Immunostimulatory oligonucleotides with modified bases and methods of use thereof

Abstract

Immunomodulatory oligonucleotide compositions are disclosed. These oligonucleotides comprise an immunostimulatory hexanucleotide sequence comprising a modified cytosine. These oligonucleotides can be administered in conjunction with an immunomodulatory peptide or antigen. Methods of modulating an immune response upon administration of the oligonucleotide comprising a modified immunostimulatory sequence are also disclosed.

We claim:  
     
         1 . An immunomodulatory oligonucleotide comprising an immunostimulatory sequence (ISS) comprising a modified cytosine.  
     
     
         2 . An immunomodulatory oligonucleotide of  claim 1 , wherein the modified cytosine comprises an addition of an electron-withdrawing group to at least position C-5.  
     
     
         3 . An immunomodulatory oligonucleotide of  claim 1 , wherein the modified cytosine comprises an addition of an electron-withdrawing group to at least position C-6.  
     
     
         4 . An immunomodulatory oligonucleotide of  claim 1 , wherein the ISS comprises a modified cytosine selected from the group consisting of azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, fluorinated cytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, halogenated cytosine, halogenated pyrimidine analogue, hydroxyurea, iodouracil, 5-nitrocytosine, 5-trifluoromethyl-cytosine, uracil, 5-fluorocytosine, 5-trifluoromethylcytosine, and 5,6-dihydrocytosine.  
     
     
         5 . An immunomodulatory oligonucleotide of  claim 1 , wherein the modified cytosine is a 5′-bromocytidine.  
     
     
         6 . An immunomodulatory oligonucleotide of  claim 1 , wherein the ISS comprises the sequence 5′-Purine, Purine, Cytosine, Guanine, Pyrimidine, Pyrimidine-3′.  
     
     
         7 . An immunomodulatory oligonucleotide of  claim 6 , wherein the modified cytosine comprises an addition of an electron-withdrawing group to at least position C-5.  
     
     
         8 . An immunomodulatory oligonucleotide of  claim 6 , wherein the modified cytosine comprises an addition of an electron-withdrawing group to at least position C-6.  
     
     
         9 . An immunomodulatory oligonucleotide of  claim 6 , wherein the modified cytosine is a 5′-bromocytidine.  
     
     
         10 . An immunomodulatory oligonucleotide of  claim 9 , wherein the cytosine at the third position from the 5′ end of the ISS octanucleotide is substituted with a 5′-bromocytidine.  
     
     
         11 . An immunomodulatory oligonucleotide of  claim 1 , wherein the ISS comprises the sequence 5′-Purine, Purine, Cytosine, Guanine, Pyrimidine, Pyrimidine, Cytosine, Cytosine-3′.  
     
     
         12 . An immunomodulatory oligonucleotide of  claim 11 , wherein the modified cytosine comprises an addition of an electron-withdrawing group to at least position C-5.  
     
     
         13 . An immunomodulatory oligonucleotide of  claim 11 , wherein the modified cytosine comprises an addition of an electron-withdrawing group to at least position C-6.  
     
     
         14 . An immunomodulatory oligonucleotide of  claim 11 , wherein the modified cytosine is a 5′-bromocytidine.  
     
     
         15 . An immunomodulatory oligonucleotide of  claim 11 , wherein the cytosine at the third position from the 5′ end of the ISS is substituted with a 5′bromocytidine.  
     
     
         16 . An immunomodulatory oligonucleotide of  claim 11 , wherein the cytosine at the third position from the 5′ end of the ISS is substituted with a 5′-bromocytidine and the cytosine at the seventh position from the 5′ end of the ISS is substituted with a 5′-bromocytidine.  
     
     
         17 . An immunomodulatory oligonucleotide of  claim 1 , wherein the ISS comprises the sequence 5′-Purine, Purine, Cytosine, Guanine, Pyrimidine, Pyrimidine, Cytosine, Guanine-3′.  
     
     
         18 . An immunomodulatory oligonucleotide of  claim 17 , wherein the modified cytosine comprises an addition of an electron-withdrawing group to at least position C-5.  
     
     
         19 . An immunomodulatory oligonucleotide of  claim 17 , wherein the modified cytosine comprises an addition of an electron-withdrawing group to at least position C-6.  
     
     
         20 . An immunomodulatory oligonucleotide of  claim 17 , wherein the modified cytosine is a 5′-bromocytidine.  
     
     
         21 . An immunomodulatory oligonucleotide of  claim 17 , wherein the Cytosine at the third position from the 5′ end of the ISS octanucleotide is substituted with a 5′-bromocytidine.  
     
     
         22 . An immunomodulatory oligonucleotide of  claim 17 , wherein the cytosine at the third position from the 5′ end of the ISS is substituted with a 5′-bromocytidine and the cytosine at the seventh position from the 5′ end of the ISS is substituted with a 5′-bromocytidine.  
     
     
         23 . An immunomodulatory oligonucleotide of  claim 1 , wherein the ISS comprises a phosphorothioate group.  
     
     
         24 . An immunomodulatory oligonucleotide of  claim 1 , wherein the ISS comprises a sequence selected from the group consisting of AACGTT, GACGTT, AACGTTCC, AACGTTCG, GACGTTCC, and GACGTTCG, wherein at least one C is substituted with a modified cytosine.  
     
     
         25 . An immunomodulatory oligonucleotide of  claim 24 , wherein the ISS further comprises a second modified cytosine.  
     
     
         26 . An immunomodulatory oligonucleotide of  claim 1 , wherein the oligonucleotide portion further comprises an RNA sequence.  
     
     
         27 . An immunomodulatory oligonucleotide of  claim 26 , wherein the ISS is an RNA sequence comprising a single-stranded or double-stranded sequence selected from the group consisting of AACGUU, GACGUU, AACGUUCC, AACGUUCG, GACGUUCC, and GACGUUCG, wherein at least one C is substituted with a modified cytosine.  
     
     
         28 . An immunomodulatory oligonucleotide of  claim 27 , wherein the ISS further comprises a second modified cytosine.  
     
     
         29 . An immunomodulatory oligonucleotide comprising the sequence SEQ ID NO:2.  
     
     
         30 . An immunomodulatory oligonucleotide comprising the sequence SEQ ID NO:5.  
     
     
         31 . An immunomodulatory oligonucleotide comprising the sequence SEQ ID NO:6.  
     
     
         32 . An immunomodulatory composition comprising 
 an immunomodulatory oligonucleotide according to  claim 1;     and further comprising an antigen.    
     
     
         33 . An immunomodulatory composition of  claim 32 , wherein the antigen is selected from the group consisting of peptides, glycoproteins, polysaccharides, and lipids.  
     
     
         34 . An immunomodulatory composition of  claim 32 , wherein the antigen is conjugated to the immunomodulatory oligonucleotide.  
     
     
         35 . An immunomodulatory composition comprising 
 an immunomodulatory oligonucleotide according to  claim 1;     and further comprising a facilitator selected from the group consisting of co-stimulatory molecules, cytokines, chemokines, targeting protein ligand, a trans-activating factor, a peptide, and a peptide comprising a modified amino acid.    
     
     
         36 . An immunomodulatory composition of  claim 35 , wherein the facilitator is conjugated to the immunomodulatory oligonucleotide.  
     
     
         37 . An immunomodulatory composition comprising 
 an immunomodulatory oligonucleotide according to  claim 1;     and further comprising an antigen;    and further comprising an adjuvant.    
     
     
         38 . An immunomodulatory composition of  claim 37 , wherein the antigen is selected from the group consisting of peptides, glycoproteins, polysaccharides, and lipids.  
     
     
         39 . An immunomodulatory composition of  claim 37 , wherein the antigen is conjugated to the immunomodulatory oligonucleotide.  
     
     
         40 . A method of modulating an immune response comprising co-administration of an immunomodulatory composition comprising an antigen and an immunomodulatory oligonucleotide according to  claim 1 .  
     
     
         41 . The method of  claim 40 , wherein the modulating of an immune response comprises induction of a Th1-type response.  
     
     
         42 . A method of modulating an immune response comprising administration of an immunomodulatory composition according to  claim 34 .  
     
     
         43 . The method of  claim 42 , wherein the modulating of an immune response comprises induction of a Th 1-type response.  
     
     
         44 . A method of modulating an immune response comprising the co-administration of an antigen, an adjuvant and an immunomodulatory oligonucleotide according to  claim 1 .  
     
     
         45 . The method of  claim 44 , wherein the modulating of an immune response comprises induction of a Th1-type response.  
     
     
         46 . A method of modulating an immune response comprising the administration of an immunomodulatory composition according to  claim 35 , wherein the components of the composition are co-administered.  
     
     
         47 . A method of modulating an immune response comprising administration of an immunomodulatory composition according to  claim 39 .  
     
     
         48 . A method of treating an individual in need of immune modulation comprising administration of a composition comprising an immunomodulatory oligonucleotide of  claim 1.