Immunostimulatory Effect Of CpG Oligodeoxynucleotides Research Articles

PSVI-3 The effect of oligodeoxynucleotides on Toll-like receptor pathway genes in goat blood

Abstract The objective of this study was to investigate the effect of two classes of oligodeoxynucleotides (ODNs) on the expression of genes involved in Toll-like receptor signaling in goat blood. Toll-like receptors play a critical role in initiating innate immune responses. Select synthetic oligodeoxynucleotides containing methylated CpG motifs (CpG ODN) have immunologic effects similar to those seen with bacterial DNA. These short synthetic single-stranded DNA molecules are being explored as adjuvants to enhance animal health. Blood samples were collected from the jugular vein of BoerXSpanish goats (n = 3) into tubes containing an anticoagulant. Blood was treated with the 10 µg/ml CpG ODN (2216, 2006) class A and B, or 200µl of PBS which served as control. Cells were incubated at 37°C with 5% CO2 and 85% humidity for 30 minutes. Total RNA was isolated using Trizol and then converted to cDNA using RETROscript kit (Qiagen). The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array was evaluated using real-time PCR. Fold change in gene expression was calculated using the 2−ΔΔCt method. The housekeeping genes GAPDH, ACTB, HPRT1, TBP, and YWHAZ was used to normalize the data. Of 84 genes tested, 74 genes were expressed in the control group. Following treatment with ODN 2006, 50 genes were expressed, 29 were up-regulated while 17 genes were down-regulated. The genes HSPD1, IFNG, MAPK8, and PTGS2 were induced by ODN 2006 treatment. Following treatment with ODN 2216, 52 genes were expressed. Treatment with ODN 2216, upregulated 26 genes and down-regulated the expression of 19 genes. The genes CLEC4E, HSPD1, IFNG, IRF1, MAPK8, PTGS2, and UBE2V1 were induced by ODN 2216. Thus specific patterns of TLR signaling may be involved in response to the immunostimulatory effect of CpG ODN in goats with implicates for their use as adjuvants to enhance animal health.

Nanodelivery systems for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Synthetic oligodeoxynucleotides containing immunostimulatory CpG motif mimic bacterial DNA and are potent activator of innate and adaptive immune responses. Therefore, CpG ODNs have significant potentials as immunotherapeutic agent for treatment of infectious diseases, allergy and cancer. Many clinical trials involving CpG ODNs either used alone or as adjuvant have been initiated. However, delivery of CpG ODNs to target sites still remains a great challenge due to their extreme susceptibility to nuclease degradation in serum and poor cellular uptake. Chemical modification of CpG ODNs backbone can protect them against degradation by nucleases, but have raised concern regarding several severe side effects. Development of efficient CpG ODNs delivery systems to address these issues and enhance their immunostimulatory effect are highly desirable. In recent years, the emergence of nanotechnology has provided unprecedented opportunities to encapsulate CpG ODN into various nanocarriers or synthesize CpG ODNs nanostructures. This review provides an overview of the delivery systems based on nanomaterials and nanostructures newly developed for enhancing the immunostimulatory effect of CpG ODNs, together with a brief discussion on perspectives for future studies in this field.

Polyethyleneimine-functionalized boron nitride nanospheres as an efficient carrier for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Polyethyleneimine-functionalized boron nitride nanospheres as an efficient carrier for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Functionalization of boron nitride nanosphere for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Functionalization of boron nitride nanosphere for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Polyethyleneimine-functionalized boron nitride nanospheres as efficient carriers for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides.

CpG oligodeoxynucleotides (ODNs) stimulate innate and adaptive immune responses. Thus, these molecules are promising therapeutic agents and vaccine adjuvants against various diseases. In this study, we developed a novel CpG ODNs delivery system based on polyethyleneimine (PEI)-functionalized boron nitride nanospheres (BNNS). PEI was coated on the surface of BNNS via electrostatic interactions. The prepared BNNS–PEI complexes had positive zeta potential and exhibited enhanced dispersity and stability in aqueous solution. In vitro cytotoxicity assays revealed that the BNNS–PEI complexes with concentrations up to 100 μg/mL exhibited no obvious cytotoxicity. Furthermore, the positively charged surface of the BNNS–PEI complexes greatly improved the loading capacity and cellular uptake efficiency of CpG ODNs. Class B CpG ODNs loaded on the BNNS–PEI complexes enhanced the production of interleukin-6 and tumor necrosis factor-α from peripheral blood mononuclear cells compared with CpG ODNs directly loaded on BNNS. Contrary to the free CpG ODNs or CpG ODNs directly loaded on BNNS, class B CpG ODNs loaded on the BNNS–PEI complexes induced interferon-α simultaneously. PEI coating may have changed the physical form of class B CpG ODNs on BNNS, which further affected their interaction with Toll-like receptor 9 and induced interferon-α. Therefore, BNNS–PEI complexes can be used to enhance the immunostimulatory effect and therapeutic activity of CpG ODNs and the treatment of diseases requiring interleukin-6, tumor necrosis factor-α, and interferon-α.

The immunostimulatory effect of CpG oligodeoxynucleotides on peripheral blood mononuclear cells of healthy dogs and dogs with atopic dermatitis

Synthetic oligodeoxynucleotides containing cytosine phosphatidyl guanine-rich DNA sequences (CpG ODN) can promote T-helper type 1 (Th1) responses, reduce T-helper type 2 (Th2) responses and/or favour regulatory T cell (Treg) responses in vitro and in vivo in humans and animals, by acting via Toll-like receptor 9 (TLR9). Since CpG ODN can be used as immune-modulators for canine atopic dermatitis (AD), the aim of the current study was to investigate their immunostimulatory potential on peripheral blood mononuclear cells (PBMC) and their subsets, from AD and healthy dogs. Expression of TLR9 and cytokine mRNA in CpG ODN-stimulated and unstimulated cells was assessed by real-time quantitative PCR.Stimulation of PBMC with CpG class C ODN upregulated mRNA expression of interleukin (IL)-6, interferon (IFN)-γ and IL-12p40 in AD dogs (P<0.05). It also stimulated IFN-γ protein secretion by PBMC of atopic and healthy dogs as measured by ELISA. In healthy dogs only, CpG class C ODN stimulated IFN-α mRNA production by CD21+ cells, and IL-10, IL-13 and IFN-γ mRNA production by CD3+ cells. Increased expression of TLR9 mRNA was only observed in CD3+ cells from AD dogs. No significantly increased gene expression was found in the CD11c+ subset upon stimulation, for those genes evaluated. The results indicate that PBMC of healthy and atopic dogs are sensitive to stimulation with CpG ODN class C, with a resulting Th1 cytokine response in AD dogs and a mixed Th1/Th2/Treg cytokine response in healthy dogs. From this study, little evidence was found to support the use of CpG ODN class C for therapeutic purposes in dogs affected with AD.

In vivo effects of oligodeoxynucleotides containing synthetic immunostimulatory motifs in the immune response to swine streptococcic septicemia vaccine in weaned piglets

The diverse immunostimulatory effects of CpG oligodeoxynucleotides (CpG ODN) have been demonstrated extensively in mice and human. Although the immunoadjuvant effects of CpG ODN in pigs were also studied in several reports, until now, little work has been carried out with regard to their effects on the adaptive immune system of newly weaned piglets. In this study, swine streptococcic septicemia killed vaccine (SSSK vaccine) was used as antigen, we assessed the in vivo immunostimulatory effects of different CpG motifs in newly weaned piglets. The proportion of CD4 +, CD8 + T lymphocytes subpopulations and proliferation of peripheral blood mononuclear cells (PBMCs), IFN-γ and IL-6 in serum, and the titre of IgG and IgG2/IgG1 isotype to SSSK vaccine in serum were tested at different time-points. The results suggested that, the CD4 +/CD8 + ratio decreased significantly in weaned piglets inoculated with phosphate buffer saline (PBS) alone, however, it was stable in CpG ODN-coinoculated newly weaned piglets. IFN-γ and IL-6 levels, the titres of specific antibodies IgG, IgG2 and proliferative responses of CpG ODN-coinjected piglets were all significantly higher than those of SSSK vaccine alone or PBS or GpC ODN-coinjected piglets. The porcine-specific ODN-induced responses were stronger in animals injected with human-specific or mouse-specific CpG ODN. These in vivo data demonstrate for the first time that CpG ODN can stimulate adaptive immune system in weaned piglets.

Immune Mechanisms and Therapeutic Potential of CpG Oligodeoxynucleotides

Unmethylated CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides activate immune cells that express Toll-like Receptor 9. Activation through this receptor triggers cellular signaling that leads to production of a proinflammatory and a Th1-type, antigen-specific immune response. The immunostimulatory effects of CpG oligodeoxynucleotides confer protection against infectious disease, allergy and cancer in animal models, and clinical trials have been initiated. However, CpG oligodeoxynucleotides may exacerbate disease in some situations. We will review current concepts in the mechanisms of activating Toll-like Receptor 9 with CpG oligodeoxynucleotides and highlight opportunities for using large animal models to better determine the mechanisms of action.

Current research on the immunostimulatory effects of CpG oligodeoxynucleotides in fish

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within the context of certain flanking bases (CpG motifs) have been shown to induce potent innate and adaptive immune responses. Recognition of CpG motifs by the innate immune system requires engagement of toll-like receptor 9 (TLR-9), a newly identified member of toll-like receptor family, which induces cell signaling and subsequently triggers an immune response. The binding CpG-ODN to its specific membrane receptor and subsequently triggering of CpG-related signaling occurred within acidified endosomes. CpG-ODNs are being harnessed for a variety of therapeutic uses, including as immune adjuvants, for cancer therapy, as anti-allergens, and as immunoprotective agents. However, potential applications for immunostimulatory CpG-ODN in fish are just being explored. This article reviews recent progress in understanding the immunostimulatory effects of CpG DNA in fish.

Strategies for enhancing the immunostimulatory effects of CpG oligodeoxynucleotides

Synthetic oligodeoxynucleotides (ODN) containing CpG sequences are recognized as a “danger” signal by the immune system of mammals. As a consequence, CpG ODN stimulate innate and adaptive immune responses in humans and a variety of animal species. Indeed, the potential of CpG ODN as therapeutic agents and vaccine adjuvants has been demonstrated in animal models of infectious diseases, allergy and cancer and are currently undergoing clinical trials in humans. While CpG ODN are potent activators of the immune system, their biologic activity is often transient, subsequently limiting their therapeutic application. Modifications in the CpG ODN backbone chemistry, various delivery methods including mixing or cross-linking of ODN to other carrier compounds have been shown to significantly enhance the biologic activity of ODN. However, the exact mechanisms that mediate this enhancement of activity are not well understood and may include local cell recruitment and activation, cytokine production, upregulation of receptor expression and increasing the half-life of ODN through creation of a depot. We will review the various approaches that have been used in enhancing the immunostimulatory effects of CpG ODN in vivo and also discuss the possible mechanisms that may be involved in this enhancement.

NADPH-oxidase may contribute to IL-12 production in macrophages stimulated with CpG phosphorothioate oligodeoxynucleotides.

CpG oligodeoxynucleotides (CpG-ODN) could be expectes to act as an adjuvant for humoral and cellular immunities. The aim of this study was to clarify the mechanism of macrophage activation induced by CpG-ODN. Macrophage activation by CpG-ODN was evaluated by measurement of IL-12 secretion. IL-12 secretion by Cp-ODN was dose-dependent and saturation was observed at 1 microM. A reactive oxygen species (ROS) scavenger, N-acetylcysteine, inhibited IL-12 secretion, suggesting that the intracellular redox state is important for the activation of macrophages by CpG-ODN. IL-12 secretion was also inhibited by NADPH-oxidase inhibitors, suggesting that ROS generated by NADPH-oxidase is involved in the immunostimulatory effect of CpG-ODN. Nuclear factor (NF)-kappaB is one of the transcriptional factors involved in IL-12 gene expression, and indeed CpG-ODN activated NF-kappaB. However, NADPH-oxidase inhibitors did not affect the activation. These results indicated that NADPH-oxidase plays a crucial role in macrophage activation by CpG-ODN and NF-kappaB does not act as a predominant transcription factor for IL-12 secretion.