A CpG oligodeoxynucleotide inducing anti-coxsackie B3 virus activity in human peripheral blood mononuclear cells
Zhongyi CongMin WanXiuli WuLi WangXiao HuFeng‐Lei YangMusheng BaoXuesong ZhangJianzhu ChenLiying WangYongli Yu
11
Citation
52
Reference
10
Related Paper
Citation Trend
Abstract:
Coxsackie B3 virus (CVB3) is the most significant pathogen causing myocarditis in humans, and antiviral therapy would be most effective in the early stages of the disease. Here we provide evidence that BW001, a C-type CpG oligodeoxynucleotide, induces anti-CVB3 activity in human peripheral blood mononuclear cells (PBMCs). In parallel, we have demonstrated that BW001 induces human PBMCs to express mRNAs of multiple types of interferon (IFN), including IFN-alpha, IFN-beta, IFN-omega and IFN-gamma, and to express mRNAs of at least 11 subtypes of IFN-alpha. The induced IFNs may contribute to the anti-CVB3 activity. The results suggest that BW001 could be developed into a medication with the potential to treat CVB3 infectious diseases by inducing natural mixed IFNs.Keywords:
CpG site
CpG Oligodeoxynucleotide
Objective While bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides with specific flanking sequences (CpG motif) are well described activators of murine immune cells, CpG motifs for activating human immune cells remain unclear. This study is to delineate CpG motifs and CpG ODN for activating human immune cells. Methods A panel of CpG ODN containing two copies of CpG motifs was screened for their capacity to stimulate human B lymphocytes to secret IgM. Then CpG ODN containing human CpG motifs was designed and tested to identify the sequence of high immunostimulatory activity for human immune cells. Results The study revealed that 5′ GTCGTT 3′ and 5′ GTCGTC 3′ were two potent CpG motifs for human immune cells, and other four CpG motifs, 5′ GACGTT 3′, 5′ GACGTC 3′, 5′ GGCGTT 3′ and 5′ GGCGTC 3′, had moderate activities. The second nucleotide in optimal human CpG motifs was thymidine, while in less active human CpG motifs or optimal murine CpG motifs, it was purines. Based on this finding, many CpG ODN were designed and identified as high immunostimulatory sequences for human B lymphocytes. Conclusion Some CpG motifs and CpG ODN are potential activators of human immune cells.
CpG Oligodeoxynucleotide
CpG site
Sequence motif
Cite
Citations (1)
CpG Oligodeoxynucleotide
CpG site
Structure–activity relationship
Immunostimulant
Cite
Citations (56)
To design and identify the CpG oligonucleotide (ODN) with immunostimulatory activities in porcine peripheral blood mononuclear cells, we had screened 38 CpG ODN The stimulatory index was determined by 3 H TdR incorporation assay and the levels of IFN γ and IL 2 in the culture supernatants were detected with the appropriate ELISA testing kids(swIFN γ kid and swIL 2 kid respectively) The results showed that CpG ODN could stimulate the proliferation of porcine PBMC and the secretion of IFN γ, with little secretion of IL 2 Among the ODN, ODN 2006 that contained 3 GTCGTT motifs could trigger the proliferation of porcine PBMC most strongly with a stimulatory index of 6 2,but only induce a low level of IFN γ On the contrary, ODN D19 with a mixed backbone of phosphodiester and phosphorothioate and a poly G tail, showed moderately immunostimulatory activities on porcine PBMC,but it could induce PBMC to release large amount of IFN γ It concludes that ODN 2006 as well as ODN D19 have different immunostimulatory activities on porcine PBMC
CpG Oligodeoxynucleotide
Phosphodiester bond
CpG site
Cite
Citations (0)
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.
CpG Oligodeoxynucleotide
CpG site
Cite
Citations (22)
Synthetic oligodeoxynucleotides (ODNs) bearing CpG dinucleotides can mimic the immunostimulatory effects of bacterial DNA in vertebrates. Besides the known CpG motifs, no other sequence motif has been shown to have independent immunostimulatory effects. Several past investigators have demonstrated that the nucleotide content or the phosphorothioate (PS) backbone may have effects independently of the sequence. However, the effect of both nucleotide content and PS backbone to stimulate human leukocytes is not well understood. We investigated the immunostimulatory activity of 34 PS-ODNs with different nucleotide contents, lengths, and methylation status on human leukocytes. The thymidine content showed strong CpG-independent contribution to immunostimulation. In contrast, ODNs rich in other nucleotides (guanosine, cytosine, or adenosine) induced no or much lower levels of immunostimulation. The observed effects were highly dependent on the PS backbone chemistry. In addition to the base content and the backbone chemistry, the length of the PS-ODN was directly related to the magnitude of its stimulatory effects, especially on B cells. In addition, methylation of CpG dinucleotides did not always cause an abrogation of the immunostimulation. Immunostimulatory effects could be observed with methylated CpG ODNs, specifically as the ODN length was increased from 18 to 24 or more nucleotides (nt). In contrast, PS-ODNs with inverted CpG dinucleotides showed some but only weak immunostimulation. Our results demonstrate that non-CpG ODNs rich in thymidine or ODNs with methylated CpG motifs have length-dependent immunostimulatory effects. Such ODNs can induce effects similar to those seen with CpG ODNs but are much less efficient in stimulating human immune cells.
CpG Oligodeoxynucleotide
CpG site
Cytosine
Thymidine
Cite
Citations (63)
Abstract: CpG oligodeoxynucleotides (CpG ODNs) bind to toll‐like receptor‐9 (TLR‐9) and activate immune cells with antigen‐presenting activity, including B cells and dendritic cells. Here we show that treatment of the latently human immunodeficiency virus (HIV)‐infected T cell line ACH‐2 with the CpG ODNs 2006 or 2040 triggers activation of viral gene expression, demonstrating that CpG‐signaling activity can also be found in T cells. The CpG ODNs g12AAC and g12GTC had no effect on virus reactivation. In contrast to the stimulating effects on viral gene expression in latently infected cells, CpG ODNs potently suppressed HIV replication in productively infected MT4 T cells or PBLs. Inhibition of virus replication was not related to the CpG motif but similarly occurred with non‐CpG phosphorothioate (PTO)‐ODNs. Thus, virus inhibition was likely caused by the PTO backbone of the CpG ODNs, probably by interfering with events prior to integration of the viral cDNA into the host genome. The ability of CpG PTO‐ODNs to trigger reactivation of latent HIV in combination with their antiviral activity on productive infection makes this substance class an interesting candidate for further test to asses their potential as supplements in HIV therapy.
CpG Oligodeoxynucleotide
CpG site
TLR9
Toll-Like Receptor 9
Cite
Citations (16)
We evaluated the innate immune response to various synthetic CpG-containing oligodeoxynucleotides (CpG ODNs) by measuring nitric oxide production in the peripheral blood monocytes from turkey poults. The results indicate that the presence of the CpG dinucleotide in ODNs was a prerequisite for activation of turkey monocytes and induction of nitric oxide (NO) synthesis. CpG motifs and sequence structure of the ODNs were also found to influence stimulatory activity greatly. The most potent CpG ODN to induce NO synthesis in turkey monocytes was human-specific CpG ODN M362, followed by CpG ODN 2006 (human), CpG ODN#17 (chicken) and CpG ODN 1826 (mouse). The optimal CpG motif for NO induction was GTCGTT. Phosphorothioate modification of CpG ODNs also significantly increased stimulatory activity. Compared with chicken monocytes, turkey monocytes appeared to be less sensitive to CpG motif variation, whereas chicken monocytes were found to respond more strictly to human-specific CpG ODNs or ODNs that contain GTCGTT motifs.
CpG Oligodeoxynucleotide
CpG site
Monocyte
Cite
Citations (26)
AIM:To investigate whether alteration of the core sequence of CpG ODN change its cellular interanalization and immunological activity.METHODS:6-FAM labeled CpG ODN was artificial synthesized and the CpG dinucleotide was altered by cytosine methylation(mCpG ODN)and/or CG sequence reversal(GpC ODN).RAW264.7 cells were cultured in vitro.After stimulated with different 6-FAM labeled ODN,the accumulation of ODN in the cells were measured by flow cytometry.RAW264.7 cells grown on glass coverslips were pretreated with each ODN and subsequently the localization of ODN in cells were observed by confocal scanning microscopy.Meanwhile,the level of TNF-α in supernatant was tested by ELISA.RESULTS:The alteration of the core sequence of CpG ODN didn't influence its accumulation in RAW264.7 cells.In vitro,6-FAM labeled ODN was internalized into RAW264.7 cells and a few green fluorescent dots were widely distributed over cytoplasm.Large amount of TNF-α were released from RAW264.7 cells stimulated by CpG ODN.However,after methylation of cytosine in CpG dinucleotide,the TNF-α release was significantly decreased(P0.01).At the same time,CG sequence reversal of CpG dinucleotide completely abolished the stimulated activity of CpG ODN.CONCLUSION:It's essential for the biological function of CpG ODN with certain CG sequence.The methylation of cytosine in CpG dinucleotide doesn't influence the internalization and localization of CpG ODN in RAW264.7 cells,but partially inhibit its immunological activity.
CpG Oligodeoxynucleotide
CpG site
Internalization
Cite
Citations (0)
CpG site
CpG Oligodeoxynucleotide
Immunostimulant
Cite
Citations (39)
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.
CpG site
CpG Oligodeoxynucleotide
Sequence motif
Cite
Citations (26)