Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection

In this report, we investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with soluble antigen (SA) from Leishmania donovani. BALB/c mice were vaccinated with the soluble antigen with or without CpG-ODN as adjuvant and then challenged with L. donovani metacyclic promastigotes. CpG-ODN alone resulted in partial protection against challenge with L. donovani. Immunization of mice with SA and CpG-ODN showed enhanced reduction in parasite load ( approximately 60%) when compared to SA ( approximately 40%) immunized mice. Immunization with SA by itself resulted in a mixed Th1/Th2 response whereas co-administration of SA with CpG-ODN resulted in a strong Th1 promoting isotype as they together promoted production of immunoglobulin G2a. Leishmania-specific Th1 cytokine response was induced by co-administering CpG-ODN and SA as they together promoted production of IFN-gamma and IL-12. In the present study, we demonstrate that immunostimulatory phosphorothioate-modified ODN are promising immune enhancers for vaccination against visceral leishmaniaisis.     

Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides

The Toll-like receptor (TLR)9 is critical for the recognition of immunostimulatory CpG motifs but may cooperate with other TLRs. We analyzed TLR1-10 mRNA expression by using quantitative real-time PCR in highly purified subsets of human PBMC and determined the sensitivity of these subsets to CpG oligodeoxynucleotides (ODN). TLR1 and TLR6 were expressed in all cell types examined. TLR10 was highly expressed in B cells and weakly expressed in plasmacytoid dendritic cells (PDC). High expression of TLR2 was characteristic for monocytes. PDC and B cells expressed marked levels of TLR7 and TLR9 and were directly sensitive to CpG ODN. In CpG ODN-stimulated PDC and B cells, TLR9 expression rapidly decreased, as opposed to TLR7, which was up-regulated in PDC and decreased in B cells. In monocytes, NK cells, and T cells, TLR7 was absent. Despite low expression of TLR9, monocytes, NK cells, and T cells did not respond to CpG ODN in the absence of PDC but were activated in the presence of PDC. In conclusion, our studies provide evidence that PDC and B cells, but not monocytes, NK cells, or T cells, are primary targets of CpG ODN in peripheral blood. The characteristic expression pattern of TLR1-10 in cellular subsets of human PBMC is consistent with the concept that TLR9 is essential in the recognition of CpG ODN in PDC and B cells. In addition, selective regulation of TLR7 expression in PDC and B cells by CpG ODN revealed TLR7 as a candidate TLR potentially involved in modulating the recognition of CpG motifs.     

Bovine and ovine blood mononuclear leukocytes differ markedly in innate immune responses induced by Class A and Class B CpG-oligodeoxynucleotide

Cytosine-phosphate-guanosine (CpG)-DNA can induce an impressive array of innate immune responses that may directly or indirectly contribute to the clearance of infectious agents. Assays, such as lymphocyte proliferative responses, have been used to demonstrate that the immunostimulatory activity of CpG-DNA is conserved among a broad range of vertebrate species, but no studies have been completed to determine if qualitative differences exist among species for CpG-oligodeoxynucleotide (ODN)-induced innate immune responses. In this study, we assessed the capacity of a Class A (ODN 2216) and a Class B (ODN 2007) CpG-ODN to induce innate immune responses in two closely related species, ovine (n = 28) and bovine (n = 29). The secretion of interferon (IFN)-alpha and IFN-gamma and non-major histocompatability complex (MHC)-restricted cytotoxic activity were assayed with CpG-ODN-stimulated peripheral blood mononuclear cells (PBMC). These investigations revealed significant interspecies and intraspecies variation in the responses. As expected, ODN 2216 was a potent inducer of IFN-alpha secretion by both bovine and ovine PBMC, but ODN 2007 also induced dose-dependent, CpG-specific IFN-alpha secretion by ovine PBMC. In contrast, a significant dose-dependent, CpG-specific IFN-gamma secretion response was only observed following ODN 2216 stimulation of bovine PBMC. Furthermore, both ODN 2216 and ODN 2007 induced CpG-specific non-MHC-restricted cytotoxicity with ovine but not bovine PBMC. Finally, there was not a single assay in which PBMC from all sheep or cattle responded at a detectable level. A striking aspect of these results is that such marked differences in CpG-ODN induced innate responses existed both between and within two closely related species.     

Preinfection treatment of resistant mice with CpG oligodeoxynucleotides renders them susceptible to friend retrovirus-induced leukemia

We recently reported that immunostimulatory oligodeoxynucleotides (CpG oligodeoxynucleotides [CpG-ODN]) were effective in postexposure treatment of retrovirus-induced disease (A. R. M. Olbrich et al., J. Virol. 76:11397-11404, 2002). We now show that the timing of treatment is a critical factor in treatment efficacy. In stark contrast to the success of postexposure treatments, we found that CpG treatment of susceptible mice prior to Friend retrovirus infection accelerated the development of virus-induced erythroleukemia. Furthermore, 70.8% of mice that were resistant to Friend virus-induced leukemia developed disease after inoculation of CpG-ODN before infection. The CpG pretreatment of these mice enhanced viral loads in their spleens and blood compared to controls that received ODN without CpG motifs. The main target cells of Friend virus, erythroid precursor cells and B cells, proliferated after CpG-ODN inoculation and provided an enlarged target cell population for viral infection. Our present findings together with our previous report demonstrate that CpG-ODN treatment of viral infections may be a double-edged sword that can result in an effective therapy but also in an acceleration of disease progression depending on the time point of treatment.     

利用小鼠模型评价含有5＇-GTCGTT-3＇特征序列的人CpG寡脱氧核苷酸的免疫刺激活性

为了寻找合适的动物模型来评价人CpG寡脱氧核苷酸（CpG-ODN）的活性,研究了CpG2006等含有5＇-GTCGTT-3＇特征序列的人CpG-ODN对小鼠的免疫刺激活性。在体外它们能够促进小鼠脾淋巴细胞转化,促进B细胞分泌IgM,但不能诱生高水平的IFN-γ。研究了CpG2006等序列在体内作为疫苗佐剂对HBsAg免疫效果的影响,发现（1）人CpG-ODN能够明显提高抗-HBs抗体水平,并逆转Al（OH）     

Human B cells express functional TRAIL/Apo-2 ligand after CpG-containing oligodeoxynucleotide stimulation

CpG-containing oligodeoxynucleotides (CpG ODN) have broad-ranging immunostimulatory effects, including the generation of antitumor immune responses. Analysis of different CpG ODN have identified two classes: CpG-A ODN, which stimulate high levels of IFN-alpha production from plasmacytoid dendritic cells and weakly activate B cells, and CpG-B ODN, which strongly activate B cells but stimulate low production of IFN-alpha from plasmacytoid dendritic cells. Previously, we observed that CpG-B ODN (2006) induces TRAIL/Apo-2 ligand (Apo-2L)-mediated killing of tumor cells by CD14(+) PBMC. In this study, we extend our investigation of CpG ODN-induced TRAIL/Apo-2L expression and activity in PBMC to include CpG-A ODN. Of the two classes, IFN-alpha production and TRAIL/Apo-2L-mediated killing of tumor cells was greatest with CpG-A ODN. Surprisingly, CD3(+), CD14(+), CD19(+), and CD56(+) PBMC expressed high levels of TRAIL/Apo-2L following CpG-A ODN stimulation. When isolated, the CD19(+) PBMC (B cells) were able to kill tumor cells in a TRAIL/Apo-2L-dependent manner. As with CD14(+) PBMC, CD19(+) sorted B cells were capable of up-regulating TRAIL/Apo-2L expression when stimulated with IFN-alpha alone. Interestingly, agonist anti-CD40 mAb further enhanced the IFN-alpha-induced TRAIL/Apo-2L expression on CD19(+) B cells. These results are the first to demonstrate human B cell-mediated killing of tumor cells in a TRAIL/Apo-2L-dependent fashion.     

Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways of type I IFN synthesis in human plasmacytoid dendritic cells

Two different CpG oligonucleotides (ODN) were used to study the regulation of type I IFN in human plasmacytoid dendritic cells (PDC): ODN 2216, a CpG-A ODN, known to induce high amounts of IFN-alpha in PDC, and ODN 2006, a CpG-B ODN, which is potent at stimulating B cells. CpG-A ODN showed higher and prolonged kinetics of type I IFN production compared with that of CpG-B ODN. In contrast, CpG-B ODN was more active than CpG-A ODN in stimulating IL-8 production and increasing costimulatory and Ag-presenting molecules, suggesting that CpG-A and CpG-B trigger distinct regulatory pathways in PDC. Indeed, CpG-A ODN, but not CpG-B ODN, activated the type I IFNR-mediated autocrine feedback loop. PDC were found to express high constitutive levels of IFN regulatory factor (IRF)7. IRF7 and STAT1, but not IRF3, were equally up-regulated by both CpG-A and CpG-B. CD40 ligand synergistically increased CpG-B-induced IFN-alpha independent of the IFNR but did not affect CpG-B-induced IFN-beta. In conclusion, our studies provide evidence for the existence of two distinct regulatory pathways of type I IFN synthesis in human PDC, one dependent on and one independent of the IFNR-mediated feedback loop. The alternate use of these pathways is based on the type of stimulus rather than the quantity of IFN-alphabeta available to trigger the IFNR. Constitutive expression of IRF7 and the ability to produce considerable amounts of IFN-alpha independent of the IFNR seem to represent characteristic features of PDC.     

Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs

Oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides trigger a strong innate immune response in vertebrates. CpG ODN show promise as vaccine adjuvants, anti-allergens, and immunoprotective agents in animal models. Their transition to clinical use requires the identification of motifs that are optimally stimulatory in humans. Analysis of hundreds of novel ODN resulted in the identification and characterization of two structurally distinct "clusters" of immunostimulatory CpG ODN. One cluster ("D") preferentially stimulates IFN-gamma production by NK cells, whereas the other ("K") stimulates cell proliferation and the production of IL-6 and IgM by monocytes and B cells. The distinct immunostimulatory properties of K and D ODN can improve the design of CpG-based products to achieve specific therapeutic goals.     

CpG-ODN Class C Mediated Immunostimulation in Rabbit Model of Trypanosoma evansi Infection

CpG oligodeoxynucleotides (CpG-ODN) stimulate immune cells from a wide spectrum of mammalian species. Class C CpG-ODN is relatively stable and has the combined immune effects of both A and B classes of CpG-ODN. Trypanosoma evansi produces the state of immuno-suppression in the infected hosts. The current chemotherapeutic agents against this parasite are limited in number and usually associated with severe side effects. The present work aimed to determine the immunostimulatory effects of CpG-ODN class C in T. evansi infected rabbits. Rabbits inoculated with CpG C and challenged with T. evansi resulted in delayed onset of clinical signs with reduced severity in comparison to that of T. evansi infected rabbits. The treatment also enhanced humoral immune responses. Histopathological findings in liver and spleen revealed enhancement of mononuclear cell infiltration and secondary B cell follicles. These results demonstrate that CpG-ODN class C, has immunostimulatory properties in rabbit model of trypanosomosis. The use of booster doses or sustained delivery of CpG-ODN will further elucidate the prolonged CpG-ODN generated immune responses.     

CpG motifs as proinflammatory factors render autochthonous tumors permissive for infiltration and destruction

In a transgenic mouse model expressing SV40 T Ag (Tag) as a de novo tumor Ag, immune surveillance fails and islet cell carcinomas grow progressively. To develop an anticancer strategy that would be effective in eradicating solid, autochthonously growing tumors, we evaluated the effectiveness of immunostimulatory oligodeoxynucleotides (ODN) with cytosine-guanine-rich (CpG) motifs (CpG-ODN). In a classical vaccination protocol, Tag was administered with CpG-ODN as adjuvant. The antitumor vaccination, however, was only effective in a prophylactic setting, despite the successful activation of a Tag-specific CTL response in vivo. Histological examination demonstrated that even primed immune cells failed to infiltrate tumors once a malignant environment was established. To ensure that effector cells were not limiting, highly activated tumor Ag-specific T cells were transferred into tumor-bearing mice. However, this treatment also failed to result in tumor infiltration and rejection. Therefore, we further tested the efficacy of CpG-ODN as a proinflammatory agent in combination with the transfer of preactivated Tag-specific CD4(+) and CD8(+) T cells. Indeed, this combination therapy proved to be highly effective, because CpG-ODN rendered insulinomas permissive for massive infiltration and destruction. The opening of tumor tissue correlated with uptake of CpG-ODN by tissue-resident macrophages and a strong up-regulation of adhesion molecules such as ICAM and VCAM on blood vessel endothelia. These data demonstrate that systemic application of proinflammatory reagents drastically enhances extravasation of effector cells into tumor tissue, an observation that is of general importance for immunotherapy of solid tumors in a clinical setting.     

Encapsulation in liposomal nanoparticles enhances the immunostimulatory,adjuvant and anti-tumor activity of subcutaneously administered CpG ODN

Immunostimulatory oligodeoxynucleotides (ODN) containing cytosine-guanine (CpG) motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through triggering of the Toll-like receptor 9. We have previously shown that encapsulation in liposomal nanoparticles (LN) enhances the immunostimulatory activity of CpG ODN (LN-CpG ODN) (Mui et al. in J Pharmacol Exp Ther 298:1185, 2001). In this work we investigate the effect of encapsulation on the immunopotency of subcutaneously (s.c.) administered CpG ODN with regard to activation of innate immune cells as well as its ability to act as a vaccine adjuvant with tumor-associated antigens (TAAs) to induce antigen (Ag)-specific, adaptive responses and anti-tumor activity in murine models. It is shown that encapsulation specifically targets CpG ODN for uptake by immune cells. This may provide the basis, at least in part, for the significantly enhanced immunostimulatory activity of LN-CpG ODN, inducing potent innate (as judged by immune cell activation and plasma cytokine/chemokine levels) and adaptive, Ag-specific (as judged by MHC tetramer positive T lymphocytes, IFN-γ secretion and cytotoxicity) immune responses. Finally, in efficacy studies, it is shown that liposomal encapsulation enhances the ability of CpG ODN to adjuvanate adaptive immune responses against co-administered TAAs after s.c. immunization, inducing effective anti-tumor activity against both model and syngeneic tumor Ags in murine tumor models of thymoma and melanoma.     

CpG-conjugated apoptotic tumor cells elicit potent tumor-specific immunity

The primary goal of cancer immunotherapy is to elicit an immune response capable of eradicating established tumors and preventing tumor metastasis. One strategy to achieve this goal utilizes whole killed tumor cells as the primary immunogen. Killed tumor cells provide a comprehensive source of tumor-associated antigens (TAAs), thereby eliminating the need to identify individual antigens. Unfortunately, killed tumor cells tend to be poorly immunogenic. To overcome this limitation, we covalently conjugated immunostimulatory CpG oligodeoxynucleotides (ODN) to apoptotic tumor cells and examined their ability to induce TAA-specific immune responses. Results indicate that CpG conjugation enhances the uptake of cell-based vaccines by dendritic cells (DCs), up-regulates co-stimulatory molecule expression, and promotes the production of immunostimulatory cytokines. Vaccination with CpG-conjugated tumor cells triggers the expansion of tumor-specific cytotoxic T lymphocytes (CTL) that reduce the growth of established tumors and prevents their metastatic spread. Thus, conjugating CpG ODN to cell-based tumor vaccines is an important step toward improving cancer immunotherapy.     

In vitro activation of chicken leukocytes and in vivo protection against Salmonella enteritidis organ invasion and peritoneal S. enteritidis infection-induced mortality in neonatal chickens by immunostimulatory CpG oligodeoxynucleotide

Unmethylated CpG oligodinucleotides (CpG-ODN) flanked by specific bases found in bacterial DNA are known to stimulate innate immune responses. In this study, synthetic CpG-ODNs were evaluated for their in vitro stimulation of leukocyte and in vivo protection against Salmonella enteritidis (SE) in neonatal chickens. Our studies showed that CpG-ODN stimulated bactericidal activities, releasing granules (degranulation) and generating reactive oxygen species (oxidative burst), in chicken heterophils and up regulated nitric oxide production in chicken peripheral blood monocytes. When day-old chickens were given (i.p.) synthetic CpG-ODNs followed by oral challenge of SE, a significant reduction (p<0.05) of organ invasion by SE was observed in chickens pretreated with CpG-ODN containing the immunostimulatory GTCGTT motif. This CpG-OND also significantly reduced mortality of chickens with acute peritoneal infection of SE. Our study provides evidence that immunostimulatory CpG-ODN stimulated innate immune activities and enhanced the resistance to infectious pathogens in neonatal chickens.     

Increase in tumor size following intratumoral injection of immunostimulatory CpG-containing oligonucleotides in a rat glioma model

The immunosuppressive environment of malignant gliomas is likely to suppress the anti-tumor activity of infiltrating microglial cells and lymphocytes. Macrophages and microglial cells may be activated by oligonucleotides containing unmethylated CpG-motifs, although their value in cancer immunotherapy has remained controversial. Following injection of CpG-containing oligonucleotides (ODN) into normal rat brain, we observed a local inflammatory response with CD8+ T cell infiltration, upregulation of MHC 2, and ED1 expression proving the immunogenic capacity of the CpG-ODN used. This was not observed with a control ODN mutated in the immunostimulatory sequence (m-CpG). To study their effect in a syngeneic tumor model, we implanted rat 9L gliosarcoma cells into the striatum of Fisher 344 rats. After 3 days, immunostimulatory CpG-ODN, control m-CpG-ODN, or saline was injected stereotactically into the tumors (day 3 group). In another group of animals (day 0 group), CpG-ODN were mixed with 9L cells prior to implantation without further treatment on day 3. After 3 weeks, the animals were killed and the brains and spleens were removed. Rather unexpectedly, the tumors in several of the animals treated with CpG-ODN (both day 0 and day 3 group) were larger than in saline or m-CpG-ODN treated control animals. The tumor size in CpG-ODN-treated animals was more variable than in both control groups. This was associated with inflammatory responses and necrosis which was observed in most tumors following CpG treatment. This, however, did not prevent excessive growth of solid tumor masses in the CpG-treated animals similar to the control-treated animals. Dense infiltration with microglial cells resembling ramified microglia was observed within the solid tumor masses of control- and CpG-treated animals. In necrotic areas (phagocytic), activation of microglial cells was suggested by ED1 expression and a more macrophage-like morphology. Dense lymphocytic infiltrates consisting predominantly of CD8+ T cells and fewer NK cells were detected in all tumors including the control-treated animals. Expression of perforin serving as a marker for T cell or NK cell activation was detected only on isolated cells in all treatment groups. Tumors of all treatment groups revealed CD25 expression indicating T cells presumed to maintain peripheral tolerance to self-antigens. Cytotoxic T cell assays with in vitro restimulated lymphocytes (⁵¹chromium release assay) as well as interferon-gamma production by fresh splenocytes (Elispot assay) revealed specific responses to 9L cells but not another syngeneic cell line (MADB 106 adenocarcinoma). Surprisingly, the lysis rates with lymphocytes from CpG-ODN-treated animals were lower compared to control-treated animals. The tumor size of individual animals did not correlate with the response in both immune assays. Taken together, our data support the immunostimulatory capacity of CpG-ODN in normal brain. However, intratumoral application proved ineffective in a rat glioma model. CpG-ODN treatment may not yield beneficial effects in glioma patients.     

Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDC) represent a highly specialized immune cell subset that produces large quantities of the anti-viral cytokines type I interferons (IFN-alpha and IFN-beta) upon viral infection. PDC employ a member of the family of toll-like receptors, TLR9, to detect CpG motifs (unmethylated CG dinucleotides in certain base context) present in viral DNA. A certain group of CpG motif-containing oligodeoxynucleotides (CpG ODN), CpG-A, was the first synthetic stimulus available that induced large amounts of interferon-alpha (IFN-alpha) in PDC. However, the mechanism responsible for this activity remained elusive. CpG-A is characterized by a central palindrome and poly(G) at the 5' and 3' end. Here we demonstrate that CpG-A self-assembles to higher order tertiary structures via G-tetrad formation of their poly(G) motifs. Spontaneous G-tetrad formation of CpG-A required the palindrome sequence allowing structure formation in a physiological environment. Once formed, G-tetrad-linked structures were stable even under denaturing conditions. Atomic force microscopy revealed that the tertiary structures formed by CpG-A represent nucleic acid-based nanoparticles in the size range of viruses. Similarly sized preformed polystyrene nanoparticles loaded with a CpG ODN that is otherwise weak at inducing IFN-alpha (CpG-B) gained the potency of CpG-A to induce IFN-alpha. Higher ODN uptake in PDC was not responsible for the higher IFN-alpha-inducing activity of CpG-A or of CpG-B-coated nanoparticles as compared with CpG-B. Based on these results we propose a model in which the spatial configuration of CpG motifs as particle is responsible for the virus-like potency of CpG-A to induce IFN-alpha in PDC.     

Novel immunostimulatory agent based on CpG oligodeoxynucleotide linked to the nontoxic B subunit of cholera toxin

In this study, we report the development of a novel, rationally designed immunostimulatory adjuvant based on chemical conjugation of CpG oligodeoxynucleotide (ODN) to the nontoxic B subunit of cholera toxin (CTB). We demonstrate that the immunostimulatory effects of CpG can be dramatically enhanced by conjugation to CTB. Thus, CpG ODN linked to CTB (CTB-CpG) was shown to be a more potent stimulator of proinflammatory cytokine and chemokine responses in murine splenocytes and human PBMCs than those of CpG ODN alone in vitro. The presence of CpG motif, but not modified phosphorothioate ODN backbone, was found to be critical for the enhanced immunostimulatory effects of CTB-CpG. Our mode-of-action studies, including studies on cells from specifically gene knockout mice suggest that similar to CpG, CTB-CpG exerts its immunostimulatory effects through a TLR9/MyD88- and NF-kappaB-dependent pathway. Surprisingly, and as opposed to CpG ODN, CTB-CpG-induced immunity was shown to be independent of endosomal acidification and resistant to inhibitory ODN. Furthermore, preincubation of CTB-CpG with GM1 ganglioside reduced the immunostimulatory effects of CTB-CpG to those of CpG ODN alone. Interestingly, conjugation of CpG ODN to CTB confers an enhanced cross-species activity to CpG ODN. Furthermore, using tetanus toxoid as a vaccine Ag for s.c. immunization, CTB-CpG markedly enhanced the Ag-specific IgG Ab response and altered the specific pattern of Ab isotypes toward a Th1 type response. To our knowledge, CTB is the first nontoxic derivative of microbial toxins discovered that when chemically linked to CpG remarkably augments the CpG-mediated immune responses.     

Transfection of human monocyte-derived dendritic cells with CpG oligonucleotides

Monocyte-derived dendritic cells (mDC), the most frequently applied DC subset in clinical studies, which can be obtained easily from peripheral blood monocytes after incubation with GM-CSF and IL-4, have not been clearly demonstrated to be activated by CpG oligodeoxynucleotides (ODN). The development of novel molecular strategies - such as the use of CpG-ODN - to increase immunological functions and thus improve the therapeutic efficiency of mDC vaccines in the treatment of malignant diseases is highly desirable. CpG-ODN need to be internalized into specific intracellular compartments to be active. Therefore, we applied electroporation and lipofection and compared these techniques with incubation to overcome possible defects in localization. Conditions of CpG-ODN transfection of these cells were optimized using fluorescein-marked ODN 2216. We were able to achieve high transfection efficiencies with various methods of delivery. However, we did not observe increased expression of maturation-associated and functionally relevant surface antigens (CD14, HLA-DR, CD40, CD83, CD80 and CD86), significant secretion of IL-12 and IFN-alpha in culture supernatant, or enhanced antitumour activation of cytokine-induced killer cells. In conclusion, our results show that non-viral transfection of CpG-ODN is not sufficient to overcome resistance of mDC to CpG activation.     

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.