Immunostimulatory DNA Research Articles

In Vivo CpG Administration Accelerates Graft-Versus-Host Disease (GVHD) Lethality by Toll-Like Receptor 9 (TLR9) Ligation of Host Antigen-Presenting Cells (APCs) and Promotes Allogeneic Bone Marrow (BM) Rejection by TLR9 Ligation of Donor APCs.

Synthetic cytosine-phosphorothioate-guanine (CpG) oligodeoxynucleotides mimic immunostimulatory bacterial DNA and bind to TLR9 present on APCs, resulting in their activation. CpG have potent anti-tumor effects and are in clinical trials. The systemic effects of CpG given in the peri-BMT period are unknown. We now report that GVHD was markedly increased by weekly B-class CpG administration in 2 distinct full MHC disparate systems. As compared to controls, CpG reproducibly resulted in a significant decrease in survival rates (37–50% vs 0%). CpG induced proinflammatory cytokine release (IL12p70; IFNγ; TNFα; IL6), indicative of APC activation, as measured on post-BMT days 2, 4, 6, and 8 in recipients of BM or BM and spleen. On day +14, in vivo imaging using donor GFP transgenic T cells revealed a profound increase in T cell infiltration in secondary lymphoid organs (spleen, Peyers patches) and in other GVHD target organs (liver, lung, skin, ileum, colon), indicating that accelerated lethality was not due exclusively to cytokine-induced tissue injury. To determine whether donor or host APCs contributed to CpG-accelerated GVHD, CpG was given to wild type (wt) or TLR9 deficient recipients of allogeneic wt or TLR9 deficient donor splenocytes. Host but not donor TLR9-expressing cells were essential for GVHD acceleration. To determine which cytokine/receptor interactions were required for CpG-induced GVHD, studies were performed with cytokine/receptor deficient hosts. Either IFNγ or TNF, but not IL-6 or IL-12p40, was required for increased GVHD. Because CpG augmented donor anti-host allogeneic responses, we studied the effects of CpG on host anti-donor mediated BM rejection. CpG given on days 0 and 7 to sublethally irradiated (6.0–6.25 Gray) recipients of full MHC disparate increased host rejection of T cell depleted (TCD) BM, resulting in aplasia seen in 50–100% of CpG-treated vs 0–10% of controls. In sublethally irradiated (5.5 Gray) recipients of single MHC disparate TCD BM, mean donor chimerism levels in peripheral blood significantly decreased from 91% to 37% (MHC class I system) and 77% to 39% (MHC class II system) for control vs CpG-treated mice, indicating CpG stimulated host CD8− and CD4− mediated rejection. A brief incubation of TCD BM with CpG prior to BMT was not sufficient to cause rejection. CpG-treated recipients deficient in IL6, IL12p40, IFNγ or TNFR1 all had increased rejection, indicating that none of these cytokines were essential for promoting graft rejection. To determine whether donor or host TLR9 ligation stimulated graft rejection, CpG were given to wt or TLR9 deficient recipients of allogeneic wt or TLR9 deficient, TCD BM. In contrast to GVHD, donor and not host TLR9 ligation was essential for stimulating BM rejection. These data indicate a role of donor APCs in allogenenic BM rejection and provide further insight into the role of TLR9 signaling in GVHD. Our studies have important implications for the testing of CpG in clinical trials.

In Vivo Control of Acute Lymphoblastic Leukemia by Immunostimulatory CpG Oligonucleotides.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While current therapy is effective for the majority of children, relapsed ALL remains the fifth most common pediatric malignancy and responds very poorly to treatment. In addition, about 50% of ALL cases occur in adults, and while remission is achieved in many patients the majority of these will succumb to relapsed disease. In most cases relapse is treated with more intensive chemotherapy or hematopoietic cell transplantation, therapies that are associated with significant toxicity. In both patient populations, therefore, there is an urgent need for novel approaches to inhibit disease progression. Immunostimulatory DNA oligonucleotides containing CpG motifs (CpG ODN) have been shown to induce strong anti-tumor immune activity in a variety of model systems and are currently being evaluated in clinical trials. We have previously reported that CpG ODN stimulation of ALL cells alters their antigen presentation ability and enhances Th1 responses by allogeneic T cells. In this study, using NOD-SCID mice bearing xenografts of primary human ALL or human ALL cell lines, we demonstrate that CpG ODN stimulate significant immune activity against ALL cells in vivo. A single intravenous injection of 300micrograms Class B CpG ODN induced a significant reduction in percentage of human ALL cells in peripheral blood (P<0.0001), spleen (P=0.0036) and liver (P=0.0004), but not bone marrow (P=0.254), compared to PBS treatment of mice. The reduced leukemia burden in the spleens of CpG ODN treated mice correlated with a significantly higher percentage of Annexin-V positive ALL cells (P=0.008 for PBS vs CpG; P=0.016 for CpG vs control ODN), indicating the induction of leukemia cell death in these mice. In addition, repeated administration of CpG ODN mediated continued disease control, and significantly improved survival of mice with established primary sample or cell line derived human ALL (P=0.002). The death of leukemia cells in vivo was independent of the ability of ALL cells to respond directly to CpG ODN, and correlated with the production of significant amounts of interferon-alpha, interferon-gamma and IL-12 in treated mice. Cell depletion studies implicate natural killer cells in the CpG ODN induced killing of human ALL. Preliminary results obtained using the Emu-ret transgenic mouse model of B cell leukemia indicate that CpG ODN treatment also induces killing of syngeneic leukemia cells, and work is ongoing to identify the immune mechanisms mediating this activity. Based on these results, we hypothesize that CpG ODN will have considerable potential as a novel agent for the prevention of ALL progression.

Oral Administration of an Immunostimulatory DNA Sequence fromBifidobacterium longumImproves Th1/Th2 Balance in a Murine Model

We have reported the antiallergic activities of the immunostimulatory oligodeoxynucleotide (ODN) BL07S, identified from genomic DNA of Bifidobacterium longum BB536 from in vitro and in vivo studies. The present study evaluated the efficiency of ODN BL07S in preventing allergic responses by oral administration. Oral administration of BL07S suppressed serum ovalbumin (OVA)-specific immunoglobulin (Ig) E levels and improved the OVA-specific IgG2a/IgG1 ratio. ODN BL07S increased Th1 cytokine and decreased Th2 cytokine production in splenocytes. These results suggest that immunostimulatory ODNs are potentially associated with the antiallergic effects of probiotics.

Modulation of immunogenicity and allergenicity by controlling the number of immunostimulatory oligonucleotides linked to Amb a 1

Immunostimulatory DNA sequences (ISS) are potent immunomodulators that can drive T(H)1 responses to antigens or allergens. This effect can be dramatically enhanced by direct linkage of ISS to the protein. Evaluate the effects of the number of ISS bound to the major ragweed allergen Amb a 1 on immunogenicity and allergenicity. Immunogenicity in mice and allergenicity using PBMC or sera from subjects with ragweed allergy were assayed. Both antibody induction in vivo and antibody recognition in vitro were highly sensitive to the number of ISSs linked. IgE recognition of Amb a 1 in competitive ELISA or histamine release assays was inhibited by ISS linkage and showed an inverse relationship to the number of ISSs bound. Type and magnitude of antibody induction in mice was also highly dependent on the number of ISS bound. At the highest ISS to protein ratios, antibody induction was very low. Moderate ISS to protein ratios induced high antibody responses in which IgG(2a) generally predominated. Low ISS to protein ratios produced the highest overall antibody responses in which IgG(1) predominated. In contrast, varied ISS to protein ratios did not affect T-cell responses. In both in vivo mouse studies and in vitro human PBMC studies, all ISS to protein ratios evaluated induced similar responses represented by high levels of IFN-gamma and low levels of T(H)2 cytokines. Controlling the number of ISS bound to a protein allows manipulation of antibody recognition and induction while retaining the potent T(H)1 properties of an ISS-linked protein. Immunostimulatory DNA sequence-linked Amb a 1 conjugate represents a safe, novel therapeutic approach for treating ragweed allergy.

Remodeling associated expression of matrix metalloproteinase 9 but not tissue inhibitor of metalloproteinase 1 in airway epithelium: Modulation by immunostimulatory DNA

Matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor of metalloproteinase 1 (TIMP-1) are hypothesized to play a role in the pathogenesis of airway remodeling in asthma. We have used a mouse model of airway remodeling to determine the pattern of expression of MMP-9 and TIMP-1 in airway epithelium and peribronchial cells, and assess whether TIMP-1, an inhibitor of MMP-9, is expressed at the same sites in the airway. In addition, we have investigated whether immunostimulatory sequences (ISSs) of DNA modulate levels of expression of MMP-9, TIMP-1, and peribronchial fibrosis. Levels of lung MMP-9 and TIMP-1 were assessed by zymography, ELISA, and immunohistochemistry. Repetitive ovalbumin challenge induced a significant increase in levels of MMP-9, TIMP-1, and peribronchial collagen deposition. The pattern of expression of MMP-9 and TIMP-1 in the remodeled airway was significantly different. MMP-9 but not TIMP-1 was expressed in airway epithelium, whereas both MMP-9 and TIMP-1 were expressed in peribronchial inflammatory cells. ISS significantly reduced expression of MMP-9 in airway epithelium (which immunostained positive for Toll receptor 9), as well as in peribronchial inflammatory cells. In vitro studies demonstrated that ISS inhibited bone marrow macrophage generation of MMP-9. Allergen-induced peribronchial fibrosis is associated with expression of MMP-9 and TIMP-1 at different anatomical sites in the remodeled airway. The ability of ISS to inhibit the expression of MMP-9 in airway epithelium (a site where its inhibitor TIMP-1 is not induced by allergen challenge) may be important in determining whether ISS contributes to reductions in airway remodeling by reducing levels of MMP-9. Immunostimulatory sequences of DNA, which are being investigated as novel therapeutics in asthma, inhibit airway remodeling in mice as well as epithelial expression of MMP-9, an enzyme that degrades the extracellular matrix proteins surrounding the airway.

Suppression of Allergic Response by CpG Motif Oligodeoxynucleotide–House-Dust Mite Conjugate in Animal Model of Allergic Rhinitis

Although there have been many therapeutic options for allergic disease, the true allergen desensitization remains a challenging goal. The classic immunotherapy has a limited efficacy, is inconvenient, and has a risk of anaphylaxis. Recent reports revealed that immunostimulatory DNA sequences (ISS-oligdeoxynucleotide [ODN], CpG motif) act as a strong Th1 response-inducing adjuvants and that DNA-based vaccination might be an effective therapeutic option. In this study, we investigate whether ISS-ODN/Dermatophagoides farinae (Der f) conjugate has antiallergic effects in the allergic rhinitis mouse model, sensitive to house-dust mites. Der f is the most common allergen-inducing allergic rhinitis in Korea. C57BL/6 mice were sensitized with crude extract of Der f. After injection of ISS-ODN or ISS-ODN/Der f conjugate, several parameters of allergic response were evaluated. Scratching and sneezing symptoms and eosinophilic infiltration into nasal mucosa were suppressed by injection with ISS-ODN only and ISS-ODN/Der f conjugate. Interleukin-5 level was decreased and interferon gamma level was increased in nasal lavage fluid by injection of ISS-ODN/Der f conjugate. Der f-specific immunoglobulin E was decreased by injection of ISS-ODN or Der f /ISS-ODN conjugate; however, these were not statistically significant. Transforming growth factor beta1 secreted by cultured splenocyte was increased significantly in ISS-ODN/Der f conjugate group. These results suggest ISS-ODN/Der f conjugate induces an antiallergic effect and induces an increase in transforming growth factor beta1 level in the allergic rhinitis model using Der f allergen. Allergic response developed by Der f allergen could be more effectively reduced by injection with ISS-ODN/Der f conjugate than by injection with ISS-ODN only.

Therapeutic Use of The Co-Administration of Immunostimulatory DNA Sequence with Allergen Extract in a Mouse Model of Timothy Allergy

Therapeutic Use of The Co-Administration of Immunostimulatory DNA Sequence with Allergen Extract in a Mouse Model of Timothy Allergy

Immunostimulatory DNA inhibits allergen-induced peribronchial angiogenesis in mice

Airway remodeling in asthma is associated with angiogenesis. We have examined whether immunostimulatory sequences of DNA (ISSs) inhibit allergen-induced airway angiogenesis and expression of angiogenic cytokines in a mouse model of airway remodeling. Mice sensitized to ovalbumin were challenged repetitively with ovalbumin for three months to develop airway remodeling and angiogenesis. Levels of angiogenesis were compared in ISS-treated and control mice. Mice challenged with ovalbumin developed significantly increased levels of peribronchial angiogenesis (increase in the number of CD31+ peribronchial small blood vessels) and an increase in the peribronchial vascular area as assessed by image analysis. Ovalbumin-induced peribronchial angiogenesis was associated with increased bronchoalveolar lavage levels of vascular endothelial growth factor (VEGF) and an increase in the number of peribronchial cells expressing VEGF. Treatment of mice with ISS before repetitive ovalbumin challenge significantly reduced the levels of peribronchial angiogenesis as well as the levels of bronchoalveolar lavage VEGF and the number of peribronchial cells expressing VEGF. ISS is unlikely to act directly on endothelial cells to inhibit angiogenesis because lung endothelial cells did not express Toll receptor 9, the receptor for ISS as assessed by RT-PCR. In vitro studies demonstrated that ISS inhibited macrophage expression of VEGF. The ability of ISS to inhibit angiogenesis in vivo is likely to be mediated by several mechanisms, including ISS reducing the number of peribronchial inflammatory cells that express VEGF, ISS inhibiting expression of TH2 cytokines such as IL-13 that promote VEGF expression, and direct effects of ISS on macrophages to inhibit VEGF expression.

Immunostimulatory oligodeoxynucleotide isolated from genome wide screening of Mycobacterium bovis chromosomal DNA

Bacterial DNA has a variety of immunostimulatory activities, such as the activation of B cells and natural killer cells, the induction of interferon-γ, and the induction of Th1-type immune responses. In contrast, mammalian DNA does not have these activities. To evaluate the genomic DNA sequences of Mycobacterium bovis that have immunostimulatory activity, we used a computer to analyze the M. bovis genome and we designed a series of synthetic, 20 base length, phosphodiester backbone oligodeoxynucleotides (ODNs) that contain CpG motifs (MB-ODNs). We screened the immunostimulatory MB-ODNs that induce the activation of the NF-κB-responsive IL-8 promoter in RAW 264.7 cells. Our experimental analyses demonstrate that the potent CpG DNA in the M. bovis genome has functional effects as a Th1-responsive adjuvant, and that it activates the transcription factor NF-κB. Moreover, we found that both the CpG motifs and the context of the sequence surrounding the CpG motif are important for the immunostimulatory activities. The identification of the potent immunostimulatory DNA sequence in a native bacterial genome may give insights to the optimal sequence for well-controlled immune responses.

Activation of Toll-Like Receptor 9 by DNA from Different Bacterial Species

Toll-like receptor 9 (TLR-9) recognizes unmethylated CpG dinucleotides which are abundant in prokaryotic DNA and yet are rare in eukaryotic DNA. Little is known about the significance of TLR-9 in terms of recognition of different bacterial DNA species. In this study HEK293 cells stably transfected with human TLR-9 were used to analyze the immunostimulatory properties of 15 bacterial DNA preparations. In addition, bacterial genome data were analyzed for the frequency of unmethylated cytosine-guanosine ([CG]) dinucleotides. We observed that DNA samples of different bacteria showed considerable differences in their potential to stimulate TLR-9. This correlated with the frequency of [CG] dinucleotides. Based upon data from our experiments the estimate of immunostimulatory bacterial DNA concentrations translated to as high as 10(9) bacteria/ml. Application of the transfection reagent DOTAP resulted in a more efficient delivery of DNA into the cell, and this went along with increased TLR-9 activation. The data indicate that bacterial DNA preparations from different species differ in their capacity to activate TLR-9, which is dependent on the individual [CG] content. Moreover, increased intracellular delivery results in a marked enhancement of immunostimulation.

Peptide crosslinked micelles: a new strategy for the design and synthesis of peptide vaccines

This report presents a new and simple methodology for the synthesis of multicomponent peptide vaccines, named the peptide crosslinked micelles (PCMs). The PCMs are core shell micelles designed to deliver peptide antigens and immunostimulatory DNA to antigen-presenting cells (APCs). They are composed of immunostimulatory DNA, peptide antigen, and a thiopyridal derived poly(ethylene glycol)-polylysine block copolymer. The peptide antigen acts as a crosslinker in the PCM strategy, which allows the peptide antigen to be efficiently encapsulated into the PCMs and also stabilizes them against degradation by serum components. Cell culture studies demonstrated that the PCMs greatly enhance the uptake of peptide antigens into human dendritic cells.

Role of Immunostimulatory DNA and TLR9 in Gene Therapy

One of the key issues of DNA-based therapies is the immunostimulatory effect caused by DNA, which it has a significant impact on the consequences of these therapies. This is a result of the recognition by Toll-like receptor-9 (TLR9); a pattern recognition receptor for unmethylated CpG motifs within DNA sequences. However, recent studies show that TLR9 can recognize non-CpG motifs and induce cellular activation when DNA is efficiently delivered to TLR9, for example, by cationic lipids. Moreover, recent research shows that TLR9-independent immunoactivation could take place. Here, we have attempted to present an overview of immunoactivation by DNA to optimize DNA-based therapies.

Modified Recombinant Allergens for Safer Immunotherapy

Molecular cloning and recombinant production of allergens offered new perspectives for the increasing problem of allergies. A variety of preparations are being developed aiming to increase safety and improve efficacy of specific immunotherapy. Recombinant-based approaches are mostly focused on genetic modification of allergens to produce molecules with reduced allergenic activity and conserved antigenicity, i.e. hypoallergens. Studies dealing with genetic modifications of allergen genes reported the production of site-directed mutants, deletion mutants, allergen fragments and oligomers, and allergen chimeras. An alternative to genetic engineering is the chemical modification of pure recombinant allergens. It has been shown that allergens modified with immunostimulatory DNA sequences (allergen-ISS conjugates), which masks IgE epitopes and adds a desirable Th1-inducing character to the allergen molecule. Other chemical modifications include oligomerization by aldehydes (allergoids) and maleylation, which seems to target allergens to particular antigen presenting cells. Several of these modified allergen preparations have been already evaluated for their safety in clinical provocation studies. So far, clinical trials showed the efficacy and safety of immunotherapy with an Amb a 1-ISS conjugate for ragweed pollen-allergic patients. In addition, a preparation consisting of hypoallergenic fragments of Bet v 1 was evaluated for immunotherapy of birch pollen-allergic patients. In parallel, several animal studies have now demonstrated the potential of genetic immunization for allergy treatment in the future.

CpG DNA/zymosan complex to enhance cytokine secretion owing to the cocktail effect

Zymosan, classified among β-(1→3)- d-glucans, is produced from the cell wall of yeast and well known to induce proinflammatory cytokines when ingested by immune cells. We found that zymosan forms a complex with immunostimulatory CpG DNA, where both zymosan and CpG DNA can induce cytokine secretion according to the different mechanisms (i.e., recognized by different receptors). The complex activated macrophages and induced cytokine secretion, more efficiently than separate administration of zymosan or CpG DNA. Microscopic observation showed that this increment of the cytokine secretion can be explained by the fact that zymosan and zymosan/CpG DNA complex are up-taken more than naked CpG DNA. Additionally, existence of two different immunostimulants in the same cells may enhance the immunoresponse. This report presents a new strategy to construct a delivering vehicle for CpG DNA and to enhance its activity with the ‘cocktail effect’ of the two immunostimulants.

Immunostimulatory oligonucleotides block allergic airway inflammation by inhibiting Th2 cell activation and IgE-mediated cytokine induction

A single treatment with a CpG-containing immunostimulatory DNA sequence (ISS) given before allergen challenge can inhibit T helper type 2 cell (Th2)–mediated airway responses in animal models of allergic asthma; however, the mechanism of this inhibition remains largely undefined. Here, we demonstrate that airway delivery of ISS before allergen challenge in Th2-primed mice acts in two distinct ways to prevent the allergic responses to this challenge. The first is to prevent induction of cytokines from allergen-specific Th2 cells, as demonstrated by the nearly complete inhibition of Th2 cytokine production, Th2-dependent functional responses, and gene induction patterns. ISS inhibits the Th2 response by rendering lung antigen-presenting cells (APCs) unable to effectively present antigen to Th2 cells, but not to Th1 cells. This loss of APC function correlates with a reduced expression of costimulatory molecules, including programmed cell death ligand (PD-L)1, PD-L2, CD40, CD80, CD86, and inducible T cell costimulator, and of major histocompatibility complex class II on CD11c+APCs from the airways of ISS-treated mice. The second important action of ISS is inhibition of immunoglobulin E–dependent release of Th2 cytokines, especially interleukin 4, from basophils and/or mast cells in the airways of Th2-primed mice. Thus, inhibition by ISS of allergic responses can be explained by two novel mechanisms that culminate in the inhibition of the principal sources of type 2 cytokines in the airways.

Fighting Food Allergy: Current Approaches

Food allergy is defined as an adverse immunologic reaction to allergens present in food and is associated with symptoms ranging from gastrointestinal discomfort to anaphylactic shock and death. The increase in prevalence and potential fatality of disease has led to increased efforts to find effective therapies and prophylaxis. While specific immunotherapy (SIT) is effective in desensitization against inhalant allergens, it is unadvised against food allergy because of the high risk of adverse side effects. A review of the recent literature shows that various approaches have been taken to develop safer and more effective SIT regimens. Here we discuss the use of recombinant allergens, peptides, DNA vaccines, immunostimulatory DNA sequences, and other bacterial products in SIT. In addition, we review nonspecific therapies such as anti-IgE administration and cytokine therapy as well as natural therapies such as probiotics and Chinese herbal medications. In conclusion, anti-IgE treatment and SIT using hypoallergenic recombinant allergens in combination with Th1-inducing adjuvants appear the most promising approaches. New initiatives to increase our understanding of the pathophysiology and immunologic mechanisms of food allergy along with the molecular characterization of food allergens should pave the way towards safer and more effective ways of combating this debilitating and potentially life-threatening disease.

Induction of interferon‐γ from natural killer cells by immunostimulatory CpG DNA is mediated through plasmacytoid‐dendritic‐cell‐produced interferon‐α and tumour necrosis factor‐α

Immunostimulatory sequences (ISS) that contain CpG motifs have been demonstrated to exert antipathogen and antitumour immunity in animal models through several mechanisms, including the activation of natural killer (NK) cells to secrete interferon-gamma (IFN-gamma) and to exert lytic activity. Since NK cells lack the ISS receptor TLR9, the exact pathway by which NK cells are activated by ISS is unclear. We determined that ISS-induced IFN-gamma from NK cells is primarily dependent upon IFN-alpha release from plasmacytoid dendritic cells (PDCs), which directly activates the NK cell. However, further analysis indicated that other PDC-released soluble factor(s) may contribute to IFN-gamma induction. Indeed, tumour necrosis factor-alpha (TNF-alpha) was identified as a significant contributor to ISS-mediated activation of NK cells and was observed to act in an additive fashion with IFN-alpha in the induction of IFN-gamma from NK cells and to up-regulate CD69 expression on NK cells. This activity of TNF-alpha, however, was dependent upon the presence of PDC-derived factors such as type I interferon. These results illustrate an important function for type I interferon in innate immunity, which is not only to activate effectors like NK cells directly, but also to prime them for enhanced activation by other factors such as TNF-alpha.

Design of Protective and Therapeutic DNA Vaccines for the Treatment of Allergic Diseases

The DNA vaccine revolution has opened a vast scope of novel approaches for protective and therapeutic treatments of type I allergy. This review gives an overview on the current status of allergy DNA vaccines and presents advances in the design of vaccine constructs. An immense number of concurring studies have proven the stimulation of Th1 cells and the induction of a balanced Th1/Th2 cytokine milieu as the fundamental mechanisms underlying the anti-allergic effects of DNA vaccines. Basic vaccine formulations thus can be optimized by improving the cellular immunogenicity via co-administration of cytokines, co-expression or co-application of immunostimulatory DNA sequences or adapting the codon usage. The latter is a frequent and major reason for impaired vaccine expression (e.g. translation of plant allergen genes in mammal cells). Because of unwanted side effects during conventional specific immunotherapy with allergen extracts, safety is increasingly demanded for both, protein and DNA vaccines for allergy treatment. We discuss the creation of hypoallergenic DNA vaccines based on deliberate allergen gene fragmentation, the use of mutations and the routine production of hypoallergenic DNA vaccines by forced ubiquitination. Furthermore, allergen-expressing DNA replicon vaccines are introduced, which enable a drastic reduction of the vaccine dose without loss of anti-allergic efficacy. Finally, the development of DNA multi vaccines and fusion vaccines for protective and therapeutic applications against certain groups of allergens is addressed.

CpG oligonucleotides partially inhibit growth of Mycobacterium tuberculosis, but not Salmonella or Listeria, in human monocyte-derived macrophages

Immunostimulatory DNA sequences and their synthetic oligonucleotide analogs (CpG-ODN) activate innate immunity and can stimulate antibacterial effects against numerous intracellular pathogens. While it has been shown previously that CpG-ODN inhibit growth of Mycobacterium avium in murine and human macrophages, we now report that Mycobacterium tuberculosis growth can be inhibited by CpG-ODN treatment of human monocyte-derived macrophages (hMDM). This inhibitory effect was reversed by IFN-γ, which has been shown repeatedly to enhance the growth of virulent M. tuberculosis in cultured hMDM. The antibacterial effect of CpG-ODN in human macrophages was specific for M. tuberculosis when compared to other intracellular pathogens including Listeria monocytogenes and Salmonella enterica serovar Dublin. These data indicate that CpG-ODN can improve the ability of hMDM to contain growth of virulent M. tuberculosis.

Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro

This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24 h of induction and cytokine mRNA expression was detected using RT-PCR at 6 h post induction. For IFN-α/β it was found that both plasmid DNA and phosphodiester ODN, containing an unmethylated CpG-motif, were able to induce IFN production in the presence of lipofectin but not without. The levels of IFN varied with individuals and were often quite low. Moreover, methylation or removal of the CpG sequence completely abolished IFN induction. CpG-containing ODN with poly-guanine (G) sequences in the 5′ and 3′ ends induced considerably higher levels of IFN, especially when the poly-G sequences had a phosphorothioate backbone. ODN with poly-G sequences also had the ability to induce IFN in the absence of lipofectin but the levels of IFN induced were radically reduced compared to those induced with lipofectin. In contrast to IFN, which was only detected upon induction, low spontaneous IL-6 production was observed in unstimulated control cultures. Nevertheless, plasmid DNA and CpG-containing ODN were able to increase the IL-6 production threefold. All the IFN inducing ODN also induced IL-6 production and the levels of IL-6 induced seemed influenced by addition of lipofectin and presence of poly-G sequences in the same way as was observed for the IFN-production. However, a complete phosphorothioate ODN with a central CpG-motif and poly-C sequences, that did not induce IFN, readily induced IL-6 both in the presence and absence of lipofectin. In addition, there was also evidence that some ODN induced increased expression of IL-12p40 mRNA. To conclude, equine PBMC were able to recognize CpG-DNA and respond with both IFN-α/β and/or IL-6 production. The levels of cytokine induced, and sometimes which cytokine induced, varied with, e.g., CpG-motifs used, the presence of poly-G sequences, ODN backbone chemistry and presence of lipofectin.