MXPA00007893A - Targeting of genetic vaccine vectors. - Google Patents

Abstract

This invention provides methods of obtaining reagents for increasing the specificity of genetic vaccines for a desired target cell or tissue type. The invention also provides delivery vehicles for use to improve genetic vaccine specificity for a target cell or tissue type.

Description

DIRECTION OF GENE VACCINE VECTORS BACKGROUND OF THE INVENTION Field of the Invention This invention belongs to the field of genetic vaccines. Specifically, the invention provides methods for improving the effectiveness of genetic vaccines by providing materials that facilitate the targeting of a genetic vaccine to a particular tissue or cell type of interest.

Background Genetic immunization represents a novel mechanism for inducing protective humoral and cellular immunity. Vectors for genetic vaccines generally consist of DNA that includes a promoter / amplifier sequence operably linked to a gene of interest (which often codes for an antigen) and a polyadenylation / transcriptional terminator sequence. After intramuscular or intradermal injection, the gene of interest is expressed followed by recognition of the resulting protein by the cells of the immune system. Genetic immunizations provide a means to produce protective immunity even in situations where the pathogens are poorly characterized or can not be isolated or cultured in the laboratory environment. The antigen is expressed in the cytoplasm of the host cell (eg, in muscle cells) or, by inclusion of a signal secretion sequence, is expressed on the surface of the host cell or is secreted from the host cell. The antigen is processed by the endogenous processes of the host cell transfected by the gene vaccine vector. When expressed cytoplasmically, it is thought that the antigen will be directed to the proteasome for proteolysis. The peptides thus derived are classified by the endogenous TAP-1 and TAP-2 and transported to the light of the rough endoplasmic reticulum (RER), where they associate with the MHC molecules of Class I to eventually move towards the cell surface as a complex. Molecular Class I, β2-microglobulin and peptide. When the antigen is released intact from the transfected cells, it is thought that it is absorbed by the endocytic pathways in APC and processed internally in them by endogenous pathways for eventual presentation on its cell surface as peptide fragments in complex with MHC molecules of the Class I or II. The effectiveness of genetic vaccination is often limited by the inefficient absorption of the genetic vaccine vectors into the cells. Generally, less than 1% of the muscle or skin cells at the injection sites express the gene of interest. Even a small improvement in the efficiency of gene vaccine vectors to enter cells can result in a dramatic increase in the level of immune response induced by genetic vaccination. A vector typically has to cross many barriers which can result as long as only a very small fraction of the DNA is expressed. Limitations to immunogenicity include: loss of vector due to nucleases present in blood and tissues; inefficient entry of DNA into a cell; inefficient entry of DNA into the cell nucleus and preference of DNA by other compartments; lack of DNA stability in the nucleus (factor that limits nuclear stability may differ from those affecting other cellular and extrecelular compartments), and, by vectors that integrate into the chromosome, the efficiency of the integration and the integration site. In addition, for many applications of genetic vaccines, it is preferable that the genetic vaccine enters a target tissue or particular target cell. Thus, there is a need for genetic vaccines that can be targeted to specific cell types and tissues of interest, and that exhibit a greater ability to enter target cells. The present invention fills those and other needs.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides methods for obtaining a specific binding molecule for a cell that is useful for increasing the absorption or specificity of a genetic vaccine by a target cell. The methods involve: creating a library of recombinant polynucleotides that by recombining a nucleic acid encoding a polypeptide comprising a nucleic acid binding domain and a nucleic acid encoding a polypeptide comprising a specific binding domain of a cells; and separating the library to identify a recombinant polynucleotide that codes for a binding molecule that can bind to a nucleic acid and a specific receptor of a cell. Target cells of particular interest include antigen-presenting and antigen-processing cells, such as muscle cells, monocytes, dendritic cells, B cells, Langerhans cells, keratinocytes and M cells. In some embodiments, the methods of the invention for obtaining a Specific binding portion of a cell useful for increasing the uptake or specificity of a genetic vaccine by a target cell involves: (1) recombining at least a first and second forms of a nucleic acid comprising a polynucleotide that codes for a binding domain of nucleic acid and at least the first and second forms of a nucleic acid comprising a ligand specific for a cell that specifically binds to a protein on the surface of a cell of interest, where the first and second forms differ from each other in two. or more nucleotides, to produce a library of the nucleic acids that it encodes n for the recombinant binding portion; (2) transfect a library of vectors to a population of host cells, each of which comprises: a) a specific binding site for the nucleic acid binding domain and 2) a member of the nucleic acid library that encode for the recombinant binding portion, wherein the recombinant binding portion is expressed and binds to the binding site to form a vector-binding portion complex; (3) lysing the host cells under conditions that do not disturb the binding of the vector-binding portion complex; (4) contacting the vector-binding portion complex with a target cell of interest; and (5) identifying the target cells that contain a vector and isolating the nucleic acids from the specific binding portion of a recombinant cell optimized from those target cells. If further optimization is desired, the methods may further involve: (6) recombining at least one nucleic acid encoding the optimized recombinant binding portion with a further form of the polynucleotide encoding a nucleic acid binding domain and / or an additional form of the polynucleotide encoding a cell-specific ligand, which are the same or different from the first and second forms, to produce an additional library of nucleic acids encoding the recombinant binding portion; (7) transfecting to a population of host cells a library of vectors comprising: a binding site specific for the binding domain of the nucleic acid and 2) the nucleic acids encoding the recombinant binding portion, where the binding portion Recombinant is expressed and binds to the binding site to form a vector-binding portion complex; (8) lysing the host cells under conditions that do not disturb binding of the vector-binding portion complex; (9) contacting the vector-binding portion complex with a target cell of interest and identifying the target cells containing the vector; (10) isolating the nucleic acids from the optimized recombinant binding portion of the target cells containing the vector; and (11) repeating (6) through (10), as necessary, to obtain a specific binding portion of an additional optimized cell useful for increasing the absorption or specificity of a genetic vaccine vector for a target cell. The invention also provides specific recombinant binding portions of a cells produced by expression in a host cell and a nucleic acid encoding the optimized recombinant binding portion obtained by the methods of the invention. In another embodiment, the invention provides genetic vaccines that include: a) an optimized recombinant binding portion comprising a nucleic acid binding domain and a specific ligand of a cell, and b) a polynucleotide sequence comprising a binding site, wherein the binding domain of the nucleic acid is capable of binding specifically to the binding site. A further embodiment of the invention provides methods for obtaining a specific binding portion of an optimized cell useful for increasing the absorption, efficacy, or specificity of a genetic vaccine for a target cell by: (1) recombining at least one first and second forms of a nucleic acid comprising a polynucleotide, which codes for a non-toxic receptor binding portion of an enterotoxin or other toxin, where the first and second forms differ from each other in two or more nucleotides, to produce a nucleic acid library recombinants 12) by transfecting vectors containing the nucleic acid library in a population of host cells, wherein the nucleic acids are expressed to form the polypeptides of the specific binding portion of a recombinant cell; (3) contacting the polypeptides of the specific binding portion of a cell, recombinants, with a receptor on the cell surface of a target cell; and (4) determining which polypeptides of the specific binding portion of a cell, recombinants, exhibit a greater ability to bind to the target cell. Methods for increasing the uptake of a genetic vaccine vector by a target cell by contacting the vector of the genetic vaccine with a specific binding portion of an optimized recombinant cell produced by those methods are also provided by the invention.

The present invention also provides methods for evolving a vehicle for delivery of a vaccine, gene vaccine vector, or a component of the vector to obtain a vehicle or an optimized release component that has, or confers a vector, enhanced ability to enter to a mammalian tissue selected after administration to a mammal. These methods involve: (1) recombinant members of a set of polynucleotides to produce a library of recombinant polynucleotides; (2) administering to a test animal a library of reproducible genetic packets, each comprising a library member of the recombinant polynucleotides operably linked to a polynucleotide encoding a display polypeptide, wherein the recombinant polynucleotide and the presentation polypeptides are expressed as a fusion protein which is presented on the surface of the reproducible genetic package; and (3) recover reproducible genetic packets that are present in the selected tissue of the test animal at an appropriate time after administration, where the retrievable reproducible genetic packets have a greater ability to enter the selected mammalian tissue after administration to the mammal . If further optimization of the delivery vehicle is desired, the method of the invention further involves: (4) recombining a nucleic acid comprising at least one recombinant polynucleotide obtained from a reproducible genetic package recovered from the selected tissue with an additional set of polynucleotides for produce an additional library of recombinant polynucleotides; (5) administering to a test animal a library of reproducible genetic packets, each comprising a member of an additional library of recombinant polynucleotides operably linked to a polynucleotide encoding a display polypeptide, wherein the recombinant polynucleotide and the presentation polypeptides are expressed with a fusion protein which is presented on the surface of the reproducible genetic package; (6) recover reproducible genetic packages that are present in the selected tissue of the test animal at an appropriate time after administration; and (7) repeating (4) through (6), as necessary, to obtain an optimized recombinant delivery vehicle that exhibits a greater ability to enter a selected mammalian tissue following administration to a mammal. Methods of administration that are of particular interest include, for example, oral, topical and inhalation. Where the administration is intravenous, the mammalian tissues of interest include, for example, the lymph nodes and the spleen. In another embodiment, the invention provides methods for evolving a vaccine delivery vehicle, a genetic vaccine vector, or a vector component to obtain a vehicle or optimized release component to obtain an optimized vector delivery component or carrier that has, or confers to a vector containing the component, greater specificity for cells representing antigens by: (1) recombining members of a group of polynucleotides to produce a library of recombinant polynucleotides; (2) producing a library of reproducible genetic packets, each comprising a member of the library of recombinant polynucleotides operably linked to a polynucleotide encoding a display polypeptide, wherein the recombinant polynucleotide and the display polypeptide are expressed as a fusion protein which is presented on the surface of the reproducible genetic package; (3) contacting the library of recombinant reproducible genetic packets with a non-APC to remove reproducible genetic packets that have non-APC-specific fusion polypeptides; and (4) contacting recombinant reproducible genetic packets that do not bind to non-APC with an ApC and recover those that bind to the APC, where the retrievable reproducible genetic packets are capable of specifically binding to APC. In a further embodiment, the invention provides methods for evolving a vaccine delivery vehicle, genetic vaccine vector or component of a vector to obtain a delivery vehicle or optimized component to obtain a delivery vehicle or optimized release component that has or confers to a vector containing a component, a greater ability to enter a target cell by: (1) recombining at least the first and second forms of a nucleic acid encoding an invasin polypeptide, where the first and second forms differ from one another yes in two or more nucleotides, to produce a library of recombinant invasin nucleic acids; (2) producing a recombinant bacteriophage library, each of which has on the surface of the bacteriophage a fusion polypeptide encoded by a chimeric gene comprising a recombinant invasin nucleic acid operably linked to a polynucleotide encoding a presentation polypeptide; (3) contacting the library of the rectal bacteriophage with the target cell population; (4) removing the unbound phage and the phage that bound to the target surface; and (5) recovering the phage that is present within the target cells, where the recovered phages are rich in the phage that has greater capacity to enter the target cells. In some embodiments, optimized recombinant gene vaccine vectors, delivery vehicles, or optimized vector components obtained using those methods exhibit better ability to enter a cell presenting antigens. These methods may involve washing the cells after the transfection step to remove the vectors that do not enter an antigen-presenting cell; culturing the cells for a predetermined time after transfection; lyse the cells that present antigens; and isolate the vector of the genetic vaccine optimized from the used cell. Antigen-presenting cells containing optimized recombinant genetic vaccine vectors can be identified, for example, by detecting the expression of a marker gene that is included in the vectors. In some embodiments, the gene vaccine vector comprises a nucleotide sequence encoding an immunogenic antigen, and optimized recombinant gene vaccine vectors are identified: transfecting individual members of a library in separate cultures of antigen-presenting cells; or by culturing APC transfected with T-lymphocytes obtained from the same individual as APC; and identify transfected APC cultures which are capable of inducing a T lymphocyte response, the lymphocyte response in these methods can be selected from the group consisting of an increase in T lymphocyte proliferation, increased cytolytic activity mediated by T lymphocytes. against a target cell and the increase in cytosine production. As an example, the vector of the genetic vaccine may be capable of inducing a TH1 response according to what is evidenced by the transfected APCs that induce a response of the T lymphocytes that implies one or more of the proliferation, production of IL-2 and production of interferon? Additional modalities of these methods involve the use of gene vaccine vectors or delivery vehicles that include a nucleotide sequence that codes for an antigen.; optimized recombinant vaccine vectors can be identified: by injecting the library of the recombinant genetic vaccine vectors in a test animal; obtaining lymphatic cells (e.g., dendritic cells) from the test animal; and recovering recombinant genetic vaccine vectors from lymphatic cells, where recovered recombinant genetic vaccine vectors exhibit better ability to enter lymphatic cells. In some embodiments, the antigen is a cell surface antigen, and before isolating the optimized recombinant gene vaccine vectors, the cells containing an optimized recombinant vector are purified to bind to an affinity reagent which selectively binds to the cell surface antigen. The invention also provides methods for evolving a vaccine delivery vehicle derived from a bacteriophage to obtain a delivery vehicle that has a greater capacity to enter a target cell. These methods involve the steps of: (1) recombining at least one first and second forms of a nucleic acid encoding an invasin polypeptide, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant invasin nucleic acids; (2) producing a recombinant bacteriophage library, each of which displays on the surface of the bacteriophage a fusion polypeptide encoded by a chimeric gene comprising a recombinant invacinate nucleic acid operably linked to a polynucleotide that encodes a presentation; (3) contacting the recombinant bacteriophage library with a population of target cells; (4) removing the unbound phage and the phage that bound to the surface of the target cells; and (5) recovering the phage that is present within the target cells, where the recovered phage is rich in phage that have a greater capacity to enter the target cells. Again, if further optimization is desired, the methods may further include the steps of: (6) recombining a nucleic acid comprising at least one recombinant invasin nucleic acid obtained from a bacteriophage which is recovered from a target cell with a set Additional polynucleotides to produce an additional library of recombinant invasin polynucleotides; (7) producing an additional library of recombinant bacteriophage, each of which has on the surface of the bacteriophage a fusion polypeptide encoded by a chimeric gene comprising a recombinant invasin nucleic acid operably linked to a polynucleotide encoding a presentation; (8) contact the recombinant bacteriophage library with a population of cells .7 objective; (9) removing the unbound phage and the phage that bound to the surface of the target cells; and (10) recovering the phage that is present within the target cells and (11) repeating (6) through (10), as necessary, to obtain an additional optimized recombinant release vehicle which also exhibits enhanced ability to enter the target cells. In some embodiments, methods for evolving a vaccine delivery vehicle derived from a bacteriophage to obtain a delivery vehicle that has a greater ability to enter a target cell may include the additional steps of: (12) inserting into the delivery vehicle recombinant optimized a polynucleotide which encodes an antigen of interest, wherein the antigen of interest is expressed as a polypeptide function comprising a second polypeptide of presentation; (13) administering the delivery vehicle to a test animal; and (14) determining whether the test vehicle is capable of inducing a CTL response in the test animal. Alternatively, the following steps may be employed: (12) inserting into the optimized recombinant release carrier a polynucleotide which codes for an antigen of interest, wherein the antigen of interest is expressed as a fusion polypeptide which comprises a second polypeptide of presentation; (13) administering the delivery vehicle to a test animal; and (14) determining whether the delivery vehicle is capable of inducing neutralizing antibodies against a pathogen comprising an antigen of interest. An example of a target cell of interest for those methods is an antigen-presenting cell.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a strategy for obtaining and using nucleic acid binding proteins that facilitate the entry of genetic vaccines, in particular, naked DNA, into target cells. The members of a library obtained by reloading the DNA are linked to a coding region of the M13 VII protein, so that a fusion protein is presented on the surface of the payment particles. The phage that efficiently enters the desired target tissue is identified, and the fusion protein is then used to coat the genetic vaccine nucleic acid. Figure 2 illustrates a strategy for separating M13 libraries for the desired direction of various tissues. The particularly illustrated example relates to the separation for improved oral delivery, but the same principle applies to libraries given by other means, including intravenous, intramuscular, intradermal, anal, vaginal or topically. After being released to a test animal, the M13 phage is recovered from the tissue of interest. The procedure can be repeated to obtain additional optimization. Figure 13 is an alignment of the nucleotide sequences encoding bacterial enterotoxins from two Escheri chia coli strains and cholera toxin B. The nucleotide sequences for enterotoxin B of E are shown. coli (SEQ ID NO: 1), enterotoxin B of E coli. (swine) (SEQ ID NO: 2), and subunit B of Cholera toxin (SEQ ID NO: 3). Figure A and Figure 4B show a protocol for the generation and transfection of human dendritic cells. Figure 4A shows the phenotype of freshly isolated monocytes (left) and cultured dendritic cells obtained by culturing blood monocytes in the presence of IL-4 and GM-CSF for seven days. Figure 4B shows a flow cytometry analysis of cultured dendritic cells after transfection by a plasmid encoding GFP.

DETAILED DESCRIPTION Definitions The term "cytokine" includes, for example, interleukins, interferons, chemokines, hematopoietic growth factors, tumor necrosis factors, and transformation growth factors. In general, these are low molecular weight proteins that regulate the maturation, activation, proliferation and differentiation of the cells of the immune system. The term "separation" describes, in general, a process that identifies optimal antigens. Several properties of the antigen can be used in the selection and separation, including antigen expression, synthesis, stability, immunogenicity and presence of epitopes of several related antigens. Selection is a form of separation in which identification and physical separation are achieved simultaneously by expression of the selection marker, which, in some circumstances, allows the cells expressing the marker to survive while the other cells die ( or vice versa) . The "separation or selection" markers include, for example, luciferase, beta-galactosidase and green fluorescent protein.The selection markers include drug and toxin resistance genes, and the like.Due to limitations in the study of immune responses In these studies, antigens are first introduced into test animals, and immune responses are subsequently studied by analyzing the protective immune responses or by studying the quality or resistance of the immune responses. induced immune response using lymphoid cells derived from immunized animals.Although spontaneous selection can and do occur in the course of natural evolution, in the methods of the present selection it is effected by man.An "exogenous DNA segment", "heterologous sequence" "or a" heterologous nucleic acid "as used herein, is one that originates from a source external to the particular host cell, or, * if it is from the same source, is modified from an original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell, but that has been modified. The modification of a heterologous sequence in the applications described here typically occurs through the use of DNA entrainment. In this way, the terms still refer to a segment of DNA which is external 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 in an ordinary manner . The exogenous DNA segments are expressed to produce exogenous polypeptides. The term "gene" is used herein broadly to refer to any DNA segment associated with a biological function. In this way, the genes include the coding sequences and / or the regulatory sequences required for their expression. Genes also include unexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of resources, including cloning a source of interest or synthesizing from information from known or predicted sequences, and can include sequences designed to have desired parameters. The term "isolated", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. This is preferably in a homogeneous state although it may be in dry form or in an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from the open reading frames that flank the gene and codes for a protein other than the gene of interest. The term "purified" denotes that the protein nucleic acid essentially gives rise to a band in an electrophoretic gel. Particularly, this means that the nucleic acid or protein is at least about 50% pure, more preferably at least about 85% pure, and even more preferably at least about 99% pure. The term "natural" is used to describe an object that can be found in nature that is distinguished from that artificially produced by man. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses, bacteria, protozoa, insects, plants or mammalian tissue) that can be isolated from a natural source and that has not been intentionally modified by man in the laboratory is natural.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in the form of a single strand or double strand. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides which have binding properties similar to those of the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and compntary sequences as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more of (or all) of the selected codons is substituted with mixed base residues and / or deoxyinosine (Batzer et al. (1991) Nucl. c Acid Res. 19: 5081; Ohtska et al. (1985) J. Biol. Chem. 260: 2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91 -98). The term "nucleic acid" is used interchangeably herein with a gene, cDNA, mRNA encoded by a gene.

"Nucleic acid derivative of a gene" refers to a nucleic acid from which the gene, or a subsequence thereof, has finally served as a standard. Thus, an mRNA, a cDNA transcribed in reverse manner from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, and a RNA transcribed from the amplified DNA, etc., are all derived from the gene and the detection of such derivative products is indicative of the presence and / or abundance of the original gene and / or the transcript of the 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 amplifier is operably linked to a coding sequence if it increases the transcription of the coding sequence. Ligation operatively means that the DNA sequences that are ligated are typically contiguous and, when necessary, bind two protein coding regions, contiguous and in the reading frame. However, since the amplifiers generally operate when several kilobases are separated from the promoter and the intronic sequences can be of varying lengths, some polynucleotide ents may be operably linked but not contiguous.

A specific binding affinity between two molecules, for example, a ligand and a receptor, means a preferential binding of one molecule by another in a mixture of molecules. The binding of the molecules can be considered specific if the binding affinity is about 1 × 10 4 M "1 to about 1 × 10 6 M 1 or greater.The term" recombinant "when used with reference to an indica cell that the cell reproduces 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 may also contain genes found in the This is the native form of the cell where the genes are modified and reintroduced into the cell by artificial means.The term also encompasses cells that contain an endogenous nucleic acid to the cell that has been modified without removing the nucleic acid from the cell; include those obtained by gene replacement, site-specific mutation, and related techniques A "recombinant expression cassette" or simply an "expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with the elements of the nucleic acid that are capable of affecting the expression of a structural gene in hosts compatible with such sequences. Expression cassettes include at least promoters and optionally, transcription termination signals. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (eg, a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or helpful in effecting the expression may also be used as described herein. For example, an expression cassette may also include nucleotide sequences that encode a signal sequence that directs the secretion of an expressed protein from the host cell. Transcription termination signals, amplifiers, and other nucleic acid sequences that influence gene expression can also be included in an expression cassette. A "multivalent antigenic polypeptide" or a "Recombinant multivalent antigenic polypeptide" is an unnatural polypeptide that includes amino acid sequences plus an original polypeptide, an original polypeptide which is typically a natural 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 original polypeptide. The original polypeptides from which the different epitopes are derived are usually homologous (ie, having the same or similar structure and / or function), and are often different isolates, serotypes, strains, species, organisms or different disease states, for example. The terms "identical" or percent of "identity", in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specific percentage of amino acid residues or nucleotides that are the same, when compared and align for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The phrase "substantially identical", in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences having at least 60%, preferably 80%, more preferably 90-95% identity of nucleotides or amino acid residues, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequence that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In some embodiments, the sequences are substantially identical throughout the coding regions. For comparison of the sequence, typically a sequence acts as a reference sequence with which the test sequences are compared. When a sequence comparison algorithm is used, the test and reference sequences are fed to a computer, after which the coordinates are designated, if necessary, and 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 relative to the reference sequence, based on the designated program parameters. The optimal alignment of the sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Ma thf 2: 482 (1981), by the homology alignment algorithm of Needleman & unsch, J. Mol. Biol. 48: 443 (1970), by the search for the similarity method of Pearson & Lipman, Proc. Na ti. Acad. Sci. USA 85: 2444 (1988), computerized implementations of those algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Package, Genetics Computer Group, 575 Science Dr., Madison, Wl) or by visual inspection (see in general Ausubel et al., Infra). An example of an algorithm that is suitable for determining the percent identity of the sequence and the similarity of the sequence is the BLAST algorithm, which is described in Ailtschul et al. , J. Mol. Biol. 215: 403-410 (1990). The programs and programming systems to carry out the BLAST analyzes are public domain and are available through the Center National Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high-grade sequence pairs (HSP) by identifying short-term words W in the sequence investigated, which are equal or satisfy some threshold qualification of positive value T when they are aligned with a word of the same length in a sequence of the database. T is known as the qualification threshold of the neighboring word (Alschul et al., Supra). It's right from the initial neighbor word acts as seed to start-searches to find larger HSPs that contain them. Word hits then extend in both directions along each sequence so that the cumulative alignment rating can be increased. The cumulative scores are calculated using, for nucleotide sequences, the M parameters (reward rating for a similar residue pair).; whenever it is > 0) and N (penalty rating for different residuals, provided it is <0). For amino acid sequences, a rating matrix is used to calculate the cumulative score. The extension of word hits in each direction stops when: the cumulative alignment qualification falls far from the amount X of its maximum value reached; the cumulative rating tends to zero or less, due to the accumulation of one or more alignments of negative rating residues; or at the end of which any sequence has been reached. The parameters of the BLAST algorithm W, T and X determine the sensitivity and speed of alignment. The BLAST program (for nucleotide sequences) uses as default errors a word length (W) of 11, an expectation (E) of 10, a cut of 100, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as a deflector values a word length (W) of 3, an expectation (E) of 10, and the qualification matrix BLOSUM62 (see Henikoff &Henikoff (1989) Proc. Na ti Acad. Sci. USA 89: 10915). In addition to calculating the percent identity of the sequence, the BLAST algorithm also performs a statistical analysis of the similarity between the two sequences (see, for example, Karlin &Altschul (1993) Proc. Na t '1. Acad. Sci. USA 90: 5873-5787). 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 a similarity occurring between two nucleotide or amino acid sequences. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01. , more preferably less than about 0.001.

Another indication that the two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specifically hybridizes to" refers to the binding, duplexing, or hybridization of only one molecule to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex (e.g., cellular total) mixture of DNA or RNA "Substantial binding" refers to the complementary hybridization between a nucleic acid probe and a target nucleic acid and encompasses minor differences that can be accommodated by reducing the severity of the hybridization medium to achieve the desired detection of the target polynucleotide sequence. "Strict hybridization conditions" and "stringent hybridization washing conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations depend on the sequence, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide for nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Acid Probes, Nucleic Part I Chapter 2"General View of the Principles of Hybridization and Strategy Testing Nucleic Acid Probes ", Elsevier, New York. Generally, the highly stringent hybridization and washing conditions are selected such that they are about 5 ° C lower than the thermal melting temperature (Tm) for the specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions" a probe will hybridize to its target sequence, but not to other sequences. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence is hybridized to a perfectly matched probe or the like. The very strict conditions are selected so that they are equal to the Tm for a particular probe. An example of stringent hybridization conditions for the hybridization of complementary nucleic acids having more than 100 complementary residues on a Southern or Northern spot filter is 50% formamide with 1 mg of heparin at 42 ° C, with the hybridization being carried to Cape during the night. An example of highly stringent washing conditions in 0.15M NaCl at 72 ° C for about 15 minutes. An example of stringent washing conditions is a wash with 0.2x SSC at 65 ° C for 15 minutes (see, Sambrook, infra., For description of the SSC buffer). Frequently, a very strict washing is preceded by a little strict washing to remove the bottom signal of the probe. An example of moderately strict washing for a duplex of, for example, more than 100 nucleotides, is 1 x SSC at 45 ° C for 15 minutes. An example of a lax wash for a duplex of, for example, more than 100 nucleotides is 4-6x SSC at 40 ° C for 15 minutes. For short probes (eg, from about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na + ion, typically a concentration of about 0.1 to 1.0 M Na + ion (or other salts). at pH 7.0 to 8.3, and the temperature is typically at least about 30 ° C. Strict conditions can also be achieved with the addition of stabilizing agents such as formamide. In general, a signal-to-noise ratio of 2x (or greater) greater than that observed for an unrelated probe in the particular hybridization assay indicates the detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical.

This occurs, for example, when a nucleic acid copy is created using the maximum codon degeneracy 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 reacts immunologically cross-linked with, or specifically binds to, the polypeptide encoded by the second nucleic acid. Thus, the polypeptide is typically, substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. The phrase "that binds specifically" (or selectively) to an antibody "or" specifically (or selectively) immunoreactive with ", when referring to a protein or peptide, refers to a binding reaction which is determinant of the presence of the protein, or an epitope of the protein, in the presence of a heterogeneous population of protein and other biological products.Thus, under the designated immunoassay conditions, the specific antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample Antibodies directed 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 be selected for its specificity by a particular protein.A variety of immunoassay formats can be used naysay to select antibodies specifically immunoreactive with a particular protein. For example, ELISA immunoassays in solid phase, spotting or Western immunoblotting, or immunohistochemistry are commonly used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Labora tory Manual, Cold Spring Harbor Publications, New York "Harlow and Lane"), for a description of the immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically a specific or selective reaction will be at least twice the signal or background noise and more typically more than 10 to 100 times the background. "Conservatively modified variations" of a particular polynucleotide sequence refers to these polynucleotides that encode identical or essentially identical amino acid sequences, or where the polynucleotide does not code for an amino acid sequence, for essentially identical sequences. Due to the generation of the genetic code, a large number of functionally identical nucleic acids encode - for any given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all code for the amino acid arginine. Thus, at each position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such variations of the nucleic acid are "silent variations", which are a kind of "conservatively modified variations". Each polynucleotide sequence described herein that codes for a polypeptide also describes every possible silent variation, except where otherwise noted. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is commonly the only codon for methionine) can be modified to produce a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid encoding a polypeptide is implicit in each described sequence.

In addition, one skilled in the art will recognize that substitutions, deletions or individual additions that alter, aggregate or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in a coded sequence are "conservatively modified variations", where the alterations result in the substitution of an amino acid with the chemically similar amino acid. The tables of conservative substitutions that provide functionally similar amino acids are known in the art. The following five groups each contain amino acids that are conservative substitutions with each other: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Containing sulfur: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acid: Aspartic acid (D), glutamic acid (E), Asparagine (N), Glutamine (Q). Also see, Creighton (1984) Proteins, W.H. Freeman and Company, for additional amino acid groupings. In addition, individual substitutions, deletions or additions that alter, or delete a single amino acid or a single percentage of amino acids in an encoded sequence are also "conservatively encoded variations." A "subsequence" refers to a nucleic acid or amino acid sequence that comprises a part of a longer sequence of nucleic acids or amino acids (e.g., polypeptide), respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides reagents for facilitating the ability of a genetic vaccine to specifically bind to and enter a target cell or tissue of interest, and methods for obtaining such agents. In particular, the invention provides methods for obtaining binding peptides and delivery vehicles which, when used in conjunction with a genetic vaccine, increase the specificity of the genetic vaccine for a particular type of target cell. The methods are also useful for obtaining components of the genetic vaccine that can confer a desired targeting specificity when used in conjunction with a genetic vaccine vector.

A. Creation of Recombinant Libraries The invention involves creating recombinant libraries of polynucleotides that are then separated to identify those members of the library that exhibit a desired property. The recombinant libraries can be created using any of the methods. The substrate nucleic acids used for recombination may vary depending on the particular application. For example, where a polynucleotide encoding a nucleic acid binding domain or a ligand for a specific receptor of a cell is to be optimized, different forms of nucleic acids encode all or part of the nucleic acid binding domain or a ligand for a specific receptor of a cell are subjected to recombination. The methods require at least two variant forms of an initial substrate. The variant forms of the candidate substrates may show a substantial sequence or secondary structural similarity to each other, but must also differ in at least two positions. The initial diversity between the forms can be the result of a natural variation, for example, the different variant forms (homologous) are obtained from different individuals or members of an organism (including geographical variants) or constitute related sequences of the same organism ( for example, allelic variations). Alternatively, the initial diversity may be induced, for example, the second variant form may be generated by an error-prone transcript, such as an error-prone PCR or the use of a polymerase that lacks proven reading activity (see Liao (1990) Gene 88: 107-111), of the first variant form, or, by reproduction of the first form in a sepa or mutant (the mutant host cells are described in more detail below). The initial diversity between the substrates is generally increased in subsequent steps of recombination of the recursive sequence. Frequently, improvements are achieved after a round of recombination and selection. However, recursive sequence recombination can be employed to achieve even greater improvements in the desired property. The sequence recombination can be carried out in many formats and swaps of different formats, as described in detail below. These formats share some common principles. Recursive sequence recombination consists of successive recombination cycles to generate molecular diversity. That is, a family of nucleic acid molecules is created that show some sequence identity to each other but differ in the presence of mutations. In any given cycle, recombination may occur in vivo or intracellular or extracellular in vi tro. In addition, the diversity resulting from recombination can be increased in any cycle by applying the above mutagenesis methods (e.g., error prone PCR or cassette mutagenesis) to any of the substrates or products of recombination. In some cases, a new property or enhanced characteristic can be achieved after only a recombination cycle in vivo or in vi tro, such as when different variant forms of the sequence are used, such as homologs of different individuals or members of an individual, or sequences related to the same organism, such as allelic variations. In a preferred embodiment up to now, recombinant libraries are prepared using DNA entrainment. The entrainment and separation or selection can be used to "evolve" individual genes, complete plasmids or viruses, multiple gene sets, or even all genomes (Stemmer (1995) Bio / Technology 13: 549-553). Repeat cycles of recombination and separation / selection can be performed to further evolve the nucleic acids of interest. Such techniques do not require the extensive analysis and computation required by conventional methods to design polypeptides. Trawling allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to the traditional pair recombination events. Thus, the sequence recombination techniques described here provide additional advantages since they provide recombination between any or all of those mutations, thus providing a faster way to explore the manner in which different combinations of mutations can affect a desired result. In some cases, however, structural and / or functional information is available which, although not required for sequence recombination, provides opportunities for modification of the technique. Exemplary formats and examples of sequence recombination, sometimes referred to as DNA drag, evolution or molecular reproduction, have been described by the inventors and co-workers herein by U.S. patent applications. copending Serial No. 08 / 198,431, filed on February 17, 1994, Serial No. PCT / US95 / 02126, filed on February 17, 1995, Serial No. 08 / 425,684, filed on April 18, 1995, No. of Series 08 / 537,874, filed on October 30, 1995, Serial No. 08 / 564,955, filed on November 30, 1995, Serial No. 08 / 621,859, filed on March 25, 1996, Serial No. 08 / 621,430 , filed March 25, 1996, Serial No. PCT / US96 / 05480, filed on April 18, 1996, Serial No. 08 / 650,400, filed on May 20, 1996, Serial No. 08 / 675,502, filed at July 3, 1996, Serial No. 08 / 721,824, filed September 27, 1996, Serial No. PCT / US97 / 17300, filed September 26, 1997, Serial No. PCT / US97 / 24239, filed in December 17, 1997; Stemmer, Sciencie 270: 1510 (1995); Stemmer et al., Gene 164: 49-53 (1995); Stemmer, Bio / Technology 13: 549-553 (1995); Stemmer, Proc. Nati Acad. Sci. U.S.A. 91: 10747-10751 (1994); Stemmer, Nature 370: 389-391 (1994); Crameri et al., Nature Medicine 2 (1): 1-3 (1996); Crameri et al. Nature Biotechnology 14: 315-319 (1996), each of which is incorporated herein by reference in its entirety for all purposes. Other methods for obtaining recombinant polynucleotides and / or for obtaining diversity in the nucleic acids used as the substrates for DNA entrainment, include homologous recombination (PCT / US98 / 05223: Publ.No W098 / 42727); mutagenesis 4 directed to the oligonucleotide (for a review see, Smith, Ann, Rev. Genet, 19: 423-462 (1985), Botstein and Shortle, Science 229: 1193-1201 (1985); Carter, Biochem. J. 237: 1- 7 (1986); Kunkel, "The efficiency of oligonucleotide-directed mutagenesis" in Nucleic acids &Molecular Biology, Eckstein and Lilley, eds., Springer Verlag, Berlin (1987)). Included among these methods is oligonucleotide-directed mutagenesis (Zoller and Smith, Nuci, Acids Res. 10: 6487-6500 (1982), Methods in Enzymol, 100: 468-500 (1983), and Methods in Enzymol. -350 (1987)) mutagenesis of phosphothiated DNA (Taylor et al., Nucí Acids Res. 13: 8749-8764 (1985); Taylor et al., Nucí Acids Res. 13: 8765-8787 (1985); Nakamaye and Eckstein, Nucí Acids Res. 14: 9679-9698 (1986), Sayers et al., Nucí Acids Res. 16: 791-802 (1988), Sayers et al., Nucí Acids Res 16: 803 -814 (1988)), mutagenesis using uracil-containing standards (Kunkel, Proc. Nat'l. Acad. Sci. USA 82: 488-492 (1985) and Kunkel et al., Methods in Enzymol. 154: 367-382 )); mutagenesis using separate duplex DNA (Kramer et al., New Acids Res. 12: 9441-9456 (1984); Kramer and Fritz, Methods in Enzymol., 154: 350-367 (1987); Kramer et al., Nuci. Res. 16: 7207 (1988)); and Fritz et al_. , Nucí. Acids Res 16: 6987-6999. Additional and suitable methods include repair of point mating mismatch (Kramer et al., Cell 38: 879-887 (1984)), mutagenesis using efficient host strains under repair (Carter et al., Nucí Acids Res. 13: 4431 -4443 (1985); ~ Carter, Methods in Enzymol 154: 382-403 (1987)), deletion mutagenesis (Eghtedarzadeh and Henikoff, Nuci Acids Res. 14: 5115 (1986)), restriction-selection and restriction- purification (Wells et al., Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986), mutagenesis by total genetic synthesis (Nambiar et al., Science 223: 1299-1301 (1984); Sakamar et al. Khorana, Nucí, Acids Res. 14: 6361-6372 (1988), Wells et al., Gene 34: 315-323 (1985), and Grundstr? M et al., Nucí. Acids Res. 13: 3305-3316 ( 1985). Equipment for mutagenesis is commercially available, (e.g., Bio-Rad, Amersham International, Anglian Biotechnology).

B. Separation Methods A recombination cycle usually followed by at least one separation or selection cycle for molecules having a desired property or characteristic. If a recombination cycle is carried out in vi tro, the products of recombination, i.e., the recombinant segments, are sometimes introduced into cells before the separation step. The recombinant segments can also be ligated to an appropriate vector or other regulatory sequences before separation. Alternatively, the recombination products generated in vitro are sometimes packaged as viruses before separation. If recombination is carried out in vivo, the products of recombination can sometimes be separated into the cells in which recombination occurred. In other applications, the recombinant segments are extracted from the cells, and optionally packaged as a virus, before separation. The nature of the separation or selection depends on what property or characteristic is to be acquired or the property or characteristic for which improvement is sought, and many examples are discussed below. It is usually not necessary to understand the molecular basis by which the recombination products (recombinant segments) have acquired new or improved properties or characteristics in relation to the initial substrates. For example, a gene vaccine vector can have many component sequences, each of which has a different intended role (eg, coding sequence, regulatory sequences, targeting sequences, stability-conferring sequences, immunomodulatory sequences, sequences that affect the presentation of antigens, and sequences that affect the integration). Each of these component sequences can be varied and recombined simultaneously. The separation / selection can then be made, for example, for recombinant segments that have a higher episomal maintenance in a target cell without the need to attribute such improvement to any of the individual component sequences of the vector. Depending on the particular separation protocol used for a desired property, the initial rounds of separation can sometimes be performed on bacterial cells due to high transfection efficiencies and ease of culture. The final rounds, and other types of separation that are not sensitive to separation in bacterial cells, are carried out in a mammalian cell to optimize the recombinant elements to be used in an environment close to the intended use. The final rounds of separation can be carried out in the precise cell type of the intended use (eg, human antigen-presenting cell). In some cases, this cell can be obtained from a patient to be treated with a view, for example, to reduce the immunogenicity problems in this patient.

The separation or selection step identifies a subpopulation of recombinant segments that have evolved towards the acquisition of a desired new property or improved properties useful in genetic vaccination. Depending on the separation, the recombinant segments can be identified as components of cells, virus components or in free form. More than one round of separation or selection can be made before each round of recombination. If a further improvement in a property is desired, at least one and usually a collection of recombinant segments surviving a first round of separation / selection are subjected to an additional round of recombination. These recombinant segments can be recombined with each other or with exogenous segments that represent the original substrate or additional variants thereof. Again, recombination can proceed in vi tro or in vivo. If the previous separation step identifies the desired recombinant segments as components of the cells, the components can be subjected to further recombination in vivo, or they can be subjected to further recombination in vi tro c can be isolated before making a round of recombination in vi tro. On the contrary, if the previous separation step identifies that the desired recombinant segments are in naked form or as components of virus, those segments can be introduced into cells to effect a round of recombination in vivo. The second round of recombination, no matter how it is carried out, generates additional recombinant segments which encompass the additional diversity that is present in the recombinant segments resulting from the previous rounds. The second round of recombination can be followed by a fourth round of separation / selection according to the principles discussed above for the first round. The stiffness of the separation / selection can be increased between rounds. Also, the nature of the separation and the property being separated may vary between rounds if an improvement in more than one property is desired or if more than one new desired property is being acquired. Additional rounds of recombination and separation can then be carried out until the recombinant segments have evolved sufficiently to acquire the desired new or improved property or function. Various separation methods for particular applications are described here. In several cases, separation involves expressing the recombinant peptides or polypeptides encoded by the recombinant polynucleotides of the library as fusions with a protein that is presented on the surface of a reproducible genetic package. For example, phage display can be used. See, for example, Cwirla et al., Proc. Nati Acad. Sci. USA 87: 6378-6382 (1990); Devlin et al., Science 249: 404-406 (1990), Scott & Smíth, Science 249: 386-388 (1990); Ladner et al., US 5,571,698. Other reproducible genetic packages include, for example, bacteria, eukaryotic viruses, yeasts and spores. The genetic packages most frequently used to present the library are bacteriophages, particularly filamentous phages, and especially phage M13, Fd and Fl. The majority of the work has involved inserting libraries that encode polypeptides to be presented in either the gilí or gVIII of that phage forming a fusion protein. See, for example, Dower, WO 91/19818; Devlin, WO 91/18989; MacCafferty, WO 92/01047 (gen III); Huse, WO 92/062074; Kang, WO 92/18619 (gen VIII). Such fusion protein comprises a sequence usual, but not necessarily, of the phage coat protein, a polypeptide to be presented and any of the gene III protein or gene VIII or fragment thereof. Exogenous coding sequences are often inserted at or near the N-terminus of gene III or gene VIII although other insertion sites are possible. Eukaryotic viruses can be used to present polypeptides in an analogous manner. For example, the presentation of human heregulin fused to gp70 of Moloney murine leukemia virus has been reported by Han et al., proc. Nati Acad. Sci. USA 92: 9747-9751 (1995). Spores can also be used as reproducible genetic packages. In this case, the polypeptides are presented from the outer surface of the spore. For example, it has been reported that B. subtilis spores are adequate. The sequences of the coating proteins of these spores are provided by Donovan et al., J. Mol. Biol. 196, 1-10 (1987). Cells can also be used as reproducible genetic packages. The polypeptides to be presented are inserted into a gene that codes for a cellular protein that is expressed on the surface of the cells. Bacterial cells include Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholearae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and Escherichia coli is especially preferred. The details of the outer surface proteins are discussed by Ladner et al., US 5,571,698 and references cited therein. For example, the lamB protein of E. coli is adequate. A basic concept of presentation methods that utilize phage or other reproducible genetic package is the establishment of a physical association between the DNA encoding a polypeptide to be separated and the polypeptide. This physical association is provided by the reproducible genetic package, which presents a polypeptide as part of a capsule enclosing the phage genome or other package, where the polypeptide is encoded by the genome. The establishment of a physical association between the polypeptides and their genetic material allows simultaneous mass separation of a very large number of phages containing different polypeptides. The phage presenting a polypeptide with affinity for an objective, for example, a receptor, binds to the target and those phages are enriched by affinity separation with the target. The identity of the polypeptides presented by those phages can be determined from their respective genomes. Using those methods a peptide identified by having a binding affinity with a desired target can then be synthesized in bulk by conventional means, or the polynucleotide encoding the peptide or polypeptide can be used as part of a genetic vaccine. The recombinant t-nucleic acid libraries that are obtained by the methods described herein are selected to identify those DNA segments that have a property which is desirable for genetic vaccination. The particular separation test employed will vary, as described below, depending on the particular property for which the improvement is sought. Typically, the entrained nucleic acid library is introduced into the cells prior to separation. If the DNA entrainment format is used in vivo, the library of the generated recombinant DNA segments already exists in a cell. If the recombination of the sequence is carried out in vitro, the recombinant library is preferably introduced into the desired cell type before separation / selection. Members of the recombinant library can be linked to an episome or virus before introduction or can be introduced directly. A wide variety of cell types can be used as receptors for evolved genes. Cells of particular interest include many types of bacterial cells that are used to release vaccines or vaccine antigens (Courvalin et al (1995) CR Acad. Sci. III 18: 1207-12), both gram negative and gram positive, such such as Salmonellas (Attridge et al. (1997) Vaccine 15: 155-62), clostridium (Fox et al. (1996) Gene Ther 3: 173-8), lactobacillus, Shigella (Sizemore et al. (1995) Science 270 : 299- 02, E. coli, streptococci (Oggioni and Pozzi (1996) Gene 169: 85-90), as well as mammalian cells, including human cells. In some embodiments of the invention, the library is amplified in a first host, and then recovered from that host and introduced into a second host more sensitive to expression, selection, separation, or any other desirable parameter. The manner in which the library is introduced into the cell type depends on the absorption characteristics of cell-type DNA, for example, having viral receptors, being able to conjugate, or being naturally competent. If the cell type is not susceptible to natural or chemically induced competition, but is sensitive to electroporation, electroporation would usually be employed. If the cell type is not susceptible to electroporation, biolistics can also be used. The biolistic PDS-1000 Genetic Cannon (Biorad, Hercules, CA) uses helium pressure to accelerate gold or tungsten microcarriers coated with DNA to target cells. The process is applicable to a wide range of tissues, including plants, bacteria, fungi, algae, intact animal tissues, tissue culture cells and animal embryos. The release of electronic pulses can be employed, which is essentially an electroporation format released for living tissues in animals and patients (Zhao, Advanced Drug Delivery Reviews 17: 257-262

[1995]). Novel methods for labeling competent cells are described in International Patent Application PCT / US97 / 04494 (Publ. No. W097 / 35957). After the introduction of the recombinant DNA gene library, the cells are optionally programmed to allow the expression of the genes to occur. In many assays, means are needed to identify cells that contain a particular vector. Vectors of genetic vaccines of all types may include a selectable gene marker. Under selective conditions, only those cells that express the selectable marker will survive. Examples of suitable markers include, the dihydrofolate reductase (DHFR) gene, the thymidine kinase (TK) gene or prokaryotic genes that confer drug resistance, gpt (xanthin-guanine phosphostibosyltransferase), which can be selected by acid mycophenolic; neo (neomycin phosphotransferase), which can be selected with G418, hygromycin or puromycin; and DHFR (dihydrofolate reductase), which can be selected with methotrexate (Mulligan &Berg (1981) Proc. Nat'l. Acad Sci. USA 78: 2072; Southern &Berg (1982) J. Mol. Appl. Genet 1: 327). As an alternative, or in addition to, a selectable marker, a genetic vaccine vector may include a selectable marker which, when expressed, confers to a cell containing the vector an easily identifiable genotype. For example, gene encoding a cell surface antigen that is not normally present on the appropriate host cell. The detection means can be, for example, an antibody or another ligand that is specifically to the cell surface antigen. Examples of suitable cell surface antigens include any CD antigen (set of differentiation) (CD1 to CD163) of a species other than the host cell that has not been recognized by host-specific antibodies. Other examples include the green fluorescent protein (GFP, see, for example, Chalfie et al (1994) Science 263: 802-805; Crameri et al (1996) Nature Biotechnol., 14: 315-319; Chalfie et al. (1995) Photochem, Photobiol, 62: 651-656, Olson et al. (1995) J.

Cell. Biol. 130: 639-650) and related antigens, several of which are commercially available. 1. Separation for Longevity or Translocation of the Vector to the Desired Fabric For certain applications, it is desirable to identify those vectors with greater longevity as DNA or to identify the vectors that end in distant tissues from the injection site. This can be achieved by administering to a mammal a population of recombinant genetic vaccine vectors by choosing the route of administration and, at several subsequent times, cutting the target tissue and recovering plasmid from the tissue by standard molecular biology procedures. The molecules in the recovered vector can be amplified in, for example, E. coli and / or by PCR in vi tro. PCR amplification may involve additional entrainment of the gene, after which the selected derivative population is used for readministration to the animals and further improvement of the vector. After several rounds of this procedure, the selected plasmids can be tested for their ability to express the antigen in the correct conformation under the same conditions in which the plasmid was selected in vivo.

Because the expression of the antigen is not part of the selection or separation process described above, not all vectors obtained are capable of expressing the desired antigen. To overcome this disadvantage, the invention provides methods for identifying a. those vectors in a population of genetic vaccine that exhibits not only the location and longevity in the desired tissue in the integrity of living DNA, but the retention of maximal antigenic expression (or expression of other genes such as cytosines, chemokines, accessory molecules) of the cell surface, MHC, and the like). The methods involve the identification in vi tro of cells that express the desired molecule using purified cells of the tissue of choice, under conditions that allow the recovery of very small numbers of cells and the quantitative selection of those with different levels of antigen expression, according to is desired Two embodiments of the invention are described, each of which utilizes a library of genetic vaccine vectors as a starting point. The goal of each method is to identify those plasmids that exhibit the desired biological properties in vivo. The recombinant library represents a population of vectors that differ in known ways (eg, a combined vector library of different functional modules), or has a randomly generated diversity generated by the insertion of random nucleocyte segments, or has been entrained in vivo to introduce low level mutations through all or part of the vectors. (a) Selection of the antigen expression located on the cell surface. In a first embodiment, the method of the invention involves the selection of the expression of the antigen located on the cell surface. The antigen gene is designed in the plasmidic library of the vaccine so that it has a region of amino acids which are directed to the cell membrane. For example, the region can code for a hydrophobic segment of C-terminal amino acids which signal the binding of a terminal phosphoinositol-glycan (PIG) on the expressed protein and direct the protein to be expressed on the surface of the transfected cell. With an antigen that is naturally a soluble protein, this method will probably not affect the three-dimensional configuration of the protein in its designed fusion with a new C-terminus. With an antigen that is naturally a transmembrane protein (eg, a surface membrane protein or pathogenic viruses, bacteria, protozoa or tumor cell) there are at least two possibilities. First, the extracellular domain can be designed to merge with the C-terminal sequence to signal the link to the PIG. Second, the protein can be expressed in toto depending on the signaling of the host cell to efficiently direct it towards the cell surface. In most cases, the antigen for expression will have an endogenous PIG terminal bond (e.g., some pathogenic protozoan antigens). The vector library is released in vivo after an appropriate time interval tissues and / or cells are collected from various target sites in the animal. The cells can be purified from the tissue using standard cellular biological procedures, including the use of monoclonal antibodies reactive with the specific cell surface as affinity reagents. It is relatively feasible to purify epithelial cells isolated from mucosal sites where the epithelium could have been inoculated or muscle neoblasts. In some embodiments, a minimal physical purification is performed before analysis. It is sometimes desirable to identify and separate specific cell populations from various tissues, such as the vessel, liver, bone marrow, lymphatic organs and blood. Blood cells can be fractionated easily by FACS to separate B, CD4 + or CD8 + cells, dendritic cells, Langerhans cells, monocytes, and the like, using various fluorescent monoclonal and antibody reagents. Those cells expressing the antigen can be identified with a fluorescent monoclonal antibody specific for the C-terminal sequence on the linked PIG forms of the surface antigen. The analysis by FACS allows the quantitative evaluation of the level of expression of the correct form of the antigen on the cell population. Cells expressing the maximum level of antigen are classified and standard molecular biology methods are used to recover the vector of the plasmid DNA vaccine that confers this reactivity. An alternative procedure that allows the purification of those cells that express the antigen (and which may be useful before loading in a cell sorter, since the cells expressing the antigen may be a minority population very small), is to form rosettes or purify by screening the cells expressing surface antigens. The rosettes can be formed between the antigen expressing the cells and the erythrocytes carrying the antibody covalently coupled with the relevant antigen. These are easily purified by gravity sedimentation. Screening of the entire cell population on Petri dishes containing immobilized monoclonal antibody specific for the relevant antigen can also be used to remove undesirable cells. Cells expressing the conformational structure of the target antigen can be identified using specific conformationally dependent mononuclonal antibodies that are known to react specifically with the same structure that is expressed on the target pathogen. Because a monoclonal antibody can not define all aspects of the correct structure of the target antigen, the possibility of an antigen which reacts with a high affinity to the diagnostic antibody but does not produce the correct conformation according to what is defined by what in which the antigen is on the surface of the target pathogen or as secreted from the target pathogen. One way to minimize this possibility is to use several monoclonal antibodies, each of which is known to react with different conformational epitopes in the folded protein correctly, in the selection process. This can be achieved by means of a secondary FACS classification, for example. The enriched plasmid population that successfully expresses sufficient antigen in the correct body site for the desired time is then used as the initial population for another selection round., incorporating the gene drag to expand diversity. In this way, the biological activity selected by the plasmid from the tissues in the animals immunized by DNA vaccine is recovered. This method can also provide the best selected vectors expressing immune accessory molecules that one may wish to incorporate into DNA vaccine constructs. For example, if it is desired to express the accessory protein B7.1 or B7.2 in antigen-presenting cells (APC) (to promote successful penetration of the antigen to T cells) APC isolated from different tissues (in or different inoculation sites) using commercially available monoclonal antibodies that recognize functional B7 proteins. (b) Selection of the expression of ajitigen / secreted ci tocxna / qvtimocins Another separation method is to identify the plasmids in a population of genetic vaccine vectors that are optimal for inducing the secretion of soluble proteins that can affect the quantitative and qualitative nature of an immune response produced. For example, vectors that are optimal for inducing the secretion of particular cytokines, growth factors and chemokines can be selected. The first step in these methods is to generate vectors containing the members of the library of the recombinant nucleic acids. These vectors can then be individually tested for their efficacy in vivo. The vector library is supplied to a test animal and, after a chosen time interval, tissue and / or cells are collected from various animal sites. The cells are purified from the tissue using standard cell biology procedures which often include the use of cell-specific surface reactive monoclonal antibodies as affinity reagents. As is the case for the cell surface antigens described above, the physical purification of separate cell populations can be carried out prior to the identification of the cells expressing the desired protein. For those studies, the target cells for the expression of cytokines will very usually be APC or B cells or T cells instead of muscle cells or epithelial cells. In such cases the separation by FACS by $ S established methods will be preferred to separate the different cell types. The different cell types described above can also be separated into relatively pure fractions using affinity separation, rosette formation or separation with magnetic beads with panels of existing monoclonal antibodies that are known to define the surface membrane phenotype of murine immune cells. The purified cells are grown on agar plates under conditions that maintain the viability of the cell. The cells that express < Required conformational structures of the target antigen are identified using conformationally dependent monoclonal antibodies that are known to react specifically with the same structure expressed on the target pathogen. The release of the relevant soluble protein from the cells is detected by incubation with monoclonal antibody, followed by a secondary reagent that gives a macroscopic signal (gold position, color development, fluorescence, luminescence). Cells expressing the maximum level of antigen can be identified by visual inspection, the cell or cell colony is removed and standard molecular biology methods are used to recover the plasmid DNA vaccine vector conferring this reactivity. Alternatively, flow cytometry can be used to identify and select cells harboring plasmids that induce high levels of gene expression. The enriched plasmid population that successfully expresses enough soluble factor in the correct body site for the desired time is then used for the initial population for another round of selection, incorporating the gene letdown to expand diversity, if further improvement is desired. . In this way, the desired biological activity encoded by the plasmid from the tissues in immunized animals is recovered with DNA vaccine. Several monoclonal antibodies, each of which is known to react with different conformational epitopes in the cytokine, chemokine or correctly folded growth factor, can be used to confirm that the initial result of selection with a monoclonal antibody reagent is still maintained with the Different conformational epitopes are probed.

In some cases the primary probe for the conformational cytokine released from the cell / cell colony on agar could be a soluble domain of the cognate receptor. (c) Flow Cytometry Flow cytometry provides a means to efficiently analyze the functional properties of millions of individual cells. The cells are passed through a zone of illumination, where they are hit by a laser beam; scattered light and fluorescence are analyzed by detectors linked to a computer. Flow cytometry provides several advantages over other methods for analyzing cell populations. Thousands of cells per second can be used, with a high degree of accuracy and sensitivity. A punishment of cell populations allows the analysis of multiple parameters of each sample. The size of the cell, viability, and morphology can be analyzed without the need for staining. When stained and labeled antibodies are used, the content of DNA, cell surface and intracytoplasmic proteins can be analyzed, and the cell type, activation status, cell cycle stage, and apoptosis detected. Up to four colonies (ie, four separate antigens stained with different fluorescent labels) and light scattering characteristics can be analyzed simultaneously (four colors require two laser instruments, one laser instrument can analyze three colors). The level of expression of the different genes can be analyzed simultaneously, and importantly, cell separation or classification based on flow cytometry ("FACS classification") allows the selection of cells with the desired phenotypes. Most libraries of vector modules, including the promoter, amplifier, intron, episomal origin of reproduction, appearance of the level of expression of the antigen, origin of the bacterium and bacterial marker, can be assayed by flow cytometry to select cells from individual human tissue culture containing the recombinant nucleic acid sequences that have the greatest improvement in the desired property. Typically, the selection is for a high level of expression of a surface antigen or substitute marker protein, as outlined schematically in Error! Reference source not found. The set of the best individual sequences is recovered from the selected cells by classification or separation based on cytometries. An advantage of this method is that very large numbers (> 107) can be evaluated in a single-bottle experiment. 2. In Vitro Separation Methods Vectors of genetic vaccines and vector modules can be separated by improved vaccination properties using various in vitro testing methods that are known to those skilled in the art. For example, the genetic vaccines used can be tested for their effect on the induction of the proliferation of a particular type of lymphocyte of interest, for example, B cells, T cells, T cell lines, and T cell clones. of separation for improved adjuvant activity and immunostimulatory properties can be carried out using, for example, human or mouse cells. A library of genetic vaccine vectors (obtained from randomized dormant DNA or from vectors harboring genes coding for cytokines, costimulatory molecules, etc.) can be separated for the production of cytokines (eg, IL-2, IL-4, IL -5, IL-6, IL-10, IL-12, IL-13, IL-15, IFN- ?, IFN-a) by B cells, cells T, monocytes / macrophages, total human PBMC, or whole blood (diluted). Cytokines can be measured by ELISA or cytoplasmic cytokine staining and flow cytometry (single-cell analysis). On the basis of the profile of cytokine production, alterations in the capacity of the vectors can be separated to direct the differentiation of TH1 / TH2 (according to what is evidenced, for example, by changes in the ratios of IL-4 / IFN-? , IL-4 / IL-2, IL-5 / IFN- ?, IL-5 / IL-2, IL-13 / IFN- ?, IL-13 / IL-2). 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), MHC class I and II, CD14, CD23, and Fc receptors, and the like. In some embodiments, gene vaccine vectors are analyzed for their ability to induce T cell activation. More specifically, spleen cells from injected mice can be isolated and the ability of cytotoxic T lymphocytes to lyse target cells can be studied. infected autologous Spleen cells are reactivated with the specific antigen in vi tro. In addition, the differentiation of T cells is analyzed by measuring the proliferation of the TH1 cytokines (IL-2 / IFN-?) And TH2 (IL-4 and IL-5) by ELISA and directly in CD4 + T cells by staining the Cytoplasmic cytokine and flow cytometry.

Genetic vaccines and vaccine components can also be tested for their ability to induce humoral immune responses, as evidenced, for example, by the induction of production of B-cell antibodies specific for an antigen of interest. These assays can be conducted using, for example, peripheral B lymphocytes from immunized individuals. Such test methods are known to those skilled in the art. Other assays involve the detection of antigen expression by the target cells. For example, selection by FACS provides the most efficient method for identifying cells that produce a desired antigen on the cell surface. Another advantage of the selection by FACS is that different levels of expression can be followed; sometimes the lowest expression may be desired. Another method involves separation using monoclonal antibodies on a plate. This method allows a large number of cells to be handled in a short time, but the method only selects the levels with the highest expression. The capture by magnetic fabrics coated with monoclonal antibodies provide another method to identify the cells that express a particular antigen. Genetic vaccines and components of the vaccine that are directed against cancer cells can be selected for their ability to inhibit the proliferation of tumor cell lines in vi tro. Such assays are known in the art. An indication of the effectiveness of a genetic vaccine against, for example, a cancerous or autoimmune disorder, is the degree of inflammation of the skin when the vector is injected into the skin of a patient or test animal. The strong inflammation correlates with a strong activation of antigen-specific T cells. Improved activation of tumor-specific T cells can lead to better tumor elimination. In the case of antioantigens, immunomodulators can be added that shift the responses towards TH2. Skin biopsies can be taken, allowing detailed studies of the type of immune response that occurs at the sites of each injection (a large number of injections / vectors can be analyzed in mice). Other suitable separation methods can detect the expression of changes in the expression of cytokines, chemokines, accessory molecules, and the like, by cells after challenge by a library by gene vaccine vectors. 3. Improved Entry of Vectors of Genetic Vaccines in Cells The method involves subjecting the polynucleotides that dormant the DNA that are involved in the entry to the cell. Such polynucleotides are referred to herein as "transfer sequences" or "transfer module". Transfer modules can be obtained that increase the transfer in a specific, cellular way or that act in a more general way. Because the exact sequences that affect the binding and transfer of DNA are not known, often the lethargy or dragging of DNA may be the only efficient method to improve the ability of DNA to enter the cytoplasm and subsequently the nucleus of cells human The methods involve recombining at least the first and second forms of a nucleic acid comprising a transfer sequence. The first and second forms differ among themselves in two or more nucleotides. Suitable substrates include, for example, transcription factor binding sites, CpG sequences, polynucleotides A, C, G, T, and random DNA fragments such as, for example, genomic DNA, human or other animal species. It has been suggested that cell surface proteins such as the 'macrophage scavenging receptor, can act as receptors for specific DNA binding (Pisetsky (1996) Immunity 5: 303). It is not known if these receptors recognize specific DNA sequences or if they bind to DNA in a sequence in a non-specific way. However, GGGG tetrads have been shown to increase DNA binding to cell surfaces (Id.). In addition to the DNA sequence, the three-dimensional structure of the plasmids may play a role in the ability of these plasmids to enter the cells. The methods of lethargy or entrainment of the DNA of the invention provide means to optimize such sequences by their ability to confer a vector the ability to enter a cell even in the absence of detailed information on the mechanism by which this effect is achieved. The library resulting from the recombinant transfer modules are separated to identify at least one optimized recombinant transfer module that increases the capacity of a vector comprising the transfer module to enter a cell of interest. For example, vectors that include a recombinant transfer module can be contacted with a population of cells under conductive conditions for entry of the vector into the cells, after which the percentage of cells 1? in the population that contains the nucleic acid vector. Preferably, the vector will contain a selectable or separable marker to facilitate identification of cells that contain the vector. In a preferred embodiment, the clonal isolates of the vectors containing the recombinant segments are used to infect separate cell cultures. The percentage of vectors that enter the cells can then be determined, for example, by counting the cells expressing a marker expressed by the vectors in the course of transfection. Typically, the recombinant process is repeated by recombining at least one optimized transfer sequence with an additional form of the transfer sequence to produce an additional library of recombinant transfer modules. The additional form may be the same or different from the first and second forms. The new library is separated to identify at least one additional optimized recombinant vector module that exhibits an improvement in the capacity of a genetic vaccine vector that includes the transfer module optimized to enter a cell of interest. The recombination and reseparation process can be repeated if necessary, until a transfer module having a sufficient capacity to improve the transfer is obtained. After one or more of the recombinations and separation, vector modules are obtained which are capable of conferring to a nucleic acid vector the ability to enter at least more than 50% of the target cells than a control vector not containing the optimized module, more preferably at least about more than 75 percent, and more preferably at least approximately more than 95 or 99 percent of the target cells that a control vector. Although for vaccine purposes, non-integral vectors are generally preferred, for some applications it may be desirable to use an integrating vector; for those applications the DNA sequences that directly or indirectly affect the integration efficiency can be included on the vector of the genetic vaccine. For integration by homologous recombination the important factors are the degree and length of the homology with the chromosomal sequences, as well as the frequency of such sequences in the genome (for example, Alu repeats). The homologous recombination that mediates the specific sequence is also important, since integration occurs more easily in the transcripcisnally active DNA. Methods and materials for constructing objective homologous constructs are described, for example, by Mansour (1988) Nature 336: 348; Bradley (1992) Bio / Technology 10: 534. For non-homologs, illegitimate and site-specific recombination is mediated by specific sites on the vector for therapy that interacts with the encoded recombinant proteins, for example, the Cre / Lox and Flp / Frt systems. See, for example, Baubonis (1993) Nucleic Acids Res. 21: 2025-2029, who report that a vector that includes a LoxP site is integrated into a LoxP site at the chromosomal site in the presence of a Cre recombinase enzyme.

C. Evolution of Union Polypeptides that Improve the Specificity and Efficiency of Genetic Vaccines The present invention also provides methods for obtaining recombinant nucleic acids encoding polypeptides which enhance the ability of genetic vaccines to enter target cells. Although the mechanisms involved in the absorption of DNA are not well understood, the methods of the invention allow to obtain genetic vaccines that exhibit better entry to cells, and to appropriate cellular compartments. In one embodiment, the invention provides methods for improving the efficiency and specificity of the uptake of a genetic vaccine nucleic acid by a given cell type by coating the nucleic acid with an evolved protein that binds to the nucleic acid of the genetic vaccine, also is able to join the target cell. The vector can be contacted with the protein in vi tro or in vivo. In the latter situation, the protein is expressed in cells that contain the vector, optionally of a coding sequence within the vector. The evolving nucleic acid binding proteins usually have nucleic acid binding activity but do not necessarily have any known ability to improve or alter the absorption of nucleic acid DNA. DNA binding proteins that can be used in those methods, include but are not limited to, transcriptional regulators, enzymes involved in the reproduction of DNA (eg, recA) and recombination of DNA, and proteins that serve for structural functions on DNA (for example, histones, protamines). Other DNA binding proteins that can be used include the repressor of phage 434, the repressor of phage lambda cl and ero, the Cap E. coli protein, myc, proteins with zippers of leucine and basic domains of DNA binding such as fos and jun; proteins with "POU" domains such as the paired Drosophila protein; proteins with domains whose structures depend on chelation with a metal ion such as zinc Cys2His2 found in TFIIIA, Zn2 (Cys) 6 clusters such as those in yeast Gal'4, the Cys His box found in retroviral nucleocapsule proteins , and the Zn2 (Cys) s clusters found in proteins of the nuclear hormone receptor type; p22 phage repressors Are and Mnt (see Knight et al (1989) J. Biol. Chem. 264: 3639-3642 and Bowie &Saur (1989) J. Biol. Chem 264: 7596-7602. RNA are reviewed by Burd &Dreyfuss (1994) Science 265: 615-621, and include the Tat and Rev of HIV.As other methods of the invention, the evolution of DNA binding proteins towards the acquisition of an efficiency Enhanced or altered absorption is effective in one or more cycles of recombination and separation Initial substances may be segments of nucleic acid encoding natural or induced variants of one or more nucleic acid binding proteins such as those mentioned above. The nucleic acid segments may be present in the vector or in isolates for the recombination step, recombination may proceed through any of the formats described herein. 8 For separation purposes, the recombinant nucleic acid segments are typically inserted into a vector, if they are not already present in such a vector during the recombination step. The vector generally codes for a selective marker that is capable of being expressed in the type of cell for which absorption is desired. If the evolving DNA binding protein recognizes a specific binding site (for example, the lacl binding protein recognizes lacO), this binding site can be included in the vector. Optionally, the vector can contain multiple cascade binding sites. Vectors containing different recombinant segments are transformed into a host cell, usually E. coli to allow the recombinant proteins to be expressed and bind to the vector coding for their genetic material. Most cells absorb only a single vector and thus the transformation results in a population of cells, most of which contain a single vector species. After an appropriate period to allow expression and binding, the cells are used under moderate conditions that do not disturb the binding of the vector to the DNA binding proteins. For example, a lysis buffer of HEPES 35 Mm (pH 7.5 with KOH). 0.1 mM EDTA, 100 mM Ma glutamate, 5% glycerol, 0.3 mg / ml BSA, 1 mM DTT, and 0.1 mM PMSF) plus lysozyme (0.3 ml to 10 mg / ml) is adequate (see Schatz et al. , Us 5,338,665). The vector binding protein and nucleic acid complexes are then contacted with cells of the type for which enhanced or altered absorption is desired under conditions that promote absorption. Suitable cells include the types of human cells that are common targets in DNA vaccination. Those cells include muscle cells, monocytes / macrophages, dendritic cells, B cells, Langerhans cells, keratinocytes, and M cells of the intestine. Mammalian cells including, for example, humans, mice and monkeys can be used for separation. The primary cells and the cells obtained from cell lines are suitable. After incubation, the cells are cultured with the selection for the expression of the selective marker present in the vector containing the recombinant segments. The cells expressing the marker are recovered. These cells are enriched in the recombinant segments that code for the nucleic acid binding proteins that increase the absorption of the vectors encoding the respective recombinant segments. The recombinant segments of the cells expressing the marker can then be subjected to an additional round of selection. Usually, the recombinant segments are first recovered from the cells, for example, by PCR amplification or by recovery of all vectors. The recombinant segments can then be recombined with one another or with other sources of DNA binding protein variants to generate additional recombinant segments. The additional recombinant segments are selected in the same manner as above. An example of a method for evolving an optimized nucleic acid protein involves the lethargy or entrainment of the histone genes. The DNA condensed with Histone may result in an increase in the transfer of the gene to the cells. See, for example, Fritz et al. (1996) Human Gene Therapy 7: 1395-1404. In this way, the lethargy or drag of the DNA can be used to evolve the histone protein, particularly the carboxy and amino-terminal peptide extensions, to increase the efficiency of DNA transfer to the cells. In this method, histone is encoded by the DNA to which it will bind. The histone library can be constructed, for example, 1) by lethargy or by dragging many of the related histone genes of a natural diversity, 2) the addition of random or partially random peptide sequences in the N- and C-terminal sequences of histone, 3) by the addition of preselected N-or C-terminal protein coding regions, such as complete cDNA libraries, nuclear protein ligand libraries, etc. These proteins can be randomized and partially linked to histone by a library of binders. In a variation of the above process, a binding site recognized by a nucleic acid binding protein can be evolved in place of, or as well as, the nucleic acid binding protein. The nucleic acid binding sites are evolved by means of a procedure analogous to nucleic acid binding proteins except that the initial substrates contain variable binding sites and the recombinant forms of those sites are separated as a component of the vector which also codes for a nucleic acid binding protein. The segments of nucleic acid that evolved, which code for the DNA binding proteins and / or the DNA binding sites that evolved can be included in gene vaccine vectors. If the affinity of the DNA binding protein is specific to a known DNA binding site, it is sufficient to include that binding site and the sequence encoding the DNA binding protein in the genetic vaccine vector together with such other coding and regulatory sequences as required to effect genetic therapy. In some cases, the DNA binding protein that evolved may not have a high degree of sequence specificity and may be unaware precisely which sites on the vector used in the separation are bound by the protein. In these circumstances, the vector should include all or most of the sequences of the separation vector together with the additional sequences required to effect the vaccination or therapy. The exemplary selection scheme employing the M13 VIII protein is shown in Figure 1. Target cells of interest include, for example, muscle cells, monocytes, dendritic cells, B cells, Langerhans cells, keratinocytes, M cells of the intestine, and similar. Cell-specific ligands that are suitable for use with each of the cell types are known to those skilled in the art. For example, proteins suitable for directing binding to antigen-presenting cells include the ligands CD2, CD28, CTLA-4, CD40 fibrinogen, factor X, ICAM-1, β-glycan (zymosan), and the Fc portion of the immunoglobulin G. (Weir's Handbook of Experimental Immunology, Eds. LA Herzenberg, DM Weir, LA Herzenberg, C. Blackwell, 5th edition, volume IV, chapters 156 and 174) because their respective ligands are present on APCs, including cells dendritic cells, monocytes / macrophages, B cells and Langerhans cells. The enterotoxins or bacterial subunits thereof are also of interest for management purposes. The ability of vectors to enter and activate APC, such as monocytes, can also be improved by coating the vectors in small amounts of lipopolysaccharide (LPS). This facilitates the interaction between the vector and the monocytes, which have a receptor on the cell surface for LPS. Due to its immunostimulatory activities, LPS probably also acts as an adjuvant, thus further enhancing immune responses. Enterotoxins produced by certain pathogenic bacteria are useful as agents that bind to cells and thus increase the release of vaccines, antigens, vectors for gene therapy and pharmaceutical proteins. In an exemplary embodiment of the invention, the receptor binding components of the enterotoxins' derived from Vibrio cholerae and enterotoxigenic strains of E. coli evolved to better binding to cell surface receptors and better entry to and transport through the cells. cells of the intestinal epithelium. In addition, they can evolve to a better binding to, and activation of, B cells different from APCs. An antigen of interest can be fused to these toxin subunits to illustrate the feasibility of the method in the oral delivery of proteins and to facilitate the separation of the subunits of enterotoxin that evolved. Examples of such antigens include growth hormone, insulin, myelin basic protein, collagen and viral envelope proteins. These methods involve recombining at least a first and second forms of a nucleic acid comprising a polynucleotide that encodes a non-toxic receptor binding portion, preferably an enterotoxin. The first and second forms differ from each other in two or more nucleotides, so that lethargy or DNA entrainment results in the production of a nucleic acid library from the binding portion of the recombinant enterotoxin. Suitable enterotoxins include, for example, an enterotoxin of V. cholerae, enterotoxins of enterotoxic strains of E. coli, Salmonella toxin, Shigella toxin and Campylobacter toxin. The vectors containing the nucleic acid library of the binding portion of recombinant enterotoxins are transfected into a population of host cells, where the nucleic acids of the binding portion of the recombinant enterotoxin are expressed to form the polypeptides of the binding portion. of the recombinant enterotoxin. In a preferred embodiment, the polypeptides of the binding portion of the recombinant enterotoxin are expressed as fusion enterotoxins on the surface of bacteriophage particles. The polypeptides of the binding portion of the recombinant enterotoxin can be separated by contacting the library with a receptor on the cell surface of a target cell and determining which polypeptides of the binding portion of the recombinant enterotoxin exhibit a greater capacity to bind to the receptor. of the target cell. The cell surface receptor may be present on the surface of a target cell itself, or it may be bound to a different cell, or the binding may be tested using a cell surface receptor that is not associated with a cell. Examples of suitable cell surface receptors include, for example, GM? - Similarly, super bacterial antigens can be evolved to alter (increase or decrease) receptor binding of T cells and MHC class II molecules. These super antigens activate the T cells in a non-specific form of the antigen. Antigens that bind to the T cell receptor / MHC class II molecules include enterotoxin B. Staphylococcal, the superantigen of Urtica dioica (Musette et al. (1996) Eur. J. Immunol. 26: 618-22) and enterotoxin A Staphylococcal (Bavari et al. (1996) J.

Infect. Dis. 174: 338-45). Phage display has been shown to be effective when selecting super antigens that bind MHC class II molecules (Wung and Gascoigne (1997) J. Immunol. Methods 204: 3-41). Cholera toxin (CT) is a protein • oligomeric of 84,000 daltons consisting of a toxic A subunit (CT-A) covalently linked to five B subunits (CT-B). CT-B functions as the receptor binding component and binds GMI ganglioside receptors on the surfaces of mammalian cells. The toxic A subunit does not necessarily serve CT function, and in the absence of CT-A, functional CT-B pentamers can be formed (Lebens and Holmgren (1994) Dev. Biol. Stand, 82: 215-227). Both CT and CT-B have been shown to have potent adjuvant activities in vivo and increase immune responses after oral administration of antigens and vaccines (Czerkinsky et al (1996) Ann. NY Acad. Sci. 778: 185 -93; Van Cott et al. (1996) Vaccine 14: 392-8). In addition, a single dose of CT and CT-B conjugated with myelin basic protein prevents the outbreak of autoimmune encephalomyelitis (EAE) is a murine model of multiple sclerosis (Czerkinsky et al., Supra.). In addition, the feeding of animals with myelin basic protein conjugated with CT-B after the onset of clinical symptoms (7 days) attenuated the symptoms in those animals. Other bacterial toxins, such as E. coli enterotoxins, Salmonella toxin, Shigella toxin and Campylobacter toxin, have structural similarities to CTs. E. coli enterotoxins have the same A-B structure as CTs since they also have sequence homology and share functional similarities. Bacterial enterotoxins can be evolved for improved affinity and entry to cells by lethargy or genetic entrainment. The similarity of the subunit of enterotoxin derived from E. coli and CT-B is 78%, and several completely conserved regions of more than eight nucleotides can be found. Subunits B of two different strains of E. coli are 98% homologous in both sequence and protein levels. In this way, it is feasible to carry the family DNA between the nucleic acids that code for the enterotoxin of different bacterial species. The libraries of the entrained toxin subunits can be expressed in a suitable host cell, such as V. cholerae. For safety reasons, strains in which toxic CT-A was suppressed are preferred. An antigen of interest can be fused to the receptor binding subunit. The secretion of chimeric proteins by V. cholerae can be selected by culturing the bacteria on agar in the presence of monoclonal antibodies specific for the antigen that was fused to the toxins and the level of secretion detected as immunoprecipitation in the agar around the colonies. Can GM ganglioside receptors also be added? to detect colonies that secrete functional enterotoxin subunits. Colonies that produce significant levels of the fusion protein are then cultured in 96-well plates, and the culture medium is tested for the presence of molecules capable of binding to cells or receptors in solution. The binding of chimeric fusion proteins to GM ganglioside receptors? on the cell surface or in solution can be detected by a monoclonal antibody specific for the antigen that was fused to the toxin. The assay using whole cells has the advantage that they can evolve to an improved binding also to receptors other than the GMi ganglioside receptor. When increasing concentrations of natural enterotoxins are added to these assays, mutants that bind to the receptors with better affinities can be detected. The affinity and specificity of the toxin binding can also be determined by surface plasmon resonance (Kuziemko et al (1996) Biochemistry 35: 6375-84). The advantage of the bacterial expression system is that the fusion protein is secreted by bacteria that could potentially be used in a large-scale production. further, because the fusion protein is in solution during selection, potential problems associated with phage display (such as the deviation towards mutant deviation that only works on the phage) can be avoided. However, phage display is useful for separation to identify enterotoxins with improved affinities. A library of carrion mutants can be expressed in phage, such as M13, and mutants with improved affinity are selected based on binding to, for example, ganglioside GMi receptors in solution or on a cell surface. The advantage of this method is that the mutants can be easily selected additionally in vivo assays as discussed below. A separation method using fusion to the M13 VIII protein is shown schematically in Figure 1. Finally, the resulting evolved enterotoxin can be fused with a DNA binding protein, and the genetic vaccine vectors coated with this protein fusion. The carrying of the DNA can be carried out separately, in which case the two domains are assembled after hauling, or in a combined reaction. Carrying results in the production of a nucleic acid library of the recombinant binding portion which can be selected by transfecting vectors which contain the library, as well as a specific binding site for the nucleic acid binding domain, in a population of host cells. The binding portion expressed in the cells and binds to the binding domain of the nucleic acid to 'form a complex vector-binding portion. The host cells can then be used under conditions that do not disturb the binding of the complex-binding portion. The vector-binding complex can then be contacted with a cell of interest, after which cells containing a vector are identified and the nucleic acids of the recombinant binding portion used are isolated from the cells. Another method for obtaining greater absorption of a target DNA with mammalian cells is also provided by the invention. Specifically, the method increases the number of copies of the target DNA absorbed in those cells that initially absorb the same DNA. The method uses cell surface expression of DNA-binding domains associated with the membrane of, for example, transcription factors, which are encoded in the DNA sequence "Objective, which also includes the recognition sequence of cognates for the binding domain. The Absorption of a target DNA molecule in a cell (by any process, passive absorption, electroporation, osmotic shock, other stresses) will drive the transcription of the gene encoding the polynucleotide binding domain. The gene encoding the binding domain is designed so that the binding domain is expressed in an anchored form to the membrane. For example, a hydrophobic amino acid fragment can be encoded at the carboxyl terminus of the binding domain, thereby leading to phosphoinositol-glycan (PIG) conjugation after partial incision of this terminal sequence. This, in turn, leads to the displacement and positioning of the binding domain on the cell surface. The same cells that absorbed the first DNA molecule will express the factor and have a greater specific affinity for the target DNA that remains extracellular. Cells that do not absorb DNA will be at a competitive disadvantage, since they do not contain the specific binding domain of the cell surface target DNA, which is required for the absorption of specifically mediated DNA. The greater binding of the target DNA to the target cell will increase the internalization efficiency of the DNA and the desired intracellular function. This process represents a positive feedback to increase the absorption of DNA towards the cells that absorbed DNA for the first time. The target DNA, wherein a circular or linear plasmid, oligonucleotide, bacterial or mammalian chromosomal fragment, is designed to contain one or more copies of a DNA recognition sequence for a factor of. transcription of mammal or bacteria. Many objective sequences will already contain one or more such motifs; those can be identified by sequence analysis. The endogenous motifs recognized by these factors can also be experimentally identified by demonstrating that the target DNA binds to one or more panels of transcription factors in an appropriate assay format. This provides practical means to determine which factor or combination of factors to use with any particular target DNA. In the case of a small oligonucleotide or a DNA plasmid (such as that used for the DNA vaccine), suitable motifs can be designed in the sequence. A particular pattern can be designed in one or more copies, cascaded or an objective sequence dispersed. Alternatively, a set of different, cascaded or separate motifs may be designed in case where more than one DNA binding protein will be expressed on the cell surface.

D. Evolution of Bacteriophage Vectors The invention provides methods for obtaining bacteriophage vectors that exhibit desirable properties for use as vectors of genetic vaccines. The principle behind the method provided by the invention is to combine the power of DNA carryover with the extraordinary power of bacteriophage genetics and the knowledge of recent advances in phage display technologies to rapidly evolve vehicles for highly genetic vaccines. innovative and powerful. Evolved vaccine vehicles can present antigens (1) natively on the surface of the APCs for the induction of an antibody response or (2) selectively invade the APCs and release DNA vaccine constructs at the APCs for intracellular expression, processing and presentation to the CTL. The most efficient methods to release pathogen antigens to professional APCs will increase the kinetics and potency of the immune response to the vaccine. Delivery vehicles or distribution of genetic vaccines that have evolved according to the methods of the invention are particularly valuable for the rapid induction of high affinity antibodies which can effectively neutralize viral epitopes or pathogenic toxins., such as superantigens or cholera toxin. High affinity antibodies are generated by somatic mutation of low affinity primary response antibodies. This so-called affinity maturation process is essential for the generation of antibodies with sufficient affinity to neutralize the pathogenic antigens. Affinity maturation occurs in the spleen at the germinal center where follicular dendritic cells (FDC), the professional antigen presenting cells, present protein antigens to B cells and processed antigen fragments to T cells. Clonally expanding cell populations B, those that have undergone somatic mutation of those mutant B cells that express antibodies with improved affinity for the antigen are selected. Thus, efficient release of the antigen to CDF will increase the kinetics and potency of the immune response to the immunizing antigen. Additionally, the processed antigen bound to the MHC is required to stimulate the antigen-specific T cells. Genetic vaccines are particularly efficient in priming the restricted MHC class I responses due to the intracellular expression of the antigen, with resultant trafficking of antigen fragments towards the MHC class I pathway. Thus, Invasive bacteriophage vectors capable of releasing genetic vaccine constructs or protein antigens to FDCs are useful. Any of several bacteriophages can evolve according to the methods of the invention. The preferred bacteriophages for these purposes are those that have been well characterized and genetically developed to present external protein epitopes; those include, for example, bacteriophage lambda, T7 and M13. Filamentous phage M13 is a particular preferred vector for use in the methods of the invention. M13 is a small filamentous bacteriophage that has been widely used to present polypeptide fragments in folded, functional form on the surface of bacteriophage particles. The polypeptides have been fused to both coat proteins of gene I and gene VIII for such presentation purposes. In this way, the M13 is a highly evolved, versatile vehicle for the efficient and targeted release of protein or DNA vaccine vehicles to target cells of interest. The following three properties are examples of the type of environments that can be achieved by using the methods of the invention to evolve bacteriophage gene vaccine vectors: (1) efficient delivery of the phage to the bloodstream by inhalation or oral administration, (2) efficient search for APCs; and (3) efficient invasion of target cells using entrained bacterial invasion proteins. Where M13 is used, fusions can be made to both coat proteins of gene III and gene VIII, so that the properties that evolve can be combined into a single phage particle. These studies can be carried out in test animals such as laboratory mice so that the constructs that have evolved can be rapidly characterized with respect to their potency as vaccine carriers. The inhalable and / or orally releasable vehicles that have evolved and the invasins that have evolved will be directly transferred for use in human cells, although the principles developed in evolution as the ability to direct them to APC from test animals are easily transferable to human cells making analogous selections about human APCs. Although these methods are exemplified by bacteriophage vectors, the methods are also applicable to other types of gene vaccine vectors. (1) Evolution of efficient release of bacteriophage vehicles by inhalation or oral delivery The invention provides methods for obtaining vectors of genetic vaccines that are capable of being released efficiently distributed to the bloodstream after administration by inhalation or by oral administration. The methods have been developed for the formulation of proteins in inhaled colloids that can be absorbed into the bloodstream through the lungs. The mechanisms by which proteins are transported into the bloodstream are not clearly understood, and thus improvements are easily exploited by evolutionary methods. Using M13 as an example, the invention involves the preparation of a library of, for example, peptide ligands, adhesion molecules, bacterial enterotoxins and randomly fragmented cDNA, which are fused to gene III, for example, of M13. Library of > 1010 individual mergers with this technology. The separation involves the preparation of patterns with a high titer (preferably> 1012 phage particles) in standard colloidal formulations, which are released or administered intranasally to test animals, such as mice. The blood samples are taken during the course of the next day and the circulating phages are amplified in E. coli. It has been established that M13 circulates for prolonged periods in the blood after being injected intravenously, and thus it is reasonable to expect that the phage that successfully enters the blood flow through the lung can be efficiently recovered and amplified in E. cells. coli In a preferred embodiment, several rounds of enrichment are applied to the initial libraries to enrich the phage that can efficiently enter the bloodstream when it is released intranasally. The candidate clones are typically individually tested to determine their relative input efficiency, and the best clones can be further characterized by sequencing to identify the nature of the fusion conferring efficient release (of particular interest to the cDNA libraries). ). The selected clones can be further evolved to improve entrainment of the whole phage genome and subjecting the phage to repeated cycles of release, recovery, amplification and entrainment. An analogous procedure was used to obtain vaccine vectors that are effective when released or orally administered. A genetic vaccine vector library was prepared by dragging the DNA. The recombinant vectors were packaged and administered to a test animal. Vectors that are stable in the stomach / intestinal environment were recovered, for example, by recovering the surviving vectors of the stomach. Vectors that efficiently enter the bloodstream and / or lymphatic tissue can be identified by recovering the vectors that reach the blood / lymph. An outline of this selection method is shown in Figure 2. (2) Evolution of bacteriophage vehicles for efficient targeting of APCs The invention also provides methods for evolving bacteriophage vectors, as well as other types of gene vaccine vectors, for efficient targeting of professional antigen presenting cells. The libraries of random peptide ligands and cDNAs used in (A) above were enriched in phage, which binds selectively to APCs by first selecting negatively for binding to non-APC cell types, and then positively selecting for binding to the APC. The selection is typically carried out by mixing high-titer phage patterns of the libraries (> 1012 phage particles) with cells (~107 cells per selection cycle) and taking the unbound phage (negative selection) or the bound phage from the cellular sediments (positive selection). An alternative selection format consists of injecting phage libraries intravenously, allow the library to circulate for several hours, collect the target organs of interest (lymph nodes, spleen), and release the phage by sonication. The positively selected phage can be amplified in E. coli and additional rounds of enrichment are made (3-5 rounds) if further optimization is desired. After choosing the number of rounds, individual phages are characterized by their ability to target lymphoid organs. The best few Candidates can undergo further evolution through iterated rounds of selection, amplification and entrainment. (3) Evolution of the bacteriophage for the invasion of APC The methods of the invention are also useful for evolving the bacteriophage and other vehicles of genetic vaccines for the invasion of target cells. This opens up the possibility of directing the MHC class I antigen processing pathways with internalized protein antigen or antigen expressed by cDNA vaccine vehicles carried by the evolved vector. The invasins comprise a large family of bacterial proteins, which interact with the integrins and promote the efficient internment of pathogenic bacteria such as Salmonella.

This embodiment of the invention involves displaying different forms of polynucleotides that encode for invasins. For example, two or more genes that code for the invasin family can be dragged. The entrained polynucleotides can be cloned as fusions to the coat protein gene of M13 gene VII, for example, high titer patterns of such libraries will be prepared. These bacteriophage libraries can be mixed with the target APC. After incubation, the cells are thoroughly washed to remove the free phage and the phage bound to the surfaces of the cells can be removed by separation in a tray against polyclonal anti-M13 antibodies. The cells are then sonicated, thereby releasing the phages that have successfully entered the target cells (thus protecting them from the anti-M13 polyclonal antiserum). These phages can, if desired, be amplified, dragged and the selective cycle will be applied interactively for example 3-5 times. The individual phage of the final cycle can then be characterized with respect to its relative invasiveness. The best candidates can then be combined with gene III fusions that code for the pathogenic epitopes of interest. That phage can be injected into mice and tested to determine their relative abilities to induce a CTL response to pathogenic antigens. Bacteriophage vaccine vehicles that evolved to have activity in mice according to the above methods will establish the principles for the evolution of similar vehicles for potent human vaccines. The ability to induce more rapid and potent CTL and neutralizing antibody responses with such vehicles is a new important tool for the evolution of improved countermeasures against the pathogens of interest.

Pharmaceutical Compositions of Genetic Vaccines and Administration Methods Release or delivery vehicles, vectors of engineered genetic vaccines, and components of the vector of the invention are useful for treating and / or preventing various diseases and other conditions. For example, genetic vaccines employing reagents obtained according to the methods of the invention are useful both in the prophylaxis and in the therapy of infectious diseases, including those caused by any bacteria, fungi, viruses or other pathogens of mammals. . The reagents obtained using the invention can also be used for the treatment of autoimmune diseases including, for example, rheumatoid arthritis, SLE, diabetes mellitus, myasthenia gravis, reactive arthritis, ankylosing spondylitis and multiple sclerosis. These and other inflammatory conditions, including IBD, psoriasis, pancreatitis, and various immunodeficiencies, can be treated using genetic vaccines that include vectors and other components obtained using the methods of the invention. Vectors of genetic vaccines and other vectors obtained using the methods of the invention can be used to treat allergies and asthma. In addition, the use of genetic vaccines is very promising for the treatment of cancer and the prevention of metastasis. By inducing an immune response against cancer cells, the body's immune system can be listed to reduce or eliminate cancer. In the preferred modalities so far, the reagents obtained using the invention are used in conjunction with a genetic vaccine. The choice of vector and components can also be optimized for the particular purpose of treating allergies or other conditions. For example, a ^ antigen for a particular condition can be optimized using methods of analogous recombination and selection those described herein. Such methods and antigens suitable for various conditions are described in U.S. Patent Application. Commonly assigned Serial No., entitled "Immunization of Antigenic Library," which was filed on February 10, 1999 as No. of Proxy File No. 18097-02871US. The polynucleotide encoding the recombinant antigenic polypeptide can be placed under the control of a promoter, for example, a high activity or specific tissue promoter. The promoter used to express the antigenic polypeptide can itself be optimized using methods of recombination and selection analogous to those described herein, as described in International Application No. PCT / US97 / 17300 (International Publication No. WO 98/13487). The vector may contain immunostimulatory sequences such as those described in U.S. Patent Application. copendiente, commonly assigned, Serial No., titled "Optimization of Immunomodulatory Molecules", presented as Proxy File No. "TTC 18097-030300US" on February 10, 1999. Reagents obtained using the methods of the invention can also be used in conjunction with multi-component gene vaccines, which are capable of designing an immune response as is most appropriate to achieve a desired effect (see, for example, the co-pending US Patent Application, commonly referred to as No.

Series, entitled "Vaccine Vector Design Genetics ", filed on February 10, 1999 as TFP File No. 18097-030100US) It is sometimes advantageous to employ a genetic vaccine that targets a particular type of target cell (e.g., an antigen-presenting cell or an antigen-presenting cell). an antigen processing cell), the appropriate methods of management are described in the commonly assigned co-pending US patent application Serial No., entitled "Vaccine Vectors Address Genetics ", filed on February 10, 1999 as TTC File No. 18097-030200US.Genetic vaccines and delivery or delivery vehicles as described herein can be released to a mammal (including humans) to induce an immunotherapeutic response or The vaccine release vehicles can be released in vivo by administration to an individual patient, typically by systemic administration (eg, by the intravenous, intraperitoneal, intramuscular, subdermal, intracranial, anal, vaginal, oral, buccal routes or can inhaled) or can be delivered by topical application Alternatively, the vectors can be released into ex vivo cells, such as explanted cells from an individual patient (eg, lymphocytes, bone marrow aspirates, tissue biopsies) or hematopoietic germ cells from universal donors, followed by the reimplantation of the cells in a This is usually after the selection of the cells that have incorporated the vector. A large number of release methods are well known to those skilled in the art. Such methods include, for example, the release of genes based on liposomes (Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6 (7): 682-691; Rose Patent U.S. No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et al. (1987) Proc. Nati Acad. Sci. I USA 84: 7413-7414, as well as the use of viral vectors (eg, adenovirals (see, for example, Berns et al. (1995) Ann. NY Acad. Sci. 772: 95-104; Ali et al. al. (1994) Gene Ther.1: 367-384; and Haddada et al. (1995) Top Curr. Microbiol. Immunol. 199 (Pt 3): 297-306 for a review) papillomavirales, retrovirals (see, for example, "Buchscher et al. (1992) J. Virol. 66 (5) 2731- 2739; Johann et al. (1992) J. Virol. 66 (5): 1635-1640 (1992); Sommerfelt et al. , (1990) Virol. 176: 58-59; Wilson et al. (1989) J. Virol. 63: 2374-2378; Miller et al., J. Virol. 65: 2220-2224 (1991); Wong-Staal et al. , PCT / US94 / 05700, and Rosenburg and Fauci (1993) in Fundamental Immunology, Paul Third Edition (ed) Raven Press, Ltd., New York and the references in it, and Yu et al., Gene Therapy (1994) supra.), And adeno-associated viral vectors (See West et al. (1987) Virology 160: 38-47; Carter et al. (1 * 989) US Patent No. 4,797,368; Carter et al., WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5: 793-801; Muzyczka (1994) J. Clin.Invest.94: 1351 and Samulski (supra) for an overview of AAV vectors, see also, Lebkowski, US Patent No. 5,173,414; Tratschin et al. 1985) Mol Cell Cell Biol. 5 (11): 3251-3260; Tratschin et al. (1984) Mol. Cell. Biol., 4: 2072-2081; Hermonat and Muziczka (1984) Proc, Nati, Acad. Sci. USA, 81: 6466-6470; McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol., 63: 03822-3828), and the like. The "naked" DNA and / or RNA comprising a genetic vaccine can be introduced directly into a tissue such as a muscle. See, for example, USPN 5,580,859. Other methods such as "biolistics" or particle-measured transformation (see, for example, # Sanford et al., USPN 4,945,050; USPN 5,036,006) are also suitable for the introduction of genetic vaccines into cells of a mammal according to the invention. These methods are useful not only for the in vivo introduction of DNA in a mammal, but also for the ex vivo modification of cells for reintroduction into a mammal. As for other methods of releasing genetic vaccines, if necessary, administration of the vaccine is repeated to maintain the desired level of immunomodulation. Genetic vaccine vectors (e.g., adenoviruses, liposomes, papillomaviruses, retroviruses, etc.) can be administered directly to the mammal for transduction of the cells in vivo. Genetic vaccines obtained using the methods of the invention can be formulated as pharmaceutical compositions for administration in any suitable manner, including palenteral (e.g., subcutaneous, intramuscular, intradermal or intravenous), topical, oral, rectal, intrathecal, buccal administration. (for example, sublingual), or local administration, such as by aerosol or transdermally, for prophylactic and / or therapeutic treatment. Pretreatment of the skin, for example, by the use of hair or hair removal agents, can be useful in transdermal delivery. 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 can be used to administer a particular composition, a particular route often provides a more immediate and more effective reaction. what another route. The pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method of administering the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention. A variety of aqueous carriers, eg, buffered saline 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. pharmaceutically acceptable auxiliaries as required for approximate physiological conditions, such as pH adjusting agents and buffers, agents for adjusting toxicity and the like, eg, sodium acetate, sodium chloride, potassium chloride, calcium chloride Sodium lactate and the like The concentration of the genetic vaccine vector in those formulations can vary widely, and will be selected mainly on the basis of the volumes and viscosities of the fluids, body weight and the like according to the particular mode of administration selected. and the needs of the Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sacks or tablets, each containing a predetermined amount of the active ingredient, such as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. The tablet forms may include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, stearate of magnesium, stearic acid and other excipients, colorants, fillers, binders, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrating agents and pharmaceutically compatible carriers. The tablet forms may comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as tablets comprising the active ingredient in an inert base, such as gelatin and glycerin emulsions or sucrose and acacia, gels and the like containing , 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 typically achieved by complexing the vaccine vector with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the vector in an appropriate resistant carrier, such as a liposome. Means for protecting 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. Nucleic acids packaged alone or in combination with other suitable components can be made in aerosol formulations (for example, they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed in acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen and the like. Formulations suitable for rectal administration include, for example, suppositories, which consist of the nucleic acid packaged with a suppository base. Suitable suppository bases include synthetic triglycerides or paraffinic hydrocarbons. In addition, it is also possible to use rectal gelatin capsules, which consist of a combination of the nucleic acid packaged with a base including, for example, liquid triglycerides, polyethylene glycols and paraffinic hydrocarbons. Formulations suitable for parenteral administration, such as, for example, intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, include sterile, isotonic aqueous and non-aqueous injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes to make the formulation isotonic with the intended recipient's blood, and sterile aqueous and non-aqueous suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. In the practice of this invention, the compositions may be administered, for example, by intravenous, oral, topical, intraperitoneal, intravesicular or intrathecally infusion. Parenteral administration and intravenous administration are the preferred methods of administration. Packaged nucleic acid formulations can be presented in a sealed container with unit doses or multiple doses, such as ampoules and flasks. The injection solutions and suspensions can be prepared with sterile powders, granules, and tablets of the type described above. The cells transduced by the packaged nucleic acid can also be administered intravenously or parenterally. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial therapeutic response to the patient over time. The dose will be determined by the efficacy of a 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 degree of any adverse side effects that accompany the administration of a particular vector, or a cell type transduced to a particular patient. The determination of the effective amount of the vector to be administered in the prophylaxis treatment of an infection or other condition, the physician assesses the toxicity of the vector, progress of the disease, and the production of anti-vector antibodies if any. In general, the equivalent dose of a naked nucleic acid from a vector is from about 1 μg to 1 mg for a typical 70 kilogram patient, and the doses of the vectors used to deliver the nucleic acid is calculated to produce an equivalent amount of therapeutic nucleic acid. The administration can be carried out via a single dose or divided dose. In therapeutic applications, the compositions are administered to a patient suffering from a disease (eg, an infectious disease or autoimmune disorder) in an amount sufficient to cure or at least partially counteract the disease and its complications. An adequate amount to achieve this is defined as "therapeutically effective dose". The effective amounts for this use will depend on the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions can be made depending on the doses and efficacy required and tolerated by the patient. In any case, the composition should provide a sufficient amount of the proteins of this invention to effectively treat the patient. In prophylactic applications, the compositions they 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 invention are determined using standard therapeutic methods in cell cultures or experimental animals. The LD50 (lethal dose for 50% of the population) and ED50 (the therapeutically effective dose in 50% of the population) can be determined using the procedures presented herein or those in other circumstances known to those skilled 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 0.1 to approximately 100 mg per patient per day can be used, particularly when the drug is administered to a programmed site and not to the blood flow, such as in a body cavity or in the lumen of an organ. Substantially higher doses are possible in topical administration. The actual methods for preparing parenterally administrable compositions will be known or apparent to 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 antigenic polypeptides of the invention, and the genetic vaccines that express the polypeptides, can be packaged in packages, devices, distributors and equipment for administering genetic vaccines to a mammal. For example, distribution packages or devices that may contain one or more unit dosage forms are provided. Typically, instructions for the administration of the compounds will be provided by the package, along with an adequate indication on the label that the compound is suitable for the treatment of an indicated condition. For example, the label may establish that the active compound within the package is useful for treating a particular infectious disease, autoimmune disorder, tumor, or for preventing or treating other diseases or conditions that are mediated by, or are potentially susceptible to, a response immune of mammal. Example 1 Improvement of Bacterial Enterotoxin Properties by DNA Drag This Example describes the use of DNA entrainment methods to evolve the enterotoxin receptor binding components derived from Vibrio cholerae and E. coli enterotoxic strains for binding Improved cell surface receptors and for improved entry and transport through intestinal epithelial cells. An antigen of interest can be provided to those toxin subunits to facilitate the separation of the subtypes of enterotoxin that have evolved, and also to facilitate the oral release of proteins. Examples of such antigens include growth hormone, insulin, myelin basic protein, collagen and viral envelope proteins. Bacterial enterotoxins are evolved to an affinity and improved entry to cells by genetic entrainment. The similarity of the subunit of the enterotoxin derived from E. coli to the cholera toxin CT-B is 78%, and several fully conserved regions of eight nucleotides are present. An alignment of the DNAs encoding CT-B and the subunits of enterotoxin B from two strains of E. coli is shown in Figure 3 to illustrate the feasibility of family DNA tracing. In one embodiment, the libraries of the entrained toxin subunits are expressed in V. cholerae. For safety reasons, strains were used in which the toxic CT-A was removed. An antigen of interest was fused to the receptor binding subunit. The secretion of chimeric proteins by V. cholerae can be separated by culturing the bacteria on agar in the presence of monoclonal antibodies specific for the antigen that was fused to the toxins, and detecting the level of secretion as immunoprecipitation in the agar around the colonies. In addition, GMi ganglioside receptors can also be added to the agar to detect colonies that secrete functional enterotoxin subunits. Colonies that produce significant levels of the fusion protein are then cultured in 96-well plates, and the culture medium is tested for the presence of molecules capable of binding to cells or receptors in solution. The binding of chimeric fusion proteins to ganglioside GMi receptors on the cell surface or in solution can be detected by means of a monoclonal antibody specific for the antigen that was fused to the toxin. The assay using whole cells has the advantage that they can be evolved to improve binding also to other receptors than the GM ganglioside receptor? <; When increasing concentrations of natural enterotoxins are added to these assays, mutants that bind to receptors with improved affinities can be detected. Enterotoxins with improved affinities can also be separated using phage display methods. A library of carrion mutants can be expressed on phage, such as M13, and mutants with improved affinity are selected based on binding to the ganglioside GMi container in solution or on cell surfaces. The advantage of this method is that the mutants can be further selected easily in in vivo assays as discussed below. The separation of the improved oral release of vaccines and proteins can be carried out both in vi tro and in vivo. The in vitro method is based on Caco-2 cells (human colon adenocarcinoma) that are cultured in tissue culture. When grown on semipermeable filters, those cells spontaneously differentiate into cells that resemble epithelium of the human small intestine both structurally and functionally (Hilgers et al (1990) Pharm. Res. 7: 902-910). The harvested toxin recombinants, fused to an antigen of interest, are placed on top of this cell layer and the beneficial mutants are detected by measuring the level of antigen transport through the cell layer. Both mutants expressed in bacteria and phage can be selected using this method. Alternatively, and additionally, the mutants are separated in vivo. When expressed on a phage, the library of entrapped enterotoxin recombinants can be separated by their better entry to the intestinal epithelium and blood flow after oral delivery. This separation system also allows the selection of mutants with the most potent adjuvant activities. The advantage of using the phage is that a large amount of phage can be given and the successful mutants can be recovered and used in subsequent rounds of trawling and selection.

Example 2 Generation and Transfection of Human Dendritic Cells; Evaluation of the Vectors that were Optimized for Those Cells The dendritic cells are the most potent antigen presenting cells known to date. This example illustrates the feasibility of using dendritic cells to separate vectors of genetic vaccines with improved properties, including efficient transfection, expression of antigens, stability, ability to present antigens. Figure 4A demonstrates the phenotype of freshly isolated monocytes and after a culture period of seven days in the presence of IL4 (400 U / ml) and GM-CSF (100 ng / ml). Cultured cells were negative for CD14, although they expressed CDla, HLA-DR, CD40, CD80 and CD86, which is a characteristic phenotype of dendritic cells (Chapuis et al. (1997) Eur. J. Immunol., 27: 431 -441). The cultured dendritic cells were then transfected with a vector encoding GTP driven by a CMV promoter. As shown in Figure 4B, the efficiency of the transfection of those cells is very low. However, a small percentage (~ 1%) of the cells expressed low levels of GFP two days after transfection under the conditions shown in the figure. These data illustrate the need for improvements in the efficiency of transfection of human dendritic cells. Very little is known about the mechanism that regulates the efficiency of transfection and transgenetic expression in dendritic cells, or how it can be improved. Therefore, DNA dragging is an ideal method, because it does not depend on an a priori assumption of the mechanisms that limit the process. The cultured dendritic cells described in this example provide the ability to select vector libraries as described here elsewhere.

Example 3 Selection of Release Vehicles Derived from Bacteriophages Which Have Greater Ability to Enter Target Cells This example describes a protocol for the use of phage display to select polypeptides that can enter dendritic cells, for example, by endocytosis mediated by the receptor. A library of recombinant polynucleotides obtained by recombination of a nucleic acid binding domain and a ligand for a dendritic cell receptor is expressed in a phage display format. The phage display library is incubated with dendritic cells for a period of time, after which the cells are washed (typically multiple washes are performed using highly concentrated saline to remove the remaining extracellular phage.) The cells are then cultured and sonicated to release the phages that have been internalized.The phages that are released are then amplified in E. coli, and the polynucleotide encoding the recombination binding portion used is obtained.If desired, the optimized polynucleotide is subjected to recombination additional to obtain additional optimization.

In a variation of this scheme, a phagemid encoding both the recombinant ligand and a selectable or separable marker (e.g., a gene encoding the green fluorescent protein operably linked to a CMV promoter) can be used. The cells that have absorbed the phage can then be identified by placing the culture under selective conditions, or by methods such as fluorescence-activated cell selection.

EXAMPLE 4 Animal Model for Separating Vectors from Genetic Vaccines This example provides a mouse model system that is useful for separating and testing vectors of genetic vaccines in human skin in vivo. Human skin pieces were xenotransplanted on the back of SCID mice. Pieces of human skin can be obtained from infants subjected to circumcision, from skin removal operations due to, for example, cosmetic reasons, or from patients who experienced amputation due to, for example, accidents. These pieces are then transplanted onto the backs of CB-17 scid / scid (SCID) mice as described by others (Deng et al. (1997) Na ture Biotechnology 15: 1388-1391; Khavari et al. (1997) Adv.

Clin. Res. 15: 27-35; Qhoate ^ and Khavari (1997) Human Gene Therapy 8: 895-901). The vector libraries are selected, for example, after topical application to the skin. However, in an analogous manner, depending on the optimal immunization route, evolved vectors may also be selected after i.m., i.v., oral, anal or vaginal release. The DNA released into the skin can be in the form of a patch, in the form of a cream, in the form of naked DNA or a mixture of DNA and agent to increase transfection (such as proteases, lipases or lipids / liposomes), and can Apply after mechanical abrasion, after removal of hair or hair, or simply by adding a drop of DNA or a mixture of DNA-lipid / liposome on the skin. Similar release methods are applied to small animals, such as mice or rats, large animals, such as cats, dogs, cows, horses or monkeys, as well as humans. Suitable proteases and lipases that increase release include, but are not limited to, individual components or mixtures of the following: a protease (such as Alcalase or Savinase) with or without alpha-amylase, a lipase (such as Lipolase) ( Sarlo et al (1997) J. Allergy, Clin Immunol., 100: 480-7).

The recovery of the optimal vectors can be effected from transfected cells, for example, by PCR, or by recovering whole vectors. Vectors can be selected based purely on their ability to enter cells or by selecting only cells expressing the antigen encoded by the vector in normal mice, monkeys or SCID mice transplanted with human skin. For example, GFP can be used as a marker gene, and after the release to detect the cells that were transfected by fluorescence microscopy or flow cytometry. Positive cells can be isolated, for example, by flow cytometry based on cell separation. This format allows the selection of vectors that optimally express antigens in and transfect human cells in vivo. Additionally, they can be separated into mice by selecting vectors that are capable of inducing effective immune responses after release into the skin. Vectors that induce more specific antibodies or CTL responses can be selected, or can be selected based on the induction of the protective immune response after challenge with the corresponding pathogen. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that they will suggest various modifications or changes in light thereof to those skilled in the art and that are included within the spirit and point of view of this application and scope of application. the attached claims. All publications, patents and patent applications cited herein are therefore incorporated by reference for all purposes. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (50)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for obtaining a cell-specific binding molecule useful for increasing the absorption or specificity of a genetic vaccine to a target cell, the method is characterized in that it comprises: creating a library of recombinant polynucleotides, recombining from a nucleic acid encoding a polypeptide comprising a nucleic acid binding domain and a nucleic acid encoding a polypeptide comprising a specific binding domain of a cell; and selecting the library to identify a recombinant polynucleotide that encodes a binding molecule that can bind to a nucleic acid and a specific receptor of a cell. 2. A method for obtaining a specific binding portion of a cell useful for increasing the absorption or specificity of a genetic vaccine by a target cell, the method is characterized in that it comprises: (1) recombining at least one first and second forms of a nucleic acid comprising a polynucleotide encoding a nucleic acid binding domain and at least the first and second forms of a nucleic acid comprising a ligand specific for a cell that specifically binds to a protein on the surface of a cell interest, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of the nucleic acids encoding the recombinant binding portion; (2) transfect a library of vectors to a population of host cells, each of which comprises: a) a specific binding site for the nucleic acid binding domain and 2) a member of the nucleic acid library that encode for the recombinant binding portion, wherein the recombinant binding portion is expressed and binds to the binding site to form a vector-binding portion complex; (3) using the host cells under conditions that do not disrupt the binding of the vector-binding portion complex; (4) contacting the vector-binding portion complex with a target cell of interest; and (5) identifying the target cells containing a vector and isolating the nucleic acids from the specific binding portion of a recombinant cell, optimized, from those target cells. The method according to claim 2, characterized in that the method further comprises: (6) recombining at least one nucleic acid encoding the optimized recombinant binding portion with a further form of the polynucleotide encoding a binding domain of nucleic acid and / or a further form of the polynucleotide encoding a cell-specific ligand, which are the same or different from the first and second forms, to produce an additional library of the nucleic acids encoding the recombinant binding; (7) transfecting in a population of host cells a library of vectors comprising: a) a binding site specific for the binding domain of the nucleic acid and 2) the nucleic acids encoding the binding portion, recombinants, where the recombinant binding portion is expressed and binds to the binding site to form a vector-binding portion complex; (8) using the host cells under conditions that do not disrupt the binding of the vector-binding portion complex; (9) contacting the vector-binding portion complex with a target cell of interest and identifying the target cells containing the vector; and (10) isolating the nucleic acids from the recombinant binding portion of the target cells containing the vector; and (11) repeating (6) through (10), as necessary, to obtain a specific, optimized cell binding portion useful for increasing the uptake or specificity of a genetic vaccine vector by a target cell. 4. The method according to claim 2, characterized in that the method further comprises identifying specific binding portions of a cell that result in greater efficiency in the transfection of the target cells. 5. The method according to claim 2, characterized in that the nucleic acid binding domain is a DNA binding domain derived from a protein selected from the group consisting of a transcriptional regulator, a polypeptide involved in the reproduction or recombination of the DNA, a repressor, a histone, a protamine, a CAP protein from E. coli, myc, a protein that has a leucine zipper, a protein that has a basic DNA binding domain, a protein that has a POU domain, a protein that has a zinc finger, and a protein that has a box of Cys3His. 6. The method according to claim 2, characterized in that the nucleic acid binding domain is an RNA binding domain derived from a protein selected from the group consisting of HIV tat and HIV rev. The method according to claim 2, characterized in that the target cell of interest is selected from the group consisting of muscle cells, monocytes, dendritic cells, B cells, Langerhans cells, keratinocytes and M cells. according to claim 7, characterized in that the cell of interest is a professional antigen presenting cell. The method according to claim 8, characterized in that the antigen presenting cell is a dendritic cell, a monocyte / macrophage, a B cell or a Langerhans cell. The method according to claim 8, characterized in that the specific ligand of the cell comprises a polypeptide selected from the group consisting of the ligands CD2, CD28, CTLA-4, CD40, fibrinogen, ICAM-1 Fc portion of the immunoglobulin G, and a bacterial enterotoxin, or a subunit thereof. 11. The method according to claim 2, characterized in that the target cell of interest is a human cell. The method according to claim 2, characterized in that the target cells containing the vector are identified by selecting the expression of a selectable label contained in the vector. The method according to claim 2, characterized in that the nucleic acid encoding the recombinant binding portion comprises a genetic vaccine vector. 14. A recombinant portion of cell-specific binding, characterized in that it is produced by expressing in a host cell a nucleic acid encoding the used recombinant binding portion obtained by the method according to claim 2. 15. A genetic vaccine, characterized in that it comprises a specific recombinant binding portion of a cell according to claim 14. 16. A genetic vaccine, characterized in that it comprises a nucleic acid encoding the optimized, recombinant binding potion obtained in accordance with claim 2. 17. A genetic vaccine, characterized in that it comprises: a) an optimized recombinant binding portion comprising a nucleic acid binding domain and a specific ligand of a cell, and b) a polynucleotide sequence comprising a binding site, wherein the domain nucleic acid binding is able to bind specifically to the union site. 18. A method for obtaining a specific binding portion of an optimized cell useful for increasing the absorption, efficacy, or specificity of a genetic vaccine for a target cell, the method is characterized in that it comprises: (1) recombining at least the first and second forms of a nucleic acid comprising a polynucleotide that encodes a non-toxic receptor binding portion of an enterotoxin, where the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant nucleic acids; (2) transfecting the vectors containing the nucleic acid library in a population of host cells, wherein the nucleic acids are expressed to form polypeptides of the specific binding portion of the recombinant cell; (3) contacting polypeptides of the specific binding portion of the recombinant cell with a receptor on the cell surface of a target cell; Y (4) determining which polypeptides of the specific binding portion of the recombinant cell exhibit a greater ability to bind to the target cell. 19. The method according to claim 18, characterized in that the cell surface receptor is present on the surface of a target cell. 20. The method according to claim 18, characterized in that the cell surface receptor is GM- ?. 21. The method according to claim 18, characterized in that the host cell is a V. cholerae cell which is unable to express CT-A. 22. A method for increasing the uptake of a genetic vaccine vector by a target cell, the method is characterized in that it comprises coating the vector of the genetic vaccine with a recombinant, optimized, specific cell binding portion, produced by the method according to claim 18. 23. The method according to claim 18, characterized in that the cell-specific binding portions, recombinants, are expressed as a fusion protein on the surface of a reproducible genetic package. 24. A method for obtaining a genetic vaccine component that confers a vector a greater capacity to enter the antigen-presenting cell, the method is characterized in that it comprises: creating a library of recombinant nucleic acids by recombining at least two forms of a polynucleotide; contacting a library of vectors, each of which comprises a member of the recombinant nucleic acid library, with a population of antigen-presenting or antigen-processing cells; and determine the percentage of cells in the population that contain the vector. The method according to claim 24, characterized in that the antigen-presenting or antigen-processing cells are selected from the group consisting of B cells, monocytes / macrophages, dendritic cells, Langerhans cells, keratinocytes, and muscle cells. 26. The method according to claim 25, characterized in that the cells are B cells, which are obtained from a B cell line. The method according to claim 24, characterized in that the separation or selection is conducted in live and the cells are monkey cells or mouse cells. 28. The method according to claim 24, characterized in that the method further comprises: culturing the cells for a predetermined time after contacting the cells with the vector library; washing the cells after the contact step to remove the vectors that did not enter an antigen-presenting cell; and isolate vectors from cells that contain a vector. 29. The method according to claim 24, characterized in that cells containing a vector are identified: transfecting individual members of the library or sets of members of the library in separate cultures of antigen cells; cocultivating the cultures of the antigen presenting cells with T lymphocytes obtained from the same individuals as the antigen-presenting cells; and identifying the cultures in which a T lymphocyte response was induced. The method according to claim 29, characterized in that the response of the T lymphocytes is selected from the group consisting of an increase in the proliferation of the lymphocytes. T, increased cytolytic activity mediated by T lymphocytes against a target cell, and an increase in cytosine production. 31. The method according to claim 24, characterized in that the vector is a reproducible genetic package and the recombinant nucleic acids are expressed as a fusion protein, which is presented on the surface of the reproducible genetic package. 32. The method according to claim 31, characterized in that the reproducible genetic package is a bacteriophage. 33. A method to obtain a component of a genetic vaccine that confers a vector an increased ability to enter cells or tissues when * administered to a mammal by means of a desired administration protocol, the method is characterized in that it comprises: creating a library of recombinant nucleic acids by recombining at least two forms of a polynucleotide; administering to a mammal "a library of vectors, each comprising a member of the library of recombinant nucleic acids, in the mammal; obtain target cells or tissues of the mammal; identify target cells or tissues that contain a vector; and recovering the vector of the identified target cells or tissues. 34. The method according to claim 33, characterized in that the target cells are lymphatic cells. 35. The method according to claim 33, characterized in that the administration is by oral ingestion, inhalation, injection, or topical application to the skin or mucous membrane. 36. The method according to claim 33, characterized in that the vector is a reproducible genetic package and the recombinant nucleic acids are expressed as a fusion protein, which is presented on the surface of the reproducible genetic package. 37. A method for evolving a vaccine delivery vehicle to obtain an optimized delivery vehicle having a greater capacity to enter a selected mammalian tissue following administration to a mammal, the method is characterized in that it comprises: (1) recombining members of a set of polynucleotides to produce a library of recombinant polynucleotides; (2) administering to a test animal a library of reproducible genetic packets, each comprising a member of the library of recombinant polynucleotides operably linked to a polynucleotide encoding a display polypeptide, wherein the recombinant polynucleotide and the polypeptide of presentation are expressed as a fusion protein, which is presented on the surface of the reproducible genetic package; and (3) recovering the reproducible genetic packets that are present in the selected tissue of the test animal at an appropriate time after administration, where the retrieved, reproducible genetic package has a greater ability to enter the selected mammalian tissue following administration of the mammal . 38. The method according to claim 37, characterized in that the method further comprises: (4) recombining a nucleic acid comprising at least one recombinant polynucleotide obtained from a reproducible genetic package recovered from the selected tissue with an additional set of polynucleotides for produce an additional library of recombinant polynucleotides; (5) administering to a test animal a library of reproducible genetic packets, each of which comprises a member of the additional library of recombinant polynucleotides operably linked to a polynucleotide encoding a display polypeptide, wherein the recombinant polynucleotide and the presentation polypeptide is expressed as a fusion protein, which is presented on the surface of the reproducible genetic package; (6) recover reproducible genetic packets that are present in the selected tissue of the test animal at an appropriate time after administration; and (7) repeating (4) to (6), as necessary to obtain an additional optimized recombinant release vehicle that exhibits enhanced ability to enter a selected mammalian tissue following administration to a mammal. 39. The method according to claim 37, characterized in that the reproducible genetic package is a bacteriophage. 40. The method according to claim 39, characterized in that the bacteriophage is M13. 41. The method according to claim 40, characterized in that the polynucleotide encoding a presentation polypeptide is selected from the group consisting of gene II and gene VIII. 42. The method according to claim 37, characterized in that the selected mammalian tissue is blood flow and the administration is by inhalation. 43. The method according to claim 37, characterized in that the intravenous administration and the selected mammalian tissue is selected from the group consisting of lymph nodes and spleen. 44. A method for evolving a vaccine delivery vehicle to obtain an optimized delivery vehicle having greater specificity for antigen presenting cells, the method is characterized in that it comprises: (1) recombining the members of a set of polynucleotides to produce a library of recombinant polynucleotides; (2) producing a library of reproducible genetic packets, each comprising a library member of recombinant polynucleotides operably linked to a polynucleotide encoding a display polypeptide, wherein the recombinant polynucleotide and the display polypeptide are expressed as a fusion protein which is presented on the surface of the reproducible genetic package; (3) contacting the library of recombinant reproducible genetic packets with a non-APC to remove reproducible genetic packets that display specific non-APC fusion polypeptides; and (4) contacting recombinant reproducible genetic packets that do not bind to non-APC with an APC and recover those that bind to the APC, where the retrievable reproducible genetic packets are able to specifically bind to the APCs. 45. The method according to claim 44, characterized in that the method further comprises the steps of: (5) recombining a nucleic acid, which comprises at least one recombinant polynucleotide obtained from a reproducible genetic package that is capable of binding specifically to APC with an additional set of polynucleotides to produce an additional library of recombinant polynucleotides; (6) producing an additional library of recombinant reproducible genetic packets, each comprising a member of the library of recombinant polynucleotides operably linked to a polynucleotide encoding a display polypeptide, wherein the recombinant polynucleotide and the presentation polypeptide are expresses as fusion proteins, which is presented on the surface of the reproducible genetic package; (7) contacting the additional library of recombinant reproducible genetic packages with a non-APC to remove those that exhibit non-APC-specific fusion polypeptides; and (8) contacting the recombinant reproducible genetic packets that did not bind to non-APC with an APC and recover the reproducible genetic packets that bind to the APC, where the retrievable reproducible genetic packets are capable of specifically binding to APC; and (9) repeating (5) to (8), as necessary, to obtain an additional optimized recombinant release vehicle that exhibits greater specificity for antigen-presenting cells. 46. A method for evolving a vaccine delivery vehicle to obtain an optimized vaccine delivery vehicle having a greater capacity to enter a target cell, the method is characterized in that it comprises: (1) recombining at least the first and second forms of a nucleic acid encoding an invasin polypeptide, wherein the first and second forms differ from each other in two or more nucleotides, to produce a library of recombinant invasin nucleic acids; (2) producing a recombinant bacteriophage library, each of which has on the surface of the bacteriophage a fusion polypeptide encoded by a chimeric gene comprising a recombinant invasin nucleic acid operably linked to a polynucleotide encoding a presentation polypeptide; (3) contacting the recombinant bacteriophage library with a population of target cells; (4) removing the unbound phage and the phage that is bound to the surface of the target cells; and (5) recovering the phages that are present within the target cells where the recovered phages are rich in phage that have a greater capacity to enter the target cells. 47. The method according to claim 46, characterized in that the method further comprises: (6) recombining a nucleic acid which comprises at least one bacteriophage recombinant invasin nucleic acid, which is recovered from a target cell with a additional set of polynucleotides to produce an additional library of recombinant invasin polynucleotides; (7) producing an additional library of recombinant bacteriophage, each of which displays on the surface of the bacteriophage a fusion polypeptide encoded by a chimeric gene comprising a recombinant invasin nucleic acid operably linked to a polynucleotide encoding a presentation; (8) contacting the recombinant bacteriophage library with a population of target cells; (9) removing the unbound phage and the phage that bound to the surface of the target cells; and (10) recovering the phages that are present within the target cells; and (11) repeating (6) to (10), as necessary, to obtain an additional optimized recombinant release vehicle which exhibits greater capacity to enter the target cells. 48. The method according to claim 47, characterized in that the method further comprises: (12) inserting into the optimized recombinant delivery vehicle a polynucleotide, which codes for an antigen of interest, where the antigen of interest is expressed as a fusion polypeptide, which comprises a second polypeptide of presentation; (13) administering the delivery vehicle to a test animal; and (14) determining whether the delivery vehicle is capable of inducing a CTL response in the test animal. 49. The method according to claim 47, characterized in that the method further comprises: (12) inserting into the optimized recombinant release car a polynucleotide, which codes for an antigen of interest, where the antigen of interest is expressed as a fusion polypeptide, which comprises a second polypeptide of presentation; (13) administering the delivery vehicle to a test animal; and (14) determining whether the delivery vehicle is capable of inducing neutralizing antibodies against a pathogen which comprises the antigen of interest. 50. The method according to claim 46, characterized in that the target cell is an APC.