Immunization-free methods for treating antigen-stimulated inflammation in a mammalian host and shifting the host's antigen immune responsiveness to a Th1 phenotype

Abstract

The invention relates to methods for preventing or reducing antigen-stimulated, granulocytemediated inflammation in tissue of an antigen-sensitized mammal host by delivering an immunostimulatory oligonucleotide to the host. In addition, methods for using the immunostimulatory oligonucleotides to boost a mammal host's immune responsiveness to a sensitizing antigen (without immunization of the host by the antigen) and shifting the host's immune responsiveness to a Th1 phenotype to achieve various therapeutic ends are provided. Kits for practicing the methods of the invention are also provided.

The invention claimed is:  
     
         1 . A method for preventing or reducing antigen-stimulated, granulocyte-mediated inflammation in a tissue of an antigen-sensitized mammalian host comprising delivering an immunostimulatory oligonucleotide (ISS-ODN) to the host; wherein a reduction in, or the absence of, a Th2 type immune response measured in the host; or a reduction in, or the absence of other clinical signs of inflammation in the host after antigen challenge, indicates that the desired prevention or reduction in granulocyte-mediated inflammation has been achieved.  
     
     
         2 . The method according to  claim 1  wherein the ISS-ODN includes a hexameric nucleotide sequence consisting of 5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.  
     
     
         3 . The method according to  claim 2  wherein the hexameric nucleotide sequence consists of AACGTT.  
     
     
         4 . The method according to  claim 2  wherein the hexameric nucleotide sequence is selected from the group of sequences consisting of AGCGTC, GACGTT, GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC, GGCGCC, AGCGCT, GACGCT, GGCGCT, TTCGAA, GGCGTT and AACGCC.  
     
     
         5 . The method according to  claim 1  wherein the ISS-ODN is conjugated to an immunostimulatory or anti-inflammatory partner selected from the group consisting of non-antigenic polypeptides, antigenic polypeptides, polysaccharides, antibodies, glycoproteins, lipids and steroids.  
     
     
         6 . The method according to  claim 1  wherein the host is suffering from an inflammatory condition induced by the sensitizing antigen selected from the group of inflammatory conditions consisting of asthma, nasal polyposis, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eosinophilic fasciitis, ideopathic hypereosinophilic syndrome and cutaneous basophil hypersensitivity.  
     
     
         7 . The method according to  claim 1  wherein the inflamed tissue is skin or mucosa.  
     
     
         8 . The method according to  claim 7  wherein the inflamed tissue is respiratory tissue.  
     
     
         9 . The method according to  claim 8  wherein the host is suffering from asthma.  
     
     
         10 . A method for boosting the immune responsiveness of a mammalian host to a sensitizing antigen without immunization of the host by the sensitizing antigen comprising delivering an immunostimulatory oligonucleotide (ISS-ODN) to the host to the host, wherein an increase in the magnitude of the host immune response to the sensitizing antigen indicates that the desired boost to the host immune responsiveness has been achieved.  
     
     
         11 . The method according to  claim 10  wherein the IS S-ODN includes a hexameric nucleotide sequence consisting of 5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.  
     
     
         12 . The method according to  claim 11  wherein the hexameric nucleotide sequence consists of AACGTT.  
     
     
         13 . The method according to  claim 1  wherein the hexameric nucleotide sequence is selected from the group of sequences consisting of AGCGTC, GACGTT, GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC, 
 GGCGCC, AGCGCT, GACGCT, GGCGCT, TTCGAA, GGCGTT and AACGCC.  
 
     
     
         14 . The method according to  claim 10  wherein the ISS-ODN is conjugated to an immunostimulatory or anti-inflammatory partner selected from the group consisting of non-antigenic polypeptides, antigenic polypeptides, polysaccharides, antibodies, glycoproteins, lipids and steroids.  
     
     
         15 . The method according to  claim 10  wherein the host is suffering from an inflammatory condition induced by the sensitizing antigen selected from the group of inflammatory conditions consisting of asthma, nasal polyposis, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eosinophilic fasciitis, ideopathic hypereosinophilic syndrome and cutaneous basophil hypersensitivity.  
     
     
         16 . The method according to  claim 10  wherein the inflamed tissue is skin or mucosa.  
     
     
         17 . The method according to  claim 16  wherein the inflamed tissue is respiratory tissue.  
     
     
         18 . The method according to  claim 17  wherein the host is suffering from asthma and the host's immune responsiveness to a respiratory allergen is boosted.  
     
     
         19 . The method according to  claim 10  wherein the antigen is presented by a pathogen and the host's immune responsiveness to an intracellular infection by the pathogen is boosted.  
     
     
         20 . The method according to  claim 19  wherein the pathogen is a virus.  
     
     
         21 . A method for shifting the immune response of a mammal host to a sensitizing antigen toward a Th1 phenotype comprising delivering an immunostimulatory oligonucleotide (ISS-ODN) to the host, wherein detection of a Th1 type immune response by the host indicates that the desired shift to the Th1 phenotype has been achieved.  
     
     
         22 . The method according to  claim 21  wherein the ISS-ODN includes a hexameric nucleotide sequence consisting of 5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.  
     
     
         23 . The method according to  claim 22  wherein the hexameric nucleotide sequence consists of AACGTT.  
     
     
         24 . The method according to  claim 22  wherein the hexameric nucleotide sequence is selected from the group of sequences consisting of AACGTT, AGCGTC, GACGTT, GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC, GGCGCC, AGCGCT, GACGCT, GGCGCT, TTCGAA, AACGTT, GGCGTT and AACGCC.  
     
     
         25 . The method according to  claim 21  wherein the ISS-ODN is conjugated to an immunostimulatory or anti-inflammatory partner selected from the group consisting of non-antigenic polypeptides, antigenic polypeptides, polysaccharides, antibodies, glycoproteins, lipids and steroids.  
     
     
         26 . The method according to  claim 21  wherein the host is suffering from an inflammatory condition induced by the sensitizing antigen selected from the group of inflammatory conditions consisting of asthma, nasal polyposis, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eosinophilic fasciitis, ideopathic hypereosinophilic syndrome and cutaneous basophil hypersensitivity, and the shift to the Th1 phenotype reduces granulocyte-mediated inflammation in the affected host tissue.  
     
     
         27 . The method according to  claim 26  wherein the inflamed tissue is skin or mucosa.  
     
     
         28 . The method according to  claim 27  wherein the inflamed tissue is respiratory tissue.  
     
     
         29 . The method according to  claim 28  wherein the host is suffering from asthma and the shift to the Th1 phenotype reduces eosinophil infiltration of the host lung tissue.  
     
     
         30 . The method according to  claim 21  wherein the host is suffering from an intracellular infection by a pathogen and the shift to the Th1 phenotype strengthens the host immune response to the pathogen.  
     
     
         31 . The method according to  claim 30  wherein the pathogen is a virus.  
     
     
         32 . The method according to  claim 21  wherein the host is suffering from reduced blood flow to a tissue and the shift to the Th1 phenotype stimulates angiogenesis in the treated tissue.  
     
     
         33 . The method according to  claim 32  wherein the host is suffering from diabetic retinopathy.  
     
     
         34 . The method according to claims  1 ,  10  or  21  wherein the desired result is measured by determining any of the following values in a sample containing lymphocytes obtained from the ISS-ODN treated host: 
 (1) a reduction in levels of IL-4, IL-5 and/or IL-10 measured before and after antigen challenge or detection of lower levels of IL-4, IL-5 and/or IL-10 in the IS S-ODN treated host as compared to an antigen-challenged control;  
 (2) an increase in levels of IL-12, IL-18 and/or IFN (α, β or γ) before and after antigen challenge or detection of higher levels of IL-12, IL-18 and/or IFN (α, β or γ) in the ISS ODN treated host as compared to an antigen-challenged control;  
 (3) IgG2a antibody production in the ISS-ODN treated host; or  
 (4) a reduction in levels of antigen-specific IgE as measured before and after antigen challenge or detection of lower levels of antigen-specific IgE in the ISS-ODN treated host as compared to an antigen-challenged control.  
 
     
     
         35 . The method according to claims  1  or  10  wherein reduction or suppression of inflammation is measured by assaying inflammatory infiltrate from the host for a reduction in granulocyte counts in inflammatory infiltrate of an affected host tissue as measured in an antigen challenged host before and after ISS-ODN administration or detection of lower levels of granulocyte counts in an ISS-ODN treated host as compared to an antigen-challenged control.  
     
     
         36 . A kit for use in-reducing or preventing inflammation in an antigen-sensitized host tissue, as well as in boosting the immune responsiveness of a host to a sensitizing antigen, comprising an immunostimulatory oligonucleotide (ISS-ODN) in a sterile vial, a device for delivering the ISS-ODN directly into a host tissue and at least one assay reagent for use in measuring any of the following values as indicators that the desired reduction or prevention of inflammation or boost in immune responsiveness has been achieved in an ISS-ODN treated host: 
 (1) a reduction in levels of IL-4, IL-5 and/or IL-10 measured before and after antigen challenge; or detection of lower (or absent) levels of IL-4, IL-5 and/or IL-10 in an ISS-ODN treated host as compared to an antigen-primed, or primed and challenged, control;    (2) an increase in levels of IL-12, IL-18 and/or IFN (α, β or γ) before and after antigen challenge; or detection of higher levels of IL-12, IL-18 and/or IFN (α, β or γ) in an ISS ODN treated host as compared to an antigen-primed, or primed and challenged, control;    (3) IgG2a antibody production in an ISS-ODN treated host; or    (4) a reduction in levels of antigen-specific IgE as measured before and after antigen challenge; or detection of lower (or absent) levels of antigen-specific IgE in an ISS-ODN treated host as compared to an antigen-primed, or primed and challenged, control.    
     
     
         37 . A kit for use in reducing or preventing inflammation in an antigen-sensitized host tissue comprising an immunostimulatory oligonucleotide (IS S-ODN) in a sterile vial, a device for delivering the ISS-ODN directly into a host tissue and at least one assay reagent for use in measuring lymphocyte proliferation, IgG2a antibody levels, serum cytokine levels and/or granuloctye counts in inflammatory infiltrate of an affected host tissue.