Immunostimulatory nucleic acid molecules

Abstract

Nucleic acids containing unmethylated CpG dinucleotides and therapeutic utilities based on their ability to stimulate an immune response and to redirect a Th2 response to a Th1 response in a subject are disclosed. Methods for treating atopic diseases, including atopic dermatitis, are disclosed.

1 - 18 . (Canceled)  
     
     
         19 . A method for preventing or suppressing antigen-stimulated, eosinophilic inflammation in an antigen-exposed subject comprising 
 administering to the subject an isolated immunostimulatory oligonucleotide comprising X1X2CGX3X4, wherein C and G are unmethylated and X1, X2, X3 and X4 are nucleotides and wherein the immunostimulatory oligonucleotide is between 6 and 100 bases in length,    in an amount to suppress a Th2 immune response, whereby eosinophilic inflammation is prevented or suppressed.    
     
     
         20 . The method of  claim 19 , wherein the immunostimulatory oligonucleotide includes a nucleotide sequence consisting of 5′-purine-purine-CG-pyrimidine-pyrimidine-3′.  
     
     
         21 . The method of  claim 20 , wherein the nucleotide sequence consists of AACGTT.  
     
     
         22 . The method of  claim 19 , wherein the immunostimulatory oligonucleotide comprises a nucleotide sequence selected from the group consisting of GTCGTT, GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT, AACGCT and AACGGT.  
     
     
         23 . The method of  claim 19 , wherein the subject has asthma, allergic rhinitis, eczema, hay fever or urticaria.  
     
     
         24 . The method of  claim 19 , wherein the eosinophilic inflammation occurs in a tissue affected by asthma, allergic rhinitis, eczema, hay fever or urticaria.  
     
     
         25 . The method of  claim 19 , wherein the eosinophilic inflammation is in the lung.  
     
     
         26 . The method of  claim 25 , wherein the subject has asthma.  
     
     
         27 . The method of  claim 19 , wherein the immunostimulatory oligonucleotide is 8-100 bases in length.  
     
     
         28 . The method of  claim 19 , wherein the immunostimulatory oligonucleotide is 8-40 bases in length.  
     
     
         29 . A method for boosting an immune response of a subject comprising 
 administering to the subject an isolated immunostimulatory oligonucleotide comprising X1X2CGX3X4,    wherein C and G are unmethylated and X1, X2, X3 and X4 are nucleotides and wherein the immunostimulatory oligonucleotide is between 6 and 100 bases in length, and    wherein an increase in activation of the subject's lymphocytes or NK cells indicates that the subject's immune response has been boosted.    
     
     
         30 . The method of  claim 29 , wherein the activation of the subject's lymphocytes or NK cells is lymphocyte proliferation.  
     
     
         31 . The method of  claim 29 , wherein the activation of the subject's lymphocytes or NK cells is IgM secretion.  
     
     
         32 . The method of  claim 29 , wherein the activation of the subject's lymphocytes or NK cells is increased expression of IL-12 and IFN-gamma.  
     
     
         33 . The method of  claim 29 , wherein the subject has an immune system deficiency.  
     
     
         34 . The method of  claim 33 , wherein the immune system deficiency is an infection.  
     
     
         35 . The method of  claim 34 , wherein the infection is a bacterial, viral, fungal or parasitic infection.  
     
     
         36 . The method of  claim 33 , wherein the immune system deficiency is a bacterial infection by bacteria having bacterial antigens and wherein the increase in lymphocyte or NK activation is activated B cell with antigen receptors specific for the bacterial antigens.  
     
     
         37 . The method of  claim 33 , wherein the immune system deficiency is cancer.  
     
     
         38 . The method of  claim 30 , wherein the immunostimulatory oligonucleotide is 8-100 bases in length.  
     
     
         39 . The method of  claim 30 , wherein the immunostimulatory oligonucleotide is 8-40 bases in length.  
     
     
         40 . The method of  claim 29 , wherein the immunostimulatory oligonucleotide is administered in conjunction with a vaccine.  
     
     
         41 . The method of  claim 29 , wherein the immunostimulatory oligonucleotide is not administered in conjunction with a vaccine.  
     
     
         42 . The method of  claim 29 , wherein the immunostimulatory oligonucleotide includes a nucleotide sequence consisting of 5′-purine-purine-CG-pyrimidine-pyrimidine-3′.  
     
     
         43 . The method of  claim 42 , wherein the nucleotide sequence consists of AACGTT.  
     
     
         44 . The method of  claim 29 , wherein the immunostimulatory oligonucleotide comprises a nucleotide sequence selected from the group consisting of GTCGTT, GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT, AACGCT and AACGGT.  
     
     
         45 . The method of  claim 29 , wherein the subject has asthma, allergic rhinitis, eczema, hay fever or urticaria.  
     
     
         46 . The method of  claim 29 , wherein the immune response occurs in a tissue affected by eczema, allergic rhinitis, hay fever or urticaria.  
     
     
         47 . The method of  claim 29 , wherein the immune response occurs in the lung.  
     
     
         48 . The method of  claim 45 , wherein the subject has asthma and the subject develops a Th1 immune response to an allergen.  
     
     
         49 . The method of  claim 29 , wherein the subject has a viral or a parasitic infection and the immune response to the infection is boosted.  
     
     
         50 . The method of  claim 49 , wherein the infection is a viral infection.  
     
     
         51 . A method for shifting the immune response of a subject to an antigen toward a Th1 immune response comprising 
 administering to the subject an isolated immunostimulatory oligonucleotide comprising X1X2CGX3X4, wherein C and G are unmethylated and X1, X2, X3 and X4 are nucleotides and wherein the immunostimulatory oligonucleotide is between 6 and 100 bases in length,    wherein detection of a Th1 type immune response by the subject indicates that the shift to the Th1 immune response has been achieved.    
     
     
         52 . The method of  claim 51 , wherein the shift to the Th1 immune response is further associated with suppression of a Th2 immune response.  
     
     
         53 . The method of  claim 51 , wherein the immunostimulatory oligonucleotide includes a nucleotide sequence consisting of 5′-purine-purine-CG-pyrimidine-pyrimidine-3′.  
     
     
         54 . The method of  claim 53 , wherein the nucleotide sequence consists of AACGTT.  
     
     
         55 . The method of  claim 51 , wherein the immunostimulatory oligonucleotide comprises a nucleotide sequence selected from the group consisting of GTCGTT, GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT, AACGCT and AACGGT.  
     
     
         56 . The method of  claim 51 , wherein the subject has asthma, allergic rhinitis, eczema, hay fever or urticaria, and the shift to the Th1 immune response prevents or suppresses eosinophilic inflammation in the subject.  
     
     
         57 . The method of  claim 56 , wherein the eosinophilic inflammation is in a tissue affected by asthma, allergic rhinitis, eczema, hay fever or urticaria  
     
     
         58 . The method of  claim 56 , wherein the eosinophilic inflammation is in the lung.  
     
     
         59 . The method of  claim 51 , wherein the subject has asthma and the shift to the Th1 immune response prevents or suppresses eosinophil infiltration into the lung of the subject.  
     
     
         60 . The method of  claim 51 , wherein the subject has a viral or parasitic infection and the shift to the Th1 immune response boosts the immune response to the infection.  
     
     
         61 . The method of  claim 60 , wherein the infection is a viral infection.  
     
     
         62 . The method of  claim 19 , wherein the desired result is measured by detecting in a sample containing lymphocyte obtained from the immunostimulatory oligonucleotide treated subject: (1) a lower level of IL-4 in the immunostimulatory oligonucleotide treated subject as compared to an antigen-challenged control; or (2) a higher level of IL-12-and/or IFN gamma in the immunostimulatory oligonucleotide treated subject as compared to an antigen-challenged control.  
     
     
         63 . The method of  claim 19 , wherein prevention or suppression of eosinophilic inflammation is measured by detecting lower levels of eosinophils in an inflammatory infiltrate in the lung in an immunostimulatory oligonucleotide treated subject as compared to an antigen-challenged control.  
     
     
         64 . The method of  claim 51 , wherein the immunostimulatory oligonucleotide is 8-100 bases in length.  
     
     
         65 . The method of  claim 51 , wherein the immunostimulatory oligonucleotide is 8-40 bases in length.  
     
     
         66 . The method of  claim 51 , wherein the immunostimulatory oligonucleotide is administered in conjunction with a vaccine.  
     
     
         67 . The method of  claim 51 , wherein the immunostimulatory oligonucleotide is not administered in conjunction with a vaccine.  
     
     
         68 . A method for preventing or reducing antigen-stimulated, granulocyte-mediated inflammation in a tissue of an antigen-sensitized subject comprising 
 administering an isolated immunostimulatory oligonucleotide to the subject,    wherein a reduction in, or the absence of, a Th2 type immune response measured in the subject, or a reduction in, or the absence of, other clinical signs of inflammation in the subject after antigen challenge, indicates that the desired prevention or reduction in granulocyte-mediated inflammation has been achieved.    
     
     
         69 . The method of  claim 68 , wherein the immunostimulatory oligonucleotide includes a hexameric nucleotide sequence consisting of 5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.  
     
     
         70 . The method of  claim 69 , wherein the hexameric nucleotide sequence consists of AACGTT.  
     
     
         71 . The method of  claim 68 , wherein the immunostimulatory oligonucleotide comprises a hexameric nucleotide sequence selected from the group consisting of GTCGTT, GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT, AACGCT and AACGGT.  
     
     
         72 . The method of  claim 68 , wherein the subject is suffering from an condition induced by the sensitizing antigen selected from the group of inflammatory conditions consisting of asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis and cutaneous basophil hypersensitivity.  
     
     
         73 . The method of  claim 68 , wherein the inflammation is in skin or mucosa.  
     
     
         74 . The method of  claim 73 , wherein the inflammation is in a respiratory tissue.  
     
     
         75 . The method of  claim 68 , wherein the subject is suffering from asthma.  
     
     
         76 . The method of  claim 68 , wherein the desired result is measured by detecting in a sample containing lymphocytes obtained from the immunostimulatory oligonucleotide treated subject: (1) a lower level of IL-4 in the immunostimulatory oligonucleotide treated subject as compared to an antigen-challenged control; or (2) a higher level of IL-12 and/or IFN gamma in the immunostimulatory oligonucleotide treated subject as compared to an antigen-challenged control.  
     
     
         77 . A method for boosting the immune responsiveness of a subject to a sensitizing antigen without immunization of the subject by the sensitizing antigen comprising administering an isolated immunostimulatory oligonucleotide to the subject, wherein an increase in the magnitude of the subject's immune response to the sensitizing antigen indicates that the desired boost to the subject's immune responsiveness has been achieved.  
     
     
         78 . The method of  claim 77 , wherein the immunostimulatory oligonucleotide includes a hexameric nucleotide sequence consisting of 5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.  
     
     
         79 . The method of  claim 78 , wherein the hexameric nucleotide sequence consists of AACGTT.  
     
     
         80 . The method of  claim 77 , wherein the immunostimulatory oligonucleotide includes a hexameric nucleotide sequence is selected from the group of sequences consisting of GTCGTT, GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT, AACGCT and AACGGT.  
     
     
         81 . The method of  claim 77 , wherein the subject is suffering from an inflammatory condition induced by the sensitizing antigen selected from the group of inflammatory conditions consisting of asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis and cutaneous basophil hypersensitivity.  
     
     
         82 . The method of  claim 81 , wherein the immune response is in skin or mucosa.  
     
     
         83 . The method of  claim 82 , wherein the immune response is in respiratory tissue.  
     
     
         84 . The method of  claim 83 , wherein the subject is suffering from asthma and the subject's immune responsiveness to a respiratory allergen is boosted.  
     
     
         85 . The method of  claim 77 , wherein the antigen is presented by a pathogen and the subject's immune responsiveness to an intracellular infection by the pathogen is boosted.  
     
     
         86 . The method of  claim 85 , wherein the pathogen is a virus.  
     
     
         87 . A method for shifting the immune response of a subject to a sensitizing antigen toward a Th1 phenotype comprising 
 administering an isolated immunostimulatory oligonucleotide to the subject,    wherein detection of a Th1 type immune response by the subject indicates that the desired shift to the Th1 phenotype has been achieved.    
     
     
         88 . The method of  claim 87 , wherein the immunostimulatory oligonucleotide includes a hexameric nucleotide sequence consisting of 5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.  
     
     
         89 . The method of  claim 88 , wherein the hexameric nucleotide sequence consists of AACGTT.  
     
     
         90 . The method of  claim 87 , wherein the immunostimulatory oligonucleotide includes a hexameric nucleotide sequence is selected from the group consisting of GTCGTT, GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT, AACGCT and AACGGT.  
     
     
         91 . The method of  claim 87 , wherein the subject is suffering from an inflammatory condition induced by the sensitizing antigen selected from the group of inflammatory conditions consisting of asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis and cutaneous basophil hypersensitivity, and the shift to the Th1 phenotype reduces granulocyte-mediated inflammation in the affected tissue.  
     
     
         92 . The method of  claim 91 , wherein the affected tissue is skin or mucosa.  
     
     
         93 . The method of  claim 92 , wherein the affected tissue is respiratory tissue.  
     
     
         94 . The method of  claim 93 , wherein the subject is suffering from asthma and the shift to the Th1 phenotype reduces eosinophil infiltration of the lung.  
     
     
         95 . The method of  claim 87 , wherein the subject is suffering from an intracellular infection by a pathogen and the shift to the Th1 phenotype strengthens the subject's immune response to the pathogen.  
     
     
         96 . The method of  claim 91 , wherein the pathogen is a virus.  
     
     
         97 . The method of  claim 87 , wherein the desired result is measured by detecting in a sample containing lymphocytes obtained from the immunostimulatory oligonucleotide treated subject: (1) a lower level of IL-4 in the immunostimulatory oligonucleotide treated subject as compared to an antigen-challenged control; or (2) a higher level of IL-12 and/or IFN gamma in the immunostimulatory oligonucleotide treated subject as compared to an antigen-challenged control.  
     
     
         98 . The method of  claim 68 , wherein reduction or suppression of inflammation is measured by assaying inflammatory infiltrate from the subject for a reduction in granulocyte counts in inflammatory infiltrate of an affected subject tissue as measured in an antigen challenged subject before and after ISS-ODN administration or detection of lower levels of granulocyte counts in an ISS-ODN treated subject as compared to an antigen-challenged control  
     
     
         99 . The method of  claim 48 , wherein the allergen is pollen, animal dander or dust.