MXPA06004891A - C-class oligonucleotide analogs with enhanced immunostimulatory potency - Google Patents

Abstract

The invention relates to a class of CpG immunostimulatory oligonucleotides containing a CpG immunostimulatory motif and a second motif which is capable of forming secondary structure, including duplex and higher order structures, in vitro and in vivo. The oligonucleotides of the invention are useful as adjuvants in vaccination. The oligonucleotides are also useful for inducing an immune response, inducing expression of a type I interferon (IFN), inducing expression of gamma interferon (IFN-&ggr;), and for treating a variety of conditions, including allergy, asthma, infection, and cancer.

Description

CLASS C OLIGONUCLEOTIDE ANALOGS WITH IMPROVED IMMUNOESTIMULATING POWER FIELD OF THE INVENTION The present invention relates in general to the immunostimulatory nucleic acids, their compositions and methods for using the immunostimulatory nucleic acids.

BACKGROUND OF THE INVENTION Bacterial DNA has immunostimulatory effects to activate B cells and natural killer cells, but the DNA of vertebrates does not have such effects. Tokunaga T et al. (1988) Jpn J Cancer Res 79: 682-6; Tokunaga T et al. (1984) JNCI 72: 995-62; Messina JP et al. (1991) J Immunol 147: 1759-64; and revision in Krieg, 1998, in: Applied Oligonucleotide Technology, C.A. Stein and A.M. Krieg, (Eds.), John Wiley and Sons, Inc., New York, NY, pp. 431-448 and Krieg AM (2002) Annu Rev Immunol 20: 709-60. It is now known that these immunostimulatory effects of bacterial DNA are the consequence of the presence of non-methylated CpG dinucleotides in particular base contexts (CpG motifs), which are common in bacterial DNA, but are methylated and have a sub-representation in the DNA of vertebrates. Krieg AM et al. (1995) Nature 374: 546-9; Krieg AM (1999) Biochim Biophys Acta 1489: 107-16. The immunostimulatory effects of the bacterial DNA can be mimicked with synthetic oligodeoxynucleotides (ODN) containing these CpG motifs. These CpG ODNs have highly stimulating effects on human and murine leukocytes, and induce the proliferation of B cells, the secretion of cytokines and immunoglobulins, the lytic activity of natural killer cells (NK) and the secretion of interferon gamma (IFN-α). ); and the activation of dendritic cells (DCs) and other cells that present antigens to express costimulatory molecules and secrete cytokines, especially the Th1-like cytokines that are important in promoting the development of Th1-like responses of T cells. These immunostimulatory effects of the natural phosphodiester CpG main chain ODN are highly specific for CpG since the effects are significantly reduced if the CpG motif is methylated, if it is changed by a GpG or if it is deleted or altered in some other way. Krieg AM et al. (1995) Nature 374: 546-9; Hartmann G and others (1999) Proc Nati. Acad Sci USA 96: 9305-10. In the first studies, it was believed that the immunostimulatory CpG motif followed the purine-purine-CpG-pyrimidine-pyrimidine formula. Krieg AM et al. (1995) Nature 374: 546-9; Pisetsky DS (1996) J Immunol 156: 421-3; Hacker H et al. (1998) EMBO J 17: 6230-40; Lipford GB et al. (1998) Trends Microbiol 6: 496-500. However, it is now known that mouse lymphocytes respond quite well to CpG phosphodiester motifs that do not follow this "formula" (Yi AK et al. (1998) J Immunol 160: 5898-906) and the same is true for B cells. and human dendritic cells (Hartmann G et al. (1999) Proc. Nati Acad Sci USA 96: 9305-10; Liang H et al. (1996) J Clin Invest 98: 1119-29).

BRIEF DESCRIPTION OF THE INVENTION The present invention relates, in part, to immunostimulatory CpG containing oligonucleotides and oligonucleotide analogues having a secondary structure with an inverted repeat at the 3 'end or near the 3' end of the molecule. The secondary structure includes the formation of double or higher order structures in certain conditions. An important feature of the oligonucleotides and oligonucleotide analogs of the invention is that the inverted repeat is not a strict Watson-Crick palindrome but can be interrupted by intervening the nucleotide sequence or analogs. Another feature of the oligonucleotides and oligonucleotide analogs of the invention is that the backbone can be modified to include nuclease-resistant, nuclease-resistant, and nuclease-sensitive links strategically located, thereby promoting activity and reducing possible toxicity. further, it is known that these oligonucleotides and oligonucleotide analogues have both class A and class B immunostimulatory activity and, consequently, are classified as novel class C immunostimulatory nucleic acid molecules. This invention is based, in part, on the discovery of the inventors of the present that immunostimulatory oligonucleotides containing CpG and oligonucleotide analogs containing an imperfect palindrome at the 3 'end or near the 3' end of the molecule have certain advantages both in terms of their preparation and their biological activity. Specifically, the oligonucleotides and class C oligonucleotide analogs of this invention are characteristically monomeric in solution. It is believed that these same nucleic acid molecules can form double intramolecular structures in vitro, which makes them stable against nuclease digestion. It is also believed that these same nucleic acid molecules can form double intermolecular structures and, possibly, even of higher order within the medium of the intraendosomal compartment, where they are believed to exert their biological activity. In one aspect, the invention provides a composition that includes an immunostimulatory nucleic acid molecule of the formula I Z [(XiYiRi) N (X2Y2R2) k Z2] p (S1 q N '(Nn) ... (N2) (N1) S2 (N1 #) (N2 #) ... (Nn #) Z3 (Formula I ) wherein each Z- ?, Z2 and Z3 is independently any sequence from 0 to 12 nucleotides in length which optionally includes a non-nucleotide linker or a d-basic spacer; each Xi and X2 is independently a nucleotide containing thymine, uracil, adenine or a substituted uracil at position 5; each Y and Y2 is independently a cytosine (C) or a modified cytosine; each Ri and R2 is independently a guanine (G) or a modified guanine; each N and N 'is independently any sequence of 0 to 12 nucleotides in length which optionally includes a non-nucleotide linker or a d-basic spacer; If it is a non-nucleotide linker, an abasic linker (spacers d), triethylene glycol units or hexaethylene glycol units, which optionally provide internucleoside linkages at the positions 2'5'-, 5'5'-, 3'3'-, 2 '2'- or 2'3'-; S2 is any non-palindromic sequence of 1 to 10 nucleotides in length or a non-nucleotide linker, an abasic linker (spacers d), triethylene glycol units or hexaethylene glycol units; every N-i, N2? ... Nn, and N -? #, N2 #, ... Nn # is any modified nucleotide or nucleotide where Ni forms a base pair with N -? #, N2 forms a base pair with N2 #, .. and Nn forms a pair of bases with Nn #; k is an integer from 0 to 5; n is an integer from 2 to 16; p is an integer from 1 to 6; yq is an integer from 0 to 10 and where, when (Nn) ... (N2) (N-?) S2 (N? #) (N2 #) ... (Nn #) is 10 to 42 nucleotides of length, S2 is 4 to 10 nucleotides in length, S2 comprises a non-nucleotide linker, an abasic linker (spacers d), triethylene glycol units or units of hexaethylene glycol, and / or (Nn) ... (N2) (N? ) S2 (N? #) (N2 #) ... (Nn #) has a GC content that is less than 2/3. In one modality, each Ni, N2, ... Nn and N -? #, N2 #, ... Nn # is chosen from C, G or modifications thereof, where C forms a pair of bases with G. In one modality, each Ni, N2, ... Nn and N -? #, N2? ... Np # is chosen among T, A or modifications thereof, where T forms a pair of bases with A. In these and in other modalities, each C, G, A and T can refer to deoxynucleotides with their corresponding bases cytosine, guanine, adenine and thymine. In a modality, each Ni, N2, ... Nn and N -? #, N2 #, ... Nn # is chosen between C, T, A, G or its modifications and C forms a pair of bases with G, T forms a pair of bases with G, A forms a pair of bases with T and A forms a pair of bases with G. In a modality, each Ni, N2? ... Nn and N? #, N2 #, ... Nn # is chosen between unmodified or modified nucleotides that form base pairs according to the Watson-Crick model, that is, each base pair N -? - N ? #, N2-N2 #, ... Nn-Nn # is a pair of Watson-Crick bases. In one embodiment, at least one of each Ni, N2 ... Nn and N1, N2 #, ... Nn # is chosen from unmodified or modified nucleotides that form base pairs that are not Watson-Crick, ie , at least a pair of bases N? -N -? #, N2 -N2 #, ... Nn-Nn- # is a base pair that is not Watson-Crick. In one embodiment, the immunostimulatory nucleic acid molecule includes a partially stabilized backbone having at least one phosphodiester linkage. In one embodiment, the immunostimulatory nucleic acid molecule includes a backbone that has at least one stabilized internucleotide linkage. In one embodiment, the intemucleotide bonds of the oligonucleotide are all phosphorothioate linkages. In one embodiment, the immunostimulatory nucleic acid molecule includes a backbone partially stabilized with a phosphodiester bond that binds at least one of Y1R1 or Y2R2. In one embodiment, Y1 is C. In one embodiment, Ri is G. In one embodiment, Y1 is C and Ri is G. In one embodiment, Xi or X2 is T. In one embodiment, X1 is T, X2 is T, Yi is C, Ri is G and k is 1. In a modality Xi is T, X2 is T, Yi is C, Ri is G, k is 1, p is 1, each N and N 'and Z3 contains zero nucleotides and Z2 is liliod (UUUU), where d (UUUU) represents dUdUdUdU, that is, (deoxyU). In one embodiment, S2 is a non-nucleotide linker. In one embodiment, S2 contains at least one residue of d-spacer spacer. In one embodiment, the oligonucleotide includes at least one non-nucleoside branched linkage. In one embodiment, the immunostimulatory nucleic acid molecule includes at least one duplicating unit, at least one triplicated unit, or at least one duplicating unit and at least one tripling unit. In one mode, Si is a duplicating unit or a tripling unit. In one embodiment, the oligonucleotide includes at least one n-nucleoside bond at the 2'5'-, 5'5'-, 3'3'-, 2'2'-, or 2'3'- position. In one embodiment, the immunostimulatory nucleic acid molecule of formula I is not an antisense nucleic acid. In one aspect, the invention provides an immunostimulatory nucleic acid molecule of formula II Zi (Nn) (Nn.1) ... (N2) (N1) S2 (N1 #) (N2 #) ... (Nn.1 #) (#) (S ^) q Z3 [(X ^ Ri ) N (X2Y2R2) k Z2] p (Formula ll) wherein each Z1 (Z2 and Z3 is independently any sequence of 0 to 12 nucleotides in length which optionally includes a non-nucleotide linker or a d-basic spacer, each Xi and X2 is independently a nucleotide containing thymine, uracil, adenine or a substituted uracil in position 5, each Yi and Y2 is independently a cytosine (C) or a modified cytosine, each R-] and R2 is independently a guanine (G) or a modified guanine, N is any sequence from 0 to 12 nucleotides in length which optionally includes a non-nucleotide linker or a d-basic spacer, Si is a non-nucleotide linker, an abasic linker (spacers d), triethylene glycol units or hexaethylene glycol units, which optionally provide intemucleoside linkages at the 2'5'- positions, 5'5'-, 3'3'-, 2'2'- or 2'3'-; S2 is any non-palindromic sequence of 1 to 10 nucleotides in length or a non-nucleotide eniazador, an abasic linker (spain) c) d), triethylene glycol units or hexaethylene glycol units; each Ni, N2, ... Nn_-t, Nn and N? #, N2 #, ... Nn -? #, Nn # is any modified nucleotide or nucleotide where Ni forms a base pair with N? #, N2 form a pair of bases with N2 #, ..., Nn-? forms a pair of bases with Nn -? # and Nn forms a pair of bases with Nn #; k is an integer from 0 to 5; n is an integer from 2 to 16; p is an integer from 1 to 6 and q is an integer from 0 to 10 and where, when (Nn) ... (N2) (N?) S2 (N? #) (N2 #) ... (Nn #) is 10 to 42 nucleotides in length, S2 is 4 to 10 nucleotides in length, S2 comprises a non-nucleotide linker, an abasic linker (spacers d), triethylene glycol units or units of hexaethylene glycol, and / or (Nn) .. . (N2) (N?) S2 (N1 #) (N2 #) ... (Nn #) has a GC content that is less than 2/3. In one embodiment, Z-i (Nn) (Nn-?) Is TYR, where Y is a cytosine or a modified cytosine and R is a guanine or a modified guanine. In one modality, each N-i, N2? ... Nn- ?, Nn and N1, N2.. Nn_1 #, Nn # is chosen from C, G or modifications thereof, where C forms a pair of bases with G. In a modality, each Ni, N2, ... Nn- ?, Np and N -? #, N2 #? ... Nn.1, Nn # is chosen between T, A or modifications of them, and T forms a pair of bases with A. In these and in other modalities, each C, G, A and T can refer to deoxynucleotides with its corresponding bases cytosine, guanine, adenine and thymine. In one modality, each Ni, N2, ... N ^, Nn, and N? #, N2 #, ... Nn -? #, Nn # is chosen from C, T, A, G or their modifications, and C forms a pair of bases with G, T forms a pair of bases with G, A forms a pair of bases with T and A forms a pair of bases with G. In a modality, each Ni, N2, ... Nn- ?, Nn, and N? #, N2 #, ... Nn -? #, Nn # is chosen between unmodified or modified nucleotides that form base pairs according to the Watson-Crick model, that is, each base pair N? -N -? #, N2-N2 #, ... Nn-Nn # it's a pair of Watson-Crick bases. In one embodiment, at least one of each Ni, N2, ... Nn- ?, Nn, and N? #, N2.. Nn -? #, Nn # is chosen from unmodified or modified nucleotides that form pairs of bases that are not of Watson-Crick, that is to say, at least a pair of bases NN? #, N2-N2.. Nn-Nn # is a pair of bases that is not of Watson-Crick. In one embodiment, the immunostimulatory nucleic acid molecule includes a partially stabilized backbone having at least one phosphodiester linkage. In one embodiment, the immunostimulatory nucleic acid molecule includes a backbone that has at least one stabilized internucleotide linkage. In one embodiment, the internucleotide linkages of the oligonucleotide are all phosphorothioate linkages. In one embodiment, the immunostimulatory nucleic acid molecule includes a backbone partially stabilized with a phosphodiester bond that binds at least one of Y1R1 or Y2R2. In one modality, Y-i is C. In one modality, Ri is G. In a modality,, Yi is C and Ri is G. In one modality, Xi or X is T. In one modality, Xi is T, X2 is T, Yi is C, Ri is G and k is 1. In a modality Xi is T, X2 is T, Yi is C, Ri is G, k is 1, p is 1, each N and N 'and Z3 contains zero nucleotides and Z2 is IIII od (UUUU), where d (UUUU) represents (deoxyU). In an S2 modality it is a non-nucleotide linker. In an S2 mode it contains at least one residue of d-spacer spacer. In one embodiment, the oligonucleotide includes at least one non-nucleoside branched linkage. In one embodiment, the immunostimulatory nucleic acid molecule includes at least one duplicating unit, at least one tripling unit or at least one duplicating unit and at least one tripling unit.

In an S-i mode it is a duplicating unit or a tripling unit. In one embodiment, the oligonucleotide includes at least one internucleoside link at the 2'5'-, 5'5'-, 3'3'-, 2'2'-, or 2'3'- position. In one embodiment, the immunostimulatory nucleic acid molecule of formula I is not an antisense nucleic acid. In one aspect, the invention provides an immunostimulatory nucleic acid molecule of the formula III (Z ') m Z3 (S3) (Formula lll) where Z 'is Z? [(X? Y-, R?) N (X2Y2R2) k Z2] p (S ^ q N '(Nn) ... (N3) (N2) (N1) S2 (N? #) (N2 #) ( N3 #) ... (Nn #); each Z- ?, Z2 and Z3 is independently any sequence from 0 to 12 nucleotides in length which optionally includes a non-nucleotide linker or a d-basic spacer, each Xi and X2 is independently a nucleotide containing thymine, uracil, adenine or uracil substituted at the 5 position, each Yi and Y2 is independently a cytosine or a modified cytosine; each Ri and R2 is independently a guanine or a modified guanine; each N and N 'is independently any sequence 0 to 12 nucleotides long which optionally includes a non-nucleotidic linker or a spacer d abasic; If a non-nucleotidic linker, an abasic linker (spacer d) units of triethylene glycol units or hexaethylene glycol, optionally provide internucleoside linkages in the positions 2'5'-, 5'5'-, 3'3'-, 2'2'- or 2'3'-; S2 is any non-palindrical sequence mica 1 to 10 nucleotides long or a non nucieotídico linker, an abasic linker (spacer d), triethylene glycol units or hexaethylene glycol units; S3 is a direct or indirect internucleoside link at the 2'5'-, 5'5'-, 3'3'-, 2'2'- or 2'3'- positions or a non-nucleotide linker, said non-nucleotide linker including abasic linkers (spacers d), triethylene glycol units or units of hexaethylene glycol that facilitate a link at position 2'5'-, 5'5'-, 3'3'-, 2'2'- or 2'3 ' - of m parts of the sequence; each Ni, N2, ... Nn and N? #, N2 #, ... Nn # is any modified nucleotide or nucleotide where Ni forms a base pair with N? #, N2 forms a base pair with N2 #, N3 forms a pair of bases with N3 ... and Nn forms a pair of bases with Nn #; k is an integer from 0 to 5; m is an integer from 2 to 10, n is an integer from 2 to 16; p is an integer from 1 to 6 and q is an integer from 0 to 10. In certain modalities, Zi [(X1Y1R1) N (X2Y2R2) k Z2] p (S?) q is a non-palindromic sequence. In certain embodiments, Z1 [(X1Y1R1) N (X2Y2R2) k Z2] p (? S) q is TCGTCGTTTT (SEQ ID NO: 40), TCGTCGTTLL, TCGA, TCGAC, TCGACGTC or TCGACGTCG, where L is a spacer d. In certain modalities Z1 [(X1Y1R1) N (X2Y2R2) k Z2] p (S?) Q is a palindromic sequence. In certain embodiments, Z [(X1Y1R1) N (X2Y2R2) k Z2] p (S?) Q is TCGACGTCGA (SEQ ID NO: 19) or TCGTCGACGA (SEQ ID NO: 34) In certain embodiments, Zi [(X1Y1R1) N (X2Y2R2) k Z2] p (S?) Q is TCGCGACGTT (SEQ ID NO: 26) or TCGCGTCGTT (SEQ ID NO: 69). In one mode (Nn) ... (N2) (N?) S2 (N? #) (N2 #) ... (Nn #) Z3 includes a sequence AGCGAAGCT, CAATATTTATTG (SEQ ID NO: 1), CCGTTTTGTGG ( SEQ ID NO: 2), CGGCGCCGTGCCG (SEQ ID NO: 19), CGGCGCCGTTGCCG (SEQ ID NO: 34), CGGCGLLCGCCG (SEQ ID NO: 5), CGGCGLLLTGCCG (SEQ ID NO: 6), CGGCGGLLCCGCCG (SEQ ID NO: 7 ), CGGCGTCGCCGCCG (SEQ ID NO: 8), CGTCGACGGGACGGG (SEQ ID NO: 10), CGTCGACGTGACGGG (SEQ ID NO: 11), GAGAGTTGGGCTCTC (SEQ ID NO: 12), GTCGAGGAGGT (SEQ ID NO: 14), TAATALLTATTA (SEQ ID NO: 15), TAATATCCATTA (SEQ ID NO: 16), or TAATATTTATTA (SEQ ID NO: 17), where L is a spacer d. In one embodiment (Nn) ... (N2) (N) S2 (N #) (N2 #) includes a GGCGCGCTGCCG (SEQ ID NO: 13) sequence in one embodiment the immunostimulatory nucleic acid molecule includes a sequence TCGACGTCGACCGTTTTGTGG (SEQ ID NO: 20), TCGACGTCGACGGGACGGG (SEQ ID NO: 21), TCGACGTCGACGTGACGGG (SEQ ID NO: 22), TCGACGTCGAGAGTTGGGCTCTC (SEQ ID NO: 23), TCGACGTCGAGCGAAGCT (SEQ ID NO: 24), or TCGACGTCGAGGAGGT (SEQ ID NO: 25) In one embodiment the immunostimulatory nucleic acid molecule includes a sequence TCGTCGTTLLACGGCGCCGTGCCG (SEQ ID NO: 37), TCGTCGTTLLACGGCGLLLTGCCG (SEQ ID NO: 38), TCGTCGTTLLCGGCGCGGCGCCG (SEQ ID NO: 39), TCGTCGTTTTACGGCGCCGTTGCCG (SEQ ID NO: 44), TCGTCGTTTTACGGCGLLLTGCCG (SEQ ID NO: 45), TCGTCGTTTTACGGCGTTTTGCCG (SEQ ID NO: 49), TCGTCGTTTTCAATATTTATTG (SEQ ID NO: 50), TCGTCGTTTTCGGCGLLCGCCG (SEQ ID NO: 52), TCGTCGTTTTCGGCGGLLCCGCCG (SEQ ID NO: 54), TCGTCGTTTTCGGCGTCGCCGCCG (SEQ ID NO: 55), TCGTCGTT "I TTAATALLTATTA (SEQ ID NO: 57), TCGTCGTTTTATATCCATTA (SEQ ID NO: 58), or TCGTCGTTTTTAATATTTATTA (SEQ ID NO: 59), where L is a spacer d.In one embodiment the immunostimulatory nucleic acid molecule includes a sequence TCGCGTCGTTCGGCGCGCTGCCG (SEQ ID NO: 30) In one embodiment the immunostimulatory nucleic acid molecule includes a sequence TCGCGACGTTCGGCGCGCTGCCG (SEQ ID NO: 27) In one embodiment the immunostimulatory nucleic acid molecule includes a sequence chosen from T * C * G * T * C * G * T * T * T * T * A * C_G * G * C_G * C * C_G * T * G * C * C * G (SEQ ID NO: 43), T * C * G * T * C_G * T * T * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 43), T * C * G * T * C * G * T * T * T * T * A * C * G * A * C * G * C * C * G * T * G * C * C * G (SEQ ID NO: 42), T * C * G * T * C * G * C * T * T * T * G * C * G * A * C * G * C * C * G * T * G * C * C * G (SEQ ID NO: 36), T * C * G * T * C * G * C * C * C * G * G * C * G * A * C * G * C * C * G * T * G * C * C * G (SEQ ID NO: 35), T * C * G * T * C * G * T * T * T * T * A * C * G * G * C * G * C * C * G * T * T * G * C * C * G (SEQ ID NO: 44), T * C * G * T * C * G * T * T * L * L * A * C * G * G * C * G * C * C * G * T * G * C * C * G (SEQ ID NO: 37), T * C * G * T * C * G * T * T * T * T * A * C * G * G * C * G * L * L * L * T * G * C * C * G (SEQ ID NO: 45), T * C * G * T * C * G * T * T * L * L * A * C * G * G * C * G * L * L * L * T * G * C * C * G (SEQ ID NO: 38), T * C * G * T * C * G * T * T * T * T * C * G * G * C * G * G * L * L * C * C * G * C * C * G (SEQ ID NO: 54), T * C * G * T * C * G * T * T * T * T * C * G * G * C * G * T * C * G * C * C * G * C * C * G (SEQ ID NO: 55), T * C * G * T * C * G * T * T * L * L * C * G * G * C * G * C * G * G * C * G * C * C * G (SEQ ID NO: 39), T * C * G * T * C * G * T * T * T * T * C * G * G * C * G * L * L * C * G * C * C * G (SEQ ID NO : 52), T * C * G * T * C * G * T * T * T * T * T * A * A * T * A * T * T * T * A * T * T * A * (SEQ | D N0. 5Q ^ T * C * G * T * C_G * T * T * T * T * T * A * A * T * A * T * T * T * A * T * T * A (SEQ ID NO: 59), t * C * G * T * C_G * T * T * T * T * C * A * A * T * A * T * T * T * A * T * T * G (SEQ ID NO: 50), T * C * G * T * C_G * T * T * T * T * T * A * A * T * A * T * C * C * A * T * T * A (SEQ ID NO: 58), T * C * G * T * C * G * T * T * T * T * T * A * A * T * A * L * L * T * A * T * T * A (SEQ ID NO: 57), T * C * G * T * C_G * T * T * T * T * A * C * G * G * C * G * L * L * L * T * G * C * C * G (SEQ ID NO: 45) , T * C * G * T * C_G * T * T * L * L * A * C * G * G * C * G * L * L * L * T * G * C * C * G * (SEQ ID NO: 38), and T * C * G * T * C_G * T * T * T * T * C * G * G * C * G * G * L * L * C * C * G * C * C * G (SEQ ID NO: 54), where L is a spacer d, * is phosphorothioate and _ is phosphodiester. In one embodiment the immunostimulatory nucleic acid molecule includes a sequence chosen from T * C * G * A * C * G * T * C * G_A_C "G * G * G * A * C * G * G * G * SEQ jD N0; 2 ^ T * C * G * A * C * G * T * C * G_A_C * G * T * G * A * C * G * G * G (SEQ ID NO: 22), T * C * G * A * C * G * T * C * G * A * C * G * G * G * A * C * G * G * G (SEQ ID NO: 21), T * C * G * A * C * G * T * C * G * A * G * G * A * G * G * T (SEQ ID NO: 25), T * C * G * A * C * G * T * C * G * A * G * C * G * A * A * G * C * T (SEQ ID NO: 24), T * C * G * A * C * G * T * C * G * A * C * C * G * T * T * T * T * G * T * G * G (SEQ ID NO: 20), and T * C * G * A * C * G * T * C * G * A * G * A * G * T * T * G * G * G * C * T * C * T * C (SEQ ID NO: 23), where * is phosphorothioate and _ is phosphodiester. In one embodiment the immunostimulatory nucleic acid molecule includes a sequence chosen from T * C * G * A * C * G * T * C * G * A * C * G * T * G * A * C * G * T * G (SEQ ID NO: 62), T * C * G * A * C * G * T * C * G * A * C * G * T * G * A * C * G (SEQ ID NO: 61), T * C_G * T * C_G * A * C_G * T * T * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 65), T * C * G * T * C_G * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 66), T * C * G * T * C_G * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 67), T * C * G * A * C * G * T * C * G * A * C * G * T * G * A * C * G * T * T (SEQ ID NO: 63), T * C * G * T * C_G * A * C_G * A * T * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 64), rC * G * T * C * G * A * C * G * A_T_C * G * G * C * G * C * C * G * rG * C * C * G (SEQ ID NO: 64), T * C * G * A * C_G * T * C * G * A * C_G * T * G * A * C * G * T * T (SEQ ID NO: 63), T * C * G * A * C_G * T * C * G * A * C * G * T_G * A * C * G * T * T (SEQ ID NO : 63), and T * C * G * T * C_G * T * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G * T (SEQ ID NO : 68), where * is phosphorothioate and _ is phosphodiester. In one embodiment the immunostimulatory nucleic acid molecule includes a sequence chosen from T * C * G * C_G * T * C * G * T * T * C_G * G * C * G * C_G * C * T * G * C * C * G (SEQ ID NO: 30), T * C * G_C * G * T * C * G * T * T * C_G * G * C * G * C_G * C * T * G * C * C * G (SEQ ID NO: 30), and T * C * G * C * G_T * C * G * T * T * C_G * G * C * G * C_G * C * T * G * C * C * G (SEQ ID NO: 30), where * is phosphorothioate and _ is phosphodiester. In one embodiment the immunostimulatory nucleic acid molecule includes a sequence T * C * G * C_G * A * C * G * T * T * C_G * G * C * G * C_G * C * T * G * C * C * G (SEQ ID NO: 27), where * is phosphorothioate and _ is phosphodiester. In one embodiment the immunostimulatory nucleic acid molecule includes a sequence chosen from T * C_G * T * C * G * T * T * T * T * A * C * G * G * C * G * T * C * G * T * G * C * C * G (SEQ ID NO: 48), T * C_G * T * C * G * T * T * T * T * A * C * G * G * C * G * T * C * G * C * G * C * C * G (SEQ ID NO: 47), and T * C_G * T * C * G * T * T * T * T * A * C * G * G * C * G * T * C * G * C * G (SEQ ID NO: 46), where * is phosphorothioate and _ is phosphodiester. In one embodiment the immunostimulatory nucleic acid molecule includes a sequence T * C_G * T * C * G * T * T * T * T * A * C * G * G * C * G * T * C * G * T * G * C * C * G (SEQ ID NO: 48), where * is phosphorothioate and _ is phosphodiester. In one embodiment the immunostimulatory nucleic acid molecule includes a sequence T * C_G * G * C * G * C * C_G * T * G * C * C * G * T * C * G * T * C_G * T * T * T (SEQ ID NO: 33), where * is phosphorothioate and _ is phosphodiester.

In an embodiment at least one nucleotide of the oligonucleotide is a substituted or modified purine or pyrimidine. In one embodiment the substituted pyrimidine is a substituted pyrimidine at the C5 or C6 position. In one embodiment the substituted purine is a purine substituted at the C8 or C7 position. In one embodiment the substituted or modified pyrimidine or pyrimidine is selected from the group consisting of substituted cytosines in the 5-position, substituted cytosines in the 6-position, substituted cytosines in the N4 position, 5-azacytosine, 2-mercaptocytosine, isocytosine, pseudoisocytosine , cytosine analogs with fused ring systems, and uracil derivatives, thymine derivatives, 7-deazaguanine, 7-deazaguanine substituted at position 7, 7-deazaguanine substituted at position 8, 7-deaza-8-azaguanine , hypoxanthine, guanines substituted at position N2, 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, substituted adenines, guanine substituted in position 8 and 6-thioguanine. In one embodiment the substituted or modified pyrimidine or pyrimidine is selected from the group consisting of 5-methylcytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, 5-iodocytosine, 5-hydroxycytosine, 6-hydroxycytosine, 5-hydroxymethylclithosine, 5-difluoromethylcytosine and 5-alkynylcytosine, N4-ethylcytosine, N, N'-propylenecytosine, fenoxacin, 5-fluorouracil, 5-bromouracil, 5-bromovinyluracil, 4-thiouracil, 5-hydroxyuracil, 5-propinyluracil, 2-thiothimine, -tiothimine, thymines substituted at position 6, 7-deaza-7-alkynyl (C2-C6) guanine, N2-methylguanine, N6-methyladenine, 8-oxoadenine, 8-hydroxyguanine and 8-bromoguanine unsubstituted or substituted. In one embodiment, the substituted or modified purine or pyrimidine is selected from the group consisting of a universal base, an aromatic ring system, an aromatic ring system and a hydrogen atom (spacer d). In one embodiment, the purified or substituted purine or pyrimidine is selected from the group consisting of 4-methylindole, 5-nitroindole, 3-nitropyrrole, base P and base K, benzimidazole, dichlorobenzimidazole, 1-methyl-1H- [1, 2,4] triazole-3-carboxylic acid, fluorobenzene and difluorobenzene. In any embodiment any N, S, X or Z is substituted by a residue selected from the group consisting of an alkyl chain of C6 to C30, bile acids, cholic acid, taurocholic acid, deoxycholate, cholesterol, oleyl-lithocholic acid, oleoyl-choline acid, glycolipids, phospholipids, sphingolipids, isoprenoids, steroids, vitamins, vitamin E, saturated fatty acids, unsaturated fatty acids, fatty acid esters, triglycerides, pyrenes, porphyrins, Texaphyrin, adamantane, acridins, biotin, coumarin, fluorescein, rhodamine, Red Texas, digoxigenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes, cyanine dye Cy3, cyanine dye Cy576, Hoechst 33258 dye, psoralen and ibuprofen. In one aspect the invention provides an immunostimulatory nucleic acid molecule that includes: (a) a 5 'end that starts with an immunostimulatory motif chosen from (TCG) nN and RDCGY? Y2N, where T is thymine, C is unmethylated cytosine, G is guanine, R is a purine, D is not C, each Yi and Y2 independently is a pyrimidine, n is an integer between 1 and 4, inclusive, and N is any sequence of 0-12 bases in length; (b) a 3 'end terminating in an inverted repeat capable of forming a hairpin or stem-and-loop structure, which includes a GC-rich stem of 2 to 6 consecutive base pairs in length and at least one base not paired or mismatched; and (c) a partially stabilized backbone that includes at least one 5'-CpG-3 phosphodiester bond. The C or the G of the CpG dinucleotide or both can be modified. In a modality, the stem rich in GC has 2 consecutive base pairs in length. In one embodiment, the stem rich in GC has 3 consecutive base pairs in length. In one embodiment, the stem rich in GC has 4 consecutive base pairs in length. In one embodiment, the stem rich in GC has 5 consecutive base pairs in length. In one embodiment, the stem rich in GC has 6 consecutive base pairs in length. In one embodiment, the GC-rich stem includes at least 2 base pairs G-C. In one embodiment, the GC-rich stem includes at least 3 base pairs G-C. In certain embodiments the unpaired or mismatched base is at least T. In one embodiment the partially stabilized backbone that includes at least one 5'-CpG-3 'phosphodiester bond also includes a plurality of phosphorothioate internucleotide linkages. In one embodiment the 5 'end has a sequence that is TCGTCGTTTTA (SEQ ID NO: 41). In one embodiment the 3 'end terminating in an inverted repeat has a base sequence which is CGGCGCCGTGCCG (SEQ ID NO: 19). In one embodiment the 3 'end terminating in an inverted repeat has a base sequence which is CGGCGTCGTGCCG (SEQ ID NO: 9). In one aspect the invention features an immunostimulatory nucleic acid having a base sequence which is TCGTCGTTTTACGGCGCCGTGCCG (SEQ ID NO: 43). In one aspect the invention features an immunostimulatory nucleic acid having a base sequence which is TCGTCGTTTTACGGCGTCGTGCCG (SEQ ID NO: 48). In one aspect the invention features an immunostimulatory nucleic acid having a base sequence which is T * C_G * T * C * G * T * T * T * T * A * C * G * G * C * G * C * C * G * T * G * C * C * G (SEQ ID NO: 43), where * represents an internucleotide link phosphorothioate and _ represents an internucleotide phosphodiester linkage. In one aspect the invention offers an immunostimulatory nucleic acid having a base sequence which is T * C * G * T * C * G * T * T * T * T * A * C_G * G * C_G * C * C_G * T * G * C * C * G (SEQ ID NO: 43), where * represents an internucleotide link phosphorothioate and _ represents an internucleotide phosphodiester linkage. In one aspect the invention offers a nucleic acid Immunostimulant that has a base sequence that is T * C * G * T * C * G * T * T * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 43), where * represents an internucleotide link phosphorothioate and _ represents an internucleotide phosphodiester linkage. In one aspect the invention provides an immunostimulatory nucleic acid having a base sequence which is T * C * G * T * C_G * T * T * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 43), where * represents an internucleotide link phosphorothioate and _ represents an internucleotide phosphodiester linkage. In one aspect the invention provides a vaccine that includes a nimostimulatory nucleic acid molecule of the invention and an antigen. In one aspect the invention provides a pharmaceutical composition that includes an immunostimulatory nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. In one aspect the invention provides a method for inducing the expression of type I interferon (IFN). The method according to this aspect of the invention includes contacting a cell capable of expressing type I IFN by an immunostimulatory nucleic acid of the invention. , in an amount sufficient to induce IFN type I expression. In an IFN type I modality is an alpha interferon (IFN-a). In one embodiment, IFN type I is an interferon beta (IFN-β). In one aspect the invention offers a method for inducing the expression of interferon gamma (IFN-γ). The method according to this aspect of the invention includes contacting a cell capable of expressing IFN-? by an immunostimulatory nucleic acid of the invention, in an amount effective to induce the expression of INF- ?. In one aspect the invention offers a method for activating the natural killer (NK) cell. The method according to this aspect of the invention includes contacting an NK cell with an immunostimulatory nucleic acid of the invention, in an amount sufficient to activate the NK cell. In one aspect the invention offers a method for treating an infection. The method according to this aspect of the invention includes administering to a subject having or at risk of developing an infection an immunostimulatory nucleic acid of the invention, in an amount effective to treat or prevent infection. In one embodiment, the subject has or is at risk of developing an infection, which may be viral, bacterial, fungal or parasitic. In one embodiment the subject has or is at risk of developing a viral infection caused by a virus which may be the hepatitis B virus (HBV), the hepatitis C virus (HCV), the cytomegalovirus (CMV), the virus of Epstein-Barr (EBV), the papillomavirus, the human immunodeficiency virus (HIV) or the herpes simplex virus (HSV). In one embodiment the subject has or is at risk of developing a bacterial infection caused by a species of bacteria that can be Leishmania, Listeria or Anthrax. In one aspect the invention offers a method for treating an allergic condition. The method according to this aspect of the invention includes administering to a subject having or at risk of developing an allergic condition an immunostimulatory nucleic acid of the invention, in an amount effective to treat or prevent an allergic condition. In one modality the allergic condition is allergic asthma. In one aspect the invention offers a method for treating cancer. The method according to this aspect of the invention includes administering to a subject having or at risk of developing a cancer an immunostimulatory nucleic acid of the invention, in an amount effective to treat or prevent cancer. In one modality, cancer can be basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, including Hodgkin's lymphoma and non-Hodgkin's, melanoma, myeloma, neuroblastoma, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, cancer of stomach, testicular cancer, thyroid cancer, uterine cancer, cancer of the urinary system, or other carcinomas and sarcomas. In one modality, cancer is a cancer sensitive to treatment with interferon alfa (IFN-a). In a modality the cancer sensitive to treatment with IFN-a can be tricholeukemia, chronic myelogenous leukemia, cutaneous T cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, Kaposi sarcoma related to AIDS, carcinoma of kidney cells, prostate carcinoma, cervical dysplasia or colon carcinoma. In one aspect the invention relates to the use of an immunostimulatory nucleic acid of the invention for manufacturing a medicament for use in the treatment of an infection. In one aspect the invention relates to the use of an immunostimulatory nucleic acid of the invention for making a medicament for use in the treatment of an allergic condition. In one aspect the invention relates to the use of an immunostimulatory nucleic acid of the invention for making a medicament for use in the treatment of allergic asthma. In one aspect the invention relates to the use of an immunostimulatory nucleic acid of the invention for manufacturing a medicament for use in the treatment of a cancer. Each of the limitations of the invention may include various embodiments of the invention. Therefore, it is expected that each of the limitations of the invention that include any element or combination of elements is included in each aspect of the invention.

BRIEF DESCRIPTION OF THE FIGURES The present invention can be understood in depth and more easily if it is analyzed together with the attached figures. These figures are only included for illustrative purposes and are not necessary to understand or to practice the invention. Figures 1A-1F are a series of graphs describing the induction of IFN-a and the signaling of TLR9 by ODN 332, 333 and 334. Figures 2A-2F are a series of graphs describing the induction of IFN. -a and the signaling of TLR9 by ODN 611, 614 and 620. Figure 3 is a graph describing the production of IFN-a in peripheral blood mononuclear cells (PBMC) stimulated by a panel of oligonucleotides. Figure 4 is a graph describing the mean titers of the group (GMT) of total IgG specific for the antigen after immunization with 1 μg of hepatitis B surface antigen (HBsAg) with the indicated amount of ODN. Figure 5 is a graph describing the GMTs of the individual isotypes of IgG specific for the antigen after immunization with 1 μg of HBsAg with the indicated amount of ODN. Figure 6 is a graph describing the responses of cytolytic T lymphocytes specific for the antigen after immunization with 1 μg of HBsAg with the indicated amount of ODN. Figure 7A is a graph describing survival according to different treatments with ODN in a murine neuroblastoma tumor model. Figure 7B is a graph describing tumor volume according to different treatments with ODN in a murine neuroblastoma tumor model. 03 91- Q l svavN? i3oap3S svioNanoas aa ouavno 63 08 DETAILED DESCRIPTION OF THE INVENTION The invention in one aspect relates to the finding that specific subclasses of CpG immunostimulatory oligonucleotides having a defined secondary structure are highly effective in mediating immunostimulatory effects. These CpG nucleic acids are useful for therapeutic and prophylactic purposes to stimulate the immune system to treat cancer, infectious diseases, allergy, asthma and other disorders and help protect against opportunistic infections after oncological chemotherapy. The strong but still balanced humoral and cellular immune responses that arise as a consequence of CpG stimulation reflect the body's own system of natural defenses against pathogens and the cancer cells that invade it. The sequences of the invention share some structural similarities with a class of CpG oligonucleotides called class C or CpG oligonucleotides of combined motifs. See the published PCT international patent application WO 03/015711. Similar to the class C oligonucleotides previously described, the CpG class C oligonucleotides of the present invention have motifs at the 5 'and 3' positions defined as part of the molecule. These class C oligonucleotides previously described have a traditional "stimulating" CpG sequence, generally located at or near the 5 'or 3' ends of the molecule, as well as a "GC-rich palindrome" motif, generally located on the other end of the molecule or near it. These combined-motive nucleic acids have immunostimulatory effects that resemble in part the effects associated with the traditional "class B" CpG ODNs, which are important inducers of B cell activation and activation of dendritic cells, and in part to the effects associated with a more recently described class of immunostimulatory nucleic acids (CpG "class A" ODN), which are important inducers of IFN-a and activation of NK cells but relatively weak inducers of cell activation B and the DC. The new CpG class C oligonucleotides of the present invention also differ structurally from the CpG class C oligonucleotides previously described. In comparison with the CpG class C oligonucleotides previously described, the immunostimulatory nucleic acid molecules of the present invention have extremely relaxed requirements with respect to the GC-rich palindrome at one end of the molecule. For example, the previously described class C oligonucleotides include in a modality a strict or perfect palindrome of at least 10 nucleotides in length and have a GC content of at least 2/3. In some embodiments, the palindrome of the class C oligonucleotides previously described may include, at most, a minimum number of consecutive mismatched nucleotides. Unlike the class C oligonucleotides previously described, the class C oligonucleotide analogs of the present invention have palindromic motifs having, in various embodiments, less than 10 nucleotides.; a GC content between zero and less than 2/3; various nucleotide and substitute analogues including those lacking a nucleobase (spacer d); an extended interspersed sequence comprising four or more consecutive nucleotides or substitutes for nucleotides that do not form Watson-Crick base pairs; and any combination thereof. In addition, in some embodiments the 3 'portions of two or more molecules can be linked together through their 3' ends. It has been discovered that this new subclass of oligonucleotides that do not have a perfect palindrome are still capable, like the CpG oligonucleotides of previously described combined motifs, of inducing high levels of IFN production, including IFN type I (e.g., IFN- a, IFN-ß) and IFN- Y- The terms "palindrome" and, likewise, "palindromic sequence", in the sense in which they are used herein, refer to a nucleic acid sequence that is its own perfect inverse complement (ie, a sequence, such as ABCDEE'D'C'B'A 'in which A and A', B and B ', C and C, D and D' and E and E 'are bases capable of forming the typical Watson-Crick base pairs, ie, GC, AT and AU In the sense in which it is used herein, a "palindrome" in a strict sense excludes the interleaved sequence or the structure does not interspersed nucleotide that does not participate in the formation of typical Watson-Crick base pairs. "tida", in the sense in which the term is used herein, refers to an imperfect palindrome, that is, to a nucleic acid sequence in which both nucleotides capable of forming the base pairs and the typical nucleotides are present. of Watson-Crick, nucleotide analogs or other structures that do not participate in the formation of typical Watson-Crick base pairs (eg, a sequence such as ABCDE-S-E'D'C'B'A ' in which A and A, B and B ', C and C, D and D' and E and E 'are bases capable of forming the typical Watson-Crick base pairs, and S is a non-palindromic sequence, or a non-nucleotide linker or an abasic linker (spacer d). In certain embodiments, nucleotides, nucleotide analogs or other structures that do not participate in the formation of typical Watson-Crick base pairs interrupt what would be a perfect palindrome. In certain embodiments, nucleotides that do not participate in the formation of typical Watson-Crick base pairs can form non-Watson-Crick base pairs with another nucleotide, for example, G-T. A pair of non-Watson-Crick bases, in the sense in which the term is used in the present, is any pair of bases other than a pair of Watson-Crick bases, including for illustrative purposes, a pair of bases of Hoogsteen and a pair of bases of so-called faltering mating (wobble). In certain embodiments, nucleotides that are not involved in the formation of typical Watson-Crick base pairs are unpaired and do not have a nucleotide base or a nucleotide base analogue with which to form a Watson-Crick base pair. or that is not from Watson-Crick, for example, G opposed to a spacer d. In certain embodiments, nucleotides that do not participate in base pair formation can form non-standard base pairs with another nucleotide, for example, diaminopyridine can form a base pair with xanthosine. In one embodiment the 5 'end of a nucleic acid starts with an immunostimulant motif chosen from (TCG) nN and RDCGY? Y2N. T is thymine, C is non-methylated cytosine, G is guanine, R is purine, D is not C, each Yi and Y2 is independently a pyrimidine, n is an integer between 1 and 4, inclusive, and N is any sequence of 0 to 12 bases in length. The 3 'end of the nucleic acid ends in an inverted repeat capable of forming a hairpin or stem and loop structure. The term "ends" refers to a structure located at or near the 3 'end. In this way, the end of an imperfect palindrome may be located at the actual 3 'end of the molecule or, alternatively, the 3"end may include 1 or more additional nucleotides that are not part of the structure of the inverted repeat. 3 'of the molecule includes 3 or less nucleotides that are not part of the structure of the inverted repetition, in a modality an "inverted repetition capable of forming a fork or stem and loop structure", in the sense in which it is used herein, it refers to a nucleotide sequence that forms a GC-rich stem or fork having 2 to 10 consecutive base pairs in length and includes at least one unpaired or mismatched base. , the stem rich in GC is 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive base pairs In some modalities, the stem rich in GC includes, at least, 2, 3 or 4 base pairs GC In one modality, an "inverted repetition capable of forming a fork or stem and loop structure", in the sense used herein, refers to a nucleotide sequence that forms an AT-rich stem or fork that has 2 to 10 pairs of consecutive bases of length and includes, at least, a base unpaired or badly paired. In individual modalities, the AT-rich stem is 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive base pairs in length. In some embodiments, the AT-rich stem includes at least 2, 3 or 4 base pairs A-T. In some cases at least one unpaired or mismatched base forms a bridge over the ends of the stem or fork. This can allow the formation of a secondary structure since it offers a flexible point in the molecule for the stems to form base pairs and a fork. Alternatively, the unpaired or mismatched base or bases may be within the stem. Preferably, if the mismatched base is within the stem, the stem is at least 3 base pairs in length. The unpaired or mismatched base or bases can be any nucleotide. In some embodiments, the unpaired or mismatched base or bases are a T. The unpaired nucleotides at the ends of the double strands are also known as protruding nucleotides or pendant ends that can significantly stabilize the formation of double structures or formation of forks. Freier SM et al. (1983) Effects of 3 'dangling end stacking on the stability of GGCC and CCGG double helixes. Biochemistry 22: 6198-206. The nucleic acid also includes a partially stabilized backbone that includes at least one 5'-CpG-3 phosphodiester bond. "In some cases, the double-stranded part of the molecule may also contain unnatural (non-standard) base pairs ( for example, diaminopyridine coupled with xanthosine) Lutz MJ et al. (1998) Recognition of a non-standard base pair by thermostable DNA polymerases Bioorg Med Chem Lett 8: 1149-52 The formulas define subgroups of the class of CpG oligonucleotides that demonstrated to have excellent immunostimulatory properties In the 5 'formulas it refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide The oligonucleotides may have one or more accessible 5 'or 3' ends. embodiments, a 3 'end may be attached to another 3' end .. Because the significance of the 5 'and 3' motifs has been discovered and described herein, it is also possible to create oligonucleotides modified substances having two such 5 'and 3' ends. This can be achieved, for example, by joining two oligonucleotides by a 3'-3 'linkage to generate an oligonucleotide having two accessible 5' ends. The 3'3 'or 5'5' linkage can be a phosphodiester, a phosphorothioate or any other modified intemucleoside bridge. Methods for achieving such links are known in the art. For example, such linkages have been described in Seliger H et al. (1991) Oligonucleotide analogs with terminal 3'-3'- and 5'-5'-intemucleotidic linkages as antisense inhibitors of viral gene expression, Nucleosides &; Nucieotides 10: 469-77 and in Jiang Z et al. (1999) Pseudo-cyclic oligonucleotides: in vitro and in vivo properties, Bioorg Med Chem 7: 2727-35. Additionally, ODNs with 3'-3 'or 5'-5' linkages where the bond between the 3 'or 5' terminal nucleosides is not a phosphodiester, a phosphorothioate or other modified bridge, can be prepared using an additional spacer, such as example a radical tri- or tetraethylene glycol phosphate (Durand M et al. (1992) Triple-helix formation by an oligonucleotide containing one (dA) 12 and two (dT) 12 sequences bridged by two hexaethylene glycol chalns, Biochemistry 31: 9197-204 U.S. Patent No. 5,658,738 and U.S. Patent No. 5,668,265). Alternatively, the non-nucleotide linker can be obtained from ethanediol, propanediol or from a deoxyribose unit (spacer d) abasic (Fontanel ML et al. (1994) Sterical recognition by T4 polynucleotide kinase of non-nucleosidic moieties 5'-attached to ollgonucleotides, Nucleic Acids Res 22: 2022-7) using the standard chemistry of phosphoramidite. The non-nucleotide linkers can be incorporated in one or multiple times, or combined together to obtain the desired distance between the 3 'ends of the two ODNs that it is desired to join. A "non-nucleotide linker", in the sense used herein, refers to any linker element that is not a nucleotide or a polymer thereof (ie, a polynucieotide), in which a nucleotide includes a purine nucleobase or pyrimidine and a sugar phosphate. Thus, a non-nucleotide linker includes an abasic nucleotide (spacer d), i.e., a phosphate-sugar unit similar to a nucleotide in which the nucleobase is replaced by a hydrogen atom. A non-nucleotide linker can be a polyethylene glycol, including by way of illustration a triethylene glycol and a hexaethylene glycol. In some embodiments, the oligonucleotide has one of the following structures: TCGTCGTTTTA (SEQ ID NO: 41), CGGCGCCGTGCCG (SEQ ID NO: 19), CGGCGTCGTGCCG (SEQ ID NO: 9), TCGTCGTTTTACGGCGCCGTGCCG (SEQ ID NO: 43), TCGTCGTTTTACGGCGTCGTGCCG (SEQ ID NO: 48), T * C_G * T * C * G * T * T * T * T * A * C * G * G * C * G * C * C * G * T * G * C * C * G * (SEQ ID NO : 43), T * C * G * T * C * G * T * T * T * T * A * C_G * G * C_G * C * C_G * T * G * C * C * G (SEQ ID NO: 43), T * C * G * T * C * G * T * T * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO : 43) and T * C * G * T * C_G * T * T * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 43). The symbol * refers to the presence of a stabilized internucleotide bond and refers to the presence of a phosphodiester bond. Immunostimulatory oligonucleotides generally have a length ranging from 6 to 100 nucleotides. In some modalities, the length ranges from 6-40, 13-100, 13-40, 13-30, 15-50 or 15-30 nucleotides or an integer scale between them. The terms "nucleic acid" and "oligonucleotide" are used interchangeably to refer to multiple nucleotides (i.e., molecules that include a sugar (e.g., ribose or deoxyribose) attached to a phosphate group and an exchangeable organic base, which is a substituted pyrimidine (eg, cytosine (C), thymine (T) or uracil (U)) or a substituted purine (eg, adenine (A) or guanine (G).) In the sense used herein, the terms "Nucleic acid" and "oligonucleotide" refer to both oligoribonucleotides and oligodeoxyribonucleotides.The terms "nucleic acid" and "oligonucleotide" also include polynucleosides (ie, a polynucleotide minus phosphate) and any other organic base containing a polymer The nucleic acid molecules can be obtained from existing nucleic acid sources (eg, genomic or cDNA), but are preferably synthetic (eg, gone by synthesis of nucleic acids). The terms "nucleic acid" and "oligonucleotide" in the sense used herein, include both the nucleic acid molecules and the oligonucleotides of the invention and the oligonucleotide analogs of the invention. The terms "oligodeoxynucleotide" and, equivalently, ODN "in the sense in which it is used herein, will include both the unmodified oligodeoxynucleotides of the invention and the oligodeoxynucleotide analogs of the invention. "oligonucleotide" also includes nucleic acids or oligonucleotides with substitutions or modifications, as occurs in bases and / or in sugars, for example, they include nucleic acids that have sugars as the main chain that are covalently bound to organic groups under molecular weight which are not a hydroxyl group at the 2 'position and which are not a phosphate group or a hydroxy group at the 5' position Thus, the modified nucleic acids can include a 2'-0-alkylated ribose group. In addition, modified nucleic acids may include sugars, such as arabinose or 2'-fluoroarabinose, in place of ribose. s can be heterogeneous in terms of the main chain composition and contain any possible combination of linked polymer units, such as peptide-nucleic acids (having a peptide-nucleic acid-like backbone). Other examples are described in more detail below. The immunostimulatory oligonucleotides of the present invention may include various modifications and chemical substitutions, as compared to natural RNA and DNA, including an internucleoside phosphodiester bridge, a β-D-ribose unit and / or a natural nucleoside base (adenine, guanine , cytosine, thymine, uracil). Those skilled in the art are aware of examples of chemical modifications, which are also described, for example, in Uhlmann E et al. (1990) Chem Rev 90: 543; "Protocols for Oligonucleotides and Analogs" Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993; Crooke ST and others (1996) Annu Rev Pharmacol Toxico! 36: 107-29; and Hunziker J et al. (1995) Mod Synth Methods 7: 331-417. An oligonucleotide according to the invention may have one or more modifications, each of which is located on an internucleoside phosphodiester bridge in particular and / or on a particular β-D-ribose unit and / or at the position of a particular natural nucleoside base, unlike an oligonucleotide of the same sequence that is made up of natural DNA or RNA. For example, the oligonucleotides may include one or more modifications, each of which is independently chosen from: a) the replacement of an internucleoside phosphodiester bridge located at the 3 'and / or 5' end of a nucleoside by a modified internucleoside bridge; b) the replacement of a phosphodiester bridge located at the 3 'and / or 5' end of a nucleoside by a dephosphine bridge; c) the replacement of a phosphate-sugar unit of the phosphate-sugar main chain by another unit; d) replacement of a β-D-ribose unit with a modified sugar unit; and e) the replacement of a natural nucleoside base with a modified nucleoside base. Some more detailed examples of the chemical modification of an oligonucleotide are the following: Oligonucleotides can include modified internucleotide linkages, such as those described in preceding points a or b. These modified bonds may be partially resistant to degradation (eg, they are stabilized). A "stabilized oligonucleotide molecule" means an oligonucleotide that is relatively resistant to degradation in vivo (e.g. through an exonuclease or an endonuclease) arising from said modifications. Oligonucleotides having phosphorothioate linkages, in some embodiments, can provide maximum activity and protect the oligonucleotide from degradation caused by exonucleases and intracellular endonucleases. An internucleoside phosphodiester bridge located at the 3 'end and / or at the 5' end of a nucleoside can be replaced by a modified intemucleoside bridge, in which the modified internucleoside bridge is chosen, for example, between phosphorothioate, phosphorodithioate, NR1R2 bridges -phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate, phosphate ester (CrC2?) - O-alkyl, phosphate - [(C6-C? 2) aryl- (C? C2?) - 0-alkyl] ester, (C? -C8) alkylphosphonate and / or (C6-C? 2) arylphosphonate, (C -C? 2) -a-hydroxymethyl-aryl (for example, disclosed in WO 95/01363), where aryl of (C6-C? 2), aryl of (C6-C20) and aryl of (C6-C? 4) are optionally substituted by halogen, alkyl, alkoxy, nitro, cyano and where R1 and R2 are, independently of each other, hydrogen, alkyl C 8), aryl of (C6-C20), aryl (C6-C? 4) -alkyl (C? -8), preferably hydrogen, (C? -C8) alkyl, preferably (C4) alkyl and /? or methoxyethyl, or R1 and R2 form, together with the nitrogen atom that It has a heterocyclic ring of 5 to 6 members that can additionally contain another heteroatom of groups O, S and N. The replacement of a phosphodiester bridge located at the 3 'and / or 5' end of a nucleoside by a dephosphine bridge ( Desfosfo bridges are described, for example, in Uhlmann E and Peyman A in "Methods in Molecular Biology", Vol. 20, "Protocols for Oligonucleotides and Analogs", S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp. 355 ff), where a dephosphine bridge is chosen, for example, between the dephosphine bridges of the formatetal, 3'-thioformacetal, methylhydroxylamine, oxime, methylenedimethyl-hydrazo, dimethylene sulfone and / or silyl groups. A phosphate-sugar unit (ie, an internucleoside bridge of ß-D-ribose and phosphodiester that together form a phosphate-sugar unit) of the phosphate-sugar backbone (ie, a phosphate-sugar backbone) it is composed of phosphate-sugar units) can be replaced by another unit, where the other unit is, for example, suitable for building a "morpholino derivative" oligomer (as described, for example, in Stirchak EP et al. (1989) Nucleic Acids Res 17: 6129-41), which is, for example, replacement by a morpholino derivative unit; or to construct a polyamide nucleic acid ("PNA", as described, for example, in Nielsen PE et al. (1994) Bioconjug Chem 5: 3-7), i.e., for example, replacement by a chain unit of PNA, for example, by 2-aminoethylglycine. The oligonucleotide may have other modifications and replacements in the carbohydrate backbone, such as peptide nucleic acids with phosphate groups (PHONA), closed nucleic acids (LNA) and oligonucleotides having sections of the backbone with alkyl linkers or amino linkers. The alkyl linker may be branched or unbranched, substituted or unsubstituted and can be chirally pure or a racemic mixture. A ß-ribose unit or a β-D-2'-deoxyribose unit can be replaced by a modified sugar unit, where the modified sugar unit is chosen, for example between β-D-ribose, aD-2 ' -deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-F-arabinose, 2'-0-alkyl (CrC6) -ribose, preferably 2'-0-alkyl (C? - C6) -ribose is 2'-0-methylribose, 2'-0-alkenyl (C2-C6) -ribose, 2'-0-alkyl (CrC6) -0-alkyI (C6C6)] ribose, 2'- NH2-2'-deoxyribose, β-D-xylo-furanose, α-arabinofuranose, 2,4-dideoxy-β-D-erythro-hexo-pyranose and carbocyclics (described, for example, in Froehler (1992) J Am Chem Soc 114: 8320) and / or open chain analogs of sugar (described, for example, in Vandendriessche et al. (1993) Tetrahedron 49: 7223) and / or bicyclo sugar analogs (described, for example, in Tarkov M and others ( 1993) Helv Chim Acta 76: 481). In some embodiments, the sugar is 2'-0-methylribose, particularly for one or both nucleotides linked by a phosphodiester bond or by an internucleoside linkage similar to a phosphodiester. The nucleic acids also include purines and substituted pyrimidines such as modified bases of C-5-pyrimidine and 7-deaza-7-substituted. Wagner RW et al. (1996) Nat Biotechnol 14: 840-4. Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine, thymine and uracil and other natural or non-natural nucleobases, and substituted and unsubstituted aromatic radicals. A modified base is any base that is chemically different from the natural bases typically found in DNA and RNA, such as T, C, G, A and U, but that share basic chemical structures with such natural bases. The modified nucleoside base may be chosen, for example, from hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-alkyl (CrC6) -uracil, 5-alkenyl (C2-C6) - uracil, 5-alkynyl (C2-C6) -uracil, 5- (hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-alkyl (C2-C6) -cytosine, 5-alkenyl (C2-C6) -cytosine, 5-alkynyl (C2-C6) -cytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 2,4-diaminopurine, 8-azapurine, a substituted 7-deazapurine , preferably 7-deaza-7-substituted and / or 7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N 4 -alkycytosine, for example, N 4 -ethylcytosine, 5-hydroxydetoxycinidine, 5-hydroxymethydedeoxycytidine, N 4 -alkyloxycytidine, for example , N4-ethyldeoxycytidine, 6-thiodeoxyguanosine and deoxynucleosides of nitropyrrole, C5-propynylpyrimidine and diaminopurine, for example, 2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chlorop urine, hypoxanthine or other modifications of natural nucleoside bases. This list is for illustrative purposes and does not constitute a limitation. In the particular formulas described herein, modified bases can be incorporated. For example, a cytosine can be replaced by a modified cytosine. A modified cytosine, in the sense in which it is used herein, is a natural or unnatural pyrimidine-based analog of the cytosine, which can replace the base domain without affecting the immunostimulatory activity of the oligonucleotide. Modified cytosines include, but are not limited to, substituted cytosines in the 5-position (eg, 5-methylcytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, 5-iodocytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-difluoromethylcytosine and substituted or unsubstituted 5-alkinylcytosine), cytosines substituted at position 6 (eg, 6-hydroxycytosine), cytosines substituted at the N4 position (eg, N4-ethylcytosine), 5-azacytosine, 2-mercaptocytosine , isocytosine, pseudoisocytosine, cytosine analogues with fused ring systems (for example, N, N'-propylenecytosine or phenoxazine) and uracil and their derivatives (for example, 5-fluorouracil, 5-bromouracil, 5-bromovinyluracil, 4-thiouracil, 5-hydroxyuracil, 5-propinyluracil). Some of the preferred cytosines are 5-methylcytosine, 5-fluorocytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine and N4-ethylcytosine. In another embodiment of the invention, the cytosine base is replaced by a universal base (e.g., 3-nitropyrrole, base P), an aromatic ring system (e.g., fluorobenzene or difluorobenzene) or a hydrogen atom (spacer d). ). A guanine can be replaced by a modified guanine base. A modified guanine, in the sense in which it is used herein, is a natural or unnatural purine-based analog of guanine that can replace this base without affecting the immunostimulating activity of the oligonucleotide. Modified guanines include, but are not limited to, 7-deazaguanine, 7-deaza-7-substituted guanine (such as 7-deaza-7-alkynyl (C2-C6) guanine), 7-deaza-8-substituted guanine, hypoxanthine, guanines substituted at the position N2 (for example, N2-methylguanine), 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidine-2,7-dione, 2,6-diaminopurine, 2 -aminopurine, purine, indole, adenine, substituted adenines (e.g., N6-methyladenine, 8-oxoadenine), guanine substituted at position 8 (e.g., 8-hydroxyguanine and 8-bromoguanine) and 6-thioguanine. In another embodiment of the invention, the guanine base is replaced by a universal base (e.g., 4-methylindol, 5-nitroindole and K base), an aromatic ring system (e.g., benzimidazole or dichlorobenzimidazole, acid amide). -methyl-1 H- [1, 2,4] triazole-3-carboxylic acid) or a hydrogen atom (spacer d). In one embodiment, both C and G of a CG dinucleotide are unmodified cytosine and guanine bases, respectively. In one embodiment, the C of a CG dinucleotide is not methylated. For use in the present invention, the oligonucleotides of the invention can be synthesized de novo using any number of methods well known in the art, for example, the β-cyanoethyl phosphoramidite method (Beaucage SL et al. (1981) Tetrahedron Lett 22: 1859); or the nucleoside H-phosphonate method (Garegg et al. (1986) Tetrahedron Lett 27: 4051-4; Froehler BC et al. (1986) Nucleic Acids Res 14: 5399-407; Garegg et al. (1986) Tetrahedron Lett 27: 4055 -8; Gaffney et al. (1988) Tetrahedron Lett 29: 2619-22). These chemistries can be carried out using a variety of commercially available automated nucleic acid synthesizers. These oligonucleotides are called synthetic oligonucleotides. An isolated oligonucleotide generally refers to an oligonucleotide that is separated from the components to which it is normally naturally associated. For example, an isolated oligonucleotide can be an oligonucleotide that is separated from a cell, a nucleus, the mitochondria or chromatin. Modified backbones, such as phosphorothioates, can be synthesized by automated techniques employing phosphoramidate or H-phosphonate chemistries. Arylphosphonates and alkylphosphonates can be made, for example, in the manner described in US Pat. No. 4,469,863; and alkyl phosphotriesters (in which the charged oxygen radical is alkylated in the manner described in U.S. Patent No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid-phase synthesis using the reagents for commercial use . Methods for making other modifications and substitutions in the DNA backbone have been described (eg, Uhlmann E et al. (1990) Chem Rev 90: 544; Goodchild J (1990) Bioconjugate Chem 1: 165). The immunostimulatory oligonucleotides may also contain one or more unusual linkages between the nucleotide or nucleotide analogue moieties. The usual internucleoside linkage is the 3'-5 'link. All other links are considered unusual internucleoside linkages, such as the bonds 2'5'-, 5'5'-, 3'3'-, 2'2'- and 2'3'-. Therefore, the nomenclature 2 'to 5' is chosen as a function of the carbon atom of the ribose. However, if unnatural sugar fractions are used, such as expanded ring sugar analogues (eg hexanose, cyclohexane and pyranose) or bicyclic or tricyclic sugar analogues, this nomenclature changes depending on the nomenclature of the monomer. In the 3'-deoxy-β-ribopyranose analogues (also called p-DNA), the mononucleotides are connected, for example, by a 4'2 'link. If the nucleotide contains a 3'3 bond, the analogue of this oligonucleotide will typically have two unbound 5 'ends. Likewise, if the nucleotide contains a 5'5'-bond, the analogue of this oligonucleotide will typically have two unbound 3'-ends. The accessibility of the unbound ends of the nucleotides can be improved through their receptors. Both types of the usual bonds (3'3'- and 5'5'-) were described by Ortigao JF et al. (1992) Antisense Res Dev 2: 129-46, where oligonucleotides having a 3'-linkage were reported. 3 'have a better stability to facilitate separation by the nucleases. You can also combine different types of bonds in a molecule, which can give rise to the branching of the oligomer. If a part of the oligonucleotide is connected at the 3 'end via a 3' 3 'link to the second part of an oligonucleotide and at the 2' end via a 2 '3 link to the third part of the molecule , there is, for example, a branched oligo-nucleotide with three 5 'ends (branched at 3'3'-, 2'3'-). In principle, the bonds between the different parts of an oligonucleotide or between different oligonucleotides, respectively, can be produced through all the parts of the molecule, as long as this does not adversely affect its recognition by its receptor. According to the nature of the nucleic acid, the linkage may include the sugar (Su) radical, the heterocyclic nucleobase (Ba) or the phosphate backbone (Ph). Thus, Su-Su type links are possible, Su-Ph, Su-Ba, Ba-Ba, Ba-Su, Ba-Ph, Ph-Ph, Ph-Su and Ph-Ba. If the oligonucleotides are further modified by means of certain non-nucleotide substituents, the linkages can be produced through the unmodified portions of the oligonucleotides. These modifications also include modified nucleic acids, for example: PNA, LNA or morpholino oligonucleotide analogues. The bonds are preferably composed of C, H, N, O, S, B, P and halogen, which contain from 3 to 300 atoms. An example of 3 atoms is an acetal bond (ODN1-3'-0-CH2-0-3'-ODN2; Froehler and Matteucci) connecting, for example, the 3'-hydroxy group of a nucleotide with the 3'-hydroxy group of a second oligonucleotide. An example of approximately 300 atoms is PEG-40 (tetraconta polyethylene glycol). Preferred bonds are the phosphodiester, phosphorothioate, methylphosphonate, phosphoramidate, boranophosphonate, amide, ether, thioether, acetal, thioacetal, urea, thiourea, sulfonamide, Schiff base and disulfide bonds. Another possibility is to use the Solulink BioConjugation System bioconjugation system (TriLink BioTechnoiogies, San Diego, CA). If the oligonucleotide is composed of two or more parts of a sequence, these parts may be identical or different. Thus, in an oligonucleotide with a 3'3'-linkage, the sequences may be identical, for example, 5'-ODN1-3'3'-ODN1-5 ', or different, for example, 5'-ODN1-3' 3'-ODN2-5 '. In addition, the chemical modification of the various parts of the oligonucleotide as well as the linker that connects them may be different. Since the uptake of the short oligonucleotides seems to be less effective than that of the long oligonucleotides, the binding of two or more short sequences improves the immunostimulation. The length of the short oligonucleotides is preferably 2 to 20 nucleotides, more preferably 3 to 16 nucleotides, but more preferably 5 to 10 nucleotides. Oligonucleotides having two or more unbound 5 'ends are preferred. The partial sequences of oligonucleotides can also be linked by non-nucleotide linkers, in particular by abasic linkers (spacers d), triethylene glycol units or units of hexaethylene glycol. Other linkers are the alkylamino, such as the C3, C6 and C12 amino linkers, and also the alkylthiol linkers, such as the C3 or C6 thiol linkers. The oligonucleotides can also be linked by aromatic residues which can be substituted by alkyl or substituted alkyl groups. The oligonucleotides may also contain a duplicating unit or a tripling unit (Glen Research, Sterling, VA), in particular oligonucleotides with a 3'3'-linkage. The one-mode duplicating unit can be based on 1,3-bis- [5- (4,4'-dimethoxytrityloxy) pentylamido] propyl-2 - [(2-cyanoethyl) - (N, N-diisopropyl)] - phosphoramidite. The triplicating unit of one embodiment may be based on the incorporation of Tr is-2,2,2- [3- (4,4'-dimethoxytrityloxy)] propyloxymethyl] ethyl - [(2-cyanoethyl) - (N, N ' -diisopropyl)] - phosphoramidite. The branching of oligonucleotides by multiple duplicating, tripling or other multipliers produces dendrimers which are another embodiment of the present invention. The oligonucleotides may also contain connectoid units arising from the reagents that modify the peptides or reagents that modify the oligonucleotides (Glen Research, Sterling, VA).

In addition, the linkers may contain one or more natural or non-natural amino acid residues, which are connected through peptide bonds (amide). Another possibility for joining the oligonucleotides is through the cross-linking of the heterocyclic bases (Verma S et al. (1998) Annu Rev Biochem 67: 99-134; page 124). Even another possibility is to perform the linkage through the sugar radical of a part of the sequence with a heterocyclic base from another part of the sequence (lyer et al. (1999) Curr Opin Mol Therapeutics 1: 344-58; page 352). The different oligonucleotides containing unusual bonds are synthesized by known methods and can be linked together on line during solid phase synthesis. Alternatively, they can be linked together after the synthesis of the individual partial sequences. CpG phosphorothioate oligonucleotides with potent stimulating activity in the mouse system tend to have lower activity in human immune cells and other non-rodent species. DNA containing these motifs (TCG) nN or RDCGY? Y2N potently stimulated human peripheral blood cells to produce IFN-a. It has been discovered according to the invention that subgroups of immunostimulatory oligonucleotides CpG possess significant immunostimulatory effects on human cells, such as PBMC, suggesting that these immunostimulatory oligonucleotides CpG are effective therapeutic agents such as human vaccines, immunotherapy for cancer, immunotherapy for asthma, general improvement of immune function, improvement of hematopoietic recovery after radiotherapy or chemotherapy and other modulating applications of the immune system. As used herein, the terms "treat", "treat" or "treatment", when used with respect to disorders such as an infectious disease, cancer, allergy or asthma, refers to a prophylactic treatment that increases the resistance of a subject. the development of the disease (for example, the infection caused by a pathogen) or, in other words, reduces the likelihood that the subject will develop the disease (for example, becoming infected with the pathogen), as well as the treatment afterwards. that the subject has developed the disease in order to fight it (for example, to reduce or eradicate the infection) or to prevent the disease from getting worse. Thus, CpG immunostimulatory oligonucleotides are useful in some aspects of the invention as a vaccine for the treatment that has or is at risk of developing allergy or asthma, an infection caused by an infectious organism, or a cancer in which an antigen has been identified. cancer specific Thus, CpG immunostimulatory oligonucleotides can be administered to a subject together with an antigen or an allergen for the treatment of an infection, allergy, asthma or cancer. Alternatively and in addition, CpG immunostimulatory oligonucleotides can also be administered alone, without the antigen or the allergen, as protection against infection, allergy or cancer or can be administered with other therapeutic agents. CpG immunostimulatory oligonucleotides can also be administered with other therapeutic agents. The administration of repeated doses may offer longer-term protection. In the sense used herein, a subject at risk is a subject who has any identifiable risk of exposure to a pathogen capable of causing an infection or an allergen or at risk of developing cancer. For example, a subject at risk of developing an infection may be a subject who is scheduled to travel to an area where a particular type of infectious agent is found, or may be a subject that because of his or her lifestyle or medical procedure is exposed to body fluids that may contain infectious organisms or directly to the body, or it can be any subject living in a region where an infectious organism or an allergen has been identified. Subjects at risk of developing an infection also include general populations to which medical agencies recommend vaccination with an antigen against a particular infectious organism. If the antigen is an allergen and the subject develops allergic responses to that particular antigen and the subject may be exposed to the antigen, for example, during the pollen season, the subject is at risk of developing an allergic response. A subject at risk of developing an allergy or asthma includes those subjects who have been identified as allergic or asthmatic but who do not suffer from active disease during treatment with the immunostimulatory oligonucleotide CpG. A subject at risk of developing an allergy or asthma also includes subjects considered at risk of developing these diseases due to genetic or environmental factors. A subject at risk of developing cancer is one who has a high probability of developing cancer. These subjects include, for example, subjects who have a genetic abnormality, and who have been shown to have a correlative relationship with a higher probability of developing cancer, subjects exposed to agents associated with cancer, such as tobacco, asbestos. or other chemical toxins, and subjects previously treated because of cancer and who are in an apparent remission stage. When a subject at risk of developing a cancer is treated with an immunostimulatory oligonucleotide CpG and optionally with a specific antigen against the type of cancer that the subject is at risk of developing, the subject may be able to fight the cancer cells as it is develop If a tumor begins to form in the subject, the subject will develop an innate immune response or a specific immune response against the tumor antigen. In addition to the use of CpG immunostimulatory oligonucleotides as a prophylactic treatment, the invention also relates to the use of CpG immunostimulatory oligonucleotides for the treatment of a subject suffering from an infection, allergy, asthma or cancer. A subject who has an infection is one who has been exposed to an infectious pathogen and has acute or chronic detectable levels of the pathogen in the organism. The CpG immunostimulatory oligonucleotides can be used with or without an antigen or other therapeutic agent to elicit a systemic or mucosal immune response innate or specific against the antigen capable of reducing the level or eradicating the infectious pathogen. In the sense used in the present, an infectious disease is one that arises as a result of the presence of a foreign microorganism in the body. It is particularly important to develop effective strategies and treatments with vaccines to protect the mucosal surfaces of the body, which are the primary place for pathogens to enter. A subject who has an allergy is one who is able to develop an allergic reaction in response to an allergen. An allergy refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include, by way of illustration, eczema, allergic rhinitis or coryza, hay fever, conjunctivitis, bronchial asthma, allergic asthma, urticaria (hives), food allergies and other atopic conditions. Allergies in general are caused by the generation of IgE antibodies against innocuous allergens. Cytokines that induce systemic or mucosal administration of CpG immunostimulatory oligonucleotides are predominantly of the so-called Th1 type (eg, IL-12, IP-10, IFN-a and IFN-α) and these induce both humoral and cellular immune responses. The other important type of immune response, which is associated with the production of cytokines IL-4 and IL-5, is called Th2 immune response. In general, allergic diseases are mediated by Th2 type immune responses. Based on the ability of the CpG immunostimulatory oligonucleotides described herein to modify the immune response of a subject, of a predominant Th2 response (which is associated with the production of IgE antibodies and allergy) to a balanced response between Th2 types / Th1 (which protects against allergic reactions), a dose of effective CpG immunostimulatory oligonucleotide can be administered to induce an immune response to a subject to treat asthma and allergy. Thus, the immunostimulatory oligonucleotides CpG have a significant therapeutic activity for the treatment of allergic conditions and asthma. Th2 cytokine levels, especially IL-4 and IL-5, are elevated in the airways of asthmatic subjects. These cytokines promote important aspects of the asthmatic inflammatory response, including the change of isotope of IgE, chemotaxis and activation of eosinophils and growth of mast cells. Th1 cytokines, especially IFN-? and IL-12, can suppress the formation of Th2 clones and the production of Th2 cytokines. Asthma refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the respiratory tract to inhaled agents. Asthma frequently, although not exclusively, is associated with atopic or allergic symptoms. Thus, asthma includes allergic asthma and non-allergic asthma. A subject who has cancer is one who has detectable cancer cells. The cancer can be malignant or non-malignant. Cancers or tumors include, but are not limited to, bile duct cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasms , lymphomas, liver cancer, lung cancer (for example, small cell and not small cell), melanoma, neuroblastomas, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, skin cancer, testicular cancer, thyroid cancer and kidney cancer, as well as other carcinomas and sarcomas. In one embodiment, the cancer is tricholeukemia, chronic myelogenous leukemia, cutaneous T lymphocyte leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder carcinoma or colon carcinoma. . Subject means any human being or vertebrate animal, including for example dog, horse, cow, pig, sheep, goat, turkey, chicken, primates, for example, monkey, and fish (aquatic culture species), for example, Salmon. In this way, the compounds can be used to treat cancers and tumors, infections and allergy / asthma in human and non-human subjects. Cancer is one of the leading causes of death in domestic animals (for example, cats and dogs). In cases where the CpG oligonucleotide is administered with an antigen, the subject may be exposed to the antigen. In the sense with which it is used herein, "exposed to" means the active step by which the subject is brought into contact with an antigen or the passive exposure of the subject to the antigen in vivo. Methods of active exposure of a subject to an antigen are known in the art. In general, the antigen is administered directly to the subject by any means, either intravenously, intramuscularly, orally, transdermally, mucosally, intranasally, intratracheally or subcutaneously. The antigen can be administered systemically or locally. The methods for administering the antigen and the immunostimulatory oligonucleotide CpG are described in more detail below. A subject may be passively exposed to an antigen if the antigen is available for exposure by the body's immune cells. A subject may be passively exposed to an antigen, for example, when a foreign pathogen enters the body or when the subject develops a tumor cell that expresses a foreign antigen on its surface. The methods by which a subject is passively exposed to an antigen may depend particularly on the timing of the administration of the immunostimulatory oligonucleotide CpG. For example, in a subject at risk of developing a cancer, an infectious disease or an allergic or asthmatic response, the immunostimulatory oligonucleotide CpG can be administered to the subject on a regular basis when the risk is greater, for example, during the allergy season or after exposure to an oncogenic agent. In addition, the immunostimulatory oligonucleotide CpG can be administered to travelers before embarking on a trip to foreign lands where they may be at risk of being exposed to infectious agents. Similarly, the immunostimulatory oligonucleotide CpG can be administered to soldiers and civilians at risk of being exposed to biological weapons to induce a systemic or mucosal immune response to the antigen in case the subject is exposed to it. In the sense in which it is used herein, an antigen is a molecule capable of eliciting an immune response. Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide imitations of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts and multicellular organisms such as parasites and allergens. In a broad sense, the term antigen includes any type of molecule that is recognized by the host's immune system as foreign. Antigens include, by way of illustration, cancer antigens, microbial antigens and allergens. A cancer antigen, as used herein, is a compound, such as a peptide or protein, associated with a tumor or with the surface of a cancer cell, capable of eliciting an immune response when expressed on the surface of a cell that presents antigens in the context of an MHC molecule. Cancer antigens can be prepared from cancer cells, either by preparing crude extracts of cancer cells, for example in the manner described in Cohen PA et al. (1994) Cancer Res 54: 1055-8, partially purifying the antigens, by means of recombinant or by de novo synthesis of the known antigens. Cancer antigens include, by way of illustration, antigens that are expressed recombinantly, to an immunogenic part thereof or to a tumor or a whole cancer cell. Such antigens can be isolated or prepared recombinantly or by any other means known in the art. As used herein, "cancer antigen" and "tumor antigen" are used interchangeably to refer to antigens that are differentially expressed by cancer cells and that can be exploited to target cancer cells. Cancer antigens are antigens that can potentially stimulate apparently tumor-specific immune responses. Normal cells encode, although not necessarily express, some of these antigens. These antigens can be characterized as those that are normally imperceptible (ie, not expressed) in normal cells, those that are expressed only in some stages of differentiation and those that are expressed temporarily, such as embryonic and fetal antigens. Other carcinogenic antigens are encoded by mutant cellular genes, such as oncogenes (for example, the activated ras oncogene), suppressor genes (for example, p53 mutant), fusion proteins that result from internal deletions or chromosomal translocations. There are even other cancer antigens that can be encoded by viral genes, such as those that contain the tumor viruses of RNA and DNA. In the sense in which it is used herein, a microbial antigen is an antigen of a microorganism that includes, by way of illustration, 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 the antigens of the natural microorganism and induce a specific immune response for said microorganism. A compound is similar to an antigen of a natural microorganism if it induces an immune response (humoral and / or cellular) to an antigen of a natural microorganism. Such antigens are used routinely in the art and are well known to those of skill in the art. Without limitation, some examples of viruses that have been found in humans are: Retroviridae (for example, human immunodeficiency virus, such as HIV-1 (also known as HTLV-III, LAV or HTLV-III / LAV or HIV) -lll); and other isolates such as HIV-LP; Picornaviridae (eg, poliovirus, hepatitis A virus, enterovirus, human Coxsackie virus, rhinovirus and ecovirus); Calciviridae (for example, strains that cause gastroenteritis); Togaviridae (eg, equine encephalitis virus, rubella virus); Flaviviridae (for example, dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (for example, vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (eg, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (for example, influenza virus); Bunyaviridae (eg, hantavirus, bunyavirus, flebovirus and nairovirus); Sand viridae (hemorrhagic fever virus); Reoviridae (for example, reovirus, orbivirus and rotavirus), Bornaviridae, Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (the majority of adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus, Poxviridae (smallpox virus, vaccine virus, syphilis virus) and Iridoviridae (for example, African swine fever virus) and unclassified viruses (for example, hepatitis delta agent (considered a defective satellite of hepatitis B virus), hepatitis C, Norwalk virus and similar viruses, such as astrovirus). Both gram-negative and gram-positive bacteria serve as antigens in vertebrate animals Gram-positive bacteria include, but are not limited to, Pasteurella spp., Staphylococci spp., And Streptococcus spp. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas spp., And Salmonella spp. For illustrative purposes, some specific examples of infectious bacteria are: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sp p. (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 spp. pathogens, Enterococcus spp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium períringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallldum, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israelii. Some examples of fungi include Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. Other infectious organisms (ie, protists) are Plasmodium spp., Such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax and Toxoplasma gondii. Plasmodium spp., Babesia microtl, Babesia divergens, Leishmania tropic, Leishmania spp., Leishmania braziliensis, Leishmania donovani, Trypanosoma gambiense and Trypanosoma rhodesiense (African narcolepsy), Trypanosoma cruzi (fungal disease) are among the blood and / or tissue transmission parasites. Chagas) and Toxoplasma gondii. A broad description of other clinically relevant microorganisms can be found in the literature, for example, see C.G.A. Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the total content of which is incorporated herein by reference. An allergen refers to a substance (antigen) capable of inducing an allergic or asthmatic response in a susceptible subject. The list of allergens is huge and may include pollens, insect venom, animal dander, dust, fungal spores, and drugs (for example, penicillin). By way of illustration, some examples of natural, animal and plant allergens include the specific proteins of the following genera: Canine (Canis familiaris); Dermatophagoides (e.g., Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia, Lolium (for example, Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica), 'Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (for example, Plantago lanceolada); Parietaria (for example, Parietaria officinalis or Parietaria judaica); Blatella (for example, Germanic Blattella); Apis (for example, Apis multiflorum); Cupressus (for example, Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (for example, Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (for example, Thuya orientalis); Chamaecyparis (for example, Chamaecyparis obtusa); Periplaneta (for example, Periplaneta americana); Agropyron (for example, Agropyron repens); Sécale (for example, Sécale cereale); Triticum (for example, Triticum aestivum); Dactylis (for example, Dactylis glomerata); Festuca (for example, Festuca elatior); Poa (for example, Poa pratensis or Poa compressa); Oats (for example, Avena sativa); Holcus (for example, Holcus lanatus); Anthoxanthum (for example, Anthoxanthum odoratum); Arrhenatherum (for example, Arrhenatherum elatius); Agrostis (for example, Agrostis alba); Phleum (for example, Phleum pratense); aris (for example, aris arundinacea); Paspalum (for example, Paspalum notatum); Sorghum (for example, Sorghum halepensis); and Bromus (for example, Bromus inermis). The antigen can be substantially purified. As used herein, the term "substantially purified" refers to an antigen, that is to say a polypeptide, which is substantially free of other proteins, lipids, carbohydrates or other substances to which it is naturally associated. Those skilled in the art can purify the polypeptide antigens using standard protein purification techniques. The substantially pure polypeptide will often yield a single major band in a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or those having several initiation codons, there may be several bands in the non-reducing polyacrylamide gel, but these form a different pattern from that which forms the polypeptide. The purity of the polypeptide antigen can also be determined by an amino-terminal amino acid sequence analysis. Other types of antigens, such as polysaccharides, small molecules, imitations, etc., are also included in the invention and may optionally be substantially pure.

The oligonucleotides of the invention can be administered to a subject with an antimicrobial agent. In the sense used herein, an antimicrobial agent refers to a natural or synthetic compound capable of eradicating or inhibiting infectious microorganisms. The type of antimicrobial agent useful according to the invention will depend on the type of microorganism with which the subject is infected or that is at risk of becoming infected. By way of illustration, antimicrobial agents include antibacterial agents, antiviral agents, antifungal agents and antiparasitic agents. Phrases such as "anti-infective agent", "antibacterial agent", "antiviral agent", "antifungal agent", "antiparasitic agent" and "antiparasitic agent" have a well-known meaning for those who have basic knowledge of the technique and are defined in the standard medical texts. Briefly, antibacterial agents eradicate or inhibit bacteria and include antibiotics and other natural or synthetic compounds that have similar functions. Antibiotics are low molecular weight molecules that cells, such as microorganisms, produce as secondary metabolites. In general, antibiotics interfere with one or more bacterial functions or structures that are specific to the microorganism and are not present in the host cells. Antiviral agents can be isolated from natural sources or synthesized and are useful for eradicating or inhibiting viruses. Antifungal agents are used to treat both superficial fungal infections and opportunistic and primary systemic fungal infections. Antiparasitic agents eradicate or inhibit parasites. By way of illustration, some examples of antiparasitic agents, also referred to as antiparasitic, useful for administration to humans, are albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, eflomitin, flurazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimonate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethamine-solfonamides, pyrimethamine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin, sodium stibogluconate (sodium antimony gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethoprim-sulfamethoxazole and triparasamide, some of which they are used as the only treatment or in combination with other agents. Antibacterial agents eradicate or inhibit the growth or function of bacteria. An important class of antibacterial agents is what constitutes antibiotics. Antibiotics, effective to eradicate or inhibit a wide variety of bacteria, are called broad-spectrum antibiotics. Other types of antibiotics are predominantly effective against Gram-positive or Gram-negative bacteria. These antibiotics are called narrow-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 usually classified based on their primary mechanism of action. In general, antibacterial agents are inhibitors of cell wall synthesis, cell membrane inhibitors, inhibitors of protein synthesis, functional inhibitors or of the synthesis of nucleic acids and competitive inhibitors. Antiviral agents are compounds that prevent cells from becoming infected with viruses or that the virus replicates within the cell. There are far fewer antiviral agents than antibacterials since the process of viral replication is closely related to the replication of DNA within the host cell that non-specific antiviral agents would be generally host-toxic. There are various stages within the process of viral infection that can block or inhibit antiviral agents. These steps include the binding of the virus to the host cell (immunoglobulin or binding peptides), exposure of the virus (eg, amantadine), synthesis or translation of viral mRNA (eg, interferon), replication of RNA or viral DNA (eg, nucleoside analogs), maturation of new viral proteins (for example, protease inhibitors) and sprouting and virus release. Nucleotide analogs are synthetic compounds similar to nucleotides, but which have an incomplete or abnormal deoxyribose or ribose group. Once the nucleotide analogues are in the cell, they are phosphorylated and produce the triphosphate form that competes with normal nucleotides to be incorporated into the DNA or viral RNA. Once the triphosphate form of the nucleotide analog is incorporated into the growing nucleic acid chain, it produces an irreversible association with the viral polymerase and, therefore, chain termination. Illustratively, nucleotide analogs include acyclovir (used for the treatment of herpes simplex virus and varicella zoster virus), ganciclovir (useful in the treatment of cytomegalovirus), idoxuridine, ribavirin (useful in the treatment of respiratory syncytial virus), dideoxyinosine, dideoxycytidine, zidovudine (azidothymidine), imiquimod and resimiquimod. Interferons are cytokines that secrete both virus-infected cells and immune cells. Interferons work by binding to the specific receptors present in the cells adjacent to the infected cells, and they cause a change in the cell that protects it from the infection produced by the virus. Interferon-a and interferon-β also induce the expression of MHC class I and class II molecules on the surface of infected cells, which increases the presentation of the antigen for recognition by the host's immune cell. Interferon-a and interferon-beta are present in recombinant forms and are used for the treatment of chronic hepatitis B and C infection. In doses effective as an antiviral treatment, interferons have serious side effects such as fever, malaise and weight loss. Antiviral agents useful in the invention include, by way of illustration, immunoglobulins, amantadine, interferons, nucleoside analogs, and protease inhibitors. By way of illustration, some specific examples of antivirals are acemannan, acyclovir, acyclovir sodium, adefovir, alovudine, alvircept sudotox, amantadine hydrochloride, aranotin, arildone, atevirdine mesylate, aviridine, cidofovir, cipamfilin, cytarabine hydrochloride, delavirdine mesylate, desciclovir, didanosine, disoxaril, edoxudin, enviradene, enviroxime, famciclovir, famotine hydrochloride, fiacitabine, fialuridine, fosarilate, foscamet sodium, fosfonet sodium, ganciclovir, ganciclovir sodium, yodoxuridine, ketoxal, lamivudine, lobucavir, memotine hydrochloride, metisazone, nevirapine , penciclovir, pirodavir, ribavarin, rimantadine hydrochloride, saquinavir mesylate, somantadine hydrochloride, sorivudine, estatolon, stavudine, tilorone hydrochloride, trifluridine, valaciclovir hydrochloride, vidarabine, vidarabine phosphate, vidarabine sodium phosphate, viroxime, zalcitabine, zidovudine and zinviroxime. The antifungal agents are useful for the treatment and prevention of infectious fungi. Antifungal agents are usually classified according to their mechanism of action. Some antifungals work as inhibitors of the cell wall by inhibiting glucose synthase. For illustrative purposes, some examples are basiungin / ECB. Other antifungal agents work by destabilizing the integrity of the membrane. Illustratively, these include imidazoles, such as clotrimazole, sertaconazole, fluconazole, itraconazole, ketoconazole, miconazole and viroconazole, as well as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimycin, UK 292, butenafine and terbinafine. Other antifungal agents act by disintegrating chitin (for example, chitinase) or immunosuppression (cream 501). The CpG immunostimulatory oligonucleotides can be combined with other therapeutic agents as adjuvants to improve immune responses. The immunostimulatory oligonucleotide CpG and another therapeutic agent can be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously, they can be administered in the same formulation or in separate formulations, but are administered at the same time. The other therapeutic agents are administered sequentially to each other and to the immunostimulatory oligonucleotide CpG, when the administration of the other therapeutic agents and the immunostimulatory oligonucleotide CpG is temporarily separated. More specifically, the immunostimulatory oligonucleotide CpG can be administered before or after the administration of (or exposure) at least one of the other therapeutic agents. The time separation between the administration of these compounds can be a matter of minutes or longer. An illustrative title, among the other therapeutic agents include adjuvants, cytokines, antibodies, antigens, etc. The compositions of the invention may also be administered with adjuvants other than nucleic acids. An adjuvant that is not a nucleic acid is any molecule or compound, except the CpG immunostimulatory oligonucleotides described herein that can stimulate the humoral and / or cellular immune response. By way of illustration, non-nucleic acid adjuvants include adjuvants that create a slow release effect, immunostimulatory adjuvants and adjuvants that create a slow release effect and stimulate the immune system. CpG immunostimulatory oligonucleotides are also useful as mucosal adjuvants. It has already been discovered that the mucosal delivery of CpG nucleic acids induce systemic and mucosal immunity. Thus, the oligonucleotides can be administered in combination with other mucosal adjuvants. Immune responses can also be induced or increased by co-administration or colinear expression of cytokines (Bueler & amp; amp; amp;; Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997; Iwasaki et al., 1997; Kim et al., 1997) or costimulatory molecules such as B7 (Iwasaki et al., 1997; Tsuji et al., 1997) with the immunostimulatory oligonucleotides CpG. The term cytokine is used as a generic name to designate a group of soluble proteins and peptides that act as humoral regulators at nanomolar to picomolar concentrations and which, under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate the processes that develop in the extracellular environment. By way of illustration, some examples of cytokines are, interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12. , IL-15, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), IFN- ?, IFN-a, IFN-β, necrosis factor tumor (TNF), TGB-ß, ligand Flt-3 and ligand CD40. In addition to cytokines, CpG oligonucleotides can be used in combination with antibodies against certain cytokines, such as anti-IL-10 and anti-TGF-β, as well as with cyclooxygenase inhibitors, ie inhibitors of COX-1 and the COX-2. Oligonucleotides are also useful for redirecting an immune response, from a Th2 immune response to a Th1 immune response. This results in the production of a relatively balanced Th1 / Th2 environment. Redirection of an immune response from a Th2 immune response to a Th1 can be assessed by measuring the levels of cytokines that are produced in response to nucleic acid (for example, by inducing monocytic cells and other cells to produce Th1 cytokines, including IFN -to). Redirecting or rebalancing the immune response of a Th2 response to a Th1 response is particularly useful for the treatment of asthma. For example, an amount effective for the treatment of asthma may be the amount useful for redirecting a Th2 type immune response that is associated with asthma to a Th1 type response or to a balanced Th1 / Th2 medium. Th2 cytokine levels, especially IL-4 and IL-5, are elevated in the airways of asthmatic subjects. The immunostimulatory oligonucleotides CpG described herein increase the Th1 cytokines, which helps to rebalance the immune system, which prevents or reduces the adverse effects associated with the predominantly Th2 immune response. The redirection of an immune response from a Th2 response to a Th1 response can also be assessed by measuring the levels of the immunoglobulin specific isotypes. For example, in mice, IgG2a is associated with a Th1 immune response and IgG1 and the IgE are associated with a Th2 immune response. CpG immunostimulatory oligonucleotides have the distinctive ability to promote cell survival, differentiation, activation and maturation of dendritic cells and are useful for in vitro, in vivo and ex vivo methods including dendritic cells. The immunostimulatory oligonucleotides CpG also increase the lytic activity of natural killer cells and the antibody-dependent cellular cytotoxicity (ADCC). ADCC can be performed using an immunostimulatory oligonucleotide CpG in combination with an antibody specific for a cell target, such as a cancer cell. When a subject is administered the CpG immunostimulatory oligonucleotide together with the antibody, the subject's immune system is induced to eradicate the cancer cell. Antibodies useful for the ADCC method include antibodies that interact with an organism cell. Many of said antibodies specific for cellular targets have been described in the art and many can be purchased commercially. The immunostimulatory oligonucleotides CpG can also be administered in conjunction with oncological treatment. Oncological treatments include oncological medications, radiation and surgical procedures. In the sense in which it is used in the present "oncological medicament" it refers to an agent that is administered to a subject for the purpose of treating cancer. In the sense in which it is used herein to "treat cancer" means to prevent the development of a cancer, reduce the symptoms of the cancer and / or inhibit the growth of an existing cancer. In other aspects, the cancer medication is administered to a subject at risk of developing a cancer in order to reduce the risk of developing it. Various types of drugs for the treatment of cancer are described herein. For the purposes of this specification, oncological drugs are classified into chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormonal therapy and biological response modifiers. In addition, the methods of the invention are intended to include the use of more than one cancer medicament together with the immunostimulatory oligonucleotides CpG. For example, when relevant, the immunostimulatory oligonucleotide CpG can be administered in conjunction with both a chemotherapeutic agent and an immunotherapeutic agent. Alternatively, the cancer medicament may include an immunotherapeutic agent and a cancer vaccine, or a chemotherapeutic agent and a cancer vaccine, or a chemotherapeutic agent, an immunotherapeutic agent and a cancer vaccine, all administered to a subject with the purpose of treating a subject who has a cancer or is at risk of developing cancer. The chemotherapeutic agent may be selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, chloroethylnitrosureas without sugar content, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragilin, GLA meglamine, valrubicin, carmustatin and poluferposan, MMI270 , BAY 12-9566, RAS farnesyltransferase inhibitor, famesyltransferase inhibitor, MMP, MTA / LY231514, LY264618 / Lometexol, Glamolec, CI-994, TNP-470, Hicamtin / topotecan, PKC412, Valspodar / PSC833, Novantrone / mitroxantrone, Metaret / suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, lncel / VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516 / marmistat, BB2516 / marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil / OK-432, AD 32 / valrubicin, Metastron / strontium derivative, Temodal / temozolomide, Evacept / liposomal doroxorubicin, 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 incogene inhibitor, BMS-182751 / oral platinum, UFT (tegafur / uracil), Ergamisol / levamisole, eniluracil / 776C85 / 5FU enhancer, capto / levamisole, Camptosar / irinotecan, Tumodex / ralitrexed, Leustatin / cladribine, Paxex / paclitaxel, Doxil / liposomal doxorubicin, Caelyx / liposomal doxorubicin, Fludara / fludarabine, Pharmarubicin / epirubicin, DepoCyt, ZD1839, LU 79553 / bis-naphthalimide, LU 103793 / dolastatin, Caetyx / liposomal doxorubicin, Gemzar / gemcitabine, ZD 0473 / anormed, YM 116, iodine seeds, inhibitors of CDK4 and CDK2, inhibitors of PARP, D4809 / dexiphosamide, Ifes / Mesnex / ifosamide, Vumon / teniposide, Paraplatin / carboplatin, Platinol / cisplatin, Vepeside / etoposide, ZD 9331, Taxotere / docetaxel, prodrug of guanine arabinoside, taxane analogues, nitrides, alkylating agents such as lelfenan and cyclophosphamide, aminoglutethimide, asparaginase, busulfan, carboplatin, chlorobuchil, cytarabine HCl, dactinomycin, danorrubicin HCl, sodium phosphate estramustine, etoposide (VP16-213), floxuridine, fluorouracil (5-FU), flutamda, hydroxyurea (hydroxycarbamide), ifosfamide, interferon alfa-2a, alpha-2b, leuprolide acetate (analog of LHRH releasing factor) , lomustine (CCNU), mechlorethamine HCl (hydrogenated mustard), mercaptopurine, mesna, mitotane (o.p'-DDD), mitoxantrone HCl, ocreotide, piicamycin, procarbazine HCl, streptozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate , amsacrine (m-AMSA), azacitidine, ertropoietin, hexamethylmelamine (HMM), interleukin2, mitoguazone (methyl-GAG, Methyl-glyoxal-bis-guanylhydrazone, MGBG), pentostatin (2'-deoxicoformycin), semustine (methyl-CCNU), teniposide (VM-26) and vindesine sulfate; This list is not limiting. The immunotherapeutic agent can be selected from the group consisting of Rituxan, Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior tß, MDX-210, MDX -11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250 , EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1 D10 Ab, SMART ABL 364 Ab and ImmuRAIT -CEA; This list is not limiting. The cancer vaccine can be selected from the group consisting of EGF, anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanin vaccine, MGV ganglioside conjugate vaccine, Her2 / neu, Ovarex, M-Vax, O-Vax, L -Vax, teratopo STn-KHL, liposomal idiotypic vaccine, BLP25 (MUC-1), Melacine, peptide antigen vaccines, toxin / antigen vaccines, MVA-based vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN , DISC-virus and ImmuCyst / TheraCyst; This list is not limiting. The use of CpG oligoestimulatory oligonucleotides in conjunction with immunotherapeutic agents, such as monoclonal antibodies, may increase long-term survival through a number of mechanisms, including the enhancement of ADCC (as stated above), the activation of NK cells and increased levels of IFN-a. Nucleic acids, used in combination with monoclonal antibodies, help reduce the dose of antibody needed to obtain a biological result. The present invention also includes methods for inducing the non-specific innate immune activation of the antigen and the broad spectrum resistance to the infectious challenge using the immunostimulatory oligonucleotides CpG. The term "innate immune activation," as used herein, refers to the activation of immune cells, other than memory B cells, and, for example, may include the activation of monocytes, neutrophils, macrophages, dendritic cells. , KN cells and / or immune cells that can respond independently of the antigen. Broad spectrum resistance to an infectious challenge is induced because the immune cells are in active form and are initiated to respond to any invading compound or microorganism. The cells must not be specifically initiated against a particular antigen. This is of particular importance in the biological wars and in the circumstances described above for travelers. The CpG oligoestimulatory oligonucleotides can be administered directly to the subject or in conjunction with a nucleic acid delivery complex. A nucleic acid delivery complex is an associated nucleic acid molecule (e.g., bound in ionic or covalent form or encapsulated within) to a target medium (e.g., a molecule that produces a binding with higher affinity for a target cell.) Examples of nucleic acid delivery complexes include nucleic acids associated with a sterol (for example; cholesterol), a lipid (e.g., a cationic lipid, a virosome or a liposome), or a specific binding agent to a target cell (e.g., a ligand recognized by the specific receptor of the target cell). The preferred complexes may be sufficiently stable in vivo to avoid significant decoupling prior to internalization by the target cell. However, the complex can be separable under appropriate conditions within the cell so that the oligonucleotide is released in functional form. The immunostimulatory oligonucleotide CpG and / or the antigen and / or other therapeutic agents can be administered alone (eg, in saline or pH buffer) or using any of the delivery vehicles known in the art. For example, the following delivery vehicles have been described: cochleates (Gould-Fogerite et al., 1994, 1996), emulsomas (Vancott et al., 1998; Lowell et al., 1997), ISCOM (Mowat et al., 1993; Carisson et al. others, 1991; Hu et al., 1998; Morein et al., 1999); liposomes (Childers et al., 1999; Michalek et al., 1989, 1992; de Haan, 1995a, 1995b); vectors of live bacteria (eg, Salmonella, Escherichia coii, 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 (eg, vaccinia, adenovirus, herpes simplex) (Galuchan et al., 1993, 1995; Moss et al., 1996; Nugent et al., 1998; Flexner et al., 1988; Morrow et al., 1999); microspheres (Gupta et al., 1998; Jones et al., 1996; Maloy et al., 1994;Moore et al., 1995; O'Hagan and others, 1994; Eldrldge 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 (Tacker et al., 1998; Mason et al., 1998; Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al., 1995; Cryz et al., 1998), particles similar to viruses (Jiang et al., 1999; Leibl et al., 1998). Other delivery vehicles are known in the art. The term "effective amount" generally refers to the amount necessary or sufficient to obtain a desired biological effect. For example, an effective amount of an immunostimulatory oligonucleotide CpG administered with an antigen to induce mucosal immunity is the amount necessary to cause the development of an IgA in response to an antigen at the time of exposure to the antigen, while the amount necessary to induce a systemic immunity is that necessary to cause the development of an IgG in response to an antigen at the time of exposure to the antigen. In combination with the guidelines contained herein, by choosing among the various active compounds and weighting factors, such as concentration, relative bioavailability, patient body weight, severity of side effects and preferred mode of administration, a treatment can be planned effective prophylactic or therapeutic that does not cause substantial toxicity but is still fully effective in treating a particular subject. The effective amount for a particular application may vary depending on factors such as the disease or condition to be treated, the particular immunostimulatory oligonucleotide CpG to be administered, the patient's weight or the severity of the disease or condition. Those of ordinary skill in the art will be able to empirically determine the effective amount of a CpG immunostimulatory oligonucleotide and / or of an antigen and / or of another particular therapeutic agent without needing to conduct undue experimentation. Doses for subjects of the compounds described herein for mucosal or local delivery typically range from 10 μg to 10 g per administration which, depending on the application, may be administered on a daily, weekly or monthly basis and any other among these or with the frequency necessary More typically, mucosal or local doses range from about 1 mg to 500 mg per administration, more typically between about 1 mg and 100 mg, with 2 to 4 administrations with a range of days or weeks between each. More typically, doses of immunostimulant range between 10 μg and 100 mg per administration and more typically between 100 μg and 10 mg with daily or weekly administrations. Doses for subjects of the compounds described herein for parenteral delivery for the purpose of inducing an antigen-specific immune response, where the compounds are delivered with an antigen but not with another therapeutic agent, are typically 5 to 10,000 times higher than the mucosal dose typical for applications such as vaccine adjuvant or immune stimulant, and more typically 10 to 1,000 times higher and more typically 20 to 100 times higher. The doses of the compounds described herein for parenteral delivery for the purpose of inducing an innate immune response, increasing ADCC or inducing an antigen-specific immune response when the CpG immunostimulatory oligonucleotides are administered in combination with other therapeutic agents or in specialized delivery vehicles ranges from approximately 100 μg to 10 g per administration which, depending on the application, it can be administered on a daily, weekly or monthly basis, and any other among them or with the necessary frequency. More typically, parenteral doses for these purposes range from about 1 mg to 5 g per administration, more typically between about 1 mg and 1 g, with 2 to 4 administrations with a range of days or weeks between each. However, in some embodiments, parenteral doses for these purposes may be used at intervals 5 to 10,000 times greater than the doses typically described herein. For any of the compounds described herein, the therapeutically effective amount can be determined initially from the animal models. The therapeutically effective dose can also be determined from human data obtained with other CpG oligonucleotides already tested in humans (there are clinical studies in humans in progress) and with compounds known to have similar pharmacological activities, such as other adjuvants , for example, LT and other antigens used in vaccines. For higher parenteral administration, higher doses may be necessary. The applied dose can be adjusted depending on the relative bioavailability and the concentration of the compound administered. The adjustment of the dose to obtain the maximum efficiency according to the methods described above and other methods is something that can be done by anyone with knowledge of the art. The formulations of the invention are administered in pharmaceutically acceptable solutions, which, routinely, contain pharmaceutically acceptable concentrations of salt, pH regulating agents, preservatives, compatible carriers, adjuvants and, optionally, other therapeutic ingredients. As a therapeutic use, the effective amount of the immunostimulatory oligonucleotide CpG and / or another therapeutic agent can be administered to a subject by any route that allows the compound to be delivered to the desired surface, eg, local, mucosal, systemic. The administration of the pharmaceutical composition of the present invention can be carried out by any means known in the art. By way of illustration, preferred routes of administration are oral, parenteral, intravenous, subcutaneous, intralesional, intratumoral, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal and rectal. For oral administration, the compounds (ie, CpG immunostimulatory oligonucleotides, antigens and / or other therapeutic agents) can be formulated easily by combining the active compound or compounds with pharmaceutically acceptable carriers well known in the art. Said vehicles make it possible to formulate the compounds of the invention in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, aqueous solutions, suspensions, etc. for oral administration to the subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipients, optionally by grinding the resulting mixture and processing the granule mixture after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, among which are included lactose, sucrose, mannitol or sorbitol, cellulose preparations such as, for example, corn starch, wheat starch, rice starch, starch potato, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as interlaced polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate. Optionally, the oral formulations can also be formulated in saline solutions or pH regulators to neutralize the internal acid conditions, or they can be administered without any vehicle. Dragee cores have a suitable cover. To that end, concentrated sugar solutions can be used, which optionally may contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and organic solvents or mixtures of suitable solvents. Dyes and pigments can be added to the covers of the tablets or dragees to identify or characterize the different combinations of the doses of active compounds. Pharmaceutical preparations that can be used orally include two-part gelatin capsules, as well as sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The two-part capsules may contain the active ingredients in a mixture with a filler such as lactose, binders such as starches and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may 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. These are well defined in the art. All formulations for oral administration must have doses suitable for such administration. For buccal administration, the compositions can be formulated in tablets or candies, in the traditional manner. The compounds can be administered by inhalation to the pulmonary tract, especially to the bronchi and more particularly to the alveoli of the lung depth, using standard inhalation devices. The compounds may be delivered in the form of aerosol spray in pressurized containers or in nebulizers, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of the pressurized aerosol, the dose unit can be determined including a valve that allows an exact dose to be delivered. An inhalation device can be used to deliver the compounds to the subject. As used herein, an inhalation device is any device for administering an aerosol, such as the dry powder form of the compounds. This type of equipment is known in the art and has been described in detail, for example, in Remington: The Science and Practice of Pharmacy, 19th Edition, 1995, Mac Publishing Company, Easton, Pennsylvania, pages 1676-1692. Many U.S. Patents, for example, U.S. Pat. No. 6,116,237, also describe inhalation devices. In the sense in which it is used herein, "powder" refers to a composition consisting of finely dispersed solid particles. Preferably, the compounds flow in a relatively free form and can be dispersed in an inhalation device and subsequently the subject can inhale them so that the compounds reach the lungs to allow their penetration into the alveoli. The term "dry powder" refers to a powder composition having a moisture content such that the particles are rapidly dispersible in an inhalation device to form an aerosol. The moisture content is generally less than about 10% by weight (% p) of water and in some embodiments it is less than about 5% p and preferably less than about 3% p.

The powder can be formulated with polymers or, optionally, it can be formulated with other materials such as liposomes, albumin and / or other vehicles. The aerosol dose and the delivery systems can be selected for a particular therapeutic application by anyone skilled in the art, according to the description, for example, of Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract", in Critical Reviews in Therapeutic Drug Carrier Systems, 6: 273-313 (1990) and Moren, "Aerosol dosage forms and formulations" in Aerosols in Medicine, Principies, Diagnosis and Therapy, Moren et al., Eds., Elsevier, Amsterdam, 1985. When it is desired to deliver the compounds systemically, they can be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion. Injectable formulations may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with the addition of a preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain agents for the formulation such as suspending, stabilizing and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In addition, suspensions of the active compounds can be prepared in the form of injectable oil suspensions. The lipophilic solvents or vehicles include fatty acids such as sesame oil, synthetic fatty acid esters, such as ethyl oleate, triglycerides or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain stabilizers or agents that increase the solubility of the compounds to make it possible to prepare highly concentrated solutions. Alternatively, the active compounds can be formulated as a powder, which must be reconstituted with a suitable vehicle, eg, sterile, pyrogen-free water, before use. The compounds may also be formulated in compositions for vaginal or rectal administration, such as suppositories or retention enemas, for example, containing conventional suppository bases, such as cocoa butter and other glycerides. In addition to the formulations described above, the compounds can also be formulated in a slow release preparation. These long-acting formulations can be formulated with suitable polymeric or hydrophilic substances (for example, an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, such as a sparingly soluble salt. The pharmaceutical compositions may also include solid phase or gel vehicles or excipients. By way of illustration, some examples of said carriers or excipients are calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers, such as polyethylene glycols. Suitable liquid or solid pharmaceutical forms are, for example, aqueous solutions or saline, microencapsulated, enriched, coated on microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for skin implantation or dried in a sharp object to be scraped over the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or prolonged release preparations of the active compounds, in which excipients and additives are commonly used and / or auxiliary agents such as disintegrants, binders, coating agents, bulking agents, lubricants, flavors, sweeteners or solubilizers, as described. The pharmaceutical compositions are suitable for use in various drug delivery systems. A brief review of the drug delivery methods can be found in Langer R (1990) Science 249: 1527-33, which is incorporated herein by reference. The CpG immunostimulatory oligonucleotides, and optionally other therapeutic agents 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 it is possible to use salts that are not pharmaceutically acceptable to prepare the pharmaceutically acceptable salts thereof. These salts include, for information purposes, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulfonic, tartaric, citric, methanesulfonic, formic, malonic , succinic, naphthalene-2-suphonic and benzenesulfonic. Also, said salts can be prepared as alkali metals or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. The agents used as pH regulators 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% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v), chlorobutanol (0.3-0.9% w / v), parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02). % p / v). The pharmaceutical compositions of the invention contain an effective amount of an immunostimulatory oligonucleotide CpG and, optionally, antigens and / or other therapeutic agents optionally included in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a human or other vertebrate animal. The term "vehicle" refers to an organic or inorganic ingredient, natural or synthetic, in which the active substance is combined to facilitate its application. The components of the pharmaceutical compositions are also capable of mixing with the compounds of the present invention, and with each other, so that there is no interaction that can negatively affect the desired pharmaceutical efficacy. The present invention is further illustrated by the following examples, which are not to be construed as limiting in any way.

EXAMPLES EXAMPLE 1 Analogs of ODN class C induce the secretion of IFN-a and the activity of human TLR9 in vitro In this series of experiments, the class C ODN analogs of the invention were analyzed in vitro for their ability to stimulate human peripheral blood mononuclear cells (PBMC), secrete INF-a and stimulate HEK293 cells, stably transfected with human TLR9 and a reporter construct of NF-? B to demonstrate TLR9 signaling. The ODNs were purchased from Biospring (Frankfurt, Germany) and their identity and purity were monitored with Coley Pharmaceutical GmbH (Langenfeld, Germany). The ODNs were diluted in phosphate buffered saline (Sigma, Germany) and stored at -20 ° C. All dilutions were carried out with pyrogen-free reagents. The test ODNs were the following: 128 T * C_G * T * C * G * T * T * T * T * A * C * G * G * C * G * T * C * G * T * G * C * C * G (SEQ ID NO : 48) 611 T * C * G * T * C * G * T * T * T * T * A * C_G * G * C_G * C * C_G * T * G * C * C * G (SEQ ID NO: 43) 614 T * C * G * T * C * G * T * T * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO : 43) 620 T * C * G * T * C_G * T * T * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 43) 331 T * C * G * C_G * A * C * G * T * T * C_G * G * C * G * C_G * C * T * G * C * C * G (SEQ ID NO: 27) 332 T * C * G * C_G * T * C * G * T * T * C_G * G * C * G * C_G * C * T * G * C * C * G (SEQ ID NO: 30) 333 T * C * G * C_G * A * C * G * T * T * C_G * G * C * G * C_G * T * C * G * C * C * G (SEQ ID NO: 28) 334 T * C * G * C_G * A * C * G * T * T * C_G * G * C * G * G * C_T * C * G * C * C * G (SEQ ID NO: 29) 335 T * C * G_C * G * T * C * G * T * T * C_G * G * C * G * C_G * C * T * G * C * C * G (SEQ ID NO: 30) 336 T * C * G * C * G_T * C * G * T * T * C_G * G * C * G * C_G * C * T * G * C * C * G (SEQ ID NO: 30) 337 T * C * G * C * G_A * C * G * T * T * C_G * G * C * G * C_G * T * C * G * C * C * G (SEQ ID NO: 28) 338 T * C * G * C * G_A * C * G * T * T * C_G * G * C * G * G * C_T * C * G * C * C * G (SEQ ID NO: 29) 339 T * C * G_C * G * A * C * G * T * T * C_G * G * C * G * C_G * T * C * G * C * C * G (SEQ ID NO: 28) 340 T * C * G_C * G * A * C * G * T * T * C_G * G * C * G * G * C_T * C * G * C * C * G (SEQ ID NO: 29) 341 T * C * G * C_G * T * C * G * T * T * C_G * G * C * G * C_G * T * C * G * C * C * G (SEQ ID NO: 31) 342 T * C * G * C_G * T * C * G * T * T * C_G * G * C * G * G * C_T * C * G * C * C * G (SEQ ID NO: 32) 343 T * C * G * C * G_T * C * G * T * T * C_G * G * C * G * C_G * T * C * G * C * C * G (SEQ ID NO: 31) 344 t * C * G * C * GT * C * G * T * T * CG * G * C * G * G * CT * C * G * C * C * G (SEQ ID NO: 32) where * represents a phosphorothioate bond and _ represents a phosphodiester bond. The 2006 CpG ODN was used (TCGTCGTTTTGTCGTTTTGTCGTT, SEQ ID NO: 56) as a positive control for the activation of the TLR9 signal. The CnG class C ODN 2429 (TCGTCGTTTTCGGCGGCCGCCG, SEQ ID NO: 53) was used as a positive control for the induction of IFN-a. The ODN no CpG 1982 (TCCAGGACTTCTCTCAGGTT, SEQ ID NO: 18) was used as a negative control. Peripheral blood buffy coat preparations were obtained from healthy human donors of male and female blood bank from the University of Dusseldorf (Germany) and, from these, PBMCs were purified by centrifugation in Ficoll-Hypaque (Sigma). The purified PBMC were resuspended in RPMI 1640 culture medium (BioWhittaker, Belgium) supplemented with 5% (v / v) heat inactivated human serum AB (BioWhittaker) or 10% (v / v) heat inactivated FCS, 2 mM L-glutamine (BioWhittaker), 100 U / ml penicillin and 100 μg / ml streptomycin (Invitrogen, Karlsruhe, Germany). Fresh PBMCs were plated in 96 well round bottom plates and incubated for 48 hours with the ODN at the indicated concentrations in a humidified 37 ° C incubator. The supernatants of the culture were collected and, if not needed immediately , they were frozen at -20 ° C until the time of use. The amount of IFN-a present in the supernatants was determined using an enzyme-linked immunosorbent assay (ELISA) developed using commercially available antibodies (Alexis GmbH, Grünberg, Germany). ODNs 128, 331-344, 611 and 620 were analyzed with PBMC from different donors and ODN 614 was analyzed with PBMC from three different donors. The HEK293 cells transfected under stable conditions used for the human TLR9 reporter gene assay expressed the human TLR9 receptor and a reporter gene construct NF-? B. The cells were incubated with the ODN for 16 h at 37 ° C in a humidified incubator. Each data point was made in triplicate. The cells were used and the assay was performed to determine the activity of the reporter gene. The stimulation indexes were calculated in reference to the activity of the reporter gene of the medium without the addition of ODN.

Results Figures 1A-1F and 2A-2F show the representative results. Figures 1A-1F show that class C ODNs 332, 333 and 334 induced large amounts of IFN-a (typically about 2000-2500 pg / ml) when they had a concentration of 1 μM. The amount of IFN-a induced by these ODNs significantly exceeded the amount of IFN-a that induced the same concentration of ODN 2006 or 1982. Figures 1A-1 F also show that the class C ODNs 332, 333 and 334 also induced a significant amount of TLR9 signaling activity (typically with a stimulation index of about 15) when they had a concentration of 10 μM. The amount of TLR9 signaling activity induced by these ODNs was approximately half of the TLR9 signaling activity that induced the same ODN 2006 concentration. Essentially, the same results were observed with ODN 128 and 335-344. Figures 2A-2F show that ODN class C 611, 614 and 620 induced even higher amounts of IFN-a (typically about 3000-4000 pg / ml) when they had a concentration of 1 μM. The amount of IFN-a induced by these ODNs significantly exceeded the amount of IFN-a that induced the same concentration of ODN 2006 or 1982. Figures 2A-2F also show that the class C ODNs 611, 614 and 620 also induced an amount Significant signaling activity of TLR9 (typically with a stimulation index of about 10-20) when they had a concentration of 10 μM. The amount of TLR9 signaling activity that these ODNs induced was also about half of the TLR9 signaling activity that induced the same ODN 2006 concentration. ODN 611 (SEQ ID NO: 43), 614 (SEQ ID NO: 43) and 620 (SEQ ID NO: 43) were also compared to ODN 2429 (one of the class C oligonucleotides containing the described original palindrome). These new class C ODNs had a higher activity in the human TLR9 assay, although the induction of IFN-a was similar to that of ODN 2429 (figure 3). Taken together, the results of these experiments demonstrate that the class C ODN analogs of the invention effectively induce the secretion of IFN-a and the activity of human TLR9 in vitro.

EXAMPLE 2 Other ODN class C analogs induce the secretion of IFN-a in vitro In this series of experiments, other class C ODN analogs of the invention were analyzed in vitro to determine their ability to induce secretion of IFN-α. The ODN class C analogs from these experiments were characterized, in part, by the presence of interrupted inverted repeats rich in AT or by the presence of repeats. interrupted inverted ones that contain spacer residues d instead of conventional nucleotide residues. The ODNs were obtained in the manner described in Example 1. The test ODNs were the following: 645 t * C * G * T * C_G * T * T * T * T * T * A * A * T * A * T * T * T * A * T * T * A (SEQ ID NO: 59) 646 j * C * G * T * C_G * T * T * T * T * C * A * A * T * A * T * T * T * A * T * T * G (SEQ ID NO: 50) 647 T * C * G * T * C_G * T * T * T * T * T * A * A * T * A * T * C * C * A * T * T * A (SEQ ID NO: 58) 649 T * C * G * T * C_G * T * T * T * T * A * C * G * G * C * G * L * L * L * T * G * C * C * G (SEQ ID NO : Four. Five) 650 T * C * G * T * C_G * T * T * L * L * A * C * G * G * C * G * L * L * L * T * G * C * C * G (SEQ ID NO : 38) 651 T * C * G * T * C_G * T * T * T * T * C * G * G * C * G * G * L * L * C * C * G * C * C * G (SEQ ID NO : 54) where * represents an internucleotide link phosphorothioate and _ represents an internucleotide phosphodiester linkage and L represents a spacer d. The human PBMC were obtained and treated in a manner analogous to that described in example 1. The amount of IFN-a in the supernatants using the enzyme-linked immunosorbent assay (ELISA) in a manner analogous to that described in example 1.

Results The class C ODN 645, 646 and 647, characterized in part by the presence of interrupted reversed repeats rich in AT, induced moderate amounts of IFN-a (typically about 1200-1500 pg / ml) when they had a concentration of 1 μM. . The amount of IFN-a that induced these ODN significantly exceeded the amount of IFN-a that induced the same concentration of ODN 2006 or 1982. The ODN class C 649, 650 and 651, characterized in part by the presence of interrupted inverted repeats rich in spacer residues d, induced large amounts of IFN-a (typically about 2000-2500 pg / ml) when they had a concentration of 1 μM. The amount of IFN-a induced by these ODNs significantly exceeded the amount of IFN-a that induced the same concentration of ODN 2006 or 1982. Taken together, the results of these experiments demonstrate that the class C ODN analogs of the invention, characterized in part by the presence of interrupted inverted repeats rich in AT or by the presence of interrupted inverted repeats containing spacer residues d instead of conventional nucleotide residues, effectively induce the secretion of IFN-a in vitro.

EXAMPLE 3 Other analogs of Class C ODN induce the secretion of IFN-a and the activity of human TLR9 in vitro In this series of experiments, the class C ODN analogs of the invention were analyzed in vitro to determine their ability to stimulate human PBMC to secrete INF-a and stimulate HEK293 cells, stably transfected with human TLR9 and a reporter construct. NF-? B to demonstrate the TLR9 signaling. The basic protocol is the one described in example 1, except that the test ODNs were the following: 664 T * C * G * A * C * G * T * C * G * A * C * G * T * G * A * C * G * T * G (SEQ ID NO: 62) 376 T * C * G * A * C * G * T * C * G * A * C * G * T * G * A * C * G (SEQ ID NO: 61) 801 T * C_G * T * C_G * A * C_G * T * T * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 65) 893 T * C * G * T * C_G * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 66) 894 T * C * G * T * C_G * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 67) 882 T * C * G * A * C * G * T * C * G * A * C * G * T * G * A * C * G * T * T (SEQ ID NO: 63) 2290 T * C * G * T * C_G * A * C_G * A * T * C_G * G * C * G * C * C_G * T * G * C * C * G (SEQ ID NO: 64) 2292 T * C * G * T * C * G * A * C * G * A_T_C * G * G * C * G * C * C * G * T * G * C * C * G (SEQ ID NO: 64) 2337 T * C * G * A * C_G * T * C * G * A * C_G * T * G * A * C * G * T * T (SEQ ID NO : 63) 2341 T * C * G * A * C_G * T * C * G * A * C * G * T_G * A * C * G * T * T (SEQ ID NO: 63) 2357 T * C * G * T * C_G * T * T * T * A * C_G * G * C * G * C * C_G * T * G * C * C * G * T (SEQ ID NO: 68) where * represents a phosphorothioate bond and _ represents a phosphodiester bond. ODNs were analyzed in different concentrations using the TLR9 reporter gene assay. The EC50 (ODN concentration with which the effect was 50% of the maximum effect) was calculated using a SigmaPlot (SigmaPlot 2002 for Windows, version 8.0). The maximum stimulation index (max SI) was calculated as the quotient between the maximum value of all the analyzed concentrations of each ODN and the control medium. The results are reproduced in table 1, where it can be observed that the stimulation indices within the range of 10 to 30 They were common.

TABLE 1 Human PBMC secreted large amounts of IFN-a after incubation for 24 hours with these CpG class C ODN. Typical amounts of IFN-a ranged between 3000 and 4000 pg / ml after incubation with ODN at equal or lower concentrations at 1 μM. The response curves of IFN-a in these experiments were very similar to those observed in Figures 2A-2F.

EXAMPLE 4 Class-C ODN analogs induce antigen-specific immune responses in vivo In this series of experiments, the CpG ODNs of the invention were analyzed in vivo in conjunction with vaccines in mice. It was observed that the class C ODNs of the invention increased the titers of antigen-specific IgG total and IgG2a (IgG similar to Th1 in mice) as well as antigen-specific cytotoxic T lymphocyte (CTL) responses in a manner at least comparable to the CpG class B ODN 2006. ODN were obtained in the manner described in Example 1. BALB / c female mice were used for all experiments (6 to 8 weeks of age). The animals were purchased from Charles River Canada (Québec, Canada) and were housed in micro-isolators in the animal care facilities of the Institute of Research of the Ottawa Hospital, Civic Site. In all in vitro assays untreated BALB / c mouse splenocytes were used. They were anesthetized with isoflurane and euthanized by cervical dislocation. The spleen was removed under aseptic conditions and placed in saline with phosphate pH regulator (PBS) + 0.2% bovine serum albumin (Sigma Chemical Company). The spleens were then homogenized and the splenocytes were resuspended in RPMI 1640 tissue culture medium (Life Technologies, Grand Island, NY) and supplemented with 2% normal mouse serum (Cedarlane Laboratories, Ontario, Canada), penicillin solution -streptomycin (final concentration 1000 U / ml and 1 mg / ml, respectively, Sigma Chemical Company) and 5 x 10"5 M ß-mercaptoethanol (Sigma Chemical Company). BALB / c mice were immunized (n = 1 O / group) with 1 μg of hepatitis B surface antigen (HBsAg) subtype ad (International Enzymes, CA) alone or in combination with 1 to 100 μg of 2006 CpG ODN, 608, 611, 618 or CpG 620 ODN. They were bled and boosted 4 weeks after the primary vaccination. Two weeks after the booster, 5 animals of each group were sacrificed and the spleen was removed for CTL analysis. Antibodies (total IgG, IgG 1 and IgG2a) specific for HBsAg (anti-HBs) were detected and quantified by the ELISA assay with the dilution of the endpoint, which was performed in triplicate in the sample of each animal. The titres of the endpoint were defined as the maximum plasma dilution that produced an absorbance value (OD 450) two times higher than that of the non-immune plasma with a cut-off value of 0.05. These were reported as mean group titles (GMT) + SEM. The CTL assays were performed following the standard method.

Briefly, spleens were removed 4 weeks after vaccination and homogenized in a single-cell suspension in RPMI 1640 tissue culture medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies ), penicillin-streptomycin solution (final concentration 1000 U / ml and 1 mg / ml, respectively, Sigma, Irvine, UK) and 5 x 10"5 M ß-mercaptoethanol (Sigma) (complete RPMI 1640). Specimens of HBsAg in the splenocyte suspensions (3 x 106 cells / ml) were restimulated for 5 days by incubation with a murine cell line (p815-S) expressing the HBsAg After the reestipulation, the potential of the lymphocytes was determined. eradicate cells expressing HBsAg using the 51 Cr release assay.The results are expressed as% specific lysis in different effect-to-objective relationships (E: T).

Results Representative results are shown in figures 4-6. As seen in Figure 4 the total IgG titers for ODN 2006 and ODN 620 were dose dependent and approximately 5 x 103 and 6.5 x 103, respectively with an ODN dose of 1 μg. The total IgG titers for ODN 2006 and ODN 620 were approximately 1 x 104 with the ODN dose of 100 μg. As seen in Figure 5, both ODN 2006 and ODN 620 significantly increased lgG2a (Th1-like IgG in mice) compared to the non-CpG ODN 2137 used as control (TGCTGCTTTTGTGCTTTTGTGCTT; SEQ ID NO: 60). Specifically, the titers of lgG2a with ODNs 2006, 620 and 2137 administered in doses of 10 μg were approximately 3 x 103, 6 x 103 and 3 x 102, respectively. As seen in Figure 6, the activity of the CTL with an E: T ratio of 100: 1 with the ODN 2006 and 620 was significantly higher than the control with doses of 10 and 100 μg. For example, the percentage of specific lysis for ODNs 2006, 260 and 2137 administered at doses of 10 μg was approximately 26%, 24% and 12%. Taken together, the results of these experiments demonstrate that the class C ODN analogs of the invention effectively induce antigen-specific immune responses in vivo.

EXAMPLE 5 Class-C ODN analogs improve survival and reduce tumor volume in a tumor model in vivo In this series of experiments, the class C ODN analogs of the invention were analyzed in vivo in a murine neuroblastoma model. It was observed that the ODN analogues of the invention significantly improved both overall survival and tumor burden. The ODNs were obtained in the manner described in Example 1. The BALB / c mice were obtained in the manner described in Example 4. On Day 0, the BALB / c mice were injected subcutaneously (sc) in the flank. left 1 x 106 neuroblastoma cells (Neuro-2a) (eg, ATCC, CCL-131, American Type Culture Collection, Manassas, VA).

The mice were administered s.c. of PBS, CpG 2006, CpG 620 or ODN 2137 non-CpG control from day 10 to day 25. The survival percentage and the tumor volume were determined.

Results Representative results are observed in Figures 7A-7B. As seen in Figures 7A-7B, mice treated with 100 μg of ODN 620 had a 50% survival rate at 80 days, compared to the corresponding 0-20% survival rates for the 2006 ODN, the ODN 2137 and the PBS. In addition, as seen in Figure 7B, mice treated with 100 μg of ODN 620 had a tumor volume that reached a maximum of about 1000 mm3 on day 28 and was reduced to 0 mm3 in 38 days. In contrast, mice treated with ODN 2137 or PBS had monotonous and faster tumor growth. Taken together, the results of these experiments demonstrate that the class C ODN analogs of the invention effectively improve survival and reduce tumor volume in a tumor model in vivo.

EXAMPLE 6 Metabolism and tissue distribution of the ODN class C analogs Adsorption / distribution studies performed with the class C ODN analogs of the invention demonstrated a metabolism and favorable elimination of organs such as the kidney and liver after the administration of a subcutaneous treatment to mice. The mice were divided into groups of 5 and each was given 250 μg of ODN in a single subcutaneous dose on day 0. At various points, the organs (liver, kidneys and spleen) were removed and the oligonucleotide and its metabolites to quantify their content.

Results The ODNs of the invention (eg, 611 and 620) showed less accumulation in the organs than the fully known phosphorothioate palindromic ODNs, such as 2429. For example, on day 3, the ODN 611, 620 and 2429 levels in the kidney they were approximately 70 + 28, 30 + 18 and 90 + 10 mg / kg, respectively. Similarly, on day 3, the levels of ODN 611, 620 and 2429 in the liver were approximately 45 + 15, 28 + 12 and 150 + 15 mg / kg, respectively. Taken together, these results demonstrate that the class C ODN analogs of the invention have a metabolism and favorable elimination of the organs in which other ODNs accumulate.

EXAMPLE 7 Physical Characteristics of Class C ODN Analogs In this group of experiments, the ODNs 2429, 611, 620, 608 were characterized (T * C * G * T * C_G * T * T * T * T * C_G * G * C_G * C * G * C_G * C * C * G, SEQ ID NO: 51) and 618 (T * C * G * T * C_G * T * T * T * T * C_G * G * C * G * G * C * C_G * C * C * G , SEQ ID NO: 53) using size exclusion chromatography, capillary gel electrophoresis (CGE), UV thermal denaturation and high pressure liquid chromatography (HPLC). When ODN 611 and ODN 620 were investigated by size exclusion chromatography (225 μM in PBS), only one peak was observed for each compound, i.e., each oligonucleotide eluted as a monomer. On the contrary, when ODN 608 and 618 (each of which contains a palindromic sequence) were investigated by size exclusion chromatography, two peaks were observed for each oligonucleotide, which is consistent with the presence of an intermolecular dimer in addition of the monomer. However, UV thermal denaturation studies indicated that ODN 611 and ODN 620 had a secondary structure in the solution, consistent with an intramolecular hairpin structure. It is believed that the hairpin structure is the result of the inverted repetition of sequences 611 and 620. In general, sharper peaks were observed in HPLC and CGE of these sequences than in sequences ODN 2429 and ODN 608.

Taken together, these results indicate that the class C ODN analogs of the invention tend to form intramolecular secondary structures and do not form intermolecular complexes in vitro at the concentration tested, while class C ODNs containing a palindrome tend to associate in complexes through intermolecular interactions. However, in vivo, it is likely that the concentrations of ODN reached in the intraendosomal compartment are sufficiently high to favor double or even higher-order complexes of ODN, including double or higher-order complexes of the ODN class C analogs. of the invention.

Equivalents The foregoing written specifications are considered sufficient to allow those skilled in the art to put the invention into practice. Those skilled in the art will see various modifications of the invention, in addition to those described herein, from the foregoing description, which fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily included in each embodiment of the invention. All references, patents and patent publications cited are fully incorporated herein by reference.

Claims (32)

NOVELTY OF THE INVENTION CLAIMS

1. - An immunostimulatory nucleic acid comprising:

TCGTCGTTTTACGGCGCCGTTGCCG (SEQ ID NO: 44). 2. The immunostimulatory nucleic acid according to claim 1, further characterized in that it consists of TCGTCGTTTTACGGCGCCGTTGCCG (SEQ ID NO: 44).

3. The immunostimulatory nucleic acid according to claim 1, further characterized in that at least one internucleotide linkage is modified phosphorothioate.

4. The immunostimulatory nucleic acid according to claim 1, further characterized in that each internucleotide linkage is modified phosphorothioate.

5. The immunostimulatory nucleic acid according to claim 1, further characterized in that at least one nucleotide in the oligonucleotide is a substituted or modified purine or pyrimidine.

6. The immunostimulatory nucleic acid according to claim 5, further characterized in that the substituted pyrimidine is a substituted pyrimidine at the C5- or C6- position.

7. The immunostimulatory nucleic acid according to claim 5, further characterized in that the substituted purine is a purine substituted at the C8 ~ or C7- position.

8. The immunostimulatory nucleic acid according to claim 5, further characterized in that the purified or substituted pyrimidine or pyrimidine is selected from the group consisting of substituted cytosines in the 5-position, cytosines substituted in the 6-position, substituted cytosines in the N4 position, 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogs with fused ring systems, and uracil derivatives, thymine derivatives, 7-deazaguanine, guanine 7-deaza- 7-substituted, 7-deaza-8-substituted guanine, 7-deaza-8-aza guanine, hypoxanthine, substituted guanines at position N2, 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d ] pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, substituted adenines, guanine substituted at position 8, and 6-thioguanine.

9. The immunostimulatory nucleic acid according to claim 5, further characterized in that the purified or substituted pyrimidine or pyrimidine is selected from the group consisting of 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 6-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine and 5-alkynyl-cytosine substituted or unsubstituted, N4-ethyl- cytosine, N, N'-propylene cytosine, phenoxazine, 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil, 2 -tiothimine, 4-thiothimine, thymines substituted in the 6-position, 7-deaza-7-alkynyl (C2-C6) guanine, N2-methyl-guanine, N6-methyl-adenine, 8-oxo-adenine, 8-hydroxyguanine and 8-bromoguanine.

10. The immunostimulatory nucleic acid according to claim 5, further characterized in that the purified or substituted pyrimidine or pyrimidine is selected from the group consisting of a universal base, a system of aromatic rings, and a hydrogen atom. (spacer d).

11. The immunostimulatory nucleic acid according to claim 5, further characterized in that the purified or substituted pyrimidine or pyrimidine is selected from the group consisting of 4-methyl-indole, 5-nitro-indole, 3-nitropyrrole, base P and K base, benzimidazole, dichlorobenzimidazole, 1-methyl-1H- [1, 2,4] triazole-3-carboxylic acid amide, fluorobenzene and difluorobenzene.

12. A vaccine comprising an immunostimulatory nucleic acid defined in any of claims 1 to 11 and an antigen.

13. A pharmaceutical composition comprising an immunostimulatory nucleic acid defined in any of claims 1 to 11 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition comprising an immunostimulatory nucleic acid defined in any of claims 1 to 11 and an anti-cancer medicane.

15. The use of an immunostimulatory nucleic acid defined in any of claims 1 to 11 for the preparation of a medicament for use in the treatment of a cancer.

16. The use as claimed in claim 15, wherein the cancer is selected from basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cervical cancer, choriocarcinoma, colon and rectal cancer , connective tissue cancer, digestive tract cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, laryngeal cancer, leukemia, liver cancer, cancer of lung, lymphoma, including Hodgkin's and non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, cancer respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, cancer of the urinary system, or other carcinomas and sarcomas.

17. The use as claimed in claim 15, wherein the cancer is a cancer sensitive to treatment with interferon alfa (IFN-a).

18. The use as claimed in claim 17, wherein the cancer sensitive to treatment with IFN-a is selected from tricholeukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, carcinoma of the squamous cells, Kaposi's sarcoma related to AIDS, renal cell carcinoma, prostate carcinoma, cervical dysplasia or colon carcinoma.

19. - The use as claimed in claim 15, wherein the medicament is for use in conjunction with an anti-cancer therapy. 20. The use as claimed in claim 19, wherein the anti-cancer therapy is radiation. 21. The use as claimed in claim 19, wherein the anti-cancer therapy is an anti-cancer drug. 22. The use as claimed in claim 21, wherein the anticancer drug is a chemotherapeutic agent. 23. The use as claimed in claim 22, wherein the chemotherapeutic agent is methotrexate, vincristine, adriamycin, cisplatin, chloroethylnitrosoureas without sugar content, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragilin, Melalamine GLA, valrublcin, 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, ISI641, ODN 698, TA 2516 / Marmistat, BB2516 / Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil / OK-432, AD 32 / Valrubicin,

Metastron / strontium derivative, Temodal / Temozolomide,

Evacet / liposomal doroxorubicin, Yewtaxan / Paclitaxel, Taxol / Paclitaxel,

Xeload / Capecitabine, Furtulon / Doxifluridine, Ciclopax / 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 / Levamisole, Camptosar / lrinotecan, Tumodex / Ralitrexed, Leustatin / Cladribine, Paxex / Paciitaxel, Doxii / liposomal doxorubicin, Caelyx / liposomal doxorubicin, Fludara / Fludarabine,

Pharmarubicin / Epirubicin, DepoCyt, ZD1839, LU 79553 / Bis-naphthalimide, LU 103793 / Dolastain, Caetyx / liposomal doxorubicin, Gemzar / Gemcitabine, ZD 0473 / Anormed, YM 116, iodine seeds, inhibitors of CDK4 and CDK2, inhibitors of PARP, D4809 / Dexiphosamide, Ifes / Mesnex / lfosamide, Vumon / Teniposide, Paraplatin / Carbopiatin, Platinol / cisplatin,

Vepeside / Etoposide, ZD 9331, Taxotere / Docetaxel, Prodrug of Guanine arabinoside, Taxano analogs, Nitrosoureas, Alkylating agents such as Melfelan and Cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chorombucil, Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Phosphate sodium estramustine, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon alfa-2a, alpha-2b, leuprolide acetate (analog of LHRH releasing factor), Lomustine (CCNU), Mechlorethamine HCl (Hydrogenated Mustard), Mercaptopurine, Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCl, Ocreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen Citrate, Thioguanine, Thiotepa, Vinblastine Sulfate, Amsacrine (m-AMSA), Azacitidine, Erythropoietin, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG, methyl-glyoxal-bis-guanylhydrazone, MGBG), Pentostatin (2'-deoxicoformycin), Semustine (methyl-CCNU), Teniposide (VM-26) or vindesine sulfate. 24. The use as claimed in claim 21, wherein the anticancer drug is an immunotherapeutic agent. 25. The use as claimed in claim 24, wherein the immunotherapeutic agent is Rituxan, Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP- 03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2 , TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1 D10 Ab, SMART ABL 364 Ab and ImmuRAIT-CEA. 26. The use as claimed in claim 21, wherein the anti-cancer drug is a cancer vaccine. 27. The use as claimed in claim 26, wherein the vaccine for cancer is EGF, anti-idiotypic anti-cancer vaccines, Gp75 antigen, GMK melanoma vaccine, ganglioside conjugate vaccine MGV, Her2 / neu, Ovarex , M-Vax, O-Vax, L-Vax, teratope STn-KHL, BLP25 (MUC-1), liposomal idiotypic vaccine, Melacine, peptide antigen vaccines, toxin / antigen vaccines, vaccine based on MVA, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus and lmmuCyst / TheraCyst.

28. - The use as claimed in claim 15, wherein the medicament is for use in conjunction with more than one anti-cancer therapy. 29.- A method to induce the expression of interferon (IFN) type I, which comprises contacting a cell capable of expressing IFN type I with an immunostimulatory nucleic acid defined in any of claims 1-11, in an amount effective to induce the expression of the IFN type I. 30.- The method according to claim 29, further characterized in that IFN type I is interferon alpha (IFN-a). 31.- A method to induce the expression of interferon gamma (IFN-?), Which comprises contacting a cell capable of expressing IFN-Y with an immunostimulatory nucleic acid defined in any of claims 1-11, in an amount effective to induce the expression of INF- ?. 32. A method for activating a natural killer (NK) cell, comprising contacting an NK cell with an immunostimulatory nucleic acid defined in any of claims 1-11, in an amount effective to activate the NK cell.