Construction of a recombinant plasmid containing multi-copy CpG motifs and its effects on the innate immune responses of aquatic animals

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides (CpG motifs) have been shown to induce potential immune responses. In this study, we designed a recombinant plasmid containing multi-copy CpG motifs, and observed its effects on innate immune responses of fish and prawn. The results showed that such plasmid DNA, compared to the vacant vector, can highly induce the activation of head kidney macrophages and the proliferation of peripheral blood leukocytes in Carassius auratus and Lateolabrax japonicus in vitro, as well as the activity of humoral defense proteins and the antibacterial activity of haemolymph in Litopenaeus vannamei in vivo. It implies that the multi-copy CpG motifs harboured in plasmid could contribute to these innate immunostimulatory effects. Therefore, the study suggested that the plasmid containing multi-copy CpG motifs might have its potential application in improving host resistance to pathogen insults in aquaculture, and have its notable advantages of high efficacy, economical cost and application to a broad range of aquatic species.     

Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.     

Bacterial DNA containing methylated CpG motifs retains immunostimulatory activity in synergy with modified lipopolysaccharides

We previously described the immunostimulatory activity of CIA07, a combination of bacterial DNA fragments and modified LPS, and demonstrated that CIA07 has antitumor activity in a mouse bladder cancer model. In this study, we investigated whether methylation of the CpG motifs on the bacterial DNA fragments affects the immunostimulatory potential of CIA07. E. coli DNA fragments were methylated with CpG methylase, and then combined with modified LPS for experiments. Our results revealed that methylated CIA07 (mCIA07) and unmethylated CIA07 were equally active in inducing cytokine secretion from human whole blood cells. In addition, both methylated DNA fragments and mCIA07 retained the ability to activate expression and nuclear translocation of NF-kappaB in RAW 264.7 cells. Finally, methylated DNA fragments and mCIA07 exhibited an antitumor activity comparable to those of their unmethylated counterparts in our mouse bladder cancer model. These data demonstrate that CpG methylation of E. coli DNA does not abrogate the immunostimulatory activity of DNA fragments or CIA07, suggesting that the synergistic activity by bacterial DNA in combination with LPS may be independent of the methylation status of CpG motifs.     

Immune stimulation in the silkworm, Bombyx mori L., by CpG oligodeoxynucleotides

Synthetic ODNs containing unmethylated CpG dinucleotides are known to stimulate immune responses in vertebrates, but so far the effect has not been studied in insects. In this report, we describe an induction of immune response following injection of oligodeoxynucleotides (ODNs) into the insect hemocoel. The fifth instar silkworm (Bombyx mori L.) larvae were injected with several synthetic ODNs containing variable number of unmethylated CpG motifs, heat-denatured genomic DNA of B. mori itself, or intact genomic DNA to observe a new induction pattern in the insect immune mechanism. When the induction of immune response was examined based on the expression rates of genes for antibacterial peptides such as attacin and cecropin, we could confirm that it was triggered upon injection of ODNs. The expression was, however, neither dependent on numbers of CpG motifs nor methylation of CpGs in ODNs. Furthermore, it was confirmed that the presence of CpG in ODN was not involved in the induction pattern of insect immunity caused by ODNs, although it has been reported that vertebrates respond in a specific manner against invading ODNs containing CpG dinucleotides. In addition, insect immunity was not stimulated by injection of intact DNA from host. In contrast, the injection of denatured genomic DNA provoked the host immune reaction. Taken together, our data suggest that foreignness of ODNs or DNA might be a key factor in the induction of insect immunity.     

Strong cytosine-guanosine-independent immunostimulation in humans and other primates by synthetic oligodeoxynucleotides with PyNTTTTGT motifs

Synthetic oligodeoxynucleotides (ODNs) containing cytosine-guanosine (CpG) motifs stimulate B and plasmacytoid dendritic cells of the vertebrate immune system. We found that in primates strong stimulation of these cells could also be achieved using certain non-CpG ODNs. The immunostimulatory motif in this case is a sequence with the general formula PyNTTTTGT in which Py is C or T, and N is A, T, C, or G. Assays performed on purified cells indicated that the immunostimulatory activity is direct. The use of a nuclease-resistant phosphorothioate backbone is not a necessary condition, since phosphodiester PyNTTTTGT ODNs are active. It was also demonstrated that ODN 2006, a widely used immunostimulant of human B cells, possess two kinds of immunostimulatory motifs: one of them mainly composed of two successive TCG trinucleotides located at the 5' end and another one (duplicated) of the PyNTTTTGT kind here described. Even though PyNTTTTGT ODNs are mainly active on primate cells, some of them, bearing the CATTTTGT motif, have a small effect on cells from other mammals. This suggests that the immunostimulatory mechanism activated by these ODNs was present before, but optimized during, evolution of primates. Significant differences in the frequency of PyNTTTTGT sequences between bacterial and human DNA were not found. Thus, the possibility that PyNTTTTGT ODNs represent a class of pathogen-associated molecular pattern is unlikely. They could, more reasonably, be included within the category of danger signals of cell injury.     

Immunostimulatory PyNTTTTGT oligodeoxynucleotides: structural properties and refinement of the active motif

It is well known that synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides within a given context stimulate B lymphocytes and plasmacytoid dendritic cells (PDCs) of the vertebrate immune system. We have reported that B lymphocyte and PDC stimulation in humans could also be efficiently achieved by using non-CpG ODNs bearing the immunostimulatory sequence (motif) PyNTTTTGT, wherein Py is C or T and N is any deoxyribonucleotide. We are now reporting a series of studies that gives further precision regarding the composition of this immunostimulatory motif. The analysis of hundreds of ODNs led us to the conclusion that the motif for optimal CpG-independent immune stimulation can be represented by a sequence of the following general formula: PyN(T/A)(T/C/G)(T/C/G)(T/G)GT, wherein Py is C or T and N is any deoxyribonucleotide and wherein at least two of the positions represented within parentheses are Ts. Requirements for optimal ODN activity are as follows: (1) at least one of the versions of the general motif must be located near the central portion of the ODN; and (2) the ODN must be 20 or more nucleotides long. PyN(T/A)(T/C/G)(T/C/G)(T/G)GT ODNs are active in a phosphorothioate or in a phosphodiester backbone. In a phosphodiester backbone a canonical motif is strongly required whereas in a phosphorothioate backbone specificity is, within certain limits, less strict. On the other hand, PyN(T/A)(T/C/G)(T/C/G)(T/G)GT oligonucleotides are inactive as a double chain or as a modified (phosphorothioate or hydroxyl-methyl modified) or unmodified RNA backbone.     

G3139 and other CpG-containing immunostimulatory phosphorothioate oligodeoxynucleotides are potent suppressors of the growth of human tumor xenografts in nude mice

Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.     

Safety of CpG oligodeoxynucleotides in veterinary species

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in particular sequence contexts (CpG ODN) are recognized as a danger signal by the innate immune system of vertebrates. For this reason, CpG ODNs have a potential application as both an adjuvant and nonspecific immune modulator and are currently being evaluated in a number of human and veterinary clinical trials. Given their potent immunostimulatory activity, CpG ODNs could possibly induce adverse reactions. As all adjuvants and immune modulators must be nontoxic to meet safety requirements, it was essential to address the safety aspects of CpG ODNs. The current review summarizes experiments carried out to date to establish the safety of CpG ODNs in animals.     

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.     

The majority of methylated deoxycytidines in human DNA are not in the CpG dinucleotide

The only natural postsynthetic modification known to occur in mammalian DNA is the methylation in the 5 position of deoxycytidines. Of the four 5'-CpN-3' dinucleotides (ie. CpG, CpC, CpA, and CpT), the dinucleotide which contains the highest proportion of deoxycytidines methylated is CpG, with 40 to 80% methylation in different mammalian genomes. It has also been shown that CpA, CpT, and CpC are methylated as well but to a much lower extent. Here we report the result of a full nearest neighbour analysis (together with quantitation of methylation levels in the 4 CpN dinucleotides) for DNA from human spleen. Using the values we have calculated the overall frequencies for all the methylated dinucleotides in the human genome. Because of the relative underrepresentation (by 7 to 10 fold) of the CpG dinucleotide, only 45.5% of total mC was present in mCpG, with 54.5% in mCpA, mCpT plus mCpC. These calculations have implications for studies into the function and significance of DNA methylation in mammalian cells.     

Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo

Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G+C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8⁺ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-γ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in particular, in diseases like herpes stromal keratitis (HSK) that involve chronic inflammatory responses in the absence of virus or viral antigens.     

Uptake, cellular distribution and novel cellular binding proteins of immunostimulatory CpG oligodeoxynucleotides in glioblastoma cells

Glioblastomas are the most malignant and most frequent brain tumors and exciting targets of gene and immunotherapy. Despite rapid development of experimental therapy little is known about the cellular behaviour of therapeutic oligodeoxynucleotides (ODNs). Here we designed uptake, cellular distribution and cellular binding proteins of immunostimulatory CpG-ODNs in glioblastoma cells by flow cytometry, fluorescence microscopy and mass spectrometry. Our data show that the phosphorothioate (PS) CpG-ODNs uptake in T98G and C6 cells is dose-, time-, temperature-dependent and independent of the CpG dinucleotides. Uptake can be inhibited by sodium azide, polyanions but not by chloroquine. After internalisation FITC labelled CpG-ODNs showed a spotted distribution in cytoplasm. Dozens of cellular binding proteins were identified using mass spectrometry. The binding of ODNs to proteins is dependent on modification and sequence but independent on CpG motif. ODNs bind to cellular proteins that are important for RNA processing and transport. Furthermore, three novel membrane proteins were identified, which might contribute to uptake of ODNs. ODNs binding to these proteins might interfere with the physiological function and thus might cause unwanted effects. Such binding also might influence the uptake efficiency or cellular distribution of therapeutic ODNs.     

Antitumor mechanisms of oligodeoxynucleotides with CpG and polyG motifs in murine prostate cancer cells: decrease of NF-kappaB and AP-1 binding activities and induction of apoptosis

Previous studies have shown that CpG oligodeoxynucleotides (ODNs) have substantial immunostimulatory effects with anticancer applications. The antitumor applications that have been described previously are mediated through the CpG-induced activation of the host immune system, not through direct antitumor effects. Using cytostasis and cell proliferation assays, we demonstrated that specific ODNs inhibit the proliferation of RM-1 cells, a murine prostate cancer cell line. Flow cytometry analysis using propidium iodide (PI) nuclear staining confirmed the direct proapoptotic effect of ODNs on prostate cancer cells. This effect was dose dependent. Further studies using Western blot analysis and electrophoresis mobility shift assay (EMSA) revealed that the treatment of prostate cancer cells with specific ODNs activated the caspase pathway(s) and decreased the binding activities of AP-1 and NF-kappaB in a time-dependent manner. Evaluation of a panel of ODNs containing different DNA motifs demonstrated that the optimal proapoptotic sequences required polyG sequences but that CpG motifs were not essential. Finally, in vivo antitumor studies showed that the proapoptotic polyG motifs significantly inhibited prostate tumor growth. PolyG motifs inhibited tumor growth, and the effects were enhanced by CpG immune activating sequences. ODN containing both polyG and CpG motifs may have enhanced efficacy in tumor therapy through multiple mechanisms of action, including direct antitumor activities and immune activation.     

A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells.

Although DNA can be extensively methylated de novo when introduced into pluripotent cells, the CpG island in the Thy-1 gene does not become methylated either in the mouse embryo or in embryonic stem cells. A 214-base-pair region near the promoter of the Thy-1 gene protects itself as well as heterologous DNA sequences from de novo methylation. We propose that this nucleotide sequence is representative of a class of important signals that limits de novo methylation in the embryo and establishes the pattern of hypomethylated CpG dinucleotides found in somatic tissues.     

Tumour class prediction and discovery by microarray-based DNA methylation analysis

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.