Role of the P2Y12 receptor in the modulation of murine dendritic cell function by ADP.

The effects of ADP on the biology of dendritic cells have been studied much less than those of ATP or adenosine. In this study, we showed that adenosine-5'-O-(2-thiodiphosphate) (ADPßS) induced intracellular Ca(2+) transients in murine dendritic cells (DCs). This effect was abolished by AR-C69931MX, a dual P2Y(12) and P2Y(13) receptor antagonist. RT-PCR experiments revealed the expression of both P2Y(12) and P2Y(13) mRNA in DCs. The Ca(2+) response to ADPßS was maintained in P2Y(13)-deficient DCs, whereas it was abolished completely in P2Y(12)(-/-) DCs. ADPßS stimulated FITC-dextran and OVA capture in murine DCs through macropinocytosis, and this effect was abolished in P2Y(12)(-/-) DCs. ADPßS had a similar effect on FITC-dextran uptake by human monocyte-derived DCs. OVA loading in the presence of ADPßS increased the capacity of DCs to stimulate OVA-specific T cells, whereas ADPßS had no effect on the ability of DCs to stimulate allogeneic T cells. Moreover, after immunization against OVA, the serum level of anti-OVA IgG1 was significantly lower in P2Y(12)(-/-) mice than that in wild-type controls. In conclusion, we have shown that the P2Y(12) receptor is expressed in murine DCs and that its activation increased Ag endocytosis by DCs with subsequent enhancement of specific T cell activation.

Transferrin receptor targeted lipopolyplexes for delivery of antisense oligonucleotide g3139 in a murine k562 xenograft model.

PURPOSE: Transferrin (Tf) conjugated lipopolyplexes (LPs) carrying G3139, an antisense oligonucleotide for Bcl-2, were synthesized and evaluated in Tf receptor positive K562 erythroleukemia cells and then in a murine K562 xenograft model. MATERIALS AND METHODS: Particle size and Zeta potentials of transferrin conjugated lipopolyplexs containing G3139 (Tf-LP-G3139) were measured by Dynamic Light Scattering and ZetaPALS. In vitro and in vivo sample's Bcl-2 downregulation was analyzed using Western blot and tumor tissue samples also exhibited by immunohistochemistry method. For athymic mice bearing with K562 xenograft tumors, tumor growth inhibition and survival rate were investigated. Nanoparticle distribution in 3-D cell cluster was observed by Laser scan confocal microscopy. IL-12 production in the plasma was measured by ELISA kit. RESULTS: In vitro, Tf-LP-G3139 was more effective in inducing down regulation of Bcl-2 in K562 cells than non-targeted LP-G3139, free G3139 and mismatched control ODN-G4126 in the same formulation. In vivo Tf-LP-G3139 was less effective than free G3139 in Bcl-2 down regulation. 3-D cell cluster model diffusion results indeed indicated limited penetration of the LPs into the cell cluster. Finally, the therapeutic efficacies of Tf-LP-G3139 and free G3139 were determined in the K562 xenograft model. Tf-LP-G3139 showed slower plasma clearance, higher AUC, and greater accumulation in the tumor compared to free G3139. In addition, Tf-LP-G3139 was found to be more effective in tumor growth inhibition and prolonging mouse survival than free G3139. This was associated with increased spleen weight and IL-12 production in the plasma. CONCLUSION: The role of the immune system in the therapeutic response obtained with the Tf-LPs is necessary and in vitro 3-D cell cluster model can be a potential tool to evaluate the nanoparticle distribution.

Dual ligand stimulation of RAW 264.7 cells uncovers feedback mechanisms that regulate TLR-mediated gene expression.

To characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-gamma, 2-methyl-thio-ATP (2MA), PGE(2), and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducibly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited; <24% of the features with significant responses to the single ligands responded nonadditively to a dual ligand pair. PGE(2) and ISO mainly attenuated, while 2MA enhanced, LPS-induced transcriptional changes. IFN-gamma and LPS cross-regulated the transcriptional response induced by each other: while LPS preferentially enhanced IFN-gamma-induced changes in gene expression at 1 h, IFN-gamma signaling primarily attenuated LPS-induced changes at 4 h. Our data suggest specific cross-talk mechanisms: 1) LPS enhances the expression of IFN-gamma-response genes by augmenting STAT1 activity and by activating NF-kappaB, which synergizes with IFN-gamma-induced transcriptional factors; 2) IFN-gamma attenuates the late LPS transcriptional response by increasing the expression of suppressor of cytokine signaling 1 and cytokine-inducible SH2-containing protein expression; 3) 2MA modulates LPS secondary transcriptional response by increasing IFN-beta and inhibiting IL-10 gene expression; 4) PGE(2) and ISO similarly regulate the LPS transcriptional response. They increase IL-10 transcription, resulting in attenuated expression of known IL-10-suppressed genes.

Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways.

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.

Cutting edge: species-specific TLR9-mediated recognition of CpG and non-CpG phosphorothioate-modified oligonucleotides.

Different DNA motifs are required for optimal stimulation of mouse and human immune cells by CpG oligodeoxynucleotides (ODN). These species differences presumably reflect sequence differences in TLR9, the CpG DNA receptor. In this study, we show that this sequence specificity is restricted to phosphorothioate (PS)-modified ODN and is not observed when a natural phosphodiester backbone is used. Thus, human and mouse cells have not evolved to recognize different CpG motifs in natural DNA. Nonoptimal PS-ODN (i.e., mouse CpG motif on human cells and vice versa) gave delayed and less sustained phosphorylation of p38 MAPK than optimal motifs. When the CpG dinucleotide was inverted to GC in each ODN, some residual activity of the PS-ODN was retained in a species-specific, TLR-9-dependent manner. Thus, TLR9 may be responsible for mediating many published CpG-independent responses to PS-ODN.

Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.

Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.

Immunostimulating capacities of stabilized RNA molecules.

Since direct injection of naked mRNA induces an immune response, we tested the capacity of RNA to signal danger. We show here that mRNA molecules that are protected from immediate degradation either through interaction with cationic proteins (trans protection) or through chemical modification of the phosphodiester backbone (phosphorothioate RNA; cis protection) act as sequence-independent danger signals on mouse DC. As opposed to CpG DNA, the cis-stabilized RNA is degraded in a few minutes, does not activate B cells and, in contrast to double-stranded RNA, requires MyD88 for activation of the DC. We postulate that phosphorothioate RNA, which mimics trans-stabilized RNA, is a new PAMP.

Amb a 1-immunostimulatory oligodeoxynucleotide conjugate immunotherapy decreases the nasal inflammatory response.

BACKGROUND: Amb a 1-immunostimulatory phosphorothioate oligonucleotide conjugate (AIC) is a novel immunotherapeutic compound consisting of purified Amb a 1 from short ragweed proteins covalently linked to an immunostimulatory phosphorothioate oligodeoxyribonucleotide. In sensitized animals AIC can stimulate an Amb a 1-specific T(H)1 response and decrease pulmonary reactivity to ragweed challenge. Clinical trials have documented reduced allergic response to AIC in comparison with licensed ragweed extract. OBJECTIVES: We sought to determine the in vivo effect of short-course immunotherapy with AIC on eosinophilia and cytokine mRNA expression in the nasal mucosa of ragweed-sensitive patients. METHODS: Ragweed-sensitive patients with allergic rhinitis were treated with 6 escalating doses of AIC (0.06-12 microg, n = 28) or placebo (n = 29) at weekly intervals immediately before the 2001 ragweed season. Symptom scores and medication use were recorded for the 2001 and 2002 ragweed seasons for all patients. A subset of patients (12 receiving AIC and 7 receiving placebo) consented to have nasal biopsy specimens taken before immunization and before and after the first ragweed season. The preseason and postseason biopsy specimens were taken 24 hours after ragweed allergen challenge and compared with the initial unchallenged biopsy specimen to assess cytokine and inflammatory cell responses by using immunocytochemistry and in situ hybridization. RESULTS: AIC was safe and well tolerated by all patients. There was no difference between the AIC and placebo groups in the number of allergen-induced major basic protein-, IL-4-, IL-5-, or IFN-gamma-positive cells in the mucosa in the first weeks after AIC immunization. On rechallenge and rebiopsy after the end of the 2001 ragweed season, however, AIC-treated patients had a significantly reduced increase in eosinophils and IL-4 mRNA-positive cells and an increased number of IFN-gamma mRNA-positive cells compared with placebo-treated patients. No difference between treatment groups was observed in symptom scores or medication use during the first ragweed season. During the second ragweed season, however, there was a significant decrease in chest symptoms and a trend toward reduced nasal symptoms in the AIC-treated group. CONCLUSION: Short-course immunotherapy with AIC can modify the response of nasal mucosa to allergen challenge by increasing T(H)1 cytokine production and decreasing T(H)2 cytokine production and eosinophilia. This modification was not immediate but was observed 4 to 5 months after completion of immunotherapy and seasonal ragweed-pollen exposure. The T-cell subset shift after immunization and seasonal exposure was followed by evidence of clinical efficacy in the second ragweed season without additional AIC immunizations.

Strong cytosine-guanosine-independent immunostimulation in humans and other primates by synthetic oligodeoxynucleotides with PyNTTTTGT motifs.

Synthetic oligodeoxynucleotides (ODNs) containing cytosine-guanosine (CpG) motifs stimulate B and plasmacytoid dendritic cells of the vertebrate immune system. We found that in primates strong stimulation of these cells could also be achieved using certain non-CpG ODNs. The immunostimulatory motif in this case is a sequence with the general formula PyNTTTTGT in which Py is C or T, and N is A, T, C, or G. Assays performed on purified cells indicated that the immunostimulatory activity is direct. The use of a nuclease-resistant phosphorothioate backbone is not a necessary condition, since phosphodiester PyNTTTTGT ODNs are active. It was also demonstrated that ODN 2006, a widely used immunostimulant of human B cells, possess two kinds of immunostimulatory motifs: one of them mainly composed of two successive TCG trinucleotides located at the 5' end and another one (duplicated) of the PyNTTTTGT kind here described. Even though PyNTTTTGT ODNs are mainly active on primate cells, some of them, bearing the CATTTTGT motif, have a small effect on cells from other mammals. This suggests that the immunostimulatory mechanism activated by these ODNs was present before, but optimized during, evolution of primates. Significant differences in the frequency of PyNTTTTGT sequences between bacterial and human DNA were not found. Thus, the possibility that PyNTTTTGT ODNs represent a class of pathogen-associated molecular pattern is unlikely. They could, more reasonably, be included within the category of danger signals of cell injury.

Inhibitory oligonucleotides block the induction of AP-1 transcription factor by stimulatory CpG oligonucleotides in B cells.

The proliferative response of primary B cells to CpG oligonucleotides (ODN) involves induction of nuclear activation promoting-1 (AP-1) transcription factor. AP-1 subunits c-Fos, Fos-B, Jun-B, and Jun-D, but not Fra-1 or Fra-2, were all induced by CpG ODNs in B cells within 30 minutes of stimulation, followed by c-Jun at 1-2 hours. c-Jun reached maximum at 6 hours. By 40 hours, Jun-B and Jun-D became dominant. Synthetic ODNs containing a single guanosine triplet/tetrad appropriately distanced from the 5' pyrimidine-rich unit, which inhibit CpG-driven cell cycle entry and apoptosis protection, blocked AP-1 induction by stimulatory ODNs when they were added simultaneously. After 30 minutes of stimulation, adding inhibitor no longer affected AP-1 at 6 hours. No AP-1 subunits escaped ODN inhibition. In a cell line transfected with an AP-1-beta-galactosidase reporter construct, CpG ODN-induced AP-1 transcriptional activity was prevented by inhibitory ODN, but lipopolysaccharide (LPS)-induced AP-1 activity was not. These data suggest that inhibitory ODNs block the CpG ODN-driven signaling pathway at a site proximal to AP-1 induction.

A phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant.

Certain oligodeoxynuclotides with CpG motifs provide enhanced immune response to co-delivered antigens. We performed a phase I, observer-blinded, randomized study in healthy anti-hepatitis B surface antigen (anti-HBsAg) antibody negative adults to explore safety and immunogenicity of co-injection of recombinant HBsAg combined with an immunostimulatory DNA sequence (ISS) 1018 ISS. Four ISS dosage groups (N=12 per group) were used: 300, 650, 1000 or 3000 microg. For each group, two controls received 20 microg HBsAg alone, two controls received ISS alone, and eight subjects received ISS+20 microg HBsAg. Subjects received two doses 8 weeks apart. Injection site reactions (tenderness and pain on limb movement) were more frequent at higher ISS+HBsAg doses but were mainly mild and of short duration. Higher anti-HBsAg antibody levels were associated with higher ISS doses. Four weeks after the first dose, a seroprotective titer (>or=10 mIU/ml) was noted for 0, 25, 75, and 87.5% of subjects by increasing ISS dose group (P<0.05) for those who received ISS+HBsAg; 1 month after the second dose this increased to 62.5, 100, 100, and 100%, respectively. Geometric mean anti-HBsAg antibody levels by increasing ISS+HBsAg dose were 1.22, 5.78, 24.75, and 206.5 mIU/ml after the first dose and 65.37, 877.6, 1545, and 3045 mIU/ml after the second dose. We conclude that 1018 ISS+HBsAg was well tolerated and immunogenic in this phase I study in healthy adults and may offer the potential for enhancement of hepatitis B virus (HBV) immunization and protection after one or two doses or in individuals who fail to respond to the standard vaccine regimen.

Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells.

Reaction to certain motifs in bacterial DNA is an important function of natural immunity. For example, single stranded oligonucleotides (ODN) containing the motif "not C, unmethylated C, G, not G" are powerful mitogens and apoptosis inhibitors for mouse spleen B cells. But replacing GCGTT or ACGTT with GCGGG or ACGGG converted a stimulatory 15-mer ODN into an inhibitory ODN. All inhibitory ODN had three consecutive G, and a fourth G increased inhibitory activity, but a deazaguanosine substitution to prevent planar stacking did not affect activity. Inhibitory ODN blocked apoptosis protection and cell-cycle entry induced by stimulatory ODN, but not that induced by lipopolysaccharide, anti-CD40 or anti-IgM+IL-4. ODN-driven up-regulation of cyclin D(2), c-Myc, c-Fos, c-Jun and Bcl(XL) and down-regulation of cyclin kinase inhibitor p27(kip1) were all blocked by inhibitory ODN. The relative potency of a series of stimulatory and inhibitory ODN was the same for all readouts measured. Interference with uptake of stimulatory ODN could not account for their inhibitory effects. Even if addition of inhibitory ODN was delayed several hours, partial inhibition of stimulatory ODN effects occurred. Inhibitory ODN hold potential as antidotes for excessive ODN stimulation in the clinical setting and provide an important tool for studying ODN recognition.

Development of an ultrasensitive noncompetitive hybridization-ligation enzyme-linked immunosorbent assay for the determination of phosphorothioate oligodeoxynucleotide in plasma.

An ultrasensitive noncompetitive hybridization-ligation heterogeneous enzyme-linked immunosorbent assay was developed for the quantitation of antisense phosphorothioate oligodeoxynucleotides in plasma using a 96-well plate format. The principle of the assay is based on heterogeneous noncompetitive binding of the analyte to a template probe, followed by addition of signal probe via ligation and detection using a fluorescence microtiter plate reader. The result showed no significant interference noted from untreated human plasma. In addition, the method is selective for the specific sequence tested (ISIS 2302) and cross-reactivity toward the 3'-metabolites is minimal (< 0.22%). A linear range of 0.05 to 2 nM (r > 0.99) was obtained in human plasma for ISIS 2302. Intraday and interday accuracy for the method was found to be within 80-120% of actual value. Intraday and interday precision has a percentage coefficient of variation less than 20%. The lower limit of quantitation of the method was 0.05 nM (0.05 pmol/ml) with 100 microL plasma or an absolute amount of 5 fmol. In summary, the assay was demonstrated to be specific, accurate, precise, and sensitive for the quantitation of ISIS 2302 in human plasma and was applied to the analysis of plasma samples in pharmacokinetic studies.

Enhancement of innate immunity against Mycobacterium avium infection by immunostimulatory DNA is mediated by indoleamine 2,3-dioxygenase.

Bacterial DNA and its synthetic immunostimulatory oligodeoxynucleotide analogs (ISS-ODN) activate innate immunity and promote Th1 and cytotoxic T-lymphocyte immune responses. Based on these activities, we investigated whether ISS-ODN could modify the course of Mycobacterium avium infection. M. avium growth in vitro was significantly inhibited by ISS-ODN treatment of human and mouse macrophages, and M. avium growth in vivo was similarly inhibited in C57BL/6 mice treated with ISS-ODN. This protective effect of ISS-ODN was largely independent of tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), nitric oxide, NADPH oxidase, alpha/beta interferon (IFN-alpha/beta), and IFN-gamma. In contrast, we found that the induction of indoleamine 2,3-dioxygenase (IDO) was required for the antimycobacterial effect of ISS-ODN. To evaluate the potential for synergism between ISS-ODN and other antimycobacterial agents, treatment with a combination of ISS-ODN and clarithromycin (CLA) was tested in vitro and in vivo. ISS-ODN significantly enhanced the therapeutic effect of CLA in both human and mouse macrophages and in C57BL/6 mice. This study newly identifies IDO as being involved in the antimicrobial activity of ISS-ODN and suggests the usefulness of ISS-ODN when used in combination with conventional chemotherapy for microbial infections.

Immunostimulatory DNA inhibits IL-4-dependent IgE synthesis by human B cells.

BACKGROUND: Immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) is a potent antiallergic immunomodulating agent in mice. However, few studies have addressed its antiallergic potential in human subjects. OBJECTIVE: We sought to determine whether a phosphoro-thioate ISS-ODN could inhibit IL-4-dependent IgE synthesis by human B cells. METHODS: Initially, nonatopic- and atopic-donor PBMCs were incubated with ISS-ODN or mutated oligodeoxynucleotide, and cytokine production and B-cell expression of IFN-gamma receptor and IL-4 receptor were measured by using ELISA and flow cytometry, respectively. In subsequent studies atopic-donor PBMCs were incubated with IL-4 alone or with ISS-ODN or mutated oligodeoxynucleotide. After 14 days, IgE production and IgM, IgG, and IgA production were determined by using ELISA. In select IgE studies cytokines were neutralized with mAbs. RESULTS: ISS-ODN induced IL-12, IFN-alpha, IFN-gamma, IL-10, and IL-6 production from both nonatopic- and atopic-donor PBMCs. ISS-ODN also increased IFN-gamma receptor and inhibited IL-4 receptor expression on B cells from both donor populations. Furthermore, ISS-ODN inhibited IL-4-dependent IgE production by atopic-donor PBMCs. Neutralization of IL-12, IFN-alpha, IFN-gamma, and IL-10, but not IL-6, attenuated the inhibitory activity of ISS-ODN on IgE production. In contrast to its inhibition of IgE synthesis, ISS-ODN stimulated the production of IgM, IgG, and IgA. CONCLUSION: These in vitro studies demonstrate that phos-phorothioate ISS-ODN elicits an innate immune response by PBMCs, which inhibits IL-4-dependent IgE synthesis. In addition, these results provide further support for consideration of ISS-ODN therapy for the treatment of allergic disease in clinical practice.

A novel function of phosphorothioate oligodeoxynucleotides as chemoattractants for primary macrophages.

Phosphorothioate cytosine-guanine oligodeoxynucleotides (CpG PS-ODNs) has been reported to induce Th1 immune responses against coadministered Ags more efficiently than phosphodiester CpG ODNs (CpG PO-ODNs). Here, we demonstrated that PS-ODNs, but not PO-ODNs, have a chemotactic effect on primary macrophages, which is independent of the CpG motif. In addition, the conjugation of a hexameric dG run (dG(6) run) at the 3' terminus reduced the concentration required for the optimal chemotactic activity of PS-ODNs by approximately 10-fold. Endosomal maturation blockers, such as monensin and chloroquine, inhibited the chemotactic effect of PS-ODNs. The inhibition of the activities of p38 mitogen-activated protein (MAP) kinase, and extracellular signal-related kinases (ERKs) as well as phosphoinositide 3-kinase with their specific inhibitors also resulted in suppressing the chemotaxis of primary macrophages induced by PS-ODNs. These results indicate that the PS-ODN-mediated chemotaxis requires the activation of ERKs, p38 MAP kinase, and phosphoinositide 3-kinase as well as endosomal maturation. In addition, the phosphorylations of the p38 MAP kinase, ERKs, and protein kinase B, Akt, were induced by PS-ODN, which were further enhanced by the presence of both a dG(6) run and CpG motifs. Our findings suggest that the chemotactic activity of PS-ODNs may be one of the mechanisms by which PS-ODNs exhibit stronger immunomodulatory activities than PO-ODNs in vivo.

Enhancement of antigen-presenting ability of B lymphoma cells by immunostimulatory CpG-oligonucleotides and anti-CD40 antibody.

Immunostimulatory oligodeoxynucleotides containing the CpG motifs (CpG-ODN) can activate antigen-presenting cells including dendritic cells, macrophages, B cells, and enhance production of Thl cytokines. So, CpG-ODN has been regarded as a promising immune adjuvant. Using the A20 B lymphoma cell model, we investigated the effect of CpG-ODN on the immunogenicity of B lymphoma cells and whether CpG-ODN could enhance the antigen-presenting ability of B lymphoma cells. After incubation with CpG-ODN, proliferation of A20 cells remained unchanged. But CpG-ODN stimulation up-regulated the expression of MHC-I, MHC-II, CD40, ICAM-1 molecules in A20 cells, enhanced the antigen uptake ability of A20 cells, and promoted A20 cell production of IgM and IgG. More importantly, A20 cells activated by CpG-ODN could stimulate allogeneic T cells in MLR and antigen-primed T cells to proliferate more efficiently, suggesting the antigen-presenting ability of A20 B lymphoma cells could be enhanced by CpG-ODN stimulation and CpG-ODN-activated B lymphoma cells might be used as a potent cellular vaccine. Although anti-CD40 mAb was as effective as CpG-ODN at activating A20 cells and A20 cells expressed more CD40 molecules after CpG-ODN stimulation, a combination of CpG-ODN and anti-CD40 mAb had no synergistic effect on A20 cell activation. Our data expanded the potential application of CpG-ODN as an immunotherapeutic agent in cancer treatment.

Prevention of acute allergic conjunctivitis and late-phase inflammation with immunostimulatory DNA sequences.

PURPOSE: To evaluate the therapeutic potential of immunostimulatory sequence oligodeoxynucleotides (ISS-ODN) administration in ocular allergy, using a mouse model of ragweed-specific conjunctivitis. METHODS: A murine model of allergic conjunctivitis involving SWR/J mice sensitized and challenged with short ragweed was used to test immunostimulatory DNA sequences for therapeutic potential. ISS-ODN or control ODN (0.1 mg/mouse) was administered intraperitoneally or topically to the conjunctiva 3 days before final allergen challenge. Multiple parameters of clinical symptoms evident during the acute-phase reaction and the cellular components of the late-phase reaction were evaluated in both groups of mice. RESULTS: All parameters of clinical symptoms were markedly inhibited after intraperitoneal injection of ISS-ODN, whereas topical application to the conjunctiva did not inhibit clinical symptoms significantly. Remarkably, a single topical treatment with ISS-ODN (as well as by intraperitoneal injection) completely inhibited both eosinophilia and neutrophilia in the late-phase reaction. CONCLUSIONS: Systemic or conjunctival administration of ISS-ODN was shown to significantly inhibit allergic responses in this mouse model. This indicates that ISS-ODN may be an effective form of immunotherapy for this class of allergic disease.

Conjugation of protein to immunostimulatory DNA results in a rapid, long-lasting and potent induction of cell-mediated and humoral immunity.

Immunostimulatory DNA sequences (ISS) are a potent Th1 adjuvant. We hypothesized that conjugation of ISS to protein antigens would strongly enhance their immunogenicity because both antigen and adjuvant (ISS) would be delivered to the same locale/antigen-presenting cell. To test this hypothesis, we conjugated a 22-mer immunostimulatory oligodeoxynucleotide (ISS-ODN) to two test antigens of differing intrinsic immunogenicity, namely Escherichia coli beta-galactosidase and the HIV-1 envelope glycoprotein gp120. We show that the antigen-ISS conjugates rapidly induce Th1 cells secreting high levels of IFN-gamma, strong CTL activity, and high titer IgG2a and HIV-neutralizing antibodies, exceeding gene and protein vaccination alone or immunization with mixtures of antigen and ISS-ODN. The data suggest that this procedure generates a novel and unique vaccine that rapidly triggers strong humoral and cell-mediated immunity.