MX2008006770A - Immunostimulatory oligoribonucleotides - Google Patents

Abstract

The invention provides immunostimulatory compositions and use of those compounds in the preparation of medicaments for the treatment of disease as well as in vitro uses. In particular, the compositions of the invention include immunostimulatory oligoribonucleotides that incorporate a sequence-dependent immunostimulatory sequence motif. Specific modifications involving phosphate linkages, nucleotide analogs, adducts, and combinations thereof are provided. Compositions of the invention, which optionally can include an antigen, can be used alone or together with other treatments to stimulate or enhance an immune response. Also provided are compositions and methods useful for treating a subject having an infection, a cancer, an allergic condition, asthma, airway remodeling, or immunodeficiency. Immnostimulatory oligoribonucleotides of the invention are believed to stimulate Toll-like receptor 8 (TLR8).

Description

IMMUNO-STIMULATORY OLIGORRIBONUCLEOTIDES FIELD OF THE INVENTION The invention relates generally to the field of immunology and more particularly to immunostimulatory molecules. More specifically, the invention relates to ribonucleic acid (RNA) molecules, including oligoribonucleotides, with immunostimulatory activity.

BACKGROUND OF THE INVENTION Toll-like receptors (TLRs) are a family of highly conserved pattern recognition (PRR) polypeptides that recognize pathogen-associated molecular patterns (PAMP) and have a critical role in innate immunity in mammals. Currently, at least ten members of the family have been identified, named TLR1 -TLR10. The cytoplasmic domains of the different TLRs are characterized by a Toll-interleukin 1 receptor (TIR) domain. Medzhitov R. et al., (1998) Mol. Cell 2: 253-8. The recognition of microbial invasion by TLR triggers the activation of a signaling cascade that is evolutionarily conserved in Drosophila and in mammals. It has been described that the adapter protein containing the TIR domain MyD88 associates with the TLRs and recruits the kinase associated with the interleukin 1 receptor (IRAK) and the factor 6 associated with the tumor necrosis factor receptor (TNF) ( TRAF6) in the TLRs. It is believed that the signaling pathway dependent on MyD88 leads to the activation of NF- transcription factors ?? and protein kinase-activated mitogen (MAPK) kinase NH2-terminal c-Jun (Jnk), critical stages in the immune activation and production of inflammatory cytokines. For a review, see Aderem A. et al. (2000) Nature 406: 782-87, and Akira S. et al. (2004) Nat. Rev. Immunol 4: 499-51 1. A series of TLR-specific ligands have been identified. Ligands for TLR2 include peptidoglycans and lipopeptides. Yoshimura A. et al., (1999) J. Immunol. 163: 1-5; Yoshimura A. et al. (1999) J. Immunol. 163: 1-5; Aliprantis A.O. and col. (1999) Science 285: 736-9. Lipopolysaccharide (LPS) is a ligand for TLR4. Poltorak A. et al., (1998) Science 282: 2085-8; Hoshino K. et al. (1999) J. Immunol. 162: 3749-52. Bacterial flagellin is a ligand for TLR5, Hayashi F. et al., (2001) Nature 410: 1099-1103. It has been described that peptidoglycan is a ligand not only for TLR2 but also for TLR6. Ozinsky A. et al. (2000) Proc. Nati Acad. Sci. USA 97: 13766-71; Takeuchi O. et al. (2001) Int. Immunol. 13: 933-40. It has recently been reported that certain low molecular weight synthetic compounds, the imidazoquinolines imiquimod (R-837) and resiquimod (R-848), are ligands of TLR7 and TLR8. Hemmi H. et al., (2002) Nat. Immunol. 3: 196-200; Jurk M. et al. (2002) Nat. Immunol. 3: 499. Starting from the recent discovery that unmethylated bacterial DNA and its synthetic analogs (CpG DNA) are ligands for TLR9 (Hemmi H. et al (2000) Nature 408: 740-5; Bauer S. et al (2001) Proa Nati. Acad. Sci. USA 98, 9237-42), it has been described that the ligands for some TLRs include certain nucleic acid molecules. Recently, it has been described that certain types of RNA are immunostimulatory in a sequence-independent or sequence-dependent manner. In addition, it has been described that these different immunostimulatory RNAs stimulate TLR3, TLR7 or TLR8.

BRIEF DESCRIPTION OF THE INVENTION The invention relates in general to immunostimulatory oligoribonucleotides (ORN) containing certain immunostimulatory RNA motifs, as well as related immunostimulatory compositions containing said immunostimulatory ORNs, and to methods for using said ORN and immunostimulatory compositions. The immunostimulatory ORNs of the invention are useful in any setting or application that requires stimulation or enhancement of an immune response. As described below, the immunostimulatory ORNs of the invention have a particular use in the preparation of pharmaceutical compositions, including adjuvants, vaccines and other medications, for use in the treatment of a variety of conditions, including infections, cancer, allergy and asthma. Therefore, the invention in certain aspects relates to immunostimulatory compositions that include immunostimulatory ORNs of the invention, as well as methods for their use. Also as described below, the immunostimulatory ORNs and immunostimulatory compositions of the invention have particular use in methods for activating an immune cell, vaccinating a subject, treating a subject having a deficiency of the immune system, treating a subject having an infection, treating a subject having an autoimmune disease, treating a subject having cancer, treating a subject having an allergic condition, treating a subject having asthma, remodeling the airway, promoting extension of epitopes and antibody-dependent cellular cytotoxicity (ADCC). As described in more detail below, the immunostimulatory ORNs of the invention are characterized in that they include at least one immunostimulatory RNA motif dependent on the sequence. The sequence-dependent immunostimulatory RNA motif in general is a short sequence of RNA, although in some embodiments the motif may also include a modification such as a modified internucleotide phosphate bond, a modified nitrogenous base, a modified sugar, a nucleotide analog or any combination thereof. As described in detail below, in one embodiment the immunostimulatory RNA motif is found in the context of an immunostimulatory ORN of the longest invention. The immunostimulatory RNA motif can also be found in the context of a DNA: chimeric RNA nucleic acid molecule.

It has been described that the sequence-dependent immunostimulatory RNA motifs and immunostimulatory ORNs incorporating said motifs are agonists for TLR8. More particularly, it has been described that at least some immunostimulatory RNA motifs dependent on sequence, immunostimulatory ORN and DNA nucleic acid molecules: chimeric immunostimulatory RNA are TLR8 agonists but not TLR7 agonists. According to some aspects of the invention the immunostimulatory RNA motif is N-U-R R2-N is a ribonucleotide and N does not include U. In some embodiments N is adenosine or cytosine (C) or derivatives thereof. U is uracil or a derivative thereof. R is a ribonucleotide in which at least one of Ri and R2 is adenosine (A) or cytosine or derivatives thereof. R is not U unless NU-R1-R2 includes at least two A. The ORN of the invention includes at least one, and in some embodiments more than one (ie, 2, 3 or 4), immunostimulatory motifs, NU- Ri-R2. ORNs do not include a TLR7 / 8 motive. The ORN preferably has a length of 4-100 and optionally includes at least one modification of the main chain. In some embodiments N-U-Ri-R2 may include at least 3 A or at least 2 C. Optionally, N-U-R R2 includes at least one G or C.

In some modalities the ORN is not ACCCAUCUAUUAUAUAACUC (SEQ ID NO .: 89). In other embodiments the ORN motif is separated from a 5 'ribonucleotide by a non-nucleotide linker. In still other embodiments the ORN motif is separated from a 3 'ribonucleotide by a non-nucleotide linker. Optionally, the ORN motif is separated from a 5 'and 3' ribonucleotide by a non-nucleotide linker. The ORN may further comprise a pharmaceutically acceptable carrier which optionally is a lipid carrier such as N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium methyl sulfate (DOTAP). In other modalities the ORN does not form complete with DOTAP. The ORN can be single-stranded or double-stranded. In other modalities, the ORN includes at least one AU. In other modalities, the ORN includes at least one CU. In some modalities, the ORN is one of the following: A * U * A * G * G * C * A * C (SEQ ID N0: 4), G * C * C * A * C * C * G * A * G * C * C * G * A * A * U * A * U * A * C * C (SEQ ID NO: l 1), AHJ * A * U * A * U * Anj + A * U * A * U * A * U * A * U * Anj * A * U (SEQ ID NO: 12),? *? *? *? *? *? *? *? *? +? +? *? *? *? *? *? *? *? *? * ? (SEQ ID NO: 13), A * A * U * A * A * U * A * A * U * A + A + U * A * A * U * A * A * U * A * A (SEQ ID NO: 16),? *? *? *? *? *? *? +? *? *? *? *? + ?, "? *? *? *? *? *? *? (SEQ ID NO: 17), A * A * A * A * U * A * A * A * A * U * A * A * A ^ * U * A * A * A * A * U (SEQ ID NO: 18), CnJ * A * C * U * A * C * U * A * C * U * A * C * U + A * C * U ^ \ * C * U (SEQ ID NO: 24), U * U * A * U * U * A * U (SEQ ID NO: 30), U * A * U * A * U * A * U (SEQ ID NO: 33), C * C * G * A * G * C * C * G * C * A * U * U * A * C * C * C (SEQ 1D N0: 48), C * C * G * A * G * C * C * G * A * U * U * G * A * A * C * C (SEQ ID NO: 76), C * C * G * A * G * C * C * G * A * A * U * A * C * C + C * C (SEQ ID NO: 42), C * C * G * A * G * C * C * A * U * A * U * A * U * A * U * C (SEQ 1D N0: 39), C * C * G * A * G * C * C * G * A * U * A * U * U * A * C * C (SEQ ID NO: 65), C * C * G * A * G * C * C * G * A * A * U * C * C * C * C + C (SEQ ID NO: 44), C * C * G * A * G * C * C * G * C * C * U * A * C * C * C + C (SEQ ID NO: 47), C * C * G ^ * G * C * C * A * U * A * U * A * U * C * C * C (SEQ ID N0: 38), C * C * G * A * G * C * C * G * C * U * A * U * A * C * C * C (SEQ ID NO: 37), C * C * G * A * G * C * C * G * A * A * U * A * A * C * C * C (SEQ ID NO: 40), C * C * G * A * G * C * C * G * C * U * A * U * C * C * C * C (SEQ 1D N0: 55), C * C * G * A * G * C * C * G * A * A * G * G * U * A * C * C (SEQ ID NO: 82), C * C * G * A * G * C * C * G * A * A * G * A * U * A * C * C (SEQ ID NO: 85), C * C * G * A * G * C * C * G * A * A * U * G * U * A * C * C (SEQ 1D N0: 63) 3 C * C * GG * C * C * G * C * C + U + A *TO* C + C + C (SEQ 1D N0: 43), C * C * G * A * G * C * C * G * C * A * U * A * U * C * C * C (SEQ ID NO: 36), C * C * G * A * G * C * C * G ^ \ * A * G * Cnj * A * C * C (SEQ ID NO: 87), C * C * G * A * G * C * C * G * C * A * U * A * C * C * C * C (SEQ ID NO: 45), C * C * G * A * G * C * C * G * C * A * U * A * A * C * C * C (SEQ ID N0) : 41)} C * C * G * A * G * C * C * G * A * A * G * G * U * G * C * C (SEQ ID ??: 83), C * C * G * A * G * C * C * G * C * A * U * C * C * C * C * C (SEQ ID NO: 46) 3 C * C * G * A * G * C * C * G * A ^ \ * G * C * U * G * C * C (SEQ ID NO: 88), C * C * G * A * G * C * C * G * C * C * G * C * C * C * C * C ( SEQ ID NO: 35), C * C * G * A * G * C * C * G ^ * A * G * C * U * C * C * C (SEQ ID NO: 84), or C * C * G * A * G * C * C * G * A * A * G * G * C * A * C * C (SEQ ID NO: 56). The ORN specifically excludes TLR7 / 8 motives. A TLR7 / 8 motif may include, for example, a ribonucleotide sequence selected from (i) 5 -C / UUG / UU-3 '(i) 5'-RURGY-3 \ (iii) 5'-GUUGB-3 ', (iv) 5'-GUGUG / U-3' and (v) 5'-G / CUA / CGGCAC-3 ', in which C / U is cytosine (C) or uracil (U), G / U is guanine (G) or U, R is purine, Y is pyrimidine, B is U, G, or C, G / C is G or C, and A / C is adenine (A) or C. In different modalities 5 - C / UUG / UU-3 'is CUGU, CUUU, UUGU, or UUUU.

In different modalities 5! -R-U-R-G-Y-3 'is GUAGU, GUAGC, GUGGU, GUGGC, AUAGU, AUAGC, AUGGU, or AUGGC, In one embodiment the base sequence is GUAGUGU. In different modalities 5'-G-U-U-G-B-3 'is GUUGU, GUUGG, or GUUGC. In different modalities 5'-G-U-G-U-G / U-3 'is GUGUG or GUGUU. In one modality the base sequence is GUGUUUAC. In different embodiments 5'-G / C-U-A / C-G-G-C-A-C-3 'is GUAGGCAC, GUCGGCAC, CUAGGCAC, or CUCGGCAC. In one aspect, the invention provides an immunostimulatory composition that includes an immunostimulatory ORN of the invention and an adjuvant. In different embodiments, the adjuvant is an adjuvant that creates a depot effect, an immunostimulatory adjuvant, or an adjuvant that creates a depot effect and stimulates the immune system. In one embodiment, the immunostimulatory composition according to this aspect of the invention is a conjugate of the immunostimulatory ORN and the adjuvant. In an embodiment according to this aspect of the invention, the immunostimulatory ORN is covalently bound to the adjuvant. In other modalities they are not conjugated. In one embodiment, the adjuvant is an TLR9 agonist. In one embodiment the adjuvant is an immunostimulatory CpG nucleic acid. The compositions of the invention may optionally include an antigen. Thus, in one aspect the invention provides a vaccine, wherein the vaccine includes an immunostimulatory ORN of the invention and an antigen. In one aspect, the invention provides a vaccine that includes a conjugate of an immunostimulatory ORN of the invention and an antigen. In one embodiment, the conjugate according to this aspect of the invention includes the immunostimulatory ORN covalently linked to the antigen. In other modalities, they are not conjugated. In different modalities, the antigen can be an antigen by itself. The antigen can be any antigen, including a cancer antigen, a microbial antigen or an allergen. In another aspect, the invention provides an immunostimulatory composition that includes a conjugate of an immunostimulatory ORN of the invention and a lipophilic moiety. In one embodiment, the immunostimulatory ORN is covalently linked to the lipophilic moiety. In one embodiment, the lipophilic moiety is selected from the group consisting of cholesteryl, palmityl and fatty acyl. In one embodiment, the lipophilic moiety is a cholesterol derivative, e.g. eg, cholesteryl. In one embodiment, the immunostimulatory ORN includes at least one deoxyribonucleotide. The at least one deoxyribonucleotide can generally be found anywhere outside the immunostimulatory RNA motif. In different embodiments, the at least one deoxyribonucleotide are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23 or 24 consecutive deoxyribonucleotides. The immunostimulatory ORN that includes non-consecutive deoxyribonucleotides is also contemplated in the invention. In different embodiments, the at least one deoxyribonucleotide is a 5 'end, a 3' end, or both a 5 'end and a 3' end of the immunostimulatory ORN. The at least one deoxyribonucleotide also corresponds to a DNA part of a DNA molecule: chimeric RNA. In one embodiment, a DNA component of the DNA: chimeric RNA molecules includes a CpG nucleic acid, i.e., a TLR9 agonist. In one embodiment, the DNA and RNA portions of the DNA: chimeric RNA molecule are covalently linked by an internucleotide phosphate linkage. In another embodiment the DNA and RNA portions of the DNA: chimeric RNA molecule are covalently linked by a linker, e.g. eg, a non-nucleotide connector. In one aspect, the invention provides an immunostimulatory composition that includes a covalently closed, partially single-stranded, dumbbell shaped nucleic acid molecule, wherein at least a single-stranded portion of the molecule includes an immunostimulatory RNA motif of the invention. In one aspect the invention provides a pharmaceutical composition that includes the composition of any of the foregoing aspects of the invention, associated with a delivery vehicle chosen from a cationic lipid, a liposome, a cochleate, a virosome, an immunostimulatory complex (ISCOM), a microparticle, a microsphere, a nanosphere, a unilamellar vesicle (LUV), a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, and a polycationic peptide, and optionally a pharmaceutically acceptable carrier. In an embodiment according to this aspect of the invention, the pharmaceutical composition includes an antigen. The ORN can be formulated in a nebulizer or an inhaler, such as a metered dose inhaler or a powder inhaler. In some embodiments, the ORN also includes an additional composition such as a chemotherapeutic agent, an antiviral agent, or a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be formulated for administration by injection or mucosa. Furthermore, in accordance with these and other aspects of the invention, in different embodiments the immunostimulatory ORN may optionally include at least one 5'-5 internucleotide linkage at least one 3'-3 'internucleotide linkage, at least one 5' internucleotide linkage. -5 'including a linking moiety, at least one 3'-3' internucleotide linkage including a linking moiety, or any combination thereof. The linking moiety in one embodiment is a non-nucleotide linking moiety. In accordance with these and other aspects of the invention, in different embodiments, the immunostimulatory ORN can optionally include at least one 2'-2 'internucleotide linkage, at least one 2'-3' internucleotide linkage, at least one 2'internetucleotide linkage. -5 'or any combination thereof. In a preferred embodiment the at least one 2'-2 'internucleotide linkage, at least one 2'-3' internucleotide linkage, or at least one 2'-5 'internucleotide linkage is outside the immunostimulatory RNA motif. Also according to these and other aspects of the invention, the immunostimulatory ORN in one embodiment includes at least one multiplier unit. Accordingly, in some embodiments the immunostimulatory ORN of the invention may have a branched structure. Branched compositions may include 3'-5, 5'-5 ', 3'-3', 2'-2 ', 2'-3', or 2'-5 'internucleotide bonds, in any combination. In one embodiment, the immunostimulatory ORN includes at least two multiplier units, resulting in a so-called dendrimer. In addition, in some embodiments the immunostimulatory ORN of the invention can include two or more immunostimulatory RNA motifs, for example arranged in tandem along a linear ORN, in different arms of a branched structure, or both in tandem along a linear ORN as in different arms of a branched structure. The branched structures, including the dendrimers, may optionally include at least one immunostimulatory CpG nucleic acid, for example as an arm separated from a branched structure. In addition, in accordance with these and other aspects of the invention, in one embodiment, the immunostimulatory ORN does not include an RNA or CG DNA dinucleotide. In one aspect, the invention provides a method for downregulating immunosuppressive CD4 + regulatory (Treg) cells. The method according to this aspect of the invention includes the step of contacting a CD4 + Treg cell with a composition containing the TLR8-specific immunostimulatory ORN of the invention in an amount effective to reduce the inhibitory effect of the CD4 + Treg cell. In one embodiment, the composition includes a TLR8 specific ORN and an immunostimulatory CpG nucleic acid, wherein the TLR8 specific ORN and the immunostimulatory CpG nucleic acid are not linked. In one embodiment, the composition includes a TLR8 specific ORN and an immunostimulatory CpG nucleic acid, wherein the TLR8 specific ORN and the immunostimulatory CpG nucleic acid are present in the form of a conjugate. In another aspect, the invention provides a method for modulating an immune response in a subject. The method according to this aspect of the invention includes the step of administering to a subject an effective amount of a composition of the invention. In some embodiments, the ORN may be delivered to the subject to treat an autoimmune disease or for remodeling the airway in a subject. The ORN can be administered with or without an antigen to the subject. Optionally, the ORN is provided by a route such as oral, nasal, sublingual, intravenous, subcutaneous, mucosal, respiratory, direct and dermal injection. The ORN can be delivered to the subject in an amount effective to induce the expression of cytokines, such as TNFa. IL-10, IL-6, IFN- ?, MCP1 and IL-12. In one aspect, the invention provides a method of vaccination of a subject. The method according to this aspect of the invention includes the step of administering to the subject an antigen and an immunostimulatory ORN of the invention. In one aspect, the invention provides a method for treating a subject that has or is at risk of having an infectious disease. The method according to this aspect of the invention includes the step of administering to the subject an effective amount of a composition of the invention. In one embodiment, the method includes the step of administering to the subject an effective amount of an immunostimulatory ORN of the invention. In one embodiment, the subject has a viral infection. The viral infection can be, for example, hepatitis B or hepatitis C. An antiviral agent can also be administered to the subject. Optionally the antiviral agent is linked to the ORN. In one aspect, the invention provides a method for treating a subject who has or is at risk of having cancer. The method according to this aspect of the invention includes the step of administering to the subject an effective amount of a composition of the invention. In one embodiment, the method includes the step of administering to the subject an effective amount of an immunostimulatory ORN of the invention. In one embodiment, the subject is also administered a chemotherapeutic or radiation product. In one aspect, the invention provides a method for treating a subject who has or is at risk of having cancer. The method according to this aspect of the invention includes the step of administering to the subject an effective amount of a composition containing a TLR8-specific immunostimulatory ORN of the invention to reduce the inhibitory effect of CD4 + Treg cells. In one embodiment, the composition includes a specific ORN of TLR8 and an immunostimulatory CpG nucleic acid, wherein the TLR8-specific ORN and the immunostimulatory CpG nucleic acid are not linked. In one embodiment, the composition includes a specific ORN of TLR8 and an immunostimulatory CpG nucleic acid, wherein the TLR8 specific ORN and the immunostimulatory CpG nucleic acid are present in the form of a conjugate. In one aspect, the invention provides a method for treating a subject who has or is at risk of having an allergic condition. The method according to this aspect of the invention includes the step of administering to the subject an effective amount of a composition of the invention. In one embodiment, the method includes the step of administering to the subject an effective amount of an immunostimulatory ORN of the invention. In one embodiment, the subject has allergic rhinitis. In one aspect, the invention provides a method for treating a subject who has or is at risk of having asthma. The method according to this aspect of the invention includes the step of administering to the subject an effective amount of a composition of the invention. In one embodiment, the method includes the step of administering to the subject an effective amount of an immunostimulatory ORN of the invention. In one modality, asthma is asthma exacerbated by a viral infection. ORN can be administered with or without an allergen.

In another aspect, the invention provides a method for treating a subject suffering from remodeling of the airways. The method according to this aspect of the invention includes the step of administering to the subject an effective amount of an immunostimulatory ORN of the invention. In one aspect, the invention provides a method for increasing antibody-dependent cellular cytotoxicity (ADCC). The method according to this aspect of the invention includes the step of administering to a subject in need of a higher ADCC, an effective amount of an immunostimulatory ORN of the invention and an antibody to increase ADCC. In one embodiment, the antibody is an antibody specific for a cancer antigen or other antigen expressed by a cancer cell. In one embodiment, the antibody is an IgG antibody. The invention in one aspect provides a method for enhancing epitope extension. The method according to this aspect of the invention includes the sequential steps of contacting a cell of the immune system with an antigen and subsequently contacting the cell with at least two doses of an immunostimulatory ORN of the invention. In one embodiment, the method is carried out in vivo. The method in one embodiment includes the steps of administering to a subject a vaccine that includes an antigen and an adjuvant and subsequently administering to the subject at least two doses of an immunostimulatory ORN of the invention, in an amount effective to induce epitope-specific immune responses. multiple The method in one embodiment involves applying a therapeutic protocol that results in exposure of the immune system to the antigen in a subject, followed by the administration of at least two doses of an immunostimulatory ORN of the invention, in an amount effective to induce immune responses. specific for multiple epitopes. In different modalities, the therapeutic protocol is surgery, radiation, chemotherapy, other medicines for cancer, a vaccine, or a vaccine for cancer. In one embodiment, the at least two doses of the immunostimulatory ORN are administered separately from each other for at least one day to one week. In one embodiment, the at least two doses of immunostimulatory ORN are administered separately from each other for at least one week to one month. In one embodiment, the at least two doses of immunostimulatory ORN are administered separately from each other for at least one month to six months. In one aspect, the invention is a method for stimulating the production of a proinflammatory cytokine, by contacting a cell expressing TLR8 with an RNA oligonucleotide (ORN) comprising, NU-R1-R2, wherein N is a ribonucleotide and N does not include U, U is uracil or a derivative thereof, and R is a ribonucleotide, wherein at least one of R1 and R2 is adenosine (A) or cytosine or derivatives thereof, and wherein R does not is U unless NU-R1-R2 includes at least two A, in which the ORN does not include a TLR7 / 8 motif and in which the ORN is 4-100 in length, in an effective amount to stimulate the production of proinflammatory cytokines, and in which the production of IFN-a in response to ORN is not significantly induced, with respect to the base value. In some embodiments, the production of IFN-a in response to ORN is less than 300 pg / ml. In one embodiment, the ORN is not ACCCAUCUAUUAUAUAACUC (SEQ ID NO .: 89). The ORN may or may not complex with N- [1 - (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium methyl sulfate (DOTAP). In some embodiments, the cell expressing TLR8 is a monocyte or a mDC. In other embodiments, the cell expressing TLR8 is in vi tro or in vivo. These and other features of the invention will now be described in more detail in relation to the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A-1 C are a set of graphs representing the production of cytokines induced by ORN after CMSP stimulation. Measuring the production of IFN-alpha and TNF-alpha cytokines, differences were seen between the motives of TLR8 and TLR7 / 8. Human PBMCs were stimulated with the indicated ORN (2 μ? With 1/3 dilution) complexed with DOTAP (25 μ9 / t t dilu dilution 1/3) or with R-848 (2 μ con with dilution 1/3) in a complete valuation curve. After 16 h the supernatants were collected and IFN-alpha (Figure 1 a) and TNF-alpha (Figure 1 b) were measured by ELISA. The data show the average of three blood donors from at least three independent experiments. DOTAP alone did not show any effect. The ORN forms complex with DOTAP, R-848 does not form complex.

DOTAP alone, it is a control. In Figure 1 c, human PBMCs were stimulated with 0.2 μ? of the indicated ORN complexed with DOTAP (2.2 μg / ml) or with R-848 (2 μ?). After 16 h, supernatants were collected and IFN-alpha (left panel) and TNF-alpha (right panel) were measured by ELISA. The data shown are the mean (± ETM) of 3 donors. Figs. 2A-2C are a set of bar graphs representing the production of cytokines induced by ORN after stimulation of pDC (Figure 2a), monocytes (Figure 2b) and mDC (Figure 2c) isolated. The cells were stimulated with ORN 0.5 μ? complexed with DOTAP 10 μg / mll ODN CpG 0.5 μ? or DOTAP or medium alone, and IFN-alpha (Figure 2a) and TNF-alpha (Figure 2b) and IL-12p40 (Figure 2c) were measured; Figs. 3A and 3B are a set of bar graphs representing the production of cytokines induced by ORN after stimulation of PBMC. Human PBMC were stimulated with the indicated ORN (ORN 0.5 μ?) Complexed with DOTAP 10 μg / ml, and IFN-alpha (Figure 3A) and TNF-alpha (Figure 3B) were measured and cytokine production was measured by the ELISA technique and compared with the Luminex technique; Figs. 4A and 4B are bar graphs showing a comparison of the maximal activities of IFN-alpha (Figure 4a) and TNF-alpha (Figure 4b) of the indicated ORN. Human PBMC were stimulated with ORN (7 concentrations, starting from 2 μ? With 1/3 dilutions) complexed with DOTAP (starting from 25 μg / ml with 1/3 dilutions) and the maximum mean activities were evaluated with 0.6 μm. from 3-6 blood donors in two individual experiments; Figs. 5A and 5B are bar graphs showing a comparison of the maximal activity of IFN-alpha (Figure 5a) versus EC50 of IFN-alpha (Figure 5b). Human PBMC were stimulated with ORN complexed with DOTAP and IFN-alpha was measured. Figs. 6A-6D are a set of graphs comparing titration curves for ORN with TLR8 (SEQ ID NO .: 13) or TLR7 / 8 (SEQ ID NO .: 21) for PBMC, isolated pDC or monocytes isolated from 3 blood donors . The cells were stimulated with ORN (4 concentrations, starting from 1 μ? With ¼ dilution) complexed with DOTAP (starting from 25 μg / m \ with ¼ dilutions). After 16 hours the supernatants were collected and the cytokine production was measured by the Luminex technique. The graphs show the production percentage of the cytokine of SEQ ID NO. 21 (with 0.3 μ?). Figs. 7A-7D show a set of bar graphs showing the maximum average activities at any concentration, of 3 blood donors for PBMC, isolated monocytes, isolated pDC and CD14-CD12-CMSP. Cells were stimulated with ORN (4 concentrations, starting from 1 μ? With VA dilution) complexed with DOTAP (starting from 25 μm / μm with VA dilutions). After 16 hours the supernatants were collected and the cytokine production was measured by the Luminex technique. The red squares indicate positive reactions against the base value of DOTAP and the medium; Figs. 8A-8D are a set of bar graphs showing the differences between the ORN of TLR8 (SEQ ID NO .: 13) and TLR7 / 8 (SEQ ID NO: 21). The cells were stimulated with ORN (4 concentrations, starting from 1 μ? With ¼ dilution) complexed with DOTAP (starting from 25 μg / m \ with ¼ dilutions). After 16 hours the supernatants were collected and the cytokine production was measured by the Luminex technique. The graph showed the mean maximum cytokine production measured at any concentration as a percentage of the ORN of TLR8 (SEQ ID NO: 13) with respect to the ORN of TLR7 / 8 (SEQ ID NO .: 21). This is shown for isolated pDC, PBMC, isolated monocytes and CMSP CD123-CD14-CMSP. Figs. 9A-9D is a set of bar graphs and curves showing the ORN reaction of TLR8 (SEQ ID NO .: 13) and TLR7 / 8 (SEQ ID NO: 21) acting by TLR8 on HEK-293 cells transfected stable. Stable transfected HEK-293 cells with NFKB and human TLR8 luciferase reading indicator gene, were stimulated for 16 hours with the indicated ORN. After 16 hours the supernatants were collected, and the cells were lysed and the luciferase activity or the cytokine level was measured. Figures 9a and 9b show the number of times of induction of luciferase N FKB after stimulation. Figure 9c shows the number of times of induction of luciferase NFKB after stimulation in the presence of inhibitors. Figure 9d shows the stimulation of IP-10 after the stimulus measured by the luciferase assay. Figs. 10A-10C are a series of graphs showing the expression of the surface marker after stimulation of human pDCs with AU-rich or GU-rich ORN. The purified CD123 + pDC (Figures 10a and 10b) or isolated monocytes (Figure 10c) were incubated with 1μN ORN? complexed with DOTAP 25 μg / ml or DOTAP alone (Figure 10a) or the indicated amounts of ORN complexed with DOTAP or DOTAP alone (Figures 10b-10c). After 16 h the cells were harvested and stained with CD123, CD1 1 c and HLA-DR antibodies (Figures 10a and 10b) or CD14 and CD19 (Figure 10c). Activation of the cell surface marker was measured by the expression of CD86 (Figures 10a and 10b) or CD80 (Figure 10c). Figure 10a shows the FACS analysis demonstrating that the AU-rich ORN (SEO ID NO .: 13) and the GU-rich ORN (SEQ ID NO .: 21) show differences in the expression of the CD86 surface marker after pDC stimulation. Figure 10b is a graph illustrating that expression of the CD86 surface marker after stimulation of human pDCs is dose dependent. Figure 10c is a graph showing that the AU-rich ORN (SEQ ID NO .: 13) and the GU-rich ORN (SEQ ID NO .: 21) show no difference in the expression of the CD80 surface marker after stimulation of human PBMCs (data not shown) and CD14-positive cells! Figs. 1 A-1 1 C are a set of bar graphs showing the differences between the ORN of TLR8 (SEQ ID NO .: 13) and the ORN of TLR7 / 8 (SEQ ID NO .: 21). The ORN of SEQ ID NO: 5 was used as a control. Bovine PBMCs were incubated with ORN (HD) 10 μ? / ??? OR ORN (LD) 2.5 μ ?? t ?? for 48 hours. The supernatants were collected and analyzed by ELISA. Figures 1 1 a-1 1 c show the level of IL-12, IFN-? and TNF-alpha, respectively; Figs. 12A-12H are a set of graphs showing that murine cells do not respond to the AU-rich ORN of SEQ ID NO: 13 in vivo or in vitro. The cells used were Raw264.7 cells (Figure 12a), J774 cells (Figure 12b), purified CD1 1 c + mouse cells (mouse sv129) (Figures 12c-12g) and mouse cells in vivo from the macrophage cell line of mouse. The concentration of cytokines was evaluated by ELISA. Fig. 13 is a graph showing that rat splenocytes do not respond to the AU-rich ORN of SEQ ID NO: 13. Splenocytes from 3 Sprague-Dawley rats were pooled and stimulated at the indicated concentrations of SEQ ID NO .: 21 , SEQ ID NO: 13 (both complexed with DOTAP 62.5 μg / p ^ \ with 1/5 dilution), R-848 or DOTAP alone (62.5 μg / ml, dilution> 1/5). The supernatants were collected after 20 hours and the TNF-alpha levels were measured by ELISA.

DETAILED DESCRIPTION OF THE INVENTION The invention relates in part to the discovery by the inventors of a series of sequence-specific immunostimulatory RNA motifs. It has now been discovered that molecules containing an immunostimulatory RNA motif are, alone or in combination with other determined components, important immunostimulatory compounds which have utility in a number of methods for treating subjects who have or are at risk of having a condition in the blood. that it would be advantageous to induce, increase or redirect an immune response. As used herein, in one embodiment, a nimostimulatory composition of the invention is an immunostimulatory ORN of the invention. It has been discovered that certain sequence-specific RNA motifs are immunostimulatory, acting through the TLR8, as opposed to other motifs (rich in GU and rich in UC) that act on TLR7 and TLR8. RNA oligonucleotides (ORN), which preferably contain AU-rich sequences, stimulate an immune response through TLR8. Production differences have been observed between IFN-alpha, TNF-alpha, IFN-gamma and IL-12 in these different classes of ORN, p. eg, the ORN that contains repetitions containing AU and GU. Interestingly, it has been found that the immunostimulatory ORN of the invention produces a strong proinflammatory cytokine response, with the exception of IFN-alpha and IFN-alpha related molecules. The production of IFN-alpha decreases or there is no after stimulation with these new ORN. According to some aspects of the invention the immunostimulatory RNA motif is N-U-R R2 N is a ribonucleotide and N does not include U. In some embodiments N is adenosine or cytosine (C) or derivatives thereof. U is uracil or a derivative thereof. R is a ribonucleotide in which at least one of Ri and R2 is adenosine (A) or cytosine or derivatives thereof. R is not U unless NU-RrR2 includes at least two A. The ORN of the invention includes at least one, and in some embodiments more than one (ie, 2, 3 or 4), immunostimulatory motifs, NU-R1- R2. ORNs do not include a TLR7 / 8 motive. The ORN is an oligonucleotide. Optionally, the oligonucleotide has a length of 4-100. The ORN may also have, for example, 8-40, 15-25 or 20-30 nucleotides in length. Optionally, the ORN includes at least one modification of the main chain. In some embodiments NU-R1-R2 may include at least 3 A or at least 2 C. Optionally, NU-R1-R2 includes at least one G or C. In some embodiments the ORN is not ACCCAUCUAUUAUAUAACUC (SEQ ID NO .: 89 ). The ORN may further comprise a pharmaceutically acceptable carrier which optionally is a lipid carrier such as N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium methyl sulfate (DOTAP). In other modalities, the ORN is not complexed with DOTAP. In other embodiments, the pharmaceutically acceptable carrier can be a peptide such as a polycationic peptide. Polycationic peptides include, for example, multiple polylysines, polyarginines and polypeptides containing more than 50% basic amino acids, especially arginine or lysine residues, in a range of more than 5, especially more than 8 amino acid residues or mixtures of they may include, for example, derivatives of a natural insect antimicrobial protein. In other modalities, the ORN includes at least one AU. In addition to being sequence specific, immunostimulatory RNA motifs are effective as single-stranded RNA, partially double-stranded RNA or fully double-stranded RNA. Clear differences were observed between the production of IFN-alpha and molecules related to IFN-alpha and other proinflammatory cytokines such as TNF-alpha, IFN-gamma, IL-10, IL-6 and IL-12, for the ORNs of the invention and ORNs that have a TLR7 / 8 motive, that is, repeats that contain GU. The ORNs of the invention having a N-U-Ri-R2 moiety, for example, those containing AU or AUU repeats (SEQ ID NO .: 12, SEQ ID NO .: 13) do not show production of the IFN-alpha cytokine after stimulation of PBMC and pDC. In contrast, ORNs having three and more U in a row (SEQ ID NO .: 14, SEQ ID NO .: 15) induced the production of IFN-alpha, despite the presence of A. It is interesting that using the same set of ORN but with G exchanged for A, a strong production of IFN-alpha was observed after the stimulation of CMSP. The data presented in this document strongly suggest the existence of two different classes of ORN: one that acts on cells expressing TLR8 such as monocytes and mDC (SEQ ID NO .: 12, SEQ ID NO .: 13, SEQ ID NO .: 16-SEQ ID NO .: 18), wherein the ORN contains the NUR R2 motifs of the invention and another which acts on cells expressing both TLR7 / 8 such as monocytes, mDC and pDC (SEQ ID NO .: 14, SEQ ID NO .: 15, SEQ ID NO: 19-SEQ ID NO.:23) which contain CU, GU and GUU sequences. Therefore, the ORNs of the invention have the ability to induce an immune response without inducing significant amounts of IFN-alpha or IFN-alpha related molecules relative to the base value. A significant amount of IFN-alpha or IFN-alpha related molecules with respect to the base value is preferably a change of less than 20% in the levels of IFN-alpha or IFN-alpha related molecules with respect to the base value. In some modalities, it is less than 1 5%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In other embodiments, the amount of IFN-alpha or IFN-alpha-related molecules that is induced is equivalent to the base value or less than the base value levels. In still other embodiments, the amount of IFN-alpha induced by the ORN of the invention is less than or equal to 20% of the IFN-alpha induced by an ORN of TLR7 / 8. The amount of IFN-alpha induced by the ORN of the invention may optionally be less than 300 pg / ml in an in vitro assay, or it may have an EC50 greater than 1.5. A molecule related to IFN-alpha, as used herein, is a cytokine or factor that is related to the expression of IFN-alpha. These molecules include, but are not limited to MIP1-beta, IP-10 and MlPI-alpha.

Recently it has been described that CD4 + Treg cells express TLR8 and that TLR8 signaling in these cells reduces or reverses their immunoinhibitory function. Peng G. et al., (2005) Science 309: 1380-4. Larger populations of CD4 + Treg cells have been observed in patients with different types of cancer, in whom immunosuppression may contribute to immune "evasion" and the unregulated growth of these cancers. Therefore, it would be expected that reversal of Treg-mediated suppression would be beneficial in the treatment of cancer. The ORN specifically excludes TLR7 / 8 motives. It has been discovered that the TLR7 / 8 motifs can produce dominant results that mask the unique immunostimulatory properties of the ORN of the invention. A TLR7 / 8 motif may include, for example, a ribonucleotide sequence such as 5'-C / UUG / UU-3 \ 5'-RURGY-3 ', 5'-GUUGB-3', 5'-GUGUG / U-3 \ or 5'-G / CUA / CGGCAC-3 '. C / U is cytosine (C) or uracil (U), G / U is guanine (G) or U, R is purine, Y is pyrimidine, B is U, G, or C, G / C is G or C, and A / C is adenine (A) or C. The sequence 5C / UUG / UU-3 'can be CUGU, CUUU, UUGU, or UUUU. In different modalities 5'-R-U-R-G-Y-3 * is GUAGU, GUAGC, GUGGU, GUGGC, AUAGU, AUAGC, AUGGU, or AUGGC. In one modality the base sequence is GUAGUGU. In different modalities 5'-G-U-U-G-B-3 'is GUUGU, GUUGG, or GUUGC In different modalities 5'-G-U-G-U-G / U-3' is GUGUG or GUGUU. In one modality the base sequence is GUGUUUAC. In different embodiments 5'-G / C-U-A / C-G-G-C-A-C-3 'is GUAGGCAC, GUCGGCAC, CUAGGCAC, or CUCGGCAC. The invention relates generally to immunostimulatory oligoribonucleotides that include one or more immunostimulatory RNA motifs, immunostimulatory compositions containing one or more immunostimulatory ORNs of the invention, and methods for using the immunostimulatory ORN and immunostimulatory compositions of the invention. As used herein, the terms "RNA" and equivalently "natural RNA" refer to two or more ribonucleotides (i.e., molecules each comprising a ribose sugar attached to a phosphate group and a base). purine or pyrimidine nitrogenous (eg, guanine, adenine, cytosine or uracil)) covalently bonded together by bond or 3'-5'-phosphodiester linkages. The immunostimulatory RNA motif can be found at the end of the immunostimulatory ORN (when the immunostimulatory ORN has free ends). For example, an immunostimulatory ORN with free ends and the immunostimulatory RNA motif placed at the end of the immunostimulatory ORN may be represented as XaM or as MXb, wherein M represents the immunostimulatory RNA motif and each of Xa and Xb independently represents one or more identical or different nucleotides of the immunostimulatory ORN exclusive of the immunostimulatory RNA motif.

Alternatively, the immunostimulatory RNA motif can be flanked at both ends by at least one additional nucleotide of the immunostimulatory ORN, whether or not the immunostimulatory ORN has free ends. For example, an immunostimulatory ORN with free ends and nucleotides flanking the immunostimulatory RNA motif can be represented as XaMXb, wherein M represents the immunostimulatory RNA motif and each of Xa and Xb independently represents one or more identical or different nucleotides. of the immunostimulatory ORN exclusive of the immunostimulatory RNA motif. In different embodiments, the immunostimulatory ORN that includes the immunostimulatory RNA motif may include a single motif or more than one immunostimulatory RNA motif. It is believed that it may be advantageous to have two or more immunostimulatory RNA motifs in a single immunostimulatory ORN, for example if the motifs are spaced so that the immunostimulatory ORN can bind two or more TLRs. For example, the immunostimulatory ORN could bind two or more TLR8 receptors by amplifying or modifying the resulting immunostimulatory effect. When the immunostimulatory ORN includes more than one immunostimulatory RNA motif, the immunostimulatory ORN can be represented in a modality such as M-iXM2, in which M-? and M2 each independently represents an immunostimulatory RNA motif and X represents one or more identical or different nucleotides of the immunostimulatory ORN unique to the immunostimulatory RNA motifs. In one embodiment X includes a non-nucleotide linker as described herein. In one embodiment, X includes a branch unit as described herein. When there is more than one immunostimulatory RNA motif in the immunostimulatory ORN, the motifs in general can be found at any position along the immunostimulatory ORN. For example, when there are two reasons, each can be found at one end of the immunostimulatory ORN. Alternatively, a motif may be found at one end and a motif may be flanked at both ends by at least one additional nucleotide of the immunostimulatory ORN. In yet another embodiment, each motif can be flanked at both ends by at least one additional nucleotide of the immunostimulatory ORN. The immunostimulatory ORN includes, but is not limited to the following, shown from 5 'to 3', read from left to right: In some embodiments, the ORN is one of the active ORNs shown in the following tables 1 and 2, such as the following: U * U * A * G * G * C * A * C (SEQ ID NO: 2), A * U * A * G * G * C * A * C (SEQ ID O: 4) 5 G * C * C * A * C * C * G * A * G * C * C * GV ^ A * U * A * U * A * C * C (SEQ ID N0: 1 1), A * U * A * U * A * U * A * U * A * U * A * U * A * U * A * U * A * U * A * U (SEQ ID N0: 12), U * U * A * U * U * A * AJ * A * U * U * A * U * U * A * U + U * Anj * U (SEQ ID NO: 13), A * A * U * A * A * U * A * A * U * A * A * U * A * A * U * A * A * U > I'A ,, 'A (SEQ N0; 16), A * A * A * U * A * A * Anj * A * A * A * U * A * A * A * U * A * A * A * U (SEQ ID NO: 17), A * A * A * A * U * A + A * A * A * U * A * A * A * A * U * A * A * A * A * \ J (SEQ ID NO: l 8), C * U + A * C * U * A * C + U * A * C * U * A * C * U * A * C * U * A + C * U (SEQ ID NO: 24), U * U * A * U * U * A * U (SEQ ID ??: 30), U * A * U * A * U * A * U (SEQ ID NO: 33) SC * C * G * A * G * C * C * G * C * A * U * U * A * C * C * C (SEQ ID NO: 48), C * C * G * A * G * C * C * G * A * U * U * G * A * A * C * C (SEQ ID NO: 76), C * C * G * A * G * C * C * G * A * A * U * A * C * C * C * C (SEQ ID NO: 42), C * C * G * A * G * C * C * A * U * A * U * A * U * A * U * C (SEQ ID NO: 39), C * C * G * A * G * C * C * G ^ * U * A * U * U * A * C * C (SEQ ID NO: 65), C * C * G * A * G * C * C * G * A * A * U * C * C * C * C * C (SEQ ID O: 44), C * C * G * A * G * C * C * G * C * C * U * A * C * C * C * C (SEQ ID NO: 47), C * C * G * A * G * C * C * A * U * A * U * A * U * C * C * C (SEQ ID NO: 38), C * C * G * A * G * C * C * G * C * U * An * A * C * C * C (SEQ ID NO: 37), C * C * G * A * G * C * C * G * A * A * U * A * A * C * C * C (SEQ ID NO: 40), C * C * G * A * G * C * C * G * C * U * A * U * C * C * C * C (SEQ] D NO: 55), C * C * G * A * G * C * C * G * A * A * G * G * U * A * C * C (SEQ ID NO: 82), C + C * G * A * G * C * C * G * A * A * G * A * U * A * C * C (SEQ ID NO: 85), C * C * G * A * G * C * C * G * A * A * U * G * U * A * C * C ( SEQ JD NO: 63), C * C * G * A * G * C * C * G * C * C * U * A * A * C * C * C (SEQ 1D NO: 43), C * C * G * A * G * C * C * G * C * A * U * A * U * C * C * C (SEQ ID NO: 36), C * C * G * A * G * C * C * G * A * A * G * C * U * A * C * C (SEQ 1D NO: 87), C * C * G * A * Gíl'C * C * G * C * A * U * A'i, C * C * C5, < C (SEQ 1D NO: 45), C * C * G * A * G * C * C * G * C * A * U * A * A * C * C * C (SEQ ID N0: 41), C * C * G * A * G * C * C * G * A * A * G * G * U * G * C * C (SEQ ID NO: 83), C * C;) tG3, 'A * G! TC < C ,,, G * C * A * U:,: C * C + C * C * C (SEQ 1 N0: 46), C * C * G * A * G * C * C * G * A + A + G * C * U * G * C * C (SEQ ID NO: 88), C * CH.G * A * G + C * C + G * C + C * G * C + C * C * C + C (SEQ ID O-.35), C * C * G * A * G * C * C * G * A * A * G * C * O * C * C * C (SEQ ID NO: 84), or C * C * G * A * G * C * C * G * A * A * G * G * C + A * C * C (SEQ JD NO: 56).

As mentioned before, RNA is a polymer of Ribonucleotides linked by 3'-5'-phosphodiester bonds. In some modalities, the immunostimulatory ORNs of the invention are RNA. However, the ORN immunostimulants of the invention are not limited to RNA, as describe below. An immunostimulatory ORN of the invention, in a modality may include one or more modified nitrogenous bases, ie derivatives of A, C, G and U. The specific modalities of these nitrogenous bases modified include, but are not limited to, 5-substituted cytosines (eg 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodocytosine, 5-hydroxy - cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and substituted or unsubstituted 5-alkynyl-cytosine), substituted cytosines in 6, cytosines substituted in N4 (eg, N4-ethyl-cytosine ), 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogues with fused ring systems (eg?,? '- propylene cytosine or phenoxazine), and uracil and its derivatives (eg 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil), thymine derivatives (e.g. , 2-thiothimine, 4-thiothimine, thymines substituted in 6), guanosine derivatives (7-deazaguanine, 7-deaza-7-substituted-guanine (such as 7-deaza-7-alkynyl (C2-C6) guanine), 7-deaza-8-substituted-guanine, hypoxanthine, guanines substituted on N2 (eg, N2-methyl-guanine), gua child substituted in 8 (p. eg, 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine), or adenosine derivatives (5-amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidine-2,7-dione , 2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substituted adenines (eg, N6-methyl-adenine, 8-oxo-adenine)). The base can also be replaced by a universal base (eg, 4-methyl-indole, 5-nitro-indole, 3-nitropyrrole, P-base, and K-base), an aromatic ring system (e.g. , benzimidazole or dichloro-benzimidazole, 1-methyl-1 H- [1, 2,4] triazole-3-carboxylic acid amide), an aromatic ring system (eg, fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer). The preferred base modifications are uracil and 7-deaza-guanine. These modified U nitrogenous bases and their corresponding ribonucleosides are available from commercial suppliers.

Specific embodiments of modified nitrogenous bases of G include N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted-guanine, 7-deaza-7-alkynyl (C2-C6) -guanine, 7- 8-substituted-guanine deaza, 8-hydroxyguanine, 6-thioguanine and 8-oxoguanine. In one embodiment, the nitrogenous base of modified G is 8-hydroxyguanine. These nitrogenous bases of modified G and their corresponding ribonucleosides are available from commercial suppliers. In some embodiments, at least one unit of β-ribose may be substituted by β-D-deoxyribose or a modified sugar unit, wherein the modified sugar unit is selected, for example from β-D-ribose, aD- ribose, ß-L-ribose (as in "Spiegelmers"), α-ribose, 2'-amino-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-alkyl (C1-) C6) -ribose, preferably 2'-0-alkyl (C1-C6) -ribose is 2'-0-methyl-ribose, 2 -0-alkenyl (C2-C6) -ribose, 2 '- [O-alkyl ( C1-C6) -O-alkyl (C1-C6)] - ribose, LNA and α-LNA (Nielsen P. et al (2002) Chemistry-A European Journal 8: 712-22), ß-D-xyl- furanose, α-arabinofuranose, 2'-fluoro-arabinofuranose and carbocyclic and / or open-chain sugar analogs (described, for example, by Vandendriessche et al (1993) Tetrahedron 49: 7223) and / or bicyclic sugar analogues ( described, for example, by Tarkov M. et al (1993) Helv. Chim. Acta 76: 481). The individual ribonucleotides and ribonucleotides of the immunostimulatory ORNs of the invention may alternatively be linked by non-nucleotide linkers, in particular non-base linkers (dSpacers), triethylene glycol units or hexaethylene glycol units. Additional connectors are the alkylamino linkers, such as C3, C6 and C12 amino connectors, and also alkylthiol linkers, such as C3 or C6 thiol linkers. The individual nucleotides and ribonucleosides of the immunostimulatory ORN of the invention may alternatively be linked by aromatic moieties which may be further substituted by substituted alkyl or alkyl groups. RNA is a polymer of ribonucleotides linked by 3'-5'-phosphodiester bonds. The nucleotides of the immunostimulatory ORN of the invention can also be linked by 3'-5'-phosphodiester bonds. However, the invention also encompasses ORN immunostimulators having non-customary internucleotide linkages, specifically including the 5'-5 \ 3'-3 ', 2'-2', 2'-3 'and 2'-5' internucleotide linkages. In one embodiment said non-routine linkages are excluded from the immunostimulatory RNA motif, even though one or more of said links may be found elsewhere in the immunostimulatory ORN. For the immunostimulatory ORN having free ends, the inclusion of a 3'-3 'internucleotide linkage can result in an immunostimulatory ORN having two free 5' ends. Conversely, for the immunostimulatory ORN having free ends, the inclusion of a 5'-5 'internucleotide linkage can result in an immunostimulatory ORN having two free 3' ends. An immunostimulatory composition of this invention may contain two or more immunostimulatory RNA motifs that may be linked by a branching unit. The internucleotide linkages can be 3'-5 ', 5'-5', 3'-3 ', 2'-2', 2'-3 'or 2'-5' bonds. Accordingly, the nomenclature 2'-5 'is chosen according to the carbon atom of the ribose. The non-usual internucleotide linkage can be a phosphodiester link, but alternatively it can be modified as phosphorothioate or any other modified linkage as described herein. The following formula shows a general structure for branched immunostimulatory ORNs of the invention by a nucleotide branching unit. In this way, Nu- ?, Nu2 and Nu3 can be linked by 3'-5 ', 5'-5', 3'-3 ', 2'-2', 2'-3 ', or 2'- bonds. 5'. The branching of the immunostimulatory ORN may also involve the use of non-nucleotide connectors and non-base spacers. In a Nu <Nu2 and Nu3 modality, they represent identical or different immunostimulatory RNA motifs. In another Nu-i embodiment, Nu2 and Nu3 comprise at least one immunostimulatory RNA motif and at least one immunostimulatory CpG DNA motif.

Nu3 X2 The immunostimulatory ORN may contain a duplicating or tripling unit (Glen Research, Sterling, VA), in particular the immunostimulatory ORNs with a 3'-3 'link. A duplicating unit in one embodiment can be based on 1,3-bis- [5- (4,4'-dimethoxytrityloxy) pentylamido] propyl-2 - [(2-cyanoethyl) - (N, N-diisopropyl)] - phosphoramidite . A tripling unit in one embodiment can be based on the incorporation of tris-2,2,2- [3- (4,4'-dimethoxytrityloxy) propyloxymethyl] ethyl - [(2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite. The branching of the immunostimulatory ORN by multiple duplicating, tripling or other multiplying units leads to the dendrimers which are a further embodiment of this invention. The branched immunostimulatory ORNs can lead to the cross-linking of receptors for the immuno-stimulatory RNA such as TLR3, TLR7 and TLR8, with different immune effects compared to the non-branched forms of the immunostimulatory ORN. In addition, the synthesis of branched immunostimulatory ORNs or otherwise multimers can stabilize RNA against degradation and can allow weak or partially effective RNA sequences to exert a therapeutically useful level of immune activity. The immunostimulatory ORNs can also contain linker units resulting from peptide modifying reagents or oligonucleotide modifying reagents (Glen Research). In addition, immunostimulatory ORNs may contain one or more natural or non-natural amino acid residues that are connected to the polymer by peptide bonds (amide). The internucleotide links 3'-5 ', 5'-5', 3'-3 \ 2'-2 ', 2'-3"and 2 -5' can be direct or indirect, direct links in this context refer to to phosphate or modified phosphate linkages as described herein, without an intermediate linker moiety An intermediate linker moiety is an organic moiety other than a modified phosphate or phosphate linkage as described herein, which may include, for example , polyethylene glycol, triethylene glycol, hexaethylene glycol, dSpacer (ie a deoxynucleotide without base), duplicator unit or triplicate unit In some embodiments, the immunostimulatory ORN is conjugated to another entity to provide a conjugate As used herein, a conjugate refers to a combination of any two or more entities linked together by any physico-chemical means, including hydrophobic interaction and covalent coupling.In another embodiment, the immunostimulatory ORN can and being conjugated to a low molecular weight ligand that is recognized by an immunomodulatory receptor. This receptor is preferably a member of the TLR family, such as TLR2, TLR3, TLR4, TLR7, TLR8 or TLR9. The ligands of low molecular weight mimic the natural ligands of these receptors. Examples include, but are not limited to, R-848 (Resiquimod), R-837 (Imiquimod, ALDARA ™, 3M Pharmaceuticals), 7-deaza-guanosine, 7-thia-8-oxo-guanosine, and 7-allyl- 8-oxo-guansosine (Loxoribine) that stimulate TLR7 or TLR8. D-glucopyranose derivatives, such as 3D-MPL (TLR4 ligand), may also be conjugated to the immunostimulatory ORN. Pam3-Cys is an example of a TLR2 ligand that can be conjugated with the immunostimulatory ORN. Oligodeoxynucleotides containing CpG motifs are ligands of TLR9, and these can also be conjugated with the immunostimulatory ORN of the invention. In one embodiment, at least one oligodeoxynucleotide comprising a CpG motif effective to stimulate TLR9 signaling is conjugated to an immunostimulatory ORN of the invention. The conjugation of ligands for different TLRs in a molecule can lead to multimerization of receptors resulting in enhanced immune stimulation or an immunostimulatory profile different from that resulting from any of said individual ligands.

In one aspect, the invention provides a conjugate of an immunostimulatory ORN of the invention and a lipophilic moiety. In some embodiments, the immunostimulatory ORN is covalently linked to a lipophilic moiety. The lipophilic moiety will generally be found at one or more ends of an immunostimulatory ORN having free ends, although in some embodiments the lipophilic moiety can be found anywhere along the immunostimulatory ORN, and thus it is not necessary that the immunostimulatory ORN have a free end. In one embodiment, the immunostimulatory ORN has a 3 'end and the lipophilic moiety is covalently linked to the 3' end. The lipophilic group in general may be a cholesteryl, a modified cholesteryl, a cholesterol derivative, a reduced cholesterol, a substituted cholesterol, cholestane, C16 alkyl chain, a bile acid, cholic acid, taurocholic acid, deoxycholate, oleyl-lithocholic acid , oleoyl-coenic acid, a glycolipid, a phospholipid, a sphingolipid, an isoprenoid, such as spheroids, vitamins, such as vitamin E, saturated fatty acids, unsaturated fatty acids, fatty acid esters, such as triglycerides, pyrenes, porphyrins, texaphyrin, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas red, digoxigenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (eg, Cy3 or Cy5), Hoechst 33258 dye, psoralen or ibuprofen . In some embodiments, the lipophilic moiety is selected from cholesteryl, palmityl and fatty acyl. In one embodiment, the lipophilic moiety is cholesteryl. It is believed that the inclusion of one or more of said lipophilic moieties in the immunostimulatory ORN of the invention confers them an additional stability against degradation by nucleases. When there are two or more lipophilic moieties in a single immunostimulatory ORN of the invention, each lipophilic moiety can be selected independently of any other. In one embodiment, the lipophilic group is linked to a 2 'position of an immunostimulatory ORN nucleotide. A lipophilic group may be linked alternatively or in addition to a heterocyclic nitrogenous base of an immunostimulatory ORN nucleotide. The lipophilic moiety can be covalently linked to the immunostimulatory ORN by any suitable direct or indirect linkage. In one embodiment, the bond is direct and is an ester or an amide. In one embodiment, the linkage is indirect and includes a spacer moiety, for example one or more non-basic nucleotide moieties, oligoethylene glycol, such as triethylene glycol (spacer 9) or hexaethylene glycol (spacer 18), or an alkanediol, such as butanediol. In one embodiment, the immunostimulatory ORN of the invention is advantageously combined with a cationic lipid or a cationic peptide. It is believed that cationic lipids and cationic peptides assist trafficking of the immunostimulatory ORN to the endosomal compartment, in which the TLR8 is located. In one embodiment, the cationic lipid is DOTAP (N- [1 - (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium methyl sulfate). It is believed that DOTAP transports the RNA oligomer within the cells and specifically leads it to the endosomal compartment, where it can release the RNA oligomer in a pH-dependent manner. Once in the endosomal compartment, the RNA can interact with some intracellular TLRs, triggering the transduction pathways of TLR-mediated signals involved in the generation of an immune response. Other agents with similar properties, including driving to the endosomal compartment, can be used instead of or in addition to DOTAP. Other lipid formulations include, for example, EFFECTENE ™ (a non-liposomal lipid with a special DNA condensation enhancer) and SUPERFECT ™ (a new action dendrimer technology). Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN ™ and LIPOFECTACE ™, which are formed from cationic lipids such as N- [1- (2, 3-dioleyloxy) -propyl] -N, N, N-trimethylammonium (DOTMA) and dimethyl-dioctadecylammonium bromide (DDAB). Methods for preparing liposomes are known in the art and have been described in many publications. Liposomes have also been reviewed by Gregoriadis G. (1985) Trends Biotechnol 3: 235-241. In one embodiment, the immunostimulatory ORNs of the invention are in the form of covalently closed, weight-shaped molecules with both primary and secondary structure. As described below, in one embodiment, said cyclic oligoribonucleotides include two single-stranded loops connected by an intermediate double-stranded segment. In one embodiment, at least one single-stranded loop includes an immunostimulatory RNA motif of the invention. Other covalently closed, weight-like molecules of the invention include chimeric DNA: RNA molecules in which, for example, the double-stranded segment is at least partially DNA (e.g., dsDNA homodimer or DNA: heterodimeric RNA) and at least one single-stranded loop includes an immunostimulatory RNA motif of the invention. Alternatively, the double-stranded segment of the chimeric molecule is RNA. In some modalities, the immunostimulatory ORN is isolated.

An isolated molecule is a molecule that is substantially pure and has no other substances with which it is normally found in nature or in vivo systems, at a practical level and suitable for the intended use. In particular, immunostimulatory NMRs are sufficiently pure and sufficiently free of other biological constituents of cells to be useful, for example, in producing pharmaceutical formulations. Since an isolated immunostimulatory ORN of the invention can be mixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the immunostimulant ORN can comprise only a small percentage by weight of the preparation. However, the immunostimulatory ORN is substantially pure in that it has substantially separated from the substances with which it may be associated in living systems. For use in the present invention, the immunostimulatory ORN of the invention can be synthesized again using or by adapting any of a number of methods well known in the art. For example, the β-cyanoethyl phosphoramidite method (Beaucage S.L. et al (1981) Tetrahedron Lett 22: 1859); nucleoside H-phosphonate method (Garegg P. et al (1986) Tetrahedron Lett 27: 4051-4; Froehler BC et al., (1986) Nuci Acid Res. 14: 5399-407; Garegg P. et al. (1986) Tetrahedron Lett 27: 4055-8, Gaffney BL et al (1988) Tetrahedron Lett 29: 2619-22). These chemical methods can be carried out by a variety of automated nucleic acid synthesizers available in the market. Additional useful synthesis methods according to the present invention are described in Uhlmann E. et al. (1990) Chem. Rev. 90: 544-84, and Goodchild J. (1990) Bioconjugate Chem. 1: 165. The synthesis of oligoribonucleotides can be carried out in solution or in a solid phase support. In solution, block coupling reactions (dimers, trimers, tetramers, etc.) are preferred, while solid phase synthesis is preferably carried out in a stepwise method using monomeric building blocks. Different chemical methods have been described, such as the phosphotriester method, the H-phosphonate method and the phosphoramidite method (Eckstein F. (1991) Oligonucleotides and Analogues, A Practical Approach, IRL Press, Oxford). While the phosphotriester method the reactive phosphorus group is in the + V oxidation state, the phosphorus + III derivatives which are more reactive are used in the coupling reactions according to the methods of phosphoramidite and H-phosphonate. In the latter two methods, the phosphorus is oxidized after the coupling step to give the stable P (V) derivatives. If the oxidant is iodine / water / base, then the phosphodiesters are obtained after deprotection. In contrast, if the oxidant is a sulfurizing agent, such as the Beaucage reagent, then the phosphorothioates are obtained after deprotection. An effective method for the synthesis of oligoribonucleotides is the combination of the synthesis in solid support using the chemistry of the phosphoramidite as originally described for oligodeoxynucleotides Matteucci and Caruthers. Matteucci M.D. and col. (1981) J. Am. Chem. Soc. 103: 3185. The synthesis of oligoribonucleotides is similar to oligodeoxynucleotides, with the difference that the 2'-hydroxy group present in the oligoribonucleotides must be protected by a suitable hydroxy protecting group. The monomers can be protected, for example, with the 2-O-t-butyldimethylsilyl group (TBDMS) in monomeric RNA building blocks. However, it has been reported that RNA synthesis using monomers containing the 2'-O-triisopropylsilyloxymethyl (TOM) group (TOM ™ Protective Group) produces a higher coupling efficiency because the TOM protecting group has less steric hindrance than the group TBDMS. While the TBDMS protecting group is removed using fluoride, rapid deprotection is achieved for the TOM group using methylamine in ethanol / water at room temperature. In the synthesis of oligo (rribo) nucleotides, chain elongation from 3 'to 5' is preferred, which is achieved by coupling a ribonucleotide unit having a 3'-phosphorus group (III) or its activated derivative with a 5'-hydroxy group free from another nucleotide unit.

The synthesis can be conveniently carried out using an automated DNA / RNA synthesizer. In this way, synthesis cyclones can be used as recommended by the providers of the synthesizers. For the ribonucleoside-phosphoramidite monomers, the coupling times are longer (eg, 400 s) compared to the deoxynucleoside monomers. As a solid support, the controlled pore glass support (CPG) of 500 to 1000 or an organic polymer support, such as the primer support PS200 (Amersham) can be used. The solid support normally contains the first nucleoside, such as 5'-O-dimethoxytrityl-N-6-benzoyladenosine, attached at its 3 'end. After cleaving the 5'-O-dimethoxytrityl group with trichloroacetic acid, elongation of the chain is achieved using, e.g. eg, 5'-0-dimethoxytrityl-N-protected-2'-0-tert-butyldimethylsilyl-nucleoside-3'-O-phosphoramidites. After successive repetitive cycles the completed oligonucleotide is cleaved from the support and deprotected by treatment with concentrated ammonia / ethanol (3: 1, v: v) for 24 h at 30 ° C. The TBDMS blocking group is finally cleaved using triethylamine / HF. The crude oligoribonucleotides can be purified by ion-exchange high pressure liquid chromatography (HPLC), ion pair reverse phase HPLC, or polyacrylamide gel electrophoresis (PAGE), and characterized by mass spectrometry. The synthesis of the 5 'conjugates is done directly by coupling a phosphoramidite of the molecule to be ligated with the 5'-hydroxy group of the terminal nucleotide in the solid phase synthesis. A variety of phosphoramidite derivatives of said ligands are available in commerce, such as cholesterol, acridine, biotin, psoralen, ethylene glycol or aminoalkyl moieties. Alternatively, aminoalkyl functions can be introduced during solid phase synthesis that allows post-synthesis derivatization by activated conjugate molecules, such as active esters, isothiocyanates or iodoacetamides. The synthesis of the conjugates by the 3 'end is normally achieved by using the correspondingly modified solid supports, such as, for example. eg, cholesterol-derivatized solid supports available commercially. However, conjugation can also be done in internucleotide bonds, nitrogenous bases or in the ribose residues, such as in the 2 'position of the ribose. For cyclic oligonucleotides, the elongation of the oligonucleotide chain can be carried out on the solid support Nucletoide PS (Glen Research) using the standard chemistry of the phosphoramidite. The cyclization reaction is then carried out on the solid support using a phosphotriester coupling method (Alazzouzi et al (1997) Nucleosides Nucleotides 16: 1513-14). In the final deprotection with ammonium hydroxide, almost the only product going to the solution is the desired cyclic oligonucleotide. The cyclic oligonucleotides of the invention include closed circular RNA forms and can include single-stranded RNA with or without double-stranded RNA. For example, in one embodiment the cyclic oligoribonucleotide includes double-stranded RNA and takes a weight conformation with the two single-stranded loops connected by an intermediate double-stranded segment. The covalently closed CpG-shaped oligodeoxynucleotides have been described in the U.S. patent. No. 6,849,725. In another embodiment, the cyclic oligoribonucleotide includes the double-stranded RNA and takes a conformation with three or more single-stranded loops connected by intermediate double-stranded segments. In one embodiment, an immunostimulatory RNA motif is located in one or more single-stranded segments. The immunostimulatory ORNs of the invention are useful alone or in combination with other agents, as adjuvants. An adjuvant as used herein refers to a substance other than an antigen that enhances activation of the immune cell in response to antigen., p. eg, a humoral and / or cellular immune response. The adjuvants promote the accumulation and / or activation of accessory cells to enhance the antigen-specific immune responses. The adjuvants are used to enhance the efficacy of the vaccines, i.e., the antigen-containing compositions are used to induce protective immunity against the antigen. Adjuvants in general include adjuvants that create a depot effect, immunostimulatory adjuvants and adjuvants that create a depot effect and stimulate the immune system. An adjuvant that creates a depot effect as used herein, is an adjuvant that causes the antigen to be slowly released into the body, thereby prolonging the exposure of immune cells to the antigen. This class of adjuvants includes, but is not limited to, alum (eg, aluminum hydroxide, aluminum phosphate); emulsion-based formulations that include mineral oil, non-mineral oil, water-in-oil or water-in-oil emulsion, oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants (eg, Montanide ISA 720; AirLiquide , Paris France); MF-59 (an emulsion of squalene in water stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.); and PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent, IDEC Pharmaceuticals Corporation, San Diego, Calif.). An immunostimulatory adjuvant is an adjuvant that produces the activation of a cell of the immune system. For example, it can cause an immune cell to produce and secrete cytokines. This class of adjuvants includes, but is not limited to, purified saponins from the bark of the Q. saponaria tree, such as QS21 (a glycolipid eluting at peak 21 with HPLC fractionation, Aquila Biopharmaceuticals, Inc., Worcester, Mass. ); poly [di (carboxylatophenoxy) phosphazene (PCPP polymer; Virus Research Institute, E.U.A.); lipopolysaccharide derivatives such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and dipeptide threonyl-muramyl (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A, OM Pharma SA, Meyrin, Switzerland); and the elongation factor of Leishmania (a purified Leishmania protein, Corixa Corporation, Seattle, Wash.). This class of adjuvants also includes CpG DNA. Adjuvants that create a reservoir effect and stimulate the immune system are the compounds that have the two functions identified above. This class of adjuvants includes, but is not limited to, ISCOMS (immunostimulatory complexes containing mixed saponins, lipids, and form virus-sized particles with pores that can contain the antigen; CSL, Melbourne, Australia); SB-AS2 (SmithKIine Beecham adjuvant system No. 2 which is an oil in water emulsion containing MPL and QS21: SmithKine Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKIine Beecham adjuvant system No. 4 containing alum and MPL; SBB, Belgium); nonionic block copolymers forming micelles such as CRL 1005 (these contain a hydrophobic polyoxypropylene linear chain flanked by polyoxyethylene chains; Vaxcel, Inc., Norcross, Ga.); and the Syntex Adjuvant formulation (SAF, an oil in water emulsion containing Tween 80 and a non-ionic block copolymer, Syntex Chemicals, Inc., Boulder, Col.). The invention in one aspect provides an adjuvant that includes an immunostimulatory ORN of the invention, by itself. In another embodiment, the invention provides an adjuvant that includes an immunostimulatory ORN of the invention and at least one other adjuvant (a combination of adjuvants). The other adjuvant may include an adjuvant that creates a depot effect, an immunostimulatory adjuvant, an adjuvant that creates a depot effect and stimulates the immune system, and a combination thereof. In one embodiment, the immunostimulatory ORN of the invention and at least one other adjuvant are covalently linked together. A combination of adjuvants according to the invention may have a synergistic immunostimulatory effect compared to the sum of the effects of the immunostimulatory ORN alone and the at least one other adjuvant alone. Additionally or alternatively, a combination of adjuvants according to the invention may have an altered immunostimulatory profile compared to that of the immunostimulatory ORN alone or the at least one other adjuvant alone. For example, the combination of adjuvants may provide a more balanced form of Th1 / Th2 immunostimulation in one embodiment, or may provide a more unbalanced form of Th1 / Th2 immunostimulation in another embodiment. Those skilled in the art will recognize how to select the individual components to promote a desired type of immunostimulation., p. eg, more balanced or more unbalanced with respect to the character of Th1 and Th2. Th1 and Th2 are described in more detail below. Also provided is a composition that includes an immunostimulatory ORN of the invention plus another adjuvant, wherein the other adjuvant is a cytokine. In one embodiment, the composition is a conjugate of the immunostimulatory ORN of the invention and the cytokine. Cytokines are proteins and soluble glycoproteins produced by many types of cells that mediate inflammatory and immune reactions. Cytokines mediate the communication between cells of the immune system, acting locally as well as systemically to recry cells and regulate their function and proliferation. The categories of cytokines include mediators and regulators of innate immunity, mediators and regulators of acquired immunity and stimulators of hematopoiesis. Interleukins are included among the cytokines (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-1 1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, and interleukins 19-32 (IL-19-IL-32), among others), chemokines (eg, ÍP-10, RANTES, ??? - 1 a, ??? - 1 ß, ??? - 3a, MCP-1, MCP-2, MCP-3, MCP -4, eotaxin, l-TAC, and BCA-1, among others), as well as other cytokines including type 1 interferons (eg, IFN-a and IFN-β), type 2 interferon (eg. , IFN-?), Tumor necrosis factor alpha (TNF-a), transforming grofactor beta (TGF-β), and different colony stimulating factors (CSF), including GM-CSF, G-CSF and M-CSF . A composition including an immunostimulatory ORN of the invention plus an immunostimulatory CpG nucleic acid is also provided. In one embodiment, the composition is a conjugate of the immunostimulatory ORN of the invention and the CpG nucleic acid, e.g. ex. an RNA: DNA conjugate. In one embodiment, the composition is a mixture of the immunostimulatory ORN of the invention and the CpG nucleic acid, in particular it is not an RNA: DNA conjugate. An immunostimulatory CpG nucleic acid as used herein, refers to a natural or synthetic DNA sequence that includes a CpG motif and that stimulates the activation or proliferation of cells of the immune system. The immunostimulatory CpG nucleic acids have been described in a series of granted 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. In one embodiment the immunostimulatory CpG nucleic acid is a CpG oligodeoxynucleotide (CpG ODN) of 6-100 nucleotides in length. In one embodiment the immunostimulatory CpG nucleic acid is a CpG oligodeoxynucleotide (CpG ODN) of 8-40 nucleotides in length. The immunostimulatory CpG nucleic acids include different classes of CpG nucleic acids. One class is potent for activating B cells but is relatively weak in the induction of IFN-a and activation of NK cells; this class has been termed class B. Class B of CpG nucleic acids are normally fully stabilized and include unmethylated CpG dinucleotide in some preferred base contexts. See, for example, the patents of E.U.A. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6.239.1 16; and 6,339,068. Another class is potent for inducing IFN-a and activating NK cells but is relatively weak in stimulating B cells; this class has been termed class A. Class A of CpG nucleic acids typically has a sequence containing palindromic CpG-phosphodiester dinucleotides of at least 6 nucleotides and a poly-G sequence stabilized at either or both ends of 5 'and 3'. See, for example, the published international patent application WO 01/22990. Still another class of CpG nucleic acids activates B cells and NK cells and induces IFN-a; this class has been designated class C. Class C of CpG nucleic acids are characterized first because they are normally fully stabilized, include a class B type sequence and a palindrome or almost palindrome rich in GC. This class has been described in the published patent application of E.U.A. 2003/0148976, the entire contents of which are incorporated herein by reference. The immunostimulatory CpG nucleic acids also include soft and semi-soft CpG nucleic acids, as described in the published patent application of E.U.A. 2003/0148976, the entire contents of which are incorporated herein by reference. Said soft and semi-soft immunostimulatory CpG nucleic acids incorporate a combination of nuclease-resistant and nuclease-sensitive internucleotide bonds, in which the different types of bonds are located according to certain standards. Also provided is a composition that includes an immunostimulatory ORN of the invention plus another adjuvant, wherein the other adjuvant is a lipopeptide such as Pam3Cys, a cationic polysaccharide such as chitosan, or a cationic peptide such as protamine. In one embodiment, the composition is a conjugate of the immunostimulatory ORN of the invention and the other adjuvant. The invention in one aspect provides a vaccine that includes an immunostimulatory ORN of the invention and an antigen. An "antigen" as used herein refers to any molecule capable of being recognized by a T cell antigen receptor or B cell antigen receptor. The term broadly includes any type of molecule that is recognized by a host immune system as strange. Antigens in general include, but are not limited to cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, polysaccharides, carbohydrates , viruses and virus extracts, and multicellular organisms such as parasites and allergens. With respect to antigens which are proteins, polypeptides or peptides, said antigens may include nucleic acid molecules encoding said antigens. Antigens more specifically include, but are not limited to, cancer antigens, including cancer cells and molecules expressed in or on cancer cells; microbial antigens, which include microbes and molecules expressed in or on microbes; and allergens. Accordingly, the invention in some embodiments provides vaccines for cancers, infectious agents and allergens. In one aspect, the invention provides a use of an immunostimulatory ORN of the invention for preparing a medicament for vaccinating a subject. The invention in one aspect provides a method for preparing a vaccine. The method includes the steps of placing an immunostimulatory ORN of the invention in intimate association with an antigen and, optionally, a pharmaceutically acceptable carrier. In different modalities the antigen is a microbial antigen, a cancer antigen or an allergen. A "microbial antigen" as used herein 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 natural isolates and fragments or derivatives thereof and also synthetic compounds that are identical or similar to antigens of natural microorganisms and induce a specific immune response for that microorganism. A compound is similar to a natural microorganism antigen if it induces an immune response (humoral and / or cellular) against an animal microorganism antigen. Such antigens are commonly used in the art and are well known to those skilled in the art. Viruses are small infectious agents that normally contain a nucleic acid nucleus and a protein coat, but they are organisms that do not live independently. Viruses can also take the form of infectious nucleic acids lacking a protein. A virus can not survive in the absence of a living cell within which it can replicate. Viruses enter specific living cells by endocytosis or by direct injection of DNA (phage) and multiply causing the disease. Viruses multiplied later can be released and infect additional cells. Some viruses are viruses that contain DNA and others are viruses that contain RNA. In some aspects, the invention also aims to treat diseases whose prions are involved in the progression of the disease, such as for example bovine spongiform encephalopathy (ie, mad cow disease, BSE) or scrapie infection in animals, or disease of Creutzfledt-Jakob in humans. Viruses include, but are not limited to, enteroviruses (including, but not limited to, viruses of the picornavi dae family, such as poliovirus, coxsackievirus, echovirus), rotavirus, adenovirus, hepatitis virus. Specific examples of viruses that have been found in humans include, but are not limited to: retroviridae (e.g., human immunodeficiency virus, such as HIV-1 (also called HTLV-IH, LAV or HTLV-III / LAV or HIV-llí; and other isolates such as HIV-LP; Picornavi dae (eg, poliovirus, hepatitis A virus, enterovirus, human coxsackie virus, rhinovirus, echovirus); Calciviridae (eg, strains) causing gastroenteritis), Togaviridae (eg, equine encephalitis virus, rubella virus), Flaviviridae (eg, dengue virus, encephalitis virus, yellow fever virus), Coronaviridae ( eg, coronaviruses), Rhabdoviridae (eg, vesicular stomatitis virus, rabies virus), Filoviridae (eg, Ebola virus), Paramyxoviridae (eg, parainfluenza virus, virus of mumps, measles virus, respiratory syncytial virus), Orthomyxoviridae (eg, influenza virus), Bunyaviridae (eg, Hantaan virus, bunyavirus , fleboviruses and nairovirus); Arenaviridae (hemorrhagic fever virus); Reoviridae (eg, reovirus, orbivirus and rotavirus); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (the majority of adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV)); Poxviridae (smallpox virus, vaccinia virus, poxvirus); Iridoviridae (eg, African swine fever virus); unclassified viruses (eg, the etiologic agents of spongiform encephalopathies, the hepatitis delta agent (which is believed to be a defective satellite of the hepatitis B virus), the hepatitis A agents, not B ( class 1 = internally transmitted, class 2 = transmitted parenterally (ie, hepatitis C), Norwalk virus and related viruses, and astroviruses) Bacteria are unicellular organisms that multiply asexually by binary fission. in its morphology, staining reactions, nutrition and metabolic requirements, antigenic structure, chemical composition and genetic homology Bacteria can be classified into three groups based on their morphological, spherical (coccus), rod (bacilli) and curved or helical ( vibrio, campylobacter, spiril and spirochetes.) Bacteria are also more commonly characterized based on their staining reactions in two classes of organisms, Gram positive and Gram negative. Gram refers to the staining method that is usually carried out in microbiology laboratories. The Gram positive organisms retain the staining after the staining method and a dark violet color appears. The Gram negative organisms do not retain the staining but take the contratinction and therefore appear pink. Infectious bacteria include, but are not limited to, Gram-negative and Gram-positive bacteria. Gram-positive bacteria include, but are not limited to, the species Pasteurella, species Staphilococci, and species Streptococcus. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species and Salmonella species. Specific examples of infectious bacteria include, but are not limited to: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (eg, M, tuberculosis, M, avium, M. intracellulare, M. kansasii, M, gordonae ), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A Streptococcus), Streptococcus agalactiae (group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, Campylobacter sp. pathogens, Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Cotynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Parasites are organisms that depend on other organisms to survive and must enter or infect another organism to continue their life cycle. The infected organism, that is, the host, provides both nutrition and habitat to the parasite. Although in its broadest sense the term parasite may include all infectious agents (ie, bacteria, viruses, fungi, protozoa and helminths), generally speaking, the term is used to refer only to protozoa, helminths and ectoparasite arthropods (p. (eg, mites, ticks, etc.). Protozoa are unicellular organisms that can replicate both intracellularly and extracellularly, particularly in the blood, intestinal tract or extracellular matrix of tissues. Helminths are multicellular organisms that are almost always extracellular (Trichinella spp. Being an exception). The helminths usually need to leave a primary host and be transmitted to a secondary host in order to replicate. Unlike these mentioned classes, the ectoparasite arthropods form a parasitic relationship with the external surface of the host body. Parasites include intracellular parasites and obligate intracellular parasites. Examples of parasites include, but are not limited to Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax, Plasmodium knowlesi, Babesia microti, Babesia divergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania braziliensis, Tropic Leishmania, Trypanosoma gambiense, Trypanosoma rhodesiense and Schistosoma mansoni. Fungi are eukaryotic organisms, of which only some produce infection in vertebrate mammals. Because fungi are eukaryotic organisms, they differ significantly from prokaryotic bacteria in size, structural organization, life cycle and multiplication mechanism. Fungi are generally classified based on morphological characteristics, reproduction modes and cultivation characteristics. Although fungi can produce different types of diseases in subjects, such as respiratory allergies after inhaling fungal antigens, fungal poisoning due to ingestion of toxic substances, such as the toxin of Amanita phalloides and phatoxin produced by poisonous mushrooms and aflatoxins, produced by Aspergillus species, not all fungi produce infectious diseases. Infectious fungi can cause systemic or superficial infections. Primary systemic infection can occur in normal healthy subjects, and opportunistic infections are found more frequently in immunocompromised subjects. The most common fungal agents that produce primary systemic infection include Blastomyces, Coccidioides, and Histoplasma. Common fungi that produce opportunistic infection in immunocompromised or immunosuppressed subjects include, but are not limited to, Candida albicans, Cryptococcus neoformans, and different species of Aspergillus. Systemic fungal infections are invasive infections of the internal organs. The body normally enters the body through the lungs, the gastrointestinal tract or intravenous catheters. These types of infections can be caused by primary pathogenic fungi or by opportunistic fungi. Superficial fungal infections involve the growth of fungi on an external surface without invasion of internal tissues. Typical superficial fungal infections include fungal skin infections involving skin, hair or nails. Diseases associated with fungal infection include asperilosis, blastomycosis, candidiasis, chromoblastomycosis, coccidiodiomycosis, cryptococcosis, fungal eye infections, fungal infections of the hair, nails and skin, histoplasmosis, lobomycosis, mycetoma, otomycosis, paracoccidioidomycosis, disseminated Penicillium marneffei, faeohifomicosis, rinosporidioisis, sporotrichosis, and zygomycosis. Other microorganisms of medical importance have been described extensively in the literature. eg, see C.G.A. Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire contents of which are incorporated herein by reference. Each of the above lists is illustrative and is not intended to be limiting. As used herein, the terms "cancer antigen" and "tumor antigen" are used interchangeably to refer to a compound, such as a peptide, protein or glycoprotein, which is associated with a cancer cell or tumor and that can elicit an immune response when expressed on the surface of an antigen-presenting cell in the context of the major histocompatibility complex molecule. Cancer antigens are differentially expressed by cancer cells and can therefore be exploited in order to target cancer cells. Cancer antigens are antigens that can apparently stimulate potentially tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those that are normally silent (that is, they are not expressed) in normal cells, those that are expressed only at certain stages of differentiation, and those that are expressed temporarily, such as embryonic and fetal antigens. Other carcinogenic antigens are encoded by mutant cell genes such as oncogenes (eg, activated ras oncogene), suppressor genes (eg, p53 mutant), fusion proteins that result from internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes, such as those carried in the RNA and DNA tumor viruses. Cancer antigens can be prepared from cancer cells by preparing crude extracts of cancer cells, for example, as described by Cohen P.A. and col. (1994) Cancer Res. 54: 1055-8, partially purifying the antigens by recombinant technology, or by de novo synthesis of known antigens. Carcinogenic antigens include, but are not limited to, recombinantly expressed antigens, an immunogenic portion or the entire tumor or cancer or cell thereof. Said antigens can be isolated or prepared recombinantly or by any other means known in the art. Examples of tumor antigens include MAGE, MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC) - C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, specific membrane antigen of prostate (PSMA), T cell / CD3-zeta chain receptor, MAGE family of tumor antigens (eg, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6 , MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A1, MAGE-A12, MAGE-Xp2 (MAGB-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE- B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE family of tumor antigens (eg, GAGE-1, GAGE-2, GAGE-3, GAGE-4 , GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21 ras, RCAS1, f etoprotein a, cadherin E, catenin a, catenin ß and catenin ?, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, polyposis adenomatous coli protein (APC), fodrin, connexin 37, idiotype Ig, p15, gp75, gangliosides GM2 and GD2, viral products such as human papillomavirus proteins, Smad family of tumor antigens, lmp-1, P1 A, EBV-encoded nuclear antigen (EBNA) -1, glycogen phosphorylase in the brain, SSX-1, SSX- 2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2. This list is not limiting. An "allergen" as used herein is a molecule that can elicit an immune response characterized by the production of IgE. An allergen is also a substance that can induce an allergy or asthmatic response in a susceptible subject. Therefore, in the context of the invention, the term allergen means a specific type of antigen that can elicit an allergic response that is mediated by the IgE antibody. The list of allergens is huge and may include pollens, insect poisons, animal scale dust, fungal spores, and drugs (eg, penicillin). Examples of allergens from natural animals and plants include specific proteins from the following Canis genera. { Canis familiaris); dermatofagoides (eg, Dermatophagoides farinae); Felis. { Felis domesticus); Ambrosia (Ambrosia artemisiifolia); Lolium (eg, Lolium perenne and Lolium multiflorum); Criptomeria (Cryptomeria japonica); Alternate (Alternaria alternata); Alder; Alnus (Alnus glutinosa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (eg, Plantago lanceolata); Parieta a (eg, Parietaria officinalis and Parietaria judaica); Blattella (eg, Blattella germanica); Apis (eg, Apis multiflorum); Cupressus (eg, Cupressus sempendrens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (eg, Juniperus sabinoides, Junipenis virginiana, Juniperus communis, and Juniperus ashei); Thuya (eg, Thuya orientalis); Chamaecyparis (eg, Chamaecyparis obtusa); Periplaneta (eg, Periplaneta americana); Agropyron (eg, Agropyron repens); Dry it (eg, Sécale cereale); Triticum (eg, Triticum aestivum); Dactilis (eg, Dactilis glomerata); Festuca (eg, Festuca elatior); Poa (eg, Poa pratensis and Poa compressa); Oats (eg, Avena sativa); Holcus (eg, Holcus lanatus); Anthoxanthum (eg, Anthoxanthum odoratum); Arrhenatherum (eg, Arrhenatherum elatius); Agrostis (eg, Agrostis alba); Phleum (eg, Phleumpratense); Phalaris (eg, Phalaris arundinacea); Paspalum (eg, Paspalum notatum); Sorghum (eg, Sorghum halepensis); and Bromus (eg, Bromus inermis). The invention in one aspect provides a conjugate of an immunostimulatory ORN of the invention and an antigen. In one embodiment the immunostimulatory ORN of the invention is covalently linked to the antigen. The covalent link between the immunostimulatory ORN and the antigen can be any suitable type of covalent bond, with the proviso that the immunostimulatory ORN and the antigen when so bound retain a measurable functional activity of each individual component. In one modality, the covalent bond is direct. In another modality, the covalent bond is indirect, p. eg, by a connecting residue. The covalently linked immunostimulatory ORN and the antigen can be processed within a cell to be released from each other. In this way the delivery to a cell of any of the components can be enhanced compared to its delivery if it were administered as a separate preparation or separate component. In one embodiment, the antigen is an antigen per se, that is, it is a preformed antigen. In one aspect the invention provides a pharmaceutical composition that includes a composition of the invention, associated with a delivery vehicle. In different embodiments, the delivery vehicle is chosen from a cationic lipid, a liposome, a cochleate, a virosome, an immunostimulatory complex (ISCOM), a microparticle, a microsphere, a nanosphere, a unilamellar vesicle (LUV), a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, and a polycationic peptide, and optionally a pharmaceutically acceptable carrier. The pharmaceutically acceptable vehicles are discussed below. The pharmaceutical composition of the invention optionally may also include an antigen. The composition of the invention, together with the antigen when present, is physically associated with the delivery vehicle using any suitable method. The immunostimulatory composition may be contained in the delivery vehicle, or may be present or associated with a surface of the delivery vehicle exposed to the solvent. In one embodiment, the immunostimulatory ORN is present on or associated with a surface of the solvent-exposed delivery vehicle, and the antigen, if present, is contained in the delivered vehicle. In another embodiment both the immunostimulatory ORN and the antigen are present or associated with a surface of the solvent-exposed delivery vehicle. In another modality more, the antigen is present or associated with a surface of the solvent-exposed delivery vehicle, and the immunostimulatory ORN is contained within the delivery vehicle. In another embodiment, both the immunostimulatory ORN and the antigen, if antigen is included, are contained in the delivery vehicle. The invention also provides methods for using the immunostimulatory compositions of the invention. In one aspect the invention provides a method of activating an immune cell. The method according to this aspect of the invention includes the step of contacting an immune cell, in vitro or in vivo, with an effective amount of a composition of the invention, to activate the immune cell. The composition of the invention may optionally include an antigen. An "immune cell" as used herein refers to any cell derived from the bone marrow that can participate in an innate or acquired immune response. Cells of the immune system include, without limitation, dendritic cells (DC), natural agressor cells (NK), monocytes, macrophages, granulocytes, B lymphocytes, plasma cells, T lymphocytes and precursor cells thereof. As used herein, the expression "Effective amount" refers to the amount of a substance that is necessary or sufficient to produce a desired biological effect. An effective amount may be, but need not be limited to an amount administered in a single administration. As used herein, the term "activates an immune cell" refers to inducing an immune cell to enter an activated state that is associated with an immune response. The expression "activates an immune cell" refers to both inducing and increasing an immune response. As used herein, the term "immune response" refers to any aspect of an innate or acquired immune response that reflects the activation of an immune cell to proliferate, perform an effector immune function, or produce a gene product. involved in an immune response. Gene products involved in an immune response may include secreted products (eg, antibodies, cytokines and chemokines) as well as intracellular and cell surface molecules characteristic of immune function (eg, some groups of differentiation antigens ( CD), transcription factors and gene transcripts). The term "immune response" can be applied to a single cell or to a population of cells. Cytokine production can be evaluated by any of a number of methods known in the art, including biological response assays, enzyme-linked immunosorbent assay (ELISA), analysis of cell separation activated by intracellular fluorescence (FACS), and reaction in polymerase chain / reverse transcriptase (RT-PCR).

In one embodiment, the immune response involves the production of an immune response of proinflammatory cytokines. An immune response of proinflammatory cytokines can include the expression of any of a number of cytokines and chemokines, including IFN- ?, TNF-a, IL-12, IL-10, IL-6, and any combination thereof. IFN-a is specifically excluded for the purposes of this invention. In one embodiment the immune response involves upregulation of cell surface markers of immune cell activation, such as CD25, CD80, CD86 and CD154. Methods for measuring the expression of said cell surface markers are well known in the art and include analysis by FACS. For the measurement of the immune response in a cell or population of cells, in one embodiment the cell or population of cells expresses TLR8. The cell can express the TLR naturally or can be manipulated to express the TLR by introducing into the cell a suitable expression vector for the TLR. In one embodiment the cell or population of cells is obtained as peripheral blood mononuclear cells (PBMC). In one embodiment, the cell or cell population is obtained as a cell line expressing the TLR. In one embodiment the cell or cell population is obtained as a transient transfectant expressing the TLR. In one embodiment the cell or cell population is obtained as a stable transfectant that expresses the TLR.

Also for use to measure an immune response in a cell or population of cells, it may be convenient to introduce into the cell or population of cells an indicator construct that is sensitive to intracellular signaling by a TLR. In one embodiment said indicator is a gene placed under the control of an NF-γ promoter. In one embodiment, the gene placed under the control of the promoter is luciferase. Under suitable activation conditions, the luciferase reporter construct is expressed and emits a detectable light signal that can be measured quantitatively using a luminometer. Said indicator constructions and other suitable indicator constructions are available commercially. The invention also contemplates the use of cell-free methods to detect activation of the TLR. In some aspects, the invention relates to compositions and methods for use in therapy. The immunostimulatory compositions of the invention can be used alone or in combination with other therapeutic agents. The immunostimulatory composition and another therapeutic agent can be administered simultaneously or sequentially. When the immunostimulatory composition of the invention and the other therapeutic agent are administered simultaneously, they can be administered in the same formulation or in separate formulations, but are administered at the same time. In addition, when the immunostimulatory composition of the invention and the other therapeutic agent are administered simultaneously, they can be administered by the same routes of administration or different routes, but are administered at the same time. The immunostimulatory composition of the invention and the other therapeutic agent are administered sequentially when the administration of the immunostimulatory composition of the invention is temporarily separated from the administration of the other therapeutic agent. The separation in time between the administration of these compounds can be a matter of minutes or it can be longer. In one embodiment, the immunostimulatory composition of the invention is administered before administering the other therapeutic agent. In one embodiment, the immunostimulatory composition of the invention is administered after administering the other therapeutic agent. In addition, when the immunostimulatory composition of the invention and the other therapeutic agent are administered sequentially, they can be administered by the same administration routes or distinct routes. Other therapeutic agents include, but are not limited to adjuvants, antigens, vaccines and drugs useful for the treatment of infection, cancer, allergy and asthma. In one aspect, the invention provides a method of vaccination of a subject. The method according to this aspect of the invention includes the step of administering to the subject an antigen and a composition of the invention. In one embodiment, administration of the antigen includes administering a nucleic acid encoding the antigen. A "subject" as used herein refers to a vertebrate animal. In different modalities the subject is a human being, a non-human primate or another mammal. In some modalities the subject is a mouse, rat, guinea pig, rabbit, cat, dog, pig, sheep, goat, cow or horse. For use in the method of vaccination of a subject, the composition of the invention in one embodiment includes an antigen. The antigen can be separated or covalently linked to an ORN of the invention. In one embodiment, the composition of the invention does not itself include the antigen. In this embodiment, the antigen can be administered to the subject separately from the composition of the invention, or together with the composition of the invention. The administration that is separated includes separate in time, separated in the site or route of administration, or separated both in time and in the site or route of administration. When the composition of the invention and the antigen are administered separately in time, the antigen can be administered before or after the composition of the invention. In one embodiment, the antigen is administered 48 hours at 4 weeks after administering the composition of the invention. The method also contemplates the administration of one or more booster doses of the antigen alone, the composition alone or the antigen and the composition, after an initial administration of the antigen and the composition. It is also contemplated in the invention that a subject can be prepared for future encounter with an unknown antigen by administering to the subject a composition of the invention, wherein the composition does not include an antigen. According to this embodiment, the subject's immune system will be prepared to mount a more vigorous response to an antigen with which the subject will meet later., for example, by environmental or occupational exposure. This method can be used, for example, by travelers, medical workers and soldiers who are likely to be exposed to microbial agents. In one aspect, the invention provides a method for treating a subject having a deficiency of the immune system. The method according to this aspect of the invention includes the step of administering to the subject an effective amount of a composition of the invention to treat the subject. An "immune system deficiency" as used herein refers to an abnormally reduced ability of an immune system to mount an immune response to an antigen. In one embodiment, a deficiency of the immune system is a disease or disorder in which the subject's immune system does not function at its normal capacity or in which it would be useful to reinforce the subject's immune response, for example, to eliminate a tumor or cancer or in an infection in the subject. A "subject having an immune deficiency" as used herein refers to a subject in which the subject's immune system has a reduced ability to mount an immune response to an antigen. Subjects who have an immune deficiency include subjects who have an acquired immune deficiency as well as subjects who have a congenital deficiency in the immune system. Subjects having an acquired immune deficiency include, without limitation, subjects having a chronic inflammatory condition, subjects having chronic renal insufficiency or renal insufficiency, subjects having infection, subjects having cancer, subjects receiving immunosuppressive drugs, subjects receiving another immunosuppressive treatment, and subjects with malnutrition. In one embodiment, the subject has a population of suppressed CD4 + T cells. In one embodiment the subject has an infection with the human immunodeficiency virus (HIV) or has the acquired immunodeficiency syndrome (AIDS). The method according to this aspect of the invention therefore provides a method for enhancing the immune response or enhancing the ability to mount an immune response in a subject in need of a more vigorous immune response. The composition and methods of the invention can be used alone or together with other agents and methods useful for the treatment of the infection. In one aspect, the invention provides a method for treating a subject having an infection. The method according to this aspect of the invention includes the step of administering to a subject having an infection an effective amount of the composition of the invention to treat the subject. In one aspect the invention provides a method for treating a subject having an infection. The method according to this aspect of the invention includes the step of administering to a subject having an infection an effective amount of the composition of the invention and a medicament for the infection to treat the subject. In one aspect the invention provides the use of an immunostimulatory ORN of the invention for preparing a medicament for treating an infection in a subject.

In one aspect the invention provides a composition useful for the treatment of an infection. The composition according to this aspect includes an immunostimulatory ORN of the invention and a medicament for infection. As used herein, the term "treating" when used in reference to a subject having a disease or condition means preventing, improving or eliminating at least one sign or symptom of the disease or condition in the subject. A "subject having an infection" is a subject having a disorder that proceeds from the invasion in the subject in a superficial, local or systemic manner, by an infectious microorganism. The infectious microorganism can be a virus, bacterium, fungus or parasite, as described above. Medications for infections include, but are not limited to, antibacterial agents, antiviral agents, antifungal agents, and antiparasitic agents. Phrases such as "anti-infective agent", "antibiotic", "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 standard medical texts. Briefly, antibacterial agents kill or inhibit bacteria, and include antibiotics as well as other synthetic or natural compounds that have similar functions. Antiviral agents can be isolated from natural or synthesized sources and are useful for killing and inhibiting viruses. The antifungal agents are used to treat superficial fungal infections as well as opportunistic and primary systemic fungal infections. Antiparasitic agents kill or inhibit parasites. Many antibiotics are low molecular weight molecules that are produced as secondary metabolites by cells, such as microorganisms. In general, antibiotics interfere with one or more functions or structures that are specific for microorganisms and that are not present in host cells. One of the problems with anti-infective therapies is the side effects that occur in the host that is treated with the anti-infective agent. For example, many anti-infective agents can kill or inhibit a broad spectrum of microorganisms and are not specific to a particular type of species. Treatment with these types of anti-infective agents results in the death of the normal microbial flora living in the host, as well as infectious microorganisms. The loss of microbial flora can lead to complications in the disease and the predisposition of the host to infection by other pathogens, since the microbial flora competes with and functions as barriers against infectious pathogens. Other side effects may come as a result of specific or non-specific effects of these chemical entities on non-microbial cells or host tissues. Another problem with the widespread use of anti-infectives is the development of strains of microorganisms resistant to antibiotics. Resistant strains of vancomycin-resistant enterococci, penicillin-resistant pneumococci, multiresistant S. aureus and multiresistant tuberculosis have already been developed and are becoming an important clinical problem. The widespread use of anti-infective agents will likely produce many strains of antibiotic-resistant bacteria. As a result, new anti-infective strategies to combat these microorganisms will be necessary. Antibacterial antibiotics that are effective in killing or inhibiting a wide variety of bacteria are called broad spectrum antibiotics. Other types of antibacterial antibiotics are effective predominantly against Gram-positive or Gram-negative bacteria. These types of antibiotics are called small-spectrum antibiotics. Other antibiotics that are effective against a single organism or disease and not against other types of bacteria are called limited-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, functional or nucleic acid synthesis inhibitors, and competitive inhibitors. Inhibitors of cell wall synthesis inhibit a stage in the process of cell wall synthesis, and in general in the synthesis of bacterial peptidoglycans. Inhibitors of cell wall synthesis include ß-lactam antibiotics, natural penicillins, semi-synthetic penicillins, ampicillin, clavulanic acid, cephalosporins and bacitracin. Β-lactams are antibiotics that contain a four-membered β-lactam ring that inhibits the last stage of peptidoglycan synthesis. The β-lactam antibiotics can be synthesized or natural. The β-lactam antibiotics produced by penicillium are natural penicillins, such as penicillin G or penicillin V. These are produced by fermentation of Penicillium chrysogenum. Natural penicillins have a narrow spectrum of activity and are generally effective against Stretococcus, Gonococcus and Staphylococcus. Other types of natural penicillins that are also effective against Gram-positive bacteria include penicillins F, X, K and O. Semisynthetic penicillins are in general modifications of the 6-aminopenicillanic acid molecule produced by a mold. The 6-aminopenicillanic acid can be modified by addition of side chains which produces penicillins having broader activity spectra than natural penicillins or different advantageous properties. Some types of semi-synthetic penicillins have broad spectra against Gram-positive and Gram-negative bacteria, but are inactivated by penicillinase. These semi-synthetic penicillins include ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, and piperacillin. Other types of semisynthetic penicillins have narrower activities against Gram-positive bacteria, but have developed properties so that they are not inactivated by penicillinase. These include, for example, methicillin, dicloxacillin and nafcillin. Some broad-spectrum semi-synthetic penicillins can be used in combination with β-lactamase inhibitors, such as clavulanic acids and sulbactam. The β-lactamase inhibitors have no antimicrobial action but work to inhibit penicillinase, thus protecting semisynthetic penicillin against degradation. Another type of β-lactam antibiotic are cephalosporins. They are sensitive to degradation by bacterial β-lactamases, and therefore are not always effective alone. However, cephalosporins are resistant to penicillinase. They are effective against a variety of Gram positive and Gram negative bacteria. Cephalosporins include, but are not limited to cephalothin, cephapirin, cephalexin, cephamandola, cefaclor, cefazolin, cefuroxin, cefoxitin, cefotaxime, cefsulodine, cefetamet, cefixime, ceftriaxone, cefoperazone, ceftazidine and moxolactam. Bacillin is another class of antibiotics that inhibit cell wall synthesis by inhibiting the release of murpeptide subunits or peptidoglycans from the molecule that supplies the subunit to the outside of the membrane. Although bacitracin is effective against Gram-positive bacteria, its use is generally limited to topical administration due to its high toxicity. Carbapenems are another class of broad-spectrum ß-lactam antibiotics, which are capable of inhibiting cell wall synthesis. Examples of carbapenems include, but are not limited to imipenems. Monobactams are also broad-spectrum β-lactam antibiotics, and include, euztrenoam. An antibiotic produced by Streptomyces, vancomycin, is also effective against Gram-positive bacteria by inhibiting the synthesis of the cell membrane. Another class of antibacterial agents are antibacterial agents that are inhibitors of the cell membrane. These compounds disrupt the structure or inhibit the function of the bacterial membranes. A problem with antibacterial agents that are inhibitors of the cell membrane is that they can produce effects in eukaryotic cells as well as in bacteria due to the similarity of phospholipids in bacteria and eukaryotic membranes. Therefore, these compounds are rarely specific enough to allow these compounds to be used systemically and to prevent the use of high doses by local administration. A membrane inhibitor clinically useful cell is polymyxin. The polymyxins interfere with the function of the membrane by binding to the phospholipids of the membrane. Polymyxin is effective primarily against Gram-negative bacteria and is generally used in several Pseudomonas infections or Pseudomonas infections that are resistant to less toxic antibiotics. The serious side effects associated with the systemic administration of this compound include damage to the kidneys and other organs. Other inhibitors of the cell membrane include amphotericin B and nystatin which are antifungal agents used mainly in the treatment of fungal infections and yeast Candida infections. Imidazoles are another class of antibiotics that are an inhibitor of the cell membrane. Imidazoles are used as antibacterial agents as well as antifungal agents, p. eg, used for the treatment of yeast infections, dermatophytic infections and systemic fungal infections. Imidazoles include, but are not limited to clotrimazole, miconazole, ketoconazole, traconazole and fluconazole. Many antibacterial agents are inhibitors of protein synthesis. These compounds prevent bacteria from synthesizing structural proteins and enzymes and therefore produce inhibition of bacterial cell growth or cell function or death. In general, these compounds interfere with transcription or translation methods. Antibacterial agents that block transcription include, but are not limited to rifampins and ethambutol. Rifampins that inhibit the RNA polymerase enzyme have a broad spectrum activity and are effective against Gram positive and Gram negative bacteria as well as Mycobacterium tuberculosis. Etamburol is effective against Mycobacterium tuberculosis. Antibacterial agents that block translation interfere with bacterial ribosomes to prevent mRNA from being translated into proteins. In general, this class of compounds include, but are not limited to tetracyclines, chloramphenicol, macrolides (e.g., erythromycin) and aminoglycosides (e.g., streptomycin). Aminoglycosides are a class of antibiotics that are produced by the Streptomyces bacteria, such as for example streptomycin, kanamycin, tobramycin, amikacin and gentamicin. Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram-negative bacteria. Streptomycin has been widely used as a main drug in the treatment of tuberculosis. Gentamicin is used against many strains of Gram-positive and Gram-negative bacteria, including Pseudomonas infections, especially in combination with tobramycin. Kanamycin is used against many Gram positive bacteria, including Staphylococci resistant to penicillin. A side effect of aminoglycosides that has limited its use clinically is that with dosages that are essential for efficacy, it has been shown that prolonged use impairs renal function and produces damage to the auditory nerves leading to deafness. Another type of antibacterial agent that inhibits translation is the tetracyclines. Tetracyclines are a class of antibiotics that are broad spectrum and are effective against a variety of Gram positive and Gram negative bacteria. Examples of tetracyclines include tetracycline, minocycline, doxycycline and chlortetracycline. They are important for the treatment of many types of bacteria, but they are particularly important in the treatment of Lyme disease. As a result of its low toxicity and minimal direct side effects, the medical community has misused and abused the use of tetracyclines, leading to problems. For example, excessive use has led to the extended development of resistance.

Antibacterial agents such as the macrolides bind reversibly to the 50S subunit of the ribosome and inhibit the elongation of the protein by peptidyl transferase or prevent the release of uncharged tRNA from the bacterial ribosome or both. These compounds include erythromycin, roxithromycin, clarithromycin, oleandomycin and azithromycin. Erythromycin is active against most Gram positive bacteria, Neisseria, Legionella and Hemophilus, but not against Enterobacteriaceae. Lincomycin and clindamycin, which block the formation of peptide bond during protein synthesis, are used against Gram positive bacteria. Another type of translation inhibitor is chloramphenicol. Chloramphenicol binds to the 70S ribosome by inhibiting the bacterial peptidyl transferase enzyme and thus preventing the growth of the polypeptide chain during protein synthesis. A serious side effect associated with chloramphenicol is aplastic anemia. The aplastic anemia develops with doses of chloramphenicol that are effective to treat bacteria in a small proportion (1 / 50,000) of patients. Chloramphenicol, which had been a highly prescribed antibiotic, is now rarely used as a result of anemic deaths. Because of its effectiveness it is still used in life-threatening situations (eg, typhoid fever). Some antibacterial agents alter the synthesis of nucleic acid or its function, for example, they bind to DNA or RNA so that their messages can not be read. These include, but are not limited to quinolones and co-trimoxazole, both synthetic chemicals and rifamycins, a natural or semi-synthetic chemical. Quinolones block the replication of bacterial DNA by inhibiting RNA gyrase, the enzyme the bacteria need to produce their circular DNA. They are broad spectrum and examples include norfloxacin, ciprofloxacin, enoxacin, nalidixic acid and temafloxacin. Nalidixic acid is a bactericidal agent that binds to the enzyme DNA gyrase (topoisomerase) that is essential for DNA replication and allows the superlices to relax and re-form, inhibiting the activity of DNA gyrase. The main use of nalidixic acid is in the treatment of lower urinary tract infections (UTI) because it is effective against several types of Gram negative bacteria such as E. coli, Enterobacter aerogenes, K pneumoniae and Proteus species that are common causes of UTI. Cotrimoxazole is a combination of sulfamethoxazole and trimethoprim, which blocks the bacterial synthesis of folic acid needed to make DNA nucleotides. Hifampicin is a derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis and meningitis caused by Neisseria meningitidis) and some Gram-negative bacteria. Rifampicin binds to the beta subunit of the polymerase and blocks the addition of the first nucleotide that is necessary to activate the polymerase, thus blocking the synthesis of the mRNA. Another class of antibacterial agents are compounds that function as competitive inhibitors of bacterial enzymes. Competitive inhibitors are mostly structurally similar to a bacterial growth factor and compete for binding but do not hold metabolic function in the cell. These compounds include sulfonamides and chemically modified forms of the sulfanilamide having even higher and broader antibacterial activity. Sulfonamides (eg, gantrisine and trimethophim) are useful for the treatment of Streptococcus pneumoniae, beta-hemolytic streptococci and E. coli, and have been used in the treatment of uncomplicated UTI caused by E. coli, and in the meningococcal meningitis treatment. Antiviral agents are compounds that prevent the infection of cells by viruses or the replication of viruses within the cell. There are far fewer antiviral drugs than antibacterial drugs because the process of viral replication is so closely related to DNA replication in the host cell, that non-specific antiviral agents will often be toxic to the host. There are several stages in the viral infection process that can be blocked or inhibited with antiviral agents. These steps include the binding of the virus to the host cell (immunoglobulin or binding peptides), virus shedding (eg, amantadine), synthesis or translation of viral mRNA (e.g., interferon), RNA replication or Viral DNA (eg, nucleoside analogs), maturation of new virus proteins (eg, protease inhibitors), and virus development and release. Another category of antiviral agents are nucleoside analogs. Nucleoside analogs are synthetic compounds that are similar to nucleosides, but that have an incomplete or abnormal deoxyribose or ribose group. Once the nucleoside analogs are in the cell, they are phosphorylated, producing the triphosphate form that competes with normal nucleotides for incorporation into the viral DNA or RNA. Once the triphosphate form of the nucleoside analogue has been incorporated into the growing nucleic acid chain, it produces the irreversible association with the viral polymerase and thus the chain termination. Nucleoside analogs include, but are not limited to acyclovir (used for the treatment of herpes simplex virus and varicella-zoster virus), ganciclovir (used for the treatment of cytomegalovirus), idoxuridine, ribavirin (useful for the treatment of syial virus) respiratory), dideoxyinosine, dicdeoxycytidine and zidovudine (azidothymidine). Another class of antiviral agents includes cytokines such as interferons. Interferons are cytokines that are secreted by cells infected by viruses as well as by immune cells. Interferons work by binding to specific receptors in the cells adjacent to the infected cells, producing the change in the cell that protects it from infection by the virus. Interferon a and β also induce the expression of MHC class I and class II molecules on the surface of infected cells, resulting in an increase in antigen presentation for the recognition of host immune cells. Interferons a and b are available as recombinant forms and have been used for the treatment of chronic infection with hepatitis B and C. With dosages that are effective for antiviral therapy, interferons have serious side effects such as fever, malaise and loss of weight.

Immunoglobulin therapy is used to prevent viral infection. Immunoglobulin therapy for viral infections is different from bacterial infections, because rather than being antigen-specific, the immunoglobulin therapy functions by binding to extracellular virions and preventing bind and enter cells that are susceptible to viral infection. The therapy is useful to prevent viral infection during the period of time in which the antibodies are present in the host. In general there are two types of immunoglobulin therapies, normal immunoglobulin therapy and hyperimmunoglobulin therapy. Normal immunoglobulin therapy uses an antibody product that is prepared from the serum of normal blood donors and are pooled. This pooled product contains low antibody titers for a wide variety of human viruses, such as hepatitis A, parvovirus, enterovirus (especially in neonates). The hyperimmunoglobulin therapy uses antibodies that are prepared from the serum of individuals who have high titers of an antibody to a particular virus. These antibodies are then used against a specific virus. Examples of hyperimmunoglobulins include zoster immune globulin (useful to prevent chickenpox in immunocompromised children and neonates), rabies immunoglobulin in humans (useful in prophylaxis following exposure of a subject bitten by a rabid animal), immune globulin hepatitis B (useful to prevent hepatitis B virus, especially in a subject exposed to the virus), and immunoglobulin against RSV (useful in the treatment of respiratory syncytial virus infections). The antifungal agents are useful in 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 imidazoles, such as clotrimazole, sertaconzol, fluconazole, itraconazole, ketoconazole, miconazole, and voriconacol and FK 463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292, butenafine, and terbinafine. Other antifungal agents work by breaking chitin (eg, chitinase) or immunosuppression (cream 501). The parasiticides are agents that kill parasites directly. Such compounds are known in the art and are generally available commercially. Examples of useful parasiticidal for human administration include but are not limited to albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, phosphate chloroquine, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, eflornithine, furazolidone, glucocorticoids, halofantrine, iodoquinol , ivermectin, mebendazole, meglumine antimonate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, parommycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethamine-sulfonamides, pyrimethamine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium (sodium antimony gluconate and), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethoprim-sulfamethoxazole, and tryparsamide. ORNs are also useful for treating and preventing autoimmune diseases. Autoimmune disease is a kind of disease in which a subject's own antibodies react with host tissues or in which immune effector T cells are self-reactive with the endogenous peptides themselves and produce tissue destruction. Thus, an immune response is mounted against the subject's own antigens, called autoantigens. 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 autoimmunity, glomerulonephritis (eg, crescentic glomerulonephritis, glomerulonephritis) proliferative), bullous pemphigus, Sjögren's syndrome, insulin resistance and autoimmune diabetes mellitus.

An "autoantigen" as used herein refers to an antigen from a normal host tissue. Normal host tissue does not include cancer cells. Therefore, an immune response mounted against an autoantigen, in the context of the autoimmune disease, is an unwanted immune response and contributes to the destruction and damage of normal tissue, whereas an immune response mounted against a cancer antigen is a Desirable immune response and contributes to the destruction of the tumor or cancer. Therefore, in some aspects of the invention directed to the treatment of autoimmune disorders it is not recommended to administer the ORN with autoantigens, in particular those that are the targets of the autoimmune disorder. In other cases, ORN can be delivered with low doses of autoantigens. A number of studies in animals have shown that administration of low doses of antigen in mucosa can result in a state of immune hyposensitivity or "tolerance". The active mechanism seems to be a cytokine-mediated immune deviation from a Th1 response to a predominantly Th2 and Th3 (ie, dominated by TGF-β). Active suppression with low-dose antigen delivery can also suppress an unrelated immune response (surrounding deletion) that has a considerable interest in the therapy of autoimmune diseases, for example, rheumatoid arthritis and SLE. The surrounding deletion involves the secretion of Th1 counterregulatory suppressor cytokines in the local environment in which the proinflammatory and Th1 cytokines are released in a specific form of antigen or non-specific antigen. "Tolerance" as used herein, is used to refer to this phenomenon. Actually, oral tolerance has been effective in the treatment of a number of autoimmune diseases in animals, which include; experimental autoimmune encephalomyelitis (EAE), experimental autoimmune myasthenia gravis, collagen-induced arthritis (CIA) and insulin-dependent diabetes mellitus. In these models, the prevention and suppression of autoimmune disease is associated with a change in antigen-specific humoral and cellular responses from a Th1 to Th2 / Th3 response. The compositions and methods of the invention can be used alone or in combination with other agents and methods useful for the treatment of cancer. In one aspect the invention provides a method of treating a subject having cancer. The method according to this aspect of the invention includes the step of administering to a subject having a cancer an effective amount of a composition of the invention for treating the subject. In one aspect the invention provides a method for treating a subject having a cancer. The method according to this aspect of the invention includes the step of administering to a subject having cancer an effective amount of the composition of the invention and an anti-cancer therapy to treat the subject.

In one aspect the invention provides a use of an immunostimulatory ORN for preparing a medicament for treating cancer in a subject. In one aspect, the invention provides a composition useful for the treatment of cancer. The composition according to this aspect includes an immunostimulatory ORN of the invention and a medicament for cancer. A subject who has a cancer is a subject who has detectable cancer cells. Cancer can be a malignant or non-malignant cancer. "Cancer" as used herein refers to an uncontrolled growth of cells that interferes with the normal functioning of the organs and systems of the body. Cancers that migrate from their original situation and plant vital organs can finally lead to the death of the subject due to the functional deterioration of the affected organs. Hematopoietic cancers, such as leukemia, can competitively overcome normal hematopoietic compartments in a subject, thus leading to hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) eventually leading to death. A metastasis is a region of cancer cells, different from the situation of the primary tumor, which results from the spread of cancer cells from the primary tumor to other parts of the body. At the time of diagnosis of the primary tumor mass, the presence of metastasis in the subject can be monitored. Metastases are most often detected by single or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms. Cancers include, but are not limited to, basal cell carcinoma, cancer of the biliary tract; bladder cancer; bone cancer; cancer of the brain and central nervous system (CNS); breast cancer; cervical cancer; choriocarcinoma; cancer of the colon and rectum; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophagus cancer; eye cancer; Head and neck cancer; intraepithelial neoplasm; kidney cancer; cancer of the larynx; leukemia; Liver cancer; lung cancer (eg, small cells and non-small cells); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; Myeloma neuroblastoma; cancer of the oral cavity (eg, lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; sarcoma; skin cancer; stomach cancer; Testicular cancer; thyroid cancer; uterine cancer; cancer of the urinary system, as well as other carcinomas, adenocarcinomas and sarcomas. The immunostimulatory composition of the invention can also be administered in conjunction with an anti-cancer therapy. Anticancer therapies include drugs for cancer, radiation and surgical methods. As used herein, a "cancer medicament" refers to an agent that is administered to a subject for the purpose of treating a cancer. As used herein, "treating cancer" includes preventing the development of a cancer, reducing the symptoms of cancer and / or inhibiting the growth of a stabilized cancer. In other aspects, the cancer drug is administered to a subject who is at risk of developing cancer in order to reduce the risk of developing the cancer. This document describes different types of drugs for the treatment of cancer. For the purpose of this specification, drugs for cancer are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormonal therapy and biological response modifiers. The chemotherapeutic agent can be selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, chloroethylnitrosoureas containing no sugar, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, ftagiline, GLA Meglamine, valrubicin, carmustaine and poliferposan. , MMI270, BAY 12-9566, RAS farnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP, MTA / LY231514, LY264618 / Lometexol, Glamolec, CI-994, TNP-470, Hycamtin / Topotecan, PKC412, Valspodar / PSC833 , Novantrone / Mitroxantrone, Metaret / Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, lncel / VX-710, VX-853, ZD0101, ISI64I, ODN 698, TA 2516 / Marmistat , BB2516 / Marmistat, CDP 845, D2163, PD183805, DX8951I, Lemonal DP 2202, FK 317, Picibanil / O -432, AD 32 / Valrubicin, etastron / strontium derivative, Temodal / Temozolomide, Evacet / Liposomal doxorubicin, Yewtaxan / Paclitaxel, Taxol / Paclitaxel, Xeload / 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 / Uracil), Ergamisol / Levamisole, Eniluracil / 776C85 / 5FU enhancer, Campto / Levamisol, Camptosar / lrinotecan, Tumodex / Ralitrexed, Leustatin / Cladribine, Paxex / Paclitaxel, Doxil / Liposomal doxorubicin, Caelyx / Liposomal doxorubicin, Fludara / Fludarabine, Farmarrubicin / Epirubicin, DepoCyt, ZD1839, LU 79553 / Bis-Naphthalimide, LU 103793 / Dolastain, Caetyx / Liposomal doxorubicin, Gemzar / Gemcitabine, ZD 0473 / Anormed, YM 16, iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809 / Dexiphosamide, Ifes / Mesnex / lfosamide, Vumon / Teniposide, Paraplatin / Carboplatin, Plantinol / cisplatin, Vepside / Etoposide, ZD 9331, Taxotere / Docetaxel, guanine-arabinoside prodrug, analog d and taxanes, nitrosoureas, alkylating agents such as melphalan and cyclophosphamide, aminoglutethimide, asparaginase, busulfan, carboplatin, chlorombucil, cytarabine HCI, dactinomycin, daunorubicin HCl, estramustine-sodium phosphate, etoposide (VP16-213), floxuridine, fluorouracil (5-FU) ), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide, interferon alfa-2a, alpha-2b, leuprolide acetate (analog of LHRH release factor), lomustine (CCNU), mechlorethamine HCI (nitrogen mustard), mercaptopurine, mesna, mitotane (op-DDD), mitoxantrone HCI, octreotide, plicamycin, procarbazine HCI, streptozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amsacrine (m-AMSA), azacitidine, erythropoietin, hexamethylmelamine (HMM), interleukin 2, mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), pentostatin (2'-deoxicoformycin), semustine (methyl-CCNU), teniposide (VM-26) and vindesine sulfate, but is not limited. The immunotherapeutic agent can be selected from the group consisting of 3622W94, 4B5, ANA Ab, anti-FLK-2, anti-VEGF, ATRAGEN, AVASTIN (bevacizumab, Genentech), BABS, BEC2, BEXXAR (tositumomab, GlaxoSmithKine), C225, CAMPATH (alemtuzumab, Genzyme Corp.), CEACIDE, CMA 676, EMD-72000, ERBITUX (cetuximab, ImClone Systems, Inc.), Gliomab-H, GNI-250, HERCEPTIN (trastuzumab, Genentech), IDEC-Y2B8, ImmuRAIT CEA, ior c5, ior egf.r3, ior t6, LDP-03, LymfoCide, MDX-1 1, MDX-22, MDX-210, MDX-220, MDX-260, MDX-447, MELIMMUNE-1, MELIMMUNE- 2, Monopharm-C, NovoMAb-G2, Oncolym, OV103, Ovarex, Panorex, Pretarget, Quadramet, Ributaxin, RITUXAN (rituximab, Genentech), SMART 1 D10 Ab, SMART ABL 364 Ab, SMART M195, TNT, and ZENAPAX (daclizurnab Roche), but it is not limited to these. The cancer vaccine can be selected from the group consisting of EGF, idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2 / neu, Ovarex, M-Vax, O-Vax , L-Vax, STn-ICHL theratope, BLP25 (MUC-1), liposomal idiotypic vaccine, melacin, peptide antigen vaccines, antigen / toxin vaccines, MVA-based vaccine, PACIS, BCG vaccine, TA-HPV, TA- CIN, DISC virus and ImmuCyst / TheraCys, but it is not limited to these. The compositions and methods of the invention can be used alone or in combination with other agents and methods useful for the treatment of allergy. In one aspect the invention provides a method for treating a subject having an allergic condition. The method according to this aspect of the invention includes the step of administering to a subject having an allergic condition an effective amount of a composition of the invention to treat the subject. In one aspect the invention provides a method for treating a subject having an allergic condition. The method according to this aspect of the invention includes the step of administering to a subject having an allergic condition an effective amount of the composition of the invention and an anti-allergy therapy to treat the subject. In one aspect the invention provides a use of an immunostimulatory ORN of the invention for preparing a medicament for treating an allergic condition in a subject. In one aspect the invention provides a composition useful for the treatment of an allergic condition. The composition according to this aspect includes an immunostimulatory ORN of the invention and an allergy medicament.

A "subject having an allergic condition" refers to a subject who currently experiences or has previously experienced an allergic reaction in response to an allergen. An "allergic condition" or "allergy" refers to the acquired hypersensitivity to a substance (allergen). Allergic conditions include, but are not limited to, eczema, allergic rhinitis or coryza, hay fever, allergic conjunctivitis, bronchial asthma, urticaria (wheal) and food allergies, other atopic conditions including atopic dermatitis; anaphylaxis; drug allergy and angioedema. Allergy is usually an episodic condition associated with the production of antibodies of a particular class of immunoglobulin, IgE, against allergens. The development of an IgE-mediated response to common aeroallergens is also a factor that indicates the predisposition towards the development of asthma. If an allergen finds a specific IgE that binds to an IgE Fe (FceR) receptor on the surface of a basophil (circulating in the blood) or mast cell (dispersed by the solid tissue), the cell becomes activated, giving as result in the production and release of mediators such as histamine, serotonin and lipid mediators. An allergic reaction occurs when the sensitizing immunoglobulin of IgE-type tissue reacts with the foreign allergen. The IgE antibody is bound to mast cells and / or basophils, and these specialized cells release chemical mediators (vasoactive amines) from the allergic reaction when they are stimulated to do so by the allergens crossing the ends of the antibody molecule. Histamine, platelet activating factor, metabolites of arachidonic acid and serotonin are among the best known mediators of allergic reactions in man. Histamine and the other vasoactive amines are normally stored in mast cells and basophilic leukocytes. The mast cells are dispersed throughout the animal tissue and the basophils circulate within the vascular system. These cells manufacture and store histamine inside the cell, unless the specialized sequence of events involving the binding of IgE, which causes its release, occurs. The symptoms of an allergic reaction vary depending on the location within the body where the IgE reacts with the antigen. If the reaction occurs along the respiratory epithelium, the symptoms in general are sneezing, coughing and asthmatic reactions. If the interaction occurs in the digestive tract, as is the case of food allergies, abdominal pain and diarrhea are common. Systemic allergic reactions, for example, after a bee sting or the administration of penicillin to an allergic subject, can be serious and often life threatening. Allergy is associated with a Th2-type immune response, which is characterized at least in part by Th2 cytokines IL-4 and IL-5, as well as the change of isotype from antibody to IgE. The Th1 and Th2 immune responses are mutually counterregulatory, so that the tendency of the immune response towards a Th1 type immune response can prevent or improve the Th2 type immune response, including allergy. Therefore, the immunostimulatory ORNs of the invention are useful in themselves to treat a subject having an allergic condition, because the immunostimulatory ORN can divert the immune response towards a Th1 type of immune response. Alternatively or in addition, the immunostimulatory ORN of the invention can be used in combination with an allergen to treat a subject having an allergic condition. The immunostimulatory composition of the invention can also be administered together with an antiallergic therapy. Conventional methods to treat or prevent allergy have involved the use of allergy medications or desensitization therapies. Some therapies in development to treat or prevent allergy include the use of neutralizing anti-IgE antibodies. Antihistamines and other drugs that block the effect of chemical mediators of the allergic reaction, they help regulate the severity of allergic symptoms but do not prevent the allergic reaction and have no effect on subsequent allergic responses. Desensitization therapies are carried out by giving small doses of an allergen, usually by injection under the skin, in order to induce an IgG-type response against the allergen. It is believed that the presence of the IgG antibody helps to neutralize the production of mediators resulting from the induction of IgE antibodies. Initially, the subject is treated with a very low dose of the allergen to avoid inducing a severe reaction and the dose is slowly increased. This type of therapy is dangerous because compounds that produce the allergic response are actually administered to the subject and severe allergic reactions can occur. Allergy medications include, but are not limited to, antihistamines, corticosteroids, and prostaglandin inducers. Antihistamines are compounds that counteract the release of histamine by mast cells or basophils. These compounds are known in the art and are commonly used for the treatment of allergy. Antihistamines include, but are not limited to acrivastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclizine, cetirizine, cetirizine analogs, chlorpheniramine, clemastine, CS 560, cyproheptadine, desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine, loratidine, metescopolamine, mizolastine, norastemizole, phenindamine, promethazine, pyrilamine, terfenadine and tranilast. Corticosteroids include, but are not limited to, methylprednisolone, prednisolone, prednisone, beclomethasone, budesonide, dexamethasone, flunisolide, fluticasone propionate, and triamcinolone. Although dexamethasone is a corticosteroid that has anti-inflammatory action, it is not commonly used for the treatment of allergy or asthma in an inhaled form because it is highly absorbed and has long-term suppressive side effects with an effective dose. However, dexamethasone can be used according to the invention to treat allergy or asthma because when administered in combination with a composition of the invention it can be administered at a low dose to reduce side effects. Some of the side effects associated with the use of corticosteroids include cough, dysphonia, oral thrush (candidiasis), and with higher doses, systemic effects such as adrenergic suppression, glucose intolerance, osteoporosis, aseptic necrosis of bone, cataract formation , suppression of growth, hypertension, muscle weakness, thinning of the skin and easy bruising. Barnes &; Peterson (1993) Am. Rev. Respir Dis. 148: S1 -S26; and Kamada A.K. et al., (1996) Am. J. Respir Crit. Care Med 153: 1739-48. The compositions and methods of the invention can be used alone or together with other agents and methods for treating asthma. In one aspect, the invention provides a method of treating a subject having asthma. The method according to this aspect of the invention includes the step of administering to a subject having asthma an effective amount of a composition of the invention to treat the subject. In one aspect the invention provides a method for treating a subject having asthma. The method according to this aspect of the invention includes the step of administering to a subject having asthma an effective amount of the composition of the invention and an anti-asthmatic therapy to treat the subject. In one aspect the invention provides a use of an immunostimulatory ORN of the invention for preparing a medicament for treating asthma in a subject.

In one aspect the invention provides a composition useful for the treatment of asthma. The composition according to this aspect includes an immunostimulatory ORN of the invention and a medicament for asthma. "Asthma" as used herein refers to a disorder of the respiratory system characterized by inflammation and narrowing of the airways, and increased reactivity of the airways to inhaled agents. Asthma is frequently, but not exclusively, associated with an atopic or allergic condition. Symptoms of asthma include recurrent episodes of asthmatic wheezing, shortness of breath, tightness in the chest and cough, which are a result of obstruction of the air flow. Inflammation of the airway associated with asthma can be detected by observing a series of physiological changes such as denudation of the airway epithelium, deposition of collagen under the basement membrane, edema, activation of mast cells, infiltration of inflammatory cells including neutrophils, eosinophils and lymphocytes. As a result of airway inflammation, asthmatic patients often experience airway hypersensitivity, airway limitation, respiratory symptoms and chronicity of the disease. Airway limitations include acute bronchoconstriction, airway edema, mucous plug formation, and airway remodeling, characteristics that often lead to bronchial obstruction. In some cases of asthma, fibrosis of the sub-basal membrane can occur leading to persistent abnormalities in lung function. Research in previous years has shown that asthma is probably the result of complex interactions between inflammatory cells, mediators and other cells and tissues residing in the airways. Mast cells, eosinophils, epithelial cells, macrophages and activated T cells all have an important role in the inflammatory process associated with asthma. Djukanovic R. et al (1990) Am. Rev. Respir. Dis. 142: 434-457. It is believed that these cells can influence the function of the airway through the secretion of preformed or newly synthesized mediators that can act directly or indirectly in the local tissue. It has also been recognized that subsets of T lymphocytes (Th2) have an important role in the regulation of allergic inflammation in the airway by the release of selective cytokines and the establishment of the chronicity of the disease. Robinson D.S. and col. (1992) N. Engl. J. Med. 326: 298-304. Asthma is a complex disorder that arises in different stages of development and can be classified based on the degree of symptoms such as acute, subacute or chronic. An inflammatory water response is associated with an early recruitment of cells in the airway. The subacute inflammatory response involves the recruitment of cells as well as the activation of resident cells producing a more persistent inflammatory motive. The chronic inflammatory response is characterized by a persistent level of cell damage and a continuous repair process, which can result in permanent abnormalities in the airway. A "subject having asthma" is a subject who has a disorder of the respiratory system characterized by inflammation and narrowing of the airways and increased reactivity of the airways to inhaled agents. Factors associated with the onset of asthma include, but are not limited to allergens, cold temperatures, exercise, viral infections, and S02. As mentioned above, asthma may be associated with a Th2-type immune response, which is characterized at least in part by Th2 cytokines IL-4 and IL-5, as well as by the change of isotype from antibody to IgE. The Th1 and Th2 immune responses are mutually counterregulatory, so that the inclination of the immune response towards a Th1 type immune response can prevent or improve a Th2 type immune response., including allergy. The modified oligoribonucleotide analogues of the invention are, therefore, useful in themselves to treat a subject having asthma because the analogs can tilt the immune response towards a Th1 type immune response. Alternatively or in addition, the modified oligoribonucleotide analogs of the invention can be used in combination with an allergen to treat a subject having asthma. The immunostimulatory composition of the invention can also be administered in conjunction with a therapy for asthma. Conventional methods for treating or preventing asthma have involved the use of antiallergic therapies (described above) and a number of other agents, including inhaled agents. Medications for the treatment of asthma in general are separated into two categories, fast relief medications and long-term control medications. Patients with asthma take long-term control medication daily to achieve and maintain control of persistent asthma. Long-term control medications include anti-inflammatory agents such as corticosteroids, cromolyn sodium and nedocromoline; long-acting bronchodilators, such as long-acting β2-agonists and methylxanthines; and leukotriene modifiers. Fast-relief medications include short-acting β2-agonists, anticholinergics, and systemic corticosteroids. There are many side effects associated with each of these drugs and none of the drugs alone or combined can prevent or completely treat asthma. Medications for asthma include, but are not limited to, PDE-4 inhibitors, bronchodilator / beta-2 agonists, K + channel opening agents, VLA-4 antagonists, neurokinase antagonists, thromboxane A2 synthesis inhibitors. (TXA2), xanthines, arachidonic acid antagonists, lipoxygenase 5 inhibitors, TXA2 receptor antagonists, TXA2 antagonists, inhibitor of lipox 5 activation proteins and protease inhibitors.

Bronchodilators / β2-agonists are a class of compounds that produce bronchodilation or smooth muscle relaxation. Bronchodilators / β2 agonists include, but are not limited to, salmeterol, salbutamol, albuterol, terbutaline, D2522 / formoterol, fenoterol, bitolterol, pirbuerol-methylxanines and orciprenaline. Long-acting β2-agonists and bronchodilators are compounds that are used to prevent long-term symptoms in addition to anti-inflammatory therapies. Long-acting β2-agonists include, but are not limited to salmeterol and albuterol. These compounds are normally used in combination with corticosteroids and are not normally used without an inflammatory therapy. They have been associated with side effects such as tachycardia, skeletal muscle tremor, hypokalemia, and prolongation of the QTc interval with overdose. Methylxanthines, including, for example, theophylline, have been used for the control and prevention of long-term symptoms. These compounds produce bronchodilation resulting from the inhibition of phosphodiesterase and the probable antagonism of adenosine. Acute toxicities related to the dose are a particular problem with these types of compounds. As a result, serum concentration should be monitored routinely in order to account for the toxicity and narrow therapeutic range that come from individual differences in metabolic clearance. Side effects include tachycardia, tachyarrhythmias, nausea and vomiting, central nervous system stimulation, headache, seizures, hematemesis, hyperglycemia, and hypokalemia. Short-acting β2-agonists include, but are not limited to albuterol, bitolterol, pirbuterol and terbutaline. Some adverse effects associated with the administration of short-acting β2-agonists include tachycardia, skeletal muscle tremor, hypokalemia, increased lactic acid, headache and hyperglycemia. Sodium cromolyn and nedocromil are used as long-term control medications to primarily prevent asthma symptoms that arise from exercise or allergic symptoms that arise from allergens. It is believed that these compounds block early and late reactions to allergens by interfering with the chloride channel function. They also stabilize the membranes of mast cells and inhibit the activation and release of mediators of the inosynophiles and epithelial cells. A period of administration of four to six weeks is normally required to achieve maximum benefit. Anticholinergics are usually used to relieve acute bronchospasm. It is believed that these compounds function by competitive inhibition of cholinergic muscarinic receptors. Anticholinergics include, but are not limited to, ipratropium bromide. These compounds only reverse bronchospasm with cholinergic mediation and do not modify any reaction to the antigen. Side effects include dry mouth and respiratory secretions, increased asthmatic whistling in some individuals, and blurred vision if it is sprayed into the eyes.

The immunostimulatory ORNs of the invention may also be useful for treating airway remodeling. The remodeling of the airway is the result of the proliferation of smooth muscle cells and / or submucosal thickening in the airways, and finally produces narrowing of the airways leading to restricted air flow. The immunostimulatory ORNs of the invention can prevent further remodeling and may even possibly reduce the buildup of tissue resulting from the remodeling process. The immunostimulatory ORNs of the invention are also useful for improving the survival, differentiation, activation and maturation of dendritic cells. Immunostimulatory oligoribonucleotides have the unique ability to promote survival, differentiation, activation and cellular maturation of dendritic cells. The immunostimulatory ORNs of the invention will also increase the lytic activity of natural aggressive cells and antibody-dependent cellular cytotoxicity (ADCC). ADCC can be produced using an immunostimulatory ORN combined with an antibody specific for a cellular target, such as a cancer cell. When the immunostimulatory ORN is administered to a subject together with the antibody, the subject's immune system is induced 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. In one embodiment, the antibody is an IgG antibody. In some aspects the invention provides a method for enhancing epitope expansion. "Epitope expansion" is used herein to refer to the diversification of epitope specificity from a dominant epitope-specific, initial targeted immune response directed against a foreign or proprietary protein to cryptic epitopes and / or subdominants in that epitope. protein (intramolecular expansion) or other proteins (intermolecular expansion). The expansion of epitopes results in specific immune responses of multiple epitopes. The immune response consists of an initial increase phase, which can be harmful, such as an autoimmune disease, or beneficial, such as vaccinations, and a subsequent reduction phase to return the immune system to homeostasis and generate memory. The expansion of epitopes can be an important component of both phases. The enhancement of the epitope expansion in the framework of a tumor allows the subject's immune system to determine additional target epitopes, not recognized initially by the immune system in response to an original therapeutic protocol, while reducing the possibility of variants of the tumor. escape in the tumor population and thus affects the progression of the disease. The oligoribonucleotides of the invention may be useful for promoting the spread of epitopes in therapeutically beneficial indications such as cancer, viral and bacterial infections, and allergy.

The method in one embodiment includes the steps of administering a vaccine comprising an antigen and an adjuvant to a subject and subsequently administering to the subject at least two doses of immunostimulatory ORN of the invention in an amount effective to induce specific immune responses of multiple epitopes. The method in one embodiment includes the steps of administering a vaccine that includes a tumor antigen and an adjuvant to a subject and subsequently administering to it. subjecting at least two doses of immunostimulatory ORN of the invention in an amount effective to induce specific immune responses of multiple epitopes. The method in one embodiment involves applying a therapeutic protocol that results in exposure to the antigen of the immune system in a subject, followed by at least two administrations of an immunostimulatory oligoribonucleotide of the invention, to induce specific immune responses of multiple epitopes, i.e. , promote the expansion of epitopes. In different modalities, the therapeutic protocol is surgery, radiation, chemotherapy, other medicines for cancer, a vaccine or a cancer vaccine. The therapeutic protocol can be implanted together with an immunostimulator, in addition to the subsequent immunostimulatory therapy. For example, when the therapeutic protocol is a vaccine, it can be administered together with an adjuvant. The combination of the vaccine and the adjuvant can be a mixture or separate administrations, i.e., injections (ie, the same drainage field). The administration is not necessarily simultaneous. If non-simultaneous injection is used, scheduling may involve preinjection of the adjuvant followed by vaccine formulation. After implementing the therapeutic protocol, immunostimulatory monotherapy begins. The optimal frequency, duration and site of administration will depend on the objective and other factors, but may be for example a monthly or bimonthly administration for a period of six months to two years. Alternatively, the administration may be daily, weekly or biweekly, or the administration may be multiple times during a day, week or month. In some cases, the duration of administration may depend on the length of therapy, e.g. For example, it may end after a week, a month, after a year or after multiple years. In other cases monotherapy may be continuous such as by an intravenous drip. The immunostimulator can be administered in a common drainage field with the objective. For use in therapy, different doses may be necessary for the treatment of a subject depending on the activity of the compound, the manner of administration, the purpose of the immunization (ie, prophylactic or therapeutic), the nature and severity of the disorder, age and body weight of the subject. The administration of a given dose can be carried out either by a single administration in the form of an individual dose unit or by several smaller dose units. The administration of multiple doses at specific intervals of weeks or months is usual to reinforce the antigen-specific immune responses.

Combined with the teachings provided herein, by choosing among the different active compounds and weight factors such as potency, relative bioavailability, patient body weight, severity of adverse side effects and preferred mode of administration, a regimen of effective prophylactic or therapeutic treatment that does not produce substantial toxicity and is still effective in treating the particular subject. The effective amount for any particular application may vary depending on factors such as the disease or condition to be treated, the particular therapeutic agent to be administered, the size of the subject, or the severity of the disease or condition. One skilled in the art can empirically determine the effective amount of a particular nucleic acid and / or other therapeutic agent without needing undue experimentation. The present doses of the compounds described herein are usually in the range of about 0.1 μg to 10,000 mg, more typically from about 1 μg / day to 8000 mg, and most typically from about 10 μg to 100 μg. Presented in terms of body weight, typical dosages are in the range of from about 0.1 μg to 20 mg / kg / day, more typically from about 1 to 10 mg / kg / day, and most typically from about 1 to 5 mg / day. kg / day. Pharmaceutical compositions containing nucleic acids and / or other compounds can be administered by any suitable route for the administration of medicaments. A variety of administration routes are available. The particular mode selected will, of course, depend on the particular agent or agents selected, the particular condition to be treated and the dosage necessary for therapeutic efficacy. The methods of this invention, generally speaking, can be practiced using any mode of administration that is medically acceptable, which means any mode that produces effective levels of an immune response without producing clinically unacceptable adverse effects. Preferred modes of administration are discussed herein. For use in therapy, an effective amount of the nucleic acid and / or other therapeutic agent can be administered to a subject by any mode that releases the agent on the desired surface, e.g. g., mucosa, systemic. The administration of the pharmaceutical composition of the present invention can be carried out by any means known to the person skilled in the art. Routes of administration include, but are not limited to, oral, parenteral, intravenous, intramuscular, intraperitoneal, intranasal, sublingual, intratracheal, inhalation, subcutaneous, ocular, vaginal and rectal. For the treatment or prevention of asthma or allergy, said compounds are preferably inhaled or ingested or administered systemically. Systemic routes include oral and parenteral routes. Inhaled medications are preferred in some modalities due to direct delivery to the lungs, the site of inflammation, mainly in asthmatic patients. Various types of devices are commonly used for administration by inhalation. These types of devices include metered-dose inhalers (MDI), respiratory-driven MDI, dry powder inhaler (DPI), spacer / container chambers combined with MDI, and nebulizers. The therapeutic agents of the invention can be delivered in a particular tissue, cell type or immune system, or both, with the help of a vector. In its broadest sense, a "vector" is any vehicle that can facilitate the transfer of the compositions to the target cells. The vector generally carries the immunostimulatory nucleic acid, antibody, antigen and / or drug specific for the disorder to the target cells with less degradation with respect to the extent of the degradation that would result in the absence of the vector. In general, the vectors useful in the invention are divided into two classes: biological vectors and chemical / physical vectors. Biological vectors and chemical / physical vectors are useful for the delivery and / or absorption of therapeutic agents of the invention. Most biological vectors are used to deliver nucleic acids, and these would be most suitable for the delivery of therapeutic agents that are or that include immunostimulatory nucleic acids. In addition to the biological vectors discussed herein, chemical / physical vectors can be used to deliver therapeutic agents including immunostimulatory nucleic acids, antibodies, antigens and drugs specific to the disorder. As used herein, a "chemical / physical vector" refers to a natural or synthetic molecule other than that derived from bacteriological or viral sources, which is capable of delivering the nucleic acid and / or another medicament. A preferred chemical / physical vector of the invention is a colloidal dispersion system. Colloidal dispersion systems include lipid-based systems, including oil in water emulsions, micelles, mixed micelles and liposomes. A preferred colloidal system of the invention is a liposome. Liposomes are artificial membrane vessels that are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vesicles (LUV) having a size in the range of 0.2 - 4.0 μ? T? they can encapsulate large macromolecules. RNA, DNA and intact virions can be encapsulated in their aqueous interior and delivered to the cells in a biologically active form. Fraley et al. (1981) Trends Biochem Sci 6:77. The liposomes can be targeted to a particular tissue by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid or protein. Ligands that may be useful for directing a liposome to an immune cell include, but are not limited to, intact molecules or fragments that interact with specific receptors of the immune cell and molecules, such as antibodies that interact with cell surface markers or immune cells.

Such ligands can be readily identified by binding assays known to those skilled in the art. In yet other embodiments, the liposome can be targeted to cancer by coupling it to one of the immunotherapeutic antibodies discussed above. In addition, the vector can be coupled to a leader nuclear peptide, which will direct the vector to the nucleus of the host cell. Lipid formulations for transfection are commercially available from QIAGEN, for example, as EFFECTENE ™ (a non-liposomal lipid with a special DNA condensation enhancer) and SUPERFECT ™ (a new-acting dendrimeric technology). Liposomes are commercially available from Gibco BRL, for example as LIPOFECTIN ™ and LIPOFECTACE ™, which are formed with cationic lipids such as N- [1- (2,3-dioleyloxy) -propyl] -N, N chloride, N-trimethylammonium (DOTMA) and dimethyldioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications. He has also reviewed the liposomes Gregoriadis G. (1985) Trends Biotechnol. 3: 235-241. It appears that some cationic lipids, including in particular N- [1 - (2,3-dioleoyloxy) -propyl] -N, N, N-thmethylammonium methyl sulfate (DOTAP), are especially advantageous when combined with modified oligoribonucleotide analogs of the invention. In one embodiment, the vehicle is a biocompatible implant or microparticle that is suitable for implantation or administration to a mammalian recipient. Exemplary bioerodible implants are described which are useful in accordance with this method in the international application PCT / US / 03307 (Publication No. WO95 / 24929, entitled "Polymeric Gene Delivery System." PCT / US / 0307 describes a polymeric matrix. biocompatible, preferably biodegradable to contain an exogenous gene under the control of a suitable promoter.The polymer matrix can be used to achieve sustained release of the therapeutic agent in the subject.The polymer matrix preferably is in the form of a microparticle such as a microsphere ( wherein the nucleic acid and / or the other therapeutic agent are dispersed by a solid polymeric matrix) or a microcapsule (in which the nucleic acid and / or the other therapeutic agent are stored in the core of a polymeric shell). Polymeric matrix forms to contain the therapeutic agent include films, coatings, gels, implants and stents. n of the polymer matrix device are selected so as to result in favorable release kinetics in the tissue into which the matrix is introduced. The size of the polymer matrix is further selected according to the delivery method to be used, usually by injection into a tissue or administration of an aerosol suspension in the nose and / or lung zone. Preferably, when an aerosol route is used the polymer matrix and the nucleic acid and / or the other therapeutic agent are integrated in a surfactant vehicle. The composition of the polymer matrix can be selected to have both favorable degradation rates and to be formed of a material that is bioadhesive, to further increase the efficiency of the transfer when the matrix is administered on a nasal and / or pulmonary surface that has suffered a wound The composition of the matrix can also be selected so that it does not degrade except for release by diffusion over a prolonged period of time. In some preferred embodiments, the nucleic acid is administered to the subject by an implant while the other therapeutic agent is administered in a limited time. Biocompatible microspheres that are suitable for delivery, such as oral or mucosal delivery, are described by Chickering et al. (1996) Biotech Bioeng 52: 96-101 and Matiowitz E. et al. (1997) Nature 386: 410-414 and PCT patent application WO97 / 03702. Both biodegradable and non-biodegradable polymer matrices can be used to deliver the nucleic acid and / or the other therapeutic agent to the subject. Biodegradable matrices are preferred. Said polymers may be natural or synthetic polymers. The polymer is selected based on the period of time along which the release is desired, in general of the order of a few hours to a year or more. Normally, the most desirable is the release over a period of between a few hours and three to twelve months, in particular for nucleic acid agents. The polymer optionally is in the form of a hydrogel which can absorb up to about 90% of its weight in water and in addition, optionally is crosslinked with multivalent ions or other polymers.

Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J.A., Hubell in Macromolecules, (1993) 26: 581-587, whose teachings are incorporated in this document. These include poly (hyaluronic acids), casein, gelatin, glutin, polyanhydrides, poly (acrylic acid), alginate, chitosan, poly (methyl methacrylates), poly (ethyl methacrylates), poly (butyl methacrylate), poly (methacrylate) isobutyl), poly (hexyl methacrylate), poly (isodecyl metachlate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (acrylate), isobutyl) and poly (octadecyl acrylate). If the therapeutic agent is a nucleic acid, the use of compaction agents may also be convenient. The compaction agents can also be used alone or in combination with a biological or chemical / physical vector. A "compaction agent", as used herein, refers to an agent, such as a histone, that neutralizes the negative charges on the nucleic acid and thus allows the compaction of the nucleic acid in a fine granule. . The compaction of the nucleic acid facilitates the uptake of the nucleic acid by the target cell. Compaction agents can be used alone, that is, to deliver a nucleic acid in a form that is more efficiently captured by the cell, or more preferably, combined with one or more of the vectors described above.

Other exemplary compositions that can be used to facilitate the uptake of a nucleic acid include calcium phosphate and other chemical mediators of intracellular transport, microinjection compositions, electroporation and homologous recombination compositions (eg, to integrate a nucleic acid into a preselected site within the chromosome of the target cell). The compounds can be administered alone (eg, in saline or buffer) or using any delivery vehicle known in the art. For example, the following delivery vehicles have been described: cochleates (Gould-Fogerite et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOM (Mowat et al., 1993, Carlsson et al., 1991, Hu et al., 1998, Morein et al., 1999); liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b); live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus Calmette-Guérin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et al. ., 1999); microspheres (Gupta et al., 3998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997); polymers (eg, carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); polymer rings (Wyatt et al., 1998); proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997); sodium fluoride (Hashi et al., 1998); transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); and virus-like particles (Jiang et al., 1999, Leibl et al., 1998). The formulations of the invention are administered in pharmaceutically acceptable solutions, which can routinely contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. The term "pharmaceutically acceptable carrier" means one or more fillers, diluents or solid or liquid encapsulating substances that are suitable for administration to a human or other vertebrate animal. The term vehicle indicates an organic or inorganic ingredient, natural or synthetic, with which the active principle is combined to facilitate the application. The components of the pharmaceutical compositions can also be mixed with the compounds of the present invention, and with each other, in a manner that there is no interaction that substantially impairs the desired pharmaceutical efficacy. For oral administration, the compounds (ie, nucleic acids, antigens, antibodies and other therapeutic agents) can be formulated easily by combining the active compound (s) with pharmaceutically acceptable carriers known in the art. Said vehicles allow the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as a solid excipient, optionally by grinding the resulting mixture and processing the mixture of granules after adding suitable auxiliary agents, if desired, to obtain tablets or dragee 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, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate. Optionally, oral formulations can also be formulated in saline or buffers to neutralize internal acidic conditions or can be administered without any vehicle. The dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which optionally may contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, suitable organic solvent and varnish solutions or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for the identification or characterization of different combinations of the doses of active compound. Pharmaceutical preparations that can be used orally include pressure setting capsules made of gelatin, as well as soft, closed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The pressure adjustment capsules may contain the active ingredients mixed with fillers 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 can also be used. Said microspheres are well defined in the art. All formulations for oral administration should be in dosages suitable for said administration. For buccal administration, the compositions may be in the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use in accordance with the present invention can be conveniently delivered in the form of an aerosol spray presentation of pressurized containers or a nebulizer, with the use of a suitable propellant, e.g. eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to supply a measured quantity. Capsules and cartridges, p. The gelatin for use in an inhaler or insufflator can be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch. When it is convenient to deliver them systemically, the compounds can be formulated for parenteral administration by injection, e.g. eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may also have forms such as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds in water soluble form. In addition, suspensions of the active compounds can be prepared in the form of oily suspensions suitable for injection. The lipophilic solvents or vehicles include fatty oils 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 suitable stabilizers or 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 to be constituted with a suitable vehicle, e.g. eg, sterile water without pyrogens, before use. The compounds can also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, containing, e.g. eg, conventional bases for suppositories such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a sustained release preparation. Said long-acting formulations can be formulated with suitable polymeric or hydrophobic materials (for example in the form of an emulsion in an acceptable oil) or with ion exchange resins, or as moderately soluble derivatives, for example as a moderately soluble salt. The pharmaceutical compositions may also comprise suitable solid phase or gel carriers 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, with cochleate, forming a coating on microscopic gold particles, contained in liposomes, nebulized, in aerosols, in granules to be implanted in the skin, or dried on a sharp object to scrape on the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with extended release of the active compounds, in which preparations are commonly used excipients and additives and / or auxiliary agents such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweeteners or solubilizers, as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of the methods of drug delivery see Langer R., (1990) Science 249: 1527-1533, which is incorporated herein by reference. The nucleic acids and optionally other therapeutic products and / or antigens can be administered on their own (pure) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but pharmaceutically unacceptable salts can be conveniently used to prepare their pharmaceutically acceptable salts. 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. In addition, said salts can be prepared as alkali or alkaline earth metal salts, such as sodium, potassium or calcium salts of the carboxylic acid group. Suitable buffering agents include: acetic acid and a salt (1-2% w / v); citric acid and a salt (1 -3% w / v); boric acid and a salt (0.5-2.5% w / v); and phosphoric acid and a salt (0.8-2% in 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% in p / v) and thimerosal (0.004-0.02% in p / v). The compositions may conveniently be presented in a unit dosage form and may be prepared by any of the methods known in the pharmacy art. All methods include the step of associating the compounds with a vehicle that constitutes one or more of the auxiliary ingredients. In general, the compositions are prepared by uniform and intimate association of the compounds with a liquid carrier, a finally divided solid support, or both, and then, if necessary, shaping the product. The liquid dose units are vials or ampoules. The solid dose units are tablets, capsules and suppositories.

Other delivery systems may include delivery systems of time release, delayed release or sustained release. Said systems can avoid repeated administrations of the compounds, increasing the convenience of the subject and the doctor. There are many types of delivery delivery systems available and known to those skilled in the art. They include polymer base systems such as poly (lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, poly (hydroxybutyric acid) and polyanhydrides. Microcapsules of the above drug-containing polymers are described, for example, in the U.S.A. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and triglycerides, hydrogel release systems; silastic systems; peptide-based systems; wax coatings; tablets using conventional binders and excipients; partially fused implants; and similar. Specific examples include, but are not limited to: (a) erodible systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Patents. 4,452,775, 4,675,189 and 5,736,152, and (b) diffusion systems in which an active component permeates at a controlled rate from a polymer as described in the U.S. Patents. No. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based delivery systems can be used, some of which are adapted for implantation. The present invention is illustrated in more detail in the following Examples, which should not be considered as limiting.

EXAMPLES EXAMPLE 1 Sensitivity of human PBMCs to oligoribonucleotides containing Methods: Luminex technology Minute pearls with Luminex color codes, called microspheres, in 100 different groups. Each group of beads can be coated with a specific reagent for a particular bioassay, allowing the capture and detection of specific analytes from a sample. Inside the Luminex compact analyzer, the lasers excite the internal dyes that identify each microsphere particle and also each indicator dye captured during the test. Many readings of each group of pearls are made, then validating the results. In this way, Luminex technology allows a multiple process of up to 100 unique tests in a single sample, both quickly and accurately.

Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors, plated and stimulated in vitro with different immunostimulatory assay and control agents for 16 hours. After 16 hours, the supernatants were collected and then analyzed by an ELISA assay. The oligoribonucleotides containing N-U-R1-R2 were tested by complexing with DOTAP and complete titration curves (7 concentrations), starting from 2 μN ORN. complexed with DOTAP 25 μg / ml and with dilution stages 1/3. Also included were some negative controls, including medium alone and DOTAP (culture well 25 μg / ml, "Liposomes") alone. Immunostimulatory control agents included the imidazoquinoline R-848 (2 μ? With dilution steps 1/3 and 7 concentrations) the ligand described for TLR7, ORN having TLR8 motifs such as AU and AUU sequences (SEQ ID NO .: 13 - SEQ ID NO .: 15), ORN having TLR7 / 8 motifs such as CU, GU and GUU sequences (SEQ ID NO .: 19 - SEQ ID NO .: 23). The results are given in Figures 1A-1 C and 3A and 3B. A similar assay was performed which tests the different ORN sequences using stimulation of isolated pDC, monocytes and mDC. The cells were stimulated with ORN 0.5 μ? complexed with DOTAP 10 μg / m \, CpG ODN 0.5 μ? or DOTAP or half alone. After 16 h the supernatants were collected and the levels of IFN-alpha, TNF-alpha and ÍL-12p40 were measured by ELISA. The results are shown in Figures 2A-2C.

Figures 1A-1C show a clear difference between the production of TNF-alpha and IFN-alpha after stimulation of PBMC, for SEQ ID NO .: 12 containing an AU sequence and SEQ ID NO .: 21 which contains a GU sequence. The subsequent sequence analysis revealed that a CUA repeat (SEQ ID NO: 24) is an additional ORN that induces TNF-alpha without IFN-alpha production. Shorter ORNs containing AU and GU repeats (SEQ ID NO .: 29- SEQ ID NO .: 34) showed similar results compared to the longer ones (SEQ ID NO .: 12 - SEQ ID NO .: 23) but with a decrease in efficacy and potency. Figures 2A-2C and 6A-6D show the analysis of AU-ORN (SEQ ID NO .: 13) and GU-ORN (SEQ ID NO .: 21) in isolated monocytes, pDC and mDC, which reflects a strong reduction of IFN-alpha production for AU-ORN (SEQ ID NO .: 13) and a clear production of TNF-alpha and IL-12p40 for both ORN. The production of IFN-alpha after stimulation with pDC ORN appears to be mediated by TLR7, whereas the production of TNF-alpha and IL-12p40 from monocytes and mDC isolates appears to be mediated by TLR8. The Luminex results reflected comparable results with the ELISA data and proved that the main difference between GU-ORN and AU-ORN is due to the production of IFN-alpha and the cytokines / genes related to IFN-alpha (Figures 3A and 3B and 8a). In addition, additional data from Luminex showed that unlike IFN-alpha and the cytokines / genes related to IFN-alpha the other cytokines / chemokines are unaffected (Figures 7A-7D and 8A-8D) with an atypical value of IL-6 of CD123-CD14 cells. This production of IL-6 may be due to the activation of B cells mediated by TLR7.

EXAMPLE 2 Comparison of the maximum activities of IFN-alpha and TNF-alpha oligonucleotides Human PBMCs were stimulated with ORN complexed with DOTAP. After 16 hours the supernatants were collected and the levels of TNF-alpha and IFN-alpha were measured. The average / maximum activities were determined with 0.6 μ? of 3-6 blood donors and two individual experiments. The results are shown in Figures 4A and 4B. These data clearly differentiate between the motifs of TLR8 and TLR7 / 8: the ORN with the NU-R1-R2 motif showed an IFN-alpha production of less than 300 pg / ml while the TLR7 / 8 ORN showed higher IFN production -alpha after stimulation of PBMC (Figure 4a). The ORN of TLR8 and TLR7 / 8 is divided by a red line. In contrast, measurements of TNF-alpha levels indicated that both the ORN with the TLR8 motif and the ORN with the TLR7 / 8 motif stimulated the production of TNF-alpha.

EXAMPLE 3 Comparison of the maximum activity of IFN-alpha with EC50 of IFN-alpha of oligonucleotides Human PBMCs were stimulated with ORN complexed with DOTAP. After 16 hours the supernatants were collected and IFN-alpha levels were measured. The average / maximum activities were determined with 0.6 μ? and the EC50 of the complete titration curves (range: 2 μ? to 0.9 nM) of 3-6 blood donors and two individual experiments. The results are shown in Figures 5A and 5B. The EC50 and maximum activities showed comparable results in relation to the motifs of TLR8 and TLR7 / 8. A low EC50 / high maximum activity represents the ORN of TLR7 / 8 (Figure 5a) while a high EC50 and low maximum activity represents the ORN of TLR8 (Figure 5b). The production of IFN-alpha and TNF-alpha after stimulation of human PBMC was tested for the ORN sequences listed in Table 1. The human PBMC were stimulated for 16 h with the indicated ORN, and the supernatants were collected and the cytokine production was measured by ELISA. Table 2 summarizes the min / max activity and EC50 of the ORN for the production of IFN-alpha and TNF-alpha.

TABLE 1: SEQ ID NO: 11 G * C * C * A * C * C * G * A * G * C * C * G * A * A * U * A * U * A * C * C - + SEQ ID NO 12 A * U * A * U * A * U * A * U * A * U + A * U * A * Ü * A * U * A * U * A * U - + SEQ ID NO 13 U * U * A * U * U * A * U * U * A + U * U + A * U * U * A * U * U * A * U * U - + SEQ ID NO 14 U * U * U * A * U + U * U * A * U + Ü + U * A * U * U * U * A * Ü + U * U * A + + SEQ ID NO 15 U * U * U * U * A * U * U * U * U * A * U * U * U * U * A * U * U * U * U * A + + SEQ ID NO 16 A * A * U * A * A * U * A * A * U + A * A * U * A * A * U * A * A * U * A * A - + SEQ ID NO 17 A * A * A * U * A * A * A * U * A * A * A * U * A * A * A * U * A * A * A * U - + SEQ ID NO 18 A * A * A * A * U * A * A * A * A * U + A * A * A * A * U * A * A * A * A * U - + SEQ ID NO 19 C * U * C * U * C * U * C * U * C * U * C * U * C * U * C * U * C * U * C * U + + SEQ ID NO 20 G * U * G * U * G * U * G * U * G * U * G * U * G * U + G * U * G * Ü * G * U + + SEQ ID NO 21 U * 0 * G * U * U * G * U * U * G * U * U * G * U * U * G * U * U * G * U * U + + SEQ ID NO 22 ü * U * U * G * ü * U * U '*: G * U * -U * U * G-iO * ü * U * G * U * ü * U * G + + SEQ ID NO 23 U * U * U * Ü * G * U * U * U * G * U * U * U * U * G * U * U * U * U * G + + SEQ ID NO 24 C + U * A * C * Ü * A * C * U * A * C * U * A * C * U * A- * C * U * A * C * U - + SEQ ID NO 25 G * U * A * G * U * A * G * U * A * G * U * A * G * U * A * G * U * A * G * U + + SEQ ID NO 26 G * U * C + G * U * C * G * U * C * G * U * C * G * U * C * G * U * C * G * U + + SEQ ID NO 27 I * U * A + I * U * A * I * U * A * I * U * A * I * U * A * I * U * A * I * U + + SEQ ID NO 28 U * U * I * U * U * I * U * U * I'UU * U * I * U * U * I * U * U * I * U * U + + SEQ ID NO 29 U * U * G * U * U + G * U + + SEQ ID NO 30 U * U * A + 0 * 0 * A * U | - + SEQ ID NO 31 U * G * U * G * U * G * U + + SEQ ID NO 32 U * C * U * C * Ü * C * U + + SEQ ID NO 33 U * A * U * A * U * A * U - + SEQ ID NO 34 G * U * A * G * U * A * G + + +: production of cytokines -: there is no production of cytokines TABLE 2: EXAMPLE 4 Synthetic ORN differentiates between the release of IFN-alpha and TNF-alpha after stimulation of human PBMCs pDC CD123 + purified (Figures 10a and 10b) or isolated monocytes (Figure 10c) were incubated with 1μ ORN? complexed with DOTAP 25 μg / ml or DOTAP alone (Figure 10a) or the indicated amounts of ORN complexed with DOTAP or DOTAP alone (Figures 10b-10c). After 16 h the cells were harvested and stained with CD123, CD1 1 c and HLA-DR antibodies (Figures 10a and 10b) or CD14 and CD19 (Figure 10c). The FACS analysis for CD86 showed that the AU-rich ORN (SEQ ID NO .: 13) and the GU-rich ORN (SEQ ID NO .: 21) show differences in the expression of the CD86 surface marker after the stimulation of pDC (Figure 10a). Stimulation with rich ORN in AU SEQ ID NO .: 13 resulted in a very small activation of CD86, whereas stimulation with ORN rich in GU SEQ ID NO .: 21 resulted in significant activation of CD86. It was determined that this activation was dose dependent (Figure 10b). The AU-rich ORN (SEQ ID NO .: 13) and the GU-rich ORN (SEQ ID NO .: 21) showed no difference in the expression of the CD80 surface marker after stimulation of CD14 and CMSP-positive cells (no the data are shown) (Figure 10 c).

EXAMPLE 5 AU-rich ORN (SEQ ID NO .: 13) and GU-rich ORN (SEQ ID NO .: 21) stimulate the specific signaling of human TLR8 in a dose-dependent manner HEK-293 cells insensitive with the expression plasmid of human TLR3 or TLR8 and the construction of the luciferase reporter gene were transfected stably. The cells were incubated with the indicated ORN sequences (10 [mu]? Complexed with DOTAP 50 [mu] lm) or control stimulation (R-848 10 [mu], poly-IC 50 [mu], ODN 10103 3.3 [mu] or DOTAP 50 μ9 / ???) for 16 hours. Activation by N FKB was measured by assaying the activity of luciferase. The results are given as the number of times of induction above the base value (average). A representative experiment of 6 independent repeats is presented (Figure 9a). Stably transfected HEK-293 cells expressing human TLR8 were stimulated with the indicated concentrations of ORN complexed with DOTAP (50 μg / G? -> 1/3 dilution) or DOTAP alone (50 μg / p \\ - > dilution 1/3) for 16 hours. Activation with N FKB was measured by analyzing the activity of luciferase. The results are given as the number of times of induction above the base value (average). A representative experiment of 3 independent repeats is presented (Figure 9b). HEK-293 cells insensitive with the human TLR8 expression plasmid and the luciferase reporter gene construction were stably transfected. The cells were incubated with the indicated ORN sequences (15 μ? Complexed with DOTAP 75 μg / ml) or control stimulation (R-848 15 μ? Or DOTAP 75 μg / vn \) and with medium (left), bafilomycin 200 nM (Baf., medium) or 1 mM chloroquine (CQ, right) for 16 hours. Activation with N FKB was measured by assaying the activity of luciferase. The results are given as the number of times of induction above the base value (average). A representative experiment of 4 independent repeats is presented (Figure 9c). RPMI 8226 cells were preincubated with Intron A 1000 U / ml for 3 hours, washed twice with medium and then stimulated for 16 hours with the indicated concentrations of ORN complexed with DOTAP (50 μg / vn \ -> 1 / dilution 3). The release of cytokines from IP-10 was measured by ELISA. The results are given as pg / ml. A representative experiment of 3 independent repeats is presented (Figure 9d). These data demonstrate the specificity of SEQ ID NO .: 13 and SEQ ID NO .: 21 for TLR8. The test was repeated with an ORN of TLR8 (SEQ ID NO .: 13), an ORN of TLR 7/8 (SEQ ID NO .: 21) and a control ORN (SEQ ID NO .: 5) (Table 3) both with a high dose (HD, 10 μg / ml) and with a low dose (LD, 2.5 μg / ml). Only the treatment with SEQ ID NO: 21 resulted in a significant production of IL-2 and TNF-alpha (Figure 1 a and 1 1 b, respectively). All the ORNs stimulated the production of IFN-? (Figure 1 1 c).

EXAMPLE 6 Mouse macrophages do not respond to AU-rich ORN (SEQ ID NO: 13) in vitro or in vivo Raw264.7 cells were isolated (FIG. 12a), J774 cells (FIG. 12b) and CD1 1 c + purified cells (Milteny, marked with magnetic beads) of mouse splenocytes sv129 (FIGS. 12c-12e) and stimulated with the indicated concentrations of ORN complexed with DOTAP (50 μg / ml and diluted with ORN), R-848 or DOTAP alone (50 μ9 / p ??). After 16 hours (Figures 12a and 12b) or 20 hours (Figures 12c-12e) the supernatants were collected and used in the ELISA for TNF-alpha (Figures 12a and 12b), IL-12p40 (Figure 12c), IFN-alpha (figure 12d) and IP-10 (figure 12e). The data represent an individual of at least three experiments (Figures 12a and 12b) and the average of 3 mice (Figures 12c-12e). To measure the ability of the AU-rich ORN to stimulate mouse cells in vivo, mice were injected with sv129 (n = 5 / group) the indicated amounts of ORN formulated with DOTAP (60, 20 or 6 μg / ml), and He drew blood after 3 hours. The production of IL-12p40 (Figure 12f), IFN-alpha (Figure 12g) and IP-10 (Figure 12h) in whole blood was measured by ELISA.

EXAMPLE 7 Purified rat splenocytes do not respond to the rich ORN in SEQ ID NO. 13 Splenocytes from 3 Sprague-Dawley rats were pooled and stimulated with the indicated concentrations of SEQ ID NO .: 21, SEQ ID NO .: 13 (both complexed with DOTAP 62.5 μg / m with 1/5 dilution), R-848 or DOTAP alone (62.5 μg / ml -> 1/5 dilution). The supernatants were collected after 20 hours and the TNF-alpha levels were measured by ELISA. As shown in Figure 1 3, stimulation with the ORN rich in GU SEQ ID NO. : 21 resulted in the production of TNF-alpha, whereas stimulation with ORN rich in AU SEQ ID NO: 1 3 did not result in the production of TNF-alpha.

EXAMPLE 8 The failure of the rodent cells to respond to the ORN rich in AU SEQ ID NO .: 13 may be a result of the polymorphism of TLR8 between the species The stimulation of human and bovine cells with the ORN rich in UA resulted in the production of cytokines, whereas the stimulation of mouse and rat cells did not. An alignment and analysis of the TLR8 sequence was carried out. Comparison of TLR8 protein sequences between different vertebrates (human, monkey, chimpanzee, dog, cow, pig, mouse and rat) showed large differences in leucine-rich repeat 3 (LRR) of domain 1. While the human being, chimpanzee and monkey are very conserved, the rat, mouse and pig showed eliminations of 4 AA in the position 106 (mouse), 103 (rat) or 102 (pig), and in the cow was demonstrated an insertion of 2 AA (105-106) compared to humans. It is interesting that in the pigs and cows another elimination of 2 AA in the same region was revealed (position 97). It is possible that the deletion in the leucine-rich repeat region of domain 1 may interfere with the binding of the AU-rich ORN.

Equivalents It is considered that the above written descriptive memory is sufficient for an expert in the field to be able to put the invention into practice. The present invention is not limited in scope by the examples provided, since the examples are only an illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims. The advantages of the invention are not necessarily met by each of the embodiments of the invention.

All references, patents and patent publications cited in this application are incorporated herein by reference in their entirety.

Claims (4)

1. NOVELTY OF THE INVENTION CLAIMS 1 .- A method for stimulating the production of a proinflammatory cytokine, comprising: contacting a cell expressing TLR8 in vitro with an RNA oligonucleotide (ORN) comprising: NU-Rr R2, in which N is a ribonucleotide and N does not include U, U is uracil or a derivative thereof and R is a ribonucleotide in which at least one of Ri and R2 is adenosine (A) or cytosine or derivatives thereof, and wherein R is not U unless NU-RrR2 includes at least two A, in which the ORN is not ACCCAUCUAUUAUAUAACUC (SEQ ID NO .: 89) and does not include a TLR7 / 8 motif and in which the ORN has a length of 4-100, in an amount effective to stimulate the production of proinflammatory cytokines and in which the production of IFN-a in response to ORN is not significantly induced with respect to the base value.
2. 2. A method for stimulating the production of a proinflammatory cytokine, comprising: contacting a cell expressing TLR8 in vitro with an RNA oligonucleotide (ORN) comprising: N-U-R R2 > wherein N is a ribonucleotide and N does not include U, U is uracil or a derivative thereof and R is a ribonucleotide in which at least one of Ri and R2 is adenosine (A) or cytosine or derivatives thereof, and wherein R is not U unless NUR R2 includes at least two A, in which the ORN does not include a TLR7 / 8 motif and does not complex with N- [1 - (2,3-dioleoyloxy) propyl] -?,?,? - t methylammonium methyl sulfate (DOTAP) and in which the ORN has a length of 4-100, in an amount effective to stimulate the production of proinflammatory cytokines and in which the production of IFN-a in Response to the ORN is not significantly induced with respect to the base value.
3. 3. The method according to claim 1 or 2, further characterized in that the production of IFN-a in response to ORN is less than 300 pg / ml.
4. 4. The method according to claim 1, further characterized in that the ORN is complexed with the N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium methyl sulfate (DOTAP) or a polycationic peptide. 5. - The method according to claim 1 or 2, further characterized in that N is adenosine or cytosine (C) or derivatives thereof. 6. - The method according to claim 1 or 2, further characterized in that N is an adenosine or cytosine. 7. - The method according to claim 1 or 2, further characterized in that U is uracil. 8. The method according to claim 1 or 2, further characterized in that the ORN includes at least one AU. 9. The method according to claim 1 or 2, further characterized in that the ORN includes at least one CU. 10. The method according to claim 1 or 2, further characterized in that the cell expressing the TLR8 is a monocyte or a dendritic cell derived from monocyte. eleven . - The method according to claim 1 or 2, further characterized in that the cell expressing the TLR8 is a mDC. 12. - The method according to claim 1 or 2, further characterized in that the ORN includes at least two AL). 13. - The method according to claim 1 or 2, further characterized in that the ORN includes at least three AU. 14. The method according to claim 1 or 2, further characterized in that NU-R1-R2 includes at least 3 A. 15. The method according to claim 1 or 2, further characterized in that NU-R1-R2 includes at least 2 C. 16. - The method according to claim 1 or 2, further characterized in that NUR R2 includes at least one G or C. 17. - The method according to claim 1 or 2, further characterized in that the ORN includes at least one modification of the main chain. 18. - The method according to claim 1 or 2, further characterized in that the ORN is single-stranded. 19. - An RNA oligonucleotide (ORN) comprising: N-U-Rr R2, wherein N is a ribonucleotide and N does not include U, U is uracil or a derivative thereof, and R is a ribonucleotide in which at least one of R1 and R2 is adenosine (A) or cytosine or derivatives thereof, and wherein R is not U except that NU- R1-R2 includes at least two A, in which the ORN is not ACCCAUCUAUUAUAUAACUC (SEQ ID NO .: 89) and does not include a TLR7 / 8 motif and in which the ORN has a length of 4-100, and includes at least one modification of the main chain. 20. The ORN according to claim 19, further characterized in that it additionally comprises a pharmaceutically acceptable carrier. twenty-one . - The ORN according to claim 19, further characterized in that the ORN is complexed with N- [1 - (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium methyl sulfate (DOTAP). 22. - The ORN according to claim 19, further characterized in that the ORN is single-stranded. 23. - An RNA oligonucleotide (ORN) comprising: N-U-R-r R2 >; wherein N is a hbonucleotide and N does not include U, U is uracil or a derivative thereof, and R is a hbonucleotide in which at least one of R1 and R2 is adenosine (A) or cytosine or derivatives thereof, and in which R is not U unless NU-R1-R2 includes at least two A, in which the ORN does not include a TLR7 / 8 motif and is not complexed with the N- [1 - (2,3- dioleoyloxy) propyl] -?,?,? - trimethylammonium methyl sulfate (DOTAP) and wherein the ORN is 4-100 in length, and includes at least one modification of the main chain and is formulated in a pharmaceutically acceptable carrier. 24. - The ORN according to claim 19 or 23, further characterized in that N is an adenosine or cytosine (C) or derivatives thereof. 25. - The ORN according to claim 19 or 23, further characterized in that N is adenosine or cytosine. 26. - The ORN according to claim 19 or 23, further characterized in that U is uracil. 27. - The ORN according to claim 19 or 23, further characterized in that the ORN includes at least one AU. 28. The ORN according to claim 19 or 23, further characterized in that the ORN includes at least one CU. 29. - The ORN according to claim 19 or 23, further characterized in that NUR R2 includes at least 3 A. 30. - The ORN according to claim 19 or 23, further characterized in that NU-RrR2 includes at least 2 C 31. - The ORN according to claim 19 or 23, further characterized in that N-U-R1-R2 includes at least one G or C. 32. - The ORN according to claim 19 or 23, further characterized in that the ORN is single-stranded. 33. The ORN according to claim 19 or 23, further characterized in that the ORN is one of the following. U * U * A * G * G * C * A * C (SEQ ID N0: 2), A * U * A * G * G * C * A * C (SEQ ID N0: 4), G * C * C * A + C * C * G * A * G * C * C * G * A * A ^ U!, IA * A: C * C (SEQ ID NO: 11), A * U * A * U * A * U * A * U * A * U * A * U * A * U * A * U * A * U * A1,, U (SEQ ID NO: 12), U * U * A * U * U * A! T; U * U ,, iA * U * Ü * A + ü * U + A * U * U ,, tA * U * U (SEQ ID NO: 13), A * A * U * A * A * U * AWU * A¾ * U * A * A * U * A * A * U * A * A (SEQ ID NO: 16), A * A * A * U * A * A * A * U * A * A * A * U * A * A * A * U * A * A * A * U (SEQ ID NO: 17), A * A * A * AnJ * A * A * A * AnJ * A WA * A * U * A * A * A * A * U (SEQ ED NO: 18), C * U * A 'C + U * AiitC * U * Alf £ C * U * A * C * U3,: A * C!, TU * A * C * U (SEQ ID NO: 24), U * U * A * U * U * A * U (SEQ ID NO: 30), or U * A * U * A * U * A * U (SEQ ID NO: 33). 34. - The ORN according to claim 19 or 23, further characterized in that it additionally comprises a nebulizer. 35. - The ORN according to claim 19 or 23, further characterized in that it additionally comprises an inhaler. 36. - The ORN according to claim 35, further characterized in that the inhaler is a metered dose inhaler. 37.- The ORN according to claim 35, further characterized in that the inhaler is a powder inhaler. 38. - The ORN according to claim 19 or 23, further characterized in that it additionally comprises a chemotherapeutic agent. 39. - The ORN according to claim 19 or 23, further characterized in that it additionally comprises an antiviral agent. 40. - The ORN according to claim 19 or 23, further characterized in that it additionally comprises an antigen. 41 - The ORN according to claim 20 or 23, further characterized in that the pharmaceutically acceptable carrier is formulated for injection. 42. - The ORN according to claim 20 or 23, further characterized in that the pharmaceutically acceptable carrier is formulated for mucosal administration. 43. - The ORN according to claim 19 or 23, further characterized in that the ORN motif is separated from a 5 'ribonucleotide by a non-nucleotide linker. 44. The ORN according to claim 19 or 23, further characterized in that the ORN motif is separated from a 3 'ribonucleotide by a non-nucleotide linker. 45. - The ORN according to claim 19 or 23, further characterized in that the ORN motif is separated from a 5 'and 3' ribonucleotide by a non-nucleotide linker. 46. - The ORN according to claim 19 or 23, further characterized in that the ORN is formulated in a polycationic vehicle. 47. The use of an ORN composition of any of claims 19-42, in the manufacture of a medicament useful for the treatment of a cancer. 48. The use as claimed in claim 47, wherein the medicament is formulated to be administrable with a chemotherapeutic product. 49. - Use as claimed in claim 47, in which the drug is formulated to be administrable with radiation. 50. - The use of an ORN composition of any of claims 19-42, in the manufacture of a medicament useful for treating asthma. 51 - The use of an ORN composition of any of claims 19-42, in the manufacture of a medicament useful for treating the allergy. 52. - The use as claimed in claim 51, wherein the allergy is allergic rhinitis. 53. The use of an ORN composition of any of claims 19-42, in the manufacture of a medicament useful for modulating an immune response. 54. The use as claimed in claim 53, wherein the medicament is useful in the treatment of an autoimmune disease. 55. - The use as claimed in claim 53, wherein the medicament is useful in the treatment of airway remodeling. 56. The use as claimed in claim 53, wherein the medicament is formulated to be administrable with an antigen. 57. - The use as claimed in claim 53, wherein the medicament is formulated to be administrable by a route selected from the group consisting of oral, nasal, sublingual, intravenous, subcutaneous, mucosal, respiratory, direct injection and dermal 58. The use as claimed in claim 53, wherein the immune response comprises the induction of cytokines. 59. The use as claimed in claim 58, wherein the cytokine is selected from the group consisting of TNFa, IL-10, IL-6, IFN-α, MCP1 and IL-12. 60. The use of an ORN composition of any of claims 19-42, in the manufacture of a medicament useful for treating asthma exacerbated by viral infection. 61 - The use of an ORN composition of any of claims 19-42, in the manufacture of a medicament useful for treating an infectious disease. 62. - The use as claimed in claim 61, wherein the medicament is useful in the treatment of a viral infection. 63. - The use as claimed in claim 62, wherein the viral infection is hepatitis B. 64. - The use as claimed in claim 62, wherein the viral infection is hepatitis C. 65. - The use as claimed in claim 62, wherein the medicament is formulated to be administrable with an antiviral agent. 66. - The use as claimed in claim 65, wherein the antiviral agent is linked to the ORN. 67. - The use as claimed in claim 61, wherein the medicament is formulated to be administrable by a route selected from the group consisting of oral, nasal, sublingual, intravenous, subcutaneous, mucosal, respiratory, direct injection and dermal 68. The use as claimed in claim 47, wherein the medicament is formulated to be administrable with an immunostimulatory CpG nucleic acid. 69. The use as claimed in claim 68, wherein the ORN and the immunostimulatory CpG nucleic acid are formulated to be administrable separately. 70. The use as claimed in claim 68, wherein the ORN and the immunostimulatory CpG nucleic acid are formulated in the form of a conjugate. 71 - The use as claimed in claim 68, wherein the ORN and the immunostimulatory CpG nucleic acid are formulated as a mixture. 72. - A method for downregulating immunosuppressive CD4 + regulatory (Treg) cells, the method comprising contacting a CD4 + Treg cell in vitro with a composition comprising an ORN of any of claims 19-42 in an effective amount to reduce the inhibitory effect of the CD4 + Treg cell. 73. The method according to claim 72, further characterized in that the composition further includes an immunostimulatory CpG nucleic acid. 74. - The method according to claim 73, further characterized in that the ORN and the immunostimulatory CpG nucleic acid are not bound. 75. The method according to claim 73, further characterized in that the ORN and the immunostimulatory CpG nucleic acid are present in the form of a conjugate.