Immunization-free methods for treating antigen-stimulated inflammation in a mammalian host and shifting the host's antigen immune responsiveness to a th1 phenotype

This invention was made with Government support under Grant No. AI37350, awarded by the National Institutes of Health. The Government may have certain rights in this invention.

The invention relates to methods and oligonucleotide compositions for use in reducing, or suppressing granulocyte-mediated inflammation in a host tissue and in modulating the host\'s immune responsiveness to an antigen.

In vertebrates, endothelial cell adhesion by granulocytes (eosinophils, basophils, neutrophils and mast cells) is followed by the release of inflammatory mediators, such as leukotrienes, major basic protein and histamine. In susceptible individuals, the resulting inflammation can damage affected host tissues.

The most common pathologic inflammatory condition is asthma, which is characterized by marked eosinophil infiltration into respiratory airways, followed by inflammation-induced tissue damage. Other pathologic inflammatory conditions associated with granulocyte infiltration into affected tissues include nasal polyposis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, eosinophilic fasciitis, idiopathic hypereosinophilic syndrome and cutaneous basophil hypersensitivity, as well as inflammation and fibrosis resulting from increased production of granulocyte-stimulatory cytokines, such as interleukin (IL)-5 and certain interferons (INF).

Routine treatment of such conditions is typically directed toward inhibiting the activity of inflammatory mediators released after granulocyte adhesion to endothelia (e.g., by delivering a corticoid composition to the affected tissues). Where the identity of an inflammation inducing antigen is known, some immune protection against further antigen challenge can be provided through immunization. However, although effective in stimulating production of neutralizing antibodies, canonical immunization does not effectively stimulate longer term cellular immunity. Moreover, antigen immunization stimulates host production of IL-4 and IL-5. IL-5 encourages granulocyte adhesion to endothelia while IL-4 induces immunoglobulin switching to the IgE isotype at the risk of anaphylaxis.

The invention provides means to rapidly suppress antigen-stimulated inflammation in a mammalian host by suppressing granulocyte infiltration into a host tissue. The invention also provides immunization-free means to provide protection to an antigen-sensitized mammalian host against subsequent antigen challenge without risk of anaphylaxis. These aims are achieved by the invention through delivery of an immunostimulatory oligonucleotide (ISS-ODN) to the host without codelivery of an immunizing antigen.

Surprisingly, ISS-ODN have anti-inflammatory properties in addition to their immunostimulatory properties. ISS-ODN are therefore useful in the treatment and prevention of inflammation associated with antigen-stimulated granulocyte infiltration of tissue, such as occurs in the respiratory passages of asthmatics during an asthma attack. Advantageously, delivery of ISS-ODN according to the invention suppresses antigen-stimulated granulocyte infiltration into host tissue even before the ISS-ODN affect the host\'s immune response to the antigen. Thus, the invention provides an antigen-independent method to reduce antigen-stimulated inflammation by suppressing cellular adhesion, thereby avoiding the release of inflammatory mediators which would be stimulated through granulocyte-binding of endothelial cells.

An example of a therapeutic application for the invention is in the control of asthma, whereby the ISS-ODN are delivered into pulmonary tissue intranasally or by systemic routes. In asthmatics, eosinophil infiltration of lung tissue occurs mainly during the late phase of an allergic response to a respiratory allergen. Canonical immunotherapy can modulate the host immune response to the allergen and eventually stem the tide of eosinophils into the host airways. However, practice of the invention suppresses eosinophil infiltration of host airways well before the host immune system responds to the respiratory allergen, thereby providing a form of protection against the airway narrowing and respiratory tissue damage which characterize an acute asthma attack.

In another aspect, the invention provides means to shift a present host cellular immune response to an antigen away from a Th2 phenotype and into a Th1 phenotype. To this end. ISS-ODN are delivered by any route through which antigen-sensitized host tissues will be contacted with the ISS-ODN. ISS-ODN administered in this fashion boost both humoral (antibody and cellular (Th1 type) immune responses of the host. Unlike canonical immunotherapy, immunity is stimulated by this method of the invention even when no additional antigen is introduced into the host. Thus, use of the method to boost the immune responsiveness of a host to subsequent challenge by a sensitizing antigen without immunization avoids the risk of immunization-induced anaphylaxis, suppresses IgE production in response to the antigen challenge and eliminates the need to identify the sensitizing antigen for use in immunization. An especially advantageous use for this aspect of the invention is treatment of localized allergic responses in target tissues where the allergens enter the body, such as the skin and mucosa.

Suppression of the Th2 phenotype according to the invention is also a useful adjunct to canonical immunotherapy to reduce antigen-stimulated IL-4 and IL-5 production. Thus, the invention encompasses delivery of ISS-ODN to a host to suppress the Th2 phenotype associated with conventional antigen immunization (e.g., for vaccination or allergy immunotherapy).

The shift to a Th1 phenotype achieved according to the invention is accompanied by increased secretion of IFN α, β, and γ, as well as IL-12 and IL-18. Each of these cytokines enhance the host\'s immune defenses against intracellular pathogens, such as viruses. Thus, the invention encompasses delivery of ISS-ODN to a host to combat pathogenic infection.

Angiogenesis is also enhanced in the Th1 phenotype (ostensibly through stimulation by IL-12). Thus, the invention encompasses delivery of ISS-ODN to a host to stimulate therapeutic angiogenesis to treat conditions in which localized blood flow plays a significant etiological role, such as in diabetic retinopathy.

Pharmaceutically acceptable compositions of ISS-ODN are provided for use in practicing the methods of the invention. The ISS-ODN of the invention include DNA or RNA oligonucleotides which are enriched with CpG dinucleotides, including those which are comprised of the primary structure 5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.

Where appropriate to the contemplated course of therapy, the ISS-ODN may be administered with other anti-inflammatory or immunotherapeutic agents. Thus, a particularly useful composition for use in practicing the method of the invention is one in which an anti-inflammatory agent (e.g., a glucocorticoid) or immunotherapeutic agent (e.g., an antigen, cytokine or adjuvant) is mixed with, or conjugated to, an ISS-ODN.

The ISS-ODN can also be provided in the form of a kit comprising ISS-ODN and any additional medicaments, as well as a device for delivery of the ISS-ODN to a host tissue and reagents for determining the biological effect of the ISS-ODN on the treated host.