enhance vaccination responses, mice were immunized intranasally (IN) with the same preparation of virus with or without CpG ODN or Escherichia coli DNA. Following IN immunization, CpG ODN or E. coli DNA promoted increased production of influenza-specific antibodies in serum, saliva and the genital tract, compared with the control groups. These studies indicate that stimulatory CpG ODN are promising new immune enhancers for vaccination applications.
Administration, Intranasal↗