MX2008008928A - Vaccines and immunotherapeutics using codon optimized il-15 and methods for using the same - Google Patents

Abstract

Nucleic acid molecules that encode IL-15 or fragments thereof, which express protein at a higher level than nucleic acid molecules with native coding sequences for IL-15 are disclosed. Nucleic acid molecules with additional modifications such as the absence of coding sequences for IL-15 signal sequences and/or the absence of IL-15 untranslated sequences and/or inclusion of non-IL-15 signal sequences are also disclosed. Vectors, including plasmids and viral vectors, comprising such nucleic acid molecules;and to host cells comprising such nucleic acid molecules are disclosed as well as methods of using such nucleic acid molecules alone or in combination with nucleic acid sequences encoding immunogens which are part of the nucleic acid molecules and/or part of a different nucleic acid molecule. Recombinant vaccines and live attenuated pathogens encoding fusion proteins, and methods of using the same, are disclosed.

Description

VACCINES AND IMMUNOTHERAPY THAT USE OPTIMIZED IL-15 OF CODONS AND METHODS TO USE THE SAME FIELD OF THE INVENTION The present invention relates to nucleic acid molecules that include a codon-optimized nucleic acid sequence encoding IL-15 and fragments thereof, improved vaccines, improved methods for prophylactically and / or therapeutically immunizing individuals against antigens, and to improve immunotherapeutic compositions and improve immunotherapy methods.

BACKGROUND OF THE INVENTION Immunotherapy refers to modulating the immune responses of the person to impart a desirable therapeutic effect. Immunotherapics refer to those compositions which, when administered to an individual, modulate the individual's immune system sufficiently to ultimately reduce the symptoms that are associated with undesirable immune responses or ultimately alleviate symptoms by increasing the desirable immune responses. In some cases, immunotherapy is part of a vaccination protocol in which the individual is administered a vaccine that exposes the individual to an antigen against which the individual generates an immune response. In such a case, the Ref .: 194765 immunotherapeutic increases the immune response and / or selectively increases a portion of the immune response (such as the cell arm or humoral arm) that is desirable to treat or prevent the particular condition, infection or disease. . The vaccines are useful for immunizing individuals against target antigens such as allergens, pathogenic antigens or antigens associated with cells involved in human diseases. Antigens associated with cells involved in human diseases include tumor antigens associated with cancer and antigens associated with cells involved in autoimmune diseases. In designing such vaccines, it has been recognized that vaccines < Those that produce the target antigen in cells of the vaccinated individual are effective in inducing the cellular arm of the immune system. Specifically, live-relieved vaccines, recombinant vaccines that use non-virulent vectors, and DNA vaccines each lead to the production of antigens in the cell of the vaccinated individual that results in the induction of the cellular arm of the immune system. On the other hand, the elimination or inactivated vaccines, and subunit vaccines comprising only proteins do not induce good cellular immune responses even though they induce a humoral response. A cellular immune response is often necessary to provide protection against pathogen infection and to provide effective immune mediated therapy for the treatment of pro pathogenic infection, cancer or autoimmune diseases. Accordingly, vaccines that produce the target antigen in cells of the vaccine subject such as live attenuated vaccines, recombinant vaccines using non-virulent vectors and DNA vaccines are often preferred. Although such vaccines are often effective to immunize individuals prophylactically or therapeutically against pro pathogen infection or human diseases, there is a need for improved vaccines. There is a need for compositions and methods that produce an increased immune response. Similarly, although some immunotherapies are useful for modulating immune responses in a patient that maintains the need to improve immunotherapeutic compositions and methods.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to nucleic acid molecules comprising nucleic acid sequences encoding IL-15 protein comprising SEQ ID NO: a fragment thereof encoding a functional fragment of the present invention. refers to nucleic acid molecules comprising SEQ ID NO: 1 or a fragment thereof encoding a functional fragment of IL-15 that are free of coding sequence for an IL-15 signal sequence and / or free region Kozak IL-15 and / or untranslated region 5 'IL-15 and / or 3' untranslated region IL-15 and / or comprises a coding sequence for a non-IL-15 signal sequence. The present invention relates to nucleic acid molecules comprising nucleic acid sequence encoding the IL-15 protein comprising SEQ ID NO: 1 or a fragment thereof encoding a functional fragment of IL-15 and further comprising sequences of coding for an antigen. The present invention relates to compositions comprising a nucleic acid molecule comprising a nucleic acid sequence comprising SEQ ID NO: 1 or a fragment thereof encoding a functional fragment of IL-15 and a nucleic acid molecule which comprises a nucleic acid sequence encoding an antigen. The present invention further relates to methods for modulating an immune response in an individual comprising administering to the individual a nucleic acid molecule comprising SEQ ID NO: 1 or a fragment thereof encoding a functional fragment of IL-15.

The present invention further relates to recombinant vaccines comprising a nucleic acid molecule comprising SEQ ID NO: 1 or a fragment thereof encoding a functional fragment of IL-15. The present invention relates to methods for inducing an immune response in an individual against an antigen comprising administering to the individual a nucleic acid molecule comprising SEQ ID NO: 1 or a fragment thereof encoding a functional fragment of IL- 15 as part of or in combination with a nucleic acid molecule encoding an antigen or in combination with an antigen.

BRIEF DESCRIPTION OF THE FIGURE Figure 1 shows SEQ ID N0: 1.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "target protein" is a means for referring to peptides and proteins encoded by gene constructs of the present invention that act as target proteins for an immune response. The terms "target protein" and "antigen" are used interchangeably and refer to a protein against which an immune response may arise. The target protein is an antigenic protein that carries at least one epitope with an undesirable pathogen or cell type protein such as a cancer cell or a cell involved in autoimmune disease against which an immune response is desired. The immune response directed against the target protein will protect the individual against and / or treat the individual for the infection or disease with which the target protein is associated. As used herein, the term "genetic construct" refers to DNA or RNA molecules comprising a nucleotide sequence that encodes a target protein or immunomodulated protein. The coding sequence includes start and end signaling operably linked to regulatory elements including a promoter and an adenylation signal capable of directing expression in the cells of the individuals to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to gene constructs that contain the necessary regulatory elements operably linked to a coding sequence that encodes a target protein or an immunomodulated protein, such that when presented in the individual's cell, the coding sequence will be expressed. As used herein, the term "carrying an epitope" refers to proteins that comprise at least one epitope that is identical to or substantially similar to an epitope of another protein. As used herein, the term "substantially similar epitope" is a means to refer to an epitope having a structure that is not identical to an epitope of a protein but nonetheless invokes a cellular or humoral immune response that reacts with such an epitope. protein. As used herein, the term "intracellular pathogen" is a means to refer to a virus or pathogenic organism that, at least part of its reproductive or life cycle, exists within a host cell and in this produces or causes occur, pathogenic proteins. As used herein, the term "hyperproliferative diseases" is a means of referring to those diseases and disorders characterized by cell hyperproliferation. As used herein, the term "hyperproliterative associated protein" is a means to refer to proteins that are associated with a hyperproliferative disease. As used herein, the term "immunomodulated protein" refers to a protein that modulates the immune system of a person to which the immunomodulatory protein is administered. Examples of immunomodulatory proteins include: IL-15, CD40L, TRAIL; TRAILrecDRC5, TRAIL-R2, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, Ox40 LIGAND, NKG2D, F4 181 MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, CD30, CD 153 (CD30L), Fos, C-jun, Sp-I, Apl5 Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, N1K, SAP K, SAPl, JNK2, JNK1B2, JNKlBl, JNK2B2, JNK2B1, JNK1A2, JNK2A1, JNK3A1 , JNK3A2, NF-kappa-B2, splice form p49, NF-kappa-B2, splice form plOO, NF-kappa-B2, splice form pl05, precursor of chain NF-kappa-B 50K, NFkB p50, IL -I human, human IL-2, human IL-4, murine IL-4, human IL-5, human IL-10, human IL-15, human IL-18, human TNF-β, human TNF-β, interleukin 12 human, MadCAM-1, NGF IL-7, VEGF, TNF-R, Fas, CD40L, IL-4, CSF, G-CSF, GM-CSF, -CSF, LFA-3, ICAM-3, ICAM-2, ICAM-I, PECAM, P150.95, Mac-1, LFA-I, CD34, RANTES, IL-8, MIP-la, E-selecton, CD2, MCP-I, L-selecton, P-selecton, FLT, Apo-1, Fas, TNFR-I5 p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4 (TRAIL), DR5, KILLER, TRAIL-R2, TRICK2, DR-6, ICE, VLA-I, and CD86 (B7.2).

Introduction The invention provides a nucleic acid sequence encoding IL-15 that provides improved protein expression relative to the native sequence. The sequence encoding improved IL-15 can be used in combination with the findings set forth in PCT application PCT / US04 / 189-62 filed June 14, 2004, Provisional Application E.U.A. Number 60 / 478,210 filed on June 13, 2003 and Provisional Application E.U.A. Number 60 / 478,205 filed June 13, 2003, which are incorporated herein by reference, particularly those that provide sequences encoding the IL-15 protein linked to non-IL-15 signal peptide, particularly igE signal peptide, and the use of such constructs in vaccines and in constructor to administer the IL-15 protein as an immunomodulated protein. In some preferred embodiments, the invention provides vectors, vaccines and immunomodulatory compositions and methods comprising nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 1 or fragments thereof encoding functional fragments of IL-15. In some preferred embodiments, such nucleic acid molecules are provided free of coding sequences for IL-15 signal sequence, and more preferably free of the Kozak IL-15 region and untranslated regions. In some preferred embodiments, the invention provides vectors, vaccines and immunomodulatory compositions and methods comprising nucleic acid molecules comprising SEQ ID NO: fragments thereof encoding functional fragments of IL-15 that are linked to coding sequences for sequence. of human IgE signal. The native sequences encoding IL-15 have been modified to improve expression. In previous improvements, elements such as coding sequences for IL-15 sequence and untranslated regions were deleted to improve expression. These previous improvements can be incorporated and used in conjunction with the improved coding sequence of the mature IL-15 protein set forth in SEQ ID NO: 1. In preferred embodiments, the nucleic acid molecule including SEQ ID NO: 1 is free of the coding sequences for IL-15 signal peptide, and preferably another signal protein such as IgE signal protein is provided at this site. On the other hand, the Kozak IL-15 region and untranslated regions are removed as well as removing inhibitory elements. The unique IL-15 sequence of such constructs preferably includes the IL-15 sequence encoding the amino acid sequence of the mature IL-15 protein free of IL-15 signal peptide. According to some embodiments, compositions are provided comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein comprising the non-IL-15 signal sequence linked to the IL-15 protein encoded by the SEQ ID NO: a fragment thereof that encodes a functional fragment of IL-15. In some preferred embodiments, the molecule is free of coding sequences for IL-15 signal sequence. In some preferred embodiments, the fusion protein is non-immunogenic in a human. According to some embodiments, compositions are provided that include the nucleic acid construct comprising SEQ ID NO: 1 or a fragment thereof encoding a functional fragment of IL-15 and optionally the other previous enhancements described above may also be included in the same nucleic acid molecule or a different nucleic acid molecule, a nucleic acid sequence encoding an antigen. Generally, the antigens, which are discussed below, can be any antigenic protein that includes allergens, pathogenic antigens, antigens associated with cancer or antigens linked to cells associated with autoimmune diseases. In preferred embodiments, the antigen is a pathogenic antigen, more preferably a pathogen selected from the group consisting of HIV, HSV, HCV, and WNV. In some preferred embodiments, the nucleic acid constructs are plasmids. In some preferred embodiments, the nucleic acid molecule is incorporated into a viral vector such as a vaccine, adenovirus, adenovirus-associated virus, retrovirus, RSV, VSV, pox virus or any other acceptable viral vector useful as a vaccine or therapy vector of genes.

The genetic constructs comprising SEQ ID NO: 1 or a fragment thereof encoding a functional fragment of IL-15 can be incorporated directly into live attenuated pathogens according to some aspect of the invention. Examples of such pathogens useful as vaccines are set forth below. In preferred embodiments, human IL-15, preferably free of IL-15 signal sequence, is ligated to the human IgE signal sequence. Compositions that include coding sequences for antigens are useful as vaccines. Compositions that do not include coding sequences for antigens may be useful as immunomodulatory compositions. In some embodiments, the protein antigens are also provided as a target for the immune response that will be increased by the expression of IL-15.

In some preferred embodiments, the nucleic acid constructs are plasmids. In some preferred embodiments, the nucleic acid molecule is incorporated into a viral vector such as a vaccine, adenovirus, adenovirus-associated virus, retrovirus, or any other acceptable viral vector useful as a vaccine or gene therapy vector.

Genetic constructs comprising SEQ ID NO: 1 or a fragment thereof encoding a functional fragment of IL-15 can be incorporated directly into live attenuated pathogens according to some aspects of the invention. Examples of such pathogens useful as vaccines are set forth below. In preferred embodiments, human IL-15, preferably free of IL-15 signal sequence, is ligated to the human IgE signal sequence. According to some embodiments of the invention, the compositions of the invention comprise genetic constructs that include coding sequences for antigens and / or immunogenic proteins. Such compositions are administered to an individual to modulate the activity of the individual's immune system and thereby increase the immune response against the antigen. When the nucleic acid molecules encoding an immunomodulatory protein are taken up by individual cells, the nucleotide sequences encoding the immunomodulatory protein are expressed in the cells and the proteins are thereby administered to the individual. Aspects of the invention provide methods for administering the coding sequences of the proteins in a single nucleic acid molecule, in compositions comprising different nucleic acid molecules that encode one or more of the various transcription factors or intermediary factors, as part of recombinant vaccines and as part of attenuated vaccines. According to some aspects of the present invention, compositions and methods are provided which prophylactically and / or therapeutically immunize an individual against a pathogen or abnormal disease-related cells. The vaccine can be any type of vaccine such as a live attenuated vaccine, a cellular vaccine, a recombinant vaccine or a nucleic acid or DNA vaccine. The present invention relates to compositions for administering the immunomodulated proteins and methods for using same. The nucleic acid molecules can be administered using any of several well known technologies including DNA injection (also referred to as DNA vaccination), recombinant vectors such as recombinant adenovirus, recombinant virus associated with adenovirus and recombinant vaccine. DNA vaccines are described in US Pat. Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, 5,676,594, and the priority applications cited herein, which are incorporated herein by reference. In addition to the administration protocols described in those applications, alternative methods of administering DNA are described in U.S. Pat. Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference. Administration routes include, but are not limited to, intramuscularly, intransally, intraperitoneally, intradermally, subcutaneously, intravenously, intraarterially, intraocularly and orally as well as topically, transdermally, by inhalation or suppository or mucosal tissue such as by lavage to vaginal, rectal, urethral tissue , buccal and sublingual. Preferred routes of administration include injection into mucosal, intramuscular, intraperitoneal, intradermal and subcutaneous tissue. Genetic constructs can be administered by means including, but not limited to, traditional syringes, needleless injection devices, or "microprojectile bombardment gene guns". When taken by a cell, the genetic constructs can be present in a cell as an extrachromosomal functional molecule and / or integrated into the chromosomal DNA of the cell. DNA can be introduced into cells where it is kept as a separate genetic material in the form of a plasmid or plasmids. Alternatively, the linear DNA that can be integrated into the chromosome can be introduced into the cell. When DNA is introduced into the cell, reagents that promote the integration of DNA into chromosomes can be added. DNA sequences that are useful for promoting integration can also be included in the DNA molecule. Alternatively, RNA can be administered to the cell. It is also contemplated to provide the genetic construct as a linear minichromosome that includes a centromere, telomeres and an origin of replication. Gene constructs can maintain part of the genetic material in living attenuated microorganisms or recombinant microbial vectors that live in the cells. Gene builders can be part of genomes of recombinant viral vaccines where the genetic material either integrates into the chromosome of the cell or remains extrachromosomal. Genetic constructs include regulatory elements necessary for the expression of genes of a nucleic acid molecule. The elements include: a promoter, a start codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for the expression of genes of the sequence encoding the target protein or the immunomodulated protein. It is necessary that these elements are operably linked to the sequence encoding the desired proteins and that the regulatory elements are operable in the individual to whom they are administered. Start codons and stop codons are generally considered to be part of a nucleotide sequence encoding the desired protein. However, it is necessary that these elements be functional in the individual to whom the gene construct is administered. The initiation and termination codons should be in a structure with the coding sequence.

The promoters and polyadenylation signals used should be functional within the cells of the individual. Examples of promoters useful for practicing the present invention, especially in the production of a human genetic vaccine, include, but are not limited to, simian virus promoters (SV40), mouse mammary tumor virus promoter (MMTV) , for its acronym in English), human immunodeficiency virus (MV) such as the BIV Long Terminal repeat promoter (LTR), Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr virus (EBV), Rous sarcoma virus (RSV) as well as promoters of human genes such as human actin, human myosin, human hemoglobin, human muscle creatine and human metallothionein. Examples of polyadenylation signals useful in practicing the present invention, especially in the production of a human genetic vaccine, include, but are not limited to, SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal that is in the pCEP4 plasmid < Invitrogen, San Diego CA), which is referred to as the SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression, other elements can also be included in the DNA molecule. Such additional elements include enhancers. The enhancer can be selected from the group including but not limited to: human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those of CMV, RSV and EBV. Genetic constructs can be provided with a mammalian origin of replication in order to maintain the extrachromosomal characteristic of the construct and produce multiple copies of the construct in the cell. Plasmids pVAXl, pCEP4 and pREP4 from Invitrogen (San Diego, CA) contain the Epstein Barr virus origin of replication and the EBNA-1 nuclear antigen coding region that produces high copy episomal replication without integration. In some preferred embodiments that relate to immunization applications, the nucleic acid molecules are administered which include nucleotide sequences that encode a target protein, the immunomodulated protein and, additionally, genes for proteins that further increase the immune response against such target proteins. . Examples of such genes are those that encode other cytokines and lymphokines such as alpha-interferon, gamma-interferon, platelet-derived growth factor (PDGF), TNF, GM-CSF, epidermal growth factor (EGF), IL-1. , IL-2, II-4, IL-6, IL-10, IL-12 and IL-15 including IL-15 having the deleted signal sequence and optionally including the IgE signal sequence. An additional element can be added, which serves as a target for cell destruction if it is desired to eliminate cells that receive the genetic construct for any reason. -A gene for herpes thymidine kinase (tk) in a form that can be expressed can be included in the genetic construct. The drug gangciclovir can be administered to the individual and such drug will cause the selective elimination of any tk produced by the cell, thereby providing the means for the selective destruction of cells with the genetic construct. In order to maximize protein production, regulatory sequences can be selected that are well suited for the expression of genes in the cells of the construct to be administered. On the other hand, the codons can be selected that are transcribed more efficiently in the cell. One of ordinary skill in the art can produce DNA constructs that are functional in cells. In some embodiments, gene constructs can be provided in order to produce coding sequences for the immunomodulatory proteins described herein linked to the igE signal peptide.

A method of the present invention comprises the steps of administering nucleic acid molecules intramuscularly, intranasally, intra-operatively, subcutaneously, intradermally, or topically or by washing mucosal tissue selected from the group consisting of inhalation, vaginal, rectal, ureter, buccal and sublingual In some embodiments, the nucleic acid molecule is administered to the cells in conjunction with administration of a polynucleotide enhancer or a genetic vaccine facilitator. Enhancers of polynucleotide function are described in Serial Number E.U.A. 5,593,972, 5,962,428 and International Application Serial Number PCT / US94 / 0899 filed January 26, 1994, each incorporated herein by reference. The genetic vaccine facilitator agents are described in Serial Number E.U.A. 021,579 filed April 1, 1994, which is incorporated herein by reference. The co-agents that are administered in conjunction with nucleic acid molecules can be administered as a mixture with the nucleic acid molecule or administered separately, simultaneously, before or after the administration of nucleic acid molecules. In addition, other agents that can function as transfection agents and / or replication agents and / or inflammatory agents and that can be co-administered with a GVF include growth factors, cytokines and lymphokines such as a-interferon, gamma-interferon, GM-CSF, platelet-derived growth factor (PDGF), TNF, epidermal growth factor (EGF), ILA, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15 as well as fibroblast growth factor, surface active agents such as immune stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analogue including monophosphoryl lipid A (WL), muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid can also be used administered in conjunction with the genetic construct. In some embodiments, an immunomodulated protein can be used as a GVF. In some embodiments, the nucleic acid molecule is provided in association with PLG to increase administration / absorption. The pharmaceutical compositions according to the present invention comprise about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions according to the present invention comprise about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain from about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 100 to about 200 micrograms of DNA. The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. In cases where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, free of pyrogens and free of particles. An isotonic formulation is preferably used. Generally, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. The stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. According to some embodiments of the invention, methods are provided for inducing immune responses against an antigen by administering the compositions of the invention to an individual. The vaccine can be a live attenuated vaccine, a cellular vaccine, a recombinant vaccine or a DNA or nucleic acid vaccine. In addition to using expressible forms of the immunomodulatory protein coding sequence to improve the genetic vaccines, the present invention relates to improved attenuated live vaccines and improved vaccines that use recombinant vectors to deliver external genes encoding antigens. Examples of live attenuated vaccines and those that use recombinant vectors to administer external antigens are described in US Pat. Nos .: 4,722,848; 5,017,487; 5,077,044; 5,110,587; 5,112,749 5,174,993; 5,223,424; 10,225,336; 5,240,703; 5,242,829 5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368 5,424,065; 5,451,499; 5,453,364; 5,462,734; 5,470,734; and 5,482,713, which are incorporated herein by reference. Gene constructs are provided which include the nucleotide sequence encoding an immunomodulated protein operably linked to regulatory sequences that can function in the vaccine to effect expression. The gene constructs are incorporated into live attenuated vaccines and recombinant vaccines to produce improved vaccines according to the invention. The present invention provides an improved method for immunizing individuals comprising the step of administering gene constructs to the cells of individuals as part of vaccine compositions that include DNA vaccines, live attenuated vaccines, and recombinant vaccines. The gene constructs comprise a nucleotide sequence that encodes an immunomodulated protein and which are operably linked to regulatory sequences that can function in the vaccine to effect expression. Improved vaccines result in an increased cellular immune response. The present invention is useful for producing enhanced immune responses against a target protein, that is, proteins specifically associated with pathogens, allergens or the "abnormal" cells of the individual. The present invention is useful for immunizing individuals against pathogenic agents and organisms in such a way that an immune response against a pathogen protein provides protective immunity against the pathogen. The present invention is useful for combating hyperproliferative diseases and disorders such as cancer by producing an immune response against the target protein that specifically associates with the hyperproliterative cells. The present invention is useful for combating diseases and autoimmune disorders by producing an immune response against a target protein that specifically associates with cells involved in the autoimmune condition. According to some aspects of the present invention, the DNA or RNA encoding a target protein and immunomodulatory proteins is introduced into the cells of an individual's tissue where it is expressed, thereby producing the encoded proteins. The DNA or RNA sequences encoding the target protein and one or both immunomodulatory proteins bind to regulatory elements necessary for expression in the cells of individuals. Regulatory elements for DNA expression include a promoter and a polyadenylation signal. In addition, other elements, such as a Kozak region, can also be included in the genetic construct. In some embodiments, the expressible forms of sequences encoding the target protein and expressible forms of sequences encoding both immunomodulatory proteins are found in the same nucleic acid molecule that is administered to the individual. In some embodiments, the expressible forms of sequences encoding the target protein are presented in a nucleic acid molecule separated from nucleic acid molecules that contain expressible forms of sequences encoding one or more immunomodulatory proteins. In some embodiments, the expressible forms of sequences encoding the target protein and expressible forms of sequences encoding one or more of the immunomodulatory proteins are presented in a nucleic acid molecule that is separated from the nucleic acid molecule containing expressible forms of sequences that encode one or more of the immunomodulatory proteins. Multiple different nucleic acid molecules can be produced and administered according to the present invention and administered to the individual. For example, in some embodiments, the expressible forms of sequences encoding the target protein are presented in a nucleic acid molecule separated from the nucleic acid molecules that contain expressible forms of sequences encoding one or more of the two immunomodulatory proteins that are present in the nucleic acid molecule separated from the nucleic acid molecules containing expressible forms of sequences encoding one or more of the immunomodulatory proteins. In such cases, the three molecules are administered to the individual. The nucleic acid molecules can be provided as plasmid DNA, the nucleic acid molecules of recombinant vectors or as part of the genetic material provided in an attenuated vaccine or cell vaccine. Alternatively, in some embodiments, the target protein and / or any or both immunomodulatory proteins can be administered as a protein in addition to the nucleic acid molecules encoding them or instead of the nucleic acid molecules encoding them. The genetic constructs may comprise a nucleotide sequence encoding a target protein or an immunomodulatory protein operably linked to regulatory elements necessary for the expression of genes. According to the invention, combinations of gene constructs are provided which include one such comprising an expressible form of the nucleotide sequence encoding the target protein and one including an expressible form of the nucleotide sequence encoding an immunomodulatory protein. The incorporation into a living cell of the DNA or RNA molecules that include the gene builder combination results in the expression of DNA or RNA and the production of the target protein and one or more immunomodulatory proteins. An increased immune response results against the target protein. The present invention can be used to immunize an individual against all pathogens such as viruses, prokaryotic and pathogenic eukaryotic organisms such as unicellular pathogenic organisms and multicellular parasites. The present invention is particularly useful for immunizing an individual against those pathogens that infect cells and that are not encapsulated as a virus, and prokaryotes such as gonorrhea, listeria and sigela. In addition, the present invention is also useful for immunizing an individual against protozoan pathogens that include a stage in the life cycle where they are intracellular pathogens.

Table 1 provides a listing of some of the viral families and genera for which vaccines according to the present invention can be made. DNA constructs comprising DNA sequences encoding peptides comprising at least one epitope identical or substantially similar to an epitope that is displayed on a pathogen antigen such as those antigens listed in the tables, are useful in vaccines. On the other hand, the present invention is also useful for immunizing an individual against other pathogens including prokaryotic and eukaryotic protozoan pathogens as well as multicellular parasites such as those listed in Table 2.

In order to produce a genetic vaccine to protect against infection by pathogens, the genetic material encoding immunogenic proteins against which a protective immune response can be mounted should be included in a genetic construct as the coding sequence for the target. If the pathogen infects intracellularly, for which the present invention is particularly useful, or extracellularly, it is impossible for all pathogenic antigens to produce a protective response. Because the DNA and RNA are both relatively small and can be produced relatively easily, the present invention provides the additional advantage of allowing vaccination with multiple pathogenic antigens. The genetic construct in the genetic vaccine may include genetic material that encodes many pathogenic antigens. For example, several viral genes may be included in the single construct thereby providing multiple targets. Tables 1 and 2 include lists of some of the pathogenic agents and organisms for which genetic vaccines can be prepared to protect an individual from infection thereof. In some preferred embodiments, methods for immunizing an individual against the pathogen are directed against HIV, HSV, HCV, WNV or HBV. Another aspect of the present invention provides a method that provides a protective immune response against hyperproliferative cells that are characteristic in hyperproliferative diseases and a method of treating individuals suffering from hyperproliferative diseases. Examples of hyperproliferative diseases include all forms of cancer and psoriasis. It has been found that the introduction of a genetic construct that includes a nucleotide sequence which encodes an associated protein - an immunogenic "hyperproliferative cell" - within the cells of an individual results in the production of those proteins in cells vaccinated from a individual. To immunize against hyperproliferative diseases, a genetic construct is administered which includes a nucleotide sequence that encodes a protein that is associated with a hyperproliterative disease to an individual. In order for the protein associated with hyperproliferation to be an effective immunogenic target, it must be a protein that occurs exclusively or at higher levels in hyperproliferative cells compared to normal cells. Target antigens include such proteins, fragments thereof and peptides, which comprise at least one epitope found in such proteins. In some cases, the protein associated with hyperproliferation is the product of a mutation of a gene that encodes a protein. The mutated gene encodes a protein that is almost identical to the normal protein except that it has a slightly different amino acid sequence which results in a different epitope that is not found in the normal protein. Such target proteins include those which are proteins encoded by the oncogenes such as myb, myc, fyn, and the transcription gene bcr / abl, ras, src, P53, neu, trk and EGRF. In addition to the oncogene products as target antigens, the target proteins for anti-cancer treatments and protective regimens include variable regions of antibodies made by B-cell lymphomas and the variable regions of T cell receptors of T-cell lymphomas., which in some modalities, are also used as target antigens for autoimmune disease. Other tumor-associated proteins can be used as target proteins such as proteins that are found at higher levels in tumor cells that include the protein recognized by monoclonal antibody 17-IA and folate binding proteins or PSA. Although the present invention can be used to immunize an individual against one or more other forms of cancer, the present invention is particularly useful for prophylactically immunizing an individual who is predisposed to develop a particular cancer or who has had cancer and for therefore be susceptible to a relapse. Developments in genetics and technology as well as in epidemiology allow the determination of the probability and risk assessment for the development of cancer in an individual. By using genetic selection and / or family health histories, it is possible to predict the likelihood that a particular individual will have to develop one of several types of cancer. Similarly, those individuals who have already developed cancer and who have been treated to remove the cancer or who are otherwise in a new treatment, are particularly susceptible to relapse and new presence. As part of a treatment regimen, such individuals can be immunized against cancer that has been diagnosed in order to combat a recurrence. Thus, once it is known that an individual has had a type of cancer and is at risk of a relapse, he can be immunized in order to prepare his immune system to combat some future appearance of the cancer. The present invention provides a method of treating individuals suffering from hyperproliferative diseases. In such methods, the introduction of genetic constructs serves as an immunotherapeutic, by directing and promoting the individual's immune system to combat hyperproliferative cells that produce the target protein. The present invention provides a method of treating individuals suffering from autoimmune diseases and disorders by conferring a broad-based protective immune response against targets that are associated with autoimmunity including cell receptors and cells which produce "self" -dirigid antibodies. Autoimmune diseases mediated by T cells include rheumatoid arthritis <; RA, for its acronym in English), multiple sclerosis (MS, for its acronym in English), Sjogren's syndrome, sarcoidosis, diabetes mellitas insulin-dependent (IDDM, for its acronym in English), autoimmune thyroiditis, reactive arthritis, spondylitis ankylosing, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease and ulcerative colitis. Each of these diseases are characterized by T cell receptors that bind to endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases. Vaccination against the variable region of the T cells would produce an immune response that includes CTLs to eliminate those cells. In RA, various variable regions specific for T cell receptors (TCRs) that are involved in the disease have been characterized. These TCRs include vß-3, vß-14, 20 Vß-17 and Va-17. Thus, vaccination with a DNA construct that encodes at least one of these proteins will produce an immune response that will target the target cells involved in the RA. See: Howell, M.D., et al, 1991 Proc. Nat. Acad. Sci. USA 88: 10921-10925; Piliard, X., et al, 1991 Science 253: 325-329; Williams, W.V. , et al., 1992 J Clin. Invest. 90: 32-6-333; each of which is incorporated herein by reference. In MS, various specific variable regions of TCRs that are involved in the disease have been characterized. These TCRs include VfP and Va-10. Thus, vaccination with an AND construct that encodes at least one of these proteins will produce an immune response that will attack the T cells involved in MS. See: Wucherpfennig, K. W., et al., 1990 Science 248: 1016-1019; Oksenberg, J.R., et. al, 1990 Nature 345: 344-346; each of which is incorporated herein by reference. In scleroderma, various specific variable regions of TCRs that are involved in the disease have been characterized. These TCRs include Vß-6, Vß-8, Vß-14 and Va-16, Va-3C, Va-7, V -14, V -15, Va-16, Va-28 and Va-12. Thus, vaccination with an AND construct that encodes at least one of these proteins will produce an immune response that will direct the T cells involved in scleroderma. In order to treat patients suffering from T-cell-mediated immune disease, particularly those for whom the variable region of the TCR has yet to be characterized, a synovial biopsy can be performed. Samples of the T cells present can be taken and the variable region of those TCRs identified using standard techniques. Genetic vaccines can be prepared by using this information. Autoimmune diseases mediated by B cells include Lupus (SLE), Grave's disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosis and pernicious anemia. Each of these diseases is characterized by antibodies that bind to endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases. Vaccination against the variable region of antibodies would produce an immune response that includes CTLs to eliminate those B cells that produce the antibody. In order to treat patients suffering from autoimmune disease mediated by B cells, the variable region of the antibodies involved in autoimmune activity must be identified. A biopsy can be performed and samples of the antibodies present at the site of inflammation can be taken. The variable region of these antibodies can be identified by using standard techniques. Genetic vaccines can be prepared by using this information. In the case of SLE, it is believed that an antigen is DNA. Thus, in patients to be immunized against SLE, their serum can be selected for anti-DNA antibodies and a vaccine can be prepared which includes DNA constructs that encode the variable region of such anti-DNA antibodies found in the sera. The common structural aspects between the variable regions of both the TCRs and antibodies are well known. The DNA sequence encoding a particular TCR or antibody can generally be found by following well known methods such as those described in Kabat, et al 1987 Sequence of Proteins of Immunological Interest U.S. Department of Health and Human Services, Bethesda MD, which is incorporated herein by reference. In addition, a general method for cloning functional variable regions from antibodies can be found in Chaudhary, V.K., et al, 1990 Proc. Nati Acad Sci. USA 87: 1066, which is incorporated herein by reference.

Table 1 Picornavirus Family Genres: Rhinovirus: (Physician) responsible for - 50% of cases of the common cold. Eterovirus: (Medical) includes poliovirus, coxsackie virus, ecovirus, and human enteroviruses such as hepatitis A virus. Aftovirus: (Veterinarian) these are foot-and-mouth disease virus. Target antigens: VPl, VP2, VP3, VP4, VPG Family of Calcivirus Genres: Norwalk Virus Group: (Medical) These viruses are an important causative agent of epidemic gastroenteritis.

Togavirus Family Genders: Alfavirus: (Medical and Veterinary) examples include Senile viruses, RossRiver virus and Eastern equine encephalitis and Western virus. Reovirus: (Medical) rubella virus.

Family Flariviridue Examples include: (Medical) dengue fever, yellow fever, Japanese encephalitis, St. Louis encephalitis and encephalitis virus that carries the tick. West Nile virus (Genbank NC001563, AF533540, AF404757, AF404756, AF404755, AF404754, AF404753, AF481864, M12294, AF317203, AF196835, AF260969, AF260968, AF260967, AF206518 and AF202541) Representative target antigens: E NS5 C Hepatitis C virus : (Medical) These viruses are not placed in a family yet but are believed to be either a togavirus or a flavivirus. The greatest similarity is with the togavirus family.

Family of Coronaviruses: (Physician and Veterinarian) Infectious bronchitis virus (poultry) Gastroenteric virus transmitted in swine (pig) Swine haemagglutin encephalitis virus (pig) Infectious peritonitis virus in cats (cats) Coronavirus enteric in felines (cat) Canine coronavirus (dog) Coronavirus associated with SARS Human respiratory coronaviruses cause -40 cases of the common cold. FORMER. 224E, OC43 Note - coronaviruses can cause hepatitis that is not A, B or C Target Antigens: The - also called M or E2 matrix protein - also called S or Spike E3 protein - also called BE or hemagglutin-elterose glycoprotein (not present in all coronaviruses) N-nucleocapsid Family of Rabdoviruses Genus: Vesiliovirus Lyssavirus: (doctor and veterinarian) Rabies Target antigen: Protein G Protein N Family Filoviridue: (Medical) Hemoragic fever virus such as Marburg and Ebola viruses Paramyxovirus family: Genus: Paramyxovirus: (Doctor and Veterinarian) Mumps virus, New Castle disease virus (important pathogen in chickens) Morbillivirus: (Medical and Veterinary) Measles, canine distemper Pneuminvirus: (Medical and Veterinary) Respiratory syncytial virus Orthomyxovirus family (Medical) Influenza virus Family of Bungavirus Genus: Bungavirus: (Medical) California encephalitis, LA Crosse Phlebovirus: (Medical) Fibere Rift Valley Hantavirus: Puremala is a virus of hemamagine fever Nairvirus (Veterinarian) sheep disease of Nairobi Also many bungaviruses do not assigned Arenavirus family (Medical) LCM, Lassi fever virus Reovirus family Genders: Reovirus: a possible human pathogen Rotavirus: acute gastroenteritis in children Orbivirus: (Doctor and Veterinarian) Colorado tick fever, Lebombo (human), equine encephalosis, blue tongue Retroyirus Family Sub-Family: Oncorivirinal: (Veterinarian) (Medical) feline leukemia virus, HTLVI and HTLVII Lentivirinal: (Medical and Veterinary) HIV, feline immunodeficiency virus, equine infections, Spumaviridae anemia virus Family of Papovavi us Sub-Family: Polyomavirus: (Medical) BKU virus and JCU Sub-Family: Papillomavirus: (Medical) many viral types associated with cancers or malignant papilloma progression.

Adenovirus (Medical) EX AD7, ARD., -O.B. - cause of respiratory disease - some adenovirus such as 275 cause enteritis Parvovirus family (Veterinarian) Feline parvovirus: causes feline enteritis Panleucopeniavirus feline Parvovirus canine Parvovirus porcine Herpes virus family Sub-Family: alphaherpesviridue Genders: Simplex virus (Medical) HSVI (Genbank XI 4112, NCOO 1806), HSVII (NC001798) Varicellovinis: (Veterinarian) pseudorabia varicella zoster Sub-Family - betaherpeeviridue Genders: Cytomegalovirus (Medical ) HCMV Muromegalovirus Sub-Family,. Gammaherpesviridue Genres: Lymphocryptovirus (Medical) EBV - (Burkitts lympho) Rhadinovirus Family of smallpox virus Sub-Family Chordopoxviridue (Medical-Veterinarian) Genres: Variola. (Smallpox) Vaccinia (Cowpox, from the nipples of cows) Parapoxivirus - Veterinarian Auipoxvirus - Veterinarian Capripoxvirus Leporipoxvirus Suipoxvirus Sub-Family: Entemopoxviridue Family of Hepadnavirus Hepatitis B Virus Not Classified Hepatitis delta Virus Table 2 Bacterial Pathogens Gram-positive pathogenic cocci include: pneumococcus; staphylococcus; and streptococcus. Gram-negative pathogenic cocci include: meningococci; and gonococci.

Enteric gram-negative pathogenic bacilli include: Enterobacteriaceae; pseudomonas, acinetobacteria and eikenella, melioidosis; ,salmonella; shigellosis; hemophilus; cancroid; brucellosis; tularemia; yersinia (pasteurela); streptobacillus mortiliformis and spirillum; listeria monocytogenes; erysipelothrix rhusiopathiae; diphtheria, cholera, anthrax; donovanosis (inguinal granuloma); and bartonellosis.

Pathogenic anaerobic bacteria include: tetanus; botulism; other Clostridia; tuberculosis; leprosy; and other mycobacteria. Pathogenic spirochetal diseases include: syphilis; - treponematosis: yaws (spirochete disease), pinta and endemic syphilis; and leptospirosis. Other infections caused by higher pathogenic bacteria and pathogenic fungi include: actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis; sporotrichosis; paracoccidiodomicosis, petrielidiosis, torulopsosis, mycetoma, and chromomycosis; and dermatophytosis.

Infections by Rickettsia include rickettsia and rickettsioses. Examples of mycoplasma and .clamidia infections include: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal infections due to chlamydia.

Pathogenic Eukaryotes Protozoa and pathogenic helminths and infections include: amoebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis; nematodes; trematodes or flatworms; and infections by tapeworms (solitary). It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (23)

1. CLAIMS < Having described the invention as above, the content of the following claims is claimed as property: 1. A nucleic acid molecule comprising SEQ ID NO: 1 or a fragment thereof, characterized in that it encodes a functional fragment of IL-15.
2. 2. The nucleic acid molecule according to claim 1, characterized in that it comprises SEQ ID NO: 1.
3. 3. The nucleic acid molecule according to claim 1 or 2, characterized in that it is free of the coding sequence for a sequence. of signal IL-15.
4. 4. The nucleic acid molecule according to claims 1-3, characterized in that it is free from the Kozak region for Kozak IL-15 and / or the 5'-untranslated region for IL-15 and / or the region not -translated in 3 'for IL-15.
5. 5. The nucleic acid molecule according to claims 1-4, characterized in that it comprises a coding sequence for a signal sequence that is not IL-15.
6. 6. The nucleic acid molecule according to claims 1-5, characterized in that it comprises a coding sequence for an IgE signal sequence.
7. 7. The nucleic acid molecule according to claim 1-6, characterized in that it further comprises a coding sequence for an antigen.
8. 8. The nucleic acid molecule according to claim 7, characterized in that the antigen is a pathogen antigen, an antigen associated with cancer or an antigen linked to cells with autoimmune diseases.
9. 9. The isolated nucleic acid molecule according to claim 8, characterized in that the antigen is a pathogen antigen from a pathogen selected from the group consisting of HIV, HSV, HCV, and WNV.
10. 10. The isolated nucleic acid molecule according to claims 1-9, characterized in that the isolated nucleic acid molecule is a plasmid.
11. 11. The nucleic acid molecule according to claims 1-9, characterized in that it is incorporated into a viral vector.
12. 12. The nucleic acid molecule according to claim 1-9, characterized in that it is incorporated into a live attenuated pathogen.
13. 13. A composition characterized in that it comprises a nucleic acid molecule according to claim 1-12, and a nucleic acid molecule comprising a nucleic acid sequence encoding an antigen. The composition according to claim 13, characterized in that the antigen is a pathogenic antigen, an antigen associated with cancer or an antigen linked to cells associated with autoimmune diseases. 15. The composition according to claim 14, characterized in that the antigen is a pathogenic antigen from a pathogen selected from the group consisting of HIV, HSV, HCV, and WNV. 16. The composition according to claims 13-15, characterized in that the nucleic acid molecules are plasmids. 17. An injectable pharmaceutical composition, characterized in that it comprises the nucleic acid molecules of claims 1-12, or the composition according to claims 13-15. 18. A method for modulating an immune response in an individual, characterized in that it comprises administering to the individual a nucleic acid molecule of claims 1-6. 19. A recombinant vaccine characterized in that it comprises a nucleic acid sequence encoding an antigen and a nucleic acid sequence of claims 1-6. 20. The recombinant vaccine according to claim 19, characterized in that the antigen is a pathogenic antigen, an antigen associated with cancer, or an antigen linked to cells associated with autoimmune diseases. 21. The recombinant vaccine according to claim 20, characterized in that the antigen is a pathogenic antigen from a pathogen selected from the group consisting of HIV., HSV, HCV, and WNV. 22. The recombinant vaccine according to claims 19-21, characterized in that the recombinant vaccine is a vaccinia recombinant vaccine. 23. A method for inducing an immune response in an individual against an antigen, characterized in that it comprises administering to the individual a nucleic acid molecule of claims 7-12, or a composition of claims 13-16, or a recombinant vaccine of the claims 19-22.