WO2002078715A1 - Methode induisant une reponse anti-inflammatoire specifique de l'antigene - Google Patents

Methode induisant une reponse anti-inflammatoire specifique de l'antigene Download PDF

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WO2002078715A1
WO2002078715A1 PCT/US2002/010528 US0210528W WO02078715A1 WO 2002078715 A1 WO2002078715 A1 WO 2002078715A1 US 0210528 W US0210528 W US 0210528W WO 02078715 A1 WO02078715 A1 WO 02078715A1
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nucleic acid
acid sequence
polypeptide
antigen
cells
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WO2002078715A8 (fr
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Ann B. Hill
Dan V. Mourich
Michael W. Munks
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Oregon Health And Science University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • This disclosure relates to the field of immunology, specifically to a method for reducing an immune response or inflammation in an animal.
  • the normal mammalian immune system responds to the introduction of "non-self antigens, such as antigens present on transplanted tissues or an infectious agent, through a variety of complex mechanisms.
  • One such mechanism is the recognition and destruction of such antigens by cytotoxic T lymphocytes (CTL cells), which selectively kill cells expressing "non-self antigens, while leaving cells expressing only self antigens unharmed.
  • CTL cells cytotoxic T lymphocytes
  • These mechanisms include cellular mechanisms (including cells of the innate immune system, T and B cells), and humoral mechanisms (including complement and antibodies).
  • T cells are converted from naive or resting state to an activated state by detecting antigen presented by specialized antigen presenting cells.
  • Antigen presenting cells play a critical role in the development of an immune response because they have the ability to recognize the presence of pathogens. In the presence of pathogens they not only present antigen (foreign material) but also impart a signal to the T cell that allows the T cell to differentiate into a pro-inflammatory effector cell. In the absence of pathogens the antigen-presenting cell may still present antigen, but the consequence for the T cell is the acquisition of tolerance. Thus the antigen presenting cells plays a key role in orchestrating the nature of the immune system's response to foreign substances. In ideal normal circumstances, a foreign pathogen is responded to with a pro- inflammatory response, whereas innocuous and self-antigens are either ignored or induce an anti-inflammatory response to a self protein or an innocuous substance.
  • APCs antigen presenting cells
  • dendritic cells are an important type of APC for stimulation with newly encountered antigens, and are likely to be important for rejection of transplants.
  • Dendritic cells are present in the skin and associated with lymphoid tissues throughout the body, and can be found in relatively low concentrations in the blood in different stages of maturation. The dendritic cells in the lymphoid tissue are found in association with T cell populations and are extremely effective presenters of antigens to these immune cells. Thus, processing and surface presentation of antigen by APCs can be thought of as a first step in the normal immune response.
  • DCs Resting dendritic cells
  • APC dendritic cells
  • phagocytosis is halted and migration to the lymph node begins. Once the DC reaches the lymph node, the level of surface molecules that provide presentation of antigen to the na ⁇ ve T cell (MHC Class I or MHC class II) increases.
  • the antigen fragments or processed peptides associate with major histocompatibility complex (MHC) proteins, specifically the class II type, and the combination "presents" on the APC surface.
  • MHC class I proteins generally associate with processed antigenic peptides produced within the cell, such as viral proteins.
  • CD 80 and CD 86 The surface expression of two co-stimulators, CD 80 and CD 86, has also proven to be necessary for effective presentation of an antigen.
  • T cells that express T cell receptors (TCRs) specific for the MHC/peptide complex and the receptor for CD 80/86, known as CD 28, are stimulated upon association with the APCs. The stimulation results in the production of cytokines and proliferation of T cells with that receptor specificity.
  • TCRs T cell receptors
  • CD 80/86 receptor for CD 80/86
  • APCs also have a function in immunization. Plasmid DNA based vaccines have historically been used to induce protective antibody and or cellular responses against disease causing pathogens (i.e. bacteria, virus and parasites).
  • pathogens i.e. bacteria, virus and parasites.
  • a gene sequence derived from the exogenous organism is cloned into a plasmid vector capable expressing a foreign protein in an eukaryotic host cell.
  • the purified plasmid DNA is then administered simply by direct injection (e.g. subcutaneous, intramuscular, intravascular, or intraperitoneal injection).
  • DNA immunization has proven to be an efficient method for inducing both antigen-specific humoral and cellular immune responses.
  • DNA may be taken up by DCs directly. Alternatively, DNA may be taken up by somatic cells (not APCs). These cells may then be phagocytosed or otherwise release antigenic material, which can be taken up by DCs and presented to T cells, through a mechanism known
  • Autoimmune diseases such as rheumatoid arthritis, multiple sclerosis or insulin dependent diabetes mellitus, are the result of a pathological immune response against self antigens.
  • Another example of autoimmunity is the condition known as sympathetic ophthalmitis, in which traumatic damage to one eye can cause an immune response to ocular tissue, which then leads to the destruction of the other eye.
  • Rejection of transplanted organs and tissues are a further example of an undesired consequence of normal immunity, which can often result in damage to and/or rejection of the transplant.
  • Hypersensitivity reactions are undesirable immune responses to harmless foreign substances.
  • An example of a hypersensitivity reaction is contact dermatitis.
  • the immune response to a foreign microorganism or virus might cause damage to surrounding healthy tissue, a phenomenon known as immunopathology.
  • Chronic hepatitis and artherosclerosis may be examples of immunopathology associated with infectious agents.
  • tolerance is often used broadly to define the extreme diminution or absences of an immune response to a particular antigen (self or foreign).
  • Thl type of immune response to pancreatic islet of Langerhan cells, which leads to their destruction and the development of diabetes mellitus.
  • a Thl response is characterized by the production of certain cytokines such as gamma interferon.
  • a Th2 type of response characterized by production of other cytokines such as interleukin (IL)-4 and IL-10, would be harmless or protective.
  • IL interleukin
  • Th3 response also called T regulatory cells. These cells are also classified by the cytokines they produce (e.g. IL-10 or transforming growth factor (TGF)- ⁇ ) which produce suppressive effects on other cells.
  • a method for inducing an immune response in a subject includes administering a therapeutically effective amount of a nucleic acid sequence containing a promoter operably linked to a nucleic acid sequence encoding an antigenic epitope and an agent that induces apoptosis.
  • Administration of the nucleic acid sequence that encodes the antigenic epitope and the agent that induces apoptosis induces the immune response upon presentation of the antigen in the subject.
  • a method for inducing immunoregulatory cells against a specific antigen includes administering a nucleic acid sequence containing a promoter operably linked to a nucleic acid sequence encoding an antigenic epitope; and an apoptotic agent and subsequently presenting the antigenic epitope to the immunoregulatory cell along with an agent that causes inflammation.
  • a nucleic acid sequence for inducing an immune response in a subject.
  • the nucleic acid sequence contains a promoter operably linked to a nucleic acid sequence encoding an antigenic epitope and an apoptotic agent.
  • a method for treating or preventing an immune-mediated disorder in a subject includes administering a therapeutically effective amount of a nucleic acid sequence to the subject.
  • the nucleic acid sequence contains a promoter operably linked to a nucleic acid sequence encoding an antigenic epitope and a nucleic acid sequence encoding an apoptotic agent.
  • the presentation of the antigenic epitope to an immune cell in the subject results in treatment or prevention of the disorder.
  • One embodiment includes the use of a nucleic acid sequence that contains a promoter operably linked to a nucleic acid sequence encoding an antigenic epitope and a nucleic acid sequence encoding an apoptotic agent to treat an immune-mediated disorder.
  • a pharmaceutical composition contains a nucleic acid sequence for reducing an immune response in a subject.
  • the nucleic acid sequence contains a promoter operably linked to a nucleic acid sequence encoding an antigenic epitope and a nucleic acid sequence encoding a polypeptide that induces apoptosis in a pharmaceutically acceptable carrier.
  • Fig. 1 is a schematic diagram of the mechanism of a DNA vaccine induced immune response. This is the model that was current in the literature before the discovery described herein.
  • cross presentation is shown. DNA transfects somatic cells, and some of these cells die. Dying cells are taken up by DCs and the antigen is cross-presented; either the dying cells or the CpG motifs in the plasmid DNA causes the DCs to mature and present antigen, leading to T cell priming.
  • Fig. 2 is a schematic diagram of the induction of apoptosis by adeno virus protein E4 orf4. Both the caspase dependent and a caspase independent mechanism are illustrated.
  • Fig. 3 is a schematic diagram of the pIRES vector.
  • an antigen is cloned into the multiple cloning site A (MCS A) of the vector, and an apoptosis-inducing gene is cloned into the MCS B site of the vector, operably linked to the IRES (internal ribosomal entry site).
  • MCS A multiple cloning site A
  • IRES internal ribosomal entry site
  • Fig. 4 shows the results of an experiment that demonstrates that priming with a plasmid co-expressing SIN nef and a pro-apoptotic gene prevented inflammation upon subsequent exposure to vaccinia virus encoding SlV-nef.
  • Fig. 4A is a bar graph showing the lymphocyte number in animals primed with p ⁇ EF.E4FWD (hatched bars) and pNEF.E4REN (white bars) and challenged with vaccinia virus expressing either nef (NN- ⁇ EF) or vaccinia virus expressing HIN-env gp 120 (NN- ENV). The lymphocyte number of na ⁇ ve animals is shown as a black bar on the left for comparison.
  • Fig. 4B is a bar graph of vaccinia titers in the ovaries of the same animals shown in Fig. 4A.
  • Fig. 5 shows that the pOVA-E4FWD plasmid induces apoptosis.
  • EL4 cells were transfected with the plasmid constructs shown, and apoptosis detected by annexin V staining and FACS analysis. The harsh transfection procedure caused damage to some cells; these were distinguished by high propidium iodide staining. Apoptotic cells were Annexin V high and propidium idodide low, as shown in the gate legend.
  • Fig. 6 is a digital image showing the spleen size observed in animals primed with various constructs and challenged with recombinant vaccinia virus expressing (ovalbumin) ova.
  • A The top two spleens were from animals primed with plasmid vaccine expressing both OVA and E4orf4. These spleens were normal size.
  • B The middle two spleens were from animals primed with vaccine expressing ova but not expressing E4orf4; these two spleens were enlarged.
  • C The bottom two spleens were from animals that were injected only with PBS before vaccinia challenge, and these spleens are also enlarged.
  • Fig. 7 shows that the anti-inflammatory vaccine is effective for a second antigen, OVA.
  • Fig. 7 A is a bar graph of the splenocyte number in animals treated with pOVA.E4FWD (hatched bar), pOVA.E4REV (white bar), PBS (stippled bar), or pENV.E4FWD (black bar), and challenged with a vaccinia virus expressing OVA. There were three animals in each group; the mean is shown and bars show the standard error. A digital image of a representative spleen from each group is shown below the graph.
  • Fig. 7B shows the vaccinia titers in the ovaries for the treatment groups shown in Fig. 7A; bars show the standard error for the 3 animals.
  • Fig. 8 is a set of graphs comparing the immune responses after vaccinia challenge to the immunizing antigen (ovalbumin) in animals receiving DNA prime with or with out E4 FWD.
  • Fig.8A is a bar graph representing a comparison of the ovalbumin specific CD4 responses (as determined by ICS FACS) induced in animals after receiving the different DNA prime treatments.
  • Fig. 8B is a bar graph representing a comparison of the ovalbumin peptide specific CD8 responses (as determined by ICS FACS) induced in animals receiving different DNA prime treatments.
  • Fig. 8C is a graph comparing the cytotoxic T cell activity induced in animals receiving the different DNA prime treatments.
  • Fig. 9 is a schematic diagram of a model for vaccination induced bystander suppression.
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non-human mammals.
  • subject includes both human and veterinary subjects.
  • Antigen A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
  • the term "antigen" includes all related antigenic epitopes.
  • An antigen can be a tissue-specific antigen, or a disease-specific antigen. These terms are not exclusive, as a tissue-specific antigen can also be a disease specific antigen.
  • a tissue-specific antigen is expressed in a limited number of tissues, such as a single tissue. Specific, non-limiting examples of a tissue specific antigen are insulin, somatostatin, collagen, and myelin basic protein.
  • a disease-specific antigen is expressed coincidentally with a disease process.
  • a disease-specific antigen are an antigen whose expression correlates with, or is predictive of, acute organ rejection following organ transplantation, a tumor specific antigen, such as tumor associated glycoprotein-72, or amyloid.
  • a disease-specific antigen may be a self antigen (autoantigen) associated with autoimmune disease.
  • autoantigen are myelin basic protein, insulin, proteolipid protein, myelin oligodendrocyte glycoprotein, type II collagen, thyroglobulin, thyrodoxin, S-antigen, acetylcholine receptor, or glutamate decarboxylase.
  • a disease specific antigen may be an antigen recognized by T cells mediating hypersensitivity reactions.
  • a specific, non-limiting example of an antigen recognized by T cells mediating hypersensitivity reactions is poison oak.
  • Apoptosis A form of programmed cell death characterized by morphological changes of a dying cell that can include plasma membrane blebbing, nuclear and cytoplasmic shrinkage, and chromatin condensation. There are two pathways of apoptotic cell death, one that is caspase-dependent, and one that is caspase-independent (see Fig. 2). Cells that undergo apoptosis often fragment into membrane-bound apoptotic bodies that are readily phagocytosed and digested by macrophages or by neighboring cells without generating an inflammatory response. See Zhu L. and Chun J., editors, Apoptosis detection and assay methods (Eaton Publishing Company/Bio Techniques Books Division, 1998).
  • Cell death is accomplished by the activation of endonucleases that fragment the cell's nuclear DNA into internucleosomal fragments. See, e.g., Zhang, J.H., and Xu, M., Cell Res., 10(3):205-11 (2000). Due to the generation of such fragments, the DNA of apoptotic cells typically migrates as a ladder of 180-200 bp multimers on an agarose gel.
  • Assays can be use to assess the presence of apoptotic cells.
  • TdT incorporation of labeled nucleotides into DNA strand breaks TUNEL assay
  • This system is a fluorescent TUNEL assay that measures apoptotic DNA fragmentation by directly incorporating fluorescein-12-dUTP at the 3'-OH DNA ends using Terminal Deoxynucleotidyl Transferase (TdT), which forms a polymeric tail.
  • TdT Terminal Deoxynucleotidyl Transferase
  • the fluorescein-dUTP-labeled DNAs from transfected cells are then visualized directly by fluorescence microscope or quantitated by flow cytometry. Samples are analyzed by confocal microscopy (Leica TCS4D) and by a FACScan Flow cytometer (Becton Dickinson).
  • Agents that induce apoptosis include, but are not limited to, Ad5E4orf4, caspase 3, Bad, or Bax.
  • necrotic cells are another form of cell death.
  • An assay for detecting necrotic cells is a DNA fragmentation assay. In contrast to the formation of DNA ladders found in apoptotic cells, necrotic cells show a smear of low molecular- weight DNA staining due to the random cleavage of DNA by nonspecific DNAses.
  • DNA is extracted from transfected cells as described by Takizawa et al., J. Gen. Virol, 74(pt. 11):2347-55 (1993). Medium and cells can be harvested from dishes and centrifuged. The pelleted cells are resuspended in cold lysis buffer (Promega) and incubated on ice for 30 min.
  • DNA is then precipitated with ethanol and treated with 1 mg/ml RNase A (Sigma) for 30 min at 37°C. Finally, aliquots of the DNA samples are electrophoresed though a 2% agarose gel and stained with ethidium bromide.
  • Autoimmune disorder A disorder in which the immune system produces an immune response (e.g. a B cell or a T cell response) against an antigen that is part of the normal host, with consequent injury to tissues.
  • An autoantigen may be derived from a host cell, or may be derived from a commensal organism such as the micro-organisms (known as commensal organisms) that normally colonise mucosal surfaces.
  • Exemplary autoimmune disorders affecting mammals include, but are not limited to, rheumatoidarthritis (RA), juvenile oligoarthritis, collagen-induced arthritis, Sjogren's syndrome, multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), inflammatory bowel disease (e.g.
  • autoimmune gastric atrophy pemphigus vulgaris, psoriasis, vitiligo, type I diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosingsialadenitis, systemic lupus erythematosis, Addison's disease, systemic sclerosis, scleroderma, polymyositis, dermatomyositis, pernicious anemia, sympathetic ophthalmitis, and the like. Rejection of transplanted autologous cells, such as pancreatic islet cells or stem cells, is also defined here as an autoimmune disorder.
  • autologous cells such as pancreatic islet cells or stem cells
  • autoimmune disorder include, but are not limited to, myelin basic protein, proteolipid protein, major oligodendrocytic protein, myelin associated glycoprotein, and beta-crystallin (multiple sclerosis and EAE); collagen type II, heatshock proteins, aggrecans, proteoglycans, f ⁇ llagrin (collagen-induced arthritis, adjuvant-induced arthritis, rheumatoid arthritis); desmin (psoriasis); S-antigen (uveitis); insulin, glutamic acid Decarboxylase (diabetes in NOD mice and in humans); tropomyosin (inflammatory bowel disease); epidermal cadherin (pemphigus vulgaris); SnRNP, histones
  • CD4+ T cell mediated immunity An immune response implemented by presentation of antigens to CD4+ T cells.
  • CD8+ T cell mediated immunity An immune response implemented by presentation of antigens to CD 8+ T cells.
  • cDNA complementary DNA: A piece of DNA lacking internal, non- coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • Cytokine Proteins made by cells that affect the behavior of other cells, such as lymphocytes. In one embodiment, a cytokine is a chemokine, a molecule whose functions include the direction of cellular trafficking.
  • cytokines are interferon gamma (IFN- ⁇ ), tumor necrosis factor-alpha (TNF- ⁇ ), TNF- ⁇ , IL-2, IL-4, IL-10, amongst others.
  • chemokines are macrophage inflammatory protein (MIP) 1 -alpha, MlPl-beta, RANTES (regulated upon activation, normal T-cell expressed, and presumably secreted), IL-8.
  • Cytokines, including chemokines may be encoded by the host or may be encoded by pathogenic organisms such as viruses. Differentiation: The process by which cells become more specialized to perform biological functions. DCs undergo maturation to become APCs.
  • Epitope An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody binds a particular antigenic epitope.
  • Expression Control Sequence A nucleic acid sequence that directs transcription of an operably linked nucleic acid sequence, hi on embodiment, an expression control sequence is a promoter. In another embodiment, and expression control sequence is an IRES (internal ribosomal entry site).
  • Sequence alterations in either an antigen epitope or an apoptotic polypeptide, that yield the same results as described herein.
  • sequence alterations can include, but are not limited to, conservative substitutions, deletions, mutations, frame-shifts, and insertions.
  • Immune response A response of a cell of the immune system, such as a B cell, T cell, macrophage or polymorphonucleocyte, to a stimulus.
  • An immune response can include any cell of the body involved in a host defense response for example, an epithelial cell that secretes interferon or a chemokine.
  • An immune response includes, but is not limited to, an innate immune response or inflammation.
  • the immune response is specific for a particular antigen (an "antigen-specific immune response").
  • an immune response is a , T cell response, such as a Thl, Th2, or Th3 response.
  • an immune response is directed to an alloantigen.
  • Alloimmune pathologies occur when tissue is transplanted from a donor whose genes are not identical to the recipient's genes.
  • the donor cells can be recognized by the recipient immune system as foreign, resulting in rejection of the transplanted tissue.
  • donor immune cells can recognize the recipient tissues as foreign, and attack the recipient (graft versus host disease).
  • a pathogenic alloimmune response can be an immune response by or against a transplanted organ or tissue (for example, pancreatic islet cells, stem cells, heart, blood vessel, valve, liver, lung, kidney, skin) or infused hematopoietic cells, such as an apheresis product or bone marrow.
  • the immune response is directed against a harmless or innocuous foreign substance, including but not limited to bee venom, pollen, nickel, talcum, ragweed, therapeutic drugs such as penicillin, and poison oak.
  • the immune response is directed against a pathogen but effector mechanisms of the immune response cause damage to bystander healthy tissue.
  • Immunoregulatory response An immune response that regulates a subsequent inflammatory response or an immune response.
  • An immunoregulatory response can be a suppressive response, which suppresses another immune response or inflammatory response.
  • a suppressive immune response involves immunoregulatory cells or T suppressor cells.
  • an immunoregulatory response involves the production of anti- inflammatory cytokines.
  • An immunoregulatory response can be an activating response, which activates another immune response or inflammatory response.
  • an activating immune response involves the up- regulation of cytokines, such as inflammation-activating cytokines.
  • Inflammation A part of the host defense response. When damage to tissue occurs, the body's response to the damage is usually inflammation. The damage may be due to trauma, lack of blood supply, hemorrhage, autoimmune attack, transplanted exogenous tissue or infection. This generalized response by the body includes the release of many components of the immune system (e.g. IL-1 and TNF), attraction of cells to the site of the damage, swelling of tissue due to the release of fluid and other processes, rnflammation can be induced by pathogens that act on a cell of the innate immune systems or can be induced by cells of the adaptive immune system. Specific, non-limiting examples of agents that induce inflammation are cytokines, chemokines and pathogens.
  • Disease states that are associated with inflammation include, but may not be limited to, autoimmune diseases as defined above; atherosclerosis; chronic hepatitis, transplanted foreign tissues; drug or other hypersensitivity reaction.
  • Isolated An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Leukocyte Cells in the blood, also termed “white cells,” that are involved in defending the body against infective organisms and foreign substances. Leukocytes are produced in the bone marrow. There are 5 main types of white blood cell, subdivided between 2 main groups: polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear leukocytes (monocytes and lymphocytes). When an infection is present, the production of leukocytes increases.
  • Mammal This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
  • Maturation The process in which an immature cell, such as dendritic cell, changes in form or function to become a functional mature cell, such as an APC.
  • Nucleic acid A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • Oligonucleotide A linear polynucleotide sequence of up to about 200 nucleotide bases in length, for example a polynucleotide (such as DNA or RNA) which is at least 6 nucleotides, for example at least 15, 50, 100 or even 200 nucleotides long.
  • a polynucleotide such as DNA or RNA
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • an IRES internal ribosomal entry site
  • operably linked DNA sequences are contiguous. Expression of two genes encoded by the same plasmid is regarded as operably linked if they are driven by one promoter and an IRES.
  • compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polynucleotide A linear nucleotide sequence, including sequences of greater than 100 nucleotide bases in length.
  • Preventing or treating a disease refers to inhibiting the full development of a disease, for example in a person who is known to have a predisposition to a disease such as an autoimmune disorder.
  • a predisposition is someone with a history of diabetes in the family, or who has been exposed to factors that predispose the subject to a condition, such as lupus or rheumatoid arthritis.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as the symptoms associated with organ transplant rejection.
  • ameliorating with reference to an immune pathology, refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms in a susceptible mammal, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, a reduction in the number or activity (such as cytokine secretion) of pathogenic T cells at the site of pathology or in the circulation, an improvement in the overall health or well-being of the individual, or by other parameters well known in the art that are specific to the particular disease.
  • Those skilled in the art can determine, based on knowledge of the expected course of the particular disease, whether there is a delayed onset of clinical symptoms.
  • Those skilled in the art can also determine whether there is an amelioration of the clinical symptoms or reduction in the number or activity of pathogenic T cells following treatment as compared with before treatment or as compared to an untreated mammal.
  • Portion of a nucleic acid sequence At least 10, 20, 30 or 40 contiguous nucleotides of the relevant sequence, such as a sequence encoding an antigen. In some instances it would be advantageous to use a portion consisting of 50 or more nucleotides. For instance, when describing a portion of an antigen (such as an antigenic epitope), it may be advantageous to remove a portion of the relevant sequence comprising at least 10, 20, 30, 40 or 50 nucleotides up to a length.
  • a promoter is an array of nucleic acid control sequences which direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • the promoter can be a constitutive or an inducible promoter.
  • a specific, non-limiting example of a promoter is the HCMV IE promoter.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
  • a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a recombinant protein is one encoded for by a recombinant nucleic acid molecule.
  • Subject Living, multicellular vertebrate organisms, a category that includes both human and veterinary subjects for example, mammals, birds and primates.
  • Therapeutically effective dose A dose sufficient to have a therapeutic efffect, for example to prevent advancement, or to cause regression of the disease, or which is capable of relieving symptoms caused by the disease, such as pain or swelling.
  • Tolerance Diminished or absent capacity to make a specific immune response to an antigen. Tolerance is often produced as a result of contact with an antigen under conditions which are not conducive to the development of a pro- inflammatory immune response.
  • a B cell response is reduced or does not occur.
  • a T cell response is reduced or does not occur.
  • both a T cell and a B cell response can be reduced or not occur.
  • an immunoregulatory or suppressive T cell response occurs.
  • a Th2 type of T cell response occurs.
  • an immunoregulatory response occurs which prevents the development of inflammation when the antigen is subsequently presented in a context which would otherwise induce inflammation.
  • a virus or vector "transduces" a cell when it transfers nucleic acid into the cell.
  • a cell is "transformed” by a nucleic acid transduced into the cell when the DNA becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication.
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • Transplantation The transfer of a tissue or an organ, or a portion thereof, from one body or part of the body to another body or part of the body.
  • An "allogeneic transplantation” or a “heterologous transplantation” is transplantation from one individual to another, wherein the individuals have genes at one or more loci that are not identical in sequence in the two individuals.
  • An allogeneic transplantation can occur between two individuals of the same species, who differ genetically, or between individuals of two different species.
  • An “autologous transplantation” is a transplantation of a tissue or a portion thereof from one location to another in the same individual, or transplantation of a tissue or a portion thereof from one individual to another, wherein the two individuals are genetically identical.
  • the tissue to be transplanted may be cells that were isolated from an individual and subjected to treatments in vitro before being transferred back into the same or another body.
  • Vector In one embodiment a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • the term "vector” includes viral vectors, such as adenoviruses, adeno-associated viruses, vaccinia, and retroviral vectors.
  • the term vector includes bacterial vectors.
  • tissue-specific antigens are myelin basic protein, insulin, and type II collagen.
  • disease associated antigens are GAD and insulin (for diabetes mellitus), myelin basic protein (for EAE and multiple sclerosis).
  • Other antigens are SIV nef and chicken ovalbumin.
  • the polypeptide that promotes cell death can be involved in a caspase-dependent or a caspase-independent apoptotic pathway.
  • the polypeptide that promotes cell death is a pro-apoptotic polypeptide (a polypeptide known to induce apoptosis, such as E4orf4, capase-3, RNA-activated protein kinase (PKR), Bax, Bad, Bak, Bik, Bcl-Xs, apoptin (VP-3) from CAV, Tat from human immunodeficiency virus (HIV), non-sturctural protein (NSP) from parvovirus, or ORF5 from PRRS).
  • a pro-apoptotic polypeptide a polypeptide known to induce apoptosis, such as E4orf4, capase-3, RNA-activated protein kinase (PKR), Bax, Bad, Bak, Bik, Bcl-Xs, apoptin (VP-3) from C
  • the agent that promotes cell death is a vertebrate gene product. In another embodiment, the agent that promotes cell death is a viral gene product.
  • the antigen and the pro-apoptotic polypeptide are encoded in a single plasmid. An example of this embodiment is depicted in Fig. 3, where the antigen (ova, SIN-nef, etc) is coupled to the pro-apoptotic gene (E4orf4, caspase 3, etc) in the pIRES vector.
  • the antigen and the pro-apoptotic polypeptide are encoded by separate plasmids, but transferred into the same cell type. Thus, both antigen expression and apoptosis is induced in the same population of cells. Without being bound by theory, antigen is thus delivered to the dendritic cells, and the necessary signals to promote or prevent dendritic cell maturation are also provided.
  • the antigenic epitope and a polypeptide that promotes apoptosis is provided in a single plasmid construct.
  • the level of expression of the antigenic epitope and the polypeptide that promotes apoptosis can be expressed at the same level or at different levels.
  • the antigenic epitope and polypeptide that promotes apoptosis are expressed equally.
  • the antigenic epitope is expressed from about 10 to about 1000 fold more than the polypeptide that promotes apoptosis, or from about 10 to about 100 fold more than the polypeptide that promotes apoptosis.
  • the antigenic epitope is expressed less than 10 fold over the polypeptide that promotes apoptosis.
  • plasmid constructs designed for differential expression of more than one polypeptide are of use herein.
  • a plasmid vector of use is the pIRES plasmid (Fig. 3).
  • Other plasmids of use are pCDNA3, pCDNA4, or any plasmid that regulates expression that includes a eukaryotic promoter.
  • a nucleic acid encoding the antigenic epitope and the polypeptide that promotes apoptosis is administered to the subject.
  • a parameter of the immune response or a disease state caused by the immune response is altered, as compared to a control.
  • subsequent exposure to the antigen is an infection with a virus expressing the antigen. Two non-limiting examples of this embodiment are described below (Examples 2 and 3).
  • subsequent exposure to the antigen occurs in the subject during the natural progression of a disease such as an autoimmune disorder.
  • subsequent exposure to the antigen occurs when the subject is exposed to transplanted autologous or allogeneic tissue, to drugs or environmental allergens, or to an infectious agent.
  • Coupling antigen expression with apoptotic induction results in induction of an immunoregulatory response.
  • the immunoregulatory response regulates another immune response that causes disease.
  • a parameter of the immunoregulatory response can be altered as compared to a control, h one embodiment, this alteration can be detected by comparing a parameter of the immunoregulatory response, or another parameter of the immune response or a parameter of the resultant disease state with a control. Both the immunoregulatory response and disease state induced by the immune response are regarded herein as parameters of the immune response.
  • control is a measurement of the parameter of the immune response in the absence of administration of nucleic acid encoding the antigenic epitope and a polypeptide that promotes apoptosis.
  • control is a measurement of the parameter of the immune response after the administration of the agent expressing the antigen in the absence of the agent causing apoptotic induction.
  • control is a measurement of the parameter of the immune response after the administration of the agent causing apoptotic induction coupled to an irrelevant antigen (i.e. an antigen that is not present in the subsequent viral challenge),
  • the control is a standard value.
  • a parameter of the immune response occurs as a result of the expression of the antigenic epitope and the polypeptide that promotes apoptosis in the subject.
  • the parameter of the immunoregulatory response is changed by about 25%o, about 50%>, about 100%, or about 200%>, as compared to the control.
  • Specific, non-limiting examples of a parameter of the immune response is the production of a specific cytokine, the number of lymphocytes in the spleen, the size of the spleen, the number of a particular type of T cell (e.g.
  • CD4+, CD8+ cell, cytotoxic T cells the number of natural killer (NK) cells or cells of the macrophage lineage, the number of B cells, or the production of a specific antibody or the number of immune cells (e.g. expressing an antigenic molecule, CD25, NK1.1, CD34, etc.).
  • Other parameters of the immune response include an alteration in the total number of lymphocytes in the spleen, other lymphoid tissue, or blood, or an alteration in a specific population of cells in the spleen, other lymphoid tissue or blood.
  • an alteration in a parameter of the immune response may include, but is not limited to, an increase in the CD4/CD25 double positive cells specific for the antigen, or a decrease in CD4 cells specific for the antigen that express jFN-gamma, as compared to a control.
  • a parameter of the immune response is reduced as compared to the control, h another embodiment, a parameter of the immune response is increased as compared to a control.
  • a parameter of the immune response that is altered by the methods disclosed herein can result in reduction of a disease.
  • measurement of reduction of a disease parameter is one way to monitor a parameter of the immune response.
  • disease states that may be altered by the methods disclosed herein include joint swelling (in rheumatoid or other arthritis); central or peripheral nervous system dysfunction (in multiple sclerosis, EAE, or autoimmune neuropathy); skin swelling or thickening; cellular infiltration of the skin, intestinal mucosa or other organ; raised serum glucose levels due to diabetes; cellular infiltration and/or destruction of pancreatic islets; atherosclerosis; and enlarged spleen or lymph nodes.
  • the methods disclosed herein can be used to treat transplant rejection.
  • MHC complexes play in triggering immune recognition has led to the development of methods by which these complexes are used to modulate the immune response.
  • isolated MHC class II molecules loaded with the appropriate antigen
  • isolated MHC/antigen complexes may be used to treat autoimmune disorders (see U.S. patent Nos. 5,194,425 and 5,284,935).
  • MHC molecules, or an MHC/antigen complex can be used to treat transplant rejection.
  • a nucleic acid molecule including a promoter operably linked to a nucleic acid sequence encoding a donor MHC, or an antigenic portion of a donor MHC, and an agent that induces apoptosis is administered to a transplant recipient.
  • Transplantation involves the transfer of a tissue or an organ, or a portion thereof, from one body or part of the body to another body or part of the body.
  • An "allogeneic transplantation” or a “heterologous transplantation” is transplantation from a donor to a recipient subject, wherein the donor and the recipient have genes at one or more loci that are not identical in sequence in the two individuals.
  • the recipient can generate an immune response against donor antigens (including donor MHC); thus immunosuprressive therapy is often used to treat transplant recipients (such as heart, lung, or kidney transplant recipients.
  • a novel method for treating transplant rejection includes administering a nucleic acid encoding a donor antigen, and an apopototic agent, such as a nucleic acid encoding a polypeptide that induces apoptosis.
  • immunosuppressive therapy can be administered in conjunction with, or subsequent to, administration of the nucleic acid encoding a donor antigen, and an apopototic agent.
  • Immunosuppressive therapy includes, but is not limited to, treatment with agents such as Cyclosporine A, FK506, or analogs thereof, or antibodies such as a monoclonal antibody that specifically binds CD3 (such as OKT3), CD4, or CD8.
  • treatment prolongs survival or improves function of the donor tissue.
  • the antigen is a donor MHC class I or class II molecule itself.
  • the donor antigen includes an ⁇ l, ⁇ 2 and or an ⁇ 3 domain of MHC class I, or an ⁇ l, ⁇ 2, ⁇ l and/or ⁇ 2 domains of donor MHC class II (see below).
  • MHC class I There are two basic classes of MHC molecules in mammals, MHC class I and MHC class II. Both classes are large protein complexes formed by association of two separate proteins. Each class includes trans-membrane domains that anchor the complex into the cell membrane. MHC class I molecules are formed from two non-covalently associated proteins, the ⁇ chain and ⁇ 2-microglobulin. The ⁇ chain comprises three distinct domains, ⁇ l, ⁇ 2 and ⁇ 3. The three-dimensional structure of the ⁇ l and ⁇ 2 domains forms the groove into which antigen fit for presentation to T-cells. The ⁇ 3 domain is an Ig-fold like domain that contains a trans-membrane sequence that anchors the ⁇ chain into the cell membrane of the APC. MHC class I complexes, when associated with antigen (and in the presence of appropriate co- stimulatory signals) stimulate CD8 cytotoxic T-cells, which function to kill any cell which they specifically recognize.
  • the two proteins which associate non-covalently to form MHC class II molecules are termed the ⁇ and ⁇ chains.
  • the ⁇ chain comprises ⁇ l and ⁇ 2 domains, and the ⁇ chain comprises ⁇ l and ⁇ 2 domains.
  • the cleft into which the antigen fits is formed by the interaction of the ⁇ l and ⁇ l domains.
  • the ⁇ 2 and ⁇ 2 domains are trans-membrane Ig-fold like domains that anchors the ⁇ and ⁇ chains into the cell membrane of the APC.
  • MHC class II complexes when associated with antigen (and in the presence of appropriate co-stimulatory signals) stimulate CD4 T- cells.
  • the primary functions of CD4 T-cells are to initiate the inflammatory response, to regulate other cells in the immune system, and to provide help to B cells for antibody synthesis.
  • MHC molecules (with the exception of class I ⁇ 2-microglobulin) are encoded in the HLA region, which is located on chromosome 6 and constitutes over 100 genes.
  • HLA-A, -B and -C There are also 3 pairs of class II MHC ⁇ and ⁇ chain loci, termed HLA-DR(A and B), HLA-DP(A and B), and HLA-DQ(A and B).
  • HLA-DR(A and B) HLA-DR(A and B)
  • HLA-DP(A and B) HLA-DP(A and B)
  • HLA-DQ(A and B) HLA-DQ
  • the vector is a plasmid.
  • a plasmid is pIRES (Clontech, Palo Alto, CA).
  • a viral vector can be utilized. These vectors include adenovirus, adeno-associated virus, or an RNA vims vectors such as a retrovirus.
  • the retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • GaLN gibbon ape leukemia virus
  • retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target specific by attaching, for example, a sugar, a glycolipid, or a protein. Targeting is also accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an R ⁇ A transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include, but are not limited to Q2, PA317, and PA12, for example. These cell lines produce empty virions, since no genome is packaged.
  • a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • ⁇ IH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • Another targeted delivery system for polynucleotides is a colloidal dispersion system.
  • Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • One colloidal system of this invention is a liposome.
  • Lippsomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. It has been shown that large uni-lamellar vesicles (LUN), which range in size from 0.2-4.0 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • LUN large uni-lamellar vesicles
  • R ⁇ A, D ⁇ A and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al., 1981, Trends Biochem. Sci. 6:77, 1981). h addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino et al, Biotechniques 6:682, 1988).
  • the composition of the liposome is usually a combination of phosphohpids, particularly high-phase-transition-temperature phosphohpids, usually in combination with steroids, especially cholesterol. Other phosphohpids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylchohne, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidyl-glycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phosphohpids include egg phosphatidylchohne, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.
  • RES reticulo-endothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand.
  • the method involves administering to a subject a therapeutically effective dose of a nucleic acid encoding an antigenic epitope and a nucleic acid encoding an apoptotic inducing agent and a pharmaceutically acceptable carrier.
  • Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan.
  • subject is meant any mammal, including a human.
  • the pharmaceutical compositions are preferably prepared and administered in dose units.
  • Solid dose units are tablets, capsules and suppositories.
  • different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • the pharmaceutical compositions are in general administered orally, nasally, by subcutaneous, intravenous, or intramuscular injection, or as implants, but any mode of administration is possible in principle.
  • Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.
  • the pharmaceutical compositions may be administered locally or systemically. Amounts effective for therapeutic use will, of course, depend on the severity of the disease and the weight and general state of the patient. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. Various considerations are described, e.g., in Gilman et al, eds., Goodman and Gilman: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.
  • a unit dosage can be about 1 ng to about 5 mg per patient per day. Dosages from 1 ng up to about 50 mg per patient per day may be used, particularly if the agent is administered to a secluded site and not in the circulatory or lymph system, such as into a body cavity, into a lumen of an organ, or directly into a tissue.
  • the dosing schedule may vary from once a week to daily depending on a number of clinical factors. It may be desirable to administer doses such as those described above by subcutaneous administration. Continuous infusion may also be appropriate.
  • purified therapeutically active molecules are generally combined with a pharmaceutically acceptable carrier.
  • Pharmaceutical preparations may contain only one type of therapeutic molecule, or may be composed of a combination of several types of therapeutic molecules.
  • the nature of the carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Pill-based forms of pharmaceutical proteins may also be administered subcutaneously, particularly if formulated in a slow-release composition.
  • Slow- release formulations may be produced by combining the target protein with a biocompatible matrix, such as cholesterol.
  • Another possible method of administering pharmaceuticals is through the use of mini osmotic pumps.
  • a biocompatible carrier would also be used in conjunction with this method of delivery.
  • the pharmaceutical compositions may be administered by any means that achieve their intended purpose. Amounts and regimens for the administration of the therapeutic molecules can be determined readily by those with ordinary skill in the clinical art of treating diseases. Typical amounts initially administered would be those amounts adequate to achieve tissue concentrations at the site of action which have been found to achieve the desired effect in an animal model.
  • a series of DNA plasmids using the adenovirus 5 E4orf4 gene were constructed to induce apoptosis.
  • a bicistronic vector was chosen containing a single CMV promoter driving the expression of two genes in separate multiple cloning sites linked by an internal ribosomal entry site (pIRES, see Fig. 3).
  • This vector expresses each of the two nucleic acid sequences with different efficiencies, hi each instance the nucleic acid sequence encoding the antigen of interest was cloned into the 'A' multiple cloning site (MCS) of the pIRES vector.
  • the nucleic acid encoding the apoptotic gene, E4orf 4 was cloned into the "B" site (under the control of the IRES) of the vector. Plasmids were produced that included the E4orf 4 in the forward (E4) direction or the in reverse (REV) orientation, which served as a control.
  • E4orf 4 the forward
  • REV in reverse
  • HM polyepitope sequence
  • SIN-nef humanized HIV-env
  • CIN-nef chicken ovalbumin
  • pIRES vector includes genes encoding both ampicillin resistance and neomycin resistance, which aid in selection.
  • mice 4-6 week old females were purchased from Jackson laboratories and maintained in appropriate animal care facilities. Mice were immunized (primed) with lOO ⁇ g of plasmid DNA encoding the priming antigen by subcutaneous (SC) injection. Ten days post DNA injection animals were challenge by intraperitoneal injection (IP) with recombinant vaccinia virus expressing antigen either homologous or heterologous to the priming antigen. Seven days post challenge animals were sacrificed. Splenocytes were isolated from the animals, enumerated, and prepared for CTL or FACS analysis. CTL Cultures and Assays:
  • spleens were remove by aseptic dissection and placed into sterile HBSS containing 1%> fetal bovine serum (FBS), penicillin, streptomycin and glutamine (PSG).
  • FBS fetal bovine serum
  • PSG penicillin, streptomycin and glutamine
  • Cell suspensions made by passing spleens through a mesh screen (Falcon) were placed into culture medium (RPMI-1640 supplemented with 10 % FBS, PSG and 5 x 10 "5 M 2-mecaptoethanol) at a density of 4 x 10 6 cells/ml.
  • the splenocyte cultures were stimulated for 5 days in the presence of the ONA peptide (SII ⁇ FEKL) [l ⁇ g/ml] and supernatant from the X63-6353 cell line as a source of IL- 2.
  • SII ⁇ FEKL ONA peptide
  • Effectors were harvested and enumerated after 5 days of peptide stimulation and used as effectors in a 51 Cr-release assay. Effectors were diluted sequentially threefold in U-bottom wells (96-wel plate Costar) to yield effector to target (E:T) ratios of 90:1, 30:1 and 10:1. Target cells were RMAS cells labeled for 1 hour with 51 Cr [100 ⁇ Ci/lxlO 6 cells]. These cells were divided into two groups, one group was pulsed with peptide [lug/ml] and one group was not pulsed with peptide. Ten thousand targets per well were added to the effectors.
  • Splenocytes were examined after viral challenge by FACS to determine specific cell populations within the spleen by direct staining with antibody (anti-CD4, CD8, B220 CD1 lc, ⁇ K1.1 and CD25) and to enumerate antigen-specific CD4 and CD8 cells by intracellular cytokine staining anti-interferon (IF ⁇ ) gamma using Cytofix/CytoPermTM with Golgi-plug as per manufacturer's instructions (Pharmigen). Briefly, cells were cultured as above for 24 hrs in the presence of peptide or antigen and the pulsed with Golgi-plug for and additional 5 hrs. The cells were then removed from culture and surface stained for CD4 or 8 with a CYC conjugate. Afterwards the cells were permeablized and stained for intracellular IFN gamma with a FITC conjugate.
  • IF ⁇ interferon
  • Ovaries were homogenized and serially diluted before plating out onto monolayers of Nero cells. When plaques were visible, the monolayers were stained with 1% crystal violet and plaques enumerated.
  • EL4 cells were transfected for 48 hours with 1.0 ⁇ g of plasmid D ⁇ A either pOVA. E4FWD, pOVAE4REV or Control (no plasmid) using Fugene ⁇ Transfection reagent (Roche Labs). Prior to staining cultured cell samples are washed twice with cold PBS and then stained with IX Binding Buffer (0.01 M HEPES, pH 7.4; 0.14 M ⁇ aCl; 2.5 mM CaCl 2 ) containing fluorescein-Annexin-V for 15 minutes. The samples are then washed once with IX Binding Buffer.
  • IX Binding Buffer 0.01 M HEPES, pH 7.4; 0.14 M ⁇ aCl; 2.5 mM CaCl 2
  • the mCK-5b Mouse Cytokine Multi-Probe Template Set contains D ⁇ A templates which can be used for the T7 R ⁇ A polymerase-directed synthesis of high- specific-activity, [ ⁇ 32 P] -labeled, anti-sense R ⁇ A probes that can hybridize with target mouse mR ⁇ As encoding Ltn (Kelner et al, Science, 266:1395-1399, 1994), RA ⁇ TES (Schall et al, Eur. J. Immunol, 22:1477-1481, 1992), Eotaxin (Ganzalo et al., Immunity, 4:1-14, 1996), MlP-l ⁇ (Sherry et al., J. Exp.
  • MlP-l ⁇ (Davatelis et al., J. Exp. Med., 167: 1939-1944, 1988), MfP-2 (Tekamp-Olson et al., J. Exp. Med, 172:911-919, 1990), MCP-1 (Rollins et al., PNAS USA, 85:3738-3742, 1988), and TCA-3 (Brown et al., J Immunol, 142:679- 687, 1989) as well as two housekeeping gene products, L32 and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (Table 1).
  • GPDH glyceraldehyde-3- phosphate dehydrogenase
  • the mCK-5b Multi-Probe Template Set is supplied in units suitable for ten probe syntheses. Each probe synthesis generates sufficient probe to analyze >20 RNA samples using PharMingen's RiboQuantTM RPA Starter Package (Cat. #45024K).
  • the probe set is hybridized in excess to target RNA isolated from the spleens, or RNA isolated from lymphocytes, in solution. Free probe and other single-stranded RNA molecules are digested with RNases. The remaining "RNase-protected" probes are purified, resolved on denaturing polyacrylamide gels according to their size, and imaged by autoradiography.
  • RNA samples are tested on a "per input cell” basis, signals from the housekeeping probes are used to monitor the degree of cell activation.
  • the multi-probe ribonuclease protection assay is a highly sensitive and specific method which can be used for the detection, quantitation and characterization of multiple RNA molecules simultaneously (Gilman et al., in Current Protocols in Molecular Biology, j. Wiley and Sons, New York, pp. 4.7.1- 4.7.8, 1988; Melton et al., Nucleic Acids Res., 12:7035-7056, 1984; Naylor et al., in Cytokines. A Practical Approach, F.R. Balkwill ed. Oxford University Press, Oxford, pp. 35-36, 1995).
  • the RPA method has been successfully applied to examining cytokine gene expression repertoires in a variety of experimental model systems for T lymphocyte differentiation (Hobbs et al., J. Immunol, 150:1262-1268, 1996; Ernst et al., Nwtr. Rev., 53:S18-25, discussion S25-16, 1995), autoimmunity (Chu et al., J. Immunol, 156:1262-1268, 1996), immunological tolerance (Phillips et al., J Exp. Med., 183:1339-1344, 1996) and anti-viral immunity (Chu et al, J.
  • RNA samples prepared from tissue samples Chou et al., J. Immunol, 156:1262-1268, 1996; Phillips et al., J Exp. Med., 183:1339-1344, 1996; Lewandowski et al., J Neuroimmunol, 55:23-34, 1994; Pharmingen, RiboQuantTMMulti-Probe Rnase Protection Assay System (Instruction Manual), 1998).
  • RNA samples prepared from tissue samples Chou et al., J. Immunol, 156:1262-1268, 1996; Phillips et al., J. Exp. Med., 183:1339-1344, 1996; Lewandowski et al., J.
  • a PCR based approach is used to assess cytokine expression. Specifically, total R ⁇ A is extracted from spleens or lymphocytes isolated from the mice. Isolated tissues or cells are used immediately or are frozen in liquid nitrogen and stored at -80°C until use. R ⁇ A extractions from spleen and lymph nodes is performed using TRIzol reagent (Life Technologies, Gaithersburg, MD) or the guanidinium isothiocyanate method (Ernst et al., Nutr. Rev., 53:S18-25, 1995).
  • Target RNA (1-5 ⁇ g) is reverse transcribed using 100U Superscriptll RT (Life Technologies, Gaithersburg, MD) at 42°C for 80 min in the presence of 50 mM Tris-HCl pH 8.3, 75 mM KC1, 3 mM MgCl 2 , 5 mM DTT, 0.5 mM dNTPs, 8 U RNasin (Promega Corp., Madison, WI) and 5 ⁇ M Oligo(dT) 16 (Perkin- Elmer/Applied Biosystems, Foster City, CA). For every reaction set, one RNA sample is performed without Superscriptll RT (RT " reaction) to provide a negative control in subsequent PCR reactions.
  • RT Superscriptll RT
  • PCR primers and fluorogenic probes for all target genes are designed using the computer program Primer Express and were purchased from Perkin-Elmer.
  • the fluorogenic probes contained a reporter dye (FAM) covalently attached at the 5 'end and a quencher dye (TAMRA) covalently attached at the 3 'end. Extension from the 3'end is blocked by attachment of a 3'phophate group. Fluorogenic probes are HPLC purified (see Article No. cyto.1998.0426, available online at the Idealibrary website for sequences of the primers useful for IL-1.
  • External controls are constructed consisting of plasmid standards for each target of interest, as well as for ⁇ -actin and GAPDH.
  • Total RNA is extracted from spleen, or from isolated lymphocytes, and cytokine cDNA fragments are generated by RT-PCR using the same primers as described above.
  • each of these amplicons is purified on silica columns (QIAquick PCR purifi-cation; Qiagen, Chatsworth, CA) and cloned into pGEM-Teasy (Promega Corp, Madison, WI). Ligated fragments are transformed into DH5a competent cells (Life Technologies, Gaithersburg, MD) and plasmid DNA is prepared using silica cartridges (Nucleobond AX plasmid purification; Macherey- Nagel, D ⁇ ren, Germany).
  • cDNA plasmid concentrations are measured by optical density spectrophotometry (Pharmacia, Uppsala, Sweden) and the corresponding copy number is calculated using the following equation:
  • Serial dilutions from the resulting clones are used as standard curves, each containing a known amount of input copy number.
  • PCR reactions are performed in the any appropriate system, such as the ABI- prism 7700 sequence detector, which contains a Gene-Amp PCR system 9600 (Perkin Elmer/ Applied Biosystems, Foster City, CA). Reaction conditions are programmed on a Power Macintosh 7200, linked directly to the Sequence Detector.
  • ABI- prism 7700 sequence detector which contains a Gene-Amp PCR system 9600 (Perkin Elmer/ Applied Biosystems, Foster City, CA). Reaction conditions are programmed on a Power Macintosh 7200, linked directly to the Sequence Detector.
  • PCR amplifications are performed in a total volume of 25 ⁇ l, containing 0.5 ⁇ l cDNA sample, 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 10 mM EDTA, 60 nM Passive Reference 1, 200 ⁇ M dATP, dCTP, dGTP and 400 ⁇ M dUTP, 3 to 9 mM MgC 12 , 100 to 200 nM of each primer, 0.625 U AmpliTaqGold and 0.25 U
  • Each PCR amplification is performed in triplicate wells, using the following conditions: 2 min at 50°C and 10 min at 94°C, followed by a total of 40 or 45 two- temperature cycles (15 s at 94°C and 1-min at 60°C) (Overbergh et al., Article No. cyto.1998.0426, available online at the Idealibrary website).
  • Example 2 Priming with a DNA vaccine encoding the antigen ovalbumin and the apoptosis-inducing gene E4orf4 reduced the subsequent inflammatory response to vaccinia virus encoding ovalbumin.
  • E4orf4 is able to induce apoptosis by both the caspase dependent and caspase independent pathways, and has been shown to be a potent inducer of apoptosis (Fig. 2).
  • SIN simian immunodeficiency virus
  • MCS multiple cloning site
  • AdE4orf4 was cloned into MCS B. (Fig. 3). This plasmid is denoted "p ⁇ EF.E4FWD".
  • a control plasmid was constructed in which SIV nef was inserted into the MCS A site and the E4orf4 gene was inserted into the MCS B site in the reverse orientation ("pNEF.E4REV"). With pNEF.E4FWD, both antigen expression and apoptosis occur, whereas with pNEF.E4REV, antigen is expressed but the E4orf4 gene is not expressed and no apoptosis occurs. A series of experiments was conducted in which these plasmids were injected subcutaneously to prime an immune response, and a recombinant vaccinia virus encoding SIV-NEF was used in an attempt to "boost" an antigen-specific CTL response.
  • Fig. 4 One experiment is shown in Fig. 4. Two groups of mice were primed with plasmid DNA constructs capable of expressing the SIN nef gene as the antigen (SrVnef). One group received plasmid encoding nef and E4orf4 in the forward orientation (p ⁇ EF.E4FWD) which allows expression of E4orf4. The other group (control) received plasmid encoding nef and E4orf in the reverse orientation (pNEF.E4REV), which does not allow the expression of E4orf4.
  • each group of animals was split into two groups and challenged with either a vaccinia virus expressing either the homologous antigen (W-NEF) or a heterologous antigen (a humanized form of HIN-1 gpl20, W-E ⁇ V).
  • W-NEF homologous antigen
  • heterologous antigen a humanized form of HIN-1 gpl20, W-E ⁇ V.
  • the lymphocyte number was then analyzed in spleens isolated from each group of animals (Fig. 4A).
  • the bar graph shows the results from treatment groups primed with either p ⁇ EF.E4 (the plasmid encoding E4 in the forward orientation), or pNEF.E4 REV (the plasmid including E4 in the reverse orientation) and subsequently challenged with a vaccinia virus encoding nef (W-NEF), or a vaccinia virus expressing HIN-1 gpl20 E ⁇ N (VV-E ⁇ V), as indicated on the x-axis of the figure.
  • W-NEF vaccinia virus encoding nef
  • VV-E ⁇ V vaccinia virus expressing HIN-1 gpl20 E ⁇ N
  • This suppression is regarded as bystander because the cells that were suppressed were responding to vaccinia, and were not responding to any antigen present in the initial DNA immunization, hi other words, the immunoregulatory cells did not specifically recognize vaccinia. They were able to suppress the response to vaccinia because the antigen that they were specific for, SIV-nef, was present in the same tissue as the vaccinia. Thus the inflammatory response to vaccinia was down-regulated in a bystander fashion as a consequence of the actions of the antigen-specific regulatory T cells.
  • pIRES constructs expressing the antigen chicken ovalbumin (OVA) in the MCS A site were constructed with E4ORF4 in FWD and REV orientations in the MCS B site.
  • OVA constructs to induce apoptosis was tested.
  • EL4 cells were transfected with the pOVA-E4FWD and pOVA-E4REV, and apoptosis detected by annexin- V staining and FACS analysis.
  • Fig. 5 The results are shown in Fig. 5. Apoptotic cells were annexin-V high and propidium iodide low. In cells transfected with ⁇ OVA-E4FWD, 3.05% of cells were apoptotic as compared to 0.28% of untransfected cells and 0.65% of cells transfected with pOVA-E4-REV. Thus the E4orf4 gene encoded in the pIRES plasmid causes apoptosis when transfected into cells.
  • pOVA-E4FWD The ability of pOVA-E4FWD to suppress inflammation in response to vaccinia expressing ovalbumin was tested.
  • One experiment is shown in Fig. 6.
  • Animals were inj ected with pOVA-E4FWD or pOV A-E4REV, and challenged with Vaccinia ovalbumin.
  • control (unimmunized) animals and animals immunized with pOVA-E4REV developed large spleens as a result of vaccinia infection.
  • animals that had been injected with pOVA-E4FWD did not develop splenomegaly in response to vaccinia expressing ovalbumin.
  • FIG. 7 A second experiment showing that pOVA-E4FWD suppressed the inflammatory response to VV-ova is shown in Fig. 7.
  • animals were also primed with plasmid constructs expressing an irrelevant antigen, HIV ENV. Mice were divided into four groups and inoculated subcutaneously with different plasmids.
  • one group was treated with a plasmid including a nucleic acid sequence encoding the OVA antigen and a nucleic acid sequence encoding E4orf4 in the forward orientation (pOVA.E4)
  • one group was treated with a plasmid including a nucleic acid sequence encoding the OVA antigen and a nucleic acid sequence encoding E4orf4 in the reverse orientation (pOVA.E4REV)
  • one group was treated with a plasmid including a nucleic acid sequence encoding a heterologous antigen (ENV) and E4orf4 in the forward orientation (pENV.E4FWD)
  • one group was treated with PBS alone.
  • mice Each group of mice was then challenged with a vaccinia virus encoding the OVA antigen.
  • Mice treated with pOVA.E4 had a reduced spleen size, and reduced numbers of lymphocytes in the spleen (Fig. 6B). Again, virus titers were measured in the ovaries and were not significantly different between groups (Fig. 7B).
  • CTL activity in the OVA.E4FWD mice was not increased as compared to the mice treated with pOVAE4REV or mock treated animals. Indeed, it is possible that there was a small reduction in CTL activity of the pOVAE4FWD primed animals as compared to the pOVAE4REV primed animals.
  • the number of CD8 positive and CD4 positive T cells specific for the immunizing antigen (ovalbuminn) was also examined by fluorescence activated cell sorting (FACS) analysis (Fig. 8 A and Fig. 8B).
  • FACS fluorescence activated cell sorting
  • the number of CD8 positive cells responding to SIINFEKL peptide as measured by FACS intracellular cytokine staining (ICS) was not different for the mice receiving the different plasmid DNA priming constructs.
  • the number of CD4 positive cells responding to whole ovalbumin as measured by FACS ICS was not different for the mice receiving the different plasmid DNA priming constructs.
  • Neutralizing antibody titers in animals primed with construct pairs pIRESgp-120.E4FWD or pIRESgp-120.E4REV were identical to normal mouse sera.
  • mice are immunized with pONA.E4FWD and pONA.E4REN, or primed with phosphate buffered saline (PBS). Half the animals are analyzed after D ⁇ A immunization. Half the animals are subsequently challenged with vaccinia virus expressing ONA, and analyzed 5 days later. Lymphocytes are isolated from spleen, lymph nodes and blood and stained with antibodies against CD4, CD8, CD25, CDl lc, B220, CTLA4 and ⁇ K1.1. The proportions of T cells expressing various markers is analyzed by FACS. It is believed that there is an increase of cells of the regulatory phenotype, co-expressing CD4, CD25 and CTLA4.
  • rmmunoregulatory cells are further detected by identifying antigen-specific cells using transgenic mice, ICS or MHC tetramer staining.
  • the cell surface phenotype of ONA-specific T cells is studied using the antibodies described above. Intracellular cytokine staining is used to detect cells that respond to antigen by making IL-10, TGF-beta, or other immunosuppressive cytokines.
  • ONA-specific CD4 cells expressing CD25 and CTLA4 are increased in response to immunization withpONA-E4FWD.
  • Immunoregulatory cells that make IL-10 or TGF-beta in response to ova antigen are increased in animals immunized with pONA-E4FWD.
  • T reg T regulatory cells
  • E4 orf4 potent apoptotic promoting agent
  • T reg T regulatory cells
  • IL-10 potent antigen-specific anti-inflammatory cells
  • the induction of antigen-specific anti-inflammatory cells can be useful in controlling the inflammatory responses associated with autoimmune disorders and transplantation. Specifically, these method are of use in studying disorders such as encephalitis, rheumatoid arthritis, insulin dependent diabetes mellitus, and islet cell transplantation.
  • cytokines in cell types within the spleens of the animals primed with pONA.E4FWD and pONA.E4REN, or primed with phosphate buffered saline (PBS), and subsequently challenged with vaccinia virus expressing ONA is assessed, h one embodiment, an mCK-5b multiprobe template system is utilized to analyze the expression of cytokines using mR ⁇ A isolated from the spleens.
  • the inflammatory agent was vaccinia virus that encoded the immunizing antigen in addition to its own (over 100) vaccinia genes.
  • the inflammatory agent is an autoantigen.
  • the immunizing antigen is either the disease epitope (autoantigen) or a normal host protein that is expressed in the tissue that is affected by the autoimmune disease (tissue-specific antigen).
  • tissue-specific antigen a normal host protein that is expressed in the tissue that is affected by the autoimmune disease.
  • the autoimmune cells or the inflammation caused by autoimmunity is suppressed in a bystander fashion by immunoregulatory cells that recognize the immunizing antigen.
  • Bystander suppression is the effect produced by regulatory cells, in most cases T cells, responding to a tissue-specific antigen that is expressed in proximity to autoantigens (see Fig. 9).
  • the regulatory cells then produce a microenvironment, most likely through the production of cytokines (including, but not limited to, TGF- beta, IL-10 or IL-13) which suppresses the response of the autoimmune cells, or suppresses the inflammation induced by the autoimmune cells.
  • cytokines including, but not limited to, TGF- beta, IL-10 or IL-13
  • the ability to induce bystander T regulatory cells by vaccination has promising potential for an immune- based autoimmune therapy, as the difficult task of determining disease specific autoantigens is no longer necessary.
  • Vaccine strategies designed to induce these antigen-specific regulatory cells only need to express antigens specific to the tissue undergoing autoimmune attack.
  • vaccination is, for example, directed to myelin basic protein
  • diabetes vaccination is, for example, directed to insulin
  • rheumatoid arthritis vaccination is, for example, directed to Type II collagen.
  • the non-obese diabetic (NOD) mouse model is an animal model system wherein animals develop diabetes with increasing age.
  • NOD non-obese diabetic
  • groups of animals at the prediabetic stage (4 weeks or younger) are vaccinated with a nucleic acid encoding insulin and a pro-apoptotic agent.
  • a plasmid is produced encoding insulin and E4orf4, or insulin and caspase-3. The number of animals developing diabetes, and the rate that the animals develop diabetes, is then analyzed.
  • the E4FWD containing vector delays the progression of the disease, or provides protection from developing the disease, when compared to animals primed with a nucleic acid encoding insulin and E4REV or as compared to untreated controls.
  • this protective effect is dependent on the route of administration.
  • subcutaneous administration of the priming nucleic acid results in delayed onset of disease (e.g. diabetes) or in protection from the development of disease.
  • the immune cell type that provides this protection is then studied by adoptive transfer studies to untreated mice (e.g in NOD mice the transplantation of specific populations of immune cells, such as CD4, CD8, NK or B cells, into NOD animals not exposed to the vector encoding the antigen and the pro-apototic agent).
  • untreated mice e.g in NOD mice the transplantation of specific populations of immune cells, such as CD4, CD8, NK or B cells, into NOD animals not exposed to the vector encoding the antigen and the pro-apototic agent.
  • a pair of plasmid DNA vectors is constructed to express the insulin beta chain (H3C) and the pro- apoptotic agent, such as E4orf4.
  • H3C insulin beta chain
  • E4orf4 the pro- apoptotic agent
  • two plasmids, pIRES IBC.E4FWD and pIRES H3C.E4REV are constructed, and sufficient plasmid DNA is produced and purified to animals for in a treatment group.
  • groups of mice receive either the pIRES IBC.E4FWD, pIRES IBC.E4REV or PBS by subcutaneous or intramuscular injection prior to 4 weeks of age. These mice are monitored for signs of disease onset, such as by monitoring glucose level using standard procedures.
  • mice are given either the pIRES BC.E4FWD, pIRES IBC.E4REV or PBS by subcutaneous or intramuscular injection. Splenocytes are harvested at 10 days post injection. CD4+, CD8+, B220 and NK1.1+ cells are isolated by immunomagnetic bead separation. These different cell types are then transferred to na ⁇ ve NOD mice by IV injection. These animals receiving the transferred cells are then observed form signs of disease onset.
  • Animals receiving pIRES IBC.E4FWD by subcutaneous injection exhibit a delayed onset or no disease progression compared to pIRES IBC.E4REV or PBS.
  • na ⁇ ve mice receiving CD4+ cells from the subcutaneously injected pIRES IBC.E4fwd animals are protected from the onset of disease as the CD4 cells have regulatory activity.
  • rat myelin basic protein is used as the antigenic epitope instead of insulin.
  • Rats are injected with the DNA constructs and monitored for signs or symptoms of the disease process. Specifically, the onset of paralysis and the degree of paralysis is monitored using a scoring system.

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Abstract

L'invention concerne une méthode qui induit, chez un sujet, une réponse immunitaire contre un site antigénique. La méthode consiste à administrer une quantité thérapeutiquement effective d'une séquence d'acide nucléique comprenant un promoteur lié de manière fonctionnelle à une séquence d'acide nucléique codant un site antigénique et à une séquence d'acide nucléique codant un agent facteur de mort cellulaire. L'administration des séquences d'acide nucléique codant un site antigénique et un agent facteur de mort cellulaire induit une réponse immunitaire lorsque, chez un sujet, le site antigénique est mis en présence d'un agent facteur d'inflammation.
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