WO2000062816A2 - Genetic tolerization - Google Patents

Abstract

The present invention provides methods for the administration of genetic vaccines to promote or induce a greater tolerance to various antigens. This will permit the treatment of patients with an allergy or autoimmunity, asthma and other immunomodulatory diseases where an undesired immune response to one or more antigens exists. In addition, methods of designing tolerizing vaccines, including methods for identifying nucleic acids that encode tolerizing antigens and cell-type specific regulatory nucleic acid sequences, are provided.

Description

DESCRIPTION

GENETIC TOLERIZATION

BACKGROUND OF THE INVENTION

The present application claims priority to Provisional Patent Application Serial

No. 60/129,753, the entire text of each of the above-referenced disclosures is specifically incorporated by reference herein without disclaimer. The government owns rights in the present invention pursuant to DARPA Federal grant MDA 972-97- 1-0013.

1. Field of the Invention

The present invention relates generally to the fields of immune tolerance to antigens. More particularly, it concerns methods of promoting or inducing immunological tolerance to various antigens in an organism using cell-type specific promoters that express antigens in non-antigen presenting cells (non-APCs). The invention also concerns methods of identifying antigens and cell-type specific promoters for use in genetic vaccines to induce or promote a greater immune tolerance to the identified antigens.

2. Description of Related Art

Allergy and autoimmunity problems are widespread and increasing in occurrence in the USA and Europe (Custovic et α/., 1998). Often the allergy or autoimmunity is elicited by an immune response to one or a few antigens. Autoimmune disease results from a breakdown in tolerance to self antigens, which involves many serious diseases such as arthritis, nephritis, thyrotoxicosis, diabetes, systemic sclerosis and systemic lupus erythematosus, etc. Allergy to the foreign antigens also causes very serious diseases such as asthma, etc. No efficient treatment methods are available clinically for these diseases. Another extremely important clinical application of tolerance is in the field of organ transplantation, and xenogeneic-organ transplantation. Much unsuccessful research has been devoted to persuading the immune system into considering specific foreign antigens on the transplant as self Although immunosuppressive drugs have been used effectively for transplantation, the side effects of such non-specific immunosuppression, i.e., increased rates of infection and of cancer, have led to a greatly interesting for searching therapies that induce tolerance to specific antigens (Sachs, 1998).

In gene therapy, repeated administration of a vectors (especially viral vectors) can induce immune responses to the virus proteins and to the encoded gene product. These responses will prevent the therapeutic activity of the subsequent doses of the vector or even causes immunopathology. This can hinder effective treatment using viral vectors when repeated administration is necessary, as in gene therapy of cystic fibrosis. Using naked DNA plasmid vector may avoid the immune response to the vectors but can not prevent the response against an encoded gene product if it is foreign to the body. (Featherstone, 1997).

Thus, methods of reducing unwanted immune responses are needed. Such methods may reduce the undesired immune reactions to transplanted organs or therapeutic agents, as well as autoimmune and allergy disorders.

SUMMARY OF THE INVENTION

The present invention overcomes these deficiencies by providing methods for delivering a "tolerizing" vaccine against one or more antigens, such as those involved in an allergy or autoimmunity, asthma, and other immunomodulatory diseases. As allergy or autoimmunity antigens are identified by expression library immunination (ELI) or other means such phage library display for binding to human IgE, etc, the simple vaccination protocols described herein could be used to tolerize an organism to the offending antigen(s). The invention also provides methods for identifying a potential tolerizing antigen, or a promoter useful for selective expression in non-APCs to be used in the vaccines of the present invention.

The methods of the present invention may also be used to tolerize a recipient patient, before an organ transplantation, to particular major histocompatibility complex antigens (e.g., MHC proteins) and other antigens causing rejections to the donor organs or tissues. Additionally, a patient could be tolerized to a transgene's expressed product to block rejection of transgene transfected cell(s) transfected in gene therapy. The invention could also be used in gene therapy to deliver therapeutic proteins (encoded in the vector) to avoid immune responses to the encoded gene product.

The invention first provides a method of attenuating or reducing an immune response to an antigen, comprising obtaining a nucleic acid segment that encodes an antigen; placing the nucleic acid segment in a non-antigen presenting cell under conditions conducive to expression of the antigen; and expressing the composition in the cell at a level that reduces an immune response the antigen in an organism. In certain embodiments, the antigen may be a protein, a polypeptide or a peptide.

In general, the term "non-antigen presenting cell" can be any cell that accomplishes the goal of the invention by allowing for tolerization against an antigen without a co-committent immune response (i.e., from the T-cell or -B-cell arms of the immune system). Such cells can be defined by those of skill in the art, using methods disclosed herein and in the art. As is understood by one of ordinary skill in the art (see for example Kuby, 1993, incorporated herein by reference), and used herein certain embodiments, a cell that displays or presents antigens normally or preferentially with a class II major histocompatability molecule or complex to an immune cell is an "antigen presenting cell." In some cases, the immune cell to which an antigen presenting cell displays or presents an antigen to is a CD4⁺T_H cell. As described herein certain embodiments, a "non-antigen presenting cell" is a cell or cell type that does not normally or preferentially present antigens associated with the type II major histocompatability complex. In preferred aspects of the invention, the non-antigen presenting cell does not normally or preferentially display or presents an antigen to a CD4⁺T_H cell. In other embodiments, a non-antigen presenting cell or cell type may also not normally or preferentially present antigens associated with a type I major histocompatability complex, although this is not required.

In certain embodiments, the nucleic acid segment is injected into the cell. In certain embodiments, the nucleic acid segment is injected by microprojectile bombardment. In certain aspects of the invention, the nucleic acid segment is placed into a cell but not integrated into the cell's genome. Of course, the nucleic acid may be placed into the cell by other means, as would be known to one of skill in the art.

In one aspect, the method to attenuating or reducing an immune response further comprises obtaining at least one additional nucleic acid segment comprising the promoter and an open reading frame that encodes at least one additional antigen; placing the at least one additional nucleic acid segment in the cell under conditions conducive to expression of the at least one additional antigen; and expressing the at least one additional antigen in the cell at a level in an organism that reduces an immune response to the at least one additional antigen. The first and the additional nucleic acid segments may be cotransfected, or transfected one after the other, into the cell.

In certain other embodiments, the nucleic acid segment may comprise a promoter. Preferably, the promoter is an eukaryotic promoter. In a particularly preferred aspect, the promoter preferentially expresses in a non-antigen presenting cell. By "preferentially expresses in a non-antigen presenting cell" the promoter does not express in other antigen presenting cells at a level that would overall stimulate an immune response to an antigen. An immune response to an antigen expressed under the control of a promoter that preferentially expresses in a non-antigen presenting cell would be as a whole down regulated, attenuated or reduced irregardless of whether detectable expression occurred in other cell types, including antigen presenting cells. A preferred promoter that is preferentially expresses in a non-antigen presenting cell is the Kerl4 promoter, though other promoters may be used as would be known to those of ordinary skill in the art.

In certain embodiments, a preferred non-antigen presenting cell is a keratinocyte. The cell may be transfected with the gene expression tolerizing vaccine and/or protein antigen tolerizing vaccine, and subsequently transferred into an organism. Alternatively, the cell may be comprised within the organism when contacted with a gene expression tolerizing vaccine and/or protein antigen tolerizing vaccine. Preferred organisms are animals, with mammals being particularly preferred, and humans being particularly preferred mammals

In some aspects, the antigen is derived from a virus, bacteria, plant, fungus or animal, though the antigen may also be derived from other sources. The antigen preferrably induces allergy, autoimmunity, asthma, an immune response against a transplanted organ, an immune response against an expressed transgene or an immunomodulatory disease. In one aspect, the immune response against a transplanted organ is stimulated by an antigen comprising a MHC protein. The antigen may be identified by any method known to those of ordinary skill in the art, including but not limited to ELI (U.S. Patent Nos. 5,989,553 and 5,703,057, each incorporated herein by reference) or phage library display (e.g., U.S. Patent Nos. 5,824,520, 5,821,047 and 5,702,892, each incorporated herein by reference).

In certain embodiments, the nucleic acid segment encodes a fusion protein. In another embodiment, the nucleic acid segment encodes a regulatory polypeptide, wherein the regulatory polypeptide induces transcription from a promoter. In a preferred aspect, the regulatory polypeptide is encoded by the Gal 4 gene. In another preferred aspect, the promoter is the Gal4 element.

The invention next provides a method of attenuating an immune response to an antigen, comprising: obtaining a first nucleic acid segment comprising a promoter that expresses in a non-antigen presenting cell and an open reading frame that encodes an antigen; placing the nucleic acid segment in a non-antigen presenting cell under conditions conducive to expression of the antigen; and expressing the antigen in the cell at a level that reduces an immune response in an organism to a second composition comprising the antigen.

The invention also provides a method of attenuating an immune response to an antigen, comprising: obtaining a first nucleic acid segment comprising a promoter and a first open reading frame that encodes a regulatory polypeptide; obtaining at least one additional nucleic acid segment comprising at least one additional promoter and at least one additional open reading frame that encodes an antigen; placing the nucleic acid segments in a cell under conditions conducive to expression of the regulatory polypeptide from the first open reading frame; and expressing the regulatory polypeptide, wherein the regulatory polypeptide enhances the expression of the antigen from the additional promoter, and wherein in the antigen is expressed in the cell at a level that reduces an immune response in an organism to a second composition comprising the antigen.

The invention provides a method of identifying a promoter that can attenuate an immune response to an antigen, comprising: obtaining a first nucleic acid segment comprising a putative promoter and a open reading frame that encodes reporter gene; placing the nucleic acid segment in a non-antigen presenting cell under conditions conducive to expression of the reporter gene from the open reading frame; and assaying for expression the reporter gene.

The invention further provides a method of identifying an antigen that can attenuate an immune response, comprising: obtaining a first nucleic acid segment comprising a promoter that expresses in a non-antigen presenting cell and a open reading frame that encodes an antigen; placing the nucleic acid segment in a non-antigen presenting cell under conditions conducive to expression of a the antigen; expressing the antigen in the cell in an organism, and assaying for a reduced immune response to the antigen. The invention additionally provides a method of attenuating an immune response to an antigen, comprising: contacting an antigen with a non-antigen presenting cell under conditions conducive to expression of the antigen by the cell; and expressing the antigen in the cell at a level that reduces an immune response the antigen in an organism.

The invention provides a tolerizing vaccine targeted to non-antigen presenting cells for use as a medicament.

The provides a use of a tolerizing vaccine expressed in non-antigen presenting cells for the manufacture or a medicament for the treatment of an allergy, an autoimmune disease, or an undesirable immune response.

As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIG. 1. Direct construction of the plasmid used the keratin promoters

(mainly K5 and K14) or Dectin 2 (Dec2) promoter strictly to control the cell-type specific expression of the genes

FIG. 2. Cascade Expression System A Dectin 2 or keratin promoter

(K5 and K14) was used to drive the cell-type specific expression of Gal4, which, in turn, binds to the Gal4 element and drives the gene expression in another plasmid in the same cell

FIG. 3. Humoral immune response against hAAT in mice The values

(N=2 for kl4-AAT group and N=3 for the others) shown have been subtracted from the concurrent mock DNA inoculated control mice (N=2) The vectors used are represented as follows Dec2i-AAT (D), dectine 2 promoter followed by a intron sequence and the sequence encoding human alphal antitrypsin (hAAT); k5i-AAT (•), keratin #5 promoter followed by a sequence of intron and hAAT (■); K14-AAT, keratin #14 promoter followed by hAAT coding sequence, and CMVi-AAT (0), cytomegalovirus immediate-early promoter followed by the sequence of an intron and hAAT

FIG. 4. Anti-hAAT antibody response in mice The values (N=4) shown have been subtracted from the concurrent mock DNA inoculated control mice (N=2) The vectors used are represented as follows CMVi-hAAT (•), kl4i-hAAT + kl4-B7 1 (■), K5-hAAT (O) and K14-hAAT (D) CMVi-hAAT is the cytomegalovirus immediate-early promoter followed by the sequence of an intron and hAAT, kl4-hAAT is the keratin #14 promoter followed by hAAT coding sequence, kl4-B7 1 is the keratin #14 promoter followed by B7 1 coding sequence, and K5-hAAT is the keratin #5 promoter followed by sequences hAAT

FIG. 5. Humoral immune response against hAAT and human growth hormone The values (N=3) shown have been subtracted from the concurrent mock DNA inoculated control mice (N=2) The vectors used are represented as follows CMV-GH GH (0) cytomegalovirus immediate-early promoter followed by the sequence of an intron and human growth hormone; and K14-AAT (•), keratin #14 promoter followed by hAAT coding sequence.

FIG. 6. Humoral immune response against hAAT in mice. Three times vaccination with K14-hAAT construct could induce the specific tolerance to the CMNi-hAAT even with two times of challenge. The values (Ν=3) shown have been subtracted from the concurrent mock DNA inoculated control mice (N=2).

FIG. 7. K14-hAAT induced tolerance to the protein challenge. hAAT- protein group was only ip 10 μg in 100 μl PBS on week 10 and the K14-hAAT protein group was pre-tolerated with K14-hAAT plasmid as shown and challenged with 10 μg protein also on week 10. The values (N=4) shown have been subtracted from the concurrent mock DNA inoculated control mice (N=2).

FIG. 8. Fas-ligand, IL10 and CTAL4Ig could inhibit the genetic immunization. Co-inoculation of CMVi-hAAT plasmid with FasL, IL10, and CTAL4Ig could potentially inhibit the immune response to the antigen in the mice but no tolerance to the antigen could be induced.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention relates to genetic immunization vector(s) which when introduced into an animal induces tolerance to a subsequent exposure to a specific antigen. Expression of an antigen in non-antigen presenting cells (non-APCs) in a host organism induces a greater immunological tolerance to the antigen. One method to specifically express of the antigen to non-APCs is to use a promoter which preferentially expresses or restricts the expression of antigen to non-antigen presenting cells (Fig. 1). Alternatively, limiting transfection of a vector to non-APCs will curtail expression of the antigen preferentially to the non-APCs. When an antigen is introduced into an animal the immune response may consist of suppressing and augmenting arms Enhancing or restricting antigen production to antigen presenting cells (e.g., dendritic cells) may augment the immune response Alternatively, enhancing or restricting expression to non-APC (e.g., keratinocytes) may tolerize to the antigen, such that, the immune response to that specific antigen would be attenuated Therefore, if a promoter was used that restricted expression of genetic vaccines to dendritic cells (e.g., the Dec2 promoter) the immune response would be augmented In contrast, and a promoter that preferentially expresses or restricts expression to keratinocytes (e.g., the Kerl4 promoter) may make the animal less responsive to subsequent exposure to the antigen

A. PROMOTERS

The methods and vectors described for tolerizing vaccines may use known cell specific promoters (e.g , non-APC cell promoters) In the examples below, the basic practice of this invention is demonstrated using a dendritic cell and keratinocyte specific promoters (Staggers et ah, 1995) It is contemplated that any number of promoters might be used, particularly if coupled to the cascade gene expression system described herein Nucleic acid sequences for various gene regulatory sequences, such as promoters and tissue specific expression elements, the amino acid encoding nucleotide sequences, and the protein, polypeptide and peptide sequences for various genes (i.e. antigenic sequences) have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art One such database, for example, is the National Center for Biotechnology Information's Genbank and GenPept databases (http //www ncbi nlm nih gov/) The regulatory regions and coding regions for these known genes may be amplified or synthesized using any technique disclosed herein or as would be know to those of ordinary skill in the art, and may be used in the immune tolerizing vaccines and methods of the invention

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon Such a promoter can be referred to as "endogenous " Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression, such as an non-antigen presenting cell. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.

The particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell, (e.g., a non-APC). To attenuate an immune response, the expression of the antigen should be minimized in antigen presenting cells. This may be achieved by selection of a promoter that preferentially expresses in non-APC cells. The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Non-limiting examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et α/., 1998), murine epididymal retinoic acid-binding gene (Lareyre et α/., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule- 1 (Almendro et l., 1996). Thus, one may select a promoter that preferentially expresses in a non-APC and/or preferentially does not express well in an APC.

It is contemplated that preferentially transfecting non-APC cells with the tolerizing vaccine construct may also attenuate an immune response. Where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter.

A particular natural cellular promoter may be too weak to produce enough antigen to consistently effect an immune response. To solve this problem a cascade expression system was developed. A regulatory protein (e.g., Gal4,) gene is put under the control of the cell specific promoter. On the same or another plasmid, the antigen-encoding gene is placed under the control of the regulatory protein. In this way a powerful activator drives the antigen expression preferentially in specific cell types. This system (diagrammed in Fig. 2) allows high level expression while retaining specificity of expression.

B. CELLS In particular embodiments, a nucleic acid for production of a tolerizing vaccine such as, for example, an antigen encoding region, a promoter or other useful nucleic acid sequence (e.g., a control sequence) may be isolated from at least one organelle, cell, tissue or organism. In other embodiments, at least one nucleic acid construct of the present invention may be transfected into at least one organelle, cell, tissue or organism. In particular aspects, the a nucleic acid construct of the present invention, such as for example a nucleic acid encoding an antigen, is transcribed, and in more specific aspects, translated into a protein, polypeptide or peptide in the at least one organelle, cell, tissue or organism. In preferred aspects, the at least one organelle, cell, tissue or organism is derived from or comprised in an animal (e.g., a human).

As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these term also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

In certain embodiments, a cell may comprise, but is not limited to, at least one skin, bone, neuron, axon, cartilage, blood vessel, cornea, muscle, facia, brain, prostate, breast, endometrium, lung, pancreas, small intestine, blood, liver, testes, ovaries, cervix, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood, embryonic or ascite cell, and all cancers thereof. An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. However, particularly preferred host cells are non-antigen presenting cells, as would be known to one of ordinary skill in the art. A preferred non-antigen presenting cell, for instance, is a keratinocyte. The host cell may be transfected in vitro or in vivo with a tolerizing vaccine, but will generally be comprised in an organism (e.g., an animal) after transfection or transformation. Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art.

Some nucleic acid constructs of the present invention may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

1. Tissues

The cell or cells from which a nucleic acid encoding an antigen, a promoter or other useful sequence, or a cell or cells to be transformed with a nucleic acid construct of the present invention may be comprised in a tissue. The tissue may be part or separated from an organism. In certain embodiments, a tissue may comprise, but is not limited to, skin, bone, neuron, axon, cartilage, blood vessel, cornea, muscle, facia, brain, prostate, breast, endometrium, lung, pancreas, small intestine, blood, liver, testes, ovaries, cervix, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood, embryonic, ascite tissue, meristematic cells, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, stalks, and all cancers thereof.

2. Organisms

In certain embodiments, the cell or tissue may be comprised in at least one organism. In certain embodiments, the organism may be, but is not limited to, an eubacteria, an archaea, an eukaryote or a virus (see webpage http : //phylogeny . arizona . edu/tree/phylogeny . html) . a. Eubacteria

In certain embodiments, the organism is an eubacteria. In particular embodiments, the eubacteria may be, but is not limited to, an aquifecales; a thermotogales; a thermodesulfobacterium; a member of the thermus-deinococcus group; a chloroflecales; a cyanobacteria; a firmicutes; a member of the leptospirillum group; a synergistes; a member of the chlorobium-flavobacteria group; a member of the chlamydia-verrucomicrobia group, including but not limited to a verrucomicrobia or a chlamydia; a planctomycetales; a flexistipes; a member of the fibrobacter group; a spirochetes; a proteobacteria, including but not limited to an alpha proteobacteria, a beta proteobacteria, a delta & epsilon proteobacteria or a gamma proteobacteria. In certain aspects, an organelle derived from eubacteria are contemplated, including a mitochondria or a chloroplast.

b. Archaea

In certain embodiments, the organism is an archaea (a.k.a. archaebacteria; e.g., a methanogens, a halophiles, a sulfolobus). In particular embodiments, the archaea may be, but is not limited to, a korarchaeota; a crenarchaeota, including but not limited to, a thermofilum, a pyrobaculum, a thermoproteus, a sulfolobus, a metallosphaera, an acidianus, a thermodiscus, a igneococcus, a thermosphaera, a desulfurococcus, a staphylothermus, a pyrolobus, a hyperthermus or a pyrodictium; or an euryarchaeota, including but not limited to a halobacteriales, methanomicrobiales, a methanobacteriales, a methanococcales, a methanopyrales, an archeoglobales, a thermoplasmales or a thermococcales.

c. Eukaryotes

In certain embodiments, the organism is an eukaryote (e.g., a protist, a plant, a fungi, an animal). In particular embodiments, the eukaryote may be, but is not limited to, a microsporidia, a diplomonad, an oxymonad, a retortamonad, a parabasalid, a pelobiont, an entamoebae or a mitochondrial eukaryote (e.g., an animal, a plant, a fungi, a stramenopiles). In particular aspects, the eukaryote is a metazoa (e.g., an animal). In particular facets the vertebrate may be a terrestrial vertebrate (e.g., a frog, a salamander, a caecilian, a reptile, a mammal, a bird) or a non-terrestrial vertebrate (e.g., a sharks, a ray, a sawfish, a chimera, a ray-finned fish, a lobe-finned fish). In additional facets, the mammal may be a monotremata (e.g., a platypus, an echidna), a multituberculata, a marsupialia (e.g., an opossum, a kangaroo), a palaeoryctoids or an eutheria (e.g., a placental mammal).

In particular embodiments, eukaryote is a fungi. A fungi may be, but is not limited to, a chytridiomycota (e.g., a water mold, an allomyces), a zygomycota (e.g., a bread mold, a rhizopus, a mucor), a basidiomycota (e.g., a mushroom, a rust, a smut) or an ascomycota (e.g., a sac fungi, a yeast, a penicillium).

In certain embodiments, the eukaryote is a green plant. A green plant may be, but is not limited to, a prasinophytes, a chlorophyceae, a trebouxiophyceae, a ulvophyceae, a chlorokybales, a klebsormidiales, a zygnematales, a streptophyta, a charales, a coleochaetales or an embryophytes (e.g., a land plant). In particular facets, the embryophytes may be, but is not limited to, a marchantiomorpha (e.g., a liverwort), an Anthoceromorpha (e.g., a hornwort), a bryopsida (e.g., a moss), a lycopsida (e.g., a lycophyte), an equisetopsida (e.g., a horsetail, a sphenophyte), a fihcopsida (e.g., a fern), a spermatopsida (e.g., a seed plant: a flowering plant, a conifer). In particular aspects, the spermatopsida may be, but is not limited to an angiosperm. An angiosperm may include, but is not limited to, a ceratophyllaceae, a nymphaeales, a piperales, an aristolochiales, a monocotyledons, an eudicots, a laurales, a chloranthaceae, a winterales or a magnoliales.

d. Viruses

In certain embodiments the organism may be a virus. Thus, a nucleic acid encoding an antigen, promoter or other useful nucleic acid sequence may be obtained from a virus. In particular aspects, the virus may be, but is not limited to, a

DNA Virus, including but not limited to a ssDNA virus or a dsDNA virus; a DNA

RNA rev transcribing virus, a RNA virus, including but not limited to a dsRNA virus, including but not limited to a -ve stranded ssRNA or a +ve stranded ssRNA; or an unassigned virus.

C. TOLERIZING VACCINES

In certain embodiments, the at least one tolerizing vaccine comprise or express an antigen. The antigen may reduce an immune response by an animal transfected or inoculated with the tolerizing vaccine encoding an antigen, or the tolerizing vaccine encoded antigen. Thus, the nucleic acid constructs may comprise a tolerizing vaccine or "gene vaccine" useful for immunization protocols. In this embodiment, tolerizing vaccines encoding antigens for allergens, autoimmune stimulating, host vs graft, or graft vs. host antigens are preferred.

The course of the immunization with the tolerizing vaccine may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays. Other immune assays can be performed and assays of protection from challenge with the pathogen can be perforned, following immunization.

1. Immunomodulators

It is contemplated that immunomodulators can be included in the vaccine to supprss the patient's response. Immunomodulators can be included as purified proteins or their expression engineered into the cells when cells are part of the composition.

Genes encoding immunomodulators can be included. The following sections list examples of immunomodulators that are of interest.

a. Cytokines

Interleukins and cytokines, and vectors expressing interleukins and cytokines are contemplated as possible vaccine components. Interleukins and cytokines, include but not limited to interleukin 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,

IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, β-interferon, α-interferon, γ- interferon, angiostatin, thrombospondin, endostatin, METH-1, METH-2, GM-CSF, G- CSF, M-CSF, tumor necrosis factor, TGFβ, LT and combinations thereof.

b. Chemokines Chemokines or nucleic acids that code for chemokines also may be used as vaccine components. Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine gene in combination with, for example, a cytokine gene, to enhance the recruitment of other immune system components to the site of treatment. Such chemokines include RANTES, MCAF, MIP1 -alpha, MIPl-Beta, and IP-10. The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines-

D. METHODS FOR DELIVERY OF A TOLERIZING VACCINE

In order to effect expression of a nucleic acid construct (e.g., a gene construct), the tolerizing vaccine expression construct must be delivered into a cell. As used herein, "gene" may refer to an encoded antigen expressed by a tolerizing vaccine construct. The tolerizing vaccine expression construct may consist only of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned which physically or chemically permeabilize the cell membrane. Some of these techniques may be successfully adapted for in vivo or ex vivo use, as discussed below.

The nucleic acid encoding the antigen may be stably integrated into the genome of the cell, or may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

The lack of substantial production of antibodies may be monitored by sampling blood of the tolerized animal at various points following administration of the tolerizing vaccine One or more additional booster doses of tolerizing vaccine may also be given The animal's ability to mount an immune response to an antigen may be measured by administration of one or more doses of the antigen, collecting the animal's serum, and tittering the serum for antibodies to the antigen, using techniques known to those of ordinary skill in the art The process of boosting and tittering is repeated until a suitable reduced antibody titer is achieved, i.e. the animal no longer mounts an effective immune response to a particular antigen

Suitable methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art Such methods include, but are not limited to, direct delivery of DNA such as by injection (U S Patent Nos 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harlan and Weintraub, 1985, U S Patent No 5,789,215, incorporated herein by reference), by electroporation (U S Patent No 5,384,253, incorporated herein by reference), by calcium phosphate precipitation (Graham and Van Der Eb, 1973, Chen and Okayama, 1987, Rippe et α/., 1990), by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985), by direct sonic loading (Fechheimer et al, 1987), by liposome mediated transfection (Nicolau and Sene, 1982, Fraley et α/., 1979, Nicolau et al, 1987, Wong et al, 1980, Kaneda et α/., 1989, Kato et al, 1991), by microprojectile bombardment (PCT Application Nos WO 94/09699 and 95/06128, U S Patent Nos 5,610,042, 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference), by agitation with silicon carbide fibers (Kaeppler et al, 1990, U S Patent Nos 5,302,523 and 5,464,765, each incorporated herein by reference), by Agrobacterium-mediated transformation (U S Patent Nos 5,591,616 and 5,563,055, each incorporated herein by reference), or by PEG-mediated transformation of protoplasts (Omirulleh et αl, 1993, U S Patent Nos 4,684,61 1 and 4,952,500, each incorporated herein by reference), by desiccation/inhibition-mediated DNA uptake (Potrykus et al, 1985). Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.

1. Particle Bombardment

A preferred method of introducing a DNA tolerizing vaccine expression construct into cells is through particle bombardment. Microprojectile bombardment techniques can be used to introduce a nucleic acid into at least one cell, tissue or organism (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT Application WO 94/09699; each of which is incorporated herein by reference). This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). There are a wide variety of microprojectile bombardment techniques known in the art, many of which are applicable to the invention.

Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et α/., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold particles or beads. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.

In certain aspects, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. An illustrative embodiment of a method for delivering DNA into a cell (e.g., a plant cell) by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with cells, such as for example, a monocot plant cells cultured in suspension The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large However, the inventors have had particular success using the following general protocol described in the specific examples, such as example 2

2. Injection In certain embodiments, a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, either subcutaneously, intradermally, intramuscularly, intervenously or intraperitoneally Methods of injection of vaccines are well known to those of ordinary skill in the art (e.g., injection of a composition comprising a saline solution) Further embodiments of the present invention include the introduction of a nucleic acid by direct microinjection Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985) The amount of tolerizing vaccine used may vary upon the nature of the antigen as well as the organelle, cell, tissue or organism used

3. Liposome-Mediated Transfection

The tolerizing vaccine expression construct may be entrapped in a liposome

Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium Multilamellar liposomes have multiple lipid layers separated by aqueous medium They form spontaneously when phospholipids are suspended in an excess of aqueous solution The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an expression construct complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).

Lipo some-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al. 1980).

In certain embodiments of the invention, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et α/., 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.

4. Electroporation

The tolerizing vaccine expression construct may be introduced into the cell via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high- voltage electric discharge.

Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al, 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al, 1986) in this manner. 5. Calcium Phosphate

The tolerizing vaccine expression construct may be introduced to the cells using calcium phosphate precipitation Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al, 1990)

6. DEAE-Dextran The tolerizing vaccine expression construct may be delivered into the cell using

DEAE-dextran followed by polyethylene glycol In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985)

7. Sonication Loading Additional embodiments of the present invention include the introduction of a nucleic acid by direct sonic loading. LTK" fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al, 1987)

8. Adenoviral Assisted Transfection The tolerizing vaccine expression construct may be introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et α/., 1992, Curiel, 1994)

9. Receptor Mediated Transfection

Tolerizing vaccine expression constructs that may be employed to deliver nucleic acid construct to target cells are receptor-mediated delivery vehicles These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in the target cells In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention Specific delivery in the context of another mammalian cell type is described by Wu and Wu, 1993 (incorporated herein by reference).

Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a nucleic acid-binding agent. Others comprise a cell receptor-specific ligand to which the nucleic acid to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al, 1990; Perales et al, 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993; incorporated herein by reference). In certain aspects of the present invention, a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of a cell-specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acid(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosyl-ceramide, a galactose-terminal asialganglioside, have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al, 1987). It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into a target cell in a similar manner. 10. Tolerizing Vaccine Delivery Using Viral Vectors

The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. Tolerizing vaccine vectors of the present invention may be a viral vector that encode one or more antigens.

a. Tolerizing Vaccine Delivery Using Adenoviral Vectors A particular method for delivery of the tolerizing vaccine expression constructs involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein.

The tolerizing vaccine expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known sero types or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

b. Tolerizing Vaccine Delivery Using AAV Vectors Adeno-associated virus (AAV) is an attractive vector system for use in the tolerizing vaccines of the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host range for infectivity (Tratschin et α/., 1984; Laughlin et α/., 1986; Lebkowski et al, 1988; McLaughlin et al, 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Patent No. 5,139,941 and U.S. Patent No. 4,797,368, each incorporated herein by reference.

c. Tolerizing Vaccine Delivery Using Retroviral Vectors Retroviruses have promise as antigen delivery vectors in tolerizing vaccines due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller, 1992).

In order to construct a tolerizing vaccine retroviral vector, a nucleic acid encoding a antigen of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983). When a recombinant plasmid containing a cDNN together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).

Gene delivery using second generation retroviral vectors has been reported. Kasahara et al. (1994) prepared an engineered variant of the Moloney murine leukemia virus, that normally infects only mouse cells, and modified an envelope protein so that the virus specifically bound to, and infected, human cells bearing the erythropoietin (EPO) receptor. This was achieved by inserting a portion of the EPO sequence into an envelope protein to create a chimeric protein with a new binding specificity.

d. Tolerizing Vaccine Delivery Using Other Viral Vectors

Other viral vectors may be employed as tolerizing vaccine expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et α/., 1988), sindbis virus, cytomegalovirus and herpes simplex virus may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et α/., 1990).

e. Tolerizing Vaccine Delivery Using Modified Viruses

The nucleic acids to be delivered may be housed within an infective virus that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors. Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989) Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al, 1989) Thus, it is contemplated that antibodies, specific binding ligands and/or other targeting moieties may be used to specifically transfect non-APC types

E. MULTIPLE ADMINISTRATIONS

In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals Periodic boosters at intervals of 1-5 years, usually three years, may be desirable to maintain tolerance to one or more antigens

The following examples are included to demonstrate preferred embodiments of the invention It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention

EXAMPLE 1 Promoters That Grant Tissue Specific Expression

The specificity of three promoters in tissue culture cells was tested The mouse (Balb/c) ears or cultured cells (1 X 10^) were (co)transfected with 1 μg reporter plasmid DNA using gene-gun method and the luciferase activity of each ear or cultured cells was measured 24 hrs later. The value of the luciferase activity is the means of four mouse ears in an arbitrary unite. The standard error in all group is within 10-30%.

The constructs used in this example included: Dec2-luc, the dectin 2 promoter followed by the luciferase open reading frame (ORF); Dec2i-luc, the dectin 2 promoter followed by an intron and the luciferase ORF; K5-luc, the keratin #5 followed by the luciferase ORF; K14-luc, the keratin #14 promoter followed by the luciferase ORF; CMVi-luc, the CMV promoter followed by an intron and the luciferase ORF; Dec2- Gal4, the dectin 2 promoter followed by the gal4 ORF; P4U-luc, four repeat of the gal4 binding element as a gal4 binding promoter followed by the luciferase ORF; and K14-Gal4, the keratin 14 promoter followed by the gal4 ORF. As evident in Table 1, the Dectin 2 (Dec2), keratin #5 (Ker5) and keratin #14 (Kerl4) promoters had varying specificity and strengths.

The Ker5 and Kerl4 promoters preferentially drove the gene expression in the keratinocyte (PAM212) cell line, with a 5 fold and a 60 fold increase compared to the non-keratinocyte cell line (fibroblast SN47), as standardized with cytomegalovirus immediate-early promoter (CMV promoter). Similarly, the Dectin 2 promoter mainly drives the gene expression in dendritic cell line (XS106) with 40 fold increase.

Using the promoters fused to green fluorescence protein (GFP), it was demonstrated that these constructs also confer cell-specific expression in skin when shot in with a gene gun. In the mouse ear, it was shown that the level of the gene expression was only 4% and less than 1% for keratin and dectin promoter respectively compared with the CMV promoter.

EXAMPLE 2 Demonstration of a Tolerizing Vaccine Against Antigens in Mammals

Mice were immunized several times with each promoter system Specific- pathogen-free (5-7 weeks old), female balb/c mice were purchased from Jackson Labs (Bar Harbor, ME) A biolistic gene gun (Rumsey-Loomis, Ithaca, NY) was used to deliver plasmid DNA into the skin of mouse ears (Williams et al, 1991, Johnston and Tang, 1993) DNA was precipitated onto gold micro-projectiles (3 ± 1 μm diameter, Metz Metallurgical Corp , South Plainfield, NJ) at 4 μg DNA/mg gold Each mouse received two shots of gold-DNA and each shots contains 1 μg plasmid DNA

The blood samples of the mice was collected from the tail vein at the time point as indicated at Fig 3 The anti-hAAT antibodies in the serum was measured using enzyme-linked immuno enzyme-linked immunosorbent assay (ELISA, Tang et al, 1992) Briefly, ninety six- well plats were coated with lμg hAAT per well in 50 μl phosphate buffered saline (PBS) (for overnight at 4°C and 100 μl blocking buffer (3% bovine serum albumin (BSA) in PBS with 0 05% tween 20) was added and incubated for 2 hrs at room temperature After 3 times washing with PBS plus 0 05% tween 20, the serum samples (diluted 1 :200 in blocking buffer) were added into the well and incubated overnight at 4°C, then further incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G and M (Jackson ImmunResearch, West Grove, PA) for 1 h at room temperature. The plate was washed and developed with TMB buffer solution (Calbiochem). The optical density was read at 450 nm and concentration of the antibodies was determined according to the standard curve. As evident in Fig. 3, the Dec2 and Ker5 promoter systems were capable of producing an immune response in at least some mice.

In an additional experiment, each mouse received two shots of gold-DNA each with 1 μg plasmid DNA. The further boosts was as indicated in Fig. 4. The mice in CMVi-hAAT group was in the same age as the others but received first shot 7 weeks later compared to the other group of mice. The mouse serum was collected and tested for anti-hAAT antibody titers in 1 :200 dilution. As before, the Dec2 and Ker5 promoter systems were capable of producing an immune response in at least some mice. However, no response was elicited by the Kerl4 promoter even after multiple boosts. Though the Dec2 promoter is less active than the Kerl4 promoter, it produced an immune response but the Kerl4 promoter did not, indicating that the type of cell expressing the antigen, not only for APCs, is important for defining the immune response.

To test whether the K14 mice were tolerized, mice were inoculated with the standard CMV-AAT plasmid. This plasmid by itself produces a strong immune response so as would be expected it boosted both the Dec2 and Ker5 inoculated mice. However, suφrisingly, the Kerl4 mice showed no response to the CMV-AAT vaccination. The mice were non-responding (tolerant) to the AAT antigen when given as a standard gene vaccine.

EXAMPLE 3 Demonstration of a Tolerizing Vaccine's Antigen Specificity

To test whether the Kerl4 immunized mice were tolerized to all proteins or specifically to the AAT antigen, the Kerl4 immunized mice were vaccinated with a plasmid encoding human growth hormone gene The each mouse received two shots of gold-DNA each with 1 μg plasmid DNA The further boosts was as indicated The mice in CMVi-GH group (N=3) was in the same age as the K14 group but received a first shot 7 weeks later The mice in K14 group received a first shot and two boost of kl4-hAAT plasmid to tolerize to the hAAT as shown in Fig 4 These mice showed no tolerization to the hGH as further vaccination with CMV-GH ensued similar immune response as the naive mice The mouse serum was collected and tested for anti-hAAT antibody titers in 1 200 dilution in first 7 weeks and 10 week and 12 week time point for anti-hGH As evident in Figure 5, the tolerization that was induced by Kerl4-AAT was specific since these mice responded normally to immunization by another antigen

To test whether the Kerl4-AAT tolerization protocol would tolerize to protein not introduced genetically, mice were first tolerized with Kerl4-AAT and then injected with lOμg of purified AAT protein in Freunds incomplete adjuvant Though mice receiving the protein did induce an immune response, this response was attenuated relative to the mice that were not tolerized and received the protein

These experiments demonstrate that restricted expression of antigen, even a secreted antigen such as hAAT, to keratinocytes induces specific tolerance to subsequent exposure to the antigen The tolerance to subsequent genetic immunization is complete and that to subsequent introduction of pure protein is partial In contrast, restricted expression to dendritic cells produces a strong immune response

EXAMPLE 4 Other Types of Tolerizing Vaccines

Additionally, it is contemplated that targeting protein vaccines to non-APC (e.g , keratinocytes) would also produce tolerance, such as antigens conjugated to a peptide that has a high affinity to the keratinocytes When injected to the dermis, these antigens will quickly bind or enter the keratinocytes and further processed and presented to the surface of the keratinocyte In this way a tolerance to the antigen may also be possible Another method involved transferred the keratinocytes cell line in vitro with a vector encoding an antigen and transfer the infected cell line to the body, which may also produce a tolerance to the transferred and expressed antigens

EXAMPLE 5

Use of a Tolerizing Vaccine in Autoimmune Disorder

A variety of diseases are caused by a host organism mounting an immune reaction to its own normal epitopes, including diseases such as arthritis, nephritis, thyrotoxicosis, diabetes, systemic sclerosis and systemic lupus erythematosus, etc Allergy to the foreign antigens also causes very serious diseases such as asthma To demonstrate that an animal may be tolerized to a host protein that is targeted in an autoimmune disease, many animal models can be used, as would be known to one of ordinary skill in the art For example, heφes stromal keratitis (Zhoa et al, 1998) was shown to be associate with a coat protein of herpes simplex virus and multiple sclerosis is induced by autoantigen myelin oligodendrocyte glycoprotein (MOG) (Genain et al, 1996) These autoantigens could be first introduced into the keratinocytes to tolerize the immune system of the animals to these antigens The animals are then further challenged with the same antigen A reduction in the immune response (i.e. antibody titer) to the antigen and/or disease level of the tolerized animals relative to naive control animal would be indicative of the tolerizing vaccine producing the desired effect

EXAMPLE 6 Use of a Tolerizing Vaccine in Allergies and Asthma Acute immune reactions to specific antigens, known as allergic reactions, result in a range of undesirable effects ranging from watery eyes and rashes to death from anaphylactic shock To demonstrate that an animal may be specifically tolerized to an allergen, an allergen's (such as, for example, the manganese superoxide dismutase of Aspergillus fumigatus MnSOD, Crameri, 1996) open reading frame may be placed under the control of Kerl4 promoter and the resulting construct injected into an animal The animal is then further challenged with the same antigen A reduction in the immune response (i.e. antibody titer) to the antigen and/or disease level of the tolerized animal relative to naive control animal would be indicative of the tolerizing vaccine producing the desired effect.

Similarly for asthma, the allergen that has been identified as causing the asthma may have its ORF placed under the control of a Kerl4 promoter and used in a similar protocol to tolerize the animal to the asthma antigen.

EXAMPLE 7 Use of a Tolerizing Vaccine in Organ Transplantation

Identification of organ donors that have compatible epitopes to a recipient is a major limiting factor in providing organ transplants. Often transplanted organs, including blood products are rejected by the host's immune system. Additionally, the host may be attacked by donor immune cells inadvertently transferred to the host with the transplanted organ.

To test whether an animal may be specifically tolerized to an epitope expressed on a transplanted organ, a donor animal's MHC molecule's ORF may be placed under the control of a Kerl4 driven vector to tolerize the recipient animal's immune system. For example, in mice, two type (H-2 K^b and H-2 K^d) of major histocompatiblity complex (MHC) molecules exist in different strains of mice. The ORF for the H-2 K^b may be used in a tolerizing vaccine and injected into a mouse that has a H-2 K^d genotype, or vice verse. The animal is then further challenged with the same antigen via transplant the organs of the donor to the recipient. A reduction in the immune response (i.e. antibody titer) to the antigen and/or symptoms of rejection of the transplanted organ by the tolerized animal relative to naive control animal would be indicative of the tolerizing vaccine producing the desired effect. EXAMPLE 8 Use of a Tolerizing Vaccine in Gene Therapy

The introduction of a gene defective or missing in a patient hold the promise of curing or alleviating the symptoms of various inherited diseases. However, the expressed gene product may provoke an immune response that may limit its therapeutic effectiveness. To demonstrate the tolerizing effect of a vaccine to a protein subsequently expressed by a gene therapy vector, for example, the growth hormone gene may be cloned into the Kerl4 promoter vector and repeatedly administered to a young, growing mouse. A reduction in the immune response (i.e. antibody titer) to the growth hormone and/or faster or superior growth of the tolerized animal relative to naive control animal would be indicative of the tolerizing vaccine producing the desired effect.

EXAMPLE 9 Antigens Expressed in Mice

Other antigens in a tolerance system can be expressed in mice by the same method as was employed for hAAT. Antigens, such as for example, human Growth Hormones, Hapatitis B surface antigens and HIV proteins and the Mycobacterium Ag85A protein, will be cloned downstream of the K14 promoter, which can control these genes expression specifically in the keratinocytes. It is expected that repeated transfection of these plasmid constructs into the animal skin will induce a tolerance to these antigen products such that subsequent introduction of the same antigen under the CMV promoter control will not produce as strong as an immune response as non- vaccinated animals, while naive animals innoculated with the CMV-antigen plasmid are expected to produce an immune response.

EXAMPLE 10

Tolerization to the alpha-myosin or

Chlamydia antigen in the Heart Disease Model Immunization with alpha-myosin or certain chlamydia antigens induces severe inflammatiory heart disease in Balb/c or A/J mice (Bachmaier K et al. Science 283 : 1335, 1999). Tolerization plasmids with the alpha-myosin or chlamydia antigens under K14 promoter control can be constructed using standard methods known to those of skill in the art. Genetic administration of these plasmids into the mouse skin is expected to induce tolerance to the antigens and such that subsequent challenge with the alpha-myosin or chlamydia antigen should show reduced inflammatory reaction in the heart tissue compared to control animals that did not receive the genetic tolerization treatment. It is contemplated that such vaccines, of course, could be used in humans.

* * * *

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press, pp. 117-148, 1986. Chen and Okayama, "High-efficiency transformation of mammalian cells by plasmid DNA," Mo/. Cell Biol 7:2745-2752, 1987 Cotten et α/., "High efficiency receptor-mediated delivery of small and large (48 kilobase) gene constructs using the endosome disruption activity of defective or inactivated adenovirus particles," Proc. Natl. Acad. Sci. USA, 89:6094-6098, 1992. Coupar et al, "A general method for the construction of recombinant vaccinia virus expressing multiple foreign genes," Gene, 68: 1-10, 1988.

Crameri et al, J. Exp. Med., 184:265-270, 1996.

Curiel, "Gene transfer mediated by adenovirus-polylysine DNA complexes," In: Viruses in Human Gene Therapy, J.-M.H. Vos (Ed.), Carolina Academic Press, Durham, N.C., pp. 179-212, 1994. Custovic et α/., Allergy, 53: 115-120; 1998.

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Claims

CLAIMS:

1. A method of attenuating an immune response to an antigen, comprising:

a) obtaining a nucleic acid segment that encodes an antigen;

b) placing the nucleic acid segment in a non-antigen presenting cell under conditions conducive to expression of said antigen; and

c) expressing the composition in said cell at a level that reduces an immune response said antigen in an organism.

2. The method of claim 1, wherein the nucleic acid segment is injected into the cell.

3. The method of claim 2, wherein said injection comprises microprojectile bombardment.

4. The method of claim 1, wherein the nucleic acid segment is placed into a cell but not integrated into the cell's genome.

5. The method of claim 1, wherein said nucleic acid segment comprises a promoter.

6. The method of claim 5, wherein the promoter is an eukaryotic promoter.

7. The method of claim 5, wherein said promoter preferentially expresses in a non-antigen presenting cell.

8. The method of claim 7, wherein the promoter is Kerl4

9. The method of claim 1, wherein the cell is a keratinocyte.

10 The method of claim 1, wherein the cell is transferred into an organism

11 The method of claim 1 , wherein the cell is comprised in an organism

12 The method of claim 11, wherein the organism is a mammal

13 The method of claim 12, wherein the mammal is a human

14 The method of claim 1, wherein said antigen is derived from a virus, bacteria, plant, fungus or animal

15 The method of claim 1, wherein said antigen induces allergy, autoimmunity, asthma, an immune response against a transplanted organ, an immune response against an expressed transgene or an immunomodulatory disease

16 The method of claim 15, wherein said immune response against a transplanted organ is stimulated by an antigen comprising a MHC protein

17. The method of claim 1, wherein said antigen is identified by ELI or phage library display.

18 The method of claim 1, wherein said nucleic acid segment encodes a fusion protein

19 The method of claim 1, wherein said nucleic acid segment encodes a regulatory polypeptide, wherein the regulatory polypeptide induces transcription from a promoter

20 The method of claim 19, wherein said regulatory polypeptide is encoded by the Gal 4 gene

21 The method of claim 19, wherein said promoter is the Gal4 element

2 The method of claim 19, further comprising

a) obtaining at least one additional nucleic acid segment comprising said promoter and an open reading frame that encodes at least one additional antigen,

b) placing the at least one additional nucleic acid segment in said cell under conditions conducive to expression of the at least one additional antigen, and

c) expressing the at least one additional antigen in said cell at a level in an organism that reduces an immune response to the at least one additional antigen

23 The method of claim 22, wherein said first and said additional nucleic acid segments are cotransfected, or transfected one after the other, into said cell

24 A method of attenuating an immune response to an antigen, comprising

a) obtaining a first nucleic acid segment comprising a promoter that expresses in a non-antigen presenting cell and an open reading frame that encodes an antigen,

b) placing the nucleic acid segment in a non-antigen presenting cell under conditions conducive to expression of said antigen, and

c) expressing the antigen in said cell at a level that reduces an immune response in an organism to a second composition comprising said antigen

25 A method of attenuating an immune response to an antigen, comprising a) obtaining a first nucleic acid segment comprising a promoter and a first open reading frame that encodes a regulatory polypeptide;

b) obtaining at least one additional nucleic acid segment comprising at least one additional promoter and at least one additional open reading frame that encodes an antigen;

c) placing the nucleic acid segments in a cell under conditions conducive to expression of said regulatory polypeptide from said first open reading frame; and

d) expressing said regulatory polypeptide, wherein said regulatory polypeptide enhances the expression of the antigen from said additional promoter, and wherein in said antigen is expressed in said cell at a level that reduces an immune response in an organism to a second composition comprising said antigen.

26. A method of identifying a promoter that can attenuate an immune response to an antigen, comprising:

a) obtaining a first nucleic acid segment comprising a putative promoter and a open reading frame that encodes reporter gene

b) placing the nucleic acid segment in a non-antigen presenting cell under conditions conducive to expression of said reporter gene from the open reading frame; and

c) assaying for expression said reporter gene. A method of identifying an antigen that can attenuate an immune response, comprising

a) obtaining a first nucleic acid segment comprising a promoter that expresses in a non-antigen presenting cell and a open reading frame that encodes an antigen,

b) placing the nucleic acid segment in a non-antigen presenting cell under conditions conducive to expression of a said antigen,

c) expressing the antigen in said cell in an organism, and

d) assaying for a reduced immune response to said antigen

A method of attenuating an immune response to an antigen, comprising

a) contacting an antigen with a non-antigen presenting cell under conditions conducive to expression of said antigen by said cell, and

b) expressing the antigen in said cell at a level that reduces an immune response said antigen in an organism