non-CpG Oligodeoxynucleotides Research Articles

Modulation of the Inflammatory Response by Pre-emptive Administration of IMT504 Reduces Postoperative Pain in Rats and has Opioid-Sparing Effects

Despite the available knowledge on underlying mechanisms and the development of several therapeutic strategies, optimal management of postoperative pain remains challenging. This preclinical study hypothesizes that, by promoting an anti-inflammatory scenario, pre-emptive administration of IMT504, a noncoding, non-CpG oligodeoxynucleotide with immune modulating properties, will reduce postincisional pain, also facilitating therapeutic opioid-sparing. Male adult Sprague-Dawley rats with unilateral hindpaw skin-muscle incision received pre-emptive (48 and 24 hours prior to surgery) or postoperative (6 hours after surgery) subcutaneous vehicle (saline) or IMT504. Various groups of rats were prepared for pain-like behavior analyses, including subgroups receiving morphine or naloxone, as well as for flow-cytometry or quantitative RT-PCR analyses of the spleen and hindpaws (for analysis of inflammatory phenotype). Compared to vehicle-treated rats, pre-emptive IMT504 significantly reduced mechanical allodynia by 6 hours after surgery, and accelerated recovery of basal responses from 72 hours after surgery and onwards. Cold allodynia was also reduced by IMT504. Postoperative administration of IMT504 resulted in similar positive effects on pain-like behavior. In IMT504-treated rats, 3 mg/kg morphine resulted in comparable blockade of mechanical allodynia as observed in vehicle-treated rats receiving 10 mg/kg morphine. IMT504 significantly increased hindpaw infiltration of mesenchymal stem cells, CD4+T and B cells, and caused upregulated or downregulated transcript expressions of interleukin-10 and interleukin-1β, respectively. Also, IMT504 treatment targeted the spleen, with upregulated or downregulated transcript expressions, 6 hours after incision, of interleukin-10 and interleukin-1β, respectively. Altogether, pre-emptive or postoperative IMT504 provides protection against postincisional pain, through participation of significant immunomodulatory actions, and exhibiting opioid-sparing effects. PerspectiveThis preclinical study introduces the noncoding non-CpG oligodeoxynucleotide IMT504 as a novel modulator of postoperative pain and underlying inflammatory events. The opioid-sparing effects observed for IMT504 appear as a key feature that could contribute, in the future, to reducing opioid-related adverse events in patients undergoing surgical intervention.

IMT504 blocks allodynia in rats with spared nerve injury by promoting the migration of mesenchymal stem cells and by favoring an anti-inflammatory milieu at the injured nerve.

IMT504, a noncoding, non-CpG oligodeoxynucleotide, modulates pain-like behavior in rats undergoing peripheral nerve injury, through mechanisms that remain poorly characterized. Here, we chose the spared nerve injury model in rats to analyze the contribution of mesenchymal stem cells (MSCs) in the mechanisms of action of IMT504. We show that a single subcutaneous administration of IMT504 reverses mechanical and cold allodynia for at least 5 weeks posttreatment. This event correlated with long-lasting increases in the percentage of MSCs in peripheral blood and injured sciatic nerves, in a process seemingly influenced by modifications in the CXCL12-CXCR4 axis. Also, injured nerves presented with reduced tumor necrosis factor-α and interleukin-1β and increased transforming growth factor-β1 and interleukin-10 protein levels. In vitro analysis of IMT504-pretreated rat or human MSCs revealed internalized oligodeoxynucleotide and confirmed its promigratory effects. Moreover, IMT504-pretreatment induced transcript expression of Tgf-β1 and Il-10 in MSCs; the increase in Il-10 becoming more robust after exposure to injured nerves. Ex vivo exposure of injured nerves to IMT504-pretreated MSCs confirmed the proinflammatory to anti-inflammatory switch observed in vivo. Interestingly, the sole exposure of injured nerves to IMT504 also resulted in downregulated Tnf-α and Il-1β transcripts. Altogether, we reveal for the first time a direct association between the antiallodynic actions of IMT504, its promigratory and cytokine secretion modulating effects on MSCs, and further anti-inflammatory actions at injured nerves. The recapitulation of key outcomes in human MSCs supports the translational potential of IMT504 as a novel treatment for neuropathic pain with a unique mechanism of action involving the regulation of neuroimmune interactions.

Intrathecal Administration of an Anti-nociceptive Non-CpG Oligodeoxynucleotide Reduces Glial Activation and Central Sensitization.

Inflammatory pain associates with spinal glial activation and central sensitization. Systemic administration of IMT504, a non-CpG oligodeoxynucleotide originally designed as an immunomodulator, exerts remarkable anti-allodynic effects in rats with complete Freund´s adjuvant (CFA)-induced hindpaw inflammation. However, the anti-nociceptive mechanisms of IMT504 remain unknown. Here we evaluated whether IMT504 blocks inflammatory pain-like behavior by modulation of spinal glia and central sensitization. The study was performed in Sprague Dawley rats with intraplantar CFA, and a single lumbosacral intrathecal (i.t.) administration of IMT504 or vehicle was chosen to address if changes in glial activation and spinal sensitization relate to the pain-like behavior reducing effects of the ODN. Naïve rats were also included. Von Frey and Randall-Selitto tests, respectively, exposed significant reductions in allodynia and mechanical hypersensitivity, lasting at least 24h after i.t. IMT504. Analysis of electromyographic responses to electrical stimulation of C fibers showed progressive reductions in wind-up responses. Accordingly, IMT504 significantly downregulated spinal glial activation, as shown by reductions in the protein expression of glial fibrillary acidic protein, CD11b/c, Toll-like receptor 4 (TLR4) and the phosphorylated p65 subunit of NFκB, evaluated by immunohistochemistry and western blot. In vitro experiments using early post-natal cortical glial cultures provided further support to in vivo data and demonstrated IMT504 internalization into microglia and astrocytes. Altogether, our study provides new evidence on the central mechanisms of anti-nociception by IMT504 upon intrathecal application, and further supports its value as a novel anti-inflammatory ODN with actions upon glial cells and the TLR4/NFκB pathway. Intrathecal administration of the non-CpG ODN IMT504 fully blocks CFA-induced mechanical allodynia and hypersensitivity, in association with reduced spinal sensitization. Administration of the ODN also results in downregulated gliosis and reduced TLR4-NF-κB pathway activation. IMT504 uptake into astrocytes and microglia support the concept of direct modulation of CFA-induced glial activation.

Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG oligodeoxynucleotides of class C

Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.

The Distinct and Cooperative Roles of Toll-Like Receptor 9 and Receptor for Advanced Glycation End Products in Modulating In Vivo Inflammatory Responses to Select CpG and Non-CpG Oligonucleotides.

Antisense oligonucleotides (ASOs) are widely accepted therapeutic agents that suppress RNA transcription. While the majority of ASOs are well tolerated in vivo, few sequences trigger inflammatory responses in absence of conventional CpG motifs. In this study, we identified non-CpG oligodeoxy-nucleotide (ODN) capable of triggering an inflammatory response resulting in B cell and macrophage activation in a MyD88- and TLR9-dependent manner. In addition, we found the receptor for advance glycation end product (RAGE) receptor to be involved in the initiation of inflammatory response to suboptimal concentrations of both CpG- and non-CpG-containing ODNs. In contrast, dosing RAGE KO mice with high doses of CpG or non-CpG ODNs lead to a stronger inflammatory response than observed in wild-type mice. Together, our data provide a previously uncharacterized in vivo mechanism contingent on ODN-administered dose, where TLR9 governs the primary response and RAGE plays a distinct and cooperative function in providing a pivotal role in balancing the immune response.

Effects of KLK Peptide on Adjuvanticity of Different ODN Sequences.

Endosomal Toll-like receptors (TLR) such as TLR3, 7, 8 and 9 recognize pathogen associated nucleic acids. While DNA sequence does influence degree of binding to and activation of TLR9, it also appears to influence the ability of the ligand to reach the intracellular endosomal compartment. The KLK (KLKL5KLK) antimicrobial peptide, which is immunostimulatory itself, can translocate into cells without cell membrane permeabilization and thus can be used for endosomal delivery of TLR agonists, as has been shown with the IC31 formulation that contains an oligodeoxynucleotide (ODN) TLR9 agonist. We evaluated the adjuvant activity of KLK combined with CpG or non-CpG (GpC) ODN synthesized with nuclease resistant phosphorothioate (S) or native phosphodiester (O) backbones with ovalbumin (OVA) antigen in mice. As single adjuvants, CpG(S) gave the strongest enhancement of OVA-specific immunity and the addition of KLK provided no benefit and was actually detrimental for some readouts. In contrast, KLK enhanced the adjuvant effects of CpG(O) and to a lesser extent of GpC (S), which on their own had little or no activity. Indeed while CD8 T cells, IFN-γ secretion and humoral response to vaccine antigen were enhanced when CpG(O) was combined with KLK, only IFN-γ secretion was enhanced when GpC (S) was combined to KLK. The synergistic adjuvant effects with KLK/ODN combinations were TLR9-mediated since they did not occur in TLR9 knock-out mice. We hypothesize that a nuclease resistant ODN with CpG motifs has its own mechanism for entering cells to reach the endosome. For ODN without CpG motifs, KLK appears to provide an alternate mechanism for accessing the endosome, where it can activate TLR9, albeit with lower potency than a CpG ODN. For nuclease sensitive (O) backbone ODN, KLK may also provide protection from nucleases in the tissues.

Therapeutic Implications of Orally Delivered Immunomodulatory Oligonucleotides

DNA has multiple and complex effects on the immune system. Bacterial DNA contains immunostimulatory CpG motifs that trigger cells expressing Toll-like receptor 9 (TLR9) to proliferate, mature, and support T helper type 1 (Th1)-biased immune responses (reviewed in ref. 1). This response confers a selective advantage to the host by improving resistance to infection. In contrast, mammalian DNA contains sequence motifs that downregulate the immune system. Synthetic oligodeoxynucleotides that mimic this inhibitory activity (iODNs) slow or prevent the development of inflammatory and autoimmune diseases, consistent with the release of DNA from dying host cells inhibiting autoaggressive immune responses (reviewed in ref. 2). As reported in this issue, Wang et al. break new ground in the study of immunomodulatory ODNs by showing that the severity of chemically induced atopic dermatitis can be reduced by oral delivery of iODNs and worsened by CpG ODNs.3

Non-CpG Oligonucleotides Exert Adjuvant Effects by Enhancing Cognate B Cell-T Cell Interactions, Leading to B Cell Activation, Differentiation, and Isotype Switching.

Natural and synthetic nucleic acids are known to exert immunomodulatory properties. Notably, nucleic acids are known to modulate immune function via several different pathways and various cell types, necessitating a complex interpretation of their effects. In this study we set out to compare the effects of a CpG motif containing oligodeoxynucleotide (ODN) with those of a control and an inhibitory non-CpG ODN during cognate B cell-T cell interactions. We employed an antigen presentation system using splenocytes from TCR transgenic DO11.10 mice and the ovalbumin peptide recognized by the TCR as model antigen. We followed early activation events by measuring CD69 expression, late activation by MHC class II expression, cell division and antibody production of switched, and nonswitched isotypes. We found that both of the tested non-CpG ODN exerted significant immunomodulatory effects on early T cell and on late B cell activation events. Importantly, a synergism between non-CpG effects and T cell help acting on B cells was observed, resulting in enhanced IgG production following cognate T cell-B cell interactions. We propose that non-CpG ODN may perform as better adjuvants when a strong antigen-independent immune activation, elicited by CpG ODNs, is undesirable.

The effects of administration of ligands for Toll-like receptor 4 and 21 against Marek's disease in chickens

Ligands for Toll-like receptors (TLRs) are known to stimulate immune responses, leading to protection against bacterial and viral pathogens. Here, we aimed to examine the effects of various TLR ligands on the development of Marek's disease in chickens. Specific-pathogen free chickens were treated with a series of TLR ligands that interact with TLR3, TLR9 and TLR21. In a pilot study, it was determined that TLR4 and TLR21 ligands are efficacious, in that they could reduce the incidence of Marek's disease tumors in infected birds. Hence, in a subsequent study, chickens were treated with lipopolysaccharide (LPS) as a TLR4 and CpG oligodeoxynucleotides (ODN) as TLR21 agonists before being challenged with the RB1B strain of Marek's disease virus (MDV) via the respiratory route. The results demonstrated that the administration of LPS or CpG ODN, but not PBS or non-CpG ODN, delayed disease onset and reduced MDV genome copy number in the spleens of infected chickens. Taken together, our data demonstrate that TLR4 and 21 agonists modulate anti-virus innate immunity including cytokine responses in MD-infected chicken and this response can only delay, but not inhibit, disease progression.

Impact of CpG oligodeoxynucleotide stimulation on percentage of T and B cells in chicken

TLR stimulation in chickens has been shown to play a role in the initiation and regulation of innate and adaptive immune responses. The aim of this study was to use flow cytometry to establish the percentage of T and B subset in blood and lymphoid organs in chicks after CpG oligodeoxynucleotide (ODN) stimulation. It was demonstrated that the percentages of CD3+, CD4+, TCRgamma delta+ cells and Bu-1+MHC class II+ cells in blood 24 h post-injection were significantly higher than in the control groups. It was also shown that the percentages of CD3+ and CD4+ cells in the spleen at 48 h post-injection were significantly higher than in control groups. The percentage of Bu-1+ cells in the bursa of Fabricius after CpG ODN stimulation (98.38 +/- 0.84) was significantly higher than that found in the non-CpG ODN control group (94.54 +/- 2.51) (p < or = 0.05). The results indicate that class B CpG ODN increases the percentage of both T (especially CD4+ cells) and B cells.

IMT504: A New and Potent Adjuvant for Rabies Vaccines Permitting Significant Dose Sparing

Background: Rabies virus infection causes encephalitis, which is almost always fatal. Vaccination can be extremely effective at preventing disease but is prohibitively costly. Vaccine formulations allowing dose-sparing and fewer inoculations with faster antibody response would be extremely desirable. IMT504, an immunostimulatory non-CpG oligo-deoxynucleotide, is a highly potent vaccine adjuvant. Methods: Human and rat antibody measurements, and rat chal-lenge studies were performed. Results: In rats, highly effective immune responses with IMT504 were observed even after diluting vaccine up to 1/625. In highly lethal, live intracerebral rabies challenge studies, protection occurred even with extremely dilute vaccine plus IMT504. In humans, antibody titers developed faster and were significantly higher with IMT504-adjuvanted diluted vaccine vs non-adjuvanted vaccine (full strength or diluted). All five administered IMT504-adjuvanted diluted vaccine reached protective antibodies (≥0.5 IU/ml) after the second injection. After the third injection, individuals receiving IMT504-adjuvanted diluted vaccine reached levels approximately 10 times higher than controls (M ± SEM: 31.0 ± 10.9 vs 3.40 ± 0.99 IU/ml). Conclusions: These data suggest that IMT504 may allow fewer inoculations, highly significant dose-sparing of vaccine, rapid antibody production and protection from rabies. Extensive clinical studies are necessary to confirm if the use of IMT504 will permit significantly greater access to highly effective life-saving rabies vaccines.

TRAIL is involved in CpG ODN-mediated anti-apoptotic signals

Synthetic oligodeoxynucleotides (ODNs) with the CpG-motifs are recognized by toll-like receptor 9 (TLR9), which elicits an immune response. Serum starvation of Raw264.7 cells increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression. However, treatment with CpG ODN reduced TRAIL expression as well as apoptosis by serum starvation. In serum starved cells, TLR9 inhibitors recovered the decreasing TRAIL expression and sub-G1 accumulation by CpG ODN. CpG ODN-regulated anti-apoptotic signals which were dependent on the Akt-FoxO3a signaling pathway. CpG ODNs activated Akt and inactivated FoxO3a in serum starved cells. Knockdown of FoxO3a by siRNA decreased TRAIL expression and apoptosis in serum-starved cells. In contrast, FoxO3a overexpression increased apoptosis by serum starvation, and CpG ODNs blocked these effects through TRAIL expression. LY294002, a PI3K-Akt inhibitor, blocked the CpG ODN effect of TRAIL expression and the sub-G1 population in serum starved cells. In contrast, overexpression of wild-type Akt reduced additional sub-G1 cells both in non-CpG ODN- and CpG ODN-treated cells. Taken together, these results demonstrate the involvement of Akt-FoxO3a signaling in TLR9-mediated downregulation of TRAIL and anti-apoptotic signals.

CpG and Non-CpG Oligodeoxynucleotides Directly Costimulate Mouse and Human CD4+ T Cells through a TLR9- and MyD88-Independent Mechanism

TLR ligands are known to activate APCs, but direct T cell responsiveness to TLR ligands is controversial. Because of their clinical relevance, we performed in-depth studies of the effects of the TLR9-associated ligands, oligodeoxynucleotides (ODNs), on highly purified T lymphocytes. Both CpG and non-CpG ODNs directly costimulate mouse and human CD4(+) T cells, resulting in activation marker upregulation, cytokine secretion, elevated TCR phosphorylation, and proliferation. Surprisingly, ODN costimulation occurred independently of TLR9 and MyD88, as well as ICOS, CD28, and TRIF. TLR9-antagonist ODNs likewise promoted T cell activation, which has important implications for the study of these "inhibitory" ODNs in inflammatory diseases. Cytokine profiling revealed that ODNs promote polarization of distinct Th subsets, and that ODNs differentially affect human naive and memory T cells. Our studies reveal a striking and unexpected ability of ODNs to directly activate and polarize T cells, presenting an opportunity to enhance the paradigm for selection of therapeutic ODNs in humans.

PyNTTTTGT Prototype Oligonucleotide IMT504, a Novel Effective Adjuvant of the FMDV DNA Vaccine

Synthetic oligodeoxynucleotides (ODNs) such as CpG can stimulate B and plasmacytoid dendritic cells in vertebrate immune systems. Several studies showed that non-CpG ODNs could also induce strong stimulation of B and T cells. PyNTTTTGT ODNs, non-CpG ODNs, can activate and cause immunoglobulin secretion by B cells and proliferation of T cells in vivo. By using PyNTTTTGT ODNs as an adjuvant for a FMDV DNA vaccine, we found that levels of antibody production, T-cell proliferation, and CTL activity were significantly increased compared with the DNA vaccine alone. Compared with the adjuvant effects of CpG ODNs on DNA vaccination, similar levels of antibody production and T-cell proliferation, and higher levels of CTL activity and IFN-gamma expression in CD8 T cells were induced by the IMT504 ODNs. On the other hand, RT-PCR results show that IMT504 ODN may activate the DNA sensor of DAI (DNA-dependent activator of IFN regulatory factors) and partially stimulate TLR9. At this point, the PyNTTTTGT prototype IMT504 ODN can reasonably be predicted to be a good adjuvant for FMDV DNA vaccine in small animals, but its efficacy in larger animals remains to be explored.

Bacterial DNA delays human eosinophil apoptosis

Oligodeoxynucleotide (ODN) sequences containing unmethylated cytidine phosphate guanosine (CpG) motifs prevalent in bacterial DNA attenuate allergic lung inflammation in experimental models of asthma but failed to inhibit eosinophilia and improve lung function in patients with asthma. Bacterial respiratory tract infections exacerbate asthma in humans. Increased eosinophil survival is a critical factor leading to persistent eosinophilic airway inflammation. Apoptosis is regarded as a key mechanism in the resolution of eosinophilic inflammation. The aim of this study was to investigate the effects of bacterial DNA and CpG ODNs on human eosinophil apoptosis in vitro and to elucidate the signalling pathway. Eosinophils were isolated from human peripheral blood by CD16- or CD16-, CD19- and CD304-negative selection. Apoptosis was determined by flow cytometric analysis of relative DNA content, Annexin-V staining and/or morphological analysis. Toll-like receptor 9 (TLR9) expression was studied by using western blotting and intracellular flow cytometry. Bacterial DNA and phosphorothioate-modified CpG ODNs, but not vertebrate DNA, were found to delay spontaneous eosinophil apoptosis. The effect of CpG ODNs was dependent on endosomal acidification and reversed by inhibitory ODN, which suggests involvement of TLR9 pathway. Furthermore, we demonstrated TLR9 expression in eosinophils derived from both atopic and healthy donors. Non-CpG ODNs had occasionally parallel but less profound effect on eosinophil apoptosis, which was not dependent on endosomal acidification. The anti-apoptotic effect of CpG ODNs was dependent on phosphatidylinositol 3-kinase (PI3K) and nuclear factor-κB (NF-κB) but not mitogen-activated protein kinases (MAPKs) as determined by inhibitor studies. Although our results suggest CpG-dependent involvement of TLR9 in the action of phosphorothioate-modified ODNs, we interestingly found that the anti-apoptotic action of native bacterial DNA in eosinophils is not dependent on unmethylated CpG motifs. This suggests that bacterial DNA contains a currently unknown recognition structure lacking from vertebrate DNA. Bacterial DNA-mediated suppression of eosinophil apoptosis is a novel mechanism for exacerbation of eosinophilic lung inflammation associated with bacterial respiratory tract infection.

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.

Intranasal immunization of mice with a formalin-inactivated bovine respiratory syncytial virus vaccine co-formulated with CpG oligodeoxynucleotides and polyphosphazenes results in enhanced protection

As respiratory syncytial virus (RSV) targets the mucosal surfaces of the respiratory tract, induction of both systemic and mucosal immunity will be critical for optimal protection. In this study, the ability of an intranasally delivered, formalin-inactivated bovine RSV (FI-BRSV) vaccine co-formulated with CpG oligodeoxynucleotides (ODN) and polyphosphazenes (PP) to induce systemic and mucosal immunity, as well as protection from BRSV challenge, was evaluated. Intranasal immunization of mice with FI-BRSV formulated with CpG ODN and PP resulted in both humoral and cell-mediated immunity, characterized by enhanced production of BRSV-specific serum IgG, as well as increased gamma interferon and decreased interleukin-5 production by in vitro-restimulated splenocytes. These mice also developed mucosal immune responses, as was evident from increased production of BRSV-specific IgG and IgA in lung-fragment cultures. Indeed, the increases in serum and mucosal IgG, and in particular mucosal IgA and virus-neutralizing antibodies, were the most critical differences observed between FI-BRSV formulated with both CpG ODN and PP in comparison to formulations with CpG ODN, non-CpG ODN or PP individually. Finally, FI-BRSV/CpG/PP was the only formulation that resulted in a significant reduction in viral replication upon BRSV challenge. Co-formulation of CpG ODN and PP is a promising new vaccine platform technology that may have applications in mucosal immunization in humans.

Control of In Vitro Immune Responses by Regulatory Oligodeoxynucleotides through Inhibition of pIII Promoter Directed Expression of MHC Class II Transactivator in Human Primary Monocytes

Ag presentation is a key step in the initiation of adaptive immune responses that depends on the expression of MHC Ags and costimulatory molecules. Immune-enhancing CpG and non-CPG oligodeoxynucleotides (ODNs) stimulate Ag presentation by stimulating the expression of these molecules and by promoting dendritic cell maturation. In this report, we identify immunoregulatory orthophosphorothioate non-CpG molecules, referred to as regulatory ODNs (rODNs), by their ability to inhibit allogeneic monocyte-stimulated T cell responses and down-regulate HLA-DR in human primary monocytes. The rODNs promoted the survival of macrophages and were able to activate IL-8 secretion through a chloroquine-resistant pathway. Messenger RNAs for HLA-DR alpha and beta and the MHC CIITA were reduced by rODNs but not by stimulatory CpG ODN2006 and non-CpG ODN2006a. CIITA transcription in monocytes was controlled primarily by promoter III and not by promoter I or IV. rODNs blocked promoter III-directed transcription of CIITA in these cells. Under conditions that induced dendritic cell differentiation, rODNs also reduced HLA-DR expression. The activity of rODNs is phosphorothioate chemistry and G stretch dependent but TLR9 independent. G tetrads were detected by circular dichroism in active rODNs and associated with high m.w. multimers on nondenaturing gels. Heat treatment of rODNs disrupted G tetrads, the high m.w. aggregates, and the HLA-DR inhibitory activity of the ODNs. The inhibition of immune responses by regulatory oligodeoxynucleotides may be useful for the treatment of immune-mediated disorders including autoimmune diseases and graft rejection.

In vitro Enhancement by CpG ODN of Phagocytic Activity of Rainbow Trout Head Kidney Phagocytes against Fish Pathogen Vibrio ordalii, but not against Polystyrene Particles

Rainbow trout (Oncorhynchus mykiss) head kidney phagocytes precultured with a synthetic cytidine-phosphate-guanosine (CpG) oligodeoxynucleotide (ODN) displayed significantly higher phagocytic activity against Vibrio ordalii than phagocytes precultured with non-CpG ODN. However, head kidney phagocytes precultured with CpG ODN did not show enhanced phagocytic activity against polystyrene particles.

Immunostimulatory PyNTTTTGT Oligodeoxynucleotides: Structural Properties and Refinement of the Active Motif

It is well known that synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides within a given context stimulate B lymphocytes and plasmacytoid dendritic cells (PDCs) of the vertebrate immune system. We have reported that B lymphocyte and PDC stimulation in humans could also be efficiently achieved by using non-CpG ODNs bearing the immunostimulatory sequence (motif) PyNTTTTGT, wherein Py is C or T and N is any deoxyribonucleotide. We are now reporting a series of studies that gives further precision regarding the composition of this immunostimulatory motif. The analysis of hundreds of ODNs led us to the conclusion that the motif for optimal CpG-independent immune stimulation can be represented by a sequence of the following general formula: PyN(T/A)(T/C/G)(T/C/G)(T/G)GT, wherein Py is C or T and N is any deoxyribonucleotide and wherein at least two of the positions represented within parentheses are Ts. Requirements for optimal ODN activity are as follows: (1) at least one of the versions of the general motif must be located near the central portion of the ODN; and (2) the ODN must be 20 or more nucleotides long. PyN(T/A)(T/C/G)(T/C/G)(T/G)GT ODNs are active in a phosphorothioate or in a phosphodiester backbone. In a phosphodiester backbone a canonical motif is strongly required whereas in a phosphorothioate backbone specificity is, within certain limits, less strict. On the other hand, PyN(T/A)(T/C/G)(T/C/G)(T/G)GT oligonucleotides are inactive as a double chain or as a modified (phosphorothioate or hydroxyl-methyl modified) or unmodified RNA backbone.