WO2016011320A1

WO2016011320A1 - Bivalent listeria-based delivery system of heterologous antigens

Info

Publication number: WO2016011320A1
Application number: PCT/US2015/040855
Authority: WO
Inventors: Anu Wallecha; Poonam MOLLI; Robert Petit
Original assignee: Advaxis, Inc.
Priority date: 2014-07-18
Filing date: 2015-07-17
Publication date: 2016-01-21

Abstract

Disclosed herein are recombinant Listeria strains comprising nucleotides encoding two or more heterologous antigens each fused to a truncated LLO, an N-terminal ActA or a PESTsequence, methods of preparing same, and methods of inducing an immune response, and treating, inhibiting, or suppressing cancer or tumors comprising administering same.

Description

BIVALENT LISTERIA-BASED DELIVERY SYSTEM OF HETEROLOGOUS

ANTIGENS FIELD OF INVENTION [0001] Disclosed herein are recombinant Listeria strains comprising nucleotides encoding two or more heterologous antigens each fused to a truncated LLO, an N-terminal ActA or a PEST-sequence, methods of preparing same, and methods of inducing an immune response, and treating, inhibiting, or suppressing cancer or tumors comprising administering same. BACKGROUND OF THE INVENTION [0002] A great deal of pre-clinical evidence and early clinical trial data suggests that the anti- tumor capabilities of the immune system can be harnessed to treat patients with established cancers. The vaccine strategy takes advantage of tumor antigens associated with various types of cancers. Immunizing with live vaccines such as viral or bacterial vectors expressing a tumor-associated antigen is one strategy for eliciting strong CTL responses against tumors. [0003] Listeria monocytogenes (Lm) is a gram positive, facultative intracellular bacterium that has direct access to the cytoplasm of antigen presenting cells, such as macrophages and dendritic cells, largely due to the pore-forming activity of listeriolysin-O (LLO). LLO is secreted by Lm following engulfment by the cells and perforates the phagolysosomal membrane, allowing the bacterium to escape the vacuole and enter the cytoplasm. LLO is very efficiently presented to the immune system via MHC class I molecules. Furthermore, Lm-derived peptides also have access to MHC class II presentation via the phagolysosome. [0004] Cancer is a complex disease and combined therapeutic approaches are more likely to succeed. Not only tumor cells, but also the microenvironment that supports tumor growth, must be targeted to maximize the therapeutic efficacy. Most immunotherapies focus on single antigens to target tumor cells and therefore they have shown limited success against human cancers. A single therapeutic agent capable of targeting multiple targets, such as tumor cells and tumor microenvironment simultaneously would have an advantage over other immunotherapeutic approaches, especially if it results in a synergistic anti-tumor effect. SUMMARY OF THE INVENTION [0005] In one aspect, the invention provided herein relates to a recombinant Listeria comprising a nucleotide molecule comprising a first and a second open reading frame each encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen or a functional fragment thereof fused to an endogenous PEST-containing amino acid sequence. In a related aspect, said nucleotide molecule further comprises a third open reading frame encoding a third polypeptide, wherein said third polypeptide comprises a heterologous antigen or a functional fragment thereof fused to an endogenous PEST-containing amino acid sequence. [0006] In a related aspect, the invention relates to a recombinant Listeria comprising a first nucleic acid molecule comprising an open reading frame encoding a recombinant polypeptide comprising a carbonic anhydrase 9 (CA9) or a fragment thereof fused to a truncated Listeriolysin O protein, and a second nucleic acid molecule comprising an open reading frame encoding a recombinant polypeptide comprising a chimeric HER2 antigen or a fragment thereof fused to an endogenous an LLO protein. [0007] In one aspect, the invention relates to a method of inducing an anti-tumor immune response in a subject comprising administering to said subject the recombinant Listeria provided herein. In a related aspect, the immune response allows treating, suppressing, or inhibiting a cancer in a subject. [0008] In one aspect, the invention relates to a recombinant Listeria strain comprising a first nucleotide molecule and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence, wherein said first nucleotide molecule is in an extrachromosomal episome or plasmid, and wherein said second nucleotide molecule is integrated into the Listeria genome. [0009] In one aspect, the invention relates to a recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence. [00010] In one aspect, the invention relates to a recombinant Listeria strain comprising a first and a second nucleic acid molecule, said first nucleic acid molecule encoding a recombinant polypeptide comprising a carbonic anhydrase 9 (CA9) antigen or a functional fragment thereof fused to a truncated Listeriolysin O (LLO), and wherein said second nucleic acid molecule encodes a chimeric HER (cHER2) protein fused to an endogenous LLO. [00011] In one aspect, the invention relates to a method of increasing the efficacy of a Listeria-based immunotherapy, said method comprising sequentially or concomitantly administering two or more recombinant Listeria strains to a subject having a tumor. In a related aspect, the Listeria strains that are administered sequentially or concomitantaly each comprise a nucleic acid molecule, said nucleic acid molecule encoding a recombinant polypeptide comprising a heterologous antigen fused to a PEST-containing polypeptide. In another related aspect, the heterologous antigen is chimeric HER (cHER2), CA9, PSA or HMW-MAA-C. [00012] In one aspect, the invention relates to a method of producing a recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence, said method comprising the steps of: a) Obtaining a plasmid

b) Recombinantly fusing in said plasmid said first and second nucleotide molecule; and

c) Transforming into said recombinant Listeria said plasmid; and d) Expressing said fusion protein by culturing said Listeria under conditions conducive to protein expression,

thereby producing a recombinant Listeria strain. [00013] In another aspect, the invention relates to a method of producing the recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST- containing amino acid sequence, said method comprising the steps of: a) Obtaining a plasmid

c) Transforming into said recombinant Listeria said plasmid; thereby producing a recombinant Listeria strain.

[00014] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS [00015] Figure 1. (A) Schematic representation of the chromosomal region of the Lmdd-143 and LmddA-143 after klk3 integration and actA deletion; (B) The klk3 gene is integrated into the Lmdd and LmddA chromosome. PCR from chromosomal DNA preparation from each construct using klk3 specific primers amplifies a band of 714 bp corresponding to the klk3 gene, lacking the secretion signal sequence of the wild type protein. [00016] Figure 2. (A) Map of the pADV134 plasmid. (B) Proteins from LmddA-134 culture supernatant were precipitated, separated in a SDS-PAGE, and the LLO-E7 protein detected by Western-blot using an anti-E7 monoclonal antibody. The antigen expression cassette consists of hly promoter, ORF for truncated LLO and human PSA gene (klk3). (C) Map of the pADV142 plasmid. (D) Western blot showed the expression of LLO-PSA fusion protein using anti-PSA and anti-LLO antibody. [00017] Figure 3. Schematic representation of monovalent and bivalent plasmids. Restriction sites that were used for cloning of antigen 1 (XhoI and SpeI) and antigen 2 (XbaI and SacI or BglII) are indicated. The black arrow represents the direction of transcription. p15 ori and RepR refers to E.coli and Listeria origin of replication. tLLO is truncated Listeriolysin O protein. Bacillus-dal gene codes for D-alanine racemase which complements for the synthesis of D-alanine in Lm ǻ dal dat strain. [00018] Figure 4. (A) Plasmid stability in vitro of LmddA-LLO-PSA if cultured with and without selection pressure (D-alanine). Strain and culture conditions are listed first and plates used for CFU determination are listed after. (B) Clearance of LmddA-LLO-PSA in vivo and assessment of potential plasmid loss during this time. Bacteria were injected i.v. and isolated from spleen at the time point indicated. CFUs were determined on BHI and BHI + D-alanine plates. [00019] Figure 5. (A) In vivo clearance of the strain LmddA-LLO-PSA after administration of 10⁸ CFU in C57BL/6 mice. The number of CFU were determined by plating on BHI/str plates. The limit of detection of this method was 100 CFU. (B) Cell infection assay of J774 cells with 10403S, LmddA-LLO-PSA and XFL7 strains. [00020] Figure 6. (A) PSA tetramer-specific cells in the splenocytes of naïve and LmddA- LLO-PSA immunized mice on day 6 after the booster dose. (B) Intracellular cytokine staining for IFN-γ in the splenocytes of naïve and LmddA-LLO-PSA immunized mice were stimulated with PSA peptide for 5 h. Specific lysis of EL4 cells pulsed with PSA peptide with in vitro stimulated effector T cells from LmddA-LLO-PSA immunized mice and naïve mice at different effector/target ratio using a caspase based assay (C) and a europium based assay (D). Number of IFNγ spots in naïve and immunized splenocytes obtained after stimulation for 24 h in the presence of PSA peptide or no peptide (E). [00021] Figure 7. Immunization with LmddA-142 induces regression of Tramp-C1-PSA (TPSA) tumors. Mice were left untreated (n=8) (A) or immunized i.p. with LmddA-142 (1x10⁸ CFU/mouse) (n=8) (B) or Lm-LLO-PSA (n=8) (C) on days 7, 14 and 21. Tumor sizes were measured for each individual tumor and the values expressed as the mean diameter in millimeters. Each line represents an individual mouse. [00022] Figure 8. (A) Analysis of PSA-tetramer⁺CD8⁺ T cells in the spleens and infiltrating T-PSA-23 tumors of untreated mice and mice immunized with either an Lm control strain or Lm-ddA-LLO-PSA (LmddA-142). (B) Analysis of CD4⁺ regulatory T cells, which were defined as CD25⁺FoxP3⁺, in the spleens and infiltrating T-PSA-23 tumors of untreated mice and mice immunized with either an Lm control strain or Lm-ddA-LLO-PSA. [00023] Figure 9. (A) Schematic representation of the chromosomal region of the Lmdd-143 and LmddA-143 after klk3 integration and actA deletion; (B) The klk3 gene is integrated into the Lmdd and LmddA chromosome. PCR from chromosomal DNA preparation from each construct using klk3 specific primers amplifies a band of 760 bp corresponding to the klk3 gene. [00024] Figure 10. (A) Lmdd-143 and LmddA-143 secretes the LLO-PSA protein. Proteins from bacterial culture supernatants were precipitated, separated in a SDS-PAGE and LLO and LLO-PSA proteins detected by Western-blot using an anti-LLO and anti-PSA antibodies; (B) LLO produced by Lmdd-143 and LmddA-143 retains hemolytic activity. Sheep red blood cells were incubated with serial dilutions of bacterial culture supernatants and hemolytic activity measured by absorbance at 590nm; (C) Lmdd-143 and LmddA-143 grow inside the macrophage-like J774 cells. J774 cells were incubated with bacteria for 1 hour followed by gentamicin treatment to kill extracellular bacteria. Intracellular growth was measured by plating serial dilutions of J774 lysates obtained at the indicated timepoints. Lm 10403S was used as a control in these experiments. [00025] Figure 11. Immunization of mice with Lmdd-143 and LmddA-143 induces a PSA- specific immune response. C57BL/6 mice were immunized twice at 1-week interval with 1x10⁸ CFU of Lmdd-143, LmddA-143 or LmddA-142 and 7 days later spleens were harvested. Splenocytes were stimulated for 5 hours in the presence of monensin with 1 M of the PSA_65- ₇₄ peptide. Cells were stained for CD8, CD3, CD62L and intracellular IFN-Ȗ and analyzed in a FACS Calibur cytometer. [00026] Figure 12. Three Lm-based vaccines expressing distinct HMW-MAA fragments based on the position of previously mapped and predicted HLA-A2 epitopes were designed (A). The Lm-tLLO-HMW-MMA_2160-2258 (also referred as Lm-LLO-HMW-MAA-C) strain secretes a ~62 kDa band corresponding to the tLLO-HMW-MAA_2160-2258 fusion protein (B). C57BL/6 mice (n=15) were inoculated s.c. with B16F10 cells and either immunized i.p. on days 3, 10 and 17 with Lm-tLLO-HMW-MAA_2160-2258 (n=8) or left untreated (n=7). BALB/c mice (n=16) were inoculated s.c. with RENCA cells and immunized i.p. on days 3, 10 and 17 with either Lm-HMW-MAA-C (n=8) or an equivalent dose of a control Lm vaccine. Mice immunized with the Lm-LLO-HMW-MAA-C impeded the growth of established tumors (C). FVB/N mice (n=13) were inoculated s.c. with NT-2 tumor cells and immunized i.p. on days 7, 14 and 21 with either Lm-HMW-MAA-C (n=5) or an equivalent dose of a control Lm vaccine (n=8). Immunization of mice with Lm-LLO-HMW-MAA-C significantly impaired the growth of tumors not engineered to express HMW-MAA, such as B16F10, RENCA and NT-2 (D). Tumor sizes were measured for each individual tumor and the values expressed as the mean diameter in millimeters ± SEM. *, P 0.05, Mann-Whitney test. [00027] Figure 13. Immunization with Lm-HMW-MAA-C promotes tumor infiltration by CD8⁺ T cells and decreases the number of pericytes in blood vessels. (A) NT-2 tumors were removed and sectioned for immunofluorescence. Staining groups are numbered (1-3) and each stain is indicated on the right. Sequential tissues were either stained with the pan-vessel marker anti-CD31 or the anti-NG2 antibody for the HMW-MAA mouse homolog AN2, in conjunction with anti-CD8Į for possible TILs. Group 3 shows isotype controls for the above antibodies and DAPI staining used as a nuclear marker. A total of 5 tumors were analyzed and a single representative image from each group is shown. CD8⁺ cells around blood vessels are indicated by arrows. (B) Sequential sections were stained for pericytes by using the anti-NG2 and anti-alpha-smooth-muscle-cell-actin (Į -SMA) antibodies. Double staining/colocalization of these two antibodies (yellow in merge image) are indicative of pericyte staining (top). Pericyte colocalization was quantitated using Image Pro Software and the number of colocalized objects is shown in the graph (bottom). A total of 3 tumors were analyzed and a single representative image from each group is shown. *, P≤ 0.05, Mann-Whitney test. Graph shows mean ± SEM. [00028] Figure 14. (A) LmddA244G/168. Listeria strain expressing chromosomal LLO- cHer2 was constructed by the method of double allelic homologous recombination between the chromosomal gene and the temperature sensitive Listeria shuttle plasmid to create LmddA-cHer2 (referred as LmddA244G). Further, to generate the bivalent strain, LmddA244G was transformed with the plasmid containing expression cassette for the fusion protein tLLO-HMC (pAdv168) (B) resulting in strain LmddA244G/168. LmddA strain was transformed with plasmid pAdv164, which contains expression cassette for tLLO-cHer2 fusion protein to create LmddA164 vaccine. LmddA backbone was transformed with plasmid pAv168, which contains expression cassette for tLLO-HMC (2160-2258 amino acid residues at the C-terminus of HMW-MAA or CSPG4) fusion protein to create LmddA168 vaccine. (C) Further, the expression and secretion of the two fusion proteins in LmddA244G/168, LLO- ChHer2 and tLLO-HMC was detected by western blot using anti-LLO and anti-FLAG antibodies respectively. [00029] Figure 15. Hemolytic activity of LmddA244G-168 was quantified using Sheep Red Blood cells. A 1.5 fold reduction in the hemolytic activity of bivalent immunotherapy LmddA244G-168 was observed when compared to 10403S. B. Intracellular growth of both bivalent and monovalent immunotherapies in J774 cell line. The intracellular growth of LmddA244G-168 was similar to monovalent immunotherapies LmddA164 and LmddA168. [00030] Figure 16. A. Established NT2 tumors were implanted with treated with mono therapies and bivalent therapy on days 6, 13 and 20. The naïve group is untreated mice. B. The percent tumor free mice in different treatment and untreated naïve group. C. The volume of established NT2 tumors after of LmddA244G-168 treatment. [00031] Figure 17. A. Generation of Her2 specific immune responses in mice after administration of monovalent (LmddA164) as well as bivalent immunotherapy (LmddA244G-168) expressing chimera Her2. The Her2 specific immune responses were evaluated in an ELIspot based assay using FvB IC1 peptide epitope -RLLQETELV (Seavey et al 2009, Clin Cancer Res. 2009 Feb 1;15(3):924-32. B. Generation of HMW-MAA-C specific immune responses in mice after administration of monovalent (LmddA168) as well as bivalent immunotherapy (LmddA244G-168) expressing HMW-MAA-C. The Her2 specific immune responses were evaluated in an ELISA based assay using affinity purified HMA- MAA-C protein fragment. [00032] Figure 18. Immunohistochemical (IHC) staining of tumors anti-CD3 antibody on day 27 of the tumor regression study. NT2 tumors were implanted on day 0 and were immunized on days 6, 13 and 20 with different immunotherapies (A) untreated naïve group; (B) mono immunotherapy (LmddA164); (C) mono immunotherapy (LmddA168); and (D) bivalent immunotherapy (LmddA244G-168). [00033] Figure 19. Immunohistochemical (IHC) staining of tumors anti-CD8 antibody on day 27 of the tumor regression study. NT2 tumors were implanted on day 0 and were immunized on days 6, 13 and 20 with different immunotherapies (A) untreated naïve group; (B) mono immunotherapy (LmddA164); (C) mono immunotherapy (LmddA168); and (D) bivalent immunotherapy (LmddA244G-168). [00034] Figure 20. Immunohistochemical (IHC) staining of tumors anti-CD4 antibody on day 27 of the tumor regression study. NT2 tumors were implanted on day 0 and were immunized on days 6, 13 and 20 with different immunotherapies (A) untreated naïve group; (B) mono immunotherapy (LmddA164); (C) mono immunotherapy (LmddA168); and (D) bivalent immunotherapy (LmddA244G-168). [00035] Figure 21. Immunohistochemical (IHC) staining of tumors anti-CD31 antibody on day 27 of the tumor regression study. NT2 tumors were implanted on day 0 and were immunized on days 6, 13 and 20 with different immunotherapies (A) untreated naïve group; (B) mono immunotherapy (LmddA164); (C) mono immunotherapy (LmddA168); and (D) bivalent immunotherapy (LmddA244G-168). [00036] Figure 22. Graph showing the individual mice and the tumor sizes on the days of tumor measurement: days 11, 18, and 21 following administration of various Listeria-based constructs. [00037] Figure 23. Established 4T1 tumors were treated with mono therapies and bivalent therapy on days 3 and 10. The naïve group is untreated mice. [00038] Figure 24. Established 4T1 tumors were treated with mono therapies and bivalent therapy on days 1, 8, and 15. The naïve group is untreated mice. [00039] Figure 25. Established NT2 tumors were treated with mono therapies, bivalent therapy, or sequential mono therapies. The naïve group is untreated mice. [00040] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE INVENTION [00041] This invention discloses, in one embodiment, a recombinant Listeria comprising a recombinant nucleic acid molecule comprising a first and a second open reading frame each encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen or a functional fragment thereof fused to a PEST-containing amino acid sequence. In another embodiment, said nucleotide molecule further comprises a third open reading frame encoding a third polypeptide, wherein said third polypeptide comprises a heterologous antigen or a functional fragment thereof fused to an endogenous PEST-containing amino acid sequence. [00042] In one embodiment of the invention, disclosed herein is a method of inducing an anti-tumor immune response in a subject comprising the step of administering to said subject the recombinant Listeria provided herein. In another embodiment, the immune response allows treating, suppressing, or inhibiting a cancer in a subject. [00043] In one embodiment of the invention, disclosed herein is a recombinant Listeria strain comprising a first nucleotide molecule and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence, wherein said first nucleotide molecule is in an extrachromosomal episome or plasmid, and wherein said second nucleotide molecule is integrated into the Listeria genome. [00044] The invention discloses, in another embodiment, a recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST-containing amino acid sequence. [00045] The invention discloses, in another embodiment, a recombinant Listeria strain comprising a first and a second nucleic acid molecule, wherein said first nucleic acid molecule encodes a recombinant polypeptide comprising a carbonic anhydrase 9 (CA9) antigen or a functional fragment thereof fused to a truncated Listeriolysin O (LLO), and wherein said second nucleic acid molecule encodes a chimeric HER (cHER2) protein fused to an endogenous LLO. [00046] The invention discloses, in another embodiment, a method of increasing the efficacy of a Listeria-based immunotherapy, the method comprising sequentially or concomitantly administering two or more recombinant Listeria strains to a subject having a tumor. In a related aspect, the Listeria strains that are administered sequentially or concomitantaly each comprise a nucleic acid molecule, said nucleic acid molecule encoding a recombinant polypeptide comprising a heterologous antigen fused to a PEST-containing polypeptide. In another related aspect, the heterologous antigen is chimeric HER (cHER2), CA9, PSA or HMW-MAA-C. In another embodiment, the method of increasing the efficacy of a Listeria- based immunotherapy enhances an antigen-specific immune response as a result of administering the same. [00047] In one embodiment, provided herein is a method of producing a recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST- containing amino acid sequence, said method comprising the steps of: a) Obtaining a plasmid

c) Transforming into said recombinant Listeria said plasmid; d) Expressing said fusion protein by culturing said Listeria under conditions conducive to protein expression.

Thereby producing a recombinant Listeria strain. [00048] In another embodiment, provided herein is a method of producing the recombinant Listeria strain comprising a bivalent expression plasmid comprising a first and a second nucleotide molecule encoding a first and a second polypeptide, wherein said first and said second polypeptide each comprise a heterologous antigen fused to an endogenous PEST- containing polypeptide, said method comprising the steps of: a) Obtaining a plasmid

c) Transforming into said recombinant Listeria said plasmid;

Thereby producing a recombinant Listeria strain. [00049] In another embodiment, provided herein is a recombinant Listeria strain comprising a bivalent episomal expression vector, the vector comprising a first, and at least a second nucleic acid molecule encoding a heterologous antigenic polypeptide or a functional fragment thereof, wherein the first and the second nucleic acid molecules each encode the heterologous antigenic polypeptide or functional fragment thereof in an open reading frame with an endogenous PEST-containing polypeptide. [00050] It will be appreciated that the term“bivalent” or“multivalent,” when in reference to a nucleotide molecule, plasmid, or vector may encompass a nucleotide molecule, nucleic acid, DNA sequence, plasmid, and the like, that expresses two (bivalent), three (multivalent), or more (multivalent) heterologous antigens each individually fused to a PEST-containing sequence (such as an N-terminal or truncated LLO, N-terminal ActA, or a PEST sequence or PEST peptide). In another embodiment, a bivalent plasmid encodes two heterologous antigens. In another embodiment, a bivalent plasmid encodes two different heterologous antigens. In another embodiment, the term“bivalent” is used interchangeably herein with “dual”. In another embodiment, a multivalent plasmid encodes three different heterologous antigens. [00051] It will be appreciated that the term“bivalent” or“multivalent,” when in reference to a recombinant Listeria strain may encompass a Listeria strain that is capable of expressing two (bivalent) or more (multivalent) heterologous antigens. It will be appreciated that the term “bivalent” or “multivalent,” when in reference to a recombinant Listeria strain may encompass a Listeria strain that expresses two (bivalent) or more (multivalent) heterologous antigens. In one embodiment a bivalent Listeria strain comprises a bivalent plasmid that expresses two heterologous antigens from a plasmid. In another embodiment, a bivalent Listeria strain expresses one heterologous antigen from the genome, (following integration of the heterologous antigen into the Listeria genome), and another heterologous antigen from a plasmid present in the cytoplasm of said Listeria strain. In another embodiment, a multivalent Listeria strain expresses three heterologous antigens from a plasmid. In another embodiment, a multivalent Listeria strain expresses three heterologous antigens, one from the genome (following integration of the heterologous antigen into the Listeria genome) and two from a plasmid present in the cytoplasm of the Listeria strain. In another embodiment, a multivalent Listeria strain expresses three heterologous antigens, two from the genome (following integration of the heterologous antigen into the Listeria genome) and one from a plasmid present in the cytoplasm of the Listeria strain. It will be well appreciated by a skilled artisan that a multivalent Listeria strain comprises the ability to express three or more heterologous antigens in total, where at least one is expressed from either the genome or from a plasmid (in any desired combination, i.e.- 3 from the plasmid and 1 from the genome, 2 from the plasmid and 2 from the genome, 1 from the plasmid and 3 from the genome, etc.). [00052] In another embodiment, a bivalent Listeria strain expresses one heterologous antigen from the genome in the context of a fusion protein with a PEST-containing polypeptide, (following integration of the heterologous antigen into the frame of the gene encoding the PEST-containing polypeptide in the Listeria genome), and another heterologous antigen from a plasmid present in the cytoplasm of said Listeria strain, also in the context of a fusion protein with a PEST-containing polypeptide. In another embodiment, a multivalent Listeria strain expresses three heterologous antigens from a plasmid, all in the context of a fusion protein with a PEST-containing polypeptide. In another embodiment, a multivalent Listeria strain expresses three heterologous antigens, one from the genome (following integration of the heterologous antigen into the Listeria genome) and two heterologous antigens from a plasmid present in the cytoplasm of the Listeria strain, all in the context of a PEST-containing polypeptide. In another embodiment, a multivalent Listeria strain expresses three heterologous antigens, two from the genome (following integration of the heterologous antigen into the Listeria genome) and one from a plasmid present in the cytoplasm of the Listeria strain, all in the context of a fusion protein with a PEST-containing polypeptide. It will be well appreciated by a skilled artisan that a multivalent Listeria strain comprises the ability to express three or more heterologous antigens in total, where at least one is expressed from either the genome or from a plasmid (in any desired combination, i.e.- 3 from the plasmid and 1 from the genome, 2 from the plasmid and 2 from the genome, 1 from the plasmid and 3 from the genome, etc.,) all in the context of a fusion protein with a PEST- containing polypeptide. [00053] In another embodiment, the PEST-containing polypeptide is a truncated LLO, an N- terminal ActA protein, or a PEST sequence. [00054] The terms“antigen,” “antigenic polypeptide,”“antigen fragment,” are used interchangeably herein and, as will be appreciated by a skilled artisan, may encompass polypeptides, or peptides (including recombinant peptides) that are loaded onto and presented on MHC class I and/or class II molecules on a host’s cell’s surface and can be recognized or detected by an immune cell of the host, thereby leading to the mounting of an immune response against the polypeptide, peptide or cell presenting the same. Similarly, the immune response may also extend to other cells within the host, including diseased cells such as tumor or cancer cells that express the same polypeptides or peptides. [00055] It will also be appreciated by a skilled artisan that the terms“antigenic portion thereof”, “a fragment thereof” and“immunogenic portion thereof” in regard to a protein, peptide or polypeptide are used interchangeably herein and may encompass a protein, polypeptide, peptide, including recombinant forms thereof comprising a domain or segment that leads to the mounting of an immune response when present in, or, in some embodiments, detected by, a host, either alone, or in the context of a fusion protein, as described herein. [00056] In one embodiment, an antigen may be foreign, that is, heterologous to the host and is referred to as a“heterologous antigen” herein. In another embodiment, a heterologous antigen is heterologous to a Listeria strain provided herein that recombinantly expresses said antigen. In another embodiment, a heterologous antigen is heterologous to the host and a Listeria strain provided herein that recombinantly expresses said antigen. In another embodiment, the antigen is a self-antigen, which is an antigen that is present in the host but the host does not elicit an immune response against it because of immunologic tolerance. It will be appreciated by a skilled artisan that a heterologous antigen as well as a self-antigen may encompass a tumor antigen, a tumor-associated antigen or an angiogenic antigen. In addition, a heterologous antigen may encompass an infectious disease antigen. [00057] In one embodiment, the terms“recombinant Listeria” and“live-attenuated Listeria” are used interchangeably here and refer to a Listeria comprising at least one attenuating mutation, deletion or inactivation that expresses a fusion protein of an antigen fused to a truncated LLO, truncated ActA or PEST sequence embodied herein. In another embodiment, a recombinant Listeria provided herein is a recombinant Listeria monocytogenes. [00058] The terms“nucleic acid,”“nucleotide,”“nucleic acid molecule,”“oligonucleotide,” or“nucleotide molecule” are used interchangeably herein and may encompass a string of at least two base-sugar-phosphate combinations, as will be appreciated by a skilled artisan. The terms include, in one embodiment, DNA and RNA. It will also be appreciated by a skilled artisan that the terms may encompass the monomeric units of nucleic acid polymers. For example, RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) and ribozymes. The use of siRNA and miRNA has been described (Caudy AA et al, Genes & Devel 16: 2491-96 and references cited therein). DNA may be in form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups. In addition, these forms of DNA and RNA may be single, double, triple, or quadruple stranded. The term may also encompass artificial nucleic acids that may contain other types of backbones but the same bases. The use of phosphothiorate nucleic acids and PNA are known to those skilled in the art, and are described in, for example, Neilsen PE, Curr Opin Struct Biol 9:353-57; and Raz NK et al Biochem Biophys Res Commun. 297:1075- 84. The production and use of nucleic acids is known to those skilled in art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and G. C. Fareed.

[00059] The terms "amino acid" or "amino acids" are understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" may include both D- and L-amino acids.

[00060] It will be appreciated by a skilled artisan that the term“open reading frame” or“ORF” may encompass a portion of an organism's genome which contains a sequence of bases that could potentially encode a protein. In another embodiment, the start and stop ends of the ORF are not equivalent to the ends of the mRNA, but they are usually contained within the mRNA. In one embodiment, ORFs are located between the start-code sequence (initiation codon) and the stop-codon sequence (termination codon) of a gene. Thus, in one embodiment, a nucleic acid molecule operably integrated into a genome as an open reading frame with an endogenous polypeptide is a nucleic acid molecule that has integrated into a genome in the same open reading frame as an endogenous polypeptide.

[00061] It will be appreciated by a skilled artisan that the term“endogenous” may encompass an item that has developed or originated within the reference organism or arisen from causes within the reference organism. For example, endogenous refers to native.

[00062] It will also be appreciated by a skilled artisan that the term“fragment” may encompass a protein or polypeptide that is shorter or comprises fewer amino acids than the full length protein or polypeptide. In one embodiment, a fragment is an N-terminal fragment. In another embodiment, a fragment is a C-terminal fragment. In yet another embodiment, a fragment is an intrasequential section of the protein or peptide. It will be understood by a skilled artisan that a fragment as provided herein is a functional fragment, which may encompass an immunogenic fragment. In one embodiment, a fragment has more than 5 amino acids. In another embodiment, a fragment has 10-20 amino acids, 20-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-350 amino acids, or 350-500 amino acids.

[00063] In an alternate embodiment, the term“fragment” refers to a nucleic acid that is shorter or comprises fewer nucleotides than the full length nucleic acid. In one embodiment, a fragment is a 5’-terminal fragment. In another embodiment, a fragment is a 3’-terminal fragment. In yet another embodiment, a fragment encodes an intrasequential section of the protein. In one embodiment, a fragment has more than 5 nucleotides. In another embodiment, a fragment has 10-20 nucleotides, 20-50 nucleotides, 50-100 nucleotides, 100-200 nucleotides, 200-350 nucleotides, 350-500 or 500-1000 nucleotides. It will be appreciated by a skilled artisan that the term“functional” within the meaning of the invention, may encompass the innate ability of a protein, peptide, nucleic acid, fragment or a variant thereof to exhibit a biological activity. Such a biological activity may encompass having the potential to elicit an immune response when used as provided herein, an illustration of which may be to be used as part of a fusion protein). Such a biological function may encompass its binding property to an interaction partner, e.g., a membrane-associated receptor, or its trimerization property. In the case of functional fragments and the functional variants of the invention, these biological functions may in fact be changed, e.g., with respect to their specificity or selectivity, but with retention of the basic biological function. [00064] It will be appreciated by a skilled artisan that the term“functional fragment” may encompass an immunogenic fragment that is capable of eliciting an immune response when administered to a subject alone or as part of a pharmaceutical composition comprising a recombinant Listeria strain expressing said immunogenic fragment. In another embodiment, a functional fragment has biological activity as will be understood by a skilled artisan and as further provided herein. [00065] In one embodiment, provided herein is a multivalent plasmid that delivers at least two antigens. In another embodiment, the plasmid is a dual or bivalent plasmid. In another embodiment, the dual, bivalent or multivalent plasmid is episomal in nature in that it remains extrachromosomal. In another embodiment, the dual, or multivalent plasmid comprises sequences for integration into the Listeria chromosome. [00066] In another embodiment, the recombinant nucleic acid backbone of a plasmid disclosed herein comprises SEQ ID NO: 1. ggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggctgcaccggtgcg tcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggc ttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagccgtttttccat aggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataaagataccaggc gtttccccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctcattcca cgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatc cggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttagaggagttagtc ttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggttcaaagagttg gtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctca agaagatcatcttattaatcagataaaatatttctagccctcctttgattagtatattcctatcttaaagttacttttatgtggaggcattaacattt gttaatgacgtcaaaaggatagcaagactagaataaagctataaagcaagcatataatattgcgtttcatctttagaagcgaatttcgcca atattataattatcaaaagagaggggtggcaaacggtatttggcattattaggttaaaaaatgtagaaggagagtgaaacccatgaaaaa aataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaataaagaaaat tcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaatcgataagta tatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaaggttacaaag atggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaatttcgagcct aacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcattaacactca gcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacgcagtaaatacatta gtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgatgacgaaatggcttacagtgaatcac aattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaatcagtgaagggaaaatgcaaga agaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctgttactaaagag cagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttgaaattatcaac taattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaacaaatatcatca aaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggcaacctcggagacttacgcgatat tttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaagacaatgaattagctgttat taaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatcactctggaggatacgttgctcaat tcaacatttcttgggatgaagtaaattatgatctcgagactagttctagatttatcacgtacccatttccccgcatcttttatttttttaaatacttt agggaaaaatggtttttgatttgcttttaaaggttgtggtgtagactcgtctgctgactgcatgctagaatctaagtcactttcagaagcatcc acaactgactctttcgccacttttctcttatttgcttttgttggtttatctggataagtaaggctttcaagctcactatccgacgacgctatggctt ttcttctttttttaatttccgctgcgctatccgatgacagacctggatgacgacgctccacttgcagagttggtcggtcgactcctgaagcct cttcatttatagccacatttcctgtttgctcaccgttgttattattgttattcggacctttctctgcttttgctttcaacattgctattaggtctgctttg ttcgtatttttcactttattcgatttttctagttcctcaatatcacgtgaacttacttcacgtgcagtttcgtatcttggtcccgtatttacctcgcttg gctgctcttctgttttttcttcttcccattcatctgtgtttagactggaatcttcgctatctgtcgctgcaaatattatgtcggggttaatcgtaatg cagttggcagtaatgaaaactaccatcatcgcacgcataaatctgtttaatcccacttatactccctcctcgtgatacgctaatacaacctttt tagaacaaggaaaattcggccttcattttcactaatttgttccgttaaaaattggattagcagttagttatcttcttaattagctaatataagaaa aaatattcatgaattattttaagaatatcacttggagaattaatttttctctaacatttgttaatcagttaaccccaactgcttcccaagcttcacc cgggccactaactcaacgctagtagtggatttaatcccaaatgagccaacagaaccagaaccagaaacagaacaagtaacattggagt tagaaatggaagaagaaaaaagcaatgatttcgtgtgaataatgcacgaaatcattgcttatttttttaaaaagcgatatactagatataacg aaacaacgaactgaataaagaatacaaaaaaagagccacgaccagttaaagcctgagaaactttaactgcgagccttaattgattacca ccaatcaattaaagaagtcgagacccaaaatttggtaaagtatttaattactttattaatcagatacttaaatatctgtaaacccattatatcgg gtttttgaggggatttcaagtctttaagaagataccaggcaatcaattaagaaaaacttagttgattgccttttttgttgtgattcaactttgatc gtagcttctaactaattaattttcgtaagaaaggagaacagctgaatgaatatcccttttgttgtagaaactgtgcttcatgacggcttgttaa agtacaaatttaaaaatagtaaaattcgctcaatcactaccaagccaggtaaaagtaaaggggctatttttgcgtatcgctcaaaaaaaag catgattggcggacgtggcgttgttctgacttccgaagaagcgattcacgaaaatcaagatacatttacgcattggacaccaaacgtttat cgttatggtacgtatgcagacgaaaaccgttcatacactaaaggacattctgaaaacaatttaagacaaatcaataccttctttattgattttg atattcacacggaaaaagaaactatttcagcaagcgatattttaacaacagctattgatttaggttttatgcctacgttaattatcaaatctgat aaaggttatcaagcatattttgttttagaaacgccagtctatgtgacttcaaaatcagaatttaaatctgtcaaagcagccaaaataatctcg caaaatatccgagaatattttggaaagtctttgccagttgatctaacgtgcaatcattttgggattgctcgtataccaagaacggacaatgta gaattttttgatcccaattaccgttattctttcaaagaatggcaagattggtctttcaaacaaacagataataagggctttactcgttcaagtct aacggttttaagcggtacagaaggcaaaaaacaagtagatgaaccctggtttaatctcttattgcacgaaacgaaattttcaggagaaaa gggtttagtagggcgcaatagcgttatgtttaccctctctttagcctactttagttcaggctattcaatcgaaacgtgcgaatataatatgtttg agtttaataatcgattagatcaacccttagaagaaaaagaagtaatcaaaattgttagaagtgcctattcagaaaactatcaaggggctaat agggaatacattaccattctttgcaaagcttgggtatcaagtgatttaaccagtaaagatttatttgtccgtcaagggtggtttaaattcaaga aaaaaagaagcgaacgtcaacgtgttcatttgtcagaatggaaagaagatttaatggcttatattagcgaaaaaagcgatgtatacaagc cttatttagcgacgaccaaaaaagagattagagaagtgctaggcattcctgaacggacattagataaattgctgaaggtactgaaggcg aatcaggaaattttctttaagattaaaccaggaagaaatggtggcattcaacttgctagtgttaaatcattgttgctatcgatcattaaattaaa aaaagaagaacgagaaagctatataaaggcgctgacagcttcgtttaatttagaacgtacatttattcaagaaactctaaacaaattggca gaacgccccaaaacggacccacaactcgatttgtttagctacgatacaggctgaaaataaaacccgcactatgccattacatttatatcta tgatacgtgtttgtttttctttgctggctagcttaattgcttatatttacctgcaataaaggatttcttacttccattatactcccattttccaaaaac atacggggaacacgggaacttattgtacaggccacctcatagttaatggtttcgagccttcctgcaatctcatccatggaaatatattcatc cccctgccggcctattaatgtgacttttgtgcccggcggatattcctgatccagctccaccataaattggtccatgcaaattcggccggca attttcaggcgttttcccttcacaaggatgtcggtccctttcaattttcggagccagccgtccgcatagcctacaggcaccgtcccgatcca tgtgtctttttccgctgtgtactcggctccgtagctgacgctctcgccttttctgatcagtttgacatgtgacagtgtcgaatgcagggtaaat gccggacgcagctgaaacggtatctcgtccgacatgtcagcagacgggcgaaggccatacatgccgatgccgaatctgactgcatta aaaaagccttttttcagccggagtccagcggcgctgttcgcgcagtggaccattagattctttaacggcagcggagcaatcagctcttta aagcgctcaaactgcattaagaaatagcctctttctttttcatccgctgtcgcaaaatgggtaaatacccctttgcactttaaacgagggttg cggtcaagaattgccatcacgttctgaacttcttcctctgtttttacaccaagtctgttcatccccgtatcgaccttcagatgaaaatgaaga gaaccttttttcgtgtggcgggctgcctcctgaagccattcaacagaataacctgttaaggtcacgtcatactcagcagcgattgccacat actccgggggaaccgcgccaagcaccaatataggcgccttcaatccctttttgcgcagtgaaatcgcttcatccaaaatggccacggc caagcatgaagcacctgcgtcaagagcagcctttgctgtttctgcatcaccatgcccgtaggcgtttgctttcacaactgccatcaagtgg acatgttcaccgatatgttttttcatattgctgacattttcctttatcacggacaagtcaatttccgcccacgtatctctgtaaaaaggttttgtgc tcatggaaaactcctctcttttttcagaaaatcccagtacgtaattaagtatttgagaattaattttatattgattaatactaagtttacccagtttt cacctaaaaaacaaatgatgagataatagctccaaaggctaaagaggactataccaactatttgttaat (SEQ ID NO: 1). [00067] In one embodiment, a bivalent plasmid backbone comprises at least two nucleic acid sequences encoding at least two antigens. In another embodiment, the bivalent plasmid backbone comprises a nucleic acid sequences having at least two open reading frames encoding at least two antigens. In another embodiment, the bivalent plasmid backbone comprises a nucleic acid sequences having two open reading frames encoding two antigens. In another embodiment, the multivalent plasmid backbone comprises a nucleic acid sequences having at least three open reading frames encoding at least three antigens. [00068] In another embodiment, the multivalent plasmid backbone comprises at least three nucleic acid sequences having at least three open reading frames encoding at least three antigens. In another embodiment, the multivalent plasmid backbone comprises a nucleic acid sequences having three open reading frames encoding three antigens. In another embodiment, the multivalent plasmid backbone comprises three nucleic acid sequences having three open reading frames encoding three antigens. [00069] In one embodiment, the antigens encoded by the bivalent Listeria strains disclosed herein include CA9, chimeric HER2 (cHER2), and HMW-MAA or a fragment thereof (see Examples 11-16 herein). In another embodiment, the HMW-MAA fragment is HMW-MAA- C (HMC). [00070] In one embodiment, a Listeria strain LmddA244G disclosed herein comprises a nucleic acid sequence comprising an open reading frame encoding a cHER2 fused to an endogenous nucleic acid comprising an open reading frame encoding an LLO protein (see SEQ ID NO: 2). [00071] atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatg catctgcattcaataaagaaaattcaatttcatccgtggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaac acgcggatgaaatcgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgc cgccaagaaaaggttacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaa gttgtgaatgcaatttcgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgt aaaacgtgattcattaacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacg ttaacaacgcagtaaatacattagtggaaagatggaatgaaaaatatgctcaagcttattcaaatgtaagtgcaaaaattgattatgatgac gaaatggcttacagtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaat cagtgaagggaaaatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagattttt cggcaaagctgttactaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggcc gtcaagtttatttgaaattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtga tgtagaactaacaaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggc aacctcggagacttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcct aaaagacaatgaattagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatc actctggaggatacgttgctcaattcaacatttcttgggatgaagtaaattatgatcctgaaggtaacgaaattgttcaacataaaaactgg agcgaaaacaataaaagcaagctagctcatttcacatcgtccatctatttgcctggtaacgcgagaaatattaatgtttacgctaaagaatg cactggtttagcttgggaatggtggagaacggtaattgatgaccggaacttaccacttgtgaaaaatagaaatatctccatctggggcac cacgctttatccgaaatatagtaataaagtagataatccaatcgaagtcgacACCCACCTGGACATGCTCCGCCAC CTCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCA CCAATGCCAGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCT CATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCG AGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGAC CCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAG CTGCAGCTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGA ACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGAATATCCAGGAGTTTGC TGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATGGG GACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGA CTCTGGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCC TGACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAAT GGCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCT CACTGAGGGAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTG CTTCGTGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTG CTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGC CACCAGCTGTGCGCCCGAGGGCAGCAGAAGATCCGGAAGTACACGATGCGGAGA CTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCC AACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGT GCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGG GAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCC AAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCC CCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGAC ACAGCTTATGCCCTATGGCTGCCTCTTAGAC (SEQ ID NO: 2), where the UPPERCASE sequences represents the nucleic acid sequence encoding a cHER2, the lower case sequences represent the sequence encoding an endogenous LLO protein and the underlined“gtcgac” sequence represents the Sal I restriction site used to ligate the tumor antigen to the endogenous LLO. In one embodiment, the endogenous LLO- CHER2 fusion is a homolog of SEQ ID NO: 2. In another embodiment, the endogenous LLO- CHER2 fusion is a variant of SEQ ID NO: 2. In another embodiment, the endogenous LLO- CHER2 fusion is an isomer of SEQ ID NO: 2. [00072] In one embodiment, the amino acid sequence of the fusion between a cHER2 and an endogenous LLO comprises SEQ ID NO: 3

[00073] In one embodiment, the amino acid sequence of endogenous LLO protein comprises SEQ ID NO: 4, M K K I M L V F I T L I L V S L P I A Q Q T E A K D A S A F N K E N S I S S V A P P A S P P A S P K T P I E K K H A D E I D K Y I Q G L D Y N K N N V L V Y H G D A V T N V P P R K G Y K D G N E Y I V V E K K K K S I N Q N N A D I Q V V N A I S S L T Y P G A L V K A N S E L V E N Q P D V L P V K R D S L T L S I D L P G M T N Q D N K I V V K N A T K S N V N N A V N T L V E R W N E K Y A Q A Y S N V S A K I D Y D D E M A Y S E S Q L I A K F G T A F K A V N N S L N V N F G A I S E G K M Q E E V I S F K Q I Y Y N V N V N E P T R P S R F F G K A V T K E Q L Q A L G V N A E N P P A Y I S S V A Y G R Q V Y L K L S T N S H S T K V K A A F D A A V S G K S V S G D V E L T N I I K N S S F K A V I Y G G S A K D E V Q I I D G N L G D L R D I L K K G A T F N R E T P G V P I A Y T T N F L K D N E L A V I K N N S E Y I E T T S K A Y T D G K I N I D H S G G Y V A Q F N I S W D E V N Y D P E G N E I V Q H K N W S E N N K S K L A H F T S S I Y L P G N A R N I N V Y A K E C T G L A W E W W R T V I D D R N L P L V K N R N I S I W G T T L Y P K Y S N K V D N P I E (SEQ ID NO: 4).

[00074] In one embodiment, LmddA164 comprises a nucleic acid sequence comprising an open reading frame encoding tLLO fused to cHER2, wherein said nucleic acid sequence comprises SEQ ID NO: 5: atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaata aagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaat cgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaag gttacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaat ttcgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcat taacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacgcagt aaatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgatgacgaaatggcttac agtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaatcagtgaaggga aaatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctg ttactaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttg aaattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaac aaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggcaacctcggaga cttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaagacaatg aattagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatcactctggagga tacgttgctcaattcaacatttcttgggatgaagtaaattatgatctcgagACCCACCTGGACATGCTCCGCCACC TCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCAC CAATGCCAGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTC ATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGA GGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACC CGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCT GCAGCTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAA CCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGAATATCCAGGAGTTTGCT GGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATGGGG ACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGAC TCTGGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCT GACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATG GCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTC ACTGAGGGAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGC TTCGTGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGC TCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCC ACCAGCTGTGCGCCCGAGGGCAGCAGAAGATCCGGAAGTACACGATGCGGAGAC TGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCA ACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGC TTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGA GAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAA AGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCC ATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACA CAGCTTATGCCCTATGGCTGCCTCTTAGAC (SEQ ID NO: 5), wherein the UPPERCASE sequences encode cHER2, the lowercase sequences encode tLLO and the underlined“ctcgag” sequence represents the Xho I restriction site used to ligate the tumor antigen to truncated LLO in the plasmid. In another embodiment, plasmid pAdv168 comprises SEQ ID NO: 5. In one embodiment, the truncated LLO-CHER2 fusion is a homolog of SEQ ID NO: 5. In another embodiment, the truncated LLO-CHER2 fusion is a variant of SEQ ID NO: 5. In another embodiment, the truncated LLO-CHER2 fusion is an isomer of SEQ ID NO: 5. [00075] In one embodiment, an amino acid sequence of a tLLO fused to a cHER2 comprises SEQ ID NO: 6:

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHA DEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNN ADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKI VVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKF GTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTK EQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSG

In one embodiment, the truncated LLO-cHER2 fusion is a homolog of SEQ ID NO: 6. In another embodiment, the truncated LLO-cHER2 fusion is a variant of SEQ ID NO: 6. In another embodiment, the truncated LLO-cHER2 fusion is an isomer of SEQ ID NO: 6. [00076] In one embodiment, an amino acid sequence of a truncated LLO (tLLO) comprises SEQ ID NO: 7: M K K I M L V F I T L I L V S L P I A Q Q T E A K D A S A F N K E N S I S S M A P P A S P P A S P K T P I E K K H A D E I D K Y I Q G L D Y N K N N V L V Y H G D A V T N V P P R K G Y K D G N E Y I V V E K K K K S I N Q N N A D I Q V V N A I S S L T Y P G A L V K A N S E L V E N Q P D V L P V K R D S L T L S I D L P G M T N Q D N K I V V K N A T K S N V N N A V N T L V E R W N E K Y A Q A Y P N V S A K I D Y D D E M A Y S E S Q L I A K F G T A F K A V N N S L N V N F G A I S E G K M Q E E V I S F K Q I Y Y N V N V N E P T R P S R F F G K A V T K E Q L Q A L G V N A E N P P A Y I S S V A Y G R Q V Y L K L S T N S H S T K V K A A F D A A V S G K S V S G D V E L T N I I K N S S F K A V I Y G G S A K D E V Q I I D G N L G D L R D I L K K G A T F N R E T P G V P I A Y T T N F L K D N E L A V I K N N S E Y I E T T S K A Y T D G K I N I D H S G G Y V A Q F N I S W D E V N Y D (SEQ ID NO: 7). [00077] In one embodiment, LmddA168 comprises a nucleic acid sequence comprising an open reading frame encoding tLLO fused to HMW-MAA-C (HMC) comprises SEQ ID NO: 8: atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaata aagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaat cgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaag gttacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaat ttcgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcat taacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacgcagt aaatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgatgacgaaatggcttac agtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaatcagtgaaggga aaatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctg ttactaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttg aaattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaac aaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggcaacctcggaga cttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaagacaatg aattagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatcactctggagga tacgttgctcaattcaacatttcttgggatgaagtaaattatgatctcgagGCCACTGAGCCTTACAATGCTGCCC GGCCCTACAGCGTGGCCCTGCTCAGTGTCCCCGAGGCCGCCCGGACGGAAGCAG GGAAGCCAGAGAGCAGCACCCCCACAGGCGAGCCAGGCCCCATGGCATCCAGCC CTGAGCCCGCTGTGGCCAAGGGAGGCTTCCTGAGCTTCCTTGAGGCCAACATGTT CAGCGTCATCATCCCCATGTGCCTGGTACTTCTGCTCCTGGCGCTCATCCTGCCCC TGCTCTTCTACCTCCGAAAACGCAACAAGACGGGCAAGCATGACGTCCAGGATTA CAAGGATGACGACGATAAG (SEQ ID NO: 8), wherein the UPPERCASE sequences encode HMC, the lowercase sequences encode tLLO, and the underlined“ctcgag” sequence represents the Xho I restriction site used to ligate the tumor antigen to truncated LLO in the plasmid. In another embodiment, plasmid pAdv168 comprises SEQ ID NO: 8. In one embodiment, the truncated LLO-HMC fusion is a homolog of SEQ ID NO: 8. In another embodiment, the truncated LLO-HMC fusion is a variant of SEQ ID NO: 8. In another embodiment, the truncated LLO-HMC fusion is an isomer of SEQ ID NO: 8. [00078] In one embodiment, an amino acid sequence of a tLLO fused to an HMC antigen comprises SEQ ID NO: 9: MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEI DKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAIS SLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVN TLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKM QEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLK LSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDL RDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGY VAQFNISWDEVNYDLEATEPYNAARPYSVALLSVPEAARTEAGKPESSTPTGEPGPMASSP EPAVAKGGFLSFLEANMFSVIIPMCLVLLLLALILPLLFYLRKRNKTGKHDVQDYKDDDDK

(SEQ ID NO: 9). In one embodiment, the truncated LLO-HMC fusion is a homolog of SEQ ID NO: 9. In another embodiment, the truncated LLO-HMC fusion is a variant of SEQ ID NO: 9. In another embodiment, the truncated LLO-HMC fusion is an isomer of SEQ ID NO: 9. [00079] In one embodiment, the sequence of HMC comprises SEQ ID NO: 10: A T E P Y N A A R P Y S V A L L S V P E A A R T E A G K P E S S T P T G E P G P M A S S P E P A V A K G G F L S F L E A N M F S V I I P M C L V L L L L A L I L P L L F Y L R K R N K T G K H D V Q D Y K D D D D K (SEQ ID NO: 10). [00080] In one embodiment, the antigens are heterologous antigens to the bacteria host carrying the plasmid. In another embodiment, the antigens are heterologous antigens to the Listeria host carrying the plasmid. [00081] In another embodiment, the recombinant episomal nucleic acid sequence encoding the plasmid backbone and at least two heterologous antigens comprises SEQ ID NO: 11. In another embodiment, the recombinant episomal nucleic acid sequence encoding the plasmid backbone and at least two heterologous antigens consists of SEQ ID NO: 11. ggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggctgcaccggtgcg tcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggc ttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagccgtttttccat aggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataaagataccaggc gtttccccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctcattcca cgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatc cggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttagaggagttagtc ttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggttcaaagagttg gtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctca agaagatcatcttattaatcagataaaatatttctagccctcctttgattagtatattcctatcttaaagttacttttatgtggaggcattaacattt gttaatgacgtcaaaaggatagcaagactagaataaagctataaagcaagcatataatattgcgtttcatctttagaagcgaatttcgcca atattataattatcaaaagagaggggtggcaaacggtatttggcattattaggttaaaaaatgtagaaggagagtgaaacccatgaaaaa aataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaataaagaaaat tcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaatcgataagta tatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaaggttacaaag atggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaatttcgagcct aacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcattaacactca gcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacgcagtaaatacatta gtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgatgacgaaatggcttacagtgaatcac aattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaatcagtgaagggaaaatgcaaga agaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctgttactaaagag cagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttgaaattatcaac taattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaacaaatatcatca aaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggcaacctcggagacttacgcgatat tttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaagacaatgaattagctgttat taaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatcactctggaggatacgttgctcaat tcaacatttcttgggatgaagtaaattatgatctcgagcatggagatacacctacattgcatgaatatatgttagatttgcaaccagagaca actgatctctactgttatgagcaattaaatgacagctcagaggaggaggatgaaatagatggtccagctggacaagcagaaccggaca gagcccattacaatattgtaaccttttgttgcaagtgtgactctacgcttcggttgtgcgtacaaagcacacacgtagacattcgtactttgg aagacctgttaatgggcacactaggaattgtgtgccccatctgttctcagaaaccataaactagtctagtggtgatggtgatgatggagct cagatctgtctaagaggcagccatagggcataagctgtgtcaccagctgcaccgtggatgtcaggcagatgcccagaaggcgggag acatatggggagcccacaccagccatcacgtatgcttcgtctaagatttctttgttggctttgggggatgtgttttccctcaacactttgatg gccactggaattttcacattctccccatcagggatccagatgcccttgtagactgtgccaaaagcgccagatccaagcaccttcaccttc ctcagctccgtctctttcaggatccgcatctgcgcctggttgggcatcgctccgctaggtgtcagcggctccaccagctccgtttcctgca gcagtctccgcatcgtgtacttccggatcttctgctgccctcgggcgcacagctggtggcaggccaggccctcgcccacacactcgtc ctctggccggttggcagtgtggagcagagcttggtgcgggttccgaaagagctggtcccagggcaccgtgtgcacgaagcagaggt gggtgttatggtggatgagggccagtccactgcccagttccctcagtgagcgcagccccagccagctgatgcccagcccttgcaggg tcagcgagtaggcgccattgtgcagaattcgtccccggattacttgcaggttctggaagacgctgaggtcaggcaggctgtccggcca tgctgagatgtataggtaacctgtgatctcttccagagtctcaaacacttggagctgctctggctggagcggggcagtgttggaggctgg gtccccatcaaagctctccggcagaaatgccaggctcccaaagatcttcttgcagccagcaaactcctggatattcttccacaaaatcgt gtcctggtagcagagctgggggttccgctggatcaagacccctcctttcaagatctctgtgaggcttcgaagctgcagctcccgcaggc ctcctggggaggcccctgtgacaggggtggtattgttcagcgggtctccattgtctagcacggccagggcatagttgtcctcaaagagc tgggtgcctcgcacaatccgcagcctctgcagtgggacctgcctcacttggttgtgagcgatgagcacgtagccctgcacctcctggat atcctgcaggaaggacaggctggcattggtgggcaggtaggtgagttccaggtttccctgcaccacctggcagccctggtagaggtg gcggagcatgtccaggtgggttctagatttatcacgtacccatttccccgcatcttttatttttttaaatactttagggaaaaatggtttttgattt gcttttaaaggttgtggtgtagactcgtctgctgactgcatgctagaatctaagtcactttcagaagcatccacaactgactctttcgccact tttctcttatttgcttttgttggtttatctggataagtaaggctttcaagctcactatccgacgacgctatggcttttcttctttttttaatttccgctg cgctatccgatgacagacctggatgacgacgctccacttgcagagttggtcggtcgactcctgaagcctcttcatttatagccacatttcc tgtttgctcaccgttgttattattgttattcggacctttctctgcttttgctttcaacattgctattaggtctgctttgttcgtatttttcactttattcga tttttctagttcctcaatatcacgtgaacttacttcacgtgcagtttcgtatcttggtcccgtatttacctcgcttggctgctcttctgttttttcttct tcccattcatctgtgtttagactggaatcttcgctatctgtcgctgcaaatattatgtcggggttaatcgtaatgcagttggcagtaatgaaaa ctaccatcatcgcacgcataaatctgtttaatcccacttatactccctcctcgtgatacgctaatacaacctttttagaacaaggaaaattcg gccttcattttcactaatttgttccgttaaaaattggattagcagttagttatcttcttaattagctaatataagaaaaaatattcatgaattatttta agaatatcacttggagaattaatttttctctaacatttgttaatcagttaaccccaactgcttcccaagcttcacccgggccactaactcaacg ctagtagtggatttaatcccaaatgagccaacagaaccagaaccagaaacagaacaagtaacattggagttagaaatggaagaagaa aaaagcaatgatttcgtgtgaataatgcacgaaatcattgcttatttttttaaaaagcgatatactagatataacgaaacaacgaactgaata aagaatacaaaaaaagagccacgaccagttaaagcctgagaaactttaactgcgagccttaattgattaccaccaatcaattaaagaagt cgagacccaaaatttggtaaagtatttaattactttattaatcagatacttaaatatctgtaaacccattatatcgggtttttgaggggatttcaa gtctttaagaagataccaggcaatcaattaagaaaaacttagttgattgccttttttgttgtgattcaactttgatcgtagcttctaactaattaat tttcgtaagaaaggagaacagctgaatgaatatcccttttgttgtagaaactgtgcttcatgacggcttgttaaagtacaaatttaaaaatag taaaattcgctcaatcactaccaagccaggtaaaagtaaaggggctatttttgcgtatcgctcaaaaaaaagcatgattggcggacgtgg cgttgttctgacttccgaagaagcgattcacgaaaatcaagatacatttacgcattggacaccaaacgtttatcgttatggtacgtatgcag acgaaaaccgttcatacactaaaggacattctgaaaacaatttaagacaaatcaataccttctttattgattttgatattcacacggaaaaag aaactatttcagcaagcgatattttaacaacagctattgatttaggttttatgcctacgttaattatcaaatctgataaaggttatcaagcatatt ttgttttagaaacgccagtctatgtgacttcaaaatcagaatttaaatctgtcaaagcagccaaaataatctcgcaaaatatccgagaatatt ttggaaagtctttgccagttgatctaacgtgcaatcattttgggattgctcgtataccaagaacggacaatgtagaattttttgatcccaatta ccgttattctttcaaagaatggcaagattggtctttcaaacaaacagataataagggctttactcgttcaagtctaacggttttaagcggtac agaaggcaaaaaacaagtagatgaaccctggtttaatctcttattgcacgaaacgaaattttcaggagaaaagggtttagtagggcgca atagcgttatgtttaccctctctttagcctactttagttcaggctattcaatcgaaacgtgcgaatataatatgtttgagtttaataatcgattag atcaacccttagaagaaaaagaagtaatcaaaattgttagaagtgcctattcagaaaactatcaaggggctaatagggaatacattacca ttctttgcaaagcttgggtatcaagtgatttaaccagtaaagatttatttgtccgtcaagggtggtttaaattcaagaaaaaaagaagcgaac gtcaacgtgttcatttgtcagaatggaaagaagatttaatggcttatattagcgaaaaaagcgatgtatacaagccttatttagcgacgacc aaaaaagagattagagaagtgctaggcattcctgaacggacattagataaattgctgaaggtactgaaggcgaatcaggaaattttcttt aagattaaaccaggaagaaatggtggcattcaacttgctagtgttaaatcattgttgctatcgatcattaaattaaaaaaagaagaacgag aaagctatataaaggcgctgacagcttcgtttaatttagaacgtacatttattcaagaaactctaaacaaattggcagaacgccccaaaac ggacccacaactcgatttgtttagctacgatacaggctgaaaataaaacccgcactatgccattacatttatatctatgatacgtgtttgttttt ctttgctggctagcttaattgcttatatttacctgcaataaaggatttcttacttccattatactcccattttccaaaaacatacggggaacacg ggaacttattgtacaggccacctcatagttaatggtttcgagccttcctgcaatctcatccatggaaatatattcatccccctgccggcctat taatgtgacttttgtgcccggcggatattcctgatccagctccaccataaattggtccatgcaaattcggccggcaattttcaggcgttttcc cttcacaaggatgtcggtccctttcaattttcggagccagccgtccgcatagcctacaggcaccgtcccgatccatgtgtctttttccgctg tgtactcggctccgtagctgacgctctcgccttttctgatcagtttgacatgtgacagtgtcgaatgcagggtaaatgccggacgcagctg aaacggtatctcgtccgacatgtcagcagacgggcgaaggccatacatgccgatgccgaatctgactgcattaaaaaagccttttttca gccggagtccagcggcgctgttcgcgcagtggaccattagattctttaacggcagcggagcaatcagctctttaaagcgctcaaactgc attaagaaatagcctctttctttttcatccgctgtcgcaaaatgggtaaatacccctttgcactttaaacgagggttgcggtcaagaattgcc atcacgttctgaacttcttcctctgtttttacaccaagtctgttcatccccgtatcgaccttcagatgaaaatgaagagaaccttttttcgtgtg gcgggctgcctcctgaagccattcaacagaataacctgttaaggtcacgtcatactcagcagcgattgccacatactccgggggaacc gcgccaagcaccaatataggcgccttcaatccctttttgcgcagtgaaatcgcttcatccaaaatggccacggccaagcatgaagcacc tgcgtcaagagcagcctttgctgtttctgcatcaccatgcccgtaggcgtttgctttcacaactgccatcaagtggacatgttcaccgatat gttttttcatattgctgacattttcctttatcacggacaagtcaatttccgcccacgtatctctgtaaaaaggttttgtgctcatggaaaactcct ctcttttttcagaaaatcccagtacgtaattaagtatttgagaattaattttatattgattaatactaagtttacccagttttcacctaaaaaacaa atgatgagataatagctccaaaggctaaagaggactataccaactatttgttaat (SEQ ID NO: 11).

[00082] In one embodiment, provided herein is a vaccine comprising a recombinant Listeria strain, wherein said Listeria further comprises a bivalent or multivalent plasmid disclosed herein and an adjuvant, cytokine, chemokine, or a combination thereof. In one embodiment, provided herein is an immunotherapy comprising a recombinant Listeria strain, wherein said Listeria further comprises a bivalent or multivalent plasmid disclosed herein and an adjuvant, cytokine, chemokine, or a combination thereof. In another embodiment, provided herein is a pharmaceutical formulation comprising a recombinant Listeria strain, wherein said Listeria further comprises the bivalent or multivalent plasmid disclosed herein and an adjuvant, cytokine, chemokine, or a combination thereof. [00083] In one embodiment of the present invention, disclosed herein is a recombinant Listeria strain comprising a first and second nucleic acid molecule, each said nucleic acid molecule encoding a heterologous antigenic polypeptide or fragment thereof, wherein the first nucleic acid molecule is integrated into the Listeria genome in an open reading frame with an endogenous LLO gene and wherein the second nucleic acid molecule is present in an episomal expression vector or plasmid within the recombinant Listeria strain. In one embodiment, the first nucleic acid molecule encodes a chimeric HER2 (cHER2) protein fused to an endogenous LLO protein and the second nucleic acid molecule encodes an HMW-MAA peptide fused to a truncated LLO, truncated ActA protein, or PEST sequence. [00084] In one embodiment, this invention provides a recombinant Listeria strain comprising a first and second nucleic acid molecule, each said nucleic acid molecule encoding a heterologous antigenic polypeptide fused to a truncated LLO, a truncated or N-terminal ActA protein or a PEST sequence. [00085] In one embodiment, the first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with an endogenous nucleic acid sequence encoding a an LLO protein, an ActA protein or a PEST sequence. In one embodiment, the first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a nucleic acid sequence encoding LLO. In another embodiment, the first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a nucleic acid sequence encoding ActA. In one embodiment, the integration does not eliminate the functionality of LLO. In another embodiment, the integration does not eliminate the functionality of ActA. In one embodiment, the functionality of LLO or ActA is its native functionality. [00086] In one embodiment, the LLO functionality is allowing the organism to escape from the phagolysosome, while in another embodiment, the LLO functionality is enhancing the immunogenicity of a polypeptide to which it is fused. In one embodiment, a recombinant Listeria of the present invention retains LLO function, which in one embodiment, is hemolytic function and in another embodiment, is antigenic function. Other functions of LLO are known in the art, as are methods and assays for evaluating LLO functionality. [00087] In one embodiment, a recombinant Listeria of the present invention has wild-type virulence, while in another embodiment, a recombinant Listeria of the present invention has attenuated virulence. In another embodiment, a recombinant Listeria of the present invention is avirulent. In one embodiment, a recombinant Listeria of the present invention is sufficiently virulent to escape the phagolysosome and enter the cytosol. In one embodiment, a recombinant Listeria of the present invention expresses a fused antigen-LLO protein. Thus, in one embodiment, the integration of the first nucleic acid molecule into the Listeria genome does not disrupt the structure nor, in another embodiment, the function of the endogenous LLO gene, ActA gene, or PEST-containing gene. In one embodiment, the integration of the first nucleic acid molecule into the Listeria genome does not disrupt the ability of said Listeria to escape the phagolysosome. [00088] In another embodiment, either the the second nucleic acid is integrated into the genome while the first is expressed from a plasmid. In another embodiment, the second nucleic acid molecule is operably integrated into the Listeria genome with said first nucleic acid molecule in an open reading frame with an endogenous polypeptide comprising a PEST sequence. Thus, in one embodiment, the first and second nucleic acid molecules are integrated in frame with a nucleic acid sequence encoding an LLO protein, while in another embodiment, they are integrated in frame with a nucleic acid sequence encoding an ActA protein. In another embodiment, the second nucleic acid molecule is operably integrated into the Listeria genome in an open reading frame with a nucleic acid sequence encoding a polypeptide comprising a PEST sequence in a site that is distinct from the integration site of the first nucleic acid molecule. In one embodiment, the first nucleic acid molecule is integrated in frame with a nucleic acid sequence encoding an LLO protein, while the second nucleic acid molecule is integrated in frame with a nucleic acid sequence encoding an ActA protein, while in another embodiment, the first nucleic acid molecule is integrated in frame with a nucleic acid sequence encoding an ActA protein, while the second nucleic acid molecule is integrated in frame with a nucleic acid sequence encoding a LLO protein. [00089] In another embodiment, this invention provides a recombinant Listeria strain comprising a first nucleic acid molecule encoding a first heterologous antigenic polypeptide or fragment thereof and a second nucleic acid molecule encoding a second heterologous antigenic polypeptide or fragment thereof, wherein said first nucleic acid molecule is integrated into the Listeria genome such that the first heterologous antigenic polypeptide and an LLO, ActA or PEST sequence are expressed as a fusion protein. In one embodiment, the first heterologous antigenic polypeptide and the LLO, ActA or PEST sequence are translated in a single open reading frame, while in another embodiment, the first heterologous antigenic polypeptide and the LLO, ActA or PEST sequence are fused after being translated separately. [00090] In one embodiment, the Listeria genome comprises a deletion of the endogenous ActA gene, which in one embodiment is a virulence factor. In one embodiment, such a deletion provides a more attenuated and thus safer Listeria strain for human use. According to this embodiment, the antigenic polypeptide is integrated in frame with LLO in the Listeria chromosome. In another embodiment, the integrated nucleic acid molecule is integrated into the ActA locus. In another embodiment, the chromosomal nucleic acid encoding ActA is replaced by a nucleic acid molecule encoding an antigen. In another embodiment, the Listeria strain comprises an inactivation of the endogenous actA gene. In another embodiment, the Listeria strain comprises an truncation of the endogenous actA gene. In another embodiment, the Listeria strain comprises a non-functional replacement of the endogenous actA gene. In another embodiment, the Listeria strain comprises a substitution of the endogenous actA gene. All of the above-mentioned modifications fall within the scope of what is considered to be a“mutation” of the endogenous actA gene. [00091] In another embodiment, the Listeria strain provided herein comprises a mutation, deletion or an inactivation of the endogenous dal/dat and actA genes and such a Listeria strain is referred to herein as an“LmddA” strain. [00092] In another embodiment, the Listeria strain provided herein comprises a mutation, deletion or an inactivation of the endogenous dal/dat/actA and prfA genes. [00093] In one embodiment, the bivalent or multivalent plasmids provided herein comprise a replication control region. In one embodiment, a recombinant nucleic acid molecule encoding the bivalent or multivalent plasmid provided herein comprises a replication control region. In another embodiment, the plasmid control region regulates replication of the recombinant nucleic acid molecule. [00094] In another embodiment, the plasmid control region comprises an open reading frame encoding a transcription repressor that represses heterologous antigen expression from the first or at least the second nucleic acid molecule. In another embodiment, the plasmid control region comprises an open reading frame encoding transcription inducer that induces heterologous antigen expression from the first or at least the second nucleic acid molecule. In another embodiment, the plasmid control region comprises an open reading frame encoding a transcription repressor that represses heterologous antigen expression from a first, second or third nucleic acid molecule. In another embodiment, the plasmid control region comprises an open reading frame encoding a transcription inducer that induces heterologous antigen expression from the first, second or third nucleic acid molecule. [00095] In another embodiment, the plasmid replication regulation region enables the regulation of expression of exogenous heterologous antigen from each of the first or at least the second open reading frame of a recombinant nucleic acid molecule comprised by the Listeria or the plasmid disclosed herein. In another embodiment, the plasmid replication regulation region enables the regulation of expression of exogenous heterologous antigen from each of the first, second or third open reading frames. [00096] In one embodiment, measuring metabolic burden in a bacteria such as a Listeria is accomplished by any means know in the art at the time of the invention which include but are not limited to, measuring growth rates of the vaccine strain, optical density readings, colony forming unit (CFU) plating, and the like. In another embodiment, the metabolic burden on the bacterial cell is determined by measuring the viability of the bacterial cell. Methods of measuring bacteria viability are readily known and available in the art, some of which include but are not limited to, bacteria plating for viability count, measuring ATP, and flow cytometry. In ATP staining, detection is based on using the luciferase reaction to measure the amount of ATP from viable cells, wherein the amount of ATP in cells correlates with cell viability. As to flow cytometry, this method can be used in various ways, also known in the art, for example after employing the use of viability dyes which are excluded by live bacterial cells and are absorbed or adsorbed by a dead bacterial cells. A skilled artisan would readily understand that these and any other methods known in the art for measuring bacterial viability can be used in the present invention. It is to be understood that a skilled artisan would be able to implement the knowledge available in the art at the time of the invention for measuring growth rates of the vaccine strain or expression of marker genes by the vaccine strain that enable determining the metabolic burden of the vaccine strain expressing multiple heterologous antigens or functional fragments thereof. [00097] In another embodiment, the integrated nucleic acid molecule is integrated into the Listeria chromosome. [00098] In one embodiment, said first nucleic acid molecule is a vector designed for site- specific homologous recombination into the Listeria genome. In another embodiment, the construct or heterologous gene is integrated into the Listerial chromosome using homologous recombination. [00099] Techniques for homologous recombination are well known in the art, and are described, for example, in Frankel, FR, Hegde, S, Lieberman, J, and Y Paterson. Induction of a cell-mediated immune response to HIV gag using Listeria monocytogenes as a live vaccine vector. J. Immunol. 155: 4766 - 4774. 1995; Mata, M, Yao, Z, Zubair, A, Syres, K and Y Paterson, Evaluation of a recombinant Listeria monocytogenes expressing an HIV protein that protects mice against viral challenge. Vaccine 19:1435-45, 2001; Boyer, JD, Robinson, TM, Maciag, PC, Peng, X, Johnson, RS, Pavlakis, G, Lewis, MG, Shen, A, Siliciano, R, Brown, CR, Weiner, D, and Y Paterson. DNA prime Listeria boost induces a cellular immune response to SIV antigens in the Rhesus Macaque model that is capable of limited suppression of SIV239 viral replication. Virology. 333: 88-101, 2005. In another embodiment, homologous recombination is performed as described in United States Patent No. 6,855,320. In another embodiment, a temperature sensitive plasmid is used to select the recombinants. [000100] In another embodiment, the construct or heterologous gene is integrated into the Listerial chromosome using transposon insertion. Techniques for transposon insertion are well known in the art, and are described, inter alia, by Sun et al. (Infection and Immunity 1990, 58: 3770-3778) in the construction of DP-L967. Transposon mutagenesis has the advantage, in one embodiment, that a stable genomic insertion mutant can be formed. In another embodiment, the position in the genome where the foreign gene has been inserted by transposon mutagenesis is unknown. [000101] In another embodiment, the construct or heterologous gene is integrated into the Listerial chromosome using phage integration sites (Lauer P, Chow MY et al, Construction, characterization, and use of two LM site-specific phage integration vectors. J Bacteriol 2002;184(15): 4177-86). In another embodiment, an integrase gene and attachment site of a bacteriophage (e.g. U153 or PSA listeriophage) is used to insert the heterologous gene into the corresponding attachment site, which can be any appropriate site in the genome (e.g. comK or the 3’ end of the arg tRNA gene). In another embodiment, endogenous prophages are cured from the attachment site utilized prior to integration of the construct or heterologous gene. In another embodiment, this method results in single-copy integrants. [000102] In another embodiment, the first nucleic acid sequence of methods and compositions as provided herein is operably linked to a promoter/regulatory sequence. In another embodiment, the second nucleic acid sequence is operably linked to a promoter/regulatory sequence. In another embodiment, each of the nucleic acid sequences disclosed herein are operably linked to a promoter/regulatory sequence. In one embodiment, the promoter/regulatory sequence is present on an episomal plasmid cmprising said nucleic acid sequence. In one embodiment, an endogenous Listeria promoter/regulatory sequence controls the expression of a nucleic acid sequence of the methods and compositions of the present invention. [000103] In another embodiment, a nucleic acid sequence disclosed herein is operably linked to a promoter, regulatory sequence, or a combination thereof that drives expression of the encoded peptide in the Listeria strain. Promoter, regulatory sequences, and combinations thereof useful for driving constitutive expression of a gene are well known in the art and include, but are not limited to, for example, the P_hlyA, P_actA, hly, and p60 promoters of Listeria, the Streptococcus bac promoter, the Streptomyces griseus sgiA promoter, and the B. thuringiensis phaZ promoter. In another embodiment, inducible and tissue specific expression of the nucleic acid encoding a peptide as provided herein is accomplished by placing the nucleic acid encoding the peptide under the control of an inducible or tissue-specific promoter/regulatory sequence. Examples of tissue-specific or inducible regulatory sequences, promoters, and combinations thereof which are useful for his purpose include, but are not limited to the MMTV LTR inducible promoter, and the SV40 late enhancer/promoter. In another embodiment, a promoter that is induced in response to inducing agents such as metals, glucocorticoids, and the like, is utilized. Thus, it will be appreciated that the invention includes the use of any promoter or regulatory sequence, which is either known or unknown, and which is capable of driving expression of the desired protein operably linked thereto. In one embodiment, a regulatory sequence is a promoter, while in another embodiment, a regulatory sequence is an enhancer, while in another embodiment, a regulatory sequence is a suppressor, while in another embodiment, a regulatory sequence is a repressor, while in another embodiment, a regulatory sequence is a silencer. [000104] In one embodiment, the nucleic acid construct used for integration to the Listeria genome contains an integration site. In one embodiment, the site is a PhSA (phage from Scott A) attPP' integration site. PhSA is, in another embodiment, the prophage of L. monocytogenes strain ScottA (Loessner, M. J., I. B. Krause, T. Henle, and S. Scherer. 1994. Structural proteins and DNA characteristics of 14 Listeria typing bacteriophages. J. Gen. Virol. 75:701– 710, incorporated herein by reference), a serotype 4b strain that was isolated during an epidemic of human listeriosis. In another embodiment, the site is any another integration site known in the art. [000105] In another embodiment, the nucleic acid construct contains an integrase gene. In another embodiment, the integrase gene is a PhSA integrase gene. In another embodiment, the integrase gene is any other integrase gene known in the art. [000106] In one embodiment, the nucleic acid construct is a plasmid. In another embodiment, the nucleic acid construct is a shuttle plasmid. In another embodiment, the nucleic acid construct is an integration vector. In another embodiment, the nucleic acid construct is a site- specific integration vector. In another embodiment, the nucleic acid construct is any other type of nucleic acid construct known in the art. [000107] The integration vector of methods and compositions disclosed herein is, in another embodiment, a phage vector. In another embodiment, the integration vector is a site-specific integration vector. In another embodiment, the vector further comprises an attPP’ site. [000108] In another embodiment, the integration vector is a U153 vector. In another embodiment, the integration vector is an A118 vector. In another embodiment, the integration vector is a PhSA vector. [000109] In another embodiment, the vector is an A511 vector (e.g. GenBank Accession No: X91069). In another embodiment, the vector is an A006 vector. In another embodiment, the vector is a B545 vector. In another embodiment, the vector is a B053 vector. In another embodiment, the vector is an A020 vector. In another embodiment, the vector is an A500 vector (e.g. GenBank Accession No: X85009). In another embodiment, the vector is a B051 vector. In another embodiment, the vector is a B052 vector. In another embodiment, the vector is a B054 vector. In another embodiment, the vector is a B055 vector. In another embodiment, the vector is a B056 vector. In another embodiment, the vector is a B101 vector. In another embodiment, the vector is a B110 vector. In another embodiment, the vector is a B1 ll vector. In another embodiment, the vector is an A153 vector. In another embodiment, the vector is a D441 vector. In another embodiment, the vector is an A538 vector. In another embodiment, the vector is a B653 vector. In another embodiment, the vector is an A513 vector. In another embodiment, the vector is an A507 vector. In another embodiment, the vector is an A502 vector. In another embodiment, the vector is an A505 vector. In another embodiment, the vector is an A519 vector. In another embodiment, the vector is a B604 vector. In another embodiment, the vector is a C703 vector. In another embodiment, the vector is a B025 vector. In another embodiment, the vector is an A528 vector. In another embodiment, the vector is a B024 vector. In another embodiment, the vector is a B012 vector. In another embodiment, the vector is a B035 vector. In another embodiment, the vector is a C707 vector. [000110] In another embodiment, the vector is an A005 vector. In another embodiment, the vector is an A620 vector. In another embodiment, the vector is an A640 vector. In another embodiment, the vector is a B021 vector. In another embodiment, the vector is an HSO47 vector. In another embodiment, the vector is an H10G vector. In another embodiment, the vector is an H8/73 vector. In another embodiment, the vector is an H19 vector. In another embodiment, the vector is an H21 vector. In another embodiment, the vector is an H43 vector. In another embodiment, the vector is an H46 vector. In another embodiment, the vector is an H107 vector. In another embodiment, the vector is an H108 vector. In another embodiment, the vector is an H110 vector. In another embodiment, the vector is an H163/84 vector. In another embodiment, the vector is an H312 vector. In another embodiment, the vector is an H340 vector. In another embodiment, the vector is an H387 vector. In another embodiment, the vector is an H391/73 vector. In another embodiment, the vector is an H684/74 vector. In another embodiment, the vector is an H924A vector. In another embodiment, the vector is an fMLUP5 vector. In another embodiment, the vector is a syn (=P35) vector. In another embodiment, the vector is a 00241 vector. In another embodiment, the vector is a 00611 vector. In another embodiment, the vector is a 02971A vector. In another embodiment, the vector is a 02971C vector. In another embodiment, the vector is a 5/476 vector. In another embodiment, the vector is a 5/911 vector. In another embodiment, the vector is a 5/939 vector. In another embodiment, the vector is a 5/11302 vector. In another embodiment, the vector is a 5/11605 vector. In another embodiment, the vector is a 5/11704 vector. In another embodiment, the vector is a 184 vector. In another embodiment, the vector is a 575 vector. In another embodiment, the vector is a 633 vector. In another embodiment, the vector is a 699/694 vector. In another embodiment, the vector is a 744 vector. In another embodiment, the vector is a 900 vector. In another embodiment, the vector is a 1090 vector. In another embodiment, the vector is a 1317 vector. In another embodiment, the vector is a 1444 vector. In another embodiment, the vector is a 1652 vector. In another embodiment, the vector is a 1806 vector. In another embodiment, the vector is a 1807 vector. In another embodiment, the vector is a 1921 /959 vector. In another embodiment, the vector is a 1921/11367 vector. In another embodiment, the vector is a 1921/11500 vector. In another embodiment, the vector is a 1921/11566 vector. In another embodiment, the vector is a 1921/12460 vector. In another embodiment, the vector is a 1921/12582 vector. In another embodiment, the vector is a 1967 vector. In another embodiment, the vector is a 2389 vector. In another embodiment, the vector is a 2425 vector. In another embodiment, the vector is a 2671 vector. In another embodiment, the vector is a 2685 vector. In another embodiment, the vector is a 3274 vector. In another embodiment, the vector is a 3550 vector. In another embodiment, the vector is a 3551 vector. In another embodiment, the vector is a 3552 vector. In another embodiment, the vector is a 4276 vector. In another embodiment, the vector is a 4277 vector. In another embodiment, the vector is a 4292 vector. In another embodiment, the vector is a 4477 vector. In another embodiment, the vector is a 5337 vector. In another embodiment, the vector is a 5348/11363 vector. In another embodiment, the vector is a 5348/11646 vector. In another embodiment, the vector is a 5348/12430 vector. In another embodiment, the vector is a 5348/12434 vector. In another embodiment, the vector is a 10072 vector. In another embodiment, the vector is a 11355C vector. In another embodiment, the vector is a 11711A vector. In another embodiment, the vector is a 12029 vector. In another embodiment, the vector is a 12981 vector. In another embodiment, the vector is a 13441 vector. In another embodiment, the vector is a 90666 vector. In another embodiment, the vector is a 90816 vector. In another embodiment, the vector is a 93253 vector. In another embodiment, the vector is a 907515 vector. In another embodiment, the vector is a 910716 vector. In another embodiment, the vector is a NN-Listeria vector. In another embodiment, the vector is a O1761 vector. In another embodiment, the vector is a 4211 vector. In another embodiment, the vector is a 4286 vector. In another embodiment, the integration vector is any other site- specific integration vector known in the art that is capable of infecting Listeria. [000111] . In another embodiment, the integration vector or plasmid of methods and compositions as provided herein does not confer antibiotic resistance to the Listeria vaccine strain. In another embodiment, the integration vector or plasmid does not contain an antibiotic resistance gene.. [000112] In another embodiment, the present invention provides an recombinant nucleic acid encoding a recombinant polypeptide. In one embodiment, the nucleic acid comprises a sequence sharing at least 80% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. In another embodiment, the nucleic acid comprises a sequence sharing at least 85% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. In another embodiment, the nucleic acid comprises a sequence sharing at least 90% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. In another embodiment, the nucleic acid comprises a sequence sharing at least 95% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. In another embodiment, the nucleic acid comprises a sequence sharing at least 97% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. In another embodiment, the nucleic acid comprises a sequence sharing at least 99% homology with a nucleic acid encoding a recombinant polypeptide disclosed herein. [000113] In one embodiment, provided herein is a method of producing a recombinant Listeria strain comprising a bivalent plasmid encoding two distinct heterologous antigens. In another embodiment, the plasmid is a multivalent plasmid that encodes 3 or more distinct heterologous antigens. In another embodiment, the plasmid is a multivalent plasmid that encodes 4 or more distinct heterologous antigens. In another embodiment, the plasmid is a multivalent plasmid that encodes 5 or more distinct heterologous antigens. [000114] In one embodiment, the recombinant Listeria provided herein expresses at leas at one antigen encoded by the plasmids disclosed herein. [000115] In one embodiment, disclosed is a method of producing a recombinant Listeria strain expressing two distinct heterologous antigens. In another embodiment, the recombinant Listeria expresses at least 3 or more distinct heterologous antigens. In another embodiment, the recombinant Listeria expresses 4 or more distinct heterologous antigens. In another embodiment, the recombinant Listeria expresses 5 or more distinct heterologous antigens. [000116] In another embodiment, the method of producing a recombinant Listeria comprises transforming said recombinant Listeria with nucleic acid comprising a bivalent or multivalent plasmid. In one embodiment, the plasmid is an episomal plasmid that remains extrachromosomal. In another embodiment, the plasmid is an integrative plasmid. In yet another embodiment, the method provided herein comprises expressing the antigens and fusion proteins disclosed herein under conditions conducive to protein expression.. [000117] It will be appreciated by a skilled artisan that the nucleic acids described herein comprise DNA vectors, RNA vectors, plasmids (extrachromosomal and/or integrative), etc., that may be used in the methods provided herein for generating any of the compositions disclosed herein. [000118] In another embodiment, the recombinant Listeria strain may express more than two antigens, some of which are expressed from one or more nucleic acid molecules integrated into the Listeria chromosome and some of which are expressed via one or more episomal expression plasmids or vectors present in the recombinant Listeria strain. Thus, as described hereinabove, in one embodiment, a recombinant Listeria strain as provided herein comprises two or more episomal expression plasmids, each of which expresses at least one distinct antigenic polypeptide. In one embodiment, one or more of the antigens are expressed as a fusion protein with LLO, which in one embodiment, is non-hemolytic LLO or truncated LLO. In one embodiment, a recombinant Listeria strain as provided herein targets tumors by eliciting immune responses to two separate antigens, which are expressed by two different cell types, which in one embodiment are a cell surface antigen and an anti-angiogenic polypeptide, while in another embodiment, a recombinant Listeria strain as provided herein targets tumors by eliciting an immune response to two different antigens expressed by the same cell type. In another embodiment, a recombinant Listeria strain as provided herein targets tumors by eliciting an immune response to two different antigens as described hereinbelow or as are known in the art. [000119] In one embodiment, a heterologous antigen disclosed herein is associated with the local tissue environment that is further associated with the development of or metastasis of cancer. In another embodiment, the heterologous antigen provided herein is associated with tumor immune evasion or resistance to cancer. In another embodiment, the heterologous antigen provided herein is an angiogenic antigen. [000120] In one embodiment, a first antigen of the compositions and methods of the present invention is directed against a specific cell surface antigen or tumor target, and a second antigen is directed against an angiogenic antigen or tumor microenvironment. In another embodiment, the first and second antigens of the compositions and methods of the present invention are polypeptides expressed by tumor cells, or in another embodiment, polypeptides expressed in a tumor microenvironment. In another embodiment, the first antigen of the compositions and methods of the present invention is a polypeptide expressed by a tumor and the second antigen of the compositions and methods of the present invention is a receptor target, NO Synthetase, Arg-1, or other enzyme known in the art. [000121] In one embodiment, provided herein is a method of producing a recombinant Listeria strain expressing two antigens, the method comprising, in one embodiment, genetically fusing a first nucleic acid encoding a first antigen and a second nucleic acid encoding a second antigen into the Listeria genome in an open reading frame with a native polypeptide comprising a PEST sequence. In another embodiment, the expressing said first and second antigens are produced under conditions conducive to antigenic expression in said recombinant Listeria strain. [000122] In one embodiment, the recombinant Listeria strain of the composition and methods as provided herein comprises an episomal expression vector comprising the second nucleic acid molecule encoding a heterologous antigen. In another embodiment, the second nucleic acid molecule encoding a heterologous antigen is present in said episomal expression vector in an open reading frame with a polypeptide comprising a PEST sequence. [000123] In one embodiment, the terms “heterologous antigen,” “antigen,” are used interchangeably herein. [000124] In another embodiment, an episomal expression vector of the methods and compositions as provided herein comprises an antigen fused in frame to a nucleic acid sequence encoding a PEST-like AA sequence. In one embodiment, the antigen is HMW- MAA, and in another embodiment, a HMW-MAA fragment. In another embodiment, the PEST-like AA sequence is KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 12). In another embodiment, the PEST-like sequence is KENSISSMAPPASPPASPK (SEQ ID No: 13). In another embodiment, fusion of an antigen to any LLO sequence that includes one of the PEST-like AA sequences enumerated herein can enhance cell mediated immunity against HMW-MAA. [000125] In another embodiment, the PEST-like AA sequence is a PEST-like sequence from a Listeria ActA protein. In another embodiment, the PEST-like sequence is KTEEQPSEVNTGPR (SEQ ID NO: 14), KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO: 15), KNEEVNASDFPPPPTDEELR (SEQ ID NO: 16), or RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR (SEQ ID NO: 17). In another embodiment, the PEST-like sequence is from Listeria seeligeri cytolysin, encoded by the lso gene. In another embodiment, the PEST-like sequence is RSEVTISPAETPESPPATP (SEQ ID NO: 18). In another embodiment, the PEST-like sequence is from Streptolysin O protein of Streptococcus sp. In another embodiment, the PEST-like sequence is from Streptococcus pyogenes Streptolysin O, e.g. KQNTASTETTTTNEQPK (SEQ ID NO: 19) at AA 35-51. In another embodiment, the PEST-like sequence is from Streptococcus equisimilis Streptolysin O, e.g. KQNTANTETTTTNEQPK (SEQ ID NO: 20) at AA 38-54. In another embodiment, the PEST-like sequence has a sequence selected from SEQ ID NO: 14-20. In another embodiment, the PEST-like sequence has a sequence selected from SEQ ID NO: 12-20. In another embodiment, the PEST-like sequence is another PEST-like AA sequence derived from a prokaryotic organism. In another embodiment, the PEST sequence is any other PEST sequence known in the art, including, but not limited to, those disclosed in United States Patent Publication No. 2014/0186387, which is incorporated by reference herein in its entirety [000126] Identification of PEST-like sequences is well known in the art, and is described, for example in Rogers S et al (Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 1986; 234(4774):364-8, incorporated herein by reference) and Rechsteiner M et al (PEST sequences and regulation by proteolysis. Trends Biochem Sci 1996; 21(7):267-71, incorporated herein by reference). “PEST-like sequence” refers, in another embodiment, to a region rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues. In another embodiment, the PEST-like sequence is flanked by one or more clusters containing several positively charged amino acids. In another embodiment, the PEST-like sequence mediates rapid intracellular degradation of proteins containing it. In another embodiment, the PEST-like sequence fits an algorithm disclosed in Rogers et al. In another embodiment, the PEST-like sequence fits an algorithm disclosed in Rechsteiner et al. In another embodiment, the PEST-like sequence contains one or more internal phosphorylation sites, and phosphorylation at these sites precedes protein degradation. In one embodiment, a sequence referred to herein as a PEST-like sequence is a PEST sequence. [000127] In one embodiment, PEST-like sequences of prokaryotic organisms are identified in accordance with methods such as described by, for example Rechsteiner and Rogers (1996, Trends Biochem. Sci. 21:267-271) for LM and in Rogers S et al (Science 1986; 234(4774):364-8). Alternatively, PEST-like AA sequences from other prokaryotic organisms can also be identified based on this method. Other prokaryotic organisms wherein PEST-like AA sequences would be expected to include, but are not limited to, other Listeria species. In one embodiment, the PEST-like sequence fits an algorithm disclosed in Rogers et al. In another embodiment, the PEST-like sequence fits an algorithm disclosed in Rechsteiner et al. In another embodiment, the PEST-like sequence is identified using the PEST-find program. [000128] In another embodiment, identification of PEST motifs is achieved by an initial scan for positively charged amino acids R, H, and K within the specified protein sequence. All amino acids between the positively charged flanks are counted and only those motifs are considered further, which contain a number of amino acids equal to or higher than the window-size parameter. In another embodiment, a PEST-like sequence must contain at least 1 P, 1 D or E, and at least 1 S or T. [000129] In another embodiment, the quality of a PEST motif is refined by means of a scoring parameter based on the local enrichment of critical amino acids as well as the motif's hydrophobicity. Enrichment of D, E, P, S and T is expressed in mass percent (w/w) and corrected for 1 equivalent of D or E, 1 of P and 1 of S or T. In another embodiment, calculation of hydrophobicity follows in principle the method of J. Kyte and R.F. Doolittle (Kyte, J and Dootlittle, RF. J. Mol. Biol. 157, 105 (1982), incorporated herein by reference. For simplified calculations, Kyte-Doolittle hydropathy indices, which originally ranged from - 4.5 for arginine to +4.5 for isoleucine, are converted to positive integers, using the following linear transformation, which yielded values from 0 for arginine to 90 for isoleucine. [000130] Hydropathy index = 10 * Kyte-Doolittle hydropathy index + 45 [000131] In another embodiment, a potential PEST motif’s hydrophobicity is calculated as the sum over the products of mole percent and hydrophobicity index for each amino acid species. The desired PEST score is obtained as combination of local enrichment term and hydrophobicity term as expressed by the following equation: [000132] PEST score = 0.55 * DEPST - 0.5 * hydrophobicity index. [000133] In another embodiment,“PEST sequence,”“PEST-like sequence” or“PEST-like sequence peptide” are used interchangeably here and refer to a peptide having a score of at least +5, using the above algorithm. In another embodiment, the term refers to a peptide having a score of at least 6. In another embodiment, the peptide has a score of at least 7. In another embodiment, the score is at least 8. In another embodiment, the score is at least 9. In another embodiment, the score is at least 10. In another embodiment, the score is at least 11. In another embodiment, the score is at least 12. In another embodiment, the score is at least 13. In another embodiment, the score is at least 14. In another embodiment, the score is at least 15. In another embodiment, the score is at least 16. In another embodiment, the score is at least 17. In another embodiment, the score is at least 18. In another embodiment, the score is at least 19. In another embodiment, the score is at least 20. In another embodiment, the score is at least 21. In another embodiment, the score is at least 22. In another embodiment, the score is at least 22. In another embodiment, the score is at least 24. In another embodiment, the score is at least 24. In another embodiment, the score is at least 25. In another embodiment, the score is at least 26. In another embodiment, the score is at least 27. In another embodiment, the score is at least 28. In another embodiment, the score is at least 29. In another embodiment, the score is at least 30. In another embodiment, the score is at least 32. In another embodiment, the score is at least 35. In another embodiment, the score is at least 38. In another embodiment, the score is at least 40. In another embodiment, the score is at least 45. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000134] In another embodiment, the PEST sequence is identified using any other method or algorithm known in the art, e.g the CaSPredictor (Garay-Malpartida HM, Occhiucci JM, Alves J, Belizario JE. Bioinformatics. 2005 Jun;21 Suppl 1:i169-76). In another embodiment, the following method is used: [000135] A PEST index is calculated for each stretch of appropriate length (e.g. a 30-35 amino acid stretch) by assigning a value of 1 to the amino acids Ser, Thr, Pro, Glu, Asp, Asn, or Gln. The coefficient value (CV) for each of the PEST residue is 1 and for each of the other amino acids (non-PEST) is 0. [000136] Each method for identifying a PEST sequence represents a separate embodiment as provided herein. [000137] In another embodiment, the PEST sequence is any other PEST sequence known in the art. [000138] In one embodiment, the present invention provides fusion proteins, which in one embodiment, are expressed by Listeria. In one embodiment, such fusion proteins comprise fusions to a tLLO, a truncated ActA or a PEST sequence. It will be understood by a skilled artisan that the term“PEST sequence” may encompass cases wherein a protein fragment comprises a PEST sequence having surrounding sequences other than the PEST sequence. In another embodiment, the protein fragment consists of the PEST sequence. Thus, in another embodiment,“fusion” refers to two peptides or protein fragments either linked together at their respective ends or embedded one within the other. It will be appreciated by a skilled artisan that the term "fused" may also encompass an operable linkage by covalent bonding. In one embodiment, the term encompasses recombinant fusion (of nucleic acid sequences or open reading frames thereof). In another embodiment, the term encompasses chemical conjugation. [000139] In another embodiment, a recombinant Listeria strain of the compositions and methods as provided herein comprises a full length LLO polypeptide, which in one embodiment, is hemolytic. [000140] In another embodiment, the recombinant Listeria strain comprises a non-hemolytic LLO polypeptide. In another embodiment, the polypeptide is an LLO fragment. In another embodiment, the oligopeptide is a complete LLO protein. In another embodiment, the polypeptide is any LLO protein or fragment thereof known in the art. [000141] In another embodiment, an LLO protein fragment is utilized in compositions and methods as provided herein. In one embodiment, a truncated LLO protein is encoded by the episomal expression vector as provided herein that expresses a polypeptide, that is, in one embodiment, an antigen, in another embodiment, an angiogenic factor, or, in another embodiment, both an antigen and angiogenic factor. In another embodiment, the LLO fragment is an N-terminal fragment. [000142] In another embodiment, the N-terminal LLO fragment has the sequence: [00054] MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIE KKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQ NNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKI VVKNATKSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGT AFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQ ALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNI IKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNE LAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYD (SEQ ID NO: 21). In another embodiment, an LLO AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID No: 21. In another embodiment, the LLO AA sequence is a homologue of SEQ ID No: 21. In another embodiment, the LLO AA sequence is a variant of SEQ ID No: 21. In another embodiment, the LLO AA sequence is a fragment of SEQ ID No: 21. In another embodiment, the LLO AA sequence is an isoform of SEQ ID No: 21. [000143] In another embodiment, the LLO fragment has the sequence: MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADEI DKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQV VNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNAT KSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVN NSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNA ENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFK AVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKN NSEYIETTSKAYTD (SEQ ID NO: 22). In another embodiment, an LLO AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID No: 22. In another embodiment, the LLO AA sequence is a homologue of SEQ ID No: 22. In another embodiment, the LLO AA sequence is a variant of SEQ ID No: 22. In another embodiment, the LLO AA sequence is a fragment of SEQ ID No: 22. In another embodiment, the LLO AA sequence is an isoform of SEQ ID No: 22. [000144] In one embodiment, the LLO protein used in the compositions and methods as provided herein comprises the following sequence: [000145] MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIE KKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSIN QNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDN KIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKF GTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQ LQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVEL TNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLK DNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQH KNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKN RNISIWGTTLYPKYSNKVDNPIE (GenBank Accession No. P13128; SEQ ID NO: 23; nucleic acid sequence is set forth in GenBank Accession No. X15127). The first 25 AA of the proprotein corresponding to this sequence are the signal sequence and are cleaved from LLO when it is secreted by the bacterium. Thus, in this embodiment, the full length active LLO protein is 504 residues long. In another embodiment, the above LLO fragment is used as the source of the LLO fragment incorporated in a vaccine as provided herein. In another embodiment, an LLO AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID NO: 23. In another embodiment, the LLO AA sequence is a homologue of SEQ ID NO: 23. In another embodiment, the LLO AA sequence is a variant of SEQ ID NO: 23. In another embodiment, the LLO AA sequence is a fragment of SEQ ID NO: 23. In another embodiment, the LLO AA sequence is an isoform of SEQ ID NO: 23. Each possibility represents a separate embodiment as provided herein. [0043] The LLO protein used in the compositions and methods as provided herein has, in another embodiment, the sequence: MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADE IDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQ VVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNA TKSNVNNAVNTLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAV NNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVN AENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSF KAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIK NNSEYIETTSKAYTD (SEQ ID NO: 24). In another embodiment, an LLO AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID NO: 24. In another embodiment, the LLO AA sequence is a homologue of SEQ ID NO: 24. In another embodiment, the LLO AA sequence is a variant of SEQ ID NO: 24. In another embodiment, the LLO AA sequence is a fragment of SEQ ID NO: 24. In another embodiment, the LLO AA sequence is an isoform of SEQ ID NO: 24. Each possibility represents a separate embodiment as provided herein. [000146] In one embodiment, the amino acid sequence of the LLO polypeptide of the compositions and methods as provided herein is from the Listeria monocytogenes 10403S strain, as set forth in Genbank Accession No.: ZP_01942330, EBA21833, or is encoded by the nucleic acid sequence as set forth in Genbank Accession No.: NZ_AARZ01000015 or AARZ01000015.1. In another embodiment, the LLO sequence for use in the compositions and methods as provided herein is from Listeria monocytogenes, which in one embodiment, is the 4b F2365 strain (in one embodiment, Genbank accession number: YP_012823), the EGD- e strain (in one embodiment, Genbank accession number: NP_463733), or any other strain of Listeria monocytogenes known in the art. [000147] In another embodiment, the LLO sequence for use in the compositions and methods as provided herein is from Flavobacteriales bacterium HTCC2170 (in one embodiment, Genbank accession number: ZP_01106747 or EAR01433; in one embodiment, encoded by Genbank accession number: NZ_AAOC01000003). In one embodiment, proteins that are homologous to LLO in other species, such as alveolysin, which in one embodiment, is found in Paenibacillus alvei (in one embodiment, Genbank accession number: P23564 or AAA22224; in one embodiment, encoded by Genbank accession number: M62709) may be used in the compositions and methods as provided herein. Other such homologous proteins are known in the art. [000148] Each LLO protein and LLO fragment represents a separate embodiment of the methods and compositions as provided herein. [000149] In another embodiment, homologues of LLO from other species, including known lysins, or fragments thereof may be used to create a fusion protein of LLO with an antigen of the compositions and methods as provided herein, which in one embodiment, is HMW-MAA, and in another embodiment is a fragment of HMW-MAA. [000150] In another embodiment, the LLO fragment of methods and compositions as provided herein, is a PEST-like domain. In another embodiment, an LLO fragment that comprises a PEST sequence is utilized as part of a composition or in the methods as provided herein. [000151] In another embodiment, the LLO fragment does not contain the activation domain at the carboxy terminus. In another embodiment, the LLO fragment does not include cysteine 484. In another embodiment, the LLO fragment is a non-hemolytic fragment. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of the activation domain. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of cysteine 484. In another embodiment, an LLO sequence is rendered non-hemolytic by deletion or mutation at another location. [000152] In another embodiment, the LLO fragment consists of about the first 441 AA of the LLO protein. In another embodiment, the LLO fragment comprises about the first 400-441 AA of the 529 AA full length LLO protein. In another embodiment, the LLO fragment corresponds to AA 1-441 of an LLO protein disclosed herein. In another embodiment, the LLO fragment consists of about the first 420 AA of LLO. In another embodiment, the LLO fragment corresponds to AA 1-420 of an LLO protein disclosed herein. In another embodiment, the LLO fragment consists of about AA 20-442 of LLO. In another embodiment, the LLO fragment corresponds to AA 20-442 of an LLO protein disclosed herein. In another embodiment, any ǻ LLO without the activation domain comprising cysteine 484, and in particular without cysteine 484, are suitable for methods and compositions as provided herein. [000153] In another embodiment, the LLO fragment corresponds to the first 400 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 300 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 200 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 100 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 50 AA of an LLO protein, which in one embodiment, comprises one or more PEST-like sequences. [000154] In another embodiment, the LLO fragment is a non-hemolytic LLO. In another embodiment, the non-hemolytic LLO comprises one or more PEST-like sequences. [000155] In another embodiment, the LLO fragment contains residues of a homologous LLO protein that correspond to one of the above AA ranges. The residue numbers need not, in another embodiment, correspond exactly with the residue numbers enumerated above; e.g. if the homologous LLO protein has an insertion or deletion, relative to an LLO protein utilized herein. [000156] In another embodiment, a recombinant Listeria strain of the methods and compositions as provided herein comprise a nucleic acid molecule operably integrated into the Listeria genome as an open reading frame with an endogenous ActA sequence. In another embodiment, a recombinant Listeria strain of the methods and compositions as provided herein comprise an episomal expression vector comprising a nucleic acid molecule encoding fusion protein comprising an antigen fused to an ActA or a truncated ActA. In one embodiment, the expression and secretion of the antigen is under the control of an actA promoter and ActA signal sequence and it is expressed as fusion to 1-233 amino acids of ActA (truncated ActA or tActA). In another embodiment, the truncated ActA consists of the first 390 amino acids of the wild type ActA protein as described in US Patent Serial No. 7,655,238, which is incorporated by reference herein in its entirety. In another embodiment, the truncated ActA is an ActA-N100 or a modified version thereof (referred to as ActA- N100*) in which a PEST motif has been deleted and containing the nonconservative QDNKR substitution as described in US Patent Publication Serial No. 2014/0186387. In one embodiment, the antigen is HMW-MAA, while in another embodiment, it’s an immunogenic fragement of HMW-MAA. [000157] In one embodiment, the present invention provides a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof. In one embodiment, the present invention provides a recombinant polypeptide consisting of an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or fused to a fragment thereof. [000158] In another embodiment, the Her-2 chimeric protein of the methods and compositions of the present invention is a human Her-2 chimeric protein. In another embodiment, the Her-2 protein is a mouse Her-2 chimeric protein. In another embodiment, the Her-2 protein is a rat Her-2 chimeric protein. In another embodiment, the Her-2 protein is a primate Her-2 chimeric protein. In another embodiment, the Her-2 protein is a Her-2 chimeric protein of any other animal species or combinations thereof known in the art. Each possibility represents a separate embodiment of the present invention. [000159] In another embodiment, a Her-2 protein is a protein referred to as“HER- 2/neu,”“Erbb2,”“v-erb-b2,”“c-erb-b2,”“neu,” or“cNeu.” Each possibility represents a separate embodiment of the present invention. [000160] In one embodiment, the Her2-neu chimeric protein, harbors two of the extracellular and one intracellular fragments of Her2/neu antigen showing clusters of MHC-class I epitopes of the oncogene, where, in another embodiment, the chimeric protein, harbors 3 H2Dq and at least 17 of the mapped human MHC-class I epitopes of the Her2/neu antigen (fragments EC1, EC2, and IC1) as described in US Patent Application Serial No. 12/945,386, which is incorporated by reference herein in its entirety. In another embodiment, the Her2-neu chimeric protein is fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O (LLO) protein and expressed and secreted by the Listeria monocytogenes attenuated auxotrophic strain LmddA. In another embodiment, the Her2-neu chimeric protein is fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O (LLO) protein and is expressed from the chromosome of a recombinant Listeria provided herein, while an additional antigen is expressed from a plasmid present within the recombinant Listeria disclosed herein. In another embodiment, the Her2-neu chimeric protein is fused to the first 441 amino acids of the Listeria-monocytogenes listeriolysin O (LLO) protein and is expressed from a plasmid of a recombinant Listeria provided herein, while an additional antigen is expressed from the chromosome of the recombinant Listeria provided herein. In another embodiment, a recombinant Listeria provided herein is a Listeria monocytogenes attenuated auxotrophic strain LmddA. [000161] In another embodiment, the HER-2 chimeric protein is encoded by a nucleic acid sequence comprising SEQ ID NO:25 [000162] acccacctggacatgctccgccacctctaccagggctgccaggtggtgcagggaaacctggaactcacctacctgccc accaatgccagcctgtccttcctgcaggatatccaggaggtgcagggctacgtgctcatcgctcacaaccaagtgaggcaggtcccac tgcagaggctgcggattgtgcgaggcacccagctctttgaggacaactatgccctggccgtgctagacaatggagacccgctgaaca ataccacccctgtcacaggggcctccccaggaggcctgcgggagctgcagcttcgaagcctcacagagatcttgaaaggaggggtc ttgatccagcggaacccccagctctgctaccaggacacgattttgtggaagaatatccaggagtttgctggctgcaagaagatctttggg agcctggcatttctgccggagagctttgatggggacccagcctccaacactgccccgctccagccagagcagctccaagtgtttgaga ctctggaagagatcacaggttacctatacatctcagcatggccggacagcctgcctgacctcagcgtcttccagaacctgcaagtaatc cggggacgaattctgcacaatggcgcctactcgctgaccctgcaagggctgggcatcagctggctggggctgcgctcactgaggga actgggcagtggactggccctcatccaccataacacccacctctgcttcgtgcacacggtgccctgggaccagctctttcggaacccg caccaagctctgctccacactgccaaccggccagaggacgagtgtgtgggcgagggcctggcctgccaccagctgtgcgcccgag ggcagcagaagatccggaagtacacgatgcggagactgctgcaggaaacggagctggtggagccgctgacacctagcggagcga tgcccaaccaggcgcagatgcggatcctgaaagagacggagctgaggaaggtgaaggtgcttggatctggcgcttttggcacagtct acaagggcatctggatccctgatggggagaatgtgaaaattccagtggccatcaaagtgttgagggaaaacacatcccccaaagcca acaaagaaatcttagacgaagcatacgtgatggctggtgtgggctccccatatgtctcccgccttctgggcatctgcctgacatccacgg tgcagctggtgacacagcttatgccctatggctgcctcttagactaa (SEQ ID NO: 25). [000163] In another embodiment, the Her-2 chimeric protein (cHER2) comprises SEQ ID NO: 26: T H L D M L R H L Y Q G C Q V V Q G N L E L T Y L P T N A S L S F L Q D I Q E V Q G Y V L I A H N Q V R Q V P L Q R L R I V R G T Q L F E D N Y A L A V L D N G D P L N N T T P V T G A S P G G L R E L Q L R S L T E I L K G G V L I Q R N P Q L C Y Q D T I L W K N I Q E F A G C K K I F G S L A F L P E S F D G D P A S N T A P L Q P E Q L Q V F E T L E E I T G Y L Y I S A W P D S L P D L S V F Q N L Q V I R G R I L H N G A Y S L T L Q G L G I S W L G L R S L R E L G S G L A L I H H N T H L C F V H T V P W D Q L F R N P H Q A L L H T A N R P E D E C V G E G L A C H Q L C A R G Q Q K I R K Y T M R R L L Q E T E L V E P L T P S G A M P N Q A Q M R I L K E T E L R K V K V L G S G A F G T V Y K G I W I P D G E N V K I P V A I K V L R E N T S P K A N K E I L D E A Y V M A G V G S P Y V S R L L G I C L T S T V Q L V T Q L M P Y G C L L D (SEQ ID NO: 26). [000164] In one embodiment, the HER2 chimeric protein or fragment thereof of the methods and compositions provided herein does not include a signal sequence thereof. In another embodiment, omission of the signal sequence enables the HER2 fragment to be successfully expressed in Listeria, due the high hydrophobicity of the signal sequence. [000165] In another embodiment, the fragment of a HER2 chimeric protein of methods and compositions of the present invention does not include a transmembrane domain (TM) thereof. In one embodiment, omission of the TM enables the HER2 fragment to be successfully expressed in Listeria, due the high hydrophobicity of the TM. [000166] In one embodiment, the nucleic acid sequence of rat-Her2/neu gene is CCGGAATCGCGGGCACCCAAGTGTGTACCGGCACAGACATGAAGTTGCGGCTCC CTGCCAGTCCTGAGACCCACCTGGACATGCTCCGCCACCTGTACCAGGGCTGTCA GGTAGTGCAGGGCAACTTGGAGCTTACCTACGTGCCTGCCAATGCCAGCCTCTCA TTCCTGCAGGACATCCAGGAAGTTCAGGGTTACATGCTCATCGCTCACAACCAGG TGAAGCGCGTCCCACTGCAAAGGCTGCGCATCGTGAGAGGGACCCAGCTCTTTGA GGACAAGTATGCCCTGGCTGTGCTAGACAACCGAGATCCTCAGGACAATGTCGCC GCCTCCACCCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGT CTCACAGAGATCCTGAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCT ACCAGGACATGGTTTTGTGGAAGGACGTCTTCCGCAAGAATAACCAACTGGCTCC TGTCGATATAGACACCAATCGTTCCCGGGCCTGTCCACCTTGTGCCCCCGCCTGCA AAGACAATCACTGTTGGGGTGAGAGTCCGGAAGACTGTCAGATCTTGACTGGCAC CATCTGTACCAGTGGTTGTGCCCGGTGCAAGGGCCGGCTGCCCACTGACTGCTGC CATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGCATTCTGACTGCCTGGCCT GCCTCCACTTCAATCATAGTGGTATCTGTGAGCTGCACTGCCCAGCCCTCGTCACC TACAACACAGACACCTTTGAGTCCATGCACAACCCTGAGGGTCGCTACACCTTTG GTGCCAGCTGCGTGACCACCTGCCCCTACAACTACCTGTCTACGGAAGTGGGATC CTGCACTCTGGTGTGTCCCCCGAATAACCAAGAGGTCACAGCTGAGGACGGAAC ACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCTCGAGTGTGCTATGGTCTGGGC ATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGACAATGTCCAGGAGTTT GATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAGAGCTTTGATG GGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTGTTCGA AACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTC CGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACG ATGGCGCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCG CTCACTGCGGGAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTC TGCTTTGTACACACTGTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCT GCTCCACAGTGGGAACCGGCCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGT AACTCACTGTGTGCCCACGGGCACTGCTGGGGGCCAGGGCCCACCCAGTGTGTCA ACTGCAGTCATTTCCTTCGGGGCCAGGAGTGTGTGGAGGAGTGCCGAGTATGGAA GGGGCTCCCCCGGGAGTATGTGAGTGACAAGCGCTGTCTGCCGTGTCACCCCGAG TGTCAGCCTCAAAACAGCTCAGAGACCTGCTTTGGATCGGAGGCTGATCAGTGTG CAGCCTGCGCCCACTACAAGGACTCGTCCTCCTGTGTGGCTCGCTGCCCCAGTGG TGTGAAACCGGACCTCTCCTACATGCCCATCTGGAAGTACCCGGATGAGGAGGGC ATATGCCAGCCGTGCCCCATCAACTGCACCCACTCCTGTGTGGATCTGGATGAAC GAGGCTGCCCAGCAGAGCAGAGAGCCAGCCCGGTGACATTCATCATTGCAACTG TAGTGGGCGTCCTGCTGTTCCTGATCTTAGTGGTGGTCGTTGGAATCCTAATCAAA CGAAGGAGACAGAAGATCCGGAAGTATACGATGCGTAGGCTGCTGCAGGAAACT GAGTTAGTGGAGCCGCTGACGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATG CGGATCCTAAAAGAGACGGAGCTAAGGAAGGTGAAGGTGCTTGGATCAGGAGCT TTTGGCACTGTCTACAAGGGCATCTGGATCCCAGATGGGGAGAATGTGAAAATCC CCGTGGCTATCAAGGTGTTGAGAGAAAACACATCTCCTAAAGCCAACAAAGAAA TTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTCCGTATGTGTCCCGCCTC CTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGCTTATGCCCTACG GCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCCAGGACCT GCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGCGG CTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACG TCAAGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTA CCATGCAGATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTC AGACGCCGGTTCACCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGG AGCTGATGACTTTTGGGGCCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCC TGATTTGCTGGAGAAGGGAGAACGCCTACCTCAGCCTCCAATCTGCACCATTGAT GTCTACATGATTATGGTCAAATGTTGGATGATTGACTCTGAATGTCGCCCGAGAT TCCGGGAGTTGGTGTCAGAATTTTCACGTATGGCGAGGGACCCCCAGCGTTTTGT GGTCATCCAGAACGAGGACTTGGGCCCATCCAGCCCCATGGACAGTACCTTCTAC CGTTCACTGCTGGAAGATGATGACATGGGTGACCTGGTAGACGCTGAAGAGTATC TGGTGCCCCAGCAGGGATTCTTCTCCCCGGACCCTACCCCAGGCACTGGGAGCAC AGCCCATAGAAGGCACCGCAGCTCGTCCACCAGGAGTGGAGGTGGTGAGCTGAC ACTGGGCCTGGAGCCCTCGGAAGAAGGGCCCCCCAGATCTCCACTGGCTCCCTCG GAAGGGGCTGGCTCCGATGTGTTTGATGGTGACCTGGCAATGGGGGTAACCAAA GGGCTGCAGAGCCTCTCTCCACATGACCTCAGCCCTCTACAGCGGTACAGCGAGG ACCCCACATTACCTCTGCCCCCCGAGACTGATGGCTATGTTGCTCCCCTGGCCTGC AGCCCCCAGCCCGAGTATGTGAACCAATCAGAGGTTCAGCCTCAGCCTCCTTTAA CCCCAGAGGGTCCTCTGCCTCCTGTCCGGCCTGCTGGTGCTACTCTAGAAAGACC CAAGACTCTCTCTCCTGGGAAGAATGGGGTTGTCAAAGACGTTTTTGCCTTCGGG GGTGCTGTGGAGAACCCTGAATACTTAGTACCGAGAGAAGGCACTGCCTCTCCGC CCCACCCTTCTCCTGCCTTCAGCCCAGCCTTTGACAACCTCTATTACTGGGACCAG AACTCATCGGAGCAGGGGCCTCCACCAAGTAACTTTGAAGGGACCCCCACTGCA GAGAACCCTGAGTACCTAGGCCTGGATGTACCTGTA (SEQ ID NO:27). [000167] In one embodiment, the nucleic acid sequence encoding the rat/her2/neu EC1 fragment is: CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGA GATCCTGAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGAC ATGGTTTTGTGGAAGGACGTCTTCCGCAAGAATAACCAACTGGCTCCTGTCGATA TAGACACCAATCGTTCCCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAA TCACTGTTGGGGTGAGAGTCCGGAAGACTGTCAGATCTTGACTGGCACCATCTGT ACCAGTGGTTGTGCCCGGTGCAAGGGCCGGCTGCCCACTGACTGCTGCCATGAGC AGTGTGCCGCAGGCTGCACGGGCCCCAAGCA (SEQ ID NO: 28). [000168] In another embodiment, the nucleic acid sequence encoding the rat her2/neu EC2 fragment is:

GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGC TCGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACC AGTGACAATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCAT TTTTGCCGGAGAGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCT GAGCAGCTCCAAGTGTTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCT CAGCATGGCCAGACAGTCTCCGTGACCTCAGTGTCTTCCAGAACCTTCGAATCAT TCGGGGACGGATTCTCCACGATGGCGCGTACTCATTGACACTGCAAGGCCTGGGG ATCCACTCGCTGGGGCTGCGCTCACTGCGGGAGCTGGGCAGTGGATTGGCTCTGA TTCACCGCAACGCCCATCTCTGCTTTGTACACACTGTACCTTGGGACCAGCTCTTC CGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGGCCGGAAGAGGATTGT GGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCACTGCTGGGGGC CAGGGCCCACCCA (SEQ ID NO: 29). [000169] In another embodiment, the nucleic acid sequence encoding the rat her2/neu IC1 fragment is: CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGG AGCTAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGG GCATCTGGATCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTT GAGAGAAAACACATCTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGT GATGGCTGGTGTGGGTTCTCCGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACAT CCACAGTACAGCTGGTGACACAGCTTATGCCCTACGGCTGCCTTCTGGACCATGT CCGAGAACACCGAGGTCGCCTAGGCTCCCAGGACCTGCTCAACTGGTGTGTTCAG ATTGCCAAGGGGATGAGCTACCTGGAGGACGTGCGGCTTGTACACAGGGACCTG GCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCAAGATTACAGATTTCG GGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAGATGGGGGCA AGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCACCCA TCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGG GCCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGG GAGAACGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGT CAAATGTTGGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCA GAATTTTCACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGG ACTTGGGCCCATCCAGCCCCATGGACAGTACCTTCTACCGTTCACTGCTGGAA

(SEQ ID NO: 30). [000170] In one embodiment, the nucleic acid sequence of human-Her2/neu gene is: ATGGAGCTGGCGGCCTTGTGCCGCTGGGGGCTCCTCCTCGCCCTCTTGCCCCCCG GAGCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTG CCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGT GGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTC CTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGA GGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGA CAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCT GTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACA GAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGG ACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACT GATAGACACCAACCGCTCTCGGGCCTGCCACCCCTGTTCTCCGATGTGTAAGGGC TCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCTGACGCGCACTGTCT GTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGA GCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTC CACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACA ACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGC CAGCTGTGTGACTGCCTGTCCCTACAACTACCTTTCTACGGACGTGGGATCCTGCA CCCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGCAGAGGATGGAACACAGC GGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGA GCACTTGCGAGAGGTGAGGGCAGTTACCAGTGCCAATATCCAGGAGTTTGCTGGC TGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATGGGGACC CAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCT GGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCTGAC CTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATGGCG CCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACT GAGGGAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTC GTGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCC ACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACC AGCTGTGCGCCCGAGGGCACTGCTGGGGTCCAGGGCCCACCCAGTGTGTCAACTG CAGCCAGTTCCTTCGGGGCCAGGAGTGCGTGGAGGAATGCCGAGTACTGCAGGG GCTCCCCAGGGAGTATGTGAATGCCAGGCACTGTTTGCCGTGCCACCCTGAGTGT CAGCCCCAGAATGGCTCAGTGACCTGTTTTGGACCGGAGGCTGACCAGTGTGTGG CCTGTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAGCGGTGT GAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCAGATGAGGAGGGCGCA TGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGGACCTGGATGACAAGG GCTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGTCCATCGTCTCTGCGGTGGT TGGCATTCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGGGATCCTCATCAAGCGAC GGCAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGC TGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGA TCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTG GCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGT GGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTT AGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTG GGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCT GCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCT GAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCT CGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTC AAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACC ATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCG CCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAG CTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTG ACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGT CTACATGATCATGGTCAAATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTC CGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGG TCATCCAGAATGAGGACTTGGGCCCAGCCAGTCCCTTGGACAGCACCTTCTACCG CTCACTGCTGGAGGACGATGACATGGGGGACCTGGTGGATGCTGAGGAGTATCT GGTACCCCAGCAGGGCTTCTTCTGTCCAGACCCTGCCCCGGGCGCTGGGGGCATG GTCCACCACAGGCACCGCAGCTCATCTACCAGGAGTGGCGGTGGGGACCTGACA CTAGGGCTGGAGCCCTCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACCCTCCG AAGGGGCTGGCTCCGATGTATTTGATGGTGACCTGGGAATGGGGGCAGCCAAGG GGCTGCAAAGCCTCCCCACACATGACCCCAGCCCTCTACAGCGGTACAGTGAGGA CCCCACAGTACCCCTGCCCTCTGAGACTGATGGCTACGTTGCCCCCCTGACCTGC AGCCCCCAGCCTGAATATGTGAACCAGCCAGATGTTCGGCCCCAGCCCCCTTCGC CCCGAGAGGGCCCTCTGCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGGC CAAGACTCTCTCCCCAGGGAAGAATGGGGTCGTCAAAGACGTTTTTGCCTTTGGG GGTGCCGTGGAGAACCCCGAGTACTTGACACCCCAGGGAGGAGCTGCCCCTCAG CCCCACCCTCCTCCTGCCTTCAGCCCAGCCTTCGACAACCTCTATTACTGGGACCA GGACCCACCAGAGCGGGGGGCTCCACCCAGCACCTTCAAAGGGACACCTACGGC AGAGAACCCAGAGTACCTGGGTCTGGACGTGCCAGTGTGAACCAGAAGGCCAAG TCCGCAGAAGCCCTGA (SEQ ID NO: 31). [000171] In another embodiment, the nucleic acid sequence encoding the human her2/neu EC1 fragment implemented into the chimera spans from 120-510 bp of the human EC1 region and is set forth in (SEQ ID NO: 32). GAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCAGG GAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCCTGCAGGA TATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTC CCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATG CCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGG GGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGATCTTG AAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTT TGTGGAAG (SEQ ID NO: 32). [000172] In one embodiment, the complete EC1 human her2/neu fragment spans from (58- 979 bp of the human her2/neu gene and is set forth in (SEQ ID NO: 33). GCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCC AGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGG TGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCCT GCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAG GCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGAC AACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTG TCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAG AGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGA CACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTG ATAGACACCAACCGCTCTCGGGCCTGCCACCCCTGTTCTCCGATGTGTAAGGGCT CCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCTGACGCGCACTGTCTG TGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAG CAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCC ACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAA CACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCC AGCTGTGTGACTGCCTGTCCCTACAACTACCTTTCTACGGACGTGGGATCCTGCAC CCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGCAGAGGAT (SEQ ID NO: 33). [000173] In another embodiment, the nucleic acid sequence encoding the human her2/neu EC2 fragment implemented into the chimera spans from 1077-1554 bp of the human her2/neu EC2 fragment and includes a 50 bp extension, and is set forth in (SEQ ID NO: 34). AATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGC CGGAGAGCTTTGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCA GCTCCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGCA TGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGG GACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAG CTGGCTGGGGCTGCGCTCACTGAGGGAACTGGGCAGTGGACTGGCCCTCATCCAC CATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGGACCAGCTCTTTCGGA ACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGG GCGAGGGCCTGGCCTGCCACCAGCTGTGCGCCCGAGGG (SEQ ID NO: 34). [000174] In one embodiment, complete EC2 human her2/neu fragment spans from 907-1504 bp of the human her2/neu gene and is set forth in (SEQ ID NO: 35). TACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAG AGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTG CCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTAC CAGTGCCAATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCA TTTCTGCCGGAGAGCTTTGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGC CAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACAT CTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAGTA ATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGCAAGGGCTGG GCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACTGGGCAGTGGACTGGCCCT CATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGGACCAGCTC TTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAG (SEQ ID NO: 35). [000175] In another embodiment, the nucleic acid sequence encoding the human her2/neu IC1 fragment implemented into the chimera is set forth in (SEQ ID NO: 36). [000176] CAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACG GAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATG CGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCT TTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTC CAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAA TCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTT CTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATG GCTGCCTCTTAGACT (SEQ ID NO: 36). [000177] In another embodiment, the nucleic acid sequence encoding the complete human her2/neu IC1 fragment spans from 2034-3243 of the human her2/neu gene and is set forth in (SEQ ID NO: 37). CAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCTG GTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATC CTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGC ACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTG GCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTA GACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGG GCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTG CCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTG AACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCTC GTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCA AAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCA TGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGC CGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGC TGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGA CCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTC TACATGATCATGGTCAAATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTCC GGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGGT CATCCAGAATGAGGACTTGGGCCCAGCCAGTCCCTTGGACAGCACCTTCTACCGC TCACTGCTGGAGGACGATGACATGGGGGACCTGGTGGATGCTGAGGAGTATCTG GTACCCCAGCAGGGCTTCTTCTGTCCAGACCCTGCCCCGGGCGCTGGGGGCATGG TCCACCACAGGCACCGCAGCTCATCTACCAGGAGTGGCGGTGGGGACCTGACACT AGGGCTGGAGCCCTCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACCCTCCGA AGGGGCT (SEQ ID NO: 37). [000178] In one embodiment, the present invention provides a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a carbonic anhydrase 9 (or carbonic anhydrase IX) protein or fused to a fragment thereof. In one embodiment, the present invention provides a recombinant polypeptide consisting of an N-terminal fragment of an LLO protein fused to a carbonic anhydrase 9 or fused to a fragment thereof. [000179] In another embodiment, the carbonic anhydrase 9 protein of the methods and compositions of the present invention is a human carbonic anhydrase 9 protein. In another embodiment, the carbonic anhydrase 9 protein is a mouse carbonic anhydrase 9 protein. In another embodiment, the carbonic anhydrase 9 protein is a rat carbonic anhydrase 9 protein. In another embodiment, the carbonic anhydrase 9 protein is a primate carbonic anhydrase 9 protein. In another embodiment, the carbonic anhydrase 9 protein is a carbonic anhydrase 9 protein of any other animal species or combinations thereof known in the art. [000180] In one embodiment, the terms “carbonic anhydrase 9”, "carbonic anhydrase IX”, and“CA9”, are used interchangeably herein. [000181] In one embodiment, the nucleic acid sequence of the human-CA9 gene is: cagaggttgccccggatgcaggaggattcccccttgggaggaggctcttctggggaagatgacccactgggcgaggaggatctgcc cagtgaagaggattcacccagagaggaggatccacccggagaggaggatctacctggagaggaggatctacctggagaggaggat ctacctgaagttaagcctaaatcagaagaagagggctccctgaagttagaggatctacctactgttgaggctcctggagatcctcaaga accccagaataatgcccacagggacaaagaaggggatgaccagagtcattggcgctatggaggcgacccgccctggccccgggtg tccccagcctgcgcgggccgcttccagtccccggtggatatccgcccccagctcgccgccttctgcccggccctgcgccccctggaa ctcctgggcttccagctcccgccgctcccagaactgcgcctgcgcaacaatggccacagtgtgcaactgaccctgcctcctgggctag agatggctctgggtcccgggcgggagtaccgggctctgcagctgcatctgcactggggggctgcaggtcgtccgggctcggagcac actgtggaaggccaccgtttccctgccgagatccacgtggttcacctcagcaccgcctttgccagagttgacgaggccttggggcgcc cgggaggcctggccgtgttggccgcctttctggaggagggcccggaagaaaacagtgcctatgagcagttgctgtctcgcttggaag aaatcgctgaggaaggctcagagactcaggtcccaggactggacatatctgcactcctgccctctgacttcagccgctacttccaatatg aggggtctctgactacaccgccctgtgcccagggtgtcatctggactgtgtttaaccagacagtgatgctgagtgctaagcagctccac accctctctgacaccctgtggggacctggtgactctcggctacagctgaacttccgagcgacgcagcctttgaatgggcgagtgattga ggcctccttccctgctggagtggacagcagtcctcgggctgctgagccagtccagctgaattcctgcctggctgctggtgacatcctag ccctggtttttggcctcctt (SEQ ID NO: 38). In one embodiment, the CA9 nucleic acid sequence is a homolog of SEQ ID NO: 38. In another embodiment, the CA9 nucleic acid sequence is a variant of SEQ ID NO: 38. In another embodiment, the CA9 nucleic acid sequence is a fragment of SEQ ID NO: 38. In another embodiment the CA9 nucleic acid sequence is any sequence known in the art including, but not limited to, those set forth in GenBank Accession nos. NM_001216.2, XM_006716867.1, XM_006716868.1, and X66839.1. [000182] In one embodiment, the amino acid sequence encoded by the human CA9 gene provided herein is: Q R L P R M Q E D S P L G G G S S G E D D P L G E E D L P S E E D S P R E E D P P G E E D L P G E E D L P G E E D L P E V K P K S E E E G S L K L E D L P T V E A P G D P Q E P Q N N A H R D K E G D D Q S H W R Y G G D P P W P R V S P A C A G R F Q S P V D I R P Q L A A F C P A L R P L E L L G F Q L P P L P E L R L R N N G H S V Q L T L P P G L E M A L G P G R E Y R A L Q L H L H W G A A G R P G S E H T V E G H R F P A E I H V V H L S T A F A R V D E A L G R P G G L A V L A A F L E E G P E E N S A Y E Q L L S R L E E I A E E G S E T Q V P G L D I S A L L P S D F S R Y F Q Y E G S L T T P P C A Q G V I W T V F N Q T V M L S A K Q L H T L S D T L W G P G D S R L Q L N F R A T Q P L N G R V I E A S F P A G V D S S P R A A E P V Q L N S C L A A G D I L A L V F G L L

(SEQ ID NO: 39). In one embodiment, the CA9 amino acid sequence is a homolog of SEQ ID NO: 39. In another embodiment, the CA9 amino acid sequence is a variant of SEQ ID NO: 39. In another embodiment, the CA9 amino acid sequence is an isomer of SEQ ID NO: 39. In another embodiment, the CA9 amino acid sequence is a fragment of SEQ ID NO: 39. In another embodiment the CA9 amino acid sequence is any sequence known in the art including, but not limited to, those set forth in GenBank Accession nos. NP_001207.2, XP_006716930.1, XP_006716931.1, and CAA47315.1. [000183] In another embodiment, the nucleic acid sequence encoding a truncated LLO-CA9 fusion comprises SEQ ID NO: 40: atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaata aagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaat cgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaag gttacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaat ttcgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcat taacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacgcagt aaatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgatgacgaaatggcttac agtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaatcagtgaaggga aaatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctg ttactaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttg aaattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaac aaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggcaacctcggaga cttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaagacaatg aattagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatcactctggagga tacgttgctcaattcaacatttcttgggatgaagtaaattatgatctcgagCAGAGGTTGCCCCGGATGCAGGAGG ATTCCCCCTTGGGAGGAGGCTCTTCTGGGGAAGATGACCCACTGGGCGAGGAGG ATCTGCCCAGTGAAGAGGATTCACCCAGAGAGGAGGATCCACCCGGAGAGGAGG ATCTACCTGGAGAGGAGGATCTACCTGGAGAGGAGGATCTACCTGAAGTTAAGC CTAAATCAGAAGAAGAGGGCTCCCTGAAGTTAGAGGATCTACCTACTGTTGAGGC TCCTGGAGATCCTCAAGAACCCCAGAATAATGCCCACAGGGACAAAGAAGGGGA TGACCAGAGTCATTGGCGCTATGGAGGCGACCCGCCCTGGCCCCGGGTGTCCCCA GCCTGCGCGGGCCGCTTCCAGTCCCCGGTGGATATCCGCCCCCAGCTCGCCGCCT TCTGCCCGGCCCTGCGCCCCCTGGAACTCCTGGGCTTCCAGCTCCCGCCGCTCCCA GAACTGCGCCTGCGCAACAATGGCCACAGTGTGCAACTGACCCTGCCTCCTGGGC TAGAGATGGCTCTGGGTCCCGGGCGGGAGTACCGGGCTCTGCAGCTGCATCTGCA CTGGGGGGCTGCAGGTCGTCCGGGCTCGGAGCACACTGTGGAAGGCCACCGTTTC CCTGCCGAGATCCACGTGGTTCACCTCAGCACCGCCTTTGCCAGAGTTGACGAGG CCTTGGGGCGCCCGGGAGGCCTGGCCGTGTTGGCCGCCTTTCTGGAGGAGGGCCC GGAAGAAAACAGTGCCTATGAGCAGTTGCTGTCTCGCTTGGAAGAAATCGCTGA GGAAGGCTCAGAGACTCAGGTCCCAGGACTGGACATATCTGCACTCCTGCCCTCT GACTTCAGCCGCTACTTCCAATATGAGGGGTCTCTGACTACACCGCCCTGTGCCC AGGGTGTCATCTGGACTGTGTTTAACCAGACAGTGATGCTGAGTGCTAAGCAGCT CCACACCCTCTCTGACACCCTGTGGGGACCTGGTGACTCTCGGCTACAGCTGAAC TTCCGAGCGACGCAGCCTTTGAATGGGCGAGTGATTGAGGCCTCCTTCCCTGCTG GAGTGGACAGCAGTCCTCGGGCTGCTGAGCCAGTCCAGCTGAATTCCTGCCTGGC TGCTGGTGACATCCTAGCCCTGGTTTTTGGCCTCCTT (SEQ ID NO: 40), wherein the UPPERCASE sequences encode cHER2, the lowercase sequences encode tLLO and the underlined“ctcgag” sequence represents the Xho I restriction site used to ligate the tumor antigen to truncated LLO in the plasmid. In one embodiment, the truncated LLO-CA9 fusion is a homolog of SEQ ID NO: 40. In another embodiment, the truncated LLO-CA9 fusion is a variant of SEQ ID NO: 40. In another embodiment, the truncated LLO-CA9 fusion is an isomer of SEQ ID NO: 40. [000184] In one embodiment, an amino acid sequence comprising a tLLO fused to CA9 comprises SEQ ID NO: 41 MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHAD EIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADI QVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKN ATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKA VNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGV NAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNS SFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAV IKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDLEQRLPRMQEDSPLG GGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSL KLEDLPTVEAPGDPQEPQNNAHRDKEGDDQSHWRYGGDPPWPRVSPACAGRFQSP VDIRPQLAAFCPALRPLELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYR ALQLHLHWGAAGRPGSEHTVEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAA FLEEGPEENSAYEQLLSRLEEIAEEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCA QGVIWTVFNQTVMLSAKQLHTLSDTLWGPGDSRLQLNFRATQPLNGRVIEASFPAG VDSSPRAAEPVQLNSCLAAGDILALVFGLL (SEQ ID NO: 41). In one embodiment, the truncated LLO-CA9 fusion is a homolog of SEQ ID NO: 41. In another embodiment, the truncated LLO-CA9 fusion is a variant of SEQ ID NO: 41. In another embodiment, the truncated LLO-CA9 fusion is an isomer of SEQ ID NO: 41. [000185] In another embodiment, the LmddA strain provided herein comprises a mutation, [000186] In one embodiment, an antigen of the methods and compositions as provided herein is fused to an ActA protein, which in one embodiment, is an N-terminal fragment of an ActA protein, which in one embodiment, comprises or consists of the first 390 AA of ActA, in another embodiment, the first 418 AA of ActA, in another embodiment, the first 50 AA of ActA, in another embodiment, the first 100 AA of ActA, which in one embodiment, comprise a PEST sequence such as that provided in SEQ ID NO: 2. In another embodiment, an N- terminal fragment of an ActA protein utilized in methods and compositions as provided herein comprises or consists of the first 150 AA of ActA, in another embodiment, the first approximately 200 AA of ActA, which in one embodiment comprises 2 PEST sequences as described herein. In another embodiment, an N-terminal fragment of an ActA protein utilized in methods and compositions as provided herein comprises or consists of the first 250 AA of ActA, in another embodiment, the first 300 AA of ActA. In another embodiment, the ActA fragment contains residues of a homologous ActA protein that correspond to one of the above AA ranges. The residue numbers need not, in another embodiment, correspond exactly with the residue numbers enumerated above; e.g. if the homologous ActA protein has an insertion or deletion, relative to an ActA protein utilized herein, then the residue numbers can be adjusted accordingly, as would be routine to a skilled artisan using sequence alignment tools such as NCBI BLAST that are well-known in the art. [000187] In another embodiment, the N-terminal portion of the ActA protein comprises 1, 2, 3, or 4 PEST sequences, which in one embodiment are the PEST sequences specifically mentioned herein, or their homologs, as described herein or other PEST sequences as can be determined using the methods and algorithms described herein or by using alternative methods known in the art. [000188] An N-terminal fragment of an ActA protein utilized in methods and compositions as provided herein has, in another embodiment, the sequence set forth in SEQ ID NO: 42: MRAMMVVFITANCITINPDIIFAATDSEDSSLNTDEWEEEKTEEQPSEVNTGPRYETAR EVSSRDIKELEKSNKVRNTNKADLIAMLKEKAEKGPNINNNNSEQTENAAINEEASG ADRPAIQVERRHPGLPSDSAAEIKKRRKAIASSDSELESLTYPDKPTKVNKKKVAKES VADASESDLDSSMQSADESSPQPLKANQQPFFPKVFKKIKDAGKWVRDKIDENPEVK KAIVDKSAGLIDQLLTKKKSEEVNASDFPPPPTDEELRLALPETPMLLGFNAPATSEPS SFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTEDELEIIRETASSLDSSF TRGDLASLRNAINRHSQNFSDFPPIPTEEELNGRGGRP (SEQ ID NO: 42). In another embodiment, the ActA fragment comprises the sequence set forth in SEQ ID NO: 42. In another embodiment, the ActA fragment is any other ActA fragment known in the art. In another embodiment, the ActA protein is a homologue of SEQ ID NO: 42. In another embodiment, the ActA protein is a variant of SEQ ID NO: 42. In another embodiment, the ActA protein is an isoform of SEQ ID NO: 42. In another embodiment, the ActA protein is a fragment of SEQ ID NO: 42. In another embodiment, the ActA protein is a fragment of a homologue of SEQ ID NO: 42. In another embodiment, the ActA protein is a fragment of a variant of SEQ ID NO: 42. In another embodiment, the ActA protein is a fragment of an isoform of SEQ ID NO: 42. Each possibility represents a separate embodiment as provided herein. Each possibility represents a separate embodiment as provided herein. [000189] In another embodiment, the recombinant nucleotide encoding a fragment of an ActA protein comprises the sequence set forth in SEQ ID NO: 43: atgcgtgcgatgatggtggttttcattactgccaattgcattacgattaaccccgacataatatttgcagcgacagatagcgaagattcta gtctaaacacagatgaatgggaagaagaaaaaacagaagagcaaccaagcgaggtaaatacgggaccaagatacgaaactgcac gtgaagtaagttcacgtgatattaaagaactagaaaaatcgaataaagtgagaaatacgaacaaagcagacctaatagcaatgttgaa agaaaaagcagaaaaaggtccaaatatcaataataacaacagtgaacaaactgagaatgcggctataaatgaagaggcttcaggag ccgaccgaccagctatacaagtggagcgtcgtcatccaggattgccatcggatagcgcagcggaaattaaaaaaagaaggaaagc catagcatcatcggatagtgagcttgaaagccttacttatccggataaaccaacaaaagtaaataagaaaaaagtggcgaaagagtca gttgcggatgcttctgaaagtgacttagattctagcatgcagtcagcagatgagtcttcaccacaacctttaaaagcaaaccaacaacca tttttccctaaagtatttaaaaaaataaaagatgcggggaaatgggtacgtgataaaatcgacgaaaatcctgaagtaaagaaagcgatt gttgataaaagtgcagggttaattgaccaattattaaccaaaaagaaaagtgaagaggtaaatgcttcggacttcccgccaccacctac ggatgaagagttaagacttgctttgccagagacaccaatgcttcttggttttaatgctcctgctacatcagaaccgagctcattcgaatttc caccaccacctacggatgaagagttaagacttgctttgccagagacgccaatgcttcttggttttaatgctcctgctacatcggaaccga gctcgttcgaatttccaccgcctccaacagaagatgaactagaaatcatccgggaaacagcatcctcgctagattctagttttacaagag gggatttagctagtttgagaaatgctattaatcgccatagtcaaaatttctctgatttcccaccaatcccaacagaagaagagttgaacgg gagaggcggtagacca (SEQ ID NO: 43). In another embodiment, the recombinant nucleotide has the sequence set forth in SEQ ID NO: 43. In another embodiment, the recombinant nucleotide comprises any other sequence that encodes a fragment of an ActA protein. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000190] An N-terminal fragment of an ActA protein utilized in methods and compositions as provided herein has, in another embodiment, the sequence set forth in SEQ ID NO: 44: MRAMMVVFITANCITINPDIIFAATDSEDSSLNTDEWEEEKTEEQPSEVNTGPRYETA REVSSRDIEELEKSNKVKNTNKADLIAMLKAKAEKGPNNNNNNGEQTGNVAINEEA SGVDRPTLQVERRHPGLSSDSAAEIKKRRKAIASSDSELESLTYPDKPTKANKRKVA KESVVDASESDLDSSMQSADESTPQPLKANQKPFFPKVFKKIKDAGKWVRDKIDENP EVKKAIVDKSAGLIDQLLTKKKSEEVNASDFPPPPTDEELRLALPETPMLLGFNAPTP SEPSSFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTEDELEIMRETAPS LDSSFTSGDLASLRSAINRHSENFSDFPLIPTEEELNGRGGRP (SEQ ID NO: 44), which in one embodiment is the first 390 AA for ActA from Listeria monocytogenes, strain 10403S. In another embodiment, the ActA fragment comprises the sequence set forth in SEQ ID NO: 44. In another embodiment, the ActA fragment is any other ActA fragment known in the art. In another embodiment, the ActA protein is a homologue of SEQ ID NO: 44. In another embodiment, the ActA protein is a variant of SEQ ID NO: 44. In another embodiment, the ActA protein is an isoform of SEQ ID NO: 44. In another embodiment, the ActA protein is a fragment of SEQ ID NO: 44. In another embodiment, the ActA protein is a fragment of a homologue of SEQ ID NO: 44. In another embodiment, the ActA protein is a fragment of a variant of SEQ ID NO: 44. In another embodiment, the ActA protein is a fragment of an isoform of SEQ ID NO: 44. [000191] In another embodiment, a truncated ActA protein comprises the sequence set forth in SEQ ID NO: 45:

A T D S E D S S L N T D E W E E E K T E E Q P S E V N T G P R Y E T A R E V S S R D I E E L E K S N K V K N T N K A D L I A M L K A K A E K G P N N N N N N G E Q T G N V A I N E E A S G V D R P T L Q V E R R H P G L S S D S A A E I K K R R K A I A S S D S E L E S L T Y P D K P T K A N K R K V A K E S V V D A S E S D L D S S M Q S A D E S T P Q P L K A N Q K P F F P K V F K K I K D A G K W V R D K (SEQ ID NO: 45).

[000192] In another embodiment, a truncated ActA sequence provided herein is further fused to an hly signal peptide at the N-terminus. In another embodiment, the truncated ActA fused to hly signal peptide comprises SEQ ID NO: 46:

M K K I M L V F I T L I L V S L P I A Q Q T E A S R A T D S E D S S L N T D E W E E E K T E E Q P S E V N T G P R Y E T A R E V S S R D I E E L E K S N K V K N T N K A D L I A M L K A K A E K G P N N N N N N G E Q T G N V A I N E E A S G V D R P T L Q V E R R H P G L S S D S A A E I K K R R K A I A S S D S E L E S L T Y P D K P T K A N K R K V A K E S V V D A S E S D L D S S M Q S A D E S T P Q P L K A N Q K P F F P K V F K K I K D A G K W V R D K

(SEQ ID NO: 46). In another embodiment, a truncated ActA as set forth in SEQ ID NO: 46 is referred to as LA229. [000193] In another embodiment, the recombinant nucleotide encoding a fragment of an ActA protein comprises the sequence set forth in SEQ ID NO: 47: atgcgtgcgatgatggtagttttcattactgccaactgcattacgattaaccccgacataatatttgcagcgacagatagcgaagattcca gtctaaacacagatgaatgggaagaagaaaaaacagaagagcagccaagcgaggtaaatacgggaccaagatacgaaactgcacg tgaagtaagttcacgtgatattgaggaactagaaaaatcgaataaagtgaaaaatacgaacaaagcagacctaatagcaatgttgaaag caaaagcagagaaaggtccgaataacaataataacaacggtgagcaaacaggaaatgtggctataaatgaagaggcttcaggagtcg accgaccaactctgcaagtggagcgtcgtcatccaggtctgtcatcggatagcgcagcggaaattaaaaaaagaagaaaagccatag cgtcgtcggatagtgagcttgaaagccttacttatccagataaaccaacaaaagcaaataagagaaaagtggcgaaagagtcagttgtg gatgcttctgaaagtgacttagattctagcatgcagtcagcagacgagtctacaccacaacctttaaaagcaaatcaaaaaccatttttcc ctaaagtatttaaaaaaataaaagatgcggggaaatgggtacgtgataaaatcgacgaaaatcctgaagtaaagaaagcgattgttgat aaaagtgcagggttaattgaccaattattaaccaaaaagaaaagtgaagaggtaaatgcttcggacttcccgccaccacctacggatga agagttaagacttgctttgccagagacaccgatgcttctcggttttaatgctcctactccatcggaaccgagctcattcgaatttccgccgc cacctacggatgaagagttaagacttgctttgccagagacgccaatgcttcttggttttaatgctcctgctacatcggaaccgagctcattc gaatttccaccgcctccaacagaagatgaactagaaattatgcgggaaacagcaccttcgctagattctagttttacaagcggggattta gctagtttgagaagtgctattaatcgccatagcgaaaatttctctgatttcccactaatcccaacagaagaagagttgaacgggagaggc ggtagacca (SEQ ID NO: 47), which in one embodiment, is the first 1170 nucleotides encoding ActA in Listeria monocytogenes 10403S strain. In another embodiment, the recombinant nucleotide has the sequence set forth in SEQ ID NO: 47. In another embodiment, the recombinant nucleotide comprises any other sequence that encodes a fragment of an ActA protein. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000194] In another embodiment, the ActA fragment is another ActA fragment known in the art, which in one embodiment, is any fragment comprising a PEST sequence. Thus, in one embodiment, the ActA fragment is amino acids 1-100 of the ActA sequence. In another embodiment, the ActA fragment is amino acids 1-200 of the ActA sequence. In another embodiment, the ActA fragment is amino acids 200-300 of the ActA sequence. In another embodiment, the ActA fragment is amino acids 300-400 of the ActA sequence. In another embodiment, the ActA fragment is amino acids 1-300 of the ActA sequence. In another embodiment, a recombinant nucleotide as provided herein comprises any other sequence that encodes a fragment of an ActA protein. In another embodiment, the recombinant nucleotide comprises any other sequence that encodes an entire ActA protein. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000195] In one embodiment, the ActA sequence for use in the compositions and methods as provided herein is from Listeria monocytogenes, which in one embodiment, is the EGD strain, the 10403S strain (Genbank accession number: DQ054585) the NICPBP 54002 strain (Genbank accession number: EU394959), the S3 strain (Genbank accession number: EU394960), the NCTC 5348 strain (Genbank accession number: EU394961), the NICPBP 54006 strain (Genbank accession number: EU394962), the M7 strain (Genbank accession number: EU394963), the S19 strain (Genbank accession number: EU394964), or any other strain of Listeria monocytogenes which is known in the art. [000196] In one embodiment, the sequence of the deleted actA region in the strain, LmddΔactA is as follows: [000197] gcgccaaatcattggttgattggtgaggatgtctgtgtgcgtgggtcgcgagatgggcgaataagaagcattaaagatcct gacaaatataatcaagcggctcatatgaaagattacgaatcgcttccactcacagaggaaggcgactggggcggagttcattataatag tggtatcccgaataaagcagcctataatactatcactaaacttggaaaagaaaaaacagaacagctttattttcgcgccttaaagtactattt aacgaaaaaatcccagtttaccgatgcgaaaaaagcgcttcaacaagcagcgaaagatttatatggtgaagatgcttctaaaaaagttg ctgaagcttgggaagcagttggggttaactgattaacaaatgttagagaaaaattaattctccaagtgatattcttaaaataattcatgaatat tttttcttatattagctaattaagaagataactaactgctaatccaatttttaacggaacaaattagtgaaaatgaaggccgaattttccttgttc taaaaaggttgtattagcgtatcacgaggagggagtataagtgggattaaacagatttatgcgtgcgatgatggtggttttcattactgcca attgcattacgattaaccccgacgtcgacccatacgacgttaattcttgcaatgttagctattggcgtgttctctttaggggcgtttatcaaaa ttattcaattaagaaaaaataattaaaaacacagaacgaaagaaaaagtgaggtgaatgatatgaaattcaaaaaggtggttctaggtatg tgcttgatcgcaagtgttctagtctttccggtaacgataaaagcaaatgcctgttgtgatgaatacttacaaacacccgcagctccgcatga tattgacagcaaattaccacataaacttagttggtccgcggataacccgacaaatactgacgtaaatacgcactattggctttttaaacaag cggaaaaaatactagctaaagatgtaaatcatatgcgagctaatttaatgaatgaacttaaaaaattcgataaacaaatagctcaaggaat atatgatgcggatcataaaaatccatattatgatactagtacatttttatctcatttttataatcctgatagagataatacttatttgccgggttttg ctaatgcgaaaataacaggagcaaagtatttcaatcaatcggtgactgattaccgagaagggaa (SEQ ID NO: 48). In one embodiment, the underlined region contains actA sequence element that is present in the LmddΔactA strain. In one embodiment, the bold sequence gtcgac represent the site of junction of the N-T and C-T sequence. [000198] In one embodiment, the recombinant Listeria strain of the compositions and methods as provided herein comprise a first or second nucleic acid molecule that encodes a High Molecular Weight-Melanoma Associated Antigen (HMW-MAA), or, in another embodiment, a fragment of HMW-MAA. [000199] In one embodiment, HMW-MAA is also known as the melanoma chondroitin sulfate proteoglycan (MCSP), and in another embodiment, is a membrane-bound protein of 2322 residues. In one embodiment, HMW-MAA is expressed on over 90% of surgically removed benign nevi and melanoma lesions, and is also expressed in basal cell carcinoma, tumors of neural crest origin (e.g. astrocytomas, gliomas, neuroblastomas and sarcomas), childhood leukemias, and lobular breast carcinoma lesions. In another embodiment, HMW-MAA is highly expressed on both activated pericytes and pericytes in tumor angiogeneic vasculature which, in another embodiment is associated with neovascularization in vivo. In another embodiment, immunization of mice with the recombinant Listeria, as provided herein, that expresses a fragment of HMW-MAA (residues 2160 to 2258), impairs the growth of tumors not engineered to express HMW-MAA (Figure 9D). In another embodiment, immunization of mice with the recombinant Listeria expressing a fragment of HMW-MAA (residues 2160 to 2258) decreases the number of pericytes in the tumor vasculature. In another embodiment, immunization of mice with the recombinant Listeria expressing a fragment of HMW-MAA (residues 2160 to 2258) causes infiltration of CD8⁺ T cells around blood vessels and into the tumor. [000200] In one embodiment, a murine homolog of HMW-MAA, known as NG2 or AN2, has 80% homology to HMW-MAA, as well as similar expression pattern and function. In another embodiment, HMW-MAA is highly expressed on both activated pericytes and pericytes in tumor angiogenic vasculature. In one embodiment, activated pericytes are associated with neovascularization in vivo. In one embodiment, activated pericytes are involved in angiogenesis. In another embodiment, angiogenesis is important for survival of tumors. In another embodiment, pericytes in tumor angiogenic vasculature are associated with neovascularization in vivo. In another embodiment, activated pericytes are important cells in vascular development, stabilization, maturation and remodeling. Therefore, in one embodiment, besides its role as a tumor-associated antigen, HMW-MAA is also a potential universal target for anti-angiogenesis using an immunotherapeutic approach. As described herein (Example 8), results obtained using an Lm-based vaccine against this antigen has supported this possibility. [000201] In another embodiment, one of the antigens of the methods and compositions provided herein is expressed in activated pericytes. In another embodiment, at least one of the antigens is expressed in activated pericytes. [000202] The HMW-MAA protein from which HMW-MAA fragments as provided herein are derived is, in another embodiment, a human HMW-MAA protein. In another embodiment, the HMW-MAA protein is a mouse protein. In another embodiment, the HMW-MAA protein is a rat protein. In another embodiment, the HMW-MAA protein is a primate protein. In another embodiment, the HMW-MAA protein is from any other species known in the art. In another embodiment, the HMW-MAA protein is melanoma chondroitin sulfate proteoglycan (MCSP). In another embodiment, an AN2 protein is used in methods and compositions as provided herein. In another embodiment, an NG2 protein is used in methods and compositions as provided herein. [000203] In another embodiment, the HMW-MAA protein of methods and compositions as provided herein has the sequence: [000204] MQSGRGPPLPAPGLALALTLTMLARLASAASFFGENHLEVPVATALTDIDLQ LQFSTSQPEALLLLAAGPADHLLLQLYSGRLQVRLVLGQEELRLQTPAETLLSDSIPHT VVLTVVEGWATLSVDGFLNASSAVPGAPLEVPYGLFVGGTGTLGLPYLRGTSRPLRG CLHAATLNGRSLLRPLTPDVHEGCAEEFSASDDVALGFSGPHSLAAFPAWGTQDEGT LEFTLTTQSRQAPLAFQAGGRRGDFIYVDIFEGHLRAVVEKGQGTVLLHNSVPVADG QPHEVSVHINAHRLEISVDQYPTHTSNRGVLSYLEPRGSLLLGGLDAEASRHLQEHRL GLTPEATNASLLGCMEDLSVNGQRRGLREALLTRNMAAGCRLEEEEYEDDAYGHYE AFSTLAPEAWPAMELPEPCVPEPGLPPVFANFTQLLTISPLVVAEGGTAWLEWRHVQ PTLDLMEAELRKSQVLFSVTRGARHGELELDIPGAQARKMFTLLDVVNRKARFIHDG SEDTSDQLVLEVSVTARVPMPSCLRRGQTYLLPIQVNPVNDPPHIIFPHGSLMVILEHT QKPLGPEVFQAYDPDSACEGLTFQVLGTSSGLPVERRDQPGEPATEFSCRELEAGSLV YVHRGGPAQDLTFRVSDGLQASPPATLKVVAIRPAIQIHRSTGLRLAQGSAMPILPAN LSVETNAVGQDVSVLFRVTGALQFGELQKQGAGGVEGAEWWATQAFHQRDVEQG RVRYLSTDPQHHAYDTVENLALEVQVGQEILSNLSFPVTIQRATVWMLRLEPLHTQN TQQETLTTAHLEATLEEAGPSPPTFHYEVVQAPRKGNLQLQGTRLSDGQGFTQDDIQ AGRVTYGATARASEAVEDTFRFRVTAPPYFSPLYTFPIHIGGDPDAPVLTNVLLVVPE GGEGVLSADHLFVKSLNSASYLYEVMERPRHGRLAWRGTQDKTTMVTSFTNEDLLR GRLVYQHDDSETTEDDIPFVATRQGESSGDMAWEEVRGVFRVAIQPVNDHAPVQTIS RIFHVARGGRRLLTTDDVAFSDADSGFADAQLVLTRKDLLFGSIVAVDEPTRPIYRFT QEDLRKRRVLFVHSGADRGWIQLQVSDGQHQATALLEVQASEPYLRVANGSSLVVP QGGQGTIDTAVLHLDTNLDIRSGDEVHYHVTAGPRWGQLVRAGQPATAFSQQDLLD GAVLYSHNGSLSPRDTMAFSVEAGPVHTDATLQVTIALEGPLAPLKLVRHKKIYVFQ GEAAEIRRDQLEAAQEAVPPADIVFSVKSPPSAGYLVMVSRGALADEPPSLDPVQSFS QEAVDTGRVLYLHSRPEAWSDAFSLDVASGLGAPLEGVLVELEVLPAAIPLEAQNFS VPEGGSLTLAPPLLRVSGPYFPTLLGLSLQVLEPPQHGALQKEDGPQARTLSAFSWRM VEEQLIRYVHDGSETLTDSFVLMANASEMDRQSHPVAFTVTVLPVNDQPPILTTNTGL QMWEGATAPIPAEALRSTDGDSGSEDLVYTIEQPSNGRVVLRGAPGTEVRSFTQAQL DGGLVLFSHRGTLDGGFRFRLSDGEHTSPGHFFRVTAQKQVLLSLKGSQTLTVCPGS VQPLSSQTLRASSSAGTDPQLLLYRVVRGPQLGRLFHAQQDSTGEALVNFTQAEVYA GNILYEHEMPPEPFWEAHDTLELQLSSPPARDVAATLAVAVSFEAACPQRPSHLWKN KGLWVPEGQRARITVAALDASNLLASVPSPQRSEHDVLFQVTQFPSRGQLLVSEEPLH AGQPHFLQSQLAAGQLVYAHGGGGTQQDGFHFRAHLQGPAGASVAGPQTSEAFAIT VRDVNERPPQPQASVPLRLTRGSRAPISRAQLSVVDPDSAPGEIEYEVQRAPHNGFLS LVGGGLGPVTRFTQADVDSGRLAFVANGSSVAGIFQLSMSDGASPPLPMSLAVDILPS AIEVQLRAPLEVPQALGRSSLSQQQLRVVSDREEPEAAYRLIQGPQYGHLLVGGRPTS AFSQFQIDQGEVVFAFTNFSSSHDHFRVLALARGVNASAVVNVTVRALLHVWAGGP WPQGATLRLDPTVLDAGELANRTGSVPRFRLLEGPRHGRVVRVPRARTEPGGSQLVE QFTQQDLEDGRLGLEVGRPEGRAPGPAGDSLTLELWAQGVPPAVASLDFATEPYNA ARPYSVALLSVPEAARTEAGKPESSTPTGEPGPMASSPEPAVAKGGFLSFLEANMFSV IIPMCLVLLLLALILPLLFYLRKRNKTGKHDVQVLTAKPRNGLAGDTETFRKVEPGQA IPLTAVPGQGPPPGGQPDPELLQFCRTPNPALKNGQYWV (SEQ ID No: 49). In another embodiment, an HMW-MAA AA sequence of methods and compositions as provided herein comprises the sequence set forth in SEQ ID No: 49. In another embodiment, the HMW-MAA AA sequence is a homologue of SEQ ID No: 49. In another embodiment, the HMW-MAA AA sequence is a variant of SEQ ID No: 49. In another embodiment, the HMW-MAA AA sequence is a fragment of SEQ ID No: 49. In another embodiment, the HMW-MAA AA sequence is an isoform of SEQ ID No: 49. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000205] In another embodiment, the HMW-MAA protein of methods and compositions as provided herein is encoded by the sequence: [000206] atgcagtccggccgcggccccccacttccagcccccggcctggccttggctttgaccctgactatgttggccagacttgc atccgcggcttccttcttcggtgagaaccacctggaggtgcctgtggccacggctctgaccgacatagacctgcagctgcagttctcca cgtcccagcccgaagccctccttctcctggcagcaggcccagctgaccacctcctgctgcagctctactctggacgcctgcaggtcag acttgttctgggccaggaggagctgaggctgcagactccagcagagacgctgctgagtgactccatcccccacactgtggtgctgact gtcgtagagggctgggccacgttgtcagtcgatgggtttctgaacgcctcctcagcagtcccaggagcccccctagaggtcccctatg ggctctttgttgggggcactgggacccttggcctgccctacctgaggggaaccagccgacccctgaggggttgcctccatgcagcca ccctcaatggccgcagcctcctccggcctctgacccccgatgtgcatgagggctgtgctgaagagttttctgccagtgatgatgtggcc ctgggcttctctgggccccactctctggctgccttccctgcctggggcactcaggacgaaggaaccctagagtttacactcaccacaca gagccggcaggcacccttggccttccaggcagggggccggcgtggggacttcatctatgtggacatatttgagggccacctgcggg ccgtggtggagaagggccagggtaccgtattgctccacaacagtgtgcctgtggccgatgggcagccccatgaggtcagtgtccaca tcaatgctcaccggctggaaatctccgtggaccagtaccctacgcatacttcgaaccgaggagtcctcagctacctggagccacgggg cagtctccttctcggggggctggatgcagaggcctctcgtcacctccaggaacaccgcctgggcctgacaccagaggccaccaatgc ctccctgctgggctgcatggaagacctcagtgtcaatggccagaggcgggggctgcgggaagctttgctgacgcgcaacatggcag ccggctgcaggctggaggaggaggagtatgaggacgatgcctatggacattatgaagctttctccaccctggcccctgaggcttggcc agccatggagctgcctgagccatgcgtgcctgagccagggctgcctcctgtctttgccaatttcacccagctgctgactatcagcccact ggtggtggccgaggggggcacagcctggcttgagtggaggcatgtgcagcccacgctggacctgatggaggctgagctgcgcaaa tcccaggtgctgttcagcgtgacccgaggggcacgccatggcgagctcgagctggacatcccgggagcccaggcacgaaaaatgtt caccctcctggacgtggtgaaccgcaaggcccgcttcatccacgatggctctgaggacacctccgaccagctggtgctggaggtgtc ggtgacggctcgggtgcccatgccctcatgccttcggaggggccaaacatacctcctgcccatccaggtcaaccctgtcaatgaccca ccccacatcatcttcccacatggcagcctcatggtgatcctggaacacacgcagaagccgctggggcctgaggttttccaggcctatga cccggactctgcctgtgagggcctcaccttccaggtccttggcacctcctctggcctccccgtggagcgccgagaccagcctgggga gccggcgaccgagttctcctgccgggagttggaggccggcagcctagtctatgtccaccgcggtggtcctgcacaggacttgacgttc cgggtcagcgatggactgcaggccagccccccggccacgctgaaggtggtggccatccggccggccatacagatccaccgcagc acagggttgcgactggcccaaggctctgccatgcccatcttgcccgccaacctgtcggtggagaccaatgccgtggggcaggatgtg agcgtgctgttccgcgtcactggggccctgcagtttggggagctgcagaagcagggggcaggtggggtggagggtgctgagtggtg ggccacacaggcgttccaccagcgggatgtggagcagggccgcgtgaggtacctgagcactgacccacagcaccacgcttacgac accgtggagaacctggccctggaggtgcaggtgggccaggagatcctgagcaatctgtccttcccagtgaccatccagagagccact gtgtggatgctgcggctggagccactgcacactcagaacacccagcaggagaccctcaccacagcccacctggaggccaccctgg aggaggcaggcccaagccccccaaccttccattatgaggtggttcaggctcccaggaaaggcaaccttcaactacagggcacaagg ctgtcagatggccagggcttcacccaggatgacatacaggctggccgggtgacctatggggccacagcacgtgcctcagaggcagt cgaggacaccttccgtttccgtgtcacagctccaccatatttctccccactctataccttccccatccacattggtggtgacccagatgcgc ctgtcctcaccaatgtcctcctcgtggtgcctgagggtggtgagggtgtcctctctgctgaccacctctttgtcaagagtctcaacagtgc cagctacctctatgaggtcatggagcggccccgccatgggaggttggcttggcgtgggacacaggacaagaccactatggtgacatc cttcaccaatgaagacctgttgcgtggccggctggtctaccagcatgatgactccgagaccacagaagatgatatcccatttgttgctac ccgccagggcgagagcagtggtgacatggcctgggaggaggtacggggtgtcttccgagtggccatccagcccgtgaatgaccac gcccctgtgcagaccatcagccggatcttccatgtggcccggggtgggcggcggctgctgactacagacgacgtggccttcagcgat gctgactcgggctttgctgacgcccagctggtgcttacccgcaaggacctcctctttggcagtatcgtggccgtagatgagcccacgcg gcccatctaccgcttcacccaggaggacctcaggaagaggcgagtactgttcgtgcactcaggggctgaccgtggctggatccagct gcaggtgtccgacgggcaacaccaggccactgcgctgctggaggtgcaggcctcggaaccctacctccgtgtggccaacggctcca gccttgtggtccctcaagggggccagggcaccatcgacacggccgtgctccacctggacaccaacctcgacatccgcagtggggat gaggtccactaccacgtcacagctggccctcgctggggacagctagtccgggctggtcagccagccacagccttctcccagcagga cctgctggatggggccgttctctatagccacaatggcagcctcagcccccgcgacaccatggccttctccgtggaagcagggccagt gcacacggatgccaccctacaagtgaccattgccctagagggcccactggccccactgaagctggtccggcacaagaagatctacgt cttccagggagaggcagctgagatcagaagggaccagctggaggcagcccaggaggcagtgccacctgcagacatcgtattctca gtgaagagcccaccgagtgccggctacctggtgatggtgtcgcgtggcgccttggcagatgagccacccagcctggaccctgtgca gagcttctcccaggaggcagtggacacaggcagggtcctgtacctgcactcccgccctgaggcctggagcgatgccttctcgctgga tgtggcctcaggcctgggtgctcccctcgagggcgtccttgtggagctggaggtgctgcccgctgccatcccactagaggcgcaaaa cttcagcgtccctgagggtggcagcctcaccctggcccctccactgctccgtgtctccgggccctacttccccactctcctgggcctca gcctgcaggtgctggagccaccccagcatggagccctgcagaaggaggacggacctcaagccaggaccctcagcgccttctcctg gagaatggtggaagagcagctgatccgctacgtgcatgacgggagcgagacactgacagacagttttgtcctgatggctaatgcctcc gagatggatcgccagagccatcctgtggccttcactgtcactgtcctgcctgtcaatgaccaaccccccatcctcactacaaacacagg cctgcagatgtgggagggggccactgcgcccatccctgcggaggctctgaggagcacggacggcgactctgggtctgaggatctg gtctacaccatcgagcagcccagcaacgggcgggtagtgctgcggggggcgccgggcactgaggtgcgcagcttcacgcaggcc cagctggacggcgggctcgtgctgttctcacacagaggaaccctggatggaggcttccgcttccgcctctctgacggcgagcacactt cccccggacacttcttccgagtgacggcccagaagcaagtgctcctctcgctgaagggcagccagacactgactgtctgcccagggt ccgtccagccactcagcagtcagaccctcagggccagctccagcgcaggcactgacccccagctcctgctctaccgtgtggtgcgg ggcccccagctaggccggctgttccacgcccagcaggacagcacaggggaggccctggtgaacttcactcaggcagaggtctacg ctgggaatattctgtatgagcatgagatgccccccgagcccttttgggaggcccatgataccctagagctccagctgtcctcgccgcct gcccgggacgtggccgccacccttgctgtggctgtgtcttttgaggctgcctgtccccagcgccccagccacctctggaagaacaaag gtctctgggtccccgagggccagcgggccaggatcaccgtggctgctctggatgcctccaatctcttggccagcgttccatcacccca gcgctcagagcatgatgtgctcttccaggtcacacagttccccagccggggccagctgttggtgtccgaggagcccctccatgctggg cagccccacttcctgcagtcccagctggctgcagggcagctagtgtatgcccacggcggtgggggcacccagcaggatggcttcca ctttcgtgcccacctccaggggccagcaggggcctccgtggctggaccccaaacctcagaggcctttgccatcacggtgagggatgt aaatgagcggccccctcagccacaggcctctgtcccactccggctcacccgaggctctcgtgcccccatctcccgggcccagctgag tgtggtggacccagactcagctcctggggagattgagtacgaggtccagcgggcaccccacaacggcttcctcagcctggtgggtgg tggcctggggcccgtgacccgcttcacgcaagccgatgtggattcagggcggctggccttcgtggccaacgggagcagcgtggcag gcatcttccagctgagcatgtctgatggggccagcccacccctgcccatgtccctggctgtggacatcctaccatccgccatcgaggtg cagctgcgggcacccctggaggtgccccaagctttggggcgctcctcactgagccagcagcagctccgggtggtttcagatcggga ggagccagaggcagcataccgcctcatccagggaccccagtatgggcatctcctggtgggcgggcggcccacctcggccttcagcc aattccagatagaccagggcgaggtggtctttgccttcaccaacttctcctcctctcatgaccacttcagagtcctggcactggctaggg gtgtcaatgcatcagccgtagtgaacgtcactgtgagggctctgctgcatgtgtgggcaggtgggccatggccccagggtgccaccct gcgcctggaccccaccgtcctagatgctggcgagctggccaaccgcacaggcagtgtgccgcgcttccgcctcctggagggacccc ggcatggccgcgtggtccgcgtgccccgagccaggacggagcccgggggcagccagctggtggagcagttcactcagcaggacc ttgaggacgggaggctggggctggaggtgggcaggccagaggggagggcccccggccccgcaggtgacagtctcactctggag ctgtgggcacagggcgtcccgcctgctgtggcctccctggactttgccactgagccttacaatgctgcccggccctacagcgtggccc tgctcagtgtccccgaggccgcccggacggaagcagggaagccagagagcagcacccccacaggcgagccaggccccatggca tccagccctgagcccgctgtggccaagggaggcttcctgagcttccttgaggccaacatgttcagcgtcatcatccccatgtgcctggt acttctgctcctggcgctcatcctgcccctgctcttctacctccgaaaacgcaacaagacgggcaagcatgacgtccaggtcctgactg ccaagccccgcaacggcctggctggtgacaccgagacctttcgcaaggtggagccaggccaggccatcccgctcacagctgtgcct ggccaggggccccctccaggaggccagcctgacccagagctgctgcagttctgccggacacccaaccctgcccttaagaatggcca gtactgggtgtgaggcctggcctgggcccagatgctgatcgggccagggacaggc (SEQ ID No: 50). In another embodiment, the recombinant nucleotide has the sequence set forth in SEQ ID NO: 50. In another embodiment, an HMW-MAA-encoding nucleotide of methods and compositions as provided herein comprises the sequence set forth in SEQ ID No: 50. In another embodiment, the HMW-MAA-encoding nucleotide is a homologue of SEQ ID No: 50. In another embodiment, the HMW-MAA-encoding nucleotide is a variant of SEQ ID No: 50. In another embodiment, the HMW-MAA-encoding nucleotide is a fragment of SEQ ID No: 50. In another embodiment, the HMW-MAA-encoding nucleotide is an isoform of SEQ ID No: 50. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000207] In another embodiment, the HMW-MAA protein of methods and compositions as provided herein has an AA sequence set forth in a GenBank entry having an Accession Numbers selected from NM_001897 and X96753. In another embodiment, the HMW-MAA protein is encoded by a nucleotide sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein comprises a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein is a homologue of a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW- MAA protein is a variant of a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein is a fragment of a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein is an isoform of a sequence set forth in one of the above GenBank entries. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000208] The HMW-MAA fragment utilized in the present invention comprises, in another embodiment, AA 360-554. In another embodiment, the fragment consists essentially of AA 360-554. In another embodiment, the fragment consists of AA 360-554. In another embodiment, the fragment comprises AA 701-1130. In another embodiment, the fragment consists essentially of AA 701-1130. In another embodiment, the fragment consists of AA 701-1130. In another embodiment, the fragment comprises AA 2160-2258. In another embodiment, the fragment consists essentially of 2160-2258. In another embodiment, the fragment consists of 2160-2258. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000209] In another embodiment, the recombinant Listeria of the compositions and methods as provided herein comprise a plasmid that encodes a recombinant polypeptide that is, in one embodiment, angiogenic, and in another embodiment, antigenic. In one embodiment, the polypeptide is HMW-MAA, and in another embodiment, the polypeptide is a HMW-MAA fragment. In another embodiment, the plasmid further encodes a non-HMW-MAA peptide. In one embodiment, the non-HMW-MAA peptide enhances the immunogenicity of the polypeptide. In one embodiment, the HMW-MAA fragment of methods and compositions as provided herein is fused to the non-HMW-MAA AA sequence. In another embodiment, the HMW-MAA fragment is embedded within the non-HMW-MAA AA sequence. In another embodiment, an HMW-MAA-derived peptide is incorporated into an LLO fragment, ActA protein or fragment, or PEST-like sequence. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000210] The non-HMW-MAA peptide is, in one embodiment, a listeriolysin (LLO) oligopeptide. In another embodiment, the non-HMW-MAA peptide is an ActA oligopeptide. In another embodiment, the non-HMW-MAA peptide is a PEST-like oligopeptide. In one embodiment, fusion to LLO, ActA, PEST-like sequences and fragments thereof enhances the cell-mediated immunogenicity of antigens. In one embodiment, fusion to LLO, ActA, PEST- like sequences and fragments thereof enhances the cell-mediated immunogenicity of antigens in a variety of expression systems. In another embodiment, the non-HMW-MAA peptide is any other immunogenic non-HMW-MAA peptide known in the art. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000211] In one embodiment, the recombinant Listeria strain of the compositions and methods as provided herein express a heterologous antigen that is expressed by a tumor cell. In one embodiment, the recombinant Listeria strain of the compositions and methods as provided herein comprise a first or second nucleic acid molecule that encodes a Prostate Specific Antigen (PSA), which in one embodiment, is a marker for prostate cancer that is highly expressed by prostate tumors, which in one embodiment is the most frequent type of cancer in American men and, in another embodiment, is the second cause of cancer related death in American men. In one embodiment, PSA is a kallikrein serine protease (KLK3) secreted by prostatic epithelial cells, which in one embodiment, is widely used as a marker for prostate cancer. [000212] In one embodiment, the recombinant Listeria strain as provided herein comprises a nucleic acid molecule encoding KLK3 protein. [000213] In another embodiment, the KLK3 protein has the sequence: [000214] MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC GGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLK NRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEE FLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCN GVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 51; GenBank Accession No. CAA32915). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 51. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 51. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 51. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 51. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000215] In another embodiment, the KLK3 protein has the sequence: [000216] IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVIL LGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAEL TDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHP QKVTKFMLCAGRWTGGKSTCSGDSGGPLVCYGVLQGITSWGSEPCALPERPSLYTK VVHYRKWIKDTIVANP (SEQ ID No: 52). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 52. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 52. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 52. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 52. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000217] In another embodiment, the KLK3 protein has the sequence: IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSL FHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVK VMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTK FMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYR KWIKDTIVANP (SEQ ID No: 53; GenBank Accession No. AAA59995.1). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 53. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 53. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 53. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 53. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000218] In another embodiment, the KLK3 protein is encoded by a nucleotide molecule having the sequence: ggtgtcttaggcacactggtcttggagtgcaaaggatctaggcacgtgaggctttgtatgaagaatcggggatcgtacccaccccctgtt tctgtttcatcctgggcatgtctcctctgcctttgtcccctagatgaagtctccatgagctacaagggcctggtgcatccagggtgatctagt aattgcagaacagcaagtgctagctctccctccccttccacagctctgggtgtgggagggggttgtccagcctccagcagcatgggga gggccttggtcagcctctgggtgccagcagggcaggggcggagtcctggggaatgaaggttttatagggctcctgggggaggctcc ccagccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattg gtgagaggggccatggttggggggatgcaggagagggagccagccctgactgtcaagctgaggctctttcccccccaacccagcac cccagcccagacagggagctgggctcttttctgtctctcccagccccacttcaagcccatacccccagtcccctccatattgcaacagtc ctcactcccacaccaggtccccgctccctcccacttaccccagaactttcttcccatttgcccagccagctccctgctcccagctgctttac taaaggggaagttcctgggcatctccgtgtttctctttgtggggctcaaaacctccaaggacctctctcaatgccattggttccttggaccg tatcactggtccatctcctgagcccctcaatcctatcacagtctactgacttttcccattcagctgtgagtgtccaaccctatcccagagacc ttgatgcttggcctcccaatcttgccctaggatacccagatgccaaccagacacctccttctttcctagccaggctatctggcctgagaca acaaatgggtccctcagtctggcaatgggactctgagaactcctcattccctgactcttagccccagactcttcattcagtggcccacattt tccttaggaaaaacatgagcatccccagccacaactgccagctctctgagtccccaaatctgcatccttttcaaaacctaaaaacaaaaa gaaaaacaaataaaacaaaaccaactcagaccagaactgttttctcaacctgggacttcctaaactttccaaaaccttcctcttccagcaa ctgaacctcgccataaggcacttatccctggttcctagcaccccttatcccctcagaatccacaacttgtaccaagtttcccttctcccagtc caagaccccaaatcaccacaaaggacccaatccccagactcaagatatggtctgggcgctgtcttgtgtctcctaccctgatccctggg ttcaactctgctcccagagcatgaagcctctccaccagcaccagccaccaacctgcaaacctagggaagattgacagaattcccagcc tttcccagctccccctgcccatgtcccaggactcccagccttggttctctgcccccgtgtcttttcaaacccacatcctaaatccatctccta tccgagtcccccagttccccctgtcaaccctgattcccctgatctagcaccccctctgcaggcgctgcgcccctcatcctgtctcggattg tgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggt gcacccccagtgggtcctcacagctgcccactgcatcaggaagtgagtaggggcctggggtctggggagcaggtgtctgtgtcccag aggaataacagctgggcattttccccaggataacctctaaggccagccttgggactgggggagagagggaaagttctggttcaggtca catggggaggcagggttggggctggaccaccctccccatggctgcctgggtctccatctgtgtccctctatgtctctttgtgtcgctttcat tatgtctcttggtaactggcttcggttgtgtctctccgtgtgactattttgttctctctctccctctcttctctgtcttcagtctccatatctccccct ctctctgtccttctctggtccctctctagccagtgtgtctcaccctgtatctctctgccaggctctgtctctcggtctctgtctcacctgtgcctt ctccctactgaacacacgcacgggatgggcctgggggaccctgagaaaaggaagggctttggctgggcgcggtggctcacacctgt aatcccagcactttgggaggccaaggcaggtagatcacctgaggtcaggagttcgagaccagcctggccaactggtgaaaccccatc tctactaaaaatacaaaaaattagccaggcgtggtggcgcatgcctgtagtcccagctactcaggagctgagggaggagaattgcattg aacctggaggttgaggttgcagtgagccgagaccgtgccactgcactccagcctgggtgacagagtgagactccgcctcaaaaaaaa aaaaaaaaaaaaaaaaaaaaaagaaaagaaaagaaaagaaaaggaagtgttttatccctgatgtgtgtgggtatgagggtatgagag ggcccctctcactccattccttctccaggacatccctccactcttgggagacacagagaagggctggttccagctggagctgggaggg gcaattgagggaggaggaaggagaagggggaaggaaaacagggtatgggggaaaggaccctggggagcgaagtggaggatac aaccttgggcctgcaggcaggctacctacccacttggaaacccacgccaaagccgcatctacagctgagccactctgaggcctcccc tccccggcggtccccactcagctccaaagtctctctcccttttctctcccacactttatcatcccccggattcctctctacttggttctcattctt cctttgacttcctgcttccctttctcattcatctgtttctcactttctgcctggttttgttcttctctctctctttctctggcccatgtctgtttctctatgt ttctgtcttttctttctcatcctgtgtattttcggctcaccttgtttgtcactgttctcccctctgccctttcattctctctgcccttttaccctcttcctt ttcccttggttctctcagttctgtatctgcccttcaccctctcacactgctgtttcccaactcgttgtctgtattttggcctgaactgtgtcttccc aaccctgtgttttctcactgtttctttttctcttttggagcctcctccttgctcctctgtcccttctctctttccttatcatcctcgctcctcattcctgc gtctgcttcctccccagcaaaagcgtgatcttgctgggtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagc cacagcttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgct gctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacct gctacgcctcaggctggggcagcattgaaccagaggagtgtacgcctgggccagatggtgcagccgggagcccagatgcctgggt ctgagggaggaggggacaggactcctgggtctgagggaggagggccaaggaaccaggtggggtccagcccacaacagtgtttttg cctggcccgtagtcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaa ggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcggtgagtcatccctactcccaagatcttgag ggaaaggtgagtgggaccttaattctgggctggggtctagaagccaacaaggcgtctgcctcccctgctccccagctgtagccatgcc acctccccgtgtctcatctcattccctccttccctcttctttgactccctcaaggcaataggttattcttacagcacaactcatctgttcctgcgt tcagcacacggttactaggcacctgctatgcacccagcactgccctagagcctgggacatagcagtgaacagacagagagcagccc ctcccttctgtagcccccaagccagtgaggggcacaggcaggaacagggaccacaacacagaaaagctggagggtgtcaggaggt gatcaggctctcggggagggagaaggggtggggagtgtgactgggaggagacatcctgcagaaggtgggagtgagcaaacacct gcgcaggggaggggagggcctgcggcacctgggggagcagagggaacagcatctggccaggcctgggaggaggggcctagag ggcgtcaggagcagagaggaggttgcctggctggagtgaaggatcggggcagggtgcgagagggaacaaaggacccctcctgca gggcctcacctgggccacaggaggacactgcttttcctctgaggagtcaggaactgtggatggtgctggacagaagcaggacaggg cctggctcaggtgtccagaggctgcgctggcctcctatgggatcagactgcagggagggagggcagcagggatgtggagggagtg atgatggggctgacctgggggtggctccaggcattgtccccacctgggcccttacccagcctccctcacaggctcctggccctcagtct ctcccctccactccattctccacctacccacagtgggtcattctgatcaccgaactgaccatgccagccctgccgatggtcctccatggct ccctagtgccctggagaggaggtgtctagtcagagagtagtcctggaaggtggcctctgtgaggagccacggggacagcatcctgca gatggtcctggcccttgtcccaccgacctgtctacaaggactgtcctcgtggaccctcccctctgcacaggagctggaccctgaagtcc cttcctaccggccaggactggagcccctacccctctgttggaatccctgcccaccttcttctggaagtcggctctggagacatttctctctt cttccaaagctgggaactgctatctgttatctgcctgtccaggtctgaaagataggattgcccaggcagaaactgggactgacctatctc actctctccctgcttttacccttagggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaac catgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaacccctg agcacccctatcaagtccctattgtagtaaacttggaaccttggaaatgaccaggccaagactcaagcctccccagttctactgacctttg tccttaggtgtgaggtccagggttgctaggaaaagaaatcagcagacacaggtgtagaccagagtgtttcttaaatggtgtaattttgtcc tctctgtgtcctggggaatactggccatgcctggagacatatcactcaatttctctgaggacacagttaggatggggtgtctgtgttatttgt gggatacagagatgaaagaggggtgggatcc (SEQ ID No: 54; GenBank Accession No. X14810). In another embodiment, the KLK3 protein is encoded by residues 401..446, 1688..1847, 3477..3763, 3907..4043, and 5413..5568 of SEQ ID No: 54. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 54. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 54. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 54. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 54. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000219] In another embodiment, the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSWVILITELTMPALPMVL HGSLVPWRGGV (SEQ ID No: 55; GenBank Accession No. NP_001025218) In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 55. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 55. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 55. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 55. Each possibility represents a separate embodiment as provided herein. [000220] In another embodiment, the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattggtg ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggc agggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgg gtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctc ctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatg ctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagag gagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgacca agttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgtgggtcattctgatcaccgaactgaccatgccagccct gccgatggtcctccatggctccctagtgccctggagaggaggtgtctagtcagagagtagtcctggaaggtggcctctgtgaggagcc acggggacagcatcctgcagatggtcctggcccttgtcccaccgacctgtctacaaggactgtcctcgtggaccctcccctctgcacag gagctggaccctgaagtcccttccccaccggccaggactggagcccctacccctctgttggaatccctgcccaccttcttctggaagtc ggctctggagacatttctctcttcttccaaagctgggaactgctatctgttatctgcctgtccaggtctgaaagataggattgcccaggcag aaactgggactgacctatctcactctctccctgcttttacccttagggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtat cacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaagga caccatcgtggccaacccctgagcacccctatcaaccccctattgtagtaaacttggaaccttggaaatgaccaggccaagactcaagc ctccccagttctactgacctttgtccttaggtgtgaggtccagggttgctaggaaaagaaatcagcagacacaggtgtagaccagagtgt ttcttaaatggtgtaattttgtcctctctgtgtcctggggaatactggccatgcctggagacatatcactcaatttctctgaggacacagatag gatggggtgtctgtgttatttgtggggtacagagatgaaagaggggtgggatccacactgagagagtggagagtgacatgtgctggac actgtccatgaagcactgagcagaagctggaggcacaacgcaccagacactcacagcaaggatggagctgaaaacataacccactc tgtcctggaggcactgggaagcctagagaaggctgtgagccaaggagggagggtcttcctttggcatgggatggggatgaagtaag gagagggactggaccccctggaagctgattcactatggggggaggtgtattgaagtcctccagacaaccctcagatttgatgatttccta gtagaactcacagaaataaagagctgttatactgtg (SEQ ID No: 56; GenBank Accession No. NM_001030047). In another embodiment, the KLK3 protein is encoded by residues 42-758 of SEQ ID No: 56. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 56. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 56. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 56. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 56. [000221] In another embodiment, the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRK (SEQ ID No: 57; GenBank Accession No. NP_001025221). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 57. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 57. In another embodiment, the sequence of the KLK3 protein comprises SEQ ID No: 57. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 57. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 57. [000222] In another embodiment, the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcacccttccgtgacgtggattggtgc tgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggca gggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaagtgagtaggggcctggggtc tggggagcaggtgtctgtgtcccagaggaataacagctgggcattttccccaggataacctctaaggccagccttgggactggggga gagagggaaagttctggttcaggtcacatggggaggcagggttggggctggaccaccctccccatggctgcctgggtctccatctgtg ttcctctatgtctctttgtgtcgctttcattatgtctcttggtaactggcttcggttgtgtctctccgtgtgactattttgttctctctctccctctcttc tctgtcttcagt (SEQ ID No: 58; GenBank Accession No. NM_001030050). In another embodiment, the KLK3 protein is encoded by residues 42-758 of SEQ ID No: 58. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 58. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 58. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 58. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 58. [000223] In another embodiment, the KLK3 protein that is the source of the KLK3 peptide has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGD SGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 59; GenBank Accession No. NP_001025220). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 29. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 59. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 59. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 59. [000224] In another embodiment, the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattggtg ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggc agggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgg gtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctc ctgaagaatcgattcctcaggccaggtgatgactccagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacct ccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggca aaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccc tgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaacccctgagcacccc tatcaaccccctattgtagtaaacttggaaccttggaaatgaccaggccaagactcaagcctccccagttctactgacctttgtccttaggt gtgaggtccagggttgctaggaaaagaaatcagcagacacaggtgtagaccagagtgtttcttaaatggtgtaattttgtcctctctgtgt cctggggaatactggccatgcctggagacatatcactcaatttctctgaggacacagataggatggggtgtctgtgttatttgtggggtac agagatgaaagaggggtgggatccacactgagagagtggagagtgacatgtgctggacactgtccatgaagcactgagcagaagct ggaggcacaacgcaccagacactcacagcaaggatggagctgaaaacataacccactctgtcctggaggcactgggaagcctaga gaaggctgtgagccaaggagggagggtcttcctttggcatgggatggggatgaagtaaggagagggactggaccccctggaagctg attcactatggggggaggtgtattgaagtcctccagacaaccctcagatttgatgatttcctagtagaactcacagaaataaagagctgtt atactgtg (SEQ ID No: 60; GenBank Accession No. NM_001030049). In another embodiment, the KLK3 protein is encoded by residues 42-758 of SEQ ID No: 60. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 60. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 60. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 60. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 60. [000225] In another embodiment, the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRKPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYAS GWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSG DSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 61; GenBank Accession No. NP_001025219). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 61. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 61. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 61. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 61. [000226] In another embodiment, the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattggtg ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggc agggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaagccaggtgatgactccagc cacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagca ctggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctcc atgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaa agcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctg cccgaaaggccttccctgtacaccaaggtggtgcattacccaaggacaccatcgtggccaacccctgagcacccctatcaacccccta ttgtagtaaacttggaaccttggaaatgaccaggccaagactcaagcctccccagttctactgacctttgtccttaggtgtgaggtccagg gttgctaggaaaagaaatcagcagacacaggtgtagaccagagtgtttcttaaatggtgtaattttgtcctctctgtgtcctggggaatact ggccatgcctggagacatatcactcaatttctctgaggacacagataggatggggtgtctgtgttatttgtggggtacagagatgaaaga ggggtgggatccacactgagagagtggagagtgacatgtgctggacactgtccatgaagcactgagcagaagctggaggcacaac gcaccagacactcacagcaaggatggagctgaaaacataacccactctgtcctggaggcactgggaagcctagagaaggctgtgag ccaaggagggagggtcttcctttggcatgggatggggatgaagtaaggagagggactggaccccctggaagctgattcactatgggg ggaggtgtattgaagtcctccagacaaccctcagatttgatgatttcctagtagaactcacagaaataaagagctgttatactgtg (SEQ ID No: 62; GenBank Accession No. NM_001030048). In another embodiment, the KLK3 protein is encoded by residues 42-758 of SEQ ID No: 62. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 62. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 62. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 62. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 62. [000227] In another embodiment, the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGIT SWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 63; GenBank Accession No. NP_001639). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 63. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 63. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 63. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 63. [000228] In another embodiment, the KLK3 protein is encoded by a nucleotide molecule having the sequence: agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtggattggtg ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggc agggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgg gtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctc ctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatg ctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagag gagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgacca agttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttc aaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggat caaggacaccatcgtggccaacccctgagcacccctatcaaccccctattgtagtaaacttggaaccttggaaatgaccaggccaaga ctcaagcctccccagttctactgacctttgtccttaggtgtgaggtccagggttgctaggaaaagaaatcagcagacacaggtgtagacc agagtgtttcttaaatggtgtaattttgtcctctctgtgtcctggggaatactggccatgcctggagacatatcactcaatttctctgaggaca cagataggatggggtgtctgtgttatttgtggggtacagagatgaaagaggggtgggatccacactgagagagtggagagtgacatgt gctggacactgtccatgaagcactgagcagaagctggaggcacaacgcaccagacactcacagcaaggatggagctgaaaacata acccactctgtcctggaggcactgggaagcctagagaaggctgtgagccaaggagggagggtcttcctttggcatgggatggggatg aagtaaggagagggactggaccccctggaagctgattcactatggggggaggtgtattgaagtcctccagacaaccctcagatttgat gatttcctagtagaactcacagaaataaagagctgttatactgtg (SEQ ID No: 64; GenBank Accession No. NM_001648). In another embodiment, the KLK3 protein is encoded by residues 42-827 of SEQ ID No: 64. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 64. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 64. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 64. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 64. [000229] In another embodiment, the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGIT SWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 65 GenBank Accession No. AAX29407.1). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 65. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 65. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 65. In another embodiment, the sequence of the KLK3 protein comprises SEQ ID No: 65. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 65. [000230] In another embodiment, the KLK3 protein is encoded by a nucleotide molecule having the sequence: gggggagccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtccgtgacgtgg attggtgctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctct cgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatc ttgctgggtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatg agcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctca cggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaa ccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaag gtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatg gtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccgg aagtggatcaaggacaccatcgtggccaacccctgagcacccctatcaactccctattgtagtaaacttggaaccttggaaatgaccag gccaagactcaggcctccccagttctactgacctttgtccttaggtgtgaggtccagggttgctaggaaaagaaatcagcagacacagg tgtagaccagagtgtttcttaaatggtgtaattttgtcctctctgtgtcctggggaatactggccatgcctggagacatatcactcaatttctct gaggacacagataggatggggtgtctgtgttatttgtggggtacagagatgaaagaggggtgggatccacactgagagagtggagag tgacatgtgctggacactgtccatgaagcactgagcagaagctggaggcacaacgcaccagacactcacagcaaggatggagctga aaacataacccactctgtcctggaggcactgggaagcctagagaaggctgtgagccaaggagggagggtcttcctttggcatgggat ggggatgaagtagggagagggactggaccccctggaagctgattcactatggggggaggtgtattgaagtcctccagacaaccctca gatttgatgatttcctagtagaactcacagaaataaagagctgttatactgcgaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID No: 66; GenBank Accession No. BC056665). In another embodiment, the KLK3 protein is encoded by residues 47-832 of SEQ ID No: 66. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 66. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 66. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 66. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 66. [000231] In another embodiment, the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGD SGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVA (SEQ ID No: 67; GenBank Accession No. AJ459782). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 67. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 67. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 67. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 67. [000232] In another embodiment, the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSVSHPYSQDLEGKGEWG

P (SEQ ID No: 68, GenBank Accession No. AJ512346). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 68. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 68. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 68. In another embodiment, the sequence of the KLK3 protein comprises SEQ ID No: 68. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 68. [000233] In another embodiment, the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGERGHGWGDAGEGASPDCQAEALSPPTQHPSPDRELGSFLSL PAPLQAHTPSPSILQQSSLPHQVPAPSHLPQNFLPIAQPAPCSQLLY (SEQ ID No: 69 GenBank Accession No. AJ459784). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 69. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 69. In another embodiment, the sequence of the KLK3 protein comprises SEQ ID No: 69. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 69. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 69. [000234] In another embodiment, the KLK3 protein has the sequence: MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGIT SWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID NO: 70 GenBank Accession No. AJ459783). In another embodiment, the KLK3 protein is a homologue of SEQ ID No: 70. In another embodiment, the KLK3 protein is a variant of SEQ ID No: 70. In another embodiment, the KLK3 protein is an isomer of SEQ ID No: 70. In another embodiment, the KLK3 protein is a fragment of SEQ ID No: 70. [000235] In another embodiment, the KLK3 protein is encoded by a nucleotide molecule having the sequence: aagtttcccttctcccagtccaagaccccaaatcaccacaaaggacccaatccccagactcaagatatggtctgggcgctgtcttgtgtc tcctaccctgatccctgggttcaactctgctcccagagcatgaagcctctccaccagcaccagccaccaacctgcaaacctagggaag attgacagaattcccagcctttcccagctccccctgcccatgtcccaggactcccagccttggttctctgcccccgtgtcttttcaaaccca catcctaaatccatctcctatccgagtcccccagttcctcctgtcaaccctgattcccctgatctagcaccccctctgcaggtgctgcaccc ctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtagcctctcgtggcagggcagt ctgcggcggtgttctggtgcacccccagtgggtcctcacagctacccactgcatcaggaacaaaagcgtgatcttgctgggtcggcac agcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaagaat cgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctatgaagg tcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttga ccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgct gtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatca cgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggaca ccatcgtggccaacccctgagcacccctatcaactccctattgtagtaaacttggaaccttggaaatgaccaggccaagactcaggcct ccccagttctactgacctttgtccttaggtgtgaggtccagggttgctaggaaaagaaatcagcagacacaggtgtagaccagagtgttt cttaaatggtgtaattttgtcctctctgtgtcctggggaatactggccatgcctggagacatatcactcaatttctctgaggacacagatagg atggggtgtctgtgttatttgtggggtacagagatgaaagaggggtgggatccacactgagagagtggagagtgacatgtgctggaca ctgtccatgaagcactgagcagaagctggaggcacaacgcaccagacactcacagcaaggatggagctgaaaacataacccactct gtcctggaggcactgggaagcctagagaaggctgtgaaccaaggagggagggtcttcctttggcatgggatggggatgaagtaagg agagggactgaccccctggaagctgattcactatggggggaggtgtattgaagtcctccagacaaccctcagatttgatgatttcctagt agaactcacagaaataaagagctgttatactgtgaa (SEQ ID No: 71; GenBank Accession No. X07730). In another embodiment, the KLK3 protein is encoded by residues 67-1088 of SEQ ID No: 71. In another embodiment, the KLK3 protein is encoded by a homologue of SEQ ID No: 71. In another embodiment, the KLK3 protein is encoded by a variant of SEQ ID No: 71. In another embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No: 71. In another embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No: 71. [000236] In another embodiment, the KLK3 protein is encoded by a sequence set forth in one of the following GenBank Accession Numbers: BC005307, AJ310938, AJ310937, AF335478, AF335477, M27274, and M26663. In another embodiment, the KLK3 protein is encoded by a sequence set forth in one of the above GenBank Accession Numbers. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000237] In another embodiment, the KLK3 protein is encoded by a sequence set forth in one of the following GenBank Accession Numbers: NM_001030050, NM_001030049, NM_001030048, NM_001030047, NM_001648, AJ459782, AJ512346, or AJ459784. Each possibility represents a separate embodiment of the methods and compositions as provided herein. In one embodiment, the KLK3 protein is encoded by a variation of any of the sequences described herein wherein the sequence lacks MWVPVVFLTLSVTWIGAAPLILSR (SEQ ID NO: 72). [000238] In another embodiment, the KLK3 protein has the sequence that comprises a sequence set forth in one of the following GenBank Accession Numbers: X13943, X13942, X13940, X13941, and X13944. [000239] In another embodiment, the KLK3 protein is any other KLK3 protein known in the art. [000240] In another embodiment, the KLK3 peptide is any other KLK3 peptide known in the art. In another embodiment, the KLK3 peptide is a fragment of any other KLK3 peptide known in the art. Each type of KLK3 peptide represents a separate embodiment of the methods and compositions as provided herein. [000241]“KLK3 peptide” refers, in another embodiment, to a full-length KLK3 protein. In another embodiment, the term refers to a fragment of a KLK3 protein. In another embodiment, the term refers to a fragment of a KLK3 protein that is lacking the KLK3 signal peptide. In another embodiment, the term refers to a KLK3 protein that contains the entire KLK3 sequence except the KLK3 signal peptide.“KLK3 signal sequence” refers, in another embodiment, to any signal sequence found in nature on a KLK3 protein. In another embodiment, a KLK3 protein of methods and compositions as provided herein does not contain any signal sequence. [000242] In another embodiment, the kallikrein-related peptidase 3 (KLK3 protein) that is the source of a KLK3 peptide for use in the methods and compositions as provided herein is a PSA protein. In another embodiment, the KLK3 protein is a P-30 antigen protein. In another embodiment, the KLK3 protein is a gamma-seminoprotein protein. In another embodiment, the KLK3 protein is a kallikrein 3 protein. In another embodiment, the KLK3 protein is a semenogelase protein. In another embodiment, the KLK3 protein is a seminin protein. In another embodiment, the KLK3 protein is any other type of KLK3 protein that is known in the art. [000243] In another embodiment, the KLK3 protein is a splice variant 1 KLK3 protein. In another embodiment, the KLK3 protein is a splice variant 2 KLK3 protein. In another embodiment, the KLK3 protein is a splice variant 3 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 1 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 2 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 3 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 4 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 5 KLK3 protein. In another embodiment, the KLK3 protein is a transcript variant 6 KLK3 protein. In another embodiment, the KLK3 protein is a splice variant RP5 KLK3 protein. In another embodiment, the KLK3 protein is any other splice variant KLK3 protein known in the art. In another embodiment, the KLK3 protein is any other transcript variant KLK3 protein known in the art. [000244] In another embodiment, the KLK3 protein is a mature KLK3 protein. In another embodiment, the KLK3 protein is a pro-KLK3 protein. In another embodiment, the leader sequence has been removed from a mature KLK3 protein of methods and compositions as provided herein. [000245] In another embodiment, the KLK3 protein that is the source of a KLK3 peptide of methods and compositions as provided herein is a human KLK3 protein. In another embodiment, the KLK3 protein is a primate KLK3 protein. In another embodiment, the KLK3 protein is a KLK3 protein of any other species known in the art. In another embodiment, one of the above KLK3 proteins is referred to in the art as a“KLK3 protein.” [000246] In another embodiment, KLK3-LLO fusions are provided in US Patent Ser. No. 9,012,141 which is incorporated by reference herein in its entirety. In another embodiment, the antigen of interest is HPV-E7. In another embodiment, the antigen is HPV-E6. In another embodiment, the antigen is Her-2/neu. In another embodiment, the antigen is NY-ESO-1. In another embodiment, the antigen is telomerase (TERT). In another embodiment, the antigen is stratum corneum chymotryptic enzyme (SCCE) and variants thereof. In another embodiment, the antigen is CEA. In another embodiment, the antigen is LMP-1. In another embodiment, the antigen is p53. In another embodiment, the antigen is carboxic anhydrase IX (CAIX). In another embodiment, the antigen is PSMA. In another embodiment, the antigen is prostate stem cell antigen (PSCA). In another embodiment, the antigen is HMW-MAA. In another embodiment, the antigen is WT-1. In another embodiment, the antigen is HIV-1 Gag. In another embodiment, the antigen is Proteinase 3. In another embodiment, the antigen is Tyrosinase related protein 2. In another embodiment, the antigen is PSA (prostate-specific antigen). In another embodiment, the antigen is selected from HPV-E7, HPV-E6, Her-2, NY- ESO-1, telomerase (TERT), SCCE, HMW-MAA, WT-1, HIV-1 Gag, CEA, LMP-1, p53, PSMA, PSCA, Proteinase 3, Tyrosinase related protein 2, Muc1, PSA (prostate-specific antigen), or a combination thereof. [000247] In another embodiment, the antigen is a tumor-associated antigen, which in one embodiment, is one of the following tumor antigens: a MAGE (Melanoma-Associated Antigen E) protein, e.g. MAGE 1, MAGE 2, MAGE 3, MAGE 4, a tyrosinase; carbonic anhydrase 9 (CA9), a mutant ras protein; a mutant p53 protein; p97 melanoma antigen, a ras peptide or p53 peptide associated with advanced cancers; the HPV 16/18 antigens associated with cervical cancers, KLH antigen associated with breast carcinoma, CEA (carcinoembryonic antigen) associated with colorectal cancer, gp100, mesothelin, EGFRvIII, a MART1 antigen associated with melanoma, or the PSA antigen associated with prostate cancer. In another embodiment, the antigen for the compositions and methods as provided herein are melanoma-associated antigens, which in one embodiment are TRP-2, MAGE-1, MAGE-3, gp-100, tyrosinase, HSP-70, beta-HCG, or a combination thereof. [000248] In one embodiment, the recombinant nucleic acid provided herein may encode two separate antigens that serve as tumor targets, which in one embodiment are Prostate Specific Antigen (PSA) and Prostate Cancer Stem Cell (PSCA) antigen. In one embodiment, the recombinant nucleic acid molecule provided herein encodes two separate antigens that serve as tumor targets, which in one embodime are cHer2 and HMW-MAA-C. In another embodiment, the recombinant nucleic acid molecule provided herein encodes two separate antigens that serve as tumor targets, which in one embodime are cHer2 and CA9. In one embodiment, the each invidiual antigen of the two or more antigens expressed by a Listeria as provided herein complement or synergize the immune response. [000249] In another embodiment, the heterologousn antigen provided herein is an angiogenic antigen that affects vascular growth. In one embodiment, the recombinant nucleic acid provided herein may encode two polypeptides each comprising an angiogenic antigen that affect vascular growth fused to a PEST-containing peptide provided herein. In one embodiment, the angiogenic antigen is any angiogenic antigen known in the art, including but not limited toEGFR-III and its related family members, VEGFR and its related family members, HMW-MAA. In one embodiment, the heterologous antigen provided herein may serve as both a tumor antigen an angiogenic factor. In one embodiment, the heterologous antigen is a tumor antigen. In another embodiment, the heterologous antigen is an inhibitor of the function or expression of ARG-1 or NOS or combination. In one embodiment, an inhibitor of NOS is N^G-mono-methyl-L-arginine (L-NMMA), N^G-nitro-L-arginine methyl ester (L- NAME), 7-NI, L-NIL, or L-NIO. In one embodiment, N-omega-nitro-L-arginine a nitric oxide synthase inhibitor and L-arginine competitive inhibitor may be encoded by the nucleic acid. In one embodiment, the second nucleic acid may encode an mRNA that inhibits function or expression of ARG-1 or NOS. [000250] In one embodiment, a heterologous antigen expressed by the Listeria of the present invention may be a neuropeptide growth factor antagonist, which in one embodiment is [D- Arg1, D-Phe5, D-Trp7,9, Leu11]substance P, [Arg6, D-Trp7,9, NmePhe8]substance P(6-11). These and related embodiments embodiments are understood by one of skill in the art. [000251] In other embodiments, the antigen is derived from a fungal pathogen, bacteria, parasite, helminth, or viruses. In other embodiments, the antigen is selected from tetanus toxoid, hemagglutinin molecules from influenza virus, diphtheria toxoid, HIV gp120, HIV gag protein, IgA protease, insulin peptide B, Spongospora subterranea antigen, vibriose antigens, Salmonella antigens, pneumococcus antigens, respiratory syncytial virus antigens, Haemophilus influenza outer membrane proteins, Helicobacter pylori urease, Neisseria meningitidis pilins, N. gonorrhoeae pilins, human papilloma virus antigens E1 and E2 from type HPV-16, -18, -31, -33, -35 or -45 human papilloma viruses, or a combination thereof. [000252] In other embodiments, the antigen is associated with one of the following diseases; cholera, diphtheria, Haemophilus, hepatitis A, hepatitis B, influenza, measles, meningitis, mumps, pertussis, small pox, pneumococcal pneumonia, polio, rabies, rubella, tetanus, tuberculosis, typhoid, Varicella-zoster, whooping cough3 yellow fever, the immunogens and antigens from Addison's disease, allergies, anaphylaxis, Bruton's syndrome, cancer, including solid and blood borne tumors, eczema, Hashimoto's thyroiditis, polymyositis, dermatomyositis, type 1 diabetes mellitus, acquired immune deficiency syndrome, transplant rejection, such as kidney, heart, pancreas, lung, bone, and liver transplants, Graves' disease, polyendocrine autoimmune disease, hepatitis, microscopic polyarteritis, polyarteritis nodosa, pemphigus, primary biliary cirrhosis, pernicious anemia, coeliac disease, antibody-mediated nephritis, glomerulonephritis, rheumatic diseases, systemic lupus erthematosus, rheumatoid arthritis, seronegative spondylarthritides, rhinitis, sjogren's syndrome, systemic sclerosis, sclerosing cholangitis, Wegener's granulomatosis, dermatitis herpetiformis, psoriasis, vitiligo, multiple sclerosis, encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis, Lambert- Eaton syndrome, sclera, episclera, uveitis, chronic mucocutaneous candidiasis, urticaria, transient hypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM syndrome, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune neutropenia, Waldenstrom's macroglobulinemia, amyloidosis, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, malarial circumsporozite protein, microbial antigens, viral antigens, autoantigens, and lesteriosis. [000253] The immune response induced by methods and compositions as provided herein is, in another embodiment, a T cell response. In another embodiment, the immune response comprises a T cell response. In another embodiment, the response is a CD8+ T cell response. In another embodiment, the response comprises a CD8⁺ T cell response. [000254] In one embodiment, a recombinant Listeria of the compositions and methods as provided herein comprise an angiogenic polypeptide. In another embodiment, anti-angiogenic approaches to cancer therapy are very promising, and in one embodiment, one type of such anti-angiogenic therapy targets pericytes. In another embodiment, molecular targets on vascular endothelial cells and pericytes are important targets for antitumor therapies. In another embodiment, the platelet-derived growth factor receptor (PDGF-B/PDGFR-ȕ) signaling is important to recruit pericytes to newly formed blood vessels. Thus, in one embodiment, angiogenic polypeptides as provided herein inhibit molecules involved in pericyte signaling, which in one embodiment, is PDGFR-ȕ. [000255] In one embodiment, the compositions of the present invention comprise an angiogenic factor, or an immunogenic fragment thereof, where in one embodiment, the immunogenic fragment comprises one or more epitopes recognized by the host immune system. In one embodiment, an angiogenic factor is a molecule involved in the formation of new blood vessels. In one embodiment, the angiogenic factor is VEGFR2. In another embodiment, an angiogenic factor of the present invention is Angiogenin; Angiopoietin-1; Del-1; Fibroblast growth factors: acidic (aFGF) and basic (bFGF); Follistatin; Granulocyte colony-stimulating factor (G-CSF); Hepatocyte growth factor (HGF) /scatter factor (SF); Interleukin-8 (IL-8); Leptin; Midkine; Placental growth factor; Platelet-derived endothelial cell growth factor (PD-ECGF); Platelet-derived growth factor-BB (PDGF-BB); Pleiotrophin (PTN); Progranulin; Proliferin; Transforming growth factor-alpha (TGF-alpha); Transforming growth factor-beta (TGF-beta); Tumor necrosis factor-alpha (TNF-alpha); Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF). In another embodiment, an angiogenic factor is an angiogenic protein. In one embodiment, a growth factor is an angiogenic protein. In one embodiment, an angiogenic protein for use in the compositions and methods of the present invention is Fibroblast growth factors (FGF); VEGF; VEGFR and Neuropilin 1 (NRP-1); Angiopoietin 1 (Ang1) and Tie2; Platelet-derived growth factor (PDGF; BB-homodimer) and PDGFR; Transforming growth factor-beta (TGF- ȕ), endoglin and TGF-ȕ receptors; monocyte chemotactic protein-1 (MCP-1); Integrins ĮVȕ3, ĮVȕ5 and Į5ȕ1; VE-cadherin and CD31; ephrin; plasminogen activators; plasminogen activator inhibitor-1; Nitric oxide synthase (NOS) and COX-2; AC133; or Id1/Id3. In one embodiment, an angiogenic protein for use in the compositions and methods of the present invention is an angiopoietin, which in one embodiment, is Angiopoietin 1, Angiopoietin 3, Angiopoietin 4 or Angiopoietin 6. In one embodiment, endoglin is also known as CD105; EDG; HHT1; ORW; or ORW1. In one embodiment, endoglin is a TGFbeta co-receptor. [000256] Examples of target antigens that may find use in the present invention include, but is not limited to: Wilm’s tumor-1 associated protein (Wt-1 ), including Isoforms A, B, C, and D; MHC class I chain-related protein A (MICA); MHC class I chain-related protein B (MICB); gastrin and peptides thereof; gastrin/CCK-2 receptor (CCK-B); Glypican-3; Coactosin-like protein; Prostate acid phosphatase (PAP); Six-transmembrane epithelial antigen of prostate (STEAP); Prostate carcinoma antigen-1 (PCTA-1); Prostate tumor-inducing gene-1 (PTI-1); Prostate-specific gene with homology to G protein-coupled receptor; Prostase; Cancer-testis antigens; SCP-1; SSX-1, SSX-2, SSX-4; GAGE; CT7; CT8; CT10; LAGE-1; GAGE-3/6, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7 GAGE-8; BAGE; NT- SAR-35; CA-125; HIP1R; LMNA; KIAA1416; Seb4D; KNSL6; TRIP4; MDB2; HCAC5; DAM family of genes; RCAS1; RU2; CAMEL; Colon cancer-associated antiges, e.g., NY- CO-8, NY-CO-13, NY-CO-9, NY-CO-16, NY-CO-20, NY-CO-38, NY-CO-45, NY-CO- 9/HDAC5; NY-CO-41/MBD2; NY-CO-42/TRIP4; NY-CO-95/KIAA1416; KNSL6; seb4D; N-Acetylglucosaminyl-transferase V (GnT-V); Elongation factor 2 mutated (ELF2M); HOM- MEL-40/SSX-2; BRDT; SAGE; HAGE; RAGE; Melanoma ubiquitous mutated (MUM-1); MUM-2 Arg-Gly mutation; MUM-3; LDLR/FUT fusion protein antigen of melanoma; NY- REN series of renal cancer antigens; NY-BR series of breast cancer antigens, e.g., NY-BR-62, NY-BR-75, NY-BR-85; BRCA-1, BRCA-2; DEK/CAN fusion protein; Ras, including with mutations in codon 12, 13, 59, or 61, e.g., mutations G12C, G12D, G12R, G12S, G12V, G13D, A59T, Q61H; K-RAS; H-RAS; N-RAS; BRAF; Melanoma antigens including HST-2; MDM-2; Methyl-CpG-binding proteins (MeCP2; MBD2); NA88-A; Histone deacetylases; Cyclophilin B (CYP-B); CA15-3; CA27.29; HsP70; GAGE/PAGE family; Kinesin-2; TATA element modulatory factor 1; tumor protein D53; NY alfa-fetoprotein (AFP); SART1; SART2; SART3; ART4; Preferentially expressed antigen of melanoma (PRAME); CAP1-6D enhancer agonist peptide; cdk4; cdk6; p16 (INK4); Rb protein; TEL; AML1; TEL/AML1; Telomerase (TERT); 707-AP; Annexin, e.g., Annexin II; CML-66; CLM-28; BLC2, BCL6; CD10 protein; CDC27; Sperm protein 17 (SP17); 14-3-3 zeta; MEMD; KIAA0471; TC21; Tyrosinase related proteins 1 and 2 (TRP-1, TRP-2); Gp-100/pmel-17; TARP; Melanocortin- 1 receptor (MC1 R); MUC-1, MUC-2; ETV6/AML1; E-cadherin; cyclooxygenase-2 (COX-2); EphA2; and infectious disease related antigens all of which are listed in US Patent publication serial no. 2014/0186387, which is incorporated by reference herein. [000257] In one embodiment, cancer vaccines as provided herein generate effector T cells that are able to infiltrate the tumor, destroy tumor cells and eradicate the disease. In one embodiment, naturally occurring tumor infiltrating lymphocytes (TILs) are associated with better prognosis in several tumors, such as colon, ovarian and melanoma. In colon cancer, tumors without signs of micrometastasis have an increased infiltration of immune cells and a Th1 expression profile, which correlate with an improved survival of patients. Moreover, the infiltration of the tumor by T cells has been associated with success of immunotherapeutic approaches in both pre-clinical and human trials. In one embodiment, the infiltration of lymphocytes into the tumor site is dependent on the up-regulation of adhesion molecules in the endothelial cells of the tumor vasculature, generally by proinflammatory cytokines, such as IFN-Ȗ, TNF-Į and IL-1. Several adhesion molecules have been implicated in the process of lymphocyte infiltration into tumors, including intercellular adhesion molecule 1 (ICAM-1), vascular endothelial cell adhesion molecule 1 (V-CAM-1), vascular adhesion protein 1 (VAP- 1) and E-selectin. However, these cell-adhesion molecules are commonly down-regulated in the tumor vasculature. Thus, in one embodiment, cancer vaccines as provided herein increase TILs, up-regulate adhesion molecules (in one embodiment, ICAM-1, V-CAM-1, VAP-1, E- selectin, or a combination thereof), up-regulate proinflammatory cytokines (in one embodiment, IFN-Ȗ, TNF-Į , IL-1, or a combination thereof), or a combination thereof. [000258] In one embodiment, the compositions and methods as provided herein provide anti- angiogenesis therapy, which in one embodiment, may improve immunotherapy strategies. In one embodiment, the compositions and methods as provided herein circumvent endothelial cell anergy in vivo by up-regulating adhesion molecules in tumor vessels and enhancing leukocyte-vessel interactions, which increases the number of tumor infiltrating leukocytes, such as CD8⁺ T cells. Interestingly, enhanced anti-tumor protection correlates with an increased number of activated CD4⁺ and CD8⁺ tumor-infiltrating T cells and a pronounced decrease in the number of regulatory T cells in the tumor upon VEGF blockade. [000259] In one embodiment, delivery of anti-angiogenic antigen simultaneously with a tumor-associated antigen to a host afflicted by a tumor as described herein, will have a synergistic effect in impacting tumor growth and a more potent therapeutic efficacy. [000260] In another embodiment, targeting pericytes through vaccination will lead to cytotoxic T lymphocyte (CTL) infiltration, destruction of pericytes, blood vessel destabilization and vascular inflammation, which in another embodiment is associated with up-regulation of adhesion molecules in the endothelial cells that are important for lymphocyte adherence and transmigration, ultimately improving the ability of lymphocytes to infiltrate the tumor tissue. In another embodiment, concomitant delivery of a tumor-specific antigen generate lymphocytes able to invade the tumor site and kill tumor cells. [000261] In one embodiment, the platelet-derived growth factor receptor (PDGF-B/PDGFR-ȕ) signaling is important to recruit pericytes to newly formed blood vessels. In another embodiment, inhibition of VEGFR-2 and PDGFR-ȕ concomitantly induces endothelial cell apoptosis and regression of tumor blood vessels, in one embodiment, approximately 40% of tumor blood vessels. [000262] In another embodiment, said recombinant Listeria strain is an auxotrophic Listeria strain. In another embodiment, said auxotrophic Listeria strain is a dal/dat mutant. In another embodiment, the nucleic acid molecule is stably maintained in the recombinant bacterial strain in the absence of antibiotic selection. [000263] In one embodiment, auxotrophic mutants useful as vaccine vectors may be generated in a number of ways. In another embodiment, D-alanine auxotrophic mutants can be generated, in one embodiment, via the disruption of both the dal gene and the dat gene to generate an attenuated auxotrophic strain of Listeria which requires exogenously added D- alanine for growth. [000264] In one embodiment, the generation of AA strains of Listeria deficient in D-alanine, for example, may be accomplished in a number of ways that are well known to those of skill in the art, including deletion mutagenesis, insertion mutagenesis, and mutagenesis which results in the generation of frameshift mutations, mutations which cause premature termination of a protein, or mutation of regulatory sequences which affect gene expression. In another embodiment, mutagenesis can be accomplished using recombinant DNA techniques or using traditional mutagenesis technology using mutagenic chemicals or radiation and subsequent selection of mutants. In another embodiment, deletion mutants are preferred because of the accompanying low probability of reversion of the auxotrophic phenotype. In another embodiment, mutants of D-alanine which are generated according to the protocols presented herein may be tested for the ability to grow in the absence of D-alanine in a simple laboratory culture assay. In another embodiment, those mutants which are unable to grow in the absence of this compound are selected for further study. [000265] In another embodiment, in addition to the aforementioned D-alanine associated genes, other genes involved in synthesis of a metabolic enzyme, as provided herein, may be used as targets for mutagenesis of Listeria. [000266] In one embodiment, said auxotrophic Listeria strain comprises an episomal expression vector comprising a metabolic enzyme that complements the auxotrophy of said auxotrophic Listeria strain. In another embodiment, the construct is contained in the Listeria strain in an episomal fashion. In another embodiment, the foreign antigen is expressed from a vector harbored by the recombinant Listeria strain. In another embodiment, said episomal expression vector lacks an antibiotic resistance marker. In one embodiment, an antigen of the methods and compositions as provided herein is genetically fused to an oligopeptide comprising a PEST sequence. In another embodiment, said endogenous polypeptide comprising a PEST sequence is LLO. In another embodiment, said endogenous polypeptide comprising a PEST sequence is ActA. [000267] In another embodiment, the metabolic enzyme complements an endogenous metabolic gene that is lacking in the remainder of the chromosome of the recombinant bacterial strain. In one embodiment, the endogenous metabolic gene is mutated in the chromosome. In another embodiment, the endogenous metabolic gene is deleted from the chromosome. In another embodiment, said metabolic enzyme is an amino acid metabolism enzyme. In another embodiment, said metabolic enzyme catalyzes a formation of an amino acid used for a cell wall synthesis in said recombinant Listeria strain. In another embodiment, said metabolic enzyme is an alanine racemase enzyme. In another embodiment, said metabolic enzyme is a D-amino acid transferase enzyme. [000268] In another embodiment, the metabolic enzyme catalyzes the formation of an amino acid (AA) used in cell wall synthesis. In another embodiment, the metabolic enzyme catalyzes synthesis of an AA used in cell wall synthesis. In another embodiment, the metabolic enzyme is involved in synthesis of an AA used in cell wall synthesis. In another embodiment, the AA is used in cell wall biogenesis. [000269] In another embodiment, the metabolic enzyme is a synthetic enzyme for D-glutamic acid, a cell wall component. [000270] In another embodiment, the metabolic enzyme is encoded by an alanine racemase gene (dal) gene. In another embodiment, the dal gene encodes alanine racemase, which catalyzes the reaction L-alanine ļ D-alanine. [000271] The dal gene of methods and compositions of the methods and compositions as provided herein is encoded, in another embodiment, by the sequence: [000272] atggtgacaggctggcatcgtccaacatggattgaaatagaccgcgcagcaattcgcgaaaatataaaaaatgaacaaa ataaactcccggaaagtgtcgacttatgggcagtagtcaaagctaatgcatatggtcacggaattatcgaagttgctaggacggcgaaa gaagctggagcaaaaggtttctgcgtagccattttagatgaggcactggctcttagagaagctggatttcaagatgactttattcttgtgctt ggtgcaaccagaaaagaagatgctaatctggcagccaaaaaccacatttcacttactgtttttagagaagattggctagagaatctaacg ctagaagcaacacttcgaattcatttaaaagtagatagcggtatggggcgtctcggtattcgtacgactgaagaagcacggcgaattga agcaaccagtactaatgatcaccaattacaactggaaggtatttacacgcattttgcaacagccgaccagctagaaactagttattttgaa caacaattagctaagttccaaacgattttaacgagtttaaaaaaacgaccaacttatgttcatacagccaattcagctgcttcattgttacag ccacaaatcgggtttgatgcgattcgctttggtatttcgatgtatggattaactccctccacagaaatcaaaactagcttgccgtttgagctt aaacctgcacttgcactctataccgagatggttcatgtgaaagaacttgcaccaggcgatagcgttagctacggagcaacttatacagca acagagcgagaatgggttgcgacattaccaattggctatgcggatggattgattcgtcattacagtggtttccatgttttagtagacggtga accagctccaatcattggtcgagtttgtatggatcaaaccatcataaaactaccacgtgaatttcaaactggttcaaaagtaacgataattg gcaaagatcatggtaacacggtaacagcagatgatgccgctcaatatttagatacaattaattatgaggtaacttgtttgttaaatgagcgc atacctagaaaatacatccattag (SEQ ID No: 73; GenBank Accession No: AF038438). In another embodiment, the nucleotide encoding dal is homologous to SEQ ID No: 73. In another embodiment, the nucleotide encoding dal is a variant of SEQ ID No: 73. In another embodiment, the nucleotide encoding dal is a fragment of SEQ ID No: 73. In another embodiment, the dal protein is encoded by any other dal gene known in the art. [000273] In another embodiment, the dal protein has the sequence: [000274] MVTGWHRPTWIEIDRAAIRENIKNEQNKLPESVDLWAVVKANAYGHGIIEV ARTAKEAGAKGFCVAILDEALALREAGFQDDFILVLGATRKEDANLAAKNHISLTVF REDWLENLTLEATLRIHLKVDSGMGRLGIRTTEEARRIEATSTNDHQLQLEGIYTHFA TADQLETSYFEQQLAKFQTILTSLKKRPTYVHTANSAASLLQPQIGFDAIRFGISMYGL TPSTEIKTSLPFELKPALALYTEMVHVKELAPGDSVSYGATYTATEREWVATLPIGYA DGLIRHYSGFHVLVDGEPAPIIGRVCMDQTIIKLPREFQTGSKVTIIGKDHGNTVTADD AAQYLDTINYEVTCLLNERIPRKYIH (SEQ ID No: 74; GenBank Accession No: AF038428). In another embodiment, the dal protein is homologous to SEQ ID No: 74. In another embodiment, the dal protein is a variant of SEQ ID No: 74. In another embodiment, the dal protein is an isomer of SEQ ID No: 74. In another embodiment, the dal protein is a fragment of SEQ ID No: 74. In another embodiment, the dal protein is a fragment of a homologue of SEQ ID No: 74. In another embodiment, the dal protein is a fragment of a variant of SEQ ID No: 74. In another embodiment, the dal protein is a fragment of an isomer of SEQ ID No: 74. [000275] In another embodiment, the dal protein is any other Listeria dal protein known in the art. In another embodiment, the dal protein is any other gram-positive dal protein known in the art. In another embodiment, the dal protein is any other dal protein known in the art. [000276] In another embodiment, the dal protein of methods and compositions as provided herein retains its enzymatic activity. In another embodiment, the dal protein retains 90% of wild-type activity. In another embodiment, the dal protein retains 80% of wild-type activity. In another embodiment, the dal protein retains 70% of wild-type activity. In another embodiment, the dal protein retains 60% of wild-type activity. In another embodiment, the dal protein retains 50% of wild-type activity. In another embodiment, the dal protein retains 40% of wild-type activity. In another embodiment, the dal protein retains 30% of wild-type activity. In another embodiment, the dal protein retains 20% of wild-type activity. In another embodiment, the dal protein retains 10% of wild-type activity. In another embodiment, the dal protein retains 5% of wild-type activity. [000277] In another embodiment, the metabolic enzyme is encoded by a D-amino acid aminotransferase gene (dat). D-glutamic acid synthesis is controlled in part by the dat gene, which is involved in the conversion of D-glu + pyr to alpha-ketoglutarate + D-ala, and the reverse reaction. [000278] In another embodiment, a dat gene utilized in the present invention has the sequence set forth in GenBank Accession Number AF038439. In another embodiment, the dat gene is any another dat gene known in the art. [000279] The dat gene of methods and compositions of the methods and compositions as provided herein is encoded, in another embodiment, by the sequence: [000280] atgaaagtattagtaaataaccatttagttgaaagagaagatgccacagttgacattgaagaccgcggatatcagtttggtg atggtgtatatgaagtagttcgtctatataatggaaaattctttacttataatgaacacattgatcgcttatatgctagtgcagcaaaaattgac ttagttattccttattccaaagaagagctacgtgaattacttgaaaaattagttgccgaaaataatatcaatacagggaatgtctatttacaag tgactcgtggtgttcaaaacccacgtaatcatgtaatccctgatgatttccctctagaaggcgttttaacagcagcagctcgtgaagtacct agaaacgagcgtcaattcgttgaaggtggaacggcgattacagaagaagatgtgcgctggttacgctgtgatattaagagcttaaacctt ttaggaaatattctagcaaaaaataaagcacatcaacaaaatgctttggaagctattttacatcgcggggaacaagtaacagaatgttctg cttcaaacgtttctattattaaagatggtgtattatggacgcatgcggcagataacttaatcttaaatggtatcactcgtcaagttatcattgat gttgcgaaaaagaatggcattcctgttaaagaagcggatttcactttaacagaccttcgtgaagcggatgaagtgttcatttcaagtacaa ctattgaaattacacctattacgcatattgacggagttcaagtagctgacggaaaacgtggaccaattacagcgcaacttcatcaatatttt gtagaagaaatcactcgtgcatgtggcgaattagagtttgcaaaataa (SEQ ID No: 75; GenBank Accession No: AF038439). In another embodiment, the nucleotide encoding dat is homologous to SEQ ID No: 75. In another embodiment, the nucleotide encoding dat is a variant of SEQ ID No: 75. In another embodiment, the nucleotide encoding dat is a fragment of SEQ ID No: 75. In another embodiment, the dat protein is encoded by any other dat gene known in the art. [000281] In another embodiment, the dat protein has the sequence: [000282] MKVLVNNHLVEREDATVDIEDRGYQFGDGVYEVVRLYNGKFFTYNEHIDR LYASAAKIDLVIPYSKEELRELLEKLVAENNINTGNVYLQVTRGVQNPRNHVIPDDFP LEGVLTAAAREVPRNERQFVEGGTAITEEDVRWLRCDIKSLNLLGNILAKNKAHQQN ALEAILHRGEQVTECSASNVSIIKDGVLWTHAADNLILNGITRQVIIDVAKKNGIPVKE ADFTLTDLREADEVFISSTTIEITPITHIDGVQVADGKRGPITAQLHQYFVEEITRACGE LEFAK (SEQ ID No: 76; GenBank Accession No: AF038439). In another embodiment, the dat protein is homologous to SEQ ID No: 76. In another embodiment, the dat protein is a variant of SEQ ID No: 76. In another embodiment, the dat protein is an isomer of SEQ ID No: 45. In another embodiment, the dat protein is a fragment of SEQ ID No: 76. In another embodiment, the dat protein is a fragment of a homologue of SEQ ID No: 76. In another embodiment, the dat protein is a fragment of a variant of SEQ ID No: 76. In another embodiment, the dat protein is a fragment of an isomer of SEQ ID No: 76. [000283] In another embodiment, the dat protein is any other Listeria dat protein known in the art. In another embodiment, the dat protein is any other gram-positive dat protein known in the art. In another embodiment, the dat protein is any other dat protein known in the art. [000284] In another embodiment, the dat protein of methods and compositions of the methods and compositions as provided herein retains its enzymatic activity. In another embodiment, the dat protein retains 90% of wild-type activity. In another embodiment, the dat protein retains 80% of wild-type activity. In another embodiment, the dat protein retains 70% of wild- type activity. In another embodiment, the dat protein retains 60% of wild-type activity. In another embodiment, the dat protein retains 50% of wild-type activity. In another embodiment, the dat protein retains 40% of wild-type activity. In another embodiment, the dat protein retains 30% of wild-type activity. In another embodiment, the dat protein retains 20% of wild-type activity. In another embodiment, the dat protein retains 10% of wild-type activity. In another embodiment, the dat protein retains 5% of wild-type activity. [000285] In another embodiment, the metabolic enzyme is encoded by dga. D-glutamic acid synthesis is also controlled in part by the dga gene, and an auxotrophic mutant for D- glutamic acid synthesis will not grow in the absence of D-glutamic acid (Pucci et al, 1995, J Bacteriol. 177: 336-342). In another rembodiment, the recombinant Listeria is auxotrophic for D-glutamic acid. A further example includes a gene involved in the synthesis of diaminopimelic acid. Such synthesis genes encode beta-semialdehyde dehydrogenase, and when inactivated, renders a mutant auxotrophic for this synthesis pathway (Sizemore et al, 1995, Science 270: 299-302). In another embodiment, the dga protein is any other Listeria dga protein known in the art. In another embodiment, the dga protein is any other gram- positive dga protein known in the art. [000286] In another embodiment, the metabolic enzyme is encoded by an alr (alanine racemase) gene. In another embodiment, the metabolic enzyme is any other enzyme known in the art that is involved in alanine synthesis. In another embodiment, the metabolic enzyme is any other enzyme known in the art that is involved in L-alanine synthesis. In another embodiment, the metabolic enzyme is any other enzyme known in the art that is involved in D-alanine synthesis. In another rembodiment, the recombinant Listeria is auxotrophic for D- alanine. Bacteria auxotrophic for alanine synthesis are well known in the art, and are described in, for example, E. coli (Strych et al, 2002, J. Bacteriol. 184:4321-4325), Corynebacterium glutamicum (Tauch et al, 2002, J. Biotechnol 99:79-91), and Listeria monocytogenes (Frankel et al, U.S. Patent 6,099,848)), Lactococcus species, and Lactobacillus species, (Bron et al, 2002, Appl Environ Microbiol, 68: 5663-70). In another embodiment, any D-alanine synthesis gene known in the art is inactivated. [000287] In another embodiment, the metabolic enzyme is an amino acid aminotransferase. [000288] In another embodiment, the metabolic enzyme is encoded by serC, a phosphoserine aminotransferase. In another embodiment, the metabolic enzyme is encoded by asd (aspartate beta-semialdehyde dehydrogenase), involved in synthesis of the cell wall constituent diaminopimelic acid. In another embodiment, the metabolic enzyme is encoded by gsaB- glutamate-1-semialdehyde aminotransferase, which catalyzes the formation of 5- aminolevulinate from (S)-4-amino-5-oxopentanoate. In another embodiment, the metabolic enzyme is encoded by HemL, which catalyzes the formation of 5-aminolevulinate from (S)-4- amino-5-oxopentanoate. In another embodiment, the metabolic enzyme is encoded by aspB, an aspartate aminotransferase that catalyzes the formation of oxalozcetate and L-glutamate from L-aspartate and 2-oxoglutarate. In another embodiment, the metabolic enzyme is encoded by argF-1, involved in arginine biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroE, involved in amino acid biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroB, involved in 3-dehydroquinate biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroD, involved in amino acid biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroC, involved in amino acid biosynthesis. In another embodiment, the metabolic enzyme is encoded by hisB, involved in histidine biosynthesis. In another embodiment, the metabolic enzyme is encoded by hisD, involved in histidine biosynthesis. In another embodiment, the metabolic enzyme is encoded by hisG, involved in histidine biosynthesis. In another embodiment, the metabolic enzyme is encoded by metX, involved in methionine biosynthesis. In another embodiment, the metabolic enzyme is encoded by proB, involved in proline biosynthesis. In another embodiment, the metabolic enzyme is encoded by argR, involved in arginine biosynthesis. In another embodiment, the metabolic enzyme is encoded by argJ, involved in arginine biosynthesis. In another embodiment, the metabolic enzyme is encoded by thiI, involved in thiamine biosynthesis. In another embodiment, the metabolic enzyme is encoded by LMOf2365_1652, involved in tryptophan biosynthesis. In another embodiment, the metabolic enzyme is encoded by aroA, involved in tryptophan biosynthesis. In another embodiment, the metabolic enzyme is encoded by ilvD, involved in valine and isoleucine biosynthesis. In another embodiment, the metabolic enzyme is encoded by ilvC, involved in valine and isoleucine biosynthesis. In another embodiment, the metabolic enzyme is encoded by leuA, involved in leucine biosynthesis. In another embodiment, the metabolic enzyme is encoded by dapF, involved in lysine biosynthesis. In another embodiment, the metabolic enzyme is encoded by thrB, involved in threonine biosynthesis (all GenBank Accession No. NC_002973). [000289] In another embodiment, the metabolic enzyme is a tRNA synthetase. In another embodiment, the metabolic enzyme is encoded by the trpS gene, encoding tryptophanyltRNA synthetase. In another embodiment, the metabolic enzyme is any other tRNA synthetase known in the art. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000290] In another embodiment, the LmddA strain provided herein comprises a mutation, deletion or an inactivation of the dal/dat and actA chromosomal genes. [000291] In another embodiment, a recombinant Listeria strain as provided herein has been passaged through an animal host. In another embodiment, the passaging maximizes efficacy of the strain as a vaccine vector. In another embodiment, the passaging stabilizes the immunogenicity of the Listeria strain. In another embodiment, the passaging stabilizes the virulence of the Listeria strain. In another embodiment, the passaging increases the immunogenicity of the Listeria strain. In another embodiment, the passaging increases the virulence of the Listeria strain. In another embodiment, the passaging removes unstable sub- strains of the Listeria strain. In another embodiment, the passaging reduces the prevalence of unstable sub-strains of the Listeria strain. In another embodiment, the passaging attenuates the strain, or in another embodiment, makes the strain less virulent. Methods for passaging a recombinant Listeria strain through an animal host are well known in the art, and are described, for example, in United States Patent Application Serial No. 10/541,614. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000292] The recombinant Listeria strain of the methods and compositions as provided herein is, in another embodiment, a recombinant Listeria monocytogenes strain. In another embodiment, the Listeria strain is a recombinant Listeria seeligeri strain. In another embodiment, the Listeria strain is a recombinant Listeria grayi strain. In another embodiment, the Listeria strain is a recombinant Listeria ivanovii strain. In another embodiment, the Listeria strain is a recombinant Listeria murrayi strain. In another embodiment, the Listeria strain is a recombinant Listeria welshimeri strain. In another embodiment, the Listeria strain is a recombinant strain of any other Listeria species known in the art. In another embodiment, the sequences of Listeria proteins for use in the methods and compositions as provided herein are from any of the above-described strains. [000293] In one embodiment, a Listeria monocytogenes strain as provided herein is the EGD strain, the 10403S strain, the NICPBP 54002 strain, the S3 strain, the NCTC 5348 strain, the NICPBP 54006 strain, the M7 strain, the S19 strain, or another strain of Listeria monocytogenes which is known in the art. [000294] In another embodiment, the recombinant Listeria strain is a vaccine strain, which in one embodiment, is a bacterial vaccine strain. [000295] In another embodiment, the recombinant Listeria strain utilized in methods of the present invention has been stored in a frozen cell bank. In another embodiment, the recombinant Listeria strain has been stored in a lyophilized cell bank. Each possibility represents a separate embodiment of the present invention. [000296] In another embodiment, the cell bank of methods and compositions of the present invention is a master cell bank. In another embodiment, the cell bank is a working cell bank. In another embodiment, the cell bank is Good Manufacturing Practice (GMP) cell bank. In another embodiment, the cell bank is intended for production of clinical-grade material. In another embodiment, the cell bank conforms to regulatory practices for human use. In another embodiment, the cell bank is any other type of cell bank known in the art. Each possibility represents a separate embodiment of the present invention. [000297]“Good Manufacturing Practices” are defined, in another embodiment, by (21 CFR 210–211) of the United States Code of Federal Regulations. In another embodiment,“Good Manufacturing Practices” are defined by other standards for production of clinical-grade material or for human consumption; e.g. standards of a country other than the United States. Each possibility represents a separate embodiment of the present invention. [000298] In another embodiment, a recombinant Listeria strain utilized in methods of the present invention is from a batch of vaccine doses. [000299] In another embodiment, a recombinant Listeria strain utilized in methods of the present invention is from a frozen stock produced by a method disclosed herein. [000300] In another embodiment, a recombinant Listeria strain utilized in methods of the present invention is from a lyophilized stock produced by a method disclosed herein. [000301] In another embodiment, a cell bank, frozen stock, or batch of vaccine doses of the present invention exhibits viability upon thawing of greater than 90%. In another embodiment, the thawing follows storage for cryopreservation or frozen storage for 24 hours. In another embodiment, the storage is for 2 days. In another embodiment, the storage is for 3 days. In another embodiment, the storage is for 4 days. In another embodiment, the storage is for 1 week. In another embodiment, the storage is for 2 weeks. In another embodiment, the storage is for 3 weeks. In another embodiment, the storage is for 1 month. In another embodiment, the storage is for 2 months. In another embodiment, the storage is for 3 months. In another embodiment, the storage is for 5 months. In another embodiment, the storage is for 6 months. In another embodiment, the storage is for 9 months. In another embodiment, the storage is for 1 year. Each possibility represents a separate embodiment of the present invention. [000302] In another embodiment, a cell bank, frozen stock, or batch of vaccine doses of the present invention is cryopreserved by a method that comprises growing a culture of the Listeria strain in a nutrient media, freezing the culture in a solution comprising glycerol, and storing the Listeria strain at below -20 degrees Celsius. In another embodiment, the temperature is about -70 degrees Celsius. In another embodiment, the temperature is about -70 - -80 degrees Celsius. [000303] In another embodiment of methods and compositions of the present invention, the culture (e.g. the culture of a Listeria vaccine strain that is used to produce a batch of Listeria vaccine doses) is inoculated from a cell bank. In another embodiment, the culture is inoculated from a frozen stock. In another embodiment, the culture is inoculated from a starter culture. In another embodiment, the culture is inoculated from a colony. In another embodiment, the culture is inoculated at mid-log growth phase. In another embodiment, the culture is inoculated at approximately mid-log growth phase. In another embodiment, the culture is inoculated at another growth phase. Each possibility represents a separate embodiment of the present invention. [000304] In another embodiment of methods and compositions of the present invention, the solution used for freezing contains glycerol in an amount of 2-20%. In another embodiment, the amount is 2%. In another embodiment, the amount is 20%. In another embodiment, the amount is 1%. In another embodiment, the amount is 1.5%. In another embodiment, the amount is 3%. In another embodiment, the amount is 4%. In another embodiment, the amount is 5%. In another embodiment, the amount is 2%. In another embodiment, the amount is 2%. In another embodiment, the amount is 7%. In another embodiment, the amount is 9%. In another embodiment, the amount is 10%. In another embodiment, the amount is 12%. In another embodiment, the amount is 14%. In another embodiment, the amount is 16%. In another embodiment, the amount is 18%. In another embodiment, the amount is 222%. In another embodiment, the amount is 25%. In another embodiment, the amount is 30%. In another embodiment, the amount is 35%. In another embodiment, the amount is 40%. Each possibility represents a separate embodiment of the present invention. [000305] In another embodiment, the solution used for freezing contains another colligative additive or additive with anti-freeze properties, in place of glycerol. In another embodiment, the solution used for freezing contains another colligative additive or additive with anti-freeze properties, in addition to glycerol. In another embodiment, the additive is mannitol. In another embodiment, the additive is DMSO. In another embodiment, the additive is sucrose. In another embodiment, the additive is any other colligative additive or additive with anti-freeze properties that is known in the art. Each possibility represents a separate embodiment of the present invention. [000306] In one embodiment, a vaccine is a composition which elicits an immune response to an antigen or polypeptide in the composition as a result of exposure to the composition. In another embodiment, the vaccine additionally comprises an adjuvant, cytokine, chemokine, or combination thereof. In another embodiment, the vaccine or composition additionally comprises antigen presenting cells (APCs), which in one embodiment are autologous, while in another embodiment, they are allogeneic to the subject. [000307] In one embodiment, a“vaccine” is a composition which elicits an immune response in a host to an antigen or polypeptide in the composition as a result of exposure to the composition. In one embodiment, the immune response is to a particular antigen or to a particular epitope on the antigen. In one embodiment, the vaccine may be a peptide vaccine, in another embodiment, a DNA vaccine. In another embodiment, the vaccine may be contained within and, in another embodiment, delivered by, a cell, which in one embodiment is a bacterial cell, which in one embodiment, is a Listeria. In one embodiment, a vaccine may prevent a subject from contracting or developing a disease or condition, wherein in another embodiment, a vaccine may be therapeutic to a subject having a disease or condition. In one embodiment, a vaccine of the present invention comprises a composition of the present invention and an adjuvant, cytokine, chemokine, or combination thereof. [000308] In another embodiment, the present invention provides an immunogenic composition comprising a recombinant Listeria of the present invention. In another embodiment, the immunogenic composition of methods and compositions of the present invention comprises a recombinant vaccine vector of the present invention. In another embodiment, the immunogenic composition comprises a plasmid of the present invention. In another embodiment, the immunogenic composition comprises an adjuvant. In one embodiment, a vector of the present invention may be administered as part of a vaccine composition. Each possibility represents a separate embodiment of the present invention. [000309] In another embodiment, a vaccine of the present invention is delivered with an adjuvant. In one embodiment, the adjuvant favors a predominantly Th1-mediated immune response. In another embodiment, the adjuvant favors a Th1-type immune response. In another embodiment, the adjuvant favors a Th1-mediated immune response. In another embodiment, the adjuvant favors a cell-mediated immune response over an antibody- mediated response. In another embodiment, the adjuvant is any other type of adjuvant known in the art. In another embodiment, the immunogenic composition induces the formation of a T cell immune response against the target protein. [000310] In another embodiment, the adjuvant is MPL. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is a TLR agonist. In another embodiment, the adjuvant is a TLR4 agonist. In another embodiment, the adjuvant is a TLR9 agonist. In another embodiment, the adjuvant is Resiquimod®. In another embodiment, the adjuvant is imiquimod. In another embodiment, the adjuvant is a CpG oligonucleotide. In another embodiment, the adjuvant is a cytokine or a nucleic acid encoding same. In another embodiment, the adjuvant is a chemokine or a nucleic acid encoding same. In another embodiment, the adjuvant is IL-12 or a nucleic acid encoding same. In another embodiment, the adjuvant is IL-6 or a nucleic acid encoding same. In another embodiment, the adjuvant is a lipopolysaccharide. In another embodiment, the adjuvant is as described in Fundamental Immunology, 5th ed (August 2003): William E. Paul (Editor); Lippincott Williams & Wilkins Publishers; Chapter 43: Vaccines, GJV Nossal, which is hereby incorporated by reference. In another embodiment, the adjuvant is any other adjuvant known in the art. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000311] In one embodiment, provided herein is a method of inducing an immune response to an antigen in a subject comprising administering a recombinant Listeria strain to said subject. In one embodiment, provided herein is a method of inducing an anti-angiogenic immune response to an antigen in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, said recombinant Listeria strain comprises a first and second nucleic acid molecule. In another embodiment, each said nucleic acid molecule encodes a heterologous antigen. In yet another embodiment, said first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with an endogenous polypeptide comprising a PEST sequence. [000312] In one embodiment, provided herein is a method of treating, suppressing, or inhibiting at least one cancer in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, said recombinant Listeria strain comprises a first and second nucleic acid molecule. In another embodiment, each said nucleic acid molecule encoding a heterologous antigen. In yet another embodiment, said first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a nucleic acid sequence encoding an endogenous polypeptide comprising a PEST sequence. In another embodiment, at least one of said antigens is expressed by at least one cell of said cancer cells. [000313] In one embodiment, provided herein is a method of delaying the onset to a cancer in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, provided herein is a method of delaying the progression to a cancer in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, provided herein is a method of extending the remission to a cancer in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, provided herein is a method of decreasing the size of an existing tumor in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, provided herein is a method of preventing the growth of an existing tumor in a subject comprising administering a recombinant Listeria strain to said subject. In another embodiment, provided herein is a method of preventing the growth of new or additional tumors in a subject comprising administering a recombinant Listeria strain to said subject. [000314] In one embodiment, cancer or tumors may be prevented in specific populations known to be susceptible to a particular cancer or tumor. In one embodiment, such susceptibilty may be due to environmental factors, such as smoking, which in one embodiment, may cause a population to be subject to lung cancer, while in another embodiment, such susceptbility may be due to genetic factors, for example a population with BRCA1/2 mutations may be susceptible, in one embodiment, to breast cancer, and in another embodiment, to ovarian cancer. In another embodiment, one or more mutations on chromosome 8q24, chromosome 17q12, and chromosome 17q24.3 may increase susceptibility to prostate cancer, as is known in the art. Other genetic and environmental factors contributing to cancer susceptibility are known in the art. [000315] In one embodiment, the recombinant Listeria strain is administered to the subject at a dose of 1 x 10⁶ - 1 x 10⁷ CFU. In another embodiment, the recombinant Listeria strain is administered to the subject at a dose of 1 x 10⁷ - 1 x 10⁸ CFU. In another embodiment, the recombinant Listeria strain is administered to the subject at a dose of 1 x 10⁸ - 3.31 x 10¹⁰ CFU. In another embodiment, the recombinant Listeria strain is administered to the subject at a dose of 1 x 10⁹ - 3.31 x 10¹⁰ CFU. In another embodiment, the dose is 5-500 x 10⁸ CFU. In another embodiment, the dose is 7-500 x 10⁸ CFU. In another embodiment, the dose is 10-500 x 10⁸ CFU. In another embodiment, the dose is 20-500 x 10⁸ CFU. In another embodiment, the dose is 30-500 x 10⁸ CFU. In another embodiment, the dose is 50-500 x 10⁸ CFU. In another embodiment, the dose is 70-500 x 10⁸ CFU. In another embodiment, the dose is 100- 500 x 10⁸ CFU. In another embodiment, the dose is 150-500 x 10⁸ CFU. In another embodiment, the dose is 5-300 x 10⁸ CFU. In another embodiment, the dose is 5-200 x 10⁸ CFU. In another embodiment, the dose is 5-15 x 10⁸ CFU. In another embodiment, the dose is 5-100 x 10⁸ CFU. In another embodiment, the dose is 5-70 x 10⁸ CFU. In another embodiment, the dose is 5-50 x 10⁸ CFU. In another embodiment, the dose is 5-30 x 10⁸ CFU. In another embodiment, the dose is 5-20 x 10⁸ CFU. In another embodiment, the dose is 1-30 x 10⁹ CFU. In another embodiment, the dose is 1-20 x 10⁹CFU. In another embodiment, the dose is 2-30 x 10⁹ CFU. In another embodiment, the dose is 1-10 x 10⁹ CFU. In another embodiment, the dose is 2-10 x 10⁹ CFU. In another embodiment, the dose is 3-10 x 10⁹ CFU. In another embodiment, the dose is 2-7 x 10⁹ CFU. In another embodiment, the dose is 2-5 x 10⁹ CFU. In another embodiment, the dose is 3-5 x 10⁹ CFU. [000316] In another embodiment, the dose is 1 x 10⁷ organisms. In another embodiment, the dose is 1.5 x 10⁷ organisms. In another embodiment, the dose is 2 x 10⁸ organisms. In another embodiment, the dose is 3 x 10⁷ organisms. In another embodiment, the dose is 4 x 10⁷ organisms. In another embodiment, the dose is 5 x 10⁷ organisms. In another embodiment, the dose is 6 x 10⁷ organisms. In another embodiment, the dose is 7 x 10⁷ organisms. In another embodiment, the dose is 8 x 10⁷ organisms. In another embodiment, the dose is 10 x 10⁷ organisms. In another embodiment, the dose is 1.5 x 10⁸ organisms. In another embodiment, the dose is 2 x 10⁸ organisms. In another embodiment, the dose is 2.5 x 10⁸ organisms. In another embodiment, the dose is 3 x 10⁸ organisms. In another embodiment, the dose is 3.3 x 10⁸ organisms. In another embodiment, the dose is 4 x 10⁸ organisms. In another embodiment, the dose is 5 x 10⁸ organisms. Each dose and range of doses represents a separate embodiment of the present invention [000317] In another embodiment, the dose is 1 x 10⁹ organisms. In another embodiment, the dose is 1.5 x 10⁹ organisms. In another embodiment, the dose is 2 x 10⁹ organisms. In another embodiment, the dose is 3 x 10⁹ organisms. In another embodiment, the dose is 4 x 10⁹ organisms. In another embodiment, the dose is 5 x 10⁹ organisms. In another embodiment, the dose is 6 x 10⁹ organisms. In another embodiment, the dose is 7 x 10⁹ organisms. In another embodiment, the dose is 8 x 10⁹ organisms. In another embodiment, the dose is 10 x 10⁹ organisms. In another embodiment, the dose is 1.5 x 10¹⁰ organisms. In another embodiment, the dose is 2 x 10¹⁰ organisms. In another embodiment, the dose is 2.5 x 10¹⁰ organisms. In another embodiment, the dose is 3 x 10¹⁰ organisms. In another embodiment, the dose is 3.3 x 10¹⁰ organisms. In another embodiment, the dose is 4 x 10¹⁰ organisms. In another embodiment, the dose is 5 x 10¹⁰ organisms. Each dose and range of doses represents a separate embodiment of the present invention. [000318] It will be appreciated by the skilled artisan that the term“Boosting” may encompass administering an additional vaccine or immunogenic composition or recombinant Listeria strain dose to a subject. In another embodiment of methods of the present invention, 2 boosts (or a total of 3 inoculations) are administered. In another embodiment, 3 boosts are administered. In another embodiment, 4 boosts are administered. In another embodiment, 5 boosts are administered. In another embodiment, 6 boosts are administered. In another embodiment, more than 6 boosts are administered. Each possibility represents a separate embodiment of the present invention.

[000319] In another embodiment, a method of present invention further comprises the step of boosting the human subject with a recombinant Listeria strain as provided herein. In another embodiment, the recombinant Listeria strain used in the booster inoculation is the same as the strain used in the initial“priming” inoculation. In another embodiment, the booster strain is different from the priming strain. In another embodiment, the same doses are used in the priming and boosting inoculations. In another embodiment, a larger dose is used in the booster. In another embodiment, a smaller dose is used in the booster. In another embodiment, the methods of the present invention further comprise the step of administering to the subject a booster vaccination. In one embodiment, the booster vaccination follows a single priming vaccination. In another embodiment, a single booster vaccination is administered after the priming vaccinations. In another embodiment, two booster vaccinations are administered after the priming vaccinations. In another embodiment, three booster vaccinations are administered after the priming vaccinations. In one embodiment, the period between a prime and a boost vaccine is experimentally determined by the skilled artisan. In another embodiment, the period between a prime and a boost vaccine is 1 week, in another embodiment it is 2 weeks, in another embodiment, it is 3 weeks, in another embodiment, it is 4 weeks, in another embodiment, it is 5 weeks, in another embodiment it is 6-8 weeks, in yet another embodiment, the boost vaccine is administered 8-10 weeks after the prime vaccine. [000320] In another embodiment, a method of the present invention further comprises boosting the human subject with a recombinant Listeria strain provided herein. In another embodiment, a method of the present invention comprises the step of administering a booster dose of an immunogenic composition comprising the recombinant Listeria strain provided herein. In another embodiment, the booster dose is an alternate form of said immunogenic composition. In another embodiment, the methods of the present invention further comprise the step of administering to the subject a booster immunogenic composition. In one embodiment, the booster dose follows a single priming dose of said immunogenic composition . In another embodiment, a single booster dose is administered after the priming dose. In another embodiment, two booster doses are administered after the priming dose. In another embodiment, three booster doses are administered after the priming dose. In one embodiment, the period between a prime and a boost dose of an immunogenic composition comprising the recombinant Listeria provided herein is experimentally determined by the skilled artisan. In another embodiment, the dose is experimentally determined by a skilled artisan. In another embodiment, the period between a prime and a boost dose is 1 week, in another embodiment it is 2 weeks, in another embodiment, it is 3 weeks, in another embodiment, it is 4 weeks, in another embodiment, it is 5 weeks, in another embodiment it is 6-8 weeks, in yet another embodiment, the boost dose is administered 8-10 weeks after the prime dose of the immunogenic composition. [000321] Heterologous "prime boost" strategies have been effective for enhancing immune responses and protection against numerous pathogens. Schneider et al., Immunol. Rev. 170:29- 38 (1999); Robinson, H. L., Nat. Rev. Immunol. 2:239-50 (2002); Gonzalo, R. M. et al., Vaccine 20:1226-31 (2002); Tanghe, A., Infect. Immun. 69:3041-7 (2001). Providing antigen in different forms in the prime and the boost injections appears to maximize the immune response to the antigen. DNA vaccine priming followed by boosting with protein in adjuvant or by viral vector delivery of DNA encoding antigen appears to be the most effective way of improving antigen specific antibody and CD4+ T-cell responses or CD8+ T-cell responses respectively. Shiver J. W. et al., Nature 415: 331-5 (2002); Gilbert, S. C. et al., Vaccine 20:1039-45 (2002); Billaut- Mulot, O. et al., Vaccine 19:95-102 (2000); Sin, J. I. et al., DNA Cell Biol. 18:771-9 (1999). Recent data from monkey vaccination studies suggests that adding CRL1005 poloxamer (12 kDa, 5% POE), to DNA encoding the HIV gag antigen enhances T-cell responses when monkeys are vaccinated with an HIV gag DNA prime followed by a boost with an adenoviral vector expressing HIV gag (Ad5-gag). The cellular immune responses for a DNA/poloxamer prime followed by an Ad5-gag boost were greater than the responses induced with a DNA (without poloxamer) prime followed by Ad5-gag boost or for Ad5-gag only. Shiver, J. W. et al. Nature 415:331-5 (2002). U.S. Patent Appl. Publication No. US 2002/0165172 A1 describes simultaneous administration of a vector construct encoding an immunogenic portion of an antigen and a protein comprising the immunogenic portion of an antigen such that an immune response is generated. The document is limited to hepatitis B antigens and HIV antigens. Moreover, U.S. Pat. No. 6,500,432 is directed to methods of enhancing an immune response of nucleic acid vaccination by simultaneous administration of a polynucleotide and polypeptide of interest. According to the patent, simultaneous administration means administration of the polynucleotide and the polypeptide during the same immune response, preferably within 0-10 or 3-7 days of each other. The antigens contemplated by the patent include, among others, those of Hepatitis (all forms), HSV, HIV, CMV, EBV, RSV, VZV, HPV, polio, influenza, parasites (e.g., from the genus Plasmodium), and pathogenic bacteria (including but not limited to M. tuberculosis, M. leprae, Chlamydia, Shigella, B. burgdorferi, enterotoxigenic E. coli, S. typhosa, H. pylori, V. cholerae, B. pertussis, etc.). All of the above references are herein incorporated by reference in their entireties. [000322] In one embodiment, the first or second nucleic acid molecule encodes a prostate specific antigen (PSA) and the method is for treating, inhibiting or suppressing prostate cancer. In another embodiment, the first or second nucleic acid molecule encodes PSA and the method is for treating, inhibiting or suppressing ovarian cancer. In another embodiment, the first or second nucleic acid molecule encodes PSA and the method is treating, inhibiting, or suppressing metastasis of prostate cancer, which in one embodiment, comprises metastasis to bone, and in another embodiment, comprises metastasis to other organs. In another embodiment, the first or second nucleic acid molecule encodes PSA and the method is for treating, inhibiting or suppressing metastasis of prostate cancer to bones. In yet another embodiment the method is for treating, inhibiting, or suppressing metastatis of prostate cancer to other organs. In another embodiment, the first or second nucleic acid molecule encodes PSA and the method is for treating, inhibiting or suppressing breast cancer. In another embodiment, the first or second nucleic acid molecule encodes PSA and the method is for treating, inhibiting or suppressing both ovarian and breast cancer. [000323] In one embodiment, the first or second nucleic acid molecule encodes a High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) and the method is for treating, inhibiting or suppressing melanoma. In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing breast cancer. In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing ovarian cancer. In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing benign nevi lesions. In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing basal cell carcinoma. In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing a tumor of neural crest origin, which in one embodiment, is an astrocytoma, glioma, neuroblastoma, sarcoma, or combination thereof. In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing a childhood leukemia, which in one embodiment, is Childhood Acute Lymphoblastic Leukemia, and in another embodiment, is Childhood Acute Myeloid Leukemia (which in one embodiment, is acute myelogenous leukemia, acute myeloid leukemia, acute myelocytic leukemia, or acute non-lymphocytic leukemia) and in another embodiment, is acute lymphocytic leukemia (which in one embodiment, is called acute lymphoblastic leukemia, and in another embodiment, is acute myelogenous leukemia (also called acute myeloid leukemia, acute myelocytic leukemia, or acute non-lymphocytic leukemia) and in another embodiment, is Hybrid or mixed lineage leukemia. In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing Chronic myelogenous leukemia or Juvenile Myelomonocytic Leukemia (JMML). In another embodiment, the first or second nucleic acid molecule encodes HMW-MAA and the method is for treating, inhibiting or suppressing lobular breast carcinoma lesions. [000324] The cancer that is the target of methods and compositions as provided herein is, in another embodiment, a melanoma. In another embodiment, the cancer is a sarcoma. In another embodiment, the cancer is a carcinoma. In another embodiment, the cancer is a mesothelioma (e.g. malignant mesothelioma). In another embodiment, the cancer is a glioma. In another embodiment, the cancer is a germ cell tumor. In another embodiment, the cancer is a choriocarcinoma. [000325] In another embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is gastric cancer. In another embodiment, the cancer is a carcinomatous lesion of the pancreas. In another embodiment, the cancer is pulmonary adenocarcinoma. In another embodiment, the cancer is colorectal adenocarcinoma. In another embodiment, the cancer is pulmonary squamous adenocarcinoma. In another embodiment, the cancer is gastric adenocarcinoma. In another embodiment, the cancer is an ovarian surface epithelial neoplasm (e.g. a benign, proliferative or malignant variety thereof). In another embodiment, the cancer is an oral squamous cell carcinoma. In another embodiment, the cancer is non small-cell lung carcinoma. In another embodiment, the cancer is an endometrial carcinoma. In another embodiment, the cancer is a bladder cancer. In another embodiment, the cancer is a head and neck cancer. In another embodiment, the cancer is a prostate carcinoma. [000326] In another embodiment, the cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the cancer is a colon cancer. In another embodiment, the cancer is a lung cancer. In another embodiment, the cancer is an ovarian cancer. In another embodiment, the cancer is a uterine cancer. In another embodiment, the cancer is a thyroid cancer. In another embodiment, the cancer is a hepatocellular carcinoma. In another embodiment, the cancer is a thyroid cancer. In another embodiment, the cancer is a liver cancer. In another embodiment, the cancer is a renal cancer. In another embodiment, the cancer is a kaposis. In another embodiment, the cancer is a sarcoma. In another embodiment, the cancer is another carcinoma or sarcoma. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000327] In one embodiment, the compositions and methods as provided herein can be used to treat solid tumors related to or resulting from any of the cancers as described hereinabove. In another embodiment, the tumor is a Wilms’ tumor. In another embodiment, the tumor is a desmoplastic small round cell tumor. [000328] In another embodiment, the present invention provides a method of impeding angiogenesis of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria encoding a heterologous antigen. In another embodiment, the antigen is HMW-MAA. In another embodiment, the antigen is fibroblast growth factor (FGF). In another embodiment, the antigen is vascular endothelial growth factor (VEGF). In another embodiment, the antigen is any other antigen known in the art to be involved in angiogenesis. In another embodiment, the methods and compositions of impeding angiogenesis of a solid tumor in a subject, as provided herein, comprise administering to the subject a composition comprising a recombinant Listeria encoding two heterologous antigens. In another embodiment, the methods and compositions of impeding angiogenesis of a solid tumor in a subject, as provided herein, comprise administering to the subject a composition comprising a mixture of two recombinant Listeria strains wherein each strain encodes a different heterologous antigens. In yet another embodiment , the methods and compositions of impeding angiogenesis of a solid tumor in a subject, as provided herein, comprise administering to the subject a composition comprising a recombinant Listeria strains encioding first heterologous antigen, followed by administering to the subject a composition comprising a recombinant Listeria strains encioding second heterologous antigen. In another embodiment, one of the two heterologous antigens is HMW-MAA. In another embodiment, the antigen is any other antigen known in the art to be involved in angiogenesis. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000329] Methods for assessing efficacy of prostate cancer vaccines are well known in the art, and are described, for example, in Dzojic H et al (Adenovirus-mediated CD40 ligand therapy induces tumor cell apoptosis and systemic immunity in the TRAMP-C2 mouse prostate cancer model. Prostate. 2006 Jun 1;66(8):831-8), Naruishi K et al (Adenoviral vector- mediated RTVP-1 gene-modified tumor cell-based vaccine suppresses the development of experimental prostate cancer. Cancer Gene Ther. 2006 Jul;13(7):658-63), Sehgal I et al (Cancer Cell Int. 2006 Aug 23;6:21), and Heinrich JE et al (Vaccination against prostate cancer using a live tissue factor deficient cell line in Lobund-Wistar rats. Cancer Immunol Immunother 2007;56(5):725-30). Each possibility represents a separate embodiment as provided herein. [000330] In another embodiment, the prostate cancer model used to test methods and compositions as provided herein is the TPSA23 (derived from TRAMP-C1 cell line stably expressing PSA) mouse model. In another embodiment, the prostate cancer model is a 178-2 BMA cell model. In another embodiment, the prostate cancer model is a PAIII adenocarcinoma cells model. In another embodiment, the prostate cancer model is a PC-3M model. In another embodiment, the prostate cancer model is any other prostate cancer model known in the art. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000331] In another embodiment, the vaccine is tested in human subjects, and efficacy is monitored using methods well known in the art, e.g. directly measuring CD4⁺ and CD8⁺ T cell responses, or measuring disease progression, e.g. by determining the number or size of tumor metastases, or monitoring disease symptoms (cough, chest pain, weight loss, etc). Methods for assessing the efficacy of a prostate cancer vaccine in human subjects are well known in the art, and are described, for example, in Uenaka A et al (T cell immunomonitoring and tumor responses in patients immunized with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein. Cancer Immun. 2007 Apr 19;7:9) and Thomas-Kaskel AK et al (Vaccination of advanced prostate cancer patients with PSCA and PSA peptide-loaded dendritic cells induces DTH responses that correlate with superior overall survival. Int J Cancer. 2006 Nov 15;119(10):2428-34). Each method represents a separate embodiment of the methods and compositions as provided herein. [000332] In another embodiment, the present invention provides a method of treating benign prostate hyperplasia (BPH) in a subject. In another embodiment, the present invention provides a method of treating Prostatic Intraepithelial Neoplasia (PIN) in a subject [000333] In one embodiment, provided herein is a recombinant Listeria strain comprising a nucleic acid molecule operably integrated into the Listeria genome. In another embodiment said nucleic acid molecule encodes (a) an endogenous polypeptide comprising a PEST sequence and (b) a polypeptide comprising an antigen in an open reading frame. [000334] In one embodiment, provided herein is a method of treating, suppressing, or inhibiting at least one tumor in a subject, comprising administering a recombinant Listeria strain to said subject. In another embodiment, said recombinant Listeria strain comprises a first and second nucleic acid molecule. In another embodiment, each said nucleic acid molecule encodes a heterologous antigen. In another embodiment, said first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a native polypeptide comprising a PEST sequence and wherein said antigen is expressed by at least one cell of said tumor. [000335] In one embodiment, "antigen" is used herein to refer to a substance that when placed in contact with an organism, results in a detectable immune response from the organism. An antigen may be a lipid, peptide, protein, carbohydrate, nucleic acid, or combinations and variations thereof. [000336] In one embodiment,“variant” refers to an amino acid or nucleic acid sequence (or in other embodiments, an organism or tissue) that is different from the majority of the population but is still sufficiently similar to the common mode to be considered to be one of them, for example splice variants. [000337] In one embodiment,“isoform” refers to a version of a molecule, for example, a protein, with only slight differences compared to another isoform, or version, of the same protein. In one embodiment, isoforms may be produced from different but related genes, or in another embodiment, may arise from the same gene by alternative splicing. In another embodiment, isoforms are caused by single nucleotide polymorphisms. [000338] In one embodiment,“fragment” refers to a protein or polypeptide that is shorter or comprises fewer amino acids than the full length protein or polypeptide. In another embodiment, fragment refers to a nucleic acid that is shorter or comprises fewer nucleotides than the full length nucleic acid. In another embodiment, the fragment is an N-terminal fragment. In another embodiment, the fragment is a C-terminal fragment. In one embodiment, the fragment is an intrasequential section of the protein, peptide, or nucleic acid. In one embodiment, the fragment is a functional fragment. In another embodiment, the fragment is an immunogenic fragment. In one embodiment, a fragment has 10-20 nucleic or amino acids, while in another embodiment, a fragment has more than 5 nucleic or amino acids, while in another embodiment, a fragment has 100-200 nucleic or amino acids, while in another embodiment, a fragment has 100-500 nucleic or amino acids, while in another embodiment, a fragment has 50-200 nucleic or amino acids, while in another embodiment, a fragment has 10- 250 nucleic or amino acids. [000339] In one embodiment, "immunogenicity" or“immunogenic” is used herein to refer to the innate ability of a protein, peptide, nucleic acid, antigen or organism to elicit an immune response in an animal when the protein, peptide, nucleic acid, antigen or organism is administered to the animal. Thus, "enhancing the immunogenicity" in one embodiment, refers to increasing the ability of a protein, peptide, nucleic acid, antigen or organism to elicit an immune response in an animal when the protein, peptide, nucleic acid, antigen or organism is administered to an animal. The increased ability of a protein, peptide, nucleic acid, antigen or organism to elicit an immune response can be measured by, in one embodiment, a greater number of antibodies to a protein, peptide, nucleic acid, antigen or organism, a greater diversity of antibodies to an antigen or organism, a greater number of T-cells specific for a protein, peptide, nucleic acid, antigen or organism, a greater cytotoxic or helper T-cell response to a protein, peptide, nucleic acid, antigen or organism, and the like. [000340] In one embodiment, a“homologue” refers to a nucleic acid or amino acid sequence which shares a certain percentage of sequence identity with a particular nucleic acid or amino acid sequence. In one embodiment, a sequence useful in the composition and methods as provided herein may be a homologue of a particular LLO sequence or N-terminal fragment thereof, ActA sequence or N-terminal fragment thereof, or PEST-like sequence described herein or known in the art. In one embodiment, such a homolog maintains In another embodiment, a sequence useful in the composition and methods as provided herein may be a homologue of an antigenic polypeptide, which in one embodiment, is CA9, cHER2 or HMW- MAA or a functional fragment thereof. In one embodiment, a homolog of a polypeptide and, in one embodiment, the nucleic acid encoding such a homolog, of the present invention maintains the functional characteristics of the parent polypeptide. For example, in one embodiment, a homolog of an antigenic polypeptide of the present invention maintains the antigenic characteristic of the parent polypeptide. In another embodiment, a sequence useful in the composition and methods as provided herein may be a homologue of any sequence described herein. In one embodiment, a homologue shares at least 70% identity with a particular sequence. In another embodiment, a homologue shares at least 72% identity with a particular sequence. In another embodiment, a homologue shares at least 75% identity with a particular sequence. In another embodiment, a homologue shares at least 78% identity with a particular sequence. In another embodiment, a homologue shares at least 80% identity with a particular sequence. In another embodiment, a homologue shares at least 82% identity with a particular sequence. In another embodiment, a homologue shares at least 83% identity with a particular sequence. In another embodiment, a homologue shares at least 85% identity with a particular sequence. In another embodiment, a homologue shares at least 87% identity with a particular sequence. In another embodiment, a homologue shares at least 88% identity with a particular sequence. In another embodiment, a homologue shares at least 90% identity with a particular sequence. In another embodiment, a homologue shares at least 92% identity with a particular sequence. In another embodiment, a homologue shares at least 93% identity with a particular sequence. In another embodiment, a homologue shares at least 95% identity with a particular sequence. In another embodiment, a homologue shares at least 96% identity with a particular sequence. In another embodiment, a homologue shares at least 97% identity with a particular sequence. In another embodiment, a homologue shares at least 98% identity with a particular sequence. In another embodiment, a homologue shares at least 99% identity with a particular sequence. In another embodiment, a homologue shares 100% identity with a particular sequence. Each possibility represents a separate embodiment as provided herein. [000341] In one embodiment, it is to be understood that a homolog of any of the sequences as provided herein and/or as described herein is considered to be a part of the invention. [000342] In one embodiment,“functional” within the meaning of the invention, is used herein to refer to the innate ability of a protein, peptide, nucleic acid, fragment or a variant thereof to exhibit a biological activity or function. In one embodiment, such a biological function is its binding property to an interaction partner, e.g., a membrane-associated receptor, and in another embodiment, its trimerization property. In the case of functional fragments and the functional variants of the invention, these biological functions may in fact be changed, e.g., with respect to their specificity or selectivity, but with retention of the basic biological function. [000343] In one embodiment,“treating” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described herein. Thus, in one embodiment, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Thus, in one embodiment,“treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. In one embodiment,“preventing” or“impeding” refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof. In one embodiment,“suppressing” or“inhibiting”, refers inter alia to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof. [000344] In one embodiment, symptoms are primary, while in another embodiment, symptoms are secondary. In one embodiment,“primary” refers to a symptom that is a direct result of a particular disease or disorder, while in one embodiment,“secondary” refers to a symptom that is derived from or consequent to a primary cause. In one embodiment, the compounds for use in the present invention treat primary or secondary symptoms or secondary complications. In another embodiment,“symptoms” may be any manifestation of a disease or pathological condition. [000345] In some embodiments, the term “comprising” refers to the inclusion of other recombinant polypeptides, amino acid sequences, or nucleic acid sequences, as well as inclusion of other polypeptides, amino acid sequences, or nucleic acid sequences, that may be known in the art, which in one embodiment may comprise antigens or Listeria polypeptides, amino acid sequences, or nucleic acid sequences. In some embodiments, the term“consisting essentially of” refers to a composition for use in the methods as provided herein, which has the specific recombinant polypeptide, amino acid sequence, or nucleic acid sequence, or fragment thereof. However, other polypeptides, amino acid sequences, or nucleic acid sequences may be included that are not involved directly in the utility of the recombinant polypeptide(s). In some embodiments, the term“consisting” refers to a composition for use in the methods as provided herein having a particular recombinant polypeptide, amino acid sequence, or nucleic acid sequence, or fragment or combination of recombinant polypeptides, amino acid sequences, or nucleic acid sequences or fragments as provided herein, in any form or embodiment as described herein. [000346] In one embodiment, the compositions for use in the methods as provided herein are administered intravenously. In another embodiment, the vaccine is administered orally, whereas in another embodiment, the vaccine is administered parenterally (e.g., subcutaneously, intramuscularly, and the like). [000347] Further, in another embodiment, the compositions or vaccines are administered as a suppository, for example a rectal suppository or a urethral suppository. Further, in another embodiment, the pharmaceutical compositions are administered by subcutaneous implantation of a pellet. In a further embodiment, the pellet provides for controlled release of an agent over a period of time. In yet another embodiment, the pharmaceutical compositions are administered in the form of a capsule. [000348] In one embodiment, the route of administration may be parenteral. In another embodiment, the route may be intra-ocular, conjunctival, topical, transdermal, intradermal, subcutaneous, intraperitoneal, intravenous, intra-arterial, vaginal, rectal, intratumoral, parcanceral, transmucosal, intramuscular, intravascular, intraventricular, intracranial, inhalation (aerosol), nasal aspiration (spray), intranasal (drops), sublingual, oral, aerosol or suppository or a combination thereof. For intranasal administration or application by inhalation, solutions or suspensions of the compounds mixed and aerosolized or nebulized in the presence of the appropriate carrier suitable. Such an aerosol may comprise any agent described herein. In one embodiment, the compositions as set forth herein may be in a form suitable for intracranial administration, which in one embodiment, is intrathecal and intracerebroventricular administration. In one embodiment, the regimen of administration will be determined by skilled clinicians, based on factors such as exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, body weight, and response of the individual patient, etc. [000349] In one embodiment, parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories and enemas. Ampoules are convenient unit dosages. Such a suppository may comprise any agent described herein. [000350] In one embodiment, sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc. Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the new compounds and use the lyophilisates obtained, for example, for the preparation of products for injection. [000351] In one embodiment, for liquid formulations, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non- aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. [000352] In one embodiment, compositions of this invention are pharmaceutically acceptable. In one embodiment, the term“pharmaceutically acceptable” refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one compound for use in the present invention. This term refers to the use of buffered formulations as well, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the compounds and route of administration. [000353] In one embodiment, a composition of or used in the methods of this invention may be administered alone or within a composition. In another embodiment, compositions of this invention admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application which do not deleteriously react with the active compounds may be used. In one embodiment, suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. In another embodiment, the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. In another embodiment, they can also be combined where desired with other active agents, e.g., vitamins. [000354] In one embodiment, the compositions for use of the methods and compositions as provided herein may be administered with a carrier/diluent. Solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. [000355] In one embodiment, the compositions of the methods and compositions as provided herein may comprise the composition of this invention and one or more additional compounds effective in preventing or treating cancer. In some embodiments, the additional compound may comprise a compound useful in chemotherapy, which in one embodiment, is Cisplatin. In another embodiment, Ifosfamide, Fluorouracilor5-FU, Irinotecan, Paclitaxel (Taxol), Docetaxel, Gemcitabine, Topotecan or a combination thereof, may be administered with a composition as provided herein for use in the methods as provided herein. In another embodiment, Amsacrine, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Gliadelimplants, Hydroxycarbamide, Idarubicin, Ifosfamide, Irinotecan, Leucovorin, Liposomaldoxorubicin, Liposomaldaunorubicin, Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin, Procarbazine, Raltitrexed, Satraplatin, Streptozocin, Tegafur-uracil, Temozolomide, Teniposide, Thiotepa, Tioguanine, Topotecan, Treosulfan, Vinblastine, Vincristine, Vindesine, Vinorelbine, or a combination thereof, may be administered with a composition as provided herein for use in the methods as provided herein. [000356] In another embodiment, fusion proteins as provided herein are prepared by a process comprising subcloning of appropriate sequences, followed by expression of the resulting nucleotide. In another embodiment, subsequences are cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments are then ligated, in another embodiment, to produce the desired DNA sequence. In another embodiment, DNA encoding the fusion protein is produced using DNA amplification methods, for example polymerase chain reaction (PCR). First, the segments of the native DNA on either side of the new terminus are amplified separately. The 5' end of the one amplified sequence encodes the peptide linker, while the 3' end of the other amplified sequence also encodes the peptide linker. Since the 5' end of the first fragment is complementary to the 3' end of the second fragment, the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction. The amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence). The insert is then ligated into a plasmid. In another embodiment, a similar strategy is used to produce a protein wherein an HMW-MAA fragment is embedded within a heterologous peptide. [000357] In one embodiment, the present invention also provides a recombinant Listeria comprising a nucleic acid molecule encoding a polypeptide comprising a heterologous antigen or fragment thereof fused to a PEST-containing sequence, wherein said nucleic acid molecule is remains episomaly in said Listeria. In one embodiment, provided herein is a recombinant Listeria capable of expressing and secreting two distinct heterologous antigens. In another embodiment, the first and second antigen are distinct. In another embodiment, said first and second antigens are concomitantly expressed. In another embodiment, said first or second antigen are expressed at the same level. In another embodiment, said first or second antigen are differentially expressed. In another embodiment, gene or protein expression is determined by methods that are well known in the art which in another embodiment comprise real-time PCR, northern blotting, immunoblotting, etc. In another embodiment, said first or second antigen’s expression is controlled by an inducible system, while in another embodiment, said first or second antigen’s expression is controlled by a constitutive promoter. In another embodiment, inducible expression systems are well known in the art. [000358] In one embodiment, provided herein is a method of preparing a recombinant Listeria capable of expressing and secreting two distinct heterologous antigens that target tumor cells and angiogenesis concomitantly. In another embodiment, said method of preparing said recombinant Listeria comprises the steps of genetically fusing a first antigen into the genome that is operably linked to an open reading frame encoding a first polypeptide or fragment thereof comprising a PEST sequence and transforming said recombinant Listeria with an episomal expression vector encoding a second antigen that is operably linked to an open reading frame encoding a second polypeptide or fragment thereof comprising a PEST sequence. In another embodiment, said method of preparing said recombinant Listeria comprises the steps of genetically fusing a first antigen into the genome that is operably linked to an open reading frame encoding a first polypeptide or fragment thereof comprising a PEST sequence and genetically fusing a second antigen that is operably linked to an open reading frame encoding a second polypeptide or fragment thereof comprising a PEST sequence. [000359] Methods for transforming bacteria are well known in the art, and include calcium- chloride competent cell-based methods, electroporation methods, bacteriophage-mediated transduction, chemical, and physical transformation techniques (de Boer et al, 1989, Cell 56:641-649; Miller et al, 1995, FASEB J., 9:190-199; Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC; Miller, 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) In another embodiment, the Listeria vaccine strain as provided herein is transformed by electroporation. Each method represents a separate embodiment of the methods and compositions as provided herein. [000360] In one embodiment, provided herein is a method of inducing an immune response to an antigen in a subject comprising administering a recombinant Listeria strain to said subject, wherein said recombinant Listeria strain comprises a first and second nucleic acid molecule, each said nucleic acid molecule encoding a heterologous antigenic polypeptide or fragment thereof, wherein said first nucleic acid molecule is operably integrated into the Listeria genome as an open reading frame with a nucleic acid encoding an endogenous polypeptide comprising a PEST sequence. [000361] In another embodiment, provided herein is a method of inhibiting the onset of cancer, said method comprising the step of administering a recombinant Listeria composition that expresses two distinct heterologous antigens specifically expressed in said cancer. [000362] In one embodiment, provided herein is a method of treating a subject having a tumor or cancer, said method comprising the step of administering a pharmaceutical composition or formulation comprising a recombinant Listeria provided hereinthat expresses two or more distinct heterologous antigens specifically expressed on said tumor. [000363] In another embodiment, the recombinant Listeria expressing two or more heterologous antigens fused to a PEST-containing sequence (such as N-terminal LLO, N- terminal ActA, or a PEST sequence or peptide), targets two or more different tumors or cancers, or metastases in a subject having said tumors or cancers or metastases. [000364] In another embodiment, provided herein is a method of ameliorating symptoms that are associated with a cancer in a subject, said method comprising the step of administering a recombinant Listeria composition that expresses two or more distinct heterologous antigens specifically expressed in said cancer. [000365] In one embodiment, provided herein is a method of protecting a subject from cancer, said method comprising the step of administering a recombinant Listeria composition that expresses two distinct heterologous antigens specifically expressed in said cancer [000366] In another embodiment, provided herein is a method of delaying onset of cancer, said method comprising the step of administering a recombinant Listeria composition that expresses two or more distinct heterologous antigens specifically expressed in said cancer. In another embodiment, provided herein is a method of treating metastatic cancer, said method comprising the step of administering a recombinant Listeria composition that expresses two or more distinct heterologous antigens specifically expressed in said cancer. In another embodiment, provided herein is a method of preventing metastatic canceror micrometastatis, said method comprising the step of administering a recombinant Listeria composition that expresses two or more distinct heterologous antigens specifically expressed in said cancer. In another embodiment, the recombinant Listeria composition is administered orally or parenterally. [000367] In another embodiment, a pharmaceutical composition comprising the recombinant Listeria provided herein is administered intravenously, subcutaneuosly, intranasally, intramuscularly, or injected into a tumor site or into a tumor. [000368] In another embodiment of the methods and compositions as provided herein, “nucleic acids” or“nucleotide” refers to a string of at least two base-sugar-phosphate combinations. The term includes, in one embodiment, DNA and RNA.“Nucleotides” refers, in one embodiment, to the monomeric units of nucleic acid polymers. RNA may be, in one embodiment, in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) and ribozymes. The use of siRNA and miRNA has been described (Caudy AA et al, Genes & Devel 16: 2491-96 and references cited therein). DNA may be in form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups. In addition, these forms of DNA and RNA may be single, double, triple, or quadruple stranded. The term also includes, in another embodiment, artificial nucleic acids that may contain other types of backbones but the same bases. In one embodiment, the artificial nucleic acid is a PNA (peptide nucleic acid). PNA contain peptide backbones and nucleotide bases and are able to bind, in one embodiment, to both DNA and RNA molecules. In another embodiment, the nucleotide is oxetane modified. In another embodiment, the nucleotide is modified by replacement of one or more phosphodiester bonds with a phosphorothioate bond. In another embodiment, the artificial nucleic acid contains any other variant of the phosphate backbone of native nucleic acids known in the art. The use of phosphothiorate nucleic acids and PNA are known to those skilled in the art, and are described in, for example, Neilsen PE, Curr Opin Struct Biol 9:353-57; and Raz NK et al Biochem Biophys Res Commun. 297:1075-84. The production and use of nucleic acids is known to those skilled in art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and G. C. Fareed. Each nucleic acid derivative represents a separate embodiment as provided herein. [000369] The terms“polypeptide,”“peptide” and“recombinant peptide” refer, in another embodiment, to a peptide or polypeptide of any length. In another embodiment, a peptide or recombinant peptide as provided herein has one of the lengths enumerated above for an HMW-MAA fragment. Each possibility represents a separate embodiment of the methods and compositions as provided herein. In one embodiment, the term“peptide” refers to native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and/or peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder. [000370] In one embodiment, “antigenic polypeptide” is used herein to refer to a polypeptide, peptide or recombinant peptide as described hereinabove that is foreign to a host and leads to the mounting of an immune response when present in, or, in another embodiment, detected by, the host. [000371] Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N- methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (-CO-CH2-), *-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)- CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. [000372] These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time. Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. [000373] In addition to the above, the peptides as provided herein may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc). [000374] In one embodiment, the term“oligonucleotide” is interchangeable with the term "nucleic acid", and may refer to a molecule, which may include, but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. The term also refers to sequences that include any of the known base analogs of DNA and RNA. [000375]“Stably maintained” refers, in another embodiment, to maintenance of a nucleic acid molecule or plasmid in the absence of selection (e.g. antibiotic selection) for 10 generations, without detectable loss. In another embodiment, the period is 15 generations. In another embodiment, the period is 20 generations. In another embodiment, the period is 25 generations. In another embodiment, the period is 30 generations. In another embodiment, the period is 40 generations. In another embodiment, the period is 50 generations. In another embodiment, the period is 60 generations. In another embodiment, the period is 80 generations. In another embodiment, the period is 100 generations. In another embodiment, the period is 150 generations. In another embodiment, the period is 200 generations. In another embodiment, the period is 300 generations. In another embodiment, the period is 500 generations. In another embodiment, the period is more than 500 generations. In another embodiment, the nucleic acid molecule or plasmid is maintained stably in vitro (e.g. in culture). In another embodiment, the nucleic acid molecule or plasmid is maintained stably in vivo. In another embodiment, the nucleic acid molecule or plasmid is maintained stably both in vitro and in vitro. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000376] In one embodiment, the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" may include both D- and L-amino acids. [000377] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved thereover for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'- N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see, e.g., Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Mulligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described, e.g., in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appi. Pharmacol. 144:189-197. Other synthetic backbones encompasses by the term include methyl-phosphonate linkages or alternating methyiphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev. 6:153-156). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product. [000378] In one embodiment of the methods and compositions as provided herein, the term "recombination site" or "site-specific recombination site" refers to a sequence of bases in a nucleic acid molecule that is recognized by a recombinase (along with associated proteins, in some cases) that mediates exchange or excision of the nucleic acid segments flanking the recombination sites. The recombinases and associated proteins are collectively referred to as "recombination proteins" see, e.g., Landy, A., (Current Opinion in Genetics & Development) 3:699-707; 1993). [000379] A "phage expression vector" or "phagemid" refers to any phage-based recombinant expression system for the purpose of expressing a nucleic acid sequence of the methods and compositions as provided herein in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. A phage expression vector typically can both reproduce in a bacterial cell and, under proper conditions, produce phage particles. The term includes linear or circular expression systems and encompasses both phage-based expression vectors that remain episomal or integrate into the host cell genome. [000380] It will be appreciated by a skilled artisan that the term“operably linked" may mean that the transcriptional and translational regulatory nucleic acid, is positioned relative to any coding sequences in such a manner that transcription is initiated. Generally, this will mean that the promoter and transcriptional initiation or start sequences are positioned 5' to the coding region. [000381] It will be understood by a skilled artisan that the term“open reading frame” or “ORF” may encompass a portion of an organism's genome which contains a sequence of bases that could potentially encode a protein. In another embodiment, the start and stop ends of the ORF are not equivalent to the ends of the mRNA, but they are usually contained within the mRNA. In one embodiment, ORFs are located between the start-code sequence (initiation codon) and the stop-codon sequence (termination codon) of a gene. Thus, in one embodiment, a nucleic acid molecule operably integrated into a genome as an open reading frame with an endogenous polypeptide is a nucleic acid molecule that has integrated into a genome in the same open reading frame as an endogenous polypeptide. [000382] In one embodiment, the present invention provides a fusion polypeptide comprising a linker sequence. In one embodiment, a "linker sequence" refers to an amino acid sequence that joins two heterologous polypeptides, or fragments or domains thereof. In general, as used herein, a linker is an amino acid sequence that covalently links the polypeptides to form a fusion polypeptide. A linker typically includes the amino acids translated from the remaining recombination signal after removal of a reporter gene from a display vector to create a fusion protein comprising an amino acid sequence encoded by an open reading frame and the display protein. As appreciated by one of skill in the art, the linker can comprise additional amino acids, such as glycine and other small neutral amino acids. [000383] In one embodiment, “endogenous” as used herein describes an item that has developed or originated within the reference organism or arisen from causes within the reference organism. In another embodiment, endogenous refers to native. [000384] In one embodiment,“heterologous” as used herein describes a nucleic acid, amino acid, peptide, polypeptide, or protein derived from a different species than the reference species. Thus, for example, a Listeria strain expressing a heterologous polypeptide, in one embodiment, would express a polypeptide that is not native or endogenous to the Listeria strain, or in another embodiment, a polypeptide that is not normally expressed by the Listeria strain, or in another embodiment, a polypeptide from a source other than the Listeria strain. In another embodiment, heterologous may be used to describe something derived from a different organism within the same species. In another embodiment, the heterologous antigen is expressed by a recombinant strain of Listeria, and is processed and presented to cytotoxic T-cells upon infection of mammalian cells by the recombinant strain. In another embodiment, the heterologous antigen expressed by Listeria species need not precisely match the corresponding unmodified antigen or protein in the tumor cell or infectious agent so long as it results in a T-cell response that recognizes the unmodified antigen or protein which is naturally expressed in the mammal. [000385] In another embodiment, a method of the present invention further comprises boosting the subject with a recombinant Listeria strain provided herein. In another embodiment, a method of the present invention comprises the step of administering a booster dose of vaccine comprising the recombinant Listeria strain provided herein. [000386] In one embodiment, "fused" refers to operable linkage by covalent bonding. In one embodiment, the term includes recombinant fusion (of nucleic acid sequences or open reading frames thereof). In another embodiment, the term includes chemical conjugation. [000387]“Transforming,” in one embodiment, refers to engineering a bacterial cell to take up a plasmid or other heterologous DNA molecule. In another embodiment,“transforming” refers to engineering a bacterial cell to express a gene of a plasmid or other heterologous DNA molecule. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000388] In another embodiment, conjugation is used to introduce genetic material and/or plasmids into bacteria. Methods for conjugation are well known in the art, and are described, for example, in Nikodinovic J et al (A second generation snp-derived Escherichia coli- Streptomyces shuttle expression vector that is generally transferable by conjugation. Plasmid. 2006 Nov;56(3):223-7) and Auchtung JM et al (Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response. Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12554-9). Each method represents a separate embodiment of the methods and compositions as provided herein. [000389]“Metabolic enzyme” refers, in another embodiment, to an enzyme involved in synthesis of a nutrient required by the host bacteria. In another embodiment, the term refers to an enzyme required for synthesis of a nutrient required by the host bacteria. In another embodiment, the term refers to an enzyme involved in synthesis of a nutrient utilized by the host bacteria. In another embodiment, the term refers to an enzyme involved in synthesis of a nutrient required for sustained growth of the host bacteria. In another embodiment, the enzyme is required for synthesis of the nutrient. Each possibility represents a separate embodiment of the methods and compositions as provided herein. [000390] It will be appreciated by a skilled artisan that the term "attenuation," may encompass a diminution in the ability of the bacterium to cause disease in an animal. In other words, the pathogenic characteristics of the attenuated Listeria strain have been lessened compared with wild-type Listeria, although the attenuated Listeria is capable of growth and maintenance in culture. Using as an example the intravenous inoculation of Balb/c mice with an attenuated Listeria, the lethal dose at which 50% of inoculated animals survive (LD.sub.50) is preferably increased above the LD.sub.50 of wild-type Listeria by at least about 10-fold, more preferably by at least about 100-fold, more preferably at least about 1,000 fold, even more preferably at least about 10,000 fold, and most preferably at least about 100,000-fold. An attenuated strain of Listeria is thus one which does not kill an animal to which it is administered, or is one which kills the animal only when the number of bacteria administered is vastly greater than the number of wild type non-attenuated bacteria which would be required to kill the same animal. An attenuated bacterium should also be construed to mean one which is incapable of replication in the general environment because the nutrient required for its growth is not present therein. Thus, the bacterium is limited to replication in a controlled environment wherein the required nutrient is provided. The attenuated strains of the present invention are therefore environmentally safe in that they are incapable of uncontrolled replication. [000391] In one embodiment, the Listeria as provided herein expresses a heterologous polypeptide, as described herein, in another embodiment, the Listeria as provided herein secretes a heterologous polypeptide, as described herein, and in another embodiment, the Listeria as provided herein expresses and secretes a heterologous polypeptide, as described herein. In another embodiment, the Listeria as provided herein comprises a heterologous polypeptide, and in another embodiment, comprises a nucleic acid that encodes a heterologous polypeptide. [000392] In one embodiment, Listeria strains as provided herein may be used in the preparation of vaccines or immunotherapies described herein. In one embodiment, Listeria strains as provided herein may be used in the preparation of peptide vaccines. Methods for preparing peptide vaccines are well known in the art and are described, for example, in EP1408048, United States Patent Application Number 20070154953, and OGASAWARA et al (Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8995-8999, October 1992). In one embodiment, peptide evolution techniques are used to create an antigen with higher immunogenicity. Techniques for peptide evolution are well known in the art and are described, for example in United States Patent 6773900. [000393] In one embodiment, the vaccines of the methods and compositions as provided herein may be administered to a host vertebrate animal, preferably a mammal, and more preferably a human, either alone or in combination with a pharmaceutically acceptable carrier. In another embodiment, the vaccine is administered in an amount effective to induce an immune response to the Listeria strain itself or to a heterologous antigen which the Listeria species has been modified to express. In another embodiment, the amount of vaccine to be administered may be routinely determined by one of skill in the art when in possession of the present disclosure. In another embodiment, a pharmaceutically acceptable carrier may include, but is not limited to, sterile distilled water, saline, phosphate buffered solutions or bicarbonate buffered solutions. In another embodiment, the pharmaceutically acceptable carrier selected and the amount of carrier to be used will depend upon several factors including the mode of administration, the strain of Listeria and the age and disease state of the vaccinee. In another embodiment, administration of the vaccine may be by an oral route, or it may be parenteral, intranasal, intramuscular, intravascular, intrarectal, intraperitoneal, or any one of a variety of well-known routes of administration. In another embodiment, the route of administration may be selected in accordance with the type of infectious agent or tumor to be treated. [000394] In one embodiment, the present invention provides a recombinant Listeria strain comprising a nucleic acid molecule encoding a heterologous antigenic polypeptide or fragment thereof, wherein said nucleic acid molecule is operably integrated into the Listeria genome in an open reading frame with an endogenous PEST-containing gene. [000395] The term "about" as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%. [000396] The term "subject" refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. In one embodiment, the term "subject" does not exclude an individual that is healthy in all respects and does not have or show signs of disease or disorder. [000397] In one embodiment, provided herein are kits comprising the pharmaceutical compositions or formulations comprising the recombinant Listeria provided herein. [000398] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. EXAMPLES [000399] A recombinant Lm that secretes PSA fused to tLLO (Lm-LLO-PSA) was developed. This strain elicits a potent PSA-specific immune response associated with regression of tumors in a mouse model for prostate cancer, wherein the expression of tLLO-PSA is derived from a plasmid based on pGG55 (Table 1), which confers antibiotic resistance to the vector. We recently developed a new strain for the PSA vaccine based on the pADV142 plasmid, which has no antibiotic resistance markers, and referred as LmddA-142 (Table 1). This new strain is 10 times more attenuated than Lm-LLO-PSA. In addition, LmddA-142 was slightly more immunogenic and significantly more efficacious in regressing PSA expressing tumors than the Lm-LLO-PSA. Table 1. Plasmids and strains

[000400] The sequence of the plasmid pAdv142 (6523 bp) was as follows: [000401] cggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggctgc accggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagc ggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagc cgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataaaga taccaggcgtttccccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgt ctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctg cgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttagag gagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggttca aagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagaccaaaa cgatctcaagaagatcatcttattaatcagataaaatatttctagccctcctttgattagtatattcctatcttaaagttacttttatgtggaggca ttaacatttgttaatgacgtcaaaaggatagcaagactagaataaagctataaagcaagcatataatattgcgtttcatctttagaagcgaat ttcgccaatattataattatcaaaagagaggggtggcaaacggtatttggcattattaggttaaaaaatgtagaaggagagtgaaacccat gaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaataa agaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaatc gataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaaggt tacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaattt cgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcatt aacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacgcagta aatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgatgacgaaatggcttaca gtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaatcagtgaagggaa aatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctgtt actaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttga aattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaaca aatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggcaacctcggagact tacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaagacaatga attagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatcactctggaggat acgttgctcaattcaacatttcttgggatgaagtaaattatgatctcgagattgtgggaggctgggagtgcgagaagcattcccaaccctg gcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatc aggaacaaaagcgtgatcttgctgggtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttccc acacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgt cagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctca ggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcg caagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggg gcccacttgtctgttatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacacca aggtggtgcattaccggaagtggatcaaggacaccatcgtggccaaccccTAAcccgggccactaactcaacgctagtagtggatt taatcccaaatgagccaacagaaccagaaccagaaacagaacaagtaacattggagttagaaatggaagaagaaaaaagcaatgatt tcgtgtgaataatgcacgaaatcattgcttatttttttaaaaagcgatatactagatataacgaaacaacgaactgaataaagaatacaaaa aaagagccacgaccagttaaagcctgagaaactttaactgcgagccttaattgattaccaccaatcaattaaagaagtcgagacccaaa atttggtaaagtatttaattactttattaatcagatacttaaatatctgtaaacccattatatcgggtttttgaggggatttcaagtctttaagaag ataccaggcaatcaattaagaaaaacttagttgattgccttttttgttgtgattcaactttgatcgtagcttctaactaattaattttcgtaagaaa ggagaacagctgaatgaatatcccttttgttgtagaaactgtgcttcatgacggcttgttaaagtacaaatttaaaaatagtaaaattcgctc aatcactaccaagccaggtaaaagtaaaggggctatttttgcgtatcgctcaaaaaaaagcatgattggcggacgtggcgttgttctgac ttccgaagaagcgattcacgaaaatcaagatacatttacgcattggacaccaaacgtttatcgttatggtacgtatgcagacgaaaaccgt tcatacactaaaggacattctgaaaacaatttaagacaaatcaataccttctttattgattttgatattcacacggaaaaagaaactatttcag caagcgatattttaacaacagctattgatttaggttttatgcctacgttaattatcaaatctgataaaggttatcaagcatattttgttttagaaac gccagtctatgtgacttcaaaatcagaatttaaatctgtcaaagcagccaaaataatctcgcaaaatatccgagaatattttggaaagtcttt gccagttgatctaacgtgcaatcattttgggattgctcgtataccaagaacggacaatgtagaattttttgatcccaattaccgttattctttca aagaatggcaagattggtctttcaaacaaacagataataagggctttactcgttcaagtctaacggttttaagcggtacagaaggcaaaa aacaagtagatgaaccctggtttaatctcttattgcacgaaacgaaattttcaggagaaaagggtttagtagggcgcaatagcgttatgttt accctctctttagcctactttagttcaggctattcaatcgaaacgtgcgaatataatatgtttgagtttaataatcgattagatcaacccttaga agaaaaagaagtaatcaaaattgttagaagtgcctattcagaaaactatcaaggggctaatagggaatacattaccattctttgcaaagct tgggtatcaagtgatttaaccagtaaagatttatttgtccgtcaagggtggtttaaattcaagaaaaaaagaagcgaacgtcaacgtgttca tttgtcagaatggaaagaagatttaatggcttatattagcgaaaaaagcgatgtatacaagccttatttagcgacgaccaaaaaagagatt agagaagtgctaggcattcctgaacggacattagataaattgctgaaggtactgaaggcgaatcaggaaattttctttaagattaaacca ggaagaaatggtggcattcaacttgctagtgttaaatcattgttgctatcgatcattaaattaaaaaaagaagaacgagaaagctatataaa ggcgctgacagcttcgtttaatttagaacgtacatttattcaagaaactctaaacaaattggcagaacgccccaaaacggacccacaact cgatttgtttagctacgatacaggctgaaaataaaacccgcactatgccattacatttatatctatgatacgtgtttgtttttctttgctggctag cttaattgcttatatttacctgcaataaaggatttcttacttccattatactcccattttccaaaaacatacggggaacacgggaacttattgta caggccacctcatagttaatggtttcgagccttcctgcaatctcatccatggaaatatattcatccccctgccggcctattaatgtgacttttg tgcccggcggatattcctgatccagctccaccataaattggtccatgcaaattcggccggcaattttcaggcgttttcccttcacaaggat gtcggtccctttcaattttcggagccagccgtccgcatagcctacaggcaccgtcccgatccatgtgtctttttccgctgtgtactcggctc cgtagctgacgctctcgccttttctgatcagtttgacatgtgacagtgtcgaatgcagggtaaatgccggacgcagctgaaacggtatct cgtccgacatgtcagcagacgggcgaaggccatacatgccgatgccgaatctgactgcattaaaaaagccttttttcagccggagtcca gcggcgctgttcgcgcagtggaccattagattctttaacggcagcggagcaatcagctctttaaagcgctcaaactgcattaagaaatag cctctttctttttcatccgctgtcgcaaaatgggtaaatacccctttgcactttaaacgagggttgcggtcaagaattgccatcacgttctgaa cttcttcctctgtttttacaccaagtctgttcatccccgtatcgaccttcagatgaaaatgaagagaaccttttttcgtgtggcgggctgcctc ctgaagccattcaacagaataacctgttaaggtcacgtcatactcagcagcgattgccacatactccgggggaaccgcgccaagcacc aatataggcgccttcaatccctttttgcgcagtgaaatcgcttcatccaaaatggccacggccaagcatgaagcacctgcgtcaagagc agcctttgctgtttctgcatcaccatgcccgtaggcgtttgctttcacaactgccatcaagtggacatgttcaccgatatgttttttcatattgc tgacattttcctttatcgcggacaagtcaatttccgcccacgtatctctgtaaaaaggttttgtgctcatggaaaactcctctcttttttcagaa aatcccagtacgtaattaagtatttgagaattaattttatattgattaatactaagtttacccagttttcacctaaaaaacaaatgatgagataat agctccaaaggctaaagaggactataccaactatttgttaattaa (SEQ ID NO: 77). This plasmid was sequenced at Genewiz facility from the E. coli strain on 2-20-08. EXAMPLE 1: Construction of attenuated Listeria strain-LmddΔactA and insertion of the human klk3 gene in frame to the hly gene in the Lmdd and Lmdda strains.

[000402] The strain Lm dal dat (Lmdd) was attenuated by the irreversible deletion of the virulence factor, ActA. An in-frame deletion of actA in the Lmdaldat (Lmdd) background was constructed to avoid any polar effects on the expression of downstream genes. The Lm dal dat ΔactA contains the first 19 amino acids at the N-terminal and 28 amino acid residues of the C-terminal with a deletion of 591 amino acids of ActA. [000403] The actA deletion mutant was produced by amplifying the chromosomal region corresponding to the upstream (657 bp-oligo’s Adv 271/272) and downstream (625 bp- oligo’s Adv 273/274) portions of actA and joining by PCR. The sequence of the primers used for this amplification is given in the Table 2. The upstream and downstream DNA regions of actA were cloned in the pNEB193 at the EcoRI/PstI restriction site and from this plasmid, the EcoRI/PstI was further cloned in the temperature sensitive plasmid pKSV7, resulting in ΔactA/pKSV7 (pAdv120).

Table 2: Sequence of primers that was used for the amplification of DNA sequences upstream and downstream of actA

[000404] The deletion of the gene from its chromosomal location was verified using primers that bind externally to the actA deletion region, which are shown in Figure 1 as primer 3 (Adv 305-tgggatggccaagaaattc, SEQ ID NO: 82) and primer 4 (Adv304-ctaccatgtcttccgttgcttg; SEQ ID NO: 83) . The PCR analysis was performed on the chromosomal DNA isolated from Lmdd and LmddΔactA. The sizes of the DNA fragments after amplification with two different sets of primer pairs 1/2 and 3/4 in Lmdd chromosomal DNA was expected to be 3.0 Kb and 3.4 Kb. On the other hand, the expected sizes of PCR using the primer pairs 1/2 and 3/4 for the LmddΔactA was 1.2 Kb and 1.6 Kb. Thus, PCR analysis in Figure 1 confirms that the 1.8 kb region of actA was deleted in the LmddΔactA strain. DNA sequencing was also performed on PCR products to confirm the deletion of actA containing region in the strain, LmddΔactA. EXAMPLE 2: Construction of the antibiotic-independent episomal expression system for antigen delivery by Lm vectors. [000405] The antibiotic-independent episomal expression system for antigen delivery by Lm vectors (pAdv142) is the next generation of the antibiotic-free plasmid pTV3 (Verch et al., Infect Immun, 2004. 72(11):6418-25, incorporated herein by reference). The gene for virulence gene transcription activator, prfA was deleted from pTV3 since Listeria strain Lmdd contains a copy of prfA gene in the chromosome. Additionally, the cassette for p60-Listeria dal at the NheI/PacI restriction site was replaced by p60-Bacillus subtilis dal resulting in plasmid pAdv134 (Figure 2A). The similarity of the Listeria and Bacillus dal genes is ~30%, virtually eliminating the chance of recombination between the plasmid and the remaining fragment of the dal gene in the Lmdd chromosome. The plasmid pAdv134 contained the antigen expression cassette tLLO-E7. The LmddA strain was transformed with the pADV134 plasmid and expression of the LLO-E7 protein from selected clones confirmed by Western blot (Figure 2B). The Lmdd system derived from the 10403S wild-type strain lacks antibiotic resistance markers, except for the Lmdd streptomycin resistance. [000406] Further, pAdv134 was restricted with XhoI/XmaI to clone human PSA, klk3 resulting in the plasmid, pAdv142. The new plasmid, pAdv142 (Figure 2C, Table 1) contains Bacillus dal (B-Dal) under the control of Listeria p60 promoter. The shuttle plasmid, pAdv142 complemented the growth of both E. coli ala drx MB2159 as well as Listeria monocytogenes strain Lmdd in the absence of exogenous D-alanine. The antigen expression cassette in the plasmid pAdv142 consists of hly promoter and LLO-PSA fusion protein (Figure 2C). [000407] The plasmid pAdv142 was transformed to the Listeria background strains, LmddactA strain resulting in Lm-ddA-LLO-PSA. The expression and secretion of LLO-PSA fusion protein by the strain, Lm-ddA-LLO-PSA was confirmed by Western Blot using anti- LLO and anti-PSA antibody (Figure 2D). There was stable expression and secretion of LLO- PSA fusion protein by the strain, Lm-ddA-LLO-PSA after two in vivo passages. EXAMPLE 3: In vitro and in vivo stability of the strain LmddA-LLO-PSA

[000408] The in vitro stability of the plasmid was examined by culturing the LmddA-LLO- PSA Listeria strain in the presence or absence of selective pressure for eight days. The selective pressure for the strain LmddA-LLO-PSA is D-alanine. Therefore, the strain LmddA- LLO-PSA was passaged in Brain-Heart Infusion (BHI) and BHI+ 100 μg/ml D-alanine. CFUs were determined for each day after plating on selective (BHI) and non-selective (BHI+D- alanine) medium. It was expected that a loss of plasmid will result in higher CFU after plating on non-selective medium (BHI+D-alanine). As depicted in Figure 4A, there was no difference between the number of CFU in selective and non-selective medium. This suggests that the plasmid pAdv142 was stable for at least 50 generations, when the experiment was terminated. [000409] Plasmid maintenance in vivo was determined by intravenous injection of 5 x 10⁷ CFU LmddA-LLO-PSA, in C57BL/6 mice. Viable bacteria were isolated from spleens homogenized in PBS at 24 h and 48 h. CFUs for each sample were determined at each time point on BHI plates and BHI + 100 μg/ml D-alanine. After plating the splenocytes on selective and non-selective medium, the colonies were recovered after 24 h. Since this strain is highly attenuated, the bacterial load is cleared in vivo in 24 h. No significant differences of CFUs were detected on selective and non-selective plates, indicating the stable presence of the recombinant plasmid in all isolated bacteria (Figure 4B). EXAMPLE 4: In vivo passaging, virulence and clearance of the strain LmddA-142

(LmddA-LLO-PSA) [000410] LmddA-142 is a recombinant Listeria strain that secretes the episomally expressed tLLO-PSA fusion protein. To determine a safe dose, mice were immunized with LmddA- LLO-PSA at various doses and toxic effects were determined. LmddA-LLO-PSA caused minimum toxic effects (data not shown). The results suggested that a dose of 10⁸ CFU of LmddA-LLO-PSA was well tolerated by mice. Virulence studies indicate that the strain LmddA-LLO-PSA was highly attenuated. [000411] The in vivo clearance of LmddA-LLO-PSA after administration of the safe dose, 10⁸ CFU intraperitoneally in C57BL/6 mice, was determined. There were no detectable colonies in the liver and spleen of mice immunized with LmddA-LLO-PSA after day 2. Since this strain is highly attenuated, it was completely cleared in vivo at 48 h (Figure 5A). [000412] To determine if the attenuation of LmddA-LLO-PSA attenuated the ability of the strain LmddA-LLO-PSA to infect macrophages and grow intracellularly, we performed a cell infection assay. Mouse macrophage-like cell line such as J774A.1 were infected in vitro with Listeria constructs and intracellular growth was quantified. The positive control strain, wild type Listeria strain 10403S grows intracellularly, and the negative control XFL7, a prfA mutant, cannot escape the phagolysosome and thus does not grow in J774 cells. The intracytoplasmic growth of LmddA-LLO-PSA was slower than 10403S due to the loss of the ability of this strain to spread from cell to cell (Figure 5B). The results indicate that LmddA- LLO-PSA has the ability to infect macrophages and grow intracytoplasmically. EXAMPLE 5: Immunogenicity of the strain-LmddA-LLO-PSA in C57BL/6 mice [000413] The PSA-specific immune responses elicited by the construct LmddA-LLO-PSA in C57BL/6 mice were determined using PSA tetramer staining. Mice were immunized twice with LmddA-LLO-PSA at one week intervals and the splenocytes were stained for PSA tetramer on day 6 after the boost. Staining of splenocytes with the PSA-specific tetramer showed that LmddA-LLO-PSA elicited 23% of PSA tetramer⁺CD8⁺CD62L^low cells (Figure 6A). [000414] The functional ability of the PSA-specific T cells to secrete IFN-γ after stimulation with PSA peptide for 5 h was examined using intracellular cytokine staining. There was a 200-fold increase in the percentage of CD8⁺CD62L^lowIFN-γ secreting cells stimulated with PSA peptide in the LmddA-LLO-PSA group compared to the naïve mice (Figure 6B), indicating that the LmddA-LLO-PSA strain is very immunogenic and primes high levels of functionally active PSA CD8⁺ T cell responses against PSA in the spleen. [000415] To determine the functional activity of cytotoxic T cells generated against PSA after immunizing mice with LmddA-LLO-PSA, we tested the ability of PSA-specific CTLs to lyse cells EL4 cells pulsed with H-2D^b peptide in an in vitro assay. A FACS-based caspase assay (Figure 6C) and Europium release (Figure 6D) were used to measure cell lysis. Splenocytes of mice immunized with LmddA-LLO-PSA contained CTLs with high cytolytic activity for the cells that display PSA peptide as a target antigen. [000416] Elispot was performed to determine the functional ability of effector T cells to secrete IFN-γ after 24 h stimulation with antigen. Using ELISpot, we observed there was a 20-fold increase in the number of spots for IFN-γ in splenocytes from mice immunized with LmddA-LLO-PSA stimulated with specific peptide when compared to the splenocytes of the naïve mice (Figure 6E). EXAMPLE 6: Immunization with the LmddA-142 strains induces regression of a tumor expressing PSA and infiltration of the tumor by PSA-specific CTLs. [000417] The therapeutic efficacy of the construct LmddA-142 (LmddA-LLO-PSA) was determined using a prostrate adenocarcinoma cell line engineered to express PSA (Tramp-C1- PSA (TPSA); Shahabi et al., 2008). Mice were subcutaneously implanted with 2 x 10⁶ TPSA cells. When tumors reached the palpable size of 4-6 mm, on day 6 after tumor inoculation, mice were immunized three times at one week intervals with 10⁸ CFU LmddA-142, 10⁷ CFU Lm-LLO-PSA (positive control) or left untreated. The naïve mice developed tumors gradually (Figure 7A). The mice immunized with LmddA-142 were all tumor-free until day 35 and gradually 3 out of 8 mice developed tumors, which grew at a much slower rate as compared to the naïve mice (Figure 7B). Five out of eight mice remained tumor free through day 70. As expected, Lm-LLO-PSA-vaccinated mice had fewer tumors than naïve controls and tumors developed more slowly than in controls (Figure 7C). Thus, the construct LmddA-LLO-PSA could regress 60 % of the tumors established by TPSA cell line and slow the growth of tumors in other mice. Cured mice that remained tumor free were rechallenged with TPSA tumors on day 68. [000418] Immunization of mice with the LmddA-142 can control the growth and induce regression of 7-day established Tramp-C1 tumors that were engineered to express PSA in more than 60% of the experimental animals (Figure 7B), compared to none in the untreated group (Figure 7A). The LmddA-142 was constructed using a highly attenuated vector (LmddA) and the plasmid pADV142 (Table 1). [000419] Further, the ability of PSA-specific CD8 lymphocytes generated by the LmddA- LLO-PSA construct to infiltrate tumors was investigated. Mice were subcutaneously implanted with a mixture of tumors and matrigel followed by two immunizations at seven day intervals with naiive or control (Lm-LLO-E7) Listeria, or with LmddA-LLO-PSA. Tumors were excised on day 21 and were analyzed for the population of CD8⁺CD62L^low PSA^tetramer+ and CD4⁺ CD25⁺FoxP3⁺ regulatory T cells infiltrating in the tumors. [000420] A very low number of CD8⁺CD62L^low PSA^tetramer+ tumor infiltrating lymphocytes (TILs) specific for PSA that were present in the both naïve and Lm-LLO-E7 control immunized mice was observed. However, there was a 10-30-fold increase in the percentage of PSA-specific CD8⁺CD62L^low PSA^tetramer+ TILs in the mice immunized with LmddA-LLO- PSA (Figure 7A). Interestingly, the population of CD8⁺CD62L^low PSA^tetramer+ cells in spleen was 7.5 fold less than in tumor (Figure 8A). [000421] In addition, the presence of CD4⁺/CD25⁺/Foxp3⁺ T regulatory cells (regs) in the tumors of untreated mice and Listeria immunized mice was determined. Interestingly, immunization with Listeria resulted in a considerable decrease in the number of CD4⁺ CD25⁺FoxP3⁺ T-regs in tumor but not in spleen (Figure 8B). However, the construct LmddA- LLO-PSA had a stronger impact in decreasing the frequency of CD4⁺ CD25⁺FoxP3⁺ T-regs in tumors when compared to the naïve and Lm-LLO-E7 immunized group (Figure 7B). [000422] Thus, the LmddA-142 vaccine can induce PSA-specific CD8⁺ T cells that are able to infiltrate the tumor site (Figure 8A). Interestingly, Immunization with LmddA-142 was associated with a decreased number of regulatory T cells in the tumor (Figure 7B), probably creating a more favorable environment for an efficient anti-tumor CTL activity. EXAMPLE 7: Lmdd-143 and LmddA-143 secretes a functional LLO despite the PSA fusion. [000423] The Lmdd-143 and LmddA-143 contain the full-length human klk3 gene, which encodes the PSA protein, inserted by homologous recombination downstream and in frame with the hly gene in the chromosome. These constructs were made by homologous recombination using the pKSV7 plasmid (Smith and Youngman, Biochimie. 1992; 74 (7-8) p705-711), which has a temperature-sensitive replicon, carrying the hly-klk3-mpl recombination cassette. Because of the plasmid excision after the second recombination event, the antibiotic resistance marker used for integration selection is lost. Additionally, the actA gene is deleted in the LmddA-143 strain (Figure 9A). The insertion of klk3 in frame with hly into the chromosome was verified by PCR (Figure 9B) and sequencing (data not shown) in both constructs. [000424] One important aspect of these chromosomal constructs is that the production of LLO-PSA would not completely abolish the function of LLO, which is required for escape of Listeria from the phagosome, cytosol invasion and efficient immunity generated by L. monocytogenes. Western-blot analysis of secreted proteins from Lmdd-143 and LmddA-143 culture supernatants revealed an ~81 kDa band corresponding to the LLO-PSA fusion protein and an ~60 kDa band, which is the expected size of LLO (Figure 10A), indicating that LLO is either cleaved from the LLO-PSA fusion or still produced as a single protein by L. monocytogenes, despite the fusion gene in the chromosome. The LLO secreted by Lmdd-143 and LmddA-143 retained 50% of the hemolytic activity, as compared to the wild-type L. monocytogenes 10403S (Figure 10B). In agreement with these results, both Lmdd-143 and LmddA-143 were able to replicate intracellularly in the macrophage-like J774 cell line (Figure 10C). EXAMPLE 8: Both Lmdd-143 and LmddA-143 elicit cell-mediated immune responses against the PSA antigen. [000425] After showing that both Lmdd-143 and LmddA-143 are able to secrete PSA fused to LLO, we investigated if these strains could elicit PSA-specific immune responses in vivo. C57Bl/6 mice were either left untreated or immunized twice with the Lmdd-143, LmddA-143 or LmddA-142. PSA-specific CD8⁺ T cell responses were measured by stimulating splenocytes with the PSA_65-74 peptide and intracellular staining for IFN-Ȗ. As shown in Figure 11, the immune response induced by the chromosomal and the plasmid-based vectors is similar. EXAMPLE 9: A recombinant Lm strain secreting a LLO-HMW-MAA fusion protein results in a broad antitumor response. [000426] Three Lm-based vaccines expressing distinct HMW-MAA fragments based on the position of previously mapped and predicted HLA-A2 epitopes were designed (Figure 12A). The Lm-tLLO-HMW-MMA_2160-2258 (also referred as Lm-LLO-HMW-MAA-C) is based on the avirulent Lm XFL-7 strain and a pGG55-based plasmid. This strain secretes a ~62 kDa band corresponding to the tLLO-HMW-MAA_2160-2258 fusion protein (Figure 12B). The secretion of tLLO-HMW-MAA_2160-2258 is relatively weak likely due to the high hydrophobicity of this fragment, which corresponds to the HMW-MAA transmembrane domain. Using B16F10 melanoma cells transfected with the full-length HMW-MAA gene, we observed that up to 62.5% of the mice immunized with the Lm-LLO-HMW-MAA-C could impede the growth of established tumors (Figure 12C). This result shows that HMW-MAA can be used as a target antigen in vaccination strategies. Interestingly, we also observed that immunization of mice with Lm-LLO-HMW-MAA-C significantly impaired the growth of tumors not engineered to express HMW-MAA, such as B16F10, RENCA and NT-2 (Figure 12D), which were derived from distinct mouse strains. In the NT-2 tumor model, which is a mammary carcinoma cell line expressing the rat HER-2/neu protein and is derived from the FVB/N transgenic mice, immunization with Lm-LLO-HMW-MAA-C 7 days after tumor inoculation not only impaired tumor growth but also induced regression of the tumor in 1 out of 5 mice (Figure 12D). EXAMPLE 10: Immunization of mice with Lm-LLO-HMW-MAA-C induces infiltration of the tumor stroma by CD8⁺ T cells and a significant reduction in the pericyte coverage in the tumor vasculature. [000427] Although NT-2 cells do not express the HMW-MAA homolog NG2, immunization of FVB/N mice with Lm-LLO-HMW-MAA-C significantly impaired the growth of NT-2 tumors and eventually led to tumor regression (Figure 12D). This tumor model was used to evaluate CD8⁺ T cells and pericytes in the tumor site by immunofluorescence. Staining of NT-2 tumor sections for CD8 showed infiltration of CD8⁺ T cells into the tumors and around blood vessels in mice immunized with the Lm-LLO-HMW-MAA-C vaccine, but not in mice immunized with the control vaccine (Figure 13A). Pericytes in NT-2 tumors were also analyzed by double staining with Į SMA and NG2 (murine homolog of HMW-MAA) antibodies. Data analysis from three independent NT-2 tumors showed a significant decrease in the number of pericytes in mice immunized with Lm-LLO-HMW-MAA-C, as compared to control (P≤ 0.05) (Figure 13B). Similar results were obtained when the analysis was restricted to cells stained for Į SMA, which is not targeted by the vaccine (data not shown). Thus, Lm-LLO-HMW-MAA-C vaccination impacts blood vessel formation in the tumor site by targeting pericytes. EXAMPLE 11: Development of a recombinant L. monocytogenes vector with enhanced anti-tumor activity by concomitant expression and secretion of LLO-PSA and tLLO- HMW-MAA_2160-2258 fusion proteins, eliciting immune responses to both heterologous antigens. Materials and Methods: [000428] Construction of the pADV168 plasmid. The HMW-MAA-C fragment is excised from a pCR2.1 -HMW-MAA_2160-2258 plasmid by double digestion with XhoI and XmaI restriction endonucleases. This fragment is cloned in the pADV134 plasmid already digested with XhoI and XmaI to excise the E7 gene. The pADV168 plasmid (Fig. 14B) is electroporated into electrocompetent the dal^(-) dat^(-) E. coli strain MB2159 and positive clones screened for RFLP and sequence analysis. [000429] Construction of Lmdd-143/168, LmddA-143/168 and the control strains LmddA- 168, Lmdd-143/134 and LmddA-143/134. Lmdd, Lmdd-143 and LmddA-143 is transformed with either pADV168 (Fig. 14B) or pADV134 plasmid. Transformants are selected on Brain- Heart Infusion-agar plates supplemented with streptomycin (250 @g/ml) and without D- alanine (BHIs medium). Individual clones are screened for LLO-PSA, tLLO-HMW-MAA_2160- ₂₂₅₈ and tLLO-E7 secretion in bacterial culture supernatants by Western-blot using an anti- LLO, anti-PSA or anti-E7 antibody. A selected clone from each strain will be evaluated for in vitro and in vivo virulence. Each strain is passaged twice in vivo to select the most stable recombinant clones. Briefly, a selected clone from each construct is grown and injected i.p to a group of 4 mice at 1x10⁸ CFU/mouse. Spleens are harvested on days 1 and 3, homogenized and plated on BHIs-agar plates. After the first passage, one colony from each strain is selected and passaged in vivo for a second time. To prevent further attenuation of the vector, to a level impairing its viability, constructs in two vectors with distinct attenuation levels (Lmdd- 143/168, LmddA-143/168) are generated. [000430] Construction of Listeria strain engineered to express and secrete two antigens as fusion proteins, LmddA244G. The antigen Her2 chimera was genetically fused to the genomic Listeriolysin O (Fig. 14A) and the second antigen HMW-MAA-C (HMC) was fused to a truncated Listeriolysin O in the plasmid. The secretion of fusion proteins LLO-ChHer2 and tLLO-HMC were detected by western blot using anti-LLO and anti-FLAG antibodies respectively (see Fig. 14C). [000431] Hemolytic assay. To determine the ability of genomic LLO to cause phagolysosomal escape a hemolytic assay was performed using secreted supernatant of control wild type 10403S and LmddA244G-168 and sheep red blood cells as target cells. [000432] In vitro intracellular replication in J774 cells. An in vitro intracellular growth assay was perfromed using a murine macrophage-like J774 cell line. Briefly, J774 cells were infected for 1 hour in medium without antibiotics at MOI of 1:1 with either one of the mono vaccines (LmddA164 and LmddA168–each generated by transforming an individual Listeria strain with pADV164 and another with pADV168, to arrive at LmddA164 and LmddA168, respectively– see Fig. 14B) or bivalent immunotherapy. At 1 h post-nfection, cells were treated with 10 μg/ml of gentamicin to kill extracellular bacteria. .. Samples were harvested at regular time intervals and cells lysed with water to quantify the number of intracellular CFU. Ten-fold serial dilutions of the lysates are plated in duplicates on BHI plates and colony-forming units (CFU) were counted in each sample. [000433] In vivo virulence studies. Groups of four C57BL/6 mice (7 weeks old) are injected i.p. with two different doses (1 x 10⁸ and 1 x 10⁹ CFUs/dose) of Lmdd-143/168, LmddA- 143/168, LmddA-168, Lmdd-143/134 or LmddA-143/134 strains. Mice are followed-up for 2 weeks for survival and LD₅₀ estimation. An LD₅₀ of >1 x 10⁸ constitutes an acceptable value based on previous experience with other Lm-based vaccines. RESULTS [000434] Once the pADV168 plasmid is successfully constructed, it is sequenced for the presence of the correct HMW-MAA sequence. This plasmid in these new strains express and secrete the LLO fusion proteins specific for each construct. These strains are highly attenuated, with an LD50 of at least 1x10⁸ CFU and likely higher than 1x10⁹ CFU for the actA-deficient (LmddA) strains, which lack the actA gene and consequently the ability of cell- to-cell spread. [000435] A recombinant Lm (LmddA-cHer2/HMC) was generated. This Lm strain expresses and secretes a chimeric Her2 (cHer2) protein chromosomally as fusion to genomic Listeriolysin O (LLO) and a fragment of HMW-MAA_2160-2258 (also named HMW-MAA C or HMC) using a plasmid as fusion to truncated LLO (tLLO), to target tumor cells and tumor vasculature concomitantly referred as LmddA244G-168. The expression and secretion of both the fusion proteins tLLO-HMC and LLO-cHer2 from LmddA244G-168 was detected by western blot using anti-FLAG and anti-LLO specific antibodies (Fig. 14B). Furthermore, the vaccine LmddA244G-168 was passaged twice in vivo in mice to stabilize the virulence of LmddA-244G and to confirm that it retained the expression of recombinant fusion proteins (Fig. 14B). The vaccine LmddA244G-168 retained its ability to express and secrete both the fusion proteins, tLLO-HMC and LLO-cHer2 after two in vivo mice passages. [000436] The strain LmddA244G-168, expresses chromosomal LLO as fusion protein LLO- cHer2 which may impact the functional ability of LLO to cause phagolysosomal escape. To determine this hemolytic assay was performed using secreted supernatant of control wild type 10403S and LmddA244G-168 and sheep red blood cells as target cells. As indicated in Figure 15A, there was a 1.5 fold reduction in the hemolytic ability of LmddA244G-168 when compared to wild type highly virulent Lm strain 10403S. [000437] Additionally, to examine if the expression of fusion protein LLO-cHer2 did not cause any deleterious effect on the ability of LmddA-cHer2/HMC to infect macrophages and its intracellular growth, a cell infection assay was performed using mouse macrophage like cells J774. The results as specified in Fig. 15B showed that intracellular growth behavior of different Listeria-based immunotherapies expressing either single or dual antigens were similar suggesting that the co-expression of two antigens did not cause any change in the ability of LmddA244G-168 to present target intracellular proteins for immunological responses.

EXAMPLE 12: Detection of immune responses and anti-tumor effects elicited upon immunization with Lmdd-244G/168. [000438] Immune responses to cHer2 and HMW-MAA are studied in mice upon immunization with Lmdd-244G-168 strain using standard methods, such as detection of IFN-Ȗ production against these antigens. The therapeutic efficacy of dual-expression vectors are tested in the NT2 breast tumor model. [000439] IFN-Ȗ ELISpot. We evaluated the ability of bivalent immunotherapy to generate immune responses specific for the two antigens Her2 and HMW-MAA in FvB mice. Mice (3/group) were immunized with different immunotherapies such as LmddA134 (Lm-control), LmddA164 and LmddA244G/168 on day 0 and boosted on day 14. Her2/neu specific immune responses were detected in the spleens harvested on day 21. The IFN-γ ELispot assay was done according to the kit instructions and spleen cells were stimulated with peptide epitope specific for the intracellular region (RLLQETELV) (SEQ ID NO: 84). [000440] IFN-γ ELISA. The generation of HMW-MAA-C specific immune responses in the splenocytes of immunized mice was determined by stimulating cells with HMA-MAA-C protein for 2 days. The IFN-γ release was detected by ELISA performed using mouse interferon-gamma ELISA kit. [000441] Anti-tumor efficacy. The antitumor efficacy was examined using mouse NT2 breast tumor model. FvB mice were implanted with 1 x 10⁶ NT2 cells on day 0 and established tumors on right flank were treated starting day 6 with three immunizations at weekly intervals with different immunotherapies. Tumors were monitored twice a week until the end of the study. Mice were euthanized if the tumor diameter was greater than 1.5 cm. RESULTS [000442] Next, the anti-tumor therapeutic efficacy of LmddA244G was examined using mouse NT2 breast tumor model. The FvB mice bearing established NT2 tumors on right flank were treated with three immunizations at one week interval with different immunotherapies expressing either mono antigens LmddA164 (ChHer2), LmddA168 (HMC) or bivalent immunotherapy LmddA244G-168. Treatment with both mono- and bivalent-immunotherapy caused a reduction of NT2 tumor as indicated in Figure 16A and 16C. However, a stronger impact on the control of NT2 tumor growth was observed after treatment with bivalent- immunotherapy. Additional analysis on the percent tumor free mice in each group confirmed that treatment with bivalent immunotherapy generated maximum tumor-free mice (70 %) when compared to mono-immunotherapy (less than 40 %) treated groups. These observations support that targeting two antigens concurrently using Listeria monocytogenes as vector for therapy resulted in enhanced anti-tumor efficacy. [000443] The ability of bivalent immunotherapy was evaluated to generate immune responses specific for the two antigens Her2 and HMW-MAA in FvB mice. Mice were immunized with different immunotherapies such as LmddA134 (irrelevant control), LmddA164 and LmddA244G/168 on day 0 and boosted on day 14. Her2/neu specific immune responses were detected using an ELISpot based assay using peptide epitope specific for intracellular region. Both mono and bivalent–immunotherapy expressing Her2 generated comparable levels of immune responses detected using ELISpot-based assay (see Figure 17). [000444] The generation and for HMW-MAA-C specific immune responses in the splenocytes of immunized mice was detected using ELISA. The expression of tumor antigen from Lm using either single copy (mono immunotherapy) or multicopy (bivalent immunotherapy) based expression generates comparable level of antigen-specific immune responses (see Figure 17). EXAMPLE 13: Immunization with either Lmdd-143/168 or LmddA-143/168 results in pericyte destruction, up-regulation of adhesion molecules in endothelial cells and enhanced infiltration of TILs specific for PSA. [000445] Characterization of tumor infiltrating lymphocytes and endothelial cell- adhesion molecules induced upon immunization with Lmdd-143/168 or LmddA-143/168. The tumors from mice immunized with either Lmdd-143/168 or LmddA-143/168 are analyzed by immunofluorescence to study expression of adhesion molecules by endothelial cells, blood vessel density and pericyte coverage in the tumor vasculature, as well as infiltration of the tumor by immune cells, including CD8 and CD4 T cells. TILs specific for the PSA antigen are characterized by tetramer analysis and functional tests. [000446] Analysis of tumor infiltrating lymphocytes (TILs). TPSA23 cells embedded in matrigel are inoculated s.c in mice (n=3 per group), which are immunized on days 7 and 14 with either Lmdd-143/168 or LmddA-143/168, depending on which one is the more effective according to results obtained in anti-tumor studies. For comparison, mice are immunized with LmddA-142, LmddA-168, a control Lm vaccine or left untreated. On day 21, the tumors are surgically excised, washed in ice-cold PBS and minced with a scalpel. The tumors are treated with dispase to solubilize the Matrigel and release single cells for analysis. PSA-specific CD8⁺ T cells are stained with a PSA65-74 H-2Db tetramer-PE and anti-mouse CD8-FITC, CD3-PerCP-Cy5.5 and CD62L-APC antibodies. To analyze regulatory T cell in the tumor, TILs are stained with CD4-FITC, CD3-PerCP-Cy5.5 and CD25-APC and subsequently permeabilized for FoxP3 staining (anti-FoxP3-PE, Milteny Biotec). Cells are analyzed by a FACS Calibur cytometer and CellQuestPro software (BD Biosciences). [000447] Immunofluorescence. On day 21 post tumor inoculation, the TPSA23 tumors embedded in matrigel are surgically excised and a fragment immediately cryopreserved in OCT freezing medium. The tumor fragments are cryosectioned for 8-10μm thick sections. For immunofluorescence, samples are thawed and fixed using 4% formalin. After blocking, sections are stained with antibodies in blocking solution in a humidified chamber at 37°C for 1 hour. DAPI (Invitrogen) staining are performed according to manufacturer instructions. For intracellular stains (ĮSMA), incubation is performed in PBS / 0.1% Tween / 1% BSA solution. Slides are cover-slipped using a mounting solution (Biomeda) with anti-fading agents, set for 24 hours and kept at 4°C until imaging using Spot Image Software (2006) and BX51 series Olympus fluorescent microscope. CD8, CD4, FoxP3, ĮSMA, NG2, CD31, ICAM-1, VCAM-1 and VAP-1 are evaluated by immunofluorescence. [000448] Statistical analysis: Non-parametric Mann-Whitney and Kruskal-Wallis tests are applied to compare tumor sizes among different treatment groups. Tumor sizes are compared at the latest time-point with the highest number of mice in each group (8 mice). A p-value of less than 0.05 is considered statistically significant in these analyses. RESULTS [000449] Immunization of TPSA23-bearing mice with the Lmdd-143/168 and LmddA-143/168 results in higher numbers of effector TILs specific to PSA and also decreases pericyte coverage of the tumor vasculature. Further, cell-adhesion markers are significantly up- regulated in immunized mice. [000450] An increased infiltration of T cells in the tumors treated with Bivalent- immunotherapy was observed using anti-CD3 and anti-CD8 staining as compared to the monovalent treated groups (Figures 18-19). [000451] In addition there was an increase in the infiltration of CD4⁺ T cells in the tumors of both LMddA168 and LmddA244-168 treated groups. Further, a reduction in the staining of blood vessels by anti-CD31 (Figure 21) was observed in the LmddA168 and LmddA244G- 168 treatment groups. EXAMPLE 14: Anti-tumor efficacy of a dual cHER2-CA9 Listeria vaccine on the growth of 4T1 tumors implanted in the mammary glands of Balb/c mice. [000452] Experimental Details: [000453] A recombinant Lm (LmddA-cHer2/CA9) was generated. This Lm strain expresses and secretes a chimeric Her2 (cHer2) protein chromosomally as fusion to genomic Listeriolysin O (LLO) and a fragment of human Carbonic Anhydrase 9 (CA9) using a plasmid as fusion to truncated LLO (tLLO), to multiply target tumor cells. [000454] Table 3

[000455] Vaccine titers:

LmddA-PSA– 6.5x10⁸

LmddA-CA9– 1.4x10¹⁰

LmddA-cHER2– 1.05x10¹⁰

Dual cHer2-CA9 (LmddA)– 1.5x10⁹ [000456] Experimental Protocols: [000457] 4T1 cells were grown in RPMI containing 10% FBS, 2mM L-Glu, 1.5g/L sodium bicarbonate, 4.5g/L glucose, 1mM sodium pyruvate, and 10mM HEPES. On the day of injection, cells were trypsinized then washed 2X in PBS. Cells were counted and resuspended

[000458] Tumors were implanted in the mammary glands of each of the mice. There are 16 mice per group. The mice were vaccinated 3 days later. On day 4, 4 mice in each group were euthanized and examined for tumor growth. Mice were given the boost of each vaccine on day 10. On day 11, 4 mice in each group were euthanized and tumors were measured. On day 18, 4-5 mice in each group were euthanized and tumors were measured. On day 21, the remaining mice in each group were euthanized and the tumors were measured. Results [000459] On day 4, the tumors are barely palpable, so no measurements were made. [000460] Table 4

[000461] The numbers in Table 4 show that the dual vaccine (recombinant Listeria expressing two heterologous antigens) initially (day 11) has a large impact on the tumor mass (Figure 21). Two of the mice euthanized had no tumors and the others were smaller than the control and around the size of the mono-CA9 and cHER2 vaccinated mice. By day 18, multiple tumors can be measured in some of the mice in several of the groups. The PBS and PSA control mice have much larger tumors than the mono-CA9 and cHER2 or the dual vaccine groups. The dual vaccine group has one outlier with a large tumor burden, otherwise the average for that group would have been the smallest. The experiment was terminated early as the mice in several groups were looking very sick and had been dying. However, at the last measurement, the mice in the dual vaccine group had the smallest tumors (Figure 21). This may be due to the level of control on tumor growth that was seen early on. [000462] In conclusion, the dual vaccine shows an initial level of tumor control in the 4T1 model that is higher than levels achieved with the mono-vaccines or the control mice as the dual vaccine groups have the smallest tumor burden at the end of the experiment (see Figure 22 and 23). EXAMPLE 15: Anti-tumor efficacy of a dual cHER2- HMW-MAA Listeria vaccine on the growth of 4T1 tumors implanted in the mammary glands of Balb/c mice. [000463] Experimental Details: [000464] A recombinant Lm (LmddA-cHer2/HMW-MAA) was generated. This Lm strain expresses and secretes a chimeric Her2 (cHer2) protein chromosomally as fusion to genomic Listeriolysin O (LLO) and high molecular weight melanoma associated antigen (HMW- MAA) using a plasmid as fusion to truncated LLO (tLLO), to multiply target tumor cells. [000465] Table 5

[000466] Vaccine titers:

LmddA-PSA– 6.5x10⁸

LmddA-HMW-MMA– 1.4x10¹⁰

LmddA-cHER2– 1.05x10¹⁰

Dual cHer2- HMW-MMA (LmddA)– 1.5x10⁹ [000467] Experimental Protocols: [000468] 4T1 cells were grown in RPMI containing 10% FBS, 2mM L-Glu, 1.5g/L sodium bicarbonate, 4.5g/L glucose, 1mM sodium pyruvate, and 10mM HEPES. On the day of injection, cells were trypsinized then washed 2X in PBS. Cells were counted and resuspended

[000469] Tumors were implanted in the mammary glands of each of the mice. There are 16 mice per group. The mice were vaccinated 3 days later. On day 8, 4 mice in each group were euthanized and examined for tumor growth. Mice were given the boost of each vaccine on day 8. On day 15, 4 mice in each group were euthanized and tumors were measured. . Mice were given another boost of each vaccine on day 15. On day 15, 21, 28 and 35, 4-5 mice in each group were euthanized and tumors were measured. On days 42, the remaining mice in each group were euthanized and the tumors were measured. Results [000470] The results are summarized in Figure 24. The graphs show that the dual vaccine (recombinant Listeria expressing two heterologous antigens) has a large impact on the tumor volume (Figure 24). The volumes of tumors in mice receiving bivalent therapy were smaller than both the control and the mono- HMW-MMA and cHER2 vaccinated mice. The PBS and PSA control mice have tumors that are comparable in volume to the mono- HMW-MMA and cHER2 groups. [000471] In conclusion, the dual vaccine shows an initial level of tumor control in the 4T1 model that is higher than levels achieved with the mono-vaccines or the control mice as the dual vaccine groups have the smallest tumor burden at the end of the experiment (see Figure 24). EXAMPLE 16: Comparative study of anti-tumor efficacy of a dual and sequential cHER2- HMW-MAA Listeria vaccine on the growth of NT2 breast tumor model. [000472] Experimental Details: [000473] The antitumor efficacy was examined using mouse NT2 breast tumor model. FvB mice were implanted with 1 x 10⁶ NT2 cells on day 0 and established tumors on right flank were treated starting day 6 with three immunizations at weekly intervals with different immunotherapies. Tumors were monitored twice a week until the end of the study. Mice were euthanized if the tumor diameter was greater than 1.5 cm. [000474] Table 6

RESULTS [000475] The anti-tumor therapeutic efficacy of different listeria vaccine regiments was examined using mouse NT2 breast tumor model. The FvB mice bearing established NT2 tumors on right flank were treated with five immunizations of 1x10⁸ at one week intervals with different immunotherapies expressing either mono antigens LmddA164 (ChHer2), LmddA168 (HMC), or combination of therapies expressing both antigens administered simultaneously (bivalent therapy) (see Table 1). In addition, a combination vs sequential therapy was carried out with different immunotherapies expressing either mono antigens LmddA164 (ChHer2), LmddA168 (HMC), a combination of therapies expressing both antigens administered simultaneously (bivalent therapy), or a combination of sequential administration of each mono antigen (cHer2 followed by HMW-MAA). In the latter, 3 weekly doses of LmddA164 (ChHer2) were administered and were followed by 3 weekly doses of LmddA168 (HMC) (see Table 1). The results are summarized in Figure 25. All the regiments caused approximately equivalent reduction of NT2 tumor volume as indicated in Figure 25. These observations show that simultaneous or sequential administration of two monovalent constructs was at least comparable to bivalent constructs in controlling tumor growth (Fig. 25). [000476] Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

What is claimed is:

1. A recombinant Listeria strain comprising a first and a second nucleic acid molecule, said first nucleic acid molecule encoding a recombinant polypeptide comprising a carbonic anhydrase 9 (CA9) antigen or a functional fragment thereof fused to a truncated Listeriolysin O (LLO), and wherein said second nucleic acid molecule encodes a chimeric HER (cHER2) protein fused to an endogenous LLO.

2. The recombinant Listeria strain of claim 1 , wherein said cHER2 is encoded by SEQ ID NO: 26.

3. The recombinant Listeria strain of claim 1, wherein said first nucleic acid molecule is in a plasmid.

4. The recombinant Listeria strain of any one of claims 1-3, wherein said second nucleic acid molecule is operably integrated into the Listeria genome.

5. The recombinant Listeria strain of any one of claims 1-4, wherein said second nucleic acid molecule encoding a recombinant polypeptide comprising cHER2 is operably integrated in frame with the endogenous gene encoding LLO in the Listeria genome.

6. The recombinant Listeria strain of any one of claims claim 1-5, wherein said recombinant Listeria strain is an auxotrophic Listeria strain.

7. The recombinant Listeria strain of claim 6, wherein said auxotrophic Listeria strain comprises a mutation, deletion or inactivation in a D-alanine racemase gene or a D- amino acid transferase gene.

8. The recombinant Listeria strain of claim 6, wherein said auxotrophic Listeria strain comprises a mutation, deletion or inactivation in a D-alanine racemase gene and a D- amino acid transferase gene.

9. The recombinant Listeria strain of any one of claims 4-8, wherein said plasmid comprises a metabolic enzyme that complements the auxotrophy of said Listeria strain.

10. The recombinant Listeria strain of claim 9, wherein said metabolic enzyme is an amino acid metabolism enzyme.

11. The recombinant Listeria strain of any one of claims 9-10, wherein said metabolic enzyme is an alanine racemase enzyme.

12. The recombinant Listeria strain of any one of claims 9-10, wherein said metabolic enzyme is a D-amino acid transferase enzyme.

13. The recombinant Listeria strain of any one of claims 1-12, wherein said Listeria comprises a mutation, deletion or inactivation in the endogenous actA gene.

14. The recombinant Listeria strain of any one of claims 1-13, wherein said recombinant Listeria strain is capable of escaping the phagolysosome.

15. The recombinant Listeria strain of any one of claims 1-14, wherein said recombinant Listeria strain has been passaged through an animal host.

16. The recombinant Listeria strain of any one of claims 1-15, wherein said recombinant Listeria strain is a recombinant Listeria monocytogenes strain.

17. An immunogenic composition comprising the recombinant Listeria strain of any one of claims 1-16 and an adjuvant, cytokine, chemokine, or a combination thereof.

18. A method of inducing an anti-tumor immune response in a subject comprising administering to said subject the recombinant Listeria of any one of claims 1-16, or the immunogenic composition of claim 17.

19. The method of claim 18, wherein said composition is administered orally or parenterally.

20. The method of any one of claims 18- 19, wherein said tumor expresses CA9.

21. The method of any one of claims 18-20, wherein said tumor expresses a HER2/neu protein.

22. A method of treating, suppressing, or inhibiting a tumor or cancer in a subject comprising administering the recombinant Listeria of any one of claims 1-16, or the immunogenic composition of claim 17 to said subject.

23. The method of claim 21, wherein said composition is administered orally or parenterally.

24. The method of any one of claims 22-23, wherein said tumor or cancer is prostate tumor or cancer, ovarian tumor or cancer, breast tumor or cancer, brain tumor or cancer, lung tumor or cancer, gastrointestinal tumor or cancer, sarcoma, pancreatic tumor or cancer, melanoma, or a combination thereof.

25. Use of the recombinant Listeria of claims 1-16, or the immunogenic composition of claim 17, for inducing an immune response against an antigen-expressing tumor or cancer.

26. Use of the recombinant Listeria of claims 1-16, or the immunogenic composition of claim 17, for treating, suppressing, or inhibiting a tumor or cancer in a subject.

27. The use of any one of claims 25-26, wherein said tumor or cancer is prostate tumor or cancer, ovarian tumor or cancer, breast tumor or cancer, brain tumor or cancer, lung tumor or cancer, gastrointestinal tumor or cancer, sarcoma, pancreatic tumor or cancer, melanoma, or a combination thereof.

28. A method of increasing the efficacy of a Listeria -based immunotherapy, said method comprising sequentially or concomitantly administering two or more recombinant Listeria strains to a subject having a tumor.

29. The method of claim 28, wherein each one of said Listeria strains comprises a nucleic acid molecule, said nucleic acid molecule encoding a recombinant polypeptide comprising a heterologous antigen fused to a PEST-containing polypeptide.

30. The method of any one of claims 28-29, wherein said heterologous antigen is chimeric HER (cHER2), CA9, PSA or HMW-MAA-C.

31. The method of any one of claims 28-30, wherein said PEST-containing polypeptide is an endogenous LLO or a truncated LLO expressed from a plasmid.

32. The method of any one of claims claim 28-31, wherein said recombinant Listeria strain is an auxotrophic Listeria strain.

33. The method of claim 32, wherein said auxotrophic Listeria strain comprises a mutation, deletion or inactivation in a D-alanine racemase gene or a D-amino acid transferase gene.

34. The method of claim 32, wherein said auxotrophic Listeria strain comprises a mutation, deletion or inactivation in a D-alanine racemase gene and a D-amino acid transferase gene.

35. The method of any one of claims 31-34, wherein said plasmid comprises a metabolic enzyme that complements the auxotrophy of said Listeria strain.

36. The method of claim 35, wherein said metabolic enzyme is an amino acid metabolism enzyme.

37. The method of any one of claims 35-36, wherein said metabolic enzyme is an alanine racemase enzyme.

38. The method of any one of claims 28-37, wherein said Listeria comprises a mutation, deletion or inactivation in the endogenous actA gene.

39. The method of claims 30-38, wherein said cHER2 is encoded by SEQ ID NO: 26.