MX2007004782A - Semi-soft c-class immunostimulatory oligonucleotides - Google Patents

Abstract

The invention relates to specific C-Class semi-soft CpG immunostimulatory oligonucleotides that are useful for stimulating an immune response. In particular the oligonucleotides are useful for treating allergy, such as allergic rhinitis and asthma, cancer and infectious disease, such as hepatitis B and hepatitis C.

Description

SEMI-SOFT IMMUNOSTIMULANT OLIGONUCLEOTIDE CLASS C FIELD OF THE INVENTION The present invention is generally related to immunostimulatory oligonucleotides with reduced renal inflammatory effects, compositions thereof and methods for the use of immunostimulatory oligonucleotides. In particular, immunostimulatory oligonucleotides are semi-soft class C oligonucleotides that are particularly effective in the treatment of allergy and asthma, cancer and infectious diseases.

BACKGROUND OF THE INVENTION Bacterial DNA has immune stimulating effects to activate B cells and natural killer cells, but vertebrate DNA does not have this effect (Tokunaga, T., et al., 1988. Jpn. J. Cancer Res. 79: 682 -686; Tokunaga, T., et al., 1984, JNCI 72: 955-962, Messina, JP, et al., 1991, J. Immunol., 147: 1759-1764, and revised in Krieg, 1998, In: Applied Oligonucleotide Technology, CA. Stein and AM Krieg, (Eds.), John Wiley and Sons, Inc., New York, NY, pp. 431-448). It is now known that these immune stimulatory effects of bacterial DNA are a result of the presence of non-methylated CpG dinucleotides in the context of particular bases (CpG motifs), which are common in bacterial DNA, but are not methylated and are sub- represented in vertebrate DNA (Rieg et al, 1995 Nature 374: 546-549; Krieg, 1999 Biochim Biophys, Acta 93321: 1-10). The immunostimulatory effects of bacterial DNA can be mimicked with synthetic oligodeoxynucleotides (ODN) containing these CpG motifs. Said CpG ODNs have highly stimulating effects on human and murine leukocytes, inducing B cell proliferation; the secretion of cytokine and immunoglobulin; the litica activity of the natural killer cell (NK) and the secretion of IFN- ?; and the activation of dendritic cells (DCs) and other antigen-presenting cells to express costimulatory molecules and secrete cytokines, especially the Th1-like cytokines that are important in promoting the development of Th1-like T cell responses. These immunostimulatory effects of native CpG ODNs with phosphodiester base structure are highly specific to CpG whereas the effects are dramatically reduced if the CpG motif is methylated, changes to a GpC, or is deleted or otherwise altered (Krieg et al. 1995 Nature 374: 546-549; Hartmann et al, 1999 Proc. Nati, Acad. Sci USA 96: 9305-10). In previous studies, the immunostimulatory CpG motif was thought to be followed by the purine-purine-CpG-pyrimidine-pyrimidine formula (Krieg et al, 1995 Nature 374: 546-549; Pisetsky, 1996 J. Immunol. 156: 421-423; Hacker et al., 1998 EMBO J. 17: 6230-6240; Lipford et al, 1998 Trends in Microbiol. 6: 496-500). However, it is now evident that mouse lymphocytes respond quite well to CpG phosphodiester motifs that do not follow this "formula" (Yi et al., 1998 J. Immunol. 160: 5898-5906) and the same is true for B cells from human and dendritic cells (Hartmann et al, 1999 Proc. Nati, Acad Sci USA 96: 9305-10, Liang, 1996 J. Clin.Invest.98: 1 1 19-1 129). Recently, several different classes of CpG oligonucleotides have been described. One class is potent for the activation of B cells but is relatively weak to induce IFN-a and NK cell activation; this class has been termed class B. CpG class B oligonucleotides are typically fully stabilized and include a non-methylated CpG dinucleotide within certain preferred base contexts. See, for example, US Patents. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6.239, 16; and 6,339,068. Another class of CpG oligonucleotides activates B cells and NK cells and induces IFN-a, this class has been termed class C. The CpG class oligonucleotides, as initially characterized, are typically fully stabilized, include a class sequence type B and a palindrome rich in GC or almost with palindrome. This class has been described in the co-pending Patent Application of E.U.A. US 10 / 224,523 filed August 19, 2002 and PCT Patent Application related PCT / US02 / 26468 published under International Publication Number WO 03/01571 1.

BRIEF DESCRIPTION OF THE INVENTION It has been found that the immunostimulatory properties of class C specific CpG oligonucleotides with the selective inclusion of one or more non-stabilized bonds between certain nucleotides have significant activity and are particularly useful in the treatment of allergy and asthma. The non-stabilized bonds are preferably natural bonds, for example, phosphodiester bonds or phosphodiester-like bonds. An unstabilized link will typically, but not necessarily, be relatively susceptible to nuclease digestion. The immunostimulatory oligonucleotides of the present invention include at least one unstabilized bond located between an adjacent 5 'C and 3' G, wherein both 5 'C and 3' G are internal nucleotides. The immunostimulatory oligonucleotides of the present invention are useful for inducing a Th1-like immune response. Accordingly, the immunostimulatory oligonucleotides of the present invention are useful as adjuvants for vaccination, and are useful for the treatment of diseases including cancer, infectious disease, allergy, and asthma. They are believed to be of particular use in any condition for prolonged or repeated administration of immunostimulatory oligonucleotides for any purpose, but are particularly useful in the treatment of asthma and allergic diseases such as allergic rhinitis.

The present invention relates in part to immunostimulatory oligonucleotides containing CpG. In one aspect the invention is an oligonucleotide having the formula: 5 'TC_GX1C_G X2Ni X3C_GN2CG 3' (SEQ ID NO: 26). The oligonucleotide includes at least 2 stabilized internucleotide linkages. "_" represents an internucleotide phosphodiester linkage or an internucleotide linkage similar to phosphodiester. i is 0-3 nucleotides in length, N2 is 0-9 nucleotides in length with N referring to any nucleotide. Xi, X2, and X3 are any nucleotide. In some embodiments X ^ X2l and X3 are T. In some embodiments the oligonucleotide may comprise 5 '.

TC_GTC_GTN1TC_GGCGCN1GCCG 3 '(SEQ ID NO: 27). In one embodiment the oligonucleotide may comprise 5 'T * C_G * T * C_G * T * N1 * T * C_G * G * C * G * CN1G * C * C * G 3' (SEQ ID NO: 27). In some N- modalities, it is 3 or 2 nucleotides in length. In other modalities Ni is 0 nucleotides in length. The immunostimulatory oligonucleotide may comprise 5 'T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3' (SEQ ID NO: 2 ), where * represents a stabilized internucleotide link. Optionally, when specifically stated, 5 'may refer to the free 5' end of the oligonucleotide and 3 'may refer to the free 3' end of the oligonucleotide. In other embodiments, the immunostimulatory oligonucleotide may comprise 5 'T * C_G * T * C_G * T * T * C_G * G * C * G * C * G * C * C * G 3' (SEQ ID NO: 3), in where * represents a stabilized internucleotide link. Optionally, when specifically stated, 5 'may refer to the free 5' end of the oligonucleotide and 3 'may refer to the free 3' end of the oligonucleotide. In another aspect, the immunostimulatory oligonucleotide has the following formula TC_G XiC_G X2C_G X3TC_GGCGC_G N33 '(SEQ ID NO: 28). N3 is 1 -5 nucleotides in length with N referring to any nucleotide. In an N3 modality it is 5 nucleotides. ??, X2, and X3 are any nucleotide. In some embodiments X1 and X3 are T. In one embodiment the oligonucleotide may comprise 5 'TC_GTC_GAC_GATC_GGCGC_GCGCCG3' (SEQ ID NO: 4), wherein the oligonucleotide includes at least 2 stabilized internucleotide linkages and _ represents an internucleotide phosphodiester link or an internucleotide linkage similar to phosphodiester. In one embodiment the oligonucleotide may comprise 5 'T * C_G * T * C_G * A * C_G * A * T * C_G * G * C * G * C_G * C * G * C * C * G 3' (SEQ ID NO : 4). Optionally, when specifically set, 5 'may refer to the free 5' end of the oligonucleotide and 3 'can refer to the free 3' end of the oligonucleotide. In accordance with another aspect of the invention an immunostimulatory oligonucleotide having the following formula: 5 'TTC_GX2C_GN1X1_GX3C_GTT 3' (SEQ ID NO: 24) is provided. The oligonucleotide includes at least 2 stabilized internucleotide bonds and represents an internucleotide phosphodiester linkage or an internucleotide linkage similar to phosphodiester. N- \ is 1 -3 nucleotides in length with N referring to any nucleotide. Xi is a pyrimidine. X2 and X3 are any nucleotide. In some embodiments X2 and X3 are T. In one embodiment the oligonucleotide may comprise 5 'TTC_GTC_GTTTX i_GTC_GTT 3' (SEQ ID NO: 25). In another embodiment the oligonucleotide may comprise 5 ' T * T * C_G * T * C_G * T * T * T * X1_G * T * C_G * T * T 3 '(SEQ ID NO: 25). In some embodiments Xi is T or C. The oligonucleotide may comprise 5 'T * T * C_G * T * C_G * T * T * T * T_G * T * C_G * T * T 3' (SEQ ID NO: 5), where * represents a stabilized internucleotide link. Optionally, when specifically stated, 5 'may refer to the free 5 * end of the oligonucleotide and 3' may refer to the free 3 'end of the oligonucleotide. The oligonucleotide may comprise 5 'T * T * T * C_G * T * C_G * T * T * T * C_G * T * C_G * T * T 3' (SEQ ID No. 6), where * represents an internucleotide linkage stabilized. Optionally, when specifically stated, 5 'may refer to the free 5' end of the oligonucleotide and 3 'may refer to the free 3' end of the oligonucleotide.

In some aspects of the invention the oligonucleotide has one of the following formulas TCGTCGTTCGGCGCGCCG (SEQ ID NO: 3), TCGTCGTCGTTCGGCGCGCGCCG (SEQ ID NO: 2), TCGTCGACGATCGGCGCGCGCCG (SEQ ID NO: 4), TTCGTCGTTTTGTCGTT. (SEQ ID NO: 5), or TTTCGTCGTTTCGTCGTT. (SEQ ID NO: 6) In other aspects of the invention the oligonucleotide has one of the following formulas TCGTCGTC, CGTCGTCG, GTCGTCGT, TCGTCGTT, CGTCGTTC, GTCGTTCG, TCGTTCGG, CGTTCGGC, GTTCGGCG, TTCGGCGC, TCGGCGCG, CGGCGCGC, GGCGCGCG, GCGCGCGC, CGCGCGCC , or GCGCGCCG. In other aspects of the invention the oligonucleotide has one of the following formulas T * C_G * T * C_G * T * C, C_G * T * C_G * T * C_G, G * T * C_G * T * C_G * T, T * C_G * T * C_G * T * T, C_G * T * C_G * T * T * C, G * T * C_G * T * T * C_G, T * C_G * T * T * C_G * G, C_G * T * T * C_G * G * C, G * T * T * C_G * G * C * G, T * T * C_G * G * C * G * C, T * C_G * G * C * G * C_G, C_G * G * C * G * C_G * C, G * G * C * G * C_G * C * G, G * C * G * C_G * C * G * C, C * G * C_G * C * G * C * C, or G * C_G * C * G * C * C * G. In other aspects of the invention there is provided an oligonucleotide comprising: T * C_G * T * C_G * T * C, where * represents a stabilized internucleotide bond and represents an internucleotide phosphodiester bond or an internucleotide linkage similar to phosphodiester. Optionally the oligonucleotide can be 5 'T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_ G * C * G * C * C 3' (SEQ ID NO .: 21 ), 5 'T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C 3' (SEQ ID NO .: 22), or 5 'T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C 3 '(SEQ ID NO .: 23), where 5' refers to the free 5"end of the oligonucleotide and 'refers to the free 3' terminus of the oligonucleotide In other respects an oligonucleotide is provided comprising: T * C_G * T * T * C_G * G, wherein * represents a stabilized internucleotide linkage and _ represents an internucleotide linkage phosphodiester or a phosphodiester-like internucleotide bond Optionally, the oligonucleotide can be 5 ' C_G * T * C_G C_G T * C_G * G * C * G * C_G * C * G * CX * G 3 '(SEQ ID NO.: 15), 5' G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 '(SEQ ID NO .: 16), 5' T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 '(SEQ ID NO .: 17), 5' C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 '(SEQ ID NO .: 18), 5' G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 '(SEQ ID NO .: 19), or 5' T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3"(SEQ ID NO .: 20) wherein 5 'refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide A pharmaceutical composition comprising an oligonucleotide of the invention and a pharmaceutically acceptable carrier is provided In some embodiments the composition It is formulated in a nebulizer or an inhaler The inhaler can be a metered dose inhaler Alternatively the inhaler is a powder inhaler.

In other embodiments, the pharmaceutical composition may include a chemotherapeutic agent. In other embodiments, the composition may include an anti-viral agent. The pharmaceutical composition may optionally include a pharmaceutically acceptable carrier formulated for subcutaneous administration, oral administration or intranasal administration. In one embodiment the oligonucleotide is in a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier. In some embodiments the oligonucleotide is formulated as an aerosol. In one embodiment the oligonucleotide additionally comprises an adjuvant or a cytokine. In one embodiment the oligonucleotide additionally comprises an antigen, wherein the oligonucleotide is an adjuvant for vaccine. In one embodiment the antigen is selected from the group consisting of: a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, and a tumor antigen. In one embodiment, the antigen is encoded by a nucleic acid vector. In one embodiment, the antigen is a peptide antigen. In one embodiment the antigen is covalently linked to the oligonucleotide or to the immunostimulatory nucleic acid molecule. In another embodiment the antigen does not covalently bind to the oligonucleotide or the immunostimulatory nucleic acid molecule.

In one embodiment the phosphodiester linkage or the phosphodiester linkage is phosphodiester. In one embodiment, the phosphodiester-like bond is dianareomerically pure boranophosphonate or phosphorothioate Rp. In one embodiment the stabilized base structure comprises a plurality of internucleotide linkages selected from the group consisting of: phosphorothioate, phosphorodithioate, methylphosphonate, methylphosphorothioate, and any combination thereof. In one embodiment the stabilized base structure comprises a plurality of phosphorothioate internucleotide linkages. In one embodiment the immunostimulatory nucleic acid molecule is 4-100 nucleotides long. In other aspects the invention is a method for the treatment of asthma by administration to a subject having or at risk of having asthma of an oligonucleotide of the invention in an amount effective to treat asthma. In still other aspects, the invention is a method for the treatment of allergy by administration to a subject having or at risk of allergy to an oligonucleotide of the invention in an amount effective to treat the allergy. In one modality the subject has allergic rhinitis. In one embodiment the oligonucleotide is administered to a mucosal surface. In other embodiments, the oligonucleotide is administered in an aerosol formulation. Optionally the oligonucleotide is administered intranasally. In accordance with another aspect of the invention, a method for inducing cytokine production is provided. The method is carried out by administering to a subject an immunostimulatory CpG oligonucleotide described in the present invention in an amount effective to induce a cytokine selected from the group consisting of IL-6, IL-8, IL-12, IL-8, TNF, IFN-a, chemokines, and IFN- ?. In another aspect the invention is a composition of the immunostimulatory CpG oligonucleotides described in the present invention in combination with an antigen or other therapeutic compound, such as an anti-microbial agent. The anti-microbial agent can be, for example, an anti-viral agent, an anti-parasitic agent, an antibacterial agent or an antifungal agent. In accordance with another aspect of the invention there is provided a composition of a device for sustained release including the immunostimulatory CpG oligonucleotides described in the present invention. The invention may optionally include a pharmaceutical carrier and / or be formulated in a device for administration. In some embodiments, the device for administration is selected from the group consisting of cationic lipids, cellular permeabilzers, proteins, and sustained release devices. In one embodiment the sustained release device is a biodegradable polymer or a microparticle. In accordance with another aspect of the invention, a method for stimulating an immune response is provided. The method includes administering an immunostimulatory CpG oligonucleotide to a subject in an amount effective to induce an immune response in the subject. Preferably the immunostimulatory CpG oligonucleotide is administered orally, locally, in a sustained release device, mucosally, systematically, parenterally, or intramuscularly. When the immunostimulatory CpG oligonucleotide is administered to the mucosal surface it can be administered in an amount effective to induce a mucosal immune response or a systemic immune response. In preferred embodiments, the mucosal surface is selected from the group consisting of an oral, nasal, rectal, vaginal, and ocular surface. In some embodiments, the method includes exposing the subject to an antigen wherein the immune response is an antigen-specific immune response. In some embodiments the antigen is selected from the group consisting of a tumor antigen, a viral antigen, a bacterial antigen, a parasitic antigen and a peptide antigen. The immunostimulatory CpG oligonucleotides are capable of eliciting a broad spectrum of immune responses. For example, these immunostimulatory CpG oligonucleotides can be used to redirect a Th2 towards a Th1 immune response. CpG immunostimulatory oligonucleotides can also be used to activate an immune cell, such as a lymphocyte (e.g., B and T cells), a dendritic cell, and an NK cell. The activation can be carried out in vivo, in vitro, or ex vivo, for example, by isolating an immune cell from the subject, contacting the immune cell with an effective amount to activate the immune cell by the immunostimulatory CpG oligonucleotide and re-administering the activated immune cell to the subject. In some modalities the dendritic cell presents a cancerous antigen. The dendritic cell can be exposed to the cancerous antigen ex vivo. The immune response produced by immunostimulatory CpG oligonucleotides can also result in the induction of cytokine production, for example, production of IL-6, IL-8, IL-12, IL-18, TNF, IFN-a, chemokines, and IFN- ?. In yet another embodiment, the immunostimulatory CpG oligonucleotides are useful for the treatment of cancer. CpG immunostimulatory oligonucleotides are also useful in accordance with other aspects of the invention in the prevention of cancer (e.g., reducing a risk for cancer development) in a subject at risk of developing a cancer. The cancer can be selected from the group consisting of cancer of the biliary tract, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, gastric cancer, intraepithelial neoplasms, lymphomas, liver cancer, lung cancer (e.g. small cell and not small cell), melanoma, neuroblastomas, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, thyroid cancer, and kidney cancer, as well as other carcinomas and sarcomas. In some important embodiments, the cancer is selected from the group consisting of bone cancer, brain cancer and CNS cancer, connective tissue cancer, esophageal cancer, eye cancer, Hodgkin's lymphoma, laryngeal cancer, oral cavity cancer, skin cancer, and testicular cancer. CpG immunostimulatory oligonucleotides can also be used to increase the response of a cancer cell to a cancer therapy (e.g., anti-cancer therapy), optionally when the immunostimulatory CpG oligonucleotide is administered in conjunction with an anti-cancer therapy. The anti-cancer therapy may be a chemotherapy, a vaccine (e.g., an in vitro dendritic cell vaccine or a cancerous antigen vaccine) or an antibody-based therapy. This latter therapy may also include the administration of an antibody specific for a cell surface antigen of, for example, a cancer cell, wherein the immune response results in antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, the antibody can be selected from the group consisting of Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, or t6 , MDX-210, MDX-1 1, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, Ab ANA, Ab SMART IDIO, Ab SMART ABL 364 and ImmuRAIT-CEA. Therefore, in accordance with some aspects of the invention, an immunostimulatory CpG oligonucleotide and an anti-cancer therapy are administered to a subject who has cancer or who is at risk of having cancer. In some embodiments, the anti-cancer therapy is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent and a vaccine for cancer. In some modalities, the cancer medication is taxol or a combination of carboplatin and paclitaxel. In yet another embodiment of the methods directed to prevent or treat cancer, the subject may be further administered interferon-a. In other aspects the invention relates to methods for the prevention of the disease in a subject. The method includes administering to the subject an immunostimulatory CpG oligonucleotide on a regular basis to promote the response of the immune system to prevent disease in the subject. Examples of diseases or conditions to be prevented using the prophylactic methods of the invention include microbial infections (e.g., sexually transmitted diseases) and anaphylactic shock from food allergies.

In other aspects, the invention is a method for inducing an innate immune response by administering to the subject an immunostimulatory CpG oligonucleotide in an amount effective to activate an innate immune response. In accordance with another aspect of the invention, a method for the treatment or prevention of a viral or retroviral infection is provided. The method includes the administration to a subject having or at risk of having a viral or retroviral infection, of an amount effective for the treatment or prevention of viral or retroviral infection of any of the compositions of the invention. In some embodiments, the virus is caused by a hepatitis virus, for example, hepatitis B, hepatitis C, HIV, herpes virus, or papillomavirus. In accordance with another aspect of the invention, a method for the treatment or prevention of a bacterial infection is provided. The method includes administration to a subject having or at risk of having a bacterial infection, an amount effective for the treatment or prevention of bacterial infection of any of the compositions of the invention. In an embodiment, the bacterial infection is due to an intracellular bacterium. In another aspect the invention is a method for the treatment or prevention of a parasitic infection by administration to a subject having or at risk of having a parasitic infection, of an amount effective for the treatment or prevention of parasitic infection of any of the compositions of the invention. In one embodiment, the parasitic infection is due to an intracellular parasite. In another modality, the parasitic infection is due to a non-helminthic parasite. In some modalities the subject is a human and in other modalities the subject is a non-human vertebrate selected from the group consisting of a dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat, mouse, and sheep. In another aspect the invention requires a method for the induction of a TH1 immune response by administering to a subject of any of the compositions of the invention in an amount effective to produce a TH1 immune response. In another aspect, the invention relates to a method for the treatment of an autoimmune disease by administration to a subject having or at risk of having an autoimmune disease of an effective amount for the treatment or prevention of autoimmune disease of any of the compositions of the invention. In other embodiments, the oligonucleotide is administered to the subject in an amount effective to induce cytokine expression. Optionally, the cytokine is selected from the group consisting of IL-6, TNF-α, IFN-α, IFN-α. and IP-10. In other embodiments, the oligonucleotide is administered to the subject in an amount effective to change the immune response toward a skewed response to Th1 from a skewed response to Th2.

In some aspects the invention is a method for the treatment of airway remodeling, comprising: administering to a subject an oligonucleotide comprising a CG dinucleotide, in an amount effective to treat remodeling of airways in the subject. In one embodiment the subject has asthma, chronic obstructive pulmonary disease, or is a smoker. In other modalities the subject is free of asthma symptoms. The use of an oligonucleotide of the invention to stimulate an immune response is also provided as an aspect of the invention. Also provided is the use of an oligonucleotide of the invention in the manufacture of a medicament for stimulating an immune response and carrying out any of the methods of the invention. Each of the limitations of the invention may comprise several embodiments of the invention. Therefore, it is anticipated that each of the limitations of the invention including any element or combinations of elements may be included in each aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a series of graphs illustrating the induction of IFN-a by human PBMC treated with CpG ODN.

Figures 2A-2B are a series of graphs illustrating the effects of the oligodeoxynucleotide CpG SEQ ID NO. 7 against an increase induced by the antigen in nasal resistance in those whose arrests were given a dose of (Figure 2A) 1 mg / kg and (Figure 2B) (0.03-0.3 mg / kg). The results are the mean ± s.e.m. ((figure 2A) = n = 5-8; (figure 2B) = n = 14-15). * P < 0.05 compared to the group tested with the antigen treated with the test vehicle (test t). Figures 3A-3B are a series of graphs illustrating the effects of the oligodeoxynucleotide CpG SEQ ID NO. 7 on antigen-induced sneezing (Figure 3A) and nasal rubbing (Figure 3B) in a model of allergic rhinitis in mice. The results are the mean ± s.e.m. (n = 10) * P < 0.05 compared to the group treated with the vehicle (Mann-Whitney test). Figure 4 is a graph illustrating a virus titration of the influence and determination of the time course of the infection. The cell numbers in the bronchoalveolar lavage fluid. The results are the mean ± s.e.m. (n = 5). Mice infected with 500 EID50 were sacrificed after 6 days due to weight loss. Figure 5 is a graph illustrating the protective effects of CpG ODN on the load of the virus in the lung. The viral load is assayed by enzyme immunoassay. The results are the average ± s.e.m. (n = 5-10). * P < 0.05 compared to group B (Kruskal-Wallis test, Dunn post-test), n.d. = no data.

Figures 6A-6C are a series of graphs illustrating the protective effects of CpG ODN on virus-induced airway inflammation by measuring total leukocytes (Figure 6A), total neutrophils (Figure 6B) and total mononuclear cells (Figure 6C) . The cell numbers in the bronchoalveolar lavage fluid. The results are the mean ± s.e.m. (n = 10) * P < 0.05 compared to group B (Kruskal-Wallis test, Dunn post-test). Figures 7A-7B are a series of graphs illustrating the protective effects of CpG ODN on cell numbers in airway inflammation induced by antigen in eosinophils (Figure 7A) and neutrophils (Figure 7B). The results are the mean ± s.e.m. (n = 10-14). * P < 0.05 compared to the group treated with the vehicle, tested by the antigen (Kruskal-Wallis multiple comparison test, Dunn post test). Figures 8A-8B are a series of graphs illustrating the protective effects of CpG ODN on the cell numbers CD3 + (Figure 8A) and CD3 + CD4 + (Figure 8B) in airway inflammation induced by antigen. The results are the mean ± s.e.m. (n = 8). * P < 0.05 compared in the group treated with the vehicle, tested with the antigen (Kruskal-Wallis multiple comparison test, Dunn post test). Figures 9A-9D are a series of graphs illustrating cell numbers in the bronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg / kg ODN (Figure 9A) 15 hours after dosing with 0.1 mg / kg from ODN (Figure 9B); 8 hours after dosing with 1 mg / kg of ODN (Figure 9C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 9D). The results are the mean ± sem (n = 10) Figures 10A-10D are a series of graphs illustrating the concentrations of IFN alpha in the bronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg / kg of ODN (FIG. 10A); 15 hours after dosing with 0.1 mg / kg ODN (FIG. 10B); 8 hours after dosing with 1 mg / kg of ODN (Figure 10C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 10D). Figures 1 1A-1 1 D are a series of graphs illustrating the concentrations of IFN gamma in the bronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg / kg ODN (Figure 1 1 A); 15 hours after dosing with 0.1 mg / kg of ODN (FIG. 1 1 B); 8 hours after dosing with 1 mg / kg of ODN (FIG. 1 1 C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 1 D). Figures 12A-12D are a series of graphs illustrating IP-10 concentrations in the bronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg / kg ODN (Figure 12A); 15 hours after dosing with 0.1 mg / kg ODN (FIG. 12B); 8 hours after dosing with 1 mg / kg of ODN (Figure 12C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 12D).

Figures 13A-13D are a series of graphs illustrating the concentrations of IL-12p40 in the bronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg / kg of ODN (Figure 13A); 15 hours after dosing with 0.1 mg / kg of ODN (Figure 13B); 8 hours after dosing with 1 mg / kg of ODN (Figure 13C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 13D). Figures 14A-14D are a series of graphs illustrating the concentrations of IL-6 in the bronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg / kg of ODN (Figure 14A); 15 hours after dosing with 0.1 mg / kg of ODN (Figure 14B); 8 hours after dosing with 1 mg / kg of ODN (Figure 14C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 14D). Figures 15A-15D are a series of graphs illustrating the concentrations of TNF alpha in the bronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg / kg of ODN (Figure 15A); 15 hours after dosing with 0.1 mg / kg ODN (FIG. 15B); 8 hours after dosing with 1 mg / kg of ODN (Figure 15C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 15D). Figures 16A-16D are a series of graphs illustrating serum IFN gamma concentrations 8 hours after dosing with 0.1 mg / kg ODN (Figure 16A); 15 hours after dosing with 0.1 mg / kg of ODN (Figure 16B); 8 hours after dosing with 1 mg / kg of ODN (Figure 16C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 16D). Figures 17A-17D are a series of graphs illustrating serum IL-6 concentrations 8 hours after dosing with 0.1 mg / kg ODN (Figure 17A); 15 hours after dosing with 0.1 mg / kg ODN (FIG. 17B); 8 hours after dosing with 1 mg / kg of ODN (Figure 17C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 17D). Figures 18A-18D are a series of graphs illustrating serum TNF alpha concentrations 8 hours after dosing with 0.1 mg / kg ODN (Figure 8A), 15 hours after dosing with 0.1 mg / kg ODN (Figure 18B); 8 hours after dosing with 1 mg / kg of ODN (Figure 18C); and 15 hours after dosing with 1 mg / kg of ODN (Figure 18D). Figures 19A-19B is a series of graphs illustrating the effects of oligodeoxynucleotides ODN CpG SEQ ID NO: 2 and ODN SEQ ID NO. 7 on the IgE-induced production of IgE (Figure 19A) and IgG2a (Figure 19B) in the mouse. The results are the mean ± s.e.m. (n = 10) * P < 0.05 compared to the group treated with the vehicle, sensitized with the antigen (Kruskal-Wallis test with Dunn's post test). Figures 20A-20D are a series of graphs illustrating the effects of ODN SEQ ID NO: 2 against the exacerbation of influenza-induced allergic airway inflammation in mice, illustrating total leukocytes (Figure 20A), eosinophils (FIG. Figure 20B), neutrophils (Figure 20C), mononuclear cells (Figure 20D), and body weight (Figure 20E). Figure 21 is an AHR protocol in which it is used in the present invention. Figures 22A-22D are a series of graphs illustrating the effect of SEQ ID NO: 7 on airway resistance and pulmonary access. For each animal, a dose response curve was obtained, and the reactivity in airways was quantified as the area under the curve. Those who were given intranasally either the vehicle (saline solution), OVA alone, or the concentrations of SEQ ID NO: 7 of 10 ul / kg, 30 ul / kg, 100 ul / kg, or 300 ul / kg, Item The data demonstrate that SEQ ID NO: 7 results in a dose-dependent reduction in AUC resistance. Figures 22A and 22B illustrate the release of histamine as a function of increased resistance of the airways (Figure 22A) or a decrease in lung access (Figure 22B). Figures 22C and 22D are bar graphs illustrating the increase in airway resistance (Figure 22C) or the decrease in lung access (Figure 22D) in response to saline treatment, OVA or SEQ ID NO 7 in the indicated doses. Figures 23A-23D are a series of graphs illustrating the effect of SEQ ID NO: 2 on airway resistance and pulmonary access. For each animal, a dose response curve was obtained, and the reactivity in airways was quantified as the area under the curve. Those who were given intranasally either the vehicle (saline), OVA alone, or the concentrations of SEQ ID NO: 2 of 10 ul / kg, 30 ul / kg, 100 ul / kg, or 300 ul / kg, Item The data demonstrated that SEQ ID NO: 2 results in a dose-dependent reduction in AUC resistance. Figures 23A and 23B illustrate the release of histamine as a function of the increased resistance of the airways (Figure 23A) or the decrease in lung access (Figure 23B). Figures 23C and 23D are bar graphs illustrating the increase in airway resistance (Figure 23C) or the decrease in pulmonary access (Figure 23D) in response to treatment with saline, OVA or SEQ ID NO 2 in the indicated doses. Figures 24A-24D are a summary of the graphs in Figures 22A-22D and 23A-23D. Figures 24A and 24B correspond to Figures 22C and 22D. Figures 24C and 24D correspond to Figures 23C and 23D. Figure 25 is a series of graphs illustrating the levels of IL-10 (pg / ml) secreted from human PBMC (3 donors) followed by exposure of these cells to the oligonucleotides listed by SEQ ID No. a length of the lower X axis of the graph (data points from 3 donors are illustrated by an A, «and x) for 48 hours. The test oligonucleotides illustrated in Figure 25 include SEQ ID NOs: 10, 9, 13, 14, 1, and 2. The concentration of oligonucleotide used to produce a particular data point is illustrated along the X axis (uM). ). The supernatant was harvested and IL-10 was measured by ELISA. The average cytokine amounts of all donors are provided.

Figures 26A-26C are a series of graphs illustrating the levels of TNF-alpha (Figure 26A), interferon-gamma (Figure 26B), and IL-6 (Figure 26C) (uM) secreted from human PBMC followed by the exposure of these cells to the oligonucleotides listed by SEQ ID NO. in the key of the graph. Each data point is the calculated average cytokine value of three donors. PBMC were incubated with the indicated concentrations of ODN. The supernatants were harvested and the cytokines were measured by ELISA. The average amounts of cytokine calculated from all donors are given. Figure 27 is a series of graphs that illustrate the levels of TNF-alpha (pg / ml) secreted from human PBMC followed by exposure of these cells to the oligonucleotides for 16 hours listed by SEQ ID NO along the bottom X axis of the graph. The oligonucleotides shown in Figure 27 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated throughout the X axis (uM). The data shown represent the values of three donors. Below the SEQ ID NO is a designation that refers to the ODN class. Css = C-semi-soft class, C = C-class, B = B-class, not CpG = an ODN without a non-methylated CpG. The supernatants were harvested and IL-6 was measured by ELISA. The average cytokine amounts of all donors are provided. Figure 28 is a series of graphs illustrating the levels of IL-6 (pg / ml) secreted from human PBMC followed by exposure of these cells to the oligonucleotides for 24 hours listed by SEQ ID NO throughout of the lower X axis of the graph. The oligonucleotides shown in Figure 28 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated as length of the X axis (uM). The data shown represent the values of three donors. Below the SEQ ID NO is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. Figure 29 is a series of graphs that illustrate the levels of IFN-gamma (pg / ml) secreted from human PBMC followed by exposure of these cells to the oligonucleotides for 48 hours listed by SEQ ID NO along the lower X axis of the graph. The oligonucleotides shown in Figure 29 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated throughout the X axis (uM). The data shown represent the values of three donors. Below the SEQ ID NO is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. Supernatants were harvested and IFN-gamma was measured by ELISA. The average cytokine amounts of all donors are provided. Figures 30A-30C are a series of graphs illustrating the levels of CD69 (MFI) expression in NK cells as an indicator of NK cell activation (Figure 30A) and CD80 (Figure 30B) and CD86 expression ( Figure 30C) on B cells followed by exposure of these cells to the oligonucleotides for 24 or 48 hours listed by SEQ ID NO. in the key of the graph. Each data point is the average fluorescence intensity of three donors. The cells were incubated with the indicated concentrations of ODN for 24, or 48 hours. The cells were stained and analyzed by flow cytometry. Figure 31 is a series of graphs illustrating the levels of expression of CD69 in NK cells as an indicator of NK cell activation followed by exposure of these cells to the oligonucleotides for 24 hours listed by SEQ ID NO. along the bottom X axis of the graph. The oligonucleotides shown in Figure 31 include SEQ ID NO: 9, 1 3, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated along the X-axis (uM). The data shown represent the values of three donors. Below the SEQ ID NO is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. The cells were stained with antibodies to CD3, CD56, and CD69 and analyzed by flow cytometry. The data presented are the mean fluorescence intensity. Figure 32 is a series of graphs illustrating the expression of CD86 in human PBMC followed by exposure of these cells to the oligonucleotides for 48 hours listed by SEQ ID NO along the lower X axis of the graph. The oligonucleotides shown in Figure 32 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated throughout the X axis (uM). The data shown represent the values of three donors. Below the SEQ ID NOs is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. The cells were stained with antibodies to CD86, CD80, CD19, and CD14 and analyzed by flow cytometry. The data presented are the mean fluorescence intensity. Figure 33 is a series of graphs illustrating the expression of CD86 in human PBMC followed by exposure of these cells to the oligonucleotides for 48 hours listed by SEQ ID NO along the lower X axis of the graph. The oligonucleotides shown in Figure 33 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated along the axis X (uM). The data shown represent the values of three donors. Below the SEQ ID NOs is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. The cells were stained with antibodies to CD86, CD80, CD 19, and CD 14 and analyzed by flow cytometry. The data presented are the mean fluorescence intensity.

Figures 34A-34C are a series of graphs illustrating the levels of expression of CD86 on plasmacytoid dendritic cells (Figure 34A) and the expression of CD80 (Figure 34B) and CD86 (Figure 34C) on monocytes followed by exposure of these cells to the oligonucleotides listed by SEQ ID NO. in the key of the graph. Each data point is the average fluorescence intensity calculated from three donors. The cells were incubated with the indicated concentrations of ODN for 48 hours. The cells were stained and analyzed by flow cytometry. Figure 35 is a series of graphs illustrating the levels of CD86 expression on monocytes after exposure of the cells to the oligonucleotides for 48 hours listed by SEQ ID NO along the lower X axis of the graph. The oligonucleotides shown in Figure 35 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated throughout the X axis (uM). The data shown represent the values of three donors. Below the SEQ ID NO is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. The cells were stained with antibodies to CD86, CD80, CD19, and CD14 and analyzed by flow cytometry. The data presented are the mean fluorescence intensity. Figure 36 is a series of graphs illustrating the expression of CD80 on monocytes followed by exposure of these cells to the oligonucleotides for 48 hours listed by SEQ ID NO along the lower X axis of the graph. The oligonucleotides shown in Figure 36 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated throughout the X axis (uM). The data shown represent the values of three donors. Below the SEQ ID NO is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. The cells were stained with antibodies to CD86, CD80, CD19, and CD14 and analyzed by flow cytometry. The data presented are the mean fluorescence intensity. Figure 37 is a series of graphs illustrating the expression of CD80 on plasmacytoid dendritic cells followed by exposure of these cells to the oligonucleotides for 48 hours listed by SEQ ID NO along the lower X axis of the graph. The oligonucleotides shown in Figure 37 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated throughout the X axis (uM). The data shown represent the values of three donors. Below the SEQ ID NO is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. The cells were stained with antibodies to CD86, CD1 1 c, CD123, and HLA-DR and analyzed by flow cytometry. The data presented are the mean fluorescence intensity. Figures 38A-38B are a series of graphs illustrating the levels of expression of intracellular IP-10 in B cells (Figure 38B) and monocytes (Figure 38A) followed by exposure of these cells to the oligonucleotides for 24 hours listed by SEQ. ID NO. in the key of the graph. Each data point is the average fluorescence intensity calculated from three donors. The cells were incubated with the indicated concentrations of ODN for 24 hours. The cells were stained and analyzed by flow cytometry. Figure 39 is a series of graphs illustrating the expression levels of intracellular IP-10 in monocytes after exposure of the cells to the oligonucleotides for 24 hours listed by SEQ ID NO along the lower X-axis of the graph . The oligonucleotides shown in Figure 39 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated along the X-axis (uM). The data shown represent the values of three donors. Below the SEQ ID NO is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. The cells were stained with antibodies to CD14, CD19, and IP-10 and analyzed by flow cytometry. The data presented are the mean fluorescence intensity.

Figure 40 is a series of graphs illustrating the levels of IP-10 intracellular expression in B cells followed by exposure of these cells to the oligonucleotides for 24 hours listed by SEQ ID NO along the lower X-axis of the graph. The oligonucleotides shown in Figure 40 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated throughout the X axis (uM). The data shown represent the values of three donors. Below the SEQ ID NO is a designation that refers to the ODN class. Css = semi-soft C-class, C = C-class, B = B-class, non-CpG = an ODN without a non-methylated CpG. The cells were stained with antibodies to CD14, CD19, and IP-10 and analyzed by flow cytometry. The data presented are the mean fluorescence intensity. Figures 41A-41F are a series of graphs illustrating a comparison of the capabilities of SEQ ID NO: 2 and fragments thereof (SEQ ID NO 15-17) to induce cytokine secretion from mouse splenocytes. The analyzed cytokines include IFN-a (Figure 41A), IFN-α? (Figure 41 B), IP-10 (Figure 41 C), IL-6 (Figure 41 D), IL-10 (Figure 4 E), and TNF-a (Figure 41 F). Figures 42A-42F are a series of graphs illustrating a comparison of the capabilities of SEQ ID NO: 2 and fragments thereof (SEQ ID NOs 18-20) to induce cytokine secretion from mouse splenocytes. The cytokines analyzed include IFN-a (FIG. 42A), IFN-? (Figure 42B), IP-10 (Figure 42C), IL-6 (Figure 42D), IL-10 (Figure 42E), and TNF-a (Figure 42F). Figures 43A-43F are a series of graphs illustrating a comparison of the capabilities of SEQ ID NO. : 2 and fragments thereof (SEQ ID NO 21-23) to induce cytokine secretion from mouse splenocytes. The analyzed cytokines include IFN-a (FIG. 43A), IFN-? (Figure 43B), IP-10 (Figure 43C), IL-6 (Figure 43D), IL-10 (Figure 43E), and TNF-a (Figure 43F).

DETAILED DESCRIPTION OF THE INVENTION A sub-population of semi-soft class-C immunostimulatory oligonucleotides are provided in accordance with the invention. The immunostimulatory oligonucleotides of the invention described in the present invention, in some embodiments have improved properties including similar potency or improved potency, reduced systemic exposure to the kidney, liver and spleen, and may have reduced reactogenicity at injection sites. Although the applicant does not abide by a mechanism, it is believed that these improved properties are associated with the strategic placement within the immunostimulatory oligonucleotides of "internucleotide bonds" phosphodiester or phosphodiester-like. The term "internucleotide linkage" as used in the present invention refers to the covalent bonding of the base structure that binds two adjacent nucleotides in a nucleic acid molecule. The covalent bond of the base structure will typically be a modified or unmodified phosphate bond, but other modifications are possible. Therefore a linear oligonucleotide that is n nucleotides long has a total of n-1 internucleotide linkages. These covalent bonds of the base structure may be modified or unmodified in the immunostimulatory oligonucleotides according to the teachings of the invention. In particular, phosphodiester internucleotide linkages or phosphodiester-like internucleotide linkages include "internal dinucleotides". An internal dinucleotide in general must mean any pair of adjacent nucleotides connected by an internucleotide linkage, in which no nucleotide in the pair of nucleotides is a terminal nucleotide, for example, no nucleotide in the nucleotide pair is a nucleotide that defines the end 5 'or 3' of the oligonucleotide. Therefore, a linear oligonucleotide that is n nucleotides long has a total of n-1 dinucleotides and only n-3 internal dinucleotides. Each internucleotide link in an internal dinucleotide is an internal internucleotide linkage. Therefore a linear oligonucleotide that is n nucleotides long has a total of n-1 internucleotide linkages and only n-3 internal internucleotide linkages. Thus, the phosphodiester internucleotide linkage or a strategically placed internucleotide linkage to phosphodiesters refers to phosphodiester internucleotide linkages or phosphodiester-like internucleotide linkages located between any pair of nucleotides in the nucleic acid sequence. In some embodiments the phosphodiester internucleotide linkages or phosphodiester-like internucleotide linkages are not placed between any pair of nucleotides closest to the 5 'or 3' end. The invention is based at least in certain aspects on the surprising discovery that the semi-soft class-C specific oligonucleotides described in the present invention have important immunostimulatory activity and are preferably useful in the treatment of allergy and asthma. These molecules have at least the same immunostimulatory activity or in many cases have a greater immunostimulatory activity, in many cases, than the corresponding fully stabilized immunostimulatory oligonucleotides having the same nucleotide sequence. A semi-soft oligonucleotide is an immunostimulatory oligonucleotide having a partially stabilized base structure, in which phosphodiester internucleotide bonds or phosphodiester-like internucleotide linkages occur only with at least one internal pyrimidine-purine dinucleotide (YZ, preferably CG). The semi-soft oligonucleotides generally possess an increased immunostimulatory potency relative to the corresponding fully stabilized immunostimulatory oligonucleotides. Due to the higher potency of the semi-soft oligonucleotides, the semi-soft oligonucleotides can be used at lower effective concentrations and have lower effective doses than conventional fully stabilized immunostimulatory oligonucleotides in order to achieve a desired biological effect. Although fully stabilized immunostimulatory oligonucleotides can exhibit a maximum dose-response, the semi-soft oligonucleotides of the present invention appear to have monotonically increasing dose-response curves (as tested by stimulation with TLR9) extending to higher concentrations beyond the optimal concentration for the corresponding fully stabilized immunostimulatory oligonucleotides. Therefore, it is believed that the semi-soft oligonucleotides of the present invention can induce greater immunostimulation than fully stabilized immunostimulatory oligonucleotides. Although fully stabilized immunostimulatory oligonucleotides less than 20 nucleotides long may have modest immunostimulatory activity compared to longer fully stabilized oligonucleotides (eg, 24 nucleotides long), semi-soft oligonucleotides as short as 16 nucleotides have been found. long have immunostimulating activity at least equal to the immunostimulating activity of fully stabilized oligonucleotides of 20 nucleotides long. In some cases where a 6-element phosphorothioate oligonucleotide appears to lack immunostimulant activity, the replacement of even an internal CG phosphodiester internucleotide linkage by a phosphorothioate linkage was found to produce a corresponding 6-element product with immunostimulatory activity. Therefore the size (e.g., the number of nucleotide residues along the length of the oligonucleotide) of the immunostimulatory oligonucleotide may also contribute to the stimulating activity of the oligonucleotide. To facilitate the capture in the cells, the immunostimulatory oligonucleotides can have a minimum length of 6 nucleotide residues. Oligonucleotides of any size greater than 6 nucleotides (even several kb in length) are capable of inducing an immune response in accordance with the invention if sufficient immunostimulatory motifs are present, since larger nucleic acids are degraded within cells. It is believed by the present inventors that semi-soft oligonucleotides as short as 4 nucleotides can also be immunostimulatory if administered to the interior of the cell. In certain preferred embodiments according to the present invention, the immunostimulatory oligonucleotides are between 4 and 100 nucleotides long. In typical embodiments the immunostimulatory oligonucleotides are between 6 and 40 or 10 and 40 nucleotides long. In certain preferred embodiments according to the present invention, the immunostimulatory oligonucleotides are between 6 and 19 or 6 and 24 nucleotides long.

It is also believed that the foregoing properties of semi-soft oligonucleotides generally increase with an increase in the "dose" of phosphodiester internucleotide linkages or phosphodiester-like internucleotide linkages that include CG internal dinucleotides. It is therefore believed, for example, that generally for a given oligonucleotide sequence with five internal CG dinucleotides, an oligonucleotide with five internal phosphodiester GC or phosphodiester-like internucleotide bonds is more immunostimulating than an oligonucleotide with four internucleotide bonds internal phosphodiester GC or the like a phosphodiester, which in turn is more immunostimulating than an oligonucleotide with three internucleotide bonds phosphodiester CG internal or similar to phosphodiester, which in turn is more immunostimulating than an oligonucleotide with two internucleotide bonds phosphodiester CG internal or similar to phosphodiester, has at its instead, it is more immunostimulating than an oligonucleotide with an internal or phosphodiester-like phosphodiester internucleotide linkage. Importantly, the inclusion of even an internal phosphodiester internucleotide linkage CG or phosphodiester-like is believed to be advantageous in comparison to a non-internal or phosphodiester-like phosphodiester internucleotide linkage. In addition to the number of phosphodiester or phosphodiester-like internucleotide linkages, the position along the length of the oligonucleotide can also affect potency. The immunostimulatory oligonucleotides of the present invention are generally protected from rapid degradation in serum. The immunostimulatory oligonucleotides of the present invention are also generally protected from rapid degradation in most tissues, with the exception of particular tissues with specific or excessive nuclease activity that are capable of degrading the immunostimulatory oligonucleotides. This results in the reduction of immunostimulatory oligonucleotides in those particular tissues, the accumulation of which could otherwise lead to undesirable effects from long-term therapy using oligonucleotides resistant to degradation. The oligonucleotides of the present invention will generally include, in addition to phosphodiester internucleotide linkages or phosphodiester-like internucleotide linkages at the preferred internal positions, the 5 'and 3' ends that are resistant to degradation. Such degradation resistant ends can include any suitable modification resulting in increased resistance ast exonuclease digestion compared to the corresponding unmodified ends. For example, the 5"and 3 'ends can be stabilized by the inclusion of at least one modification of the phosphate of the base structure In a preferred embodiment, the at least one modification of the phosphate of the base structure at each end is independently a internucleotide linkage of phosphorothioate, phosphorodithioate, methylphosphonate, or methylphosphorothioate In another embodiment, the endpoint resistant to degradation includes one or more nucleotide units connected by peptide or amide linkages at the 3 'end.Even other stabilized ends, including but not limited to Those additionally described below are intended to be included by the invention As described above, the oligonucleotides of the present invention include phosphodiester bonds or phosphodiester-like bonds in and optionally adjacent to the internal CG dinucleotides.These CG dinucleotides are frequently of immunostimulant motifs. However, it is not necessary for an oligonucleotide to contain phosphodiester bonds or phosphodiester-like bonds in each immunostimulant motif. Additional phosphodiester bonds or phosphodiester-like bonds can also be maintained for even more rapid renal digestion of these "oligonucleotides stabilized" in another way. An internucleotide phosphodiester linkage is the type of nucleic acid binding characteristics found in nature. The internucleotide phosphodiester linkage includes a phosphorus atom flanked by two oxygen atoms that form a bridge and also linked by two additional oxygen atomsone loaded and the other not loaded. The phosphodiester internucleotide linkage is particularly preferred when it is important to reduce the half-life of the oligonucleotide in the tissue. An internucleotide linkage similar to phosphodiester is a phosphorus-containing bridging group that is chemically and / or diastereomerically similar to the phosphodiester. Measurements of similarity to the phosphodiester include susceptibility to nuclease digestion and the ability to activate RNAse H. Thus, for example, phosphodiester oligonucleotides, but not phosphorothioate oligonucleotides, are susceptible to nuclease digestion, although both oligonucleotides of phosphodiester and phosphorothioate activate RNase H. In a preferred embodiment, the phosphodiester-like internucleotide linkage is a boranophosphate link (or equivalently, boranophosphonate). The Patent of E.U.A. No. 5, 177, 198; Patent of E.U.A. No. 5,859,231; Patent of E.U.A. No. 6, 160, 109; Patent of E.U.A. No. 6,207,819; Sergueev et al., (1998) J Am Chem Soc 120: 9417-27. In another preferred embodiment the phosphodiester-like internucleotide linkage is diastereomerically pure phosphorothioate Rp. It is believed that the diastereomerically pure Rp phosphorothioate is more susceptible to nuclease digestion and is better for activating the RNAse H compared to the mixed or diastereomerically pure phosphorothioate Sp. It should be mentioned that for purposes of the present invention, the term "phosphodiester-like internucleotide linkage" specifically excludes the internucleotide linkages of phosphorodithioate and methylphosphonate. The immunostimulatory oligonucleotide molecules of the present invention have a chimeric base structure. For purposes of the present invention, a chimeric base structure refers to a partially stabilized base structure, wherein at least one internucleotide linkage is phosphodiester or phosphodiester-like, and wherein at least one different internucleotide linkage is a stabilized internucleotide linkage, where the at least one phosphodiester or phosphodiester-like linkage and the at least one stabilized linkage are different. Since it has been reported that boranophosphonate linkages are stabilized relative to phosphodiester linkages, for purposes of the chimeric nature of the base structure, the boranophosphonate linkages can be classified either as phosphodiester-like or as stabilized, depending on the context . For example, a chimeric base structure in accordance with the present invention could in one embodiment include at least one phosphodiester (phosphodiester or phosphodiester-like) and at least one boranephosphonate (stabilized) linkages. In another embodiment a chimeric base structure in accordance with the present invention could include the boranophosphonate (phosphodiester or phosphodiester-like) and phosphorothioate (stabilized) linkages. A "stabilized internucleotide link" should mean an internucleotide linkage that is relatively resistant to in vivo degradation (eg, via an exo- or endo-nuclease), as compared to an internucleotide phosphodiester link. Preferred stabilized internucleotide linkages include, without limitation, phosphorothioate, phosphorodithioate, methylphosphonate, and methylphosphorothioate. Other stabilized internucleotide linkages include, without limitation: peptide, alkyl, dephosphoryl, and others as described above. Modified base structures such as phosphorothioates can be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. The aryl- and alkyl phosphonates can be made, for example, as described in the U.S. Patent. No. 4,469,863; and the alkyl phosphotriesters (in which the oxygen-laden portion is alkylated as described in U.S. Patent No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. The methods for the elaboration of other modifications and substitutions of the DNA base structures have been described. Uhlmann E et al. (1990) Chem Rev 90: 544; Goodchild J (1990) Bioconjugate Chem 1: 165. Methods for the preparation of chimeric oligonucleotides are also shown. For example, the patents issued to Uhlmann et al describe such techniques. The modified mixed base structures of ODN can also be synthesized using a commercially available DNA synthesizer and standard phosphoramidite chemistry. (FE Eckstein, "Oligonucleotides and Analogues - A Practical Approach" IRL Press, Oxford, UK, 1991, and MD Matteucci and MH Caruthers, Tetrahedron Lett.21, 719 (1980)) After coupling, PS bonds are introduced by sulfurization using the Beaucage reagent (RP lyer, W. Egan, JB Regan and SL Beaucage, J. Am. Chem. Soc. 12, 1253 (1990)) (0.075 M in acetonitrile) or phenyl acetyl disulfide (PADS) followed by end modification with acetic anhydride, 2,6-lutidine in tetrahydrofuran (1: 1: 8; v: v: v) and N-methylimidazole (16% in tetrahydrofuran). This end modification step is carried out after the sulfurization reaction to minimize the formation of undesirable phosphodiester (PO) bonds at the positions where a phosphorothioate linkage is to be located. In the case of the introduction of a phosphodiester linkage, for example into a CpG dinucleotide, the phosphorus-III intermediate is oxidized by treatment with an iodine in water / pyridine solution. After cleavage from the solid support and final deprotection by treatment with concentrated ammonia (15 hours at 50 ° C), the ODNs are analyzed by HPLC on a Gen-Pak Fax column (Millipore-Waters) using a NaCl gradient. (eg pH A regulator: 10 mM NaH2PO4 in acetonitrile / water = 1: 4 / v: v pH 6.8, pH regulator B: 10 mM NaH2P04, 1.5 M NaCl in acetonitrile / water = 1: 4 / v: v 5 to 60% B in 30 minutes at 1 ml / min) or capillary gel electrophoresis. The ODN can be purified by CLAR or by FPLC on a Source High Performance column (Amersham Pharmacia). The homogeneous fractions for HPLC are combined and desalted via a C18 column or by ultrafiltration. The ODN was analyzed by MALDI-TOF mass spectrometry to confirm the calculated mass. Oligonucleotides of the invention may also include other modifications. These include nonionic analogs of DNA, such as alkyl and aryl phosphates (in which the oxygen-charged phosphonate is replaced by an alkyl or aryl group), phosphodiester and alkyl phosphotriesters, in which the oxygen-laden portion is alkylated. It has been shown that oligonucleotides which contain diol, such as tetraethylene glycol or hexaethylene glycol, at either end or both ends are substantially resistant to nuclease degradation. The oligonucleotides of the present invention are nucleic acids that contain specific sequences that have been found to induce an immune response. These specific sequences that induce an immune response are referred to as "immunostimulatory motifs", and oligonucleotides containing immunostimulatory motifs are referred to as "nucleic acid immunostimulatory molecules" and, equivalently, "immunostimulatory nucleic acids" or "immunostimulatory oligonucleotides". Therefore, the immunostimulatory oligonucleotides of the invention include at least one immunostimulatory motif. In a preferred embodiment the immunostimulatory motif is an "internal immunostimulatory motif". The term "immunostimulatory internal motif" refers to the position of the motif sequence within a longer sequence of nucleic acids, which is longer in length than the motif sequence in at least one nucleotide associated both to the 5 'ends and 3 'of the immunostimulatory motif sequence. Immunostimulatory oligonucleotides include immunostimulatory motifs which are "CpG dinucleotides". A CpG dinucleotide can be mediated or non-methylated. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is a nucleic acid molecule containing a non-methylated cytosine-guanine dinucleotide sequence (eg, an unmethylated 5'-cytidine followed by 3 'guanosine and associated by a phosphate linkage) and which activates the immune system; said immunostimulatory oligonucleotide is a CpG oligonucleotide. CpG oligonucleotides have been described in numerous issued patents, published patent applications, and other publications, including US Patents. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6.239.1 16; and 6,339,068. An immunostimulatory oligonucleotide containing at least one methylated CpG dinucleotide is an oligonucleotide containing a methylated cytosine-guanine dinucleotide sequence (eg, a methylated 5 'cytidine followed by a 3' guanosine and associated by a phosphate linkage) and which activates the immune system. Recently it has been described that there are different classes of CpG oligonucleotides. One class is potent to activate B cells but is relatively weak to induce IFN-a and NK cell activation; this class has been termed class B. CpG class B oligonucleotides are typically fully stabilized and include a non-methylated CpG dinucleotide with certain preferred base contexts. See, for example, US Patents. Nos. 6, 194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another class is potent for inducing IFN-a and NK cell activation but it is relatively weak to stimulate B cells; this class has been termed class A. CpG class A oligonucleotides typically have poly-G sequences stabilized at the 5 'and 3' ends and a central palindromic sequence containing a phosphodiester dinucleotide sequence of at least 6 nucleotides. See, for example, published patent application PCT / US00 / 26527 (WO 01/22990). Even another class of CpG oligonucleotides activates B cells and NK cells and induces IFN-a; this class has been called the C-class. The CpG class-C oligonucleotides, as initially characterized, are typically fully stabilized, and include a class B-type sequence and a GC-rich palindrome or almost a palindrome. This class has been described in the co-pending Patent Application of E.U.A. 10 / 224,523, filed August 19, 2002 and published as US2003 / 0148976 and US10 / 978,283 filed October 29, 2004, with a related PCT application published as WO2005 / 042018 the total contents of which are incorporated herein invention as references. Class C oligonucleotides are also referred to as CpG C ODN. In certain embodiments and CpG C ODNs include a combination of motifs wherein one motif is a CG-rich palindrome or a neutralizing motif, and another motif is a stimulatory motif, for example , a CpG motif or the TCGTCG sequence. The CpG C ODN can have the formula: 5 'X1DCGHX2 3'. ^ and X2 are independently any sequence from 0 to 10 nucleotides long. D is a nucleotide other than C. C is cytosine. G is guanine. H is a nucleotide other than G. The nucleic acid sequence also includes a nucleic acid sequence selected from the group consisting of P and N located immediately toward 5 'to X1 or immediately toward 3' to X2. N is a B cell neutralizing sequence that begins with a CGG trinucleotide and is at least 10 nucleotides long. P is a GC-rich palindrome containing a sequence of at least 10 nucleotides long.

In some embodiments, the immunostimulatory nucleic acid is 5 'NX- | DCGHX2 3', 5 'XT DCGHX ^ 3', 5 'PX ^ CGHXz 3', 5 'X1DCGHX2P 3', 5 'X! DCGHXjPXa 3', 5 'X ^ CGHPXa 3 ', 5' DCGHX2PX3 3 \ 5 'TCGHX2PX3 3', or 5 'DCGHPX3 3'. X3 is any sequence from 0 to 10 nucleotides long. In other embodiments, the immunostimulatory nucleic acid is 5 'DCGHP 3'. Optionally D and / or H are thymine (T). In other modalities H is T and X2 is CG, CGT, CGTT, CGTTT, or CGTTTT. H is T and X2 is CG or CGTTTT in accordance with other modalities. In accordance with other modalities, C is not methylated. N includes at least four CG dinucleotides and no more than two CCG trinucleotides in some embodiments. Optionally P includes at least one inosine. The nucleic acid may also include a poly-T sequence at the 5 'end or the 3' end. Altematively the CpG C ODN can have the formula: 5 'N1PyGN2P 3'. G is guanine. N-i is any sequence of 1 to 6 nucleotides long. In some embodiments, Ni is at least 50% pyrimidines and preferably at least 50% T. In other embodiments, Ni includes at least one CG motif, at least one TCG motif, at least one Cl motif, at least one TC I motif, at least one IG reason, or at least one TIG reason. Neither is TCGG or TCGH in other modalities. H is a nucleotide other than G.

Py is a pyrimidine. In some modalities Py is a non-methylated C. N2 is any sequence of 0 to 30 nucleolides long. In some embodiments N2 is at least 50% pyrimidines or at least 50% T. In other embodiments N2 does not include any poly G or poly A motif. P is a GC-rich palindrome containing a sequence of at least 10 nucleotides long. In some modalities P is completely palindromic. In other modalities P is a palindrome that has between 1 and 3 consecutive nucleotides interposed. Optionally the interposed nucleotides can be TG. In other embodiments P includes at least 3, 4, or 5 nucleotides C and at least 3, 4, or 5 nucleotides G. In accordance with other embodiments P includes at least one inosine. In one embodiment, the GC-rich palindrome has a base content of at least two thirds of G and C. In another embodiment, the GC-rich palindrome has a base content of at least 81 percent of G and C. In some embodiments the GC-rich palindrome is at least 12 nucleotides long. The GC-rich palindrome can be made exclusively from C and G. In some embodiments the GC-rich palindrome can include at least one nucleotide that is neither C nor G. In some embodiments the GC-rich palindrome includes at least one CGG trimer. , at least one CCG trimer, or at least one CGCG tetramer. In some embodiments, the GC-rich palindrome includes at least four CG dinucleotides. In certain preferred embodiments, the GC-rich palindrum has a central CG dinucleotide. In certain embodiments, the GC-rich palindrome is CGGCGCGCGCCG (SEQ ID NO: 58), CGGCGGCCGCCG (SEQ ID NO: 59), CGACGATCGTCG (SEQ ID NO: 60) or CGACGTACGTCG (SEQ ID NO: 61). In certain embodiments, the GC-rich palindrome is CGCGCGCGCGCG (SEQ ID NO: 62), GCGCGCGCGCGC (SEQ ID NO: 63), CCCCCCGGGGGG (SEQ ID NO: 64), GGGGGGCCCCCC (SEQ ID NO: 65), CCCCCGGGGG (SEQ ID NO. : 66) or GGGGGCCCCC (SEQ ID NO: 67). In some embodiments N1PyGN2 is a sequence selected from the group consisting of TTTTTCG, TCG, TTCG, TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT, and TCGTCGT. An immunostimulatory nucleic acid of 1 3-100 nucleotides in length is provided in accordance with other aspects of the invention. The nucleic acid has the formula: 5 'NiPyG / IN2P 3'. G / l refers to a particular nucleotide which is either a G or an I. G is guanine, and I is Inosine. ? ? is any sequence of 1 to 6 nucleotides long. Py is a pyrimidine. N2 is any sequence from 0 to 30 nucleotides long. P is a palindrome containing a sequence of at least 10 nucleotides long. In some modalities P is a palindrome rich in GC. In other modalities P is a palindrome rich in HF. NiPylN2 in some modalities is TCITCITTTT (SEQ ID NO: 62).

One class of oligonucleotides referred to in the present invention as modified class-C oligonucleotides are characteristically monomeric in solution. It is believed that these nucleic acid molecules can form intramolecular duplex structures in vitro, rendering them stable against nuclease digestion. It is also believed that these same nucleic acid molecules can form an intermolecular duplex and possibly even structures with a higher order within the environment of the intraendosomal compartment, where they are believed to exert their biological activity. Modified oligonucleotides class-C have general formula. The formula IZ, [(X1Y1R1) N (X2Y2R2) k Z2] p (S q N '(Nn) ... (N2) (N1) S2 (N1 #) (N2 #) ... (Nn #) Z3 (formula I) wherein each of Zi, Z2, and Z3 is independently any sequence of 0 to 12 nucleotides long which optionally includes a non-nucleotide linker or abasic Separator, each of Xi and X2 is independently a thymidine, deoxyuridine , deoxyadenosine or a 5-substituted deoxyuridine, each of Yi and Y2 is independently a cytosine (C) or a modified cytosine, each of and R2 is independently a guanine (G) or a modified guanine, each of N and N 'is independently any sequence of 0 to 12 nucleotides long which optionally includes a non-nucleotide linker or abasic Separator, ST is a non-nucleotide linker, with the abasic spit (d Separators), triethylene glycol units or hexaethylene glycol units, which are provided optionally for the internucleotide links 2'5'-, 5'5'-, 3'3'-, 2'2'-, or 2'3 S2 is any non-palindromic sequence of 1 to 10 nucleotides long or a non-nucleotide linker, an abasic linker (d Separators), triethylene glycol units or hexaethylene glycol units; each of Ni, N2,. .. Nn, and N1 #, N2 #; ... Nn # is any nucleotide or a modified nucleotide where the base pairs N-? with N #, the base pairs N2 with N2 #, ... and the base pairs Nn with Nn #; k is an integer from 0 to 5; n is an integer from 2 to 16; p is an integer from 1 to 6; yq is an integer from 0 to 10, and where when (Nn) ... (N2) (N) S2 (? 1 #) (? 2 #) ... (?? #) is 10 to 42 nucleotides long, S2 is 4 to 10 nucleotides long, S2 comprises a non-nucleotide linker, an abasic linker (d Separators), triethylene glycol units or hexaethylene glycol units, and / or (Nn) ... (N2) (N1) S2 (N1 #) (N2 #) ... (Nn #) has a GC content that is less than 2/3. In a modality each of Ni, N2, .... Nn, and N1 #, N2 #, ... Nn # is chosen from C, G, or modifications thereof, where C forms base pairs with G In one modality each of N2, .... Nn, and N #, N2 #, ... Nn # is chosen from T, A, or modifications thereof, and T forms base pairs with A.

In these and other embodiments each of C, G, A, and T may refer to deoxynucleotides with corresponding bases of cytosine, guanine, adenine, and thymine. In a modality each of Ni, N2, .... Nn, and Ni #, N2 #, ... Nn # is chosen from C, T, A, G, or modifications thereof, and C forms pairs of bases with G, T forms base pairs with G, A forms base pairs with T, and A forms base pairs with G. In a modality each of N2, .... Nn, and N1 #, N2 #, ... Nn # is chosen from unmodified or modified nucleotides that form Watson-Crick base pairs. In one modality each of N t N2 Nn, and Ni #, N2 #, ... Nn # is chosen from unmodified or modified nucleotides that form non-Watson-Crick base pairs. In one embodiment the immunostimulatory nucleic acid molecule includes a partially stabilized base structure with at least one phosphodiester bond. In one embodiment the immunostimulatory nucleic acid molecule includes a base structure with at least one stabilized internucleotide linkage. In one embodiment the intemucleotide bonds of the oligonucleotide are all phosphorothioate linkages.

In one embodiment the immunostimulatory nucleic acid molecule includes a partially stabilized base structure with a phosphodiester bond linking at least one of or Y2R2 In a modality Yi is C. In one embodiment it is G. In a modality Yt is C and R1 is G In one modality or X2 is T. In a modality Xi is T, X2 is T, Y is C, is G, and k is 1. In an X mode! is T, X2 is T, is C,? is G, k is 1, p is 1, N and N 'and Z3 each contains zero nucleotides, and Z2 is M i l or d (UUUU). In an S2 modality it is a non-nucleotide linker. In an S2 mode it contains at least one residue d abasic Separator. In one embodiment the oligonucleotide includes at least one non-nucleoside branched linkage. In one embodiment the nucleic acid immunostimulatory molecule includes at least one doubler unit, at least one trebler unit, or at least one doubler unit and at least one trebler unit. In a modality If it is a doubler unit or a trebler unit. In one embodiment the oligonucleotide includes at least one internucleoside link 2.5'5'5'-, 3'3'-, 2'2"or 2'3'-.

In one aspect, the invention provides an immunostimulatory nucleic acid molecule of formula II? (Nn) (Nn.i) ... (N2) (N1) S2 (N1 #) (N2 #) ... (Nn-1 #) (Nn #) (Yes) q Z3 [(X1Y1R1) N ( X2Y2R2) k Z2] p (formula II) wherein each of Zi, Z2, and Z3 is independently any sequence of 0 to 12 nucleotides long which optionally includes a non-nucleotide linker or abasic separator; each of Xi and X2 is independently a thymidine, deoxyuridine, deoxyadenosine or a 5-substituted deoxyuridine; each of Y1 and Y2 is independently a cytosine (C) or a modified cytosine; each of R i and R 2 is independently a guanine (G) or a modified guanine; N is any sequence of 0 to 12 nucleotides long which optionally includes a non-nucleotide linker or d-abasic Separator; S1 is a non-nucleotide linker, an abasic linker (d Separators), methylene glycol units or hexaethylene glycol units, which are optionally provided for the internucleoside linkages 2'5'-, 5'5'-, 3'3'-, 2'2 '-, or 2'3'-; S2 is any nonpalindromic sequence of 1 to 10 nucleotides long or a non-nucleotide linker, an abasic linker (d Separators), triethylene glycol units or hexaethylene glycol units; each of Ni, N2, .... Nn-1, Nn, and Ni #, N2 #, ... Nn-i #, Nn # is any modified nucleotide or nucleotide where N forms base pairs with N1 # , N2 forms base pairs with N2 #, ... Nn.i forms base pairs with Nn. #, and Nn forms base pairs with Nn #; k is an integer from 0 to 5; n is an integer from 2 to 16; p is an integer from 1 to 6, and q is an integer from 0 to 10, and where when (Nn) ... (N2) (N) S2 (N #) (N2 #) ... (Nn #) is 10 to 42 nucleotides long, S2 is 4 to 10 nucleotides long, S2 comprises a non-nucleotide linker, an abasic linker (d Separators), triethylene glycol units or hexaethylene glycol units, and / or (Nn) ... ( N2) (N1) S2 (N1 #) (N2 #) ... (Nn #) has a GC content that is less than 2/3. In a Z-i (Nn) (Nn-i) modality it is TYR, where Y is a cytosine or a modified cytosine and R is a guanine or a modified guanine. In a modality each of N2 Nn-i, Nn, and N #I N2 #, ...

Nn-i #, Nn # is chosen from C, G, or modifications thereof, where C forms base pairs with G. In one mode each of Ni, N2 Nn.i, Nn, and N1 # l N2 #, ...

Nn-i #, Nn # is chosen from T, A, or modifications thereof, and T forms base pairs with A. In these and other modalities each of C, G, A, and T may refer to deoxynucleotides with corresponding bases of cytosine, guanine, adenine, and thymine. In a modality each of N2, .... Nn-, Nn, and N1 #, N2 #, ... Nn-1 #, Nn # is chosen from C, T, A, G, or modifications thereof , and C forms base pairs with G, T forms base pairs with G, A forms base pairs with T, and A forms base pairs with G.

In one modality each of Ni, N2, .... N ^, Nn, and N1 #, N2 #, ... Nn-i #, Nn # is chosen from unmodified or modified nucleotides that form Watson base pairs -Crick. In one embodiment each of Ni, N2 Nn-1, Nn, and N1 #, N2 #, ... Nn.i #, Nn # is chosen from unmodified or modified nucleotides that form non-Watson-Crick base pairs. In one embodiment the immunostimulatory nucleic acid molecule includes a partially stabilized base structure with at least one phosphodiester bond. In one embodiment the immunostimulatory nucleic acid molecule includes a base structure with at least one stabilized internucleotide linkage. In one embodiment the internucleotide linkages of the oligonucleotide are all phosphorothioate linkages. In one embodiment the immunostimulatory nucleic acid molecule includes a base structure partially stabilized with a phosphodiester bond that binds at least one of Y1R1 or Y2R2. In a modality Yi is C. In a modality Ri is G. In a modality Yi is C and Ri is G. In a modality Xi or X2 is T. In a modality Xi is T, X2 is T, Yi is C, is G, yk is 1.

In a modality Xi is T, X2 is T,? t is C, Ri is G, k is 1, p is 1, N and N 'and Z3 each contains zero nucleotides, and Z2 is TTTT or d (UUUU). In an S2 modality it is a non-nucleotide linker. In an S2 mode it contains at least one residue d abasic Separator. In one embodiment the oligonucleotide includes at least one non-nucleoside branched linkage. In one embodiment the immunostimulatory nucleic acid molecule includes at least one doubler unit, at least one trebler unit, or at least one doubler unit and at least one trebler unit. In a modality If it is a doubler unit or a trebler unit. In one embodiment the oligonucleotide includes at least one 2.5'-, 5'5'-, 3'3'-, 2'2'-, or 2'3'- internucleoside linkage. In one aspect the invention provides an immunostimulatory nucleic acid molecule of formula III (U) m Z3 (S3) (formula III) wherein U is Z, [(X1Y1 R1) N (X2Y2R2) k Z2] p (S ^ NT (Nn) .... (NsXNzXN S2 (N1 #) (N2 #) (N3 #) ... (Nn #); each of Z1 f Z2, and Z3 is independently any sequence from 0 to 12 nucleotides long which optionally includes a non-nucleotide linker or d-abasic Separator; each of X-? and X2 is independently a thymidine, deoxyuridine, deoxyadenosine or a 5-substituted deoxyuridine; each one of ?? and Y2 is independently a cytosine or a modified cytosine; each of R1 and R2 is independently a guanine or a modified guanine; each of N and N 'is independently any sequence of 0 to 12 nucleotides long which optionally includes a non-nucleotide linker or d-abasic Separator; If it is a non-nucleotide linker, an abasic linker (d Separators), triethylene glycol units or hexaethylene glycol units, which are optionally provided for the internucleoside linkages 2'5'-, 5'5'-, 3'3'-, 2'2 '-, or 2'3'-; S2 is any nonpalindromic sequence of 1 to 10 nucleotides long or a non-nucleotide linker, an abasic linker (d Separators), triethylene glycol units or hexaethylene glycol units; S3 is a direct or indirect internucleoside link 2'5'-, 5'5'- 3'3 '. 2'2'-, or 2'3'-, or a non-nucleotide linker, said non-nucleotide linker including abasic linkers (d Separators), triethylene glycol units, or hexaethylene glycol units facilitate 2'5'-, 5'5 'linkage -, 3'3'-, 2'2'-, or 2'3'- of m parts of the sequence; each of Ni, N2, .... Nn, and N #, N2 #; ... Nn # is any modified nucleotide or nucleotide where? T forms base pairs with N #, N2 forms base pairs with N2 #, N3 forms base pairs with N3 #, ... and Nn forms base pairs with Nn #; k is an integer from 0 to 5; m is an integer from 2 to 10; n is an integer from 2 to 16; p is an integer from 1 to 6; and q is an integer from 0 to 10. In certain modalities ?? [(XiYi Ri) N (X2Y2R2) k Z2) p (Yes) q is a non-palindromic sequence.

In certain embodiments Zi ((X1Y1 R1) N (X2Y2R2) k Z2] p is TCGTCGTTTT (SEQ ID NO: 29), TCGTCGTTDD (SEQ ID NO: 30), TCGA, TCGAC, TCGACGTC, or TCGACGTCG, where D is a d Separator In certain modalities [(X1Y1 R1) N (X2Y2R2) k Z2] p (If) q is a palindromic sequence In certain modalities Z1 [(X1Y1 R1) N (X2Y2R2) k Z2] p (S q is TCGACGTCGA (SEQ ID NO: 31) or TCGTCGACGA (SEQ ID NO: 32) In certain embodiments Z1 [(X1Y1 R1) N (X2Y2R2) k Z2] p (Yes) q is TCGCGACGTT (SEQ ID NO: 33) or TCGCGTCGTT ( SEQ ID NO: 34) In one mode (Nn) .. - (N2) (N S2 (N1 #) (N2 #) ... (Nn #) Z3 includes a sequence AGCGAAGCT, CAATATTTATTG (SEQ ID NO: 35 ), CCGTTTTGTGG (SEQ ID NO: 36), CGGCGCCGTGCCG (SEQ ID NO: 37), CGGCGCCGTTGCCG (SEQ ID NO: 38), CGGCGDDCGCCG (SEQ ID NO: 39), CGGCGDDDTGCCG (SEQ ID NO: 40), CGGCGGDDCCGCCG (SEQ ID NO: 41), CGGCGTCGCCGCCG (SEQ ID NO: 42), CGTCGACGGGACGGG (SEQ ID NO: 43), CGTCGACGTGACGGG (SEQ ID NO: 44), GAGAGTTGGGCTCTC (SEQ ID NO: 45), GTCGAGGAGGT (SEQ ID NO: 46) , TAATADDTATTA (SEQ ID NO: 47), TAATATCCATTA (SEQ ID NO: 48), or TAATATTTATTA (SEQ ID NO: 49), where D is d Separator. In one embodiment (Nn) ... (N2) (N1) S2 (N1 #) (N2 #) ... (Nn #) includes a sequence GGCGCGCTGCCG (SEQ ID NO: 50). In one embodiment the 5 'end of the nucleic acid begins with an immunostimulatory motif chosen from (TCG) nN and RDCGY- | Y2N. T is thymine, C is not methylated cytosine, G is guanine, R is a purine, D is not C, each of Yi and? 2 independently is a pyrimidine, n is an integer between 1 and 4, inclusive, and N is any sequence of 0-12 bases long. The 3 'end of the nucleic acid ends in an inverted repeat capable of forming a hairpin or stem structure. The term "ends" refers to a structure at or near the 3 'end. Therefore, the end of the close palindrome can be located at the actual 3 'end of the molecule or alternatively the 3' end can include 1 or more additional nucleotides that are not part of the inverted repeat structure. Preferably the 3 'end of the molecule includes 3 or fewer nucleotides that are not part of the inverted repeat structure. In one embodiment a "repeated inverted capable of forming a fork or stem structure" as used in the present invention refers to a nucleotide sequence that forms a GC-rich stem or fork that is 2 to 10 base pairs consecutive long, and includes at least one mismatched or coincident base. In individual modalities the GC-rich stem is 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive base pairs long. In some modalities in stem rich in GC includes at least 2, 3, or 4 base pairs G-C. In a "repeated inverted form capable of forming a fork or stem structure" as used in the present invention, it refers to a nucleotide sequence that forms an AT-rich stem or fork that is 2 to 10 base pairs consecutive long, and includes at least one mismatched or coincident base. In individual modalities, the AT-rich stem is 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive base pairs long. In some embodiments, the AT-rich stem includes at least 2, 3, or 4 base pairs A-T. In some cases the at least one mismatched or coincident base forms a bridge with the ends of the stem or fork. This can allow the formation of the secondary structure by providing a flexible point in the molecule so that the stems form base pairs and form a fork. Alternatively, the mismatched or coinciding base (s) may be within the stem. Preferably if the mismatched base is on the stem, then the stem is at least 3 base pairs long. The mismatched or coincident base (s) can be any nucleotide. In some modalities the mismatched or coincident base is a T. The mismatched nucleotides at the end of the double strands are also known as pendant nucleotides. or pending ends that can significantly stabilize the formation of the duplex or the formation of the fork. Freier SM et al. (1983) Effects of 3 'dangling end stacking on the stability of GGCC and CCGG double helixes. Biochemistry 22: 6198-206. The nucleic acid also includes a partially stabilized base structure ncluding at least one 5'-CpG-3 'phosphodiester linkage. In some cases the part of the double chain of the molecule may also contain unnatural (non-standard) base pairs (for example, diaminopyridine coupled with xanthosine). Lutz MJ et al. (1998) Recognition of a non-standard base pair by thermostable DNA polymerases. Bioorg Med Chem Lett 8. 1 149-52. The formulas define subpopulations of the class of CpG oligonucleotides that demonstrate excellent immune stimulating properties. In the 5 'formulas it refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide. The oligonucleotides may have one or more accessible 5 'or 3' ends. In some modalities with 3 'end it can be associated to another 3' end. Since the importance of the 5 'and 3' motifs has been discovered and described in the present invention, it is also possible to create modified oligonucleotides having two of said 5 'or 3' ends. This can be achieved, for example by joining two oligonucleotides through a 3 '-3' link to generate an oligonucleotide having one or two accessible 5 'ends. Said structure can have a formula such as 5'-RDCGYiY2N- Y2YiGCDR-5 '(where D represents no C; SEQ ID NO: 51) or 5'- (TCG) nN-N (GCT) n-5 '(SEQ ID NO: 52). The 3'3'- or 5'5'- linkage can be a phosphodiester, phosphorothioate, or any other modified internucleoside bridge. Methods for achieving such links are known in the art. For example, such linkages have been described in Seliger H et al. (1991) Oligonucleotide analogs with terminal 3 '-3'- and 5'-5'-internucleotidic linkages as antisense inhibitors of viral gene expression, Nucleosides & nucleotides 10: 469-77 and Jiang Z et al. (1999) Pseudo-cyclic oligonucleotides: in vitro and in vivo properties, Bioorg Med Chem 7: 2727-35. In some embodiments the oligonucleotide has one of the following structures: TCGTCGTTTTA (SEQ ID NO: 53), CGGCGCCGTGCCG (SEQ ID NO: 54), CGGCGTCGTGCCG (SEQ ID NO: 55), TCGTCGTTTTACGGCGCCGTGCCG (SEQ ID NO: 56), TCGTCGTTTTACGGCGTCGTGCCG ( SEQ ID NO: 57), The invention in one aspect includes the finding that a specific subclass of class C immunostimulatory CpG oligonucleotides having a chimeric base structure is highly effective in mediating immune stimulatory effects. These CpG oligonucleotides are therapeutically and prophylactically useful for stimulating the immune system for the treatment of cancer, infectious diseases, allergy, asthma, autoimmune disease, and other disorders and help protect against opportunistic infections after chemotherapy for the treatment of cancer. . The strong and balanced humoral and cellular immune responses that result from the stimulation of CpG that reflects the body's own natural defense system against invading pathogens and cancer cells. The invention includes, in one aspect, the discovery of a subpopulation of immunostimulatory CpG oligonucleotides that have improved immunostimulatory properties and reduced renal inflammatory effects. In some cases, renal inflammation has been observed in subjects who have been administered with an oligonucleotide completely phosphorothioate. It is believed that the chimeric oligonucleotides described in the present invention produce less renal inflammation than the completely phosphorothioate oligonucleotides. Additionally, these oligonucleotides are highly effective in stimulating an immune response. Therefore, the phosphodiester region of the molecule does not reduce its effectiveness. The preferred immunostimulatory CpG oligonucleotides fall within one of the following 7 general formulas: 5 'TTC-GX2C-GN1X1-GX3C-GTT 3' (SEQ ID NO: 24) where N ^ is 1 -3 nucleotides in length with N referring to any nucleotide, X is a pyrimidine, X2 and X3 are any nucleotide. 51 TTC_GTC_GTTTXi_GTC_GTT 3 '(SEQ ID NO. 25), where Xi is a pyrimidine. 5 'T * T * C_G * T * C_G * T * T * T * X1_G * T * C_G * T * T 3' (SEQ ID NO: 25), where X-i is a pyrimidine. 5 'TC_GX! C_G? 2? T X3C_GN2CG 3' (SEQ ID NO .: 26), where i is 0-3 nucleotides in length, N2 is 0-9 nucleotides in length with N referring to any nucleotide and Xi, X2 , and X3 are any nucleotide. 5 'TC GTC GTN TTC GGCGCNI GCCG 3' (SEQ ID NO .: 27), where ?? is 0-3 nucleotides in length. 5 'T * C_G * T * C_G * T * N1 * T * C_G * G * C * G * CN1G * C * C * G 3' (SEQ ID NO .: 27), where Ni is 0-3 nucleotides at length. 5 'TC_G X C_G X2C_G X3TC_GGCGC_G N33"(SEQ ID NO .: 28), where N3 is 1 -5 nucleotides in length with N referring to any nucleotide and Xi, X2, and X3 are any nucleotide.

Optionally, when specified in the formula, 5 'refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide. The symbol * used in the formulas refers to the presence of a stabilized internucleotide linkage. The symbol _ in these structures refers to the presence of an internucleotide phosphodiester linkage. Internucleotide bonds not marked with an * can be stabilized or not stabilized, as long as the oligonucleotide includes at least 2-3 phosphodiester or phosphodiester-like internucleotide bonds. In some embodiments it is preferred that the oligonucleotides include 3-6 phosphodiester or phosphodiester-like bonds. In some cases the bonds between the CG motifs are phosphodiester and in other cases they are phosphorothioate bonds or other stabilized bonds. In some embodiments the oligonucleotide has one of the following structures: T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G (SEQ ID NO: 2) T * C_G * T * C_G * T * T * C_G * G * C * G * C * G * C * C * G (SEQ ID NO: 3) TC_GTC_GAC_GATC_GGCGC_GCGCCG (SEQ ID NO: 4) T * C_G * T * C_G * A * C_G * A * T * C_G * G * C * G * C_G * C * G * C * C * G (SEQ ID NO: 4) T * T * C_G * T * C_G * T * T * T * T_G * T * C_G * T * T (SEQ ID NO: 5) T * T * T * C_G * T * C_G * T * T * T * C_G * T * C_G * T * T (SEQ ID NO 6) TCGTCGTTCGGCGCGCCG (SEQ ID NO: 3) TCGTCGTCGTTCGGCGCGCGCCG (SEQ ID NO: 2) TCGTCGACGATCGGCGCGCGCCG (SEQ ID NO: 4) TTCGTCGTTTTGTCGTT (SEQ ID NO: 5) TTTCGTCGTTTCGTCGTT (SEQ ID NO: 6) TCGTCGTC CGTCGTCG GTCGTCGT TCGTCGTT CGTCGTTC GTCGTTCG TCGTTCGG CGTTCGGC GTTCGGCG TTCGGCGC TCGGCGCG CGGCGCGC GGCGCGCG GCGCGCGC CGCGCGCC GCGCGCCG. T * C_G * T * C_G * T * CCG * T * CG * T * CGG * T * C_G * T * C_G * T "rC_G * T * C_G * T * T C_G * T * C_G * T * T * CG * T * C_G * T * T * C_G T * C_G * T * T * C_G * G C_G * T * T * C_G * G * CG * T * T * C_G * G * C * GT * T * C_G * G * C * G * CT * C_G * G * C * G * C_G C_G * G * C * G * C_G * CG * G * C * G * C_G * C * GG * C * G * C_G * C * G * CC * G * C_G * C * G * C * CG * C_G * C * G * C * C * G The terms "nucleic acid" and "oligonucleotide" also comprise nucleic acids or oligonucleotides with substitutions or modifications, such as in bases and / or sugars For example, these include oligonucleotides having sugar base structures that are covalently linked to organic groups with a low molecular weight different from a hydroxy group at the 2 'position and a non-phosphate group or a hydroxy group In the 5 'position, therefore, the modified oligonucleotides can include a 2'-0-alkylated ribose group In addition, the modified oligonucleotides can include sugars such as arabinose or 2'-fluoroarabinose in place of ribose. otides can be heterogeneous in the composition of the base structure thus containing any possible combination of polymer units associated together such as peptide-nucleic acids (having an amino acid base structure with nucleic acid bases). The nucleic acids also include purines and substituted pyrimidines such as modified bases of C-5-pyrimidine and 7-deaza-7-substituted purine. Wagner RW et al. (1996) Nat Biotechnol 14: 840-4. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymine, 5-methylcytosine, 5-hydroxycytosine, 5-fluorocytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other portions of nucleobases that occur naturally and that do not occur naturally, substituted and unsubstituted. Other such modifications are well known to those skilled in the art. The immunostimulatory oligonucleotides of the present invention may comprise various modifications and chemical substitutions, as compared to RNA and natural DNA, which includes an internucleotide phosphodiester bridge, a β-D-ribose unit and / or a natural nucleotide base (adenine, guanine, cytosine, thymine, uracil). Examples of chemical modifications are known to the person skilled in the art and are described, for example, in Uhlmann E et al. (1990) Chem Rev 90: 543; "Protocols for Oligonucleotides and Analogs" Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993; Crooke ST et al. (1996) Annu Rev Pharmacol Toxicol 36: 107-129; and Hunziker J et al. (1995) Mod Synth Methods 7: 331-417. An oligonucleotide according to the invention may have one or more modifications, wherein each modification is located at a particular phosphodiester internucleotide bridge and / or at a particular β-D-ribose unit and / or at a particular natural nucleotide base position. in comparison with an oligonucleotide of the same sequence that is composed of natural DNA or RNA. For example, the invention relates to an oligonucleotide which may comprise one or more modifications and wherein each modification is independently selected from: a) replacement of a phosphodiester internucleotide bridge located at the 3 'end and / or the 5' end of a nucleotide by a modified internucleotide bridge, b) the replacement of the phosphodiester bridge located at the end 3 'and / or the 5' end of a nucleotide by a defosfo bridge, c) the replacement of a sugar phosphate unit from the sugar phosphate base structure by another unit, d) the replacement of a β-unit D-ribose for a modified sugar unit, and e) the replacement of a natural nucleotide base with a modified nucleotide base.

The most detailed examples for the chemical modification of an oligonucleotide are the following. A phosphodiester internucleotide bridge located at the 3 'end and / or the 5' end of a nucleotide can be replaced by a modified internucleotide bridge, where the modified internucleotide bridge, for example, is selected from phosphorothioate, phosphorodithioate, NR1R2- bridges phosphoramidate, boranophosphate, a-hydroxybenzyl phosphonate, phosphate- (C1-C21) -O-alkyl ester, phosphate - [(C6-C12) aryl- (C1-C21) -O-alkyl-ester, (C1-C8) alkyl-phosphonate and / or (C6-C12) arylphosphonate, (C7-C12) -a-hydroxymethyl-aryl (for example, described in WO 95/01363), wherein (C6-C12) aryl, (C6-C20) aryl and (C6-) C14) aryl are optionally substituted by halogen, alkyl, alkoxy, nitro, cyano, and wherein Ri and R2 are, independently of each other, hydrogen, (C1-C18) -alkyl, (C6-C20) -aryl, (C6-) C14) -aryl- (C1-C8) -alkyl, preferably hydrogen, (C1-C8) -alkyl, preferably (C1-C4) -alkyl and / or methoxyethyl, or Ri and R2 form, together with the nitrogen atom that the holders, a ring 5-6 membered heterocyclic which may additionally contain an additional heteroatom from the group O, S and N. The replacement of a phosphodiester bridge located at the 3 'end and / or the 5' end of a nucleotide by a defosfo bridge ( defosfo bridges are described, for example, in Uhlmann E and Peyman A in "Methods in Molecular Biology", Vol. 20, "Protocols for Oligonucleotides and Analogs", S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16 , pp. 355 ff), in which a phosphorylated bridge is selected, for example, from the formalo-dephosphonate bridges, 3'-thoformacetal, methylhydroxylamine, oxime, methylenedimethyl-hydrazo, dimethylenesulfone and / or silyl groups. A sugar phosphate unit (eg, an internucleotide bridge of β-D-ribose and phosphodiester together forms a sugar phosphate unit) from the sugar phosphate base structure (eg, a sugar phosphate base structure is composed of sugar phosphate units) can be replaced by another unit, where the other unit is, for example, suitable for making a "morpholino derivative" oligomer (as described, for example, in Stirchak EP et al. (1989) Nucleic Acids Res. 17: 6129-41), i.e., for example, replacement by a unit derived from morpholino; or to construct a nucleic acid polyamide ("PNA", as described for example, in Nielsen PE et al. (1994) Bioconjug Chem 5: 3-7), ie, for example, replacement by a base structure unit of PNA, for example, by 2-aminoethylglycine. A β-ribose unit or a pD-2'-deoxyribose unit can be replaced by a modified sugar unit, wherein the modified sugar unit is for example selected from β-D-ribose, aD-2 ' deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-F-arabinose, 2'-O-(C1-C6) alkyl-ribose, preferably 2 -O-alkyl of ( C1-C6) -ribose is 2'-0-methylribose, 2'-0-alkenyl of (C2-C6) -ribose, 2 '- [O-alkyl of (C1-C6) -0-alkyl of (C1-6) C6)] - ribose, 2'-NH2-2'-deoxyribose, β-D-xylo-furanose, α-arabinofuranose, 2,4-dideoxy ^ -D-erythro-hexo-pyranose, and carbocyclic (described, for example, in Froehler J (1992) Am Chem Soc 114: 8320) and / or open-chain sugar analogues (described, for example, in Vandendriessche et al. (1993) Tetrahedron 49: 7223) and / or bicyclic sugar analogs. (described, for example, in Tarkov M et al. (1993) Helv Chim Acta 76: 481). In some preferred embodiments the sugar is 2'-0-methylribose, particularly for one or both of the nucleotides associated by an internucleotide phosphodiester linkage or an internucleotide linkage similar to phosphodiester. A modified base is any base which is chemically distinct from the naturally occurring bases typically found in DNA and RNA such as T, C, G, A, and U, but which share basic chemical structures with these bases that occur naturally. The modified nucleotide base can be, for example, selected from hypoxanthines, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C 1 -C 6) -alkyluracil, 5- (C 2-) C6) - alkenyluracil, 5- (C2-C6) -alkynyluracil, 5- (hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5- (C1-C6) -alkylcytosine, 5- (C2-C6) - alkenylcytosine, 5- (C2-C6) -alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 2,4-diamino-purine, 8-azapurine, a 7-deazapurine substituted, preferably 7-deaza-7-substituted and / or 7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine, for example, N4-ethylcytosine, 5-hydroxideoxycytidine, 5-hydroxymethildeoxycytidine, N4-alkyloxycytidine, example, N4-ethyldeoxylcytidine, 6-thiodeoxyguanosine, and nitropyrrole deoxyribonucleotides, C5-propynylpyrimidine, and diaminopurine for example, 2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurin a, 2-amino-6-chloropurine, hypoxanthines or other modifications of a natural nucleotide base. It is intended that this list be exemplary and not to be interpreted as limiting. The oligonucleotides may have one or more accessible 5 'ends. It is possible to create modified oligonucleotides having two of said 5 'ends. This can be achieved, for example by joining two oligonucleotides through a 3'-3 'link to generate an oligonucleotide having one or two accessible 5' ends. The 3'3 'link can be a phosphodiester, phosphorothioate or any other modified internucleotide bridge. Methods for achieving such bonds are known in the art. For example, such bonds have been described in Seliger, H.; et al., Oligonucleotide analogs with terminal 3'-3'- and 5'-5'-intemucleotidic linkages as antisense inhibitors of viral gene expression, Nucleotides & Nucleotides (1991), 10 (1 -3), 469-77 and Jiang, et al., Pseudo-cyclic oligonucleotides. in vitro and in vivo properties, Bioorganic & Medicinal Chemistry (1999), 7 (12), 2727-2735. Additionally, the 3 * 3 'linked oligonucleotides wherein the bond between the 3' terminal nucleotides is not a phosphodiester, phosphorothioate or other modified bridge, can be prepared using a additional spacer, such as a tri- or tetra-ethylene glycol phosphate portion (Durand, M. et al, Triple-helix formation by an oligonucleotide containing one (dA) 12 and two (dT) 12 sequences bridged by two hexaethylene glycol chains, Biochemistry (1992), 31 (38), 9197-204, U.S. Patent No. 5658738, and U.S. Patent No. 5668265). Alternatively, the non-nucleotide linker can be derived from ethanediol, propanediol, or from an abiotic deoxyribose unit (d Separator) (Fontanel, Marie Laurence et al., Sterical recognition by T4 polynucleotide kinase of non-nucleosidic moieties 5 '). -attached to oligonucleotides; Nucleic Acids Research (1994), 22 (11), 2022-7) using standard phosphoramidite chemistry. The non-nucleotide linkers can be incorporated one or multiple times, or can be combined with each other allowing any desirable distance between the 3 'ends of the two ODNs to be associated. It has recently been reported that CpG oligonucleotides appear to exert their immunostimulatory effect through interaction with the Toll-like receptor 9 (TLR9). Hemmi H et al. (2000) Nature 408: 740-5. The TLR9 signaling activity can then be measured in response to the CpG oligonucleotide or to another immunostimulatory oligonucleotide by measuring the signals related to NF-α, NF-KB, and suitable events and intermediates upstream or downstream of NF-KB. . For use in the present invention, the oligonucleotides of the invention can be synthesized de novo using any of numerous methods well known in the art. For example, the b-cyanoethyl phosphoramidite method (Beaucage, S.L., and Caruthers, M.H., Tet.Let.22: 1859, 1981); and the H-phosphonate nucleotide method (Garegg et al, Tet.Let.27: 4051-4054, 1986, Froehler et al., Nuci.Acid.Res.14: 5399-5407, 1986; Garegg et al, Tet. Let., 27: 4055-4058, 1986, Gaffney et al, Tet.Let.29: 2619-2622, 1988). These chemistries can be carried out by a variety of automated nucleic acid synthesizers available in the market. These oligonucleotides are referred to as synthetic oligonucleotides. An isolated oligonucleotide generally refers to an oligonucleotide that is separated from the components with which it is normally associated in nature. As an example, an isolated oligonucleotide may be one that is separated from a cell, from a nucleus, from a mitochondrion or from chromatin. The oligonucleotides are partially resistant to degradation (eg, they are stabilized). A "stabilized oligonucleotide molecule" should mean an oligonucleotide that is relatively resistant to degradation in vivo (for example via an exo- or endo-nuclease). Stabilization of the nucleic acid can be achieved via modifications of the basic structure. Oligonucleotides having phosphorothioate linkages provide maximum activity and protect the oligonucleotide from degradation by intracellular exo- and endo-nucleases. Other modified oligonucleotides include phosphodiester-modified oligonucleotides, combinations of oligonucleotides with phosphodiester and phosphorothioate, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof.

Modified base structures such as phosphorothioates can be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. The aryl- and alkyl phosphonates can be made, for example, as described in the U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which the oxygen-laden portion is alkylated as described in U.S. Patent No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other modifications and substitutions to the base structure of DNA have been described (eg, Uhlmann, E. and Peyman, A., Chem. Rev. 90: 544, 1990; Goodchild, J., Bioconjugate Chem 1: 165, 1990). Other stabilized oligonucleotides include: nonionic analogs of DNA, such as alkyl- and aryl phosphates (in which the phosphonate charged with oxygen is replaced by an alkyl or aryl group), phosphodiester and alkyl phosphotriesters, in which the oxygen-laden portion to rent. It has also been shown that diol-containing oligonucleotides, such as tetraethylene glycol or hexaethylene glycol, at either end or both ends show to be substantially resistant to nuclease degradation. It has been discovered in accordance with the invention that subpopulations of immunostimulatory CpG oligonucleotides have dramatic immune stimulatory effects on human cells such as PBMC cells, suggesting that these immunostimulatory CpG oligonucleotides are effective therapeutic agents for vaccination in humans, immunotherapy of the cancer, asthma immunotherapy, general improvement of immune function, improvement of hematopoietic recovery after radiation or chemotherapy, autoimmune disease and other immune system modulating applications. It has also been demonstrated that subpopulations of immunostimulatory CpG oligonucleotides are useful in the treatment of asthma and allergic rhinitis. A subpopulation that has an allergy is a subject that has or is at risk of developing an allergic reaction in response to an allergen. An allergy refers to an acquired hypersensitivity to a substance (allergen). Allergic conditions include but are not limited to eczema, allergic rhinitis or coryza, hay fever, conjunctivitis, bronchial asthma, urticaria (urticaria) and food allergy, and other atopic conditions. Allergies are usually caused by the generation of IgE antibody against harmful allergens. Cytokines that are induced by systemic or mucosal administration of immunostimulatory CpG oligonucleotides are predominantly of a class called Th1 (examples are IL-12, IP-10, IFN-a and IFN-α) and these induce both humoral and immune responses. cell phones. The other major type of immune response, which is associated with the production of the cytokines IL-4 and IL-5, is called a Th2 immune response. In general, it seems that allergic diseases are mediated by Th2-type immune responses.

Based on the ability of immunostimulatory CpG oligonucleotides to change the immune response in a subject from a predominantly Th2 response (which is associated with the production of IgE antibodies and with allergy) towards a balanced Th2 / Th1 response (which is protective against allergic actions), an effective dose for inducing an immune response of an immunostimulatory CpG oligonucleotide as a therapy alone is maintained without allergen or in combination with an allergen can be administered to a subject to treat or prevent asthma and the allergy Therefore, immunostimulatory CpG oligonucleotides have a significant therapeutic utility in the treatment of allergic conditions such as asthma and allergic rhinitis. Th2 cytokines, especially IL-4 and IL-5, are elevated in the airways of asthmatic subjects. These cytokines promote important aspects of the asthmatic inflammatory response, including change of isotype IgE, chemotaxis and eosinophil activation and growth of the barley cell. The Th1 cytokines, especially IFN-? and IL-12, can suppress the formation of Th2 clones and the production of Th2 cytokines. Asthma refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently associated, although not exclusively, with atopic or allergic symptoms. Asthma can be exacerbated by viral infections. The combination of asthma and viral infections significantly worsens symptoms in a subject. The oligonucleotides used in the present invention provide significant benefits for the treatment of exacerbation of asthma induced by viruses. Below are several examples of such therapy. Allergic rhinitis is a disorder that results in inflammation of the nasal mucosa caused by allergens such as pollen or dust. The term includes rhinitis medicamentosa, rhinitis sicca, and atrophic rhinitis. There are two general types of allergic, temporary and perennial rhinitis. Temporary allergic rhinitis is usually referred to as hay fever and is usually caused by mold or pollen. Perennial allergic rhinitis is usually caused by an inherent sensitivity to one or more types of allergens. This condition usually continues throughout the year or for the time the patient is exposed to the allergen. Both types of allergic rhinitis include a type 1 hypersensitivity (mediated by IgE) that leads to inflammation. It is thought that this inflammation is caused by excessive degranulation of mast cells and basophils of blood origin in response to certain allergens. This leads to increased levels of IgE and the concomitant release of mediators of inflammation, such as histamine, and chemotactic factors, such as cytokines, prostaglandins and leukotrienes, which results in a localized inflammation reaction. The immunostimulatory oligonucleotides can be administered as a single therapy is maintained without an additional medication or anti-allergy / asthma therapy or in combination with said therapy or medication.

Drugs and anti-allergy / asthma typical therapies include the use of intranasal vasoconstrictors, intranasal and systemic antihistamines, intranasal glucocorticoids, mast cell stabilizers such as cromolyn compounds, and oral decongestants. An allergen refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject. The list of allergens is huge and can include pollens, less than insects, animal dander, fungal spores and drugs (for example penicillin). Examples of natural, animal and plant allergens include but are not limited to specific proteins of the following genera: Canine (Canis familiaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia); Lolium (for example Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternate (Alternaria alternata); Alder, Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (for example Plantago lanceolata); Parietaria (for example Parietaria officinalis or Parietaria judaica); Blattella (for example Blattella germanicd); Apis (for example Apis multiflorum); Cupressus (for example Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (for example Juníperus sabinoídes, Juniperus virginiana, Juniperus communis and Juníperus asheí); Thuya (for example Thuya orientalis); Chamaecyparis (for example Chamaecyparis obtusa); Periplaneta (for example Periplaneta americana); Agropyron (for example Agropyron repens); Sécale (for example Sécale cereale); Triticum (for example Triticum aestivum); Dactylis (for example Dactylis glomerata); Festuca (for example Festuca elatior); Poa (for example Poapratensis or Poa compressa); Oats (for example Avena sativa); Holcus (for example Holcus lanatus); Anthoxanthum (for example Anthoxanthum odoratum); Arrhenatherum (for example Arrhenatherum elatius); Agrostis (for example Agrostis alba); Phleum (for example Phleum pratense); Phalaris (for example Phalaris arundinacea); Paspalum (for example Paspalum notatum); Sorghum (for example Sorghum halepensis); and Bronms (for example Bromus inermis). Oligonucleotides are also useful for redirecting an immune response from a Th2 immune response to a Th1 immune response. This results in the production of a relatively balanced Th 1 / Th 2 environment. The re-direction of an immune response from a Th2 to a Th1 immune response can be assayed by measuring the levels of cytokines produced in response to the nucleic acid (e.g., by inducing monocytic cells and other cells to produce Th1 cytokines, including IL-12, IFN-α and GM-CSF). The redirection or rebalance of the immune response from a Th2 response to a Th1 response is particularly useful for the treatment or prevention of asthma. For example, an effective amount for the treatment of asthma may be that amount; useful for redirecting a Th2-type immune response that is associated with asthma to a Th1-type immune response or a balanced response by the Th1 / Th2 environment. Th2 cytokines, especially IL-4 and IL-5, are elevated in the airways of asthmatic subjects. Nmunoestimulantes CpG oligonucleotides of the invention cause an increase in Th1 cytokines which the helps to rebalance the immune system, preventing or reducing the adverse effects associated with a predominately Th2 immune response. The CpG immunostimulatory oligonucleotides are also useful in some aspects of the invention as a vaccine for treating a subject at risk of developing an infection with an infectious organism or a cancer in which we have identified a cancer-specific antigen, in addition to the allergy or asthma. CpG immunostimulatory oligonucleotides can also be provided without the antigen or allergen for protection against infection, allergy or cancer, and in this case repeated doses may allow longer term protection. A subject at risk as used in the present invention is a subject which is at risk of exposure to a pathogen that causes an infection or a cancer or an allergen or is at risk of developing cancer. For example, a subject at risk may be a subject who plans to travel to an area where a particular type of infectious agent is found or may be a subject who due to his lifestyle or due to medical procedures is exposed to bodily fluids that They may contain infectious organisms or directly to the body or may even be any subject that lives in an area where an infectious organism or an allergen has been identified. Subjects at risk of developing the infection also include general populations to which a medical agency recommends vaccination with an antigen from a particular infectious organism. If the antigen is an allergen and the subject develops allergic responses to that particular antigen and the subject can be exposed to the antigen, for example, during the pollen season, then that subject is at risk of exposure to the antigen. A subject at risk of developing an allergy or asthma includes those subjects who have been identified as having an allergy or asthma but who do not have the disease active during treatment with the immunostimulatory CpG oligonucleotide as well as the subjects considered to be at risk of develop these diseases due to genetic or environmental factors. A subject at risk of developing a cancer is one that has a high probability of developing cancer. These subjects include, for example, subjects that have a genetic abnormality, the presence of which has been shown to have a correlated relationship with a higher probability of developing a cancer, and subjects exposed to cancer causing agents such as tobacco, asbestos, or other chemical toxins, or a subject who has been previously treated for cancer and is in apparent remission. When treating a subject at risk of developing a cancer with an antigen specific for the type of cancer to which the subject is at risk of developing and with an immunostimulatory CpG oligonucleotide, the subject may be able to kill the cancer cells as appropriate. develop. If a tumor begins to form in the subject, the subject will develop a specific immune response against the tumor antigen. In addition to the use of immunostimulatory CpG oligonucleotides for prophylactic treatment, the invention also comprises the use of immunostimulatory CpG oligonucleotides for the treatment of a subject having an infection, allergy, asthma, or cancer. A subject who has an infection is a subject who has been exposed to an infectious pathogen and has detectable acute or chronic levels of the pathogen in the body. The immunostimulatory CpG oligonucleotides can be used with or without an antigen to mount a systemic or mucosal immune response specific to the antigen that is capable of reducing the level of or eradicating the infectious pathogen. An infectious disease, as used in the present invention, is a disease that is generated from the presence of a foreign microorganism in the body. It is particularly important to develop effective vaccine strategies and treatments to protect the mucosal surfaces of the body, which are the main site of entry of the pathogen. A subject who has a cancer is a subject who has detectable cancer cells. Cancer can be a malignant or non-malignant cancer. Cancers or tumors include but are not limited to cancer of the biliary tract; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms, lymphomas, liver cancer; lung cancer (for example, small cell and not small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; Testicular cancer; thyroid cancer; and kidney cancer, as well as other carcinomas and sarcomas. In one embodiment, the cancer is hairy cell leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma. , or colon carcinoma. A subject must mean a human or vertebrate animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, turkey, chicken, primate, for example, monkey, and fish (aquaculture species), for example Salmon. Therefore, the invention can also be used to treat cancer and tumors, infections, and allergy / asthma in no less subjects. Cancer is one of the leading causes of death in companion animals (eg, data and dogs). As used in the present invention, the term "treating, treating, or treating" when used with respect to a disorder such as an infectious disease, cancer, allergy, or asthma refers to a prophylactic treatment which increases a subject's resistance to development of the disease (e.g., infection with a pathogen) or, in other words, decreases the likelihood that a subject will develop the disease (e.g., become infected with the pathogen) as well as a treatment after the subject has developed the disease in order to fight the disease (for example, reduce or eliminate the infection) or prevent the disease from getting worse. In cases when the CpG oligonucleotide is administered with an antigen, the subject can be exposed to the antigen. As used in the present invention, the term "exposed to" refers to either the active contact step of the subject with an antigen or the passive exposure of the subject to the antigen in vivo. Methods for active exposure of a subject to an antigen are well known in the art. In general, an antigen is administered directly to the subject by any means such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The antigen can be administered systematically or locally. The methods for administering the antigen and the immunostimulatory CpG oligonucleotide are described in greater detail below. A subject is passively exposed to an antigen if an antigen is available for exposure to immune cells in the body. A subject can passively be exposed to an antigen, for example, by the entry of a foreign pathogen into the body or by the development of a tumor cell that expresses a foreign antigen on its surface. Methods in which a subject is passively exposed to an antigen may depend particularly on the time of administration of the immunostimulatory CpG oligonucleotide. For example, in a subject at risk of developing a cancer or an infectious disease or an allergic or asthmatic response, the subject may be administered with the immunostimulatory CpG oligonucleotide on a regular basis when that risk is greatest, eg, during the season. of allergies or after exposure to an agent that causes cancer. Additionally, the immunostimulatory CpG oligonucleotide can be administered to travelers before they travel to foreign countries where they are at risk of exposure to infectious agents. Similarly, immunostimulatory CpG oligonucleotides can be administered to soldiers or civilians at risk of exposure to biological warfare to induce a systemic or mucosal immune response to the antigen when and if the subject is exposed to it. An antigen as used in the present invention is a molecular capable of eliciting an immune response. Antigens include but are not limited to cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptides and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts and multicellular organisms such as parasites and allergens. The term antigen broadly includes any type of molecule which is recognized by a host immune system as a foreign molecule. Antigens include but are not limited to cancerous antigens, microbial antigens, and allergens. A cancerous antigen as used in the present invention is a compound, such as a peptide or protein, associated with a tumor or cancer cell surface and which is capable of eliciting an immune response when expressed on the surface of a host cell. of the antigen in the context of an MHC molecule. Cancerigenic antigens can be prepared from cancer cells either by preparing unpurified extracts of cancer cells, for example, as described in Cohen, et al., 1994, Cancer Research, 54: 1055, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. Cancer antigens include but are not limited to antigens that are expressed recombinantly, an immunogenic portion of, or a total tumor or cancer. Said antigens can be isolated or prepared recombinantly or by any other means known in the art. A microbial antigen as used in the present invention is an antigen of a microorganism and includes but is not limited to viruses, bacteria, parasites, and fungi. Said antigens include the intact microorganism as well as isolates and natural fragments or derivatives thereof and also synthetic compounds which are identical to or similar to the antigens of natural microorganisms and induce a specific immune response for that microorganism. A compound is similar to an antigen of a natural microorganism if it induces an immune response (humoral and / or cellular) with respect to an antigen of a natural microorganism. Such antigens are routinely used in the art and are well known to those skilled in the art.

The term "substantially purified" as used in the present invention refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify viral or bacterial polypeptides using standard techniques for protein purification. The substantially pure polypeptide will often produce a larger particular band in a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or those having several start codons, several bands may exist in a non-reducing polyacrylamide gel, but these will form a distinctive pattern for that polypeptide. The purity of the viral or bacterial polypeptide can also be determined by the amino terminal amino acid sequence analysis. Other types of antigens not encoded by a nucleic acid vector such as polysaccharides, small molecules, mimics etc. are included within the invention. The oligonucleotides of the invention can be administered to a subject with an antimicrobial agent. An antimicrobial agent, as used in the present invention, refers to a compound that occurs naturally or synthetically that is capable of eliminating or inhibiting infectious microorganisms. The type of antimicrobial agent useful in accordance with the invention will depend on the type of microorganism with which the subject becomes infected or is at risk of becoming infected. Antimicrobial agents include but are not limited to antibacterial agents, antiviral agents, antifungal agents and anti-parasitic agents. The phrases such as "anti-infective agent", "antibacterial agent", "antiviral agent", "antifungal agent", "antiparasitic agent" and "parasiticide" have well-established meanings for those skilled in the art and are defined in the texts standard doctors. Briefly, antibacterial agents eliminate or inhibit bacteria, and include antibiotics as well as other synthetic or natural compounds that have similar functions. Antibiotics are low molecular weight molecules that are produced as secondary metabolites in cells, such as microorganisms. In general, antibiotics are involved with one or more bacterial functions or structures that are specific to the microorganism and that are not present in the host cells. The anti-viral agents can be isolated from natural sources or they can be synthesized and are useful to eliminate or inhibit the viruses. Anti-fungal agents are used to treat superficial fungal infections as well as opportunistic and primary systemic fungal infections. Antiparasitic agents eliminate or inhibit parasites. Examples of anti-parasitic agents also refer to parasiticides useful for administration in humans, including but not limited to albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, eflornithine, furazolidone, glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimonate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethamine- sulfonamides, pyrimethamine-sulfadoxine, quinacrine HCI, quinine sulfate, quinidine gluconate, spiramycin, sodium stibogluconate (sodium antimony gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethoprim-sulfamethoxazole, and triparsamide some of which are used alone or in combination with others. Antibacterial agents eliminate or inhibit the growth or function of bacteria. A great class of antibacterial agents are antibiotics. The antibiotics, which are effective in eliminating or inhibiting a wide range of bacteria, are referred to as broad spectrum antibiotics. Other types of antibiotics are predominantly effective against bacteria of the gram-positive or gram-negative type. These types of antibiotics are referred to as narrow-spectrum antibiotics. Other antibiotics, which are effective against a particular organism or disease and not against other types of bacteria, are referred to as narrow-spectrum antibiotics. Antibacterial agents are sometimes classified based on their main mode of action. In general, antibacterial agents are inhibitors of cell wall synthesis, cell membrane inhibitors, inhibitors of protein synthesis, inhibitors of synthesis or nucleic acid function, and are competitive inhibitors.

Antiviral agents are compounds that prevent the infection of cells by viruses or that prevent the replication of viruses within the cell. There are far fewer antiviral drugs than antibacterial drugs because the process of viral replication is very closely related to DNA replication in the host cell, so that non-specific antiviral agents could be frequently host-toxic. There are several stages within the viral infection process which can be blocked or inhibited by antiviral agents. These steps include, binding of the virus to the host cell (immunoglobulin or binding peptides), removal of the virus coat (for example amantadine), synthesis or translation of the viral mRNA (for example interferon), replication of the RNA or viral DNA (for example nucleotide analogs), maturation of new viral proteins (for example protease inhibitors), and budding and release of the virus. Nucleotide analogs are synthetic compounds that are similar to nucleotides, but that have an incomplete or abnormal deoxyribose or ribose group. Once the nucleotide analogues are found in the cell, they are phosphorylated, producing the formed triphosphate that competes with normal nucleotides for incorporation into viral DNA or RNA. Once the triphosphate form of the nucleotide analog is incorporated into the growing strand of nucleic acid, this results in irreversible association with the viral polymerase and thus the termination of the strand. Nucleotide analogs include, but are not limited to, acyclovir (used for the treatment of herpes simplex virus and varicella-zoster virus), gancyclovir (used for the treatment of cytomegalovirus), idoxuridine, ribavirin (used for the treatment of respiratory syncytial virus), dideoxyinosine, dideoxycytidine, zidovudine (azidothymidine), imiquimod, and resimiquimod. Interferons are cytokines that are secreted by cells infected by the virus as well as by immune cells. Interferons work by binding to specific receptors in cells adjacent to infected cells, causing the change in the cell that protects against infection by the virus. Interferon a and β also induces the expression of MHC class I and class II molecules on the surface of infected cells, which results in the increased presentation of the antigen for recognition of the host immune cell. Interferons a and b are available as recombinant forms and have been used for the treatment of chronic hepatitis B and C infection. In doses that are effective for anti-viral therapy, interferons have several side effects such as fever, malaise and weightloss. Antiviral agents useful in the invention include but are not limited to immunoglobulins, amantadine, interferons, nucleotide analogs, and protease inhibitors. Specific examples of antivirals include but are not limited to Acemannan; Acyclovir; Acyclovir sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine hydrochloride; Aranotin; Arildona; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril, Edoxudin; Enviradeno; Enviroxime; Famciclovir; Famotine hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet sodium; Fosfonet sodium; Ganciclovir; Ganciclovir sodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir, Memotine hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir; Ribavirin; Rimantadine hydrochloride; Saquinavir Mesylate; Somantadine hydrochloride; Sorivudine; Statolon; Stavudina; Tilorone hydrochloride; Trifluridine; Valacyclovir hydrochloride; Vidarabina; Vidarabine phosphate; Sodium Vidarabine phosphate; Viroxime; Zalcitabine; Zidovudine; and Zinviroxime. Antifungal agents are useful for the treatment and prevention of infectious fungi. Antifungal agents are sometimes classified by their mechanism of action. Some antifungal agents function as inhibitors of the cell wall by inhibiting glucose synthase. These include, but are not limited to, basiungin / ECB. Other antifungal agents work by destabilizing the integrity of the membrane. These include, but are not limited to, immidazoles, such as clotrimazole, sertaconazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconacol, as well as FK 463, amphotericin B5 BAY 38-9502, MK 991, pradimycin, UK 292, butenafine, and terbinafine. Other antifungal agents work through the degradation of chitin (for example chitinase) or immunosuppression (cream 501). The immunostimulatory CpG oligonucleotides can be combined with other therapeutic agents such as adjuvants to improve immune responses. The immunostimulatory CpG oligonucleotide and another therapeutic agent can be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same formulation or in separate formulations, but are administered at the same time. The other therapeutic agents are sequentially administered to each other and to an immunostimulatory CpG oligonucleotide, when the administration of the other therapeutic agents and the immunostimulatory CpG oligonucleotide is temporarily separated. The separation in time between the administration of these compounds can be a matter of minutes or it can be longer. Other therapeutic agents include but are not limited to adjuvants, cytokines, antibodies, antigens, etc. The compositions of the invention can also be administered with adjuvants that are not nucleic acids. The adjuvant that is not nucleic acid is any molecule or compound except for the immunostimulatory CpG oligonucleotides described in the present invention which can stimulate the humoral and / or cellular immune response. Non-nucleic acid adjuvants include, for example, adjuvants that create a depot effect, immune stimulatory adjuvants, and adjuvants that create a depot effect and that stimulate the immune system. CpG immunostimulatory oligonucleotides are also useful as mucosal adjuvants. It has been previously reported that both systemic immunity and mucosal immunity are induced by mucosal administration of CpG oligonucleotides. Therefore, the oligonucleotides can be administered in combination with other mucosal adjuvants. Immune responses can also be induced or increased by co-administration or co-linear cytokine expression (Bueler & amp; amp; amp;; Mulligan, 1996; Chow et at, 1997; Geissler et al, 1997; Iwasaki et al, 1997; Kim et al, 1997) or co-stimulant molecules B-7 (Iwasaki et al, 1997; Tsuji et al, 1997) with immunostimulatory CpG oligonucleotides. The term cytokines is used as a generic name for a diverse group of proteins and soluble peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate procedures that take place in the extracellular environment. Examples of cytokines include, but are not limited to, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18. , granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interferon-? (? -IFN), IFN-a, tumor necrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40 ligand. Oligonucleotides are also useful for improving the survival, differentiation, activation and maturation of dendritic cells. The immunostimulatory CpG oligonucleotides have the unique ability to promote the cell survival, differentiation, activation and maturation of dendritic cells. The immunostimulatory CpG oligonucleotides also increase the lithic activity of the natural killer cell and the antibody-dependent cellular cytotoxicity (ADCC). ADCC can be carried out using an immunostimulatory CpG oligonucleotide in combination with a specific antibody to a cell target, such as a cancer cell. When the immunostimulatory CpG oligonucleotide is administered to a subject in conjunction with the antibody, it is induced to the subject's immune system to kill the tumor cell. Antibodies useful in the ADCC method include antibodies that interact with a cell in the body. Many of said antibodies specific for cell targets have been described in the art and many are commercially available. Immunostimulatory CpG oligonucleotides can also be administered in conjunction with an anti-cancer therapy. Anti-cancer therapies include drugs for cancer, radiation and surgical procedures. As used in the present invention, a "cancer medicament" refers to an agent which is administered to a subject for the purpose of treating a cancer. As used in the present invention, "cancer treatment" includes preventing the development of a cancer, reducing the symptoms of cancer, and / or inhibiting the growth of an established cancer. In other aspects, the cancer drug is administered to a subject at risk of developing a cancer for the purpose of reducing the risk of developing the cancer. Various types of drugs for the treatment of cancer are described in the present invention. For the purpose of this specification, drugs for cancer are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormone therapy, and biological response modifiers. In one embodiment, the cancer medicament is a chemotherapeutic agent selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, chloroethylnitrosoureas containing no sugar, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragylina, GLA meglamine, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RAS famesyl transferase inhibitor, famesil transferase inhibitor, MMP, MTA / LY231514, L Y264618 / Lometexol, Glamolec, CI-994, TNP- 470, Hicamtin Topotecan, PKC412, Valspodar / PSC833, Novantrone / Mitroxantrone, Metaret / Suramin.a Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, IncelA / X-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516 / Marmistat, BB2516 / Marmistat, CDP 845, D2163, PD183805, DX8951I, Lemonal DP 2202, FK 317, Picibanil / OK-432, AD 32 / Valrubicin, Metastron / strontium derivative, Temodal / Temozolomide, Evacet / liposomal doxorubicin, Yewtaxan / Placlitaxel, Taxol / Paclitaxel, Xel oad / Capecitabine, Furtulon / Doxifluridine, Cyclopax / oral paclitaxel, oral Taxoid, SPU-077 / Cisplatin, HMR 1275 / Flavopiridol, CP-358 (774) / EGFR, CP-609 (754) / RAS oncogene inhibitor, BMS-182751 / oral platinum, UFT (Tegafur / Uracilo), Ergamisol / Levamisol, Eniluracil / 776C85 / potentiator of 5FU, Campto / Levamisol, Camptosar / lrinotecan, Tumodex / Ralitrexed, Leustatin / Cladribine, Paxex / Paclitaxel, Doxil / liposomal doxorubicin, Caelyx / Liposomal doxorubicin, Fludara / Fludarabine, Pharmarubicin / Epirubicin, DepoCyt, ZDI 839, LU 79553 / Bis-Naphthalimide, LU 103793 / Dolastain , Caetyx / liposomal doxorubicin, Gemzar / Gemcitabine, ZD 0473 / Anormed, YM 116, iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809 / Dexiphosamide, Ifes / Mesnex / lfosamide, Vumon / Teniposide, Paraplatin / Carboplatin, Plantinol / cisplatin, Vepeside / Etoposide, ZD 9331, Taxotere / Docetaxel, guanine arabinoside prodrug, Taxano analogue, nitrosoureas, alkylating agents such as melphelan and cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin, Daunorubicin HCI, Estramustine sodium phosphate, Etoposide (VP 16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alpha-2b, Leuprolide aceta to (analog of LHRH releasing factor), Lomustine (CCNU), Mechlorethamine HCI (mustard nitrogen), Mercaptopurine, Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate , Thioguanine, Tiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Ertiropoietin, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanilhydrazone, MGBG), Pentostatin (2'deoxicoformycin), Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate. In one important modality, the drug for cancer is taxol. In another form, the cancer medication is a combination of carboplatin and paclitaxel. In another embodiment, the cancer medicament is an immunotherapeutic agent selected from the group consisting of Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP- 03, ior t6, MDX-210, MDX-1 1, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb- G2, TNT, Gliomab-H, GNI-250, E D-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, Ab ANA , Ab SMART IDIO, Ab SMART ABL 364 and ImmuRAIT-CEA. Additionally, the methods of the invention are intended to encompass the use of more than one cancer drug together with immunostimulatory CpG oligonucleotides. As an example, when appropriate, immunostimulatory CpG oligonucleotides can be administered with either a chemotherapeutic agent or an immunotherapeutic agent. Alternatively, the cancer medicament may encompass an immunotherapeutic agent and a cancer vaccine, or a chemotherapeutic agent and a cancer vaccine, or a chemotherapeutic agent, an immunotherapeutic agent and a cancer vaccine all administered to a subject for the purpose of treatment of a subject who has cancer or who is at risk of developing cancer.

The use of immunostimulatory CpG oligonucleotides in conjunction with immunotherapeutic agents such as monoclonal antibodies is capable of increasing long-term survival through numerous mechanisms including the significant improvement of ADCC (as discussed above), activation of natural killer cells ( NK) and an increase in IFN-a levels. Oligonucleotides when used in combination with monoclonal antibodies serve to reduce the dose of antibody required to achieve a biological result. As used in the present invention, the terms "cancer antigen" and "tumor antigen" are used interchangeably to refer to antigens that are differentially expressed by cancer cells and can therefore be exploited for the purpose of to direct them towards the cancer cells. Cancer antigens are antigens that apparently can potentially stimulate the specific immune responses of the tumor. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those that are normally silent (eg, are not expressed) in normal cells, those that are expressed only in certain stages of differentiation and those that are expressed temporally such as embryonic and fetal antigens. Other cancerous antigens are encoded by mutant cell genes, such as oncogenes (eg, activated ras oncogene), suppressor genes (eg, mutant p53), fusion proteins that result from internal deletions or chromosomal translocations. Even other cancer antigens can be encoded by viral genes such as those that are carried in tumor viruses of RNA and DNA. Immunostimulatory CpG oligonucleotides are also useful for the treatment and prevention of autoimmune disease. Autoimmune disease is a kind of disease in which the subject's own antibodies react with the host tissue or in which the immune effector T cells are self-reactive to the endogenous own peptides and cause tissue destruction. Therefore, an immune response is mounted against the antigens proper to the subject, referred to as auto antigens. Autoimmune diseases include but are not limited to rheumatoid arthritis, Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus (eg, pemphigus vulgaris) ), Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, infertility associated with autoimmune processes, glomerulonephritis (eg, crescentic glomerulonephritis) , proliferative glomerulonephritis), bullous pemphigoid, Sjogren's syndrome, insulin resistance, and autoimmune diabetes mellitus.

A "self-antigen" as used in the present invention refers to an antigen of a normal tissue of the host. The normal tissue of the host does not include cancer cells. Therefore an immune response is mounted against a self-antigen, in the context of an autoimmune disease, is an undesirable immune response and contributes to the destruction and damage of normal tissue, while an immune response is mounted against A cancerous antigen is a desirable immune response and contributes to the destruction of the tumor or cancer. Therefore, in some aspects of the invention which have the aim of treating autoimmune disorders it is not recommended that immunostimulatory CpG oligonucleotides be administered with self antigens, particularly those which are the target of the autoimmune disorder. In other cases, immunostimulatory CpG oligonucleotides can be administered with low doses of self antigens. Numerous animal studies have shown that mucosal administration of low doses of the antigen can result in a state of immune hyporesponse or "tolerance". The active mechanism seems to be a cytokine-mediated immune shift away from a Th1 response predominantly towards a Th2 and Th3 response (eg, dominated by TGF-β). Active suppression with the administration of a low dose of antigen may also suppress an unrelated immune response (surrounding deletion) which is of considerable interest in the therapy of autoimmune diseases, for example, rheumatoid arthritis and SLE. The surrounding deletion includes the regulatory secretion against Th1, suppressor cytokines in the local environment where the proinflammatory and Th1 cytokines are released either in a specific manner from the antigen or non-specific antigen. The "tolerance to" as used in the present invention is used to refer to this phenomenon. In fact, oral tolerance has been effective in the treatment of numerous autoimmune diseases in animals including: experimental autoimmune encephalomyelitis (EAE), experimental autoimmune myasthenia gravis, collagen-induced arthritis (CIA), and insulin-induced diabetes mellitus. In these models, the prevention and suppression of immune disease is associated with a change in humoral and cellular responses specific to the antigen from a Th1 response to a Th2 / Th3 response. The invention also includes a method for the induction of a non-specific innate immune activation of the antigen and the broad spectrum resistance to the infectious test using the immunostimulatory CpG oligonucleotides. The term "non-specific innate immune activation of the antigen" as used in the present invention refers to the activation of immune cells other than B cells and for example may include the activation of NK cells, T cells or other immune cells that can respond independently of the antigen or a certain combination of the cells. Broad-spectrum resistance to the infectious test is induced because the immune cells are in active form and primed to respond to any invading compound or microorganism. The cells do not have to be primed specifically against a particular antigen. This is particularly useful in biological warfare, and the other circumstances described above such as for travelers. The immunostimulatory CpG oligonucleotides can be administered directly to the subject or can be administered in conjunction with a complex for nucleic acid administration. A "complex for administration of nucleic acid" means a nucleic acid molecule associated with (eg, ionically or covalently linked to or encapsulated in) a medium for administration (eg, a molecule that results in a higher binding affinity to the target cell) . Examples of complexes for nucleic acid administration include nucleic acids associated with a sterol (e.g. cholesterol), a liquid (for example a cationic lipid, virosome or liposome), or a specific binding agent to the target cell (for example a ligand recognized by a specific target cell receptor). The preferred complexes may be stable enough in vivo to prevent significant decoupling prior to internalization by the target cell. However, the complex can be cleaved under appropriate conditions within the cell such that the oligonucleotide is released in a functional form. The vehicles for administration or devices for administration have been described for the administration of an antigen and oligonucleotides to the surfaces. The immunostimulatory CpG oligonucleotide and / or the antigen and / or other therapeutics can be administered alone (eg, in saline or pH buffer) or using any carriers known in the art. The term "effective amount" of an immunostimulatory CpG oligonucleotide refers to the amount necessary or suitable to carry out a desired biological effect. For example, an effective amount of an immunostimulatory CpG oligonucleotide administered with the antigen to induce mucosal immunity is that amount necessary to cause the development of IgA in response to an antigen after exposure to the antigen, while that amount required to induce the antigen. Systemic immunity is that amount necessary to cause the development of IgG in response to an antigen after exposure to the antigen. Combined with the teachings provided in the present invention, by choosing between the various active compounds and considering factors such as potency, relative bioavailability, patient's body weight, severity of adverse side effects and preferred mode of administration, a effective prophylactic or therapeutic treatment regimen which does not cause substantial toxicity and yet is completely effective in treating the particular subject. The effective amount for any particular application may vary depending on such factors as the disease or condition to be treated, the particular immunostimulatory CpG oligonucleotide to be administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular immunostimulatory CpG oligonucleotide and / or antigen and / or other therapeutic agent without need for experimentation. The subject doses of the compounds described in the present invention for mucosal or local administration typically have a range of about 0.1 ug to 10 mg per administration, which depends on the application that may be provided daily, weekly, or monthly and any other amount of time between these. More typically, the mucosal or local dose ranges from about 0 ug to 5 mg per administration, and more typically from about 100 ug to 1 mg, with 2-4 administrations being separated by days or weeks. More typically, immune stimulant doses have a range of 1 ug to 10 mg per administration, and more typically from 10 ug to 1 mg, with administrations daily or weekly. The subject doses of the compounds described in the present invention for parenteral administration for the purpose of inducing an antigen-specific immune response, wherein the compounds are administered with an antigen but not with another therapeutic agent are typically from 5 to 10,000 times greater than the effective mucosal dose for adjuvant or immune stimulant applications for vaccine, and more typically 10 to 1,000 times greater, and more typically 20 to 100 times higher. The doses of the compounds described in the present invention for parenteral administration for the purpose of inducing an innate immune response or for increasing ADCC or for inducing an antigen-specific immune response when the immunostimulatory CpG oligonucleotides are administered in combination with other therapeutic agents or in specialized administration vehicles typically in the range of about 0.1 ug to 10 mg per administration, which depends on the application that can be provided daily, weekly, or monthly and any other amount of time between them. More typically parenteral doses for these purposes have a range of about 10 ug to 5 mg per administration, and more typically from about 100 ug to 1 mg, with 2-4 administrations being separated by days or weeks. However, in some embodiments, parenteral doses for these purposes may be used at a range of 5 to 10,000 times greater than the typical doses described above. For any compound described in the present invention the therapeutically effective amount can be determined initially from animal models. A therapeutically effective dose can also be determined from human data for CpG oligonucleotides that have been evaluated in humans (clinical trials have been initiated in humans) and for compounds known to exhibit similar pharmacological activities, such as other adjuvants, for example , LT and other antigens for vaccination purposes. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the compound administered. The adjustment of the dose to achieve maximum efficiency based on the methods described above and other methods as is well known in the art is well within the capabilities of the person skilled in the art. The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, pH regulating agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. For use in therapy, an effective amount of the immunostimulatory CpG oligonucleotide can be administered to a subject by any mode that delivers the oligonucleotide to the desired, eg, mucosal, systemic surface. The administration of the pharmaceutical composition of the present invention can be achieved by any means known to the person skilled in the art. Preferred routes of administration include but are not limited to oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, and rectal. For oral administration, compounds (e.g., immunostimulatory CpG oligonucleotides, antigens and other therapeutic agents) can be formulated readily by combining the active compound (s) with pharmaceutically acceptable carriers well known in the art. Said vehicles allow the compounds of the invention to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, watered pastes, suspensions and the like, for oral indigestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as a solid excipient, optionally by grinding a resulting mixture, and processing the granule mixture, after adding suitable auxiliary elements, if desired, to obtain tablet or graft cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol.; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally, oral formulations can also be formulated in saline or pH regulators, for example EDTA to neutralize internal acid conditions or can be administered without said vehicles. The oral dosage forms of the aforementioned component or components are also specifically contemplated. The component or components can be chemically modified so that oral administration of the derivative is efficient. Generally, the chemical modification contemplated in the binding of at least a portion to the molecule of the component itself, wherein said portion allows (a) the inhibition of proteolysis; and (b) takes it into the bloodstream, from the stomach or intestine. An increase in the overall stability of the component or components and an increase in circulation time in the body is also desired. Examples of such portions include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts" In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-lnterscience, New York, NY, pp. 367-383; Newmark, et al, 1982, J. Appl. Biochem. 4: 185-189. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-thioxocane. Preferred for pharmaceutical use, as indicated above, are the polyethylene glycol portions. For the release location component it can be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has formulations available which do not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protecting the oligonucleotide or by releasing the biologically active material beyond the environment of the stomach, such as in the intestine. To ensure total gastric resistance, a coating impermeable to at least pH 5.0 is essential. Examples of the most common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Those coatings can be used as mixed films. A coating or mixture of coatings can also be used in tablets, which are not intended to protect against the stomach. This may include sugar coatings, or coatings that make the tablet easier to swallow. The capsules may consist of a hard shell (such as gelatin) for the administration of a dry therapeutic element, for example; powdered; For liquid forms, a soft gelatin shell can be used. The seal cover material could be coarse starch or other edible paper. For pills, tablets, molded tablets or crushable tablets, most mass forming techniques can be used. The therapeutic element can be included in the formulation as multiple fine particles in the form of granules or concentrates of a particle size of about 1 mm. The formulation of the material for the administration of the capsule could also be as a powder, slightly compressed caps or even as tablets. The therapeutic element could be prepared by compression. All colorants and flavoring agents may be included. For example, the oligonucleotide can be formulated (such as by liposome or microsphere encapsulation) and then can be further contained within an edible product, such as a chilled beverage containing dyes and flavoring agents.

One can dilute or increase the volume of the therapeutic element with an inert material. These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain organic salts can also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell. The disintegrants can be included in the formulation of the therapeutic element towards a solid dosage form. Materials used as disintegrants include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramilopectin, sodium alginate, gelatin, orange peel, carboxymethylcellulose acid, natural sponge and bentonite can all be used. Another form of disintegrants are insoluble cation exchange resins. Powdered gums can also be used as disintegrants and as binders and these can include powdered gums such as agar, karaya or tragacanth. Alginic acid and its sodium salt can also be used as disintegrants. The binders can also be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethylcellulose (CMC). Both polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) can both be used in alcoholic solutions to granulate the therapeutic element. An anti-friction agent can be included in the formulation of the therapeutic element to prevent sticking during the formulation process. Lubricants can be used as a layer between the therapeutic element and the die wall, and these can include but are not limited to; Stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants can also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. Slippers that can improve the flow properties of the drug during formulation and can be added to help to the rearrange during compression. The glidants can include starch, talc, fumed silica and hydrated silicoaluminate. To aid in the dissolution of the therapeutic element in the aqueous environment a surfactant may be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents can be used and could include benzalkonium chloride or benzethonium chloride. The list of potential non-ionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid, methyl cellulose and carboxymethylcellulose. These surfactants could be present in the oligonucleotide formulation or derived either alone or as a mixture in different proportions. Pharmaceutical preparations that can be used orally include pressure-adjusted capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Pressure-adjusting capsules may contain the active ingredients in admixture with the filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. Microspheres formulated for oral administration may also be used. Said microspheres have been well defined in the art. All formulations for oral administration should be in doses suitable for said administration. For buccal administration, the compositions may take the form of tablets or tablets formulated in conventional manner.

For administration by inhalation, the compounds for use in accordance with the present invention can conveniently be administered in the form of an aerosol spray presentation from pressurized packings or a nebulizer, with the use of a suitable propellant, for example , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dose unit can be determined by providing a value for administering a measured amount. Capsules and cartridges of eg gelatin for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. The pulmonary administration of the oligonucleotides (or derivatives thereof) is also contemplated in the present invention. The oligonucleotide is administered to the lungs of a mammal while inhaling and traversing the epithelial lining of the lung into the bloodstream. Other reports of inhaled molecules include Adjei et al. , 1990, Pharmaceutical Research, 7: 565-569; Adjei et al. , 1990, International Journal of Pharmaceutics, 63: 135-144 (leuprolide acetate); Braquet et al., 1989, Journal of Cardiovascular Pharmacology, 13 (suppl 5): 143-146 (endothelin-1); Hubbard et al. , 1989, Annals of Infernal Medicine, Vol. III, pp. 206-212 (α1-antitrypsin); Smith et al. , 1989, J. Clin. Invest. 84: 1 145-1 146 (α-1-proteinase); Oswein et al. , 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J. Immunol. 140: 3482-3488 (interferon-g and tumor necrosis factor alpha) and Platz et al., U.S. Patent. No. 5,284,656 (stimulating factor of the granulocyte colony). A method and composition for the pulmonary administration of drugs for the systemic effect is described in the U.S. Patent. No. 5,451, 569, filed September 19, 1995 to Wong et al. Contemplated for use in the practice of this invention is a wide range of mechanical devices designed for the pulmonary administration of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powdered inhalers, all of which are familiar to those experts in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, developed by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer, developed by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, prepared by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts. All these devices require the use of suitable formulations for the dispersion of the oligonucleotide (or derivative). Typically, each formulation is specific to the type of device employed and may include the use of a suitable propellant material, in addition to the usual diluents, adjuvants and / or vehicles used in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of vehicles is contemplated. The chemically modified oligonucleotide can also be prepared in different formulations depending on the chemical modification type or the type of device used. Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise oligonucleotide dissolved in water at a concentration of about 0.1 to 25 mg of biologically active oligonucleotide per ml of solution. The formulation may also include a pH regulator and a simple sugar (for example, for stabilization of the oligonucleotide and regulation of the osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent the oligonucleotide-induced surface aggregation caused by the atomization of the solution in the formation of the aerosol. Formulations for use with a metered dose inhaler device will generally comprise a finely divided powder containing the oligonucleotide suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material used for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1, -tetrafluoroethane, or combinations thereof. . Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid may also be useful as a surfactant. Formulations for dispersion from a powder inhaler device will comprise a finely divided dry powder containing oligonucleotide and may also include a dough-forming agent, such as lactose, sorbitol, sucrose, or mannitol in amounts that facilitate dispersion of the powder to from the device, for example, 50 to 90% by weight of the formulation. The oligonucleotide should be prepared more advantageously in the form of a particle with an average particle size of less than 10 mm (or microns), more preferably 0.5 to 5 mm, for the most effective delivery to the distal lung. Nasal administration of a pharmaceutical composition of the present invention is also contemplated. Nasal administration allows the passage of a pharmaceutical composition of the present invention into the bloodstream directly after administering the therapeutic product to the nose, without the need to deposit the product in the lung.

Formulations for nasal administration include those with dextran or cyclodextrin. For nasal administration, a useful device is a small, hard bottle to which a measured dose thickness is attached. In one embodiment, the measured dose is administered by extraction of the pharmaceutical composition of the present invention in solution within a chamber of defined volume, said chamber having an aperture sized to form in aerosol and formulated in aerosol when forming a spray when compressed a liquid in the chamber. The chamber is compressed to administer the pharmaceutical composition of the present invention. In a specific mode, the camera is in a piston arrangement. These devices are commercially available. Alternatively, a compressible plastic bottle with an aperture or aperture sized to aerosolize an aerosol formulation was compressed to form a spray when compressed. The opening is usually located on the top of the bottle, and the top is generally tapered to fit partially in the nasal passages for the efficient administration of the aerosol formulation. Preferably, the nasal inhaler will provide a measured amount of the aerosol formulation for the administration of a metered dose of the drug. The compounds, when it is desirable to administer them systematically, can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain agents for formulation such as suspending, stabilizing and / or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate in oily suspensions for injection. Suitable lipophilic solvents or carriers include fatty acids such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous suspensions for injection may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain stabilizers or suitable agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions. Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, eg, sterile, pyrogen-free water, before use. The compounds can also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations previously described, the compounds can also be formulated as depot preparations. Such long-lasting formulations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The pharmaceutical compositions may also comprise suitable solid phase or gel containers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous solutions or salines for inhalation, microencapsulated, mixed, coated on microscopic gold particles, contained in liposomes, nebulized, aerosols, concentrated for implant in the skin, or dried on a cutting object to be scraped on the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops and preparations with protrusion release of the active compounds, in which excipients and additives for preparation and / or auxiliaries such as disintegrants, binders, coating agents, wetting agents, lubricants, flavo rings, sweeteners or solubilizers are used in a customary manner as described above. The pharmaceutical compositions are suitable for use in a variety of systems for drug administration. For a brief review of the methods for drug administration, see Langer, Science 249: 1527-1533, 1990, which are incorporated herein by reference. The immunostimulatory CpG oligonucleotides and optionally other therapeutics and / or antigens can be administered per se (pure) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but the non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts thereof. Said salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, malonic, succinic , naphthalene-2-sulphonic, and benzenesulfonic. Also, said salts can be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium or calcium salts of the carboxylic acid group. Suitable pH regulating agents include: acetic acid and a salt (1-2% w / v); citric acid and a salt (1-3% w / v); boronic acid and a salt (0.5-2.5% w / v); and phosphoric acid and a salt (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); Parabens (0.01 -0.25% w / v) and thimerosal (0.004-0.02% w / v). The pharmaceutical compositions of the invention contain an effective amount of an immunostimulatory CpG oligonucleotide and optionally antigens other therapeutic agents optionally included in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to a human or other vertebrate animal. The term vehicle denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions are also capable of being mixed with the compounds of the present invention, and with each other, so that there is no interaction that could substantially alter the desired pharmaceutical efficiency. The present invention is further illustrated by the following examples, which should not be further considered as limiting. The total contents of all references (including references to the literature, patents filed, published patent applications, and co-pending patent applications) cited throughout this application are expressly incorporated herein by reference.

EXAMPLES Oliqodeoxynucleotides (ODNs) The following ODNs are used in the examples. SEQ ID. DO NOT. Sequence 1 T * C_G * T * C_G * A * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 2 T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 T * C_G * T * C_G * T * T * C_G * G * C * G * C * G * C * C * G 4 T * C_G * T * C_G * A * C_G * A * T * C_G * G * C * G * C_G * C * G * C * C * G T * T * C_G * T * C_ G * T * T * T * T_ G * T * C_ G * T * T 6 T * T * T * C_G * T * C_G * T * T * T * C_G * T * C_G * T * T 7 T * C_G * T * C_G * T * T * T * T * G * A * C_G * T * T * T * T * G * T * C_G * T * T 8 TCG TCG TTT TGT CGT TTT GTC GTT (all links *) 9 T * C_G * C_G * A * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G TCG TCG TTT TGA CGT TTT GTC GTT (all links *) 14 TCCAGGACTTCTCTCAGGTT (all links *) 5 C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 6 G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 17 T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 18 C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 19 G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 20 T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 21 T * CG * T * CG * T * CG * T * T * CG * G * C * G * CG * C * G * C * C 22 T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C 23 T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C Materials and methods: Oligodeoxynucleotides (ODNs) The ODNs were obtained from Biospring (Frankfurt, Germany) or Sigma-Ark (Darmstadt, Germany), and were checked for identity and purity by Coley Pharmaceutical GmbH (Langenfeld, Germany). The ODNs were diluted in phosphate buffered saline (Sigma, Germany), and stored at -20 ° C. All dilutions were carried out using pyrogen-free reagents. The ODNs SEQ ID NO. 15-23 were synthesized by Trilink Biotechnologies.

Cellular purification The preparations of the peripheral blood yellow coat from healthy male and female human donors were obtained from the Blood Bank of the University of Dusseldorf (Germany) and from these, the PBMC were purified by centrifugation on Ficoll. -Hypaque (Sigma). Purified PBMCs were used either recently obtained (for most tests) or suspended in freezing medium and stored at -70 ° C. When required, aliquots of these cells were thawed, washed and resuspended in RPMI 1640 culture medium (BioWhittaker, Belgium) supplemented with 5% (v / v) of heat inactivated human AB serum (BioWhittaker, Belgium) or 10% (v / v) FCS inactivated by heat, 2 mM L-glutamine (BioWhittaker), 100 U / ml penicillin and 100 ug / ml streptomycin (Invitrogen (Karlsruhe, Germany)).

Cytokine Detection Thawed or freshly obtained PBMCs were seeded in 48-well flat bottom plates, or in 96-well round bottom plates, and incubated with ODN at concentrations as indicated in a humidified 37 ° C incubator. The supernatants of the cultures were harvested and if not used immediately, they were frozen at -20 ° C until required. The amounts of cytokines in the supernatants were evaluated using commercially available ELISA (Diaclone, EUA) or ELISAs developed in the laboratory using commercially available antibodies (from Becton Dickinson / Pharmingen or PBL). The studies for example 13 were carried out as follows: Spleens were removed from 6 mice (male, BALB / c). Splenocytes from each spleen were separated by gentle pressing through a cell sieve (pore size 70 um), and then pooled. Splenocytes were added to the wells of the culture plates. 1x107 cells in a volume of 900 ul of medium to each well. The medium was RPMI 1640 containing 0% fetal bovine serum. 100 ul of aliquots of the CpG ODN solutions in medium were added to each well to produce a final concentration in the wells of 0.01 - 10 ug / ml. After incubation for 36 hours (37C, 5% C02 in incubator), the culture supernatants were harvested and tested for cytokine concentrations using either the Luminex multiplex assay (IFN- ?, IP-10, IL- 10, IL-6, TNF-a) or ELISA (IFN-a).

Induction of an increase induced by the antigen in the nasal resistance in those whose the (males, Hartley) were sensitized with the antigen (ovalbumin, 5 mg both intraperitoneal and subcutaneous) on the day of study 0. Reinforcement sensitization was provided (5 mg, intraperitoneal) on the day of study 4. Those who were tested with the antigen by exposure to the antigen administered intranasally twice a week for two consecutive weeks. The first test was on study day 14. SEQ ID NO: 7 (lot number AQE-03J-001-M, 0.03-1 mg / kg in 150 ul / kg of saline) was administered intranasally once a day. week, two days before the first antigen test of the week. With the exception of the final test on study day 24, the antigen test was with ovalbumin (1.5 mg / kg in 150 ul / kg saline). The animals were pre-treated with mepyramine (10 mg / kg, intraperitoneal) 30 minutes before the test to protect against histamine-induced anaphylaxis. On study day 24, those who were anesthetized to allow the measurement of nasal resistance using a Buxco breathing mechanics system and software. The final antigen test was then performed with ovalbumin (2.5 mg in 250 ul saline) administered within the nasopharynx. The animals were pretreated with mepiraminq (3 mg / kg, intraperitoneal) 30 minutes before the test. Nasal resistance was measured for 40 minutes after the test. Induction of antigen-induced increase in nasal resistance in mice Mice (BALB / c males) were sensitized on study days 0 and 7 with the antigen (ovalbumin, 100 ug, ip) with adjuvant aluminum hydroxide (Pierce Alum Lp.). Mice were tested daily with the antigen by exposure to the antigen administered nasally (1 mg in 10 ul of saline). The first test was on study day 14. SEQ ID NO: 7 was administered intranasally twice. The first dose was given 2 days before the first test of the antigen. The second dose was given 7 days later. Alternatively, budesonide (an anti-inflammatory, synthetic corticosteroid) was administered intranasally daily. The first doses were given 2 days before the first test with the antigen. Each day, the dose of budesonide was administered intranasally 4 hours before the test with the intranasal antigen. Endpoints were measured on study day 26 (for example, 7 days after the second dose of SEQ ID NO: 7-2, nasal tissues were taken for histopathological evaluation of inflammation.) Separated mice received a final test of the antigen and the incidences of sneezing and nasal rubbing were recorded for a period of 10 minutes after the test.

Statistical analysis The Mann-Whitney test was used to compare the series of data in which the populations that were started to compare were not normal. Dunnitt's multiple comparisons test was used for the comparison of the data series with respect to a particular control.

Induction of cytokine in the mouse in vivo Mice (males, BALB / c) were dosed with ODN CpGs (0.1 and 1 mg / kg) or control vehicle (saline, 25 ul) by intranasal instillation. The bronchoalveolar lavage fluid and serum (separated from the blood obtained by cardiac puncture) were collected 8 hours and 15 hours after dosing. The cell numbers in the bronchoalveolar lavage fluid were counted with an Advia automated cell counter. The concentrations of cytokines and chemokines in the bronchoalveolar lavage fluid and in the serum were assayed using either ELISA (IFNa, IL-12p40) or the multiple cytokine system Luminex (IFN- ?, IP-10, IL-1 a, IL-? ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-0, IL-13, IL-15, IL-17, GM-CSF, RANTES , TNFa). The minimum detectable concentrations were different for each analyte, but were found in the range of 0.3 -12 pg / ml.

Induction of inflammation by the influenza virus in mouse airways. The Influenza virus (influenza type A, its type H1 N1, from mouse adapted to strain PR8) was a gift from David Woodhouse, Trudeau Institute, Saranac Lake, NY. As a preliminary study to titrate the influenza dose and determine the time course of infection, BALB / c mice (female) were infected with the influenza virus by intranasal instillation on study day 0. The mice received 50, 200 or 500 infectious doses in eggs (EID) 50 of virus in 40 ul of saline. Inflammation of airways was evaluated 1, 3, 6, 9 and 14 days after infection.

Intranasal administration of CpG ODN and measurement of airway inflammation Mice received a CpG ODN (0.03, 0.3 or 3 mg / kg) by nasal instillation in 25 ul of saline. Each mouse was dosed twice, 6 days and 2 days before infection with influenza virus (200 EID50, intranasal). This dose of virus was selected from the preliminary study. Inflammation in airways and viral load in the lung were evaluated 6 days after infection. This time point was selected from the preliminary study. Cells in the airways were recovered by bronchoalveolar lavage. The total leukocyte counts were carried out in an automated Advia cell counter (Adíva, Bayer Diagnostics, Zurich, Swiza). Differential cell counts were carried out by light microscopy of cytocentrifuged preparations stained with Wright-Giemza stain. The lungs were removed and homogenized with 300 ul of sterile PBS. The supernatants were collected and the viral load was assayed using an enzyme immunoassay kit (Takara Biomedical, Shiga, Japan) used according to the manufacturer's instructions. This assay uses a monoclonal antibody against a nuclear protein of an influenza A virus as a solid phase, and a polyclonal antibody for detection of anti-influenza virus: EXAMPLE 1 Effects on secretion of IFN-g by human PBMC treated with CpG ODN Methods: Peripheral human blood mononuclear cells for any three (SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6) or seven (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO. : 3) donors were incubated with CpG ODN for 48 hours at the indicated concentrations. The secretion of interferon alpha was measured by PBMC from human. Results: Figure 1 demonstrated an increased production of IFN-a after incubation with CpG ODN. The data represents the mean +/- SEM. Note that the absolute levels in pg / ml do not know in directly comparing, since PBMC were used from different donors and the results from each donor are variable.

EXAMPLE 2 Stimulation of transfected cells with TLR9 in vitro Methods: HEK 293 cells transfected with human TLR9 were incubated with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 for 16 hours. The signal was determined by a luciferase reading. Results: The results are shown in table 1. The EC50 was calculated using Sigma Plot (SigmaPlot 2002 for Windows version 8.0). The maximum stimulation index (max SI) was calculated as the quotient between the highest value of all the concentrations evaluated for any ODN and the control medium.

TABLE 1 EXAMPLE 3 Effects of the oligodeoxynucleotide CpG SEQ ID NO: 7 against the antigen-induced increase in nasal resistance in which Methods: To investigate the effects of the oligodeoxinucleotide CpG SEQ ID NO: 7 against antigen-induced increases in nasal resistance in which, those whose antigen was sensitized, were then tested with the antigen nasally. Nasal resistance was measured 40 minutes before the test.

TABLE 2 Summary of the study protocol sensitization test with antigen test with antigen with antigen? 4 Dosage with Dosage with SEQ ID NO: 7 SEQ ID NO: 7 F? Day: 4 12 14 19 24 Measurement of the increase in nasal resistance induced by antigen Results: Figure 2 shows that the antigen test caused a progressive increase in nasal resistance during 40 minutes that was significantly suppressed in those who had been treated with SEQ ID NO: 7 (0.03-1 mg / kg).

EXAMPLE 4 Effects of the oligonucleotide CpG SEQ ID NO: 7 in a rhinitis model allergic in mouse Methods: BALB / c mice were used to study the effects of SEQ ID NO: 7 on the symptoms of allergic rhinitis. After sensitization and testing with the antigen, tissues were taken to Histopathological evaluation of inflammation. The separated mice received a final load of the antigen and the incidences of sneezing and nasal rub for a period of 10 minutes after the test.

TABLE 3 Summary of the study protocol: mice treated with oligonucleotide SEQ ID NO: 7 SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 8? ? Awareness raising daily with anti-aging f f f f f f f? f f f f f f Day: 0 7 12 13 14 15 16 17 18 19. 20 21 22 23 24 25 26? terminal points TABLE 4 Summary of the study protocol: mice treated with budesonide Daily dose with budesonide Sensitization Daily tests with antigen? Day: 0 7 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Terminal points Results: The test with the antigen caused sneezing and rubbing of the nose. As shown in Figure 3, the incidences of both were suppressed in the mice treated with SEQ ID NO: 7.

EXAMPLE 5 Airway Inflammation Influenced by Virus Influence on Mouse Lung: Effects of CpG class C oligodeoxynucleotides Introduction: CpG oligodeoxynucleotides (ODNs) induce immune-modified cytokines that could provide anti-asthma effects. The CpG class C ODN induces high titers of IFN-a compared to the previous class B ODNs. CpG class C ODNs may offer the additional benefit of suppressing exacerbations induced by the asthma virus. This study investigates the ability of three CpG class C ODNs to suppress the burden of virus (influenza) in mouse lungs and on airway inflammation induced by the virus. Methods: Influenza virus was used to induce airway inflammation in BALB / c mice. CpG ODN were administered intranasally and the protective effect was measured. Results: Figure 4 shows that in a preliminary study to titrate the influenza dose and determine the time course of the infection, the influenza virus causes an accumulation of leukocytes in the airways. The peak inflammation was achieved after 6-9 days. Mice infected with 500 EID50 of virus showed marked weight loss after 6 days and were sacrificed. Figure 5 shows the protective effects of CpG ODN on viral load and airway inflammation induced by the virus. Pretreatment with ODN CpGs before infection with the influenza virus (200 EID50) reduced the burden of virus in the lung as tested 6 days after infection. Figure 6 shows that infection of the influenza virus causes an accumulation of leukocytes in the airways 6 days later. These were predominantly neutrophils and mononuclear cells (monocytes, macrophages and lymphocytes). Vfew eosinophils were found. Cell accumulation was significantly suppressed in mice pretreated with any of the CpG ODN.

EXAMPLE 6 Effects of CpG class C oligodeoxynucleotides against airway inflammation induced by antigen in the mouse The activity of three CpG class oligodeoxynucleotides was compared C (ODNs). The CpG ODN class B SEQ ID NO: 7 were included in the study for comparison. Methods: The mice (BALB / c males) were sensitized on study days 0 and 7 with the antigen (cockroach, 10 ug, intraperitoneal) with aluminum hydroxide adjuvant (Pierce Alum, intraperitoneal). Mice were tested with the antigen by exposure to the antigen administered intranasally (10 ug in 40 ul of saline), twice each week for two consecutive weeks. The first test was on study day 21. The CpG ODN (1, 10 and 100 ug / kg) were administered intranasally once a week, two days before the first antigen test of the week. Airway inflammation was evaluated 48 hours after the last test. The cells in the airways were recovered by bronchoalveolar lavage. Differential cell counts were performed by means of an automated Advia cell counter. The numbers of T cells (total CD3 + cells, and CD3 + CD4 + cells) were counted by flow cytometry.

TABLE 5 Summary of the protocol study Sensitization with antigen test Antigen test with antigen? < Dosage with Dosage with CpG ODN CpG ODN F Day: 19 21 24 26 28 31 33 Inflammation evaluation of airways TABLE 6 ODN CpG evaluated Results: The antigen test caused accumulations of eosinophils and T cells in the airways (Figures 7 and 8). There was no accumulation of neutrophils. Each of the CpG ODNs caused a significant suppression of eosinophil accumulation at the highest dose evaluated (100 mg / kg) (Figure 7). The numbers of T cells were also low, although the reductions were generally not statistically significant (figure 8).

EXAMPLE 7 Induction of cytokine by CpG class C oligodeoxynucleotides in the mouse in vivo The activities of three oligodeoxynucleotides were compared CpG class C (ODNs). The CpG ODN class B SEQ ID NO: 7 were included in the study for comparison. Methods: The concentrations of cytokine and chemokines in the bronchoalveolar lavage fluid and in the serum were tested as described in materials and methods.

TABLE 7 Treatment groups Results: Figure 9 shows cell numbers in the bronchial-alveolar lavage fluid. Intranasal instillation of each of the class C ODN, especially at 1 mg / kg, showed a tendency to cause a moderate accumulation of leukocytes in the bronchoalveolar lavage fluid. Figures 10-15 show the cytokine concentrations in the bronchoalveolar lavage fluid. Intranasal instillation of each CpG ODN induced measurable titers of IFN-a, IFNy, IP-10, IL-12p40, IL-6 and TNF-a in the bronchoalveolar lavage fluid. The titers of the other analytes measured did not reach detectable concentrations or the concentrations were below the background level (typically <20 pg / ml, data not shown). Class C ODNs were more potent than ODN class B SEQ ID NO: 7 as inducers of IFN-a, IFNy, IP-10, IL-12p40, IL-6 and TNF-a. The increased potency of the class C ODN was especially evident at the dose level of 0.1 mg / kg. Of particular interest was the observation that only class C ODNs were able to induce any measurable titers of IFN-a and IFNy after dosing at 0.1 mg / kg (Figure 10). Figures 15-18 show serum cytokine concentrations. Intranasal instillation of each of the CpG ODN induced measurable titers of IFN-? IL-6 and TNF-a in serum. The titers of the other analytes measured did not reach the detectable concentrations (typically <20 pg / ml, data not shown). When compared to the CpG class B SEQ ID NO: 7, each of the three class C ODNs were more potent inducers of the immune modified cytokines IFN-α, IFN- ?, IP-10, IL-12p40, IL-6 and TNF-a.

EXAMPLE 8 Effects of oligodeoxynucleotides CpG SEQ ID NO: 2 and SEQ ID NO: 7 on the production of IgE induced by antigen in the mouse Methods: The mice (BALB / c males) were sensitized on study days 0 and 7 with the antigen (ovalbumin, 10 ug, i.p.) and aluminum hydroxide, adjuvants (Pierce Alum, Lp). Mice received SEQ ID NO: 2 or SEQ ID NO: 7 at study days -2, 0, 5 and 7 (for example two days before each sensitization and at the day of sensitization). Mice were bled by cardiac puncture on day 18 of study. Serum was collected by centrifugation and assayed by ELISA for IgE and IgG2a specific to ovalbumin.

TABLE 8 Treatment groups: TABLE 9 Summary of the study protocol Results: The sensitization of the antigen resulted in serum titers of IgE antigen IgG and IgG2a specific to (ovalbumin). The production of IgE was suppressed in the mice treated with any SEQ ID NO: 2 or SEQ ID NO: 7, although the production of IgG2a was enhanced (FIG. 19). Conclusions: The data of Example 8 show that SEQ ID NO: 2 and SEQ ID NO: 7 suppressed in IgE production associated with Th2 in response to antigen sensitization, and potentiated lgG2a production associated with Th1. The results of this study provide additional evidence that these CpG oligos can suppress a Th2-type response to antigen exposure in the mouse.

EXAMPLE 9 Effects of SEQ ID NO: 2 against exacerbated airway inflammation induced by the combined influence infection and 8th antigen test Introduction: The CpG class C oligodeoxynucleotide SEQ ID NO. 2 can suppress the burden of influenza virus and airway inflammation induced by the virus in mice. The present study investigated the protective effects of SEQ ID NO: 2 against exacerbated airway inflammation induced by the combination of virus infection and antigen test. Methods: Antigen and virus administrations. Mice (BALB / c males) were sensitized on study days 0 and 7 with antigen (cockroach, 10 ug, intraperitoneal) with aluminum hydroxide as an adjuvant (Pierce Alum). Mice were tested for antigen by exposure to the antigen administered intranasally (10 ug in 40 ul of saline), twice each week for three consecutive weeks. The first test was on study day 21. The mice were infected with influenza virus (influenza type A, subtype H1 N1, mouse adapted to strain PR8, 200 EID50 in 40 ul of saline) by intranasal instillation on study day 34 (for example before the last pair of tests with the antigen). Alternatively, separate groups of mice received the antigen test alone or the virus infection alone.

SEQ ID NO: 2 (100 ug / kg) was administered intranasally once a week, two days before the first antigen test of the week. Inflammation of the airways was evaluated 48 hours after the last antigen test. The cells in the airways were recovered by bronchoalveolar lavage. Differential counts of cells were carried out by light microscopy from cytocentrifuge preparations stained with Wright-Giemza stain.

TABLE 10 Summary of the study protocol Results: Figure 20 shows that infection with the influenza virus alone or the antigen test only causes an increase in the total number of leukocytes in the bronchoalveolar lavage fluid. In the mice infected with the virus, this accumulation of cells included a marked neutrophilia, whereas in the mice tested with the antigen, the accumulation included a marked eosinophilia. When compared to the mice they received the antigen test alone, compared to those that were tested with the antigen and those that were infected with the virus showed an exacerbated accumulation of leukocytes in the bronchoalveolar lavage fluid induced by the virus. This increased accumulation included both neutrophils and mononuclear cells. However, these mice showed reduced eosinophilia. Similarly, other investigators have shown that infection with influenza can suppress airway eosinophilia in mice that have been tested with the antigen, and have hypothesized that this is a Th 1 -mediated effect (eg, Wohlleben et al., 2003) . The treatment with SEQ ID NO: 2 (100 ug / kg) does not suppress the neutrophilia induced by the virus (figure 20). This negative finding was expected since, in a previous study, a higher dose of 300 ug / kg was more desirable to show anti-virus effects. In addition, SEQ ID NO: 2 (100 ug / kg) did not significantly suppress the eosinophilia induced by the antigen. This positive finding was in agreement with previous studies. SEQ ID NO. 2 (100 ug / kg) significantly suppressed the exacerbated airway inflammation induced in mice that were both infected with the virus and tested with the antigen. The exacerbated accumulations of neutrophils and mononuclear cells were suppressed. In addition to the exacerbated inflammation of the airways, mice that were both infected with the virus and tested with the antigen showed a marked loss of body weight. This was suppressed significantly in the mice treated with SEQ ID NO: 2.

Conclusions: Both children and adults with asthma, infections with respiratory tract virus important elements that precipitate airway obstruction and panting breathing. The inflammatory processes involved are complex. However, neutrophilia induced by the virus and the recruitment and activation of mononuclear cells are implicated in the aggravation of airway obstruction that contributes to these exacerbations of asthma (reviewed by Gern and Busse, Nature Immunology, 2002) . The data of Example 9 demonstrate that SEQ ID NO: 2 markedly suppresses the exacerbated accumulations of neutrophils and mononuclear cells induced in mice by combining the viral infection with the antigen test.

EXAMPLE 10 Studies with which EXAMPLE 10a Summary of an AHR protocol in which Those who were sensitized on study days 0 and 4 with the antigen (ovalbumin, 0.5 ml, 1% OVA i.p./s.c.) With aluminum hydroxide as adjuvant. Those whose antigen was tested by inhalation ovalbumin aerosol exposure twice a week for two consecutive weeks. The first test was on study day 13. CpG ODN or vehicle (saline, 20 ul) were administered intranasally once a week, two days before the first antigen test of the week. The airway hyperreactivity was evaluated 24 hours after the last antigen test by measuring the bronchial constriction (increased resistance of the airways) to intravenous methacholine. For each animal, a dose-response curve to methacholine was used, and the reactivity in the airways was quantified as the area under the curve. Figure 21 shows a scheme of the procedure.

EXAMPLE 10b Effect of SEQ ID NO: 7 on airway resistance and lung access in which Method: Those who were sensitized as described in example 10. The first test was on study day 13. Those who were given intranasally either the vehicle (saline solution), OVA alone, of the concentrations of SEQ ID NO: 7 of 10 ul / kg, 30 ul / kg, 100 ul / kg, or 300 ul / kg, it Results: Figure 22 shows that SEQ ID NO: 7 causes a dose-dependent reduction in the AUC resistance.

EXAMPLE 10c Statistical analysis of the effect of SEQ ID NO: 7 on airway resistance and lung access in which Method: Dunnett's multiple comparison test was used to analyze the data from the experiments in Example 10b. Dunnett's multiple comparison test allows comparison of all samples with a particular control group. Results: Tables A and B show that SEQ ID NO: 7 causes a dose-dependent reduction in the AUC resistance.

TABLE A Analysis of one-way variance (ANOVA) The P value is 0.0032, considered very significant. The variation between the means of the columns is significantly higher than expected Dunnett's multiple comparison test Control column: OVA Value of q is greater than 2,632 then the P value is less than 0.05 Assumption test, are the standard deviations of the groups equal? TABLE B Analysis of one-way variance (ANOVA) The P value is 0.0001, considered very significant. The variation between the means of the columns is significantly higher than expected Dunnett's multiple comparison test Control column: OVA Value of q is greater than 2,632 then the P value is less than 0.05 Assumption test: are the standard deviations of the groups equal? EXAMPLE 10d Effect of SEQ ID NO: 2 on airway resistance and lung access in which Method: Those who were sensitized as described in example 10. The first test was on study day 13. Those who were given intranasally either the vehicle (saline solution), OVA alone, of the concentrations of SEQ ID NO: 2 of 10 ul / kg, 30 ul / kg, 100 ul / kg, or 300 ul / kg, it Results: Figure 24 shows that SEQ ID NO: 2 causes a dose-dependent reduction in the AUC resistance.

EXAMPLE 10e Statistical analysis of the effect of SEQ ID NO: 2 on airway resistance and lung access in which Method: Dunnett's multiple comparison test was used to analyze the data from the experiments in Example 10d. Dunnett's multiple comparison test allows comparison of all samples with a particular control group. Results: Tables C and D show that SEQ ID NO: 2 results in a dose-dependent reduction in AUC resistance.

TABLE C Analysis of one-way variance (ANOVA) The P value is <0.0001, considering very significant. The variation between the means of the columns is significantly higher than expected Dunnett's multiple comparison test Control column: OVA Value of q is greater than 2,620 then the P value is less than 0.05 Assumption test: are the standard deviations of the groups equal? TABLE D Analysis of one-way variance (ANOVA) The P value is 0.0003, considered very significant. The variation between the means of the columns is significantly higher than expected Dunnett's Multiple Comparison Test Control column: OVA Value of is 2.620 then the P value is less than 0.05 Assumption test: are the standard deviations of the groups equal? The levels of IL-0, TNF-alpha, interferon-gamma, and IL-6 (pg / ml) produced by human PBMC followed by exposure of these cells to the CpG oligonucleotides described in the present invention are shown in annexed figures 25-29. The test oligonucleotides illustrated in Figure 25 include SEQ ID NOs: 10, 9, 13, 14, 1, and 2. The concentration of oligonucleotide used to produce a particular data point is illustrated along the X axis (uM). ). As shown in Figure 25, each of the oligonucleotides evaluated in the assays were able to produce different levels and secretion patterns of IL-10. From those ODN SEQ ID NO 1 and 2 evaluated, a dramatically increased induction of IL-10 resulted. Figures 26A-26C illustrate data that relate to TNF-alpha, interferon-gamma, and IL-6 at three representative doses. The more detailed graphs of these cytokines are illustrated in Figures 27-29 with additional doses of oligonucleotide.

EXAMPLE 12 The levels of the B cell cell, plasmacytoid dendritic cell and monocyte activation followed by exposure of these cells to the CpG oligonucleotides described in the present invention are shown in the attached figures 30A-30C to 40. The oligonucleotides examined are illustrated in FIGS. Figures by SEQ ID NO and include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The concentration of oligonucleotide used to produce a particular data point is illustrated along the axis X (uM). As demonstrated in Figures 30A-30C, 32, 33, 38A-38B, and 40 the CpG oligonucleotides evaluated in the assays were able to activate B cells, as represented by the tested markers. In Figures 30A-30C and 31 the CpG oligonucleotides tested in the assays were able to activate NK cells, as represented by the tested markers. In Figures 34A-34C, 35, 38A-38B, and 39 the CpG oligonucleotides tested in the assays but capable of activating monocytes, as represented by the tested markers. In Figures 34A-34C and 37 the CpG oligonucleotides tested in the assays were able to activate the plasmacytoid dendritic cells, as represented by the tested markers. The five ODNs having a semi-soft base structure that were tested in the assays showed increased potency in the assays (IFN-alpha, IP-10, IL-10) compared to SEQ ID NO. 9 (structure completely of phosphorothioate). The potency of these semi-soft ODNs was also increased in: activation of monocytes (expression of CD80, CD86), activation of pDC (expression of CD86), intracellular IP-10 (monocytes and B cells), secretion of IL- 6, and B cell activation (expression of CD80, CD86). For example, at approximate equivalents or lower concentrations in most of the ODNs tested, this resulted in a better induction of the cell surface markers compared to the completely phosphorothioate element SEQ ID NO. 9.

EXAMPLE 13 The objective of this study was to investigate the biological activity of the selected fragments (putative metabolites) of SEQ ID NO: 2. The activity was determined by measuring the ability of each of the fragments to induce the secretion of cytokines associated with TLR9 from mouse splenocytes in vivo.

The stimulation of cytokine secretion by the fragments of SEQ ID NO: 2 is shown in the attached figures 41A-41 F to 43A-43F. The oligonucleotides examined are illustrated in the appended figures by SEQ ID NO and include SEQ ID NO: 15-23. The concentration of oligonucleotide used to produce a particular data point is illustrated along the X axis (ug / ml). As demonstrated in Figures 41A-41 F-43A-43F SEQ ID NO: 2 and the fragments (putative metabolites, SEQ ID NOs: 15-23) tested induce the cytokines associated with TLR9 IFN-α, IFN- ?, IP -10, IL-6, IL-10 and TNF-a from mouse splenocytes in vitro (Figures 41A-41 F, 42A-42F and 43A-43F). These data show that each of the fragments retains biological activity.

Claims (4)

1. NOVELTY OF THE INVENTION CLAIMS 1 .- An oligonucleotide comprising: 5"5 ' TC_GTC_GTN1TC_GGCGCNiGCCG 3 '(SEQ ID NO: 27), wherein the oligonucleotide includes at least 2 stabilized internucleotide bonds and _ represents an internucleotide phosphodiester linkage or an internucleotide linkage similar to phosphodiester and wherein Ni is 0-3 nucleotides in length, with N referring to any nucleotide.
2. 2. The oligonucleotide according to claim 1, further characterized in that it is 3 nucleotides in length.
3. 3. The oligonucleotide according to claim 1, further characterized in that the oligonucleotide comprises 5 'T * C_G * rC_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 '(SEQ ID NO 2), where * represents a stabilized internucleotide link.
4. 4. The oligonucleotide according to claim 3, further characterized in that the oligonucleotide is 5 '5' T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 '(SEQ ID NO: 2), where * represents a stabilized internucleotide link, where 5' refers to the free 5 'end of the oligonucleotide and 3' refers to the free 3 'end of the oligonucleotide. 5. - The oligonucleotide according to claim 1, further characterized in that N-i is 0 nucleotides in length. 6. - The oligonucleotide according to claim 1, further characterized in that the oligonucleotide comprises 5 'T * CG * T * CG * T * T * CG * G * C * G * C * G * C * C * G 3 '(SEQ ID NO: 3), where * represents a stabilized internucleotide link. 7. - The oligonucleotide according to claim 6, further characterized in that the oligonucleotide is 5 'rC_G * T * C_G * T * T * C_G * G * C * G * C * G * C * C * G 3' ( SEQ ID NO: 3), wherein * represents a stabilized internucleotide bond, where 5 'refers to the free 5' end of the oligonucleotide and 3"refers to the free 3 'end of the oligonucleotide 8. - An oligonucleotide comprising: 5 'TC_GTC_GAC_GATC_GGCGC_GCGCCG3' (SEQ ID NO: 4), wherein the oligonucleotide includes at least 2 stabilized internucleotide bonds and - represents an internucleotide phosphodiester linkage or an internucleotide linkage similar to phosphodiester 9. - The oligonucleotide according to claim 8, further characterized in that the oligonucleotide is 5 'T * C_G * T * C_G * A * C_G * A * T * C_G * G * C * G * C_G * C * G * C * C * G 3' (SEQ ID NO: 4), where * represents a stabilized internucleotide link 10. - An oligonucleotide comprising: 5 'TTC_GTC_GTTTX _GTC_GTT 3' (SEQ ID NO: 25), wherein the oligonucleotide includes at least 2 stabilized internucleotide bonds and represents an internucleotide phosphodiester linkage or an internucleotide linkage similar to phosphodiester and wherein i is a pyrimidine. 1. The oligonucleotide according to claim 10, further characterized in that Xi is T. 12. The oligonucleotide according to claim 1, further characterized in that the oligonucleotide comprises 5 'T * T * C_G * T * C_G * T * T * T * T_G * T * C_G * T * T 3 '(SEQ ID NO: 5), where * represents a stabilized internucleotide link. 13. The oligonucleotide according to claim 1, further characterized in that the oligonucleotide is 5 'T * T * C_G * T * C_G * T * T * T * T_G * T * C_G * T * T 3' (SEQ ID NO: 5), wherein * represents a stabilized internucleotide linkage, wherein 5 'refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide. 14. - The oligonucleotide according to claim 10, further characterized in that Xi is C 15. The oligonucleotide according to claim 14, further characterized in that the oligonucleotide comprises 5 'T * T * T * C_G * T * C_G * T * T * T * C_G * T * C_G * T * T 3 '(SEQ ID NO: 6), where * represents a stabilized internucleotide link. 16. - The oligonucleotide according to claim 14, further characterized in that the oligonucleotide is 5 'T * T * T _G * T * C_G * T * "TTX_G * T * C_G * T * T 3' (SEQ ID NO: 6), where * represents a stabilized internucleotide link, where 5 'refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide 17. An oligonucleotide comprising: TCGTCGTTCGGCGCGCCG (SEQ ID NO: 3). 18. - An oligonucleotide comprising: TCGTCGTCGTTCGGCGCGCGCCG (SEQ ID NO: 2). 19. - An oligonucleotide comprising: TCGTCGACGATCGGCGCGCGCCG (SEQ ID NO: 4). 20. An oligonucleotide comprising: TTC GTC GTTTTGTC GTT (SEQ ID NO: 5). 21. - An oligonucleotide comprising: TTTCGTCGTTTCGTCGTT (SEQ ID NO: 6). 22. An oligonucleotide comprising: T * C_G * T * C_G * T * C, wherein * represents a stabilized internucleotide bond and - represents an internucleotide phosphodiester linkage or an internucleotide linkage similar to phosphodiester. 23. The oligonucleotide according to claim 22, further characterized in that the oligonucleotide is 5 'T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_ G * C * G * C * C 3"(SEQ ID NO .: 21), where 5 'refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide. 24. - The oligonucleotide according to claim 22, further characterized in that the oligonucleotide is 5 'T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C 3' (SEQ ID NO. : 22), where 5 'refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide. 25. - The oligonucleotide according to claim 22, further characterized in that the oligonucleotide is T * C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C 3 '(SEQ ID NO: 23), where 5' refers to the free 5 'end of the oligonucleotide and 3' refers to the free 3 'end of the oligonucleotide. 26. - An oligonucleotide comprising: T * C_G * T * T * C_G * G, where * represents a stabilized internucleotide bond and _ represents a phosphodiester bond or a phosphodiester-like bond. 27. The oligonucleotide according to claim 26, further characterized in that the oligonucleotide is 5 '. C_G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3"(SEQ ID NO.: 15), where 5 'refers the free 5 'end of the oligonucleotide and 3' refers to the free 3 'end of the oligonucleotide 28. The oligonucleotide according to claim 26, further characterized in that the oligonucleotide is 5'. G * T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 '(SEQ ID NO .: 16), where 5' refers the free 5 'end of the oligonucleotide and 3' refers to the free 3 'end of the oligonucleotide. 29. - The oligonucleotide according to claim 26, further characterized in that the oligonucleotide is 5 'T * C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 '(SEQ ID NO: 17), where 5' refers to the free 5 'end of the oligonucleotide and 3' refers to the free 3 'end of the oligonucleotide. 30. The oligonucleotide according to claim 26, further characterized in that the oligonucleotide is 5 'C_G * T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 '(SEQ ID NO: 18), where 5' refers to the free 5 'end of the oligonucleotide and 3' refers to the free 3 'end of the oligonucleotide. 31. - The oligonucleotide according to claim 26, further characterized in that the oligonucleotide is 5 'G * T * C_G * ~ PT * C_G * G * C * G * C_G * C * G * C * C * G 3' (SEQ ID NO .: 19); wherein 5 'refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide. 32. - The oligonucleotide according to claim 26, further characterized in that the oligonucleotide is 5 'T * C_G * T * T * C_G * G * C * G * C_G * C * G * C * C * G 3 * ( SEQ ID NO: 20), where 5 'refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide. 33. - A pharmaceutical composition comprising an oligonucleotide of any of claims 1-32 and a pharmaceutically acceptable carrier. 34. - The pharmaceutical composition according to claim 33, further characterized in that it additionally comprises a nebulizer. 35. - The pharmaceutical composition according to claim 33, further characterized in that it additionally comprises an inhaler. 36. - The pharmaceutical composition according to claim 35, further characterized in that the inhaler is a metered dose inhaler. 37. - The pharmaceutical composition according to claim 35, further characterized in that the inhaler is a powder inhaler. 38. - The pharmaceutical composition according to claim 33, further characterized in that it additionally comprises a chemotherapeutic agent. 39. - The pharmaceutical composition according to claim 33, further characterized in that it additionally comprises an antiviral agent. 40. - The pharmaceutical composition according to claim 33, further characterized in that the pharmaceutically acceptable carrier is formulated for subcutaneous administration. 41. - The pharmaceutical composition according to claim 33, further characterized in that the pharmaceutically acceptable carrier is formulated for oral administration. 42. - The pharmaceutical composition according to claim 33, further characterized in that the pharmaceutically acceptable carrier is formulated for intranasal administration. 43 - The use of an oligonucleotide as claimed in any of claims 1-32, in the preparation of a medicament useful for stimulating an immune response. 44.- The use of an oligonucleotide as claimed in any of claims 1-32, in the preparation of a medicament useful for the treatment of asthma 45.- The use as claimed in claim 44, in where asthma is exacerbated by viral infection. 46. The use of an oligonucleotide as claimed in any of claims 1 -32, in the preparation of a drug useful for the treatment of allergy. 47. The use claimed in claim 46, wherein the allergy is allergic rhinitis. 48. The use of an oligonucleotide as claimed in any of claims 1-32, in the preparation of a drug useful for the treatment of cancer. 49. - The use as claimed in claim 48, wherein the medicament is adapted to be administrable with a chemotherapeutic agent. 50. The use as claimed in claim 48, wherein the medicament is further adapted to be administrable with radiation. 51. The use of an oligonucleotide as claimed in any of claims 1-32, in the preparation of a medicament useful for the treatment of an infectious disease. 52. The use of an oligonucleotide as claimed in any of claims 1-32, in the preparation of a drug useful for the treatment of a viral disorder. 53. The use as claimed in claim 52, wherein the viral disorder is hepatitis. 54.- The use as claimed in claim 53, wherein the hepatitis is hepatitis B. 55 - The use as claimed in claim 53, where hepatitis is hepatitis C. 56.- The use as claimed in claim 51, wherein the medicament is further adapted to be administrable with an anti-viral agent. 57 -. 57 - The use of an oligonucleotide as claimed in any of claims 1-32, in the preparation of a medicament useful for the treatment of autoimmune disease. 58. The use of an oligonucleotide as claimed in any of claims 1-32, in the preparation of a medicament useful for the treatment of airway remodeling. 59. The use as claimed in claim 43, wherein the medicament is adapted to be administrable without an antigen. 60. - The use as claimed in claim 43, wherein the medicament is adapted to be administrable with an antigen. 61. - The use as claimed in claim 43, wherein the medicament is adapted to be administered by a route selected from the group consisting of oral, nasal, sublingual, intravenous, subcutaneous, mucosal, respiratory, injection direct, and dermally. 62. The use as claimed in claim 43, wherein the medicament is adapted to be administered in an amount effective to induce cytokine expression. 63. - The use as claimed in claim 62, wherein the cytokine is selected from the group consisting of IL-6, TNF-α, IFN-cc, IL-10, IL-12, IFN- and and IP- 0. 64. The use as claimed in claim 43, wherein the medicament is adapted to be administered in an effective amount to change the immune response towards an immune response towards a skewed response to Th1 from a skewed response to Th2.