Treatment of infectious diseases with immunostimulatory oligodeoxynucleotides containing CpG motifs

Bacterial DNA with CpG motifs can efficiently stimulate the vertebrate immune system. Thus, synthetic oligodeoxynucleotides that contain such CpG motifs (CpG-ODN) are currently used in preclinical and clinical studies to develop new allergy or cancer therapies and vaccine adjuvants. Recent animal studies indicate that CpG-ODN therapies can also be used for successful treatment of infections caused by bacteria, parasites or viruses. In these experiments, innate and adaptive immune responses against pathogens were augmented by CpG-ODN and subsequently induced resistance against infectious diseases. The stimulation of dendritic cells played a central role for the therapeutic effect of CpG-ODN. However, CpG-ODN can also have negative side effects, which accelerate disease progression in some viral infections. Clinical studies with CpG-ODN will determine their potential for the therapy of infectious diseases in humans.     

Effective postexposure treatment of retrovirus-induced disease with immunostimulatory DNA containing CpG motifs

Therapeutic strategies for the treatment of acute retroviral infections have relied mainly on antiviral drugs. In this study we used the Friend virus model system to demonstrate that enhancement of the immune system can also have dramatic therapeutic effects. Since resistance to Friend virus-induced leukemia in mice is associated with T helper cell type 1 (Th1) immune responses, we enhanced these responses in susceptible mice by treatment with synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN). Treatments begun at 4 days postinfection increased recovery from 6% in the control group to 74% in the CpG-treated group. CpG-mediated recovery was associated with a significant reduction of viral loads in the blood and spleens of treated mice compared to those of control animals. The treatment promoted Th1-type cytokine production by splenocytes of Friend virus-infected mice and augmented Friend virus-specific cytotoxic T-cell responses, but no influence on the virus-specific neutralizing antibody response was observed. Friend virus-specific CD8(+) T cells were critical for effective treatment with CpG-ODN, since in vivo depletion of these cells from treated mice prevented their recovery. Our results demonstrate that CpG-ODN therapy can significantly enhance virus-specific cellular immune responses and prevent retrovirus-induced disease. These findings may have implications for antiviral therapy in general.     

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.     

Reduction of 1-methyl-1-nitrosourea-induced mammary gland carcinoma by in vivo application of immunostimulatory CpG motifs in Sprague-Dawley rats

In the present work the role of 13-cis retinoic acid and CpG oligodeoxynucleotides (CpG-ODN) in a 1-methyl-1-nitrosourea (MNU)-induced mammary gland carcinoma animal model was investigated. Treatment with both components, applied either alone or in combination, induced a significant decrease of the tumour burden and the volume of tumours only in rats that received CpG-ODN (p = 0.046, compared to the MNU control group). The data indicate that the Th-1 biased immunostimulatory capacities of CpG motifs may play a significant role in induction of protective immune responses against mammary gland tumours in Sprague-Dawley rats.     

Requirement of nucleobase proximal to CpG dinucleotide for immunostimulatory activity of synthetic CpG DNA

Synthetic oligodeoxyribonucleotides containing CpG dinucleotides exhibit potent immunostimulatory activity in vertebrates. Although the molecular mechanisms of recognition and interaction of CpG DNA sequences with receptors are not well understood, the current evidence suggests that the receptor shows considerable selectivity for CpG DNA sequences with different preferences in mouse (GACGTT) and human (GTCGTT) species. In our continued effort to understand the chemical and structural characteristics of CpG DNA required for the immunostimulatory activity and thereby for the recognition of receptors in the immunostimulatory pathway, we examined the requirement of nucleobases in the two adjacent nucleotide positions on the 5'- and the 3'-side to the CpG dinucleotide (P(1)P(2)CGP(3)P(4)) for the immunostimulatory activity. These studies, in which a natural nucleoside is substituted with an abasic nucleoside (X), suggest that a nucleobase is absolutely required in C, G, P(3), and P(4) positions for immunostimulatory activity. Surprisingly, an abasic nucleoside is permitted at either P(1) or P(2) depending on the neighboring base. It was found that 'GXCGTT' motif has an intermediate immunostimulatory activity between those of 'GACGTT' and 'GTCGTT' in the mouse cells.     

Binding and uptake of immunostimulatory CpG oligodeoxynucleotides by human neuroblastoma cells

Oligodeoxynucleotides (ODNs) that contain unmethylated CpG dinucleotides (CpG-ODN) trigger a strong innate immune response in vertebrates. They have been used to eradicate experimental neuroblastoma, but a direct interaction of CpG-ODN with neuroblastoma cells has not been investigated. We have analyzed uptake, binding, and intracellular distribution of CpG-ODN in the neuroblastoma cells line SKNSH. Our results indicate that cellular uptake of CpG-ODN is dose, time, temperature, and energy dependent but independent of the CpG motif. After internalization, CpGODN localized to the cytoplasm and showed a typical speckled distribution pattern. The intracellular distribution pattern and binding proteins are CpG motif independent as well. Thus, CpG-ODNs are taken up by neuroblastoma cells by a nonspecific transfer mechanism for oligonucleotides and interact with intracellular proteins. These mechanisms might help us to understand the biodistribution of oligo within tumors and might be helpful in evaluating the therapeutic effects of oligonucleotides and rational drug design.     

Modulation of immunostimulatory activity of CpG oligonucleotides by site-specific deletion of nucleobases

The effect of nucleobase deletion in the 3'- or the 5'-flanking sequence to a CpG-motif on immunostimulatory activity of CpG-containing oligonucleotides was examined by cell proliferation, secretion of IL-12 and IL-6 in mouse spleen cell cultures, and by spleen enlargement in mice. Deletion of one or two nucleobases in the 3'-flanking sequence to a CpG-motif at certain positions did not affect immunostimulatory activity, while similar deletions in the 5'-flanking sequence increased immunostimulatory activity compared with the parent oligo.     

CpG序列的免疫激活机理及应用

ＣｐＧ序列是一些在动物体内具有强烈的免疫激活功能的寡聚脱氧核苷酸。本文探讨了ＣｐＧ 序列在动物体内激活免疫反应的作用机理,概述了ＣｐＧ应用研究的进展,展望了ＣｐＧ 序列的应用前景     

CpG motif的免疫调节作用及机制

CpGmotif是以CpG二核苷酸为核心的,未甲基化的特殊的DNA序列,在微生物基因组中的出现频率高于在脊椎动物中的出现频率,近年来发现这种特殊的基序具有许多免疫调节作用。如刺激淋巴细胞增殖,分泌细胞因子,产生IgG2a类抗体,诱导Th1类应答,本介绍这种独特的CpGmotif的特征,可能的调节机制及应用前景。     

Synovial cytokine mRNA expression during arthritis triggered by CpG motifs of bacterial DNA

Our results show that cytokines derived from macrophages play an important role in pathogenesis of arthritis triggered by CpG oligodinucleotide (CpG ODN). IL-12 is in this respect an important immunomodulator during the development of joint inflammation.     

Suppression of TLR9 immunostimulatory motifs in the genome of a gammaherpesvirus

Multiple receptors within the innate immune system have evolved to recognize nucleic acids as signatures of viral infection. It is believed that this specificity is essential for viral detection, as viruses often lack other invariant features that can serve as suitable targets for innate receptors. One such innate receptor, TLR9, has been implicated in the detection of many dsDNA viruses. In this study, we investigate the detection of murine gammaherpesvirus 68 (MHV68) by TLR9. We find that the genomic DNA of the murine CMV, a very potent inducer of innate responses. Genome-wide analysis of the number of stimulatory versus nonstimulatory CpG motifs present in the genome of each virus reveals that the MHV68 genome contains only a fraction of the number of immunostimulatory motifs present in murine CMV. Notably, MHV68 appears to have selectively suppressed the number of stimulatory motifs through cytosine to thymine conversion. These data suggest that certain viruses may have evolved and modified their genomic content to avoid recognition by nucleic acid-sensing receptors of the innate immune system.     

Synovial cytokine mRNA expression during arthritis triggered by CpG motifs of bacterial DNA

Our results show that cytokines derived from macrophages play an important role in pathogenesis of arthritis triggered by CpG oligodinucleotide (CpG ODN). IL-12 is in this respect an important immunomodulator during the development of joint inflammation.     

CpG基元的免疫学进展及基因治疗策略

CpG诱发免疫反应的能力对免疫预防是一大优点 ,但在基因治疗中 ,质粒中所含的CpG基元能够阻止基因转移或导致巨大副作用 (炎症和毒性 )。本文简单综述了CpG基元的免疫学活性、主要作用和信号转导通路 ,并且针对它在基因治疗中存在的问题及可能的解决办法进行探讨。     

Requirements for mammalian carboxylesterase inhibition by substituted ethane-1,2-diones

Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many clinically used drugs. Hence, the activity of these agents is likely dependent upon the levels and location of CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study, we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different classes of molecules were assayed: one containing different atoms vicinal to the carbonyl carbon atom and the benzene ring [PhXC(O)C(O)XPh, where X=CH?, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibitory potency (when X=N), or conversion of the compounds into substrates for the enzymes (when X=S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Subsequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH? to C?H??] and 1-phenyl-2-alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH? to C?H??], demonstrated that the potency of enzyme inhibition directly correlated with the hydrophobicity (clogP) of the molecules. We conclude from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group.     

Site of chemical modifications in CpG containing phosphorothiate oligodeoxynucleotide modulates its immunostimulatory activity

Phosphorothioate oligodeoxynucleotides containing CpG motifs have immunostimulatory activity. Appropriate substitution of deoxynucleosides in the flanking region of CpG-containing phosphorothioate oligodeoxynucleotides with 2′-O-methylribonucleosides results in significant decreases or increases in their immunostimulatory activities. The results provide insights in how to chemically modify phosphorothioate oligodeoxynucleotides containing CpG motifs to suppress or enhance their immunostimulatory activity for different therapeutic uses.     

Site of chemical modifications in CpG containing phosphorothioate oligodeoxynucleotide modulates its immunostimulatory activity

Phosphorothioate oligodeoxynucleotides containing CpG motifs have immunostimulatory activity. Appropriate substitution of deoxynucleosides in the flanking region of CpG-containing phosphorothioate oligodeoxynucleotides with 2'-O-methylribonucleosides results in significant decreases or increases in their immunostimulatory activities. The results provide insights in how to chemically modify phosphorothioate oligodeoxynucleotides containing CpG motifs to suppress or enhance their immunostimulatory activity for different therapeutic uses.     

CpG motifs as proinflammatory factors render autochthonous tumors permissive for infiltration and destruction

In a transgenic mouse model expressing SV40 T Ag (Tag) as a de novo tumor Ag, immune surveillance fails and islet cell carcinomas grow progressively. To develop an anticancer strategy that would be effective in eradicating solid, autochthonously growing tumors, we evaluated the effectiveness of immunostimulatory oligodeoxynucleotides (ODN) with cytosine-guanine-rich (CpG) motifs (CpG-ODN). In a classical vaccination protocol, Tag was administered with CpG-ODN as adjuvant. The antitumor vaccination, however, was only effective in a prophylactic setting, despite the successful activation of a Tag-specific CTL response in vivo. Histological examination demonstrated that even primed immune cells failed to infiltrate tumors once a malignant environment was established. To ensure that effector cells were not limiting, highly activated tumor Ag-specific T cells were transferred into tumor-bearing mice. However, this treatment also failed to result in tumor infiltration and rejection. Therefore, we further tested the efficacy of CpG-ODN as a proinflammatory agent in combination with the transfer of preactivated Tag-specific CD4(+) and CD8(+) T cells. Indeed, this combination therapy proved to be highly effective, because CpG-ODN rendered insulinomas permissive for massive infiltration and destruction. The opening of tumor tissue correlated with uptake of CpG-ODN by tissue-resident macrophages and a strong up-regulation of adhesion molecules such as ICAM and VCAM on blood vessel endothelia. These data demonstrate that systemic application of proinflammatory reagents drastically enhances extravasation of effector cells into tumor tissue, an observation that is of general importance for immunotherapy of solid tumors in a clinical setting.     

Plasmid DNA induces increased lymphocyte trafficking: a specific role for CpG motifs

Bacterial DNA, primarily through immunostimulatory cytosine-guanine (CpG) motifs, induces the secretion of cytokines and activates a variety of effector cells. We investigated the possibility that CpG motifs might also modulate immunosurveillance by altering cell trafficking through a regional lymph node. Intradermal injection of plasmid DNA induced rapid and prolonged increases in the number of lymphocytes collected in efferent lymph. This effect on cell trafficking was not dependent on the expression of an encoded reporter gene but varied with plasmid construct and required a circular form. Injection of synthetic oligodeoxyribonucleotides containing CpG motifs did not alter lymphocyte trafficking but CpG-enhanced plasmid induced a dose-dependent increase in cell trafficking. Phenotypic analyses revealed that the increase in cell trafficking involved all lymphocyte subpopulations and represented a mass movement of cells. These observations reveal that bacterial DNA, through immunostimulatory CpG motifs, alters immunosurveillance by increasing cell recruitment to a regional lymph node.