Classes Of Oligodeoxynucleotides Research Articles

Discrepant antitumor efficacies of three CpG oligodeoxynucleotide classes in monotherapy and co-therapy with PD-1 blockade

Unmethylated CpG oligodeoxynucleotides (ODNs) activate plasmacytoid dendritic cells (pDCs) and B cells to induce humoral and cellular immunity, and are under development for the treatment of multiple cancers. However, the specific differences in antitumor effects among the three CpG ODN classes when administered as a monotherapy or in co-therapy with the anti-PD-1 antibody are unclear. We compared the immunostimulatory effects in vitro and antitumor effects in a CT26 subcutaneous mouse tumor model among the three CpG ODN classes. We found that CpG-A slightly suppressed tumor growth but possessed no synergistic antitumor effects with the anti-PD-1 antibody. CpG-B at low doses significantly inhibited tumor growth and possessed synergistic antitumor effects with the anti-PD-1 antibody. A high dose of CpG-C was required to achieve antitumor effects comparable to those of CpG-B, which was consistent with the immunostimulatory effects in B-cell proliferation and TLR9-NF-κB activation. Importantly, CpG-C in combination with anti-PD-1 antibody inhibited tumor growth more quickly and effectively than CpG-B because CpG-B significantly upregulated PD-L1 expression on multiple host immune cells to promote tumor immune escape. Moreover, co-therapy increased the infiltration of effector memory T cells. In summary, CpG-B and CpG-C with different optimal concentrations possessed strong antitumor effects, while CpG-C was more rapid and effective for co-therapy with the anti-PD-1 antibody.

Synthetic Oligodeoxynucleotides in the Regulation of Leukotriene Synthesis in Human Neutrophils

Polymorphonuclear leukocytes (PMNLs, neutrophils) play a central role in the defense against microbial infections. In the immune response to bacterial and fungal infections neutrophils eliminate pathogens through phagocytosis. During phagocytosis neutrophils produce physiologically active compounds called leukotrienes. Neutrophil production of leukoriene B4 is responsible for a second wave of neutrophil recruitment during inflammation. In the activation of neutrophils, not only the bacteria themselves play a role, but also pathogen associated molecular patterns, in particular, bacterial DNA and its fragments. Neutrophil priming with bacterial products makes neutrophils more responsive to activating stimuli. Synthetic CpG‐oligodeoxynucleotides (CpG‐ODNs) mimic microbial DNA and are now widely used to study DNA‐mediated cellular events. In our study we compared the effects of synthetic oligodeoxynucleotides (ODNs) of different structure on leukotriene synthesis in neutrophils during Salmonella bacteria phagocytosis. We treated PMNLs with opsonized S. typhimurium at a ratio of bacteria:PMNLs in the range 20–25:1. Leukotrienes and other 5‐lipoxygenase products were detected after priming neutrophils by 30 min incubation with 1 μM ODN and further stimulation for 15 min with opsonized Salmonella bacteria. The neutrophil exposure to A‐class CpG‐ODN, with phosphodiester inner core, inhibited leukotriene synthesis in neutrophil interaction with bacteria Salmonella typhimurium. Sequences, containing the human telomeric (TTAGGG)n repeats, on the contrary, activated leukotriene synthesis. Effects of A‐class CpG‐ODNs were sensitive to inhibitors of endosomal acidification and probably induced intracellular signaling through binding to toll like receptor TLR9. Synthetic DNA fragments containing stimulating CpG motifs initiate TLR9 signals, which lead to activation of the transcription factor NF‐kB. In our assays NF‐kB inhibitor BAY11‐7082 abolished the effects of A‐class CpG ODNs and increased the effects of telomeric ODNs on leukotriene synthesis. The results of our study revealed that the signal to inhibition of leukotriene synthesis by A‐class CpG‐ODNs and to activation of leukotriene synthesis by telomeric ODNs may be due to interaction of both classes of ODNs with TLR9 on neutrophils.Ethics statement. The authors prepared neutrophils from the blood of healthy volunteers. Blood was collected via venous puncture, as approved by the Ministry of Public Health Service of the Russian Federation. Experimental and the subject consent procedures were approved by the Ethics Committee of the A. N. Belozersky Institute of Physico‐Chemical Biology, Moscow State University. Fully informed consent was obtained, and all investigations were conducted according to the principles laid down in the Declaration of Helsinki.Support or Funding InformationThis study was funded by a grant from the Russian Foundation for Basic Research (17‐04‐01491)

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.

DNA–polymer conjugates for immune stimulation through Toll-like receptor 9 mediated pathways

Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide motifs are agonists of Toll-like receptor 9 and are currently being investigated for use as vaccine adjuvants through the promotion of type I immunity. Several classes of ODN have been developed which differ in their propensity to aggregate, which in turn alters cytokine profiles and cellular subsets activated. Although aggregation state is correlated with the change in cytokine response, it is unknown if this results from a change in the number of ODNs available for binding and/or the possible engagement of multiple TLR9 molecules. Here, we examined the role of ligand valency on the activation of TLR9 through the synthesis of ODN–poly(acrylic acid) (PAA) conjugates. The compositions and size of the conjugates were characterized by UV-vis spectroscopy, proton nuclear magnetic resonance, gel permeation chromatography and dynamic light scattering. Enzyme-linked immunosorbent assays of cytokine secretion by murine-like macrophages indicate that these ODN–PAA polymer conjugates show enhanced immunostimulation at 100-fold lower concentrations than those required for ODN alone, for both TNF-α and IL-6 release, and are more potent than any other previously reported multivalent ODN constructs. Increasing valency was shown to significantly enhance cytokine expression, particularly for IL-6. Knockdown by siRNA demonstrates that these polymer conjugates are specific to TLR9. Our results define valency as a critical design parameter and polymer conjugation as an advantageous strategy for producing ODN immunomodulatory agents.

A Novel Class of Immune-Stimulatory CpG Oligodeoxynucleotides Unifies High Potency in Type I Interferon Induction with Preferred Structural Properties

Unmethylated deoxycytidyl-deoxyguanosin dinucleotide (CpG)-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists for Toll-like receptor 9. We here describe a new class of CpG ODNs, the so-called P-Class, which combines preferred properties of known CpG ODN classes. This P-Class contains two palindromic sequences, enabling it to form concatamers, multimeric units, where each molecule is bound via Watson-Crick basepairing to a second and a third palindrome. The type I interferon-inducing potency and efficacy of the double-palindromic P-Class ODN is substantially higher than that of previously described C-Class ODNs, and they stimulate superior cytokine production upon in vivo application. The multimeric structures of the P-Class can be resolved to monomers and dimers by formulation in low-salt buffer, retaining the strong and potent immune effects. Taken together, we have discovered a novel class of CpG ODNs, the P-Class, with promising superior activity for disease application.

Differential Effects of P-Class Versus Other CpG Oligodeoxynucleotide Classes on the Impaired Innate Immunity of Plasmacytoid Dendritic Cells in HIV Type 1 Infection

Abstract Human plasmacytoid dendritic cells (PDC) are the major producers of type I interferons (IFN) after stimulation with CpG oligodeoxynucleotides (ODN). HIV-1-infected patients show a deficit in PDC numbers and function with progression of disease. CpG ODN appear to be attractive therapeutics to support the impaired innate immunity in HIV-1 infection. PDC counts, phenotype, and function were analyzed in 23 HIV-infected untreated individuals and 16 controls. Markers for migration (CCR7), activation (CD80), maturation (CD83), and endocytosis (BDCA2) were evaluated at baseline and 20 h after in vitro stimulation with class A, B, C, and P ODN. PDC counts and the expression of BDCA2 on these cells were significantly lower in HIV-1-infected subjects compared to controls (both p < 0.001). After stimulation with CpG ODN, CD80 and CD83 were upregulated to a similar extent in patients and controls, whereas CCR7 was upregulated more efficiently by CpG-P and CpG-C than CpG-A in HIV-1-infected individuals compared to controls. The IFN-alpha induction significantly differed for the CpG ODN classes (A > P > C > B) in patients and controls (p < 0.05). Functional PDC deficits in IFN-alpha and TNF-alpha induction were particularly evident in subjects with less than 500 CD4(+) cells/mul. CpG-P ODNs not only induced remarkable IFN-alpha production in patient PBMCs, but also significantly upregulated the antibacterial and antiviral CXC chemokine IP-10. In conclusion, PDC counts, phenotype, and function are significantly impaired in HIV-1-infected subjects. Optimized P-class ODN may be effective in reversing this innate immune defect, which should be further evaluated in vivo.

Attenuated cytokine responses in porcine lymph node cells stimulated with CpG DNA are associated with low frequency of IFNα-producing cells and TLR9 mRNA expression

The immune stimulatory effects of synthetic CpG DNA, on porcine peripheral blood mononuclear cells (PBMC) have been reported, but little is known about CpG-induced responses in other lymphoid tissues of pigs. We investigated innate immune responses induced by CpG DNA in cells from blood, lymph nodes (LN) and spleens of pigs. Porcine PBMC and lymph node cells (LNC) were stimulated in vitro with three classes (A-, B- and C-class) of CpG oligodeoxynucleotides (ODNs), and a non-CpG control ODN. All three classes of CpG ODNs induced significant production of IFNα, TNFα, IL-1, IL-6 and IL-12 in PBMC. In contrast, in LNC, only IL-12 was stimulated by all three classes of CpG ODNs, while IFNα, and IL-6 were induced by A- and C-class ODNs. No TNFα was induced in LNC by any of the ODNs. Significant lymphocyte proliferation was induced in PBMC by all three classes of CpG ODNs and non-CpG control. However, in LNC, B- and C-class ODNs induced significant proliferation, while no proliferation was seen with A-class and non-CpG control ODN. All three classes of ODNs induced NK-like cytotoxicity in PBMC and spleen cells, but were less effective in inducing NK cytotoxicity in LNC. We then investigated the reasons for the relatively poor CpG-induced responses in LNC. Our investigations revealed that LNC had a lower frequency of IFNα-secreting cells and expressed low levels of TLR9 mRNA compared to PBMC. We conclude that the lower number of IFNα-secreting cells and receptor expression may contribute to the attenuated responses in LNC following stimulation with CpG ODN.

Oligonucleotides Suppress IL-8 in Skin Keratinocytes In Vitro and Offer Anti-Inflammatory Properties In Vivo

DNA codes for genetic information. Furthermore, recent findings suggest that DNA offers additional function, particularly in the recognition of microorganisms. In this study, we investigated two classes of oligodeoxynucleotides (ODN) in skin keratinocytes; namely, an ODN comprising two cytidine-phosphate-guanosine (CpG) motifs (CpG-1-phosphorothioate (PTO)) and a poly-cytidine (Non-CpG-5-PTO) as control. Both fluorescence-tagged ODN were rapidly taken up by cells and accumulated already after 5 minutes in perinuclear compartments. In order to test whether ODN convey immunological effects in keratinocytes, secretion of IL-8 was measured. Interestingly, both CpG-1-PTO and Non-CpG-5-PTO suppressed basal and tumor necrosis factor alpha-induced IL-8 levels measured in cell culture supernatants. Experiments using deletion mutant revealed a critical length of approximately 16 nucleotides conveying IL-8 suppression. Studies regarding the ODN backbone offered that PTO bondings are critical for significant IL-8 suppression. In order to substantiate the anti-inflammatory response, a contact hypersensitivity mouse model was utilized. Topical application of Non-CpG-5-PTO-containing ointments reduced ear thickness in sensitized mice. Taken together, these findings suggest an anti-inflammatory effect of ODN in epithelial cells in vitro and in vivo, indicating that DNA molecules offer distinct biological activities restricted to the physiological compartment applied. This effect seems to be independent from Toll-like receptor 9.

CpG-B ODNs potently induce low levels of IFN-αβ and induce IFN-αβ-dependent MHC-I cross-presentation in DCs as effectively as CpG-A and CpG-C ODNs

Deoxycytidyl-deoxyguanosine [(CpG)3] oligodeoxynucleotides (ODNs) signal through TLR9 to induce type-I IFN (IFN-alphabeta) and IFN-alphabeta-dependent MHC-I cross-presentation of exogenous antigens by dendritic cells (DCs). A puzzle was presented by our observation that three ODN classes, CpG-A, CpG-B, and CpG-C, had similar efficacy for induction of IFN-alphabeta-dependent MHC-I antigen cross-presentation by myeloid DCs despite greatly differing for induction of IFN-alphabeta (CpG-A>CpG-C>>CpG-B). All ODN classes similarly enhanced plasmacytoid DC (pDC) presentation of exogenous MHC-I-restricted peptide, although pDCs did not cross-process protein antigen. MHC-I and the transporter for antigen presentation were induced by all ODN classes or IFN-alpha. CpG-B ODNs were slightly more potent than CpG-A or CpG-C ODNs for induction of low levels of IFN-alphabeta but less efficacious at high concentrations than CpG-A or CpG-C ODNs. Low levels of IFN-alphabeta induced by CpG-B ODNs sufficed for full induction of MHC-I cross-presentation. Thus, CpG-B ODNs are slightly more potent but less efficacious than CpG-A and CpG-C ODNs for induction of IFN-alphabeta. High sensitivity to IFN-alphabeta allows CpG-B ODNs to be equally efficacious for induction of MHC-I cross-presentation. CpG-B ODNs may be effective for inducing therapeutic responses that require low levels of IFN-alphabeta and may avoid unnecessarily high induction of IFN-alphabeta.

CpG‐B ODNs induce low levels of IFNαβ and induce IFNαβ‐dependent MHC‐I cross‐presentation in DCs as effectively as CpG‐A, CpG‐C ODNs, and TLR agonists

CpG oligodeoxynucleotides (ODNs) and single stranded RNAs (ssRNA) signal through TLR9 and TLR7, respectively, to induce type-I IFN (IFNαβ) and IFNαβ-dependent MHC-I cross-presentation of exogenous antigens (Ags) by dendritic cells (DCs). A puzzle was presented by our observation that three ODN classes, CpG-A, CpG-B and CpG-C, and TLR7 agonists ssRNA40 and R837, had similar efficacy for induction of IFNαβ-dependent-MHC-I Ag cross-presentation by myeloid DCs (mDCs) despite greatly differing for induction of IFNαβ (CpG-A > CpG-C ~ TLR7 agonists >> CpG-B). All ODN classes similarly enhanced plasmacytoid DC (pDC) presentation of exogenous MHC-I-restricted peptide, although pDCs did not cross-process protein Ag. MHC-I and the transporter for antigen presentation were induced by all ODN classes or IFNα. CpG-B ODNs were slightly more potent than CpG-A or CpG-C ODNs for induction of low levels of IFNαβ, but less efficacious at high concentrations than CpG-A or CpG-C ODNs. Low levels of IFNαβ induced by CpG-B ODNs sufficed for full induction of MHC-I cross-presentation. Thus, CpG-B ODNs are slightly more potent but less efficacious than CpG-A, CpG-C ODNs or TLR7 agonists for induction of IFNαβ. High sensitivity to IFNαβ allows CpG-B ODNs to be equally efficacious for induction of MHC-I cross-presentation. CpG-B ODNs may be effective for inducing therapeutic responses that require low levels of IFNαβ and avoid unnecessarily high induction of IFNαβ.

C-Class CpG ODN: sequence requirements and characterization of immunostimulatory activities on mRNA level

Synthetic oligodeoxynucleotides (ODN) containing unmethylated deoxycytosine–deoxyguanosine (CpG) motifs are very potent inducers of the innate immune system, mimicking the effects of bacterial DNA. CpG ODN are recognized by Toll-like receptor 9 (TLR9). Three classes of TLR9 agonists have been described: B-Class CpG ODN that induce strong B- and NK-cell activation and A-Class ODN that induce very high levels of IFN- α by plasmacytoid dendritic cells. The recently described C-Class ODN combine most efficiently properties of A- and B-Class ODN in that they induce strong B-cell activation comparable to B-Class ODN together with IFN- α secretion comparable to A-Class ODN. Here, we investigate sequence requirements of C-Class ODN regarding optimal IFN- α secretion. Sequence as well as backbone modifications like 2′- O-methyl modifications especially in the 5′ part of the ODN influence IFN- α-producing capacity. Kinetic studies on mRNA level for CD69, IFN- γ, IP-10 and IL-18 by semi-quantitative PCR demonstrated differences in mRNA transcription for some cytokines suggesting different regulatory mechanisms for different ODN classes. High amounts of IP-10 mRNA and protein as well as up-regulation of IL-18 mRNA were observed especially for the A- and C-Classes. According to these data, C-Class ODN can be described as strong Th1 inducers with the stimulation of type I and II interferon as well as IP-10 production and strong NK activation. These characteristics can be availed to induce potent anti-tumor or anti-viral effects. Consequently, C-Class CpG ODN represent ideal drug candidates for anti-viral and/or anti-tumor therapy.

Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities.

Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine (CpG) dinucleotides (CpG ODN) mimic the immunostimulatory activity of bacterial DNA and are recognized by the Toll-like receptor 9 (TLR9). CpG ODN of the B-Class stimulate strong B cell and NK cell activation and cytokine production. The highest degrees of NK stimulation as well as IFN-alpha secretion by plasmacytoid DC were found to occur only with A-Class ODN. A third class of CpG ODN combines the immune effects of A- and B-Class CpG ODN. C-Class ODN strongly stimulate B cell or NK cell activation and IFN-alpha production. In contrast to the A-Class, the C-Class is wholly phosphorothioate, has no poly-G stretches, but has palindromic sequences combined with stimulatory CpG motifs. All classes stimulate TLR9-dependent signaling, but with strikingly different dose-response relationships that are quite in contrast to those observed for IFN-alpha. Effects similar to those on human cells were observed on mouse splenocytes. In contrast, splenocytes from TLR9-deficient mice did not show any response to the three CpG ODN classes. In vivo studies demonstrate that C-Class ODN are very potent Th1 adjuvants. C-Class ODN may represent new therapeutic drugs that combine the effects of A- and B-Class ODN for broad applications in infectious disease or cancer therapy.