MXPA00007889A

MXPA00007889A - Optimization of immunomodulatory properties of genetic vaccines.

Info

Publication number: MXPA00007889A
Application number: MXPA00007889A
Authority: MX
Inventors: Juha Punnonen
Original assignee: Maxygen Inc
Priority date: 1998-02-11
Filing date: 1999-02-10
Publication date: 2002-09-18
Also published as: EP1053343A2; EP1053312A2; WO1999041402A3; EP1056842A2; MXPA00007893A; CA2320431A1; JP2002507392A; JP2002503478A; AU2674199A; WO1999041369A3; MXPA00007891A; CA2320960A1; AU3291099A; WO1999041368A2; JP2002503461A; WO1999041369A2; CA2320626A1; WO1999041402A2; WO1999041368A3; AU2674299A

Abstract

This invention provides methods for obtaining molecules that can modulate an immune response, and immunomodulatory molecules obtained using the methods. The molecules find use, for example, in the tailoring of an immune response induced by a genetic vaccine for a desired purpose.

Description

OPTIMIZATION OF THE IMMUNOMODULATING PROPERTIES OF GENE VACCINES CampQ of the Invention. The present invention pertains to the field of modulation of immunological responses, such as those induced by genetic vaccines.

Antecedents of the Invention Antigen processing and presentation is only one factor which determines the effectiveness of the vaccination, whether it is carried out with genetic vaccines or with more classical methods. Other molecules included in the determination of vaccine effectiveness include cytokines (interleukins, interferons, chemokines, hematopoietic growth factors, tumor necrosis factors, and growth-transforming factors), which are proteins with a small molecular weight that regulate the maturation, activation, proliferation and differentiation of cells in the immune system. The characteristics of cytokines are, the pliotropy and the redundancy; that is, that a cytokine frequently has several functions and a certain function, is frequently mediated, by more than one cytokine. In addition, several cytokines have additive or synergistic effects with other cytokines, and a number of cytokines, also have components that the receptor shares. Due to the complexity of cytokine networks, studies on the physiological significance of a given cytokine have been difficult, although recent studies using mice with cytokine-deficient genes have significantly improved our understanding of the functions of cytokines. of the cytokines in vivo. In addition to soluble proteins, several membrane binding co-stimulatory molecules play a fundamental role in the regulation of immune responses. These molecules include CD40, ligand CD-40, CD27, CD80, CD86 and CD150 (SLAM), and these are typically expressed in lymphoid cells after activation, by means of antigen recognition or through cell interactions. a cell. Auxiliary T cells (TH), which are key regulators of the immune system, which have the ability to produce a large number of different cytokines and based on their cytokine synthesis pattern, TH cells are divided into two subsets (Paul and Seder (1994), Cell 76: page 241 to 251). TH 1 cells produce high levels of I L-2 and IFN-β cells and no or minimal levels of IL-4, I L-5 and IL-13. In contrast, TH2 cells produce high levels of I L-4, IL-5 and IL-13, and the production of I L-2 and IFN-α. It is minimal or absent. TH 1 cells activate macrophages, dendritic cells and increase the cytolytic activity of [cytotoxic CD8 +, T lymphocytes and NK cells (Id.), While TH2 cells provide efficient support to B cells and are also mediators in allergic responses due to the ability of TH2 cells to induce the change of the IgE isotype and the differentiation of B cells into IgE secretory cells (De Vries and Punnonen (1996) in cytokine regulation of humoral immunity: basic and clinical Editors Snapper, CM., John Wiley &Sons, Limited, West Sussex, UK, pages 195 to 215). The exact mechanisms that regulate the differentiation of T helper cells are not fully understood, but it is believed that cytokines play an important role. It has been shown that I L-4 directs the differentiation of T 2, whereas I L-12 induces the development of TH 1 cells (Paul and Seder, supra.). In addition, it has been suggested that membrane binding co-stimulatory molecules, such as CD80, CD86 and CD150, can direct the development of TH 1 and / or TH 2 cells, and that the same molecules that regulate differentiation of TH cells, also affect the activation, proliferation and differentiation of B cells in Ig-secreting plasma cells (Cocks and Associates (1995) Nature 376: pages 260 to 263; Lenschow and Associates (1996) Immunity 5: pages 285 to 293; Punnonen and Associates (1993) Proc. Nat'l Acad. Sci USA 90: pages 3730 to 3734; Punnonen and Associates (1997) J. Exp. Med. 185: pages 993 to 1004).

Studies, both in man and in mice, have shown that the synthesis profile of the cytokines of the T (TH) helper cells plays a crucial role in the determination to solve several infections caused by viruses, bacteria and parasites. The high frequency of TH 1 cells generally protects against lethal infections, while the dominant T 2 phenotype frequently results in disseminated and chronic infections. For example, the TH 1 phenotype is observed in its tuberculoid (resistant) form of leprosy and the T 2 phenotype in leprosy and multibacillary (susceptible) lesions (Yamamura and Associates (1991) Science 254: pages 277 to 279). Similarly, end-stage VI H patients have cytokine synthesis profiles similar to TH2 and it has been proposed that the TH 1 phenotype protects against the AIDS virus (Maggi y Asociados (1994) J. Exp. Med 180: pages from 489 to 495). In addition, the survival of meningococcal septisemia is determined genetically, based on the ability of peripheral blood leukocytes to produce TN F-a and I L-10. Individuals from families with high IL-10 production have an increased risk of fatal meningococcal disease, while members of families with a high production of TNF-a are more likely to survive the infection (Westendorp et al. Associates (1997) Lancet 349: pages 170 to 173). Cytokine treatments can dramatically influence the differentiation of TH 1 / TH 2 cells and the activation of macrophages and, therefore, can cure infectious diseases. For example, a BALB / c mouse infected with Leishmania Major, develop a fatal disseminated disease with a TH2 phenotype, but when treated with anti-I L-4 mAbs or IL-12, the frequency of TH 1 cells in the mouse increases, and therefore can counteract pathogenic invasion (Chatelain and Associates (1992) J. Immunol. 148: pages 1 182 to 1 187). In a similar way, the I FN-? protects mice from lethal Herpes Simplex virus (HSV) and MCP-1 prevents lethal infections by Pseudomonas aeruginosa or Salmonella typhimurium. In addition, cytokine-based treatments, such as recombinant I L-2, have shown beneficial effects in humans with a common immunodeficiency variable (Cunningham-Rundles and Associates (1994) N. Engl. J. Med. 331: pages 918 to 921. The administration of cytokines and other molecules to modulate the immunological response, in a more appropriate manner for the treatment of a particular disease, may provide an important tool for the treatment of the disease, however, immunomodulatory treatments. currently available, they may have several disadvantages, such as, the insufficient specific activity, the induction of immunological responses against, the immunomodulator that is administered, and other potential problems.Therefore, there is a need for immunomodulators that exhibit improved properties. in relation to those that are currently available.

The present invention has the purpose of covering this and other needs.

Summary of the Invention The present invention provides methods for obtaining a polynucleotide having a modulating effect on an immune response, which is induced by a genetic vaccine, either directly (for example, in the form of an immunomodulatory polynucleotide) or indirectly ( for example, by translating the polynucleotide, to create an immunomodulatory polypeptide). The methods of the present invention comprise: the creation of a library of recombinant polynucleotides; and selection in the library to identify at least one optimized recombinant polynucleotide, which exhibits, either by itself or through the encoded polypeptide, an increased ability to modulate a immunological response that has created a nucleic acid form from of the library that was created. The examples include, the CpG sequences rich in polynucleotides, the polynucleotide sequences encoding a co-stimulator (for example, B7-1, B7-2, CD1, CD40, CD154 (ligand for CD40), CD 150 (SLAM) or a cytokine) . The selection step used in these methods may include, for example, the introduction of gene vaccine vectors, which comprise the library of recombinant nucleic acids within a cell, and the identification of cells that exhibit an increased capacity to modulate an immunological response of interest or an increased capacity, to express an immunomodulatory molecule. For example, a library of recombinant nucleic acids with cytokine encoders can be selected by testing the ability of cytokines encoded by nucleic acids to activate cells that contain a receptor for the cytokine. The receptor for the cytokine can be native to the cell or can be expressed from a heterologous nucleic acid encoding the cytokine receptor. For example, optimized co-stimulants can be tested to identify those by which the cells or culture medium have the ability to induce a predominantly immunological response of TH2, or a predominantly TH1 immune response. In some embodiments, the polynucleotide having a modulatory effect on an immunological response, is obtained by means of: (1) the recombination of, at least, a first and second forms of a nucleic acid, which is, or encodes a molecule which is, comprised in the modulation of an immunological response, wherein the first and second forms differ from each other, by two or more nucleotides, to produce a library of recombinant polynucleotides; and (2) selecting the library to identify at least one optimized recombinant polynucleotide that exhibits, either by itself or through the encoded polypeptide, an increased ability to modulate an immune response than a nucleic acid form, from which library was created. If further optimization is desired, the method may further comprise: (3) the recombination of at least one optimized recombinant polynucleotide with an additional form of nucleic acid, which is in the same form or in forms other than the first and the second, to produce an additional library of recombinant polynucleotides; (4) selection of the additional library, to identify at least one optimized recombinant polynucleotide exhibiting an increased ability to modulate an immune response, than a form of nucleic acid from which the library was created; and (5) repeating (3) and (4), as necessary, until the optimized recombinant polynucleotide exhibits an additional augmented ability to modulate an immune response to a form of nucleic acid from which the library was created. In some embodiments of the present invention, the library of recombinant polynucleotides is selected by means of: the expression of the recombinant polynucleotides so that the encoded peptides or polypeptides are produced as fusions with a protein displayed on the surface of a genetic package that can be replicated; contacting the genetic packets that can be replicated with a plurality of cells that show the receptor, and identifying the cells that exhibit a modulation of the immune response mediated by the receptor.

The present invention also provides methods for obtaining a polynucleotide, which encodes an accessory molecule that improves the transport or presentation of the antigens by a cell. These methods comprise the creation of a library of recombinant polynucleotides, subjecting them to recombination nucleic acids that encode all or part of the accessory molecule; and selecting the library to identify an optimized recombinant polynucleotide that encodes a recombinant accessory molecule that confers on the cell an increased or decreased ability to carry or present an antigen on the surface of the cell compared to an accessory molecule encoded by the non-recombinant nucleotide acid . In some embodiments, the selection step comprises: introducing the recombinant polynucleotide library into a gene vaccine vector encoding an antigen to form a vector library; the introduction of the vector library into mammalian cells; and the identification of mammalian cells that exhibit increased or decreased immunogenicity to the antigen. In some embodiments of the present invention, the cytokine that is optimized is interleukin-12 and the selection is made by culturing mammalian cells, which contain genetic vaccine vectors in a culture medium, and detecting whether the proliferation of the cell T or differentiation of the T cell is induced by contact with the culture medium. In another embodiment, the cytokine is interferon-a and the selection is made, expressing the recombinant vector module as a fusion protein, which is displayed on the surface of a bacteriophage to form a phage sample library, and identifying the members of the phage library, which have the ability to inhibit the proliferation of the B cell line. Another modality, uses B7-1 (CD80) or B7-2 (CD86), as the co-stimulant and the cell or medium of culture, is tested to determine its ability to modulate an immune response. The present invention provides methods for the use of DNA entrainment, to obtain optimized recombinant vector modules encoding cytokines and other co-stimulants that exhibit reduced immunogenicity, compared to a corresponding peptide encoded by a non-optimized vector module. Reduced immunogenicity can be detected by introducing a cytokine or a co-stimulant encoded by the recombinant vector module into a mammal and determining whether an immune response is induced against the cytokine. The present invention also provides methods for obtaining optimized immunomodulatory sequences that encode a cytokine antagonist. For example, suitable cytokine agonists include a soluble cytokine receptor and a transmembrane cytokine receptor having a sequence of defective signs. Examples include? I L-10R and? I L-4R, and the like.

Brief Description of the Drawings Figure 1 shows an example of a cytotoxic T cell induction sequence (CTIS), obtained from the HBsAg polypeptide (PreS2 plus S regions). Figure 2 shows a CTIS that has heterologous epitopes attached to the cytoplasmic portion. Figure 3 shows the derivation of immunogenic agonistic sequences (IAS), as described in Example 3. Specific death (percent) is shown for an effector: target (E: T) ratio of five. Figure 4 shows a method for the preparation of immunogenic agonist sequences (IAS). Natural type (WT) and mutated forms of nucleic acids encoding a polypeptide of interest, as assembled and subjected to DNA entrainment in order to obtain nucleic acid coding and a polyepitope region containing the potential agonist sequence . Figure 5 shows a scheme for improving immunostimulatory sequences by means of DNA entrainment. Figure 6 is a diagram of a procedure, by means of which, recombinant libraries of human genes IL-12 can be selected to identify the entrained I L-12 genes encoding recombinant I L-12 which has an increased ability to induce T cell proliferation. Figure 7 shows the results of a high performance functional assay for vectors encoding IL-12 variants obtained using the method of the present invention. Figure 8 shows the induction of T cell proliferation in transfection of T cells by means of individual vectors encoding the IL-12 variants. Figure 9 shows the results of an experiment, which demonstrates that a dragged I L-1 chimera, obtained using the methods of the present invention, exhibits an improved ability to activate human T cells. Figure 10 shows a model of how activation of the T cell or anergy can be induced by means of gene vaccine vectors encoding different variants of B7-1 (CD80) and / or B7-2 (CD86) . Figure 11 shows a method for using DNA entrainment, in order to obtain variants of CD80 / CD86, which have an improved ability to induce the activation or anergy of the T cell. Figure 12 shows the results obtained in a test of selection of the altered function of B7. . Figure 13 provides experimental results, which demonstrate that entrained B7 chimeras produce potent activation of the T cell. Figure 14 presents an alignment of nucleotide sequences for human and mouse IL-10 receptor sequences.

Figure 15 shows an alignment of nucleotide sequences of B7-1 (CD80) genes from humans, rhesus and rabbit monkeys.

Detailed Description of the Invention. Definitions The term "cytokine" includes, for example, interleukins, interferons, chemokines, hematopoietic growth factors, tumor necrosis factors, and growth transformation factors. In general, these are proteins, with a small molecular weight that regulates the maturation, activation, proliferation and differentiation of cells of the immune system. The term "screening" describes, in general, a process that identifies the optimal immunomodulatory molecules. Various properties of the respective molecules can be used in the selection including, for example, the ability to induce a desired immune response in a test system. The selection is a form of "screening", in which the identification and physical separation are achieved simultaneously through the expression of a selection marker, which, in some genetic circumstances, allows the cells expressing the marker, survive, while other cells die (or vice versa). Selection markers include, for example, luciferase beta-galactosidase, and green fluorescent protein. The selection markers include genes resistant to drugs and toxins, and the like. Due to limitations in the study of primary immunological responses in vitro, in vivo studies are particularly useful screening methods. In these studies, genetic vaccines that include nmodulatory molecules are first introduced for testing in animals, and immune responses are subsequently studied by means of protective immune responses, which are analyzed or by means of the study of quality or strength. of the induced immune response, using lymphoid cells derived from an immunized animal. Although spontaneous selection can and does occur in the course of natural evolution, in the methods of the present invention, selection is made by man. An "exogenous segment of DNA", "heterologous sequence" or a "heterologous nucleic acid", as used in the present invention, is one that originates from a source external to the particular host cell, or, if it comes from the same source is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell, but that has been modified. Modification of the heterologous sequence in the applications described in the present invention generally occurs through the use of DNA carryover. Therefore, the terms refer to a segment of DNA, which is foreign or heterologous to the cell, or homologous to the cell but at a position within the nucleic acid of the host cell, in which the element is not found usually.

The exogenous DNA segments are expressed to produce exogenous polypeptides. The term "gene" is used widely to refer to any DNA segment associated with a biological function. Thus, the gene includes coding sequences and / or the regulatory sequences required for its expression. The genes also include unspecified DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning a source of interest or synthesizing known or anticipated sequence information, and can include sequences designed to have the desired parameters. The term "isolated", when applied to a nucleic acid or protein, indicates that the nucleic acid or protein is essentially free of other cellular components with which it is associated in its natural state. A homogenous state is preferable, although it may be, either in a dry state or in an aqueous solution. Purity and homogenicity are usually determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein, which is the predominant species present in a preparation, is substantially purified. In particular, an isolated gene is separated from the open reading frames, which flank the gene and encode a protein different from the gene of interest. The term "purified" indicates that a nucleic acid or protein essentially originates from a band on an electrophoretic gel. Particularly, it means that the nucleic acid or protein, is at least about 50% pure, more preferably, at least about 85% pure, and even more preferably at least about 99% pure. The term "occurring naturally" is used to describe an object that can be found in nature as different from that which is artificially produced by man. For example, a polypeptide or polynucleotide sequence that is present in an organism (including, viruses, bacteria, protozoa, insects, plants or mammalian tissues) that can be isolated from a source of nature, and which has not been intentionally modified by man in the laboratory, it is something that happens naturally. The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof, either in the form of single or double braid. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to those of reference nucleotide acids and which are metabolized in a manner similar to nucleotides occurring from natural way Unless otherwise indicated, a particular nucleic acid sequence also implicitly comprises conservatively modified variants thereof (e.g., substitution of codon degeneracy) and complementary sequences, as well as, the sequences explicitly indicated. . Specifically, degenerate codon substitutions can be achieved by the degeneracy sequences, in which, the third position of one or more selected (or all) codons, is substituted with deoxyinosine, basic mixed and / or deoxyinosine residues (Batzer and Associates (1 991) Nucleic Acid Res. 19: page 5081, Ohtsuka and Associates (1985) J. Biol. Chem. 260: pages 2605 to 2608, Cassol and Associates (1992), Rossolini and Associates (1994) Mol. Probes 8: pages 91 to 98). The term nucleic acid is used interchangeably with gene, cDNA and mRNA encoded by a gene. "Nucleic Acid Derived from a Gene", refers to a nucleic acid, for whose synthesis the gene, or a subsequence thereof, has finally served as a template. In this way, a mRNA, a reverse transcribed cDNA of a mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the simplified DNA, etc. , are all derived from the gene, and the detection of said derivative is an indicator of the presence and / or abundance of the original gene and / or of the gene transcribed in. a sample. A nucleic acid is "operably linkable", when placed within a functional relationship with another nucleic acid sequence, for example, a promoter or enhancer is operably linked to a coding sequence, if the transcription of said frequency increases. encoder. Operably linked, it means that the DNA sequences being linked are generally contiguous and, when necessary to join two protein coding regions, contiguous and in the reading frame. However, some enhancers generally function when they are separated from the promoter by several kilobases and intronic sequences that can be of variable length, some polynucleotide elements can be operably linked, but not contiguous. A specific binding affinity between two molecules, for example, a ligand and a receptor, means a preferential bond of one molecule to the other in a mixture of molecules. The binding of the molecules can be considered specific, if the binding affinity is from about 1 x 104 M "1 to about 1 x 106 M" 1 or greater. The term "recombinant", when used with reference to a cell, indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. The recombinant cells may contain genes that are not found within the native form of the cell (not recombinapte). The recombinant cells may also contain genes found in the native form of the cell where the genes are modified or reintroduced into the cell, by artificial means. The term also includes cells that contain an endogenous nucleic acid to the cell that has been modified without removing the nucleic acid from the cell; said modifications include those obtained by the replacement of the gene, specific mutation of the site and related techniques. A "recombinant expression cassette" or simply, "an expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that have the ability to affect the expression of a structural gene in hospitality compatible with these sequences. Expression cassettes include at least promoters and optionally, transcription termination signals. Generally, the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Also, additional factors necessary or useful to effect expression may be used, as described in the present invention. For example, an expression cassette may also include nucleotide sequences that encode a signal sequence that directs the secretion of a protein expressed from the host cell. Termination transcription signals, enhancers and other nucleic acid sequences that influence gene expression can also be included in an expression cassette. A "recombinant polynucleotide" or a "recombinant polypeptide" is a polynucleotide or polypeptide that does not occur naturally and that includes nucleic acid or amino acid sequences respectively, from one or more nucleic acid sources or polypeptides, whose nucleic acids or source polypeptides may be naturally occurring nucleic acids or polypeptides, or they may themselves have undergone mutagenesis or other modification. The source polynucleotides or polypeptides, from which the different nucleic acid or amino acid sequences are derived, are sometimes homologous (eg, they have, or encode a polypeptide that encodes, a structure and / or similar function), and that , for example, frequently they are of isolated, serotypes, deformations, species, organisms or different disease conditions. The terms "identical" or "percent identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or that have a specified percentage of amino acid residues. or nucleotides that are the same, when they are compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The phrase "substantially identical", in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences having at least 60%, preferably 80%, more preferably 90 to 95% identity of nucleotide or amino acid residues, when they are compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, there is substantial identity over a region of the sequences that is at least 50 residues in length, more preferably over a region of at least about 100 residues and more preferably, the sequences are substantially identical over at least 150 waste. In some embodiments, the sequences are substantially identical over the entire length of the coding regions. For the comparison of the sequence, generally a sequence acts as a reference sequence, with which the test sequences are compared. When a sequence comparison algorithm is used, the test and reference sequences are entered into a computer, the subsequent coordinates are designated, if necessary, and the program parameters of the sequence algorithm are also designated. The comparison sequence algorithm then calculates the percentage of the sequence identity for the test sequence relative to the reference sequence based on the designated program parameters. Optimal alignment of the sequences for comparison can be conducted, for example, by the local Smith & Homology algorithm; Waterman, Adv. Appl. Math. 2: page 482 (1981), by the alignment homology algorithm of Needleman & Wunsh, J. Mol. Biol. 48: page 443 (1970), by the search for the similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci USA 85: page 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, in the Genetics Software Package of Winsconsin, Genetics Computer Group, 575 Science Dr., Madison, Wl) , or by visual inspection (see generally Ausubel and Associates, infra). An example of an algorithm that is suitable for determining the percent identity of the sequence and the similarity of the sequence in the BLAST algorithm, which is described in Altschul and Associates, J. Mol. Biol. 215: pages 403 to 410 (1990). The software for conducting the BLAST analysis is publicly available through the National Center for Biotechnology Information (http: //www.ncbi.nlm.nih.gov/). This algorithm comprises, first, the identification of highly qualified sequence pairs (HSPs), identifying short words of length W in the investigation sequence, which, either match or satisfy a threshold valued positively of the T rating when they are aligned with a word of the same length in a database sequence. A T, we refer to as a neighborhood word qualification threshold (Altschul and Associates, supra). These initial word hits of neighborhood, act as seeds for the initiation of searches to find larger HSPs that contain them. This word is then used extended in both directions along each sequence, to the extent that the cumulative alignment qualification can be increased. The cumulative ratings are calculated using, for nucleotide sequences, the parameters M (reward rating of a pair of matching residues, always> 0) and N (penalty rating for residues that do not match, always <0). For the amino acid sequences, a rating matrix is used to calculate the cumulative score. The extent of the word hits in each direction are interrupted when the cumulative alignment score fails for the amount X from its maximum achieved value; the accumulation score goes to zero or lower, due to the accumulation of one or more negative qualifying residue alignments, or that the level of any of the sequences has been reached. The W, T and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) is used as defaults to the length of the words (W) of 1 1, an expectation (E) of 10, a cut of 100, M = 5, N = -4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults the word length (W) of 3, an expectation (E) of 10, and a qualification matrix BLOSUM62 (see Henikoff &Henikoff (1989) Proc. Natl). Acad. Sci. USA 89: page 10915). In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin &Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90 : pages 5873 to 5787). A measure of the similarity provided by the BLAST algorithm is the smallest probability of sum (P (N)), which provides an indication of the probability by means of which, a union between two nucleotide or amino acid sequences, it would possibly happen. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the nucleic acid test with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and more preferably less than about 0.001. Another indication that two nucleic acid sequences are substantially identical, is that the two molecules hybridize one to the other, under extreme conditions. The phrase "specifically hybridizes to" refers to a binding, duplexing, or hybridization of a molecule only to a particular sequence of nucleotides, under severe conditions when said sequence is present in a complex (eg, total cellular) mixture of DNA or RNA "Substantially binds or binds" refers to the complementary hybridization between a test nucleic acid and a target nucleic acid, and comprises minor disparities that can be accommodated by reducing the severity of the hybridization medium to achieve detection desired of the target polynucleotide sequence. "Severe Hybridization Conditions" and "Severe Hybridization Wash Conditions", in the context of nucleic acid hybridization experiments, such as, northern and southern hybridizations, are dependent sequences, and different, under different environmental parameters . To longer sequences, and hybridize specifically at higher temperatures. An extensive guide for the hybridization of nucleic acids, is in the article Laboratory techniques in Biochemistry and Molecular Biology - Hybridation with Nucleic Acid Probes part 1 chapter 2 of Tijssen (1993) 'Overview of principles of hybridization and the strategy of nucleic acid probé assays ", Elsevier, New York, Generally, highly severe hybridization and washing conditions are selected to be about 5 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength. Generally, under "severe conditions", a sample will hybridize to its target subsequence, but not to other sequences.Tm, is the temperature (defined ionic strength and pH) in which, 50% of the target sequence hybridizes to a perfectly matched sample.The very severe conditions are selected to be equal to the Tm of a particular sample.An example of the conditions of hybridization for the hybridization of complementary nucleic acids, which have more than 100 complementary residues in a filter in a spot of the south or north, is 50% formamide with a milligram of heparim at a temperature of 42 ° C, being made Hybridization during the night. An example of the highly severe washing conditions is 0.15M of ÑaCI, at a temperature of 72 ° C, for about 15 minutes. An example of severe washing conditions is a 0.2 x SSC wash at a temperature of 65 ° C, for 15 minutes (see, Sambrook, infra, for a description of the SSC regulator). Frequently, a high severity wash is preceded by a low severity wash to remove the bottom of the test signal. An example of a mean severity wash for a duplex of, say, more than 100 nucleotides, is 1 x SSC to one. temperature of 45 ° C, for 15 minutes. An example of low severity washing for a duplex is, for example, more than 100 nucleotides, 4-6 X SSC at a temperature of 40 ° C, for 15 minutes. For short samples (eg, from about 10 to 50 nucleotides), severe conditions generally comprise salt concentrations of less than about 1.0% M Na +, generally about 0.01 to 1.0% M Na + (and other salts), at a pH of 7.0 to 8.3, and the temperature is generally at least about 30 ° C. Severe conditions can be achieved with the addition of destabilizing agents, such as formamide. In general, a signal at a noise ratio of 2x (or greater) than that observed for an unrelated test in the particular hybridization assays indicates the detection of a specific hybridization. Nucleic acids that do not hybridize to one another, under severe conditions, are still substantially identical, if the polypeptides they encode, are substantially identical. This happens, for example, when a copy of nucleic acid is created, using the maximum degeneracy of the codon allowed by the genetic code. An additional indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is reactive, immunologically cross-linked with or specifically binds to the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is generally substantially identical to a second polypeptide, for example, when the two polypeptides differ only by conservative substitutions. The phrase "specifically (or selectively) binds an antibody" or "specifically (or selectively) immunoreactive with", when referring to a protein or peptides, which refers to a binding reaction which is determinative of the presence of the protein or an epitope from the protein, in the presence of a heterogeneous population of proteins and other biological agents. Thus, the designated conditions of the immunoassay, the binding of the specified antibodies to a particular protein and, do not bind in a significant amount to other proteins present in the sample. Antibodies raised against a multivalent antigenic polypeptide will generally bind to proteins from which one or more of the epitopes were obtained. Specific binding to an antibody under such circumstances may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry, are routinely used to select monoclonal antibodies, specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies. A laboratory Manual, Cold. Spring Harbor Publications, New York "Harlow and Lane"), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Generally, a specific or selective reaction will be at least twice the background signal or noise and more generally more than 10 to 100 times the background. The "conservatively modified variations" of a particular polynucleotide sequence refers to those polynucleotides that are encoded with identical or essentially identical amino acid sequences, or where the polynucleotide is not encoded with an amino acid sequence, to sequences essentially identical. Due to the degeneracy of the genetic code, a large number of nucleic acids of identical functionality encode any given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA and AGG, all encode amino acid arginine. Thus, in each position, when an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. These variations of the nucleic acid are "silent variations", which are a kind of "conservatively modified variations". Each sequence of polynucleotides described in the present invention, which encodes a polypeptide, also describes every possible silent variation, except where otherwise indicated. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to produce a molecule of identical functionality, by standard techniques. Consequently, each "silent variation" of nucleic acid, which encodes a polypeptide, is implicit in each described sequence. In addition, an expert in the art will recognize that individual substitutions, withdrawals or additions which alter, aggregate, or withdraw a single amino acid or a small percentage of amino acids (generally, less than 5%, more generally less than 1%) in an encoded sequence, are "conservatively modified variations" ", wherein the alterations result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables that provide amino acids of similar functionality are well known in the art. The following five groups each contain amino acids that are conservative substitutions of another amino acid: Aliphatic: Glycine (G), Ayanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Containing sulfur: Methionine (M), Cysteine (C) Basics: Arginine (R), Lysine (K), Histidine (H); Acids: Aspartic Acid (D), Glutamic Acid (E), Asparagine (N), Glutamine (Q). To obtain additional groups of amino acids, see also, Creighton (1984) Proteins, W. H. Freeman and Company. In addition, individual substitutions, withdrawals or additions that alter, aggregate or withdraw a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservatively modified variations". A "subsequence" refers to a nucleic acid or amino acid sequence, comprising a part of a longer sequence of nucleic acids or amino acids (eg, polypeptide) respectively.

Description of the Preferred Modalities of the Present Invention. The present invention provides methods for obtaining polynucleotide sequences which can be modulated, either directly or indirectly (for example, by encoding a polypeptide) the immune response, when present in a genetic vaccine vector. In another embodiment, the present invention provides methods for optimizing the transport and presentation of antigens. The polynucleotides, optimized immunomodulators obtained using the methods of the present invention, are particularly suitable for use in conjunction with vaccines, including genetic vaccines. One of the advantages of genetic vaccines is that one can incorporate immunomodulatory molecules that encode genes, such as cytokines, co-stimulant molecules and molecules that improve antigen transport and the presentation of genetic vaccine vectors. This provides opportunities for modulated immune responses that are induced against the antigenes expressed by the genetic vaccines.

A Creation of Recombinant Libraries. The present invention comprises the creation of recombinant libraries of polynucleotides which are then selected to identify those members of the library, which exhibit a desired property. Recombinant libraries can also be created using any of several methods. The substrate nucleic acids, used for recombination, may vary depending on the particular application. For example, when a polynucleotide encoding a cytokine, chemokine or other accessory molecule is to be optimized, different forms of nucleic acid encoding all or part of the cytokine, chemokine or other accessory molecules are subjected to recombination. The methods require, at least, two variant forms of a starting substrate. The variant forms of the candidate substrates may show substantial sequences or secondary structural similarity with one another, but they must also differ in at least two positions. The initial diversity between the forms can be the result of natural variations, for example, the different variant forms (homologous), are obtained from individuals from different formations of an organism (including geographical variants) or constitute related sequences of the same organism (for example, allelic variations). Alternatively, the initial diversity may be induced, for example, the second variant form may be generated by error propensity transcription, such as a PCR-propensity by mistake, or the use of a polymerase, which lacks test activity reading (see Liao (1990) Gene 88: page 107 to 1 1 1), of a first variant form, or by replication of the first form in a mutant strain (the mutant host cells, will be discussed in more detail, below) . The initial diversity between the substrates is increased considerably in the subsequent steps of the combination resource sequence. Frequently, improvements are achieved after a round of recombination and selection. However, recursive sequence recombination can be used to achieve still further improvements in a desired property. The sequence recombination can be achieved in many different formats and permutations of formats, as will be described in more detail below. These formats share some common principles. The sequence recourse recombination comprises successive recombination cycles to generate the molecular diversity. That is, one creates a family of nucleic acid molecules that show some sequence identity with another, but that differs in the presence of mutations. In any given cycle, recombination may occur in vivo or in vitro, and intracellular or extracellular. In addition, the diversity resulting from recombination, can be increased in any cycle, by means of the application of previous methods of mutagenesis (for example error prone PCR or cassette mutagenesis) to any of the substrates or products for recombination . In some cases, a new property or an improved or characteristic property can be achieved after only one cycle of recombination in vivo or in vitro, as when different variant forms of the sequence are used, such as homologues of individuals or different deformations of a organism, or related sequences, from the same organism as allelic variations. In a currently preferred embodiment, the recombinant libraries are prepared, using DNA carry over. Trawling and screening can be used to "develop" individual genes, plasmids or whole viruses, accumulations of multiple genes, or even whole genomes (Stemmer (1995) Bio / Technology 13: pages 549 to 553). The repetitive recombination cycles and selection can be performed to further develop the nucleic acids of interest. These techniques do not require the extensive analysis and computation required by conventional methods for the engineering of polypeptides. Trawling allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to recombination events in the form of traditional pairs. Thus, the sequence recombination techniques described in the present invention provide particular advantages, in that they provide the combination between mutations in any and all of these, providing by them, a very fast means of exploring, the way in which which may affect the combinations of different mutations, a desired result. In some cases, however, it is available < ? the structural and / or functional information, which, although not required for the recombination of the sequ, provides opportunities for the modification of the technique. The formats and examples of sequ recombination, which we sometimes refer to as DNA drag, evolution, molecular breeding, have been described by the present inventors and co-workers in the pending applications as well. U.S. Patent Application Serial No. 08 / 198,431 filed February 17, 1994, serial patent number PCT / US95 / 02126 filed on February 17, 1995, serial patent number 08 / 425,684 filed on April 18, 1995, the patent series number 08 / 537,874 filed on October 30, 1995, the serial patent number 08 / 564,995 filed on November 30, 1995, the serial patent number 08/621, 859 filed on March 25, 1996, the serial patent number 08/621, 430 filed on March 25, 1996, patent number PCT / US / 96/05480 filed on April 18, 1996, serial patent number 08 / 650,400 filed on May 20, 1995, the serial patent No. 08 / 675,502 filed on July 3, 1996, serial patent number 08/721, 824 filed on September 27, 1996, serial patent number PCT / US97 / 17300 filed on September 26, 1997 and serial number patent PCT / US97 / 24239 filed on September 17, 1997; Stemmer, Sci 270: page 1510 (1995); Stemmer and Associates, Gene 164: pages 49 to 53 (1995); Stemmer, Bio / Technology 13: pages 549 to 553 (1995); Stemmer, Proc. Natl. Acad. Sci. E. U.A. 91: pages 10747 to 10751 (1994); Stemmer, Nature 370: pages 389 to 391 (1994); Crameri and Associates, Nature Medicine 2 (1): pages 1 to 3 (1996); Crameri and Associates, Nature Biotechnology 14: pages 315 to 319 (1996), each of which is incorporated herein by refer in its entirety as a refer for all uses. Other methods for obtaining recombinant polynucleotides and / or for obtaining diversity in nucleic acids used as substrates for entrainment, include, for example, homologous recombination (PCT / US98 / 05223, publication number W098 / 42727); directed oligonucleotide mutagenesis (for review, see, Smith, Ann. Rev. Genet, 19: pages 423 to 462 (1985); Botstein and Shortle, Sci 229: pages 1 193 to 1201 (1985); Carter, Biochem. 237: pages 1 to 7 (1986), Kunkel's article, "The Effici of Directed Oligonucleotide Mutagenesis" in Nucleic acids &Molecular Biology, Editors Eckstein and Lilley, Springer Verlag, Berlin (1987). Included among these methods is the directed oligonucleotide mutagenesis (Zoler and Smith, Nucí Acids, Res. 10: pages 6487 to 6500 (1982), Methods in Enzymol 100: pages 468 to 500 (1983), and Methods in Enzymol 154: pages 329-350 (1987)) mutagenesis of phosphothiated DNA (Taylor et al., Nucí Acids Res. 13: pages 8749 to 8764 (1985); Taylor et al., Nucle. Acids. Res. 13: pages 8765 a 8787 (1985); Nakamaye and Eckstein, Nucí. Acids Res. 14: pages 9679 to 9698 (1986); Sayers and Associates, Nucí. Acids Res. 16: - pages 791 to 802 (1988); Sayers and Associates, Nucí. Acids Res. 16: pages 803 to 814 (1988)), templates for mutagenesis using uracil content (Kunkel, Proc. Nat'l Acad. Sci. USA 82: pages 488 to 492 (1985); and Kunkel and Associates, Methods in Enzymol 154: pages 367 to 382)); mutagenesis using exposed duplex DNA (Kramer and Associates, Nuci Acids, Res. 12, pages 9441 to 9456 (1984), Kramer and Fritz, Methods in Enzymol, 154: pages 350 to 367 (1987), Kramer and Associates, Nuci. Acids Res. 16: page 7207 (1988) and, Fritz and Asociados Nucí Acids, Res. 16: pages 6987 to 6999 (1988)). Additional suitable methods include repair of the decoupling point (Kramer and Associates, Cell 38: pages 879 to 887 (1984)), mutagenesis using deficient host / repair deformations (Carter and Associates, Nuci Acids, Res. 13: pages 4431 to 4443 (1985); Carter, Methods in Enzymol. 154: pages 382 to 403 (1987)), removal mutagenesis (Eghtedarzadeh and Henikoff, Nucí Acids, Res. 14: page 51 15 (1986)), restriction-selection and restriction-purification (Wells and Associates, Phil. R. Soc. Lond A 317: pages 415 to 423 (1986)), mutagenesis by total synthesis of the gene (Nambiar and Associates, Science 223: pages 1299 to 1301 (1984); Sakamar and Khorana, Nuci, Acids. 14: pages 6361 to 6372 (1988), Wells and Associates, Gene 34: pages 315 to 323 (1985), and Grundstrom and Associates, Nuci Acids, Res. 13: pages 3305 to 3316 (1985)). The equipment for mutagenesis can be obtained in the market (for example, Bio-Rad, Amersham International, Anglian Biotechnology).

B. Selection Methods. Usually a recombination cycle is followed by at least one cycle of selection of molecules having the desired properties or characteristics. If an in vitro recombination cycle is performed the recombination products, for example, recombinant segments, are sometimes introduced into the cells before the selection step. The recombinant segments can also be linked to an appropriate vector or other regulatory sequences before selection. Alternatively, the recombination products generated in vitro are sometimes packaged as viruses before selection. If the recombination is performed in vivo, the products of recombination can sometimes be selected in the cells in which the recombination occurred. In other applications, the recombinant segments are extracted from the cells, and optionally packaged as viruses, before selection. The nature of the selection depends on the property or characteristic to be acquired or the property or characteristic for which improvement is sought, and many examples will be presented below. Generally, it is not necessary to understand the molecular basis, which is the particular product of recombination (recombinant segments) have acquired new or improved properties or characteristics related to the starting substrates. For example, a gene vaccine vector can have many component sequences, each having a different intended role (eg, coding sequence, regulatory sequences, target setting sequences, stability-granting sequences, sequences).

Immunomodulators, antigen presentation that affects the sequences and integration that affects the sequences). Each of these component sequences can be varied and recombined simultaneously. Then, selection can be made, for example, for recombinant segments having increased episomal maintenance in a target cell, without the need to attribute said improvement to any of the individual component sequences of the vector. Depending on the particular selection protocol used for a desired property, the initial round of selection may sometimes be performed on bacterial cells, due to high transfection efficiencies and ease of culture. Subsequent rounds, and other types of selection, which are not accessible for selection in bacterial cells, are performed in mammalian cells to optimize the recombinant segments to be used in an environment close to the intended use. The final rounds of selection can be made in the precise type of the cell, for the intended use (for example, a human antigen presentation cell). In some cases, this cell can be obtained from a patient who is to be treated with a view, for example, to minimize the immunogenicity problems in this patient. The selection step identifies a subpopulation of recombinant segments that have been developed towards the acquisition of a desired new or improved property or properties useful in genetic vaccination. Depending on the selection, the recombinant segments can be identified as components of cells, components of viruses or in their free form. More than one round of selection can be made after each round of recombination. If a further improvement in one property is desired, at least one, and generally, a collection of surviving recombinant segments from the first round of selection, is subjected to an additional round of recombination. These recombinant segments can be recombined with each other or with exogenous segments representing the original substrates or additional variations thereof. Again, recombination can be carried out in vitro or in vivo. If the previous selection step identifies the desired recombinant segments as cell components, the components can be subjected to further recombination in vivo, or they can be subjected to further in vitro recombination or they can be isolated before performing a round of in vitro recombination. On the contrary, if the previous selection step identifies desired recombinant segments in their naked form or as components of viruses, these segments can be introduced into the cells, to perform a round of recombination in vivo. The second round of recombination, regardless of how it is carried out, generates additional recombinant segments, which comprise a diversity additional to that present in the recombinant segments that is the result of the previous rounds.

The second round of recombination may be followed by an additional round of selection, in accordance with the principles discussed above in the first round. - The severity of the selection can be increased between rounds. Also, the nature of the selection and the property being selected may vary between rounds, if an improvement is desired in more than one property or if it is required that it acquires a desired additional property. Then, additional rounds of recombination and selection can be made, until the recombinant segments have been developed enough to acquire the new or improved property or function. In the present invention, various selection methods are described for particular applications. In many cases, the selection comprises the expression of recombinant peptides or polypeptides, encoded by the recombinant polynucleotides of a library, in the form of fusions with a protein that is shown on the surface of a replicable genetic package. For example, phage display can be used. See, for example, Cwirla y Asociados, Proc. Natl. Acad. Sci. E. U .A. 87: pages 6378 to 6382 (1990); Devlin and Associates, Science 249: pages 404 to 406 (1990); Scott & Smith, Science 249: pages 386 to 388 (1990); Ladner and Associates, North American patent 5,571, 698. Other genetic packages that can be replicated include, for example, bacteria, eukaryotic viruses, yeasts and spores.

The most frequently used genetic packages for screen libraries are: bacteriophages, particularly filamentous phages and especially phages M 13, Fd and F 1. Most of the work has included the introduction of libraries that # encode peptides to be shown in any of the formations of these phages and protein fusions gl l l or gVI I I.

See, for example, Dower, WO 91/19818; Devlin, WO 91/18989; MacCafferty, WO 92/01047 (gene 11); Huse, WO 92/06204; Kang, WO 92/18619 (gene VI I I). Each fusion protein comprises a signal sequence, generally, but not necessarily, of the protein of the phage layer, a polypeptide to be displayed and, either the gene protein or the VI II gene or a fragment of the same. Exogenous coding sequences are often inserted at or near the N terminus of the I l l gene or the VI I I gene, although other insertion sites are possible. Eukaryotic viruses can be used to display polypeptides in an analogous manner. For example, the sample of a human heregulin fused to gp70 from Moloney murine leukemia virus has been reported by Han and Associates, Proc. Natl. Acad. Sci. E. U.A. 92: pages 9747 to 9751 (1995). The spores can also be used as genetic packages that can be replicated. In this case, the polypeptides are shown from the outer surface of the spore. For example, spores from B. subtilis have been reported to be suitable. The coating protein sequences of these spores are provided by Donovan and Associates, in J. Mol. Biol. 196, pages 1 to 10 (1987). Also, cells can be used as genetic packages that can be replicated. The polypeptides to be displayed are inserted into a gene encoding a cellular protein that is expressed on the surface of the cells. Bacterial cells, including Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and especially, Escherichia coli, are preferred. . The details of the outer surface proteins, are explained by Ladner and Associates, in the North American patent number 5,571, 698 and in the references that we have cited in the present invention. For example, the E. coli lamB protein is suitable. A basic concept of sample methods that use phage or other genetic packages that can be replicated, is the establishment of a physical association between the DNA encoding a polypeptide to be selected and the polypeptide. This physical association is provided by the replicable genetic package, which shows a polypeptide as part of a cipher enclosing the phage genome or other package, wherein the polypeptide is encoded by the genome. The establishment of a physical association between the polypeptides and their genetic material allows the simultaneous mass selection of very large amounts of phages carrying different polypeptides. The display of the phage as a polypeptide with affinity to an objective, for example, a receptor that binds to the target and these phages are enriched by the selection of affinity to the target. The identity of the polypeptides shown, coming from this phage, can be determined from their respective genomes. By using these methods, a polypeptide identified as having a binding affinity for a desired target, then it can be synthesized in bulk by conventional means, or the polynucleotide encoding the peptide or polypeptide can be used as part of a genetic vaccine .

C. Evolution of Improved Immunomodulatory Sequences. Cytokines can dramatically influence the activation of the macrophage in the differentiation of TH 1 / TH 2 cells, and therefore, in the result of infectious diseases.

In addition, recent studies suggest in an important way that the DNA itself can act as an adjuvant, by activating the cells of the immune system. Specifically, the DNA sequences rich in non-methylated CpG were shown to improve the differentiation of the T 1 cell, activate cytokine synthesis by means of monocytes and induce the proliferation of B lymphocytes. The present invention, therefore, provides methods to improve the immunomodulatory properties of genetic vaccines (a), by developing the stimulating properties of DNA itself, and (b) by developing genes that code for cytokines and related molecules that are involved in the regulation of the immune system . So, these genes are useful in gene vaccine vectors. Of particular interest are the I FN-a and the I L-12, which promote the immunological responses towards an auxiliary cell phenotype T1 (TH 1) and, through this, improve the capacity of the host cell to counteract the pathogenic invasions. Also, methods are provided for obtaining improved immunomodulatory nucleic acids, which have the ability to inhibit, or augmented activation, differentiation or anergy of specific antigen T cells. Due to limited information about the structures and mechanisms that regulate these events, the molecular breeding techniques of the present invention provide much faster solutions than rational design. The methods of the present invention generally comprise the use of DNA entrainment or other methods to create a library of recombinant polynucleotides. Then, the library is selected to identify the recombinant polynucleotides in the library, when they are included in a gene vaccine vector or administered in conjunction with a genetic vaccine, they have the ability to increase or otherwise alter an immune response induced by the vector . The selection step, in some embodiments, may comprise the introduction of a gene vaccine vector, which includes the recombinant polynucleotides into the cells of mammals and determines whether the cells, or the culture medium obtained by cell culture, has the ability to modulate an immune response. Optimized recombinant vector modules, obtained through the recombination of polynucleotides, are useful, not only as components of genetic vaccine vectors, but also for the production of polypeptides, for example, modified cytokines and the like, which can be administered to a mammal, to increase or change an immune response. The polynucleotide sequences obtained using the DNA entrainment methods of the present invention can be used as a component of a genetic vaccine, or they can be used for the production of cytokines or other immunomodulatory polypeptides that are used, themselves, as reactants Therapeutic or prophylactic If so desired, the optimized immunomodulatory polynucleotide sequence encoding the polypeptide can be determined and deduced the amino acid sequence used to produce polypeptides using methods known to those skilled in the art. 1 .- Immunostimulatory DNA sequences. The present invention provides methods for obtaining polynucleotides that are immunostimulatory when introduced into a mammal. Oligonucleotides, containing hexamers with a central CpG, flanked by two 5 'purines (GpA or ApA), and three 3' pyrimidines (TpC or TpT), efficiently induce cytokine synthesis and proliferation of B cells (Krieg and Associates, (1995) Nature 374: page 546; Klinman and Associates (1996) Proc. Nat'l. Acad. Sci. E. U.A. 93: page 2879; Pisetsky (1996) Immunity 5: pages 303 to 310), in vitro and act as adjuvants in vivo. Genetic vaccine vectors, in which oligos are inserted, which contain immunostimulatory sequence (ISS), have an increased capacity to improve the responses of specific antigen antibodies, after vaccination with DNA. The minimum length of an ISS oligonucleotide for an in vitro functional activity is 8 (Klinman and Associates, supra). It was discovered that the twenty-three with three CG motifs were significantly more efficient in the synthesis of cytokine induction than one quixer with two CG motifs (Id). It has been suggested that GGGG tetrads are involved in DNA binding to cell surfaces (receptors of expressed macrophages, eg cleansing receptors, which bind DNA) (Pisetsky and Associates, supra). According to the present invention, a library is generated, by subjecting the DNA of random recombination (eg fragments of human, murine or other genomic DNA), or oligonucleotides containing known ISS, poly A, C, G or T sequences, or combinations thereof. The DNA, which includes at least the first and second forms, which differ from each other, in two or more nucleotides, are recombined to produce a library of recombinant polynucleotides.

Then, the library is selected to identify those recombinant polynucleotides that exhibit immunostimulatory properties. For example, the production of cytokine induction in vitro can be selected at the introduction of the library, in a suitable cell type that can be selected. A diagram of this procedure is shown in Figure 5. Among the cytokines that can be used as an indicator of immunostimulatory activity are, for example, IL-2, I L-4, I L-5, I L-6 , I L-10, IL-12, I L-13, IL-15, and I FN- ?. Also, changes in the proportions of IL-4 / IFN- ?, I L-4 / I L-2, IL-5 / I FN- ?, IL-5 / IL-2, I L-13 can be tested. / I FN- ?, IL-13 / I L-2. An alternative screening method is the determination of the ability to induce cell proliferation, comprised in the immune response, such as B cells, T cells, monocytes / macrophages, total PBL and the like. Other selections include detection of the induction of APC activation, based on changes in the expression levels of the surface antigens, such as B7-1 (CD80), B7-2 (CD86), MHC class I and II, and CD14. Other useful selections include the identification of recombinant polynucleotides, which induce T cell proliferation. Because ISS sequences induce B cell activation, and because of the various homologies between the surface antigens expressed by the T cell and B cell, polynucleotides can be obtained that have stimulating activities in T cells.

Libraries of recombinant polynucleotides can also be selected by enhanced CTL and antibody responses in vivo, and improved protection from infections, cancer, allergy and autoimmunity. Recombinant polynucleotides that exhibit the desired property can be recovered from the cell, and if further improvement is desired, entrainment and selection can be repeated, optimized ISS sequences can be used as an adjuvant separately from a current vaccine, or the DNA sequence of interest can be fused to a genetic vaccine vector 2. Cytokines, Chemokines, and Accessory Molecules. The present invention also provides methods for obtaining cytokines, cytokine antagonists, chemokines and other optimized accessory molecules that direct, inhibit, or augment immune responses. For example, the methods of the present invention can be used to obtain genetic vaccines and other reagents (e.g., optimized cytokines and the like), which when administered to a mammal, improve or alter an immune response. These optimized immunomodulators are useful for the treatment of infectious diseases, as well as other conditions, such as inflammatory disorders, in a non-specific antigen fashion. For example, the methods of the present invention can be used to develop optimized immunomodulatory molecules for the treatment of allergies. Optimized immunomodulatory molecules can be used alone or in conjunction with specific antigen gene vaccines to prevent or treat allergies. There are four basic mechanisms, by means of which allergy-specific immunotherapy can be achieved. First, a reagent can be administered that causes a decrease in allergen-specific TH2 cells. Second, a reagent that causes an increase in specific allergen-specific TH 1 cells can be administered. Third, an increase in suppressive CD8 + T cells can be directed. Finally, the allergy can be treated by inducing anergy of the specific allergen T cells. In this example, cytokines are optimized using the methods of the present invention, to obtain reagents that are effective in achieving one or more of these immunotherapeutic goals. The methods of the present invention are used to obtain antiallergic cytokines having one or more properties, such as, enhanced specific activity, improved secretion after introduction into target cells, are effective at lower doses than natural cytokines and have fewer side effects. The targets of particular interest include interferon-a / ?, I L-10, I L-12, and the antagonists of IL-4 and IL-13. The optimized immunomodulators, or optimized recombinant polynucleotides encoding the immunomodulators, can be administered alone, or in combination with other accessory molecules. The inclusion of optimal concentrations of appropriate molecules can increase a desired immune response, and / or lead to the induction or suppression of a particular type of immune response. The polynucleotides encoding the optimized molecules can be included in a genetic vaccine vector, or the optimized molecules encoded by the genes can be administered as polypeptides. In the methods of the present invention, a library of recombinant polynucleotides encoding immunomodulators is created by subjecting substrate nucleic acids to a recombination protocol, such as a DNA blot or other method known to those skilled in the art. Nucleic acid substrates are generally two or more forms of a nucleic acid encoding an immunomodulator of interest. Cytokines are among the immunomodulators that can be improved using the methods of the present invention. Cytokine synthesis profiles play a crucial role in the ability of the host to counteract viral, bacterial or parasite infections and cytokines can dramatically influence the effectiveness of genetic vaccines and the outcome of infectious diseases. It has been shown that several cytokines, for example, IL-1, IL-2, I L-3, IL-4, IL-5, IL-6, I L-7, IL-8, I L-9, IL -10, I L-1 1, IL-12, IL-13, IL-14, IL-15, I L-16, IL-17, I L-18, G-CSF, GM-CSF, I FN- a, I FN- ?, TGF-β, TN Fa, TNF-β, IL-20 (M DA-7), and ligand ftl-3, stimulate immune responses in vitro or in vivo. Immunological functions that can be improved using appropriate cytokines, include, for example, B cell proliferation, Ig synthesis, Ig isotype change, T cell proliferation, and cytokine synthesis, TH 1 and TH 2 cell differentiation. , activation and proliferation of CTL, activation and production of cytokine by means of dendritic cells / monocytes / macrophages, and differentiation of dendritic cells from monocytes / macrophages. In some embodiments, the present invention provides methods for obtaining optimized immunomodulators that can direct an immune response toward a TH1 or TH2 response. The ability to influence the direction of the immune response in this way is of great importance in the development of genetic vaccines. By altering the response, of the HT type, the result of an infectious disease can be fundamentally changed. The high frequency of TH 1 cells generally protects against lethal infections with intracellular pathogens, while a dominant TH2 phenotype frequently results in chronic disseminated infections. For example, in humans, the TH1 phenotype is present in the tuberculoid (resistant) form of leprosy, whereas the TH2 phenotype is found in lepromatous, multiple-bacillus (susceptible) lesions (Yamamura and Associates (1991)). Science 254: page 277). The patients of SI DA in terminal stage, have the TH2 phenotype. Studies in family members indicate that survival to meningococcal septicemia depends on the cytokine synthesis profile of PBL, with high synthesis of I L-10 associated with a high risk of lethal outcome, and being associated high TNF-a with a low risk.

• Similar examples have been discovered in mice. For example, a - BALB / c mouse, is susceptible to the infection of leishmania major, these mice develop fatal disseminated diseases with the TH2 phenotype. Treatment with anti-IL-4 monoclonal antibodies, or with I L-1, induces a TH 1 response, resulting in a cure. Anti-interferon monoclonal antibodies exacerbate the disease. For some applications, it is preferable to direct an immune response in the direction of a TH2 response. For example, where increased mucosal immunity, including protective immunity, is desired, the increased TH2 response can lead to increased production of antibodies, particularly IgA. The T (TH) helper cells are probably the most important regulators of the immune system. TH cells are divided into two subsets, based on their cytokine synthesis pattern (Mosmann and Coffman (1989) Adv. Immunol. 46: page 1 1 1). TH 1 cells produce high levels of the cytokine IL-2 and IFN-γ, and minimal or no levels of IL-4, IL-5 and 1L-13. In contrast, TH2 cells produce high levels of IL-4, I L-5, and IL-13, and a minimal or no production of I L-2 and IFN-? T 1 cells activate macrophages, dendritic cells and increase the cytolytic activity of T lymphocytes, CD8 + cytotoxic and natural killer (NK) cells (Paul and Seder (1994) Cell 76: page 241), whereas T cells 2, provide an efficient aid for B cells, and are also mediators in allergic responses, due to the ability of TH2 cells, to induce the change of the IgE isotype and the differentiation of B cells into IgE-secreting cells ( Punnonen and Associates (1993) Proc. Nat'l. Acad. Sci. USA 90: page 3730). The methods of selection of cytokines, chemokines and other improved accessory molecules are generally based on the identification of modified molecules that exhibit enhanced specific activity in the target cells that are sensitive to the cytokine, chemokine or other respective accessory molecule. A cytokine, chemokine or recombinant accessory molecule nucleic acid library can be expressed, for example, in phage or as a purified protein and tested using in vitro cell culture assays. Importantly, when recombinant nucleic acids are analyzed as components of DNA vaccines, the most optimal DNA sequences (in addition to the functions of the protein products) can be identified in terms of their immunostimulating properties, transfection efficiency, and its capacity to improve the stability of vectors. The identified recombinant and optimized nucleic acids can then be subjected to new rounds of entrainment and selection. In one embodiment of the present invention, cytokines are developed that direct the differentiation of TH1 cells. Due to their ability to promote immune responses towards a TH 1 genotype, the genes coding for interferon-a (IFN-a.) And interleukin-12 (I L-12), are preferred substrates for recombination and selection with the aim to obtain activities and maximum specific capacities to act as adjuvants in genetic vaccinations. I FN-a is a particularly preferred target for optimization, using the methods of the present invention, due to its effects on the immune system, the growth of tumor cells, and viral replication. Due to these activities, I FN-a was the first cytokine to be used in clinical practice. Currently I FN-a is used for a wide variety of applications, including various types of cancers and viral diseases. I FN-a also efficiently directs the differentiation of human T cells into the TH 1 phenotype (Parronchi et al. (1992) J. Immunol 149: page 2977). Nevertheless, it has not been fully investigated in the vaccination models, because in contrast to human systems, it does not affect the differentiation of TH 1 in mice. The species difference was recently explained by data indicating that, like IL-12, IFN-a induces STAT4 activation in human cells, but not in murine cells, and STAT4 has been shown to be required in an IL-12 differentiation mediated by TH 1 (Thierfelder and Associates (1996) Nature 382: pages 171).

Dragging the DNA family is a preferred method for the optimization of I FN-a, using as a substrate the IFN-a gene of mammals, which are homologs of 85% to 97%. Larger amounts of recombinants other than 1026 can be generated from the natural diversity of these genes. To allow rapid parallel analysis of recombinant interferons, high throughput methods can be used for their expression and biological assay as protein fusion in bacteriophages. Recombinants with improved potency and selectivity profiles are being selectively cultured to obtain improved activity. Variants can be selected that demonstrate an enhanced binding to I FN-a receptors for further analysis, using a selection of mutants with optimal ability to direct TH 1 differentiation. More specifically, the abilities of I FN-a mutants to induce the production of IL-12 and IFN-? in human T lymphocyte cultures in vitro, which can be studied by cytokine-specific ELISA tests and by cytokine cytokine staining and flow cytometry. I L-12 is, perhaps, the most potent cytokine that directs TH 1 responses, and has also shown that it acts as an adjuvant and increases T 1 responses after genetic vaccination (Kim and Associates (1997). ) J. Immunol. 158: page 816). L-12 is a cytokine that has a unique structure and functionality. It is the only heterodimeric cytokine known to date, composed of a light chain of 35 kD (p35) and a heavy chain of 40 kD (p40) (Kobayashi and Associates (1989) J. Exp. Med. 170: page 827; Estern and Associates (1990) Proc. Nat'l. Acad. Sci. USA 87: page 6808). Recently, Lieschke and Associates ((1997) Nature Biotech, 15: page 35) demonstrated that a fusion between the p35 and p40 genes results in a single gene that has activity comparable to that of two genes expressed separately. These data indicate that it is possible to drag the IL-12 genes as an entity, which is beneficial in the design of the entrainment protocol. Due to its promoter activity of the T cell growth, normal human peripheral blood T cells can be used in the selection of the most active I L-1 genes, making possible the direct selection of mutants of I L-12 with the activities more potent in human T cells. The mutants of IL-12 can be expressed in CHO cells for example, and the ability of the floaters to induce the proliferation of determined T cells (Figure 6). The concentrations of I L-1 in the floating ones can be normalized based on a specific ELISA test that detects the card fused to the entrained I L-1 molecules. The incorporation of I FN-a and / or IL-12 genes developed into gene vaccine vectors is expected to be safe. The safety of I FN-a has been demonstrated in numerous clinical studies and in the daily practice of hospitals. A phase II trial of I L-12 in the treatment of cancer patients in renal cells resulted in several unexpected adverse effects (Tahara and Associates (1995) Human Gene Therapy 6: page 1607. However, gene I L-12 as a component of genetic vaccines, aids in high levels of local expression, while the levels observed in the circulation are minimal compared to those observed after systemic bolus injections.Also, some of the adverse effects of the systemic treatments based on I L-1, it is probable that they are related to their unusually long half-life (up to 48 hours in monkeys) .DNA trawling may allow the selection of a shorter half-life, thus reducing toxicity, even at high doses of boluses.In other cases, genetic vaccines that can induce TH2 responses are preferred, especially when improved anti-HIV production is desired. bodies, as an example, IL-4 has been shown to direct the differentiation of TH2 cells (which produce high levels of IL-4, I L-5 and IL-13, and mediated immune responses for allergies). Immune responses that are promoted to the TH2 phenotype are preferred when genetic vaccines are used to prophylactically immunize against autoimmune diseases. TH 1 responses are also preferred when vaccines are used to treat and modulate existing autoimmune responses, due to autoreactive T cells, are generally of the T 1 phenotype (Libiau and Associates (1995) Immunol.

Today 16: pages 34 to 38). I L-4 is also the most potent cytokine in the induction of IgE synthesis; Mice with IL-4 deficiency lack the ability to produce IgE. Asthma and allergies are associated with an increased frequency in I L-4 producing cells, and are genetically linked to the locus encoding IL-4, which is on chromosome five (in close proximity to the coding genes). of IL-3, I L-5, IL-9, I L-13 and GM-CSF). I L-4, which is produced by activated T cells, basophils and mast cells, is a protein that has 153 amino acids and two potential sites of N-glycocylation. Human IL-4 is only approximately 50% identical to the I L-4 of the mouse, and the activity of IL-4 is specific to the species. In humans, I L-13 has activities similar to those of IL-4, but IL-13 is less potent than I L-4 in the induction of IgE synthesis. The I L-4, is the only known cytokine, which directs the differentiation of TH2. Enhanced I L-2 agonists are also useful for directing the differentiation of the TH2 cell, while the improved IL-4 antagonist can direct the differentiation of the TH1 cell. The improved agonist IL-4 and the antagonist can be generated by entraining the I L-4 or the soluble receptor of I L-4. The IL-4 receptor, consists of a chain of IL-4Ra (140 kD, high affinity binding unit) and a chain of I L-2R? (These cytokine receptors share a common chain). The chain of IL-4Ra is shared by the complex receptors of I L-4 and I L-13. Both, 1L-4 and I L13 induce phosphorylation of the I chain L-4Ra, but the expression of the I chain L-4Ra alone in the transplants is not sufficient to provide a functional IL-4R. The soluble IL-4 receptor is currently in clinical trials for the treatment of allergies. The use of the DNA entrainment methods of the present invention can develop a soluble receptor IL-4, which has an improved affinity for IL-4. Such receptors are useful for the treatment of asthma and other diseases mediated by the TH2 cell, such as severe allergies. The entrainment reactions can take advantage of the natural diversity present in the cDNA libraries, coming from the activated T cells of humans and other primates. In a typical embodiment, a library of an entrained IL-4Ra chain is expressed in a phage, and mutants that bind to IL-4 with improved affinity are identified. The biological activity of the selected mutants is then tested, using cell-based assays. I L-2 and I L-15 are also of particular interest for use in genetic vaccines. IL-2 acts as a growth factor for activated B and T cells, and also modulates the functions of NK cells. IL-2 is produced predominantly by TH 1 clones similar to the T cell and therefore, is mainly considered to function in delayed reactions of the hypersensitivity type. However, I L-2 has direct potent effects on the proliferation and synthesis of Ig by B cells. The complex immunoregulatory properties of IL-2 are reflected in the mouse phenotype with IL-2 deficiency, the which has a high mortality at young age and multiple effects on its immunological functions, including the spontaneous development of inflammatory bowel disease. IL-15 is a more recently identified cytokine, which is produced by multiple cell types. The I L-15 shares several, but not all, of the IL-2 activities. Both I L-2 and IL-1 5 induce B cell growth and differentiation. However, assuming that the production of IL-15 in the mouse with IL-2 deficiency is normal, it is clear that, IL-15 can not substitute the function of IL-2 in vivo, since these mice have immunodeficiencies multiple The IL-2, has synergistically shown the increase in human Ig production induced by I L-10 in the presence of anti-CD40 mAbs, but antagonized with the effects of IL-4. IL-2 also increases IL-4 dependent on IgE synthesis by purified B cells. On the other hand, IL-2 showed, murine IgGI-dependent inhibition of IL-4 and Ig synthesis both in vitro and in vivo. Similarly, I L-2 inhibited human IgE synthesis dependent on IL-4, by unfractionated human PBMC, but the effects were less significant than those of IFN-a or IFN-α. . Due to their ability to activate both B cells and T cells, IL-2 and I L-15 are useful in vaccination. Indeed, IL-2, as a protein and as a component of a genetic vaccine, has been shown to improve the effectiveness of vaccinations. The improvement in the specific activity and / or levels / syn- thetic expression of IL-2 and I L-15 through the use of the DNA entrainment methods of the present invention, increases the advantageous effects compared with the IL- 2 and I L-15 of the natural type. Another cytokine of particular interest for the optimization and use in genetic vaccines, according to the methods of the present invention, is interleukin-6. IL-6, is a cytokine derived from monocyte that was originally described as a B cell differentiation factor or a factor 2, B cell stimulator due to its ability to increase the levels of Ig secreted by activated B cells . I L-6 has also been shown to increase IgE synthesis induced by I L-4. Also, it has been suggested that IL-6 is a mandatory factor for the synthesis of Human IgE, because the neutralization of the anti-I L-6 mAbs completely blocked the IgE synthesis induced by IL-4. Mice with IL-6 deficiency have an uneven ability to produce IgA. Due to its potent activities in the differentiation of B cells, IL-6 can improve the levels of specific antibodies produced after vaccination. It is particularly useful as a component of DNA vaccines, because high local concentrations can be achieved, thereby providing more potent effects in the cells adjacent to the transfected cells expressing the anti-immunogenic gene. IL-6, with improved specific activity and / or with improved expression levels, obtained by means of DNA entrainment, will have more beneficial effects than the wild-type I L-6. Interleukin-8 is another example of a cytokine that, when modified according to the methods of the present invention, is useful in genetic vaccines. IL-8 was originally identified as a neurophilic chemotactic derived from a monocyte and an activation factor. Subsequently, IL-8 has also been shown to be a chemotactic for T cells and to activate basophils, resulting in improved histamine and leukotriene release from these cells. In addition, IL-8 inhibits the adhesion of neutrophils to monolayers of cytokine-activated endothelial cells, and protects these cells from neutrophil-mediated damage. Therefore endotolium cells, derived from IL-8, were suggested to decrease the inflammatory events that occur in the vicinity of the walls of blood vessels. IL-8 also modulates the production of immunoglobulin, and inhibits IgG4 induced by IL-4 and IgE synthesis by both unfractionated human PBMC and purified B cells in vitro. This inhibitory effect was independent of IFN-a, IFN-α Prostagrandin E2. In addition, I L-8 inhibited the spontaneous synthesis of IgE by PBMC derived from atopic patients. Because of its ability to attract inflammatory cells, I L-8, like other chemotactic agents, is useful in potentiating the functional properties of vaccines, including DNA vaccines (which act as adjuvants). The beneficial effects of IL-8, can be improved using the entrainment methods of the present invention, to obtain an IL-8 with improved specific activity and / or with improved expression in the target cells. Interleukin 5, and antagonists thereof, can be optimized using the methods of the present invention, for use in genetic vaccines. The I L-5 is produced mainly by TH2 type T cells, and seems to play an important role in the pathogenesis of allergic disorders, due to its ability to induce eosinophilia. IL-5 acts as a eusinophilic differentiation and survival factor, both in mice and in humans. The blocking activity of IL-5, by the use of monoclonal neutralizing antibodies, strongly inhibits pulmonary eusinophilia and hyperactivity in mouse models, and mice with IL-5 deficiency do not develop eosinophilia. These data also suggest that I L-5 antagonists may have a therapeutic potential in the treatment of allergic eosinophilia. It has also been shown that I L-5 increases both the proliferation of, and the synthesis of Ig, the activated B cells of the mouse and human. However, other studies suggested that IL-5 has no effect on the proliferation of human B cells, while activating the encinophils. The I L-5, apparently is not crucial for the maturation or differentiation of conventional B cells, due to their antibody responses in mice with deficiency of I L-5, are normal. However, these mice have a developmental defect in CD5 + B cells, which indicate that IL-5 is required for normal differentiation of this subset of B cells in the mice. At sub-optimal concentrations of IL-4, IL-5 was shown to improve IgE synthesis by human B cells in vitro. In addition, a recent study suggested that the effects of IL-5 on human cells depend on the mode of stimulation of the B cell. IL-5 significantly increased the synthesis of IgN by B cells stimulated with Moraxella catarrhalis . In addition, I L-5 energized by suboptimal concentrations of IL-2, but had no effect on Ig synthesis by B cells activated with SAC. Activated human B cells also expressed I L-5, suggesting the mRNA that IL-5 can also regulate B cell function, including the synthesis of IgE by autocrine mechanisms. The present invention provides methods for developing an IL-5 antagonist that efficiently binds to, and neutralizes IL-5 or its receptor. These antagonists are useful as components of vaccines used for the prophylaxis and treatment of allergies. Nucleic acids encoding I L-5, for example, from humans and other mammalian species, are entrained and selected for binding to immobilized IL-5R, for initial screening. Polypeptides that exhibit the desired effect in the initial screening assays, then can be selected for the highest biological activity, using assays such as, inhibition of IL-5 growth of the cultured dependent cell lines, in the presence of I L-5 recombinant natural. Alternatively, the entrained IL-5Ra chains are selected for the enhanced binding to IL-5. Tumor necrosis factors (a and ß.) And their receptors are also suitable targets for modification and use in genetic vaccines. TNF-a, which was originally described as cachecttin due to its ability to cause tumor necrosis, is a 17 kDa protein, which is produced in low amounts in almost all cells in the human body after activation. TNFa acts as an endogenous pyrogen and induces the synthesis of several proinflammatory cytokines, stimulates the production of acute phase proteins, and induces the proliferation of fibroblasts. TN Fa plays an important role in the pathogenesis of endotoxin shock. A form of membrane binding of TNF-a, (mTNF-a.) which is involved in interactions between B and T cells, is rapidly up-regulated in a period of 4 hours of T cell activation, the mTN Fa plays a role in the polyclonal activation of the B cell, observed in patients infected with HIV. Monoclonal antibodies specific for mTNFa or p55 TNFa receptor, significantly inhibit IgE synthesis, induced by clones of CD4 + T cells activated by their membranes. Mice deficient in p55 TNF-aR are resistant to endotoxic shock, and soluble TNF-aR prevents autoimmune diabetes mellitus in NOD mice. The phase I1 trials are being conducted using mTNF-aR in the treatment of rheumatoid arthritis, after the promising results obtained in the phase I trials. The methods of the present invention can be used, for example, to develop a TNF-aR, which has an improved affinity, and therefore, has the ability to act as an antagonist for TNF activity. encode TNF-aR and exhibit sequence diversity, such as, the natural diversity observed in the cDNA libraries of activated human T cells and other primates are entrained. Trailed nucleic acids are expressed, for example, in phages, after which mutants that bind to TNF-α are selected as an improved affinity. If desired, the improved mutants can be subjected to additional assays using biological activity, and the entrained genes can be subjected to one or more rounds of entrainment or selection. Another objective of interest for the application of the methods of the present invention is the interferon-α and the evolution of the antagonist of this cytokine. The IFN-α receptor consists of a glycoprotein binding component of 90 kD, an extracellular portion of 228 amino acids, a transmembrane region, and an intracellular region of 222 amino acids. For functional activity, glycolization is not required. A simple chain provides a high affinity link (10"9 -10" 10 M), but it is not sufficient for signaling. The receptor components dimerize the bond of the binder. The IFN-? of the mouse is 53% identical to that of the mouse at the amino acid level. The human and mouse receptors only link IFN-? of human and mouse, respectively. The vaccinia, cowpox and camelpox viruses have homologues of sI FN-γ R, which have a relatively low amino acid sequence similarity (-20%), but have the ability to efficiently neutralize IFN-? in vitro These homologs link the IFN-? of the human, bovine, rat (but not mouse) and may have activity in vivo as IFN-α antagonists. All eight sites are retained in the human and mouse myxoma and the Shope fibroma virus (6 in the vaccinia virus) the polypeptides of I FN-? R that indicate structures similar to 3-D. An extracellular portion of the mlFN-βR with a kD of 100 to 300 pM has been expressed in insect cells. The treatment of the NZB / W mouse (a human SLE model mouse) with msI FN-? receptor, (100 milligrams, three times a week i.p.) inhibits the presentation of glomerolonephritis. All mice treated with that sIFN-? or anti-I FN-? mAbs, were alive four weeks, after the treatment was discontinued, compared with 50% of the placebo group and 78% of killed mice treated with IFN-α. The methods of the present invention can be used to develop I FN-? R receptor polypeptides with improved affinity, and to develop IFN-α? with enhanced specific activity and improved ability to activate cellular immune responses. In each case, the nucleic acids encoding the respective polypeptides, and which exhibit a sequence diversity (for example, that observed in the cDNA libraries of activated T cells of humans and other primates), are subjected to recombination and selection to identify those recombinant nucleic acids that encode a polypeptide having an enhanced activity. In this case of the entrained I FN-? R, the entrained nucleic acid library can be expressed in phages, which are selected to identify the mutants that bind to the I FN-? with improved affinity. In the case of I FN- ?, the dragged library is analyzed for its enhanced specific activity and enhanced activation of the immune system, for example, using the activation of monocytes / macrophages as an assay. The IFN-α molecules developed, can improve the effectiveness of vaccinations (for example, when they are used as adjuvants). Diseases that can be treated using the high affinity sIFN-αR polypeptides, using the methods of the present invention, include, for example, multiple sclerosis, systemic lupus heritomatous (SLE), organ rejection after treatment, graft against host. Multiple sclerosis, for example, is characterized by the increased expression of I FN-? in the brain of patients, and the increased production of IFN-? by the T cells of the patient in vitro. Treatment of IFN-α has been shown to significantly exacerbate the disease (in contrast, with EAE in the mouse). The growth transforming factor (TGF) -β is another cytokine that can be optimized for use in genetic vaccines using the methods of the present invention. TGF-β, has regulatory activities of growth in, essentially all types of cells, and has also been shown to have complex modulatory effects on the cells of the immune system. TGF-β inhibits the proliferation of both B cells and T cells, and also suppresses the development of, and differentiation of, cytotoxic T cells and NK cells. TGF-β has been shown to direct the change of IgA, both in murine and in human B cells. It has also been shown to induce the germ line in a transcript in murine and B cells, supporting the conclusion that TGF-β can induce IgA change specifically. Because of its ability to direct the change of IgA, TGF-β is useful as a component of DNA vaccines, which aids in the induction of potent mucosal immunity, eg, vaccines for diarrhea. Also, due to its potent antiproliferative effects, TGF-β is useful as a component of therapeutic vaccines against cancer. TGF-β, with improved specific activity and / or improved / kinetic expression levels, will have increasing beneficial effects, compared to natural TGF-β. Cytokines that can be optimized using the methods of the present invention also include the granulocyte colony stimulating factor (G-CSF) and the granulocyte / macrophage colony stimulating factor (GM-CSF). These cytokines induce differentiation of the stem cell from bone marrow into granulocytes / macrophages. The administration of G-CSF and GM-CSF significantly improves the recovery of bone marrow transplantation (BM) and radiotherapy, reducing infections and the time patients have to spend in the hospital. GM-CSF increases the production of antibodies, after vaccination with DNA. G-CSF is a protein of 175 amino acids, while GM-CSF has 127 amino acids. The human G-CSF is 73% identical at the amino acid level to the murine G-CSF and the two proteins show cross-reactivity of species. G-CSF has a homodimeric receptor (dimer with kD of symbol 200 pM, monomeric ~ 2-4nM), and the receptor for GM-CSF, is a complex of three subunits. Cell lines transfected with the cDNA, which encodes G-CSF R, proliferate in response to G-CFS. The cell lines are dependent on the available GM-CSF (such as TF-1). G-CSF is not toxic and currently works very well as a drug. Nevertheless, the treatment is expensive, and the more powerful G-CFS, could reduce the cost for the patients and for the institutions to the health care. Treatments with these cytokines are generally of short duration, and it is likely that patients will never need the same treatment again, reducing the likelihood of problems with immunogenicity. The methods of the present invention are useful for the development of G-CSF and / or GM-CSF, which have an enhanced specific activity, as well as other polypeptides having G-CSF and / or GM-CSF activity. Nucleic acids G-CSF and / or GM-CSF, which have sequence diversity, for example, those obtained from the cDNA libraries, of various species, are entrained to create a G-CSF and / or GM-gene library. CSF dragged. These libraries can be selected, for example, by collecting colonies, transfecting plasmids into suitable host cells (e.g., CHO cells) and floating assays using positive receptor cell lines. Alternatively, related techniques or the phage sample can be used, again using positive receptor cell lines. Still another method of selection, comprises the transfection of the genes entrained in the cell lines dependent on G-CSF / GM-CSF. The cells are cultivated at the rate of one cell per container and / or in large flasks, of very low density; and the cells that grow fastest are selected, the entrained genes from these cells are isolated; if desired, these genes can be used for additional rounds of trawling and selection.

The neurotrophic ciliary factor (CNTF) is another suitable objective for the application of the methods of the present invention. CNTF has 200 amino acids, which exhibit 80% frequency identity between the rat and rabbit CNTF polypeptides. CNTF, has inflammatory effects similar to I L-6, and induces the synthesis of acute phase proteins. CNTF, is a cytosolic protein, which belongs to the M-family of IL-6 / IL-1 1 / LI F / oncostatin, and becomes biologically active, only after being available, either by cellular injury or by an unknown release mechanism. CNTF is expressed by the Schwann cells of myelination, astrocytes and sciatic nerve. Structurally, CNTF is a dimeric protein, with a new antiparallel distribution of subunits. Each subunit adopts a four-helix bending of double crossover, in which two propellers contribute to the dimera interface. The Lys-155 mutants lose activity, and some Glu-153 mutants have a biological activity of 5 or 10 times higher. The CNTF receptor consists of a specific CNTF receptor chain, gp130 and an LI F-β receptor. The CNTFRa chain lacks a transmembrane domain portion, instead of being an anchored GPI. At a high concentration, the CNTF can mediate the CNTFR-independent response. The soluble CNTFR binds to the CNTF and subsequently, it can bind to the LIFR and induce signaling through the gp130. CNTF increases the survival of several types of neurons, and protects neurons in an animal model of Huntington's disease (in contrast to NGF, the neurotrophic factor, and neurotrophin 3). Mice touched by the CNTF receptor, have severe deficiencies of motor neurons birth, and beaten by CNTF mice exhibit these deficiencies after birth. The CNTF also reduces obesity in mouse models. The diminished expression of CNTF is sometimes observed in psychiatric patients. Phase 1 studies in patients with ALS (annual incidence -1 / 100,000, family cases 5%, death 90% within 6 years) found significant side effects after dosages greater than 5mg / kg / day, for subcutaneous administration (including anorexia, weight loss, reactivation of herpes simplex virus (HSV1), cough, and increased oral secretions). Antibodies against CNTF were detected in almost all patients, illustrating in this way the need for an alternative CNTF with different immunological properties. The recombination and selection methods of the present invention can be used to obtain modified CNTF polypeptides, which exhibit decreased immunogenicity in vivo; The highest specific activity can also be used using these methods. The entrainment is performed, using the nucleic acids encoding the CNTF. In a preferred embodiment, a hybrid I L-6 / LIF / (CNTF) is obtained by entrainment, using an excess of oligonucleotides encoding the CNTF receptor binding sites. The phage sample can then be used to test the lack of binding to the IL-6 / LI receptor. The initial selection is followed by a high affinity binding test to the CNTF receptor, and if desired, functional tests. using cell lines that respond to the CNTF. The entrained CNTF polypeptides can be tested to identify those that exhibit reduced immunogenicity at the time of administration to a mammal. Another method in which the means of recombination and selection of the present invention which can be used to optimize CNTF can be used, is to improve the secretion of the polypeptide. When a cDNA of a CNTF, is linked operably to a leader sequence of hNGF, only 35 to 40% of CNTF produced is secreted. Objective diseases for treatment with optimized CNTF, which use either the gene entrained in an expression vector in the form of a DNA vaccine, or a purified protein, include obesity, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), diabetic neuropathy, shock and brain surgery. The polynucleotides encoding the chemokines can also be optimized, using the methods of the present invention and included in the genetic vaccine vectors. At least, three classes of chemokines are known, based on structure: C chemokines (such as lymphocycline), CC chemokines (such as, MCP-1, MCP-2, MCP-3, MCP-4, M IP- 1 a, M IP-1 b, RANTES), CXC- chemokines (such as, IL-8, SDF-1, ELR, Mig, IP10) (Premack and Schall (1996) Nature Med. 2: page 1 174). The chemokines can attract other cells that mediate the immunological and inflammatory functions, thus potentiating the immune response. Cells that are attracted to different types of chemokines include, for example, lymphocytes, monocytes, and neutrophils. Generally, C-X-C chemokines are chemoattractant for neutrophils, but not for monocytes; C-C chemokines attract monocytes and lymphocytes, but not neutrophils; C chemokines attract lymphocytes. Genetic vaccine vectors may also include optimized recombinant polynucleotides that encode accessory surface binding molecules, such as those that comprise modulation and enhancement of immune responses. These molecules, which include, for example B7-1 (CD80), B7-2 (CD86), CD40, ligand for CD40, CTLA-4, CD28 and CD150 (SLAM), can be subjected to DNA entrainment to obtain variants who have altered and / or improved activities. The optimized recombinant polynucleotides encoding the CD1 molecules are also useful in a genetic vaccine vector for certain applications. CD 1 are non-polymorphic molecules that are structurally and functionally related to MHC molecules. It is important to note that, CD1 has activities similar to M HC, and that they can function as a molecule that presents an antigen (Porcelli (1995) Adv. Immunol. 59: page 1). CD 1 is highly expressed in dendritic cells, which are highly efficient cells that present antigens. The simultaneous transfection of the target cells with the DNA vaccine vectors encoding CD 1 and an antigen of interest, are likely to promote the immunological response. Because CD1 cells, in contrast to MHC molecules, exhibit limited allelic diversity in an uncultivated population (Porcelli, supra.), Large populations of individuals with different genetic backgrounds can be vaccinated with a CD1 allele. The functional properties of the CD1 molecules can be improved by the DNA entrainment methods of the present invention. Optimized recombinant TAP genes and / or gene products can also be included in a genetic vaccine vector. The TAP genes and their optimization are explained in more detail below for several purposes. In addition, heat shock proteins (HSP), such as HSP70, can also be developed for improved presentation and processing of antigens. HCP70 has been shown to act as a coadjuvant for the induction of CD8 + T cell activation and increases the immunogenicity of specific antigenic peptides (Blachere and Associates (1997) J. Exp. Med 186: pages 1315 to 1322). When HSP70 is encoded by the genetic vaccine vector, the presentation and processing of antigenic peptides is likely to increase, thereby improving the efficacy of the genetic vaccines. DNA entrainment can be used to further enhance the properties, including the adjuvant activity, of heat shock proteins, such as HSP70. The cytokines, chemokines and polypeptides of accessory molecules produced recombinantly, as well as the antagonists of these molecules, can be used to influence the type of immune response to a given stimulus. However, the administration of polypeptides sometimes has disadvantages, including a short half-life, high cost, difficulty in storing it (it must be stored at a temperature of 4 ° C) and a requirement of high volumes. Also, bolus injections can sometimes cause side effects. The administration of polynucleotides encoding recombinant cytokines and other molecules results in most or all of these problems. DNA, for example, can be prepared in high purity, is stable, resistant to temperature, non-infectious and easy to manufacture. In addition, the cytokine polynucleotide-mediated administration can provide long-term, consistent expression, and administration of polynucleotides in general is considered safe. The functions of cytokines, chemokines and accessory molecules are redundant and pleiotropic, and therefore, it can be difficult to determine which cytokines and combinations of cytokines are the most potent in the induction and increase of specific immunological antigen responses after vaccination.

In addition, the most useful combination of cytokines and accessory molecules generally depends on different types of immune responses that are desired after vaccination. As an example, IL-4, has been shown to direct the differentiation of TH2 cells, (which produces high levels of IL-4, I L-5 and IL-13, and mediated allergic immune responses), whereas I FN-? and I L-1, direct the differentiation of TH 1 cells (which produce high levels of IL-2 and IFN-α), and immunological responses mediated delayed type. In addition, the most useful combination of cytokines and accessory molecules, is also likely to. depend on the antigen used in the vaccination. The present invention provides a solution to this problem of obtaining an optimized genetic vaccine cocktail. The different combinations of cytokines, chemokines and accessory molecules, are assembled into vectors using the methods of the present invention. These vectors are then selected for their ability to induce immune responses in vivo and in vitro. Large vector libraries, generated by gene entrainment, and by combination molecular biology, are selected to see the maximum capacity to direct the immunological responses towards, for example, as desired, the TH 1 or TH 2 phenotype. A library of different vectors can be generated, assembling the different evolved promoters, cytokines (evolved), cytokine antagonists (evolved), chemokines (evolved), accessory (evolved) molecules and immunostimulatory sequences, and each of which can be prepared using the methods of the present invention. DNA sequences and compounds that facilitate transfection and expression can also be included. If the pathogen (s) is known, specific DNA sequences encoding the immunogenic antigens of the pathogen can be incorporated into these vectors, providing a protective immunity against the pathogen (s) (as in genetic vaccines). The initial selection is preferably carried out in vitro. For example, the library can be introduced into cells that are tested for their ability to induce differentiation of T cells, capable of producing cytokines that are indicative of the type of immune response desired. For a TH 1 response, for example, the library is selected to identify recombinant polynucleotides that have the ability to induce T cells to produce IL-2 and IFL-α, while inducing the production of T cells from I L-4, IL-5 and IL-13, to identify the recombinant polynucleotides that favor the TH2 response. The selection can also be carried out in vivo, using animal models. For example, vectors produced using the methods of the present invention can be tested for their ability to protect animals against lethal infections. The other selection method includes the injection of Leshmania major parasites, in stages in the BALB / c mice (non-curative). The sets of plasmids i.v. are injected. , i.p. , or in stages for these mice and the size of the remaining stages are followed. Still another method of selection in vivo, comprises the detection of IgE levels, after infection with Nippostrongylus brasiliensis. High levels indicate a TH2 response, while low IgE levels indicate a TH 1 response. Successful results in animal models are easy to verify in humans. The in vitro selection can be carried out, to test the human TH 1 or TH 2 phenotype, or for other desired immune responses. The ability of vectors to induce protection against infections in humans can also be tested. Because the principles of immunological functions are similar in a wide variety of infections, immunostimulatory DNA vaccine vectors are not only useful in the treatment of a number of infectious diseases, but also in the prevention of infections, when the vectors are administered to the entry sites of the pathogen (for example, the lung or the intestine). 3. Cellular Receptor Agonists or Antagonists. The present invention also provides methods for obtaining optimized recombinant polynucleotides, which encode a peptide or polypeptide that can interact with a cellular receptor that is involved in the mediation of an immune response. The optimized recombinant polynucleotides can act as an agonist or a receptor antagonist. Cytokine antagonists can be used as components of genetic vaccine cocktails. Immunosuppressive blocking cytokines, instead of adding simple proinflammatory cytokines, are likely to potentiate the immune response in a more general way, because several trajectories are enhanced at the same time. By appropriate selection of the antagonist, the immune response induced by a genetic vaccine can be designed, in order to obtain the response that is most effective to achieve the desired effect. Antagonists against any cytokine can be used as appropriate; Particular cytokines of interest to include blocking are, for example, IL-4, I L-13, IL-10 and the like. The present invention provides methods for obtaining cytokine antagonists, which have a greater effectiveness in blocking the action of the respective cytokine. Polynucleotides encoding improved cytokine antagonists can be obtained by the use of gene screening to generate a recombinant library of polynucleotides, which are then selected to identify those that encode an improved antagonist. They can be used as substrates for DNA screening, for example, polynucleotides that encode receptors for the respective cytokine. In the recombination reaction, at least two forms of substrates will be present, each form deviating from the other, in at least one position of the nucleotide. In a preferred embodiment, the different forms of nucleotides are homologous cytokine receptors of different organisms. The resulting library of recombinant polynucleotides is then selected to identify those encoding the cytokine antagonist with the desired affinity and biological activity. The I L-10, will be explained as an example of the effect that can be achieved by including a cytokine antagonist in a genetic vaccine cocktail, as well as how the effect can be improved using the DNA trapping methods of the present invention. Interleukin 10 (IL-10) is perhaps the most potent anti-inflammatory cytokine known to date. IL-10 exhibits a number of trajectories that effect the potentiation of inflammatory responses. The biological activities of I L-10 include, inhibition of M HC class II expression in monocytes, inhibition of the production of I L-1, IL-6, IL-12, TNF-a by monocytes / macrophages and the inhibition of proliferation and production of IL-2 by T lymphocytes. The importance of I L-10, as a regulatory molecule of immunological and inflammatory responses, was clearly demonstrated in a mouse with I L deficiency. -10. These mice are bred, retarded, anemic and spontaneously develop inflammatory bowel disease (Kuhn and Associates (1993) Cell 75: page 263). In addition, both innate and acquired immunity to Listeria monocytogenes were shown to be elevated in mice with I L-10 deficiency (Dai and Associates (1997) J. Immunol., 158: page 2259). It has also been suggested that genetic differences in the levels of IL-10 production may affect the risk of patients dying from meningococcal infections. Families with a high production of I L-10, had the risk increased by a probability of 20, of a fatal result of meningococcal disease (Westendorp and Associates (1997) Lancet 349: page 170). It has been shown that IL-10, activates normal and malignant B cells in vitro, but does not appear to be a major promoter of cytokine growth for in vivo cells, because mice deficient in I L-10 have normal levels of B lymphocytes and Ig in your circulation. In fact, there is evidence that IL-10 can down-regulate the function of the B cell through the inhibition of the accessory cell function of monocytes. However, it seems that I L-10 plays a role in the growth and expansion of malignant B cells. It has been shown that monoclonal IL-10 antibodies and I L-10 anti-perception oligonucleotides inhibit the transformation of B cells by EBV in vitro. In addition, B-cell lymphomas are associated with EBV, and the majority of EBV + lymphomas produce high levels of IL-10, which is derived from both human genes and coded I L-10 homologs. by the EBV. The B-cell lymphomas, related to AIDS, also secrete high levels of I L-10. In addition, patients with an IL-10 serum detectable at the time of lymphoma diagnosis, which is not intermediate / high-grade Hodgkin's, have a short survival, suggesting additionally a role of I L-10 in the pathogenesis of the cells B malignant Antagonization of I L-10 in vivo can be beneficial in several infections and in malignant diseases and in vaccination. The effect of blockade of I L-10, is an improvement of the immune response that depends on the specificity of the response. This is useful in vaccinations and in the treatment of serious infectious diseases. In addition, an I L-10 antagonist is useful in the treatment of B cell diseases, which exhibit a production of IL-10 and viral I L-10, and may also be useful in promoting the overall anti-immune response. - tumors in cancer patients. The combination of an I L-10 antagonist with gene therapy vectors may also be useful in the genetic therapy of tumor cells in order to obtain a maximal immune response against the tumor cells. If entrainment of I L-10 results in an IL-10 with enhanced specific activity, this IL-10 molecule would have potential in the treatment of autoimmune diseases and inflammatory bowel diseases. IL-10 with enhanced specific activity may also be useful as a component of gene therapy vectors in reducing the immune response against vectors, which are recognized by cell memory and may also reduce the immunogenicity of these vectors. An I-L-10 antagonist has been made, through the generation of a soluble form of the I-L-10 receptor (slL-10R; Tan and Associates (1 995) J. Biol. Chem. 270: page 12906). However, the sIL-I OR is linked in I L-10 with 50 pM kD, while the wild-type linked surface receptor has an affinity of 35 to 200 pM. As a result, the molar excess of 151 folds of sIL-10R, is required for a maximum average inhibition of the biological function of I L-10. In addition, the affinity of viral IL-10 (monologue of I L-10 encoded by Epstein-Barr virus), to sIL-10R is moved 1000 fold less than hl L-10, and in some situations, such as when treating malignant B cells associated with EBV, it can be beneficial if the function of viral IL-10 can be blocked. Taken together, this soluble form of I L-10R is not likely to be effective in antagonizing I L-10 in vivo. To obtain an IL-10 antagonist having sufficient affinity and antagonist activity to function in vivo, DNA entrainment can be performed using polynucleotides encoding the I L-10 receptor. The IL-10 receptor with higher than normal affinity will function as an I-L-10 antagonist, because it significantly reduces the amount of I L-10 available for binding to the wild type I L-10R. functional. In a preferred embodiment, I L-10R is entrained, using a homologous cDNA encoding I L-10R derived from human and other mammalian species. An alignment of IL-10 receptor sequences from humans and mice is illustrated in Figure 14 in order to illustrate the possibility of DNA family entrainment when developed in IL-10 receptors with improved affinity. A phage library of recombinant IL-10 receptors can be selected for the enhanced binding of IL-10R entrained in humans or viral IL-10. The natural IL-10 and / or viral IL-10 are added in increasing concentrations upon request, to have a greater affinity. The phage bound to I L-10 can be recovered using anti-IL-10 monoclonal antibodies. If desired, entrainment can be repeated one or more times, after which the developed soluble L-10R is analyzed, in functional tests to determine its ability to neutralize the biological activities of IL-10 / IL-10. viral. More specifically, the soluble I L-10R developed, is studied to determine its ability to block the inhibitory effects of IL-10 in the synthesis of cytokine and M HC class II expression, by monosites, T cell proliferation, and for its ability to inhibit the increasing effects of IL-10, on the proliferation of B cells activated by anti-CD40 monoclonal antibodies. An antagonist of I L-10 can also be generated by developing IL-10 to obtain variants that bind to the IL-10R, with greater affinity of the wild type, but without activation of the receptor. The advantage of this method is that an I L-0 molecule can be developed with an improved specific activity using the same methods. In a preferred embodiment, I L-10 is entrained using homologous cDNAs, which purify I L-10 derived from human and other mammalian species. In addition, a gene encoding viral IL-10 can be included in the entrainment. A library of recombinants of IL-10 is selected seeking the enhanced binding to the human I L-10 receptor. Members of the library linked to I L-10R can be recovered by anti-IL-10R monoclonal antibodies. This selection protocol is likely to result in molecules I L-10 with both antagonist or agonist activities. Due to the demands of initial selection for a higher affinity, it is likely that a proportion of the agonists have an enhanced specific activity, when compared to natural human IL-10. The functional properties of the mutant I L-10 molecule are determined in biological assays similar to those described above for the ultra high affinity I L-10 receptors (cytokine synthesis and MHC class II expression by monosites, proliferation of B cells and T). A mutant IL-4 antagonist has been generated previously, illustrating the general possibility of the method (Kruse and Associates (1992) EMBO J. 1 1: pages 3237 to 3244). A mutation of the amino acid in IL-10 resulted in a molecule that binds efficiently to the I chain L-4Ra, but has a minimal agonist activity such as I L-4.

Another example of an IL-10 antagonist is I L-207 / MDA-7, which is a secreted protein of 206 amino acids. This protein was originally characterized as mda-7, which is a melanoma cell derivative of the growth regulator of tumor cells (Jiang and Associates (1995) Oncogene 1 1: page 2477; (1996) Proc. Nat ' 1. Acad. Sci. USA 93: page 9160). IL-20 / mda-7, is structurally related to I L-10, and antagonizes several functions of I L-10 (extract from the thirteenth European immunology convention, Amsterdam, June 22 to 25, 1997). In contrast to IL-10, IL-20 / mda-7, increases the expression of CD80 (B7-1) and CD86 (B7-2), in human monocytes and regulates the production of TNF-a and I L- 6 The I L-20 / mda-7 also improves the production of I FN- ?, by means of the PBMC activated by PHA. The present invention provides methods for improving genetic vaccines by incorporating I L-20 / mda-7 genes into the genetic vaccine vectors. The methods of the present invention can be used to obtain variants of IL-20 / mda-7 that exhibit improved capacity for IL-10 antagonist activity. When a cytokine antagonist is used as a component of DNA vaccine or gene therapy vectors, the maximum local effect is desirable. Therefore, in addition to a soluble form of the cytokine antagonist, a transmembrane form of the antagonist can be generated. The soluble form can be provided in the form of a purified polypeptide to the patient, by, for example, intravenous injection. Alternatively, a polynucleotide encoding the cytokine antagonist can be used as a component in a genetic vaccine or a gene therapy vector. In this case, either or both of the forms, soluble and transmembrane, can be used. In cases where both the soluble and transmembrane forms of the antagonist are encoded by the same vector, target cells express both forms, resulting in maximum inhibition of cytokine function on the surface of the target cell and its immediate neighborhood. The peptides or polypeptides obtained by the use of these methods can replace the natural ligands of the receptors, such as cytokines or other molecules customary in their ability to exert an effect on the immune system., through the receiver. A potential disadvantage of the administration of cytokines or other customary molecules themselves, is that the autoimmune reaction could be induced against the natural molecule, either due to breakage tolerance (if a natural cytokine or other molecule is used) or inducing cross-reactive immunity (humoral or cellular) when related molecules are used, but different. Through the use of the methods of the present invention, agonists or antagonists can be obtained which avoid these potential disadvantages. For example, relatively small peptides can be used, such as agonists that can mimic the activity of the natural immunomodulator, or antagonize activity activity, without inducing cross-reactive immunity in the natural molecule. In a preferred embodiment of the present invention, the optimized agonist or antagonist obtained through the use of the methods of the present invention is about 50 amino acids of length or less, preferably of about 30 amino acids or less, and more preferably of about 20 amino acids of length or less. The agonist or antagonist peptide is preferably at least about 4 amino acids in length, and more preferably at least about 8 amino acids in length. The polynucleotides that flank the mimetic peptide coding sequence can also be optimized using the methods of the present invention, in order to optimize the expression, conformation or activity of the mimetic peptide. Optimized agonist or antagonist peptides or polypeptides are obtained by generating a library of recombinant polynucleotides and selecting the library to identify those that encode a peptide or polypeptide that exhibits an improved ability to modulate an immune response. The library can be produced, using methods such as DNA entrainment or other methods described in the present invention or otherwise known to those skilled in the art. The selection is conveniently performed, by expressing the peptides encoded by the members of the library on the surface of a population of replicable genetic packets, and identifying those members that bind to a member of interest, eg, a receptor. The optimized recombinant polynucleotides that are obtained using the methods of the present invention can be used in various ways. For example, the polynucleotide in a genetic vaccine vector, under the control of the appropriate expression control sequences, so that the mimetic peptide is expressed at the introduction of the vector in a mammal. If desired, the polynucleotide can be placed in the vector, supported on the coding sequence of the surface protein (for example, gene 11 or gene VI I I), in order to preserve the conformation of the mimetic. Alternatively, the polynucleotide encoding the mimetic can be inserted directly into an antigenic coding sequence of the genetic vaccine to form a coding sequence for a "mimotope on antigen" structure. The polynucleotide encoding the mimotope on the structure of the antigen can be used within the genetic vaccine, or it can be used to express a protein that is administered by itself as a vaccine. As an example of this type of application, the coding sequence of a mimetic peptide is introduced into a polynucleotide encoding the "M circuit" of the hepatitis B surface antigen (HBsAg). The M circuit is a sequence of six amino acid peptides linked by system residues, which is found in amino acids 139 to 147 (numbering within the S protein sequence). The M circuit, in the natural HBsAg protein, is recognized by the monoclonal antibody RFHB7 (Chen and Associates, Proc. Nat'l. Acad. Sci. E. U.A. , 93: pages 1997 to 2001 (1996)). According to Chen and Associates, circuit M forms an epitope of HBsAG, which does not overlap and separates from at least four other H BsAg epitopes. Due to the probable Cys-Cys disulfide bond, in the hydrophilic part of the protein, the cyclic conformation of the amino acids from 139 to 147 is likely. Therefore, this structure is similar to that found in the filamentous protein regions. phage plll and pVI II, where the mimotope sequences are placed. Therefore, a mimotope can be inserted, using the methods of the present invention in this region of the amino acid sequence HBsAg. The chemokine receptor CCR6 is an example of a suitable target for a mimetic peptide obtained using the method. The CCR6 receptor is a 7 transmembrane domain protein (Dieu and Associates, Biochem Biophys, Res. Comm. 236: pages 212 to 217 (1997) and J. Biol. Chem. 272: pages 14893 to 14898 (1997)) which is involved in the chemoattraction of immature dendritic cells, which are found in the blood and migrate to antigenic sites (Dieu and Associates, J. Exp. Med. 188: pages 373 to 386 (1998)). CCR6 binds the chemokine MI P-3a, so that a mimetic peptide that has the ability to activate CCR6, can provide an additional chemoattractant fusion to a particular antigen and, thus, promote taking by means of dendritic cells after immunization, with the antigen fusion of the mimetic antigen, or a DNA vector that expresses the antigen. Another application of this method of the present invention is to obtain molecules that can act as agonists for the cleaning macrophage receptor (MSR; see, Wloch and Associates, Hum. Gene Ther. 9: pages 1439 to 1447 (1998)). The MSR is involved in mediating the effects of various immunomodulators. Among these bacterial DNAs, the plasmids used in DNA vaccination and oligonucleotides are included, which are often potent immunostimulants. Oligonucleotides of a certain chemical structure (eg, posfotio-oligonucleotides) are particularly potent, whereas bacterial or plasmid DNA must be used in relatively large amounts to produce an effect. The ability of oligonucleotides containing dG residues to stimulate B cells and increase the activity of immunostimulatory CpG motifs and lipopolysaccharides to activate macrophages is also mediated by MSR. Some of these activities are toxic. Each of these immunomodulators, together with a variety of polyanionic ligands, bind to the MSR. The methods of the present invention can be used to obtain mimetics of one or more of these immunomodulators that bind to the MSR with high affinity, but suffer from toxic properties. Said mimetic peptides are useful as immunostimulants or adjuvants. The MSR is a trimeric integral membrane glycoprotein. The three extracellular regions of terminal C, rich in cysteine, are connected to the transmembrane domain, by means of a fibrous region that is composed of a helical-a tube and a triple helix similar to collagen (see, Kodama and Associates, Nature 343 : pages 531 to 535 (1990)). Therefore, the selection of the library of recombinant polynucleotides can be carried out, expressing the structure of the extracellular receptor and artificially attaching it to plastic surfaces. Libraries can be expressed, for example, by phage sample, and selected to identify those that bind to receptors with high affinity. The optimized recombinant polynucleotides, identified by means of this method, can be incorporated into antigenic coding sequences to evaluate their modulatory effect on the immune response. 4. - Co-stimulant molecules capable of inhibiting or increasing the activation, differentiation or anergy of antigen-specific T cells. Also provided are methods for obtaining optimized recombinant polynucleotides which, when expressed, have the ability to inhibit or increase the activation, differentiation or anergy of antigen-specific T cells.

Activation of the T cell is initiated, when the T cell recognizes its specific antigenic peptides in the context of the MHC molecule in the plasma membrane of the antigen presenting cells (APC), such as monocytes, dendritic cells (DC), Langerhands cells or B cells. Activation of CD4 + T cells requires recognition by the T cell receptor (TCR) of an antigenic peptide, in the context of MHC class II molecules, while CD8 + T cells, recognize the peptides, in the context of MHC class I molecules. However, it is important to know that the recognition of antigenic peptides is not sufficient for the induction of T cell proliferation and cytokine synthesis. An additional costimulant signal is required, "the second signal". The co-stimulatory signal is mediated by means of CD28, which binds to its ligands B7-1 (CD80) or B7-2 (CD86), generally expressed in the antigen presented by the cells. In the absence of the co-stimulatory signal, the activation of the T cell does not occur, or the T cells become anergic. In addition to CD28, CTLA-4 (CD 152) also functions as a ligand for B7-1 and B7-2. However, in contrast to CD28, CTLA-4 mediates a negative regulatory signal for T cells and / or induces anergy and tolerance (Walunas and Associates (1994) Immunity 1: page 405; Karandikar and Associates (1996) J Exp. Med. 184: page 783). It has been shown that B7-1 and B7-2, have the ability to regulate several immune responses, and have been implicated as important in the immunological regulation in vaccines, allergy, autoimmunity and cancer. Genetic therapy or gene vaccine vectors expressing B7-1 and / or B7-2 have also shown that they have a therapeutic potential in the treatment of the aforementioned diseases and to improve the effectiveness of genetic vaccines. Figure 10 illustrates the interaction of APC and CD4 + T cells, but the same principle is true with CD8 + T cells, with the exception that T cells recognize antigenic peptides in the context of MHC class I molecules. Both B7-1 and B7-2 are linked to CD28 and CTLA-4, even though the sequence similarities between these four molecules are very limited (from 20 to 30%). It is desirable to obtain mutations in B7-1 and B7-2, which only influence the binding to one ligand, but not the other, or improve the activity through a ligand, while decreasing the activity through the other . Furthermore, since the affinities of the B7 molecules to their ligands appear to be relatively low, it would be desirable to discover mutations that improve / alter the activities of the molecules. However, rational designs do not make predictions of useful mutations possible due to the complexity of the molecules. The present invention provides methods for solving these difficulties, making it possible to generate and functionally identify the different B7 molecules, with relative altered capacities to induce T cell activation, differentiation, cytokine production, anergy and / or tolerance. Through the use of the methods of the present invention, mutations can be found in B7-1 and B7-2, which only influence the binding to one ligand but not the other, or that improve the activity through a ligand. while they decrease the activity through the other. It is likely that DNA entrainment is the most powerful method in the discovery of new variants of the B7, with link capabilities relatively altered to CD28 and CTLA-4. The variants of B7, which act through CD28 with enhanced activity (and with decreased activity through CTLA-4), are expected to have improved ability to induce T cell activation. In contrast, the variants of B7, which are linked and act through CTLA-4 with enhanced activity (and with decreased activity through CD28) are expected to be potent negative regulators of T cell functions and induce tolerance and the anergy. DNA drag or other combination method is used to generate variants of B7 (for example, B7-1 / CD80 and B7-2 / CD86), which have altered relative capacity to act through CD28 and CTLA -4, when compared to the wild-type B7 molecules. In a preferred embodiment, the different forms of the substrate used in the recombination reaction are B7 CADs, coming from several species. Said cDNAs can be obtained by methods known to those skilled in the art, including RT-PCR.

Generally, the genes encoding these variant molecules of B7 are incorporated into the gene vaccine vectors encoding an antigen, so that the vector can be used to modify the responses of specific antigen T cells. The vectors that harbor the B7 genes that act efficiently through CD28 are useful in the induction, for example, protective immune responses, while the vectors harboring the genes that encode the B7 gene, which act efficiently through the CTLA -4, are useful in the induction, for example, tolerance and anergy of allergen-specific or self-antigenic T cells. In some situations, such as in tumor cells or cells that induce antimmunological reactions, the antigen may already be present on the surface of the target cell, and the variant B7 molecules may be transfected in the absence of an antigen gene. additional exogenous Figure 11, illustrates a selection protocol that can be used to identify variants B7-1 (CD80) and / or B7-2 (CD86) that have an increased / ability to induce activation or anergy of the T cell, and the application of this strategy is described in greater detail in Example 1. Several methods can be used for the selection of this variant. For example, flow selection systems, based on cytometry, can be used. The library of molecules B7-1 and B7-2 is transfected into cells that do not generally express these molecules (for example, COS-7 cells and any cell line from species other than humans with limited or non-crossed reactivity, with respect to the linkage). of ligand B7). An internal marker gene can be incorporated in order to analyze the number of copies per cell. The soluble CTLA-4 and CD28 molecules can be generated for use in the flow cytometry experiments. Generally, these will be fused with the Fc portion of the IgG molecule to improve the stability of the molecules and to make spotting easy by anti-IgG mAbs, as described by van der Merwe and Associates (J. xp. 185 to page 393, 1997). The cells transfected with the library of B7 molecules can then be stained with the soluble CTLA-4 and CD28 molecules. Cells that demonstrate an increased or decreased binding ratio of CTLA-4 / CD28 will be classified. Then, the plasmids are recovered, and the coding sequences of the variant B7 entrained are identified. These selected B7 variants can then be subjected to further rounds of trawling and selection, and / or can be further analyzed, using functional tests, as described below. The B7 variants can also be selected directly, based on their functional properties. For in vivo studies, B7 molecules can also be developed to work in mouse cells. Bacterial colonies with plasmids with mutant B7 molecules are selected and the plasmids are isolated. These plasmids are then transfected into cells that present the antigen, such as dendritic cells, and the capacities of these mutants are analyzed to activate T cells. One of the advantages of this method is that assumptions are not made with regarding binding affinities or specificities to known ligands, and possibly, new activities can be discovered, through the ligands that are still to be identified. In addition to dendritic cells, other cells that are relatively easy to transfect (eg, U937 or COS-7), can be used in selection, as long as the "first T-cell signal" is induced by, for example, anti-CD3 monoclonal antibodies. Activation of the T cell can be analyzed by methods known to those skilled in the art, including, for example, the measurement of proliferation, cytokine production, CTL activity or expression of activation antigens, such as the IL-2 receptor, CD69 or the HLA-DR molecules. Through the use of antigen-specific T cell clones, such as the T cells specific for the antigen of the tiny powder of the Der p I house, they will allow the analysis of the activation of antigen-specific T cells (Yssel and Associates (1992)). J. Immunol., 148: pages 738 to 745). One can identify those mutants that increase or exhibit T cell proliferation, or increase or exhibit the CTL response. In a similar way, variants that have altered the ability to induce cytokine production or the expression of activation antigens, as measured, for example, by the cytokine-specific ELISA test or the cytometry The variants of B7 are useful in the modulation of immunological responses in autoimmune diseases, allergy, cancer, infectious diseases and vaccination. Variants of B7, which act through CD28 with enhanced activity (and with decreased activity through CTLA-4) will have an enhanced ability to induce T cell activation. In contrast, the variants of the B7, which are linked and act through CTLA-4 with enhanced activity (and with decreased activity through CD28), will be potent negative regulators of T cell functions and to induce tolerance and anergy. Thus, by incorporating the genes encoding these variants of B7 molecules into the gene vaccine vectors encoding an antigen, it is possible to modify the response of antigen-specific T cells. The vectors harboring B7 genes that act efficiently through CD28 are useful for induction, for example, protective immunological responses, while vectors harboring genes encoding B7 genes that efficiently act through CTLA-4 are useful. for the induction, for example, tolerance and anergy of the allergen T cells or self antigen. In some situations, such as in tumor cells or in cells that induce autoimmune reactions, the antigen may already be present on the surface of the target cell, and the variant of the B7 molecule may be transfected in the absence of a gene additional exogenous antigen. The methods of the present invention are also useful for obtaining variants of B7 that have an increased effectiveness in driving the differentiation of either TH1 or TH2 cells. Differential roles have been observed for B7-1 and B7-2 molecules in the regulation of the differentiation of helper T cells (TH) (Freeman and Associates (1995) Immunity 2: page 523; Kuchroo and Associates (1995) Cell 80: page 707). Differentiation of the TH cell can be measured by analyzing the cytokine production profiles induced by any particular variant. High levels of I L-4, IL-5 and / or IL-13 are indicative of efficient differentiation of the TH2 cell while high levels of I FN-? or IL-2, can be used as a marker of differentiation of the TH 1 cell. The variants of B7, with altered capacity to induce the differentiation of TH 1 or TH 2 cells, are useful, for example, in the treatment of allergic, malignant, autoimmune and infectious diseases and in vaccination. The present invention also provides methods for obtaining variants of B7, which have an increased ability to induce the production of IL-10 by specific antigen T cells. The high production of I L-10, is a characteristic of regulatory T cells, which can suppress the proliferation of antigen-specific CD4 + T cells (Groux and Associates (1997) Nature 389 to page 737). DNA entrainment is carried out as described above, after which the recombinant nucleic acids encoding the B7 variants, which have an increased induction capacity of IL-10, can be identified by, for example , ELISA test or cytometry flow using intracytoplasmic cytokine staining. Variants that induce high levels of I L-10 production are useful in the treatment of allergic and autoimmune diseases.

D.- Optimization of Transport and Presentation of Antigens. The present invention also provides methods for obtaining genetic vaccines and accessory molecules that can improve the transport and presentation of antigenic peptides. A library of recombinant polynucleotides is created and selected to identify those that encode the molecules that have improved properties compared to their wild-type counterparts. The polynucleotides themselves, can be used in genetic vaccines, or the products of the polynucleotide gene, can be used for therapeutic or prophylactic applications. 1. Proteasomes. The peptides of class I presented in complex molecules of major histocompatibility are generated by cellular proteasomes. The gamma interferome can stimulate the presentation of the antigen, and part of the mechanism of action of the interferon may be due to the induction of the proteasome beta, subunits LMP2 and LMP7, which replace the homologous subunits beta Y (delta) and X ( epsilon). Said replacement changes the cleavage specificity of the proteasome peptide and may increase the immunogenicity of the class I epitope. The Y (delta) and X (epsilon) subunits, as well as other newly discovered proteasome subunits, such as the homologous MECL-1 of MC14, are characteristic of cells that are not specialized in antigen presentation. Therefore, incorporation into cells by the transfer of DNA from LMP2, LMP7, M ECL-1 and / or other specific epitopes of presentation and potentially interfering subunits of interferon, may increase epitope presentation. It is likely that the peptides generated by the proteasome containing the interferon-inducing subunits are transported to the endoplasmic reticulum by means of the TAP molecules. The present invention provides methods of obtaining proteasomes, which have an increased or decreased ability to specifically process the MHC class I epitopes. According to the methods, DNA entrainment is used to obtain developed proteins that can either have new specificities which could increase the immunogenicity of some proteins and / or increase the activity of the subunits, once they are linked to the proteasome Because the transition from a nonspecific proteasome to a class I epitope-specific proteasome may pass through several conditions (in which some, but not all, interferon-inducing subunits are associated with the proteasome), potentially achieve many different proteolytic specificities. Therefore, the development of specific subunits similar to LMP, can create new compositions of proteasomes, which have improved functionality for the presentation of epitopes. The methods comprise the performance of DNA entrainment, using as substrates two or more forms of polynucleotides, which encode the proteasome components, wherein the forms of the polynucleotides differ by at least one nucleotide. The entrainment is carried out as described in the present invention, using polynucleotides that encode any one or more of the different proteasome components, including, for example, LMP2, LMP7, MECL-1 and other individual proteasome components that they are specifically included in the presentation of the class I epitope. Examples of suitable substrates are described in, for example, Stohwasser and Associates (1997) Eur. J. Immunol. 27: pages 1 182 to 1187 and Gaczynska and Associates (1996) J. Biol. Chem. 271: pages 17275 to 17280. In a preferred embodiment, family trawling is used, in which, the different substrates are components of proteasomes which encode polynucleotides of different species.

After the recombination reaction has been terminated, the resulting library of recombinant polynucleotides is selected to identify those encoding the proteasome components having the desired effect in the production of the class I epitope. For example, recombinant polynucleotides can be introduced into a genetic vaccine vector, which also encodes a "4 <; -. particular of interest. The vector library can then be introduced into mammalian cells, which are then selected to identify cells that exhibit increased specific antigenic immunogenicity. Methods of analysis of proteasome activity are described in, for example, Groettrup and Associates (1997) Proc. Nat'l. Acad Sci. E. U.A. 94: pages 8970 to 8975 and Groettrup and Associates (1997) Eur. J. Immunol. 26: pages 863 to 869. Alternatively, the methods of the present invention can be used to develop proteins, which bind strongly with proteasomes, but which have a decreased activity or do not have activity, thus antagonizing the activity of the proteasome and decreasing the ability of cells to present class I molecules. Said molecules can be applied to genetic therapy protocols in which it is desirable to decrease the immunogenicity of exogenous proteins expressed in cells as a result of genetic therapy, and which would otherwise be processed for the presentation of the Class I, allowing the cell to be recognized by the immune system. Said subunits of high affinity and low activity, similar to the LMP, will demonstrate immunosuppressive effects, which are also useful in other therapeutic protocols, where the cells that express a protein that is not of themselves, need to be protected from a response immunological The specificity of the proteasome and the TAP molecules (will be explained below), may have developed together naturally. Therefore, it may be important that two trajectories of the processing system of class I be collated in a functional manner. A further aspect of the present invention comprises carrying out DNA tracing simultaneously in two gene families, followed by random combinations of the two, in order to discover the appropriately collated proteolytic and transport specificities. 2. Antigen transport. The present invention provides methods for improving the transport of antigenic peptides from the cytosolic compartment to the endoplastic reticulum, and thereby to the surface of cells in the context of MHC class I molecules. Improved expression of antigenic peptides , results in improved immune response, particularly in the enhanced activation of CD8 + cytotoxic lymphocytes. This is useful in the development of DNA vaccines and in gene therapy. In one embodiment, the present invention comprises the development of TAP genes (carriers associated with antigen processing) to obtain genes that exhibit enhanced antigen presentation. The TAP genes are members of the ATP binding cassette family of the membrane transceivers. These proteins transport the antigenic peptides to the M HC class I molecules and are included in the expression and stability of the MHC class I molecules on the cell surface. To date, two TAP genes have been cloned, TAP 1 and TAP2 (Powis and Associates (1996) Proc. Nat'l. Acad. Sci. USA 89: pages 1463 to 1467; Koopman and Associates (1997) Curr. Opin Immunol., 9: pages 80 to 88; Monaco (1995) J. Leukocyte Biol. 57: pages 543 to 557). TAP1 and TAP2 form a heterodimer, and these genes are required for the transport of the peptides in the endoplasmic reticulum, where they bind to MHC class I molecules. The essential role of TAP gene products in the presentation of antigenic peptides was demonstrated in mice with disordered TAP genes. A deficient mouse of TAP1 has drastically reduced levels of surface expression of MHC class I molecules, and a positive selection of CD8 + T cells in the thymus, which is greatly reduced. Therefore, the number of CD8 + T lymphocytes in the periphery of the TAP deficient mouse is extremely low. Re-transferring the TAP genes to these cells restores the level of expression of the MHC class I molecule. The TAP genes are a good target for genetic trawling, due to the natural polymorphism since these genera of several mammalian species have been cloned and sequenced, including humans (Beck and Associates (1992) J. Mol. Biol. 228: pages 433 to 441, Genbank Accession No. Y13582, Powis and Associates, supra.), TAP1 of the gorilla (Lute and Associates (1996) Humman Immunol.50: pages 91 to 102), mouse (Reiser and Associates (1998) Proc. Nat'l. Acad. Sci. USA 85: pages 2255 to 2259; Marusiña and Associates (1997) J. Immunol. 158: pages 5251 to 5256, TAP1: Genbank Accession numbers U60018, U60019, U60020, U60021, U60022 and L76468- L67470; TAP2: Genbank Accession numbers U60087, U60088, U60089, U60090, U60091 and U60092), hamster (TAP1, Genbank Accession numbers AF001 154 and AF001 157, TAP2, Genbank Accession numbers AF001 156 and AF001 155). In addition, it has been shown that the point of mutations in the TAP genes can result in the specificity of the altered peptide and in the presentation of the peptide. Also, functional differences in TAP genes, derived from different species have been observed. For example, the human TAP and the rat TAP, which contain the rTAP2a allele, are rather promiscuous, whereas the mouse TAP is restrictive and selects against the peptides with small C-terminus, polar, hydrophobic or positively charged with amino acids. . The basis for this selectivity is unknown.

The methods of the present invention comprise carrying out the DNA tracing of the TAP 1 and TAP2 genes, using as substrates, at least, two forms of TAP 1 and / or TAP2 polynucleotide sequences, which differ in, at least, a position of the nucleotide. In a preferred embodiment, the TAP sequence derived from different mammalian species are used as substrates for entrainment. The natural polymorphism of the genes can provide additional diversity of the substrate. If desired, TAP optimized genes, obtained from a trawl and selection round, can be subjected to additional rounds of trawl / selection, to obtain optimized nucleotides that encode the TAP. To identify the optimized polynucleotides encoding the TAP of a library of recombinant TAP genes, the genes can be expressed in the same plasmid as a target antigen of interest. If this step is limiting the point of antigen presentation, then it will result in an improved presentation for CD8 + CTL. The TAP mutants can selectively act to increase the expression of a particular antigen peptide fragment, for which expression levels are being otherwise limited or to cause the transport of a peptide, which would normally never be transferred into a peptide. RER and made available to link with the M HC class I. When used in the context of gene therapy vectors in cancer treatments, the developed TAP genes provide a means to improve the expression of the M HC class I molecules in the tumor cells and obtain an efficient presentation of the antigenic peptides. specific to the tumor. In this way, vectors containing the developed TAP genes can induce potent immune responses against malignant cells. Trailed TAP genes can be transferred to malignant cell lines that express low levels of MHC class I molecule using vectors or retroviral electroporation. The efficiency of transfection can be monitored using marker genes, such as the green fluorescent protein, encoded by the same vector as the TAP genes. Cells that express equal levels of green fluorescent protein, but higher levels of MHC class I molecules, as an efficient marker of TAP genes, are then classified, using the flow cytometry, and the developed TAP genes are then recovered from these cells, by means of, for example, PCR or by the recovery of the complete vectors. These sequences can then be subjected to new rounds of entrainment, selection and recovery, if further optimization is desired. The molecular evolution of TAP genes can be combined with the simultaneous evolution of the desired antigen. The simultaneous evolution of the desired antigen can further improve the presentation efficiency of the antigenic polypeptide, after DNA vaccination. The antigen can be developed, using gene entrainment, to contain structures that allow optimal presentation of the desired antigenic peptide, when the optimal TAP genes are expressed. The TAP genes that are optimal for the presentation of the antigenic peptides of a given antigen may be different from the TAP genes that are optimal for the presentation of the antigenic peptide of another antigen. The technique of dragging the gene, is an ideal method and perhaps the only one to solve this type of problems. The efficient presentation of the desired antigenic peptides can be analyzed using specific cytotoxic T lymphocytes, for example, by measuring cytokine production or CTL activity of T lymphocytes, using methods known to those skilled in the art.

Inductor Sequences of Cytotoxic T Cells and Immunogenic Agonist Sequences.

Certain proteins are more likely than others to transport the MHC class I epitopes, because they are more easily used by the cellular machinery, included in the processing necessary for the presentation of the class I epitope. The present invention provides methods for identifying the expressed polypeptides, which are particularly efficient to 'pass through various biosynthetic and degrading steps leading to the presentation of the class I epitope, and the use of these polypeptides to improve the presentation of the CTL epitopes from of other proteins. In one embodiment, the present invention provides the inductive sequences of cytotoxic T cells (CTIS), which can be used to transport the heterologous epitopes of class I, for the purpose of vaccination against the pathogen from which the heterologous epitopes are derived. An example of CTIS is obtained from the hepatitis B surface antigen (HBsAg), which has been shown to be an effective transporter of its own CTL epitopes, when administered as a protein under certain conditions. Immunization of DNA with plasmids employing HBsAg also induces high levels of CTL activity. The present invention provides a shorter truncated fragment of the HBsAg polypeptide, which works very efficiently in the induction of CTL activity, and achieves CTL induction levels that are higher than those achieved with the protein HBsAg or with the plasmid encoding the HBsAg polypeptide. The synthesis of a CTIS, derived from the HBsAg is described in Example number 3, and in Figure 1, a diagram of a CTIS is illustrated. ER localization of the truncated polypeptide may be important to achieve adequate proteolytic release of the peptide containing the CTL epitopes (see, Cresswell &Hughes (1997) Curr. Biol. 7: pages R552 to R555; Craiu and Associates (1997) Proc. Nat'l. Acad. Sci. USA 94: pages 10850 to 10855). The pre-S2 region and the transmembrane region provide T-helper epitopes, which may be important for the induction of a strong cytotoxic immune response. Because the truncated CTIS polypeptide has a simple structure (see Figure 1), it is possible to adhere one or more heterologous sequences of the class I epitope to the C-terminal end of the polypeptide, without having to maintain the specific conformation of the protein. Said sequences are then available for the processing mechanisms of the class I epitope. The size of the polypeptide is not subject to the normal restrictions of the structure of the native HBsAg. Therefore, the length of the heterologous sequence, as well as the number of included CTL epitopes, is flexible. This is shown schematically in Figure 2. The ability to include a long sequence that contains either multiple and distinct sequences of class I or alternative variations from a single CTL sequence allows the drag methodology to be applied. DNA The present invention also provides methods for obtaining immunogenic agonist (IAS) sequences, which induce the ability of the specific CTL of the cells expressing the natural epitope sequence In some cases, the reactivity is greater than if the CTL response is induced by the natural epitope (see, Example 3 and Figure 3), said CTL, induced by IAS, can be rotated from a repertoire of the T cell different from that induced by the natural sequence. the ability to respond poorly to a given epitope can be overcome by recruiting T cells from a larger set In order to discover said IAS, the amino acid at each position of a peptide that induces CTL (perhaps including residue positions called anchor) can be varied in a range of 19 amino acids that are not normally present in the position.The DNA entrainment methodology can be used, to explore a large range of sequence possibilities. A synthetic segment of the gene, which contains multiple copies of the original epitope sequence, can be prepared, so that each copy possesses a small amount of nucleotide changes. The gene segment can be dragged to create a diverse range of CTL epitope sequences, some of which should function as IAS, this process is illustrated in Figure 4. In practice, oligonucleotides are generally constructed in accordance with the previous design, and are enzymatically polymerized to form the synthetic segment of the concatenated epitope gene. Restriction sites can be incorporated into a fraction of oligonucleotides to allow the division and selection of certain size ranges of concatenated epitopes, most of which will have different sequences and, therefore, will be a potential IAS . The segment of the gene containing the epitope can be linked, by appropriate methods of cloning to a CTIS, as well as that of HBsAg. The resulting plasmid constructs can be used for the immunization based on the DNI and the induction of the CTL.

E. Pharmaceutical Compositions of Genetic Vaccine and Methods of Administration.

The improved immunomodulatory polynucleotides and polypeptides of the present invention are useful for the treatment and / or prevention of various diseases and conditions with which the respective antigens are associated. For example, genetic vaccines employing reagents obtained according to the methods of the present invention are useful both in the prophylaxis and in the therapy of infectious diseases, including those caused by any bacteria, fungus, virus or other pathogen. of mammals. The reagents obtained using the present invention can also be used for the treatment of autoimmune diseases, including, for example, rheumatoid arthritis, SLE, diabetes mellitus, myasthenia gravis, reactive arthritis, ankylosing spondylitis and multiple sclerosis. These, and other inflammatory conditions, including I BD, psoriasis, pancreatitis and various immunodeficiencies, can be treated using genetic vaccines that include vectors and other components obtained using the methods of the present invention. Genetic vaccine vectors and other reagents obtained using the methods of the present invention can be used to treat allergies and asthma. In addition, the use of genetic vaccines is a great promise for the treatment of cancer and the prevention of metastasis. By inducing an immune response against cancer cells, the body's immune system can be considered to be prepared to reduce or eliminate cancer. In the preferred embodiments of the present invention, the reagents obtained using the methods of the present invention are used in conjunction with a genetic vaccine vector. The selection of vector and components can also be optimized for the particular purpose of treating allergy or other conditions. For example, an antigen associated with the treatment of a particular condition can be optimized using recombination and selection methods analogous to those described in the present disclosure. Said methods and the appropriate antigens for the different conditions are described in the North American patent application, series number, entitled "Immunization of the Antigen Library", which is also pending, and has been commonly assigned, which was filed on April 10, 2006. February 1999, in accordance with file number 18097-028710US. The polynucleotide encoding the antigenic recombinant polypeptide can be placed under the control of a promoter, for example, a high activity or tissue specific promoter. The promoter used to express the antigenic polypeptide by itself can be optimized using the methods of recombination and selection analogous to those described herein, as described in International application number PCT / US97 / 17300 (International Publication Number W098 / 13487). The reagents obtained using the methods of the present invention can also be used in conjunction with multi-component genetic vaccines, which have the ability to design a immunological response as the most appropriate to achieve a desired effect (see, for example, the patent application North American Series number, entitled "Design of the Vector of Genetic Vaccine ", filed on February 10, 1999, with the file number 18097-030100US) also pending and commonly assigned.It is sometimes advantageous to use a genetic vaccine, which is elaborated for a particular type of target cell (for example, an antigen presentation cell or an antigen processing cell) methods for achieving the appropriate objectives are described in the North American patent application serial number, entitled "Preparation of Vectors Objectives of Genetic Vaccines", filed on February 10, 1999, with file number 18097-030200US, also pending and commonly assigned. Genetic vaccine vectors that include optimized recombinant polynucleotides, obtained as described in the present application, can be administered to a mammal (including humans) to induce a therapeutic or prophylactic immune response. The vaccine administration vehicles can be administered in vivo, by administration to an individual patient, generally, by means of systemic administration (eg, intravenous, intraperitoneal, intramuscular, subdermal, intracranial, anal, vaginal, oral, oral route or can be inhaled), or can be administered by means of topical application. Alternatively, the vectors can be administered to ex vivo cells, such as the explanted cells of an individual patient (e.g., lymphocytes, spinal cord aspirates, tissue biopsy) or to the universal trunk systems of the hematopoietic donor, followed by reimplantation. of the cells in a patient, generally, after the selection of cells, which have been incorporated into the vector. A large number of administration methods are well known to those skilled in the art. Such methods include, for example, the administration of genes based on the liposome (Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6 (7): pages 682 to 691; Rose, US Patent Number 5,279,833; Brigham (1991) patent WO 91/06309; and Felgner and Associates (1987) Proc. Natl. Acad. Sci. E. U.A. 84: pages 7413 to 7414), as well as the use of viral vectors (eg, adenoviral (see, for example, Berns and Associates (1995) Ann. NY Acad. Sci. 772: pages 95-104; Ali and Associates ( 1994) Gene Ther. 1: pages 367 to 384, and Haddada and Associates (1995) Top Curr. Microbe, Immunol. 199 (Pt 3): pages 297 to 306 for review)), papalomaviral, retroviral (see, for example , Buchscher and Associates (1992) J. Virol 66 (5) pages 2731 to 2739, Johann and Associates (1992) J. Virol 66 (5): pages 1635 to 1640 (1992), Summerfeit and Associates (1990) Virol. 176: pages 58 to 59, Wilson and Associates (1989) J. Virol 63: pages 2374 to 2378, Miller and Associates, J. Virol 65: pages 2220 to 2224 (1991), Wong-Staal and Associates, PCT patent / US / 94/05700, and Rosenburg and Fauci (1993) in Fundamental Immunology, third edition, Paul (ed) Raven Press, Limited, New York and the references found therein and, Yu and Associates, Gene Therapy ( 1994) supra), and associated viral vectors with adeno (see, West and Associates, (1987) Virology 160: pages 38 to 47; Carter and Associates (1989) North American patent number 4,797,368; Carter and Associates, patent WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5: pages 793 to 801; Muzyczka (1994) J. Clin. Invst. 94: page 1351 and Samulski (supra) for an overview of AAV vectors; see also Lebkowski, North American patent number 5, 173,414; Tratschin and Associates (1985) Mol. Cell. Biol. 5 (11): pages 3251 to 3260; Tratschin and Associates (1984) Mol. Cell. Biol., 4: pages 2072 to 2081; Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. E. U.A. 81: pages 6466 to 6470; McLaughlin and Associates (1988) and Samulski and Associates (1989) J. Virol. , 63: pages 03822 to 3828) and the like.

The "naked" DNA and / or RNA comprising a genetic vaccine can be introduced directly into a tissue, such as a muscle. See, for example, North American patent number 5,580,859. Other methods such as "biolistic" or particle-mediated transformation (see, for example, Sanford and associates, U.S. Patent No. 4,945,050; U.S. Patent No. 5,036,006), are also suitable for the introduction of genetic vaccines into the cells of a mammal. , according to the present invention. These methods are useful, not only for the in vivo introduction of DNA in a mammal, but also for the ex vivo modification of cells, for reintroduction to a mammal. As in the other methods of administration of genetic vaccines, if necessary, the administration of the vaccine is repeated in order to maintain the desired level of immunomodulation. Genetic vaccine vectors (eg, adenoviruses, liposomes, papillomaviruses, retroviruses, etc.) can be administered directly to the mammal by transduction of the cells in vivo. The genetic vaccines obtained using the methods of the present invention can be formulated as pharmaceutical compositions for administration in any suitable manner, including parenteral (eg, subcutaneous, intramuscular, intradermal or intravenous), topical, oral, rectal, intrathecal administration , oral (for example, sublingual), or local administration, such as by means of an aerosol or transdermally, for prophylactic and / or therapeutic treatments. Pre-treatment of the skin, for example, by the use of hair-removing agents, may be useful for transdermal administration. Suitable methods of administration, such as packaged nucleic acids, are available and are well known to those skilled in the art, and although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. The pharmaceutically acceptable carriers are determined in part, by the particular composition that is being administered, as well as, by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention. A variety of aqueous vehicles can be used, for example, regulated salt and the like. These solutions are sterile and are generally free of undesirable matters. These compositions can be sterilized, by means of conventional, well-known techniques. The compositions may contain pharmaceutically acceptable excipients, as required to approximate physiological conditions, such as, a pH adjustment and regulatory agents, toxicity adjusting agents and the like, eg, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the genetic vaccine vector in these formulations can vary widely, and will be selected, based primarily on fluid volumes, viscosities, body weight and the like, according to the particular administration modality selected and, the patient's needs . Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid, suspended in diluents, such as water, saline or PEG 400; (b) capsules, pills or tablets, * each containing a predetermined amount of the active ingredient, such as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. The forms of tablets may include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, silicon colloidal dioxide, croscarmellose sodium, talcum, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, regulating agents, wetting agents, preservatives, agents flavors, dyes, disintegrating agents, and pharmaceutically compatible vehicles. The tablet forms may comprise the active ingredient in a flavor, generally sucrose and acacia or tragacanth, as well as, tablets comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia in emulsions, gel, and the like containing, in addition to the active ingredient, vehicles known in the art. It is recognized that genetic vaccines, when administered orally, should be protected for digestion. This is achieved, generally, either by making complexes of the vaccine vector with a composition to produce an acid-resistant enzymatic hydrolysis or by packaging the vector in an appropriately resistant carrier, such as a liposome. Means to protect digestion vectors are well known in the art. The pharmaceutical compositions can be encapsulated, for example, in liposomes, or in a formulation that provides a sustained release of the active ingredient. Packaged nucleic acids, alone or in combination with other suitable components, can be manufactured in aerosol formulations (for example, they can be "nebulized") to be administered by means of inhalation. The aerosol formulations can be placed in acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen and the like. Formulations suitable for rectal administration include, for example, suppositories, which consist of the nucleic acid packaged with a suppository base. Suitable suppository bases include natural or synthetic triglycerides, or paraffin hydrocarbons. In addition, it is also possible to use rectal gelatin capsules, which consist of a combination of the nucleic acid packaged with a base, including, for example, liquid triglycerides, polyethylene glycols and paraffin hydrocarbons. Formulations suitable for parenteral administration, such as, for example, intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous solutions for sterile isotonic injection, which may contain antioxidants, regulators, bacteriostats, and dissolved materials that produce the isotonic formulation with the blood in the recipient intended for the same and, sterile aqueous and non-aqueous suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers, and Conservatives In the practice of the present invention, the compositions may be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral administration and intravenous administration are the preferred methods of administration. Packaged nucleic acid formulations can be presented in unit dose or multiple dose containers, such as ampules and vials. The injection and suspension solutions can be prepared from sterile powders, granules and tablets of the type described above. Cells transduced by the packaged nucleic acid can also be administered intravenously or parenterally.

The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose will be determined by the efficacy of the particular vector employed and the condition of the patient, as well as by the body weight and vascular surface area of the patient to be treated. The size of the dose will also be determined by the existence, nature, and to the extent of any adverse side effects that accompany the administration of a particular vector, or type of cell transduced in a particular patient. To determine the effective amount of the vector to be administered in the treatment or prophylaxis of an infection or other condition, the physician evaluates the toxicities of the vector, the progress of the disease, and the production of antibodies against the vector, if any. . In general, the equivalent dose of a naked nucleic acid of a vector, is from about one μg to one mg, for a typical 70 kilogram patient, and the doses of the vectors used to administer the nucleic acid, are calculated to produce a equivalent amount of therapeutic nucleic acid. The administration can be carried out by means of a single dose or divided doses. In therapeutic applications, the compositions administered to a patient suffering from a disease (eg, an infectious disease or an autoimmune disorder) in an amount sufficient to cure or at least partially reduce the disease and its complications. An adequate amount to do this is defined as a (therapeutically effective dose). The effective amounts for this use will depend on the severity of the disease and the general condition of the patient's health. Single or multiple administrations of the compositions can be administered depending on the dose required and tolerated by the patient. In any case, the composition must provide a sufficient amount of the proteins of the present invention to effectively treat the patient. In prophylactic applications, the compositions are administered to a human or other mammal, in order to induce an immune response that can help protect against the establishment of an infectious disease or other condition. The toxicity and therapeutic efficacy of the genetic vaccine vectors provided by the present invention are determined using standard pharmaceutical methods in cell cultures or experimental animals. The LD50 (the lethal dose at 50% of the population) and the ED50 (therapeutically effective dose in 50% of the population) can be determined using the methods presented in the present invention, and those known in another way by those skilled in the art. the art. A typical pharmaceutical composition for intravenous administration would be from about 0.1 to 10 mg per patient per day. Dosages from 0.1 to approximately 100 mg per patient per day can be used. Particularly, when the drug is administered at a remote site, and not in the bloodstream, such as, within a body cavity or within a lumen of an organ. Substantially higher dosages are possible in topical administration. Current methods for the preparation of compositions that can be administered parenterally will be known and appreciated by those skilled in the art, and are described in greater detail in publications, such as, Remington's Pharmaceutical Science, 15a. Edition, Mack Publishing Company, Easton, Pennsylvania (1980). The multivalent antigenic polypeptides of the present invention, and the genetic vaccines that express the polypeptides, can be packaged in packages, supplying devices, and equipment for the administration of genetic vaccines to a mammal. For example, the packages or supplying devices that contain one or more dosage units. Generally, instructions for the administration of the compounds will be provided with the packages, together with a suitable indication on the label, that the compound is suitable for the treatment of an indicated condition. For example, the label may state that the active compound within the package is useful for the treatment of a particular infectious disease, autoimmune disorder, tumor, or to prevent or treat other diseases or conditions that are mediated by, or potentially susceptible to, the immunological response of the mammal.

EXAMPLES The following examples are offered to illustrate, but not to limit the present invention.

Example 1 . Altered specificity of the ligand of B7-1 (CD80) and / or B7-2 (CD86), by s of DNA entrainment This Example describes the use of the DNA entrainment methods of the present invention, in order to obtain B7-1 and B7-2 polypeptides having altered biological activity.

Drag DNA DNA tracing is used to generate a library of variants of B7 (B7-1 / CD80 and B7-2 / CD86) that have an altered relative capacity to act through CD80 and CTLA-4, when compared with type B7 molecules of the wild type. Generally, the B7 cDNAs of different species are generated by s of RT-PCR, and these sequences are dragged, using the DNA family trawl. The B7-1 nucleotide alignments of human, rhesus monkey and rabbit are shown in Figure 15, demonstrating that the DNA family trapping method is possible when B7 molecules are developed.

Selection of variants B7. The library is then selected to identify those variants that are useful in the modulation of immune responses in autoimmune diseases, allergy, cancer, infectious diseases and vaccination. You can use any of the different methods of selecting the variants.

A. Selection System Based on the Cytometric Flow. The library of molecules B7-1 and B7-2 is transfected into cells that do not generally express these molecules (for example, COS-7 cells and any cell line of different species, with limited or no cross-reactivity with humans). with respect to the binding of ligand B7). In order to analyze the number of copies per cell, an internal marker gene can be incorporated. The soluble CTL-A and CD28 molecules are generated in order to facilitate cytometric flow experiments. Generally, these soluble polypeptides are fused with the Fc portion of the IgG molecule to improve the stability of the molecules and to make spotting possible, by s of anti-IgG monoclonal antibodies, labeled, as described by Van der Merwe. and Associates ((1997) J. Exp. Med. 185: page 393). The cells transfected with the libraries of B7 molecules "are then stained with the soluble CTLA-4 and CD28 molecules.The cells that show a ratio of CTLA-4 / CD28, increased or decreased, are classified, then the plasmids are retrieved and the entrained sequences are identified.These selected B7 variants can then be subjected to further rounds of trawling and selection, and can be further analyzed, using functional assays as described below.

B. Selection Based on the Functional Properties. The colonies of bacteria containing the plasmids, which include the mutant B7 molecules, are collected and the plasmids are isolated. These plasmids, are then transfected, in the cells that present the antigen, such as the dendritic cells, and the capacities of these mutants to activate the T cells, are analyzed. One of the advantages of this method is that no assumptions are made regarding affinities or binding specificities with the known ligands, and possibly new activities can be discovered through the ligands that are still to be identified. Activation of the T cell can be analyzed by uring proliferation, cytokine production, CTL activity or expression of activation antigens, such as the IL-2 receptor, the CD69 or HLA-DR molecules. The use of specific antigen T cell clones, such as the T cells specific for the tiny dust antigen of the house Der p I, allows the analysis of the activation of the T cell of the specific antigen. Mutants are identified that can increase or inhibit T cell proliferation, or increase or inhibit CTL responses. In a similar manner, variants that have the altered ability to induce cytokine production or the expression of activation antigens, as ured, for example, by the cytokine-specific ELISA test or the flow, can be selected. of cytometry The results obtained using the proliferation-based assay are shown in Figure 13.

C. Ability to Direct Differentiation of Cells, whether TH1 or TH2. Because the differential roles for the B7-1 and B7-2 molecules have been identified in the differentiation of the auxiliary cell T (Freeman and Associates (1995) Immunity 2: page 523; Kuchroo and Associates (1995) Cell 80: page 707), the B7 variants that are most effective in driving the differentiation of the cells, either TH 1 or TH 2, can be selected. Differentiation of the TH cell can be measured by means of the analysis of the cytokine production profiles induced by each particular variant. High levels of IL-4, IL-5 and / or IL-13 are an indication of efficient differentiation of the TH2 cell, while high levels of IFN-α or IL-2, can be used as a marker of TH 1 cell differentiation. Variants of B7 that have altered capacity to induce differentiation of TH 1 or TH 2 cells are more likely to be useful in the treatment of allergic, malignant, autoimmune and infectious diseases and in vaccination.

D. Increased Production of IL-10. The high production of I L-10, is a characteristic of regulatory T cells, which can suppress the proliferation of CD4 + T cells, specific for antigens (Groux and Associates (1997) Nature 389: page 737), therefore , B7 variants can be selected to identify those that have an increased capacity to induce the production of I L-10 by antigen-specific T cells. The production of IL-10 can be measured, for example, by the ELISA or cytometry flow assay, using intracytoplasmic cytokine staining. Variants that induce high levels of I L-10 production are useful in the treatment of allergic or autoimmune diseases.

Example 2. The Evolution of Cytokines for Enhanced Specific Activity and / or Improved Expression Levels.

This Example describes a method for developing a cytokine, for enhanced specific activity and / or improved expression levels, when the genetic vaccine is transfected into cells of a mammal. I L-12 is the most potent cytokine for directing responses of TH 1 cells, and improves the effectiveness of genetic vaccination. The developed IL-12 molecules are useful for use as components of genetic vaccines. IL-12 is a heterodimeric cytokine, composed of a light chain of 35 kD (p35) and a heavy chain of 40 kD (40) (Kabayashi and Associates (1989) J. Exp. Med. 170: page 827; Stern and Associates (1990) Proc. Nat'l. Acad. Sci. USA 87: page 6808). Recently, Lieschke and Associates (Nature Biotechnol. (1997) 15: page 35), demonstrated that a fusion between the p35 and p40 genes, results in a single gene having an activity comparable to that of two genes expressed separately. Therefore, a gene I L-12, is dragged as an entity that encodes both subunits, which is beneficial in the design of the entrainment protocol. The IL-12 subunits can also be expressed separately in the same expression vector, or the subunits can be expressed separately and selected using co-transfections of the two vectors, providing additional entrainment strategies. IL-12 plays several important roles in the regulation of allergic responses, for example, IL-12 induces differentiation of TH 1 cells, and down-regulates the response of TH2. IL-12 exhibits an IgE synthesis, both in vivo and in vitro, and also induces the production of I FN- ?. Accordingly, it is desirable to obtain an optimized L-12 that has a better ability to perform these functions at the time of its administration to a mammal. The cytokine genes, including the I L-12 genes, from humans and non-human primates, are generally 93 to 99% homologous, (Villínger and Asociados (1995) J. Immunol. 155: pages 3946 to 3954), providing a good starting point for family drag. A library of dragged IL-12 genes was obtained by entraining subunits p35 and p40, derived from human, rhesus monkey, cat, dog, cow, pig and goat, and was incorporated into vectors, and the floating ones of these transfectants , are analyzed to determine their biological activity, as illustrated in Figure 6. Due to their activities promoting the growth of the T cell, it is possible to use normal peripheral human blood T cells, in the selection of IL genes. -12 more active, making possible the direct selection of mutants of IL-12, with more potent activities in human T cells. As illustrated in Figure 7, a functional selection test has been established successfully. In this assay, COS-7 cells were first transfected with vectors encoding IL-12 subunits. Forty-eight hours after transfection, the ability of these culture floaters to induce the proliferation of activated human peripheral blood T cells was studied. Figure 8 indicates the consistency of the level of T cell proliferation induced in this assay, indicating that the assay can be used to distinguish the activities among the floaters that have different capacities to induce T cell activation. In other words , the assay provides means to select the improved activities, similar to those of I L-12 in the culture floats of the transfected cells. The ability of a vector with an optimized polynucleotide encoding I L-2 was tested to induce the activation of human T cells. The results, shown in Figure 9, show that entrained IL-12 has a significantly improved ability to induce T cell activation, compared to natural IL-12. Figure 6 illustrates a general strategy for the selection of developed cytokine genes. The specific example is provided for I L-12, but a similar method is applicable for all cytokines, when sensitive cell types are used for each cytokine. For example, the same method can be used to develop GM-CSF using the TF-1 of the cell line sensitive to GM-CSF in the selection. In addition, although in this example, the vectors are transfected into the CHO cells, any mammalian cell that can be transfected in vitro can be used as a host cell. In addition to the CHO cell, other cells good to serve as hosts, include cell lines WI-26, COS-1, COS-7, 293, U937, and recently isolated human antigens that present cells, such as monocytes, cells B and dendritic cells.

Example 3. Cytotoxic T Cells Inducing Sequences Derived from the Hepatitis B Surface Antigen and T Cell Epitopes Agonists, Strongly Immunogenic.

This Example describes the preparation of a polypeptide sequence capable of the efficient presentation of T cell epitopes and a strategy for the application of DNA entrainment, to detect highly immunogenic agonistic T cell epitopes. The HBsAg polypeptide (PreS2 plus S regions) was truncated by the introduction of a stop codon, at position 103 of the amino acid (counting from the start of the methionine of the PreS2 primer), transforming a codon of TGT system into the TGA of codon arrest. The amino acid sequences of the truncated protein were, therefore: QWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVN PVLTTASPLSSIFSR IGDPALNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLG GTTV * where the one-letter key standard for amino acids is used. The methionine residues at the beginning of the PreS2 and S regions are underlined, and the CTL epitope, restricted by the mouse Ld, is double underlined, the asterisk (*) represents the stop codon artificially introduced. A similar structure for this truncated polypeptide is shown in Figure 1. During protein biosynthesis, the N-terminal region of the HBsAg polypeptide is transported through the endoplasmic reticulum (ER) membrane. The first part of the S region is a transmembrane structure, which closes the polypeptide in the ER. The remaining C-terminal region of the polypeptide is located in the cytoplasmic compartment where it can be accessed by means of the epitope processing mechanism of the cell. This structure forms, what we refer to as the Cytotoxic Inductor Sequence of the T Cell (CTIS). A CTIS is preferably used in conjunction with an agonistic immunogenic sequence (IAS). As an example of an IAS, each position of an epitope of class I, restricted by Ld of twelve amino acids from the HBsAg, was replaced by an alanine codon encoding the DNA sequence of the epitope. The results show that, in some cases, the reactivity is greater than if the CTL response is induced by the normal epitope (Figure 3).

Example 4 Treatment of Obesity, Anorexia, and Cachexia, using Optimized Immunomodulatory Molecules.

The optimized immunomodulatory molecules that are obtained, using the methods of the present invention, find use in a wide variety of applications, in addition to their use in vaccination. For example, there is growing evidence that certain forms of obesity are associated with dysfunction of the immune system, and that molecules that regulate immune responses, for example, cytokines, can induce or inhibit obesity. The invention provides methods for the optimization of immunological regulatory molecules, for the treatment of obesity, anorexia and cachexia. The leptin and ciliary neurotrophic factor (CNTF) are examples of cytokines that have been shown to play a role in the development of obesity. The congenital deficiency of leptin, results in an early presentation of severe obesity in humans (Montague and Associates (1997) Nature 387: pages 903 to 908), and it has been shown that the CNTF, corrects obesity and diabetes associated with deficiency and resistance of leptin (Gloaguen and Associates (1997) Proc. Nat'l. Acad. Sci. USA 94: pages 6456 to 6461). Antagonists of CNTF and / or leptin are useful in the treatment of anorexia and / or cachexia. The methods of the present invention are used to generate molecules of leptin and / or CNTF, which have an improved specific activity. The methods are also useful for obtaining improved cytokines that exhibit reduced immunogenicity in vivo. Immunogenicity is a particular concern for the CNTF, because natural CNTF is highly antigenic, which results in the production of high levels of anti-CNTF antibodies, when administered to humans. The improved cytokine molecules prepared using the methods of the present invention are administered as polypeptides, or the entrained nucleic acids encoding the improved leptin and / or CNTF polypeptides are useful in genetic vaccine vectors. The present invention also provides methods for the generation of vectors that induce the production of increased levels of leptin and / or CNTF. The methods of the present invention can also be used to obtain reagents that are useful for the treatment of anorexia, cachexia and related diseases. In this modality, the leptin and / or CNTF antagonists are developed using the DNA entrainment methods. For example, a leptin receptor can be developed to obtain a soluble form that has an improved affinity for leptin. The leptin receptor in mice has been found in the hypothalamus (Mercer and Associates (1996) FEBS Lett 387: page 1 13), a known region comprising the maintenance of energy balance, and in the choroid plexus and Ieptomeninges, which are part of the blood / brain barrier.

With the understanding that the examples and modalities described in the present application are for the purpose of illustration only and that various modifications or changes in view of them will be suggested to the persons skilled in the art, and that they are included within the spirit and the content of the present application, as well as the scope of the appended claims. All publications, patents and patent applications cited in the present description are incorporated therein, as a reference for all purposes.

Claims

R E I V I N D I C A C I O N S Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A method for obtaining a polynucleotide having a modulatory effect on an immune response, or encoding a polypeptide having a modulatory effect on a immunological response, which is induced by a genetic vaccine vector, said method comprising: creation of a library of recombinant polynucleotides; and selecting the library to identify an optimized recombinant polynucleotide that has, or encodes a polypeptide having, a modulatory effect on an immune response induced by a gene vaccine vector; Wherein, the optimized recombinant polynucleotide or the polypeptide encoded by the recombinant polynucleotide exhibits an increased ability to modulate an immune response, compared to a non-recombinant polynucleotide from which the library was created. 2. The method, as described in claim 1, further characterized in that the optimized recombinant polynucleotide is incorporated into a gene vaccine vector. 3. The method, as described in claim 1, further characterized in that the optimized recombinant polynucleotide or a polypeptide encoded by the optimized recombinant polynucleotide is administered in conjunction with a gene vaccine vector. 4. The method, as described in claim 1, further characterized in that the library of recombinant polynucleotides is created by means of a process selected from the group consisting of DNA entrainment, error-prone PCR, oligonucleotide-directed mutagenesis, mutagenesis. Uracil-mediated, and repair-deficient host mutagenesis. 5. The method, as described in claim 1, further characterized in that the polynucleotide has a modulatory effect on an immune response, which is obtained by means of: (1) The recombination of at least the first and second forms of a nucleic acid that is, or encodes, a molecule that is comprised in the modulation of an immune response, wherein, the first and second forms differ, from one another, in two or more nucleotides, to produce a library of recombinant polynucleotides; and (2) The selection of the library to identify, at least, an optimized recombinant polynucleotide that exhibits, either by itself, or through the encoded molecule, an improved ability to modulate an immune response than a nucleic acid form of which the library was created. 6. The method, as described in claim 5, further characterized in that the method, optionally comprises the steps of: (3) The recombination of at least one optimized recombinant polynucleotide, with an additional form of nucleic acid, the which is the same or different from the first and second forms, to produce an additional library of recombinant polynucleotides; (4) The selection of the additional library to identify, at least, an additional optimized recombinant polynucleotide, which exhibits an improved ability to modulate an immune response than a nucleic acid form from which the library was created; and (5) Repetition of steps (3) and (4), as necessary, until the additional optimized recombinant polynucleotide exhibits an additional enhanced ability to modulate a immunological response than a nucleic acid form from which it was created. library. 7. The method, as described in claim 1, further characterized in that the optimized recombinant polynucleotide encodes a peptide or polypeptide that can interact with a cellular receptor involved in the mediation of an immune response, wherein the peptide or polypeptide acts as an agonist or antagonist of the receptor. 8. The method, as described in claim 7, further characterized in that the cellular receptor is a macrophage cleansing receptor. 9. The method, as described in the claim 7, further characterized in that the cellular receptor is segregated from the group consisting of a cytokine receptor and a chemokine receptor. 10. The method, as described in the claim 9, further characterized in that the chemokine receptor is CCR6. The method, as described in claim 7, further characterized in that the peptide or polypeptide mimics the activity of a natural ligand for a receptor, but does not induce immunological reactivity to the natural ligand. 12. The method, as described in claim 7, further characterized in that the library is selected by: The expression of recombinant polynucleotides, such that the encoded peptides or polypeptides are produced as fusions with a protein displayed on the surface of a Replicable genetic package; The contact of the replicable genetic packages, with a plurality of cells that show the receptor; and Identification of cells that exhibit a modulation of an immune response mediated by the receptor. 13. The method, as described in the claim 12, further characterized in that the replicable genetic package is selected from the group consisting of a bacteriophage, a cell, a spore and a virus. 14. The method, as described in the claim 13, further characterized in that the replicable genetic package is n M 13 bacteriophage, and the protein is encoded by the gene l l l or the gene Vi l. 15. The method, as described in claim 7, further characterized in that the method further comprises introducing the optimized recombinant polynucleotide into a genetic vaccine vector, and administering the vector to a mammal, wherein, the peptide or polypeptide is expressed, and acts as an agonist or antagonist of the receptor. 16. The method, as described in claim 7, further characterized in that it additionally comprises the production of the peptide or polypeptide encoded by the optimized recombinant polynucleotide, and the introduction of the peptide or polypeptide into a mammal, in conjunction with a genetic vaccine vector. . 17. The method, as described in claim 7, further characterized in that the optimized recombinant polynucleotide is inserted into a nucleotide sequence encoding the antigen of a gene vaccine vector. 18. The method, as described in claim 17, further characterized in that the optimized recombinant polypeptide is introduced into a nucleotide sequence encoding an M circuit of an HBsAg polypeptide. 19. The method, as described in claim 1, further characterized in that the optimized recombinant polynucleotide comprises a nucleotide sequence, rich in unmethylated CpG. 20. The method, as described in claim 1, further characterized in that the optimized recombinant polynucleotide encodes a polypeptide that inhibits an allergic reaction. twenty-one . The method, as described in claim 20, further characterized in that the polypeptide is selected from the group consisting of interferon-a, interferon- ?, I-L-10, IL-12, an antagonist of IL-4, an antagonist of I L-5, and an I L-13 antagonist. 22. The method, as described in claim 1, further characterized in that the optimized recombinant polynucleotide encodes an I L-10 antagonist. 23. The method, as described in claim 22, further characterized in that the antagonist of I L-10 is soluble or a defective I-L-10 receptor or I L-20 / MDA-7. 24. The method, as described in claim 1, further characterized in that the optimized recombinant polynucleotide encodes a co-stimulant. 25. The method, as described in claim 24, further characterized in that the co-stimulant is B7-1 (CD80) or B7-2 (CD86), and the selection step comprises the selection of variants with altered activity through of CD28 or CTLA-4. 26. The method, as described in claim 24, further characterized in that the co-stimulant is CD 1, CD40, CD154 (ligand for CD40) or CD 150 (SLAM). 27. The method, as described in the claim 24, further characterized in that the co-stimulant is a cytokine. 28. The method, as described in the claim 27, further characterized in that the cytokine is selected from the group consisting of IL-1, I L-2, IL-3, I L-4, IL-5, I L-6, IL-7, IL-8, IL-9, I L-10, IL-1 1, I L-12, IL-13, IL-14, IL15, I L-16, IL-17, IL-18, GM-CSF, G-CSF, TNF-a, IFN-a, IFN-α, and IL-20 (MDA-7). 29. The method, as described in claim 28, further characterized in that the library of recombinant polynucleotides is selected by means of the test of the ability of the cytokines encoded by the recombinant polynucleotide to activate cells, which contain a receptor for the cytokine. 30. The method, as described in claim 29, further characterized in that the cells contain a heterologous nucleic acid encoding the receptor for the cytokine. 31 The method, as described in claim 28, further characterized in that the cytokine is interleukin-12 and the selection is made by: Culturing mammalian cells, which contain the genetic vaccine vector in a culture medium; and Detection, if T cell proliferation or differentiation of the T cell, is induced by contact with the culture medium. 32. The method, as described in claim 28, further characterized in that the cytokine is interferon-a and the selection is carried out by: Expression of the recombinant polynucleotides, so that the encoded peptides or polypeptides are produced as fusions with a protein shown on the surface of a replicable genetic package; The contact of replicable genetic packages, with a plurality of B cells; and Identification of the members of the phage library that have the ability to inhibit the proliferation of B cells. 33. The method, as described in claim 28, further characterized in that the immunological response of interest, is differentiation of T cells to TH 1 cells, and the selection is made by contacting the population of T cells, with the cytokines encoded by members of the recombinant polynucleotide library, and identifying members of the library that encode a cytokine that induces the T cell to produce IL-2 and interferon-? 34. The method, as described in the claim I 27, further characterized in that the cytokine encoded by the optimized recombinant polynucleotide, exhibits reduced immunogenicity compared to a cytokine encoded by a non-optimized polynucleotide, and the reduced immunogenicity is detected by the introduction of a cytokine encoded by the recombinant polynucleotide in a mammal, and the determination of whether an immune response is induced against the cytokine. 35. The method, as described in claim 24, further characterized in that the co-stimulant is B7-1 (CD80) or B7-2 (CD86) and the cell is tested for its ability to co-stimulate an immune response. 36. The method, as described in claim 1, further characterized in that the optimized recombinant polynucleotide encodes a cytokine antagonist. 37. The method, as described in claim 36, further characterized in that the cytokine antagonist is selected from the group consisting of a soluble cytokine receptor and a transmembrane cytokine receptor having a defective signal sequence. 38. The method as described in claim 36, further characterized in that the cytokine antagonist is selected from the group consisting of? I L-10R and? IL-4R. 39. The method, as described in the claim 1, further characterized in that the optimized recombinant polynucleotide encodes a polypeptide capable of inducing an immunological response predominantly of TH 1. 40. The method, as described in the claim 1, further characterized in that the optimized recombinant polynucleotide encodes a polypeptide capable of inducing an immunological response predominantly of TH2. 41 A method for obtaining a polynucleotide that encodes an accessory molecule that improves the transport or presentation of antigens by a cell, said method comprising: Creation of a library of recombinant polynucleotides by subjecting it to recombination nucleic acids encoding all or part of the molecule accessory; and Selection of the library to identify an optimized recombinant polynucleotide encoding a recombinant accessory molecule that confers a cell an increased or decreased ability to transport or present an antigen on a cell surface, compared to an accessory molecule encoded by nucleic acids non-recombinants 42. The method, as described in the claim 41, further characterized in that the selection comprises: introducing the recombinant polynucleotide library into a gene vaccine vector encoding an antigen to form a vector library; The introduction of the vector library in mammalian cells; and Identification of mammalian cells, which exhibit increased or decreased immunogenicity to the antigen. 43. The method, as described in claim 41, further characterized in that the accessory molecule comprises a proteasome or a TAP polypeptide. 44. The method, as described in the claim 41, further characterized in that the accessory molecule comprises a cytotoxic T cell-inducing sequence. 45. The method, as described in claim 44, further characterized in that the cytotoxic T cell-inducing sequence is obtained from a hepatitis B surface antigen. 46. The method, as described in claim 41, further characterized in that the accessory molecule comprises an immunogenic agonist sequence.