MXPA00007892A - Antigen library immunization - Google Patents

Abstract

This invention is directed to antigen library immunization, which provides methods for obtaining antigens having improved properties for therapeutic and other uses. The methods are useful for obtaining improved antigens that can induce an immune response against pathogens, cancer, and other conditions, as well as antigens that are effective in modulating allergy, inflammatory and autoimmune diseases.

Description

IMMUNIZATION OF ENTIGENS LIBRARY Field of the Invention The present invention pertains to the field of methods for the development of immunogens that can induce efficient immune responses against a wide range of antigens.

Antecedents of the Invention The interactions between pathogens and hosts are the result of millions of years of evolution, during which, the immune system of mammals has developed sophisticated means to counteract pathogenic invasions. However, bacterial and viral pathogens have simultaneously gained a number of mechanisms to improve their virulence and survival in the hosts, presenting a major challenge for the research and development of vaccines despite the power of modern molecular and cellular biology techniques. . In a manner similar to the evolution of pathogenic antigens, it is likely that several cancer antigens have gained means to down-regulate their immunogenicity as a mechanism for escaping the host immune system. The efficient development of the vaccine is also hampered by the heterogeneity of the different deformations of the pathogens, generated in part by the forces of evolution as means for the pathogens to escape from the. immunological defenses. Pathogens also reduce their immunogenicity by selecting antigens that are difficult to express, process and / or transport in host cells, thereby reducing the possibility of immunogenic peptides to molecules that initiate and modulate immune responses. The mechanisms associated with these challenges are complex, with multiple variations and poorly characterized. Accordingly, there is a need for vaccines that can induce a protective immune response against bacterial and viral pathogens. The present invention fulfills this purpose and other needs.

Summary of the Invention The present invention provides multivalent antigenic recombinant polypeptides that include a first antigenic determinant of a first polypeptide associated with a disease and at least one second antigenic determinant of a second polypeptide associated with a disease. Polypeptides associated with the disease can be selected from a group consisting of cancer antigens, antigens associated with autoimmune disorders, antigens associated with inflammatory conditions, antigens associated with allergic reactions, antigens associated with infectious agents and other antigens that are associated with a disease condition.

In another embodiment, the present invention provides a library of recombinant antigens containing recombinant nucleic acids encoding the antigenic polypeptides. Generally, libraries are obtained by recombining at least a first and a "second form of a nucleic acid, which includes a sequence of polynucleotides that encode an antigenic polypeptide associated with a disease, wherein the first and second forms differ from one another. the other in two or more nucleotides, to produce a library of recombinant nucleic acids Another embodiment of the present invention provides methods for obtaining a polynucleotide encoding a recombinant antigen that has improved ability to induce an immune response to a disease condition These methods comprise: (1) the recombination of at least one first and second forms of a nucleic acid, which comprises a polynucleotide sequence that encodes an antigenic polypeptide that is associated with the disease condition, wherein the first and second forms differ from each other in two or more nucleotides os, to produce a library of recombinant nucleic acids; and (2) selecting the library to identify at least one recombinant nucleic acid encoding an optimized recombinant antigenic polypeptide that has enhanced the ability to induce an immune response to the disease condition.

In addition, these methods optionally comprise: (3) the recombination of at least one recombinant nucleic acid! optimized with an additional form 'of the nucleic acid, which is the same or different from the first and second forms, to produce an additional library of recombinant nucleic acids; (4) the I selection of the additional library! to identify at least one additional optimized recombinant nucleic acid encoding an I polypeptide that has an improved ability to induce an immune response to the disease condition; and (5) repeat steps (3) and (4), as necessary, until the nucleic acid additional optimized recombinant encodes a polypeptide that has an improved ability to induce an immune response to the condition of illness. In some embodiments, the optimized recombinant nucleic acid encodes a multivalent antigenic polypeptide and the! selection is made, expressing the nucleic acid library recombinants in an expression of the phage display, which is shown on a phage particle surface; making contact ! the phage with a first antibody that is specific for a first serotype of the pathogenic agent and selecting those phages that bind to the first antibody; and make contact with those phages that bind to the first antibody with a second antibody that is specific for a second serotype of the pathogenic agent and selecting those phages that bind to the second I antibody; wherein those phages that bind to the first antibody and the second antibody express a multivalent antigenic polypeptide. The present invention also provides methods for obtaining the recombinant viral vector, which has increased the ability to induce an antiviral response in a cell. These methods may include the steps of: (1) recombining at least a first and a second forms of a nucleic acid which comprises a viral vector, wherein the first and second forms differ from each other in two or more nucleotides , to produce a library of recombinant viral vectors; (2) transfection of the library of recombinant viral vectors in a population of mammalian cells; (3) staining of the cells to determine the presence of the Mx protein; and (4) isolation of recombinant viral vectors from the positive staining of cells exhibiting an increased capacity to induce an antiviral response.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic representation of a method for generating a chimeric multivalent antigen that has immunogenic regions of multiple antigens. The antibodies for each of the non-chimeric immunogenic paternal polypeptides are specific for the respective organisms (A, B, C). However, after carrying out the recombination and selection methods of the present invention, a chimeric immunogenic polypeptide is obtained which is recognized by the antibodies raised against each of the three paternal immunogenic polypeptides. Figure 2 shows the principle of DNA family drag. A family of antigen genes from the related pathogens are subjected to entrainment, which results in a library of chimeric and / or mutated antigens. Selection methods are employed to identify those recombinant antigens that are the most immunogenic and / or cross-protective. These may, if desired, be subjected to additional rounds of haulage and selection. Figures 3 A and 3B illustrate a scheme for a method by means of which recombinant polypeptides can be obtained which can induce a broad-spectrum immune response. In Figure 3A, the natural immunogenic polypeptides from pathogens A, B and C provide protection against the corresponding pathogen from which the polypeptide is derived (left panel). After the drag, a chimeric A / B / C polypeptide is obtained which can induce a protective immune response against all three types of pathogens (right panel). In Figure 3B, the entrainment with nucleic acids of substrates of the two pathogenic deformations (A, B), which encode polypeptides that are protective against only the corresponding pathogen, is used. The resulting chimeric polypeptide after entrainment can induce an immune response that is effective against, not only the two strains of the paternal pathogen, but also against the third deformation of the pathogen (C). Figure 4 is a diagram of some of the possible factors that can determine whether a particular polynucleotide encodes an immunogenic polypeptide having a desired property, such as increased immunogenicity and / or cross-reactivity. Those regions of the frequency that positively affect a particular property are indicated as plus (+) signs throughout the antigen gene, while those regions of the sequence that have a negative effect are shown as a minus sign ( -). A set of related antigen genes are entrained and selected to obtain the recombinant nucleic acids that have gained regions of positive sequence and have lost regions of negative sequence. Prior knowledge is required regarding which regions are positive or negative for a particular characteristic. Figure 5 illustrates a schematic representation of the selection strategy for selecting a library of antigens. Figure 6 illustrates a schematic representation of the set-up and unwinding strategy, as used in the selection of the antigen library. Figure 7 is an alignment of the glycoprotein D (gD) nucleotide sequences of HSV-1 (SEQ ID NO: 1) and HSV-2 (gD-1 (SEQ ID NO: 2) and gD- 2 (SEQ ID NO: 3)).

Figure 8 A shows a diagram of a gp120 HIV expression method using gene vaccine vectors and generation of a library of gp120 entrained genes. Figure 8B shows the PCR primers that are useful for obtaining gp120 nucleic acid substrates for DNA entrainment reactions. Suitable primers for the generation of substrates include 602SF (I D. SEC.NO: 4), 7773R (I D. SEC.NO.:5), and suitable primers for the amplification of the entrained nucleic acid include 6196F (I D. SEC.NO: 6) and 7746R (SEQ ID NO: 7). The preparer BssH2-6205F (I D. SEC.NO: 8) can be used to clone the resulting fragment into a gene vaccine vector. Figure 9 illustrates the hepadnavirus envelope gene domain structure. Figure 10 illustrates a schematic representation of the use of trawling to obtain hepadnavirus proteins in which the immunogenicity of an antigenic domain was improved. Figure 11 shows a strategy in which genes encoding hepadnavirus proteins having an antigenic domain having improved immunogenicity are entrained to obtain recombinant proteins in which all three domains have enhanced immunogenicity. Figure 12 shows the transmembrane organization of the H BsAg polypeptide.

Figure 13 shows a method for using the phage display to obtain recombinant allergy genes that do not bind to the previously existing IgE. Figure 14 illustrates a strategy for the selection of recombinant allergy genes to identify those that are effective in the activation of TH cells. The clones of the PBMC and T cells from atopic individuals are exposed to the antigen-presenting cells that display the antigen variants obtained using the methods of the present invention. In order to identify those variants of the allergic genes that are effective in the activation of the T cells, the induction of the proliferation of T cells of the cultures or of a cytokine synthesis pattern that are indicative of a particular activation of T cells is tested. type T that is desired. If desired, variants of the allergy gene that test positive in the in vitro assay can be subjected to the in vivo test. Figure 15 shows a strategy for the selection of recombinant cancer antigens to identify those that are effective in the activation of T cells of cancer patients. Figure 16A and Figure 16B illustrate two different strategies for the generation of vectors containing multiple T cell epitopes obtained, for example, by DNA entrainment. In Figure 16A, each individual nucleic acid encoding the entrained epitope is linked to a single promoter, and the multiple gene constructs of the promoter epitope can be placed in a single vector. The scheme shown in Figure 16B comprises multiple nucleic acid linkers encoding the epitope to a single promoter. Figure 17 shows the sequences of the coding regions PreS2-S (I D. SEC.NOS: 9 and 11) and the corresponding amino acid sequences (I D. SEC.NOS: 10 and 12) of antigens different from the surface of hepatitis B (HBsAg) or proteins (WHV) of hepatitis B of the marmot of America (SEQ ID NOS: 15 and 16). Suitable primers for the amplification of this region are also illustrated (HBV, SEQ ID NOS: 13 and 14, WHV, I D. SEC NOS: 17 and 18). Figure 18 illustrates preparers (SEQ ID NOS: 19 to 22) which are suitable for the amplification of large fragments containing S2S coding sequences. The primers hybridize regions that are approximately 200 bp of the desired sequences. Figure 19 illustrates an alignment of the amino acid sequences of the antigens on the surfaces of different subtypes of HVB (I D SEC NOS: 10 and 12). Figure 20 illustrates a diagram of multimeric particles that are assembled when an appropriate number of chimeric polypeptides and native S HBsAg monomers are mixed.

Detailed Description of the Invention Definitions The term "selection" describes, in general, a process that identifies optimal antigens. Various properties of the antigens can be used in the selection, including antigen expression, folding, stability, immunogenicity and presence of epitopes of several related antigens. Selection is a form of classification in which identification and physical separation are achieved simultaneously by the expression of a selection marker, which, in some genetic circumstances, allows the cells that express the marker to survive while the other cells die (or vice versa). Selection markers include, for example, luciferase, beta-galactosidase and green fluorescent protein. Selection markers include genes for resistance 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, antigens are first introduced into test animals, or by means of the study of the quality or resistance of the induced immune response using lymphoid cells derived from immunized animals. Although the spontaneous response can occur, and indeed occurs in the course of natural evolution, in the methods of the present invention, it is performed by man. An "exogenous DNA segment", "heterologous sequence" or a "heterologous nucleic acid", as used in the present invention, is one that originates from a source foreign to the particular host cell, or, if the same source is modified from its original form. Therefore, a heterologous gene is a host cell that includes a gene that is endogenous to the particular host cell, but that has been modified. The modification of a heterologous sequence in the applications described in the present invention generally occurs through the use of DNA carryover. Thus, the term refers to the 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 regularly . The exogenous DNA segments are expressed to produce the exogenous polypeptides. The term "gene" is used broadly to refer to any DNA segment associated with a biological function. In this way, the genes that include coding sequences and / or the regulatory sequences required for their expression. The genes also include the non-expressed DNA segments which, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including the cloning of a source of interest, or synthesized from information of known or anticipated sequences, 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 the natural state. It is preferably in a homogeneous condition although it can be either dry or an aqueous solution. Purity and homogeneity are determined using generally chemical analytical 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 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, give rise to essentially a band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is pure by at least about 50%, more preferably it is at least about 85% pure, and more preferably is about 99% pure. The term "occurs naturally" is used to describe an object that can be found in nature, which is different from being artificially produced by man. For example, a sequence of polypeptides or polynucleotides that is present in an organism (including viruses, bacteria, protozoa, insects, plants or tissues of mammals) that can be isolated from a source of nature and which, have not been intentionally modified by man in the laboratory, it is one that occurs naturally. The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in their form, either single or double braided. Unless specifically limited, the term includes nucleic acids that contain known analogs of natural nucleotides, which have binding properties similar to those of the reference nucleic acids and are metabolized in a manner similar to the nucleotides that occur Natural way. Unless otherwise indicated, a particular nucleic acid sequence also implicitly comprises conservatively modified variants thereof (eg, degenerative codon substitutions) and complementary sequences, as well as the sequence indicated explicitly. Specifically, degenerative codon substitutions, in which the third position of one or more selected (or all) codons, is substituted with mixed base residues and / or deoxyinosine (Baltzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka and associates (1985) J. Biol. Chem 260: pages 2605 to 2608; Cassol and associates (1992); Rossolini and associates (1994) Mol. Cell. 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, whose synthesis of the gene, or a subsequence thereof, has finally served as a template. Thus, a mRNA, an inverse transcribed cDNA of a mRNA, an RNA transcribed from a cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc. , all are derivatives of the gene and the detection of said derivative products, it is indicative of the presence and / or abundance of the original gene and / or the transcribed gene in a sample. A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. Operably linked, it means that the DNA sequences that are being linked are generally contiguous and, where it is necessary to join two coding regions of proteins, contiguous and in the reading frame. However, since the enhancers generally function when they are separated from the promoter by several kilobases and the intronic sequences can be of varying lengths, some elements of the polynucleotide can be linked in an operable manner, but not in a contiguous manner. A specific binding affinity between two molecules, for example, a ligand and a receptor, means a preferential bond of one molecule for another in a mixture of molecules. The bonding of the molecules can be considered specific, if the binding affinity is 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 (non-recombinant) form of the cell. The recombinant cells can also contain the genes found in the native form of the cell where they are modified or introduced back into the cell by artificial means. The term also contemplates, cells containing an endogenous nucleic acid to the cell that has been modified without removing the nucleic acid from the cell; Many modifications include those obtained by gene replacement, site-specific mutations, and related techniques. A "recombinant expression cassette" or simply an "expression cassette" is nucleic acid constructed, generally recombinantly or synthetically, with nucleic acid elements that have the ability to effect the expression of a structural gene in the host compatible with said 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 the expression may be used, as described in the present description. For example, an expression cassette may also include nucleotide sequences that encode a signal sequence and drive the secretion of an expressed protein from the host cell. Transcription termination signals, enhancers and other nucleic acid sequences that influence gene expression can also be included in an expression cassette. A "multivalent antigenic polypeptide" or a "multivalent antigenic recombinant polypeptide" is a polypeptide that does not occur naturally, which includes amino acid sequences of more than one source polypeptide, which source polypeptide is generally a naturally occurring polypeptide. At least some of the regions of different amino acid sequences constitute epitopes that are recognized by antibodies found in a mammal that has been injected with the source polypeptide. For example, the source polypeptides from which the different epitopes are derived are generally homologous (eg, have the same structure or a similar structure and / or function), and often come from isolates, serotypes, strains, different species, of the organism or of different disease conditions.

The terms "identical" or percentage of "identity" in the context of two or more nucleic acid 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 means of 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 the nucleotide or amino acid residue, when they are compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by means of visual inspection. Preferably, the substantial identity exists in a region of the sequences that is at least about 50 residues in length, more preferably in a region of at least about 100 residues, and more preferably the sequences are substantially identical in each other. less about 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 sequences acts as a reference sequence, with which the test frequencies are compared. When a sequence comparison algorithm is used, the test and reference sequences are entered into the computer, the coordinates of the subsequence are designated, if necessary, the program parameters of the sequence algorithm are designated. The sequence comparison algorithm then calculates the percent identity of the sequence for the test sequence (s) related to the reference sequence, based on the designated program parameters. Optimal alignment of the sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: page 482 (1981), by means of the alignment homology algorithm of Needleman & Wunsch, J. Mol. Biol. 48: page 443 (1970), using the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: page 2444 (1988), through the computerized implementations of these algorithms (GAP, BESFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575, Science Dr., Madison, Wl) , or by means of 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 is the BLAST algorithm, which is described in the article by Altschul and associated in J. Mol. Biol. 215: pages 403 to 410 (1990).

The software to carry out the BLAST analysis is marketed to the public through the National Center of Biotechnology Information (http // www.ncbi, nlm.nih.gov/.). This algorithm first comprises the identification of the pairs of high qualification sequences (HSO), by identifying short words of length W in the investigation sequence, which, either match or satisfy any qualification T of the positive valued threshold aligned with a word of the same length in a database sequence. We refer to T as the neighborhood threshold of the word qualification (Altschul and associates, supra). These initial hits in the word neighborhood, act as seeds for the initiation of searches to find longer HSPs that contain them. Word hits are then extended in both directions along each sequence, up to where the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the M parameters (award rating for a pair of co-incidental residues); always > 0) and N (penalty rating for residues that do not match, always < 0). For amino acid sequences, a rating matrix is used to calculate the cumulative score. The extension of word hits in each direction is interrupted when: the cumulative alignment score fails for the amount X of its maximum value achieved; the cumulative rating goes to zero or less, due to the accumulation of one or more waste alignments with a negative rating; or that the end of each sequence has been reached. The W, T and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLAST program (for nucleotide sequences) uses as default a word length (W) of 1 1, an expectation (E) of 10, a cut of 100, M = 5, N = 4, and a comparison of both braids . For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the qualification matrix BLOSUM62 (See Henikoff &Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: page 10915). In addition to calculating the percent identity of the sequence, the BLAST algorithm also performs the statistical analysis of the similarity between two sequences (see, for example, Karlin &Altschul (1992) Proc. Nat'l. Acad. Sci. USA 90 : pages 5873 to 5787). One measure of the similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability of coincidental coincidence between two nucleotide or amino acid sequences. For example, a nucleic acid is considered to be similar to the reference sequence, if the smallest sum probability in a comparison of the nucleic acid test to 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 with each other under severe conditions. The phrase "specifically hybridizing to" refers to the binding, duplexing or hybridization of a molecule only to a particular sequence of nucleotides, under severe conditions when that sequence is present in a complex mixture of DNA or RNA (e.g., total cell ). "Substantially binding" refers to the complementary hybridization between a test nucleic acid and a target nucleic acid, comprising a minor unbalance that can be accommodated by reducing the severity of the hybridization medium to achieve the desired detection of the polynucleotide sequence objective. "Severe hybridization conditions" and "severe hybridization washing conditions" in the context of nucleic acid hybridization experiments, such as South and North hybrids, are sequence dependent, and are different under different environmental parameters. longer sequences hybridize specifically at higher temperatures.An extensive guide for nucleic acid hybridization is found in Laboratory Techniques in Biochemistry and Molecular Biology -Hybridization with Nucleic Acid Probes part 1, chapter 2"Overview of principles of hybridization and the strategy of nucleic acid probé assays "from Tijssen (1993), Elsevier, New York It is generally selected that the highly severe hybridization and washing conditions are approximately 5 ° C lower than the thermal melting point (Tm) for the specific sequence in a defined ionic strength and pH, usually under "severe conditions" a sample It will ridicule its target subsequence, but not other frequencies. The Tm is the temperature (under defined ionic strength and pH) in which 50% of the target sequence is hybridized to a perfectly matched sample. It is selected that the very severe conditions are equal to the Tm for a particular sample. An example of severe hybridization conditions for complementary nucleic acids which have more than 100 complementary residues in a filter in a South or North zone, is 50% formamide with 1 mg of heparin at a temperature of 42 ° C, Hybridization is carried out at night. An example of highly severe washing conditions is 0.15M NaCl 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 1 5 minutes (for a description of the SSC regulator, see Sambrook, infra). Frequently, a wash of high severity is preceded by a wash of low severity, in order to remove the signal from the bottom of the sample. An example of a mean severity wash for a duplex of, for example, greater than 100 nucleotides, is 1 x SSC at a temperature of 45 ° C for 15 minutes. An example of low severity washing for a duplex is, for example, greater than 100 nucleotides, it is 4-6x SSC at a temperature of 40 ° C for 15 minutes. For short tests (eg, approximately 10 to 50 nucleotides), severe conditions generally comprise salt concentrations less than about 1.0 M Na + ion, generally from about 0.01 to 1.0 M Na + ion concentration (u other salts) at a pH of 7.0 to 8.3, and the temperature is generally at least about 30 ° C. Severe conditions can also be achieved by the addition of destabilizing agents, such as formamide. In general, a noise ratio signal of 2x (or greater) than that observed for an unrelated sample in the particular hybridization assay indicates the detection of a specific hybridization. Nucleic acids that do not hybridize to each other under severe conditions are still substantially identical, if the polypeptides they encode are substantially identical. This happens, for example, when a copy of a nucleic acid is created using a 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. Therefore, a polypeptide is generally substantially identical to a second polypeptide, for example, wherein two peptides differ only by conservative substitutions. The phrase "binds specifically (or selectively) to an antibody" or "immunoreactive specifically (or selectively) with", when referring to a protein or peptide, refers to the binding reaction which determines the presence of the protein, or an epitope of the protein, in the presence of a heterogeneous population of proteins or other biological products. Thus, under the designated conditions of the immunological assay, the specified antibodies bind to a particular protein and do not bind-in a significant amount to the 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 conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunological assay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid phase ELISA, western blot, or immunological histochemistry immunological assays are routinely used to screen for monoclonal antibodies that are specifically immunoreactive with a protein. See, Antibodies (1988) to Harlow and Lane Laboratory Manual, Cold Spring Harbor Publications, New York "Harlow and Lane"), for a description of the immunological assay 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), Alanine (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. The present invention provides a new method for the development of vaccines, which is called "immunization of the antigen library". There are no other available technologies for the generation of libraries of related antigens or for the optimization of known protective antigens. The most powerful existing methods for the identification of vaccine antigens, such as the production of high-throughput sequences or the immunization of expression libraries, only explore the space of the sequence provided by the pathogenic genome. These methods are likely to be insufficient, because they generally only attack the simple deformations of the pathogen, and because natural evolution has led pathogens to the down regulation of their own immunogenicity. In contrast, the immunization protocols of the present invention, which use libraries of entrained antigens, provide the means to identify the new antigen sequences. Those antigens that are the most protective can be selected from these pools by means of in vivo challenge models. Immunization of the antigen library dramatically expands the diversity of available immunogenic sequences, and therefore, these libraries of chimeric antigens can also provide means to defend them against newly emerging pathogen variants of the future. The methods of the present invention make it possible to identify individual chimeric antigens that provide effective protection against a variety of existing pathogens, producing improved vaccines for troops or civilian populations. The methods of the present invention provide a method based on evolution, such as a DNA drive in particular, which is an optimal method for improving the immunogenicity of many types of antigens. For example, the methods provide means for obtaining optimized cancer antigens, which are useful for the prevention and treatment of malignant diseases. In addition, an increasing number of auto-antigens have been characterized, which cause autoimmune and allergenic diseases, which cause atopy, allergy and asthma. The immunogenicity and processing of these antigens can be improved in a similar manner with the methods of the present invention.

The immunization methods of antigen libraries of the present invention provide a means, by which a recombinant antigen can be obtained which has the improved ability to induce an immunological response to a pathogenic agent. A "pathogenic agent" refers to an organism or virus, which has the ability to infect a host cell. Pathogenic agents generally include and / or code for a molecule, generally a polypeptide, that is immunogenic in that an immunological response to the immunogenic polypeptide is generated. Frequently, the immune response raised against an immunogenic polypeptide from a serotype of the pathogenic agent has no ability to recognize, and therefore, to protect against, a serotype different from the pathogenic agent, or other related pathogenic agents. In other situations, the polypeptide produced by a pathogenic agent is not produced in sufficient quantities, or is not sufficiently immunogenic, for the infected host to elicit a sufficient immune response against the pathogenic agent. These problems are solved by the methods of the present invention, which generally comprise the recombination of two or more forms of nucleic acid encoding a polypeptide of the pathogenic agent, or the antigen involved in another disease or condition. These recombination methods, referred to in the present application as "DNA entrainment", use as substrates, nucleic acid forms that differ from one another in two or more nucleotides, so that a library of recombinant nucleic acids. The library is then selected to identify at least one optimized recombinant nucleic acid encoding an optimized recombinant antigen that has the improved ability to induce an immunological response to the pathogenic agent and another condition. The resulting recombinant antigens are often chimeric in that they are recognized by antibodies (Abs) that react against multiple deformations of the pathogen, and generally, they can also cause a broad spectrum of immune responses. It is known that specific neutralizing antibodies are mediators of protection against several pathogens of interest, although other additional mechanisms, such as cytotoxic T lymphocytes, are probably also involved. The concept of multivalent chimeric antigens that widely induce reaction antibody responses is illustrated further in Figure 1. In the preferred embodiments of the present invention, the different forms of the nucleic acids encoding the antigenic polypeptides are obtained from members of a family of related pathogenic agents. This embodiment of DNA tracing using nucleic acids from related organisms, known as "family trawling", is described by Crameri and associates ((1998) in Nature 391: Pages 288 to 291) and illustrated schematically in Figure 2. Polypeptides of different deformations and serotypes of pathogens generally vary between 60 and 98%, which will allow the efficient carryover of the DNA family. Therefore, DNA family entrainment provides an effective method for generating multivalent cross-protection antigens. The methods are useful for obtaining individual chimeras that effectively protect most or all variants of the pathogen (Figure 3 A). In addition, immunizations using whole libraries or pools of antigen-entrained chimeras can also result in an identification of chimeric antigens that protect against pathogen variants that were not included in the starting antigen population (e.g. against deformation C by means of the dragged library of chimeras / mutants of deformations A and B in Figure 3B). Accordingly, the antigen library immunization method makes possible the development of immunogenic polypeptides that can induce immunological responses against the variants of the newly emerging, poorly characterized pathogens. The recombination of sequences can be achieved in many different formats and format swaps, as described in more detail below. These formats share some principles. common. For example, the objectives for the modification vary in the different applications, as well as the property that is expected to be acquired or improved. Examples of the candidate objectives for the acquisition of a property or the improvement of a property, include genes that encode proteins which have immunogenic and / or toxigenic activity when introduced into a host organism. The methods use at least two variant forms of a starting target. The variant forms of the candidate substrates may show the similarity of secondary or substantial structural sequence with one another, but must also differ in at least one and preferably at least two positions. The initial diversity between the forms can be the result of natural variation, for example, the different forms of variants (homologs) are obtained from different individuals or deformations of a different organism, or constitute related sequences of the same organism (for example, allelic), or constitute homologues of different organisms (interspecific variants). Alternatively, initial diversity can be induced, for example, variant forms can be generated by error-propensity transcription, such as an error-prone PCR and the use of a polymerase, which lacks test-reading activity (see, Liao (1990) Gene 88: pages 107 to 1 1 1), or of the first variant form, or, by replication of the first form in a deformation effecting the mutation (the host cells that effect the mutation are explained in more detail later, and they are generally well known). A deformation that effects mutation, can include any mutants in any organism affected in the functions of uneven repair. These include mutant products of the mutS gene, mutT, mutH, mutL, ovrD, dem, vsr, umuC, umuD, sbcB, recJ, etc. The disparity is made by genetic mutation, allelic replacement, selective inhibition by an added reagent, such as a small compound or an expressed anti-perception RNA, and other techniques. The disparity can be of the mentioned genes, or of homologous genes in any organism. Other methods for generating initial diversity include methods well known to those skilled in the art, including, for example, the treatment of a nucleic acid with a chemical or other mutant gene, through spontaneous mutation, and by system induction. of error-prone repair (for example, SOS) in a cell that contains the nucleic acid. The initial diversity between the substrates is increased significantly in the subsequent steps of recombination for the generation of the library.

Properties comprised in immunogenicity The effectiveness of an antigen in the induction of an immune response against a pathogen may depend on several factors, many of which are not well understood. Most of the previously available methods for increasing the effectiveness of antigens depend on the understanding of the molecular basis for these factors. However, DNA entrainment and immunization of the antigen library according to the methods of the present invention are effective even when the molecular bases are unknown. The methods of the present invention do not depend on a priori assumptions. Polynucleotide sequences that can positively or negatively affect the immunogenicity of an antigen encoded by the polynucleotide are frequently dispersed throughout the entire antigen gene. Several of these factors are shown in dramatic fashion in Figure 4. By recombination of different forms of polynucleotides encoding the antigen using DNA entrainment, followed by the selection of those chimeric polynucleotides that encode an antigen that can induce an immune response improved, you can obtain mainly sequences that have a positive influence on the immunogenicity of the antigen. Those sequences that negatively affect the immunogenicity of the antigen (Figure 4), It is not necessary to know the particular sequences included.

DNA Trapping Methods Generally, the methods of the present invention include the realization of DNA recombination ("entrainment"), and screening or selection, can be used to "develop" individual genes, plasmids or whole viruses, accumulations of genes multiple, 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 natural pairs (for example, as occurs during sexual replication). 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 to explore, the manner in which which may affect the combinations of different mutations, a desired result. In some cases, however, structural and / or functional information is available, which, although not required for recombination of the sequence, provides opportunities for the modification of the technique. The DNA entrainment methods of the present invention may comprise at least one of at least four different methods for enhancing immunogenic activity, as well as for extending specificity. First, DNA entrainment can be carried out in a single gene. Second, several highly homologous genes can be identified by means of sequence comparison with known homologous genes. These genes can be synthesized and dragged as a family of homologs, to select the recombinants with the desired activity. The entrained genes can be cloned into suitable host cells, such as E. coli, yeast, plants, fungi, animal cells and the like, and those encoding the antigens having the desired properties, can be identified by the methods described then . Third, total genome entrainment can be carried out to entrain genes encoding the antigenic polypeptides (together with other genomic nucleic acids). For full-genome entrainment methods, it is not yet necessary to identify which genes are being dragged. Instead, for example, bacterial cells or viral genomes are combined and entrained to acquire recombinant polypeptides having an increased capacity to induce an immune response, as measured by any of the assays described below. Fourth, the antigenic genes encoding polypeptides can be modified codons to access the diversity of mutation that is not present in genes that occur in any natural way. The details of these procedures are found later. The formats and examples of sequence 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 No. PCT / US95 / 02126 filed on February 17, 1995, Serial Patent No. 08 / 425,684 filed on April 18 of 1995, the Serial Patent No. 08 / 537,874 filed on October 30, 1995, the Serial Patent No. 08 / 564,995 filed on November 30, 1995, the Serial Patent No. 08/621, 859 filed on March 25 of 1996, Serial Patent No. 08/621, 430 filed on March 25, 1996, Patent Number PCT / US / 96/05480 filed on April 18, 1996, Serial Patent No. 08 / 650,400 filed on April 20, 1996. May 1995, Serial Patent No. 08 / 675,502 filed July 3, 1996, Serial Patent No. 08/721, 824 filed September 27, 1996, Patent Series No. PCT / US / 97 / 17300 filed on September 26, 1997 and Serial Patent No. PCT / US97 / 24239 filed on September 17, 1997; Stemmer, Science 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 et al., Nature Medicine 2 (1): pages 1 to 3 (1996); Crameri et al., Nature Biotechnology 14: pages 315 to 319 (1996), each of which is incorporated in the present description in its entirety as a reference for all uses. Other methods for obtaining recombi 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); oligonucleotide-directed mutagenesis (for review, see, Smith, Ann, Rev. Genet, 19: pages 423 to 462 (1985), Botstein and Shortle, Science 229: pages 1 193 to 1201 (1985), Carter, Biochem. 237: pages 1 to 7 (1986), Kunkel's article, "The Efficiency 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 n 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., Nucí. Acids. Res. pages 8765 to 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, Nuci Acids Res. 16: pages 803 to 814 (1988)), mutagenesis templates using uracil content (Kunkel, Proc. Nat'l Acad. Sci. E. U.A. 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, Nucí Acids, Res 12, pages 9441 to 9456 (1984), Kramer and Fritz, Methods in Enzymol 154: pages 350 to 367 (1987), Kramer and associates, Nucí Acids Res. 16: page 7207 (1988)); and, Fritz and associates Nucí. Acids Res. 16: pages 6987 to 6999 (1988)). Suitable additional methods include repair of the decoupling point (Kramer et al., 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, Nuci Acids, Res. 14: page 51 15 (1986)), restriction- selection and restriction-purification (Wells and associates, Phil., Trans. R. Soc. Lond A 31 7: pages 415 to 423 (1986)), mutagenesis by total gene synthesis (Nambiar et al., Science 223: pages 1299 to 1301 (1984), Sakamar and Khorana, Nucí Acids, Res 14, pages 6361 to 6372 (1988), Wells and associates, Gene 34: pages 315 to 323 (1985), and Grundstrom and associates, Nucí 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). The aging process begins with at least two substrates, which generally show some degree of frequency identity with each other (eg, sequence identity of at least 30%, 50%, 70%, 80% or 90%), but differ from each other in certain positions. The difference can, make any type of mutation, for example, substitutions, insertions or deletions. Often the different segments differ from each other in approximately five to twenty positions. In order for the recombination to generate an increased diversity, in relation to the starting materials, the starting materials must differ from each other, in at least two nucleotide positions. That is, if there are only two substrates, they must be in at least two divergent positions. If there are three substrates, for example, one substrate may differ from the second in a single position and the second may differ from the third in only one position. The starting DNA segments may be natural variants of one another, for example, allelic variants or species. The segments can also come from non-allelic genes, which show some degree of structural and generally functional relativity (for example, different genes within a superfamily, such as the family of Yersinia V antigens, for example). The starting DNA segments can also be variants induced from one another. For example, one segment of DNA can be produced, by replicating error-prone PCR from the other, the nucleic acid can be treated with a chemical or another mutant gene, or by replacing a mutagenic cassette. Induced mutants can also be prepared, by propagating one (or both) of the segments in a mutagenic strain, or by inducing the error-prone repair system in the cells. In these situations, strictly speaking, the DNA segment is not a single segment, but a large family of related segments. The different segments that form the starting materials are often of the same length or substantially the same length. However, it is not necessary that this be the case; for example, one segment can be a subsequence of others. The segments may be present as part of larger molecules, such as vectors, or they may be in an isolated form. The starting DNA segments are recombined, by any of the sequence recombination formats, provided in the present disclosure, for the purpose of generating a diverse library of recombinant DNA segments. This library can vary widely in size, from having just under 10, to more than 105, 109, 1012 or more members. In some embodiments, the generated starting segments and recombinant libraries will include full-length coding sequences and any essential regulatory sequences, such as, a promoter and a polyadenylation sequence, required for expression. In another embodiment, the recombinant DNA segments in the library can be inserted into a common vector, providing the sequences necessary for expression, before making the selection.

Substrates for the Evolution of Recombinant Antigens Optimized The present invention provides methods for obtaining recombinant polynucleotides encoding antigens that show an improved ability to induce an immune response to a pathogenic agent. The methods can be applied to a wide variety of pathogenic agents, including, potential biological combat agents and other organisms and polypeptides that can cause disease and toxicity in humans and other animals. The following examples are only illustrative and not limiting. 1 . Bacterial Pathogens and Toxins. In some embodiments, the methods of the present invention are applied to bacterial pathogens, as well as to toxins produced by bacteria and other organisms. The methods can be used to obtain recombinant polypeptides, which can induce an immunological response against the pathogen, as well as recombinant toxins that are less toxic than the polypeptides native to the toxins. Frequently, the polynucleotides of interest encode the polypeptides that are present on the surface of the pathogenic organism. Among the pathogens for which the methods of the present invention are useful for the production of protective immunogenic recombinant polypeptides, are the Yersinia species. Yersinia Pestis, the causative agent of the pest, is one of the most virulent bacteria known to have LD50 values in mice with less than ten bacteria. The pneumonic form of the disease is spread rapidly among humans by aerosol or infectious droplets and can be lethal in a few days. A particularly preferred target for obtaining a recombinant polypeptide that can protect against Yersinia infection is V antigen, which is a virulence factor of 37 kDa, which induces a protective immune response and is currently being evaluated as a subunit vaccine (Brubaker (1991) Current I nvestigations of the Microbiology of Yersinae, 12: page 127). The V Yersinae antigen only, is not toxic, but the Y. Pestis isolates, which lack the V antigen, are avirulent. The Yersinia V antigen has been successfully produced in E. coli by several groups (Leary and associates (1995) Infect. Immun.3: page 2854). Antibodies that recognize the V antigen can provide passive protection against homologous deformations, but not against heterologous deformations. In a similar way, immunization with purified V antigen protectors protects only the homologous deformations. To obtain the cross-protection recombinant V antigen, in a preferred embodiment of the present invention, the V antigen genes, coming from several Yersinia species, are subjected to family trawling. The genes encoding the V antigen of the Y. Pestis V antigen, Y. Enterocolitica and Y. Pseudotuberculosis, for example, are identical at the DNA level of 92 to 99%, making them ideal for optimization using trawl families, according to the methods of the present invention. After entrainment, the recombinant nucleic acid library is selected to determine those encoding the recombinant V antigen polypeptides, which can induce an improved immune response and / or have enhanced cross-protection. Bacillus Anthracis is the causative agent of anthrax, which is another example of a bacterial target, against which the methods of the present invention are useful. Anthrax protective antigen (PA) provides a greater protective immune response in test animals, and some antibodies against PA also provide some protection. However, the immunogenicity of PA is relatively poor, so that multiple injections are generally required when PA is used in its wild type. Co-vaccination with lethal factor (LS) can improve the effectiveness of natural PA vaccines, but toxicity is a limiting factor. Accordingly, the DNA carryover and immunization methods of the antigen libraries of the present invention can also be used in order to obtain non-toxic LF polynucleotides, which encode the LF, from various B. anthracis deformations that are subjected to family drag. The library resulting from the recombinant LF nucleic acids can then be selected to identify those encoding the recombinant LF polypeptides, which show reduced toxicity, for example, tissue culture cells can be inoculated with the recombinant LF polypeptides, in the presence of the PA, and select those clones for which the cells survive. If desired, the non-toxic LF polypeptides can be re-crossed to retain the immunogenic epitopes of LF. Those who are selected through the first selection, then may be subjected to a secondary selection. For example, the ability of recombinant non-toxic LF polypeptides can be tested to induce an immune response (e.g., CTL or antibody response) in a test animal, such as a mouse. In the preferred embodiments of the present invention, the recombinant non-toxic LF polypeptides are then tested for their ability to induce protective immunity in test animals, against challenge, by different deformations by B. anthracis. The protective antigen (PA) of B. anthracis is also a suitable target for the methods of the present invention. The nucleic acids that code the PA, of different deformations of B. Anthracis, are subjected to the DNA drag. Then, the appropriate fold can be selected, for example, E. coli, using polyclonal antibodies. The ability to induce broad-spectrum antibodies is selected in a test animal that is generally used, either alone or in addition to a preliminary screening method. In the preferred embodiments of the present invention, those recombinant polynucleotides that exhibit the desired properties can be cross-referenced such that the immunogenic epitopes are maintained. Finally, the selected recombinants are tested for their ability to induce a protective immunity against different deformations of B. anthracis, in test animals. Staphylococcus aureus and streptococcus are other toxins that can serve as an example of the target polypeptide, which can be altered using the methods of the present invention. The deformations of Staphylococcus aureus and the group Streptococci A, are comprised in a wide range of diseases, including food poisoning, toxic shock syndrome, scarlet fever, and various diseases of the autoimmune system. They secrete a variety of toxins, which include at least five cytolytic toxins, a coagulase, and a variety of enterotoxins. Enterotoxins are classified as superantigenic because they cross-link MHC class I molecules with T-cell receptors to cause constitutive activation of the T cell (Fields et al. (1996) Nature 384: page 188). This results in the accumulation of pathogenic levels of cytokines, which can lead to multiple organ failure and disease. At least thirty related but different enterotoxins have been sequenced and can be grouped phylogenetically into families. Crystal structures have been obtained from different members alone and in compounds with the MCH class I molecules. Certain mutations in the binding sites of MHC class I molecules of the toxins significantly reduce their toxicity and can form the basis for attenuated vaccines (Woody and associates (1997) Vaccine 15: page 133). However, a successful immune response to one type of toxins, can provide protection against closely related family members, while a small protection against toxins from other families can be observed. The entrainment of families of enterotoxin genes from different members of the family can be used to obtain recombinant toxin molecules, which have a reduced toxicity and which can induce a protective cross-immune response. Trailed antigens can also be selected to identify the antigens that elicit the neutralizing antibodies, in an appropriate animal model, such as a mouse or a monkey. Examples of such assays can include ELISA formats, in which the antibodies evoked prevent the binding of the enterotoxin, the M HC complex and / or T cell receptors, in the cells or purified forms. These assays may also include formats in which the added antibodies would prevent the T cells from cross-linking with the appropriate antigen presented by the cells. Cholera is an old disease, potentially lethal, caused by the bacterium vibrio cholerae, and still, an effective vaccine for its prevention is not available. Much of the pathogenesis of this disease is caused by cholera enterotoxin. Ingestion of microgram quantities of cholera toxin can induce severe diarrhea, which causes the loss of tens of liters of fluid. Cholera toxin is a complex of a single catalytic subunit A, with a pentameric ring of identical subunits B. Each subunit is inactive on its own. The B subunits bind to specific ganglioside receptors on the surface of the epithelial cells of the intestine and detonate the entry of the A subunit into the cells. The ADP ribosylates of subunit A, which are a regulatory G protein that initiates a cascade of events that cause a massive sustained electrolyte flow and water within the intestinal lumen, resulting in extreme diarrhea. B subunit of cholera toxin is an attractive target for the vaccine, for a number of reasons. It is a major target of protective antibodies generated during cholera infection, and contains epitopes for neutralizing antitoxin antibodies. It is non-toxic without subunit A, is orally effective, and stimulates the immune response, by producing a strong intestinal mucosa, dominated by IgA, which is essential in the protection against cholera and cholera toxin. Subunit B is also being investigated for use as an adjuvant in other vaccine preparations, and therefore, the toxins developed can provide general improvements for a variety of different vaccines. Heat-labile enterotoxins (LT), from enterotoxigenic E. coli strains, are structurally related to cholera toxin, and are 75% identical at the DNA sequence level. To obtain optimized recombinant toxin molecules, which show reduced toxicity and an increased capacity to induce an immune response that is protective against V. cholerae and E. Coli, the genes encoding the related toxins are subjected to DNA entrainment. . The recombinant toxins are then tested for one or more of the desirable characteristics. For example, improved cross-reactivity of the antibodies raised against the polypeptides of the recombinant toxin can be selected, by the lack of toxicity in a cell culture assay, and by the ability to induce a protective immune response against pathogens and / or against the toxins themselves. The dragged clones can be selected by phage display and / or selected by ELISA phage assays and ELISA assays for the presence of epitopes of the different serotypes. So, variants of proteins with multiple epitopes and used to immunize mice or other test animals can be purified. The animal serum is then assayed by the antibodies, for the different B-chain subtypes and the variants that provoke a broad reactive cross-response, which will be further evaluated in a virulent challenge model. The toxins of E. coli and V. cholerae can also act as adjuvants, which have the ability to increase mucosal immunity and oral administration of vaccines and proteins. Accordingly, the library of recombinant toxins can be tested to determine the increase in adjuvant activity. The improved expression levels and the stability of the B-chain pentamer can also be selected from the entrained antigens, which may be less stable than when they are in the presence of the A chain in the hexameric complex. The addition of a heat treatment step or denaturing agents, such as salts, urea and / or guanidine hydrochloride, may be included before the ELISA tests, to measure the fields of the molecules correctly bent by the appropriate antibodies . At times, it is desirable to select stable B-chain monomer molecules, in an ELISA format, for example, using antibodies that are monomerically linked, but not pentameric chains. Additionally, the ability of the entrained antigens can be selected to elicit neutralizing antibodies in an appropriate animal model, such as a mouse or monkey. For example, antibodies that bind to the B chain and prevent its binding to specific ganglioside receptors on the surface of intestinal epithelial cells can prevent disease. In a similar way, antibodies that bind to the B chain and prevent pentamerization or binding of the A chain block, can be useful in preventing disease. Bacterial antigens, which can be improved by DNA entrainment, for use as vaccines, also include, but are not limited to, the CagA and VacA antigens of Helicobacter pylori (Blaser (1996) Aliment. Pharmacol. pages 73 to 77; Blaser and Crabtree (1996) Am. J. Clin. Pathol. 106: pages 565 to 567; Censini and associates (1996) Proc. Nat'l. Acad. Sci. E. U .A. 93: pages 14648 to 14643). Other suitable antigens of H. Pylori, include, for example, the antigens for four immunoreactive proteins of 45-65 kDa, as reported by: Chatha et al. (1997) I ndian J. Med. Res. 105: pages 170 to 175; and the homologue (HspA) of H. Pylori GroES (Kansau and associates (1996) Mol. Microbiol. 22: pages 1013 to 1023). Other suitable bacterial antigens include, but are not limited to, the 43-kDa proteins and fimbrilin (41 kDa) of P. Gingivalis (Boutsl et al. (1996) Oral. Microbiol. Immunol. 1 1: pages 236 to 241); protein A from surface pneumococcal (Briles et al. (1996) Ann. NY Acad. Sci. 797: pages 1 18 to 126); Chlamydia psittaci antigens, 80-90 kDa protein and 1 10 kDa protein (Buendía and associates (1997) FEMS Microbiol, Lett 150: pages 1 13 to 1 19); the exolglycolipid chlamydial antigen (GLXA) (Whittum-Hudson et al. (1996) Nature Med. 2: pages 1 1 16 to 1 121); the specific antigens of the Chlamydia pneumoniae species, in molecular weight ranges of 92-98, 51 -55, 43-46, and 31.5-33 kDas, and genus-specific antigens in the ranges of 12, 16 and 65- 70 kDa (Halme et al. (1997) Scand.J. Immunol. 45: pages 378 to 384); proteins of variable opacity (Opa), phase of Neisseria gonorrhoeae (GC) or Escherichia coli (Chen and Gotschlich (1996) Proc. Nat'l. Acad. Sci. E. U.A. 93: pages 14851 to 14856) , in any of the twelve immunodominant proteins of Shistosoma mansomi (in a molecular weight range of 14 to 208 kDa), as described by Cutts and Wilson (1997) Parasitology 1 14: pages 245 to 255; the 17 kDa protein antigen of Brucella Abortus (De Mot and associates (1996) Curr. Microbiol 33: pages 26 to 30), a homolog of the 17 kDa protein antigen gene of the Gram negative pathogen of Brucella Abortus, identified in the form of the actinomycete myocardium Rhodococcus sp N 186/21 (De Mot and associates (1996) Curr. Microbiol 33: pages 26 to 30), the staphylococcal enterotoxins (SES) (Wood and associates (1997) FEMS Immunol. Med. Microbiol 17: page 1 to 10), the R2 reductase protein of the 42-kDa Mn Hyperneumoniae ribonucleotide Nrdf or the 15-kDa subunit protein of M. hyopneumoniae (Fagan et al. (1997) Infect. Immun. 65: pages 2502 to 2507), the PorA protein of the meningococcal antigen (Feavers and associates (1997) Clin Lab. Immunol 3: pages 444 to 450), protein A (PspA) from the pneumococcal surface (McDaniel et al. associates (1997) Gene Ther 4: pages 375 to 377), the FopA protein of the outer membrane of S. T ularensis (Fulop and associates (1996) FEMS Immunol. Med. Microbiol. 13: pages 245 to 247); the outer main membrane protein, within the deformations of Actinobacillus genus (Hartmann and associates (1996) Zentrabl, Bakteriol, 248: pages 255 to 262); the p60 or (Hly) listeriolisin antigen from Listeria monocytogenes (Hess and associates (1996) Proc. Nat'l Acad. Sci. E. U.A. 93: pages 1458 to 1463); the flagellar antigens (G) observed in Salmonella enteritidis and S. pullorum (Holt and Chaubal (1997) J. Clin Microbiol 35. Pages 1016 to 1020); the protective antigen of bacillus anthracis (PA) (Ivins and associates (1995) Vaccine 13: pages 1779 to 1784); antigen 5 of Echinococcus granulosus (Jones and associates (1996) Parasitology 1 1 3: pages 213 to 222); the role genes of Shigella Dysenteriae 1 and Escherichia coli K12 (Klee et al. (1997) J. Bacteriol 179: pages 2421 to 2425); the Rib and Alpha cell surface proteins of the Streptococcus B group (Larsson and associates (1996) Infect. Immun 64: pages 3518 to 3523); the secreted 37 kDe polypeptide, end in the virulence plasmid of 70kd of the pathogenic Yersinia spp (Leary and associates (1995) Contrib.Microbiol.Immunol.13: pages 216 to 217 and Roggenkamp and associates (1997) Infect.Immun. : pages 446 to 451); the spirochete of Lyme disease Ospa (protein A of the outer surface) of Borrelia Burgdorferi (Li and associates (1997) Proc. Nat'l. Acad. Sci. E. U.A. 94: pages 3548 to 3589, Padilla and associates (1996) J. Infect. Dis. 174: pages 739 to 746, and Wallich and associates (1996) Infection 24: pages 396 and 397); the brucella melitensis group 3 antigen, coding for Omp28 (Lindler and associates (1996) Infect. Immun 64: pages 2490 to 2499); the Pac antigen of Streptococcus mutans (Murakami and associates (1997) Infect, Immun 65: pages 794 to 797); adhesin A from pneumolisin, pneumococcal neuraminidases, autolysin, hyaluronidase, and the pneumococcal surface of 37 dDas (Paton et al. (1997) Microb. Drug Resist.3: pages 1 to 10); antigens from 29 to 32, 41 to 45, 43 to 71 x 10 (3) of Salmonella typhy (Perez and associates (1996) Imunology 89: pages 262 to 267); the K antigen as a marker of Klebsiella pneumoniae (Priamukhina and Morozova (1996) Klin, Lab. Diagnoses 47 to 49); Neocardial antigens of molecular mass of approximately 60, 40, 20 and 15-10 kDa (Prokesova and associates (1996) Int. J.Immunopharmacol. 18: pages 661 to 668); the ORF-2 antigen of Staphylococcus aureus (Rieneck and associates (1997) Biochim Biophys Acta 1350: pages128 to 132); the GlpQ antigen from Borrelia Hermsii (Schwan and associates (1996) J Clin Clinical Microbiol 34: pages 2483 to 2492); Cholera protective antigen (CPA) (Sciortino ( nineteen ninety five) . J. Diarrhoeal Dis. Res. 14: pages 16 to 26); the antigen of the 19kDa protein of Streptococcus mutans (Senpuku and associates (1996) Oral Microbiol, Immunol.1 1: pages 121 to 128); Antigen toxin protector antigen (PA) (Sharma and Associates (1996) Protein Expr. Purif. 7: pages 33 to 38); the antigens and toxoids of Clostridium perfringens (Strom et al. (1995) Br. J. Rheumatol 34: pages 1095 to 1096); the SEF14 fimbrial antigen of Salmonella enteritidis (Thorns and associates (1996) Microb. Pathog 20: pages 235 to 246); the capsular antigen of Yersinia pestis (F1 antigen) (Titball and associates (1997) Infect, Immun 65: pages 1926 to 1930); the 35 kilodalton protein of Mycobacterium leprae (Triccas and associates (1996) Infect. Immun. 64: pages 5171 to 5177); the main protein of the outer membrane, CD, extracted from the Moraxella (Branhamella) Catarrhalis (Yang and associates (1997) FEMS Immunol.Med Microbiol. 17: pages 187 to 199); the pH6 antigen (PsaA protein) of Yersinia pestis (Zav'yalov and associates (1996) FEMS Immunol.Med. Microbiol. 14: pages 53 to 57); the major surface glycoprotein, gp63, of Leishmania major (Xu and Liew (1994) Vaccine 12: pages 1 534 to 1 536; Xu and Liew (1995) Immunology 84: pages 173 to 176); 65 mycobacterial heat shock protein, mycobacterial antigen (Mycobacterium leprae hsp65) (Lowrie and associates (1994) Vaccine 12: pages 1537 to 1540; Ragno and associates (1997) Arthritis Rheum. 40: pages 277 to 283; Silva (1995) Braz. J. Med. Biol. Res. 28: pages 843 to 851); antigen 85 of Mycobacterium tuberculosis (Ag85) (Huygen and associates (1996) Nat. Med. 2: pages 893 to 898); the 45/47 kDa antigen complex (APA) of Mycobacterium tuberculosis, M. Bovis and BCG (Horn and associates (1996) J. Immunol. Methods 197: pages 151 to 159); the mycobacterial antigen, the 65kDa heat shock protein, hsp65 (Tascon and associates (1996) Nat. Med. 2: pages 888 to 892); the mycobacterial antigens MPB64, MPB70, MPB57 and the alpha antigen (Yamada and associates (1995) Kekkaku 70: pages 639 to 644); the 38 kDa protein of M. Tuberculosis (Vordermeier and associates (1995) Vaccine 13: pages 1576 to 1582); the antigens MPT63, MPT64 and MPT59 of Mycobacterium tuberculosis (Manca and associates (1997) Infecí Immun.65: pages 16 to 23; Oettinger and associates (1997) Scand. J. Immunol. 45: pages 499 to 503; Wilcke and associates (1996) Tuber Lung, Dis 77: pages 250 to 256); the 35 kilodalton protein of Mycobacterium leprae (Triccas and associates (1996) Infect Immun 64: pages 5171 to 5177); the ESAT-6 antigen of the virulent microbacterium (Brandt and associates (1996) J. Immunol. 157: pages 3527 to 3533; Pollock and Andersen (1997) J. Infect. Dis. 175: pages 1251 to 1254); the 16 kDa antigen of Mycobacterium tuberculosis (Hsp 16.3) (Chang and associates (1996) J. Biol. Chem. 271: pages 7218 to 7223); and the 18 kilodalton protein of Mycobacterium leprae (Baumgart and associates (1996) Infecí Immun 64: pages 2274 to 2281). 2. Viral Pathogens. The methods of the present invention are also useful for obtaining nucleic acids and recombinant polypeptides, which have an increased capacity to induce an immune response against viral pathogens. While the recombinants of baclerias described anleriorly are generally administered in the form of polypeptides, the recombinants that confer viral protection are preferably administered in the form of nucleic acids, such as genetic vaccines. An illustrative example is the Hantaan virus. The glycoproteins of this virus usually accumulate in the membranes of the Golgi apparatus of the infected cells. This poor expression of the glycoprotein, prevents the development of sufficient genetic vaccines against these viruses. The methods of the present invention solve this problem by carrying out the entrainment of DNA in the nucleic acids encoding the glycoproteins, and identifying those recombinants that show an increased expression in a host cell and / or improved immunogenicity, when administered in the form of a genetic vaccine. A convenient method of selection for these methods is to express the recombinant polynucleotides as fusion proteins to the PIG, which results in the display of the polypeptides on the surface of the host cell (Whitehorn et al. (1995) Biotechnology (NY. ) 13: pages 1215 to 1219). The classification of fluorescent activated cells is then used to classify and recover those cells that express an increased amount of the antigenic polypeptide of the cell surface. This preliminary screening can be followed by immunogenicity tests in mammals, such as mice. Finally, in the preferred embodiments, those recombinant nucleic acids are tested as genetic vaccines, for their ability to protect a test animal against the challenge of the virus. Flaviviruses are another example of a viral pathogen for which the methods of the present invention are useful for obtaining a recombinant polypeptide or a genetic vaccine that is effective against a viral pathogen. The flaviviruses, consist of three accumulations of antigenically related viruses: Dengue 1 -4 (identity of 62 to 77%), Japanese encephalitis viruses, San Luis and Murray Valley (identity of 75 to 82%), and encephalitis virus carried by tick (identity from 77 to 96%). The Dengue virus can induce protective antibodies against SLE and yellow fever (identity of 40 to 50%), but very few efficient vaccines are available. In order to obtain genetic vaccines and recombinant polypeptides that exhibit increased cross-reactivity and immunogenicity, the polynucleotides encoding the envelope proteins of the related viruses are subjected to DNA entrainment. The resulting recombinant polynucleotides can be tested, either as genetic vaccines, or through the use of the expressed polypeptides, by their ability to induce the response of neutralizing, largely reactive antibodies. Finally, those clones that are favorable in preliminary selections can be tested for their ability to protect a test animal against the rest of the virus. Viral antigens that can be developed by DNA entrainment, for improved activity such as vaccines include, but are not limited to, influenza A virus N2 neuraminidase (Kilbourne et al. (1995) Vaccine 13: pages 1799 to 1803); Dengue virus (E) and premembrane (prM) envelope antigens (Feighny et al. (1994) Am. J. Trop.Med. Hyg. 50: pages 322 to 328); Putnak and associates (1996) Am. J.

Trop. Med. Hyg. 55: pages 504 to 510); the VI H antigens Gag, Pol, Vif and Nef (Vogt and associates (1995) Vaccine 13: pages 202 to 208); antigens of VI H gp120 and gp 160 (Achour and associates (1995) Cell, Mol. Biol. 41: pages 395 to 400; Hone and associates (1994) Dev. Biol. Stand 82: pages 1 59 to 162); the gp41 epitope of the human immunodeficiency virus (Eckhart et al. (1996) J. Gen. Virol. 77: pages 2001 to 2008); the rotavirus VP4 antigen (Mattion et al. (1995) J. Virol. 69: pages 5132 to 5137); the rotavirus VP7 or VP7sc protein (Emslie and associates (1995) J. Virol. 69: pages 1747 to 1754; Xu and associates (1995) J. Gen. Virol. 76: pages 1971 to 1980); Herpes simplex virus (HSV) glycoproteins gB, gC, gD, gE, gG, gH and gl (Fleck and associates (1994) Med. Microbiol. Immunol. (Berl) 183: pages 87 to 94 [Mattion, 1995]; Ghiasi and associates (1995) Invest Ophthalmol, Sci 36: pages 1352 to 1360, McLean and associates (1994) J. I nfect, Dis 170: pages 1 100 to 1 109); the immediate early ICP47 protein of the herpes simplex virus type 1 (HSV-1) (Banks and associates (1994) Virology 200: pages 236 to 245); the immediate early proteins (I E) ICP27, ICP0 and ICP4 of the herpes simplex virus (Manickan and associates (1995) J. Virol 69: pages 471 1 to 4716); the nucleoprotein and hemagglutinin of the influenza virus (Deck and associates (1997) Vaccine 15: pages 71 to 78; Fu and associates (1997) J. Virol. 71: pages 2751 to 2721); VP1 proteins of parvovirus capsid B 19 (Kawase and associates (1995) Virology 21 1: pages 359 to 366) or VP2 (Brown and associates (1994) Virology 198: pages 477 to 488); the nucleus and e antigen of the Hepatitis B virus (Schodel et al. (1996) Intervirology 39: pages 104 to 106); the surface antigen of hepatitis B (Shiau and Murray (1997) J. Med. Virol. 51: pages 159 to 166); hepatitis B surface antigen fused to the virus core antigen (Id.); the particles of the preS2 nucleus of the hepatitis B virus (Nemeckova and associates (1996) Acta Virol. 40: pages 273 to 279); the HBV preS2-S protein (Kutinova and associates (1996) Vaccine 14: pages 1045 to 1052); glycoprotein I of VZV (Kutinova and associates (1996) Vaccine 14: pages 1045 to 1052); the rabies virus glycoproteins (Xiang and associates (1994) Virology 199: pages 132 to 140; Xuan and associates (1995) Virus Res. 36: pages 151 to 161) or the ribonucleocapsid (Hooper et al. (1994) Proc. Nat'l. Acad. Sci. EU .A. 91: pages 10908 to 10912); human cytomegalovirus (HCMV) glycoprotein B (UL55) (Britt and associates (1995) J. Infect. Dis. 171: pages 18 to 25); the protein of the nucleocapsid (HCV) of the hepatitis C virus, in a secreted or non-secreted form, or as a fusion protein with the antigens of the medium surface (Pre-S2 and S) or principal (S) of the hepatitis virus B (H BV) (Inchauspe and associates (1997) DNA Cell Biol. 16: pages 185 to 195; Major and associates (1995) J. Virol. 69: pages 5798 to 5805); the antigens of the hepatitis C virus: the core protein (pC); E 1 (pE1) and E2 (pE2), alone or as fusion proteins (Saito et al. (1997) Gastroenterology 1 12: pages 1321 to 1330); The gene encoding the fusion protein of the respiratory syncytial virus (PFP-2) (Falsey and Walsh (1996) Vaccine 14: pages 1214 to 1218; Piedra and associates (1996) Pedriatr. Infect. Dis. J. 15: pages 23 to 31); the VP6 and VP7 genes of the rotaviruses (Choi and associates (1997) Virology 232: pages 129 to 138; Jin and associates (1996) Arch. Virol. 141: pages 2057 to 2076); proteins E1, E2, E3, E4, E5, E6 and E7 of the human papillomavirus (Brown and associates (1994) Virology 201: pages 46 to 54; Dillner and associates (1995) Cancer Detect. Prev. 19: pages 381 to 393 Krul and associates (1996) Cancer Immunol Immunother 43: pages 44 to 48, Nakagawa and associates (1997) J Infect. Dis 175: pages 927 to 931); the human T lymphotropic virus type I human gag protein (Porter and associates (1995) J. Med. Virol, 45: pages 469 to 474); Epstein-Barr virus (EBV) gp340 (Mackett and associates (1996) J. Med. Virol 50: pages 263 to 271); the LMP2 protein, latent in the membrane of the Epstein-Barr virus (EBV) (Lee and associates (1996) Eur. J. Immunol., 26: pages 1875 to 1883); the nuclear antigens 1 and 2 of the Epstein-Barr virus (Chen and Cooper (1996) J. Virol 70: pages 4849 to 4853; Khanna and associates (1995) Virology 214: pages 633 to 637); the nucleoprotein of the measles virus (N) (Fooks et al. (1995) Virology 210: pages 456 to 465); and the glycoprotein gB of cytomegalovirus (Marshall and associates (1994) J. Med. Virol 43: pages 77 to 83) or the glycoprotein gH (Rasmussen and associates (1994) J. Infect. Dis. 170: pages 673 to 677) . 3. Parasites The antigens from the parasites can also be optimized by means of the methods of the present invention. These include, but are not limited to, schistosome antigens associated with the intestine CAA (circulating anodic antigens) and CCA (circulating cathodic antigens), in Schistosoma mansoni, S. haematobium or S. japonicum (Deeider and associates (1996) Parasitology 12: pages 21 to 35); a multiple antigen peptide (MAP) composed of two different protective antigens, derived from the Schistosoma mansoni parasite (Ferru et al. (1997) Parasite Immunol., 19: pages 1 to 11); the molecules of the surface of the Leishmania parasite (Lezama-Davila (1997) Arch. Med. Res. 28: pages 47 to 53); larvae antigens of the third stage (L-3) of L. loa (Akue et al. (1997) J. Infect. Dis. 175: pages 158 to 163); the Tams1 -1 and Tams1 -2 genes, which encode the major surface antigens of the 30 and 32 kDa merozoite of Theileria annulata (Ta) (d'Oliveira et al. (1996) Gene 172; pages 33 to 39); antigen 1 or 2, from the merozoite surface of Plasmodium falciparum (al-Yaman and associates (1995) Trans. R. Soc. Trop.Med. Hyg, 89: pages 555 to 559; Beck and associates (1997) J. Infect. Dis 175: pages 921 to 926; Rzepczyk and associates (1997) Infect. Immun 65: pages 1098 to 1 100); the epíioppes B, based on the proiein (CS) of the circumsporozoile, the Plasmodium berghei (PPPPN PND) 2 (I D. SEC NO: 23) and the Plasmodium yoelli (QGPGAP) 3QG (SEQ ID NO: 24) June with an epitope of the auxiliary cell T of P. Berghei KQI RDSITEEWS (I D. SEC.NO: 25) (Reed and associates (1997) Vaccine 15: pages 482 to 488); Plasmodium falciparum antigens coded NYVAC-PÍ7, sporozoite derivatives (circumporozoite protein and sporozoite surface protein 2), liver (antigen I, liver stage), blood (prolein 1 of the surface merozoite, repeated antigen of serine, and antigen of the apical membrane 1), and sexual (antigen of sexual elapa of 25 kDa) stages of the life cycle of the parasite that were inserted ^ in a single genome NYVAC to generate the NYVAC-PÍ7 (Tine and associates (1996 ) Infect. Immun. 64: pages 3833 to 3844); the Plasmodium falciparum Pfs230 antigen (Williamson et al. (1996) Mol. Biochem. Parasitol. 78: pages 161 to 169); the antigen of the apical membrane of Plasmodium falciparum (AMA-1) (Lal and associates (1996) Infect. Immun 64: pages 1054 to 1059); the Pfs28 and Pfs25 proleins of Plasmodium falciparum (Duffy and Kaslow (1997) Infec. Immun 65: pages 1 109 to 1 1 13); the prolein on the surface of the merozoite of Plasmodium falciparum MSP1 (Hui et al. (1996) Infect. Immun 64: pages 1502 to 1509); the Pf332 antigen of malaria (Ahlborg and associates (1996) Immunology 88: pages 630 to 635); Plasmodium falciparum erythrocyte membrane protein I (Baruch et al. (1995) Proc. Nat'l. Acad. Sci. E. U.A. 93: pages 3497 to 3502; Baruch et al. (1995) Cell 82: pages 77 to 87); Plasmodium falciparum merozoite surface antigen PfMSP-1 (Egan and associates (1996) J. Infect. Dis. 173: pages 765 to 769); Plasmodium falciparum antigens SERA, EBA-175, RAP1 and RAP2 (Riley (1997) J. Pharm, Pharmacol .. 49: pages 21 to 27); schistosoma japonicum paramyosin (Sj97) or fragments thereof (Yang et al. (1995) Biochem. Biophys. Res. Commun. 212: pages 1029 to 1039); and Hsp70 in the parasites (Maresca and Kobayashi (1994) Experientia 50: pages 1067 to 1074). 4. ' Allergy. The present invention also provides methods for obtaining reagents that are useful for the treatment of allergy. In one embodiment, the methods comprise the preparation of a library of recombinant polynucleotides that encode an allergen, and the selection of the library to identify those recombinant polynucleotides, which exhibit improved properties, when they are used as immunotherapeutic reagents for the treatment of allergy. For example, allergy-specific immunotherapy using natural antigens carries a risk of inducing anaphylaxis, which can be initiated by the cross-linking of IgE receptors in the major cells. Therefore, allergens that are not recognized by the previously existing IgE are desirable. The methods of the present invention provide methods by means of which said allergenic variants can be obtained. Another improved property of interest is the induction of broader immune responses, and increased safety and efficacy.

The synthesis of specific polyclonal and allergenic IgE requires multiple interactions between B cells, T cells and professional cells that present the antigen (APC). The activation of the unprepared natural B cells is initiated when the specific B cells recognize the allergen by the cell surface immunoglobulin (slg). However, costimulatory molecules expressed by T cells activated in both soluble and membrane-bound forms are necessary for the differentiation of B cells into IgE-secreting plasma cells. The activation of the T helper cells requires the recognition of an antigenic peptide in the context of the M HC class I I molecules in the plasma membrane of APC, such as monocytes, dendritic cells, Langerhans cells or prepared B cells. The professional APC can efficiently capture the antigen and the peptide complexes and MHC class II molecules are formed in an intracellular proteolytic compartment subsequent to the Golgi, and are subsequently exported to the plasma membrane, where they are recognized by the receptor. T cell (TCR) (Whitton (1998) Curr Top, Microbiol, Immunol 232: pages 1 to 13). In addition, activated B cells express CD80 (B7-1) and CD-86 (B7-2, B70), which are the counter receptors for CD28 and which provide a co-stimulatory signal for T cell activation resulting from the proliferation of the T cell and the synthesis of the cytokine. As the allergen-specific T cells from atopic individuals generally belong to the subset of TH2 cells, the activation of these cells also leads to the production of IL-4 and I L-12, which, together with the costimulatory molecules linked to the membrane expressed by activated helper T cells, lead to the differentiation of B cells into IgE-secreting plasma cells. Principal cells and eosinophils are the key cells in the induction of allergic symptoms in the target organs. Recognition of the allergen results in the cross-linking of receptors that lead to the degranulation of cells and the release of mediating molecules, such as histamine, prostaglandins and leukotrienes, which cause allergic symptoms. The immunotherapy of allergic diseases currently includes hyposensitization treatments using increasing doses of the allergen injected to the patient. These treatments result in the bias of the immune responses to the TH 1 phenotype and increases the proportion of IgG / IgE antibodies specific for allergens. Because these patients have specific IgE antibodies circulating for allergens, these treatments include a significant risk of anaphylactic reactions. In these reactions, the freely circulating allergen is recognized by the IgE molecules linked to the high affinity IgE receptors in the main and eosinophil cells. Recognition of the allergen results in cross-linking of receptors that leads to the release of mediators, such as histamine, prostaglandins, and leukotrienes, which cause allergic symptoms and, occasionally, anaphylactic reactions. Other problems associated with hyposensitization include the low efficacy and difficulties in the production of allergenic extracts in a reproducible manner. The methods of the present invention provide a means to obtain allergens which, when used in genetic vaccines, provide a means to solve the problems that have limited the usefulness of previously known hyposensitization treatments. For example, by means of the expression of antigens on the surface of cells, such as muscle cells, the risk of anaphylactic reactions is significantly reduced. This can be conveniently achieved using gene vaccine vectors that encode the transmembrane forms of the allergens. Allergens can also be encoded in such a way that they are expressed efficiently in transmembrane forms, further reducing the risk of anaphylactic reactions. Another advantage provided by the use of genetic vaccines for hyposensitization is that genetic vaccines can include cytokines and accessory molecules, which additionally drive the immune responses to the TH 1 phenotype, thereby reducing the amount of IgE antibodies produced and increasing the effectiveness of the treatments. To further reduce the production of IgE, entrained allergens can be administered using vectors that have been developed to induce mainly IgG and IgM responses, with little or no IgE response (see, for example, the US Patent Number Series). 09/021, 769, filed on February 1, 1988). In these methods, the polynucleotides encoding the known allergens, or homologs or fragments thereof (eg, immunogenic peptides) are inserted into the DNA vaccine vectors and used to immunize allergic and asthmatic persons. Alternatively, entrained allergens are expressed in the manufacture of cells, such as E. coli or yeast cells, and subsequently purified and used to treat patients or avoid allergic disease. DNA entrainment or another method of recombination can be used to obtain allergens that activate T cells but can not induce anaphylactic reactions. For example, a library of recombinant polynucleotides that encode allergenic variants can be expressed in cells, such as antigen-presenting cells, which are then contacted with PBMC or T cell clones from atopic patients. Those members of the library who efficiently activate TH cells from atopic patients can be identified by performing T cell proliferation assays, or cytokine synthesis (eg, synthesis of I L-2, IL-4, I FN -?) Those recombinant allergenic variants that are positive in in vitro tests can then be tested in vivo. Examples of allergies that can be treated include, but are not limited to, allergies against small dust particles in the home, grass pollen, birch pollen, herbal pollen, hazel pollen, cockroaches, rice, pollen. olive tree, mushrooms, mustard, bee venom. Antigens of interest include those of animals, including microparticles (e.g., Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis), such as allergens der. p1 (Scobie and associates (1994) Biochem. Soc. Trans. 22: page 448S; Yssel and associates (1992) J. Immunol. 148: pages 738 to 745), der p2 (Chua et al. (1996) Clin. Exp. Allergy 26: pages 829 to 837), der p3 (Smith and Thomas (1996) Clin. Exp. Allergy 26: pages 571 to 579 ), der p5 and der p V (Lin and associates (1994) J. Allergy Clin. Immunol. 94: pages 989 to 996), der p6 (Bennett and Thomas (1996) Clin. Exp. Allergy 26: pages 1 150 a 1 154), der p7 (Shen and associates (1995) Clin. Exp. Allergy 25: pages 416 to 422), der f2 (Yuuki and associates (1997) Int. Arch. Allergy Immunol. 1 12: pages 44 to 48) , der f3 (Nishiyama and associates (1995) FEBS Lett 377: pages 62 to 66), der f7 (Shen and associates (1995) Clin. Exp. Allergy 25: pages 1000 to 1006); Mag 3 (Fujikawa and associates (1996) Mol Immunol 33: pages 31 1 to 319). Also of interest as antigens, the allergens of the dust mite of the house Tyr p2 (Eriksson and associates (1998) Eur. J. Biochem. 251: pages 443 to 447), Lep d1 (Schmidt and associates (1995) FEBS Lett 370: pages 1 1 to 14), and glutathione S-transferase (O'Neill and associates (1995) Immunol Lett 48: pages 103 to 107); amino acid polypeptide 219, 25.589 Da, homologous with glutathione S-transferases (O'Neill and associates (1994) Biochim, Biophys. Acta. 1219: pages 521 to 528); Blo t 5 (Arruda and associates (1995) Int. Arch. Allergy Immunol. 107: pages 456 to 457); Phospholipase A2 from bee venom (Carballido ^ and associates (1994) J Allergy Clin. Immunol.93: pages 758 to 767; Jutel and associates (1995) J. Immunol. 154: pages 4187 to 4194); skin antigens / bovine dander BDA 1 1 (Rautiainen and associates (1995) J. Invest. Dermatol.105: pages 660 to 663) and BDA20 (Mantyjarvi and associates (1996) J. Allergy Clin. Immunol. 97: pages 1297 a 1303); the main horse allergen Equd (Gregoire and associates (1996) J. Biol. Chem. 271: pages 32951 to 32959); the allergen Myr p I of the jumping ant M. pilosula and its homologues the allergenic polypeptides Myr p2 (Donovan and associates (1996) Biochem Mol. Biol. Int. 39: pages 877 to 885); the allergens from 1 to 13, 14, 16 kD of the mite Blomia trppicalis (Caraballo and associates (1996) J. Allergy Clin.Immunol.98: pages 573 to 579); the allergens Blag Bd90K of the cockroach (Helm and associates (1996) J. Allergy Clin.Immunol.98: pages 172 to 180); and Bla g 2 (Arruda et al. (1995) J. Biol. Chem. 270: 19563 to 19568); the cockroach Cr-PI allergens (Wu and associates (1996) J. Biol. Chem. 271: pages 17937 to 17943); the allergen of fire bee venom, Sol i 2 (Schmidt and associates (1996) J.

Allergy Clin. Immunol. 98: pages 82 to 88); the major allergen of the insect Chironomus thummi Chi t 1 -9 (Kipp and associates (1996) Int. Arch. Allergy Immunol. 1 10: pages 348 to 353); dog allergen Can f 1 or cat allergen Fel d 1 (Ingram and associates (1995) J Allergy Clin. Immunol.96: pages 449 to 456); albumin derived, for example, from the horse, dog or cat (Goubran Botros et al. (1996) Immunology 88: pages 340 to 347); deer allergens with molecular mass of 22 kD, 25 kD or 60 kD (Spitzauer and associates (1997) Clin. Exp. Allergy 27: pages 196 to 200); and the main allergen of 20 kd of cow (Ylonen and associates (1994) J Allergy Clin. Immunol. 93: pages 851 to 858). Pollen and grass allergens are also useful in vaccines, particularly after optimization of the antigen by means of the methods of the present invention. Such allergens include, for example, Hor v9 (Astwood and Hill (1996) Gene 182: pages 53 to 62, Lig v1 (Batanero et al. (1996) Clin. Exp. Allergy 26: pages 1401 to 1410); 1 (Muller and associates (1996) I nt., Arch. Allergy Immunol., 109: pages 352 to 355), Lol p II (Tamborini and associates (1995) Mol. Immunol., 32: pages 505 to 513); Lol pVA , Lol pVB (Ong and associates (1995) Mol Immunol 32: pages 295 to 302), Lol p 9 (Blaher and associates (1996) J Allergy Clin Immunol 98: pages 124 to 132); Par JI (Costa et al. (1994) FEBS Lett 341: pages 182 to 186; Sallusto and associates (1996) J. Allergy Clin. Immunol. 97: pages 627 to 637), Par j 2.0101 (Hard and associates ( 1996) FEBS Lett 399: pages 295 to 298), Bet v1 (Faber and associates (1996) J. Biol. Chem. 271: pages 19243 to 19250), Bet v2 (Rihs and associates (1994) Int. Arch Allergy Immunol, 105: pages 190 to 194), Dac g3 (Guerin-Marchand and associates (1996) Mol. Immunol 33: pages 797 to 806); Phl p 1 (Petersen and associates (1995) J. Allergy Clin.Immunol.95: pages 987 to 994); Phl p 5 (Muller and associates (1996) Int. Arch. Allergy Immunol. 109: pages 352 to 355); Phl p 6 (Petersen and associates (1995) Int. Arch. Allergy Immunol. 108: pages 55 to 59); Cry j I (Soné et al. (1994) Biochem Biophys., Res. Commun. 199: pages 619 to 625); Cry j I I (Namba and associates (1994) FEBS Lett.353: pages 124 to 128); Cor 1 (Schenk et al. (1994) Eur. J. Biochem. 224: pages 717 to 722); cyn d 1 (Smith and associates (1996) J Allergy, Clin. Immunol., 98: pages 331 to 343); the cyn d7 (Suphioglu and associates (1997) FEBS Lett. 402: pages 167 to 172); Pha a 1 and isoforms Pha a 5 (Suphioglu and Singh (1995) Clin. Exp. Allergy 25: pages 853 to 865); Cha or 1 (Suzuki and associates (1996) Mol Immunol 33: pages 451 to 460); profilin derived, for example, from timothy grass or from birch pollen (Valenta et al. (1994) Biochem. Biophys., Res. Commun. 199: pages 106 to 1 18); P0149 (Wu et al. (1996) Plant, Mol. Biol. 32: pages 1037 to 1042); the Ory s1 (Xu and associates (1995) Gene 164: pages 255 to 259); and Amb a V and Amb t 5 (Kim et al. (1996) Mol.I mmunol., 33: pages 873 to 880; Zhu et al. (1995) J. Immunol. 155: pages 5064 a Vaccines against food allergens. they can also be developed using the methods of the present invention .. Antigens suitable for entrainment include, for example, profilin (Rihs et al. (1994) I nt. Arch. Allergy Immunol. 105: pages 190 to 194); rice allergen cDNAs belonging to the family of alpha-amylase / trypsin inhibitor genes (Alvarez and associates (1995) Biochim Biophys Acta 1251: pages 201 to 204), the main allergen of olive, Ole and I (Lombardero and associates ( 1994) Clin. Exp. Allergy 24: pages 765 to 770), Sin a I, the main allergen of mustard (González de la Peña et al. (1996) Eur. J. Biochem. 237: pages 827 to 832); parvalbumin, the main allergen of salmon (Lindm and associates (1996) Scand., J. Immunol., 44: 335-3 pages 44); apple allergens, such as the main allergen Mal d 1 (Vanek-Krebitz and associates (1995) Biochem. Biophys. Res. Commun. 214: pages 538 to 551); and peanut allergens, such as Ara h 1 (Burks et al. (1995) J. Clin.Invest.96: pages 1715 to 1721). The methods of the present invention can also be used to develop recombinant antigens that are effective against fungal allergies. The fungal allergens useful in these vaccines include, but are not limited to, the allergenic Cía h l l l, Cladosporium herbarum (Zhang et al. (1995) J. Immunol. 154: pages 710 to 717); the allergen Psi c 2, a fungal cyclophilin, from the Psilocybe cubensis basidiomycete (Horner et al. (1995) Int. Arch. Allergy Immunol. 107: pages 298 to 300); hsp 70 cloned from the cDNA library of Cladosporium herbarum (Zhang et al. (1996) Clin. Exp. Allergy 26: pages 88 to 95); the 68 kD allergen of Penicillium notatum (Shen et al. (1995) Clin. Exp. Allergy 26: pages 350 to 356); aldehyde dehydrogenase (ALDH) (Achatz et al. (1995) Mol Immunol., 32: pages 213 to 227); enolasa (Achatz et al. (1995) Mol Immunol., 32: pages 213 to 227); YCP4 (Id.); ribosomal acid protein P2 (Id.). Other allergens that can be used in the methods of the present invention include latex allergens, such as the major allergen (Hev b 5) of the natural rubber latex (Akasawa et al. (1996) J. Biol. Chem. 271: pages 25389 to 25393; Slater and associates (1996) J. Biol. Chem. 271: pages 25394 to 25399). The present invention also provides a solution to another disadvantage of vaccination as a treatment for allergy and asthma. While genetic vaccination primarily induces CD8 + T cell responses, the induction of allergen-specific IgE responses depends on CD4 + T cells and their B helper cells. TH2 cells are particularly efficient at inducing synthesis of IgE because they secrete higher levels of IL-4, IL-5 and I L-13, which lead to the change of the Ig isotype to the IgE synthesis. I L-5 also induces eosinophilia. The methods of the present invention can be used to develop recombinant antigens that efficiently induce the responses of CD4 + T cells, and drive the differentiation of these cells towards the TH1 phenotype.

. Inflammatory and Autoimmune Diseases Autoimmune diseases are characterized by the immune response that attacks the tissues and cells of the body itself, or the immunological responses to the specific pathogen that are also dangerous for the tissues of the cells, or the non-specific immunological activation of the which is dangerous for one's tissues or cells. Examples of autoimmune diseases include, but are not limited to, 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 the antigens that are optimized using the methods of the present invention. These conditions are frequently characterized by an accumulation of inflammatory cells, such as lymphocytes, macrophages and neutrophils, at sites of inflammation. Altered levels of cytokine production are observed, generally, with increased levels of cytokine production. Several autoimmune diseases include diabetes and rheumatoid arthritis, which are linked to certain M HC haplotypes. Other autoimmune disorders, such as reactive arthritis, have been shown to be activated by bacteria such as Yersinia and Shigella, and evidence suggests that some other autoimmune diseases, such as diabetes, multiple sclerosis, rheumatoid arthritis , they can also be initiated by viral or bacterial infections in individuals that are genetically susceptible. Current treatment strategies generally include anti-inflammatory drugs, such as NSAIDs or ciclosporin, and antiproliferative drugs, such as methotrexate. These therapies are not specific, so that there is a need for therapies that have a higher specificity, and be administered in means to conduct the immunological responses towards the direction that inhibits the autoimmune process. The present invention provides several strategies by means of which these needs can be met. First, the present invention provides methods for obtaining antigens that have a higher tolerogenicity and / or have improved antigenicity. In a preferred embodiment, the antigens prepared in accordance with the present invention exhibit improved induction of tolerance by means of oral administration. Oral tolerance is characterized by the induction of immunological tolerance after oral administration of large amounts of the antigen. In animal models, this method has proven to be a very promising means for the treatment of autoimmune diseases, and clinical trials to treat the effectiveness of this method in the treatment of human autoimmune diseases are in progress, such as, rheumatoid arthritis and multiple sclerosis. It has also been suggested that induction of oral tolerance against viruses used in gene therapy could reduce the immunogenicity of gene therapy vectors. However, the required amounts of the antigen for the induction of oral tolerance are very high and the methods of the present invention provide a means for obtaining antigens that exhibit a significant improvement in the induction of oral tolerance. Immunization of expression libraries (Barry and associates (1995) Nature 377: page 632) is a particularly useful method of selecting optimal antigens for use in genetic vaccines. For example, to identify the autoantigens present in Yersinia, Shigella, and the like, it can be selected by looking for the induction of T cell responses in individuals that are HLA-B27 positive. Complexes that include epitopes of bacterial antigens and MHC molecules associated with autoimmune diseases, for example, HLA-B27 in association with Yersinia antigens can be used in the prevention of reactive arthritis and ankylosing spondylitis in individuals who are HLA-B27 positive. The selection of optimized antigens can be made in animal models, which are known to those skilled in the art. Examples of suitable models for different conditions include collagen-induced arthritis, the NFS / sId mouse model of human Sjogren syndrome; a 120 kD antigen specific for an organ recently identified as an analogue of the human cytoskeletal protein (a-fodrin (Haneji and associates (1997) Science 276: page 604); the New Zealand mouse model White / Black F 1 hybrid , from human SLE, in mouse NOD, a mouse model of human diabetes mellitus, a mutant mouse of fas / fas ligand, which has spontaneously developed autoimmune and lymphoproliferative diseases (Watanabe-Fukunaga and associates (1992) Nature 356: page 314), and experimental autoimmune encephalomyelitis (EAE), in which the basic myelin protein induces a disease resembling human multiple sclerosis.Autoantigens that can be entrained according to the methods of the present invention and used in vaccines for the treatment of multiple sclerosis include, but are not limited to, myelin basic protein (Stinissen and associates (1996) J. Neurosci. Res. 45: pages 500 to 51 1) or a fusion protein of the basic myelin protein and the proteolipid protein (Elliott and associates (1996) J. Clin. Invest. 98: pages 1602 to 1612), proteolipid protein (PLP) (Rosener and associates (1997) J. Neuroimmunol.75: pages 28 to 34), the 2 ', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) ( Rosener et al. (1997) J. Neuroimmunol., 75: pages 28 to 34), the Epstein Barr virus (EBNA-1) nuclear antigen-1 (Vaughan et al. (1996) J.

Neuroimmunol. 69: pages 95 to 102), HSP70 (Salvetti and associates (1996) J. Neuroimmunol .. 65: pages 143 to 153; Feldmann and associates (1996) Cell 85: page 307). Target antigens which, after entrainment according to the methods of the present invention, can be used for the treatment of scleroderma, systemic sclerosis, and systhemic lupus erythematosus include, for example, (-2-GPI, glycoprotein 50 kDa (Blank and associates (1994) J. Autoimmun.7: 441 to 455 pages), Ku (p70 / p80) autoantigen, or its 80-kd subunit protein (Hong and associates (1994) Invest. Ophthalmol .Vis. Sci 35: pages 4023 to 4030; Wang and associates (1994) J. Cell Sci. 107: pages 3223 to 3233), the nuclear self-antigens La (SS-B) and Ro (SS-A) (Huang and associates (1997)). J. Clin. Immunol., 17: pages 212 to 219; Igarashi et al. (1995) Autoimmunity 22: pages 33 to 42; Keech and associates (1996) Clin. Exp. Immunol. 104: pages 255 to 263; Manoussakis and associates ( 1995) J. Autoimmun 8: pages 959 to 969; Topfer and associates (1995) Proc. Nat'l. Acad. Sci. USA 92: pages 875 to 879), the proteasome (subunit type C9 (Fe ist and associates (1996) J. Exp. Med. 184: pages 1313 to 1 318), antigens of Scleroderma Rpp 30, Rpp 38 or Scl-70 (Eder and associates (1997) Proc. Nat'l. Acad. Sci. USA 94: pages 1 101 to 1 106; Hietarinta and associates (1994) Br. J. Rheumatol. 33: pages 323 to 326), the centrosome autoantigen PCM-1 (Bao et al. (1995) Autoimmunity 22: pages 219 to 228), the autoantigen polymyositis-scleroderma (PM-Scl) (Kho et al. (1997) J. Biol. Chem. 272: pages 13426 to 13431), scleroderma (and other systemic autoimmune diseases) the CENP-A autoantigen (Muro et al. (1996) Clin. Immunol. Immunopathol 78: pages 86 to 89), U5, a small nuclear ribonucleoprotein (snRNP) (Okano and associates (1996) Clin Immunol, Immunopathol 81: pages 41 to 47), the 100-kd protein of the PM autoantigen. -Scl (Ge and associates (199.6) Arthritis, Rheum 39: pages 1588 to 1595), Th (7-2) nucleolar ribonucleoproteins U3- (Verheijen and associates (1994) J. I mmunol. ginas 173 to 182), the ribosomal L7 protein (Neu et al. (1995) Clin. Exp. Immunol. 100: pages 198 to 204), hPopl (Lygerou and associates (1996) EMBO J. 15: pages 5936 to 5948), and the 36-kd nuclear antigen matrix protein (Deng and associates (1996) Arthritis Reum. 39: pages 1300 to 1307). Autoimmune liver diseases can also be treated using the improved recombinant antigens that are prepared according to the methods described in the present invention. Among the antigens that are useful in such treatments are the cytochromes P450 and the transferase-glucuronosyl-UPD (Obermayer-Straub and Manns (1996) Baillieres Clin.Gostroenterol.10: pages 501 to 532), the cytochromes P450 2C9 and P450 1 A2 (Bourdi and associates (1996) Chem: Res. Toxicol 9: pages 1 159 to 1 166; Clement and associates (1997) J. Clin Endocrinol, Metab.82: pages 1353 to 1361), the LC-1 antigen (Klein and associates (1996) J Pediatr Gastroenterol, Nutr 23: pages 461 to 465), and the 230-kDa protein associated with Golgi (Funaki and associates (1996) Cell Struct. Funct. 21: pages 63 to 72). For the treatment of autoimmune diseases of the skin, useful antigens include, but are not limited to, the 450 kD human epidermal autoantigen (Fujiwara et al. (1996) J. Invest. Dermatol. 106: pages 1 125 to 1 130), the pemphigoid bullous antigens of 230 kD and 180 kD (Hashimoto (1995) Keio, J. Med. 44: pages 1 15 to 123; Murakami and associates (1996) J. Dermatol. Sci. 13: pages 1 12 to 17), pemphigus leaf antigens (desmoglein 1), pemfigus vulgaris antigen (desmoglein 3), BPAg2, BPAg l, and Vi l type collagen (Batteux and associates (1997) J. Clin. Immunol. 17: pages 228 to 233; Hashimoto and associates (1996) J. Dermatol. Sci. 12: pages 10 to 17), a 168 kDa mucosal antigen in a subset of pemphigoid healing patients (Ghohestani et al. (1996) J. Invest. Dematol. 107: pages 136 to 139), and a 218-kd nuclear protein (218-kd Mi -2) (Seeling and associates (1995) Arthritis Rheum 38: pages 1389 to 1399). The methods of the present invention are also useful for obtaining improved antigens by the treatment of insulin-dependent diabetes mellitus, using one or more of the antigens, which include, but are not limited to, insulin, proinsulin, GAD65 and GAD67, heat shock protein 65 (hsp65), and island 69 cell antigen (ICA69) (French and associates (1997) Diabetes 46: pages 34 to 39; Roep (1996) Diabetes 45: pages 1 147 to 1 156; Schloot et al. (1997) Diabetology 40: pages 332 to 338), the homologs of viral proteins to GAD65 (Jones and Crosby (1996) Diabetology 39: pages 1318 to 1324), the protein tyrosine phosphatase related to the antigen of the Island cell (PTP) (Cui et al. (1996) J. Biol. Chem. 271: pages 24817 to 24823), ganglioside GM2-1 (Cavallo et al. (1996) J. Endocrinol 150: pages 1 13 to 120; Dotta and associates (1996) Diabetes 45: pages 1 193 to 1 196), glutamic acid decarboxylase (GDA) (Nepom (1995) Curr. Opin. Immunol., 7: pages 825 to 830; Panina-Bordignon and associates (1995) J. Exp. Med. 181: pages 1923 to 1927), island cell antigen (ICA69) (Karges et al. (1997) Biochim Biophys. Acta 1360: pages 97-101; Roep et al. (1996) Eur. J. Immunol. 26: pages 1285 to 1289), the Tep69, an epitope of the simple T cell, recognized by the T cells of diabetes patients (Karges and associates (1997) Biochim. Biophys, Acta 1360: pages 97-101), ICA 512, a type I antigen of diabetes (Solimena et al. (1996) EMBO J. 15: pages 2102 to 21 14), a tyrosine phosphatase of the island cell protein and the 37-kDa autoantigen derived therefrom in type I diabetes. (including IA-2, IA-2) (La Gasse and associates (1997) Mol. Med. 3: pages 163 to 173), the 64 kDa protein from human ln-1 1 1 cells or follicular thyroid cells which are immunologically precipitated with sera from patients with similar island surface antibodies, (ICSA) (Igawa and associates (1996) Endocr. J. 43: pages 299 to 306), fogrin, a homologue of protein tyrosine phosphatase human transmembrane, an autoantigen of type 1 diabetes (Kawasaki and associates (1996) Biochem Biophys Res. Commun. 227: pages 440 to 447), the 40 kDa and 37 kDa tryptic fragments and their precursors IA-2 and IA- 2 in IDDM (Lampasona et al. (1996) J. Immunol., 157: pages 2707 to 271 1; Notkins and associates (1996) J. Autoimmun., 9: pages 677 to 682), cholera insulin conjugate, or insulin (Bergerot and associates (1996) Proc. Nat'l. Acad. Sci. USA. 94: pages 4610 to 4614), carboxypeptidase H, the human homolog of gp3 30, which is a renal epithelial glycoprotein involved in the induction of Heymann nephritis in rats, and the island mitochondrial autoantigen of 38-kD; (Arden et al. (1996) J. Clin. I nvest 97: pages 551 to 561). Rheumatoid arthritis is another condition that can be treated using the optimized antigens prepared according to the present invention. Antigens useful for the treatment of rheumatoid arthritis include, but are not limited to, the 45 kDa DEK nuclear antigen in the particular presentation of juvenile rheumatoid arthritis and iridocyclitis (Murray et al. (1997) J. Rheumatol. : pages 560 to 567), glycoprotein-39 from human cartilage, an autoantigen in rheumatoid arthritis (Verheijden and associates (1997) Arthritis, Rheum 40: pages 1 15 15125), a 68k autoantigen in arthritis rheumatoid (Blass and associates (1997) Ann. Rheum, Dis. 56: pages 317 to 322), collagen (Rosloniec et al. (1995) J. Immunol. 155: pages 4504 to 451 1), collagen type II (Cook et al. (1996) Arthritis, Rheum 39: pages 1720 to 1727, Trentham (1996) Ann, NY Acad. Sci. 778: pages 306 to 314), the cartilage binding protein (Guerassimov et al. (1997) J. Rheumatol. 24: pages 959 to 964), ezrin, radixin and moesin, which are autoimmune antigens in rheumatoid arthritis (Wagatsuma et al. (1996) Mol. Immunol. 33: pages 1 171 to 1 176), and the heat shock microbacterial protein 65 (Ragno et al. (1997) Arthritis, Reum 40: pages 277 to 283). Also among the conditions for which improved antigens suitable for treatment can be obtained, are the autoimmune diseases of the thyroid. Antigens that are useful for these applications include, for example, thyroid peroxidase and the thyroid stimulating hormone receptor (Tandon and Weetman (1994) J, R. Coll. Physicians Lond. 28: pages 10 to 18), Thyroid peroxidase of human Graves' thyroid tissues (Gardas et al. (1997) Biochem. Biophys. Res. Commun. 234: pages 366 to 370; Zimmer and associates (1997) Histochem. Cell. Biol. 107: pages 1 15 to 120), a 64-kDa antigen associated with ophthalmopathy associated with the thyroid (Zhang and associates (1996) Clin Immunol.Immunopathol.80: pages 236 to 244), the human TSH receptor (Nicholson and associates (1996) J, Mol Endocrinol, 16: pages 1 59 to 170), and the 64 kDa protein from I n-1 1 1 cells or human follicular thyroid cells that are immunologically precipitated with sera of patients with antibodies on the surface of the island cell (ICSA) (Igawa and associates (1996) Endocr. J. 43: pages 299 to 306). Other conditions and associated antigens include, but are not limited to, Sjogren's syndrome (-fodrin, Haneji and associates (1997) Science 276: pages 604 to 607), myasthenia gravis (the human M2 acetylcholine receptor or fragments thereof). , specifically the second extracellular circuit of the human acetylcholine receptor M2, Fu and associates (1996) Clin.I mmunol.Immunopathol.78: pages 203 to 207), vitiligo (tyrosinase, Fishman and associates (1997) Cancer 79: pages 1461 a 1464), a 450 kD human epidermal autoantigen recognized by the serum of the individual with diseases of skin blisters, and ulcerative colitis (chromosomal proteins HMG 1 and HMG2, Sobajima and associates (1997) Clin. Exp. Immunol. 135 to 140). 6. Cancer Immunotherapy holds great promise for the treatment of cancer and the prevention of metastasis. Through the induction of an immune response against cancer cells, the body's immune system can be considered on the list to reduce or eliminate cancer. The improved antigens obtained using the methods of the present invention provide immunotherapies for cancer with increased effectiveness, compared to those that are currently available. One method for cancer immunotherapy is vaccination using vaccines that include or code for antigens that are specific for tumor cells or by injecting patients with purified recombinant cancer antigens. The methods of the present invention can be used to obtain antigens that show an increase in the immunological responses against specific antigens of known tumors, and also to look for new protective antigenic sequences. Antigens that have optimized expression, processing, and presentation can be obtained as described in the present invention. The method used for each cancer in particular may vary. For the treatment of hormone-sensitive cancers (e.g., breast cancer and prostate cancer), the methods of the present invention can be used in order to obtain optimized hormone antagonists. For highly immunogenic tumors, including melanoma, the recombinant antigens that optimally drive the immune response against the tumor can be selected. Breast cancer, in contrast, has a relatively low immunogenicity and progresses slowly, so that individual treatments can be designed for each patient. The prevention of metastasis is also a goal in the design of cancer vaccines.

Among tumor-specific antigens, which can be used in the antigenic entrainment methods of the present invention are: bullous pemphigoid antigen 2, mucin prostate antigen (PMA) (Beckett and Wright (1995) Int. J. Cancer 62: pages 703 to 710), the Thomsen-Friedenreich antigen associated with the tumors (Dahlenborg and associates (1997) Int. J. Cancer 70: pages 63 to 71), the prostate specific antigen (PSA) (Dannull and Belldegrun (1997) Br. J. Urol. 1: pages 97-103), luminal epithelial antigen (LEA.135) of breast carcinoma and transitional cell vesicle carcinoma (TCC) (Jones and associates (1997 ) Anticancer Res. 17: pages 685 to 687), serum antigen associated with cancer (CASA) and cancer antigen 125 (CA 125) (Kierkegaard et al. (1995) Gynecol. Oncol. 59: pages 251 to 254 ), the epithelial glycoprotein 40 (EGP40) (Kievit and associates (1997) I nt. J. Cancer 71: pages 237 to 245), the squamous cell carcinoma antigen (SCC) (Lozza et al. (1997) Anticancer Res. 17: pages 525 to 529), cathepsin E (Mota et al. (1997) Am. J. Pathol. 150: pages 1223 to 1229), tyrosinase in melanoma (Fishman and associates (1997) Cancer 79: pages 1461 to 1464), the cellular nuclear antigen (PCNA) of brain caverns (Notelet and associates (1997) Surg. 47: pages 364 to 370), the breast cancer antigen DF3 / MUC1 (Apostolopoulos and associates (1996) Immunol.Cell.Biol.74: pages 457 to 464; Pandey and associates (1995) Cancer Res. 55: pages 4000 to 4003), the embryonic carcinoma antigen (Paone et al. (1996) J. Cancer Res. Clin. Oncol. 122: pages 499 to 503; Schlom et al. (1996) Breast Cancer Res. Treat. 38: pages 27 a 39), the antigen associated with the tumor CA 19-9 (Tollivier and O'Brien (1997) South Med. J. 90: pages 89 to 90; Tsuruta and associates (1997) Urol. I nt. 58: pages 20 a 24), the human melanoma antigens MART-1 / Melan-A27-35 and gp100 (Kawakami and Rosenberg (1997) Int. Rev. I mmunol., 14: pages 173 to 192; Zajac and associates (1997) Int. J. Cancer 71: pages 491 to 496), the T and Tn (CA) pancreaticoma epitopes of glycopeptides (Springer (1995) Crit. Rev. Oncol. 6: pages 57 to 85), an autoantigen associated with the 35 kD tumor in the papillary thyroid carcinoma (Lucas et al. (1996) Anticancer Res. 16: pages 2493 to 2496), adenocarcinoma antigen KH-1 (Deshpande and Danishefsky (1997) Nature 387: pages 164 to 166), microbacterial antigen A60 (Maes and associates (1996) J. Cancer Res. Clin. Oncol. 122: pages 296 to 300), heat shock proteins (HSPs) (Blachere and Srivastava (1995) Semin. Cancer Biol. 6: pages 349 a 355), and the oncogenic products of MAGE, tyrosinase, melan-A and gp75 and mutants (for example, p53, ras and HER-2 / neu (Bueler and Mulligan (1996) Mol. Med. 2: pages 545 to 555; Lewis and Houghton (1995) Cancer Seminal Biol. 6: pages 321 to 327; Theobald and associates (1995) Proc. Nat'l. Acad. Sci. USA 92: pages 1 1993 to 1 1997). 7. Contraception.

Genetic vaccines containing the optimized antigens obtained by means of the methods of the present invention are also useful for contraception. For example, genetic vaccines can be obtained in a way that they code the specific antigens of the sperm cells, and in this way, induce immunological responses against sperm. Vaccination can be achieved by, for example, administration of recombinant bacterial strains, for example Salmonella and the like, which express a sperm antigen, and as well as by the induction of anti-hCG neutralizing antibodies by means of vaccinating vaccines. DNA encoding human chorionic gonadotropin (hCG), or a fragment thereof. Sperm antigens that can be used in genetic vaccines include, for example, dehydroxygenase lactate (LDH-C4), galactosyltransferase (GT), rabbit sperm autoantigen SP-10 (RSA) guinea pig ( g) PH-20, wash signal protein (CS-1), human HSA-63 (h) PH-20, and AgX-1 (Zhu and Naz (1994) Arch. Androl. 33 pages 141 - 144), the synthetic sperm peptide, P10G (O 'Rand and associates (1993) J. Reprod Immunol 25 pages 89-102), the sperm proteins of 135kD, 95kD, 65kD, 47kD, 41kD and 23kD , and the FA-1 antigen (Naz et al. (1995) Arch. Androl, 35 pages 225-231), and the 35 kD fragment of cytokeratin 1 (Lucas et al. (1996) Anticancer Res. 16 pages 2493-2496 ).

The methods of the present invention can also be used to obtain genetic vaccines that are specifically expressed in the testis. For example, polynucleotide sequences that drive the expression of genes that are specifically for the testis (e.g., fertilization antigen 1 and the like) can be used. In addition to sperm antigens, antigens expressed in oocytes or reproductive regulating hormones may be useful targets for contraceptive vaccines. For example, genetic vaccines can be used to generate antibodies against gonadotropin releasing hormone (GnRH) or zona pellucida proteins (Miller et al. (1997) Vaccine 15 pages 1858-1862). Vaccinations using these molecules have been shown to be effective in animal models (Miller and associates (1997) Vaccine 15 pages 1858-1862). Another example of a useful component of a genetic contraceptive vaccine is the ZP3 glycoprotein of the zona pellucida of the ovaries (Tung and associates (1994) Reprod. Fertil, Dev 6 pages 349-355).

Methods of Selection and Identification of Optimized Recombinant Antigens. Once DNA entrainment has been performed to obtain a library of polynucleotides encoding recombinant antigens, the library is screened to identify those members thereof that encode the antigenic peptides that have an enhanced ability to induce a response. immunological to the pathogenic agent. The selection of recombinant polynucleotides encoding polypeptides having an improved ability to induce an immune response may comprise methods, either in vivo or in vitro, but more frequently, comprises a combination of these methods. For example, in a typical embodiment, members of a recombinant nucleic acid library are collected, either individually or as pools. The clones can be subjected to the analysis directly or can be expressed to produce the corresponding polypeptides. In a preferred embodiment of the present invention, an in vitro selection is performed to identify the best candidate sequences for in vivo studies. Alternatively, the library can be submitted directly to challenge studies in vivo. The assays can employ either the nucleic acids themselves (e.g., in the form of genetic vaccines) or the polypeptides encoded by the nucleic acid. A schematic diagram of a typical strategy, is shown in Figure 5. Both in vitro and in vivo methods are described in more detail below. If a recombination cycle is performed in vivo, the products of the recombination, for example the 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 vivo are sometimes packaged in viruses before selection (eg, bactereophagous). If 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. Frequently, improvements are achieved after a round of recombination and selection. However, recursive recombination of the sequence can also be used to achieve still further improvements of a desired property, or to bring up new (or "different") properties. Recursive recombination of the sequence comprises the successive recombination cycles to generate the molecular diversity. That is, a family of nucleic acid molecules that show some sequence identity with one another but that differ in the presence of mutations is created. In any given cycle, recombination may occur in vivo or in vitro, intracellularly or extracellularly. In addition, the diversity resulting from recombination can be increased in any cycle by means of the application of the previous methods of mutagenesis (for example, error-propensity PCR or cassette mutagenesis) either to substrates or recombination products. .

In a currently preferred embodiment, the polynucleotides encoding the recombinant antigens are subjected to a new molecular crossover, which provides a means for raising chimeras / mutants entrained back to a paternal or wild-type sequence, while retaining the mutations which are critical for the phenotype that provides the optimized immune responses. In addition to removing neutral mutations, the new molecular crossover can also be used to characterize which of the many mutations in an improved variant, contribute most to the improved phenotype. This can not be done in a poor library mode by any other method. The new crossing is performed by means of the entrainment of the improved sequence with a large molar excess of the paternal sequences. The nature of the selection depends on the property or characteristic that is to be acquired or the property or characteristic for which an improvement is sought, and many examples are provided below. Generally, it is not necessary to understand the molecular basis by means of which particular products of recombination (recombinant segments) have acquired new or improved properties or characteristics related to the starting substrates. For example, a gene encoding an antigenic polypeptide can have many component sequences, each having a different intended role (see, for example, Figure 4). Each of these component sequences can be varied and recombined simultaneously. The selection can then be made, for example, for recombinant segments that have an improved ability to induce an immune response to a pathogenic agent without the need to attribute said improvement to any of the individual component sequences of the recombinant polynucleotide. Depending on the particular selection protocol used for a desired property, the initial rounds of selection can sometimes be performed using bacterial cells due to their high transfection deficiencies and ease of cultivation. However, especially for the test of. Immunogenic activity, test animals are used for library expression and selection. In a similar way, other types of selection which are not easy to select from bacterial cells or simple eukaryotic libraries are performed on selected cells for use in a closed environment different from their intended use. The final rounds of selection can be performed on cells or organisms that are as close as possible to the precise cell type or organism that is intended to be used. If additional improvements are desired in one property, at least one, and generally a collection of surviving recombinant segments in a first round of selection, are subjected to a round of additional recombination. These recombinant segments can be recombined with each other or with exogenous segments representing the original substrates or additional variants thereof. Again, recombination may proceed in vivo or in vitro. If the above selection step identifies the desired recombinant segments as cell components, the components may be subjected to further recombination in vivo, or they may be subjected to further recombination in vitro, or they may be isolated before performing a round of recombination in vitro. On the contrary, if a 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 performed, generates additional recombinant segments, which comprise the additional diversity to which the recombinant segments resulting from the previous rounds are present. The second round of recombination can be followed by additional rounds of selection according to the principles explained above for the first round. The severity of the selection can be increased between rounds. As wellI PEPPER. , the nature of the selection and the property that is being selected may vary between rounds, if improvement is desired in more than one property or if more than one new property is acquired. Additional rounds of recombination and selection can then be performed until the recombinant segments have been developed sufficiently to acquire the desired new property or enhanced function. The practice of the present invention comprises the construction of recombinant nucleic acids and the expression of genes in transfected host cells. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of in vitro cloning and amplification methods suitable for the construction of recombinant nucleic acids, such as expression vectors are well known to those skilled in the art. General texts describing molecular biological techniques useful in the practice of the present invention include mutagenesis, including Berger and Kimmel, Guide to Molecular Clonin Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger ); Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Ed.), Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular Biology, F. M . Ausubel and associates, editors, current protocols, a capital partnership between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented until 1998) ("Ausubel")). Examples of techniques sufficient to direct those skilled in the art through in vitro amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), replicase Q amplification and other techniques mediated by polymerase RNA (e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987) U.S. Patent Number 4,683,202; A Guide to PCR Protocols for Methods and Applications (Innis and associates, eds) Academic Press I nc. San Diego CA (1990) (Innis); Arnheim & Levínson (October 1, 1990) C & EN pages 36 to 47 The Journal of N I H research (1991) 3, pages 81 to 94; (Kwoh and associates (1989) Proc. Natl. Acad. Sci. E. U.A 86 page 1 173; Guatelli and associates (1990) Proc. Natl. Acad. Sci. USA 87 page 1874; Lomoll and associates (1989) J. Clin Chem 35, page 1826; Landegren et al. (1988) Science 241, pages 1077-1080; Van Brunt (1990) Biotechnology 8, pages 291-294; Wu and Wallace (1989) Gene 4, page 560 Barringer and associates (1990) Gene 89, page 1 17; and Sooknanan and Malek (1995) Biotechnology 13, pages 563-564.) Improved methods of in vitro cloning of amplified nucleic acids are described in US Patent Number 5,426,039 issued to Wallace and associates The improved methods for the amplification of large nucleic acids by means of PCR are summarized in Cheng et al. (1994) Nature 369 pages 684-685, and references therein, in which PCR applications up to 40kb are generated.An expert in the art will appreciate that essentially any RNA can be c Onverted into a double-pressed DNA, suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, Ausubel, Sambrook and Berger, all above.

Oligonucleotides to be used as samples, such as, for example, in vitro amplification methods, for use as gene samples, or entrainment targets (eg, synthetic genes or gene segments) are chemically synthesized, usually from According to a solid phase tri-esterdephosphoramidite method described by Beaucage and Caruthers (1981) Tetrahedron Letts. , 22 (20) pages 1859-1862, for example, using an automated synthesizer, as described in Needham-VanDevanter and associates (1984) Nucleic Acids Res., 12 pages 6159-6168. The oligonucleotides can also be custom designed and ordered from a variety of commercial sources known to those skilled in the art. Undoubtedly, essentially any nucleic acid as a known sequence, can be designed according to the needs from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com). The Great American Gene Company (http://www.genco.com). ExpressGen Inc. (www.expressgen.com). Operon Technoloigies Inc. (Alameda, CA) and many others. In a similar way, the peptides and antibodies can be ordered according to need from any of a variety of sources such as PeptidoGenic (pkim@ccnet.com). HTI Bio-products, Inc. (http://www.htibio.com). BMA Biomedicals Ltd (U.K.), Bio Synthesis, Inc., and many others. 1 . Purification and in vitro analysis of Nucleic Acids Recombinants and Polypeptides. Once the DNA carryover has been performed, the resulting library of recombinant polynucleotides can be subjected to purification and a preliminary in vitro analysis, in order to identify the most promising candidates for recombinant nucleic acids. Advantageously, the assays can be practiced in a high performance format. For example, in order to purify individual recombinant antigens, the clones can be collected by means of robots in 96-deposit, cultured formats, and if so desired, frozen for storage. Total cell lysates (V antigen), periplasmic extracts, or floating cultures (toxins) can be tested directly by means of the ELISA tests, as described below, but sometimes high performance purification is also needed. . Affinity chromatography using immobilized antibodies or the incorporation of a small non-immunogenic affinity tag, such as a hexahistidine peptide, with immobilized metal affinity chromatography, will also allow rapid purification of the protein. High bonding reagents with 96-well filter bottom plates provide a high-throughput purification process. The scale of culture and purification will depend on the production of protein, but the initial studies will require less than 50 micrograms of protein. Antigens that show improved properties can be purified on larger scales by means of FPLC for a new test and challenge studies in animals. In some embodiments, the entrained polynucleotides encoding the antigen are tested as genetic vaccines. Genetic vaccine vectors containing the entrained antigen sequences can be prepared using robotic colony collection and subsequent purification of the robotic plasmids. The robotic purification protocols of plasmids are available so that they allow the purification of 600 to 800 plasmids per day. The amount and purity of the DNA can also be analyzed, for example, in 96-well plates. In a currently preferred embodiment, the amount of DNA in each sample is robotically normalized, which can significantly reduce the variation between different batches of vectors. Once the proteins and / or nucleic acids are collected and purified as desired, these can be subjected to any of a number of in vitro analysis methods. Each selection includes, for example, a phage display, flow cytometry, and ELISA assays to identify antigens that are efficiently expressed and that have multiple epitopes and an appropriate fold pattern. In the case of bacterial toxins, libraries can also be screened for reduced toxicity in mammalian cells.

As an example, to identify recombinant antigens that are cross-reactive, a panel of monoclonal antibodies can be used for selection. A humoral immune response generally establishes targets for multiple regions of antigenic proteins. Accordingly, monoclonal antibodies can be raised against several regions of immunogenic proteins (Alving and associates (1995) Immunol Rev. 145 Page 5). In addition, there are several examples of monoclonal antibodies that only recognize a deformation of a particular pathogen, and by definition, the serotypes of pathogens are recognized by different sets of antibodies. For example, a panel of monoclonal antibodies has been raised against the VEE envelope proteins, thereby providing a means to recognize the different subtypes of the virus (Roehring and Bolin (1997) J. Clin.Microbiol 35 page 1887). Such antibodies, combined with phage display and ELISA selection, can be used to enrich recombinant antigens that have multiple pathogen strain epitopes. Flow cytometry based on cell sorting will additionally allow the selection of variants that are expressed more efficiently. Phage display provides a powerful method for the selection of proteins of interest from large libraries (Bass and associates (1990) Proteins: Struct. Funct. Genet 8 page 309; Lowman and Wella (1991) methods: A Companion to Methods Enz . 3 (3); 205-216. Lowman an Wells (1993) J. Mol. Biol. 234; 564-578) some recent reviews in the technique of phage display, for example, McGregor (1996) Mol Microbiol 20 (4) pages 685-92; Phage Display of Peptides and Proteins: A Laboratory Manual. BK Kay, J. Winter, J, McCafferty eds. , Academic Press 1996; O'Neil and associates (1995) Curr. Opin. Struct. Biol. 5 (4) pages 443-9; Phizicky et al. (1995) Microbiol Rev. 59 (1) pages 94-123; Clackson and associates (1994) Trends Biotechnol. 12 (5) pages 173-84; Felici and associates (1995) Biotechnol. Annu. Rev. 1 pages 149-83; Burton (1995) Immunotechnology 1 (2) pages 87-94; See also, Cwirla and associates, Proc. Natl. Acad. Sci. USA 87 pages 386-388 (1990); US Patent 5,571, 698 granted to Ladner and associates. Each phage particle deploys a unique variant protein on its surface and packages the decoder gene of that particular variant. The genes carried by the antigens are fused to a protein that is expressed on the surface of the phage, for example, gene 3 of phage M 13, and phage vectors within the cloned ones. In a currently preferred embodiment, a suppressor codon stop (eg, an amber stop codon) separates the genes so that in an E. coli suppressor strain, the antigen-gl l lp fusion is produced and is reached to be incorporated into the phage particles at the time of infection with the phage helper M 13. The same vector can lead to the production of a non-fused antigen only in a non-suppressor E. coli, for purification of the protein.

In the genetic packages most often used for display libraries are bacteriophages, particularly filamentous phage, and especially phage M 13, Fd and F1. Most of the work has involved inserting polypeptide coding libraries to be deployed within either the GL l or the VI VI of these fusion protein-forming phages. See, for example, WO 91/19818 granted to Dower; WO 91/18989 granted to Devlin; WO 92/01047 granted to McCafferty (Gen l l l); WO 92/06204 granted to Huse; WO 92/18619 granted to Kang (Gen VI I I). Said fusion protein comprises a signal sequence, generally but not necessarily, of the phage coat protein, a polypeptide to be deployed and, either the gene 11 or the VI I I gene of the protein or a fragment thereof. The exogenous coding sequences are frequently inserted at or near the N terminus of the gene l l l or of the .gen VI I I although other insertion sites are possible. Eukaryotic viruses can be used to display the polypeptide in an analogous manner. For example, the sample of a human erogulin fused to gp70 of Moloney murine leukemia virus has been reported by Han et al., Proc. Natl. Acad. Sci. USA 92 pages 9747-9751 (1995). The spores can also be used as replicable genetic packages. In this case, the polypeptides are shown from the outer surface of the spore. For example, spores from B. subtilis have been reported as adequate. The coating protein sequences of these spores are provided by Donovan and associates, J. Mol. Biol. 196, 1-10 (1987). The cells can also be used as replicable genetic packages. The polypeptides to be displayed are inserted into the gene encoding a cell protein that is expressed on the cell surface. Bacterial cells which include Salmonella typhimurium, Bacillus Subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseri 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 U.S. Patent 5,571, 698 and references are cited in the present disclosure. For example, the E. coli lamB protein is adequate. The basic concept of deployment methods using phage or other replicable genetic packages 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 capsid 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 phage showing a polypeptide with affinity to an objective, for example, a receptor, binds the target to these phages that are enriched by the affinity selection to the target. The identity of the polypeptide shown from these phages can be determined from their respective genomes. Using these methods, a polypeptide identified as having a binding affinity for a desired target can then be synthesized by volume, by conventional means, or the polynucleotide encoding the peptide or polypeptide can be used as part of a genetic vaccine. Variants with specific binding properties, in this case, the binding of specific antibodies for families, are easily enriched by means of separation with immobilized antibodies. Single-family-specific antibodies are used in each round of separation to rapidly select for variants that have multiple epitopes of the antigen families. For example, antibodies specific to family A can be used to select those dragged clones that show specific epitopes A in the first round of separation. A second round of separation with antibodies specific B, will select from clones A, those that show the specific epitopes A and B. A third round of separation with specific antibodies C, will select the variants with epitopes A, B and C. There is a selection It continues during this process for clones that express themselves well in E. coli and that are of a stable selection production. Improvements in factors such as transcription, translation, secretion, folding and stability are frequently observed and will increase the usefulness of the clones selected for use in the production of vaccines. ELISA phage methods can be used to rapidly characterize individual variants. These tests provide a rapid method of quantifying variants, without requiring the purification of each of the proteins. The individual clones are accommodated in 96-well plates, cultured and frozen for storage. The cells in the duplicated plates are infected with the auxiliary phage, cultured overnight, and pelleted by means of centrifugation. The floaters containing the phage displaying the particular variants are incubated with immobilized antibodies and the linked clones are detected by means of the conjugates of anti-M 13 antibodies. The titration series of the phage particles, immobilized antigen, and / or The competitive antigen competition studies of the soluble antigen, all are highly effective means to quantify the binding of the protein. Variant antigens that show multiple epitopes will be further studied in appropriate animal challenge models. Several groups have reported a system that shows the ribosome in vitro, for the selection of mutant proteins with desired properties from large libraries. This technique can be used in a manner similar to phage display to select or enrich the variant antigens with improved properties, such as broad cross-reactivity for the antibodies and enhanced bending (see, for example, Hanes et al. (1997) Proc, Nat'l. Acad. Sci. USA 94 (10) pages 4937-42; Mattheakis and associates, (1994) Proc. Nat'l. Acad. Sci. USA 91 (19) pages 9022-6; He and associates (1997) Nucí. Acids Res. 25 (24) pages 5132-4; Nemoto and associates (1997) FEBS lett. 414 (2) pages 405-8). There are other deployment methods for selecting improved properties, such as increased expression levels, broad cross-reactivity, increased fold and stability. These include, but are not limited to, the deployment of proteins in intact E. coli or other cells (eg, Francisco et al. (1993) Proc. Nat'l. Acad. Sci. USA 90 pages 1044-1048; Lu and associates (1995) Bio / Technology 13 pages 366-372). Fusions of antigens entrained with proteins bound to DNA can bind the antigen protein to its gene in an expression vector (Schatz et al. (1996) Methods Enzymol, 267 pages 171-91; Gates et al. (1996) J. Mol. Biol. 255 pages 373-86). Different deployment methods and ELISA assays can be used to select entrained antigens with improved properties, such as multiple epitope display, improved immunogenicity, increased expression levels, increased fold ranges and efficiency, increased stability to such factors such as temperature, regulators, solvents, improved purification properties, etc. The selection of entrained antigens with improved expression, fold, stability and purification profile under a variety of chromatographic conditions, can be very important improvements to incorporate them for vaccine manufacturing processes. In order to identify recombinant antigenic polypeptides that exhibit improved expression in a host cell, flow cytometry is a useful technique. Flow cytometry provides a method to efficiently analyze the functional properties of millions of individual cells. The levels of expression of several genes can be analyzed simultaneously and the classification of cells based on the flow of cytometry, also allows the selection of cells showing antigenic variants expressed correctly on the surface of the cell or in the cytoplasm. Very large numbers of cells (> 107) can be evaluated in a single-vial experiment, and the set of the best individual sequences can be recovered from the sorted cells. These methods are particularly useful in the case of, for example, the glycoproteins of the Hantaan virus, which are generally expressed in a very poor way in the cells of mammals. This method provides a general solution for improving the expression levels of pathogen antigens in mammalian cells, a phenomenon that is critical to the function of genetic vaccines.

In order to use flow cytometry to analyze polypeptides that are not expressed on the cell surface, the recombinant polynucleotides can be designed in the library, so that the polynucleotide is expressed as a fusion protein having an amino region. acids which is established as an objective for the membrane of the cell. For example, the region can encode a hydrophobic stretch of the C-terminus of the amino acids, which signals the adhesion of a phosphoinositol-glycan (PIG) term on the expressed protein and leads to the protein to be expressed on the surface of the transfected cell (Whitehorn et al. (1995) Biotechnology (NY) 13 pages 1215-9). With an antigen that is naturally soluble in the protein, this method will probably not affect the three-dimensional folding of the protein in this designed fusion with a new term C. With an antigen that is naturally a transmembrane protein (eg, a membrane surface protein) on pathogenic viruses, bacteria, protozoa or tumor cells) there are at least two possibilities. First, the extracellular domain can be designed to be fused with the sequence of the C terminal for PIG link signaling. Second, the protein can be expressed in toto dependent on the signaling of the host cell to drive it efficiently to the surface of the cell. In the minority of cases, the antigen for expression will have an endogenous link of the PIG terminal (for example, some antigens of pathogenic protozoa).

Those cells expressing the antigen can be identified with a fluorescent monoclonal antibody specific for the C-terminal sequence in the PIG-linked forms of the surface antigen. The FACS analysis allows the quantitative evaluation of the level of expression of the correct form of the antigen of the cell population. The cells expressing the maximum level of antigens are classified and the standard methods of molecular biology are used to recover the plasmid vectors of the DNA vaccine to which this reactivity has been conferred. An alternative procedure that allows the purification of all those cells that express the antigen (and that can be useful before loading it into a cell sorter, because the antigen that expresses the cells can be from a population of a very small minority), is, for the distinctive, or purification by separation of the cells that express the antigen on the surface. The tags can be formed between the antigens expressing the cells and the erythrocytes containing the antibodies covalently coupled to the relevant antigen. These are easily purified by means of gravity sedimentation. The separation of the population of cells on petri dishes, which contain the immobilized monoclonal antibody specific for the relevant antigen, can also be used to remove unwanted cells. In the high throughput assays of the present invention, it is possible to select up to several thousand different entrained variants in a single day. For example, each reservoir of a microtitre dish can be used to perform a separate assay, or if the concentration or effects of the incubation time are to be observed, every 5 to 10 tanks can be tested for a single variant. Therefore, a simple standard microtiter dish can test approximately 100 reactions (eg, 96). If plates of 1536 deposits are used, then a single plate can easily test from about 100 to about 1, 500 different reactions. It is possible to try several different dishes separated per day; the assay selections for up to about 6,000 to 20,000 different assays (eg, comprising different nucleic acids, coding proteins), concentrations, etc.), it is possible using the integrated systems of the present invention. More recently, microfluidic methods for handling reagents have been developed, for example, by Caliper Technologies (Palo Alto, CA). In one aspect, members of the library, e.g., cells, viral plaques, or the like are separated into a solid medium to produce individual colonies (or plaques). Colonies or plaques are identified, using an automatic colony collector (eg, the Q-bot, Genetix, UK), collected and innoculated up to 10,000 different mutants within the 96-well microtiter plates, which optionally obtain glass beads in deposits to avoid aggregation. The Q-bot does not collect a complete colony, but rather inserts a pin through the center of the colony and leaves with a very small cell sample (or viruses in plate applications). The time in which the pin is in the colony, the number of plates to innoculate the culture medium, the time in which the pin is in the middle, half of each size that carries out the innoculation, and each can be controlled and optimized. The uniform process of the Q-bot decreases the error of human manipulation and promotes the range for the establishment of culture (approximately 10,000 / 4 hours). These cultures are then stirred at a temperature and an incubator with controlled humidity. The glass balls in the plates of the microtitrator act to promote uniform aeration of the dispersion of cells or the like, similar to the blades of a fermentor. The clones of the crops of interest can be cloned by means of the limiting dilution. The plates or cells constituting the libraries can also be directly selected for the production of proteins, either by means of detection of hybridization, activity of the protein, binding of the protein to antibodies or the like. The ability to detect a subtle increase in the performance of a member of the library drawn on parental deformations depends on the sensitivity of the assay. The opportunity to find organisms that have an improvement in the ability to induce an immune response is increased by the number of individual mutants that can be selected by the assay. To increase the chances of identifying a set of sufficient size, a pre-selection can be used to increase the number of mutants processed by bending-10. The goal of the previous selection will be to quickly identify mutants that have product titers equal to or better than the parental strain (s) and move only these mutants to the liquid cell culture for subsequent analysis. A number of well-known robot systems have also been developed for the solution phase chemistries useful in the test systems. These systems include automatic work stations similar to automatic synthesis devices developed by Takeda Chemical Industries, LTD. (Osaka, Japan), and many systems that use robot arms (Zymate I I, Zymark Corporation, Hopkinton, Mass.; Orea, Hewlett-Packard, Palo Alto, Calif. ), which simulate manual synthetic operations performed by a scientist. Any of the foregoing apparatuses are suitable for use with the present invention, for example, for the high-throughput screening of molecules encoded by nucleic acids altered by codon. Those skilled in the relevant art will appreciate the nature and implementation of modifications for these apparatuses (if they exist), so that they can operate as mentioned in the present description with reference to the integrated system.

High-throughput screening systems are commercially available (see, for example, Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Bechman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA; , etc). These systems are usually automatic complete procedures, including all sample and reagent pipetting, fluid supplies, scheduled incubations, and final microplate readings at the appropriate detector (s) for the assay. These configurable systems provide high performance and fast start-up, as well as a high degree of flexibility and design.

The manufacturers of such systems provide detailed protocols of several high yields. Thus, for example, Zymark Corp., provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding and the like. Microfuidic methods have also been developed for the handling of reagents, for example, by Caliper Technologies (Palo Alto, CA). In any of the embodiments of the present invention, viewed (and, optionally, recorded) images are additionally and optionally processed by a camera or other recording apparatus (e.g., a photodiode and data storage apparatus), for example, by digitizing the image and / or storing and analyzing the image on a computer. As noted above, in some applications the signals resulting from the assays are fluorescent, performing, in these examples, the appropriate optical detection methods. A variety of peripheral equipment and commercially available software is valid for digitizing, storing and analyzing a digitized or digitized optical image, for example, using a PC (DOS-based machines, OS2 WI NDOWS, WI NDOWS NT or WINDOW95 compatible with chips from Intel x86 or Pentium), MACI NTOSH or UNIX-based computers (for example, SUN workstation). In common use within the art, a conventional system transports light from the test apparatus to a cooled charge coupled device (CCD) camera. A CCD camera includes a formation of photo elements (pixels). The light from the specimen is reflected in the CCD. The particular pixels corresponding to the regions of the specimen, (for example sites of individual hybridization in a formation of biological polymers) are shown to obtain readings of light intensity for each exposure. Multiple pixels are processed in parallel to the increase in speed. The apparatus and methods of the present invention are readily used to observe any sample, for example, by fluorescent and dark field microscopic techniques. The integrated systems for analysis in the present invention typically include a digital computer with high performance liquid control software, image analysis software, data interpretation software, a liquid robot control armature for the transfer of solutions from a source to an operable destination linked to the digital computer, an input device (for example a computer keyboard), to input data to the digital computer, for high-performance liquid transfer using the liquid robot control armature, and optionally , an image scanner for the digitization of brand signals from the marked component of the test. The image scanner interacts with the image analysis software to provide a measure of optical intensity. Normally, the intensity measurement is interpreted by the data interpretation software, to show whether optimized recombinant antigen polypeptide products are produced. 2. Immunization of Antigen Library In a currently preferred embodiment, immunization of antigen library (ALI), is used to identify optimized recombinant antigens, which have improved immunogenicity. In an animal for testing, ALI comprises the introduction of the nucleic acid library encoding recombinant antigens, or the recombinant antigens encoded by the entrained nucleic acids. Subsequently, the animals are subjected to live stimuli, using live pathogens. Neutralization of antibodies and cross-protective immunological responses are studied after immunization with whole libraries, pools and / or variants of individual antigens. Methods of immunizing animals for testing are well known to those skilled in the art. In the presently preferred embodiments, the test animals are immunized two or three times at two week intervals. One week after the last immunization, the animals are stimulated with live pathogens (or mixtures of pathogens), and the survival and symptoms of the animals are followed. Immunizations that use animals for testing, are described, for example in, Roggenkamp and associates (1997) Infecí. Immun. 65: 446; Woody and associates (1997) Vaccine 2: 133; Agren and associates (1997), J.

Immul. 158: 3936; Konisi et al. (1992), Virology 190: 454; Kinney and associates (1988), J. Virul. 62: 4697; lacono-Connors and associates (1996) Virus Res. 43: 125; Kochel and associates (1997) Vaccine 15: 547, and Chu and associates (1995), J. Virol. 69: 6417. Immunizations can be carried out by injecting either the integrated recombinant polynucleolides, for example, as a genetic vaccine, or by immunizing animals with polypeptides encoded by the recombinant polynucleotides. Bacterial antigens are usually selected first as recombinant proteins, where viral antigens are preferably analyzed using genetic vaccines. To dramatically reduce the number of experiments required to identify antigens having improved immunogenic properties, a conjunction and unwinding can be used, as illustrated in diagram form in Figure 6. The sets of recombinant nucleic acids, or polypeptides encoded by the recombinant nucleic acids, are used to immunize the animals for testing. These sets that result in a protection against the stimulus to pathogens, are subsequently subdivided and subjected to additional analysis. The high throughput in vitro methods described above can be used to identify the best candidates for sequences for in vivo studies. Stimulation models that can be used to select protective antigens include pathogen and toxin models, such as Yersinia bacteria, bacterial toxins (such as Staphylococcal and Streptococcal enterotoxins, E. coli / V. cholerae enterotoxins), Venezuelan equine encephalitis virus (VEE), Flaviviruses (Japanese encephalitis virus, tick borne encephalitis virus, Dengue virus), Hantaan virus, Herpes simplex, influenza virus (for example, Influenza A virus), Vesicualr Stotatitis virus, Pseudomonas aeruginosa, Salmonella tiphimurium, Escherichia coli, Klebsiella pneumoniae, Toxoplasma gondii, Plamodiium yoelii, Hepes simplex, influenza virus (for example, Influenza A virus), and Vesicular Stomatitis Virus. However, animals for testing can also be stimulated with tumor cells to make possible the selection of antigens that efficiently protect against malignancies. Individual antigens or pools of antigens are introduced into the animals intradermally, intramuscularly, intravenously, intratracheally, anally, vaginally, orally or intraperitoneally, and the antigens that can prevent the disease are chosen, when desired, to Drag laps and additional selection.

Eventually, the most potent antigens based on in vivo data in animals for testing and comparative in vitro studies in animals and men, are chosen for experiments in humans, and their ability to prevent and treat diseases in humans is investigated. In some embodiments, the immunization of antigen libraries and the conjunction of individual clones is used to immunize against a pathogenic strain, which was not included in the sequences that were used to generate the library. The level of transverse protection provided by different deformations of a given pathogen can be significant. However, the homologous titrant is always superior to the heterologous titrant. The conjunction and unwinding is specifically efficient in models where minimal protection is provided by the wild-type antigens used as starting material for entrainment (e.g., minimal protection by antigens A and B against deformation C in Figure 3B). This method can be taken, for example, when the V-antigen of Yersinae or Hantaan virus glycoproteins is understood.

In some embodiments, the desired selection comprises the analysis of the immune response based on immunological assays known to those skilled in the art. Normally, test animals are first immunized and blood or tissue samples are collected, for example, once or twice a week, after the last immunization. These studies make it possible to measure, one by one, the immune parameters that correlate with protective immunity, such as the induction of specific antibodies (particularly IgG) and the induction of specific T-lymphocyte responses, in addition to determining whether an antigen or Antigen set provides protective immunity. The basal cells or peripheral blood mononuclear cells can be isolated from the immunized test animals, and the presence of antigen-specific T cells and induction of cytokine synthesis can be measured. The staining of ELISA, ELISPOT and cytoplasmic cytokine combined with cytometry, can provide this information at a simple cell level. Common immunological tests that can be used to identify the efficacy of immunization include antibody measurements, neutralization assays, and analysis of activation levels or frequencies of antigen or lymphocyte display cells that are specific for the antigen or pathogen. Test animals that can be used in such studies include, but are not limited to, mice, rats, guinea pigs, hamsters, rabbits, cats, pigs and monkeys. The monkey is a particularly useful animal for testing, since the MHC molecules of monkeys and humans are very similar. The virus neutralization assays are useful for the detection of antibodies that not only bind specifically to pathogens, but also neutralize the function of the virus. These assays are usually based on the detection of antibodies in the serum of immunized animals and on the analysis of these antibodies for their ability to inhibit viral growth in tissue culture cells. Such assays are well known to those skilled in the art. In the publication by Dolin R (J. I nfect, Dis. 1995, 172: 1 175-83), an example of a virus neutralization assay is described. Virus neutralization assays provide means for selecting antigens. They also provide protective immunity. In some embodiments, the entrained antigens are selected for their ability to induce T cell activation in vivo. More specifically, peripheral blood mononuclear cells or basal cells of injected mice can be isolated and the ability of cytotoxic T lymphocytes to lyse infected autologous target cells is studied. The basal cells can be reactivated with the specific antigen in vitro. In addition, the helper cell activation and differentiation T, is analyzed by measuring the proliferation or production of TH1 (I L-2 and IFN-?) And TH2 (I L-4 and I L-5) cytokine cells through ELISA and directly in CD4 + T cells, by staining cytoplasmic cytokine and flow cytometry. Based on the cytokine production profile, alterations in the ability of the antigens to direct TH 1 / TH 2 differentiation can also be selected (as evidenced, for example, by changes in proportions I L-4 / I FN- ?, IL-4 / IL-2, IL-5 / IFN- ?, I L-5 / I L-2, I L-13 / v, IL-13 / IL-2). The analysis of T cell activation induced by antigen variants is a very useful screening method, since the potent activation of specific T cells in vivo correlates with the induction of protective immunity. The frequency of antigen-specific CD8 + T cells in vivo can also be analyzed directly, using tetramers of MHC class I molecules expressing specific peptides derived from the corresponding pathogen antigens (Ogg and McMichael, Curr Opin. Immunol. : 393-6; Altman et al., Science 1996, 274: 94-6). The binding of the tetramers can be detected using flow cytometry and will provide information regarding the effectiveness of the entrained antigens to induce the activation of specific T cells. For example, cytometry and staining of tetramers, provide a method of efficient identification of T cells that are specific for a given antigen or peptide. Another method comprises washing, using plates covered with tetramers with the specific peptides. This method allows large numbers of cells to be handled in a short time, but the method only selects the highest expression levels. The higher the frequency of antigen-specific T cells in vivo, the greater the efficiency of immunization, allowing the identification of antigen variants that have the most potent capacity to induce immune protective responses. These studies are particularly useful when conducted in monkeys, or other primates, since MHC class I molecules of humans resemble those of other primates more than those of mice. Another useful screening method is the measurement of the activation of antigen presenting cells (APC) in response to immunization by antigen variants. The induction of APC activation can be detected based on changes in the surface expression levels of activation antigens, such as B7-1 (CD80), B7-2 (CD86), M HC class II and II, CD14, CD23 and Fc receptors and the like. Trapped cancer antigens that induce cytotoxic T cells that have the ability to kill cancer cells can be identified by measuring the ability of T cells derived from immunized animals to kill cancer cells in vitro. Normally, cancer cells are first labeled with radioactive isotopes and the release of radioactivity is an indication of killing of tumor cells after incubation, in the presence of T cells from immunized animals. Such cytotoxicity assays are known in the art.

Also an indication of the efficiency of an antigen to activate specific T cells for, for example, carcinogenic, allergenic or autoantigen antigens, when the antigen is injected into the skin of a patient or an animal for testing, is the degree of inflammation on the skin, . The strong inflammation is correlated with the strong activation of antigen-specific T cells. Activation of tumor-specific T cells can lead to improved killing of tumors. In the case of autoantigens, immunomodulators can be added that deviate the responses towards TH2, where in the case of allergens, a TH 1 response is desired. Skin biopsies can be taken, allowing detailed studies of the type of immune response that occurs at the sites of each injection (in mice and monkeys, large amounts of injections / antigens can be analyzed). Said studies include the detection of changes in the expression of cytokines, chemokines, accessory molecules and the like, by cells when the antigen is injected into the skin. In order to select the antigens that have optimal capacity to activate antigen-specific T cells, mononuclear cells from previously infected or immunized human individuals can be used. This is a particularly useful method, since the MHC molecules that will be present in the antigenic peptides are human HCM molecules. Peripheral blood mononuclear cells or purified professional antigen presentation cells (APCs) can be isolated from vaccinated or previously infected individuals or from patients with acute infection with the pathogen of interest. Because these individuals have increased the frequencies of T-cells specific for circulating pathogens, the antigens expressed in PBMCs or APCs purified from these individuals will induce proliferation and cytokine production by antigen-specific CD4 + and CD8 + T cells. Therefore, antigens that simultaneously contain epitopes of several antigens, can be recognized for their ability to stimulate T cells from several patients infected or immunized with different pathogenic antigens, carcinogenic antigens, autoantigens or allergens. A bright coat derived from a blood donor can be obtained, which contains lymphocytes from 0.5 liters of blood, and up to 10 4 PBMC, making possible very large selection experiments using T cells from a donor. When healthy vaccinated individuals (laboratory volunteers) are studied, B cell lines transformed by EBV can be made from these individuals. These cell lines can be used as antigen presenting cells in subsequent experiments, using blood from the same donor; this reduces the variation of internal trials and from donor to donor. In addition, antigen-specific T cell clones can be made, after which, the antigen variants are introduced to the B cells transformed by EBV. Subsequently, the efficiency with which transformed B cells induce the proliferation of specific T cell clones is studied. When working with specific T cell clones, proliferation and cytokine synthesis responses are significantly higher, than when total PBMCs are used, because the frequency of antigen-specific T cells among PCMC is very low. CTL epitopes can be presented by most types of cells, since the glycoproteins on the surface of the major histocompatibility class I (MHC) complex are widely expressed. Therefore, transfection of cells in culture by libraries of antigen sequences entrained in suitable expression vectors can lead to class I epitope presentation. If the specific CTLs directed to a given epitope, have been isolated from an individual, then the co-culture of the transfected presentation cells and the CTLs, can lead, if the epitope occurs, to trigger the release by the CTLs of cytokines, such as IL-2, I FN- ?, or TNF. The higher amounts of TNF released will correspond to the more efficient processing and presentation of the class I epitope from the developed, entrained sequence. Trailed antigens that induce cytotoxic T cells have the ability to kill infected cells that can also be identified by measuring the ability of T cells derived from immunized animals to kill infected cells in vitro. Normally the target cells are first labeled with radioactive isotopes and the release of radioactivity is an indication of killing of the target cell after incubation in the presence of T cells from immunized animals. Such cytotoxicity assays are known in the art. A second method to identify optimized CTL epitopes does not require the isolation of reactivating CTLs with the epitope. In this method, the cells expressing the MHC class 1 surface glycoproteins are transfected with the library of developed sequences, as indicated above. After a suitable incubation is left for processing and presentation, a soluble detergent extract is prepared from each cell culture and after a partial purification of the epitope-MHC complex (perhaps optional), the products are subjected to mass spectrometry (Henderson and associates (1993) Proc. Nat'l. Acad. Sci. USA 90: 10275-10279). Since the sequence of the epitope is known, the presentation of which will be increased, the mass spectrogram can be calibrated to identify this peptide. In addition, a cellular protein used for internal calibration could be the integrated M HC molecule. Therefore, the amount of peptide epitope binding can be measured as a ratio of MHC molecules.

Use of Recombinant Multivalent Antigens The multivalent antigens of the present invention are useful for treating and / or preventing different diseases and conditions, with which the respective antigens are associated. For example, multivalent antigens can be expressed in a suitable host cell and are administered in polypeptide form. Formulations and formulation regimens suitable for the administration of vaccines are well known to those skilled in the art. In the currently preferred embodiments, optimized recombinant polynucleotides encoding improved allergens are used in conjunction with a gene vaccine vector. The choice of vector and components can also be optimized for the particular purpose of treating allergies. For example, the polynucleotide encoding the antigen polypeptide can be replaced under the control of a promoter, for example, a tissue-specific or high-activity promoter. The promoter used to express the antigen polypeptide can be optimized using methods of recombination and selection analogous to those described in the present invention. The vector may contain immunostimulatory sequences, such as described in the commonly assigned US Patent Application, also pending Series No., entitled "Optimization of Molecules Immunomodulators ", filed as TTC File No. 18097-030300US on February 10, 1999. For many of the immune responses mediated by the antigens described in the present invention, a vector constructed to drive a TH 1 response is preferred (see example, the commonly assigned US Patent Application, also pending Series No., entitled "Construction of Genetic Vaccine Vector", presented as File TCC No. 18097-030100US on February 10, 1999). Sometimes it is convenient to employ a genetic vaccine that is targeted to a particular target cell (eg, an antigen presenting cell or an antigen processing cell); Appropriate management methods are described in the commonly assigned, also pending, US Patent Application, Series No., entitled "Genetic Vaccine Vector Address", filed as TCC File No. 18097-030200US on February 10, 1999. Genetic vaccines encoding the multivalent antigens described in the present disclosure 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, usually by systemic administration (eg, intravenous, intraperitoneal, intramuscular, subdermal, intracranial, anal, vaginal, oral, buccal route or a route of administration that can be inhaled) or via a route of administration of topical application. Alternatively, the vectors can be administered to ex vivo cells, such as explanted cells from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or hematopoietic stem cells from universal donors, followed by reimplantation of the cells in a patient, usually after the selection of cells that have incorporated the vector. A large number of administration methods are well known to those skilled in the art. Such methods include, for example, liposome-based gene administration (Debs and Zhu (1993), WO 93/24640, Mannino and Gould-Fogerite (1988) Bio Techniques 6 (7): 682-691, US Patent No. 5,279,833 for Rose; Brighan (1991) WO 91/06309; and Felgener and associates (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414, as well as the use of viral vectors (eg, adenoviral (see, for example. Berns and associates (1995) Ann. NY Acad. Sci 772 95-104; AM and associates (1994) Gene Ther. 1: 267-384; and Haddada and associates (1995), Curr. Top. Microbiol. Immunol. 199 (Pt 3): 297-306 for review), papillomaviral, retroviral (see for example, Buchscher and associates (1992) J. Virol 66 (5) 2731-2739; Johann and associates (1992) J. Virol 66 ( 5): 1635-1640 (1992), Sommerfelt and associates, (1990) Virol 176: 58-59, Wilson and associates (1989) J. Virol 63: 2374-2378, Miller and associates, J. Virol. : 2220-2224 (1991), -Staal and associates, PCT / US94 / 05700 and Rosenburg and Fauce (1993) in Fundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd, New York and the references thereof, and Yu and associates, Gene Therapy (1994) supra.), and adeno-associated viral vectors (see, West and associates (19887) Virology 160: 38-47; Carter and associates (1989) American Patent No. 4,797,368; WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5: 793-801; Muzyczka (1994) J. Invst. 94: 1351 and Samulski (supra) for a review of AAV vectors; see also, US Patent No. 5, 173,414, by Lebkowski, Tratschin already Partners (1985) Mol. Cell. Biol. 5 (11): 3251-3260; Tratschin and associates (1984) Mol. Cell. Biol., 4: 2072-2081; Hermonaat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81: 6466-6470; McLaughlin and associates (1988) and Samulski and associates (1989) J. Virol. , 63: 03822-3828), and the like. DNA and / or RNA "Nude", which comprises a genetic vaccine, can be introduced directly into a tissue, such as muscle. See for example, USPN 5,580,859. Other methods such as biolistics or particle-mediated transformation (see for example, Sanford and associates, USPN 4,945,050; USPN 5,036,006), for the introduction of genetic vaccines into cells of a mammal according to the present invention are also suitable. These methods are useful not only for the live introduction of DNA in a mammal, but also for the ex vivo modification of cells for reintroduction in a mammal: As in 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 cell transduction in vivo. Genetic vaccines obtained using these methods of the present invention, can be formulated as pharmaceutical compositions for administration in any suitable form, including parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical, oral, rectal, intrathecal, buccal (for example, sublingual), or local administration, such as by aerosol or transdermally, for prophylactic and / or therapeutic treatment. In the transdermal administration, pretreatment of the skin may be useful, for example, by the use of hair removal agents. Suitable methods of administration of such packaged nucleic acids are available, and are well known to those skilled in the art, and although more than one route of administration can be used for a particular composition, often, a particular administration route can be used. provide a more immediate and more effective reaction than another. The pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Therefore, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention. A variety of aqueous carriers, for example regulated salt and the like, can be used. These solutions are sterile and are generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients, as required for approximate physiological conditions, such as pH adjusting and regulating agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and Similar. The concentration of the genetic vaccine vector in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like, according to the particular mode of administration selected and the needs of the patient. Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, pills or tablets, each containing a predetermined amount of active ingredient, such as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. The tablet forms may include one more excipients of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, stearate of magnesium, stearic acid and other excipients, colorants, fillers, binders, diluents, regulating agents, wetting agents, preservatives, flavoring agents, inks, disintegrating agents, and pharmaceutically compatible carriers. The tablet forms may comprise active ingredients within a flavoring, typically sucrose and acacia or tragacanth, as well as the tablets comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels and the like. they contain, in addition to the active ingredient, carriers known in the art. It is recognized that genetic vaccines, when administered orally, should be protected from digestion. This is usually carried out either by complicating the vaccine vector with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the vector in a suitably 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 slow release of the active ingredient. Packed nucleic acids can be made, alone or in combination with other suitable components, in aerosol formulations (for example, they can be "nebulized"), to be administered through inhalation. Aerosol formulations can be placed in acceptable pressurized impellers, such as dichlorodifluoromethane, propane, nitrogen and the like. Said formulations suitable for rectal administration, include, for example, suppositories, which consist of nucleic acid packaged with a suppository base. Suitable suppository bases include natural or synthetic trlglycerides or paraffin hydrocarbons. Formulations suitable for parenteral administration, such as, for example, intra-articular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, include sterile aqueous and non-aqueous isotonic injection solutions, which contain antioxidants, regulators, bacteriostats and solutes which reproduce the isotonic formulation with the blood of the intended recipient, and sterile aqueous and non-aqueous suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. In the practice of the present invention, 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. The packaged nucleic acid formulations can be presented in sealed unit dosage or multiple dosage containers, such as ampoules and flasks. The injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the type described above. The cells transduced by the nucleic acid can also be administered intravenously or parenterally.

The dose administered to a patient, within the context of the present invention, must be sufficient to effect, over time, a beneficial therapeutic response in the patient. The dose will be determined by the efficacy of the particular vector employed and the condition of the patient, as well as the body weight or vascular surface area of the patient to be treated. The size of the dose will also be determined by the existence, nature and extent of any adverse side effects that accompany the administration of a particular vector, or type of cell transduced in a particular patient. In determining the effective amount of the vector that will be administered in the treatment or prophylaxis of an infection or other condition, the medical specialist evaluates vector toxicities, disease progression, and the production of anti-vector antibodies, if at all. exist. In general, the equivalent dose of a naked nucleic acid from a vector is from about 1 μg to 1 mg for a normal 70 kilogram patient, and the doses of vectors used to administer the nucleic acid are calculated to produce an amount of equivalent therapeutic nucleic acid. The administration can be carried out by means of simple or divided doses. In therapeutic applications, the compositions are 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 alleviate the disease and its complications. A suitable amount to accomplish 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 state of health of the patient. Simple or multiple administrations of the compositions can be administered, depending on the dose and frequency, as required and tolerated by the patient. In any case, the composition must provide a sufficient amount of the proteins of the present invention to treat the patient effectively. In prophylactic applications, the compositions are administered to a human or other mammal 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 pharmaceutical methods in cell cultures or animals for experiment. The LD5o (the lethal dose for 50% of the population) can be determined, and the ED5o (the therapeutically effective dose in 50% of the population), using the procedures presented in the present invention, and those known procedures of another way by experts in the art.

A typical pharmaceutical composition for intravenous administration would be from about 0.1 to 10 mg. per patient per day. Doses of from 0.1 to about 100 mg per patient per day can be used, particularly when the medicament is administered to an isolated site and not within the bloodstream, such as within a body cavity or within a lumina of an organ. In topical administration, substantially higher doses are possible. Current methods of preparing 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, 15th. Edition, Mack Publishing Company, Easton, Pennsylvania (1980). The multivalent antigen polypeptides of the present invention and the genetic vaccines that express the polypeptides can be packaged in packages, delivery apparatus and equipment for administering genetic vaccines to a mammal. For example, packages and delivery apparatus containing one or more dosage unit forms are provided. Typically, instructions for administering the compounds will be provided with the packages, along with an appropriate 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, a mammalian immune response.

EXAMPLES The following examples are presented by way of illustration but not to limit the present invention.

EXAMPLE 1 Development of Ampli-Spectrum Vaccines against Pathogens and Bacterial Toxins A. Evolution of Yersinia V Antigens This Example describes the use of DNA entrainment to develop immunogens that produce strong protective cross-immune responses against a variety of Yersinia strains. Passive immunization with antigen-anti-V antibodies or active immunization with purified V antigen may provide protection against stimulation with a virulent autologous Yersinia species. However, protection against heterologous species is limited (Motin et al., (1994) Infect. Immun. 62: 4192). As described in the present description, the V-antigen genes of a variety of Yersinia strain, including serotypes of Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis, are subjected to DNA entrainment. The Yersinia pestis V antigen coding sequence is used, for example, as a challenge in a database investigation to identify homologous genes that can be used in a family tracing format to obtain improved antigens.

In Table 1, the results of a BLAST investigation of the GenBank and EMBL database are shown, in which each line represents a single sequence entry that lists the database, access number, name of the position, bit marker and value E. (See, Altschul and associates (1997) Nucleic Acids Res. 25: 3389-3402, for a description of the research algorithm). Homologous antigens have been cloned and sequenced from a number of distinct yet related Yersinia strains and additional natural diversity is obtained by cloning antigen genes from other strains. These and other genes or fragments thereof are cloned by methods such as PCR, entrained and selected for enhanced antigens.

The entrained clones are selected by phage display and / or selected by ELISA to identify those recombinant nucleic acids encoding polypeptides having multiple epitopes corresponding to the different serotypes. The entrained antigen genes are cloned into a filamentous phase genome for the deployment of polyvalent phage or a phagemid vector suitable for the deployment of monovalent phage. A typical protocol for washing antigens by phage display is as follows: • Cover a suitable surface at night (for example, Nunc Maxisorp Multi-Purpose Reservoir Plate) at a temperature of 4 ° C with a target antibody, usually at a concentration of 1 -10 μg / ml in PBS or another suitable regulator. • Rinse and Block with PBSM (PBS + 3% dry milk without fat) for 1 to 2 hours, at a temperature of 37 ° C. • If necessary, pre-block the phage (PBSM, RT 1 hr). • Rinse the tube and leave the phage for binding (usually 1 hr @ 37 ° C) «You can vary the time, temperature, regulator, addition of a competitive inhibitor, etc. • Thoroughly wash (15x) with PBS (PBS + 0.1% TWEEN20), then with PBS • Elute bound phage with 100 mM triethylamine, competitive ligand, low pH protease (for example, 10 mM glycine), etc. , and then, if necessary, neutralize the pH. • Infect E. coli with eluted phage to transduce the expression of phagonid in a new host. Holder and plate for colonies on medication plates.

• Collect colonies in developed middle cells and infect them with auxiliary phage to produce phage for the next round.

Phage ELISA assays are a useful method to quickly evaluate clones alone after library washing. The colonies alone are collected in the individual reservoirs of a multi-deposit plate containing a 2YT medium and developed as a master plate. A duplicate plate is infected with auxiliary phage and developed so that the phage of a single reservoir will display a single antigen variant. A suitable protocol for phage ELISA assays is described below: • Cover the microtiter plate overnight with 50 μl of 1 μg / ml of target antibody at a temperature of 4 ° C. • Rinse and block with PBSM for 2 hours @ 37 ° C. • Rinse, add the previously blocked phage and leave for binding for one hour @ 37 ° C. • Wash the plates with PBST 3X, then PBS 3X with 2 minute soaks. • Add, for 1 hr @ 37 ° C, anti-M 13 antibodies conjugated by H RP (or AP). • Add substrate and measure absorption. • Identify positive clones for further evaluation.

ELISA assays can also be used to select individual antigens with multiple epitopes or increased expression levels. The colonies alone, are collected in individual deposits of a plate of multiple deposits that contain a medium and adequate development, as a master plate so that the antigens produced from a single deposit, are a variant of only antigen. A duplicate plate is developed and induced to produce proteins, for example, by the addition of 0.5 mM I PTG for systems and developments based on Lac repressor, for a time suitable for the antigen that will be produced. At this point, a crude antigen preparation is made, which depends on the antigen and where it is produced. The secreted proteins can be evaluated by floating-cell assay after centrifugation. Periplasmic proteins are often easily released from cells, by simple extraction into hyper- or hypo-tonic regulators. Proteins produced intracellularly require some form of cell lysate, such as detergent treatment to release them. A suitable protocol for ELISA assays is described below: • Cover the microtiter plate overnight with 50 μl of 1 μg / ml of target antibody at a temperature of 4 ° C. • Rinse and block with PBSM for 2 hrs. @ 37 ° C.

• Rinse, add antigen preparation and leave for binding for 1 hr @ 37 ° C. • Wash the plates with PBST 3x • Add secondary antibody conjugated by HRP (or AP) and incubate for 1 hr @ 37 ° C. • Add the appropriate substrate and measure absorption. • Identify positive clones for further evaluation.

Antibodies specific to many of the different antigens are commercially available (eg, Toxin Technology, Inc., Sarasota, FL) or can be generated by immunizing suitable animals with purified antigens. Sepharose Protein A or Protein G (Pharmacy) can be used to purify serum immunoglobulins. Various affinity purification schemes can be used, to further purify, if necessary, specific antibody families, such as immobilization of specific antigens for NHS activated sepharose beads, CNBr or epoxy. Other related antigens, including soluble forms, can be included to prevent binding and immobilization of cross reactive antibodies. The multivalent polypeptides that are identified by the initial screening protocol are purified and subjected to live selection. For example, the entrained antigens selected by a combination of any or none of these methods are purified and used to immunize animals, initially mice, which are subsequently evaluated for improved immune responses. Typically, 10 micrograms of protein is injected to a suitable site with or without an appropriate adjuvant, for example, alhydrogel (EM Seargent Pulp and Chemical, Inc.) and the animals are promoted with an additional dose after 2 to 4 weeks. At this point, serum samples are extracted and evaluated by the ELISA assay for the presence of antibodies that cross-react against multiple parental antigens. In this ELISA assay format, the antigens are covered in plates of multiple deposits, then the serial dilutions of each serum are allowed to bind. After washing antibodies without binding, a secondary antibody conjugated by H RP or AP is ligated against the constant region of the test antibody, for example, IgG Fc (Sigma). The absorbance of each deposit is read by a plate reader at the appropriate wavelength (eg 490 nm for OPD), and those that produce higher antibody titers to multiply antigens, are selected for further evaluation. Additionally, the ability of the antigens to generate neutralizing antibodies can be evaluated in an appropriate system. The antigenic variants that provoke a broad cross-reactive response are further evaluated in a virulent stimulus model, with the appropriate pathogenic organism. For example, multivalent polypeptides are used to immunize mice, which are subsequently stimulated with live Yersinia bacteria. Those multivalent polypeptides that protect against stimulation are identified and purified.

B. Evolution of ampli-spectrum vaccines against bacterial toxins This Example describes the use of DNA entrainment, to obtain multivalent polypeptides, which are effective to induce an immune response against a broad spectrum of bacterial toxins. 1 . Staphylococcus Group A Streptococci, which can cause diseases such as food poisoning, toxic attack syndrome and autoimmunity disorders, is highly toxic by inhalation. The toxin family of Streptococcus Group A numbers around 30 related members, making this group a suitable target for family trawling. Therefore, this Example describes the use of family DNA carryover to create chimeric proteins that have the ability to elicit broad spectrum protection. As described in the present disclosure, nucleic acids encoding many diverse attenuated toxins are subjected to DNA entrainment. Table 2 shows the output of a BLAST investigation of GenBank, PDL, EMBL and Swissprot, using protein B enterotoxin aureus S, to identify homologous genes that can be used in a family tracing format to obtain improved antigens. The entrained recombinant clones are initially selected by phage display and / or selected by ELISA for the presence of multiple epitopes of the different families. Variant proteins with multiple epitopes are purified and used for in vivo selection, as described above. The specific antibodies for different subtypes are analyzed and the variants of toxins that provoke widely crossed reactive responses, will be further evaluated in the stimulus models. 2. Escherichia coli and Vibrio cholerae This Example, describes the use of DNA entrainment to obtain cross-reactive multivalent polypeptides, which induce an immune response against the labila toxin-by heating E. coli (LT), cholera toxin (CT), and verotoxin (VT) The nucleic acids encoding the B-chains of cholera toxin and LT, are subjected to DNA entrainment. Table 3 shows the results of a BLAST investigation that uses the B chain of cholera toxin V, to identify homologous genes that can be used in a family tracing format, to obtain improved antigens. Homologous antigens have been cloned and sequenced from a number of related but distinct Vibrio and E. coli strains. These and other genes or fragments thereof can be cloned by methods such as PCR, entrained and selected for enhanced antigens.

Chimeric toxins that cause high levels of neutralization of antibodies against both toxins and have improved adjuvant properties are identified. For example, the dragged clones are selected by phage display and / or are selected by ELISA assays for the presence of epitopes of the different parental B-chains. Variants with multiple epitopes are purified and further studied for their ability to act as adjuvants and to elicit cross protective immunological responses in stimulus models.

Example 2 Evolution of Amplio-Spectrum Vaccines against Borrelia burgdorferi Lyme disease is currently one of the fastest developing infectious diseases in the United States. This is caused by infection of the spirochete bacterium Borrelia burgdorferi, which is carried and spread by the infected tick picket. Early signs of infection include skin rash and flu-like symptoms. If Lyme disease is left untreated, it can cause arthritis, heart disorders and facial paralysis. Early treatment of Lyme disease with antibiotics can stop the infection, but a lasting immunity can not develop, making it possible to contract the infection again. To acquire immunity, a current vaccine requires three immunizations over a period of one year. Both passive and active immunization with protein from outer surface protein B. Burgdorferi (OspA), have been useful in protection against infection with B. burgdorferi, but have no effect against infections in progress, since this antigen is not expressed in vertebrate hosts. The OspA protein, normally anchored on the outside of the cell, by a portion of lipid covalently adhered through a terminal amine cysteine residue. In contrast, the outer surface protein C (OspC), which is highly expressed by spirochetes in vertebrate hosts and vaccination of individuals infected with OspC protein, can be an effective therapy in the cure of infection (Zhong et al. (1997). ) Proc. Nat'l. Acad. Sci. USA 9412533-12538 A recent BLAST investigation (Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402) of GenBank, PDB, SwissProt, databases was used. Spupdate and PI R non-redundant, to identify homologs of the outer surface protein gene OspA.This resulted in the identification of around 20 OspA-related entries.Table 4 shows in each case one hundred entries under the different deformations of B. burgdorferi, B. garinii, B. afzelli, B. tanukii and B. turdi, which share at least 83% of the identity of the DNA sequence with the OspA protein Borrelia burgdorferi.The ospA genes from these and other def Ormations, provide a source of diversity for family trawling, to obtain antigens for the prevention of Lyme disease. These genes are cloned by methods such as PCR, entrained and selected for enhanced antigens.

A BLAST investigation with the OspC B. burgdorferi protein gene revealed about 200 related entries. In Table 5 below, the entries for one hundred sequences that share at least 82% of the identity of the DNA sequence are shown, wherein said Table provides a source of diversity for the carryover of families, for get improved therapies in the treatment of Lyme disease. These genes are cloned through methods such as PCR, entrained and selected for enhanced antigens.

Example 3 Evolution of Amplio-Spectrum Vaccines against Mycobacterium Tuberculosis is an ancient bacterial disease, originated by Mycobacterium tuberculosis, which continues to be a major public health problem worldwide, and efforts have been made to make the best effort in its eradication (Morb. Mortal Wkly Rep (1998 August 21; 47 (RR-13): 1 - 6) In infections of around 50 million people, around 3 million people will die this year, it was discovered that the currently available vaccine, Bacille Calmette-Guerin (BCG), is less effective in developing countries and has been isolated an increasing amount of multiple drug resistant strains (MDR) .The main immunodominant antigen of M. tuberculosis is the 30-35 kDa (aka antigen, alpha-antigen) which is usually a lipoglicoprotein on the surface of the cell. protective antigens, include a 65-kDa heat-impact protein, and a 36-kDa proline-rich antigen (Tascon et al. (1996) Nat. Med. 2: 888-92.) Table 6 shows the output of a investigation B LAST, using the coding sequence of the main antigen 30-35 kDa of M. Tuberculosis (antigen a.k.a. 85, alpha-antigen), to identify homologous genes that can be used in a family tracing format, to obtain improved antigens. Many homologous antigens of a large number of related but distinct microbacterial deformations have been cloned and sequenced. These genes are cloned through methods, such as PCR, entrained and selected for enhanced antigens.

Example 4 Evolution of Amplio-Spectrum Vaccines against Helicobacter pylori Chronic infection of the gastroduodenal mucosa by Helicobacter pylori bacteria is responsible for chronic active gastritis, peptic ulcers and gastric cancers, such as adenocarcinoma and low-grade B-cell lymphoma. An increase in the occurrence of deformations resistant to antibiotics is limiting this therapy. The use of vaccines, which both avoid and treat infections in progress, is being actively pursued (Crabtree JE (1998) Gut 43: 7-8; Axon AT (1998) Gut 43 Suppl 1: S70-3; Dubois and associates (1998) Infecí. Immun. 66: 4340-6; Tytgat GN (1998) Aliment Pharmacol. Ther. 12 Suppl 1: 123-8, Blaser MJ (1998) BMJ 316: 1 507-10; Marchetti and associates (1998) Vaccine 16: 33-7; Kleanthous et al. (1998) Br. Med. Bull. 54: 229-41; Wermeille and associates (1998) Pharm. World Sci. 20: 1-17 The identification of Helicobacter antigens suitable for use in preventive and therapeutic vaccines may include two two-dimensional gel electrophoresis, sequence analysis and serum profiling (McAtee et al. (1998) Clin. Diagn. Lab.

Immunol. 5: 537-42; McAtee and associates (1998) Helicobacter 3: 163-9). Antigenic differences between species and related Helicobacter deformities may limit the use of vaccines for the prevention and treatment of infections (Keenan and associates (1998) FEMS Microbiol Lett.161: 21 -7). In this Example, the entrainment of the DNA family of immunologically related but distinct antigens allows the isolation of chimeric antigens that can provide a broad cross-reactive protection against many deformations and related Helicobacter species. Models of persistent infection by H. pylori deformation adapted for mouse, which have been used to evaluate the therapeutic use of vaccines against infections, are used to evaluate dragged antigens (Crabtree J E (1998) Gut 43: 7-8; Axon AT (1998) Gut 43 Suppl 1: S70-3). The evacuation of products (CagA) of the gene associated with cytotoxin (VacA) and cytotoxin, has been evaluated as a vaccine against H infection. Pilori in animal models, which support the application of this method in humans. Table 7 shows the results of a BLAST investigation using the VacA H. Pylori gene to identify homologous genes that can be used in a family tracing format to obtain improved antigens. The homologous antigens have been cloned and sequenced from a strain number H. pylori distinct yet related and you can obtain an additional natural diversity, by cloning genes antigens from other deformations. These and other genes or fragments thereof, are cloned through methods such as PCR, entrained and selected for enhanced antigens.

Table 8 shows the results of a BLAST investigation, using the CagA H gene. Pylori, to identify homologous genes that can be used in a family tracing format to obtain improved antigens. The homologous antigens have been cloned and sequenced from a number of H deformations. Pylori related although distinct and can obtain an additional natural diversity, by cloning of antigen genes from other deformations. These and other genes or fragments thereof, will be cloned through methods such as PCR, entrained and selected, for improved antigens.

Example 5 Development of Amplitude-Spectrum Vaccines Against Malaria This Example describes the use of DNA entrainment to generate improved vaccines against malaria infection. An excellent target for evolution by DNA entrainment is the merozoite surface protein Plasmodium falciparum, MSP 1 (Hui et al., (1996) Infect. Immun. 64: 1502-1509). MSP 1 is expressed on the surface of merozoites as an integral membrane protein. This is separated by parasite proteases just before and concomitant with the breakdown and release from infected cells. The separation seems to be mandatory for the total function in MSP 1 linking to RBC receivers. The separated fragments remain attached to the merozoite membrane. Other membrane proteins in merozoites also participate in adhesion and cases of specific invasion. MSP 1 is a proven candidate for inclusion in a vaccine against the asexual blood stage of malaria. The genes that encode MSP 1 can be isolated from several strands of Plasmodium falciparum merozoites using PCR technology. Additional naturally occurring related genes can be used to increase the diversity of the starting genes. An MSP1 gene library is generated by DNA entrainment, and this library is selected for induction of efficient immune responses. The selection can be made by injecting individual variants into animals for testing, such as mice or monkeys. Any of the purified recombinant proteins or DNA vaccines or viral vectors encoding the relevant genes are injected. Normally, a promoter injection is given 2 to 3 weeks after the injection. Subsequently, the sera of the animals are collected for testing and, in these sera, the presence of antibodies that reduce the invasion of merozoites within erythrocytes (RBC) is analyzed. The RBCs are infected by merozoite, immediately inside the RBCs, the merozoites differentiate into a ring and this mature to a schizont containing several nascent daughters merozoites, which later explodes out of the cell, destroying it, and continues to cling and invade another RBC. In vivo the merozoite is probably extra cellular only for a few seconds. In vitro, any blockage in this case can dramatically reduce the level of reinfection. Antibodies against MSP 1, bind on the surface of merozoites that are released from the infected RBC or schizont, when they break down and thereby reduce the ability of these merozoites to adhere and bind to related RBC receptors found in the RBC surface not infected. The addition of merozoites is reduced, the entry of merozoite into a new RBC is reduced, and therefore the numbers of recently invaded cells detected in the early ring stage are reduced, if the culture is examined a few hours after the blockade of the invasion test. In some assay formats, a substitute inhibition of merozoite invasion is to notice the appearance of agglutinated merozoites, although this is an indirect measure of antibodies that causes reduced invasion. The dragged antigens that induce the most potent antibody responses that reduce the invasion of merozoites in uninfected erythrocytes, are selected to elaborate additional tests and can be subjected to new rounds of drag and selection. In subsequent studies, the ability of these antigens to induce antibodies in man is investigated. Again, any of the purified recombinant antigens or DNA vaccines or viral vectors encoding the relevant genes are injected and protective immunological responses are analyzed.

EXAMPLE 6 Development of Wide Spectrum Vaccines Against Vial Pathogens This Example describes the use of DNA entrainment to obtain vaccines that can induce an immune response against multiple isolates of viral pathogens. A. Venezuelan equine encephalitis virus (VEE). The VEE belongs to the genera of alphaviruses, which are transmitted generally by means of mosquitoes. However the VEE is an unusual alphavirus in that it is highly infectious through aerosol inhalation for both humans and rodents. The manifestations of the disease in human ranges, ranging from subclinical or febrile benign diseases to infection or severe inflammation of the central nervous system. The clearance of the virus coincides with the production of specific anti-VEE antibodies, which are considered to be the main mediators of protective immune responses. (Schmaljohn et al. (1982) Nature 297: 70). VEE s an unusual virus also because its main target outside the central nervous system is the lymphoid tissue, and therefore, the replication of variants of the defect can provide means for target vaccines or for pharmaceutically useful proteins for the immune system . It is known that at least seven subtypes of VEE can be identified genetically and serologically. Based on the epidemiological data, the isolation of the virus falls into two main categories: deformations l-AB and lC, which are associated with epizootic / epidemic VEE, and the remaining serotypes, which are mainly associated with the vertebrate / mosquito cycles enzootic and circulating in specific ecological zones (Johnston and Peters, in Fields Virology, Third Edition, eds, BN Fields and associates, Líppincott-Raven Publishers, Philadelphia, 1996). The enveloped protein (E) appears to be the main antigen in the induction of neutralization of Abs. Therefore, DNA entrainment is used to obtain a library of recombinant E proteins, by shuffling the corresponding genes derived from various strains of VEE. These libraries and individual chimeras / mutants thereof are subsequently selected for their ability to broadly induce cross-reactive and protective Ab responses.

B. Flaviviruses. Japanese encephalitis virus (JE), tick borne encephalitis virus (TBE) and Dengue virus are viruses carried by anthropods belonging to the Flavivirus family, which includes 69 related viruses. The heterogeneity of the viruses within the family is a major challenge for the development of the vaccine. For example, there are four serotypes of the major Dengue virus, and a tetravalent vaccine that induces the neutralization of Abs against the four serotypes is necessary. In addition, non-neutralizing antibodies induced by infection or vaccination by a Dengue virus may cause the disease to increase during a subsequent infection by another serotype. Therefore, the broad-spectrum, cross-protection vaccines for TBE and JE would provide significant improvements for existing vaccines. In this Example, the ability of DNA entrainment to efficiently generate chimeric, and mutated, genes is used to generate cross-protection vaccines. 1 . Japanese encephalitis virus. Japanese encephalitis virus (JE) is a prototype of the JE antigen complex, which includes St. Louis encephalitis virus, Murray Valley encephalitis virus, Kunjin virus, and West Nile virus (Monath and Heinz, In Fields Virology, Third Edition, BN Fields and associates, Lippincott-Raven Publishers, Philadelphia, pages 961-1034, 1996). Infections caused by JE are relatively rare, but the fatality of the case is 5-40% because no specific treatment is available. J E is widely distributed in China, Japan, the Philippines, Russia and India from the Far East, providing a significant threat to those who travel in these areas. Currently the available J E vaccine is produced from mouse brain tissues infected with simple virus isolation. Side effects are observed in 10% to 30% of vaccines. To obtain chimeric and / or mutated antigens that provide a protective immune response against all or most of the viruses within the JE complex, DNA entrainment is carried out in viral envelope genes. The identity of the amino acid within the JE complex varies between 72% and 93%, in addition, significant antigen variation has been observed among JE strains by neutralization assays, agar gel diffusion, antibody uptake and antibody analysis monoclonal (Oda (1976) Kobe J. Med. Sci. 22: 123; Kobyashi and associates (1984) Infect. Immun. 44: 1 17). In addition, the amino acid divergence of the envelope protein gene among 13 strains from different Asian countries is as much as 4.2% (Ni and Barrett (1995) J. Gen. Virol 76: 401). The library resulting from the recombinant polypeptides encoded by entrained genes is selected to identify those that provide a protective cross-immune response. 2. Encephalitis virus carried by tick. Tick borne encephalitis virus complex comprises 14 antigenically related viruses, eight of which cause disease to humans, including Powassan virus, Louping ill and tick borne (TBE) '(Monath and Heinz, in Fields Virology, Third Edition, eds BN Fields and associates, Lippincott-Raven Plublishers, Philadelphia, pages 961-1034, 1996). The TBE has been recognized in all the countries of Central and Eastern Europe, Scandinavia and Russia, where the Powassan virus occurs in Russia, Canada and the United States. Symptoms range from a flu-like illness to meningitis, meningoencephalitis and severe meningoencephalitis with a fatality rate of 1% to 2% (Gresikova and Calisher, in Monath edition., The arboviruses: ecology and epidemiology, vol. IV, Boca Raton, FL, CRC Press, pages 177-203, 1988). The DNA family tracing is used to generate chimeric envelope proteins derived from the TBE complex to generate protective cross protection antigens. The envelope proteins within the family are from 77% to 96% homologous, and the viruses can be distinguished by specific mAbs (Holzmann et al., Vacuna, 10, 345, 1992). The envelope protein of the Powassan virus is 78% identical to the amino acid level of the TBE, and cross-protection is unlikely, although epidemiological data are limited. The Langat virus is used as a model system to analyze protective immune responses in vivo (lacono-Connors and associates (1996) Virus Res. 43: 125). The Langat virus belongs to the TBE complex, and can be used in stimulation studies and BSL3 facilities. Serological studies based on recombinant envelope proteins are carried out to identify immunogenic variants that induce high levels of antibodies against envelope proteins derived from most or all of the TBE complex viruses. 3. Viruses of Dengue. Dengue viruses are transmitted through mosquito bites, and are a significant threat to groups and populations of civilians particularly in tropical areas. There are four serotypes of the main Dengue virus, named Dengue 1, 2, 3 and 4. A tetravalent vaccine is required that neutralizes the antibodies against the four strains of Dengue, to avoid the antibody-mediated increase of the disease, when the individual finds the virus of the other deformation. The enveloped protein of the Dengue virus has been shown to provide an immunological response that protects from a future stimulus with the same virus deformation. However, the levels of neutralization of antibodies produced are relatively low and protection from the stimulation of the live virus is not always observed. For example, mice injected with genetic vaccines encode the enveloped protein of Dengue-2 virus, developed neutralizing antibodies when they were analyzed by in vitro neutralization assays, but the mice did not survive the stimulation with the live Dengue-2 virus (Kochel and associates (1997) Vaccine 15: 547-552). However, protective immunological responses were observed in mice immunized with recombinant vaccinia virus expressing the structural proteins of Dengue-4 virus (Bray et al. (1989) J. Virol. 63: 2853). These studies indicate that E-protein vaccines work, but significant improvements in the immunogenicity of protective antigens are required. In this Example, the DNA carryover is carried out in the genes encoding the envelope protein (E), for the four Dengue viruses and their antigenic variants. The DNA family tracing is used to generate variants of chimeric E protein that induce superior titration neutralizing antibodies against all Dengue serotypes. The E proteins of the different Dengue viruses share from 62% to 77% of their amino acids. Dengue 1 and Dengue 3, are the most closely related (homologous to 77%), followed by Dengue 2 (69%) and Dengue 4 (62%). These homologies are well within the range that allows efficient family trawling (Crameri et al. (1998) Nature 391: 288-291). The entrained antigen sequences are incorporated into gene vaccine vectors, and into purified plasmas and are subsequently injected into mice. The sera are collected from the mice and analyzed for the presence of high levels of cross reactive antibodies. The best antigens are selected for further studies using in vivo stimulation models, to select chimeras / mutants that induce cross protection against all Dengue deformities.

C. Expression and Enhanced Immunogenicity of Glycoproteins of Hantaan virus One of the advantages of genetic vaccines is that vectors expressing pathogenic antigens can be generated even when the pathogen provided can not be isolated in culture.

An example of this potential situation was an epidemic of severe respiratory disease among rural residents of the West of the United States, which was caused by a previously unknown hantavirus, Sin Nombre virus (Hjelle and associates (1994) J. Virol. 68: 592). Much RNA sequence information was obtained from the virus, before the virus could be isolated and characterized in vitro. In these situations, genetic vaccines can provide means to generate efficient vaccines in a short period of time, creating vectors encoding antigens encoded by the pathogen. However, genetic vaccines can only work if these antigens are adequately expressed in the host. The hantaan virus belongs to the Bunyavirus family. A characteristic of this family is that its glycoproteins normally accumulate in the membranes of the Golgi apparatus when they are expressed by cloned cDNAs, thereby reducing the efficacy of the corresponding genetic vaccines (Matsouka and associates (1991) Curr. Top. Microb. Immunol. 169: 161-179). The different expression of the Hantaan virus proteins on the surface of the cell is also an explanation for the poor immune responses that follow the injections of Hantaan virus genetic vaccines. In this Example, DNA family tracing is used to generate the recombinant Hantaan virus derived from glycoproteins which are efficiently expressed in human cells, and which can induce protective immune responses against the wild-type pathogen. The nucleic acids encoding the Hantaan virus protein are carried with the genes encoding other homologous Bunyavirus glycoproteins. The resulting library is selected to identify proteins that are easily expressed in human cells. The selection is carried out using a double marker expression vector, which allows the simultaneous analysis of the efficiency and expression of transfection of the fusion proteins, which are linked by PIG to the cell surface (Whitehorn et al. 1995) Biotechnology (NY) 13: 1215-9). Flow cytometry based on cell sorting is used to select for Hantaan virus glycoprotein variants that are expressed efficiently in mammalian cells. The corresponding sequences are subsequently obtained by PCR or plasma recovery. These chimeras / mutants are further analyzed for their ability to protect wild mice against Hantaan virus infections.

Example 7 DNA Dragging of Glycoproteins B and / or D HSV-1 and HSV-2 as a Means to Induce Improved Protective Immunological Responses This Example describes the use of DNA entrainment to obtain B polypeptides of HSV glycoprotein (gB) and polypeptides D of glycoprotein (gD), which exhibit an enhanced ability to induce protective immune responses upon administration to a mammal. Epidemiological studies have shown that previous HSV-1 infections provide partial protection against HSV-2 infections, indicating the existence of cross reactive immunological responses. Based on previous vaccination studies, the major immunogenic glycoproteins in HSV appear to be gB and gD, which are encoded by 2.7 kb and 1.2 kb genes, respectively. The gB and gD genes of HSV-1 are approximately 85% identical to the corresponding gene of HSV-2, and the gB genes of each share a small sequence identity with the gD genes. Baboon HSV-2 gB is approximately 75% identical to HSV-1 or -2 gB of human, with long tensions of almost 90% identity. In addition, the identity of 60 to 75% is found in portions of the equine and bovine herpes virus genes.

Family trawling is used using nucleic acids from substrates that encode gB and / or gD of HSV-1 and HSV-2. Preferably, the homologous genes are obtained from HSVs of various deformations. Figure 7 shows an alignment of nucleotide sequences gD from HSV-1 and two strains of HSV-2. The antigens encoded by entrained nucleic acids are expressed and analyzed in vivo. For example, it can be selected by enhanced induction of neutralizing antibodies and / or CTL responses against HSV-1 / HSV-2. Protective immunity can also be detected by stimulating mice or guinea pigs with viruses. The selection can be carried out using sets of individual clones or clones.

Example 8 Evolution of Proteins H IV Gp120 for I nduction of Broad Spectrum Neutralization Ab Answers. This Example describes the use of DNA entrainment to generate immunogens that cross-react between different deformations of viruses, different from the wild-type immunogens. The entrainment of two types of envelope sequences can generate immunogens that induce neutralization antibodies against a third deformation. The antibody-mediated neutralization of H IV-1 is strictly type-specific. Although the neutralization activity is extended in infected individuals over time, the induction of said antibodies by vaccination has proven to be extremely difficult. Antibody-mediated protection of H IV-1 infection in vivo correlates with antibody-mediated neutralization of virus in vitro. Figure 8 illustrates the generation of dragged gp 120 gene libraries. The gp 120 genes derived from H IV-1 DH 12 and HIV-1 111 B (N L43) are entrained. The gp 120 genes / mutants are subsequently analyzed for their capacity to induce antibodies that have a broad spectrum capacity to neutralize different deformations of HIV. The individual gp 120 genes are incorporated into gene vaccine vectors, which are then introduced into mice by injection or topical application on the skin. These antigens can also be delivered in the form of purified recombinant proteins. Immunological responses are measured by analyzing the ability of mouse sera to neutralize the growth of H IV in vitro. The neutralization tests are carried out against H IV-1 DH 12, HIV-1 I IB and H IV-189.6. Chimeras / mutants that demonstrated broad spectrum neutralization are chosen for additional rounds of trawling and selection. Additional studies are being conducted on monkeys to illustrate the ability of the gp 120 genes entrained to provide protection for subsequent infection with immunodeficiency virus.

Example 9 Drag Antigen of the Hepadnavirus Wrapper Protein.

The Hepatitis B virus (HBV) is one of a member of a family of viruses called hepadnaviruses. This Example describes the use of individual genomes and genes of this family that are used for DNA entrainment, which results in antigens having improved properties. A. Wrap-up of envelope protein genes Hepadnavirus. The envelope protein of HBV, gathers to form particles that carry antigenic structures collectively, known as the Hepatitis B surface antigen (HBsAg, this term is also used to designate the protein by itself).

Antibodies to the main antigen site, designated as the "a" epitope, (which is found in the envelope field called S), have the ability to neutralize the virus. Therefore, immunization with the HBsAg-containing protein serves as a vaccine against viral infection. The envelope of H BV also contains other antigen sites that can protect against viral infection and are potentially vital components of an improved vaccine. The epitopes are part of the envelope protein fields known as preS1 and preS2 (Figure 9). The entrainment of DNA from the envelope gene from several members of the hepadnavirus family, it is used to obtain more immunogenic proteins. Genes derived from the following hepatitis viruses are specifically traced: • Viruses of human HBV, subtypes ayw and adw2 • A hepatitis virus isolated from chimpanzee. • A hepatitis virus isolated from gibbon. • A hepatitis virus isolated from marmot of America. If desired, genes from other genotypes of human viruses are available for inclusion in the DNA entrainment reactions. Similarly, other animal hepadnaviruses are available. Some artificial genes are elaborated, to promote the efficiency of the formation of the chimaeras that result from the DNA drag: • In one case, a synthetic gene that is elaborated contains the HBV envelope sequence, except for those codons whose specific amino acids are found in the chimpanzee and gibbon genes. For these codons, the chimpanzee or gibbon sequence is used. • In a second case, a synthetic gene that is synthesized in which the sequence of the gene preS2 coming from the deformation adw2 HBV of human, is fused with the S region of the marmot of America. • In a third case, all the oligonucleotides required to chemically synthesize each of the hepadnavirus envelope genes, are mixed in approximately equal amounts and allowed to harden to form a sequence library.

After the DNA shedding of the hepadnavirus envelope genes, either or both of the two strategies are used to obtain improved HBsAg antigens. Strategy A: Antigens are selected by immunization of mice using two possible methods. The genes are injected into the forms of DNA vaccines, for example, entrained envelope genes carried by a plasma comprising the genetic regulatory elements required for the expression of the envelope proteins. Alternatively, the protein is prepared from the entrained genes and used as the immunogen. Sequences that give rise to a greater immunogenicity of any of the HBV antigens carried by preS1 -, preS2-, are selected for a second round of entrainment (Figure 10). During the second round, the best candidates are chosen based on their improved antigenicity and their other properties, such as higher expression level or more efficient secretion. The selection and additional drag returns continue until a maximum optimization is obtained for one of the antigenic regions. The individually optimized genes are subsequently used as a combination vaccine for the induction of optimal responses for epitopes carried by preS 1 -, preS2- and -S.

Strategy B: After the isolation of individually optimized genes such as in Strategy A, the preS 1, preS2 and S candidates are dragged together or in pairs, in additional turns to obtain genes that encode proteins that show improved immunogenicity for at least two regions containing HBsAg epitopes (Figure 11).

B. Use of HBsAg to transport epitopes from unrelated antigens. Several of the characteristics of HBsAg, make a useful protein to transport epitopes extracted from other unrelated antigens. The epitopes can be either B epitopes (which induce antibodies) or T epitopes extracted from the class I type (which stimulates CD8 + T lymphocytes and induced cytotoxic cells) or class II type (which induces T lymphocytes and are important to provide immune memory responses). 1 . B cell epitopes. The amino acid sequences of the potential B epitopes are chosen from any pathogen. Such sequences are often known to induce antibodies, but the immunogenicity is weak or otherwise unsatisfactory for the preparation of a vaccine. These sequences can also be mimotopes, which have been selected based on their ability to have a certain antigenicity or immunogenicity. The amino acid coding sequences are added to a hepadnavirus envelope gene. The heterologous sequences may either replace certain envelope sequences, or be aggregated in addition to all the envelope sequences. The heterologous epitope sequences can be placed at any position in the envelope gene. A preferred position is in the region of the envelope gene encoding the major "a" epitope of HBsAg (Figure 12). It is likely that this region is exposed in the lateral part of the particles formed by the envelope protein, and therefore will expose the heterologous epitopes. DNA entrainment is carried out in the envelope gene sequences, maintaining the sequence of the heterologous constant epitopes. The selection is carried out to select candidates that are secreted in the culture medium after the transfection of plasmas from the library entrained in cells in tissue culture. Clones that encode a secreted protein are subsequently tested for immunogenization in mice, either as a DNA vaccine or as a protein antigen, as described above. Clones that provide enhanced induction of antibodies for the heterologous epitopes are chosen for additional turns of DNA entrainment. The process continues until the immunogenicity of the heterologous epitope is sufficient to be used as a vaccine, against the pathogen from which the heterologous epitopes were derived. 2. Class I Epitopes MHC Class I epitopes are relatively short linear peptide sequences, which are generally between 6 and 12 amino acids in length, more often 9 amino acids in length. These epitopes are processed by antigen presenting cells, either after the epitope synthesis inside the cell (usually as part of a larger protein) or after the understanding of the soluble protein by the cells. The polynucleotide sequences that encode one or more Class I epitopes are inserted into the sequences of a hepadnavirus envelope gene, either by replacing certain envelope sequences, or by inserting the epitope sequences into the envelope gene. This is usually carried out by modifying the gene before DNA entrainment or by including in the entrainment reaction certain fragments of oligonucleotides encoding the heterologous epitopes, as well as sequences of hepadnaviruses of sufficient flanking which will be incorporated in the entrained products. Preferably, heterologous class I epitopes are placed at different positions in the various genes of hepadnaviruses used for the DNA entrainment reaction. This will optimize the opportunities to find chimeric genes that transport the epitopes in an optimal position for efficient presentation.

Epitopes Class I I.

MHC Class I epitopes are generally required to be part of a protein which is taken up by the antigen presenting cells, instead of being synthesized within the cell. Preferably, said epitopes are incorporated into a carrier protein, such as the H BV casing which can be produced in a soluble form or which can be secreted if the gene is delivered in the form of a DNA vaccine. The polynucleotides encoding heterologous class I epitopes are inserted into regions of the hepadnavirus envelope genes that are not included in the transmembrane structure of the protein. DNA entrainment is carried out to obtain a segregated protein that also transports class I I epitopes. When injected in the form of a protein, or when the gene is delivered as a DNA vaccine, the protein can be taken up by the antigen presenting cells for the processing of the class I epitopes.

Example 10 Evolution of broad spectrum vaccines against Hepatitis C virus. The antigenic heterogeneity of different deformations of the Hepatitis C virus (HCV) is a major problem in the development of efficient vaccines against HCV. Antibodies or CTLs specific for a deformation of HCV, usually do not protect against other deformations. Multivalent vaccine antigens that simultaneously protect against several strains of HCV could be of great importance when developing efficient vaccines against HCV. The envelope genes of. HCV, which encode envelope E1 and E2 proteins, have been shown to induce both antibody and lymphoproliferative responses against these antigens (Lee et al. (1998) J. Virol. 72: 8430-6), and these responses can be optimized for DNA trawls. The hypervariable region 1 (HVR1) of the HCV envelope E2 protein is the most variable antigen fragment in the whole viral genome, and is the main responsible for the inter and intra-individual heterogeneity of the infection virus (Puntoriero et al. (1998) EMBO J. 17. 3521 -33). Therefore, the gene encoding E2 is a particularly useful target for evolution by DNA entrainment. DNA tracing of HCV antigens, such as proteins E1, E2, of nucleocapsid envelope, provides a means to generate multivalent HCV vaccines that simultaneously protect against various HCV deformities. These antigens are dragged using the DNA family trapping method. The starting genes, will be obtained from several natural isolates of HCV. In addition, related genes from other viruses can be used to increase the number of different recombinants that are generated. Subsequently, a library of related chimeric variants of HCV antigens is generated, and this library will be selected to induce immunological responses with a broad cross-reaction. The selection can be made directly in vivo, by injecting individual variants into animals for testing, such as mice or monkeys. Any of the purified recombinant proteins or DNA vaccines encoding the relevant genes are injected. A propellant injection is usually given 2 to 3 weeks after the first injection. Subsequently, the sera of the animals for testing are collected and these sera are tested for the presence of antibodies that react against multiple isolates of HCV virus. Before the development of the in vivo test is initiated, the antigens can be previously enriched in vitro for antigens that are recognized by polyclonal antisera derived from patients or animals for previously infected test. Alternatively, monoclonal antibodies that are specific for various HCV strains are used. The selection is made using a phage display or ELISA assays. For example, antigen variants are expressed in bacteriophage M 13 and the phages are subsequently incubated in plates coated with antisera derived from patients or animals for testing infected with various isolates of HCV. The phages that bind to the antibodies are subsequently eluted and further analyzed in animals for testing, for induction of cross-reactive antibodies.

Example 1 1 Evolution of chimeric allergens that induce broad immunological responses and that have a reduced risk of induction of anaphylactic reactions. Allergy-specific immunotherapy is performed by injecting increasing amounts of the allergens provided to patients. Normally the therapy alters the types of allergen-specific immune responses, from a dominant response of the auxiliary cell T of type 2 (T 2) to a dominant response of the T cell type 1 (TH1). However, because allergic patients have increased levels of IgE antibodies, specific for allergens, allergy immunotherapy comprises a risk of anaphylactic reactions mediated by the IgE receptor. The T (TH) helper cells have the capacity to produce a large number of different cytokines, and based on their TH cells of cytokine synthesis pattern, they are divided into two subgroups (Paul and Seder (1994) Cell 76. 241 - 251). TH 1 cells produce high levels of IL-2 and I FN-gamma and do not produce levels or produce minimal levels of IL-4, IL-5 and IL-13. In contrast, TH2 cells produce high levels of IL-4, IL-5 and IL-13, while the production of IL-2 and I FN-gamma is minimal or non-existent. TH1 cells activate macrophages, dendritic cells, and increase the cytolytic activity of CD8 + cytotoxic T lymphocytes and NK cells (Id.), Where TH2 cells provide efficient support for B cells, and also mediate allergic responses due to ability of the T 2 cells to induce the change of the IgE isotype and the differentiation of the B cell, in the IgE segregation cell (De Vries and Punnonen (1996) in Cytokine regulation of humoral immunity: basic and clinical aspects.) Eds. Snapper , CM., John Wiley &Sons, Ltd., West Sussex, UK, pages 195 through 215). This Example describes methods for generating chimeric allergens that can broadly modulate allergic immune responses. This can be achieved as by dragging DNA from related allergenic genes to generate chimeric genes. In addition, chimeric / mutated allergens are less likely to be recognized by pre-existing IgE antibodies in patients. Importantly, allergenic variants that are not recognized by IgE antibodies can be selected using patient sera and negative selection (Figure 13). As an example, allergenic chimeric allergen variants of Der p2, Der f2, .Tyr p2 Lep d2 and Gly d2 are generated. These household dust acaric acid allergens are very common in allergic and asthmatic symptoms of exacerbation, and improved means are desired to regulate said allergic immune responses. Domestic dust acarids can be used as sources of genes. The corresponding genes are dragged using the DNA family drag and a drag library is generated. Phage display is used to exclude allergens that are recognized by antibodies from allergic individuals. It is particularly important to exclude variants that are recognized by IgE antibodies. The phage expressing the allergenic variants are incubated with sets of sera derived from allergic individuals. The phages that are recognized by the IgE antibodies are removed and the remaining allergens are further tested in vitro and in vivo, for their ability to activate allergen-specific human T cells (Figure 14). Because allergy immunotherapy is believed to work through the induction of a TH 1 dominant response, as compared to the TH2 allergic response, efficient T cell activation and the induction of a TH 1 response by Allergenic variants, is used as a measure of the effectiveness of allergens to modulate T cell responses., the optimal allergenic variants are tested additionally in vivo, studying skin responses after injections to the skin. A strong inflammatory response around the site of the injection is an indication of efficient activation of the T cell, and the allergenic variants that induce the response of the most efficient delayed type T cell (usually observed 24 hours after the injection), are the best candidates for additional in vivo studies to identify allergens that effectively regulate allergic immune responses. Therefore, these allergenic variants are analyzed for their ability to inhibit allergic responses in allergic, atopic and asthmatic individuals. The selection of allergenic variants is illustrated further in Figure 13 and Figure 14.

Example 12 Evolution of cancer antigens that induce efficient anti-tumor immune responses Several cancer cells express antigens that are present at significantly higher levels in malignant cells than in other cells of the body. These antigens provide excellent targets for preventive vaccines against cancer and cancer immunotherapy. The immunogenicity of said antigens can be tested by DNA entrainment. In addition, DNA entrainment provides means to improve the expression levels of cancer antigens. This Example describes methods for generating cancer antigens that can efficiently induce antitumor immune responses by entraining DNA from related cancer antigen genes. Libraries of glycoprotein genes associated with entrained melanoma (gp 100 / pmel 17) are generated (Huang et al. (1998) J. Invest. Dermatol.1 1: 662-7). The genes can be isolated from the melanoma cells obtained from several patients, who have gene mutations, increasing the diversity in the starting genes. In addition, a gp100 gene can be isolated from other mammalian species, to further increase the diversity of the starting genes. A typical method for the isolation of genes is RT-PCR. The corresponding genes are dragged using a simple DNA gene drag or a DNA family drag and a drag library is generated.

The entrained gp100 variants, either sets of clones or individual clones, are subsequently injected into animals for testing, and immune responses are studied (Figure 15). Antigens that are expressed either in E. coli or recombinant purified proteins, are injected, or the antigen genes are used as components of DNA vaccines. The immune response can be analyzed, for example by measuring the anti-gp100 antibodies, as previous studies indicate that the antigen can induce specific antibody responses (Huang et al., Supra.). Alternatively, test animals that can be stimulated by malignant cells express gp100. Animals that have been immunized efficiently will generate cytotoxic T cells specific for gp100, and survive the stimulus, whereas in animals not immunized or poorly immunized, malignant cells will grow efficiently resulting in eventual, lethal expansion. of the cells. In addition, antigens that induce cytotoxic T cells that have the ability to kill cancer cells can be identified by measuring the ability of T cells derived from immunized animals to kill cancer cells in vitro. Normally cancer cells are first labeled with radioactive isotopes and the release of radioactivity is an indication of the killing of tumor cells after incubation in the presence of T cells from immunized animals. Such cytotoxicity tests are known in the art. Antigens that induce higher levels of specific antibodies and / or that can protect against a larger number of malignant cells can be chosen for additional rounds of entrainment and selection. Mice are useful test animals, since a large number of antigens can be studied. However, monkeys are the preferred test animal because the MHC molecules of monkeys are very similar to that of humans.

Peripheral blood mononuclear cells from human individuals infected or previously immunized can be used to select the antigens that have optimal capacity to activate antigen-specific T cells. This is a particularly useful method, because the M HC molecules that will be present in the peptide antigens, are HCM molecules of human. Trapped cancer antigens that induce cytotoxic cells that have the ability to kill cancer cells, can be identified by measuring the ability of T cells derived from immunized animals to kill cancer cells in vitro. Normally cancer cells are first labeled with radioactive isotopes, and the release of radioactivity is an indication of the killing of tumor cells after incubation in the presence of T cells from immunized animals. Such cytotoxicity assays are known in the art.

Example 13 Evolution of autoantigens that induce efficient immunological responses. The autoimmune diseases are characterized by an immune response directed against the antigens expressed by the host. The autoimmunity response is usually mediated by TH 1 cells that produce higher levels of I L-2 and I FN-gamma. Vaccines that can direct cells T-specific autoantigens towards the TH2 phenotype that produce increased levels of IL-4 and IL-5. For these vaccines to work, vaccine antigens must have the ability to efficiently activate specific T cells.

DNA entrainment can be used to generate antigens that have such properties. To optimally induce differentiation of the TH2 cell, it may be beneficial to co-administer the cytokine, which has been shown to increase the activation and differentiation of the TH2 cell, such as I L-4 (Racke et al. (1994) J. Exp. Med. 180: 1961 -66). This Example describes methods for generating autoantigens that can induce immunological responses efficiently. DNA entrainment is carried out in genes of related autoantigens. For example, libraries of entrained myelin basic proteins, or fragments thereof, are generated (Zamvil and Steinman (1990) Ann. Rev. Immunol.8: 579-621); Brocke et al. (1996) Nature 379: 343-46). MBP is considered an important autoantigen in patients with multiple sclerosis (MS). The genes encoding MBP from at least bovine, mouse, rat, guinea pig and human pigs have been isolated providing an excellent starting point for family trawling. A typical method for the isolation of genes is RTPCR. Trailed MBP variants, either sets of clones or individual clones, are subsequently injected into animals for testing, and immune responses are studied. Trailed antigens are expressed either in E. coli and the purified recombinant proteins are injected, or the antigen genes are used as components of DNA vaccines or viral vectors. The immune response can be analyzed, for example, by measuring anti-MBP antibodies by ELISA. Alternatively, lymphocytes derived from test animals immunized with MBP are activated, and T-cell proliferation or cytokine synthesis is studied. A sensitivity assay for cytokine synthesis is ELISPOT (McCutcheon and associates (1997) J. Immunol. Methods 210: 149-66). Mice are useful test animals due to the large number of antigens that can be studied. However, monkeys are the preferred test animal because the MHC molecules of monkeys are very similar to that of humans. Peripheral blood mononuclear cells from MS patients can also be used to select antigens that have optimal capacity to activate MBP-specific T cells. This is a particularly useful method, because the MHC molecules that will be present in the antigen peptides are human MHC molecules. Trailed antigens that activate MBP-specific T cells can be identified by measuring the ability of T cells derived from MS patients to proliferate or produce cytokines in culture, in the presence of antigen variants. Such assays are known in the art. One trial is ELISPOT (McCutcheon et al., Supra.). An indication of the efficiency of a MBP variant to activate specific T cells, is also the degree of inflammation of the skin when the antigen is injected into the skin of a patient with MS. The strong inflammation is correlated with the strong activation of T cells specific for the antigen. It is likely that enhanced activation of MBP-specific T cells particularly in the presence of IL-4, resulting in improved TH2 cell responses, which are beneficial in the treatment of MS patients.

Example 14 Method for Optimizing the Immunogenicity of the Hepatitis B Surface Antigen This Example describes the methods by which the envelope protein sequence of the hepatitis B virus can be enveloped to provide a more immunogenic surface antigen. This protein is important for the lower responder vaccine and for chronic hepatitis B immunotherapy.

Background Current HBV vaccines (Merck, SKB) are based on the immunogenicity of the viral envelope protein and contain the major (or minor) form of the envelope protein produced as particles in yeast. These particles induce antibodies to the main surface antigen (HBsAg) which can protect against infection with antibody levels that are at least 10 milli-International Units per milliliter (mU / ml). These preparations of recombinant proteins do not have the capacity to induce humoral immunity in chronic transporters (about 300 million cases worldwide), which would be important to control the spread of the virus. In addition, certain individuals respond poorly to the vaccine (up to 30 to 50% of the vaccines in some groups), and do not develop levels of antibody protection. The inclusion of the natural epitope sequences contained in the Medium or Large forms of the viral envelope protein has been used as a method to increase the immunogenicity of the vaccine preparations. An alternative method is to introduce new helper T cell epitopes (eg, not present in the wild-type virus sequence), into the HBsAg sequence, using DNA trickle technology.

Method DNA sequences of HBsAg from different subtypes of HBV (for example ayw and adr) and related marmot hepatitis viruses are prepared for entrainment. Comparison of the genes that encode these proteins suggests that recombination would occur 10 times within 850 base pairs, when DNA sequences from ayw and America's marmot hepatitis virus (WHV) are crawled. In Figure 17, nucleotide and amino acid sequences of portions of different HBV subtypes are shown. The sequence of the main HBsAg B-cell antigen site (the "a" epitope) can be retained in the protein sequence, including the outer "a" circuit coding sequences in the preparation of the final protein. The peptide analog (s) for the "a" B HBsAg epitope has been described by (Neurath and associates (1984) J. Virol. Methods 9: 341-346), and the immunogenicity of the "a" epitope has been demonstrated by (Bhatnagar and associates (1982) Proc. Nat'l. Acad. Sci. USA 79: 4400-4404). HBsAg and WHsAg share the principal "a" determinant, and chimpanzees can be protected by both antigens (Cote et al. (1986) J. Virol. 60: 895-901). Similarly, CTL epitopes included in the protein can be included in a defined manner. It is also possible to easily introduce B or T epitopes (auxiliary or CTL) from other antigens in the entrained HBsAg sequence. This can be focused on the immune response, for certain epitopes, independent of other potentially dominant epitopes from the same protein. In addition, the availability of an "a" loop in H BsAg may provide a region of the envelope protein in which other antigens or artificial mimitopes could be included. In all cases where a new HBV envelope sequence is prepared to include a specific epitope (from HBV, another pathogen or another tumor cell), entrainment of the surrounding HBsAg envelope sequences will serve to optimize the expression of the protein and help ensure that the immune response is directed to the desired epitope. Next, several methods of analysis and use of entrained HBsAg sequences are described.

A. Modulation of the expression of HBsAg levels The entrained HBsAg sequences are introduced into the cells in culture, and the ability to direct the expression of secreted HBsAg (measured with clinical kits for expression of HBsAg). This can be used to identify the sequence of entrained HBsAg, which exhibits optimized HBsAg expression levels. Said coding sequences are particularly interesting for DNA vaccination.

B. Rodeo of low sensitivity to HBsAg The entrained HBsAg sequences are evaluated for their ability to induce an immunological response to clinically relevant HBsAg epitopes. This can be done using mice of the aphotypes H-2s and H-2f, which respond poorly or do not respond at all to the immunization of the HBsAg protein. In these experiments, it can be verified that the antibodies are generated for the major epitope "a" in the S protein, and a second protective epitope in the PreS2 region (a linear sequence). The PreS2 and S coding sequences for the envelope protein (HBsAg) from the subtype HBV ayw (plasma pCAG-M-Kan, Whalen) and WHV (plasma pWHV8 of ATCC) are amplified for the two plasmas by PCR and entrained. In Figure 18, examples of suitable markers for PCR amplification are illustrated. The library entrained from the sequences is cloned into an HBsAg expression vector and individual colonies are chosen for the preparation of the plasma DNA. The DNA is administered to the test animals and the vectors that induce the desired immune response are identified and recovered.

C. Presentation of natural HBsAg CTL epitopes by enveloped HBsAg proteins This example describes methods for using the enveloped HBsAg protein to present native H BsAg CTL epitopes. The entrainment is used to increase the overall immunogenicity of the HBsAg protein, as mentioned above. However, some of the enveloped HBsAg sequences are replaced with class I or class I epitope sequences from the native HBsAg protein, in order to stimulate immunoreactivity specifically for these natural viral epitopes. Alternatively, the natural viral epitopes can be added to the wrapped protein, without loss of immunogenicity of the enveloped HBsAg.

D. Expression of the CTL epitope derived from a tumor by enveloped HBsAg proteins This example describes methods for using the enveloped HBsAg protein which is used to express CTL epitopes derived from a tumor. The overall immunogenicity of the HBsAg protein is increased by entrainment. However, some enveloped HBsAg sequences are replaced with class I or class I I epitope sequences from tumor cells, in order to stimulate the specific immunoreactivity for these natural viral epitopes. Alternatively, tumor cell epitopes can be added to the envelope protein, without the loss of immunogenicity of the HBsAg.

E. Expression of mimitope sequences by HBsAg This example describes the use of a wrapped H BsAg protein for the expression of mimitope sequences. Again the wrapped HBsAg protein is used to increase the overall immunity of the protein. However, some of the enveloped HBsAg sequences are replaced with the mimotope sequences to stimulate immunoreactivity specifically for the natural sequence, which cross-reacts with the mimotope. Alternatively, the mimotope sequences can be added to the enveloped protein without loss of immunogenicity of the enveloped HBsAg.

Example 15 Fusion of HBsAg Polypeptide Proteins and HIV gp120 Protein This Example describes the preparation of the protein fusion ( "chimeras") formed from the HBsAg polypeptide and the extracellular fragment 120 of the HIV envelope protein and its use as a vaccine.

Background When used as a vaccine, the monomeric recombinant gp120 has failed to induce antibodies that have a strong neutralizing activity with major isolates of the H IV virus. It has been suggested that the oligomeric forms of the H IV envelope protein, which exposes certain regions of the tertiary structure, would be better able to obtain virus-using antibodies (Parrin et al. (1997) Immunol., Lett 57: 105- 12, VanCott and associates (1997) J. Virol 71: 4319-4330) In this Example, the DNA carryover is applied to this problem in order to obtain gp120 polypeptides which adopt slightly different confirmations than those of the previous preparations of recombinant gp 120. To allow the individual gp120 molecules to interact with the oligomers, there is a fusion between gp120 sequences (at the -N-terminus of the fusion) and H BsAg sequences (at the -C-terminus of the fusion). The N-terminal peptide sequence of the S region of the HbsAg polypeptide is a transmembrane structure which is ensured within the membrane of the endoplasmic reticulum. The current -N terminal of the S region as well as the PreS2 sequences are located in the luminus part of ER. These sequences are located on the outside of the H BsAg. By placing the gp120 sequences at the -N terminal of the H BsAg preS2 or S sequences, the gp120 sequences are also located on the outside. Therefore, the gp120 molecules can be brought together in three-dsional space to interact as in the virus. Since the exact conformation of the final cha, which will have the highest possible immunogenicity, can not be anticipated, DNA entrainment is employed. The HBsAg polypeptide sequences, which function as a platform, and gp120 are carried. The selection of entrained products can be carried out by ELISA using antibodies (polyclonal or monoclonal), which have been previously determined to have virus neutralizing activity.

Method The sequences encoding the gp120 fragment of the envelope protein H IV are preferably prepared as a synthetic gene to include codons, which are optimal for expression of the gene in mammals. The gp120 sequence will normally include a signal sequence at its terminal end -N. The gp120 sequences are inserted in the PreS2 region of an H BsAg expression plasma. In the preS2 region of plasma pMKan and its derivatives, an EcoRI site and an Xhol site are available for cloning. The gp120 sequences can be inserted between these two sites, which brings the gp120 closer to the start of the S coding sequences or within the EcoRI site alone, which leaves a space sequence of about 50 amino acids between the gp120 sequences and the start of the S region of HBsAg.

These two different cloning strategies will provide for the emergence of chic molecules in which the gp120 sequences are located at different distances from the transmembrane region of the HBsAg sequence. This may be convenient to allow the gp120 sequences to adopt conformations that are more suitable immunogens than the monomeric gp120. The DNA entrainment of the entire chic sequence is carried out. Family traction is preferred; this comprises the preparation of several gp 120 -HBsAg fusion proteins, in which different sequences of gp120 and HBsAg (or WHV) are used. In Figure 19, an alignment of HBsAg nucleotide sequences is shown. After the entrainment of the different sequences, the products are cloned into an expression vector such as pMKan. The sets of clones from the library of entrained products are transfected into cultured cells and the secretion of chic proteins is tested with antibodies highly reactive for gp120. Positive clones can be further evaluated with particular antibodies that have demonstrated activity in H IV neutralization, for example, the anti-CD4 binding field recombinant human monoclonal antibody, IgG 1 b12 (Kessler et al. (1997) AIDS Res. Hum Retroviruses 1: 13: 575-582; Roben and associates (1994) J. Virol. 68: 4821 -4828). Subsequently, the candidate clones can be used to immunize mice and the antiserum obtained is evaluated for the neutralization activity of H IV virus in in vitro assays.

Because the gp120 molecule (approximately 1100 amino acids) is larger in size than the monomeric preS2 + S HBsAg protein (282 amino acids), it is likely that not all HBsAg monomers in an aggregated particle will contain a gp120 sequence. The internal initiation of protein synthesis can take place in the HBsAg coding sequences in the initiating methionine that marks the beginning of the S region. Therefore, the chimeric molecule (which contains the gp120 sequences) will be mixed in the cell with the S region and the multimeric particles must be combined with an appropriate amount of chimeric polypeptides and native HBsAg monomers. Alternatively, an S expression plasma can be mixed with the plasma expressing the chimera, or with a simple plasma which expresses the chimera and the S-form can be constructed. In Figure 20, a diagram of the resulting particles is shown.

EXAMPLE 16 DNA Dragging of B-glycoproteins and / or HSV-1 and HSV-2 as a Means to Induce Enhanced Protective Immune Responses This Example describes the use of DNA drift to obtain B-polypeptides of HSV glycoprotein (gB) and D-glycoprotein polypeptides (gD) that exhibit an enhanced ability to induce protective immune responses when administered to a mammal. Epidemiological studies have shown that previous infections with HSV-1 provide partial protection against HSV-2 infections, indicating the existence of cross reactive immunological responses. Based on previous vaccination studies, the major immunogenic glycoproteins HSV appear to be gB and gD, which are encoded by 2.7kb and 1.2kb genes, respectively. The gB and gD genes of HSV-1 are approximately 85% identical to the HSV-2 gene, and the gB genes of each share a small sequence identity with the gD genes. Baboon HSV-2 gB is approximately 75% identical to HSV-1 or -2 gB of human, with long tensions of almost 90% identity. In addition, the identity of 60% to 75% is found in portions of the equine and bovine herpes virus genes. Family tracing is employed using nucleic acids encoding gB and / or gD HSV-1 and HSV-2 as substrates. Preferably the homologous genes are obtained from HSVs of various deformations. In Figure 7, an alignment of gD nucleotide sequences from HSV-1 and two HSV-2 strains is shown. The antigens encoded by the entrained nucleic acids are expressed and analyzed in vivo. For example, it can be selected by the enhanced induction of neutralizing antibodies and / or CTL responses against HSV-1 / HSV-2. Protective immunity can also be detected by stimulating mice or Guinea pigs with the viruses. The selection can be made using sets of individual clones or clones.

EXAMPLE 17 Evolution of HIV gp120 Proteins for Induction of Broad Spectrum Neutralization Ab Answers This Example describes the use of DNA entrainment to generate immunogens that cross-react between different virus deformations, in a manner different from natural type immunogens. . The entrainment of two types of envelope sequences can generate immunogens that induce neutralizing antibodies against a third strain. The antibody-mediated neutralization of H IV-1 is strictly type-specific. Although the neutralization activity in infected individuals is extended over time, the induction of said antibodies by vaccination has proven to be extremely difficult. Antibody-mediated protection from HIV-1 infection in vivo correlates with neutralization mediated by virus antibodies in vitro. Figure 8 illustrates the generation of dragged gp120 gene libraries. The gp120 genes derived from H IV-1 DH 12 and H IV-1 I I IB (NL43) are entrained. The chimeric / mutant gp120 genes are subsequently analyzed for their ability to induce antibodies that have a broad spectrum ability to neutralize different H IV deformities. The individual gp120 genes dragged, are incorporated into vectors of genetic vaccines, which are then introduced into mice through injection or topical application on the skin. These antigens can also be delivered in the form of purified recombinant proteins. Immunological responses are measured by analyzing the ability of mouse sera to neutralize H IV growth in vitro. The neutralization tests are carried out against H IV-1 DH 12, H IV-1 I I I B and HIV-189.6. Chimeras / mutants that demonstrate broad-spectrum neutralization are chosen for additional laps of entrainment and selection. Additional studies in monkeys are carried out to illustrate the ability of the entrained gp120 genes to provide protection for subsequent infection with immunodeficiency virus. It is clear that the examples and embodiments described in the present invention are for illustrative purposes only and that various modifications and changes thereof will be suggested to those skilled in the art, and that they are included in the spirit and scope of the present application and of the appended claims. All publications, patents, and patent applications cited in the present invention are incorporated therein as reference for all purposes.

Claims (53)

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 recombinant multivalent antigen polypeptide comprising a first determinant antigen and a first polypeptide, and at least a second determinant antigen from a second polypeptide.

2. The multivalent antigen polypeptide as described in claim 1, further characterized in that the polypeptide comprises at least a third determinant antigen from a third polypeptide.

3. The multivalent antigenic polypeptide as described in claim 1, further characterized in that the first and second are selected from the group consisting of carcinogenic antigens, antigens associated with autoimmunity disorders, antigens associated with inflammatory conditions, antigens associated with allergic reactions, and antigens from infectious agents.

4. - The multivalent antigen polypeptide as described in Claim 3, further characterized in that the antigens are derived from a virus, a parasite or a bacterium.

5. - The multivalent antigen polypeptide as described in claim 4, further characterized in that the antigens are from a virus selected from the group consisting of a Venezuelan equine encephalitis virus or a related virus, a Japanese Encephalitis complex virus , a virus of a tick borne encephalitis virus complex, a Dengue virus, a Hanta virus, an H IV, a Hepatitis B virus, a Hepatitis C virus or a Herpes simplex virus.

6. - The multivalent antigen polypeptide as described in Claim 5, further characterized in that the antigens are envelope proteins.

7. - The multivalent antigen polypeptide as described in claim 4, further characterized in that the antigens are from a bacterium and are selected from a group consisting of a Yersinia antigen, an enterotoxin Staphylococcus aureus, an enterotoxin Streptococcus pyogenes, a Vibrio cholera toxin, a labile enterotoxin Escherichia coli, an Osp A polypeptide and an OspC polypeptide from a Borrelia species, an Antigen 85 polypeptide from a Mycobacterium species, a VacA polypeptide and a CagA polypeptide from Helicobacter pylori, and a MSP antigen from Plasmodium falciparum.

8. The multivalent antigen polypeptide as described in claim 1, further characterized in that the multivalent antigen polypeptide exhibits a reduced affinity with IgE from a mammal, compared to the first and second polypeptides.

9. The multivalent antigen polypeptide as described in claim 1, further characterized in that the first determining antigen and the second determining antigen are from different serotypes of a pathogenic organism.

10. The multivalent antigen polypeptide as described in claim 1, further characterized in that the first determining antigen and the second determining antigen are from a different species of pathogenic organism.

1. The multivalent antigen polypeptide as described in claim 1, further characterized in that the first polypeptide and the second polypeptide are allergenic.

12. The multivalent antigen polypeptide as described in claim 1, further characterized in that the allergens are allergenic dust mites, grass pollen allergens, birch pollen allergens, ragweed pollen allergens, hazel pollen allergens, cockroach allergens, rice allergens, olive tree pollen allergens, fungal allergens, mustard allergens, and bee venom.

13. - The multivalent allergenic polypeptide as described in claim 1, further characterized in that the first polypeptide and the second polypeptide are associated with an inflammatory or autoimmune disease.

14. The multivalent antigen polypeptide as described in claim 13, further characterized in that the first polypeptide and the second polypeptide are self antigens associated with a disease selected from the group consisting of multiple sclerosis, scleroderma, systemic sclerosis, systemic lupus erythematosus, disorder of auto-immunity Hepatic, skin auto-immune disorder, insulin-dependent diabetes mellitus, thyroid autoimmunity disorder and rheumatoid arthritis.

5. The multivalent antigen polypeptide as described in claim 1, further characterized in that the first polypeptide and the second polypeptide are carcinogenic antigens or sperm antigens.

16. A library of recombinant antigen comprising recombinant nucleic acids encoding antigen polypeptides, wherein the library is obtained by recombining at least the first and second forms of a nucleic acid, which comprises a polynucleotide sequence encoding an associated antigen polypeptide with the disease as in wherein the first and second forms differ from each other in two or more nucleotides to produce a library of recombinant nucleic acids.

17. - The recombinant antigen library as described in Claim 16, further characterized in that the first and second polypeptides are toxins.

18. - A method for obtaining a polynucleotide encoding a recombinant antigen that has improved ability to induce an immune response to a disease condition, wherein the method comprises: (l) recombination of at least the first and second forms of a nucleic acid which comprises a polynucleotide sequence encoding an antigen polypeptide that is associated with the disease condition, wherein the first and second forms differ from each other, in two or more nucleotides to produce a library of recombinant nucleotide acids; (2) selecting the library to identify at least one optimized recombinant nucleic acid, which has improved ability to induce an immune response for the disease condition.

19. The method as described in Claim 18, further characterized in that the method further comprises: (3) recombining at least one optimized recombinant nucleic acid with a further form of the nucleic acid, which is the same or different from the first and second forms, to produce an additional library of recombinant nucleic acids; (4) selecting the additional library to identify at least one additional optimized recombinant nucleic acid encoding a polypeptide having improved ability to induce an immune response for the disease condition; and (5) repeating subsections (3) and (4), as necessary, until the additional optimized recombinant nucleic acid encodes a polypeptide having improved ability to induce the immune response to the disease condition.

20. The method as described in Claim 18, further characterized in that the polypeptides associated with the disease are selected from the group consisting of the cancer antigen, antigens associated with autoimmune disorders, antigens associated with inflammatory conditions, antigens associated with reactions allergic, and antigens associated with infectious agents.

The method as described in claim 18, further characterized in that the disease condition is an infectious disease and the first and second forms of the nucleic acid, each encoding an antigen of a different serotype of a pathogen.

22. The method as described in claim 21, further characterized in that the first and second forms of the nucleic acid are each derived from a different species of pathogen.

23. The method as described in claim 21, further characterized in that the selection is carried out: introducing into the animal for testing either: a) the library of recombinant nucleic acids, or b) recombinant polypeptides encoded by the library of recombinant nucleic acids; introduce the pathogen into the test animal; determine if the test animal is resistant to stimulus by the pathogen.

24. The method as described in Claim 23, further characterized in that the pathogen introduced in the test animal is of a serotype which was used as a source of the first and second forms of the nucleic acid.

25. The method as described in Claim 23, further characterized in that the library is subdivided into a plurality of sets, each of which is introduced into an animal for testing to identify pools that include an optimized recombinant nucleic acid encoding a polypeptide, which has improved ability to induce an immune response to the pathogen.

26. The method as described in Claim 25, further characterized in that the sets including an optimized recombinant nucleic acid that are further subdivided into a plurality of subsets, each of the subsets is introduced into an animal for testing to identify those sets which include an optimized recombinant nucleic acid, which encodes a polypeptide which has improved ability to induce an immune response to the pathogen.

27. - The method as described in claim 18, further characterized in that the optimized recombinant nucleic acid encodes a multivalent antigen polypeptide and the selection is carried out by: expression of the recombinant nucleic acid library in a display expression vector of phage, so that the recombinant antigen is expressed as a fusion protein with a phage polypeptide that is displayed on a phage particle surface; contacting the phage with a first antibody that is specific for a first serotype of the pathogen and selecting those phages that bind to the first antibody; contacting those phages that bind to the first antibody with a second antibody that is specific for a second serotype of the pathogen, and selecting those phages that bind to the second antibody; wherein said phage that binds to the first antibody and the second antibody, expresses a multivalent antigen polypeptide.

28. The method as described in claim 27, further characterized in that the selection further comprises contacting said phage which binds to the first and second antibodies with one or more additional antibodies, each of which is specific for a serotype. additional pathogen, and selecting that phage that binds to the respective additional antibodies.

29. The method as described in Claim 27, further characterized in that the phage display expression vector, comprises a stop codon of suppression between the recombinant nucleic acid and the phage polypeptide, whereby the expression of the one cell or host which comprises a corresponding suppressor tRNA results in the production of the fusion protein and expression in a second host, which lacks a corresponding suppressor tRNA that results in the production of the recombinant antigen not as a fusion protein .

30. - The method as described in Claim 18, further characterized in that the optimized recombinant antigen exhibits an improved level of expression of host cells, and the selection is carried out by the expression of each recombinant nucleic acid in the host cell, and subjecting the host cells to cell sorting based on flow cytometry to obtain those host cells that exhibit the recombinant antigen on the surface of the host cell.

The method as described in claim 18, further characterized in that the improved property is selected from the group consisting of: improved immunogenicity; improved cross-reactivity against different forms of polypeptide antigens associated with disease; reduced toxicity; in vivo activity of the improved adjunct agent; improved production of the immunogenic polypeptide.

32. The method as described in claim 31, further characterized in that the improved property is increased cross-reactivity against different forms of polypeptides associated with the disease, and the first and second forms of the nucleic acid are from a first and second forms of polypeptide associated with the disease.

33. - The method as described in Claim 32, further characterized in that the first and second forms of the polypeptide associated with the disease are obtained from at least a first and second species of a pathogen, and the optimized recombinant nucleic acid encodes a recombinant polypeptide that induces a protective response against both species of the pathogen.

34. The method as described in claim 33, further characterized in that the recombinant polypeptide induces a protective response against at least one additional species of the pathogen.

35. - The method as described in claim 33, further characterized in that the pathogen is a toxin.

36. - The method as described in claim 33, further characterized in that the pathogenic agent is a virus or a cell.

37. - The method as described in Claim 33, further characterized in that the polypeptide associated with the disease is an antigen - Yersinia V.

38. The method as described in claim 37, further characterized in that at least the first and second forms of a -nucleic acid are obtained from at least one first and second Yersinia species.

39. The method as described in Claim 38, further characterized in that the Yersinia species are selected from the group consisting of Y. Pestis, Y. Enterocolitica and Y. Pseudotuberculosis.

40. - The method as described in claim 33, further characterized in that the pathogen is a bacterial toxin.

41. The method as described in Claim 18, further characterized in that the disease condition is cancer and the selection step comprises the introduction of the optimized recombinant nucleic acids into a genetic vaccine vector, and the members of the vaccine are tested. the library for its ability to inhibit the proliferation of cancer cells or induce the death of cancer cells.

42. The method as described in Claim 41, further characterized in that the optimized recombinant nucleic acid comprises a nucleotide sequence encoding a tumor specific antigen.

43. The method as described in claim 41, further characterized in that the optimized recombinate nucleic acid comprises a nucleotide sequence that encodes a molecule, which has the ability to inhibit the proliferation of cancer cells.

44. - The method as described in Claim 18, further characterized in that the disease condition is an inflammatory response, which has an unknown or non-antigenic specificity, and the selection step comprises one or more of the steps that are found then: a) determining the ability of the genetic vaccine vector to induce cytokine production by PBMC, synovial fluid cells, purified monocyte / macrophage T cells, dendritic cells or T cell clones; b) determining the ability of the genetic vaccine vector to induce the activation or proliferation of the T cell; and c) determining the ability of the genetic vaccine vector to induce differentiation of the T cell to TH 1 or TH 2 cells.

45. - The method as described in Claim 18, further characterized in that the disease condition is an autoimmune response.

46. The method as described in Claim 45, further characterized in that the optimized recombinant antigen polypeptide changes the immune response of a T 1 -mediated response to a T 2 -mediated response.

47. - The method as described in claim 18, further characterized in that the disease condition is an allergic immune response.

48. The method as described in claim 47, further characterized in that the optimized recombinant antigen polypeptide changes the immune response of a T 2 -mediated response to a response mediated by TH 1.

49. The method as described in claim 47, further characterized in that the optimized recombinant antigen polypeptide induces an immune response characterized by the expression IgG and IgM and by a reduced IgE expression.

50. The method as described in claim 47, further characterized in that the optimized recombinant antigen polypeptide is not recognized by the pre-existing IgE molecules present in the sera of atopic mammals.

51. The method as described in Claim 50, further characterized in that the r-optimized recombinant antigen polypeptide retains T cell epitopes, which are involved in the modulation of a T cell response.

52. - A method for obtaining a recombinant viral vector, which has an improved ability to induce an antiviral response in a cell, wherein the method comprises the steps of: (1) recombining at least the first and second forms of a nucleic acid which comprises a viral vector, wherein the first and second forms differ from one another in one or more nucleotides, to produce a library of recombinant viral vectors; (2) transfect the library of recombinant viral vectors and a population of mammalian cells; (3) staining the cells for the presence of the Mx protein, and (4) isolating the recombinant viral vectors from the cells staining positive for the Mx protein, wherein the recombinant viral vectors from the positive staining cells exhibit an enhanced ability to induce an antiviral response.

53. The method as described in Claim 52, further characterized in that the viral vector comprises an influenza viral genomic nucleic acid. SUMMARY The present invention is directed to library immunization, which provides methods for obtaining antigens having improved properties for therapeutic use and other uses. The methods are useful for obtaining improved antigens that can induce an immune response against pathogens, cancer or other conditions, as well as antigens that are effective in modulating allergic, inflammatory and autoimmune diseases.