AU1387299A - Expression plasmids for multiepitope nucleic acid-based vaccines - Google Patents

Description

WO99/24596 PCT/US98/23745 1 DESCRIPTION Expression Plasmids For Multiepitope Nucleic Acid-Based Vaccines 5 Statement of Related Applications This application is related to U.S. Patent Application entitled "IL-12 Gene Expression and Delivery Systems and Uses", filed October 10, 1997, Serial No. not yet assigned, by Jeff Nordstrom, Bruce Freimark and Deepa Deshpande, Lyon 10 & Lyon Docket No. 226/285 and U.S. Patent Application entitled "Gene Expression and Delivery Systems and Uses", filed October 10, 1997, Serial No. not yet assigned, by Jeff Nordstrom, Bruce Freimark and Deepa Deshpande, Lyon & Lyon Docket No. 226/284, both of which are incorporated herein by 15 reference in their entirety, including any drawings. Introduction The invention relates generally to gene therapy, in particular, the invention relates in part to improved 20 plasmids and methods for nucleic acid based vaccines. Background of the Invention The following discussion of the background of the invention is merely provided to aid the reader in 25 understanding the invention and is not admitted to describe or constitute prior art to the present invention. Plasmids are an essential element in genetic engi neering and gene therapy. Plasmids are circular DNA molecules that can be introduced into bacterial cells by 30 transformation which replicate autonomously in the cell. Plasmids allow for the amplification of cloned DNA. Some plasmids are present in 20 to 50 copies during cell growth, WO99/24596 PCT/US98/23745 2 and after the arrest of protein synthesis, as many as 1000 copies per cell of a plasmid can be generated. (Suzuki et al., Genetic Analysis, p. 404, (1989).) Current non-viral approaches to human gene therapy 5 require that a potential therapeutic gene be cloned into plasmids. Large quantities of a bacterial host harboring the plasmid may be fermented and the plasmid DNA may be purified for subsequent use. Current human clinical trials using plasmids utilize this approach. (Recombinant DNA 10 Advisory Committee Data Management Report, December, 1994, Human Gene Therapy 6:535-548). Studies normally focus on the therapeutic gene and the elements that control its expression in the patient when designing and constructing gene therapy plasmids. Generally, therapeutic genes and 15 regulatory elements are simply inserted into existing cloning vectors that are convenient and readily available. Plasmid design and construction utilizes several key factors. First, plasmid replication origins determine plasmid copy number, which affects production yields. 20 Plasmids that replicate to higher copy number can increase plasmid yield from a given volume of culture, but excessive copy number can be deleterious to the bacteria and lead to undesirable effects (Fitzwater, et al., EMBO J. 7:3289-3297 (1988); Uhlin, et al., Mol. Gen. Genet. 165:167-179 (1979)). 25 Artificially constructed plasmids are sometimes unstably maintained, leading to accumulation of plasmid-free cells and reduced production yields. To overcome this problem of plasmid-free cells, genes that code for antibiotic resistance phenotype are included 30 on the plasmid and antibiotics are added to kill or inhibit plasmid-free cells. Most general purpose cloning vectors contain ampicillin resistance (P-lactamase, or bla) genes.

WO99/24596 PCT/US98/23745 3 Use of ampicillin can be problematic because of the potential for residual antibiotic in the purified DNA, which can cause an allergic reaction in a treated patient. In addition, P-lactam antibiotics are clinically important for 5 disease treatment. When plasmids containing antibiotic resistance genes are used, the potential exists for the transfer of antibiotic resistance genes to a potential pathogen. Other studies have used the neo gene which is derived 10 from the bacterial transposon TnS. The neo gene encodes resistance to kanamycin and neomycin (Smith, Vaccine 12:1515-1519 (1994)). This gene has been used in a number of gene therapy studies, including several human clinical trials (Recombinant DNA Advisory Committee Data Management 15 Report, December, 1994, Human Gene Therapy 6:535-548). Due to the mechanism by which resistance is imparted, residual antibiotic and transmission of the gene to potential pathogens may be less of a problem than for P-lactams. In addition to elements that affect the behavior of the 20 plasmid within the host bacteria, such as E. coli, plasmid vectors have also been shown to affect transfection and expression in eukaryotic cells. Certain plasmid sequences have been shown to reduce expression of eukaryotic genes in eukaryotic cells when carried in cis (Peterson, et al., Mol. 25 Cell. Biol. 7:1563-1567 (1987); Yoder and Ganesan, Mol. Cell. Biol. 3:956-959 (1983); Lusky and Botchan, Nature 293:79-81 (1981); and Leite, et al., Gene 82:351-356 (1989)). Plasmid sequences also have been shown to fortu itously contain binding sites for transcriptional control 30 proteins (Ghersa, et al., Gene 151:331-332 (1994); Tully and Cidlowski, Biochem. Biophys. Res. Comm. 144:1-10 (1987); and Kushner, et al., Mol. Endocrinol. 8:405-407 (1994)). This WO99/24596 PCTIUS98/23745 4 can cause inappropriate levels of gene expression in treated patients. Nucleic acid vaccines, or the use of plasmid encoding antigens, has become an area of intensive research and 5 development in the last half decade. Comprehensive reviews on nucleic acid vaccines have recently been published [M.A. Liu, et al.(Eds.), 1995, DNA Vaccines: A new era in vaccinology, Vol. 772, Ann. NY. Acad. Sci., New York; Kumar, V., and Sercarz, E., 1996, Nat. Med. 2:857-859; 10 Ulmer, J.B., et al., (Eds.) Current Opinion in Immunology; 8:531-536. Vol. 772, Ann. NY. Acad. Sci., New York]. Protective immunity in an animal model using plasmid encoding a viral protein was first observed in 1993 by Ulmer et al. [Ulmer, J.B., et al., 1993, Science 259:1745-17491. 15 Since then, several studies have demonstrated protective immunity for several disease targets and human clinical trials have been started. Summary 20 The use of epitopes, small immunologically relevant protein sequences that are capable of inducing both cellular and humoral responses that result in a protective or therapeutic immune response against large and complex pathogens, is an attractive and amenable strategy provided 25 by the present invention for incorporation into nucleic acid-based vaccines. If multiple epitopes are expressed in the context of a synthetic gene construct, immunity against many antigenic targets, multiple strain variants or multiple pathogens is possible. This disclosure describes the 30 structures and characteristics of gene expression systems that are capable of expressing multiple epitopes.

WO99/24596 PCTIUS98/23745 5 Thus, in one aspect the invention provides a method of genetic immunization comprising the step of presenting multiple epitopes to an organism in need of said immunization. 5 In preferred embodiments, the multiple epitopes are presented with one or more augmenting cytokines and/or are presented with a delivery vehicle selected from the group consisting of cationic lipids, delivery peptides, and polymer based deliver systems. 10 In another aspect, the invention features a plasmid for expression of multiple epitopes comprising a nucleic acid sequence encoding multiple epitopes, wherein each of said epitopes is capable of creating an immune response. In preferred embodiments, the plasmid includes a 15 promoter, a 5' UTR sequence, and a 3' UTR sequence, a nucleic acid sequence encoding polyubiquitin, there are spacers between the nucleic acid regions encoding each of said epitopes, there are proteolytic cleavage sites between each of said epitopes, there are ER targeting signals between 20 each of said epitopes, there are lysosomal and/or endosomal targeting sequences between each of said epitopes. In other aspects, the invention provides a multivalent expression system as shown in Figure 8 and selected from the group consisting of two plasmids, two genes, IRES, and 25 alternative splicing and a method of making a plasmid producing the appropriate nucleic acid sequence. The summary of the invention described above is non limiting and other features and advantages of the invention will be apparent from the following detailed description of 30 the preferred embodiments, as well as from the claims.

WO99/24596 PCT/US98/23745 6 Brief Description of The Drawings Figure 1 shows a plasmid for multiple epitopes using a beads on a string approach. Figure 2 shows a plasmid for multiple epitopes using 5 beads on a string fused to polyubiquitin. Figure 3 shows a plasmid for multiple epitopes using beads on a string with spacers between epitopes. Figure 4 shows a plasmid for multiple epitopes using beads on a string with proteolytic cleavage sites between 10 epitopes. Figure 5 shows a plasmid for beads on a string epitopes with ER targeting sequences. Figure 6 shows a plasmid for multiple epitopes with ER targeting sequences. 15 Figure 7 shows a plasmid for multiple epitopes with lysosomal/endosomal targeting sequences. Figure 8 shows types of multivalent expression systems. Figure 9 shows a DNA vaccine expression plasmid with two genes. 20 Figure 10 shows a design of a drug-controlled DNA vaccine expression plasmid. Detailed Description of the Preferred Embodiments Various exemplary plasmids and methods for multiepitope 25 nucleic acid based vaccines are described below. The following explanation of the invention is to aid in understanding various aspects of the invention. The following explanation does not limit the operation of the invention to any one theory. 30 WO99/24596 PCT/US98/23745 7 I. Expression Plasmid for Epitopes Arranged as Beads-on-a String In expression plasmids of this type, the multiple epitopes are directly linked to each other. No spacer 5 sequences between the epitopes are included. The epitope sequences themselves are sufficient for the formation of a functional "pseudo" protein that can be processed into individual peptide epitopes via proteosome cleavage. This concept, i.e. beads-on-a-string, is supported by data that 10 shows full CTL responses to numerous epitopes when they are placed into novel locations within different proteins (Nomura M, Nakata Y, Inoue T et al., J. Immunol. Methods, 193:41-9 (1996) and Weidt G, Deppert W, Buchhop S, Dralle H, Lehmanngrube F., J. Virol., 69:2654-8 (1995)). 15 An and Whitton, J. Virol., 71:2292-302 (1997) have described that a beads-on-a-string approach is feasible with a recombinant vaccinia virus vector. They appear to have demonstrated that a linear array of B-cell, CTL and Th epitopes was able to induce the corresponding immune 20 response. Gilbert et al., Nature Bio., 15:1280-84 (1997) has demonstrated that the beads-on-a-string approach is feasible with a recombinant Ty-VLP vector. They described that a linear array of 15 defined malaria epitopes induced protective CTL responses in mice, and that neither epitope 25 order nor flanking sequences influenced the processing of the epitopes. Multiple epitopes expressed from a recombinant vaccinia virus vector as a string of 10 contiguous minimal CTL epitopes, which were restricted by five MHC alleles and 30 derived from five viruses, a parasite, and a tumor model, induced a primary CTL response in vivo in the appropriate mouse strain. This illustrates that multiple CTL epitopes WO99/24596 PCT/US98/23745 8 can be effectively delivered in a beads-on-a-string array (Thomson et al., J. Immunol., 157:822-6 (1996)). The present invention provides an exemplary expression system for beads-on-a-string as shown below (See Figure 1): 5 Promoter / 5' UTR / intron / AUG / (Epitope)n / stop codon / 3' UTR / poly(A) signal Table I below provides a description of each of these genetic elements. Table I. Description of genetic elements. 10 Element Description Promoter CMV, tissue-specific (e.g. APC specific), or synthetic promoter 5' UTR Optimized to assure mRNA stability and 15 translatability. Current optimal sequences are UT11 (from human loricrin gene) or UT12 (from CMV). Intron Synthetic intron that has optimized 5' ss, 3' ss and branch point sequences. 20 Current optimal sequence is IVS 8. Initiation codon AUG is placed in the context of the Kozak sequence to ensure optimal initiation of translation. (Epitope)n String of epitopes, each having a length 25 of 9-10 amino acid residues in length for class I presentation, or >10 amino acid residues for class II presentation. It appears that at least 15 epitopes may be strung together. One of the main 30 considerations will be to avoid the placement of glycine or proline adjacent to the desired epitope termini.

WO99/24596 PCTILUS98/23745 9 Stop codon For termination of translation. To ensure efficient termination, it is desirable to string two stop codons in tandem. 5 3' To ensure efficient processing of the UTR/poly(A) mRNA an efficient poly(A) signal, such signal as from human growth hormone, is required. 10 II. Expression Plasmid for Multiple Epitopes as Beads-on-a String Linked to a Polvubiquitin Chain Degradation of many eukaryotic proteins requires their prior ligation to polyubiquitin (Ub) chains, which target the substrates to the 26S proteasome, an abundant cellular 15 protease. Thus, it is advantageous to encode a Ub chain that is fused to the N-terminus of the multiple epitopes that are arranged as beads-on-a-string. Expression plasmids with Ub fused to the antigen have been used to achieve class I presentation (Gueguen and Long, Proc. Natl. Acad. Sci. USA 20 93:14692-97 (1996)). Illustrated below is an expression system for beads-on a-string fused to polyubiquitin (Ub) (See Figure 2): Promoter / 5' UTR / intron / AUG / Ub / (Epitopes-Spacer)n / 25 stop codon / 3' UTR / poly(A) signal III. Expression Plasmid for Multiple Epitopes as Beads-on-a String with Spacers Between Epitopes An expression system for beads-on-a-string with spacers 30 is shown below (See Figure 3): Promoter / 5' UTR / intron / AUG / (Epitopes-Spacer)n / stop codon / 3' UTR / poly(A) signal WO99/24596 PCT/US98/23745 10 Some investigators have shown that flanking sequence can profoundly influence the generation of epitopes (Yellenshaw et al., J. Immunol., 158:1727-33 (1997); Shastri et al., J. Immunol., 155:4339-46 (1995); Del-Val et al., 5 Cell, 66:1145-93 (1991); Eggers et al., J. Exp. Med. 182:1865-70 (1995); Niedermann et al. Immunity, 2:289-95 (1995); Lippolis et al. J. Virol. 69:3134-46 (1995)). In particular, glycine or proline residues adjacent to the minimal epitope should to be avoided, since peptide bonds to 10 these residues are known to be resistant to protease activity (Niedermann et al., 1995). Spacers may facilitate the formation of epitopes that induce immunity. Ideally, the spacer sequence should be one that does not conform at all to the rules for class I or II 15 epitope. However, it may be desirable to include a hydrophobic, basic or acidic residue at the C-terminus of the spacer to facilitate cleavage between the spacer and the adjacent epitope. The length of the spacer that would be optimal is not known and would have to be determined 20 empirically. IV. Expression Plasmid for Multiple Epitopes as Beads-on-a String with Proteolytic Cleavage Sites Between Epitopes An expression system for beads-on-a-string with 25 proteolytic cleavage sites is diagramed in summary form below (See Figure 4): Promoter / 5' UTR / intron / AUG / (Epitopes-Cleavage Site)n / stop codon / 3' UTR / poly(A) signal 30 Many viruses (e.g. retroviruses, flaviviruses) generate mRNAs that encode polyproteins that must undergo proteolytic cleavage to form the mature protein products. Cleavage, which occurs at specific sites, is catalyzed by host WO 99/24596 PCT/US98/23745 11 proteinases or by virally encoded proteinases. For example, the polyprotein from hepatitis C virus is structured as follows: H2N-C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. Host cell signal peptidases cleave the junctions in the 5 region between C and NS2. The viral proteinase (NS2-3 proteinase)cleaves the junction between NS2 and NS3. Another viral proteinase (NS3 proteinase) cleaves the junctions between NS3 and NS5B. One approach is to insert host cell cleavage sites 10 between the epitope sequences. This may be achieved by insertion of the sequences that are located at the junctions between the C-El-E2-p7-NS2 proteins of the hepatitis polyprotein. Other possibilities are to utilize the recognition site for specific cellular proteases. 15 A second approach is to insert cleavage sites for the viral proteinase between the epitope sequences. Thus, the site for the sequence recognized by the NS3 proteinase, Asp/Glu-X-X-X-X-Cys/ThriSer/Ala (Koch and Bartenschlager, Virology, 237:78-88 (1997)), may be inserted. However, for 20 this approach to work, the NS3 proteinase must be also encoded by the expression plasmid. Thus, two transcription units are required, one for the multiepitopes, one for the viral proteinase. See Section V. Multivalent expression plasmids for nucleic acid-based vaccines. 25 V. Expression Plasmids for Multiple Epitopes With Targeting The classical pathway for antigen presentation in the context of class I involves the partial degradation of 30 antigenic proteins into peptides by the proteasome. The peptides are then transported into the endoplasmic reticulum by peptide transporters (TAP-1 and TAP-2). It is within the WO99/24596 PCT/US98/23745 12 lumen of the ER or cis-Golgi that the peptides are loaded into the binding pocket of the MHC class I molecules. Ciernik et al., J. Immnunol. 2369-75 (1996) have demonstrated that the immunogenicity of an epitope may be enhanced if the 5 epitope sequence is fused in frame with the adenovirus E3 leader sequence and expressed from a plasmid delivered by particle bombardment. An epitope fused to the E3 leader yielded greater protection from tumor challenge than an epitope without the leader. 10 ER targeting signals have also been used by Overwijk et al., Identification of a Kb-restricted Ctl Epitope of Beta galactosidase: Potential Use in Development of Immunization Protocols for "Self" Antigens, Methods 12: 117-23 (1997). The signal sequence allows the epitope to be targeted to the ER 15 by the standard protein translocation process. The N terminal leader sequence targets the peptide to the ER by first binding, as a nascent sequence, to the 54 kDa subunit of the SRP particle. The leader sequence subsequently binds to the b-subunit of the membrane-bound transporter protein, 20 Sec61p. Following entry into the lumen of the ER through a putative channel, a peptidase cleaves the peptide to remove the leader sequence. This mechanism is independent of the TAP transporter system. This alternative mechanism may be advantageous if epitope formation by proteasome cleavage, or 25 epitope transport by the TAP system, are limiting steps in antigen presentation. For ER targeting, a leader sequence preferably needs to be attached to the N-terminus of each epitope. Adding a leader sequence to the multiple epitopes that are arranged 30 as beads-on-a string concept is unlikely to work, since the leader will be attached only to the first epitope sequence.

WO99/24596 PCT/US98/23745 13 Placing an individual targeting sequence on each of the epitopes that is arranged in a bead-on-a-string assembly is a possibility. However, this strategy will depend on accurate proteolytic cleavage at the N-terminus of the 5 leader sequence and at the C-terminus of the adjoined epitope sequence. A gene expression system that utilizes alternative splicing will yield individual epitopes with their own leader sequences. The peptide epitopes produced by this strategy will not depend on random degradation of a 10 protein precursor. The only processing that is required is N-terminal processing that is associated with protein translocation. The C-terminal ends of the epitopes are defined by the stop codons that are designed into the system. The preprotein 15 products may be incompletely synthesized until protein translocation through the pore into the ER has occurred. Alternatively, the prepeptides may be synthesized in their entirety prior to ER translocation. This may expose the prepeptide to the proteasome and transport of proteins that 20 transport epitopes to the ER by the standard pathway. A. Expression Plasmid for Multiple Epitopes with ER Targeting Sequences An expression system for beads-on-a-string epitopes 25 with targeting sequence is shown below (See Figure 5): Promoter / 5' UTR / AUG / ER signal sequence / epitope-1 / stop codon / ER signal sequence / epitope-2 / stop codon / ER signal sequence / epitope-3 / stop codon / 3' UTR/poly(A) signal 30 An alternative splicing system for multiple epitopes with targeting sequence is shown below (See Figure 6): Promoter / 5' UTR / AUG / ER signal sequence / 5'ss / internal intron sequence / 3'ss-1 / epitope-1 / stop codon-1 WO99/24596 PCTIUS98/23745 14 / 3'ss-2 / epitope-2 / stop codon-2 / 3'ss-3 / epitope-3 / stop codon-3 / 3' UTR/poly(A) signal Table II below describes the genetic elements used in the alternative splicing strategy. 5 Table II. Description of genetic elements for the alternative splicing strategy. Element Description 10 ER Signal The N-terminal leader sequence from sequence 5'ss adenovirus E3 or preprolactin Strong 5' splice site, one that exactly matches the consensus sequence. Such a sequence is found in the synthetic 15 intron, IVS8. Internal This sequence is derived from the intron synthetic intron, IVS8. It extends from sequence the 3' end of the 5' splice site to the 5' end of the polypyrimidine tract of 20 the 3'splice site. Alternative 3' ss-1, 3' ss-2, 3' ss-3: These splice 3' splice sites sites will be designed to be used equally. Thus, their relative strengths need to be mathed. This will be 25 accomplished by introducing purines within the polypyrimidine regions of the splice site sequences. In the alternative RNA splicing system, the strengths 30 of the 3' splice sites must be balanced to splicing from the 5'ss to each of the 3' splice sites (3'ss-1, to 3'ss-2, to 3'ss-3, etc.). Balanced splicing will be achieved by controlling the purine content of the pyrimidine-rich WO99/24596 PCT/US98/23745 15 sequences of the 3' splice sites. In general, the greater the purine content, the weaker the splice site. There are model systems to follow. For example, the major late transcript of adenovirus is alternately spliced into 5 5 families of transcripts that are produced in roughly equivalent amounts. Thus, one way to design an appropriate alternatively spliced system for epitopes is to model the 3' splice sites of adenoviral late transcripts. Another key feature is that, after splicing, the leader 10 sequence must be fused in frame with the peptide sequence of each epitope. Also, by altering the strengths of the 3' splice sites, the relative amounts of the epitopes may be varied. This may important if certain epitopes are more dominant than others. 15 Table III below shows an example of a balanced set at 3' splice sites derived from the adenoviral late transcript. Table III. Example of a balanced set of 3' splice sites (derived from the adenoviral late transcript) WO 99/24596 PCT/US98/23745 16 5' ss Source Alternative 3' ss i / Source 2/ CAGkGTAAGT IVS8 TTTGCTTTTCCCCAGIG Ad2 (11039) Consensus 5' ss) 5 TTGTATTCCCCTTAGPT Ad2 (14149) GTTGTATGTATCCAG4C Ad2 (16515) GTAACTATTTTGTAG4A Ad2 (17999) CCATGTCGCCGCCAG4-A Ad2 (18801) ATGTTTTGTTTGAAG4T Ad2 (21649) 10 TTCCTTCTCCTATAG4G Ad2 (24094) 1/ The consensus sequence for a 3'ss is YYYYYYYYYYYNYAGG. Y = C or T, and 4 = intron/exon junction. 15 2/ Adenovirus 2 (Ad2) sequences are from the Genbank entry, ADRCG. Numbers in parentheses indicate the nucleotide position of each 3' splice site. Note the locations of the purines (A or G) that interrupt the polypyrimidine (C or T) region.

WO99/24596 PCT/US98/23745 17 B. Expression Plasmid for Multiple Epitopes with Endosomal/Lysosomal Targeting Sequences An expression system structure is shown below (See Figure 7): 5 Promoter / 5' UTR / AUG / ER signal sequence / 5'ss / internal intron sequence / 3'ss-i / epitope-i / LAMP-1 transmembrane-cytoplasmic tail / stop codon-1 / 3'ss-2 / epitope-2 / LAMP-1 transmbrane-cytoplasmic tail / stop codon-2 / 3' ss-3 / epitope-3 / LAMP-1 transmbrane 10 cytoplasmic tail / stop codon-3 / 3' UTR/poly(A) signal To target class II antigen presentation, it may be desirable to directly target the peptide epitope to the endosomes or lysosomes. One strategy employs the transmembrane and cytoplasmic tail sequences from a one of 15 the lysosomal-associated membrane glycoproteins, such as LAMP-1. Wu et al., (1995) have used such a sequence, in combination with an N-terminal ER targeting sequence, to target an antigen to the endosomal and lysosomal compartments for class II antigen presentation. Thus, each 20 epitope is preceded by an N-terminal leader sequence (e.g. adenovirus E3) and followed by the C-terminal endosomal/lysosomal targeting sequence (e.g. the transmembrane and cytoplasmic tail region of LAMP-1). Another sequence that may be employed for endosomal 25 targeting is the cytoplasmic tail of membrane immunoglobulin (Weiser et al., Science 276:407-9 (1997); Achatz et al., Science 276:409-11 (1997)). Since the transmembrane/cytoplasmic tail can be added to some, but not necessarily all epitopes, it would be 30 possible to target some epitopes to the ER for class I presentation and others to the endosomes/lysosomes for class II presentation.

WO99/24596 PCT/US98/23745 18 VI. Multivalent Expression Plasmids for Nucleic Acid-Based Vaccines For effective nucleic acid-based vaccines, it may be important to have the capability of expressing multiple gene 5 products. For example, expression of multiple intact antigens or multiepitope gene product may enhance the potency of the these vaccines. Co-expression of costimulatory proteins, such as IL-2, IL-6, IL-12, GM-CSF, B7.1 or B7.2, have been demonstrated to enhance the immune 10 response to an encoded antigen (Geissler et al., J. Immunol. 158:1231-1237 (1997), Irvine et al., (1996); Kim et al., Vaccine 15:879-83 (1997); Okada et al., J. Immunol. 159:3638-47(1997); Barry and Johnston, Scand. J. Immunol., 45:605-12(1997)). Co-expression of proteins that facilitate 15 peptide epitope formation, such as proteolytic enzymes (e.g. the NS3 protease from hepatitis C (Koch and Bartenshlager, 1997)) or chaperone proteins (e.g. heat shock protein Hsp65 (Wells et al., Scand. J. Immunol., 45:605-12 1997)), may also enhance the response. 20 The various types of multivalent expression plasmids are described in Figure 8. They include (1) multiple complete genes, or transcription units, on a single plasmid, (2) generation of polycistronic mRNAs using a internal ribosome entry site (IRES) sequence, and (3) generation of 25 multiple mRNAs by alternative RNA splicing. The design of a nucleic acid-based vaccine expression plasmid that has two genes is shown in Figure 9. The details of these systems are described in the related patent applications incorporated herein by reference 30 on page 1.

WO99/24596 PCT/US98/23745 19 Examples The present invention will be more fully described in conjunction with the following specific examples which are not to be construed in any way as limiting the scope of the 5 invention. Geneswitch Example The GeneSwitch is a chimeric protein that consists of human progesterone receptor with a modified ligand binding 10 domain, a DNA binding domain from yeast GAL4, and an activator domain from Herpes Simplex VP16. When a synthetic steroid, mifepristone, is administered, the GeneSwitch protein becomes activated (binds the synthetic steroid, presumably dimerizes, and translocates to the nucleus). The 15 activated GeneSwitch then binds to a target sequence (multiple GAL4 binding sites linked to a minimal promoter) and thereby stimulates the transcription of the desired transgene (Wang et al., Proc. Natl. Acad. Sci. USA 91:8180 84 (1994); Wang et al., Nature Biotechnology 15:239-243 20 (1997a); Wang et al., Gene Therapy 4:432-41 (1997b)). The GeneSwitch may be used to regulate the expression of a plasmid for nucleic acid-based vaccines. It is possible that the timing of expression may influence the immune response. Thus, with a GeneSwitch regulated system, 25 the genes that encode the multiepitopes may be turned on at a defined time after DNA delivery by the administration of the ligand (mifepristone) to the animal. If the expression plasmid persists in vivo for a long enough time, the GeneSwitch system also can be used to provide pulsatile 30 expression of the multiepitope gene products. An example of the design of a system that is regulated by the GeneSwitch is shown in Figure 10.

WO99/24596 PCTIUS98/23745 20 One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The molecular complexes and 5 the methods, procedures, treatments, molecules, specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which 10 are encompassed within the spirit of the invention are defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the 15 scope and spirit of the invention. All patents and publications mentioned in the speci fication are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the 20 same extent as if each individual publication was specific ally and individually indicated to be incorporated by reference. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, 25 limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions 30 which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any WO99/24596 PCT/US98/23745 21 equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention 5 has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this 10 invention as defined by the appended claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of 15 members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described. Other embodiments are within the following claims.

Claims (12)

1. A method of genetic immunization comprising the step of presenting multiple epitopes to an organism in need of said immunization. 5

2. The method of claim 1 wherein said multiple epitopes are presented with one or more augmenting cytokines. 10

3. The method of claim 1 wherein said multiple epitopes are presented with a delivery vehicle selected from the group consisting of cationic lipids, delivery peptides, and polymer based deliver systems. 15

4. A plasmid for expression of multiple epitopes comprising a nucleic acid sequence encoding multiple epitopes, wherein each of said epitopes is capable of creating an immune response. 20

5. The plasmid of claim 4 further comprising a promoter, a 5' UTR sequence, and a 3' UTR sequence.

6. The plasmid of claim 5 further comprising a nucleic acid sequence enclosing polyubiquitin. 25

7. The plasmid of claim 5 wherein there are spacers between the nucleic acid regions encoding each of said epitopes. 30

8. The plasmid of claim 5 further comprising proteolytic cleavage sites between each of said epitopes. WO99/24596 PCT/US98/23745 23

9. The plasmid of claim 5 further comprising ER targeting signals between each of said epitopes.

10. The plasmid of claim 5 further comprising 5 lysosomal and/or endosomal targeting sequences between each of said epitopes.

11. A multivalent expression system as shown in Figure 8 and selected from the group consisting of two plasmids, 10 two genes, IRES, and alternative splicing.

12. A method of making a plasmid of anyone of claims 4-10 comprising the step of producing the appropriate nucleic acid sequence. 15