• Biotechnology and Bioprocess Engineering:BBE
• /
• v.10 no.1
• /
• pp.29-33
• /
• 2005

A DNA vaccine methodology using eukaryote expression vectors to produce immunizing proteins in the vaccinated hosts is a novel approach to the development of vaccine and immuno-therapeutics, and it has achieved considerable success over several infectious diseases and various cancers. To further enhance its efficiency, attempts were made to develop novel plasmid vectors containing multiple immunostimulatory CpG motifs, for rapid and strong immune response. First, a 2.9 kb compact plasmid vector (pVAC), containing CMV promoter, polycloning site, BGH poly(A) terminator, ampicillin resistance gene and pBR322 origin was constructed. A pVAC-hEPO was also constructed, which contained a human erythropoietin gene, for evaluating the transfection efficiency of naked plasmid DNA both in vitro and in vivo. To examine the adjuvant effect of multi-CpG motifs on naked plasmid DNA, 22 and 44 enriched and unmethylated CpG motifs were introduced into pVAC to generate pVAC-ISS1 and pVAC-ISS2, respectively. $100{\mu}g$ of pSecTagB, pVAC, pVAC-ISS1 or pVAC-ISS2 were each injected intramuscularly into the tibilias anterior muscle of Balb/c mice. The level of interleukin-6 induced in the mice injected with pVAC-ISS1 and pVAC-ISS2 were significantly elevated after 12 hours, which were almost 2 and 2.5 times higher than that in the mice injected with pSecTagB, respectively. These results suggest that DNA vaccine plasmids with enriched CpG motifs can induce rapid secretion of interleukin-6 by lymphocytes. In conclusion, these vectors can contribute to the development of adjuvant-free DNA vaccinations against infectious diseases and various cancers.

• Fisheries and Aquatic Sciences
• /
• v.6 no.3
• /
• pp.125-129
• /
• 2003

Effects of synthetic oligodeoxynucleotides (ODNs) containing cytidine-phosphate-guanosine(CpG) motif(s) on nonspecific immune responses of olive flounder (Paralichthys olivaceus) and on protection against lethal infection with Edwardsiella tarda were investigated. Respiratory bunt activities of the head kidney phagocytes in the fish injected either 0.25 or 0.5 ${\mu}g/fish$of ODNs containing CpG motifs (ODN 1826 and ODN 1670) were significantly higher than those injected with an ODN containing a guanosine-phosphate-cytidine (GpC) motif (ODN 1720) or with hanks balanced salt solution (HBSS, control) at 3, 5 and 7 days after injection. The serum lysozyme activities of fish injected with 0.25${\mu}g$ of ODN 1826 were significantly higher than those injected with ODN 1720 or HBSS at 1 and 7 days after injection. At 7 days after injection, the group of fish injected with CpG ODNs showed higher serum lysozyme activities than fish injected with ODN 1720 or control. The group of fish injected 0.25 or 0.5${\mu}g$ of CpG ODNs showed higher survival rates than those treated with GpC ODN and the control group after challenge with Edwardsiella tarda. The present study proved the ability of synthetic CpG ODN to increase nonspecific immune responses and disease resistance in olive flounder.

• Clinical and Experimental Pediatrics
• /
• v.48 no.3
• /
• pp.251-259
• /
• 2005

Atopy is a highly prevalent and serious health problem. The prevalence and severity of asthma and allergic diseases have increased over recent decades, particularly in industrialized nations. Early life infections may protect against the development of atopy and allergic diseases like asthma. The inverse relationship between the incidence of atopy and childhood infections has led to the 'hygiene hypothesis', which suggests that diminished exposure to childhood infections in modern society has led to decreased Th1-type responses. Th1 and Th2 responses are counter-regulatory. Reduced Th1 may lead to enhanced Th2-type inflammation, which is important in promoting asthma and allergic disease via up-regulation of IL-4, IL-5, and IL-13. It is now widely accepted that altered regulation of Th2 responses(and possibly the balance between Th1 and Th2 responses) is an important factor in the development of atopy. CpG DNA represent a novel class of drugs with substantial immunomodulatory properties. CpG DNA contain unmethylated motifs centered on the CpG dinucleotides, like bacterial DNA. These CpG DNA promote Th1 and regulatory type immune responses and suppress Th2 responses. In murine studies, CpG DNA are effective in prevention and treatment of asthma and allergic diseases. CpG DNA are just beginning to be tested in human asthma. While its precise mechanisms continue to be fully studied, CpG DNA offers considerable promise as a novel treatment for atopic inflammation. It may prove to be an important disease modifying therapy, or even curative therapeutic agent for asthma and allergic diseases.

• Food Science and Biotechnology
• /
• v.17 no.6
• /
• pp.1285-1288
• /
• 2008

The purpose of this study was to characterize and investigate the immune activity of CpG oligodeoxynucleotides (ODNs) from Bifidobacterium longum. Bacterial CpG motifs have attracted considerable interests because of their immunomodulatory activities. Genomic DNA from B. longum was prepared and amplified for 4 different 180-188-mer double-stranded ODNs (BLODN1-BLODN4). When immune cells (RAW 264.7 murine macrophages and JAWS II dendritic cells) with these ODNs were treated, BLODN4 induced the highest immune activity. To assess the effectiveness of the CpG sequences within BLODN4, single-stranded 40-mer ODNs containing CpG sequences (sBLODN4-1, sBLODN4-2) were synthesized. sBLODN4-1 induced higher level of cytokines such as interleukin (IL)-12p40 and tumor necrosis factor (TNF)-$\alpha$ by macrophage and IL-6 and TNF-$\alpha$ by dendritic cells than did sBLODN4-2. The results suggest that CpG ODNs-enriched components of B. longum might be useful as an immunomodulatory functional food ingredient.

• BMB Reports
• /
• v.40 no.4
• /
• pp.486-493
• /
• 2007

Atopic dermatitis (AD) is a chronic inflammatory skin disease and the pathogenesis of AD is associated with the release of various cytokines/chemokines due to activated $Th_2$ immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide in the context of particular base sequence (CpG motifs) are known to have the immunostimulatory activities in mice and to convert from Th2 to Th1 immune responses in AD. We aimed to investigate that CpG ODN, especially phosphodiester form, can stimulate the protective immunity in NC/Nga mice with AD. We isolated BMDCs from NC/Nga mice and then, cultured with GM-CSF and IL-4 for 6 days, and treated for 2 days by either phosphorothioate ODN or phosphodiester ODN. CpG ODN-treated DCs resulted in more production of IL-12. When CpG ODN-treated DCs were intravenously injected into the NC/Nga mice, the NC/Nga mice with CpG ODN-treated DCs showed significant improvement of AD symptoms and decrease of IgE level. Histopathologically, the NC/Nga mice skin with CpG ODN-treated DCs showed the decreased IL-4 and TARC expression comparing with non-injected mice. These results may suggest that phosphodiester CpG ODN-treated DCs might function as a potent adjuvant for AD in a mouse model.

• Journal of fish pathology
• /
• v.16 no.2
• /
• pp.103-110
• /
• 2003

• Molecules and Cells
• /
• v.39 no.12
• /
• pp.888-897
• /
• 2016

Stable expression of Foxp3 is ensured by demethylation of CpG motifs in the Foxp3 intronic element, the conserved non-coding sequence 2 (CNS2), which persists throughout the lifespan of regulatory T cells (Tregs). However, little is known about the mechanisms on how CNS2 demethylation is sustained. In this study, we found that Ten-Eleven-Translocation (Tet) DNA dioxygenase protects the CpG motifs of CNS2 from re-methylation by DNA methyltransferases (Dnmts) and prevents Tregs from losing Foxp3 expression under inflammatory conditions. Upon stimulation of Tregs by interleukin-6 (IL6), Dnmt1 was recruited to CNS2 and induced methylation, which was inhibited by Tet2 recruited by IL2. Tet2 prevented CNS2 re-methylation by not only the occupancy of the CNS2 locus but also by its enzymatic activity. These results show that the CNS2 methylation status is dynamically regulated by a balance between Tets and Dnmts which influences the expression of Foxp3 in Tregs.

• BMB Reports
• /
• v.43 no.4
• /
• pp.250-256
• /
• 2010

Natural phosphodiester bond CpG-DNA that contains immunomodulatory CpG motifs (PO-DNA) upregulates the expression of proinflammatory cytokines and induces an Ag-driven Th1 response in a CG sequence-dependent manner in mice. In humans, only phosphorothioate backbone-modified CpG-DNA (PS-DNA) and not PO-DNA has immunomodulatory activity. In this study, we found that liposome-encapsulated PO-DNA upregulated the expression of human $\beta$-defensin-2 (hBD-2) and major histocompatibility class II molecules (HLA-DRA) in a CG sequence-dependent and liposome- dependent manner in human B cells. Of the three different liposomes, DOTAP has the unique ability to enhance the immunomodulatory activity of PO-DNA. In contrast, HLA-DRA and hBD-2 promoter activation can be induced by liposome-encapsulated PS-DNA in a CG sequence-independent manner, depending on the CpG-DNA species. Our observations demonstrate that, when encapsulated with a proper liposome in the immune system, natural PO-DNA has the potential to be a useful therapy for the regulation of the innate immune response.

• Clinical and Experimental Pediatrics
• /
• v.45 no.7
• /
• pp.875-883
• /
• 2002

Purpose : Airways eosinophilia and increased IgE, characteristic features of asthma, result from a predominant Th2 response. In this study, we investigated the effect of CpG oligodeoxynucleotides (ODNs) on the inhibition of airways eosinophilia in mice with established airway inflammation. We also investigated the immunological mechanisms involved. Methods : Groups of BALB/c mice were sensitized intradermally with ovalbumin(OVA). At week 10, airway inflammation was induced by intranasal challenge of the mice with OVA. At week 14, the mice were challenged intranasally again with OVA in the presence and without the presence of CpG ODNs. Mice with saline administration served as negative controls. Bronchoalveolar lavage fluids(BALF) were obtained and eosinophils were counted. Th1 and Th2 cytokines in the spleen cell cultures were measured by ELISA. Serum OVA-specific IgE and IgG2a antibodies were also measured by ELISA. Results : BALF eosinophils were significantly inhibited in the CpG ODNs-treated mice(P<0.01). IgE and IgG2a levels increased significantly in both CpG ODNs-treated and untreated groups as compared to the negative control group; there was, however, no significant difference between the two groups four days after intranasal administration of CpG ODNs. Cytokine analysis revealed decreased production of IL-4, IL-5, and IL-13 and increased production of IL-12 in the CpG ODNs-treated group as compared to the untreated group. Interestingly, $IFN-{\gamma}$ levels were not upregulated in the CpG ODNs-treated group. Conclusion : CpG ODNs vaccination is a potentially useful approach for reversing airways eosinophilia in mice with established airways inflammation.

• BMB Reports
• /
• v.39 no.6
• /
• pp.788-793
• /
• 2006

Bacterial DNA containing immunostimulatory CpG motifs can stimulate antigen-presenting cells to express co-stimulatory molecules and to produce various cytokines in vivo and in vitro. In this study, we fragmented macromolecular E.coli genomic DNA with DNase I, and analyzed the ability of the resulting DNA fragments to induce the NF-${\kappa}B$ activation and humoral immune response. Furthermore, using computational analysis and luciferase assay for synthetic ODNs based on the sequence of the immunostimulatory DNA fragments (DF-ODNs), an active component of DF-ODNs sequences was investigated. Experimental results demonstrated that DF-ODN is optimal for the NF-${\kappa}B$-responsive promoter activation in the mouse macrophage cell line and the humoral immune response in vivo. In agreement with the activity of the DF-ODNs processed by DNase I, a synthetic ODN based on the DF-ODN sequences is potent at inducing IL-12 mRNA expression in primary dendritic cells. These results suggest that the discovery and characterization of a highly active natural CpG-ODN may be achieved by the analyses of bacterial DNA fragments generated by a nuclease activity.