CpG frequency-dependence in the activity of eukaryotic DNA methyltransferase: in vitro methylation of CpG-rich islands.
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CpG islands in vertebrate genomes.
Although vertebrate DNA is generally depleted in the dinucleotide CpG, it has recently been shown that some vertebrate genes contain CpG islands, regions of DNA with a high G+C content and a high frequency of CpG dinucleotides relative to the bulk genome. In this study, a large number of sequences of vertebrate genes were screened for the presence of CpG islands. Each CpG island was then analysed in terms of length, nucleotide composition, frequency of CpG dinucleotides, and location relative to the transcription unit of the associated gene. CpG islands were associated with the 5' ends of all housekeeping genes and many tissue-specific genes, and with the 3' ends of some tissue-specific genes. A few genes contained both 5' and 3' CpG islands, separated by several thousand base-pairs of CpG-depleted DNA. The 5' CpG islands extended through 5'-flanking DNA, exons and introns, whereas most of the 3' CpG islands appeared to be associated with exons. CpG islands were generally found in the same position relative to the transcription unit of equivalent genes in different species, with some notable exceptions. The locations of G/C boxes, composed of the sequence GGGCGG or its reverse complement CCGCCC, were investigated relative to the location of CpG islands. G/C boxes were found to be rare in CpG-depleted DNA and plentiful in CpG islands, where they occurred in 3' CpG islands, as well as in 5' CpG islands associated with tissue-specific and housekeeping genes. G/C boxes were located both upstream and downstream from the transcription start site of genes with 5' CpG islands. Thus, G/C boxes appeared to be a feature of CpG islands in general, rather than a feature of the promoter region of housekeeping genes. Two theories for the maintenance of a high frequency of CpG dinucleotides in CpG islands were tested: that CpG islands in methylated genomes are maintained, despite a tendency for 5mCpG to mutate by deamination to TpG+CpA, by the structural stability of a high G+C content alone, and that CpG islands associated with exons result from some selective importance of the arginine codon CGX. Neither of these theories could account for the distribution of CpG dinucleotides in the sequences analysed. Possible functions of CpG islands in transcriptional and post-transcriptional regulation of gene expression were discussed, and were related to theories for the maintenance of CpG islands as "methylation-free zones" in germline DNA.
Animals↗
Signal transduction pathways activated by CpG-DNA.
While more and more attention has been paid to CpG-DNA with respect to its usefulness as an adjuvant, its molecular mechanism of action is less well defined. Over the last few years, at least two major signalling pathways have been shown to be utilized by CpG-DNA: the NF-kappa B activation pathway and the stress-kinase pathway. Direct downstream events of these pathways are induction of transcriptional activity of NF-kappa B and transcriptional activity of AP-1. As far as investigated, CpG-DNA uses signal transduction pathways originally described for other stimuli, such as LPS, IL-1 or TNF. Therefore, to us, the prime question is: where does CpG-DNA-induced signalling enter these known pathways? This raises questions about the existence of a CpG-DNA-sequence-specific receptor. Several points of evidence support the probability of the existence of a cellular receptor: There is a strong motif (unmethylated CpG) dependency for CpG-DNA-induced signalling. There is cell-type specificity. Dendritic cells, macrophages and B cells respond to CpG-DNA, but other cell types, such as fibroblasts and T cells, do not. In addition, classic signal-transduction pathways are rapidly switched on in a parallel manner, as is known for other receptors. Using competing non-CpG ODNs and inhibitors of endosomal acidification, some evidence has been obtained that CpG ODNs are taken up into endosomes by a CpG-independent receptor, followed by a pH-dependent step before signalling starts. A model based on these findings is proposed in Fig. 4. Nevertheless, other receptor-independent activities of CpG-DNA cannot yet be ruled out. Although unlikely, we should consider the possibility that CpG-DNA directly interacts with cellular nucleic acids either by direct hybridization with complementary nucleotides or by formation of DNA triplexes (VASQUEZ and WILSON 1998). While these possibilities have been explored by antisense technology, using a huge variety of ODNs, there is no experimental evidence that such interactions are important for the activity of CpG-DNA. In this context, it is important to note that DNA, especially phosphothioate-stabilized ODNs with poly-G stretches, have substantial CpG-independent activities, although these activities seem not to depend on specific, antisense-like DNA-DNA interactions (PISETSKY 1996). One good example comes from experiments using ODNs on primary T cells. Co-stimulation of CD3-primed T cells with CpG ODN leads to a significant increase of IL-2 secretion and proliferation; however, these effects are CpG independent (K. Heeg, personal communication). Remarkably, these poly-G stretches seem to be inactive when transferred to double-stranded DNAs, such as plasmid DNA (WLOCH et al. 1998). In contrast, to my knowledge, no immune-stimulatory effect of bacterial DNA has been described that can not be abolished by CpG-specific methylation. Taken together, CpG-dependent and CpG-independent activities must be distinguished from one another. Among these effects, CpG-dependent signalling is better defined. Much effort is going into the investigation of the pharmacological applications of CpG-DNA. Once CpG-receptor-like structures are known, the question of the physiological role of CpG-DNA can be tackled.
Animals↗
Immunotherapy with CpG DNA conjugated with T-cell epitope peptide of an allergenic Cry j 2 protein is useful for control of allergic conditions in mice.
Immunotherapy using T-cell epitope peptides or CpG DNA conjugated with allergenic protein is useful, although the mechanisms of these therapies differ. However, the combination of CpG DNA and peptide, but not protein, had not been documented. Therefore, we investigated CpG DNA conjugated with peptide to obtain positive synergistic effects. In the first experiment, mice were vaccinated with a conjugate of CpG DNA and Cry j 2 T-cell epitope peptide p246-259 (CpG-peptide); a mixture of CpG DNA and peptide (CpG+peptide); peptide alone, or PBS alone, and immunized with Cry j 2. In the second experiment, mice were immunized with Cry j 2 and injected with CpG-peptide, CpG+peptide, peptide only, or PBS only. In both experiments, Cry j 2-specific IgE, IL-4, and IL-5 were significantly lower in mice given CpG-peptide, versus those given CpG+peptide, peptide alone, or PBS alone. However, IgG2a, IgG2b and IFN-gamma did not increase in mice injected with CpG-peptide. In the third experiment, CpG-peptide significantly attenuated nasal symptoms (sneezing and nasal rubbing) compared to CpG+peptide, peptide alone, or PBS alone. Mice were also injected with a conjugate of CpG DNA and Cry j 2 protein (CpG-Cry j 2) or CpG-peptide to compare prime responses. Mice vaccinated with CpG-Cry j 2 generated Cry j 2-specific IgG1, whereas those vaccinated with CpG-peptide did not produce IgG1. This study demonstrated, for the first time, that immunotherapy with CpG DNA conjugated with a T-cell peptide is useful in preventing and treating allergic conditions.
Animals↗
CpG 7909: PF 3512676, PF-3512676.
CpG 7909 [PF-3512676] is an immunomodulating synthetic oligonucleotide designed to specifically agonise the Toll-like receptor 9 (TLR9). It is being developed for the treatment of cancer [ProMune] as a monotherapy and in combination with chemotherapeutic agents, and it is also under development as an adjuvant [VaxImmune] for vaccines against cancer and infectious diseases. CpG 7909, acting through the TLR9 receptor present in B cells and plasmacytoid dendritic cells, stimulates human B-cell proliferation, enhances antigen-specific antibody production and induces interferon-alpha production, interleukin-10 secretion and natural killer cell activity. Coley Pharmaceutical Group originally developed CpG 7909 using its CpG DNA technology. In March 2005, Coley granted Pfizer an exclusive global license to develop and commercialise CPG 7909 [ProMune] for the treatment, control and prevention of multiple cancer indications. Coley licensed CpG 7909 [VaxImmune] to Chiron Corporation for adjuvant use with Chiron's prophylactic vaccine candidates against infectious diseases in December 2003. Chiron was acquired by and merged into Novartis in April 2006. In 2002, GlaxoSmithKline (GSK) was granted a worldwide, non-exclusive licence to Coley's CpG immunostimulatory oligonucleotides, including CpG 7909 [VaxImmune], for their use as adjuvants for cancer vaccines. In 2000, Coley entered into a co-exclusive licensing agreement with GSK for the development of therapeutic and prophylactic vaccines against infectious diseases. This licensing agreement included CpG 7909 [VaxImmune] and other CpG-based immunostimulatory oligonucleotides. In September 2004, Coley Pharmaceuticals was awarded a 16.9 million US dollars, 5-year contract from the National Institute of Allergy and Infectious Diseases (NIAID), one of the National Institutes of Health (NIH), to support the development of novel immune-activating drugs for defense against bioterror agents. This contract will be used to expand Coley's proprietary line of TLR Therapeutic products. Together with prior awards, the new contract brings the total committed biodefense funding for Coley to 35 million US dollars. During the first quater of 2006, Pfizer disclosed its intention to develop CpG 7909 for breast cancer. A phase I/II trial in patients with NHL has also been conducted in 24 patients with relapsed or refractory disease at the University of Iowa. Pfizer initiated two international phase III trials under the special protocol assessment (SPA) procedure of the US FDA. These trials are evaluating CpG 7909 in combination with chemotherapy versus chemotherapy alone as a first-line treatment for patients with advanced (stage IIIb or IV) non-small-cell lung cancer (NSCLC). Approximately 800 patients will be enrolled in each trial. The primary endpoint is overall survival time. In 2005, Coley completed a multinational phase IIb trial of CpG 7909 in combination with chemotherapy in 112 patients with NSCLC. The goal of the study was to improve the outcome of standard first-line chemotherapeutic regimens (taxane and platinum) for NSCLC by adding CpG 7909. Coley has been granted 11 US patents, covering key aspects of the company's CpG TLR9 antagonist technologies. Three US patents relating to CpG 7909 and the use of certain oligonucleotides for treating cancer are due to expire in 2014. Coley also has pending US patent applications covering the specific sequence of CpG 7909 and its use to treat cancer, which, if issued, would be expected to expire between 2014 and 2017. In April 2004, Coley received US Patent No. 6,727,230 covering the use of oligonucleotides containing at least one phosphorothioate linkage to stimulate cellular immune responses. In November 2003, Coley received US Patent No. 6,653,292, which protects the use of TLR9-containing oligonucleotides to treat or prevent cancer or to enhance multimodal cancer treatment regimens. In July 2002, Coley received US Patent No. 6,406,705 covering its CpG oligonucleotide immunostimulants for use in combination with conventional adjuvants. In April 2001, Coley was issued with US Patent No. 6,207,646 covering the composition and use of its immune stimulants including CpG 7909. In September 2001, Coley was granted US Patent No. 6,214,806, which expands the coverage on the use of CpG oligonucleotides in the treatment of certain respiratory disorders.
Adjuvants, Immunologic↗
Requirements for effective inhibition of immunostimulatory CpG motifs by neutralizing motifs.
The DNA of bacteria and many viruses contain unmethylated CpG dinucleotides in particular sequence contexts that activate vertebrate immune cells. A subset of these CpG motifs was previously found to oppose the effects of immunostimulatory (CpG-S) motifs and has been termed neutralizing (CpG-N) motifs. Here we show that oligodeoxynucleotides (ODNs) composed of clusters of CpG-N motifs could partially inhibit the induction of interleukin-12 (IK-12) from mouse spleen cells by ODN containing CpG-S motifs. However, non-CpG-containing ODN were also inhibitory, suggesting that neutralization of CpG-S ODNs by CpG-N ODNs in trans was nonspecific. Neutralization of CpG-S motifs by CpG-N motifs in cis was specific, but the degree of inhibition was strongly dependent on the particular CpG-S motif being neutralized, with motifs having an A residue 5' to the CG being much more resistant to inhibition than motifs having a T residue 5' to the CG. The degree of inhibition was dependent on the spacing between the CpG-S and CpG-N motifs, with the ability to neutralize inversely correlating with distance. In addition, whereas ODNs containing extended clusters of CpG-N motifs were nonstimulatory, isolated CpG-N motifs remained stimulatory in most sequence contexts. Finally, CpG-N ODNs were shown to be nonstimulatory when instilled into the lungs of BALB/c mice, but the ability of CpG-N motifs to neutralize CpG-S motifs in cis was not observed. These results show that there are precise and fairly complex interactions between immunostimulatory and inhibitory sequence motifs that govern whether a given DNA is able to activate the vertebrate immune system.
Adjuvants, Immunologic↗
CpG ODNs treatments of HIV-1 infected patients may cause the decline of transmission in high risk populations - a review, hypothesis and implications.
The Joint United Nations Program on HIV-1/AIDS (UNAIDS) announced its goal to stop HIV-1 transmission by antiviral (HAART) treatment of patients since at the end of 2003 the number of people living with HIV-1 was 38 million, 25 million in the sub-Saharan region of Africa. The present review deals with a new approach to simultaneously treat HIV-1/AIDS patients in HIV-1 endemic regions with CpG oligodeoxynucleotides (ODNs) and people at high risk of infection with a vaccine containing CpG ODNs combined with synthetic HIV-1 peptides by intranasal and intradermal applications. During HIV-1 infection a gradual increase in the levels of IL-4 and IgE in the patients' serum, was reported. It was suggested that such an increase of the cytokine IL-4 and the IgE immunoglobulin are interconnected and may serve as indicators for the coming stage of AIDS. It was also suggested that the IL-4 and IgE increase in the serum of HIV-1 infected people resemble the increase of IL-4 and IgE levels in allergic patients that were exposed to endogenous or environmental allergens [Becker, Virus Genes 28, 5--18, 2004]. Indeed, it was reported that the HIV-1 virions' shed gp120 molecules, which contain a superantigen (superallergen) domain that enables the viral glycoprotein to bind the V(H)3 domain of IgE molecules that are bound to FcepsilonRI+ hematopoietic cells [basophils, mast cells, dendritic cells (DCs) and plasmacytoid DCs (pDCs)]. Such interaction was reported to induce the hematopoietic cells to release large amounts of Th2 cytokines IL-4, IL-5, IL-10 and IL-13. These findings led to the hypothesis [Op. cit.] that the cure of HIV-1/AIDS patients requires the induction of endogenous synthesis of type I interferons (INF alpha and beta) with a bacterial CpG rich DNA that will induce the patients' pDCs to release large amounts of type I IFNs. Under these conditions HIV-1 replication in polarized to Th2 cells is inhibited. Type I IFNs reactivate the patients' inhibited Th1 cells to synthesize IL-2 and IL-12 cytokines that activate the maturation of CTL precursors. The unmethylated bacterial DNA activates B synthesis to switch to IgG and IgA synthesis. The novel drug CpG ODNs is being tested for the prevention and the treatment of allergic humans and in the experimental system of allergic mice. It was also reported that treatment of mice with CpG ODN prior to or after retrovirus infections protected and cured, respectively, the retrovirus infection. It was also reported that CpG ODNs treatments of mice exposed to allergen protected them against the development of the allergic response. Phase I treatment of healthy people with CpG ODNs provided information on the safety of these compounds. The CpG ODNs A and B bind to Toll like receptors that are present in pDCs and B cells, respectively, CpG ODN - A is the ligand for TLR9+ pDCs and induce the release of large amounts of IFN-alpha, beta. CpG ODN-B is the ligand for TLR9+ in B cells and induce the synthesis of IgG and IgA. CpG ODN-C contains motifs from CpG ODNs A and B and is more active. The present review is based on findings from studies that reported that CpG ODNs treatment of retrovirus infected mice, monkeys and allergic mice prevented the virus and allergens caused diseases, respectively. Based on these studies, a hypothesis is presented that treatment of HIV-1 infected and AIDS patients with CpG ODN-A and B or CpG ODN-C have the potential to inhibit IL-4 synthesis and release from FcrepsilonRI+ hematopoietic cells by inducing TLR9+ pDCs to release large amounts of type I IFNs. TLR9+ B cells are induced by CpG ODN-B to switch from IgE to IgG, IgA synthesis. In addition, type I IFNs (alpha, beta) have the capacity to inhibit HIV-1 replication in polarized Th2 cells. Type I IFNs reactivate the patients' Th1 cells to synthesize IL-2 and IL-12 cytokines, activators of the precursor cytotoxic T cells (CTLs), leading to the reactivation of the inhibited adaptive immune response. Antiviral CTLs have the ability to clear the virus infection. The present novel approach to the treatment and of HIV-1/AIDS patients with CpG ODNs may prevent HIV-1 transmission and the AIDS pandemic if controlled studies on the treatments with CpG ODNs of HIV-1 infected people will be done by international and private agencies and companies to define the effective treatment regime and the efficacy of the treatments to HIV-1 infected people at different times post-infection. It is also hypothesized that in order to stop HIV-1 transmission in HIV-1 endemic regions the people at high risk of HIV-1 infection should be treated at the same time as HIV-1 infected people with a vaccine containing synthetic CpG-ODNs combined with synthetic HIV-1 peptides, compatible with the major HLA haplotypes of the regional population. The vaccine may be self-applied by people at high risk of infection by the intra-nasal route and by intra-dermal application as a "peplotion vaccine". The stimulation of the antiviral CTL response by HIV-1 infected people and the active antiviral immune response in the vaccinated population may lead to a decline in HIV-1 transmission and may be a model for control of the HIV-1/AIDS pandemic.
Adjuvants, Immunologic↗
Novel immunostimulatory agent based on CpG oligodeoxynucleotide linked to the nontoxic B subunit of cholera toxin.
In this study, we report the development of a novel, rationally designed immunostimulatory adjuvant based on chemical conjugation of CpG oligodeoxynucleotide (ODN) to the nontoxic B subunit of cholera toxin (CTB). We demonstrate that the immunostimulatory effects of CpG can be dramatically enhanced by conjugation to CTB. Thus, CpG ODN linked to CTB (CTB-CpG) was shown to be a more potent stimulator of proinflammatory cytokine and chemokine responses in murine splenocytes and human PBMCs than those of CpG ODN alone in vitro. The presence of CpG motif, but not modified phosphorothioate ODN backbone, was found to be critical for the enhanced immunostimulatory effects of CTB-CpG. Our mode-of-action studies, including studies on cells from specifically gene knockout mice suggest that similar to CpG, CTB-CpG exerts its immunostimulatory effects through a TLR9/MyD88- and NF-kappaB-dependent pathway. Surprisingly, and as opposed to CpG ODN, CTB-CpG-induced immunity was shown to be independent of endosomal acidification and resistant to inhibitory ODN. Furthermore, preincubation of CTB-CpG with GM1 ganglioside reduced the immunostimulatory effects of CTB-CpG to those of CpG ODN alone. Interestingly, conjugation of CpG ODN to CTB confers an enhanced cross-species activity to CpG ODN. Furthermore, using tetanus toxoid as a vaccine Ag for s.c. immunization, CTB-CpG markedly enhanced the Ag-specific IgG Ab response and altered the specific pattern of Ab isotypes toward a Th1 type response. To our knowledge, CTB is the first nontoxic derivative of microbial toxins discovered that when chemically linked to CpG remarkably augments the CpG-mediated immune responses.
Adaptor Proteins, Signal Transducing↗
Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-alpha induction in plasmacytoid dendritic cells.
Two different types of CpG motif-containing oligonucleotides (CpG ODN) have been described: CpG-A with high induction of IFN-alpha in plasmacytoid dendritic cells; and CpG-B with little induction of IFN-alpha, but potent activation of B cells. In this study, we demonstrate that CpG-A fail to activate B cells unless plasmacytoid dendritic cells are present. We identified a new set of CpG ODN sequences which induces high levels of IFN-alpha in plasmacytoid dendritic cells but remains capable of directly activating B cells. These new CpG ODN (termed CpG-C) are more potent stimulants of B cells than CpG-B due to their ability of directly and indirectly (via plasmacytoid dendritic cells) activating B cells. The sequence of CpG-C combines structural elements of both CpG-A and CpG-B. The most potent sequence, M362, contains a 5'-end 'TCGTCG-motif' and a 'GTCGTT-motif', both of which are present in CpG-B (ODN 2006); a palindromic sequence characteristic for CpG-A (ODN 2216); but no poly G motif required for CpG-A. In conclusion, we defined the first CpG-containing sequences that potently activate both TLR9-expressing immune cell subsets in humans, the plasmacytoid dendritic cell and the B cell. CpG-C may allow for improved therapeutic immuno-modulation in vivo.
Adjuvants, Immunologic↗
Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells.
Plasmacytoid dendritic cells (PDC) represent a highly specialized immune cell subset that produces large quantities of the anti-viral cytokines type I interferons (IFN-alpha and IFN-beta) upon viral infection. PDC employ a member of the family of toll-like receptors, TLR9, to detect CpG motifs (unmethylated CG dinucleotides in certain base context) present in viral DNA. A certain group of CpG motif-containing oligodeoxynucleotides (CpG ODN), CpG-A, was the first synthetic stimulus available that induced large amounts of interferon-alpha (IFN-alpha) in PDC. However, the mechanism responsible for this activity remained elusive. CpG-A is characterized by a central palindrome and poly(G) at the 5' and 3' end. Here we demonstrate that CpG-A self-assembles to higher order tertiary structures via G-tetrad formation of their poly(G) motifs. Spontaneous G-tetrad formation of CpG-A required the palindrome sequence allowing structure formation in a physiological environment. Once formed, G-tetrad-linked structures were stable even under denaturing conditions. Atomic force microscopy revealed that the tertiary structures formed by CpG-A represent nucleic acid-based nanoparticles in the size range of viruses. Similarly sized preformed polystyrene nanoparticles loaded with a CpG ODN that is otherwise weak at inducing IFN-alpha (CpG-B) gained the potency of CpG-A to induce IFN-alpha. Higher ODN uptake in PDC was not responsible for the higher IFN-alpha-inducing activity of CpG-A or of CpG-B-coated nanoparticles as compared with CpG-B. Based on these results we propose a model in which the spatial configuration of CpG motifs as particle is responsible for the virus-like potency of CpG-A to induce IFN-alpha in PDC.
Adolescent↗
CpG islands: features and distribution in the genomes of vertebrates.
We have investigated the distribution of unmethylated CpG islands in vertebrate genomes fractionated according to their base composition. Genomes from warm-blooded vertebrates (man, mouse and chicken) are characterized by abundant CpG islands, whose frequency increases in DNA fractions of increasing % of guanine + cytosine; % G + C (GC), in parallel with the distribution of genes and CpG doublets. Small, yet significant, differences in the distribution of CpG islands were found in the three genomes. In contrast, genomes from cold-blooded vertebrates (two reptiles, one amphibian, and two fishes) were characterized by an extreme scarcity or absence of CpG islands (detected in these experiments as HpaII tiny fragments or HTF). CpG islands associated with homologous genes from cold- and warm-blooded vertebrates were then compared by analyzing CpG frequencies, GC levels, HpaII sites, rare-cutter sites and G/C boxes (GGGGCGGGGC and closely related motifs) in sequences available in gene banks. Small, yet significant, differences were again detected among the CpG islands associated with homologous genes from warm-blooded vertebrates, in that CpG islands associated with mouse or rat genes often showed low CpG and/or GC levels, as well as low numbers of HpaII sites, rare-cutter sites and G/C boxes, compared to homologous human genes; more rarely, CpG islands were just absent. As far as cold-blooded vertebrates were concerned, a number of genes showed CpG islands, which exhibited a much lower frequency of CpG doublets than that found in CpG islands of warm-blooded vertebrates, but still approached the statistically expected frequency; none of the other features of CpG islands associated with genes from warm-blooded vertebrates were present. Other genes did not show any associated CpG islands, unlike their homologues from warm-blooded vertebrates.
Animals↗
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.
Adjuvants, Immunologic↗
CpG oligodeoxynucleotides inhibit tumor growth and reverse the immunosuppression caused by the therapy with 5-fluorouracil in murine hepatoma.
AIM: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.
Animals↗
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.
Adjuvants, Immunologic↗
Immunostimulatory CpG-oligodeoxynucleotides induce a factor that inhibits macrophage adhesion.
Selected phosphorothioate oligodeoxynucleotides containing CpG (CpG-ODN) activate immune responses, including immunoglobulin synthesis, B cell proliferation, and cytokine production by monocytes. We examined the effect of a CpG-ODN (#1760) on the adhesion of macrophages derived from human blood monocytes in vitro. CpG-ODN (6 microg/mL) completely inhibited the adherence of macrophages to plastic or glass during 7 or more days of culture. A non-CpG control ODN (#1814) was without effect. Two other CpG-ODNs (#1826 and #1842) also completely inhibited macrophage adherence. The specific inhibitor of CpG-ODN, quinacrine (0.1 micromol/L), blocked this action. CpG-ODN reduced the rate of senescence and cell death of monocytes in culture but did not influence their phagocytosis, procoagulant activity, or support of the mixed lymphocyte response. Four days of exposure of monocytes to CpG-ODN up-regulated the expression of the endotoxin receptor CD14 and down-regulated the mannose (scavenger) receptor, a result that is consistent with blocking the maturation of monocytes to macrophages. Incubation of peripheral blood monocytes (PBMCs) with CpG-ODN resulted in the generation of a heat labile factor that inhibited macrophage differentiation and accounts for the efficacy of the CpG-ODN. T cells selected from PBMCs by magnetic beads generated the majority of this factor. Cytokines (interleukin-3 (IL-3), IL-4, IL-6, IL-10, interferon-gamma, tumor necrosis factor-alpha, transforming growth factor-beta, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1) did not inhibit macrophage adherence like CpG-ODN did. Antibodies to IL-6 or IL-10 did not block the activity of CpG-ODN. Dexamethasone inhibited macrophage adherence, and lipopolysaccharide had a minor effect. We conclude that immunostimulatory CpG-ODNs inhibit macrophage adherence by provoking the production of an unidentified heat-labile factor.
Adjuvants, Immunologic↗
Enhancement of immune responses by co-delivery of a CpG oligodeoxynucleotide and tetanus toxoid in biodegradable nanospheres.
Synthetic oligodeoxynucleotides (ODN) consisting of unmethylated bacterial DNA sequences with CpG motifs are potent immunological adjuvants. Immunostimulatory CpG sequences are species-specific. Optimal CpG sequences specific for humans, rodents, livestock, and companion animals have been reported. Nearly all of these reports describe the use of soluble forms of CpG ODN and antigens. We investigated the co-delivery of CpG ODN and antigens in biodegradable nanospheres as an alternative approach for immunization using tetanus toxoid (TT) as the model antigen and ODN #1826 as the model CpG sequence. TT and CpG ODN were co-encapsulated in poly(D,L-lactic-co-glycolic acid) nanospheres. Separate groups of C57BL/6 mice were subcutaneously immunized twice with TT and CpG ODN in nanospheres (test group), TT alone in nanospheres, TT alone in nanospheres mixed with CpG ODN in solution, TT and CpG ODN both in solution (reference group), TT alone in solution, and alum adsorbed TT. T cells isolated from the test group showed strong antigen-specific T cell proliferation ex vivo (stimulation index=45). This was significantly (P<0.0001) higher than that observed for T cells isolated from the reference group. The T cell proliferation of the test group was associated with higher levels of interferon gamma secretion (IFN-gamma 2694.7+/-41.1 pg/ml) than that of the reference group (814.7+/-50.2 pg/ml). Interleukin 4 (IL-4) secretion, if any, was below the detection limit (<13 pg/ml) in all the groups. Anti-sera obtained from the test group also showed very high total IgG titers (end point titers, 2560000) that were 16 times higher than the reference group. Similarly, differences of 8-fold for IgG1 and IgG3, and 5-fold for IgG2b titers were observed. Noticeably, the antibody response induced in the alum-TT group was far less (total IgG, end point titers 160000) than that obtained in the TT-CpG ODN nanospheres group. Overall, the results show that co-delivery of CpG and TT resulted in induction of both T helper type 1 and type 2 (Th1 and Th2) immune responses with a bias towards Th1 type. These results suggest that the co-delivery of CpG ODN adjuvants and antigens in nanospheres is a more efficient approach for immunization than the use of CpG ODN and TT in solution.
Adjuvants, Immunologic↗
The arthritogenic and immunostimulatory properties of phosphorothioate oligodeoxynucleotides rely on synergy between the activities of the nuclease-resistant backbone and CpG motifs.
Experiments with immunostimulatory unmethylated CpG-containing DNA are usually conducted with nuclease-protected phosphorothioate oligodeoxynucleotides (S-ODNs), rather than phosphodiester oligodeoxynucleotides (O-ODNs). We compared the murine immune responses to S-ODNs and O-ODNs that either contained or lacked CpG motifs. Both CpG and non-CpG S-ODNs induced synovitis, as did sequence-matched CpG O-ODN, but not GpC O-ODN. There was a minimum length requirement for arthritogenic S-ODNs since a CpC dinucleotide S-ODN did not induce arthritis. There were both sequence- (CpG > non-CpG) and backbone-dependent (S-ODN > O-ODN) differences in the levels of DNA-induced arthritis upon intra-articular injection with the ODNs. However, CpG O-ODN being an exception, induced more severe arthritis than the GpC S-ODN. The levels of in vitro proliferation and production of IL-6, TNF-alpha, IL-12, and RANTES by splenocytes following exposure to CpG S-ODN were significantly higher than those induced by CpG O-ODN. In addition, both proliferative responses and cytokine production induced by S-ODN-stimulated splenocytes increased significantly when the S-ODN contained a CpG motif. Transcription factor NFkappaB was activated by both CpG S-ODN and CpG O-ODN but interestingly not by GpC S-ODN. This indicates that the NFkappaB signal pathway modulates CpG-mediated immunostimulation, while sequence-independent immune activation by the phosphorothioate backbone is probably signalled via a different pathway.
Adjuvants, Immunologic↗
Unmethylated CpG-containing oligodeoxynucleotides inhibit apoptosis in WEHI 231 B lymphocytes induced by several agents: evidence for blockade of apoptosis at a distal signalling step.
Certain oligodeoxynucleotides (ODN) containing cytosine followed by guanosine (CpG) protect B cells from apoptosis, and induce B-cell proliferation and cytokine production. We investigated the effect of phosphorothioate CpG-containing ODNs (5'-ATAATCGACGTTCAAGCAAG-3' or 5'-TCCATGACGTTCCTGACGTT-3') and control ODNs (which did not contain CpG) on apoptosis and cell growth in WEHI 231 murine B lymphoma cells. Anti-surface (alpha-s)IgM antibody induces 40-60% DNA degradation and growth arrest of WEHI 231 cells in 24 h. Both of these effects were substantially reversed by 30 ng/ml CpG-ODN added up to 8 hr after alpha-sIgM. Control ODNs not containing the CpG motif were without effect. We explored various hypotheses to account for these effects. The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate, inhibits apoptosis induced by alpha-sIgM, but the anti-apoptotic effect of CpG-ODN was not affected by inhibitors of protein kinase C, indicating that CpG-ODN does not act via protein kinase C. CpG-ODN inhibited apoptosis and growth arrest induced by C2- and C8-ceramide, sphingomyelinase and an intracellular Ca2+ pump inhibitor thapsigargin, indicating that inhibition is not mediated via suppression of the ceramide cycle or suppression of Ca2+ mobilization. CpG-ODN partially inhibited apoptosis induced by okadaic acid, a protein phosphatase inhibitor, and by menadione, a free radical generator. CpG-ODN also inhibited apoptosis and growth arrest induced by ultraviolet-irradiation, glucocorticoid, vinca alkaloids, and doxorubicin. CpG-ODN significantly protected cells from DNA fragmentation induced by alpha-sIgM in the presence of cycloheximide, but cycloheximide itself induces apoptosis which was unaffected by CpG-ODN. These results suggest that CpG-ODNs powerfully modulate the process by which immune cells are committed to death or proliferation by a mechanism acting on distal cell signalling events. CpG-ODNs may be able to decrease immunosuppression in patients undergoing cancer chemotherapy.
Animals↗